In Vitro Culture Of Bovine Preantral Follicles In Serum-free System And Their Ultrastructural Study

A new meachanical method for the isolation of preantral follicles from bovine ovaries was developed and the relationships between the size and developmental status of bovine ovaries and the number of isolated preantral follicles from them were disscussed. A serum-free culture system has been established by comparing the growth .development and secretion of E2 of the preantral follicles cultured in vitro in the presence or absence of FCS. By this system, effects of gonadotrophins (FSH and LH) and bovine follicular fluid(BFF) on in vitro development of preantral follicles, secretion of E2 , and the ultrastructural changes during culture were studied. The results showed that:1 A simple, rapid mechanical method can be used effectively to isolate intact preantral follicles from adult cow ovaries. The mean numerber of preantral follicles isolated from each ovary of cows was 46.9 18.8. The processing time was 25.4?.7 min for every follicle . The diameters of the preantral follicles isolated were 60~150, most of which were secondary follicles and could be cultured in vitro.2 The number of preantral follicles was positively correlated with the ovarian weight in the ovaries with normal external appearance. The presence or absence of corpus lutea did not affect the number and distribution status of isolated preantral follicles.The distribution of superficial antral follicles from ovaries was associated with the number of preantral follicle isolated. Large number of preantral follicles could be aquired from ovaries with balanced distribution of superficial antral follicles, whether corpus lutea existed or not. But seldom or no preantral follicles could be collected from fatly bloodly ovaries, and immature ovaries, or ovaries with fat corpus lutea.3 There was no obvious difference on growth and development of preantral follicles in thepresence or absence of of FCS in culture medium. In the serum-free culture system ,the follicles could grow continuously, synthesizing measurable level of oestradiol and maintaining their normal ultrastructral morphology.4 The quality evalution for preantral follicles before or after culture can be done by contrast phase microscope combined withTEM.5 Culture medium supplemented with gonadotrophins (FSH and LH) was helpful for the in vitro development and growth of preantral follicles. The addition of BFF in the presence of gonadotrophins promoted the in vitro development and growth of preantral follicles and the secretion of E2 significiantly. The level of oestradiol arrived the highest value(48.24pg/ml) in the sixth day of culture of follicles with a diameter of 100 m above.6 The transmission electron microscope(TEM) analysis showed that the mechanically isolated preantral follicles were with 1 to 3 layers of granulose cells tightly enclosing the oocyte and intact basement membrane. The ultrastructural characteristics of the preantral follicles fresh isolated or cultured in vitro were mostly similar to those presented in situ.