Reducing the effects of heat stress and ageing on sperm DNA in Thoroughbred stallions

Every breeding season, thousands of hours are spent ensuring a mare visits the stallion at exactly the right time, to maximise the chances of pregnancy on the first cover. This not only prioritises the welfare of both horses, it also minimises the costs for breeders.

But breeders know that it doesn’t work every time. Many factors can impact the likelihood of a successful pregnancy, and the AgriFutures Thoroughbred Horses program has been investing in research to better understand these factors and identify ways to reduce their impact.

One project completed earlier this year focused on the stallion’s equally important role in breeding. A mare might be ready, but compromised stallion fertility can mean the breeding window is missed.

As we discussed last year, stallion fertility in particular can be impacted by hot, humid conditions, and can decline further as the stallion ages. With Australian conception rates lower than those in comparable breeding countries, understanding these impacts and how to identify and manage declines in fertility is vital to the breeding industry’s success.

AgriFutures can now share the results of this research which provides practical advice to help stallion managers and owners maximise their success this season.

Too hot to handle

Heat exposure, particularly without opportunities to cool down, is known to reduce stallion fertility, although the underlying mechanisms have not been well understood. Some stallions also appear more susceptible than others. While heat stress has been linked to oxidative stress in humans and other species, its specific effects on stallion fertility had received little research.

Collecting blood and dismount semen samples from commercial breeding stallions allowed a team based at the University of Newcastle - consisting of Narantsatsral Sandagdorj, Róisín Griffin, Ceilidh Jenkins, Dr Zamira Gibb, and Dr Aleona Swegen - to identify heat susceptible stallions and compare biomarkers to understand what part of the process was being damaged by oxidative stress from heat.

“Oxidative stress happens when unstable, damaging molecules called free radicals outpace the body’s antioxidant defences,” said Dr Swegen. “It can happen in the whole body or in an isolated organ or cell. Oxidative stress occurs as a result of things like inflammation or exposure to toxins or radiation - but also, as we’re learning, with heat stress.”

Sperm are particularly susceptible to oxidative stress, because they are both very small and have a very powerful metabolism. They also have very limited ability for antioxidant defence.

The full effects of a heat event might not be noticed for several weeks when breeding Thoroughbreds, as the sperm most sensitive to these events may take up to three weeks to be ejaculated. The additional two-week wait for the first pregnancy scan can further delay detection, resulting in valuable time being lost using potentially compromised sperm.

As the team discovered in this study, stallions who were more susceptible to heat stress had a reduced capacity for antioxidants in their seminal plasma, increased oxidative degrading of lipids, and more systemic inflammation, all of which correlated with lowered sperm motility. Additionally, oxidative stress was damaging the DNA in the sperm themselves.

“Oxidative stress can kill sperm cells or make them incapable of fertilising an egg, showing up as reduced fertility,” Dr Swegen said.

“There’s a grey zone where the cells are alive and still capable of fertilisation, but their DNA has been damaged by the oxidative attack, usually during sperm development. You may still get a pregnancy, but if the DNA is compromised, this can lead to pregnancy loss or compromised offspring health.”

Approximately one in five of the stallions in the study were identified as heat susceptible, which sends a stark warning to studs to seriously consider closer assessment of their own stallions so they can catch this kind of issue early.

Under the microscope

All observant stallion managers will routinely have their stallion’s sperm assessed under the microscope for obvious physiological problems, but DNA damage isn’t always so easy to spot.

“Damaged sperm cells can be motile and have normal morphology,” Dr Swegen said. “Specific assays - laboratory tests - are needed to assess DNA damage, and there are several assays that have been developed for this purpose. These are used routinely in IVF/human fertility medicine when a sperm related problem is suspected.”

All of the different assays work slightly differently and detect different types of DNA damage, so part of the project was dedicated to working out which one would be most useful to detecting the type of damage that could impact a successful pregnancy.

“We found that the comet assay was the most sensitive assay in stallions and was related to fertility in the field,” said Dr Swegen. “It is called a comet because the assay uses an electric field to spread out the fragmented DNA and it looks like a comet under the microscope. Damaged DNA travels further and makes a longer ‘comet tail’. Alkaline comet assays measure both single- and double-strand DNA breaks.”

Narrowing the test range down to comet assays gives stud farms a definitive tool to reach for when sperm appears fine under the microscope but they are still experiencing fertility problems.

Keeping your cool

Heat stress can be combatted with simple adaptations to a stallion’s routine and environment. Giving stallions the opportunity to get respite from heat is critical to reducing the risk of heat stress.

“Earlier in the study, we found that on some farms, stallions were housed in barns overnight and in the paddock during the day, meaning they were exposed to the heat of the day outside, and then going into barns that held the heat in during the night, while the air outside was much cooler,” Dr Swegen said.

“So stallions were missing out on that overnight respite from the heat, which we have found is really important.”

Monitoring inside temperatures and utilising fans, air conditioners, misters, and evaporative cooling with hosing stallions off are all still useful methods for reducing the risk of heat stress.

In addition, Dr Swegen and the team have developed a validated equine THI (temperature humidity index) to assist studs and stallion managers with anticipating the likelihood of heat stress.

“The concept of THI has been around for a long time,” Dr Swegen said. “It’s essentially an equation that combines temperature and relative humidity into one number and is used quite widely in assessing heat stress in livestock, but it has not really been looked at in horses.”

The study concluded that a THI of 80 or more indicated a high risk of heat stress, but Dr Swegen stressed that susceptibility to heat-induced subfertility is very stallion dependent.

“In terms of stallion fertility, we would recommend monitoring cumulative heat exposure and inadequate overnight cooling, rather than relying exclusively on the temperature or THI recorded on the day of breeding,” she said. “Sudden increases in temperature, and especially nights when temperatures don’t come down during the night to provide respite, tend to be the most likely things to affect a stallion’s fertility.”

Age is not just a number

Increasing age has been linked to decreased fertility before, but the team were keen to turn the lens on the Thoroughbred population. Jenkins, Griffin, Dr Swegen, and Dr Gibb along with Dr Natasha Hamilton and Rose Upton examined the performance of 33,546 offspring of 35 stallions in relation to the sire’s age at conception, and how the stallions’ fertility changed over their lifespan.

It can take years to determine whether a stallion will become a high-value sire. With around three years between conception and a foal’s racetrack debut at age two, a stallion may need five or more years to produce enough racing offspring for his performance as a sire to be assessed.

Increasing age was associated with with poorer outcomes across all measures. Stallion fertility began to decline from age 14, with the sharpest drop in stallions aged 19 or older. Stallions aged eight or younger were significantly more likely to produce a live foal than those aged 19 or older.

Stallions aged eight and under were significantly more likely to produce a live foal than stallions 19 or older.

The proportion of offspring that reached the racetrack also declined with stallion age. On average, 81.2% of offspring from stallions aged eight or younger raced, compared with 66.7% of offspring from stallions aged 19 or older.

Success on the track was linked in a very similar way. The youngest bracket resulted in 71.2% winners to runners, whereas for those born to the stallion aged 19 or older, the percentage dropped to 57.2%, with that percentage also dropping incrementally across each intermediary age bracket. The proportion of stakes performers, stakes winners, and Group winners also declined with stallion age.

This does not mean older stallions cannot remain fertile or produce champions. Many continue to do so throughout their breeding careers and are champions in their own right.

The late Snitzel serves as one such example; he achieved an end of season fertility rate of 82.7% in 2023 as a 21-year-old, with 109 2-year-olds ready to run for him this year. Last season, the 2-year-olds he conceived as a 20-year-old included two Group 1 winners and five further stakes winners. It is just important to acknowledge that age has a quantifiable relationship to racetrack success.

The mare also plays an important role, although her contribution may be harder to quantify.

“In the published study, we did look at mare age to make sure it wasn’t confounding our results, but we couldn't really look at things like mare quality or performance because these are subjective,” Dr Swegen said.

“The mare side is really interesting too and our team is working on a study where we crunch the numbers on mare factors. We will be looking at the effects of mare age on fertility and offspring performance, and how these interact with the stallion effects.

“This has been a little bit more challenging because a single mare never has quite as many offspring as a stallion might, so there are fewer data points to work with, and it's harder to generate the robust datasets like the one we built for the stallion ageing study.”

Dr Swegen is hopeful the team will be able to share the results of their study into the mare’s role in the near future,

The next steps

The study found a clear link between older paternal age and poorer racetrack performance across all measures, but the data did not reveal the underlying cause. One possible explanation is sperm damage caused by oxidative stress.

“There can be increased oxidative stress in the body as we get older because of tissues ageing, reduced antioxidant capacity, and altered function in our organs and in our cells,” Dr Swegen said.

“If oxidative stress is affecting the DNA in the sperm and older animals are less able to repair that damage, the damage is more likely to be carried through to the offspring.

“But there could be a few different explanations for the reduced offspring performance - for example epigenetic effects or increased mutational load in the developing sperm cells. So it will be really exciting to dig deeper into these processes in our future studies.”

An area the team explored to support fertility while they examine other factors in sub and declining fertility was supplementation of NT Factor Lipids + L-carnitine, which together have shown promise in a laboratory setting.

“Combined supplementation of the two reduced sperm head defects significantly, suggesting a potential synergistic effect,” Dr Swegen said. “NT Factor Lipids alone significantly reduced sperm DNA fragmentation and reduced lipid membrane scrambling compared with the other treatments.

“In short, the supplements improved sperm quality parameters, the results are promising - but we have yet to test them in the field to confirm improved fertility.”

Building on these two studies, the team is preparing to test the supplements in the field as part of the next phase of research.