Antioxidant effect of quercetin and gallic acid in domestic dog epididymal sperm cryopreservation

Abstract

Cryopreservation induces negative effects on spermatozoa due to oxidative stress. The aim of this study was to evaluate the effect of the addition of quercetin and gallic acid on sperm characteristics under the cryopreservation process of domestic dog epididymal semen. Samples were collected from sexually mature males by direct epididymal collection technique after orchiectomy and diluted with a commercial extender. Treatments included Control, DMSO (0.005 %), Ethanol (0.05 %), Q25 (quercetin 25 μM), GA45 (gallic acid 45 μM), and a combination of Q25 + GA45. Motility parameters were determined using a Computer-Assisted Sperm Analysis (CASA) system. Viability, mitochondrial membrane potential, acrosomal integrity, DNA fragmentation and reactive oxygen species (ROS) production were analyzed using flow cytometer. Variables were analyzed by ANOVA. Gross and progressive motility in Control and Q25 were similar, and higher than the rest of treatment groups (p < 0.05). Conversely, ethanol showed the lowest mitochondrial membrane potential among all treatments, except for Q25 + GA45, which showed lower than Control and DMSO groups (p < 0.05). For DNA fragmentation all treatments were similar (p > 0.05). Ethanol showed a negative effect on viability and acrosomal integrity compared to Control (p < 0.05). Regarding antioxidant capacity, all treatments showed lower ROS production when compared to Control (p < 0.05). In conclusion, the addition of quercetin and gallic acid reduced ROS production but did not improve sperm parameters during cryopreservation of domestic dog epididymal semen. Furthermore, ethanol used as a solvent for gallic acid adversely affected these parameters, although antioxidants and their combination partially mitigated this negative effect.