Repro in the Rockies
by: Les Sellnow
February 2003
Article # 4087

The center of the Colorado State University (CSU) veterinary school's equine reproductive universe is its 22,000-square-foot Animal Reproduction and Biotechnology Laboratory and a smaller satellite, the Equine Reproduction Laboratory. In these facilities, faculty members, graduate students, post-doctorate fellows, and visiting scientists from around the world work on a daily basis to unveil the mysteries and solve the problems of equine reproduction. A particular focus is on horses whose organs have been compromised by age and illness.

The veterinary school is also known for its research and accomplishments in the field of orthopedics, and a new research center devoted to that field of study has been constructed.

Reproduction Accomplishments

Colorado State University, of course, is not the only research center heavily involved in equine reproduction. There are a number of others around the country where studies are ongoing to better understand the reproductive systems of both stallions and mares. However, the successes achieved at CSU allow it to lay claim to being one of the leaders in the field.

Here is a capsule look at the school's "Top 11" in equine reproductive accomplishments in recent years that backs that assertion:

Leading the Way

Accomplishments like these don't just happen. They result from a program that remains focused on its objectives. Strong leadership is required to maintain that focus.

Providing that leadership, along with three fellow faculty members, has been Ed Squires, MS, PhD, Dipl. ACT (Hon), professor of biomedical sciences and supervisor of the Equine Reproduction Laboratory. Squires and his colleagues are on a mission, of which a key part is to help valuable, but subfertile or infertile, older mares and stallions pass on their genes.

The Preservation of Equine Genetics Program (PEG) was created with collaboration between Squires and Neil and Barbara Chur, who own nursing homes, health care facilities, and Strawberry Banks Arabian Farm, all located in or near Buffalo, N.Y. The program has been responsible for raising about $2 million for reproductive research at CSU (members pledge to donate $10,000 per year for at least five years).

The initial 11 goals for the program have been condensed to 10 (two goals involving semen freezing were combined). As Squires reviews them today, he can proudly point to significant accomplishments in some areas, and a frustrating lack of results in others.

Embryo freezing--This technique permits mare owners to have embryos collected on the farm, then cooled and shipped to CSU where they can be frozen and stored for later use. The secret to successful freezing of embryos, says Squires, is size. The best success is with Day 6 embryos that are less than 300 microns in size. The process could open the door for more international trade, since transportation costs and disease control are cheaper and easier with frozen embryos than with live horses, Squires says. Research is also continuing on the effort to successfully freeze embryos that are older and larger than the Day 6 variety because they are easier to collect.

Freezing oocytes--A technique has been developed at CSU to freeze eggs. The success rate has been 30-50% for eggs being alive when thawed. The first foals from frozen, thawed eggs have been born at CSU. Work in this area--and in oocyte collection and transfer--has helped researchers learn what constitutes a healthy oocyte or egg.

Improved semen freezing techniques--Cryoprotectants other than glycerol (the most commonly used) are being studied since glycerol is toxic to some stallion sperm. Progress also has been made in the ability to assess damage to sperm as the result of the freezing process. Sperm from certain stallions survives freezing and thawing far better than that from others; identifying problem stallions prior to shipping and insemination will be an economic benefit to horse owners. Squires says success rates for shipped frozen semen will go up when only semen that withstands the freezing and thawing process is used. A key process in assessing sperm damage involves the flow cytometer, which can evaluate if the sperm plasma membranes, acrosomes, or mitochondria have been damaged during freezing and thawing.

Multiple ovulations--A current research project is aimed at determining the effects of multiple flushings. With only a single embryo with which to work, the whole fertilization and harvesting process must be started over if the embryo is lost or destroyed during recovery or transfer. With multiple embryos from a single flush, the odds of a successful transfer would increase a great deal. Multiple ovulations, says Squires, also can be an economic benefit. "The single biggest cost in embryo transfer is maintaining the recipient mare while we wait for an embryo," he says. Multiple ovulations, he believes, could conceivably reduce the cost of embryo transfer by half.

Additionally, a drug to produce multiple ovulations has been developed at the school and turned over to the Canadian firm Bioniche (formerly Vetrapharm Company), located near Toronto. A group of mares at CSU were injected with the drug in the summer of 2002, and each mare ovulated an average of three follicles.

Gamete intrafallopian tube transfer--This technique involves first harvesting an egg, or oocyte, from a mare which is unable to conceive or carry a foal to term, then transferring it into the oviduct of a recipient. A side benefit to the approach--because the sperm is placed in the oviduct rather than the uterus--is that only a small number of sperm are required, thus making it a beneficial approach with sub-fertile stallions. The technique is in place, but is being fine-tuned through ongoing research.
Oocyte collection/maturation--The goal is to collect eggs from the mare's follicles at various stages of development before ovulation, mature them in a test tube, then fertilize them. The problem has been in fertilizing the eggs in vitro, or outside the mare's body. (More about that later). The spectacular advance has come with the removal of entire ovaries from mares which have died, then shipping those ovaries to CSU where the eggs are harvested and transferred to recipients. Eggs harvested with this technique were successfully transferred to a recipient which was then bred, and the recipient mare gave birth to a normal, healthy foal.
Oocytes from many deceased mares' ovaries have been harvested at CSU and transferred to recipients. An important part of the ongoing research, Squires says, is to determine the best temperature for shipping ovaries. At present, it appears that room temperature is best as cooling can destroy the eggs. Research continues.

In-vitro fertilization--This is an area where little progress has been made. Back in 1990, says Squires, French scientists produced the first "test tube" foals and no one, including the French, has done it since. For some reason, fertilization doesn't normally take place when an equine egg and equine sperm are combined in a petri dish or test tube.

Intracytoplasmic sperm injection (ICSI)--This procedure, which is a form of in-vitro fertilization, shows promise. The technique involves injecting one sperm into an egg. The first ICSI foal was produced at CSU in 1996.
Oocyte transfer--This technique is approaching routine at CSU, with about 30 mares successfully involved in the program during 2002. Eggs are collected from the donor mare--normally a mare unable to get pregnant or incapable of carrying a foal to term--and transferred to a recipient. The recipient is then bred so that fertilization occurs naturally.

Sexed semen--A technique has been developed to separate sperm carrying X and Y chromosomes. Sex is determined by the stallion, which carries both X and Y chromosomes--X for female, Y for male--while the mare carries only X chromosomes. A foal receiving an X chromosome from the stallion will be a filly, but if it gets a Y chromosome from the stallion, it will be a colt. By separating spermatozoa with X and Y chromosomes in the stallion's semen, the sex of a foal can be determined in advance. Pregnancy rates with sexed semen runs between 50-70% at present, says Squires. The technique of separating X- and Y-bearing sperm by flow cytometry is owned by XY Inc., of Fort Collins, Colo.

CSU-Developed Products

In addition to these research areas, CSU researchers are involved with developing drugs that assist the reproductive process. One of the first and perhaps the best known is Regumate (altrenogest), a synthetic form of progesterone that suppresses estrous behavior and helps a mare maintain pregnancy if she can't produce enough of the hormone on her own.
One of the newer drugs is Ovuplant (deslorelin), a gonadotropin-releasing hormone (GnRH) that insures ovulation within 36-48 hours after being implanted once a mare develops a preovulatory follicle.

Giving and Getting

While some of the research involves techniques that are state of the art and require sophisticated facilities and personnel, some of the others, Squires believes, have practical adaptation for many horse farms. These include semen freezing, embryo freezing, and multiple ovulations.

Efforts to share information with horse owners has been an emphasis at CSU since 1967, when B.W. Pickett, DVM, PhD, and J.L. Voss, DVM, first launched the Equine Reproduction Program. Utilized in this effort have been press releases, seminars that are open to the public, and publication of informational bulletins. Research results have been published in a multitude of scientific publications.

One of the school's seminars is responsible for recruiting a major financial contributor. In 1988, Lucy Whittier, a prominent and progressive Arabian breeder from California with a band of some 60 Arabians at her Los Colinas Arabian Ranch near the small town of Pilot Hill, Calif., decided to attend a seminar at CSU on artificial insemination that was being taught by Pickett and Squires. She was highly impressed with Squires and the two became friends.

She established the Lucy G. Whittier Foundation with her donation of $1 million for reproductive research at CSU. She also is a member of the PEG program.

Another big supporter is Gail Holmes, a breeder and exhibitor of cutting horses raised on her Double Dove farm in Longmont, Colo. "I read an article in U.S. News and World Report that Colorado State was rated as the number two veterinary school in the country," she says. "I called Ed and asked him to give me a tour." She quickly became part of the PEG program, and began a stallion service auction where the stallion owner donates a breeding fee that is sold to the highest bidder. The money from the sale is then donated to CSU.

" I called 25 of the leading cutting horse stallion owners in the country," Holmes said, "and only two turned me down." The auctions have been a rousing success, raising more than $100,000 per year since their inception some six years ago. One of the auctions netted $250,000 for CSU.
In addition, Holmes has made other personal financial contributions aimed at improving CSU facilities and has been a major contributor toward the orthopedic research center that has been developed.

Rules Driving Research

A recent change in American Quarter Horse Association (AQHA) registration rules just might stimulate more interest in reproductive research from that segment of the horse population. Since being established as a registry, AQHA has stipulated that only one foal from a mare could be registered in a given year. A couple of years ago, several cutting horse breeders who owned embryo transfer foals which weren't eligible for registration filed a lawsuit against AQHA, charging that the rule was basically a restraint of trade. A judge recently ruled in their favor and, as a result, AQHA established a new rule that imposes no limit on the number of foals from a particular dam that can be registered in a given year. Because progress--even in research--is often driven by demand, this monumental rule change by the largest equine breed registry in the world just might focus more attention on research involving multiple ovulations.

Whether that is true remains to be seen, but in the meantime, CSU, with the financial help of many horse owners across the country and around the world, will continue its efforts to unveil the mysteries surrounding equine reproduction.