Post-thaw quality of canine spermatozoa exposed to hydroxychalcones: a double-edged sword

Hydroxychalcones are phenolic chalcone derivatives with antioxidant activity and affinity for lipid-rich membranes, enabling modulation of oxidative processes such as lipid peroxidation. Because cryopreservation exposes canine spermatozoa to severe oxidative stress and membrane damage, these compounds represent potential candidates for redox modulation during cryogenic stress. This study evaluated the effects of selected chloro- and methyl-substituted hydroxychalcones on the post-thaw functional quality of canine spermatozoa. Semen collected from 5 Beagle dogs was pooled prior to cryopreservation. The experiments were conducted in six independent replicates using a Tris-fructose-egg yolk extender supplemented with chlorinated (2ClCH, 4ClCH; Experiment 1) or methylated (2CH3CH, 4CH3CH; Experiment 2) hydroxychalcones at 0.1 or 0.4 mM. Frozen-thawed spermatozoa were evaluated after thawing and after 2 and 4 h of incubation using CASA and flow cytometry. In Experiment 1, chlorohydroxychalcones did not affect total or progressive sperm motility, although 4ClCH significantly reduced average path velocity after 4 h post-thaw (P < 0.01). Incubation time was the predominant factor influencing most sperm characteristics, and none of the chlorohydroxychalcone treatments differed significantly from the control. In Experiment 2, methyl-substituted hydroxychalcones induced time-dependent reductions in sperm motility and velocity parameters at 2 and 4 h post-thaw. Flow cytometric analysis revealed a consistent reduction in lipid peroxidation-positive sperm subpopulations, while other functional parameters remained unaffected. Although hydroxychalcones reduced lipid peroxidation in frozen-thawed canine spermatozoa, this antioxidant effect did not translate into improved sperm motility. These findings indicate that hydroxychalcones were not beneficial under the conditions of this study.