In vitro maturation and embryonic development of vitrified and intracytoplasmically inseminated porcine oocytes

本研究的目的：1)探讨半胱胺或对硫醇在猪卵母细胞成熟培养中的作用；2)建立猪卵母细胞的玻璃化冷冻方法；3)建立卵母细胞胞浆内单精子注射(ICSI)的新方法；结果表明：1)ICSI后添加100μM半胱胺(37%)或对硫醇(27%)后的囊胚率显著高于含PVA的对照组(16%)，与使用含卵泡液的培养液(31%)相似。2)ICSI用于猪卵母细胞的体外成熟，不需要高水平的多精受精，可获得有效的囊胚率(30%以上)。用30%乙二醇(EG)+0.5M蔗糖的冷冻法对体外成熟卵母细胞进行体外成熟培养，获得37%的体外成熟卵母细胞，ICSI法获得的囊胚率为14%。4)猪未成熟卵母细胞玻璃化冷冻时，形态正常的卵母细胞比例与有无卵丘细胞显著相关；5)对于猪卵母细胞ICSI后的胚胎发育，直径为1 mm的WOW培养系统在特定的培养液中是有效的。6)结果表明，猪卵母细胞的体外成熟是通过无血清玻璃化冷冻液获得的，然后使用指定的成熟介质进行体外成熟，尽管成熟率很低(不到10%)。

The present study investigated 1) to examine effects of cysteamine or p-mercaptoethanol in a defined maturation medium for porcine ooctytes, 2) to establish a vitrification method of porcine oocytes, 3) to establish a Novel method for intracytoplasmic sperm injection (ICSI) to in vitro matured porcine oocytes and to produce blastocysts in a serum-free, defined culture medium, and 4) to examine a serum-free vitrification solution for porcine oocytes. The following results were obtained.1) The addition of 100 μM cysteamine (37%) or p-mercaptoethanol (27%) resulted in significantly higher blastocyst rates following ICSI than that of the PVA containing control medium (16%), and were similar to that using a medium containing follicular fluid (31%).2) For ICSI to in vitro matured porcine oocytes, a Novel method is effective without a high polyspermic fertilization and produce an efficient rate (over 30%) of blastocysts following ICSI.3) For vitrification of porcine immature oocytes, a solution of 30% ethylene glycol (EG) + 0.5 M sucrose using a Cryotop method produced 37% of in vitro matured oocytes after in vitro maturation culture, and a blastyoicyst rate of 14% was obtained following ICSI.4) For vitrification of porcine immature oocytes, there was a significant correlation in the proportion of normal morphological oocytes between equilibration times and the presence or absence of cumulus cells; 4 min equilibration for cumulus-enclosed oocytes and 1 min for cumulus-denuded oocytes.5) For embryonic development of porcine oocytes following ICSI, the Well of the Well (WOW) culture system using 1 mm in diameter was effective in a defined culture medium.6) It was shown that in vitro matured porcine oocytes were obtained by a vitrification solution without serum and followed by in vitro maturation using a defined maturation medium, although the maturation rates was low (less than 10%).10.