229 SHORT-TERM EXPOSURE OF MATURE OOCYTES TO A NITRIC OXIDE DONOR FOR INDUCING OXIDATIVE STRESS RESISTANCE ON IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

Recent studies have shown that short-term exposure of oocytes to stressors such as hydrostatic pressure, osmotic stress, and oxidative stress might induce stress tolerance in embryos. In this research we studied the effect of short-term exposure of bovine in vitro-matured cumulus-oocyte complexes (COC) with a nitric oxide donor (SNP) on IVF, embryo development, embryo quality, and relative gene expression related to cell redox state regulation. The COC were selected and matured in TCM 199 supplemented with 10% inactivated FBS, 6 mg mL^–1 of LH, 6 mg mL^–1 of FSH, 1 mg mL^–1 of oestradiol, and 0.2 mmol of pyruvate and then incubated for 22 to 24 h at 38.5°C in 5% CO₂ in a humidified atmosphere (n = 12). Before IVF, mature COC were incubated during 1 h with different concentration of sodium nitroprusside, SNP (control without SNP, 10^–6 M, 10^–5 M, and 10^–4 M SNP) in maturation media at 38.5°C and 5% CO₂ in a humidified atmosphere. For IVF procedure, oocytes of each treatment and sperm of one bull were co-incubated for 18 to 20 h at 38.5°C and 5% CO₂. Presumptive zygotes were separately cultured until Day 7 under mineral oil at 38.5°C and 5% CO₂, 5%O₂, and 90% N₂ in a humidified atmosphere. Embryo quality was analysed by staining with CDX2 antibody for trophectoderm cells and compared with total embryo cells stained with Hoechst 33342. Relative gene expression for each treatment were evaluated after RNA extraction and cDNA synthesis in Stratagene MX 3000P real-time equipment with Agilent qPCR software MX pro 4.1 version. Differences between experimental groups (n = 12) were measured using a one-way ANOVA test in the STATGRAPHICS plus 5.1 version software. P < 0.05 was considered statistically significant. Cleavage percentage at 72 h post-insemination was significantly different between the control and 10^–4 M SNP group (82 ± 8.4% v. 77 ± 7.1%, respectively) and between 10^–5 M and 10^–4 M SNP group (84.9 ± 4.1% v. 77 ± 7.1%, respectively). Blastocyst percentage at 7 days of culture was significantly different between control and 10^–4 M SNP group (34.1 ± 7.8% v. 26.2 ± 4.9%, respectively). Embryo development between control group and treatments was similar within early, expanded, and hatched blastocyst percentage. Embryo quality of expanded blastocyst was similar between control group and treatments (ICM: TE). No significant differences in gene expression after SNP exposure was observed (iNOS, eNOS, nNOS, PRDX5, HSP70, HSP90, HIF1A, BCL2A). Oocytes incubated with a high concentration of SNP showed lower cleavage and blastocyst rates, showing that this treatment was deleterious for in vitro embryo production in bovine. However, there were no significant differences on embryo quality assessed by ICM : TE ratio and/or in gene expression pattern of 7-day cultured expanded blastocysts.