Effect of different mechanical isolation techniques on developmental competence and survival of buffalo ovarian preantral follicles

The present study aimed to analyze different methods of mechanical isolation of buffalo preantral follicles (Experiment I), in vitro culture of isolated follicles in different groups of culture medium over collagen gel matrix (Experiment II) and subsequent in vitro development and survival of isolated preantral follicles (PFs) (Experiment III). In Experiment I, ovarian cortical pieces were separated and PFs isolated by different mechanical methods. In Experiment II, isolated follicles were divided into three groups and cultured in TCM-199 having 10% FBS, 1% ITS, and 20 ng/ml EGF (Group A, control), addition of 0.5 μg/ml FSH (Group B) or FSH + 100 ng/ml IGF-I (Group C). Follicles were incubated at 38.5 °C in 5% CO2 with maximum humidity. In Experiment III, based on the outcome of Experiment I and II, PFs were cultured from those isolation method and treatment group, showing better growth and developmental pattern to analyze the impact of growth factors on in vitro growth of follicles in long term culture. It was found that micro-dissected PFs showed higher survival rate and growth after 15 days of culture compared to PFs isolated by other methods. Follicles cultured with FSH + IGF-1 on collagen gel matrix, showed significantly (P < 0.05) higher survival rate and mean diameter of follicles on day 15 of culture compared to control. In summary, it has been shown that isolation of follicles by micro-dissection has advantages over other methods, being relatively simple, inexpensive and less harmful to follicles. Micro-dissected buffalo PFs maintained the architecture, showed antrum formation in presence of FSH and IGF-I over the collagen gel matrix.