Seasonal variation in blood biochemistry of long term captive Mediterranean tortoises (Testudo graeca and T hermanni)

Research in Veterinary Science 1987, 43, 379-383

Seasonal variation in blood biochemistry of long term captive Mediterranean tortoises (Testudo graeca and T hermanni) K. LAWRENCE, 23 Woodside Gardens, Chineharn, Basingstoke, Hampshire RG24 OEU

Reference values for five blood chemistry parameters hibernating tortoises, especially in respect of underin 18 Mediterranean tortoises of two species, Testudo standing conditions such as post hibernational graeca and T hermanni, were determined on up to 10 anorexia (Lawrence and Jackson 1983), the situation occasions during the year. Statistically significant has been reviewed. The previously published data has seasonal variations were demonstrated in blood urea been re-examined and three other blood biochemical and blood glucose. Seasonal variations were demon- parameters have been measured, so that a meaningful strated in blood urea and blood glucose. Seasonal comparison with findings from tortoises presented variations in total plasma proteins, lipids and choles- for clinical examination at different times of year can terol, however, were limited to gravid females. The be made. In addition aninsight into the use of stored study also suggested that three energy sources were energy sources during hibernation is gained, which available to the tortoise during hibernation, lipids may reflect possible energetic problems exacerbated stored in the fat body, endogenous protein degrada- by captivity in the UK. tion and glucose derived from hepatic glycogen. TORTOISES have been imported into the United Kingdom from the Mediterranean region since the 1890s. Numbers imported gradually increased, because of the popularity of tortoises as pets, until by 1937 as many as 200,000 were arriving annually at the London Docks. The outbreak of the second world war curtailed the tortoise trade until the early 1950s. The number of animals imported peaked in 1967 when licenses authorising the importation of 476,186 tortoises were issued. The mass trade in tortoises finally ceased on January I, 1984. However, nearly three million tortoises were imported into the United Kingdom between 1965 and 1979 with estimates of up to six million since the early 1950s (Lambert 1980). Therefore it is interesting that little basic physiological data has been collected on an animal that has been maintained in captivity in such large numbers , for nearly 40 years. Two papers have been published illustrating a seasonal variation in blood and plasma urea levels in Testudo hermanni (Gilles-Baillien and Schoffeniels 1965, Gilles-Baillien 1969). A cyclic variation in blood glucose concentrations was also shown in T hermanni by Kuchling (1981). Seasonal variations in haematological values have been demonstrated in T graeca and T hermanni by Gilles-Baillien (1973) and Lawrence and Hawkey (1986). In view of the physiological implications of seasonal changes in the blood of

Materials and methods Eighteen healthy, long term captive tortoises, 12 spur-thighed (T graeca) and six Hermann's (T hermann i), were maintained in captivity as described by Collins (1980). The animals ranged in weight from 700 to 2625 g. Further details of the animals are recorded by Lawrence and Hawkey (1986). Blood samples were taken on IOoccasions as part of a health screening programme, although not all samples were examined for all blood components. The tortoises were sampled up to six times during hibernation, on leaving hibernation, one month after leaving hibernation, midsummer (this coincided with egg laying in some females) and on entering hibernation. Blood samples were collected from the dorsal coccygeal vein (Samour et al 1984) and were mixed with lithium heparin at a concentration of 0·75 mg rnl: I blood. A total of 180samples were collected and 630 analyses were undertaken. Samples were examined using the Unitest 300 System (Boehringer Corporation) for blood urea (catalogue number 288730), blood glucose (catalogue number 288586), total plasma protein (catalogue number 288560), total plasma lipids (catalogue number 288799) and plasma cholesterol (catalogue number 288497). The mean, standard deviation and range for each parameter was determined and the data were tested for the effect of season using Student's paired I test.

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The blood biochemistry findings from clinically normal adult T graeca and T hermanni are listed in Table. 1 and presented graphically in Fig I. Data from gravid females during the period April to October is shown in Fig 2. Seasonal trends were similar in both species for all data and both sexes for blood urea and glucose. However, there were statistically significant increases in total plasma protein, plasma lipids and cholesterol in gravid females around the time of oviposition in August. Both blood urea and glucose showed a statistically significant peak in April at the time of arousal from hibernation. While the blood urea levels had started to rise in March the blood glucose peak was much more obviously associated with awakening. Total plasma protein, lipids and cholesterol in general showed little seasonal variation with individual variation within a month being greater than the overall seasonal trends. However, there was a well defined rise in total plasma protein, lipids and cholesterol in sexually active females of both species in association with egg laying.

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Gilles-Baillien and Schoffeniels (1965) presented data showing the monthly variation of blood urea levels in recently imported T hermanni. They showed a peak level in April of 103' 7 mmol litre - I in comparison with 103' 3 mmollitre- I for T hermanni and 109·7 for T graeca in this study. They demonstrated a gradual fall in blood levels to a low of 3·9 mmol litre ' ! in July before the levels gradually built up again. However, the 'hibernation' levels from October to February were three times greater than those shown in Table I or Fig 17although the March levels were similar. In the second study reported by Gilles-Baillien (1969), again in T hermanni, peak levels were again found in April (93' 9 mmol litre -I) with a low in June and-July. The 'hibernation' levels, however, did not start to rise until December when they reached 22 mmol litre-I. The blood urea levels then remained steady until the beginning of April before they rose sharply to peak in late April. The pattern of change shown in Fig 1 is similar to the two published reports but the blood urea levels between October and February are much lower. Previously published studies involved recently imported individuals entering their first hibernation since importation. This first hibernation was assoiciated with weight losses of at least 20 per cent (GillesBaillien 1973), far in excess of the average weight loss in the long term captives of 5 .22 per cent for T graeca and 4· 55 per cent for T hermanni (Lawrence and Hawkey 1986). The increased weight loss (in associa-

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tortoises between May and October was 3' 10 mmol litre-I. Kuchling does not report blood glucose levels associated with arousal from hibernation. However hibernating animals that were 'stressed' by being warmed to room temperature before killing showed a sudden rise in blood glucose to 19'66 mrnol litre-I. This mirrors the situation when tortoises became active at the end of hibernation. This sudden peak of glucose may act as a trigger to arousal and it appears to he associated with a rise in environmental temperature. In the winter of 1984-85 a veryrnild period in January 1985 caused the arousal of tortoises throughout the United Kingdom. In a small survey involving IS of these tortoises all showed a rise in blood glucose to more than 10 mmol litre - I. Of the 15 tortoises, nine did not show a peak when aroused again in March 1985 and subsequently developed post hibernational anorexia (PHA) (Lawrence and Jackson 1983). Total plasma protein

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tion with a lower weight to length ratio) showed that the tortoises were in poor condition before hibernation. The increased blood urea levels indicated that protein catabolism provided a large proportion of the energy requirements during hibernation in the recently imported tortoise. In contrast, the long term captives in the present study were heavier in relation to length and had, in consequence, larger energy stores in the form of the fat body. This reduced the .dependence on protein catabolism as an energy source during hibernation. The variation in blood glucose levels throughout the year showed a statistically significant increase in April and May. Kuchling (1981) reported glucose levels in active tortoises between April and October of 1·43 mmol litre-I. However, this was an average figure for 31 individuals each sampled on a single occasion with no indication of monthly variation. The mean level reported in this study for active

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It appears that as the environment warms up in protein. Indeed Gilles-Baillien (1969) suggested that a early spring a rise in blood glucose levels is triggered, metabolic arousal precedes total awakening which this rise being caused by the conversion of liver implies an enhanced catabolism of protein in late glycogen. In hibernating reptiles the degree of hibernation. This increase in protein catabolism is not glycaemia in the winter is inversely correlated with the reflected in the total plasma protein in April at amounts of glycogen stored (Gregory 1982). If the arousal because samples were not analysed. liver glycogen is depleted by a period of unusually high temperature during hibernation a second arousal Plasma lipids and plasma cholesterol peak may not occur in March or April. These animals may then not be stimulated to recommence feeding. The final two parameters, plasma lipids and cholesIf tortoises become active during hibernation, it terol, showed little seasonal variation except in gravid would seem wise to recommend that they should be females, where levels peaked around the time of ovikept awake and active until the spring. This will give position. Seasonal variations have not been studied in the owner the chance to initiate feeding in synchrony many reptiles and Gregory (1982) concluded that the with the glucose surge. The arousal peak may also be relationship between plasma lipid levels and fat use reduced or indeed abolished if an underweight-for- during hibernation is unclear. Marked increases in length tortoise enters hibernation (Jackson 1980), the plasma lipids and protein have been reported in animal being light in weight because of inadequate turtles accompanying oestrus (Laskowski 1936, Clark energy reserves stored in the fat body or in the liver as 1967)so that the peaks in plasma lipid and cholesterol glycogen. The speculation that high blood glucose in egg-laying females are not unexpected. Why is levels represent a source of readily available energy on there no obvious peak in total plasma protein in arousal from hibernation is not novel. August? The mean figure presented for August in Hutton and Goodnight (1957) demonstrated a Fig 1 is 82· 2 g litre - I. However if the gravid females similar relationship in two species of chelonia, are examined separately from the non-pregnant Terrepena carolina and Pseudemys scripta elegans, as females and males the figures are 96'14 g litre- 1 and did Peters (1959) in Terrepena ornata. 72· 43 g litre -1, respectively, a statistically significant There were no statistically significant seasonal difference (P<0·02). changes in total plasma protein except in egg-laying The absolute cholesterol levels in both species of females in August. However, there was a trend for a tortoise reported here are low compared with studies gradual reduction in levels during hibernation with a in other species. Jackson and Legendre (1967) showed rise in March. The rise, however, may only reflect a cholesterol levels between 5 and 13 mmol litre"! in reduction in the volume of circulating blood as the chelonians. However, in the only two vegetarian March increase in total plasma protein is associated species listed, the cholesterol levels were around with an increase in packed cell volume (Lawrence and 2 mmol Iitre" '. Therefore the apparently low levels in Hawkey 1986). The pattern of seasonal change is the tortoises were associated with the predominantly similar to that reported for the turtle Malaclemys geo- vegetarian diet offered during the investigation. The study also suggests that three energy sources graphica (Campbell and Turner 1937). The use of protein as an energy source during are available to the tortoise duririg hibernation. (i) Fat stored in the fat body. This source of stored hibernation has not been investigated thoroughly in reptiles (Gregory 1982). However, Aleksuik and energy meets the majority of the hibernating Stewart (1971) suggested that protein may be an tortoise's needs for most of the hibernation. If, important energy source during the hibernation of however, the tortoise enters hibernation with a low Thamnophis sirtalis. The respiratory quotient (RQ) of weight-to-length ratio endogenous proteins may hibernating tortoises (T graeca) is reported as 0'73 supercede as the energy source. (Kayser 1940) and it was suggested by Bennett and (ii) Degradation of endogenous protein. It has been Dawson (1976) that this be accepted as indicating fat suggested by Gilles-Baillien (1969) that metabolic catabolism rather than protein. This is too simplistic a arousal precedes total awakening and that an statement as Roberts (1968) showed that an RQ of enhanced catabolism of protein is reflected by the rise 0'71 was associated with both fat and protein in blood urea concentration in late hibernation. The catabolism in the lizard Uta stansburiana. It was rise may be sooner if fat stores are prematurely suggested that a low RQ could also be associated with depleted. (iii) Glucose derived from hepatic glycogen. The protein catabolism in uricotelic reptiles. The tortoise, T graeca, is to a large extent uricotelic with up to 52 sudden increase in blood glucose levels on arousal per cent of the total nitrogen excretion being in the from hibernation represent a readily available source form of uric acid (Schmidt-Neilsen 1980). The rise in of energy. In tortoises in which this surge of glucose the blood urea concentration during hibernation can does not occur classic post hibernational anorexia only result from a degradation of endogenous occurs. The arousal peak will be depressed or

Tortoise blood biochemistry eliminated by periods of high temperature during hibernation or if animals hibernate with insufficient stored energy resources. The second cause has profound implications when considering the mortality of recently imported tortoises. References ALEKSUIK, M. & STEWART, K. W. (1971) Ecology 52, 485-490 BENNETT, A. F. & DAWSON, W. R. (1976) Biology of the Reptilia. Eds C. Gans and W. R. Dawson. Volume 5A. London, Academic Press. p 172 CAMPBELL, M. L. & TURNER, A. H. (1937) Biological Bulletin of the Marine Biology Laboratory, Woods Hole 73,504-510 CLA RK, N. B. (I %7) Comparative Biochemistry and Physiology 20, 823-834 COLLINS, P. (1980) Testudo I, 27-40 GILLES-BAILLlEN, M. (1969) Archives Internationales de Physiologie et de Biochimie 77, 427-440 GILLES-BAILLlEN, M. (1973) Archives lnternationales de Physiologie et de Biochimie 81, 723-732 GILLES-BAILLlEN, M. & SCHOFFENIELS, E. (1965)Annales de la Societe Royale Zoologique de Belgique 95,75-79 GREGORY, P. T. (1982) Biology of the Reptilia. Eds C. Gans and F. H. Pough. Volume 13. London, Academic Press. pp 53-154

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HUTTON, K. E. & GOODNIGHT, C. J. (1957) Physiological Zoology 30, 198-207 JACKSON, C. G. & L~GENDRE, R. C. (1967) Comparative Biochemistry and Physiology 20, 311-312 JACKSON, O. F. (1980) Journal of Small Animal Practice 21, 409-416 KAYSER, C. (1940) Annales de Physiologie et Physiochimie Biologique 16, 1-68 KUCHLlNG, G. (1981) Amphibia-Reptilia 2, 235-241 LAMBERT, M. R. K. (1980) Proceedings of the European Herpetological Symposium, Cotswold Wildlife Park, Oxford. pp 17-23 LASKOWSKI, M. (1936) Biochemie Zoologie 248, 318-321 LAWRENCE, K. & HAWKEY, C. (1986) Research in Veterinary Science 40,225-230 LAWRENCE, K. & JACKSON, O. F. (1983) Veterinary Record 112, 487-488 PETERS, K. (1959) Transactions of the Kansas Academy of Science 62,15-20 ROBERTS, L. A. (1968) Ecology 49,809-819 SAMOUR, H. J., RISLEY, T., MARCH, T., SAVAGE, B., NIEVA, O. & JONES, D. M. (1984) Veterinary Record 114, 472-476 SCHMIDT-NEILSEN, K. (1980) Animal Physiology: Adaptation and Environment. 2nd edn. Cambridge University Press. p 36

Received August 28, 1986 Accepted January 23, 1987