Environmental and biochemical factors can compromise the DNA integrity of equine spermatozoa. Several sperm DNA damage assays exist, but their relative prognostic value has not been assessed in the horse. This study compared four assays for detecting DNA damage in stallion spermatozoa: the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis (comet) assay, and the 8-hydroxy-2′-deoxyguanosine (8-OHdG) ELISA. Experiment 1 assessed the effect of hydrogen peroxide (H₂O₂), a driver of oxidative stress, on sperm DNA damage across increasing concentrations and incubation times. Experiment 2 assessed arachidonic acid (AA), an upstream driver of endogenous oxidative stress. Experiment 3 evaluated the prognostic value of each assay using semen from Thoroughbred stallions during periods of high and low fertility. Across experiments, the comet assay detected DNA damage at lower stressor doses and shorter incubation times than the other assays and was the only assay to distinguish semen from low-fertility stallions from that of high-fertility stallions. The SCSA, SCD, and 8-OHdG ELISA were comparatively less sensitive in detecting significant differences under the same conditions. In conclusion, the comet assay demonstrated the most consistent responsiveness in detecting sperm DNA damage in stallions under the tested conditions.

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.

Reproductive ageing influences fertility and offspring fitness across mammals. In humans and model organisms, paternal age has been associated with changes in sperm quality and genomic integrity, including oxidative stress, DNA damage, and epigenetic alterations; however, these mechanisms have not been directly examined in large equine populations. Thoroughbred racehorses provide a system to investigate paternal age effects at scale using detailed breeding records. In this retrospective observational study, with data from 35 retired stallions and 33,546 progeny, we assessed associations between sire age at conception, reproductive outcomes and progeny racing performance using generalized linear mixed-effects models. Progeny conceived when stallions were ≤ 8 years were more likely to race (odds ratio [OR] = 2.21, 95% CI 1.93–2.52), place in a stakes race (OR = 3.71, 95% CI 2.74–5.04), and win a stakes race (OR = 3.58, 95% CI 2.43–5.29) than those conceived at ≥ 19 years. These findings demonstrate population-level associations between paternal age and reproductive outcomes.