Our results demonstrate that addition of glucose at any concentration during the first 2 days of IVC permitted some of the embryos to develop to the blastocyst stage under both oxygen tensions. However, the provision of only glucose as an energy substrate in IVC media failed to give rates of blastocyst development comparable to that achieved in the pyruvate-lactate-containing medium. Culturing of porcine embryos at early developmental stages with pyruvate and lactate in the present study also resulted in an increase of total number of cells in blastocysts developed under 5% oxygen tension. Taken together, these results indicate that pyruvate and lactate appear to be important energy substrates for both early embryonic development in vitro and improvement in the number of cells in the blastocyst. Our observation confirms the previous reports, which reported that pyruvate and lactate seem to be predominant energy substrates for the first cleavage division for porcine embryos [23, 31]. However, there were evidences that concentration of pyruvate and lactate when both were present in the medium could affect embryo development [32, 33], thus the correct ratio of pyruvate to lactate should be noted. Because the ratio is important for maintenance of the intracellular NAD+:NADH ratio and redox equilibrium, thus regulating the oxidation-reduction equilibrium between the cytoplasm and mitocondria [32, 34]. In this study, culturing of early porcine embryos in medium with 0.17 mM pyruvate and 2.73 mM lactate for the first 2 days of culture may provide the embryos with more suitable conditions for cellular oxidation reduction equilibrium, resulting in viable embryo growth and development.
In this study, we found that the developmental ability of embryos exposed to 1.5 mM glucose was lower than those of embryos in the other glucose groups. In contrast, when the concentration of glucose was increased to 3.5 mM, a significant increase in blastocyst formation was observed under 5% oxygen tension, and a slight increase was observed under 20% oxygen tension. The rate, however, did not differ from that of Gluc-5.5 group. These results indicate that the efforts to improve blastocyst formation by culturing the embryos in media with glucose at concentrations lower than 5.5 mM was found to be ineffective. At a concentration of 1.5 mM glucose, the necessary energy substrates may not have been present in sufficient concentrations for development of the embryos, resulting in the failure of the embryos to develop to the blastocyst stage. In addition, we also found that the developmental rates of embryos in the Gluc-10 and -20 groups, regardless of the oxygen tension, did not differ from those in other glucose groups, except for Gluc-1.5 group. These results suggest that the concentration of glucose in the medium that can be used by the Day 1–2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development. The lack of response to the high glucose concentration in this study could be explained as the limited ability of early porcine embryos to utilize glucose  or the embryos use only a fraction of the glucose that they take up . It is known that metabolic activity and substrates preferences of embryos appear to change between early and late cleavages with elevated glucose and oxygen consumption as they approach cavitation . It has been reported that the optimal concentration of glucose varies according to the stage of development [36, 37]. In pigs, the first marked increase in glucose metabolism occurs at or about the time of activation of the embryonic genome [38, 15]. Therefore the susceptibility of the embryos exposed to the high glucose appears to be depended on the developmental stage.
In this study, we found that H2O2 level of Day 1 embryos in the Pyr-Lac group embryos at either oxygen tension was lower than that at any of the glucose concentrations tested. Generation of ROS induced by glucose utilization was assumed to be caused by the activation of NADPH oxidase, an enzyme that catalyzes the oxidation of NADPH, generates NADP that serves as a coenzyme of the oxidative arm of the pentose phosphate pathway (PPP) [4, 39–42]. Production of superoxide anion and H2O2 via NADPH oxidase has been described on a rabbit blastocyst surface , and the incubation of mouse embryos with an inhibitor of NADPH oxidase induces a dose-dependent reduction in H2O2 production . Since activity of PPP was higher at zygotes and embryos at the 2-cell stage compared with later developmental stages [9, 11], thus enhanced ROS production in embryos cultured with glucose in this study may be associated with the glucose utilization by the early developmental stage of porcine embryos through the pentose phosphate pathway (PPP). Taken together, results from the present study indicate that at least a part of glucose toxicity may be caused by the formation of ROS in Day 1 embryos via glucose metabolism through PPP, resulting in a reduction of both cleavage (data not shown) and blastocyst formation rates. On the other hand, NADPH generated as a result of the PPP activity is also reported to act for the reduction of intracellular GSH, a tripeptide thiol compound that plays a major role in regulating ROS concentrations within cytoplasm . Therefore, we measured the GSH content of early embryos after culturing them with glucose. However, we did not find any effects of glucose on GSH content in both Day 1 and Day 2 embryos. Moreover, we observed a decrease in GSH content of Day 1–2 embryos in all experimental groups. Although the reason was not clear at present, the most likely reason is that it may be due to the inability of early porcine embryos to synthesize GSH. This hypothesis was consistent with a previous work on mouse embryos, which found that preimplantation embryos could not synthesize GSH de novo until the morula or blastocyst stage . During early development, the embryos seem to be dependent on the stored GSH that may be packaged during oocytes development in ovary . Gardiner and Reed  observed that the GSH content of mouse embryos decreases continuously from 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 . Other study demonstrated that the hyperglycemia condition, through the generation of ROS, might lead to the production of ceramide and activation of apoptosis . An exposure of bovine embryos to high concentrations of glucose (10–30 mM) during development from the one-cell 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 . Wongsrikeo et al.  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 . Therefore, our results suggested that increasing glucose-induced 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  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 the positive effect of decreasing the oxygen concentration in this study was mostly seen when the embryo was cultured with pyruvate-lactate.