Study On Optimizing The Procedure Of Early Porcine Embryonic Cell Nuclear Transfer

Early embryonic cell has the developmental totipotency. Every embryonic blastomere cell can develop to an intact individual separately in a proper condition. One embryonic cell can be separated into many blastomere cells. As be the nuclear donor of the embryonic cell nuclear transfer(ECNT), it can breed the number of eminent species largely, increase the number of endangered animals, supply large number of supply same genotype animals, improve the efficiency of gene transfer and sex control and offer same genotype apparatus or organ for human transplant.But the efficiency of ECNT is still relatively low. One important factor is the excessive of nuclear transfer(NT) detail and complication of NT procedure. With the development of IVM(in vitro maturation)-IVF(in vitro fertilization)-IVC(in vitro cultivation) embryonic engineering technology, the procedure of ECNT will be optimized and reducted. The experiment was done in order to grope the procedure of ECNT which was fit for producing embryos in the lab largely and optimize the procedure of ECNT.Porcine was acted as the experimental animal. It was researched on problem of the origin of obtaining early embryonic cells, methods of separating blastomere cells, best parameter and order of ECNT embryo electric activation and system of ECNT embryo IVC, which was to find a scientific method of fitting ECNT and a procedure of fitting the lab producing embryos largely, optimize the procedure of ECNT and improve the ECNT embryo in vitro. According to the analysis of the experimental data, the research results were as follows:Exp.1, optimizing the procedure of obtaining early embryonic cells. In the experiment, IVC 44~48h matured oocytes were IVF with different kinds of semen(fresh, epididymis, freeze-thawy). From the difference of cleavage rate, rate of 4~8-cell embryo and rate of embryo of more than 8-cell, a best kind of semen for getting early embryonic blastomere cells in the lab could be screened out. The results showed that the cleavage rate of semen of fresh, of epididymis and of freeze-thawy IVF was 40.83%(49/120), 39.47%(45/114), 34.68%(43/124), respectively, there were significant differences between them (P<0.05). Rate of 4~8-cell embryo was 16.67%(20/120), 15.79%(18/114), 15.32%(19/124), respectively, there were no significant differences between them (P>0.05). Rate of embryo of more than 8-cell was 2.50%(3/120), 1.75%(2/114), 0.81%(1/124), respectively, there were no significant differences between them (P>0.05). There were terribly significant differences between electric activation parthenogenesis and IVF group on cleavage rate(52.22%(47/90)), rate of 4~8-cell embryo(28.89%(26/90)) and rate of embryo of more than 8-cell(6.67%(6/90)) (P<0.01). The experimental results showed that the effect of IVF with the semen of freeze-thawy was quite the same while IVF with the semen of fresh and epididymis. It could get early embryos by IVF instead of multiovulating embryos in vivo. So it could save the time and money of...