【作者】 朱广香；

【导师】 马恒东；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2003， 硕士

【摘要】 近年来，胚胎移植的商业应用和各种胚胎工程等方面的研究和开发都需要大量的优质早期胚胎。胚胎的来源和成本已成为制约胚胎工程实用化的重要因素。把卵泡卵母细胞进行体外成熟培养和体外受精后，便可获得大量廉价的优质早期胚胎，既克服了超数排卵技术的局限性，又可满足科研和生产对胚胎的需求。 本研究以屠宰奶山羊的新鲜卵巢为材料，利用抽吸法和切割法，回收可培养有腔卵母细胞。对120个卵巢的卵母细胞回收率及其回收可培卵数进行统计，平均每个卵巢可获得卵母细胞4.72个，但是可以用于培养的只有1.43个。从每个卵巢获得可用卵数量看，切割法优于抽吸法。结果表明卵母细胞采集方法对卵母细胞数量和质量有显著影响，而切割法是采集山羊卵巢卵母细胞的一种简单而有效的方法。 实验所得的山羊卵巢卵母细胞体外培养成熟的总成熟率为82.5％(301／365)，说明本研究所采用培养方案是适合于山羊卵巢卵母细胞体外培养成熟的。 血清添加量的不同，对卵母细胞体外成熟率有很大的影响。本实验比较了基础培养液中添加FBS体积分数为0％、10％和15％时的成熟率。结果证明：不添加FBS组的成熟率极显著(P＜0.01)低于其它两组，而10％FBS组与15％FBS组差异不显著(P＞0.05)。体外成熟时间的长短对卵母细胞成熟有很大的影响，本研究结果表明培养16h与培养24h、26h的卵母细胞成熟率分别为67.6％、81.7％和81.7％，前者与后两者的差异极显著(P＜0.01)，而培养24h与26h时的成熟率差异不显著(P＞0.05)。 不同血清成分对山羊卵巢卵母细胞体外培养成熟有一定的影响。本研究在基础培养液中分别添加10％标准胎牛血清(FBS)和新生犊牛血清(NCS)，其成熟率分别为81.7％和83.8％，两者之间差异不显著(P＞0.05)。而NCS价格较便宜，在实际生产中可考虑用NCS代替FBS用于大批量体外培养山羊卵巢卵母细胞。 本实验分别在38.0℃、38.5℃、39.0℃的不同温度下，对卵母细胞进行体外培养成熟，成熟率分别为82.1％、81.7％、83.3％。结果表明：培养温度只要在山羊正常体温及其变动范围内，则成熟率无显著差异(P＞0.05)，最佳温度以39.0℃为宜。 培养液内一般情况下添加青霉素、链霉素用于防止细菌污染，，本研究表明未添加抗菌素组的成熟率(84.6％)高于添加组(81.7％)，但两组差异不显著(P＞0.05)。说明实际生产中，在条件相对比较简陋的情况下进行卵母细胞的体外培养是可行的。更多还原

【Abstract】 In recent years, the business that embryo transplant application and the research of every kind of embryo engineering development all need a mass of high-quality earlier period embryo. The source and the cost of the embryo has become the important factor that practical turn of embryo engineering. Making use of the oocytes from Slaughtered Goats FVM (in vitro maturation), joining together the Bore goat sperm capacitation and the embryo transplants, we could investigate the low cost technique route that improves the goat species of our country.Ovaria from slaughtered goats were carried to a laboratory at 37.0℃~39.0℃ in 2-3 hour. From an ovary could be obtained 1.43 Cumulus oocytes complexes (COCs). The result showed that slicing obtained significantly more useable oocytes than aspiration.There were 365 oocytes collected from the Graafian follicles (2~6mm in diameter) of newly slaughtered goat have been grouped into three type: Type A, the oocytes surrounded by cumulus cells; Type B, the oocytes surrounded by partly by cumulus cells; Type C, denuded oocytes. Goat oocytes of grade A& B are selected for in vitro maturation (FVM).These oocytes have been cultured in three different kinds of medium. 1), Ml99 + Pencillin(75^g/ml)+Streptomycin(60ug/ml)+10%FBS+LH(20μg/ml)+FSH(10IU/ml);2),M 199+Pellcillin(75μg/ml)+Streptomycin(60μg/ml)+15%FBS+LH(20μg/ml)+FSH(10IU/ml); 3),M199+10%FBS+LH(20ng/ml)+FSH(10IU/ml),at 38.0℃~39.0℃under water- satured atmosphere of 95% air-5% CO2 for 24~26 hour. The percentage of oocytes maturation canbe calculated under the microscope. Out of a total number of 365 cultured oocytes, 301(82.5%) matured oocytes were obtained. The results suggested that an addition of 10%~15%FBS, 10IU/ml FSH, and 20ug/ml LH was effective to improve oocyte maturation, and to extrude the first polar body.In the present experiments, the function of NCS on goat oocytes for IVM was studied. The results were as follows: the in vitro maturation rate of the follicular oocytes in the medium with FSH and LH supplemented with 10% NCS (83.8%) was higher than that of with 10%FBS (81.7%, P>0.05). This research established groundwork for investigating suitable mature system of the oocyte IVM for actual production demand.Follicular oocytes were collected from new slaughtered goat’s ovaries. 345 cumulus-oocyte combinations (COCs) of grade A &B were cultured respectively in the mediums of M199+Pencillin(75μg/inl)+Streptomycin(60μg/inl)+LH(20μg/ml)+FSH(10IU/ml),M199+ 10%FBS+Pencillin(75μg/ml)+Streptomycin(60μg/ml>l-LH(20μg/ml)+FSH (10 IU/ml), M199+15%FBS+Pencillin(75μg/ml)+Streptomycin(60μg/ml)+LH(20μg/ml)+FSH(10IU/ ml).The matur -ation rates were 0%, 81.7% and 81.7% respectively. It is concluded that 10%~15% FBS is beneficial to the maturation in vitro of goat follicular oocytes.In this experiment, the effects of temperature on goat oocytes IVM were studied. The results suggested that the effects of the temperature of 38.0℃ > 38.5℃ and 39.0℃ were not different on the IVM rate of the follicular oocytes (P>0.05). The effects of culture time on goat oocytes IVM were studied. The maturation rate of oocytes cultured for 24,26h was significantly higher than that of 16h (P<0.05).In the present experiments, the ability of bacteriophage on goat oocytes FVM was studied.The results suggested that an addition of 75ug/ml Pencillin+60ug/ml Streptomycin reduce the oocytes maturation rate. But the effect was not prominent (P<0.05). This result indicated that the Goat oocytes IVM is could put in practice for actual production.更多还原