L-carnitine or melatonin supplementation during vitrification and warming mitigates oxidative stress and improves cryotolerance in immature bovine cumulus-oocyte complexes

Abstract

Vitrification of immature cumulus-oocyte complexes (COCs) is a valuable tool in assisted reproductive technologies, but it often induces oxidative stress, reduces viability, and compromises developmental potential. This study investigated the effects of supplementing vitrification and warming solutions with non-enzymatic antioxidants — glutathione (GSH, 5 mM), L-carnitine (LC, 3.03 mM), or melatonin (MLT, 10⁻⁶ mM) — on nuclear maturation, oxidative stress, early apoptosis, cryosurvival, and gene expression. COCs were vitrified and warmed in the presence or absence (negative control) of antioxidants; non-vitrified COCs served as fresh controls. Vitrification significantly reduced (P < 0.05) viability, survival, and nuclear maturation rates compared to fresh COCs. However, MLT increased viability, and LC enhanced nuclear maturation (P < 0.05 vs negative control). Gap junction activity and early apoptosis were not significantly affected by antioxidant treatment. Intracellular GSH and ROS levels were restored in antioxidant-treated groups, comparable to fresh controls (P > 0.05), whereas the negative control had increased ROS and decreased GSH (P < 0.05). In cumulus cells, expression of mitochondrial genes ATP6 and ATP8—key for oxidative phosphorylation—was downregulated in the negative control. Moreover, LC and MLT supplementation counteracted this effect, upregulating SOD2 (mitochondrial superoxide dismutase) and SOD1 (cytosolic isoform), respectively. In oocytes, both LC and MLT upregulated SOD1 (P < 0.05). In summary, these findings suggest that LC and MLT supplementation improve cryotolerance and mitigate oxidative stress in vitrified immature bovine COCs by modulating antioxidant defenses and mitochondrial gene expression.