Study Of Parthenogenesis Or Enucleation Method Of Mouse Oocyte And Zygote Cultured In Vitro

Mouse oocyte was lesser comparatively, micromanipulation induced break of cytoplast. It was difficult to establish the mouse nuclei transfer technology which was affected seriously by culture medium, activated condition and enucleated method. In order to establish mouse nuclei transfer technology, mouse zygote culture medium and activated conditions were optimized in vitro and enucleated method of MII oocyte were investigated in this study.1.To optimize the in vitro culture system of mouse pronuclear zygote and to investigate the effect of FBS, PVA, hLIF on mouse zygote development in vitro, we did four experiments separately. Test I:M16, mM16, KSOM, Mksom and CZB were selected as putative culture medium to carry out in vitro culture test on mouse pronuclear zygote development to filtrate a optimal cultural system ..Based on the first test, we added FBS(Test II), PVA(Test III) or hLIF(Teast IV) to the optimized culture medium to study the effect of these additive on the mouse embryo development. Result of test I was that the mM16 was selected as the best culture medium, the next was mKSOM. The 4-cell development rate were 94.7%(91/96) and 85.8.%(73/85) and the morula/blastocyst development rate were 84%(80/96) and 81.1%(69/85) in mM16 and mKSOM, respectively. The ratios were obviously higher than those in other mediums, the difference were significant(p﹤0.05). Result of test II was that, BSA was replaced by 5%FBS, 10% FBS or 15% FBS in mM16, the ratio of embryo development to morula/blastocyst declined significant(32.6%,35.8%), the difference were significant compared with control group (82.1%)(p<0.05). Result of test III was that, BSA was replaced by 0.05 mg/mL PVA, 0.1 mg/mL PVA, 0.5 mg/mL PVA or 1 mg/mL PVA in mM16, no embryos developed to morula/blastocyst , the difference were significant compared with control group(p<0.05). Result of test IV was that, all three hLIF concentrations improveed the viability of embryo remarkably in vitro, and when the concentration of hLIF was 1000 IU/mL, the effect of hLIF on embryo viability was the best. It was indicated that the ratio of blastocyst could reach to 84% merely adding 0.1 mmol EDTA,0.5 mmol taurine and1000 IU/mL hLIF to the M16 without changing other components. BSA could not be substituted effectively by FBS or PVA in mM16.2. To study parthenogenesis of mouse oocyte by the different activation methods.thirteen hours after donor mice treatment with hCG, oocytes were collected and cultured in vitro for 4...