Equine Reproductive Research: Focus on the Stallion
There have been many advances in preserving, evaluating and utilizing stallion semen.
Cryopreservation of semen allows stallions to cover more mares, but not all semen freezes well. | Arnd Bronkhorst Photography

Be it artificial insemination, embryo transfer or intracytoplasmic sperm injection, quality sperm is required for any assisted reproductive technique (ART) to be successful. Consider the effect of cooling and freezing on stallion semen, for example.  

“Cryopreservation of spermatozoa still proves challenging because the semen from different stallions can react very differently such that freezing extenders and protocols need to be individually tailored,” advised Tom Stout, VetMB, PhD, of the Department of Equine Sciences at Utrecht University in The Netherlands. 

Further, he feels that assessing the quality of fresh, chilled/cooled or cryopreserved samples also needs improvement.  

Working toward a common goal of optimizing pregnancy rates, several research groups have recently published studies looking at how best to cryopreserve sperm, analyze semen to identify quality samples, and predict the chances of a successful AI. These studies are summarized in this article.  

Advances in Cryopreservation 

Cryoprotectants are contained in freezing extenders to preserve stallion semen.
Cryoprotectants, which are contained in freezing extenders, include milk, egg yolk and 2-5% glycerol. | Arnd Bronkhorst Photography

Cryopreserving sperm allows more than one mare to be inseminated by the same ejaculate without needing to use the sample within 48 hours of collection. Samples can be used for any of the assisted reproductive techniques, and high-value genetic banks can be created by storing samples.  

“The cryopreservation process starts by centrifuging the semen to remove seminal plasma and concentrate the sperm pellet,” described Stout. “The pellet is then diluted in an extender containing cryoprotectants (such as glycerol or dimethyl formamide) and membrane protectants (such as egg yolk). Finally, cooling is performed either in a programmable freezing machine (nitrogen vapor) or at a set height above liquid nitrogen.” 

Cryopreserving stallion semen maintains biological samples in a state of suspended animation, maintaining the fine structure of cells. That is in theory, at least. Like other aspects of animal reproduction, horses do not follow the same rules as other species.  

For example, bull semen has 60% to 70% motility post-thaw, but stallions have 40% to 60% at best (Contreras et al. 2023). In fact, some stallions have such poor post-thaw motility they are classified as “poor freezers.” 

Much effort has therefore gone into finding ways to improve equine sperm cryopreservation. But, as relayed by Contreras et al., individual stallions respond differently to cryopreservation, which makes creating a cryopreservation “recipe” difficult. 

“As semen has a high individual variability, no two stallions have the same chemical composition, and the reaction of sperm for each individual to the freezing process varies,” wrote Contreras and colleagues.  

Traditional semen freezing extenders are comprised of milk, egg yolk and 2-5% glycerol, which are different types of cryoprotectants. Some stallions, however, are sensitive to glycerol. Therefore, alternative cryoprotectants are required, such as ethylene glycol, propylene glycol, amides (e.g., dimethylformamide) or dimethyl sulfoxide.  

According to Contreras et al., “The highest cryosurvival rates are obtained with 500 mM dimethylformamide, 250 mM glycerol and 500 mM ethylene glycol.” 

Stout said, “Cryoprotectants are contained in commercial freezing extenders, usually glycerol or a combination of dimethylformamide and glycerol. But some commercial semen freezing centers/practitioners prefer to make their own extenders, adding their own choice of cryoprotectants.” 

Choice of nonpenetrating cryoprotectants can also affect post-thaw survival of sperm. Studies show that using a higher percent egg yolk generates better post-thaw results. Commercial products with either 4% egg yolk or 20% egg yolk plus lactose-EDTA are available.  

Stout added, “You can also often find extenders with or without the penetrating cryoprotectants and can add them yourself.” 

Contreras et al. suggested that in an ideal world, the cryoprotectants—both the penetrating and nonpenetrating components—would be optimized for each stallion given the individual variation of each stallion to the cryopreservation process. 

According to Stout, many practitioners attempt to optimize cryoprotectant concentrations.  

“They often have two or three preferred extenders that they test on a new stallion and then select the one that works best,” Stout explained.  

It is well known that cryogenically preserved sperm cells are damaged by free radicals such as reactive oxygen species. While some free radicals are needed for capacitation, for example, excess reactive species can cause extensive cellular damage and induce apoptosis (programmed cell death). Many of the naturally occurring antioxidants are found in the seminal fluid, but this fluid is removed prior to freezing as some of its components have a negative effect on cryopreservation. 

“Adding antioxidants to the cryopreservation mix can improve post-thawing motility and other functionality parameters,” wrote Contreras et al. “Such antioxidants can include coenzyme Q10, bovine serum albumin, zinc sulfate, epigallocatechin-3-gallate and resveratrol.” 

The antioxidant cholesterol-loaded cyclodextrin (CLC) also functions as a membrane stabilizer, improving cryosurvival and motility rates.    

Stout added that, “Some antioxidants may be included in the freezing extender, but what they are is often not known because commercial cryoprotectants are proprietary products. Antioxidants can be added to cryoprotections, which some practitioners already do.”  

Advances in Assessing Sperm Quality 

Woman analyzing stallion semen motility under microscope.
Stallions have 40-60% post-thaw motility at best, and research has focused on improving cryopreservation. | Arnd Bronkhorst Photography

When performing AI with either cool-stored or frozen-thawed semen, the quality of the sperm inseminated must be determined. Under field conditions, sperm motility and other indicators of sperm quality such as morphology and plasma membrane intactness (i.e., viability) also need to be recorded and analyzed. 

“Both type and incidence of the sperm morphologic features are important for predicting the stallion’s fertility potential,” said Camilo Hernández-Avilés, DVM, PhD, an equine theriogenology resident at the Department of Large Animal Clinical Sciences, Texas A&M University. “Given the subjective nature of sperm morphology analysis, the technique utilized—and the evaluator’s experience—will play an important role in the analysis and interpretation of sperm morphology.”  

Hernández-Avilés and colleagues therefore conducted a study (Theriogenology 2023) in which they used four different techniques to analyze morphologic abnormalities in stallion sperm:  

  1. Eosin/nigrosin staining (EN) 
  2. Dip-Quick staining (DQ) 
  3. Phase-contrast microscopy (PH) 
  4. Differential interference contrast microscopy (DIC).  

Using these techniques, samples from stallions with high-quality semen (>57% morphologically normal sperm), moderate quality (23-56% morphologically normal sperm), or low-quality samples (<22% morphologically normal sperm) were included. Each sample was analyzed by three different evaluators.  

The ultimate goal was to determine if the technique and/or the intrinsic sperm quality level affected the evaluators’ ability to evaluate sperm morphologic abnormalities using three commonly used techniques, explained Hernández-Avilés. 

They found that the level of agreement between EN, DQ, PH and the gold standard (DIC) varied depending on the sperm quality level. In the high-quality sperm group that had few morphologically abnormal sperm, the evaluators consistently identified normal sperm.  

Disagreements between evaluators in terms of sperm morphology, however, were greatest in the moderate quality group.  

“This, in part, could be due to the lower severity of abnormalities compared to the low-quality sperm group,” explained Hernández-Avilés. “When intrinsic sperm quality is low, then the abnormalities may be more severe and therefore easier to recognize regardless of the technique used.”  

The study authors also found that, across all sperm quality levels, the agreement between PH and DIC was higher than either EN or DQ and DIC. Specifically, DIC and PH appear more “sensitive” at detecting subtle changes in sperm morphologic abnormalities such as abnormal or bent midpieces. DQ and EN underestimated the frequency of these defects.  

“Both preparation technique and evaluator ability are important because underestimating sperm morphologic abnormalities can greatly alter the clinical interpretation of the stallion’s fertility potential,” concluded Hernández-Avilés.  

That said, both DIC and PH are less commonly used in private practice settings because of the expenses associated with equipment.  

“While EN or DQ might underestimate certain sperm morphologic abnormalities, it is always better to analyze sperm morphology than not do it at all,” stated Hernández-Avilés. 

“Texas A&M University provides sperm morphology analysis via PH or DIC,” he said. “Samples can be fixed and shipped through regular mail services providing an opportunity for practitioners that do not have the capabilities to perform their own sperm morphology analysis to not miss very important information regarding the intrinsic sperm quality of stallions.” 

Sperm Motility and Metabolic Parameters As Predictors of AI Success 

Predicting the probability of successful artificial insemination (AI) based on semen parameters alone would benefit the equine industry immensely.  

“If a low success rate was predicted, then a second insemination during the same estrous cycle could greatly improve the likelihood of pregnancy,” said Ashlee Medica, Bachelor of Biotechnology (Hons), PhD candidate from the School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Australia.  

In a recent study, Medica’s research team sought to determine if evaluating semen motility coupled with sperm metabolic activity could predict the outcome of AI. They collected semen samples from seven Standardbreds from a pacing stud in New South Wales, Australia. Semen parameters were measured in fresh samples and again in samples that had been cooled for 24 hours and rewarmed to 37oC (chilled).  

“These two conditions mimic protocols used in clinical AI practice and were used to determine the most appropriate time to perform sperm analysis prior to insemination,” Medica said.   

Sperm parameter measurements included ejaculate volume, sperm concentrations and sperm motility. Motility was measured using an iSperm Equine portable computer-assisted sperm analysis device.  

“Motility of all samples was also evaluated by their ability to migrate through a Samson™ System isolation chamber,” said Medica. 

The Samson system is a prototype device consisting of a sample chamber separated from a reaction chamber by a 5-micrometer polycarbonate membrane. The Samson System has been designed as a motility “challenge” to isolate the sub-population of sperm that have the potential to ascend the mare’s reproductive tract and initiate fertilization.  

Medica explained, “The Samson device works by placing semen into the sample chamber, and the highly motile sperm migrate through the membrane into the reaction chamber containing WST-1. (“WST-1 is a membrane-impermeant probe, which in the presence of equine spermatozoa becomes reduced to a formazan product that can be readily detected spectrophotometrically.” This quote is from the study “Predicting the outcome of Thoroughbred stallion matings on the basis of dismount semen sample analyses” published in Reproduction, March 2023.) Within the reaction chamber, only sperm with high metabolic activity (and hypothesized to have high fertilizing potential) are able to reduce WST-1 into formazan, a blue-black compound. The reduction of WST-1 to formazan can be easily measured spectrophotometrically.”  

In total, 22 of 46 inseminations (47.8%) resulted in early positive pregnancy performed at 14 days post ovulation as determined via rectal ultrasound. 

Medica said that this is a normal outcome for AI performed with chilled semen. However, Stout said that larger studies typically report pregnancy rates of about 55-60% for chilled semen. 

An algorithm was created that incorporated all sperm parameters, WST-1 reduction, and mare and stallion information. The overall accuracy of predicting pregnancy was 87.9% for fresh semen and 95% for chilled semen.  

Based on these results, Medica et al. concluded that sperm analysis using the rapid field techniques iSperm and the Samson System can yield valuable information for predicting the outcome of AI. 

Future Directions 

Despite these advances, continued research to achieve desirable outcomes such as Day 14 and Day 42 pregnancies (per first cycle, ideally) that progress to live foals with low rates of early embryonic losses is needed.  

“We still don’t really have a way to identify ‘fertile’ sperm and therefore still cannot accurately predict fertility,” said Stout. “Available tests may work well for some individuals but will be way off the mark for others.”

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