Abstract：The sealed culture of embryos is the fundation of space embryonic development study. In this special culture system, appropriate oxygen content in culture medium is essential to the development of sealed culture embryos.The purpose of present study is to research the influence of two reference gas containing different percentage of O2 and two different aeration time on mouse(Mus musculus) 2-cell embryos developmental competence in sealed culture system. We firstly aerated culture medium with high purity reference gas(5% O2, 5% CO2, 90% N2 or 7.5 % O2, 5 % CO2, 87.5 % N2) for 120 or 150 min, respectively. Control groups were the embryos cultured in medium of no gas aeration and micro-drop under conventional open condition. Peroxide accumulation in cultured embryos was detected after culture for 24 and 48 h; The expression of hypoxia-inducible factor-1α (HIF-1α) in embryos was detected after culture for 96 and 72 h. Furthermore, the blastocyst rate and hatching rate were statistical analyzed and the total cell number of blastocyst were counted after culture for 72 and 96 h. As the results showed, the peroxide production could be detected in embryos cultured for 24 h in medium aerated with 7.5% O2 for 120 and 150 min. The expression of HIF-1α was detectable after culture for 96 h in medium aerated with 5% O2 for 120 min, 150 min and 7.5% O2 for 120 min group, while the accumulation of HIF-1α in no gas group was detectable since cultered for 48 h. Mouse 2-cell embryo could grow well with a pretty high blastocyst rate and hatching rate in 5% O2 aeration 120 min, 5% O2 aeration 150 min, 7.5% O2 aeration 120 min and 7.5% O2 aeration 150 min group. The blastocyst rate of mouse 2-cell embryo cultured for 72 h with 5% O2 aeration 120 min(92.63±0.89)% was higher than the other three aeration sealed culture group, but no significant difference showed(P＞0.05). The blastocyst rate of no gas group(57.04±10.04)% was significantly lower than each aeration sealed culture group(P＜0.05). The blastocyst rate of micro-drop culture group(98.67±1.33)% was significantly higher than 7.5% O2 aeration 120 min group((87.15±2.35)%, P＜0.05). After culture for 96 h, the blastocyst and hatching rate of each aeration sealed culture group and micro-drop culture group had no significant difference(P＞0.05), but all significantly higher than no gas group(P＜0.05). The total cell number of blastocyst of each aeration sealed culture group cultured for 72 h had no significant difference(P＞0.05). The total cell number of blastocyst in 5% O2 aeration 120 min group(114.12±3.66) at 96 h was significantly higher than other aeration group and no gas group(P＜0.05), but there was no significant difference between micro-drop culture group(110.56±5.24, P＞0.05). Taken together, we could conclude that aerating embryo culture medium with high purity reference gas containing 5% O2, 5% CO2, 90% N2 for 120 ~150 min can support mouse 2-cell embryos developing to blastocyst and hatching in sealed culture system. This study determines the appropriate proportion of O2 in reference gas and appropriate aeration time for sealed culture of mouse 2-cell embryos, and perfects the sealed culture system, and accumulates data for the establishment of a suitable culture system for space embryonic development study.
