This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the
quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years,
were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106
sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with
MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and
equilibration. After thawing, semen was evaluated for motility and kinetics at different time points. Sperm
viability, plasma membrane, acrosome, DNA integrity, mitochondrial membrane potential, apoptosis, and
intracellular reactive oxygen species (ROS) concentrations were evaluated. The results revealed that MitoQ at
concentrations of 25, 50, and 100 nM improved (P< 0.01) the total sperm motility after 30 minutes of incu-
bation. In addition, 25 nM MitoQ improved the sperm amplitude of lateral head displacement values (P< 0.01)
after 30 minutes of incubation. Conversely, negative effects on sperm motility, kinetics, and viability were
observed with the highest tested concentration of MitoQ (200 nM). The various concentrations of MitoQ did not
affect the plasma membrane, acrosome, and DNA integrity, or the mitochondrial membrane potential and
intracellular ROS concentrations. In conclusion, supplementation of MitoQ during cryopreservation, had a mild
positive effect on sperm motility and kinetics especially at a concentration of 25 nM, while the highest con-
centration (200nM) has a detrimental effect on motility and viability parameters of frozen-thawed stallion sperm.