| The technique of somatic cell nuclear transfer(SCNT) and the combination with genetargeting technique offers new opportunities and idea for genetic engineering and genomepreservation in mammalian animal species.Pigs are considered as ideal organ donor for future human xenotransplantation. SCNTprovides an oppommity for producing HAR and Endoviruses free pigs. Up to date, theefficiency of cloned piglets production has been very low, with less than 1% of transferredembryos surviving to term.This research focused on establishing a more efficient system for producing pigs bySCNT. Systematical study had been carried on factors affecting on efficiency of SCNT byoptimizing NT technique and culture conditions. The aim is to produce enough clonedembryos,which were transferred to surrogate gilts or sows.Fetal fibroblast cell lines were set up from fetuses of Guangxi Bama Xiang-pigs at 40d ofgestation and adult ear skin fibroblast cell lines was set up by using explant-seeding method.We also established pig oviduct epithelium cell lines by scraping and cumulus cells fromfollicular fluid. Effects of freezing and thawing on the fetal fibroblast cells, cell grow curveand chromosome analysis were discussed. Results showed that freezing and thawing didn'tinfluence the fetal fibroblast cells proliferation,cell grow curve submitted typical S-shape,92%ploidy of one cell lines maintained normal when cultured up to passage 10.To study the effects of a few factors such as the state of COCs (the cumulus oocytescomplexes), the culture medium, the different maturation times and the addictives of culturemedium on the in vitro maturation (IVM) of oocytes in pigs, the ovaries of pigs are used as asource of oocytes for IVM research: First,COCs which are prepared from follicles in differentdiameters or different grades have significantly different maturation rate in IVM of porcineoocytes. The maturation rate of COCs from 3～6mm diameter follicles is 81.2%, and that of COCs from over 6mm diameter follicles is 80.7%, which is significantly higher than that ofCOCs from less than 3mm,diameter follicles which is 65.5%. Oocytes maturation of Grade A(80.7%) and B COCs (79.3%) is very significantly different from Grade C (26.0%,P＜0.01).Second, culture medium of TCM199 and NCSU-23 are used for IVM of porcine oocytes, theyshow the same culture ability in IVM (The maturation rate in TCM199 is 79.8%, NCSU-23 is80.6%), but NCSU23 appears better culture quality in IVM maturation than TCM199. Third,the effect of different maturation times of COCs on in vitro mature of oocytes shows that: thematuration rate of 44h significantly higher than that of 40h and 48h, which is 81.4%,61.2%,66.2% (P＜0.05), this rate is significantly different from 36h,7.3% (P＜0.01),Forth,NCSU-23medium with or without epidermal growth factor (EGF) and Recombinant Porcine GrowthHormone (rPGH) had no much difference. Addition of porcine follicular fluid (PFF) to maturemedium promotes the in vitro maturation of oocytes. The maturation rate of Group PFF is84.2%,83.7%, which is significant higher than Group Without PFF (55.8%,P＜0.05).Repeated parthenogenetic activation experiments confirmed that sorbicolan was moresuccessful as an activation medium compared with mannitol. In the different activationconditions, 300V/mm,20μs,3DC and 200V/mm,60μs, 1DC were suitable to improve cleavagerate and blastocyst rate.After electrical activation, the oocytes cultured in the medium with2mmol/L 6-DMAP or 2mg/ml CB were not evidently different in subsequent development,however, more dead oocytes in the medium with CB than anther. The experiment evaluatedthe effects of ionomycin concentration and treatment time on parthenogenetic activation, andas the result,20μmol/L ionomycin treating 40min obtained the most blastocyst.Oocytescultured in vitro (IVC) for 44h and 48h profited parthenogenetic activation,meanwhile,thehighest cleavage rate for 44h was 85.5% and the highest blastocyst rate for 48h was 44.9%.Compared with McGrath-Solter method and pressurization method, the enucleation rateof spindle-view (95.5%) seemed safer and more efficient. Fetal fibroblast cells and adult earskin fibroblast cells as donor cells were fit for development of reconstructed embryos.The fetal fibroblast cell before passage 10 had more advantages in reconstructed embryosdevelopment as donor cells than after passage 10. In the activation conditions of200V/mm,60μs, 1DC,B2 medium was more adequate than NCSU-23 for reconstructed cellsactivated electrically to develop, but the difference between them was not significant.Considering the experiment cost, NCSU-23 medium was more suitable.Constructed embryo transfer was carried by oviduct and 16 surrogate gilts were used.Ingroup 1, six were re-estrus until 20～22d.In group 2,three often were not reestrus until 45d.
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