In Vitro Culture Of Bovine Preantral Follicles In Serum-Free System

In vitro culture of the preantral follicles from bovine ovaries is one of new reporductive biotechniques with great prospects, which could supply large numbers of oocytes for In vitro production of embryos. A new mechanical method for the isolation of preantral follicles from bovine ovaries and a serum-free culture system using McCoy's 5a medium (pH7.4)supplemented with 20mM Hepes, 3mM L-glutamine, 0.1%BSA, 29ng/mL testosterone, 2.5u, g/mL transferrin, 100ng/mL insulin and 4ng/mL selenium for culture of bovine preantral follicles were developed .By this the system, bovine preantral follicles in different diameters were cultured in vitro;and the effects of the number of follicles in each well of culture plate, different concentrations of NGF(nerve growth factor), Vitamin E, and Vitamin C on in vitro development of preantral follicles were studied. The results showed that: 1.under sterile condition, ovarian cortical slices at the thickness of 0.6-lmm were dissected from ovaries with skin-graft razor. The dissected ovaries and the cortical slices were washed with PBS,and the washed PBS was collected into petri dishes.Then the cortical slices were clipped into pieces of 2mm in the culture medium by ophthalmic surgical clippers.The pieces were filtrated through nylon mesh filters(450um)and the medium was collected into petri dishes.The remnants on the filter were clipped further into smaller fragments and filtrated through 300um nylon mesh filters .The medium was collected into petri dishes.Fifteen minutes after collection,all the washed PBS and filtrated medium mentioned above were sought out for preantral follicles under a stereomicroscope at >90 x magnification .The recovery rate of preantral follicles was 57.23 + 10.21 per hour).2.With 250u1 McCoy's 5a culture medium in each well of 96-well culture plates, by incubating in a humidified atmosphere with 5%CO2 at 37Cand changing the culture medium every 5 days, the serum-free culture system is successful for the development of preantral follicles. In this system the follicles grew continuously maintaining their normal morphology. Antrum formation occurred on about the 7th day of culture of preantral follicles with pre-culture diameter of 130um or more, and the rate of antrum formation was about 56.25%(9/16).3. A new method for assessment of viability of follicles by staining with Trypan blue and hoechst33342 was developed..4.Culture medium supplemented with 12uM Vitamin E and 96uM Vitamin C was helpful for the in vitro development and growth of follicles. The addition of NGF(4-8IU/ml) to the culture medium promoted the in vitro development and growth of preantral follicles(<60u m).