Effect of quercetin, L-ergothioneine and H89 on sperm motility and kinematic pattern, plasma membrane functionality and in vitro heterologous fertilizing capacity of cryopreserved equine semen

•Quercetin increases beat-cross frequency and reduce curvilinear velocity (VCL).•H89 decrease curvilinear velocity and amplitude of lateral head displacement (ALH).•H89 plus quercetin decrease total and progressive motility and VCL.•H89 plus L-ergothioneine decrease VCL and ALH. Semen cryopreservation causes extensive chemical and physical damage to sperm structure, which generates premature aging and reduces viability and fertility of spermatozoa. The addition of antioxidants to freezing extenders can reduce the oxidative damage caused by excessive generation of reactive oxygen species (ROS), and the premature aging could be reduced by adding an enzyme inhibitor that prevents an anticipated capacitation. The aim of this study was to evaluate the in vitro effect of quercetin (Q), L-ergothioneine (E) and H89 addition to cryopreserved equine spermatozoa. Six experimental groups were stablished: control, Q, E, H89, H89Q and H89E. The analyzed parameters were sperm motility and kinematic using computer assisted sperm analysis (CASA), plasma membrane functionality with the hypoosmotic swelling test (HOST) and fertilizing capability with in vitro heterologous fertilization. Quercetin reduced curvilinear velocity (VCL) and increased beat-cross frequency (BCF), while its combination with H89 (H89Q) reduced total motility, progressive motility, VCL and hyperactive sperm (HA). Likewise, H89 and its combination with E (H89E) decreased VCL and amplitude of lateral head displacement (ALH). No significant differences were observed among treatments for membrane functionality and fertilizing capacity of sperm. In conclusion H89 in combination with Q and E reduced sperm motility or some kinematic parameters. However, they did not influence plasma membrane functionality and in vitro fertilizing capacity of frozen-thawed equine semen.