The Art of  BREEDING
By Sue Piscopo, DVM, PHD

Successful breeding of horses is not always as easy as presenting a receptive mare to a fertile stallion.  There are many things that can go awry with the reproductive process.  Broodmares that were once fertile and produced many excellent foals might one day face the realities of repetitive foaling injuries, chronic uterine infections, or simply old age.  Stallions also can experience a decrease in sperm number and quality as the years pass.  Pain associated with arthritic hocks or chronic back problems can interfere with a stallion’s ability to mate.  When a mare or stallion develops any kind of problem that interferes with its ability to perform reproductively, it could be time to investigate the options of assisted reproductive technology (ART).  These are the techniques that offer the hope of producing foals from mares or stallions that might no longer be able to pass along their genes to future generations any other way.
The Subfertile Stallion:
Research has shown that maximum pregnancy rates are achieved when a stallion’s ejaculate is sufficient quality to allow a mare to be inseminated with 500 million progressively motile sperm every other day during the five or seven days of heat.  Problems arise when semen evaluation reveals either insufficient numbers or decreased quality of sperm, with more abnormal sperm or fewer sperm that can swim vigorously in one direction (progressively motile).  Therefore, methods have been developed to make the best use of limited numbers of good-quality sperm.
Fertility With Fewer Sperm:
Deep Uterine and Hysteroscopic Insemination – As the name implies, deep uterine insemination involves artificial insemination of a mare deep within the uterus, inside the uterine horn adjacent to the ovary with a pre-ovulatory follicle identified via ultrasound.  Only five to 25 million progressively motile sperm are required for this method, because the long journey from uterine body to uterine horn, during which many sperm divert from the path to the egg are lost, has already been completed.
Sperm are taken even farther in the female tract during hysteroscopic insemination, during which sperm are deposited at the uterotubal junction, or the very edge of the oviduct.  Here, the sperm need only travel a short distance to meet the egg or oocyte within the oviduct.  Only one to 10 million progressively motile sperm are required.
Gamete Intrafallopian Transter (GIFT) – GIFT is a new technique in horses that involves the transfer of both sperm and oocytes directly into a recipient mare’s oviduct.  Obviously, the sperm needn’t travel far, but must have the ability to penetrate and fertilize an oocyte.  A mere 200,000 – 500,000 progressively motile sperm are required.  Pregnancy rates ranging from 27 to 82% have been reported.
Intracytoplasmic Sperm Injection (ICSI) – When a subfertile stallion is known to be the problem in a breeding pair and various insemination procedures as well as GIFT have failed, there is another ART technique that has produced several foals and requires only minimal numbers of sperm.  During intracytoplasmic sperm injection (ICSI), a single sperm is injected into a mature oocyte (one ready for fertilization).  Injected oocytes are then cultured in the laboratory until newly formed embryos can be transferred to recipient mares.  The ICSI technique can provide a means of obtaining foals from stallions with very low sperm numbers or poor-quality sperm.
Understanding Ovulation
It is important to understand the processes of a normal estrus, or heat, in order to understand what can go wrong with ovulation and interfere with fertility.  Mares are seasonally polyestrus, starting to cycle each spring as daylight lengthens and continuing to ovulate approximately every three weeks until fall, when the days get shorter.  After a transition period in the spring, the first ovulation is marked by the appearance of a dominant follicle on one ovary, reaching a size of approximately 35 mm.  This follicle secretes estrogens, which signal the start of an estrus period lasting five to seven days.  The dominant follicle continues to grow during estrus, reaching a maximum diameter of about 45mm.  Toward the end of estrus, there is a surge of luteinizing hormone (LH), which stimulates ovulation.  The empty follicle forms the corpus luteum (CL), which secretes progesterone to support a new pregnancy that might be developing.  If no pregnancy occurs, 14 – 16 days after ovulation the CL is destroyed in a process called luteolysis, and the cycle begins again.

The Subfertile Mare:
Unlike the subfertile stallion, identifying a subfertile mare is not as easy as doing a semen evaluation.  Most subfertile mares have a history of occasional reproductive underperformance or outright failure.  The veterinarian will thus perform a thorough history and physical examination to try to diagnose the problem.  What is the mare’s age?  We know that fertility decreases after age 13 and drops precipitously after age 20.  Is she in good health?  Is there evidence of Cushing’s disease or poor nutrition?
The mare’s reproductive history is also very important.  If she has foaled in the past, she might have stretching or scarring that has allowed pneumovagina, or air in the vagina, to develop.  This can promote bacterial entry into the cervix and uterus.  Examination with rectal palpation and ultrasound might reveal interior tears, adhesions, scars, and retained fluid that could be interfering with successful breeding.
A complete examination will require samples for uterine culture (to identify any bacteria present) and cytology (evaluation of cells such as inflammatory cells that might be present) as well as uterine biopsy specimens to examine the endometrial lining.  The uterus must be capable of supporting the placenta and the developing fetus.
In terms of treatment, less serious problems can be corrected or managed with remedies such as careful selection of a highly fertile stallion, insemination close to the time of ovulation, and perhaps the timed use of oxytocin after insemination to help clear fluids from the uterus and prevent infection.  More serious problems might require ART.
Younger mares often have time on their side.  In other words, many of the problems causing subfertility in mares 6 to 10 and even up to 15 years of age are related to the tubular genitalia (vulva, vagina, cervix, etc.) and anatomical changes of aging that cause the reproductive tract to shift in position, allowing fluids and contaminants to interfere with breeding.  Luckily, many of these problems can be managed conservatively.  
Even when more severe problems develop, younger mares are usually better candidates for ART because fertility is maintained in the ovary and oviduct, where many ART techniques are aimed.  Unfortunately, the same is not true for the older mare.

Problems in the Older Mare:
When a mare reaches 20 years or more, the decrease in fertility experienced is directly related to structural and functional changes in her reproductive tract.  Owners are sometimes confused when a productive broodmare reaches her 20s and suddenly can no longer conceive, even after delivering many foals in the last few years.  But these reproductive changes are due solely to aging and are independent of past reproductive performance.
For example, the oviduct is critical during the first five to six days of pregnancy.  It collects the oocyte from the follicle and sperm from the uterus, and provides the environment for fertilization and early embryonic development.  Yet ther is evidence that collection of the oocyte from the follicle is less effective in older mares, disturbing the rhythm of the entire process.
The ovary also suffers from aging.  During the ovulatory season, older mares have been shown to have sporadic ovulations instead of  regular ovulations at 21-day intervals.  Research has shown that during these sporadic cycles, aging mares begin a process where the ovaries fail to grow large follicles, perhaps due to depleted reserves of oocytes.  In one study, only 50% of mares aged greater than 20 years had three or more sequential ovulations during one season.  Once cycles become sporadic like this, it is typically only a few years before reproductive senescence, or the end of a mare’s reproductive life, becomes a reality.
Embryo Viability – Problems with oocyte and ovary viability have prompted the question of whether aged mares are reasonable candidates for embryo transfer.  For example, aged mares have had embryos removed for transfer to younger recipient mares.  Unfortunately, in general, few embryos are collected from older mares during each attempt, and these embryos tend to have more morphologic (structural) abnormalities.  Research has also shown that these embryos tend to be delayed in their development, producing fewer pregnancies and more embryonic loss.
Efforts are underway to determine if waiting an additional day before flushing the embryos from the uterus allows time for aged mares’ embryos to compensate for delayed development.  Even so, it is likely that there are still defects in embryos collected from older mares because of problems with the aged oocytes.
Oocyte Viability – There is growing evidence that oocyte defects might be of great concern for subfertility in aged mares.  These defects haven’t been specifically identified, but they are considered intrinsic and age-related.  Research has shown that when the same stallion is used, oocytes from young mares (6 to 10 years old) are significantly more likely to develop into embryonic vesicles by Day 12 (92%) than those from mares older than 20 years (31%), as evidenced by ultrasound examination.
Another study reported pregnancy rates of 54 – 83% for oocytes from young mares (less than 13 years old) and 27 -40% for oocytes from aged mares (older than 20 years).  These older oocytes were often transferred into subfertile recipient mares in a commercial program, yet still obtained about 30% pregnancy of higher.  These data again suggest that older oocytes have lower fertility.  However, even a 40% pregnancy rate offers hope, and suggests that transfer of oocytes from older mares into healthy young recipients could hold promise.



ART Update for Subfertile Mares.
New techniques are constantly evolving in the field of assisted reproduction.  Many times, the practical  applications arise from laboratory techniques developed for experimental purposes.
Oocyte Transfer – Compared to embryo transfer, which requires a donor mare to conceive and support an embryo for the first five to eight days of pregnancy, oocyte transfer requires only the growth of a pre-ovulatory follicle with a viable oocyte.  Once transferred to a recipient mare, fertilization and embryonic and fetal development all take place within the recipient mare.  Although data suggest that mares greater than 20 years of age have oocytes with decreased fertility, resulting in perhaps 30% pregnancy rate, this might be one of the best options for obtaining foals from aged mares.
Salvaging Oocytes – In the US, there is a limited source of ovaries from mares (such as slaughterhouses) to collect oocytes from follicles for experimentation.  Therefore, when ovaries become available from a mare through donation after euthanasia or some other means, the immature oocytes can be collected, matured in culture, and transferred to recipients.  This technique is now being applied more practically to salvage oocytes from ovaries of valuable mares that die suddenly or must be euthanized.
The ovaries can be surgically removed and shipped to a facility at Colorado State University for oocyte recovery and recipient transfer.  Three foals have been born since the technique was first attempted in 1999.  Research continues to improve the protocol for transfer of ovaries and maturation of immature oocytes, some of which was presented at the 2004 AAEP Convention.
Cryopreservation and Vetrification – The cryopreservation (cold storage) of oocytes or young embryos is problematic, since there is a high surface-to-volume ratio of these cells.  The relatively slow process of traditional freezing can disrupt the cellular structure, rendering it unable to develop once thawed.  Yet the use of frozen equine embryos has resulted in successful pregnancies, and the equine embryo structure plays an important role in that success.  For example, the equine embryo is unique in that it forms a type of membranous protein capsule around itself that prevents cryoprotectant from penetrating and water from leaving.  Smaller embryos (less than 300 micrometer) have been frozen most successfully, yet many breed registries won’t register a foal that is produced this way.
Vitrification is a process of rapid freezing, and it has allowed oocytes to be successfully preserved for later transfer.  In 2001, the first two foals were born from vitrified oocytes.  One of the more practical aspects of vetrification is that it allows for direct transfer of frozen oocytes into a recipient, since the thaw is so rapid.  In addition, immature oocytes that are vitrified can not only survive vitrification as well as mature oocytes, but can continue the maturation process afterward with good results.

Future Directions:
There are some thing that are still out of reach for subfertile horses, even in the age of ART.  Success with in vitro fertilization (IVF) for example, long a hope of the equine industry, has simply not materialized.  Superovulation (stimulating multiple follicles to ovulate at once) in mares has been problematic.  But embryo transfer continues to improve and is in wide use.  Oocyte transfer is now providing the hope of producing foals from older mares.  Insemination techniques are becoming more refined and allow greatly decreased numbers of sperm to achieve pregnancy.
The techniques of GIFT and ICSI are successfully achieving pregnancies.  Cloning has been achieved in the equid.  As with many other species, multiple laboratories are working to produce pregnancies from cloned equine embryos.  A clone is simply an identical twin, so if this technique becomes a reality, particularly valuable horse might be candidates for cloning.  Regardless, ART continues to advance, improving upon older techniques and adapting new ones to improve reproductive success in horses.