Abstract

Introduction 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 (COCs) with a nitric oxide donor (SNP) on in vitro fertilization (IVF), embryo development and relative gene expression related with cell redox state regulation. Methods Mature oocytes (COCs) were incubated during 1 hour with different concentration of sodium nitroprusside, SNP (Control without SNP, 10–6M, 10–5M and 10–4M SNP) in maturation media at 38.5°C and 5% CO2, 5% O2 in humidified atmosphere. For IVF procedure, oocytes of each treatment and sperm of one bull were co-incubated for 18–20 hours at 38.5°C and 5% CO2, 5% O2. Presumptive zygotes were separately cultured in KSOM-FBS media until day 7 under mineral oil at 38.5°C and 5% CO2, 5% O2 and 90% N2 in a humidified atmosphere. Relative gene expression analyzes of 3 pools of embryos for each treatment were evaluated after RNA extraction and cDNA synthesis in a stratagene MX 3000P real time equipment with the Agilent’s qPCR software MX pro 4.1 version. Results Cleavage percentage at 72 hours post-insemination were significantly different between control group and 10–4M SNP (82 ± 8.4% vs. 77 ± 7.1%, respectively) and between 10–5M and 10–4M SNP (84.9 ± 4.1% vs. 77 ± 7.1%, respectively). Blastocyst percentage at 7day culture were significantly different between control group and 10–4M SNP (34.1 ± 7.8 % vs. 26.2 ± 4.9%, respectively). Embryo development between control group and treatments were similar within early, expanded and hatched blastocyst percentage. Embryo relative gene expression shown that some genes were up-regulated (iNOS, PRDX5, HSP70) or down-regulated (iNOS, nNOS, HSP90, HIF1A, BCL2A) after SNP exposure. Conclusion Oocytes incubated with high concentration of SNP shown lower cleavage percentage and lower blastocyst percentage, being deleterious for in vitro embryo production in bovine. SNP modulates gene expression pattern on 7 day cultured expanded blastocyst, and these changes are associated with stress tolerance induction process. Grants: Supported by FONDECYT 1130888, CONICYT, Gobierno de Chile, Chile.