Unless you work exclusively with Thoroughbreds, if you’re involved in breeding horses, you’re probably familiar with cooled semen.

So noted Lorenzo G. Segabinazzi, DVM, MS, PhD, assistant professor of theriogenology at the Ross University School of Veterinary Medicine, in St. Kitts and Nevis, during his presentation on the topic at the 2022 American Association of Equine Practitioners Convention, held Nov. 18-22 in San Antonio, Texas.

Cooled semen has made breeding horses that live long distances from each other much simpler and cheaper. But semen quality remains important to a successful breeding program.

In a stallion deemed a “good cooler,” the sperm quality “almost doesn’t change” in the 24 to 48 hours—and sometimes as long as 72 hours—after semen collection, Segabinazzi said. However, “bad cooler” stallions can see sperm quality drop 12 to 24 hours after collection.

What’s a mare owner to do when they’ve induced ovulation and the semen that shows up is of poor quality? Skip the breeding cycle? Breed using poor-quality semen? Or try to improve the semen?

In his study, Segabinazzi hypothesized that poor-quality cooled semen could be improved via processing and re-extension—a process that can improve the longevity of sperm motility. To find out, his team assessed semen parameters and fertility of cooled-stored semen processed and re-extended in three types of extenders.

“The aim is to develop an alternative method for improving semen quality and fertility after the cooling process,” he said.

Segabinazzi’s research team classified 21 stallions as good coolers (8), moderately bad coolers (6), and severely bad or no coolers (7), defined as having less than 25% total motility 24 hours after cooling.

They began by cooling semen in a simple milk-based extender for 24 and 48 hours. Then, they processed the semen by centrifugation or SpermFilter (a sperm concentrator that does not involve centrifugation) to remove the supernatant (excess fluid). They then resuspended the semen in skim milk (BotuSemen), skim milk with pentoxifylline (BotuSemen Turbo), or egg yolk (BotuCrio) based extenders.

In addition to motility, the researchers assessed plasma membrane integrity (PMI) and mitochondrial membrane potential (HMMP)—two indicators of semen quality—in all samples. Segabinazzi reported that sperm motility increased in all samples with the resuspended egg yolk extender. Both the egg yolk and skim milk with pentoxifylline extenders improved HMMP. Semen processed via SpermFilter had better PMI than centrifuged semen.

They also looked at pregnancy rates of 18 mares (66 cycles) inseminated with semen from nine stallions characterized as bad coolers, said Segabinazzi. Mares inseminated with SpermFilter/skim milk pentoxifylline semen had 50% pregnancy rates, while mares inseminated with SpermFilter/egg yolk semen had 68% pregnancy rates. The control group (unprocessed semen) had a pregnancy rate of 14%.

The results showed that both SpermFilter and centrifugation can improve bad and no-cooler semen, though Segabinazzi said he prefers SpermFilter out of concern for mechanical damage centrifugation can potentially cause. He said his first choice for an extender is egg yolk, though skim milk with pentoxifylline might also be an effective option.