Seminal plasma, and not sperm, induces time and concentration‐dependent neutrophil extracellular trap release in donkeys

Abstract

Background In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen‐thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. Objectives To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in response to frozen‐thawed semen. Study design In vitro experiments. Methods Jenny PMNs were exposed to jackass fresh or frozen‐thawed semen, isolated sperm or seminal plasma (SP), over the course of three experiments. NET formation in response to different treatments was assessed through manual quantification of stained slides. A one‐way analysis of variance (ANOVA), followed by a post hoc Sidak test, was carried out to determine statistical significance. Results NET release occurred in a semen concentration‐ and incubation‐time‐dependent manner. Surprisingly, frozen‐thawed donkey sperm did not increase NETosis rate in comparison with the control (23 ± 2.5% vs. 31 ± 3.7%; P > .05), whereas fresh semen exposure did (78 ± 5.7% vs. 26 ± 3.2%, P < .01). NETosis increased in the presence of SP, regardless of the presence or absence of sperm, in comparison with the control in both fresh (84 ± 5.2% and 77 ± 5.0% vs. 12 ± 2.7%, respectively; P < .01) and frozen (95 ± 2.2% and 94 ± 2.9% vs. 14 ± 3.8%, respectively; P < .01) samples. Moreover, exposure of PMN to viable and motile sperm, in the absence of SP, did not increase NETosis rates (P > .05). Conclusions Donkey SP, and not sperm‐intrinsic factors, is able to trigger NETosis in both time‐ and semen concentration‐dependent manner. The physiological relevance of such response against semen in the donkey remains to be elucidated.

Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance

“…Despite advances in cryopreservation, the use of frozen-thawed semen has been limited in donkeys due to the low conception rates obtained when jennies (0 to 36%) are inseminated compared to mares (33% to 56%) [ 7 , 8 , 9 , 10 ], which is attributed to the intense endometrial inflammatory response that occurs when the jenny is inseminated with frozen-thawed sperm [ 11 , 12 , 13 ]. In vivo and in vitro experiences have shown that SP, which is removed during the semen-freezing process because of its harmful effects on equids’ sperm [ 14 ], may play a key role in modulating this response [ 12 ].…”

Adverse Effects of Single Neutrophil Extracellular Trap-Derived Components on Bovine Sperm Function

“…During natural coitus and AI in bovines, semen is deposited into the uterus. The NET response usually occurs in utero; is regulated by seminal plasma in horses [16], pigs [17], and donkeys [18]; and has a minor role in the bovine [19]. Irrespective of semen deposition, sperm will migrate through the FRT to fertilise the oocyte; however, a large proportion of spermatozoa remain behind [20].…”

Clinical insights: Assisted reproductive techniques: More than a solution to subfertility?

“…Uterine neutrophils are thought to be released primarily in response to the presence of the spermatozoa themselves and, as well as binding directly to sperm, have been reported to capture spermatozoa by extruding their DNA to produce "neutrophil extracellular traps". 4 Fortunately, stallion seminal plasma has been shown to contain proteins that inhibit the binding or capture of live sperm (eg CRISP3) and others that enhance the binding of dead sperm (eg lactoferrin) by neutrophils. 5 Surprisingly, it appears that in donkey semen the seminal plasma, rather than the spermatozoa, is the primary stimulator of neutrophil extracellular trap formation.…”

“…5 Surprisingly, it appears that in donkey semen the seminal plasma, rather than the spermatozoa, is the primary stimulator of neutrophil extracellular trap formation. 4 Clearly, there is a need for further research into the proteins present in seminal plasma, or that could be added to semen extenders or introduced into the uterus of the mare prior to breeding, that can protect high-quality spermatozoa from elimination by neutrophils as they pass through the uterus towards the oviduct.…”