Oral Presentation ESA-SRB-ANZOS 2025 in conjunction with ENSA

Mediating immune tolerance of sperm in sheep: Identifying factors in ram seminal plasma responsible for facilitating sperm neutrophil evasion (128638)

Sophie Warr 1 , Taylor Pini 2 , Simon de Graaf 1 , Jessica Rickard 2
  1. School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia
  2. School of Veterinary Science, The University of Queensland, Gatton, QLD, Australia

Seminal plasma (SP) is a complex immune modulator within the female reproductive tract of several species [1]. In sheep, it can restore the fertility of cryopreserved sperm [2] and is necessary for the fertility of epididymal sperm [3] following cervical artificial insemination. We have also shown it capable of protecting cryopreserved sperm from polymorphonuclear leukocyte (PMN) binding in vitro [4]. Thus, we hypothesise SP, and its components confer immune protection to sperm following deposition in the ovine cervix and that sperm cryopreservation disrupts this delicate sperm-female interaction.

To delineate the influence of soluble or extracellular vesicle (EV) derived SP components on sperm-PMN binding, a series of PMN binding assays were conducted using frozen-thawed ram spermatozoa (FT; n=3 rams). Firstly, blood-derived PMNs from Merino ewes (n=2) were incubated (120min;37ºC) with FT supplemented with either whole SP, EV-depleted SP, isolated EVs or PBS (n=3 replicates).  EVs were separated from a pool of SP (n=24 rams x 3 ejaculates) via ultracentrifugation [5]. While SP treatments (24.17±0.29%, 23.91±0.21%, 20.15±0.23%) reduced PMN-binding compared to the control (38.22±0.47%; p<0.01), there was no significant difference between SP treatments, suggesting the protective effect is both soluble and of EV origin. 

To further elucidate the role of soluble SP components, FT was incubated (120min;37ºC) with SP fractionated into 5 molecular weight treatments (<10, 10–30, 30–50, 50–100, >100 kDa), whole SP and PBS (n=3 replicates).  Binding was significantly reduced in 30–50 (22.44±0.65%), and >100 (21.67±0.55%) kDa fractions, compared to PBS (58.83±1.36%), but was similar to whole SP (20.67±0.49%), suggesting these fractions may harbour key immunoregulatory factors.

Mass spectrometry will now identify quantitative differences between treatments, linking protein and/or vesicle cargo expression with protective effect. These results enhance our understanding of SP-mediated immune modulation in sheep and may identify candidates to restore immunotolerance and fertility of cryopreserved ram sperm.

  1. 1. Schjenken, J.E. and S.A. Robertson, The female response to seminal fluid. Physiological reviews, 2020. 100(3): p. 1077-1117.
  2. 2. Salamon, S. and W. Maxwell, Storage of ram semen. Anim Repro Sci, 2000. 62(1-3): p. 77-111.
  3. 3. Rickard, J.P., et al., Seminal plasma aids the survival and cervical transit of epididymal ram spermatozoa. Reproduction, 2014. 148(5): p. 469-478.
  4. 4. Warr, S., et al., Seminal plasma protects frozen-thawed ram spermatozoa from neutrophil attack: a complement mediated dynamic. Reproduction, 2025. 170(2).
  5. 5. Leahy, T., et al., Quantitative Proteomic Analysis of Seminal Plasma, Sperm Membrane Proteins, and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane. Proteomics (Weinheim), 2020. 20(12): p. e1900289-n/a