Establishment of a system for in vitro fertilization and in vitro culture of bovine oocytes using the synthetic oviduct fluid medium

合成输卵管液体培养基体外受精和体外培养牛卵母细胞系统的建立

基本信息

批准号：
09660295
负责人：
TAKAHASHI Yoshiyuki
金额：
$ 2.05万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660295/
关键词：
cattle oocytes in vitro fertilization in vitro culture synthetic oviduct fluid medium 合成卵管液

项目摘要

This study was performed to establish a reliable system for the in vitro production of bovine embryos using in vitro matured, fertilized and cultured ovarian oocytes. Factors that affect the in vitro fertilization and subsequent embryonic development were investigated using the synthetic oviduct fluid (SOF) as a basic culture medium. Results revealed that reducing the oxygen concentration from 20 to 5% during in vitro insemination did not affect the fertilization rate, but improved the subsequent embryonic development in vitro. Optimal concentrations of glucose in the in vitro insemination medium depend on the composition of the basic medium for insemination. Glucose at 0 to 1 mM gave higher fertilization rate when SOF was used, while addition of 5 to 13.9 mM glucose showed higher fertilization rate when BO medium was used. It is worthy to note that SOF requires less concentration of sperm to obtain high and reliable fertilization rate as compared to BO medium. Subsequent experiments for embryo culture demonstrated that amino acid supplementation, especially addition of glycine and/or taurine improved the embryonic development to the blastocyst stage. Amino acids added to the culture medium degraded, and produced ammonium ion. However, an increase in the ammonium concentration did not affect the embryonic development under the present culture conditions. Results also revealed that both insulin and insulin-like growth factor-I (IGF-I) stimulate the in vitro development of bovine embryos by acting synergistically with amino acids through IGF-I receptor. Optimal concentration of glucose during in vitro culture of bovine embryos was around 1 mM when zygotes were cultured throughout 6 days to the blastocyst stage, although glucose requirement for bovine embryos changed between 8- to 16-cell stages. The in vitro culture system established in the present study was successfully applied for the production of clone embryos using nuclear transplantation technique.

进行了这项研究以建立一个可靠的系统，用于使用体外成熟，受精和培养的卵巢卵母细胞体外生产牛胚胎。使用合成卵巢液（SOF）作为基本培养基研究了影响体外受精和随后的胚胎发育的因素。结果表明，在体外授精过程中，将氧气浓度从20％降低到5％不会影响受精率，而是改善了随后的体外胚胎发育。体外授精培养基中葡萄糖的最佳浓度取决于基本培养基的成分。当使用SOF时，0至1 mm处的葡萄糖给出了更高的受精率，而使用BO培养基时，添加5至13.9 mm的葡萄糖显示出更高的受精率。值得注意的是，与BO培养基相比，SOF需要更少的精子浓度才能获得高可靠的受精率。随后进行胚胎培养的实验表明，补充氨基酸，尤其是甘氨酸和/或牛磺酸的添加将胚胎发育改善到胚泡阶段。将氨基酸添加到降解的培养基中，并产生铵离子。但是，在当前培养条件下，铵浓度的增加并不影响胚胎发育。结果还表明，胰岛素和胰岛素样生长因子-I（IGF-I）均通过通过IGF-I受体协同作用于氨基酸来刺激牛胚胎的体外发育。在整个6天培养合子阶段时，在牛胚胎体外培养期间，葡萄糖的最佳浓度约为1 mm，尽管牛胚胎的葡萄糖需求在8至16细胞阶段发生变化。本研究中建立的体外培养系统成功地用于使用核移植技术生产克隆胚胎。