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

本研究探讨了半胱胺和巯基乙醇对猪卵母细胞体外成熟的影响，建立了猪卵母细胞的玻璃化冷冻方法，建立了猪卵母细胞体外成熟的卵母细胞胞浆内单精子注射（ICSI）方法，和4）检查猪卵母细胞的无血清玻璃化冷冻液。结果表明：（1）添加100 μM半胱胺（37%）或p-巯基乙醇（27%）的ICSI囊胚率显著高于添加PVA的对照组（16%），与添加卵泡液的对照组（31%）相似。一种新的方法是有效的，没有高多精受精，并产生有效率（超过30%）的囊胚。3）对于猪未成熟卵母细胞的玻璃化冷冻，使用Cryotop方法的30%乙二醇（EG）+0.5M蔗糖的溶液在体外成熟培养后产生37%的体外成熟卵母细胞，4）对于猪未成熟卵母细胞的玻璃化冷冻，平衡时间与卵丘细胞的存在与否之间，形态正常的卵母细胞比例存在显著的相关性;卵丘封闭的卵母细胞平衡4分钟，卵丘裸露的卵母细胞平衡1分钟。5）对于ICSI后猪卵母细胞的胚胎发育，使用直径为1 mm的Well of the Well（WOW）培养系统在确定的培养基中是有效的。6）显示通过不含血清的玻璃化溶液，然后使用确定的成熟培养基进行体外成熟，获得体外成熟的猪卵母细胞，尽管成熟率低（小于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.