Climate, Fertility and Oxidative Stress: Systemic and Localized Responses Associated with Ambient Heat-Induced Subfertility in Stallions

Ambient heat exposure reduces male fertility in mammals with scrotal testes. Our previous work has demonstrated that some stallions are more susceptible to ambient heat-related subfertility than others, yet the mechanism for heat-induced subfertility remains uncertain, limiting both diagnosis and preventative measures. This study sought to define how the phenotype of stallions susceptible to heat-induced subfertility differs from that of more resilient animals, by measuring the systemic (blood plasma) and localized (reproductive tract) inflammatory and oxidative stress markers of sperm concentration, sperm motility assessments, total antioxidant capacity (TAC; in blood and seminal plasma), malondialdehyde (MDA; in blood and seminal plasma), oxidized guanine species (8-OH-2dG; in blood plasma and spermatozoa DNA), sperm DNA damage (assessed via Halo, SCSA (Sperm Chromatin Structure Assay) and CMA3 (Chromomycin A3)), and c-reactive protein (CRP; in blood plasma). Post-breeding dismount semen samples (n = 357) and blood plasma samples (n = 97) were collected from 31 stallions at commercial thoroughbred studs throughout one breeding season (NSW, Australia). A subset of stallions (16%) was deemed heat-induced subfertility-susceptible (HISS) stallions. These animals showed reduced seminal plasma antioxidant capacity, increased systemic and localized lipid peroxidation, and distinct systemic inflammatory response. Seminal antioxidant capacity was found to be strongly associated with impaired sperm motility (r = 0.739 * vs. r = −0.059). The plasma c-reactive protein of heat-susceptible stallions correlated to heat exposure (r = 0.597 *) and affected sperm motilities (r = −0.527 **, r = −0.434 *). Systemic oxidative DNA damage (8-OH-2dG) also increased following heat events (r = 0.862 ***) and correlated with fertility losses (FCP: r = −0.740 **, PCP: r = −0.603 *). Non-HISS stallions displayed greater variability in systemic antioxidant status and robust response following heat exposure (r = 0.307 *) and localized antioxidant capacity was more strongly correlated to systemic antioxidant capacity than in the heat-susceptible group (r = 0.897 *** vs. r = 0.482 **). We demonstrate that impaired antioxidant responses, altered redox balance and suppressed acute-phase inflammatory signalling are key features associated with heat-induced subfertility in stallions and highlight biomarkers that could be used to identify animals with heat-susceptible fertility.