Dorado , JesúsSaa, Luis RodrigoGalarza Lucero, Diego AndrésSantiago Moreno, JuliánTamay Siguenza, Erika FabiolaPalacios Aguilar, María PaulaPelaez Zhingri, Gabriela Micaela2023-02-282023-02-2820221947-5535http://dspace.ucuenca.edu.ec/handle/123456789/41161https://www.liebertpub.com/doi/10.1089/bio.2022.0097This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96 , and finally frozen with INRA-Freeze  with either no supplementation (as control), 1 mM ME, or 2mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution  2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater ( p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences ( p < 0.01) between stallions (1–4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.es-ESPeruvian paso horseSpermatozoaOxidative stressCaffeineMelatoninARTÍCULO10.1089/bio.2022.0097