Identifying Heat-Susceptible Stallions Using a Novel Environmental and Statistical Modelling System

Systemic heat stress is detrimental to sperm production and function, via the induction of oxidative stress and DNA damage. However, this phenomenon is yet to be thoroughly examined in a field setting relevant to commercial breeding horses. The duration of spermatogenesis makes direct observation of the heat-fertility relationship challenging, but anecdotal evidence suggests that some stallions are more susceptible to heat-stress than others. This study aimed to design a model capable of identifying heat-susceptible stallions, by utilising environmental monitoring, statistical modelling, and oxidative DNA damage assessments. Temperature and humidity loggers were installed in the stables and paddocks of 46 Thoroughbred stallions during the 2017 and 2018 breeding seasons. Dismount semen samples were collected weekly from these stallions (2017 n = 486; 2018 n = 318). Samples were diluted (2:1, extender:semen) and sperm concentration and motility were recorded (iSperm™). Samples were then snap frozen for oxidative DNA damage assessment, using a novel 8OHdG assay. This assay can inform on both total DNA damage, and the abundance of 8-hydroxy-2’-deoxyguanosine (8OHdG) DNA adducts—thereby measuring the proportion of damage caused by oxidative stress. Fertility results were also collated at the conclusion of each breeding season. A polynomial distributed lag model was used to identify stallions whose fertility was adversely affected by heat events, based on correlations between fertility rates (per-cycle conception and first cycle conception rates) and maximal ambient temperature and humidity. A subpopulation of 18 stallions were identified as being susceptible to heat stress (r≥ 0.50; p ≤ 0.05). Of these, six stallions (accounting for 13% of the total population) demonstrated consistent negative correlations between sperm DNA damage and fertility, and consistent positive correlations between DNA damage and ambient temperature and humidity (r ≥ 0.50; p ≤ 0.05). These findings indicate that there is a subpopulation of stallions that are particularly susceptible to heat-induced subfertility, with a further subset affected by heat- induced sperm DNA damage. This study presents a novel protocol that can effectively identify heat-sensitive stallions and could easily be implemented on commercial stud farms.