Effect of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries

The objective of this study was to compare enzymatic and mechanical methods, at distinct fetal ages, on isolation of different developmental stages of preantral follicles from bovine ovaries. Fetal ovaries were obtained from pregnant cattle at 150 to 270 d of gestation, and 135,521 preantral follicles at different stages of development were studied. The dissociation of ovaries with a mechanical procedure resulted in an average of 938.16 preantral follicles. In contrast, 3,715.56 follicles were obtained when enzymatic digestion was used (P=0.0001). Histological evaluation confirmed follicular stages and demonstrated that both mechanical and mechanical-enzymatic procedure did not affect the cellular integrity of the follicles. Granulosa cell-oocyte complexes surrounded by a basal membrane, were considered preantral follicles in this study. The ratio of different stages of isolated preantral follicles was significantly (P=0.0001) correlated to fetal age. The earliest fetal age at which tertiary follicles were identified was at 210 d of gestation. The results confirm previous observation that follicular development and atresia are initiated during fetal development. These data provide information on methodologies to isolate intact bovine preantral follicles for investigating the control and regulation of follicular, development and the growth of preantral follicles in vitro.