猪卵母细胞体外成熟及乙醇孤雌激活研究

本试验探讨了不同基础培养液(mTCM-199、NCSU23)和PMSG、HCG浓度、作用时间对猪卵母细胞体外成熟的影响和乙醇及乙醇与各种化学试剂联合处理对猪体外成熟卵母细胞孤雌激活、孤雌生殖胚胎发育的影响,最终找到了最佳的猪卵母细胞体外成熟培养体系,并确定了乙醇孤雌激活的最佳浓度、作用时间和与不同化学试剂联合处理的最佳组合。试验结果得出:最佳的猪卵母细胞培养体系为:NCSU23+20IU/mlPMSG+20IU/mLHCG+10%PFF,激素作用时间为45h,成熟率可达到70.3+3.9%;乙醇的最佳激活浓度为8%,激活率为29.6+2.5%;乙醇激活的最佳处理时间为10min,激活率为32.6+4.1%;乙醇与CB+CHX联合处理的激活率最高,激活率为60.8+3.2%,卵裂率、3～4细胞率、8细胞及8细胞以上率分别为45.1+3.0%、28.6+3.7%、8.7+1.2%;乙醇与CB+6-DMAP联合处理的激活率为51.7+1.9%,卵裂率、3￣4细胞率、8细胞及8细胞以上率分别为41.7+3.6%、30.3+4.3%、9.9+1.8%,虽然激活率、卵裂率低于乙醇与CB+CHX处理组,但3￣4细胞率、8细胞及8细胞以上率均高于乙醇与CB+CHX处理组(30.3+4.3%vs28.6+3.7%,9.9+1.8%vs8.7+1.2%)。因此,乙醇激活的最佳方法为:8%乙醇处理10min,CB+6-DMAP处理7h。

Study On In Vitro Maturation Of Porcine Oocytes and Ethanol Activation

The objective of the present study was to investigate the effect of two different basic in vitro maturation(IVM) mediums(mTCM-199、NCSU23),concentration and treatment time of pregnant mare serum gonadotropin(PMSG) and human chorionic gonadotropin(HCG) on IVM of porcine oocytes;furthermore,to examine the effect of ethanol and ethanol plus various chemical agents on parthenogenetic activation and the development of parthenogenetic embryos. The rational IVM maturation of porcine oocytes system is NCSU23+20IU/mlPMSG+20IU/mlHCG+10%PFF,of which the maturation rate was 70.3+3.9%.The best parthenogenetic activation concentration of ethanol was 8%( activation rate : 29.6+2.5%).The best activation length of exposure by 8% ethanol was 10 min(activation rate : 32.6+4.1% ). Activation by ethanol associated with CB+CHX got the highest activation rate(60.8+3.2%),and the rate of cleavage,3~4cells,8cells and more than 8cells were 45.1+3.0%, 28.6+3.7%, 8.7+1.2%,respectively.Treated by ethanol associated with CB+ 6-DMAP,the rates of activation, cleavage, 3~4 cells, 8cells and more than 8cells were 51.7+1.9%, 41.7+3.6%, 30.3+4.3%, 9.9+1.8%, respectively; although the rates of activation and cleavage were lower than that of ethanol plus CB+CHX, the rates of 3~4 cells, 8cells and more than 8cells were higher. These results indicate that the best method of activation of pig oocytes by ethanol plus various chemical agents was 8% ethanol treating for 10 min,and then oocytes treated by CB+6-DMAP for another 7 h.