Stallion Semen Freezing 101

Once the semen is frozen, it can be stored indefinitely in liquid nitrogen tanks until it’s time to breed a mare.

Photo: Courtesy Sandro Barbacini, DVM

Nowadays, an actively competing stallion in the United Kingdom can sire a foal in California without the mare ever leaving the state. International distribution is just one of the ways frozen semen is helping breeders around the world. But how can veterinarians take fresh semen, freeze it solid, ship it, and then wake the sperm back up? It’s a complex process.

At the 2016 Western Veterinary Conference, Heath King, DVM, Dipl. ACT, shared how veterinarians can implement frozen semen processing in their practices by outlining the steps and reviewing techniques. King is an assistant clinical professor of theriogenology at the Mississippi State University College of Veterinary Medicine, in Starkville.

In addition to creating a global breeding market, frozen semen allows breeders to keep genetic lines alive after a stallion dies or is unable to physically breed any longer, eliminates the need to transport mares long distances for insemination, and eases scheduling difficulties that arise with fresh or cooled semen or live cover, King said.

But it’s not without its drawbacks. For example, processing, shipping, and storing frozen semen can be costly, and not all stallions’ semen can be frozen successfully.

“Tremendous variations in freezability exist among stallions, the cause of which is not known,” King explained. “Generally, when post-thaw motility (or movement capability) is compared, 25% of stallions freeze well, 50% freeze fair, and 25% freeze poorly.

“There is also a subset of stallions that appear to freeze well based on their post-thaw motility, but are unable to produce a pregnancy with frozen semen,” he added.

Because of these challenges, it’s essential that frozen semen is processed properly to allow for the best possible pregnancy rates.

Evaluating Semen

After collection and before freezing, King said, it’s important to evaluate the semen’s concentration (how many sperm are in the ejaculate), progressive motility (movement), and morphology (structure).

“Cryopreservation does not improve semen quality,” he stressed. “Therefore, a decision not to freeze a poor-quality ejaculate can be made before proceeding further.”

In addition to evaluating the semen after collection, King recommended examining it after dilution with a semen extender (more on that in a moment) and again after thawing to “ensure that an adequate number of sperm are available for insemination.”

Once the practitioner is satisfied with the semen quality, it’s time to concentrate it.

Concentrating Sperm

It’s crucial to concentrate the sperm before freezing by separating them from the seminal plasma. The most common method to do so, King said, is with centrifugation. In this process, the semen is placed in a tube and spun at 400 to 600 G for 10 to 15 minutes, he said, for a sperm recovery rate of about 80%.

“The recovery rate can be improved without mechanical damage to the sperm cells by the utilization of a commercially available cushion medium, which allows centrifugation at 1,000 G for 20 minutes,” King added.

A “soft sperm pellet” should result, he said, which can then be easily separated from the seminal plasma.

King noted another method of separating sperm from plasma that doesn’t require a mechanical centrifuge. The Sperm Filter (marketed by Biotech Botucatu, in Brazil), he said, “utilizes a synthetic hydrophilic membrane that allows the passage of seminal plasma only,” keeping the concentrated sperm on top of the membrane.

Concentrated sperm can then be added to an extender.

Semen Extenders

King said extenders—simply, a product added to semen to preserve its fertilizing ability—generally include several components:

  • A pH buffer, which helps keep the semen at a stable pH during processing, freezing, and thawing;
  • A cryoprotectant, most commonly glycerol, which prevents damage to the sperm during freezing;
  • A lipoprotein source, typically an egg yolk, which also serves as a protective coating for the sperm during freezing;
  • A sugar source, which the sperm use as an energy source;
  • Detergents to enhance the protective effects of the lipoproteins.
  • Antibiotics, to prevent bacteria overgrowth.

Veterinarians might observe that not all stallions’ semen responds the same to the different extenders available.

“The logical approach is to test-freeze a stallion in several different extender formulations to determine which produces the best results,” King said. This will allow the practitioner to see which product works best for each individual stallion, troubleshoot if none of the extenders are successful, or make the decision to refer a “problem stallion” to a specialist for further assistance.

King recommended using two extenders when freezing semen: a centrifugation extender, which is also used when preparing cooled semen, and a freezing extender. “The freezing extender is similar to a cool storage extender, but with a higher lipoprotein content and the addition of a penetrating cryoprotectant,” which provides further protection for the sperm during the freezing process, he said.

He recommended adding the freezing extender in a stepwise approach to prevent overdilution.

Packaging and Freezing

Prior to freezing, the semen is cooled to about 5°C (41°F). He cautioned that it’s important to control the cooling rate to prevent the sperm from becoming damaged due to cold shock. He added that the freezing extender manufacturer generally provides cooling recommendations.

Also before freezing, the semen must be packaged in straws, King said. Most commonly semen is packaged in 0.5 mL straws, but he noted that some opt to use 5 mL straws. He noted, however, that the smaller straws generally allow for more accurate control of the cooling and thawing rates, potentially reducing the likelihood of damaging the sperm.

“Straws should be labeled with the horse’s name, registration number, name of the freezing facility, and the freeze code,” King said. A freeze code includes the date of freezing and an identifying letter if more than one stallion is collected in a day, he said.

There are two options when it comes time to freeze the semen, King said: a programmable freezer or a vapor box.

“A programmable freezer provides more precise control of the cooling rate, but it is expensive and has not been shown to consistently improve the post-thaw quality of the semen,” he explained. A vapor box, on the other hand, is less expensive and can be constructed with a Styrofoam box.

“Semen is frozen in a vapor box by placing straws on floating racks, exposing them to liquid nitrogen vapor for 15 to 20 minutes prior to plunging them directly into the liquid nitrogen,” King said.

Once the semen is frozen, it can be stored indefinitely in liquid nitrogen tanks until it’s time to breed a mare. At that point, it can be carefully thawed, reevaluated, and placed in the ovulating mare’s uterus.

Take-Home Message

Freezing semen is an effective way to store genetic material until it’s needed to impregnate a mare. While not all stallions’ sperm can be frozen, carrying the process out carefully can help the semen have the highest fertility possible after freezing and thawing.

About the Author

Erica Larson, News Editor

Erica Larson, news editor, holds a degree in journalism with an external specialty in equine science from Michigan State University in East Lansing. A Massachusetts native, she grew up in the saddle and has dabbled in a variety of disciplines including foxhunting, saddle seat, and mounted games. Currently, Erica competes in eventing with her OTTB, Dorado.

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