Study On Goat Somatic Nuclear Transplantation And Embryo Transfer

The goat was used as a model for somatic nuclear transfer and embryotransfer.In the experiment,it had been made a systematic research on in vitro maturation of goat oocyte,enucleation of the oocytes,microinjection of somatic cell,eletrofusion of the cytoplast and somatic cell,activation and in vitro culture of the reconstructed embryos.and embryo transfer.The results were as follows:1 .There were 2886 usable cumulus-oocyte complexes(COCs) recovered from follicles of slaughtered goat ovaries (1938) in breeding season and nonbreeding season.lt was demonstrated that more usable COCs can be recovered during the breeding season than the nonbreeding season.And oocytes were able to mature on a higher maturation rate.2.The oocytes had been cultured in several different cultural systems.The results showed that more than the percentage of 87.3 oocytes were matured in Ml99 supplemented liu/mlhCG,lug/ml 17 P -E2,15-20%HGS.And there was a high and stable maturation rate (75%) in M199 supplemented liu/ml hCG,lug/ml 17 P -E2,10mg/ml BSA.which is no-serum cultural system.3.The diameter of follicle has a certain influence on maturation of oocytes.The experiment show that the maturation rate of the goat oocytes with diameter 2-5mm and >6mm was 89% and 4.4%,respectively.Cumulus has an significant influence on muratation of oocytes .The maturation rateof oocytes with more than three layers cumulus around the oocyte, partly surrounded,and with nothing was 92%,56% and O.In addition ,maturation rate of oocytes follicular phase was significantly higher than that of luteal phade.4.1n the culture of somatic cell,method was established for culture of goat oviduct epithelium cells in vitro.And the passaged cultures were obtained from goat oviduct epithelium cells.The primary cultured cumulus cells from Boer goat COCs and the passaged cultures of slaughtered white Goat oviduct epithelium cells were prepared for goat somatic nuclear transfer as donor cells.5.In the experiment of parthenogemc activation,the goat matured oocytes were activated by electrical stimulus alone ,treated by lonomycin alone ,or treated by ionomycin followed by 6-DMAP.The results demontrated that latter had a better effect on goat oocytes parthenogenic activation than the the two formers both of which can acitvate oocytes.6.Goat activated oocytes were cultured in three medium systems Ml99 supplemented 10%FCS, M199 Supplemented 10%FCS with cumulus monolayer, and SOFaaBSA. The results indicate that the goat early embryos can be cultured in SOF and Ml99.7.In the experiment of adult somatic nuclear transfer,90 reconstructed embryos was performed by enucleated metaphase II cytoplasts injected fresh boer cumulus cells.The cleavage rate of cloned embryos was 5.7% after 48h in M199 supplemented 10%FCS with cumulus momolayer.And the cloned embryos merely developed to 8-cell stage.With the same method,64 cloned embryos were successfully reconstructed by oviduct epithelium cells without cryopreservation. The cleavage rate of cloned embryos was 7.1% after 48h .And the cloned embryos can develop to 16-cell stage .On the whole,the latter had a better effect on the cloning than the former.8.In the embryo transfer of boer goat ,40 sexually mature does were superovulated with two methods .The first used FSH and PG.The average total number of usable morula or blastocyst was 4.5.But the average total number of morula or blastocyst from the donor treated by vaginal plug was 3.5.The former was better than the latter.The early embryos were transferred to 90 recipient does.The rate of pregnancy was 62.2%.