Studies Related To Isolation And Culture Of Animal Preantral Follicles

1. The distribution, histological and ultrastructural characterization of buffalo and mouse preantral follicles in ovaries was studied. Primordial follicles, which had one layer of squamous granulosa cells, were always located in the outer part of ovary cortex. Primary follicles, which had a single layer of cubical granulosa cells, were found in the middle cortex. Secondary follicles with two or more layers of cubical granulosa cells in symmetric distribution were located only within the inner region of the cortex with many large blood vessels. The diameter of primordial, primary and secondary follicles and their oocyte diameter in adult buffalo ovaries was larger than those in heifer and fetal ovaries. Adult buffalo primordial, primary and secondary follicles also had more ganulosa cells in comparison with heifer and fetal follicles. The diameter of primordial follicles and oocytes and granulosa cell number from 7d, 14d and 28d of mice did not differ significantly, but the diameter of primary and secondary follicles and their oocytes from 7d mice was shorter than that of 14d and 28d mice, granulosa cell number of primary and secondary follicles from 7d mice was also less than that of 14d and 28d mice (p<0.05). The basement membrane of buffalo and mice follicles was made of two layers of thin and smooth fiber-like material. Mitochondria had cap ultrastructure. Organelle of oocyte distributed near the nucleus.2. Methods for isolation of buffalo and mouse preantral follicles were investigated. Comb-scrape method harvested significantly more preantral follicles (152.35+44.81/ovary) than cutting (32.62+ 14.81/ovary) and micromanipulation method (8.95+3.44/ovary, p<0.05). The average manipulative time for each ovary (39.05+4.27min) was also less than cutting (46.43+4.15min) and micromanipulation method (44.55+7.82min, p<0.05). Majority of preantral follicles isolated by comb-scrape method were primordial follicles (41.25%) and primary follicles (38.79%), and very few of secondary follicles (19.95%). These results indicated that comb-scrape method was suitable for isolating buffalo preantral follicles.3. Fetal ovaries harvested significantly more preantral follicles (943.43+143.13/ovary) than heifer (249.50+40.11 /ovary) and adult ovaries (128.50+19.99/ovary) (p<0.01) , indicating that more preantral follicles can be isolated from buffalo fetal ovaries.4. Digestion of small ovary tissue pieces with 0.02% 0.04% and 0.08% collagenase harvested significantly more preantral follicles in comparison with without digestion (40.38+6.12 47.75+6.84 and 52.69+6.15 vs 33.56+6.15 per ovary, p<0.05), indicating that combination of cutting and enzymatic method can harvest more buffalo preantral follicles than cutting method alone.5. Comb-scrape method harvested significantly more mouse preantral follicles (61.00+9.12/ovary) than cutting (26.30+5.06/ovary) and micromanipulation method (14.10+4.16/ovary, p<0.05). The average manipulative time for each ovary (30.80+3.91min) was also less than cutting (39.10+4.01min) and micromanipulation method (38.40+3.50min, p<0.05). Majority of preantral follicles isolated by comb-scrape method were secondary follicles (83.61%) and primary follicles (12.13%), and very few of primordial follicles (4.26%). These results indicate that comb-scrape method is also suitable for isolating mouse preantral follicles.6. Effects of isolation methods and buffalo ages on the growth of preantral follicles in vitro culture were studied. The survival rate of preantral follicles isolated using the comb-scrape method at 48 and 72h after in vitro culture did not differ significantly from that of cutting and micromanipulation methods (p>0.05). The survival rate of buffalo fetal preantral follicles was significantly lower than that of heifer and adult preantral follicles (34.69% vs 52.67% and 52.03%, p<0.05) after in vitro culture of 72h. The survival rate of buffalo preantral follicles isolated by 0.08% collagenase treatment was significantly lower than the follicles insolated by 0, 0.02% and...