This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
Author's personal copy Micron 41 (2010) 1005–1010
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Morphological, morphometrical and ultrastructural characterization of Phrynops geoffroanus’ (Testudines: Chelidae) blood cells, in different environments Carlos Eduardo Saranz Zago c , Tiago Lucena da Silva c , Maria Isabel Afonso da Silva c , Larissa Paola Rodrigues Venancio c , Priscila Pasqüetto Mendonc¸a c , Luiz Roberto Falleiros Junior a , Luiz Dino Vizotto c , Sebastião Roberto Taboga a , Claudia Regina Bonini-Domingos c , Maria Tercília Vilela de Azeredo-Oliveira c,∗ , Classius de Oliveira b a
Departamento de Biologia, Centro de Microscopia e Microanálise – UNESP-IBILCE-São José do Rio Preto, SP, Brazil Departamento de Biologia, Laboratório de Anatomia Comparada – UNESP-IBILCE-São José do Rio Preto, SP, Brazil c Departamento de Biologia, Centro de Estudos de Quelônios – UNESP-IBILCE-São José do Rio Preto, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 23 February 2010 Received in revised form 2 June 2010 Accepted 19 June 2010 Keywords: Phrynops geoffroanus Turtles Blood cells Morphology Ultrastructure
a b s t r a c t The aim of this study was to evaluate the formed elements in the periferical blood of two amostral groups of Phrynops geoffroanus: one from an urban environment under domestic sewage dumping, and another from a non-contaminated environment. Blood samples of 36 animals (females and males) were collected through cardiocentesis. Sixteen specimens were from the urban environment, and 20 were from a control environment. Samples of blood tissue were used for light microscopy analysis, and also for morphometric analysis of red blood cells. For the ultrastructural analysis, blood samples of 2 animals were used. The formed elements found, using morphological and ultrastructural analysis were: nucleated red blood cells; thrombocytes; neutrophils, lymphocytes, monocytes, basophils; eosinophils; heterophils, and azurophils. The morphometric analysis of all red blood cells parameters examinated in females showed a statistically significant difference, but in males just the nuclear area showed difference between the specimens of the two environments. The elements identified by light microscopy were elucidated by electron transmission microscopy. This P. geoffroanus study is the first one that makes a correlation between these environments and the description of turtle’s blood cells, thus contributing to the identification of the hematological characteristics of this group. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Geoffroy’s side-necked turtle (Phrynops geoffroanus) belongs to the order Testudines and family Chelidae. They are small-sized and diurnal animals, which are frequently found in rivers, lakes and ponds with slow currents, and have a wide distribution, in South America countries (Gans, 1980; Goulart, 2004; Pritchard and Trebbau, 1984). The circulating blood of turtles has several primitive cells, such as nucleated red blood cells, responsible for oxygen transporting, and the thrombocytes, which are involved in clotting process. The production of the blood cells occurs in the spleen and bone marrow. The white blood cells observed in turtles peripheral blood are: neutrophils, lymphocytes, basophils, monocytes, eosinophils,
∗ Corresponding author at: CEQ, IBILCE-UNESP, Rua Cristóvão Colombo, 2265; Jd. Nazareth; CEP: 15054-000, Brazil. Tel.: +55 17 3221 2378; fax: +55 17 3221 2390. E-mail address:
[email protected] (M.T.V. de Azeredo-Oliveira). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.06.006
heterophils and azurophils, this cells are responsible for defense mechanisms (Andrew, 1965; Canfield, 1998; Frye and Murphy, 1991; Garcia-Navarro and Pachaly, 1994; Stacy and Whitaker, 2000). Environmental degradation linked to human population growth results in ecological niches variation for several species. The human impact on the environment becomes a major threat to the organisms that lives there; however, some species can survive even in completely impacted environments (Souza and Abe, 1999). São José do Rio Preto is located in the southeast region of Brazil, at the following geographic coordinates: 20◦ 49 12 S 49◦ 22 44 E. The city is physically divided by the Rio Preto river and its tributaries, including the Piedade and Felicidade stream. The Rio Preto river is approximately 120 km long, and is a tributary of the Turvo river, which flows into the Rio Grande river, and is a component of the Turvo Grande basin (Zago et al., 2010). With approximately 450 thousand inhabitants, the city is a constant source of water pollution from dozens of streams that run though the city.
Author's personal copy 1006
C.E.S. Zago et al. / Micron 41 (2010) 1005–1010
The aim of this study were to analyze the blood cells of the animals from the urban environment, which had been contaminated by domestic sewage, as well as blood cells of the animals from the control environment using morphological, ultrastructural and morphometric analysis.
were mixed in uranyl acetate and lead citrate. The material was analyzed under a Zeiss – Leo IN 910 transmission electronic microscope operated at 80 kV.
2. Material and methods
One hundred red cells were analyzed for each animal under an Olympus BX 60 light microscope (objective of 100×). The images were captured using the Image – Pro Plus computer program. The cell parameters used were: larger diameter, smaller diameter, nuclear area and total area of red cells (m).
2.1. Animals We used blood samples of 36 P. geoffroanus specimens, 10 females and 10 males from the “Reginaldo Uvo Leone” breeding farm in Tabapuã, São Paulo (20◦ 59 47.4 S, 49◦ 07 16.6 W), and 8 females and 8 males, from the Felicidade stream, located in São José do Rio Preto city (20◦ 46 20.6 S, 49◦ 21 18.0 W). This stream runs through an urban area and is a dump site of domestic sewage and has flat banks interspersed with predominantly herbaceous vegetation, slopes and a large number of P. geoffroanus. The “Reginaldo Uvo Leone” breeding farm works with wild and exotic reptiles, amphibians and birds with commercial purpose. They work with a larger number of turtles species, including P. geoffroanus. The animals are fed with fish food (45% of protein), and the water of the artificial lakes is from an artesian well, devoid of contaminations. Because of that, these animals were chosen as a control group. 2.2. Sample collection We collect 3 mL of blood by cardiocentesis, between the humeral and pectoral plastron scutes in each animal. We used a 40:12 size needle (BD Precision Glide® ) and sodium heparin as anticoagulant. After collecting the blood, the orifice was sealed with surgical glue (Vetband® ), and the animals were kept under observation for 72 h before being returned to their habitats. The blood smears were made in less than 24 h, in order to avoid further color impregnation caused by heparin (Brites and Rantin, 2004; Mader, 1996). 2.3. Morphological analysis Three blood smears of each animal were fixed in methanol and stained by Panotic (Hematocor® , Biology), according to the manufacturer’s instructions. These slides were used for analysis and identification of thrombocytes, white and red blood cells under light microscopy (Olympus BX 60), using the objective of 100×. 2.4. Ultrastructural analysis We analyzed blood samples of two P. geoffroanus males from the urban environment using a electron transmission microscopy (Zeiss – Leo IN 910) from the Center of Microscopy and Microanalysis in the Instituto de Biociências, Letras e Ciências Exatas – IBILCE/UNESP, São José do Rio Preto, Brazil. We used this technique in order to characterize the ultrastructural aspects of this specie, having no intention to make correlations with structural data related to the environment, only the description of ultrastructural morphology, because of that we used only two animals. The blood sample was centrifuged at 1000 rpm for 10 min. The plasma was discarded and the layer of leukocytes was set at 3% glutaraldehyde in a 0.1 M phosphate buffer for 2 h at 4 ◦ C. The material was post fixed and then put into osmium tetroxide 1% for 2 h at 4 ◦ C. It was then dehydrated in an increasing concentration series of acetone and embedded in Araldite resin (Pellizzon et al., 2002). Ultrathin sections (75 nm) were made using a diamond knife in ultramicrotome LKB, and mounted on copper grids and the cuts
2.5. Morphometric analysis
2.6. Statistical analysis Statistical data from morphometric analysis were performed using the software BioEstat 3.0. The resulting values were tested to normality using the Shapiro–Wilk test and homoscedastic distribution. The parametric test used to compare averages was Student’s t-test, and the non-parametric test used was the Mann–Whitney U-test, with the alpha of 5% (Ayres et al., 1998). 3. Results 3.1. Morphological analysis Red blood cells, thrombocytes, neutrophils, lymphocytes, monocytes, basophils, eosinophils, heterophils and azurophils were found in the peripheral blood of P. geoffroanus (Fig. 1B). The red cells were numerous, oval or elliptical in shape, and presented one or more irregular nuclei, with thick and densely granular chromatin (Fig. 1A and B). In the peripheral blood immature forms of red blood cells were found, which were usually smaller than the mature forms (data not showed). The thrombocytes had an elliptical shape, basophilic nucleus, cytoplasm weakly stained by Panotic and smaller than the red cells, this cells are originated from thromboblasts (Fig. 1C). Neutrophils presented a basophilic and unsegmented nucleus, similar to the ones found in mammals. Fibrillar chains and basophilic, eosinophilic and azurophilic granulations were observed in the cytoplasm (Fig. 1D). The lymphocytes were smaller than the monocytes, with a slightly basophilic cytoplasm (Fig. 1E). The basophils had a segmented nucleus and basophilic granulations, which were always spherical, stick-like, or intracytoplasmatic (Fig. 1F). The monocytes found had curved nuclei, with a condensed chromatin and a clear cytoplasm (Fig. 1G-m). The other cells found in the peripheral blood were azurophils with basophilic nucleus and cytoplasm (Fig. 1G-a). The heterophils (granulocytes) had slightly eccentric lobed or bi-lobed nucleus and intracytoplasmatic inclusions in the form of granules or spindle poles (Fig. 1H-h). The eosinophils (acidophilus) nuclei were simple or lobulated with spherical cytoplasmic granulations that were red or orange in color (Fig. 1H-e). 3.2. Ultrastructural analysis The ultrastructural analysis of the red blood cells revealed nuclei with peripheral heterochromatin, evenly electron-dense cytoplasm with few mitochondria and Golgi complex near the nucleus (Fig. 2A and B). The thrombocytes had a nucleus with peripheral heterochromatin and electron-dense and homogenous cytoplasm, with cytoplasmatic projections, secretion vesicles, few granules and mitochondria (Fig. 2C). In neutrophils a single large nucleus was found, with peripheral chromatin and cytoplasm rich in mitochondria, granules of secretion and endoplasmic reticulum (Fig. 2D).
Author's personal copy C.E.S. Zago et al. / Micron 41 (2010) 1005–1010
1007
Fig. 1. Morphological analysis of blood cells of Phrynops geoffroanus. Panotic staining: (A) red cells; (B) bi-nuclear red cells (arrow); (C) thrombocytes (arrows); (D) neutrophil (arrow); (E) lymphocytes (arrows); (F) basophil (arrow); (G) azurophils (a) and monocytes (m); (H) heterophils (h) and eosinophils (e). Bars: 10 m.
The lymphocytes had a nucleus occupying almost all the cells, with dense chromatin in the periphery and nucleolus not evident. The cytoplasm, restricted to a small portion of the cell, revealed the presence of a well-developed endoplasmic reticulum, ribosomes and few mitochondria (Fig. 2E). In basophils, we observed a peripheral nucleus with peripheral heterochromatin, and cytoplasm presenting lipids and electron-dense and homogeneous secretion granules (Fig. 2F). The monocytes had U-shaped nuclei with peripheral heterochromatin and cytoplasm with mitochondria, secretion vesicles, secretion granules and endoplasmatic reticulum (Fig. 3A). The ultrastructural analysis of heterophils revealed the presence of a nucleus with peripheral heterochromatin and cytoplasm rich in grains. Some of the grains were electron-dense, while others had varying degrees of electron density and several shapes (round and stick-like) and sizes. We also observed secretion vesicles (Fig. 3B). The eosinophils had a large nucleus with slightly dense heterochromatin. In the cytoplasm electron-dense secretory granules was found, with rounded or oval shape, and several sizes (Fig. 3C).
The azurophils, cells with irregular shape, had nuclei with peripheral heterochromatin and cytoplasm with a moderate amount of organelles, especially mitochondria and secretion vesicles of the Golgi complex (Fig. 3D). 3.3. Morphometric analysis Table 1 shows the values of the parameters examined in the red blood cells (larger diameter, smaller diameter, area and total area of the nucleus) of females and males of P. geoffroanus collected in two different environments (urban and control). Morphometric analysis of red blood cells of P. geoffroanus females from the two environments showed a difference statistically significant in all parameters analysed, females from the urban environment showed a larger values than the females from the control environment (Table 1). Morphometric analysis of red blood cells of P. geoffroanus males from the two environments showed a difference statistically significant only in the area of the nucleus, the males from the controlled
Author's personal copy 1008
C.E.S. Zago et al. / Micron 41 (2010) 1005–1010
Fig. 2. Ultrastructural analysis of the blood cells of Phrynops geoffroanus: (A and B) red blood cells; (C) thrombocytes; (D) neutrophil; (E) lymphocytes; (F) basophil. Golgi complex (GC); secretion vesicles (SV); mitochondria (M); secretion granule (SG). Magnification: A (7750×); B (21,560×); C–E (12,930×) and F (10,000×).
environment showed a larger area of the nucleus in relation to the males from the environment (Table 1). 4. Discussion The analysis performed in the blood cells of P. geoffroanus, using light and electron transmission microscopy helped to confirm several morphological and ultrastructural characteristics, confirming the data available for other species of turtles (Pellizzon et al., 2002; Pitol et al., 2008; Ugurtas et al., 2003; Wood and Ebanks, 1984). The red blood cells observed in P. geoffroanus had the same characteristics of Chelonia mydas (as described by Wood and Ebanks,
1984; Canfield, 1998) and other reptiles (Frye and Murphy, 1991; Goulart, 2004), with nuclei with peripheral heterochromatin and electron-dense and uniform cytoplasm. The thrombocytes of P. geoffroanus also had characteristics as many mitochondria and the nucleus has peripheral heterochromatin, similar to those described by other authors (Canfield, 1998; Frye and Murphy, 1991; GarciaNavarro and Pachaly, 1994; Goulart, 2004; Pellizzon et al., 2002). Neutrophils of P. geoffroanus have the same morphological characteristics of neutrophils of C. mydas, as described by Wood and Ebanks (1984). However, these authors did not analyze the ultrastructural characteristics of these cells. The lymphocytes of P. geoffroanus have the same characteristics of the lymphocytes of C.
Table 1 Red blood cells morphometric analysis of Phrynops geoffroanus females and males from an urban and control environment. Parameters
p ≤ 0.05
Females Control
Larger diameter Smaller diameter Total area Nucleus area *
17.1 9.9 142.6 14.7
± ± ± ±
Urban 1.4 1.1 23.4 3.1
Significant statistical differences—t-test.
18.6 10.7 165.9 15.6
p ≤ 0.05
Males Control
± ± ± ±
1.5 1.2 24.6 3.4
0.0001* 0.0001* 0.0001* 0.0001*
17.2 10.4 147.8 17.7
± ± ± ±
Urban 1.3 4.7 21.4 5.3
17.2 10.3 146.1 14.8
± ± ± ±
1.4 0.8 18.8 2.2
0.6586 0.5544 0.0933 0.0001*
Author's personal copy C.E.S. Zago et al. / Micron 41 (2010) 1005–1010
1009
Fig. 3. Ultrastructural analysis of the blood cells of Phrynops geoffroanus: (A) monocyte; (B) heterophile; (C) eosinophils; (D) azurophils. Secretion vesicles (arrows). Magnification: A and D (10,000×); B (7750×) and C (12,930×).
mydas and Phrynops hilarii (Pitol et al., 2008; Wood and Ebanks, 1984). Nucleoli were not observed, and few mitochondria and several projections were found. The basophils of P. geoffroanus have segmented nuclei and strongly basophilic granules similar to C. mydas and P. hilarii (Wood and Ebanks, 1984; Pitol et al., 2008). These cells contain secretory granules that line the nucleus, similar to those of Gallotia simonyi (Martínez-Silvestre et al., 2005). The monocytes have the same characteristics that have been described by several authors for reptiles (Frye and Murphy, 1991; Garcia-Navarro and Pachaly, 1994; Goulart, 2004; Pitol et al., 2008). The azurophils had the same morphological characteristics that had been described by Frye and Murphy (1991) for other reptiles, as irregular shape, nucleus with periferical heterochromatin and presence of mitochondria, secretion vesicles and Golgi complex. The eosinophils of P. geoffroanus had a peripheral nucleus with rounded shape granulations of acidophilous color, similar to the characteristics observed in C. mydas and P. hilarii (Pitol et al., 2008; Wood and Ebanks, 1984). This group of cells has homogeneous granules with moderate electron density, similar to those found in G. simonyi (Martínez-Silvestre et al., 2005). The heterophils showed red or brown color in Panotic staining and are found in reptiles, birds, and mammals, such capybara (Hydrochoerus hydrochaeris) and domestic rabbit (Oryctolagus cuniculus). These cells have lobed or bi-lobed nuclei, containing cytoplasmatic granules with several shapes (Canfield, 1998; Frye and Murphy, 1991; Garcia-Navarro and Pachaly, 1994; Goulart, 2004; Pitol et al., 2008). Granules of different shapes, sizes and electron density similar to those found in this study have also been found in G. simonyi (Martínez-Silvestre et al., 2005). Hematological studies of many species of reptiles showed the presence of nucleated red blood cells, white blood cells and thrombocytes (Millan et al., 1997; Moura et al., 1999). Studying the New Guinea snapping turtle (Elseya novaeguineae), Anderson et al. (1997) found the same cells that we found in P. geoffroanus, with the exception of the neutrophils, that showed variations in the blood profile of both male and female specimens in geographic regions with small differences in temperature.
In the peripheral blood of the swamp alligator (Caiman crocodilus yacare) nucleated red blood cells and thrombocytes, heterophils, eosinophils, basophils, lymphocytes and monocytes azurophils were found and described by Moura et al. (1999), Stacy and Whitaker (2000). These cells were observed in Crocodylus palustris and Crocodylus porosus yearlings (Millan et al., 1997). Those studies made it possible for researchers to highlight the differences in the leukocytes between saurians and turtles. In this study, light microscopy analysis revealed the presence of red blood cells, thrombocytes and seven types of leukocytes in the peripheral blood of P. geoffroanus. The analysis of transmission electronic microscopy allowed us to characterize the nuclei and the cytoplasmatic organelles of each cell type, expanding the knowledge of the functions and characteristics of turtles blood cells. For the morphometric analysis, the average size of the red blood cells found in P. geoffroanus is consistent with the values that have been previously described for reptiles (Garcia-Navarro and Pachaly, 1994). A comparison of the morphometric parameters of larger diameter and area of the nucleus of cells of P. geoffroanus showed larger values than those found to some freshwater turtles as Emys orbicularis hellenica and Mauremys rivulata, and terrestrial turtles (Testudo hermanni hermanni and Testudo graeca Iberia) from Turkey (Ugurtas et al., 2003). However, when we compared the parameters of smaller diameter and total area of the nucleus, the values observed for P. geoffroanus were lower than those found in E. orbicularis hellenica, M. rivulata, and terrestrial turtles as T. hermanni hermanni and T. graeca iberia. These differences could be a result of physiological adjustments to different environments. The morphometric analysis of red blood cells of females revealed differences between animals of the two environments in all parameters analysed (larger diameter, smaller diameter, total area and core area). The males of the control environment showed difference statistically significant in the area of the nucleus when compared with the males of the control environment, which presented smaller averages. Such data may indicate that red cells undergo changes in the presence of adverse situations, which
Author's personal copy 1010
C.E.S. Zago et al. / Micron 41 (2010) 1005–1010
cytoplasmatic is likely because of the constant interference of environmental change. The morphometric analysis of red blood cells of P. geoffroanus allowed us to standardize the diameter and the total area of the nucleus, as well as the difference between these parameters in terms of the environment from where the animal was collected. This P. geoffroanus study is the first one that makes a correlation between these environments and the description of turtle’s blood cells, thus contributing to the identification of the hematological characteristics of this group. Acknowledgment This study was funded by the Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) of Brazil. References Anderson, N.L., Wack, R.F., Hatcher, R., 1997. Hematology and clinical chemistry reference ranges for clinically normal, captive New Guinea snapping turtle (Elseya novaeguineae) and the effects of temperature, sex, and sample type. Journal of Zoo and Wildlife Medicine 28 (4), 394–403. Andrew, W., 1965. Comparative Hematology. Grune & Stratton, New York and London. Ayres, M., Junior, M.A., Ayres, D.L., Santos, A.S., 1998. Bioestat, third ed. MPEG/CNPq. Brites, V.L.C., Rantin, F.T., 2004. The influence of agricultural and urban contamination on leech infestation of freshwater turtles, Phrynops geoffroanus, taken from two areas of the Uberabinha river. Environmental Monitoring and Assessment 96, 273–281. Canfield, P.J., 1998. Comparative cell morphology in the peripheral blood film from exotic and native animals. Australian Veterinary Journal 76 (12), 793–800. Frye, F.L., Murphy, J.B., 1991. Health and Welfare of Captive Reptiles. Clifford Warwick. Gans, C., 1980. Répteis do Mundo. Universidade de São Paulo, São Paulo.
Garcia-Navarro, C.E.K., Pachaly, J.R., 1994. Manual de Hematologia Veterinária, first ed. Livraria Varela Ltda. Goulart, C.E.S., 2004. Herpetologia, herpetocultura e medicina de répteis, first ed. L.F. Livros de Veterinária LTDA. Mader, D.R., 1996. Reptile Medicine and Surgery. W.B. Saunders Company. Martínez-Silvestre, A., Marco, I., Rodriguez-Dominguez, M.A., Lavín, S., Cuenca, R., 2005. Morphology, cytochemical staining, and ultrastructural characteristics of the blood cells of the giant lizard of El Hierro (Gallotia simonyi). Research in Veterinary Science 78, 127–134. Millan, J.M., Janmaat, A., Richardson, K.C., Chambers, L.K., Fomiatti, K.R., 1997. Reference ranger for biochemical and haematological values in farmed saltwater crocodile, Crocodylus porosus yearlings. Australian Veterinary Journal 75 (11), 814–817. Moura, W.L., Matushima, E.R., Oliveira, L.W., Egami, M.I., 1999. Aspecto morfológico e citoquímico dos glóbulos sangüíneos de Caiman crocodilus yacare (Daudin, 1802) (Reptilia, Crocodilia). Brazilian Journal of Veterinary Research and Animal Science 36 (1), 01–09. Pellizzon, C.H., Azevedo, A., Casaletti, L., Lunardi, L.O., 2002. The thrombocyte aggregation process in the turtle Phrynops hilarii (Chelonia). An ultrastructural study. Journal of Submicroscopic Cytology and Pathology 34 (3), 323–327. Pitol, D.L., Issa, J.P.M., Caetano, F.H., Lunardi, L.O., 2008. Radioautographic study of the seasonal distribution of leukocytes in turtles Phrynops hilarii (Chelonia Chelidae). Micron 1228, 1–6. Pritchard, P.C.H., Trebbau, P., 1984. The Turtles of Venezuela, Fundación de Internados Rurales (Venezuela). Society for the study of Amphibians and Reptiles. Stacy, B.A., Whitaker, N., 2000. Hematology and blood biochemistry of captive mugger crocodiles (Crocodylus palustris). Journal of Zoo and Wildlife Medicine 31 (3), 339–347. Souza, F.L., Abe, A.S., 1999. Um sobrevivente em rios poluídos. Ciência Hoje 147, 01–09. Ugurtas, I.H., Sevinc¸, M., Yildirimhan, H.S., 2003. Erythrocyte size and morphology of some tortoises and turtles from Turkey. Zoological Studies 42 (1), 173–178. Wood, F.E., Ebanks, G.K., 1984. Blood cytology and hematology of the green sea turtle, Chelonia mydas. Herpetologica 40 (3), 331–336. Zago, C.E.Z., Ferrarezi, A.L., Vizotto, L.D., Oliveira, C., Cabral, S.R.P., Tabaga, S.R., Bonilla-Rodriguez, G.O., Venancio, L.P.R., Bonini-Domingos, C.R., 2010. Hemoblobin polymorphism and hematological profile of Geoffroy’s side-necked turtle (Phrynops geoffroanus, Testudines) in the northwestern region of São Paulo State, Brazil. Genetics and Molecular Research 9, 721–726.
