应用猪冷冻精液进行卵子胞质单精子注射生产胚胎

发布时间：2018-09-10 06:48

【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(P0.05)。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。
[Abstract]:In recent years, the scientific research and animal production based on porcine (Sus scrofa) embryos have developed rapidly, and the demand for embryos has increased. However, pig embryos and gametes are immature in cryopreservation, and the rate of development of IVF embryos is low, and polysperm fertilization is easy to occur. Therefore, it is necessary to establish a rapid and efficient technique system for the production of healthy embryos in vitro. In this study, we integrated and optimized porcine semen freezing, oocyte maturation and oocyte cytoplasmic sperm injection (intracytoplasmic sperm injection,ICSI) techniques, and established a set of in vitro efficient production of porcine embryos. Firstly, a stable maturation system of porcine oocytes was established. The cleavage rate and blastocyst development rate of parthenogenetic activated embryos were 79.51% and 32.30%, respectively. The cleavage rate of embryo was 49.89% and 28.30%, and the blastocyst development rate was 31.44% and 10.55%, respectively. The cleavage rate and blastocyst development rate of IVF with fresh semen were significantly higher than that of frozen semen (P0.05). Further ICSI test showed that cleavage rate and blastocyst development rate of porcine embryos produced by oocyte cytoplasmic injection of frozen sperm in vitro were 83.00% and 23.32%, respectively, while the cleavage rate of fresh semen routine IVF fertilized eggs was 83.00% and 23.32%, respectively. The blastocyst development rate was 49.89% and the blastocyst development rate of 31.44%.ICSI embryos was lower than that of fresh semen IVF fertilized oocytes and parthenogenetic embryos, but the overall blastocyst rate was obtained. The in vitro production of porcine embryos by oocyte cytoplasmic injection of frozen spermatozoa was significantly higher than that of fresh semen IVF. The research provides an effective and inexpensive preparation system for pig healthy embryos for scientific research and production.
【作者单位】：北京农学院动物科技学院;中山大学孙逸仙纪念医院;中国农业大学农业生物技术国家重点实验室;
【基金】：国家自然科学基金青年科学基金(No.31601941) 国家重点基础研究发展计划(973)项目(No.2016YFA0100202) 北京市自然科学基金(No.6152004) 北京市教委一般项目(No.PXM2016_014207_000008) 北京市大学生科研训练项目(No.5046516564/003) 2015校级科研水平提高经费-高级别科研项目培育(No.1086716274)
【分类号】：S828

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