One plus one plus one equals one. The stallion, dam, and surrogate dam probably never met, but all three contributed to produce a foal. Today's assisted breeding technologies can overcome the constraints of distance, competitive involvement, reproductive health, and the calendar.

As a horse breeder, you can employ embryo transfer to produce better foals (in most breeds except Thoroughbreds). In your goal of improving your stock, you can influence the quality of the new generation. Your valuable mare is able to reproduce many more foals than possible through traditional breeding regimens.

You benefit from two decades of equine research, much of it pursued at Colorado State University. In CSU's Equine Reproduction Laboratory, research has generated a clinical service for horse breeders across the United States. Much of this equine research follows procedures pioneered in the larger bovine industry.

The laboratory works with mare owners in an active equine embryo transfer program. To assist the three "parents" of a planned foal, a network of veterinarians and technicians cooperate with you, the breeder. For your convenience, the network extends nationwide, eliminating the need to transport your mare (and her nursing foal) to another facility.

Owner's Involvement

Embryo transfer is based on your mare conceiving a foal, then donating the embryo to a surrogate dam. The surrogate carries the foal to term and delivers it.

Your donor mare must be capable of ovulation, and her egg must become fertilized by the stallion's sperm. When she donates the developing embryo, removed through a collection process, her task is complete.

You need to be aware of your mare's estrous cycle, specifically when the mare ovulates. CSU's Dirk Vanderwall, DVM, PhD, said, "We base all the procedures on the day the mare ovulated, as the age of the embryo is based on the day ovulation occurred, or Day Zero."

Most mares have an estrous cycle of 21 to 22 days, with the cycle beginning on the day of ovulation. Hormonal changes determine the cycle. Progesterone, important for maintaining pregnancy, rises at the time of ovulation. It reaches a peak level in the middle of the cycle, when the mare does not demonstrate estrus behavior. Beginning on Day 14 or 15 after ovulation, the uterus secretes the hormone prostaglandin F2 alpha. As the progesterone level falls, the next estrous cycle begins. A shot of prostaglandin can begin the next cycle to bring the mare back in heat. (For a more complete text on mare and stallion hormones, see The Horse of December 1995, page 41.)

Timing is crucial for successful embryo transfer. When a mare conceives, the embryo first develops in the oviduct, then enters the uterus from 5 to 6 days after ovulation. Day 6 is the earliest date for embryo collection, when the embryo has most likely entered the uterine horn. At this date, the embryo is called a morula.

"It's about the size of a dust particle," said Vanderwall. "The embryo has a tough outer shell, the zona pellucida. Its central area is a compact mass of developing embryonic cells, no more than 64 to 128 cells. Over the next 24 hours, the morula will develop to the next gestational stage, the blastocyst."

In the blastocyst, the compact mass of cells has changed to a fluid-filled chamber of several hundred cells. By Day 7, the now-expanded blastocyst measures 300 microns, or .3 millimeters in diameter. It is visible to the naked eye and shows a mass of cells that will develop into the fetus. The remaining tissue will form the placenta.

For embryo transfer from your mare, you should document her reproductive history. She should be cycling normally.

Embryo Transfer In The Laboratory

The process benefits valuable progenitors. You might own a mare which conceives, but cannot carry or deliver a live foal. She may be unsound, aged, injured, or needed to perform as an athlete. A likely candidate would be an older mare with a history of conceiving and later aborting. She is likely to provide a fertilized egg for embryo collection.

Vanderwall noted how embryo transfer decreases foaling risk: "In some older mares, the uterine artery ruptures and hemorrhages at the time of foaling. It can be fatal in the immediate post-foaling period. Some mares survive that first episode of uterine artery tear, but they're at greater risk of suffering rupture at a subsequent foaling. Embryo transfer minimizes the mare having to carry successive pregnancies."

Embryo transfer also allows your per-
formance mare to continue her athletic career while reproducing. For example, Colorado sport horse breeder Patty Arnett was able to breed four foals in one year from her Dutch Warmblood mare Amerens. The champion hunter delivered one foal herself, while surrogate dams carried the other three.

In the performance mare, lactation limits the mare's ability to continue showing or racing.

"There's no reason why a mare can't get pregnant and still compete well into the seventh or eighth month of gestation," said Vanderwall. "When she does have the foal on the ground, then you have disruption of athletic performance from four, five, to six months, until you wean the foal. She then won't be able to be in a conditioning program, so you lose the mare's ability to keep performing when she's lactating."

The collection process is non-surgical, performed by flushing the embryo from the uterine lumen. CSU technicians generally perform the uterine flush on Day 7.

"We use a container full of a sterile salt solution, with some protein added," said Vanderwall. "We place a catheter through the vagina and through the cervix, and an inflatable cuff on the catheter provides a fluid-tight seal. The solution passes down through a tubing system into the uterine lumen.

"As the fluid swirls throughout the lumen and drains back out through gravity, it collects the embryo, which is swept back out. The fluid and embryo pass out through the tubing system, into and through an embryonic filter."

He added that the process uses four liters of fluid, running four times in one-liter amounts. The filter traps the embryo, and a technician examines the filter under a stereo dissection microscope. When the embryo is identified, it is removed into a more enriched medium until the time of transfer.

"With a smaller embryo at Day 6, the embryo collection rate is lower," said Vanderwall. "First, the embryo may still be in the oviduct, and we can't recover it. Second, it's small, which could cause us to miss the embryo. That doesn't happen very often."

Usually a failed attempt at recovery means the mare was not pregnant or the six-day embryo was still in the oviduct instead of the uterus.

The surrogate dam must be ready to accept the embryo--her estrous cycle needs to be synchronized with the donor mare's cycle. The surrogate ovulates either one day prior, the same day, or one to three days after the donor mare. CSU acquires and maintains a substantial number of recipient mares, certified through their age (three to 12) and a thorough reproductive examination. Their estrous cycles are recorded to match them with expected donor mares. If the cycles don't match, the laboratory uses hormonal therapy to synchronize ovulation.

CSU transfers embryos using both surgical and non-surgical methods, with the surgical method resulting in higher pregnancy rates. Veterinarians perform a standing surgical process with the tranquilized surrogate mare standing in stocks. Under sterile conditions, the surgeon makes an incision in the recipient's flank, locates the uterus, and exteriorizes the uterine horn.

The transfer technician makes a small puncture hole into the uterine lumen and threads a small plastic pipette into the lumen. The pipette contains the embryo for deposit.

After being sutured, the mare receives procaine penicillin for five consecutive days. She is housed separately and her condition monitored through Day 14 post-surgery, after which she returns to the herd.

Non-surgical transfer resembles the artificial insemination process.

"We use a disposable artificial insemination catheter that holds a one-half cc semen-freezing straw," described Vanderwall. "The embryo is placed in that straw at the end of the insemination rod. The transfer technician inserts the sterile transfer catheter through the vagina and through the cervix, into the mare's reproductive tract, and the embryo is deposited into the uterine lumen."

At CSU, the first pregnancy check of the recipient mare is Day 12, or five days after transfer. The pregnancy rate one week after surgical transfer runs 75%. Mares are checked at regular intervals, and the pregnancy rate at Day 50 is 65%. Vanderwall noted that a 1995 project for non-surgical transfer resulted in a 73% pregnancy rate, almost equal to the surgical transfers.

Long-Distance Embryo Transfer

Collecting embryos for shipment has sparked interest in the procedure. Your mare can remain at home for the entire breeding process, if you breed her through artificial insemination and collect the embryo for shipment to the CSU Equine Reproduction Laboratory.

This procedure is possible through local veterinarians trained in embryo collection. CSU conducts courses for equine practitioners, and the Laboratory handles embryos collected from approximately 30 veterinarians across the United States.

Vanderwall explained that the Laboratory has developed methods for short-term storage. The technique involves packaging and shipment in controlled temperatures.

"The embryo is placed in a special culture medium, Ham's F10, which is a modified salt solution containing nutrients. That has all the factors that an embryo requires for short-term storage during transport to Fort Collins.

"The (collecting) veterinarian puts five milliliters of the culture medium into a small plastic tube and deposits the embryo into the tube. He caps the tube and places it into a larger tube that contains the same culture medium."

The tube is packaged in an Equitainer, the same container designed for shipping semen. The container will maintain the components at a refrigerated temperature of five degrees Celsius for more than 24 hours, but CSU recommends 24 hours as the maximum time between packaging and receipt.

The collecting veterinarian ships the Equitainer to the Laboratory via overnight shipment or counter-to-counter through an airline's freight service.

Business Aspects

Embryo transfer requires advance planning. First, you decide how many foals you want from your donor mare. Instead of one per year, you can aim for three, four, or even five foals in a year from one valuable broodmare.

Regulations of each breed association limit the number of foals registered from a mare in a year. CSU reports most mares represent the American Saddlebred, Arabian, Quarter Horse, and European Warmblood breeds. The American Saddlebred Horse Association modified its regulations in 1995, allowing registration of up to four foals from one donor mare in a year. Both the Arabian Horse Registry and American Quarter Horse Association restrict the number to one foal per mare.

CSU offers two types of embryo transfer contracts, for the donor mare onsite or shipped embryos. On each form, you specify the number of embryos requested to be transferred, which allows the Laboratory to manage the number of recipient mares purchased. The Laboratory handled a total of 250 embryo transfer mares in 1995, up from 130 in 1994. The breeding season runs from March 1 to Sept. 1.

Most mare owners request two or three embryos be collected. For donor mares boarded at the Laboratory, the mare is administered prostaglandin after embryo collection. This ensures a shorter estrous cycle, and a shorter stay at CSU. The interval between successful embryo collections is about 18 days. The prostaglandin also prevents pregnancy in the donor mare if an embryo remained uncollected.

Current costs run $1,500, plus board, for a mare shipped to CSU. This non-refundable nomination fee covers collection of embryos through three cycles. Semen shipping and shoeing (recommended by the Laboratory) are extra. The nomination fee for shipped embryos is $1,000, covering up to four embryo shipments. In both cases, as owner of the donor mare, you purchase the in-foal surrogate dams at a price of $2,500 each. These mares are confirmed 50 days pregnant, and you're responsible for moving them from CSU for foaling.

As a breeder, you must balance the cost against the value of the expected foal. Another consideration is the age of your broodmare, which can affect the fertility of her eggs.

"There's a dramatic loss in fertility after 15 to 18 years of age," said Vanderwall. "It's more severe in a mare 18 to 21 years. Our embryo transfer research indicates that the eggs produced by older broodmares may be less viable than the eggs produced by younger mares."

One economic benefit is adjusting a mare's breeding schedule. Through a broodmare's lifetime, her conception schedule can move her to later foaling dates in successive years. If she slips to a summer foaling date, you might choose to wait to breed her early the next season, to move her back to a desirable schedule. You lose a year with a barren mare.

"If your breed association allows embryo transfer, you can breed the mare after she foals in May or June and transfer the embryo into a surrogate recipient mare," suggested Vanderwall. "You will have her foal on the ground the following year, but your mare won't be carrying that pregnancy. You can breed her early in the next season and catch her back up in the breeding schedule."

IVF Prospects

In vitro fertilization has had limited success in the horse. Vanderwall named two barriers: egg maturation and sperm cell capacitation.

"The oocyte, or unfertilized egg, needs to undergo a maturation process that prepares it for fertilization," explained Vanderwall. "We have made tremendous progress in our ability to artificially mature horse eggs. Our main limitation is the sperm treatment. Capacitation is a necessary change that sperm cells must undergo in order for them to be able to fertilize an egg."

He described two advanced reproductive techniques that CSU hopes to offer as complements to embryo transfer: GIFT and ICSI.

GIFT, which represents Gamete Intrafallopian Transfer, is the transfer of an unfertilized egg from a donor to a surrogate mare. The egg is then fertilized in the surrogate's reproductive tract.

"A veterinarian and technician non-surgically harvest the unfertilized egg directly from the follicle of the donor mare. With an ultrasound probe in the vagina of the mare, they take the egg out of the donor mare prior to ovulation--they artificially ovulate the mare."

The surrogate dam is bred, and the egg is surgically transferred into her oviduct for fertilization. This process assists the donor mare that might not ovulate normally, or in which the egg doesn't become transported to the normal site of ovulation.

Intracytoplasmic Sperm Injection, or ICSI, is a newer procedure, in which fertilization occurs outside the mare, in a modification of in vitro fertilization. A mature, unfertilized egg is penetrated by a single sperm cell.

"The egg is held up against a pipette, and it is aspirated so suction holds it in place," said Vanderwall. "A microsurgical needle contains the single sperm cell, which we physically inject into the egg."

He noted that CSU currently has a mare in foal as a result of this technique. The embryo began to develop in vitro, and it was transferred into the recipient mare.

About the Author

Charlene Strickland

Award-winning writer Charlene Strickland lives in Bosque Farms, N.M. She has published 8 books and over 600 magazine articles, and is a member of the International Alliance of Equestrian Journalists.

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