Background. We previously reported that heat-induced subfertility-susceptible (HISS) stallions show reduced seminal antioxidant capacity, elevated lipid peroxidation and markedly reduced C-reactive protein (CRP), pointing to a suppressed acute-phase response consistent with heat acclimation. Here, we aim to further characterise this phenotype and investigate upstream inflammatory and heat-shock mediators driving susceptibility.
Methods. Post-coital dismount semen (n=357) and blood plasma (n=97) were collected from 31 thoroughbred stallions across four studs over one breeding season (NSW, Australia). HISS (n=5) and non-susceptible (n=19) stallions were classified by significant negative correlations between weekly first-cycle and per-cycle pregnancy rates and peak climate indices over the preceding two weeks. Blood plasma was assayed by ELISA for IL-6, HSP70/27/90, STAT3, CRISP3, creatinine, cortisol and seminal plasma for IL-6, CRISP3, STAT3, and cortisol. Groups were compared by Mann–Whitney tests; correlations used mixed-effects models with animal as a random effect.
Results. HISS stallions showed elevated blood plasma IL-6 (3.9-fold; p=0.0002), HSP70 (p=0.02), HSP27 (p=0.02) and STAT3 (2.1-fold; p=0.02), with reduced CRISP3 (p=0.01), cortisol (p=0.005) and CRP (66-fold; p=0.0001); HSP90, creatinine and all seminal-plasma equivalents were unchanged. Inflammatory marker associations were different between groups: IL-6 tracked STAT3 only in HISS (p<0.001) and CRP only in non-HISS (p<0.001), while STAT3 tracked HSP70 only in non-HISS (p=0.008). IL-6 and STAT3 were inversely related to seminal antioxidant capacity, and cortisol tracked heat exposure, in HISS alone.
Conclusion. Susceptibility is associated with a modified, rather than suppressed, systemic response: CRP is uncoupled from an elevated IL-6/STAT3 signal (a dissociation with precedent in impaired hepatic acute-phase states). Reduced seminal antioxidant capacity in these animals may therefore reflect antioxidant consumption by reactive oxygen species generated through the elevated IL-6/STAT3 response, itself consistent with disinhibition by reduced CRISP3.