Studies On Bovine Oocyte In Vitro Maturation And Its In Vitro Fertilization With Sorted Sperm

The research was carried out to study oocytes in vitro maturation (IVM),in vitro fertilization (IVF) with sorted sperm and in vitro culture (IVC),to optimize in vitro production (IVP) system of bovine sex control embryo and provide theoretic basis of this technique for practical purpose out of laboratory, as well as establish theoretic basis for somatic cell nuclear transfer and transgentic animal engineering. To attain those objectives the research was performed in three parts as under:In order to optimize the bovine oocytes in vitro maturation system, the factors effect IVM of bovine oocytes was investigated in this study. The cumulus oocyte complexes (COCs) were collected by aspiration from the follicles on the ovaries which were picked off from the local slaughter house, and the COCs were incubated in different concentration of FSH,LH and 17β-estrogen (E2), respectively. It was found that the optimal concentrations were FSH 10μg/ml, LH 10μg/ml and E2 1μg/ml, respectively; Compared with 5% FCS/OCS and 10% FCS/OCS in TCM-199 medium, the latter 10% FCS/OCS is better for the maturation of bovine oocytes, whereas there no significant difference between each other(P>0.05).However, It was found that 10% OCS is more suitable to IVM of oocytes if without exogenous hormone in TCM-199 medium, so adding 10% OCS is practicable and more economical.Through the experiment we studied the effect of diffrent fertilization media,sperm density and capacitation solution on bovine oocyte in vitro fertilization,to improve the environments of fertilization of sorted sperm and to select a convenient and high-performance methods as well as provide theoretic basis of fertilization quality and quantity for IVM. The result indicates that adding l0μg/ml Caffein and 5 mM heparin is applicable to IVF of sorted sperm capacitation;The sperm density of 1.0×106 pieces/ml is more suitable to IVF of sorted sperm fertilization; Adopting BO and TALP medium with heparin as sperm in vitro capacitation and fertilization medium to research the effect of the sperm capacitation and fertility between BO and TALP,The result showed there no significant difference between BO and TALP in sorted sperm IVF(P>0.05), both of them could be acceptable for the sorted sperm fertilization, while compare to TALP, the effect of BO to earlier embryo's growth is in slight extent.In order to establish an suitable earlier embryo cultivation system, the experiment was carried out to study conditions of co-culture system,different culture medium and the effect of sorted and unsorted sperm in IVF.The result indicates that SOF or CR1(with BSA) is more suitable for the co-culture system than TCM199; co-culture system was much better than unco-culture system(P<0.05),whereas there no significant difference between GCM and BOECM group(P>0.05); The rate of cleavage and blastocyst between sorted sperm and unsorted sperm also no significant difference (P>0.05) in the condition of co-culture system, however, the effect of unsorted sperm is little good for sorted sperm..