Minerals in Animal and Human Nutrition, 2nd Edition

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands © 2003 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier's Health Science Rights Department, Elsevier Inc., 625 Walnut Street, Philadelphia, PA 19106, USA; phone: (+ I) 215 238 7869, fax: (+ I) 215 238 2239, E-mail: healthpermissions@ elsevier.com. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com). by selecting 'Customer Support' and then 'Obtaining Permissions'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+ I) (978) 7508400, fax: (+ I) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLP, UK; phone: (+44) 207 631 5555; fax: (+44) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier's Health Science Rights Department, at the phone, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2003 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguing in Publication Data A catalogue record from the British Library has been applied for. ISBN: 0 444 51367 I

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This book is dedicated, with appreciation, to my parents; my wife, Lorraine; my daughters, Suzannah, Joanna, and Teresa and their husbands and children; and to three great animal nutritionists (Raymond B. Becker, Tony J. Cunha and Jack K. Loosli) at the University of Florida for their practical knowledge of the livestock industry and for encouragement to write books.

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Preface The second edition of Minerals in Animal and Human Nutrition contains under one cover 19 chapters of concise, up-to-date information on mineral nutrition for livestock and poultry, with comparative aspects to laboratory animals and human nutrition. The first chapter is an introduction dealing with historical considerations, distribution in the body, general functions, mineral requirements and tolerances, methods of analysis, mineral bioavailability, status detection, and incidence of mineral deficiencies and toxicities. Chapters 2 through 14 discuss the properties and distribution, metabolism, functions, requirements, natural sources, deficiency, supplementation, and toxicity of the established and most common minerals. Chapter 15 is concerned with five toxic elements and their significance to various species and methods of control. In Chapter 16, chromium and newly discovered essential and other trace elements are discussed. Chapter 17 covers mineral sources, while Chapter 18 deals with maximum tolerance levels. The final chapter discusses mineral supplementation concepts. The present second edition has been completely and vigorously revised. Since the first edition in 1992, a great deal of new information has been generated in the field of minerals; this is reflected by the fact that more than half of all the references in the majority of chapters have been published since the first edition. The new edition continues to provide a large number of classic photographs that illustrate mineral deficiencies and toxicities that have been provided by distinguished scientists in the mineral research field. The purpose of this book is to provide, as both a college textbook and a reference source, a comprehensive text that contains current information on mineral nutrition. Most emphasis is centered on minerals in which naturally occurring deficiencies or excesses are of economic importance. A unique feature of this book is the practical implications of mineral deficiencies and excesses and the conditions under which they might occur in various animal species and humans. It is hoped that this book will be of worldwide use and will continue, as the first edition, to be used as a textbook and as an authoritative reference book for use by research and extension specialists, in the animal, poultry, and veterinary sciences fields and for feed manufacturers, teachers, students, and others. A comparison between the balance of chemical, metabolic, and functional aspects of minerals and their practical and applied considerations has been made. Unlike other textbooks, this one places strong emphasis on practical aspects of mineral supplementation in each chapter and devotes the last chapter to this subject. When preparing the two editions of this book, I obtained numerous suggestions from eminent scientists in both the United States and other countries. I wish to express my sincere appreciation to them and to those who supplied photographs and other material used. I am especially grateful to C. B. Ammerman, J. D. xv

xvi

Preface

Arthington, R. B. Becker, D. K. Beede, G. D. Butcher, G. E. Combs, J. H. Conrad, G. K. Davis, G. L. Ellis, P. Henry, J. F. Hentges, W. E. Kunkle, J. K. Loosli, P. G. Mallonee, R. M. Mason, R. D. Miles, R. O. Myer, W. M. Neal, E. A. Ott, A. L. Shealy, R. L. Shirley, H. D. Wallace, A. C. Warnick, and S. N. Williams (Florida); O. Balbuena and B. J. Carrillo (Argentina); B. Hetzel (Australia); E. Espinosa (Bolivia); N. dos Santos Fernandez, Jiirgen Dobereiner, Francisco Megale, and C. H. Tokarnia (Brazil); T. Ma (China); W. J. Miller and N. W. Neathery (Georgia); M. Anke (Germany); U.S. Garrigus (Illinois); S. P. Arora (India); W. M. Beeson (Indiana); D. V. Catron and V. C. Speer (Iowa); C. W. Absher, J. A. Boling, G. L. Cromwell, V. W. Hays, and D. E. Miksch (Kentucky); J. O. Evans (Kenya); J. Mtimuni (Malawi); G. F. Combs and W. Mertz (Maryland); C. Garcia Bojalil (Mexico); A. T. Forrester, E. R. Miller, and D. E. Ullrey (Michigan); L. E. Carpenter and H. S. Teague (Minnesota); B. O'Dell and R. L. Preston (Missouri); J. Kubota, M. L. Scott, and S. E. Smith (New York); K. M. Hambidge and J. D. Latshaw (Ohio); J. Adair, O. H. Muth, J. E. Oldfield, and F. M. Stout (Oregon); J. Zorrilla-Rios (Panama); R. M. Leach (Pennsylvania); M. Echevarria (Peru); O. E. Olson (South Dakota); B. D. H. Van Niekerk (South Africa); O. M. Mahmoud (Sudan); H. S. Ergun and K. Goksoy (Turkey); A. E. Olson and J. L. Shupe (Utah); J. C. Montero, D. Morillo, and E. A. Velasco (Venezuela); I. A. Dyer, J. W. Kalkus, and R. C. Piper (Washington); G. Bohstedt and M. L. Sunde (Wisconsin); and O. A. Beath (Wyoming). I am particularly grateful to Nancy Wilkinson and Pamela Miles for working on various sections and tables of the book and along with my wife (Lorraine McDowell) for their thorough editing assistance and useful suggestions. I wish to thank Mary Schemear, Shirley Levi, Patricia French and Sabrina Robinson for skillful typing. Finally, I am indebted to the Animal Sciences Department of the University of Florida for providing the opportunity and support for this undertaking. Lee Russell McDowell

xvi

Chapter 1

General Introduction

I. INTRODUCTION

All forms of living matter require inorganic elements, or minerals, for their normal life processes. All animal tissues and all feeds contain inorganic or mineral elements in widely varying amounts and proportions. Some confusion exists in use of the terms "minerals" and "elements" in nutrition and feeding. In practical nutrition, the term "mineral" is generally used to denote all the mineral inorganic elements. However, not all the elements are minerals (i.e., carbon, hydrogen, oxygen, and nitrogen), and minerals frequently found as salts (e.g., carbonates, oxides and sulfates) can be a combination of different inorganic elements. For the purpose of this book, the terms "mineral," "element," and "mineral element" are used interchangeably. The mineral elements are solid, crystalline, chemical elements, which cannot be decomposed or synthesized by ordinary chemical reactions. These inorganic elements constitute the ash that remains after ignition of organic matter. The common method of determining the total mineral or inorganic content of feeds consists merely of measuring the total ash remaining after high-temperature burning of the organic matter. This analysis is of little value either for expressing mineral requirements or for indicating the useful mineral content of foods, for two basic reasons. In the first place, body requirements are specific for certain inorganic elements. Secondly, ash may not be a measure of total inorganic matter present, because some organic carbon may be bound as carbonate and some inorganic elements, such as sulfur (S), selenium (Se), iodine (I), fluorine (F), and even sodium (Na) and chlorine (Cl) may be lost during combustion. In practice, the most important reason for the determination of total ash in a food is to permit calculation of the nitrogen-free extract by difference, as required in the proximate analysis of foodstuffs. Also, the ash analysis can be used in forages to estimate the amount of dust and soil that has been harvested with the feed. II. CLASSIFICATION OF MINERALS

Minerals are classified in a number of ways, with some classification schemes having a place in understanding their requirements and/or nutritional roles. Minerals that are needed in relatively large amounts are referred to as major or 1

General Introduction

2

TABLE 1.1 Essential Mineral Elements Traditional Established Minerals Macrominerals Calcium (Ca) Potassium (K) Sodium (Na) Sulfur (S)

Phosphorus (P) Magnesium (Mg) Chlorine (CI)

Microelements (trace minerals) Copper (Cu) Cobalt (Co) Chromium (Cr) Iron (Fe) Manganese (Mn) Iodine (I) Molybdenum (Mo) Selenium (Se) Zinc (Zn)

Newer Microelements (Discovered since 1970) Aluminum (AI) Boron (B) Fluorine (F) Lead (Pb) Nickel (Ni) Silicon (Si) Vanadium (V)

Arsenic (As) Bromine (Br) Germanium (Ge) Lithium (Li) Rubidium (Rb) Tin (Sn)

macrominerals. Others that are needed in very small amounts are referred to as trace minerals or microminerals. These terms do not imply any lesser role for the trace minerals. Rather, they represent quantity designations based on the amounts required in the diet and their generally low or "trace" concentrations in tissues. The major minerals are required in concentrations of greater than 100 ppm (parts per million) and often as a percentage of the diet (or g per kg), while trace elements are required at less than 100 ppm and are expressed as ppm and sometimes as ppb (parts per billion). Twenty-nine elements are known to be required by at least some animal species (Table 1.1). Seven elements are macrominerals and 22 can be referred to as microminerals or trace elements. The listing of some of the trace elements as essential is difficult and sometimes tentative. An essential element is one that is required to support adequate growth, reproduction, and health throughout the life cycle, when all other nutrients are optimal. Essentiality is less certain when there is only a small change in the rate of growth, when the environment is suboptimal, or when there is a microbial infection (O'Dell and Sunde, 1997). Observed improvements in performance upon supplementation with a mineral may be due to changes in the intestinal microflora, to a pharmacologic effect, or to interactions with other elements. The proof that each element is essential rests upon experiments with one or more species. In these experiments, clinical signs produced by diets adequate in all nutrients, except the mineral in question, have been prevented or overcome by adding that mineral to the diets. All the elements mentioned have not been tested with all species, but it is highly probable that there are few exceptions to the need for all of them by all higher animals. There is no disagreement concerning the essentiali ty of the trace elements chromium (Cr), cobalt (Co), copper (Cu), I, iron (Fe), manganese (Mn), molybdenum (Mo), selenium (Se), and zinc (Zn) although not all would present practical nutritional supplemental problems for livestock or humans.

Classification of Minerals

3

Whether an element is considered essential would depend on the criteria used. A viewpoint in human nutrition is that nutritional requirements should include consideration of the total health effects of nutrients, not just their roles in preventing deficiency pathology (Nielsen, 1996). Therefore, the terms "beneficial element" and "apparent beneficial intake (ABI)" are in use. In humans, for example, the ABI for maximal benefit of F relates to its proven benefits for dental health and its suggested role in maintaining bone integrity. The ABI seems more appropriate for the elements with beneficial, if not essential, actions that can be extrapolated from animals to humans; these elements include, in addition to F, arsenic (As), lithium (Li), nickel (Ni), silicon (Si), and vanadium (V). More recently discovered trace elements since 1970 are referred to as "new trace elements." These newer trace minerals are elements with an established or highly suspected requirement for one or more species, and include aluminum (AI), As, boron (B), bromine (Br), F, germanium (Ge), Li, Ni, lead (Pb), rubidium (Rb), Si, tin (Sn), and V. The essentiality of these last 13 elements is based on growth and other effects with animals under highly specialized conditions, such as improved procedures for purification of diets and use of metal-free isolator systems for raising animals. Furthermore, more precise and accurate methods of determining minute quantities of trace elements have been developed. Although markedly different in their chemistry, mode of action, and effective levels, the newer essential trace elements have in common the facts that they were first known for their toxic effects and that induction of a dietary deficiency is often difficult. An additional 20 to 30 trace elements occur regularly in feeds and animal tissue, and it is unknown whether they serve some useful purpose or are merely incidental contaminants. It is likely using advanced methodology that some of these elements one day will be considered essential. It is also possible that some of the more tentatively established essential elements may be declared non-essential with further studies. Eight mineral elements can also be classified as cations, including calcium (Ca), magnesium (Mg), potassium (K), Na, Fe, Mn, Cu, and Zn. Six other elements are either anions or are usually found in anionic groupings. These are chloride (CI-), iodine (1-), phosphate (PO~), molybdate (MoO;), selenite (SeO) and sulfate S04'. Likewise, they can be classified on the basis of valence number and on their group position in the periodic chart of the elements. These classifications can be useful because they describe physical and chemical attributes of importance in nutrition (Miller, 1979). For example, the monovalent cations, K and Na have a very high absorption percentage and major interrelationships exist between them. In contrast, the absorption percentage of the divalent cations (Ca, Mg and Zn) is much lower. Numerous factors may alter the availability of the essential anions and cations. The most soluble and absorbable form of any of the elements should be the simple ionic state of the atom or ionic group of atoms (for example, as Ca++, Mg++, Mn++). However, many electronegative compounds in nature are looking for a cation with which it can share its electrons, thereby forming a stable compound (Leeson and Summers, 200 I). Often the resultant compound is highly insoluble in water but nevertheless dissociates to a sufficient extent in the intestinal tract to

4

General Introduction

allow absorption of the essential cations. This is influenced by the gastric acidity of hydrochloric acid in the stomach, which converts the cations temporarily into chloride salts, which allows good absorption from the intestinal tract. Therefore, even Mn oxide, Cu sulfide, or Zn oxide, which are highly insoluble chemical compounds, are converted to Mn chloride, Cu chloride, or Zn chloride which are forms more easily absorbed.

III. mSTORY The purpose of this section is to provide an overview of the historical development of knowledge concerning the essential nature of mineral elements as related to deficiency and excess. Table 1.2 summarized chronologically the history of important events relating to the nutrition of mineral elements. Most research accomplishments will not be cited in the Literature Review, and the reader should consult additional reports (i.e., Maynard, 1937; McCollum, 1956; Underwood, 1966, 1981; McCay, 1973; Loosli, 1978, 1991; Georgievskii et al., 1981; McDowell, 1985; Underwood and Mertz, 1987) for a more comprehensive treatment of the subject. Likewise, historical treatment of the various minerals is covered in chapters 2 through 16 of this book. Mineral nutrition of domestic animals was considered to be of limited importance as late as the early 1900s (Ammerman and Goodrich, 1983). Armsby (1880) had concluded in his book, Manual of Cattle Feeding, that "In practice, in the feeding of mature animals intended to be kept in a medium condition, or to be fattened, a lack of the necessary mineral matters is scarcely ever to be feared. They are, indeed, generally in excess. Only common salt is in certain respects, an exception ..." The history of deficiency diseases must date from antiquity. However, before the middle of the 19th century, only the most nebulous ideas existed as to the nature, origin, and functions of the mineral constituents of plant and animal tissues (Underwood, 1981). In 1874, Forster observed that the minerals in the ash of tissues are required to support animal life (McCollum, 1956). This observation helped establish the dietary essentiality of mineral elements. It should be recognized that only when methods were devised to identify and measure mineral elements in body tissues and feeds and to characterize responses to pure elements, was it possible to replace supposition with facts about the essential nature of any nutrient. Much of the earlier knowledge about nutrition resulted from systematic observations stimulated by a need to solve critical health problems with people and their domestic animals. Often, a new scientific discovery has proven to be a confirmation of common beliefs of native people and an explanation of why the beliefs are true (Loosli, 1974). Much information about mineral needs of animals gained by trial and error over centuries was never recorded, and there is no way of learning what was practiced (Loosli, 1978). There is a view that the "fall of Thebes was hastened by heavy livestock mortalities caused by unidentified agents when grazing apparently luxuriant pastures" (Underwood, 1966). There is also the suggestion that part of

History

5

TABLE 1.2 History of Nutritional Importance of Mineral Elements 29 BC 40-120 AD 23-79 AD 1295 1669 Before 1680

1747 1748 1770 1784 1791 1811-1825 1823 1842 1847 1847 1850-1854 1869 1873 1880 1893-1899 1905 1919 1920 1922 1922 1924 1926 1928-1933 1931 1931-1933

The "Fall of Thebes" was hastened by heavy livestock mortalities caused by unidentified agents while grazing luxuriant pastures. Salt fed to domestic animals during the time of Plutarch. Virgil and Pliny recommended salts for milk production. Clinical signs of Se toxicosis were apparently described by Marco Polo as affecting grazing livestock in China. Brand isolated phosphorus from urine. Sydenham treated anemia with iron filings. Menghini found iron in blood. Gahn reported phosphorus present in bones. Scheele reported that bones contain calcium phosphate. Scheele reported sulfur in proteins. Fordyce showed that canaries need "calcareous earth" supplemented to grain diets. Work by Courtois, Coindet, and Boussingault led to the discovery of iodine, the effectiveness of iodine in burnt sponges and specifically that iodine was the only cure for goiter. Proust reported chlorine in the hydrochloric acid in gastric juice. Chossat found pigeons required calcium for bone growth. Liebig reported potassium in animal tissues. Boussingault conducted the first experiment that cattle need common salt. Chatin published studies relating environmental iodine deficiency to incidence of endemic goiter in man and animals. Raulin discovered the essentiality of zinc for the microorganism Aspergillus niger. Von Bunge put forward the hypothesis of antagonism between sodium and potassium and between sodium and chlorine. Forster demonstrated that animals require minerals, and feeding dogs only meat resulted in deficiencies. Von Bunge and Abderhalden showed that young animals receiving milk require supplemental iron. Babcock studied salt requirements of cattle, noting particular importance for lactating cows. Kendall isolated and named thyroxin from thyroid gland; the hormone was found to contain 65% iodine. Bertrand in France and McHargue in the United States initiated the use of purified diets to study the need and function of various minerals Bertrand and Berzon showed zinc was necessary for rat growth and hair development. McCollum and co-workers found that in addition to calcium and phosphorus, rickets is caused by vitamin D deficiency. Theiler and co-workers illustrated phosphorus deficiency for grazing cattle and found that supplementation corrected bone chewing, prevented death loss from botulism. and increased growth and reproductive rates Leroy showed that magnesium increases the growth of mice. Warburg established that respiratory enzymes in animals contain an iron porphyrin group. Neal, Becker and Shealy established copper as an essential element for ruminants. Kemerer and McCollum showed manganese was essential for rats and mice, a deficiency causing tetany. Sjollema related a licking disease in cattle to copper deficiency. (Continued)

General Introduction

6

TABLE 1.2 (Continued)

1935 1935 1935 1936-1937 1937

1938 1938-1942 1940 1946 1948 1950-1954 1953 1954 1955 1957 1958-1959 1959 1970-1997

Franke and Potter identified selenium as the factor in forage responsible for alkali disease in farm animals. Duncan and Huffman observed tetany in calves due to low magnesium content of milk. Underwood and Filmer and, independently, Marston and Lines found that enzootic marasmus in sheep was a cobalt deficiency. Wilgus, Norris and Houser reported that manganese deficiency resulted in a perosis in chicks. Becker and co-workers established that the "salt sick" condition of cattle in Florida (USA) was caused by a combination of pasture deficiencies of cobalt, copper and iron. Bennets and Chapman demonstrated that enzootic ataxia of newborn lambs resulted from ewes receiving insufficient copper during pregnancy. Ferguson, Lewis and Watson showed that molybdenum toxicity resulted in a severe diarrhea for grazing cattle. Hevesy and others began to use radioisotopes to study mineral metabolism. Keilin and Mann reported zinc as a component of the enzyme carbonic anhydrase. Moulton established that small concentrations of fluorine in drinking water prevented dental caries. Rickes and co-workers and, independently, Smith showed that Co is an integral part of vitamin B12 • Dick noted metabolic interrelationships among copper, molybdenum and inorganic sulfates in ruminants. Richert and Westerfield isolated molybdenum from the metalloenzyme xanthine oxidase. Needy and Harbaugh found that high fluorine concentrations in drinking water resulted in mottling of tooth enamel. Tucker and Salmon discovered that parakeratosis, a severe skin disease, was a zinc deficiency for swine. Schwartz and Foltz identified selenium as a factor that prevents liver necrosis in rats. Scott prevented exudative diathesis in poultry with selenium, while Muth, Oldfield, Remmert, McLean, Thompson, Claxton and others prevented white-muscle disease in ruminants with this element. Schwarz and Mertz showed that chromium was essential for glucose metabolism. The most recently discovered elements ("new trace elements") were established using highly purified diets and metal-free isolator systems. These elements included aluminum, arsenic, boron, bromium, fluorine, germanium, lead, lithium, nickel, rubidium, silicon, tin, and vanadium.

'Compiled from a number of sources, including Maynard, 1937; McCollum, 1956; Underwood, 1966, 1981; McCay, 1973; Loosli, 1978; Georgievskii et al., 1981; McDowell, 1985; Underwood and Mertz, 1987.

the reason for the "fall of Rome" was related to infertility caused by Pb toxicosis of the upper class due to use of metal versus clay cooking utensils. Common salt was an item of trade before recorded history to satisfy the salt cravings of grazing animals and for use to flavor foods. Wars were even fought and children were sold into slavery to obtain the precious commodity, salt. Feeding "salts" to domestic animals can be traced to the time of Plutarch (40 to 120 A.D.). Virgil and Pliny (23 to 79 A.D.) recommended salts for milk production. There are many references to the feeding of salt in Britain after 1750 following land enclosure. Phosphorus was isolated from urine in 1669 by Brand, and both Ca and P were shown to be constituents of bone by Gahn in 1748. In 1842, Chossat demonstrated

History

7

that Ca was a necessary supplement to a grain diet for bone development in birds. Many experiments were carried out in Europe and North America during the following 50 years on Ca and P metabolism and requirements as reviewed by Forbes and Keith (1914). After the importance of the Ca: P ratio became recognized, as well as the actual levels of these elements in the diet and the discovery of vitamin D in 1922, it became possible to prevent or cure rickets. This had been a serious disease of children and young animals produced in the winter when they were kept indoors to protect them from the cold, thus also preventing exposure to sunlight (Loosli, 1978; McDowell, 1985). In the early 1800s, mineral constituents of plants were shown to vary with soil type and stage of maturity of forages, and these changes were considered to be important for animals, a view Boussingault later demonstrated to be true. Bone chewing by cattle was recorded in Africa in 1780 and in Paraguay in 1838, but the relation of osteophagia to P deficiency in cattle was not clarified until Theiler et al. (1924) of South Africa published their classical experiments showing that the dried grasses were critically low in P and that P supplements corrected bone chewing, overcame death losses from botulism, and markedly increased growth rates and reproductive levels. Iron was shown to be present in blood by Menghini in 1747. As long ago as 1680, Sydenham is credited with having treated anemia with Fe solutions prepared by steeping steel filings in wine, but he did not know why it was effective. In 1867, Boussingault published data showing the Fe content of a number of different animals and various foods and beverages, since Fe was generally accepted by that date to be an essential element. An excellent example of the usefulness of common practices to alleviate nutritional deficiencies relates to I in South America. In 1824, Alexander Humboldt described goiter in Colombia and stated that native Indians knew of a salt deposit that was an effective remedy, which was not true for other salt sources. A sample taken to France was shown by Boussingault to contain I, which was absent from the other salts. In 1831, Boussingault advised the Colombian government to provide general distribution of the naturally iodized salt to the population. Iodine had been discovered by Courtois in 1811, and nine years later, he began prescribing it as a cure for goiter. Over dosages of I caused injury to some patients, and physicians condemned its use. It was a 100 years later before the general use of iodized salt was started in the United States with medical authority support (McCollum, 1956). In 1926, Leroy showed that Mg increased the growth of mice. McCollum and associates reported in 1931 that deficiency caused tetany in rats, and Duncan and Huffman observed similar signs in 1935 in calves fed milk as the only food. As a result of this discovery, the etiology of grass tetany, which had been described earlier, could now be clarified. Potassium was discovered in 1847, but due to its general abundance in most natural feeds, it is only since the 1960s that K deficiencies have been reported for livestock under practical conditions. Except for Fe and I, the trace elements were not studied until about 1928. Copper was shown to be essential for hemoglobin formation and Fe utilization in rats by

8

General Introduction

Hart and Elvehjem in 1928. Following this discovery, there was more than a decade of intensive research interest, which resulted in the discovery of other essential elements. Sjollema reported Cu deficiency in cattle and sheep in the Netherlands in 1933. Bennets and Chapman reported that enzootic ataxia of newborn lambs was Cu deficiency. Molybdenum was found to cause a severe diarrhea in cattle in 1938 by Ferguson and co-workers, with Dick in the early 1950s establishing the metabolic interrelationships among Cu, Mo, and inorganic sulfate for ruminants. Cobalt deficiency was first identified as the cause of a "wasting disease" and anemia in cattle and sheep in Australia in 1935 by Filmer and Underwood, and Marston and Lines. From Florida (USA), the first report of a Co deficiency in cattle was in 1937, associated with a condition known as "salt sick" (Becker et al., 1965). The "salt sick" condition in Florida was prevented only if all three of the deficient elements, Co, Cu, and Fe, were supplied in adequate quantities. Previously, in Florida Cu was established as essential to ruminants (Neal et al., 1931). Manganese stimulated growth of mice and reproduction of rats in studies by Kemerer and McCollum in 1931. In 1937, Wilgus and co-workers showed that chick perosis was caused by Mn deficiency, and in the same year, Lyons and Insko found that chondrodystrophy in chicks was caused by lack of Mn in the diets of laying hens. However, the importance of Mn for cattle was shown much later, in the early 1950s. A similar situation existed with Zn, which was shown to be essential for growth and hair development of mice and rats in 1922, but its need by pigs was not shown until 1955 and for calves not until 1957. Zinc deficiency for grazing cattle was reported in 1960 by Legg and Sears. In 1954, Neely and Harbaugh found that mottling of tooth enamel, a disease affecting humans and livestock, was produced by high F concentrations in drinking water. Evidence that F is essential for livestock is limited to recent reports showing that a deficiency causes skeletal abnormalities in female goats and poor growth in their offspring after 10 generations of low F diets (Anke et al., 1997b). Earlier tests demonstrated that traces in the drinking water (0.7 to 1.0 ppm) help to prevent dental cavities in children, but 2.0 ppm or more causes mottling of the teeth enamel. Higher intakes (4 to 6 ppm in water) increase bone density and help to prevent osteoporosis and associated incidence of collapsed or distorted vertebrae in people over 55 years of age. There is also evidence that extra F decreases the aorta calcification often seen in low-F areas. However, it is the toxicity of F that is of greater importance in animal nutrition (Shupe et al., 1974). Marco Polo apparently described clinical signs due to Se toxicosis while traveling in China late in the 13th century. It was not until 1935 that Franke and Potter identified the toxic principle causing "alkali disease" and "blind staggers" in grazing animals was due to excess Se. In 1957, Se was shown to be essential by preventing liver necrosis in rats. The following year Se was shown to prevent exudative diathesis in poultry and white muscle disease in young ruminants. Shortly after this, Se in combination with vitamin E, was shown to prevent certain specific muscular dystrophy conditions and other forms of tissue degeneration in a large number of species. Up to the early 1950s 13 minerals had been identified as essential; these being the major elements Ca, P, K, Na, CI, Sand Mg, and the trace (micro) elements;

Mineral Distribution in Body

9

Fe, I, Cu, Mn, Zn and Co. By 1959, Mo, Se, and Cr had been added to this list, as well as beneficial aspects for supplemental F for humans. Starting in the 1970s, 13 additional elements were shown to be required for animals. Most of these elements were known only for their toxic properties. The "new trace elements" were discovered using highly purified diets and metal-free isolator systems. To date, with few exceptions, these newer elements have not been shown to be essential for livestock or humans that are consuming typical diets. One of the latest elements to be established as essential is rubidium (Rb). Female goats fed < 280 ppb (~g/kg) Rb had abortions, lower birth weight and increased mortality among kids (Anke et al., 1997a).

IV. MINERAL DISTRIBUTION IN BODY It should be noted that 96% of body weight consists of the four organically bound elements (carbon, hydrogen, oxygen, and nitrogen). The principal cations and anions together account for 3.5% of body weight, the remainder comprising additional elements (Table 1.3). The percentages of the macromineral constituents of the body are indicated by the following data showing the average analyses of 18 steers of varying ages exclusive of the contents of the digestive tract (Hogan and Nierman, 1927). Element Calcium Phosphorus Potassium Sodium Sulfur Chlorine Magnesium

Percent

1.33 0.74 0.19 0.16 0.15 O.ll 0.04

Typically Ca represents about 46% and P about 29% of total body minerals. Potassium, S, Na, Cl and Mg together account for about 25%, while essential trace elements constitute less than 0.3% of the total. Mineral distribution within the body's tissues is not uniform, since some tissues selectively concentrate specific elements. However, the proportions of each mineral, expressed as amount of fatfree dry body substance, are very similar among species in adult mammals and poultry (Scott et al., 1982). Each organ, in accordance with its function, has a characteristic mineral composition, which again is very similar in all mammals. However, after a period of undernutrition or water deprivation, there is quite a sharp rise in the mineral content [fat-free dry matter (OM)]. It should be noted that the Na, K, and Cl concentrations of the body are constant during all stages of development from embryo to full

General Introduction

10

TABLE 1.3

Elemental Composition of Human Body" Element Oxygen Carbon Hydrogen Nitrogen

Percent 65.00 18.0

10.0 3.0

Macrominerals Calcium Phosphorus Potassium Sulfur Sodium Chlorine Magnesium

1.5 1.0 0.35 0.25 0.15 0.15 0.05

Trace Minerals Iron Zinc Manganese Copper Iodine

0.004 0.003 0.0003 0.0002 0.00004

"The major elements oxygen, carbon, hydrogen and nitrogen comprise 96% of the body; macrominerals are 3.45% and trace and other minerals, 0.55%.

development, whereas the Mg, Ca and P contents in the embryo are only one half of the respective concentration in the adult animal. Bone is the primary storage site for many of the essential elements. Between 80% and 85% of the total body mineral matter, or ash, of the body is located in the skeletal tissues and consists mainly of salts of Ca, P and Mg. Thus, 99% of the total Ca, 80 to 85% of P and some 70% of Mg occur in bone (Underwood, 1981). The thyroid gland is the most specific storage site for I, and no less than 80% of the total body I is normally found there. In contrast to Ca, P, and Mg in bone, I in thyroid tissue, and Co as part of vitamin B12, most minerals are distributed more evenly throughout the body where they exist in a variety of functional combinations and in characteristic concentrations. These elements must be maintained within quite narrow limits if the functional and structural integrity of the tissues is to be safeguarded, and health and production optimized.

V. GENERAL FUNCfIONS OF MINERALS

Unlike other nutrients; mineral elements cannot be synthesized by living organisms. Minerals have four broad functions: structural, physiological, catalytic, and hormonal or regulatory. The most obvious function of mineral elements in the body is to provide structural support (skeleton). Bone is formed through the

General Functions of Minerals

11

deposition of Ca and P as hydroxyapatite into a protein matrix. Calcium, P, Mg, F, and Si in bones and teeth all contribute to the mechanical stability. Another example of structural function is the use of Ca by birds to produce eggshells. The presence of P and S in muscle proteins further illustrates the function of structural components of body tissue for these minerals. Minerals such as Zn and P can also contribute structural stability to the molecules and membranes of which they are part. Only small fractions of the Ca, Mg, and P, and most of the Na, K, and CI are present as electrolytes in the body fluids and soft tissues. Electrolytes present in body fluids, such as blood or cerebrospinal fluid, serve important functions in maintaining acid-base and water balance, and osmotic pressure; they regulate membrane permeability and exert characteristic effects on the exitability of muscles and nerves. For example, a certain balance between Ca, Na, and K in the fluid which bathes the heart muscle is essential for the normal relaxation and contraction that constitute its beating. Also, profound disturbances in neuromuscular function arise in the animal when the levels of Ca and Mg in the blood plasma fall below certain limits. Calcium is also directly involved in the coagulation of blood. Mineral salts are sometimes fed to dairy and feedlot cattle beyond the established requirement because of their role as buffers. Buffers have been reported to improve feed intake, milk production, milk composition, and animal health (Rogers et al., 1982). Mineral salts used as buffers function to control the excess hydrogen ion concentration in the rumen, intestines, tissues and body fluids, or increase the rate of passage of liquids from the rumen, or both (NRC, 2001). In addition to its bone function, P participates in a multiplicity of metabolic reactions involving energy transfer. Phosphorus also is an integral part of the nucleic acids. In addition to P, several trace metals, such as Fe, Cr, Ni, Mn, and Zn are components of ribonucleic acid (RNA), the compound vital to all protein synthesis and, therefore, to life itself (Underwood, 1981). Essential trace elements are integral components of certain enzymes and of other biologically important compounds, such as Se in glutathione peroxidase, Fe in hemoglobin, Co in vitamin B12 and I in the thyroid hormones thyroxine and triiodothyronine. Also, the insulin molecule contains both Zn and S. Certain minerals have regulatory functions in that they exert some control on cell replication and differentiation: Ca, for example, influences signal transduction and Zn influences transcription, adding to long-established regulatory roles, such as that of the element I as a constituent of the thyroid hormones (Underwood and Suttle, 1999). Functions of minerals are interrelated and balanced against each other and most often cannot be considered as single elements with independent and self-sufficient roles in the organized bodily processes. The definite relationship of Ca and P in the formation of bones and teeth and the interrelationships of Fe, Cu, and Co (in vitamin B12) in hemoglobin synthesis and red blood cell formation serve as examples. Sodium, K, Ca, P, and CI serve individually and collectively in the body fluids. A number of trace elements (e.g., Cu, Zn, Fe, and Se), in addition to certain

12

General Introduction

vitamins (e.g., vitamins A, D, E, B6, and folacin) and other nutrients, are strongly related to adequate immune response. These nutrients act together and/or separately for different components of an active immune response.

VI. MINERAL REQUIREMENTS AND TOLERANCES It is not the purpose of this chapter to provide detailed information on mineral requirements for each area of livestock production. The two main sources of information on mineral requirements for various species are the U.S. National Research Council's (NRC) "Nutrient Requirements" series and the British (ARC) "Nutrient Requirements of Farm Livestock" series. For human mineral requirements, the latest edition of "Recommended Dietary Allowances" (RDA) and "Dietary Reference Intakes" (2001) should be consulted. The NRC publication "Mineral Tolerance of Domestic Animals" provides suggested toxic levels of minerals for livestock (NRC, 1980). Mineral requirements and tolerances for livestock and humans for specific minerals are presented in the present book in chapters 2 through 16 and in the appendix tables. Likewise, Chapter 18 deals with mineral tolerances for livestock. Mineral requirements are generally expressed in several ways, in amounts per day or per unit of product, such as milk or eggs, or in proportions of the dry matter of the diet consumed. The former method is more precise, but expressing minerals as proportions of diet dry matter has obvious practical advantages. The requirements are meant to define the lower limits of adequacy in each case and are arrived at by relating the growth, health, production, or other relevant criteria in the animal with varying dietary mineral concentrations. Dietary recommendations can be stated as ranges, rather than as single figures of intake. The statement of a range can take into account the differences of intake that are required to meet the requirement when supplied by typical diets of different bioavailability. That presentation takes into account the homeostatic regulation of higher organisms that tends to buffer marginally deficient or marginally excessive intake by changing the efficiency of absorption and excretion (Underwood and Mertz, 1987). The actual amount of mineral in the diet may also influence utilization. For example, if the diet contains more Ca than required, homeostatic mechanisms are brought into play with the efficiency of absorption being decreased. The mineral status of the animal may also influence absorption. A Fe-deficient animal is more efficient in the absorption of Fe than an animal with adequate Fe stores. Minimum mineral intakes must be sufficient to ensure the long-term maintenance of the mineral reserves of the body tissues and the amounts of those minerals in the edible products of the animal. Through homeostatic mechanisms, the animal body has the capacity to make some adjustment to suboptimal intakes by reducing the amount of the mineral in its products. As an example, most trace minerals are substantially reduced in milk when dietary intakes are low. However, for

Mineral Requirements and Tolerances

13

macrominerals such as Ca, P, Na, and K the concentration in milk remains constant so that these minerals are spared only by reducing milk production. To conserve Ca for egg-laying poultry, the shell strength can be reduced in order to maintain production. Therefore, the assessment of mineral needs has come to include determination of the minerals in the tissues, fluids and products, as well as such gross criteria as weight gains, milk yields and so on. Many factors affect mineral requirements, including kind and level of production, age, level and chemical form of elements, interrelationships with other nutrients, mineral intake, breed and animal adaptation. Mineral requirements are highly dependent on the level of productivity. Highproducing dairy cows require much more dietary Ca and P than low-yielding cows because of the richness of milk in those elements. However, the necessary percentages in the diet do not rise to the expected extent because total dry matter intakes increase with rising productivity of the cow almost as rapidly as do mineral requirements. The P requirements of laying hens tend to follow a similar pattern with increasing egg production but those of Ca do not. For example, a non-laying hen can normally meet its Ca needs from a diet containing 0.2 to 0.3% Ca on a DM basis, whereas some 8 to 10 times this concentration is necessary for a hen approaching maximum egg production (Underwood, 1981). Improved practices that lead to improved milk, egg and wool production and growth rates for poultry and livestock will necessitate more attention to mineral nutrition. Mineral deficiencies, often marginal under low levels of production, are likely to become important, and previously unsuspected nutritional deficiency signs may occur as production level increases (Long et al., 1969; Thornton et aI., 1969; Underwood, 1981). Mineral requirements vary also with the criteria of adequacy employed. As the amount of a mineral available to the animal becomes deficient as a result of inadequate intake or depletion of body reserves, certain processes fail in the competition for the inadequate supply. The priority of demand exerted by these processes for the mineral vary among different animal species and within species, with the age of the animal and the rapidity with which the deficiency develops (Underwood, 1981). For instance, in sheep, the processes of pigmentation and keratinization of wool appear to be the first to be affected by a low Cu status. Thus, if wool quality is taken as the criterion of adequacy, the Cu requirement of the sheep is higher than if growth rate is used as criteria. The criterion of adequacy is also important, as illustrated by the fact that minimum Zn requirements for spermatogenesis and testicular development in male sheep are higher than for growth (Underwood and Somers, 1969), and Mn requirement is similarly lower for growth than for fertility (Underwood, 1981). Important differences in mineral metabolism can be attributed to breed and to individual animal variation. This can be illustrated by only a certain percentage of growing pigs suffering from tissue degeneration after being fed the same Se-vitamin E deficient diets (McDowell et al., 1974). Likewise, the l-deficient clinical sign of goiter in calves and skin lesions of Zn-deficient cattle are sometimes seen in a small percentage of animals, while most animals in the herd appear normal

14

General Introduction

Fig. 1.I Parakeratosis from Zn deficiency showing genetic variation. Above: Pigs showing varying degrees of parakeratosis from Zn deficiency. Below: Same pigs after 16 days of receiving a diet containing 40 ppm of Zn. (Courtesy of V.W. Hays and V.C. Speer, Iowa State University, Ames)

(McDowell, 1985). Fig. 1.1 illustrates three pigs that had received a Zn-deficient diet, with skin lesions varying in pigs from severe to completely unaffected. Adequate intake of feed by animals is essential in meeting mineral requirements. For example, factors which greatly reduce forage intake, such as low protein « 7.0%) content and increased degree of lignification, likewise reduce the total minerals consumed by grazing animals. Information concerning the toxicity or tolerance of minerals is incomplete (NRC, 1980). According to available information, the toxic level of most major minerals is about 4 to 10 times the recommended level for young, growing livestock. The toxic levels of trace minerals appear to be highly variable, ranging between 4 and 1500

Methods of Mineral Analyses

15

times the recommended level (See Chapter 18). As was true with mineral requirements, a series of "safe" dietary levels of potentially toxic elements has been established, depending on the extent to which other elements that affect their absorption and retention are present (NRC, 1980).

VII. METHODS OF MINERAL ANALYSES Research findings with minerals have been greatly facilitated by the development of several sophisticated, powerful, analytical tools. Atomic absorption spectrophotometry has been especially helpful, since its cost is within the budget of most laboratories (See Fig. 1.2). Other methods, such as emission spectrometry and neutron activation analysis, have many advantages, although equipment needed is expensive. These methods have resulted in a marked reduction in time and labor required for analyses, thus encouraging scientists to carry out more complete programs of experimentation. More importantly, these analytical techniques have increased both sensitivity and accuracy of mineral analysis.

A. Sampling for Mineral Analyses Due to very low levels of most trace elements in biological samples, there is a high potential for gross contamination during sampling, storage, handling, and analysis. Precautions should be taken to use non-metal sampling devices, tools, and containers. Plastics are recommended, with Teflon and polyethylene being the most popular. The use of certified standards will quickly identify most errors but not necessarily those from laboratory milling, because the standard comes ready-milled (Underwood and Suttle, 1999). Significant levels of trace elements can be found in reagents, air, hair, skin, clothing, etc. The analyst must evaluate and eliminate potential sources of contamination in every step of the analytical methodology.

B. Analytical Techniques In an extensive world survey, Iyengar and Woittiez (1988) indicated that both atomic absorption spectrophotometry (AAS) and neutron activation analysis (NAA) are the most frequently used analytical techniques. Other techniques such as inductively coupled plasma optical emission spectroscopy (ICP-oES), x-ray fluorescence (XRF), isotopic dilution mass spectrometry (lDMS), proton induced x-ray emission (PIXE), and near infrared reflectance spectroscopy (NIRS) have been used less extensively. 1. ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)

This is a very specific technique with few interferences, has good sensitivity and precision and is relatively low cost. The main drawbacks are limited linear calibration range and the fact that it is a single-element technique.

16

General Introduction

Fig. 1.2 Sample preparation and analysis. The upper photograph illustrates a wet digestion technique. The most widely used method of mineral analysis utilizes flame atomic absorption spectrophotometry (lower photo) (University of Florida).

The element to be analyzed is introduced into a flame where it becomes dissociated from its chemical bonds into an unexcited, un-ionized ground state as individual atoms. The element in this state is capable of absorbing radiation at discrete lines of narrow wavelength. When a light beam at one of these wavelengths is directed through the flame, the amount of this light absorbed as it passes through the flame is proportional to the concentration of the element being analyzed.

Methods of Mineral Analyses

17

Two mam types of atomization sources are used: flame and graphite furnace (GF-AAS). The graphite furnace improves the sensitivity of atomic absorption. With this device many elements can be determined at concentrations 1000 times lower than what can be detected by flame atomic absorption. This technique can be used in situations where only a small amount of sample is available. 2. NEUTRON ACTIVATION ANALYSIS (NAA)

The NAA procedure is a multi-element technique, but is almost exclusively limited to centers of analytical expertise due to the radiochemical techniques involved. This technique bombards the sample with neutrons so that the elements present become radioactive and can then be quantitatively detected. 3. INDUCTIVELY COUPLED PLASMA OPTICAL EMISSION SPECTROSCOPY (ICP-DES)

This is a technique using inductively coupled plasma generators as an atomization source for optical emission spectrometry. It has a high analytical sensitivity for about 70 elements with detection limits frequently in the ppb (ngjml) range, and calibration graphs rectilinear over 5 orders of magnitude with respect to analyte concentration (Dean et al., 1989). Some advantages of ICP-OES technology are the high temperature (5500 to 8000 K) that allows for the complete ionization of elements; therefore, minimizing chemical interferences, a wider linear working range, and detection limits lower than flame AAS, but poorer than GF-AAS. One disadvantage of ICP-OES is that it is less precise with a coefficient of variation of 2 to 3% versus 0.3% for flame AAS. 0

4. INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS)

The coupling of an ICP with a mass spectrometric detector was achieved by Houk et al. (1980). It is a particularly promising method because it allows not only for total metal concentrations to be determined, but also for isotopic information to be elucidated. This ability allows the technique of isotope dilution analysis (IDA) to be applied and tracer studies to be performed using stable nonradioactive isotopes (Dean et al., 1989). ICP-MS has a similar sensitivity to AAS and GF-AAS and requires less sample preparation time and in the case of GF-AAS, less analysis time. 5. ISOTOPE DILUTION MASS SPECTROMETRY (ID-MS)

This procedure is one of the most widely used definitive methods, and is the only mass spectrometric method which produces highly accurate analytical results for a great number of elements (Heumann, 1985). In 10 analysis, the sample is treated with an accurately known amount of a stable enriched isotope, for example, the 206Pb isotope. From a knowledge of the isotope ratios in the fortified and unfortified samples and the degree of fortification, the amount of analyte in the sample can be calculated (Dean et al., 1989).

General Introduction

18

6. X-RAY FLUORESCENCE SPECTROMETRY (XRF) A more recent development in mineral analyses has been x-ray fluorescence. Used commonly in the mining industry, this technique seems to have great potential for rapid assay of minerals in feed, following minimal sample preparation (Valdes and Leeson, 1990). This procedure provides the means for the identification of an element by measurement of its characteristic x-ray emission wavelength or energy. The method allows the quantification of a given element by first measuring the emitted characteristic line intensity and then relating this intensity to elemental concentration. Today nearly all x-ray spectrometers use the fluorescence excitation method and employ a sealed x-ray tube as the primary excitation source (Jenkins, 1988). 7. NEAR INFRARED REFLECTANCE SPECTROSCOPY (NIRS) The use of NIRS for determining simple components in grains and oilseeds has been used routinely over the last few years. This procedure quickly became an ideal laboratory technique because it is very fast, inexpensive, there is no sample preparation except grinding, and the technique does not require trained people after the calibrations are developed. Several nutrients in a sample can also be analyzed simultaneously (Leeson and Summers, 2001). Near infrared reflectance spectroscopy (NIRS) can provide quick, nondestructive and quantitative analyses of an enormous range of organic and inorganic constituents of homogeneous plant and animal tissues. Near infrared spectra depend on the number and type of C-H, N-H, and 0-H bonds in the material being analyzed. The spectral features are then combined with reliable compositional or functional analyses of the material in a predictive statistical model. This model is then used to predict the composition of new or unknown samples. The accuracy and precision of the reference values for the calibration data set in part determines the quality of the predictions made by NIRS. However, NIRS analyses are often more precise than standard laboratory assays. 8. CONVENTIONAL METHODS For analysis of feeds for Ca, P, Na, K, Cl, Mg, Zn, and Mn, conventional methods (i.e., NFIA, 1991) have been in routine use in feed laboratories for many years. These methods are fully described in the official methods of analysis of the Association of Official Analytical Chemists (AOAC, 1997). Whetter and Ullrey (1978) developed an improved method for determining Se by a fluorometric procedure that is sensitive and accurate. No single technique is applicable to all trace element analyses. There are several factors to consider when choosing methodology and instrumentation: 1. 2. 3. 4.

the requirement for single-element or multi-element analysis, type (matrix) of biological samples and potential interferences, number of samples and time required per sample (automated or manual), sample size required,

Methods for Estimating Mineral Bioavailability and Requirements

19

5. availability, size, cost, ease of operation, and service of instrumentation, 6. skill, training, and experience of available personnel, and 7. whether the instrumentation will be shared with other laboratories and/or used by different personnel (WHO, 1996; Miles et al., 2001). C. Sample Digestion Analytical techniques such as NIRS, NAA, and XRF can sometimes be used with minimal sample preparation; however, atomic spectrometric techniques require a pretreatment to remove organic matter. This is usually accomplished by either a dry ashing or wet oxidation-digestion. High-temperature dry ashing consists of placing the sample into a suitable dish, drying the sample, and placing it into a muffle furnace. Often ashing aids and/or posttreatment with small amounts of acid are required to destroy the organic material completely (Miles et al., 2001). Wet digestion is usually faster than dry ashing and can be set up to do a number of digestions simultaneously (Fig. 1.2). Acids and reagents used must be of the highest purity, since large amounts are sometimes required. For ultratrace analysis, the control of the blank is often the determinant factor in the level of analysis that can be performed. The analyst must ascertain the purity of each batch of acid or reagents to be used. This is especially true if the acids are shipped or stored for any length of time in glass bottles.

D. Calibration Standards Atomic spectroscopic analytical methods determine the concentration of samples by comparing their analytical signals to those of a series of calibration standards. The analytical determinations are only as accurate as the standards. Matrix matching is the most accurate but is time-consuming and requires detailed knowledge of the sample matrix. Dilution of the samples and standards into a common matrix can eliminate the most obvious interferences and is suitable for large numbers of samples. Every analytical method must be validated for accuracy. This is best done by verifying results using a standard reference material (SRM) of the same composition as the samples to be analyzed. The SRM are available from the National Institute of Standard and Technology (U.S.), the International Atomic Energy Agency (Vienna, Austria), Community Bureau of Reference (Brussels), National Institute for Environmental Studies (Tsukuba), World Health Organization (Solna). They come with a certificate, which gives reference values of components plus confidence levels. VIII. METHODS FOR ESTIMATING MINERAL BIOAVAILABILITY

AND REQUIREMENTS Not all sources of elements are created equally. Different sources of the same element can vary from zero availability to a highly available element. Ammerman

General Introduction

20

(1995) has reviewed the most suitable methods for determining mineral bioavailability.

A. Absorption and Chemical Balance bsornti intake - total fecal excretion 100 A pparent a sorption = . k x mta e Digestion and absorption of mineral elements by an animal provides an estimate of its bioavailability. It is assumed that once a mineral is absorbed from the gastrointestinal tract, it is available for physiological processes or for storage. Absorption, however, cannot always be equated to bioavailability. For example, I in the form of 3,5-diiodosalicyclic acid was shown to be well utilized by rats but was an ineffective source of I for cattle (Miller et al., 1965). The I-containing compound was readily absorbed by both species, but cattle had a very limited capacity to remove the mineral from the organic part of the molecule. Absorption studies of several days in length in which intake and fecal excretion is measured have often been carried out with the macrominerals Ca, P, and Mg. Very few absorption studies have been carried out with microelements due to the large errors resulting from even the slightest contamination and unknown endogenous sources. 1.

ApPARENT ABSORPTION

Apparent absorption is used in the evaluation of sources of certain mineral elements and is defined as total intake minus total fecal excretion of the element. Values are usually expressed as a percentage of intake. The difference between intake and excretion represents net disappearance of the element from the gastrointestinal tract and does not correct for the portion of the element present in feces that resulted either from abrasion of mucosal cells or from excretion of the element back into the gastrointestinal trace (Ammerman, 1995). Apparent absorption is of limited value for elements where feces is the major pathway of excretion (e.g., Ca, P, Zn, Mn, and Cu). 2.

TRUE ABSORPTION

. T rue a b sorption

= (intake -

tot.fecal exc. - tot.endogenous fecal exc.) 100 . k x mta e

True absorption corrects for the portion of the element which has been absorbed into the animal's body and subsequently is excreted back into the gastrointestinal tract. This portion of the total fecal excretion can be designated as "total endogenous fecal excretion." Minimum endogenous fecal loss represents the minimal or inevitable loss from the animal's body (ARC, 1980). True absorption represents total intake minus total fecal excretion (tot. fecal exc.) from which total endogenous fecal excretion has been subtracted.

Methods for Estimating Mineral Bioavailability and Requirements

21

The value for true absorption is greater than that for apparent absorption and is a more valid estimate of the amount of an element available for physiological purposes. Total endogenous fecal excretion can be estimated by use of appropriate radioisotopes (Underwood, 1981). 3.

URINARY EXCRETION

Urine is the major pathway for Mg, I, and K excretion, but minor for Mn, Fe, Zn, and Cu. Urinary excretion can be a useful indicator of absorption for Mg and K and other elements with similar excretion characteristics. 4.

NET RETENTION

Net retention is defined as total intake minus total excretion (total fecal plus total urinary) of the mineral. Collection of urine during absorption studies allows net retention to be calculated. Although this information may be useful in interpreting results, net retention probably has limited value in determining bioavailability of a mineral. In many situations, the mineral excreted in the urine represents a portion that was potentially nutritionally effective and that has been involved in, or was available for use in metabolism (Ammerman, 1995).

B. Growth and Specific Tissue Response 1. GROWTH

Growth response resulting from lack of or adequacy of a specific element is often used to establish the need and requirement for a particular mineral. A disadvantage of growth rate assays lies in the fact that, for many elements, the method requires use of semipurified diets, which increases cost and which also may yield results not entirely applicable when practical diets containing natural ingredients are fed (Ammerman, 1995). The young chick is an ideal assay animal because of (a) limited nutrient stores; (b) lack of or minimal coprophagy; (c) rapid rate of growth and; (d) high nutrient demand. 2.

BONE DEVELOPMENT

Bone development, as usually measured by bone ash response in the very young chicken, has been considered for years as one of the most critical tests for estimating bioavailability of Ca and P compounds, as well as vitamin D. In general, the bone of choice has been the tibia, and bone ash has been expressed as either total tibial ash or as tibial ash concentration of the dry, fat-free bone. Bone ash and bone breaking strength (force required to fracture the bone) have also been used widely in swine for both Ca and P. Bones used most commonly are the metacarpals and metatarsals. Stone and Mcintosh (1977) reported that femur breaking strength in pigs was a much more sensitive indicator of skeletal development and susceptibility to bone fracture than was bone ash or dimensional characteristics. Williams et al.

General Introduction

22

(1991) demonstrated that chemical, physical, and mechanical properties of bone could be used to evaluate the P status of cattle. Noninvasive bone techniques such as dual photon absorptiometry, radiographic photometry, and ultrasound can be used to estimate bone mineral content and bone strength and thus status of P and Ca (Williams et al., 1991). 3. ESSENTIAL COMPOUNDS OR ENZYMES

Functional assays for bioavailability in which the mineral element is necessary for an essential compound have been used. Cobalt is needed for vitamin B12 production as Fe is required for hemoglobin synthesis. The enzymes glutathione peroxidase and cytochrome C oxidase are influenced by Se and Cu, respectively. Iodine is an essential component of the thyroid hormones (i.e., thyroxin and triiodothyronine). 4.

TISSUE ACCUMULAnON

Accumulation of specific mineral elements in various target organs (e.g., liver, bone) has been used for many years as a response criterion. It was reported by Watson et al. (1970) that bone Mn concentrations in chicks fed with semipurified diets were more directly related to dietary concentrations of the element than were growth rate or leg development. For evaluation of P status in ruminants, a rib biopsy procedure is available (McDowell, 1997). Trace element concentrations of Cu, Co, and Se in liver tissue (e.g., biopsy) has been used to evaluate the status of these elements for ruminants in many tropical countries (McDowell, 1997, 1999). Biological availability of several microelements for ruminants and poultry have been estimated by tissue uptake following high dietary levels, and short-term supplementation (Henry et aI., 1986). The advantages of this method are fewer animals are required to test for significant differences, due to higher dietary levels and there is no need for purified diets. A disadvantage with this method is that homeostasis mechanisms are not considered. As an example, an animal often will utilize a nutrient much more efficiently at a deficiency or low status level than when the nutrient is in excess. 5.

USE OF ISOTOPES

Accumulation of radioactive or stable isotopes in target organs can be used to estimate absorption. Reviews of methods for assessment of mineral utilization in humans and laboratory animals, including the use of stable isotopes and intrinsic and extrinsic labeling with radioisotopes, are available (O'Dell, 1984).

IX. DETECTION OF MINERAL STATUS

The detection of mineral element deficiencies or excesses involves clinical, pathological, and analytical criteria as well as response from specific element

Incidence of Mineral Deficiencies and Toxicities

23

supplementation. Clinical signs of mineral deficiencies along with soil, water, plant, and animal tissue analyses have all been used with varying degrees of success to establish mineral deficiencies and toxicities (McDowell, 1985, 1997; Mills, 1987; Suttle, 1988; Underwood and Suttle, 1999). Since mineral analyses are complicated and expensive, it is important to select and analyze the minimum number of plant and animal tissues (or fluids) that are most indicative of mineral status in animals. Methods of diagnosis of mineral deficiencies or toxicities and appropriate critical levels have been reviewed (NCMN, 1973; Miller and Stake, 1974; Egan, 1975; Underwood, 1979, 1981; McDowell, 1985, 1997, 1999). The publication "Minerals for Grazing Ruminants in Tropical Regions" lists analyses of considerable value and critical levels for assessment of mineral status of ruminants (McDowell, 1997). The most reliable method to confirm mineral deficiencies is response derived from specific mineral supplementation. However, supplementation studies are costly in time and resources if conducted with adequate control and assessment. For several decades, a major goal in mineral research has been to discover and/or develop simple and accurate biochemical measurements of the status of animals for the minerals in which there are important practical problems (Miller and Stake, 1974). Like soils and plants, animal tissue mineral concentrations are influenced by many factors. Nevertheless, when appropriate interpretation is made, animal tissue concentrations are often better indicators of the mineral status of livestock than either plant or soil concentrations (McDowell, 1976, 1985, 1997, 1999). When the evidence obtained from clinical, pathological, and biochemical examinations of the animal and from chemical analysis of the diet and its components is combined and assessed, it is usually possible to detect and define any nutritional abnormality of mineral origin, even when it is mild (Underwood and Suttle, 1999).

X. INCIDENCE OF MINERAL DEFICIENCIES AND TOXICmES Mineral deficiencies and imbalances for livestock are reported from almost all world regions. Wasting diseases (Fig. 1.3), loss of hair, depigmented hair, skin disorders, non-infectious abortion, diarrhea, anemia, loss of appetite, bone abnormalities, tetany, low fertility, and pica (Fig. 1.4) are clinical signs often suggestive of mineral deficiencies throughout the world. There is good evidence that a wasting disease in Colombia (Fig. 1.5), a periodontal disease in Brazil (Fig. 1.6) and paralysis condition in Venezuela (Fig. 1.4) are the result of mineral deficiencies or imbalances. The extent to which a lack of sufficient energy and protein is responsible for these clinical signs and disease conditions is still largely unanswered. However, numerous investigators have observed that livestock sometimes deteriorate in spite of an apparent adequate feed supply (Sutmoller et al., 1966). Ruminants grazing forages in a severe Co- or Cu-deficient area are even more limited by lack of these elements than either that of energy or protein.

24

General Introduction

Fig. 1.3 Animal in poor condition even though pasture is of good quality (top). Same animal 2 \I, months later (bottom) after access to a high-quality free-choice mineral supplement. (Courtesy of Juan Carlos Montero. Programa NUTRILUZ, University of Zulia, Maracaibo, Venezuela)

Incidence of Mineral Deficiencies and Toxicities

25

Fig. 1.4 Signs of mineral deficiencies. Pica is illustrated as bone chewing (A and B) and eating of bark (C). Often it is a characteristic of phosphorus deficiency. Photo D is a devastating disease condition in Venezuela referred to as "sind rome paraplejico" (bovine paraplegic syndrome). (Courtesy of: A ~ David Morillo, FONAIP, Estacion Experimental Zulia, Venezuela; B - Jiirgen Dobereiner and Carlos H. Tokarnia, EMBRAPjUFRRJ, Rio de Janeiro, Brazil; C - Juan Carlos Contero, Programa NUTRILUZ, University of Zulia, Maracaibo, Venezuela; D - L.R. McDowell, University of Florida, Gainesville)

26

Fig. 1.4

General Introduction

Continued.

Mineral nutrition disorders range from acute mineral deficiency or toxicity diseases, characterized by well-marked clinical signs and pathological changes to mild and transient conditions difficult to diagnose and expressed as a vague unthriftiness or unsatisfactory growth and reproduction. The latter assume great importance because they occur over large areas and affect a large number of animals. Mineral deficiency signs can be confusing, as the observed conditions can

Incidence of Mineral Deficiencies and Toxicities

27

Fig. I.S A wasting disease ("secadera") of cattle in the Llanos of Colombia. Animals are characterized by an emaciated condition in spite of good-quality available forage. (L.R. McDowell, University of Florida, Gainesville)

Ten-month-old Zebu (Gir) (left) in the region of Jaciara, Mato Grosso, Brazil, affected by Fig. 1.6 "cara inchada". Bi- or unilateral swelling of the maxillary bones in the advanced stage of periodontal disease is the reason for its popular name that means "swollen face". Right: Deep, mostly symmetrical lesions at the site of the Papilla interdentalis lingualis between the maxillary Pd 3 and Pd 4 characterize the progressing periodontal disease. (Courtesy of Jiirgen Dobereiner, EMBRAPjUFRRJ, Rio de Janeiro, Brazil)

28

General Introduction TABLE 1.4 Mineral Deficiencies or Toxicities of Ruminants in Tropical Counttes"

Required elements Calcium Argentina, Bolivia, Brazil, Colombia, Costa Rica, EI Salvador, Guatemala, Guyana, India, Malawi, Mexico, Panama, Peru, Philippines, Senegal, Surinam, Uganda, Venezuela, Zaire Argentina, Brazil, Chile, Colombia, Costa Rica, Guatemala, Guyana, Haiti, Magnesium Honduras, Jamaica, Kenya, Malawi, Peru, Surinam, Trinidad, Uganda, South Africa, Uruguay, Venezuela Antigua, Argentina, Bolivia, Botswana, Brazil, Ceylon, Chile, Colombia, Costa Rica, Phosphorus Cuba, Dominican Republic, Ecuador, El Salvador, Egypt, Ghana, Guatemala, Guyana, Haiti, Honduras, India, Indonesia, Jamaica, Kenya, Malagasy Republic, Malawi, Malaysia, Mexico, Nicaragua, Nigeria, Panama, Paraguay, Peru, Philippines, Puerto Rico, Senegal, Somalia, South Africa, Surinam, Swaziland, Tanzania, Trinidad, Uganda, Uruguay, Venezuela, Zaire, Zimbabwe Brazil, Haiti, Nigeria, Panama, Swaziland, Uganda, Venezuela Potassium Bolivia, Brazil, Chad, Colombia, Dominican Republic, Guatemala, Kenya, Malawi, Sodium New Guinea, Nigeria, Panama, Philippines, Senegal, Somalia, South Africa, Surinam, Swaziland, Thailand, Uganda, Uruguay, Venezuela, Zimbabwe Brazil, Colombia, Ecuador, Uganda Sulfur Argentina, Brazil, Colombia, Costa Rica, Cuba, Egypt, EI Salvador, Guyana, Cobalt Haiti, India, Indonesia, Katanga, Kenya, Malaysia, Mexico, Nicaragua, Northern Africa, Peru, Philippines, South Africa, Surinam, Uganda, Uruguay, Zaire Argentina, Bolivia, Brazil, Colombia, Costa Rica, Cuba, Dominican Republic, Copper (or molybdenum Ecuador, EI Salvador, Ethiopia, Guatemala, Guyana, Haiti, Honduras, India, toxicity) Indonesia, Kenya, Malaysia, Malawi, Mexico, Panama, Peru, Philippines, Senegal, South Africa, Sudan, Surinam, Swaziland, Tanzania, Trinidad, Uruguay, Venezuela, Zaire, Zimbabwe Worldwide Iodine Brazil, Costa Rica, India, Panama Iron Manganese Argentina, Brazil, Burma, Costa Rica, Panama, South Africa, Uganda Bahamas, Bolivia, Brazil, Colombia, Costa Rica, Dominican Republic, Ecuador, Selenium Guyana, Honduras, Indonesia, Malawi, Mexico, Paraguay, Peru, South Africa, Swaziland, Thailand, Uganda, Uruguay, Venezuela Zinc Argentina, Bolivia, Brazil, Colombia, Costa Rica, Dominican Republic, Ecuador, EI Salvador, Guatemala, Guyana, India, Indonesia, Kenya. Malawi, Mexico, Panama, Peru, Philippines, Puerto Rico, South Africa, Sudan, Swaziland, Uganda, Uruguay, Venezuela Toxic Elements: Fluorine Algeria, Argentina, Ecuador, Guyana, India, Kenya, Mexico, Morocco, Saudi Arabia, South Africa, Tanzania, Tunesia Manganese Brazil, Costa Rica, Indonesia, Peru, Surinam Selenium Argentina, Brazil, Central African Republic, Chad, Chile, Colombia, Ecuador, Honduras, India, Iran, Kenya, Madagascar, Mexico, Nigeria, Northern Africa, Peru, Puerto Rico, South Africa, Sudan, Upper Volta, Venezuela "McDowell (1976,1985); Fick et 1/1. (1978); McDowell et al. (1984).

Incidence of Mineral Deficiencies and Toxicities

29

involve more than one mineral and be combined with the effects of energy-protein deficiencies, various types of parasitism, toxic plants, and infectious diseases. Mineral deficiencies result most often when animals (and also humans) are confined within a given area and are thus closely dependent upon the structure of the soil and the plant life in a very limited space. They no longer have recourse to migrations in order to compensate for the insufficiencies of the soil or the climate. Various studies have shown that certain trace mineral deficiencies can be prevented when ruminants are allowed greater grazing opportunities and when monogastric species are given access to feeds produced from more than one geological region (McDowell, 1985). In human nutrition, the Se-deficient conditions in China of Keshan and Kaschin-Beck diseases are attributed to the people in the affected region consuming a monotonous locally produced diet (i.e., rice) with no foods introduced from more Se-rich regions. For both humans and animals, deficiencies of trace minerals (also true for vitamins) are less likely when a variety of foods or feeds produced from different geological regions are available. Allman and Hamilton (1949) gathered information from various parts of the world on locations of livestock nutritional deficiencies. Russell and Duncan (1956) and Underwood (1981) have reported selected world locations of mineral deficiencies and toxicities. Information on mineral deficiencies and excesses specifically for grazing livestock in Latin America was updated (Phillips, 1956; De Alba, 1971; McDowell, 1985, 1997, 1999). Table 1.4 lists reports of mineral deficiencies or toxicities for grazing livestock in tropical African, Latin American and Asian countries. Most information in Table 1.4 is a combination of reviews on the reported incidence of mineral deficiencies or toxicities in the developing tropical countries of the world. The numerous references to support Table 1.4 for reported incidences of deficiencies and toxicities are listed elsewhere (McDowell, 1976, 1985, 1997; Fick et al., 1978). An additional tropical country not listed in Table 1.4 is Australia, where reported mineral deficiencies for grazing livestock are P, Ca, S, Co, Cu, I, and Se (Stobbs and Minson, 1980; Minson, 1990). A wide range of mineral deficiencies and excesses has been established in many countries of the world on the basis of forage analysis (See Chapter 17 of this volume.). A summary of mineral concentrations of 2615 forage samples included in the 1974 "Latin American Tables of Feed Composition" indicated that mineral deficiencies were severe and widespread (McDowell et al., 1977). Based on mineral requirements for grazing beef cattle, the percentage of forage samples deficient were as follows: Ca, 31%; P, 73%; Na, 60%; Mg, 35%; Co, 43%; Cu, 47%; Fe, 24%; Mn, 21%; and Zn, 75%. Molybdenum was over 3 ppm in 14% of the samples. Both I and Se were known to be widely deficient, based on other criteria, but few analyses were available. Analyses from the past 20 years illustrate the widespread areas of Se deficiencies throughout the world (McDowell, 1997, 1999; Oldfield, 1999).

30

General Introduction

XI. REFERENCES Allman, R. T., and Hamilton, T. S. (1949). "Nutritional Deficiencies in Livestock," FAO Agriculture Studies No.5, Washington, D.C. Ammerman, C. B. (1995). In "Bioavailability of Nutrients for Animals" (C. B. Ammerman, D. H. Baker, and A. J. Lewis, eds.) p. 83, Academic Press, San Diego. Ammerman, C. D. and Goodrich, R. D. (1983). J. Anim. Sci. 57(Suppl. 2), 519. Anke, M., Arnhold, W., Muller, M., IlIing, H., Schafer, U, and Jaritz, M. (I 997a). In "Handbook of Nutritionally Essential Mineral Elements" (B. L. O'Dell, and R. A. Sunde, eds.) p 465. Dekker, New York. Anke, M., Gurtler, H., Neubert, E., Glei, M., Anke, S., Jaritz, M., Freytag, H., and Schafter, U. (I 997b). In "Proc. Ninth Symp. on Trace Elements in Man and Animals" (P. W. F. Fischer, M. R. L'Abbe, K. A. Cockell, and R. S. Gibson, eds.) p. 192. NRC Research Press, Ottawa, Canada. AOAC (1997). "Methods of Analyses." Association of Official Analytical Chemists. 16th Ed. Publ. AOAC. Washington, D.C. Armsby, H. P. (1880). "Manual of Cattle-Feeding." John Wiley and Sons, New York. ARC (1980). Agriculture Research Council. "Nutrient Requirements of Farm Livestock," No. 2 Ruminants. Her Majesty's Stationary Office, London, England. Becker, R. B., Henderson, J. R., and Leighty, R. B. (1965). "Mineral Malnutrition in Cattle." Bull.699, Fla. Agri. Exp. Stn., Gainesville, FL. Dean, J. R. Crews, H. M., and Ebdon, L. (1989). In "Applications ofInductively Coupled Plasma Mass Spectrometry" (A. R. Date, and A. L. Gray, eds.), p. 141. Chapman and Hall, New York. De Alba, J. (1971). "Feeding of Livestock in Latin America," 2nd Ed. La Prensa Medica Mexicana, Mexico. DRI (Dietary Reference Intakes). (2001). Panel on Micronutrients of Food and Nutrition Board. National Academy Press, Washington, D.C. Egan, A. R. (\975). In "Trace Elements in Soil-Plant-Animal Systems" (D. J. Nicholas, and A. R. Egan, eds.), p. 371. Academic Press, New York. Fick, K. R., McDowell, L. R., and Houser, R. H. (1978). In "Proceedings Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 149. Univ. Florida Press, Gainesville, FL. Forbes, E. B., and Keith, M. H. (\914). In "Phosphorus Compounds in Animal Metabolism," Ohio Agr. Exp. Sta. Tech. Bul. 5, p. 746, Wooster, OH. Georgievskii, V. I., Annenkov, B. N, and Samokhin, V. T. (1981). "Mineral Nutrition of Animals." Butterworths, London, England. Henry, P. R., Ammerman, C. B., and Miles, R. D. (1986). Poult. Sci. 65, 983. Heumann, K. G. (1985). Biomed. Mass Spectrom. 12,477. Hogan, A. G., and Nierman, J. L. (1927). In "Studies of Animal Nutrition - VI the Distribution of the Mineral Elements in the Animal Body as Influenced by Age and Condition." Missouri Agr. Exp. Res. Bul. 107, Columbia, MO. Houk, R. S., Fassel, V. A., Flesch, G. D., Svec, H. J., Gray, A. L., and Taylor, C. E. (1980). Anal. Chem. 52,2283. Iyengar, V., and Woittiez, J. (\988). Clin. Chem. 34(3), 474. Jenkins, K. (1988). In "X-Ray Fluorescence Spectrometry" (J. D. Winefordner, ed.), p. 51, John Wiley, New York. Leeson, S., and Summers, J. D. (2001). In "Nutrition of the Chicken," 4th Ed., University Books, Guelph, Canada. Legg, S. P., and Sears, L. (1960). Nature (London) 186, 1061. Long, M. I. E., Ndyanabo, W. K., Marshall, B., and Thornton, D. D. (1969). Trinidad Trop. Agr. 46, 201. Loosli, J. K. (\974). Proc. Nigerian Soc. Anim. Prod. 1,74. Loosli, J. K. (\978). In "Proc. Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 5. Univ. Florida Press, Gainesville, FL. Loosli, J. K. (1991). In "Handbook of Animal Science" (P.A. Putnam, ed.), p. 25, Academic Press, San Diego. Maynard, L. (1937). "Animal Nutrition," McGraw-Hill Book Co., New York. McCay, C. M. (1973). "Notes on the History of Nutrition Research" (F. Vergar, ed.), Hans Huber Publisher, Berne, Stuttgart, Vienna. McCollum, E. V. (1956). "A History of Nutrition." Houghton Millin, Boston, MA.

References

31

McDowell, L. R. (1976). In "Beef Cattle Production in Developing Countries" (A. 1. Smith, ed.). p. 216, Univ. of Edinburgh Press, Edinburgh, Scotland. McDowell, L. R. (1985). "Nutrition of Grazing Ruminants in Warm Climates." Academic Press, New York. McDowell, L. R. (1997). "Minerals for Grazing Ruminants in Tropical Regions", (3rd Ed.) University of Florida, Gainesville, FL. McDowell, L. R. (1999). "Minerais para Ruminantes sob Pastejo em Regi6es Tropicais, Enfatizando 0 Brasil." University of Florida, Gainesville, FL. McDowell, L. R., Conrad, J. H., and Ellis, G. L. (1984). In "Symposium on Herbivore Nutrition in SubTropics and Tropics - Problems and Prospects" (F. M. C. Gilchrist, and R. I. Mackie, eds.) p. 67. Pretoria, South Africa. McDowell, L. R., Conrad, 1. H., Thomas, J. E., Harris, L. E., and Fick, K. R. (1977). Trap. Anim. Prod. 2,273. McDowell, L. R., Kroening, G. H., Froseth, 1. A., and Haller, W. A. (1974). NUlI'. Rep. Int. 9, 359. Miles, P. H., Wilkinson, N. S., and McDowell, L. R. (2001). "Analysis of Minerals for Animal Nutrition Research," Department of Animal Sciences, University of Florida, Gainesville, FL. Miller, 1. x., Swanson, E. W., and Hansen, S. M. (1965). J. Dairy Sci. 48, 888. Miller, W. J. (1979). "Dairy Cattle Feeding and Nutrition." Academic Press, New York. Miller, W. J. and Stake, P. E. (1974). In "Proceedings Georgia Nutrition Conference for Feed Industry," p. 25. Univ. of Georgia, Athens, GA. Mills, C. F. (1987). J. Anim. Sci. 65, 1702. Minson, D. J. (1990). "Forage in Ruminant Nutrition." Academic Press, New York. NFIA (1991). "NFIA Laboratory Methods Compendium. Vol. I. Vitamins and Minerals." National Feed Ingredients Association, West Des Moines, IA. NCMN - Netherlands Committee on Mineral Nutrition (1973). "Tracing Mineral Disorders in Dairy Cattle." Centre for Agricultural Publishing, Wageningen, The Netherlands. Neal, W. M .. Becker, R. B.. and Shealy, A. L. (1931). Science 74, 418. Nielsen, F. H. (1996). J. NlIIr. 126, 2377S. NRC. (1980). "Mineral Tolerance of Domestic Animals." National Academy of Sciences - National Research Council, Washington, D.C. NRC. (200 I). "Nutrient Requirements of Domestic Animals, Nutrient Requirements of Dairy Cattle." 7th Ed. National Academy of Sciences - National Research Council, Washington, D.C. O'Dell, B. L. (1984). Nutr. Rev. 42, 301. O'Dell, B. L.. and Sunde, R. A. (1997). In "Handbook of Nutritionally Essential Mineral Elements," Marcel Dekker, Inc .. New York. Oldfield, J. E. (1999). In "Selenium World Atlas," Selenium-Tellurium Development Association, Grimbergen, Belgium. Phillips, R. W. (1956). "Recent Developments Affecting Livestock Production in Americas," p. 83. FAO Agriculture Development Paper No. 55, Washington, D.C. Rogers, 1. A., Davis, C. L., and Clark, 1. H. (1982). J. Dairy Sci. 65, 577. Russell, F. C; and Duncan, D. L. (1956). "Minerals in Pasture: Deficiencies and Excesses in Relation to Animal Health." Technical Communication No. 15, Rowett Institute, Aberdeen, Scotland. Scott, M. L. Nesheim, M. C.. and Young, R. J. (1982). "Nutrition of the Chicken." M.L. Scott and Associates, Ithaca, NY. Shupe, 1. L.. Ammerman, C. Boo Peeler, H. T., Singer, L., and Suttie, J. W. (1974). "Effects of Fluorides in Animals." National Academy of Sciences - National Research Council, Washington, D.C. Stobbs, T. H. and Minson, D. J. (1980). In "Digestive Physiology and Nutrition of Ruminants" 3rd Ed., (D.C. Church, ed.), p. 257. 0 & B Books, Corvallis, OR. Stone, B. A., and Mclntosh, C. H. (1977). Aust, J. Agric. Res. 28, 543. Sutmoller, P., Vahia de Abreu, A. van der Grift,1., and Sombroek, W. G. (1966). "Mineral Imbalance in Callie in the Amazon Valley." The Netherlands Communication No. 53. Department of Agricultural Research, Royal Tropical Institute, Amsterdam, The Netherlands. SUllie, N. F. (1988). S. Afr. J. Anim. Sci. 18(1), 15. Theiler, A., Green, H. H., and Du Toit, P. J. (1924). Union S. Afr. J. Dep. Agric. 8, 460. Thornton, D. D., Long, M. I. E., and Marshall, B. (1969). Trinidad Trap. Agr. 46, 269. Underwood, E. J. (1966). "Mineral Nutrition of Livestock." FAO Commonwealth Agricultural Bureaus, London, England. Underwood, E. 1. (1979). In "Proc, of the Florida Nutrition Conference," p. 203. Univ. of Florida, Gainesville, FL.

32

General Introduction

Underwood, E. J. (1981). "The Mineral Nutrition of Livestock." Commonwealth Agricultural Bureau, London, England. Underwood, E. J., and Mertz, W. (1987). In "Trace Elements in Human and Animal Nutrition" 5th Rev. Ed., Vol. I (W. Mertz, ed.) p. 1. Academic Press, New York. Underwood, E. J., and Somers, M. (1969). Aust. J. Agric. Res. 20, 889. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" 3rd Ed., Midlothian, UK. Valdes, E. V., and Leeson, S. (1990). Poult. Sci. 69, 1803. Watson, L. T., Ammerman, C. B., Miller, S. M., and Harms, R. H. (1970). Poult. Sci. 49, 1548. Whetter, P. A., and Ullrey, D. E. (1978). J. Assoc. Off Anal. Chern. 61(4),930. WHO (1996). In "Trace Elements in Human Nutrition." World Health Organization, Geneva. Williams, S. N., Lawrence, L. A., McDowell, L. R., Wilkinson, N. S., Ferguson, P. W., and Warnick, A. c., (1991). J. Anim. Sci. 69, 1232.

Chapter 2

Calcium and Phosphorus I. INTRODUCTION Calcium (Ca) and phosphorus (P) are considered together because they constitute the major part of the mineral content of bone. They are very closely related; a deficiency or an excess of one wi11 interfere with the proper utilization of the other. The Ca: P ratio in the bone is slightly greater than 2: 1 and is approximately constant. In young animals and humans, shortage of Ca, P, or vitamin D results in rickets (Fig. 2.1), and in the adult or more mature animal, osteomalacia. Calcium and P are the two most abundant mineral elements in the animal body. They are frequently found in insufficient quantities in common feedstuffs to meet requirements of livestock. Phosphorus deficiency is predominantly a condition of grazing ruminants, especially cattle, whereas Ca deficiency is more a problem of animals fed mostly on concentrates, especially pigs and poultry, and also feedlot cattle finished on high-grain diets. For humans, owing to the consumption oflow Cadiets by both infants and adults, Ca deficiency is second only to iron (Fe) deficiency.

n.

mSTORY

Rickets in man and animals has been known since antiquity; however, the relationships of Ca, P, and vitamin D to rickets were not known until more modern times. In the 1800s, the association ofCa and P to rickets was becoming clear, but the relationship of the disease to vitamin D was not established until the 1920s. Dr. Clive M. McCay of Cornell University has written a very comprehensive review of the history of nutrition research, including that of Ca and P. Unless stated otherwise, early citations for Ca and P noted herein are presented in his review (McCay, 1973). Sir Humphrey Davey is credited with the discovery of Ca in 1808, but during the 1700s, a number of experiments were carried out with animal bones. In 1736, an English surgeon, John Belchier, found that boiling dyes with bran and feeding this to pigs led to the dyes being deposited in the bone. This discovery led to the study of bone calcification. Brandt, a German alchemist, first isolated P from human urine in 1669. By 1769, a Swedish chemist, Gahn, recognized P as an essential part of bone, and large quantities of P were prepared from bone ash in 1771 by Scheele. Bone composition was studied with growing interest during the eighteenth century. It was known that burning bones left an ash. Scheele and Gahn around 33

34

Calcium and Phosphorus

Fig. 2.1 Calves and young bulls with rickets. (Courtesy of Francisco Megale, Universidade Federal de Minas Gerais, Escola de Veterinaria, Belo Horizonte, MG, Brazil)

1770 discovered that the earthy matter of bone was calcium phosphate. By 1803, lime phosphate was being fed to children with rickets, and it was claimed to improve teeth and heal fractures. It was known by 1817 that the inorganic part of bone was largely calcium phosphate with a small amount of carbonate and some magnesium (Mg). In 1841, Boussingalt first stated, "The bones as we have seen, contain a large quantity of lime: it is required, therefore, that the elements of this salt, phosphoric acid and lime, should form part of the diet." He noted that missionaries observed South American Indian children eating soil, which was evidence of depraved appetite, but their typical foods such as corn were very low in Ca. This represents the attempts (although of little or no value) of primitive people to provide Ca supplementation. In 1842, Choussat reported the first direct experiment in which Ca was shown to modify the composition of bone. Pigeons fed a diet of wheat and water died with very fragile bones, particularly the sternum. When CaC0 3 was added to the diet, bones were normal. In 1973, Nessler found that fragile cattle bones had thin shaft walls, were light in weight and contained less Ca and P than did bones of healthy animals. In the late 1800s, rickets was produced in swine fed low-Ca diets. Rickets in calves was prevented or cured by feeding alfalfa hay. The need for adequate supplies of Ca for the nursing mother was well established by 1900. Every dentist knew that gestation and lactation weakened the mother's teeth. In the early 1900s,

Chemical Properties and Distribution

35

it was also becoming apparent that Ca alone was not responsible for rickets. By 1909 researchers were producing rickets by feeding low-P diets. In actuality, at the turn of the twentieth century, the greatest cause of rickets was from lack of sunlight or vitamin D. Phosphorus deficiency was observed and described in cattle as early as 1785. A great deal of the early work on P deficiencies was carried out in South Africa, where it was intimately associated with the occurrence of lamsiekte (literally lame sickness) and botulism. Le Vaillant (1976; cited by Butterworth, 1985), in his book entitled Travels into the Interior Part ofAfrica, noted lamsiekte and mentioned bone eating or osteophagia. It was left to Theiler (1920, 1927) finally to elucidate the etiology of lamsiekte, botulism, and P deficiency. This South African researcher studied cattle exhibiting subnormal growth, low reproduction, and a depraved appetite or pica illustrated by bone chewing. Theiler concluded that vegetation containing low levels of P induced a deficiency in the animal, which in turn provoked a depraved appetite (pica), causing the animal to become infected with the toxicogenic organism Clostridium botulinum when bone was consumed. Chicco and French (1959) cite Azara (1838) as reporting osteophagia in cows in Paraguay and claim that this was the first mention of P deficiency in Latin America. In the Gulf Coast area of Texas, Schmidt (1926) reported that a fatal disease of cattle, creeps, could be prevented by bonemeal and salt supplementation. Later, the disease stiffs or sweeney was shown to be caused by a deficiency of P in Florida (Becker et al., 1933). In recent years, a great deal of the research with Ca and P is associated with more accurately determining requirements of these nutrients, interrelationships with other minerals (and other nutrients), and metabolism. A new phase of Ca and P research began in the late 1960s with discovery of how active forms of vitamin 0 influence Ca and P metabolism.

III. CHEMICAL PROPERTIES AND DISTRmUTION

A. Calcium Calcium is a soft, silvery white metallic element found most widely in rocks as chalk, limestone, and marble. It is much harder than sodium (Na) but softer than aluminum (AI) or Mg. Calcium is an alkaline earth metal with an atomic weight of 40.08, and an atomic number of20. Its occurrence in the earth's crust is 3.64% (fifth element in order of abundance). It is found naturally only in compounds, chiefly as limestone (calcium carbonate), calcium fluoride, and calcium sulfate. More than 80% of the Ca found in the crust of the earth is in the form of limestone. Calcium is the most abundant mineral element in the animal body (I to 2%), with 99% of it occurring in bone and teeth and the remainder, constituting the physiologically active pool of free Ca, is found in the extracellular fluid and within cells. Variable amounts of Ca are present in almost all feedstuffs (see Section VII).

36

Calcium and Phosphorus

Calcium is generally deficient in grains and abundant in most forages. Its content in natural feeds varies widely, depending on the species of plant and plant part analyzed. Grains such as barley, com, sorghum, oats, and wheat are very low in Ca (0.02 to 0.10%). The nonlegume roughages such as grass hay and mature range forages are intermediate in Ca content (0.31 to 0.36%), and legume forages such as alfalfa and clover hay contain 1.2 to 1.7% Ca (NRC, 1980).

B. Phosphorus Phosphate is anyone of a number of chemical compounds that contain P and oxygen in the phosphate radical P04-3. Phosphorus has an atomic weight of 30.97, and its atomic number is 15; it has one naturally occurring isotope, 31p. It forms about 0.12% of the earth's crust. Phosphorus does not occur free in nature, as it is much too reactive. Essentially, all of the naturally occurring P compounds are phosphates and always occur on the surface of the earth in the form of orthophosphates. The ultimate source of P is igneous rocks (formed by solidification of molten rock), in four principal types of large deposits: (1) igneous apatites, (2) marine phosphorites, (3) phosphatized rock, and (4) guano. Two major groups of sedimentary deposits important for the production of feed phosphates are pellet phosphorite and guano. The major phosphate deposits are found in a form called pellet phosphorite in Florida (USA), Morocco, Israel, and North and South Carolina (USA). Guano phosphate originates from the action of P in the excrement of birds and bats with limestone beds, principally in Curacao and Christmas Island. In Mexico, the same general type of phosphate was formed from bat guano in caves. Most phosphates in nature occur as apatite, CalO(P04MF,CI,OHh or 3Ca3(P04h·Ca(F,CI,OHh The high level of fluoride (F) in the natural rock phosphates limits their effectiveness as P sources for animal nutrition (see Chapter 14). This is relatively unimportant for fertilizer-grade phosphates, as there is little plant uptake of F from soil. Phosphorus is the second most abundant mineral element found in the animal body, and 80 to 85% is in bones and teeth. Phosphorus is present in all common feedstuffs (see Section VII). Seeds are uniformly higher in P than are roughages and seed by-products, such as wheat bran and oil meals, are especially rich in P. Feeds containing milk and bone are high in both P and Ca.

IV. METABOLISM

A. Absorption Many factors influence Ca and P absorption, utilization and metabolism, including adequate levels of one to the other. A Ca : P ratio of I : I to 2: I is usually recommended, with a close ratio most critical if P intake is marginal or inadequate.

Metabolism

37

The absorption of Ca and P is throughout most of the intestinal tract with the duodenum and jejunum being the most active absorptive sites. The large intestine contributes to Ca absorption, with an estimation of total absorption of 11% for the rat (Bronner and Pansu, 1999). In rats, Ca was shown to be mostly absorbed in the small intestine, but when an insoluble Ca salt is not sufficiently absorbed in the small intestine, the large intestine compensates by absorbing greater quantities of Ca (Shiga et al., 1998). In the rat, 7% of the total vitamin D-dependent calcium binding protein (calbindin) is found in the large intestine (Escoffier, 1996) indicating a controlled Ca absorption. Contrary to many species, in the horse, the colon is the major site of absorption and reabsorption of P (Frape, 1998). Small amounts of Ca may be absorbed from the rumen (Yano et al., 1991). Generally, only 30 to 50% of ingested Ca is normally absorbed, whereas 70 to 80% of dietary P is absorbed (Arnaud and Sanchez, 1996; Ternouth and Coates, 1997). Calcium is absorbed according to need up to the limits set by the absorbability of the mineral in the diet; this is close to 90% for milk and probably rarely oats> barley> rye. Magnesium levels of wheat were the lowest, whereas those of rye were the highest. For tropical herbage, Mg concentrations were relatively low, with slightly more than one-third of 288 forages included in the 1974 Latin American Tables of Feed Composition containing 0.2% Mg or less (McDowell et al., 1977). Forage mineral analysis from a large number of farms in four Latin American tropical countries indicated a considerable number of forages contained less than 0.20% Mg (dry basis): Bolivia (64%), Colombia (56%), Dominican Republic (33%), and Guatemala (76%) (McDowell, 1985). Temperature and light may affect forage Mg concentrations and grass tetany etiology. Overall shading probably reduces forage Mg availability; the incidence of grass tetany is greater when daily radiation levels are low (Mayland et al., 1976). Magnesium concentrations generally decline as the plant matures (Aleroft, 1954), but this lowered concentration is often less dramatic than for many other minerals. Stem tissue contributes appreciably to total plant Mg; that of early maturity stage of growth forms the bulk of plant dry matter, Magnesium concentrations were greater in stems than in leaves of dwarf elephant grass (Pennisetum purpureum) (Montalvo et al., 1987). Magnesium fertilizers can significantly increase pasture concentrations, usually without influencing yields. Calcined magnesite (MgO) at the very high rate of 3180 kg/ha doubled the Mg content of an English pasture, from an average of 0.2 to 0.4%, while lower and more economic rates of application resulted in intermediate increases (Aleroft, 1961). High fertilization with K and/or N, especially in soils with low Mg content, reduced the Mg content in grasses. Mayland and Grunes (1979) reported that high K application rates reduced Mg concentration by an average of 15 to 20% compared to that of untreated forage. The detrimental effects of N fertilization may be because N increases K concentrations. Rosero et al. (1975) fed orchard grass and a fescue-rye hybrid to sheep and found that N fertilization lowered intake, percentage Mg absorption, and Mg balance in lambs. Biological availability of different sources of Mg for ruminants varies considerably. Peeler (1972) reported that the availability of Mg ranged from 10 to 25% in forages and from 30 to 40% in grains and concentrates. Kemp et al. (1961) reported that, contrary to most minerals, Mg availability improves with increasing maturity of grasses and may be decreased by heavy K and N fertilization. Perdomo et al. (1977) studied the apparent digestibility in sheep of five minerals at three stages of regrowth for three tropical forages. Digestibility and retention of Ca, P, and Na by sheep tended to decline with maturity, while availability of Mg tended to increase at 56 days of age for the three species. Usually Mg in preserved forages is more available than in pastures. Apparent availability of Mg was significantly higher in ensiled than in frozen or artificially dried grass (Powley and Johnson, 1977). The availability of Mg may range from 5 to 33% in succulent feeds and from 10 to 40% in hay and concentrates (Wilkinson and Stuedemann, 1979). For human diets, excellent sources of Mg include nuts, legumes, and unmilled grains. Removal of the germ and outer layers from cereal grains, however, results in

160

Magnesium

a Mg loss of about 80%. Green vegetables are good sources of the element for humans, whereas milk, fish, and meat contain relatively low Mg concentrations.

VIII. DEFICIENCY

A. Effects of Deficiency

Magnesium deficiency is manifested clinically by retarded growth, hyperirritability and tetany, peripheral vasodilation, anorexia, muscular incoordination, and convulsions. Most practical diets contain adequate Mg to promote optimal performance. The exception is grazing ruminants and especially mature lactating cattle, which are most susceptible to Mg deficiency and/or abnormal Mg metabolism. Because of adequacy of Mg for most species consuming typical diets, special dietary ingredients (i.e., purified diets) are used to study both requirements and deficiency in nonruminants. 1.

SWINE

Metabolically, Mg is an extremely important nutrient, but its requirement is so low, or its recycling is so efficient relative to other macrominerals that deficiency must be produced by use of diets not containing cereal grains and oilseed meals. The signs of a Mg deficiency in order of appearance are hyperirritability, muscular twitching, reluctance to stand, weak pasterns, loss of equilibrium, tetany, and finally death. There was a high mortality rate in baby pigs fed low-Mg diets. The Mg-deficient pigs also exhibit stepping syndrome (Fig. 5.1), which causes them to keep stepping or lifting their hind legs almost continuously while standing (Mayo et al., 1959). Miller et al. (1965) reported that pigs fed 25 or 75 ppm Mg showed slow growth, poor feed conversion, weakened pasterns, stepping syndrome, tetany, and death. Pigs fed 125 ppm showed all these signs except tetany and death. High dietary manganese (Mn) in addition to low Mg intakes greatly increased convulsive seizures and death in growing pigs (Miller et al., 2000). The high dietary Mn was found to greatly depress heart concentrations of Mg. 2.

POULTRY

Magnesium deficiency seldom occurs in poultry fed practical diets. When highly purified feedstuffs are fed to young chicks, one observes poor growth, lethargy, convulsions, and death, with rather large differences in response within a chick group. Deficient chicks often pant and gasp, and there is poor feathering, decreased muscle tone, ataxia, progressive incoordination, and convulsions followed by death (Bird, 1949). Newly hatched chicks fed a diet devoid of Mg lived only a few days. Ducklings fed no supplemental Mg showed signs of deficiency and died within 10 to 16 days (Van Reen and Pearson, 1953). Plasma Mg and Ca are reduced by Mg deficiency. Marginal deficiency may allow near-normal growth, but plasma Mg will be reduced, and when disturbed chicks show signs of neuromuscular

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Fig. S.l Magnesium deficiency in swine and sheep. Five-week-old pig (A) showing stepping syndrome; the pig keeps stepping almost continuously while standing. Weakness of pasterns is apparent. Magnesium deficiency in a lamb (8) illustrating unthriftiness. Note the stiff legs. (A - courtesy of: E. R. Miller and D. E. Ullrey, Michigan State University, East Lansing; 8 - courtesy of U. S. Garrigus, University of Illinois, Urbana)

hyperirritability. Chicks may have brief episodes of convulsions followed by coma, from which they usually recover. Mahoney et al. (1992) noted an 80% reduction in growth of broilers fed 0.02 vs 0.06% Mg. A deficiency of Mg in the diet of laying hens results in rapid decline in egg production, blood hypomagnesemia, and a marked withdrawal of Mg from bones of Mg-deficient hens. Egg size, weight of shell, and Mg content of yolk and shell are decreased owing to Mg deficiency (Leeson and Summers, 2001). Feeding a semipurified diet containing 56 ppm Mg decreased egg production and reduced serum Mg within 10 to 14 days (Sell et al., 1967). Fertilization of eggs was not affected, but hatchability of fertile eggs was reduced markedly. A decline in hatchability precedes decreases in egg production and diet consumption.

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RUMINANTS

Grass tetany (or hypomagnesemia) is a complex ruminant metabolic disorder affected by mineral composition of forage species, soil properties, fertilizer practices, season of the year, temperature, animal species, breed, and age. Grass tetany is quite rare for livestock consuming predominantly legumes, as they are generally higher in Mg than grasses. Grass tetany is not an entirely appropriate name because the disorder is not limited to animals receiving grass and is characterized by convulsions rather than tetany. A number of clinical syndromes in cows, sheep. and goats are included in the grass tetany syndrome. Grass tetany is also referred to as lactation tetany, grass staggers, wheat pasture poisoning, winter tetany, and milk tetany in calves. Signs of hypomagnesemic tetany are encountered both in grazing ruminants and in calves reared too long on milk without access to other feeds. The spring-calving cow is usually more susceptible to development of tetany within a few weeks after calving. Susceptibility to grass tetany is increased in older ruminants because of the decreased ability to mobilize skeletal Mg with increasing age (see Section IV). Consequently, an abrupt change from a normal diet to one with inadequate available Mg can result in hypomagnesemia within 2 to 18days, even though the previous feed was high in Mg (Dishington and Tollersrud, 1967). It seems to be most predominant in cows subsequent to the second lactation (third to fifth lactation). In the United States grass tetany is a greater problem with beef than with dairy cattle, probably because of greater Mg availability in concentrates, along with the substantial amounts of grain usually fed to lactating dairy cows. In many developing tropical countries, dairy cows often do not receive concentrates in substantial quantities. Usually, only female animals are affected with grass tetany, although it has been reported in calves and steers (Crookshank and Sims, 1955). Cows are particularly susceptible to tetany when nursing a calf or producing milk. Sometimes pregnant animals die from the condition. Grass tetany generally occurs during early spring, or a particularly wet autumn, among older animals grazing grass or small grain forages in cool weather. Cows are most likely to get grass tetany soon after they are turned out on spring pasture. However, there is also a winter type of tetany that affects cattle fed winter diets (sometimes poor quality hay) in confinement. Grass tetany has also been reported in fall-calving beef cows. In addition to low blood Mg, the fall tetany or winter tetany syndrome is often associated with ketosis (Boling, 1982). In New Zealand, where cows are pastured throughout the year, the disorder occurs most frequently in late winter and early spring (Underwood and Suttle, 1999). Tetany is seen where ruminant production is highly developed, high quality pastures are available, and high-yielding, quick-maturing stock are raised. Voisin (1963) suggested that farming methods have caused an imbalance in the soil and herbage, upsetting the Mg metabolism of grazing ruminants. The incidence of a Mg deficiency is influenced as much or more by management considerations as by geographical location.

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Although it is not characterized by death, nonclinical hypomagnesemia is far more common than clinical tetany, and economic consequences of lowered production are substantial (NCMN, 1973). The subclinical stage of Mg deficiency may be followed by spontaneous recovery from hypomagnesemia and does not invariably progress to the acute disorder (Underwood and Suttle, 1999). Chronic marginal deficiency of Mg will result in reduced feed intake and performance. Suboptimal performance can be related to a decline in cellulose digestion. Studies with lambs and steers suggest that feed intake and cellulose digestion decline faster than does serum Mg when Mg-free diets are fed (Chicco et al., 1973; Emery, 1976). Boling (1982) has described the external or visible signs of grass tetany syndrome in cows. Initially, the cow may have a depressed appetite and exhibit a dull, lethargic appearance. As the condition progresses, signs of stiffness as she walks, and ultimately, a staggering gait may become apparent. As the condition progresses, the cow becomes highly excitable and nervous and has readily visible muscular tremors. The head is held high with staring eyes with movements becoming stiff and stilted. Chewing, hypersalivation, and blinking of the third eyelid are particularly characteristic of hypomagnesemic tetany. In the most severe stage, the animal collapses to the ground with continuation of the tetanic muscular spasms. The legs will usually thrash the ground around the cow, uprooting forage. Death occurs after collapse if the animal does not receive medical treatment (Fig. 5.2). Crookshank and Sims (1955) note that 6 to 10hr are usually required from the time of the first clinical signs until the animal passes into a comatose condition. If treatment is not initiated before coma, there is little chance of recovery. McCoy et al. (2000) reported that hypomagnesemic tetany was associated with alterations in regional brain monoamine concentrations in cattle. Monoamines are important brain neurotransmitters. The cerebral cortex and cerebellum regions in the brain play an important role in both voluntary and involuntary motor function, and therefore these alterations may playa role in the etiology of hypomagnesemic tetany. Preconvulsive clinical signs of hypomagnesemia in sheep are less clearly defined than in cattle and can be confused with those of hypocalcemia or pregnancy toxemia. Hypomagnesemia tetany in sheep is almost exclusively a disease of the first 8 weeks of lactation, with ewes nursing twins most susceptible. The incidence is highest I to 4 weeks after lambing (Herd, 1966). In Australia, however, a high incidence of hypomagnesemia tetany in breeding ewes has been correlated with periods of rapid winter growth of pastures (Underwood and Suttle, 1999). A lamb with hypomagnesemic tetany may fall on its side with its legs alternately rigidly extended and relaxed. Frothing at the mouth and profuse salivation are evident, and death may occur. The signs of Mg deficiency in adult ewes are similar to those in younger animals, but death may occur more rapidly after convulsions. Clinical tetanies in cattle, milk-fed calves, and sheep can combine hypomagnesemia and hypocalcemia (low blood Ca). A high incidence of hypocalcemia has been reported with wheat-pasture poisoning (Bohman et al., 1983). Both Ca and Mg metabolism are interrelated in this malady.

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Fig.5.2 Cow in collapse stage of tetany and death from tetany. Note area around forelegs where ground has been thrashed during convulsions. (Courtesy of I.A. Boling, C.W. Absher, and D.E. Miksch, University of Kentucky, Lexington)

The economic importance of grass tetany arises from its high death rate and its sudden occurrence. Grass tetany causes an estimated $50 to 150 million in livestock production losses each year in the United States (Wood, 1999). In the United States the mortality among untreated clinical cases is 30% or more (Grunes et al., 1970). Clinical tetany is endemic in some countries, affecting only a small proportion of

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cattle (l to 2%). However, individual herds may report incidence of tetany as high as 20%. Hypomagnesemic tetany occurs in most European countries, North America, Australia, South Africa, and New Zealand. McDowell (1985) listed 17 developing tropical countries where Mg tetany is encountered or highly suspected. Reports of Mg deficiency are much more prevalent in temperate than in tropical regions. Some of the reasons for fewer reports of grass tetany in tropical regions include the prevalence of the condition during cooler temperatures (8 to 14°C), and for many tropical countries, the generally low productivity of pastures and of herds, and lack of fertilization of pasture with Nand K. Nevertheless, grass tetany can be a problem for grazing ruminants in tropical countries, since forages are often low in Mg. 4. HORSES

Horses grazing pastures that produce tetany in cattle are seldom affected. Foals fed a purified diet containing 8 ppm Mg developed clinical signs including hypomagnesemia, nervousness, muscular tremors, and ataxia followed by collapse, with hyperapnea (hard breathing), sweating, convulsive paddling of legs, and, in some cases, death (Harrington, 1974). Mineralization of arteries, elastic tissue, spleen, lungs, and heart were observed at necropsy. Foals fed Mg-deficient diets showed degeneration in the lung, spleen, skeletal muscle, and heart (Harrington, 1975). Skeletal muscle degeneration was consistently found in all the foals fed the Mg-deficient diet for 71 days or longer, although it was not extensive. In this study, a sharp decrease in blood serum Mg levels was detected within 24 to 48 hr after the foals were given the Mg-deficient diet; at necropsy there was a reduction in bone Mg concentration. 5. OTHER ANIMAL SPECIES

a. Dogs. Anorexia, vomiting, decreased weight gain, and hyperextension of the front legs were observed in puppies (7 to 9 weeks of age initially) that were fed a purified diet containing less than 5 ppm Mg for 3 weeks (Kahil et al., 1966). By 4 to 6 weeks the puppies fed this diet showed irritability, ataxia of hind legs, and convulsive seizures. In another study with puppies, similar clinical signs were observed, and at necropsy, aortas of these animals contained extreme mineralized lesions, primarily Ca and P deposits (NRC, 1985a). b. Cats. Magnesium deficiency has been reported in cats (Chausow et al., 1985). At 50 ppm of dietary Mg, kittens grew poorly and exhibited muscular weakness, hyperirritability, convulsions, anorexia, reduced bone and serum Mg concentration, and calcification of the aorta. c. Laboratory Animals. Deficiency of Mg was first studied intensively in the rat. In rats, lowering dietary Mg to 1.8 ppm resulted in vasodilation, hyperirritability, convulsions, and death (NRC, 1995). Renal calcification is common and may be

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detected within two days after initiating a markedly deficient diet. Tufts and Greenberg (1938) reported that lactating female rats fed a deficient diet were bred successfully but did not suckle their young. Magnesium deficiency has been shown to induce bone loss in the rat (Rude et al., 1998). Laurant et al. (1999, 2000) have reported that dietary Mg intake can affect mechanical properties of rat carotid artery. These mechanical alterations could contribute to the development of atherosclerosis, hypertension, and cardiovascular diseases. Rock et al. (1995) suggested that dietary Mg deficiency in rats enhances free radical production in skeletal muscle. Magnesium deficiency has been established in mice and guinea pigs. Alcock and Shils (1974) reported that Mg-deficient mice, without showing previous hyperirritability, developed rapid and usually immediately fatal convulsions. Clinical signs of deficiency in young guinea pigs fed low-Mg purified diets include poor weight gains, hair loss, decreased activity, poor muscular coordination and stiffness of rear limbs, elevated serum P, and anemia (NRC, 1995).

d. Rabbits. Kunkel and Pearson (1948) found Mg deficiency in rabbits caused poor growth and hyperexcitability with convulsions. Inadequate Mg may result in fur chewing, alopecia, blanching of the ears, and alteration of fur texture and luster in rabbits fed a diet containing 5.6 ppm Mg (NRC, 1977). e. Fish. Magnesium deficiency causes anorexia, reduced growth, lethargy, and reduced tissue Mg content in fish. Deficiency signs in rainbow trout included loss of appetite, decreased growth, lethargy, reduced bone ash, spinal curvature, and histological changes in muscle, pyloric caeca, and gill filaments (Cowey et al., 1977). Trout fed Mg-deficient diets (40 ppm) developed renal calcification at dietary Ca levels of 2.7% and a dietary Ca:P ratio of 1:1. Dietary Mg deficiency in channel catfish and common carp causes poor growth, anorexia, sluggishness, muscle flaccidity, high mortality, and depressed tissue Mg levels (NRC, 1993). Magnesium deficiency has not been demonstrated in fish in a seawater environment, where they obtain Mg by drinking the water.

f Nonhuman Primates. Vitale et al. (1963) induced Mg deficiency (6 ppm dietary Mg) in growing cebus monkeys (Cebus apella), characterized by weight loss, hyperirritability, and convulsions. Serum Mg fell from about 1.3 to 0.7 mg/dl, and at necropsy, marked sudanophilia and connective tissue plaques were observed in the aortas of the deficient animals. Magnesium deficiency in rhesus monkeys resulted in hypomagnesemia, hypocalcemia, and hyperirritability (NRC, 1978). 6.

HUMANS

For patients fed a low-Mg formula, urinary Mg fell sharply to levels no longer detectable within a week, and plasma Mg fell continuously. The most prominent and consistent symptoms and signs were nausea, muscle weakness, irritability, mental derangement, and myographic changes.

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Magnesium deficiency with or without symptoms has been reported in numerous disease states (Shils, 1997), including severe malabsorption, diabetes, chronic alcoholism, and malnutrition. Deficiency of Mg is associated with prolonged infusions of Mg-free parenteral fluids - usually in association with prolonged losses of gastrointestinal secretions - renal tubular dysfunction occurring in acute or chronic kidney diseases or secondary to drugs, childhood malnutrition, familial disorders or renal or intestinal conservation, hyperaldosteronism and hyperparathyroidism, especially in the immediate post-pararthyroidectomy period. In recent years the literature has been concerned with the possible preventive and therapeutic rolls of Mg in relation to coronary artery disease (CAD) (Shils, 1997; Ford, 1999). The argument has been advanced that the American public has a significant amount of asymptomatic Mg deficiency and that this is a contributing factor in the prevalence of CAD. Serum Mg concentrations were inversely associated with mortality from CAD and all-cause mortality (Ford, 1999). Magnesium deficiency may affect heart function through changes of K, Na, and Ca concentrations in extracellular and intracellular fluids. The clinical situation becomes more complex with coexisting myocardial disease and when primary electrolyte abnormalities are exacerbated by anoxemia and/or diuretic therapy (Shils, 1997). High blood pressure is a major risk factor for stroke and a recent study has identified K, Mg, and fiber as significant modulators of stroke for men (Suter, 1999; see also Chapter 4). Dietary Mg depletion can be induced in otherwise healthy women; it results in increased energy needs and adversely affects cardiovascular function during submaximal work (Lukaski and Nielsen, 2002).

B. Assessment of Magnesium Status For all species with a Mg deficiency, concentrations in serum, erythrocytes, and urine are depressed. Increasing dietary Mg for lambs resulted in a linear increase in serum Mg (Chester-Jones et al., 1989). Peak serum Mg levels were 3.0, 3.2,4.2, and 5.5 mg/dl for lambs fed 0.2, 0.6, 1.2, and 2.4% Mg, respectively. Bone concentrations of Mg are significantly reduced with a deficiency. This is less pronounced for older ruminants, as they are less able to mobilize Mg than are younger animals. Serum Mg is a good indicator of Mg status of various species, but Mg urinary excretion and erythrocyte concentrations are better indicators. The Mg content of the erythrocytes decreases to about one-half the normal amount during the early phase of depletion, but Mg concentration in serum does not decrease until there is a severe deficiency. In contrast, an excess or a lack of Mg is immediately reflected in daily excretion of Mg in urine; hence, daily urinary excretion is a better criterion of Mg supply than is serum Mg concentration. If Mg deficiency is suspected and if the serum values are normal, erythrocyte Mg content and 24-hr urinary excretion should be measured. When these values are normal, Mg deficiency is very unlikely. For humans with suspected Mg deficiency, Mg concentrations are evaluated in serum, urine, and blood mononuclear cells (Shils, 1996). The concentration of Mg in human mononuclear cells has been claimed to be a better guide to Mg nutriture

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than is the serum concentration. A load test, which is the urinary Mg concentration after an infusion of a Mg salt, is an accurate measurement of Mg status. For grazing ruminants, confirmation of grass tetany is justified only when blood or urine samples are low in Mg. A reduction in Mg content of cerebrospinal fluid « 1.6 mg/dl) is an accurate indicator of deficiency (Meyer, 1976). The ranges in serum or plasma Mg level (mg/lOO ml) for cattle and sheep are as follows: normal values, 1.8 to 3.2; slight hypomagnesemia, 1.2 to 1.8; and severe hypomagnesemia, 1.2 or less (NCMN, 1973). In calves showing the typical signs of Mg deficiency, serum Mg is frequently as low as 0.1 mg/dl compared with a normal of around 2.5 mg. Tentative criteria for Mg in urine are as follows: more than 10.0 mg/dl, adequate to liberal; 2.0 to 10.0 mg/dl, inadequate; less than 2.0 mg/dl, severe deficiency and danger of tetany. A rough assessment of supply for grazing animals can be obtained from the content of Mg, N, and K in pasture. This approach is more accurate when the pasture is sampled close to the date of grazing. If the dates are more than a week apart, the assessment in unreliable. This method can be used only for grazing cattle, whereas the urine method is reliable on indoor diets as well as pasture (NCMN, 1973).

IX. SUPPLEMENTATION Magnesium supplementation is most important for ruminants for the prevention and cure of hypomagnesemia tetany. Berger (1992) calculated that, in a 100-cow herd, preventing the loss from grass tetany of a single cow every three years would more than pay the cost of Mg supplementation. For other species Mg deficiency is uncommon and is brought about by special diets and various disease conditions. Normally, adequate Mg is present in practical diets to meet the requirements of poultry. Since feedstuffs commonly used for poultry contribute Mg levels of 0.11 to 0.24% to the diet, and the estimated requirement varies between 0.03 to 0.06, a need for Mg supplementation seems unlikely, but little research has been done (Sell, 1980). Assuming that swine utilize Mg from feedstuffs with about the same efficiency as chickens, the Mg supplied by common feed ingredients should meet their requirements. Nevertheless, some feed industry nutritionists feel that supplemental Mg may help to prevent hyperirritability and tail biting in confined pigs. Female ruminant livestock that develop tetany should receive medical treatment immediately by intravenous injections or enemas. Treatment can include s.c. injection of a single dose of 200 to 300 ml of a 20% solution of magnesium sulfate or i.v. injection of a similar dose of magnesium lactate (Underwood and Suttle, 1999). These treatments will restore serum Mg of an affected cow to near normal within about 10 min and are almost always followed by disappearance of signs of tetany. Serum Mg concentrations will decrease again unless the cow is immediately removed from the tetany-producing pasture and fed Mg-adequate diets. Most treatments include Ca in addition to Mg salts because hypocalcemia usually accompanies Mg deficiency. Some veterinarians use i.v. injections of chloral

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hydrate or magnesium sulfate to calm excited animals, and then follow with a Ca-Mg gluconate solution. If the animal again goes into convulsions, a second dose of Ca-Mg gluconate solution may be required. Intravenous injections should be administered slowly because there is danger of heart failure if given too rapidly (Grunes and Mayland, 1984). In acute cases, Mg enemas can be used to treat animals with tetany. An enema of 100 ml of 20% MgCh is effective because Mg absorption takes place also in the rectum and colon. The enema may be given with the probe inserted 25 em into the anus. Magnesium chloride is used instead of MgS04 for enemas because colonic transport of Mg requires a simultaneous transport of an anion (CI in this case). The colonic epithelium is impermeable to the sulfate ion, as evidenced by the cathartic action of magnesium sulfate in the gut (Bell and Oluokum, 1977). Treatment effectiveness depends on the time between development of clinical signs and treatment, but those given within an hour have a much greater chance of success. Irreversible pathological changes (possibly of the central nervous system) may develop, and the animal may die if treatment is delayed. Dietary supplementation of Mg in place of intravenous injections or enemas has not been effective in treating tetany cases since too much time is required for the Mg to reach that part of the GI tract where it can be absorbed. For follow-up treatment, the animal should be removed from the tetanyproducing pasture and fed hay and concentrates. Also, 30 g Mg (e.g., MgO) should be given daily (Grunes and Mayland, 1984). Force-feeding ofMg may be necessary, but after a week the amount can be greatly reduced. Cows that get tetany are likely to get it again. Commonly, the recommendation is to take the entire herd off the tetany-prone pasture, if it can be done without unduly exciting the cows. It is often better to bring Mg supplements to the cattle under range conditions. Since the cause of grass tetany is a metabolic deficiency of Mg resulting from either a simple deficiency of Mg or factors that lower the efficiency of utilization of the element, increasing Mg intake should prevent the disturbance (Fontenot, 1980). Several safe and practical means of raising the Mg intakes of animals enough to prevent losses from tetany have been devised. There is some agreement that 50 to 60 g MgO/day is the minimum secure prophylactic dose for adult dairy cattle; with 7 to 15 g/day for calves, and 7 g/day for lactating ewes (Underwood, 1981). When a diagnosis of grass tetany is made, one or more of the following practices may be useful in preventing new cases from occurring. A. Magnesium Fertilization Fertilizing pastures with relatively high levels of Mg or liming with dolomitic limestone for several years increases Mg in the forage. Incorporating dolomitic limestone below the soil surface will increase its effectiveness. The amount of fertilizer Mg required may vary considerably, depending on the soil pH, texture, and K content. Local recommendations should be followed to avoid using too much Nand K fertilizer. Fertilizer Mg and limestone (dolomite) as methods of control have limitations on many soil types and usually have to be accompanied by

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other means of supplying additional Mg. Plant breeders have also improved forage Mg concentrations. A new grass referred to as "Hi-Mag" in the United States is a tall fescue that contains 20% more Mg than other plants in the tested regions (Wood, 1999).

B. Foliar Mg Application Foliar dusting of pastures with fine calcined magnesite (MgO) before or during tetany prone periods has proved effective, provided it is applied at not less than 17 kgjha at not more than IO-day intervals (Rogers, 1979). Foliar application is more rapid and more effective in increasing the level of plant Mg than fertilization and, therefore, prevents tetany in the majority of the herd if applied when early signs of tetany are observed. A disadvantage of foliar Mg application is that the element is easily washed off by rain. To help keep rain from washing the MgO off the forage, a water slurry of 10% MgO and 1.5% bentonite can be applied to the grass with a suspension fertilizer applicator. Neither fertilization nor foliar application of Mg are the methods of choice where forage yields are low under extensive grazing conditions. particularly in developing countries, because of unfavorable cost-benefit relationships.

C. Oral Magnesium Supplementation For calves that are being fed concentrates, provision of 50 g of MgOjday in 300 to 400 g of concentrate mixture is adequate. Other nutritionists have recommended from 10 to 20 g of Mg daily per head of mature cattle. In New Zealand, the method of choice is to give 10 g Mg as magnesium chloride daily at milking time (Underwood and Suttle, 1999). Unlike common salt (NaC\), most Mg salts are quite unpalatable. Supplemental Mg, as part of a concentrate mixture, is the best way of ensuring adequate intake by ruminants. When concentrates are not fed, freechoice Mg feeding is recommended. The provision of special high-Mg mineral blocks or mineral salt mixtures on pasture was more effective in raising blood Mg levels quickly after the initial drop than was the Mg fertilization treatment (Reid et al., 1976). Various combinations of MgO with salt, protein supplements, molasses, other concentrate ingredients, and other feeds have been used to obtain optimal Mg intakes (Miller, 1979). From West Virginia average consumption of Mg by beef cows given a free-choice mixture of 40% salt, 40% dicalcium phosphate, and 20% MgO ranged from 1.3 to 4.2 g/head/ day (Reid et al., 1976). This compared to an intake level of 5 to 109 Mg from a similar mixture containing 20% dried molasses, or 4.1 to 8.8 g Mg from commercial molasses -- MgO blocks (15% Mg). Several relatively successful free-choice consumption formulas of both liquid and dry supplements are as follows: (1) MgO plus molasses at a ratio of I: 1; (2) 97% molasses plus 3% MgCI2 (often with urea and a source of P); (3) equal parts of MgO, salt, bonemeal, and grain; and (4) a 1:1 ratio of salt and MgO. In the southeastern United States, a complete mineral mixture with 25% MgO (14% Mg)

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has been effective in preventing grass tetany in beef cattle (Cunha, 1973). Licking wheels or licking belts are sometimes used to slowly dispense MgO or MgS0 4 in molasses. An oral Mg supplement is of value only during seasonal occurrences of grass tetany (Aleroft, 1961). Unfortunately, many commercial Mg-containing, freechoice mineral supplements are often of little value because (1) they contain inadequate quantities of Mg to protect against tetany during susceptible periods, and (2) provision of such supplements to normal animals during nonsusceptible periods is useless as a prophylactic measure, since additional Mg will not provide a depot of readily available Mg for emergency use. Some producers feed Mg supplements about a month before the Mg tetany season, to decrease the amount of Mg needed daily during the susceptible period. The success of cobalt (Co) bullets in providing supplemental Co led to the development of Mg alloy bullets. Different size bullets are available for cattle and sheep. Bullets are presumably retained by the ruminoreticular fold and release a limited amount of Mg (see Chapter 19). For cattle, Ritchie and Hemingway (1968) used bullets 3 inches in length, one inch in diameter and weighing approximately 100 g. These were composed of a metal Mg alloy containing 86% Mg, 12% AI, and 2% Cu. These were designed to release about I g Mg per day for about 50 days. The three treatment levels used by these workers were zero, two, and four bullets per cow. There were 16 cases of tetany (six deaths) from 169 untreated cows, and no tetany from those treated with two or four bullets. Egan (1969) reported that Mg alloy bullets controlled an outbreak of hypomagnesemia in ewes. Some studies have shown Mg bullets ineffective in preventing grass tetany (Kemp and Todd, 1970; Stuedemann et al., 1984). The main disadvantage of Mg bullets is that often the daily Mg released is insufficient. Also, some bullets are regurgitated, and bullet decomposition is variable (Fontenot, 1980).

D. Magnesium Supplemented in Water Addition of a soluble Mg salt to water has been successful in both increasing blood Mg and preventing grass tetany (Rogers and Poole, 1976). Magnesium sulfate has been used. Some diarrhea may occur, but this has not been a problem. Magnesium acetate or magnesium chloride may be used instead of magnesium sulfate. For this treatment to be effective, the drinking trough must be the only source of water. In addition to supplementation, some management procedures lessen the risk of grass tetany. Animals should be adapted slowly to tetany causing pastures in the spring; as an example, feed animals hay before turning them out on new pastures. Pastures that are most likely to cause grass tetany should be available to steers and dry stock versus lactating animals. Feed more legume hay and high-legume pastures to milking cows and cows nursing calves, since legumes are higher in Mg than are grasses. Supplemental Mg salts are frequently used orally as saline purgatives (cathartics). The main forms of Mg used as laxatives are MgS04 (Epsom salts) and Mg(OHh

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(milk of magnesia). Magnesium sulfate has been used externally to give relief to patients suffering from joint problems. Magnesium is also given to dairy and beef cattle as a buffer. Feeding large amounts of concentrate with small amounts of forage, or with forages that have been finely chopped, will decrease a cow's saliva output and increase the acid load on lactating dairy cows and beef cattle on finishing diets. Feeding dairy cows limited roughage decreases the percentage of milk fat. Supplemental MgO or sodium bicarbonate increases milk fat. Supplementing with either 0.36 kg sodium bicarbonate or 0.18 kg MgO per day prevented depression in milk fat (Emery et al., 1965).

Supplementation of Mg to cattle has been shown to reduce calving difficulties (Villalba, 1999). Administration of two subcutaneous injections (10 ml magnesium gluconate) of Mg during the last 45 days of pregnancy significantly reduced difficult calvings. Recent information has shown that Mg supplementation may have beneficial effects on meat quality. Stress before slaughter can lead to pale, soft, and exudative (PSE) pork by stimulating the rate of immediate postmortem acidification. Magnesium supplementation prior to slaughter resulted in calmer pigs with significantly improved meat quality and reduced incidence of PSE meat (D'Souza et al., 1998; Apple et al., 2000). Increasing dietary Mg levels beyond current recommendations increased marbling scores in cattle fed fat-supplemented diets (Ramirez and Zinn, 2000). A number of sources of Mg salts are available for dietary supplementation. The effectiveness of different chemical forms of the element have mostly been compared in ruminants (Henry and Benz, 1995). Magnesium phosphate, a calciummagnesium-phosphate and magnesium-ammonium-phosphate are all satisfactory sources. For sheep the oxide and hydroxide forms of Mg had lower bioavailability than magnesium sulfate (Henry and Benz, 1995). Previously, Ammerman et al. (1972) found that the biological availabilities of Mg supplements in reagent-grade magnesium carbonate, magnesium oxide, and magnesium sulfate were 43.8, 50.9, and 57.6°;{., respectively (see Chapter 19 of this volume). Storry and Rook (1963) studied Mg availability from various salts by measuring increase in urinary Mg excretion when the salts were fed to nonlactating dairy cows. Availability was highest for the citrate form, intermediate for the oxide, lactate, acetate, and nitrate forms, and lowest for sulfate, silicate, and chloride forms. Availability (apparent absorption) of Mg for cattle was much higher for MgO than for dolomitic limestone, 51.1 versus 14.3%, respectively (Gerken and Fontenot, 1967). Considering cost per unit and biological availability, magnesium oxide generally is the best form of Mg for supplementation (Wilkinson and Stuedemann, 1979). However, if supplementation is in water, more soluble forms of Mg including sulfate, chloride, and acetate must be used. Physiological state of animals, source, particle size, and processing of the supplement all have been shown to influence Mg availability from MgO. Magnesium oxide appeared to have higher Mg bioavailability than magnesium sulfate for pre-calving cows, but Mg bioavailability was not different postcalving

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(Grings and Males, 1988). For steers fed MgO, a smaller particle size was more available (Noller et al., 1987). Availability of Mg varies for different sources of MgO (Van Ravenswaay et al., 1989), and processing temperature, both inadequate and excessive, affect Mg availability (Beede and O'Connor, 1986). Magnesium deficiency may be fairly common in acutely or chronically ill patients. Inadequate intake or provision of Mg is associated with alcoholism, protein-calorie malnutrition (usually with infection), and incorrectly formulated parenteral preparations (Shils, 1996). Magnesium loss with the urine is increased by alcohol and by various diuretic medicines. Disease conditions requiring Mg supplementation include malabsorption disorders, renal tubular dysfunction, endocrine disorders (e.g., hyperaldosteronism and hyperparathyroidism), and genetic disorders. Stress, whether physical (i.e., exertion, heat, cold, traumaaccidental, or surgical burns), or emotional (i.e., pain, anxiety, excitement, depression), and dyspnea as in asthma increases need for Mg (Seelig, 1994). Genetic differences in Mg utilization may account for differences in VUlnerability to Mg deficiency and differences in body responses to stress. High blood pressure is a major risk factor for stroke. Potassium, Mg and fiber are modulators of stroke (Suter, 1999). The best strategy to achieve a high intake of Mg, K, and fiber is a diet rich in fruits and vegetables.

X. TOXICITY Magnesium toxicosis due to ingestion of natural feedstuffs has not been reported and does not appear likely, but would be most likely to occur from using excess levels of supplementary Mg (NRC, 1980). Certain levels of Ca and P in the diet protect the animals from toxicosis (Nugara and Edwards, 1963), as does K in ruminant diets (Fontenot, 1980). Clinical signs of Mg toxicosis for various species are lethargy, disturbance in locomotion, diarrhea, lowered feed intake and performance, drowsiness, and death. The tolerance of poultry and swine for high concentrations of dietary Mg is not well defined. With growing chicks, 0.64% Mg depressed growth and increased mortality, while 0.32% had no effect (Nugara and Edwards, 1963). In feeding layers 0.8% Mg, Hess and Britton (1997) described loss in egg production and body weight as a consequence of reduced feed intake. Decline in shell quality was associated with a 30% decrease in plasma Ca and a 100% increase in plasma Mg. Plasma P was increased by up to 80% suggesting that the bird was mobilizing bone in an attempt to meet demands for Ca. The use of dolomitic limestone (i.e., 10% Mg), especially in the diets of laying hens, may decrease egg production, produce eggs with thinner shells, and cause wet droppings. Increasing dietary Mg from 0.16 to 0.22% lowered rate and efficiency of gain in growing or finishing swine when they weighed 20 to 45 kg, but had no effect thereafter (Krider et at., 1975). Cattle and sheep should be able to tolerate 0.5% Mg (NRC, 1980). Oral administration of 0.5% Mg to wethers did not produce toxicosis, but 0.8% or higher resulted in signs of toxicosis. Chester-Jones et at. (1989) reported levels as

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low as 0.6% Mg produced diarrhea in wether lambs and depressed nutrient utilization; at 2.4% Mg the element was elevated in selected tissues. Severe diarrhea was reported in wethers receiving 0.30% magnesium sulfate (P. R. Henry, personal communication). Increasing dietary Mg (2.4 to 4.7%) in steers caused a progressive degeneration of the stratified squamous epithelium of ruminal papillae (ChesterJones et al., 1990). Although 0.5% Mg is a suggested tolerance level for ruminants, about 0.6% supplemental Mg (as MgO) has been used in low-roughage diets to correct milk fat depression without apparent harm, except for occasional diarrhea (Miller et al., 1972). Intensity of diarrhea is closely related to dietary Mg, with reduced feed consumption and gains in evidence for the higher (2 and 4%) Mg concentrations (Gentry et al., 1978). Seventy percent of calves fed 0.6% Mg and 30% of calves at 0.6% Mg plus NaC1 had stones in their kidneys consisting primarily of Ca apatite (Peters son et al., 1988). Christiansen and Webb (1990) reported that the feeding of high levels of MgO (1.5%) reduced intestinal absorption of amino acids in lambs. The presence of high Mg levels in water (about 1%) was reported to cause a weakening effect on humans and livestock in some areas of the United States (Allison, 1930). Generally large oral intakes of Mg are not harmful to people with normal renal function, but impaired renal function resulting in Mg retention is often associated with hypermagnesemia (Shils, 1997). Early symptoms of hypermagnesemia for humans include nausea, vomiting, and hypotension. At the most severe level of hypermagnesemia, respiratory depression, coma, and asystolic cardiac arrest may occur (Mordes and Wacker, 1978). Calcium infusion can counteract Mg toxicity. Avoidance of Mg-containing medications in patients with significant renal disease is recommended unless there is good reason and close monitoring (Shils, 1997).

XI. REFERENCES Aikawa, J. K. (1959). Proc. Soc. Exp. Bioi. Med. 100, 293. Alcock, N., and MacIntyre, I. (1962). C/in. Sci. 22, 185. Alcock, N., and Shils, M. E. (1974). Proc. Soc. Exp. Bioi. Med. 146, 137. Al-Ghamdi, S. M., Cameron, E. c., and Sutton, R. A. (1994). Am. J. Kidney Dis. 24, 737. Alcroft, R. (1954). Vet. Rec. 66, 517. Alcroft, R. (1961). Vet. Rec. 73, 1255. Allison, I. S. (1930). Science 71, 559. Ammerman, C. B., Chicco, C. F., Loggins, P. E., and Arrington, L. R. (1972). J. Anim. Sci. 34, 122. Apple, J. K., Maxwell, C. V., deRodas, B., Watson, H. B., and Johnson, Z. B. (2000). J. Anim. Sci. 78, 2135. Beede, D. K., and O'Connor, M. A. (1986). In "Proc. Florida Nutriton Conference" p. 191. Daytona Beach, FL. Bell, M. c., and Oluokum, J. A. (1977). Tenn. Farm Home Sci. Prog. Rep. 104,22. Berger, L. L. (1992). Salt Trace Minerals 24, 12. (Salt Institute, Alexandria, VA). Bird, F. H. (1949). J. Nutr. 39, 13. Blaxter, K. L., and McGill, R. F. (1956). Vet. Rev. Annot. 2,35. Bohman, V. R., Horn, F. P., Littledike, E. I., Hurst, 1. G., and Griffin, D. (1983). J. Anim. Sci. 57,1364. Bohman, V. R., Lesperance, A. L., Harding, G. D., and Grunes, D. L. (1969). J. Anim. Sci. 29,99. Boling, J. A. (1982). Anim. Nutr. Health 37, 20.

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Brink, E. J., and Beynen, A. C. (1992). Prog, Food Nutr. Sci. 16, 125. Brink, E. J., Dekker, P. R., VanBeresteijn, E. C., and Beynen, A. C. (1992). Br. J. Nutr. 68, 271. Brody, T. (1999). In "Nutritional Biochemistry," 2nd Ed., p. 794. Academic Press, San Diego. Chausow, D. G., Forbes, R. M., Czarnecki, G. L., and Corbin, J. E. (1985). Proc. Am. Soc. Anim. Sci. 295 (Abstr.). Chester-Jones, H., Fontenot, J. P., and Veit, H. P. (1990). J. Anim. Sci. 68,4400. Chester-Jones, H., Fontenot, J. P., Veit, H. P., and Webb, K. E., Jr. (1989). J. Anim. Sci. 67, 1070. Chicco, C. F., Ammerman, C. B., Hillis, W. G., and Arrington, L. R. (1972). Am. J. Physiol. 222, 1496. Chicco, C. F., Ammerman, C. B., and Loggins, P. E. (1973). J. Dairy Sci. 56, 822. Christensen, D. A., Nesheim, M. c., Hutt, F. B., and Cole, R. K. (1964). Poult. Sci. 43, 1307. Christensen, M. L., and Webb, K. E., Jr. (1990). J. Anim. Sci. 68,2095. Coudray, C; Feillet-Coudray, C; Grizard, D., Tressol, J. C, Gueux, E., and Rayssiquier, Y. (2002). J. Nutr. 132, 2043. Cowey, C. B., Know, D., Adron, J. W., Geroge, S., and Pirie, B. (1977). Br. J. Nutr. 38, 127. Crookshank, H. R., and Sims, F. H. (1955). J. Anim. Sci. 14,964. Cunha, T. J. (1973). Feedstuffs 45(20),27. Dalley, D. E., Isherwood, P., Sykes, A. R., and Robson, A. B. (1997). J. Agric. Sci., Cambridge 129, 107. Dennis, E. J. (1971). Fert. Solutions 15, 44. Dishington, J. W., and Tollersrud, S. (1967). Acta Vet. Scand. 8, 19. DRI (Dietary Reference Intakes). (2001). Panel on Micronutrients of Food and Nutrition Board. National Academy Press, Washington, D.C. D'Souza, D. N., Warner, R. D., Leury, B. J., and Dunshea, F. R. (1998). J. Anim. Sci. 76, 104. Dugmore, T. J., Lesch, S. F., and Walsh, K. P. (1987). S. Afr. J. Anim. Sci. 17(4), 183. Egan, D. A. (1969). Irish Vet. J. 23, 8. Emanuele, S. M., Staples, C. R., and Wilcox, C. J. (1991). J. Anim. Sci. 69, 801. Emery, R. S. (1976). In "Symposium: Magnesium in Ruminant Nutrition," p. I. Israel Chemicals Ltd., Tel Aviv, Israel. Emery. R. S., Brown, L. D., and Bell, J. W. (1965). J. Dairy Sci. 48, 1647. Fontenot, J. P. (1980). "NFIA Literature Review on Magnesium in Animal Nutrition." National Feed Ingredients Association, West Des Moines, Iowa. Fontenot, J. P., Allen, V. G., Bunce, G. E., and Goff, J. P. (1989). J. Anim. Sci. 67, 3445. Fontenot, J. P., Bunce, G. E., Webb, K. E., and Allen, V. G. (1983). "Role of Magnesium in Animal Nutrition," Virginia Polytechnic Institute and State University, Blacksburg, VA. Ford, E. S. (1999). Int. J. Epidemiol. 28,645. Gentry, R. P.. Miller, W. J., Pugh, D. G., Neathery, M. W., and Bynum, J. B. (1978). J. Dairy Sci. 61, 1750. Gerken, H. J., Jr., and Fontenot, J. P. (1967). J. Anim. Sci. 27, 1404. Giduck, S. A., and Fontenot, J. P. (1987). J. Anim. Sci. 65, 1667. Grace, N. D., and MacRae, J. C. (1972). Brit. J. Nutr. 27, 51. Greene, L. W., Baker, J. F., and Hardt, P. F. (1989). J. Anim. Sci. 67, 3463. Greene, L. W., Soils, J. c., Byers, F. M., and Schelling, G. T. (1986). J. Anim. Sci. 63, 189. Grings, E. E., and Males, J. R. (1988). J. Anim. Sci. 66, 566. Grunes, D. L., and Mayland, H. F. (1984). "Controlling Grass Tetany." Agricultural Research Service and Extension Service, United States Department of Agriculture Leaflet No. 561, Washington, D.C. Grunes, D. L., Stout, P. R., and Brownell, J. R. (1970). Adv. Agron. 22, 331. Guenter, W., and Sell, J. L. (1973). J. Nutr. 103,875. Guenter, W., and Sell, J. L. (1974). J. Nutr. 104, 1446. Hardwich, L. L., Jones, M. R., and Brautbar, N. (1991). J. Nutr. 121, 13. Hajj, R. N., and Sell, J. L. (1969). J. Nutr. 97,441. Hakansson, J. (1975). Swedish J. Agric. Res. 5, 145. Harrington, D. D. (1974). Am. J. Vet. Res. 35, 503. Harrington, D. D. (1975). Br. J. Nutr. 34, 45. Heaton, F. W. (1960). Biochem. J. 77, IP (Abstr.). Henry, P. R., and Benz, S. A. (1995). In "Bioavailability of Nutrients for Animals" (C. B. Ammerman, D. H. Baker, and A. J. Lewis, eds.), p. 239. Academic Press, San Diego. Herd, R. P. (1966). Aust. Vet. J. 42, 160. Hess, J. B., and Britton, W. M. (1997). Poult. Sci. 76, 703. Kahil, M. E., Parrish, J. E., Simons, E. L., and Brown, H. (1966). Diabetes 15, 734. Karbach, U. (1989). Gastroenterology 96, 1282.

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Kemp, A. (1983). In "Role of Magnesium in Animal Nutrition" (J. P. Fontenot, G. E. Bunce, K. E. Webb, Jr., and V. G. Allen, eds.), p. 143.Virginia Polytechnic Inst. and State Univ., Blacksburg, VA. Kemp, A., Deijs, W. B., Hemkes, O. J., and Van Es, A. J. H. (1961). Neth. J. Agric. Sci. 9, 134. Kemp, A., Deijs, W. B., and Kluvers, E. (1966). Neth. J. Agric. Sci. 14,290. Kemp, A., and Todd, J. R. (1970). Vet. Rec. 86,463. Krider, J. L., Albright, J. L., Plumlee, M. P., Conrad, J. H., Sinclair, C. L., Underwood, L., Jones, R. G., and Harrington, R. B. (1975). J. Anim. Sci. 40, 1027. Kruse, H. D., Orent, E. R., and McCollum, E. V. (1932). J. Bioi. Chem. %, 519. Kunkel, H. 0., and Pearson, P. B. (1948). J. Nutr. 36, 657. Larvor, P. (1983). In "Role of Magnesium in Animal Nutrition," (J. P. Fontenot, G. E. Bunce, K. E. Webb, Jr., and V. G. Allen, eds.), p. 81. Virginia Polytechnic Inst. and State Univ., Blacksburg, VA. Laurant, P., Daile, M., Berthelot, A., and Rayssiguier, Y. (1999). Br. J. Nutr. 82, 243. Laurant, P., Hayoz, D., Brunner, H., and Berthelot, A. (2000). Br. J. Nutr. 84, 757. Laurenz, J. C.; Green, L. W., Byers, F. M., and Schelling, G. T. (1988). J. Anim. Sci. 66,463 (Abstr.). Leeson, S., and Summers, J. D. (2001). In "Nutrition of the Chicken." University Books, Guelph, Ontario, Canada. Littledike, E. T., Wittam, J. E., and Jenkins, T. G. (1995). J. Anim. Sci. 73, 2113. Lukaski, H. C., and Nielsen, F. H. (2002). J. Nutr. 132, 930. Mahan, D. c., and Shields, R. G. (1998). J. Anim. Sci. 76, 506. Mahoney, C. P., Alster, F. A., and Carew, L. B. (1992). Poult. Sci. 71, 1669. Martens, H., and Rayssiguier, Y. (1980). In "Digestive Physiology and Metabolism in Ruminants" (Y. Ruckebusch and P. Thivend, eds.), p. 447. AVI Publishing, Westport, CT. Mayland, H. F., and Grunes, D. L. (1979). In "Proc, Grass Tetany" (M. Stelly, ed.), p. 123. American Society of Agronomy, Madison, WI. Mayland, H. F., Grunes, D. L., and Lazar, V. A. (1976). Agron. J. 68, 665. Mayo, R. H., Plumlee, M. P., and Beeson, W. M. (1959). J. Anim. Sci. 18.264. McAleese, D. M., Bell, M. C., and Forbes, R. M. (1961). J. Nutr. 74, 505. McCoy, M. A., Young, P. B., Hudson, A. J., Davison, G., and Kennedy, D. G. (2000). Res. Vel. Sci. 69, 301. McDowell, L. R. (1985). "Nutrition of Grazing Ruminants in Warm Climates." Academic Press, New York. McDowell, L. R., Conrad, J. H., Thomas, J. E., Harris, L. E., and Fick, K. R. (1977). Trop. Anim. Prod. 2,273. Meyer, H. (1976). In "Symposium: Magnesium in Ruminant Nutrition" p. 35. Israel Chemicals Ltd., Tel Aviv, Israel. Milia, P. J., Aggett, P. J., Wolff, O. H., and Harries, J. T. (1979). Gut 20, 1028. Miller, E. R., Ullrey, D. E., Zutaut, C. L., Baltzer, B. V., Schmidt, D. A., Hoefer, J. A., and Luecke, R. W. (1965). J. Nutr. 85. 13. Miller, K. B., Caton, J. S., Schafer, D. M., Smith, D. J., and Finley, J. W. (2000). J. NUlr. 130,2032. Miller, W. J. (1979). "Dairy Cattle Feeding and Nutrition." Academic Press, New York. Miller, W. L Britton, W. M., and Ansari, M. S. (1972). In "Magnesium in the Environment" (J. B. Jones, Jr., M. C. Blount, and S. R. Wilkinson, eds.). Taylor County Publishing Co., Reynolds, GA. Montalvo, M. I., Viega, J. V., McDowell, L. R., Ocumpaugh, W. R., and Mott, G. O. (1987). Nutr. Rev. Int. 35, 157. Mordes, J. P., and Wacker, W. E. (1978). Pharmacol. Rev. 29, 273. Mosley, G., and Jones, D. I. H. (1974). J. Agric. Sci. 83, 37. NCMN (Netherlands Committee on Mineral Nutrition). (1973). "Tracing Mineral Disorders in Dairy Cattle." Centre for Agricultural Publishing, Wageningen, The Netherlands. Noller, C. H., Wheeler, L. J., and Patterson, J. A. (1987). J. Dairy Sci. 70(Suppi. 1),200 (Abstr.). NRC. (1980). "Mineral Tolerance of Domestic Animals." National Academy of Sciences-National Research Council, Washington, D.C. NRC. (l982b). "United States-Canadian Tables of Feed Composition" 3rd Ed. National Academy of Sciences-National Research Council, Washington, D.C. NRC. "Nutrient Requirements of Domestic Animals." National Academy of Sciences-National Research Council, Washington, D.C. (1977). Nutrient Requirements of Rabbits, 2nd Ed. (1978). Nutrient Requirements of Nonhuman Primates. (I 982a). Nutrient Requirements of Mink and Foxes. (l985a). Nutrient Requirements of Dogs, 2nd Ed. (l985b). Nutrient Requirements of Sheep, 5th Ed.

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(1986). Nutrient Requirements of Cats, 3rd Ed. (1989). Nutrient Requirements of Horses, 5th Ed. (1993). Nutrient Requirements of Fish. (1994). Nutrient Requirements of Poultry, 9th Ed. (1995). Nutrient Requirements of Laboratory Animals. (1996). Nutrient Requirements of Beef Cattle, 7th Ed. (1998). Nutrient Requirements of Swine, 10th Ed. (2001). Nutrient Requirements of Dairy Cattle, 7th Ed. Nugara, D., and Edwards, H. M. (1963). J. Nutr. SO, 181. Osborne, T. B., and Mendel, L. B. (1918). J. Bioi. Chem. 34, 131. Peeler, H. T. (1972). J. Anim. Sci. 35,695. Perdomo, J. T., Shirley, R. L., and Chicco, C. F. (1977). J. Anim. Sci. 45, 1114. Petersson, K. H., Warner, R. G., Kallfelz, F. A., and Crosetti, C. F. (1988). J. Dairy Sci. 71, 3369. Phillipson, A. T., and Storry, J. E. (1965). J. Physiol. 181, 130. Phipps, D. A. (1976). "Metals and Metabolism." Clarendon Press, Oxford, England. Powley, G., and Johnson, C. L. (1977). J. Agric. Sci. 88,477. Ram, L., Schonewille, J. T, Martens, H., Van't Klooster, A. T., and Beynen, A. C. (1998). J. Dairy Sci. 81, 2485. Ramirez, J. E., Alvarez, E. G., Montano, M., Shen, Y., and Zinn, R. A. (1998). J. Anim. Sci. 76,1753. Ramirez, 1. E., and Zinn, R. A. (2000). J. Anim. Sci. 78, 2072. Reffett, J. K., and Boling, J. A. (1985). J. Anim. Sci. 61, 1004. Reid, R. L., Jung, G. A., and Gross, C. F. (1976). In "Proc. Hill Lands International Symposium" p. 640. West Virginia University, Morgantown, WV. Reis, M. A., Reyes, F. G. R., Saad, M. A. J., and Velloso, L. A. (2000). J. Nutr. 130, 133. Rendig, V. V., and Grunes, D. L. (1979). "Symposium: Grass Tetany." Am. Soc. Agron., Madison, WI. Ritchie, N. S., and Hemingway, R. G. (1968). Vet. Rec. 82, 87. Robson, A. B., Field, A. Sykes, A. R., and McKinnon, A. E. (1997). Brit. J. Nutr. 78, 975. Rock, E., Astier, C., Lab, c., Vignon, X., Gueux, E., Motta, c.. and Rayssiguier, Y. (1995). J. Nutr. 125, 1205. Rogers, P. A. M. (1979). Irish Vet. J. 33, 115. Rogers, P. A. M., and Poole, D. B. R. (1976). Irish Vet. J. 30, 129. Rook, J. A. F., and Storry, J. E. (1962). Nutr. Abstr. Rev. 32, 1055. Rosero, 0., Tucker, R. E., Mitchell, G. E., Schelling, G. T., and Templeton, W. C. (1975). J. Anim. Sci. 41,415 (Abstr.). Rude, R. K., Kirchen, M. E., Gruber, H. E., Stasky, A. A., and Meyer, M. H. (1998). Miner. Electrolyte Metab. 24, 314. Salih, Y., McDowell, L. R., Hentges, J. P., Mason, R. M., Jr., and Wilcox, C. J. (1987). J. Dairy Sci. 70, 608. Schonewille, J. T., Van't Klooster, A. T, Wouterse, H., and Beynen, A. C. (1999a). J. Dairy Sci. 82, 1824. Schonewille, J. T, Beynen, A. C., Van't Klooster, A. T, Wouterse, H., and Ram, L. (1999b). J. Nutr. 129,2043. Seelig, M. S. (1994). J. Am. Call. Nutr. 13,429. Sell, J. L. (1980). "NFIA Literature Review on Magnesium in Animal Nutrition." National Fertilizer Ingredients Association, West Des Moines, IA. Sell, J. L., Hajj, R., Cox, A., and Guenter, W. (1967). Br. Poult. Sci. 8, 55. Shils, M. E. (1996). In "Present Knowledge in Nutrition" (E. E. Ziegler, and L. J. Filer, eds.) ILSI Press, Washington, D.C. Shils, M. E. (1997). In "Handbook of Nutritionally Essential Mineral Elements" (B. L. O'Dell and R. A. Sunde, eds.) Marcel Dekker, Inc., New York. Shockey, W. L., Conrad, H. R., and Reid, R. L. (1984). J. Dairy Sci. 67, 2594. Sjollema, B. (1930). Vet. Rec. 10,425. Standish, J. F., Ammerman, C. 8., Palmer, A. Z., and Simpson, C. F. (1971). J. Anim. Sci. 33,17. Starnes, S. R., Spears, J. W., Froetschel, M. A., and Croom, W. J. (1984). J. Nutr. 114,518. Storry, J. E. (1961). Nature 190,1197. Storry, J. E., and Rook, J. A. F. (1963). J. Agric. Sci. 61, 167. Stuedemann, J. A., Wilkinson, S. R., and Lowrey, R. S. (1984). Am. J. Vet. Res. 45, 698. Suter, P. M. (1999). Nutr. Rev. 57, 84. Suttle, N. F. (1987). In "The Nutrition of Herbivores" (J. B. Hacker and J. H. Ternouth, eds.), p. 333. Academic Press, Sydney.

c.

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Tufts, E. V., and Greenberg, D. M. (1938). J. Bioi. Chern. 122,715. Underwood, E. J. (1981). "The Mineral Nutrition of Livestock." Commonwealth Agricultural Bureaux, London, UK. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" (3rd Ed.), Midlothian, UK. Valdivia, R., Ammerman, C. B., Wilcox, C. J., and Henry, P. R. (1978). J. Anim. Sci. 47,1351. Van Ravenswaay, R. 0., Henry, P. R., Ammerman, C. B., and Littell, R. C. (1989). J. Dairy Sci. 72, 2968. Van Reen, R., and Pearson, P. B. (1953). J. Nutr. 51, 191. Verbeek, M. J., Van den Berg, G. J., Lemmens, A. G., and Beynen, A. C. (1993). J. Nutr. 123, 1880. Villalba, M. del Pilar (1999). In "Eficacia de la Suplementaci6n con Magnesio Parenteral en la Reducci6n de Partos Distocicos en Bovinos" Thesis, Faculty of Veterinary Sciences, Tandil, Argentina. Vitale, J. J., Velez, H., Guzman, c., and Correa, P. (1963). Circ. Res. 12, 642. Voisin, A. (1963). "Grass Tetany." Charles C. Thomas, Springfield, IL. Wachirapakorn, C., Sykes, A. R., and Robson, A. B. (1996). Proc. N. Z. Soc. Anim. Prod. 56, 133. Wacker, W. E. C. (1969). Ann. N. Y. Acad. Sci. 162,717. Watson, W. S., Lyon, T. D. B., and Hilditch, T. E. (1980). Metabl. Clin. Exp. 29, 397. Whittemore, C. T., and Manson, J. M. (1995). Vet. Rec. 139,218. Wilkinson, S. R., and Stuedemann, J. A. (1979). In "Symposium Grass Tetany" (V. V. Rending and D. L. Grunes, eds.), p. 93, Am. Soc. Agron., Madison, WI. Wood, M. (1999). Agri. Res. April, 15. Wu, C. L., and Britton, W. M. (1974). Poult. Sci. 53, 1645.

Chapter 6

Sulfur I. INTRODUCTION

Sulfur (S) is one of the more abundant elements in nature; however, a shortage of S-containing amino acids is a worldwide problem in human and animal nutrition. In most animals it represents about 0.15 to 0.25% of the body weight. It is difficult to divorce the dietary requirements of inorganic S from dietary levels of S-amino acids since the supply of tissue sulfate may be derived entirely from S-amino acid catabolism. The greater part of S in animals is present in the two S-containing amino acids, methionine and cysteine, or in the double form of the latter, cystine. Of the many S compounds, all can be synthesized in vivo from the essential amino acid methionine with the exception of S-containing vitamins thiamin and biotin. However, microorganisms in the ruminant digestive tract are capable of synthesizing S-containing amino acids and thiamin and biotin from inorganic S sources. Nonruminant animals have few assimilatory microorganisms so that the major proportion of their S requirement must be in the form of amino acids. Diets for ruminants that are low in protein or that contain a large proportion of the nitrogen (N) requirement as nonprotein N (NPN) (e.g., urea) may be deficient in S. Sulfur is a more critical nutrient for ruminants than nonruminants from the standpoint of potential deficiency. In recent years, S toxicosis has become more common, partly due to its high concentration in many byproduct feeds. The disease conditions in ruminants of polioencephalomalacia (PEM) and copper (Cu) deficiency are both favored by high dietary S intakes. Sulfur nutrition has been reviewed by Muth and Oldfield (1970), Goodrich et al. (1978), NRC (1980), Baker (1987), Griffith (1987), and Underwood and Suttle (1999).

II. mSTORY

Sulfur has been used in several forms since antiquity, but not until recently has there been a real understanding of its biological significance. In 1784, Scheele reported S in proteins. This information was not greatly expanded until the 1930s when the essential amino acid, methionine, was shown to contain one atom of S per 179

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molecule (McCollum, 1956). Most of the nutritional history of S relates more to the requirements and metabolism of the S-amino acids. For ruminant nutrition, Loosli et al. (1949) first demonstrated that ruminal microorganisms of sheep fed urea as the sole nitrogen source were able to synthesize methionine from inorganic S and that the sheep grew and remained in positive nitrogen balance. This was expanded when researchers showed that dietary radiolabeled S as sodium sulfate (Na2S04) was incorporated into cystine and methionine in the rumen and utilized to form milk protein (Block et al., 1951). During the period 1945 to 1954, Dick (1956) noted metabolic interrelationships among Cu, molybdenum (Mo), and inorganic sulfates in ruminants, which resulted in Cu deficiency.

III. CHEMICAL PROPERTIES, DISTRIBUTION, AND USES Sulfur is a solid, nonmetallic element. It has an atomic number of 16 and an atomic weight of 32.064. Sulfur is brittle and has almost no taste. When it is rubbed or melted, it gives off a "rotten egg" odor. It does not dissolve in water but dissolves readily in carbon disulfide. Sulfur ignites at a low temperature and burns very quickly. It burns in air with a pale-blue flame and gives off sulfur dioxide, a colorless gas. When sulfur dioxide is exposed to moist air, it mixes with the moisture in the air and forms sulfurous acid. Both sulfur dioxide and sulfurous acid are constantly being formed in the air in cities that burn large amounts of coal and gas. Large quantities of S are found in the earth's crust, both in a pure state and in combination with other substances. Sulfur occurs in a pure state in volcanic regions. It combines with metals to form valuable metal ores. Gypsum, also called calcium sulfate, is an important mineral that contains S. Sulfur has many commercial uses. Pure S is used to make up a group of valuable substances known as S compounds. These S compounds include sulfuric acid, the sulfite salts, and sulfur dioxide. Sulfur mixes with saltpeter and charcoal to form gunpowder, and is used to some extent in the manufacture of matches. About 80% of the annual production of S is used for production of sulfuric acid, which is used in the manufacture of phosphate fertilizer, purification of gasoline, production of synthetic fibers (e.g., rayon), pesticides, steel processing, bleaching agents for paper pulp, sugars and vegetable oils, preservation of beverages and food, and producing rubber products and synthetic rubber (NRC, 1980). Large amounts of sulfuric acid are also used in storage batteries. Sulfur is used in various medicines, most importantly as sulfa drugs or sulfonamides. These medicines fight bacteria and other organisms and are effective for many diseases including: pneumonia, dysentery, meningitis, blood poisoning, urinary tract infections, and some venereal diseases. Normally, sulfa drugs do not actually kill bacteria. Instead, they prevent the bacteria from multiplying. Many bacteria need a chemical called para-aminobenzoic acid (PABA) to multiply. This acts like a necessary vitamin for these bacteria. The sulfonamide drugs have a chemical structure similar to PABA, but they have S atoms where PABA has

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carbon atoms. Bacteria cannot tell the difference between the two and absorb the sulfa drug rather than the PABA. The S atoms then stop one or more of the growth processes of the bacter~a, and the bacteria cannot multiply.

IV. METABOLISM

The metabolism of S differs markedly between monogastrics and ruminants, and an understanding of this difference is basic to an appreciation of the S cycle and of the nutritional value of S compounds. The major terrestrial source of S is mineral sulfide, which is converted to inorganic sulfate by weathering and to organic S by microbial action in the soil (Young and Maw, 1958). Sulfur enters into metabolic pathways at oxidation levels as sulfate or sulfide; and forms of S must be either oxidized to sulfate or reduced to sulfide before they are utilizable by ruminants. Other naturally occurring forms of S such as thiosulfate, polythionates, polysulfides, and elemental S must be either oxidized to sulfate or reduced to sulfide before they are available for biosynthetic reactions. Biologically, the interconversion of sulfate and sulfide is a reversible process and can be considered as two broad phases: sulfate reduction, the reduction of sulfate to sulfide; and sulfide oxidation, the oxidation of sulfide to sulfate. Sulfate reductions and sulfide oxidation constitutes an overall process termed the "S cycle." It is largely a microbial process; but both animals and plants make some application of the cycle. Because several groups of bacteria utilize sulfate reduction or sulfide oxidation in their energy metabolism, the involvement of inorganic S compounds in the nutrition of microorganisms is more extensive than in the nutrition of either plants or animals. Plants can reduce sulfate to sulfide as indicated by their growth with sulfate as their only source of S. Plants generally reduce only enough sulfate to sulfide to meet nutritional requirements. A limited group of anaerobic sulfate-reducing microbes produces large amounts of sulfide during growth with adequate sulfate available. This has been considered a respiratory sulfate reduction in that the sulfate serves as a terminal acceptor similar to oxygen respiration (Peck, 1970). Mammals are unable to reduce sulfate to sulfide for the biosynthesis of S-containing amino acids and the cofactors such as biotin, thiamin, and coenzyme A. However, mammals are generally capable of incorporating sulfate as such into various lipids, carbohydrates (e.g., mucopolysaccharides), and phenols and metabolizing and incorporating to a limited extent reduced S compounds (Peck, 1970). Inorganic sulfate is taken up by higher plants and converted to organic S in the form of the S-containing amino acids, which in turn serve as an organic S source for both monogastric and ruminant animals. Many bacteria, including the microbial flora of the ruminant, are also able to convert inorganic S to organic S in the form of methionine, cysteine, and cystine and hence for the many functions of S in the body. Monogastric animals have few, if any, intestinal assimilatory bacteria to form organic S from inorganic sources and, therefore, must rely upon the S-amino acid sources for their requirement of organic S.

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The proportion of total S flow into the rumen which is 'captured' as ruminal microbial protein varies widely and is determined by factors such as the S source (methionine being less degraded than other S-amino acids) (Bird, 1972) and by the coavailability of other substances (chiefly degradable N) (Beever, 1996). Optimal microbial synthesis and S capture occur when fermentable energy, degradable S, N, and phosphorus (P) are supplied at rates which match the synthetic capacity of the ruminal microbial biomass (Underwood and Suttle, 1999). In animals, S absorption occurs in the rumen and small intestine, with substantial amounts absorbed through the ruminal wall (Bray, 1969); absorption by active transport of the inorganic sulfate takes place in the small intestine, especially the ileum (Dziewiatkowski, 1970). Organic forms of S are readily absorbed in the small intestine. The absorption mechanism is very efficient. Morrow et al. (1952) demonstrated that rats excreted, in the urine, 41 to 64% of an oral dose of inorganic sulfate 35S within 8 hours of administration. Humans also rapidly excreted sulfate in the urine. An additional source of S to the animal is S secreted in saliva. In saliva S is a mixture of both inorganic and organic forms, with salivary S higher in cattle than sheep (Underwood and Suttle, 1999). Sulfur is recycled to the rumen, with similarities to the recycling system for the urea-N system. The amount of S recycled in sheep is much less than for cattle (Kennedy et al., 1975). Under grazing conditions particularly with mature grasses rather than with legumes, and possibly under more intensive conditions, recycled S becomes nutritionally very significant (Moir, 1979).

V. RELATIONSHIPS OF SULFUR TO OTHER ELEMENTS Sulfur is closely associated with N and the dietary elements Cu, Mo, and selenium (Se) (see Chapters 8 and 13).

A. Sulfur, Copper, and Molybdenum An interrelationship between S, Cu, and Mo was first reported by Dick and Bull (1945). They reported that dietary Mo decreased liver Cu storage. Dick (1953a) recognized that there was a third factor in alfalfa hay, but not in oat hay, that potentiated the Cu-Mo antagonism, and concluded that the factor was inorganic sulfate. Dick (1953b) also showed that sulfate influenced Mo excretion in the urine and level in the blood. In the presence of S, high intakes of Mo can induce a Cu deficiency due to formation of insoluble Cu-Mo-S complexes (e.g., thiomolybdates) in the digestive tract that reduce the absorption of Cu (Mason, 1986, 1990). Several pathways exist by which Cu-Mo-S interactions mediate Cu deficiency (Dick, 1956; Ryan et al., 1987). Some thiomolybdates are absorbed and impair the metabolism ofCu in the body. Larson et al. (1995) cited research suggesting that Cu-thiomolybdate-albumin

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183

complexes cause Cu metabolism failure in the body. Price et al. (1987) showed that tri- and tetrathiomolybdates were the S-Mo complexes responsible for reducing Cu absorption, while the di- and trithiomolybdates had the greatest effect on Cu metabolism in the body. Sulfur also reduces Cu absorption by the formation of insoluble copper sulfide in the rumen, independent of formation of thiomolybdates. The effect of S alone may be greater than the S-dependent effects of Mo (Underwood and Suttle, 1999). Ruminant animals are much more susceptible to MojCu imbalance than are nonruminant animals. The primary effect probably occurs in the rumen through the involvement of sulfide-generating bacteria and the consequent formation of unavailable compounds such as cupric thiomolybdate. Provision of additional dietary Cu overcomes adverse clinical responses by inhibiting absorption of thiomolybdate or its derivatives. Formation of thiomolybdates also affects the kinetics of S metabolism by affecting sulfide formation and absorption. Thiomolybdates rapidly react with particulate matter and proteins to form complexes that bind Cu strongly, reducing its solubility, decreasing the hydrogen sulfide (H 2S) concentration and thereby the rate of sulfide absorption (Gawthorne et al., 1985). 1.

SULFUR-SELENIUM INTERRELATIONSHIPS

Selenium and S are members of Family VI of the periodic table and they share many physical and chemical properties. Competition between Se and S is due to similarity in chemical structure. Selenoamino acids are molecules with similar chemical structure to the S-containing amino acids (Se is substituted for S) and they compete for reactive sites on enzymes. Schwarz and Foltz (1957) were first to demonstrate an interrelationship between Se and S-containing amino acids in animals. Shrift (1958) reported that Se interfered with enzymes of S metabolism which in some instances was nullified when S metabolites were added to the diet. Methionine and cysteine reduced the toxic effect of their Se analogs. Sulfate was shown to reduce the effectiveness of Se in preventing white muscle disease in lambs by Hintz and Hogue (1964). Schubert et al. (1961) suggested an antagonistic relationship between dietary Se and S after field studies had shown considerable muscular dystrophy in lambs born of ewes fed alfalfa and grass hay high in both Se and S. In a more recent study with sheep, van Ryssen et al. (1998) reported that increasing dietary S from 0.2 to 0.4% reduced hepatic Se. For dairy cattle, dietary S from sulfate reduced Se balance especially when cows were fed diets with less than 0.3 ppm Se (Ivancic and Weiss, 2001). Lane et al. (1979) fed Torula yeast diets to rats with four levels of Se (0.01, 0.06, 0.11, and 0.61 ppm) and three levels ofS (267,3567, and 5267 ppm). When the two high levels of S were fed, 0.11 and 0.61 ppm levels of Se were necessary to obtain maximum glutathione peroxidase (a Se-containing enzyme) activity in small intestinal tissue of rats. Glutathione peroxidase activity was depressed in the tissue when rats were fed corn-soybean meal diets low in Se (0.03 ppm) and supplemented with S. Selenium at 0.11 ppm in the diet was adequate to prevent the

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high levels ofS from depressing the enzyme activity. Van Vleet (1982) reported that ducklings fed over four weeks with a commercial starter mash having "adequate" levels of Se and vitamin E developed a Se deficiency syndrome if the diet was supplemented with 0.5% S. Perdomo et al. (1966) reported that as S in fertilizer was increased, the Se content in clover, millet, and corn decreased. Holstein steers grazing S-fertilized herbage had lower blood glutathione peroxidase activity than those grazing unfertilized herbage (Murphy and Quirke, 1997). Dietary S from sulfate reduced Se balance especially when lactating cows were fed diets with less than 0.3 ppm of Se dry basis (Ivancic and Weiss, 2001). Both the feces and urine are paths of S excretion. However, S is excreted principally in urine. In the urine three forms occur: inorganic sulfate, the principal fraction, which represents the final stage of oxidation of organic S; ethereal S, which is present in complex detoxication products; and neutral S, which occurs as cystine, taurine, thiosulfates, and other compounds. Sulfur excreted via feces, is largely in inorganic forms. Since excreted S arises primarily from protein catabolism, there is a rather constant ratio between S and the N in the urine. There is evidence that the excretion of neutral S is proportional to the basal metabolism.

VI. FUNCfIONS Strictly speaking, S is only an essential nutrient for plants and microbes, because only they can synthesize S-amino acids and hence proteins from degradable inorganic S sources (Underwood and Suttle, 1999). Sulfur is required for the formation of the many S-containing compounds found in essentially all body cells and, therefore, is an essential nutrient. The important body S compounds include the S-containing amino acids, other S-containing molecules, hormones, and vitamins. A. Sulfur Amino Acids Sulfur is an essential element for all animals as S-containing compounds have vital metabolic functions in all living cells. Methionine, cystine, cysteine, homocysteine, cystathionine, and taurine are S-containing amino acids. Liver enzymes are able to produce cystine and cysteine from methionine; but all living animals require methionine. Methionine may be demethylated to form homocysteine; then combined with serine to form cystathionine, which upon cleavage produces cysteine and homoserine. Thus cysteine and cystine are nonessential amino acids, but a large part of the S-amino acid requirement of animals can be met by cystine and cysteine. The S-amino acids play an important function in protein structure due to their incorporation into polypeptide chains. Also, free sulfhydryl groups participate in hydrogen (H) binding, and covalent disulfide bonds between cysteine structures of the protein molecule. Disulfide bonds contribute to the biological activity of

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enzymes and many proteins (Boyer, 1959). Sulfhydryl groups provide sites for H bonding, as well as sites for the attachment of prosthetic groups of enzymes to substrate and for binding of substrates to active sites of enzymes.

B. Other Sulfur-Containing Molecules Hemoglobin, cytochromes, coenzyme A, coenzyme M, lipoic acid, Sadenosylmethionine, glutathione, heparin, penicillin G, metallothionein, and sulfate polysaccharides including chondroitin all contain S. Chondroitin is a key component of cartilage, bone, tendons, and blood vessel walls. Hemoglobin is an oxygen carrier and the cytochromes are used in electron transport. Coenzyme A serves as a carrier of acyl groups in enzymatic reactions of fatty acid oxidation and pyruvate oxidation, and is involved in the acylation of choline to form acetylcholine. Coenzyme M is essential for growth of a strain (Methanobacterium ruminatum) of methane-producing bacteria (McBride and Wolfe, 1971). Coenzyme M is a 2-mercaptoethane-sulfonic acid, i.e., HS-CH rCHrS0 3; and is required for the formation of methane from methylcobalamin (Taylor and Wolfe, 1974). Lipoic acid is a coenzyme involved in the decarboxylation of pyruvic acid and other keto acids. S-adenosylmethionine is a methylating agent in the synthesis of methyl-containing substances such as N-methyl nicotinamide, creatine, choline, epinephrine, anserine, and glycocyamine. Glutathione participates in the maintenance of proper redox potentials in cells. Heparin is a blood anticoagulant and penicillin is an antibiotic. Cysteine-rich molecules, such as metallothionein, playa vital role in protecting animals from excesses of Cu, cadmium (Cd), and zinc (Zn) while others influence Se transport and protect tissues from Se toxicity (Underwood and Suttle, 1999). Dietary S has also been shown to enhance the humoral immune response of goats (Rao et al., 1999).

C. Hormones and Vitamins Sulfur-amino acids are prominent structural features of some hormones. Insulin has disulfide bonds both within and between each of its two-polypeptide chains. Oxytocin has a disulfide linkage between cysteine residues, which helps form an internal ring structure. Sulfur is a part of the vitamins thiamin and biotin (McDowell, 2000). Thiamin pyrophosphate participates as a coenzyme in the decarboxylation of both alpha-ketoglutarate and pyruvate, and in transketo lase reactions. Biotin participates in the metabolism of aspartic acid, in the decarboxylation of oxalacetic, oxalosuccinic and succinic acids, and acts as a carrier for carbon dioxide in carboxylation reactions.

VII. REQUIREMENTS Considerable research has and will continue to be conducted on the S-amino acid requirements of monogastric species, rather than S requirements. Monogastric

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species do not require S, but rather organic S (sulfur-amino acids, thiamin, biotin, etc.) sources in their diets. With the exception of thiamin and biotin, all other organic sources can be derived from methionine. The total absence of inorganic S from the diet may, however, increase the S-amino acid requirement, which suggests that S from the amino acids is used to synthesize other organic compounds containing S. For ruminant species, S requirements need to be established for both the ruminal microbes and the ruminant animal. A. Sulfur Requirement of Ruminal Microbes The microbes of the rumen can incorporate inorganic S into organic compounds. Ruminal microbes utilize inorganic S to make S-containing amino acids, which are incorporated into microbial protein, and following digestion of the microbes in the lower alimentary tract may become a part of the body tissue. Block et al. (1951) infused radioactive 35S into the rumen and measured the activity of the isotope in the protein of milk from ewes and goats. The milk proteins contained 80% of the 35S. Lewis (1954) found that sulfate was reduced to sulfide in the rumen of sheep, and that other oxidized S compounds were also reduced to sulfide. Kulwich et al. (1957) administered radioactive 35S to sheep and found it was absorbed into the blood rapidly and peaked at six hours. Most of the dose was excreted within four days, 31% in the feces and 49% in urine. Labeled S was found in all tissues sampled and much of the isotope was present in cystine and cysteine. Anderson (1956) concluded that sulfate-S, protein-S, and free amino acid-S was reduced to sulfide by ruminal microbes and the sulfide was incorporated into microbial amino acids. Cattle ruminal fluid was incubated in vitro with labeled sulfate and substrates representative of concentrate and forage diets by Emery et al. (1957). They observed that cystine synthesis was twice as rapid as methionine formation and that S incorporation into amino acids was more rapid with the concentrate than the forage substrate. Without an adequate amount of S the ruminal microbes have a decreased ability to function normally, and reduced digestibilities (e.g., cellulose) of feedstuffs and N retention occur (Thomas et al., 1951). The artificial rumen was utilized by Hunt et al. (1954) to show that elemental S, sulfate, cysteine, and methionine stimulated the synthesis of riboflavin and vitamin B 12 • Martin et al. (1964) found that in vitro cellulose digestion was less (1.6%) when ruminal inoculum from cattle fed a low-S diet was used than that which occurred (33.5%) when the inoculum was from cattle fed a S-adequate diet. Whanger and Matrone (1967) observed large amounts of lactate produced in vitro when ruminaI fluid from sheep fed a S-free diet was utilized. They stated that as much as 30% of the lactate was converted to propionate via this pathway by microbes from S-deficient sheep. It appears that the microbes of sheep fed the S-deficient diet did not have the ability to hydrogenate acrylate. All ruminal bacteria require S, but not all bacteria can utilize inorganic S. Emery et al. (1957) reported that only 5 of 10 strains studied utilized significant amounts of inorganic sulfate to synthesize organic S compounds, and only three strains

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incorporated inorganic sulfate into microbial protein when cysteine was present. Hume and Bird (1970) observed that ruminal protein synthesis increased from 82.1 to 86.9 g/day as the S content of the diet was increased from 0.075 to 0.237%. Cystine and sulfate gave similar quantities of microbial protein synthesis. Slyter et al. (1986) reported that when purified diets deficient in S were supplied to cultures in vitro, less methane (3.2 vs 32.6 mmol/I daily) was produced and fewer cellulytic microbes were present than in sheep ruminal fluid supplemented with S. This was interpreted to be due to recycling of S to the rumen where it is efficiently scavenged by ruminal bacteria. Evans and Davis (1966) observed the optimum S level to be 0.29% for cellulose digestion in the rumen of fistulated steers. Unfortunately, beef cattle requirements have been determined with high-starch diets, resulting in unreasonably low recommended S requirements (Bull, 1979).

B. Dietary Sulfur Requirement of Ruminant Animals Determining S requirements of ruminants has involved supplementing diets with methionine, cystine, sulfate salts, or elemental S in balance trials, feedlot trials, or by radioisotope studies. Loosli and Harris (1945) increased the growth rate of lambs fed a diet containing 6.55% crude protein by raising the level to 10.28% with urea plus sulfate, or urea plus methionine. Lofgreen et al. (1947) increased N retention when 0.2% methionine was added to a 10% crude protein basal diet in which 40% of the N was supplied from urea. Nitrogen retention and wool production were improved in sheep by supplementing their diets with methionine or with methionine and cystine (McLaren et al., 1965). Digestion of cellulose decreased when steers were fed purified diets deficient in S (Martin et 01., 1964). Bull and Vandersall (1973) demonstrated that sodium (Na) sulfate, calcium (Ca) sulfate, DL-methionine and methionine hydroxy analog (MHA) were equivalent in promoting cellulose digestion in vitro, and concluded that the optimum level of S was from 0.16 to 0.24% for ruminants. Yearling wethers fed 0.155% total S in diets had good rates of growth (Rendig and Weir, 1957). A sheep diet that contained II % protein was calculated to require 0.176% S by Moir et. al. (1968) based on a need for a N:S ratio of 10:I. Chalupa et al. (1973) found better weight gains by Angus steers when sodium sulfate or elemental S were added to increase S from 0.05 to 0.13%; and feeding these forms of S in steer diets up to about 0.6% had no deleterious effects. However, Bouchard and Conrad (1973) found that when dietary S as calcium sulfate exceeded 0.3% dairy cows had reduced feed intake; and Johnson et 01. (1968) reported that the addition of 0.5% S as calcium sulfate decreased gains of lambs. Bouchard and Conrad (1973) found that dry matter intake and dry matter digestibilities were improved when basal diets with 0.10 or 0.06% S were supplemented to provide 0.15 or 0.18%, respectively. It was estimated by these workers that diets with 0.12% S would result in S balance in cows producing between 9 and 37 kg milk daily. Johnson et 01. (1971) calculated endogenous fecal loss of S to be 0.15 g/Iamb/day and an equal amount of endogenous S was lost in

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the urine daily. The maintenance requirement for S, determined from metabolic urinary and fecal losses and content of wool growth, was calculated to be 0.48 g of retainable S daily. This amount of S would be supplied by 1.79, 0.86, or 0.69 g of S as elemental S, sodium sulfate or methionine, respectively. Lambs gaining 100 g daily with 0.15% dietary S would require 0.63 g of retain able S. Sulfur requirements for grazing ruminants appear to be between 0.10 and 0.32% (McDowell, 1985). Sulfur requirements according to ruminant and horse NRC publications are presented in Table 6.1 and range from 0.14 to 0.32%.

c. Dietary Nitrogen:Sulfur (N:S) Ratios

in Relation to Sulfur Requirements

Ruminants through their ruminal microbes may satisfy their metabolic requirements for dietary Nand S compounds from cellulosic materials that are inadequate nutritionally for monogastrics. Products of ruminants such as beef, lamb, and milk have N:S ratios of about 15:I, while wool has a ratio of 5:I. Plants have a ratio of approximately 13:I. Loosli (1952) suggested that S requirements be expressed relative to those for N, and recommended a N:S ratio of 15:I based on the relatively constant ratio observed in animal tissues. For maximal growth, dry matter intake and feed efficiencyof Alpine and Angora goats, the S requirement was 0.21 to 0.24% and the N:S ratio was 9.5 to 11.1(Qi et al., 1992). The N:S ratio may have little use in some situations such as grasses with low S bioavailability or heat damaged forages (Goodrich and Garrett, 1986). Moir et al. (1968) working with sheep and utilizing S additions that narrowed the N:S ratio from 12:1 for the basal diet to 9.5:1 for supplemented diets observed an improvement in N retention from 28.8 to 36.0%. As the N:S ratio of recycled Nand S in the rumen of sheep is between 70 and 80:I, Moir et al. (1968) suggested that there are instances where animals will not benefit from N recycling due to the concurrent S deficiency; and supplementation with nonprotein N is of no value unless additional S is provided. The N:S ratio of a feedstuff protein is not important in itself if the protein is totally degraded in the rumen. If it is completely degraded, then the ratio of N to total S is the critical value. Bouchard and Conrad (1973) reported that lactating dairy cows did not

TABLE 6.1

Dietary Sulfur Requirements" Animal species

Requirements (%)

Reference

0.15 0.20 0.14-0.18 0.18--0.26 0.16--n.32 0.15

NRC (1996) NRC (2001) NRC (1985)

Beef cattle Dairy cattle Sheep, mature ewes young ewes Goats Horses "Expressed as per unit animal feed, OM.

NRC (1981) NRC (1989)

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respond when N:S ratios were changed from 14:1 to 8.2:1, 12.0:1 to 6.6:1, 11.0:1 to 6.9: 1, and 14:1 to 8.4: 1 in several trials.

Sulfur requirements are higher and S deficiencies are more prevalent in wooltype sheep and Angora goats (Qi, 1988, 1992). Cattle needed less S than sheep to digest a urea-supplemented, low quality diet (Kennedy and Siebert, 1972). Both protein and S requirements are high for mohair and wool growth, because efficiency of use of microbial (or undegraded dietary) protein for fleece growth is only 26% compared with around 80% for most other purposes (Underwood and Suttle, 1999). Also, Merino flocks selected for high fleece production show greater responses to supplements of S-amino acids than those selected for low fleece weight (Williams et al., 1972; Qi et al., 1994). Anaerobic fungi may be particularly sensitive to S concentration in the rumen; additional S increased the fungal ruminaI concentration and S-amino acids leaving the rumen (Weston et aI., 1998).

YIn. NATURAL SOURCES

There is a wide variation of S concentrations in feeds (Appendix Table II). Sulfur concentrations in pastures and conserved forages can vary widely from less than 0.05 to higher than 0.50% (McDowell, 1985; Underwood and Suttle, 1999). Various types of forages have different nutrient S requirements for optimum yields. The critical S content of sugar cane is 0.04% or less (Stanford and Jordan, 1966) compared to approximately 0.22% for alfalfa (Pumphrey and Moore, 1965). The wide variations in S content of plants are due largely to the amount of S in plant protein (Allaway and Thompson, 1966). Genetic factors determine the amino acid composition of plant proteins. It appears that sulfate S does not build up in the plant to a significant extent until the S requirement of the plant for protein formation has been met. The S status of the plant may be indicated by sulfate S content. McClung et al. (1959) found that when S was not applied to soils of the central plateau of Brazil, plant growth was only 4 to 30% of that obtained when a complete fertilizer was applied. Forages grown on soils so deficient in S that yield is depressed will have very low concentrations of S, far below those needed by animals (Rendig, 1986). Hay yield of bermudagrass was lower when it contained less than 0.14% S, and crimson clover responded to S fertilization when it also had less than 0.14% S (Kamprath and Jones, 1986). The type of forage in the diet may also influence S requirement. Sulfur requirements may be higher for cattle grazing sorghum Sudan grass because S is required in the detoxification of the cyanogenic glucosides found in most sorghum forages. Sulfur bioavailability varies with the type of forage; fescue has a lower S availability than other grasses. Cattle consuming fescuehay will often respond with improved intake and fiber digestion following S supplementation (Berger, 1999). Ruminants consuming large quantities of corn silage will likely be receiving inadequate S. The amount of S in corn silage samples analyzed in the Georgia Forage Testing Laboratory in 1973-1974 averaged 0.07% in dry matter, with a

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range of 0.005 to 0.19%. More than 80% of corn silages had less than one-half the estimated 0.20% S needed for lactating cows (Miller, 1979). With S fertilization, total S concentrations in corn silage were increased by approximately 32% (Buttrey et al., 1986). Cereal grains are quite low in S, with corn, rice, rye, sorghum, and wheat ranging from 0.05 to 0.18% (Appendix Table II). Oilseed S concentrations were moderately high with cottonseed meal, rapeseed meal, and soybean meal ranging from 0.41 to 0.58% S. Molasses is a good source, ranging from 0.47 to 0.60% S. High-sulfur feed ingredients such as feathers, viscera, and fecal waste used in livestock diets or as a fertilizer are organic sources of S. Forages grown on biosolids (municipal sewage sludge)-amended soil frequently have increased S contents (McBride et al., 2000; O'Connor et al., 2001). As a result of a three-year experiment using biosolid fertilization, forage S was elevated to 0.4% and Cu deficiency was in evidence in cattle (Tiffany et al., 2000, 2002). Sulfates in the water can be a major source of S intake. For example, in one of the cases cited by Kung et al. (l998b), sulfates in the drinking water ranged from 2200 to 2800 ppm. When the water S intake was expressed as a percentage of the dry matter consumed, it averaged 0.67%. Smart et al. (1986) demonstrated that cows receiving sulfated water (0.35% total dietary S) had plasma Cu concentrations lower than cattle drinking nonsulfated water (0.20% total dietary S), and a dietary Cu concentration of 10 ppm was not enough to correct the problem. Digesti and Weeth (1976) proposed the maximum safe concentration of sulfates in drinking water for cattle is 2500 ppm. Water sulfate concentrations as high as 5000 ppm have been reported (Veenhuizen and Shurson, 1992).

IX. DEFICIENCY A. Effects of Deficiency 1.

MONOGASTRIC SPECIES

Despite the fact that S is a key mineral in many compounds essential for life, dietary inorganic S is not necessary for the health of monogastric animals. Pigs and poultry can do quite well with only organic S (S-amino acids, thiamin, biotin, etc.) sources in their diets. However, the total absence of inorganic S from the diet may increase the S-amino acid requirement, because the breakdown of S-amino acids is used to synthesize other organic compounds containing S. The dietary requirements of S are not stated for monogastric species but rather the requirement for methionine. Sulfur deficiency for monogastrics is more correctly a protein deficiency (i.e., methionine). Present evidence indicates that in humans, the requirement for synthesis of S-containing compounds can be met by a single, S-containing amino acid, methionine. It is possible that in infants, as in felines, there is an additional requirement for taurine, an amino sulfonic acid.

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Breast-fed infants have higher plasma levels of taurine than infants fed casein-based formulas (Sturman et al., 1976). Clinical signs of deficiency for monogastric species are typical for protein deficiency in the various monogastric species. The following discussion on the effects of supplemental inorganic S will be limited to swine and poultry. a. Swine. Two-week-old pigs fed purified diets containing 0.8% methionine tolerated half the methionine being replaced with inorganic sulfate without affecting weight gain, feed efficiency or the collagen content in tendons (Robel, 1976). Karunskii et al. (1982) reported that pigs fed trace element-deficient diets supplemented with normal levels of Fe, Cu, cobalt (Co), Zn, and manganese (Mn) had increased weight gains and feed efficiencies compared to controls. The improved gains of pigs due to the trace elements were significantly higher when the elements were given as sulfates rather than as chloride or carbonate salts. The NRC (1998) for swine concludes that the S provided by S-containing amino acids seems adequate to meet the pig's needs for synthesis of S-containing compounds with exception of thiamin and biotin.

b. Poultry. Gordon and Sizer (1955) suggested that inorganic sulfate was of importance in poultry nutrition. They fed a basal diet to chicks that was deficient in cystine (0.08% cystine, 0.51% methionine) and sulfate-free and found that by adding 0.5% sodium sulfate there was a 31.4% growth increase (372 vs 488 g) over the basal diet by the end of the fifth week. However, inorganic sulfate could not replace cystine or methionine for protein synthesis. Simultaneous supplementation with 0.5% sodium sulfate and 0.22% methionine gave a 66.1% growth response over the basal diet (375 vs 617 g) indicating that poultry could satisfy part of their total S requirement with inorganic sulfate. Hinton and Harms (1972) utilized a similar basal diet fed to chicks and found that supplementation with 0.2% sodium sulfate resulted in a 14.5% growth increase over the basal diet. Sasse and Baker (1974) concluded that there is no dietary requirement for sulfate in chicks per se since they found no response to sulfate in the presence of adequate S-containing amino acids. Miles et al. (1983a,b) demonstrated a three-way interrelationship between methionine, choline, and sulfate in poultry. They concluded that inadequate dietary choline in studies involving methionine and sulfate would result in a slightly higher methionine requirement since more of the methionine would have to be used to meet the methyl-group requirement. Also, inadequate sulfate in diets designed to study the interrelationship between methionine and choline would lead to higher methionine requirement because methionine would be used to provide S. When the interrelationship between S-containing amino acids and inorganic sulfate is studied the choline level of the diet should be given proper attention. 2.

RUMINANTS

Outward signs of S deficiency (Fig. 6.1) include loss of appetite, reduced weight gain, reduced wool growth in sheep, excessive lacrimation, cloudy eyes, dullness, weakness, emaciation, and death (Thomas et aI., 1951; Kincaid, 1988; Qi, 1988,

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Fig. 6.1 Lambs fed a low-S diet. The lamb on the left received 3 g of S per pound of diet, whereas the other lamb received none. The deficient lamb exhibited excessivesalivation, lacrimation. and shedding of wool. (Courtesy of U.S. Garrigus, University of IIlinois, Urbana)

1992). With a deficiency wool or hair can be shed; adding S to deficient animals increased grease and clean mohair production, grease and clean mohair staple strength, and staple length (Qi et al., 1992). Rakes and Clark (1984) suggested that lameness in dairy cattle may be associated with S deficiency, as characterized by slower-growing, less flexible hooves. With a S deficiency, a number of workers have observed a reduction in ruminal bacterial numbers as well as changes in the types of bacteria present (e.g., fewer cellulytic microbes). It has been suggested (Kennedy et al., 1968) that the S requirement for cellulose digestion is greater than that for starch. Therefore, grazing ruminants that are consuming high-cellulose diets will more quickly develop a S deficiency. Due to lack of cellulose digesting bacteria, S-deficient forages characteristically have longer retention times in the reticulo-rumen and lower intake than S-adequate forages (Rees and Minson, 1978; Hegarty et al., 1994; Mathews et al., 1994). On S-deficient diets, ruminaI sulfide concentrations are low ( < 1 ug sulfide Simi of ruminal fluid), and much of this may be held by the cellular fraction. The type of fermentation is different when S-deficient diets were fed, lactic acid concentration of the rumina I fluid increased, and the production of butyric and higher chain acids was depressed (Whanger, 1972; Slyter et al., 1988). In vitro studies indicated that added sodium sulfide (31 mg suifide-S/IOO ml fluid) decreased methane production but increased the molar ratio of carbon dioxide to methane from 1.98 to 4.49 (Whanger, 1972). Sulfur-deficient regions are worldwide, with low herbage S being reported in the six continents (Tabatabai, 1986). Improved ruminant animal production has resulted following S supplementation (McDowell, 1976; Stobbs and Minson, 1980; Qi et al., 1994). From China, vast regions of the northeast, southwest, and southern pastoral regions are considered S-deficient for grazing sheep (Qi et al., 1994).

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Requirements by sheep and goats for S, expressed as a percentage of S in dietary DM, were not met by any of the 27 dominant forages in these regions (Hou, 1982). Sulfur deficiency was recognized in the U.S. in the 1900s. In the United States, S deficiency occurs most frequently in temperate regions where S contributions from precipitation and irrigation water are low and soils have originated from moderately weathered volcanic parent materials (Qi et aI., 1994). Both these conditions exist in major portions of the western U.S. Sulfur deficiency seldom occurs in arid regions because in arid regions, productivity of plants is low and soluble S04 accumulates in the soil from ground water evaporation. Deficiencies occurred most frequently in sub-humid regions and humid regions where rainfall leaches soluble S below the rooting zone of plants. Soils of the tropics generally have low levels of S compared to those of temperate regions. Responses to fertilizer S are widespread in the tropics and have been recorded in 40 tropical countries with 23 different crops (International Fertilizer Development Center (IFDC), 1979). A limited amount of analysis has indicated that many tropical forages contained considerably less than an optimum S concentration of 0.20%. Sulfur analyses of 10 forage samples from the llanos rangelands of both Colombia and Venezuela were low, ranging from 0.032 to 0.088% (Miles and McDowell, 1983). Some Brazilian studies found very low levels of S «0.1 % S, DM) in at least 30% of tropical grasses analyzed, principally during the dry season (Cavalheiro and Trindade, 1992). From these regions, severe leaching of soils and frequent burning of grasslands led to the assumption that many, if not most, llanos rangeland forages will befound deficient in S. McClung et al. (1959) also suggested that very low soil S levels in many tropical regions are due to repeated burning of dry grass which caused losses of 75% of the S by volatization.

B. Assessment of Sulfur Status in Ruminants Since S deficiency in ruminants relates to the well-being of the ruminal microflora, the best diagnosis may be afforded by obtaining samples of ruminaI fluid by stomach tube and determining whether or not they contain sufficient sulfide for unrestricted microbial protein synthesis (Underwood and Suttle, 1999). A suggested critical level would be between 1.0 to 3.8 pg S/I (Weston et al., 1988; Hegarty et al., 1991). Low serum sulfate « 10 mg/l) has been suggested as an indicator of S deficiency. Whiting et al. (1954) found a normal serum sulfate level in range ewes fed a basal diet that contained 0.09% S. Since serum sulfate may be of endogenous origin its use as an indicator of dietary S status is questionable. If another factor such as N, is limiting, serum S values for a given S intake are increased (Underwood and Suttle, 1999). A lack of S also results in a microbial population that does not utilize lactate; therefore, lactate accumulates in the rumen, blood, and urine. Plasma concentrations of amino acids are influenced by a S deficiency. Calves fed a S-deficient purified diet had elevated levels of serine, citrulline, alanine, cystine and total dispensable amino acids, but less glycine and tyrosine in blood plasma (Chalupa et al., 1971). Concentrations of methionine in plasma and liver fell when

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calves were given a diet sufficiently low in S (0.04%, OM) to retard growth (Slyter et al., 1988). It appears that the best indicator of S status of ruminants is the dietary S content and animal performance (e.g., growth and wool growth) after supplementation of S.

X. SUPPLEMENTATION For monogastric species S needs are met by selecting feeds to meet methionine requirements. Next to lysine, methionine is often the second most limiting amino acid. Synthetic methionine is available and is frequently added to swine and poultry diets. With the exception of low S-containing forages, natural protein feedstuffs for ruminants normally contain sufficient S to meet the S requirement of ruminal microbes and additional S need not be added to protein-adequate diets. However, if NPN sources are utilized such as urea then S may be needed for microbial protein synthesis. Unlike natural protein sources, urea contains no S. Loosli and Harris (1945) increased the growth rate of lambs fed a diet containing 6.55% crude protein by raising the level to 10.28% with urea plus sulfate, or urea plus methionine. Lofgreen et al. (1947) increased N retention by adding 0.2% methionine to a crude protein basal diet in which 40% of the N was supplied from urea. Lambs fed urea-containing diets without added S lost weight and were in negative N balance, while those supplemented with S were in positive N balance (Thomas et al., 1951). Nitrogen retention and wool production in sheep were improved by supplementing their diets with methionine or with methionine and cystine (McLaren et al., 1965). Hill et al. (1985) working with beef calves fed diets of corn silage supplemented with urea, with and without added S, found that the urea-supplemented diets with added S resulted in greater gains and a trend toward improved feed efficiency compared to those not receiving S. A review (Miles and McDowell, 1983) summarized four cattle S-supplementation trials in which control diets contained between 0.04 and 0.10% S. From these studies, intake by supplemented cattle increased between 7 and 260%, and production of milk and meat increased anywhere from 6 to more than 400%. There is no need to supply excess S when N in the diet exceeds the animal's requirement, and most feeds have adequate S. However, when high levels of NPN are added to diets to make up a N deficiency in the diet or supplement, then additional S is needed. Diets low in S (less than 0.1% OM) should be supplemented with 3 g of inorganic Sf 100g urea, which is the equivalent of 1 part S to 15 parts of NPN (NRC, 1976). A popular way of providing S (e.g., calcium sulfate) to grazing cattle is inclusion of S as part of a molasses supplement, in a "lick wheel" or as a molasses-urea-bran block. These molasses mixtures have the advantage of providing energy, protein, S, and other needed nutrients such as phosphorus (P), Co, and Cu. For ruminants in feedlot systems or high concentrate feeding dairy operations, dietary S supplementation can be provided as ruminal escape protein

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or ruminally-protected S-containing amino acids. There are advantages in providing S in the form of methionine analogues (e.g., malyi and methylmalyl methionine), which are not degraded by ruminal microbes. Where ruminal microbial protein synthesis cannot provide sufficient methionine to meet the needs of the animal and methionine is the most limiting amino acid, it may be more economical to feed such "protected" S sources than to feed S in expensive escape protein (bypass protein), such as fish meal (Underwood and Suttle, 1999). A major use of methionine analogues is for types of animal and production with high methionine requirements (e.g., wool production) (Coetzee et al., 1995). Bassett et al. (1981) reported that rumen-protected methionine increased feed intake, grease, and clean fleece weight of Angora goats. Rumen-protected methionine at 0.54% of dietary DM produced maximum clean mohair production. A major method of providing supplemental S to grazing livestock is by S fertilization of forage crops. The incidence of S deficiency in many countries is increasing due to the increasing use ofhigh-N-P-K, low-S-containing fertilizers and the increased need for S brought about by increases in yields (Coleman, 1966). Shifting from the use of ordinary superphosphate (12% S) to triple superphosphate (1 % S) eliminates a source of available S (lFDC, 1979). Although economically prohibitive in many tropical regions, S fertilization is an effective way of increasing forage S, as well as crop yields. An additional value of S fertilization is that some reports from tropical regions have indicated that S fertilization may increase forage intake by improving the palatability of less palatable species (Rees et al., 1974; Centro International de Agricultura Tropical (CIAT), 1981). With S-deficient Pangola grass, applying fertilizer S increased intake by 44%, compared with only 28% when the sheep were drenched with sodium sulfate (Rees et al., 1974). Sulfur fertilization to Desmonium ovalifolium in the Colombian llanos increased foliar S, N, and biomass production, decreased tannin content in leaves and, most importantly, increased intake of the forage (CIAT, 1981). Sulfur supplementation of early vegetative Kenhy tall fescue hay diets improved its utilization (Muntifering et al., 1984). Chestnut et al. (1986) observed that in orchard grass, S fertilization increased S content, changed plant tissue composition, and increased digestibility of phenolic constituents. Morrison et af. (1990) reported that dietary S affected DM intake and digestibility by sheep. The source of supplemental S can influence its bioavailability. Goodrich et al. (1978) gave the following ran kings from the most available to the least available: L-methionine > calcium sulfate> ammonium sulfate> sodium sulfate> molasses S > sodium sulfide> lignin sulfonate> elemental S. Most bioavailability experiments have been conducted with either sheep or cattle in growth or absorption studies or with in vitro ruminal fermentation techniques. Assuming these criteria, including S absorption, are valid indicators of bioavailability, most sources of S have been well utilized when compared with sodium sulfate as the standard. The biologically available S in L-methionine found by Johnson et al. (1971) and in DLmethionine by Albert et af. (1956) was higher than in other forms of S studied. Methionine hydroxy analog (MHA) compares favorably with methionine as a source ofS for ruminal microbes in vitro (Gil et al., 1973a,b). In concentrate feedlot

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diets, where considerable feed bypasses the ruminal fermentation, the methionine in feed may be absorbed by the lower tract and utilized. Bouchard and Conrad (1974) reported that sodium sulfate, calcium sulfate and a mixture of potassium (K) sulfate and magnesium sulfate were equally usable as supplementary S for lactating dairy cows. They recommended a levelof 0.17 to 0.20% S for lactating dairy cow diets. Approximately three times more S was needed in the elemental form for lambs than as methionine (Albert et al., 1956; Johnson et al., 1971). However, Chalupa et al. (1973) reported that elemental S and sulfate S were equivalent sources for cattle. The S in corn and corn silage basal diets was found to be less available than S in sodium sulfate, methionine, and MHA (Bull and Vandersall, 1973). Bouchard and Conrad (1973) found S in molasses was 65 to 75% digestible with dairy cows; which was 15 to 20% less available than in Na and Ca sulfates. Inorganic compounds including ammonium bisulfate, ammonium sulfate, calcium sulfate, sodium bisulfate, and sulfuric acid were generally equal to sodium sulfate as a source of S (Henry and Ammerman, 1995). However, elemental S was not utilized as well as sodium sulfate when tested in both cattle and sheep. Sulfur, as the highly insoluble elemental S or lignin sulfonate, is much less available, and it is suggested that elemental S (flowers of S) is utilized about one-third as efficiently as the sulfate or methionine forms (McDowell, 1985). Sulfur in corn and corn silage has been found to be less available than that in sodium sulfate, methionine, and MHA. An additional supplementation consideration relates to reducing odor in manure. Formulating diets to contain reduced S concentrations will likewise reduce S excretion, with a reduction of hydrogen sulfide gas and odor (Shurson et al., 1999).

XI. TOXICITY The toxicity of S is dependent upon its form and route of administration. Whereas elemental S is considered one of the least toxic elements, hydrogen sulfide rivals cyanide in toxicity (NRC, 1980). The variable tolerances to different amounts and sources of S in the literature partly reflect differences in the rate of ingestion of degradable S, the rate of sulfide absorption across the ruminal wall (which is pH-dependent) and the rate of sulfide capture by ruminal microbes (Underwood and Suttle, 1999). Simultaneous addition of urea can lessen the depression of appetite and digestibility caused by Salone. The toxic effects of dietary inorganic sulfate are believed due to its conversion to hydrogen sulfide by the gastrointestinal flora in both ruminants and non-ruminants. In the rumen S from various forms and sources can be readily converted to sulfide. Sulfur is much less toxic to monogastrics due to the limited ability of their microflora to generate sulfide from S products. In monogastrics, S is relatively inert and can therefore be tolerated at relatively high levels. In ruminants, the ingestion of large amounts of S can lead to acute S toxicosis resulting in death. The immediate signs of distress include thrashing, kicking at stomach, staggering, and moaning followed by subsequent death within 48 hours, suggesting a fairly high capacity to produce sulfide. High concentrations of sulfide in ruminal gas have been

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197

reported (McAllister et al., 1992) and have resulted in respiratory distress, reduced feed intake, and reduced ruminal motility (Bird, 1972). For ruminants, in the normal process of eructation (belching of gases), 60% of eructated gases are inhaled and enter the respiratory tract (Bulgin et al., 1996). Therefore, inhalation of hydrogen sulfide from high sulfate has been implicated in enhancing sulfide toxicity. The margin between S requirement and toxic concentrations for ruminants is extremely small. The requirement is suggested to be between approximately 0.1 to 0.25%. However, maximum dietary tolerable levels of S for ruminants were suggested at 0.4% by the National Research Council in 1980 (NRC, 1980). However, more recent research indicates the toxicity to be less, ranging from 0.3 to 0.4%. Zinn et al. (1999) suggested an even lower tolerance; steers on a concentrate diet were adversely affected when ammonium sulfate was used to increase dietary S from 0.2 to 0.25%. Bouchard and Conrad (1973) suggested that this level should be less than 0.30% for lactating cows. If high levels of S inhibit intake, extreme caution should be taken during the close-up and early lactation stages where DM intake is lower than desired. Sulfide is readily absorbed through the ruminal wall into the bloodstream (Bray, 1969). Once absorbed, sulfide inhibits the functions of carbonic anhydrase, dopa oxidases, catalases, peroxidases, dehydrogenases, and dipeptidases, adversely affecting oxidative metabolism and the production of ATP (Short and Edwards, 1989). Specifically, sulfide is also thought to block the enzyme cytochrome C oxidase. Sulfide also binds to hemoglobin, creating sulfhemoglobin, reducing the oxygen carrying capacity to tissues. Sulfide also has a paralyzing effect on the carotid body and therefore may also inhibit normal respiration (Bulgin et al., 1996). Acute reactions in response to increased levels of ingested elemental S have been reported in sheep (Bulgin et al., 1996). These animals had grazed on an alfalfa field that had been sprayed with elemental S (60 kgjha). Within two hours after being released onto this field, some of the animals began to show signs of distress and quickly died. Upon necropsy, it was noted that the rumina I pH was 6 to 6.5, there was an odor of rotten eggs and pulmonary edema was observed. Immediate deaths were probably from acute sulfide toxicosis. Excess S can also impair animal performance by reducing the availability of other minerals. For example, hydrogen sulfide in the rumen binds with Mo to form thiomolybdates. Thiomolybdates bind with Cu in the rumen to form an insoluble complex. Sulfur also reduces Cu absorption by the formation of insoluble Cu sulfide in the rumen, independent of the formation of thiomolybdates (see Section V-I). In addition to detrimentally affecting Cu metabolism, excess S interferes with Se metabolism. Fertilizing pastures with ammonium sulfate resulted in increased forage S and lower liver Cu in cows grazing the forage (Arthington et al., 2002). Excess fertilizer S can promote a Se deficiency resulting in a higher probability of white muscle disease (see Section V-2). Another problem that can occur when high dietary S leads to the production of excess sulfides in the rumen is polioencephalomalacia, or PEM (Lowe et al., 1996; Gould, 1998; McDowell, 2000). The disease affects the central nervous system.

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Clinical signs in mild cases include dullness, blindness, muscle tremors (especially of the head), and opisthotonos. The condition is characterized by circling, head pressing, and convulsions, and in severe cases, the animal collapses within 12 to 72 hours after onset of the disease. Gould et al. (1991) reported that steers with the highest ruminal fluid sulfide concentrations coincided with the onset of clinical signs of PEM. Several cases of PEM occurred when gypsum had been used as a feed intake limiter. It would appear that the sulfate ion of gypsum, during its conversion to sulfide, must pass through sulfite, which may destroy thiamin. The sulfite ion apparently will cleave thiamin at the methylene bridge, mimicking thiaminase. In lambs, PEM was induced by administration of a sulfide solution; neurological clinical signs included stupor, visual impairment, and seizures (McAllister et al., 1992). After 12 weeks of feeding a high-S diet (0.8% S) to sheep, all animals had developed PEM and the availability of Cu and Zn was depressed (Krasicka et al., 1999). The exact interaction between dietary S, thiaminase production, and PEM is not well understood; Kung et al. (1998b) postulated sulfates in the feed or water are converted to hydrogen sulfide in the rumen. When the hydrogen sulfide is eructated with the other ruminal gases, it is inhaled and can damage lung and brain tissues. In some studies, thiamin status was within normal ranges and giving thiamin injections did not prevent the signs of PEM in all cases. There is the suggestion that PEM from excess S differs in fine pathology and responsiveness to thiamin from that attributed entirely to a thiamin deficiency (Underwood and Suttle, 1999). Maximum tolerable levels for monogastrics are not definitive. Sulfur is less toxic for monogastrics because intestinal absorption of inorganic S compounds is low and much less sulfide is formed in the intestinal tract than for ruminants. Smith (1973) concluded that 0.69% S (inorganic and organic) for rat diets is the optimal level. Reduced growth for poultry resulted from 1.4% S (NRC, 1994). Paterson et al. (1979) reported that weanling pigs consuming drinking water that contained 600 ppm S as sodium sulfate had loose feces and diarrhea, but no effect on weight gain or feed conversion. Sows provided water containing up to 664 ppm S as sodium sulfate from 30 days post-breeding to 28 days of lactation had no problems with reproduction. Corke (1981) described accidental S poisoning in horses when flowers of S were fed to 14 horses ranging in age from 5 to 12 years. Consumption was 0.2 to 0.4 kg per horse. Twelve horses appeared dull and lethargic followed by purgation within 12 hours; the other two had mild signs 48 hours after administration. Two horses died from respiratory failure associated with cyanosis and terminal convulsions 48 hours after consuming the S. Many common feeds and sources of water can contain high levels of Sand/or sulfate. Some common feeds have moderate to high levels of S. For example, corn gluten meal, molasses (cane and beet), and brassicas (e.g., turnips) are high in S (0.43 to 0.72% S) (Kung et al., 1998a). Other feeds that contain high concentrations of S include, fish, feather, meat, and blood meals that are common sources of rumen undegradable intake protein. Condensed molasses fermentation solubles is a high sulfate byproduct that contains 5.75% S (Hannon and Trenkle, 1990). Water can also be very high in sulfates with levels in excess of 5000 ppm (Veenhuizen and

References

199

Shurson, 1992). Digesti and Weeth (1976) suggested that it was safe for cattle to consume water containing 2500 ppm of sulfate. Recently, Wagner et al. (1998) reported lower intake and gains in steers fed water with 2000 ppm sulfate. In recent years, there has been increased incidence of excess S in ruminant diets. Part of this is due to fertilization practices. High forage S is the result of aggressive fertilization with ammonium sulfate. Ammonium sulfate production has increased significantly and it is often priced competitively, both as a fertilizer and feed ingredient. Laboratory analysis of Michigan forages grown in 1996 and 1997 had 1.5 to 3.0 times greater S than forages grown in 1994 and 1995 (Beede, 1999). Cattle grazing ammonium sulfate-fertilized bahia grass containing 0.50% S were less able to respond to Cu supplementation (Arthington et al., 2002). Upon removal from high-S forages, Cu-deficient cattle were able to rapidly respond to Cu supplementation.

XII. REFERENCES Albert, w. W., Garrigus, U. S., Forbes, R. M., and Norton, H. W. (1956). J. Anim. Sci. 15,5. Allaway, W. H., and Thompson, J. F. (1966). Soil Sci. 101,240. Anderson, C. M. (1956). N. Z. J. Sci. Tech. Ser. A. 37, 379. Arthington, J. D., Rechcigl, J. E., Yost, G. P., McDowell, L. R., and Fanning, M. D. (2002). J. Anim. Sci. SO, 2507. Baker, D. H. (1987). Meth. Enzy. 143,297. Bassett, J. W., Baldwin, D. B., Calhoun, M. C., and Stobart, R. H. (1981). Res. Rep. Texas Agric. Exp. Sta., p. 68. Beede, D. (\999). Michigan Dairy Review. EIU436, East Lansing, MI. Beever, D. E. (1996). S. Afric. J. Anim. Sci. 26, 20. Berger, L. L. (1999). Sail and Trace Minerals 31(2), I. Bird, P. R. (\972). Aust. J. Bioi. Sci. 25, 1087. Block, R. J., Stekol, J. A., and Loosli, J. K. (1951). Arch. Biochem. Biophys. 33, 353. Bouchard, R., and Conrad, H. R. (1973). J. Dairy Sci. 45, 1429. Bouchard, R., and Conrad, H. R. (1974). Can. J. Anim. Sci. 54, 587. Boyer, P. D. (1959). The Enzymes. Academic Press, New York. Bray, A. C. (\969). Aust. J. Agric. Res. 20, 734. Bulgin, M. S., Stuart, S. D., and Mather, G. (\996). JAVMA. 208,1063. Bull, L. S. (\ 979). In "The 2nd Annual International Minerals Conference," p. Ill. International Minerals & Chemical Corporation, Mundelein, IL. Bull, L. S., and Vandersall, J. H. (1973). J. Dairy Sci. 56, 105. Buttrey, S. A., Allen, V. G., Fontenot, J. P., and Reneau, R. B. (1986). J. Anim. Sci. 63, 1236. Cavalheiro, C. L., and Trindade, D. S. (1992). "Os minerals para bovines e ovinos criados em pastejo," Fund. Banco do Brasil, Porto Alegre. Centro International de Agricultura Tropical (CIAT) (1981). CIAT Annual Report 1981, Cali, Colombia. Chalupa, W., Oltjen, R. R., and Dinius, D. A. (1973). J. Anim. Sci. 37, 340 (Abstr.). Chalupa, W., Oltjen, R. R., Slyter, L. L., and Dinius, D. A. (1971). J. Anim. Sci. 33, 278(Abstr.). Chestnut, A. B., Fafey, G. c., Berger, L. L., and Spears, J. W. (1986). J. Anim. Sci. 63, 1926. Coetzee, J., de Wet, P. 1., and Burger, W. J. (1995). S. Afric. J. Anim. Sci. 25,87. Coleman, R. (\ 966). Soil Sci. 104, 230. Corke, M. J. (1981). Vel. Rec. 109,212. Dick, A. T. (l953a). Aust. Vel. J. 29, 233. Dick, A. T. (1953b). Nature 172,637. Dick, A. T. (1956). Soil Sci. 81, 229. Dick, A. T., and Bull, L. B. (1945). Aust, Vel. J. 21, 70. Digesti, R. D., and Weeth, H. J. (1976). J. Anim. Sci. 42, 1498.

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Dziewiatkowski, D. D. (1970). In "Symposium: Sulfur in Nutrition" (0. H. Muth and J. E. Oldfield, eds.), p. 97. AVI Publishing Co., Westport, CT. Emery, R. S., Smith, C. K., and Huffman, C. F. (1957). Appl. Microbiol. 5, 360. Evans, J. L., and Davis, G. K. (1966). J. Anim. Sci. 25, 1014. Gawthorne, J. M., Allen, J. D., and Nader, C. J. (1985). In "Trace Element Metabolism in Man and Animals (TEMA-3)." (C. F. Mills, I. Bremner, and J. K. Chesters, eds.), p. 346. Aberdeen, Scotland. Gil, L. A., Shirley, R. L., and Moore, J. E. (l973a). J. Dairy Sci. 37, 159. Gil, L. A., Shirley, R. L., and Moore, J. E. (I973b). J. Dairy Sci. 56, 757. Goodrich, R. D., and Garrett, J. E. (1986). In "Sulfur in Agriculture," (M. A. Tabatabai, ed.). Agron, Monogr. 27, Madison, WI. Goodrich, R. D., Kahlon, T. S., Pamp, D. E., and Cooper, D. P. (1978). "Sulfur in Ruminant Nutrition." National Feed Ingredients Association, West Des Moines, IA. Gordon, R. S., and Sizer, I. W. (1955). Science 122, 1270. Gould, D. H (1998). J. Anim. Sci. 76, 309. Gould, D. H., McCallister, M. M., Savage, J. C., and Hamar, D. W. (1991). Am. J. Vet. Res. 52, 1164. Griffith, Owen W. (1987). Meth. Enzy. 143,366. Hannon, K., and Trenkle, A. (1990). J. Anim. Sci. 68, 2634. Hegarty, R. S., Nolan, J. V., and Leng, R. A. (1991). Archiv. Anim. Nutr., Berlin, 41, 725. Hegarty, R. S., Nolan, J. V., and Leng, R. A. (1994). Aust. J. Agri. Res. 45, 1215. Henry, P. R., and Ammerman, C. B. (1995). In "Bioavailability of Nutrients for Animals", (c. B. Ammerman, D. H. Baker, and A. J. Lewis, Eds.) p. 127. Academic Press, San Diego. Hill, G. M., Boling, J. A., and Bradley, N. W. (1985). Int. J. Vitam. Nutr. Res. 55,439. Hinton, C. F., and Harms, R. H. (1972). Poult. Sci. 51, 701. Hintz, H. F., and Hogue, D. E. (1964). J. Nutr. 82, 495. Hou, X., (1982). "The Geography of Plant Colony and its Chemical Composition of Dominant Plant Species in China." Scientific Publishing Unit, p. 51. Beijing, China. Hume, J. D., and Bird, P. R. (1970). Aust, J. Agric. Res. 21, 315. Hunt, C. H., Bentley, O. G., Hershberger, T. V., and Cline, J. H. (1954). J. Anim. Sci. 13,570. International Fertilizer Development Center (IFDC) (1979). "Sulfur in the Tropics." The Sulfur Institute, Muscle Shoals, AL. Ivancic, J., and Weiss, W. P. (2001). J. Dairy Sci. 84, 225. Johnson, W. H., Goodrich, R. D., and Meiske, J. C. (1971). J. Anim. Sci. 32,778. Johnson, W. H., Meiske, J. C., and Goodrich, R. D. (1968). J. Anim. Sci. 27, 1166 (Abstr.). Kamprath, E. J., and Jones, U. S. (1986). In "Sulfur in Agriculture,"(M. A. Tabatabai, ed.), p. 323. American Society of Agronomy. Madison, WI. Karunskii, A. I., Povysh, R., Korml, E., Zhivotin, S. K.H. (1982). 57 Chern. Abstr. 97, 161413. Kennedy, L. G., Mitchell, G. E., and Little, C. O. (1968). Sulphur Inst. J. 4(1),8-9. Kennedy, P. M., and Siebert, B. D. (1972). Aust, J. Agr. Res. 23, 45. Kennedy, P. M., Williams, E. R., and Seibert, B. D. (1975). Aust, J. Bioi. Sci. 28, 31. Kincaid, R. (1988). Macro elements for ruminants. In "The Ruminant Animal - Digestive Physiology and Nutrition," p. 326. Prentice Hall, NJ. Krasicka, B., Gralah, M. A., Sieranska, B., and Kulaseh, G. (1999). Repro. Nutr. Development 39, 625. Kulwich, R., Struglia, L., and Pearson, P. B. (1957). J. Nutr. 61, 113. Kung, L., Bracht, J. P., Hession, A. 0., and Tavares, J. Y. (1998a). Feedstuffs, Nov. 16, 1998, p. 12. Kung, L., Hession, A. 0., Bracht, J. P. (1998b). J. Dairy Sci. 81, 2251. Lane, H. W., Shirley, R. L., and Cerda, J. J. (1979). J. Nutr. 100,444. Larson, B. L., Arthington, J., and Corah, L. R. (1995). Vet. Med. June 1995, p. 61. Lewis, D. (1954). Biochem. J. 56, 301. Lofgreen, G. P., Loosli, J. K., and Maynard, L. A. (1947). J. Anim. Sci. 6, 344. Loosli, J. R. (1952). Feed Age 2, 44. Loosli, J. K., and Harris, L. E. (1945). J. Anim. Sci. 4,435. Loosli, J. K., Williams, H. H., Thomas, W. E., Ferris, F. H., and Maynard, L. A. (1949). Science 110,144. Lowe, J. c., Scott, P. R., Howie, F., Lewis, M., Fitzsimons, J., and Spence, J. A. (1996). Vet. Rec. 138,327. Martin, J. E., Arrington, L. R., Moore, J. E., Ammerman, C. B., Davis, G. K., and Shirley, R. L. (1964). J. Nutr. 83, 60. Mason, J. (1986). Toxicology 42, 99. Mason, J. (1990). Irish Vel. J.43, 18. Mathews, B. W., Sollenberger, L. E., and Staples, C. R., (1994). J. How. Pat. Agric. 5,21. McAllister, M. M., Gould, D. H., and Hamar, D. W. (1992). J. Compo Pathol. 106,267.

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Schwarz, K., and Foltz, C. M. (1957). J. Amer. Chern. Soc. 78, 3292. Short, S. B., and Edwards, W. C. (1989). Vet. Human Toxicol. 31, 451. Shrift, A. (1958). Bot. Rev. 24, 550. Shurson, J., Whitney, M., and Nicolai, R. (1999). Feedstuffs 71(4),12. Slyter, L. L., Chalupa, W., and Oltjen, R. R. (1988). J. Anim. Sci. 66, 1016. Slyter, L. L., Chalupa, W., Oltjen, R. R., and Weaver, J. M. (1986). J. Anim. Sci. 63, 149. Smart, M. E., Cohen, R., Christensen, D. A., and Williams, C. M. (1986). Can. J. Anim. Sci. 66, 669. Smith, J. T. (1973). J. Nutr. 103, 1008. Stanford, G., and Jordan, H. V. (1966). Soil Sci. 101, 258. Stobbs, T. H., and Minson, D. J. (1980). In "Digestive Physiology and Nutrition of Ruminants" (D. C. Church, ed.), p. 357. 0 & B. Books, Corvallis, OR. Sturman, J. A., Rassin, D. K., and Gaul\, G. E. (1976). Pediatr. Res. 10,415. Tabatabai, M. A. (ed.) (1986). "Sulfur in Agriculture." Amer. Soc. Agron. Madison, WI. Taylor, C. D., and Wolfe, R. S. (1974). J. Bioi. Chern. 249, 4879. Thomas, W. E., Loosli, J. K., Williams, H. H., and Maynard, L. A. (1951). J. Nutr. 43, 515. Tiffany, M. E., McDowell, L. R., O'Connor, G. A., Martin, F. G., Wilkinson, N. S., Cardoso, E. C., Percival, S. S., and Rabiansky, P. A. (2000). J. Anim. Sci. 78, 1331. Tiffany, M. E., McDowell, L. R., O'Connor, G. A., Martin, F. G., Wilkinson, N. S., Percival, S. S., and Rabiansky, P. A. (2002). J. Anim. Sci. 80, 260. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" (3rd Ed.), Midlothian, UK. van Ryssen, J. B. J., van Malsen, P. S. M., and Hartmann, F. (1998). J. Agric. Sci. (Camb.) 130, 107. Van Vleet, J. F. (1982). Am. J. Vet. Res., 43, 851. Veenhuizen, M. F., and Shurson, G. C. (1992). J. Am. Vet. Med. Assoc. 201,487. Wagner, J. J., Loneragan, G. H., Gould, D. H., and Thoren, M. (1998). J. Anim. Sci. 751(Suppl. 1),272. Weston, R. H., Lindsay, J. R., Purser, D. B., Gordon, G. L. R., and Davis, P. (1988). Austr. J. Agric. Res. 39, 1107. Whanger, P. D. (1972). World Rev. Nutr. Diet 15,225. Whanger, P. D., and Matrone, G. (1967). Biochim. Biophys. Acta. 136,27. Whiting, F., Sen, S. B., Bezeau, L. M., and Clark, R. D. (1954). Can. J. Agric. Sci. 34, 261. Williams, A. J., Robards, G. E., and Saville, D. G. (1972). Austr. J. Bioi. Sci. 25, 1269. Young, L., and Maw, G. A. (1958). "The Metabolism of Sulphur Compounds", John Wiley & Sons, New York. Zinn, R. A., Alvarez, E., Monano, M., and Ramirez, E. (1999). J. Anim. Sci. 77(Suppl. 1),115.

Chapter 7

Iron

I. INTRODUCTION

Iron (Fe) deficiency is one of the most common deficiency diseases of swine and humans. Baby pigs fed only milk and reared in concrete-floored pens are highly vulnerable to Fe deficiency. Iron deficiency is one of the most common human deficiency diseases in the world. More people suffer from Fe deficiency anemia than any other nutrient deficiency. In many developing countries, nearly 40% of the female population is anemic. Females are most likely deficient owing to menstrual Fe losses and added requirements for pregnancy. Recent studies indicate that excess Fe can be a problem for humans who are beyond the growth and reproductive years. For livestock other than baby pigs, Fe deficiency is rarely a practical concern, except in circumstances involving blood loss or disturbances resulting from parasitic infestation or disease.

n, mSTORY From antiquity, man has recognized the special role of the metal Fe in health and disease (Loosli, 1978; Yip and Dallman, 1996; Beard and Dawson, 1997). Iron compounds were used by the Egyptians as early as 1500 BC, and by the Hindus, Greeks, and Romans somewhat later. The early Greeks recognized anemia and treated it by drinking water in which a sword had been allowed to rust. Hippocrates used Fe therapeutically for such diverse maladies as dermatitis, hemorrhoids, wounds, gout, diarrhea, vomiting, weakness, edema, fever, and cystitis. Guggenheim (1995) has reviewed the historical significance of chlorosis as it related to Fe deficiency. Chlorosis (hypochromic anemia) was a name used in 1615, from the Greek word chloros (meaning green), as patients had a greenish tint to their skin. It was also called green sickness or weak blood. The first description of hypochromic anemia was in 1554, when Lang described a girl who was weak and her face, which in the last years was distinguished by rosiness of cheeks and redness of lips, is somehow as if exsanguinated, sadly paled, the heart trembles with every movement of her body, trouble dancing and stair climbing. Lang called the disease Morbus virgineus, a condition peculiar to virgins. Clark (1887) was often struck by frequent occurrences of anemia or chlorosis in girls... among the rich and poor, 203

Iron

204

among the cultured and the rude ... among the idle and occupied ... independent of country and town. Clark also believed wearing tight corsets may have displaced the abdominal organs and possibly, purely by mechanical pressure, decreased the volume of food eaten. Also, the bowels became either obstructed, confined, or inadequately relieved. In the sixteenth century, Monarde was the first to suggest a relationship between Fe and blood. Menghini drew attention to the Fe content of blood by lifting particles of dried, powdered blood with a magnet (Yip and Dallman, 1996). The role of Fe in blood formation became apparent in the seventeenth century when it was shown that Fe salts were of value in the treatment of Fe-deficiency anemia in young women. In 1664, Syndenham showed that administration of salts of Fe would restore the pink color in the cheeks of people suffering from anemia. The mechanism involved was not understood until Zinoffsky discovered in 1886 that hemoglobin (horse) crystals contained 0.335% Fe. Similar concentrations of Fe were demonstrated for a range of animal species. Convincing proof that inorganic Fe could be used for hemoglobin synthesis came in 1932 from Castle and co-workers, who found that the amount of Fe given parenterally to patients with hypochromic anemia corresponded closely to the amount of Fe in circulating hemoglobin (Yip and Dallman, 1996). Boussingault (1872) was probably the first to recognize Fe as a nutrient for animals, and he determined the Fe content of carcasses of a number of species and of food sources. In 1981, Braasch described anemia in suckling pigs; however, McGowan and Chrichton (1923) first published evidence associating Fe deficiency with baby pig anemia. They successfully treated the anemia with large doses of ferric oxide. For many years, nutritional interest in Fe was focused on its role in hemoglobin formation and oxygen transport. Keilin and others established the presence of Fe in the hemeprotein enzymes, the cytochromes, and the role of these enzymes in the oxidative mechanisms of all cells (Underwood and Suttle, 1999).

m.

CHEMICAL PROPERTIES AND DISTRIBUTION

Iron is the second most abundant metal in the earth's crust after aluminum, about 5%. Most plant materials used in the feeding of farm animals contain large and variable concentrations of Fe. Iron is silvery-white or gray and is somewhat magnetic. It holds magnetism only after hardening (as alloy steel). It is stable in dry air but readily oxidizes in moist air, forming rust (chiefly oxide, hydrated). Iron is alloyed with other elements in forming steel. Iron is a transition metal with an atomic weight of 56. It has two stable oxidation states (+ 2 and + 3) and several unstable oxidation states in aqueous solutions, and widely variable redox potentials depending on the ligands (Conrad et al., 1980). A special property of Fe is how easily it changes between the two oxidation states, Fe2 + (ferrous) and Fe3+ (ferric). This property makes Fe complexes useful in electron-transfer reactions.

Metabolism

205

Iron is a component of every living organism. The Fe content of animals varies from birth to maturity. Adult humans (70 kg) are estimated to contain 4 to 5 g Fe or 60 to 70 ppm of the whole body (Bothwell et al., 1979). Most body Fe exists in complex forms bound to protein, either as porphyrin or heme (Fig. 7.1) compounds, particularly hemoglobin and myoglobin, or as nonheme protein-bound complexes such as transferrin, ferritin, and hemosiderin (see Sections IV and V).

IV. METABOLISM

A. Absorption and Transport

Various aspects of Fe metabolism are reviewed by Yip and Dallman (1996), Beard and Dawson (1997), and Brody (1999). A schematic outline of Fe metabolism is shown in Fig. 7.2. Animals have a limited capacity to excrete Fe; Fe homeostasis in the body is largely controlled by absorption. The absorption of Fe is affected by (1) the age, Fe status, and state of health of the animal or individual; (2) conditions within the gastrointestinal tract; (3) the amount and chemical form of the Fe ingested; (4) the amounts and proportions of various other components of the diet, both organic and inorganic; and (5) genetic control, at least for excess absorption (Burk et al., 2001). Iron is poorly absorbed, but is better absorbed from animal (heme form) than from plant foods (non-heme forms) (South et al., 2000; Lynch, 2002). Generally, as the level of dietary Fe increases, the percentage absorbed decreases (Wood and Han, 1998). The absolute amount may increase as dietary levels increase, but the Fe status of the animal has a greater influence on the amount of Fe absorbed (Van Campen, 1974). In Fe-deficient rats, 80% of the dietary Fe was absorbed, while in Fe-adequate rats, only 7 to 10% was absorbed. For adult humans, only 5 to 15% of food Fe is absorbed from ordinary mixed diets, but may increase to twice this level or more in children and in cases of Fe deficiency. Josephs (1958) reported that

HOOC-CHiCHi

CH, Fig. 7.1 The structure of the iron protoporphyrin complex heme.

206

Iron

Urine, Bile, Feces

Fig.7.2 Iron metabolism. (Courtesy of M.L. Scott, Cornell University, Ithaca, NY)

2 to 20% of an oral dose of radio-Fe was absorbed in normal human subjects, compared with 20 to 60% in patients with Fe-deficiency anemia. Iron absorption occurs throughout the gastrointestinal tract. The major sites are the duodenum and jejunum. Although not absorbed in the stomach, the stomach contributes hydrochloric acid, which not only helps to remove protein-bound Fe by protein denaturation but also helps in Fe solubilization and reduction of ferric Fe to the ferrous state. Iron is absorbed in the ferrous state, in the ferric form in feed, and also in combination with organic compounds. Ascorbic acid and cysteine in food may aid in reduction of Fe from the ferric to the ferrous state and enhance Fe absorption (Cook and Reddy, 2001). The mucosal block theory (Hahn et aI., 1943) states that only enough Fe is absorbed in normal animals to meet needs, and is rejected when stores are adequate. Iron taken into mucosal cells is converted into ferritin, and when the cells become physiologically saturated with ferritin, further absorption is impeded until the Fe is

Metabolism

207

released from ferritin and transferred to plasma. This theory has been modified since the ultimate regulator of Fe absorption appears to be the Fe concentration in the mucosal epithelial cells of the duodenum. Decreased mucosal Fe induces an increase in the intestinal Fe absorption in the early stage of Fe-deficiency. Thus, Fedeficient animals absorb ingested Fe almost directly into the blood with very little remaining in the musosal cells; Fe-adequate animals transfer only a small portion of Fe absorbed by mucosal cells to the blood (Conrad and Crosby, 1963). Iron absorption is favored by more acid conditions. Normal gastric secretion is necessary for optimal absorption of Fe by rats (Murray and Stein, 1970). Inorganic Fe forms complexes with normal gastric juice at a low pH. These complexes remain soluble when the pH is raised to neutrality. Decreased stomach acidity, due to overconsumption of antacids, ingestion of alkaline clay, or pathologic conditions such as achlorhydria or partial gastrectomy, may lead to impaired Fe absorption (Beard and Dawson, 1997). Absorption of nonheme forms of Fe is greatly influenced by various dietary chelates. Ascorbic acid promotes Fe absorption, and ethylenediaminetetraacetic acid (EDTA) inhibits its absorption. Histidine, lysine, and cysteine enhance ferrous Fe uptake. High dietary levels of phosphorus (P) reduce Fe absorption, presumably by the formation of insoluble ferric phosphate and phytate. Dietary phytate reduces bioavailabilities of Fe and other minerals and thus absorption (Stahl et al., 1999; Kamao et al., 2000) while a phytase will release Fe and other minerals from this complex (Stahl et al., 1999). High dietary levels of copper (Cu), manganese (Mn), lead (Pb), and cadmium (Cd) increase Fe requirements by competing for absorption sites in the intestinal mucosa. The enterocyte is a highly specialized, polarized, absorptive cell found on the intestinal villus that controls the passage of dietary Fe into the body. The passage of Fe through the enterocyte entails transport of the metal across the following three formidable cellular barriers: the apical membrane, intracellular translocation across the cytosol, and release of Fe across the basolateral membrane and thence into the circulation (Wood and Han, 1998). Ferrous Fe entering the blood plasma is quickly oxidized to the ferric state. The ferric form immediately complexes with a specific B1-globulin (transferrin), and is transported throughout the body. Plasma transferrin links various cycles of Fe metabolism and thus regulates body Fe distribution. Transferrin accepts Fe that is absorbed from the intestinal tract and released from sites of storage and from hemoglobin destruction. The second phase of transferrin-Fe transport is delivery to the bone marrow for hemoglobin synthesis, to the placenta for fetal needs, and to cells for Fe-containing enzymes. More than 70% of plasma Fe turnover goes to erythroid cells in bone marrow for hemoglobin synthesis. ' The discovery of iron regulatory proteins (lRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of Fe metabolism. Cellular Fe homeostasis is achieved through the controlled synthesis of several proteins involved in the movement, storage, and utilization of Fe (Piero, 2001). These IRPs bind to Fe-responsive elements in specific mRNAs and regulate their utilization. Because IRPs are key modulators of the uptake and metabolic fate

208

Iron

of Fe in cells, they are focal points for the modulation of cellular Fe homeostasis in response to a variety of agents and circumstances (Eisenstein and Blemings, 1998). Placental Fe transport is unidirectional and increases rapidly as pregnancy progresses. In animals with the hemochorial type of placenta, which includes the rat, rabbit, guinea pig, and human, the rate of Fe transfer across the placenta from maternal plasma transferrin is sufficient to account for all Fe accumulated by the fetus (Bothwell et al., 1979). A progesterone-inducible purple protein possessing phosphatase activity, uteroferrin, has been isolated from porcine placental tissue and is proposed to have a role in transfer of Fe to the fetal piglet (Ducsay et al., 1984). Hemoglobin Fe conservation is achieved by reticuloendothelial cells of the liver, spleen, and bone marrow, which recruit Fe from senescent or nonviable erythrocytes. They phagocytize the erythrocytes for the purpose of breaking down hemoglobin and releasing Fe. Released Fe is either rapidly returned to the circulation via plasma transferrin or held in a slowly exchanging pool of storage Fe in the reticuloendothelial cell (Fillet et al., 1974). This system of Fe reutilization is an efficient mechanism whereby a constant source of Fe is available daily for resynthesis of body hemoglobin.

B. Storage Ferritin concentration in tissues, together with that of hemosiderin, reflects the animal's Fe status. These Fe storage compounds are present primarily in the liver, reticuloendothelial cells, and bone marrow. The liver contains about 60% of the body ferritin (Beard and Dawson, 1997). Apoferritin, ferritin without Fe, is synthesized in the presence of a positive Fe balance and becomes loaded with Fe deposits of ferric hydroxide and ferric phosphate. Ferritin is a nonheme protein (globulin) compound (containing up to 20% Fe), which is present throughout the body and particularly in the liver. A high positive correlation exists between human serum ferritin concentrations and body Fe stores (Walters et al., 1973). Hemosiderin is relatively amorphous, containing as much as 35% Fe primarily as colloidal ferric hydroxide with very little protein (Shoden and Sturgeon, 1961). In most species, hemosiderin is the predominant form at high tissue levels, and ferritin predominates at lower levels. Exchange of Fe from ferritin to transferrin is reversible, giving the body access to its Fe stores and permitting redistribution of body Fe. This process involves the reduction of ferritin-Fe'' " and can be accomplished by riboflavin, ascorbic acid, glutathione, or cysteine, and to a lesser extent by zanthine oxidase (acting as a dehydrogenase) (Conrad et al., 1980). Mobilization of Fe from Fe stores also requires the Cu-containing enzyme of the plasma ceruloplasmin (ferroxidase I), as discussed in Chapter 8. Iron storage in the newborn is influenced by the maternal diet during gestation; most storage occurs late in gestation. If the number of young born is larger than usual, for example, twins in humans and extra-large litters in hogs, the individuals's supply tends to be smaller. Even if the store is normal, a long nursing period

Physiological Functions

209

without supplementary Fe-rich food will exhaust it (Maynard et al., 1979; Yip and Dallman, 1996). C. Excretion and Blood Loss Absorbed Fe is retained with great tenacity and therefore is not readily lost from the body except through hemorrhage. Iron is released from hemoglobin during erythrocyte breakdown, carried to the liver, and secreted in the bile. Most bile Fe is reabsorbed and used again to form hemoglobin. Although absorbed Fe is retained with great tenacity and, in the absence ofbleeding, excretion is very small, the amounts lost are of nutritional importance, particularly for growing or pregnant animals. Iron losses from bleeding from injury or menstrual flow can be substantial. The normal menstrual flow is about 35 ml per period. This is equivalent to about 18 mg Fe, as blood contains 0.5 mg of Fe/ml. Excessive menstrual blood loss is the most common cause of Fe deficiency in women. The upper limit of the normal period is about 80 ml; excessive losses may rise over 200 ml per period (Brody, 1999). Iron is excreted in the feces and urine, in addition to losses through sweat, hair, and nails. Most of the total Fe present in feces is nonabsorbed food Fe; probably less than 3% is endogenous Fe. Although Fe excreted in feces and urine is the major excretory loss, there is a continual dermal loss in the sweat, hair, and nails. Most of this occurs in desquamated cells, but cell-free sweat contains some Fe.

V. PHYSIOLOGICAL FUNCI'IONS

Iron plays a key role in many biochemical reactions. It is present in several enzymes responsible for electron transport (cytochromes), for activation of oxygen (oxidases and oxygenases), and for oxygen transport (hemoglobin and myoglobin). The cytochrome system is a series of reactions in which oxidation occurs with production of adenosine triphosphate (ATP) and formation of water. Iron has oxidation-reduction activity and transports electrons. Bound Fe changes oxidation state and functions at the active sites of numerous oxidation-reduction enzymes and oxygen-binding proteins. Iron exists in the animal body mainly in complex forms bound to protein (hemoproteins) as heme compounds (hemoglobin or myoglobin), as heme enzymes (mitochondrial and microsomal cytochromes, catalase, and peroxidase), or as nonheme compounds (flavin-Fe enzymes, transferrin, and ferritin). Lactoferrin is an Fe-containing glycoprotein secreted by mammary cells that has an important role as an antibiotic agent in the gland (Troost et al., 2002). Calves fed lactoferrin consumed more calf starter and improved performance (Joslin et al., 2002). The hemoproteins contain Fe in which four of the six coordination positions around the Fe atom are occupied by the nitrogen atoms of a porphyrin, most frequently protoporphyrin (Fig. 7.1). Hemoglobin and the catalases contain four heme groups per molecule, whereas myoglobin, the cytochromes, and peroxidases contain one heme group per molecule.

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The biosynthesis of heme begins with synthesis of d-aminolevulenic acid, the precursor to porphyrins, and ends with incorporation of Fe 2 + into the porphyrin ring by the enzyme ferrochetalase. Heme synthesis is impaired in Fe deficiency because of substrate insufficiency for ferrochetalase, and also in Cu deficiency owing to decreased activity of the Cu-dependent enzyme cytochrome oxidase, which reduces Fe3+ to Fe2+ before incorporation into the porphyrin molecule (Williams et al., 1976). Hemoglobin (blood) Fe represents approximately 60% of total body Fe, whereas myoglobin represents about 4% of total Fe (Brody, 1999). The molecular weight of hemoglobin is 68,000, and each molecule contains four atoms of Fe. Hemoglobin is a tetramer composed of four globin moieties each containing a heme unit bound loosely by noncovalent bonding of Fe and the imidazole nitrogen of a histidine residue in each protein chain. Myoglobin is an Fe-porphyrin protein of one Fe atom per molecule, with a molecular weight of about 17,000. It is present in muscle cells and has a higher affinity for oxygen than does hemoglobin, facilitating the transfer of oxygen from oxyhemoglobin to sites of oxidation in muscle cells (Fruton and Simmonds, 1958). Hemoglobin is compartmentalized in red blood cells (erythrocytes), and accounts for over 90% of the total protein of those cells (Davies, 1961). Erythrocytes are formed in the bone marrow; this process is called hematopoiesis. Erythrocytes are continuously destroyed and replaced, with an average life span of 120 days. Iron released by the normal bloodcell destruction can be used again to form hemoglobin, practically without loss. Hemoglobin binds oxygen; after release of its oxygen to the tissues, hemoglobin binds carbon dioxide in the venous blood. Carboxyhemoglobin releases carbon dioxide in the lung in exchange for oxygen. Enzymes containing Fe include catalase, cytochrome A, B, and C, lactoperoxidase in milk, and verdoperoxidase in leucocytes, succinate dehydrogenase, nicotinamide-adenine dinucleotide, reduced (NADH)-coenzyme QIO reductase, a phosphatase in swine uterine fluid, and glutamate formimino-transferase (Fruton and Simmonds, 1958; Conrad et al., 1980). Enzymes activated by Fe ions include tryptophan peroxidase-oxidase, aconitase, homogentisic oxidase, hyroxyanthranilate cleavage enzyme, phenylalanine hydroxylase, and histidine decarboxylase (Fruton and Simmonds, 1958). Catalase and peroxidase enzymes break down peroxide molecules in the presence of reducing agents. In normal feather pigmentation for certain breeds of poultry and coloration in mink, Fe may be an essential component of an enzyme involved in melanin formation. The cytochromes contain heme as the active site, with the Fe-porphyrin ring functioning to reduce ferrous Fe and ferric Fe with the acceptance of electrons. The ability of Fe to change between the divalent and trivalent state allows the cytochromes A, B, and C to participate in the electron transfer chain. The cytochromes function as electron carriers, linking the oxidation of substrate with the reduction of molecular oxygen in aerobic metabolism. Iron plays a significant role in the tricarboxylic acid (Krebs) cycle, as all of the 24 enzymes in this cycle contain Fe either at their active centers or as essential cofactors.

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211

Iron functions have been shown to be related to normal thyroid hormone production (Beard et al., 1989) and disease resistance (Weinberg, 1984; Kuvibidila et al., 2001). Iron is needed for learning and cognitive function in laboratory animals (Kwik-Uribe et al., 2000; Piero et al., 2001), monkeys (Golub et al., 2000) and humans (Krebs, 2000; Youdim, 2001). Some behavioral consequences of Fe deficiency relate to dopamine transporter functioning (Hunt et al., 1994; Erikson et al., 2000).

VI. REQUIREMENTS The net requirements for Fe are the sum of the amounts laid down in the blood and tissues in the process of growth and the amounts lost in feces, urine, and sweat, in blood loss, in parturition, and in milk and eggs. Conversion of physiological requirements into dietary requirements is made difficult by variations among individuals in absorptive capacity and among foods and food combinations in the Fe bioavailability. The position is further complicated by the ability of the body to increase Fe absorption during Fe deficiency (Brody, 1999; Underwood and Suttle, 1999). Iron requirements are influenced by the chemical form or combination in which the mineral is ingested and by the amounts and proportions of other components of the whole diet. Feeds of animal origin such as meat meal and fish meal are better sources of Fe per unit than feeds of plant origin. The availability of Fe in plant foods such as beans, peas, corn, bread, and rice are poor ranging from less than 1 to 10% (Brody, 1999). However, the non heme in meat, fish, chicken, and liver may be about 20% available. The Fe of simple Fe salts is better absorbed than the Fe in ordinary feedstuffs (see Sections V and IX). High levels of dietary P and phytate reduce Fe absorption, presumably by the formation of insoluble ferric phosphate and phytate; and high dietary levels of several divalent metals, notably Cu, Mn, Pb, and Cd, increase Fe requirements by competing for absorption sites in the intestinal mucosa (Underwood and Suttle, 1999). Estimated Fe requirements for various animal species and humans are presented in Table 7.1. The suggested Fe dietary (dry basis) requirement for ruminants is between 15 and 50 ppm. Young animals have higher requirements than do adults. Young ruminants fed on exclusive whole-milk diets (milk is low in Fe) can develop Fe-deficiency anemia within 2 to 3 months. Although quite variable, reserves of the calf generally are sufficient to prevent serious anemia if dry feeds are fed beginning in the first few weeks. Swine have the greatest problem maintaining Fe status because of low stores at birth and rapid growth rate. Because of the definite need to supply Fe to baby pigs born in confinement (see Sections VIII and IX), more accurate requirements are available for this class of animal than for other livestock species. The Fe requirements of swine decrease with age because of a decrease in the blood volume per unit of body weight and increased feed consumption (NRC, 1998). There is a large Fe requirement in chickens during high egg production. A chicken egg

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TABLE 7.1 Iron Requirement for Various Species" Species

Purpose

Requirement

Reference

Chickens

Leghorn-type (}-6 wk Leghorn-type 6-18 wk Leghorn-type laying Leghorn-type breeding Broilers-all classes All classes All classes All classes Growing All classes All classes Growing Breeding, lactating Channel Catfish Growing All classes All classes Growing Adults, male Adults, female Adults, pregnant

80 mg/kg 60 rng/kg 50 mg/kg 60 mg/kg 80 mg/kg 60-120 rug/kg 50-80 mg/kg 50 mg/kg 15--43 mg/kg 30-50 mg/kg 40-50 mg/kg 40-100 mg/kg 80 rug/kg 30 mg/kg 80 mg/kg 35 mg/kg 35 mg/kg 50 rug/kg 8-11 mg/day 8-18 mg/day 27 rug/day

NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (1985b) NRC (1989) NRC (1998) NRC (1998) NRC (1993) NRC (1986) NRC (1995) NRC (1995) NRC (1995) DRI (2001) DRI (2001) DRI (2001)

Japanese quail Turkeys Beef cattle Dairy cattle Sheep Horses Swine Fish Cats Rats Mice Guinea pigs Humans

"Expressed as per unit animal feed either on as-fed (approximately 90% DM) or dry basis (see Appendix Table I).

contains about 1.1 mg Fe. A minimal Fe dietary requirement of 35 to 45 ppm was needed for laying hens to maintain a normal hematocrit, but for optimum hatchability, was somewhat higher at 55 ppm (Leeson and Summers, 2001). Typical diets (except milk) are generally high enough in Fe to supply requirements for farm livestock. During the reproductive years, adult women lose an average of 20 mg Fe monthly as a result of menstruation and approximately 800 mg Fe for each pregnancy. These losses are in addition to an approximately I mg daily Fe loss from exfoliation of intestinal epithelial cells (Fairbanks, 1978). Daily Fe requirements are 8 to II mg for adult males, 8 to 18 mg for females, 27 mg for pregnant females, and 6 to 12 mg for children (DRI, 2001).

VII. NATURAL SOURCES The Fe content of most feed ingredients is highly variable, reflecting differences in soil and climatic conditions as well as differences in variety or processing procedures. The Fe level in herbage plants is basically determined by the species and type of soil on which the plants grow and can be greatly affected by soil

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TABLE 7.2 Mean Forage Iron Concentratlons" Country Argentina"

Colombia" Dominican Republic" Bolivia" Guatemala' Malawi"

Season

Mean (ppm)

Critical concentration (cc)(%)

Percentage below cc

Wet Wet Dry Dry Wet Dry Wet Dry Wet Dry

384 139 171 154 \78 \22 378 661 207 195

50 30 30 30 30 30 30 30 50 50

3 0 0 0 0 0 0 0 6 3

'Critical concentrations are based on ruminant needs (McDowell, 1985). "Based on 340 samples (Balbuena et al., 1989). 'Based on 35 and 36 samples for the wet and dry seasons, respectively (Vargas et al., 1984). dBased on 69 samples (Jerez et al., 1984). "Based on 16 samples for the wet season (McDowell et al., 1982) and 84 for the dry season (Peducasse et al., 1983). 'Based on 84 samples for both the wet and dry seasons (Tejada et al., 1987). sBased on 48 and 21 samples for the wet and dry seasons, respectively (Mtimuni et al., 1990).

contamination. Mitchell (1963) indicated that soil contains 20 to 100 times the Fe content found in pastures grown on that particular soil. Acid soil conditions favor availability and plant uptake of Fe. Even plants grown on neutral or slightly alkaline soils often contain quite high levels of Fe. Typical Fe contents of livestock feedstuffs are given in Appendix Table II. Most forages contain Fe concentrations considerably in excess of the requirements of herbivorous animals (Table 7.2). Beeson (1941) reported that cultivated grasses and legumes range from 100 to 700 ppm Fe, although values in excess of 1000 ppm have been noted. Of 256 forage averages in the 1974 Latin American Tables of Feed Composition, only 3.5% contained less than 30 ppm Fe (McDowell et al., 1974). Of 192 and 120 forage samples collected during the dry and wet seasons in northern Mato Grosso, Brazil, mean Fe concentrations were 212 and 263 ppm, respectively (Sousa et al., 1981). However, other reports from Panama note that 7 out of 28 locations averaged less than 30 ppm Fe for samples of Hyparrhenia rufa (Chicco, 1972). Most cereal grains contain 30 to 60 ppm Fe, and species differences appear to be small, although 10 and 20 ppm have been recorded for Egyptian-grown corn and barley, respectively (Abou-Hussein et al., 1970). Legumes and oilseeds are richer in Fe than the cereal grains and may contain 100 to 200 ppm Fe. Bioavailability of Fe in cereal grains and oil seeds is reduced because ofphytate in these feeds (Hurrell et al., 1992; Zhou and Erdman, 1995). Supplemental microbial phytase is highly effective in releasing phytate-bound Fe (Stahl et al., 1999). Feeds of animal origin, other than milk and milk products, are rich sources of Fe. Meat meals and fish meals commonly contain 400 to 600 ppm, and blood meals,

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Iron

more than 3000 ppm Fe (Morris, 1987). Dried skimmed milk, whey, and buttermilk powders used in normal feeding practices vary greatly in Fe content because of variable contamination during processing and storage. In one study (Blaxter et al., 1957), milk, as it comes from the udder, varied from 0.18 to 0.31 ppm Fe. In contrast, values after the contamination of usual handling may be substantially higher, at an average of about 0.5 ppm (Underwood, 1977). In a study of Brahman beef cows, Fe concentration in colostrum milk was relatively high at 1.04 ppm, but declined to 0.51 after 3 months (Salih et al., 1987). Many of the minerals used to supply the Ca and P needs of animals contain Fe. Ground limestone, oyster shell, and many forms of calcium phosphate used as mineral supplements frequently contain 200 to 500 ppm Fe. Drinking water can also be a nutritionally important source of Fe, with considerable variation in Fe content (see also Chapter 17 of this volume). The richest Fe sources for humans are the organ meats (liver and kidney), egg yolk, dried legumes, cocoa, cane molasses, and parsley. Poor sources include milk and milk products, white sugar, white flour and bread (unenriched), polished rice, potatoes, and most fresh fruit (Morris, 1987). Boiling in water can reduce the levels of Fe in vegetables by as much as 20% (Skeets et al., 1931), while milling lowers the Fe content in white flour. Only limited information is available on Fe bioavailability of natural feed sources. Feedstuffs in which Fe is complexed to heme (i.e., animal by-products) show better biological availability than to those containing nonheme Fe (e.g., chlorophyll-poor plant ingredients) (see Section 6). However, meat was also shown to enhance nonheme Fe absorption in swine diets (South et al., 2000). Swain et al. (2002) suggest that enhancement of nonheme Fe by beef may be due to peptides produced during gastrointestinal digestion and that histidine content may be important. Fritz et al. (1970) found that Fe in fish-protein concentrates had relative biological values ranging from 8 to 53%. Relative biological values for animal byproduct feedstuffs may be estimated to be 50 to 60%, and probably higher for blood meals (Conrad et al., 1980).

VIII. DEFICIENCY

A. Effects of Deficiency Iron deficiency affects many systems through the reduction in tissue oxygenation resulting from decreased hemoglobin concentration. Anemia may occur whenever the available supply of Fe becomes deficient relative to the needs for hemoglobin formation. Signs of a lack of Fe, in addition to anemia and related blood changes, include lower weight gains, listlessness, inability to withstand circulatory strain, labored breathing after mild exercise, reduced appetite, and decreased resistance to infection. For humans and other species, Fe deficiency in the young can result in learning and cognitive limitation as well as behavioral consequences. Iron

Deficiency

215

deficiency is to be expected primarily in young, rapidly growing animals that have limited access to Fe in their environment and in their feed, and particularly during the suckling period, since milk is very low in Fe. Iron deficiency is of limited practical significance in farm animals other than suckling pigs. For humans, it is one of the most common deficiencies in both industrialized and developing countries. 1.

SWINE

Piglet anemia is an uncomplicated Fe deficiency, completely preventable by farrowing piglets in conditions that permit access to soil or pasture, or by direct administration of supplemental Fe to the newborn pigs (Kleinbeck and McGlone, 1999). Piglets denied access to sources of Fe other than sows milk develop anemia within 2 to 4 weeks of birth. The anemia is typically described as a hypochromicmicrocytic type. Blood hemoglobin levels fall from a normal of about 10 g/dl to as low as 4 g/dl, Mortality at this time is high, but surviving piglets begin a slow spontaneous recovery at 6 to 7 weeks, when they begin to eat the sows food and undertake foraging. Iron deficiency in the nursing pig may vary from a borderline chronic anemia to acute anemia. One of the first signs of chronic anemia is a roughness of haircoat. The hair is dull, coarse, and stands erect. The skin becomes wrinkled (Fig. 7.3), and the normally pink mucous membranes became pale. Pigs are listless; the head and upper eyelids droop; the ears and tail hang limp. Subcutaneous edema may appear in neck, shoulder, and limb areas (Conrad et al., 1980), and anemic pigs do not have the characteristic pink ears and snout. This may be seen before any other signs are evident (Miller, 1981). A sign of a more acute anemia is labored breathing or a spasmodic movement of the diaphragm muscles following exercise, referred to as thumps. Fast-growing pigs

Fig.7.3 Iron deficiency. Left, anemic pig. Note listlessness and wrinkled skin. These signs, along with paleness about eyelids, ears, and nose, as well as low hemoglobin, are typical of baby pig anemia. Right, normal pig given iron. (Courtesy of H.D. Wallace, University of Florida, Gainesville)

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Iron

may die suddenly of anoxia. Iron deficiency may lower the resistance of the pig to disease, and respiratory problems and enteritis may appear more frequently in chronically anemic pigs. Many reports have also demonstrated a reduced rate of weight gain by anemic pigs (Hannon, 1971; Conrad et al., 1980). Necropsy findings further confirm Fe deficiency (Conrad et al., 1980). Clear fluid is found in body cavities; the heart is dilated, pale, and soft; the lungs are edematous and may be collapsed. The liver is enlarged, mottled, and infiltrated with fat. All organs have a lighter than normal color. The blood is thin and watery. The baby pig develops a physiological anemia caused by a rapid increase in blood volume within the first day and a half of life. This poor start is compounded by an Fe deficit brought about by a combination of the following factors: (1) unusually low Fe stores at birth, compared with the newborn of most other species; (2) absence of the polycythemia of birth common to other animal species; (3) low levels of Fe in sows milk; and (4) very rapid early growth rate compared to other mammalian species (Underwood and Suttle, 1999). Piglets normally reach four to five times their birth weight at the end of 3 weeks and eight times their birth weight by 8 weeks, imposing Fe demands much greater than can be supplied by the sow's milk. Such a rapid growth rate requires the retention of 7 to II mg Fe/day, whereas only about I mg/day is obtained from milk alone (Venn et al., 1947). The young pig is therefore highly dependent on endogenous sources of Fe when consuming only milk. Anemia can occur in older pigs fed diets very high in Cu to promote growth and increase the efficiency of feed use, unless these diets are supplemented with Fe well above levels that are otherwise adequate (Gipp et al., 1974). Gipp and co-workers (1974) showed that the hypochromic, microcytic anemia induced by high dietary Cu is the result of an impairment of Fe absorption; this impairment is ameliorated by ascorbic acid. Dietary Cu levelsup to 60 ppm had no effect on liver Fe concentration, but 120 ppm Cu resulted in 50% decrease in liver Fe (Bradley et al., 1983). Iron deficiency in growing pigs has been found when a high dietary concentration of cottonseed meal is fed (Kornegay et al., 1961). Gossypol, a toxic component of cottonseed meal, forms a chelate complex with Fe, thereby severely reducing Fe absorption. Iron supplementation (see Section IX) counteracts gossypol toxicity. 2.

POULTRY

Most practical feedstuffs contain sufficient Fe to meet the nutritional requirements for poultry. A number of studies, however, have clearly demonstrated the need for Fe supplementation of certain semipurified diets (Conrad et al., 1980; Leeson and Summers, 2001). Hill and Matrone (1961) found that chicks fed a skim milk-sucrose-based diet containing 7 ppm Fe experienced anemia associated with increased abnormally shaped erythrocytes. For both chicks and poults, Fe deficiency results in a hypochromic macrocytic anemia. Birds deficient in Fe have lowered growth rates and reduced hemoglobin and hematocrit concentrations (AI-Ubaidi and Sullivan, 1963). Hill and Matrone (1961) also observed achromatrichia among Fe-deficient Rhode Island Red chicks, which normally produce red-brown plumage. For

Deficiency

217

turkeys, AI-Ubaidi and Sullivan (1963) reported that the Fe-deficient Broad Breasted Bronze poults also showed poor feathering and impaired plumage pigmentation. The loss of the normal feather color in poultry was the result both of the loss of an Fe-containing red pigment and of impairment in melanin synthesis in the feathers. For mature poultry, low-Fe diets did not affect body weight; with deficient diets, there were significant reductions in blood hemoglobin and hematocrit concentrations (Morek, 1978). Also by 3 weeks, Fe-deficient hens were found to deposit less than 50% as much Fe in egg yolk as control hens. Although Fe deficiency was not associated with a marked or consistent effect on egg production, the 36 to 46 week-old Fe-deficient hens were reported to experience a decrease in egg production sooner than did hens fed the Fe-supplemented diet. Working with laying hens, heart hypertrophy was observed in birds fed less than 55 ppm (Aoyagi and Baker, 1995). Iron deficiency in breeding hens is characterized by embryonic mortality during the ninth through fifteenth days of incubation. The peak in mortality was associated with severe hypochromic anemia in embryos surviving to 10 days of incubation (Morek, 1978). The few chicks that survived to hatching were anemic and had only about one-half of the hematocrit and hemoglobin concentrations of day-old chicks from Fe-adequate hens. 3.

RUMINANTS

Iron deficiencies are most likely to occur in young ruminant animals because milk is low in Fe. Hibbs et al. (1961) reported that calves may be deficient in Fe at birth since 30% of those born in a 12-year U.S. study had low hemoglobin values « 9 gj100 ml). Calves fed milk diets may develop anemia within 8 to 10 weeks. Newborn calves receiving milk have increased hemoglobin formation as a result of supplemental Fe (Thomas et al., 1954). Experimentally, Fe-deficiency anemia has also been produced in milk-fed lambs and in lambs raised on slotted wooden floors and fed a semipurified diet (NRC, 1985b). Twin calves are more likely to develop anemia than single calves, because they compete for a limited maternal supply of Fe (Kume and Tanabe, 1994). Young calves fed an exclusive milk diet exhibit a microcytic, normochromic, or hypochromic anemia with marked decreases in liver nonheme Fe concentration and serum Fe (Furugouri, 1978). Clinical signs include anemia, lower weight gains, listlessness, inability to withstand circulatory strain, labored breathing after mild exercise, reduced appetite, atrophy of the papillae of the tongue, and blanching of visible mucous membranes. In addition to low performance, calves with Fe deficiency have high susceptibility to disease (Mollerberg et al., 1975). The incidence of scours is higher in anemic than in normal calves. With Fe treatment, anemic calves respond promptly in performance, and hematological and tissue parameters for Fe are restored. Light-colored veal is associated with low levels of muscle myoglobin and restricted Fe intakes. An objective in the rearing of veal calves is to produce light pink meat to satisfy a growing consumer demand in the United States. Meat from

218

Iron

anemic veal calves contains less hemoglobin Fe, myoglobin, and cytochrome C than that from normal calves. Hemoglobin levels can fall to 50% of normal even with normal growth (Underwood and Suttle, 1999). Thus, there is a conflict between healthy veal calves and veal that appeals to consumers (Furugouri, 1978). Heavy infestation with intestinal parasites results in Fe-deficiency anemia in lambs and calves (Campbell and Gardiner, 1960). Anemia can result from the direct loss of blood via blood-sucking parasites, increased rate of degradation of blood cells, and a depression of hematopoiesis from toxic substances produced by the parasites. Iron deficiency seldom occurs in older ruminants unless there is considerable blood loss from parasitic infestations or disease. Iron deficiency is considered rare for grazing livestock due to generally adequate pasture concentrations and contamination of plants by soil (see Chapter 17). Soil contamination of forages and direct soil consumption often provide excess quantities of dietary Fe. In New Zealand, annual ingestion of soil can reach 75 kg for sheep and 600 kg for dairy cows (Healy, 1974). Iron deficiency may be a problem when ruminants are fed low quality forages, such as straw, for extended periods (Sen and Ray, 1964). These investigators noted that Fe supplementation reduced weight losses in lactating cattle and produced more rapid gains in suckling calves. Iron deficiency has been reported in Florida (Fig. 7.4) when cattle grazed forages grown on white and gray, sandy loam and fine

Fig.7.4 This weak (Fe-deficient) 12-year-old cow, which was grazing on a Blanton fine sand (yellow) soil in Florida, had to be helped up. Her hemoglobin was only 4.8 g/ I00 ml of whole blood. After being given supplemental Fe (ferric ammonium citrate), the hemoglobin value increased to 12.6 g/IOO ml, and she regained body condition and strength. (Courtesy of the late R.B. Becker. University of Florida. Gainesville)

Deficiency

219

sand soils (Becker et al., 1965). When Florida cattle are pastured on light, sandy soils, and have heavy insect or parasite infestations, the additional Fe has been useful. The hemoglobin levels, as well as the condition of the animals, have improved under such treatment (Davis, 1951). Other investigators who fed supplemental Fe revealed no production benefit when older animals had been consuming typical diets. However, Fe deficiency exists and, in some cases, supplemental Fe has produced marked improvement (Thomas, 1970). 4.

HORSES

The primary signs of Fe deficiency in horses are microcytic and hypochromic anemia. In severe cases of anemia, the horse will breathe hard in an effort to get enough oxygen to the various tissues of the body, and to make the available hemoglobin carry as much oxygen as possible. Inadequate oxygen is especially critical to working or racing horses. An anemic horse is more susceptible to stress factors and diseases (Cunha, 1990) as well as reduced growth, scouring, and pneumonia. Anemic horses lack a healthy pink color, and their blood looks watery at necropsy. The horse also becomes weak, inactive, develops a rough hair coat, and tires very quickly. Although young, milk-fed foals are most susceptible to this anemia, Fe deficiency is not a practical problem in foals or mature horses at any performance level (NRC, 1989). An exception may occur when horses are heavily parasitized (Cunha, 1990). 5.

OTHER ANIMAL SPECIES

a. Dogs and Cats. For both puppies and kittens, a hypochromic, microcytic type of anemia is produced on low-Fe diets (NRC 1985a, 1986). After weaning, animals that receive a certain quantity of Fe-rich feeds (e.g., meat) should not develop anemia, unless blood loss is a problem.

b. Laboratory Animals. Iron deficiency results in a microcytic, hypochromic anemia in both rats and mice (NRC, 1995). Mice show reduced birth weights and litter sizes, and rats have white incisor teeth, cardiomegaly, splenomegaly, and enlarged cecum (Cusack and Brown, 1965). Black-haired rats fed a Fe- (or Cu-) deficient diet developed achromotrichia. Rats deficient in Fe have a compromised immune system including impaired phagocytosis and natural killer cell activity, and reduced antibody production (Hallquist et al., 1992). Mice that received a low Fe diet (2 to 10 ppm) for 30 days were characterized by low body weights, anemia, and suppressed T-Iymphocyte-dependent functions associated with antibody production (NRC, 1995). Marginal Fe-deficient mice demonstrated significantly lower grip strength and cognitive function (Kwik-Uribe et al., 2000). c. Rabbits. Iron deficiency in rabbits produces microcytic, hypochromic anemia (NRC, 1977). At birth, rabbits have a very large Fe reserve (liver), so the newborn

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Iron

are not dependent on a supply of Fe in the milk. Iron deficiency is unlikely in rabbits under practical conditions owing to the generous distribution of the mineral in feedstuffs.

d. Faxes and Mink. In mink, Fe-deficiency anemia occurs if diets contain high levels of certain kinds of fish, e.g., coalfish, whiting, blue whiting, and hake (Furugouri, 1978). These diets cause an impairment of Fe absorption following overt Fe-deficiency anemia. Clinical signs of Fe deficiency include microcytic, hypochromic anemia, severe emaciation, growth retardation, high mortality, roughened fur, and lack of underfur pigmentation (cotton-fur syndrome, Fig. 7.5). For foxes, Fe-deficiency signs also include anemia and depigmentation of underfur (Rimeslatten, 1959). Both trimethylamine oxide and formaldehyde, found in the fish digestive tract, have been identified as causative factors of cotton fur (NRC, 1982). e. Fish. Iron deficiency has been shown to cause hypochromic, microcytic anemia in common carp, red sea bream, yellowtail, eel, and brook trout (NRC, 1993). For catfish, the onset of mortality was earlier for fish fed an Fe-deficient diet (Lim et al., 2000).

f Nonhuman Primates. Fitch et af. (1964) reported that diets containing soybean protein resulted in a microcytic, hypochromic anemia characteristic of Fe deficiency in monkeys. Amine et al. (1972) reported Fe-deficiency anemia in squirrel monkeys fed modified cow milk diets. For adolescent Rhesus monkeys, a combined Fe and zinc (Zn) deficiency affected behavior that was characterized by reduced activity, reduced participation in behavioral testing and slower response (Golub et al., 2000).

Fig. 7.5 Pelts of cotton (left) and normal (right) mink, parted to show underfur. The condition can be overcome by supplying iron parenterally. (Courtesy of F.M. Stout, J.E. Oldfield, and J. Adair, Oregon State University, Corvallis)

Deficiency

6.

221

HUMANS

Iron deficiency is one of the most common nutritional disorders in the world, affecting nearly two billion people (WHO, 1994), and the deficiency affects especially infants, young children, and women of the reproductive years. When Fe deficiency is sufficiently severe, red blood cell synthesis becomes impaired, and anemia results. Prolonged Fe deficiency results in the development of hypochromic, microcytic anemia accompanied by a normoblastic, hyperblastic bone marrow containing little or no hemosiderin. Iron deficiency in human adults is manifested clinically by listlessness and fatigue, palpitations on exertion, and sometimes by a sore tongue, angular stomatitis, erythema at the corners of the mouth (cheilitis), dysphagia, and koilonychia (spoon nail). In children, anorexia, reduced growth, and decreased resistance to infection are commonly observed, but oral lesions and nail changes are rare (Morris, 1987). Low Fe status among adolescents may limit their growth spurt (Brabin and Brabin, 1992). Iron deficiency results in a significant reduction in physical activity and performance. A prolonged cardiorespiratory recovery period after exercise has been observed in anemic women (Anderson and Barkue, 1970). Low income postpartum women bear a substantially greater Fe deficiency risk than never pregnant women (Bodnar et al., 2002). The prevalence of Fe deficiency anemia is estimated to be 25% in infants and children worldwide and 50% of women and children in the less developing countries (DeMaeyer and Adiels-Tegman, 1985). According to data from the third National Health and Nutrition Examination Survey (Alaimo et al., 1994), the prevalence of Fe-deficiency anemia in children I to 2 years old in the United States was 3% and the prevalence of Fe deficiency without anemia was 9%. In many tropical regions, anemia, Fe deficiency, malaria, and multiple helminth infections coexist and are interrelated. Sub-Saharan Africa epitomizes this situation, although similar situations exist in equatorial South America, and south and southeast Asia. In these communities, anemia is typically prevalent and severe, especially in pregnant women and young children, and is often an important cause of mortality (Stoltzfus, 1997). For example, the case fatality rate for children admitted with severe anemia to one hospital in rural Tanzania was 6.1 % (Alonso Gonzalez et al., 2000). The consequences of maternal anemia may be serious, with reported associations ranging from preeclampsia to low birth weight and increased risk of maternal deaths (Spinillo et al., 1994; Sapre and Joshi, 1996). The relationship between anemia or Fe deficiency anemia and increased risk of preterm delivery «37 wk gestation) has been supported by several studies (Scholl and Hediger, 1994; Zhou et al., 1998; Scholl and Reilly, 2000). Behavioral disturbances such as pica, characterized by abnormal consumption of nonfood items such as soil (geophagia) and ice (pagophagia), are often present in Fe deficiency. Physiologic manifestations of Fe deficiency have also been noted in immune function, cognitive performance and behavior, thermoregulatory performance, energy metabolism, and exercise or work performance (Beard and Dawson, 1997; Haas and Brownlie, 2001). Iron-deficiency anemia alters the ability

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of humans to maintain body core temperature during acute cold exposure. Investigations have documented clear alterations in thermoregulation, the thyroid system, and the sympathetic nervous system (Beard and Dawson, 1997). Symptoms of Fe deficiency include lethargy, lack of concentration, lower intelligence scores, and decreased cognitive and attentional processes (ldjadinata and Pollitt, 1993; Pollit, 1993;Walter, 1993).There is evidence that Fe plays a role in neurobiological processes. Various studies have proposed changes in neurotransmitter metabolism (Beard et al., 1994; Chen et al., 1995), myelin formation (Larkin and Rao, 1990) and hippocampal functioning (Rao et al., 1999). Recent studies in humans clearly demonstrate that in early life, the brain is quite susceptible to Fe deficiency (Kwik-Uribe et al., 1999; Rao et al., 1999; Piero et al., 2000). Verbal learning, memory, lower intelligence, and physical performance may be impaired in Fe-deficient adolescent girls (Bruner et al., 1996). A 21-week experiment showed that volunteers with borderline anemia, as measured by blood hemoglobin, were less able to concentrate than those with higher hemoglobin (Kretsch and Green, 2001). Girls between the ages of 12 and 16 with Fe-deficiency anemia posted significantly lower math scores than did non-anemic females or males (Lord, 2001). Infants and children in developing countries are particularly vulnerable to Fe deficiency (Fig. 7.6). Often nursing infants receive only their mother's milk, which is extremely low in Fe, until the next child comes along. The Fe reserve of the human infant is usually exhausted before the end of the sixth month. To complicate this, the weaned child frequently receives very little protein and Fe-rich food (e.g., meat), but rather high-carbohydrate, low-Fe foods including cassava, potatoes, bread, and white rice. Not only infants but whole populations, particularly in developing countries, have blood-sucking parasites, especially hookworms (Stoltzfus et al., 2000). The amount of blood lost varies with the type of parasite and with the number present. One hookworm may consume 1 ml of blood daily, and an infestation of over 100 results in devastating blood loss. In developing countries not only are Fe intakes low but the bioavailability of Fe from the diets is often very low, owing mainly to the low availability of factors facilitating nonheme Fe absorption (fish, meat, and ascorbic acid). An Fe-poor diet and rapid growth are prime causes of Fe deficiency in infants and preschool children. However, in many such children, intestinal bleeding (from hookworm infestation or from bovine milk sensitivity) may also be a factor. Similarly, the suboptimal Fe content of the diet of young women contributes to the high prevalence of Fe deficiency in this group, although blood loss is the major factor (Fairbanks, 1978). Iron need is increased when there is either rapid growth or accelerated Fe loss e.g., from bleeding, hemoglobinuria, pregnancy, or lactation. In adults, Fe deficiency must be taken as evidence of Fe loss, which is usually through gastrointestinal bleeding.

Assessment of Iron Status The characteristic Fe-deficiency anemia is of a hypochromic, microcytic type. However, hypochromic anemia may also occur when the total Fe content of the

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Fig. 7.6 A child with iron deficiency anemia in Montero, Bolivia. The vast majority of children in this tropical region suffered from the deficiency as a result of carbohydrate-rich diets, low in iron, as well as high infestations of the blood sucking hookworm parasite. Hematocrits of the population were very low, being in the 20s, with several values as low as 6. (L.R. McDowell, University of Florida, Gainesville)

body is normal. In livestock or humans with a typical Fe-deficiency anemia, the hemoglobin level is reduced, the red cells are smaller in size and contain less hemoglobin than normal. The concentration of Fe in plasma-bound transferrin is reduced, but the level of transferrin itself is increased (Hallberg, 1984). The serum ferritin level, which is related to the size of the Fe stores, is usually very low. Departure from normal levels of serum Fe, total Fe-binding capacity, percentage transferrin saturation value, and hemoglobin and hematocrit values can all be used to diagnose Fe deficiency in livestock. The most commonly used screening methods for the presence of Fe deficiency in the population are the measurements of hemoglobin or hematocrit concentration for the presence of anemia (WHO, 1994). These measurements are relatively simple and cheap, can be carried out under field conditions, and values below a certain cut-off point indicate or define that anemia is likely to exist. However, low hemoglobin and hematocrit values are not sensitive indicators of early Fe deficiency stages because they only occur when storage Fe is severely depleted. Their use is often limited to diagnosis and confirmation of Fe deficiency (Miller and Stake, 1974). Evaluation of bone marrow hemosiderin Fe is usually regarded as the reference standard for assessment of Fe stores (Holyoake et al., 1993). It is considered as the

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most sensitrve and specific indicator of body Fe stores and unlike other conventional laboratory measures of Fe status, it is not confounded by infection or inflammation. This technique, however, is costly, invasive, painful. and impractical for routine examinations (Ahuwalia et al., 2000). Serum ferritin and percentage saturation of transferrin are early indicators of Fe deficiency. For Fe overload plasma Fe, transferrin saturation and plasma ferritin are good diagnostic criteria (Beard and Dawson, 1997). An assessment of Fe deficiency in ruminants can be made using reduced transferrin saturation « 13 to 15%), serum Fe (