Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils

SIMPLE SUMMARY: Donkey Artificial Insemination (AI) with frozen/thawed semen results in poor fertility outcomes. Jennies show a significant post-AI endometrial reaction, with a large amount of defense cells—polymorphonuclear neutrophils (PMN)—migrating to the uterine lumen. Seminal plasma (SP) has a detrimental effect on sperm conservation and its removal is a necessary step in the semen freezing protocol. However, several SP proteins seem to control sperm-PMN binding. Single layer centrifugation (SLC) with colloids, which has been used to select spermatozoa and improve reproductive performance in different species, is known to remove SP proteins attached to the sperm membrane. In this study, two experiments were performed. The first one compared the quality of SLC-selected and non-selected fresh donkey spermatozoa. In the second experiment, PMN obtained from the peripheral blood were co-incubated with selected and unselected spermatozoa, and the interaction between PMN and spermatozoa was analyzed. In conclusion, SLC of fresh donkey semen increases the proportion of functionally intact spermatozoa and appears to remove the SP proteins that inhibit sperm-PMN binding, thus increasing sperm phagocytosis by PMN. ABSTRACT: This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.