The Laboratory Swine

www.crcpress.com preface Laboratory swine is a relatively unknown species, although the use of swine in research date...

Author: Peter J. A. Bollen | Axel Kornerup Hansen | Helle J. Rasmussen | P. J. A. Bollen

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preface Laboratory swine is a relatively unknown species, although the use of swine in research dates back centuries. However, the number of swine used in research stands no comparison with the number of rodents used. Recently, the pattern of animal use has changed because of a move toward replacement, reduction, and refinement in animal experimentation. This has caused an increase in the number of swine, because swine, and miniature swine in particular, are seen as an alternative to other nonrodent species such as dogs and primates. This book in the Laboratory Animal Pocket Reference Series was written to provide a source of information on laboratory swine. It is aimed at animal caretakers, technicians, and investigators, but the laboratory animal veterinarian may also want to use this book as a reference. This book provides an overview, not an in-depth review of the use of swine. Extensive references are given for further study. It is our hope that this book will contribute to the humane use of laboratory swine with the housing, husbandry, veterinary care, and experimental techniques that are appropriate to the species-specific physiology and behavior of swine.

©1999 CRC Press LLC

the authors Peter Bollen, B.Sc., M.Sc., has studied veterinary medicine at the University of Utrecht, the Netherlands, turning to laboratory animal science during the preclinical part of this study. He graduated as an experimental zoologist, after which he continued his studies of laboratory animal science at the Royal Veterinary College in London, England, obtaining his M.Sc. degree in 1994. He joined Ellegaard Göttingen Minipigs, a laboratory swine breeding station, where he is currently the scientific manager. In 1998, he began a research project determining the nutritional requirements of miniature swine, leading to a Ph.D. thesis. The work is supported by the Danish Academy of Technical Sciences, and is being carried out at the Biomedical Laboratory of the University of Southern Denmark in Odense, Denmark. Axel Kornerup Hansen, Dr.Vet.Sci., DVM, is professor of Laboratory Animal Science and Welfare at the Royal Veterinary and Agricultural University in Copenhagen, Denmark, from which he also graduated in veterinary medicine in 1985. After a period in small animal practice and a laboratory animal breeding center, he was appointed assistant director at the Department of Experimental Medicine at the University of Copenhagen, where he was in charge of the health monitoring laboratory. In 1996 he earned his doctorate with a thesis about health monitoring and microbiological quality of laboratory rats. In his present position, Dr. Hansen's main research interest is in laboratory animal infections, as well as methods for improving the welfare of animals used for research. Dr. Hansen has published


more than 100 scientific papers and chapters in textbooks, and has been involved in setting up European guidelines for health monitoring of a range of animal species.

Helle Juul Rasmussen, DVM, is appointed as a veterinary surgeon at the Biomedical Laboratory of the University of Southern Denmark in Odense, Denmark. She graduated in veterinary medicine from the Royal Veterinary and Agricultural University in Copenhagen, Denmark, in 1991, and worked in veterinary practice from 1991 to 1996. Since 1997 she has worked in the field of laboratory animal science, and was appointed to the Department of Experimental Medicine at the University of Copenhagen, Denmark. In 1998 she joined the Biomedical Laboratory of the University of Southern Denmark in Odense, Denmark. She is currently involved in research and teaching programs dealing with surgery and microsurgery, anesthesia, and pain relief of laboratory animals.


contents 1 IMPORTANT BIOLOGICAL FEATURES Breeds The Landrace The Yorkshire or Large White The Duroc The Hampshire The Piétrain Other Breeds Hybrid Breeds Miniature Breeds The Vietnamese Potbelly Pig Nomenclature Behavior Anatomical and Physiological Features Integument and Skeleton Digestive System Abdominal Organs Cardiovascular System Pulmonary System Lymphatic and Endocrine System Normative Values 2 HUSBANDRY Housing Environmental Conditions Temperature and Humidity Ventilation Illuminaton


Noise Environmental Enrichment Group Housing Individual Housing Nutrition Nutrient Requirements Feeding Levels Sanitation Frequency Methods Transportation Record Keeping 3 MANAGEMENT AND QUALITY ASSURANCE Microbiological Management and Quality Impact of Infections on Animal Research using Swine Zoonotic Diseases Legal Regulations Precautions to Prevent Infections Common Findings in Health Monitoring Genetic Monitoring Accreditation AAALAC ISO 9000 Other Organizations 4 VETERINARY CARE Clinical Examination of Swine Microbiological Sampling Diseases of Swine Therapeutic Treatment Pathological Examination Anesthesia and Analgesia Reactions of Swine to Sedation and Anesthesia Routes of Administration of Sedatives and Anesthetics Preanesthetic Management Premedication Anesthetic Induction Endotracheal Intubation General Anesthesia


Anesthetic Maintenance and Assessment of Anesthetic Depth Drugs used in Swine Anesthesia Analgesia Pain Control Pain Recognition in Swine Drugs used in Swine Analgesia Monitoring during Anesthesia Anesthetic Support Anesthetic Emergencies Malignant Hyperthermia/Porcine Stress Syndrome Postoperative Management Euthanasia 5 EXPERIMENTAL TECHNIQUES Restraint Sampling Techniques Blood Sampling Urine and Feces Cerebrospinal Fluid Bile and Pancreatic Excretions Administration of Compounds Basic Surgical Procedures Catheterization Catheter Maintenance Laparotomy Safety Testing of Chemicals and Drugs Necropsy 6 RESOURCES Associations Books Journals Internet Resources Swine Diet and Equipment BIBLIOGRAPHY


1 important biological features Swine are increasingly used as models in biomedical research. Developments in the field of immunology and xenotransplantation prospects have renewed interest in this species, the anatomy and physiology of which have notable similarities to those of man. Since their development at the beginning of the 1950s, miniature swine have been used in toxicity testing as a nonrodent species. The small size of miniature swine is obviously advantageous for housing and handling, but also for the quantity of test compound, which usually is available in only small amounts. Moreover, pressure from society to reduce the number of monkeys and dogs in biomedical research has caused an increase in the use of (miniature) swine.

breeds The wild boar, Sus scrofa, is the ancestor of all modern breeds of swine. First evidence of domestication in Europe is some 3500 years old, although the cradle of the domesticated swine is claimed to have been in China, about 10,000 years ago1. The American continent has no indigenous wild swine, except the distantly related Tayassuidae (peccaries). Sus scrofa is a member


TABLE 1.1: CLASSIFICATION

OF

SUIFORMES.

Order

Suborder

Family

Artiodactyla

Suiformes

Hippopotamidea

Genus

Tayassuidae

Suidae

Babyrousa

Phacochoerus

Potamochoerus

Hylochoerus

Sus


Species Hippopotamus amphibius (hippopothamus) Hexaprotodon liberiensis (pygmy hippopotamus) Pecari tajacu (collared peccary) Tayassu pecari (white-lipped peccary) Catagonus wagneri (Chacoan peccary) Babyrousa babyrussa (babirusa) Phacochoerus africanus (common warthog) Phacochoerus aethiopicus (Somali warthog) Potamochoerus porcus (red river hog) Potamochoerus larvatus (bushpig) Hylochoerus meinertzhageni (giant forest hog) Sus scrofa (Eurasian wild boar) Sus salvanius (pygmy hog) Sus verrucosus (Javan warty pig) Sus barbatus (bearded pig) Sus celebensis (Sulawesi warty pig) Sus phillippensis (Philippine warty pig) Sus cebifrons (Visayan warty pig)

of the swine family (Suidae), which includes species of nonruminant, even-toed ungulates. Suidae are omnivores, and are divided into five genera, with a total of 13 species. Through selection, numerous breeds of swine have been developed. Existing breeds are relatively young in comparison to the history of domestication of swine. Local Celtic swine with lop ears were widespread in European countries in the 18th and 19th centuries, and are considered to be the primogenitors of the Landrace. Crosses with Chinese breeds in the early 1800s,

Fig. 1.1 Landrace (Photo Leif Schack-Nielsen/Biofoto) and with English breeds in the late 1800s, established the Danish Landrace, which was widely used for the derivation of other national Landrace breeds. The Yorkshire or Large White, a breed with pointed, standing ears, originated in England and was exported to many countries around the world during the last half of the 19th century. The breed association for the American Yorkshire was established in 1893. Swine are numerous worldwide. In 1997, annual swine production counted 56 million in the United States. The total figure for the 15 countries of the European Union was 119 million, with Denmark, the


Netherlands, France, Spain, and Germany as the leading swine producing countries. These figures have not changed much during the last five years.2 A short description of common breeds follows, based on descriptions from Porter (1993)1 and Jones (1998)3, whereas the average weights are from Sambraus (1992).4

The Landrace The Landrace swine has a white-colored, long body and lop ears. It has a long head, with a slightly concave nose line. The Landrace breed is extremely variable in type and has its own distinct characteristics in different countries. The average weight of sows is 273 kg (601 lbs), and boars weigh 312 kg (687 lbs).

The Yorkshire or Large White The Yorkshire or Large White is a large, white breed of medium length. It has pointed, standing ears and a concave nose line. The sow is very fertile, with a litter size of 11, and weighs an average of 280 kg (617 lbs). The average weight of boars is 320 kg (705 lbs).

Fig. 1.2 Yorkshire (Photo Leif Schack-Nielsen/Biofoto)


Fig. 1.3 Duroc (Photo Leif Schack-Nielsen/Biofoto)

The Duroc The Duroc is a red swine with a large frame and semilop ears. The color varies from light to deep red. The Duroc is goodnatured. The average weight of sows is 300 kg (661 lbs), and boars weigh 350 kg (771 lbs).

The Hampshire The Hampshire is black with a white belt over the shoulder. Its body is compact and the legs relatively short. Its ears are pointed and standing, and its nose line is concave. The average weight of sows is 280 kg (617 lbs), and boars weigh 320 kg (705 lbs).

The Piètrain The Piètrain is a short breed with a broad, deep body. Its color is white with gray or black patches. It has short, pointed, standing ears. This breed is mainly used to improve meat quality in hybrid breeds. The Piètrain is very susceptible to stress. The average weight of sows is 266 kg (586 lbs), and boars weigh 277 kg (610 lbs).


Fig. 1.4 Hampshire (Photo Leif Schack-Nielsen/Biofoto)

Other Breeds In 1990, the National Association of Swine Records had registered only eight pure-bred swine. Besides the Landrace, Yorkshire, Hampshire, and Duroc, the Berkshire, Chester White, Poland China, and Spotted were registered.1

Hybrid Breeds Purebred swine are found almost exclusively in breeding herds. Modern production herds, however, utilize cross-breeding to produce hybrid grower swine. Hybrid breeds currently dominate the market; consequently, swine procured for the laboratory from an agricultural producer will most likely be of mixed breed. Triple-crossed, such as Yorkshire x Landrace (YL) sows with a Duroc (D) boar, are common.

Miniature Breeds Miniature swine are especially bred for research purposes. Many of the present breeds of miniature swine have their origin in the Minnesota (Hormel) minipig, which was bred from 1949 at the


Fig. 1.5 Göttingen minipig

Fig. 1.6 Yucatan micropig (Photo courtesy of Charles River Laboratories)


Fig. 1.7 Sinclair minipig (Photo courtesy of Sinclair Research Center)

Fig. 1.8 Hanford minipig (Photo courtesy of Charles River Laboratories)


Hormel Institute in Austin, Texas. Miniature swine derived from this population are the Göttingen minipig (1961) and the Sinclair minipig (1965). Other breeds are the closely related Hanford (1958) and Pitman-Moore (1969) miniature swine. A Mexican feral swine was introduced into the laboratory in 1960 and later referred to as the Yucatan minipig. From 1972, a smaller subline of the Yucatan was selected, the Yucatan micropig. Several other breeds have been established, including the Ohmini and Clawn minipig in Japan. There are two weight categories of minipigs: a 35-55 kg (77121 lbs) and a 70-90 kg (154-198 lbs) adult weight category. To the first category belong the Göttingen minipig and the Yucatan micropig, whereas the Hanford and Yucatan minipigs belong to the latter category.

The Vietnamese Potbellied Pig This is a small Asian black breed with wrinkled skin, especially on the face. Its ears are short and standing, and its abdomen is round, almost touching the ground. The Vietnamese potbellied pig is not well suited for the laboratory because of its vigorous character.

Nomenclature Since many different breeds of swine exist, each bred and selected for prevailing characteristics, it is essential to report in scientific publications which breed was used for a study in order to facilitate a true comparison of experimental results among laboratories. In commercial breeding, breeds generally are referred to with capitals, but this may not be desirable for the scientific community because of unfamiliarity with this system. A nomenclature system, as the one used for rodents, is not known for laboratory swine. Until international rules for swine nomenclature have been established, the scientifically correct way of presenting data includes breed, gender, age, and weight, besides supplier and housing conditions.5 The definitions of common terms used for swine, are given below: Swine, hog, or pig Denotation for the species Sus scrofa; the first two terms are commonly used in American


literature, whereas the latter is mainly used in British literature. Sow

Sexually mature female

Gilt

Sexually immature female

Boar

Sexually mature male

Barrow

Castrated male

Piglet

Juvenile swine

Porcine

Adjective used in relation to swine

behavior An important reason why swine became the subject of domestication is their behavior. Their feeding, sexual and social behavior, and typically docile nature and general adaptability, favored domestication. Compared to wild swine, domesticated swine are calmer, quieter, and less active, although they still share many behavioral characteristics with wild swine, such as social feeding and explorative behavior.6 Wild swine live in small social groups formed by sows and their offspring. Young males live in bachelor groups, whereas adult boars tend to be solitary. In groups of females a social dominance order is quickly established. Generally, the level of aggression, mainly expressed by butting or biting the neck and ears, quickly subsides in stable social groups. Sexually mature males may fight fiercely, especially in the presence of females. Newborn swine establish a social order, the teat order, within a few days after birth. As soon as a teat order has been established, each piglet consistently suckles the same teat. Dominant piglets generally occupy the more productive anterior teats. If young swine from different litters are mixed after weaning, a new dominance hierarchy will be established. Ranking is the result of aggressive interaction, but may take place without overt aggression. It exists as long as a group is together. Subordinates which have been separated from a group will be attacked after reintroduction, whereas a dominant animal may be separated and reintroduced without any trouble.7


Wild swine spend most of the day rooting and collecting food. Their feeding and explorative behavior are closely linked. During a day, swine eat many small portions. Swine are omnivorous and diurnal, with elevated activity during the evening. In the laboratory, activity of swine is related to the presence and activity of humans, rather than the light-dark cycle.8

anatomical and physiological features9, 10, 11, 12 Integument and Skeleton The skin of swine has anatomical and physical similarities with human skin; consequently, swine are often used as models for percutaneous absorption, skin toxicology, and wound-healing studies. Swine have short, sturdy bones. The number of ribs varies greatly, from 13 to 17, but most commonly 14 or 15 ribs are present. The bony structure of the thorax is considerably smaller than the external dimensions suggest. The skull is extremely sturdy and has a largely extended frontal sinus. Swine are used in osteoporosis research and cartilage-repair studies.

Digestive System Swine have an extensive dentition, with 44 elements. For this reason they are often used in dental research. The formula for the permanent teeth is i3/3, c1/1, p4/4, m3/3. The canine teeth or tusks can be found in both boars and sows, but are most developed in boars. The boar’s tusks grow throughout the animal's life. Swine are born with 8 teeth, the needle teeth (i1/1, c1/1). After two months, two more deciduous incisors and three of the deciduous premolars have erupted (i3/3, c1/1, p3/3). At 6 months, the first permanent teeth erupt. The swine is at least 18 months old before all permanent teeth have erupted. Swine are monogastric, and the stomach has a large fundus and a diventriculum. The area where the esophagus enters the stomach has nonglandular mucosa and is prone to peptic ulcers. The bile duct and pancreatic duct enter the duodenum. Similar to man, swine have an unbranched pancreatic duct, which makes swine good models for studying pancreatic secretion. The jejunum and ileum contain numerous patches of lymph nodes.


TABLE 1.2: BASIC

(RANGE) OF SWINE, AND 35-55 KG AND 70-90 KG.

BIOLOGICAL PARAMETERS

MINIATURE SWINE WITH ADULT WEIGHTS OF

Parameter

Swine

Miniature swine 35-55 kga 70-90 kgb

lifespan (yrs) body temperature (oC) chromosomes (2n)14 weight at birth (kg) weight at 6 mths (kg) weight at 1 yr (kg) weight at 2 yrs (kg) energy intakec (MJ/day)15 food intakec,d (kg/day) water intake (ml/kg/day)16

10-15 38-39

10-15 37-38

10-15 38-39

38 1.3-1.9 90-110

38 0.4-0.7 12-22

38 0.6-1.0 25-40

150-180 200-300 49-56

25-40 35-55 13.8-20.2

45-70 70-90 21.9-29.2

3.6-4.1

1.0-1.5

1.6-2.1

80-120

80-120

80-120

a) Yucatan micropig, Göttingen and Sinclair minipig b) Yucatan and Hanford minipig c) voluntary intake (ad libitum); metabolizable energy (ME) = 1.2 x W0.75 (MJ/day), calculated for the body weight (W) at 6 months of age d) voluntary intake (ad libitum); energy concentration (ME) = 13.6 MJ/kg (3265 kcal/kg)

The large intestines are coiled and voluminous. Because of the structure and large volume, the cecum lies to the left.

Abdominal Organs The liver has much fibrous tissue, giving the surface a net-like appearance. The pancreas has two lobes, which are difficult to identify. The kidneys have multiple papillae protruding into a central renal pelvis via the calyces. In structure and size they are similar to human kidneys. Lymph nodes are located along all major arteries in the abdomen. The uterine horns in sows are very long, and the cervix is tightly closed with mucosal ridges. The boar has large testes and various accessory genital glands. The ejaculate is voluminous, 200-500 ml. The penis is fibrous and has a corkscrewed tip. The preputial diverticulum produces a strong odor.


TABLE 1.3: HEMATOLOGICAL

PARAMETERS


Parameter


Swine17 18

Miniature swine 35-55 kga

RBC (106/mL) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) MCH (pg) MCHC (%) WBC (103/mL) Segmented neutrophils (%) Band neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%) Platelets (103/ml)

19 20

70-90 kgb

4.4-8.6 9.0-16.2 33.9-45.9 17.6-79.6 15.2-56.3 29.4-35.9 6.3-21.1 22.0-60.6

5.30-9.25 9.0-15.8 32-61 40.0-73.0 15.2-26.4 29.4-37.9 4.4-26.4 10.0-80.0

5.6-8.8 13.1-17.0 36.3-53.7 58.2-72.5 18.9-24.3 31.1-34.5 6.9-21.2 18.0-94.0

0-4.2 38.1-73.1 0-15 0-7.7 0-1.3 220-665

0-4.0 14.0-87.0 0-13.0 0-8.0 0-3.0 148-898

0-2.0 21.0-71.0 2.0-15.0 0-13.0 0-5.0 217-770

21

a) Yucatan micropig, Göttingen and Sinclair minipig b) Yucatan and Hanford minipig

Cardiovascular System The heart of the swine is small in relation to body size. The coronary distribution is similar to that of humans, but the left azygous vein ends directly in the heart instead of in the caval vein. Swine develop atherosclerosis of the major arteries after ingestion of a lipidous diet over a relatively short period.

Pulmonary System Swine have a tracheal bronchus to ventilate the right cranial lobe; consequently, endotracheal intubation should not continue as far as the tracheal bifurcation.

Lymphatic and Endocrine System The lymph nodes of swine are inverted, with the follicular centers in the medulla. The parathyroids are not located near the thyroids, but are related to the thymus. The right adrenal lies in close contact with the caudal caval vein, which disables total adrenalectomy.


Normative Values Biological, hematological, clinical chemical, and respiratory and cardiovascular parameters are presented in Tables 1.2-1.6. When comparing data, it is important to compare data from agematched, rather that weight-matched swine.13 However, variation between individuals and breeds may occur due to genotype, environment, and experimental procedures.

TABLE 1.4: CLINICAL

Parameter


CHEMICAL PARAMETERS


Swine

22

Miniature swine 35-55 kga

Glucose (mg/dL) Creatinine (mg/dL) Bilirubin (mg/dL) Cholesterol (mg/dL) Total protein (g/dL) Albumin (g/dL) Globulin (g/dL) A:G ratio Sodium (meq/L) Potassium (meq/L) Chloride (meq/L) Calcium (mg/dL) Phosphorus (mg/dL) AST (IU/L) ALT (IU/L) CK (IU/L) GGT (IU/L) LDH (IU/L)

48-135 1.2-2.0 0.0-0.3 50-140 2.25-8.15 0.5-4.3 1.4-3.6 1.1-3.5 133-153 3.1-6.2 96-117 5.5-15.7 4.8-9.8 14-56 5-78 52-326 14-34 140-1155

19 20

43-133 0.5-1.6 0.0-0.2 39-131 6.0-8.8 2.9-3.8 1.5-5.2 0.9-1.7 132-146 3.5-7.4 94-140 8.6-12.6 4.9-9.8 13-47 40-106 105-6000 25-78 462-1800



70-90 kgb 21 23 56-153 1.2-2.0 0.0-0.3 47-173 6.3-9.4 4.1-5.6 1.4-3.6 1.1-3.5 142-153 3.9-5.2 95-114 9.3-11.6 5.0-8.3 15-53 20-48 37-270 41-86 389-727

TABLE 1.5: RESPIRATORY AND CARDIOVASCULAR FUNCTION (MEAN + SD) OF CONSCIOUS SWINE, AND MINIATURE SWINE WITH ADULT WEIGHTS OF 35-55 KG AND 70-90 KG. Parameter

heart rate mean arterial blood pressure (mmHg) respiration rate arterial pH arterial pCO2 (mmHg) arterial pO2 (mmHg)

Swine

24

Miniature swine 35-55 kga 25

70-90 kgb 13 26

105 ± 10.6 102 ± 9.3

83 ± 15 97 ± 14

105 ± 7 89 ± 3

20 ± 7.48 40 ± 71 ±

20 ± 9 7.43 ± 0.03 40 ± 3 109 ± 17

25 ± 7.48 43 ± 84 ±

2.9 ± 0.03 2.3 3

4 ± 0.04 4 4


TABLE 1.6: REPRODUCTION PARAMETERS OF SWINE, AND MINIATURE SWINE WITH ADULT WEIGHTS OF 35-55 KG AND 70-90 KG. Parameter sexual maturity (months) minimum breeding age (months) estrus cycle (days) length of estrus (days) length of pregnancy (days) litter size weight at birth (kg) age at weaning (days)

Swine

7

Miniature swine

7

5-6

7

14 3 114

14 3 114

14 3 114

10-14 1.30 28-35

5-8 0.45-0.60 28-35

5-8 0.60-1.00 28-35



27 28

6

35-55 kga 4-5

70-90 kgb 6

29

1035/ch02 Page 17 Tuesday, November 9, 1999 1:37 PM

2 husbandry An important factor for the successful use of swine in biomedical research is knowledge about the specific husbandry requirements of swine. Housing, feeding, and care should be appropriate to the anatomy, physiology, and behavioral characteristics of this species. Changes in environmental conditions, feeding, and care may have an impact on experimental results in swine 30 studies.

housing At a typical commercial breeding establishment, separate quarters are provided for: • Young stock from weaning to breeding age (growing quarters) • Dry sows and gilts, and breeding boars (mating quarters) • Pregnant sows (waiting quarters) • Parturient and nursing sows (farrowing quarters) In the growing quarters, swine are housed in groups of the same sex. Groups will live in good harmony when enough space per animal is available. They can move around freely to avoid aggressive interaction. Large groups or groups in a confined



Fig. 2.1 Group pen at a breeding facility with solid concrete floor in the back, and grids in the front. The minipigs are fed from the floor, and chains are hanging from the sides for environmental enrichment. space are not able to maintain a stable dominance order, result31 ing in a higher level of aggression. Stable social groups are best established after weaning. Young gilts and pregnant sows are group housed. In the farrowing quarters, sows are caged in special farrowing crates equipped with bars, preventing them from lying on their young. 7 Breeding boars are housed individually. Pigs kept at the laboratory can be housed individually or in small groups in floor pens. The floor can be made of solid concrete or raised grids. Concrete floors should have a rough surface for secure footing, and may be coated with an epoxy layer. Bedding should be provided, e.g., wood shavings or straw. This gives good rooting and nesting possibilities for the animals, but is more laborious during sanitation. Grid floors provide good sanitation since usually no bedding is provided. Grid floors can be made of galvanized steel, plastic-coated steel, or fiberglass, and may consist of parallel bars or have a mesh structure. Galvanized steel is less suitable since it conducts heat too well,



Fig. 2.2 Single pen at a research facility with solid concrete floor in the back, and grids in the front. The floor is covered with wood shavings, and the minipigs are offered hay for environmental enrichment (Photo courtesy of Scantox). whereas plastic-coated steel and fiberglass preserve heat more satisfactorily. However, when using raised grid floors room temperature is more critical, as compared to concrete floors with bedding since the animals have no possibility of nesting and creating an insulated microenvironment. Attention should be paid to the spacing of the grids so that hooves will not be damaged. When using parallel bars, a bar width of approximately 10 mm and a spacing of approximately 12 mm is appropriate for most sizes of swine. Plastic-coated, diamond-shaped grids (Tenderfoot®) with a spacing of 10-15 mm is a very satisfactory floor type for swine. When swine are housed on grid floors, hooves need to be trimmed at regular intervals since they do not wear down on grid floors as they do on concrete floors. Pens should be robust, since swine are forceful animals with strong snouts. They also rub their sides along the sides of the pen, pushing with considerable weight. Concrete or brick walls are most often used in combination with a fencing of galvanized



or stainless steel. No sharp edges or protruding bolts should be present. Especially when animals are single housed, fencing should be placed in such a way as to make it possible for the animals to see, smell, and hear each other. Swine are social animals and need to have visual, olfactorial, and auditory contact if physical contact is not possible. Young swine and miniature swine can be housed in dog facilities without major adaptions to the pens. Usually, only lowering automated watering valves and feeding bowls is necessary. Automated watering valves are preferred to water bowls since swine drink considerable amounts of water during a day, and water should be constantly available. Feeding bowls should be secured to the cage, and should be made of a smooth material that is easy to clean. When group housing swine, troughs are commonly used. Attention should be paid to competition; there should be enough space at the trough for each individual animal. Nevertheless, differences in feed uptake may occur when feeding from troughs, with a differentiation in body-weight development as a result. Hence, trough feeding is less suitable for the laboratory. Sheltered and outdoor housing is unsuitable in a research setting because of uncontrolled environmental conditions and the risk of infection. In regions with mild climatic conditions, raising pigs in huts or kennels on pastures is gaining in popularity. When obtaining swine from agricultural sources, inquiries must be made about housing type since outdoor swine without exception need to be treated with antihelminthics when arriving at the laboratory. Space guidelines for swine are described by the National 32 33 Research Council and the European Council Directive. Since no consensus exists among the guidelines, common sense has to be applied when housing swine. The animals should be "provided with housing, environment, degree of movement, food, water and care which are appropriate to their health and wellbeing. Restrictions to the physiological and behavioral needs of experimental animals should be limited to the absolute mini33 mum." In general, less space is given to swine in agriculture than is recommended for research swine. Space recommendations for research swine are given in Table 2.1.



Fig. 2.3 Feeding trough for group feeding of newly weaned miniature swine TABLE 2.1: SPACE Number of swine per enclosure 1

2-5

>5


RECOMMENDATIONS FOR SWINE.32

Weight (kg)

Floor area per animal (ft2)

Floor area per animal (m2)

200 200 200

8.0 12.0 15.0 24.0 48.0 >60.0 6.0 10.0 20.0 40.0 >52.0 6.0 9.0 18.0 36.0 >48.0

0.72 1.08 1.35 2.16 4.32 >5.40 0.54 0.90 1.80 3.60 >4.68 0.54 0.81 1.62 3.24 >4.32


environmental conditions Elements of environmental conditions are temperature and humidity, ventilation, illumination and noise.

Temperature and Humidity Since swine have relatively sparse hair and lack sweat glands, they are sensitive to temperature fluctuations and extreme temperatures. Especially when swine are housed on grid floors without bedding, flawless temperature control of the facility is necessary. Optimum environmental temperatures for swine vary with age. Newborn swine thrive best at temperatures of 30-38° C (86-100° F). This is achieved by partial floor heating in the 7 farrowing pen, with the addition of a heat lamp. Swine are best housed at the thermoneutral temperature. At this temperature, the relative humidity appears to be unimportant. Energy conversion is not influenced by the relative humidity at the thermoneutral temperature, which was found to be 29° C (84° F) in juvenile (6-8 weeks old), 24° C (75° F) in young (14-16 weeks old), and 17.4° C (63° F) in adult (34-36 weeks 34 old) miniature swine. These figures apply to swine generally, but swine housed on raised grid floors without bedding should not have temperatures lower than 20°C (68° F) because of an expected higher heat loss. The relative humidity should be maintained at 50-70%.

Ventilation Ventilation is essential to keep the concentration of potentially harmful gases low, especially in densely stocked quarters. Concentrations of NH3 and H2S should not exceed 10 ppm and 5 ppm, respectively, and CO2 levels should not rise beyond 0.15 vol-%. However, air circulation should not exceed 0.2 to 0.3 m/s 7 for adult animals, or 0.1 m/s for piglets. Generally, a ventilation rate of 10-15 changes per hour with fresh air is recommended, but the dimensions of the room and the number of animals present need to be considered, especially to prevent overventilation as swine are sensitive to draft. On the other hand, underventilation will lead to a raised environmental temperature and increased levels of noxious gases.



Illumination A light-dark cycle of 12 hours is usually applied to swine, with the lights switched on from 6 a.m. to 6 p.m. A light intesity of 100 lux is appropriate, but in the mating quarters of breeding facilities, light intensity should be 200 lux, with the lights on from 6 a.m. to 8 p.m.

Noise Swine are relatively insensitive to noise. As a matter of fact, they are quite noisy themselves, and it is recommended that staff wear hearing protection when handling swine. However, sudden, explosive noises should be avoided since these induce fright.

environmental enrichment Swine are lively animals. Although they spend 70 to 80 percent of their time lying or sleeping, during the remaining time they are actively strolling around, exploring, and rooting. When given the space, swine are clean in their habits, choosing specific sites for defecation and urination while keeping their sleeping areas dry. The availability of clean, dry straw contributes substantially to the well-being of swine. Straw provides comfortable bedding, and keeps the animals occupied with rooting and chewing activities. Unless experimental conditions require otherwise, the best way of accommodating swine and miniature swine is by housing them in groups of up to 10 or 15 animals in spacious pens with straw-covered floors.

Group Housing Social grouping and the establishment of a social hierarchy are behavioral traits typical for swine. These behavioral patterns should be considered when housing swine. If possible, individual housing of swine should be avoided. When group housing swine, uniformity of the group must be observed, and trough space must be sufficient to permit all animals to feed at the same time. Ideally, partitions should be provided, enabling weaker individuals to avoid the more aggressive ones. Group size should not exceed 10 to 15 animals, otherwise no lasting hierarchy will



be established. If possible, groups should be kept together. When the animals have to be regrouped, the extent of fighting may be minimized by bringing unfamiliar animals together just before feeding or sleeping time, preferably in a newly cleaned pen. The individual scent of all animals of the group may be camouflaged with a strong fragrance (mint oil, cresol), and in critical cases animals can be tranquilized before being brought together. Adult boars are solitary, and individual housing is appropriate. Nonetheless, it is possible to group house boars, with the exception of particularly aggressive individuals, provided they have been reared together or have been given the opportunity to get accustomed to each other under unconfined conditions. Barrows can be group housed under similar conditions to those of females.

Fig. 2.4 Woodshavings or straw and a hard plastic ball are excellent environmental enrichment for swine since they stimulate rooting behavior (Photo courtesy of Pfizer Central Research)

Individual Housing At the laboratory, individual housing is common. As described before, individually housed animals should have visual, olfactorial, and auditory contact with other swine to prevent them



from being socially deprived. To a large extent, swine become 35,36 attached to humans, especially when housed individually, and daily positive contact with staff should be included in the 37 animal care program. Most important to swine is the provision of items that can be manipulated, especially when no bedding is provided, to satisfy the needs of chewing and rooting. The following items can be considered as elements of environmental enrichment: • a chain suspended from the side of the pen • a hayrack with straw or hay • a nylon brush or broom head attached to fencing • a heavy ball, plastic crate, or plastic turf mat placed on the floor A chain and straw or hay in a hayrack primarily satisfy the need for chewing, but they also provide material for manipulation with the snout. A brush or broom head attached firmly to the fencing is used for rubbing against, but also for biting, whereas balls, crates, or turf mats are used for satisfying the need for rooting. For all elements of enivronmental enrichment it is essential to allow a good level of sanitation. The animals quickly lose interest if the items get soiled, especially items placed on the floor of the pen. To avoid soiling and preserve the novelty of the items, balls, crates, or turf mats should only be given to the animals for a limited period daily.

nutrition Most publications relating to swine nutrition focus on optimal utilization of nutrients with an eye on efficient growth, since feed accounts for two-thirds of the costs of producing marketweight swine.38 Although laboratory swine nutrition is basically similar to production swine feeding, it may be desirable to control weight gain. Therefore, restricted feeding is often applied at the laboratory. Moreover, miniature swine are prone to obesity and, consequently, restricted feeding is necessary to maintain normal physiological conditions in miniature swine. There are indica-



Fig. 2.5 Pressed and extruded feed pellets tions that nutrient requirements of miniature swine differ from those of other swine,39 but until further evidence has been produced, nutrient requirements of swine should be used as a basis for miniature swine nutrition.40, 41

Nutrient Requirements The National Research Council (NRC) provides detailed information on the composition of swine diets, including energy, protein and amino acids, minerals and vitamins, and diet formulation. However, the given nutrient requirements are minimum standards when feeding ad libitum and should not be considered recommended allowances.16 Table 2.2 gives an overview of the nutrient requirements of swine with body weights between 5 and 80 kg. Swine are able to utilize high-fiber diets because of fermentation in the cecum and large intestine. However, fiber levels over 7-10% inhibit growth. Dietary fiber also has an effect on the passage of food through the intestines, and diets with a high fiber content prolong the gastrointestinal transit time. fiber levels of up to 15% have no effect, but levels exceeding 15% will



TABLE 2.2: NUTRIENT FROM 5 TO 80 KG.16

REQUIREMENTS OF SWINE WITH BODY WEIGHTS

Body weight (kg)

MEa content of diet (MJ/kg)

Expected feed intake (g/day)

Crude protein (%)

5-10 10-20 20-50 50-80

13.6 13.6 13.6 13.6

500 1,000 1,850 2,575

23.7 20.9 18.0 15.5

a) Metabolic energy; 1 MJ = 240 kcal (13.6 MJ/kg = 3,265 kcal/kg)

increase the transit time. Especially when roughage, such as straw or hay, is fed, gastric emptying and intestinal transit time are significantly prolonged.42 Low energy diets, especially meant as maintenace diets for miniature swine, usually have a relatively high fiber content of 13-15%. The minimum level of minerals and vitamins is well defined,16 and commercial diet producers may be expected to have included sufficient mineral and vitamin premix to cover the requirements of swine. However, when comparing levels from catalogue values and analysis reports, a disturbing discrepancy may be found within and between brands.39

Feeding Levels The feed intake of swine, as in all mammals, depends on the energy concentration of the feed. However, when the equation for estimated energy intake with ad libitum feeding of swine is compared to the equation for energy intake used for interspecies comparison, based on metabolic weight, swine have a much larger energy intake than expected from this equation. The general practice in swine feeding is ad libitum feeding. With this, modern production swine grow 2-3 kg (4.4-6.6 lbs) per week, so that they weigh 80-100 kg (175-225 lbs) at the age of 5-7 months when, generally, they are slaughtered. This enormous weight increase may be undesirable at the laboratory. Therefore, restricted feeding is often applied at research facilities. In miniature swine, restricted feeding is essential since they



Fig. 2.6 The expected metabolizable energy (ME) intake for growth can be calculated with the equation for interspecies 15 0.75 based on metabolic weight (ME=1.20*W ). comparison, 38 0.63 However, the best fitting equation for swine (ME=2.52*W ) results in a higher expected energy intake easily become obese when fed without restriction. From studies in rodents, it has become clear that a feeding level of 60% of the ad libitum food intake is sufficient to prevent obesity, fulfills the nutrient needs, and leads to a longer, healthier life as compared to ad libitum feeding.43 This may be applied to laboratory swine as well, but more often restricted feeding with a low energy diet is applied to allow the animals a satisfying food volume intake. Whereas a standard swine diet has a metabolic energy content of 13.6 MJ/kg (3,265 kcal/kg), a typical lowenergy diet has a metabolic energy content of 9.5 MJ/kg (2,275 kcal/kg). Feeding levels can be found in Table 2.3. Clean drinking water should be available at all times. Therefore, automated watering systems (nipple or bowl type) are recommended. Although water is essential, suprisingly little research has focused on the water requirements of swine. True water consumption is often overestimated since swine like to play with water, and wastage is often not taken into account.16 Approxi-



TABLE 2.3: FEEDING LEVELS. Daily Expected Body weight Energy Intake Feeding (g/day)b (kg) (MJ/day)a

Restricted Low Energy Feeding Feeding (g/day)c (g/day)d

5 10 20 50 80

175 300 500 995 1,415

4.00 6.75 11.35 22.55 32.10

290 500 835 1,660 2,360

255 425 715 1,425 2,025

a) ME = 1.2 W0.75 (MJ/day)15 b) metabolizable energy density of diet = 13.6 MJ/kg (3,265 kcal/kg) c) 60% of expected energy intake with a diet of ME=13.6 MJ/kg (3,265 kcal/kg) d) 60% of expected energy intake with a diet of ME= 9.5 MJ/kg (2,275 kcal/kg)

mately 120 ml/kg water are used by growing swine fed a dry pelleted diet.16, 44 The delivery rate of water via automated watering devices should be about 0.5 l/min. If automated watering devices are not available, the usual practice is to mix water with the diet and refill the trough with water after the swine have finished eating.

sanitation Sanitation is important to maintain the health of swine. Excreta and food remains are a substrate for microbial growth and attract insects and rodents which could act as vectors for diseases. Therefore, pens should be cleaned frequently, but sanitation should be carried out with minimal use of disinfectants to prevent chemical contamination of the animal environment.45

Frequency Daily removal of excreta is good practice. When swine are housed on solid floors with bedding, removal of soiled bedding is essential. Soiled bedding represents the largest volume of waste material, and could lead to capacity problems when disposing waste in bags or containers.



Fig. 2.7a Watering device for young miniature swine.

Fig. 2.7b Watering device for adult miniature swine.



Swine housed on grid floors usually press feces through the grids with their hooves, whereas urine runs through. Under the grid floor, a sewer canal may be present through which the excreta can be flushed away. When larger numbers of animals are present, animal rooms for swine should be constructed with a manure cellar under the grid floors. Here, excreta accumulate and are pumped to a large basin outside the facility at regular intervals. Grid floors should be checked daily, and feces accumulated in corners should be scraped away. Pens should be washed weekly, although pens with grid floors are usually washed at longer intervals to prevent excessive amounts of liquid manure. A mild disinfectant may be used. Disinfectants should be rinsed away and solid floors swabbed dry before allowing the animals to return to their pens. Pens should be sanitized thoroughly on a monthly basis. High-pressure cleaning is commonly used in swine facilities. Because of aerosol formation, high-pressure cleaning should be carried out without additives to the water. A detergent or disinfectant is usually added to low-pressure water to rinse with after high-pressure cleaning. Detergents or disinfectants should be rinsed away and solid floors swabbed dry before allowing the animals to return to their pens.

Methods Sweeping should be minimized to prevent dust formation. Floors of pens, corridors, and utility rooms should be washed and swabbed dry after gross debris has been removed. Staff working with soiled bedding, especially wood shavings, should wear dust masks for protection. High-pressure cleaning should include floors, walls, fencing, bowls and troughs, drinking devices, and items for environmental enrichment. Detergents or disinfectants should not be used before high-pressure cleaning, nor should they be added to high-pressure water because of the risk of exposure to aerosols during high pressure cleaning. Staff should wear protective clothing (watertight suits or aprons), fluid shield masks, and hearing protection. A wide range of detergents and disinfectants are available, and suitable detergents and disinfectants should be chosen based on experience and what is already present at the facility. An overview of disinfectants and disinfection is given by Russell et al. (1984).46



transportation Transportation is stressful for swine, and gentle handling is necessary. Since swine suffer from travel sickness, animals should be deprived of food 12 hours prior to, and during, transportation. If animals are transported over long periods, breaks should be included to offer the animals water at least every 6 hours. Hot, humid conditions during transportation should be avoided, as well as low temperatures and drafts. Ideally, climatized vehicles should be used to maintain environmental conditions similar to those at the animal facility.

Fig. 2.8 Vari Kennel® transport kennel for swine under 25 kg Swine are generally transported freely standing in a truck or trailer. A space of 0.5 m2/100 kg (2.5 ft2/100 lbs) body weight should be allowed. Floors must not be slippery; a slatted floor covered with wood shavings is commonly used. Large loading surfaces should be avoided, or should be subdivided by partitions.



Young swine or miniature swine can be transported in portable dog kennels (e.g. Vari Kennel®) or custom-made crates. Floors should be covered with wood shavings. Straw is not as suitable as wood shavings since it does not absorb as well. There should be openings for fresh air, and sufficient space to lie down comfortably. Requirements for swine transportation are described in manuals for transportation of live animals by road47 and air.48

Fig. 2.9 Identification of swine with ear notches and ear marks. To the left, the identification system for Göttingen minipigs consists of an earnotch system which allows numbering from 0-99, plastic earmarks, and an intramuscularly implanted transponder. To the right, an animal marked number 54.

record keeping Record keeping requires individual identification of animals. Swine may be identified with ear notches, ear tatoos, ear tags, neck bands, or intramuscular electronic transponders. For temporary marking of swine, colored markers or spray can be applied on the skin. Individual animal records should contain the animal's identification number, identification numbers of the parents (if known), and pen number. Body-weight development and medical treatment should be recorded as well, and preferably also information about feed intake, abnormalities, and research procedures.


3 management and quality assurance Management of swine at the laboratory should aim at achieving the highest possible scientific outcome of research on swine. Therefore, it is essential to standardize production and maintenance of swine, as well as to assure the quality. Quality assurance of laboratory swine has especially been concerned with microbiological and genetic factors. Furthermore, the status of the swine as an important production animal requires that certain legal demands must be fulfilled.

microbiological management and quality Impact of Infections on Animal Research using Swine In Chapter 4, information about important infections in laboratory swine is given. Such infections may have an impact on experiments with swine in various ways. Basically, the impact may be divided as follows:

Pathological changes, clinical disease and mortality. Clinically apparent disease is mostly due to bacterial infections, such as Actinobacillus pleuropneumoniae, which causes severe lung disease. Animals suffering from such a disease are seldom used for experiments, but infections may have a severe economic


impact on laboratory swine breeding and maintenance. However, some infections induce severe pathological changes in the animals which may not be discovered until during the experiment. Mycoplasma spp. and Haemophilus parasuis may both cause polyser49 ositis, and surviving swine consequently have adhesions in the thorax and abdomen which are often not discovered until the swine has already been anesthetized and has undergone laparatomy.

Immunomodulation. The immune system may be modulated by spontaneous infections in the absence of clinical disease, an effect which may be suppressive or activating or both at the same time, but on different parts of the immune system. As a general rule of thumb, all viruses should be regarded as possibly being immunosuppressive, one of the reasons being the viremic phase in the pathogenesis of many viral infections during which cells of the immune system may be infected. The immunological impact of African swine fever virus has been intensively studied, and it has been shown that changes 50 in the immune system persist after recovery from infection. African swine fever is not common in herds supplying swine for experiments, but more frequently occurring viruses such as influenza viruses may have a serious impact on the immunology of 51 the animals, a phenomenon which is well-known from other animal species.

Physiological modulation. Some microorganisms have a specific effect on enzymatic, hematological, and other parameters monitored in the animal during an experiment. Organic function disturbances may change the outcome of the experiment without the knowledge of the scientist, e.g., the altered function of the liver caused by some spontaneous hepatic infections may influence pharmacokinetics. Infection with Streptococcus suis, for instance, decreases single dose clearance 52 but increases multiple dose clearance of sulfadimidine.

Competition between microorganisms within the animal. Spontaneous infections in an experimental infection model may compete with the experimental infection, which in the worst cases may fail. Gnotobiotic- or microbiologically-defined swine


can be experimentally infected with Helicobacter pylori, while 53 conventional swine cannot. Such a difference between microbiological categories of animals may be explained by natural infection with related agents, as species-specific Helicobacter 54 spp. have been isolated in swine. Infections with species other than Helicobacter may also play a role.

Interference with reproduction. For the breeder, changes in the fertility of the animals may be the cause of serious trouble. Infections giving rise to clinical disease in a major part of the population are very likely to reduce the fertility, and it is also quite obvious that infections such as 55 rotaviruses leading to high mortality in neonates, will reduce the outcome of breeding or disturb experiments using newborn swine. Direct effects of the infection on reproduction, such as the change in sex hormones, pathological anatomical changes in the reproductive tract, or infection of the embryo causing abortion and stillbirths are also observed, e.g., with common viruses such as porcine parvovirus and the enterovirus SMEDI.56 Reproduction studies, in which the absence of such infections have not been documented, may be prone to criticism.

Interference with oncogenesis. Infectious agents may either induce cancer, enhance the carcinogenic effect of certain compounds, or reduce the incidence of cancer in laboratory animals, and opportunistic pathogens may become pathogenic in animals with cancer, e.g., swine with lymphosarcomas are more likely to die from colibacillosis than swine without lymphosarcomas.57

Contamination of biological products. Microorganisms present in the animal may contaminate samples and tissue specimens such as cells, sera, etc. This may complicate in vitro maintenance of cell lines, and may interfere with experiments performed on cell cultures or isolated organs. Furthermore, the reintroduction of such products into an animal laboratory will cause a risk to the animals kept in that laboratory. In fact, some of the mycoplasmae which complicate maintenance of cell lines are porcine mycoplasmae originating from


either the use of porcine serum in the media, or the porcine origin of the cells to be grown.58

Zoonotic Diseases Some important zoonoses are listed in Table 3.1. It should be noted that zoonoses of porcine origin in laboratory animal staff are rare. The major risk seems to be development of erysipelas after contact with swine infected with Erysipelothrix rhusiopathiae.59 The potential future use of swine as organ donors for human xenotransplantation has created a new term, xenozoonosis,60 i.e., infections which may pass with the organ from the animal donor to the human recipient. More information about the risk of porcine retroviruses for humans is needed to evaluate the risk connected with porcine-human xenotransplantation. It is obvious that porcine infections already known to possess a zoonotic potential should be a matter of concern. However, these are well-defined and diagnostic and preventive tools are welldeveloped, while there is little knowledge of the risks connected with the xenotransplantation entry port. It is conceivable that porcine viruses may mutate into a human variant, maybe with a lethal effect. If so, this may have implications not only for the individual patient, but for mankind as such. Obviously, emphasis has mostly been placed on retroviruses. Xenotransplant recipient patients need immunosuppression, and, therefore, it is of vital interest to know whether immunosuppression will allow non human retroviruses to propagate in humans. Little is known about exogenous retroviruses in swine but, in fact, type C retroviruses have been found in the blood of certain strains of miniature swine after exposure to radiation,61 and it is known that porcine retroviruses may infect human cell lines.62 Herpes viruses may be another concern. The maccaque herpes virus type B can cause fatal encephalitis in humans, and this may also be so for other types of herpes viruses, e.g., the porcine Aujeszky’s virus. Porcine Aujeszky’s virus is known to be fatal in other animal species, such as ruminants. Also, rotaviruses, which are extremely common, even in purpose-bred miniature swine,63 may be potential xenozoonotics. Gnotobiotic piglets can be experimentally infected with rotaviruses of a human nature,64 and it has been shown that some rotaviruses may be shared by


TABLE 3.1: PORCINE

INFECTIONS WITH A ZOONOTIC POTENTIAL.

Agent

Symptoms In pigs

Brucella suis

Mostly no symptoms. Classical signs are abortions, infertility, weak piglets at birth, infection of the testes and arthritis

Campylobacter jejuni

Diarrhea or no symptoms

Eschericia coli (only certain strains causing porcine edema disease) Erysipelothrix rhusiopathiae

Neurologic symptoms, edema of the eyelids, occassionally edema of the neck and abdominal skin Fever, arthritis, often squarish erysipeloid lesions (pinpoints to > 3 inches)

Leptospira spp.

Normally none, in some cases abortions


Transmission to man

Risk for research staff

In man Severe fever and disabling Direct contact with lesions of the spine which tissues, blood, urine, vaginal discharges, or may become lethal aborted fetuses and placentas from infected animals Diarrhea Fecal contamination of human food Fecal contamination of Similar to the pig, human food occassionally damage to the heart valves, or sudden death Contact with infected Similar to the pig, pigs occassionally damage to the heart valves, or, sudden death Haemorrhagic fever Contact with porcine urine

Low

Low Low

High

Low

TABLE 3.1: PORCINE

INFECTIONS WITH A ZOONOTIC POTENTIAL.

Agent

Symptoms In pigs

Transmission to man In man

Salmonella spp.

Diarrhea

Diarrhea

Taenia solium

None

Trichinella spiralis

None

Brain seizures, muscle pain and weakness, heart failure, sometimes sudden death due to development of cysticercs. Muscle encapsulations resulting in weakness and muscle pain

Mycobacterium bovis, M. avium, and M. tuberculosis

None (granulomas may be found during necropsy)


Risk for research staff

Tuberculosis

Fecal contamination of human food Intake of insufficiently cooked pork

High

Intake of insufficiently cooked pork

Low

Inhalation and ingestion

Low

Low

swine and humans, e.g., the Gottfried porcine rotavirus.65 Similar questions may be raised about bacteria such as Streptococci.

Legal Regulations Most Western countries have regulations concerning import and export of live animals. OIE (Office International de Épizooties), the International Office of Epizootics, is an organization of individual member countries that registers infectious diseases worldwide, sets standards, and issues guidelines on how to prevent the spread of infectious animal diseases.66 OIE is responsible for the administration of the International Animal Health Code, according to which the member countries have certain responsibilites such as warning each other if any of the diseases listed in Table 3.2 are diagnosed in a member country. Swine are especially subject to strict regulations since pork production is essential for the national economy in several countries. According to U.S. regulation, the Secretary of Agriculture has "authority to make such regulations and take such measures as he may deem proper to prevent the introduction or dissemination of the contagion of any contagious, infectious, or communicable disease of animals ... from a foreign country into the United States or from one State ... to another." Such rules are a common part of agricultural legislation in most Western countries. In order to achieve these goals, national departments of agriculture in the OIE member countries run mandatory health programs aimed at irradication of OIE-reportable diseases such as brucellosis, foot-and-mouth disease, and swine fever, only allowing import and export of swine and porcine products under strict state veterinary control. Therefore, prior to import and export of swine, one should always contact the national or local veterinary authorities to ensure that licenses are granted and that the swine are provided with all necessary certificates. Regulations that cover swine in biomedical research in the USA are: • Animal Welfare Act (U.S. Department of Agriculture, 7USC 2131) and their associated regulations (9 CFR: Parts 1,2, and 3)


TABLE 3.2: REPORTABLE INFECTIOUS DISEASES OF SWINE REGISTERED BY THE INTERNATIONAL OFFICE OF EPIZOOTICS (OIE). List A Diseases

List B Diseases

Definition:

Definition:

Transmissible diseases which have the potential for very serious and rapid spread, irrespective of national borders, which are of serious socio-economic or public health consequence and which are of major importance in the international trade of animals and animal products. Foot and mouth disease Vesicular stomatitis Rift Valley fever Bluetongue African swine fever Classical swine fever

Transmissible diseases which are considered to be of socio-economic and/or public health importance within countries and which are significant in the international trade of animals and animal products. Anthrax Aujeszky's disease Leptospirosis Rabies Avian diseases Atrophic rhinitis of swine Enterovirus encephalomyelitis Porcine brucellosis Porcine cysticercosis Porcine reproductive and respiratory syndrome Transmissible gastroenteritis Trichinellosis

• Public Health Service Policy on Humane Care and Use of Laboratory Animals (Health Research Extension Act of 1985) for studies funded by the U.S. Government The principles for implementation of the Public Health Service policy are those described in the Guide for the Care and Use of Laboratory Animals.32 The Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals do not apply to swine used in agricultural research. Here, the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching can be used as guidance.67

Precautions to Prevent Infections In several countries, organizations to ensure the health of farm swine exist. National organizations such as the National SPF Swine Accreditation Agency in the United States have, since the


late fifties, set up systems for swine production according to a three-step principle: 1.

Production of germ-free animals by rederivation techniques, i.e., Caesarean section or embryo transfer

2.

Upstart of a breeding colony on the basis of the rederived animals in a protected (barrier) environment

3.

Regular screening of the colony for the presence of certain agents (health monitoring)

Rederivation. Germ-free swine may be produced by removing them aseptically from the uterus by hysterectomy and rearing them in isolators.68 In the isolators they may be associated with a specific flora. The term gnotobiotic covers germ-free animals as well as animals with a specific flora. Gnotobiotic swine are used for immunological studies such as natural and adaptive immunity and early antibody synthesis, radiation studies, studies of monoassociation with certain bacteria, and studies of parenteral nutrition.69, 70,71 Germ-free swine have a lower peripheral blood leucocyte count than conventional swine, although they have an increased number of peripheral T-cells, a lower percentage of neutrophilic granulocytes, lower total serum protein, and higher serum albumin and beta-globulin levels.72 An alternative to Caesarean section is embryo transfer.73 The piglets will receive the microbiological status of the recipient mother.

Barrier protection. As long as the swine are kept in isolators the gnotobiotic status can be maintained. If the swine are used for the upstart of a breeding colony, housed under conditions protecting them against certain agents for which a monitoring system is operated, terms such as minimal disease (MD), specific pathogenfree (SPF), and microbiologically defined swine are used. Meat-producing SPF systems consist of various levels of herds; i.e., animals derived from Caesarean sections are used for the upstart of a primary SPF herd, which may deliver swine for breeding in a secondary SPF herd. No further animals are introduced into primary herds.


Swine produced under barrier protection equivalent to what is applied when breeding laboratory rats and mice are referred to as microbiologically defined.27,74 Microbiologically defined swine have improved the possibilities of using swine for research, not only in toxicology,75,76,77 but also in more specific research such as studies of Helicobacter pylori infection.78 Microbiologically defined swine seem to differ in serum and hematology values from conventional swine in the same way as described for gnotobiotic swine above.20 Some quarantine and barrier demands are also fulfilled in the production of SPF and MD swine, although in general not as strict as for microbiologically defined swine (Table 3.3). The SPF production method was first applied in agricultural swine farming, where it was used to eliminate infections with a negative impact on the farmer's economy. However, SPF swine have also been widely used for experimental purposes because they are free of mycoplasmae. The term minimal disease (MD) often covers SPF swine colonies with reinfection of some of the regulated pathogens. This term may be used for former SPF colonies infected with, for example, Mycoplasma hyopneumonia. Swine that are neither SPF nor microbiologically defined are called conventional. TABLE 3.3: TYPICAL

HOUSING CONDITIONS FOR DIFFERENT CATEGORIES

OF PIGS FOR EXPERIMENTAL PURPOSES.

Caesarian originated Barrier: Quarantine regulation Change of dress Shower in Decontamination of: Diet Equipment Water Absolute filtered ventilation Health monitored


Conventional

Specific Pathogen Free (SPF) Minimal Disease (MD)

Microbiologically defined

-

+

+

-

+

+

-

+ -

+ +

-

-

+ + + +

-

+

+

Health monitoring. Table 3.4 gives some examples of infections covered by the SPF term, although it should be remembered that organizations in different countries work with slightly different lists. Some associations of laboratory animal science, e.g., the Scandinavian Society for Laboratory Animal Science79 or the Federation of European Laboratory Animal Science Associations (FELASA)80 have issued guidelines for advanced health monitoring of swine for experiments. Such guidelines recommend monitoring for a range of agents. Some commercial vendors specialized in purpose-breeding of swine for research have set up their own systems which may differ slightly from the guidelines (Table 3.5). TABLE 3.4: INFECTIONS WHICH MAY BE COVERED UNDER SPECIFIC PATHOGEN FREE (SPF).81,82

THE TERM

Disease

Pathogen

Morbus Aujeszky (Pseudorabies)

Herpesvirus (Aujeszky’s virus)

Pneumonia Actinobacillus pleuropneumonia

Mycoplasma hyopneumoniae

Swine dysentery

Serpulina hyodysenteria

Transmissible gastroenteritis

Coronavirus (TGE)

Atrophic rhinitis

Pasteurella multocida

Sarcoptic mange

Sarcoptes scabiei

Porcine Reproductive and Respiratory Syndrome

PRRS virus

In breeding colonies, health monitoring is based on random representative samples. This means it is assumed that a few animals can be sampled for examination but the results can be used to describe the entire colony. If one animal is found to be infected with a certain organism, the entire colony is considered infected with that particular organism. Often the opposite is also assumed, i.e., if no animals are found to be infected with a certain organism, the entire colony is considered free of that particular organism. This, however, requires the use of a statistically valid


sample size.83 The sample size may be calculated using the equation in Figure 3.1. As soon as a sample has been taken, it becomes historical. By comparing two samplings taken at a certain interval, the last sampling will visualize whether changes have occurred between the two samplings. A random sample of animals is only predictive for the microbiological status of other animals if these animals have some kind of contact with each other. Therefore, it is essential to define the microbiological entity, i.e., a definition of the group of animals for which a sample is predictive. A simple one-room unit used for breeding in a commercial establishment will normally have its own staff and barrier to separate it from all other units; each of these separate units should therefore be regarded as a separate microbiological entity. However, if the unit is separated into different rooms that are all served by the same staff and get their supplies through a common barrier, it is hard to define the microbiological entity; it should be noted that the more subunits included, the higher the uncertainty of the sampling. If the infection is only found in one subunit, the prevalence is "diluted," which may not be taken into consideration when calculating theI sample size.85 Most standards for health monitoring also demand regular clinical inspections by a trained veterinarian. In some systems vaccines and/or antiparasitics are banned, while they are frequently applied in other systems. This should be checked with the vendor. Prior to any purchase of swine for experimetal purposes a health report should be retrieved from the vendor.

Common Findings in Health Monitoring In the majority of conventional swine, antibodies against parvo and influenza viruses as well as H. parasuis are found. Furthermore, although less common, antibodies against porcine coronaviruses, hemagglutinating encephalomyelitis virus, porcine reproductive and respiratory disease viruses, and all serotypes except Types 7 and 8 of A. pleuropneumoniae and Mycoplasma hyopneumoniae are found. Bacteriological, fungal, and parasitological examinations often reveal the presence of A. pleuropneumoniae, Bordetella bronchiseptica, Clostridium perfringens, Eubacterium suis, H. parasuis, Pasteurella multocida, Salmonellae,


TABLE 3.5: INFECTIONS

INCLUDED IN SOME PROGRAMS FOR HEALTH MONITORING OF MICROBIOLOGCIALLY DEFINED PIGS.

VIRAL INFECTIONS Africian swine fever Morbus Aujeszky Encephalomyocarditis virus Hemagglutinatinating Encephalomyelitis Porcine coronaviruses Porcine cytomegalovirus Porcine Influenza Porcine Parvovirus Porcine Reprodutive & Respiratory Syndrome Porcine Rotavirus SMEDI Swine fever Teschener disease BACTERIAL AND FUNGAL INFECTIONS Actinobacillus pleuropneumoniae Actinomyces pyogenes Bordetella bronchiseptica Brucella suis Campylobacter spp. Clostridium perfringens Erysipelothrix rhusiopathiae Eubacterium suis Helicobacter spp.


Ellegaard Göttingen Minipigs85

Charles River Laboratories86

country country country country

Cons. abs. Yes No Yes

Cons. abs. Yes No No

in in in in in

country country country country country

Yes No Yes Yes Yes

No No No No Cons. abs.

in in in in

country country country country

Yes Cons. abs. Yes Cons. abs.

No No Cons. abs. Cons. abs.

All types On request Yes On request No On request Yes Yes

All types No Yes Cons. abs. Yes Yes Yes Yes

All types Yes Yes Yes Yes Yes No No

No

Yes

No

ScandLAS79

FELASA80

No Yes No Yes

If If If If

present present present present

in in in in

No No Yes Yes No

If If If If If

present present present present present

Yes Yes Cons. abs. Yes

If If If If

present present present present

Type 2,3,4 On request Yes On request Yes Yes Yes Yes

No

TABLE 3.5: INFECTIONS

INCLUDED IN SOME PROGRAMS FOR HEALTH MONITORING OF MICROBIOLOGCIALLY DEFINED PIGS.

(continued)

ScandLAS79

FELASA80

Ellegaard Göttingen Minipigs85

Charles River Laboratories86

Yes On request On request Yes Toxin prod. Yes No Yes Yes Yes Yes On request Yes

Yes No On request Yes Toxin prod. Yes No Yes Yes No Yes On request Yes

Yes No Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes

Yes Yes No Yes Yes Yes Yes No Yes No No No No

Arthropods Helminths Enteric protozoa Toxoplasma gondii

Yes Yes Only coccidiae Yes

Only sarcoptes Yes Only coccidiae Yes

Yes Yes Only coccidiae Yes

Yes Yes Yes Yes

On request: Cons. abs.:

The guidelines demand that the producer screens for the agent if requested by the user. Considered absent; some agents are not included in the program, because they are irrellevant due to national health programs The infection should only be included in the program if it has not been eliminated by national health programs

BACTERIAL AND FUNGAL INFECTIONS

Haemophilus parasuis Klebsiella pneumoniae Microsporum spp Mycoplasma hyopneumoniae Pasteurella multocida Salmonellae Staphylococcus aureus Staphylococcus hyicus b-hemolytic streptococci Streptococcus pneumoniae Streptococcus suis Trichophyton spp Yersinia enterocolitica PARASITOLOGICAL INFECTIONS

If present in country:


Fig. 3.1 Sample sizes for health monitoring in relation to the expected prevalence of the monitored infection. If the colony size is small, i.e. less than 10.000, or the expected prevalence low, i.e. under 20%, the population size is of some impect on the sample size. If this is not the case, the sample size (S) can be 83 calculated from following equation : S > log C. (1-(p*N1)) C= accepted risk of a false negative diagnosis; N1= sensitivity of the applied test; p= expected prevalence Staphylococcus hyicus, S. suis, as well as helminth eggs and coccidiae.87 SPF swine do not harbour infections such as A. pleuropneumoniae and Mycoplasma hyopneumoniae, which are mentioned in the guidelines for this category of swine, but concerning nonlisted agents there is no guarantee for the absence of the other abovementioned agents. Swine housed according to the principles for microbiologically defined animals have been shown to harbour a lower number of infections than swine housed according to agricultural SPF guidelines, but also in microbiologically defined swine, health monitoring may reveal


the presence of certain agents with a research interference potential, e.g., rotaviruses.63

genetic monitoring Genetic monitoring, used as a tool for standardization in laboratory rodent breeding, has not been applied in the same way to the breeding of swine for research. On the contrary, most genetic programs are aimed at the development of a research swine more applicable than those presently available, such as the selection for a smaller size of Yucatan miniature swine.88 Breeders register certain parameters on a routine basis in order to use these for selection. Such parameters may include:27,74 • weight at birth • growth rate • number of visible ear veins • temper • number of teats • malformations Recently, genetic maps of the swine genome have been made.89,90

accreditation Certain bodies may accredit laboratory animal facilities with respect to production quality or animal welfare.

AAALAC International AAALAC International, the Association for Assessment and Accreditation of Laboratory Animal Care, runs a voluntary accreditation program in which research institutions demonstrate that they not only meet the minimums required by law, but are taking the extra step to achieve and showcase excellence in animal care and use. Apart from accreditation, AAALAC also offers independent assessments of animal research programs to


help institutions to improve their animal care and use practices continuously. AAALAC International accreditation is common in the United States, but animal facilities and breeders in other parts of the world are increasingly seeking AAALAC International accreditation. Breeding of common laboratory animal species in European Union member states is subject to regulations concerning destinational breeding of laboratory animals for research, which means that all breeders must be licensed by and work under inspection of the local animal welfare authorities. These regulations, however, do not regulate swine breeding, and therefore, swine breeders may want to seek AAALAC accreditation instead.

ISO 9000 ISO 9000 is a set of five universal standards for a Quality Assurance system issued by the International Standardization Organization (ISO), a worldwide associaton of national standards bodies. Currently, 90 countries have adopted ISO 9000 as national standards. The system aims to assure compliance between the quality of the product and the expectations of the customer. The less comprehensive standard ISO 9002, the socalled "Quality System—Model for Quality Assurance in Production, Installation, and Servicing," may be applied to breeders of laboratory animals. ISO 9000-registered vendors have ensured that they have a proper quality assurance system. This system is becoming increasingly important. The overall accreditation is granted by a national ISO member organization. In the U.S. this is the American National Standards Institute.

Other Organizations Several organizations other than AAALAC International and ISO 9000, issue quality directives relevant for laboratory animal facilities.91 Studies submitted to the Food and Drug Administration (FDA) or Environmental Protection Agency (EPA) should comply with Good Laboratory Practice (GLP). The EN 45000 directive, issued by the ISO, is a quality guide for test, inspection, and calibration laboratories, whereas production plants and laboratories working with food and food additives should comply with the Hazard Analyses Critical Control Points (HACCP) standards.


4 veterinary care The veterinary care of swine starts with the arrival of the animals at the facility. Clinical examination should be a standard procedure upon arrival. Swine purchased from agricultural sources usually have no health record, and microbiological sampling should be included as part of the clinical examination. Swine originating from specialized laboratory swine breeders are usually provided with a health monitoring report (HMR). The HMR should be studied for positive findings, and it should be considered whether positive findings pose a risk for the existing swine population in the facility. Until results from the clinical examination are available, swine should be housed in quarantine facilities.

clinical examination of swine92,93 A good clinical examination starts with the history of the individual animal. Breed, sex, age, weight, and the animal's identification number should be recorded, and existing HMR or animal certificates should be added to the record. Swine are usually not accustomed to handling, so to avoid misleading observations of physiological parameters, swine should be observed as much as possible without handling or restraint. However, swine arriving at the facility are heavily influenced by transportation, and it should be taken into account


that the animals are excited by the new environment. Posture, behavior and respiration should be observed prior to restraint. Diseased swine may have long, rough-haired coats, with the hair standing up (piloerection). A dog-sitting posture is associated with respiratory difficulty. Hypothermic swine usually lie in sternal recumbency with the legs tucked under the body. Normally, swine lie on their sides, but this is avoided by animals with heart disease or abdominal pain. Lame swine are reluctant to rise. Swine in good nutritional states should neither be too fat nor have observable bony structures in the body. The normal body temperature is 38-39° C (100-102° F). A check list for clinical examination of swine is given in Table 4.1. TABLE 4.1: CHECK

LIST OF CLINICAL EXAMINATION OF SWINE.

Condition

Desirable

Undesirable

body weight

normal for breed, age and sex flat hair coat with a normal thickness for the breed, white-pink color in unpigmented areas of the skin no discharge, pink mucosa, normal dentition for age normal size, pink mucosa, not fouled; vulva may show watery, clear to whitish discharge

thin, fat, or obese

skin

mouth, nose, and eyes anus, vulva

pale, anemic skin, thickened keratinized patches, wounds and sores, any lesion discharge, blindness, lesions, ulcers

feet, legs, and gait

normal stance and movement

respiration*

no coughing or sneezing, normal respiration

cardiovascular system* feces

normal pulse rate

diarrhea, excessive or purulent discharge from vulva, prolapses; red, swollen vulva is a sign of estus foot lesions, swollen joints, difficulty in lying down and getting up, lameness frequent coughing or sneezing, forced or superficial respiration collapse during restraint

solid, brownish-yellow to brownish green color, depending on feed

watery or extremely hard; reddish, black, or yellow color

*Auscultation in swine is difficult because of the course hair, and often because of lack of cooperation and loud vocalization of the animal.


microbiological sampling63,92 Blood samples for serology are taken from the jugular vein or cranial caval vein. Usually, not more than a few drops of blood can be taken from ear veins, but this may be sufficient for some tests. Methods of blood sampling are described in Chapter 5. Sterile, cotton-tipped swabs should be used for sampling nasal secretions. The swab is introduced into one of the nares, and inoculated onto a plate or kept in Stuart's transport medium for bacterial culture. A fecal sample is collected from the rectum and divided into two parts. One part is kept in a sterile tube for microbiological examination, while the other is used for parasitology and is kept in a plastic container for fecal flotation. A skin scraping for fungi and mites is taken with a sterile scalpel blade from an area with lesions or the shoulder, and kept in a sterile tube for microscopic inspection. The samples are immediately sent to a health monitoring laboratory for serological, microbiological, and parasitological examination. Infectious agents that can be included in the examination are listed in Table 3.5.

diseases of swine A complete overview of diseases of swine is not possible within the scope of this book. If a detailed description of disease is 94 required, it is recommended to use Leman's Diseases of Swine 95 as a reference handbook. An atlas of swine diseases is a useful tool in diagnosing disease. A summary of swine diseases is given in Table 4.2. It should be stressed that prevention of disease by procuring swine from reliable sources is essential. Use of health-monitored swine, preferably with an SPF or microbiologically defined status, reduces the risk of introducing infectious disease into the laboratory facility. Swine from sources with differing health statuses should not be housed together. Thorough sanitation and disinfection of animal facilities is essential for disease control. Pathogens may be harboured in the animal facility and,


Fig. 4.1 Iron dextran (100 mg/kg IM)

Fig. 4.2 Administration of iron to neonatal swine


consequently, nondiseased swine may be infected, even though no swine have been present in the facility for a long period.

therapeutic treatment Medical treatment starts early in the life of swine. All newborn swine are treated with a prophylactic iron medication. Leaving 96 out iron treatment will lead to severe anemia. This is due to the rapid growth rate of newborn swine and the low iron content of sow's milk. Most common is intramuscular treatment with 97 100 mg/kg iron-dextran, but oral dosing is also applied to prevent neonatal anemia. Swine have often been exposed to therapeutic treatment, and one has to consider that this may have an influence on the experiment. Iron treatment will often lead to siderosis, with iron 97,98 But swine deposits in the liver, kidneys and lymph nodes. are also routinely treated with antimicrobial and antihelminthic agents, especially swine originating from agricultural sources. This could lead to disease after experimental procedures since infection, suppressed with antimicrobial treatment, may progress to disease as the animal may be immunosuppressed 99 due to the experimental procedure. A relatively small number of pharmaceutical compounds are available for swine. Antimicrobial feed additives make up by far the largest volume of chemotherapeutic agents administered to swine. They are administered at low, often subtherapeutic levels, 100 for disease control and improvement of growth rate; hence, they are often called growth promoters. Growth promotors should not be used in purpose-bred laboratory swine, and are not used in miniature swine at the breeding station. Antimicrobial and antihelminthic therapy may be used in all swine for the treatment of clinical disease. Table 4.3 lists antimicrobial drugs for swine, along with guidelines for dosing. It is recommended to review drug labels for specific doses.

pathological examination Diseased swine with no prospects of natural recovery or recovery after medical treatment must be euthanized. Methods of euthanization


TABLE 4.2: DISEASES System Cardiovascular

OF SWINE92,94

Condition Myocarditis

Pericarditis

Vasculitis

Hemorragic conditions


Cause Streptococci Encephalomyocarditis virus Foot-and-mouth Swine vesicular disease Vit. E/selinium deficiency, iron deficiency Malignant hyperthermia Pasteurella spp. Mycoplasma spp. Hemophilus parasuis Actinibacillus pleuropneumoniae Streptococci African swine fever Hog cholera Erysipelas Hemophilus parasuis Actinibacillus pleuropneumoniae Intoxication Factor VIII deficiency African swine fever Platelet disorders (thrombocytopenia)

Remarks Increased heart rate, sudden death

Increased heart rate, sudden death

Blood loss, bleedings, anemia

Blood loss, bleedings, anemia

TABLE 4.2: DISEASES

System Digestive

Respiratory

OF SWINE92,94

Condition Enteritis

Atrophic rhinitis

Pneumonia

Pleuritis Viral airway infections


Cause Rotavirus Coronavirus Isospora suis (coccidiosis) Escherichia coli Clostridium perfringens Serpulina hyodysenteriae Salmonella spp. Trichuris suis Campylobacter spp. Pasteurella multocida

Remarks Watery to pasty diarrhea Watery diarrhea

Bordetella bronchiseptica Mycoplasmae Mycoplasma hyopneumoniae Actinobacillus pleuropneumoniae Bordetella bronchiseptica Influenza H1N1 and H3N2

Nasal discharge, sneeze Nasal discharge Dry cough Forced respiration, fever, dogsitting position Cough, forced respiration, fever Dry cough, superficial respiration

Aujeszki's disease

Cough, periodical fever

Hemorrhagic diarrhea Mucohemorrhagic diarrhea

Variable Nasal discharge, snout deformities

TABLE 4.2: DISEASES System Skin and skeletal

OF SWINE

Clinical sign Lameness Arthritis Foot lesions Exudative dermatitis Erysipelas Abcesses

Parakeratosis

Erysipelothrix rhusiopathiae Sterptococci, Actinomyces pyogenes, multiple agents Candida albicans Microsporum, Trichphyton Swine poxvirus Foot-and-mouth, Swine vesicular disease, Parvovirus Zinc deficiency

Hyperkeratinization

Unknown

Dermatitis Ringworm Swine pox Vesicular diseases


Cause Osteochondrosis (genetic, Vit. D deficiency, nutrient imbalances) Streptococci, Mycoplasmae Housing conditions (concrete floors, grids) Staphylococcus hyicus

Remarks Weak legs, kneeling position Swollen, hot joints Lesions on hooves, lameness Greasy pig disease, black greasy spots Red spots, generalized Enlargement under skin, purulent inflammation Circular spots, grey exudate Circular red spots Palules and pustules Vesicular lesions especially on the snout and edge of the hooves Eryhtematous areas, overlaid with scales Brownish greasy scales on the back

TABLE 4.3: ANTIMICROBIAL Drug Amoxicillin Ampicillin Erythromycin Lincomycin Neomycin Oxytetracycline Oxytetracycline Sulfachlorpyridazyne Sulfamethazine Sulfathiazole Tiamulin Tylosin Ceftiofur Enrofloxacin

DRUGS FOR SWINE100

Maximum daily dose (mg/kg)a 11-13 20 11-20 11 11 7-11 44-55 77-110 240 220 9 18-22 3 2.5

Intervalb

Routec

SID TID SID SID SID SID SID SID SID SID SID BID SID SIC

PO SC, IM IM IM PO IM PO PO PO PO PO IM IM IM

a) Review drug labels for specific dosages b) SID = once a day; BID = twice a day; TID = three times a day c) PO = oral (per os); SC = subcutaneous; IM = intramuscular

are described later in this chapter. General conditions in which euthanization is advised are: • • • • • • • • •

rectal or vaginal prolapses with tissue damage wounds of such severity that healing is in doubt multiple sores and wounds, including tail-biting wounds cardiac failure lameness from a broken leg or arthritis paralysis or neurological disorders strangulated hernias emaciation severe septicemia, pneumonia, or enteritis The procedure for necropsy is described in Chapter 5.

anesthesia and analgesia Swine are considered challenging animals to work with. Lack of cooperation when physical restraint is applied often hinders


examination and treatment procedures in these animals. They vocalize loudly and struggle hard when restrained. Furthermore, massive restraint imposed on swine may profoundly stress cardiopulmonary function. Therefore, chemical restraint or anesthesia are often needed for even minor procedures. Anesthesia of swine presents some special problems compared to other animal species. There are few accessible superficial veins and arteries. Tracheal intubation can be difficult because of anatomical peculiarities (i.e., small oral orifice, dorsal protrusion of the tongue, and ventral sloping of the larynx) which make the laryngeal opening difficult to see. Also, there is a high incidence of malignant hyperthermia.

Reactions of Swine to Sedation and Anesthesia During development of sedation and anesthesia, swine first become ataxic. Soon after they lose their righting reflex and become immobile and unresponsive to nonpainful manipulations. At this stage, purposeful movement will occur in response to a painful stimulus, and the swine may vocalize and try to right itself. When anesthesia continues to deepen, the animal will still make purposeful movements in response to painful stimuli but will not vocalize or attempt to right itself. With further deepening of anesthesia the response to painful stimulation is limited to the specific region stimulated, whereas with deep anesthesia local movement in response to a painful stimulus is either slight or completely absent.

Routes of Administration of Sedatives and Anesthetics Sedatives are usually administered by intramuscular (IM) route, whereas anesthetics are generally administered intavenously (IV). Routes of administration are described in Chapter 5.

Preanesthetic Management Like all other animals, swine should be acclimatized for preferably one to two weeks in the experimental facility before being submitted to anesthesia and surgical procedures. Clinical examination as described in Table 4.3 should be performed before any procedure.


The animals should not be fed 12-24 hours prior to anesthesia to prevent vomiting during and after anesthesia. Piglets are prone to hypoglycemia and should be held from sucking for only 1-2 hours prior to anesthetic induction. The fasting time should only be decreased in emergency situations, as fed swine are prone to develop gaseous distention and are at an increased 101 risk of vomiting with aspiration. Animals should not be deprived of water at any time unless gastric surgery is being performed, and in these cases water should only be withheld for a few hours prior to surgery. The animals should be weighed in order to calculate the correct anesthetic dosage.

Fig. 4.3 Drugs for premedication (azaperone 4 mg/kg, midazolam 0.5 mg/kg, atropine 0.04 mg/kg IM)

Premedication Animals are premedicated in order to sedate and relax them before the anesthetic procedure. The premedication will also reduce the dosage of the anesthetic drug. Often the premedication will include an analgesic drug (preemptive analgesia). In most cases atropine should be administered to reduce bronchial


and salivary secretions, and to protect the heart from vagal inhibition which might occur during endotracheal intubation or during surgical procedures, particularly if the viscera are handled.

F i g . 4 . 4 IV access to the ear vein with a 22-gauge Venflon® cannula

Anesthetic Induction Delivery of inhalation anesthetics via a face mask is very stressful and should be avoided if possible. The induction should be performed in quiet surroundings, as sedated animals are susceptible to noise. An IV access of the ear veins is prepared on the dorsal surface of the ear and the skin is rinsed with alcohol. While the vein is being compressed at the base of the ear, a 22gauge Venflon® cannula, or similar cannule, with a length of 25 mm (1 inch) is introduced into the vein and secured with adhesive tape. The Venflon® cannula should be flushed with heparinised saline. Two common anesthetics for induction are: 1.

Propofol (5 mg/kg IV)


Half of the dose is given relatively rapidly, and after one to two minutes the remaining propofol is slowly injected until the animal is reflexless, i.e., relaxation of the jaw muscle and total absence of muscular contractions of the tongue. At this point the animal should still breathe spontaneously. (The more rapidly the anesthetic is injected into the vascular system, the more rapidly the animal goes through the different anesthetic stages. Passing the different anaesthetic stages should not be too slow, but not too rapid, either.) 2.

Thiopentone (6 mg/kg IV) The same procedure is used as for propofol, but not as rapidly. Inject half of the dose during the first minute, and after a few minutes inject the remaining thiopentone until the animal is reflexless.

Endotracheal Intubation Endotracheal intubation should be performed as soon as anesthesia is induced. It provides a means of alveolar ventilation and protects the airway and lungs in the event of regurgitation. Endotracheal intubation can present some difficulties, but once the anatomy is understood the procedure is easily carried out. Proper placement of the tube is confirmed by feeling expired air moving through the tube or by visual confirmation via laryngoscopy. The size of the tubes range from Nos. 3.0-8.0 for swine under 50 kg, and Nos. 8.5–14.0 for swine over 50 kg. Once the tube is in place, it should be secured using tape or gauze around 101 the lower jaw. Endotracheal intubation is usually followed by apnea, so assisted ventilation is critical at this stage, but excessive ventilation will decrease blood carbon-dioxide concentration and may prolong apnea. The tube is connected to a Magill circuit with oxygen, and the reservoir bag is depressed once or twice to ventilate the animal manually. This should be repeated until the animal has a regular spontaneous ventilation. The intubation procedure may be performed with the animal in dorsal, lateral, or sternal recumbency.


Dorsal recumbency The swine is placed in dorsal recumbency. No assistant is needed. The laryngoscope is positioned with the handle turned

Fig. 4.5 Endotracheal intubation, schematic. The laryngoscope (1) first presses the soft palate (3) to dorsal, and thereafter the tongue to ventral, to reveal the epiglottis (2). When the laryngeal opening is visible, a tube can be inserted. A= oropharynx; B= nasophanynx; 1= laryngoscope; 2= epiglottis; 3= soft palate; 4= pharyngeal diventicle; 5= laryngeal diventricle; 6= trachea; 7= esophagus upward and passed into the pharyngeal cavity. The tip of the laryngoscope is used to displace the epiglottis from the soft palate. Now the epiglottis and the laryngeal aperture are visible and can be sprayed with a topical local anesthetic (lidocain) to prevent laryngeal spasm and coughing when intubating lightly anesthetized animals. The epiglottis is depressed with the tip of the laryngoscope upwards against the base of the tongue, and firm pressure with the blade of the laryngoscope presses the maxilla against the surface of the table. Now the vocal cords can be observed. The laryngoscope is tilted 45º, which will result in the larynx being flattened against the ventral surface of the neck. As a result the laryngeal passage will be straightened. The


endotracheal tube is passed into the trachea during expiration while being gently rotated in a screw-like fashion. This will facilitate the passage through the apertura without undue 102,103,104 force.

Lateral recumbency The swine is placed in left lateral recumbency with the assistant extending the neck by holding the upper jaw with the left hand and the lower jaw and the tongue with the right hand. A laryngoscope with a 20-25 cm straight blade is placed at the base of the tongue. With the tip of the laryngoscope, the soft palate is lifted upwards so that the epiglottis becomes visible. The larynx is sprayed with a topical local anesthetic. The endotracheal tube, stiffened by a guide, is used to press the epiglottis forward onto the base of the tongue, and the tip of the laryngoscope blade is placed on the epiglottis to bring the vocal cords into view. The tube is now advanced into the trachea during expiration. A slight rotation of the tube will facilitate the introduction.

Fig. 4.6 Endotracheal intubation, lateral recumbency. The assistant holds the mouth open


Sternal recumbency The swine is placed in sternal recumbency with the assistant holding the mouth open. The procedure is the same as described for lateral recumbency.

Fig. 4.7 Endotracheal intubation, lateral recumbency. After the laryngeal opening is visualized with a laryngoscope, a tube is inserted

General Anesthesia General anesthesia can be maintained by either inhalation anesthesia or continuous infusion.

Inhalation anesthesia • Halothane(1-2%) in an oxygen/nitrous oxide (1/1) mixture. • Isoflurane(1.5-2.5%) in an oxygen/nitrous oxide (1/1) mixture. • Sevoflurane(2.5-3.5%) in an oxygen/nitrous oxide(1/1) mixture. Respiration should be assisted by artificial respiration with a tidal volume of 8-12 ml/kg body weight, and a frequency of 101 15-20 strokes per minute.


TABLE 4.4: ANESTHETIC

PROTOCOLS.

Premedication Combinations Azaperone Midazolam Atropine

4 mg/kg IM 0.5 mg/kg IM 0.04 mg/kg IM

Moderate to deep sedation; no analgesia; moderate muscle relaxation; non-painful manipulation possible.

Ketamine Medetomidine Butorphanol

6-10 mg/kg IM 0.06-0.08 mg/kg IM 0.2 mg/kg IM

Deep sedation, or short term surgical anesthesia; muscular relaxation; laryngeal relaxation makes intubation possible; effectively reversed by yohimbine.110 111

Tiletamine/Zolazepam Xylazine

4.4 mg/kg IM 2.2-4.4 mg/kg IM

Reliable and rapid anesthetic induction, or light to medium short term anesthesia for minor procedures; laryngeal relaxation makes intubation possible; apnea may occur.102 106 108 112 113

Anesthetic Induction Propofol

1.5–5.0 mg/kg IV

Thiopental

6.6-25 mg/kg IV

Inhalation Anesthesia Nitrous oxide

MAC= 195%

Halothane

MAC= 0.91-1.25%


Rapid induction, rapid recovery; rapid injection may cause apnea.102 104 105 Dilute to 5% solution; prolonged recovery; apnea may occur.102 104 105 Low potency in swine; not as sole agent; reduces the requirement of other inhalant agents.103 105 Potent myocardial depressant (dose-dependant); lower safety margin than isoflurane and sevoflurane.103 105

TABLE 4.4: ANESTHETIC

PROTOCOLS.

Isoflurane

MAC= 1.2-2.04%

Sevoflurane

MAC= 2.3-2.66%

Infusion Anesthesia Propofol infusion

4-7 mg/kg/h IV


More rapid induction and recovery than halothane; dosedependant depression of cardiovascular system less than with halothane, and at higher doses; combination with nitrous oxide reduces the dose required; no hepatic injury; minimal biotransformation.103 105 New agent; very rapid induction and recovery; minimal biotransformation; minimal cardiovascular depression.

Poor analgesia; must be combined with potent analgesic for major surgical procedures.

Infusion anesthesia • Propofol infusion: 4-7 mg/kg/hr IV, in combination with 104 a potent analgesic, e.g., fentanyl (30-100 m g/kg/hr IV).

Anesthetic Maintenance and Assessment of Anesthetic Depth During a specific anesthetic regimen it is important to assess the anesthetic depth continuously. Autonomic responses have been assumed to be of value in animals, but properly validated studies still need to be done. In spite of this, it seems reasonable to assume that i neuromuscular blockers are not used, the loss of consciousness and unresponsiveness to painful stimuli will indicate adequate anesthesia for most drugs. It is generally accepted that purposeful movement in response to painful stimulus must be taken as indicating conscious awareness of pain. During surgical anesthesia the animal should not react to painful stimuli. A surgical rat-tooth forceps should be used to apply the stimulus in order to predict the absence of response to a surgical stimulus.105 Different areas of the body lose their sensitivity to noxious stimuli at different planes of anesthesia. Loss of response in the tail occurs first, followed by loss of pedal withdrawal, and finally loss of response to stimulus to the nasal septum and perianal region. Stimulus to a dew claw, applied at full pressure with a forceps at the level of the coronary band, will be followed by pedal withdrawal or muscle twitch when the anesthetic level is insufficient. Stimulus to the nasal septum or the lateral lip fold, applied at moderate pressure, will be followed by head shaking, jaw clamping, or vocalization. Stimulus to the perianal region (anal sphincter), also applied at moderate pressure, will be followed by contraction of the sphincter muscle. Laxity of jaw tone and a stable heart rate indicate that a surgical plane of anesthesia has been achieved.102 Autonomic responses (e.g. increase in heart rate or blood pressure of more than 10%) may indicate inadequate anaesthetic depth.105

Drugs used in Swine Anesthesia Anticholinergics Atropine: • Reduces bronchial and salivary secretions


• Protects the heart from vagal inhibition, which may occur during intubation or surgical procedures101, 103 Glycopyrrolate: • Reduces bronchialand salivary secretions • Protects the heart from vagal inhibition • Longer duration of action than atropine • Glycopyrrolate is the drug of choice in swine, since they occasionally vomit during the recovery period. The drug tends to decrease gastric fluid secretion and acid contents, and promotes gastric emptying.103

Tranquilizers and sedatives Phenothiazine: acepromazine • Produces sedation, but no analgesia • Acts synergistically with other anaesthetics and narcotic analgesics, and reduces the required dosages of these drugs • The duration of action may extend into the postoperative phase, making recovery smooth. Moderate hypotension may occur.108 Butyrophenones: azaperone, fluanisone • Sedation but no analgesia • More potent than phenothiazines • Some hypotension may occur101,103 Benzodiazepines: diazepam, midazolam, zolazepam • Induce mild hypnosis, sedation and muscle relaxation, but little or no analgesia • Potentiate the action of most anesthetics and narcotic drugs • Midazolam, being water soluble, may be more reliable in uptake. Midazolam is more potent than diazepam and hence requires a smaller dose, but has a shorter duration of action.


• Antagonists exist (Flumazenil)103,108 Alpha-2-agonists: xylazine, medetomidine • Should not be used as a sole agent. • Alpha-2-agonists have transient analgesic activity and produce hypotension. • In combination with other agents, good sedation and muscle relaxation is provided. • Specific antagonists exist (Atipamezole, Yohimbine).101,102,108, 109,110

Anesthetics Ketamine: • Produces a state of cataleptoid sedation with variable analgesia, increased muscle tone and amnesia. • Minimal depression of the cardiovascular system. • Should not be used as a monoanesthetic since it does not provide adequate analgesia and muscle relaxation.102,105, 107 Tiletamine/zolazepam: • Produces heavy sedation and immobilization for approximately 20 minutes • In combination with other drugs (e.g., xylazine) endotracheal intubation is possible and adequate analgesia is provided.101, 102, 106, 107, 111,112 Barbiturates: pentobarbitone, thiopental • Dose-dependent sedation advancing into hypnosis. • Poor or no analgesic activity. • Swine are prone to apnea secondary to the administration of these drugs and assisted ventilation is necessary. • Severe cardiovascular depression and prolonged recovery may occur. • Should not be used as a sole agent.101,102,108


Isopropyl derivative: Propofol • Hypnotic agent providing effective sedation, muscle relaxation, and moderate analgesia with a minimal depression of cardiovascular function. • Rapid onset and short duration of action • Apnea may occur, so assisted ventilation must be at hand. Must be combined with adequate analgesic for major surgery. • No preservative is added, therefore the entire vial must be used shortly after opening to avoid the risk of bacterial contamination.101,102,103

Antagonists Atipamezole: • A potent, selective, specific alpha-2-antagonist. • Effectively reverses medetomidine and xylazine sedation. Flumazenil: • Specific benzodiazepine antagonist. • Antagonizes lingering sedation and muscle relaxation.103 • Nalbuphine: • Specific opioid agonist-antagonist. • Antagonizes the effects of m-agonists (i.e., fentanyl), while maintaining an analgesic effect at k-receptors.108 Naloxone: • Pure opioid antagonist with effect at m and k-receptors. Yohimbine: • A specific alpha-2-antagonist. • Rapidly reverses the effects of xylazine.113 Doxapram: • Analeptic with specific stimulatory effect on the respiratory system. • Short duration of action, therefore repeated doses may be required.108


Analgesia Pain is a severe, unpleasant sensory and emotional experience accompanied by fear, anxiety, and panic—hence suffering of the animal. Even though there is still a lack of objective means of pain recognition and assessment in animals, it is reasonable to assume that pain is of significance to animals and is a sensation they would rather avoid if they could. Until we know more about pain perception in animals, we must assume that conditions which would cause pain in man also lead to the perception of pain in animals – thus it is appropriate to administer analgesics to prevent pain where it is likely to occur.103,114

Pain Control The goal is to weaken or interrupt the nociceptive process at one or more points between the peripheral nociceptor and the cerebral cortex. This is done by using balanced analgesia, where analgesic drugs are administered in combination and at multiple sites to induce analgesia by altering more than one part of the nociceptive process. An opioid analgesic combined with a nonsteroidal antiinflammatory drug (NSAID) and a local anesthetic could be used. A local anesthetic (e.g., bupivacaine) infiltrated at the site of incision will provide analgesia for 10-12 hours, whereas a NSAID will weaken transduction of the noxious stimuli by decreasing production of endogenous algogenic substances (e.g., prostaglandins) at the site of injury. Opioids will weaken the perception of pain in the brain. Preemptive analgesia refers to the application of balanced analgesic techniques prior to exposing the patient to noxious stimuli. Administration of drugs prior to a painful procedure will provide the optimal analgesia.102 Surgically induced pain may require the use of opioids for 2448 hours postoperatively (72 hours after major surgery).103, 108 NSAIDs may be used in combination with opioids postoperatively for 24-36 hours, and can be continued when opioids are no longer necessary for a further 24-36 hours.108


Pain Recognition in Swine It is important that swine recover rapidly after surgical procedures. Rapid recovery will without doubt influence wound healing positively. Since there still are no objective tools for pain assessment in swine, the animals' behavior serves as a standard for subsequent pain evaluation. Normal behavioral observations in swine include interest in the surroundings, including staff; willingness to move around; explorative behavior; tail wagging; reaction to handling; vocalization when presented with feed; and willingness to eat. When swine react differently from this pattern, pain and distress might be the cause; e.g., when swine lie unresponsive in sternal recumbancy, it is a typical indication of pain. Also, when swine are reluctant to eat, pain or distress should always be considered.115

Drugs Used in Swine Analgesia Opiates Opiates are potent analgesics with relatively short half-lives that are used to alleviate severe and acute pain.103 Butorphanol: • Potent mixed agonist-antagonist that has three to five times the potency of morphine. • Its m-antagonist properties can be used to reverse the action of fentanyl while maintaining some analgesic effects by its agonist action at k-receptors.103 Buprenorphine: • Potent partial m-agonist. • Prolonged duration of action (6-12 hours).103 Fentanyl, Sufentanil: • Potent analgesics, about 80-100 times more potent than morphine. • Respiratory depression may occur. • Short duration of action.


• Part of balanced anesthesia during surgery; administered as continuous IV infusions.108

Non-steroidal anti-inflammatory drugs (nsaids) Good analgesia for post-surgical pain, or where pain is induced by inflammation or is chronic and of integumentary or musculoskeletal origin. NSAIDs should not be combined with steroids or other NSAIDs, but can be combined with opioids or local anesthetics. Adverse reactions such as disturbed renal function, impaired platelet function, and gastrointestinal ulceration and bleeding may occur.103 Carprofen: • Good analgesic for orthopedic and soft-tissue postoperative pain. • Can be administered preoperatively. • No nephrotoxicity, gastro-intestinal bleeding, or hemostatic problems. Flunixin: • Should not be administered for longer than three days. Ketoprofen: • postoperative and chronic treatment of pain. Keterolac: • Should not be administered for longer than three days. • Moderate to strong analgesic properties.116 Phenylbutazone: • Mild analgesic properties.

Monitoring during Anesthesia The cardiovascular system, the pulmonary system, and body temperature are important parameters to monitor during anesthesia. Monitored values should be recorded every ten minutes. Evaluation of the cardiovascular system can be performed by palpation of peripheral pulse, blood pressure measurement (invasive or noninvasive), heart rate, and ECG.101 One should remember that ECG monitoring only assesses the electrical activity of the heart and


not the mechanical activity.102 Evaluation of the respiratory system can be perfomed by evaluation of mucous membrane color, blood gas analysis, pulse oximetry, capnography measures, and blood pH.

Anesthetic Support The swine is very prone to hypothermia because of inefficient thermoregulatory mechanisms and very little body hair, as well as a large body-surface area. Thermal support is therefore of vital importance. Circulating warm-water blankets as well as blankets during surgery will minimize the heat loss. Intravenous fluid administration during the whole anesthetic period with a warmed, balanced electrolyte solution administered at a rate of 5-10 ml/kg/hour should be standard.101 Postoperative apnea is often a problem. When a swine is taken from the respirator, the ability to breathe should be carefully monitored.102

Anesthetic Emergencies Laryngospasm Insufficient anesthetic level, excessive manipulation, numerous unsuccessful attempts to intubate, and extubation may all lead to laryngospasm. Succinylcholine 1-2 mg/kg IV and immediate ventilatory support may be attempted. Local spraying of the lanynx with 2% lidocain prior to intubation will decrease the risk of laryngospasm.101

Edema of the larynx Difficulties in connection with intubation and overinflation of the endotracheal cuff can cause swelling and edema of the airways, and can lead to airway obstruction. Such tissue trauma does not become apparent until the swine is extubated. Corticosteroids, NSAIDs or diuretics may be given prophylactically or symptomatically.101

Respiratory system Insufficient ventilation of the animal can lead to respiratory arrest and death. Whenever there is insufficient ventilation or respiratory arrest, the oxygen supply should be checked. Admin-




istration of anesthetic, including nitrous oxide, should be stopped and 100% oxygen given. Ventilation must be assisted, manually or mechanically. The airways must be checked to see if the tongue is obstructing the larynx. The respiratory tract should be aspired to remove bronchial secretions, blood, or vomit. A specific respiratory stimulative such as Doxapram can be used in a dosage of 5-10 mg/kg IV. The action of this drug is of relative short duration, so repeated doses may be required every 15-20 minutes. A specific antagonist should be considered.

Cardiovascular system Most anesthetic drugs depress the cardiovascular function. In case of cardiovascular failure, administration of anesthetic should be stopped and ventilation assisted with 100% oxygen, 12-20 breaths/min. Rapid intravenous infusion of fluids (isotonic saline, plasma expanders, or whole blood) is given. Any hemorrhage is controlled, and if bradycardia occurs, this is treated with atropine 0.02 mg/kg IV (or intratracheally). In case of cardiac arrest, external cardiac massage, 80-100 compressions/min, should be given. Adrenaline 0.01 mg/kg is administered intracardially, but IV administration or administration into the endotracheal tube can also be performed.

Malignant Hyperthermia/Porcine Stress Syndrome Malignant hyperthermia/Porcine Stress Syndrome is a hereditary condition where the animal, when exposed to halothane, shows increased blood lactate concentration followed by metabolic acidosis and hyperkalemia. The symptoms are tachycardia, stiffness of the muscles, tachypnea, and hyperventilation, which rapidly progresses to apnea, cyanosis of the skin, and a rapid rise in body temperature. The treatment consists of IV administration of dantrolene 1-3 mg/kg. The condition can be prevented by injecting dantrolene 3.5-5 mg/kg IV prior to administration of halothane.102

Postoperative Management Postoperative care is of vital importance, and frequent monitoring of the animal cannot be overemphasized. Recovery should always take place in a separate room where the animal cannot injure


itself. A room temperature of 25° C (77° F), a heating lamp, and circulating warm-water blankets can be used to prevent hypothermia. Extubation should only be performed when a strong swallowing reflex is apparent. In case of postoperative vomiting or excessive secretions, the respiratory passages should be cleared by suction. Fluids and antibiotics should be administered when necessary.

euthanasia Swine are euthanized humanely by IV injection of 20% solution of sodium pentobarbitone, either directly into the cranial caval vein or into an ear vein after sedation. If the use of sedatives and anesthetics is excluded, a captive bolt can be used. This method must be followed by exsanguination. With any method, cessation of respiration and heartbeat, as well as loss of reflexes, are good indicators of irreversible death. Death should always be confirmed by exsanguination.115

Fig. 4.8 Captive bolt pistol; the bolt pisol is placed on the skull, at the crossing of two immaginary lines from the ears to the eyes


5 experimental techniques Swine have been used as experimental animals throughout history, mainly in anatomical and physiological research. In his De Humani Corporis Fabrica from 1543, Vesalius referred to swine experiments performed by the Roman physician Galen (130-200 AD), but made observations in swine himself as well. Also, Harvey, in his Anatomica de Motu Cordis et Sanguinis Animalibus (1628), and Bernard, in his Introduction à l'étude de la médicine expérimental (1865), described observations made in swine. At present, swine are used in a wide range of disciplines.

restraint Restraint techniques in swine fall into three categories: manual 11,12, 94 restraint, mechanical restraint and chemical restraint. An often forgotten but simple method of restraint of placid sows is stroking the abdomen while talking softly to the animal. This will allow general examination. In any case, the animals have to be approached first. Swine have to be approached quietly, and talking softly will calm the animal. Crouching is less threatening to the animal than standing and bending over it. Usually, the swine will face the approaching person in an awaiting attitude. When swine feel threatened, they face toward the source of threat to avoid


Fig. 5.1 Illustration of Galen from De Humani Corporis Fabrica (1543) being attacked from the side, since the natural offensive behavior of swine is butting and biting on the shoulder and neck region. Young swine and miniature swine may be seized by the front leg in this situation and lifted up onto the arms. If the animal is facing away or tries to escape, a hind leg may be grabbed instead. The tail and ears should not be used to lift the animal. Larger swine should preferably be trained to walk from their pens, and they can be driven in the right direction by walking behind them. A board can be used to drive the animal, and can also be used to restrain large swine against a wall. Young swine and miniature swine under 15-20 kg (33-44 lbs) can be lifted and manually restrained by holding them in one's arms. One arm is held under the abdomen or hindquarters, while the other arm supports the bow. This method is suitable for a single IM administration in the cervical muscles or a short examination. For examining the abdomen or feet, a swine can


be positioned on its back in the lap of a sitting person. Alternatively, the animal can be held by both hind legs, head down. When blood sampling is needed, drugs are to be administered, or a short examination is necessary, large swine are often restrained with a nose snare. A loop, usually a steel cable, is placed around the upper jaw behind the tusks to hold the animal. In large swine, this is often the only method of restraint, other than chemical restraint. It is a stressful method, and should be avoided in young and miniature swine. Swine under 50 kg (110 lbs) can be restrained in dorsal recum122 bency in a V-shaped trough, or on the abdomen in a hammock. Both methods are mainly used for short restraint for blood sampling, but trained, conscious swine may be restrained in a hammock over longer periods, e.g., for infusion studies or ECG recording. In contrast to dogs being restrained in hammocks, swine should hang freely without touching the floor, since they cannot be trained to stand still. Mechanical restraint methods for specific purposes may be developed individually, such as restraint 123 in a bucket for intranasal inoculation or swabbing.

Fig. 5.2 Restraint method using a V-trough


Fig. 5.3 Swine restrained in hammocks during a 6-hour infusion study (Photo courtesy of Scantox) Inactivation of swine by sedatives is referred to as chemical restraint. The most common sedative in swine is azaperone (Stresnil, Sedaperone), which should be dosed at 4 mg/kg IM (see Table 4.4). A single IM administration in large swine can be carried out in the animal's pen by using a syringe with an extension tube. The hypodermic needle is darted into the cervical muscles and the syringe emptied. The extension tube allows some degree of movement of the animal.

sampling techniques Blood Sampling There are few percutaneous access sites to veins in swine, since veins and arteries lie either under thick skin and subcutaneous 101,103 fat, or deep between muscles. Possible sampling sites are: • cranial caval vein • jugular vein


• femoral vein • saphenous vein • cephalic vein • carotid artery • femoral artery • cardiac puncture The cranial caval vein and femoral vein are most often used, and the procedure will be described. The ear veins are not suited for withdrawal of large blood samples since these veins are too small and will collapse upon withdrawal of blood. Arteries should be avoided for blood sampling since puncture may lead to the formation of hematoma. The best location for blood sampling is from the cranial caval vein, with the swine in dorsal recumbency. Large swine have to be restrained standing, with a snare. For dorsal recumbency, the swine may be placed on a table top or in a V-shaped trough, the latter being preferable as it limits the movements of the animal. One or two assistants are required to restrain the animal in addition to the person taking the blood sample. The front legs are bent caudally and the neck is stretched, holding the animal by its snout. A second person may hold the hind legs. To prevent a rightening reflex when placing the animal on its back, the animal should not be rolled over the longitudal axis into the Vshaped trough, but over the transversal axis. When this is done in a quiet manner the animal will be relaxed and easy to restrain. The manubrium of the sternum is now palpated, and a 21-gauge hypodermic needle inserted into the medial plane at an angle of 45-60° to an imaginary horizontal line, a few millimeters cranial to the manubrium of the sternum. Creating a slight vacuum in the syringe while inserting the needle will facilitate easy detection when a vein is punctured since blood will stream into the syringe. A vacutainer blood-sampling system will ease the procedure. For swine up to 10-15 kg (22-33 lbs), a hypodermic needle of 25 mm (1 inch) can be used. Larger swine, 6 from 15-50 kg (33-110 lbs), require a needle of 45 mm (1 /8 inches), whereas swine over 50 kg (110 lbs) require a needle 1 size of 18 gauge/65 mm (2 /2 inches). The hypodermic needle should not be inserted at too flat an angle, i.e., under 45°,


because of the risk of puncturing the pericardium or the heart. Alternatively, a hypodermic needle can be inserted into the jugular furrow. The point of the needle should be directed toward the medial plane, dorsal to the manubrium of the sternum. The angle to an imaginary horizontal line should be 45°. The two methods described will puncture the brachiocephalic plexus, cranial to the cranial caval vein. The caval vein may also be punctured since it is unsure exactly which vessel has been punctured. When inserting a needle into the jugular furrow in the sagittal plane, the jugular vein will be punctured. However, in young swine and miniature swine, this method is not as successful as puncturing the brachiocephalic plexus.

Fig. 5.4 Sites for blood sampling. The needle should be inserted under 45-60°, cranial to the sternum (A), or in the jugular furrow (B), and directed toward the medial plane. To the right, the figure shows the location og the needle in a large swine, restrained with a snare. A= insertion point cranial of the manubrium of the sternum; B= jugular furrow; 1= briachiocephalic vein; 2= external jugular vein; 3= internal jugular vein


Fig. 5.5 Blood sampling from the brachiocephalic plexus in a miniature swine in dorsal recumbancy The blood volume of a single sample should not exeed 10% of 124 the total blood volume. Since swine have a blood volume of 75 ml/kg, a maximum volume of 7.5 ml/kg can be taken. An interval of 2 weeks between single samples should be respected. In repeated sampling, as used in pharmacokinetic studies, it is recommended to not take more than a total volume of 7.5 ml/kg. For a 10-kg swine this corresponds to 15 samples of 5 ml within 24 hours, whereafter the animal is not sampled for two weeks. Unlimited access to drinking water should be secured to allow the animal to rehydrate itself. The site of sampling should be varied in repeated sampling to reduce the risk of hematoma. For accessing the brachiocephalic plexus, sampling could be varied between the right and left jugular fossa and the medial plane. If repeated sampling is necessary over a long period, vascular 101,125,126 catheterization may be considered. Methods are described later in this chapter.

Urine and Feces Only in sows can the urethra be catheterized since boars have a cork-screwed penis tip and a sigmoid curve in the body of the


penis. However, urine sampling in swine by catheterization of the urethra is seldom performed since it requires an anesthetized animal. A large suburethral diverticulum disables catheterization in conscious swine since catheterization is difficult without the help of a speculum. Urine and feces are often collected by means of a metabolic cage. Cages designed for dogs may be used for small or miniature swine. Feces will stay behind on the grid floor of the metabolic cage, while urine is collected via a pan with a funnel. Contamination of urine with feces is unavoidable, but can be minimized by collecting the feces regularly.

Cerebrospinal Fluid Cerebrospinal fluid can be collected from the anesthetized swine by puncturing the cisterna magna. Sampling cerebrospinal fluid in conscious swine is not possible because of the non-cooperative behavior of swine. The swine should be placed in lateral recumbency and the head fully flexed. The cervical area should be clipped and disinfected, e.g., with 1% chlorhexidine in 70% ethanol. A 20-gauge spinal needle with a length of 90 mm (3 1/2 inches) provided with a stylet should be inserted about 5 cm (2 inches) caudal to the occipital protuberance and advanced slowly toward the mouth. The needle should be kept parallel to the table surface. The atlanto-occipital membrane and dura mater of the cisterna magna lie about 7 cm (2 6/8 inches) deep in a 40-kg (88 lbs) swine. A volume of 5-10 ml can be collected at intervals of four days.127

Bile and Pancreatic Excretions Bile and pancreatic excretions can be collected in a chronically catheterized animal.101,128 Methods are described later in this chapter.

administration of compounds Small needle sizes (19-21 gauge) can be used for most swine for SC, IM, or IV injections.104 Subcutaneous administration (SC) is not easily executed since the swine is a fixed-skin animal, like humans. Small


Fig. 5.6 SC administration volumes may be given on the lateral side of the neck immediately caudal to the ear or in the flank.104,105 In fat swine, the injection is often administered into the subcutaneous fat. Intramuscular administration (IM) is preferably given in the cervical muscle groups, in the region overlaid by the ear, close to the transition from hairy to hairless skin. This region has the thinnest layer of fat, with the muscles closest to the skin. The semimembranous, semitendinosus, and gluteal muscles in the hind leg may also be used, but cause more resentment to injection than the neck region. The drugs are administered through a 19- to 21-gauge needle, with a minimum length of 34 cm (1 1/8 - 1 6/8 inches) to ensure that the injection is given into the muscle. The needle can be connected to an extension tube, which will allow the swine to move freely while the drug is being injected. Intravenous administration (IV) often requires a sedated animal. The central or ventrolateral ear veins are most commonly used. The artery is located on the dorsolateral aspect of the ear.105 A 20- to 22-gauge hypodermic needle can be used. The use of a butterfly needle has the advantage that the needle


Fig. 5.7 IM administration in the neck

Fig. 5.8 IM administration in the hind leg


Fig. 5.9 IV administration; puncture of marginal ear vein with a butterfly needle can be taped to the ear. When a Venflon® over-the-needle cannula or similar cannula is used, the cannula can be kept in the ear vein for two to three days. The use of a stylet will prevent coagulation inside the cannula in this situation. For an emergency situation the cranial caval vein can be used. This vein can be accessed as described earlier. Intraperitoneal administration (IP) is seldom used in swine.

basic surgical procedures101, 103 All surgery should be performed using aseptic procedures and techniques. Anything that comes into contact with the surgical site should be sterile, i.e., instruments, suture materials, syringes, and needles. To prepare the animal for surgery the hair should be removed from the surgical site with a clipper, and the skin scrubbed with soap and water, then disinfected with alcohol and an iodine solution. The surgical site should be


Fig. 5.10 IV administration; administration of a compound. The butterfly needle is kept in place with adhesive tape isolated with sterile drapes. The surgeon and assisting staff should wear caps, face masks, sterile gowns, and sterile surgical gloves.

catheterization Carotid artery and the internal jugular vein A 5-cm (2-inch) longitudinal incision is made in the midline approximately 3 cm (1 1/8 inches) cranial to the manubrium of the sternum. A longitudinal incision is made in the sternohyoideus muscle down to the trachea. The vessels are located on either side of the trachea. The carotid artery and the jugular vein are isolated for a length of 2 cm (6/8 inch) using blunt dissection. There should be as little manipulation of the vein as possible since it contracts easily, which makes catheterization more difficult. After isolation of the vessel, a length of suture material is placed around the vessel at the distal end and at the proximal end. The distal suture is tied. The proximal suture is placed loosely around the vessel.


Carotid artery An arterial clamp is positioned on the artery right after the proximal suture. A small cut approximately 1/3 of the diameter of the arterial wall is made with a pair of fine scissors (preferably microscissors) close to the distal ligature. A catheter is placed in the artery, using a vascular guide to hold the lumen of the artery open and while the arterial clamp is slowly loosened, the catheter is introduced into the artery. The proximal suture is tied around the catheter and the artery, and the catheter is fixed using the distal ligature ends.

Internal jugular vein A catheter is placed in the vein in the same manner as in the artery, except that it is not necessary to use the clamp. The proximal suture is lifted slightly to empty the vessel, and the catheter is introduced into the vein using a vascular guide. It might be helpful to loosen the proximal suture a little just prior to entering the vascular lumen in order to fill the vessel with blood.

External jugular vein A 5-cm (2-inch) longitudinal incision is made approximately 5 cm lateral to the midline in the same area as for the carotid artery. The vein is located deep between the brachiocephalic and the sternocephalic (sternomastoideus) muscles. The vein is cannulated in the same manner as described for the internal jugular vein.

Cephalic vein The cephalic vein joins the external jugular vein and lies superficially in the axillary furrow. A 3-cm (11/8-inch) incision is made in the furrow perpendicular to the midline and beginning 4 cm (16/8 inches) from the manubrium of the sternum. The vessel is dissected bluntly for a length of 2 cm (6/8 inch). It is cannulated in the same manner as above.

Femoral artery and vein The femoral canal can be located by palpating the pulse in the fold between sartoric and gracilic muscles. An incision is made starting cranial to the origin of the pectineal muscle and extend-


Fig. 5.11 IV Catheterization of the jugular vein; isolation of the vein ing along the femoral canal distally on the leg. The subcutaneous tissue is dissected and the two muscle groups are located and separated bluntly. The two vessels are located in the deep, right next to the saphenus nerve, which lies cranial to the artery. The vessels are carefully separated from the nerve and then dissected over 2-3 cm (6/8 - 1 1/8 inches). Beware of the side-branches of both the artery and the vein. These must be carefully ligated and transsected. Both vessels are cannulated as described above. Other vessels, such as the subcutaneous mammary veins and the median coccygeal artery, are also suitable for cannulation.

Catheter Maintenance Catheters need to be flushed daily using heparinized saline (2 U/mL). If an animal is receiving a continuous IV infusion, flushing is not necessary during the infusion period. The end of the injection cap should be cleaned with isopropyl alcohol before each use to decrease the risk of introducing bacteria into the catheter site. The catheter exteriorization site should be cleaned daily with an antibacterial solution or ointment.


Fig. 5.12 IV Catheterization of the jugular vein; introduction of the catheter

Laparotomy A 15-cm (6-inch) long midline incision is made from the xiphoideal proceeding of the sternum through the skin, subcutaneous layer, and muscle layer in the linea alba. The peritoneum is grasped with a tissue forceps, lifted, and incised with a small hole using a scalpel. The blunt tip of a pair of scissors is inserted and the peritoneum is incised, lifting the tissue to make sure not to incise the underlying organs.

Portal vein cannulation The spleen is exposed and the gastrolienalic veins are located between the large curve of the stomach and the spleen. The vein is dissected carefully, without traumatizing the spleen tissue. Two sutures are placed around the vessel. The distal suture is tied. A small cut is made in the vein and a soft silicone tube is placed in the vein and secured very loosely with the proximal suture. Too tight a ligature will prevent the further insertion of the catheter. The tube is carefully directed into the splenic vein (sharp bend), and introduced 30-35 cm (12-14 inches) further


until it can be palpated in the portal vein. The catheter has a tendency to turn downward into the gastroduodenal vein. To prevent this, the tube should be palpated by putting one’s fingers into the epiploic foramen and behind thd tube. Here, one should be able to guide the tube gently in the cranial direction.

The hepatic vein The left lobe of the liver is identified. An intestinal clamp is put on the lobe to control bleeding, and a transverse incision is made a few centimeters from the edge. A central vein is identified, and a silicone rubber tube is introduced approximately 8 cm (3 1/8 inches) into the liver after removal of the clamp. The wound is closed by putting a hemostatic material into the wound to control bleeding, and then suturing the wound.

Pancreatic duct The pancreatic duct is identified near the proximal end of the pancreas, close to the duodenum. The peritoneum is transsected and the duct is dissected. Two ligatures are placed, and the one next to the duodenum is ligated. The duct is cut with micro scissors and the catheter introduced using a vascular guide.

Safety Testing of Chemicals and Drugs Miniature swine are increasingly used as the nonrodent species in safety testing of chemicals and drugs. Large swine are used to a lesser extent, with the exception of toxicity testing of agrochemicals or other compounds available in large quantities, since body weight, and thus dosing, of large swine are the limiting factors. Miniature swine are used in all fields of safety testing, including dermal toxicology,129,130 and acute and chronic systemic toxicology by oral dosing or continuous infusion.76 131 Miniature swine have also been used in specialized fields such as immunotoxicology,74 reproductive toxicology,132 and teratology.133

Dermal toxicology The procedures for acute dermal toxicity and acute dermal irritation are described in OECD Guidelines 402 and 404, respectively, issued by the Organization for Economic Cooper-


ation and Development (OECD).134,135 The animals should be prepared as follows: • The back and sides of the swine are clipped one day in advance. • The back is divided into 6 or 8 squares on both sides of the spine. The total surface of the squares should correspond to 10% of the total body surface. • The total body surface can be estimated using:

BS = (70 x BW0.75)/1000, where BS = body surface (m2) and BW = body weight (kg). The equation is based on the fact that animals have a basal metabolism of 70 kcal/kg and a heat production of 1000 kcal/m2. The use of metabolic body weight (BW0.75) makes the equation applicable for interspecies comparison. • A prescribed amount of test compound is applied to the skin so that all squares are covered, and dressed with gauze pads. The pads are kept in place with adhesive tape. • The duration of exposure should be in accordance with the relevant regulatory guideline. After the period of exposure, the pads are removed and the compound is washed off the skin. The skin is then examined for erythema and edema.130

Acute and chronic systemic toxicology Guidelines for acute and chronic systemic toxicology relevant for swine are, among others, described by OECD in guidelines 401, 409 and 452. Guideline 409, subchronic oral toxicity, nonrodent: 90 days, mentions miniature swine explicitly as an acceptable species.136 Oral administration of the test compound can be done by: • Mixing the compound directly into the feed. • Administering the compound in capsules into the oropharynx via a balling gun.


• Administering the compound directly into the stomach via gastric intubation. Gastric intubation is the most controlled method of oral administration. Although swine are ready eaters, feed may have a reduced palatability after compound has been mixed into it. Capsule dosing has the uncertainty of capsules not being swallowed. Swine have to be restrained for gastric intubation. The procedure is as follows: • A technician sitting on a table top holds the swine in an upright position sitting on its hindquarters; the swine is held between the legs of the technician by firm pressure. • The front legs of the swine are bent caudally and held with one hand. The other hand stretches the neck of the swine, and the animal is pressed against the chest of the technician. A seat fixed to the table top in which the swine can sit will ease the restraint. • A soft gastric tube of 3-4 mm in diameter can be used for miniature swine of 5-15 kg (11-33 lbs). For larger swine, a 6 mm gastric tube can be used. The length of the tube should reach from the mouth to past the last rib of the restrained animal. • The tube is inserted into the mouth and introduced. Normally, the swine will protest and vocalize loudly. When the swine stops vocalizing, it should be checked if the trachea has been intubated instead of the esophagus by listening to the respiration. The compound is injected into the stomach when the tube is in place. No guidelines for maximum volumes are available, but a typical volume is 10-20 ml. Intravenous administration of test compound can be by single injection, repeated administration, or continuous infusion. Methods of single IV administration were described in earlier. Repeated administration and continuous infusion require a surgically prepared animal. Methods for catheter implantation were also described earlier. A vascular access port can be used for repeated administration as well as for blood sampling.125,137 For continuous infusion, the catheter should be connected to an


Fig. 5.13 Oral administration by gastric intubation (gavaging) ambulatory infusion pump which is placed in the pocket of a jacket on the back of the swine. The procedure is as follows: • After the animal has been catheterized, the catheter is connected to the infusion pump. The catheter should be exteriorized on the shoulder, at the same level as where the pocket of the jacket will be located. The reservoir of the infusion pump should be filled with saline. • A well-fitting jacket is put on the swine while it is still anesthetized. The infusion pump is placed in a pocket of the jacket, and a counterweight is placed into the other pocket to balance the jacket. • When the swine wakes from the anesthesia with the jacket in place, it will easily accept the jacket. • The battery and reservoir should be changed daily. Seven days postoperatively the study can commence, and the saline is replaced with test compound.


A typical repeated dose study lasts for 28 days, whereas subchronic toxicity studies last for 90 days.

Fig. 5.14 Miniature swine with jacket for continuous infusion (Photo courtesy of Scantox)

necropsy92 ,93 Necropsy is an important tool in the diagnosis of disease, but also in evaluating the pathological effects of an experimental procedure. Necropsy is an essential part in the safety testing of chemicals and drugs. Whereas for the diagnosis of disease only affected organs are sampled for histopathological examination, in safety testing a narrowly described list of organs is weighed and sampled. Organs which have to be examined are described by regulatory bodies such as the Organization for Economic Cooperation and Development (OECD) and the Food and Drug Administration (FDA). Table 5.1 gives an overview of organs to be sampled for histopathological examination.

Equipment. Necessary equipment includes rubber gloves and boots, a knife with a five to seven inch blade, scalpel blades and holder, scissors, and forceps. To collect tissues and take samples for microbiological examination, containers with formalin, plastic bags, strings, and culture swabs are essential. Helpful equipment


TABLE 5.1: ORGANS

TO BE SAMPLED FOR HISTOPATHOLOGICAL

EXAMINATION IN SAFETY TESTING OF CHEMICALS AND DRUGS.

Organ/tissue

FDA Redbooka

OECDb

Adrenals Aorta Bone marrow Brain

left + right microscopy microscopy three specified sections microscopy microscopy microscopy microscopy microscopy left + right microscopy microscopy microscopy microscopy left + right microscopy microscopy only one node only one gland left + right microscopy microscopy microscopy microscopy left left left two specified sections microscopy microscopy left + right microscopy including parathyroids microscopy microscopy microscopy

left + right microscopy microscopy three specified sections

Bronchi Cecum Colon Duodenum Esophagus Eyes Femur Gall bladder Heart Ileum Jejunum Kidneys Liver Lungs Lymph nodes Mammary gland Ovaries Pancreas Pituitary Prostate Rectum Salivary glands Sciatic nerves Skeletal muscle Skin Spinal cord Spleen Stomach Testes Thymus Thyroid Trachea Urinary bladder Uterus

microscopy microscopy microscopy microscopy left + right if signs of toxicity are present microscopy microscopy microscopy microscopy left + right microscopy microscopy only one node only one gland left + right, including cervix microscopy microscopy microscopy left + right left left if signs of toxicity are present three specified sections microscopy microscopy left + right microscopy including parathyroids microscopy microscopy microscopy

a) Food and Drug Administration, Redbook, 1982. b) Organization for Economic Co-operation and Development, Guidelines for testing of chemicals, Section 4: Health Effects, 1981.


includes knife, rib cutters, microscope slides, labels, ruler, syringes, and hypodermic needles.

Preservation. The only necessary preservative is a buffered 4% formaldehyde solution (formalin). This can be prepared by adding 100 ml 40% formaldehyde to 900 ml water, and adding 6.5 g diphasic anhydrous sodium phosphate and 4.0 g monobasic sodium phosphate. Specimens should not be thicker than 5 mm to allow proper preservation. The correct tissue-formalin ratio is 1:10. Specimens meant for microbiology should be kept in a plastic bag in a refrigerator; they should not be kept in formalin or frozen.

Procedure Start by examining the external features, such as described for a clinical examination. If the animal is found dead, and the cornea is cloudy and the abdomen discolored green, a necropsy is often less valuable since the animal has undergone autolysis. The swine is placed in left lateral recumbency, then: • Cut deep into the right axilla; extend the incision to the point of the mandible and to the anus (insert the knife into the subcutis and cut outward); reflect the skin; cut the right hip joint; open the stifle and reflect the limb; examine inguinal lymph nodes. • Open the abdomen with a paracostal cut; extend to the pelvis with a paralumbar cut; reflect abdominal wall ventrally. • Stab diaphragm and incise; cut ribs and remove. • Examine peritoneal and thoracic cavities; if exudates, adhesions, fibrin, or other pathological changes are present, collect tissue for culture and histopathology; blood and urine should be collected at this point. • Open the pericardium in situ, and examine. • Cut close to the bone on the medial side of both mandibles, and free the tongue; pull the tongue caudally; cut


hyoid bones; cut soft tissue along vertebrae and free cervical and thoracic viscera intact. • Examine oral cavity; examine regional lymph nodes; incise tongue, and open the esophagus. • Palpate lungs; open trachea and major airways; cut bronchial lymph nodes; open heart. • Squeeze gallbladder to test patency. • Compress feces from rectum; place a ligature and cut distal from the ligature; free entire intestinal tract using blunt and sharp dissection. • Remove both adrenals and incise with scalpel. • Remove the kidneys by pulling the ureter caudally; peel capsules; cut to the pelvis and examine; open the urinary bladder. • Examine and remove the reproductive tract. • Remove the spleen from the stomach and incise. • Remove skin and muscle from the head; flex the head to locate the atlanto-occipital joint and cut to the ventral surface of the joint; collect cerebrospinal fluid if necessary; cut the joint and neck muscles, and remove the head. • Remove the skull cap and isolate the brain; examine the meninges; collect tissue for culture and histopathology. • Spread intestines, and open stomach, duodenum, sections of the jejunum, ileum, cecum, ascending colon and descending colon; collect tissue if necessary*. * Usually, the gastrointestinal tract is examined last in necropsies. If the clinical problem is related to the GI tract, it should be examined as early as possible since it rapidly undergoes autolysis.


6 resources This chapter is meant to give an overview of laboratory swinerelated resources without the aim of being exhaustive. It should be realized that resources are time sensitive, especially Internet resources.

associations AAALAC International The Association for Assessment and Accreditation of Laboratory Animal Care International supports the use of animals to advance medicine and science when there are no nonanimal alternatives, and when it is done in an ethical and humane way. When animals are used, AAALAC works with institutions and researchers to serve as a bridge between progress and animal well-being. This is done through a voluntary accreditation program in which research institutions demonstrate that they are not only meeting the minimums required by regulations, but are going the extra step to achieve and showcase excellence in animal care and use. AAALAC International 11300 Rockville Pike, Suite 1211, Rockville, MD 20852-3035 Phone: 301.231.5353 - Fax: 301.231.8282 E-Mail: [email protected] - Internet: http://www.aaalac.org


AALAS The American Association for Laboratory Animal Science is a forum for the exchange of information and expertise in the care and use of laboratory animals. Since 1950, AALAS has been dedicated to the humane care and treatment of laboratory animals, and to the quality research that leads to scientific gains that benefit mankind and animals. AALAS has more than 8500 members, including clinical veterinarians, technicians, technologists, educators, researchers, administrators, animal producers, and national and international experts. American Association for Laboratory Animal Science 9190 Crestwyn Hill Drive, Memphis, Tennessee 38125 Phone: 901-754-8620 — Fax: 901-753-0046 E-mail: [email protected] — Internet: http://www.aalas.org

AASP The American Association of Swine Practitioners is a nonprofit, educational, professional society organized to increase the knowledge of veterinarians in the field of swine medicine; elevate the standards of swine practice; promote the relationship between swine practice, the swine industry, and the public interest; promote the interests of swine veterinarians; improve the public stature of swine veterinarians; cooperate with veterinary and agricultural organizations and regulatory agencies; and promote goodwill among AASP members. AASP has approximately 2000 members hailing from practice, industry, and academia. American Association of Swine Practitioners 902 1st St, Perry, Iowa 50220-1703 Phone: 515-465-5255 — fax: 515-465-3832 E-mail: [email protected] — Internet: http://www.aasp.org

ACLAM The American College of Laboratory Animal Medicine is an organization of board-certified veterinary medical specialists who are experts in the humane, proper, and safe care and use of laboratory animals. ACLAM establishes the standards of education, training, experience, and expertise necessary to become qualified as a specialist, and recognizes that achievement through board certification. ACLAM actively promotes the advancement


of knowledge in this field through professional continuing education activities, the development of educational materials, and the conduct of research in laboratory animal medicine and science. ACLAM fosters the recognition of its members who contribute to human and animal health improvements by being the leaders of the veterinary medical specialty known as laboratory animal medicine. The American College of Laboratory Animal Medicine does not have a permanent office address, but can be contacted through its president or secretary. Internet: http://www.aclam.org

ASLAP The American Society of Laboratory Animal Practitioners promotes the acquisition and dissemination of knowledge, ideas, and information among veterinarians and veterinary students with an interest in laboratory animal practice. The society does so for the benefit of laboratory animals, other animals, and society in general. ASLAP believes that laboratory animals do not differ from other animals in their need for proper preventive and therapeutic medical care, nutrition, physical and psychological comfort, and all other elements that contribute to their health and well-being. The American Society of Laboratory Animal Practitioners does not have a permanent office address, but can be contacted through its president or secretary on the Internet: http://www.aslap.org

FELASA The Federation of European Laboratory Animal Science Associations was founded in 1978. Its members are the European national laboratory animal science associations from England (LASA), Scandinavia (Scand-LAS), Germany (GV-Solas), France (SFEA), Spain (SECAL), Italy (CISAL), the Netherlands (NVP), Belgium (BCLAS), and Switzerland (SGV). As an international body, FELASA represents the common interests of the constituent associations by coordinating the development of education, animal welfare, health monitoring, and other aspects of laboratory animal science. Within the Council of Europe and the European Union, FELASA has a political role through offering authorative advice to these bodies.


The Federation of European Laboratory Animal Science Associations does not have a permanent office address, but can be contacted through its president or secretary at the following Internet address: http://www.felasa.org

ICLAS The International Council for Laboratory Animal Science was founded in 1956. Its aim is to promote and coordinate the development of laboratory animal science throughout the world, and promote international collaboration. One of its main activities is the harmonization of health monitoring programs, and to realize this it has established reference health monitoring laboratories. The International Council for Laboratory Animal Science does not have a permanent office address, but can be contacted through its president or secretary at the following Internet address: http://www.iclas.org

ILAR The Institute for Laboratory Animal Research is a division of the Commission on Life Sciences, one of eight major units within the National Research Council. The National Research Council is the working arm of the National Academy of Sciences, a private, nongovernmental, nonprofit organization chartered by Congress in 1863 to "investigate, examine, experiment, and report upon any subject of science." ILAR works under the direction of a 15-member council composed of experts in laboratory animal medicine, zoology, genetics, medicine, ethics, and related biomedical sciences. The council provides advice on all aspects of ILAR's program, and formulates plans for the initiation of new programs. Institute for Laboratory Animal Research 2101 Constitution Avenue, NW, Washington, DC 20418 Phone: 202-334-2590 - Fax: 202-334-1687 E-mail: [email protected] .Internet: http://www4.nas.edu/cls/ilarhome.nsf


National SPF Swine Accrediting Agency The National SPF Swine Accrediting Agency, Inc., dates back to May 1952. Its aim is to maintain and monitor hysterectomyderived SPF swine colonies of accredited swine producers, to eliminate and prevent chronic growth retarding diseases, and to enlarge the economic profit of swine producers. The National SPF Swine Accrediting Agency can be contacted to obtain adresses of accredited swine producers in order to procure SPF swine for the laboratory. National SPF Swine Accrediting Agency, Inc. PO Box 280, Conrad, Iowa 50621 Phone: 515-366-2124 - Fax: 515-366-2232 E-mail: [email protected] - Internet: http://www.nationalspf.com

books A wide range of reference books on laboratory swine is available. Two general books are: 1.

Mount, L.E. and D.L. Ingram, The Pig as a Laboratory Animal, Academic Press, London, 1971.

2.

Pond, W.G. and K. Houpt, Biology of the Pig, Comstock Publishing Associates, Ithaca, NY, 1978.

The symposium "Swine in Biomedical Research" has been organized three times during the last 35 years, after which the proceedings were published as handbooks: 1.

Bustad, L.K., R.O. McClellan, and M.P. Burns, Eds., Swine in Biomedical research, Frayn & McClellan, Seattle, 1966.

2.

Tumbleson, M.E., Ed., Swine in Biomedical Research, vol. 1-3, Plenum Press, New York, 1986.

3.

Tumbleson, M.E. and L.B. Schook, Eds., Advances in Swine in Biomedical Research, vol. 1-2, Plenum Press, New York, 1996.


One author, Michael Swindle, has published several handbooks on laboratory swine, anesthesia and surgery, and animal models: 1.

Swindle, M.M., Basic Surgical Exercises Using Swine, Praeger Press, Philalelphia, 1983.

2.

Swindle, M.M., Swine as Models in Biomedical Research, Iowa State University Press, Ames, 1992.

3.

Swindle, M.M., Surgery, Anesthesia, and Experimental Techniques in Swine, Iowa State University Press, Ames, 1998.

A book on cardiovascular research using swine has been published. According to the authors, this book was especially prepared because "information on cardiovascular studies in swine is scattered widely throughout biomedical and agricultural literature, and few comprehensive reviews have been prepared": Stanton, H.C. and H.J. Mersmann, Eds., Swine in Cardiovascular Research, vol. 1-2, CRC Press, Boca Raton, 1986.

journals Contemporary Topics in Laboratory Animal Science Contemporary Topics in Laboratory Animal Science is a bimonthly publication that serves as the official communication vehicle for the American Association for Laboratory Animal Science (AALAS). Contemporary Topics includes a section of refereed articles on clinical, technical, management, and philosophical topics. CT also highlights AALAS news, upcoming meetings, and activities. It also provides updates on other issues related to laboratory animal science. American Association for Laboratory Animal Science 9190 Crestwyn Hill Drive, Memphis, Tennessee 38125 Phone: 901-754-8620 — Fax: 901-753-0046 Internet: http://www.aalas.org/pubs/ctonline.htm


ILAR Journal ILAR Journal is the quarterly, peer-reviewed publication of the Institute for Laboratory Animal Research (ILAR), which is a unit of the National Research Council, National Academy of Sciences. ILAR Journal provides thoughtful and timely information for all those who use, care for, and oversee the use of laboratory animals. The audience of ILAR Journal includes investigators in biomedical and related research, institutional officials for research, veterinarians, and members of animal care and use committees. Institute for Laboratory Animal Research 2101 Constitution Avenue, NW, Washington, DC 20418 Phone: 202-334-2590 - Fax: 202-334-1687 Internet: http://www4.nas.edu/cls/ijhome.nsf

Lab Animal Lab Animal is a peer-reviewed journal for professionals in animal research, emphasizing proper management and care. Editorial features include: new animal models of disease, breeds and breeding practices, lab animal care and nutrition, new research techniques, personnel and facility management, facility design, new lab equipment, education and training, diagnostic activities, clinical chemistry, toxicology, genetics, and embryology as they relate to laboratory animal science. Lab Animal PO Box 5054, Brentwood, TN 37024-5054 Phone: 800-524-0384 or 615-377-3322 - Fax: 212-696-9591 Internet: http://www.labanimal.com

Laboratory Animals Laboratory Animals is an international journal with a strong base in Europe, but with subscriptions in 49 countries. In 1997, papers from 19 countries were published in the journal. The editorial board includes members from Europe and North America. Laboratory Animals is published by Laboratory Animals, Ltd., and is the official journal of the Federation of European Laboratory Animal Science Associations (FELASA).


The Royal Society of Medicine Press, Ltd. 1 Wimpole Street, London, W1M 8AE, United Kingdom Phone: (+44) 171-290-2927 - Fax: (+44) 171-290-2929 Internet: http://www.rsmpress.co.uk/

Laboratory Animal Science Laboratory Animal Science, an international journal of comparative and experimental medicine, is the leading English-language publication in the field. It is ranked by the Science Citation Index in the upper third of all scientific journals. LAS is published six times a year and presents refereed manuscripts on topics such as diseases of laboratory animals, animal models of human diseases, and research methodology. Laboratory Animal Science serves as a forum for the exchange of information on etiopathogenesis, epidemiology, diagnosis, therapy, and prevention of infectious and noninfectious diseases of laboratory animals; naturally occurring and induced models of human disease; research applications of animals; and the biology of laboratory animals. American Association for Laboratory Animal Science 9190 Crestwyn Hill Drive, Memphis, Tennessee 38125 Phone: 901-754-8620 — Fax: 901-753-0046 Internet: http://www.aalas.org/pubs/laonline.htm

Pig International Pig International is an international journal for swine producers. It contains information on health topics, nutrition and breeding, and has many product resources. Pig International is published monthly. Watt Publishing, Co. 122 S. Wesley Av., Mt. Morris, Illinois 1054-1497 Phone: 815-734-4171 - Fax: 815-734-4201

internet resources Many of the associations mentioned previously have links to other Internet resources relevant for laboratory animal science. Special types of resources are electronic databases for searching


literature or scientific data. Only a small fraction of what is available is mentioned below.

Literature resources: PubMed (MedLine) http://www.ncbi.nlm.nih.gov/pubmed Grateful Med (MedLine) http://www.nlm.nih.gov/services/igm.html

Scientific resources: PiGBASE, the genome database of the pig http://www.projects.roslin.ac.uk/pigmap/ecpigmap.html US National Coordination Program for Swine Gene Mapping http://www.genome.iastate.edu/maps/index.html

swine diet and equipment Resources of swine diet and equipment are so diverse that a summary is not possible. The majority of swine used in research originate from agricultural sources, and local directories should be used. If SPF swine are required, the National SPF Swine Accrediting Agency can be contacted for local adresses. Miniature swine are generally supplied by specialized laboratory animal breeding companies. At least two buyer guides specialized in the laboratory animal field are available: Lab Animal Buyers' Guide 1999, Lab Animal, 27 (11), 1998. Published yearly by Lab Animal. Laboratory Animals Buyers' Guide 1999. Published every three years by Laboratory Animals, Ltd.

Miniature Swine Resources Charles River Laboratories (Yucatan, Hanford) 251 Ballardvale Street, Wilmington, MA 01887 Phone: 978-658-6000 - Fax: 800-992-7329 Charles River France (Yucatan) 59 Rue de la Paix, F-76410 Saint Aubin Les Elbeuf, France Phone: (+33) 2-3296-9110 - Fax: (+33) 2-3577-0999 Harlan Sprague Dawley (Sinclair, Yucatan)


PO Box 29176, Indianapolis, IN 46229-0176 Phone: 317-984-7521 - Fax: 317-894-1840 Ellegaard Göttingen Minipigs (Göttingen) Soroe Landevej 302, DK-4261 Dalmose, Denmark Phone: (+45) 58185818 - Fax: (+45) 58185880 Several other breeders in the U.S. produce miniature swine, among others Panepinto and Associates, S&S Farms, and Lone Star Laboratory Swine.

Diet Resources Diet for miniature swine can be obtained from: Bio-Serv 1 Eighth St., Frenchtown, NJ 08825 Phone: 908-996-2155 - Fax: 908-996-4123 Harlan Telkad PO Box 44220, Madison, WI 53744-4220 Phone: 608-277-2070 - Fax: 608-277-2066 Purina Mills PO Box 66812, St. Louis, MO 63166-6812 Phone: 314-768-4592 - Fax: 314-768-4859 Zeigler Bros. PO Box 95, Gardners, PA 17324 Phone: 717-677-6181 - Fax: 717-677-6826 Special Diet Services PO Box 705, Witham, Essex CM8 3AB, United Kingdom Phone: (+44) 1376-511-260 - Fax: (+44) 1376-511-247 Diets can be obtained from several other companies.


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Jones, G.F., Genetic aspects of domestication, common breeds and their origin. In M.F. Rothschild and A. Ruvinsky, eds., The Genetics of the Pig, CAB International, Wallingford, 1998.

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21.

Radin, M.J.. M.G. Weiser, and M.J. Fettman. Hematologic and serum biochemical values for Yucatan miniature swine. Lab. Anim. Sci., 36 (4), 425, 1986.


22.

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24.

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26.

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27.

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29.

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30.

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