供体细胞培养处理方法对水牛核移植效果的影响

Development of Buffalo Nuclear Transfer Embryos Derived from Donors Cultured in Different Ways

以经常规培养法 (DMEM+10 % FCS)、血清饥饿法 (DMEM+0 .5 % FCS培养 5～ 10 d)和 Apidicolin- APD结合血清饥饿法 (0 .1mg/ L APD培养 2 4 h,DMEM+0 .5 % FCS培养 1～ 18d)培养处理的水牛卵巢颗粒细胞和水牛成体耳部成纤维细胞作供核 ,分别采取带下注核法和胞质内注核法进行核移植。同一供核细胞各处理组间的核移植胚融合率 (以颗粒细胞作供核 )以及重组胚的囊胚发育率无明显差异 (P>0 .0 5 ) ,但经 APD+0 .5 % FCS培养处理供体细胞核移植后的分裂率显著高于其他组 (P<0 .0 5 )。用 7%乙醇处理的成体耳部成纤维细胞进行核移植 ,其重组胚的分裂率和囊胚发育率与对照组 (不含乙醇 )均无明显差异 (P>0 .0 5 )。结果表明 ,(1)血清饥饿处理水牛供体细胞对其核移植效果没有影响 ;(2 ) DNA合成抑制剂 APD结合血清饥饿培养处理水牛颗粒细胞和成体耳部成纤维细胞 ,可提高其核移植效果 ;(3)乙醇预激活处理水牛成体耳部成纤维细胞 。

Effects of donor cell treatments on the development of nuclear transfer(NT) embryos were explored.Granulosa cells and adult ear fibroblasts cultured in normal medium(DMEM+10% FCS),serum starvation medium(0.5% FCS for 5-10 d) or APD plus 0.5% FCS(in 0.1 mg/L APD for 24 h and following in 0.5% FCS for 1-18 d) were transferred into enucleated oocytes by electric fusion or direct injection.There was no significant difference in either fusion rate(granulosa cells as donor cells) or percentage of reconstructed embryos developing to blastocysts(P>0.05) among the culture treatments within each cell types.However,the cleavage rate of reconstructed embryos derived from donor cells treated with APD+0.5% FCS was significantly higher than that of other groups(P<0.05).In addition,pre-treatment of donor cells with 7% ethanol in manipulation medium did not affect the cleavage and blastocyst development rate of reconstructed oocytes(P>0.05).In conclusion:(1)treating buffalo donor cells with APD and serum starvation may improve their NT efficiency but serum starvation alone is inefficient in our system;(2)pre-treatment of adult buffalo ear fibroblasts with 7% ethanol have no significant effect on the development of NT embryos.