Sheep - Cattle Interspecies Somatic Cell Nuclear Transfer,

Sheep-bovine inter-species nuclear transplantationDoctoraI candidate: Li YuqiangAdvisor: Pro f. Zhang YOng(College of Animal Science and Veterinary Medicine, Northwest Sci-tech University of Agriculture andForestry Yangling, Shaanxi, PR China, 7l2100)ABSTRACTInter-species nuclear transfer embryos have been proved could complete full-term development and givebirth of offspring. But its efficiency was stiIl very low To develop high efficient inter-species nucleartransplantation technology is very important fOr fast production of rare/endangered animals. The presentexperiment studied on the sheep-bovine inter-species nuclear transplantation for the purpose of improvinginter-species nuclear transfer efficiency. The enucleation and activation of bovine oocytes, the proper methodof embryo construction by direct oocyte intraplasmic nuclear injection were first studied. Based on theseconditions, the preimplantation development of inter-species nuclear transplanted embryos constructed fromsheep skin fibrob]asts and bovine oocytes were studied with the fOcus on the treatment of the donor ce1ls. Itwas indicated thattl). The primary cell cultures of sheep skin fibroblast can weIl established through attachment culture oftissue fragments. The passaged ceII viability and proliferation abiIity cou1d be improved when attachment celItrypsiniZaion were performed at room temperature (20-22) and cell suspension were obtained by oscillate; it'sa good method to get nucIear transfer donor cells. Before expIanting, thawed cells equilibrized twice, each30min or once l0h could improve cell attachment rate significantly Serum-free culture media Ml99 withaddition of ITS could support cell proliferation slowly, but could not support ceIl attachment; BSA additioncould promote its ability of supporting cell proliferation and attachment, but could not reach the efficiency ofl0%FBS. It was found that large bovine folIicular fluid (BFF) added into Ml99 at 20% concentration couldobtained higher quality cell attachment and proliferation than l0%FBS. It was also found that the so-calledattachment inhibition of cultured cell was just a relative phenomenon. When culture cells long times withoutpassaging and with refreshing the culture media frequently, the ceIl could grow on the surface of attached cellslayer forming cells cluster (before cryopreservation) or as dispersing single cells (after cryopreservation).These cells couId attach and proliferate when explanted to a new culture bottle.2). There were about 7 pieces of cumulus oocyte compIexes (COCs) could get from per ovary collectedfrom local abattoiL And the numbers of grade A, B COCs were slightly under 5. Only those COCs that have$$-4#rNWmm&8taroFRrtcompact cumulus cells and homogeneous oocyte plasma (A, B grade) could mature and develop well. Thehigher rate of grade A, B COCs in batches, the higher rate of maturation and development rate of grade A, Boocytes. The cumulus celIs dispersed inferiorly when COCs cuItured in Ml99 added with 20% estrous calfserum (ECS, self made) as compared with Ml99+FBS+gonodtropin, the maturation rate were not different,but the parthenogenetic development rate significantly lower. BECMaa + BSA could not get as equal effect asMl99 on maturation rate and parthenogenetic development rate. lnsulin addition cou1d promote the ability ofBECMaa + BSA to support oocytes maturation, but no effect in M l99 + FBS.3). The activation efficiency of bovine oocytes by Ionomycin + 6-DMAP or ethanol + 6DMAP/ CHXwere significantly higher than Ionomycin or ethanol alone. The parthenogenetic development was more stablewhen bovine oocytes activated by Inonomycin + 6DMAP than ethanol + 6-DMAP/CHX, and it mainlyexhibited in the higher deveIopment rate from 8/l6 cells stage to blastocyst.4). The Ionger time of in vitro maturation culture, the far distance of the first polar body from thechromosome. Bovine oocytes ought to enucleated at/before lVM 20h when more than...