Studies on Preservation of Enucleated Oocytes and Nuclear Transplantation in Cattle and Horses

去核的卵母细胞可以支持转移的核的重编程和由核转移产生的合子的发育。本研究的主要目的是通过对牛去核卵母细胞冷冻保存方法的研究，提高牛去核卵母细胞的克隆效率。结果总结如下：（1）在去核卵母细胞的制备过程（IVM、去核和活化）中，向培养基中添加亚油酸-白蛋白（其提高了体外产生的牛胚胎的冷冻耐受性）可有效地产生耐冷冻的卵母细胞（Hochi et al.，兽医医学科学杂志，2000年）。（2）从常规IVF判断，以3，000 - 5，000 ℃/min的冷却速率玻璃化冷冻体外成熟的牛卵母细胞不会导致活力损失（Hochi等人，Cryobiology，2001）。本研究的第二个目的是建立体外生产马！胚胎，为未来通过核移植克隆马的进展。结果总结如下：（3）使用精子穿透去透明带仓鼠卵母细胞的试验，发现用0.1 μM的离子载体A23187处理种马精子2 min可有效诱导精子获能和顶体反应（Matsukawa et al.，J. Reprod.偏差：2002年）。（4）在A23187处理后，种马精子的胞质内注射导致65%的正常受精率和体外囊胚期胚胎的成功产生（Matsukawa等人，2002年IETS会议上的海报展示）。

Enucleated oocytes can support reprogramming of transferred nuclei and development of the zygotes produced by the nuclear transfer. The fitst aim of this study was to improve cloning efficiency in cattle, throughout the development of cryopreservation methods for enucleated oocytes. Results are summarized as follows:( 1 ) Addition of linoleic acid-albumin, which improved freezing tolerance of in vitro-produced bovine embryos, to the media during the preparation process of enucleated oocytes ( IVM, enucleation, and activation ) was effective to produce oocytes tolerable to freezing ( Hochi et al., J. Vet. Med. Sci., 2000 ).( 2 ) Vitrification of in vitro-matured bovine oocytes at a cooling rate of 3,000-5,000℃/min resulted in no loss of the viability when judged from routine IVF ( Hochi et al., Cryobiology, 2001 ). The second aim of this study was to establish the system for in vitro production of equine!embryos, for the future progress in horse cloning by nuclear transfer. Results are summarized as follows:( 3 ) Using sperm penetration assay into zona-free hamster oocytes, treatment of stallion spermatozoa with ionophore A23187 at 0.1 μM for 2 min was found to induce sperm capacitation and acrosome reaction effectively ( Matsukawa et al., J. Reprod. Dev., 2002 ).( 4 ) Intracytoplasmic injection of stallion spermatozoa following the A23187 treatment resulted in the normal fertilization rate of 65 % and successful production of blastocyst-stage embryos in vitro ( Matsukawa et al., Poster presentation at IETS meeting, 2002 ).