【作者】 卫恒习；

【导师】 桑润滋；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2005， 硕士

【摘要】 本研究系统地探讨了体外受精中的诸多影响因素,以期建立一套比较完善的绵、山羊体外受精的操作体系。研究的主要内容为:绵羊体外受精影响因素和山羊体外受精影响因素。 在绵羊体外受精中,研究了不同体外受精培养体系（SOFaa和CR1aa）、不同卵巢保存温度（25℃、37℃）和采卵方法、不同成熟培养时间（21h、24h、27h）、成熟液中不同FSH剂量和激素组合、FBS的灭活处理、10%新鲜PFF的添加、不同受精液种类和精子制备方法以及发育培养是否换液对绵羊IVM/IVF/IVC的影响,结果表明:SOFaa体系的卵裂率和桑-囊胚发育率（17.22%（47/273）和19.15%（9/47））均高于CR1aa体系（10.68%（22/206）和9.09%（2/22））,得出在本实验室条件下,SOFaa体系在绵羊IVF的效果优于CR1aa体系;在运输时间为6～9h时,37℃保存会显著降低受精卵裂率,25℃是绵羊卵巢较合适的保存温度;抽吸法每个卵巢平均获得1.43±0.36枚可用卵,抽吸后的卵母细胞再用切割法采卵,还可以获得与抽吸法数量相当的可用卵（1.16±0.3枚）,但即使两种方法综合运用,仍显著低于切割法获得的可用卵数（2.59±0.82和5.47±1.43,P<0.01）,而且切割法的受精卵裂率显著高于抽吸法和抽吸后切割法的受精卵裂率（59.93%（193/339）:43.90%（162/369）:38.86%（129/332）,P<0.01）,表明切割法是最佳的采卵方法;在SOFaa体系的基础上,绵羊卵母细胞IVM时间以21h～24h的效果较好:FSH对绵羊卵母细胞体外成熟是必不可少的,添加FSH显著提高受精卵裂率（P<0.01）,以添加20μg/ml FSH效果最佳;LH和E₂对绵羊卵裂率和桑-囊胚发育率无显著作用（P>0.05）;血清灭活处理对卵裂率无显著影响（P>0.05）,但有提高胚胎发育率的趋势;在10%灭活FBS的基础上再添加10%新鲜PFF对卵裂率没有影响,但能显著提高桑-囊胚发育率（41.38%（12/29）:14.71%（5/34）,P<0.05）;SOF受精液的桑-囊胚发育率显著高于BO受精液（32.81%（21/64）:12.5%（5/40）,P<0.05）;密度梯度离心法制备精子的受精卵裂率和胚胎发育率均好于上浮离心法;应用SOFaaBSA混合气培养绵羊IVF胚过程中换液操作显著提高了胚胎发育阻滞率（P<0.05）,降低了桑-囊胚发育率。 在山羊体外受精研究中,比较了不同厂家FSH、不同FSH剂量（0 AU/ml、0.02AU/ml、0.04 AU/ml、0.06 AU/ml）、不同激素组合、不同成熟培养时间（21h、24h、27h）、氨基酸、不同受精液种类（mSOF、SOF、BO）、不同发育培养液及培养方法、负压培养等对山羊IVM/IVF/IVC的影响。结果表明,日本FSH与宁波FSH差异不显著（P>0.05）;添加FSH显著提高了山羊体外受精卵裂率（P<0.01）,添加剂量以0.02～0.04AU/ml（日本FSH）效果最佳;LH对山羊的IVM/IVF无显著影响;E₂的添加对山羊的IVM/IVF有负面影响,使桑-囊胚发育率显著降低（P<0.05）;山羊卵母细胞成熟时间以24～27h为宜;在成熟液TCM199中添加EAA和NEAA能显著提高受精卵裂率（P<0.05）,桑-囊发育率也有所提高（P>0.05）;不同受精液的受精卵裂率和桑-囊胚发育率差异不显著（P>0.05）,改良的无糖mSOF受精液可能具有提高受精卵裂更多还原

【Abstract】 Many influence factors within IVF were experimented systematically in this study for the sake of establishing an acceptable IVF system on sheep and goat. The main contents of this study are: effect of different factors on sheep IVF technique and the effect of different factors on goat IVF technique.In the study of sheep IVF technique, the effects of SOFaa and CR1aa systems, the different ovary keep temperature (25℃, 37℃ ) and different methods of collecting oocytes, the different IVM time(21hs, 24hs, 27hs), the different supplemental dose of FSH, the different hormone combination strategy, the FBS heat-inactived or not and the supplement of 10% fresh PFF in IVM medium, the different IVF media and different sperm preparation methods, the influence of changing IVC medium during IVC with a SOFaa mixed gas system to the effect of sheep IVM/IVF/IVC were studied. The results shows, the cleavage rate and morula-blastocyst (M-B) development rate of SOFaa system are higher than those of CR1aa system(17.22%(47/273) and 19.15%(9/47) vs 10.68%(22/206) and 9.09%(2/22)). So the SOFaa system is better than CRlaa system on sheep IVF research in our Lab; as the transport time between 6-9hs, 25 ℃ is a suitable temperature to keep the ovary compared to 37 ℃; the aspiration method can obtain 1.43±0.36 good quality oocytes per ovary, a comparative number of good oocytes can also obtained by slicing the ovary aspirated(1.16+0.3), but even the two methods are synthetically used, the number of good oocytes is still sifnificantly lower than that of the direct-slicing method(2.59±0.82 vs 5.47±1.43, P<0.01), the cleavage rate of the direct-slicing method is significantly higher than that of aspiration method and the indirect-slicing method(59.93%(193/339) vs 43.90%(162/369) vs 38.86%(129/332), P<0.01), So the direct-slicing method is the best way to collecting oocytes; with SOFaa system, the better IVM time is 21hs-24hs; the supplement of FSH is essential to the IVM of sheep, the supplement of FSH in IVM medium can significantly increase the cleavage rate(P<0.01), the best dose of FSH is 20μg/ml for the sake of M-B development rate; LH and E2 has no obvious function to improve the cleavage rate and the M-B rate (P>0.05); the FBS heat-inactived have no obvious influence on the cleavage rate(P>0.05); The supplement of 10% fresh PFF to the更多还原