Study On The Optimization Of In Vitro Maturation And Fertilization Of Sheep In Summer Anestrous Season

This study was to optimiza the technologies for production of embryo in vitro, including in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) in sheep to establish a perfect system for in vitro embryo production (IVP) and to make these technologits going out of laboratory to be used in the fields of embryonic engintering.Experiment one was designed to study the IVM methods for increasing the maturation rate of oocytes. The effects were studied which included methods of IVM of oocytes, supplementation of different density hormones, temperature of ovaries transportation and culture time of oocytes in sheep. The result showed that oocytes of grade A with uniform and compact cytoplasm and parceled by at least three layers of cumulus cells were cultured which obviously improved the maturation rate of oocyted in sheep when the ovaries transport temperature was 35-38°C(69.57% vs 50.85% vs 67.81%, 67.19% vs 38.33% vs 44.07%). FSH 12μg/ml, LH 10μg/mlL, E₂ 1μg/ml is proper to sheep oocytes IVM in summer anestrous period. The maturation rate, cleavage rate, blastocyst rate of Flat ware mothed higher than that of Droplet method in sheep. But there was no singnificant differences between two methods. The maturation rate, cleavage rate was significant higher for 27h than 24h, 20h in sheep oocytes (68.64% vs 51.01% vs 44.12%).Experiment two was designed to study the optimization of IVF conditions for increasing the quality and quantity in vitro matured of sheep oocytes in summer anestrous period. The effect of fertilization media, which in eluded BO medium and BO+TCM-199 mixture in proportion (3 : 2, BM) and oocyte-sperm interaction time and water quality of medium preparation on the fertilization and embryonic development in sheep. The result showed two types of fertilization media had the similar effect on the cleavage rate of fertilized embryos. But, the blastocyst stage in BM medium had significant higher than those fertilized in BO (31.96% vs 20.45%). oocyte-sperm interaction in 15¹8h interaction had significant higher blastocyst rate than that for 10h, 24h interaction (24.76% vs 29.59% vs 3.70% vs 10.89%). The types of water used in the experiment: three-distilled water and Milli-Q water. It indicated that the cleavage rate had no significant difference betweent medium prepared with two types of water. However, the frequency of development to the blastocyst stage in medium prepared with Milli-Q water was significant higher than with three-distilled water (30.48% vs 19.75%).