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 invitro 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 500μL of TCM-199 medium+50mg mL−1 gentamycin+20ng mL−1 epidermal growth factor, for 22h at 38.5°C in 5% CO2 in air and then supplemented with (1) 0.2μM lycopene, (2) 5μM menadione, (3) 0.2μM lycopene+5μM 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 5μM CellROX® Green (ThermoFisher Scientific) for 1h. 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 [50µL of synthetic oviductal fluid (SOF)+(5g mL−1 insulin, 5g mL−1 transferrin, 5ng 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 invitro oocyte maturation effectively scavenged free radicals, lowering oxidative stress and improving embryo quality post-vitrification and warming.

Lycopene Improves Maturation Rate and Antioxidant Status of in vitro Matured Mouse Oocytes

“…Supplementation of bovine COCs with 0.2µM lycopene during their IVM at 40.5°C resulted in higher maturation rate than those matured at the same temperature with no lycopene (Sidi et al 2021). Remarkably, maturation rate of heat-stressed lycopene treated oocytes was comparable to those of matured at standard laboratory temperature (38.5°C) (Sidi et al 2021). Similar trends in oocyte maturation rates under elevated temperature have been observed by the present work, suggesting a conserved effect of lycopene on heat-challenged oocytes.…”

“…Protective effect of lycopene on thermally challenged oocytes has been suggested (Kang et al 2021;Sidi et al 2021). Supplementation of bovine COCs with 0.2µM lycopene during their IVM at 40.5°C resulted in higher maturation rate than those matured at the same temperature with no lycopene (Sidi et al 2021). Remarkably, maturation rate of heat-stressed lycopene treated oocytes was comparable to those of matured at standard laboratory temperature (38.5°C) (Sidi et al 2021).…”

“…Oocyte response to heat shock usually involves generation of high amounts of ROS which were reduced by administration of antioxidants both in vitro and in vivo (Lawrence et al 2004;Roth et al 2008). Protective effect of lycopene on thermally challenged oocytes has been suggested (Kang et al 2021;Sidi et al 2021). Supplementation of bovine COCs with 0.2µM lycopene during their IVM at 40.5°C resulted in higher maturation rate than those matured at the same temperature with no lycopene (Sidi et al 2021).…”