Poa * J be ar ean, Ss % { ‘ be fi J ee ~ ad co ‘ ae cae ne b . ed j et ‘ = Vy oe my iv i Ss a bs rae | 7 _4 | be aN i reer aa ane of — 1 haya Frye and , eos: Hea Murphy ] A pay kal Fi de Oe | ; 4 oui - Pore { me k, rl : =: CHAPMAN & HALL atte 2 Ss .Health and Welfare of Captive Reptiles Edited by Clifford Warwick Director of the Institute of Herpetology Worcester, UK Fredric L. Frye Director of the Fund for Clinical Research Davis, California USA and James B. Murphy Curator of the Department of Herpetology Dallas Zoo Texas, USA CHAPMAN & HALL London : Glasgow : Weinheim - New York - Tokyo - Melbourne - MadrasPublished by Chapman & Hall, 2-6 Boundary Row, London SEI 8HN, UK Chapman & Hall, 2-6 Boundary Row, London SE1 8HN, UK Blackie Academic & Professional, Wester Cleddens Road, Bishopbriggs, Glasgow G64 2NZ, UK Chapman & Hall GmbH, Pappelallee 3, 69469 Weinheim, Germany Chapman & Hall USA, One Penn Plaza, 41st Floor, New York NY 10119, USA. Chapman & Hall Japan, ITP-Japan, Kyowa Building, 3F, 2-2-1 Hirakawacho, Chiyoda-ku, Tokyo 102, Japan Chapman & Hall Australia, Thomas Nelson Australia, 102 Dodds Street, South Melbourne, Victoria 3205, Australia Chapman & Hall India, R. Seshadri, 32 Second Main Road, CIT East, Madras 600 035, India First edition 1995 © 1995 Chapman & Hall Typeset in 10/12 Times by Photoprint, Torquay, Devon Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the UK Copyright Designs and Patents Act, 1988, this publication may not be reproduced, stored, or transmitted, in any form or by any means, without the prior permission in writing of the publishers, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to the publishers at the London address printed on this page. The publisher makes no representation, express or implied, with regard to the accuracy of the information contained in this book and cannot accept any legal responsi ity for any errors or omissions that may be made ‘A catalogue record for this book is available from the British Library Library of Congress Catalog Card Number: 94-7266 ISBN 1-4020-0403-6 Transferred to Digital Print 2001 (©9) Printed on permanent acid-free text paper, manufactured in accordance with ANSI/NISO Z39.48-1992 and ANSI/NISO Z39.48-1984 (Permance of Paper).Contents List of contributors Acknowledgements Introduction: Health and welfare of captive reptiles Clifford Warwick, Fredric L. Frye and James B. Murphy 1 Physiology and functional anatomy Harvey B. Lillywhite and Robert E. Gatten Jr 1.1 Introduction 1.2. Body temperature, energetics and ectothermy 1.3 Light and photoreception 1.4 Water exchange and humidity 1.5 Digestive physiology and nutrition 1.6 Respiration and circulation 17 Pain and stress 18 Conclusion Acknowledgements References 2 _ Biology of stress: interactions with reproduction, immunology and intermediary metabolism Louis J. Guillette Jr, Alison Cree and Andrew A. Rooney 2.1 Introduction 2.2 Reproduction 2.3 Immunity 2.4 Corticosteroids, intermediary metabolism and growth 2.5 Implications for captive husbandry of reptiles and future research Acknowledgements References xiii RERERSBEBau 8 338 KEBbvi CONTENTS, 3. Nutritional considerations Fredric L. Frye 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 Introduction Provision of an adequate water supply Selection of food Apprehension of prey and gathering of fodder Initial processing Assimilation Elimination Miscellaneous factors and their effects on nutrition Concluding remarks References and further reading 4 Veterinary perspectives and techniques in husbandry and research John E. Cooper and David L. Williams 41 42 43 4.4 Introduction Captive reptiles Free-living reptiles Conclusions Acknowledgements References and recommended reading 5 Naturalistic versus clinical environments in husbandry and research Clifford Warwick and Catrina Steedman 5.1 5.2 5.3 5.4 5.5 5.6 Introduction Context: Welfare in husbandry and research Terminology for wild and captive reptile environments Naturalistic versus clinical environments Conclusions Recommendations Acknowledgements References 6 Normal behaviour James C. Gillingham 6.1 6.2 6.3 6.4 6.5 Introduction Maintenance behaviours Distance-reducing behaviour Agonistic behaviour Conclusions References BSBSSSLSBALE B 98 105 110 111 11 113 113 115 115 116 126 127 128 129 131 131 132 139 143 150 153CONTENTS vii 7 ° Effects of ontogenetic processes and rearing conditions Gordon M. Burghardt and Donna Layne 7.1 Introduction 7.2 The prenatal period 7.3 Parental care 7.4 Handling and novel environments 7.5 Cage size and structure 7.6 Social arrangements 7.7 Feeding 7.8 Defensive behaviour 7.9 Long-term influence of captive regimes 7.10 Conclusions Acknowledgements References Behavioural consequences of husbandry manipulations: indicators of arousal, quiescence and environmental awareness David Chiszar, W. Thomas Tomlinson, Hobart M. Smith, James B. Murphy and Charles W. Radcliffe 8.1 Introduction 8.2 Cage cleaning and exploratory behaviour 8.3 Use of familiar artificial chemical cues 8.4 Chemical recognition of self 8.5 Sensitivity to spatial considerations 8.6 General discussion Acknowledgements References Psychological and behavioural principles and problems Clifford Warwick 9.1 Introduction 9.2 Living wild and noticing captivity 9.3. Recognizing and interpreting signs of psychological and ethological well-being and poor welfare 9.4 Specific psychological and ethological problems 9.5 General considerations 9.6 Conclusions Acknowledgements References 165 165 166 168 170 171 172 173 175 179 180 180 180 186 186 187 191 192 193 198 201 205 205 206 208 215 224 233 235 235CONTENTS 10 Ethologically informed design in husbandry and research Neil Greenberg 11 10.1 10.2 10.3 10.4 Introduction Ethologically informed design Case studies: ethologically integrated designs Recommendations and conclusions Acknowledgements References Miscellaneous factors affecting health and welfare Phillip C. Arena and Clifford Warwick 11.1 11.2 11.3 11.4 11.5 11.6 11.7 11.8 Introduction Stress, pain and sensitivity Thermal factors, thermoregulation and light Growth Electromagnetism in the artificial environment Reintroductions to nature Euthanasia and killing Conclusions Acknowledgements References 239 239 240 255 263 263 B BBSS3sss8a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.RESPIRATION AND CIRCULATION } [2] Figure 1.5 Scanning electron micrograph of a portion of the faveolar parenchyma of a lung from a lizard, Tupinambis nigropunctatus, Trabeculae of first, second, third and fourth order are indicated as TI-T4. Favaolie (F) are grouped into niches (N). Scale bar = 500 pm. (Reproduced from Perry (1989), p. 197, by courtesy of Marcel Dekker, Inc.) reptiles generally, the conducting airways and respiratory surfaces are not as elaborate as comparable structures in mammals, a condition that correlates with the lower metabolic rates of ectotherms. The complexity and diversity of reptilian lung structure, together with considerations of habitat and behavioural specializations, possibly pre- dispose reptiles to infection by lung parasites. Reptilian lungs frequently contain parasites and the load may worsen if captive animals are stressed or subjected to inappropriate physical conditions. Severe parasitic pulmonary lesions can lead to lethal haemorrhage or oedema if blood pressures are increased by activity or gravity stress (as in upright posture) (H.B.L., unpub. obs.). These considerations possibly contribute to the fact that many health problems in captive reptiles are related, or lead, to respiratory problems. Lung ventilation is powered by muscular expansion and contraction of the thoraco-abdominal space; and, in some species of turtles and squamates, pulmonary smooth muscle may contribute to air movements within the lung spaces. Both the depth and frequency of lung ventilations vary greatly, even within a species or in individual animals. Therefore changes in ventilatory behaviour (aside from extreme alterations such as panting)PHYSIOLOGY AND FUNCTIONAL ANATOMY are not usually reliable indicators of serious changes in health. Temperat- ure change, excitement, activity, diving and skin shedding can produce marked ventilatory changes. The skin, pharyngeal lining and cloacal bursae of aquatic snakes and turtles may serve as accessory respiratory surfaces (Feder and Burggren, 1985). However, the majority of species exchange virtually all O2 and most CO, across the lung. Except for hibernating turtles, most aquatic reptiles depend on lung breathing and require periodic access to air. For reptiles that are kept in underground burrow systems, precaution should be taken to provide adequate ventilation that prevents accumulation of high levels of CO, (Ultsch and Anderson, 1986). Reptiles that burrow in sand, soils or leaf litter behaviourally achieve adequate O, uptake and CO, release (Pough, 1969a, 1969b). If reptiles become hypoxic, they tend to seek lower temperatures when choices are available (Hicks and Wood, 1985; Wood, 1991). 1.6.2 BLOOD CIRCULATION All reptiles circulate blood by means of a central heart which, except in crocodilians, consists of a single ventricle that is filled from two atria. All species appear to have the capacity for shunting blood between the systemic and pulmonary circuits according to physiological requirements (Burggren, 1987; Heisler, 1985; White, 1976). Furthermore, blood flow varies substantially between periods of apnoea and periods of active ventilation of the lungs (Burggren, 1977, 1987; Burggren and Shelton, 1979). Thus, one should expect blood flow in both the systemic and pulmonary circuits to fluctuate considerably over time. Heart rates of reptiles are generally lower than those of mammals, except when reptiles have achieved high body temperatures when rates can exceed 100 beats per min. Arterial blood pressures vary with species, activity and physio- logical state, with values for mean pressure ranging typically from 15-20 mm Hg to 50-70 mm Hg (Axelsson et al., 1989; Burggren, 1977; Burggren and Johansen, 1982; Lillywhite, 1985; Seymour and Lillywhite, 1976). As in most vertebrates (and all ‘higher’ taxa), fluid, respiratory gases and other metabolically important molecules are exchanged between blood and tissues at capillaries. The capillary interface with interstitial fluids appears to be comparatively ‘leaky’ and fluids shift readily between the vascular and extravascular compartments, subject to physiological controls. Partly because of these attributes, reptiles can regulate blood (plasma) volume quite well (Lillywhite and Smith, 1981; Smits and Kozubowski, 1985). On the other hand, animals may be subject to oedema due to excess capillary filtration when capillary pressures are high because of arterial hypertensionRESPIRATION AND CIRCULATION 23 or gravity stress. Large snakes should not be held in vertical positions for long periods (>2-3 min) because gravitational pooling of blood and oedema in dependent tissues compromise adequate blood circulation (Lillywhite, 1988; Lillywhite and Smits, 1992). The lymphatic system is not well studied but is extensive and important, at least in some reptiles (Ottaviani and Tazzi, 1977). Lymphatic spaces may provide significant stores of body fluid in some species (Smits, 1985). Circulating blood volumes of reptiles vary from a few per cent to about 14% of body mass, with most values typically around 6% of body mass (Pough and Lillywhite, 1984; Thorson, 1968). Blood volume, as a propor- tion of body mass, decreases as body size increases, at least in some turtles (Hutton, 1961). The plasma fraction comprises roughly two-thirds to three- quarters of the total blood volume but values vary with species, activity, temperature and health (Lillywhite and Smits, 1984; Thorson, 1968). Un- usually low values for haematocrit are generally indicative of haemorrhage or poor health. Few studies have examined how blood or plasma volume change, or how well blood circulates, in dormant or hibernating reptiles with low body temperature, reduced heart rate and increased blood viscosity (due to the low temperature) (Huggins, 1961; Huggins and Percoco, 1965; Snyder, 1971). Blood can be sampled for clinical or research purposes from veins or various sinuses that are accessible by needle (for example, orbital sinuses of lizards) (McDonald, 1976; Olson et al., 1975). Only trained persons should attempt to obtain blood samples by cardiac puncture because of the potential for extensive damage to the heart. Furthermore, blood drawn by cardiac puncture may be inadequate for precise measurement of blood gases (Kerr and Frankel, 1972). In addition, samples of blood taken from cut tissue (for example, tail tip) are likely to be diluted with interstitial fluids and may yield erroneous values for measurements such as haematocrit. Handling of crocodilians induces glycolysis and results in blood lactate values that remain elevated for up to 24 hours (Coulson and Hernandez, 1964; Seymour er al., 1985); experimenters should not assume that an animal that has been handled is truly ‘at rest.’ The pH or acid-base status of the blood and interstitial fluids depends on metabolic acid production, respiratory gas exchange, ion levels and kidney function. Unlike the case in mammals, which regulate constant pH, the plasma pH of reptiles varies with changes in body temperature (approx- imately —0.016 pH unit per 1 °C increase in temperature) (Howell and Rahn, 1976). Such shifts of pH in relation to temperature appear to be regulated largely by adjustments of ventilation (and thus CO, levels) (Ackerman and White, 1980; Davies et al., 1982; Lutz et al., 1989; Stinner, 1987). Thus, it seems possible that behaviours associated with temperature change may, in some circumstances, be causally related to acid-base regulation rather than thermoregulation per se.24 PHYSIOLOGY AND FUNCTIONAL ANATOMY 1.7 PAIN AND STRESS The perception of pain and the physiological consequences of acute and chronic stress are only beginning to be understood in reptiles. Comparatively little is known about pain perception in reptiles (Liang and Terashima, 1993); most of our understanding is derived from indirect observations of responses to pharmacological agents and anaesthetics (Frye, 1991a). Both pain and stress are difficult to measure in any vertebrate animal. In the absence of more quantitative understanding of these subjects, we suggest that common sense be adopted as the appropriate guideline in determining considerate care in reptiles. As one example for consideration here, rough handling of snakes can obviously inflict pain (as judged from behavioural responses) and is always stressful. If a large snake is held vertically, writhing movements or the mere mass of the animal can damage the vertebral column; in addition, the gravitational stress of upright posture can impair circulation. Stress can be reduced significantly, and any pain or bodily damage associated with handling can be avoided, if a captured snake is lifted gently on a hooked stick and deposited slowly into a container rather than being grabbed behind the neck with a force-grip instrument. Following this recommenda- tion may not be possible during capture of all species, but most viperids, for example, can be handled in this way. Snakes procured in a non-aggressive manner are more likely to do better in captivity because the experience of a traumatic capture conceivably induces chronic stress. Handling stress can cause dramatic hormonal changes even in reptiles that are habituated to humans (Lance, 1992). Improper handling, as well as crowding and poor sanitation, produce physiological deterioration leading to reduced growth, suppressed reproductive capacity and increased mortality from disease. Further research is needed to understand the physiological basis of, and interactions with, stressful states and to evaluate how stress can be ameliorated in captive animals. 1.8 CONCLUSION Investigators and animal care technicians responsible for the husbandry of captive reptiles must understand not only the general principles of the physiology and functional anatomy of reptiles but also the specific physiological, morphological and behavioural characteristics of the species under their care. Successful husbandry requires an appreciation of both the scientific literature pertinent to the physiology of the species in question and the features of the habitat(s) normally occupied by the animals. In the absence of precise information regarding the proper care of a particular species, those responsible should provide a varied environment where theREFERENCES 25 animals can select the conditions of temperature, light, humidity, food and so on that they prefer. Careful observation and reporting of the behaviour of such animals will lead to a better understanding of their physiological needs while in captivity. ACKNOWLEDGEMENTS We are grateful to Belinda Davis for her help in the preparation of this manuscript. REFERENCES Ackerman, R.A. and White, F.N. (1980) The effects of temperature on acid-base balance and ventilation of the marine iguana. Respiration Physiology, 39, 133- 47. Avery, R.A. (1982) Field studies of body temperatures, in Biology of the Reptilia, Vol. 12, Physiology C, Physiological Ecology, (eds C. Gans and F.H. Pough), Academic Press, New York, pp. 93-166. Axelsson, M., Holm, S. and Nilsson, S. (1989) Flow dynamics of the Crocodilian heart. American Journal of Physiology, 256, R875-9. Bartholomew, G.A. (1966) A field study of temperature relations in the Galapagos marine iguana. Copeia, 1966, 241-50. Bartholomew, G.A. (1982) Physiological control of body temperature, in Biology of the Reptilia, Vol. 12, Physiology C, Physiological Ecology, (eds C. Gans and F.H. Pough), Academic Press, New York, pp. 167-211. Bjorndal, K.A. (1989) Flexibility of digestive responses in two generalist herbivores, the tortoises Geochelone carbonaria and Geochelone denticulata. Oecologia, 78, 317-21. Bjorndal, K.A. (1991) Diet mixing: non-additive interactions of diet items in an omnivorous freshwater turtle. Ecology, 72, 1234-41. Bjorndal, K.A. and Bolten, A.B. (1990) Digestive processing in a herbivorous freshwater turtle: consequences of small-intestine fermentation. Physiological Zoology, 63, 1232-47. Bjorndal, K.A., Bolten, A.B. and Moore, J.E. (1990) Digestive fermentation in herbivores: effect of food particle size. Physiological Zoology, 63, 710-21. Brattstrom, B.H. (1978) Learning studies in lizards, in Behavior and Neurology of Lizards: An Interdisciplinary Colloquium, (eds N. Greenberg and P.D. MacLean), US Dept of Health, Education, and Welfare Publ. No. ADM 77-491, pp. 173-81. Burggren, W.W. (1977) Circulation during intermittent lung ventilation in the garter snake Thamnophis. Canadian Journal of Zoology, 55, 1720-5. Burggren, W.W. (1987) Form and function in reptilian circulations. American Zoologist, 27, 5-19. Burggren, W.W. and Johansen, K. (1982) Ventricular haemodynamics in the monitora You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.REFERENCES acid-base regulation in the salt-water crocodile, Crocodylus porosus, at rest and after exhaustive exercise. Journal of Experimental Biology, 118, 143-59. Smits, A.W. (1985) Correlates of activity, diet, and body water flux in the chuckwalla lizard Sauromalus hispidus. Physiological Zoology, 58, 166-74. Smits, A.W. and Kozubowski, M.M. (1985) Partitioning of body fluids and cardiovascular responses to circulatory hypovolaemia in the turtle, Pseudemys scripta elegans. Journal of Experimental Biology, 116, 237-50. Snyder, G.K. (1971) Influence of temperature and haematocrit on blood viscosity. American Journal of Physiology, 220, 1667-72. Stevenson, R.D., Peterson, C.R., and Tsuji, J.S. (1985) The thermal dependence of locomotion, tongue flicking, digestion, and oxygen consumption in the wandering garter snake. Physiological Zoology, 58, 46-57. Stinner, J.N. (1987) Thermal dependence of air convection requirement and blood gases in the snake Coluber constrictor. American Zoologist, 27, 41-7. Summers, C.H. and Norman, M.F. (1988a) Chronic low humidity stress in the lizard Anolis carolinensis: changes in diurnal corticosterone rhythms. Journal of Experimental Zoology, 247, 271-8. Summers, C.H. and Norman, M.F. (1988b) Chronic low humidity stress in the lizard Anolis carolinensis: effects of ovarian and oviductal recrudescence. Journal of Experimental Zoology, 248, 192-8. Thorson, T.B. (1968) Body fluid partitioning in Reptilia. Copeia, 1968, 592-601. Townsend, C.R. and Cole, C.J. (1985) Additional notes on requirements of captive whiptail lizards (Cnemidophorus), with emphasis on ultraviolet radiation. Zoo Biology, 4, 49-55. Tracy, C.R. (1982) Biophysical modeling in reptilian physiology and ecology, in Biology of the Reptilia, Vol. 12, Physiology C, Physiological Ecology, (eds C. Gans and F.H. Pough), Academic Press, New York, pp. 275-321. Troyer, K.E. (1984a) Structure and function of the digestive tract of a herbivorous lizard Iguana iguana. Physiological Zoology, 57, 1-8. Troyer, K.E. (1984b) Diet selection and digestion in Iguana iguana: the importance of age and nutrient requirements. Oecologia, 61, 201-7. Troyer, K.E. (1987) Small differences in daytime body temperature affect digesti of natural food in a herbivorous lizard (Iguana iguana). Comparative Bio- chemistry and Physiology, 87A, 623-6. Ultsch, G.R. and Anderson, J.F. (1986) The respiratory microenvironment within the burrows of gopher tortoises (Gopherus polyphemus). Copeia, 1986, 787-95. White, F.N. (1976) Circulation, in Biology of the Reptilia, Vol. 5, Physiology A, (eds C. Gans and W.R. Dawson), Academic Press, New York, pp. 275-334. Wood, S.C. (1991) Interactions between hypoxia and hypothermia. Annual Review of Physiology, 53, 71-85. Yokota, S.D., Benyajati, $., and Dantzler, W.H. (1985) Renal function in sea snakes I. Glomerular filtration rate and water handling. American Journal of Physiology, 249, R228-36. Zimmerman, K. and Heatwole, H. (1990) Cutaneous photoreception: a new sensory mechanism for reptiles. Copeia, 1990, 860-2. Zug, G.A. and Dunson, W.A. (1979) Salinity preference in fresh water and estuarine snakes (Nerodia sipedon and N. fasciata). Florida Scientist, 42, 1-8. 31Biology of stress: interactions with reproduction, immunology and intermediary metabolism Louis J. Guillette Jr, Alison Cree and Andrew A. Rooney 2.1 INTRODUCTION Animals must maintain a steady physiological state, homeostasis, in response to a changing environment. An understanding of the stress response is essential if one is to appreciate the complex physiological mechanisms maintaining homeostasis in vertebrates. Moreover, a compre- hensive understanding of the biology of stress is essential if we are to reduce stress in the care of any vertebrate species, including those that are not domesticated and traditional laboratory animals. The goals of this chapter are to review our knowledge of the phenomenon of the physiological stress response in vertebrates generally, and then to describe in detail the limited information available on the biology of stress in reptiles. We shall not deal with psychic or emotional considerations of stress in reptiles. Specifically, we focus on the interactions between the physiological stress response and reproduction, immune function and intermediary metabolism. These three areas are critical to any husbandry and captive management programme. If not prevented or countermanded, the detrimental aspects of the stress response mounted by, for example, Health and Welfare of Captive Reptiles. Edited by Clifford Warwick, Fredric L. Frye and James B. Murphy. Published in 1995 by Chapman & Hall, London. ISBN 0 412 55080 6INTRODUCTION 33 the adrenal gland can block effective reproduction and growth and, if severe, can lead to death. We end by outlining some of the major gaps in our knowledge and by suggesting future research needs for increasing our limited understanding of the biology of stress in reptiles. 2.1.1 THE ADRENAL AND THE STRESS RESPONSE A brief consideration of terminology is important at this point. In our everyday lives as humans, we commonly use the word stress to refer both to the external forces applied to us and to the state of hardship or strain that such forces produce. As scientists we need a more precise terminology that distinguishes between the ‘force’ and the ‘response’. In this chapter, we will follow previous scientific usage in using the word stressors to refer to the external forces applied to animals that threaten their homeostasis. We will refer to the stress response as the combination of responses mounted by an animal toward such a stressor that involve increased activity of the adrenal gland. The adrenal gland of reptiles exhibits a consistent anatomical association with the gonad. The adrenal is composed of two types of tissue, interrenal and chromaffin (Hebard and Charipper, 1955). The majority of the adrenal gland is composed of cells of mesodermal origin, the steroidogenic interrenal cells, which secrete corticosteroids and are homologous with the cells of the mammalian adrenal cortex (Gabe, 1970; Chester-Jones, 1977). The chromaffin cells, derived from the neural crest, secrete adrenaline (epinephrine) and noradrenaline (norepinephrine) and correspond to the cells of the adrenal medulla in mammals. Unlike in mammals, these cell types are intermingled and do not form discrete regions (Figures 2.1 and 2.2). An excellent review of the anatomy and development of the adrenal has previously been published (Gabe, 1970). In this review, we will be primarily concerned with the effects of the corticosteroid hormones produced by the interrenal cells. We will focus on the effects of cortico- steroid hormones on reproduction, immunity, intermediary metabolism and growth. Little will be said about the effects of the hormones adrenaline and noradrenaline produced by the chromaffin tissue. The adrenal gland of all vertebrates studied to date responds to environmental stressors by secreting corticosteroids, either corticosterone or cortisol. The relative concentrations of plasma corticosterone and cortisol differ among vertebrate classes: corticosterone is primarily secreted in amphibians, reptiles and birds and cortisol in fish and most mammals (Greenberg and Wingfield, 1987). The synthesis of vertebrate cortico- steroids from the interrenal tissue (adrenal cortex of mammals) is under the control of an anterior pituitary hormone, adrenocorticotropin (ACTH). ACTH is in turn regulated by corticotropin-releasing hormone34 BIOLOGY OF STRESS } Figure 2.1 Photomicrographs of the adrenal gland from an adult Mountain spiny lizard, Sceloporus jarrovi. Note the islands of dark staining chromaffin cells (A, B) surrounded by steroidogenic interrenal cells. Due to normal histological techniques, the lipid is removed from the vacuole of the steroidogenic interrenal cells (C); thus, they appear clear with a large round nucleus. C = chromaffin cells; E = epididymis; I = interrenal cells. Bar = 50 um. H & E staining. (Photos by J. Matter.)INTRODUCTION 35 chromaffin cells stain ganglion; I = interrenal cells. Bar = 50 um. H & E staining. (Photos by J. Matter.)BIOLOGY OF STRESS Hypothalamus Pituitary Adrenal gland Figure 2.3 Schematic representation of the control of stress steroid hormone release. Corticosterone is released from the adrenal interrenal cells due to stimulation by the pituitary hormone adrenocorticotropin (ACTH). ACTH release is controlled by corticotropin (CRH) released from the hypothalamus. Various stressors can elicit the release of CRH from the hypothalamus. This system appears to function in all reptiles studied to date. See text for additional details. (CRH) that is synthesized by the paraventricular nucleus of the hypo- thalamus (Figure 2.3). Various factors influence plasma corticosteroid concentrations including genetic variability, age, sex, nutritional state, the type of stressor and the frequency of stressor presentation (Selye, 1973). Selye (1936, 1937) pioneered the field of study we today refer to as ‘stress research’. He described a series of general, non-specific anatomical and physiological modifications characteristic of the animal exposed to various stressors (e.g. environmental extremes). He termed this syndromea You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.38 BIOLOGY OF STRESS 2.2 REPRODUCTION Most workers acknowledge the fact that stress can influence the health and reproductive activity of wild or captive reptiles, but almost no published data are available on this. Although we understand the basic mechanisms controlling reproduction and stress, we still have a relatively poor compre- hension of the interactions between these systems in reptiles. As in other vertebrates, reproductive activity in reptiles is controlled by the neuro- endocrine system (see Licht, 1985, for an extensive review of this topic). Various factors, such as environmental conditions (photoperiod and temperature), nutritional state and social behaviour influence the nervous system and control the release of a neuropeptide, gonadotropin releasing hormone (GnRH), from the hypothalamus. GnRH is released into the hypothalamo-hypophysial portal system, a vascular connection between the hypothalamus and pituitary (hypophysis), and is transported to the pituitary where it stimulates the secretion of two gonadotropins: follicle stimulating hormone (FSH) and luteinizing hormone (LH). All reptiles studied appear to possess both these hormones, except squamates that possess a single FSH-like gonadotropin as determined by functionality testing (Ishii, 1991). The gonadotropins function at the level of the gonad in stimulating gametogenesis (FSH) and steroidogenesis (LH) (Ishii, 1991). Typically, the reptilian testis synthesizes large concentrations of the androgen testosterone with lesser amounts of dehydrotestosterone (Bourne, 1991), whereas the ovary synthesizes primarily estradiol-17B during the follicular growth (vitellogenic) phase of the cycle and progesterone during the luteal phase of the reproductive cycle (Jones and Baxter, 1991). These steroid hormones are essential for the maturation of the repro- ductive tract of both females and males during the reproductive cycle (Jones and Baxter, 1991; Palmer and Guillette, 1991). Additionally, steroid hormones stimulate many species-specific secondary sexual charac- teristics (e.g. coloration) and modify reproductive behaviours in reptiles (Moore and Lindzey, 1992; Whittier and Tokarz, 1992). 2.2.1 SEX AND STRESS HORMONES Do male and female reptiles respond differently to a stressor? The available data suggest that the sex of the individual can influence the stress response. Adult male alligators (Alligator mississippiensis) had signifi- cantly higher corticosterone levels than adult female alligators in captive and wild populations (Elsey et al., 1990b). A difference in plasma cortico- sterone concentration is also apparent between the sexes in turtles. For example, male loggerhead (Caretta caretta) and olive ridley (Lepidochelys olivacea) turtles had elevated plasma corticosterone concentrations when compared with females collected under similar conditions (Schwantes,REPRODUCTION 39 1986), whereas no difference was observed between juvenile male and female loggerhead turtles studied in Florida (Gregory, 1994). Female whiptail lizards (Cnemidophorus sexlineatus) exhibited greater concentra- tions of plasma corticosterone compared with males in response to a similar stressor (Grassman and Hess, 1992a, b). However, this difference was reversed when the females were ovariectomized and males were castrated (Grassman and Hess, 1992b). Further support for the reciprocal influences between the gonadal and adrenal axes has been provided by Greenberg et al. (1984), who showed that castrated male Anolis carolinensis did not exhibit elevated plasma corticosterone concentration in response to a dominant conspecific. Variation in the adrenal response of males and females to exogenous hormone treatment has also been reported. Injections of follicle stimulat- ing hormone (FSH), luteinizing hormone (LH), pregnant mare serum gonadotropin (PMSG) and estradiol-17B stimulate increased plasma corticosterone concentration in females but were not effective in male painted turtles, Chrysemys picta (Callard and Callard, 1978). In female Dipsosaurus dorsalis, LH and PMSG were both effective in stimulating increased plasma corticosterone concentrations, but in males only estradiol-17B was effective (Callard and Callard, 1978). Likewise, estradiol-17B increased plasma corticosterone in male lizards of the genus Sceloporus (Callard and Callard, 1978). 2.2.2 EFFECTS OF STRESS ON SEX STEROIDS Most studies examining the effects of stress on reproduction have done so by examining the influence of a stressor on circulating concentrations of various sex steroid hormones. The vast majority of these studies have examined acute, short-term responses to a stressor and have not docu- mented long-term, chronic responses. One of the most common acute stressors used is capture (Table 2.1). Wild male alligators exhibited a 50% reduction in plasma testosterone at 4h after capture with a further decline to less than 10% of initial values at 24h after capture (Lance and Elsey, 1986). Coincident with decreasing plasma testosterone levels, plasma corticosterone increased dramatically during the first 12 h, declined between 12 and 24 h, and then rose again at 40 h (Lance and Elsey, 1986). As in male alligators, stress has been shown to suppress plasma androgen concentrations in male lizards, tuatara and turtles. Wild male tree lizards, Urosaurus ornatus, exposed to acute capture stress (held in a bag) exhibited a 50% reduction in plasma testosterone concentration at 4h after capture (Moore ef al., 1991). In male tuatara (Sphenodon punctatus), capture, individual confinement in a cloth bag and repeated blood sampling significantly decreased plasma testosterone at 24h after the initial sample (Cree et al., 1990a). In wildBIOLOGY OF STRESS (1661) 79 2 enedeyeyw = uw Og 2qeus YNpy vivDUNd meLuNd sKuessry ‘utes sowsossdAy Jo uortsofuy (261) meyspeag Ps paypodsun, snypuso smioydowat ‘uyes atuorodAy jo suonsafut (0661) 20ue] 1M soptuaang — stsuarddississnu sow3iyyy 49)@M Jo saruyjes snouRA 0} aansodxs] (861) voiney mT sojiuannt — sysuarddississiut sow8q)y sayem outjes 07 aunsodxs] (0661) soup pure uewissein) we 2yeway ainpy suazodiun sruoydopruau ‘Aworoureag (3861) UeMON pue srowmNS ym ED yews uMPY sysuauyjouv9 syouy Aupuuny 07] (0661) S219 pue wessei nu s2-S1 >yewa} ynpy suavndiun snuoydopruauy Suruure £q pamoyjoy aunsodxo ploy (0661 sour {(8 0661) “70 19 Aosta Pel sajuaang —sysuarddissyssyu sow3yyy Ausuap y8tH (a6) "70 12 B19qu91D POOL sojeu ayeurpsoqns sysuauyjos2 syouy (sojeus weuiwop Jo souasasd) Aysseso1y [BOS (g61) aie] pue 200] a1 souoang — stsuarddhssissiu 40703 y (S261) preHeD uy 0g payrsodsun) prod skwashsy) Suyjdwes-poojq pareada pue suypuct sojeuiay pur Suyduzes-poojq (€661) 4108915 Ye sajew apy ~ aqruoang mnzsv> vn2103 pareadas pur yuresysas yeoiskyd “oamdecy Suydwres-poojq (1661) “7 22 Kasia uP aqeway npy —_sysuarddyssyssyus soww8 yyy poveadas pue quyesisos yeaskyd ‘oumided Suyduzes-pooq (9861) Aastq pue sour] 4s ayew ynpy — sisuarddississyu sow8iyyy Payeadas pur quresysaz jeorskyd ‘amide paziworapeuod pue (2661) S82H pur uewssess luyw og “sayeway pur soi yy snimautyras snuoydopruauy — urseys-puey war pue quowauyuod (0661) Sm24 pur ueWsseI = UI OE soe Y suasvdiun smioydopruauy — ulsey>-puey ua} ULsDIUI puE JoWI2UyUC> s Vv pivdsaa 02007] soGeo jenpupur ut juoWaUyUOD 2 soyew 3inpy snymizund wopouayds ‘eq pop u! jUsWIOUYUOD pu aimed sojeus ynpy ‘snypuso snunvsoup) eq moj ut jwowauyUoo pue oimide>) poyrsodsun, ——sypmyaonu smoydouary eq ioj9 ut jwowauyuoo pur aimde) ans sapads 4078 Aaoysyy-ayy pue x25 “sojndas ut aseojo1 2uOsD}S0O1I09 DE[NWINS 0} UMOUY SION] 1°7 FG8LREPRODUCTION 41 male painted turtles (Chrysemys picta), capture and repeated bleeding induced a significant decline in plasma testosterone at 24 h after capture to 10% of the initial concentration (Licht et al., 1985a). Male green turtles (Chelonia mydas: Licht et al., 1985b) and male musk turtles (Sternotherus odoratus: Mendonga and Licht, 1986) show a similar decline in plasma T concentration, although the response in both species is less than that observed in painted turtles. In the snapping turtle, Chelydra serpentina (Mahmoud et al., 1989), adult males exhibited an initial rise of plasma testosterone at 6h after capture followed by a significant decline by 24-48 h. Plasma concentrations remained below initial values at 7 days after capture when the final blood sample was obtained. The month of capture influenced both the pattern of the stress response and the degree to which the animals responded. For example, males captured in early summer (June) during the reproductive season showed a marked increase in testosterone at 6 h after capture whereas those caught in late summer (August) following the reproductive season showed a slight decrease within the same time period. This may reflect seasonal differences in the ability of the testes to synthesize androgens (Mahmoud et al., 1989). In contrast to these reports, Bourne et al. (1986) observed that captivity did not suppress androgens in male shingleback lizards (Tiliqua rugosa). Acute changes in plasma testosterone concentration may not have a long-lasting or permanent inhibitory effect on the male reproductive system, but long- term studies are needed to verify this conclusion. Capture stress has also been reported to modify plasma progesterone concentration in male tuatara. The adrenal gland of reptiles secretes not only corticosteroids but also progesterone (Callard, 1975; Dauphin- Villemant and Xavier, 1987). Capture stress without repeated blood sampling stimulated a significant rise in plasma progesterone concentration at 3 h after capture in wild male tuatara (Cree et al., 1990a). The functional role of progesterone in male reptiles is still poorly defined. Interestingly, it stimulates the expression of sexual behaviour in male Cnemidophorus inornatus (Lindzey and Crews, 1988) whereas it is inhibitory in male Anolis carolinensis (Young et al., 1991). Very few studies have examined the influence of stress on plasma sex hormone concentrations in female reptiles. No effect on plasma sex steroid concentrations (estradiol-17B, progesterone, testosterone) was detected in female garter snakes (Thamnophis sirtalis parietalis) in response to captivity (Whittier et al., 1987). Similarly, plasma concentrations of estradiol-17B and testosterone were not significantly different in wild vitellogenic tuatara confined for 3 h, compared with vitellogenic females sampled immediately after capture, whereas plasma progesterone did increase significantly in the confined females (Cree et al., 1990b). Plasma estradiol-17B and progesterone concentrations increased significantly at 6h after capture in female snapping turtles (C. serpentina: Mahmoud42 BIOLOGY OF STRESS etal., 1989). Female plasma concentrations of estradiol-17B and progester- one then decreased, but did not fall to levels below the initial values by the 7th day. The response of the females to capture was also influenced by the stage of reproduction. Gravid animals showed a larger increase in plasma estradiol-17B concentrations and lower concentrations of plasma proges- terone at 24 h after capture when compared with gravid turtles (Mahmoud et al., 1989). These data suggest that the acute stress of capture can dramatically modify plasma concentrations of a reproductive hormone. Moreover, they suggest that the stress response may be modified by sex or stage of reproductive activity. However, they do not indicate the persistence of the modified plasma hormone concentrations, nor do they indicate an effect on actual reproductive activity. The only available data set supporting a chronic effect comes from a captive study. Captive adult alligators maintained at high stocking densities had plasma corticosterone concentra- tions significantly higher than those maintained at lower stocking densities (Elsey et al., 1990b) and elevated plasma corticosterone was correlated with decreased growth in juvenile alligators (Elsey et al., 1990a). Managers of crocodile farms have observed poor reproductive activity following the transport of animals from one pond to another or after extensive modifica- tion of a pond (J. Seaman, pers. comm.). Both activities are considered stressful and many farmers have concluded that stress limits the repro- ductive activity of their animals. This conclusion warrants study, for at this time no data are available to support or reject the hypothesis that stress is directly inhibiting reproductive activity in wild or farmed reptiles. (See Summers and Norman, 1988, for data showing that stressful housing conditions do inhibit reproduction in the lizard Anolis carolinensis in a laboratory setting.) 2.2.3 SEASON, REPRODUCTIVE CYCLES AND ADRENAL FUNCTION Variation in the histological appearance of the adrenal gland due to season has been reported in various species (Gabe, 1970). These changes are based on weight and volume change, cytology and histochemistry. It appears that the adrenal gland undergoes seasonal changes in morphology in all vertebrates (Lofts and Bern, 1972). Several studies have demon- strated that reptilian interrenal tissue hypertrophies and cell volume increases during the spring and summer (for examples see Gabe, 1970). Additionally, long-term studies of several desert lizards of Australia have clearly demonstrated that adrenal responsiveness to ACTH stimulation varies with season, as does plasma corticosterone concentration (Brad- shaw, 1986). Further, these studies have demonstrated that stress due to a combination of reproductive activity and extreme climatic conditions actually leads to death in at least one lizard species, Ctenophorusa You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.a You have either reached 2 page thts unevalale fer vowing or reached your ievina tit for his book.IMMUNITY 45 integrate our understanding of the stress response to an animal's repro- ductive biology. Reproduction is also precluded if an animal has poor nutritional reserves or serious illness. The influence of stress on these parameters must also be considered. 2.3 IMMUNITY Historically, a physical change in the immune system has been one of the major morphological indications of stress. Selye’s indices of acute stress in mammals are enlargement of the adrenals, atrophy of lymphoid tissues, and ulcers in the gastro-intestinal tract (Selye, 1936). The effects of stress on the reptilian immune system are obscured by several complicating factors. The first problem with investigating stress- mediated effects on any physiological parameter is that the manipulations necessary to study the animal may in fact induce a stress response (Licht et al., 1985a). This difficulty is inherent to the study of stress and is not unique to reptiles. The second problem is that many basic aspects of the immune system of reptiles have not been thoroughly characterised (Cooper et al., 1985). A third difficulty is created by the seasonal nature of immune function in reptiles (indeed in all ectotherms). Mammalian and avian studies are relatively unencumbered by the annual fluctuations in immune function that characterize ectotherms. Exploring this character- istic ectothermic trait, however, may aid in understanding the general control of immune function. The mechanisms that regulate the immune systems of ectotherms (in particular those that depress immune reaction or create seasonality in immune function) may help to illuminate the mechan- isms that control the stress-induced suppression of the immune response in endotherms as well as ectotherms. 2.3.1 THE REPTILIAN IMMUNE SYSTEM The immune system of all vertebrates shares several features including the possession of lymphocytes, the ability to synthesize immunoglobulins that are fundamentally identical throughout the subphylum and the ability to mount T cell functions (Saad, 1988). All vertebrates possess the above immune attributes but the immune system of agnathans, cartilaginous fish and urodele amphibians appears to be significantly more primitive than that of other vertebrates (Zapata et al., 1992). Reptiles share many additional immune features with teleost fish, anuran amphibians, mammals and birds (Zapata et al., 1992). Only reptiles and other vertebrates possessing similar immune systems will be utilized in the following discussion.BIOLOGY OF STRESS 2.3.2 MORPHOLOGY The immunologically advanced vertebrates listed above possess a thymus, spleen, gut-associated lymphoid tissue (GALT) and bone marrow (Zapata et al., 1992). Bone marrow is a source of stem cells — cells that eventually reside in peripheral immune organs after functional or developmental modifications. Mammalian peripheral lymph organs include lymph nodes (Cooper et al., 1985); however, lymph nodes have not been found in ectotherms (Zapata et al., 1992). Birds possess a bursa of Fabricius, an additional immune organ in the cloacal region that mammals do not have. Within reptiles, bursal-equivalent regions are present in crocodilians and chelonians, whereas sphenodontids and squamates appear to lack a bursa of Fabricius (Cooper, 1973). There is, however, evidence that at least one lizard, Chalcides ocellatus, has a bursa-like lymphoepithelial macroorgan (Hussein et al., 1978a). 2.3.3 CELL TYPES Blood cells of the reptilian immune system include granulocytes, mono- cytes, plasma cells, thrombocytes and lymphocytes. The functional role of granulocytes and monocytes in reptilian immune systems is almost unknown, although their presence suggests relative importance (Cooper et al., 1985). Evidence for a major histocompatibility complex (MHC) in ectotherms is largely based upon functional studies such as allograft rejection (Afifi et al., 1993) and mixed leucocyte reaction (Saad et al., 1986). Reptiles synthesize at least two classes of immunoglobulin (Ig): a high molecular weight Ig related to mammalian IgM, and a more distantly related Ig, termed IgY, of lower molecular weight (El Ridi, 1992). Although few species have been investigated to date, available data suggest that Igs vary widely between reptilian taxa; for example, one low molecular weight Ig was found in the lizard Calotes versicolor (Natarajan and Muthukkaruppan, 1984) whereas two low molecular weight Igs were found in the snake Thamnophis ordinoides (Coe et al., 1976). In fact, much of the basic research on reptilian immunity and immune cells remains to be done. Lymphocytes are the best studied cells of the reptilian immune system, yet even the delineation into B and T lymphocyte subsets remains unclear (Cuchens et al., 1976). There are several indirect lines of evidence for the existence of distinct B and T lymphocyte subsets in reptiles. For example, two lymphocyte populations have been demonstrated within the lizard Chalcides ocellatus: thymus-derived lymphocytes that share cell-specific antigens with thymocytes (T-like cells), and non-thymus derived lympho- cytes that possess surface membrane immunoglobulin determinants (B-like cells) (El Ridi and Kandil, 1981; Saad and El Ridi, 1988). PhylogeneticIMMUNITY 47 comparison with other animals that have T and B cells, including fish (Cuchens and Clem, 1977) and mammals, also indicates the likelihood of these cells in all reptiles. Phylogenetic differences illustrate the limitations of such comparisons. For instance, the maturational sequence of B lymphocytes (B cells) involves different peripheral lymphoid organs in mammals and birds (Grossman, 1984). Direct evidence for lymphocyte developmental sequences in reptiles is poor but leucopoiesis in bone marrow and peripheral blood has been described in one species (Cordylus vittifer: de V. Pienaar, 1962). The sequence of B-like cell development remains obscure in reptiles (Cooper ef al., 1985). 2.3.4 SEASONAL FLUCTUATIONS IN REPTILIAN IMMUNE SYSTEMS The immune system of reptiles, indeed in all ectotherms, is not maintained at a stable functional level throughout the year. There are seasonal fluctuations in both structure and function of ectothermal immune systems (Zapata et al., 1992). Seasonal changes have been reported in multiple levels of the reptilian immune system: lymphoid organ structure, number of rosette-forming cells and plaque-forming cells, cell viability, proportion of T-like and B-like cells, antibody titres, response to mitogens and mixed leucocyte reactions (Saad, 1988). 2.3.5 SEASONAL CHANGES IN MORPHOLOGY The area of the immune system best studied for seasonality is probably lymphoid tissue morphology. The ectotherm trend is for a transient regression in lymphoid tissue during the breeding period and again in the winter (Zapata et al., 1992). The lymphoid complex of squamates and turtles atrophies and regenerates in a periodic, species-specific manner (El Ridi et al., 1988). In reptiles, two general patterns of seasonal immune tissue modification have been found. There is either a single seasonally- associated peak in lymphoid organ development or there are two peak seasons of increased development of the lymphoid organs during any given year. For the lizards Chalcides ocellatus (Hussein et al., 1978a), Mabuya quinquetaeniata, Uromastyx aegyptia (Hussein et al., 1978b) and Scincus scincus (Hussein et al., 1979b), summer represents the season of maximal development of the lymphoid organs and the greatest number of lympho- cytes. In winter, these lizards exhibit an involuted thymus and a spleen depleted of white pulp — strong evidence that the amount of lymphoid tissue in both of these organs was significantly reduced. A turtle, Mauremys caspica, also displays a single peak in thymus and spleen condition, which differs from the lizard pattern primarily in the timing of maximal lymphoid development (Leceta and Zapata, 1985). In contrast, the immune complex of the snakes Spalerosophis diadema (Hussein et al., 1979a), Psammophis