Comparative Sensitivity of Canine Swim-Up Sperm Fractions Treated with Antifreeze Protein and Trehalose Following Freeze–Thaw

Sperm cryopreservation is widely used in reproductive biotechnology, but the freeze–thaw process often reduces sperm quality by impairing motility, viability, and DNA integrity. This study assessed how antifreeze protein III (AFP III; 1.5 µg/mL) and trehalose (25 mM), alone or combined, influence post-thaw sperm quality in swim-up fractions. After routine semen evaluation, ejaculates were separated into upper, native, and lower fractions and diluted in a Tris–fructose–egg yolk extender containing the additives. Post-thaw analyses covered motility, kinematic parameters, apoptosis, intracellular hydrogen peroxide (H2O2), mitochondrial activity, lipid peroxidation, viability, and DNA fragmentation. Because all fractions originated from the same ejaculate, differences among native, upper, and lower sperm populations were evaluated using a mixed-effects model with sperm fraction as a fixed effect and dog/ejaculate as a random effect. Native semen showed significant differences among fractions (p < 0.05). The upper fraction showed the highest TM and PM, together with clearly superior kinematic performance in VCL, VSL, VAP, and ALH. All these parameters were significantly higher than those measured in the lower fraction (p < 0.05). In contrast, the native fraction displayed intermediate values for TM, PM, VCL, VSL, VAP, and ALH, showing a significant difference compared with the upper fraction (p < 0.05) but no significant difference relative to the lower fraction (p > 0.05). Beat cross frequency (BCF) did not differ among groups (p > 0.05). AFP III improved motility and reduced H2O2-derived ROS (p < 0.05), whereas trehalose decreased motility (p < 0.05) but increased mitochondrial activity in the lower fraction (p < 0.05). After freezing, the upper fraction showed higher lipid peroxidation and DNA fragmentation, while the lower fraction exhibited more apoptosis and reduced viability (p < 0.05). Overall, AFP III provided the most consistent protection, and trehalose produced mixed, fraction-dependent effects, confirming that sperm subpopulation characteristics strongly influence freeze–thaw outcomes.