【作者】 高立功；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 发育生物学， 2006， 硕士

【摘要】 本实验通过改变以下试验条件:卵母细胞体外成熟时间的不同、卵母细胞去核时操作液成分的不同,来研究其对山羊核移植效率的影响,希冀寻找一个有效的核移植方案。1、将山羊卵母细胞经过不同的体外成熟时间后:18 h、22 h、26 h,比较其成熟率发现,经三种成熟时间的卵母细胞成熟率差异不显著。将这三种情况下的卵母细胞去核后,比较其去核率时发现,随卵母细胞成熟时间的延长去核率下降(87.1%, 84.4%, 76.5%)。但是对核移植胚的体外发育情况进行进一步研究发现,18h时的卵裂率(71.8%)明显低于22 h和26 h(81.1%,82.5%);22 h时的囊胚率(19.6%)高于18 h和26 h时的囊胚率(12.5%, 15.7%)。所以,卵母细胞体外成熟时间以22 h左右为宜。2、本研究比较了判断山羊卵母细胞去核效果的指标:将具有第一极体的卵母细胞放入含细胞松弛素B(cytochalasin-B,CCB)浓度分别为5 mg/L、7.5 mg/L、10 mg/L、15 mg/L的操作液中盲吸去核,经荧光染色后检测去核率,再构建重组胚观察重组胚的发育情况;在含7.5 mg/L CCB的操作液中分别再加入质量浓度为2%、2.5%、3%的蔗糖进行去核,再用相同方法确定去核率,再构建重组胚观察重组胚情况。结果:CCB浓度为7.5 mg/L时,去核成功率(84.4%)明显高于对照组(70.7%),也高于其他各组;平均操作时间随CCB浓度的升高而降低;含CCB浓度为7.5 mg/L与5 mg/L的操作液去核卵母细胞所构建的重组胚卵裂率(81.1%、82.4%)显著高于对照组和15 mg/L的卵裂率(69.8%、71.8%);含CCB浓度为7.5 mg/L与5 mg/L的操作液去核卵母细胞所构建的重组胚囊胚率(19.6%、18.8%)显著高于对照组的囊胚率(9.5%)。蔗糖质量浓度为2.5%时的去核成功率(91.4%)明显高于对照组(84.4%);平均操作时间随蔗糖质量浓度的升高而降低。结论:去核时在如下条件下,可以取得最佳去核效率:(1)去核时操作液中CCB浓度7.5mg/L;(2)操作液中蔗糖质量浓度为2.5%。更多还原

【Abstract】 In order to find an effective nuclear transfer program, this thesis investigated three factors affecting the efficiency of somatic nuclear transfer on goats: the in vivo mature time of oocytes, the component of operation liquid for oocyctes enucleating and the parthenoactivation mode of oocytes.1. Goat oocytes differently matured in vivo 18 h, 22 h and 26 h and the maturing rates were not remarkable. Then these 3 kinds of oocytes were enucleated, the result showed enucleating rates declined as maturing time prolonged. These were 87.1%, 84.4% and 76.5%, respectively. After nuclear transferring, we observed the cleavage rates and the blastula rates of nuclear-transfer embryos. The cleavage rate of embryos corresponding 18 h (71.8%) was obviously lower than ones 22 h (81.1%) and 26 h (82.5%). The blastocyst rate corresponding 22 h (19.6%) was remarkable higher than 18 h (12.5%) and 26 h (15.7%).The conclusion is that the suitable time of oocyte maturation in vivo is about 22 h.2. Putting oocysts with first polarbody into operation liquid which containing cytochalasin-B (CCB), 5 mg/L, 7.5 mg/L, 10 mg/L and15 mg/L ,respectively. After blind enucleating, we detected enucleating rates by fluorescent staining and then constructed recombinant embryos, observing their developmental capacity. Adding sucrose, whose mass concentration was 2%, 2.5% and 3%, respectively, to the operation liquid containing 7.5 mg/L CCB and after enucleating, we detected enucleating rates in the same way and then constructed recombinant embryos, observing their developmental capacity. The result was that achievement ratio of enucleating in operation liquid with 7.5 mg/L CCB (84.4%) was remarkable higher than control group(70.7%) and other groups. Average operating time decreased as CCB concentration increasing. The cleavage ratios of recombinant embryos constructed from oocytes enucleated in operation liquid with 7.5 mg/L and 5 mg/L CCB were 81.1% and 82.4% respectively, remarkable higher than control group(69.8%) and 15 mg/L CCB group(71.8%). The blastocyst ratios of recombinant embryos constructed from oocytes enucleated in operation liquid with 7.5 mg/L更多还原