| In order to consummate culture system in vitro of zona-free embryo, increase the developmental rate and quality of zona-free embryo culturing in vitro, this experiment studied four basic medium (KSOM,CZB,M16 and M199)'s effects on the development of zona-free embryo culturing in vitro through statistical analysis of 2-cell,4-cell,morulaes and blastocysts developmental rate and blastocysts cell count, in order to select the best nutrient medium, at the same time, examined after adding fetal calf serum and seralbumin how that affect on the all index to finally decide the dose. We took the result of selection as the basal medium, which were respectively added into the different density of Trehalose and Hypotaurine, then studied their effects on mouse zona-free embryo culturing in vitro. Chose 2-cell and blastocysts stage culture solution of zona-free embryo, measured the activities of superoxide dismutase(SOD), glutathion peroxidase(GSH-Px) and the content of Malondialdehyde(MDA), studied the antioxidation of trehalose and hypotaurine on zona-free embryo culturing in vitro. Specific test results are showed as follow :Put the mouse zona-free embryo into KSOM,CZB,M16 and M199 nutrient medium, added the different dose of fetal calf serum and seralbumin respectively and proceeded many time culturing selection, after combining all the indexes, we eventually decided to take KSOM nutrient medium which was added to 20% fetal calf serum as the standard medium in this study, its blastocysts rate was 31.58%.Trehalose could promote the development in vitro of mouse zona-free embryo in some degree, especially the effects on morulaes and blastocysts rate compared with control group were extremely significant(P<0.01), among them the 4μg/ml group was the most efficient one, morulaes, blastocysts rate and blastocysts recovery rate were respectively 44.97%,31.95% and 84.36%.The activity of SOD and GSH-Px in embryo culture solution which had been added trehalose would decrease gradually as culturing time passing by, while the content of MDA increased. Compared with control group the activity of SOD and GSH-Px in all treatment groups significantly increased(P<0.05), the difference between the treatment groups were not significant(P>0.05), 2-cell group (added 6μg/ml trehalose) and blastocysts groups (the dose of trehalose were respectively 4, 6 and 8μg/ml) had extremely significant difference with control group(P<0.01). The content of MDA in all groups of 2-cell stage did not have significant change compared to control group (P>0.05), blastocysts groups (the dose of trehalose were respectively 6 and 8μg/ml) significantly decreased compared with control group (P<0.05).Adding a certain density of hypotaurine into KSOM nutrient medium which contained 20% fetal calf serum could increase developmental rate of mouse zona-free embryo in vitro. 2-cell,4-cell,morulaes and blastocysts rate of 6μg/ml hypotaurine group were respectively 86.61%,76.53%,44.99% and 32.21%, and they were all higher than the other test groups(P<0.01). The rate of blastocysts recovery was the highest which reached 81.25% in 4μg/ml hypotaurine group, compared with the other groups its difference was significant(P<0.05). The promotive effect of hypotaurine would decrease following increasing density, compared with control group, developmental rate of 8μg/ml group's all developmental stages had no significant difference (P>0.05), but it also did not appear inhibition phenomenon.The activity of SOD and GSH-Px in embryo culture solution which had been added hypotaurine would decrease gradually as culturing time passing by, while the content of MDA increased. Compared with control group, the activity of SOD and GSH-Px in all treatment groups significantly increased (P<0.05), but the differences between all treatment groups were not significant(P>0.05). The activity of SOD and GSH-Px in blastocysts stage of 6 and 8μg/ml had extremely significant difference with control group(P<0.01); The content of MDA in 2-cell stage's all treatment groups had no significant difference with control group, but all treatment groups of blastocysts stage significantly decreased compared with control group(P<0.05).
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