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.

为了建立一个无需合并体细胞培养的可靠的牛胚胎培养系统，在去除积云细胞后，在化学定义的培养基中培养了牛的一细胞胚胎，并研究了影响胚胎发育的因素。经过改性的含聚乙烯醇代替BSA（MSOF）的合成产卵培养基被用作基础培养基。（1）补充了基础培养基（BME）中包含的20种氨基酸的结果。除了这20种氨基酸外，补充牛磺酸和/或增加甘氨酸剂量进一步改善了体外的胚胎发育。（2）在存在氨基酸和/或葡萄糖的情况下，补充了胰岛素或胰岛素样生长因子-i（IGF-I）增强了早期胚胎发育，胚泡中细胞数量的增加。（3）气体大气中的氧浓度是影响胚胎发育Invitro的最关键因素之一。在5％氧气的气体大气下，补充氨基酸和没有体细胞的胰岛素的MSOF中的胚胎发育优于与TCM-199+FCS中的牛卵形卵巢e骨细胞共培养。培养基还影响了体外的胚胎发育。用Milli-Q水制备的化学定义培养基中的胚胎发育优于蒸馏水和离子交换水的胚胎发育。目前的研究清楚地表明，牛一单细胞胚胎可以成功地培养到胚泡阶段，直至胚泡阶段使用纯水制备的化学定义培养基，并补充氨基酸和胰岛素或IGF-I的气氛。