Effect of quercetin, L-ergothioneine and pentoxifylline in post-thaw stallion semen

Abstract

Cryopreservation produces an oxidative stress that alters the stallion semen quality. Different antioxidants have been added to cooled, frozen or post-thawed semen, but in many cases, they have had limited results and even deleterious effects. In recent years, promising results on conventional antioxidants have been reported. This research is aim at evaluating the effect of quercetin, L-ergothioneine and pentoxifylline in the post-thaw quality conservation of stallion semen. The semen from five horses (Equus caballus) was cryopreserved by a conventional freezing protocol. Post-thaw and reactive oxygen species (ROS) production and inhibition were assessed by H₂DCFDA assay; and concentrations of semen supplementation were defined for L-ergothioneine 150 µM, quercetin 150 µM and pentoxifylline 3,0 mM. At 0, 6, 12 and 24 hours, sperm motility and kinetics were assessed by SCA^® system; vitality and mitochondrial activity by SYBR14 / IP and JC-1 probes; membrane integrity by hypoosmotic swelling test (HOS) and lipid peroxidation by TBARS assay. The regression analysis and adjustment of the generalized linear models (GLM)were performed. ROS maximum inhibition was 73%, 20% and 37% for quercetin, L-ergothioneine and pentoxifylline, respectively. The progressive motility (PM), curvilinear velocity (VCL) and average path velocity (APV) were higher at 6 hours for L-ergothioneine and quercetin, as well as VCL at 12 hours for L-ergothioneine (p<0,05). It is concluded that L-ergothioneine and quercetin have a favorable post-thaw effect on PM, VCL and APV of stallion semen.

Key words: antioxidants, cryopreservation, spermatozoa, fertility.