Culture of Coldwater Marine Fish

CULTURE OF COLD-WATER MARINE FISH Edited by E. Moksness E. Kjørsvik and Y. Olsen Fishing News Books An imprint of Bla...

Author: Erlend Moksness | E. Kjorsvik | Y. Olsen

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CULTURE OF COLD-WATER MARINE FISH Edited by

E. Moksness E. Kjørsvik and

Y. Olsen

Fishing News Books An imprint of Blackwell Science

CULTURE OF COLD-WATER MARINE FISH Edited by

E. Moksness E. Kjørsvik and

Y. Olsen

Fishing News Books An imprint of Blackwell Science

© 2004 by Blackwell Publishing Ltd Editorial Offices: Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Tel: +44 (0)1865 776868 Iowa State Press, a Blackwell Publishing Company, 2121 State Avenue, Ames, Iowa 50014-8300, USA Tel: +1 515 292 0140 Blackwell Publishing Asia Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia Tel: +61 (0)3 8359 1011 The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 2004 by Blackwell Publishing Ltd Library of Congress Cataloging-in-Publication Data Culture of cold-water marine fish / editors, E. Moksness, E. Kjørsvik, and Y. Olsen. p. cm. Includes bibliographical references (p. ). ISBN 0-85238-276-6 (hardback : alk. paper) 1. Marine fishes. 2. Fish-culture. I. Moksness, Erlend. II. Kjørsvik, E. III. Olsen, Yngvar. SH163.C85 2004 639.34¢2—dc21 2003002265 ISBN 0-85238-276-6 A catalogue record for this title is available from the British Library Set in 10 on 13 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed and bound in Great Britain using acid-free paper by Bath Press, Bath For further information on Blackwell Publishing, visit our website: www.blackwellpublishing.com

Contents

Preface List of Contributors 1 Introduction The Editors 1.1

References

2 Abiotic Factors B.R. Howell and S.M. Baynes 2.1 2.2 2.3 2.4

Introduction Oxygen and oxygen consumption Ammonia Temperature 2.4.1 Seasonal temperature cycle and spawning 2.4.2 Egg and larval development 2.4.3 Sex ratio 2.4.4 Growth and metabolism 2.5 Salinity 2.6 Hydrogen sulphide 2.7 Light 2.7.1 Growth and development 2.7.2 Reproduction 2.8 Algae blooms 2.9 Site selection 2.10 References 3 Microbial Interactions, Prophylaxis and Diseases O. Vadstein, T.A. Mo and Ø. Bergh 3.1

Fish–microbe interactions and implications in aquaculture 3.1.1 Disease-causing organisms 3.1.2 Normal fish–microbe interactions, infection pathways and pathogenesis 3.1.3 The immune system of fish

xiv xv 1 5 7 7 7 10 12 13 13 14 14 15 17 18 18 20 21 23 26 28 28 28 29 33

iv

Contents

3.2

3.3

3.4

3.5

3.6

3.7

3.8 3.9

Viral diseases: diagnosis 3.2.1 Infectious pancreatic necrosis virus (IPNV) 3.2.2 Nodaviruses 3.2.3 Other viruses Bacterial diseases: diagnosis 3.3.1 Vibrio species 3.3.2 Aeromonas species Parasitic protists and metazoans: diagnosis, prophylaxis and treatment 3.4.1 Protists 3.4.1.1 Amoebae 3.4.1.2 Apicomplexans 3.4.1.3 Microsporidia 3.4.1.4 Ciliates 3.4.1.5 Flagellates 3.4.2 Metazoans 3.4.2.1 Myxosporidia (parasitic Cnidarians) 3.4.2.2 Monogeneans 3.4.2.3 Cestodes 3.4.2.4 Trematodes 3.4.2.5 Nematodes 3.4.2.6 Acanthocephalans 3.4.2.7 Leeches 3.4.2.8 Crustaceans A strategy for microbial control 3.5.1 General considerations 3.5.2 A strategy for microbial control and important elements in such a strategy Improving environmental conditions 3.6.1 Non-selective reduction of microbes 3.6.2 The use of probiotics 3.6.3 Selection for desirable bacteria Improving the resistance of the fish 3.7.1 Modulation of specific immunity—vaccination 3.7.2 Modulation of non-specific immunity 3.7.3 The effect of nutrition and genetics on resistance against microbes Closing remarks References

4 Live Food Technology of Cold-Water Marine Fish Larvae Y. Olsen 4.1 4.2

Introduction Cultivation systems

35 35 36 37 38 39 39 40 41 41 41 42 43 44 44 44 45 46 46 47 48 48 49 50 51 55 57 57 58 59 61 61 62 63 63 64 73

73 75

Contents

4.3

4.4

4.5 4.6 4.7

Production of rotifers 4.3.1 Biological characteristics 4.3.1.1 General biology and life history 4.3.1.2 Feeding kinetics of B. plicatilis 4.3.1.3 Growth, mortality and egg ratio 4.3.2 Cultivation feed and feed treatments 4.3.3 Cultivation of rotifers 4.3.3.1 Maintenance of stock cultures 4.3.3.2 Inoculation phase 4.3.3.3 Early growth phase 4.3.3.4 Late growth phase—harvesting strategies 4.3.3.5 Production in batch culture 4.3.3.6 Production in continuous culture 4.3.4 High-intensity rotifer cultivation 4.3.5 Problems in rotifer cultivation 4.3.5.1 Feeding-related problems 4.3.5.2 Environmentally related problems 4.3.5.3 Disease and contamination 4.3.5.4 Problem identification—diagnostic criteria 4.3.5.5 Counter-measures against undesirable situations 4.3.6 Biochemical composition during steady-state feeding and growth 4.3.6.1 Proteins and essential amino acids 4.3.6.2 Lipids and essential fatty acids 4.3.6.3 Vitamins and minerals 4.3.7 Short-term enrichment techniques to improve nutritional value 4.3.7.1 Proteins 4.3.7.2 Lipids and fatty acids 4.3.8 Stability of nutritional value Production of Artemia 4.4.1 Feeding and growth 4.4.2 Biomass and biochemical composition 4.4.3 Pre-enrichment cultivation 4.4.3.1 Disinfection of cysts 4.4.3.2 Decapsulation of cysts 4.4.3.3 Hatching of cysts 4.4.4 Enrichment and stability of n-3 fatty acids 4.4.4.1 n-3 HUFA enrichment 4.4.4.2 Stability of n-3 fatty acids post-enrichment 4.4.5 n-3 HUFA of Artemia juveniles 4.4.6 Vitamins and minerals Marine copepods Concluding remarks References

v

76 76 76 77 79 80 81 81 82 83 86 88 92 96 96 97 97 98 98 99 100 100 102 105 106 106 106 108 111 111 113 114 114 114 115 116 116 119 121 122 122 124 125

vi

Contents

5 Brood Stock and Egg Production D. Pavlov, E. Kjørsvik, T. Refsti and Ø. Andersen 5.1 5.2

5.3

5.4

5.5

Reproductive strategies Gonad maturation 5.2.1 Females 5.2.2 Males 5.2.3 Spawning once or many times? 5.2.4 Endocrine regulation Brood-stock management and egg production 5.3.1 Brood-stock nutrition 5.3.1.1 Ration size 5.3.1.2 Feed composition 5.3.1.3 Fatty acids 5.3.1.4 Micronutrients 5.3.1.5 Pigments and minerals 5.3.2 Photoperiod 5.3.3 Temperature 5.3.4 Present husbandry practices and egg collection 5.3.4.1 Cod 5.3.4.2 Turbot 5.3.4.3 Atlantic halibut 5.3.4.4 Wolf-fish Egg quality 5.4.1 Assessment of egg quality 5.4.1.1 Egg morphology 5.4.1.2 Fertilisation success and cortical reaction 5.4.1.3 Blastomere morphology 5.4.1.4 Egg size 5.4.1.5 Chemical content 5.4.1.6 Cytology 5.4.1.7 Oxygen consumption 5.4.1.8 Evaluating mammalian embryo quality 5.4.2 Factors affecting egg quality 5.4.2.1 Over-ripening 5.4.2.2 Viability of ovulated eggs in vivo 5.4.2.3 Viability of ovulated eggs in vitro 5.4.2.4 Changes in the eggs 5.4.3 Change in egg quality over the spawning season 5.4.4 Maternal effects 5.4.5 Conclusions Sperm production and quality 5.5.1 Features of sperm production and quality

129

129 132 134 135 136 138 142 143 144 145 146 147 149 150 152 152 153 154 154 155 156 157 159 159 161 164 166 167 168 168 168 168 169 170 170 172 173 174 175 175

Contents

5.6

5.7

5.8

5.5.1.1 Morphology 5.5.1.2 Gonadosomatic index and ejaculate volume 5.5.1.3 Concentration 5.5.1.4 Motility 5.5.1.5 Fertilising capacity 5.5.1.6 Biochemistry and oxygen consumption 5.5.2 Influence of environmental factors on sperm quality 5.5.3 Sperm storage Selective breeding 5.6.1 Expected benefits 5.6.2 Phenotypic value and variance 5.6.3 Genotype by environmental interaction 5.6.4 Breeding goal 5.6.5 Growth rate 5.6.6 Feed efficiency 5.6.7 Disease resistance 5.6.8 Quality 5.6.9 Age at sexual maturation 5.6.10 Base population and brood-stock development 5.6.11 Inbreeding 5.6.12 Selection methods 5.6.12.1 Individual selection (mass selection) 5.6.12.2 Family selection 5.6.12.3 Progeny testing 5.6.12.4 Combined selection 5.6.13 Response to selection 5.6.14 Multi-trait selection Modern biotechnology and aquaculture 5.7.1 Molecular pedigree analysis 5.7.2 Genetic mapping and QTL analysis 5.7.3 Transgenic fish 5.7.4 Future prospects References

6 From Fertilisation to the End of Metamorphosis—Functional Development E. Kjørsvik, K. Pittman and D. Pavlov 6.1 6.2 6.3 6.4

Intervals of fish ontogeny and definitions of the organism 6.1.1 Relative duration of the various stages of development Egg classification 6.2.1 Egg structure and composition Insemination and fertilisation Embryonic development and hatching

vii

175 176 177 178 179 179 180 181 182 183 183 184 185 185 185 185 186 186 186 186 186 186 187 187 187 188 188 188 189 191 192 193 193 204

204 207 208 209 212 214

viii

Contents

6.5

6.6

6.7

6.8 6.9

6.4.1 Cod (Gadus morhua) 6.4.2 Wolf-fish (Anarhichas lupus) 6.4.3 Embryo growth and yolk absorption From hatching to metamorphosis 6.5.1 To be a larva . . . 6.5.2 . . . or not to be a larva 6.5.3 The yolk-sac period—preparation for real ‘real life’ 6.5.4 Metamorphosis Functional development of organ systems from hatching to metamorphosis 6.6.1 Sensory system 6.6.1.1 Vision and the oculovestibular system 6.6.1.2 Chemosensory system 6.6.1.3 Lateral line 6.6.2 Digestive system 6.6.2.1 Gut, pancreas and liver differentiation 6.6.2.2 Digestive enzymes 6.6.2.3 Digestive physiology—lipids, proteins and carbohydrates 6.6.2.4 Stomach development and metamorphosis 6.6.3 Muscle and body skeleton 6.6.3.1 Swimming capacity and muscle development 6.6.3.2 Musculature changes during metamorphosis 6.6.3.3 Skeletal changes 6.6.4 Swim-bladder 6.6.5 Osmoregulation 6.6.6 Respiration and excretion 6.6.7 Neuroendocrine systems 6.6.8 Growth hormone, prolactin and cortisol 6.6.9 The immune system 6.6.10 Skin and pigmentation 6.6.11 Larval feeding behaviour 6.6.12 Larval growth 6.6.12.1 How can we express larval growth? 6.6.13 Influence of diet 6.6.14 Juvenile quality Hatchery design ` 6.7.1 The demersal eggs of wolf-fish 6.7.2 Pelagic eggs (cod, turbot, halibut) 6.7.2.1 Cod 6.7.2.2 Turbot 6.7.2.3 Halibut Critical aspects of larval cultivation References

215 221 224 225 225 226 226 228 229 230 230 232 232 233 234 236 239 240 241 241 243 244 245 247 248 250 255 256 256 257 260 261 262 264 265 265 265 266 266 266 267 269

Contents

7 First Feeding Technology Y. Olsen, T. van der Meeren and K.I. Reitan 7.1 7.2

7.3

7.4

7.5

Introduction Nutritional requirements of marine fish larvae 7.2.1 Essential fatty acids 7.2.2 Main lipid classes 7.2.3 Physiological basis of n-3 HUFA requirements 7.2.4 Protein and essential amino acids 7.2.5 Protein versus lipid nutrition Definitions and system description 7.3.1 Extensive systems: large closed nature-like systems 7.3.2 Semi-intensive systems: large suspended mesocosms, enclosures or outdoor tanks 7.3.3 Larval rearing in relatively small tanks: classical intensive hatchery techniques 7.3.3.1 Water treatment and supply 7.3.3.2 Production lines for live feed 7.3.3.3 Larval rearing systems 7.3.3.4 Automation and process control Larval rearing in ‘nature-like systems’ 7.4.1 Pioneer work 7.4.2 The ‘lagoon method’ as a production system 7.4.3 Larval food and feeding in mesocosms 7.4.3.1 Initiation of exogenous feeding: the ‘green gut’ 7.4.3.2 Prey selection 7.4.3.3 Feeding, growth and survival Larval first feeding in intensive systems 7.5.1 Physical chemical environment 7.5.2 Feeding characteristics of fish larvae 7.5.2.1 Food selection 7.5.2.2 Feeding and functional response 7.5.2.3 Larval feeding rate of live feed 7.5.2.4 Larval feeding on microalgae 7.5.3 Feeding regime components for cold-water larviculture 7.5.3.1 Microalgae 7.5.3.2 Rotifers 7.5.3.3 Artemia naupli 7.5.3.4 Juvenile Artemia 7.5.4 Tentative feeding regimes for common species 7.5.4.1 Stocking densities 7.5.4.2 Live food rations 7.5.4.3 Atlantic cod and haddock 7.5.4.4 Atlantic halibut

ix

279

279 280 280 280 282 284 284 285 287 289 290 291 292 293 294 295 295 296 297 298 299 300 301 301 302 303 303 304 305 306 306 307 307 308 308 308 309 310 310

x

Contents

7.6 7.7

7.5.4.5 Turbot 7.5.4.6 Sole, wolf-fish and hake 7.5.5 Growth-rate characteristics during first feeding 7.5.6 Nutritional challenges and conflicts 7.5.6.1 Criteria of nutritional value for live feed 7.5.6.2 Lipids and n-3 HUFA 7.5.6.3 Essential amino acids and proteins 7.5.6.4 Synergetic importance of lipids and proteins 7.5.6.5 Vitamins and minerals 7.5.6.6 General recommendations on larval nutrition 7.5.7 Microbial conflicts and challenges 7.5.7.1 Methods of non-selective reduction of bacteria 7.5.7.2 Methods for selective enhancement of favourable bacteria 7.5.8 Use of ‘green water’ techniques 7.5.8.1 Effects of ‘green water’ 7.5.8.2 Nutritional effects 7.5.8.3 Microbial effects 7.5.8.4 Live feed retention time in larval tanks Concluding remarks References

8 Weaning and Nursery J. Stoss, K. Hamre and H. Otterå 8.1 8.2 8.3

8.4 8.5

Introduction Developmental aspects of digestion in marine fish larvae Nutrition 8.3.1 Macronutrient composition 8.3.2 Composition of dietary protein 8.3.3 Composition of the lipid fraction 8.3.4 Vitamin supplementation Microparticulate diets Weaning and nursery stage, practical aspects 8.5.1 General 8.5.1.1 The role of early start feeding 8.5.1.2 Early weaning and co-feeding 8.5.1.3 Uptake and ingestion of formulated diets 8.5.1.4 Availability of particulate food 8.5.1.5 Tank hygiene 8.5.1.6 Rearing temperature and light 8.5.1.7 Vaccination against bacterial diseases 8.5.1.8 Handling of fish 8.5.2 Cod

312 312 313 314 314 315 319 322 323 324 325 326 327 328 329 331 332 332 333 333 337

337 338 341 341 342 344 344 345 346 346 346 347 348 348 348 349 350 351 352

Contents

8.6

8.5.2.1 8.5.2.2 8.5.2.3 8.5.2.4 8.5.2.5 8.5.3 Turbot 8.5.3.1 8.5.3.2 8.5.3.3 8.5.3.4 8.5.3.5 8.5.4 Halibut 8.5.4.1 8.5.4.2 8.5.4.3 8.5.4.4 References

Early weaning Weaning Cannibalism Gas bubble formation Nursery Early weaning Weaning Nursery Rearing density Tanks Growth Early weaning Weaning Nursery

9 On-Growing to Market Size M. Jobling 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10 9.11 9.12

Introduction Analysis of feeds and feedstuffs Protein requirements and sources Lipids and lipid requirements Carbohydrates Micronutrients: vitamins and minerals Feed types and formulations Feeding regimes and practices Growth and feed conversion Nutrient deposition and body composition Concluding comments References

10 The Status and Perspectives for the Species T. Svåsand, H.M. Otterå, G.L. Taranger, M. Litvak, A.B. Skiftesvik, R.M. Bjelland, D.A. Pavlov, J.Chr. Holm, T. Harboe, A. Mangor-Jensen, B. Norberg and B. Howell 10.1

Atlantic cod 10.1.1 Introduction 10.1.2 Brood stock, egg production and incubation 10.1.3 Extensive production 10.1.4 Intensive production 10.1.5 On-growing

xi

352 353 354 354 354 355 355 356 357 358 358 359 359 360 360 361 361 363 363 364 367 377 383 385 391 396 402 413 422 423 433

433 433 433 435 436 440

xii

Contents

10.2

10.3

10.4

10.5

10.6

10.7

10.1.6 Future prospects 10.1.7 References 10.1.8 Further reading Haddock 10.2.1 Introduction 10.2.2 Brood stock, egg production and incubation 10.2.3 Larval rearing 10.2.4 Weaning and on-growing 10.2.5 Health 10.2.6 Commercial development 10.2.7 Future prospects 10.2.8 Further reading Hake 10.3.1 Introduction 10.3.2 Egg production and incubation 10.3.3 Larval rearing 10.3.4 Weaning and on-growing 10.3.5 Future prospects 10.3.6 References Wolf-fish 10.4.1 Introduction 10.4.2 Brood stock, egg production and incubation 10.4.3 Larval rearing and on-growing 10.4.4 Future prospects 10.4.5 References Halibut 10.5.1 Introduction 10.5.2 Brood stock, egg production and incubation 10.5.3 Larval rearing 10.5.4 Weaning and on-growing 10.5.5 On-growing systems 10.5.6. References Turbot 10.6.1 Introduction 10.6.2 Brood stock, egg production and incubation 10.6.3 Larval rearing 10.6.4 Weaning and on-growing 10.6.5 Future prospects 10.6.6 References Sole 10.7.1 Introduction 10.7.2 Brood stock, egg production and incubation 10.7.3 Larval rearing 10.7.4 Weaning and on-growing

442 442 443 444 444 446 447 448 449 449 450 450 451 451 451 452 453 454 454 454 454 455 456 458 459 461 461 462 464 466 466 466 467 467 468 468 469 470 471 471 471 472 472 473

Contents

10.7.5 10.7.6

xiii

Future prospects References

473 474

11 Marine Stock Enhancement and Sea-Ranching T. Svåsand and E. Moksness

475

11.1 11.2

11.3 11.4

11.5 11.6

Introduction Stock enhancement and sea-ranching in Europe and North America 11.2.1 Atlantic cod 11.2.2 Other cold-water species Stock enhancement and sea-ranching in Asia Prospects and limitations of enhancement and sea-ranching 11.4.1 Biological constraints 11.4.2 Economic constraints Recommendations and guidelines References

12 New Species in Aquaculture: Some Basic Economic Aspects R. Engelsen, F. Asche, F. Skjennum and G. Adoff 12.1

Introduction 12.1.1 Markets, productivity and production growth 12.1.2 The economics of a market and productivity 12.1.3 The evolution of the salmon industry 12.1.4 The evolution of the sea bass and sea bream industry 12.1.5 The evolution of the American catfish industry 12.2 Cod 12.3 Haddock 12.4 European hake 12.5 Wolf-fish 12.6 Halibut 12.7 Turbot 12.8 Sole 12.9 Conclusions 12.10 References Index

475 477 477 479 480 482 482 483 485 485 487 487 489 491 493 495 496 496 501 501 503 504 507 511 512 515 517

Preface

Mariculture has a long history worldwide, but intensive production, as seen with salmon today, is a relatively new approach. The global production and marketing of salmon is a success story, and many other species have, partly because of that success, caught interest as potential candidates for future mariculture. In Norway, the attention on species other than salmonides increased in the 1980s, and significant research efforts were made to develop future production technology for cold-water marine fish species. The work focused very much on early life histories and the main goal was to obtain a high and stable number of juveniles for further on-growing. As a result of this research activity, efforts were increased to educate students from high school to university. Besides our own research on mariculture, we have also been involved in the teaching of students at university level. Over the years we have all produced compendiums as textbooks for the students and when Blackwell Publishing approached us some years ago to ask if we were willing to turn our compendiums into a book, we accepted their offer immediately. This was mainly because we saw a great need for such a textbook for our students. We also realised that each of the sections covered in the book had developed so quickly that we needed to invite expert authors for the chapters to improve the quality of the book. All the invited authors responded positively, and we are very grateful to them for their contribution to the book. We would also like to thank all the students, technicians and colleagues, from all research institutions, who have contributed to the comprehensive research and technological developments, that have made this book possible. The book is dedicated to the late Professor Arne Jensen who was an enthusiastic driving force for research in developing aquaculture in Norway until his death in August 2000. The Editors

List of Contributors

Adoff, Grethe Rønnevik Bergen Aqua AS, Bredalsmarken 15–17, Møhlenpris, Box 2604, 5836 Bergen, Norway Andersen, Øivind Institute of Aquaculture Research, PO Box 5010, N-1432 Aas, Norway Asche, Frank Stavanger University College, Box 2557 Ullandhaug, N-4091 Stavanger, Norway Baynes, Stephen M. CEFAS Weymouth Laboratory, Banach oad, The Nothe, Weymouth, Dorset DT4 8UB, UK Bergh, Øyvind Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Bjelland, Reidun M. Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Engelsen, Rolf Bergen Aqua AS, Bredalsmarken 15–17, Møhlenpris, Box 2604, 5836 Bergen, Norway Hamre, Kristin Fiskeridirektoratets ernæringsinstitutt, PO Box 185 Sentrum, N-5804 Bergen, Norway Harboe, Torstein Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Holm, Jens Chr. Directorate of Fisheries, Strandgt. 229, PO Box 185, N-5804 Bergen, Norway Howell, Bari R. CEFAS Weymouth Laboratory, Banach oad, The Nothe, Weymouth, Dorset DT4 8UB, UK. Present address: 73 Plasturton Avenue, Pontcanna, Cardiff CF11 9HN, UK Jobling, Malcolm NFH, University of Tromsø, Dramsveien 201, N-9001 Tromsø, Norway Kjørsvik, Elin Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway

xvi

List of contributors

Litvak, Matt Centre for Coastal Studies and Aquaculture, University of New Brunswick, PO Box 5050, Saint John, NB, E2L 4L5, Canada Mangor-Jensen, Anders Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Meeren, Terje van der Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Mo, Tor Atle National Veterinary Institute, Ullevålsvn. 68, N-0454 Oslo 4, Norway Moksness, Erlend Institute of Marine Research, Department of Coastal Zone, Flødevigen Marine Research Station, N-4817 His, Norway Norberg, Birgitta Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Olsen, Yngvar Trondhjem Biological Station, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondhjem, Norway Otterå, Håkon M. Institute of Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5017 Bergen, Norway Pavlov, Dimitri A. Moscow State University, Faculty of Biology, Department of Ichthyology, Moscow 119899, Russia Pittman, Karin Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology, N-5020 Bergen, Norway Refstie, Terje Akvaforsk, PO Box 203, N-6600 Sunndalsøra, Norway Reitan, Kjell I. SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway Skiftesvik, Anne B. Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Skjennum, Finn Chr. Bergen Aqua AS, Bredalsmarken 15–17, Møhlenpris, Box 2604, 5836 Bergen, Norway Stoss, Joachim Stolt Seafarm AS, Øyeslatta 63, N-4484 Øyestranda, Norway Svåsand, Terje Institute of Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5017 Bergen, Norway Taranger, Geir L. Institute of Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5017 Bergen, Norway Vadstein, Olav Trondhjem Biological Station, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondhjem, Norway

Chapter 1

Introduction The Editors

The annual global production from aquaculture exceeded 30 million metric tons in 1998, representing more then 20% of the total annual yield from fisheries and mariculture. Asia is by far the largest producer of marine products, with respect to both fisheries and aquaculture, and China has the dominant position by contributing more than 32% of the total yield. Aquaculture production in Europe is approximately 5% of that in Asia, and is dominated by anadromous salmonids (Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss) and the marine species sea bass (Dicentrarchus labrax), sea bream (Sparus aurata) and turbot (Scophthalmus maximus). While these three marine species are mainly farmed in southern Europe, the main production of salmonids is in northern European countries, particularly Norway and Scotland. At the turn of this century, the total world production of salmonids reached more than 1 million metric tons, of which Norway produced 50%. The doubling of production over the last 10 years is evidence of the success of this form of farming, and is indicative of the potential that may exist for farming other species in the cold-water environment (FAO-statistics, www.fao.org). Environmental conditions are the natural limiting factors for aquaculture activity. In northern Europe the temperature can vary between 0 and 20°C during the season, whereas the salinity can vary between 10 and 34‰ in coastal waters. In this environment there is potential for farming several marine fish species, such as cod (Gadus morhua), haddock (Melanogrammus aeglefinus), hake (Merluccius merluccius), wolf-fish (Anarhichas spp.), halibut (Hippoglossus hippoglossus), turbot and sole (Solea solea). The enhancement of natural stocks and sea ranching are important current and future issues in northern Europe as well as in other regions of the world. In fact, these activities were the driving force behind the first initiatives in the cultivation of cold-water marine fish, which started in 1882. A former ship’s officer, Captain G.M. Dannevig, took the initiative and established a cod hatchery in southern Norway in the 1880s, with the main goal of improving and stabilising the local cod fishery (‘Flødevigen Utklekningsanstalt’, now the Institute of Marine Research, Department of Coastal Zones, Flødevigen Marine Research Station). At the same time, the American S.P. Baird convinced the American Congress to build a hatchery for cod in Woods Hole, and the two hatcheries were in operation at approximately the same time. Dannevig collected brood stock of cod in the winter, transferred the fish to a larger spawning basin, and collected the newly fertilised eggs for further incubation in the laboratory. After hatching, the

2

Culture of cold-water marine fish

yolk-sac larvae were transported from the hatching boxes in the laboratory to different locations along the coast of southern Norway and released into the sea. A similar activity took place off the north-eastern coast of the USA, and during the period between 1920 and 1950 a large number of newly hatched cod larvae were released in the coastal waters of the two countries. In Norway, the activity started in 1883 and did not end until 1971 (Solemdal et al., 1984). In 1886, as part of the verification that the cod larvae hatched in the Flødevigen hatchery were viable and able to grow and survive in nature through to the juvenile stage, yolksac larvae of cod were stocked in a 2500-m3 outdoor concrete enclosure. The experiment was a success, and generated the first artificially produced juvenile cod. It was another 50 years before the next major achievement. Gunnar Rollefsen, who was in charge of the hatchery at the Trondhjem Biological Station, succeeded in feeding plaice (Platessa platessa) larvae, a local candidate for stock enhancement, on newly hatched Artemia. This was a major breakthrough in the development of intensive production methods for juvenile marine fish using readily produced live food. Rollefsen then became the first director of the Institute of Marine Research in Bergen and was not able to continue this work. No further major progress was made in the field during the next couple of decades, but an important step forward was the discovery by Japanese scientists (Ito, 1960, see review by Nagata & Hirata, 1986) that a ubiquitous brackish water rotifer, Brachionus plicatilis, could also be an effective live food for marine fish larvae. In Europe, British scientists pioneered the development of mass-rearing techniques for marine flatfish initially using Artemia nauplii to feed plaice and sole (Shelbourne, 1964), and subsequently demonstrating the utility of the smaller rotifer, B. plicatilis, for the smaller larvae of lemon sole (Microstomus kitt) (Howell, 1973) and turbot (Jones et al., 1974). Success with turbot was not achieved before the early 1970s. The use of Brachionus sp. was a key factor contributing to this success, but the crucial discovery was that significant survival of turbot larvae could only be obtained if certain species of microalgae were added to the tanks along with rotifers and Artemia (Howell, 1979). This was also found to be important for other species, and led to an appreciation of the importance of dietary sources of long-chain polyunsaturated fatty acids for these species (Scott & Middleton, 1979). This was paralled by work in Japan that demonstrated that n-3 fatty acids were essential for marine fish (Fukusho, 1977; Kitajima & Koda, 1976), and that marine larvae in particular had high dietary requirements (Watanabe et al., 1978). These findings resulted in cultivation techniques that ensured a high n-3 fatty acid level in the live feed. Considerable efforts have been made worldwide during the last decade to improve and adapt the Japanese methods to species with commercial potential. This has resulted in major activity in mariculture worldwide for marine cold-water fish species. The challenges of developing suitable technology to rear marine fish are multidisciplinary, and the crucial factors involved in the process, from brood stock maintenance to the market place, are illustrated in Fig. 1.1. The production chain involves several steps, and the general challenges during production involve knowledge about: (1) the general biology of the fish species; (2) the chemical and physical environmental requirements;

Introduction

3

Figure 1.1 Schematic diagram of the production process and the knowledge needed for economically feasible mariculture production.

(3) the nutritional requirements; (4) the effects of the microbial environment. Comprehensive knowledge of all these fundamental issues, and on their interactions in fish culture, is needed to develop a feasible mariculture industry of cold-water fish species. The production of viable juveniles is still the main constraint on the development of new cold-water species for aquaculture. Early experience suggests that the on-growing stages are more straightforward for most species considered, and that it will be possible to take advantage of the infrastructure and services that are already established for Atlantic salmon. This has been possible to a limited extent for juvenile production. One reason for the difficulty in rearing marine fish larvae is illustrated in Fig. 1.2, which shows that the larvae are very tiny and immature at the time of hatching compared with those of salmon. The larvae are 3–22 mm in length when they start feeding. Larvae of wolf-fish, which is the only species able to feed on dry pellets from the very beginning, are the largest. Other species have to be fed live prey during the larval stage. However, this is also the case for other marine species such as sea bass and sea bream that are successfully farmed in southern Europe. An additional challenge with the cold-water species has been their high requirements of n-3 fatty acids, and the complicated life cycle of some targeted species such as the Atlantic halibut. It is clear, however, that major improvements have been made over the last few decades, and that further progress is made each year. Another issue that has delayed the development of the culture of marine species is the success of the Atlantic salmon industry in the northern hemisphere. Salmon aquaculture has

4


Hake, Turbot, Sole Cod, Haddock Halibut

Wolf-fish

Salmon

5

10

15 20 Length (MM)

25

30

Figure 1.2 Relative size of the larvae of cold-water fish species that are considered as candidates for mariculture. Table 1.1 Year 1886 1976 1980 1983 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001

Overview of number of juveniles produced until 2001 of five selected cold-water marine fish species. Cod

Haddock

Wolf-fish

Halibut

Turbot

2 500 4 400 2 60 000 130 000 50 000 266 000 422 000 513 000 330 000 390 000 86 000 175 000 320 000 230 000 53 000 156 000 113 000 100 000 500 000 1 000 000

400 300 5 000 5 000 5 000 2 000 18 000 79 000 95 000 165 000

25 000 31 000 50 000

2 100 1 000 2 000 4 000 8 000 35 000 130 000 316 000 794 000 175 000 255 000 520 000 1 520 000 1 620 000 1 740 000 1 300 000

40 000 150 000 352 000 430 000 615 000 350 000 510 000 380 000 460 000 300 000

1 705 000 ~6 000 000 ~7 000 000

been very successful and is economically feasible, and has attracted private investment to the degree that this has inhibited developments with marine species. The key to successful cultivation of any fish species is to produce high and stable numbers of high-quality juveniles annually. Table 1.1 shows the number of juveniles produced annu-

Introduction

5

ally for the seven marine fish species which are being considered for future aquaculture. Turbot have been cultured for some years, and juvenile production is high and stable from one year to another, while haddock and hake still are at an early stage. The production of juvenile sole is not a problem, but the rather slow growth rate so far achieved during the on-growing stages has inhibited commercial developments. Both turbot and sole have been included in this book because they are important as model fish for the other species. Cod has been cultivated for more then 100 years, but the juvenile production is still too low and too unreliable. The fish is well known in the market, and the yield from fisheries has declined significantly over the years. These facts, combined with increasing prices in the market, mean that its potential in aquaculture is currently considered to be high. The key for future production is intensive production. Similarly, juvenile production of halibut has been too unstable to support a significant industry. However, with a better understanding and control of intensive juvenile production, farmed halibut will soon be on the market in increasing numbers. Experiments with wolf-fish have taken place during the past 15 years, and of the two species considered, the spotted wolf-fish (A. minor) has been favoured because of its much higher body growth rate compared with that of the common wolf-fish (A. lupus). The production of hake juveniles has been very limited, and a lot of work still remains to be done before any form of commercialisation can be realised. This textbook describes the current state of knowledge on the cultivation of cold-water marine fishes, and considers problems and solutions in the rearing of all life stages.

1.1 References Fukusho, K. (1977) Nutritional effects of the rotifer, Brachionus plicatilis, raised by baking yeast on larval fish of Oplegnathus fasciatus, by enrichment with Chlorella sp. before feeding. Bull. Nagasaki Pref. Inst. Fish, 3, 152–4 (in Japanese). Howell, B.R. (1973) Marine fish culture in Britain. VIII. A marine rotifer, Brachionus plicatilis Muller, and the larvae of the mussel, Mytilus edulis L., as foods for larval flatfish. J. Cons. Int. Explor. Mer, 35, 1–6. Howell, B.R. (1979) Experiments on the rearing of larval turbot, Scophthalmus maximus L. Aquaculture, 18, 215–25. Ito, T. (1960) On the culture of mixohaline rotifer Brachionus plicatilis O.F. Muller. Rep. Fac. Fish. Mie Pref. Univ., 3, 708–40 (in Japanese). Jones, A., Alderson, R. & Howell, B.R. (1974) Progress towards the development of a successful rearing technique for larvae of the turbot, Scophthalmus maximus L. In: The Early Life History of Fish (ed J.H.S. Blaxter), pp. 731–7. Springer, Berlin, Heidelberg, New York. Kitajima, C. & Koda, T. (1976) Lethal effects of the rotifer cultured with baking yeast on the larval sea bream, Pagrus major, and the increase rate using the rotifer recultured with Chlorella sp. Bull. Nagasaki Pref. Inst. Fish, 2, 113–16 (in Japanese). Nagata, W.D. & Hirata, H. (1986) Mariculture in Japan: past, present, and future prospectives. Mini Rev. Data File Fish. Res., 4, 1–38. Scott, A.P. & Middleton, C. (1979) Unicellular algae as a food for turbot (Scophthalmus maximus) larvae—the importance of dietary long-chain polyunsaturated fatty acids. Aquaculture, 18, 227–40. Shelbourne, J.E. (1964) The artificial propagation of marine fish. Adv. Mar. Biol., 2, 1–83.

6


Solemdal, P., Dahl, E., Danielssen, D.S. & Moksness, E. (1984) The cod hatchery in Flødevigen— background and realities. In: The Propagation of Cod Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 17–45. Flødevigen Rapportserie, 1. Watanabe, T., Kitajima, C., Arakawa, T., Fukusho, K. & Fujita, S. (1978) Nutritional quality of rotifer, Brachionus plicatilis, as a living feed from the viewpoint of essential fatty acids for fish. Bull. Jpn. Soc. Sci. Fish, 44, 1109–14 (in Japanese).

Chapter 2

Abiotic Factors B.R. Howell and S.M. Baynes

2.1 Introduction The abiotic environment is of critical importance in determining the performance of cultured fish. Marine waters are mainly characterised by their temperature and salinity, and these factors will largely determine the range of species that can be grown. Light also has a considerable impact on performance, but is rather more readily manipulated than either temperature or salinity. Fish growth and survival is also affected by a range of other water-quality factors, many of which are influenced directly or indirectly by the metabolic activity of the fish themselves. In intensive culture systems, for example, growth may be particularly impaired by sub-optimal oxygen and ammonia levels. Less direct adverse effects may arise from exposure to hydrogen sulphide or algae blooms, both of which may be a consequence of high environmental organic loadings arising from the activities of the fish farm. This chapter reviews the impacts of the major environmental factors affecting the performance of fish, and concludes with a review of the factors to be considered in site selection.

2.2 Oxygen and Oxygen Consumption Oxygen content is probably the most important aspect of water quality because of its central role in driving metabolic processes and the profound effects of deprivation on both the performance and the welfare of the fish. Sub-optimal dissolved oxygen (DO) levels can increase stress and disease susceptibility, reduce food intake, growth and food conversion efficiencies, and, of course, under extreme conditions cause mortalities. Regular monitoring of oxygen levels in culture systems is a clear imperative, but changes in behaviour may also provide an indication of the development of sub-optimal conditions. These may include reduced feeding activity, swimming near the surface and increased ventilation rates. In extensive systems such as ponds, photosynthetic activity plays an important role in determining DO levels and consequently considerable diel variations can occur which can threaten fish survival. In productive fish ponds in which dense concentrations of algae have developed, diel variations in DO can be as great as 7–8 mg l-1, with super-saturation occurring during the day and sub-saturation during the night (Boyd, 1979). In northern latitudes, however, intensive culture methods predominate, and in these systems the supply of oxygen

8


OXYGEN CONCENTRATION (mg/l)

12 o

10 C o

20 C

10

8

6 0

10

20

30

40

SALINITY (g/l) Figure 2.1 The relationship between oxygen solubility and salinity at 10 and 20°C.

is dependent on water flow to a much greater extent than that arising from photosynthetic activity. In intensive systems, stocking levels will be determined by the ability to maintain adequate oxygen levels. Maximum stocking densities in cages will be limited by water exchange rates, and with careful management diel variations rarely exceed 2–3 mg l-1, although these may still prove stressful (Beveridge, 1987). However, extreme reductions in DO may occur as a result of dense algae blooms, and these can result in fish kills. Particularly high stocking densities can be achieved in tanks with high and more stable DO levels achieved through the use of a diverse range of aeration devices or direct injection of oxygen. However, damaging fluctuations may occur unless adequate control and fail-safe systems are in place. The solubility of oxygen in water depends on a variety of factors, but is largely determined by temperature, salinity and altitude, since solubility decreases as each of these factors increases. In a marine environment, altitude has negligible impact. The effect of temperature and salinity (Fig. 2.1) shows that saturation oxygen concentrations are higher in freshwater than in seawater at 35 p.p.t., and that there is a reduction in saturation oxygen concentration in each medium with increasing temperature. Pressure also has a significant effect on DO levels. The saturation concentration of freshwater at sea level is 11.0 mg l-1 compared with 8.9 mg l-1 at an altitude of 2000 m (Shepherd & Bromage, 1988). Water acquires oxygen from the air by a process of diffusion at a rate which is dependent on water temperature, salinity, the degree of saturation of the water and the level of turbulence at the air–water interface. The last two factors influence the concentration gradient, which is the main driving force for oxygen transfer (Wheaton, 1977). The transfer of oxygen within a water mass is almost entirely dependent on water movement, since the diffusion of oxygen within water is extremely slow. For example, it has been calculated that to raise the oxygen content at a depth of 10 m from zero to 0.4 mg l-1 by diffusion alone would take about 600 years (Wheaton, 1977)! This emphasises the need to ensure

Abiotic factors

9

good mixing within culture systems to avoid the development of areas of low DO as a result of both the respiratory activity of the fish and the microbial degradation of organic material such as food, faeces and dead fish. The biological oxygen demand of organic material can be highly significant in intensive culture systems, and measures that ensure its regular removal are an important imperative. Tolerance to DO levels varies considerably both within and between species. In general, levels of 5 mg l-1 are considered to be acceptable to aquatic organisms, although many species, such as some tilapias, can survive and grow well at DO levels below 2 mg l-1. Most fish species can tolerate 1–2 mg l-1 for short periods, but mortalities would occur if this continued for more than a few hours. For salmonids, minimum DO levels are considered to be 5.0–5.5 mg l-1 for fish and 7 mg l-1 for eggs (Shepherd & Bromage, 1988). For those species that have a relatively high oxygen requirement, conditions under which oxygen solubility is reduced, particularly high temperature and salinity, can place the fish at risk. High temperatures not only reduce oxygen solubility, but also increase oxygen demand from enhanced levels of feeding and swimming activity. Taking such factors into account, it has been calculated that salmon, Salmo salar, should never be fed at temperatures above 20°C (Shepherd & Bromage, 1988). The proportionately higher oxygen consumption of small fish relative to large fish is also an important factor that should be taken into account in this respect. Models have been developed that take these factors into account in estimating oxygen consumption, and they provide a valuable management tool that not only minimises the risk of fish experiencing extreme situations, but allow near optimal conditions to be maintained in order to maximise efficiency and hence profitability. Actual DO levels in culture systems depend on the balance of the rate of consumption and the rate of supply. Rates of oxygen consumption vary not only with fish size, but also between species, and are strongly influenced by environmental conditions, particularly temperature. Rates of oxygen consumption shortly after fertilisation may range from 3.7 ng h-1 (cod, Gadus morhua) to 70 ng h-1 (rainbow trout, Oncorhynchus mykiss), but on average will increase 20-fold during the period from fertilisation to hatch (Rombough, 1988). Thus, early embryos may be relatively unaffected by low oxygen concentration, whereas in older embryos the retarding effect increases progressively (Kamler, 1992). At optimum temperatures, oxygen consumption is relatively high because of increased growth rates and levels of activity. At temperatures above the optimum, fish are stressed and their warning and defence systems are mobilised, thereby further significantly increasing oxygen consumption (Wheaton, 1977). Thus, at temperatures above the optimum, further temperature increases cause stress that increases oxygen demand more rapidly than the initial increases in temperature. Similarly, and regardless of temperature, external sources of stress significantly increase oxygen demand. This is why, for example, fish are easily asphyxiated during harvesting operations. The relationship between oxygen consumption and temperature, fish size and feeding rate is illustrated by recent experiments on the common wolf-fish (Anarhichas lupus) carried out by Steinarsson & Moksness (1996). At 7°C, the oxygen consumption of juveniles (0.5 kg) ranged from 37 to 62 mg O2 kg-1 h-1, whereas that of adults (6.9 kg) ranged from 29 to 44 mg O2 kg-1 h-1. This illustrates the decrease in weight-specific oxygen consumption with increasing fish size. These authors also demonstrated that oxygen consumption was not uniform


OXYGEN CONSUMPTION (mg/kg/h)

10

70

60

FEEDING FEEDING

50

40

30 9 12 15 18 21

0

3

6

9 12 15 18 21

0

3

6

TIME OF DAY

Figure 2.2 Diel rhythm of oxygen consumption of wolf-fish fed daily to satiation. Redrawn from Steinarsson & Moksness (1996).

throughout a 24-h period, showing a distinct diel rhythm (Fig. 2.2). Oxygen increased steadily after feeding at 0900 h, and decreased markedly during the night. This diel rhythm in oxygen consumption was also evident on non-feeding days, indicating an association with photoperiod as well as with feeding times.

2.3 Ammonia In fish, the majority of nitrogenous excretion occurs at the gill surface, with ammonia and ammonium ions being the main excretory products. Because of its high solubility and small molecular size, ammonia diffuses extremely rapidly. It can be lost through any surface which is in contact with water, and need not be excreted by the kidney. Ammonia is the most toxic form of inorganic nitrogen. Other products of nitrogen metabolism (e.g. urea and creatine) are produced in smaller quantities and may be excreted in the urine, through the skin or via the gills. Some of the ammonia is produced in the liver and is transported to the gills by the blood, but some may also be produced in the gills themselves by deamination of plasma amino acids. Spotte (1979) provides a comprehensive account of the mechanism of ammonia excretion and the toxic effects that may be induced. The toxicity of ammonia is largely controlled by pH through its effect on the hydrolysis of ammonium ions (NH4+), unionised ammonia (NH3) being the most toxic form. The proportion of unionised ammonia (PUIA) is described by Equation 2.1, which represents the concentrations of ammonia and ammonium ions.

(

[

PUIA = [NH 3 ] [NH 3 ] + NH 4

+

])

(2.1)

This is influenced most importantly by pH, temperature and salinity, which affect the equilibrium and the constant KaS that is determined from the concentrations.

Abiotic factors

[

S

K a = [NH 3 ][H + ] NH 4

+

]

11

(2.2)

The pH has the greatest effect on the PUIA, which can be calculated from the pH and this constant at a given temperature and salinity:

[

(

S

PUIA = 1 + antilog pK a - pH

)]

-1

(2.3)

where pKaS = -log KaS An increase of one pH unit (e.g. from pH 7 to pH 8) causes the proportion of unionised ammonia to increase about ten-fold. An increase in temperature from 10 to 20°C produces approximately a doubling in the proportion of the unionised form. However, as the salinity increases there is a slight fall in the proportion of the unionised form, the change being about 10% for a difference in salinity of 15 practical salinity units (p.s.u.). The buffering capacity of seawater provides a relatively stable pH, usually above pH 7, whereas freshwater tends to have a less stable pH, often below 7. Therefore saltwater systems will have a greater proportion of dissolved ammonia in the toxic unionised form than equivalent freshwater systems. As a result, if fish are held with a limited exchange of water, the risk of ammonia toxicity may be greater in saltwater than in freshwater. This would be the case, for example, when fish are reared in recirculation systems or when they are transported in closed systems. The lack of buffering of freshwater is to some extent an advantage in these circumstances. Any increase in dissolved CO2 from respiration will reduce the pH of freshwater and lead to an increase in the proportion of ionised ammonia. This would tend to counteract the toxic effects of a rise in total ammonia as waste products accumulate in the system. On the other hand, the natural buffering capacity of seawater minimises any pH change due to CO2 levels, and provides less compensation for any increase in toxic ammonia. In general, however, the instability of the pH of freshwater normally poses a greater risk since surface waters can rapidly become alkaline if high rates of photosynthesis reduce the carbonate levels. In addition, the toxic effects of ammonia may be increased under conditions of low oxygen levels. This may be caused by elevated levels of ammonia interfering with the ability of haemoglobin to retain oxygen. Tolerance to ammonia in water is 30% less at a dissolved oxygen level of 5 mg O2 l-1 than it is at 8.5 mg O2 l-1. There is some uncertainty as to the way in which environmental levels of ammonia exert their effects. It seems unlikely that ammonia enters the animals across the gills, and more probable that the effects are indirect. As the concentration gradient between environmental and tissue levels of ammonia decreases, the rate of ammonia loss is reduced, causing elevated levels in the tissues. Whether toxicity is due to diffusion into the gills or retention of metabolic ammonia, the effects are the same. Broadly, these effects are histopathological changes in the gills and other organs, decreased resistance to disease and impairment of growth. The effects on the gills may include necrosis, thickening of the epithelium, increased mucus production and epithelial rupturing and haemorrhage. Any fusing of lamellae reduces the surface area of the gills with a consequent impairment of gaseous (O2 and CO2) exchange. Ammonia has also been implicated in non-specific conditions such as fin and tail rot, anaemia and bacterial gill disease.

12


The estimation of the lethal limits of ammonia is hampered by a number of factors and is known to vary with species, other water-quality parameters, experimental methods, and the age, acclimation history and the condition of the test animals. For example, it has been shown that rainbow trout and coho salmon (Oncorhynchus kisutch) acclimated to sub-lethal levels of ammonia become resistant to otherwise lethal concentrations. Lethal limits for teleosts appear to range from about 0.07 mg NH3-N l-1 for rainbow trout fry to 1.4 mg NH3-N l-1 for juvenile striped bass (Morone saxatilis). Much of the early work on ammonia toxicity was concerned with rainbow trout and other freshwater species. The increased level of farming marine fish in the last decade or so has stimulated a number of studies of ammonia toxicity in commercially important species such as seabass (Dicentrarchus labrax), seabream (Sparus aurata) and turbot (Scophthalmus maximus). These illustrate inter-species differences in ammonia toxicity. Person-Le-Ruyet et al. (1995) used a continuous-flow method to determine the LC50s of unionised ammonia under optimal conditions of temperature, salinity, pH and dissolved oxygen concentrations for juveniles (6–163 g) of all three species. Median LC50s ranged from 1.7 mg NH3-N l-1 for seabass to 2.5–2.6 mg NH3-N l-1 for seabream and turbot. These values did not change significantly from 24 to 96 h and were not related to fish size. These authors also found that blood plasma levels of ammonia were positively correlated with external concentrations, and that a 50% mortality occurred after a 4-day exposure when the increase in total ammonia nitrogen was four times the initial level in seabass, but ten times the initial level in seabream and turbot. This showed that seabass have a lower threshold of physiological disturbance than both seabream and turbot, and explains their greater sensitivity to ammonia. Similar studies with the larval stages of seabass and Senegal sole (Solea senegalensis) demonstrated inter-species differences, 24-h LC50s being 0.28 mg NH3-N l-1 for seabream and 1.32 mg NH3-N l-1 for Senegal sole. These data also support the view that the larval stages are more sensitive to ammonia than the juvenile stages. Twenty-four-hour LC50 values are by no means safe limits. If fish are exposed to fluctuating levels of ammonia that span the 24-h LC50 levels, the resulting toxicity may be higher than expected from continuous exposure to such concentrations and more difficult to predict. While the concentration of total ammonia in marine fish hatcheries is generally below 0.7 mg TAN l-1, in some cases it may be as high as 2 mg TAN l-1.

2.4 Temperature A change in temperature affects the rate of biological processes and consequently will affect an animal’s metabolic rate and activity. Between lower and upper thermal limits, the rate generally increases to a maximum as the temperature increases to the optimum, but above the optimum deleterious effects become more significant and the rate falls. Animals are adapted to the niche they occupy in their environment. A species’ distribution and the seasonal changes are reflected in the characteristics of the life cycle and in optima for various processes such as growth, activity and aspects of reproduction. For particular species, optimal temperatures very commonly differ with the stage of development (viz. egg, larva, juvenile and adult) and even in individuals, related processes such as

Abiotic factors

13

appetite, digestion and growth may have different optima. This section will highlight particular examples from this variety of effects, with emphasis on their importance for cultivation.

2.4.1 Seasonal Temperature Cycle and Spawning The annual cycle of temperature change in north temperate surface waters lags behind the annual cycle of daylength by a month or two, with the minimum in February and March and the maximum in August and September. The range between the winter minimum and the summer maximum varies with latitude and the depth of the mixed water column. This annual change in ambient temperature is important with regard to the selection of species for particular sites, although water temperature can be controlled at a cost in pump-ashore sites (see Section 2.8). However, for broodstock fish, some exposure to low winter temperatures and increasing temperatures in spring is considered to be important. A period of low temperature when vitellogenesis is taking place is thought to improve egg quality in several species. Egg diameter is greater in cod held at low temperatures, and fecundity is considered to be better in common sole (Solea solea) that have experienced a temperature of less than 10°C. Female wolf-fish must be kept at a temperature below 10°C for at least 4 months before ovulation for normal egg maturation. Rising temperatures may help to initiate spawning, although reaching a threshold temperature does not necessarily trigger spawning in wild stocks. The manipulation of temperature together with photoperiod enables spawning period to be extended and good egg quality to be maintained, for example in cod and haddock (Melanogrammus aeglefinus). However, temperature affects the timing of spawning by influencing gonad development and growth rather than by providing a specific cue to set the time.

2.4.2 Egg and Larval Development Water temperature affects the efficiency with which yolk is converted into embryo tissues. Within the range of thermal tolerance of a species, eggs tend to demonstrate greater efficiency at lower temperatures than they do at higher temperatures (Kamler, 1992). Optimal temperatures for embryonic development are not necessarily the same for larval growth. If eggs or larvae are reared at the extremes of their temperature range, they very often have developmental abnormalities such as the poor articulation of the jaw of Atlantic halibut (Hippoglossus hippoglossus) when reared at 9°C instead of 6°C. Even within the range at which growth of the yolk-sac larva appears normal, a change in incubation temperature of a few degrees Celsius may cause significant changes in the relative timing of organogenesis. Such changes may have consequences for the fitness of the larvae, and care must be taken in evaluating an optimum. A temperature difference of a few degrees at egg incubation and the early larval stage (e.g. 5–8°C in Atlantic halibut) has a profound influence on the number and size of white muscle fibres, which ultimately determines the muscle cross-sectional area. This can have long-term effects on the muscle growth in juvenile stages, and makes avoiding unplanned temperature changes during early rearing very important.

14


2.4.3 Sex Ratio For several species, there is evidence that the temperature during early rearing can influence the phenotypic or functional sex of fish. It is unclear how widespread this effect is in gonochoristic ‘coldwater’ marine fish, but one species of interest, the hirame or Japanese flounder (Paralichthys olivaceus), does demonstrate what appears to be thermolabile sex determination. It has been shown that a normal 50 : 50 male : female sex ratio occurs if juveniles are reared through the first 120 days after hatching at 18°C. Higher water temperatures (20, 23 or 25°C) during the same period lead to approximately 75% males in the brood, although it is also reported that slower-growing fish became predominantly male and so the effect may not be entirely due to temperature.

2.4.4 Growth and Metabolism The many studies on the growth of fish show a marked influence of temperature. The overall effect on growth rate depends on the interaction of the effect of temperature on the appetite and digestion, and the difference in the effects on standard and active metabolic rates. Brett (1979), and Brett & Groves (1979) provide extensive reviews of how temperature, amongst other factors, affects metabolic rate and growth, and should be consulted for detailed accounts. The optimum temperature for growth rate very often changes as fish grow, but this relationship varies with the stage of development. In cod, for example, the optimum has been shown to increase from 9.7 to 13.4°C as larvae grow from 73 to 251 mg. The optimum for small (50–1000 g), immature fish is higher (11–15°C), but for large (1.5–2.5 kg), sexually mature cod the optimal temperature range is 9–12°C, which is slightly less than for larvae. This pattern of change with ontogeny is also common in other species. The most significant relationships involved in on-growing fish are those between temperature and the rate of food uptake and between temperature and growth rate. The change in the efficiency of feed conversion (i.e. growth per unit ration) with temperature depends on how these two relationships interact. Fish fed a maintenance ration do not increase in size: the energetic equivalence of the diet is fully utilised in the swimming involved in feeding, digesting the food, excretion, osmoregulation, renewing tissue and so on, thus maintaining the status quo. Fish fed to satiation (when appetite no longer drives a feeding response) have an excess in energetic terms over what is needed for maintenance, and that provides scope for growth. Temperature does not affect the level of the ration required for maintenance in the same way as it affects appetite and the ration required for satiation. As temperature changes, so does the scope for growth. Figure 2.3 shows generalised curves that indicate the energetic value of the maximum and maintenance rations and the difference is that available for growth: this increases with temperature up to a maximum before decreasing rapidly. The change in the scope for growth is comparable to the pattern of the change in growth rate with temperature, and the peak occurs close to the optimum temperature for growth. If a situation is considered where the higher ration is less than satiation but greater than maintenance, the scope for growth is reduced and the peak occurs at a lower temperature. The optimum temperature for growth decreases as the ration is reduced.

Abiotic factors

15

Satiation ration

5

Maintenance ration Scope for growth

ENERGY (k cal)

4

3

2

1

0 0

5

10

15

20

25

30

TEMPERATURE (°C) Figure 2.3 Generalised curves that indicate the relation between temperature and ration plotted as the energetic value of the quantity consumed at satiation and maintenance levels. The difference between them is the energetic equivalence that is available for growth and is plotted as ‘scope for growth’. The temperature at which the maximum value in scope for growth is reached approximates to the optimum temperature for growth.

The efficiency with which food is converted into growth very often increases as the temperature is reduced, and it is not uncommon to have an optimum temperature for feed conversion efficiency which is somewhat lower than the optimum for growth. Much of the published work in this area is for salmonids, but the relatively small amount of information for cold-water marine fish supports these generalisations.

2.5 Salinity The salinity of seawater varies little offshore, and in general marine fish are adapted to live in this stable environment. There are a limited number of species that naturally occupy the more variable conditions found in estuaries, and their physiology is better suited to tolerate the daily changes associated with each tidal cycle. Salinity is measured on the practical salinity scale, which relates the conductivity of a sample to that of a standard potassium chloride solution. If seawater at 15°C has a conductivity equal to that of the standard, it is said to have a salinity of 35 p.s.u. Values given in p.s.u. are approximately equivalent to those recorded in parts per thousand (S‰), the units more commonly used in earlier literature. A salinity of 35 p.s.u. is generally accepted as the norm for offshore seawater, while in coastal waters, although the salinity is still quite stable, run-off from the land may lead to the norm being 32 or 33 p.s.u., for example. In estuaries, however, depending on the tidal

16


range, fluctuations measured in tens of units may occur twice a day. The difference in density of freshwater and seawater means that unless there is adequate mixing, the freshwater tends to float over the salt water forming an oblique halocline, i.e. a discontinuity between the different salinity waters, that moves up and down the estuary with the tidal flow. The location of a water intake or the position of cages in an estuary in relation to these salinity changes are important considerations when selecting the site of a marine fish farm or the choice of species for estuarine waters. The salts in the body fluids of fish are maintained by the osmoregulatory system at a concentration of about one-third of that of full-strength seawater. This differential means that fish in seawater passively lose water by osmosis through the gills and body surfaces, whilst salts enter the body. To maintain homeostasis, the fish has to drink seawater continually and excrete the excess ions. Specialised chloride cells in the gills remove ions such as sodium and chloride, while the kidneys produce small volumes of very concentrated urine. Fish that live in estuaries in brackish water at less than 10–11 p.s.u. are subject to the opposite fluxes: salts leave the body and water enters by osmosis. Fish that tolerate both environments are able to do so because chloride cells can take up monovalent ions to replace those lost, and the kidneys produce copious amounts of dilute urine to counteract the osmotic flux. Unless the surrounding water is isotonic with the body fluids, energy is constantly used to maintain the body fluids’ composition. Embryos and larval fish are less well adapted to control the flux of water and ions in and out of the body, and therefore do not tolerate changes in salinity as well as adult fish. The conditions required for successful reproduction are thus more closely defined. Gametes within the adult’s body are in osmotic balance with body fluid blood plasma, but at the time of spawning those of oviparous species are exposed to the osmotic stresses of their surroundings. The ways that species may be affected by, or are able to tolerate, the osmotic changes that occur during embryogenesis and later larval development have been reviewed by Alderdice (1988). The energy required for osmoregulation affects yolk utilisation efficiency and the growth rate of larvae. In some euryhaline species this can lead to larger larvae hatching at intermediate salinities compared with those reared in either fully fresh or saline water (e.g. newly hatched striped bass are longer at 5 p.s.u. than at either 1 or 10 p.s.u.). Survival is affected by salinity, and the salinity to which larvae are expected to be adapted is not necessarily optimal. For example, newly hatched Atlantic halibut survive best at 29–34 p.s.u., which is slightly lower than full-strength seawater, although there is no benefit of reduced salinity after 30 days. The salinity of the water also affects the buoyancy of the eggs and larvae, and this may have consequences for survival that are not directly associated with osmoregulation. In species that need to gulp air to fill the swim bladder, low salinities may mean that the larvae are not sufficiently buoyant to reach the surface and survival is reduced. In a fish-farming context, salinity can be manipulated to some advantage. The large eggs of Atlantic halibut, for example, are fragile and can be protected during embryo development by incubating them at their salinity of neutral buoyancy. Salinity and temperature do not have independent effects on development and ideally should be considered together. The effect of temperature influences the rate of reactions, and

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17

the salinity influences the energy required to regulate the body composition. Where the interactions of these influences have been analysed rigorously, the optima for growth do not necessarily coincide with those for the most efficient food conversion, and compromises in setting conditions must be made.

2.6 Hydrogen Sulphide Hydrogen sulphide is a noxious gas and is a product of anaerobic decomposition of organic material. Anoxic conditions develop when the rate of deposition is high and the supply of oxygen is insufficient to meet the metabolic demands of the plant and animal communities inhabiting the sediments. Under such anoxic conditions, benthic communities dominated by low-oxygen-tolerant and anaerobic species develop, and potentially toxic chemicals, such as hydrogen sulphide, are produced. Almost all the hydrogen sulphide in the environment is produced by a specialised group of micro-organisms. The most widely distributed sulphate reducer is Desulfoxibrio desulfurcans, which is found in freshwater environments. A closely related species, D. estuarli, is its marine equivalent. The main requirements for bacterial sulphate reduction to hydrogen sulphide are the absence of oxygen, the presence of sulphate, the presence of oxidizable organic substrates to supply hydrogen atoms, and the presence of organic nutrients to support the growth of the bacteria, including vitamins, amino acids and nucleotides. Only a narrow range of organic molecules can be oxidised by the sulphatereducing bacteria. These include acetic and lactic acid, although some strains can oxidise hydrogen. The range of nutrient molecules used by sulphate-reducing bacteria is also narrow, and includes lactate, pyruvate, fumarate and malate. The sulphate reduction reaction in anaerobic environments can be represented by the following equation: 2CH 2O + 2H + + SO 4

2-

fi H 2S + 2CO2 + 2H 2O

(2.4)

In this equation, CH2O represents carbohydrates; the equation becomes much more complicated if it is written more accurately to show the consumption of glucose (C6H12O6) or similar molecules. Hydrogen sulphide is highly soluble in water and is readily precipitated as ferrous sulphate (FeS2), producing the black colour characteristic of anoxic sediments. Hydrogen sulphide is highly toxic to fish, but is readily oxidised to a harmless form by exposure to oxygen. The toxicity is increased at higher temperatures and at pH values less than 8, when the largest percentage of hydrogen sulphide is in the toxic unionised form. The toxicity is based on the capacity of the molecule to inhibit the reversible binding of oxygen to haemoglobin by binding to, and inactivating, cytochrome oxidase. In addition, the release of hydrogen sulphide has been implicated as a causative agent of gill damage in caged Norwegian salmon stocks. Damage to the gills of brown trout fry, Salmo trutta, following exposure to low, chronic concentrations (2–5 mg l-1) of hydrogen sulphide includes thickened gill lamellae and bulbous tips. The 96-h LC50 for this species has been estimated to be 7 mg l-1. In an aquaculture context, hydrogen sulphide may present a problem in a wide variety of situations. It may be present in water sourced from wells, but this rarely presents problems under conditions of adequate aeration. In general, the source of hydrogen sulphide is more

18


likely to be organic materials generated by the farming activities themselves than the water sources unless the siting of the farm has been less than judicious. Thus, any situation where organic material is permitted to accumulate is likely to become a source of hydrogen sulphide. This may occur, for example, in fish ponds, beneath fish cages or in recirculation systems. The severity of any problem will depend both on the rate of accumulation of the organic materials and the rate at which oxygen is supplied. The greatest problems may occur when anaerobic sediments are disturbed, for example during husbandry or harvesting operations, when large amounts of hydrogen sulphide may be released. In northern Europe, the accumulation of organic material beneath salmon cages has perhaps given the greatest cause for concern. Under normal conditions, hydrogen sulphide generated in the sediments would become oxidised back to sulphate at the sea/sediment interface. Under more extreme conditions of organic input, however, the boundary between the reduced and oxidised zone may lie much higher in the water column, and hydrogen sulphide gas may escape to the atmosphere before being oxidised. Despite the solubility of hydrogen sulphide, it has been detected 9 m above the bottom in the vicinity of salmon cages. Water flows and the management of the operation will principally determine the extent of the problem.

2.7 Light The quality, intensity and photoperiod are the characteristics of light that affect fish, and the conditions needed to obtain optimum performance in culture differ with species and stage of development. The effects can be considered under two headings: the influence on growth and development, which is particularly important during the larval stages, and the influence on reproduction.

2.7.1 Growth and Development Boeuf & Le Bail (1999) have recently reviewed this subject. This section will concentrate on the information available for marine species. Light can have a direct influence on the hatching process. The hatching of Atlantic halibut, for example, is inhibited by light. After the eggs have been held in constant light until after embryo development is complete, a transfer to darkness results in rapid and synchronous hatching. It appears that light influences the control of the secretion of the hatching enzyme. This is also the case for other species, although the response is not the same: in some, hatching can be more common during the light phase. In some species, it is possible that even the rate of embryonic development before hatching can be affected by light. Walleye pollock (Theragra chalcogramma), a deep-water species, has embryos that develop more rapidly under constant darkness than under diel light conditions. The larvae of the marine fish being considered for aquaculture are primarily visual feeders, and thus light plays a significant role in determining the success of early feeding. Larval vision is characterised by limited spectral sensitivity, although the range of wavelengths to which the larval eye is sensitive normally increases with age. Hatchery rearing of

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embryos and larvae is generally under artificial white light, and it is the intensity that is particularly important for the success of rearing. Different species have different requirements. Atlantic halibut yolk-sac larvae held at 10 lux show significantly better growth and survival than those kept at 1000 lux, probably because their activity is greater at the higher light intensity, resulting in less of the yolk reserves being available for growth. Yolk-sac cod larvae develop faster in constant darkness than larvae kept under a diel light cycle, since swimming activity is 6–10 times less in darkness. Once exogenous feeding begins, a suitable light level is necessary for active feeding to be successful. For example, first-feeding larvae of greenback flounder (Rhombosolea tapirina) held in total darkness die within 20 days of hatching. The optimum intensity varies with species. Cod larvae show maximum feeding incidence at 1 lux and a clear inhibition of feeding at light levels above 12 lux, and Atlantic halibut are reported to have a higher feeding success at 0.5 lux than at 50 lux. Turbot, a species that has a surface-orientated feeding behaviour, feeds best at light levels of 860 lux and higher, but feeds poorly at 12 lux. The optimum light level does vary with the type of food offered. Whitefish (Coregonus sp.) larvae fed an inert diet at 20 lux do not grow as well as those held at 300 or 500 lux, whereas there is no significant difference under these different light intensities when the larvae are fed live Artemia nauplii. The movement of the swimming nauplii may enhance their visibility and attractiveness to the larvae even at low light levels. There is evidence that the colour of the tank in combination with the light intensity is important. Low light intensity in black tanks may give poorer growth and survival than in lighter coloured tanks because of the lack of contrast between the prey and the background. The light intensity can also influence feeding by affecting the distribution of larvae within the rearing tank, and hence the spatial overlap with the distribution of food. It has been suggested that Atlantic halibut larvae aggregate around the walls, the bottom and the surface in rearing tanks owing to a phototactic response to reflected light. Supplying additional UV light can result in a more even vertical distribution and a greater ingestion of Artemia. Diffuse light reduces the tendency of Artemia to swarm, and so generates a more even distribution of food within a tank, but an absence of high-density patches may lead larvae to ingest less. Daylength has been shown to affect the growth of some species; longer days increase the period over which visually feeding larvae are able to take food. However, 24-h light does not necessarily improve survival as well as growth. Larvae of sea bass show better growth, but poorer survival, in continuous light compared with a 9-h light period. On the other hand, for haddock, there appears to be no effect of photoperiod, although a period of inactivity during a dark phase may benefit digestion and assimilation. However, a dark period is not only associated with feeding and growth. A diel light cycle appears to facilitate swim-bladder inflation, and therefore can contribute to better survival. In some species the swim-bladder is filled by the early larvae gulping air at the surface during the dark period, and in others gas secretion is frequently stimulated nocturnally. Where light is natural rather than artificial, the high intensity of sunlight and its spectral composition may have adverse effects on young fish unless it is filtered. Ultraviolet radiation can cause biological damage to the cells of an organism. Proteins and nucleic acids absorb short-wave ultraviolet light, and the photochemical damage that results can disrupt

20


protein synthesis and cell replication. Eggs and early larvae generally lack protective pigments and are particularly sensitive. The damage caused can depend on the cumulative dose received, and this will vary with the time of year and the weather conditions, as well as water depth and clarity. The effects may cause particular problems in extensive shallow-water rearing systems or where fish are held in cages near the surface without shading. The effect of photoperiod on juvenile growth is generally less pronounced than for larvae. A few species show a positive relationship between longer photoperiods and growth (e.g. turbot), but others do not (e.g. yellowtail flounder, Pleuronectes ferrugineus). It is likely that the effect on growth is more pronounced when feeding is restricted. Daylength also plays a role in determining the age of first maturity in turbot (as well as other species; see below). Exposure to an extended photoperiod during the first winter decreases the proportion of males maturing in their first year of growth compared with those in a natural photoperiod. This illustrates that growth and reproduction do affect each other, and the separation of these subjects in this account is rather artificial.

2.7.2 Reproduction The reproduction of cold-water fish is generally an annual event synchronised with the time of year when the chances of survival of the progeny are maximised. In most species changing daylength, in addition to the annual cycle of temperature change, provides the main cue that regulates the hormonal control of gonadal recrudescence, maturation and spawning. There has been considerable study of the effect of altered seasonal cycles of daylength or fixed photoperiods on salmonid reproduction (Bromage et al., 1993), and most of the work with marine species is based on experience with salmonids. The ability to shift or prolong the spawning season is a great advantage for commercial fish cultivation provided that water temperatures allow early developmental stages to be reared out of their normal season. This allows hatchery facilities to be used more efficiently and production to be increased. Various approaches have been used to alter spawning time, and the simplest to understand is the use of an annual cycle of changing daylength that follows the normal pattern, but is phase-shifted by several months. For example, the hatchery could have three different groups of broodstock fish, one under a normal light cycle, one that is delayed by 4 months and another delayed by 8 months, leading to peak spawning by the three stocks in March, July and November, respectively (Fig. 2.4). Constant photoperiods and sudden changes from short days to long days will also advance or delay spawning and can be simpler to implement practically, but the timing of the change and the photoperiod history of the fish are very important. Bromage et al. (1993) summarise this succinctly. They suggest that the change in photoperiod entrains an endogenous clock that controls reproduction, and it is the perception of changing daylength rather than absolute photoperiod that is significant. An abrupt change from a photoperiod of 18 h to one of 14 h light advances spawning by a period similar to that following a change from 10 h light to a 6-h photoperiod. Although this work is based mainly on studies with rainbow trout (Oncorhynchus mykiss), similar mechanisms operate in other species that exhibit an influence of photoperiod. Examples include work with Atlantic halibut using different phase-shifted annual cycles to either delay or advance spawning; compressed annual cycles can result in

Abiotic factors

4 months delay

LIGHT PERIOD (h)

18:00

8 months delay

21

Normal light cycle

14:00

10:00

06:00 Mar

May

Jul

Sep

Nov

Jan

Mar

May

Figure 2.4 Generalised curves showing the normal change in daylength during the year (at a latitude of about 51°N) and two comparable cycles, one phase-shifted by 4 months, the other by 8 months. Maintaining different groups of a species that spawn shortly after a period of increasing daylength (such as turbot) under each light cycle would provide gametes from the different stocks in July, November and March, respectively.

cod spawning twice in one year; spawning in turbot can be induced by a sudden change from a short-day to a long-day photoperiod regime. Not only are annual reproductive cycles affected by photoperiod, the initial maturation of fish can also be influenced. It is often convenient in commercial fish farming to delay the onset of sexual maturity and obtain better growth performance in the early years. It has been shown, for example, that exposure of cod age 1+ years to continuous light from the midsummer solstice onwards will delay gonad development and inhibit spawning at 2 years (see Section 10.1). This treatment also reduces the proportion of females in the group that spawn at 3 years of age, and somatic growth in the second year is better than normal. Data in the published literature has inconsistencies in the effects of photoperiod manipulation, and species-specific differences do exist. However, some reported variation within species may be attributable to the differences in the time of year the treatments were begun, or the previous photoperiod regime to which the fish were subjected prior to being switched to the treatment regime of interest.

2.8 Algae Blooms An algae bloom is the name given to the development of an occasional high concentration of planktonic algal cells that forms rapidly when water conditions are suitable. The presence of high cell concentrations, usually dominated by one species, may cause the water to appear coloured, leading to so-called ‘red tides’, usually caused by species of dinoflagellate, or ‘brown tides’, which tend to be associated with diatom blooms. Such blooms are not always

22


toxic, and the name red tide is often used for any algal bloom, but the same names are often used to describe blooms which result in a build-up of toxins in the water. These toxins can cause fish kills, or become concentrated in fish and shellfish rendering them toxic to humans. Blooms that lead to the release of toxins are frequently referred to as harmful algal blooms, or HABs. However, apart from producing toxins, algal blooms can be detrimental to fish and shellfish in several ways. Their physical presence can damage fish gills, dissolved oxygen in the water can be severely depleted, and the flavour of the fish can be adversely affected if they are harvested within a few days of being exposed to a bloom. The environmental conditions that can lead to blooms include high light levels, relatively warm water and sufficient nutrients to allow rapid growth. Therefore, they often occur in spring and summer in temperate regions, and usually in coastal waters during relatively calm periods when the population of phytoplankton cells can multiply without being dispersed. They are frequently derived from the development of resting cells as conditions become suitable, leading to a rapid increase in cell numbers. The main impact of algae blooms on cultivated fish is often at cage-farm sites where there is no simple way of isolating the fish in the cages from the developing algae population. Even in pump-ashore farms, toxins and algae can reach the fish tanks before the problem has been recognised. Many of the serious problems caused have been associated with salmonids in net-pens, or in the culture of warmer-water fish such as striped bass. Even so, there is little reason to believe that cold-water marine fish will not be affected at some time as the industry expands. The information that follows provides a broad overview of the types of bloom that can form. Several different groups of algae have species that may form blooms. In marine waters, the most important groups are the dinoflagellates (Dinophyta), diatoms (Bacillariophyceae) and members of the Haptophyta. Species of Cyanobacteria (blue–green algae) that form blooms are uncommon in marine waters and rarely lead to fish kills, although some freshwater species are a significant source of the toxins that can kill fish. The most important group, in terms of effects from toxins, is the Dinophyta. Many different dinoflagellate species may produce blooms, but only about a dozen produce toxins. In some species, toxins may be produced only if there is some imbalance in the nutrients in the water, such as a phosphate depletion. Significant fish kills or contamination of fish as a result of HABs have tended to be in warm temperate coastal waters, but some of the species involved occur in cold temperate waters, and if conditions prove suitable HABs could develop. Several different classes of dinoflagellate toxins exist (see Stickney, 2000, for more details of their effects). Most are known through their effects on people who consume shellfish that have concentrated the toxin; the most familiar names describing these are paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP). PSP is caused by a saxotoxin produced by species of the genera Alexandrium, Pyrodinium and Gymnodinium. DSP is caused by okadaic acid produced by species of Dinophysis and Prorocentrum. Despite the names, the toxins can also accumulate in fish. Neurotoxic shellfish poisoning is caused by brevetoxins that are produced by Ptychodiscus brevis, and these toxins have also been associated with fish kills along the east coast of the USA. Several genera produce gambiertoxin and ciguatoxin, which can lead to ciguatera in humans. This is poisoning as a result of eating

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contaminated fish, which are usually of tropical origin, although it has been reported in saupe from the Mediterranean. Although the location of most HABs is largely outside the areas of culture of cold-water marine fish, algal species are prime candidates for transfer from one area to another in ballast water of ships, and their distribution can change rapidly. In addition, new algal species and new toxins are still being isolated, and it is possible that more may be found in cold temperate waters. As recently as the 1990s, the dinoflagellate Pfiesteria piscicida was first recognised as the causative agent in major fish kills in estuaries of the south-eastern United States. It is most likely to cause problems at temperatures above 26°C, but the toxin is effective as low as 12°C. The release of the toxin is actually stimulated by the presence of live fish. The toxin leads to ulcerative diseases at cell densities of 100–250 cells ml-1 and is lethal if the concentration of the P. piscicida is 250–300 cells ml-1 . There are a few diatom species that can cause HABs, particularly those of the genus Pseudonitzschia. They produce the toxin domoic acid, which is the cause of amnesic shellfish poisoning. As well as occurring in shellfish, it can accumulate in planktivorous fish and could therefore be a problem for consumers of the fish, but there are no reports of a direct effect causing significant fish kills. Some non-toxic diatoms can cause one of the more significant problems affecting cagecultured fish in cold temperate coastal water. High concentrations cause physical damage that contributes to fish mortalities. The silicaceous theca of species such as Skeletonema costatum and Chaetoceros convolutus have setae with small spines that can seriously damage the lamellae of fish gills. At concentrations of more than 5 cells ml-1 this causes mucus buildup and impaired gas exchange, that has resulted in mortalities of Pacific salmon in cagefarm sites. At concentrations of less than 5 cells ml-1, the damage is sufficient to give rise to increased mortality from vibriosis. Of the Haptophytes, Phaeocystis, Prymnesium and Chrysochromulina are the familiar genera that are associated with algal blooms and fish kills. Prymnesium parvum is mainly a problem of fresh water, but it does occur in low-salinity brackish waters. It produces a potent ichthyotoxin called prymnesin. Mass mortalities of salmon and trout have occurred as a result of blooms in Norwegian fjords, where the salinity at the surface was about 5 p.s.u. and toxin production was enhanced because of a phosphorus limitation in the water. Phaeocystis pouchetii has long been considered to be a problem when blooms of the colonial form occur, mainly through the massive increase in biological oxygen demand that occurs when the bloom collapses and the decaying colonies settle out of the water column. However, recent work has shown that a chemical is released by the cells that is toxic to cod larvae and remains in filtered seawater. Production of the toxin increases when the cells are exposed to increased light levels.

2.9 Site Selection Decisions regarding the choice of site for an aquaculture enterprise are among the first and the most important in determining the success of the operation. The characteristics of the chosen site can have a significant bearing on the capital outlay, the running costs of the

24


operation and the rate of production and mortality of the target species. Although the selection of the site is of fundamental importance to all forms of culture, Beveridge (1987) argues that the decisions are perhaps more crucial for water-based culture operations than those based on land, reasoning that there is some potential for improving the latter. For example, more bore holes may be drilled to increase the water supply, or sediment traps or filters can be installed to reduce suspended solids. There are fewer options for such manipulations with water-based systems, and consequently it is important to get it right from the outset. Treece (2000) points out that before a detailed appraisal of potential sites is made, it is important to determine the overall objectives of the enterprise. First and foremost, the market for the product needs to be assessed. It is important to know what the market channels are, and how the product is to be delivered to the market in good condition and without excessive cost. Having considered the market and other factors, the required production level and the pattern of that production need to be determined. The area of land required can then be estimated; this is a function of the number of crops per year, the total production and the production strategy. It should also be remembered that a farmer can select a site to suit a particular species, or conversely select a species to suit a particular site. The final step of these preliminary considerations should be to assess the financial returns of the project. Treece (2000) expresses the view that a return of 20%, or preferably 30–50%, per annum over a 12-year horizon should be required before progressing further with the site-selection procedure. If the likely return is less than that level, the project should be abandoned before further resources are committed. However, such decisions will depend on the goals of the company. Having decided to proceed, the characteristics of available sites need to be evaluated. Beveridge (1987) provides a comprehensive review of the factors to be considered in selecting a site. Although he focuses particularly on those of importance to cage culture, the majority are common to both cage and land-based tank or pond culture. The following overview is based largely on this detailed review unless otherwise attributed. It is self-evident that by far the most important resource to be considered for an aquaculture project is water supply. The availability and physical, chemical and biological characteristics of the water will be the principal determinant of the production characteristics of the site. A record of seasonal changes over a significant time-period should be known in order to assess the degree to which the site characteristics are likely to match the requirements of the target species. Of prime importance is temperature, since this is the physical attribute of the water that will have the greatest controlling influence on growth rate. Seasonal changes in temperature may be important in planning production strategies, since it would be desirable, for example, for peak temperatures to coincide with high stock holdings to obtain the greatest weight gain from the most favourable conditions. The avoidance of temperature extremes will also be important, during both the summer and the winter months, to avoid prolonged periods of low growth and low food conversion efficiencies, and even mortalities under the most severe conditions. In this respect, any changes in the temperature preferences of the species over its life-cycle will be an important consideration. Although most marine fish are euryhaline, salinity can vary considerably in coastal areas, and extreme conditions can arise from a combination of excessive run-off from the land and poor mixing and flushing rates. Freshwater run-off can also have a significant impact on temperature. In

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temperate regions, freshwater will have a cooling effect in the winter and a warming effect in the summer. Oxygen levels are rarely limiting, although areas of high organic input should be avoided. The pH of the water is an important factor because extreme values can directly damage gill surfaces. It is also important because it affects the toxicity of several pollutants, including ammonia and cyanide, and heavy metals such as aluminium. The ideal pH for most fish species is 6–8.5. This is not a problem at most marine sites because seawater is naturally alkaline, with a pH typically within the range 7.5–8.5, and because it is well buffered and is less prone to fluctuations than freshwater. The effects of turbidity can be significant, but depend to a large extent on the nature of the suspended particles. Some may have toxic properties, such as the salts of various metals, whereas those of an organic nature can have a significant oxygen demand and cause oxygen depletion. High levels of certain types of suspended solids can cause gill damage and have also been implicated in diseases such as fin-rot. In general, it is considered that levels below 100 mg l-1 have little effect, while areas where levels are significantly above that should be avoided. Water contaminated with pollutants should clearly be avoided, although this is difficult to guarantee. At the very least, sites should not be chosen which are close to industrial complexes or in areas susceptible to such developments. Organically polluted areas may increase the risk of disease since they seem to harbour more disease agents than unpolluted areas. For example, the pathogenic bacterium Vibrio parahaemolyticus has been found in exceptionally large numbers in sewage-polluted waters. Similarly, areas prone to phytoplankton blooms, which can generate adverse effects by clogging gills, reducing DO levels, tainting the flesh or producing toxins, should be avoided, but may be difficult to identify. Susceptibility should be assessed from discussions with local people and the relevant authorities, and nutrient-rich sites and those where the exchange period is more than a few days should be avoided. Good water exchange at a site is important to avoid the accumulation of wastes in the vicinity of the farm. This is unlikely to be a problem in relatively open-coast situations, but cage farms are often situated well within sea lochs or fjords in areas where exchange rates may not be adequate. Estimating the exchange rate is a complex process, and in lochs or fjords is dependent on the size and topography of the basin, the number, location and depth of sills, the magnitude of freshwater inputs and the tidal range. Beveridge (1987) reviews the calculation of exchange rates and recommends that marine sites should have good bottom as well as surface currents, and that the exchange period should be in days rather than weeks. The exchange rate of the water mass in which the fish are held is a critical factor, since this will, to a large extent, determine the rate at which oxygen is supplied and solid and dissolved wastes are removed. In tanks, the exchange rate is largely under the control of the farmer, who can vary flow rates in accordance with the biomass loading, or more precisely the metabolic activity, of the fish to maintain near-optimal conditions. In contrast, exchange rates in cages are much less controllable, since they are almost entirely dependent on tideand wind-generated currents, and to some extent the activity of the fish. Current velocities in coastal marine sites vary widely, ranging from 0 cm s-1 at slack water to more than 250 cm s-1 at full flood and ebb tides. It is clearly desirable that the period of slack water is as short as possible. Current velocity during the ebb and flood tides will determine the density

26


of fish that can be stocked, and hence will have an important bearing on the economics of the operation. However, excessive currents can adversely affect the behaviour of the fish and deform the cage, thus reducing its volume. In practice, it is recommended that current velocities should be below 100 cm s-1, and preferably between 10 and 60 cm s-1. Other environmental factors are also important determinants of site suitability for cages. Weather can be of considerable importance in that excessive rainfall can have a marked effect on salinity, and excessive wind can cause considerable damage to structures and limit access to the stock. One of the most damaging effects of wind is through wave generation, and the probable conditions at potential sites need to be carefully considered. Beveridge (1987) describes how this can be estimated from information on the long-term frequency and direction of surface wind speeds, fetch lengths corresponding to the directions of the strongest prevailing winds, and the depth of water along the fetch. The depth of water directly beneath the cages is of less importance, although it is important that cages should be well clear of the bottom at all states of tide to allow effective dispersal of waste food and faeces, and to avoid local deoxygenation and toxic effects (e.g. H2S) from the accumulation of these waste organic materials. Other factors of no less importance which must be considered in evaluating sites relate to legal issues, including access and rights, the provision of essential utilities such as electricity, freshwater and telephones, and the availability in the region of labour, contractors and possibly even research support. The nature and area of the land is of particular importance. In the case of a cage farm, sufficient land should be available for the construction of buildings to house, for example, laboratories, stores, offices and accommodation for staff, preferably within sight of the cages. Mooring or launching facilities for boats will also be important. With regard to pump-ashore tank systems, the land should be as close to sea level as possible (but without risks of flooding) to minimise pumping costs.

2.10 References Alderdice, D.F. (1988) Osmotic and ionic regulation in teleost eggs and larvae. In: Fish Physiology. Vol 11. The Physiology of Developing Fish. Part A. Eggs and Larvae. (eds W.S. Hoar & D.J. Randall), pp. 163–251. Academic Press, San Diego. Beveridge, M.C.M. (1987) Cage Aquaculture. Fishing News Books, Farnham. Boeuf, G. & Le Bail, P.Y. (1999) Does light have an influence on fish growth? Aquaculture, 177, 129–52. Boyd, C.E. (1979) Water Quality in Warm Water Fish Ponds. Auburn University Agricultural Experiment Station, AL. Brett, J.R. (1979) Environmental factors and growth. In: Fish Physiology. Vol VIII. Bioenergetics and Growth. (eds W.S. Hoar, D.J. Randall & J.R. Brett), pp. 599–675. Academic Press, San Diego. Brett, J.R. & Groves, T.D.D. (1979) Physiological energetics. In: Fish Physiology. Vol VIII. Bioenergetics and Growth. (eds W.S. Hoar, D.J. Randall & J.R. Brett), pp. 279–352. Academic Press, San Diego. Bromage, N., Randall, C., Duston, J., Thrush, M. & Jones, J. (1993) Environmental control of reproduction in salmonids. In: Recent Advances in Aquaculture. Vol. 4. (eds J.F. Muir & R.J. Roberts), pp. 55–65. Blackwell Scientific, Oxford.

Abiotic factors

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Kamler, E. (1992) Early Life History of Fish: An Energetics Approach. Chapman & Hall, London. Person-Le-Ruyet, J., Chartois, H. & Quemener, L. (1995) Comparative acute ammonia toxicity in marine fish and plasma ammonia response. Aquaculture, 136, 181–94. Rombough, P.J. (1988) Respiratory gas exchange, aerobic metabolism, and effects of hypoxia during early life. In: Fish Physiology. Vol XI. The Physiology of Developing Fish. Part A. Eggs and Larvae (eds W.S. Hoar & D.J. Randall), pp. 59–161. Academic Press, San Diego. Shepherd, J. & Bromage, N. (1988) Intensive Fish Farming. BSP Professional Books, Oxford. Spotte, S. (1979) Seawater Aquariums. Wiley, New York, Chichester, Brisbane, Toronto. Steinarsson, A. & Moksness, E. (1996) Oxygen consumption and ammonia excretion of common wolf-fish, Anarhichas lupus Linnaeus 1758, in an experimental-scale, seawater, land-based culture system. Aquaculture Res., 27(12), 925–30. Stickney, R.R. (2000) Dissolved oxygen. In: Encyclopaedia of Aquaculture (eds R.R. Stickney), pp. 229–32. Wiley, New York. Treece, G.D. (2000) Site selection. In: Encyclopaedia of Aquaculture (ed R.R. Stickney), pp. 869–79. Wiley, New York. Wheaton, F.W. (1977) Aquaculture Engineering. Wiley, New York.

Chapter 3

Microbial Interactions, Prophylaxis and Diseases O. Vadstein, T.A. Mo and Ø. Bergh

This chapter deals with microbes and infectious agents and their interactions with the fish at all developmental stages. Whereas several texts on this topic look primarily at diseasecausing organisms, we also try to deal with the natural interactions between microbes and hosts. Thus, we try to have both a veterinary and a microbial ecology perspective on the issue. The chapter starts with a general presentation of fish–microbe interactions (including both mutualistic/commensalistic and parasitic relationships, and some general immunology), followed by known problem organisms (virus, bacteria and parasitic proto- and metazoa). The second half of the chapter presents a general strategy for the control of infectious agents, and discusses how to improve environmental conditions and the resistance of the fish. The ontogeny of the immune system of the fish, and the differences between open and closed systems, are used throughout as a back-drop to a discussion of countermeasures.

3.1 Fish–Microbe Interactions and Implications in Aquaculture Compared to life in air, life in water exists in a far more hostile environment in a microbial sense. A fish has to handle typical bacterial concentrations of approximately 1 000 000 bacteria ml-1, of which 0.1–1% can be cultured on a non-selective agar. Concentrations of virus are one to two orders of magnitude higher than bacterial densities. However, only a few of these micro-organisms are harmful to higher organisms. By far the majority of viruses in aquatic environments are infective to other micro-organisms. Of the large numbers of different types of marine bacteria, only relatively few are known to be able to cause infections leading to disease.

3.1.1 Disease-Causing Organisms If an organism spends part or the whole of its life in association with another species it is called a symbiont. The majority of symbiotic relationships are neutral or even beneficial for the host (cf. the importance of the normal flora below). A relationship in which one symbiont benefits while the other (the host) is neither helped or harmed is called commensalism, whereas when both symbionts benefit the relationship is termed mutualistic. However, if the symbiont lives at the expense of, or harms, the host, it is a parasitic organ-

Microbial interactions, prophylaxis and diseases

29

ism. The parasitic way of life is so successful that it has evolved independently in most groups of organisms. Although viruses and bacteria are parasitic organisms, the term parasite usually refers to parasitic protists and metazoans. The fish-pathogenic bacteria, i.e. the bacteria that are able to cause disease in fish, are commonly divided into two different ecological categories. Obligate pathogens are specialised organisms that cannot survive by any other means than parasitising another organism. Conversely, opportunistic pathogens, sometimes referred to as facultative pathogens, possess a variety of other survival strategies and are not dependent on parasitising other organisms in order to ensure their own survival. Opportunistic pathogens are often naturally present in the environment, such as the water-column or sediments, and are characteristically able to take advantage of opportunities such as weakened fish or environmental conditions that may be favourable to the opportunist but unfavourable to the fish. Most fish-pathogenic bacteria belong to this category. In addition to bacteria considered to be pathogenic in a classical sense, some bacteria may colonize a host owing to either a weakened host and/or their presence at high concentrations. Because of over-colonization, these bacteria may be harmful for the host. Such bacteria may be termed opportunistic bacteria. Fish viruses are dependent on fish cells in order to reproduce, and thus should be regarded as obligate pathogens. In many cases, carrier states may be found where viruses are reproducing in a host (fish) with no external signs of disease. This is also true for many bacteria, including obligate pathogens. Fungi have generally not been associated with major problems for cultured marine fish. However, reports on Ichthyophonus spp. in several wild species suggest that one may expect problems caused by this group of organisms. In fact, Ichthyophonus have recently caused problems in reared salmon fed untreated, fresh marine fish. Eucaryotic parasites possess a wide variety of different life cycles. Often very complicated life cycles may be found where the fish is only an intermediate or final host. Parasites may live both on the surface (ectoparasite) or inside (endoparasite) of its host. Virulence is a quantitative term that refers to the relative ability of a pathogen to cause disease, i.e. its degree of pathogenicity. Virulence factors are thus factors that contribute to the virulence, such as specific abilities to adhere to host cells or enter the host, or abilities to survive within the host, produce certain toxins etc. For example, pathogenic strains of Vibrio anguillarum, the causative agent of vibriosis, produce a number of proteases, hemolysins, cytotoxins and dermatotoxins. These compounds contribute to the ability of the bacterium to exploit the resources provided by the host, and their effects can be observed as lesions. Different strains of pathogens may differ with respect to which virulence factors are present, and non-pathogenic strains (of e.g. V. anguillarum) are common.

3.1.2 Normal Fish–Microbe Interactions, Infection Pathways and Pathogenesis Because parasitic organisms, by definition, are dependent on their host, the relationship between parasite and host is dynamic. In most cases the dynamics are not determined by host and parasite only, but also by other symbiotic organisms of the host. Either a commensalistic (neutral) or mutualistic (beneficial) relationship with the host will influence the dynamics (Fig. 3.1). Shifts between the various types of symbiotic organisms will influence

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the health status of the host. The competitive ability of the mutualistic and commensalistic organisms, the number of parasites, the virulence of the parasite and the susceptibility of the host are decisive for whether the parasite will grow and multiply on or within the host, which is termed an infection. In order to cause disease in a fish, an infective organism must be successful through several stages of the infection cascade (Fig. 3.2). First, the pathogen must establish physical contact with the host and colonise epithelial cells or mucosa. The ability to adhere to the host, colonize the host and eventually enter into the host’s various tissues are thus vital to fish pathogens. There is considerable evidence that bacteria or viruses which are able to initiate infection are able to adhere specifically to epithelial cells. Many bacterial pathogens have been shown to be capable of adhering to fish cells and to grow in mucus in vitro. Generally, the gills and intestinal surfaces are important sites of adhesion and colonisation of fish pathogens, but entry through the skin is also possible. The second stage of infection involves cell damage, and may also include cell penetration and intracellular proliferation. Not all pathogens invade the host cells to cause damage and therefore enter the third stages, that entail systemic spread. The gills are constantly flushed with water, and only possess a thin structure separating the blood from the water surrounding the fish. This makes the gill an important site of entry for parasitic organisms such as bacteria or viruses. The surface of the gills is a natural habitat for bacteria. Phagocytosis by macrophages occurs in the gills, and thus bacteria may be phagocytosed in this organ. Furthermore, the gill is an important organ of antigen uptake, particularly of particulate antigens such as vaccines. The intestine of fish is a natural habitat for bacteria, and many are useful to the fish. The intestinal microflora may be beneficial to fish in three different ways: (1) They may participate in digestion, (2) they may synthesise essential growth factors and nutrients, and (3) they

MUTUALISM

When relationships move in this direction, the infectious disease process begins

COMMENSALISM

When relationships move in this direction, reestablishment of healthy host occurs

PARASITISM Figure 3.1 Symbiotic relationships between microorganisms and their host. Such relationships are highly dynamic, and shifts among them may occur due to shifts at the community level. Mutualism is most beneficial, and occurs when both symbionts benefit from the relationship. The most destructive is parasitism, in which one symbiont lives at the expense of, or harms, the host. Commensalism is the relationship in which one symbiont benefits while the other (the host) is neither helped or harmed. Redrawn from Prescott et al. (1999).


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Access to mucosa

Stage 1

Stage 2

Stage 3

Resistance to host defences

Competition with commensals Cell damage

Adherence

Colonisation

Lateral spread

Intracellular proliferation

Cell penetration

Entry into blood-stream and lymph system

Mucus

Epithelium

Laminaria propria

Resistance to host defences

Proliferation

Systemic spread Figure 3.2 The three stages of infection by pathogens (redrawn from Williams et al., 1988).

may play an important role in protection against pathogens. Bacteria and viruses are engulfed by endocytosis in the intestinal epithelium. Intestinal bacteria may take part in the degradation of various compounds such as complex carbohydrates to molecules that may be taken up and utilised by the fish, or in a synthesis of molecules which are of benefit to the fish, such as essential fatty acids or vitamins (Hansen & Olafsen, 1999; Ringø & Birkbeck, 1999). Intestinal bacteria may also play a part in protection against disease (cf. Ringø & Gatesoupe, 1998). In adult fish, a bacterium entering the fish via the intestine must first pass through the stomach. The secretion of lethal substances and the low pH in the stomach are important barriers against pathogens using this route. In larval fish, however, the stomach is not well developed and the intestine is more accessible. Live food organisms such as rotifers and Artemia nauplii spp. naturally filter bacteria, and may contain large amounts of bacteria (Skjermo & Vadstein, 1993; Makridis et al., 2000a). Pathogens present in the live feed cultures may thus be presented to larval fish intestines in concentrated live-feed packages. Bacteria colonise the fish intestine even before active feeding commences. In halibut, the mouth is not developed when the larva hatches, but the intestine is still available to aquatic bacteria. In this case, the pseudobranchs is the site of entry. Bacteria are taken up from the water at rates that exceed drinking rates by one order of magnitude (Reitan et al., 1998). This uptake is therefore active and regulated by some unknown mechanism. Prior to the onset of exogenous feeding, larvae generally possess a non-fermentative intestinal flora, whereas the onset of feeding generates processes which probably include a reduction in oxygen levels in parts of the intestine, leading to a flora dominated by fermentative bacteria (Bergh et al., 1994).

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One of the main functions of fish skin is the separation of the internal tissues from the environment. The mucus secreted by cells in the skin contains immunoactive compounds. Specialised eucaryotic parasites, such as lice, may penetrate the skin, thus making it possible for bacterial pathogens to enter through the lesions. Salmon lice (Lepeophtheirus salmonis) infecting Atlantic salmon (Salmo salar) may act as a vector for the infectious salmon anaemia (ISA) virus (Nylund et al., 1994). It is likely that many parasites are important vectors transmitting pathogens, and that by causing damage to the fish skin they open the way for other pathogens. In some cases a bacterium can remain localised at mucosal surfaces and initiate damage by liberating toxins. This mode of action may be important for opportunistic bacteria. However, in most cases the pathogenic bacteria penetrate the epithelium and spread to other parts of the body, where growth is initiated. Viruses must enter host cells in order to reproduce, and are normally dependent on specific receptor molecules that fit surface structures of the virus particle in order to enter the host cell. From the site of entry, pathogens are spread mainly via the blood or lymphatic systems. The ability to survive in blood and the action of phagocytes is of vital importance to the pathogen, although the actual mechanisms by which pathogens survive or even multiply are poorly understood. Survival in serum has been documented for several pathogens. Although bacteria may be phagocytosed, several pathogens have been shown to survive and multiply within the phagocytes. The outer layers of the fish egg, chorion and zona radiata, seem to be highly protective against bacteria. To date only Tenacibaculum ovolyticum (previously named Flexibacter ovolyticus), a pathogen to halibut eggs, has been shown to be able to penetrate these structures and thus get access to the resources inside the egg (Hansen et al., 1992). However, many other bacteria colonise the surface of the egg, and pathogens may be able to infect the larvae post-hatch (Bergh et al., 1992). Special attention should be paid to some bacteria that are spread intra ovum, i.e. within eggs. A fish pathogen that has been shown to occupy this ecological niche is Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salmonids. Interestingly, this is an obligate pathogen that seems highly specialised for the niches it occupies, which include growth within host macrophages. It is a very slow-growing bacterium, both in laboratory cultures and within the fish. The progress of the disease is slow, making it possible for the host to reproduce, thus spreading the genes of its parasite. It is likely that intra-ovulary pathogens are also present in marine fish, but they remain to be found. Viruses can also be transmitted vertically. This is the case for the nodavirus causing viral encephalopathy and retinopathy in Atlantic halibut (Grotmol et al., 1997). It is not known whether the virus is transmitted intra ovum or on the surface of eggs, but successful application of disinfectants suggests that the virus may be present externally, i.e. in the ovary fluid. The possibility of an outbreak of disease is dependent on a large number of factors which interact with each other. The fish, the pathogen, and the physical and chemical environment may all influence the status of any of the others (Fig. 3.3). When trying to improve survival in aquaculture, work should be aimed at all these factors. Improvements in physio/chemical environmental conditions and strengthening the resistance of the fish will contribute to


A: Factors determining viability (P V )

33

B: Manipulation to increase

Genetics, Nutrition, Immune system Larvae

Larvae

Pv

Microbial environ. Numbers Composition

Pv

Physiochemical environ.

Microbial environ.

Physiochemical environ.

Chemicals, Physical stress

Figure 3.3 A. The three factors of significance for the probability of viable larvae (PV) and the conditions which influence these factors. B. The probability of viable larvae (PV) may be increased by using methods that push the circles towards each other. Redrawn from Vadstein (1997). Reprinted with permission from Elsevier Science.

increases in survival and viability, as will the elimination of pathogens and strengthening of the commensal microflora. However, microbial control cannot be regarded as absolute, but is more a question of probabilities. This is evident if we consider the three interacting factors that are decisive for the development of conditions that ensure good viability of the larvae (Fig. 3.3). The fish, and the biological and physiochemical environments are in turn influenced by several factors, and manipulating these conditions may increase the probability of viable larvae. However, it is clear that manipulation of only one of these three factors may have a limited effect. Therefore, in any strategy to achieve microbial control and thus improve the viability of the fish, it is important to use a range of counter-measures directed towards the different aspects shown in Fig. 3.3.

3.1.3 The Immune System of Fish The main function of the immune system is to protect the animal against disease-causing organisms. The immune system of fish shows clear similarities to that of mammals, and several reviews have covered various aspects of this subject (cf. Vadstein, 1997). The immune system comprises both non-specific and specific components, and involves both cellular and humoral factors (Fig. 3.4). This division into four constituents is somewhat misleading because all components are interwoven and mutually dependent. The non-specific defence mechanisms are part of all normal fish, and do not require prior contact with an antigen/pathogen to elucidate a response. On the other hand, the specific immune system requires activation, and there is thus a time-lag between the first introduction to the antigen and the activation. This process is also temperature-dependent (Bly & Clem, 1992). It is believed that non-specific immunity is phylogenetically older than specific immunity, and one might therefore speculate that fish are more reliant on non-specific defence than higher vertebrates.


Cellular

Humoral

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Non-specific (innate)

Specific (acquired)

Lytic enzymes, e.g. lysozyme Complement Agglutinins and precipitins Enzyme inhibitors Growth inhibitors

Antibodies

Macrophages/monocytes Granulocytes Non-specific cytotoxic cells

B-cells T-cells

Figure 3.4 Simplified figure showing the main components of the non-specific and specific immune system.

The immune defence systems of the fish mature during the development of eggs, larvae and fry. Unlike mammals, for instance, that are born at comparatively advanced developmental stages, most fish hatch at ontogenetically primitive stages which are analogous to the early developmental stages of embryos of higher vertebrates. This is also true with respect to the immune system. However, the ontogenetic stage at which hatching takes place is highly variable between species. Halibut larvae hatch at a very primitive stage and the wolf-fishes are relatively advanced at hatching, whereas cod and turbot, for instance, may be viewed as intermediate cases with respect to developmental stage at hatching. It is believed that the larvae of most fish species do not have the ability to develop specific immunity during the early stages of development. In this respect, they are reliant on passive immunisation from maternal antibodies. Although it has been reported that maternal transfer of specific immunity does not occur in salmonids (Ellis, 1988a), this mechanism has been experimentally demonstrated in tilapias (Mor & Avtalion, 1990; Sin et al., 1994). In any given species, size rather than age seems to be most critical for when specific immunity may be developed. Larvae of many species go through a process of self-recognition after hatching, where the specific immune system ‘learns’ to recognise the tissue of the individual. This is of importance in aquaculture, as specific immunostimulation, i.e. vaccination, when performed at these immature developmental stages, could induce immunosuppression rather than immunoprotection, with reduced survival as a result (Joosten et al., 1995). Vaccination of early life stages must therefore be carefully evaluated with respect to the maturation and status of the immune system of the species at different developmental stages (Fig. 3.5). The non-specific immune system is probably the major defence against micro-organisms in larvae. Although our understanding of the components of the innate defence system of fish is growing, relatively little is known about the functioning and ontogeny of the general immune system in marine larvae. In the few fish species that have been studied, the major lymphoid organs are not fully developed at the time of hatching, and the phagocytic activ-


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Immunoprotection

No protection Immunosuppression

Larval ontogenesis (time) Vaccination possible Protection induced by vaccination

Figure 3.5 Theoretical protection by vaccination as a function of the larval ontogenetic stage at which vaccination is carried out. Vaccination at early ontogenetic stages generally induces immunosuppression rather than immunoprotection, with a reduced survival rate as a result. Thus, vaccination should only be done after the fish has become sufficiently immunologically mature to ensure positive immunoprotection. Following vaccination, there is a time delay before protection is achieved because of the time that is necessary before the immune reaction has induced protection.

ity is mainly associated with gills, skin and gut. It is therefore possible that during the stages when the lymphoid organs are developing, the main cellular defence is by the phagocyte populations within the integument. The non-specific or innate immune system is regarded as the first line defence of animals. Furthermore, it seems that the bacterial problems in larviculture are more often due to opportunistic bacteria than specific pathogens (Vadstein et al., 1993; Munro et al., 1995). This emphasises how reliant the larvae are on their non-specific defence under intensive hatchery conditions.

3.2 Viral Diseases: Diagnosis 3.2.1 Infectious Pancreatic Necrosis Virus (IPNV) Juvenile stages of several marine species, particularly halibut and turbot, are susceptible to infections by an aquatic birnavirus, infectious pancreatic necrosis virus (IPNV) (Biering et al., 1994; Mortensen et al., 1993). Only minor sero- and genotypic differences have been found between isolates from halibut and turbot and the N1 or Sp strain from Atlantic salmon (Biering et al., 1997). Moreover, isolates from salmonids have been found to establish an infection in halibut, and thus transfer of virus between salmon and marine species cannot be excluded. From challenge experiments with halibut fry and yolk-sac larvae, it can be concluded that the virus is the causative agent of disease. Temperature influences IPN

36


mortality, as well as the developmental stage of the fish. In general, smaller fry are more susceptible to infection. IPNV infections in wild cod have been observed in Denmark and the Faroe Islands. Fish of 2–10 g have developed the disease, which is associated with high mortalities. The most characteristic clinical signs of disease in challenged halibut are distended stomach, uncoordinated swimming and trailing, and white faecal casts (Biering et al., 1994). The symptoms are most prominent in small fry, but were also observed in larger individuals. Pathological findings included focal necrosis of the liver, kidney and intestine, but the pancreatic tissue was unaffected. The absence of pathological findings in pancreatic tissue of challenged fish reported by Biering et al. (1994) was in disagreement with the findings from naturally infected halibut fry (Mortensen et al., 1990), where severe necrosis of the pancreatic acinar cells was found, together with nuclear pycnosis. Reports from turbot vary on this point, although samples from natural outbreaks in Norway demonstrated pathological findings in pancreatic tissue (Mortensen et al., 1993). The intestine may be the primary organ for virus entry and replication, as indicated by immunohistochemical observations in a challenge experiment with yolk-sac larvae (Biering & Bergh, 1996). However, these authors also pointed out that differences in pathology and susceptibility to IPNV infection generally occur between different developmental stages. As the term infectious pancreatic necrosis virus, by definition, implies necrosis of the pancreas, a condition not always found in affected halibut, the term aquatic birnavirus is probably more correct (Biering et al., 1994).

3.2.2 Nodaviruses Nodaviruses, a family of neuropathogenic viruses first described from insects, are known to cause infections in many marine fish species in many parts of the world (reviewed by Munday & Nakai, 1997), including turbot and halibut. The virus has been known to cause disease in turbot fry since an outbreak in an extensive production lagoon in 1989, and the virus was originally described as a picornavirus-like agent (Bloch et al., 1991). Mortality appeared during weaning onto moist pellets. Diseased fish became lethargic, often lying abdomen-up on the bottom. Atypical swimming, such as rotating, spinning and horizontal looping, was observed when the fish were disturbed. The clinical signs indicated a disturbance of the central nervous system, and were followed by 100% mortality. Under electron microscopy, vacuolated cells were found in the brain and medulla of the diseased fish, with large numbers of crystalline virus particles. In halibut aquaculture, problems with nodaviruses have caused a decrease in Norwegian juvenile halibut production since 1995 (Bergh et al., 2001). An outbreak of a nodavirusrelated disease, viral encephalopathy and retinopathy (VER), in halibut was first recorded in the summer of 1995, when two major hatcheries in western Norway were severely affected (Grotmol et al., 1995, 1997). Most larvae died in the period of early metamorphosis, approximately 60 to 70 days post-hatch. The first clinical signs of VER seen in the larvae were reduced skin pigmentation and an empty, transparent intestine due to reduced food intake. Darkening of the skin could be seen. Abnormal behaviour such as spiral swimming and looping was observed in the early stages of a disease outbreak. Severely diseased larvae and


37

juveniles became lethargic, often lying upside down on the bottom. Lesions and vacuolisation in the retina, brain and spinal cord, and in ganglia of the peripheral nervous system, were typical findings, with no lesions detected in other organs. There is no doubt that the nodavirus is the causative agent of the disease, as in challenge experiments the strain from halibut fry was able to replicate and cause VER in halibut yolksac larvae (Grotmol et al., 1999). Monitoring the progression of the infection following challenge suggested that the portal of entry into the larvae may have been the intestinal epithelium, while the route of infection to the central nervous system (CNS) may have been axonal transport to the brain stem through cranial nerves such as the vagus nerves. Diagnosis of nodavirus infections has so far been dependent on histological and immunohistochemical investigations (Grotmol et al., 1997, 1999). A reverse transcriptase–polymerase chain reaction (RT–PCR) assay based on the capsid protein nucleotide sequence of the halibut nodavirus strain has been developed (Grotmol et al., 2000). Vertical transmission occurs, but may be successfully counteracted by ozone disinfection of eggs (Grotmol & Totland, 2000). There are differences between nodavirus isolates from different fish species. Comparing the pathogenicity of a Norwegian strain from halibut and a Japanese strain from striped jack Pseudocaranx dentex, Totland et al. (1999) found that the halibut strain, which caused high mortality to halibut larvae, was incapable of replicating or inducing mortality in striped jack larvae, and vice versa. However, this difference might be the result of either hostspecificity or the difference in rearing temperature. Nodavirus strains affecting cold-water species from Norway and Japan are closely related, belonging to a separate clade, whereas nodavirus strains from temperate species, such as striped jack, may be genetically more distant. VER is by far the most important disease problem in present-day halibut aquaculture (Bergh et al., 2001). The outbreak in Norway coincides with a decline in halibut fry production, and is probably the major reason why Norway’s production of halibut fry has levelled out since 1995. No vaccine is currently commercially available. However, a recombinant vaccine has been shown to give significant protection in challenge experiments with turbot, Scophthalmus maximus (Húsgarð et al., 2001). At present, one is left with traditional prophylactic countermeasures such as egg disinfection and improved hygiene.

3.2.3 Other Viruses The rhabdovirus VHSV, the causative agent of the so-called Egtved disease or viral heamorrhagic septicaemia in rainbow trout, may also cause problems in marine fish aquaculture, as there are reports of the isolation of VHSV-like viruses from cod and turbot. Nucleotide sequencing of the glycoprotein gene of VHSV from different geographical areas has confirmed a link between VHS in farmed salmonids and viruses isolated from cod (Stone et al., 1997). Two virus isolates recovered from wild-caught cod off Shetland and from farmed turbot in Scotland showed 99.4% nucleotide sequence similarity with a virus associated with VHS in rainbow trout. Challenge experiments with turbot has confirmed the aetiology of the disease. Typical signs of the disease in challenged turbot included darkening of the skin and the presence of haemorrhaging around the head and fin bases

38


(Snow & Smail, 1999). VHSV has also been found in wild-caught Pacific cod, Gadus macrocephalus (Meyers et al., 1992). Another related virus, the hirame rhabdovirus, is known to cause disease in the hirame, or Japanese flounder, Paralichthys olivaceus (Oseko et al., 1988). The signs of infection were congestion of the gonad, focal haemorrhage of skeletal muscle and fins, and accumulation of ascitic fluid. Histopathologically, the kidney indicated necrotic changes by nuclear degeneration of haematopoietic cells and haemorrhage in the interstitial tissue. The spleen showed necrosis and haemorrhage in the pulp, and skeletal muscle revealed hyperaemia and haemorrhage of capillary vessels. Hyperaemia and haemorrhage were observed in the interstitial tissue of the seminiferous duct and ovarian lamella, and in the connective tissues around the seminal duct and oviduct of the testis and ovary. Mucosa of the alimentary tract showed hyperaemia and haemorrhage. An iridovirus-like agent has been described in association with systemic infection in cultured turbot fry in Denmark (Bloch & Larsen, 1993). The initial signs of disease were reduced feed intake, lethargy and darkening in pigmentation, especially of the tail and fins. Later, one may observe atypical swimming and spasms in the terminal stages. Investigation by electron microscopy of samples of fin, gill, liver, kidney, spleen, heart, pancreas and intestinal collagen, and in one of three brain samples investigated, confirmed the presence of an iridovirus-like agent ca. 170 nm in diameter. Another virus, Herpesvirus scophthalmi, which is 200–230 nm in diameter, has only been described under electron microscopy from turbot in Scotland and Wales (Buchanan & Madeley, 1978). An affected fish is lethargic, often lying with head down and tail up on the bottom. A virus suggested to be a member of the aquareovirus group was demonstrated by Lupiani et al. (1989), but the disease condition was described as of mixed bacterial and viral aetiology.

3.3 Bacterial Diseases: Diagnosis Bacteria may be present in large numbers on the surface of fish eggs. This epiflora seems to be dominated by members of the Cytophaga/Flavobacterium/Flexibacter group, while Vibrio spp. are not frequent (Hansen & Olafsen, 1989; Keskin et al., 1994; Bergh, 1995). The composition of the intestinal bacterial flora associated with yolk-sac larvae resembles the egg epiflora, whereas a shift in the intestinal microflora from a generally non-fermentative towards a fermentative flora dominated by the Vibrio/Aeromonas group coincides with the onset of exogenous feeding (Bergh et al., 1994; Bergh, 1995). The psychrotrophic Tenacibaculum ovolyticum (formerly named Flexibacter ovolyticus) was isolated from halibut eggs with high mortality (Hansen et al., 1992). It resembles the fish pathogen T. maritimum, but differs in several biochemical and physiological characteristics. Challenge experiments confirmed that the bacterium is able to cause mortality to halibut eggs and yolk-sac larvae by penetrating the eggshell (Bergh et al., 1992). This bacterium has not been found on hosts other than halibut, despite the fact that it is able to cause mortality to cod, Gadus morhua, eggs and larvae (Bergh, 2000). Pathogenic Flexibacter- or Cytophaga-like organisms have also been described from cultured turbot (Mudarris &


39

Austin, 1989) and wild-caught cod (Hilger et al., 1991). Other bacterial pathogens described from cod are Streptococcus parauberis (Romalde, 1999) and a Mycobacterium sp. (B. Hjeltnes, National Veterinary Institute, personal communication, 2002).

3.3.1 Vibrio Species Several species of Vibrio are able to cause mortality to yolk-sac larvae. V. anguillarum, V. splendidus and a V. salmonicida-like strain were tested in a challenge experiment with halibut larvae by Bergh et al. (1992). Unlike Tenacibaculum ovolyticum, the Vibrio spp. did not cause mortality to eggs, but infected the larvae post-hatch, causing mortality during the yolk-sac stage. Of these species, V. anguillarum, serotype O1 and O2, are the most potent, whereas the others must be present in relatively large numbers in order to cause significant mortality (Ø. Bergh, unpublished results, 2002). Opportunistic bacteria affecting yolk-sac larvae typically induce changes in larval behaviour, reducing the ability of the larva to initiate exogenous feeding and decreasing their buoyancy, as demonstrated on halibut and turbot larvae by Skiftesvik and Bergh (1993). Thus, even though the opportunists may not kill the larva directly, the probability of the larva surviving the first feeding period may be reduced. Investigating the pathogenesis of vibriosis in turbot larvae, Grisez et al. (1996) utilised V. anguillarum-enriched Artemia franciscana as vector organisms. The authors concluded that the anterior part of the intestine was the major port of entry, and that the bacteria were transported through the intestinal epithelium by endocytosis, after which the bacterium was released in the lamina propria. From there, the bacterium was transported by the blood to different organs, eventually leading to septicaemia and mortality. Apart from V. anguillarum, two more species within this genus have been described as causative agents of vibriosis in turbot: V. damsela (Fouz et al., 1992), and V. splendidus (Farto et al., 1999; Gatesoupe et al., 1999). In cod, V. anguillarum dominates among pathogenic isolates (Knappskog et al., 1993; Wiik et al., 1995). Isolates of V. anguillarum from Norwegian marine fish with vibriosis were found to be free of plasmids, strongly indicating that their virulence properties were chromosome-mediated (Wiik et al., 1989).

3.3.2 Aeromonas Species The causative agent of furunculosis in Atlantic salmon, Aeromonas salmonicida subsp. Salmonicida, is for most practical purposes apathogenic to halibut. In a field survey during a major outbreak of furunculosis in the Atlantic salmon stock, all dead halibut at the station were subjected to investigation (Hjeltnes et al., 1995), but no indications of a transfer were found. An experimental challenge with typical A. salmonicida subsp. salmonicida administered to yolk-sac larvae gave a more complex result (Bergh et al., 1997). Significant mortality did take place, but this was probably a result of the production of toxic exudates by the bacterium, as histological and immunohistochemical examinations of the larvae revealed no evidence of bacteria in affected tissues. In contrast to the A. salmonicida subsp. salmonicida, which comprises a homogenous group of strains, atypical A. salmonicida strains are heterogenous with respect to serologi-

40


cal and biochemical characteristics (Wiklund & Dalsgaard, 1998). However, atypical strains of A. salmonicida are occasionally isolated from diseased halibut and turbot suffering from septicaemia, although at present it cannot be ruled out that some of these infections may be secondary. Recently, Ingilæ et al. (2000) reported significant mortality in a challenge experiment with an atypical strain isolated from halibut administered to juvenile halibut and spotted wolf-fish, confirming the pathogenicity of this bacterium. Mortality of turbot following challenge with atypical furunculosis has also been reported (Perez et al., 1996). Comparing the pathogenicity of one atypical and one typical strain of A. salmonicida to subadult halibut (weight range 154–254 g), Bricknell et al. (1999) found no mortality of halibut as a result of a bath challenge. Minimum lethal doses per halibut after intraperitoneal injection of bacterium were 106 (typical A. salmonicida) and 107 (atypical A. salmonicida). Following the challenge, a stress test of the survivors gave the result that 9 of the 87 halibut died, but all were culture-negative for A. salmonicida, indicating that no carrier state was present.

3.4 Parasitic Protists and Metazoans: Diagnosis, Prophylaxis and Treatment A parasite lives at the expense of another organism, a host, and is usually dependent on this host to complete its life cycle. The term parasite usually refers to unicellular and multicellular eucaryotes, and thus excludes bacteria, fungi and viruses. The damage caused by a parasite can vary from minor to serious and even life-threatening. Usually, fish that carry relatively few parasite specimens cannot be considered to be diseased. Disease occurs when a fish is infected with so many parasites, of one or more species, that its normal life functions are disturbed. There is no exact number of parasites that will cause disease. This depends on several parameters such as parasite virulence, host susceptibility and resistance, host size and age, relative parasite size compared to host, and many more. Some parasites may occur in hundreds or even thousands without causing severe pathology, while only one or two Lernaeocera branchialis may cause the death of a small Atlantic cod. Many parasites are present in farmed fish without ever causing clinical disease, but they can significantly reduce fish growth. Such parasites can sometimes cause greater economic loss for the farmer than more pathogenic parasites, as the former will easily be overlooked while the latter will be treated at an early stage in a disease outbreak. Parasite infections may also facilitate infections by other organisms, both by creating injuries, which serve as infection routes, and by reducing the general fish disease resistance. Thus, prophylactic measurements to keep the number of parasites low may also have beneficial effects against viral, bacterial, fungal and other parasitic infections. The identification of parasites, at least to family or genus level, is often necessary to ensure that correct prophylactic methods and chemotherapeutic compounds are chosen. Many multicellular parasites can be observed macroscopically. The majority of parasites, however, can only be demonstrated by the use of a dissection or light microscope, or even an electron microscope. Identification of fish parasites is still mainly based on morphology


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(shape) and morphometry (size), but an increasing number of parasites are identified by the use of molecular techniques. A large number of parasites can be found in farmed and wild marine fish. In this text, the most common parasite groups in fish are mentioned. For each group, only one or a few species causing disease are mentioned. At present, examples of parasitic diseases in cultured cold-water marine fish are few. That is why examples of parasitic diseases in cultured warmwater marine fish are given. It is likely that the cold-water marine fish farms will experience the same kind of parasitic diseases as production increases.

3.4.1 Protists 3.4.1.1 Amoebae Diagnosis/Identification Amoeba is a generic term for protists that move with the aid of pseudopodia (temporary projections of protoplasm and cell membrane for moving and feeding). The term amoeba no longer represents a single taxon, as many different protists have amoeboid developmental stages. Most protists with amoeboid development stages belong to the phylum Rhizopoda. Multiplication mainly occurs by division into two equal parts, a process known as binary fission. Occasionally, a sexual process associated with flagellated or amoeboid gametes occurs. Identification is based on locomotive form and behaviour, the presence of flagellated stages, cyst structure, and nuclear structure and division. Symptoms/Pathology Amoebas may be found in many organs. Amoeboid gill disease seems to be the most common. The pathology includes elevated mucus production, epithelial hyperplasia and metaplasia, and fusion of primary and secondary lamellae. Agents causing amoebic gill disease in clinically diseased turbot, Scophthalmus maximus, were recently identified (Dyková et al., 1999), and a similar amoeboid gill disease has also been observed in farmed halibut, but the causative organism(s) has not yet been identified. Prophylaxis/Treatment Lower stocking densities and net cleaning may be of preventative value. Treatment with freshwater has been effective against Paramoeba pemaquidensis associated with proliferative gill disease of farmed salmonids in seawater. Chemical baths have been relatively ineffective. 3.4.1.2 Apicomplexans Diagnosis/Identification Most apicomplexans live intracellularly. In at least one of the stages in the life cycle, the apicomplexan cell has a set of organelles at the apex, the apical complex, used for

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penetration into a host cell. The complex life cycle usually involves both asexual and sexual reproduction. Fish apicomplexans are divided into three groups. Coccidia mainly develop in cells of the intestinal wall and are spread by direct transmission or the use of paratenic hosts. Adeleids and piroplasmids are found in the blood and are spread by blood-sucking invertebrates, mainly leeches. Identification of coccidia is mainly based on the shape and size of oocysts containing sporozoites usually enveloped in sporocysts, while adeleids and piroplasmids are identified in fresh and stained blood smears. Symptoms/Pathology A number of apicomplexans cause pathology and mortality in wild marine fish, but also in farmed marine fish. The blood-living apicomplexan Haemogregarina sachai has caused severe pathology in farmed turbot (Kirms, 1980). Prophylaxis/Treatment Little is known about prophylaxis, but it could involve elimination of the leeches or parasitic crustaceans needed for transmission. Some chemical compounds have been effective. 3.4.1.3 Microsporidia Diagnosis/Identification Microsporidians live intracellularly. The transmission stage is a spore that contains a hollow, evertible polar tube. Under an appropriate stimulus from a host, the polar tube is everted and the sporoplasm is propelled through the tube. The parasite penetrates host tissue and follows circulatory vessels to the final site. Both asexual and sexual reproduction is involved in the life cycle. During the asexual phase, parasites may spread to a large number of host cells and a large part of an organ may be affected. Some microsporidians stimulate the infected host cell to enormous hypertrophy, resulting in a xenoma or ‘cyst’, often macroscopically visible (Fig. 3.6, see colour plate section). Microsporidians are transmitted directly between fish. Spores can retain their infectivity in water at 40°C for at least 1 year. Identification is usually based on the shape and size of the spores, which are usually less than 7 mm in length. Symptoms/Pathology Tetramicra brevifilum has caused disease in farmed turbot in Spain and Great Britain. The outbreaks were associated with a drop in the water temperature. Affected fish showed erratic swimming behaviour, swelling of different parts of the body, darkening of the dorsal surface and overproduction of mucus. Heavily infected fish had jelly-like muscles (Figueras et al., 1992). A species of Nucleospora (formerly Enterocytozoon) has been found in dead and moribund halibut, Hippoglossus hippoglossus, in Norway (Nilsen, 1999). The spleen and kidney were swollen. The excretory and haematopoietic tissues in the kidney were degener-


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ated. In the head kidney, more than 50% of the lymphoblasts were infected with microsporidians in different developmental stages (Nilsen, 1999). An unidentified microsporidian has caused mortality in farmed sea bream Sparus aurata (Abela et al., 1996).

Prophylaxis/Treatment Because of the longevity of spores and direct transmission, infections by microsporidians are usually difficult to prevent. Few chemical compounds are effective (Schmahl & Mehlhorn, 1989).

3.4.1.4 Ciliates Diagnosis/Identification Ciliates can be found in any fish organ and on external surfaces. They are partly or completely covered by a large number of cilia, which are used for locomotion and feeding. They have a vegetative macronucleus and a generative micronucleus. Most species multiply by binary fission. Identification is mainly based on structure of the oral apparatus, combined with some ultrastructural features.

Symptoms/Pathology Ciliates are commonly found as ectoparasites or endoparasites in farmed and wild marine fish. Many species are not strictly parasitic, as they only use fish for attachment, and feed on bacteria and small protists in the water. However, these ciliates may become true parasites when they occur in large numbers (Fig. 3.7, see colour plate section), often as a consequence of increased organic burden, bacteria or protist numbers in the seawater, and/or reduced host resistance and immune responses. Some examples are species of the genus Trichodina (Figs. 3.8 and 3.9, see colour plate section), and ‘permanently’ attached ciliates such as members of the genera Apiosoma, Epistylis and Riboscyphidia. A Uronema-like ciliate has repeatedly caused severe systemic ciliatosis (Fig. 3.7) in farmed turbot in Norway (Sterud et al., 2000), and could be the same species that has been found in farmed turbot in Spain (Dyková & Figueras, 1994). Another scuticociliatid, Philasterides dicentrarchi, has caused a similar systemic infection in farmed sea bass, Dicentrarchus labrax (Dragesco et al., 1995). Reduced growth in farmed turbot due to Trichodina sp. has been documented (Sanmartin Durán et al., 1991), while heavy infections with T. hippoglossi have been observed in farmed halibut larvae (Nilsen, 1995). Prophylaxis/Treatment Prophylactic measures include reduced host stress and reduced stocking density. A formalin bath (usually 1 : 4000) for half-an-hour is commonly used to treat ectoparasitic ciliatosis. Treatment with freshwater, reducing the salinity to 10 p.p.t. or lower, for 30–60 min may be effective. Oral drug administration has also been shown to be effective (Rapp, 1995).

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3.4.1.5 Flagellates Diagnosis/Identification Parasitic flagellated protists belong to several distinct phyla. They all have one or several flagella used for locomotion and host attachment. Multiplication is usually by binary fission. Many parasitic species have morphologically different free-living stages for transmission between hosts. The cell, excluding flagella, in most species is between 10 and 40 mm. Identification is based on the number and location of flagella, the shape of the cell, the shape and size of nuclei, and other ultrastructural features. Symptoms/Pathology The most serious flagellated protists in farmed fish are ectoparasites, which often cause skin and gill epithelial hypertrophy/hyperplasia and necrosis. Species of the genus Ichthyobodo have a special disc for attaching to the host cell, while species of the genus Cryptobia use flagella for attachment. I. necator is a well-known pathogen from freshwater and marine cultures of salmonids. Similar but morphologically different species have been found on 25 marine fish species (Urawa et al., 1998). The genus Cryptobia includes both ectoparasitic (skin and gills) and endoparasitic (blood and gut) species. It is suggested that the endoparasitic ones should be included in the genus Trypanoplasma. The ectoparasitic Cryptobia have a direct transmission, while the endoparasitic ones need a leech. The blood flagellate C. bullocki has caused pathology and disease in wild flatfishes in Chesapeake Bay, USA (see Woo & Poynton, 1995). Dinoflagellates such as Amyloodinium spp. and Piscioodinium spp. often cause suffocation due to severe damage of the gill epithelium. Prophylaxis/Treatment A formalin bath (1 : 4000) for about 30 min has been the most commonly used treatment against ectoparasitic flagellates. Sometimes it has been necessary to use a higher concentration (1 : 3000), possibly because of a high organic content in the cages.

3.4.2 Metazoans 3.4.2.1 Myxosporidia (Parasitic Cnidarians) Diagnosis/Identification Previously myxosporidians were classified as protists, but recent scientific studies have shown that they are parasitic cnidarians (Siddall et al., 1995) and are thus metazoans (Smothers et al., 1994). Myxosporidians can be found in any fish organ. They undergo several vegetative (pre-sporogonic) stages before reaching the transmission stage, which is a multicellular spore (Figs. 3.10 and 3.11, see colour plate section). Probably most, but not all, myxosporidians alternate between two hosts, a fish and an invertebrate, in their life cycle. Most myxosporidians have spores between 10 and 20 mm in length. Identification is mainly based on spore morphology.


45

Symptoms/Pathology Many species can cause severe pathology, either in their pre-sporogonic stages or due to the spores. Spores of coelozoic species are mainly found in the gallbladder and in the urinary bladder and associated tracts, while spores of histozoic species, living in host tissue, may be found in large ‘cysts’ or spread in the skeletal muscles, resulting in an unappetising fish. Species of the genus Kudoa may elicit post-mortem myoliquefaction, and thus reduce the market value of infected fish products (Moran et al., 1999). Myxidium leei has caused severe pathology in farmed sea bream Sparus aurata (Diamant et al., 1994). Prophylaxis/Treatment Myxosporean spores are very resistant and can tolerate extended freezing. Different disinfectants can be used to kill free spores, while treatment by medicated pellets may reduce the infection. 3.4.2.2 Monogeneans Diagnosis/Identification Monogeneans are mainly ectoparasites on fish fins, skin and gills (Figs. 3.12 and 3.13, see colour plate section). They are attached to the host by a specialised attachment organ called an opisthaptor, which includes hold-fast structures such as hooks and/or clamps. The life cycle is direct, involving only one host. With the exception of the viviparous Gyrodactylids, monogeneans lay eggs which hatch into a swimming, infective oncomiracidium. This larvae attaches directly to, or moves to, the final site where it feeds. Most monogeneans are between 0.5 and 20 mm in length. Identification is mainly based on the shape and size of sclerites in the opisthaptor and structures associated with the male sex organ, which is present in all adult specimens as monogeneans are hermaphrodites. Symptoms/Pathology Because of their direct life cycle, many monogeneans are troublesome parasites in fish mariculture. This is especially true of the viviparous species of the genus Gyrodactylus. Different species of this genus have caused severe pathology and mortality in many Norwegian marine fish farms, including Atlantic and spotted wolf-fish, cod, halibut and plaice (personal observations, 1990–2000). A Microcotyle sp. has caused pathology and mortality in farmed sea bream (Sanz, 1992), and Diplectanum aequans has caused gill pathology in farmed sea bass (Cognetti Varriale et al., 1992). The relatively large Entobdella hippoglossi is easily observed macroscopically, and may cause skin irritation and ulcers in halibut. Prophylaxis/Treatment Reduced host density and increased water flow may reduce infection abundance, especially for egg-laying species. A large number of drugs have been shown to be effective, although

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rarely completely (Santamarina et al., 1991; Cognetti Varriale et al., 1992; Schmahl, 1993). Most commonly used has been a formaldehyde bath (in 1 : 4000) for about 30 min. Freshwater may also be effective against marine monogeneans. Large monogeneans, such as E. hippoglossi, can effectively removed from the skin of a resting host by a pair of forceps. 3.4.2.3 Cestodes Diagnosis/Identification Cestodes are endoparasites. They are long, flattened and usually whitish worms with complex life cycles, using fish as final or intermediate hosts. In the former case, the segmented adult worms occur in the digestive tract and may reach several decimetres in length (Fig. 3.14, see colour plate section). Larval cestodes, usually non-segmented, may be free-living or encapsulated in skeletal muscles, internal organs or the abdominal cavity. Cestodes found in fish usually use crustaceans as their first intermediate host. Cestodes lack an alimentary canal and food is absorbed through the outer surface. Identification is mainly based on the shape of the attachment organ at the anterior end, the scolex, and the shape and arrangement of the sex organs in the segments, the proglottids. Symptoms/Pathology Most cestodes using fish as final hosts are not regarded as pathogens. However, when they occur in large numbers in the digestive tract, they can significantly reduce the growth of the fish. Larval cestodes in viscera may cause severe pathology. Organs may be destroyed and/or important metabolic processes may be altered or reduced. Fish mortality due to larval cestodes is usually associated with intensity of infection. The occurrence of cestode larvae may cause extensive visceral adhesions. Prophylaxis/Treatment Oral treatment by adding chemicals to dry pellets is commonly used against cestodes in the digestive tract. Treatment against larval cestodes in fish tissue is generally not effective. 3.4.2.4 Trematodes Diagnosis/Identification Trematodes, also known as flukes, are endoparasites using fish as their final or intermediate host (Fig. 3.15, see colour plate section). Adult trematodes usually have two suckers, one anteriorly and one at the mid-body. The anterior one is associated with the mouth opening. Blood flukes usually lack suckers. Larvae are typically encapsulated in subepidermal tissue in gills, skin or fins. Most flukes are hermaphrodites, having both male and female sex organs. Trematodes have complex life cycles including two, but usually more, hosts. Most commonly flukes use molluscs, especially snails, as their first intermediate host, but other


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invertebrates such as oligochaetes may also be involved. Most fish trematodes are between 2 and 20 mm in length. Identification is based on several structures such as the shape and position of suckers, external appendages, and the shape and size of sex organs and eggs. Symptoms/Pathology Adult trematodes mainly occur in the digestive tract and are generally not very harmful to the host. The exception is blood flukes. Serious disease and mass mortality due to blood flukes of the genus Paradeontacylix has been observed in the 0+ age group in farmed species of amberjack (Seriola spp.) (Ogawa & Egusa, 1986; Crespo et al., 1992). A related species, Aporocotyle simplex, is commonly found in the vascular system of several pleuronectid flatfish in Europe. The occurrence of large numbers of encapsulated trematode larvae, such as Cryptocotyle lingua, in the skin of farmed fish (Figs. 3.16 and 3.17, see colour plate section) may reduce fish growth, while the associated host response, including melano-macrophages which result in black spots, may reduce the market value of the fish. Prophylaxis/Treatment For prophylaxis, farms could be moved to locations with fewer first intermediate hosts in the environment. Treatment against adult flukes in the digestive tract is not usually carried out. Effective treatment against blood flukes or larvae is difficult. 3.4.2.5 Nematodes Diagnosis/Identification Nematodes are endoparasites found in any host organ. They are thread-like and tapered at both ends, and covered by a rigid cuticle. Parasitic nematodes have complex life cycles involving several hosts. Fish can act as final or intermediate hosts. The first intermediate host is usually an invertebrate, primarily a crustacean. Most adult nematodes live in the fish intestine, while larval stages are mainly found in the flesh and viscera. Identification is based on external structures, mouth-associated structures, and internal organs such as the digestive tract. Symptoms/Pathology Adult nematodes in the intestine are not considered to be important pathogens, but larvae in the flesh and viscera may cause disease and economic problems. To date, nematodes have rarely been found in marine fish reared in floating cages. In northern European mariculture, Hysterothylacium aduncum is occasionally found in intestine or viscera; this parasite uses fish as both a final and a second intermediate host. Recently, Hysterothylacium sp. larvae were commonly found in small halibut just after their first feeding on natural zooplankton filtered from seawater. The relatively large larvae were coiled 2–3 turns in the abdominal cavity. Very few fish had more than one larva, which could indicate that two or more specimens are lethal to small halibut.

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Prophylaxis/Treatment Prophylaxis could include measures to prevent potential intermediate hosts entering the fish tanks or cages. Chemical treatment against larval nematodes is usually very difficult. If necessary, some orally administrated anthelmintics are effective against adult nematodes in the intestine. 3.4.2.6 Acanthocephalans Diagnosis/Identification Acanthocephalans are endoparasites using fish as their final or intermediate host. Adult worms are found in the intestine, while juvenile worms occur in the viscera, especially the mesentery and the liver when the fish is used as an intermediate host. Fish acanthocephalans use crustaceans as an intermediate host. Acanthocephalans lack an alimentary canal, and food is absorbed through the outer surface. They have an invaginable proboscis with hooks used for attachment to the host intestine (Fig. 3.18, see colour plate section). The size of the adult worm varies from a few millimetres to several centimetres, but most species are about 10 mm. Identification is based on body shape, and on the hooks and spines of the proboscis, among other things. Symptoms/Pathology Acanthocephalans are generally not considered to be important fish pathogens. Fish can be heavily infected without showing signs of clinical disease, and it is not unusual to find acanthocephalans that have penetrated the intestinal wall and protrude into the abdominal cavity of healthy fish. However, such infections probably cause reduced growth. A negative correlation between the number of Echinorhynchus gadi present and energy stores in cod, Gadus morhua, has been demonstrated (Buchmann, 1986). Prophylaxis/Treatment Acanthocephalans are good colonisers, and the prevention of exposure is the most effective method of limiting infections. Orally administered drugs mixed in food pellets have been shown to be effective (Taraschewski et al., 1990). 3.4.2.7 Leeches Diagnosis/Identification Leeches are ectoparasites mainly found on the body and gills, and in the oral cavity. They are thread-like and usually have two disk-shaped suckers, one in the anterior and one in the posterior end. Many leeches are obligate parasites, but after a blood meal they may leave the host for a long period to digest the meal and to deposit cocoons containing eggs. Identification is based on many different structures such as pigmentation, spots, setaes in cephalic segments, protrusible proboscis, and a pharynx with or without jaws.


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Symptoms/Pathology Leeches alone are generally not considered to be important fish pathogens. The effects are usually localised, and restricted to attachment and/or feeding sites. However, leeches are important vectors for many blood-living (haematozoic) protists such as flagellates and apicomplexans, but also some bacteria and viruses. Johanssonia arctica is important as a vector for a variety of haematozoa in northern seas. It transmits Trypanosoma murmarensis to a number of commercially important hosts, including Atlantic cod and American plaice, Hippoglossoides platessoides, and it also transmits intra-erythrocyte parasites of the genus Haemohormidium (Burreson, 1995). Prophylaxis/Treatment To date, there are only a few reports of leeches found on fish in mariculture, and little has been written about prevention and treatment. However, several bath treatments have been used effectively against freshwater leeches, mainly Piscicola geometra, and similar treatments could possibly be effective against marine leeches. 3.4.2.8 Crustaceans Diagnosis/Identification Parasitic crustaceans are ectoparasites mainly found on the body and gills, and in the oral cavity. They have an exoskeleton, and are characteristically segmented with several appendages used for attachment to the host. Most parasitic crustaceans are similar in appearance to their free-living relatives, but some are so modified that only experts are able to identify them as crustaceans. The life cycle is mostly direct, involving only one host. It usually includes several stages, both free-living and parasitic, with a moult between each stage. Parasitic stages are anchored or can move freely on the host surface. The size of adult parasitic crustaceans ranges mostly between 1 and 30 mm. Larval stages can be less than 1 mm and difficult to observe macroscopically. Identification is mainly based on the shape and size of segments and appendages. Symptoms/Pathology Because of their direct life cycle, many parasitic crustaceans are troublesome parasites in fish mariculture. This especially refers to parasitic copepods moving freely on the host surface, such as the caligid genera Lepeoptheirus and Caligus (Figs. 3.19 and 3.20, see colour plate section), but also to many other species in different crustacean groups. When they are relatively numerous, parasitic crustaceans may cause the death of the host if untreated, but even a relatively low number of adult parasites may significantly reduce the growth of the host. Anchored copepods may be a potential problem in mariculture. Adult females of Lernaeocera branchialis, anchored to a gill arch of cod and other gadids, have experimentally caused host disease and mortality, primarily as the result of anorexia, stress and blood loss (Khan, 1988). Moreover, it has been reported that wild fish infected by L. branchialis are

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20–30% underweight (Mann, 1952). Isopods of the genus Ceratothoa have caused reduced growth and mortality in farmed sea bass and sea bream (Sarusic, 1999). Prophylaxis/Treatment Prophylactic measures could include the production of 1-year-old fish only, and allowing the site to lie fallow for a period after slaughtering. In salmonid mariculture, cleaner-fish (mostly wrasse species) have been used successfully as a prophylactic measure against L. salmonis. Several chemical compounds are effective against parasitic crustaceans. Treatments are given orally in feed or as bath treatments. New compounds have been introduced almost yearly in the last decade.

3.5 A Strategy for Microbial Control Microbial problems seem to be qualitatively different for larval and on-growing stages, and these differences are discussed below. For most species, the production of juveniles is a major bottleneck. The main symptoms are poor reproducibility in terms of survival, growth and quality, and the problem seems to be the same for temperate and warm-water species of both fish and crustaceans. The symptoms indicate a lack of control of at least one factor. Nutritional factors and egg quality may be ruled out as the principal cause, because the lack of reproducibility is also manifested in replicate tanks with full sibling groups that are given the same treatment. This does not mean that nutritional factors and egg quality are optimal (cf. Chapters 4, 5 and 7). Recent scientific data and accumulating experience in commercial hatcheries indicate that the bacteria normally selected for in hatcheries may be the principle cause of problems associated with the production of juveniles (Vadstein et al., 1993; Bergh et al., 1992; Skjermo & Vadstein, 1999; Bergh, 2000). This is most probably due to opportunistic bacteria, because the reported incidences of specific pathogens are low (Munro et al., 1995), even though some pathogens may be overlooked as they have not yet been described. Also for on-growing stages, microbial problems are occasionally very severe. However, these have different symptoms, and traditional disease outbreaks are the normal phenomenon. Whether or not opportunistic bacteria or specific pathogens cause problems will have strong implications for the choice of strategy for microbial control. This is because of the different ways of controlling the import of microbes to cultivation systems, and how the immune system of the fish can be used prophylactically. Another important difference between larval and on-growing fish is that for many species the on-growing stages takes place in open or semi-open systems, whereas larval stages are kept under controlled conditions in tanks indoors. Obviously, the openness of the system has strong implications for the possibilities for microbial control. We have previously (Vadstein et al., 1993) proposed a strategy for microbial control that consists of three different elements (Fig. 3.21). Two of the elements involve environmental factors, whereas the third considers the fish itself. Different methods have been proposed for


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Improvement of larval resistance

Figure 3.21 Outline of the three elements in the strategy to obtain microbial control in the rearing of marine fish (redrawn from Vadstein et al., 1993). © Swets & Zeitlinger. Used with permission.

Non-selective reduction of bacteria

Selective enhancement of bacteria

the different elements of the strategy (cf. below). Before presenting the strategy in detail (Section 3.5.2.), together with examples of the use of the various methods (Sections 3.6. and 3.7), we present some important general considerations. However, the first line of defence with animal diseases should always be legislative control, thus ensuring that pathogens are not introduced into new areas and environments. It must be emphasised that many outbreaks of disease are the unfortunate result of such introductions. Among the most well-known examples is the introduction of furunculosis to Norway by the import of smolts infected with Aeromonas salmonicida subsp. salmonicida from Scotland, leading to an epizootic event with large economic consequences. Most probably, the disease was originally imported to Europe from North America. Another example is the probable introduction to European crayfish of the so-called crayfish plague. The causative agent is a fungus which is not pathogenic to American crayfish, but is lethal to European crayfish. Hygiene routines are often underestimated as a method for avoiding microbial problems. There are numerous examples of aquaculture facilities that have significantly improved their stability of production by simply implementing good hygiene practice. The advantage is that this does not require R&D or large investments. Moreover, the implementation of good hygiene routines will increase the awareness of microbe–fish interactions, and may improve the state of awareness regarding an emerging microbial problem. General hygiene rules are presented in Table 3.1. There is no further discussion about either legislative control or hygiene routines in relation to animal diseases in this chapter.

3.5.1 General Considerations As mentioned above, whether or not opportunistic bacteria or specific pathogens cause microbial problems, and also the degree of openness of the culture system, have strong implications for the choice of strategy for microbial control. Generally, microbes can be transferred to, or interact with, fish in a number of ways, which are illustrated for larvae in Fig. 3.22. These sources can be divided into two main categories, external and internal, which strongly affect each other. To what extent external sources influence the microbial conditions in the rearing environment depends on the openness of the system. For a cage system, the

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Table 3.1

General hygienic rules for aquaculture facilities.

General hygiene and disinfection The facility should be well organised with good cleaning routines and fixed storage places for all equipment Hygienic zones should be established in an intelligent manner. Transport of people and equipment should be avoided All equipment should be washed, disinfected and dried after use. Disinfection cannot compensate for normal cleaning Infection hygiene Establish the possible sources of pathogens (biological materials, water, equipment, feed, personnel, visitors, including dogs, birds, etc.) Establish where pathogens could proliferate inside the facility Evaluate and establish possible counter-measures Establish and implement regular control routines. This will promote early detection Establish routines for documentation of hygienic practice Minimise stress and handling

External Internal live feed

water

microalgae

Figure 3.22 Important bacterial sources interacting with mucosal surfaces of larval fish. Modified from Salvesen (1999).

external factors are important because of the open contact with the sea, and the large input of food which has its own microflora. For indoors, first-feeding tank systems, microbes from the live feed cultures are the main external input. Traditionally, there has been considerable attention to microbes in the intake water, where water treatment to reduce the density of bacteria is a more or less standard procedure. On the other hand, control measures directed towards microbes associated with the live feed have mostly been neglected. This is in spite of the fact that detrimental effects due to bacteria associated with the live feed are well known (Benavente & Gatesoupe, 1988). There are also strong interactions among the internal sources. Whereas the outer surface of the fish is colonised by bacteria in the water, the intestine is generally affected by bacteria entering the intestine by active uptake from the water, and by bacteria associated with the feed. The number of bacteria associated with dry feed pellets is fairly low, but the number may exceed 1010 cells per gram live feed (Skjermo & Vadstein, 1993). Live feed represents the heaviest bacterial load to the larvae, except for the first days of the larval stage when active uptake directly from the water is significant (Reitan et al., 1998). For on-growing


53

stages, the density of bacteria in the feed will determine what is the most significant source. The processing and storage methods may cause considerable variation in bacterial content, and soft feed is likely to be more vulnerable to bacterial growth than dry pellets. Even though the main sources of microbes may be identified, the various internal sources in a rearing environment will still interact with each other (see Fig. 3.22). In particular, defecation processes may be a significant source of bacteria in the water. This is from fish, and also from live feed in the case of larval rearing. It is also well established that the bacterial flora of live feed changes after their transfer to the tanks. This includes both a reduction in numbers and a change in the composition of the bacterial flora (Øie et al., 1994; Olsen et al., 2000). The direct input of bacteria into a system is not the only manner in which the density of bacteria in a rearing system is affected. The direct or indirect input of organic matter is also decisive, since it serves as a substrate for bacterial growth. Bacterial growth in first-feeding tanks may be fairly high, with production in the range of 0.5–2 divisions per day. The above discussion has mainly considered quantitative aspects, but the species of microbes that are present and dominant are more important than the actual number. However, numbers and composition cannot be discussed as separate issues, as the total bacterial community (numbers) is the sum of the individual populations (composition). It is obvious that the composition of the externally introduced microflora will influence the composition of the microflora of the rearing system. This influence will be stronger the more open the rearing system is, and open systems therefore have less possibility of effective management. Generally, the microbial management of open systems is connected to all aspects related to localisation, and to disease management strategies for early detection. Early detection may prevent a problem reaching epizootic proportions, and the initiation of countermeasures at an early stage is of vital importance. In more closed rearing systems, the impact of external sources of bacteria is still significant, but they are more manageable because water sources and live feed can be treated before they enter the fish tanks. In more closed rearing systems, the internal sources are increasingly important. The internal sources may affect the bacterial composition in both a positive and a negative way. The presence of haemolytic bacteria in larvae may seed the water with these bacteria (Skjermo & Vadstein, 1999), which affect the water flora negatively. On the other hand, the ingestion of algae by live feed may change their flora to a more diverse one, which may have a positive impact on larval viability in the next stage (Olsen et al., 2000). To understand such interactions may be decisive in the design of a microbial management strategy. Our comprehension of interactions between bacteria at the species level and fish is inadequate, except perhaps for a few fish pathogens. However, this research field is developing rapidly, and several reviews have recently been published (Ringø & Birkbeck, 1999; Gatesoupe, 1999; Hansen & Olafsen, 1999). Because of our limited knowledge of normal flora and normal fish–microbe interactions, it may be adequate to classify bacteria based on a general ecological scheme. A differentiation between opportunistic and non-opportunistic species may be appropriate, as may also be the division between parasitic and mutualistic species (cf. Fig. 3.1). The former division has been applied to aquaculture with some success (cf. Section 3.6.3), and is theoretically formalised as the r/K-concept (cf. Andrews & Harris,

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Table 3.2 Some key characteristics of organisms with r- and K-strategies, and pioneer and mature (climax) communities. Characteristics

Maximum growth rate Biomass at carrying capacity Effect of enrichment Competitive ability at a low supply of substrate per individual Mortality Affinity to substrate

r-strategist

K-strategist

High Unstable Rapid growth

Low Stable Slow growth

Poor Density-dependent, often catastrophic Low

Good Density-independent High

Characteristics

Biological control Stability to perturbations Diversity (species, biochemical) Niche width Specialisation

Pioneer community

Mature community

Low Poor Low Wide Low

High Good High (?) Narrow High

1986). The r/K-concept does not, of course, entail an either/or. r and K represents the two extremes in a continuum: r-selection occurs in an uncrowded environment with a large supply of substrate per capita (Table 3.2), and r-strategists are considered to be opportunistic species; K-selection, on the other hand, occurs in crowded environments with a low supply of nutrients per cell. Several characteristic properties of r- and K-selected species are given in Table 3.2, and these properties can be used both as a selective force and for diagnostic purposes, e.g. percentage opportunistic species (Salvesen & Vadstein, 2000). Pathogens are often characterised as r-strategists (Andrews, 1984), but the group probably contains a large number of non-pathogenic, opportunistic species that may cause some of the problems experienced in the rearing of marine larvae. At the community level, r- and K-selected species dominate in pioneer (developmental) and mature (climax) communities, respectively (Odum, 1971). These two types of community also have distinctly different characteristics (Table 3.2). Pioneer communities will generally be systems with low stability against perturbations and with low biological control. On the other hand, matured water inhabited by K-strategists will be stable systems, with high biological control and high resistance to perturbations below a specific level. The current intensive production methods used for marine fish tend to increase the carrying capacity of the system and to select for opportunistic microbes. This tendency is stronger the more intensive and closed the system is. The reasons for such an unfavourable development are a high load of organic matter, large oscillations in this load, and direct perturbations of the bacterial community. Critical factors that sustain such a development are decimation of bacteria in in-flowing water without controlled recolonisation of the microbial


55

community, high loads of bacteria and organic matter together with feed, and an internal load of organic matter as faeces from live feed and larvae, or as dead larvae. Intensive larval rearing systems may serve as an example of a system with major negative perturbations, whereas open cages with good water circulation are an example of the opposite.

3.5.2 A Strategy for Microbial Control and Important Elements in such a Strategy The multiple aspects discussed above, which need to be considered when developing a strategy to obtain microbial control in the cultivation of marine fish, emphasise the necessity of not focusing on one counter-action only. Solving a multidimensional problem requires a multidimensional strategy. In the case of microbial control, this is even more important because control cannot be regarded as absolute, but is a matter of probability. Thus, by increasing the number of counter-actions, an increased probability of obtaining and maintaining microbial control is achieved. The proposed strategy for microbial control consists of three different elements (see Fig. 3.21). Two of the elements involve environmental factors, whereas the third relates to the fish itself. Both quantitative and qualitative aspects of the bacterial flora are included. These two aspects are strongly dependent on each other, and it is not obvious which of these affect the fish larvae. Several different methods have been proposed for different elements of the strategy (Table 3.3), and there are examples in the literature on the use of some of them (Sections 3.6 and 3.7). Non-Selective Reduction of Bacteria The methods used for non-selective control can be put into two groups. Some methods are aimed at a non-permanent or non-stable reduction (disinfection and grazer control), whereas

Table 3.3 Examples of methods that can be used for the different elements in the microbial management strategy suggested. Modified from Vadstein et al. (1993). © Swets & Zeitlinger. Used with permission. Non-selective reduction of microbes Surface disinfection of eggs Reduction in input of organic matter Removal of organic matter Grazer control of bacterial biomass Selective enhancement of microbes Selection for desirable bacteria Addition of selected bacteria to tanks Incorporation of selected bacteria in feed Improvement of resistance against microbes Stimulation of general immune system Stimulation of specific immune system (vaccination) Modulation of general and specific maternal immunity Nutritional supplements to improve susceptibility to microbes and wound healing

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others are aimed at a reduction in the carrying capacity of the system (reduction in input and removal of organic matter). Selective Enhancement of Bacteria The application of probiotic bacteria in livestock production and for humans has aroused considerable interest (Vanbelle et al., 1990), and to some extent this is also true in aquaculture (Gatesoupe, 1999). Probiotics can be defined as micro-organisms that are able to colonise the digestive tract, to maintain or increase the natural gut flora to prevent colonisation by pathogenic organisms, and to secure optimal utility of the feed (Vanbelle et al., 1990). Their use can improve the health and productivity of farmed animals. Probiotics can be administrated both by direct addition of the selected organisms to the tanks, or by incorporation of the organisms into live or formulated feed. Regulation of the composition of the bacteria by selective measures differs from the probiotica concept by the fact that no organisms are added to the system. Instead, a physical, chemical or biological factor is used as a selective force. Thus, the selection is based on the statement of the pioneering microbiologist Martinus W. Beijerinck that ‘everything is everywhere, the environment selects’. It must be emphasised that this statement should not be interpreted in such a way that attempts to hinder the spread of pathogenic agents are neglected. Biogeographical differences with respect to the occurrence of pathogens are well documented. Improvement of Resistance Against Bacteria The natural ability of animals to resist potentially harmful bacteria, and the possibility of stimulating this ability, are used extensively in both human medicine and animal husbandry. Generally, such approaches are called immunotherapy, and include methods that utilise immunological principles to prevent or treat diseases. The best-known method is vaccination, which is the stimulation of the specific immune system, and has also been applied in aquaculture with some success (Ellis, 1988b; Anderson, 1992). In addition to specific stimulation targeted at a specific pathogen (i.e. vaccination), it is also possible to stimulate the non-specific immune system (Vadstein, 1997). An example of non-specific immunostimulation is macrophage activation. Fish larvae do not have the ability to develop specific immunity, as their immune system is not mature. For specific immunity they rely on immunoglobulins from the mother (maternal immunity). Therefore, one possibility is to manipulate specific antibody composition and levels in eggs and larvae through immunisation of the mother. The nutrition of fish has an indirect impact on their resistance against pathogens, as several nutritional factors influence the immune system. These include fatty acids, minerals and vitamins (Landolt, 1989; Blazer, 1992). Resistance to pathogens also has a genetic component, with significant differences between families and strong heredity of such differences (Gjedrem et al., 1991; Fjalestad et al., 1995). Examples of the use of various methods included in Table 3.3 are presented in Sections 3.6 and 3.7.


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3.6 Improving Environmental Conditions The physical, chemical and biological environments significantly affect the health status, and hence the viability, of reared fish. We treat only biological environmental conditions in this chapter, and give examples of methods used for microbial management (see Chapter 2).

3.6.1 Non-Selective Reduction of Microbes Bacteria colonise the surface of fish eggs, and these microbes may serve as a reservoir for the horizontal transfer of pathogens from brood stock facilities to hatcheries. A safe and effective method to disinfect the surface of fish eggs is therefore a prerequisite for the establishment of a hygienic barrier between brood stock and first feeding. Salvesen & Vadstein (1995) tested the potential of four different chemicals for use as surface disinfectants of marine fish eggs. These were two chlorine-releasing compounds (sodium hypochlorite and chloramine-T), an iodophore (buffodine) and an aldehyde (glutaraldehyde). The experiments included eggs of place, cod and Atlantic halibut, and revealed that glutaraldehyde was the most promising of the four chemicals. When eggs were treated for 5–10 min at a concentration of 400–800 mg l-1, a high bactericidal effect was observed without adverse effects to eggs or yolk-sac larvae. Survival at the egg stage and the viability of yolk-sac larvae was improved. The other three chemicals caused adverse effects on eggs and larvae at the concentrations where a satisfactory bactericidal effect was obtained. A further evaluation of glutaraldehyde (Harboe et al., 1994; Salvesen et al., 1997) confirmed these results and revealed several positive effects in addition to providing a hygienic barrier. These positive effects included increased hatchability (Fig. 3.23), more synchronised hatching, increased stress-tolerance and survival of larvae, and improved growth during first feeding. Surface disinfection of eggs with glutaraldehyde has been implemented in many marine fish facilities. It should be noted that the activity of glutaraldehyde is very temperaturesensitive (see Salvesen & Vadstein, 1995), and the procedures applied for cold-water fish cannot be used directly for other temperature regimes. The mechanisms of the differences

100

Halibut Turbot Cod Plaice

% hatch

80

Figure 3.23 Hatchability after disinfection with glutaraldehyde in seven egg batches of different marine fish species (redrawn from Salvesen, 1999).

60 40 20 0

Not disinfected

Disinfected

58


observed between disinfected and non-disinfected eggs are not clear. Both the easier exchange of gases and matter between the egg and the environment, and the reduced probabilities of over-colonisation by microbes or unfavourable colonisation of the larvae are possible explanations. Recent investigations concluded that aldehydes have little effect against fish pathogenic viruses (Arimoto et al., 1996). Therefore, Grotmol & Totland (2000) studied the effects of ozonation of halibut eggs, and concluded that this procedure efficiently hindered the transfer of nodavirus to the larvae both in cases of naturally infected broodstock and in experimentally infected eggs. Because nodaviruses are the main disease problem in Norwegian halibut aquaculture, ozonation has replaced glutaraldehyde disinfection in some facilities. There are not many examples of other non-selective reduction methods in the literature. Maeda & Nogami (1989) demonstrated the significant role of grazers in controlling the density of bacteria during cultivation of crab larvae (Portunus tridentatus). No other studies treating this subject are known. Both Brachionus plicatilis (Rotifera) and Artemia franciscana (Branchiopoda) are able to graze bacteria to some extent (Vadstein et al., 1993; Makridis & Vadstein, 1999), and could therefore affect the biomass of bacteria in rearing tanks. With densities of five and three individuals per millilitre of B. plicatilis and A. franciscana, respectively, these organisms may clear between 5% and 140% of the tank volume of bacteria per day. Thus, we may conclude that whereas B. plicatilis does not seem to have a great impact on bacterial density, A. franciscana may do so.

3.6.2 The Use of Probiotics Gatesoupe (1999) has recently reviewed the use of probiotics in aquaculture. To date limited information exists on this topic, but existing data and experience are promising, particularly when one considers that the first work was done only 15 years ago (Kozasa, 1986). Knowledge of terrestrial organisms indicates that the application of probiotics has to follow some basic principles (Fuller, 1989). These include the facts that the probiotic is part of the autochthonous flora, has the ability to establish itself and proliferate in the intestine, and is able to resist the environmental conditions in the intestinal tract (e.g. lytic enzymes, low pH and bile salts). Frequently, this knowledge has not been fully implemented in the limited number of attempts to apply the probiotica concept in aquaculture. Generally, probiotic bacteria may be supplied either by direct addition to the water or encapsulated in feed. Encapsulation is possible in both formulated feed and live feed. Whereas direct addition to the water is possible for larval stages or other situations with tank rearing at low flow-through rates (Ringø et al., 1996; Ringø & Vadstein, 1998; Makridis et al., 2000b), encapsulation in feed is the only possible method of administration in open or high-flow-through systems. It has been reported that it is possible to incorporate selected bacteria in both B. plicatilis and A. franciscana, and that added bacteria persist for some time in association with the live feed after transfer to tank conditions (Makridis et al., 2000a). However, there are differences between various strains of bacteria, and therefore each different case requires validation.


59

It has been shown that administered bacteria colonise the intestine for a significant period of time (Strøm & Ringø, 1993; Olsson, 1995; Munro et al., 1995; Andlid et al., 1995; Austin et al., 1995; Ringø et al., 1996; Ringø & Vadstein, 1998; Makridis et al., 2000b). These studies include several hosts (fish and shellfish) and several probiotic bacteria candidates. Moreover, these studies were conducted at both larval and juvenile stages, and by administration via water and encapsulated in feed. However, still there is limited knowledge on the stability and persistence of such manipulations of the mucosal flora. A few studies have investigated the effect of probiotic bacteria in challenge tests with a pathogen. Some of these studies showed increased survival, but the effects are not always reproducible (cf. Gatesoupe, 1999). In some studies the mortality rate was not truly reduced as only a delay in mortality was observed. Also, there are few reports to date of an improvement in survival (Ringø & Vadstein, 1998) or growth (Byun et al., 1997; Gatesoupe, 1997). However, the application of probiotic bacteria in aquaculture seems promising, but considerable research is still needed for a full evaluation of the possibilities and constraints. The main obstacles are limited knowledge about their function and normal microbial interactions in the intestinal tract of marine fish, and a good strategy for the selection of candidate bacteria.

3.6.3 Selection for Desirable Bacteria The application of a rearing technology where a selective force is used to promote beneficial bacteria has been reported in the literature (Vadstein et al., 1993; Skjermo et al., 1997; Salvesen et al., 1999). Such selection in relation to, for example, the r/K-concept, is possible by applying a selective force such as a low supply rate of organic matter per bacteria, and produces a community with slowly growing bacteria and a strong ability to resist high pulses of organic matter (Salvesen et al., 1999; Salvesen & Vadstein, 2000; O. Vadstein, unpublished results, 2002). These authors have termed this concept for microbial maturation of water. In an experiment with yolk-sac larvae of Atlantic halibut, the effect of applying microbially matured water or filtered seawater for maintaining yolk-sac larvae at high densities was compared (20–30 larvae per litre; Vadstein et al., 1993). Both types of water were applied with or without the addition of an antibiotic (25 p.p.m. final concentration of oxytetracycline). Whereas similar average survival rates were observed for the two treatments with matured water and the treatment with filtered water with added antibiotic (range 87–90%), the average survival rate was only 45% for the treatment with filtered water without antibiotic. The addition of antibiotics to filtered water considerably reduced the variability between replicates, with a coefficient of variation of 15 and 75% for the treatments with and without oxytetracycline, respectively. Much lower variability was observed for the two treatments with microbially matured water (CV 3 and 8%, respectively). In first feeding experiments with turbot (Skjermo et al., 1997; Salvesen et al., 1999), microbially matured water produced a significantly bigger larval size just 5 days after hatching (Fig. 3.24). At this stage the larvae had been eating for 2–3 days, and on average larvae in matured water were approximately 20% larger in size. The differences in size were main-

60


µg C larva-1

35 Exp.I Exp.II Exp.III

30 25 20 15 10 F

F+A

M

M+A

Figure 3.24 Effect of microbially matured water on the size of turbot larvae on day 5 after hatching in three first-feeding experiments. Larvae were reared in filtered water (F), filtered water with added microalgae (F + A), matured water (M) and matured water with added microalgae (MA). Data from Skjermo et al. (1997) and Salvesen et al. (1999).

tained until metamorphosis, and clearly demonstrate the effect of water quality on the viability of larvae during first feeding. Improved survival in first-feeding experiments was also observed for larvae reared in matured water, but the differences were not statistically significant because of low statistical power. The mechanism for these effects is not understood, but several explanations are possible. The bacteria in the matured water may be beneficial from a nutritional perspective by providing nutrients or digestive enzymes (Ringø & Birkbeck, 1999), or they may support the establishment of a beneficial primary intestinal flora that efficiently prevents colonisation by detrimental bacteria through competition (Tannock, 1984; Dopazo et al., 1988; Westerdahl et al., 1991; Olsson et al., 1992). Based on the theoretical background of the approach, the second hypothesis is the most likely. A large number of studies have concluded that the use of algae in larval rearing improves growth and survival, and several hypotheses have been proposed as the mechanism for these observations (cf. Reitan et al., 1997). One of the hypotheses is the selective effect that algae have on the bacterial community (Skjermo & Vadstein, 1993). It is well known from studies with algal cultures that each alga has its specific bacterial flora (e.g. Bell, 1983; Salvesen et al., 2000). Moreover, fish larvae reared in tanks with algae present have a bacterial colonisation pattern that is different from the one that exists without the addition of algae (Skjermo & Vadstein, 1993; Skjermo et al., 1997; Salvesen et al., 1999). Although some of the positive effects due to the addition of algae are caused by other factors, the bacterial hypothesis is most likely a part of the ‘green water’ complex, and represents one way of selecting for beneficial bacteria. The positive effect of algae on the magnitude and composition of the bacterial flora has also been demonstrated by the ‘treatment’ of live feed in algal suspensions (Olsen et al., 2000). By the short-term incubation of Artemia franciscana in water to which the green algae Tetraselmis sp. had been added, bacterial numbers associated with A. franciscana were dramatically reduced and the composition of the bacterial community changed in a positive way. The change in composition was from a low-diversity community dominated by one Vibrio species and haemolytic bacteria to a higher diversity and a reduced dominance of haemolytic


61

strains. These differences were also manifested in Atlantic halibut larvae during first feeding by feeding the larvae with untreated or algal-treated A. franciscana.

3.7 Improving the Resistance of the Fish Imperfect rearing conditions easily induce stress (cf. Angelidis et al., 1987), stress influences the immune system of the animals, and stressed animals are more susceptible to intestinal infections than healthy animals (Tannock, 1984). This clearly emphasises the relevance of including resistance to infections in a microbial management strategy. This can be done by stimulation of the immune system, either directly by the use of immunostimulants, or indirectly by nutritional factors. Both specific and non-specific immunostimulation involves the use of immunostimulants. An immunostimulant may be defined as an agent that stimulates the non-specific (innate) immune mechanisms when given alone, or the specific immune system when given together with an antigen (vaccination). Substances used as immunostimulants include bacteria and bacterial products, muramyl dipeptides, polysaccharides and synthetic chemicals (Vadstein, 1997).

3.7.1 Modulation of Specific Immunity—Vaccination Scientifically based vaccines have had tremendous significance for humans since Louis Pasteur and Charles Chamberland developed the first vaccine in 1884: an anthrax vaccine. In aquaculture, effective vaccines have been developed during the last few decades (Gudding et al., 1999), and vaccination is one of the most important prophylactic measures against disease. The salmon industry in Norway is a good example of the great impact vaccination may have. In 1987, almost 50 000 kg of antibiotics was used to produce 50 000 tons of salmon. In the same year, a vaccine against cold-water vibriosis was introduced, and later on programmes for vaccination against yersiniosis and furunculosis were implemented. The impact of these vaccination programmes was tremendous, and in 1997 only 746 kg of antibiotics was used to produce 316 000 tons of fish. Thus, whereas 1000 g antibiotics was used per ton produced in 1987, this was reduced to 2 g per ton produced in 1997. To date, relatively few pathogens that cause disease in marine fish have been discovered (cf. Section 3.5). However, this is most likely due to the fact that aquaculture of cold-water fish is still in its infancy. As the industry develops, new pathogens will be discovered. To date, it seems that the knowledge developed for salmonids may also be used to some extent for cold-water fish. However, there are clear indications that vaccines should be designed according to species, because different pathogens affect different species. The obvious positive effect of vaccination is reduced mortality, but for sustainable biological production the reduced need for medication is also significant. One side-effect of vaccination by injection is local reactions in the peritoneal cavity (Midtlyng et al., 1996). The magnitude of such side-effects is dependent on the formulation of the vaccine. Studies of the vaccination of marine species are scarce, and little has been published. Ingilæ et al. (2000) demonstrated protection in challenge experiments with halibut and

62


spotted wolf-fish vaccinated intraperitoneally with oil-emulsified vaccines against atypical Aeromonas salmonicida. Vaccination of cod against Vibrio anguillarum has been shown to increase survival in challenge experiments as well as in field trials (B. Hjeltnes, National Veterinary Institute, personal communication, 2002), and the same has been shown for halibut (Bergh et al., 2001). Several vaccines designed specifically for marine fish species are now available. Vaccines against viral diseases are presently at the experimental stage. Húsgarð et al. (2001) showed protection in turbot in challenge experiments with nodavirus following vaccination with a recombinant vaccine. For salmonids the change from a freshwater stage to a seawater stage creates some benefits for disease control. For marine fish that stay in seawater during their whole life cycle, it is therefore desirable to vaccinate at an early stage. However, the stress induced by vaccination at a young age may entail immunosuppression and increased susceptibility to pathogens, and it may also reduce performance in other areas such as growth (Lillehaug et al., 1999). The problems connected with vaccination at early stages, and the fact that the specific immune system is not fully developed at this stage, may be counteracted by the fact that egg/larvae ‘inherit’ specific immunity from the mother: maternal immunity. It has been demonstrated that it is possible to manipulate specific antibody composition and levels in eggs and larvae through immunisation of the mother. Immunisation of tilapia (Oreochromis aureus) broodstock with different proteins resulted in a considerable increase in antibody activity (Mor & Avtalion, 1990). The maximum increase was 10–13 log2 units in embryos that hatched 15–35 days after immunisation. In a second study with the same species, vaccination of broodstock with live tomites of a ciliated protozoa 1 month before hatching resulted in protection in challenge experiments of >75% (Sin et al., 1994). Atlantic salmon broodstock vaccinated against yersiniosis showed maternal transfer of specific antibodies to eggs and yolk-sac lavae, but at low levels which were insufficient to protect the offspring against yersiniosis (Lillehaug et al., 1996). Too little is known to evaluate the full potential of the stimulation of maternal immunity as a method in microbial management. However, it is reasonable to believe that at least for some diseases, vaccination or the secondary stimulation of mothers with appropriate vaccines before the spawning season could protect the larvae against disease in the first period after hatching.

3.7.2 Modulation of Non-Specific Immunity There are two situations where stimulation of the non-specific immune system is appropriate. First, for larval stages which have not developed specific immunity, but which have a functional general immune system. Second, in a situation with a general microbial problem (no specific pathogens identified) or with a high stress level. In fact, both these criteria seem to be fulfilled during first feeding. A large number of substances are known to act as immunostimulants, and with variable specificity and activation mechanisms (Vadstein, 1997). Since the first publication in 1985 (Oliver et al., 1985), considerable data have been accumulating on stimulation of the non-specific immune system of fish (Anderson 1992; Secombes, 1994; Vadstein, 1997). Moreover, non-specific stimulation has been detected over a wide range of complexity levels, including humoral, cellular and organism parameters


63

Table 3.4 Type of effects reported after the stimulation of the nonspecific immune system of fish. Summarised from Vadstein (1997). Humoral effects Increased levels of lytic enzymes Increased levels of complement Production of interleukine-like molecules Cellular effects Increased phagocytosis and killing of bacteria Increased production of oxidative compounds Organism-level effects Increased survival in challenge test with bacteria Increased growth rates Counteraction of immune suppression

(Table 3.4). It is interesting to note that at the organism level, increased resistance to pathogens, improved growth and a reversal of immune suppression have been documented. Positive effects (Table 3.4) have been obtained by baths, injections and oral administration. In these studies, a large number of fish species have been investigated. The fact that nonspecific immune stimulation has been demonstrated with a variety of stimulants, administration methods, response parameters and species strongly demonstrates the robustness of the method. However, to date, most studies have not provided sufficient information for industrial application. This includes most aspects of the administration regime, including concentrations needed, administration pathway, time and frequency of administration, and what type of conditions that are appropriate for treatment. However, one may conclude that stimulation of the non-specific immune system will be part of the microbial management regime aimed at reducing mortality in cold-water aquaculture.

3.7.3 The Effect of Nutrition and Genetics on Resistance Against Microbes As mentioned above, both nutritional and genetic factors have indirect influences on the immune system, wound healing and resistance to infections. In addition, some components in commercial diets have been shown to reduce the resistance to infections (Tacon, 1985). For more details on nutrition, see Chapters 7 and 9.

3.8 Closing Remarks This chapter has tried to emphasise both beneficial/neutral and detrimental interactions between fish and microbes/infectious agents, and that the prevention of problems through good routines and appropriate and sustainable countermeasures is the best strategy for building an industry. Man has a tendency to search for ‘the solution’, but with such a complex and diverse problem, no ‘magic solution’ exists. The problem organisms (cf. Sections 3.2–3.4) have a high diversity of infection strategies, the vulnerability of fish species and

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developmental stage varies, and open and closed cultivation systems need different control strategies. In fact, this complex problem requires a diversity of countermeasures. Therefore, we believe that to minimise microbial problems, a general strategy for microbial management is required (see Section 3.5). Such a strategy should involve two steps. First, to analyse the expected or realised problem. Second, to decide on at least two or three countermeasures that could effectively reduce the probability of the problem developing, or prevent the expansion of the problem (see Table 3.3). Once again, the focus should always be on preventive efforts. Aquaculture of cold-water marine species is both a fairly new scientific discipline and an industry in its infancy. As a result, there are large gaps in the knowledge required for establishing an economically sound and sustainable industry. Disease problems have been identified as a major bottleneck, in particular in the production of juveniles. However, the various microbial problems of different species and developmental stages have not yet been fully identified, and only a limited number of organisms causing disease in cold-water species have been described. As a consequence, the topics dealt with in this chapter are part of an area that is developing very quickly, and which requires considerable future research efforts.

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Romalde, J.L. (1999) Genetic analysis of turbot pathogenic Streptococcus paruberis strains by ribotyping and random amplified polymorphic DNA. FEBS Microbiol. Lett., 179, 297–304. Salvesen, I. (1999) Microbial ecology in early life stages of marine fish: Development and evaluation of methods for microbial management in intensive larviculture. Dr. Sci. Dissertation, Norwegian University of Science and Technology. Salvesen, I. & Vadstein, O. (1995) Surface disinfection of eggs from marine fish. Evaluation of four chemicals. Aquacult. Int., 3, 155–71. Salvesen, I. & Vadstein, O. (2000) Evaluation of plate count methods for determination of maximum specific growth rate in mixed microbial communities, and its possible application for diversity assessment. J. Appl. Bacteriol., 88, 442–8. Salvesen, I., Øie, G. & Vadstein, O. (1997) Surface disinfection of Atlantic halibut (Hippoglossus hippoglossus L.) and turbot (Scophthalmus maximus L.) eggs with glutaraldehyde: evaluation of concentrations and contact times. Aquacult. Int., 5, 249–58. Salvesen, I., Skjermo, J. & Vadstein, O. (1999) Growth of turbot (Scophthalmus maximus L.) during first feeding in relation to the proportion of r/K-strategists in the bacterial community of the rearing water. Aquaculture, 175, 337–50. Salvesen, I., Reitan, K.I., Skjermo, J. & Øie, G. (2000) Microbial environments in marine larviculture: impacts of algal growth rates on the bacterial load in six microalgae. Aquacult. Int., 8, 275– 87. Sanmartin Durán, M.L., Fernandez Casal, J., Tojo, J.L., Santamarina, M.T., Estevez, J. & Ubeira, F. (1991) Trichodina sp.: effect on the growth of farmed turbot (Scophthalmus maximus). Bull. Eur. Assoc. Fish Pathol., 11, 89–91. Santamarina, M.T., Tojo, J., Ubeira, F.M., Quinteiro, P. & Sanmartin, M.L. (1991) Anthelmintic treatment against Gyrodactylus sp. infecting rainbow trout, Oncorhynchus mykiss. Dis. Aquat. Org., 10, 39–43. Sanz, F. (1992) Mortality of cultured seabream (Sparus aurata) caused by an infection with a trematode of the genus Microcotyle. Bull. Eur. Assoc. Fish Pathol., 12, 186–8. Sarusic, G. (1999) Preliminary report of infestation by isopod Ceratothoa oestroides (Risso, 1826), in marine cultured fish. Bull. Eur. Assoc. Fish Pathol., 19, 110–12. Schmahl, G. (1993) Up-to-date chemotherapy against Monogenea: a review. Bull. Fr. Peche Piscic., 328, 74–81. Schmahl, G. & Mehlhorn, H. (1989) Treatment of fish parasites. 6. Effects of Sym. Triazinone (Toltrazuril) on developmental stages of Glugea anomala Moniez, 1887 (Microsporidia): a light and electron microscopic study. Eur. J. Protistol., 24, 252–9. Secombes, C.J. (1994) Enhancement of fish phagocyte activity. Fish Shellfish Immunol., 4, 421– 36. Siddall, M.E., Martin, D.S., Bridge, D., Cone, D.M. & Desser, S.S. (1995) The demise of a phylum of protists: Myxozoa and other parasitic Cnidaria. J. Parasitol., 81, 961–7. Sin, Y.M., Ling, K.H. & Lam, T.J. (1994) Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus. Aquaculture, 120, 229– 37. Skiftesvik, A.B. & Bergh, Ø. (1993) Changes in behaviour of halibut (Hippoglossus hippoglossus) and turbot (Scophthalmus maximus) yolk-sac larvae induced by bacterial infections. Can. J. Fish. Aquat. Sci., 50, 2552–7. Skjermo, J. & Vadstein, O. (1993) The effect of microalgae on skin and gut bacterial flora of halibut larvae. In: Fish Farming Technology (ed H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 61–7. A.A. Balkema, Rotterdam.


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Skjermo, J. & Vadstein, O. (1999) Techniques for microbial control in the intensive rearing of marine larvae. Aquaculture, 177, 333–43. Skjermo, J., Salvesen, I., Øie, G., Olsen, Y. & Vadstein, O. (1997) Microbially maturated water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae. Aquacult. Int., 5, 13–28. Smothers, J.F., von Dolen, C.D., Smith, L.H. Jr. & Spall, R.D. (1994) Molecular evidence that the Myxozoan protists are metazoans. Science, 265, 1719–721. Snow, M. & Smail, D.A. (1999) Experimental susceptibility of turbot, Scophthalmus maximus, to viral haemorrhagic septicaemia virus isolated from cultivated turbot. Dis. Aquat. Org., 38, 163–8. Sterud, E., Hansen, M.K. & Mo, T.A. (2000) Systemic infection with Uronema-like ciliates in farmed turbot, Scophthalmus maximus (L.). J. Fish Dis., 23, 33–7. Stone, D.M., Way, K. & Dixon, P.F. (1997) Nucleotide sequence of the glycoprotein gene of viral hemorrhagic septicaemia (VHS) viruses from different geographical areas. A link between VHS in farmed fish species and viruses isolated from North Sea cod (Gadus morhua L.). J. Gen. Virol., 78, 1319–26. Strøm, E. & Ringø, E. (1993) Changes in bacterial flora of cod, Gadus morhua (L.), larvae after inoculation of Lactobacillus plantarum in the water. In: Physiological and Biochemical Aspects of Fish Larval Development (eds B. Walther & H.J. Fyhn), pp. 226–8. University of Bergen, Bergen. Tacon, A.G.F. (1985) Nutritional Fish Pathology. Morphological Signs of Nutrient Deficiency and Toxicity in Farmed Fish. ADCP/REP/85/22 FAO/UNDP, Rome. Tannock, G.W. (1984) Control of gastrointestinal pathogens by normal flora. In: Current Perspectives in Microbial Ecology (eds M.J. Klug & C.A. Reddy), pp. 374–82. American Society for Microbiology, Washington. Taraschewski, H., Mehlhorn, H. & Raether, W. (1990) Loperamid, an efficacious drug against fish-pathogenic acanthocephalans. Parasitol. Res., 76, 619–23. Totland, G.K., Grotmol, S., Morita, Y., Nishioka, T. & Nakai, T. (1999) Pathogenicity of nodavirus strains from striped jack, Pseudocaranx dentex, and Atlantic halibut, Hippoglossus hippoglossus, studied by waterborne challenge of yolk-sac larvae of both teleost species. Dis. Aquat. Org., 38, 169–75. Urawa, S., Ueki, N. & Karlsbakk, E. (1998) A review of Ichthyobodo infection in marine fishes. Fish Pathol., 33, 311–20. Vadstein, O. (1997) The use of immunostimulation in marine larviculture: possibilities and challenges. Aquaculture, 155, 401–17. Vadstein, O., Øie, G., Olsen, Y., Salvesen, I., Skjermo, J. & Skjåk-Bræk, G. (1993) A strategy to obtain microbial control during larval development of marine fish. In: Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 69–75. A. A. Balkema, Rotterdam. Vanbelle, M., Teller, E. & Focant, M. (1990) Pro-biotics in animal nutrition: a review. Arch. Anim. Nutr., 40, 543–67. Westerdahl, A., Olsson, J.C., Kjelleberg, S. & Conway, P.L. (1991) Isolation and characterization of turbot (Scophthalmus maximus)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl. Environ. Microbiol., 57, 2223–8. Wiik, R., Hoff, K.A., Andersen, K. & Daae, F.L. (1989) Relationships between plasmids and phenotypes of presumptive strains of Vibrio anguillarum isolated from different fish species. Appl. Environ. Microbiol., 55, 826–31. Wiik, R., Stackebrandt, E., Valle, O., Daae, F.L., Rødseth, O.M. & Andersen, K. (1995) Classification of fish pathogenic vibrios based on comparative 16S rRNA analysis. Int. J. Syst. Bacteriol., 45, 421–8.

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Wiklund, T. & Dalsgaard, I. (1998) Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: a review. Dis. Aquat. Org., 32, 49–69. Williams, P.H., Roberts, M. & Hinson, G. (1988) Stages in bacterial invasion. J. Appl. Bacteriol. Symp. Suppl., 131S–47S. Woo, P.T.K. & Poynton, S. (1995) Diplomonadida, Kintoplastida and Amoebida (Phylum Sarcomastigophora). In: Fish Diseases and Disorders. Vol. 1. Protozoan and Metazoan Infections (ed P.T.K. Woo), pp. 27–96. CAB International, Wallingford.

Chapter 4

Live Food Technology of Cold-Water Marine Fish Larvae Y. Olsen

4.1 Introduction Successful and economically feasible production of marine fish juveniles is highly multidisciplinary, requiring adequate competence in both larval and live-food technology. The live-feed developments include zootechnical, nutritional and microbial aspects, and these have been a major challenge (see also Chapter 7). Only two zooplankton families have so far shown that they can be produced regularly at an acceptable cost: the rotifer Brachionus sp. and the brine shrimp Artemia sp. (e.g. Lubzens, 1987; Sorgeloos et al., 1986). The most commonly used species within these groups are Brachionus plicatilis and Artemia franciscana, which are used alone or in appropriate combinations as live feed for most marine fish species in culture. There is an increasing use of the smaller rotifer species Brachionus rotundiformis in marine fish larviculture, but this species has only recently been used for cold-water species, and will be covered only briefly in this chapter. There is a well-established international live-food production technology for Brachionus and Artemia. This technology has been modified for use for marine cold-water species of fish, and was taken up in the 1970s in northern countries (e.g. Howell, 1979). This chapter describes the basic cultivation techniques and methods for manipulation of nutritional value that are suitable for marine cold-water fish larvae. In the developmental work, the biochemical composition, or nutritional value, of the cultivated live feed was inspired by that of natural zooplankton, since marine copepods are believed to be important as a natural food for many larval fish species. In particular, the chapter will present the developments and findings of recent Norwegian research programmes. Methods for rotifer production and nutritional manipulation that are suitable at low temperatures will be the main focus. Both nutritional and microbial aspects are covered, and marine copepods have served as a nutritional reference. These cold-water methods deviate from the normal procedures used world-wide on some points: the production and n-3 HUFA (highly unsaturated n-3 fatty acids; see Section 7.2) enrichment of rotifers can readily been done simultaneously and not in successive steps. This has been found to be feasible for cold-water species. The international Artemia technology forms the basis of the cold-water adaptations, and these methods are already well described (see manuals by Sorgeloos et al., 1986; Lavens & Sorgeloos, 1996). In this chapter, the focus on Artemia will primarily involve specific problems of n-3 HUFA enrichment and stability. These problems

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Feeding system

Oxygen monitoring system, optional Seawater

Aeration tube

Heating Temperature control

Freshwater

Mixing Disinfection Temperature acclimatisation Aeration Electrical control, security

Water exchange tube

Drainage valve

Figure 4.1 Schematic view of the cultivation equipment and supporting functions needed for rotifer and Artemia production and enrichment.

have become evident during the rearing of Atlantic halibut (Hippoglossus hippoglossus) larvae, an important species for aquaculture in northern countries (Olsen et al., 1999; Shields et al., 1999b). Microalgae are important components of fish larval diets, either directly, or indirectly as food for Brachionus and Artemia. The methods of cultivation and further use during rearing of cold-water fish larvae are not different from those used for rearing other species. Other authors have provided detailed descriptions of the production technology of microalgae (see papers in Fulks and Main, 1991; Coutteau, 1996), and such methods will not be covered in this chapter. The specific use of microalgae during first feeding is covered in Chapter 7.

Live food technology of cold-water marine fish larvae

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4.2 Cultivation Systems There is no unified technical design of cultivation systems for rotifers and Artemia, and all producers tend to use their own approach. However, there is a common core of system design and function that is generally useful during live feed production (see Lavens & Sorgeloos, 1996). The specific pre-treatments needed for the process water of live-feed cultures depend on the local water quality. Figure 4.1 illustrates one possible technical arrangement, including water supply and pre-treatment, tank design, feeding system and control functions. A vital requirement for the process water is the complete removal of zooplankton and larvae of marine organisms. Standard filter units (e.g. sand filters) will not retain zooplankton individuals as small as 20–30 mm (e.g. many ciliates). This group can create serious problems in rotifer cultures. Specific disinfection treatments may be needed as a protection against disease and contamination by small zooplankton species. Most strains of B. plicatilis have their optimum for growth in brackish water (e.g. 10–15 p.p.t.). If reduced salinity is used during cultivation, freshwater should be added and mixed with the seawater before disinfection and temperature acclimation. It is important that the process water is temperature-acclimated and thoroughly aerated before being added to the culture tanks in order to avoid super-saturation of nitrogen. B. plicatilis and Artemia can be cultivated in almost all types of tank. Cylindrical–conicalshaped tanks allow efficient precipitation and easy removal of organic wastes from the bottom during intensive feeding and high animal densities, but other types of concrete or glass-fibre tanks of various shapes and sizes have been used in commercial hatcheries. Some companies may use tanks larger than 100 m3. The volume of the cultivation units used is accordingly highly variable, but cultivation and lipid enrichment in very small units (e.g. 1000 rotifers ml-1). Air can be supplied by airstones or another diffuser

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system suspended close to the walls and bottom of the tanks. A 200 l culture will need one small air outlet, a 4-m3 culture will need more than four big airstones. Equipment for monitoring and controlling culture conditions is not strictly necessary for low densities of rotifers and Artemia, but very intensive cultures will require monitoring. Adequate aeration of cultures is probably among the main problems of producing rotifers in large and intensive units (>2 m3) and of n-3 HUFA enrichment of Artemia. The biotechnology industry has developed very intense aeration systems for very large production units of yeast and bacteria. The full adoption of this technology to rotifers and Artemia is still incomplete. Commercial large-scale production of live feed requires the installation of partly automated feeding systems, a cleaning and washing system for the live feed, enrichment systems, and systems for the cooling, storage and concentration of cultures. Labour costs may constitute over two-thirds of the live-feed costs. Appropriate equipment will therefore reduce the costs of production considerably, and allow the staff to concentrate on the more important tasks of rotifer and Artemia production. Transport can be mediated by gravity, vacuum, or thoroughly tested pumps.

4.3 Production of Rotifers In the early 1960s, the Japanese pioneered the use of Brachionus plicatilis as live feed for cultured marine larvae (Ito, 1960; review by Nagata & Hirata, 1986). The rotifer technology for cold-water species was mainly developed during the early 1990s. The descriptions involve general biology, cultivation techniques, and methods that were established to manipulate the biochemical composition of rotifers. These methods deviate to some extent from the methods normally used for temperate and warm-water fish larvae. The use of rotifers during first feeding is treated in Chapter 7, and the importance of the microbial communities of livefeed cultures is treated in Chapter 3.

4.3.1 Biological Characteristics 4.3.1.1 General Biology and Life History Rotifers are small metazoa, and most species live in fresh water. A layer of keratin-like proteins forms their epidermis and is termed the lorica. The form and characteristics of the lorica are important taxonomic criteria. A rotary organ or corona, recognised by its annular ciliation, ensures locomotion in a whirling water movement that facilitates the rotifers feeding on small organisms in the water. The rotifer strains most commonly used as live feed for marine fish larvae are Brachionus plicatilis and Brachionus rotundiformis. The former morphotype was previously termed the L-type Brachionus, and is characterised by a lorica length of 130–340 mm. The latter morphotype has been termed the S-type, and is characterised by a typical lorica length of 90–210 mm. These strains are apparently not genetically isolated, but the S-type seems to have higher optimum temperatures for growth than the L-type (see Fu et al., 1991; Dhert, 1996). B. plicatilis is by far the most commonly used species in aquaculture. Most of the examples in this chapter are representative of the L-strain isolate of B. plicatilis (termed the


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SINTEF strain, lorica length 250–330 mm) that has been thoroughly studied and used in firstfeeding trials with turbot, Atlantic halibut and cod. The strain has both an asexual (amictic or parthenogenetic) and a sexual (mictic) life cycle, but males and females carrying resting eggs have only been observed on a very few occasions. The normal production temperature and salinity is 20–22°C and 20 p.p.t., respectively. Under these conditions, the average amictic females produce their first eggs 1.4 days after hatching. Thereafter, they produce 21 eggs during the following 6.7 days. The developmental time of the eggs is 0.41 days. The post-reproductive period of the rotifer is 2.4 days, giving a total average life span of 10.5 days (Korstad et al., 1989a). 4.3.1.2 Feeding Kinetics of B. plicatilis B. plicatilis can feed on a wide range of food particles, including bacteria, microalgae, protozoa and dead organic material. The upper size level of food particles that can be ingested by the rotifer depends on the shape and nature of the food. B. plicatilis is able to ingest bacteria 0.16 day-1 if the inoculum contains a high fraction of senile rotifers. This calls for careful use of Equation 4.8 under non-steady-state growth conditions. The egg ratio (ER, eggs per rotifer) of rotifer cultures has proved to be a very useful tool in mass cultivation. Eggs per rotifer can easily be counted using a microscope, and the value can be interpreted as a dynamic variable reflecting the growth and production rates of the cultures. The linear relationship between the average egg ratio and the net specific growth rate of steady-state cultures is illustrated in Fig. 4.2C. This figure indicates that an egg ratio of 0.18 eggs rotifer-1 is to be expected for zero net growth rate of the culture, and that cultures growing at the maximum net growth rate of 0.45 day-1 is expected to have an average egg ratio of 0.68 eggs rotifer-1. Figure 4.2D shows the terms of Equation 4.8 as a function of the culture egg ratio. The net growth rate relationship is simply the inverse of the curve in Fig. 4.2C extrapolated to zero egg ratios. The curve suggests a negative population growth rate of -0.16 day-1 for cultures of rotifers without eggs. Because the gross growth rate must be zero for animals without eggs, this value can be interpreted as the mortality rate for starved cultures. The mortality rate of well-fed cultures is 0.1 day-1 (see above), and the mortality curve in Fig. 4.2D assumes linearity between mortality rate and egg ratio. The gross growth rate is the sum of mortality and net growth (Equation 4.8), and the following equations describe growth and mortality rates as functions of the egg ratio of the present rotifer strain. m net = 0.90 ER - 0.16

(4.9)

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m = 0.16 - 0.087 ER

(4.10)

m gross = 0.81 ER

(4.11)

Again, the equations are only strictly valid for steady-state conditions. The relationship between net growth, gross growth and mortality is important in practical production, for example during attempts to predict future production and to diagnose problems, because both reduced gross growth rate and enhanced mortality rate may affect the net rotifer production. Even though both events will result in low production, the counter-measures needed to overcome these problems may be quite different. The coefficients in Equations 4.9–4.11 are believed to be independent of the food used, because the egg developmental time is independent of the food quality and quantity (King & Miracle, 1980). If for some reasons the rotifers exhibit a higher net growth rate with another feed, the egg ratio is expected to increase proportionally. The above equations are valid for the temperature range 20–22°C. However, changes in temperature will most likely affect the developmental time of the eggs, and in turn the relationship between egg ratio and specific growth rate. It will also affect the metabolic activity of the rotifer.

4.3.2 Cultivation Feed and Feed Treatments B. plicatilis will consume most food particles of an appropriate size for consumption. Efficient cultivation feeds must also cover the nutritional demands of the rotifers and secure proper hygienic conditions in the cultivation tanks. Appropriate live feeds are microalgae, baker’s yeast, used alone or in combination with dispersed, emulsified (or even crude) marine oils, and formulated diets that are commercially available. Crude and cheap diets such as micronised fishmeal and dried yeast powder are not efficient, probably because of extensive leakage of organic compounds that create losses in nutritional value, poor hygiene conditions and enhanced rotifer mortality. Many species of microalgae are excellent food for rotifers, but their production costs are high. Some producers give microalgae as a component of the diet (1–5%), normally together with formulated diets or live baker’s yeast. Small supplements of microalgae can contribute to better rotifer health and viability, and thereby reduce risks. It is a common belief that even a moderate use of microalgae, in an appropriate combination with another principal feed, will tend to make the rotifer production more predictable and the rotifers more viable. Japanese pioneers learned that live baker’s yeast was an efficient and cheap feed for rotifers (Nagata & Hirata, 1986). To overcome an essential n-3 fatty acids deficiency in fish larvae, they later suggested that yeast should be supplied together with emulsified marine oil (Fukusho, 1977; Kitajima & Koda, 1976). For many reasons, this principal technique has proved feasible for producing rotifers for cold-water species of fish. The mixture of yeast and oil can be added in controlled, high-quantity rations, which is not very easy with microalgae. This is important in order to run controlled and predictable large-scale production of rotifers. It is also noteworthy that baker’s yeast and crude marine oils are very cheap diets, and that the rotifer feed costs will also remain low if a commercial formulated emulsified diet is used instead of the crude marine oil (e.g. Selco-type, INVE Aquaculture, Belgium).


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Before addition to the rotifer tank, the yeast should be mixed with lukewarm fresh water (30 rotifers ml-1) may be fed normal feed rations. The risk of severe rotifer mortality is highest during the first phase of the cultivation process. The risk may be reduced if the rotifers are fed >1–2 mgC l-1 of microalgae (visible colour) during the first 1–2 days after inoculation. Another way to reduce risks is to increase the initial inoculation density to >100 rotifers ml-1. A combination of high initial rotifer density, careful rinsing of the inoculum, and initial feeding by microalgae will normally secure success. The number of rotifers that is available is low if the inoculum is taken from stock cultures. A suitable method is then to grow the rotifers with microalgae only during the first few days (densities >2 rotifers ml-1). This is easily done by inoculating them with microalgae in illuminated rotifer tanks (light tubes or other sources, >100 W m-3) at 10% strength of a normal algal medium (formulations in Smith et al., 1993; Coutteau, 1996). The main production feed should be added before the algae become grazed down by the rotifers, but the rotifers should be fed algae only for ca. 2 days. This method is also appropriate if the quality and viability of the production cultures are poor. The green algae Tetraselmis spp. have been shown to support culture self-cleaning quite efficiently. Many contaminating micro-zooplankton species cannot ingest the large Tetraselmis cells very efficiently, and will probably be out-competed by rotifers after the change in food source. 4.3.3.3 Early Growth Phase The first 2–6 days of the cultivation process, when food rations and rotifer densities are raised (see Fig. 4.3), has been shown to be the most critical phase of rotifer production. As mentioned above, prophylactic measures to counteract potential problems are high initial rotifer densities and thorough quality evaluation of the inoculum. A further serious problem of the initial phase is related to a mismatch between the food ration offered and the rotifers’ food requirements for growth. Feeding a high specific food ration (SFR, food per rotifer per day) is important in order to obtain a rapid growth rate and viable rotifers. On the other hand, overfeeding may cause unfavourable environmental conditions (low oxygen, high reactive ammonia and extensive bacterial growth) and enhanced rotifer mortality. Feeding during the early phase of growth must be based on the rotifers’ actual food requirements for growth. The specific growth rate of rotifer cultures for a given feed is a function of the specific food ration (SFR, e.g. mg yeast rotifer-1 day-1) supplied. The relationships between growth rate and specific feeding rate obtained during batch and continuous cultivation are shown in Fig. 4.4A. Figure 4.4B shows the same relationship, but using feeding rate per day (day-1, food C per rotifer C and day), which is assumed to be less straindependent than the former expression. The figures reveal that the rotifer cultures must be fed approximately 0.5 mg baker’s yeast (plus 0.05 mg oil) per rotifer per day, or 0.5 day-1 (oil included) in order to maintain a positive net growth rate. The growth response is comparable for batch and continuous cultures when the feeding rate is below 1 mg yeast per rotifer per day, yielding growth rates below 0.2 day-1. For higher food rations, the growth response for a given food ration becomes dependent of the cultivation method used. The lower spe-

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Growth rate, day-1 0.6

Growth rate, day-1 0.6 0.5

A

0.4

0.4

0.3

0.3

0.2

0.2

0.1

B

0.5

Continuous Batch

0.1

Continuous Batch

0

0

0

1 3 5 0 2 4 Feeding rate, mg Yeast (rotifer day)-1

0.5

1 1.5 2 2.5 Feeding rate, day-1

3

3.5

Figure 4.4 Relationship between rotifer feeding and growth rates. Specific population growth rate (day-1) as a function of feeding rate expressed (A) in terms of mg wet yeast rotifer-1 day-1, and (B) in terms of day-1 (mgC day-1 mg rotifer-C-1).

cific growth rates obtained in continuous cultures are primarily the result of higher feed losses compared with those of closed batch cultures. Cultures harvested continuously at a high rate are characterised by high water turbidity (see below), which means high losses during water exchanges. The batch cultures are closed and the food is better utilised under high feeding conditions. The curves shown in Fig. 4.4A can be described by the following empirical equations: m net = 0.377 (SFR - 0.488) SFR

(continuous cultures)

(4.12)

m net = 0.305[ln(SFR ) + 0.715]

(batch cultures)

(4.13)

where mnet is the net specific growth rate of the rotifer cultures (day-1), and SFR is the specific food ration (mg yeast rotifer-1 day-1). The equations relate the growth rate and food rations given to the rotifers, and the net growth rate together with measured rotifer densities per unit volume (RD) are needed for predictions of production (P). P = m net RD

(4.14)

Equations 4.12 and 4.13 can be rearranged to express the specific food ration needed to sustain a given net growth rate. SFR = 0.184 (0.377 - m net ) SFR = 0.489(26.6)

m net

(continuous cultures)

(4.15)

(batch cultures)

(4.16)


85

Table 4.1 Detailed procedures of rotifer feeding during the early phase of growth. Equations and definitions are given in the text. Internal control method 1 Make decision on culture growth rate (for example 0.3 day-1, the recommended range is 0.2–0.4 day-1) 2 Estimate the corresponding specific food ration (SFR) using Equation 4.16 (mnet = 0.3 day-1 requires 1.3 mg yeast individual-1 day-1, SFR range for the recommended mnet-range (0.2–0.4 day-1) is 0.94–1.8 mg yeast individual-1 day-1) 3 Count rotifer density (RD) per unit volume 4 Estimate the culture food ration (FR, food per unit volume and day) (FR = SFR · RD) 5 Feed the estimated ration to the culture (in one, two or several portions) 6 Repeat steps 3–6 with constant SFR for the next few days until the maximum sustainable feed ration (FRmax), which has already been determined, is reached. Feed the cultures their maximum sustainable feed ration from that day External control method 1 Make decision on culture growth rate (for example 0.3 day-1, the recommended range here is 0.2–0.4 day-1) 2 Estimate the corresponding SFR using Equation 4.16 (mnet = 0.3 day-1 requires 1.3 mg yeast individual-1 day-1, SFR range for the recommended mnet-range (0.2–0.4 day-1) is 0.94–1.8 mg yeast individual-1 day-1) 3 Count initial rotifer density per unit volume (RD0) 4 Estimate the initial culture food ration (FR0, food per unit volume and per day on day 0) (FR0 = SFR · RD0) 5 Feed the estimated day 0 culture ration (FR0) to the culture (in one, two or several portions) 6 For the next day (day 1) and later (days 2, 3, . . .), estimate the food ration for day n according to Equation 18. The daily percentage increase in FR depends on the predetermined growth rate. A growth rate of, for example, 0.3 day-1 requires a daily increase in the food ration (IR) of 35% per day (Equation 4.17) 7 If the quality of the cultures is acceptable (see below), feed the day n dose 8 Repeat steps 6–8 until the maximum sustainable feed ration, (FRmax) which has already been determined, is reached. Feed the cultures their maximum sustainable feed ration from that day

A thorough knowledge of rotifer food requirements is the basis for safe and reproducible production. Two basically different methods, which are operationally quite close, may be used to raise rotifer densities of cultures from low to high levels during the early phase of production in batch culture (Table 4.1). Internal Control Method The rotifers are fed a constant specific food ration (SFR, mg food individual-1 day-1) all through the early growth phase. The estimated culture food ration (FR, g food m-3 day-1) will then depend on the actual growth response of the rotifer population. This means that the actual growth response of the rotifers controls feeding. The cultures must be counted daily. External Control Method The initial feeding conditions are estimated in the same way as for the internal control method, but thereafter the rotifers are fed according to a fixed feeding regime where the culture food ration (FR) is increased exponentially to sustain the expected exponential increase in rotifer density. Further control is therefore mainly in the hands of the producer. The cultures do not need to be counted daily, but the quality of the cultures should be evaluated visually before the food is added. The exponential rate of increase in food rations (IR, % day-1) must be kept within

86


the growth capabilities of the rotifers. A net growth rate range of 0.20–0.40 day-1 corresponds to an IR range of 22–49% day-1, as estimated by Equation 4.17. IR = (e mnet - 1)100%

(4.17)

The food ration for day n (FRn) can be calculated as FR n = FR 0 ◊ (IR 100 +1)

n

(4.18)

where FR0 is the food ration at the initial day (n = 0) and RI is the daily increase rate of the food ration. For both methods, cultures should be inspected visually and only be fed if the food is consumed and the culture quality is satisfactory (e.g. low turbidity caused by small particles). Cultures of poor quality should be treated specifically, and optionally brought out of production (see below). It is also important to realise that there will always be a critical upper value for the culture food ration (FRmax, g m-3 day-1). This critical value for sustainable rotifer production will depend on cultivation method, system design, feeding mode, food quality and oxygen supply. The maximum sustainable ration for the simplest techniques and small production units (1000 rotifers ml-1). It should be noted that anything other than one daily water exchange and one daily feeding require partial automation of the cultivation system. Under conditions of steady oxygen supply, there is a marked drop in the ambient oxygen of the cultures, which reaches a minimum 4–6 h after the addition of food. This drop is probably related to increased defecation and microbial activity in the period after food supply and increased feeding rate. This oxygen drop is highly reproducible and is related to the food dose, and the culture may collapse if the concentration becomes lower than the critical value for the strain (e.g. 2–3 mg O2 ml-1). Continuous feeding will reduce this potential problem. Models for Growth and Production in Continuous Cultures The variation in rotifer density with time (dRD/dt) in cultures that are harvested (diluted) and fed at constant rates can be expressed as


dRD dt = RD(m net - D)

93

(4.26)

where RD is the rotifer density, mnet is the net specific growth rate, and D is the specific dilution rate, defined as dV/(dt V) if dV/dt is the daily volume exchanged and V is the culture volume. Cultures of rotifers, or any other microorganisms maintained in continuous culture, tend to reach a steady state of growth characterised by constant numbers of organisms independent of time. This implies that dRD/dt = 0, meaning that the specific growth rate of the rotifers is identical to the culture dilution rate. m net = D = dV (dt V )

(4.27)

The mathematical relationships become somewhat more complicated if the process of dilution is not strictly continuous in time. If the cultures are diluted at a frequency of t days, the growth rate is given by the following general equations: m net = ln [V (V - dV )] t

(4.28)

m net = - ln (1 - dV V ) t

(4.29)

which is equivalent to

Reorganisation of Equation 4.29 gives dV V = 1 - e - mnett

(4.30)

where t = 1 for one dilution per day. The above equations relate the operational variable (harvested volume, dilution rate) to the growth rate of the cultures. It must be emphasised that the equations are only strictly valid for steady-state conditions. The rotifer density just before harvesting should then be constant and independent of time. The harvesting rate in continuous cultures (F%) is a practical term that can be defined as F% = dV V 100%

(4.31)

The steady-state relationship between harvesting rate and the specific net growth rate of the rotifers is F% = 100%(1 - e - mnett )

(4.32)

m net = - ln(1 - F% 100) t

(4.33)

which is equivalent to

The above equations are fundamental for predicting the growth and production of rotifers in continuous steady-state cultures, and are valid for all harvesting frequencies (t is days between dilutions).

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The daily net production in continuous cultures (Pd) can be predicted as rotifer density multiplied by the net growth rate: Pd = RDad (e mnett - 1)

(4.34)

Pd = RDbd (1 - e - mnett )

(4.35)

or

where RDad and RDbd are rotifer densities just after and before dilution, respectively. Alternatively, the daily rotifer production of continuous cultures can be estimated as rotifer density at the time of harvesting (RDbd) multiplied by the harvested volume (dV). Pd = dV ◊ RDbd

(4.36)

The theoretical optimal harvesting rate of continuous cultures is the rate that corresponds to half the maximum growth rate of the rotifer culture, as in batch culture. The maximum specific growth rate is variable (temperature, salinity, strain), but is easily measured at given conditions. This fact makes the optimisation of production in continuous rotifer cultures fairly easy. The optimal production can be estimated by substituting 0.5 mmax for mnet in Equations 4.34 and 4.35. The replaced volume (dV) that results in optimal production efficiency (dVopt) can be estimated as dVopt = V (1 - e -0.5mmaxt )

(4.37)

Finally, it should be noted that the production at a given harvesting rate will also depend on the food ration given, because this food ration will affect the rotifer density (RD). As a firstorder approximation, the rotifer density, and therefore also the production, can be assumed to be proportional to the food ration at any dilution rate (Equation 4.24). Dynamics of Growth in Continuous Rotifer Cultures As mentioned above, the rotifer density of the culture will become constant after some time (a few days or a week), if a constant fraction of the culture is harvested and replaced by seawater each day. This means that the net growth balances the losses from harvesting, and that the specific growth rate of the rotifers is constant (steady state of growth). Typical time-courses found in cultures harvested at rates of 5, 18 and 27% day-1 are illustrated in Fig. 4.8A. The average rotifer densities are inversely related to the harvesting rate or the culture growth rate. Figure 4.8C and D shows an inverse, linear relationship between the specific growth rate and the rotifer biomass expressed in terms of numbers and carbon biomass. Low growth rates result in relatively dense cultures. The carrying capacity can be found if the curves are extrapolated to a zero harvesting rate. High growth rates result in low rotifer density, and the maximum specific growth rate is indicated if the curves are extrapolated to zero density.


Rotifer density, mill m-3

Rotifer weight, ng ind-1

500

700

300

600

200

500

95

B

400 100

300 200

50

100

A

30 20

0

5

10

0 0

15

0.1

0.2

0.3

0.4

Growth rate, day-1

Time, days

DW ind-1 Prot. ind-1

5% day-1 18% day-1 27% day-1

Rotifer biomass, gC m-3

Rotifer density, mill m-3

C

300

60

D

50 40

200 30 20

100

10 0

0 0

0.1

0.2

0.3

0

0.4

0.1

0.2

0.3

0.4

Growth rate, day-1

Growth rate, day-1

Rotifer production, mill m-3 day-1

Rotifer production, gC m-3 day-1

40

10

E

F

8

30

6 20 4 10

Measured

2

Simulated

Mesaured Simulated

0

0 0

0.1

0.2

0.3

0.4

Growth rate, day-1

0

0.1

0.2

0.3

0.4

Growth rate, day-1

Figure 4.8 Characteristics of B. plicatilis grown at various rates in continuous cultures. A. Development of rotifer densities with time in cultures diluted at various rates (mill individual m-3). B. ng dry matter and protein per individual rotifer. C. Steady-state rotifer densities (mill individual m-3). D. Steady-state rotifer biomass (gC m-3). E. Steady-state rotifer production rates (mill individual m-3 day-1). F. Steady-state carbon production rates (gC m-3 day-1).

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Figure 4.8E and F shows the measured and predicted production of the same cultures (see legend). The optimum production level for intermediate growth rates is clearly indicated. The measured and predicted values are very close for growth rates below the optimum level, but deviations become more pronounced for the higher range of values. The nutritional value of the rotifers increases with growth rate (Fig. 4.8B), with intermediate values at the optimum growth rate for production. It is important to recognise that the rotifer growth rate, their nutritional value, and their production efficiency can all be controlled during continuous production simply through selection of the dilution rate of the culture. Continuous production of rotifers therefore allows a more efficient control of both production and food quality than production in batch culture. Finally, the equations presented for batch and continuous production allow a wide range of further simulations and calculations that cannot be further elaborated here.

4.3.4 High-Intensity Rotifer Cultivation The next generation of rotifer production technology with very high rotifer densities is now being developed in research laboratories. Japanese scientists were the pioneers of this developmental work, and they have been able to produce and maintain B. rotundiformis in densities higher than 100 000 individuals per ml in 1000-l batch cultures. This has been made possible by feeding the cultures concentrates of Chlorella, and the implementation of strict environmental control of the cultures (Yoshimura et al., 1997). A second attempt to develop highly intensive rotifer cultivation systems was made with B. plicatilis fed with the formulated feed Culture Selco. Belgian scientists were the first to maintain the bigger B. plicatilis in densities of 7–10 thousand in stable cultures that were harvested throughout many weeks. Environmental control in the cultures is achieved by circulating the culture water in an external loop in which it is thoroughly purified. The rotifers remain in the culture tank, and can be harvested in batch or continuous modes independently of the water circulation rate (200–500% day-1). The circulating water is first sterilised with ozone, then treated several times by protein skimmers, and finally treated and recolonised by bacteria in a biofilter before being transferred back to the culture. Feasibility studies have shown that the technology reduces the costs of rotifer production compared with traditional techniques (Suantika et al., 2000). Industry will always be hesitant to change their live-food technology, and it is not surprising that high-density cultivation systems for rotifers are still not being used in commercial hatcheries. Both of the cultivation methods and systems described above have been thoroughly tested. The technologies will be further improved, and will probably be implemented in commercial hatcheries in the coming years. Skilled staff is a precondition, and lower production costs are a driving force.

4.3.5 Problems in Rotifer Cultivation A common problem in rotifer cultivation is overfeeding, which in turn may cause acute oxygen deficiency, high concentrations of reactive ammonia, or the invasion of microzooplankton (e.g. ciliates). Another problem is inadequate environmental control that results


97

in bad water and culture quality. These problems may sometimes be difficult to diagnose. A third main problem is specific infections or contamination that results in fatal diseases and enhanced mortality. 4.3.5.1 Feeding-Related Problems Two types of cultivation problems are related to the food and the feeding process. The food may be nutritionally inadequate, which means that limitations in the supply of essential compounds will occur during long-term cultivation. The second main problem is indirect, and is the result of a disparity between food supply and food consumption. Baker’s yeast and marine oil, microalgae and commercial formulated feeds have all been shown to support rotifer growth and reproduction. The use of dried yeast and other crude powder diets is not recommended because of their high losses of organic compounds in seawater. A high organic load may create microbial problems, and physical/chemical stability in seawater is an important requirement for a formulated rotifer diet. Emulsified marine oils create fewer microbial problems than powder diets. In general, the oil droplets are probably less available as a bacterial substrate than dissolved organic compounds and organic particles, and emulsified oils tend to be more inert than crude oils. A disparity between food ration and food consumption is probably the most common problem in rotifer cultivation, particularly during the critical early phase of growth. The rotifers may occasionally be either over-fed or starved. Severe mortality is likely to occur with sudden changes in the specific food ration (SFR). Sudden decreases in SFR will often result in reduced viability and enhanced population mortality. On the other hand, sudden increases to levels above the maximum consumption capacity of the rotifers may cause very unfavorable hygiene conditions and harmful invasions of micro-zooplankton in the cultures. The feeding frequency of the rotifer cultures may also affect the quality of the cultures. There is an obvious limit to the maximum single food ration that can be added (see above). The present strain has tolerated 250 g yeast plus 25 g oil m-3 day-1 without experiencing a fatal oxygen deficiency or abnormal feeding activity, but such a high dose is not generally recommended. The mode of food supply may also affect the competition between rotifers and micro-zooplankton species, but the general mechanisms are not well understood. 4.3.5.2 Environmentally Related Problems Pollution of the process water may cause severe toxicity problems, the nature of which is beyond the scope of this chapter. Many problems which are related to water quality are difficult to diagnose precisely unless a specific hypothesis on the nature of the problem can be established. However, most of the problems that originate with bad water quality will become expressed in reduced rotifer egg ratio or birth rate. Acute oxygen deficiency may occur when the food ration becomes high, and maintenance of an adequate oxygen supply is more challenging in large cultures than in small. The wall and bottom zones of tanks may rapidly become anaerobic. This may, in turn, inhibit population growth in the entire culture. Continuous feeding is a way to avoid fatal oxygen defi-

98


ciency. Reactive ammonia may also accumulate in the late growth phase, but B. plicatilis apparently has a relatively high tolerance to reactive ammonium. However, some care should be taken to ensure that the pH does not become low while the cultivation temperature is high. 4.3.5.3 Disease and Contamination Parasites, bacteria or viruses that may cause rotifer death, as well as pathogens for the fish larvae, may infect the rotifer cultures. Specialists must carry out the diagnostic work in the case of serious infections (see Chapter 3), and detailed knowledge is important in choosing an efficient counter-measure, which may involve complete disinfection of the entire production system for live feed and fish. Some micro-zooplankton species are harmful to rotifers, at least temporarily. Some ciliate species tend to stress the rotifers by becoming attached to their filtering system. The rotifers react by rapid swimming movements (rotating and swimming in circles) and enhanced mortality. Some of the common ciliates in rotifer cultures are too big, or they swim too fast, to be eaten efficiently by the rotifers. Many the species normally cannot compete efficiently for suspended food particles because they are benthic species that are only able to survive in pelagic waters if the food concentration is high. Some ciliate species, which amongst others colonise and eat dead rotifers in the sediments, may cause serious problems during cultivation. The most harmful species are those which interact physically with the rotifers, either by disturbing their filtering activity, or by colonising and killing them directly. There are some indications that these attacks primarily occur when the rotifers are in bad shape for some reason. 4.3.5.4 Problem Identification—Diagnostic Criteria Some easily measurable characteristics of rotifer cultures can be used to diagnose problems. These are:

• egg ratio • swimming speed • turbidity of culture • abundance of suspended micro-zooplankton, mostly ciliates • abundance of attached micro-zooplankton If the net increase in rotifer numbers throughout is lower than the net growth rate predicted based on the egg ratio, it may indicate an enhanced mortality rate in the cultures. On the other hand, if an unexpected reduction in net growth rate is accompanied by a correspondingly low egg ratio (birth rate), the problem is normally related to sub-optimal food quality/quantity or unsatisfactory environmental conditions. Some rotifer producers use swimming speed as a criterion for rotifer viability (Snell et al., 1987). The biological basis for this method is the fact that old post-productive rotifers tend to swim more slowly than younger individuals. The average swimming speed can therefore give information on the age distribution and growth potential of the culture.


99

The culture turbidity is a fast and useful predictor of culture quality and performance. The rotifers may perform very well with a relatively high abundance of larger suspended organic particles in the culture, but a high abundance of small particles can be an early sign of coming problems (unless there is obvious over-feeding). Both a high abundance of bacteria and micro-zooplankton, as well as leftovers of feed, may cause high turbidity, and a closer examination under a microscope may then be useful. If the turbidity is caused by yeast cells and oil droplets, and if the egg ratio of the rotifers is high, the situation may not be threatening. However, high turbidity along with an egg ratio that is lower than expected indicates reduced food consumption and birth rate (gross growth rate). The situation may be threatening, and the environmental conditions may be unsatisfactory (e.g. low oxygen, high reactive ammonia, and other pollution). Inadequate food quality is another explanation. However, this is unlikely if the food has already proved to be satisfactory in rotifer production. If high culture turbidity caused by food particles combined with low rotifer egg ratio is a common problem, nutritional factors should also be considered. A high abundance of micro-zooplankton (e.g. ciliates) in the rotifer cultures is an indication of over-feeding or inadequate cultivation routines. The situation is not necessarily threatening, but immediate measures should be taken to reduce the abundance of these organisms. A high abundance of ciliates that are attached to the rotifers is threatening. In any event, the situation is undesirable and will require some kind of action. 4.3.5.5 Counter-Measures Against Undesirable Situations Appropriate counter-measures against identified problems may involve changes in feeding procedures, direct rinsing or treatment of the cultures, the termination of cultures, or measures of a more prophylactic nature. An efficient cultivation system, along with thorough monitoring, is probably the best way to avoid problems. A prophylactic measure already recommended during cultivation is to use microalgae as a supplementary food just after inoculation. Another prophylactic measure is to rinse the inoculum taken from production cultures very thoroughly. When cultures remain turbid, the normal measure will be to skip feeding. This treatment is also recommended if the abundance of suspended micro-zooplankton is high. Some other measures may also be efficient against a high abundance of most micro-zooplankton species and bad hygiene conditions in general. These measures, which may be used alone or in combination, are:

• change of cultivation tank • change to algal-based feeding • starvation and cooling • freshwater treatment Infected cultures may become healthy by simply changing the cultivation tank. Starvation or an appropriate change in food source can be combined with this treatment. Cooling (10–15°C) in combination with starvation may work, but washing harvested rotifers for 5

100


–10 min in fresh water is probably more efficient. B. plicatilis will survive this treatment, whereas some of its competing ciliates will die. However, it is difficult to get rid of harmful contaminants by washing, starvation and cooling alone. The problems may be temporarily reduced, but repeated treatments will normally be required. An application of microalgae as the only feed may be surprisingly efficient in many cases.

4.3.6 Biochemical Composition During Steady-State Feeding and Growth The biochemical composition of rotifers used as live feed for marine larvae, and in particular their content of essential compounds, is paramount for their ability to sustain larval growth and survival. It is important to recognise that the biochemical composition is highly dynamic and variable throughout the phases of cultivation, nutrient enrichment and postenrichment. To control live feed quality throughout production, it is important to understand the fundamental biological mechanisms which mediate these changes. The overall integrated nutritional and environmental status of the rotifers is most efficiently expressed through their specific growth rate. Another important issue is the terms used to express nutritional value, e.g. essential amino acids and protein. The nutritional value may be expressed by:

• percentage of amino acid or total amino acids (weight or molar, the so-called profile) • weight of amino acid per prey biomass (dry matter or other biomass expression) • weight of amino acid per individual prey The same applies to fatty acids and other compounds. The last expression relates nutrients to the individual prey, which is also the item that the larvae relate to during feeding. This term is the most variable and is sensitive to treatment, and may also be the most adequate term to express nutrient quality. Nevertheless, it may be a sound approach to include all terms in research work (Øie et al., 1997; Øie & Olsen, 1997; Makridis & Olsen, 1999). 4.3.6.1 Proteins and Essential Amino Acids Nitrogen-based estimates of protein derived using the common conversion factor of 6.25 mg protein/mg N yields overestimates, whereas amino acid-based estimates are likely to be slight underestimates. A conversion factor of 4.2 mg protein/mg N has been established through testing and analysis of amino acid profiles. This factor yields more equal results for estimates made for both zooplankton and fish larvae (Øie & Olsen, 1997). Protein is a major component of the biomass, and the content will to some extent reflect the energy level of the organism. The protein content per individual rotifer is a dynamic variable that is related to food availability and specific growth rate (Fig. 4.9A, modified from Øie & Olsen, 1997). The values may be relatively scattered, but several studies confirm that fast-growing rotifers typically contain twice as much protein as slow-growing, starved rotifers, and a common range of variation for the present strain is 100–200 ng protein per individual. A positive relation to growth rate is also found for protein per dry matter, but this


Protein contents

4 00

101

A

3 00

2 00

1 00 m g Pr o t g DW - 1

ng P ro t in d-1

0 0

0.1

0.2

0.3

0.4

Growth rate, day-1

Figure 4.9 Protein contents and amino acid distribution of B. plicatilis grown at various rates. A. Protein contents expressed in terms of dry matter (open symbols) and individuals (solid symbols). B. Relationship between single amino acid (AA) contents in starved (m = 0.05 day-1) and well-fed (m = 0.22 day-1) rotifers. Solid symbols show amino acid values in percentage AA of total AA (profiles, slope 1.02, not significantly different from 1, P < 0.05). Open symbols show amino acid values in terms of ng AA individual-1 (slope 1.53). The dotted line shows a 1 : 1 relationship.

Amino acids of well fed rotifers

14

B

12 10 8 6 4 2

% o f to ta l A A

A A ro tifer -1

1:1 -lin e

0 0

2

4

6

8

10

12

14

Amino acids of starved rotifers

relationship is often not statistically significant because protein is also a substantial part of the dry matter (Øie et al., 1997; Øie & Olsen, 1997). A very common misunderstanding is that the protein and amino acid contents of rotifers are constant and independent of growth conditions. This belief originates in their very stable amino acid profiles (Lubzens et al., 1989). Figure 4.9B illustrates that the percentage amino acid content of total amino acids is independent of feeding conditions (solid symbols, close to the 1 : 1 line), whereas protein per rotifer is significantly higher in the well-fed than in the starved animals (open symbols, slope of curve 1.53) (data from Makridis & Olsen, 1999). The amino acid content is 53% higher in well-fed rotifers grown at 0.22 day-1 than in starved

102


rotifers grown at 0.05 day-1. It is therefore an oversimplification to conclude that protein and amino acid contents are constant. The contents per dry matter and per rotifer are indeed dynamic variables. Some authors have emphasised the importance of free amino acids during the very early larval stages (Fyhn, 1990, 1993). B. plicatilis exhibits relatively high levels of free amino acids (4–6% of the total amino acids, Øie et al., 1997; Øie & Olsen, 1997). In addition, its protein tends to disintegrate rapidly in the larval guts (Hjelmeland et al., 1993). 4.3.6.2 Lipids and Essential Fatty Acids The main fraction of the rotifer lipids is storage lipids with minor physiological functions (40–80%). This is different from the proteins, and it is therefore to be expected that lipids will relate differently to growth rate than protein. Figure 4.10A illustrates that the lipid contents per dry matter of rotifers shows a reduction with increasing growth rate, whereas the amount per individual remains constant. The range of lipid contents for fast-growing and starved rotifers, cultured and fed as described above, is typically 100 and 150 mg per g DW, respectively. The content per individual is typically 50 ng. This pattern is the opposite to that found for proteins, and the result is a more variable ratio of protein to lipid, which is positively related to the growth rate of the cultures (Fig. 4.10C). Although lipid per individual remains constant and is independent of the growth rate, the fatty acid percentage composition shows a substantial variation. The percentage n-3 HUFA becomes reduced when the growth rate increases, whereas the percentages of saturated and mono-unsaturated fatty acids become higher (Fig. 4.10B). Looking more closely into the group of n-3 HUFA, it is notable that DHA (22:6 n-3) is reduced twice as fast as EPA (20:5 n-3). The corresponding quantitative reduction of DHA is substantial. The characteristic patterns of lipids and fatty acids that are shown in Fig. 4.10 are the result of lipid metabolism, and not of variable dietary lipids. The higher lipid level per gram dry matter of severely starved animals is in apparent conflict with their severe food limitation. This must therefore imply that rotifers cannot utilise lipids efficiently during severe starvation, and that starved rotifers retain essential n-3 HUFA more efficiently than well-fed ones. This finding suggests that starved rotifers are nutritionally beneficial from an n-3 HUFA perspective, which conflicts with their protein contents and the need for efficient production. This is important knowledge that may have to be considered. The enrichment studies described below are all representative for rotifers grown at a rate of 0.1–0.2 day-1, which is a sub-optimal rate from a production efficiency perspective, but an optimal rate from an n-3 HUFA and lipid retention perspective. For low growth rates, the lipid content of the rotifers is positively related to the lipid content of their food, as shown in Fig. 4.10D. The response of enhanced food lipids on rotifer lipids is relatively moderate for naturally occurring lipid levels (10 days (0.1–0.2 day-1). The fatty

104


B

ASaturated

Saturated Monounsaturated

Monounsaturated

n-6

n-6

18:3 n-3

18:3 n-3

18:4 n-3

18:4 n-3

20:4 n-3

20:4 n-3

20:5 n-3

20:5 n-3

22:5 n-3

22:5 n-3

22:6 n-3

22:6 n-3

Sum n-3

Sum n-3 0 10 20 30 40 50 60

Dietary lipids

0 10 20 30 40 50 60 Rotifer lipids

Sum n-3 in rotifers, % of total FA 80

C 60

40

20

0 0

20 40 60 80 Sum n-3 in emulsion, % of total FA

100

Figure 4.11 Fatty acid distribution of steady-state rotifers grown at rates of 0.05–0.2 day-1. A & B Steady-state relationship between dietary (A) and rotifer (B) percentage fatty acid distribution. C. Steady-state percentage of n-3 fatty acids of rotifers as a function of the percentage n-3 fatty acid distribution of their dietary lipids (the dotted curve shows the 1 : 1 ratio).

acid profiles are almost identical; percentage DHA is slightly lower, while percentage DPA (22:5 n-3) and saturated fatty acids are slightly higher in the rotifer than in the emulsion. The total percentage of n-3 fatty acids remains almost equal. This close relationship between dietary and rotifer n-3 fatty acids is relatively independent of the n-3 content of the food, as illustrated in Fig. 4.11C. The response is close to unity and linear for n-3 contents equilibrium time, 8–10 days). 4.3.6.3 Vitamins and Minerals Information about the vitamins and minerals in B. plicatilis is scarce, but accumulating (e.g. Sandnes et al., 1994; Merchie et al., 1995; Lie et al., 1997). A high number of components are involved, and fundamental knowledge about fish larval requirements is indeed very limited. This calls for a pragmatic approach, and the use of general knowledge and indexes established for cultured fish (e.g. Lall, 1989; NRC, 1993; see also Table 8.3 in Chapter 8). As for protein and lipids, there is a relationship between the vitamin and mineral contents of the feed and the resulting contents of the rotifer. This is exemplified in Table 4.2 for the vitamin contents of rotifers grown on a number of different diets (data from Sandnes et al., 1994; Lie et al., 1997). None of the rotifers are obviously deficient in vitamins, and the water-soluble ascorbic acid and thiamin become enhanced in rotifers that feed on the microalga I. galbana in the larval tanks (see ‘green water’ technique, Chapter 7). Minerals and trace elements are also found in reasonable amounts in the rotifers. A sub-optimal supply

Table 4.2 Vitamin contents (mg g DW-1) of B. plicatilis fed different diets (data from Sandnes et al., 1994; Lie et al., 1997).

Ascorbic acid Thiamin Riboflavin Pantothenic acid Niacin Pyridoxine Biotin Vitamin B12 Vitamin A (retinol) Vitamin E (alpha-tocopherol)

1 Baker’s yeast + capelin oil

2 Baker’s yeast + Super-Selco

3 Baker’s yeast + DHA Super-Selco

4 Diet 3 + Isochrysis galbana, 72 h

267 16 23 118 167 2 3 1 2–3 mg l -1). The Artemia nauplii are harvested using suitable harvesting gear before the enrichment diet has been completely removed (12–24 h, longer enrichment periods require further additions of food). Animals are carefully rinsed, and used as live feed immediately. Prolonged post-harvest storage will require cooling facilities.

Typical response patterns for lipids and fatty acids during the first 2 days of enrichment are shown in Fig. 4.15. The lipid diets used in these two training trials, Super Selco (SS, Fig. 4.15A and B) and DHA Super Selco (DSS, Fig. 4.15C and D) differ in their contents of saturated and monounsaturated fatty acids (S + M), DHA and n-3 HUFA (the right-hand panels express percentage composition of emulsion). Lipids and fatty acids show a similar pattern of accumulation in Artemia independently of the diet used (Fig. 4.15A and C), which is to be expected because the diet rations are identical. The quantitative accumulation of DHA was highest with the use of DSS, the enrichment diet that was richest in DHA. The general pattern of variation in percentage fatty acid contents (Fig. 4.15B and D) is similar to that found for rotifers. The fatty acid composition approaches the composition of the dietary oil. Two distinct features illustrate the main differences between rotifers and Artemia. The DHA level of the Artemia tends to level-off at a value less than 50% of the level of the oil, and the response to an enhanced DHA level in the oil is therefore relatively moderate, although positive. The situation is different for EPA, which, unlike DHA, reaches

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Figure 4.15 Lipid, fatty acids and n-3 HUFA contents of Artemia franciscana as a function of enrichment time (two trials). A (Trial 1, enrichment diet Super-Selco). Total lipids, total fatty acids and quantitative contents of EPA and DHA. B. Percentage distribution of n-3 HUFA and other groups of fatty acids from trial 1. C. (Trial 2, enrichment diet DHA Super-Selco). Total lipids, total fatty acids and quantitative contents of EPA and DHA. D. Percentage distribution of n-3 HUFA and other groups of fatty acids from trial 2.

the same percentage composition in Artemia as in the feed. The differences in n-3 HUFA accumulation between the species are therefore to a great extent a result of low DHA accumulation. The fact that EPA reaches levels higher than those in the oil must be a result of animal metabolism, and experiments suggest that DHA is efficiently catabolised to EPA in n-3 HUFA-enriched Artemia during starvation. The percentage saturated plus monounsaturated fatty acids (Sat + Mono) in Artemia are reduced for both diets, but do not reach the lower level of the diets during the enrichment period, and equilibrium in fatty acid distribution is never reached. The general features and variability in Fig. 4.15 illustrate the extent of predictability and the enrichment levels that may be obtained during short-term n-3 HUFA enrichment of Artemia. It is important to note, in this regard, that short-term enrichment should be run for >12 h, but never for >48 h. There is reasonable room for manipulation of the Artemia lipid


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composition, but there are also clear constraints, in particular for DHA and total n-3 HUFA. Artemia differs from the natural food organisms of cold-water fish larvae such as marine copepods on this point (Olsen, 1999a) (see below). The combined effect of DHA catabolism and EPA accumulation in Artemia results in a markedly lower DHA : EPA ratio in Artemia than in rotifers and copepods. Further developments in enrichment techniques, the use of DHA-rich oils, and the application of extremely DHA-rich/EPA-poor oils containing phospholipids or salts of fatty acids (Tocher et al., 1997; Han et al., 2000; Harel et al., 1999) have resulted in higher percentage DHA levels and higher DHA:EPA ratios. These modifications may be feasible as long as the expenses are kept within an acceptable level for commercial larval rearing. 4.4.4.2 Stability of n-3 Fatty Acids Post-Enrichment As for rotifers, most nutritional components of Artemia decrease during starvation, and the quantitative loss rates of most components are comparable to those found for rotifers (Olsen et al., 1993; Evjemo et al., 2001). The fact that Artemia is hatched from cysts characterised by a reproducible initial biochemical composition and high energy content should imply that Artemia will not become nutritionally and energetically inadequate unless starved for a long time. However, the common commercially available strain of Artemia (A. franciscana), and most other identified strains as well, have one negative and critical property from a larval nutrition point of view. It is well documented that by some mechanism, these Artemia strains lose DHA very rapidly from their tissues, not only from their gut, after being enriched by n-3 HUFA (e.g. Dhert et al., 1993; Evjemo et al., 1997, 2001). This pattern seems to be independent of the enrichment diet and takes place for all developmental stages of the animal (Olsen, 1999b). These losses must originate in selective DHA catabolism. An enhanced lossrate of DHA compared with other fatty acids is more clearly expressed for Artemia than for B. plicatilis, which also exhibits a moderate catabolic preference for DHA over other fatty acids during starvation (see Fig. 4.13D). The rate of DHA catabolism is positively related to the temperature. Losses are very high at high temperatures, and it is notable that virtually no DHA is left after 1 day at a temperature of 25°C, which is a common temperature for warm-water species. This fact is not normally considered during larval rearing, but may be critical for some species, in particular for fast-growing carnivores in the very early stages when food is not consumed immediately after being added. Artemia cultures must be maintained at temperatures 2.5 mg O l . -3

-3

2

-1

It appears to be difficult to produce >4-day-old juveniles which also exhibit a high DHA content, but the feeding procedure above works well for younger stages, which are the optimal size groups for halibut larvae. Three-day-old A. franciscana juveniles show a satis-

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factory lipid and fatty acid content (19% lipid, about 19 mg DHA g DW-1) and a protein to lipid ratio of 3.4. The survival is obviously lower and more variable than for short-term enrichment, but is still acceptable under skilled management. The biomass per prey is more than twice as high as that of short-term enriched nauplii. High DHA catabolism is also a characteristic feature for juvenile stages of A. franciscana, and the nutritional problems encountered using juvenile Artemia are therefore more or less identical to those described for nauplii. It remains to be seen if juvenile Artemia will become used more frequently for feeding marine juvenile fish larvae such as Atlantic halibut.

4.4.6 Vitamins and Minerals Information on vitamins and minerals in Artemia is available (Léger et al., 1986; Lavens & Sorgeloos, 1996; Olsen et al., 2000). Fundamental knowledge on fish larval requirements is very limited, and information and indexes for cultured fish are needed as a reference (e.g. Lall, 1989; NRC, 1993). The mineral content of Artemia is believed to be adequate, but a recent study revealed that the selenium in cysts may be insufficient. This was reported by Van Stappen et al. (1996), who also provided data suggesting that the vitamin content of Artemia cysts is sufficient. Vitamins such as ascorbic acid may be efficiently enriched in Artemia nauplii through techniques comparable to those for lipids and n-3 HUFA. Van Stappen et al. (1996) and Olsen et al. (2000) reported that ascorbic acid and vitamin B6 are lost when starving A. franciscana. The thiamine content, however, remained, constant throughout severe starvation. The general pattern of variation for vitamins is comparable to that of n-3 HUFA, with clear effects of selective retention for some single components, and selective and faster catabolism for others. As for rotifers, a sub-optimal supply of vitamins and minerals is unlikely, but it cannot be ruled out completely. It is also clear that enhanced levels of ascorbic acid for growth may have positive effects on larval viability (Merchie et al., 1995).

4.5 Marine Copepods It is generally believed that the natural food for larvae of fish species such as turbot, cod and halibut consists of small species and young stages of marine copepods. These groups of zooplankton are available in the coastal waters of many countries, and an obvious approach in early efforts to feed marine fish larvae was to supply harvested copepods of an appropriate prey size for the fish larvae as live feed. This approach was successful to some extent in the pioneering period when the rotifer and Artemia technologies were immature, and the copepod technology was further developed and scaled-up to be applicable to the commercial production of fish juveniles (see Chapter 7). Harvested copepods are still being used in the commercial production of fish fry in northern countries, in particular for Atlantic halibut (van der Meeren & Naas, 1997). The


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zooplankton is cultured and harvested from coastal locations using automated pumping and concentration systems. The harvested, mixed zooplankton stock is further treated to isolate appropriate size fractions that are transferred to fish larvae maintained in suspended firstfeeding enclosures. This technology of first feeding has worked to some extent, but it must be regarded as premature with a restricted potential. There are several possible pitfalls using copepods, such as the transient availability of suitable prey for the larvae, and the probable transfer of parasites and diseases. However, experience to date has supported the assumption that copepods, which are believed to be the natural food of the larvae, can meet the nutritional requirements of fish larvae, and in particular the requirements for n-3 HUFA or DHA (Shields et al., 1999a). A common and sound approach has therefore been to use such copepods as a main reference during efforts to develop rotifer and Artemia technology for marine cold-water species of fish. There is a considerable database available on the biochemical composition of marine copepods (e.g. Båmstedt, 1986; Sargent & Henderson, 1986; Mauchline, 1998), and specific sampling programmes have been run to characterise the harvested and isolated copepods in hatcheries that use the copepod technology. Some examples of the fatty acid contents of common species which are actually being used are illustrated in Fig. 4.17. The values obtained for newly hatched and short-term-enriched A. franciscana and B. plicatilis enriched by different techniques are shown for comparison. The cases of rotifers illustrated are extreme: B1 is the lower extreme value for rotifers enriched during cultivation, whereas B2 shows the high extreme value for rotifers successively enriched during short-tem enrichment (see Fig. 4.12). The lipid level of the younger stages of copepods is low and relatively independent of species, although it is slightly higher for C. finmarchicus. Additional studies have clearly shown that this is a robust conclusion (Evjemo et al., 2003). The low lipid level implies a high fraction of phospholipids, which is in agreement with a ubiquitous high percentage of DHA and EPA (45–60% of total fatty acids). The high percentage n-3 HUFA in the copepods is very stable because these fatty acids, as the main functional components of membrane phospholipids, are selectively retained during starvation (see Fig. 4.16C). Artemia may incorporate larger quantities of DHA and n-3 HUFA than copepods through efficient enrichment procedures, but its tissues are notoriously fatter than those of copepods. It is difficult to obtain both a low lipid level and a high n-3 HUFA level in Artemia using the established techniques for enrichment, and the percentage contents of n-3 HUFA are lower than in copepods. The consequence is that with an adequate n-3 HUFA content, Artemia will exhibit twice the lipid content of copepods. It is not clear how this will affect larval fish growth, survival and performance. Rotifers that are enriched according to the methods presented in this chapter can, in principle, be almost identical to marine copepods. B1 and B2 (Fig. 4.17) represent normal rotifers enriched with different normal commercial emulsified oils. The use of an emulsified oil with an identical fatty acid composition as in Temora sp. and Eurytemora sp. would have resulted in rotifers with a similar fatty acid composition (see Fig. 4.11). Additional short-term enrichment could have been used to adjust the quantitative level of fatty acids and lipid if necessary (see Fig. 4.12).

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Figure 4.17 Fatty acid contents of cultivated live feed and some marine copepods commonly used as live feed for marine fish larvae (copepod data from Evjemo and Olsen, 1997). A1, newly hatched Artemia nauplii; A2, Artemia nauplii short-term enriched for 24 h by DHA Selco; B1, rotifers enriched by Super-Selco during production (a lower range of values is normally obtained); B2, rotifers enriched during growth and thereafter during shortterm enrichment with DHA Super-Selco; C. Calanus finmarchicus, copepodid stages I, II and III; T, Temora longicornis; E, Eurytemora sp.

4.6 Concluding Remarks This chapter has described experience in production techniques and methods to manipulate the nutritional value of rotifers and Artemia to be used as live feed for marine fish larvae. Some further aspects of these issues that are more closely related to first feeding are treated in Chapter 7. In addition, the microbial aspects of live feed cultures are treated in Chapters 3 and 7. It is important to emphasise that the microbial characteristics of a live feed culture are just as important as the nutritional characteristics for the successful breeding of marine


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larvae. Larval rearing is highly multidisciplinary, and success can only be obtained if all the important aspects of the live feed are adequately covered. Rotifer technology for marine cold-water fish species is well established, but the general principles and techniques will always have to be adapted to meet the requirements of a given species of fish larvae. The nutritional value of the rotifers can be controlled during production, during successive short-term enrichment, and in the phase of first feeding. The main tools have been developed, but fine-tuning of the nutritional as well as the microbial treatments for each species will be needed. The main developmental challenge is to intensify and automate the cultivation techniques. This may contribute towards a reduction in costs and the risks of production. A preliminary Artemia technology for cold-water species has been established, but further improvements are needed for some species of fish larvae. Unstable DHA after enrichment and the low efficiency of DHA incorporation from Artemia in halibut larvae are problems in the establishment of a controlled and economically feasible rearing of Atlantic halibut larvae that must be fed Artemia from the very beginning of first feeding. The commercialisation of an Artemia strain which can retain DHA in its tissues more efficiently is one countermeasure, but introducing co-feeding, or inhibiting DHA catabolism by some means, are other options. The established technology is probably optimal for cod and turbot, which are fed rotifers in the initial phase.

4.7 References Båmstedt, U. (1986) Chemical composition and energy content. In: The Biological Chemistry of Marine Copepods (eds E.D.S. Corner & S.C.M. O’Hara), pp. 1–58. Clarendon Press, Oxford. Coutteau, P. (1996) Micro-algae. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 7–48. FAO Fisheries Technical Paper No. 361. Dhert, P. (1996) Rotifers. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 49–78. FAO Fisheries Technical Paper No. 361. Dhert, P., Sorgeloos, P. & Devresse, B. (1993) Contributions towards a specific DHA enrichment in the live food Brachionus plicatilis and Artemia sp. In: Proceedings from the International Conference on Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 109–15. Trondheim, 9–12 August 1993. Balkema, Rotterdam. Evjemo, J.O. & Olsen, Y. (1997) Lipid and fatty acid content in cultivated live feed organisms compared to marine copepods. Hydrobiologia, 358, 159–62. Evjemo, J.O. & Olsen, Y. (1999) Effect of food concentration on the growth and production rate of Artemia franciscana feeding on algae (T. iso). J. Exp. Mar. Biol. Eco., 242, 273–96. Evjemo, J.O., Coutteau, P., Olsen, Y. & Sorgeloos, P. (1997) The stability of docosahexanoic acid in two Artemia species following enrichment and subsequent starvation. Aquaculture, 155, 135–48. Evjemo, J.O., Vadstein, O. & Olsen, Y. (2000) Feeding and assimilation kinetics of Artemia franciscana fed Isochrysis galbana (clone T. iso). Mar. Biol., 136, 1099–109. Evjemo, J.O., Danielsen, T.L. & Olsen, Y. (2001) Losses of lipid, protein and n-3 fatty acids in enriched Artemia franciscana starved at different temperatures. Aquaculture, 193, 65–80. Evjemo, J.O., Reitan, K.I. & Olsen, Y. (2003) Copepods as live food organisms for marine fish larvae with special emphasis on nutritional value. Aquaculture, in press.

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Fu, Y., Hirayama, K. & Natsukari, Y. (1991) Morphological differences between two types of the rotifer Brachionus plicatilis OF. Müller. J. Exp. Mar. Biol. Ecol., 151, 29–41. Fukusho, K. (1997) Nutritional effects of the rotifer Brachionus plicatilis raised by baking yeast on larval fish of Oplegnathus fasciatus, by enrichment with Chlorella sp. before feeding. Bull. Nagasaki Pref. Inst. Fish, 3, 152–4 (in Japanese). Fulks, W. & Main, K.L. (eds) (1991) Rotifer and Microalgae Culture Systems. Proceedings of a US–Asia Workshop. Honolulu, Hawaii, 28–31 January. Argent Laboratories, Washington. Fyhn, H.J. (1990) Energy production in marine fish larvae with emphasis on free amino acids as a potential fuel. In: Animal Nutrition and Transport Processes. 1. Nutrition in Wild and Domestic Animals (ed J. Mellinger), pp. 176–92. Karger, Basel. Fyhn, H.J. (1993) Multiple functions of free amino acids during embryogenesis in marine fishes. In: Physiological and Biochemical Aspects of Fish Development (eds B.T. Walther & H.J. Fyhn), pp. 299–308. University of Bergen, Bergen. Han, K.M., Geurden, I. & Sorgeloos, P. (2000) Enrichment strategies for Artemia using emulsions providing different levels of n-3 highly unsaturated fatty acids. Aquaculture, 183(3–4), 335–47. Harel, M., Ozkizilcik, S., Lund, E., Behrens, P. & Place, A.R. (1999) Enhanced absorption of docosahexaenoic acid (DHA, 22:6 n-3) in Artemia nauplii using a dietary combination of DHA-rich phospholipids and DHA-sodium salts. Comp. Biochem. Physiol., 124B(2), 169–76. Hjelmeland, K., Uglestad, I. & Olsen, Y. (1993) Proteolytic activity and post-mortem autolysis in prey for marine fish larvae. In: Physiological and Biochemical Aspects of Fish Development (eds B.T. Walther & H.J. Fyhn), pp. 229–32. University of Bergen, Bergen. Holling, C.S. (1966) The functional response of invertebrate predators to prey density. Mem. Entomol. Soc. Can., 48, 1–85. Howell, B.R. (1979) Experiments on the rearing of larval turbot, Scophthalmus maximus L. Aquaculture, 18, 215–25. Ito, T. (1960) On the culture of mixohaline rotifer Brachionus plicatilis O. F. Muller. Rep. Fac. Fish. Mie Pref. Univ., 3, 708–40 (in Japanese). King, C.E. & Miracle, R.M. (1980) A perspective on aging in rotifers. Hydrobiologia, 73, 13–19. Kitajima, C. & Koda, T. (1976) Lethal effects of a rotifer cultured with baking yeast on the larval sea bream, Pagrus major, and the increase rate using the rotifer recultured with Chlorella sp. Bull. Nagasaki Pref. Inst. Fish., 2, 113–16 (in Japanese). Korstad, J.E., Olsen, Y. & Vadstein, O. (1989a) Life history of Brachionus plicatilis fed different algae. Hydrobiologia, 186/187, 43–50. Korstad, J.E., Vadstein, O. & Olsen, Y. (1989b) Feeding kinetics of the rotifer Brachionus plicatilis fed Isochrysis galbana. Hydrobiologia, 186/187, 51–7. Lall, S.P. (1989) The minerals. In: Fish nutrition, 2nd edn (ed J.E. Halver), pp. 219–57. Academic Press, New York. Lavens, P. & Sorgeloos, P. (eds) (1996) Manual on the Production and Use of Live Food for Aquaculture. FAO Fisheries Technical Paper No. 361, pp. 295. Léger, P., Bengtson, D.A., Simpson, K.L. & Sorgeloos, P. (1986) The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biopl. Annu. Rev., 24, 521–623. Lie, Ø., Haaland, H., Hemre, G.I., Maage, A., Lied, E., Rosenlund, G., Sandnes, K. & Olsen, Y. (1997) Nutritional composition of rotifers following a change in diet from yeast and emulsified oil to microalgae. Aquacult. Int., 5(5), 427–38. Lubzens, E. (1987) Raising rotifers for use in aquaculture. Hydrobiologia, 147, 245–255. Lubzens, E., Tandler, A. & Minkov, G. (1989) Rotifers as food in aquaculture. Hydrobiologia, 186/187, 387–400.


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Makridis, P. & Olsen, Y. (1999) Protein depletion of the rotifer Brachionus plicatilis during starvation. Aquaculture, 174, 343–53. Mauchline, J. (1998) The biology of calanoid copepods. In: Advances in Marine Biology, Vol. 33 (eds J.H.S. Blaxter, A.J. Southward & P.A. Tyler), 710 pp. Academic Press, New York. Merchie, G., Lavens, P., Dhert, P., Deshasque, M., Nelis, H., De-Leenheer, A. & Sorgeloos, P. (1995) Variation of ascorbic acid content in different live feed organisms. Aquaculture, 134(3–4), 325–37. Nagata, W.D. & Hirata, H. (1986) Mariculture in Japan: past, present, and future perspectives. Mini. Rev. Data File Fish. Res., 4, 1–38. NRC (1993) Nutrient Requirements for Coldwater Fishes. Sub-Committee on Coldwater Fish Nutrition, National Research Council, Washington, DC. Øie, G. & Olsen, Y. (1997) Protein and lipid content of the rotifer Brachionus plicatilis during variable growth and feeding conditions. Hydrobiologia, 358, 251–8. Øie, G., Makridis, P., Reitan, K.I. & Olsen, Y. (1997) Protein and carbon utilization of rotifers (Brachionus plicatilis) in first feeding of turbot larvae (Scophthalmus maximus L.). Aquaculture, 153, 103–22. Olsen, Y. (1999a) Lipids and essential fatty acids in aquatic food webs. What can freshwater ecologists learn from mariculture? In: Lipids in Freshwater Ecosystems (eds M.T. Arts & B.C. Wainman), pp. 161–202. Springer, New York. Olsen, A.I. (1999b) Development of production technology of juvenile Artemia optimal for feeding and production of Atlantic halibut fry. D. Phil. Thesis, Norwegian University of Science and Technology, Department of Biotechnology, Trondheim. Olsen, A.I., Mæland, A., Waagbø, R. & Olsen, Y. (2000) Effect of algal addition on stability of fatty acids and some water-soluble vitamins in juvenile Artemia franciscana. Aquacult. Nutr., 6(4), 263–73. Olsen, Y., Reitan, K.I. & Vadstein, O. (1993) Dependence of temperature on loss rates of rotifers, lipids, and w3 fatty acids in starved Brachionus plicatilis cultures. Hydrobiologia, 255/256, 13–20. Olsen, Y., Evjemo, J.O. & Olsen, A.I. (1999) Status of the cultivation technology for production of Atlantic halibut (Hippoglossus hippoglossus) juveniles in Norway/Europe. Aquaculture, 176, 3–13. Reitan, K.I., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1993) Nutritional effects of algal addition in first feeding of turbot (Scophthalmus maximus L.) larvae. Aquaculture, 118, 257–75. Rothhaupt, K.O. (1990a) Differences in particle size-dependent feeding efficiencies of closely related rotifer species. Limnol. Oceanogr., 35(1), 16–23. Rothhaupt, K.O. (1990b) Changes of the functional responses of the rotifers Brachionus rubens and Brachionus calyciflorus with particle sizes. Limnol. Oceanogr., 35(1), 24–32. Sandnes, K., Lie, Ø., Haaland, H. & Olsen, Y. (1994) Vitamin contents of the rotifer Brachionus plicatilis. Fisk. Dir. Skr. Ernœring, 6(2), 117–19. Sargent, J.R. & Henderson, R.J. (1986) Lipids. In: The Biological Chemistry of Marine Copepods (eds E.D.S. Corner & S.C.M. O’Hara), pp. 59–108. Clarendon Press, Oxford. Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R. & Sargent, J.R. (1999a) Natural copepods are superior to enriched Artemia nauplii as feed for larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr., 129(6), 1186–94. Shields, R.J., Gara, B. & Gillespie, M.J.S. (1999b) A UK perspective on intensive hatchery rearing methods for Atlantic halibut (Hippoglossus hippoglossus). Aquaculture, 176, 15–25. Smith, L.L., Fox, J.M. & Granvil, D.R. (1993) Intensive algal culture techniques. In: CRC Handbook of Mariculture. Vol. 1. Crustacean Aquaculture, 2nd edn. (ed J.P. McVey), pp. 3–13. CRC Press, Boca Raton, FL.

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Snell, T.W., Childress, M.J. & Boyer, E.M. (1987) Assessing the status of rotifer cultures. J. World Aquacult. Soc., 18, 270–77. Sorgeloos, P., Lavens, P., Léger, P., Tackaert, W. & Versichele, D. (1986) Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Manual prepared for the Belgian Administration for Development Cooperation and the Food and Agriculture Organization of the United Nations. Artemia Reference Center, Faculty of Agriculture, State University of Ghent, 319 pp. Suantika, G., Dhert, P., Nurhudah, M. & Sorgeloos, P. (2000) High-density production of the rotifer Brachionus plicatilis in a recirculation system: consideration of water quality, zootechnical and nutritional aspects. Aquacult. Eng., 21, 201–14. Tocher, D.R., Mourente, G. & Sargent, J.R. (1997) The use of silages prepared from fish neural tissues as enrichers for rotifers (Brachionus plicatilis) and Artemia in the nutrition of larval marine fish. Aquaculture, 148, 213–31. Vadstein, O., Øie, G. & Olsen, Y. (1993) Particle-size-dependent feeding by the rotifer Brachionus plicatilis. Hydrobiologia, 255/256, 261–7. van der Meeren, T. & Naas, K.E. (1997) Development of rearing techniques using large enclosed ecosystems in the mass production of marine fish fry. Rev. Fish. Sci., 5, 367–90. van Stappen, G., Merchie, G., Dhont, J., Lavens, P., Baert, P., Bosteels, T. & Sorgeloos, P. (1996) Artemia. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 79–136. FAO Fisheries Technical Paper No. 361. Watanabe, T., Kitajima, C., Arakawa, T., Fukusho, K. & Fujita, S. (1978) Nutritional quality of rotifer, Brachionus plicatilis, as a living feed from the viewpoint of essential fatty acids for fish. Bull. Jpn. Soc. Sci. Fish, 44, 1109–14 (in Japanese). Watanabe, T., Kitajima, C. & Fujita, S. (1983) Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture, 34, 115–43. Yoshimura, K., Usuki, K., Yoshimatsu, T. & Hagiwara, A. (1997) Recent development of a high-density mass culture system for the rotifer Brachionus rotundiformis Tschugunoff. Hydrobiologia, 358, 139–44.

Chapter 5

Brood Stock and Egg Production D. Pavlov, E. Kjørsvik, T. Refsti and Ø. Andersen

Good brood-stock management and good farming practices are necessary to obtain highquality offspring in fish aquaculture. The primary requirement for the successful mass cultivation of fish is the availability of eggs and sperm of good quality. A reliable, large quantity of healthy and normal juveniles can only be obtained from brood stock which is kept under adequate environmental and nutritional conditions. In order to obtain successful gonadal growth, gamete maturation and spawning of captive fish, it is important to understand the reproductive physiology and the spawning processes of the fish. These processes are sensitive to changes in environmental conditions and to physiological stress, and how external factors may modify reproduction is important knowledge for effective brood-stock management. However, we have little or no information on controlled reproduction and recruitment for many of the approximately 1000 fish species that are cultivated. The scope of this chapter is to describe the reproductive biology of fish, with special emphasis on factors of importance for brood-stock husbandry and offspring quality of cold-water marine species in aquaculture.

5.1 Reproductive Strategies Fish reproductive patterns may be very species-specific. The more than 20 000 different fish species in the world inhabit a larger variety of habitats than any other vertebrate group. Fish can be found in polar seas and lakes, in tropical swamps, in the greatest depths of the oceans or at high altitudes in freshwater streams. They have subsequently evolved into a wide variety of forms and life styles, and so have their reproductive styles, developmental patterns and environmental needs. These different developmental patterns and reproductive styles may be classified according to spawning tactics and ecological niches for development. The Russian scientist Sergei Kryzhanovskii (1949) was the first to propose a classification based on the spawning features of some freshwater fish. Five ecological groups were classified according to their spawning substrates: lithophils (rock and gravel spawners), phytophils (plant spawners), psammophils (sand spawners), ostracophils (egg deposition inside mussels) and pelagophils (pelagic spawners). According to the main idea of Kryzhanovskii and his followers, the concept of ecological groups should not be regarded simply as

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distinct adaptations of eggs and young to environmental conditions. The adaptation of reproductive styles is reflected in the whole ontogeny, and will determine the features of adult ecology, migration and distribution. Later several classification schemes were constructed for freshwater and marine fish, and these are described in reviews by Pavlov (1989) and Balon (1990). The most comprehensive evolutionary classification of reproductive styles (or reproductive guilds, according to the author) was created by the Slovenian–Canadian scientist Eugene Balon, based on the ideas of Kryzhanovskii. The majority of known reproductive styles of fish are now included in three ethological sections, with two ecological groups in each (Table 5.1). Several reproductive groups are characterised by spawning substrate, and associated characters of eggs, embryos and larvae are included in each ecological group. Each ecological group includes several guilds (or sub-groups). For example, the ecological group (A.1) is composed of seven guilds: pelagic spawners (pelagophils), rock and gravel spawners with pelagic larvae (lithopelagophils), rock and gravel spawners with benthic larvae (lithophils), non-obligatory plant spawners (phytolithophils), obligatory plant spawners (phytophils), sand spawners (psammophils), and terrestrial spawners with the eggs scattered out of water on damp sod (aerophils). The concept of reproductive guilds reflects evolutionary lines to a certain extent. Both the succession of groups within the ethological sections and the succession of guilds in each ecological group represent a trend from a life style characterised by small unprotected eggs and high fecundity to a life style with larger eggs, lower fecundity and more complex protection of eggs and offspring. The eggs are spawned and develop independently from the parents for most fish species. Freshwater fish eggs are mostly demersal (develop on the bottom or on a substrate), whereas almost all pelagic eggs (floating freely in the water) are marine. However, parental care has developed in many species (in 3000–5000 species of teleost fishes), and care of the offspring may continue after hatching (e.g. mouth-brooding). Increased parental investment in the individual offspring will generally lead to decreased fecundity, less larval specialisations and more advanced development at hatching (see also Chapter 6). Many littoral species guard their eggs (cottids, blennies, gobies), and care is more often carried out by the male than by the female. Nests may also be built and/or one of the parents may ventilate the egg mass, e.g. the wolf-fishes (family Anarhichadidae) and the lumpsucker Cyclopterus lumpus. Viviparity has evolved independently in several groups of fish. It is a dominant mode of reproduction in cartilaginous fish. In teleosts, viviparity is widespread, but not so prevalent; about 510 species are known to be viviparous. The gestation (or ‘pregnancy’) may occur within the follicle (intrafollicular gestation) or within the ovarian cavity (intraluminal gestation). In species such as the redfish (Sebastes) and guppies (Poecilia), embryonic nutrition depends solely on yolk reserve in the eggs, and first-feeding larvae are extruded from the mother’s body (ovoviviparity). However, the redfish possess intraluminal gestation, and guppies intrafollicular gestation. Another form of parental care is viviparity, where nourishment is supplied by maternal structures, and the offspring are often born as juveniles. The majority of the species used for cold-water marine aquaculture (from the orders Pleuronectiformes and Gadiformes) produce pelagic eggs, which are scattered in the water during spawning and not protected, and they can therefore be placed in the guild A.1.1 of

Brood stock and egg production

Table 5.1

Classification of reproductive styles (guilds) in fish (modified after Blaxter, 1988 and Balon, 1990).

Ethological section

Ecological group

Reproductive guilds

A Non-guarders 1

Open and substratum spawners

1 2 3 4 5 6 7 1 2 3 4 5

Pelagic spawners Rock and gravel spawners with pelagic larvae Rock and gravel spawners with benthic larvae Non-obligatory plant spawners Obligatory plant spawners Sand spawners Terrestrial spawners, damp conditions Beach spawners, above waterline at high tides Annual spawners, eggs estivate Rock and gravel spawners Cave spawners Spawners in live invertebrates

1 2 3 4 1 2 3 4 5 6 7 8

Pelagic spawners, at surface of hypoxic waters Above-water spawners; male splashes around Rock spawners Plant spawners Froth nesters Miscellaneous substratum and materials nesters Rock and gravel nesters Glue-making nesters Plant material nesters Sand nesters Hole nesters Anemone nesters; at base of host

2

B Guarders 1

2

C Bearers 1

2

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Brood hiders

Substratum spawners

Nest spawners

External bearers

Internal bearers

1 Transfer brooders; eggs carried before deposition 2 Auxiliary brooders; adhesive eggs carried on skin under fins, etc. 3 Mouth brooders 4 Gill-chamber brooders 5 Pouch brooders 1 Facultative internal bearers; occasional internal fertilisation of normally oviparous fish; eggs rarely retained long 2 Obligate lecithotrophic live bearers; no maternal– embryonic nutrient transfer 3 Matrotrophous oophages and adelophages; one or a few eggs developing at the expense of other eggs or embryos 4 Viviparous trophoderms; nutrition partially or entirely from female via ‘placental’ structures

pelagic spawners (pelagophils). The perciform genus of wolf-fish Anarhichas, which is regarded as promising for cold-water marine farming in northern Europe and Atlantic Canada (Brown et al., 1995), possesses internal fertilisation, and the fertilised eggs are released several hours after ovulation and copulation between spawners. Owing to the internal insemination and protection of the egg mass by a parent during the development of embryos, the species of this genus should be placed in guild C.2.1, as they can be consid-

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Table 5.2

Features of egg production in different marine fish species.

Species

Fertilisation Duration of spawning season of a female (days)

Number of spawned eggs

Atlantic salmon1 Wolf-fish2 Cod3 Turbot4 Halibut5

External

10 000–15 000

Internal External External External

1 1 50–60 12–38 50–60

Number Number of Periodicity of of egg eggs in a releasing egg batches batch (¥103) batches (days) 1

5 000–50 000 1 2.5–14.5 million 10–20 10–15 million 5–16 0.5–1.5 million 4–16

1.3–6.8 10–530 – 10–350

Egg diameter (mm)

–

5–7

– 2.5–3.1 2.0–4.0 2.9–3.8

4.31–6.38 1.16–1.89 0.97–1.10 3.00–3.80

1

Gjedrem (1993). Falk-Petersen et al. (1999); Tveiten and Johnsen (1999); Moksness & Pavlov (1996). 3 Kjesbu (1989); Kjesbu et al. (1991); Iversen and Danielssen (1984); Kjørsvik (1994); Olsen (1997). 4 Jones (1974); Howell (1979); Bromley et al. (1986); Bromley et al. (2000); Fauvel et al. (1993). 5 Haug et al. (1984); Norberg & Kjesbu (1991); Kjørsvik & Holmefjord (1995); B. Norberg, personal communication, 2002. 2

ered ethologically as bearers, and ecologically as facultative internal (or lecithotrophic) live bearers. According to this classification, a short retention of fertilised eggs within the oviduct should not be considered as viviparity. The features of egg production in the different species considered in this book are summarised in Table 5.2. The species which do not protect their eggs, e.g. cod (Gadus morhua L.), turbot (Psetta maxima L.) and Atlantic halibut (Hippoglossus hippoglossus L.), show very high total fecundity or egg yield (more than 14 million, 18 million and 1.5 million eggs, respectively) and comparatively small egg diameters (Fig. 5.1a). The egg diameter of spotted wolf-fish (Anarhichas minor Olafsen) is substantially higher (5.0–6.0 mm), and absolute fecundity is less than 35 000 eggs.

5.2 Gonad Maturation In many fish species, the gonads in females are represented by paired ovaries attached to the dorsal surface of the body cavity. However, in some species only one ovary is developed. A single ovary, or paired ovaries fused in their caudal parts (as in wolf-fish), are common for species with internal insemination. In ovaries of the open type, the oocytes are moved into the body cavity after ovulation, and then are released into the water through the genital pore. This arrangement can be found in less derived teleost fish, e.g. salmonids. Ovaries of the closed type possess a cavity for the storage of oocytes after ovulation. The caudal parts of the paired ovaries transform into the oviducts, which join before reaching the genital pore. This arrangement is usual for the majority of teleost fish. In males of teleost fish, the genital system includes paired testes, and the urinary canal which runs from the urinary bladder joins with the spermiduct to form a canal opening into the urogenital pore. The testes consist of the testicular canals surrounded by a dense membrane. Two types of testes, cyprinoid and percoid, are defined, based on the position


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A

B

Figure 5.1 (a) Absolute fecundity and egg diameter (before swelling), and (b) average total length at hatching and at the beginning of the larval and juvenile states in five marine fish species with different types of ontogeny.

of the testicular canals and spermiduct. Testes of the first type have an oval form in crosssection, and the testicular canals begin from the gonad periphery and reach the spermiduct at the dorsal or dorso-medial part of the gonad. In cross-section, the testicular canals appear as ampoules of different shapes. This type is common for cyprinids, herrings, salmonids and other fish. Testes of the second type have a form which is similar to a triangle in crosssection. Radial testicular canals reach the spermiduct, which is located in the deep part of the gonad close to its dorsal surface. Testes of the latter type are found in perciform species, in some flatfish (Soleidae) and in several other species. Testes of a transitory type (between the two basic types) are also known (Makeyeva, 1992). It is therefore clear that the capacity of a fish species to produce gametes, and their fecundity, will be influenced by their reproductive styles. However, the basic principles of gonadal development and gamete maturation can be somewhat generalised. In the developing gonads, the germinal tissue will differentiate at an early stage in the life of the fish into oogonia in females and spermatogonia in males. The species considered in this book are all gonochoristic, i.e. they have the same sex throughout their whole life cycle. However, sex change is not uncommon in the fish world, whereby they first differentiate into one sex, and then later

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into the other. Species maturing first as males are called protandrous, and species maturing first as females are called protogynous.

5.2.1 Females Primordial germ cells will migrate into the ovary and form oogonia during embryonic development. When the gonad maturation process (oogenesis) begins, the female oogonia will undergo numerous mitotic cleavages and develop into previtellogenic primary oocytes (Fig. 5.2). These primary oocytes will enter the first meiotic cleavage, which is arrested at the prophase. The oocytes will then undergo a long growth period with endocytotic accumulation of yolk material. This is due to the hepatic (liver) synthesis of yolk vitellogenin (Vtg), which is a large lipoprotein molecule (MW up to 600 000). This process is called vitellogenesis. Vtg may also be modified and act as a carrier molecule for lipids, carbohydrates, phosphates and several other yolk nutrient components. The Vtg is transported by the blood to the ovary, where it is taken up by the developing oocytes through the ovarian follicle cells that surround each oocyte. Vtg is enzymatically transformed into lipovitellin and phosvitin in the oocytes. Most of the increase in gonad growth and oocyte size takes place during the vitellogenic phase (Le Menn et al., 2000). Vtg is the main component of the yolk material, being up to 80–90% of the egg yolk dry matter. Lipoproteins other than Vtg probably also contribute to the incorporation of material such

Figure 5.2 (A) Different stages of oocyte growth and maturation during the ovarian cycle of fish, and (B) follicular layers enclosing a growing oocyte. FOM, final oocyte maturation; GV, germinal vesicle (nucleus); GVM, GV migration; GVBD, GV breakdown; OD, oil droplet (Khan & Thomas, 1999). Reproduced with permission from Elsevier Science.


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as lipids into the oocytes. Vitellogenesis stops when the maturing oocytes have reached a certain size and a certain stage in development. Oocyte growth and maturation is illustrated in Fig. 5.2, and the development is well described by Khan & Thomas (1999). The final oocyte maturation (FOM) in fish is associated with the resumption of meiosis via a hormonal signal. A polar body containing a haploid set of chromosomes is shed from the primary oocyte, resulting in a haploid secondary oocyte after the completion of the first meiotic division (Wallace & Selman, 1990). The nucleus (also called the germinal vesicle) of the secondary oocyte enlarges and migrates from the centre to the animal pole of the oocyte, adjacent to the micropyle opening in the oocyte membrane (see e.g. Goetz, 1983; Kjesbu et al., 1996). The micropyle is the only opening in the egg envelope, and is just large enough to let one sperm cell penetrate during insemination. A ‘fertilisation cone’ is formed around the first spermatozoan and blocks the entrance to the micropylar canal, thus protecting the egg against polyspermi. The nucleus migration is followed by a continuation of meiosis, which proceeds to the metaphase of the second meiotic division, before it is again arrested. This stage is characterised by the breakdown of the nuclear membrane (the nucleus becomes invisible). The lipid and yolk droplets will generally coalesce and the oocyte will appear more homogenous. One or several lipid droplets may also be formed at this stage. The cytoplasm will be situated at the periphery of the oocyte, in a layer between the yolk and the eggshell (see also Chapter 6). Numerous cortical alveoli in the cytoplasm can be found towards its outer membrane (the vitelline membrane). The completion of the second meiotic division, where the secondary oocyte will transform into an egg cell, will not take place until egg activation, i.e. the discharge of cortical alveoli, causing the separation of the egg envelope (also called zona radiata, chorion or eggshell) from the vitelline membrane. The polar bodies contain a haploid nucleus but very little cytoplasm, and will degenerate rapidly. In many marine fish species, especially in forms with pelagic eggs, oocytes enlarge substantially due to hydration (a massive influx of water) during the final maturation phase (Craick & Harvey, 1987; Mangor-Jensen, 1987). This seems to be regulated by the proteolysis of the yolk proteins into free amino acids by the lysosomal enzyme cathepsin L, which results in an osmotic pressure for hydration and a dramatic increase in oocyte diameter, especially in pelagic eggs (Thorsen et al., 1993; Carnevali et al., 1999). The water content in pelagic eggs increases from 50–70% in vitellogenic oocytes to 85–95% in hydrated oocytes (Craick & Harvey, 1987; Thorsen et al., 1996; Finn et al., 2000). The ovarian follicle will rupture when the oocytes have completed the final maturation, and the oocytes are then ovulated into the ovarian lumen, or, as in salmonids, into the body cavity, where they are surrounded by ovarian fluid until spawning. The eggs are ready to be fertilised after completion of the final hydration. Both maturation and ovulation are triggered by hormonal control, but represents two comparatively independent processes (see Section 5.2.4).

5.2.2 Males Spermatogenesis is divided into four periods: (I) division, (II) growth, (III) maturation and (IV) formation of spermatozoa. Two meiotic divisions occur in the maturation period,

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resulting in the appearance of haploid spermatocytes of the second order, and then spermatids. During the final (IV) period of spermatogenesis, a spermatozoon is formed from each spermatid. When male sexual maturation approaches, primary spermatocytes are formed by several mitotic divisions of the spermatogonia. The basal membrane in the sperm-producing tubuli consists of glycogen-rich Sertoli cells, which surround and support the increasing numbers of developing spermatocytes. The primary spermatocytes will undergo the first meiotic division to haploid secondary spermatocytes, which will rapidly divide into the second meiotic division and form spermatids. Each of the primary spermatocytes will thus divide into four haploid spermatids. During the final spermiogenesis, the spermatids will develop into spermatozoa, and the mature spermatozoa are released to the sperm ducts (spermiation). This process is generally followed by milt hydration, in which there is an increase in the water content of the seminal fluid–spermatozoa suspension. Cell size decreases to approximately half from the secondary spermatocyte to the spermatid, with a subsequent decrease to the spermatozoon. The sperm can be released in a short time in some species, or continuously as several spermatogenic waves in others. The latter type is more common for batch-spawning species (Makeyeva, 1992). In most fish with internal insemination, the spermatozoa are grouped into spermatozeugmas or spermatophores. These are transferred through the anal fin that is transformed into a gonopod. In some species, including fish from the Anarhichadidae family, the spermatozoa remain free and are transferred through a primary (urogenital papilla) copulative organ (Billard, 1986; Makeyeva, 1992). Fish spermatozoon morphology is very species-specific, and depends on the taxonomy, as well as on the reproductive biology of the species (Ginsburg, 1968; Turdakov, 1972; Baccetti, 1984; Emelyanova & Makeyeva, 1985; Jamieson, 1991; Mattei, 1991; Makeyeva, 1992). The spermatozoon includes an egg-shaped head, which consists mainly of the haploid nucleus. It also has a middle section with numerous mitochondria to produce the necessary energy for swimming, and a flagellum (tail), which for teleost fish has a typical flagellatestructure (two central tubuli surrounded by nine double tubuli). The teleosts lack an acrosome for the penetration of the spermatozoon through the egg envelope because of the presence of a micropyle, but possess specialised structures on the plasma membrane at the top of the spermatozoon head (Billard et al., 1995). Once the sperm has been shed, or as soon as it comes in contact with the water surrounding the fish, it will become activated and is very short-lived. Therefore, great care has to be taken not to allow the sperm to come into contact with water during the stripping procedure or during sperm storage.

5.2.3 Spawning Once or Many Times? Different species have different spawning strategies, and this will be reflected in variations in the gonad maturation patterns. Some, such as the Pacific salmon or the anguillid eels, may spawn once in a life-time (semelparous species). Others may spawn more than once (iteroparous species), and most of these iteroparous fish will typically have one spawning season per year.


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Figure 5.3 Patterns of gamete development in fish with synchronous, group synchronous or multiple group synchronous ovarian development (Pankhurst, 1998).

Depending on the different spawning modes of the species, three different types of gonad development can be recognised: synchronous, group synchronous and multiple group synchronous (the latter is also called asynchronous) (reviewed by Tyler & Sumpter, 1996; Pankhurst, 1998). The gonad development pattern of males will follow the female type of oocyte development. The semelparous species only need to develop gonads once in their lifetime. The females all have one homogenous group of developing oocytes in the ovary, and this is described as synchronous ovarian development (Fig. 5.3). The iteroparous species will develop gonads several times during their life-span, and consequently their ovaries will contain a population of previtellogenic oocytes as well as developing oocytes that will be ovulated during the forthcoming spawning season. Such gonad development is called group synchronous (Fig. 5.3), and is found in a number of high-latitude and temperate species as well as in many deep-water fish. The wolf-fish (Anarhichas), the species of the genus Macrozoarces, the Atlantic herring (Clupea harengus), and the iteroparous salmonids all have group synchronous oocyte development: all vitellogenic oocytes grow in unison, particularly in the few weeks before ovulation, causing ovulation of all vitellogenous oocytes in a short time period, and the subsequent release of a single egg clutch. However, there is a further difference between fish spawning once per year and those spawning several batches of eggs per spawning season. This last group demonstrates multiple group synchrony (they are also called asynchronous) (Fig. 5.3), where previtellogenic

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oocytes are present together with several different stages of maturing vitellogenic oocytes. This is the most common pattern of gonad development among teleost fish, and is typical for tropical and warm temperate marine and fresh-water fish, as well as for some cold temperate species. The majority of marine fish species with pelagic eggs (e.g. cod and flatfish) have multiple group synchronous oocyte development, with subsequent spawning of multiple egg batches over a period of time (see Table 5.2 and references therein). A high variation in the number of egg batches is observed in different species and in different females of one species. The ovulatory rhythms are species-specific, and individual variations are observed in some species. The varying numbers of egg batches and the variable fecundity within a species are mostly due to fish size and environmental conditions, such as temperature, feeding conditions, the presence of stress factors, etc. The fecundity of fish is generally inversely correlated to the egg size; the more eggs a species produces, the smaller the eggs will be. Demersal eggs are generally larger than pelagic eggs, and the mean size of a pelagic egg is around 1 mm in diameter (Blaxter, 1988; Kendall et al., 1984). The halibut therefore produces some of the larger pelagic eggs (3 mm). Within a species, a large female will generally produce larger eggs than a smaller female. One factor complicates this picture: species producing multiple spawnings per season generally produce progressively decreasing egg sizes towards the end of the spawning season. For cod, a large fish will also produce larger eggs than a small fish, but the seasonal difference in egg size within a single female is generally larger than differences in egg size between females of different size. If egg size is to be studied in relation to fish size, the same spawning stage of the fish must be compared.

5.2.4 Endocrine Regulation Reproduction is controlled by internal (endogenous) rhythms, which are stimulated by environmental influences such as light, temperature and nutritional factors. In temperate waters, the seasonal changes in daylength are important cues for the onset of gonad maturation, thus making it possible to produce offspring during the season offering the best chances of nutrition and survival for the larvae and juveniles. The reproductive cycle is regulated mainly by hormonal production, and is controlled by the so-called brain–pituitary–gonad axis. Gonad maturation is really an orchestrated cascade of events, and it is also a beautiful example of how finely tuned and vulnerable physiological processes are. Light affects the internal rhythms of fish through the eyes and through the pineal gland in the uppermost part of the forebrain. The pineal gland secretes the hormone melatonin, and the levels fluctuate according to light levels, with the greatest production during darkness. Fish in temperate waters are generally sensitive to changes in daylength (and in tropical areas even to lunar cycles), and the resulting seasonal changes in melatonin will probably act together with the endogenous rhythm of the fish to stimulate the production of gonadotropin-releasing hormones (GnRH) in the hypothalamus. GnRH is a key regulator in reproduction for all vertebrates, although the possible role of melatonin in fish reproduction is still speculative (Mayer et al., 1997; Bromage et al., 2001). The GnRH is transported to the pituitary gland (which is attached to the hypothalamus), where it will induce sexual


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maturation by stimulating special neuro-secretory cells in the pituitary to produce and secrete two different gonadotropin hormones, namely follicle stimulating hormone (FSH, also called Gth-I) and luteinising hormone (LH, also called Gth-II). FSH and LH are structurally similar to mammalian gonadotropins (Swanson, 1991; Tyler et al., 2000). These hormones are transported by the blood to the gonads, where in females they are bound to specific surface receptors for the different hormones in the follicle cells surrounding the oocytes. FSH appears to be involved in vitellogenesis (Tyler et al., 2000), whereas LH seems to be responsible for oocyte maturation and ovulation (Swanson, 1991; Nagahama, 2000). Thus, FSH is present in the blood during the early stages of gonad development, whereas LH is present during the maturational phase. The secretion of these two gonadotropins is also regulated differently. The FSH hormone regulates the production of maturing oocytes, and is secreted during most of the maturation cycle, with increasing blood plasma levels up to final maturation. FSH will stimulate the ovarian follicle cells to synthesise the androgens and oestradiol-17b. Rising levels of oestradiol-17b and testosterone thus signal active development of the ovaries. Oestradiol-17b and testosterone are synthesised in a twocell model (Fig. 5.4), where the theca cells synthesise testosterone in response to FSH, and oestradiol-17b is subsequently produced in the granulosa cells from testosterone, which is aromatised by cytochrome P450 aromatase (Kagawa et al., 1982; Nagahama, 2000; Patiño et al., 2001). Oestradiol-17b is transported by the blood to the liver and stimulates hepatic production and secretion of the yolk protein vitellogenin (Vtg) and ‘zona radiata’ proteins (material for making the egg envelope, or chorion). These proteins are transported from the liver by the blood to the ovaries. The ‘zona radiata’ proteins are deposited around the oocyte (OppenBerntsen et al., 1992; Tyler et al., 2000), and Vtg is actively sequestered by receptor-mediated endocytosis through the oocyte membrane (Wallace & Selman, 1990; Specker & Sullivan, 1994; Mommsen & Walsh, 1998). The secretion of FSH and LH is regulated by feedback mechanisms of oestradiol-17b and testosterone (Peter & Yu, 1997). In addition, oestradiol17b will stimulate the differentiation of female sex characters, whereas testosterone also functions in the final maturation of the oocytes and as a sexual pheromone both for females and males. The largest increase in oocyte size occurs during vitellogenesis, and this process may last for several months. Blood plasma levels of Vtg, oestradiol-17b and testosterone will increase gradually up to final maturation. The final maturation, ovulation and spawning are regulated by the luteinizing hormone (LH), and plasma levels of LH increase rapidly up to spawning. Ovulation can be described as the release of a mature oocyte from the follicle wall in the ovary. LH stimulates the gonads to produce progesterone hormones called the maturationinducing steroids (MIS), which initiate the final oocyte hydration (Nagahama, 2000). The stimulation of final maturation by MIS seems to occur by the activation of a maturation-promoting factor that consists of cyclin B and a catalytic kinase. MIS stimulates the synthesis and action of cyclin B by binding to a membrane-bound receptor on the oocyte surface (see e.g. Nagahama, 1987). MIS also stimulates the follicle cells in the ovary to produce prostaglandins. Prostaglandin F2a will induce contraction of the ovaries, and thus be responsible for ovulation. Prostaglandin and MIS may also stimulate the spawning

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Liver Vitellogenin & Zona radiata proteins

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Figure 5.4 Oocyte growth and final maturation (FOM). During oocyte growth, oestradiol-17b and testosterone are synthesised in a two-cell model, where the theca cells in the ovarian follicle synthesise testosterone in response to the follicle stimulating hormone (FSH, produced in the pituitary), and oestradiol-17b is subsequently produced in the granulosa cells (also in the follicle) from testosterone, which is aromatised by cytochrome P450 aromatase. Oestradiol-17b is transported by the blood to the liver, and stimulates hepatic production and secretion of the yolk protein vitellogenin (Vtg) and zona radiata proteins (ZRP, material for making the egg envelope or chorion). These proteins are transported from the liver by the blood to the ovaries. The ZRP is deposited around the oocyte and Vtg is actively incorporated into the oocyte by receptor-mediated endocytosis. The final oocyte maturation (FOM), ovulation and spawning is regulated by the luteinizing hormone (LH), and plasma levels of LH increase rapidly up to spawning. LH stimulates the gonads to produce progesterone hormones, which initiate the final oocyte hydration. See text for a further explanation. Figures by Geir Lasse Taranger, Institute of Marine Research, Bergen.


Two cell model - oocyte growth


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Figure 5.5 Plasma levels of vitellogenin (VTG), oestradiol-17b (E2) and testosterone (T) in laboratory-held mature female halibut prior to and during the reproductive period. Values are means +1 SE. Missing standard error bars are too small for presentation (Methven et al., 1992).

behaviour of both females and males (by acting as a post-ovulatory pheromone), and thus synchronising the spawning act between the sexes. Female release of MIS and prostaglandins into the water is registered by the males, which are reacting with increasing levels of male LH and a subsequent increased production and release of sperm. Several other factors are also up- and down-regulated in the ovaries around ovulation, and these are described by Goetz and Garczynski (1997). In fish with synchronous or group synchronous oocyte development, such as salmonids and wolf-fish, there is only one peak in the levels of Vtg and of the different steroid hormones, and then the levels decline gradually during the month preceeding ovulation (Jobling, 1995; Tveiten & Johnsen, 1999). For species with multiple group synchronous gonad maturation, such as cod and Atlantic halibut, the levels will increase up to the commencement of spawning. The levels of Vtg and the different hormones will then oscillate during the spawning season, indicating that vitellogenic oocytes are developing in the ovary at the same time as mature oocytes are ready to undergo final maturation (Fig. 5.5). During male maturation, the pituitary gonadotropins LH and FSH will regulate steroidogenesis and spermatogenesis by activating receptors in the Leydig cells (LH receptors) and in the Sertoli cells (FSH receptors) (Schulz et al., 2001). The Leydig cells are responsible for the production of the steroid testosterone and its derivative 11-ketotestosterone (Nagahama, 1994). In male teleosts, the growth and development of the testes is thus associated with rising plasma levels of testosterone and 11-ketotestosterone. 11-ketotestosterone seems to be responsible for regulation of the spermatogenesis (development of sperm cells), it stimulates the development of secondary male sexual characters, and may also have growth-promoting effects (Borg, 1994; Nagahama, 1994). Males are very sensitive to the female secretion of MIS, which will stimulate sperm maturation and release, and also stimulate male production of MIS rather than 11-ketotestosterone. Male MIS is probably synthesised in the sperm cells, although the surrounding testicular tissue is necessary.

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Induced ovulation and spawning include injections of pituitary extracts, chorionic gonadotropin (HCG) and synthetic hypothalamic-releasing hormones (see e.g. Zohar & Mylonas, 2001). The injections of gonadotropin-releasing hormone agonists (GnRHa) is an effective alternative to pituitary extracts and HCG in the induction of oocyte growth, maturation and ovulation in a range of fish species. The gonadotropins can be under the control of an inhibitory hormone (dopamine). Treatment with antagonists of dopamine together with injections of synthetic analogues of GnRH leads to an enhanced release of gonadotropins. It is important to note that hormonal stimulation often leads to maturation and ovulation, but does not induce natural spawning. For the latter process, special environmental conditions are required. The majority of cold-water marine fish species which are regarded as interesting for aquaculture will undergo ovulation without hormonal stimulation, under controlled artificial environments in captivity. Implants of GnRHa were applied to males of the Atlantic halibut to synchronise the time of spermiation and egg ovulation (Vermeirssen et al., 1998). Without such injections, spermiation often commences about 1–2 months prior to female ovulation. However, the most interesting application of the GnRH implants was that they improved milt fluidity late in the spawning season, when milt is often difficult to use because of its very high viscosity.

5.3 Brood-Stock Management and Egg Production The environmental conditions of fish (e.g. temperature and feeding) may be not optimal for their development and growth in the wild, at least not during part of the year, but they should be close to optimal in captivity. However, the artificial conditions (unusual environment, including crowding, light, temperature, feeding and the absence of caves) may represent a stressful situation, which is most severe at the beginning of the establishing phase of the brood stock from wild populations. Conditions suitable for spawning (e.g. special habitats) are almost never reached in marine farms, which causes problems for natural spawning in captivity (i.e. courtship behaviour and spawning rituals, followed by the release of fertilised eggs). The term ‘natural spawning’is used in this chapter to describe the ovulation and release of eggs and milt by the fish without hormone treatment or stripping of the fish to obtain gametes. Stress may affect the reproductive process in fish, and was recently reviewed by Schreck et al. (2001).The stress response depends on the species of fish, the stage of maturity and the duration of the stress factor(s). According to the definition of Schreck et al. (2001), stress is ‘the response of the body, i.e. a physiological cascade of events that occurs when the organism is attempting to resist death or re-establish homeostatic norms in the face of an insult’. The stressors induce the pituitary synthesis and secretion of corticotropic hormones, which in turn stimulate the synthesis and secretion of cortisol, a glucocorticoid hormone. There is a possible direct link between gonadotropins (and their influence on egg maturation and reproduction) and stress hormones. The influence of stress can be manifested directly in the reduced survival of adult fish. The secondary effect of stress is the immunosupression that renders the fish vulnerable to pathogens owing to the action of cortisol, which depresses the ability of fish leucocytes to form antibodies. Under adverse conditions, a female can select between energy allocated for


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somatic growth and energy for reproduction. In the first case, the resorption of the ovary and egg atresia can occur. There are several indications of reduced egg quality associated with unfavourable maintenance of brood fish (see review by Brooks et al., 1997). In particular, for spawning cod separated into pairs in small tanks, up to one-third of the fish showed signs of stress resulting in irregular spawning intervals and poor egg quality caused by over-ripening (Kjesbu, 1989; Kjørsvik & Holmefjord, 1995). Nevertheless, the females apparently possess a buffering mechanism which protects their eggs from the negative effects of stress (Schreck et al., 2001). For instance, a number of eggs can be resorbed, and the energy allocated to the rest of the developing oocytes. Parental stress may also cause reduced viability and survival of the offspring (see e.g. Morgan et al., 1999). However, little is known about such problems, and much more work remains to be done in this area.

5.3.1 Brood-stock Nutrition Brood-stock nutrition will affect the viability and health of the offspring as well as that of the brood-stock fish, and feeding the brood-stock a diet to fulfil its optimal reproductive potential is therefore of vital importance. Most work on the nutrient requirements of broodstock fish is limited to a few species (mainly salmonids and sparids). However, it is clear that many problems in fish culture, including low fertilisation rate and poor egg and larval quality, are directly related to the diet composition of the brood stock. Sexually maturing fish (and other animals) generally have increased requirements for specific nutrients (i.e. somewhat different nutritional demands than during the grow-out phase), and on-growing feeds are often not adequately covering the dietary requirements of broodstock. Only a few commercial brood-stock diets have been developed for cold-water marine fish aquaculture owing to the short history of this industry, and to the present small production volume of these species. It is often reported that brood-stock fish fed on ‘natural diets’ produce eggs of better quality than those on formulated commercial diets (Brooks et al., 1997). The nutrition of the brood-stock can be improved by feeding marine fish solely on fresh marine organisms (squid, cuttlefish, mussels, krill and small crustaceans), or marine organisms in combination with commercial diets. However, the use of unprocessed products does not always provide adequate levels of nutrients, and also increases the risk of disease transmission. At the same time, the nutritional quality of commercially formulated feed designed for each species could be substantially improved. At present, feed is the largest production cost for commercial aquaculture, and research to develop substitutes for fish oil and fish meal is now focused on oil seeds (especially soybeans), meat by-products and microbial proteins (see also Chapter 9). However, vegetable proteins have an inappropriate amino acid balance, and they contain phytoestrogens. A brood-stock diet based on soybean meal could thus reduce nutrients which are essential for reproduction (Izquierdo et al., 2001), and phytoestrogens may have an adverse effect on reproductive development in both males and females. For fish, the primary driving force for reproduction is generally reflected in the investments made during gonad growth, and this is especially so for females. Many species, such

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as salmon and cod, tend to reduce (or stop) their feed intake during gonad maturation and/or spawning, and the energy and nutrients necessary for gonadal growth must be taken from their body reserves. A wild female salmon uses almost 90% of her fat and 50% of her muscle protein to build up the gonads. Other species, such as the gilthead seabream (Sparus aurata L.), will continue to feed throughout gonad maturation and the spawning period, and will therefore rely on both body reserves and diet to produce eggs (the egg biomass is often greater than their own body weight). Brood-stock nutrition is important in terms of both the quality and the quantity of the feed, but we know relatively little of specific brood-stock requirements for each species. When considering the effects of brood-stock nutrition on egg and sperm quality and offspring viability, knowledge of the reproductive pattern of the species in question is very important. It is of particular importance to know the timing and duration of the gonad maturation period (especially vitellogenesis), as this is the time when most nutrients and energy are incorporated into the oocytes. Also important is the reproductive strategy (synchronous, group synchronous or multiple group synchronous), the duration of the spawning period, and the feeding pattern of the fish during gonad maturation and spawning. These parameters will regulate how and when dietary brood-stock nutrients must be available to build up the gonads, and whether we must rely on the body reserves of the fish, or if we may use direct feeding during maturation and spawning to ensure optimal egg quality. Fish will then not be limited to using dietary nutrients during gonad maturation and spawning, and body reserves can be mobilised from different tissues for gonadal growth. A good brood-stock diet and feeding regime must therefore result in a good status of the body stores before the beginning of vitellogenesis. The cod, sea bass (Dicentrarchus labrax L.), gilthead seabream and wolf-fish species are all different types of spawner, and have thus different requirements in terms of when the nutrients required for gonad development should be available. Wolf-fish vitellogenesis starts approximately 3 months prior to spawning, and all eggs are shed in one batch (Tveiten & Johnsen, 1999). In cod, which is a continuous spawner with no feeding activity during spawning, ovarian development starts 8–9 months prior to the onset of spawning, and nutrients are incorporated into the oocytes up to the final maturation of the different egg batches (Kjesbu et al., 1991). A similar pattern is seen for halibut and turbot. Gilthead seabream is also a continuous spawner (although not in cold water), but will continue its feeding activity during the spawning period. In this species, ovarian development starts 2 months prior to spawning, and feeding high-quality diets for 60 days prior to and throughout spawning has resulted in a positive effect on spawning results. However, even this period seems too short to fully alter the egg fatty acids composition, and it is now recommended to feed bream a special brood-stock diet for at least 3 months prior to, and throughout, the spawning period (Almansa et al., 1999). 5.3.1.1 Ration Size Food restriction may lead to an inhibition of gonad maturation, and feeding rations and growth rate have a significant effect on the size and potential fecundity of the fish. As shown for cod,


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feeding conditions have a major influence on the fecundity and proportion of pre-vitellogenic oocytes, whose number is regulated by means of atresia. In addition, egg size decreases more rapidly during spawning in starving fish, and the egg viability is lower (Kjesbu et al., 1991; Kjørsvik, 1994; Karlsen et al., 1995). Growth during the months prior to spawning has been found to affect the time of spawning in cod, with well-fed fish commencing spawning earlier than less well-fed fish (Kjesbu et al., 1996, 1998). Cod with high condition factors produce more pre-vitellogenic oocytes than fish of the same size deprived of food. In general, rations less than 100% will not affect egg viability and fry survival, but will decrease fecundity, possibly delay the time of spawning, and reduce the proportion of fish that mature (Springgate et al., 1985; Cerdá et al., 1994). Rainbow trout egg size and initial larval growth seem to be affected by brood-stock ration size, but not egg and fry survival. In cod, however, female fecundity was affected by ration size, but no effects were found in egg size. The degree of atresia is correlated to the condition factor of the fish (Kjesbu et al., 1991). When cod were exposed to periodic starvation during vitellogenesis, with weights being reduced to around 60% of those fed full rations, the observed differences in fecundity were only due to differences in fish size, and the relative fecundity was the same for all fish. In other experiments, in pre-spawning starvation of recruit spawners of cod for up to 9 weeks, almost all fish matured (Karlsen et al., 1995). Starvation for 9 weeks did not affect spawning time or the relative weight of the testes, but did affect ovary weight. In addition, the mean fecundity of the fish starved for 9 weeks was significantly lower than that in the well-fed control group. The difference in the results of the effect of starvation on cod egg production between this study and the previous investigations can be explained by a different sensitivity to food variation during vitellogenesis of repeat and recruit spawners. In several marine fish species, reduced fecundity is related to the influence of a nutrient imbalance on the endocrine system, or to a restriction in the availability of biochemical components for egg formation. In sea bass, reduced rations seem to result in a reduced fecundity and delayed spawning period, but not in reduced egg quality and larval viability (Cerdá et al., 1994). Feeding of brood-stock should be to satiation, according to the daily appetite of the fish. Depending on the species, it is important to maintain an adequate food supply until vitellogenesis or spawning is completed. Reductions in feed rations during the later stages of maturation or spawning may result in atresia (resorption of oocytes), and thus reduced fecundity. 5.3.1.2 Feed Composition Fish seem to be very flexible in their ability to adapt to different levels of macro-nutrients such as protein and fat without a reduction in reproductive performance. However, this flexibility seems to have certain limits, which are different from species to species. The important components of the diet, which may determine egg viability, are the lipids, essential polyunsaturated fatty acids (PUFA), protein, vitamins, carotenoids and various trace elements (see reviews by Kjørsvik et al., 1990; Bromage, 1995; Brooks et al., 1997). For rainbow trout, the dietary protein levels should be at least 33%, whereas for red sea bream (Pagrus major (Temmink et Schlegel)), gilthead seabream and seabass it is recom-

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mended to keep the brood-stock dietary protein level at a minimum of 45% to prevent a reduced egg quality (Watanabe, 1985; Cerdá et al., 1994; Tandler et al., 1995; FernándezPalacios et al., 1997). Cod and halibut have a protein demand that is more similar to that seabream and seabass than to salmonids (Lie et al., 1988). Large variations in dietary lipid levels seem to have little effect on reproductive performance for salmonids or for bass and bream (10–30%). However, there are specific requirements for certain essential fatty acids during gonad maturation, and therefore very low lipid levels in the feed may not be adequate. An elevated carbohydrate level may have a positive effect on reproductive performance. Glucose is an important energy source for maturing gonad tissues, and an improved glucose tolerance has been observed in fish during vitellogenesis. Dietary carbohydrate levels between 5 and 28% did not affect cod brood-stock growth, feed conversion rate or gonadal development, possibly because the requirements for protein and lipids were met (Hemre et al., 1995). However, too high dietary carbohydrate levels seems to affect egg quality negatively, by increasing the ovarian lipid stores. 5.3.1.3 Fatty Acids The (n-3) and (n-6) essential fatty acids (EFA) are very important for normal reproduction and the egg development, and the egg viability of several marine fish species have been improved by altering the lipid composition of brood-stock diet (see Fig. 5.6, with data from Watanabe, 1985; and reviews by Sargent, 1995; Rainuzzo et al., 1997). Such a deficiency will affect fecundity, egg quality, hatching success and the number of normal larvae, and will lead to deformations in the juveniles that are produced. When brood-stock of gilthead seabream were fed an EFA-deficient diet for 2 months prior to spawning, the spawning period

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Figure 5.6 Red seabream and gilthead seabream egg and larval quality from brood-stock fed different levels of essential fatty acids (EFA) and different protein levels. Values are related to results obtained in fish fed a control high-protein and high-EFA diet. Buoyant eggs were defined as fertilised, and normal eggs were classified according to the number of oil droplets. (a) Gilthead seabream, from the last part of the spawning period (data from Almansa et al., 1999); (b) red seabream (data from Watanabe, 1985). See a further explanation of the results in the text.


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was reduced by 50%, and egg quality was significantly reduced towards the end of the spawning season (Fig. 5.6a). In general, a certain level and a correct balance between the three essential fatty acids docosahexaenoic acid (DHA, 22:6(n-3)), eicosapentaenoic acid (EPA, 20:5(n-3)) and arachidonic acid (ARA, 20:4(n-6)) seem to be important for successful reproduction and embryonic development (Watanabe, 1985; Luquet & Watanabe, 1986; Hardy et al., 1989; Thrush et al., 1993; Harel et al., 1994; Fernández-Palacios et al., 1995, 1997; Almansa et al., 1999; Navas et al., 1997; Bell et al., 1997). DHA plays a fundamental role in embryonic development, especially for the development of membranes and of brain and nerve tissues. A high DHA supply seems particularly important for the rapidly growing pelagic marine fish eggs and larvae, as these larvae have a high percentage of neural tissues in a relatively small body mass. Certain dietary nutrients, in particular EPA and ARA levels, show a correlation with fertilisation rates. Two possible explanations of this effect are (a) sperm fatty acid composition (and sperm motility) depends upon the essential fatty acid content of the brood-stock diet, and (b) both EPA and ARA modulate steroidogenesis in the testis, and the timing of spermiation may be delayed and the fertilisation rate reduced in conditions of a deficiency of these fatty acids (Izquierdo et al., 2001). ARA has recently been recognised as an essential fatty acid for egg quality in fish. ARA is a precursor for prostaglandins, which are important in the final maturation of oocytes. Prostaglandins also act as a pheromone for the stimulation of male sexual behaviour, and for the synchronisation of male and female spawning. The dietary ARA level may thus have a direct impact on spawning behaviour, and thus fertilisation success, in fish. However, high EPA levels compared with ARA levels may inhibit the production of eiconoids derived from ARA, which exemplifies the importance of a good balance between EPA and ARA in marine brood-stock diets. 5.3.1.4 Micronutrients Several vitamins and minerals are important for fish reproduction, and the most important are discussed in this section. Vitamin E is important for the control of reproduction, testes function, macrophage function and intracellular oxidation in mammals and fish. A deficiency of dietary vitamin E will affect the number of spawning fish as well as hatching success and juvenile survival (Watanabe, 1985). This vitamin is the most important of the cellular fatsoluble antioxidants in the body, and has a stabilising effect on the embryonic membranes. Vitamin E is transported to the oocytes by lipoproteins, and the main carrier seems to be the low-density lipoproteins (LDL) and not vitellogenin. Varying vitamin E levels in brood-stock feed has received special attention, because this vitamin is closely linked to lipid metabolism. The level of vitamin E in the gonads increases during vitellogenesis, and this level reflects that in brood-fish muscle prior to the start of gonad growth in salmon and turbot (Lie et al., 1994; Hemre et al., 1994). The egg concentration of vitamin E (measured as a-tocopherol) closely resembles the dietary levels in the brood-stock feed, and a positive effect from vitamin E on egg hatching success and juvenile viability has been demonstrated for several species. This is shown in

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Vitamin E supplement in broodstock feed (mg/kg) Figure 5.7 An appropriate content of vitamin E is necessary in the brood-stock feed to ensure normal embryo development and larval survival in Atlantic salmon. A low content of vitamin E in relation to the total content of n-3 HUFAs in the brood-stock feed resulted in decreased hatching success and larval survival from hatching to start-feeding (data from Rønnestad & Waagbø, 2001).

Fig. 5.7 for salmon (data from Rønnestad & Waagbø, 2001), where dietary vitamin E levels increased from 50 to 250 mg/kg resulted in significantly improved egg and fry survival when the fish were fed high levels of polyunsaturated fatty acids. Marine brood-stock diets normally contain high levels of polyunsaturated fatty acids, and should thus contain vitamin E levels that secure effective prevention of in vivo lipid oxidation. Vitamin C (ascorbic acid) requirements tend to increase during sexual maturation, and the deposition of ascorbic acid in the growing oocytes is important for the hydroxylation of protein-bound proline and lysine to give optimum collagen strength throughout the embryonic stages (Waagbø et al., 1989; Blom & Dabrowski, 1995). This vitamin is an important anti-oxidant, and is vital for bone and cartilage tissue formation and for the non-specific immune system. A deficiency will result in reduced egg production and reduced egg and sperm quality. The hatching success of rainbow trout, seabass and seabream were found to be dependent on the brood-stock dietary level of ascorbic acid, and vitamin C in the eggs may be transferred to the larva and support normal development if the start-feed is deficient in this vitamin (Sandnes et al., 1984; Soliman et al., 1986; Blom & Dabrowski, 1996). Despite observed differences in the free amino acid profiles of eggs, egg strength and neutral buoyancy, no effect on fertilisation or hatching rates were detected for cod eggs from brood stock fed a low vitamin C diet (Mangor-Jensen et al., 1994). Ascorbic acid also has an important role in sperm quality, as it prevents oxidative damage to sperm cell DNA and thereby maintains the genetic integrity of the sperm cells (Dabrowski & Ciereszko, 1996). Too low a level of ascorbic acid will reduce sperm concentration and motility, which will reduce fish fertility, and it has been associated with a high percentage


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of abnormal offspring in rainbow trout (Ciereszko & Dabrowski, 1995; Ciereszko et al., 1999). 5.3.1.5 Pigments and Minerals Fish are unable to synthesise carotenoids, and so obtain them from their diet. The yellow, orange or red colours of the eggs of teleost fish are caused by the presence of carotenoids in the yolk, and intuitively the most intensely coloured eggs are often regarded as eggs of high quality. Within a species, the eggs of fish from the wild often have more intensive coloration and larger carotenoid content than the eggs from cultured brood stocks. However, carotenoid content measured quantitatively by a spectrophotometric method does not necessarily relate to the visual intensity of the pigment. For example, the eggs of wild Atlantic salmon, which contain astaxantin as their only pigment detectable by thin-layer chromatography, seem more intensively coloured to the eye than do the eggs of farmed Atlantic salmon, which contain only canthaxantin. Pelagic fish eggs are usually almost colourless, and demersal eggs possess more or less intensive coloration of the yolk. The difference is often explained as possibly based on the respiration function of the carotenoids: the oxygen supply of pelagic eggs is much better, and the presence of a large amount of pigment is not required. However, there is an alternative explanation of this phenomenon. In pelagic eggs, the colour of carotenoids represents a disadvantage by attracting predators, and they become transparent by converting the carotenoids into a colourless form. For example, the unripe ovaries of cod are bright orange or yellow due to the presence of carotenoids, but mature eggs are colourless. Vitamin A and astaxanthin are important for cell proliferation (growth), for the function of epithelial cells in the ovaries and testes (and other organs), and for the vision. Deficiencies in the brood-stock may lead to reduced fertility and offspring deformities. Little is known about the effects of brood-stock nutrition with this vitamin, but it is suggested that requirements for vitamin A can be covered by carotenoids, and in particular astaxanthin. Astaxanthin is a pro-vitamin A, and is an important antioxidant. It is also suggested that astaxanthin plays a role in the respiration processes in eggs. The possible functions of carotenoids in fish eggs have been discussed over a prolonged period (Craik, 1985; Mikulin, 2000). There are some obvious functions of carotenoids in eggs. They are the source of external pigment for chromatophores of the skin in larvae and juveniles, and the precursors of the vitamin A involved in light reception in the eye. The carotenoids may play some part in oxidative metabolism under conditions of environmental shortage of oxygen. The connection between carotenoid content and egg quality has mainly been studied on salmonid fish. High fertilisation and hatching rates can be achieved over a wide range of carotenoid levels. However, the egg quality substantially decreases when carotenoid content reaches a ‘critical level’. Astaxanthin is effectively transferred from brood-stock feed to eggs in salmonids and in cod (Grung et al., 1993), and positive effects of dietary astaxanthin on egg quality were found for red seabream and yellowtail (Watanabe & Miki, 1993; Verakunpiriya et al., 1997a). In rainbow trout (Oncorhynchus mykiss Walbaum), the carotenoid content may exceed 13 mg g-1, but the critical level seems to be 1–3 mg g-1. The carotenoid composition obviously has a great influence on egg quality,

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but the nature of the carotenoids necessary for the maintenance of high egg quality has not yet been fully defined. Minerals are required for normal reproduction (Watanabe, 1985), but requirements above those needed for normal growth have not been established for fish.

5.3.2 Photoperiod Fish are governed by endogenous rhythms that make them able to spawn at approximately annual intervals, even under constant conditions of light and other environmental factors. Changes in daylight length regulates the reproductive cycle of our temperate fish species to a large extent, and manipulation of the photoperiods may be used to change to spawning periods which are not normal in order to provide egg production over an extended period of the year. This is shown in Fig. 5.8 for halibut. However, the temperature will also vary during the normal changing daylight cycles of the year, and the temperature range is critical for a successful maturation and spawning of viable eggs. Temperature control is therefore necessary for successful manipulation of photoperiods. The photoperiod is the principal determinant of the timing of maturation, and other environmental factors (e.g. temperature and nutritional status) act in a permissive way to enable maturation to proceed.

Figure 5.8 Duration of spawning, from first to last observed egg batch, of Atlantic halibut females on simulated natural (open boxes), four-month advanced (hatched boxes) and 4-month delayed (filled boxes) annual photoperiod cycle during four sequential spawning seasons. Vertical dashed lines are drawn at 4-month intervals as a visual aid (after Björnsson et al., 1998). Reproduced with permission from Elsevier Science.


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The mechanism for the determination of the timing of final maturation under the influence of photoperiod is not well understood. It is known that melatonin levels (indoleamine hormone from the pineal gland) rise during the night and in the autumn, and that the seasonal changes in melatonin levels correlate with changes in the levels of GnRH and gonadal steroids. A possible link between the pineal function and the endocrine cascade was recently reviewed by Bromage et al. (2001), who concluded that ‘the clear effects of photoperiod on the timing of reproduction on the one hand, and on the diel and seasonal patterns of melatonin on the other, provide strong circumstantial evidence that melatonin is the intermediary in these processes. Direct experimental evidence for this involvement is, however, far less convincing’. An extended spawning period by the use of several brood-stock groups with different maturation cycles may be beneficial for the hatchery economy, as a higher number of production cycles per year will result in higher production and a more effective use of the facilities. The fish farms should use out-of-phase seasonal light cycles at least until the response of the fish to the photoperiod can be ascertained. For example, the seasonal light cycle may be compressed into periods of time shorter than 1 year. Once a degree of advance or delay is achieved, the stock can be maintained at 12-month seasonal cycles. Then portions of light cycles can be substituted with constant photoperiods, which are easier to apply at commercial farms. Light can also be used to prevent early maturation in young on-growing fish. The exposure of Atlantic salmon to continuous light affects the number of fish matured as grilse, and lighting techniques are widely used to reduce maturity levels and increase the growth of fish in cages (Taranger et al., 1998). Recent experiments showed that photoperiod manipulation changed the incidence of sexual maturation, spawning time, fecundity and egg size in cod (Hansen et al., 2001). Cod reared under a natural photoperiod spawned between January and April. Cod that were transferred from a natural photoperiod to continuous light in December spawned earlier, and had a lower fecundity and smaller eggs than cod reared under a natural photoperiod. Oocytes of females reared under continuous light from June were arrested in the cortical alveoli stage, and even in the second year of continuous light, very few females matured. The pattern of sexual maturation influenced the somatic growth pattern. At the age of 26 months, the weight of cod reared under a natural photoperiod and continuous light was 1.5 and 2.5 kg, respectively. A reduction in daylength is thus a vital environmental signal regulating the maturation and spawning of cod, and sexual maturation may be arrested or considerably delayed in its absence. Turbot eggs are now routinely produced commercially throughout the year by means of photoperiod manipulations, and several larger cod and halibut hatcheries have established brood-stock groups for the year-round production of eggs (see also Chapter 2). The effects of changing light regimes on the spawning cycle of Atlantic halibut is shown in Fig. 5.8 (from Bjørnsson et al., 1998). Preliminary experiments with common wolf-fish show that photoperiod apparently has a major influence on the ovulation time: the majority of females exposed to a 6 hours light and 18 hours dark (6L:18D) photoperiod from May did not mature in the following spawning season (Moksness & Pavlov, 1996). A review of literature devoted to the influence of photoperiod on the egg quality of salmonids and marine fish species showed that results were controversial (Brooks et al.,

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1997). Present knowledge indicates that the extent to which egg quality is affected by manipulating the photoperiod may depend on how well other environmental parameters are controlled, and possibly the time of year at which the advance/delay in spawning occurs.

5.3.3 Temperature Temperature and feed availability are two of the most important factors affecting fish fecundity, and the optimal temperature for the feeding and growth of brood fish is not necessarily suitable for normal gonad development and spawning. In many species, the best egg quality is obtained if spawning occurs in a certain temperature range. The optimal temperature during gonad maturation is very important, and temperatures which are too high during vitellogenesis or final maturation may reduce fecundity and result in a high degree of atresia and poor egg quality. The time of final maturation can be changed under the influence of temperature manipulations, and a small reduction in temperature during vitellogenesis may delay spawning significantly. As seen for cod, this may not be critical for fecundity and offspring viability. The ovulation of females of common wolf-fish kept at a constant photoperiod (18L:6D) was observed over the entire year, and showed a peak in January. Maintaining the fish at a lower temperature (8–9°C vs. 10–14°C) for 3 months prior to spawning did not change ovulation time, but led to a substantial increase in egg quality, with the average proportion of normally developed eggs being 6.4% and 69.2%, respectively (Pavlov & Moksness, 1996a). More synchronous ovulation (within 1.5 months) was registered with a natural photoperiod. Females exposed to 8 or 12°C from May to September, and kept at 4°C afterwards, delayed ovulation by 4 or 5 weeks, respectively, compared with fish held continuously at 4°C (Tveiten & Johnsen, 1999). Water temperature seems to be a comparatively less important factor for the final maturation of males: good-quality sperm was obtained from males kept at several temperatures ranging from 2 to 12°C (Pavlov & Moksness, 1994a). Turbot and common sole are especially susceptible to a comparatively high temperature (above 14–15°C) during gametogenesis and the beginning of the spawning period, and a decrease of about 3°C leads to a substantial improvement in egg quality (Devauchelle et al., 1987, 1988). In Atlantic halibut, the viability of eggs from females kept at a constant temperature of 6°C over a spawning season was consistently higher than those from females maintained under fluctuating ambient temperature (Brown et al., 1995, cited from Brooks et al., 1997). This observation is apparently connected to the natural spawning habitat of Atlantic halibut in deep waters with a stable temperature (Haug, 1990).

5.3.4 Present Husbandry Practices and Egg Collection All species considered in this book will undergo normal oocyte maturation, final egg maturation and ovulation without the use of hormonal injections. However, conditions which are suitable for normal courtship and spawning behaviour (e.g. special habitats in the spawning grounds) are almost never achieved in fish farms, which causes problems. Of the species which could be used for cold-water aquaculture, only a few will spawn naturally in captivity. Fertilised eggs from two species, cod and common sole (Solea vulgaris Quensel), are


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normally collected from naturally spawning brood stock. Natural spawning of Atlantic halibut and turbot brood stocks has been observed occasionally, but it is not common and the fertilisation and hatching rates are highly variable, apparently due to inadequate conditions in captivity. For common wolf-fish, natural spawning with subsequent guarding of the eggs by the male was observed once at the Tromsø Sea Aquarium, after the spawners had adapted to the artificial conditions over several years (Ringø & Lorentzen, 1987). In captivity, males are often inactive and females may shed unfertilised eggs. Species such as the salmonids, Atlantic halibut, turbot and the wolf-fish must therefore be stripped for a regular supply of eggs and milt, with subsequent artificial insemination of the eggs. Care must then be taken to strip the fish at the correct time in relation to ovulation in order to avoid ageing (or over-ripening) of the eggs. As will be described in Section 5.4, over-ripening of eggs before fertilisation is an important reason for poor egg viability in marine fish. When fish are stripped for gametes, a clean, dry container should be used for each of the batches of eggs and milt, and contact with (sea)water must be avoided to prevent activation of the sperm. Often, several fish will be stripped at the same time, and the gametes can be stored for a short period (at the same temperature as for fish, or colder) before they are fertilised. For pelagic marine eggs, ‘wet’ fertilisation is normally used by mixing the gametes with seawater for fertilisation. The male wolf-fish has a very small volume of semen, and the mature fish exhibits internal fertilisation. To fertilise wolf-fish eggs, the gametes are therefore mixed together for at least 4 h before the addition of seawater. Insemination is a process during which gametes are brought together. Fertilisation, in its broadest sense, is a process started with insemination, continued with egg activation and cortical reaction, and terminated with the fusion of male and female pronuclei. Only the latter process can be considered as fertilisation in a strict sense, and a further description of the fertilisation process can be found in Chapter 6. 5.3.4.1 Cod Most cod brood-stocks are still caught from the wild. Adult cod is normally caught in the autumn/winter and the fish are maintained in cages or large tanks. The volume of the cages ranges from 125 to 1000 m3, and the depth is about 2 m or more. Cod brood-stocks are normally held at temperatures between 5 and 14°C, and the temperature optimal for spawning is 4–6°C (Rosenlund et al., 1993; Jobling & Pedersen, 1995). The density of fish in the cages is about 30–35 kg m-3. The brood-stock fish are fed standard dry pellets, moist pellets or fresh fish to satiation two or three times a week. If they are kept in cages, the fish are transferred to spawning pens (80–150 m3 in volume) or to large tanks (e.g. 7–10 m cylindrical tanks) a few weeks before spawning. The sex ratio is normally maintained at three females to one or two males, and the stocking density is about 5 kg m-3. The brood-stock can be used for spawning over several years, and the fish are usually tagged before their first spawning. Males and females can be distinguished by the appearance of the genital pore 2–4 weeks before spawning (not very reliable), or by ultrasound inspection of the developing gonads (most reliable). Cod showing courtship behaviour spawn naturally in captivity. Courtship may be observed at almost any time of the day, but spawning occurs mostly at night. As the spawning season

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progresses, the number of eggs per batch tends to decrease. The fertilised pelagic eggs will float to the surface, and are collected daily with an air-lift system or separated from the outflow water of the tank. The number of eggs may vary between 400 000 and 750 000/l, with an average of about 500 000/l. Typically, a female in good condition produces about 1–1.5 l of eggs/kg body weight. The collected eggs are usually disinfected with either glutaraldehyde (400 p.p.m. for 10 min) or Buffodine (100 ml in 10 l water for 10 min) and rinsed thoroughly before being transferred to the incubators. Usually over 95% of the eggs are fertilised, but as many as 10–35% of the embryos show malformations (Jobling & Pedersen, 1995; Kjørsvik, 1994). 5.3.4.2 Turbot Turbot brood-stock are kept in large tanks (20 m2, 1 m depth), at a temperature ranging from 9 to 14°C (Suquet et al., 1998a). The upper temperature limit for normal maturation of turbot is approximately 16°C. If the temperatures are higher from 1.5 months prior to spawning, the eggs will not be viable (Devauchelle et al., 1988). The brood-stock fish are generally fed a mixture of moist pellets and fish, or commercial dry pellets. Turbot ovulate egg batches every 2–4 days over a period ranging from 12 to 38 days in different females. The ovulatory period differs between females, and, in addition, may differ from year to year, and even within a single season. However, a lack of constancy during the spawning season may be explained by unstable water temperatures. Experience with egg production from turbot shows that at the time of stripping, only a variable fraction of the eggs obtained will be buoyant, and of these only a variable fraction will be fertilised. Low viability of eggs is connected with a rapid post-ovulatory overripening of the eggs. A low ratio of floating eggs may be due either to the presence of ‘old’ eggs from a previous ovulation, or to over-ripening of the last ovulated batch (or both). However, the low fertilisation rate in some egg batches is compensated by intensive egg production in this species (with annual relative fecundity reaching 285–463 g eggs kg-1 body weight). Fertilisation success and egg viability will thus increase with closer monitoring of the brood-stock, as was also shown by McEvoy (1984). 5.3.4.3 Atlantic Halibut Atlantic halibut brood-stocks are normally maintained in circular tanks (diameter 3.5–15 m, 1–2-m depth, light-protected, 34‰ salinity), and at temperatures below 8°C during gonad maturation and spawning (Olsen, 1997; Shields, 2001). An increase in temperature during the spring, and changes in water temperature in general will affect spawning rhythm and egg quality negatively. Female brood-stock fish normally range between 20 and 80 kg, whereas males are much smaller. Stocking density is up to approximately 11 kg m-3, with sex ratios typically ranging from 1–2 males per female. Brood-stock fish are normally fed to satiation at least three times each week, and the diets are usually based on fresh fish, either presented whole, or in a ‘sausage mix’ with fish meal, oils and micro-nutrients. However, there has recently been a widespread transfer to formulated diets owing to concerns about batch variations in diet


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quality, and because of the risk of disease transmission through the fresh-fish diet. The prevailing method of diet preparation involves mixing fish meal with fish oil and water, then extruding it into a sausage. The development of dry pelleted diets is taking place separately. No feed is offered to fish during the spawning season. In Atlantic halibut, the egg viability is relatively stable for approximately 6–8 h postovulation, but declines thereafter due to post-ovulatory deterioration. If not stripped in time, females may shed their eggs into the tank. Females are therefore monitored by recording the timing and quantity of egg releases (in tank overflow collectors), and by observing the progressive distension of the abdomen between ovulations. An ultrasonographic technique (7.5-MHz linear transducer) may be used to observe the appearance of hydrating oocytes in the ovary. In Atlantic halibut, at least 90% of the stripped eggs will normally be fertilised. Although hatching success varies widely between egg batches, up to 75–80% of the fertilised eggs may hatch. However, egg batches with a high fertilisation success may have a very poor hatching success (see Section 5.4). 5.3.4.4 Wolf-fish Common wolf-fish brood-stock is maintained in tanks of 2.4 m3, at stocking densities lower than 14 fishes m-3. The oxygen content is above 6 mg O2 l-1 in the outlet water, and the salinity fluctuates between 32.0 and 34.7‰ (Moksness, 1994). The brood-stocks of spotted wolffish are kept in outdoor, partly covered tanks at ambient sea temperature (3–10°C), and in indoor facilities using a supply of cooled water during the warm months (before and during final sexual maturation). The optimal temperature in this period should be close to 4°C. In brood-stock maintained in a simulated natural photoperiod, egg ovulation is registered from July to January, with a peak in October (Falk-Petersen et al., 1999). Common wolf-fish are fed commercial dry salmon pellets. Before final maturation, 1–4 weeks prior to egg ovulation, females are kept in tanks about 600 l each at densities of 1–6 fish per tank, at a low light intensity, and they are not fed (Pavlov & Moksness, 1996a; Moksness & Pavlov, 1996). The presence of males does not seem to influence the maturation of females or the egg quality (Pavlov & Moksness, 1994a). In wolf-fish species, which all have internal insemination, the size of the female’s abdomen increases rapidly 1–2 days before ovulation. Opening of the genital pore indicates the time of ovulation. In females with a swollen abdomen, the time of the opening of the genital pore should be checked several times a day. Ovulated eggs can be inseminated in vivo by injecting sperm into the opened genital pore of the female. Following insemination in vivo, the eggs are released into water within 24 h and stick together in a clutch representing an egg-ball. This is formed by the female, whose curving body surrounds the clutch for at least 1–2 h (Pavlov & Radzikhovskaya, 1991). Such a clutch might be eaten by the female, and incubation of the eggs is difficult: unfertilised and dead eggs cannot be removed, and all the eggs may die due to bacterial infection. Therefore, a method of insemination in vitro has been developed for common wolf-fish (Pavlov, 1994b; Moksness & Pavlov, 1996). After stripping of the eggs, the excess slimy ovarian fluid is removed. To increase the probability of contact between gametes without damaging them, a special procedure involving cylindrical vessels is used for the insemination (Fig. 5.9). After an inversion of the vessel,

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Figure 5.9 Schematic representation of several operations for artificial insemination and the prevention of adhesiveness of common wolf-fish eggs. (a) Pouring sperm into a cylindrical vessel. (b) Placing the eggs in the vessel (minimum egg/spermatozoa ratio 1 : 200 000). (c) Covering the upper part of the vessel. (d) Repeated mixing of eggs with sperm by inversions of the vessel kept at 2–7°C for 4–6 h (note that liquid ovarian fluid with the sperm appears in the upper part of the vessel, while eggs sink to the bottom). (e) Distribution of the eggs on the bottom of large trays with stagnant seawater to prevent contact between eggs for at least 6 h. (f) Transfer of eggs into upwelling incubators (Moksness & Pavlov, 1996, modified).

the eggs settle to the bottom after passing through the layer of sperm, and thus good mixing of gametes is achieved. The average fertilisation rate of wolf-fish eggs is about 95%. The wolf-fish eggs will become adhesive and will stick to each other immediately after contact with seawater. To prevent this stickiness, which lasts until the hardening process is completed, the eggs may be distributed in trays with stagnant marine water during the hardening process (Moksness & Pavlov, 1996).

5.4 Egg Quality Fish juvenile production is generally characterised by variable egg mortality, high and variable larval mortalities, and variable juvenile quality. The poor or variable output in juvenile production can often be linked to problems with egg and larval quality. A strengthened focus on these aspects and how they are related is therefore necessary to obtain a better understanding of the biological mechanisms and implications involved in offspring quality. Variations in egg quality leading to variable hatching rates are often encountered, and are well demonstrated in salmonids such as Atlantic salmon and rainbow trout, as well as in several marine species, e.g. Atlantic cod, turbot, halibut, gilthead seabream and wolf-fish. A study of rainbow trout egg batches from commercial hatcheries (Bromage, 1995) showed that the best egg batch gave a fry survival higher than 80% after 120 days, whereas the


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FERT EYE HATCH SWIM UP

100

Survival (%)

best

75

50 mean

25 Figure 5.10 Rainbow trout: fertilisation rates and survival at eyeing, hatch and swim-up of egg batches (n = 15) (after Bromage, 1995).

worst

0

20 30 50

120

Time (days)

poorest egg batch did not survive to hatching (Fig. 5.10). The mean fry survival 120 days after fertilisation was less than 30% (data from 15 egg batches). This situation is even more variable for marine fish, and especially among the multiple spawners. The eggs from the spawning fish stock may be regarded as the seed for the fish harvest, and egg and larval quality (or viability) is important for fish recruitment in the sea as well as for optimal juvenile production in aquaculture. Owing to the very high variability in captive fish, there has been an increasing interest in egg quality problems in aquaculture (see reviews by Kjørsvik et al., 1990; Bromage, 1995; Brooks et al., 1997). Egg quality in its strict sense can be defined as the egg’s potential to produce viable fry (Kjørsvik et al., 1990). A more practical version of this definition would be that egg quality is the potential of the ovulated egg batch to produce viable fry. According to this definition, egg properties depend on the genotype of the mother, as well as on the morphological, chemical and physiological processes occurring in the egg. Fish egg quality can be affected by many factors (Fig. 5.11), e.g. maternal age and condition factor, the timing of the spawning cycle, over-ripening processes, genetic factors and also the intrinsic properties of the egg itself (Bromage et al., 1994). These egg-quality characteristics and properties, and the influence of some environmental factors on the egg’s potential, are discussed in this section.

5.4.1 Assessment of Egg Quality An important aspect of hatchery management is to assess whether your egg batches seem viable or not. Such an evaluation should be at an early stage in development, in order to avoid wasting valuable resources on a poorly performing group. Important questions for such an assessment will thus be:

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Ovulation Parental effects (mostly maternal) - age, size, condition factor - spawning stage - parental stress - genetics

Fertilisation

Viable embryo

Egg/embryo

Oocyte

Nutrition Induced spawning Overripening Environmental conditions - husbandry practices - temperature - light regimes - water quality

Non-viable embryo

Figure 5.11 Known causes for varying egg quality in fishes.

• Are there any possible predictive criteria for egg quality which can be applied at an early stage? • If gametes are stripped from the mature fish, how long after ovulation may the unfertilised eggs be viable? • How may poor egg quality affect the viability of surviving larvae and juveniles? Much effort has been put into evaluation criteria for marine egg and larval quality, and during the last two decades, some reasonably reliable criteria for egg quality and the effects of egg over-ripening have been established based on empirical research. Egg quality is usually assessed after fertilisation despite the contribution of the paternal genes. However, if a large enough quantity of good-quality sperm is used for the insemination, the paternal effect can be excluded. Moreover, as is known (Ginsburg, 1968), paternal genes can be expressed mainly from the step of gastrulation. In hatcheries the quality of pelagic eggs is usually assessed by their ability to float or sink in seawater. However, buoyancy is not a good criterion of egg quality for a number of marine fish species, in particular for Atlantic halibut. A preliminary assessment of egg quality can be made based on visual characteristics such as easy or hard stripping of the eggs, the amount and consistency of the ovarian fluid, the elasticity and colour of the eggs, and the presence of damaged eggs. For example, in common wolf-fish, good quality eggs are usually accompanied by a comparatively large amount of ovarian fluid (up to 100 ml in large females), which is essential for good mixing of eggs and sperm during internal insemination in the wild (Moksness & Pavlov, 1996). The resistance of the eggs to bacterial contamination may to a certain degree indicate their quality: poorquality eggs are more prone to bacterial contamination (Pavlov & Moksness, 1993; Kjørsvik et al., 1990). However, to date, for many marine species in fish farming, egg morphology,


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fertilisation success and the ratio of normal blastomeres at early stages of cleavage seem to be the most useful general tool for the assessment of egg quality, and these are also promising tools for prediction of the potential viability of developing embryos, larvae and juveniles for several species. 5.4.1.1 Egg Morphology The preliminary assessment of egg quality just after stripping can be illustrated for common wolf-fish eggs stripped from the brood-stock and inseminated in vitro. In mature unfertilised eggs, oil droplets have a comparatively low density, and they move freely to the upper side of the yolk. The presence of several oil droplets attached to the yolk cytoplasmic membrane (Fig. 5.12a) may indicate that some of the egg granules are not fused, and the egg is thus immature (Pavlov et al., 1992). The presence of a perivitelline space and blastodisc in the ovulated egg before exposure to seawater (Fig. 5.12b) means that a cortical reaction has occurred inside the female’s body and that fertilisation of this egg is impossible (see Chapter 6 for a more detailed description of fish egg terminology and development). Damage to the yolk cytoplasmic membrane leads to the outflow of the yolk (Fig. 5.12c), and all the contents of the egg become homogenous and whitish (Fig. 5.12d). Large numbers of whitish eggs indicate bad egg quality, but, in addition, damage to the yolk membrane may be caused by the stripping procedure. Several eggs have a deep–yellow or brown colour due to the presence of large oil structures that appear after the fusion of oil droplets (Fig. 5.12e). The presence of eggs covered by a follicular layer at the beginning of resorption (Fig. 5.12f) indicates a deterioration in the process of egg ovulation. Similar observations may be carried out for pelagic fish eggs, since whitish, non-transparent eggs that rapidly sink to the bottom when mixed with seawater are non-viable. However, it is quite common to obtain a small percentage of non-viable eggs when multiple spawners such as halibut and turbot are stripped, and eggs that are transparent and floating may be viable and of good quality (but not always!). The multiple spawners regularly ovulate new batches of eggs, and when stripped, some eggs from a previous batch may be present, together with the new egg batch. Eggs from several species contain oil globules; some have one and others have several. The specific number of oil globules seems to be a good measure of egg quality in some species, such as the striped jack (Pseudocaranx dentex) (Vassallo-Agius et al., 2001). 5.4.1.2 Fertilisation Success and Cortical Reaction Fertilisation success is widely used in commercial hatcheries to predict egg survival. Many species in captivity show a very high variability in fertilisation success. For some species, fertilisation success seems to be related to embryo viability and hatching success, and this seems to be valid, for example, for demersal freshwater salmonid eggs as well as for pelagic marine eggs from yellowtail flounder (Manning & Crim, 1998), gilthead seabream (Almansa et al., 1999) and the striped jack (Pseudocaranx dentex, Fig. 5.13). For yellowtail, a relation was found between fertilisation success and the percentage of normal larvae, but not between fertilisation and hatching success (Verakunpiriya et al., 1997a,b). Such a clear correlation is

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Figure 5.12 Morphology of eggs just after stripping from a female of common wolf-fish. (a) Egg which appears normal, but the presence of several oil droplets distributed outside the upper part of the yolk and attached to the yolk indicate that the egg is immature; (b) cortical reaction and the formation of the blastodisc took place inside the female’s body; (c) constriction of yolk due to damage of the yolk membrane; (d) egg with damaged yolk membrane; (e) egg with the oil structure and constricted yolk; (f ) resorbing egg covered by a follicular layer. bl, blastodisc; mc, micropyle; od, oil droplets; os, oil structure; ps, perivitelline space; yl, yolk. The natural orientation of the eggs with a view from the side is given (Pavlov & Moksness, 1994a, modified). Reproduced with permission from Elsevier Science.

not always found for other species such as cod (Kjørsvik & Lønning, 1983; Kjørsvik et al., 1984), halibut (Shields et al., 1997; E. Kjørsvik, unpublished data, 1996), turbot (McEvoy, 1984; Kjørsvik et al., 2003) or seabass (Saillant et al., 2001). Low fertilisation rates are usually related to very poor hatching success. However, a high fertilisation success does not necessarily lead to a high egg and larval viability for all species. Although the fertilisation rate seems to be a reliable indicator of egg quality in salmonids


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H a tc h in g (% )

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R = 0,7662

20

0 0

20

40

60

80

100

F e rtilis a tio n (% )

Figure 5.13 Fertilisation success and hatching in striped jack, Pseudocaranx dentex (figure made from data in Vassallo-Agius et al., 2001).

and several other species, it is a necessary, but not a sufficient, egg quality criterion for marine fish eggs of many species (see reviews by Kjørsvik et al., 1990; Bromage, 1995; Brooks et al., 1997). For example, in several females of common wolf-fish brood-stock, the fertilisation rate was close to 100%, but the proportion of normally cleaved eggs ranged from 0 to 20% (Pavlov & Moksness, 1994a). In halibut egg batches with fertilisation rates close to 100%, the percentage of normally cleaved eggs and the hatching success may vary between 10 and 90% (Bromage et al., 1994; E. Kjørsvik et al., unpublished results, 1994–2002). The features of the cortical reaction, which is closely linked to the fertilisation process, may also be used as part of an egg quality evaluation. In cod and wolf-fish, it has been observed that the duration of the cortical reaction may vary in eggs from different females. In eggs of poor quality, the duration of this process was prolonged, and the cortical reaction was often incomplete, resulting in less hardening of the egg envelope and a smaller perivitelline space (Kjørsvik & Lønning, 1983; Kjørsvik et al., 1984; Pavlov & Moksness, 1996a,b). Remnants of visible cortical alveoli in the cytoplasm and in the blastomeres after cleavage are observed for several marine species in connection with a high incidence of abnormal and incomplete cleavage (Kjørsvik et al., 1990; Pavlov et al., 1992; E. Kjørsvik, unpublished results, 1994–2002). These observations suggest a similarity in the morphological deterioration of the eggs of different marine fish species. 5.4.1.3 Blastomere Morphology Kjørsvik et al. (1990) suggested that an assessment of cell symmetry at the early stages of cleavage (normal blastomeres) might be a possible general indicator of egg quality for marine fish. This morphological criterion seems to be the most reliable so far, and significant positive correlations have been observed between normal blastomeres in the earliest cleavage stages, hatching rates and the viability of yolk-sac larvae hatching in species such as Atlantic cod, turbot and halibut (Shields et al., 1997; E. Kjørsvik, unpublished data, 1994–2002).

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Figure 5.14 Irregular blastomere cleavage of Atlantic halibut (an irregular cell number (7) and cell shape; no symmetry), cod (irregular cell size and cell shape), and turbot (4 cells, poor contact between cells). Photograph Elin Kjørsvik.

Figure 5.15 Morphology of cells at the stage of 16 blastomeres in common wolf-fish. (a) Normal and (b–j) abnormal cleavage.

As noted in the previous section, there should be no (or very few) inclusions of vacuoles (cortical granules) in the cytoplasm. Pelagic eggs are generally easiest to observe during the 4- to 16-cell stages, because after subsequent cleavages, the blastomeres are divided into several cell layers and become too small to observe individually. These early cleavages should be synchronous, and cell divisions should be complete and result in the correct number of blastomeres. The early blastomeres should be regular in shape, with clear cell margins and good contact between adjacent cell membranes. Examples of irregular blastomere cleavages are shown in Fig. 5.14. Similar observations on blastomere morphology have been made for the demersal common wolf-fish eggs, where the patterns of cleavage can easily be assessed at the stages from eight blastomeres to morula. In normal eggs at the stage of 16 blastomeres, all cells can be distinguished clearly (Fig. 5.15a). In poor-quality eggs (Fig. 5.15b–j) cleavage is incomplete, the number of blastomeres is different, the blastomeres have irregular shapes, or the borders of some cells are not visible. Then empty spaces form in the blastodisc at morula stage (Pavlov & Moksness, 1994a), and the cells totally destroy before blastulation (Pavlov et al., 1992).


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A word of caution is necessary regarding the assessment of blastomere symmetry. In many fish species, the size of blastomeres may be different from the first cell division. In good-quality eggs of cod, the cells may be of unequal size at the stage of two blastomeres (Makhotin, 1982). In good-quality eggs of common wolf-fish obtained from wild-caught spawners, at the four-blastomere stage, some cells may be twice as large as others, and at morula stage, internal cells are substantially smaller than the cells on the periphery of the blastodisc (Pavlov et al., 1992). According to Doronin (1985), asymmetrical cleavage due to uneven cell size in teleost fish is the rule rather than the exception. Therefore, an asymmetry of cleavages due to normal differences in the relative size of cells in the blastodisc may not indicate egg quality in many fish species, and the most reliable criteria are the correct cell number, regularly shaped cells, and clear margins between them. In addition, for Atlantic halibut eggs, Shields et al. (1997) described such parameters of egg quality as bilateral symmetry about the axes of the eight blastomeres (which seems a reliable criterion for this species), proximity of adjacent cell membranes, and the absence of inclusions of vacuoles between adjacent blastomere membranes or on the periphery of the blastodisc. Previous studies have revealed that hatching success is normally more reliably correlated to the rate of abnormal blastomeres (early cell development) than to the fertilisation rate in marine fish such as Atlantic cod (Kjørsvik & Lønning, 1983; Kjørsvik et al., 1984, 2003; E. Kjørsvik, unpublished data, 1994–2002), halibut (Shields et al., 1997; E. Kjørsvik et al., unpublished data, 1994–2002), wolf-fish (Pavlov & Moksness, 1994a) and turbot (Kjørsvik et al., 2003), and in planktonic samples of wild fish eggs (Westernhagen et al., 1988; Cameron et al., 1989). For most species investigated so far, the correlation between the proportion of normally developing early embryos (or blastomeres) and hatching success or survival rates is usually high, regardless of whether the eggs are spawned and fertilised in the wild or in captivity. For cod, it has also been demonstrated that normal blastomeres are related to larval survival in the yolk-sac stage (Fig. 5.16), and to larval viability when exposed to a 100 Larval survival day 6 Hatching

Percentage (%)

80

2

R = 0,6295

60

40

2

R = 0,7799

20

0 0

20

40

60

80

100

Normal blastomeres (%) Figure 5.16 Normal blastomeres of cod in relation to hatching success and survival of yolk-sac larvae up to startfeeding (6 days after hatching) at 5°C. Each point represents one egg batch from different females, either stripped immediately after trawl capture or obtained from brood-stock spawning (E. Kjørsvik, A. Thorvik and R. van der Braak, unpublished data, 1994).

164


100 2

R = 0,5421

Percentage (%)

80

60 2

R = 0,3712

40

Hatching 20

Norm 230 dd Normal newly hatched

0 0

20

40

60

80

100

Normal blastomeres (%) Figure 5.17 Normal blastomeres of halibut in relation to hatching success and percentage of normal larvae at hatching and at 230 day-degrees at 5°C. Each point represents one egg batch from a brood-stock female (E. Kjørsvik, T.E. Leren, Ø. Aaleskjær and T.F. Galloway, unpublished data, 2000–2002).

high-salinity functionality test. However, the significance of normal blastomeres as an egg quality criterion is not always apparent in relation to hatching success. In halibut, normal blastomeres are not usually very well correlated to hatching success, but rather to the ratio of normal yolk-sac larvae (Fig. 5.17). Hatching success is therefore not necessarily a good measure of egg quality for all species. A recent study of egg quality in turbot further demonstrates a long-term effect of poor egg quality (Fig. 5.18, from Kjørsvik et al., 2003). Normal blastomere morphology was related to hatching success and larval viability (tested by a high-salinity functionality test). When larvae were reared to metamorphosis, there was also a significant correlation between normal blastomeres, survival to the juvenile stage, and juvenile quality (measured as the completion of metamorphosis and correct pigmentation). Survival to the end of metamorphosis for these groups varied between 2 and 30%, but if only normally metamorphosed and normally pigmented juveniles were included, the juvenile production yield for these groups varied between 1.5 and 27% of the original number of larvae stocked in the start-feeding tanks. For some of these groups, only about half of the surviving juveniles had a normal appearance. For a farmer, the resources needed to rear all these larval groups will be the same, but the difference in income will be very large. It may therefore be of great economic importance to assess the egg quality in the hatchery. 5.4.1.4 Egg Size Egg size is generally positively correlated with the size of the larva. It is important to note that in the egg after swelling, the yolk diameter and the relationship between the cytoplasm volume (measured between the beginning of cleavage and the beginning of epiboly) and the


165

100

Hatching (%) Time to 50% mortality (minutes)

Hatching Time to 50% larval mortality in stress test

80

2

R = 0,8587

60

40 2

R = 0,743

20

0 0

20

40

60

80

100

Normal blastomeres (%)

a

Survival day 37

100

Normal metamorphosis

2

R = 0,5146

Normal pigmentation

Percentage (%)

80 2

R = 0,639

60

40

20 2

R = 0,3406

0

b

0

20

40

60

Normal blastomeres (%)

80

100

Figure 5.18 Turbot egg quality. (a) Normal blastomeres in relation to hatching success and larval survival in a high-salinity functionality test on day 3 after hatching. (b) Normal blastomeres in relation to survival by the end of metamorphosis and in relation to success of normal pigmentation and completion of metamorphosis (from Kjørsvik et al., 2003). Reproduced with permission from Elsevier Science.

volume of the yolk are more important in the determination of larval parameters than egg size itself (Pavlov, 1989; Balon, 1999). In the wild, larger larvae are less vulnerable to predators, they eat a wider variety of food items, have a survival advantage owing to larger yolk reserves, and may have a higher growth rate. However, larger offspring would be more noticeable as prey (see reviews by Kjørsvik et al., 1990; Blaxter, 1992; Kamler, 1992; Brooks et al., 1997). The size of the eggs seems to have different importance for different species. In salmonids, larger eggs produce significantly larger larvae, but this size advantage is generally lost shortly after first-feeding (Bromage & Cumaranatunga, 1988). Experiments with offspring obtained from cod collected in the wild revealed positive relationships between egg and larval size

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and some larval viability characteristics. Larvae hatched from the largest eggs (>1.5 mm diameter) initiated feeding earlier and expressed a higher incidence of feeding during the first days of exogenous feeding, they showed higher frequencies of swim-bladder occurrence (percentage of larvae with a developing swim-bladder on day 10 after hatching) and had a higher growth rate, at least during the first 2 weeks (Marteinsdottir & Steinarsson, 1998). In cod, as in many other species, the smallest eggs are produced towards the end of the spawning cycle, and a more variable egg quality has also been observed during the last part of spawning (Kjørsvik, 1994). Since egg size is linked to fish size as well as to the spawning cycle, it may be difficult to use egg size per se as a criterion for egg quality. In the brood-stock of common wolf-fish maintained at a comparatively high temperature, poor egg quality was associated with smaller egg diameter. However, in fish kept at a more appropriate temperature, egg diameter was not correlated with the proportion of normally cleaved eggs, but the coefficient of variation (CV) of egg diameter was significantly negatively correlated with this proportion. In addition, negatively skewed frequency distributions of egg diameter were registered, with a higher skewness for the poor-quality egg group (Pavlov & Moksness, 1994a, 1996a). These results suggest that an analysis of the value of the CV and the parameters of egg size distribution may be useful for the assessment of egg quality for some species. According to data from many authors obtained mainly for freshwater species, a high CV reaching 10–15% is associated with poor egg quality (see review by Zhukinskii & Gosh, 1988). 5.4.1.5 Chemical Content A biochemical evaluation of egg quality parameters shows that certain components are ‘essential’ for an organism, while other components are species-specific and may indicate a positive egg quality criterion for one species and poor egg quality for another. The most commonly studied chemical parameters are pigments and vitamins, as well as some inorganic and organic components. The role of these parameters in the assessment of the quality of mainly fresh-water fish and salmonids is discussed by several authors (Kjørsvik et al., 1990; Kamler, 1992; Bromage & Cumaranatunga, 1988). In general, differences in levels of mineral ions, amino and fatty acids, vitellogenin and carotenoids did not show any substantial relationship with egg quality (Bromage & Cumaranatunga, 1988), and the egg composition will, to a large extent, reflect the composition of the fish diet. As many other articles also demonstrate, differences in egg viability may be found if essential components in the brood-stock diet are below certain levels, and the possible effects of nutrition on egg quality are discussed in Section 5.3.1. However, several investigations have shown that in fish fed a uniform diet, egg viability may be connected with their chemical content. Devauchelle et al. (1988) reported greater total lipid percentages of the dry weight in non-viable (unfertilised) versus viable (fertilised) turbot egg batches from a brood-stock. In Atlantic halibut, a comparison of lipid class and fatty-acid compositions for viable and non-viable egg batches (determined according to fertilisation and hatching rates, cell symmetry and percentage of larvae to first feeding) revealed that they were similar between egg categories with the exception of cholesterol, whose level was significantly greater in the non-viable eggs (Bruce et al., 1993). A relationship between


167

the egg quality of two first-time spawners and of a repeat-spawning female of Atlantic halibut and their biochemical composition has been reported (Evans et al., 1996). Eggs from firsttime spawners with lower fertilisation rates were significantly lower in total lipid, triacylglycerol and sterol contents. These eggs also had significantly lower percentages of two essential fatty acids, i.e. docosahexaenoic acid and arachidonic acid. A reduced egg quality was also observed in first-time spawners of cod compared with older spawning fish (Solemdal et al., 1995). Several recent papers have been devoted to the investigation of the chemical characteristics of fish eggs, which can be useful in an evaluation of their quality. Such studies may deal with variations in egg composition during spawning periods, egg maturation processes and composition in relation to mechanisms that are possible determinants of egg quality, and all are contributing to our increasing knowledge of how biological mechanisms are linked to offspring viability. In carp (Cyprinus carpio L.), for example, fertilisation rates were correlated to ovarian fluid pH and protein content, egg respiration rate, pyruvate kinase activity and malate dehydrogenase activity. These enzymes are linked to the cell energy metabolism, and their activity level may thus affect normal development (Lahnsteiner et al., 2001). In turbot, the pH values of the ovarian fluid were positively correlated with egg viability and fertilisation success (Fauvel et al., 1993). Thus, ovarian fluid pH may be used as a predictor of over-ripening and fertilisation success in turbot. Likewise, in a study of egg quality in Perch (Perca fluviatilis (L.)) (Kestemont et al., 1999), the cathepsin- activity in 7-day-old eggs increased during the spawning season, and the mean hatching success and larval viability declined during the spawning season. 5.4.1.6 Cytology Cytological methods are used to reveal chromosomal anomalies during the final maturation of oocytes and at earlier stages of developing embryos. Ovulated oocytes of starred sturgeon (Acipenser stellatus Pallas) obtained by hormonal stimulation were at different stages of meiosis, showing desynchronisation between maturation and ovulation (Faleeva, 1987), and a clear correlation was found between chromosomal anomalies and fertilisation rate. According to the author, different sizes and shapes of the chromosomal spindle at metaphase II may indicate errors in meiosis. Desynchronisation of maturation and ovulation, as well as other cytological anomalies leading to reduced egg quality, have been described for several freshwater species bred in captivity (Detlaf, 1977; Detlaf et al., 1981; Korovina, 1986; Makeyeva et al., 1987). In fish embryos at early stages of development (before gastrulation), three types of chromosomal aberrations have been described: (1) delayed anaphases caused by delayed division of some of the centromeres, but the chromosomes in most cases reach the poles at late telophase; (2) chromosomes or their fragments remain in or near the equatorial plane; (3) some of the chromosomes do not divide, or do not divide properly, and remain in the equatorial plane forming a bridge between the dividing chromosomes (Kjørsvik et al., 1984). Correlations between the survival of the eggs and cytogenetic status are found for several species, but the number of cytological studies for marine fish is small, and is restricted mainly to toxicological investigations (see review by Kjørsvik et al., 1990).

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5.4.1.7 Oxygen Consumption In carp, the oxygen consumption rate was measured just after ovulation for 3–8 min using a polarographic method (Zhukinskii & Gosh, 1988). The oxygen consumption rate was substantially lower in over-ripe eggs and in eggs with different types of deterioration. Significant positive correlations between oxygen consumption rate and the subsequent survival of embryos and larvae were registered. According to the authors, this method of egg quality assessment could be used in fish culture. A substantially lower oxygen consumption rate (61–70% of the rate in the control group of high-quality eggs) was registered in over-ripe eggs of smelt, Osmerus eperlanus (L.), at the initial stages of egg development (from two blastomeres to blastula) (Korovina, 1986). 5.4.1.8 Evaluating Mammalian Embryo Quality Embryo quality is also a well-known problem in human medicine as well as in animal husbandry. In vitro fertilisation (IVF) and embryo transfer techniques are often characterised by variable embryo quality and low pregnancy rates (Giorgetti et al., 1995), and in mice and in humans, 15–50% of embryos die during the pre-implantation period from mechanisms that are largely unknown (see Warner et al., 1998). Therefore several eggs may be implanted in order to increase the chances of pregnancy, resulting in high multiple-pregnancy rates. In order to avoid these problems, much work on estimating embryo quality has been undertaken for humans and for other mammals. The most frequently used embryo quality codes are also based on blastomere symmetry and blastomere size, colour and density, and on embryo developmental rate (Stringfellow & Seidel, 1998). Several studies have examined the mechanisms regulating cell cycles and development, and results related to embryo mitotic activity (Wurth et al., 1994) and ATP content (Blerkom et al., 1995) have suggested that embryo quality is linked to physiological mechanisms. There also seems to be a genetic basis for pre-implantation egg and embryo survival. Mouse and human embryos of poor quality exhibit a very high degree of apoptosis compared with normal embryos, and two genes that regulate apoptosis (programmed cell death) were expressed differently in embryos of different quality (Warner et al., 1998). Factors that stimulate oocyte maturation and embryo development, such as insulin-like growth factors (IGFs) and insulin, may also be important. Expression of IGFs and their receptors is now a potential marker for human embryo quality (Liu et al., 1997), as the activity of several of these genes correlates well with a morphological assessment of embryo quality. There are indications that the same may be true for fish embryos.

5.4.2 Factors Affecting Egg Quality 5.4.2.1 Over-ripening The process of over-ripening (or egg ageing) can be defined as the deterioration of eggs that are retained after ovulation in the ovarian fluid within the female’s body (in vivo) or after stripping (in vitro). Even in the early stages of modern fish culture history, over-ripe eggs were observed to be a problem (Grimm, 1916), and since then over-ripening has been studied


169

in a large number of fish species (see review by Korovina, 1986). Over-ripening of eggs may occur due to stress both in captivity and in the wild if the requirements for spawning are not met, or the usual spawning habitats are destroyed. The problem of over-ripening is especially important for many batch-spawning marine species, which do not spawn naturally in captivity, and where the eggs should be stripped at the correct time. For example, Howell and Scott (1989) observed that in turbot, on average, 60% of eggs were not viable due to postovulatory deterioration in the female’s body. The deterioration registered in the ovulated eggs may be connected with an oxygen deficit occurring after the release of the eggs from follicles surrounded by blood vessels. This hypothesis can be supported by the viability of ovulated eggs in living and dead fish: over-ripening is much faster in dead individuals (Korovina, 1986). 5.4.2.2 Viability of Ovulated Eggs In Vivo The viability of eggs retained in the female’s body in different fish species ranges from several hours to several days. The longest period of viability of ovulated eggs is registered in autumn-spawning fishes, in particular salmonids. For instance, a high proportion of normally developing eggs (>90%) was registered in coho salmon (Oncorhynchus kisutch (Walbaum)) 20 days after ovulation (Fitzpatrick et al., 1987). However, in carp (Cyprinus carpio (L.)), a substantial decrease in egg quality (with a fertilisation rate 10–15% lower than that just after ovulation) was observed 1.5–2 h after ovulation (Korovina, 1986). A very short viability period for ovulated eggs (about 1 h) is also reported for striped bass (Morone saxatilis (Walbaum); Stevens, 1966). The optimum time for fertilisation of the ovulated eggs retained in the female’s body seems to be both species-specific and temperature-dependent. For instance, eggs of rainbow trout (Oncorhynchus mykiss) stripped between 4 and 6 days after ovulation at 10°C consistently achieved the highest rates of fertilisation (Bromage, 1995). The lower fertility of eggs stripped immediately after ovulation is not well understood. In some species, the eggs may need a maturation period after ovulation, as the presence of slightly immature or ‘underripened’ oocytes has been observed (Bromage & Cumaranatunga, 1988). However, in many batch-spawning marine fish species with a short period of egg viability after ovulation, the highest egg quality seems to occur just after ovulation. The periods of viability of ovulated eggs in several cold-water marine fish species are given in Table 5.3. In cod, which will spawn naturally in captivity, over-ripening may occur if the fish are stressed. When the mature cod were separated in pairs in smaller tanks, some spawned at irregular intervals, and low fertilisation rates and the occurrence of abnormal embryos were registered (Kjesbu, 1989). In turbot, post-ovulatory over-ripening of the eggs was registered even when the fish were stripped daily. A clear indication was obtained of the effect of temperature on the rate at which eggs lost their ability to be fertilised (Howell & Scott, 1989). The fertilisation rate 24 h after ovulation was reduced to about 80% at 14°C and to 30% at 18°C. Thus, maintaining turbot brood stock at a comparatively low constant temperature during the spawning period would reduce the rate of over-ripening and allow eggs of better quality to be obtained. In Atlantic halibut, accurate monitoring of the ovulatory cycles may allow the eggs to be stripped at times close to ovulation. A wide variation

170

Table 5.3


Period of viability of ovulated eggs (h) in some marine fish species.

Species

Cod Turbot Halibut Wolf-fish

In vivo

In vitro

Viability

°C

Source

Viability

°C

Source

–

– 12–14 4.0 4.0

– McEvoy (1984) Bromage et al. (1994) Moksness & Pavlov (1996)

9 – 6 13

5.0 – 4.0 4.0

Kjørsvik & Lønning (1983) – Bromage et al. (1994) Moksness & Pavlov (1996)

10 12 >24

in the periods of over-ripening of eggs from different females is often observed, and indications of ‘under-ripeness’ have been reported for the eggs of female Atlantic halibut stripped very close to ovulation (Bromage et al., 1994; Holmefjord, 1996; E. Kjørsvik, unpublished data, 1996). In wolf-fish, the over-ripening of eggs has not been registered owing to the comparatively rapid (within 24 h) deposition of ovulated eggs by females. At the same time, eggs stripped just after the opening of the genital pore, indicating the onset of ovulation, were often of poor quality, and showed some incidence of ‘under-ripening’ (D.A. Pavlov, unpublished data). 5.4.2.3 Viability of Ovulated Eggs In Vitro Eggs stripped and stored in the ovarian fluid seem to undergo over-ripening in a similar way to that which occurs in the female’s body. However, for the ovulated eggs of many species, the rate of their deterioration is higher when they are stored in vitro, apparently owing to a lower oxygen supply than in the female’s body, or to the influence of the components of atmospheric air. For example, ovulated eggs of rainbow trout retain viability for at least 10 days in vivo, but they may be kept in an external medium for a few hours only before fertilisation, and the best results are achieved if eggs are fertilised within 1 h of being stripped from the female (Bromage & Cumaranatunga, 1988). In Atlantic halibut, the periods of viability of ovulated eggs in vivo and in vitro seem to be similar (see Table 5.3). The eggs from multiple batch spawners such as cod, halibut and turbot must all be fertilised within a few hours after ovulation (see Fig. 5.20). 5.4.2.4 Changes in the Eggs The structural changes which are responsible for the deterioration of eggs during overripening are not well researched. In their review, Kjørsvik et al. (1990) noted that changes due to over-ripening were often described as visible discoloration or non-transparency, the fusion of cortical alveoli and a ‘dimpled’ appearance of the cytoplasm. However, such changes are visible only after the viability of the eggs is already significantly reduced. Repeated inseminations of eggs stored in the ovarian fluid show a decrease in the proportion of normally cleaved eggs within a few hours, and a steady increase in the proportion of eggs with abnormal cleavage and uncleaved blastodiscs (Fig. 5.19). During the storage of eggs for a longer time, the proportion of unactivated eggs also increases. The eggs with


171

Figure 5.19 Morphology of common wolf-fish eggs before and after insemination. (a, a1) Egg before insemination; (b, b1) fertilized egg at 3 h after insemination; (c, c1) unfertilised egg 24 h after release into water. bl, blastodisc; ca, cortical alveoli; fs, fibrous structure; gr, granule; mc, micropyle canal; mf, micropyle funnel; mp, micropyle pit; od, oil droplet; ps, perivitelline space; yl, yolk (Pavlov & Moksness, 1996a). Reproduced with permission from Elsevier Science.

an uncleaved blastodisc are activated by spermatozoa, but fertilisation (i.e. a fusion of male and female pronuclei) does not take place. At the same time, apparently abnormal cleavage is possible only after fertilisation. Abnormal cleavage of over-ripened eggs, as well as the formation of abnormal embryos, have been described in many fresh-water fish (see review by Korovina, 1986), as well as in cod (Kjesbu, 1989), Atlantic halibut (Bromage et al., 1994; Shields et al., 1997) and wolf-fish (Moksness & Pavlov, 1996). For cod and halibut, the effects of over-ripening (in vitro) are shown in Fig. 5.20, which shows that 8 h storage of unfertilised eggs in ovarian fluid resulted in a significant decline in normal blastomeres and hatching success. It is also worth noting that fertilisation success for halibut eggs remained reasonably high throughout 24 h egg storage in ovarian fluid, thus illustrating that fertilisation rate is not a good criterion for halibut egg quality. The changes which are responsible for the appearance of unactivated eggs have been described in common wolf-fish. Most of the eggs kept in ovarian fluid were unchanged, but in approximately 10%, a small perivitelline space had formed after 24 h at 7.0°C. The cortical reaction in these eggs was not complete: only some of the cortical alveoli were broken at the animal pole of the egg (Pavlov & Moksness, 1996b). Apparently, these eggs could not be fertilised. After being placed into water, the perivitelline space increased slightly, but the cortical alveoli remained, and the blastodiscs did not appear (Fig. 5.19c, c1). Therefore, the breakdown of at least some of the cortical alveoli in the egg represented an event which was associated with over-ripening.

172


Cod

Halibut

100

Percentage

80 60 40 Fertilisation Normal blastomeres Hatching

20 0 0

5

10

15

20

25 0

5

10

15

20

25

Storage of unfertilised eggs in ovarian fluid (h) Figure 5.20 Effects of egg ageing (over-ripening) on fertilisation success, percentage of normal blastomeres and hatching success in cod and halibut. Eggs and milt were stripped from newly caught mature cod and from broodstock halibut. Over a time-period of 24 h, unfertilised eggs were stored in ovarian fluid, and milt were stored dry (5°C). Samples were fertilised in seawater at set time intervals. Arrows indicates when the ageing effects become significant, (E. Kjørsvik, A. Thorvik and L. Schei, unpublished data, 1996/1997).

However, the events leading to the appearance of abnormal cleavage remain unknown. Mechanisms regulating cell cycles and embryonic development will clearly be involved, and it has been established from several studies that embryo quality is linked to physiological mechanisms. One important aspect of normal cell cycles is the available energy charge in a cell, as measured by the adenylated phosphates (ATP, ADP, AMP). Recent investigations of egg ageing in cod (in vitro) showed that the ATP content of eggs during the process of over-ripening declined faster than that of any other parameter observed (E. Kjørsvik et al., unpublished data, 1996). There was a significant reduction in the ATP content of the egg after only 4 h storage in ovarian fluid before fertilisation, and fertilisation success and percentage of normal blastomeres were significantly reduced after 8 h storage. Other biochemical and physiological changes in the eggs which occur during overripening are described in Section 4.4.1, and in the reviews by Bromage & Cumaranatunga (1988) and Kjørsvik et al. (1990).

5.4.3 Change in Egg Quality Over the Spawning Season Egg quality is often lowest in females that spawn at the beginning of the spawning season. However, the general trend is the highest egg quality at the beginning (except the first batches in batch-spawning fish) or in the middle of the spawning season, and a decline in quality towards the end. For example, the eggs from several cod earlier spawners were smaller than eggs from later batches, and these eggs were characterised by lower quality (Kjesbu et al., 1990; Marteinsdottir & Steinarsson, 1998). At the same time, the mean egg size shows a


173

steady decline throughout the spawning season, from approximately 1.4–1.5 mm to 1.2– 1.3 mm. This has been reported for fish from a brood-stock (Kjørsvik, 1994; Mangor-Jensen et al., 1994) and for those collected in the wild (Marteinsdottir & Steinarsson, 1998). According to the observations of the latter authors, egg size seems to be positively correlated with egg quality. Poor egg quality in the last batches of cod eggs was also observed by Kjesbu (1989). A decrease in egg diameter and an increasing proportion of abnormal eggs were registered over the spawning season of cod from the White Sea, based on ichthyoplankton samples (D.A. Pavlov, unpublished data). Substantial decreases in the dry weight of egg batches have been registered over the spawning season of captive Atlantic halibut (Evans et al., 1996). In common sole, egg size tended to decline during the spawning season both in captivity and in the wild, and the shortest larvae that hatched from the smallest eggs might not be able to accept Artemia nauplii as a first food (Baynes et al., 1993). The smallest mean egg diameters (5.0 and 5.2 mm) with the largest CVs (7.8 and 5.8%) were observed in two common wolf-fish females which spawned at the beginning of the breeding season. For these females, no eggs cleaved normally (Pavlov & Moksness, 1996a). In the brood stock of common wolf-fish, the diameter of the eggs of repeat-spawning females was lowest at the beginning of the breeding season (November), increased towards the middle of the season (January–February) and decreased towards its end (April), but egg quality was stable throughout the season. The lower quality of the eggs towards the end of the spawning season in batch-spawned fishes can be explained by an exhaustion of the energetic reserves of the females.

5.4.4 Maternal Effects In general, an improvement in egg quality from the first to the second and third spawning seasons has been reported for several fish species (see review by Brooks et al., 1997). A decreased egg mortality in second spawners was shown in the same individuals of coastal cod from a brood stock (Solemdal et al., 1995). In this species, egg size increased significantly from the first to the second spawning season, with a smaller increase in third-time spawners kept at the same level of condition (Kjesbu et al., 1996). In cod collected in the wild, eggs of the largest size (>1.5 mm diameter) and with the highest quality tended to be produced by the largest females and, in general, by the older females. The female’s characteristics (length, weight, age, condition factor) were significantly related to egg size, which in turn, was highly correlated with the components of larval viability (Marteinsdottir & Steinarsson, 1998). In Atlantic halibut, the fertilisation rate of eggs from a repeat spawner and two first-time spawners was 81% and 56%, respectively. In addition, these two groups of eggs had different biochemical compositions (Evans et al., 1996). However, egg quality was not different in first and repeat spawners of common wolf-fish (Pavlov & Moksness, 1996a). Preliminary data indicate that in a single population of rainbow trout, females that produce better-quality eggs in their first spawning season (year) also do so in the subsequent season, suggesting that there are genetic influences on egg quality (Brooks et al., 1997). In turbot, the largest fish tended to produce the largest eggs, and in both subsequent years the same females produced the largest or the smallest eggs (Howell & Scott, 1989).

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5.4.5 Conclusions In conclusion, fertilisation success and blastomere morphology generally show good correlation with embryo and larval viability, and blastomere morphology in particular seems to be a valuable tool for the assessment of egg quality in fish. The principles of such morphological criteria are very similar in fish and in mammals, and these criteria represent the only non-invasive scoring system for egg or embryo quality evaluation. Egg quality is clearly linked to physiological mechanisms, and there seems to be an important genetic basis for offspring viability. Only few studies have examined the possible long-term effects of poor egg quality on juvenile viability, and more studies should aim at understanding the mechanisms controlling egg viability and the long-term effects of varying egg quality on the development and functionality of the growing fish. Owing to the very high variability in egg quality and offspring viability from brood-stock fish, egg-quality parameters should be included in future quality certification from producers of juveniles. For the species considered in this book, a careful assessment of egg quality should be carried out when embryos are at the 8–16-cell stage (preferably) of cleavage, and the eggs should be divided into several categories, as shown in Fig. 5.21. In this scheme, the eggs with cortical alveoli without blastodiscs are referred to as ‘unactivated’ despite the breakdown of some cortical alveoli. Eggs with a non-cleaved blastodisc (one cell) are referred to as activated and unfertilised. Eggs with abnormal cleavage are apparently fertilised, and the fertilisation rate may therefore be much higher than the proportion of eggs which are able to undergo normal development. A normal good-quality egg batch may thus be described as exhibiting a high fertilisation rate, pelagic eggs have good buoyancy, the eggs are transparent, with no or very few visible cortical alveoli in the cytoplasm, the early cleavages are synchronous and produce the correct number of cells, and the early blastomeres are regular in shape with clear margins between them and good contact between adjacent cell membranes. An assessment of fertilisation success and the proportion of normal blastodiscs currently seems to be the only ‘universal’ criterion for egg quality in cold-water marine fish, and the quality characteristics used for fish are very similar to those used in the present assessment of embryo quality for in-vitro fertilisation of mammals (including humans). This procedure

Eggs

Activated

Normal cleavage (Fig. 5.15a)

Abnormal cleavage (Fig. 5.15b–j)

F e r t i l i s ed

Unactivated

Incomplete cortical reaction (Fig. 5.19c, c1)

Uncleaved blastodisc (Fig. 5.12b)

U

n

f

e

r

Damaged yolk membrane, whitish (Fig. 5.12d)

t

i

l

i

Yellow or brown oil structure (Fig. 5.12e)

s

e

Resorbing, with follicular layer (Fig. 5.12f)

d

Figure 5.21 Categories of artificially inseminated eggs obtained in common wolf-fish from a brood-stock.


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may be done in a few minutes per egg batch, and is easy to perform with a simple stereomicroscope.

5.5 Sperm Production and Quality Sperm quality and its productive characteristics, including mainly ejaculate volume, concentration and motility, has a decisive influence on the success of artificial reproduction in fish. As in spermatozoon morphology, these characteristics show a great variation in different species, depending on the mode of reproduction. In general, sperm quality is less related to husbandry conditions and environmental factors than egg quality.

5.5.1 Features of Sperm Production and Quality 5.5.1.1 Morphology Sperm composition and malformations in the structure of spermatozoa have been studied in a restricted number of species. In the sperm of turbot stripped from 47 to 80 days before the beginning of the spawning period of the females, numerous spermatids were present. Some spermatozoa had slightly condensed chromatin and a middle piece containing numerous vesicles, while others had condensed chromatin and a middle piece containing only a few vesicles. Mature cells had dense chromatin. After the end of the spawning season of the females, no spermatids were present and the middle piece always contained very few vesicles. Different degrees of chromatin expression were connected with the decondensation caused by the ageing process. In addition, some degenerated spermatozoa appeared in which the plasma membrane was broken or had disappeared. Thus, the sperm collected after the spawning period of the females was not suitable for artificial fertilisation (Suquet et al., 1998a). In Atlantic halibut, observations of spermatozoa at the end of the motility phase showed drastic distortions of the flagellum. A spermatozoon of common wolf-fish from the brood-stock normally has one flagellum, but in some spermatozoa two basal plates with two axonemes are observed. This structure is regarded as abnormal (Pavlov et al., 1997). However, the spermatozoa of two closely related species, Zoarces elongatus and ocean pout (Macrozoarces americanus), normally possess biflagellar tails (Koya et al., 1993; Yao et al., 1995). As in turbot, spermatids at different stages of transition to spermatozoa are found in the ejaculate of common wolf-fish mainly at the beginning of the breeding season. Large numbers of spermatids together with motile mature spermatozoa are observed in the seminal lobule lumen of Zoarces elongatus, a viviparous fish with internal fertilisation, and in several other fish species with ‘semi-cystic’ asynchronous spermatogenesis occurring partly outside cysts (Mattei et al., 1993). The presence of spermatids in the ejaculate is apparently not normal, and can be caused by the stripping procedure. In addition to spermatids and normal spermatozoa, smaller cells without flagella showing agitated movements are observed in common wolf-fish ejaculate (Pavlov et al., 1997). The proportion increases towards the end of the breeding season, and they are probably represented by degraded spermatozoa.

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Table 5.4

Main sperm parameters in some marine fish species.

Species

Maximum GSI (%)

Ejaculate volume (ml)

Cod Turbot

16 0.8

– 0.2–2.2

– 0.7–11.0

Halibut

–

1–92

11.9–37.2

0.2–10.6

0.012–1.198

Wolf-fish

0.1

Ocean pout 2.2

5–20

Sperm concentration (¥109 spz ml-1)

0.00756–0.215

Spermatocrit (%) 59 40

Sperm motility

1 s–120 min 1–17 min

40–100

60–70 s

–

>48 h

0.8–1.8

>24 h

References

Suquet et al. (1994) Suquet et al. (1994, 1995) Billard et al. (1993); Suquet et al. (1994) Johannessen et al. (1993); Moksness & Pavlov (1996) Yao & Crim (1995); Yao et al. (1995)

5.5.1.2 Gonadosomatic Index and Ejaculate Volume In species with seasonal reproductive cycles, the gonadosomatic index (GSI, i.e. (gonad weight/body weight) ¥ 100) indicates the efficiency of spermatogenesis. GSI is related to spawning behaviour, and in the case of external insemination it is lowest in species spawning in stagnant water and in couples when a close contact between the genital openings of the male and female can be achieved. Among cold-water marine fish species with external insemination, the lowest GSI is registered in common sole and in turbot (0.2% and 0.8%, respectively), and these low values are apparently connected with the spawning behaviour of the species. As a rule, species with internal insemination possess a comparatively low GSI. For example, it is lowest in common wolf-fish in comparison with other marine and fresh-water species (Table 5.4). In addition, the common wolf-fish differs from the majority of fish with seasonal spawning by having a relatively stable GSI and spermatozoa production over the entire year. In the males of ocean pout, another fish with internal insemination, the values of the GSI and ejaculate volume are much higher than in common wolf-fish, and this may be connected with the different amounts of energy put into sperm production owing to features of parental care (the eggs are protected by the male in wolf-fish and by the female in ocean pout). The volume of sperm released by stripping (or collected with a catheter, as in ocean pout) is comparatively low in species with a low GSI, and depends on the phase of the breeding season and the stripping frequency. In turbot, both fortnightly and weekly stripping result in the release of decreasing sperm volumes per stripping as a function of time. However, an increase in collection frequency has no effect on the total sperm volume collected during a 2-month period (Suquet et al., 1994). In males of common wolf-fish stripped at approximately monthly intervals, ejaculate volume was minimal at the beginning and at the end of the breeding season and reached maximum values in August, before egg ovulation in the majority of females (Fig. 5.22a).


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a Ejaculate volume (ml)

‡

Sperm concentration (ⴛ106 ml–1)

b

Ap r May Jun

Jul

Aug

Sep

Oct

Nov

Dec Ja n

Feb Mar

Figure 5.22 (a) Volume of ejaculate, and (b) concentration of sperm in 20 males of common wolf-fish repeatedly stripped during the breeding season. Error bars are the standard errors (Moksness & Pavlov, 1996).

5.5.1.3 Concentration The concentration of spermatozoa is lowest in species with internal insemination (Table 5.4). For example, in common wolf-fish it is about 66 times lower than in Atlantic salmon, Salmo salar L. (Pavlov & Moksness, 1994b). In marine fish species with external insemination, sperm concentration is low in turbot, and this concentration is similar to that in tilapia (Oreochromis spp.), which has one of the lowest concentration values. Sperm concentration depends strongly on the phase of the breeding season and the stripping frequency. In turbot, sperm concentration increases during the spermiation period. However, after a sampling period of 2 months, the sperm concentration observed at the last stripping was significantly lower in fish stripped weekly or fortnightly than in individuals stripped monthly (Suquet et al., 1994). In common wolf-fish, the maximum sperm concentration is registered during the peak of egg ovulation in females (Fig 5.22b). Stripping the sperm at different frequencies showed that sperm concentration did not decrease if the sperm was sampled every 12–14 days (Pavlov & Radzikhovskaya, 1991). Spermatocrit can reflect sperm concentration in the majority of species. For instance, spermatocrit of ocean pout with a very low sperm concentration is also low (see Table 5.4). In Atlantic halibut, a positive relationship between sperm concentration and spermatocrit has been reported (Tvedt et al., 2000). However, in turbot, despite a low sperm concentration,

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comparatively high spermatocrit values are found (see Table 5.4), which may be caused by the presence of spermatids and degenerated spermatozoa in the ejaculate. Total sperm production (i.e. the total number of spermatozoa in the ejaculate) does not seem to depend much on the weight, age or number of spawnings in males of marine species, e.g. cod, turbot and common wolf-fish. However, a high variation in sperm production is observed in many species, and some males are characterised by very low production characteristics over the entire breeding season, possibly showing the genetic effects of sperm quality. 5.5.1.4 Motility The motility of activated spermatozoa is usually assessed based on its duration (when all or most of the spermatozoa stop exhibiting progressive forward movement), or using ranked scores for percentages of motile spermatozoa. The rank ranges used by the authors are different (see reviews by Trippel & Neilson, 1992; Billard et al., 1995). The swimming speed of sperm was measured in cod using a video-recorder of enlarged images of sperm against a haemacytometer grid pattern. The average swimming speed of motile spermatozoa was 75 mm 30 s-1, and the fastest recorded swimming speed was 1000 mm 30 s-1 (Trippel & Neilson, 1992). Stroboscopic illumination and dark-field microscopy was used to measure flagellar beat frequency, cell velocity and the distance covered by spermatozoa of salmonids (Cosson et al., 1985). The intensity and duration of spermatozoa motility indicates the quality of the males, and can be used to predict fertilisation rate. However, high sperm motility is not always essential for successful fertilisation. For example, immotile vibrating spermatozoa obtained from some male cod showed a similar fertilisation level to that found when exclusively motile sperm was used (Trippel & Neilson, 1992). In marine fish with external insemination, the dilution of sperm in a hypertonic medium initiates motility in the spermatozoa. In turbot, sperm motility is triggered by dilution in both hypertonic and isotonic media. The duration of movement is lower in isotonic diluents, but the reasons for sperm activation in these diluents are not understood. A decreased percentage of motile turbot spermatozoa was registered after increasing the dilution of the sperm, and to protect spermatozoa against dilution the addition of proteins is necessary (Suquet et al., 1994). In marine fish, the duration of sperm motility is generally higher than that in salmonids and fresh-water species. In Atlantic herring, Clupea harengus L., and Pacific herring, C. pallasi Val., spermatozoa show vibrating movements, and intensive forward movements are observed only in the micropyle area. The spermatozoa of these species remain viable for 7 and 1 days after release into water, respectively (Makeyeva, 1992). In Atlantic halibut at 18–20°C, the percentage of motile spermatozoa declines slowly in the first minute of motion and abruptly thereafter. The duration of spermatozoa motility is associated with the decrease in flagellum beat frequency and spermatozoa velocity. The beat frequency remains fairly stable (40–50 Hz) over the first 55 s after dilution, but then drops suddenly to values around 10–15 Hz. Over the same period, spermatozoa velocity declines gradually. The possible physiological and biochemical reasons for the sudden drop in beat frequency of Atlantic halibut spermatozoa have been discussed (Billard et al., 1993, 1995). Spermatozoa of common wolf-fish and ocean pout, which are both characterised by internal insemination, are motile in undiluted ejaculate. They are apparently immotile in the genital tract of the male, and the reasons for their activation in the


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external medium are not known. Their motility lasts more than 1 day (see Table 5.4). Longlived spermatozoa are connected with the need for the sperm and the eggs to be very well mixed in the female’s ovarium in order to increase the probability of contact between gametes. Spermatozoa motility may depend on the phase of the spawning season and the stripping frequency. The motility of turbot spermatozoa decreased significantly at the end, and for 2–3 months after the end, of the spawning period of the females (Suquet et al., 1998a). In this species, an increase in stripping frequency from monthly to weekly did not influence the duration of spermatozoa motility (Suquet et al., 1994). However, high-frequency stripping (every 3–14 days) in males of common wolf-fish led to a substantial decrease in the duration of spermatozoa motility (assessed after their dilution in marine water) during the breeding season (Pavlov & Radzikhovskaya, 1991). 5.5.1.5 Fertilising Capacity Fertilisation rate is the most reliable indicator of sperm quality. However, for an appropriate use of this test, the minimal sperm-to-egg ratio and the minimal contact time between gametes required for successful insemination should be assessed. These parameters are known for a limited number of species. The results of experiments with the insemination of turbot and common wolf-fish eggs using various dilutions and contact times are shown in Fig. 5.23. In turbot, when high-quality egg batches are used, about 6000 spermatozoa per egg are required to obtain maximum fertilisation success. This value is low compared to that in other fish species, and it is connected to the high fertilisation capacity of turbot spermatozoa. Owing to this capacity, sperm production in turbot is comparatively low. In addition, the sperm requirements of turbot eggs seem to depend on egg quality: more sperm is needed for the insemination of egg batches with lower viability. In common wolf-fish, the minimal sperm-to-egg ratio to achieve high fertilization rates depends on the contact time between gametes, and is approximately 200 000. This value is similar to that reported for salmonids. The large number of spermatozoa required for the successful (internal) fertilisation of wolf-fish eggs may be connected to their low velocity in the viscous ovarian fluid of the female, and to the comparatively large diameter of the eggs. In turbot, to reach a high fertilisation rate, the contact time between gametes should be at least 3 min at a spermatozoa-to-egg ratio of 6000, and this time should be increased to 4–5 min at a ratio of 1500 spermatozoa per egg. An extremely long contact time between gametes (>2 h) is required for common wolf-fish. This contact time depends on the spermto-egg ratio: if this ratio is low, the contact time should be increased to 7 h. Thus, the spermto-egg ratio and the contact time between gametes are inversely related, and apparently an increase in the contact time is accompanied by a higher probability that the spermatozoa will reach the micropyle. However, in the majority of fish with external insemination, the contact time is restricted by the short life of spermatozoa in water. 5.5.1.6 Biochemistry and Oxygen Consumption The biochemical composition of sperm and seminal fluid in fish is reviewed by Piironen & Hyvärinen (1983) and Billard et al. (1995). The biochemical composition and oxygen con-


s

s

180

b

s

s

a

c

d

Figure 5.23 Fertilisation rate in relation to sperm-to-egg ratio and contact time between gametes in (a,b) turbot and (c,d) common wolf-fish. (a) Vertical dashed line indicates a ratio of 6000 spermatozoa per egg. The eggs are from different breeders with high fertilisation rates. (b) The eggs from five batches; sperm-to-egg ratio 6000. (c) The eggs from one female; contact time between gametes 2, 7 or 12 h. (d) The eggs from one female; sperm-toegg ratio >200 000 (Pavlov, 1994a, b; Suquet et al., 1995, modified). Reproduced with permission from Elsevier Science.

sumption of spermatozoa in relation to their quality were described by Gosh (1989). In particular, the level of lactic acid may indicate sperm quality: it is higher in the sperm of older fish, and a negative correlation was observed between this level and sperm concentration on the one hand and fertilisation rate on the other in some fresh-water fish species. An intensive forward movement of spermatozoa is possible only at a certain level of ATP. A restriction of the oxygen supply leads to a sudden decrease in the motility of spermatozoa. The resumption of good conditions for gas exchange is associated with an increasing synthesis of ATP, and the motility increases again. However, in turbot, no significant decrease in ATP content in the sperm was recorded during the spermiation period despite observed differences in spermatozoa concentration and motility (Suquet et al., 1998a).

5.5.2 Influence of Environmental Factors on Sperm Quality Sperm quality depends on the husbandry and feeding conditions, but this relationship has rarely been investigated. The duration of spermatozoa motility depends on the temperature:


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it decreases at higher temperatures. For example, in Atlantic salmon, the duration of forward movements is 53 s at 5°C and only 22 s at 30°C (Makeyeva, 1992). The photoperiod during cultivation of the brood-stock seems to have little influence on sperm quality. In particular, in turbot males receiving an annual light and temperature cycle compared with those receiving a 6-month contracting schedule, the sperm parameters (ejaculate volume, sperm concentration and motility) were similar (Suquet et al., 1994). The difference in sperm volume and sperm concentration between common wolf-fish males kept in different light cycles (18L:6D and 6L:18D) for a year was not significant (Moksness & Pavlov, 1996). The influence of osmotic pressure and pH on the motility of spermatozoa was studied in Atlantic halibut. The spermatozoa were motile in a range of osmotic pressure from 300 to 1150 mOsmol kg-1, and the percentage of motile cells increased from pH 6.5 to 8.5 and declined above pH 8.5. The duration of forward movement reached a maximum at pH 7.5–8.0 (Billard et al., 1993).

5.5.3 Sperm Storage In aquaculture practice, sperm storage is necessary when the periods of egg and sperm maturation in fish brood-stock are different, and this can be used for selective breeding of fish from different stocks. In general, fresh sperm collected from males can be stored in vitro for a short period of time. One reason for the fast deterioration of spermatozoa is an insufficient oxygen supply, resulting in a lower level of ATP. During sperm storage, the amount of carbohydrates decreases and the level of lactic acid increases, indicating intensive glycolysis. An increasing amount of lactic acid has a negative effect on the viability of spermatozoa (see reviews by Gosh, 1989; Billard et al., 1995). Sperm storage can be improved by stocking fewer sperm with a larger volume of available air, and sperm concentration can be reduced by diluting the sperm with an extender (rich in K+ or sucrose) that does not activate spermatozoon motility (Billard, 1988). In Atlantic halibut semen stored on ice for 24 h, the percentage of motile spermatozoa did not exceed 40%. In this species, the fertilisation rate declined by about 20% over 16 h after the storage of sperm at 1–3°C (Martin-Robichaud & Rommens, 2000). The experiments showed that Atlantic halibut sperm remained viable after short-term freezing and thawing of diluted sperm in the presence of 10% propanediol. The capacity of the sperm to be frozen declined rapidly after collection and storage in vitro for a period of 7 h (Billard et al., 1993). In common wolf-fish, sperm stored in undiluted ejaculate at 4°C had a high fertilising ability for at least 10 h after collection. This ability might be longer because of the long duration of spermatozoa motility (over 2 days). However, to date only the combined effect of egg and sperm storage has been reported (Moksness & Pavlov, 1996). Sperm storage capacity may depend on the phase of the spawning season. For example, in seabass stored at 4°C, this capacity decreases from 70 h at the beginning of the spawning season to 30 min in the middle and at the end of this season (Billard et al., 1977). In turbot, the short- and long-term storage capacities of sperm decreased as the spermiation period progressed (Suquet et al., 1998a). Cryopreservation is used for the long-term storage of sperm, and the sperm from more than 200 fish species have been cryopreserved. Although few practical applications have

182


been achieved to date (Billard et al., 1995; Chao & Liao, 2001), such techniques are in use in ‘gene banks’ which have been established to preserve the genes of different strains of wild salmon. Such techniques may also secure some sperm availability if sperm quality declines towards the end of the spawning period. During sperm collection for cryopreservation, several factors are essential in order to maintain sperm quality: (1) collect the sperm without any contamination by faeces, blood or scales; (2) hold the sperm by providing air or oxygen for respiration; (3) maintain the temperature of collected milt at 4°C. Collected sperm is diluted in a freezing medium containing extenders and cryoprotectants. In addition to the universal cryoprotectant dimethyl sulfoxide (DMSO), other single cryoprotectants, combined cryoprotectants, and cryoprotectants with egg yolk or sucrose have been used. The milt mixture is placed in a tube, frozen and stored in liquid nitrogen. An alternative method of freezing sperm is the formation of pellets in hollows on the surface of dry ice. The sperm is thawed in a waterbath with the addition of diluting solutions with an appropriate ion content and pH. The freezing and thawing protocols vary with species, and higher quantities of thawed milt are needed to obtain fertilisation rates which are comparable to those obtained when fresh milt is used. The results obtained for some species allow the use of cryopreservation in routine aquaculture practices. For example, turbot spermatozoa can be cryopreserved in DMSO mixed with egg yolk, or in a sucrose solution with 10% DMSO and 10% egg yolk. Sperm of this species was stored during a 9-month period in liquid nitrogen. No significant differences in spermatozoa motility, fertilisation rate, hatching rate, survival and wet weight of larvae were observed using fresh or frozen–thawed sperm (Suquet et al., 1998b).

5.6 Selective Breeding In farm animals and plants, selective breeding has played an important role in domestication, increasing yields, survival rates and improving product quality. Today, one cannot really imagine any commercial animal husbandry or plant production without genetically improved livestock or plants. Quantitative genetics is the theoretical basis on which animal and plant breeding programmes are founded. Although it has been applied in agriculture since around 1920, the theory has only recently been applied to aquaculture. Prospects for genetic improvements in aquaculture species are even more promising than for domestic animals (reviewed by Gjedrem, 1975, 1992; Kinghorn, 1983; Gjerde, 1986), mainly owing to higher reproduction capacities and the large additive genetic variation seen for traits of economic importance (e.g. growth rate) in many species. Because of the high reproduction capacities, a small number of new breeders are needed in each generation, and high selection intensity can be consistent. The expected selection response also depends on the choice of selection method, of which several are available for obtaining additive genetic improvement. The methods will differ according to which type of breeder will provide the information needed for selection decisions. The objective of all methods is to maximise the probability of correctly ranking the animals with respect to their breeding value, which is


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an estimate of each individual’s ability to produce high/low-performing offspring. Which method to choose depends on several factors. Among the most important is the heritability of the trait(s), the nature of the trait (e.g. normally distributed or binary; whether records can be obtained on live individuals, etc.) and the reproductive capacity of the species. Owing to the high fecundity of aquaculture species, the selection methods usually applied are individual selection (mass selection), family selection or a combination of the two (combined selection). The last method is the most efficient, where the best families, and subsequently the best individuals within the best families, are selected according to the breeding goal defined. Several large-scale breeding experiments and breeding programmes have demonstrated a substantial response to selection for growth rate. The following estimates of genetic gain per generation have been reported (reviewed by Gjerde, 1986). Coho salmon 10% Rainbow trout 13% Atlantic salmon 10–25% Channel catfish 12–20% Nile tilapia 17% The average of these estimates is 15% genetic gain per generation, indicating that the growth rate can be doubled in less than seven generations of selection. Marine species are still new in intensive fish farming, and to date no selection experiment or results from a commercial selection scheme have been reported. To optimise a breeding programme, knowledge about genetic parameters such as phenotypic, genetic variation and heritability for the relevant trait(s) is needed, together with estimates about genetic correlations between traits and in-breeding depression. Until this information is available, the construction of a selection scheme for marine species has to be based on knowledge of other fish species. These will be mainly salmonids, where large-scale breeding programmes have been applied since the early 1970s.

5.6.1 Expected Benefits The benefits of a genetic improvement in growth rate are reductions in both fixed and variable production costs. The latter is due to a reduced energy requirement for maintenance throughout the production process. It is generally assumed that an improved feed conversion rate will be obtained as a correlated response to increased growth rate.

5.6.2 Phenotypic Value and Variance The value observed when a character is measured or scored for an individual is called the phenotypic value of the individual. The phenotypic value (which is the only component which can be measured) can be partitioned into two components, one attributable to the influence of genotype, i.e. the particular assemblage of genes possessed by the individual, and

184


one attributable to the influence of the environment, i.e. all non-genetic circumstances that influence the phenotypic value. Symbolically, this is P =G+E

(5.1)

where P is the phenotypic value, G is the genotypic value and E is the environmental deviation. Thus, we can think of the genotype conferring a certain value on the individual, and the environment causing a deviation from, and masking, the genetic value of that individual. The mean environmental deviation in the population as a whole is then taken to be zero, so that the mean phenotypic value is equal to the mean genotypic value. The genetics of a character centre around a study of its variation. The variance in phenotypic values is the phenotypic variance (VP), which is also termed the total variance. The variance in genotypic values is termed the genetic variance (VG), and the variance caused by environmental deviation is the environmental variance (VE). Symbolically, VP = VG + VE + VE = VA + VD + VL + VES + VER

(5.2)

In the equation 5.2, VG is further partitioned into three separate components: VA is the additive genetic variance, or the variance due to the average additive value of the gene. VD is the dominance genetic variance, or the variance due to the value of intra-locus interaction among genes. VL is the interaction genetic variance, or the variance due to the value of interlocus interaction among genes. The sum of VD and VL is termed the non-additive genetic variance, and cannot be used in a selection scheme based on pure breeding for additive genetic improvement. The environmental, or non-genetic, component is divided into two separate components: VES is the variance due to the value of systematic recognisable environmental causes. Examples of systematic causes that are at least partly under experimental control in fish farming are age, nutritional factors, water temperature, tank, cage or pond effects, and sex effects. VER is the variance due to the value of unknown or random environmental causes, which therefore cannot be eliminated by experimental or testing design. The value of VG, which is the breeding value, cannot be measured but only estimated, and all other factors included in the model more or less mask the breeding value. The ratio VG/VP express the extent to which an individual’s phenotypes are determined by their genotypes. This is heritability (h2) in the broad sense. The ratio VA/VP expresses the extent to which an individual’s phenotypes are determined by the additive value of their genes. This is heritability in the narrow sense. Heritability (h2) determines the degree of resemblance between relatives, and is of the greatest importance in the additive genetics of animals. If the heritability for a trait is low, the most effective course is selection based on family selection, while individual selection might be effective when the heritability is medium or high.

5.6.3 Genotype by Environmental Interaction Genotype by environmental interaction implies that strains, progeny groups or individuals rank differently when kept under different environmental conditions. If the interaction is substantial, a separate breeding population may be needed for each particular environment.


185

For both Atlantic salmon and rainbow trout, a significant genotype by farm interaction has been reported for growth rate (Gunnes & Gjedrem, 1978, 1981). However, the interaction accounted for a relatively small proportion of the total phenotypic variance. The authors therefore concluded that only one breeding population of Atlantic salmon and one of rainbow trout are needed in Norway. A more serious genotype by environment interaction was reported when sibling groups of rainbow trout were reared in quite different temperatures (McKay et al., 1984) and production systems (Sylven et al., 1991). It is important that the genotype by environment interaction is investigated when a breeding scheme is planned for marine species.

5.6.4 Breeding Goal The traits to be improved through a selection programme have to be specified for each species, production condition and market. For a trait to be included as a breeding goal, the following prerequisites must hold:

• the trait must be of economic importance • it must be possible to measure or judge (score) the trait • the trait must show genetic variation between individuals When planning a breeding programme, the following breeding goals should be evaluated.

5.6.5 Growth Rate Growth rate is recognised as a principal factor in most aquaculture production, and this trait is included in most genetic improvement programmes for aquaculture species. An increased growth rate means shorter production cycles, reduced risks and the possibility of rearing bigger fish before they reach sexual maturity. Growth rate also shows a positive genetic correlation with feed conversion efficiency, which has a major impact on overall production efficiency.

5.6.6 Feed Efficiency Feed efficiency is probably the factor of highest economic importance in intensive production. This trait cannot be measured directly, but it is genetically linked to growth rate. This leads to a positively correlated response in feed-conversion efficiency when selection on growth rate is applied.

5.6.7 Disease Resistance An improved natural resistance against diseases might be an important breeding goal. Improving natural resistance will give a higher survival rate until harvesting. If a specific disease is stated as a breeding goal, it has to be combined with an adequate protocol for the testing and ranking of families or individuals.

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5.6.8 Quality Quality traits which can be improved through selection will be different for different species and have to be stated in accordance with the market.

5.6.9 Age at Sexual Maturation For some species, sexual maturation will develop before harvest size. Maturation will decrease the growth rate, and for some species maturation is also followed by reduced meat quality and increased mortality. The general breeding objective for this trait should therefore be a fish that reaches marked size before first sexual maturation.

5.6.10 Base Population and Brood-Stock Development For many species, the base population has to be founded from a wild population. If so, the base population should have as broad a genetic variation as possible. If domesticated breeders are available, own brood-stock should be compared with other available stocks before forming the base population. However, a selection within the present brood stock should be initiated as soon as possible.

5.6.11 In-breeding In-breeding generally results in in-breeding depression, which is noticeable as reduced performance, particularly for traits connected with reproductive capacity and viability. However, in-breeding might also depress the growth rate (Kincaid, 1976a,b; Gjerde et al., 1983). It is therefore important to keep the rate of in-breeding at a low level in a breeding programme. Within a population under selection, the rate of in-breeding per generation is a function of the number of sires and dams used as parents for each new generation. Assuming there is no genetic relationship among sires and dams, 50 sires and 50 dams will give an increase in in-breeding coefficient of 0.5% per generation. This is probably an acceptable rate of inbreeding for most traits.

5.6.12 Selection Methods 5.6.12.1 Individual Selection (Mass Selection). Individual selection is easy and inexpensive to practise, and its demand for test capacity is low. It represents a relatively efficient selection method for traits of high heritability which display continuous phenotypic variation, and which can be recorded in live animals. Growth rate is the only trait that fulfils these requirements and thus can be improved efficiently through individual selection. A breeding programme based on individual selection should be designed to restrict the accumulation of in-breeding in the population under selection in order to avoid the negative effects of in-breeding depression and loss of genetic variability. This can only be done by securing an effective population size which is large enough to prevent in-breeding. To secure


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an effective population size, a fixed number of progeny per selected pair can be stocked together for grow-out testing. Assuming a heritability for growth rate in the range 0.20–0.30, a selection scheme based on individual selection can be designed to keep the expected accumulation of in-breeding below 1% per generation. This will be sufficient to avoid inbreeding depression and maintain the genetic variation, and hence ensure a steady genetic selection response. Another strategy is to develop two separate genetic lines, where selection is applied within each line. The dissemination of genetic material should then be achieved by crosses between lines. 5.6.12.2 Family Selection Family selection will be more efficient than individual selection except for traits of very high heritability. When applying family selection, it is also possible to improve binary (either–or) traits (e.g. age at maturity, mortality) and traits which can only be recorded on dead animals (e.g. quality traits). To apply family selection, the relationships between all individuals in the population have to be known. To achieve this, families have to be reared separately until they can be tagged, or they have to be identified by genetic markers before selection. 5.6.12.3 Progeny Testing This method of selection is widely applied for farm animals such as cattle, sheep and goats. The selection is made among the parents on the basis of information from their tested progeny. As for family selection, the relationships between all individuals in the population have to be known, and the families have to be produced and identified. Progeny testing cannot be applied for species where mortality after spawning is total or high. This is not the case for the majority of marine species, and for marine species progeny testing can be applied in combination with family selection in large-scale breeding programmes. 5.6.12.4 Combined Selection This method optimally combines all the information that can add to our knowledge of the breeding value of an individual; it would include recorded information about the individual itself, and information about full sibs, half sibs and progeny, as well as pedigree information. It represents the general solution for obtaining the maximum rate of genetic gain, and other simpler methods are special cases of this method. Therefore, it is always the most efficient method. Selection indices are the most efficient way to combine information about an individual and its relatives, as well as information about specific traits. For combined selection, all the information is combined into an index of merit, where the traits are weighted according to their relative economic value. The index gives the genetic value of all individuals in the population, and breeders are selected in accordance with the index.

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5.6.13 Response to Selection The expected genetic gain (DG) or response to selection per generation depends on four parameters. The formula for individual selection is DG = i ¥ h2 ¥ sP

(5.3)

where i is the standardised selection differential (also called the selection intensity), h2 is the heritability of the trait and sP is the phenotypic standard deviation, i.e. the square-root of the phenotypic variance (VP). A more general formula, which is applicable to all methods of selection, is DG = i ¥ rTI ¥ sG

(5.4)

where i is as described above, rTI is the accuracy of selection, i.e. the correlation between the true and estimated breeding value, and sG is the additive genetic standard deviation, i.e. the square-root of the additive genetic variance (VA). The expected response to selection is directly proportional to the accuracy of selection and the selection intensity. The majority of marine species have a very high reproduction potential in culture, and a small number of individuals are needed as spawners. It is therefore possible to achieve high selection intensity, and thereby a high level of genetic gain.

5.6.14 Multi-Trait Selection When several traits are included as breeding goals, multi-trait selection is the most effective way to achieve genetic improvement in all traits. Multi-trait selection is based on selection indices (breeding value) estimated for each trait. The selection indices combine information about an individual and its relative, as well as information about several traits. All information is combined into an index of merit, where the traits are weighted according to their relative economic value. For all species in aquaculture where reproduction is controlled, full- and half-sib groups with a large number of individuals can be produced. This makes it possible to estimate breeding values and indexes with great accuracy. This fact, together with the probable high selection intensity, gives great opportunities for genetic progress. These opportunities should be used in effective selection schemes for all farmed species.

5.7 Modern Biotechnology and Aquaculture The genetic improvement of fish stocks represents a major challenge for aquaculture in order to enhance the use of available feed and land resources. In most, if not all, fish species the biological potential is considerable owing to the high heritable variation for production traits such as growth rate and disease resistance. Large-scale breeding programmes have successfully been implemented for several aquaculture species. Modern biotechnology will become increasingly more valuable as a supplementary tool towards achieving the growing demands


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of world aquaculture. Some potential applications of these sophisticated molecular methods are pedigree analysis by DNA fingerprinting, gene-mapping studies, and the identification of genetic markers or specific genes associated with desirable traits. All these approaches involve the amplification of nuclear or mitochondrial DNA, and make use of the polymerase chain reaction (PCR). With this technique, the gene or genetic marker of interest is easily amplified to visible amounts for a reasonable price within less than 2 h (Fig. 5.24). Since tiny amounts of tissue containing only a few copies of the desirable gene are sufficient as a template in this DNA replication process, the PCR technique has revolutionised all aspects of molecular studies, including those within aquaculture.

5.7.1 Molecular Pedigree Analysis Selective breeding programmes in aquaculture make use of family information, which requires that families are kept separately until the fish are large enough to be physically tagged. This imposes major economic and practical problems, and can induce environmental effects common to full-sibs. The identification of family groups by their specific DNA fingerprint may dramatically improve this situation. Fish from different families can then be reared together in the same tank even from the egg stage. This also allows larger numbers of families to be tested, and thus facilitates the use of higher selection intensities without a rapid increase in in-breeding. Repetitive DNA sequences, or so-called microsatellites, have successfully been used empirically to reconstruct pedigrees in fish populations with families mixed from hatching. Microsatellites are useful markers for genetic tagging, owing to their high number and variability within the genome. Hundreds of polymorphic microsatellites have already been identified in many fish species of economic value, but the numbers of potential microsatellite loci present in the fish genome are likely to be of the order of 100 000. In particular, AC dinucleotide repetitions were estimated to be present in numbers of 2.34 ¥ 105 at intervals of about 7000 base pairs in the genome of Atlantic cod (Brooker et al., 1994). The four most informative microsatellites identified in the Atlantic salmon genome were predicted to be sufficient to assign at least 99% of the offspring to the correct pair with 100 crosses involving 100 males and 100 females. An additional polymorphic microsatellite was required to correctly assign 99% of the offspring when the 100 crosses were produced with 10 males and 10 females (Villanueva et al., 2002). This indicates that parental assignment is feasible with the genetic markers currently available in several fish species. Both the efficiency and the costs of microsatellite-based pedigree analysis should be considered before this method is included in a breeding programme. In practice, the parents and the mixed offspring are genotyped by PCR amplification of the appropriate microsatellite loci from crude DNA extracts from small non-destructively sampled quantities of tissue such as fish scales, mucus or a fin clipping. Using this protocol, 2000 fish from a mixture of 500 families can be screened for 10 markers in less than a month, allowing 99% of the fish to be parentally assigned. For the time being, offspring have been assigned to parents of known genotype. However, with sufficient levels of variability, the identification of families may also be achievable in the absence of parental information. It should be noted that substantially more markers need to be screened for resolving individuals compared with family

190 Culture of cold-water marine fish

Figure 5.24 Detection of microsatellite markers by polymerase chain reaction (PCR). This microsatellite consists of tandem repeats of the dinucleotide CA. The number of repeats in such microsatellites varies with the individual (e.g. four repeats in fish I, and eight repeats in fish II). These variations can be detected by PCR amplifying the repeated DNA fragment with the flanking primers 1 and 2. This process yields amplified DNA products of different lengths as visualised by gel electrophoresis.


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discrimination. The steadily decreasing costs of genetic tagging may still not compete with traditional physical tagging. Because the genotype information is detached from the individual, genetic tagging implies that the fish has to be retyped each time its performance is evaluated or individuals are selected. In a selective breeding programme, each fish needs to be tested many times during its life cycle, and selection must be carried out at several stages, so this approach is not economic with current technology. Hopefully, the use of microchipbased genotyping may solve this problem. DNA fingerprinting may also have several other applications within fish management, including evaluating in-breeding levels, stock identification, and the movements of released fish and their possible genetic interactions with wild stocks. The rapid evolutionary rate of mitochondrial DNA makes these markers ideal for fish population studies.

5.7.2 Genetic Mapping and QTL Analysis All selective breeding programmes depend on genetic variation in the breeding stock, but no information about the functional genes influencing the phenotype is needed. Even though a quantitative trait is influenced by multiple genes, a variation in a few genes may be responsible for a major part of the variation in the phenotype. By quantitative trait loci (QTL) analysis, it is possible to identify the region(s), or QTLs, of the genome in which these so-called major genes are localised, still without knowing the identity of these genes. A QTL analysis is carried out by searching for linkages between variations in performance of experimental crosses and the alleles of genetic markers, which have been mapped to their positions or loci (singular locus) on the genome. The numerous, hypervariable microsatellites are ideal markers for creating genetic maps, since they are evenly spaced throughout the genome. Genes are also useful markers, but they are widely spaced with large gaps between them, and only a fraction of the total number of genes seems to exist in allelic forms. The more markers utilised and the more polymorphic the markers, the greater the likelihood of detecting an association with variation in a quantitative trait. A genetic map is established by studying the inheritance or segregation of markers in cross-breeding experiments. Based on this linkage analysis, the markers are put together within linkage groups, each group corresponding to a single chromosome. The mapping of fish genomes, including hundreds of genetic markers, has been worked out for several commercial fish species, including salmonids, Atlantic cod, Pacific herring, sea bass and tilapia. At present, zebra fish show the most complete genetic map, including more than 2000 markers (Woods et al., 2000). The methodology for the detection of QTLs in fish is currently still being developed, and very few QTL markers have been identified. In rainbow trout, microsatellite markers linked to two QTLs for upper temperature tolerance have been reported (Jackson et al., 1998). The key role of the major histocompatibility complex (MHC) in immune defence, and the highly polymorphic nature of MHC, make this complex a candidate QTL for disease resistance in fish. Indeed, a strong association between MHC alleles and resistance against furunculosis has been identified in Atlantic salmon (Langefors et al., 2001). In tilapia, the length of two microsatellites located within the promoter of the prolactin gene was shown to be associated with prolactin expression and growth of salt-challenged fish (Streelman & Kocher, 2002). This knowledge of linkages between desirable phenotypic traits and QTL markers can be

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used for marker-assisted selection (MAS), which will improve the accuracy of selection and thus lead to increased selection responses. Even without knowing the identity of the genes within a QTL, we can obtain information about their actions and interactions, and estimate how much of the total variation is accounted for by the QTL variation. In the future, QTL analyses and MAS will certainly not replace traditional selective breeding, but they should be implemented in the breeding programmes as supplementary tools to estimate breeding values and evaluate breeding candidates.

5.7.3 Transgenic Fish The high fecundity of most fish and external fertilisation and embryonic development make them especially suitable for transferring specific genes. The production of transgenic fish is aimed at dramatically improving traits such as growth, disease resistance and environmental tolerance. Because they are influenced by multiple genes, the nature of such quantitative traits makes them difficult to manipulate by gene transfer techniques. However, significant growth enhancement of several fish species has been demonstrated after the introduction of the growth hormone gene as the major gene under the control of a strong promoter element (Melamed et al., 2002). It should be noted that the increased growth rate is mainly due to the large amounts of growth hormone produced in large tissues such as the liver, and not by the transfer of millions of gene copies. Surprisingly, the growth of transgenic wild-strain rainbow trout was shown not to surpass that of a non-transgenic domesticated strain being selected for fast growth (Devlin et al., 2001). Furthermore, introducing the growth hormone construct into this fast-growing domestic strain did not cause further growth enhancement. These results indicate that similar alterations in growth rate can be achieved both by selective breeding and by transgenesis, but that the effects are not additive, at least not in rainbow trout. There are several problems to be overcome before transgenic animals can be produced on a large scale. Indeed, in over 90% of the microinjected eggs, the transgene is not efficiently integrated into the genome at the one-cell stage. The result is a highly mosaic transgenic fish and low frequencies of germ-line transmission, since only the tissues developing from the transformed cell will carry the transgene. Furthermore, the injected DNA integrates at single or multiple random sites in the genome of the recipient embryo, and each develops into a unique hemizygous fish. Hence, the establishment of a stable transgenic broodstock will be a costly endeavour, requiring several generations. On the other hand, attempts to combat viral and bacterial pathogens which threaten commercial stocks by utilising DNA vaccines have been promising. This technique is based on the injection of DNA encoding part of the antigen, usually a bacterial outer membrane or viral capsid protein, in the fish muscle, where the protein will be synthesised and the production of antibodies induced. A significant degree of protection against infectious hematopoietic necrovirus (IHNV) was found in Atlantic salmon after vaccination with a gene construct containing an IHNV glycoprotein (Traxler et al., 1999). Similarly, protection against viral haemorrhagic septicaemia virus (VHS) was induced in vaccinated rainbow trout (Lorentzen et al., 1999). The main disadvantage of these approaches is that they require quite detailed information about the structure, conformation and encoding sequence of the pathogen’s protein.


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An alternative way to increase the resistance of fish to pathogens is to target the non-specific immune response through the use of antimicrobial peptides, which are found in both vertebrates and invertebrates. Short peptides consisting of 30–50 amino acids with strong antimicrobial activity have been isolated from the skin mucus of several fish species, including Atlantic halibut and winter flounder. Furthermore, the encoding sequences of the histonederived ‘hippoglossin’ of halibut have been determined (Birkemo et al., 2003). To date known attempts to produce transgenic fish carrying genes encoding antimicrobial peptides all relate to the lysozyme gene, which has a non-specific antibacterial effect.

5.7.4 Future Prospects The applications of molecular techniques in aquaculture are promising, but still somewhat uncertain. While high costs seems to be the only hindrance to the widespread application of genetic markers for identification purposes and MAS, the situation regarding the commercial use of genetically modified fish is more complex. Although the potential importance of gene transfer technology is large, a major concern relates to the possible impact which release or accidental escape of gene-modified individuals may have on natural ecosystems. Other controversial aspects are related to animal welfare, food safety and the public perception of gene manipulation in general. To what extent such issues will constrain the future use of transgenic animals in applied aquaculture production remains to be seen.

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Chapter 6

From Fertilisation to the End of Metamorphosis—Functional Development E. Kjørsvik, K. Pittman and D. Pavlov

Adult fish show a large variation in morphology and ecology, and so do their offspring. When studying the appearance of the early life stages of fish, one is immediately struck by their diversity and morphological dissimilarity to adults, and that they most often inhabit totally different habitats from their parents. A knowledge of reproductive and developmental biology and ecology is therefore crucial for producing offspring of optimal quality, and for the successful mass rearing of juvenile fish fry. The early life stages of fish also have species-specific and stage-specific environmental and nutritional requirements. Developments in rearing technology and start-feeding have benefitted significantly from studies of the developmental biology and ecology of fish eggs and larvae. The following chapter will focus on the functional egg and larval development of cold-water marine aquaculture species, and on aspects that are particularly important for their cultivation. Their nutritional and environmental needs are thoroughly covered elsewhere in this book.

6.1 Intervals of Fish Ontogeny and Definitions of the Organism The basic developmental mechanisms are similar in all teleost species, but differences exist with regard to the relative timing of growth and the development of specialised cells and organs. Genetic differences, yolk size and environmental conditions all influence the developmental stage and larval size at hatching and at the transition to exogenous feeding. Some common criteria are needed to define the stages of early fish development in general. The variety of developmental patterns should be considered, and the definitions should apply to as many patterns as possible. In this chapter, we use the operational definitions listed below. Egg: Yolk-sac larva:

Larva:

an encapsulated embryo (from spawning to hatching). a free-living embryo with a yolk-sac which may or may not be absorbed before exogenous feeding (i.e. from hatching to the start of exogenous feeding). from the start of exogenous feeding to the completion of metamorphosis.

From fertilisation to the end of metamorphosis

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Metamorphosis: the transitional stage between larva and juvenile. Juvenile: having the final phenotype, but not sexually mature. Each of the stages can be divided into several substages (Fig. 6.1). In developmental biology, the offspring is generally regarded as an embryo up to the point where exogenous feeding begins, i.e. the embryonic stage includes the egg stage and the yolk-sac larva stage (Balon, 1999).

Figure 6.1 Early life-history stages of fish larvae, as shown for the jack mackerel (Trachurus symmetricus). From the original drawings of Ahlstrom & Ball, 1954.

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Ontogeny is the entire life cycle of an organism from egg activation (and subsequent fertilisation) to death, and it seems natural to distinguish intervals of fish development. However, the principles for the definition of these intervals have been discussed for at least half a century (Vasnetsov, 1953). The main problem for the subdivision of the ontogeny lies with the divergent answers to this question: Do fish develop gradually, or is their development non-gradual (saltatory) with distinguishable natural intervals separated by thresholds? According to the ‘gradualists’, ontogeny is a gradual process with continuous changes in the form, structure, physiology and behaviour of an organism. According to the concept of non-gradual ontogeny of fish (Vasnetsov, 1953; Kryzhanovskii et al., 1953) and the theory of saltatory (step-wise) ontogeny (Balon 1985, 1990), the life-history of fish can be separated into periods which may be defined as the longest intervals of ontogeny separated by the most decisive thresholds (Table 6.1). This means that the organism develops through a series of rapid, almost sudden, changes in form and/or function, which are separated from each other by longer intervals during which changes are smaller, more continuous and rather insignificant. Between these periods of rapid change, the organism will prepare for the next rapid change, and the organs and tissues necessary for the next big level of change will develop to converge functionally at a certain threshold. Whether the functional development of fish larvae is saltatory or not is still a question of much debate among scientists. The main factor to remember is that larval requirements and environmental responses change during their development.

Table 6.1 Terminology of life history stages. From Kendall et al. (1984), in which the references can be found. Reproduced with permission of the American Society of Ichthyologists and Herpetologists

END POINT EVENTS Spawning

Blastopore closure

Tailbud free

Hatching

Yolk-sac absorbed

Full finray complement Attains Attains juvenile present, adult squamation body body begun, loss proportions, proportions, Notochord Notochord pigment, pigment, flexion Metamorph- of larval starts to complete osis begun characters habits flex habits

TERMINOLOGY Primary developmental stages

Transformation larva

Yolk-sac larva

Transitional stages Subdivisions

Juvenile

Larva

Egg

Early

Middle

Preflexion larva

Late

Flexion larva

Postflexion larva

Pelagic or special juven

OTHER TERMINOLOGIES Hubbs, 1943, 1958

Embryo

Postlarva

Prolarva

Prelarva

Hattori, 1970

Snyder, 1976, 1981 (phases)

Postlarva

Embryo

Nikolsky, 1963

Balon, 1975 (phases)

Prejuvenile

Larva

Sette, 1943

Cleavage egg

Embryo

Eleutheroembryo

Protopterygiolarva

Protolarva

Pterygiolarva Mesolarva

Metalarva


207

The terminology applied to fish ontogeny, which has been used in most practical research, is based mainly on the concept of ontogeny as a sequence of ‘normal stages’, and has evolved from fisheries and ichthyoplankton ecology (Table 6.1). The egg phase includes several ‘stages’, and the hatched free organism with a yolk can be called a yolk-sac larva or a prelarva. The larval phase consists of the ‘stages’ where a finfold is present and where the fins are forming. The embryonic and yolk-sac periods are characterised by endogenous feeding. The transition to exogenous feeding (and not hatching) is therefore regarded by many as one of the most critical periods for survival.

6.1.1 Relative Duration of the Various Stages of Development The type of early ontogeny is related to the relative duration of the main developmental intervals. The relative durations in five species are presented in Fig. 6.2. As a reference point for a comparison of different developmental styles, reaching the ‘juvenile state’ (i.e. the stage of the disappearance of larval characteristics and the formation of the most juvenile characteristics, including skeletal ossification) is used. Cod, turbot and Atlantic halibut possess a comparatively short embryonic phase of development inside the egg-shell, and a prolonged larval period. Among them, the Atlantic halibut has a prolonged yolk-sac stage of about 44 days at 9°C. The common wolf-fish is characterised by a prolonged embryonic phase ranging from 104 days in captivity under the influence of a comparatively high temperature to 9.5 months in nature. In addition, larval exogenous feeding in this species begins just after normal hatching.

Egg state

Yolksac larva

Larval state

Wolffish

Salmon

Halibut

Turbot

Cod

0%

20 %

40 %

60 %

80 %

100 %

Duration from fertilization to juvenile state

Figure 6.2 Scheme representing comparative features of early ontogeny in cod, turbot, Atlantic halibut, Atlantic salmon and common wolf-fish. The duration of development from egg activation to onset of juvenile state = 100% (approximately 59, 90, 108, 112 and 177 days, respectively).

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6.2 Egg Classification There is a great variability in the reproductive styles of fish (see also Chapter 5), and these styles are determinants for the species differences in fecundity, egg size and egg type. The Norwegian scientist G.O. Sars (1869) was the first to discover that fish may have pelagic eggs (i.e. eggs floating freely in seawater). When he described the pelagic nature of Atlantic cod eggs and larvae for the first time, he shed new light on the wide variety of reproductive styles and early life history patterns of fish. Until then, all known fish eggs were demersal, i.e. developing on the bottom or attached to a substrate. The eggs of all fish have a polar distribution of yolk and cytoplasm and belong to the telolecithal type. The concentration of yolk at the vegetative pole of the egg is different in species from the various taxonomic groups. The eggs also have different features of yolk distribution in the cytoplasm, and based on this characteristic, two egg subtypes are known (Makeyeva, 1992): (1) (2)

Eggs with no separation of yolk Eggs with separation of yolk

The egg cleavage pattern varies according to the different fish groups, and the two subtypes are characterised primarily by different developmental patterns. In eggs of the first subtype, the yolk is distributed in the cytoplasm as granules or conglomerates. These eggs will undergo full (holoblastic) cleavage, i.e. the whole egg goes through cell cleavage after fertilisation (with uneven cell sizes). Eggs of this subtype are common for lower bony fish such as lungfish (e.g. Neoceratodus), Chondrostei (sturgeons) and Holostei (Amia and Lepisosteus). Fish with this egg type all spawn in fresh water. All teleosts, most of the sharks (Chondrichthyes) and the living coelacanth, Latimeria chalumnae, produce eggs of the second subtype, where the yolk is separated from the cytoplasm. Only the cytoplasm (and not the yolk) is subject to cleavage. Therefore, the cleavage is called meroblastic or discoidal. Most marine fish spawn pelagic eggs that are fertilised externally and float individually near the surface of the sea. Pelagic eggs are generally small; the egg size may vary from about 0.6 to 4.0 mm in diameter, with a mean diameter of 1 mm (Kendall et al., 1984). However, there are many exceptions to this pattern. For example, the halibut has exceptionally large pelagic eggs with a diameter of about 3 mm. Halibut eggs also differ from typical pelagic eggs in buoyancy. The halibut spawn at great depths, and the eggs develop in the mesopelagic layer, at about 150–250 m deep (Haug et al., 1984), while most pelagic eggs float in the upper surface layers. Pelagic eggs are generally spherical and transparent, as is shown for cod and halibut eggs in Fig. 6.3. This is the normal pattern for pelagic eggs, regardless of systematic position, whether the adult fish has pelagic or demersal habits, or if it lives in coastal or oceanic waters, or in tropical or boreal areas. Many coastal, freshwater fish and some marine lay demersal eggs, which are generally larger than pelagic eggs. Demersal eggs are often adhesive, and may be laid in some sort of nest or cluster. Most demersal eggs are also less transparent, and have a much thicker egg-shell (or egg envelope or chorion) than pelagic ones (Fig. 6.3).


209

Figure 6.3 Cod, halibut and wolf-fish eggs shown according to their relative size. Cod eggs (in the middle) are the typical size of a pelagic egg with a diameter of about 1.3 mm, whereas halibut (3 mm, on the left) has very large eggs for its pelagic nature. The large demersal egg of the common wolf-fish (5.5 mm) is comparable in size to salmonid eggs. Pelagic eggs are more transparent than demersal eggs because they have a much thinner shell. Photographs: Elin Kjørsvik and Inger-Britt Falk-Petersen.

Within a species, there is little variation in egg characteristics such as size, number and size of oil globules, pigmentation and the morphology of the developing embryo. The development time is highly temperature-dependent and species-specific. Between species, egg size and fecundity tend to be inversely related (Blaxter, 1988). Species with larger eggs also generally have a longer incubation time, their larvae are much more developed at hatching, and their development may often go direct from hatching to the juvenile stage. The time-scale from fertilisation to hatching may be days for species with small pelagic eggs, the large demersal eggs of salmonids and wolf-fish tend to take several weeks or months, whereas the very large eggs of elasmobranchs may take even longer. The large difference in size between salmonids and wolf-fish versus marine pelagic eggs and larvae (see Fig. 1.2, Chapter 1; Table 6.2) illustrates how much more advanced the functionality and viability of the larger eggs and larvae may be. These differences in size, embryo development time and developmental stage at hatching are the main reasons for the difficulties experienced in marine larval rearing, as pelagic larvae are much less developed at the time of start-feeding than the larger demersal ones.

6.2.1 Egg Structure and Composition In general, mature unfertilised teleost eggs are soft, and do not tolerate much mechanical pressure. A fish egg (Fig. 6.4) consists of a large mass of yolk material surrounded by a thin layer of cytoplasm and an outer egg envelope (egg-shell or chorion). Numerous small

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Spawning and egg characteristics of some cold-water aquaculture species.

Species

Fertilisation

Egg diameter (mm)

Egg type

Number of spawned eggs

External

5–7

Demersal

10–15 thou.

Wolf-fish

Internal

5.2–6.0

Demersal

Cod3

External

1.1–1.7

Pelagic

External

0.90–1.2

Pelagic

10–15 mill.

External

3.0–3.8

Mesopelagic

0.5–1 mill.

Atlantic salmon1 2

Turbot4 Atlantic halibut 1

5

Preferred developmental temperature (°C)

Hatching time (d°)

Size of newly hatched larvae (mm)

Hatching to start-feeding (d°)

6–8

510

17–20

300

5–50 thou.

5–8

900

23

2.5–14.5 mill.

5–12

90

4

27

13–18

75–102

2.7–3.1

36

4–7

82–85

6

Gjedrem, 1993. Falk-Petersen et al., 1999; Tveiten & Johnsen, 1999. 3 Kjesbu, 1989; Kjesbu et al., 1991; Iversen & Danielssen, 1984; Galloway, et al., 1999a. 4 Jones, 1974; Howell, 1979; Bromley et al., 1986; Bromley et al., 2000; Fauvel et al., 1993. 5 Haug et al., 1984; Pittman et al., 1990; Norberg et al., 1991; B. Nordberg, personal communication, 2002; Galloway et al., 1998. 2

0–30

220–270


Table 6.2


211

Egg shell

Cytoplasm Oil droplets Cortical alveoli

Yolk

Micropyle Figure 6.4 Schematic drawing of an ovulated, unactivated fish egg. Most of the egg content is yolk, which contains all the necessary nutrients for the development of the embryo and yolk-sac larva. In some species, the yolk may contain one or several oil droplets. The micropyle is situated at the animal pole of the egg, and it is the only opening in the egg shell (egg envelope) where a spermatozoon may enter to fertilise the egg cell. The nucleus is not visible in the mature egg. The narrow opening is a protection against polyspermi, and it becomes clogged after the entrance of the first sperm cell.

granules or cortical alveoli are distributed in the peripheral cytoplasm of the egg. These alveoli contain the enzymes and macromolecules necessary for the egg activation. The cytoplasm of a mature oocyte contains a nucleus (the germinal vesicle) located at the animal pole directly under the micropyle, which is a small opening in the egg-shell. The micropyle is obstructed after the entrance of the first sperm cell. The eggs of many fish species contain oil (lipid) droplets. Turbot eggs have one large oil droplet, whereas cod and halibut have none. Salmonid and wolf-fish eggs contain several lipid droplets. These droplets are fixed at the animal pole of the egg in salmonids, and move freely within the yolk in turbot and wolffish species. The egg envelope is a non-living part of the egg, and consists mainly of latticed proteinaceous (keratin-like scleroproteins) concentric layers. The egg envelope is also the hardest part of the egg, and it provides physical protection to the developing embryo. Its structure is therefore related to the ecological conditions during egg development. Thus, pelagic eggs generally have thin egg envelopes relative to their diameter, whereas the envelopes of demersal eggs are thicker, more robust and are often supplied with extra outer layers that may be adhesive.The primary egg envelope (originating from the superficial protoplasm of the oocyte) contains many tiny radial canals and is called the zona radiata. The

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Table 6.3


Egg weight and composition of some cold-water aquaculture species.

Species

Egg wet weight (mg)

Atlantic salmon1 Common wolf-fish2 Cod3 Turbot4 Halibut5

53–142 115 1 0.6 17

Ash content (% of DW) 4

Water content (%)

Lipid content (% of DW)

Protein content (% of DW)

60–67

30

66–74

FAA content (nmoles/egg)

950 13 10 9

90–93 90 87–90

5–13 25–32 12–15

47 40–52 55

250 65 2300

1

Kamler, 1992; Sargent, 1995; Berg et al., 2001. H.J. Fyhn, unpublished data, 2002. 3 Lønning et al., 1988; Finn et al., 1995a, b; Fyhn & Serigstad, 1987. 4 Falk-Petersen et al., 1989; Finn et al., 1991; Finn, 1994; Rønnestad et al., 1995. 5 Finn et al., 1991; Rønnestad & Fyhn, 1993. 2

secondary egg envelope is formed by the follicle cells and is called the chorion. The egg envelope forms an outer adhesive coat in demersal eggs of many fish species, e.g. the lumpfish (Cyclopterus lumpus) and the wolf-fish. The nature of such adhesion is still unknown. The yolk contains the material necessary for development and metabolism during the embryo and yolk-sac larva stages. A comparison of the egg characteristics of various species is given in Table 6.3. The general pattern is that species with larger eggs contain more energy, have longer incubation times and produce larger larvae at hatching than species with smaller eggs. If egg size is similar, larvae hatching from demersal eggs will generally be larger and more developed than larvae hatching from pelagic eggs. This is also reflected in the egg composition, as demersal eggs generally contain less water and higher absolute levels of lipids (especially neutral lipids) than pelagic eggs. About 90–95% of the yolk mass in pelagic eggs is water, which makes them buoyant. However, the yolk must also contain all the necessary nutrients and energy for normal embryonic development, such as proteins, free amino acids, lipids (many species have oil droplets in the yolk), vitamins and minerals. The different yolk components are used sequentially, according to the changing needs of the developing embryo.

6.3 Insemination and Fertilisation Insemination is the process of mixing eggs and sperm to obtain close contact between the gametes. According to the definition of Balon (1990), fertilisation in its broadest sense is a process which starts with insemination, continues with activation and cortical reaction, the formation of the perivitelline space and bipolar differentiation, and ends with the fusion of male and female pronuclei. Only the latter process can be considered to be fertilisation in a strict sense. The fertilisation process contributes to profound changes in the egg characteristics, and the eggs are very vulnerable during this interval. Egg activation includes a complex of changes. First, the developmental block of meiosis at the metaphase of the second meiotic division is released. In addition, the permeability of


213

the egg membrane increases. A transitory rise in intracellular Ca2+ occurs, starting at the animal pole and spreading towards the vegetal pole (Polzonetti et al., 2002). This Ca2+ wave seems to be necessary for metabolic activation and cell-cycle control in the egg. The cortical alveoli are activated by the Ca2+ wave, and their membranes fuse with the plasma membrane, resulting in an exocytosis of the alveolar content (cortical reaction or cortical wave, see Fig. 6.5) beneath the egg envelope (chorion). This process seems to be dependent on the presence of Ca2+ in the water (Lønning et al., 1984). In fish, egg activation is induced by the

Figure 6.5 Cortical reaction in a cod egg. The egg is activated by the entrance of a sperm cell through the micropyle, which can be seen as a small indentation in the upper part of the egg (the animal pole). The content of the cortical alveoli are released in a wave-like action starting in the micropyle area and spreading over the egg surface. This can be observed as the disappearance of the cortical alveoli, as these time-lapse photographs during the first 10 min after sperm addition demonstrate. From Davenport et al., 1981.

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penetration of a spermatozoon, by contact with water (as in salmonids), or even by mechanical stimulation (Ginzburg, 1972). In the majority of marine fish species, eggs are activated by spermatozoa: without insemination, the cortical reaction either does not take place or is incomplete. The release of the cortical colloidal macromolecules contribute to an influx of (sea)water through the chorion, leading to formation of the perivitelline space (a fluid-filled space between the egg envelope and the embryo). The perivitelline space is normally narrow in pelagic eggs, but it may be wide in some species such as the long rough dab Hippoglossoides platessoides. In addition, the formation of the perivitelline space may be caused by partial shrinkage of the yolk, as has been observed in cod and in common wolf-fish. After this initial activation, the cytoplasm starts to become concentrated towards the animal pole, in preparation for the first cell cleavage. The cytoplasm (cell material) is heavier than the yolk, and in pelagic eggs the developing cells can be observed below the yolk. In the egg, the second meiotic division is then completed and the female pronucleus appears. The spermatozoon head transforms into the male pronucleus. The pronuclei fuse, causing a resumption in diploid chromosome numbers. The egg envelope (or chorion) is highly permeable to small molecules such as water, ions and amino acids. It is impermeable to larger molecules, and is therefore an effective barrier to bacteria and viruses. It also undergoes chemical and structural changes during the activation and the fertilisation process. Owing to the uptake of water, the egg swells, and at the same time the egg-shell hardens, a process that results in a thinner membrane with a much higher rigidity. Swelling of wolf-fish eggs in seawater is accompanied by hardening of the egg envelope and adhesion to other eggs, but not to other substrates. In marine fish eggs, there seems to be a good correlation between egg hardness and envelope thickness. The demersal lumpsucker eggs, which have very thick egg envelopes, may resist a mechanical force of about 2 kg before breaking, whereas other pelagic eggs may only tolerate forces ranging from 100 to 400 g before breaking (Lønning et al., 1988). The egg envelope structure is species-specific, and its characteristics may be used for species identification.

6.4 Embryonic Development and Hatching All the fish species which are regarded as promising candidates for marine aquaculture are teleosts. For the teleosts, embryogenesis from fertilisation to hatching tends to follow the same basic pattern (e.g. Kendall et al., 1984; Blaxter, 1988). Therefore, in this section, a brief description of the general patterns of embryonic development is given exclusively for eggs with a separated yolk showing meroblastic cleavage, using cod as a model (see Figs. 6.6 and 6.7). Other descriptions of cod development can be found in Sars (1869) and Fridgeirsson (1978), for example. The intervals of embryonic development are given according to Makhotin et al. (1984) and Makeyeva (1992). Comparative data will be presented for common wolf-fish, a species with an alternative type of early ontogeny (see Figs. 6.8 and 6.9).


215

6.4.1 Cod (Gadus morhua) Mature cod eggs are spherical and transparent, often with a light orange colour. Their diameter ranges from 1.4 to 1.8 mm. After spawning, the eggs collect together just below the water surface. A narrow (0.4 mm) primary egg envelope (zona radiata) encases the thin cytoplasmic layer surrrounding the yolk. The animal pole with the micropyle is located in the lower part of the egg.

Step I. Activation, Hardening of the Egg Envelope, Formation of Perivitelline Space and Blastodisc Egg activation can be observed as a consecutive breakdown of the cortical alveoli from the animal to the vegetal pole, and the formation of the perivitelline space between the thin cytoplasmic layer surrounding the yolk and the internal surface of the egg envelope. The perivitelline space is small: the egg volume remains almost the same, and the wet weight of the egg increases to 15–20%. Then the cytoplasm aggregates below the micropyle forming the blastodisc (Fig. 6.6a). The second meiotic division is then completed, and female and male pronuclei fuse. The hardening of the egg shell is observed during the first few hours after fertilisation.

Step II. Cleavage The concentration of cytoplasm after fertilisation has the appearance of one cell before the first cleavage. The dividing cells are called blastomeres, and the first series of cell cleavages transforms the single egg cell into a multicellular body called the blastodisc on the surface of the yolk. Cleavage represents a series of mitotic cell divisions in the blastodisc, leading to the formation of a semispherical cap above the yolk, composed of many blastomeres. The size of the cells decreases with each subsequent division. In many fish species, including cod, one cell appears to be slightly larger than the other after the first cleavage (Fig. 6.6b). The second cleavage is perpendicular to the first. The two sections of the third cleavage are parallel to each other, forming eight blastomeres. During the first four cleavages, the cell divisions are incomplete, leaving a thin layer of uncleaved cytoplasm at the bases of the cells, thus forming the syncytial layer. The cell divisions become asynchronous from the 5 to 7th cleavage cycles. The 5th cleavage is often latitudinal (parallel to the egg equator). As a result, the 32 blastomeres are separated into upper cells, which are not connected to the yolk, and lower blastomeres, which join to each other by means of the syncytial layer (Fig. 6.6c). Later the lower blastomeres form a specific layer, the periblast, with giant nuclei surrounding the base of the blastodisc. The periblast is central during the process of blastoderm epiboly and later for yolk utilisation. The stages between 64 and 256 blastomeres are called large-cell morula (Fig. 6.6d), and as the blastomere size decreases, the stages may be called middle-cell or small-cell morula (Fig. 6.6e,f ). The entire cell mass is the blastoderm. In some species, periodic motoric contraction waves in the cytoplasm surrounding the yolk can be observed. This

Figure 6.6 Stages of the embryonic development of cod at 1.8°C. Scale bar = 1 mm. (Makhotin et al., 1984; with additional drawings by V.V. Makhotin). Note that the natural position of the animal pole with the blastodisc is below the yolk. (a) Formation of blastodisc, age 8 h from egg activation; (b) two blastomeres, age 10 h; (c) 32 blastomeres, age 20 h; (d) large-cell morula, age 1 day 6 h; (e) small-cell morula, age 1 day 16 h; (f ) small-cell morula, the periblast nuclei at the periphery of the blastodisc, age 1 day 21 h; (g) beginning of blastulation, age 2 days 2 h; (h) the middle of blastulation, age 3 days 3 h; (i, j) beginning of gastrulation, formation of the dense sector and the germ ring in the blastodisc, age 3 days 23 h; (k, l) appearance of the embryonic shield, age 4 days 9 h; (m) beginning of blastoderm epiboly, age 4 days 19 h; (n) end of gastrulation, blastoderm epiboly 30% of the yolk surface, age 5 days 10 h; (o) organogenesis, blastoderm epiboly 60% of the yolk surface, age 6 days 1 h; (p) first somites, blastoderm epiboly 70% of the yolk surface, age 6 days 6 h; (q) eight somite pairs, formation of the yolk plug, age 6 days 11 h; (r) 28 somite pairs, Kupffer’s vesicle, age 8 days 13 h; (s) end of segmentation of the caudal part of the embryo, 50 myomeres, age 14 days 14 h; (t) appearance of pigment spots on the body of the embryo, age 15 days 5 h.


217

motoricity leads to mixing of the perivitelline fluid and better respiration of the blastoderm inside the egg envelope. Step III. Blastulation As the blastomere divisions continue, the first cell differentiation becomes visible, and the embryo is called a blastula. The surface cells flatten and become polygonal, forming an epithelial layer, the periderm. The contact between the deeper cells becomes weaker and spaces can be formed between them. However, the typical blastocoel found in sturgeons or lungfish does not appear. The deep cells form short protrusions, called lobopodia, and show restricted movements relative to each other. At the blastula stages, the blastoderm begins to protrude downward into the yolk (Fig. 6.6g), and later the blastodisc has a spindle-like form (Fig. 6.6h). At the late blastula, both the upper and the lower blastoderm surfaces flatten. The blastodisc diameter decreases to 0.6 mm (compared with 0.8–0.9 mm at the previous stage). The periblast zone around the blastodisc becomes narrower. This zone transforms the yolk nutrients used by the blastoderm cells. With each successive reduction in cell size with cleavage, the content of nuclear material (DNA) remains unchanged, and thus the ratio of DNA to cytoplasm increases. Step IV. Gastrulation Gastrulation is the process by which the initially uniform cells of the blastoderm separate into primary germ layers: the ectoderm (the outer layer) and the endoderm (the inner layer). Later, a third middle layer, the mesoderm, appears between the ectoderm and the endoderm. The ectoderm will develop into the epidermis and the nervous system, the endoderm into the alimentary canal and other digestive organs, and the mesoderm leads to the formation of muscles, the circulatory system and the sex organs. Gastrulation in teleost fish differs from that in other vertebrates (Ballard, 1973, 1982). Generally, gastrulation is associated with two types of cell movement: epiboly (the distribution of cells towards the vegetal pole of the egg) and axial latitudinal convergence to the anlage of the embryo (the embryonic shield). During gastrulation, the properties of the deep cells of the blastoderm change. The lobopodia transform into long protrusions, called philopodia, and the cells adhere to each other. In early gastrulation, some of the deep cells migrate to the blastodisc periphery, forming the embryonic shield and germ ring (Fig. 6.6i,j). In the germ ring, the deep cells of the blastoderm are separated into two layers: the epiblast adjacent to the internal surface of the periderm and the hypoblast lying on the periblast. The epiboly of the blastoderm begins from the migration of the periblast towards the vegetal pole of the egg. The marginal cells of the periderm become elongated and adhere to the periblast as it begins to migrate. During epiboly, the caudal end of the embryonic shield remains at the edge of the germ ring (Fig. 6.6m,n). At the end of epiboly, the margin of the spreading periderm forms the yolk plug (not homologus to blastopore in amphibians and sturgeons). Incubation temperatures can determine the developmental stages reached by the

218


embryo at yolk plug closure. In many demersal eggs, the blastoderm epiboly terminates at more advanced stages of embryo development than in pelagic eggs. During epiboly, the cells of the hypoblast migrate towards the embryonic shield (axial convergence), and the cells of the epiblast migrate longitudinally with the periderm. The hypoblast cells are often separated into two layers: the lower layer (presumptive endoderm) composed of cells with intracellular spaces, and the upper layer (presumptive mesoderm) represented by several layers of tightly packed cells. When they reach the embryonic shield, the cells have predetermined locations in the ectoderm, mesoderm or endoderm. The embryo is covered by the flattened peridermal cells. The determination of the cells occurs during blastulation, and the fate maps of various organ anlages are known for several fish species. Step V. Organogenesis Organogenesis starts with the formation of the notochord anlage. This anlage is seen as an inflation in the yolk. Depending on the fish species, organogenesis can begin before or after yolk plug closure. In cod, organogenesis starts when blastoderm epiboly reaches approximately 50% of the yolk surface and the embryo is clearly seen at 60% of the epiboly (Fig. 6.6o). The formation of optic vesicles, the neural keel and three brain vesicles (forebrain, midbrain and hindbrain) can be observed. The first somites appear at 70% of blastoderm epiboly (Fig. 6.6p). Invagination of the optic vesicles forms when eight somite pairs appear in the embryo body (Fig. 6.6q). The yolk plug closes when 20 somite pairs are visible. After blastopore closure, the lenses of the eyes, the optic vesicles, the heart, the olfactory capsules, Kuppfer’s vesicle (in the caudal, ventral part of the trunk) and the first melanophores on the dorsal surface of the head can be seen (Fig. 6.6r). Step VI. First Muscle Contractions of the Embryo In cod, weak movements of the caudal part of the embryo begin before the onset of heart contractions. The body length increases, and its segmentation reach the maximum of approximately 50 myomeres (Fig. 6.6s). The anlages of pectoral fins, branchial arches and hatching glands (on the surface of the eyes and the head) can be seen at this stage. Towards the end of this period, melanophores on the embryo body are concentrated into several groups and the eyes become pigmented (Fig. 6.6t). The embryo sometimes moves inside the egg. Step VII. Preparation for Hatching and Hatching This period is characterised by intensive secretory activity of the hatching glands, which can be seen as many slightly granular circles in the head region long before hatching. The localisation of hatching glands differs between species. In cod, they are found in the head (Adoff, 1987), whereas in halibut they form a circular ring around the yolk sac (Helvik et al., 1991). These glands secrete an enzyme (chorionase) which weakens the egg envelope. A special hydrostatic organ, hydrosinus, is formed in the area from the head to the beginning of the dorsal part of the finfold. Guanine glossy pigment appears in the eyes. The swim-


219

bladder, urinary bladder, liver and gallbladder can be seen. The embryo often rotates inside the egg. A high oxygen concentration in the water can lead to inhibited release of the chorionase, and to delayed hatching and even death of the embryo. A decreasing oxygen concentration stimulates secretion of the enzyme by means of both signals from the central nervous system and increasing embryonic movements. These motoric movements are important for the mixing of the perivitelline fluid, which stimulates chorionase secretion. The destruction is observed in several areas of the egg envelope, and its strength decreases. The embryo hatches by energetic movements of the body. Normal hatching is associated with the appearance of the caudal part of the embryo from the egg envelope. During the hatching of abnormal embryos with restricted body movements, the head appears first due to the local action of chorionase on the egg envelope in the area of the head region. Exposure to different environmental conditions may cause a large variation in the degree of development at hatching (even within a single egg batch). However, despite a high variability, the stage of hatching is species-specific, and the hatching event leads to a substantial change in the relation of the organism to its environment. Step VIII. The Endogenous Yolk-sac Period The embryo at hatching has a large yolk sac and hydrosinus in the dorsal pre-anal part of the body (Fig. 6.7a). There are 11–12 myomeres before the anus and 38–39 after the anus. The melanophores are distributed in several transverse rows on the body (including three rows in the caudal part) and on the dorsal surfaces of the midbrain and hindbrain. Several melanophores are seen on the swim-bladder and liver. The jaws are well developed, but the mouth is closed. The newly hatched yolk-sac larvae gather together near the surface of the water with the yolk sac uppermost. Sometimes they can move in a horizontal direction by means of undulations of the caudal part of the body. Phototaxis is absent. During the rapid absorption of the yolk, the ventral part of the head separates from the yolk-sac and the mouth opens (Fig. 6.7b). The first blood cells begin to circulate in the main blood vessels of the body. There are no blood vessels on the surface of the yolk-sac. The embryos can move in different directions with the yolk-sac underneath, but they float to the surface and rotate when they are still. The jaws of the embryo become mobile when the yolk-sac volume decreases to 60% of its volume at hatching, the eyes have some restricted movement. The swim-bladder fills with gas during the yolk-sac period. The volume of the hydrosinus increases, and yolk-sac larvae have a positive phototaxis. Their ventral part is always directed downwards, and they possess neutral buoyancy when they are still. Step IX. Mixed (Endogenous and Exogenous) Feeding The yolk-sac volume decreases to 70% between hatching and the first exogenous feed uptake, and becomes narrow in the anterior area (Fig. 6.7c). The intestinal cavity is markedly expanded and the rectum is formed. The gall bladder is filled with gall and becomes green. The stomach and pyloric caecae are absent. Peristaltic waves can be seen along the gut. The

220


Figure 6.7 Stages of the subsequent development of cod at 7.2°C (Makhotin et al., 1984, modified; drawings f and g are by S.G. Soin). (a) Embryo at hatching, TL (total length) of the embryo = 4.3 mm, age 19 days from egg activation; (b) beginning of blood circulation, TL = 4.6 mm, age 1 day 8 h from hatching; (c) transition to mixed (exogenous) feeding, TL = 4.8 mm, age 4 days; (d) movements of the pectoral fins, TL = 5.2 mm, age 7 days; (e) transition to exclusively exogenous feeding, TL = 5.5 mm, age 10 days; (f ) formation of rays in the pectoral, caudal, anal and two dorsal fins, TL = 6.7 mm; (g) formation of rays in all fins except pelvic fins, TL = 9.5 mm.

larvae are actively swimming and feeding. Movements of the pectoral fins are registered when the yolk is almost completely resorbed (Fig. 6.7d). Nine paired primary neuromasts can be seen along the presumptive lateral line, but secondary neuromasts appear at later developmental steps. The mouth is open, with well-developed jaws. Step X. Exclusively Exogenous Feeding, Finfold Differentiation The yolk is totally resorbed (Fig. 6.7e). The hydrostatic organs of the embryo are represented by the hydrosinus, wide finfold and swim-bladder. The lower jaw is longer than the upper one. Diffuse yellow pigment appears in the body. The digestive tract still has a low degree of differentiation. Its separation into oesophagus, stomach, middle intestine and rectum is registered only during the juvenile period.


221

Step XI. Development of Rays in the Fins The rays develop in all fins with the exception of the first dorsal fin (Fig. 6.7f). The anlages of the pelvic fins (below the pectoral fins) can be seen. The melanophores are scattered on the body without marked spots. At 9.5 mm total length, the rays are formed in all fins with the exception of pelvic fins (Fig. 6.7g). The intestine is covered by an intensive internal black pigment.

6.4.2 Wolf-fish (Anarhichas lupus) Mature oocytes of White Sea common wolf-fish from a wild population, 5.0–5.7 mm (average 5.3 mm) in diameter, have a light yellow colour. The lipid droplets, up to 0.4 mm in diameter, are concentrated mainly in the upper area of the yolk and possess more intensive coloration. These droplets can move freely in the yolk. The egg envelope includes the zona radiata covered by a thin layer (zona pellucida). The eggs are inseminated and fertilised internally after copulation between the spawners, and they are released into the water 8–15 h before the first cleavage. The swelling of the egg envelopes in the water is accompanied by stickiness, and the eggs form a ball-like clutch after the female has coiled around them for several hours. The eggs adhere to each other, but do not stick to any substratum. They develop in holes, and are protected by the male during their whole embryonic development. In general, the first steps of embryonic development in common wolf-fish are similar to those in cod (Fig. 6.8a–c). The perivitelline space is very narrow at the beginning of embryonic development, but it increases during the process of yolk absorption. Organogenesis begins when blastoderm epiboly reaches 50% of the yolk surface (Fig. 6.8d). Yolk plug closure is observed when three somite pairs appear in the embryo (2.3 mm in length). The lipid droplets begin to fuse into larger ones when the number of somite pairs has increased to 19–21 (Fig. 6.8e). The embryo is located mainly in the lower part of the egg. A large lipid droplet can be seen at step VI (the beginning of muscle contractions in the embryo, and the formation of blood cells) (Fig 6.8f). At this step, the circulation of blood plasma without blood cells can be observed in the vessels of the embryo and in a vessel (the hepatic vitelline vein) located to the left of the embryo. At step VII (the appearance of a large number of blood cells), the right part of the hepatic vitelline vein is formed, and the zone of yolk-sac vascularisation increases (Fig. 6.8g). The last step of embryonic development (VIII, preparing to hatch and hatching) is characterised by intensive movements of the pectoral fins, jaws and body of the embryo. The vascularisation of the yolk sac is almost totally complete (Figs. 6.8h, 6.9a,b). At later stages, fin rays appear in all fins (with the exception of the pelvic fins, which are absent and represented only by skeletal rudiments), and the intensity of body pigmentation increases (Fig. 6.9c–f). Well-developed segmental blood vessels continue into the dorsal and anal fins forming a complex respiration net. In addition, a special respiration organ, the pseudobranch, is developed behind each eye. The eggs become opaque several days before hatching. The embryos hatch at a total length (TL) of between 19 and 24 mm, with the yolk sac almost totally resorbed and many juvenile characters (Fig. 6.9g). They are pelagic and show

222


Figure 6.8 Stages of the initial embryonic development of common wolf-fish at 4.9°C. Scale bar = 1 mm (Pavlov, 1986). (a) Formation of the perivitelline space and blastodisc, age 8 h; (b) early gastrulation, formation of the dense sector in the blastodisc, blastoderm diameter 3.4–3.6 mm, age 10 days; (c) gastrulation, formation of the embryonic shield, blastoderm epiboly 30% of the yolk surface, blastoderm diameter 3.8–4.3 mm, age 12 days; (d) early organogenesis, appearance of the neural keel, blastoderm epiboly 50% of the yolk surface, age 14 days; (e) 20 somite pairs in the embryo body, formation of the gill cover, fusion of the lipid droplets, age 23 days; (f ) 26 myomeres before the anus, 32 myomeres after the anus, heart pulsation, beginning of blood circulation, formation of a large lipid droplet, TL = 4 mm, age 36 days; (g) 26 myomeres before the anus, 49 myomeres after the anus, beginning of blood flow in the right part of the hepatic vitelline vein, TL = 9 mm, age 57 days; (h) beginning of the jaw and pectoral fin movements, diameter of the zone without blood vessels on the surface of the yolk sac = 2 mm, TL = 11 mm, age 84 days. bl, blastodisc; dp, pit-like depression in the yolk; ds, dense sector of the blastodisc; es, embryo shield; fs, fibrous structure; pz, periderm zone; sd, small lipid droplets.

a positive phototaxis. The transition to mixed (exogenous) feeding is observed just after hatching. The juvenile period begins at approximately 32 mm TL and coincides with the transition to a mainly demersal mode of life. Ossification of the majority of the skeletal elements is complete in specimens of this size. Individuals of a larger size have a body shape and colouration which are similar to those in adult fish (Fig. 6.9h). Thus, cod are characterised by a typical form of indirect development, with a pronounced metamorphosis during the transition from larvae with a low degree of morphologic development to juveniles. In wolf-fish, which have a transitory type of early ontogeny, the larvae possess a much higher degree of morphologic development and metamorphosis is almost absent. Owing to a prolonged period of development inside the egg envelope, the sub-period


223

Figure 6.9 Stages of the subsequent development of common wolf-fish at 4.9°C for 83 days and 7.8°C afterwards. The embryos (a–f ) have been removed from the egg envelopes (Pavlov, 1986; modified). (a, b) The stage shown in Fig. 6.8h; (c) formation of fin rays in the pectoral fins and caudal fin, gill filaments on the branchial arches, beginning of continuous movements of the pectoral fins, TL = 14 mm, age 93 days; (d) formation of teeth on the jaws and rays in the dorsal and anal fins, TL = 17 mm, age 100 days; (e) appearance of pigment spots on the body of the embryo, continuous movements of the jaws, TL = 17.8 mm, age 107 days; (f ) formation of rays in the dorsal part of the caudal fin, TL = 18.3 mm, age 113 days; (g) larva at hatching, transition to mixed feeding, TL = 21 mm, age 135 days; (h) juvenile, appearance of pigment spots at the bases of the dorsal and anal fins, TL = 45 mm, age 196 days. ps, pseudobranch.

of development outside the egg envelope (free-embryo phase or yolk-sac period) in this species is absent. In general, wolf-fish are much more protected from the external environment than cod. However, this protection has required several adaptations directed mainly towards solving the respiration problem of the embryo developing inside the egg envelope, including a complex blood circulation system and functioning pectoral fins and branchial apparatus.

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6.4.3 Embryo Growth and Yolk Absorption Yolk is the major source of energy and the material for body formation in developing embryos of oviparous species. At the initial developmental stages, the transport of nutritional substances from the yolk to the blastodisc occurs by means of cytoplasmic threads. The periblast has a major role in the trophic function of the embryo, and this function continues until the yolk has been totally absorbed. In many fish species, the blood system at the surface of the yolk sac is used for the transportation of nutrients from the yolk to the embryo. Yolk is absorbed by the phagocytic activity of the inner part of the periblast (vitellolysis zone), where it is degraded into substances with a low molecular weight and transported into the blood. The initial growth of the embryo mass (without the yolk) and yolk absorption can be described by an exponential curve and a linear equation, respectively (Novikov, 2000). Thus, the comparative independence of the regulation processes governing embryo growth and yolk absorption can be expected. In cod, at the steps of cleavage and blastulation, the protein content in the blastoderm remains at a low and constant level, and begins to increase from mid-gastrulation. In salmonids, the beginning of protein growth is registered from organogenesis (Novikov, 2000). The growth rate of the embryo decreases until the beginning of exogenous feeding, and a negative growth (due to tissue absorption) can be observed in unfed larvae. In some species (e.g. wolf-fish), the rate of yolk absorption is more or less constant over the entire interval of development using yolk reserves. In other species, such as Atlantic salmon, yolk is consumed mainly after hatching, which coincides with an abrupt increase in breathing intensity and the energetic demands of the embryo. Embryo growth and yolk absorption rates increase at higher temperatures within the range which is suitable for normal development. However, the effect of temperature on the embryo size and the amount of yolk at identical developmental stages can be especially important for aquaculturists. An increase followed by a slowing down of embryo growth rate in eggs incubated at higher temperatures is often registered at the steps of gastrulation and organogenesis (e.g. cod), or at later steps (e.g. salmonids). During the second half of embryonic development, embryos at the same developmental stage at lower temperatures are larger and have less yolk than those at higher temperatures. In embryos of rainbow trout, the difference in embryo size between ‘cold’ and ‘warm’ groups can reach 10% by length and 50–100% by protein content (Novikov, 2000). At the same time, a difference in embryo size at hatching could be the result of a different relationship between embryo growth and yolk absorption at different temperatures, or because of the transition of the hatching event to a later developmental stage at lower temperatures. As a result of the first reason, larvae from ‘cold’ groups are often larger at the stage of total yolk absorption and at later stages than those from ‘warm’ groups, and this may be an advantage in their subsequent growth and survival. However, the incubation of eggs at temperatures below a certain threshold can lead to a slowing down of embryo growth rate. This change in the correlation between the rates of embryo growth and yolk absorption at different temperatures does not occur in common wolf-fish: embryo size and yolk content at identical developmental stages remain the same regardless of incubation temperature (Pavlov & Moksness, 1995).


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6.5 From Hatching to Metamorphosis Embryos can hatch at various sizes and degrees of development, depending mostly on the size of the yolk (see Fig. 1.2, Chapter 1). As stated previously, newly hatched yolk-sac larvae from demersal eggs are generally more advanced in development than larvae from pelagic eggs of a comparable size. In species with large eggs, hatching may be delayed until the yolk sac is absorbed and the larvae are ready to feed at hatching. Larvae from pelagic marine eggs with small yolks are thus less well developed at hatching than larvae hatching from eggs with larger yolks. Newly hatched pelagic larvae generally lack a functional mouth, eye pigments, digestive system and differentiated fins. Yolk-sac larvae (free embryos) rely on their yolk supplies until their sensory, circulatory, muscular and digestive systems develop to such a degree that they are able to capture food. They are either transparent or lightly pigmented, and they possess species-specific characters (pigment patterns, body shape and size) which are useful for identification in field investigations and for systematic studies.

6.5.1 To Be a Larva . . . Direct or indirect development makes a difference to when and how larvae will change to the adult form (Balon, 1985, 1990). A larva is a transitory form of life, which often inhabits an entirely different niche than the adult form. Larvae may have a different body shape from the adults, and they are characterised by temporary larval organs and tissues such as an unpaired finfold with respiratory blood vessels and a dorsal sinus. Spines and filamentous appendages to give buoyancy may be present. The process of metamorphosis, or remodelling of the organism, is often connected with substantial changes in the larval morphology owing to the transition to a new environment. For example, the transition from pelagic to demersal life in flatfish is associated with a flattening of the body and the migration of one eye to the opposite side of the head, which becomes the upper side of the body. The body shape of the European eel (Anguilla anguilla) changes from leaf-like to eel-like during a prolonged migration from the Sargasso sea to the coast of Europe. These are typical examples of indirect development from larva to juvenile. Thus, a fish larva may be defined as a free-living, non-reproductive post-embryonic stage that is markedly different in form from the adult, and which undergoes metamorphosis into the adult form. Many of the species currently farmed have indirect developmental patterns (all flatfish and cod) which are associated with high fecundity, small eggs, larval stages, prolonged metamorphosis and high mortality. One probably important reason why many marine fish species have numerous small eggs, resulting in small, vulnerable larval stages, is their ability to make use of the rich planktonic habitat. The open sea is an excellent place for dispersal of the offspring, and the larvae are literally hatched into a very nutrient-rich ‘soup’ of small zooplankton prey organisms. The larval characteristics change during development, and the transformation to the juvenile stage may be gradual or abrupt. The latter is often the case when they move from pelagic to demersal habitats.

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6.5.2 . . . or Not to Be a Larva Not all fish pass through a real larval stage. Ovoviviparous and viviparous species, and many species with large eggs, a large yolk/blastodisc and/or parental care may omit the larval stage completely. In oviparous species, the embryo may hatch at an advanced stage of development, and transit to exogenous feeding as a juvenile after a short period of time. Thus, the direct ontogeny is characterised by the absence of metamorphosis, and allows development without using energy to produce transient larval tissues. In wolf-fish, the onset of larval exogenous feeding coincides with hatching, and their ontogeny is regarded as direct by some and intermediate by others. In these species, the period between the start of exogenous feeding and the loss of larval characters (e.g. the yolk sac) is a very short interval of development. The intermediate mode of early ontogeny is a transitory type. At the transition to exogenous feeding, the organism possesses many juvenile characters, and as a result, the metamorphosis is not pronounced and the period to the loss of larval characters is comparatively short. For example, in salmonids the organism has both larval and juvenile characters in the period from the beginning of exogenous feeding to when it reaches the juvenile state, and this period is called the ‘alevin’ period (Balon, 1980, 1985). Salmonids can be said to go through two metamorphoses, one at the end of the larval stage and one at smoltification (note that smoltification is reversible but metamorphosis is not). The alevin period is analogous to a short larval period, and the distinction between these two terms is very slight, if it exists at all. At the start of exogenous feeding, salmonids and wolf-fish have a clear skin pigmentation, possess functional gills and fins, and can be fed commercial pelleted diets from startfeeding because their digestive system is as well developed as in juveniles. The major difference between early ontogeny in salmonids and wolf-fish is that salmonids have a prolonged yolk-sac larval phase, which is absent in wolf-fish because they develop for a longer period in the egg, hatch at an advanced developmental stage and begin to feed externally just after hatching (Fig. 6.10). In viviparous fish, the young organism may be born as a juvenile. For example, the eelpout (Zoarces viviparus L.) and the redfish (Sebastes sp.) possess juvenile characters and transit to exogenous feeding just after parturition. Despite the presence of a small yolk sac, these organisms can be defined as juveniles. Species with direct ontogeny generally have low fecundity, with a low mortality in their large offspring, and they hatch at advanced stages of development. Thus, the transition from indirect to direct ontogeny represents a gradient to a more specialised type of development with a shortening of the most vulnerable larval period.

6.5.3 The Yolk-Sac Period—Preparation for ‘Real Life’ During the yolk-sac stage, the organism must develop in order to be able to eat, grow and survive. When yolk-sac reserves are depleted, the larva must be able to catch prey and avoid predators, and to digest the ingested food. To become a functional first-feeding larva, several larval qualities must develop and become functional before yolk nutrients are depleted (e.g. Blaxter, 1988). Their visual acuity, activity level, swimming pattern, muscular development,


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Figure 6.10 Comparative development of Atlantic salmon (1) and common wolf-fish (2) at 10°C. A, formation of blastodisc (1.5 days); B, start of finfold formation (40 days); C, formation of dorsal, anal and caudal fin rays (60 days); D, first feeding (salmon 90 days, wolf-fish at hatching 100 days); E, start of juvenile period (102 and 110 days, respectively). Drawing from Pavlov and Moksness, 1994.

jaw development and digestive capacity must all be correctly in place in order to catch food and survive. At the time for first feeding, fish larvae therefore have well-developed heads, brains, eyes and jaws, which are all essential features in order to survive and grow. The body is slender, with somatic muscles around the notochord. Pelagic fish larvae have no differen-

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Table 6.4 Organs that are present/not present in pelagic marine fish larvae such as cod, turbot and Atlantic halibut at the start of exogenous feeding. Present

Not present

Gut (no stomach) Liver and pancreas Large eyes and brain Well-developed olfactory and taste senses Neuromasts Finbuds Median finfold and subcutaneous space

Stomach (i.e. less efficient protein digestion) Calcified skeleton

Lateral line Functional gills Real fins and fin rays Skin and scales

tiated skin, but a median finfold filled with fluid surrounds the body and yolk sac. Its function is probably both for buoyancy and for regulatory purposes. The gut is more or less a straight tube, which is connected to the liver and a discrete pancreas. A summary of the organs that are present/not present in pelagic larvae is shown in Table 6.4. A characteristic feature of larval development is allometric growth, which means that the growth rate of one body part or tissue may differ from the growth rate of the whole organism (Osse et al., 1997). Developing larvae have to use their limited energy resources economically, and allometric growth ensures that growth is concentrated in those tissues and functions that are most important for the survival of the larva. Allometric growth is typical of larval stages, whereas older fish grow isometrically (i.e. all body parts have similar growth rates). For a fish larva, it is important to grow as quickly as possible, as a larger size generally increases its survival potential. A larger larva will be able to catch larger prey (more energy in one bite), and it will swim faster and thereby be more succesful in hunting prey and avoiding predators. Swimming capacity is thus crucial for larval survival, and the early development of swimming organs must therefore have a high priority. Systems for feeding and swimming should develop simultaneously and in balance with each other. Recent experiments clearly demonstrate that the allometric growth and function of organs and structures suggests a functionally optimised growth that matches the expected necessary priorities for larval survival and growth. Thus, fish larvae apparently spend their available energy on the most important functions for survival.

6.5.4 Metamorphosis Metamorphosis means transformation, and has been used to describe rapid morphological and physiological changes following a stable period of slow development. In fish, as in frogs, metamorphosis marks the transition from larval to adult form. This functional transition affects almost every system from internal physiology and neurology to external phenotype, but not the reproductive structures (even though these may be indirectly affected by external conditions during this phase). When does metamorphosis start? The literature shows various answers, not all of which are defined or compatible:


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• when larval allometric growth changes to isometric growth, indicating that this is a species-specific application; • when the primordial finfold, or the larval median finfold, is absorbed and median fins are visible (species-specific); • when axial skeletal vertebrae develop, which requires histological examination or clearing and staining for degree of ossification (species-specific); • when endocrine levels are driven by the larva´s own neuroendocrine stimuli, rather than reflecting hormones deposited by the female fish (species-specific).

There is general agreement about what metamorphosis involves, despite the lack of concensus on specifics. In general, it involves changes in the non-reproductive structures between the period of embryonic growth and sexual maturation, excluding embryonic development, growth, sexual maturation and ageing. This may include such features as the resorption of the median finfold, the development of the axial skeleton, the formation of a proper stomach, changes in neurology, vision and behaviour, changes in dermal structure and pigmentation, changes in the basis of respiration and osmoregulation, and the development of specialised muscles. These changes are not necessarily rapid, and are usually triggered by an external or internal cue. They also imply that during metamorphosis the fish occupies a different ecological niche from the preceding or adult stages. By convention, metamorphosis is now divided into at least four ‘stages’. (1) Pre-metamorphosis: the larval stage before changes in muscle and head structure are visible, and close to first feeding. (2) Prometamorphosis: when allometric growth of the body commences and some structural changes can be seen. (3) Climax metamorphosis: when there is strong allometric growth of the body, and structural changes are well underway. (4) Post-climax metamorphosis: when the early juvenile form has been attained. This terminology will be used throughout the rest of this chapter. The process of metamorphosis is best understood by examining the changes in specific systems, and then looking at triggers that may stimulate, control or modify the course of change. In the following sections we will look at the individual larval organ systems and how each one changes.

6.6 Functional Development of Organ Systems from Hatching to Metamorphosis At the time of mouth opening, the fish larvae will soon deplete the relatively small supply of energy from the yolk sac, and must learn to catch food from their surroundings. Larvae from larger eggs have more time and energy available before irreversible starvation occurs (the so-called point of no return, PNR). If larvae are starving, or if the nutritional quality of the food is inadequate, the larvae may quickly reach PNR, be unable to ingest and digest

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prey, and die. In cod, the PNR appears to be just before 9 days after hatching, and exogenous feeding usually starts 4–5 days after hatching (Kjørsvik et al., 1991).

6.6.1 Sensory System Functional sense organs such as optical receptors, mechanoreceptors and chemoreceptors are vital for larval detection of prey. The sensory organs of pelagic larvae are incomplete at hatching, and the timing of their development may differ from species to species. This section is divided into vision and the oculovestibular system, chemosensory systems such as the nares, and the lateral lines. The eyes of fish with direct, indirect or intermediate development differ with respect to the timing of the formation of the photoreceptors (rods and cones), although there are many species-specific differences. The formation of visual cells is also related to the optical environment which the larvae and juveniles inhabit. In general, the pigmentation of the previously colourless eyes occurs simultaneously with first feeding. This is actually the development of the pigment epithelium, which is a layer of dark pigment that can expand over the visual cells (cones), reacts to light intensity, and significantly increases the conemediated acuity (sharpness of vision) during bright light (Evans & Fernald, 1990). In general, marine fish larvae inhabiting the pelagic are visual predators. They have retinas comprising cones that require high light intensities for vision and see in colour. The rest of the retina is not fully differentiated prior to metamorphosis, but visual images are nonetheless translated into neural impulses. Most fish larvae seem to have a pure cone retina at first feeding, which may explain why so many teleost larvae feed at relatively high light levels. It is not known to what extent meso- and bathypelagic larvae have a pure cone retina, but this is the case for halibut, whose larvae are assumed to ascend from the mesopelagic to the epipelagic zone prior to metamorphosis (Fig. 6.11). Before metamorphosis the lens may be directly on the retina, but as the fish grows the lens separates. The field of focus may be short and fairly fixed, which affects the distance at which prey can be perceived. 6.6.1.1 Vision and the Oculovestibular System Fish regulate the amount of light entering the retina (not the eye, as mammals do) by extending or contracting the pigment epithelium over the tips of the cones and rods. The rods and cones themselves can extend or contract according to the light conditions. This is called retinomotor action, since the cells in the retina move relative to each other. Fish have another distinction from other visual systems in that they focus by moving the lens with small muscles (retractor lentis), not by changing the shape of the eye. The ecology of marine fish dictates the form and function of the eye. All elements of the eye and retina, including physical placement, photoreceptor specificity, biochemistry and physiology, can be affected by a change in the environment and the amount of illumination. As the fish moves from the epipelagic to deeper in the water column (which is generally the case), the eyes become larger in the need to capture the ever-decreasing amounts of light, which itself becomes more monochromatic (goes towards one colour; in the ocean, this colour is usually blue).


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Figure 6.11 Development of the halibut (Hippoglossus hippoglossus) retina from hatching through to metamorphosis. Initially, the neuroblastic cells are in contact with the lens. The pigment epithelium and the retinal layers develop at around 150 day-degrees. Lens separation, the cone mosaic, rod recruitment and the retractor lentis muscle appear at around metamorphosis (Kvenseth et al., 1996).

In prometamorphosis, in addition to the cones, rods begin to be recruited to the retina (the fish can see at lower light levels). Coincident with rod neurogenesis, phototaxy changes. A larva which previously avoided light will be drawn to it, while a larva which was positively phototaxic may now prefer lower light levels. It is thus possible that rods signal a physical migration of the fish from one area to another. In freshwater fish, rods generally appear before hatching, whereas in salmon, rods occur at ‘swim-up’ just prior to exogenous feeding (a good example of such a shift in phototaxy). Other muscles in addition to the retractor lentis are added, meaning that the eye has a greater range of movement and a wider field of view. The distance at which prey may be detected also extends. The section of the visual field in which most prey are taken may change with both the physical movement of the eyes relative to the cranium (migrating more anteriorly, or slowly migrating from one side as in flatfish) and the number of photoreceptors. The photoreceptors each contain a pigment which is photoreactive. In some species, these visual pigments may change during metamorphosis, and the change can be induced by thyroxin. In salmonids, there is a transient UV-sensitive cone associated with smoltification.

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At climax metamorphosis, the cones reorganise and a mosaic is formed in a regular pattern of single and double cones, and sometimes accessory cones. The retina is fully formed, and further growth involves stretching the retinal surface and some recruitment of new cells. As the fish continues to grow, the distance between the lens and the retina extends, and is compensated for by the growth of the lens. Flatfish metamorphosis involves the spectacular migration of one eye to the other side of the head, leaving the fish with an ocular and an abocular side. This migration of the eye necessitates a major reorganisation of the vestibulo-ocular pathways (nerves going from the eyes to the otolith labyrinths to the brain, which are meant to indicate which way is up, among other things). The optic nerve of the migrating eye begins to coil and elongate prior to eye migration. The otolith labyrinth nerves are recoupled with both the muscles around the eye and the optic tectum. In flatfish, even though the eye migrates, the corresponding vestibular system (otolith and labyrinth) does not. During this metamorphic change there is a substantial increase in the number of neurons projecting from the vestibular nerve to the vestibular neural complex in the brain stem, and many more of the vestibulo-ocular neurons project bilaterally to the rostral eye motor nuclei after metamorphosis in turbot (Jansen & Enger, 1996). The otoliths remain on either side of the head, but the neural reorganisation means that the fish has changed its neural definition of the physical meaning of ‘up’. There may be a permanent change in the optic tectum. The period of eye migration may be shorter than the period of metamorphosis, and is variable. 6.6.1.2 Chemosensory System Many larvae from demersal eggs have olfactory receptors at hatching, whereas in many pelagic larvae the olfactory organ (nares, or olfactory plates) usually develops shortly after hatching. The nares are initially flat structures on the surface of the larva, with exposed chemosensory cells. These perceive impulses (detect amino acids) from any direction. In addition, pear-shaped taste buds in the mouth and pharyngeal region, as well as elsewhere, appear early in larval development. During development, they increase in number and size. The larvae are probably adapted to a certain taste and palatability of food, and these requirements have to be met in order to improve the acceptance of formulated diets. As the larva becomes prometamorphic, the nares (olfactory plates) descend and skin flaps rise laterally. These flaps fuse at around climax metamorphosis and form a tunnel over the chemosensory cells, which thus detect amino acids and other molecules from the direction of the water flow entering the channel. It is possible that a change in chemoreception occurs during metamorphosis as different cells recruit and specialise. 6.6.1.3 Lateral Line Larval marine fish generally have numerous, relatively large, free neuromasts. Initially these neuromasts are single, and are mostly concentrated around the eye and olfactory cup, with some along the body and the head. These gradually increase in number and aggregation, with more external free neuromasts developing along the body axis prior to the development of the familiar lateral line of juvenile fish. These external neuromasts comprise small sensory


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hair cells at the base covered by a large gelatinous cupula that extends outward. The neuromasts perceive currents or vibrations in almost any direction. The number and complexity of neuromast assemblages increases with larval age, going from single to doublets to triplets in juveniles. Neuromasts also become encased in the developing lateral line canals at around metamorphosis. The canals on the head close over the exposed neuromasts first (at around climax metamorphosis), followed by the gradual closure of the trunk (body) canal. While the density of neuromasts decreases, the directional specificity of their sensory signal increases owing to their enclosure in a canal aligned along the body. In flatfish, the position and number of neuromasts changes according to whether they are on the ocular or the abocular side. The increase in the directional specificity of the response accompanies the development of a species-specific behaviour pattern (Poling & Fuiman, 1997).

6.6.2 Digestive System By the end of the yolk-sac period, larvae must obtain exogenous food in order to have energy for the synthesis of new tissue (growth) and for their maintenance metabolism. This includes capture, ingestion and digestion of the prey, and absorption and assimilation of the nutrients. In aquaculture, finding the larval nutritional requirements for optimal growth is also imperative. The dietary needs of developing larvae are different from those of the adult fish, and therefore larval nutrition should always be considered according to the ontogenetic development of their digestive systems, their nutritional requirements and their behaviour. It is possible to distinguish three types of larva based on the development of their digestive functions (Govoni et al., 1986). (1) (2) (3)

A functional stomach is developed before they start feeding. No stomach develops during the larval stage, but it is developed at a later stage. No stomach is ever developed.

Salmonids and the wolf-fish are typical examples of fish that possess a functional stomach at the time of first feeding. Both salmonids and wolf-fish can be fed formulated diets from the first feed uptake, owing to efficient protein digestion in their stomach. The pelagic marine larvae of cod, turbot and halibut are typical of larvae that develop their stomach later, and most larvae in this group gain a functional stomach during metamorphosis. Fish that have no stomach throughout their whole life are generally herbivores, often with a very long gut compared with species with functional stomachs. Their diet consists mainly of carbohydrates, which are less digestible and consequently need a longer gut passage time. The lack of a functional stomach is one of the main reasons why start-feeding has been a bottleneck in marine fish larval rearing, and why we have had problems in developing formulated feeds that are suitable for these pelagic marine larvae. The digestive capacity of pelagic marine fish larvae has therefore been classified as ‘undeveloped’, ‘simple’ or ‘primitive’, and some theories have maintained that these fish larvae are dependent on exogenous digestive enzymes from their prey organisms.

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However, the natural diet of marine pelagic larvae consists of zooplankton and phytoplankton, and the larvae are already raptorial carnivores from the end of the yolk-sac stage. More recent studies have shown that these larvae do indeed develop a digestive apparatus that is very well adapted and specialised to their life-style and feeding patterns. 6.6.2.1 Gut, Pancreas and Liver Differentiation For animals in general, the enzymatic digestion of food occurs in the lumen of the stomach and the intestine. This is a complex chemical process, where several digestive enzymes catalyse the breakdown of large food molecules into simpler compounds (nutrients) that can be absorbed through the gut epithelial cells. The stomach contents are acidic, and the intestinal lumen is slightly alkaline. The enzymes have different optimal pH values according to their site of action in the digestive system. Digestive enzymes are secreted from the gastric glands in the stomach, from the exocrine pancreas, where the enzymes are secreted to the gut lumen through the pancreatic duct, or from the intestinal epithelial cells. At hatching, the digestive tract of pelagic marine larvae is a closed straight tube (the mouth and anus are not yet formed), and it is histologically undifferentiated throughout its length. The mouth and anus will form during the yolk-sac phase, and the gut will lengthen and differentiate into a buccopharynx, fore-gut, mid-gut and hind-gut (Figs. 6.12 and 6.13).

M N Sw E Mg

L

Hg

Fg

Figure 6.12 Gut differentiation in cod larvae. The drawing shows a cod larva at the onset of first feeding. The lower picture shows a longitudinal histological section through the digestive tract of a 17-day-old cod larva. Drawing from Galloway et al., 1999a; the histological section was made by E. Kjørsvik. L, liver; Fg, foregut; Mg, midgut; Hg, hindgut; E, oesophagus; Sw, swim-bladder; N, notochord; M, muscle.


Yolk sac larva

Final yolk absorption

Larva

Transformation

235

Juveniles & adults Oesophagus

Foregut Incipient gut

Stomach

Midgut

Anterior intestine

Hindgut

Posterior intestine

Figure 6.13 Development of the alimentary canal in larval fish. At hatching, the gut is an undifferentiated straight tube which is closed at mouth and anus. The liver and pancreas are formed at hatching, and become functional at the time of exogenous feeding. The yolk is absorbed through the yolk syncytium, which is connected to the liver. The gut is differentiated into three distinct regions by the start of feeding. During larval development, the gut length and thickness increase, and the stomach is formed as an expansion from the fore-gut. From Govoni et al., 1986.

During the first feeding period, the gut will increase in length and width, thus considerably increasing its absorptive surface. The gut wall (mucosae) is a thin layer of smooth muscle, connective tissue and squamous epithelium, lined with numerous absorptive microvilli towards the gut lumen. The gut epithelial cells are called enterocytes. A small expansion may appear posterior to the oesophagus which is called a ‘post-oesophageal swelling’, and this is where the stomach will later develop. There are no digestive gland structures in the larval digestive system, and thus no pepsinogen is present (i.e. a digestive enzyme secreted in the stomach for breaking down complex proteins). The gut mucosa is very dynamic tissue which is involved in the hormonal and nervous activation of enzyme and bile synthesis and their subsequent secretion from the pancreas and liver. It is the main site of the digestion and absorption of nutrients (Fig. 6.14), and the epithelial cells of the gut are renewed more often than cells in other tissues. Ingested food particles are transported through the digestive tract by peristaltic movements in the gut smooth muscle. Even though the larval digestive system is less elaborate than that in adult fish, the functions and tissues which are essential for nutrient digestion, absorption and metabolism are developed at the beginning of exogenous feeding. The gut will continue to increase in volume and length throughout the whole larval phase, the intestinal folds become more obvious, and gut passage rates will increase. The liver, with its bile system, and the pancreas develop during late embryogenesis and the yolk-sac stage, and these organs are functional before mouth opening and first feeding. The liver rises from the periblast, with a clear connection to the gall bladder and to the yolk. Unlike in most adult fish, the larval pancreas is most commonly described as a distinct organ, which will develop to a diffuse tissue towards the end of the larval phase or during the juvenile phase. This is the case for cod (Kjørsvik et al., 1991; Morrison, 1993), halibut (Kjørsvik & Reiersen, 1992), turbot (Segner et al., 1994) and common wolf-fish (Falk-Petersen & Hansen, 2001). The general sequence of digestive system development is shown in Fig. 6.15 for the Japanese flounder (Paralichthys olivaceus). The developmental sequence of cell differentiation, like the synthesis of enzymes, appears to be genetically programmed at first, while a

236


Hindgut

Midgut

Proteins

Foregut

Lipids

Absorption Ingestion PI Extinction

Pv Transport

Digestion

Resynthesis

Accumulation LI

Accumulation GA

Transport

Ly N

a

GA

N

b

Figure 6.14 (a) Histological section of a turbot larva, showing mid-gut epithelium with lipid vacuoles (E. Kjørsvik and T. Bardal, Department of Biology, NTNU, unpublished data, 2002). (b) Schematic drawing of enterocytes in the different parts of the larval gut. Lipids are mostly absorbed in the mid-gut epithelial cells (enterocytes), and macromolecules from proteins are taken up by pinocytosis in the hind-gut epithelial cells. From Govoni et al., 1986.

dietary influence has been detected on both the organ structure and the enzyme levels once feeding has started. 6.6.2.2 Digestive Enzymes The pancreatic cells start to synthesise enzymes well before the onset of exogenous feeding, and this serves as conditioning for the first feed intake (Hjelmeland et al., 1984; HoehneReitan et al., 2001a). All the digestive enzymes (except the stomach enzymes) seem to be present in pelagic marine larvae at the onset of first feeding (Hoehne-Reitan & Kjørsvik, in press). These include pancreatic enzymes such as lipases, trypsin, chymotrypsin and amylase, which are responsible for the luminal digestion of food macromolecules, and enterocytic brush-border bound enzymes such as aminopeptidase, maltase and alkaline phosphatase, which complete the breakdown into absorbable monomeres. The digestive enzymes and the activities detected during first feeding in larval turbot are shown in Fig. 6.16. Pancreatic and intestinal enzyme activities are generally low at the onset of first feeding, but will increase exponentially after exogenous food intake has been established if the quality and quantity of food is satisfactory (Hoehne-Reitan et al., 2001a; Hoehne-Reitan & Kjørsvik, in press). Some of the most important larval digestive enzymes are proteolytic trypsin-like enzymes and lipolytic enzymes such as lipases. These are produced in the exocrine pancreas and are secreted to the gut, where they become active in the alkaline environment produced by the


237

Figure 6.15 Schematic development of the diffuse pancreas in Japanese flounder, Paralichthys olivaceus. Shaded areas indicate the distribution of the pancreatic tissue from a distinct to a diffuse organ. (a) 3 dph (days post-hatch); (b) 10 dph; (c) 20 dph; (d) 30 dph; (e) 45 dph (= metamorphosed). bd, bile duct; es, oesophagus; gb, gall bladder; hd, hepatic duct; in, intestine; li, liver; pa, pancreas; ph, porta hepatis; py, pyloric appendages; re, rectum; st, stomach (from Kurokawa and Suzuki, 1996). Reprinted with permission from Elsevier Science.

Trypsin Aminopeptidase Alkaline phosphatase Maltase Esterase Carboxypeptidase A, B Acid phosphatase Amylase Phosphodiesterase Lipase

Pepsin

Phospholipase A2

1 Hatching

3

6

23

28

Life prey Weaning

Figure 6.16 Digestive enzymes and activities detected during first feeding in the intestinal tract of larval turbot (from Hoehne-Reitan & Kjørsvik, in press). Dph denotes the time (days) after hatching when the enzyme or the enzyme activity was detected for the first time. Data from Segner et al., 1994, Ueberschär, 1993, Munilla-Morán et al., 1990 and Cousin et al., 1987.

238


e

-1 Lipase (mg larva )

10

Artemia d cd

Rotifers bc Hatching 1

bc abc abc ab

a ab

*

ab Algae

0.1

-5

0

5 10 15 Days after hatching

20

25

Figure 6.17 Content of bile salt-dependent lipase in developing turbot larvae determined by an ELISA assay. Different letters indicate significant differences between means of the fed larvae; * indicates a significant difference between fed (䊉) and starved (䉱) larvae. An immunoreaction was also found in the egg stage (䊊) (from HoehneReitan et al., 2001b).

bile. Some of the few quantitative studies of larval digestive enzymes have shown that the pancreatic cells start the synthesis of trypsinogen (the inactive form of trypsin) and ‘bilesalt-dependent lipase’ (the most important lipase in fish) well before the onset of exogenous feeding, as is shown for cod and turbot in Fig. 6.17. The presence of these enzymes follow a similar pattern of development. Larval synthesis, content and activity of pancreatic enzymes are generally low at hatching. The yolk-sac phase is characterised by a food-independent increase in enzymes, which may be interpreted as an imprinted physiological event during development (Hjelmeland et al., 1984; Zambonino Infante & Cahu, 1994; Hoehne-Reitan et al., 2001b; Hoehne-Reitan & Kjørsvik, in press). During the first days of exogenous feeding, a plateau, or even a decline, in larval enzymatic content has been observed in both fed and starved larvae, and larval development and the initial responses to feeding conditions seem critical during this short period (Hoehne-Reitan & Kjørsvik, in press). This phase is also generally characterised by increased mortality and low growth rates (Blaxter, 1988). In older larvae, the synthesis of enzymes increases exponentially if the larvae are fed adequate food, and the amount of enzyme secretion seems to be dependent on the larval feed intake, and possibly on the diet composition (e.g. Pedersen et al., 1987; Zambonino Infante & Cahu, 2001; Hoehne-Reitan et al., 2001a, b; Hoehne-Reitan & Kjørsvik, in press). It has been shown for several species that fish larvae produce enough digestive enzymes to be able to digest the ingested food. The contribution of digestive enzymes from the prey organisms seems to be very low during the start-feeding period, and generally does not exceed 6% of the total larval enzyme activity (Hoehne-Reitan et al., in press). A significant contribution of exogenous enzymes from the prey organisms to fish larval digestion thus seem very unlikely in older larvae, although they could play a role in the initial phase of first feeding (Table 6.5).


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Table 6.5 Contribution of exogenous enzyme activity from prey to the total activity found in marine fish larvae or the larval gut (% of total activity in the larvae/intestine). Modified from Hoehne, 1999. Enzyme

% activity of total

Days

Lipase

0

Lipase PLA2 WEH

0.5–1.1 (of larvae) 1.7–5.8 (of larvae) 0.1 (of larvae)

Lipase Amylase Trypsin

0.6–4.5 (of intestine) 2.5–23.4 (of intestine) 0.9–5.9 (of intestine)

Protease

0.6 (of intestine)

Trypsin

3 (of intestine)

Esterase Exonuclease Amylase Protease

89–94 (of intestine) 79–88 (of intestine) 17–24 (of intestine) 43–60 (of intestine)

Neutral lipase

3.9–5.2 (of larva) 1.6–2.3 (of larva) 2.6–4.1 (of intestine)

6 12/13

Phospholipase

7.4–9.9 (of larva) 1.7–2.9 (of larva)

6 12/13

Prey

Fish species 1

–

Rotifers

–

7–20

Artemia

Striped bass2

40 days) compared with other pelagic marine larvae. They are also relatively fragile, and are sensitive to suboptimal environmental influences (temperature, salinity, mechanical disturbance, light and microbial activity). The larvae are usually kept in silos, as previously described, at 5–6°C in darkness until exogenous feeding starts. The general idea of silo management is to provide a stable environment with good water quality, but without mechanical disturbance of the larvae. Variable survival rates and high mortalities are still common problems, and the larvae often develop with serious malformations such as gaping jaws and yolk-sac oedema. The water flow rate may be adjusted according to the vertical position of the larvae in the silo, usually 0.8–1.5 tank volumes per day for the larger silos, and more often for the smaller ones. The larvae are kept in the silos until they start exogenous feeding. They are transferred to start-feeding tanks at 220–270 d°, and the temperature is gradually changed to 9–12°C.

6.8 Critical Aspects of Larval Cultivation From the information given in this chapter, it is possible to deduce several consequences for aquaculture based on our general knowledge of egg and larval development. Sensitivity varies during embryonic development, and the embryo is especially sensitive to environmental effects during gastrulation (or epiboly). If halibut eggs are transported during this stage, for example, severe mortality generally occurs. It is therefore a good rule

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of thumb to transfer marine fish eggs only after the yolk plug has closed, although it should be done a least a few days before hatching, and before the hatching enzymes have weakened the egg-shell. Salmonids and wolf-fish should only be transported after the ‘eyeing stage’, i.e. after pigmented eyes have become visible in the embryo. The most critical aspect of larviculture is generally the very variable survival rate during the larval rearing stages. In turbot, for example, catastrophic mortalities may occur in the diet transition phase between the first and second week after hatching. For flatfish, developmental abnormalities such as incomplete eye migration, malpigmentation, skeletal deformities or incomplete operculum also often appear at metamorphosis, and this may be due to inadequate nutrition, microbial conditions or other physical factors. It is still a challenge to fully understand the interaction between the larvae and the prey, and between the larvae and the environment. Environmental factors may also affect muscle fibre recruitment in fish embryos and larvae, but little is known about how the environment might influence the muscle cell precursor population, or how maternal investment may affect egg and larval growth mechanisms. Identifying the developmental stages at which muscle precursor cells are most sensitive to external factors, and how egg quality, and maternal and environmental factors might affect muscle differentiation and growth in larval fish should therefore be further studied. The changes in the digestive system have a direct impact on rearing protocols because the fish can eat and utilise different feeds at different stages. In flatfish and some pelagic fish, metamorphosis is accompanied by settling to near the bottom of the tank, and the feeds no longer need to be live organisms. However, if the stomach is not fully differentiated, there is a risk of the feed being too complex. In such cases, despite an adult external appearance, the immature internal development will leave the fish unable to break down or absorb complex feeds, or there will be a risk of gut damage due to dry feeds of inadequate quality. The high ‘weaning mortalities’ often observed in cultured fish changing from live prey to formulated feeds may partially be the result of an inadequate understanding of speciesspecific digestive development at this stage. However, the digestive capacity of start-feeding marine larvae is high, and there is a growing understanding that formulated feeds may change (partly or completely) the present need for live prey organisms. Much research in this field is still necessary to obtain such a goal. Prior to the development of functional gills and visible erythrocytes, larval marine fish require high and stable oxygen levels in the hatchery water. The development of gills and adult-type erythrocytes with haemoglobin for binding oxygen means that metamorphosed fish can physically occupy environments with lower or more variable oxygen conditions than was possible during the larval stage. The tight correlation between temperature, the oxygencarrying capacity of the water, and the narrow temperature optimum for normal growth of most fish larvae indicate that conditions for larval growth must be well controlled. The larva’s initial inability to focus means that high prey densities are necessary for them to have a chance of entering the field of focus of a marine fish larva. It is also necessary to have reasonably high light and contrast levels between the prey and the background colours. However, once rods recruit, usually at around metamorphosis, the light levels may be lowered and the fish may actively avoid or seek out lighted areas, depending on the species. The


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attachment of muscles to the lens during metamorphosis means that the field of focus extends and prey densities may be lowered without affecting the rate of capture. Farmed fish usually need higher light levels during larval feeding than at later stages. Prey densities need to be high to enter the field of focus. During and after metamorphosis, more directionally specific responses to the environment are possible when the nares have formed, and the cephalic and trunk lateral lines have formed as almost closed canals. With increasing visual acuity, fish become more sensitive to their environment. Malformations and endocrine effects during metamorphosis may be a response to factors occurring much earlier in the life history, such as the period of first feeding. Any factor (nutrition, environment, maternal, viral) impinging on the development of organs and tissues such as the brain, and hence the pituitary–thyroid axis, will affect the ability of the developing fish to complete metamorphosis. Much more research is necessary in this area to fully understand the importance of larval nutrition, neural development and functional differentiation. Larvae generally have only a non-specific defence system. The specific immune system generally starts developing before climax metamorphosis, and is often mature only long after the completion of metamorphosis. This means that hygiene is very important for the early life stages, especially around first feeding, when the bacterial challenge can be high and the immune system is still very immature. Vaccination too early can cause tolerance rather than immunity. An intimate knowledge of these factors is necessary for the establishment of any species-specific immunisation programme. The transition from the larval stage to the juvenile stage is marked by metamorphosis. Metamorphosis involves every organ system, and is a complex process involving internal and external rhythms and stimuli. There may be several overlapping critical periods during which the appropriate nutrition and environment will contribute to the quality of the developing juvenile. When any critical stimulus is insufficient, viable but unusual postmetamorphic forms may occur. Further studies of the molecular basis of morphogenesis will aid in determining the ways in which rearing protocols affect juvenile quality.

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Chapter 7

First Feeding Technology Y. Olsen, T. van der Meeren and K.I. Reitan

7.1 Introduction It is quite a few years since early pioneers such as Dannevig first succeeded in culturing fry of marine fish in enclosed pelagic ecosystems which simulated ‘nature-like’ conditions with natural zooplankton as live feed (Rognerud, 1887). However, half a century had passed before Rollefsen (1940) first demonstrated that plaice larvae could be fed a zooplankton species that was not a natural component of the marine food web. This zooplankton species was Artemia, which is available from cysts all the year round (see Chapter 4). In this way, Rollefsen may have taken the first step towards developing the hatchery methods for marine fish larvae that are now used world-wide. Further major improvements in feeding techniques did not take place until Japanese scientists cultured a smaller zooplankton species, the rotifer Brachionus plicatilis, and later learned that fish larvae had high requirements for highly unsaturated n-3 fatty acids (n-3 HUFA). The artificial food chain of rotifers and Artemia was not nutritionally adequate, but the n-3 HUFA content of the zooplankton could be enhanced by feeding procedures later known as n-3 HUFA enrichment techniques. The final key to successful larval rearing was an understanding of the strong interactions between normal non-pathogenic bacteria and the fish larvae. These interactions were widely accepted during the 1990s (see Chapter 3). The production of fish larvae and viable fry has been a major obstacle in the development of a marine aquaculture industry. The challenges of larval first feeding are highly multidisciplinary, and the physical–chemical, nutritional and microbial conditions of the larvae must all meet their requirements. The main interacting factors are:

• The conditions in the rearing environment, the physical–chemical cultivation regime and the technology used to maintain that regime • Larval nutritional requirements and the feeding regime • Larval interactions with normal and pathogenic bacteria, and microbial hatchery management.

As well as these interacting factors, the quality and viability of eggs and yolk-sac larvae are paramount for rearing larvae successfully. A number of other fundamental aspects become crucial during first-feeding, where the process line from brood stock, to egg and yolk-sac larvae meets the process lines of the live food components in a completely new environment.

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This chapter describes the cultivation systems and methods that have been used to feed marine cold-water fish larvae from post-hatching to metamorphosis, or to weaning onto formulated feed (see Chapter 8). A wide variety of methods are described, but the main focus is on methods based on traditional rotifer and Artemia technology (see Chapter 4). The nutritional and microbial factors and their interactions during first feeding are of particular importance, and the fundamental aspects of these issues are presented in Chapters 3 and 4, respectively. Physical factors that are important in mariculture are generally addressed in Chapter 2, and are only covered briefly here. The most fundamental issues of larval morphological and physiological development that are important to an understanding of the complex nature and constraints of larval feeding are presented in Chapter 6. However, this chapter includes a short section on lipid and protein requirements, since these are essential to any discussion of the nutritional aspects of cultivation.

7.2 Nutritional Requirements of Marine Fish Larvae 7.2.1 Essential Fatty Acids Fatty acids are characterised by the number of their carbon atoms and double bonds. Fatty acids that can be synthesised de novo by animals (non-essential fatty acids) include saturated and monounsaturated fatty acids, with no and one double bond, respectively. Animals cannot synthesise fatty acids with two or more double bonds (polyunsaturated fatty acids, PUFA), although these are essential for growth and development. These fatty acids must be supplied in the food, and are known as essential fatty acids (EFA). EFA are grouped into two families: the linoleic acid (n-6) and the linolenic acid (n-3) families (Fig. 7.1). Highly unsaturated fatty acids (HUFA) are PUFA with 20 or more carbon atoms. Both animal and plant cells can catabolise, elongate and desaturate fatty acids through successive steps. However, animal cells cannot desaturate fatty acids closer than carbon 9–10 from the methyl end (n-9). This implies that all n-6 and n-3 fatty acid bonds of marine origin have been formed by organisms of other kingdoms, among which marine algae are the most important. Many carnivorous fish species, including species that are attractive for aquaculture, have little metabolic capacity to elongate and desaturate the shorter C18-precursors of the n-6 and n-3 families. However, they are able to modify HUFA through catabolic chain shortening reactions (Sargent et al., 1991). High contents of n-3 HUFA, e.g. eicosapentaenoic acid (20 : 5 n-3, EPA) and docosahexaenoic acid (22 : 6 n-3, DHA), are found only in aquatic plants and animals. These fatty acids are essential components for neural tissues, cell membrane functioning and many regulatory functions. Generally, n-3 fatty acids dominate n-6 fatty acids by a factor 5–20 in marine food webs, but the n-6 HUFA arachidonic acid (ARA, Fig. 7.1A) is also essential for marine fish.

7.2.2 Main Lipid Classes Lipids are grouped as neutral or polar lipids depending on their polarity (Fig. 7.1B). Triacylglycerides (TAGs) and wax esters (WEs) are neutral and abundant storage lipids which

First feeding technology

A

281

FAMILIES OF ESSENTIAL FATTY ACIDS Linolenic acid family, n-3

Linoleic acid family, n-6

linolenic acid, 18:3 n-3

linoleic acid, 18:2 n-6

H C 3

COOH

HC 3

COOH

eicosapentaenoic acid, 20:5 n-3 (EPA)

arachidonic acid, 20:4 n-6 (ARA) COOH

HC 3

H C 3

COOH

docosahexaenoic acid, 22:6 n-3 (DHA) COOH HC 3

B

MAIN CLASSES OF LIPIDS IN MARINE ANIMALS

NEUTRAL LIPIDS: Tri-acylglycerids (di-, mono-, glyco-)

: COOH

: OH

POLAR LIPIDS: Phospholipids

Wax esters

P

R

Figure 7.1 Characteristics of essential fatty acids and abundant lipid classes. (A) Schematic overview of the main fatty acids of the n-3 and n-6 families of essential fatty acids. (B) Main lipid classes of marine animals.

provide metabolic energy through oxidative catabolism. The TAG molecule is the dominant energy and carbon storage product in higher animals. Many zooplankton species populating cold waters, either in the deep parts of the oceans or at high latitudes, store their surplus energy as WE, which is formed by a long-chain monounsaturated fatty alcohol moiety bound to a fatty acid moiety. WEs have a lower melting point and a higher energy content per unit weight than TAGs. Zooplankton that store WE may exhibit very high contents towards the end of the growth season (>50% of dry weight, Sargent & Henderson, 1986). The basic unit of phospholipids (PL) is a diacylglyceride molecule with a phosphate group bound to an organic group in a terminal position of the glycerol (R, Fig. 7.1B). Common organic groups of PL are ethanolamine, choline and inositol, and the respective PLs are denoted phospatidylethanolamine, phospatidylcholine and phospatidylinositol. Aquatic coldblooded animals are characterised by having PL containing a high fraction of PUFA. PL, together with cholesterol and sphingolipids (not shown), are ubiquitous constituents of cell membranes, and are therefore both structurally and functionally important.

282


Feed lipids

Ingestion Digestion

Intestine

Assimilation

Tissues Cell membrane Metabolism (chain elongation synthesis and desaturation) Growth Regulation Exchange of molecules/ions

20:5 n-3 (EPA)

Synthesis of tissue hormones Prostaglandin G3 Prostaglandin G2

20:4 n-6 (ARA)

Regulatory functions

Figure 7.2 Schematic view of the uptake, assimilation, metabolism and functional roles of essential n-3 and n-6 fatty acids in animals.

7.2.3 Physiological Basis of n-3 HUFA Requirements Marine coldwater fish larvae need high proportions of n-3 HUFA to meet their requirements for growth and development. The experimental determination of the n-3 HUFA requirements for the early stages of marine fish larva fed live zooplankton has proved to be quite complicated. Therefore, major questions regarding their n-3 HUFA requirements still exist for many species. EFAs supplied in the food are crucial for the following metabolic and physiological processes (Fig. 7.2):

• Growth or de novo formation of biomass: tissue formation and differentiation • Membrane activity and metabolism: membrane transports, respiration and activities • Regulation of metabolism: prostaglandins and their hormonal functions

enzyme

The physiological justification for the relatively high n-3 HUFA requirements of marine cold-water fish is associated with PL synthesis and the formation of cell membranes. PL is


283

synthesised by enzyme systems that exhibit a higher affinity for polyunsaturated fatty acids (e.g. n-3 HUFA) than for other fatty acids. Under conditions of excess supply, these fatty acids are preferentially esterified to the sn2 position of the glycerol in both TAG and PL, whereas saturated and monounsaturated fatty acids are more frequently esterified to the terminal sn1 and sn3 positions. This discrimination explains why one out of two fatty acids of PL is expected to be polyunsaturated, whereas one out of three tends to be polyunsaturated in TAG (Sargent et al., 1991). This enzyme mechanism, along with other differences in enzyme specificity, secure some degree of genetic control in the composition of the membranes. This is critical from a functional point of view. However, the enzymes may incorporate enhanced levels of monounsaturated and saturated fatty acids in the membrane PL under conditions of n-3 HUFA deficiency. This is expected to result in reduced n-3 HUFA contents in the membranes (Sargent et al., 1991), and will, at some point which is species-specific, result in the gradually reduced activity of all membrane-bound enzymes. As a consequence, physiological capacity or general health is reduced. Inadequate dietary n-3 HUFA supplies may therefore have a general impact on animal function and health. Larval requirements of n-3 HUFA are strongly related to growth and tissue differentiation, which include eye, brain and neural development. Neural tissues contain very high amounts of DHA (Mourente et al., 1991), implying that this fatty acid is particularly important for very young and fast-growing stages of fish larvae. The specific growth rates of fish larvae are normally much higher than those for the adult fish. Larvae of cold-water species, e.g. cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) will double their biomass within a week or two, and some fast-growing species such as turbot (Scophthalmus maximus) will do so in 3 days. From an evolutionary point of view, we may assume that the essential components that are found in high quantities in the eggs are important in order to maintain normal growth and development during the early stages of life, and eggs of marine species do contain high levels of DHA. It is also easy to imagine that an inadequate brain function and vision, as demonstrated for DHA-deficient herring larvae (Sargent et al., 1993), will be fatal for marine larvae and other animals. It is likely that pronounced species-dependent differences exist in the n-3 HUFA requirements of marine fish larvae. Our quantitative knowledge of the n-3 HUFA requirements of marine fish larvae is inadequate, and there is no general conceptual understanding of the factors which determine these requirements. It is assumed that fish living at lower temperatures at high latitudes or in deep water will exhibit higher n-3 HUFA requirements than fish living in warmer water. Independent of temperature, it is further assumed that fast-growing species will have higher n-3 HUFA demands in order to develop functional membranes. Another factor is the ability of the species to elongate and desaturate shorter n-3 fatty acids to form EPA and DHA. A high metabolic flexibility in order to modify short-chain n-3 fatty acids may contribute to reduced requirements for n-3 HUFA. Such metabolic flexibility is apparently characteristic of many salmonids, including rainbow trout. Conversely, it is commonly believed that carnivorous marine fish have a lower flexibility than omnivorous and detritivorous species (Olsen, 1999a). Presumably it has not been important for carnivorous fish species to retain the facility for fatty acid elongation and desaturation during evolution because they naturally eat a well-balanced diet according to their specific requirements. Most

284


of the marine species of interest for mariculture are indeed carnivorous with high n-3 HUFA requirements. The optimisation of diets with respect to n-3 HUFA contents is therefore an important issue in marine larval fish nutrition. The C20-fatty acids EPA and ARA of the membrane phospholipids are precursors in prostaglandin synthesis (see Fig. 7.2). The prostaglandins are precursors for a number of regulating compounds known as tissue hormones. Prostaglandin G3 is synthesised from EPA and acts as a regulatory antagonist to prostaglandin G2, which is synthesised from ARA. Both ARA and EPA are derived from the membrane PL through the actions of phospholipases. The G3/G2 ratio, which is believed to modify many cellular processes, will depend on the ratio of EPA to ARA in the membranes, and ultimately in the diet. The relatively poor enzymatic control (i.e. genetic control) of PL composition (i.e. the ratio EPA to ARA), and the successive prostaglandin synthesis (G3/G2 ratio), implies that the fatty acid composition of the food will directly affect the regulatory hormonal processes of fish larvae and other animals.

7.2.4 Protein and Essential Amino Acids Amino acids are the fundamental components of enzymes, other proteins and nucleotides, and they are important precursors or N-sources for a wide range of biomolecules. Protein is quantitatively more important than lipids for larval growth, and protein malnutrition was understood long before lipid malnutrition. The amino acids that are assumed to be essential, or conditionally essential, for fish are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane, valine, cystine and tyrosine. Dietary proteins are the main source of essential amino acids. This chapter only presents results that clearly demonstrate the importance of protein nutrition during first feeding (Section 7.5.6). The protein requirements of fish are covered thoroughly in Chapter 9, and the requirements and digestion capabilities of specific fish larvae are discussed in Chapter 6.

7.2.5 Protein Versus Lipid Nutrition There is a fundamental biological difference between protein and lipid nutrition. The sequence of amino acids of enzymes, and the further synthesis of biomolecules mediated by these enzymes, are strictly genetically controlled. This means that the regulatory functions of proteins are under the strict control of genes. In addition, the percentage amino acid composition shows little variability between species, and is only moderately affected by nutrition. This means that the amino acid composition of the prey will be relatively close to that of the predator. This is contrary to the situation for lipids, which have regulatory cell functions, e.g. prostaglandin precursors. The fatty acid composition of these lipids is only moderately controlled by genes, and the fatty acid composition in the food is far more variable than the amino acid composition. This is why deficiencies in specific n-3 HUFA is more readily expressed than deficiencies in specific EAA. Because of these fundamental differences in (1) variability in distribution and (2) genetic control of lipid and protein metabolism, there is a greater probability that marine fish larvae


285

will encounter a deficiency of specific EFA than of EAA. However, proteins may still be supplied in sub-saturating quantities compared with growth requirements. An inadequate supply of protein is likely to occur, and will be expressed as a reduction in the specific growth rate of the larvae. Other disorders may occur with severe deficiencies. Conversely, symptoms and disorders caused by n-3 HUFA deficiency develop more gradually (see above), and a severely reduced growth rate is more likely.

7.3 Definitions and System Description The principal differences in the methods used by the pioneers of marine larviculture can still be seen in more recent methods of raising cold-water fish species. Two extreme cultivation concepts, and one intermediate one, can be defined (Fig. 7.3). Concept 1. Larval Feeding in Large Closed Nature-like Systems Larval densities are low (

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