【作者】 蔡春花；

【导师】 罗光彬；

【作者基本信息】 延边大学 ， 动物遗传育种与繁殖， 2002， 硕士

【摘要】 本研究分析了牛卵巢保存温度、外源激素（LH、17β-E2）和不同培养时间对牛卵母细胞体外成熟的影响。结果表明，卵巢保存温度为25～29℃、30～35℃、36～40℃时，牛卵母细胞的体外成熟率分别为55.3%、70.1%、60.7%。经统计分析牛卵巢保存温度对牛卵母细胞体外成熟的影响不大（P >0.05）；成熟培养基中未添加激素组、只添加17β-E2和添加LH+17β-E2组时，成熟率分别为57.8%、64.3%和70.1%。经统计分析添加LH+17β-E2与未添加激素组差异显著（P <0.05）；牛卵母细胞体外培养不同时间22～25 h、26～30 h、31～34 h时，牛卵母细胞的体外成熟率分别为56.3%、65.4%和68.4%，三组间差异不显著（P >0.05）。本研究还对成熟分裂中期（MⅡ）的牛卵母细胞分别用不同浓度的乙醇和不同浓度钙离子载体A23187进行处理，观察其激活率；筛选出激活卵子最佳乙醇浓度和A23187浓度，再与亚胺环己酮复合活性化处理，观察其激活率和孤雌发育能力。结果表明，用不同浓度的乙醇5%、7%、9%、11%、13%活性化处理后，所得的激活率分别为41.2%、47.2%、53.1%、39.7%、23.4%。经统计分析7%和9%组与其他组差异显著（P<0.05），两组之间差异不显著（P >0.05），因此选用乙醇浓度为9%组做下一步试验；用不同浓度（2.5 μM、5μM、7.5 μM）的钙离子载体A23187活性化处理后，所得的激活率为分别为29.3%、51.8%、45.4%。钙离子载体A23187浓度 5μM与7.5μM组分别与2.5μM组差异显著（P<0.05），但5μM和7.5 μM组之间差异不显著（P>0.05），因此选用5 μM组做进一步实验；9%的乙醇和5 μM<WP=4>的钙离子载体单一活性化处理和分别与亚胺环己酮复合活性化处理时，所得的单一活性化处理（53.1%，51.8%）低于复合活性化处理的激活率（77.3%，76.8%），经统计分析差异显著（P<0.05），并且乙醇单一活性化处理和复合活性化处理时2PN形成率为44.1%和72.1%，差异显著（P<0.05）；A23187单一活性化处理和复合活性化处理时2PN形成率为40.2%和 72.9%，差异显著（P<0.05）。9%的乙醇和5μM的钙离子载体分别与亚胺环己酮复合活性化处理卵子后，进行了孤雌发育能力的比较，卵子发育为2-8细胞的胚胎占71.0%和62.9%，两种处理方法差异不显著（P>0.05）。更多还原

【Abstract】 The effects of preserving ovary temperature, exogenous hormone（LH、17β-E2）, and various culturing time （22-25h,26-30h, and 31-34h）to the bovine oocytes maturating in vitro were analyzed in this research . The outcome suggested that , the effects of bovine ovary which maturated in vitro preserving temperature（25-29℃,30-35℃, and 36-40℃） to oocytes were not very clear（P>0.05）,after statistically analyzed, and the bovine oocytes maturating rates were 55.3%,70.1%,and 60.7%,respectively;the maturity rates of the bundles which were not added hormone, added 17β-E2 , and added LH+17β-E2 were 57.8%,64.3% and 70.1% respectively, and the difference between the bundle which was added LH+17β-E2 and the bundle not added hormone was significance（P<0.05）,after statistically analyzed; the mature rate of bovine oocytes which had been cultured in vitro for 31-34h was 68.4%, which was higher than the two bundles which had been cultured for 22-26h(56.3%) and 27-30h (65.4%) respectively, but the differences among the three bundles were not significance（P>0.05）,after statistically analyzed. Bovine oocytes which were in maturation mephase （MⅡ）were treated by various concentration of ethanol (5%, 7%, 9%, 11%, and 13%) or calcium ionophore A23187 （2.5μM , 5μM, and 7.5μM）in this research , and observed its activating rate. The best concentrations of ethanol and A23781, which were required to activate ova ,were screened . The bovine oocytes were treated by the compound activity of cycloheximide , and observed their activity rates and the ability of parthenogenesis.The outcome suggested that , the bundles which contained the ethanol 7%, and 9% had differences with other bundles ,and the difference between the two bundles was not significance （P>0.05）<WP=6>after statistically analyzed .But the activity rate (53.1%) of the bundle which contained 9% ethanol was higher than that of the bundle which contained 7% ethanol , so the bundle which contained 9% ethanol was chosen in next step . The difference between the two bundles whose calcium ionophore A23187 concentrations are 5μM or 7.5μM was not significance （P>0.05）, but the difference between the two bundles and the bundles whose concentration was 2.5μM was significance （P<0.05）after statistically analyzed, and the activity rate (51.8%) of the bundle whose concentration was 5μM was chosen in next step. Not only was the difference between the single activating treatment and compound activating treatment which contained 9% ethanol with cycloheximide or 5μM calcium ionophore significance, but also the activating rates (77.3%, 78.8% ,respectively) of compound treatment was higher than that of single treatment (53.1%, 51.8% ,respectively), and the difference between the formation rates of 2PN which were treated by single activating treatment and that of the 2PN treated by compound activating treatment was significance （P<0.05）(40.2%,72.9% respectively) after statistically analyzed. The ova which had been disposed by 9% ethanol with cycloheximide were compared in the ability of parthenogenesis ,and the embryos which were developed to 2-8 cells were 71.0% , 62.9% respectively, but the difference between two ways was not significance （P>0.05）,after statistically analyzed.更多还原