Effect of resveratrol supplementation in freezing and vitrification media on cryosurvival of dog epididymal spermatozoa

Resveratrol (RES), a natural polyphenol, may reduce oxidative damage to sperm during cryopreservation due to its antioxidant properties. This study evaluated the impact of RES supplementation in media used for conventional slow (CS) freezing and kinetic vitrification (KV) on the cryosurvival of dog epididymal spermatozoa. Sixty epididymides were retrieved from 30 orchiectomized adult dogs, and sperm samples were recovered by retrograde flushing using TCG-EY extender (tris, citric acid, glucose +20% egg yolk), resulting in 20 pooled samples. Four treatments were formed from each pooled sample based on the cryopreservation method and RES addition (50 and 0 [Control, Co] μM): CS-RES, CS-Co, KV-RES, and KV-Co. The CS freezing involved exposing 0.25 mL straws containing sperm samples (plus 5% glycerol) to static liquid nitrogen (LN2) vapors, while KV process was done by directly dropping 30-μL drops of samples (plus 250 mM sucrose) into LN2. Results indicated that CS-RES treatment increased (p < 0.05) motility (TM), plasma and acrosome membrane integrity (IPIA), and reduced oxidative stress (OS) post-thawing compared to their control, CS-Co (respectively, TM: 36.9 ± 1.54 vs. 25.8 ± 1.71%; IPIA: 49.7 ± 1.27 vs. 40.4 ± 1.31%; OS: 4.7 ± 1.02 vs. 12.7 ± 1.38%). Meanwhile, KV-RES treatment only improved (p < 0.05) beat-cross frequency than their control, KV-Co (6.7 ± 0.18 vs. 6.1 ± 0.17 Hz) without significant differences in IPIA (35.7 ± 1.13 vs. 36.0 ± 1.24%), TM (32.1 ± 1.79 vs. 28.4 ± 1.85%) or OS (12.6 ± 1.55 vs. 10.9 ± 2.1%). In summary, RES supplementation enhanced motility and membrane protection and reduced oxidative stress during and after the freezing process of dog epididymal sperm. However, its efficacy was limited in the vitrification process.