DNase Promotes the Release of Sperm Trapped by NETs and Induces Important Changes in Donkey Sperm Motility Patterns

A deeper understanding of the donkey's reproductive strategy is essential to improve reproductive efficiency in this species. In the jenny's uterus, sperm selection relies on the post-insemination endometrial reaction, characterized by the influx of polymorphonuclear neutrophils (PMNs), which form extracellular traps (NETs) that capture spermatozoa. Of these, some are liberated through the action of a deoxyribonuclease (DNase) present in the seminal plasma. This study aimed to develop an in vitro model to evaluate the effects of NETs and DNase action on donkey sperm viability and motility. For this purpose, donkey ejaculates were collected, PMNs were isolated from the jenny's blood and free spermatozoa were selected using a Silica gel filtration (EquiPure^®, EP, Nidacon International AB, Mölndal, Sweden) to form five experimental groups: control (semen), SPZ-PMN, SPZ-PMN-DNase, SPZ-PMN-EP, SPZ-PMN-DNase-EP. Sperm viability and motility were assessed in all experimental groups using eosin-nigrosin staining and CASA sperm analysis system, respectively, at 1, 2 and 3 h of incubation. Sperm viability remained stable in the groups containing PMN, while it significantly (p < 0.05) decreased in the control group, suggesting a protective effect of PMN on sperm viability. Regarding kinetic parameters, spermatozoa initially captured by NETs and later liberated by DNase exhibited higher velocity (VCL, VSL, VAP), linearity (LIN), straightness (STR) and oscillation (WOB). These changes in sperm motility may be related to the physiological changes occurring before sperm capacitation. This study can improve the understanding of the unique reproductive strategy of donkeys and could be useful to improve in vitro sperm capacitation and embryo production.