Production Of Yak (Bos Grunniens) & Takin (Budorcas Taxicolor) Embryos By Interspecies Nuclear Transfer

Interspecies cloning might be used as an effective method to conserve endangered species and to support the study of nuclear-cytoplasm interaction. In this study, we describe the development of yak-bovine embryos (or takin-bovine embryos) in vitro produced by fusing yak (or takin) ear fibroblasts with enucleated bovine oocytes, and blastocyst-stage yak-bovine embryos transferred into bovine surrogate mothers established pregnancies, One of which would produce an apparently normal yak in next month.1 We established two takin somatic cell lines and nineteen yak somatic cell lines from the donor animals coming from the Beijing Animal Park, Qinhuangdao Wild Animal Park and Qinghai province. They respectively belonged to the ear fibroblast cell lines, cumulus cells and oviduct cells lines. We found cell lines were normal after the tissues had stayed at 0℃ for 48 hours. These results made for collecting the tissue from endangered animal at the field2 Somatic cell nuclear transfer: we firstly established our enucleated methods by combining H33342 staining method and blind sucked method. Secondly, the parthenogenetic activation of bovine oocytes with chemical stimulation was investigated. The activation rate of the complex chemical activation was higher than the single chemical ones.The complex of A23187 + 6-DMAP got the best result (cleaved 83.6%). At last, we compared the development of the bovine parthenogenetic blastocysts cultured in M199 and CRlaa and found bovine embryos was suitable to be cultured in CR1aa.3 Interspecies cloning yak: we got higher blastocysts development in vitro (35%) by fusing these yak cells with enucleated bovine oocytes. The results suggest that oocytes were the basis for cloning success. When the maturated rate was lower than 40%, the induced blastocyst rate was lower (3% versus 25%, P<0.01). Further, delaying activation following nuclear transfer showed the potential for greatly improving embryo development in vitro. The yak blastocyst development rates increased from 21% to 35% by increasing the activation delayed period from 0.5hr to 1.5hr. No difference was observed among the various sources and the various preparations of donor cells.4 We also compared the blastocyst development of takin-bovine embryos with yak-bovine and bovine-bovine embryos and compared the cell numbers of the blastocyst. Our results indicated that: (1) takin-bovine cloned embryos could develop to the blastocyst stage in vitro (5%), (2) using the same nuclear transfer protocol, development efficiency was significantly different between bovine-bovine cloning, yak-bovine, and takin-bovine cloning (48% versus 28% versus 5%, P<0.01), and (3) cell numbers in the blastocysts of the three species of embryos were not significantly different. These results suggested that the bovine oocytes could reprogram the takin, yak, and bovine fibroblast nuclei. However, the development efficiency of intra-species cloning tended to be higher than inter-species cloning; the closer the species of the donor cell was to the recipient oocyte (yak versus takin), the greater the blastocyst development in vitro.5 Testing the genomic and mitochondrial DNA of yak (or takin) blastocysts: blastocyst genomic DNA only came from the yak (or takin) donor cells while the mitochondria DNA came mainly from bovine oocyte. These suggested the blastocysts come from interspecies cloning.6 Yak-bovine blastocysts transfer: 55 percent surrogate bovine delayed estrus, three of sixty surrogate bovines get pregnancy at 60th day. Now one continues gestation longer than 210 days.7 Although inter-species somatic nuclear transfer (SNT) embryos of evolutionary close species can develop to term, our results fould yak-bovine blastocyst got low pregnancy in surrogate bovine. It was important to analyze a wide variety of genes in individual cloned embryos. But using conventional RT-PCR strategies permitted analysis of only a few genes in one embryo. In this study, a global RT-PCR strategy has been used, allowing the analysis of almost hundre...