145 Lycopene as a promising in vitro maturation supplement to increase bovine in vitro fertilisation efficiency

Oocyte calcium oscillation is required for cortical granule exocytosis in order to block polyspermy, and it depends on well-orchestrated interactions between mitochondria and the endoplasmic reticulum. Thus, mitochondrial quality directly affects calcium homeostasis. Lycopene, a potent mitochondria-targeted antioxidant, protects against stimulus-induced oxidative mtDNA damage, mitochondrial membrane depolarisation, and loss of mitochondrial ATP synthesis. Therefore, the aim of this study was to evaluate the effect of lycopene supplementation during bovine in vitro maturation (IVM) on fertilisation and polyspermy rate. To do so, cumulus-oocyte complexes from slaughterhouse-derived ovaries were matured in groups of 60 in 500 μL modified bicarbonate-buffered TCM-199 (supplemented with 50 μg/mL gentamycin and 20 ng/mL epidermal growth factor in 5% CO₂ in air for 22 h at 38.5°C). The maturation medium was supplemented with 3.6 μM lycopene (treatment group), 2.4% (v/v) DMSO (solvent group), or no supplementation (control group). Subsequently, oocytes were fertilised with frozen-thawed semen of a tested bull with a high polyspermy rate. Sperm mitochondria were labelled with 200 nM MitoTracker™ Green FM (ThermoFisher) after 45/90% Percoll (Cytiva) gradient selection to evaluate fertilisation. After 18 h of fertilisation, the presumed zygotes were vortexed for 3 min in 2.5 mL HEPES-TALP to remove the zona-attached cumulus and sperm cells. Afterwards, zygotes were incubated in 2.5% (w/v) pronase to completely remove the zona pellucida, fixed in 4% paraformaldehyde (v/v), and stained with Hoechst. Monospermy was considered when zygotes exhibited two pronuclei and one tail while zygotes with more than two tails were categorised as polyspermic. Zygotes in which the differences between the number of pronucleus and tails were distinct to one were excluded to avoid parthenogenic activated and/or miscount of pronucleus/tails. Fertilisation rates were fitted in generalised mixed effects models, and the replicate was set as random. In all, 302 zygotes were analysed in three replicates. Results are expressed as least squares means ± standard errors. Lycopene supplementation increased the in vitro fertilisation (IVF) efficiency (monospermic zygotes/total oocytes) (41.8 ± 9.4%) compared with control (25.8 ± 7.9%; P = 0.04) and DMSO (22.0 ± 7.6%; P = 0.02). Although there was no significant difference, lycopene supplementation tended to decrease polyspermy rates (polyspermic/penetrated zygotes) compared with control (57.6 ± 10.0% vs 72.7 ± 8.6%; P = 0.07, respectively) and improved compared to DMSO: 76.9 ± 8.2%; P = 0.04). Interestingly, lycopene treatment increased pronucleus decondensation rates in penetrated zygotes (85.3 ± 2.2%) compared with control (73.6 ± 2.6%; P = 0.0009) and DMSO (72.2 ± 2.7%; P = 0.0009). In conclusion, lycopene supplementation during IVM seems to improve oocyte quality, since higher IVF efficiency with increased decondensation rates were achieved. In order to validate these results, further studies should be conducted with different bulls and mitochondrial function and structure should be assessed.