【作者】 武彩红；

【导师】 侯加法； 芮荣；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2006， 博士

【摘要】 哺乳动物卵母细胞冷冻保存，在动物种质资源保护等方面已日益显示出强大的应用潜力。诚然，关于卵母细胞的冷冻保存，业已形成众多的技术方案，这些方案大多在小鼠、牛和人卵母细胞上应用并获得良好的冷冻效果。迄今为止，猪卵母细胞的冷冻保存尚未取得突破性进展；适用于猪卵母细胞冷冻保存的技术方案仍十分有限。因此，建立有效的猪卵母细胞冷冻保存技术平台依然是当前最为迫切的研究任务。此外，有关猪卵母细胞冷冻耐受性的机理研究，报道仍然偏少。本研究采用OPS(open pulled straw)法对猪未成熟(GⅤ期)和体外成熟(MⅡ期)卵母细胞进行玻璃化冷冻保存，研究其冻后存活力和发育能力所受的影响；在优化冷冻程序的基础上，对猪卵母细胞的超微结构和细胞骨架结构进行系统研究，以期加深对影响猪卵母细胞冻后发育能力的机理的认识，为建立适合于猪卵母细胞冷冻保存的技术方案提供参考，为进一步完善猪卵母细胞的冷冻保存技术奠定基础。本研究共包括以下7个部分： 试验一、猪卵母细胞的采集与体外成熟研究 本试验以屠宰场采集的猪卵巢卵母细胞为研究对象，旨在优化猪卵母细胞回收方法及其体外成熟培养体系，为进一步研究提供适用的猪GⅤ期和MⅡ期卵母细胞。以抽吸法采集猪卵巢中直径为2～5mm及直径大于5mm卵泡卵母细胞，再用切割法采集直径小于2mm卵泡卵母细胞。结果表明，切割法所获卵母细胞数及每只卵巢平均采卵数均显著高于抽吸法(分别为1300枚对570枚和31.0枚对13.6枚；P＜0.05)；但切割法所获卵母细胞可用率仅为33.8％，显著低于抽吸法67.5％的可用率(P＜0.05)。用抽吸法采集直径为2～5mm的卵泡卵母细胞，分4组进行体外成熟培养，研究激素和猪卵泡液(pFF)对卵母细胞体外成熟的影响：Ⅰ组(对照组)，成熟培养液为TCM-199(含10％NCS)+pFF+PMSG+hCG+17β-E2+L-半胱氨酸，卵母细胞在其中培养44h；Ⅱ组(无激素组)，成熟培养液为TCM-199(10％NCS)+pFF+L-半胱氨酸，卵母细胞在其中培养44h；Ⅲ组(无卵泡液组)，成熟培养液为TCM-199(10％NCS)+PMSG+hCG+17p-E2+L-半胱氨酸。卵母细胞在其中培养44h；Ⅳ组(基础培养液组)，卵母细胞在成熟培养液(同Ⅰ组)中培养24h后，转入基础培养液TCM199(10％NCS)+L-半胱氨酸组中，继续培养20h。结果表明，卵母细胞在添加激素和pFF的成熟培养液中培养24h后，转入无激素、无pFF的基础培养液中继续培养20h，体外成熟率达78.8％，明显高于另外3组更多还原

【Abstract】 The cryopreservation of mammalian oocytes increasingly displayed its huge potential of application in animal resource protection. Although various of protocols for oocyte freezing have been developed, most of them were applied in mouse, cattle and human oocyte cryopreservation successfully. To date, cryopreservation of porcine oocytes has not yet made any breakthrough. Suitable protocols used in cryopreservation of porcine oocytes were quite limited. Therefore, it became urgent to establish a high efficient cryopreservation platform for porcine oocytes. In addition, study on the mechanisms of cryotolerance against cryopreservation of porcine oocytes was also limited. In the present study, porcine germinal vesicle (GV) stage and metaphase II (MII) stage oocytes were vitrified with open pulled straw (OPS) method in order to investigate the effects of cryopreservation on their post-warming survival and developmental competence. On the basis of the optimization of freezing protocol, the ultrastructure and cytoskeletal structure of porcine oocytes were investigated systematically so as to enhance the understanding of mechanisms on influencing viability of vitrified porcine oocytes, to furnish a reference for porcine oocyte cryopreservation, and to establish a basis for further improvement of cryopreservation of porcine oocytes. This study contained the following 7 parts.Experiment 1. Collection and maturation in vitro of porcine oocytes. The purpose of the experiment was to optimize the collection and in vitro maturation procedures of porcine oocytes from ovaries acquired in an abattoir. The aspiration method was used to collect oocytes from 2-5 mm and >5 mm follicles in diameter and the cutting method was adopted to collect oocytes from follicles < 2 mm in diameter. The result showed that either total number or average number per ovary of oocytes obtained with cutting method were significantly higher than those from aspiration method (1300 versus 570 and 31.0 versus 13.6, respectively, P<0.05). However, the percentages of available oocytes from cutting method were 33.8%, significantly lower than the percentage of aspiration method (67.5%,更多还原