【作者】 蔡令波；

【导师】 王锋；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2002， 硕士

【摘要】 本试验以猪为研究对象，利用屠宰场废弃的猪卵巢，从中获取卵母细胞后，进行体外成熟培养和冷冻保存研究。主要研究了促性腺激素、血清类型、猪卵泡液对猪卵巢卵母细胞体外成熟的影响，比较了从卵巢上卵泡φ＜2mm、2～5mm、＞5mm分别获得的卵母细胞体外成熟能力的差异，以及培养过程中激素的存在时期对卵母细胞体外成熟的影响；并对不同发育阶段猪卵母细胞的冷冻保存进行了研究。为下一步猪的克隆和转基因研究提供卵母细胞来源，并为其它动物特别是濒危野生动物的保种提供技术参考。结果表明： 1．促性腺激素在卵母细胞体外成熟过程中起着重要作用。培养液中添加10IU／mlPMSG和10IU／ml hCG组卵母细胞的总成熟率(88.78％)极显著高于对照组(60.01％)(P＜0.01)。 2．培养液中添加10％NCS和10％ECS均能有效地促进猪卵母细胞的体外成熟，其中，添加ECS的成熟率(72.86％)显著高于NCS(62.21％)(P＜0.05)。 3．在体外成熟培养液中添加10％pFF抑制卵母细胞的体外成熟。不添加pFF组的A级成熟率(53.65％)和总成熟率(79.60％)均显著高于添加pFF组(分别为29.81％和68.51％)(P＜0.05)。 4．不同直径大小卵泡中的卵母细胞体外成熟能力不同。大卵泡(φ＞5mm)和中等卵泡(φ2～5mm)中抽取的卵母细胞体外成熟率显著高于小卵泡(φ＜2mm)卵母细胞(P＜0.05)。中等卵泡卵母细胞体外成熟率最高(79.39％)，但与大卵泡卵母细胞(75.30％)差异不显著(P＞0.05)。 5．在猪卵母细胞体外成熟过程中，成熟前期激素的存在对卵母细胞的成熟是必需的，而在成熟培养后期去掉激素可以促进卵母细胞成熟。在猪卵母细胞体外培养48h时，培养前24h培养液中加入激素，后24h去掉激素，卵母细胞的A级成熟率(51.73％)和总成熟率(83.25％)最高，极显著高于前24h不加激素，后24h添加激素培养的成熟率(P＜0.01)，也显著高于不含激素的培养液连续培养48h的成熟率(P＜0.05)，但与添加激素连续培养48h组成熟率差异不显著(P＞0.05)。 6．本实验对不同发育阶段的猪卵母细胞的冷冻保存进行了研究。结果表明，用1.6MPROH作为冷冻保护剂，采用慢速冷冻保存方法，培养成熟后冷冻保存的卵母细 南京农业大学硕士学位论文：猪卵母细胞体外成熟与冷冻保存的研究 胞成活率（35．59O）高于培养前（24．64O）和培养24h后（23．36O）冷冻保存组， 差异显著（P＜0．05）。培养 24h（77．22O）和培养成熟后（72．slo）冷冻保存组， 其卵母细胞解冻后的形态完整率均显著高于培养前冷冻保存组（53二4％） （P＜0．05）。从而说明，猪 M 11期卵母细胞的抗冻能力高于 GV期和培养 24h的卵 母细胞。更多还原

【Abstract】 In this paper, porcine ovaries collected from slaughters were used. Factors affecting oocytes IVM, such as addition of hormones, different type of serums, follicular fluid and size of follicle were studied. Cryopreservation of oocytes in different growth phase was also studied in this paper. The results were as follows:1. Addition of PMSG (10 IU/ml) and hCG (10 IU/ml) in the maturation medium had significantly effected on the maturation rate of IVM, the maturation rate of experimental group was very significant higer than control group (P<0.01).2. The maturation rate of IVM was very significantly higher in the culture medium contain 10%ECS (72.86%) than 10%NCS (62.21%) (P<0.05).3. Addition of 10% porcine follicular fluid (pFF) to mature medium inhabits the in vitro maturation of oocytes. The maturation rate in medium without pFF (79.60%) was significant higher than medium contain 10% pFF (68.51%) (P<0.05).4. Oocytes from different size of follicular had different development ability. Oocytes collected from big ( >5mm) and medium ( 2 ~ 5mm) follicles had a higher maturation rate than oocytes from little (<2mm) follicles (P<0.05).5. When oocytes maturation for 48h in vitro, removal of hormone supplements from mature media at 24h after culture enhance oocytes maturation and cumulus expansion. And the maturation rate (83.25%) was very significant higher than oocytes that culture for 24hin the media without hormone subsequent added hormone for another 24h (63.94%) (P<0.01). also higher than oocytes culture for 48h in media without hormones (60.01%) (P<0.05).6. The experiment was studied the cryopreservation of porcine oocytes in different growth stages. The results showed that oocytes in MII stage had higher antifreeze ability than GV stage and oocytes culture for 24h. The survival rate of oocytes after cryopreservation and thawing was 35.59%, significant higher than oocytes cryopreservation in GV stages (24.64%) and culture for 24h (23.36%) (P<0.05). The rate of morphological normality after thawing in group of MII stage (72.81%) and culture for 24h (77.22%) was significantly higher than in group of GV stages (53.24%) (P<0.05).更多还原