应用猪冷冻精液进行卵子胞质单精子注射生产胚胎

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摘要摘要近年来以猪(Sus scrofa)胚胎为材料的科学研究和动物生产快速发展，胚胎需求量增大，而猪胚胎、配子冷冻保存不成熟，体外受精胚胎发育率低，容易出现多精受精，因此需要建立一套快捷、高效的猪体外生产健康胚胎的技术体系。本研究集成并优化了猪精液冷冻、卵母细胞成熟和卵母细胞胞质单精子注射(intracytoplasmic sperm injection, ICSI)技术，建立了一套体外高效生产猪胚胎的技术体系。首先建立了稳定的猪卵母细胞成熟体系，其孤雌激活胚胎卵裂率和囊胚发育率分别为79.51%和32.30%。然后利用新鲜精液和冷冻精液进行体外受精(in vitro fertilization, IVF)，胚胎卵裂率分别为49.89% 和28.30%，囊胚发育率分别为31.44%和10.55%。利用新鲜精液进行IVF获得的胚胎卵裂率和囊胚发育率均明显高于冷冻精液(P＜0.05)。进一步进行了ICSI实验，卵母细胞胞质注射冷冻精子单精子体外生产的猪胚胎卵裂率、囊胚发育率分别为83.00%和23.32%，而新鲜精液常规IVF受精卵卵裂率、囊胚发育率分别为49.89%和31.44%。ICSI胚胎囊胚发育率低于新鲜精液IVF受精卵和卵母细胞孤雌激活胚胎的囊胚发育率，但对于总体囊胚获得率，卵母细胞胞质注射冷冻精子体外生产猪胚胎明显高于新鲜精液IVF方法。研究工作为科研和生产所需猪健康胚胎提供了有效的、廉价的制备技术体系。

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	曹素英
	李瑞岐
	张少鹏
	师丙波
	魏晴晴
	曹安
	韩建永

关键词 ：猪, 卵母细胞, 体外受精, 卵母细胞胞质单精子注射

Abstract：Abstract Pig (Sus scrofa) is a good animal model for human disease and stem cell study, also is one of the important domestic species in the husbandry. In recent years, more and more scientific research needs pig preimplantation embryos for experiment material, due to the development and application of pig embryo engineering and related scientific research on porcine. Because of immaturation of the pig semen and embryo cryopreservation techniques, most of pig preimplantation embryos used in laboratory are obtained by in vitro fertilization (IVF) with fresh semen or frozen-thawed semen. Collection and transportation of fresh semen is inconvenient, and IVF with frozen semen is inefficient. Polyspermy is also easily to happen, which can lead to result mistake. Therefore, the establishment of a fast and efficient technological system for producing high-quality porcine embryos in vitro needs urgent resolve for scientists in the related research fields. In this study, we integrated and optimized porcine semen cryopreservation, oocyte in vitro maturation and oocyte intracytoplasmic injection (ICSI) techniques to establish a highly efficient pig embryo in vitro production system. First, we established stable system of oocyte in vitro maturation, the cleavage rate of parthenogenetic activation and blastocyst developmental rates were 79.51% and 32.30% respectively. And then, we performed IVF with fresh semen and frozen semen, embryo cleavage rates were 49.89% and 28.30% respectively, blastocyst developmental rates were 31.44% and 10.55% respectively. Embryo cleavage rate and blastocyst developmental rate of IVF with fresh semen were significantly higher than those of freeze-thawing semen (P<0.05). In addition, further test was conducted by in vitro production of pig embryo using the method of oocyte ICSI with frozen semen, the cleavage rate and blastocyst developmental rates were 83.00% and 23.32% respectively, while the embryo cleavage rate and blastocyst rates were 49.89% and 31.44% respectively by IVF with fresh semen. Blastocyst developmental rate of ICSI was lower than that of IVF and parthenogenetic activation. But for total blastocyst yield rate, it was significantly higher by oocyte ICSI than that of fresh semen IVF method. This study sets up an effective, economical system to produce embryos for scientific research and animal reproduction.

Key words： Porcine Oocyte In vitro fertilization Intracytoplasmic injection

收稿日期: 2016-12-27 出版日期: 2017-05-02

基金资助:国家自然基金青年项目;国家重点研发计划;北京市自然基金;北京市教委项目;北京市大学生科研训练项目;2015科研水平提高经费--高级别科研项目培育

通讯作者: 曹素英 E-mail: 20137602@bua.edu.cn

引用本文:

曹素英李瑞岐张少鹏师丙波魏晴晴曹安韩建永. 应用猪冷冻精液进行卵子胞质单精子注射生产胚胎 [J]. , 2017, 25(5): 813-819.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I5/813

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