Comparative Haematology International (1998) 8:174-177 ~) 1998 Springer-Verlag London Limited COMPARATIVE HAEMATOLOGY INTERNATIONAL Original Article Normal Blood Chemistry of Free-living Green Sea Turtles, Chelonia mydas, from the United Arab Emirates C. R. H a s b d n ~'3, A. J. L a w r e n c e 3, J. N a l d o 2, J. H. S a m o u r 2 and S. M. A1-Ghais ~ 3 1Marine Environment Research Institute, 2Wildlife Veterinary Research Institute, Environmental Research and Wildlife Developmenl Agency, Abu Dhabi, United Arab Emirates; ~Dept. of Biological Sciences, University of Hull, Hull, UK Abstract. Blood chemistry values were obtained from 62 clinically normal, free-living green sea turtles, Chehmia mydas, in order to establish normal blood chemistry values for this species in the Arabian Gulf. Sampled individuals were classed by sex and size for analysis. Keywords: Arabian Gulf; Blood chemistry; Chelonia mydas; Clinically normal; Green sea turtle Introduction The green sea turtle, Chelonia mydas, a herbivorous chelonian (Mortimer 1995) distributed in warm tropical waters, is one of the most studied species of sea turtles (Hughes 1995). Their occurrence in the waters of the Arabian Gulf has been documented (Anderson 1979; Miller 1989). Through qualitative observations made by the Environmental Research and Wildlife Development Agency (ERWDA) from the United Arab Emirates, it has been recorded that the waters of Ras A1-Khaimah (RAK), maintains a large population of this species. These waters, as well as the Arabian Gulf as a whole, possess unique environmental characteristics, due to its semi-enclosed nature (Basson et al. 1977) and extreme arid climatic conditions (B6er 1997). Water salinity is higher than that of open ocean waters as high evaporation rates and low freshwater input persists (Miller 1989). In addition, the risk of oil contamination in this region is high due to the existence of oil refineries Correspondence and offprint requests to. Carlos Roberto Hasbfin, Dept. of Biological Sciences, University of Hull, HU6 7RX, UK. (Krupp et al. 1996; Gasperetti et al. 1993; Aspinall 1995). In order to assess normal health parameters for this species in the Arabian Gulf, and therefore develop future comparisons from individuals detected in adverse conditions, ERWDA performed a green sea turtle health assessment study in RAK, located at 25°42'53"N and 55°47'45"E, throughout the month of May, 1997. As a component of this health assessment, blood was collected to establish normal blood chemistry values for ti"ee-living individuals, the findings of which are presented here. Materials and Methods The study was performed in Ras A1 Khaimah in the north-east of the UA, bordering the Sultanate of Oman near the Strait of Hormuz. Blood was collected from green sea turtles from the coastline between Yazirah A1 Hamrah (25°43'N-55°48'E) and Ar Rams, approximately 20 km north-east. Throughout the study area, fishermen cast their 2000 m beach seine approximately 3 km away from the shore. Using two boats, one at each end of the seine, they trawl towards the shore for fish. Turtles captured accidentally using this fishing technique were pulled out from the seine by the research team and turned on their carapaces for restraint. Turtles with fibropapillomas, characterised by multiple benign fibroepithelial tumours (Herbst et al. 1995) or other apparent abnormalities were not encountered. Each sampled individual was measured with a flexible plastic tape over the curve carapace length (CCL, nuchal notch to posterior notch), over the curve width, straight lengths B l o o d C h e m i s t r y o f the G r e e n S e a T u r t l e 175 urea nitrogen, triglycerides, Na +, K + and C1-. Quality control of all procedures was carried out using reference plasmas (Precinorm U, Precipath U, Precinorm UPX). Statistics comparing results between large females and large males and between large and small turtles were carried out using a Student's unpaired t-test (Microsoft Excel Analysis Toolpack, Microsoft Corporation, USA). A value of p<0.05 was taken to be significantly different. between plastron and cloaca (SPCL) and straight lengths between plastron and tail tip (SPTL). Since there are no strict guidelines using morphological and/or behavioural characteristics for the assessment of adulthood in large turtles, nor sex from smaller sea turtles, they were classed into groups of large females (CCL, 89), large males (CCL, 89), medium sized turtles (CCL, 88-70) and small turtles of undetermined sex (CCL, 69). Within 1 h of capture, and after tagging each individual for proper identification, turtles were bled from the dorsal cervical sinus, the jugular vein or the ventral caudal sinus using 5 ml syringes and 19 x 1.5 inch, 21 x 1.5 inch or 23 x 1 inch gauge needles. After collection, blood was immediately mixed in lithium heparin in commercially available storage tubes, and were stored in an ice box. Within 1 h of collection, samples were centrifuged at 10000g for 10 min using a bench-top microcentrifuge. A total of 62 blood plasma samples from clinically normal turtles were sent to the Central Veterinary Research Laboratory, Dubai, UAE. Blood chemistry was analysed using a Hitachi 90011 Analyser (Boehringer Mannheim, Germany). All commercial test kits and reference plasma were also supplied by Boehringer Mannheim. Total plasma protein and albumin were measured using the biuret and bromocresol green methods respectively, globulin and A/G ratio being derived from these values. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), lactate dehydrogenase (LDH) and creatinine kinase (CK) were measured at 37°C using commercial kits. Commercial kits were also used for magnesium (xylidyl blue reaction), calcium (o-cresolphthalein method), uric acid and cholesterol (enzymatic colorimetric assay) and for creatinine, iron, blood Results Results obtained from clinically normal sea turtles are presented in Table 1. When comparing values between large females and large males, there were no significant differences for most values with the exception of lactic dehydrogenase (LDH), higher in females (p<0.001); aspartate aminotransferase (AST), higher in males (/)<0.05); iron, higher in females (/)<0.05) and calcium, higher in males (p < 0.001). Discussion Blood chemistry values for green sea turtles have been established in free-living juveniles from Hawaii (Aguirre et al. 1995), the Bahamas (Bolten and Bjorndal, 1992), and from captive individuals (Norton et al. 1990). Bolten and Bjorndal (1992) found no differences between juvenile males and females except for phosphorus (male, 7 _+ 1.2; female, 6.5 _+ 1.2). In the present study, although large males had a lower phosphorus concentration than large females or small turtles of undetermined sex (7.11 _+ 1.92, 8.06 __ 1.96 and 8.53 T a b l e 1. B l o o d c h e m i s t r y v a l u e s f r o m clinically n o r m a l , f r e e - l i v i n g g r e e n s e a turtles Assay (Chelonia mydas) Large females (CCL~>89 cm) Large males (CCL~>89 cm) Medium turtles (88~<CCL~>70) Small turtles (CCL~>69 cm) Mean _+ SD n Mean -+ SD Mean _+ SD Mean _+ SD ALP(U/L) LDH (U/L) 27.21_+9.65 211.66_+139,39 19 18 10-43 50-491 29.75+16.59 85.08_+39.15 16 13 11-62 21 153 28.92_+9.9 99.92_+72.59 13 12 15-51 24 223 33.92+17.25 86.17+51.24 12 12 12 64 18-173 AST (U/L) Phos (mg/dl) 153.5_+48.48 8.06_+1.96 18 14 65-243 3.3 1 1 . 7 182.35_+46.71 7.11_+1,92 17 12 109-267 4.4 1 1 . 1 176.92_+47.87 7.55+1.61 13 10 136-303 4.4-9.9 178.25_+61.02 8.53_+1.6 12 9 112 336 6.4-11.2 ALB (g/dl) TP (g/db Crea(mg/dl) Iron (,ug/dl) CK (U/L) BUN (mg/dl) Ca (mg/dl) 1.91 _+0.32 5.73 _+0.55 0.43_+0.11 77.75_+35.72 702-+830.11 12.28_+9.53 6.86_+3.0 11 17 I6 12 12 18 13 1.5-2.5 4.7-6.8 0.26~0,64 50-163 122 3075 1-42 3.02-11.03 1.97_+0.30 6.34_+ 1.7 0.51_+0.14 52.2_+13,7 930.67_+901.33 7.15_+5.63 10.46+2,6 9 17 17 9 9 13 11 1.4-2.4 3-9.7 0.27~).77 39-78 227-3013 3-25 5.12-14.8 1.96_+0,3 5.9_+0.9 0.5+_0.1 64.29+_20 383.86_+162.8 7.83_+6,66 8.99_+2.98 7 13 I3 7 7 12 10 1.6-2.5 4.5 7,5 0.35-0.67 35 98 228-706 4-28 3.99-12.57 1.31 _+0,46 4.54_+ 1,05 0.42_+0.12 41.17-+15.2 425_+176.7 19.55+29.3 8.18_+1.96 6 11 li 6 6 11 9 0.6 2 2.8-6,5 0.21 0.58 23 62 173-619 2 87 4.3-9.83 Mg (mg/dl) Chol (mg/dl) 7.6_+0.24 226.08-/- 123.06 12 13 7.24-7.96 110~19 7.75_+0.22 181.8-+51.87 9 10 7.28-8.02 118 257 7.79_+0.18 167.71-+26.46 7 7 7.56-8.02 127~06 7.37_+0.57 121.17+43.07 6 6 6.31 7.82 70-/99 Trig (mg/db Ua (mg/dl) 433.85 -+633.52 0.69_+0.49 13 16 55-2289 0 1.6 163.4 _+72.84 0.53_+0.4 10 12 82-294 0-1.3 197.57 _+81.1 0.5+0.42 7 l0 71-282 0-1.5 113.5 _+83.9 0.83_+0.56 6 10 25 273 0.1-1.9 Na/mEq/1) K (mEq/1) 146_+5.4 6.61 _+2.22 9 9 135-153 4.5 10.9 3 3 136-151 4.5-6.2 151 _+1.41 5.2_+0 2 2 15~152 5.2-5.2 148.4_+6.91 4.78+0.42 5 5 138 157 4.2-5.2 CL (mEq/l) 93.78 _+ 10.46 9 79-105 3 84-104 97.5 +4.95 2 94-101 101.6_+4.04 5 97 108 SD, standard deviation; n, no. of samples. Range 144.33_+7.64 5.1 _+0.95 92_+ 10.58 n Range n Range n Range 176 ± 1.6, respectively), these differences were not statistically significant. In order to consider size-related differences, the blood chemistry values from all large turtles, independent of sex, were grouped and compared with those values from small turtles of undetermined sex. Differences were observed on half of the examined values. Significantly higher values were found in large turtles for total protein ( p = < 0 , 0 0 1 ) , albumin, LDH, iron, and cholesterol (p= <0.01), and for triglycerides and creatine kinase ( p = <0.05). However, chloride and phosphorus were higher in small turtles at p = <0.01 and p = <0.05, respectively. The lower results for calcium in females (6.86 ± 3) as compared with males (10.46 ± 2.6), medium-sized turtles (9.88 + 2.98) and small turtles (8.18 ± 1.96) may be attributed to the egg formation process which adult females undergo as they initiate nesting in the months of June and July in the Arabian region. It has been widely documented that calcium levels fall in domestic cattle alter parturition as they require calcium for milk production, whereas in birds, calcium level rises with egg production (Lemeij 1996). Lutz and DunbarCooper (1987), while working with seasonal variations in blood chemistry from loggerhead sea turtles (Caretta caretta) in Cape Canaveral, suggested that there were no seasonal trends for calcium. However, their study was performed on wild, accidentally captured turtles, which where examined independently of their age and sex. To the knowlege of the authors, no data are available on the seasonal variation of blood calcium in female chelonians to allow proper comparison. With the exception of sodium, mean blood chemistry values from small turtles from this study (CCLs ranging from 31 to 69 cm) fell within the range recorded in the Bahamas by Bolten and Bjorndal (1992) from 100 juvenile turtles with a straight-line carapace length ranging from 24.8 to 67.9 cm. Sodium values were lower in this study (148.8 ± 6.91) compared to the Bahamas population (172 ± 5), but were similar to the values obtained from three green turtles from Florida (150 + 4) (Norton et al. 1990). All other values from Florida turtles fell within the range reported here. Similarly, values presented by Aguirre et al. (1995) fell within the present ranges, with the exception of blood urea nitrogen, which was higher in this study (19.55 + 29.3, range = 2-87) compared to the lower values of the Hawaiian sample (1.0 ± 1.0, range = O-2.O). However, some discrepancies are noted in the definition of normal and abnormal values by other authors. In a diseased juvenile green sea turtle affected with fibropapillomas and renal myxofibroma, Norton et al. (1990) defined a calcium level of 3.4 mg/dl as hypocalcaemic and a blood urea nitrogen of 58 mg/dl as elevated. These observations were made by comparing the debilitated turtle with three clinically normal individuals from the same species. On the other hand, Bolten and Bjorndal (1992), recorded the normal value for calcium as 9.1 ± 2.1 with a range (r) of 1.6-12.2, c.R. Hasbtin et al. using sample size of 100. Thus the low calcium value of 3.4 mg/dl in the debilitated turtle fell within the normal range as observed by these authors. However, it fell outside the normal range established in this study (8.18 + 1.96), r = 4.36-9.83) and by Aguirre et al. (1995) (8.42 + 1.02 r = 7,2-9.7). Similarly, the values reported by Norton et al. for blood urea nitrogen from the debilitated turtle fell within the range recorded in this study for small turtles (19,55 _+ 29,3, r = 2-87). However, when compared to Bolten and Bjornda1"s results (7 _ 5, r = 2-37) and to Aguirre's data (1.0 ± 1.0, r = 0-2.0), the value for blood urea nitrogen fell outside the range. Caution should therefore be observed when determining abnormal blood chemistry results, especially if the comparison is made with ranges obtained from a small sample size. In this study, the extreme arid conditions of the Arabian Gulf do not seem to affect the blood chemistry values of green sea turtles when compared to those of other populations from other areas. It is noted, however, that these comparisons generally consider only juvenile turtles. It is hoped that the data presented here, including normal blood chemistry values for both sexes of large turtles, presumably adult, and for sizes ranging from 31 to 105.5 cm, contribute to the present knowledge on sea turtle biochemistry. Acknowledk,ement.~. The authors express their sincere gratilude to Shell Gas, Abu Dhabi and Shell Marketing Middle East, Dubai for their financial support and to Ms Judith Howlett, Dr Tom Bailey, Mr Christudas Silv:mose and Mr Mohammed Nafeez for their technical assistance. References Aguirre AA, Balasz GH, Spraker TR and Gross TS (1995) Adrenal and hematological responses to stress in juvenile green turtles (Chelonia mydas) with and without fibropapillomas. Physiol Zool 68:831-854 Anderson SC (1979) Synopsis of the turtles, crocodiles and amphisbaenians of Iran. Proc California Acad Sci XLI 22:501-528 Aspinal S (1995) United Abrah Emirates. In: Scott DA (ed) A directory of wetlands in the Middle East. IUCN, Gland, Switzerland and IWRB, Slimbridge, UK, pp 560. Basson P, Burchard J, Hardy J, Price A (1977) Biotopes of the Western Arabian Gulf. Aramco Dhahran, p. 284. B6er B (1997) An introduction to the climate of the United Arab Emirates. J Arid Environ 35:3-16. Bolten AB, Bjorndal KA (1992) Blood profiles for a wild population of green turtles (Chelonia mydas) in the southern Bahamas: sizespecific and sex-specific relationships. J Wild Dis 28(3):407~-13 Gasperetti J, Stimson AF, Miller JD, Ross JP, Gasperetti PR (1993) Turtles of Arabia. In: Butticker W, Krup F (eds) Fauna of Saudi Arabia, vol. 13. Natural History Museum, Basle, Switzerland and NCWCD, Riyadh, Kingdom of Saudi Arabia, p. 435. Herbst LH, Jacobson ER, Klein PA (1995) Green turtle fibropapillomatosis: evidence for a viral etiology. Proceedings Joint Conference AAZV/WDA/AAWV,12-17 August p. 224 Hughes GR (1995) Nesting cycles in sea turtles-typical or atypical? In: Bjorndal KA (ed) Biology and conservation of sea turtles. Smithsonian Institution Press, Washington DC, p. 583. Krupp F, Abuzinada AH, Nader IA (1996) A marine wildlife sanctuary for the Arabian Gulf. Environmental research and Blood Chemistry of the Green Sea Turtle conservation following the 1991 Gulf War Oil Spill. NCWCD, Riyadh, Kingdom of Saudi Arabia and Senckenberg Research Institution, Frankfurt, Germany, p. 511 Lemeij JT (1996) Biochemistry and sampling. In: Beynon PH, Forbes NA, Harcourt Brown NH (eds) Manual of raptors, pigeons and waterfowl. British Small Animal Veterinary Association. Cheltenham, UK, pp. 63-67 Lutz PL, Dunbar-Cooper A (1987) Variations in the blood chemistry of the logerhead sea turtle, Caretta caretm. Fish Bull 85:37-42 177 Miller JD (1989) An assessment of the conversation status of marine turtles in Saudi Arabia. MEPA Coastal and Marine Management Series 1, report no. 9, p. 202 Mortimer J (1995) Feeding ecology of sea turtles. In: Bjorndal KA (ed) Biology and conservation of sea turtles. Smithsonian Institution Press, Washington, DC, p. 583. Norton TM, Jacobson ER, Sundberg JP (1990) Cutaneous fibropapillomas and renal myxofibroma in a green turtle, Chelonia mydas. J Wild Dis 26:265-270