Study On Handmade Somatic Cell Cloning Technology In Cattle

In this study,we used high-yielding Holstein dairy cow's cumulus cells, ear fibroblasts cells and oviduct epithelium cells for nuclear cells, the zona-free of cattle oocytes with somatic cell nuclear transplantation of factors were discussed, and optimized the cattle's zona-free oocytes nuclear transfer technology programme for the further study of transgenic cattle and from the embryonic stem cell research laid the foundation. The results were as follows:1. The cell number of blastocyst of the zona-free reconstructed embryo by handmade enucleation (was significant higher than that by the traditional enucleation(124.40vs194.0)(P>0.05).2. The fusion rate increased significantly with the decreasing of AC voltage,and the fusion rate when AC voltage was 3V was significantly higher than others(55.51% vs 72.17% vs 93.83%)(P<0.05).3. The rate of cleavage and blastocyst of reconstructed embryos treated with A23187 + 6-DMAP was significantly higher than that treated with Ionomycin + 6-DMAP(88.7%vs43.6%,37.4% vs 19.9%)(P<0.05).4. The interval of the fusion between the activation were chosen 1h,2h and 3h, the rates of cleavage and blastocyst of reconstructed embryos had no significant difference(88.05%vs78.79%vs76.62%,31.45% vs43.29 % vs32.95%) (P>0.05).5. The rate of cleavage of the zona-free reconstructed embryo in mCR1aa or CR1aa was significantly higher than that in SOFaaci(83.17% and 65.56%vs37.85%)(P<0.05).6. The total number of COCs collected by negative-pressure aspiration or by cutting ovaries was significantly higher than that by injector aspiration(360 and 361vs219),and the COCs of grade 1-2 had significantly higher number by cutting ovaries(233 vs 86 and 208)(P<0.05).7. The rates of cleavage of the 6th and 10th of the fibroblast cells for nuclear transplantation was significantly higher than the 17th(80.26% and 93.1%vs33.96%) (P <0.05).8. We used high-yielding Holstein dairy cow's cumulus cells, ear fibroblasts cells and oviduct epithelium cells for nuclear cells,there was no significant differencein the rate of cleavage and blastocyst among the three cell lines(89.12 %vs80.26% vs79.12%,33.65 %vs25.77 %vs21.19%) (P> 0.05).9. The reconstruction embryo cleavage and blastocyst rate by the frozen cumulus cell or by the non-frozen group were not significantly different((78.61% vs89.12%,29.42% vs33.65%) (P> 0.05). 10. The proportion between fresh and frozen cells of Grade 3 injuried had significant difference (5.91% vs1.35%).