Resveratrol supplementation during cryopreservation improves the post-thaw quality of alpaca (Vicugna pacos) epididymal sperm

Cryopreservation of alpaca (Vicugna pacos) epididymal spermatozoa is an important strategy for preserving valuable genetic resources; however, freezing and thawing can induce oxidative and cellular alterations that compromise post-thaw sperm quality. This study evaluated the effects of resveratrol supplementation during cryopreservation on the post-thaw quality and functional characteristics of alpaca epididymal spermatozoa. A total of 210 cauda epididymides were collected, and samples with ≥ 50% progressive motility were considered eligible. Sixty samples met this criterion. Experiment 1 included 10 samples from different males and was conducted as a preliminary dose-response experiment to select the resveratrol concentration for subsequent evaluation. Experiment 2 included 50 samples from different males and evaluated the effects of the selected concentration of 50 μM resveratrol on post-thaw sperm quality and functional characteristics. Spermatozoa in Experiment 2 were evaluated before cryopreservation and after thawing following cryopreservation in a TES-Tris egg yolk extender without resveratrol (YM) or supplemented with 50 μM resveratrol (YM + RES). Compared with YM, resveratrol supplementation resulted in higher progressive motility, plasma membrane functionality, mitochondrial membrane potential, and homologous sperm-zona pellucida binding, together with lower percentages of ROS-positive spermatozoa and early apoptosis-like spermatozoa and a lower DNA fragmentation index (P < 0.05). No significant differences between YM and YM + RES were detected for sperm viability, oxidoreductase activity, or the late apoptosis-like/non-viable sperm population. In conclusion, supplementation with 50 μM resveratrol attenuated several cryopreservation-associated alterations and improved selected post-thaw functional characteristics of alpaca epididymal spermatozoa, supporting its potential application in sperm cryopreservation and post-mortem germplasm rescue.