In Vitro Maturation And Fertilization Of Porcine Oocytes And No-surgical Embryo Transfer Of Porcine

Pig is the optimal animal donor of tissue and organ for xenograft of human beings for its high level homologous gene ,similar sizes of organs and metabolism of physiology and biochemistry. We should modify the gene of the early embryos of porcine in order to investigate xenograft. Until now, the total efficiency of in vitro production of porcine embryos is not high and most researchers use the surgical method to transfer porcine embryos, this method does harm to the pigs and the procedure is very complicated. Therefore, it is very important to optimize the micro-enviroment and conditions for in vitro maturation,fertilization and build a matured procedure for porcine non-surgical embryo transfer. In the present study, we use three collection methods :stab--broken,slicing and aspiration method to collect porcine oocytes from 3~6mm ovary follicles to investigate if the collection method can influent the oocytes collection efficiency ,nuclear maturation rate and fertilization result ; We also use a new culture method: nest dish culture method and a classical culture method: micro-drop with mineral oil culture method to culture porcine oocytes comparatively, and hypothesized that the new culture method has influence in the procedure of culture; Finely, we investigate the non-surgical embryo transfer technique of porcine to check the successful rate of transfer and pregnancy. Our results showed that: 1.The A and B class oocytes'collection efficiency of stab-broken and slicing method was significant higher than the aspiration method (55.3%, 50.0% vs 30.2%, P < 0.05), but there was no significant difference between stab-broken and slicing method; The collection efficiency of C class oocytes , stab-broken method was very significant lower than slicing and aspiration method (5.9% vs 15.2%, 15.9%, P<0.01), no significant difference can be seen between slicing and aspiration method; These was no significant difference between stab-broken and slicing method in total collection efficiency, but the two method were significant high than aspiration method (81.3%, 75.0% vs 53.4%; P < 0.05). 2.Stab-broken method's maturation rate was the highest one of all the three methods of A and B class oocytes, but there was no significant difference when compared with slicing and aspiration method. The maturation rate of A class oocytes was significant higher than the B class oocytes of all the collection methods(P<0.05). 3.The collection method can influent the fertilization results, although no significant different cleavage rate can be seen between Stab-broken method (33.1%) and Slicing method (22.2%), the cleavage rate of Stab-broken method was significant higher than the aspiration method (21.0%). 4.No significant difference can be seen on the sperm penetration ratio,monospermic ratio ,sperm per oocyte and cleavage rate between nest dish group and single dish group, but the male pronuclear formation and blastocyst rate of nest group were significant higher than the single dish group (P<0.05). 5.we used 2~4 cell mouse embryos transferred to the sows by non-surgical transfer(NS), the results showed that the successful rate of NS was 75% (3 / 4); when transferred morulae and blastocysts to the other 7 recipients , none of them was pregnant (0 / 7), but the contrast group's pregnant rate was 66.7%(2/3).