Membrane and mitochondrial antioxidants improve post-thaw functional quality of stallion spermatozoa

Cryopreservation of stallion semen induces oxidative stress that compromises sperm motility, membrane integrity, and mitochondrial function. Given the high mitochondrial reliance of equine spermatozoa and their susceptibility to oxidative damage, compartment-specific antioxidant strategies may improve post-thaw functional resilience. This study evaluated the effects of astaxanthin, a membrane-active carotenoid, and MitoTEMPO, a mitochondria-targeted superoxide scavenger, on the functional and oxidative status of frozen-thawed stallion spermatozoa. Ejaculates from nine stallions were cryopreserved in BotuCrio® extender supplemented with astaxanthin (10, 25, 50 µM) or MitoTEMPO (5, 10, 25 µM). Post-thaw quality was assessed using computer-assisted sperm analysis and multiparametric flow cytometry to evaluate motility, viability, apoptosis-like changes, acrosomal integrity, mitochondrial activity, reactive oxygen species (ROS) production, and intracellular calcium levels. Both antioxidants improved selected post-thaw functional parameters compared with controls (P < 0.05), including total and progressive motility, kinematic parameters, viability, and acrosomal integrity, and were associated with reduced intracellular and mitochondrial ROS levels. Astaxanthin was primarily associated with improved plasma membrane stability, whereas MitoTEMPO was linked to enhanced mitochondrial activity. The most consistent improvements were observed at intermediate concentrations (25 µM astaxanthin and 10 µM MitoTEMPO). These findings indicate that membrane- and mitochondria-targeted antioxidants exert complementary protective effects during stallion semen cryopreservation and may enhance post-thaw functional competence of spermatozoa. Further studies including fertility assessment are warranted to confirm their practical relevance in equine artificial insemination.