Semen extender and seminal plasma alter the extent of neutrophil extracellular traps (NET) formation in cattle

During artificial insemination in bovine, the deposition of semen into the uterus results in an immune reaction which is based on polymorphonuclear neutrophils activity, including the formation of neutrophil extracellular traps. The formation of neutrophil extracellular traps as a reaction of neutrophils to spermatozoa was recently described. However, it is not completely clear which components of the semen are responsible for this reaction. The objective of this study was to quantify and compare the formation of neutrophil extracellular traps following in vitro incubation of bovine polymorphonuclear neutrophils with semen and extenders of different origins and conditions. We investigated the interactions between bovine polymorphonuclear neutrophils and different semen extenders, various seminal plasma concentrations from young and old bulls as well as sexed and non-sexed semen and their corresponding extenders. Three different semen extenders from two companies in fresh and frozen-thawed conditions were compared. Fresh semen extenders showed higher neutrophil extracellular traps induction than did frozen-thawed ones. The formation of neutrophil extracellular traps were also dependent on the presence of seminal plasma. We could show that seminal plasma alone, without any sperm cells, induced the reaction and that the addition of at least 1% seminal plasma already resulted in the formation of neutrophil extracellular traps. Furthermore, seminal plasma from young bulls led to significant higher neutrophil extracellular traps induction. No difference between non-sex-sorted and sex-sorted sperm and its extenders was observed. Taken together, different semen extenders as well as the amount and origin of seminal plasma influence neutrophil extracellular traps formation, whereas sex-sorted sperm did not affect the reaction.