A Preliminary Study On The Isolation And Culture Of Buffalo Preantral Follicles

The present studies were conducted to investigate the distributing and the histological character of buffalo preantral follicles in ovary, and explore a method for isolation and culture of buffalo preantral follicles.(1) The distributing and histological character of buffalo preantral follicles in ovary was studied by means of paraffin section. The distributing of different stage follicles had distinctly region. Primordial follicles, which had one layer of squamous granulosa cells, were always located in the outer part of the cortex; Primary follicles, which had a single layer of cubical granulosa cell, were found in the middle cortex. Secondary follicles, which distributed many large blood vessels, had two or more layers of cubical granulosa cells and were distributed only within the inner region of the cortex. Follicles had two or 4 layers of granulosa cells, which distributed asymmetricly. The diameter of primordial, primary and secondary follicles was 36.9 ± 7.7,48.7±8.9 and 92.0 ± 27.3μm respectively and the diameter of corresponding oocytes was 19.6 ± 6.0, 27.3 ± 7.1 and 49.1 ± 17.4μm respectively. The number of granulosa cells in primordial and primary follicles were 10.8 ± 2.3 and 18.1±3.1 respectively.(2) Methods for isolation of buffalo and cattle preantral follicles were investigated. The comp scrape method (Ml) showed harvested significantly more buffalo preantral follicles (152.4± 44.8/ovary) than cutting (M2) (32.6 ± 14.8/ovary) and micromanipulation M3 (9.0 ± 3.4/ovary)(p<0.05). The needle gap of comp was proved to be 500 and 750μm in the further experiment. The basement membrane of follicles isolated by M1 remained integrity as identified by electron microscope. Ml was also proved to be effective in isolating cattle preantral follicles (1195.2±685.0/ovary). Results indicate that Ml can isolate buffalo and bovine preantral follicles effectively.(3) Effects of ascorbic acid (Vc), FSH and EGF on the in vitro development of buffalo preantral follicles were examined. Ascorbic acid can maintain the follicular morphologic integrity during culture but does not affect the survival and growth of follicles. FSH can promote the growth of follicles, but does not affect the survival and morphology of follicles. The growthpromotive role of FSH is enhanced by the presence of Vc. EGF can accelerate the diameter increasing of follicles in the early culture stage, and this action is enhanced in the presence of FSH.(4) The in vitro growth of follicles in different diameters (60~180μm) was examined. As the follicular diameter increases, the survival rate and growing extent of follicles increases, the morphological abnormity of follicles decreases.(5) Conventional, two dimension (agarose coating) and three dimension (agarose embed) culture system were compared in their efficiency of supporting follicle growth. Three dimensions culture system is proved to be suitable for cultunng the follicles in diameter of 60~100μm, while the two dimension culture system is proved to be suitable for culturing the follicles in diameter of 100-140μm.