Establishment Of In Vitro Culture System In Bovine Oocyte And Embryo

In this study, the aim is to establish and optimize the systems of in vitro operation technologyof bovine oocyte and embryo in new laboratory, furthermore promote embryo biology engineerdevelopment, and also found the basis for study of cell clone and transgenic in bovine. For thisobjective, we collected the bovine oocytes of ovaries whose surface follicles diameters were 2-6mm from the slaughter house, and they were matured in the base maturing medium TCM199supplemented with 20% FBS. First, We sudied the effects on IVM of bovine oocytes of FSH, LH,1713-E₂ and EGF concentrations, the carrying times and temperatures of ovaries, the method ofcollecting bovine oocyte, the physiology condition of bovine ovaries; Second, we studied theeffects on cleavage rates of parthen-activation system and the reactiive time of oocytes in medicine;Third, we studied the effects on cleavage rates of fertilization system and the treated methods ofmatured oocytes before IVF; At last, we studied the effects on blastula rate of in vitro culturesystem. The main results are as follows:1. The system of bovine oocytes in vitro maturation was established in the new embryolaboratory. We chose cow and cattle ovaries in follicular phase from the slaughter house. Theovaries were stored in isotonic Na chloride. The carrying temperature was controlled at about 30℃and carrying time was controlled in 4h. The suction method was used to take out of oocytes. Themedicine that maturated bovine oocyte included Tissue Culture Medium-199 (Earle's), 20%FBS,50μg/ml LH, 50μg/ml FSH, and 20μg/ml 17β-E₂, and the maturing rate could reach 80%.2. The maturing rates of bovine oocytes were increased remarkably by adding EGF, and thematuring rates were increased continually as increasing working concentration of EGF which was50μg/ml, the maturing rate reached 82.2%. Because EGF is too expensive, we did not add largedose.3. The parthen-activation reactive condition was that 5mmol/L ionomycin treated for 5min,2mmol/L 6-DAMP and 5μg/ml CB treated for 4h. 4h was the best time of bovine oocyte activationin the 6-DAMP, and the cleavage rates and blastula rates could reach 85.1% and 26.8%.4. IVF-100 was used in vitro fertilization, because IVF-100 was not needed to prepare usually,store conveniently, and gain a higher cleavage rates. After the oocytes have matured for 20h, theywere digested by permease and treated with suctionpipe. It made the cleavage rate reach 76.8%.5. The co-culture system that was B₂ and vero, had a higher cleavage rate (80.6%) and blastularate (32.1%).