Hot Topics in Equine Reproduction
By Erica Larson, News Editor • Apr 08, 2013 • Article #31660
Photo: Anne M. Eberhardt
Assisted reproduction has opened a whole new world to some horse breeders. A Kentucky mare who's never left her home farm, for instance, could produce a foal from a European sire who's competing at the top of his sport. And while artificial insemination isn't a new concept, researchers are continually making advancements that improve the practice.
At the 2013 Western Veterinary Conference, held Feb. 17-21 in Las Vegas, Nev., Heath King, DVM, Dipl. ACT, an assistant clinical professor at the Mississippi State University College of Veterinary Medicine, reviewed some current hot topics in assisted equine reproduction with veterinary attendees.
Testicle Processing for Sperm Recovery
The death of a stallion with a bright stud career ahead of him can have a devastating economic impact on his owner. Fortunately, veterinarians and researchers have learned to recover sperm from recently deceased stallions' epididymis (the duct that stores sperm as it matures).
Some veterinarians routinely freeze semen and have experience extracting epididymal sperm, but many prefer to harvest the stallion's testicles and ship them to another facility for processing. While immediate sperm collection and processing is generally preferred, King noted that one study showed no change in motility of sperm harvested from testicles immediately and after 24 hours refrigeration in six of nine stallions.
According to King, there are several important steps practitioners should take during testicle processing:
- Castrate the stallion either prior to or immediately after euthanasia or death;
- Double ligate (bind or tie off) the spermatic cord—the combined structure extending from the groin area to the testes, through which the vas deferens (sperm's transport conduit from the testes to the urethra) and a number of vessels and nerves run—as close to the groin as possible before cutting it;
- Once the testicles have been removed, ligate the vas deferens and rinse the testicles with sterile saline; and
- Wrap the testicles in paper towels and seal them in separate plastic bags.
If sending the testicles elsewhere, practitioners should ship them overnight in a cooling device commonly used to transport semen, King said.
King noted that pregnancy rates in mares inseminated with frozen epididymal sperm are generally lower than in mares inseminated with frozen semen; however, the reason behind this difference remains unknown.
Low Dose, Deep Uterine Horn Insemination
Veterinarians generally use 200 million to 500 million progressively motile sperm to impregnate a mare using conventional (uterine body) artificial insemination. But using deep uterine horn insemination (which involves placing semen directly into the uterine horn adjacent to the ovary containing the follicle), some veterinarians have successfully used as few as 1 million sperm, King said.
He explained that veterinarians use deep horn insemination in a variety of scenarios, such as with frozen semen known to have limited progressively motile sperm numbers and when managing subfertile stallions that ejaculate low sperm numbers.
He noted that scientists have reported conflicting results on mare pregnancy rates when bred with uterine body insemination and deep uterine horn insemination.
Cushioned Semen Centrifugation
Moving forward, King described the use of a cushion media during semen centrifugation, a measure he says can improve sperm recovery rates.
"Semen is routinely centrifuged at a force of 400 to 600G for 10 to 15 minutes," he explained. "Increasing the gravitational force will increase recovery rate, but can injure sperm."
To protect the sperm from injury when the gravitational force increases, he said, veterinarians can add a commercially available cushion media to the semen prior to centrifugation. After centrifugation, he noted, most of the cushion media should be removed via aspiration.
King then reviewed how adding the anticoagulant drug heparin to sperm prior to insemination could help increase pregnancy rates when breeding mares that have already ovulated. Veterinarians breeding mares with cooled, shipped semen might have a shipment arrive after the mare has ovulated. Fertility declines as the oocyte ages, and after the oocyte is 12-18 hours old (post-ovulation) few mares conceive. Furthermore, sperm cannot fertilize a mare's oocyte until they've undergone a process in the mare's reproductive tract called capacitation—a series of changes on the sperm's surface that allows for the first step of the fertilization process. Researchers aren't sure exactly how long it takes for sperm to undergo capacitation, but believe it is approximately seven to eight hours.
"Heparin has been used to induce capacitation of sperm," King said, explaining that the drug increases the “fertility window” after ovulation.
He cited one study that showed a significant increase in pregnancy rates when mares were bred post-ovulation with heparinized semen (100%) compared to non-heparinized semen (43%).
King said veterinarians should add heparin to semen at a rate of 1.2 units per milliliter of semen before incubating the combination at 37°C (98.7°F) for about 30 minutes.
Finally, King reviewed the use of a combination of drugs to induce ejaculation in stallions, a procedure that can be used to collect semen from horses no longer able to mount mares or dummy mounts.
King explained the horse's "sympathetic nervous system controls erection, emission, and ejaculation through the stimulation of alpha adrenergic receptors," and that veterinarians often use a tricyclic antidepressant called imipramine combined with ?-2 agonist to induce ejaculation. He said administering oral imipramine two hours prior to an intravenous dose of xylazine produce the most consistent results (with 50% or more of stallions ejaculating).
"The ejaculate produced by the imipramine/xylazine protocol is typically low in volume, but highly concentrated," he said.