| In order to take advantage of resource of Anhui province and establish an appropriate procedure for IVEP, the present dissertation studied the in vitro production of bovine embryos with bovine ovaries derived from slaughterhouse. The bovine ovaries were transported for about 12 h at 30-38°C and 20-25°C, respectively, and then the oocytes were cultured in vitro. The results showed that 2.42 and 3.33 cumulus-oocyte complexes (COCs) per ovary were harvested respectively (P>0.05), and the maturation rate was 70.75 % and 79.55% respectively (P>0.05). After in vitro fertilization, the cleavage rates were 48.06% and 65.21% respectively (P<0.05), while the morula and blastula rates were 20.08% and 30.12% respectively (P<0.05). The cleavage rate was most significant higher when the cumulus cells of COCs after in vitro maturation were no removal than that of removal during in vitro fertilization (63.21% vs 32.33%, P<0.01). The morula and blastula rates were also significantly higher (31.66% vs 15.29%, P<0.05).Different concentrations (0, 10%, 20% and 40%) of bovine follicle fluid (bFF) from over 4mm were supplemented to the maturation medium. The results showed that the maturation rate was 73.28%, 74.16%, 69.88% and 63.68% respectively. The maturation rate of 40% bFF group was significantly lower than that of no bFF group (P<0.05). The cleavage rate and morula and blastula rate of 10%, 20% amd 40% group were significantly higher than those of no bFF group (P<0.05). Different concentrations (0, 10 ng/ml, 20 ng/ml and 50 ng/ml) of EGF were supplemented to the maturation medium. The results showed that the maturation rate had no difference among them (75%, 77.72%, 82.96% and 80.42% respectively, P>0.05) but the the cleavage rate and morula and blastula rate of EGF addition groups were significantly higher than no EGF group (P<0.05). Comparative experiment was carried out between swimming-up method and washing method of frozen/thawed semen in order to observe the respective effect of two methods on the efficiency of in vitro production of bovine embryos. The results showed that the cleavage rate was 63.81% and 31.20% respectively (P<0.05) while the morula and blastula rate was 33.21 % and 22.82% respectively (P<0.05).When bovine fertilized zygotes were cultured in medium M199 supplemented 10% FBS, in CR1aa with 3 mg/tnl BSA, or in SOFaa with 3 mg/ml BSA without cumulus monolayes, the cleavage rate was 47.28%, 63.15% and 60.94% respectively, and the blastulas rate was 0, 15.52% and 19.20% respectively, and the hatched blastulas rate was 0, 58.33% and 62.50% respectively. The cleavage rate, the blastulas rate and the hatched blastulas rate was significantly lower than that of the others (P<0.05). When bovine fertilized oocytes were cultured in medium M199 supplemented 10% FBS, CR1aa supplemented 3mg/ml BSA and SOFaa supplemented 3mg/ml BSA with cumulus monolayes, the cleavage rate was 48.20%, 58.61% and 59.78% respectively (P>0.05), the blastulas rate was 24.39%, 22.37% and 20.88% respectively (P>0.05), and the hatched blastulas rate was 62.50%, 66.67% and 75% respectively (P>0.05). The ovaries were divided into two groups according to the age of slaughtered cattle, young group (<8 mon old) and adult group (>8 mon). The results showed that 2.50 and 4.02 COCs per ovary were harvested respectively, and the maturation rates were 39.66 % and 87.27 % respectively. The results showed that the cleavage rates were 39.70% and 71.37% respectively, and the morula and blastula rates were 10.06% and 31.81% respectively. There was significant difference between the young group and adult group (P<0.05).In conclusion, a comparatively feasible and effective procedure of bovine IVEP was established. The bovine ovaries collected from slaughterhouse were transported for about 12h at 20-25°C. Bovine cumulus-enclosed oocytes were matured in M199 supplemented with 10% FBS, 10% bFF or 20ng/ml EGF for 24h. Frozen/thawed semen was treated by swimming-up in Sp-TALP solution. Mature oocytes and treated semen were incubated all together for 18-20 h in IVF-TALP supplemented 10μg/ml heparin. The presumed zygotes were transferred to SOFaa culture medium supplemented 3mg/ml BSA, till that zygotes developed to morula or blastocyst.
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