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

为了建立一种可靠的牛胚胎培养体系，将体外受精获得的牛单细胞胚胎在去除卵丘细胞后的化学成分中进行培养，并对影响胚胎发育的因素进行了研究。用含聚乙烯醇的改良合成输卵管液体培养液代替牛血清白蛋白(MSOF)。本研究结果如下：(1)基础培养液(Eagle‘s，BME)和最低必需培养液(Minimum Essential Medium，MEM)中添加20种氨基酸显著促进了囊胚期的发育。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促进了胚胎的体外发育。(2)在氨基酸和/或葡萄糖存在的情况下，补充胰岛素或胰岛素样生长因子-I(IGF-I)可以促进早期胚胎的发育，使囊胚中的细胞数量增加。(3)气体中的氧气浓度是影响体外胚胎发育的最关键因素之一。在5%氧气气氛下，添加氨基酸和胰岛素的mSOF在无体细胞的情况下的胚胎发育优于在Tcm-199+FCS中与牛输卵管上皮细胞共培养的胚胎发育。(4)配制培养液的水质也影响胚胎的体外发育。用Milli-Q水配制的化学限定培养液的胚胎发育优于蒸馏水和离子交换水。本研究清楚地表明，在5%氧气的气体气氛下，用纯水配制的添加氨基酸和胰岛素或IGF-I的化学限定培养液可以成功地培养牛单细胞胚胎直到囊胚期。

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.