Hochi Shinichi
Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan.
J Reprod Dev. 2003 Feb;49(1):13-21. doi: 10.1262/jrd.49.13.
Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.
对马和牛这两个物种中影响植入前胚胎和卵泡卵母细胞冷冻敏感性的因素进行了分析。(1) 在以甘油作为冷冻保护剂 (CPA) 的两步冷冻法中，马囊胚的存活受到胚胎囊发育的影响。使用乙二醇 (EG) 和蔗糖作为CPA可提高解冻后囊胚的存活率，并使得在不移除CPA的情况下将胚胎移植到受体母马体内成为可能。此外，当胚胎逐步暴露于玻璃化溶液时，通过玻璃化冷冻保存的早期囊胚能够在体外和体内发育。玻璃化程序也应用于相对较大的扩张囊胚。(2) 体外产生的牛胚胎被认为对冷冻过程高度敏感。为了解决这个问题，在无血清系统中产生的第7天囊胚在投入液氮之前以0.3℃/分钟而不是0.6℃/分钟的速度冷却，解冻后活力没有损失。在IVM/IVF培养基或IVC培养基中添加LAA分别有效地产生了对冷冻相对耐受的原核期合子或桑椹胚期胚胎。(3) 对未成熟马卵母细胞的透射电子显微镜观察表明，细胞损伤发生在卵丘细胞与卵母细胞之间的缝隙连接部位附近。在牛中，当卵母细胞在IVM的中间阶段（冷冻时为GVBD，玻璃化时为Met-I）进行冷冻保存时，可获得更高的受精率。发现以超快速冷却速率（3000-5000℃/分钟）为特征的开放拉制玻璃毛细管中牛Met-II卵母细胞的玻璃化可避免玻璃化和复温的任何有害影响。
