139 Effect of lycopene supplementation in maturation medium on production of reactive oxygen species and post-vitrification quality of bovine blastocysts

Abstract

Excessive production and accumulation of reactive oxygen species (ROS) may cause embryo damage associated with oxidative stress. Lycopene, a natural antioxidant, can scavenge singlet oxygen and is one of the most effective antioxidants among carotenoids. We evaluated the effects of supplementation of lycopene (antioxidant), menadione (prooxidant), and their combination during in vitro oocyte maturation on ROS generation in matured oocytes and the quality of vitrified-warmed embryos. Cumulus–oocyte complexes, collected from the slaughterhouse, were matured in groups of 60 in 500 μL of TCM-199 medium + 50 mg mL⁻¹ gentamycin + 20 ng mL⁻¹ epidermal growth factor, for 22 h at 38.5°C in 5% CO₂ in air and then supplemented with (1) 0.2 μM lycopene, (2) 5 μM menadione, (3) 0.2 μM lycopene + 5 μM menadione (L+M), or (4) not supplemented (control). Fertilization and embryo culture were performed similarly for all the groups. In the first experiment, ROS measurement (n = 236; via fluorescent microscopy) was performed in denuded, matured oocytes incubated in 5 μM CellROX® Green (ThermoFisher Scientific) for 1 h. Fluorescent intensity was measured in Image-J. In the second experiment, embryos in the blastocyst stage (n = 143) were vitrified as previously described by Ortiz-Escribano et al. (2017 Biol. Reprod. 96, 288-301). Vitrified blastocysts were then warmed and washed in decreasing concentrations of sucrose and incubated for 2 days in culture medium [50 µL of synthetic oviductal fluid (SOF) + (5 g mL⁻¹ insulin, 5 g mL⁻¹ transferrin, 5 ng mL⁻¹ selenium)]. The quality of vitrified-warmed blastocysts was assessed using a differential staining as described by Wydooghe et al. (2011 Anal. Biochem. 416, 228–230). The effects of pro- and antioxidant supplementation on oocyte fluorescent intensity and embryo quality parameters were fitted in linear mixed-effects models, and results are expressed as least squares means and standard errors. The fluorescent intensity for ROS was lower (P < 0.05) in lycopene (10.06 ± 2.92) than in menadione (16.8 ± 2.92). No differences (P > 0.05) in ROS intensity values were found among the other groups [control (13.5 ± 2.92) and L+M (13.7 ± 2.90)]. Total cell number (TCN) was similar (P > 0.05) in lycopene (153 ± 2.95), L+M (143 ± 4.59), and control (145 ± 3.67) but lower (P < 0.05) in menadione (134 ± 6.08). Lesser numbers of apoptotic cells (AC) and AC/TCN values (P < 0.05) were recorded in lycopene (4.12 ± 3.07 and 2.71 ± 2.21) compared with control (6.18 ± 3.82 and 4.31 ± 2.75), L+M (6.00 ± 4.79 and 4.22 ± 3.45), and menadione (7.75 ± 6.33 and 5.82 ± 4.56). For the remaining embryo quality parameters, no differences were found (P > 0.05). In conclusion, lycopene supplementation during in vitro oocyte maturation effectively scavenged free radicals, lowering oxidative stress and improving embryo quality post-vitrification and warming.