Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm–PMN Binding in the Donkey

SIMPLE SUMMARY: Previous research demonstrated that seminal plasma, the fluid that together with sperm composes the semen, modulates the interaction between sperm and polymorphonuclear neutrophils (PMN) within the donkey uterus. This study sought to evaluate whether a specific set of proteins or the entire seminal plasma is involved in that modulation. Seminal plasma was fractioned into six portions: <3, 3–10, 10–30, 30–50, 50–100, and >100 kDa. Sperm incubated with two specific fractions, containing 30–50 kDa and 50–100 kDa proteins, exhibited the highest motility and viability after co-incubation with PMN for 4 h, and the highest sperm–PMN interaction at the beginning of the experiment. These results suggest that proteins of 30–50 and 50–100 kDa, rather than the entire seminal plasma, appear to be involved in the inflammatory reaction within the donkey endometrium. ABSTRACT: While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. Remarkably, sperm interacts with PMN, but the presence of seminal plasma reduces that binding. As seminal plasma is a complex fluid made up of different molecules, including proteins, this study aimed to evaluate how different seminal plasma fractions, separated by molecular weight (<3, 3–10, 10–30, 30–50, 50–100, and >100 kDa), affect sperm–PMN binding. Sperm motility, viability, and sperm–PMN binding were evaluated after 0 h, 1 h, 2 h, 3 h, and 4 h of co-incubation at 38 °C. Two seminal plasma fractions, including 30–50 kDa or 50–100 kDa proteins, showed the highest sperm motility and viability. As viability of sperm not bound to PMN after 3 h of incubation was the highest in the presence of 30–50 and 50–100 kDa proteins, we suggest that both fractions are involved in the control of the jenny’s post-breeding inflammatory response. In conclusion, this study has shown for the first time that specific fractions rather than the entire seminal plasma modulate sperm–PMN binding within the donkey uterus. As several proteins suggested to be involved in the control of post-AI endometritis have a molecular weight between 30 and 100 kDa, further studies aimed at determining the identity of these molecules and evaluating their potential effect in vivo are much warranted.