兔卵母细胞孤雌激活方法的研究  

Study on the parthenogenetic activation of rabbit oocytes

作　　者：向舒[1] 杨金姬[1] 石金月[1] 张顺[1] 王建[1] 陆凤花[1] 石德顺[1] 

机构地区：[1]广西大学动物繁殖研究所,广西南宁530005

出　　处：《中国兽医学报》2011年第7期1059-1062,1075,共5页Chinese Journal of Veterinary Science

基　　金：国家"863"高科技研究发展计划资助项目(2008AA10-Z160)

摘　　要：对兔卵母细胞的孤雌激活方法进行探讨,为兔的体细胞核移植奠定良好的技术平台。体外成熟培养20～22h的兔卵母细胞随机分组,分别进行以下试验:采用不同脉冲电压、脉冲次数和脉冲时间,电激活兔卵母细胞,摸索最佳电激活参数;比较不同的激活方法处理兔卵母细胞的效果,Ⅰ:Ion 5min+6-DMAP 3h,Ⅱ:Ion 5min+CHX 3h,Ⅲ:Ion 5min+6-DMAP联合CHX 3h,Ⅳ:电激1次联合6-DMAP+CHX 3h;优化6-DMAP与CHX作用时间。结果显示:当电激参数为:电压40V/mm,脉冲时间20μs和脉冲次数3次时,兔孤雌胚的分裂率和囊胚率最高,可达80.64%和14.51%;采用第Ⅳ种方法激活兔的卵母细胞,胚胎的分裂率和囊胚率(80.64%,13.71%)均显著高于Ⅰ组(66.67%,8.6%)和Ⅱ组(43.21%,1.24%,P<0.05),略高于第Ⅲ组(77.59%,12.07%);当兔孤雌胚分别在含6-DMAP和CHX的CM中培养1h和3h时,1h处理组在分裂率(80.68%vs 77.59%)和囊胚率(15.91%vs 12.07%)上都略高于3h处理组。结果表明:兔卵母细胞经Ion处理5min,或先电激1次(参数为电压40V/mm,脉冲时间20μs,脉冲3次),再用2mmol/L 6-DMAP+5mg/L CHX处理1～3h,均可获得较好的激活效果,有利于兔孤雌胚的发育。Methods for activating rabbit oocytes were investigated in this study to establish a favorable foundation for rabbit somatic cell ntlclear transfer. Rabbit oocytes were matured in vitro for 20-22 h, then carried on experiments as follows. Exploring to the optimal condition of direct current （DC）field intensity and pulse width for rabbit oocytes electro-activation; comparing to the effect of the ooctyes activated by different ways, Ⅰ：Ion5min＋6-DMAP3h,Ⅱ：Ion5min＋CHX3h,Ⅲ：Ion5min＋6-DMAP and CHX 3 h,Ⅳ ：Electro-activation （1 time） ＋6-DMAP and CHX 3 h;aptimizing the joint action time of 6-DMAP and CHX. The result showed that when using 40 V/mm,20 μs electrical pulse to rabbit oocytes,the cleavage and blastocyst rate of the 3 treatments can reach 80.64% and 14.51%. In experiment 4, the development ability of embryos treated with IV group （80.64%,13.71% ） were significantly higher than that of oocytes treated with Ⅰ group （66.67% ,8.6%） or Ⅱ group （43.24% ,1.35% ,P〈0.05） ,but no significant difference comparing with Ⅲ group （77.59 %, 12.07 %, P〉 0.05）. These results indicated that the comparatively suitable electro-activation condition for rabbit oocyte was that of 40 V/mm, 20 μs and 3 times of direct current pulses; and the methods of Ion 5 min or electric pulse （1 time）,then 6-DMAP＋CHX 1-3 h could activate the rabbit oocytes more effectively.

关 键 词：兔卵母细胞 电激活 离子霉素 6-DMAP 放线菌酮 

分 类 号：S826.1[农业科学—畜牧学]