These studies were carried out to investigate the effects of the size of follicles, semen types, capacitation methods, and additions of hormones, estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) to the medium on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3∼5㎜.
The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24∼48hrs. in an incubator with 5% CO₂ in air at 38.5℃ and then matured oocytes were again cultured for 18∼20 hrs, with motile capacitated spermatozoas the TCF (Tyrode calcium-free) solution containing 100㎍/㎖ uf heparin. The results obtained in these experiments were summarized as follows:
1. The oocytes classified as "A,B,C,D and Degenerative" depending morphological integrity and those 61.4%, 12.1%, 19.2%, 4.2% and 3.O% of the total oocytes recovered, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 66.1, 33.3%, respectively.
2. The average number of the follicular oocytes recovered from follicles size, 1∼2㎜, 3∼5㎜ and above 5㎜ in diameter were 67, 98 and 63, respectively. The maturation and fertilization rate of the follicular oocytes, cultured in the TChi-199 medium were 56.7%, 82.5%, 46.0% and 44.8% 71.4%, 28.6%, respectively.
3. The fertilization and cleavage rate of the follicular oocytes, inseminated with spermatozoas of epididymal cauda, neat and frozen semen were 63.3%, 73.3%, 70.0% and 32.7%, 37.8% 38.3%, respectively.
4. The fertilization and cleavage rate of follicular oocyces, fertilized with capacitated spermatozoas by heparin, BFF and HIS methods were 70.0% 53.8%, 34.2% and 38.3%, 23.1%, 17.1%, respectively. And the fertilization and cleavage rate were higher method of heparin.
5. The maturation and fertilization rate of follicular oocytes, cultured in the TCM-199 medium supplemented with 5%, 10%, 15%, 20% and FSH, HCG, 17 β-estradiol were 76.0∼82.3% and 26.2∼70.0%, and those values were higher the supplementation than non-supplementation.
6. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5∼20% ECS and FCS were 7 4.O∼80.6%, 26.2∼30.0% and 71.7∼76.9%, 51.9∼58.0% and the values were higher the supplement of ECS than FCS.
7. The maturation rate (68.0∼64.6%) and fertilizatiou rate(59.6∼60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with l0% FCS and 20∼30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF.
8. The maturation rate(76.5%) and fertilization rate (61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1×10^6/㎖ MCC were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with l0% FCS and 1×10^(4∼5)/㎖ and 1×10^8/㎖ cumulus cells.
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