Functional Additives in Equine Semen Extenders for Cryopreservation: Current Evidence and Future Perspectives

Cryopreservation of equine semen is an important technique for effective modern breeding programs.Stallion spermatozoa are inherently sensitive to cooling and cryopreservation, during which they are exposed to various stressors that affect both viability and post-thaw functional capabilities.In particular, oxidative stress, osmotic stress, membrane destabilization, and mitochondrial dysfunction are wellestablished critical issues, and thus numerous compounds have been evaluated as additives in semen extenders to support protective preservation and maintain postthaw sperm quality and fertility.The current knowledge about protective components in seminal plasma and the effects of several bioactive compounds as alternatives or adjuncts to conventional extenders for equine semen extenders have been discussed in the present review.The main groups of additives for equine semen extenders, their modes of action, biological efficacy, and limitations were also examined.Antioxidants (enzymatic and non-enzymatic), and mitochondrialtargeted antioxidants (MTAs), such as melatonin and MitoQ, provide a recent perspective as antioxidants that protect sperm mitochondria and sustain their bioenergetics.Structural additives such as trehalose, cholesterol, and cholesterolloaded cyclodextrin have also shown encouraging results.Despite significant advances in seminal extender development, extender additives generally lack consistency in achieving improved sperm characteristics and protection.However, some novel additives are on the horizon with great potential.Recently, advances in nanotechnology have led to the development of novel delivery systems for reproductive additives.Although mostly at experimental stage, these methodologies have the potential for targeted delivery and sustained protection of stallion spermatozoa.Besides optimizing semen storage conditions, further improvement of semen preservation relies on additive optimization of semen extenders and, on the development of well-balanced, species-specific, and individually optimized semen extenders, supplemented by current research methods and delivery systems.