| The majar object of this paper is to research some factors on bovine somatic cell nuclear transfer effiency and to develop a technical system to produce bovine embryos in vitro by SCNT , which will be used to clone bovine and isolate bovine ntESCs. Ear fibroblasts cell was used as donor cell, in vitro matured enucleated bovine oocytes were employed as recipients .The results obtained were as follows:1. Preparation of donor cellsWe established 2 ear skin fibroblasts cell lines (BEF422 and BEF274) from ear skin tissues of two Holstein cows by tissue culture. And the cells showed great viability . The number of chromosomes counted in BEF422 of passage 13 showed a normal karyotype,being female 60 XX with no visible abnormality. It was a simple mathed ro isolate bovine cumulus cell from COCs after maturation in vitro .2. Establishment of a system for in vitro maturation (IVM) of bovine oocytesBovine ovaries were obtained from a local abattoir and transported to laboratory in physiological saline at 23~25℃within 6h after collection . Cumulus-oocytes complexes (COCs) with more than two layers of cumulus cells and uniform cytoplasm were selected and IVM.Different maturation media were compared in present experiments.The results showed that:⑴When four serum and serum surrogate, FBS, NBS, BSA and PVA, were added in basic IVM culture medium (TCM-199+2.5μg/mL Sodium Pyruvate+10mM/L HEPES + 2mM/L Glutamine + 50μL/mL ITS + 0.1 IU/mL hMG +1μg/mL E2 +10 ng/mL EGF), the oocytes maturation rate between group 10mg/ml BSA and 10%FBS were not significantly different, but higher significantly than group 10%NBS and 1.0%PVA(P < 0.05);after activation , the cleavage rate of 10mg/ml BSA was highest in four groups (P < 0.05), the cleavage rate between group 10%FBS and 1.0%PVA were not significant, the cleavage rate of 10%NBS was lowest;the blastocyst rate of 1.0%PVA was highest in four groups(P < 0.05), the blastocyst rate between group 10mg/ml BSA and 10%FBS were not different, the blastocyst rate of 10%NBS was lowest. So the IVM rate and blastocyst rate between FBS and BSA were not significantly different, indicating that BSA could replace FBS in bovine IVM medium.⑵Uracil was added in bovine IVM medium without serum(+10mg/ml BSA ), The result showed that the 50μg/ml uracil significantly improve the maturation rate , cleavage rate, and blastocyst rate comparing to control group and the group with uracil (100μg/ml, 150μg/ml).⑶ATP was added in bovine IVM medium without serum(+10mg/ml BSA ) ,The result showed ATP has not significant effect on the maturation rate of the oocytes, however it can enhance the viability of the oocytes. especially 500μg/ml ATP can improve significantly the blastocyst rate. Therefore ,protain-free IVM medium, which is M199 containing 2.5μg/mL Sodium Pyruvate, 10mM/L HEPES ,2mM/L Glutamine ,50μL/mL ITS ,0.1 IU/mL hMG ,1μg/mL E2 , 50μg/ml uracil ,10 ng/mL EGF and 10mg/ml BSA could improve the rate of bovine oocytes IVM and developmental ability of parthenogenetic embryos after activation .3. SCNT in bovine⑴For improving fusion and development of bovine cloned embryos ,factors affecting the electrofusion of bovine somatic cell nuclear transfer were investigated,The results showed in four telectrofusion programs, Program③( 1.9KV/cm,10μs,one pulse) was higher than the other three in electrofusion rate and blastocyat rate(P <0.05) , which were 70.05% and 37.6% respectively;⑵For optimization of Program③telectrofusion condition , pulsed voltage and pulse duration time were investigated respectively, The results showed the telectrofusion condition(1.9KV/cm, 15μs) could get great fusion and development of bovine cloned embryos.⑶The cleavage (91.6%vs 88.04%,87.5%,89.91%, P <0.05) and blastocyst (37.61%vs 34.93%,26.37%,32.65%, P <0.05)rate of Ino +6-DMAP were the highest in four acativation methods ,which was significant. It was showed that Ino +6-DMAP could improve bovine cloned embryos developmental ability , which is great activation method to bovine cloned embryos .⑷The cleavage rate (90.75% vs 83.51%,86.27%, P <0.05) and blastocyst rate (32.41% vs 27.28%vs16.05%, P <0.05) of bovine cloned embryos when were activated at 3h after electrolfusion were the highest than delaying 1h and 2h activation .the result showed that delaying 3h activation after electrolfusion could improve bovine cloned embryos developmental ability.⑸For improving the development of bovine cloned embryos , affect of G-1/G-2 sequential culture system on the development of bovine somatic cell nuclear transfer were investigated in this study.The results showed G-1/G-2 sequential culture system coculture with bovine cumulus improved the development of bovine cloned embryos greatly,the Rate of cleavage (90.91% vs 85.95% vs 73.50%,P<0.05)and blastocyt (36.67% vs 27.88% vs 18.60%, P<0.05)were higher than SOFaa and CR1aa culture systems , the result showed that beyond three culture medium, G-1/G-2 sequential culture system could improve the bovine cloned embryos developmental ability .⑹Comparing the effect of G-1/G-2 sequential culture system coculture with or without bovine cumulus on bovine cloned embryos, developmental ability in G-1/G-2 sequential culture medium coculture with bovine cumulus was higher significantly than that in G-1/G-2 sequential culture medium coculture without bovine cumulus in cleavage rate and blastocyst rate (P<0.05).So G-1/G-2 sequential culture medium coculture with bovine cumulus was a good culture system for bovine somatic nuclear transfer.⑺Effect of different donor cell on bovine nuclear transfer was investigated and the result showed that among three donor cell types BEF422,BEF 274 and cumulus, the fusion rate ,cleavage rate and blastocyst rate of cumulus were higher significantly than BEF422 and BEF 274(P<0.05). The fusion and the cleavage rates between ear skin fibroblasts cell BEF422 and BEF 274 were not significantly different, but blastocyst rate of BEF422 was significantly higher than that of BEF274 (P<0.05).It indicated that the bovine cloned embryos developmantal ability of culumus was higher than ear skin fibroblasts cell, and the bovine cloned embryos developmental ability of different ear skin fibroblasts cells isolated from different Holstein cow was different.⑻Effect of different passage of donor cell on bovine nuclear transfer were investigated ,and the results showed that the fusion rate of BEF422 P20~21 cell was lower than P6~7 cell(68.02% vs 73.72%), but the cleavage rate and blastocyst rate of BEF422 P20~21 cell was higher singnificantly than P6~7 cell (P<0.05), indicating that the cloning rate of high passage cell was higher than low passage cell.⑼Effect of different breed cow recipients on bovine cloned embryos pregnancy rate was investagated. Total 214 reconstructed embryos from blastocyst were transferred into 43 recipient(32 Holstein cow and 11 Qinchuan cattle) in 20 experimants of embryo trasnfer. checked by rectal examination 75d afer transfer, seven out of 34 were pregnant ,the pregnancy rate was 20.59%, the pregnancy rate between Holstein recipients and Qinchuan recipeints were not singnificantly different (P>0.05), indicating that different breed recipients of bovine cloned embryos had no effects on pregnancy rate .
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