Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano

• Autor(a) principal: Jimenez, Carolina Rodriguez
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1877
• topic: Biotecnologia; Bovinos; Folículo pré-antral; Foliculogênese; Ovário in situ.; Biotechnology; Cattle; Pre-antral follicle; Folliculogenesis; Ovary situ; CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL::FISIOPATOLOGIA DA REPRODUCAO ANIMAL

The objectives of this study were (I) to determine the best basic culture medium for bovine preantral follicles culture enclosed in ovarian cortical tissue (in situ), using the minimum essential medium (α-MEM), tissue medium culture 199 (TCM-199) and medium McCoy s (McCoy s); (II) to evaluate different concentrations of Insulin like growth factor 1 (IGF-1) and growth hormone (GH) in the development of bovine preantral follicles in situ cultured and (III) to evaluate a sequential medium containing GH and, or, IGF-1 on long-term in situ culture of bovine preantral follicles. Ovaries of cows without defined breed standard were collected from local slaughterhouse and transported to the laboratory in TCM-199 supplemented with HEPES, sodium bicarbonate, pyruvic acid, penicillin and streptomycin. In the laboratory the ovarian cortex was fragmented and was taken for short-term cultures (7 days) on phases I, II and long-term (14 days) on phase III. The culture was performed in an incubator at 37 °C and 5 % CO2 in a 24 well plate with complete medium exchange every 2 days. Uncultured (control) and cultured ovarian fragments were processed for histological evaluation (morphological and morphometric) and evaluated the percentage of normal morphologically follicles, primordial and developing (primary and secondary) follicles, beyond the follicular and oocyte diameter. In addition, viability was assessed by trypan blue staining, the follicles classified as being stained and unstained, dead and living, respectively. The morphological and morphometric variables were analyzed by ANOVA followed by comparisons of means by Tukey and Dunnett s test and viability variables were subjected to chi-square (χ2) test. The results of Phase I showed that α- MEM supplemented (α-MEM+) preserved follicular morphology and it highlighted in morphometric and viability analysis. Consolidating the results of phase I, on phase II we used the α-MEM+ with different concentrations of recombinant human IGF-1 (0, 30, 70, 100 ng/mL) or recombinant human GH (0, 10, 25, 50 ng/mL), composing the experiment 1 and 2, respectively. The results showed that IGF-1 (30 ng/ml) and GH (50 ng/mL) treatments were the most effective concentration to maintain morphological and morphometric functionality, as well as the viability of in situ cultured of bovine preantral follicles. On phase III ovarian fragments were cultured in α-MEM+ in the absence or presence of GH (50 ng/mL) and, or, IGF-1 (30 ng/mL) added as follows: (α- MEM+) for 14 days, (GH→IGF-1) GH from 1 st day to 7th day and IGF-1 from 8th day to 14th day, (GH→IGF-1) IGF-1 from 1st day to 7th day, and GH from8 th day to 14th day and (GH+IGF-1) for 14 days. The treatment (GH→IGF-1) maintained the percentage of normal morphology follicles during the whole culture period and the percentage of developing follicles increased compared to fresh control after 7 days of culture, while follicular diameter increased in both 7th and 14th days of culture. Therefore, it is concluded that in situ cultured bovine preantral follicles in sequential medium treated with GH→IGF-1 improved the development and follicular diameter until the first half of the culture and kept these parameters with normality and viability until the end of in vitro culture.