BLOOD CLINICAL BIOCHEMISTRY AND PACKED CELL VOLUME OF THE CHINESE THREE-STRIPED BOX TURTLE, CUORA TRIFASCIATA (REPTILIA: GEOEMYDIDAE)

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1 BLOOD CLINICAL BIOCHEMISTRY AND PACKED CELL VOLUME OF THE CHINESE THREE-STRIPED BOX TURTLE, CUORA TRIFASCIATA (REPTILIA: GEOEMYDIDAE) Author(s): Alessandro Grioni, D.V.M., M.V.S. (Conservation Medicine), M.R.C.V.S., Kevin K. Y. Ho, B.Sc., Ph.D., Nancy E. Karraker, B.Sc., M.Sc., Ph.D., and Kenneth M. Y. Leung, B.Sc., M.Phil., Ph.D. Source: Journal of Zoo and Wildlife Medicine, 45(2): Published By: American Association of Zoo Veterinarians DOI: URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Journal of Zoo and Wildlife Medicine 45(2): , 2014 Copyright 2014 by American Association of Zoo Veterinarians BLOOD CLINICAL BIOCHEMISTRY AND PACKED CELL VOLUME OF THE CHINESE THREE-STRIPED BOX TURTLE, CUORA TRIFASCIATA (REPTILIA: GEOEMYDIDAE) Alessandro Grioni, D.V.M., M.V.S. (Conservation Medicine), M.R.C.V.S., Kevin K. Y. Ho, B.Sc., Ph.D., Nancy E. Karraker, B.Sc., M.Sc., Ph.D., and Kenneth M. Y. Leung, B.Sc., M.Phil., Ph.D. Abstract: Reference intervals for blood biochemical analytes and packed cell volume are useful to veterinarians and conservation programs for monitoring the health of threatened species. Populations of the critically endangered Chinese three-striped box turtle, Cuora trifasciata (Bell, 1825), are heavily depleted in the wild because of overharvesting; and many individuals are now maintained in captive breeding programs. Reference intervals of biochemical analytes and packed cell volume were established for individuals of this species held in a captive breeding program at Kadoorie Farm and Botanic Garden, Hong Kong. Blood samples were obtained from 86 healthy individuals from a captive population during annual health examinations between 2006 and Packed cell volume and 17 biochemical analytes were measured, and data were analyzed using both multivariate and univariate statistical analyses. Significant differences in eight analytes between males and females were identified. Ten analytes were found to increase or decrease significantly with the body weight of the turtles. The reported plasma reference intervals, based on 2.5th and 97.5th percentiles, may serve as benchmarks for clinical assessment and conservation of this critically endangered species. Key words: Biochemistry, blood, Cuora trifasciata, plasma, reference interval, turtle. INTRODUCTION From Kadoorie Farm and Botanic Garden, Lam Kam Road, Tai Po, New Territories, Hong Kong, The People s Republic of China (Grioni); Department of Natural Resources Science, University of Rhode Island, 105 Coastal Institute at Kingston, Kingston, Rhode Island 02881, USA (Karraker); and The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, The People s Republic of China (Ho, Leung). Correspondence should be directed to Dr. Leung (kmyleung@hku.hk). The Chinese three-striped box turtle, Cuora trifasciata (Bell, 1825), inhabits hillside streams and riparian woodlands of southern China, Laos, and Vietnam. 5,7,56,57 This species feeds on small fishes, shrimps, crabs, earthworms, and aquatic insects. 7,29,38 Being a long-lived animal, it can reach a maximum life span of.30 yr. 50 Nevertheless, it shows delayed sexual maturity, with males maturing at 4 yr of age and females maturing at 7 yr of age. 39,53 In summer, mature females lay only three to seven small eggs; thus, this species is particularly vulnerable to exploitation. 38 Like many freshwater turtles, C. trifasciata is threatened with extinction because of overcollection, habitat destruction, and illegal trade. 5,27,32,57 Wild populations have declined to,1,000 individuals; thus, the species is currently listed as critically endangered by the International Union for Conservation for Nature based on its level of exploitation and projected population reduction. 33 Cuora trifasciata is also classified as a grade II stateprotected animal in China where hunting of this species is prohibited. 57 Populations have become so heavily depleted throughout its range that the Convention on International Trade in Endangered Species of Wild Fauna and Flora recently enacted a zero export quota on this appendix IIlisted species, effectively eliminating legal commercial trade in the species. 16 Cuora trifasciata, also known as the golden coin turtle symbolizes longevity and wealth for Chinese people. 17,30 It has been widely used in traditional Chinese medicine for.3,000 yr, because many people believe that it contains cancercuring properties. 5,36,58 Turtle jelly (or Gui Ling Gao, made of the plastron boiled with herbs) is an example of one of its medicinal uses, whereas its meat, dried carapace, and plastron are also eaten. 36,37 This species is seen in the pet trade and is sold in the black market in China because it is believed to bring good fortune to a family. 5,25,30,52 Decline of this species in China has become so severe that the high demand 5,58 and the everincreasing market price (up to US$2,000/kg) 25,26 are currently sustained by importing wild turtles from Vietnam and Laos or from breeding turtles in captivity. 5 The continued harvesting of turtles from the wild makes the species scarce; thus, they become more valuable to collectors. 23 Ongoing extirpations and declines in populations suggest that this species is at severe risk of extinction in the wild. 5,49,52 Conservation actions to protect C. 228

3 GRIONI ET AL. CLINICAL BIOCHEMISTRY FOR CUORA TRIFASCIATA 229 trifasciata are immediately needed. 38 Little is known, however, regarding the health conditions of wild and captive individuals, and no assessment methods have been established. Blood reference intervals are often used to assess the health of animal populations. For measured characteristics, this refers to values within upper and lower bounds in which a healthy population is contained. 2,24 A reference interval has not been previously established for C. trifasciata and would be useful for assessing the health of individuals from the wild and held in captive breeding programs. Thus, this study aims to establish plasma reference intervals for packed cell volume (PCV) and 17 biochemical analytes for C. trifasciata and to identify factors (time, sex, and body weight) associated with differences in blood biochemistry and hematology of the species. The results will provide benchmarks for health diagnosis and monitoring for C. trifasciata and hence facilitate its conservation and the sustainability of wild populations. 46,54 MATERIALS AND METHODS Testing individuals All individuals of C. trifasciata included in this study are held in captivity as part of a joint program of the Agriculture, Fisheries, and Conservation Department (AFCD) of the Hong Kong Special Administrative Region Government and Kadoorie Farm and Botanic Garden (KFBG). The KFBG holds the animals as part of an ex situ captive breeding program. The program commenced in July 2001, and at present it includes 113 C. trifasciata. The group is composed of captive-bred and wild hatchlings, juveniles, and breeding adults, as well as turtles confiscated by the authorities from illegal trade. All hatchlings are maintained in indoor tanks, and juveniles and adults are held in outdoor enclosures. Two to seven individuals are held in an outdoor breeding unit that is designed to have a dry area and a freshwater pool. All turtles are fed on the same diet composed of freshwater turtle pellets (Mazuri, USA), fresh vegetables, fruit and fish. All fresh items are supplemented with Nutrobal calcium powder (VETARK Professional, Winchester, XN, United Kingdom). Each individual is identified via a microchip, carapace notch, or both that relate to a unique identification number according to the computerized Animal Records Keeping System (ISIS, Eagan, Minnesota , USA). All resident turtles undergo annual health examinations that take place in September or October to avoid handling turtles during the egg-laying season from May to August. 39 The examination for all turtles consists of an external physical examination, coelomic palpation for eggs, blood sampling, and treatment for internal parasites and also includes three radiographic projections on a selected group of animals. Blood collection and sample processing Blood samples were collected from 86 healthy juveniles and adults (38 males and 48 females) of C. trifasciata during the examinations between 2006 and All animals examined had a carapace length.12 cm, the minimum length at which individuals are moved from indoor holding tanks to the outer enclosure. Turtles were not fasted before blood collection. The venipuncture site was either the jugular vein or the subcarapacial (subvertebral) sinus. Blood was collected with a 1-ml tuberculin syringe (BD Biosciences, Singapore, ) and a 25-ga 5/8 needle (BD Biosciences) for the jugular sites, whereas the same syringe and a 22-ga 1½ needle (BD Biosciences) were used at the subcarapacial site. The needle was bent to about 708 on the needle hub and was then inserted into either jugular vein, just caudally to the animal s tympanum, at an angle of about 458. The subcarapacial venipuncture was performed by inserting the needle dorsal to the neck and positioned immediately caudal to the attachment of the skin to the carapace. The needle was inserted with a dorsocaudal direction, angled at about 608 aiming to the margin between the nuchal and first vertebral scutes. Approximately ml of blood was collected from each turtle, and this was no more than 1% of the body weight of the animal. Samples were immediately placed in a lithium-heparin blood tube (Sarstedt, Nümbrecht, 51582, Germany) after collection and labeled with the appropriate turtle identification number. Tubes were kept temporarily refrigerated during collection by means of a thermal bag and ice boxes. After animals were sampled or after 2 hr had elapsed, whichever came first, blood samples were transferred to a refrigerator and maintained at a temperature of 3 58C until analysis for clinical biochemistry. Biochemical and hematologic analysis Blood samples were analyzed within 2 days after sampling. Up to April 2009, biochemical analyses were performed at Pathlab Medical

4 230 JOURNAL OF ZOO AND WILDLIFE MEDICINE Laboratories Ltd., Hong Kong, using a Vitros 5.1 FS analyzer (Ortho Clinical Diagnostics, Johnson & Johnson, Raritan, New Jersey 08869, USA). Blood samples were separated within 24 hr of collection using the reagent Vitros Slide (Ortho Clinical Diagnostics). Biochemical profiles were determined for the following analytes: albumin (ALB), amylase (AMY), aspartate aminotransferase (AST), Ca, cholesterol (CHOL), creatine phosphokinase (CPK), c-glutamyl transferase (GGT), lactate dehydrogenase (LDH), P, K, Na, total protein (TP), triglyceride (TRYGL), and uric acid (UA). After April 2009, blood samples were separated between 4 and 6 hr after collection and biochemical analyses were performed using Avian/Reptilian Profile Plus (Abaxys, Union City, California 94587, USA) with an in-house blood analyzer (VetScan VS2, Abaxys). Profiles included ALB, AST, bile acids (BA), Ca, CPK, globulin (GLOB), glucose (GLU), P, K, Na, TP, and UA. PCV, the only hematologic analyte in this study, was determined using a StatSpin centrifuge (Iris Company, Westwood, Massachusetts 02090, USA) and a graduated scale. All biochemical and hematologic analytes were evaluated at 378C. In some cases, unequal sample sizes were found among analytes due to insufficient blood volumes. Statistical analysis Identifying outliers: Data were examined for outliers using Dixon s outlier range statistic. 21 Extreme values were identified as outliers if D/ R. 0.3, where D is the absolute difference between the most extreme and the next nearest value, and R is the range of all values. Extreme values were removed before further analyses. Identifying temporal and sex differences: Nondimensional scaling was used to elucidate temporal differences among biochemical analytes. An analysis of similarities was used to compare these differences. A similarity matrix was obtained using the Bray Curtis index, and the measured values of each analyte were logarithmically transformed to minimize the influence of relatively large values. Tests were performed using Primer (Primer-E Ltd., Ivybridge, PL21 9RH, United Kingdom). Because the analysis failed to meet the assumptions of normality and equal variances, a Mann-Whitney U-test was used to examine temporal differences of PCV and sex differences among the medians of all analytes. A Spearman rank correlation test was used to determine whether relationships existed between each plasma analyte and turtle body weight. If sex differences were identified previously, correlation analyses were conducted separately by sex. A sequential Bonferroni correction was used to minimize the cumulative type-i error incurred in the multiple correlation tests. Estimation of reference intervals: Reference intervals of each analyte were presented as 2.5th and 97.5th percentiles. These percentiles indicated the central 95% of values in which the values for healthy populations were expected to be located. This method has been commonly adopted in establishing reference intervals in humans 41 and in other turtles and tortoises. 3,13,24 When analyte levels were sex dependent, reference intervals were calculated separately for males and females. All univariate statistical analyses were performed using EXCEL version 2003 (Microsoft, Redmond, Washington , USA) and PASW Statistics 18.0 (SPSS Inc., Chicago, Illinois 60606, USA). RESULTS Temporal and sex differences Significant temporal and sex differences were noted for some biochemical analytes and temporal differences in PCV (Table 1). Temporal differences were documented in BA, GLOB, and GLU (ANOSIM, R ¼ 0.031, P, 0.007). Conversely, there were no temporal differences in levels of the following biochemical analytes: ALB, AMY, AST, Ca, CHOL, CPK, GGT, LDH, P, K, Na, TP, TRYGL, and UA (ANOSIM, R ¼ 0.045, P, 0.159). PCV differed between 2009 and 2010 (Mann-Whitney test, U ¼ , P, 0.05). Thus, only 2010 data for BA, GLOB, GLU, and PCV were used in further analyses. Sex differences were identified in eight biochemical analytes including AMY (P, 0.05), Ca (P, 0.001), CHOL (P, 0.001), GGT (P, 0.05), LDH (P, 0.001), P (P, 0.01), TRYGL (P, 0.001), and UA (P, 0.001) (Fig. 1). No sex differences were observed in the other analytes (P. 0.05). Correlation with body weight Nine biochemical analytes and PCV were significantly correlated with turtle body weight. For sex-independent cases, ALB (P, 0.001), AST (P, 0.001), GLOB (P, 0.01), and TP (P, 0.001) were positively correlated with body weight. CPK (P, 0.001), GLU (P, 0.001), and PCV (P, 0.05) were negatively correlated with body weight (Fig. 2).

5 GRIONI ET AL. CLINICAL BIOCHEMISTRY FOR CUORA TRIFASCIATA 231 For sex-dependent cases, Ca was positively correlated with the body weight of female turtles (P, 0.001), but not with that of males (P. 0.05). P was negatively correlated with the body weight of males (P, 0.05), but positively correlated with that of females (P, 0.001). UA was negatively correlated with the body weight of males (P, 0.001) and females (P, 0.001) (Fig. 3). DISCUSSION Blood biochemistry and hematology often vary with sex, age, and diet of turtles, venipuncture sites, and time of survey. 1,4,44 In the present study, blood profiles differed between the sexes of C. trifasciata in eight biochemical analytes. Female turtles had higher values for AMY, Ca, CHOL, P, and TRYGL, whereas males had higher levels of GGT, LDH, and UA. As expected, higher Ca occurred in female turtles 3,8,48 because Ca is essential for eggshell formation 15,42,45 and other body functions, such as hormone action, cardiac and skeletal muscle contraction, smooth muscle contraction, and nerve impulse transmission. 28 Female turtles also showed elevated CHOL 6,14,47 that is associated with estrogen production. 3 Being an important component of egg yolk, CHOL also plays a critical role in egg development and vitellogenesis. 18,29 Elevated P and TRYGL levels in females were also attributed to egg production and vitellogenesis. 13 Higher TRYGL level in females facilitates ovarian growth and interacts with progesterone. 12 Studies in other turtle species have shown that mature females often have higher levels of TRYGL, constituents that are essential to supporting egg production. 14,34 LDH increases with metabolic rate in other turtle species, 13,14 suggesting that male C. trifasciata probably have higher metabolic rates than females. In addition, males exhibited higher levels GGT and UA, and females had elevated levels of AMY. Further research is needed to elucidate the different profiles of these plasma analytes between the sexes in C. trifasciata. This study indicated that levels of many plasma analytes were body-size dependent. Body weight of C. trifasciata correlated positively with the analytes ALB, AST, GLOB, and TP and negatively with CPK and GLU, and also PCV. The correlations were found to be sex dependent in Ca, P, and UA. ALB and TP have also been found to increase with age in loggerhead turtles (Caretta caretta). 34 Plasma levels of ALB and TP are usually related to dietary intake wherein larger turtles may consume more food that elevates analyte levels. 10 Ca is bound to plasma proteins, such as ALB, in the blood. 43 Healthy adult female turtles at KFBG are considered to be reproductively active. Therefore, a physiological increase of Ca corresponds to an increase in ALB. TP may increase in response to estrogen-induced hyperproteinemia because of the increase in GLOB, a type of protein used for the production of yolk or vitellogenesis. 9 Ionized Ca, considered the most reliable indication of physiologically active Ca in circulation, 9 was not assessed in this study because of equipment limitations. It was found that P decreased in males and increased in females with increasing body weight, and similar observations have been documented in other turtle species. 22 Mild hyperphosphatemia in reproductive females may be due to bone remodeling and folliculogenesis. 22 Because the mechanism underlying changes in AST with turtle size is still unknown, further research in this area is needed. CPK, an enzyme responsible for energy metabolism, is associated with tissue activities and metabolic rate. 51 A decrease was reported in loggerhead sea turtles.12 mo old. This was suspected to be related to a decrease in population density, and therefore animal interactions, resulting in a reduction of stress and fighting wounds. 34 Increased CPK levels may also be related to infections, muscle injuries, and exertion, especially during animal handling (e.g., during the venipuncture procedure). 9,55 In C. trifasciata, plasma levels of GLU and PCV also decreased with body weight, and such trends are likely to be associated with age-dependent differences in their metabolism. In reptiles, lymph hemodilution may occur at all venipuncture sites, and it is still not known how it influences results of blood analyses. 4,31 Therefore, the results obtained from blood taken from both the subcarapacial sinus and the jugular site were combined. However, samples obtained from the subcarapacial sinus were discarded if hemodilution was considered excessive. During summer, turtles were always fed in the afternoon every other day, and they usually ingested the food immediately after it was provided. Blood was collected in late morning regardless of the feeding practice of the previous day. Blood lipemia could occur as a dietary consequence or metabolic problem, whereas lipemic blood has been reported to cause artifacts, especially for Ca and UA levels. 19,55 In this study, however, no plasma was found to be lipemic.

6 232 JOURNAL OF ZOO AND WILDLIFE MEDICINE Table 1. Reference intervals (RI) of 17 biochemical analytes and packed cell volume (PCV) for blood samples of C. trifasciata. If there are significant differences between the sexes (P, 0.05), the values are provided for each sex. The 17 biochemical analytes measured include albumin (ALB), amylase (AMY), aspartate aminotransferase (AST), bile acids (BA), Ca, cholesterol (CHOL), creatine phosphokinase (CPK), c-glutamyl transferase (GGT), globulin (GLOB), glucose (GLU), lactate dehydrogenase (LDH), P, K, Na, total protein (TP), triglyceride (TRYGL), and uric acid (UA). Lower reference Upper reference Analyte n Mean SD Median Minimum Maximum RI a limit (90% CI) b limit (90% CI) Period of data collection ALB (g/dl) AMY (U/l) Male Female AST (U/l) BA lmol/l 73 n.d. c n.d.,35,35 61,35 n.d. n.d Ca (mg/dl) Male Female CHOL (mg/dl) Male Female CPK (U/l) GGT (U/l) Male Female GLOB (g/dl) to GLU (mg/dl) LDH (U/l) Male 34 4, , , , , , , , , , , Female 37 3, , , , , , , , , , , P (mg/dl) Male Female K (mmol/l) Na (mmol/l) TP (g/dl) TRYGL (mg/dl) Male to Female , to , ,

7 GRIONI ET AL. CLINICAL BIOCHEMISTRY FOR CUORA TRIFASCIATA 233 Table 1. Continued. Period of data collection Lower reference Upper reference Analyte n Mean SD Median Minimum Maximum RI a limit (90% CI) b limit (90% CI) UA (mg/dl) Male Female PCV (%) a RI is defined as 2.5th percentile to 97.5th percentile of values. b CI, confidence interval. c n.d., not determined. The time of survey within a year may also affect the results. Annual health examinations, including blood collection, were performed at the end of each year to reduce disturbance during the egglaying period. Timing of blood sampling is also appropriate because venipuncture is performed just before the winter when these animals enter into a dormant phase. During the winter, folliculogenesis and vitellogenesis are likely to cease in C. trifasciata. Although establishing reference intervals is beneficial to animal health and conservation, previous studies indicate that there may be several limitations to this approach. 20,35,40 Laboratories vary with equipment, techniques, and sampling methodologies and may yield different reference intervals for the same blood sample. Repeated sampling and careful interpretation are therefore recommended. Blood samples collected over the span of a year would enable us to capture temporal variation in blood chemistry and hematology. Larger sample sizes would ensure that variation in PCVs and analyte levels among individuals in population is captured, thus resulting in the development of more precise reference intervals. This would also reduce the impact on reference intervals of individuals suffering from subclinical diseases or abnormalities. 11 Reference intervals may differ between animals in natural habitats and captive facilities. Therefore, caution should be used in extrapolating the results from captive to wild populations. 6 However, for many species of endangered turtles, such as C. trifasciata, extremely small sizes of wild populations may make it nearly impossible to establish statistically sound blood profiles for wild populations. Because populations of this species are declining and more captive breeding projects have been set up in different countries, our results will enhance diagnostic procedures for the growing captive populations worldwide. This study serves as the first step of a long-term study of the blood biochemistry and hematology of C. trifasciata that will enable finetuning of plasma reference intervals and thus facilitate further studies that assess the health of captive and wild C. trifasciata populations. Additional study is needed to evaluate the blood profile of this species in different seasons, among various age groups and in different captive conditions, and should include additional hematologic analytes, such as blood cell counts and ionized Ca.

8 234 JOURNAL OF ZOO AND WILDLIFE MEDICINE Figure 1. Boxplots showing biochemical analyte values of blood samples of C. trifasciata with significant differences between sexes (M, male, F, female). Analytes include (a) amylase (AMY), (b) Ca, (c) cholesterol (CHOL), (d) c-glutamyl transferase (GGT), (e) lactate dehydrogenase (LDH), (f ) P (PHOS), (g) triglyceride (TRYGL), and (h) uric acid (UA). CONCLUSIONS This study has established reference intervals for various plasma biochemical analytes and PCV for the critically endangered C. trifasciata and evaluated analytes by sex and body size. These reference intervals will be useful to veterinarians and wildlife conservationists for monitoring the health of this turtle species, particularly in captive

9 GRIONI ET AL. CLINICAL BIOCHEMISTRY FOR CUORA TRIFASCIATA 235 Figure 2. Significant relationships between measured analytes of blood samples of C. trifasciata and their body weights. Analytes include (a) albumin (ALB), (b) aspartate aminotransferase (AST), (c) creatine phosphokinase (CPK), (d) globulin (GLOB), (e) glucose (GLU), (f) total protein (TP), and (g) packed cell volume (PCV).

10 236 JOURNAL OF ZOO AND WILDLIFE MEDICINE Figure 3. Significant sex-dependent relationships between measured analytes of blood samples of C. trifasciata and body weight. Analytes include (a) Ca, (b) P (PHOS), and (c) uric acid (UA). Left column indicates males, and right column indicates females. breeding programs, and they may facilitate both clinical and conservation actions. Further studies should investigate age and seasonal variation in blood biochemistry and hematology of C. trifasciata and evaluate how venipuncture sites may influence reference intervals. Acknowledgments: We thank the AFCD of the HKSAR Government and the Fauna Conservation Department of the KFBG for supporting this project, Dr. Gary Ades for proofreading a draft of this paper, Ms. Christine Cheung for technical assistance, and Dr. Danny Lau for advice on some of the statistical analysis. KKYH thanks The University of Hong Kong for partially supporting his Ph.D. studentship. LITERATURE CITED 1. Aguirre AA, Balazs GH. Blood biochemistry values of green turtles, Chelonia mydas, with and without fibropapillomatosis. Comp Haematol Int. 2000;10:

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