亮甲酚蓝对猪卵母细胞体外成熟及核移植胚胎发育能力的影响  

Effect of BCB Staining on in vitro Maturation and Development of Nuclear Transfer Embryo of Pig Oocyte

作　　者：徐国旗[1] 华再东[2] 许生成[1] 郑新民[2] 

机构地区：[1]青海大学农牧学院动物医学系,西宁810016 [2]湖北省农业科学院,动物胚胎工程及分子育种湖北省重点实验室,武汉430064

出　　处：《中国畜牧兽医》2016年第3期754-760,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家转基因生物新品种培育重大专项(2015ZX08010-003、2015ZX08006-003);湖北省农业科技创新中心课题(2011-620-001-003)

摘　　要：试验旨在研究亮甲酚蓝(brilliant cresyl blue,BCB)染色对卵母细胞体外成熟及后期胚胎发育潜力的影响。本研究利用13、26、39、52μmol/L BCB对成熟培养前的卵丘-卵母细胞复合体(cumulus-oocyte-complexes,COCs)染色90min,比较各组卵母细胞的着色率、成熟率及孤雌激活胚胎和核移植胚胎的发育情况。结果表明,随着BCB浓度的增加,COCs着色率依次增加(20.00%、46.39%、51.66%和59.03%),但猪卵母细胞体外成熟率逐渐降低(74.03%、72.16%、70.53%和48.61%);不同浓度BCB染色后所得BCB+组卵的成熟率均明显高于BCB-组。试验结果发现,BCB浓度在26μmol/L时,经染色的COCs既有较高的着色率,且不影响其体外成熟的效率。基于此,研究选取26μmol/L BCB作为最佳浓度对猪卵母细胞进行染色筛选,然后进行体外培养、孤雌激活及核移植试验。结果显示,筛选的BCB+组卵母细胞的孤雌胚和核移植胚的卵裂率和囊胚率均显著高于BCB-组(P<0.05),而与对照组间无显著差异(P>0.05)。胚胎移植试验挑选BCB+组中发育较好的1-2细胞期重组胚对5头代孕母猪进行了移植,其中2头怀孕,1头顺利产下了6头健康胎儿。综合以上试验结果表明,利用BCB染色可作为一种有效的方法筛选体外成熟质量较高的猪卵母细胞,同时提高胚胎体外生产效率。This experiment aimed to study the effect of brilliant cresyl blue（BCB）on in vitro maturation of pig oocytes and the developmental capacity of pig SCNT embryos.The cumulusoocyte complexes（COCs）were stained with different concentrations of BCB（13,26,39and52μmol/L）for 90 min,and then we divided the COCs into BCB^＋and BCB^-for in vitro culture 42 to 44h.The results showed that,with the concentration of BCB increased,the staining rate（20.00%,46.39%,51.66% and 59.03%）raised gradually while the maturation rate of oocytes（74.03%,72.16%,70.53% and 48.61%）reduced,the percentages of oocytes staining by26μmol/L BCB for 90 min were higher than that of other groups in staining rate and maturation rate.However,the nuclear maturation rate of BCB＋groups were higher than that of BCB-group.Therefore,26μmol/L BCB was selected as the most effective concentration dying the oocytes（BCB＋）,which were used as parthenogenetic activation and nuclear transfer embryos.The cleavage and blastocyst rates of parthenogenetic activation and SCNT embryos in BCB＋group were significantly higher than that of BCB-group（P〈0.05）,but there were no significant differences between the cleavage and blastocyst rates in the groups of BCB^＋and control（P〉0.05）.Reconstructed embryos derived from the COCs stained with BCB were transferred to five surrogates,and six cloned piglets were obtained from one of the two pregnant pigs.These results showed that COCs stained with BCB was an effective method to select high-quality oocytes,which could improve the efficiency of in vitro embryo production.

关 键 词：猪 卵母细胞 体外成熟 亮甲酚蓝染色 孤雌激活 核移植 

分 类 号：S814.8[农业科学—畜牧学]