【作者】 靳双星；

【导师】 李跃民；

【作者基本信息】 西南农业大学 ， 临床兽医学， 2005， 硕士

【摘要】 卵泡是哺乳动物卵巢上的基本结构和功能单位,是卵母细胞生长和成熟的微环境。在卵泡发生过程中,卵巢上尽管有大量的原始卵泡存在,但真正能发育到排卵前的卵泡很少,造成遗传资源的巨大浪费。为了开发利用这一卵泡资源,我们对猪腔前卵泡分离培养方法及腔前卵泡卵母细胞的体外成熟培养进行了研究,目的是为了进一步挖掘猪的遗传潜力,为体外受精、动物克隆和转基因等胚胎生物技术研究提供大量的优质卵源。 分离方法研究:本研究以分离到的卵泡数量、分离时间和卵泡在体外培养三天后的存活率为指标比较了两种机械方法分离猪腔前卵泡的效果。结果表明:用针刮法分离猪腔前卵泡的数量(φ<50μm:203600±59000;50μm≤φ≤1501μm:29.40±10.34;φ>150μm:9.30±3.29)显著高于剪碎法(φ<50μm:38900±12000;50μm≤φ≤150μm:13.954±3.70;φ>150um:4.954±1.61)(p<0.01),而且针刮法分离时间比剪碎法短(16.16±1.43min vs 20.30±1.48min,p<0.01)。体外培养三天后,腔前卵泡存活率在两种方法之间没有明显差异(81.00% vs 78.97%,p>0.05)。说明应用针刮法分离猪腔前卵泡能有效保护卵母细胞和颗粒细胞的正常形态以及基膜的完整性,从而使分离到的猪腔前卵泡维持正常的生理活性。 基础培养液选择研究:本研究以卵泡生长快慢、卵泡腔形成状况和卵泡存活率为指标比较了M199和NCSU23两种基础培养液的培养效果,结果表明,培养在M199中的腔前卵泡生长速度显著快于培养在NCSU23中的腔前卵泡(5d:31.01±15.79 vs 21.73±12.69,p<0.01:10d:44.69±25.90 vs 33.25±17.35,p<0.05),培养在两组的猪腔前卵泡前5d的生长率都极显著地高于后5d的生长率(M199:31.01±15.79 vs 13.68±11.50,p>0.01;NCSU23:21.734±12.69 vs 11.52±9.19,p<0.01)。而腔前卵泡的成腔率和存活率没有明显差异。因此,选择M199培养液来研究不同培养方法和不同FSH浓度对腔前卵泡体外培养的影响。 培养方法研究:采用96孔培养板法、凹窝培养法和颗粒细胞铺层培养法对猪腔前卵泡进行体外培养。结果表明,培养在凹窝培养体系的腔前卵泡生长率显著低于96孔培养板法(5d:26.44±17.38 vs 19.18±9.37,p<0.05),但凹窝培养体系可以显著增加腔前卵泡的存活率(81.58% vs 55.88%,p<0.05),同时也证明颗粒细胞铺层培养法对腔前卵泡生长没有影响。 FSH添加剂量研究:本实验比较了M199培养液中三种FSH浓度(0.5、1、2IU/mL)对腔前卵泡的影响。结果表明:FSH浓度为2IU/mL时腔前卵泡的生长率最高,0.5IU/mL和2 IU/mL两组之间,在卵泡生长率(5d:25.96±19.15 vs 36.59±16.28,p<0.05;10d:40.87±33.02 vs 60.73±26.58 p<0.01)、成腔率(9.09% vs 31.70%,p<0.05)和存活率(54.55% vs 81.48%,p<0.05)上存在着显著差异。三个处理组腔前卵泡生长率,前5d和后5d相比差异极显著(0.5IU/mL组:25.69±19.15 vs 14.91±13.91,p<0.01;1IU/mL组:33.00±20.35 vs 17.65±15.17.p<0.01;2IU/mL组:36.59±16.28 vs 24.14±11.48,p<0.01)。 猪腔前卵泡卵母细胞体外成熟培养研究:为考察猪腔前卵泡卵母细胞最佳成熟方式,我们以极体排出情况为判断标准,采用一步法和两步法成熟猪腔前卵泡卵母细胞。结果表明:更多还原

【Abstract】 The ovarian follicle is the basic structural and functional unit of mammalian ovary and it provides the microenvironment for the growth and maturation of oocyte. Despite the large population of primordial follicles present in the ovary during folliculogenesis, many follicles undergo atresia, and only a few of them develop to the preovulatory stage, which is just a waste of hereditary resources. The isolation, culture and the in vitro maturation (JVM) of porcine preantral follicles were studied in order to empolder and utilize these resources. The objective of this study is to empolder further porcine genetic potential and provide large number of oocytes in high quality for embryo biotechnology such as in vitro fertilization, cloning and transgenic technology.This study compared the effects of two mechanical methods on the isolated porcine preantral follicles regarding the number, handling times of isolation preantral follicles and their livability after three days culture in vitro as indexes. The result showed that needle scraping method harvested significantly more porcine preantral follicles(Φ < 50um: 203600±59000, 50μm□Φ□150μm: 29.40±10.34, Φ>150μm: 9.30±3.29) than cutting(Φ<50um: 38900±12000, 50μm□Φ□50μm: 13.95±3.70, Φ150μm: 4.95±1.61) ( p<0.01). The average handling time of needle scraping method for each ovary (16.16±1.34min) was less than that of cutting (20.30±1.48min) ( p<0.01). The livability of preantral follicles after three days in-vitro culture was not significant different between the two groups (81.00% vs 78.97%, p>0.05). The results indicated needle scraping mechanical isolation could be used to maintain normal physiological functions of preantral follicles as it kept the natural figures of oocytes and granulosa cells and preserved the integrity of basement membrane.Two basic medium of Ml99 and NCSU23 were compared as their effects on the grow speed, antrum formation and follicular livability. These results demonstrated that preantral follicles cultured in M199 medium grew faster than those mNCSU23 (5d: 31.01±15.79 vs21.73±12.69, p<0.01,10d: 44.69±25.90 vs 33.25 ±17.35, p<0.05) . The follicular growth rate during the first 5 days of culture was higher than that during the latter 5 days in both of the two culture-medium groups (M199: 31.01±15.79 vs 13.68±11.50, p>0.01;NCSU23:21.73±12.69 vs 11.52±9.19, p< 0.01) . However, antrum formation and follicular livability had no difference between the two groups. Therefore, M199 was chosen to investigate the effects of different culture methods and various concentrations of FSH on the in vitro culture (IVC) of porcine preantral follicles.96-well plates, hollowness and granulose cells coating culture systems were adopted in culturing porcine preantral follicles in vitro. The results showed that the follicular growth rate in hollowness culture system was lower than that in 96-well plates(5d: 26.44±17.38 vs 19.18±9.37, p更多还原