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In Vitro culture of bovine early embryos using chemically defined media.

使用化学成分确定的培养基对牛早期胚胎进行体外培养。

基本信息

批准号：
04660331
负责人：
TAKAHASHI Yoshiyuki
金额：
$ 1.34万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

To establish a reliable bovine embryo culture system without co-culture of somatic cells, bovine one-cell embryos derived from in vitro fertilization were cultured in chemically defined media after removal of cumulus cells, and factors affecting embryonic development were investigated. Modified synthetic oviductal fluid medium containing polyvinyl alcohol instead of BSA (mSOF) was used as a basal medium. Results from the present study are summarized as follows.(1) Supplementation of 20 amino acids contained in basal medium Eagle's (BME) and minimum essential medium (MEM) improved development to the blastocyst stage dramatically. In addition to these 20 amino acids, supplementation of taurine and/or increasing the dose of glycine further improved the embryonic development in vitro.(2) In the presence of amino acids and/or glucose, supplementation of insulin or insulin-like growth factor-I (IGF-I) enhanced early embryonic development with an increase in the cell numbers in blastocysts.(3) Oxygen concentration in the gas atmosphere was one of the most critical factors which affect embryonic development invitro. Under a gas atmosphere of 5% oxygen, embryonic development in mSOF supplemented with amino acids and insulin without somatic cells was superior to that of co-culture with bovine oviductal epitherial cells in TCM-199+FCS.(4) Quality of water for preparing culture media also affected the embryonic development in vitro. The embryonic development in a chemically defined medium prepared with Milli-Q water was superior to those for distillled water and ion-exchanged water.The present study clearly demonstrates that bovine one-cell embryos can be successfully cultured up to the blastocyst stage under a gas atmosphere of 5% oxygen using a chemically defined medium prepared with pure water and supplemented with amino acids and insulin or IGF-I.

为建立一种可靠的无体细胞共培养的牛胚胎培养体系，将体外受精获得的牛单细胞胚胎去除卵丘细胞后，在化学成分确定的培养基中进行培养，并对影响胚胎发育的因素进行研究。使用含有聚乙烯醇代替BSA的改良合成输卵管液培养基（mSOF）作为基础培养基。本研究的结果总结如下。(1)在基础培养基Eagle's（BME）和最低必需培养基（MEM）中添加20种氨基酸可显著提高囊胚发育。除了这20种氨基酸外，补充牛磺酸和/或增加甘氨酸的剂量进一步改善了体外胚胎发育。(2)在存在氨基酸和/或葡萄糖的情况下，补充胰岛素或胰岛素样生长因子-I（IGF-I）增强早期胚胎发育，囊胚中细胞数量增加。(3)气体环境中的氧浓度是影响胚胎体外发育的重要因素之一。在含5%氧气的气氛中，在不添加体细胞的情况下，添加氨基酸和胰岛素的mSOF中胚胎发育的效果优于TCM-199 + FCS中与牛输卵管上皮细胞共培养的胚胎发育的上级效果。(4)培养液的水质也影响胚胎的体外发育。用Milli-Q水配制的化学成分确定的培养基中的胚胎发育优于蒸馏水和离子交换水。本研究清楚地表明，牛单细胞胚胎可以成功地培养到囊胚期，在5%的氧气气氛下，使用纯水配制的化学成分确定的培养基，并补充氨基酸和胰岛素或IGF-I。