Aged during oocytes development in ovary [38]. Gardiner and Reed [45] observed that the GSH content of mouse embryos decreases continuously from PubMed ID: unfertilized oocytes to the blastocyst stage. Thus, it is likely that the observed decrease in GSH content could be due to a depletion of GSH stored in the oocytes combined with an inability of the embryo to synthesize GSH [44,45]. It has been reported that glucose induced cell death through a free radicals-mediated mechanism [16]. Other study demonstrated that the hyperglycemia condition, through the generation of ROS, might lead to the produc-Page 10 of(page number not for citation purposes)Reproductive Biology and Endocrinology 2006, 4: of ceramide and activation of apoptosis [46]. An exposure of bovine embryos to high BMS-214662 site concentrations of glucose (10?0 mM) during development from the onecell to the blastocyst stage resulted in a decrease in the total number of cells in the blastocysts and an increase in the frequency of apoptotic cells [47]. Wongsrikeo et al. [48] reported that replacement of glucose with fructose enhanced embryo quality by increasing the total cell number of blastocysts and decreasing the index DNA fragmented nucleus in blastocysts. In this study, a reduced total cell numbers in blastocysts were observed when the embryos were cultured with glucose for the first 2 days of culture at any concentration as compared with that in the Pyr-Lac group. However, we were unable to detect a significant increase in the number of DNA-fragmented nuclei in blastocysts cultured with increased concentrations of glucose (except for Gluc-20 group that developed under 5 oxygen tension). It has been reported that the response of the cell against oxidative stress can differ widely depending on the intensity of the stress and its duration, and this response varies from the stimulation of cell proliferation to cell arrest or to cell death by apoptosis or necrosis [49]. Therefore, our results suggested that increasing glucoseinduced ROS in Day 1 embryos cultured with glucose may have contributed only to cell cycle arrest, as evidenced by reduction of the cleavage rate and blastocyst formation. The oxygen environment could influence the metabolism [50,51] and the oxidation-reduction potential [32,52] in the embryo. Decreasing the oxygen concentration during culture in vitro has been reported to be beneficial for mammalian embryo development [53-55]. Our results show that, except for the Gluc-20 group, the total number of cells in blastocysts developed under 20 oxygen tension with any energy substrate was lower than that in blastocysts developed under 5 oxygen tension. Although no interaction was found between the oxygen concentration and the energy supplement groups (except for the Pyr-Lac group), blastocyst formation rates of embryos developed under 5 oxygen tension were slightly higher than those in embryos cultured under 20 oxygen tension. These results were consistent with our previous findings [30] and those of other recent reports [53-55] that indicate the beneficial effects of a low oxygen tension during culture in vitro in improving embryonic development and embryo quality. In addition, as mentioned above, since the oxygen effect was suggested is due to an alteration in oxidation-reduction potential in early developmental stages of embryos; at which their development is most dependent on the NAD+:NADH ratio as well as pyruvate:lactate ratio [32,52], thus.