Abstract

Age modifies the redox balance of stallion sperm and may determine post-thaw performance. We compared cryopreserved sperm from younger (3–5 year), mature (6–15 year), and older (16–24 year) Arabian stallions using imaging flow cytometry for total reactive oxygen species (ROS) (DCFH-DA) and superoxide (DHE), Annexin V/PI for apoptotic-like changes, JC-1 for mitochondrial membrane potential (MMP), and photometric assays for oxidative protein modifications (OMP), malondialdehyde, catalase, and superoxide dismutase. Older stallions showed higher OMP, higher catalase activity, more Annexin V-positive cells, and lower MMP, with reduced post-thaw motility; younger stallions exhibited minimal oxidative damage yet low progressive motility (PM); mature stallions had the highest total/PM consistent with a balanced redox state. These findings suggest prioritizing semen collection from mature stallions (6–15 years) to improve cryopreservation outcomes. Antioxidants are recommended for older stallions to reduce oxidative damage, while energy-boosting compounds are advised for younger stallions to improve motility. Validation of age-tailored interventions and definition of operational ROS/MMP thresholds are warranted to optimize dosing and prevent reductive stress.

Key words: sperm cryopreservation, age, stallions, oxidative stress, imaging flow cytometry