Cryopreservation protocol and antioxidant supplementation determine post-thaw functional quality of goat sperm

Goat sperm cryopreservation remains a major challenge because freeze-thaw procedures induce structural and functional damage that compromises post-thaw sperm quality. Although antioxidant supplementation has been widely investigated to reduce oxidative stress, its effectiveness remains inconsistent and may depend on the cryopreservation protocol. This study was conducted in two phases. In Phase 1, three antioxidants-ascorbic acid (2.5, 5.0 and 10.0 mg/mL), cysteamine (50, 100 and 200 μM), and glycine (5, 10 and 20 mM)-were screened using semen from six bucks (three Alpine-French and three Murciano-Granadina). Post-thaw assessments via the AndroVision® CASA system (Minitube, Germany; software version 0.26), viability (eosin-nigrosine staining), plasma membrane integrity (HOST), and thermoresistance test (TTR) revealed no statistically significant effects of antioxidant treatment on motility and kinematic parameters (P > 0.05). However, numerical differences favored glycine at 20 mM, which yielded the highest estimated marginal means for total motility (60.35 ± 3.51%), progressive motility (59.97 ± 3.55%), and fast motility (47.74 ± 3.98%), surpassing the control group. The TTR proved more discriminative: ascorbic acid at 10.0 mg/mL produced the most severe decline, whereas cysteamine at 100 μM achieved the highest values among treated groups. In Phase 2, glycine at 20 mM-selected for its numerically favorable kinematic profile-was validated in an independent experiment comparing two cryopreservation protocols: Programmable and Manual. Evaluations included CASA motility and kinematic parameters, plasma membrane integrity, acrosome integrity, mitochondrial activity, thermoresistance test, cold shock resistance test, and gene expression analysis (SOD1, HSP70, CAT). Results demonstrated that the cryopreservation protocol exerted a significantly greater effect than antioxidant supplementation. The manual protocol consistently produced superior values for total motility, progressive motility, fast motility, kinematic parameters (VCL, VAP, VSL, BCF, ALH, HAC), acrosome integrity, and cold shock resistance (P < 0.05). In contrast, glycine supplementation at 20 mM did not significantly affect any evaluated parameter (P > 0.05), nor did the Protocol × Antioxidant interaction. Relative expression of SOD1, HSP70, and CAT did not differ between protocols, treatments, or their interaction (P > 0.26). Overall, these findings indicate that, under the conditions evaluated, optimization of the cryopreservation protocol had a greater impact on post-thaw sperm functional quality than glycine supplementation, highlighting the importance of optimizing cryopreservation procedures before evaluating antioxidants as complementary strategies.