THE GUINEA PIG: BIOLOGY, CARE, IDENTIFICATION, NOMENCLATURE, BREEDING, AND GENETICS Ron Banks USAMRIID Seminar Series 17 February 1989 NOTE: A shaded statement indicates that an ACLAM boarded indivigual has identified that material as having a high probability of being on the boards, or having been on past boards! Use this material as a starting point for study. I. INTRODUCTION A. History and Origin 1. Origin of the guinea pig is unclear. a. Wild guinea pig is Cavia aperea b. Widely distributed in Argentina, Uraguay, and Brazil c. Cavia cutleri is still wild in Peru 2. Spanish found Andean Indians had domesticated Cavia cutleri a. Used as food and for religious sacrifices b. Used for food around Spanish Colonial Empire. 3. Paintings such as the last supper have included the guinea pig as main course meal. 4. Guinea pigs are often apartment reared in Peru today. 5. 1500's: Dutch sailors introduced the guinea pig into Europe. 6. 1770's: Probably reached the United States as pets and fancy animals. 7. Origin of "guinea pig" name is vague. Common name used by fanciers is "cavy". a. Does resemble a suckling pig b. Is prepared for eating by scalding/scraping c. Some suggest "guinea" was derived from the fact that trading ships may have travelled via Guinea in West Africa, or via Guiana d. Adult females called sows, and adult males called boars. Parturition referred to as farrowing. B. Taxonomy Kingdom - Animal Phylum - Chordata (with notochord and gills) Subphylum - Craniata (Vertebrata): Chordates with organized head region Class - Mammalia (Warm-blooded craniates with hair coat. Young nourished from mammary glands) Subclass - Theira (Viviparous Mammals = Live Young) Infraclass - Eutheria (Placental mammals, versus Metatherial marsupials, and Protherial egg layers) Order - Rodentia (single row of upper and lower paired incisors which grow continuously; no canine teeth) Suborder - Hystricomorpha ("porcupine like") All except Coypu have (1) a vaginal closure membrane and (2) a masseter muscle insertion that passes through the large infraorbital foramen. Other suborders include Sciuromorpha: "squirrel-like" ... such as squirrels, marmots, gophers, beavers, and Kangaroo rats and the suborder Myomorpha: "rat- like" ... house mouse, norway rat, hamsters, voles, gerbils, etc.) Family - Caviidae (tailless South American rodents with (1) one pair of mammae and (2) four digits on front feet and three digits on hindfeet. Genus - Cavia species - porcellus C. Varieties ("types" or "breeds") - Characterized by length, texture, and direction of growth of hair) 1. ENGLISH: a. Short, smooth, straight hair (3.8 cm) b. Solid colors: albino, white, black, agouti, sandy, red, chocolate, cream, etc. c. Bicolored and tricolored animals also. d. Most inbred and outbred laboratory guinea pigs are the English variety. 2. ABYSSINIAN: a. Short, course hair that radiates from multiple centers on the body to form rosettes. b. Variety of colors. 3. PERUVIAN: a. Has long silky hair up to 15 cm (6 in) long. b. Absence of two hip rosettes are referred to by fanciers as "angora" or "shelties". D. Laboratory Stocks and Strains 1. Listings of available stocks and strains are provided in the ninth edition of the ILAR publication "Animals for Research", and the "NIH Rodents 1980 Catalogue" 2. Outbred English stocks listed in above sources: a. Dunkin-Hartley--("albino") b. Hartley--("albino") c. Pirbright-Hartley d. Shorthair 3. Inbred Strains: a. Strain 2 and 13 are the only two inbred guinea pig strains used to any extent. b. Only remaining strains developed by Wright at the U.S. Bureau of Animal Industry in 1915. 4. Mutant Stocks/Strains: a. Complement 4 Deficient (C4D/N): (1) Developed from spontaneous mutation in NIH multipurpose guinea pig stock in 1970. (2) Partially dominant mode of inheritance; i.e., heterozygotes have intermediate levels of C4 activity. (3) Most immunologic reactions are normal. b. Waltzer (Wz) (1) Wz mutation occurred spontaneously in NIH guinea pig stock in 1953. (2) "Waltzing" or circling and deafness develop due to atrophy of organ of Corti cells. (3) Inherited as an autosomal dominant gene with full penetrance. (4) Homozygous condition results in high perinatal mortality. c. Hairless and Immunodeficient Mutant (LAS 29(6): 744-748, 1979): (1) Spontaneous mutant strain from Hartley stock at Eastman Kodak Company in 1979. (2) At birth, are smaller, have tannish skin with numerous wrinkles, and have stunted vibrissae. (3) Shortened lifespan; many die during first week, one lived for 9 months. Body hairs rare, short, and poorly retained; a few have temporary fuzzy hairs. (4) No grossly visible thymic tissue, cystic spaces found where thymus should have been. Germinal follicles reduced or absent in lymph nodes and intestinal lymphoid tissues. (5) Hypogammaglobulinemic. (6) Deaths due to systemic cytomegalovirus (intranuclear inclusion bodies in cardiac fibers), systemic balantidiasis, and Pneumocystis carinii. d. Hairless Euthymic Guinea Pigs: (1) Now available from Charles River Laboratories. (2) Hair bulb, erector pili, and sebaceous glands do exist. (3) Produces defective hair shaft (4) Reduced number of shafts (5) Normal functional thymus II. BIOLOGY A. Anatomy 1. Skin and external features a. Hair and skin: (1) Consist of coarse large guard hairs surrounded by undercoat of fine hair. (2) Each hair follicle has associated sebaceous gland (3) Sudoriferous glands absent (4) 5-6 rows of tactile hair (vibrissae) on lateral nose (5) Prominent hairless area (1-1.5 cm diam) just caudal to pinna of each ear. (6) Perineal Sac: Bilateral diverticula in circumanal region containing large accumulation of sebaceous glands. White waxy accumulations termed "scrotal plugs" in males. b. Mammary Glands: (1) Both males and females have a single pair of inguinal teats surrounded by a hairless area. (2) A single large papillary duct opens to exterior. 2. Dental Formula: 1 01 3 2(I-C-PM-M-) = 20 teeth. 1 01 3 a. Diastemal space between incisors and premolars. b. A11 teeth continue to grow for lifetime of the animal. c. The molars are hypsodontic = prism shaped teeth with high crowns d. With medial inclinement of the teeth and continuous growth, encasement of the tongue may occur e. Only one set of teeth (monophydont) 3. Bones a. Vertebral formula:C7,T13-14,L6,S2-3,Co4-6 6 sternebrae. 13 or 14 pairs of ribs: 1st-6th sternal 7th-9th contribute to costal arch 10th-14th are floating b. Feet: Forefeet have 4 digits each: (3 phalanges in each digit except 4th which has 2) Rear feet have 3 digits each. c. Vestigial clavicles present. d. Pelvic Girdle: (1) Consist of ilium, ischium, acetabulum, and pelvis bones. (2) Pubic symphysis generally remains fibrocartilage throughout life of animal. (3) The pubic symphysis degenerates 2 weeks prior to parturition resulting in complete destruction by parturition (4) Palpation of separation can be used to estimate parturition. 4. Muscular System a. Well developed masticatory muscles (Masseter and digastricus) b. Reflects the gnawing behavior and corresponding mastication by grinding (versus chewing). 5. Cardiovascular/Respiratory System a. Pharynx: (1) Soft palate is continuous with base of the tongue and lateral walls of the oral cavity (2) Opening into larynx is a small intrapharyngeal ostium (AALAS Abstract #96, 1983) b. Lungs: (1) 3 left lobes (cranial, middle, caudal) and 4 right lobes (cranial, middle, caudal, accessory) (2) Pleural cavities are continuous (LAS 16(5): 411, 1966) c. Arteries: 3 deviations from normal mammalian vascular pattern (Amer. J. Anat. 139, 269- 284. 1974): (1) There may be 2 or 3 pairs of renal arteries (versus normal 1 pair in most mammals). (2) The abdominal aorta gives rise to a celiomesenteric trunk instead of separate celiac and cranial mesenteric arteries. (3) A bronchoesophageal artery passes from the right subclavian, the right internal thoracic, or the brachiocephalic trunk instead of from the aorta. 6. Hemolymphatic System a. Thymus: Present in immature animal (1) Gradually involutes as the animal matures. (2) In adults may be completely gone or persist in caudal cervical or cranial mediastinum. (3) Thymic tissue is replaced primarily with fat as age progresses. (4) In immature animals it is composed of 2 compressed lobulated glands on each side of the ventral cervical midline. (5) Extends from the angle of the mandible approximately halfway to the thoracic inlet (Cooper and Schiller, 1975). (6) Accessory thymic lobes in most guinea pigs; usually paired and adjacent to the parathyroid gland or fused to it (Cooper and Schiller, 1975). (7) Guinea Pig used extensively for immunologic studies because the cervical thymus is easily removed (LAS 25, 82-84, 1975). b. Parathymic Lymph Nodes: (1) Located in cervical region. (2) Receive lymph from the thymus. (3) Studied to determine the immunologic inter-relationship between the thymus and lymph nodes. 7. Gastrointestinal System a. Tongue: (1) Rostral one-third is free. (2) Remainder attached to floor of oral cavity. (3) Small filiform papillae anteriorly, large fungiform papillae posteriorly. b. Stomach: (1) No keratinized non-glandular portion present i.e., all glandular. c. Small Intestine: (1) About 125 cm (50 in) in length (2) Common bile duct enters duodenum 1 cm caudal to pylorus. d. Cecum: (1) Occupies left side of abdominal cavity (2) Large thin-walled sac 15-20 cm long. (3) Accounts for about 15% of body weight. (4) Has 3 taenia coli (dorsal, ventral and medial) (5) Produces out-pouchings called haustra. e. Peyers patches: (1) About nine flat white 1 mm diameter area of lymphocyte aggregates on mucosal surface. f. Colon: (1) Ascending, spiral ascending, transverse, and descending portions. g. Liver: (1) 6 lobes: right and left lateral, right and left medial, caudate and quadrate. (2) Cystic duct from gallbladder joins common hepatic duct to form common bile duct. h. Pancreas: (1) Divided into cranial and caudal lobes. (2) Pancreatic duct enters duodenum 7 cm distal to common bile duct. i. Salivary Glands: 4 pairs present Parotid Mandibular Sublingual Molar (Zygomatic). j. Microscopic Features of GI tract: (1) Pseudohemosiderosis: normal accumulation of hemosiderin in the lamina propria of the villa of small and large intestines. 8. Reproductive System a. Male: (1) Os penis present (2) Inguinal canal remains open throughout lifetime (3) Vesicular glands (seminal vesicles): large, coiled. tubular, long (10 cm) (a) Intra-abdominal glands (b) Contain a creamy thick white fluid that forms concretion (vaginal plug) when mixed with coagulating gland secretions. (4) Other accessory sex glands include: (a) Coagulating gland: i) Duct opens into the calyculus seminalis of the urethra ii) A small white median papilla which protrudes into the urethral lumen. (b) Bulbourethral glands (c) Prostate b. Female: (1) Ovaries: (a) Corpora lutea produced each 16-17 day cycle (b) Grossly visible as small pink structures. (2) Vaginal closure membrane: (a) Usually perforate only at estrus mid-gestation, and parturition. (3) Cervix: (a) Each horn of the uterus opens into the cervix (b) A single os cervix opens to the vagina. 9. Urinary System: a. Kidney: Has a single longitudinal renal papilla with lateral calyces. b. External urethral orifice in female opens independently from the vagina onto the perineum. 10. Adrenal Glands: a. Triangular in shape; bilobed; with lateral lobe larger than medial. b. Larger in males than females c. Strain 2's have significantly larger adrenals than outbred stocks (Am.J. Anatomy 63(2):273- 295, 1938) d. Are the largest adrenals relative to body weight among animal species; the larger size is due to a thicker cortex e. Guinea pig and human secrete cortisol as their main glucocorticoid hormone (vs. corticosterone in most rodent species) B. Physiology (Reference BGP 63-98) 1. Hemo-Lymphatic System a. White Blood Cells (1) Neutrophils 38% (2) Lymphocytes 55% (3) Monocytes 3% (4) Others 4% b. Chemistries (1) Glucose 60 - 100 mg% (2) BUN 8 - 20 mg% (3) Plasma Protein 5.2 - 6.0 (4) Serum Calcium 4.5 - 6.0 c. Erythrocytes: (1) RBC Numbers: 4.5-7.0 x 106/mm3. (2) PCV: 37-48%. (3) Mean Corpuscular Vol. (MCV):70.3-85.0 (4) RBC Life Span: 60-80 days (5) RBC's are fairly fragile. (6) Hemoglobin: 11.0-15.2 gm/100 ml blood. (a) Resistant to oxidation by nitrites to methemoglobin (also rat, mouse, hamster, and gerbil) versus species very susceptible to nitrite oxidation (man, monkey, dog, and rabbit) (b) Has high oxygen affinity because of high levels of 2,3-DPG (as do rat, rabbit, dog, horse, man, and guinea pig) versus those species with low oxygen affinity (cat and ruminants) (7) MCHC = 30.5% (8) Acute hemolytic anemia in response to excess dietary cholesterol (versus cardiovascular lesions in most animals). d. Kurloff Cells: (1) Unique mononuclear leukocytes containing round or ovoid cytoplasmic inclusions called Kurloff bodies: from 1-8 microns in diameter. (2) Inclusion is prob. a mucopolysaccharide substance secreted by the cell itself. (3) Numbers increase markedly during: (a) Pregnancy (b) Exogenous estrogen treatment (c) Increases are rare in fetus and neonates. (4) 0.24% of WBC's in males (5) Number in females varies with estrous cycle. (6) High numbers found in placenta. They may constitute a physiologic barrier separating fetal antigens from immunologically competent maternal cells (7) Originate from spleen and thymus e. Lymphocytes: Lymphomyeloid complex (LMC) studied because: (1) The neonatal guinea pig possesses a very mature lymphocyte, similar to human infants (2) The guinea pig more nearly resembles man immunologically and hormonally than rats and mice (3) Cervical thymus is readily accessible for thymectomy / modification. f. Thymus: (1) At one year, most thymic tissue is involuted with fat deposits. (2) Thymectomy reduces lymphoid organ weights and produces lymphopenia. (3) Guinea pig (also ferret, monkey and man) is considered a corticosteroid-resistant species because steroid treatment does not readily affect lymphocyte count or thymus. (Other species respond with decreased thymus weights and lymphocyte counts). g. Bone Marrow: Guinea pig marrow similar to that of man: (1) Easily dispensed (2) Stains well (3) Actively erythropoietic at birth (unlike rat). 2. Cardiovascular System a. Blood Distribution: (1) Plasma volume: 3.88% of body weight (2) Blood volume: 6.96% of body weight b. Heart Rate: resting (by telemetry) was 275 (229-319) per minute. c. Blood Pressure: systolic rarely greater than 100 mm Hg. d. ECG: similar to human. 3. Reproductive Physiology a. General: (1) Guinea pig a good animal model because: (a) It is easy to handle (b) Has distinct signs of estrus (vaginal membrane opening) (c) Predictable reproductive behavior. (2) Most closely resembles woman of all small laboratory animals because: (a) Has a long cycle (15-17 days) (b) Ovulates spontaneously (c) Has an actively secreting corpus luteum. (3) In the laboratory, guinea pigs are polyestrous, nonseasonal breeders. b. Puberty: (1) Female: First estrus at 30-134 days (mean 67.8+21.5 SD) (2) Male: 56-70 days c. Estrous Cycle: (1) Length: 13-20 days average is 16 days. (2) Proestrus: (lasts 1-1.5 days) (a) Signs include increased activity and vigorous pursuit of cagemates (b) Vigorous mounting in the ten hours prior to estrus. (3) Vaginal membrane: (a) Opening precedes estrus (b) Open for about 2 days during cycle. (c) Open for average 11 days during 1st cycle; 5 days on future cycles (d) Closure occurs after ovulation. (4) Estrus: (lasts 9-11 hours): (a) Exhibit copulatory reflex (lordosis or opisthotonos) (b) Receptivity occurs during darkness. 5 p.m. to 5 a.m. (5) Ovulation: Occurs 10 hours after onset of estrus and is spontaneous. (6) Postpartum estrus: (a) Occurs with a 12-15 hours postpartum (b) Lasts approx. 3.5 hours. (7) Vaginal Smears: (a) A better indicator of estrus than vaginal opening. (b) Onset of estrus indicated by rounded cornified cells. (c) Influx of leukocytes indicates ovulation. d. Hormonal Control of Ovarian Activity: (1) Corpus luteum (CL): (a) Secretes progesterone, which increases rapidly after ovulation. (b) Luteal cells hypertrophy between days 3 and 9 (2) FSH (follicle-stimulating hormone): (a) Increases about day 13 (b) This promotes estrogen synthesis by the developing follicle. (3) An LH (luteinizing hormone) surge effects ovulation. e. Fertilization and Implantation: (1) Fertilization occurs in fallopian tubes: (a) Must occur within 20 hours of ovulation. (b) Usually only 3.4 ova (range 1-5) ovulated. (2) Implantation: (a) 8-12 cell stage enters uterus on day 3 (b) Implants on day 6-7. (3) Postestrus insemination (a) Within 0-16 hours (b) Intraperitoneal AI of semen reported to be as successful as natural mating during estrus. f. Gestation: (59-72 days; mean = 63 days) (1) Length is inversely related to number of fetuses carried. (2) Females may double weight due to fetal mass (3) Hormonal Control of Pregnancy Involves: (a) CL of pregnancy continues to grow until day 18-20 (b) CL remains functional throughout the pregnancy. (c) Placenta begins endocrine (Progesterone) activity after day 15 (d) Pregnancy can be completed subsequent to bilateral ovariectomy after day 21. (e) Progesterone: plasma levels increase rapidly after 15 day post- coitus; peak between days 30-45. (f) Estrogens: Appear at day 20; peak at day 56-60; undetectable following parturition. g. Placentation: (1) Discoidal, Labyrinthine Hemomonochorial (a) Has a single layer fetal capillary membranes that are in direct contact with maternal bloodstream (b) Very similar to humans. ARM Hemochorial: also found in rat, mouse, hamster, rabbit, & armadillo). h. Parturition: (1) Symphysis pubis begins to relax in response to relaxin (2) Returns to normal within 24 hours postpartum. (3) Parturition lasts 10-30 minutes with an average interval between deliveries of 7.4 min (range = 1-16 minutes). (4) Average age of sow at first litter is 175 days (range 93-420). (5) Litter Size: Ranges from 1-8; Ave. is 3. (6) Neonate Viability, Size Growth: (a) Optimal survival if litter size is 2-4. (b) Stillbirth incidence increases with size of litter. (c) Lowest mortality with 69 day gestation. (d) Asphyxia from fetal membranes a frequent cause of death. (e) Fetalphagia not observed in the guinea pig. (f) Stillbirth incidence reported as high as 45% in some colonies; especially Strain 13's. (g) Weight at birth inversely related to litter size; average is 80 g, 60-130 g range. i. Lactation: (1) Estrous cycle continues normally after parturition regardless of whether sow is or is not lactating. (2) Most offspring will survive if no nursing, but weak, runty pigs may result. (3) Peak lactation period is during days 5- 8; agalactia by day 18-23, or 24 hours after pups removed. (Weaning usually at 14 - 21 days). (4) Rate of pup growth directly related to milk yield of sow. j. Male Reproduction: (1) Puberty (presence of sperm in semen) at around 10 weeks: sexual maturity (adult concentrations of sperm) usually not until 14-19 weeks. (2) Ejaculation occurs in the first or second intromission; followed by a refractory period of one hour before copulation can reoccur. (3) Electroejaculation has been performed with lumbar and rectal electrodes. (4) Liquification of coagulated semen can be done with 0.1% chymotrypsin phosphate. (5) Vaginal plug is the portion of ejaculate secreted by seminal vesicles which coagulates instantaneously on emission (characteristic of ORDER Rodentia). Plug falls out of the vagina a few hours after its formation. (6) Cobayin is a toxin found in the seminal fluid of guinea pigs. Produces death in 0.5-2.0 hours when given IP to rabbits. C. Behavior (Ref. BGP pp. 31-52) 1. Behavior in the Wild a. C. porcellus, C. aperea, C. cutleri: (1) Do not dig burrows, but use burrows excavated by other animals, crevices in rocks, or shrubbery. (2) The alpha male is intolerant of sexual activity by lower ranking males. (3) Mutual grooming not excessive. b. Diet: (1) Green grass and vegetables (2) Are especially fond of alfalfa (3) Do not store food c. Activity: (1) Most active in morning and evening (2) Little evidence of parental defense of young (3) Response to danger is either immobility, or sudden explosive scattering of animals. 2. Perception and Individual Behavior Patterns a. Perception (1) Vision: Eyes open at birth, but poorly developed depth of vision and object discrimination (2) Audition: Maximum sensitivity between 500 and 8000 Hz. Range: 125-40 or 50,000. (3) Gustation: (a) Avoid sweet foods, especially if more than 0.5M glucose (b) Reject overly bitter, salty, or chemically pure diets (4) Other: Olfaction (pheromones esp.) probably important in behavior. b. Activity Patterns: (1) Under constant illumination, almost continual activity mostly feeding and self-grooming. (2) Less activity with excessive heat (75- 85oF). c. Responses to Dangers: (1) "Immobility response": (a) Tend to freeze in response to un- familiar sudden sounds (b) A self-protection response; last a few seconds, or up to 20 minutes (c) Electrical shock may also evoke this immobility response; thus not good subjects for pain or shock avoidance studies. (2) "Scatter Response": (a) Usually elicited by sudden movements (b) Involves stampeding in all directions, jumping from a cage, trampling young, or rapid circling. (3) Preyer reflex: Cocking of the pinna of the ears in response to a short signal from a Galton whistle (Clapped hands will work) d. Feeding and Elimination Behavior: (1) Feeding: (a) Generally occurs during daylight hours especially at dawn and dusk (b) Laboratory-reared animals feed both at night and during the day. (c) Newborn animals can eat solid food within the first day after birth. (2) Drinking: (a) With sipper tubes guinea pigs don't lick, draw out water by grasping tube and pulling head back. (b) With ball bearing antidrip devices in tubes, gnawing occurs. (c) With a water dish the head is tilted back after each sip. (d) Newborns can learn to drink from sipper tube within 48 hours of birth. (3) Elimination: (a) Newborn guinea pigs usually incapable of voluntary micturition for several days postpartum (up to 1 week) (b) Maternal licking stimulates both defecation and urination. (c) Coprophagy is commonly observed in adults and weanling age young. Usually eat fecal pellets directly from the anus. e. Social Behavior: (1) Tolerate close physical contact with each other at rest (2) Seem to seek contact when moving about (3) Lactation: mothers assume a sitting position to allow young to crowd under the mother. f. Grooming: (1) Little mutual grooming (2) Self-grooming observed as early as birth; usually after feeding. (3) Hairpulling is common in pups pulling hair from mother during or shortly after end of lactation. (4) Hair pulling and ear-chewing can be exaggerated under crowding and stress. g. Communication (1) Olfactory: (a) Scent marking with anal glands is common (b) Males mark with supracaudal gland by rubbing rump. (2) Auditory: (a) "chutts" - brief vocalizations (.05 sec.); single or paired (b) "chutter" - 2 to 4 components (c) "whine" - during flight, discomfort or evasion (d) Also have whistle, tweet, purr, drr, scream, squeal. h. Dominance Relations: (1) Conflict arises over space, access to food, drink, objects to gnaw on. (2) Offensive responses: (a) Range from vocal response to chasing to deep bite wounds (b) Male adults: when caged with females may result in death of the less dominant males 3. Reproductive Behavior: a. Mating Behavior: (1) Male Behavior: Anogenital investigation by sniffing or licking, mounting with a forepaw clasp, pelvic thrust, intro- mission and ejaculation. (2) Female Behavior: (a) Female displays lordosis, wide stance of hind feet, and perineal dilation (opening of vaginal closure membrane). (b) Estrous cycle: i) Behavioral estrus is 8 hours ii) Best indicator of receptivity is lordosis posture in response to a male or gentle stroking toward head over rump iii) Occurs nocturnally b. Female Parental Behavior: (1) Parturition (a) First pup born after 5 minutes of labor (b) Born head first; the mother then licks and nibbles until fetal membranes are consumed and pup is cleaned. (c) Interval between pups usually less than 3-4 minutes (d) Placenta usually expelled after pups are born; most are eaten by mother or others in pen. (2) Maternal Solicitude: (a) Once the dam enters postpartum estrus, shows little interest in offspring till mating is completed. (b) Maternal care is limited i) Passively allows nursing ii) Neonatal grooming limited to anogenital stimulation. III. CARE AND MANAGEMENT OF GUINEA PIGS A. Environment 1. Temperature a. Optimal temperature range is 65-75oF (17- 24oC). Slightly higher for the hairless GP (75 - 79oF) b. Above 90oF may see: (1) Heat prostration and deaths especially in advanced pregnancy (2) Abortions (3) Sterile matings from males c. Poor growth in young if below 55oF (13oC) d. Guinea pigs tolerate cold better than hot temperatures e. Type of caging affects optimal room temperature; i.e., higher temperature needed for wire-bottom cages. f. Control of temperature fluctuations more important that actual temperature as guinea pigs are more sensitive to variations than other laboratory species. 2. Relative Humidity: a. 40-60% (50% optimal) b. Survival of airborne organisms is lowest at this level. 3. Ventilation a. 10-15 air changes per hour. b. Air velocity and direction of flow should be designed to produce a draftless and even distribution of air. c. Auxiliary power should be available for the environmental control system. 4. Space requirements for guinea pigs a. Animal Welfare Act (1) Minimum height: 6.5 inches (2) Minimum floor space: size space (square inches) under 350 gm 60 over 350 gm 90 breeders 180 b. NIH ("Guide for the Care and Use of Laboratory Animals, 1985) (1) Minimum height : 7 in (17.8cm) (2) Minimum floor space: size space (square inches) under 350 gm 60 over 350 gm 101 5. Caging a. Usually constructed of stainless steel, fiberglass, polycarbonate, or plastic. b. Usually in batteries on portable racks (some breeders us fixed tiered compartments, but regular disinfection difficult) c. Solid bottom, bin type cages are most common; especially for breeding animals. d. Wire floors should be welded, rather than interwoven mesh; recommended sizes vary from 1/2" to 1 1/2" square; (smaller size should be avoided to preclude broken legs). e. Tumblebrook cage is commonly used for breeding. It is a portable 5 cage rack: each solid bottom cage is 30 x 30 x 10 inches. 6. Bedding a. Peat moss, dried corn cobs, cedar shavings, pine shavings, straw, hardwood chips b. Pine shavings are popular c. Any bedding should be free of dust d. Sawdust has been reported to cause infertility in guinea pigs due to adherence to genital mucosa (vulva or prepuce) and interference with copulation. e. Cereal straw and wood chips may produce upper G.I. (mouth, esophagus) injuries with resultant infection and mortality. 7. Cage Sanitation a. High concentrations of mineral salts in guinea pig urine result in accumulation of mineral scale b. Weekly rotation of acid and alkaline detergents has been effective in removing this scale c. Should be sanitized, i.e., water heated to 180oF, live steam, or effective chemical disinfectant B. Feeding 1. Vitamin C a. Guinea pigs and primates are the only commonly used laboratory animals which require an exogenous dietary source of Vitamin C (ascorbic acid). Vitamin C is also required by fruit-eating bats and some birds. b. Are unable to convert glucose to ascorbic acid because they lack the microsomal enzyme L-gulonolactone oxidase. c. Ascorbic acid required to: (1) Prevent scurvy (2) Maintain natural disease resistance (3) To maintain blood complement (4) Essential coenzyme in the hydroxylase reactions forming hydroxyproline and hydroxylysine, which are components of collagen. d. Deficiency results in: (1) Formation of defective collagen (2) Enlarged costochondral junctions (3) Disturbed epiphyseal growth centers (4) Hemorrhages in various tissues (5) Delayed wound healing (6) Impaired bone and tooth formation. e. Daily Ascorbic Acid requirements: (1) Immature guinea pigs: 0.5 mg/100 g body weight (2) Maintenance (over 300 g BW): 6 mg (3) Breeding females: 20 mg f. Sources of Vitamin C: (1) Commercial Guinea Pig feed: (a) Feed within 4-6 weeks of manufacture (although under "proper" storage conditions, adequate ascorbic acid levels should be present for 90 days). Commercial feeds usually contain 1000 mg/kg. (b) Should store at 50oF or less for best preservation of ascorbic acid. (c) Ascorbic acid deteriorates rapidly; accentuated by heat. (2) Supplementation with ascorbic acid in drinking water: (a) Should be added only to distilled or deionized water since chlorinated tap water will inactivate ascorbic acid. (b) Add at the rate of 200-400 mg/L (c) Undergoes rapid oxidation and requires frequent preparation (or use of automatic dispenser) (d) Copper and other metals should not be used in the watering system as they catalyze ascorbic acid. Stainless steel is satisfactory. (3) Green Vegetable Supplementation: (a) Kale: i) 115 mg ascorbic acid per 100g ii) Should be washed in a hyperchlorinated solution to minimize bacterial contamination. (b) Carrots, cabbage, sprouted grains, chicory, marigolds have also been used. (c) Lettuce is a poor source of Vit C. (d) 10-44% average increase in weaned offspring with the use of green vegetable supplements has been reported. 2. Feeders: a. Standard "J" type feeders or wall-mounted feeders with a baffle work well in keeping guinea pigs from climbing into the feeders. b. Bowls are not desirable because guinea pigs tend to sit, sleep and defecate in the bowls. 3. Feed Consumption: a. Growing guinea pigs eat about 8% of their body weight in feed daily. Adults eat about 6% body weight daily b. Pelleted diets preferred (label and date secondary feed containers to avoid confusing with rabbit chow) c. Alteration in composition of a feed or introduction of a new feed may result in a sharp decline in feed consumption to the point of starvation or signs of vitamin deficiency occur. 4. Protein: a. Have an usually high requirement for certain amino acids (arginine, methionine, and tryptophan) b. Requirements can be met by providing 18-20% protein diet usually of plant origin. 5. Roughage: a. Crude fiber should be 13.5% (ILAR, 1967) b. Many breeders advocate feeding hay; may reduce boredom and decrease "barbering" c. Has been reported to decrease hair thinning often associated with late pregnancy. 6. Experimental Diets: a. Guinea pigs discriminate between feeds within a few days of birth. Dietary preparations for nutritional studies should therefore be introduced within a few days of birth. b. Acceptance can be enhanced by pelleting powdered diets in gelatin, moistening feed, or transitory mixing. C. Water 1. Requirements (Adults): a. With "greens" supplement - 50-100 ml per day b. Without "greens" supplement - 250-1000 ml per day (excess amount required because of spillage). 2. Spillage: a. Occurs because guinea pigs play with sipper tubes and rapidly empty water bottles b. Can be controlled by: (1) Fitting drinking bottles with ball- bearing equipped sipper tubes. (2) Mounting automatic watering systems slightly (3/4") outside the cage (must adjust valve pressure to prevent water from squirting into the cage). 3. Water bowls not recommended because guinea pigs rapidly foul the water with excreta. 4. Guinea pigs will blow food and other materials from their mouth up through the sipper tubes. 5. Sipper tubes should be made from stainless steel or other hard material since guinea pigs chew rather than lick the end of the tube D. Breeding 1. Males: a. Fertile matings before 8-10 weeks of age b. Usually mated at 3-5 months (450-600 gm) c. Many breeders mate at weaning. Efficient reproduction until 27-30 months of age. 2. Females: a. First estrus as early as 4-5 weeks of age b. Usually mated between 2.5-3 months of age, or when reach 450-600 gm. c. Should be bred by 6 months of age to lessen likelihood of the animal: (1) Becoming excessively fat (2) Having firm fusion of the symphysis pubis, both of which may result in contributing to dystocias or pregnancy toxemia. d. Efficient reproduction until 27-30 months of age. e. Females should have produced five or more litters. E. Breeding systems: 1. Monogamous pairs: a. One male and one female are mated in a single cage. b. Most commonly used to maintain pedigreed nucleus or foundation colonies of inbred strains which require brother x sister matings. \A Intensive: (1) Male remains with female throughout parturition and breeds during the postpartum estrus which occurs 3-4 hours after parturition (2) Results in an average of 5 litters per year per sow. d. Non-intensive: (1) Female separated from the male just prior to parturition. (2) Female bred during the first regular estrus following postpartum estrus. (3) Results in 3.5 litters per year per sow. 2. Polygamous Mating (Harem Breeding) a. Used for outbred production b. One boar mated with 4-6 sows. Can be maintained as: (1) Intensive groups: (a) The group is maintained as a stable colony and held intact throughout their useful breeding life (b) Maximizes advantage of postpartum breeding and communal rearing of young (2) Non-intensive groups: (a) Sows in advanced pregnancy are isolated either individually or in groups for farrowing (b) After weaning the sows are returned to the mating pens. 3. Reproductive Index: The number of weanlings per female per month. a. Outbreds: (1) Average litter size 3-4 (2) Reproductive index is 1.0 for most commercial breeders b. Inbreds: (1) Average litter size 2-3 (2) Reproductive index is 0.7 weanlings per month per female. (3) Lower index is attributed to a decline in vigor and fertility displayed by a reduced frequency of farrowing and smaller litters. 4. Breeding Colony Mortality: a. Abortions in one report of 8.3% b. Stillbirth incidence increases with larger litters. Stillbirths are frequent in primiparous animals. c. Other causes include embryonic resorptions, uterine hemorrhage, pregnancy toxemia, dystocias, and exhaustion from prolonged labor are common causes of death in breeding colonies. 5. Birth Weights and Growth: a. Depend on nutrition, genetics, a litter size, gestation length, and litter interval. b. Average weight in litter of 3-4 is 85-95 gm c. Young weighing less than 50 g usually die d. Should weigh 165-240 g at 21 days e. Weaning usually between 15-21 days, or 180 g weight f. Gain 2.5-3.5 g per day during first 2 months g. Weight gains slow but continue to 12-15 months of age (1) Adult sows 700-850 gms (2) Adult boars 950-1200 gms F. Handling 1. The guinea pig is easily excited 2. Vocalize frequently when excited 3. Should be approached and handled in a gentle quiet manner. 4. Are docile, easily handled and seldom bite. 5. Guinea pigs are very susceptible to stressful situations. a. Transportation and handling may result in significant weight losses (up to 50g) within 24-48 hours. b. This weight is regained rapidly if conditions are stabilized but may alter experimental data. 6. Abortions are not uncommon in late pregnancy following movement or frightening events. 7. Restraint: a. Should be grasped firmly and quickly around the thorax from above with one hand. b. The thumb should be behind a front leg and fingers forward of the opposite limb. c. As the animal is lifted, the free hand should be placed under the hindquarters to support and control the caudal portion of the body; especially late pregnancy females. G. Sexing 1. In young guinea pigs, females exhibit a "Y"-shaped fold in the genital area. The female also has an external urethral orifice separate from and ventral to the vagina. 2. Young males have a slit in the anogenital area. The penis is easily extruded from the prepuce with gently pressure and is located just anterior to the "slit". EM Note: the anogenital distance is the same for females and males. H. Identification: 1. Multicolored animals may be identified by their natural markings. 2. Ear tags: Not too satisfactory because they are easily torn from the ear or fall out due to ear- chewing by cagemates. Used successfully by some breeders. 3. Ear notching: Notches may heal closed or the ears become shredded from fighting, obliterating the notches. 4. Ear tatoos: Use a cutting edge suture needle or special pliers. Use black ink on white and green ink on pigmented ears. 5. Fur Dyes: a. Used temporarily for up to 6 weeks b. Clean fur with 70% alcohol to remove fur grease and allow to dry c. Use any of the following solutions, made up in 70% alcohol: (1) Saturated picric acid (yellow) (2) 3-5% acidic, basic, or carbol-fuchsin (red) (3) 3-5% methyl or gentian violet (purple) (4) 3-5% brilliant, ethyl, or malachite green (5) 3-5% trypan blue I. Drug Administration 1. Intramuscular injections: a. Give in the posterior lateral thigh 0.5 to 1 cm above the stifle. b. In large guinea pigs, the lumbar musculature, although caution should be exercised. 2. Intraperitoneal injections: a. Inject slightly to the right of the midline about one inch anterior to the pubis at a low (30o) angle. b. Elevate rear of animal to shift viscera anteriorly. 3. Subcutaneous injections: a. Easily made in dorsal location (nape of neck). b. The flank mat also be used, but caution should be exercised. 4. Intravenous injections: a. Use 23-27 gauge needle. b. Facilitated by sedation (e.g., Ketamine HC1) various routes used, including: (1) Caudal auricular vein (LAS, Feb 1981, pp 85-86) (2) Cephalic vein (3) Femoral vein (4) Lateral metatarsal vein - largest superficial vein (5) Dorsolateral penile veins (6) Lingual vein 5. Chronically implanted catheters: a. carotid artery b. jugular vein 6. Oral Route: a. One report suggest this is easily accomplished by using: (1) Bulbed needles (2) 5 french infant feeding tubes b. This may damage the mouth (due to small intrapharyngeal ostium), so use a small syringe and feed the medication per os J. Blood Sampling 1. Anterior vena cava (Lab Animal, 11(6): 66-68, 1982) a. Lightly anesthetize guinea pig b. Use 23 gauge, 1 inch needle inserted at notch between manubrium and first left rib c. Yields several cc's blood rapidly 2. Femoral Triangle (artery and vein) (LAS, 25(2), 1975, pp 216-217) a. Use 23 ga, 3/4 inch needle b. Yield 3 cc or more blood 3. Cardiac Puncture: a. Requires light anesthesia b. Insert needle at the intercostal space and apical beat, or lay animal on back and insert at 30o angle in the xiphoid area. c. Should be used as a terminal procedure only. 4. Vacuum assisted bleeding (LAS, 25(1), 1975, pp 106-107) a. Nick the superficial surface of the lateral metatarsal vein distal to malleolus. Coat the leg with silicone grease petroleum jelly. b. Use - 0.5mm Hg vacuum over leg. c. Yields 3 cc blood per minute. 5. Orbital Sinus puncture a. Microhematocrit capillary tubes introduced into medical canthus of the eye. b. Should be lightly anesthetized. 6. Toenail clipping - small volumes 7. Ear Veins: (LAS, 33(1), 1983, pp. 70-71) a. Punctured with a mechanically-activated lancet: blood collected in capillary tubes b. Allows frequent, multiple small volume samples to be collected K. Anesthesia of Guinea Pigs 1. General considerations a. Fasting for 12 hours prior to anesthesia recommended to: (1) Prevent regurgitation of stomach contents (2) So that the weight measured just prior to anesthesia is more apt to represent a true value. b. Stage 3 surgical anesthesia plane characterized by: (1) Flaccid skeletal muscles (2) Disappearance of reflex responses (3) Shallow, regular respiration. c. During stage 3, may exhibit peculiar squirming movements in one leg or entire body which cease spontaneously (additional anesthetic at this point may be fatal). d. Atropine sulfate may be used as a preanesthetic to decrease salivation. 2. Inhalation Anesthetics: a. Methoxyflurane (Metofane) (1) Excellent anesthetic for guinea pigs (2) 1% for induction, 0.3% for maintenance (3) Induction takes 15-18 minutes; recovery 10 or more minutes. b. Halothane, Isoflurane (1) Considered "high risk" because guinea pigs first hold their breath and then breathe deeply (2) Halothane: reported to be hepatotoxic in guinea pigs following repeated dosages. Causes a 35-40% decrease in blood pressure. c. Administration of Inhalation Agents: (1) Anesthesia chamber (2) Nose cone (3) Bell jar (4) Endotracheal intubation: EMA Use a canine otoscope to visualize larynx (b) A 5 french urinary catheter can be used as a tracheal tube (JAVMA 179(11), 1981, p 1295). (5) Tidal volume of 3.8 ml/kg body weight. 3. Injectable Anesthetics: a. Sodium Pentobarbital: (1) Dose: 28-35 mg/kg I.P. gives 30-100 minutes of surgical anesthesia. (2) Should be diluted 50% in normal saline. b. Thiopental (Pentothal): (1) Dose: 20-55 mg/kg IP; 20 mg/kg IV (2) Dilute to 1% concentration c. Thiamylal Sodium (Surital): 20 mg/kg IP d. Ketamine Hydrochloride: (1) Dose: 22-44 mg/kg IM gives excellent sedation for venipuncture, tattooing, etc. (2) Combine with 5-10 mg/kg xylazine IM to produce light anesthesia (3) Surgical anesthesia with 44 mg/kg Ketamine plus 5 mg/kg xylazine IM (Abstract No. 88, LAS 32(4), 1982, p. 434) (4) Self-mutilation has been associated with IM Ketamine in guinea pigs, (LAS, 34(5), 1984, p. 516); (IP injection should be considered) e. Fentanyl-Droperidol (Innovar-Vet): (1) Dose: 0.22 ml to 0.88 ml/kg IM (2) High dose may produce nerve, vessel, and muscle necrosis and self-mutilation of digits at 7-10 days post-injection. (3) Causes vasodilation which may aid in venipuncture at peripheral vessels. f. Telazol: (1) Dose: 10 - 140 mg/lb plus 8 mg\kg IM mixed (2) Provides good anesthesia for most procedures (3) Gives about an hour of working time L. Antibiotic Therapy 1. Many antibiotics apparently cause an alteration in the intestinal flora which leads to diarrhea or death within 3-5 days. Clostridium difficile toxin has been suggested as the cause (LAS 31(2): 156-160, 1981)(Med Micro & Imm 169:187-196, 1981) P5 "Toxic" antibiotics include: a. Penicillin b. Bacitracin c. Erythromycin d. Clindamycin e. Lincomycin f. Chlortetracycline g. Oxytetracycline h. Streptomycin 3. "Safe" antibiotics include: (from Williams, C.S.F., 1976): a. Chloramphenicol palmitate: 60 mg orally daily for 5-7 days b. Chloramphenicol succinate: 10-30 mg/kg BID IM c. Sulfamethazine: 4 ml of 12.5% solution/500 ml of drinking water for 1-2 weeks d. Cephaloridine: 10 mg/animal/day M. Euthanasia 1. Carbon dioxide in a closed container 2. Barbiturate overdose IP 3. T-61 Euthanasia Solution IV. GUINEA PIG GENETICS A. Background: 1. 1900-1960's genetic research was concerned with: a. The genetic analysis of coat colors b. The texture c. Studies on the effects of inbreeding. 2. Genetic research today is directed at the analysis of biochemical, physiological and immunologic characteristics. 3. Many mutants that have been described are no longer obtainable. 4. Diploid chromosome number is 2N=64, with one pair of sex chromosomes and 31 pairs of autosomes. 5. Sex determination is xx (female) and xy (male). B. Pigmentation: 1. 15 loci are known to influence coat color in guinea pigs. Most are known to have a single gene mode of inheritance. 2. Individual loci: a. Agouti locus (A): (1) This locus controls amount and distribution of: :\W Eumelanin (black or brown pigment) (b) Phaeomelanin (yellow and reddish pigment) both in individual hairs and in the coat. Two alleles recognized. (2) Light-bellied agouti allele (A): Agouti with light belly. This is the wild type for Cavia porcellus with terminal or subterminal yellow band (phaeomelanin) and rest of the hair black or brown (eumelanin). (3) Non-agouti allele (a): Eliminates the phaeomelanin band from individual hairs and animals are therefore black (or brown, or variation). b. Brown locus (B): (1) Wild type allele (B): Black color (eumelanin) (2) Brown allele (b): changes eumelanin in hair, skin, and eyes brown black to brown. When combined with agouti wild type, a cinnamon-agouti color results. c. Color, or albino locus (C): (1) A series of mutants at this locus reduces the amount of pigment. There is no true albino allele homologous to that in mice(c). (2) Wild type allele (C): full pigmentation. Examples include blacks, reds, golden agoutis. All of these have black eyes. (3) Acromelanic albino (ca): Black (eumelanin reduced to patches on extremities (nose, feet, ears). Yellow (Phaeomelanin) eliminated completely. Eyes are pink due to lack of pigment in the iris and retina. (4) The ca allele interacts with the extension allele (e) at the E locus to give a synthetic albino (ca/ca e/e) color in the common laboratory Dunkin- Hartley"albino" guinea pig. C. Pelage Variations: 1. Rough (R) locus: the dominant gene R is necessary for any "roughness" rosette patterns. a. "Roughness" or rosettes: this hair pattern is characteristic of the Abyssinian breed and is regulated by four loci. b. The action of this gene is modified by the incompletely dominant genes M and Re. 2. Long Hair (l): a. Autosomal recessive character b. Hair length up to 8, 12. or 16 cm at 3, 5, and 7 months c. Peruvian breed: has long hair (1/1) and rough (R) alleles, resulting in 2 hip rosettes which throw hair forward over head. d. Smooth coated long haired type referred to as "angoras" or "shelties" V. "GERM FREE AND SPECIFIC PATHOGEN FREE" GUINEA PIGS (BGP, pp. 21-30) A. Background 1. Germ-free Guinea Pigs a. Not commercially available b. Impractical to maintain breeding colonies because enlarged cecum contributes to poor breeding performance. c. Usually obtained by hysterotomy / hysterectomy of conventional animals. d. Germ-free neonates are marked by precocious behavior requiring little or no handfeeding (sterile milk unnecessary), easily reared to maturity. e. First germ-free pigs derived by cesarean section in 1895 (Nuttall and Thierfelder). 2. Gnotobiotic Guinea Pigs a. Preferred term for disease-free guinea pigs. b. Obtained by germ-free derivation and then fed a defined bacterial flora. 3. Germ-free Husbandry-- Often individually housed to: a. Avoid tendency to nurse at the anal end of cagemates b. To avoid hair pulling and chewing B. Germ-Free vs. Conventional 51 Cecal distension a. Conventional animals' cecum is 15% of body weight, 30% in germ-free. b. Same weights as conventional when corrected for increased cecal size. c. Attributed to lack of resistance to stretching in the cecal wall. d. Predisposes to cecal rupture, herniation, torsion, volvulus, and uterine prolapse. e. Anal and rectal prolapse has increased incidence of about 5%. 2. Kurloff bodies not observed in germ-free guinea pigs--reason unknown (observed however with induced protozoal infections). 3. Hypoplastic GI lymph nodes and lymphatics 4. WBC: a. Total WBC: 1900 in germ-free vs. 5 -12,000 in conventional b. Neutrophils: 55% compared to 10-20% in conventional c. Lymphocytes: 42% vs. 80% in conventional 5. Immunoglobulins: a. Gamma globulins same until 8-10 weeks of age, then decrease in germfree to 20-50% of normal. b. Hemolytic complement activity same. 6. Pregnancy a. Frequent in germ-free, but rarely reach term b. Sows abort; prolapse uterus, often death c. Cecal distention postulated as cause of reproductive problems 7. Germ-free are slower to develop signs of scurvy. 8. Miscellaneous Abnormalities a. Enlarged gall bladder b. Decreased muscle tone c. Thin gut wall d. Reduced lymphoid tissue VI. SELECTED BIBLIOGRAPHY: GUINEA PIGS 1. Altman, P.L. and Katz, D.D (eds.), "Inbred and Genetically Defined Strains of Laboratory Animals, Part 2: Hamster, Guinea Pig, Rabbit, and Chicken", Federation of American Societies for Experimental Biology, Bethesda, MD, 1979. 2. Clark, J.D. and Baker, H.J., The Guinea Pig: I. Introduction and Husbandry (V-6085); II. Biology (V-6086), and III. Diseases (V-6087) (2x2 slide/ cassette tape programs), ACLAM and Washington State University. 3. Cooper, G. and Schiller, A.L., "Anatomy of the Guinea Pig", Harvard University Press, Cambridge, 1975. 4. Festing, M.F.W., The Guinea Pig In "The UFAW Handbook on the Care and Management of Laboratory Animals", 5th ed., edited by UFAW, Churchill Livingstone, NY, 229-247 (1976) 5. Holmes, D.D., "Clinical Laboratory Animal Medicine": Iowa State University Press, Ames, IA, 1984. 6. Manning, P.J., Wagner, J.E., and Harkness, J.E., Biology and Diseases of Guinea Pigs, In "Laboratory Animal Medicine", Fox, J.G., Cohen, B.J., and Loew, F.M., eds, pp. 149-181 Academic Press, 1984. 7. National Academy of Sciences, National Research Council, Institute of Laboratory Animal Resources, "Guide for the Care and Use of Laboratory Animals", National Academy of Sciences, Washington, D.C., 1985. 8. Obeck, D.K., The Guinea Pig, In: Aeromedical Review 4- 74, Selected Topics in Laboratory Animal Medicine, Volume 22, USAF School of Aerospace Medicine, Brooks Air Force Base, TX, 1974. 9. Wagner, J.E. and Harkness, J.E., "The Biology and Medicine of Rabbits and Rodents", 2nd ed. Lea and Febiger, Philadelphia, 1983. 10. Wagner, J.E. and Manning, P.J. (eds), "The Biology of the Guinea Pig", Academic Press, NY (1976). 11. Williams, C.S.F., "Practical Guide to Laboratory Animals", C.V. Mosby Co., Saint Louis, MO, 1976. 12. Stokes, W. S., USARIID Seminar Series, 1986. 13. Rothman, S. W., "Presence of Clostridial difficile toxin in guinea pigs with penicillian-associated colitis. Med J Immun, 169(1981)187-196 14. Charles Rivers Update, "Euthymic Hairless Guinea Pigs for Dermatological Studies". Vol 1, Number 2, Spring 1986 15. Mallort V. T., "Comparative Delayed Contact Hypersensitivity in Haired versus Hairless Guinea Pigs". Pharmakon Research International, Waverly, Pa. July 1987 16. Flecknbell P. A., "Laboratory Animal Anesthesia". Harcourt Brace Jovanovich, New York, NY. 1987