Addition of cholesterol linked to cyclodextrin in processing of cryopreserved equine spermatozoa and addition of motility stimulants post-thaw.

Abstract: This study investigated the effects of cholesterol-loaded cyclodextrin (CLC) before cryopreservation and the use of FertTalp (FT) after thawing on the structural and functional quality of equine spermatozoa. Methods: Two ejaculates from twelve stallions were divided into four groups: G1 (0 mg CLC), G2 (1.0 mg), G3 (1.5 mg), and G4 (2.0 mg). Post-thaw semen quality was evaluated through computer-assisted sperm analysis and flow cytometry. Results: In Experiment I, supplementation with 1.5 mg (G3) significantly improved total motility (64.9 ± 10.1 %) and progressive motility (40.5 ± 8.8 %) compared with the control group (G1) (55.5 ± 15.7 % and 31.9 ± 10.8 %; P < 0.05). No differences were observed for mitochondrial potential or membrane lipid disorder. Cholesterol supplementation did not influence overall longevity during the thermoresistance test, although G3 maintained higher velocity parameters (VSL, VCL, VAP) at several time points compared with G2 (P < 0.05). In Experiment II, FT did not alter sperm kinetics during the first 40 min after thawing; however, at 80 and 120 min, FT-treated samples presented higher proportions of rapid spermatozoa, greater VSL and VAP, and increased linearity (P < 0.05). FT also increased the proportion of viable spermatozoa without intracellular H₂O₂, although the magnitude of this effect was small. Conclusions: Supplementation with 1.5 mg of CLC before freezing improved initial post-thaw motility without affecting mitochondrial activity or membrane organization. Post-thaw FT enhanced velocity-related parameters and supported sperm functional longevity. The combined approach may represent a practical strategy to optimize the post-thaw performance of stallion semen.