【作者】 陈江凌；

【导师】 王新庄；

【作者基本信息】 河南农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 本研究利用屠宰牛的卵巢,先用抽吸法收集卵巢表面直径为2～8mm卵泡液,然后,用切割法获取卵巢表面直径2mm以下的小腔卵泡卵母细胞。对获得的A、B级小腔卵泡卵母细胞进行体外成熟培养、体外受精培养和受精卵的体外培养的研究。实验结果如下:1.本研究探讨了牛卵巢保存温度以及育成牛和经产牛卵巢对牛小腔卵泡卵母细胞体外成熟的影响。结果表明:卵巢保存温度在31～37℃时,可用卵母细胞数和成熟的小腔卵泡卵母细胞数均较多;经产牛小腔卵泡卵母细胞体外成熟效果好于育成牛。2.比较不同的激素组合(FSH/LH、FSH/hCG和hCG/PMSG)对牛小腔卵泡卵母细胞体外成熟的影响。结果表明:对照组1牛小腔卵泡卵母细胞的成熟率最低(6.52%),且与第2、3、4相比均差异显著(P<0.05);第3组卵母细胞的成熟率最高(23.00%),但与第2、4两组牛小腔卵泡卵母细胞的成熟率间差异不显著(P>0.05)。3.在成熟培养液中分别添加0%、5%、15%和20%的FCS。结果表明:在成熟培养液中添加适量的FCS显著促进牛小腔卵泡卵母细胞体外成熟(P<0.05),其中添加15% (v/v)的FCS组的牛小腔卵泡卵母细胞体外成熟率最高,添加10%(v/v)的FCS组次之,但两者间差异不显著(P>0.05)。4.添加0.05、0.1μmol/mL的β-巯基乙醇均能促进牛小腔卵泡卵母细胞体外成熟(P>0.05),但添加0.2μmol/mL的β-巯基乙醇组显著抑制牛小腔卵泡卵母细胞体外成熟率(P<0.05),以0.1μmol/mL的β-巯基乙醇组牛小腔卵泡卵母细胞体外成熟率最高。5.比较不同的直径的卵泡液对牛小腔卵泡卵母细胞体外成熟的影响。结果表明:与对照组1相比,第2组在培养液中添加直径≤2 mm的卵泡卵泡液,卵母细胞的成熟率降低,但二者差异不显著(P>0.05);第3组在培养液中添加直径2～6mm的卵泡卵泡液显著促进牛小腔卵泡卵母细胞的成熟(P<0.05),卵母细胞的成熟率最高。6.比较在体外成熟培养液中分别添加不同浓度的LIF和(或)胰岛素对牛小腔卵泡卵母细胞体外成熟的影响。结果表明:在体外成熟培养液中同时添加LIF和胰岛素的I-8组牛小腔卵泡卵母细胞的体外成熟率最高(35.78%),而单独添加胰岛素的I-6组牛小腔卵泡卵母细胞的体外成熟率最低(22.12%);I-8组与I-6组间牛小腔卵泡卵母细胞的成熟率差异显著(P<0.05),但与对照组I-1以及其它各组间成熟率均差异不显著(P>0.05)。7.比较了上浮法和Percoll密度梯度离心法两种精子离心方法对卵泡卵母细胞体外受精的影响,结果表明:Percoll密度梯度离心法预处理的精子卵泡卵母细胞的卵裂率(19.17%)高于上浮法(17.48%),但两者间没有显著的差异(P>0.05)。8.与对照组相比,和输卵管上皮细胞单层共培养系统的卵泡卵母细胞的卵裂率虽然高一些,但二者间卵裂率没有显著的差异(P>0.05)。更多还原

【Abstract】 In this study, oocytes of cattle were collected from follicles below 2mm in surface of ovary of butchered cattle after the follicles 2~8mm were pumped , and the oocytes of A、B ranking levels were used for in vitro maturation (IVM), in vitro fertilization(IVF) and in vitro culture (IVC),the result showed that:1.This experiment studied the effects of various storage temperature、growing cattle and delivered cow on in vitro maturation of bovine oocytes derived from small antral follicles.Results showed that: Ovaries reserved temperature 31~37℃, the useable and matured oocytes derived from small antral follicles were more than other temperature; The maturated effect of oocytes derived from small antral follicles by delivered cow was better than growing cattle.2.To compared the the effects of different hormone combination on in vitro maturation of bovine oocytes derived from small antral follicles,the results showed that: the maturation rate of oocytes was lowest in control group 1th and it’s significant differences to compared with 2th,3th and 4th group(P<0.05);the maturation rate of oocytes is highest in group 3(23.00%),but it’s not significant differences to compared with 2th and 4th(P>0.05).3.Supplementation of 0%、5%、15% and 20% FCS in the maturatin medium, the results showed that:Supplementation of sufficient FCS significantly improved the maturation rate of oocytes(P<0.05). The maturation rate of oocytes was highest with 15% FCS and second with 10%FCS, but there was no significant difference(P>0.05).4.To contrast with control group, supplementation of 0.05μmol/mL and 0.1μmol/mLβ-ME could improved the maturation rate of oocytes (P>0.05) and supplementation of 0.2μmol/mLβ-ME could inhibited the maturation rate of oocytes significantly(P<0.05),the maturation rate was highest with 0.1μmol/mLβ-ME in the culture medium .5.To compared the the effects of fluid coming from different diameters, the results showed that: To contrast with control group, the maturation rate of oocytes derived from small antral follicles in 2th group is lowest,but it’s not significant differences between them;it’s significant differences between control group and the 3th group in which fluid of 2~6mm diameter was added to the culture medium.6.To compared the the effects of different LIF concentration and(or) insulin concentration on in vitro maturation of bovine oocytes derived from small antral follicles,the results showed that: Maturation rate of oocytes was highest (35.78%)with the supplementation of LIF and insulin in I-8 group in the same time ,and it’s lowest in I-6 group(22.12%);Maturation rate of oocytes in I-8 group was significant difference with that in I-6 group(P<0.05),but it’s not significant difference with other groups (P>0.05).7. To compared the the effects of swim-up and percoll density gradient centrifugation treated sperms on in vitro fertilization.The results showed that:The cleavage rate of oocytes by percoll density gradient centrifugation treated sperms is higher than that of oocytes by swim-up treated sperms,but there wasn’t significant difference(P>0.05).8. To contrast with control group, The cleavage rate of oocytes cultured with MOEC was higher,but there isn’t significant difference(P>0.05).更多还原