Reproduction: An Emerging Focus at North Carolina State

Many of the United States' leading equine reproduction research institutions, such as Colorado State University, the University of Idaho, Utah State University, and Texas A&M University, are located in the western half of the country. But North Carolina State University is working to make sure horse owners in the eastern half of the country have the same caliber of reproductive research and services available nearby.

Says Carlos Pinto, DVM, PhD, Dipl. ACT, assistant professor of theriogenology, "North Carolina has traditionally not been well known for equine reproduction research. When I came here four years ago, I was charged to try to do more. The most important initiative was creating the satellite repro clinic at Southern Pines (formerly called the Veterinary Equine Research Center, now termed the Equine Health Center, 62 miles from the Raleigh college). 2004 was our first breeding season there; we started small, but our caseload tripled in 2006 from last season. And now we do more than 100 semen collections per year for clinical, teaching, and research purposes."

The 83-acre Equine Health Center (which also offers ophthalmology, podiatry, and lameness diagnostic services) has reproductive clinicians (board certified theriogenologists) at the facility at least three days per week, and their services include more than just basic exams, palpations, and inseminations. Southern Pines' many paddocks and laid-back atmosphere are home to about 20 broodmares available as surrogate mothers in the clinical embryo transfer program. Oocyte transfer and gamete intrafallopian transfer are also on the menu of services offered at NC State.

Working on client horses is important at both clinics, but research to better understand equine reproduction and find more effective ways of assisting reproduction is a major focus.

Recent Research

The following studies done at NC State were all presented at the 9th International Symposium on Equine Reproduction in Kerkrade, The Netherlands, in August 2006, and published in Animal Reproduction Science (volume 94).

Semen vials to replace straws? One simple concept has found great practical success at NC State--using 3.6-mL plastic semen vials rather than 0.5-mL or 1-mL straws to store semen. The shorter, fatter vials are much easier to use and they provide nearly three times as much semen storage volume in a tank as straws, says Pinto.

"Semen quality is almost the same; motility is slightly lower, but the breeding dose is way above the minimum we need, and the vials are much easier to work with before and during freezing," he explains. "Rather than using multiple straws (to get one full breeding dose), you just use the one and unscrew the top (instead of dealing with plugged straws)."

He also noted that the slightly lowered motility might be improved with variations in freezing protocols, which were designed for straws, not vials.

Detecting progesterone hormone in mares with a canine assay "It's hard to find kits to detect progesterone levels in mares (this helps determine if the mare is in the luteal phase of her estrous cycle; if she is not, she is ready for breeding)," says Pinto. Pointing to a photo of a progesterone assay kit, he comments, "This one's designed for dogs (Ovucheck's Premate 10) and we validated its use for horses." He reported an overall accuracy of 98.8% for the test's ability to report high (more than 3 ng/mL) vs. low (less than 3 ng/mL) progesterone levels in mares.

Synbiotics, the company that manufactures the Premate 10, is working on an equine version of the test for field and clinic use, but they don't yet have an estimated date of availability.

Pinto notes that determining progesterone levels might have many different applications depending on the reproductive status of the mare. He gave the following examples:

  • Confirming a mare has ovulated (progesterone rises about two days post-ovulation);
  • Confirming a mare has adequate luteal function (high progesterone levels to support the pregnancy during the first trimester); and
  • Determining whether a mare is no longer transitional (from winter anestrus, or lack of cycling, to spring estrous cycling). Mares cycle erratically at the beginning of the breeding season, he noted; this kit might help detect when a mare begins to have ovulations (because progesterone rises two days post-ovulation and remains high for about 14 days).

Investigating stallion fertility This study "looked at a sperm membrane protein (SP22) to see if there was any variation in its concentration across seasons," Pinto explains. "We don't know exactly what this protein controls, but in rats we know that if you block the protein, you affect fertility. Results of previous studies headed by Dr. Gary Klinefelter (a research biologist at the U.S. Environmental Protection Agency), a collaborator in the current equine study, indicate it has a pivotal role in fertilization and is thus a biomarker of sperm quality."

The study found that this protein tends to be higher for spermatozoa collected in the fall (which represent sperm cells produced during summer, a period of peak testicular function). Analysis of spermatozoa collected during winter and spring are currently underway and preliminary results point to a seasonal expression of the protein SP22. He suggested that this protein could serve as a marker of fertility with more research.

Prostaglandin dosage Another study evaluated various dosages of a commercial brand of prostaglandin F2alpha (Lutalyse) to cause mares to short cycle. The study dosages ranged from the recommended dose of 2 cc down to one-eighth of that--0.25 cc administered in the muscle.

"This work has been done before, but what hasn't been done before is to see how normal the next cycle is (as assessed by ultrasonography of the ovaries and uterus)," says Pinto. "Some people say, 'I don't want to induce changes in my mare, I want a normal cycle.' Well, it is a normal cycle. The results of our study showed that the reproductive cycles induced by prostaglandins were characterized by normal follicular dynamics, follicle size, rate of growth, and ovulation pattern."

"Some of these studies are simple, but that's good for two things--they get students' interest engaged, and make it easier for horse breeders to understand what you've learned," he comments. "Many of current studies are performed in great part by veterinary students with special interest in research and by those participating in summer research internships."

Ongoing Research

Embryo transfer timing Normally, an embryo donor mare is flushed at Day 7 or 8 post-breeding; at that age, embryos are often too small to see in the mare with ultrasound. Pinto has about three years of data from holding off until later. "I was waiting for an embryo to be visible (on ultrasound) at about 9.5-10 days," he says. "At that stage, they're about 3 cm, and that's about as small as I can see them. So this is like pregnancy checking a mare and only flushing her if you see an embryo and can confirm that she's pregnant. And then when you transfer it into another mare, you can see if it was successful." Pregnancy rates are about 30%, compared to 50-60% at the end of the season with smaller embryos, he notes. He has gotten a live foal from this schedule as well.

But there are drawbacks: "It's not ready to be applied yet, because it's hard to manipulate those large embryos," he adds. "We don't have a tool designed to handle and manipulate them. We hope to have in the near future an ultrasound machine that will enable us to identify these embryos at 1.5 to 2.5 mm in diameter; that would help with research and maybe clinical application, as you have better success manipulating smaller embryos."

Future Plans

Pinto says his main goal is to build a program at NC State that aims not only for excellence at clinical reproductive services, but also to train high-qualified veterinary specialists through intense teaching and participation in clinical research studies, such as summer research projects. "It is an excellent chance to train veterinary students in special procedures while translating the importance of scientific investigation and its application to clinics," he comments.

As far as upcoming clinical services, Pinto says intracytoplasmic sperm injection (ICSI) will soon be offered for clients. He's targeting the 2008 breeding season for 2009 foals. In keeping with NC State's goal of becoming a premier equine reproductive service provider, he adds, "Only a small percentage of breeders utilize it, but if anyone should offer it, it should be a tertiary care center like us that has the mission to teach and train future veterinary specialists."

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About the Author

Christy M. West

Christy West has a BS in Equine Science from the University of Kentucky, and an MS in Agricultural Journalism from the University of Wisconsin-Madison.

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