Serum biochemistry profile determination for wild loggerhead sea turtles nesting in Campos dos Goytacazes, Rio de Janeiro, Brazil/Determinacao do perfil bioquimico serico de tartarugas marinhas de vida livre da especie em nidacao em Campos dos Goytacazes, Rio de Janeiro, Brasil.
The first sea turtles appeared in the late Jurassic, about 65 million years ago. They have adapted their anatomical features to life in the marine environment. Nowadays there are seven living species of sea turtles worldwide. Five of the seven species are found in Brazilian waters: Lepidochelys olivacea (olive ridley), Eretmochelys imbricata (hawksbill), C. caretta (loggerhead), Chelonia mydas (green) and Dermochelys coriacea (leatherback) (BAPTISTOTTE et al., 2003).
Throughout their highly migratory lifecycles, sea turtles play important ecological roles in the marine and terrestrial ecosystems. If these reptiles were to become extinct, the negative impacts on the environment would be potentially significant (BJORNDAL & JACKSON, 2003).
Loggerheads can be found in temperate, tropical and sub-tropical areas from both hemispheres. In Brazil, about 80% of nests belong to this species (BAPTISTOTTE et al., 2003). The reproductive range of C. caretta on the Brazilian coast extends from Rio de Janeiro to Sergipe States (MARCOVALDI & CHALOUPKA, 2007).
The data in the literature about sea turtles biochemical analyses are scarce and most of the studies used small sample sizes, being the majority of animals in captivity (KAKIZOE et al., 2007). According to CAMPBELL (1996), variables including sampling site, gender, age, diet and seasonal changes will influence blood parameters. Beyond that, comparisons among studies are hindered by the differences in analytical techniques.
The present study aimed to establish baseline biochemical profile values for free-ranging nesting female loggerheads, and to correlate these parameters with the size of animals.
MATERIAL AND METHOD
Twenty-eight nesting females were sampled from Farol de Sao Thome (21[degrees]45'15"S-41[degrees]19'28"W), city of Campos dos Goytacazes, Rio de Janeiro, between November 2004 and January 2005. The region is characterized by the presence of restinga habitat, which consists of low lying salt-tolerant vegetation on coarse sandy soils. Campos dos Goytacazes has a wet and dry tropical climate, with average annual temperatures between 20[degrees]C and 32[degrees]C, and a average annual rainfall of 1300mm, unevenly distributed through the year (FEEMA, 1993). The turtles received physical examination and then, were tagged with Inconel metal tags on the front flippers, measured (curved carapace length-CCL and curved carapace width-CCW) with a flexible tape measure, and weighed during egg laying; a digital scale Oswaldo Filizola, model BI-100 D (Tecnica Industrial Oswaldo Filizola Ltda., Sao Paulo, Brazil), with a maximum capacity of 500kg, was used for weighting.
Five milliliters of blood were collected from the dorsal cervical venous sinus into tubes without anticoagulant and kept at room temperature for 20-30 minutes to allow complete clot formation and retraction. The whole blood was centrifuged at 560g for 10 minutes, and then the serum was separated and frozen at -20[degrees]C for subsequent biochemical analysis. The samples were processed within 15 days of blood collection.
Both serum and plasma samples could be employed for all the assays carried out. However, serum was preferred because of its convenience and lower cost. Additionally, in plasma samples, the total protein is 3% to 5% higher due to the presence of fibrinogen (KRATZ et al., 2002), which could possibly interfere with the results.
The biochemistry analyses were processed at CAD (Centro de Apoio e Diagnostico Veterinario) in Rio de Janeiro. Blood biochemistry variables urea, creatinine, ALT (Alanine aminotransferase), AST (Aspartate aminotransferase), GGT (Gamma-glutamyl transferase), ALP (Alkaline phosphatase), cholesterol and triglycerides, were processed on Ciba Express 550[R] autoanalyzer. The calcium values were obtained through Bio 2000[R] semi automatic analyzer. The biochemical kits used for all tests were from Bio Tecnica[R] provided by Quality SA. Sodium and potassium dosages were determined by flame photometer Corning series 400[R].
RESULTS AND DISCUSSION
Mean, standard deviation and degree of variance were performed for each parameter using Microsoft Excel[R] (Table 1). An evaluation was made of the linear relationship between biochemical parameters and turtle's size using the Pearson and Spearman's coefficients, with assistance from SPSS[R] software. The average weight was 119.2kg, average CCL was 101cm and average CCW was 92.5cm (Table 2).
The average values for total protein reported here (3.9g d[L.sup.-1] [+ or -] 0.72) fall within the range found by GICKING et al. (2004), for C. Caretta, and by AGUIRRE & BALAZS (2000) for C. mydas. However, serum albumin values (1.3g d[L.sup.-1] [+ or -] 0.40) were higher than those found by WHITAKER & KRUM (1999) for immature loggerheads from captivity. Albumin levels are often elevated during vitellogenesis, due to increased protein synthesis demands (CAMPBELL, 1996). According to WILKINSON (2004), hemodilution by lymph is a possible cause of iatrogenic hypoalbuminemia. That author reported the diluted and pale appearance of blood in case of lymphatic contamination. In this study all samples were examined at time of collection and appeared thick and dark red, thus apparently lymph free.
Relatively higher levels of globulin (2.6g d[L.sup.-1] [+ or -] 0.59) were found when compared to WHITAKER & KRUM (1999) for captive juvenile turtles and to AGUIRRE & BALAZS (2000) for C. mydas. Higher serum globulin values have been reported by BOLTEN & BJORNDAL (1992) for juvenile C. mydas from the Bahamas (3.6g d[L.sup.-1] [+ or -] 0.7) and by GICKING et al. (2004) (3.8g d[L.sup.-1]). According to them, this hyperglobulinemia may be associated to chronic parasite infection, suggesting that, based on body condition and nest-building activity, the general health of the turtles in this study was good.
The average of Albumin:Globulin (A:G) ratio (0.5 [+ or -] 0.20) was similar to the results obtained by AGUIRRE & BALAZS (2000) for healthy green turtles and not far from GICKING et al. (2004) for loggerheads. However, the A:G relation is not a widely used parameter in the clinical evaluation of sea turtles.
Urea values ranged from 11 to 70mg d[L.sup.-1] (mean 35.25mg d[L.sup.-1] [+ or -] 13.50) and were similar to those reported by CAMPBELL (1996) (20-80mg d[L.sup.-1]) for adult individuals of C. caretta, C. mydas, E. imbricata and L. kempi, outside reproductive period. Based on this, it can be assumed that reproduction itself does not alter the urea values. WYNEKEN et al. (2006) reported higher urea values for young loggerheads. Breeding females leave foraging areas, thus reduce or even cease feeding, in the period preceding nesting activity, only to return at the end of the breeding season. This may explain the lower urea values found in this study. BONNET (1979) correlated the hyporexia observed in this period with lower urea values.
The creatinine concentration (0.5mg d[L.sup.1] [+ or -] 0.15) is normally low in reptiles (<1mg d[L.sup.-1]) (WYNEKEN et al., 2006), since it is not actively secreted nor reabsorbed by the kidney tubules (WILKINSON, 2004). The mean sodium levels were 147mEq [L.sup.-1] ([+ or -] 14.05), similar to reports from STAMPER et al. (2005) for migratory loggerheads in North Carolina, as a result, in both cases, of decreased food intake. The sodium and potassium values obtained by WHITAKER & KRUM (1999) for captive juvenile loggerheads were higher as the animals were fed daily. The mean potassium concentration was 4.2mEq [L.sup.-1] ([+ or -] 0.91), similar to CAMPBELL (1996) and BOLTEN et al. (1992). Sodium and potassium levels may be altered by the intermittent activity of salt glands. Additionally, WILKINSON (2004) suggested decreased food intake as an explanation for reduced potassium levels.
Concerning mean calcium values (9.9mg d[L.sup.-1] [+ or -] 1.11), they were high when compared to other sea turtles. However, according to WILKINSON (2004), calcium is mobilized for egg formation during the reproductive period. DEEM et al. (2006) also found elevated calcium and phosphorus levels in a population of nesting leatherbacks.
The mean phosphorus values (7.9mg d[L.sup.-1] [+ or -] 1.85) were similar to CAMPBELL (1996) for adults from four different species of sea turtles. The average of Ca:P ratio (1.3) was compatible with DIVERS (2000), who established a Ca:P ratio greater than 1.0 for healthy reptiles.
ALP activity (13.5IU [L.sup.-1] [+ or -] 4.69) was similar to that suggested by BOLTEN et al. (1992) for loggerheads. However, WHITAKER & KRUM (1999) found higher serum activities for captive juvenile loggerheads (73.9IU [L.sup.-1] [+ or -] 37), and so did BOLTEN & BJORNDAL (1992) for wild juvenile C. Mydas (43IU [L.sup.-1] [+ or -] 16). According to CAMPBELL (1996), ALP is associated with increased osteoblastic activity. Based on this information, it is probable that animals in development stage show higher enzymatic levels, since osteoblasts are responsible for bone matrix synthesis. Sick L. kempi sampled by WHITAKER & KRUM (1999) at the New England Aquarium had extremely high ALP activities (465.3IU [L.sup.-1] [+ or -] 306) due to their clinical conditions. Although ALP is widely distributed in reptile body, literature suggests that it can be found in organs such as bones and reproductive tract (WILKINSON, 2004). Thus damage to these systems can raise the enzyme levels. The ALT concentration (14IU [L.sup.-1] [+ or -] 6.35) agreed with literature (CAMPBELL, 1996; WILKINSON, 2004). However, the AST activity (151IU [L.sup.-1] [+ or -] 61.86), was low. Meanwhile, STAMPER et al. (2005) found even lower AST activities for migratory animals (129IU [L.sup.-1]) and higher values for resident turtles (206IU [L.sup.-1]). According to these authors, 33% of the resident animals had carapace lesions and thickening of the skin, whereas only 10% of the migratory turtles showed these injuries. As such, they attributed the higher AST values in the resident population to tissue injuries. The present study observed a positive correlation between size variables (weight, curved carapace length and curved carapace width) and ALT (P<0.05, n=28) and AST (P<0.05; n=28) enzymes, suggesting that animals with higher hepatic volume have greater enzyme activity. It is relevant to mention that no studies correlating these parameters were found in literature. In chelonians, factors expected to have a significant effect upon hepatic transaminases includes liver or muscular diseases (WILKINSON, 2004; DIVERS, 2000). These factors, however, have not influenced AST and ALT values in this study, since nesting females are generally in good health and capable of reproducing. Additionally, health assessment should take into account the relationship between biochemistry values and body size in order to obtain reliable results and avoid erroneous interpretation of the animals general condition.
The serum GGT activity ranged between 0.1 and 2.4IU [L.sup.-1] (mean 0.72IU [L.sup.-1] [+ or -] 0.67). These values are similar to DIVERS (2000), although GGT is not a parameter frequently used to evaluate sea turtles health conditions (WILKINSON, 2004), since it is normally low (DIVERS, 2000).
Mean amylase activity was 627IU [L.sup.-1] ([+ or -] 232), falling within the range from DEEM et al. (2006) for leatherback nesting females from the Republic of Gabon. The average activity found by DEEM et al. (2003) for loggerhead nesting females in the Caribbean was 623.5IU [L.sup.-1] ([+ or -] 150.1). Additionally, DEEM et al. (2009) found higher amylase activities for nesting loggerheads (407IU [L.sup.-1] [+ or -] 131) than for foraging (263IU [L.sup.-1] 99) or stranded (180IU [L.sup.-1] [+ or -] 106) individuals of the same species. This enzyme could possibly be influenced by hormonal factors during the reproductive period. However, further research is required in order to confirm this hypothesis.
The Cholesterol (247mg d[L.sup.-1] [+ or -] 48) and triglycerides (580mg d[L.sup.-1] [+ or -] 230) concentrations were significantly higher than in literature. DERICKSON (1976) noted that, due to vitellogenesis, endogenous cholesterol and triglycerides levels are typically increased in nesting females. BONNET (1979) observed that fasting sea turtles showed increased concentrations of blood lipids associated to the catabolism of energy reserves. As such, it was suggested that increased levels of serum lipids are a result of reduced food intake and vitellogenesis by nesting females.
Mean uric acid values ranged between 0.3mg d[L.sup.-1] and 1.2mg d[L.sup.-1] (mean 0.62mg d[L.sup.-1] [+ or -] 0.21). BOLTEN & BJORNDAL (1992) found higher values in juvenile green turtles (1.5mg d[L.sup.-1] [+ or -] 0.6) because young C. mydas are primarily carnivores and according to literature, carnivorous reptiles have higher uric acid blood levels.
It was concluded that the free-ranging nesting C. caretta showed significant variation in their biochemical profile due to reproductive output. Changes in calcium, phosphorus, cholesterol and triglyceride levels were related to vitellogenesis and egg formation. The obtained values were compared with reference physiological levels for breeding and non-breeding turtles.
Although it is important to monitor health status of a population, there is little published information regarding sea turtles biochemical profiles. Further studies are needed to provide more understanding of this subject.
In summary, this study has characterized, for the first time, the biochemical profile of C. caretta nesting females from the northern state of Rio de Janeiro. The data presented here may be useful as reference in the assessment of health and disease of loggerhead populations from southeastern Brazil. In addition, this study might contribute to the rehabilitation of sick individuals and hence, to their reintroduction in the wild.
We acknowledge Projeto TAMAR-ICMBio and CAD for the technical support and CAPES for the financial support.
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Daphne Wrobel Goldberg (I, II) Jucara Wanderlinde (II) Isabel Maria Alexandre Freire (III) Luiz Cesar Pereira da Silva (III) Nadia Regina Pereira Almosny (IV)
(I) Departamento de Bioquimica, Universidade Estadual do Rio de Janeiro (UERJ), Av. 28 de setembro, 87 Fds, 4o Andar, Vila Isabel, 20551-030, Rio de Janeiro, RJ, Brasil. E-mail: email@example.com. Autor para correspondencia.
(II) Fundacao Centro Brasileiro de Protecao e Pesquisa das Tartarugas Marinhas (Fundacao Pro-Tamar), Campos dos Goytacazes, RJ, Brasil.
(III) Laboratorio de Experimentacao Animal de Bio-Manguinhos, Rio de Janeiro, RJ, Brasil.
(IV) Faculdade de Medicina Veterinaria, Universidade Federal Fluminense (UFF), Niteroi, RJ, Brasil.
Received 06.13.10 Approved 10.28.10 Returned by the author 12.13.10 CR-3714
Table 1--Mean, standard deviation, minimum and maximum reference interval of the biochemical profile of free-ranging nesting females C. caretta (n=28), Campos of Goytacazes, RJ. Standard Biochemical Parameters Mean Deviation Minimum Maximum Urea (mg [dL.sup.-1]) 35.25 13.50 11 70 Creatinine 0.50 0.15 0.2 0.9 ALT (U [L.sup.-1]) 14 6.35 2 26 AST (UI [L.sup.-1]) 151.25 61.86 46 330 ALP (UI [L.sup.-1]) 13.53 4.69 4 25 GGT (UI [L.sup.-1]) 0.72 0.67 0.1 2.4 Cholesterol (mg [dL.sup.-1]) 247.75 48.52 145 317 Triglicerydes (mg [dL.sup.-1]) 580.28 232.08 143 995 TP (g [dL.sup.-1]) 3.96 0.72 2.3 5.3 Albumine (g [dL.sup.-1]) 1.30 0.40 0.5 2.1 Globuline (g [dL.sup.-1]) 2,66 0.59 1.4 3.7 A:G 0.51 0.20 0.15 1 Phosphorus (mg [dL.sup.-1]) 7.96 1.85 3.9 12.1 Calcium (mg [dL.sup.-1]) 9.98 1.11 7.9 12.9 Ca:P 1.33 0.41 0.77 2.4 Amylase (UI [L.sup.-1]) 627.75 232.93 311 1112 Sodium (mEq [L.sup.-1]) 147.28 14.05 108 168 Potassium (mEq [L.sup.-1]) 4.27 0.91 2 6.6 Uric Acid (mg [dL.sup.-1]) 0.62 0.21 0.3 1.2 Table 2--Mean, standard deviation and reference interval of the variables CCL (curved carapace length), CCW (curved carapace width) and WEIGHT of free-ranging nesting females C. caretta (n=28), Campos of Goytacazes, RJ. Standard Variables Mean deviation Minimum Maximum CCL (m) 1.01 0.05 0.86 1.12 CCW (m) 0.92 0.04 0.82 1.02 Weight (kg) 119.20 19.04 71.7 168.1