Mitochondria-targeted antioxidants (MitoQ and Mito-TEMPO) versus untargeted antioxidants (SOD and CoQ10) improve post-thaw quality of rooster sperm by mitigating mitochondrial oxidative damage.

Sperm freezing is a key technique in poultry reproductive technologies, especially for genetic improvement and conservation. Nevertheless, rooster sperm are highly sensitive to freezing-induced damage, resulting in significant declines in sperm quality and fertility. This damage primarily arises from excessive reactive oxygen species (ROS) production, mitochondrial structural disruption, mitochondrial membrane potential loss, lipid peroxidation, and apoptosis pathways activation. This study compared the effects of mitochondria-targeted antioxidants (MitoQ and Mito-TEMPO) and untargeted antioxidants (SOD and CoQ10) on frozen-thawed rooster sperm quality. Semen was diluted with extenders supplemented as follows: control (no antioxidant), SOD (100 IU/mL), CoQ10 (1 μM), MitoQ (50 and 100 nM), Mito-TEMPO (50 and 100 μM), and a combination of MitoQ + Mito-TEMPO (50 nM + 50 μM). Both antioxidant types, especially mitochondria-targeted compounds, significantly improved total and progressive motility, viability, plasma membrane and acrosome integrity, reduced lipid peroxidation, DNA fragmentation, and apoptotic like changes (P ≤ 0.05). The combined MitoQ/Mito-TEMPO treatment showed the greatest protective effects and significantly increased fertility rates compared to control (P ≤ 0.05), indicating enhanced preservation of mitochondrial structure and function. MitoQ reduces electron leakage in the electron transport chain and prevents release of pro-death factors, while Mito-TEMPO scavenges superoxide radicals and maintains mitochondrial redox balance. These results suggest that specific mitochondrial targeting with MitoQ and Mito-TEMPO is an effective strategy to enhance frozen-thawed rooster sperm quality and improve reproductive performance in poultry breeding programs.