【作者】 胡勇策；

【导师】 雷安民；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2009， 硕士

【摘要】 本研究以牛卵母细胞为研究材料,在不同的体外培养体系中进行卵母细胞体外成熟培养,以探索研究影响卵母细胞体外成熟培养的因素,并建立牛卵母细胞无血清体外成熟培养体系,为以后如孤雌激活胚胎发育,体细胞核移植,转基因动物等相关研究提供参考。同时,筛选微卫星引物对体细胞核移植动物进行分析鉴定,为克隆动物亲缘关系的确认提供参考。具体试验内容如下:1.采集挑选牛卵母细胞进行体外成熟培养(1)在基础培养液也中添加10% FBS和10mg/ml的BSA,这两组的卵母细胞成熟率、卵裂率及囊胚率都没有显著差异(P>0.05),试验显示10mg/mlBSA可以代替10% FBS,对牛卵母细胞进行无血清体外成熟培养。(2)在四组不同的体外成熟培养体系中培养卵母细胞,试验结果显示在基础培养液中添加10mg/ml的BSA后再添加50μL/mL的ITS和50μg/ml的尿嘧啶,卵母细胞成熟率比其他三组都高,且差异显著(0.01<P<0.05),而在卵裂率与囊胚率上差异不显著(P>0.05),试验显示在基础培养液中添加10mg/ml的BSA同时再添加50μL/mL的ITS和50μg/ml的尿嘧啶有利于卵母细胞的成熟并获得更多的高质量成熟卵母细胞以发育到囊胚。2.筛选出10对微卫星引物BMS875,BMS574,BM2113,BM203,BMS1290,BMS1004,ETH225,FCB11,TGLA227和BM1824鉴定本实验室2008年12月出生的体细胞克隆牛,试验结果显示出生小牛和核供体细胞这10个STR位点的PAGE基因型完全一致,而与出生小牛无血缘关系的代孕母牛和试验对照母牛均不相同,证实本实验室获得的克隆牛来源于核供体细胞。更多还原

【Abstract】 Bovine oocytes were cultered in different culture systems for in vitro maturation in this pager.The major object of this paper is to research some factors on bovine oocytes in vitro maturaton and established a serum-free culture system for bovine oocytes IVM .While,Microsatellite DNA were chosen to identify the born SCNT cloned bovine.The results obtained were as follows:1.Bovine oocytes were obtained and chosen for IVM.(1)10% FBS and 10mg/ml BSA were added in basic IVM culture medium. The results show that the oocytes maturation rate, cleavage rate and blastocyst rate between groups 10mg/ml BSA and 10%FBS were not significantly different(P>0.05),indicating that 10%FBS could be replaced by 10mg/ml BSA in bovine oocytes IVM.(2)Bovine oocytes were cultured in four different groups of serum-free culture systems for oocyters IVM.The results show that the maturation rate of oocytes that cultured in the basic IVM medium that contain 10mg/ml BSA , 50μL/mL ITS and 50μg/ml Uracil was higher significantly than that of the other three groups and were significantly different(0.01<P<0.05). While the cleavage rate and blastocyst rate between this group and the other three groups were not significantly different (P>0.05),indicating that the basic IVM medium that contain 10mg/ml BSA , 50μL/mL ITS and 50μg/ml Uracil was benefit for the oocytes IVM and could obtained more the higher quality maturationed oocytes to develop to blastocyst.2. Ten microsatellite markers including BMS875,BMS574,BM2113,BM203,BMS1290,BMS1004,ETH225,FCB11,TGLA227 and BM1824 were selected to identify the born SCNT cloned bovine from our laboratory in 2008 Dec. The results showed that the gene types of the the ten STR loci are identify between the born bovine and nuclear donor cell,and different with the recipient cow and control cow.we confirmed that the born bovine is a SCNT clone bovine.更多还原