In vitro embryo development can be affected by many different factors, such as incubation atmosphere, which can cause oxidative stress. In this study the effect of 2 gas mixtures and the addition of the antioxidant agent N-acetylcysteine (NAC) on porcine embryos produced in vitro and lipoperoxidation (LPO) were evaluated. Ovaries collected from slaughtered gilts were aspirated to obtain oocytes, to be matured and fertilized in vitro. In experiment 1 (E1), putative zygotes were cultured in NCSU-23 medium at 38.5°C in humidified air and randomly allocated to each of the following atmospheres: conventional atmosphere (CA: 5% CO2 in air) and modified atmosphere (MA: 5% CO2, 5% O2, 90% N2). In experiment 2 (E2), zygotes were incubated in CA or MA and cultured in NCSU-23 with 2.5 mM NAC. In experiment 3 (E3), embryos produced in E1 and E2 were used to evaluate LPO with the TBARS method: embryos were sonicated, then 300 μL of distilled water and 680 μL of thiobarbituric acid were added and heated at 95°C for 20 min in a water bath. The resulting reaction was read by spectrophotometry at 532 nm. Final concentration was expressed in micromoles of TBARS/embryo (μMTE). Five days after IVF, embryos were evaluated to determine their development to the morula stage. Data from each experiment were transformed using arcsine square root function before analysis, and means were compared using ANOVA. For E1, 954 oocytes were used, 475 for CA and 479 for MA, in 5 replicates. The percentage of cleaved embryos was significantly higher in MA (300/479, 63%) compared with CA (245/475, 52%) (P < 0.05). The percentage of morulae obtained was higher in MA (127/479, 27%) than in CA (80/475, 17%) (P < 0.05). In E2, 936 oocytes were used, 470 for CA NAC and 466 for MA NAC, in 5 replicates. There were no differences in the percentages of cleaved embryos in MA NAC (250/466, 54%) compared with CA NAC (243/470, 52%), and morulae in MA NAC (86/466, 18%) compared with CA NAC (81/470, 17%) (P > 0.05). In E3, during the first 24 h of culture the embryos in CA NAC had lower LPO concentrations (0.029μMTE) (P < 0.05) than in CA (0.059μMTE), MA (0.057μMTE), and MA NAC (0.054μMTE). In morulae, a higher concentration of LPO was observed in the CA group (0.103μMTE) compared with MA (0.083μMTE), CA NAC (0.085μMTE), and MA NAC (0.081μMTE). A correlation analysis between morulae development and LPO in CA and MA, with or without NAC, did not show a significant relationship (r = 0.20; P > 0.05). It is concluded that MA improves the development of in vitro-derived porcine embryos; that NAC treatment did not cause differences in any stage of embryo development, probably meaning that the embryo has its own defensive mechanisms against LPO, and therefore that NAC is not a good antioxidant for porcine embryos, or that it does not work well at the dosage and/or in the culture medium used; that the embryos exposed to CA and MA showed higher LPO that the groups with NAC, indicating that the addition of NAC tends to decrease LPO; and that LPO could be a good indicator of embryo viability.
We thank CONACYT Mexico for the graduate student’s scholarship.
An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes