Age-related DNA damage in stallions: an ongoing investigation

Abstract

Foals sired by younger stallions record significantly higher racing speeds (Sharman.et.al. Royal Society Open Science. 2022;9:220691), and more race wins than foals sired by older stallions (Brazil.et.al. Honours Dissertation. University of Limerick. 2016). We hypothesize that this may be due to an age-associated increase in sperm DNA damage, as has been demonstrated in other species. Therefore, this study aimed to investigate the relationship between stallion age and sperm DNA damage. Post-coital dismount semen samples (n=75) were collected weekly from 46 commercial Thoroughbred stallions in the Hunter Valley (Australia). Samples were immediately diluted (2:1, EquiPlus:semen), and the high-density spermatozoa were isolated using EquiPure centrifugation to reduce contaminants (somatic cells and other debris). Any samples that were contaminated with urine were excluded from the study. Sperm count and motilities were recorded onsite (iSperm™), and samples were snap frozen for DNA damage analyses. Management information, including pregnancy scan results, were also collected. For this study, stallions aged ≤9 years were categorized as “young” while those aged ≥12 were categorized as “old”. Stallions between 9 and 12 years were excluded to improve the likelihood of observing an age-related difference. Mare age and fertility status were similar across both groups. All data were checked for normality (Shapiro-Wilk) and analyzed using unpaired t-tests. Sperm count and motility did not differsignificantly between age cohorts. DNA strand breaks were significantly higher in spermatozoa from older stallions compared to younger stallions (alkaline comet assay: 19.7±0.55% vs 15.4±0.57% mean tail intensity; P≤0.05, and sperm chromatin dispersion or ‘Halo’ assay; 0.48±0.045in vs 0.70±0.074in ‘halo’ area; P≤0.05, respectively). These breaks may be attributed to poor chromatin packaging, as older stallions had a concomitant deficiency of sperm protamines compared to young stallions (11.6±1.53 vs 6.9±1.07 chromomycin A3 positive cells; P≤0.05, respectively). Although younger stallions had lower levels of DNA strand breakage, they had more oxidized DNA adducts (8-hydroxy-2′-deoxyguanosine (8-OHdG) assay; P≤0.05). Interestingly, regardless of age, positive correlations between the proportion of oxidized DNA adducts and fertility were observed (R2=0.269 for younger, R2=0.483 for older stallions; P≤0.05). This is likely a reflection of the increased use of oxidative phosphorylation, and subsequent ROS production, in higher fertility ejaculates (Gibb.et.al. Biology of Reproduction. 2014;91(3):77). In conclusion, while stallion sperm DNA damage rises with age, it does not appear to influence fertility outcomes. However, further research is needed to determine if age-related sperm DNA damage compromises the next generation's genome. In addition, we must identify the most vulnerable genomic loci for sperm DNA damage and determine how these DNA lesions may contribute to reduced racing performance and health in offspring.