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

本研究旨在建立一套可靠的利用体外成熟、受精和培养的卵巢卵母细胞体外生产牛胚胎的体系。以人工合成输卵管液(SOF)为基本培养液，研究了影响体外受精和胚胎发育的因素。结果表明，在体外受精过程中，将氧气浓度从20%降至5%不会影响受精率，但会促进后续的体外胚胎发育。体外受精培养液中葡萄糖的最佳浓度取决于受精基础培养液的组成。当使用SOF时，0~1 mM葡萄糖的受精率较高，而使用BO培养液时，添加5~13.9 mM葡萄糖的受精率较高。值得注意的是，与BO液相比，SOF需要更少的精子浓度才能获得高而可靠的受精率。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，可以促进胚胎发育到囊胚期。添加到培养基中的氨基酸被降解，并产生铵离子。然而，在目前的培养条件下，氨浓度的增加并不影响胚胎的发育。结果还表明，胰岛素和胰岛素样生长因子-I(IGF-I)都通过IGF-I受体与氨基酸协同作用，促进牛胚胎的体外发育。当受精卵培养6天至囊胚期时，牛胚胎体外培养的最适葡萄糖浓度为1 mM左右，尽管牛胚胎的葡萄糖需要量在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克隆胚胎的生产。

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.