Combined effects of pre-freeze cholesterol-loaded cyclodextrin and post-thaw pentoxifylline on the functional longevity of cryopreserved stallion sperm.

BACKGROUND: Cryopreservation causes structural and metabolic damage to stallion spermatozoa, impairing post-thaw functional competence.
AIMS/OBJECTIVES: This study evaluated whether cholesterol-loaded cyclodextrin (CLC) supplementation before freezing, combined with pentoxifylline (PTX) after thawing, improves the functional quality, oxidative status, mitochondrial activity, and preliminary fertility outcomes of cryopreserved stallion semen.
METHODS: Semen from twelve stallions was assigned to four CLC concentrations (0, 1, 1.5, and 2 mg), frozen, thawed, and divided into subgroups with or without PTX supplementation (7.18 mM). Sperm kinetics were assessed during a 120-minute thermoresistance test, and flow cytometry was used to evaluate mitochondrial membrane potential, membrane destabilization, and intracellular hydrogen peroxide. A preliminary in vivo assessment was performed using semen from the control, 1.5 mg CLC, and 1.5 mg CLC + PTX groups.
RESULTS: CLC was associated with improved structural preservation and selected immediate post-thaw kinetic responses, whereas PTX reduced velocity, linearity, and motility-related parameters throughout incubation. PTX also increased intracellular hydrogen peroxide and reduced the JC-1 aggregate/monomer ratio in groups with lower CLC concentrations, indicating impaired mitochondrial membrane potential. The combined protocol produced concentration-dependent responses, with higher CLC concentrations partially preserving mitochondrial membrane potential under PTX exposure. Pregnancy occurrence appeared comparable among groups, but the limited number of mares and cycles precluded statistical inference.
CONCLUSION: CLC and PTX influenced different components of post-thaw sperm performance, with no clear evidence of synergistic improvement under the evaluated conditions. Further studies should refine PTX dosing and explore alternative strategies to improve cryopreserved stallion semen.