ABSTRACT. 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 nonbanded 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 Method for peripheral blood lymphocyte culture able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.
The seminal ducts (efferent ductule, epididymis, and deferent duct) in adults of Phrynops geoffroanus were examined using light microscopy. A series of tubules (efferent ductules) connect the testes to the epididymides. The efferent ductules are formed by a rete of small tubules of varying diameters, with simple columnar epithelium formed by the ciliated cells, nonciliated cells, and few basal cells. The epididymis is a simple, long and highly convoluted tubule that receives the efferent ductules throughout its extension. It is covered by a pseudostratified columnar epithelium with three cellular types: the principal cells, which are the most abundant, basal cells, and a small narrow cell. The histological differences in the epididymis region (cranial, medial, and caudal), as well as the differences in the epithelium throughout the reproductive cycle, are discussed. The deferent ducts consist of a low pseudostratified epithelium with two cellular types: the principal and basal cells. During the months analyzed, spermatozoa were stored in the epididymis, and deferent ducts were found. Anat Rec, 294:145-155, 2011. V V C 2010 Wiley-Liss, Inc.
The number of species of reptiles has decreased since the time that they ruled the Earth until the present days. Turtles have been little studied, particularly regarding their cytogenetics. The present study investigated, using classical and molecular cytogenetic techniques, the chromosomes of Chelonoidis carbonaria, a terrestrial species, and Phrynops geoffroanus, an endangered species that lives along the rivers, both in South America. Blood samples were collected from animals at the breeding farm Reginaldo Uvo Leone in Tabapuã, SP. All procedures were approved by the Animal Ethics Committee (No. 50/07-CEEA, Botucatu, SP) and IBAMA/ RAN (No. 14729-1). The metaphases were prepared from blood incubated in culture medium for lymphocytes, which were stimulated to divide by the addition of phytohemagglutinin. After this procedure, colchicine was added to inhibit the formation of spindle fibers, maintaining the cells in metaphase. We assessed male and female C. carbonaria, which had the same number of chromosomes, 2n = 52, and specimens of P. geoffroanus, which were described for the first time and showed females with 2n = 58 and males with 2n = 57 chromosomes. Using the Gbanding technique it was possible to identify pairs of homologous chromosomes and with the C-banding technique, the presence of regions of constitutive heterochromatin in macrochromosomes and microchromosomes. Silver impregnation detected the nucleolar organizer regions (NORs) in two microchromosomes in males and only one of the microchromosomes in females in C. carbonaria. The same pattern was found in P. geoffroanus. The FISH technique revealed the presence of rRNA sites showing positive staining for NORs. This study showed the ability of classical and molecular cytogenetic techniques to identify the chromosomes of males and females of C. carbonaria and P. geoffroanus, terrestrial and aquatic species, respectively, making it possible to use them as a model for genetic and evolutionary studies.
The reproductive system of male Phrynops geoffroanus adults is macroscopically described and the variation in testicular biometry is evaluated. A pair of oval testes is connected by the efferent ductules to the epididymis, which continue as deferent ducts, which emerge in the penis. The volume of the gonads showed the highest averages during spring and summer months. GSI varied significantly throughout the year, with the highest averages observed in the months that correspond to the end of spring and the beginning of summer, when the reproduction of the species takes place, and the lowest averages were seen in winter, suggesting a cyclical testicular activity.
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