VITAMIN A REQUIREMENTS OF YOUNG RED-EARED SLIDER TURTLES (PSEUDEMYS SCRIPTA ELESANS) Thesis fbrth'e Degree am. 3, 7 Mlcnm STATE umvmm I MARILYN PATRisCIA ANDERSM . I) .1972. ‘ ‘ mu 1mmammmmmnn‘mmmm11nl 3 1293 91074 9210 Y C PM r r A Tue vi t taming re ABSTRACT VITAMIN A REQUIREMENTS OF YOUNG RED—EARED SLIDER TURTLES (PSEUDEMYS SCRIPTA ELEGANS) By Marilyn Patricia Anderson The vitamin A requirement for growth and maintenance of health of hatchling red-eared slider turtles, Paeudémys scripta elegans, was studied. Two lots of 40 turtles each were divided into groups of 10 turtles each. One group in each lot was killed at the beginning of the experiment to serve as controls for normal histology and initial liver vitamin A levels. The remaining 3 groups in each lot were maintained in separate tanks of distilled water and were provided with a wood float and ap25 watt reflectorized lamp. The turtles were fed experimental, lyOphilized diets based on the nutrient requirements of the chick. One group (I, Lot 1; IV, Lot 2) in each lot was fed the basal diet composed of hog heart supplemented with glucose, corn oil, minerals and vitamins exclusive of vitamin A. Two groups (II and III) in Lot 1 were fed the basal diet supplemented with 140 and 280 IU of retinyl palmitate per kg of fresh hog heart, respectively. Two groups (V and VI) in Lot 2 were fed the basal diet supplemented with 900 and 1800 IU of all-trans retinal per kg of fresh hog heart, respectively. All groups fed the diets developed similar clinical signs and lesions characterized by keratinizing squamous metaplasia of nasal, oral, tympanic, ocular and 5:315 tract met-i2 3,333, bronchi i was tissue ve‘. arctic and mine' in: vitamin A l eilot 2 initial :egectively. Th :32: l were 2.9 itain A values :35 P513111. respe erei slider turl his possible u filed water On I "$5 t° Vitamin A Marilyn Patricia Anderson urinary tract membranes. In contrast the mucous membranes of the trachea, bronchi and eSOphagus were normal. The pancreas, bones and nervous tissue were unaffected. Renal tubule epithelia were degenerate, necrotic and mineralized. Urinary tract calculi occurred frequently. Liver vitamin A levels at necrOpsy, in ug/gm of fresh liver, for Lot 1 and Lot 2 initial control groups (0 and VII) were 10.45 and 8.74, respectively. The liver vitamin A values for Groups I, II and III of Lot 1 were 2.91, 1.76 and 9.10 ug/gm, respectively. The liver vitamin A values for Groups IV, V and VI of Lot 2 were 1.92, 3.40 and 2.48 ug/gm, respectively. The livers of 6 older, wild-caught, red- eared slider turtles had an average of 19.55 ug of vitamin A per gram. It is possible that depletion of body ions and osmotic action of dis- tilled water on some membranes initiated lesions which mimicked and/or led to vitamin A deficiency. VITAMIN A REQUIREMENTS OF YOUNG RED-EARED SLIDER TURTLES (PSEUDEMYS SCRIPTA ELEGANS) By Marilyn Patricia Anderson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1972 I extend spec Sager, for his g‘ academic pro gr fieDepartment of Lie academic envi 33911115 project My sincere a :L-e Animal Husb a1 1?: execution of aifvTECiated was Siinterest in ACKNOWLEDGMENTS I extend special thanks to my major professor, Dr. Vance L. Sanger, for his guidance and encouragement during this project and my academic program. I also thank Dr. C. C. Merrill, Chairman of the Department of Pathology, Michigan State University, for providing the academic environment, research facilities and scholarship that made this project possible. My sincere appreciation is extended to Dr. Duane E. Ullrey of the Animal Husbandry Department for his enthusiasm and guidance during the execution of this project from its beginning to its end. Also appreciated was financial support from the Detroit Zoological Society and interest in the project by Mr. James Langhammer, Curator of Reptiles at the Detroit 200. I am also indebted to the Animal Husbandry Department for computer time and to Dr. William Magee for his assistance in the statistical analysis of my data and its processing for computerized readout. Thanks are also extended to Mrs. Rosemary Covert and Dr. William Ely for guidance in liver vitamin A analysis. ii ED: ION O I 0 Statement Objectives 37.11.3135 REVI BC Vitamin A. PhysiolOgy Vitamin A Vitamin A Calcmm 31 Sodium Dej REptilian Turtle An; (Duncan: N ectops: TABLE OF CONTENTS INTRODUCTION 0 o o o o o o o o o o o o o o o I. o 0 Statement of the Problem . . . . . . . . . Obj ec tives O O O O O O O O O O O O O O O O LITEMTURE REVIEW 0 0 O O O O O O O O 0 O O O O 0 Vitamin A. . .-. . . . . . . . . . . . . Physiology of Vitamin A. . . . . .'. . Vitamin A Deficiency in Mammals and Birds. Vitamin A Deficiency in Turtles. . . . . . Calcium and the Urinary Tract. . . . . . . Sodium Deficiency (Follis, 1958) . . . . . Reptilian Nutrition. . . . . . . . . . . . Turtle Anatomy and Physiology. . . . . . . Skeletal. . . . . . . . . . . . . . Sense Organs. . . . . . . . . . . . Cephalic Vasculature (Bruner, 1907) Osmoregulation. . . . . . . . . .-. Natural History of Pseudemys . . . . . MATERIALS AND METHODS . . . . . . . . . . . . . AniIMls o o o o o o '0 o o o o o a o o o o 0 Group Assignment. . . . . . . . . . Turtle Groups 0 o o o o o o o o 'o o Diets I O O O O O O O O 0 O O O O O O O O 0 Housing. . . . . . . . . . . . . . . . . . Sanitation . . . . . . . . . .-. . . .'. . Lot 1 O 0 O O O O O O O O O O O 0 0 Lot 2 O O O 6 O O O O 0 O O O O O 0 Parameters Monitored . . . . . . . . . . . Food Consumption. . . . . . . . . . Body Weight . . . . . . . . . . . . BOdy Length 0 O O 0 O 0 O O O O O 0 Clinical Signs. . . . . . . . . . . NecrOPSieS O O O O O O f O O O O I O O O 0 iii Page NH O: Oxoxoooumww 15 15 15 l6 17' 18 l9 l9 19 20 20 '20 20 20 21 RESULTS DISCUSS SUMMARY Liver Vitamin A Analysis . Body Weight, Plastron and Carapace Lengths . Organ and Body Weights at NecrOpsy . Food Consumption . Tissue Vitamin A Levels at NecrOpsy. Clinical Signs and Gross Lesions . Postmortem Examination . . . Kidneys . Urinary Bladder and Larynx. . Trachea and Bronchi . Buccal Cavity . . Tympanic Cavity . . . Nasal Cavity. Ocular Area . Lungs . Miscellaneous ION. . . . BIBLIOGRAPHY. . . APPENDI CBS 0 O O O Accessory Observations. iv Urinary Bladders. Page 21 23 23 23 29 29 36 38 42 44 46 46 46 48 48 48 51 51 54 6O 62 66 75 :318 10 H 12 13 ‘1 N 15 16 Body we Turtle Formula Day 0 b 4 1 .’ «My ‘ 6‘7ch A. U Shell 1 from Da Shell 1 from Da Shell 1 at net: Shell 1 at necr Organ V l and 1 Food c: Food Cc Liver t 10: 1’ Liver ; and 2 1 Liver ‘ IHCide] Incifie: LOt l i Table 10 ll 12 13 14 15 16 LIST OF TABLES Body weight-carapace length relationships in turtles . . Turtle groups and dietary treatments . . . . . . . . . . Formula for the basal diet . . . . . . . . .'. . . . . . Day 0 body measurements of hatchling Pseudemys scripta ezegans I C C U U C I O O O 9 I C C C I. . . O O O I O C 0 Shell length and body weight changes in Lot 1 turtles from Day 0 to Day 76 . . . . . . . . . . . . . . . . . . Shell length and body weight changes in Lot 2 turtles from Day 0 to Day 191. I O O O O O O I O I O C O . O O 0 Shell length, organ and body weights of Lot 1 turtles at necropsy. O 0 O O O O O 9 O O O O 0 O O O O O O O O 0 Shell length, organ and body weights of Lot 2 turtles at necropsy. O O I O O 9 I t I O I O I O O I O O O O O 0 Organ weight as percent of body weight at necropsy, Lot 1 and Lot 2 turtles. . . . . c . . . . . . -.- . . . . . Food consumption at the p.m. feeding, Lot 1 turtles. . . Food consumption at the p.m. feeding, Lot 2 turtles. . . Liver weights and liver vitamin A levels at necrOpsy in Lot 1, Lot 2 and 6 wild red-eared slider turtles . . . . Liver and kidney vitamin A levels in 4 juvenile female and 2 mature male red-eared slider turtles . . . . . . . Liver vitamin A levels at necropsy . . . . . . . . . . . Incidence of squamous metaplasia . . . . . . . . . . . . Incidence of lesions other than squamous metaplasia in Lot 1 and Lot 2 turtles. . . . . . . . . . . . . . . . . Page l4 16 17 .24 25 26 27 28 30 .' 31 32 33 34 39- 41 Isle Body measure: 1) or Day 19] Body measure: turtle at Daj Mineral ton: Table B-1 C-l Body measurements of each turtle at Day 0 and Day 76 (Lot 1) or Day 191 (Lot 2). . . . . . . . . . . . . . . . . . Body measurements and liver vitamin A values of each turtle at Day-N (day of necrOpsy). . . . . . . . . . . . Mineral content of Lot 1, Lot 2, and adult turtle shells . vi Page . 72 32139 I 1 Turtle with e: distention an Turtle with d membrane . . Cloudy Shelli tubule With u inclusions . Calcification in the absenc CAICUli from Squamous meta lumen is fill des‘lllielInated, Cross sectiox Of the phat-y] evident. , CIOSS sectio lining (a) h and has been tentIOn 0f t Figure 10 LIST OF FIGURES Turtle with ex0phthalmos caused by periocular lymphatic distention and distended by keratinized debris . . . . . . . Turtle with dry conjunctiva and protruding nictitating membrane . . . . . . . . . . . . . . . . . . . . . . . . . Cloudy swelling of the epithelium in the proximal convoluted tubule with unidentified single and multiple cytOplasmic inclusions . . . . . . . . . . . . . . . . . . . . . . . . . Calcification and squamous metaplasia of renal tubules in the absence of inflammation . . . . . . . . . . . . . . .' Calculi from the urinary bladder . . .q. . . . . .-. '.' .1. Squamous metaplasia of the urinary bladder mucosa. The lumen is filled with keratinized debris and masses of desquamated, degenerating epithelial cells . . . . . . -.- .- Cross section of the larynx in which squamous metaplasia of the pharyngeal (a) and laryngeal (b) membranes is eVidEnt I I I I I I I I I I I I I I I I . I I I I I I I I I I Cross section of the tympanic cavity (A) in which the lining (a) has undergone keratinizing squamous metaplasia and has been detached by lymphatic distention. . . .j. . . . Cross section through the eye. Notice the lymphatic dis- tention of the palpebrum (p), hyperkeratosis of the cornea (c) and keratinizing squamous metaplasia of the nictitating membrane (n) and palpebral.conjunctiva . . . . . . . . -.- . Partial resorption of the cartilaginous cone in the medullary cavity of the femur indicating normal bone development I I I I I I I j I I I I I I I I I I I I . I I VI I I I ' vii Page 37 .’ 37 43 43 45 45 47 49 50 53 C IN '7“ . . “-318. S I 1.; I! .9“ any.“ -—..- “a - MW,” >< #655.54 p: 5,. a} IUI _ “HM“ 1. l bk,“ '5 . 1 mg INTRODUCTION The role of reptiles in research is steadily increasing, especially in areas of biochemistry and physiology. Also many varieties of I reptiles are sold as pets which includes about 15,000,000 turtles i annually in the United States (Anon., 1972). Zoological parks also -. _._- AI ‘7.- i. find reptiles to be a great attraction to the visiting public. 4 The 2 major problems of reptilian adaptation to captivity are L nutrition and environment. Most reptiles are known to be either omnivorous, carnivorous or herbivorous and a few qualitative nutrient requirements have been established empirically. However, quantitative nutrient requirements are not known for any reptile, nor is the role of trace elements known. Statement of Problem Pseudémys scripta eZegans is the small red-eared slider turtle commonly sold in pet Shops. Many of these turtles die within several months of purchase. They become anorectic, their eyelids swell shut and their shells become deformed. The problem appears to be nutrition- ally and/or environmentally related and is occasionally complicated by seCondary infections. Dietary deficiency of the vitamins A and D is thought to be partly responsible for the ocular and shell lesions, respectively, since specific vitamin therapy improved the condition of many afflicted turtles (Elkan and Zwart, 1967; Wallach, 1971a). Since the red-eared slider and its close relatives, Chrysemys, Grqptemys, Clemmys, Emys, Chinemys and Pseudbmedusa are not only kept l .— I (T) iii :lze 1 ~ ~.,. ‘ _ "O I. :‘fi'tl‘v "Vth I. :ai-eare 2 as pets but are also important as research animals and zoological park speciumms, there is a need for establishing the qualitative and quanti- tative nutrient requirements of these turtles. Objectives The objectives of this study were to describe the clinical signs and the macroscopic and microscopic changes in vitamin A deficient turtles and to determine the quantitative vitamin A requirement for support of growth and maintenance of health of the recently-hatched red-eared slider turtle. LITERATURE REVIEW Vitamin A A.fat-solub1e accessory factor (fat-soluble A or vitamin A) was found to be an essential nutrient in 1913 (McCollum and Davis, 1913). Acmmprehensive review of literature on vitamin A.was made by Moore (1957). Sebrell and Harris (1967) and DeLuca and Suttie (1970) were mfitors of multiple author books in which the recent developments in \dtamin A research were reviewed. Vitamin A is a series of fat-soluble isomers. The all-trans retinol is the predominant naturally occurring and most biologically mnive form. Vitamin A is unstable to oxygen and light especially in the absence of antioxidants. Powdered vitamin A, in the palmitate and acetate ester forms, is readily available for commercial purposes. The- Powder may be stabilized and made water dispersible by the addition of a.gelat1n and sugar coating. Freezing also prolongs the storage life of vitamin A. Physiology of Vitamin A Absorption of vitamin A occurs in the small intestine. It is decreased in protein deficiency and it is facilitated by the presence of bile due to emulsifying effects and/or antioxidant prOperties (Bernhard, Ritzel and Steiner, 1954). Storage of vitamin A is mainly in the liver (70 to 90%, Jones, 1965) i 11 both hepatocytes and Kupffer cells, and in normal animals significant 4 concentrations of vitamin A are also found in the kidneys, lungs, blood and milk. Goodman (1970) found that retinol circulates in the plasma in association with a specific retinol transport protein complexed with a prealbumin protein. Vitamin A is not excreted from the body of most animals, even with high levels of absorbed vitamin A, unless there is infection or chronic renal disease. However, depletion .of liver vitamin A, especially with high stores, normally occurs at rates greater than that calculated for physiological needs while with low stores the rate of depletion is slow (Moore, 1957). In rats stress of cold temperature increases depletion of normal liver stores (Sundaresan, Winters, and Therriault, 1967) but not of low stores (Phillips, 1962) and dietary levels of vitamin E inversely modifies the biological utilization of vitamin A (Davies and Moore, 1941). Growth restriction from any cause decreases liver vitamin A depletion (Hayes, 1971; Johnson and Bauman, 1948). A The function of vitamin A, other than invision, is unknown. Dingle (1961) and Roels (1967) suggest that vitamin A is a membrane active substance. Fell (1970) suggested-thatvitamin A promotes the fusion Of lysosomal and cell wall membranes which enables release of PI‘OteOlytic enzymes from the cell. One of these proteolytic enzymes, cathePSin, promotes the resorption of bone and cartilage matrices similar to that observed, inclinical hypervitaminosis A. Amino- c“-‘Ptoic acid, cortisol and antiserum to cathepsin D inhibits the action of Vitamin A on in vitro cartilage matrix breakdown. (Fell, 1970) . Cort1801 is thought to stabilize membranes and amino-caproic acid inhibits a lysosomal protease. Parnell and Sherman (1962) found that different epithelial tissues h ave different threshold levels of response to vitamin A, the 5 gastrointestinal tract having one of the lowest and the skin the highest. Fell (1957) found that skin in vitm formed mucus-secreting cells under the influence of excess vitamin A. Wolf and DeLuca (1970) said that cells of epithelia in general form unmus-secreting cells when vitamin A levels are adequate and form keratinized cells when vitamin A levels are inadequate. They hypothe- size that vitamin A is involved in glyc0protein (mucus) synthesis which is mediated by a specific transfer ribonucleic acid (t-RNA) which in turn translates a specific messenger ribonucleic acid (m-RNA). When \dtamin A.is inadequate the t-RNA for glycoprotein synthesis is not 1noduced.which allows another t-RNA to form and translate its own uhRNA‘which in turn allows the formation of a different cell type, i.e., a keratinized cell forms from a mucus-secreting cell type. This bioChemdcal explanation supports Hayes' suggestion that-the switch of one cell type to another type depends upon the stage of differentiation of that cell (Hayes, 1971). Thompson (1970) found that retinoic acid could functionally replace retinol except in vision and reproduction. He suggested that testicular andrOSEn secretion was reduced inxvitamin A deficiencybecause of redUCEd circulating gonadotrOpin without reduced production of gonado- tr°P1n-by the pituitary. He also suggested that retinol acts directly Iqmn the testicular germinal epithelium and that retinol or retinal but no; IEtinoic acid maintains pregnancy by direct action on the uterus and/or by action of vitamin A on steroid biosynthesis. Vitamin A Deficiency in Mammals and Birds. Lesions and clinical signs of vitamin A deficiency in mammals and lxlrds are well documented (Moore, 1957; Sebrell and Harris, 1967; 1) €flL“¢a.and Suttie, 1970; Follis, 1958; Barnicot and Datta, 1956). The 1. Va ‘1 's .t‘ -l (I 6 main clinical signs and lesions include: 1) night blindness, 2) ataxia, 3) increased cerebrospinal fluid pressure, 4) xerophthalmia, 5) arrest of skeletal growth, 6) deafness (dogs) and 7) blindness (calves). The main histopathologic lesion is keratinizing squamous metaplasia of various ectodermal, mesodermal and endodermal epithelia and occurs in the following approximate order: 1) ducts of salivary and accessory oral and pharyngeal glands, 2) respiratory epithelium of the nares, trachea and bronchi, 3) conjunctiva, cornea and ducts of paraocular glands and 4) pancreatic ducts, enamel organ and e80phagea1 gland ducts. Other microscopic lesions associated with vitamin A deficiency include: 1) atr0phy of seminiferous tubules, 2) kidney tubule degeneration with or without squamous metaplasia and 3) formation of calculi in the urinary bladder and kidney tubules (Beaver, 1961; Elvehjem and Neu, 1932; VanLeersum, 1928). Jungherr (1943) found in chickens that there was an inverse rela- tionship between the level of dietary vitamin A and the severity of vitamin A deficiency lesions in nasal epithelia. He also found that 5-week-old chicks had low storage of vitamin A in the liver regardless 0f high, medium or low levels of dietary vitamin A whereas in 7-month- °1d Chickens there was an approximate direct correlation of liver 8t:01'38e and dietary levels of vitamin A. He considered 15 IU of vitamin A/gm‘of liver as borderline between vitamin A deficiency and insufficiency in growing chicks and suggested that 250 to 350 IU of vitemit: A/head/day in the diet be the minimum chick requirement up to 8 wefiks of age. ‘2‘}! 7 Vitamin A Deficiency in Turtles Elkan and Zwart (1967) reviewed the literature on a common ocular disease of turtles that is characterized by swollen eyes and blindness. They described the clinical signs and lesions in 32 turtles which had this. ocular disease and concluded that the turtles were affected by a "metabolic disorder dominated by vitamin A deficiency." They saw keratinizing squamous metaplasia, accompanied by infiltration with eosinophilic granulocytes, in the lacrimal and harderian glands, con- junctiva, nictitating membrane, pancreatic ducts, kidney collecting tubules, rureters, urinary bladder and bile ducts. Corneas were hyper- keratotic and fatty degeneration of the liver occurred frequently. The livers of 2 affected turtles (Graptemys 8p.) had 9 and 19 IU of vitamin A/gm, respectively. Other sporadic lesions seen were: 1) acute pro- liferative glomerulonephritis, 2) eosin0philic granulocytic infiltra- tion of kidney interstitial tissue, 3) chronic nephritis, 4) acute eosin0philic granulocytic thyroiditis, 5) enlarged thyroid follicles with hyperplastic epithelium and 6) perivascular eosin0philic granulo- cytic infiltration in the liver. Some turtles could not dive due to uncontrolled buoyancy and many sought the highest and driest location in their environment. Some affected turtles recovered with oral vitamin A therapy. Elkan and Zwart suggested that the eosinophilic granulo- CYtictinfiltrations might be a peculiarity of chelonians in regardto Vitamin A deficiency. The liver vitamin A values Elkan and Zwart .found were extremely low Vcompared to values for other reptiles and amphibians which ranged from 35 to 8000 IU/gm of liver (Gillam, 1938). 8 Calcium and the Urinary Tract Calcification of renal tubules and formation of calculi in the urinary tract are not only common with vitamin A deficiency but also with ion deficiencies. In chloride deficient rats calcium salts pre- cipitate in convoluted and collecting tubules, often resulting in tubule obstruction (Follis, 1958). In magnesium deficient rats tubules and glomeruli degenerate'and become calcified and calculi form in tubule lumens (Follis, 1958). Smith and Williams (1971) stated that all renal stones contain a matrix of complex muc0proteins and thatrover 90% contain calcium. Theyalso listed a few conditions which contribute to the formation of calcium-containing calculi sich as oliguria, urine stasis, alkaline urine, phosphaturia, increased calcium and/or ammonia excretion and foreign bodies. They also state that calcium content of urine increases with excessive dietary calcium and with metabolic acidosis (but not respiratory acidosis); excretion of calcium may be above or equal to normal in humans with calcium-containing calculi. Epstein (1971) said that: l) excretion of calcium in urine depends directly upon glomerular fnitration rate and/or blood calcium levels, 2) calcium and sodium reabsorption takes place in the proximal renal tubule and urinary calcium is directly influenced by the amount of‘urinary sodium, 3) after excessive dietary intake of calcium, urinary calcium loss may coMinus for several months after withdrawal of dietary calcium, 4), calcium will precipitate in dead or degenerate renal tissue and 5) leaions ofhypercalcemia, from any. cause, includes degeneration and neel‘osis of renal tubules, exclusive of proximal tubules, and calcified, celIUlar debris which may form obstructing calculi in tubules. 8 Calcium and the Urinary Tract Calcification of renal tubules and formation of calculi in the urijmary tract are not only common with vitamin A deficiency but also with.ion deficiencies. In chloride deficient rats calcium salts pre- cipitate in convoluted and collecting tubules, often resulting in tubule obstruction (Follis, 1958). In magnesium deficient rats tubules and glomeruli degenerate and become calcified and calculi form in tubule lumens (Follis, 1958). Sudth and Williams (1971) stated that all renal stones contain a matrix of complex muc0proteins and that over 90% contain calcium. They also listed a few conditions which contribute to the formation of calcium-containing calculi sich as oliguria, urine stasis, alkaline urine, phosphaturia, increased calcium and/or ammonia excretion and foreign bodies. They also state that calcium content of urine increases with excessive dietary calcium and with metabolic acidosis (but not respiratory acidosis); excretion of calcium may be above or equal to rwrmal in humans with calcium-containing calculi. Epstein (1971) said that: l) excretion of calcium in urine depends directly upon glomerular filtration rate and/or blood calcium levels, 2) calcium and sodium reabsorption takes place in the proximal renal tubule and urinary calcium.is directly influenced by the amount of urinary sodium, 3) after excessive dietary intake of calcium, urinary calcium loss may continue for several months after withdrawal of dietary calcium, 4) caleiumwill precipitate in dead or degenerate renal tissue and 5) lesions of hypercalcemia, from any cause, includes degeneration and necrOsis of renal tubules, exclusive of proximal tubules, and calcified Celllufir debris which may form obstructing calculi in tubules. 9 Sodium Deficiency (Follis, 1958) Corneal epithelium becomes hyperkeratotic in sodium deficient rats. Sodium deficient rats also have retarded growth and subsequent weight loss, swollen eyelids and hair loss. The swollen eyelids result from tarsal and meibomian gland duct blockage and cystic dilatation of acini which undergo squamous metaplasia. Sodium depletion can be produced by excessive water intake, dietary restriction and inhibition of sodium reabsorption. Reptilian Nutrition Nutrition or poikilothermic animals is intimately related to environment. Ambient temperatures affect body temperature and thus in Ldvo enzymatic reaction rates. Energy requirements to maintain body temperature also depend on the ambient temperature. Seasonal influences include not only temperature changes but also daylight length-hormonal balances which in turn affects appetite and nutrient requirements for Senoral daily activity, reproduction, hibernation and estivation. Even if the preper temperature, humidity, lighting and food are adequate, a reptile in captivity may still suffer from inadequate nutrition as a result-of its peculiar behavior, anatomy and physiology (Wallach, 1971b; King, 1971). For example, an inferior individual may starve to death under the intimidation of a dominant one, especially if there is overew crowding or inadequate furniture such as perches or hiding places. Wallach (1971b) and Marcus (1971) reviewed and described the environmental and nutritional and infectious diseases of reptiles, respectively. Some of the major nutritional problems are: 1) steatitis ofVitamin E deficiency, 2) goiter of iodine deficiency, 3) skeletal diseases of calcium—phosphorus-vitamin D imbalance and 4) edema in ,m’ 10 chelonians due to vitamin A deficiency. In all in-tances these disease- nutrient relationships are established on empirical bases only. Turtle Anatomy and Physiology Skeletal Cans, Bellairs and Parsons (1969) edited a comprehensive review of reptilian osteology and dentition and chelonian shell formation. The cortex of chelonian long bones is mainly primary bone without haversian systems and near the epiphysis it is thin and of endosteal origin. The medullary space contains cancellous bone and hemopoietic tissue. The epiphyses do not form secondary centers of ossification in turtles. At the ends of long bones, unique to young turtles, crocodiles and birds, is a large mass (cone) of cartilage which becomes eroded from all sides, sometimes leaving temporary islands of cartilage in the medulla (Figure 10). The outer border of the epiphysis is composed of fibrocartilage becoming undifferentiated cartilage internally. Flat- tcued cartilage cells in the growth zone form columns and hypertrophy; these columns may be poorly develOped in young turtles. Irregular cavities are eroded into the cartilaginous masses at the ends of the long bones but once columns are formed the erosion is regular, follow- ing the columns.' Some endochondral bone formation in turtles appears to involve a true metaplasia of cartilage to bone instead of osteo— blastic bone formation after cartilage destruction. Suzuki (1963) described the skeletal system of Pseudemys scripta elegans hatchlings (carapace length of 29 to 35 mm), juveniles and adults and correlated histologic differences with age, sex and seasons. He found that hatchling femurs had an ossified endosteal layer and a periosteal collar of osteoid whereas turtles a few months older had ___.l _._ _E 11 femurs with both layers well calcified. At the ends of hatchling long bones was a large medullary cone of cartilage which was removed by the time the turtles attained a carapace length of 40 to_65 mm. He also found that calcification of cartilage and osteoid occurred primarily in periods of active growth which coincided with adequate food intake. Clark and Gibbons (1969) found a positive correlation between plastron length and calcium content of the plastron and carapace. Sense Organs Gans and Parsons (1970a) edited a comprehensive review of ocular, nasal and auditory structures in reptiles. The nasal vestibule in Pseudémys is lined by keratinized stratified squamous epithelium. The external nasal gland enters the dorsolateral wall of the posterior vestibulum. The nasal cavity proper has sensory olfactory epithelium posterodorsally, nonsensory respiratory epithelium anteroventrally and an area of sensory, perhaps vomeronasal, epithelium.anteroventrally (McCotter, 1917). Bowman's glands, which occur only in the subepie thelial sensory olfactory epithelium, are alveolar in form and lined by columnar or pyramidal cells which may become_cuboida1 in the ducts. Turtles have a lacrimal gland temporally and a harderian gland nasally in-the orbital-cavity. With the light microscOpe these glands are indistinguishable except as to location (Elkan and Zwart, 1967). The optic nerve passes between these glands. In some species of.turtles the lacrimal gland cell is almost identical to reptilian renal tubule cells, both microscopically and ultrastructurally (Cowan, 1971). Both glands empty into the base of the conjunctival sac through numerous ducts. There is no nasolacrimal canal. 12 The middle ear of Paeudémys is connected with the pharynx by a narrow eustachian tube which is lined by a mucous membrane. The tympanum is lined by a thin squamous epithelium and the tympanic cavity is lined with several types of epithelium varying from simple squamous to cuboidal with scattered mucus-secreting cells. Cephalic Vasculature (Bruner,il907) There are extensive intracranial and extracranial venous sinuses which have numerous bidirectional anastomoses. Their function is thought to be equalization of blood pressure in the head region. The orbital sinus receives most of the blood from the anterior cephalic region and the internal jugular vein is its only outlet. The internal jugular vein which drains over 90% of the cephalic region is equipped with a special constrictor muscle which is responsible for the swell mechanism of cephalic skin molting.r Muscular tone keeps a turtle's eyelids open and prevents orbital sinus distention. Closing of the lids Operates by localized increases in blood pressure. The cerebrospinal and extracellular spaces in the brain of Pseudémys are described by Heisey (1970). The spaces are large compared to mammals. Osmoregulation Blood osmotic pressure is regulated primarily by the kidneys. Dantzler and Schmidt-Nielsen (1966) found that changes in the glomerular filtration rate of turtles varied only with the number of functioning nephrons which work to full capacity or not at all. Extrarenal osmoregulation occurs in the-mucosa of the pharynx and cloaca (Dunson, 1967a), in the urinary.bladder and paired accessory D a.” 2.",1. 'vna‘ t 4. salt! ”‘71 ‘th' :EII ‘PH. 4,... l3 urinary bladders (Rosen, 1970; Steinmetz, 1967; Steinmetz, Omachi and Frazier, 1967) and in most reptiles in specialized salt secreting glands (lacrimal glands in turtles) located in the cephalic area (Dessauer, 1970; Cowan, 1969; Cowan, 1971). Trobec and Stanley (1971) found that Chrysemys held in distilled water for 40 days were depleted of their sodium stores by 166 ueq/kg of body weight/day (or about 0.2%/day of their total sodium). They _t also found that turtles held in-tap water for 48 days had a net loss of-sodium without clinically apparent ill effects. Natural History of Pseudémys Cagle (1950) described in detail the life history of Pseudemys scripta. This turtle is found in quiet, shallow, fresh water where there is abundant sunlight and vegetation. Cagle found the turtles active between temperatures of 10 to 35 C. Egg laying occurs from April to July and hatching from July to September; some turtles over— winter in the nest. The yolk mass helps shape the carapace and plastron when it is drawn into the body shortly after hatching. Measurements. of 86 hatchlings from Louisiana were: Carapace lengty, mm E = 32.46; range, 28.0-35.8 Plastron length, mm 2 =.30.89; range; 27.1-33.8 Body weight, gm i = 8.07; range, 5.4-10.0 Yolk‘uass, cm 1-3 No hatchlings started growing earlier than the following April whether or not they overwintered in the nest. In Illinois the growing season is from May to October and in Louisiana from April to November. Hatchlings approximately double their size in the first growing season then continue to grow at reduced rates in succeeding seasons (Carr, 1952). 14 Clark and Gibbons (1969) examined stomach contents in Pseudémys scripta and found the turtles to be omnivorous. They found a shift in hatchlings and juveniles during the growing season from a basically carnivorous diet to a more herbivorous diet, the latter typical of adults. Body weight-carapace length relationships were published for 3 species of turtles. The following table summarizes the weight-length (W-L) formulas : Table 1. Body weight-carapace length relationships in turtles Genus Formula W units L units Authority -4 2.79 Chrysemys W - 4.4 x 10 L mm Graham, 1971 Trionyx w - 0.1202 L2°95 gm cm Dunson, 1967b Chelydra W = 1.6 x 10-4 L3°06 lb in Lagler and Applegate, 1943 Chelydra W a 3.36 x 10-.4 L2°93 gm cm * * From the data of Lagler and Applegate (1943) converted from the avoirdupois system to the metric system by Mosimann (see Dunson, 1967b). MATERIALS AND METHODS Animals Eighty hatchling, red-eared slider turtles, Pseuflbmys scripta elegans, were acquired from a commercial dealer. Forty of these turtles (Lot 1, Groups 0, I, II and III) were received in May 1971 and may have been holdovers from the 1970 hatching season. Nine of these turtles had ulcers on the forefeet and/or tail, probably the result of canni- balism and/or trauma. One healthy turtle had a crimp in its shell at the plastron-bridge junction. The other 40 turtles (Lot 2, Groups IV, V, VI and VII) were received in August 1971; all had an egg tooth and 10 had some yolk material adherent to the plastron. The egg teeth were lost within 2 weeks indicating that all turtles in this lot were received within a few days of hatching. For the purpose of identification, the plastron of each turtle was photographed to record the unique pattern of spots and ocelli. Group Assignment Lg£_l. The 9 turtles with skin ulcers and the l with the crimped shell were placed in Group 0. The remaining 30 turtles were ranked by ‘weight. Each of the 3 heaviest turtles was randomly assigned to treat- ment Group I, II or III. Each turtle in succeeding groups of 3 was similarly assigned to one of the 3 treatment groups. 15 16 ‘Lg£_2. The 40 turtles were ranked by weight and each turtle in Stumessive groups of 4 was randomly assigned to Group IV, V, VI or VII. Turtles in Groups IV, V and VI were in turn randomly assigned, by similar weight outcome groupings, to subgroup A or B. Turtle Groups Table 2. Turtle groups and dietary treatments Vitamin A * Zinc Lot. Group Diet Supplement Supplement** 1 0 none none none 1 I Basal 0 0 1 II Supplemented 140+ 0 1 III Supplemented 280+ 0 2 IV A Basal 0 0 2 IV B Supplemented 0 100 2 V A Supplemented 900++ 0 2 V B Supplemented 900++ 100 2 VI A Supplemented 1800++ 0 2 VI B Supplemented 1800++ 100 2 VII none none none * IU (International Units) of vitamin A/kg of fresh hog heart. ** ‘ ppm (parts per million) of elemental zinc. 1. Retinyl palmitate, gelatin coated. ++ All-trans retinyl acetate. l7 Diets The basal diet was based on the chick nutrient requirements (NAB, 1971). Table 3. * Formula for the basal diet Table 3 lists the basal diet ingredients and formula. Ingredient Amount Fresh ground hog heart, gm** 500 MnSO4'H20, mg 19.1 KIO3, mcg 38.0 Vitamin D3, ICU 22.5 a-toc0pheryl acetate, IU 4.5 Folic acid, mcg 135 L—ascorbic acid, mg 50 Ca(H2P04)2°H20, gm 2.78 CaCOB, gm 5.84 Glucose monohydrate, gm 1.12 Corn oil, gm 2.25 Ethoxyquin (antioxidant), mg 13.5 * Contained 4.9 ug of vitamin A/gm (dry basis) after 1y0philization. ** Contained 3.4 pg of vitamin A/gm (wet basis). The dry ingredients were combined to form a premix for the basal diet. The premix for Lot 2 was equal to that of Lot 1 minus the KIO 3 which was later added to the hog heart as an aqueous solution (2 pg KIO3/ml distilled water). Hearts from lOO-kg hogs were trimmed of fat, large blood vessels and coarse connective tissue. After at least 1 coarse and 2 fine mechanical l8 grindings, the hog hearts were placed in aluminum pans for hand mixing with premix (9.83 gm premix/500 gm fresh ground hog heart). The ethoxyquin was mixed with the corn oil and hand mixed with the heart. When Lot 1 diets were prepared, a vitamin A premix was made by adding 140 mg of gelatin coated retinyl palmitate (500,000 USP units/gm) to 500 gm of glucose monohydrate. The following amounts of vitamin A premix and balancing glucose were added per 500 gm of hog heart: Diet, group I II III Premix, gm 0 0.5 l Glucose, gm 1 0.5 0 When Lot 2 diets were prepared all-trans retinyl acetate in cotton- seed 011 base (100 IU/mg) was added to the corn oil before adding the ethoxyquin. Zinc was added to subgroup B diets in an aqueous solution which was prepared by adding distilled water, qs to 1000 gm, to 4.40 gm of ZnSO4'7H20. The amount of zinc solution added to the hog heart (hh) was-determined by the following formula: (hh,gm) x (0.20) x (100)/1000 3 (solution, gm). All diet ingredients were thoroughly hand mixed and then_mechanica11y reground at least 8 times. The diets were frozen in 5- to lO-mm thick layers and lyophilized. The diets were stored at -70 0. Only 1- to 2-week quantities were kept at refrigeration temperatures for daily use. Housing * Each group of turtles was housed in a separate lO-gallon aquarium that was half filled with distilled water. Each tank was provided with * Metaframe stainless steel aquarium, Metaframe Corporation, Maywood, N.J. 07607. 19. a 1 x 10 x 15 cm.wood float, an 8-inch 25-watt lamp with reflector and a plastic water filter box containing nylon fiber and activated charcoal. The lights and air-driven water filters Operated 24 hours a day. Room temperature was between 21 and 30 C. Sanitation Lot 1 Twice a week the aquariums and filter boxes were washed with detergent* and thoroughly rinsed under pressure with tap water. Immediately after the second semi-weekly wash the aquariums and filter boxes were soaked for 10 minutes in iodine solution** and thoroughly rinsed with tap water. Filter fiber was rinsed semidweekly and changed bidweekly. The wood floats were autoclaved weekly. The turtles were ** rinsed in iodine solution before return to cleaned aquariums. as; THIe aquariums and filter boxes were washed weekly with detergent and thoroughly rinsed with tap water. Semi-weekly the aquariums were thorougally rinsed with tap water. At weekly intervals the wood floats were a.thoclaved and the filter fiber changed. The turtles were rinsed ** weekly :ln.iodine solution before being returned to the cleaned aquariums . x * Pyroneg, Diversey Chemical Company, Chicago, 111. 60606. 1** Wescodyne, West Chemical Products, Inc., 42-16 West Street, L098 18 land, N.Y. 20 Parameters Monitored Food Consumption Each turtle group was fed in a separate 2-quart plastic box* containing enough distilled water to just cover the turtles. They were fed twice a day for 30 minutes at each feeding. Twice a week afternoon food consumption determinations were run for each group. Approximately 0.5 gm of food was offered to each group. The uneaten food (orts) was filtered back using a 12-cm, #4 "Whatman" filter paper, a Buechner funnel and vacuum. The filters with orts were dried overnight at 54 C and reweighed. Food consumed = food offered (filter with orts — filter). Simultaneous wet and dry control determinations revealed a 30% weight loss of food offered resulting from leaching, drying and handling. Body Weight Each turtle was weighed twice a week in midafternoon in conjunction with the late afternoon food consumption determinations. Body Length Bi—weekly, in conjunction with body weight determinations, the plastron and carapace of each turtle was measured along the midline using a vernier caliper. Clinical Signs Turtles were observed at least twice daily and notes made describing behavior, clinical signs and gross lesions. * Freezette Food Container (Style 159), Republic Molding Corp., Chicago, Ill. 40648. 21 NecrOpsies Turtles in Groups 0 and VII were killed on Day 0 for their respect- ive experimental lot and served as controls for normal histology and gross anatomy. Turtles were killed by decapitation when body weight decreased and/or anorexia was evident. Fresh organ weights were recorded for heart, liver, kidneys (together) plastron and carapace. An attempt was made to remove the adrenal gland and gonad from each kidney before weighing. The following tissues were placed in neutral buffered 10% formalin until histologic examination was made: thyroid, heart, trachea, lungs, spleen, neck retractor muscles, kidneys, urinary bladders, esophagus, stomach, intestines, pancreas, piece of liver, right and/or left femur, fat bodies and the entire head with the dorsal meninges exposed. After fixation with formalin the heads were placed in decalcifying solution* for approximately 30 minutes and then returned to formalin after a tap water rinse. The plastron, carapace and remain- ing liver were frozen. Tissues were sectioned at 6 u and stained with hematoxylin and eosin. Liver Vitamin A Analysis The method used for quantitating vitamin A levels in the liver utilized the Carr-Price reaction in which vitamin A in chloroform with antimony trichloride to form a transiently stable blue solution with absorption maximum at 620 mu (Carr and Price, 1926). A homogenate of 1 part liver to 2 parts deionized distilled water was refluxed in 45 m1 ** of alcoholic potassium hydroxide for 20 minutes and then diluted to * Cal—Ex, Fisher Scientific Company, Fair Lawn, N.J. ** 9 ml of 11 N KOH plus 91 ml of absolute ethyl alcohol. 22 100 ml with deionized distilled water. Duplicate 30-ml aliquots were taken and extracted with petroleum ether.* Three- to five-milliliter aliquots of extract were read for OD at 440 mu in a Coleman Jr. II spectrophotometer to check for carotene and then evaporated under 25 psi (1.75 kg/cmz) vacuum at 55 C. A few samples with readings at 440 mu were checked at various mu settings before evaporation. The residue was suspended in 1 m1 of chloroform after which 2 ml of antimony tri- chloride solution** were added. An immediate reading of the transiently stable OD at 620 mu was made to determine vitamin A content (plus caro- tene if present). Micrograms of vitamin A in the evaporated extract was determined from a standard curve. Micrograms of vitamin A per gram of liver was mathematically determined.‘ * Skellysolve F, Skelly Oil Company, 605 West 47th Street, Kansas City, Mo. *9: 200 mg of SbCl qs to 1 liter with chloroform. 3 RESULTS Body Weight, Plastron and Carapace Lengths These parameters for Lot 1 and Lot 2 turtles on Day 0 of the respective experiments are summarized in Table 4. The Day 0 body weight- carapace length relationship for Lot 1 turtles is W = 0.567 L2°13; for Lot 2 turtles, W = 0.202 L3°Ol. "W" is body weight in grams and "L" is carapace length in centimeters. Table 5 summarizes changes in the above parameters in Lot 1 turtles between Day 0 and Day 76 of the experiment. There was a statistically significant difference (P<0.05) for each of the above parameters between Groups I and III but not between Groups I and II. Table 6 summarizes changes in the above parameters inLot 2turtles between Day 0 and Day 191 of the experiment. There was no statistically 81gum-"£8111: difference (P>0.05) for each of the above parameters between any of the groups. Since there was no statistically significant dif- ference (P>0.05) between the no-zinc and zinc (A and B) subgroups, the A and 3 data were combined in each group. Organ and BodLWeights at Necropsy A. cOl‘rection for the age, in days, for each turtle was made in the Statistical analysis of these parameters. Tables 7 and 8 summarize these data for Lot 1 and Lot 2, respectively. Statistically there were high simple correlations between heart, liver and coIllbined kidney weights and between body weight and weights of these organs . 2 3 24 No.~m wo.~m om.qm «N.mm om.mm mm.mm mm.oa I Hm.o co.w oe Ham mm.om mm.om Ho.qm em.mm NH.wN oa.mm mw.m I NN.m ,mm.n oq Ham Nq.mm mo.~m .Hm.qm mm.¢m om.m~ oo.~m mw.m I mm.m ao.m 0H HH> No.5m oe.mm mm.em m~.¢m wq.Hm mm.~m mm.m I 00.5 oo.m oa H> mw.om oq.m~ mm.em mq.em ow.om mm.~m =mm.0H I mn.m .mm.m OH > mm.om nm.~m ma.qm em.mm oa.om mm.~m om.m I Hm.o cm.w 0H >H mq.om mo.~m mm.mm em.mm mq.om mm.mm om.m I mm.m mm.m 0H HHH qw.mm om.mm om.mm mm.em NH.mN mo.mm mw.m I NN.m mm.“ 0H HH mm.om mN.om ~H.¢m mm.qm om.m~ oa.mm mo.m I mm.m ow.n OH H co.om mo.mm wo.¢m mo.qm oa.om mw.mm om.m I mo.m mm.m OH 0 swoon m owumu m omens m moauusk macaw as «nag 833mm 3m: c0333 mo umaasz am Jews; boom mrommmm numsfinm mmsmfizmmm wcaanoumn mo munmamuammma kvon 0 Ann .q manna 25 Table 5. Shell length and body weight changes in Lot 1 turtles from Day 0 to Day 76 Group II III Number of Turtles 9 7 Vitamin A/kg wet diet 140 280 :t SE Body weight (Day 0), gm 8.01 8.01 8.39 0.38 * Body weight (Day 76) gm 7.86 8.82 12.01 1.06 * Body.weight gain, mg/day 2.0 10.7 47.6 11.2 Plastron length (Day 0), mm 32.27 32.30 32.92 0.52 ** Plastron length (Day 76), mm 33.03 33.40 35.95 0.92 * Plastron length gain, u/day 10.0 14.5 39.9 7.4 Carapace length (Day 0), mm 34.25 34.34 34.76 0.46 ** Carapace length (Day 76), mm 34.98 35.64 38.58 1.14- * Carapace length gain, u/day 9.6 17.1 50.3 10.8 * . Significantly greater than the least 2 values (P<.05). ** Significantly greater than the least value (P<.05). Table 7. Shell length, organ and body weights of Lot necrOpsy 27 l turtles at Group I II III Number of turtles 10 10 10 Vitamin A, IU/kg wet diet 0 140 280 iZSE * Body weight, gm 7.57 6.93 9.64 0.72 ** Plastron weight, mg 608 500 802 62 ** Carapace weight, mg 1220 1029 1704 160 ** Liver weight, mg 436 363 638 67 ** Heart weight, mg 33 28 44 0.4 ** Kidney weight, mg 48 44 72 0.7 * Plastron length, mm 33.10 31.90 34.86 0.81 Carapace length, mm 35.01 33.73 37.48 1.00 1. **+ * Significantly less than the greatest value (P<.05). Significantly greater than the least 2 values (P<.05). +Correction for differences in the age of turtles was made. ”V \ fl..___ _ 28 Table 8. Shell length, organ and body weights of Lot 2 turtles at necrOpsy Group* IV V VI Number of turtles 10 10 10 Vitamin A IU/kg wet diet 0 900 1800 .i SE Body weight, gm 10.80 11.61 13.44 0.78 Plastron weight, mg 828 929 1023 67 Carapace weight, mg 1733 1996 2356 194 Liver weight, mg 841 825 1071 74 Heart weight, mg 36 44 51 4 Kidney weight, mg 145 137 163 10 Plastron length, mm 33.48 35.11 35.11 0.75 Carapace length, mm 36.26 37.57 38.76 0.91 * No significant differences between the groups (P>.05); correction for differences in the age of turtles was made. 29 Table 9 is a summary of organ weights as a percent of body weight at necrOpsy. These data were not statistically analyzed. Only Lot 2, Group VII, turtles had large yolk sacs in the abdominal cavity at the time of necrOpsy. The dimensions of these yolk sacs were: 3.76-7.13 mm Diameter E 5.60 mm; range Length 2 11.27 mm; range 6.73-18.6l mm Food Consumption Tables 10 and 11 summarize afternoon food consumption of Lot 1 and Lot 2, respectively, expressed as: 1) mg of food consumed/gm of turtle weight and 2) mg of food consumed/turtle. Values for food consumed were reduced by 30% before calculating the above data to correct for loss of food offered due to leaching, drying and handling. Tissue Vitamin A Levels at Necropsy Livers from all Lot 1 and Lot 2 turtles were analyzed for vitamin A and carotene. In addition livers from 6 wild-caught red-cared slider turtles (4 juvenile females and 2 mature males) were analyzed for vitamin A and carotene; the kidneys from 3 were also analyzed. Table 12 summarizes liver weights and liver vitamin A levels in the experimental and wild turtles. All livers and kidneys examdned were negative for carotene. No correction for age differences of the turtles was made. Table 13 summarizes the liver and kidney vitamin A data from the 6 wild turtles. Table 14 (A) summarizes liver vitamin A levels in Lot 1 turtles after statistical correction for age differences of the turtles. Table 14 (B) summarizes liver vitamin A levels in Lot 2 turtles without cor- rection for age differences. A few of the green leg-pit fat bodies from 1 turtle (Lot 2, Group V) were analyzed for carotene and vitamin A. The fat was negative for H> m.NMIm.oH N.©N m.NNIN.HH N.mH n.0HIo.n o.m wN.HIHm. on. mo.IwN. mo. ¢.wIN.¢ H.o om .>.>H N m.©NI¢.MN w.qN o.mHIN.oH «.mH N.w Iw.o ¢.m _H<.OION. Hm. mm.I©N. om. o.¢IH.m m.m OH HH> N AU .3 m.NmIm.oH m.mN m.NNIN.HH o.mH N.OHI©.m m.m mN.HION. we. mo.IoN. Hq. «.mIH.m o.m cc HHm N HHH o.mNIm.wH o.MN w.ONI¢.NH a.mH m.a Im.m 0.5 NN.HIem. mo. No.ImN.. me. «.mIN.N o.m om .HH.H H o.OMIm.NH q.qN n.0NIo.HH m.oH w.m In.m H.w Hn.OImN. on. N¢.IMN. Hm. v.0Iw.H w.m m o H o.OMIm.nH m.MN w.ONIo.HH o.oH w.m Im.m w.m NN.HImN. No. No.IMN. ow. «.mlw.H H.m mm HHm H omens m swoon m omens m swoon x omens N swoon x moHuu:H macho uOH ooomdumo + couummHm oommwumo couumMHm mmoanM unmom uo>HH mo .oz mmHuusu N uOH was H sou .hmmouoos us uanoB moon mo unmouom mo u£MHoB cameo .m oHan 3 1 Table 10. Food consumption at the p.m. feeding, Lot 1 turtles Group I Group II Group III Month i range I range i range A. mg of food/gm of turtle weight June. 3.2 2.1-4.1 2.8 1.6-3.6 3.1 1.1-4.6 July 2.2 0.3-3.6 1.3 0.0-4.4 1.9 0.1—4.8 August 2.4 1.6-3.5 1.2 0.5-1.9 1.0 0.0-1.9 September 1.6 0.4-3.4 1.4 0.0-4.0 1.0 0.6-1.9 October - - 3.2 1.7-4.5 - - November - - 3.9 2.6-6.5 - - B. mg of food/turtle June 24.5 16.0-31.0 21.9 12.2-27.9 24.5 8.6-37.4 July 17.5 2.2-28.7 11.0 0.0-35.5 18.9 1.3-43.7 August 19.0 12.8-28.3 10.9 4.5-17.7 12.0 0.0-24.5 September 12.4 2.8-25.9 12.6 0.0-32.2 14.7 8.6-27.6 October - - 26.1 14.2-37.4 - - November - - 31.0 20.3-57.3 - - 32 Table 11. Food consumption at the p.m. feeding, Lot 2 turtles Group IV Group V Group VI Month i range I range E range A. mg of food/gm of turtle weight September 0.9 0.0-2.1 1.2 0.0-2.5 1.0 0.1-2.0 October 1.2 0.2-2.5 0.8 0.3-2.1 1.0 0.0-2.2 November 0.9 0.3—2.1 1.0 0.0-2.3 0.8 0.1-1.5 December 0.6 0.0—1.4 0.6 0.0-1.8 0.6 0.3-1.0 January 0.3 0.0—0.7 0.5 0.0-1.3 0.5 0.0-1.2 February 0.6 0.1-2.0 0.2 0.0-1.0 0.4 0.0-1.4 March 0.4 0.0-1.2 0.2 0.0-0.3 0.2 0.0-0.8 April 0.1 0.0-0.6 1.6 0.0-4.6 0.4 0.0-1.7 B. mg of food per turtle September 8.1 0.0-17.8 10.4 0.0-21.5 8.6 0.9-17.4 October_ 10.6 1.9-22.4 5.4 1.9-12.2 8.8 0.0-19.0 November 8.3 2.8-19.0 9.6 0.0—22.8 7.8 0.5-13.9 December 5.1 0.1-11.4 5.6 0.0-18.1 5.6 2.9-9.9 January 2.7 0.0- 5.9 4.8 0.0-13.7 5.0 0.0-12.7 February 5.1 0.8-16.9 2.4 0.0-10.4 4.1 0.0-14.0 March 3.9 0.0-10.8 1.9 0.0- 8.6 2.2 0.0- 8.8 1.0 0.0- 3.3 13.5 0.0-38.5 4.8 0.0-19.6 April 33 Table 12. Liver weights and liver vitamin A levels at necropsy in Lot 1, Lot 2 and 6 wild red-eared slider turtles* Number of _Liver weight, gm pg; vitamin A/gm liver Group Turtles x range x range 0 10 0.30l 0.122-0.579 10.45 0.36-6l.45 I l0 0.395 0.120—0.688 2.9l+ 0.44-10.10 II 10 0.466 0.144-0.924 l.76+ 0.42— 2.98 III 10 0.575 0.126-l.229 9.10+¢ 0.43-34.00 H IV 10 0.552 0.322-l.074 1.92 0.00- 3.00 H V 10 0.540 0.293-l.047 3.40 1.10- 4.35 H VI 10 0.752 0.425-l.310 2.48 0.54- 4.78 VII 10 0.335 0.227-0.389 8.74- 4.01-13.54 wild**' 6 3.278 2.05944.582 19.55 l.70‘39¢94 *Uncorrected for age differences of turtles. **4 juvenile females and 2 mature males. +No significant differences (P>.05). ++No significant differences (P>.05). ¢3 turtles died early. 34 Table 13. Liver and kidney vitamin A levels in 4 juvenile female and 2 mature male red-eared slider turtles* Liver _Kidneys Sex weight, gm ug of vitamin A/gm weight, gm ug of vitamin A/gm F 1.62 ' 14.30 0.14 9.32 F 1.37 1.70 0.30 1.10 F 0.46 39.94 - _ F 0.87 8.19 - - M 1.31 14.50 0.32 5.28 M 0.57 38.67 - - E 1.03 19.55 0.25_ 5.23 * Negative for carotene. fable 1 11‘." g: 35 * Table 14. Liver vitamin A levels at necropsy A. Lot 1++ ** ** ** Vitamin A Group 0 Group I Group II Group III IU/kg of wet diet - 0 140 280 ;: SE+ ug/gm of fresh liver 7.83. 3.45 3.47. 8.65 3.46 IU/gm of fresh liver 26.1 11.5 11.6 28.8 11.5 B. Lot 27H: Group Group. Group Group Vitamin A IV** v** VI** VII IU/kg of wet diet 0 . 900 1800 - + SE ug/gm of fresh liver 1.92 3.40 2.48 8.74 0.44 IU/gm of fresh liver 6.4 11.3 8.3 29.1 1.47 No carotene present. ** No significant difference (P>.05); Lot 1 - corrected for age differences of the turtles; Lot 2 - uncorrected for age differences of the turtles. 1- Lot 1 - excludes Group 0;_Lot 2 - excludes Group VII. 1'Ten turtles per group. 36 carotene but had 8.33 ug of vitamin A/gm. The liver from this turtle had 0.75 pg of vitamin A/gm. The weight of fat analyzed was 0.11 gm; the total liver weight was 1.074 gm. At the time of necrOpsy, yolk sacs from Lot 2, Group VII, turtles were mistakenly placed in formalin (10%, neutral, buffered). They were stored in the formalin for 11 months at room temperature which varied from 21 to 40 C. Two of these yolk sacs were negative for carotene, although they were yellow in color, but had 6.02 pg and 2.54 ug of vitamin A/gm, respectively. Clinical Signs and Gross Lesions Groups I through VI had similar clinical signs and gross lesions characterized by severe palpebral distention with fluid, dyspnea, and anorexia, with weight loss and lethargy. Palpebral distention was the most constant lesion, usually bilateral and usually the first sign of disease. A few turtles appeared to have ex0phthalmos (Figure 1). Many eyelids became baggy when the distention subsided and often exposed dry, granular conjunctivae, corneas and nictitating membranes (Figure 2). The xerophthalmia was seen as early as 2 days and as late as 3 weeks after onset of palpebral distention. However, 1 Group II turtle with swollen palpebra, which continued to eat, had glistening corneas and normal conjunctiva when killed after 40 days. Weight loss, from decreasing appetite, followed onset of palpebral distention by a few days to 1 week. Anorexia was confirmed by the_ seventh to twenty-first day when turtles had drastic weight loss and/or defecated only mucus with or without bile. Affected turtles that con- tinued to eat grabbed at the floating food after bumping into it with 37 Figure l. Turtle with exophthalmos caused by peri— ocular lymphatic distention and distended by keratinized debris. x 4. Figure 2. Turtle with dry conjunctiva and protrud- ing nictitating membrane. x 4. their heads. noses near t eats often the food. I gas-filled : laser intes Dyspne ing, snicki Immine- I Of yellow : turtles her youth brea the Surfac the jaw (1 along the Anore day and n: CalEed thI Many dysp. tended to Seve one Side to the SL‘ 15 (A) I I tiSsueS 38 their heads. Some nudged the food with their nose and/or held their noses near the food before grabbing it for ingestion. Grabbing move— ments often fell short of the food or were aimed off to the side of the food. At necrOpsy anorectic turtles frequently had empty and/or gas-filled intestines. Some had only a few pellets of bile in the lower intestine. Dyspnea was characterized by sneezing, gasping, whistling, squeak- ing, snicking and mouth breathing assisted by vigorous abdominal muscle pumping.‘ Dyspnea was due to laryngeal obstruction with soft plaques of yellow material and/or to obstruction of the external nares. Many turtles had bulging snouts in the area of the external nasal glands. Mouth breathing turtles had small, firm, yellow plugs of material on the surface of the tongue (in lingual glands), at the dorsal angle of the jaw (in dorsal buccal glands) and on the floor of the oral cavity along the tongue (in sublingual glands). Anorectic turtles became lethargic, tended to remain on the float day and night and did not respond rapidly, if at all, to stimuli that caused the other turtles to flee the float for safety of the water. Many dyspneic turtles spent normal amounts of time underwater but they tended to surface frequently and noisily. Several turtles could not regulate their buoyancy. A few listed to one.side or head down.. Others could not stay submerged without bobbing to the surface after paddling movements ceased.’ Postmortem Examination Postmortem findings in Groups I through VI were equivalent. Tables 15 (A), (B) and 16 summarize the incidence of lesions in the various tissues from Lot 1 and Lot 2 turtles. All further remarks on postmortem H Iable 15. I Group Eeithelia Lacrinal gla " rderian g] Conjunctiva Coma" Eustachian 1 Dorsal bucca Sublingual l lingual glal Buccal mucos Taryn}; Trachea Bronc'ni Enernal na. N35511 reces ReSpiratory Olfactoq II ESCphaguS Fancreatic Urinary bla ACcessozy u Table 15. Incidence of squamous metaplasia 39 A. Lot 1 Group 0 I II III Epithelia Lacrimal gland 0/10 7/9 5/10 6/10 Harderian gland 0/10 5/8 3/9 5/8 Conjunctiva 0/10 7/8 7/8 6/9 Cornea* 0/10 7/7 5/9 6/7 Eustachian tube 0/10 6/9 4/10 6/9 Dorsal buccal glands 0/9 7/8 2/6 4/5 Sublingual glands 0/9 6/6 4/7 5/6 Lingual glands 0/10 4/6 3/5 5/6 Buccal mucosa 0/10 8/9 6/9 5/8 Larynx 0/8 3/6 3/7 4/6 Trachea 0/7 0/9 0/5 0/7 Bronchi 0/8 0/10 0/7 0/9 External nasal gland 0/3 3/5 2/7 1/4 Nasal recess 0/10 2/6 4/8 2/6 Respiratory nasal mucosa 0/10 6/7 5/10 6/10 Olfactory nasal mucosa 0/10 0/6 1/6 1/10 Esophagus 0/4 0/8 0/4 0/5 Pancreatic ducts 0/5 0/7 0/5 0/8' Urinary bladder 0/3 2/7 0/6 2/5 Accessory urinary bladders 0/6 1/5 0/4 1/5 B. Lot 2 Group IV V VI VII Epithelia Lacrimal gland 4/9 7/8 6/10 0/8 Harderian gland 3/5 4/6 - 0/4 Conjunctiva 10/10 9/10 10/10 0/10 Cornea* 3/7 7/8 7/9 0/10 Eustachian tube 8/9 9/10 9/10 0/10 .Dorsal buccal glands 4/5 4/4 7/7 0/1 Sublingual glands~ 4/5 1/1 5/6 - Lingual glands 6/8 6/7 7/8 0/4 Buccal mucosa 10/10 9/10 8/10 0/10 Table 15 (CC Group Lerynx Trachea Bronchi External na. Hasal reces Respiratory Olfactory n Esophagus Pancreatic Urinary bla Accessory 'u \ * H5 Hr Ft Table 15 (cont'd.) 40 . Lot 2 Group IV V VI VII Larynx 6/6 5/6 9/9 0/7 Trachea 0/8 0/5 0/9 0/8 Bronchi_ 0/8 1/10** 0/8 0/9 External nasal gland 0/2 4/4 2/4 0/1 Nasal recess 2/2 2/2 - 0/4 Respiratory nasal mucosa 8/8 8/9 7/8 0/9 Olfactory nasal mucosa 0/6 4/4 0/1 0/3 ESOphagus 0/6 0/5 0/5 0/5 Pancreatic ducts 0/4 0/5 0/8 0/3 Urinary bladder 1/9 2/9 1/7 0/5 Accessory urinary bladders 2/4 1/2 0/6 0/5 * Hyperkeratosis. as Focal, associated with focal pneumonia. Iable l6. ‘. Lesion Renal mine Swollen pa Urolithia 41 Table 16. Incidence of lesions other than squamous metaplasia in Lot 1 and Lot 2 turtles Lot 1 Lot 2 Lesion Group 0 I II III IV V VI VII Renal mineralization 0/1 5/5 7/7 6/6 7/9 8/9 4/10 0/3 Swollen palpebra 0/10 8/10 7/10 7/10 7/10 7/10 8/10 0/10 Urolithiasis 0/10 3/10 3/10 3/10 0/10 4/10 0/10 0/10 u emanation are V chemise. No lesions trachea, pantree cranium, femur , The kidney disease had de proximal tubul proximal, dist and cellectin; glm‘nuli wer Proximal tion, but wit The bodies w and Stained . Clear halo 3 Cells c pyknotic um fragmented material Si clumps of n netaplasia some 1 ing Squaw)1 42 examination are valid for only Groups I through VI unless indicated otherwise. NO lesions were observed in any Of the following tissues: esophagus, trachea, pancreatic ducts, Optic nerve, retina, brain, peripheral nerves, cranium, femur, thyroid, neck retractor muscles and stomach. Kidneys The kidneys from turtles that had the previously described clinical disease had degenerative changes characterized by cloudy swelling of proximal tubule cells (Figure 3) and atrOphy.and mineralization of proximal, distal and collecting tubule cells (Figure 4). Some distal and collecting tubule epithelia had squamous metaplasia (Figure 4). The glomeruli were normal. Proximal tubule cells in many kidneys with little or no mineraliza- tion, but with cloudy swelling, had distinct intracytoplasmic bodies. The bodies were small, round, homogeneous to concentrically laminated and stained magenta. These bodies were frequently surrounded by a clear halo and were solitary or multiple per cell (Figure 3). Cells of tubules undergoing mineralization had deeply stained walls, pyknotic nuclei and lightly stained cyt0p1asm. Many similar cells were fragmented and collapsed. Others formed rings of amorphous, clumped material similar to that described by VanLeersum in rats (1928). Solid clumps of mineralized material (calculi) filled many tubules. Squamous metaplasia of tubule epithelium was seen in a few turtles in these areas. Some renal pelvises and their respective ureters had extreme keratiniz- ' ing squamous metaplasia which was always associated with presence of mineralized material in the lumen, with or without inflammatory cells from an ascending inflammation. , 'f “V" ”Nor;4\oobd.ro 1. I4. gr L. II Ill *3” Figure 3. Cloudy swelling of the epithelium in the proximal convoluted tubule with unidentified single and multiple cytOplasmic inclusions. H & E stain; x 560. Figure-4. Calcification and squamous metaplasia of renal tubules in the absence of inflammation. H & E stain; x 140. Hang seve ritualized tI scne affected interstitial Specifically had moderate PWXinal tu‘t tissue. Om tration of moderate mi observatior r I W Each one Cilcol a side am morn. Home mo: Ihree 0t} are Show] . Mic aCCe380r 6) and a metaplas Pseude me 44 Many severely affected kidneys, those with large numbers of mineralized tubules, had no evidence of inflammation (Figure 4). In some affected kidneys there were a few eosinOphilic granulocytes in interstitial and subcapsular areas, but they did not appear to be specifically associated with affected tubules. Other affected kidneys had moderate numbers of eosinophilic granulocytes in the base of proximal tubule cells and/or within the interstitial and subcapsular tissue. One clinically normal turtle (in Group VI A) had heavy infil- tration of eosinophilic granulocytes throughout the kidney as well as moderate mineralization of proximal tubules. Kidney lesions preceded observation of squamous metaplasia in the lower urinary tract. Urinary Bladder and Accessory UrinarygBladders Each of 7 turtles had a large calculus in the urinary bladder. One calculus was yellow and triangular in shape and measured 5 mm on a side and was 2 mm.thick. Its surface was finely granular, almost smooth.‘ The other 6 calculi were yellow, rough and 2 to 4 mm in diameter. Three more turtles had small, green-yellow grit in the urinary bladder. Three Others had similar grit in the ureters. Representative calculi are shown in Figure 5. '. Microscopically about one-fourth of the urinary bladders and/or accessory urinary bladders had keratinizing squamous metaplasia (Figure 6) and about 50% of these were accompanied by calculi. Grossly the metaplastic epithelia appeared as thick, soft, yellow mucosal pseudomembranes. The bladder walls were never inflamed regardless of the degree of squamous metaplasia. However, there were mixed inflammatory cell types and occasionally bacteria among the keratinized and mineralized debris 45 z IIIIIIIIIIIIIII ‘éflv‘ Figure 5. Calculi from the urinary bladder. ..-:'.T‘:‘..\J - - O . ’, ‘.‘ ' ”I . _ ‘ ‘ . . ..O Q a ' ’ _ ' .‘ - . . "~) ‘ I ‘I 0' o'- .- o ' , .- V s.._’ I ' . : l . {fi' . , . . I . ‘ 0 . ' ~ ~ .1. .‘ l . x. , Y ._ | \ a . ‘ . . \ e) ‘ i“ f u I; / Figure 6. Squamous metaplasia of the urinary bladder. ‘mucosa. The lumen is filled with keratinized debris and masses of desquamated, degenerating epithelial cells. H & E stain; x 55. found in t had simila Focal was freque More Of squamo Plaques. the lazy] along th. andIniXe Th nbmfll fOCal C Pneumol tended epithe B: . Higfii game: buccé Of 8. Stru 1538‘ 46 found in the lumens of the bladders (Figure 6). Ureters and cloacas had similar concomitant lesions. Focal to diffuse squamous metaplasia of cloacal and penile mucosas was frequent, with or without the presence of bladder lesions. Larynx More than 50% of the laryngeal mucosas examined had varying degrees of squamous metaplasia (Figure 7) which grossly appeared as yellow plaques. The lesions never extended beyond the anterior portion of the laryngeal tube and were most severe on the pharyngeal surface and along the fissure of the epiglottis. Occasionally fibrinous exudate and mdxed types of inflammatory cells covered the affected mucosa. Trachea and Bronchi The mucosa of these 2 structures had adequate mucus secretion and normal columnar to cuboidal cells with abundant cilia. There was no focal or diffuse squamous metaplasia except in l turtle which had focal pneumonia. Several bronchi and bronchioles in this turtle were dis- tended with inflammatory cells and lined by a thin stratified squamous epithelium; other portions of the lung were normal. Buccal Cavity With few exceptions the turtles had squamous metaplasia of the general buccal mucosa, lingual glands, sublingua1,glands and dorsal buccal glands. The ducts of the glands were affected first. Many glands of severely affected turtles had squamous metaplasia of the entire structure without inflammation. Atrophy of the acini soon followed the. resulting accumulation of secretion, cellular debris and/or inflammatory 47 Figure 7. Cross section of the-larynx-in which squamous metaplasia of the pharyngeal (a) and laryngeal (b) membranes is evident. H & E stain; x 55. 48 cells. Unless the glands were ruptured, no inflammation of the gland wall of its surrounding tissue was seen. Tympanic Cavity Over two-thirds of the turtles had keratinizing squamous metaplasia or hyperkeratosis of the tympanic cavity mucosa. Mildly affected turtles had focal undermining of mucus epithelial cells-by squamous cells. MOre severely affected turtles had thick layers of keratinized material filling the tympanic cavity (Figure 8). Nasal Cavity The nasal vestibulum'was hyperkeratotic in dyspneic turtles. MOst turtles had varied degrees of keratinizing squamous metaplasia of the external nasal glands, ventral and lateral nasal cavity (respiratory and non-Olfactory sensory epithelia) and/or internal choanal mucosa. The dorsal epithelium of the nasal cavity (olfactory sensory epithelium) and Bowman's glands were unaffected. Turtles with bulging snouts had external nasal glands that were distended with layers of keratinized material. Less severely affected glands had squamous metaplasia only in the ducts. In the nasal cavity proper the first structures to have squamous metaplasia were ridges and protruding structures.‘ There was little if any inflammation of the epithelia. Some cavities and acini of the external nasal gland contained mixed types of inflammatory cells in dabris and/or serofibrinous exudate. Ocular Area The swollen palpebra seen grossly were due to distended lymphatics without interstitial edema (Figure 9). 49 cross section of the tympanic cavity H & E stain; x 55. Figure 8. _(A) in which the lining (a) has undergone keratinizing squamous metaplasia and has been detached by lymphatic distention. 50 Figure 9. Cross section through the eye. Notice the lymphatic distention of the palpebrum (p), hyper- keratosis of the cornea (c) and keratinizing squamous metaplasia of the nictitating membrane (n) and palpebral conjunctiva. H & E stain; x 55. 01 01 OI II I] U... 51 With few exceptions all turtles had squamous metaplasia of the conjunctiva and nictitating membrane mucosa (Figure 9). Over two-thirds of the turtles had hyperkeratosis of the cornea (Figure 9) without evidence of primary inflammation or edema. Mere than 50% Of the turtles had some degree of squamous metaplasia of lacrimal and/or harderian glands. The lesions started in the ducts of the glands and the deep acinar epithelia remained normal, atrophied or underwent squamous metaplasia. Secretions, sometimes mixed with eosinophilic granulocytes, tended to accumulate in the acini. Severely. affected glands had large cavities lined with stratified squamous epi- thelium.and packed with layers of keratinized material. No inflamma- tory cells were seen within the gland walls or interstitially unless glands had ruptured, in which case a granulomatous inflammation with eosinophilic granulocytes was seen. Lungs Seven of 60 turtles (3 in Lot 1 and 4 in Lot 2) had pneumonia. One had interstitial pneumonia with infiltration of eosinophilic granulo- cytes.‘ The others had bronchOpneumonias with eosinophilic granulocytes and serofibrinous exudate. Bronchial epithelium was normal except in 1 turtle that had focal squamous metaplasia secondary to inflammation. Only 1 turtle had a buoyancy difficulty clinically. The others were lethargic and stayed out of the water. Two turtles had a large, encapsulated granuloma with a caseous center, on the anterior pleura. No etiologic agent was seen. Miscellaneous Observations Three turtles in Lot 1 had trematode ova in 1 or more of these tissues: lung, nasal respiratory submucosa, pancreas, spleen, liver, wall of th walls. TI sorroundi Sew accumule other p unknown A} due to Varyir. apparI 0f eo Unkno in t1. lag at; 01" reg; 52 wall of the urinary bladder, esophageal wall, intestinal and gastric walls. The only host reaction to the ova was 1 layer of macrOphages surrounding each ovum. Several pancreases had slight periductular and/or interstitial accumulations of eosinOphilic granulocytes for unknown reasons. Several other pancreases had focal vacuolar degeneration of exocrine cells, for unknown reasons. About 10% of the livers had diffuse fatty degeneration, probably due to fat mobilization secondary to anorexia. All livers contained varying amounts of melanin-like pigment in the Kupffer cells (this apparently is normal in turtles). A few livers had slight infiltration of eosinOphilic granulocytes in sinusoidal and periductular areas, for unknown reasons. Anorectic turtles had serous atrOphy of fat bodies. Fat bodies in the anterior leg pits were affected before those in the posterior leg pits. Femurs of all turtles were normal. The cartilaginous mass (cone) at the ends of the medullary cavity (Figure 10) tended to be smaller or totally reabsorbed in the_proximal end in turtles that lived longest, regardless of their treatment group. 53 Figure 10. Partial resorption of the cartilaginous cone in the medullary cavity of the femur indicating normal bone development. H & E stain; x 55. DISCUSSION When the clinical signs and lesions in Lot 1 turtles appeared to be equivalent in all dietary treatment groups, a second experiment, with Lot 2 turtles, was conducted. The form of vitamin A for Lot 2 diets was oil-based all-trans retinol to insure a more diffuse and even distribution in the food mixture than was possible with the gelatin-coated retinyl palmitate used for Lot 1 diets. Half of the turtles in each Lot 2 treatment group were supplemented with zinc in the diet to rule out the possibility of parakeratosis of zinc deficiency due to high dietary calcium. The zinc supplement had no influence on the disease. Although direct comparison Of Lot 1 and Lot 2 results is not valid because of seasonal differences and differences in ages of the turtles, the disease syndrome was equivalent in both lots of turtles. Lot 1 had earlier onset of the disease (by 2 months), perhaps due to overwintering utilization of body stores and/or to being on experiment during the season when rapid growth commences in nature. The disease syndrome observed in these turtles is consistent with vitamin A deficiency in turtles as described by Elkan and Zwart (1967). The lesions are also consistent with those in vitamin A-deficient birds and mammals as summarized by Meore,(l957), Sebrell and Harris (1967) and Follis (1958). The corneal hyperkeratosis and squamous metaplasia of epithelia in the.urinary tract, the oral, nasal and tympanic cavities and the ocular structures are typical of vitamin A-deficient animals. The distention of palpebral and periorbital lymphatics might be a 54 55 manifestation of equalization of pressure in the cephalic area. Since the turtles have extensive bidirectional vascular anastomoses, any increase in cerebrospinal fluid pressure might be diverted to the less confined structures, the palpebra. This might also account for the absence of clinically apparent blindness, ataxia or other central nervous system disturbance associated with increased cerebrospinal fluid pressure. Changes in the lacrimal and harderian glands did not contribute greatly to the palpebral swelling but did contribute to exophthalmos. Lymphatic and venous-sinus distention in the periorbital areas and accumulation of keratinized material in the conjunctival sac also contributed to exophthalmos. The role of the thyroid appeared insignificant. In contrast to Elkan and Zwart (1967) there were no inflammatory, hyperplastic or degenerative lesions seen in any of the thyroids examined micro- scOpically (6/30 in Lot 1 and 13/30 in Lot 2). No gross abnormalities of the thyroid were seen in any of the turtles. Elkan and Zwart (1967) did not mention calculi in the urinary tract of their turtles or mineralization of kidney tubules. These lesions were frequent in the turtles in this study. Similar lesions were reported in.birds (Elvehjem and Neu, 1932) and mammals (Beaver, 1961; VanLeersum, 1928) that were deficient in vitamin A. In contrast to Elkan and Zwart (1967) the turtles in this study had very little squamous metaplasia within the kidney, glomeruli were normal-and there was no evidence of chronic interstitial nephritis. Some turtles had a few eosinOphilic granulocytes (EG) at the base of renal tubule cells, a lesion also reported by Elkan and Zwart.(l967). In addition the turtles had kidneys with extensive degeneration of 56 proximal tubule cells characterized by cloudy swelling and the presence of homogeneous to laminated intracyt0plasmic bodies. The lower tubules .were either normal or had become severely mineralized. Few intermediate stages were seen. Squamous metaplasia was only observed in a few kidneys. These had extensive mineralization of tubules and intratubular calculi. Elkan and Zwart (1967) did not mention findings in the nasal and oral cavities, trachea and bronchi or esophagus. The turtles in this study had extensive squamous metaplasia of nasal and oral epithelia but the other 3 tissues were normal. In contrast to Elkan and Zwart these turtles did not have squamous metaplasia of pancreatic and bile ducts nor extensive perivascular infiltration of the liver by EC. However, the livers had fatty degeneration, probably due to fat mobili- zation accompanied by anorexia. Compared to Elkan and Zwart, infiltra— tion of EC in areas of squamous metaplasia was less frequent. The EC were either present for no apparent reason, because of focal infection or because keratinized material entered subepithelial tissues. The turtles frequently rubbed their swollen or dry eyes, which may have ruptured affected paraocular glands. The lesions seen in the turtles of this study.and the lesions reported by Elkan and Zwart (1967) appear similar but may not be entirely comparable. Their turtles came from many private sources with no standardization of age or diet. With 4 exceptions, the vitamin A liver levels in the 86 Pseudémys scripta elegans turtles examined in this study were low like those reported by Elkan and Zwart (1967) for 2 turtles (2.7 and 5.7 pg of vitamin A/gm of liver). One Group 0, one Group III and 2 wild turtles had the following respective fresh liver vitamin A values: 61, 34, 57 39 and 40 ug/gm. The rest of the turtles had from 0 to 20 ug/gm with most having below 10. In contrast Gillam (1937) found the following liver vitamin A values in 3 other species of reptiles: 2500 and 650 ug/gm in 2 giant monitor lizards (zoo specimens), 860 ug/gm in a 25- foot python (Zoo specimen) and 35 ug/gm in an alligator. He also found 10 ug/gm in.a frog's liver. The average liver vitamin A values in Groups 0 and,VII were high compared to their respective dietary treatment groups. It appears that turtles in Groups I through VI depleted their liver vitamin A stores regardless of the level of dietary vitamin A supplementation. Perhaps they were unable to ingest sufficient quantities of the diet and/or to absorb the-vitamin A. Any decrease in vitamin-A absorption was not due to insufficient bile since bile secretion was abundant even in turtles which were anorectic. Renal lesions might have contributed to loss of vitamin A by leakage through injured tubules. Perhaps young turtles are similar to chicks, which Jungherr (1943) found incapable of vitamin A storage until they attained an age of about 2 months. There is an alternate hypothesis to explain the observed lesions. Since the turtles were.on a relatively high calcium diet there might have been a persistent hypercalciuria. Secondly, since the-turtles were maintained in distilled water, ion depletion of the body could have occurred similar to that reported by Trobec and Stanley (1971). Ion depletion, especially of chloride, could produce renal tubule degeneration (Follis, 1958) and with hypercalciuric calcification of the tubules could occur thus initiating a vicious cycle of ion loss and tubule degeneration. A possible change from a normal isotonic or hypotonic urine to an abnormally hypertonic urine could have stimulated squamous metaplasia of the lower urinary tract epithelia. Calcified 58 tubule cells would slough and serve as nidi for calculus formation which in turn would be an irritatnt to epithelia. These changes would facilitate urine stasis and produce a favorable environment for bac- teria. Ascent of bacteria to the kidney could account for the infil- tration of eosinOphilic granulocytes into the renal tubule cells and interstitium. Since the cloaca and urinary bladders participate in ion conser- vation squamous metaplasia could result in_further ion depletion and acid-base imbalance. With a need for conservation of chloride, gastric hydrochloric acid secretion might be curtailed initiating anorexia. Similarly, with need for salt conservation the lacrimal gland might. curtail sodium chloride secretion. With concomitant osmotic action of distilled water on the eye and lack of hypertonic secretion from the lacrimal gland, the ocular structures would undergo squamous metaplasia and/or hyperkeratosis. Osmotic action of distilled water might also account for the squamous metaplasia of the nasa1,.oral and otic cavity membranes. Loss of pharyngeal ion conservation mechanisms might also contribute to ion depletion. This second hypothesis is supported by the fact that renal lesions. preceded lower urinary tract lesions.and that those structures exposed to osmotic action of distilled water were most severely affected. The second hypothesis to explain the disease syndrome of the turtles in this study appears most correct. If the disease was primary vitamin A deficiency changes would be expected in additional tissues such as the trachea, bronchi, pancreatic ducts and bones. No signifi- cant lesions were seen in.any of these tissues. Lack of esophageal lesions might be explained on anatomdc grounds. In contrast to mammals, the esophagus does not contain accessory tubuloacinar glands and the 59 epithelium resembles that of the trachea. It has ciliated, pseudo- stratified columnar epithelium that contains abundant periodic acid- Schiff positive cytOplasmic granules. Perhaps the physiological needs of vitamin A by the eSOphageal, tracheal and bronchial epithelium were minimal compared to other epithelia exPeriencing osmotic action of distilled water and thus would be the last to undergo squamous meta- plasia. The lack of bone lesions might be due to the slow rate of growth in turtles and the seasonal and individual variation. For example, after 8 months 1 turtle in Group VI gained 3% of its original length whereas another turtle in the same group gained 20%. A Group III turtle gained 24% of its original length in 3 months. It appears that osmotic action of distilled water on some membranes resulted in lesions that mimicked and/or led to vitamin A deficiency. SUMMARY Forty red-eared slider turtles, Pseudemys scripta elegans, in each of 2 lots were used in an attempt to determine the vitamin A requirement for growth and maintenance of health of captive turtles. Thirty turtles E—:? in each lot were.fed diets composed of ground hog heart supplemented . with corn oil, glucose, minerals and vitamins. The remaining 10 turtles in each lot were killed on Day 0 of their respective experiment to £5; serve as controls for normal histology, gross anatomy and initial liver vitamin A stores. Three equal groups of turtles in each lot were kept in separate tanks of distilled water and provided with a wood float, a reflectorized lamp and an air—driven water filter containing activated charcoal and nylon fiber. The turtles were fed twice a day for 30 minutes-at each feeding. Lot 1 turtles were fed similar diets containing either no vitamin A supplement or 140 or 280 IU of vitamin A/kg of fresh hog heart. Lot 2 turtles were fed similar diets containing either no vitamin A supplement or 900 or 1800 IU of vitamin A/kg of fresh hog heart. All turtles fed the diets had similar signs and lesions character- ized by swollen eyelids, dyspnea, anorexia, lethargy, weight loss and keratinizing squamous metaplasia of nasal, oral, tympanic, ocular and urinary tract membranes. Urinary calculi-occurred frequently. Renal lesions were primarily degeneration of proximal tubule cells and minerali- zation of distal tubules and collecting ducts. In contrast the trachea, bronchi, esOphagus, pancreatic ducts, bones and nervous tissue were normal. 60 61 Liver vitamin A values at necr0psy in Lot 1 and Lot 2 standard groups were 10.45 and 8.74 pg of vitamin A/gm of fresh liver, respectively. Liver vitamin A values in the Lot 1 and Lot 2 groups fed diets without vitamin A supplementation were 2.9l-and 1.92 IU/gm, respectively. Liver values were 1.76 and 9.10 IU/gm for Lot 1 groups fed the diets supple- mented with 140 and 280 IU/kg, respectively. Liver values were 3.40 and 2.48 IU/gm for Lot 2 groups fed the diets supplemented with 900 and 1800 IU/kg, respectively. rr‘? It appeared that depletion of body ions and osmotic action of distilled water on some membranes initiated lesions that mimicked and/or led to vitamin A deficiency. BIBLIOGRAPHY BIBLIOGRAPHY Anon.: Prohibition on importation of pet turtles. J.A.V.M.A., 160, (1972): 1482-1483. Barnicot, N. A., and Datta, S. P.: Vitamin A and bone. In The Bio- chemistry and Physiology of’Bone. 'G. H. Bourne (ed.), Academic Press, New York, 1956: 507-538. Beaver, D. L.: Vitamin A deficiency in the germ-free rat. Am. J.‘ Path., 38, (1961): 335-357. Bernhard, K., Ritzel, G., and Steiner, K. U.: Helv. Chim Acta, 37, f (1954): 306. Cited in Vitamin A, by Moore, 1957, p. 194. Bruner, H. L.: The cephalic veins and sinuses of reptiles. Am. J. Anat., 7, (1907): 1-117. 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S.: Biology of’the Reptilia, Vol. 3. Mbrphology C. Academic Press, New York, 1970b. Gillam, A. E.: The vitamin A and A2 contents of mammalian and other animal livers. Biochem. J., 32, (1938): 1496-1500. Goodman, DeW. 8.: Retinol transport in human plasma. In The Fat- Sbluble Vitamins. H. F. DeLuca and J. W. Suttie (ed.), The Univ. of Wisconsin Press, Madison, 1970: 203-212. 64 Graham, T. E.: Growth rate of the red-bellied turtle, Chrysemys rubriventris, at Plymouth, Massachusetts. Copeia, (1971): 353-356. ‘ Hayes, K. C.: 0n the pathOphysiology of vitamin A deficiency. Nutri- tion Review, 29(1), (1971): 3-6. Heisey, S. R.: Cerebrospinal and extracellular fluid spaces in turtle brain. Am. J. Physiol., 219, (1970): 1564-1567. Johnson, R. M., and Bauman, C. A.: Relative significance of growth and metabolic rate upon the utilization of vitamin A by the rat. J. Nutr., 35, (1948): 703-715. Jones, L. M.: Fat-soluble vitamins. Ianeterinary Pharmacology and Therapeutics, 3rd ed., L. M. Jones (ed.), Iowa State Univ. Press, Ames, Iowa, 1965: 872-902. Jungherr, E.: Nasal hist0pathology and liver storage in subtotal vitamin A deficiency of chickens. Bull. No. 250. Storrs Agricultural Experiment Station (Conn.), 1943. King, F. W.: Housing, sanitation, and nutrition of reptiles. J.A.V.M.A., 159(11), (1971): 1612-1615. Lagler, K. F., and Applegate, V. C.: Relationship betweeen.the length and weight in the snapping turtle Chelydma serpentina Linnaeus. Am. Nat., 77, (1943): 476-478. Marcus, L. C.: Infectious disease of reptiles. J.A.V.M.A., 159(11), (1971): 1626-1631. McCollum, E. V., and Davis, M.: The necessity of certain lipins in the diet during growth. J. Biol. Chem., 15, (1913): 167-175. McCotter, R. E.: The vomero—nasal apparatus in Chrysemys punctata and Rana catesbiana. Anat. Rec., 13, (1917); 51—67. Moore, T.: Vitamin A. Elsevier Publishing Co., New York, 1957. National Academy of Sciences: Nutrient requirements of poultry, 6th revised ed. National Research Council, Washington, D.C., 1971. Parnell, J. P., and Sherman, B. 3.: Effect of vitamin A on keratiniza- tion in the A-deficient rat. In Fundamentals of'Keratinization. E. 0. Butcher and R. F. Sognnaes (ed.), Publication No. 70. Am. Assn. for the Advancement of Science, Washington, D.C., 1962: 113. Phillips, W. E. J.:' Low-temperature environmental stress and the metabolism of vitamin A in the rat. Can. J. Biochem. Physiol., 40, (1962): 491-499. Roels, 0. A.: Biochemical systems. D. Effect on membranes. In The Vitamins, Vol. 1. W. H. Sebrell, Jr., and R. S. Harris (ed.), Academic Press, New York, 1967: 193-209. 65 Rosen, S.: The turtle bladder. I. MOrphological studies under vary- ing conditions of fixation. Exp. Molec. Path., 12, (1970): 286-296. Sebrell, W. H., and Harris, R. S.: The Vitamins, Vol. 1.‘ Academic Press, New York, 1967. Smith, L. H., Jr., and Williams, H. E.: Kidney stones. In Diseases of' the Kidney, 2nd ed., Vol. II. M. B. Strauss and L. G. Welt (ed.), Little, Brown and Co., Boston, 1971: 973-996. Steinmetz, P. R.: Characteristics of hydrogen ion transport in urinary bladder of water turtle. J. Clin. Invest., 46(10), (1967): 1531-1540. Steinmetz, P. R., Omachi, R. S., and Frazier, H. S.:' Independence of hydrogen ion secretion and transport of other electrolytes in turtle bladder. J. Clin. Invest., 46(10), (1967): 1541-1548. Sundaresan, P. R., Winters, V. G., and Therriault, D. C.: Effect of low environmental temperature on the metabolism of vitamin A (retinol) in the rat. J. Nutr., 92, (1967): 474-478. Suzuki, H. K.:. Studies on the osseous system of the slider turtle. Ann. N.Y. Acad. Sci., 109, (1963): 351-410. Thompson, J. N.: The role of vitamin A in reproduction. In The Fat- Sbluble Vitamins. H. F. DeLuca and J. W. Suttie (ed.), The Univ. of Wisconsin Press, Madison, 1970: 267-281. Trobec, T. N., and Stanley, J. G.: Uptake of ions and water by thev painted turtle, Chrysemys picta. COpeia, (1971): 537-542. VanLeersum, E. C.: Vitamin A deficiency and calcification of the epi- thelium of the kidney. J. Biol. Chem., 79, (1928): 461-464. Wallach, J. D.:' Medical care of reptiles. J.A.V.M.A., 155(7), (1971a): 1017-1034. Wallach, J. D.: Environmental and nutritional diseases of captive reptiles. ‘J.A.V.M.A., 159(11), (1971b): 1632-1643. Wolf, G., and DeLuca, L.: Recent studies on some metabolic functions of vitamin A. In The Fat-Soluble Vitamins. H. F. DeLuca and J. W. Suttie (ed.), The Univ. of Wisconsin Press, Madison,. 1970: 257-265. APPENDICES i APPENDIX A BODY MEASUREMENTS OF EACH TURTLE AT DAY 0 AND DAY 76 (LOT 1) OR DAY 191 (LOT 2) 66 Table A-1. Body measurements of each turtle at Day 0 and Day 76 (Lot 1) or Day 191 (Lot 2) Plastron Carapace Day of Length Length Turtle Necropsy Body weight, gm. 11128 inch 1/128 inch Lot Group No. (Day-N) Day 0 Day 76 'Day 0 Day 76 Day 0 Day 76 1 0 3-82 0 9.56 ,- 175 - 180 - 1 0 3-83 0 9.10 - 164 - 174 - l 0 3-84 0 8.93 - 166 - 175 - 1 0 3-85 0 8.85 - 172 - 182 - l 0 3-86 0 7.53 - 166 - 167 - l 0 3-87. 0 7.50 - 163 - 176 - l 0 3-14 0 7.02 - 162 - 172 - 1 0 3-15 0 6.95 - 153 - 162 - l 0 3-16 0 6.75 - 155 - 165 - 1 0 3-17 0 5.63 - 152 - 168 - 1 I 3-52 105 9.69. 9.92- 172 177 180 185 l I 3-53 38 8.84 - 173 - 186 - l . I 3-54 130 8.68 7.46 169 169 179 179 1 I 3-55 93 8.44 8.06 170 173 '175 179 1 I 3-56 72 8.04 - 165 - 179 - l I 3-57 130 7.66 7.23 161 162 168 168 1 1 3-58 ' 73 7.32 - 156 - 167 - 1 I 3-59 84 6.92 5.19 150 150 163 159 1 I 3-60 114 6.67 9.31 156 170 173 190 1 I 3-61 28 5.78 - 149 - 153 - 1 11 3-62 130 9.83 12.24 164 178 181 197 1 11 3-63 119 9.11 11.21 173 184 180 198 1 11 3-64 104 8.69 10.65 169 178 177 189 1 11 3-65 119 8.29. 8.54~ 160 161 176 177 1 II 3-66 186 8.26 10.70 167. 175 174 187 1 11 3-67 99 7.59 6.63 165 165 173 171 1 II 3-68 105 7.07 5.99 161 160 173 169 1 11 3-69 182 6.83 6.95 155‘ 156 164 163 1 11 3-70 104 6.43- 6.50 154 161 163 169 1 11 3-71 39 5.22 - 142 - 149 - Table A-1 (cont'd.) 67 Plastron Carapace Day of Length Length Turtle NecrOpsy Body weight, gm 11128 inch 1/128 inch Lot Group No. (Day-N) Day 0 Day 76 Day 0 Day 76 Day 0 Day 76 1 111 3-72 105 9.30 13.30 172 188 184 205 1 III 3-73 114 9.24 18.71 178 211 183 228 1 III 3-74 86 8.66 9.41 161 168 176 182 1 III 3-75 104 8.27 10.25 166 175 172 183 1 III 3-76 130 8.23 14.89. 163 187 175 209 1 III 3-77 104 8.00 11.12- 166 184 173 192 1 III 3-78 103 7.02 6.38 158 158 166 165 1 III 3-79 18 6.70 - 154 - 163 - 1 III 3-80 31 6.38' - 156 - 162 - 1 111 3-81 25 5.89 Da- 191 155 Da- 191 162 Da- 191 2 IV A 5-34 245 9.96 10.18 178 183 182 186 2 IV B 5-35 217 9.40 9.66 166 169 186 191 2 IV A 5-36 163 9.01 - 172 - 178 - 2 IV B 5-37 245 8.94 8.37 168 169 183 183 2 IV A 5-38 245 8.70 9.00 164 169 177 182' 2 IV B 5-39 152 8.29 - 154 - 166 - 2 IV B 5-40 141 8.25 - 166 - 180 - 2 IV A 5-41 87 8.15 - 159 - 174 - 2 IV B 5-42 221 8.02 7.59 154 154 172 173 2 IV A 5-43 217 6.91 7.25 152 157 168 177 2, V A. 5-44 206 10.37 12.20 169 189 186 209 2 V B 5-45 60 9.80. - 165 - 179 - 2 V A 5-46 245 9.10 8.62 174 175 186 189 2 v B 5-47 181 a.s4 - 164 - 176 - 2 V B 5-48 197 8.60 8.50 167 175 175 183 2 V A 5-49 223 8.47 10.10 162 170 172 182 2 V A 5-50 152. 8.19, - 165 - 176 - 2 V B 5-51 201 8.09 14.62 156 215 169 222 2 V A 5-52 209 7.99 8.86 160 169 176 188 2 V B 5-53 209- 7.73 7.40 162 166 171 177 Table A-1 (cont'd.) 68 Plastron Carapace Day of Length Length Turtle Necr0psy Body weight, g§_ 11128 inch 1/128 inch Lot Group No. (Day-N) Day 0 Day 76 Day 0 Day 76 Day 0 Day 76 Day 191 Day 191 Day 191 2 VI B 5-54 245 9.95 14.18. 170 187 187 216 2 VI A 5-55 246 9.68 11.10 173 181 190 202 2 VI B 5-56 212 9.13 9.48 163 165 173 178 2 VI*A 5-57 246 8.96 13.63 164 184 183 208 2 VI'A 5-58 216 8.51 13.05 161 188 175 205 2 VI B 5-59. 245 8.42 9.75 159 170 173 186 2 VI A 5-60 216 8.19 8.37- 160 164 172 179 2 VI B 5-61 216 8.15 9.71 160 169 172 186 2 VI'B 5-62 245 7.99 13.40 159 184 169 202 2 VI A 5-63 246 7.66 8.31 159 164 171 179 2 VII 5-64 ' o 7.52 - 152 - 162 - 2- VII 5-65 0 8.80 - 171 - 189 - 2 VII 5-66 0 8.26 - 161 - 171 - 2 VII 5-67 0 8.15, - 157 - 178 - 2 - VII 5-68 0 9.89_ - 165 - 179 - 2 VII 5-69 0 9.39 - 173 - 183 - 2 VII 5-70 0 8.90 - 151 - 170 - 2 VII 5-71 0 9.04 - 167 - 179 - 2 VII 5-72. 0 8.40 - 170 - 180 - 2 v11 5-73 0 8.021 - 149 - 162 - APPENDIX B BODY MEASUREMENTS AND LIVER VITAMIN A VALUES OF EACH TURTLE AT DAY-N (DAY OF NECROPSY)' Table B-1. 69 Body measurements and liver vitamin A values of each turtle at Day-N (day of necropsy) Plas- Cara- Plas- Cara- 2 Kid- ug Vit. tron pace. Body tron pace Heart ney Liver A/gm Turtle Day- Length Length Wt. Wt. Wt. Wt. Wt. Wt. Fresh Group No. N 1/128" 1/128" gm gm gm gm gm gm Liver 0 3-82 0 175 180 9.088 0.816 1.667 0.025 0.046 0.579 10.042 0 3-83 0 164 174 8.631 0.685 1.462 0.020 0.041 0.400 8.212 0 3-84 0 166 175 8.249 0.783 1.688 0.027 0.032 0.294 2.304 0 3-85 0 172 182 8.663 0.645 1.566 0.029 0.026 0.479 2.698 0 3-86 0 166 167 7.491 0.692 1.267 0.021 0.024 0.276 9.053 0 3-87 0 163 176 6.882 0.678 1.082 0.024 0.030 0.214 61.454 0 3-14 0 162 172 7.016 0.495 0.985 - - 0.194 7.653 0 3-15 0 153 162 6.875 0.525 1.030 0.021 0.020 0.236 1.773 0 3-16 0 155 165 6.758 0.386 0.781 0.018 0.024 0.122 0.356 0 3-17 0 152 168 5.876 0.468 0.931 0.025 0.028 0.207 0.926 I 3-52 105 177 184 9.048 0.685 1.535 0.046 0.058 0.601 1.135 I 3-53 38 174 185 7.821 0.637 1.122 0.036 0.056 0.399 0.742 I 3-54 130 170 178 7.469 0.619 1.158 0.039 0.051 0.482 2.747 I 3-55 93 173 177 7.516 0.575 1.134 0.030 0.040 0.408 1.278 I 3-56 72 172 185 8.772 0.752 1.618 0.032 0.058 0.488 1.458 I 3-57 130 162; 167 7.284 0.587 1.080 0.026 0.034 0.688 0.438 I 3-58 73 156 164 6.105 0.414 0.864 0.022 0.043 0.191 3.768 I 3-59 84 151 159 5.268 0.441 0.811 0.024 0.034 0.191 2.424 I 3-60 114 170 189 7.805 0.723 1.475 0.039 0.059 0.378 10.100 1 3-61 28 144 152 5.130 0.349 0.689 0.019 0.029 0.120 5.000 11 3-62 130 175 193 11.156 0.740 2.050 0.044 0.046 0.924 1.782 11 3-63 119 184 200 9.660 0.770 1.695 0.041 0.090 0.692 0.755 II 3-64 104 178 189 10.208 0.866 1.702 0.040 0.035 0.560 0.422 11 3-65 119 161 176 7.936 0.626 1.145 0.035 0.034 0.482 1.569 II 3-66 186 174 188 9.170 0.652 1.327 0.037 0.063 0.500 2.714 II 3-67 99 164 171 7.171 0.477 0.937 0.030 0.047 0.407 2.203 11 3-68 105 161 170 16.325 0.476 0,919 0.027 0.057 0.324 1.234 II 3-69 182 156 163 6.110 0.455 0.758 0.024 0.031 0.402 2.976 II 3L70 104 159 168 6.019 0.439 0.994 0.030 0.034 0.227 2.624 II 3-71 39 144 144 4.429 0.260 0.572 0.021 0.054 0.144 1.334 70 Table B-1 (cont'd.) Plas-. Cara- Plas- Cara- 2 Kid- pg Vit° tron pace Body tron pace Heart ney Liver A/gm Turtle Day- Length Length Wt. Wt. Wt. Wt. Wt. Wt. Fresh Group No. N 1/128" 1/128" gm gm gm gm gm gm Liver III 3-72 105 189 207 11.964 0.949 2.128 0.064 0.079 0.792 33.999 III 3-73 114 213 231 16.568 1.433 3.002 0.075 0.152 1.229 0.430 III 3-74 86 162 182 9.177 0.740 1.652 0.057 0.046 0.578 1.714 III 3-75 104 175 184 9.009 0.720 1.467 0.044 0.070 0.684 0.968 III 3-76 130 190 212 13.266 1.087 2.764 0.053 0.088 1.005 1.282 111 3-77 104 183 192 9.707 0.842 1.819 0.048 0.060 0.646 3.040 III 3-78 103 157 165 6.530 0.537 0.933 0.025 0.080 0.366 1.052 III 3-79 18 153 160 5.845 0.427 0.846 0.016 0.031 0.126 11.905 III 3-80 31 154 159 4.329 0.388 0.660 0.015 0.028 0.139 19.608 III 3-81 25 152 160 4.719 0.440 0.684 0.018 0.051 0.189 16.970 IV A 5-34 245 183 187 9.985 0.802 1.779 0.043 0.041 0.623 2.686 IV B 5-35 217 169 190 9.332 0.711 1.576 0.041 0.041 0.450 0.000 IV A 5-36 163 172 178 9.368 0.745 1.563 0.035 0.040 0.715 2.632 IV B 5-37 245 169 182 8.064 0.541 1.283 0.044 0.037 0.436 1.882 IV A 5-38 245 169 184 9.060 0.697 1.525 0.037 0.038 0.618 3.004 IV B 5-39 152 159 171 7.278 0.601 1.181 0.030 0.032 0.325 2.778 IV B ,5-40 141 171 184 6.970 0.580 1.253 0.035 0.046 0.322 2.916 IV A 5-41 87 184 204 13.005 1.130 2.709 0.042 0.166 1.074 0.748 IV B 5-42 221 154 172 6.701 0.539 1.078 0.027 0.057 0.561 1.158 IV A 5-43 217 157 175 7.170 0.514 1.272 0.025 0.034 0.393 1.436 V A 5-44 206 189 207 14.981 1.081 2.716 0.090 0.102 1.047 1.101 V B 5-45 60 165 174 9.378 0.528 1.055 0.026 0.029 0.429 6.150 V A 5-46 245 176 188 8.138 0.641 1.460 0.032 0.033 0.346 4.348 V B 5-47 181 174 188 8.655 0.924 1.926 0.035 0.046 0.483 3.283 V B 5-48 197 173 182 8.218 0.705 1.621 0.037 0.038 0.475 2.778 V A 5-49 223 170 180 9.605 0.873 1.760 0.040 0.042 0.649 3.455 V A 5-50 152 187 200 9.223 0.875 2.055 0.053 0.049 0.644 5.172 V B 5-51 201 201 213 12.777 1.158 2.724 0.057 0.068 0.696 1.144 V A 5-52 209 169 186 7.448 0.574 1.510 0.032 0.023 0.334 3.166 V B 5-53 209 165 175 6.934 0.530 1.048 0.036 0.037 0.293 3.431 '7: .94 71 Table B-1 (cont'd.) Plas- Cara- Plas- Cara- 2 Kid- pg Vit. tron pace Body tron pace Heart ney Liver A/gm Turtle Day- Length Length Wt. Wt. Wt. Wt. Wt. Wt. Fresh Group No. N 1/128" 1/128" gm gm gm gm gm gm Liver VI B 5-54- 245 187 216 14.100 1.020 2.773 0.049 0.055 0.980 0.544 VI A 5-55 246 181 201 10.428 0.867 2.116 0.055 0.063 0.662 2.724 VI B 5-56 212 165 184 9.003 0.692 1.495 0.044 0.067 0.726 2.551 VI A 5-57 246 201 228 16.025 1.280 3.392 0.079 0.143 1.310 1.258 VI A 5-58 216 186 201 12.451 0.995 2.694 0.039 0.056 0.722 4.784 VI B 5-59 245 170 184 10.087 0.762 1.716 0.047 0.039 0.560 4.815 VI A 5-60 216 164 177 7.765 0.687 1.423 0.039 0.041 0.425 1.593 VI B 5-61 216 169 184 10.494 0.782 1.726 0.066 0.063 0.700 0.694 VI B 5-62’ 245 184 202 12.803 0.949 2.556 0.058 0.064 0.917 3.047 VI A 5-63 246 164 178 8.075 0.637 1.337 0.040 0.031 0.520 2.778 VII 5-64 0 152 162 7.377 0.530 1.194 0.025 0.015 0.227 8.974 VII 5-65 0 171 189 8.929 0.605 1.504 0.023 0.037 0.316 13.377 VII 5-66 0 161 171 8.252 0.619 1.349 0.024 0.027 0.381 7.870 VII 5-67 0 157 178 8.053 0.658 1.435 0.021 0.031 0.336 9.792 VII 5-68 0 165 179 9.904 0.722 1.712 0.026 0.032 0.360 7.532 VII 5-69 0 173 183 9.680 0.674 1.748 0.026 0.025 0.347 7.812 VII 5-70 0 151 170 8.640 0.693 1.562 0.030 0.025 0.389 4.012 VII 5-71 0 167 179 8.983 0.613 1.640 0.029 0.027 0.364 7.372 VII 5-72 0 170 180 8.182 0.665 1.523 0.021 0.020 0.278 7.083 VII 5-73 0 149 162 7.876 0.561 1.284 0.025 0.026 0.351 13.541 Adult‘ ‘ (Sex) F 6-19 10-4- 355 386 69.87 5.89014.636 0.212 0.186 2.059 14.30 71 F 6-20 10-4- 449 481 134.4613.26525.609 0.303 0.269 3.488 1.70 71 F 6-21 13-3- 463 489 142.4414.60930.788 0.337 0.361 3.054 39.94 1 F 6-22 13-3- 444 501 146.1012.66931.642 0.493 0.436 3.142 8.19 1 M 6-23 lgIB- 488 539 188.5513.60232.915 0.409 0.415 3.345 14.50 M 6-24 10-3- 519 554 184.6818.80241.942 0.431 0.494 4.582 38.67 71 APPENDIX C MINERAL CONTENT OF LOT 1, LOT 2, AND ADULT TURTLE SHELLS 72 Mineral content of Lot 1, Lot 2, and adult turtle shells 28:1 Shell Calcium Magnesium gghosphorus Turtle free Ash ppm of % of ppm of % of ppm of % of No . Wt . , gm gm ash ash ash ash ash ash 3-82‘ .6714 .3822 488.93 12.79 7.24 0.19 201.15 5.26 3-83 .6480 .1240 510.65 41.18 7.27 0.59 193.1 15.57 3-84 .7428 .1122 487.58 43.46 7.74- 0.69. 206.55~ 18.41 3-85 .8341 .1826 795.30 43.55 5.42 0.30 179.78 9.84 3-86 .5475 .0726 331.99 45.73. 4.33 0.60 144.50 19.90 3-87 .5519 .0879 389.65 44.33 6.49 0.74 167.86 19.10. 3-14 .4281 .0752? 296.59 39.44 4.33 0.58 144.98 19.28- 3-15, .4575 .0717 289.34 40.35 5.45 0.76 143.04 19.95 3-16 .3321 .0579 251.85 43.50 4.92 0.85 125.60 21.69 3-17 .3740 .0614 256.51 41.78 4.421 0.72. 123.66 20.14 3-52 .8341 .1826 407.87 22.34 7.97. 0.44 324.96 17.80 3-53 .6073 .1376 586.52 42.62 6.92- 0.50 212.44 15.44 3-54 .5991 .1384 614.88 44.43, 6.11 0.44 264.4 19.10 3-55 .5552 .0846 407.87 46.39 4.84 0.57 161.52 19.09 3-56 .8612 .1677 709.55 42.31 9.22 0.55 320.43 19.11- 3-57' .7181 .1872 845.61- 45.17 11.07- 0.59. 337.74 18.04 3-58 .3814 .0839 341.52 40.70 4.24 0.50 472.58 56.33 3-59 .4705 .1200 573.72 47.81 8.67 0.72 261.66 21.81~ 3-60 .8691 .1709 730.55 42.75 5.76 0.34 255.15 14.93 3-61 1.6189_ .3803 1729.12 45.47 22.99 0.60 633.91 16.69 3-62 1.1510 .2519 1074.44 42.65 14.30 0.57! 470.14 18.66 3-63 1.0429 .1958 910.9 46.52~ 8.73 0.44 354.76 18.12- 3-64 .9930 .2025 950.43 46.96 9.14 0.45 401.09 19.81 3-65 .7573 .2206 601.97 27.29 8.19 0.37 250.99 11.38, 3-66 .7093 .1920 849.05 44.22 12.96 0.68 301.44 15.70 3-67 .4957 .0944 423.72 44.88 4.42, 0:47 172.25 18.25 3-68 .4875 .1337 592.42 44.31 6.93 0.52 343.8 18.24 3-69 .4890 .1175 507.77 43.21 7.39 0.63 215.40 18.33, 3-70 .4969 .0820 348.70 42.52 4.24 0.52 161.79 19.73. 3-71 .2573 .0462 209.61 45.37 1.54 0.33 96.65 20.92 Table C-l (cont'd.) 73 2::1 Shell .Calcium Magnesium Phosphorus Turtle free Ash ppm of Z of ppm of Z of ppm of Z of No. Wt.,gm gm ash ash ash ash ash ash 3-72 1.1182 .2041 856.75 41.98 13.70 0.67 308.84 15.13 3-73 1.6798 .2840 1150.33- 40.50 17.76 0.62 478.71' 16.86 3-74 .8971 .1888 840.03 44.49 10.78 0.57 338.58 17.93 3-75 .9100 .1921 823.42_ 42.86 5.42 0.28 365.92 19.05 3-76 1.5180 .2866 1278.5 44.61 15.49. 0.54' 483.14 16.86 3—77 1.1433 .2423 983.52 40.59 12.10 0.50 407.75 18.28 3—78 .4270 .1031 404.99 39.28 5.65 0.55 158.70- 15.39 3-79 .4792_ .1292 599.40 46.39 9.14. 0.71 224.5 17.38 3-80 .3330 .0870 389.65 44.79 5.26 0.60 136.51‘ 15.69 3-81 .3271 .0610 250.17 41.01 4.96 0.81 126.56 20.75 5-34 1.1172 .2629 1342.66 51.07- 18.76 0.71 158.6 6.03 5-35 1.8426 .1978 854.86 43.22 10.55 0.53 297.03- 15.02 5-36 .9344 .1945 887.64 45.64 9.53 0.49 355.77 18.29 5-37 .6498 .0886 404.21 45.621 8.00 0.90. 178.11 20.10 5-38 .9935 .2235 999.90 44.744 12.26 0.55 373.55 16.71 5-39 .6423 .1201 331.99 27.64 - 4.33- 0.36 220.33 18.35 5-40 .7342 .1690 733.51 43.40 9.14~ 0.541. 309.87 18.34 5-41 1.4535 .2891 250.17- ' 8.65 18.02 0.62 538.12 18.61 5-42 .6855 .1507‘ 656.0 43.53 . 7.76 0.51 265.39 17.61 5-43 .6905 .1495 637.2 42.62 7.28 0.49 223.62 14.96 5-44 .3165 .0776 324.14 41.77 4.43 0.57 151.79 19.56 5-45 .4847 .0742' 307.58 41.45 4.741 0.64~. 144.01 19.41 5-46 .9160 .2186 988.12 45.20- 11.49 0.52 384.24 17.58 5-47 1.2418 .2698 1240.50 45.98 13.044 0.48 441.82 16.38 5-48 .9496 .2274 854.87 37.59 13.89 0.61 351.22_ 15.45 5-49 1.1306 .3200 1548.7 48.40 16.29 0.51‘ 603.89. 18.87 5-50 1.2134 .2232 1026.80 46.00 11.08 0.50 320.89 14.38 5-51 1.6763 .1954 823.42 42.14 13.60 0.70 296.54 15.18 5—52 .7980 .2040 899.39 44.09. 9.02 0.44 313.83 15.38 5-53 .5276 .1020 349.3 34.24 4.74 0.46 202.31 19.83 Table C-l (cont'd.) 74 ::Z: Shell Calcium Magnesium Phosphorus Turtle free Ash ppm of z of ppm of Z of ppm of Z of No. Wt.,gm gm ash ash ash ash ash ash 5-54 1.4547 .2609 1156.59 44.33 15.96 0.61~ 434.98 16.67 5-55 1.1603 .2006 886.62- 44.20 12.81 0.64 327.53. 16.33 5-56 .8484 .1283 503.70 39.26 7.36 0.57 199.77. 15.57 5-57 1.8389 .4302 1989.31 46.24 26.29 0.61' 748.27 17.39 5-58 1.4748 .3008' 1366.06 45.41 13.21 0.44 511.86 17402 5-59 1.0122 .2498 1113.50 44.58 12.23 0.49 454.60 18.20 5-60 .9810 .1632 718.34- 44.02 9.50 0.58 328.73 20.14- 5-61 .9513 .1789 755.08 42.21 11.65 0.65 334.04- 18.67 5-62 1.1883 .1634 697.08 42.66 8.74 0.53 290.61 17.79 5-63 ..8578 .2267 1038.19 45.80. 13.20 0.58 459.78 20.28 5-64 .5031 .0605 242.02 40.00 4.33 0.73 114.00 18.84 5-65 .5532 .0508 190.82_ 37.56 4.27 0.84 107.25 21.11 5-66 .5737 .0551 220.39 40.00. 4.62- 0.84. 118.82 21.57 5-67 .5347 .0642 276.57 45.07- 4.71 0.73. 135.74 21.14 5-68 .6467 .0764 307.58 40.26 6.58 0.86 154.22 20.19 5-69 .6490 .0777 303.51~ 39.06 6.96 0.90 161.79 20.82 5-70 .5073 .0581 185.47 31.92 3.83 0.66 122.69 21.12 5—71 .6075 .0643 254.19 39.53 6.02. 0.94 137.71 21.42 5-72 .5737 .0640 284.63 47.44 5.73 0.90 100.91‘ 15.77 5-73 .5313 .0551 228.61 41.49 6.11 1.11 122.70 22.27 6-19 .3396 .2045 950.40 46.47 11.84 0.58 392.92 19.21 6-20 .3883 .2352 1144.16 48.65 15.86 0.67 476.40 20.26 6-21 .5451 .3429 1557.91 45.43 22.22 0.65 635.52 18.53 6-22 6-23 .4882 .3017 1552.60 51.46 15.22 0.50 547.91 18.16 6-24 .6209 .3841 1762.41 45.88 20.49 0.53 668.665 17.41 VITA Marilyn P. Anderson was born in Detroit, Michigan, on June 26, 1944. She received her primary and secondary education in the public 57. schools of Detroit and Pontiac, Michigan, and Arlington, Virginia. She graduated from Pontiac Northern High School in 1962. She attended Michigan State University receiving a BS in 1966 and a DVM in 1967. In 1968 she entered the Peace Corps, serving in Brazil until ii 1970 as veterinary advisor to ACARES, the agricultural extension agency in the State of Espirito Santo. In 1971 she reentered Michigan State University to pursue a Master of Science degree in veterinary pathology. 75 "1111111111111111“