247 EFFECTS OF SILDENAFIL OR SNAP SUPPLEMENTATION, OR BOTH, DURING IN VITRO MATURATION ON LIPID AND REACTIVE OXYGEN SPECIES ACCUMULATION IN BOVINE OOCYTES AND EMBRYOS

Abstract

Previous studies have demonstrated that a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillmaine, SNAP) and a phosphodiesterase inhibitor (Sildenafil, SILD) delay the meiotic resumption of oocytes removed from the follicular environment, and therefore could be used to improve the quality of in vitro-matured (IVM) oocytes. However, it has been reported that SILD-treated cells have increased lipid metabolism and that NO supplementation can modulate the oxidative stress. This study aims to determine the effects of SNAP or SILD supplementation, or both, during IVM on embryo developmental rates, on lipid accumulation of IVM oocytes and on reactive oxygen species (ROS) and lipid accumulation of embryos derived from IVM oocytes. Bovine oocytes were cultured in TCM199 containing 1.0 μg mL^–1 of FSH, 50 μg mL^–1 of hCG, 1.0 μg mL^–1 of oestradiol, 0.2 mM pyruvate, 83.4 μg mL^–1 of amikacin, 10% FBS (control group; GCONT), supplemented with 10 µM SILD (GSILD), 0.1 µM SNAP (GSNAP) or both (GS+S). After 24 h of IVM, matured oocytes were assessed for lipid quantification (approximately 49 per group) or used for in vitro embryo production (IVP; approximately 340 oocytes per group). For lipid quantification, denuded oocytes were fixed with 5% triton in 4% paraformaldehyde (PFA) for 30 min and stained with 1 ng mL^–1 of Nile Red for 30 min. Embryo lipid analyses (approximately 55 per group) were performed as described for oocytes. For ROS assessment (approximately 58 per group), IVP embryos were stained with 10 µM of H₂DFFDA for 1 h and fixed for 30 min in 4% PFA. Stained oocyte and embryo assessments were performed on epifluorescence microscopy, and captured images were analysed on ImageJ (NIH, Bethesda, MD, USA) to quantify the fluorescence intensity (f.i). Statistical analyses were performed with data from 3 replicates for oocytes and 4 for embryos: statistical differences were assessed for lipid and ROS quantity and development rates by split-plot ANOVA. Variables considered in the model were SNAP (presence/absence) and SILD (presence/absence). Means were compared by Student's t at P < 0.05. Regarding oocyte lipid accumulation, groups with SILD (GSILD and GS+S) presented higher lipid quantity (f.i: 52.11 and 47.24, respectively) compared with GCONT and GSNAP (f.i: 38.86 and 41.86, respectively). Supplementation during IVM did not affect development rates (cleavage of 88.1, 88.2, 88.8, and 89.5% and blastocyst rates of 41.2, 38.6, 40, and 41.2% for GCONT, GSNAP, GSILD, and GS+S, respectively). Regarding embryo lipid quantity, similar to oocyte results, SILD groups (GSILD and GS+S) presented higher lipid accumulation (f.i: 68.9 and 68.5, respectively) compared with GCONT (f.i: 55.8) and GSNAP (f.i: 58.9). Considering embryo ROS quantity, GCONT (f.i: 35.9) and GS+S (f.i: 34.2) had the highest levels; however, GS+S did not differ from GSNAP (f.i: 32.85), which was similar to GSILD (f.i: 30.4). In conclusion, SILD had a negative effect on lipid accumulation, which could be due to increased lipid synthesis without increasing lipid oxidation because no increase of embryo ROS levels was observed.