【作者】 胡文举；

【导师】 张英汉； 王新庄；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 南阳牛是我国五大黄牛良种之一,有着十分重要的保种和育种价值。以南阳牛卵泡卵母细胞为材料通过体外成熟培养和体外受精技术建立南阳牛胚胎基因库是进行南阳牛保种的一个新途径。但是,迄今为止还未见到对南阳牛体外受精技术进行系统性研究的报道。本研究以屠宰后南阳牛的卵巢为材料,用抽吸法收集卵巢表面2～8mm卵泡中的卵母细胞,通过体外成熟、体外受精及受精卵体外培养的研究,探讨了影响南阳牛体外受精的因素。建立了适合南阳牛胚胎体外生产的可行性程序。试验结果如下:1.探讨了卵母细胞周围的卵丘细胞层数对卵母细胞体外成熟和发育潜力的影响。结果显示,具有2～8层卵丘细胞的卵母细胞的第一极体率（87.5%）、卵裂率（74.2%）和囊胚率（35.1%）均显著的高于8层以上组、2层以下组和裸卵组;2层以下组的第一极体率（67.5%）、卵裂率（48.8%）和囊胚率（23.8%）,显著高于8层以上组和裸卵组;裸卵组的效果最差,囊胚率为0%。2.卵母细胞成熟培养22h以上时卵母细胞的第一极体率（87.5%、87.5%）虽然高于培养18～22h组,但是,卵裂率（41.3%、24.3%）和囊胚率（26.3%、11.1%）均明显低于培养18～22h组（75.3%、36.2%）,差异极显著（P<0.01）;培养18h以下时,卵母细胞的第一极体率、卵裂率和囊胚率显著降低。3.在培养液中添加10%的OCS,卵母细胞的第一极体率（87.5%）、卵裂率（74.2%）和囊胚率（35.1%）最高,与添加FCS组和BSA组之间差异极显著（P<0.01）;添加FCS组的第一极体率,卵裂率和囊胚率与添加BSA组之间差异极显著（P<0.01）;添加BSA组的第一极体率（52.5%）、卵裂率（32.9%）和囊胚率（15.6%）最低。4.比较了成熟培养基础液中添加0%（对照组,添加10%FCS）、10%、20%、30%、40%BFF对卵母细胞体外成熟的影响。结果表明,添加10%、20%BFF组的卵母细胞第一极体率（82.5%、85.0%）、卵裂率（76.9%、77.9%）及囊胚率（30.1%、35.8%）显著高于对照组、30%和40%组。卵泡液的浓度增加到30%和40%时与添加20%组相比卵母细胞的第一极体率和卵裂率分别降低了42.5、75.0和44.9、64.0个百分点,但添加30%组的囊胚率比添加20%组提高了5.3个百分点。5.在卵母细胞成熟培养液中分别单独添加FSH、LH、E₂与对照组相比,对卵母细胞的第一极体率、卵裂率和囊胚率的影响各组之间差异不显著（P>0.05）;添加复合激素E₂+FSH+LH对卵母细胞的第一极体率和卵裂率没有明显的影响,但添加复合激素E₂+FSH+LH时卵母细胞的囊胚率显著的其他各组（P<0.01）。6.用直接离心法、Percoll密度梯度离心法和上浮法处理的南阳牛精子进行体外受更多还原

【Abstract】 As one of five excellent breeds of cattle in China, Nanyang cattle have great value in breed conservation and in breeding. It is a new approach for breed conversation of Nanyang cattle via foundation of Nanyang cattle embryo gene pool by in vitro maturation（IVM） and in vitro fertilization （IVF）of follicular oocyte. So far, there has not been report about systematic research on （IVF） of Nanyang cattle. In this study, ooctytes of Nanyang cattle were collected from 2 to 8 mm follicles in surface of ovary of butchered Nanyang cattle, and the oocytes were used for in vitro maturation （IVM）, in vitro fertilization（IVF） and in vitro culture （IVC）, the objective is to explore factors that affected IVF of oocytes of Nanyang cattle and establish a feasible program to in vitro production of embryos in Nanyang cattle. The results were as follows:1.The number of cumulus cell layer around oocytes influenced IVM and IVF of oocytes. The results showed that the first polation rate（87.5%） ,cleavage rate（74.2%） and blastocyst rate （35.1%）of oocytes rounded by 2—8 layers of cumulus cells were obviously higher than groups with more than 8 layers and less than 2 layers. The group with less than 2 layers had higher first polation rate（67.5%） ,cleavage rate（48.8%） and blastocyst rate（23.8%） than group with more than 8 layers and group of nude oocystes. The group of nude oocytes performed worst, blastocyst rate was zero.2. 10% ocs was added to culture medium and fisrt poleation rate （87.5%）, cleavage rate（74.2%）, blastocyst rate（35.1%） of oocytes were significant different （P<0.01）from groups that added FBS and BSA respectively. The FBS group and BSA group had dramatic discrepancy in first poleation rate ,cleavge rate and blastocyst rate（P<0.01）. The first poleation rate （52.5%）,cleavage rate （32.9%）and blastocyst rate（15.6%）in BSA group were the lowest.3.Mature medium added with 0% （control group, 10%FBS）, 10%BFF, 20%BFF, 30%BFF, and 40%BFF respectively were compared in the effect on IVM, the results showed that oocytes in 10%BFF group and 20%BFF group were significantly higher in first poleation rate （82.5%,85.0%）,cleavage rate （76.9%,77.9%）and blastocyst rate （30.1%,35.8%）than contrast group, 30%BFF group and 40%BFF group. When concentration of liquid in oocytes came to 30% and 40%, first poleation rate ,cleavage rate of oocytes declined 42.5,75.0 and 44.9,64 percent compared to 20% group, but blastocyst rate in 30% group increased 5.3 percent than 20% group.4.FSH, LH and E₂ were added to mature medium respectively, the first poleation rate, cleavage rate and blastocyst rate of oocytes between the three group had no significant difference（P>0.05） when compared with control group. E₂+FSH+LH group had no effect on更多还原