The Latest in Breeding

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With frozen semen old-hat and frozen embryos beginning to gain commercial viability, it was only natural for researchers to turn their petri dishes and microscopes to new pursuits. For many equine reproductive scientists today, that means studying frozen oocytes (eggs). Just last year, the first horses born from this technology were foaled. So far, they’re still the only two. But researchers believe this latest high-tech take on breeding could reach your barn within a few years.

What It’s All About

The impact of this new technology could be huge. Cryopreservation of oocytes—deep-freezing eggs to preserve them for future use—will give mare owners more options and give mares nearly the same reproductive flexibility as a stallion.

“[It] provides a method to preserve valuable female genetics for future generations,” says Elaine Carnevale, DVM, an assistant professor of physiology at Colorado State University (CSU) who has been working on the school’s frozen egg project, which produced those two foals in 2001. “With further development, this will allow mare owners to preserve a limited number of eggs (and potential foals) when a valuable mare dies or ages.”

And there are other benefits. Oocyte freezing also allows a performance mare to continue in competition while simultaneously launching her broodmare career years earlier than usual. However, embryo transfer provides the same opportunity, and has advantages over frozen oocytes in many cases, notes Carnevale. But, frozen oocyte breeding has the advantage when it comes to breeding mares that are poor embryo donors, she says.

In addition, oocyte freezing allows the mare owner to collect eggs from a young, fertile mare, then save them until the mare has proven herself as a performer. Even if the mare is aged and less fertile at that point—or if she dies prematurely—her eggs can still be used.

Furthermore, unlike frozen embryos, frozen oocytes aren’t fertilized until they’re placed in the recipient mare. Since only the female line is cryopreserved, you don’t pay a stud fee for the semen only to have it (or, to be precise, the embryo) sit in cold storage for an indeterminate time. And, it means that you can take your time waiting for just the right stallion to come along.

Eventually, the technology could also help preserve not only rare equine genes, but also endangered species around the globe.

How It Works

Although not yet perfected, frozen oocyte technology is definitely on its way to private breeding barns in the near future. Here’s what the process looks like:

1. Choose the donor mare. As with embryo transfer, this is the foal’s actual dam, although she likely will not carry the foal herself. Ideally, says Carnevale, the donor mare will be young—between three and 10 years of age—and healthy. “As mares get older,” she explains, “they have more problems associated with health of the eggs.” If your aim is to preserve valuable genetic material from aged mares, though, those guidelines may be ignored when the process enters the “real world” of breeding.

2. Collect the egg. Using an ultrasound probe, the veterinarian or breeding tech will gather the contents of the mare’s ovarian follicle. “We prefer to collect eggs from follicles within 12 to 16 hours before ovulation, because the success of the collection procedure is higher,” says Carnevale. “In the freezing experiment, we remove as many [eggs] as the mare has for that cycle that would be ready to ovulate.”

3. Freeze the egg. Under a microscope, the single-cell oocyte is separated from the rest of the follicle contents. Next, in a process called vitrification, the egg is placed onto a sewing needle-sized cryoloop, then flash-frozen with a quick dip in liquid nitrogen.

4. Store the egg. In the CSU study, the eggs were stored in liquid nitrogen for up to two months. but it’s likely that eggs could be preserved for thousands of years.

5. Thaw the egg. To actually use the egg for breeding, explains Carnevale, “The oocyte is removed from liquid nitrogen and placed in a medium designed to help it gradually lose the components (cryoprotectants) of the freezing medium.” The process takes only minutes to complete.

6. Prepare the recipient mare. The vet or breeding tech will remove the recipient mare’s own oocyte, so it cannot be fertilized, says Carnevale, noting that the mare should be in estrus. The mare is then inseminated. (While the CSU experiment used artificial insemination, Carnevale notes that the recipient mare could also be bred by live cover.)

7. Implant the egg. To complete the process, the egg is placed into the mare’s oviduct about 12 hours after insemination, which gives the semen time to enter the oviduct (where fertilization occurs) before the transfer.

Stacking Up the Odds

At this point, the breeding becomes much like any other, with the owner (or, in this case, the research team) waiting to see if the mare conceives. If so, it’s business as usual.

That said, it’s worth noting that certain challenges of frozen oocyte technology do reduce the odds of conception. “In the research that was done at CSU, a 12 percent embryo development rate was obtained, whereas oocytes that were transferred without being cryopreserved had an approximately 80 percent development rate,” says Carnevale.

Part of the reason for this substantial difference is the nature of an egg: Unlike an embryo, which has roughly a thousand cells, the oocyte is a single fragile cell. That means even a slight problem during freezing or thawing can easily damage it. And, says Carnevale, a damaged oocyte is less likely to result in pregnancy.

Improving those conception rates is a primary focus of the CSU research, and probably the largest stumbling block between taking this technology from the lab to the real world. Still, Carnevale estimates that frozen oocyte breeding may be a reality for private horse breeders within the next few years. “A lot depends on what risks the owners are willing to take, e.g., what success rate they want before they are willing to spend money for the procedure,” she says.

For breeders willing to take some calculated risks, frozen oocyte technology could soon give them one more tool to use in their efforts to get healthy, top-quality foals on the ground—even when a mare is busy competing or past the age of bearing her own offspring.

What's Next?

Here’s a look at other equine reproductive technologies that could reach the commercial level soon:

Post-mortem egg transfers. In this technique, eggs are recovered from mares that have died, then cultured (placed in an incubator to “mature”) and transferred into recipient mares. CSU has worked on five of these cases, with the deceased mares’ ovaries sent by mail to the school. Four pregnancies resulted, but three were lost. The fourth resulted in a healthy foal, born in 2002.

In vitro fertilization. “This is where sperm is combined with an oocyte in vitro (in the lab) in order for fertilization to take place,” explains Dale Paccamonti, DVM, MS, Dipl. ACT, professor at the Louisiana State University school of Veterinary Medicine. Despite the 1996 birth of the first “test tube” foal produced from this method, in vitro fertilization remains in the experimental stage. The technique could eventually be used to help subfertile mares and stallions produce offspring.

Sperm injection. This technique was used as part of the in vitro fertilization project that produced the first test-tube foal. In this procedure, a single sperm cell is injected directly into an egg that has been removed from the ovary. Although used in humans, the success rate in horses is not too high. However, both CSU and LSU have produced foals using this method, which could prove useful in breeding subfertile stallions, since it would work regardless of low sperm count or low sperm motility.

Cloning. CSU’s Ed Squires predicts that a horse will be cloned in the next year or two. However, he also expects the process would be both difficult and expensive. Paccamonti notes that several researchers in the U.S. and Europe are all working on this project, with two pregnancies at the moment. The mares are carrying cloned mules. —SDW

Making Ethyl

During the summer of 2000, researchers in the Animal Reproduction and Biotechnology Laboratory at Colorado State University in Ft. Collins, Colorado, collected and froze 26 equine oocytes. The eggs were removed within two months, thawed and transferred to multiple recipients, with about four oocytes placed in each mare. The mares had previously been bred via artificial insemination.

Three of the original 26 frozen oocytes were fertilized and resulted in embryos (imaged by ultrasound). One embryo was manually crushed to prevent the danger of twins. In July 2001, two healthy, normal foals were born. The colt, named Vitreous, and the filly, dubbed Ethyl, became the first foals ever produced using frozen oocyte technology. They are now living with their “adoptive” families, and both are healthy and happy, reports Carnevale.

Future work on the project, Carnevale notes, will examine different ways of cryopreserving eggs and of fertilizing the thawed eggs in order to improve conception rates and make the process more commercially viable. —SDW