权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

牛、马去核卵母细胞保存及核移植的研究

基本信息

批准号：
12660252
负责人：
HANADA Akira
金额：
$ 2.3万
依托单位：
Shinshu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660252/
关键词：
NUCLEAR TRANSFER CLONE CRYOPRESERVATION ENUCLEATION LAA ETHYLENE GLYCOL CATTLE HORSES

项目摘要

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 ).

去核卵母细胞可以支持移植核的重编程和核移植产生的受精卵的发育。本研究的首要目的是提高牛的克隆效率，通过发展冷冻保存无核卵母细胞的方法。结果表明：(1)在去核卵母细胞（IVM、去核、活化）制备过程中，在培养基中添加亚油酸-白蛋白，可提高体外培养的牛胚胎的冷冻耐受性（Hochi et al.， J. Vet.）。医学科学。， 2000)。(2)将体外成熟的牛卵母细胞以3000 - 5000℃/分钟的冷却速度玻璃化，从常规试管婴儿（IVF）的角度来看，没有丧失活力（Hochi et al., Cryobiology, 2001）。本研究的第二个目的是建立体外生产马！胚胎，为将来通过核移植克隆马取得进展。(3)通过对无带仓鼠卵母细胞的精子渗透实验，发现离子载体A23187在0.1 μM下作用2 min可有效诱导精子获能和顶体反应（Matsukawa et al.， J. repd .）。Dev., 2002)。(4)在A23187处理后，将种马精子胞浆内注射，正常受精率为65%，体外成功产生囊胚期胚胎（Matsukawa et al.，在IETS会议上的海报展示，2002）。