February 2002

Where human medicine leads, veterinary medicine soon follows. Years ago, the cattle industry was at the forefront of assisted reproduction with their acceptance of artificial insemination, but equine breed organizations were slower to jump on the bandwagon. But times have changed. Today, assisted reproduction increasingly is accepted among horse owners and breed organizations. The American Morgan Horse Association registers foals conceived via artificial insemination as well as through embryo transfer. These were the best technologies available a decade ago, but now, increasingly sophisticated techniques are pushing equine reproduction to new levels. Some of the techniques being explored in university research centers are not, at this time, commercially available. But, if history is a guide, as researchers develop protocols that work consistently, private practice equine specialists may soon be using these new breeding techniques.

ICSI: Intracytoplasmic Sperm Injection
Probably the most technically complex equine breeding procedure is intracytoplasmic sperm injection, or ICSI for short. To date, ICSI has produced two live foals at Colorado State University (C.S.U.). Although parts of the procedure are used in other breeding techniques, the success of ICSI depends on all parts perfectly working together. This technique will help breeders with valuable mares who can produce eggs, but can't develop embryos.

The procedure begins with eggs being taken from one of the donor mare's ovaries, then placed in a special incubator to mature. When the egg is ready to be fertilized, scientists collect semen from the stallion and inject each egg with one sperm. While a sperm may look healthy, it may have some abnormality that prohibits fertilization. In the first ICSI procedure at C.S.U., four eggs were injected with sperm and only one was fertilized. Scientists grow the fertilized egg in the laboratory unit it is ready to be implanted. Then the embryo is placed in a recipient mare and the 11-month wait begins.

Scientists at C.S.U. first turned to this human method of sperm injection when they had difficulty in getting sperm to penetrate and fertile eggs held in a liquid medium. And like the human procedure, ICSI is expensive. A price tag of up to $10,000 is not out of line for this assisted reproduction. On the surface the procedure sounds simple, but replicating a natural process is never easy. The challenge with ICSI is that it bypasses part of the fertilization process that takes place in the mare's reproductive track before the egg and sperm unite. When sperm are first ejaculated, they are not mature. They require the chemical and physiological stimulant of the mare's reproductive track to reach maturity. What scientists must do in the ICSI procedure is to replicate that natural function in a clinical setting.

For more information about C.S.U.'s equine reproduction services call (970) 491-8626.

Deep Insemination
Dr. Mary Scott, D.V.M., Ph.D., a member of the clinical faculty in equine reproduction service at the Veterinary Medical Teaching Hospital, University of California, Davis campus and her colleagues are looking at ways to reduce the number of sperm needed for fertilization in their assisted reproduction program. Currently, the rule of thumb is 300 million to 500 million sperm per dose for optimal fertilization. But a new technique called deep insemination may be able to provide acceptable fertilization rates using only 50 millions healthy, vigorously moving (called progressively motile), previously frozen sperm per insemination. Fresh semen can live up to six days in the mare's reproductive track, but frozen and thawed semen has a much shorter life. Placing the semen closer to the preovulatory follicle increases chances for pregnancy.

Dr. Scott and her colleagues are placing two doses of 50 million progressively motile thawed sperm, 24 hours apart, deep into the uterine horn that has a preovulatory follicle. The procedure is done onsite using a pipette and stylette designed to reach the uterine horn. Placement is by manual palpation, so surgery is not required.

"Preliminary results are good," said Dr. Scott. "We have a success rate exceeding 60 percent." However, Dr. Scott acknowledges they are using a stallion with high fertility, excellent motility, and good sperm structure for this procedure and that lower sperm quality might easily produce fewer pregnancies.

Deep penetration places the sperm in the uterine horn near the oviduct, where fertilization actually takes place, but by doing so, it bypasses part of the natural process. "Nature has designed sperm to move along the lining of the uterus in a process called the hydrodynamic capturing effect," said Dr. Scott. "Sperm move more efficiently along surfaces, but it is only normal sperm that are 'captured' by the mare's uterine lining."

This aspect of natural selection, called capacitation, ensures that only a highly functional population of sperm moves toward the egg. It's nature's way of screening out poor genetics.

"Normally, there is a significant reduction in sperm numbers as they move from the site of semen deposition in the body of the uterus to the oviduct," explained Dr. Scott. "But with deep insemination, the natural process of sperm transport is short-circuited, so fewer inseminated sperm are needed to achieve pregnancy."

By dividing semen into smaller quantities, Dr. Scott points out that a stallion can pass on good genetics by inseminating many more mares during a breeding ovulation.

For more information about the U.C. Davis assisted reproduction program call (530) 752-0290.

Oocyte Transfer
Dr. Elaine Carnevale, D.V.M., Ph.D., and Assistant Professor at the Equine Reproduction Laboratory and Animal Research and Biotechnology Laboratory at Colorado State University, has been pioneering oocyte transfer in horses.

"Oocyte [non-fertilized egg] transfer is much like embryo transfer," Dr. Carnevale said, "but we take it back a few steps and don't ask as much of the donor mare."

This procedure, like ICSI, is used with mares who are able to produce eggs, but are unable to grow embryos. However, there are major differences. Oocyte transfer places the egg directly into the recipient mare's oviduct prior to breeding either through natural cover or artificial insemination. The advantage to oocyte transfer over ICSI is that the sperm go through natural capacitating as they move through the mare's reproductive tract. An additional advantage of oocyte transfer is that it also bypasses any problems with the donor mare's oviducts, uterus, and reproductive tract. For example, oocytes can be collected from mares with chronic uterine infections or a badly scarred cervix and placed in a healthy recipient mare. The recipient mare would then be inseminated and hopefully carry a healthy foal to term.

Currently, the success rate in the commercially assisted reproduction program at C.S.U. is between 30-40 percent in older mares who have been infertile using other methods of breeding, including embryo transfer. However, Dr. Carnevale says that by the end of the breeding season at C.W.U., 7- to 80 percent of the donor mares in the program will have one ore more pregnant recipients going home.

Two factors significantly affect the success of oocyte transfer. "In older mares, the oocyte quality is not ideal," Dr. Carnevale said. "Although pregnancies are often obtained using oocytes from mares older than 20 years of age, it may take extra transfers per pregnancy."

The second factor affecting success, Dr. Carnevale said, is the quality of the semen used. "We love to use fresh semen," she stated, "but many successful transfers are done using cooled, transported semen."

Researchers prefer fresh semen simply because the process of cooling and shipping semen often decreases motility. But, said Dr. Carnevale, by using young, fertile mares and fresh or cooled semen from stallions with high fertility, she and her colleagues have been able to achieve more than 80 percent success per transfer.

"The procedure isn't cheap." Dr. Carnevale warned. "It takes more time, equipment, and personnel. A foal can cost up to $10,000." But to reproduce the genes of a superior mare, this price may be inconsequential.

The C.S.U. Equine Reproduction Laboratory can be reached by phoning (970) 491-8626.

Oocyte Cryopreservation
Cryopreservation uses freezing to preserve living tissue. Until recently, only frozen semen has been available to breeders. But all that is changing. Two foals were born this year at C.S.U. using previously frozen eggs. Dr. Lisa Maclellan, Ph.D., collected eggs (oocytes) from donor mares and froze them using a special process called vitrification. Later, the eggs were thawed and placed into the oviducts of recipient mares where, after insemination, successful fertilization occurred. Two healthy foals, named "Vitreous," after the procedure, and "Ethyl" after the chemical ethylene glycol used as a cyroprotectant, were born. In years to come, owners will be able to preserve eggs from mares and continue to produce foals after the mare becomes too old to produce healthy eggs, or as in the case now with some stallions, is no longer alive.

Embryo Cryopreservation
Dr. Maclellan is also conducting research on embryo freezing. The challenge lies in raising the viability of larger embryos for freezing. Dr. Maclellan has found that small embryos collected on day six after ovulation freeze well, while larger, more mature embryos collected on days seven or eight, do not. Currently, the use of small, frozen, and thawed embryos is resulting in a pregnancy rate of 71 percent while larger embryos result in a success rate of 31 percent.

GIFT: Gamete Intrafallopian Transfer
Researchers at Colorado State are taking oocyte transfer one step further with gamete intrafallopian transfer (GIFT). The procedure for oocyte transfer is followed with one important difference. When the egg is placed in the oviduct via glass pipette, a small amount of semen is added as well. The success rate has been 60 percent using fresh semen from stallions with high fertility and egg placement in young mares. According to Dr. Carnevale, the next step is to use fresh sperm from sub-fertile stallions. Research, under the direction of Ph.D. candidate Dr. Marco Coutinho da Silva, D.V.M., M.S., will continue exploring the viability of using cooled, shipped semen as well as frozen semen in the procedure.

Sperm Separation
Well, it's about time we started separating the good from the bad…sperm, that is. Dr. Juan Samper, D.V.M., MSc, Ph.D., and Associate Professor at Kansas State University's College of Veterinary Medicine is pioneering a separation technique designed to identify sperm that are abnormal. This research is especially beneficial in evaluating semen that has been frozen for storage and rewarmed later for insemination, but could be used to assess fresh or cooled semen as well. Some stallions produce sperm that do not tolerate freezing well, but standard microscope evaluation of motility does not identify morphologically (structurally) abnormal sperm. Sperm that are abnormal often have membrane damage. This damage prohibits the sperm from binding to the oviductal cells. It is the binding of the sperm and the oviductal cells that causes the sperm to mature. Immature sperm are not capable of fertilization. Dr. Samper has found that the number of sperm in each semen sample that binds to oviductal cells correlates to fertility.

Dr. Samper developed a specially designed filter that traps sperm with membrane damage. This allows him to examine semen for capacitation ability. Evaluating semen for the ability to mature is critical in stallions with low semen output. This information will allow veterinarians to adjust the number of sperm needed to optimize fertilization. In addition, this filtration technique could be an important part of the evaluation of a stallion prior to sale as it's is currently the most accurate way of assessing fertility. However, at this time, the filtration evaluation is not commercially available.

For more information about the K.S.U. commercial freezing and embryo transfer services call (785) 532-5700.

Sex Selection of Sperm
One of the most exciting new breeding operations is the sex selection of foals. C.S.U. is in partnership with XY Inc. and Cytomation, both of Fort Collins, Colorado. XY has the worldwide rights to sperm sorting used for sex selection in non-human mammals. Cytomation produces the computerized equipment that actually sorts the sperm. And there's an important and close Morgan connection to XY - Dr. Mervyn Jacobson and Gail Bratz own Stove Prairie Ranch, a well-known Colorado Morgan farm. And expert in flow cytometry, the high-speed cell sorting process used in sex selection of sperm, Dr. Jacobson also has been the chairman of XY Inc. since 1997.

The U.S.D.A. actually developed and patented the protocol and technology for sperm sorting. Its first application was in the dairy industry, where millions of male calves are slaughtered each year. But when Dr. Jacobson became CEO of XY, the company expanded into equine reproduction as well. XY and C.S.U. staff use a flow cytometer in the sex selection process. A key researcher, Professor of Physiology in Biomedical Science in the College of Veterinary Medicine and Biomedical Science at C.S.U., Dr. Ed Squires, Ph.D., says the theory is simple although the process itself is challenging.

Once sperm is collected, it is stained with a florescent dye. The sperm are then passed through the flow cytometer. Sperm with "x" chromosomes, which produce females, appear more deeply stained because they possess more DNA than sperm with "y" chromosomes that produce males. The sperm receive an electrical charge that allows them to be sorted by "x" and "y" chromosomes.

"At this time, the flow cytometer is unable to sort about 90 percent of the sperm in each sample," said Dr. Squires. "However, the 10 percent that are sorted are accurate to about 90 percent."

This means that although the probability of getting the sex desired in a foal is substantially increased over unsorted sperm use, it is not foolproof. And aside from sex determination, no other selection of genetic material takes place: size, color, and conformation are still unknown variables.

Scientists at C.S.U. and XY have found that accurate placement of the sperm allows them to use a much lower dose, effectively off-setting the smaller amount of sperm available through this process. The standard in traditional AI is 500 million progressively motile sperm. At C.S.U., a form of deep insemination is used to with excellent results. A small, concentrated dose of 20 million sperm is placed, with the help of a video-endoscope at the uterotubal junction: this is called historoscopic insemination. This non-surgical AI procedure is efficient and safe. In an exciting breakthrough, XY and S.S.U. researches produced sex-selected foals last year using only five million sperm, or 1 percent of the 500 million used in typical AI.

In 2001, XY and C.S.U. scored another first. They produced nine sex-selected foals using shipped semen that was 18 hours old prior to the sorting process. "It was important for our research and for the commercial application of this process to be sure shipped semen could survive the sorting process and successfully impregnate mares," said Dr. Jacobson.

Gail Bratz sees sex selection as a real boon to breeders. "This process eliminates the time and cost of breeding and raising foals you don't want. To the small breeder especially, sex selection is a great benefit," she said.

An interesting note to sex selection is that preference for stallions or mares runs just about even. "About half the breeders want stallion and half want mares," said Gail. "It depends on what genetics they want to preserve and pass on."

Initial worries about too many mares or too many stallions being produced with this method simply haven't materialized.

XY plans to make the process commercially available in 2002. "The cost is between $4,000 and $5,000 for up to three heat cycles of the mare you want to breed," says Dr. Squires.

However, XY plans to license the rights to the technology used in the sex selection of sperm to veterinarians, universities, and breeders throughout the world. "Our first license is in Argentina where they will sex-select fillies for polo ponies," said Dr. Jacobson.

Research and technology have changed the way breeding programs are being managed around the world. Techniques being pioneered at universities and private laboratories today will provide an increasing number of breeding options in the years ahead. This new era is, for many breeders, a dream come true.