Seminal plasma protects frozen-thawed ram spermatozoa from neutrophil attack: a complement mediated dynamic

Abstract

In brief: Frozen-thawed ram spermatozoa show reduced fertility following cervical artificial insemination, likely attributed to an elevated immune response in the ewe. This paper identifies the immunoprotective effect of ram seminal plasma (SP) against polymorphonuclear neutrophil binding, highlighting potential for fertility improvement.

Abstract: Following cervical artificial insemination, frozen-thawed (FT) ram spermatozoa display reduced fertility compared to fresh spermatozoa, irrespective of sperm motility and viability, likely attributed to an elevated immune response in the ovine cervix. SP has previously been shown to be beneficial to sperm transport in the ovine cervix, yet the components responsible for this protective effect remain unknown. As such, the present study examined the immune dynamics of frozen-thawed ram spermatozoa with polymorphonuclear neutrophils (PMNs), utilising a neutrophil binding assay. The effect of 25% v/v SP supplementation on binding susceptibility was also investigated. A portion of SP was heat-treated before incubation (HTSP) to isolate the impact of SP proteins. The presence of SP significantly reduced sperm-PMN binding (37.15 ± 0.02%, 46.59 ± 0.02%, 38.83 ± 0.02%) compared to FT alone (62.83 ± 0.02%, 75.74 ± 0.02%, 56.0 ± 0.03%) across all serum groups (no serum, serum and heat-treated serum, respectively). HTSP showed comparable binding susceptibility to the FT treatments, indicating that the protective effect of SP is attributed to a heat-labile factor. Serum significantly increased sperm-neutrophil binding irrespective of SP treatment. However, this was reduced to serum-free levels following heat-treatment, suggesting sperm-neutrophil dynamics are further mediated by protein components within ewe serum, likely complement proteins. The viability of sperm or the presence of freezing medium did not influence PMN binding. Furthermore, PMN viability and therefore binding were not influenced by the presence of serum or SP. Together, the current study presents new evidence for the immunoprotective properties of SP in sheep, which could be leveraged to aid transit through the cervix.

Keywords: cervical AI; cryopreservation; immune; sheep.

Figure 1

DIC images of ram spermatozoa interactions with polymorphonuclear neutrophils (PMNs) at 40× magnification following Wright’s staining. Arrowheads indicate the ram spermatozoon head (red), midpiece (blue) and tail (black). Solid brackets indicate inactive PMNs while dashed brackets indicate activated PMNs. NETs are denoted by a solid black arrow. (A) Inactive PMN not bound to spermatozoa, characterised by a defined multi-lobular nucleus and intact plasma membrane.(B) Active PMN unbound to spermatozoa, displaying early stages of extrusion of NET, characterised by filaments extending outward from the cell, derived from DNA from the cell’s nucleus. (C) Activated PMN phagocytosing ram spermatozoa via attachment at the head. (D) Activated PMN with fully extruded NET networks with two captured ram spermatozoa.

Figure 2

Percentage of neutrophils bound to ≥1 spermatozoa. Sperm incubated with isolated PMNs (no serum), 7.5% serum (serum), 7.5% heat-treated serum (HT serum). Data are presented as an average over ram (n = 3) and replicate (n = 3). Columns with different superscripts denote significant differences (P < 0.05) across treatments.

Figure 3

Percentage of neutrophils bound to ≥1 spermatozoa. Frozen-thawed spermatozoa (FT) and FT spermatozoa treated with 25% seminal plasma (FTSP) or heat-treated seminal plasma (FTHTSP) were incubated 1:1 (v/v) with isolated PMNs (no serum), 7.5% serum (S), 7.5% heat-treated serum (HTS). Data are presented as an average over ram (n = 3) and replicate (n = 4). Columns with different superscripts denote significant differences (P < 0.05) across treatments.

Figure 4

Levels of complement C5 protein in the supernatant of the neutrophil binding assay. Frozen-thawed sperm (FT), frozen-thawed sperm supplemented with 25% (v/v) seminal plasma (FTSP), and frozen-thawed sperm supplemented with 25% (v/v) heat-treated seminal plasma (FTHTSP) were incubated 1:1 (v/v) with isolated PMNs (no serum), 7.5% serum, 7.5% heat-treated serum (HT serum) for 120 min. Supernatant was then assessed for C5 concentration using ELISA. Data are presented as an average over pooled rams (n = 3) and replicates (n = 2).

Figure 5

Viability of PMNs. Isolated PMNs were incubated with PBS, SSF, 25% v/v SP or serum, and viability was assessed via Trypan blue exclusion. Data are presented as an average over replicate (n = 4) and treatment. Columns with asterisks denote significant differences (P < 0.01) across time points.