The Effect of Different Extenders and Testicular Anaesthesia During Orchiectomy on Post‐Thawed Spermatological Parameters of Stallion Epididymal Spermatozoa

ABSTRACT

Cryopreservation of epididymal stallion spermatozoa is a valuable tool for fertility preservation, particularly after orchiectomy. However, the potential interactions between local anaesthesia and extender composition on post‐thaw spermatozoal quality remain poorly understood. This study investigated the effects of intratesticular lidocaine injection and the efficacy of various extender (egg yolk [EY] vs. lecithin [LC]) and cryoprotectant (glycerol [GLY] vs. dimethyl sulfoxide [DMSO]) combinations on stallion epididymal spermatological parameters. Using a paired factorial design, epididymal spermatozoa were collected from both lidocaine‐injected (right) and non‐injected (left) testes of eight stallions. Samples were cryopreserved in Tris‐based extenders supplemented with EY + GLY, EY + DMSO, LC + GLY or LC + DMSO. Spermatozoal motility (fresh, post‐equilibration and post‐thaw), viability, plasma membrane and acrosome integrity (PMAI), mitochondrial reactive oxygen species (MROS), spermatozoa malondialdehyde (MDA) and glutathione (GSH) levels were evaluated. Intratesticular lidocaine administration had no significant effect on spermatozoal motility at any evaluation stage, and no differences were detected in any biochemical or spermatologic parameters assessed post‐thaw ( p  > 0.05). Regarding cryopreservation media, a significant main effect of extender type was observed for spermatozoal viability ( p  < 0.05) and MROS levels ( p  < 0.001), with EY‐based extenders providing superior protection compared to LC‐based formulations. A significant Extender × Cryoprotectant interaction was detected for GSH levels ( p  < 0.05), with the EY + GLY combination yielding the highest antioxidant capacity. No significant differences were observed in MDA levels among treatment groups ( p  > 0.05). Intratesticular lidocaine administration during orchiectomy does not compromise epididymal spermatozoal quality after cryopreservation. Among the evaluated cryopreservation strategies, the combination of egg yolk and glycerol in a Tris‐based extender provides the most effective protection against oxidative stress and membrane damage in stallion epididymal spermatozoa.