A successful breeding calls for the latest gadgets and technology.

Whether reproduction occurs through natural breeding or artificial insemination, techniques and tools exist to help mare and stallion owners achieve their final goal: a complication-free conception, a problem-free pregnancy, and a healthy foal. A successful one-time breeding is the best scenario; however, Mother Nature often has other plans. When fertility problems occur, a veterinarian with a specialty in equine reproduction can help provide the tools--both high- and low-tech--to ensure breeding success.

On the Mare Side ...

Inducing Ovulation Regardless of breeding method, inseminating the mare only once during her estrous cycle is ideal. Because there is a very limited window of time for insemination, the mare must be ready (ovulating) at the same time that the stallion is available. "The only way to effectively do this is to induce ovulation," says Ed Squires, DVM, MS, PhD, Dipl. ACT, executive director of the University of Kentucky's Gluck Equine Research Foundation. To induce ovulation, veterinarians can inject the mare with deslorelin, a synthetic hormone that stimulates the development of the ovarian follicle and release of the oocyte (egg).

Detecting Ovulation Recent innovations such as color Doppler ultrasound, which illustrates velocity and direction of blood flow, have been useful for breeding. The ultrasound machine is used to detect increased blood flow around the follicle as the follicle prepares for ovulation. "Looking at blood flow as a predictor of ovulation is still in its infancy," says Squires, "but the thought is that if a follicle that is close to ovulating has a difference in blood flow, then you can more accurately predict ovulation."

Insemination The technique of inseminating the mare with a very low dose of semen has been optimized and incorporated more in breeding programs over the last 10 years, according to Squires. A method called deep-horn insemination places the semen directly into a horn of the uterus, close to the uterine papilla (the site where the fallopian tube enters the uterus), instead of in the body of the uterus where the sperm must migrate to the oviduct (fallopian tube). "Instead of stopping at the body of the uterus you thread a long, specialized catheter to the tip of the horn and deposit the semen," says Squires. "This technique allows the use of lower numbers of sperm placed closer to the site of fertilization." This is helpful when the semen is frozen-thawed or when there is not a large number of sperm--either the stallion is not producing many viable sperm or there is a limited dose available.

Fertilization Another technique, called GIFT (gamete intrafallopian tube transfer), places the egg and the sperm together in the oviduct. Transvaginal (via the vagina) ultrasound guides the aspiration of the follicle for transfer of the oocyte to the fallopian tube, where fertilization occurs.

Transvaginal ultrasound also is used during intracytoplasmic sperm injection (ICSI), where sperm is injected directly into the egg.

Ultrasound bounces sound waves off soft tissues and records the resulting signals, which helps veterinarians visualize internal structures. Ultrasonography is an increasingly inexpensive technology that has become a standard tool in the breeding industry for monitoring follicular growth and changes in the uterus, detecting pregnancy, and observing the fetus.

On the Stallion Side ...

When fertility problems arise, many breeders just focus on the mare. Her barren (nonpregnant) state is the most obvious indicator of the success or failure of a breeding. However, the stallion is just as important. "The stallion doesn't get as much attention," says Karen Berk, owner and operator of Equine Reproduction Services in Dunnellon, Fla. "Each stallion is different, and mare owners must become educated on what they should ask for and what they should be receiving."

Collecting and Evaluating Semen If the mare cannot be shipped to the stallion or if artificial insemination (AI) is your breeding method of choice, a breeding phantom (dummy mount) must stand in for the real thing. The breeding phantom can have an effect on the quality of the semen, according to Berk. One size does not fit all, and Berk has obtained better sperm samples with a dummy mount that is tailored to the stallion's size and mating position.

"When the stallion ejaculates ... the front legs go straight down, the legs lock, and the neck arches over the mare," says Berk. "Not all breeding phantoms allow this to happen." Some mounts are too wide or round and put pressure on the withers by spreading the stallion's front legs apart when he mounts. The wrong size or shape mount can make the stallion very sore and stiff. Mounts that are too low put undue pressure on the hocks and pasterns. Berk reports a 30% increase in ejaculate sperm cells when the stallion is physically comfortable during semen collection.

Sperm Quantity Evaluation of sperm quantity is critical for estimating a stallion's breeding potential. Accurate sperm counts in ejaculates are essential to ensure sufficient cells are present in shipments to mare owners. "It takes 200-500 million progressively motile cells upon insemination to get a mare pregnant," says Berk. "It never takes one cell unless you are fertilizing in vitro (in the lab)."

A hemocytometer often is used to measure sperm numbers and is considered the gold standard for counting sperm in a sample. A hemocytometer is a specialized glass slide with wells and a grid marked into it. A solution containing the ejaculate is injected into chambers in the slide, covered with a coverslip, and examined under a phase contrast microscope.

Other devices determine sperm cell concentration by measuring the amount of light that is diffracted away from the sample. These devices are about the size of a postal scale or small printer and are used in laboratories and breeding operations. In a study reported at the 2001 AAEP Convention, scientists at Texas A&M University's College of Veterinary Medicine tested the performance of several photometric devices against the hemocytometer. According to the authors, the photometric devices tended to overestimate or underestimate the sperm concentrations when compared to the hemocytometer standard.

Sperm Quality also affects the stallion's ability to impregnate the mare. "The important parameters of semen quality are motility, volume, and density (concentration)," says Berk. Reproduction specialists often use a phase contrast microscope to determine motility--the ability of the sperm to move in a straight, forward motion. Phase contrast microscopy provides a dark background for viewing live cells that would appear transparent under a regular microscope.

Counting sperm cells in a known volume, such as with the hemocytometer, provides overall sperm concentration. This serves as a baseline for calculating the amount of semen extender needed to maintain the sperm during shipment and the number of doses possible from an ejaculate.

Breeding specialists use the following formula to determine the number of progressively motile sperm available in the sample: Volume x Concentration x Percentage of motile sperm = the number of progressively motile sperm available.

The volume is the number of milliliters of fluid obtained in the ejaculate. The concentration represents the number of progressively motile sperm in the sample.

For stallions with low sperm density, a technique called density gradient centrifugation might improve the quality and potency of semen prior to shipping. In this procedure the semen is placed in a tube and spun at relatively low speeds in a centrifuge (a rotating apparatus) to separate liquid and solid sample portions.

"The old thinking was that centrifugation damages sperm cells," says Jose Len, MVZ, MS, Dipl. ACT, instructor in theri-ogenology at Louisiana State University. But Len and his colleagues found that at very low forces (900 g for 10 minutes) the sperm's plasma membrane (the cell membrane), ability to swim, and the integrity of the acrosome (the enzyme-producing area on the head of the sperm cell) were not damaged. "This technique is used more and more to improve the fertility of certain stallions," says Len. A fairly recent advancement is the use of a silica-based solution to remove poor-quality spermatozoa and, thus, provide a higher-quality sample.

Researchers also are investigating the use of a "cushion" medium that is used with centrifugation to help preserve fertility of frozen semen. The cushion allows the semen to be centrifuged at high centrifugal forces for a longer period of time and not damage the sperm. This allows a higher recovery of sperm (less sperm is lost in the process).

Semen Preservation and Transport Cryopreservation (freezing) of sperm allows stallion owners to store sperm indefinitely. This method is being used more often to ship semen around the world. "Frozen semen is like buying an insurance policy for the stallion owner," says Berk. "However, mare owners also need to be aware that it can be used only under certain circumstances. For example, you should not use cryopreservation with a mare that has a history of reproductive problems." Berk advises owners to discuss breeding plans for mares in their teens or older with a veterinarian, as conception rates for these horses are lower.

"Assisted reproduction is becoming more and more common," says Squires. "We are using more and more frozen semen than we were in the past, because the technology seems to be improving. Cold-shipped semen lasts for a maximum of only about 48 hours at 5��C (41��F). Frozen semen, if it is stored properly in liquid nitrogen, should last 50,000 years without a decrease in quality or fertility. This allows you to have something that lasts forever, and you can export it around the world."

"There is a lot of work being done in this area," says Len. "We are still trying to improve cryopreservation protocols." Because stallions' sperm plasma membrane is composed differently than that of other animals, equine sperm might require different freezing protocols than animals such as cows and pigs.

Take-Home Message

Before you breed your mare or stallion, educate yourself. Says Karen Berk, "If you are going to spend the money--and it costs a lot--become educated about what you should be getting."

Also seek the expertise of someone who knows what they are doing, says Len. You can find an equine theriogenologist (a veterinarian who specializes in reproduction) through the AAEP website (www.aaep.org), through the American College of Theriogenologists (www.theriogenology.org), or by asking your veterinarian for a referral.

About the Author

Nancy Zacks, MS

Nancy Zacks holds an M.S. in Science Journalism from the Boston University College of Communication. She grew up in suburban Philadelphia where she learned to ride over fields and fences in nearby Malvern, Pa. When not writing, she enjoys riding at an eventing barn, drawing and painting horses, volunteering at a therapeutic riding program, and walking with Lilly, her black Labrador Retriever.

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