Establishing physiological blood parameters in the loggerhead sea turtle (Caretta caretta)

Transcription

1 DOI /s ORIGINAL PAPER Establishing physiological blood parameters in the loggerhead sea turtle (Caretta caretta) D. Gelli & V. Ferrari & A. Zanella & P. Arena & L. Pozzi & S. Nannarelli & C. Vaccaro & D. Bernardini & S. Romagnoli Received: 12 February 2008 /Revised: 18 June 2008 /Accepted: 19 June 2008 # Springer-Verlag 2008 Abstract The loggerhead sea turtle (Caretta caretta), one of the three sea turtle species inhabiting the Mediterranean Sea, is an endangered species. However, although frequently treated in marine animal rescue centers, and the subject of several studies in literature, as yet, few studies have been conducted on large numbers of loggerhead sea turtles in order to establish physiological reference ranges that enable the identification of pathological values. The lack of studies on reference parameters probably depends on the fact that marine turtles treated in wildlife rescue centers are usually in a critical conditions, thus precluding the collection of data on healthy animals and compromising the reliability of any data obtained from them. The present biological study was therefore conducted in order to obtain a database from healthy animals in natural conditions. Serum biochemistry and serum protein electrophoresis were performed on blood Communicated by F.-J. Kaup D. Gelli (*) : V. Ferrari : C. Vaccaro : D. Bernardini : S. Romagnoli Department of Veterinary Clinical Science, University of Padua, Viale dell Università 16, Legnaro, Padua, Italy donatella.gelli@unipd.it A. Zanella College of Agriculture, University of Padua, Viale dell Università 16, Legnaro, Padova, Italy P. Arena : L. Pozzi : S. Nannarelli Linosa Sea Turtle Rescue Center, Island of Linosa, Agrigento, Italy P. Arena : L. Pozzi : S. Nannarelli CTS Sezione Conservazione Natura via A: Vesalio, Rome, Italy samples obtained from 65 healthy adult loggerhead sea turtles captured and delivered to the Sea Turtle Rescue Center of Linosa (Italy); the blood samples were collected during the clinical examination of rescued animals. Laboratory analyses of serum samples were made in order to establish reference parameters, commonly required for laboratory diagnoses in mammals and diseased animals. Keywords Loggerhead. Sea turtle. Electrophoresis. Protein. Biochemistry. Caretta caretta Introduction The Mediterranean sea is inhabited by three species of sea turtle: the leatherback turtle (Dermochelys coriacea), the loggerhead turtle (Caretta caretta), the green turtle (Chelonia mydas) and the leatherback turtle (Dermochelys coriacea) However, despite the fact that the loggerhead turtle, the only species to nest on Italian coasts, is the most common casualty in marine wildlife rehabilitation centers in Italy, little is known about its physiological conditions. The paucity of data on this aspect depends mainly on the difficulty involved in collecting data on healthy animals in their natural habitat. Around the Pelagie (Pelagie is the geographic name of these islands) Islands (Italy), fishing activities with nets cause the accidental trapping of healthy turtles that are, as a rule, brought to rescue centers. In the present study, data were collected from 65 turtles on 80 captured specimens of C. caretta. Following selection criteria were: age (only sub-adults were considered), good general physical condition (absence of ectoparasites, weight of kg) and alert behaviour (based on level of reactivity, attempts at freeing from the net, physical strength). The purpose of this study was to establish

2 physiological serum reference ranges both for biochemistry and protein fractions of C. caretta. Materials and methods During September 2005, 80 sub-adult sea turtles (C. caretta) were captured in the Mediterranean Sea (Pelagie Islands, Italy) by biologists and fishermen and delivered to the Marine Rescue Center of the island of Linosa (Sicily, Italy). 1 This study was conducted over a relatively short time span in order to avoid variations in findings due to the influence of seasonal changes on physiological conditions of the animals. Following a clinical examination, performed in all 80 turtles, 15 animals were excluded from the study for one or more of the following reasons: presence of ecto- and endoparasites, presence of hook inside the coelomic cavity and/or poor body condition. Blood collection (jugular vein) was performed in the remaining 65 animals; 10-ml blood samples were drawn from each animal into vacutainer tubes, which were then centrifuged at 2,500 rpm for 10 min. The serum was removed, pipetted into 1.5-ml Eppendorf tubes and stored at 20.0 C. The protein fraction was evaluated in serum, since fibrinogen can alter the electrophoretogram in the beta gamma region (Thomas 2000). analysis was 20 C. Diluted samples (5 μl serum with 120 μl water) were introduced by pressure injection (5 or 10 s at 0.8 psi) followed by protein separation at a constant voltage of 25 kv. The total run time was 10 min, and the serum proteins were detected online at 200 nm. Between runs, the capillary was washed for 1 min with 1 N NaOH, 0.5 min with water and 1 min with running buffer. Results Mean values, SD and ranges for all parameters considered are reported in Table 1. On comparing representative serum protein electropherograms obtained in this study (Figs. 1 and 2) with a standard electrophoretic pattern of sea turtles found in literature (Gicking et al. 2004) and at last considering the albumin level found on using the colorimetric method, serum proteins in the loggerhead turtle of our study were divided into five fractions: the first (right-hand peak) corresponds to an albumin fraction, the second to the alpha-globulin migration zone and the third (left-hand peak) to the betagamma globulin migration zone. In the electrophoretogram shown in Fig. 2, six peaks appear: The fifth and sixth peaks on the right are albumin fractions, and beta-gamma fractions always appear as a bridged peak. The values of serum protein fractions are reported in Table 2. Analytical assay Serum biochemistry was determined in each sample by means of a Hitachi 912 automatic analyzer (Roche Diagnostics GmbH, D Mannheim, Germany) using assays validated and warranted by Roche Diagnostics. The following biochemical analyses were performed on serum samples: glucose, gamma-glutamyltranferase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine kinase, lactate dehydrogenase, cholesterol, triglyceride, calcium, phosphorus, total bilirubin, urea, uric acid, creatinine, total protein and albumin. Serum protein separations were made using the P/ACE MDQ capillary electrophoresis system (Beckman Coulter, Inc., 4300 N. Harbor Blvd., Fullerton, CA, 92835, USA) equipped with automatic constant volume sample injection devices and temperature control for capillary. System control, data collection and analysis were done with the respective software of the equipment. Untreated fused silica capillaries of 50-cm length and 50-μm i.d. were used. The running buffer was 60 mm sodium tetraborate, ph 9.4 (Sigma Chemical), and the temperature of capillary during the 1 Authorized by the Ministry of Environment to rescue, treat and perform studies on marine animals. Discussion Haematological and serum biochemical investigations are the first and most important steps when ascertaining physiological vs pathological conditions of patients, including reptiles. In all 65 turtles sampled in the present study, glucose levels were within the range previously reported as normal (Campbell 1996). The individual variations in serum glucose observed in our cases were probably related to the stress of capture, transportation and restraint before clinical examination. To our knowledge, this is the first study in literature to report on alanine aminotransferase, gamma-glutamyl transferase, creatine kinase, cholesterol, triglycerides and total bilirubin values in healthy loggerhead turtles. Moreover, since the physical condition of animals considered was good, it would be reasonable to consider the values obtained in all these cases as valid reference ranges for future studies. In most of our cases, aspartate aminotransferase and lactate dehydrogenase values were higher than those reported in literature (Campbell 1996; Wilkinson 2004). On the contrary, triglycerides values appeared lower than what reported by Campbell (1996). Overall, alkaline phosphatase levels were slightly lower than some values reported in literature, but were in agreement with the low

3 Table 1 Mean values, SD and ranges for all parameters Parameter Max Min Mean±SD Value a Value b Value c Glucose ± mmol/l 131.4±10.2 mg/dl Gamma-glutamyl ±1.12 transferase (U/I) Alanine ± IU/l aminotransferases (U/I) Aspartate ± IU/l 123±38 IU/l 122.5±37.8 IU/l aminotransferases (U/I) Alkaline phosphatase (U/I) ± /60 IU/l 74±37 IU/l 73.9±36.9 IU/l Creatine kinase (U/I) ,703.89±3, ,614.5±1,677 IU/l Lactate ± IU/l 108.8±63.5 IU/l dehydrogenase (U/I) Cholesterol ± mmol/l Triglycerides ±0.54 <3.2 mmol/l Calcium ± mmol/l 1.8±0.2 mmol/l 7.1±0.8 mg/dl Phosphorus ± mmol/l 1.7±1.1 mmol/l 5.3±3.5 mg/dl Total bilirubin ( mol/l) ±0.68 <1.7 mol/l 7 Urea (mol/l) ±22.27 Uric acid (mol/l) ± <119 mmol/l 41±18 mol/l Creatinine ( mol/l) ±35.36 <26 mol/l Total protein (g/l) ± g/l 20±8 g/l 2±0.8 g/dl Albumin (g/l) ± ±2.6 g/l 1±0.26 g/dl a Campbell (1996) in Mader Reptile Medicine and Surgery : suggested serum biochemistry reference values for mature sea turtles (C. mydas, C. caretta, Eretmochelys imbricate and Lepidochelys kempi). b Wilkinson (2004) in Mcarthur Medicine and Surgery of Tortoises and Turtles c Wyneken (2006) in Mader Reptile Medicine and Surgery : medical care of sea turtles values for this enzyme found in most chelonians (Campbell 1996; Stein 1996). Serum urea concentrations were evaluated because of its role in the metabolism of reptile (Stein 1996). Creatinine, until now never reported in literature for C. caretta, was present in small amounts in the serum samples. In the present study, serum creatinine concentrations were lower than what reported for most chelonians, whereas serum uric acid concentrations were higher than Fig. 1 Representative serum protein electropherograms

4 Fig. 2 Representative serum protein electropherograms: appearance of six peaks those reported by other authors (Campbell 1996; Wilkinson 2004). Serum calcium concentrations were lower than those reported in literature (Campbell 1996; Wilkinson 2004), whereas serum phosphorus concentrations were higher (Campbell 1996). In the present study, moreover, the overall serum total protein level was slightly higher than what reported by other authors (Campbell 1996; Wilkinson 2004). The data reported by Campbell refer to four different species of sea turtle, no distinction being made between them. One author (Wyneken 2006) reports values specific for C. caretta, but the data provided were obtained in young turtles weighing less than 2 kg, and it is reasonable to assume that the metabolism and eating habits of these younger animals must be different from those of the adults. Electrophoresis is a powerful diagnostic tool, which is often of fundamental diagnostic importance when used in conjunction with other laboratory techniques. In veterinary practice, it is known that disease-related changes occur in biochemical values and electrophoretogram findings. In wild animals, these laboratory investigations are still in their infancy due to the difficulty in collecting data; this explains the scarcity of reference values which compromises the reliability of diagnoses made in turtles in critical conditions. Several blood parameters have been reported in literature for C. caretta, and some authors (Masat and Dessauer 1968; Gicking et al. 2004) observe that in chelonians, changes in biochemical values and plasma proteins may be also be related to physiological conditions (age, sex and season). Data in the present study were collected in a large number of specimens, and some of the values obtained were comparable to those reported in literature despite differences in the analytical methods used. This further suggests that the values found by us might be used as reliable physiological parameters for this species. Although pathological value of biochemical parameters and further studies are needed to gain information on normal and electrophoretic patterns in the loggerhead sea turtle, the findings reported in the present study may be considered a first step in establishing physiological reference ranges. Table 2 Values of serum protein fractions Protein fractions Max Min Mean±SD Values a (g/dl) Albumin 1 (g/l) ±0.99 Albumin 2 (g/l) ± ±0.17 Alpha-globulin (g/l) ± ±0.10 Beta-globulin (g/l) ± ±0.20 Gamma-globulin (g/l) ± ±0.62 Total protein (g/l) ± ±0.72 Albumin/Globulin (g/l) ± ±0.10 a Gicking from: Journal of Herpetological Medicine and Surgery (2004)

5 Acknowledgements The authors would like to thank Doctor De Battisti R., College of Agriculture and Forestry Sciences, University of Padua, Corpo Forestale dello Stato, Padua; Doctor Rallo G.P., Museo delle Valli, Oasi di Valle Averto Venice; Galante I., CTS Settore Conservazione della Natura, Rome. This experiment comply with the current laws of the Italian country. All the healthy turtles subject of the study were released after collecting data. References Campbell TW (1996) Sea turtle rehabilitation. In: Mader DR (ed) Reptile medicine and surgery, 1st edn. Saunders, Philadelphia, pp Gicking J, Foley AM, Har KE, Raskin RE, Jacobson E (2004) Plasma protein electrophoresis of the Atlantic loggerhead sea turtle. J Herpe Med Surf 14(3):14 18 Masat R, Dessauer H (1968) Plasma albumins of reptiles. Comp Biochem Physiol 25: doi: / x(68) Stein G (1996) Hematologic and blood chemistry values in reptiles. In: Mader DR (ed) Reptile medicine and surgery, 1st edn. Saunders, Philadelphia, pp Thomas J (2000) Overview of plasma proteins. In: Feldman BF, Zinkl JG, Jain NC (eds) Schalm s veterinary hematology, 5th edn. Lippincott, Williams & Wilkins, Philadelphia, pp Wilkinson R (2004) Clinical pathology. In: McArthur S, Wilkinson R, Meyer J (eds) Medicine and surgery of tortoises and turtles. Blackwell, Oxford, UK, pp Wyneken J (2006) Medical care of sea turtles. In: Mader DR (ed) Reptile medicine and surgery, 2nd edn. Saunders, Philadelphia, pp