不同激活方法对绒山羊卵母细胞孤雌发育的影响  

Effects of Different Activation Protocols on Development of Cashmere Goat Parthenogenetic Embryos

作　　者：金永[1] 袁建龙[1] 朱兵[1] 郭旭东[1] 刘东军[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010021

出　　处：《农业生物技术学报》2011年第3期495-500,共6页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2008ZX08008-002)资助

摘　　要：为了进一步探索绒山羊卵母细胞孤雌发育的方法,进而为绒山羊的体细胞核移植技术提供高效的体外激活方法,本实验以屠宰场绒山羊卵巢为材料,采用切割法收集卵母细胞,并进行体外成熟。将体外成熟的卵母细胞分别经IA23187+6-DMAP、离子霉素(Ionomycin)+6-二甲基氨基嘌呤(6-DMAP)、7%乙醇+6-DMAP、IA23187+放线菌酮(CHX)、Ionomycin+CHX、7%乙醇+CHX处理,体外发育48h后统计卵裂率、8-cell率,7～9d后统计囊胚率和囊胚细胞数。结果表明,IA23187+6-DMAP处理组的卵裂率(89.6±4.9%)显著低于Ionomycin+6-DMAP(99.1±0.8%)、IA23187+CHX(97.6±1.2%)、Ionomycin+CHX(99.3±0.6%)及7%乙醇+CHX(97.5±1.4%)处理组(P<0.05),而与7%乙醇+6-DMAP(96.3±0.5%)处理组没有显著差异;IA23187+CHX(81.3±5.8%)和Ionomycin+CHX(82.5±6.6%)处理组的8-细胞率显著高于IA23187+6-DMAP(48.4±4.3%)、Ionomycin+6-DMAP(42.5±1.8%)、7%乙醇+6-DMAP(38.2±2.1%)及7%乙醇+CHX(62.5±2.2%)处理组(P<0.05);IA23187+6-DMAP(22.8±2.4%)和Ionomycin+6-DMAP(20.6±4.8%)处理组的囊胚率显著高于7%乙醇+6-DMAP(10.5±1.8%)、IA23187+CHX(8.4±1.2%)、Ionomycin+CHX(11.8±2.0%)及7%乙醇+CHX(8.8±2.5%)处理组(P<0.05);囊胚细胞数(200.0±14.0、169.6±11.4、187.0±8.8、201.3±16.5、179.6±5.2和177.6±20.8个)各组间没有明显差异(P>0.05)。利用IA23187、Ionomycin及乙醇联合6-DMAP和CHX,对山羊卵母细胞进行激活均能获得较高的卵裂率,IA23187、Ionomycin及乙醇与CHX联合激活处理组可获得较高的8-cell率,IA23187+6-DMAP和Ionomycin+6-DMAP处理组获得较高的囊胚率,不同处理组的囊胚均具有较高的细胞数量。IA23187或Ionomycin联合6-DMAP是激活山羊卵母细胞最适方法,是体细胞核移植技术过程中的较为高效的体外激活方法,该研究结果为山羊卵母细胞孤雌发育及体外激活等相关研究提供了借鉴。For deeply researching parthenogenetic development of cashmy goat oocytes,and offerring an efficient in vitro activated method for the somatic cell nuclear transfer of cashmy goat,the ovaries from slaughterhouse were used as material and cutted to collect oocyte,then the oocytes were matured in vitro.In this experement the matured oocytes were treated with IA23187＋ 6-dimethylaminopurine（6-DMAP）,Ionomycin＋ 6-DMAP,7% ethanol＋ 6-DMAP,IA23187＋ CHX,Ionomycin＋ CHX and 7% ethanol ＋ cycloheximide（CHX） respectively.Activated oocytes were cultured in vitro in modified synthetic oviductal fluid（mSOF） medium and assessed for cleavage,8-cell stage（48 h） and the blastocyst stage（7~9 d）.The cleavage rates were lower significantly in IA23187＋ 6-DMAP（89.6±4.9%） compared to Ionomycin＋ 6-DMAP（99.1± 0.8%）,IA23187＋CHX（97.6±1.2%）,Ionomycin＋ CHX（99.3±0.6%） and 7% ethanol＋ CHX（97.5±1.4%）（P0.05）,but no significantly different with 7% ethanol＋ 6-DMAP（96.3±0.5%,P 0.05）;The 8-cell stage rates were higher significantly in IA23187＋ CHX（81.3±5.8%） and Ionomycin＋CHX（82.5±6.6%） compared to other treatments（48.4± 4.3,42.5± 1.8,38.2± 2.1 and 62.5± 2.2%,respectively,P0.05）;The blastocyst rates were higher significantly in IA23187＋ 6-DMAP（22.8± 2.4%） and Ionomycin＋ 6-DMAP（20.6±4.8%） compared to other treatments（10.5±1.8,8.4±1.2,11.8±2.0 and 8.8±2.5%,respectively,P0.05）;The cell number of blastocysts were not significantly different among treatments 200.0 ±14.0,169.6 ± 11.4,187.0 ± 8.8,201.3±16.5,179.6±5.2 and 177.6±20.8,respectively,（P 0.05）.All groups of IA23187,Ionomycin and the combination of ethanol and CHX used to activate goat oocyte could obtain higher rate of oocyte cleavage and higher 8-cell rate,the combination of IA23187 and 6-DMAP group and the combination of Ionomycin and 6-DMAP group could obtain higher blastocyst rate,and the blastocyst from different actived group all had higher cell number,the group of

关 键 词：绒山羊 卵母细胞 孤雌激活 胚胎发育 

分 类 号：S828.36[农业科学—畜牧学]