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

本研究旨在建立一种可靠的牛卵巢卵母细胞体外成熟、受精和培养的胚胎生产系统。以人工合成输卵管液（SOF）为基础培养液，研究了影响体外受精及胚胎发育的因素。结果表明，在体外授精过程中，将氧浓度从20%降低到5%并不影响受精率，但改善了随后的胚胎体外发育。体外授精培养基中葡萄糖的最佳浓度取决于授精基础培养基的组成。当使用SOF时，0至1 mM的葡萄糖产生较高的受精率，而当使用BO培养基时，添加5至13.9mM的葡萄糖显示较高的受精率。值得注意的是，与BO培养基相比，SOF需要更低的精子浓度以获得高且可靠的受精率。随后的胚胎培养实验表明，补充氨基酸，特别是添加甘氨酸和/或牛磺酸，可以改善胚胎发育到囊胚阶段。添加到培养基中的氨基酸降解，并产生铵离子。然而，在本培养条件下，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）通过IGF-I受体与氨基酸协同作用，促进牛胚胎的体外发育。牛胚胎体外培养过程中葡萄糖的最佳浓度为1 mM左右，受精卵培养6天至囊胚期，尽管牛胚胎对葡萄糖的需求在8- 16细胞期之间变化。本研究建立的体外培养体系已成功应用于核移植技术生产克隆胚胎。

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.