Broodmare and Stallion

Reproductive Management Techniques

- An Overview

by Brad Ray, M.S.
www.raystallionservices.com

Introduction

The purpose of this article is to give the reader an abbreviated summary of broodmare and stallion reproductive management techniques. While it will give an overview of current management practices, equine reproductive management is a field in which scientific investigation is ongoing and advancing steadily. New information, gleaned from the use of advanced techniques in molecular/cellular biology and endocrinology, is improving our understanding of the basic science of how the horse reproduces itself. Using this information, new techniques in the field known as "assisted reproduction" or "AR" emerge and are made available to help broodmare and stallion owners and managers.

What is "assisted reproduction"?

In short, the ways in which we humans intervene in, and hopefully improve, the reproductive processes of many species comprise the field of Assisted Reproduction (AR). These include techniques such as artificial insemination, transported semen, embryo collection and transfer, gamete (sex cell) and embryo preservation, and fertility/infertility management. While many of these techniques were first developed for species other than the equine, horse breeders quickly recognized the potential value of AR in their businesses. Even the hobbyist has something to gain from incorporating AR into their breeding program.


AR allows us to improve the reproductive efficiency of our mares and stallions, allowing them to reproduce more reliably, frequently, safely and over a greater portion of their lifetime. Using these powerful techniques, we can extend our horse's reproductive lifespan —even beyond their passing.

A Very Brief Overview of Basic Mare Reproductive Physiology Before discussing AR techniques, a bit of background on mare reproductive physiology is in order. The mare's reproductive tract consists of the vagina, cervix, uterus and two ovaries. It's basic purpose is to provide the environment, as well as the mare's genetic contribution— the egg (or "oocyte"), in which conception can occur and the resulting embryo can develop until it is capable of living on the outside. Mares are "seasonal breeders" — typically during the spring and summer months of March through September (in the northern hemisphere). During these months, the normal mare will cycle with an average of 19 to 21 days between ovulations. Each cycle can be divided into at least two distinct periods: diestrus and estrus.

Diestrus follows ovulation and lasts around 14 days. During diestrus, the mare's cycle is primarily under the influence of progesterone and she is externally and internally unreceptive to the stallion. Externally, she exhibits aggressive behavior toward an interested stallion, strongly discouraging him from any attempt to breed her. Internally, her cervix (which is the passageway between the vaginal vault and the uterus) is tightly closed and is therefore impenetrable by semen. Her ovaries are only in the early stages of developing "follicles"— fluid-filled capsules containing a single egg.

At approximately 12 days after ovulation, progesterone levels fall and, as a result of increasing follicular activity, estrogen levels rise and the mare enters the estrus phase of her cycle around 14 days after ovulation. The estrus phase will last 6-7 days. She becomes increasingly receptive to the stallion. By this time, she has developed 1-2 "dominant" follicles on her ovaries. The dominant follicle(s) continue to grow and the remaining non-dominant follicles undergo a process known as "atresia" and will not go on to ovulate.

The dominant follicle(s) will mature to approximately 35-50 mm in diameter, undergo maturational changes in response to the hormone LH, and ovulate at approximately day 20. A mare will typically remain receptive to the stallion for at least 12 hours after ovulation, during which time she is still fertile.

How can we control a mare's cycle?

Because the natural onset of cycling in early spring is the result of increased perception of light due to increased daylength, we can artificially hasten the onset of cycling by putting a mare "under lights" in early December.

A typical stall lit with a 120-200W bulb at 5:00 am until daylight and from 4:00 pm until 10:00 pm will stimulate early cycling activity. Most mares under this lighting regimen will have regular cycles by early to mid-February.

This allows the mare to be bred for an early January foaling date. While this is not an ideal time of the year to foal a mare, many breed registries dictate January 1 to be the "birthday" of their member horses. Young horses competing in weanling, yearling and two-year-old events are at a distinct advantage if they are born earlier in the year.


"Short Cycling"

This diestrus phase of a mare's cycle can be shortened by approximately one week by administering a lutealytic agent which ablates the corpus luteum, the ovarian gland responsible for progesterone production during diestrus.

This is sometimes done at the mare owner's request in order to shorten a mare's stay at a breeding facility or to synchronize the cycles of several mares so that they may be bred at approximately the same time. Conversely, some mares are given supplemental progesterone during diestrus to prolong diestrus, also to aid in the synchronization of multiple mares or to provide additional hormonal support during early gestation.

Induction of Ovulation

As a dominant ovarian follicle matures during the estrus phase of a mare's cycle, it develops the ability to "see" leuteinizing hormone, or LH by developing receptors for this protein hormone. LH is the hormone signal responsible for triggering the final maturational changes which a follicle must undergo prior to ovulation. The egg within the follicle must also undergo changes in order to be capable of normal fertilization and subsequent embryonic development. A mare will naturally produce LH and initiate this process on her own.

We can, however, exert some control on this process ourselves through the use of human chorionic gonadotropin or "hCG". hCG looks, structurally, like LH and therefore is able to trigger the same physiologic response by binding LH receptors associated with the follicle. A mare will generally ovulate within 36-48 hrs of hCG administration as long as the follicle has developed the receptors to "see" it. This is generally the case after 4 days of estrus and a dominant follicle 35 mm or larger in diameter. By intervening in the mare's cycle with hCG, we can control when she ovulates to better coincide with a stallion's breeding schedule, a frozen semen breeding protocol, synchronization of several mare's ovulations, even our own schedules. Induction of ovulation is commonplace in today's breeding facilities.


Artificial Insemination

Artificial insemination refers to the process of introducing semen into the uterus manually, without a natural breeding. Semen is collected from a stallion using an artificial vagina (AV) and is transferred into a mare's uterus via a long pipette which is passed through the cervix and into the uterus.

Advantages of an artificial insemination program include: 1) decreased risk of injury to mares, | stallions and handlers 2) the ability to breed multiple mares with semen from a single ejaculate 3) decreased risk of infection to the mare and stallion 4) the ability to breed a larger book of mares to a stallion 5) semen can be transported to mares off the stallion farm (transported semen—see later) and 6) increased pregnancy rates overall.

Artificial insemination programs also eliminate the need to breed a mare on an every-other-day basis as is the standard procedure in a "backyard" breeding program. Instead, ultrasound imaging of the mare's reproductive tract is used (ideally, on a daily basis) to follow the progression of follicular development as well as endometrial (uterine lining) changes during estrus. Ultrasound can also be used to detect any uterine or ovarian abnormalities which can then be factored into the management of the cycle. Such information is generally undetectable by rectal palpation. Using this information, possibly in conjunction with the above-mentioned cycle management techniques, ovulation can be predicted and insemination performed shortly before (within 24 hours). Pregnancy rates in a well-managed artificial insemination program typically exceed those obtained in a natural service program due to the higher level of attention paid to the mare's reproductive changes as well as a decreased risk of infection and ability to manage otherwise undetectable pathology. Semen collection and evaluation is best done in a facility designed for such procedures, with an appropriately equipped laboratory staffed with well-trained personnel. As mentioned above, stallion semen is collected with an artificial vagina (AV) consisting of a rigid cylindrical shell lined with a bladder filled with warm water ending in a sterile collection bottle. Most stallions in an artificial insemination program are trained to mount an artificial mare known as a "phantom" and are collected on an every-other-day basis. A tease mare is often present to provide additional stimulation for the stallion.

Once collected, the semen should be evaluated for volume, concentration, motility, total spermatozoa count, and overall character. Proper handling of the semen is critical as semen is extremely sensitive to temperature and contamination. The evaluation procedure allows the breeding manager to determine the necessary volume of semen to be introduced into the mare's uterus. As each collection is different, an evaluation should be performed for every collection. Ideally, a mare should be inseminated with enough semen to provide 500 million progressively motile spermatozoa. Most stallions provide collections yielding 4 to 12 billion spermatozoa, allowing for multiple inseminations.

Before insemination, the semen should be diluted in an "extender" (a nutrient mixture typically formulated from skim milk, glucose and antibiotic) to extend its' longevity and decrease the risk of infection.

Transported Semen

The advent of shipped semen technology has drastically changed the horse breeding business. In years past, a broodmare had to be transported to the stallion in order to be bred. This involved considerable risk, expense and inconvenience to the mare owner. Consequently, many mare owners chose to breed their mare to a stallion within a relatively short distance of their farm, sometimes having to overlook faults in quality and greatly limiting their choice of stallions. Transported semen, whether cooled or frozen, has allowed the breeder to select from stallions anywhere in the world. As a result, the overall quality of foals produced has improved greatly as breeders become more educated in a breeding market which has become highly competitive and specialized.


Cooled-shipped semen is collected and evaluated according to the above method and diluted into extenders specially formulated for maintaining the spermatozoa for up to 72 hours. A minimum of 1 billion progressively motile spermatozoa should be provided in each insemination dose, allowing for some loss during transit. The semen is packaged into a shipping container which has been designed to cool the semen slowly from 37 degrees C to approximately 5 degrees C over a period of 8 hours. The "industry standard" is the Equitainer, a very durable unit meant for use over many years, costing around $350.00. More recently, disposable units have become available and are adequate for most stallion's semen. They are designed to be used a limited number of times (generally 5) and are considerably less expensive to purchase and transport. The semen is transported to the mare via overnight or same-day delivery and inseminated upon arrival. Insemination should occur within 12-24 hours prior to ovulation. Shipped semen conception rates vary among stallions but are, overall, quite good in a well managed program. When considering breeding your mare with shipped semen, it is important to ask the stallion owner/facility about the stallion's shipped semen conception rates (per cycle), expected motility at 24 and 48 hours after collection, and charges associated with collection, packaging and shipment. A reputable facility should be willing to provide references.

Also, sending your mare to a good receiving station to manage the breeding will improve your chances of success.


Stallion semen may be frozen and stored at very low temperature for an extended period of time. It is stored submersed in liquid nitrogen at -196 C, a temperature so low that metabolic reactions in the spermatozoa occur at an extremely slow rate. In essence, the spermatozoa are kept in a state of suspended animation. The term "frozen" is not completely accurate to describe the state of the cells. Rather, the cytoplasm of the sperm cell has been treated with a cryoprotectant —a sort of antifreeze— which prevents the contents of the cell from truly solidifying. Without the cryoprotectant, the expansion forces due to freezing would shear intracellular structures as well as the cell membrane which surrounds them, destroying the cell and rendering the semen useless. These cryoprotectants and membrane stabilizers are contained in a number of specialized extenders formulated specifically for freezing semen. The methods used to freeze equine semen are highly technical, necessitating very specialized equipment and instrumentation. Protocols dictate very precise handling methods, packaging, cooling and thawing rates and temperatures. Even for those who do it frequently, there is a sense of awe when a dose of frozen semen is thawed and the previously inanimate cells come to life under a microscope, resuming their mission completely unaware that they have been frozen for many months or years.


Conception rates with frozen semen are generally lower than with fresh or cooled semen. The rigors of the freezing and thawing process invariably result in the loss of a portion of the motile spermatozoa. Post-thaw motility rates in the 40%-50% range are likely, with rates above 60% considered above-average. Also, most frozen semen has a decreased longevity in the mare's reproductive tract after insemination,  requiring  a  more exacting breeding protocol.

Best results are seen when a mare is inseminated within a 4 to 6 hour window around ovulation. hCG is often administered to better control/predict the timing of ovulation. An average of two cycles per pregnancy should be expected, though fertility is variable among stallions. For this reason, frozen semen is most often used when a stallion is unavailable due to injury or death, he resides in a foreign country, or has a busy show schedule during the breeding season.

Embryo Transfer

Embryo transfer (ET) entails the collection of a 7-8 day old embryo from a "donor" mare's uterus and transferring it into the uterus of a reproductively sound "recipient" mare. The cycles of the donor and recipient are synchronized with one another, often through the use of the above-mentioned hormonal modulation techniques. Once in estrus, the donor mare's cycle is not managed any differently than if she was to carry the foal on her own. However, at 7-8 days after ovulation, her uterus is flushed with several liters of an embryo collection medium with the fluid recovered and filtered through a very fine mesh which traps the embryo. At this time point in development, the embryo is an "expanded blastocyst" and is 500-1500 microns (0.5-1.5 mm) in diameter. The filtered fluid is searched under high magnification for the embryo which is then transferred into a special holding medium, where it will remain until transferred into the recipient. Embryo recovery rates are quite high (approximately 75% per attempt) when a fertile mare is bred to a fertile stallion, Problem mares will yield embryos at a lower rate depending upon their unique reproductive profile.


Once identified, the embryo is transferred, either surgically or non-surgically through the cervix, into the recipient's uterus. Most embryo transfer facilities currently use the non-surgical transfer procedure due to its greater ease and good rate of success (approximately 75-80% per transfer).

Recipient mares are often given supplemental progesterone after the transfer, improving the chances of success.


The recipient mare makes no genetic contribution to the developing foal, though her size can impose limits on the growth of the foal in utero. Studies have shown that embryos derived from large breeds transferred into smaller breed recipients may result in foals which are smaller at birth than they might have been, otherwise. However, the consensus is that these foals will "catch up" to their genetic potential. Regardless, an effort is made to more closely match donor and recipient size.

Embryo transfer is an excellent means of obtaining multiple foals from a donor mare and several registries now allow registration of more than one foal from a mare in a single season. "Superovulation" techniques allow breeders to collect and transfer multiple embryos from a mare on a single cycle—2 to 4 on average. ET also allows many mares which are incapable of carrying a pregnancy to term to continue producing. Show mares can produce foals with a minimal layoff from training and showing.

Oocyte Transfer

A variation on the embryo transfer theme is oocyte transfer. Ooctye (egg) collection involves the use of an ultrasound-guided needle to puncture the donor's preovulatory follicle. The contents of the follicle, ideally including the egg, are then aspirated into a collection bottle and searched under high magnification for the oocyte. Equine oocytes are quite small (100 microns or 0.1 mm in diameter) and the search and handling process is a delicate one.

Once recovered, the oocyte is surgically transferred into the oviduct of a synchronized recipient. The recipient has been artificially inseminated with semen from the stallion of choice an hour, or so, before the transfer. The spermatozoa have traveled into the oviduct into which the oocyte is transferred and conception occurs in the recipient. She then carries the developing foal as if it were her own. Again, Superovulation is often employed in order to yield multiple oocytes from the donor.

Equine oocyte transfer is a relatively new offering for the breeder, available commercially since 1995. Pregnancy rates have improved considerably and approach those of embryo transfer. It is a very valuable tool for obtaining foals from mares which are incapable of producing an embryo (due to oviductal blockages) or whose uterine environment is incapable of sustaining an embryo to the 7-8 day time-point when an embryo can be collected.

New Horizons

Much of the current research in equine assisted reproduction focuses on commercially viable methods for freezing equine embryos and oocytes for later transfer, separation of X and Y chromosome bearing spermatozoa for controlling the sex of the foal, in vitro fertilization, and even cloning. Likewise, basic scientific research is ongoing in the effort to better understand, at the molecular and cellular level, how horses reproduce.

Brad Ray, M.S. is general manager of Premier Breeding Services, LLC in Sedalia, CO. He earned a bachelor's degree in Molecular, Cellular, and Developmental Biology from the University of Colorado, Boulder and a master's degree in Reproductive Physiology from Colorado State University, where he was general manager of the Equine Reproduction Laboratory. He has worked in private industry since 1995. During the "off-season ", he operates a mobile laboratory offering stallion semen freezing services nationwide as well as breeding management consultation services.

He can be contacted at: Premier Breeding Services 303-681-9717 (lab) 719-240-1598 (cell) 5501 West Dakan Rd., Sedalia, CO 80135

 

GLOSSARY

atresia

loss of function, regression

cytoplasm

fluid within the cell confined by the cell membrane or wall (bacteria); contains the components for, and acts as the environment in which, the biochemical reactions necessary to cell function can occur :

endocrinology

the study of hormones and their physiologic actions.

expanded blastocyst

an early embryonic developmental stage in which the embryo consists of a hollow sphere of cells (the trophoblast) and includes a small cluster of cells which will ultimately develop into the fetus.

human chotionic gonadotropin (hCG)

a hormone produced by the human chorion (a fetal tissue) which can be administered to mares to initiate follicle maturation and ovulation.

lutealytic agent

an agent capable of causing "luteolysis" or j regression of luteal tissue.    The luteal: tissue of the corpus luteum is responsible for the production of progesterone during the diestrus phase of a mare's cycle.

luteinizing hormone (LH)

a reproductive hormone, produced by the anterior pituitary, which, in the female, triggers maturational changes in the ovarian follicle and oocyte during late estrus.

motility

a term which connotes the ability to move; "progressive" motility implies movement with clear direction and purpose.

oocyte

unfertilized egg/ovum

oviduct

or fallopian tube; the passageway through which the egg travels, after ovulation, to reach the uterus

progesterone

a reproductive hormone, produced by the corpus luteum during diestrus and early pregnancy. Though it acts on many tissue types, one of its principle functions is to increase uterine and cervical tone through smooth muscle contraction. Both are important in maintaining pregnancy.

superovulation

a term used to describe the hormonal super-stimulation of the ovary to cause the production of a greater than normal number of ovarian follicles and ovulations.



Text Copyright 2007 by Brad Ray
Photos Copyright 2007 by Horse Connection Magazine