| Oocyte recovered from ovaries after slaughter, displayed a wide range of quality. However, up to the present there is no non-invasive method to select competent oocyte accurately. It is now widely recognised that apoptotic cell death is an underlying mechanism of cell loss during follicular atresia. Many studies have showed that follicular atresia is associated with the apoptosis of granulosa cells and the subsequent loss of oocyte competence. But there are many different viewpoints on the relationship between the follicular atresia and the oocyte developmental competence. To solve these problems, it's necessary to establish an individual culture system without impairing oocyte developmental competence. In the present study, we compared several apoptosis assays, studied and established a new individual culture system using goat ovaries after slaughter as experimental material. Under this system we studied the effect of granulosa cell apoptosis on cumulus expansion, oocyte developmental competence and other questions. Finally, we establish a new method to estimate the developmental competence of oocyte based on the density of granulosa cell in follicle fluid. The results obtained are as follows:1. Annexin V assay, Hematoxylin assay and H33342 assay was used to determinate granulosa cell apoptosis. There was significant correlation among the three assays, but H33342 was economic and convenient.2. The drop size and culture density had no impact on oocyte maturaion in vitro. As to development in vitro, too low or too high culture density was proved detrimental to embryo development. Thus, it was determinated that 10 wells per 100μl drop for IVM, and 20 wells per 50μl drop for IVC in wells in drop (DIP) system.3. Compared with the group culture and the individual culture, we found that the efficiency of DIP was the same as the group culture, whereas the individual culture impaired the oocyte developmental competence severely and oocytes did not develop past the 16-cell stage.4. Oocyte recovery from dissected follicles can make use of ovary more sufficiently.5. The morphology of COC and cumulus expansion was both worse with the increasing percentage of granulosa cell apoptosis gradually. Cumulusexpansion had great effect on oocyte developmental competence. Oocytes with Grade 0 of cumulus expansion were hard to mature and few of them cleaved and developed to 8-cell stage. In 1.0-1.4mm follicles, cumulus expansion had no effect on maturation. Whereas in 2.0-3.9mm follicles, oocytes with Grade 3 of cumulus expansion had higher cleavage rate and morular/blastocyst rate than other groups.6. As to 1.0-1.9mm follicles, oocytes maturation benefited from high granulosa cell apoptosis. But in 2.0-3.9mm follicles, granulosa cell apoptosis had no influence on oocyte maturation.7. Granulosa cell apoptosis affect oocyte developmental competence significantly. In 2.0-3.9mm follicles, oocytes from slightly atresia (percentage of apoptosis 5-30%) follicles have better developmental competence, resulting a higher morular/blastocyst rate. But this effect was not observed in 1.0-1.9mm follicles.8. Follicle size had great effect on cumulus expansion and oocyte developmental competence. If granulosa cell apoptosis did not be considered, the cumulus expansion was better and oocyte developmental competence increased with follicle diameter.9. Most than 70% of granulosa cells apoptosis in follicle fluid, and the density of granulosa cells in fluid can reflect the follicle atresia. Oocytes from follicle of intermediate density (7.5-12.5 X 106/ml) of granulosa cells obtained higher blastocyst rate. As high percentage of basal atresia (25.9-40.0%) occurred in <2.5mm follicles, this method is suitable for >2.5mm follicles. However, oocytes from follicles of this portion have better developmental competence, thus the practicability of this method didn't be impaired.
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