RESUMO
We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.
Assuntos
Técnicas de Cultura de Células , Cromossomos/genética , Meios de Cultura/química , Linfócitos/citologia , Tartarugas/genética , Animais , Corantes Azur/análise , Brasil , Bandeamento Cromossômico , Cromossomos/química , Colchicina/farmacologia , Feminino , Fixadores , Humanos , Linfócitos/efeitos dos fármacos , Masculino , Mitógenos/farmacologia , Índice Mitótico , Fito-Hemaglutininas/farmacologiaRESUMO
Complete blood counts and hemoglobin isoform data were gathered from 36 specimens of the turtle species Phrynops geoffroanus from the northwestern region of São Paulo State, Brazil. They were collected in an urban area. The hemoglobin profiles were obtained after red blood cell lysis and by electrophoretic migration in alkaline pH, acid pH, and neutral pH buffer. The hemoglobin components were confirmed using high-performance liquid chromatography (HPLC). Erythrogram analysis included hematocrit, total hemoglobin concentration, total red blood cell count, and red blood cell indices. The leukogram included a total white blood cell count and a calculation of the percent values of neutrophils, lymphocytes, monocytes, basophils, eosinophils, heterophils, and azurophils. HPLC analysis revealed three hemoglobin components; the first with a concentration of 5.5%, the second was a major component with an average concentration of 67.1%, and the third with a concentration of 28.5%. The hematological profile obtained for these specimens allowed us to establish a pattern for P. geoffroanus in São Paulo State Northwestern region. The average hematocrit values were 22.5% for females and 24.0% for males. For total hemoglobin, we found average values of 6.66 g/dL in females and 7.22 g/dL in males. The number of white blood cells was 2725 x 10(3)/microL for females and 2775 x 10(3)/microL for males. There was a predominance of heterophils, eosinophils, and monocytes in both sexes. No significant differences were found between males and females for hematological profile. The hematological results were compared to literature data for other Chelonia. They were similar to what is known for fresh water turtles.