Untimely End--Early Embryonic Death
Breeding horses can be both exhilarating and devastating. Wrapped up in the process are hopes, dreams, and visions of future greatness and glory. The hopes and dreams begin with the selection of a stallion for your special mare. You assess her strengths and weaknesses, then select that "just right" stallion to complement her. Even before the mating occurs, you can envision the foal which will be the product of this union. It will be the best one you've ever bred!
Then there is that tense time after the mating occurs, either via artificial insemination or by natural cover. Will she settle? Anxiously, you wait the required time period, then call for the veterinarian to do an ultrasound examination at or shortly after Day 16.
The veterinarian carefully inserts the transducer rectally to scan the uterus. You wait, and wait, and wait while the vet maneuvers the instrument, all the while keeping an eye on the ultrasound screen.
Then, the magic moment and the words you hoped for. "She's pregnant."
This is an exhilarating moment whether one has been involved in the breeding of one mare or hundreds. The miracle of nature has occurred once again. A tiny egg and an even tinier sperm have united. A new life is on the way.
Unfortunately, there are times where that exhilaration turns to despair.
Here's a possible scenario. The mare is bred. She is examined via ultrasound and declared to be in foal. You turn her out with a mixed group and one day, several weeks or more later, you glance over and see a gelding nuzzling the mare. She, in turn, is squatting and urinating, her tail cranked up and to one side, her vulva winking.
You quickly call the veterinarian. Another ultrasound exam. This time the news is not good tidings. "Nothing there. She lost it."
Questions assail your mind. Why? What happened? When did it happen? Will it happen again if she is rebred?
There will be more questions than answers. We are dealing with a problem that, unfortunately, is all too common--early embryonic death (EED). While your veterinarian won't have much trouble telling you what EED is, he or she might have a harder time determining just how it happened and why. The vet will be in good company. Some of the world's leading researchers in the field of equine reproduction are struggling to unravel all of the mysteries involved with EED.
They know a lot about it, but there is much more that is speculated, and just as much is unknown. What they do know beyond a doubt is that there is a significant disparity between mares which are declared to be pregnant and those which actually give birth to a live foal.
What Is EED?
Early embryonic death is defined as loss of the conceptus or embryo before organogenesis (origin and development of the organs) is complete at about 40 days. More often EED occurs before 35 days have elapsed, and it might be earlier--before the embryo even enters the uterus from the fallopian tube.
The causes are myriad.
Before we discuss them in some detail, however, we must set the stage by taking a look at the complex process that occurs during those first 35 to 40 days of gestation. Along the way, we will attempt to identify potential occurrences during those early days that are implicated in EED.
We will be using varying terms to identify that little package of life that is microscopically small during those first 35 to 40 days--zygote, morula, blastocyst, conceptus, embryo. Don't be confused. They all describe something that is better known in the early stages as an embryo, and in later stages as the fetus.
The information that follows doesn't come from a single source, nor is it technically complete. We will attempt to steer through the murky technical waters and provide a relatively clear picture of what is going on as this minuscule package of cells and genetic material begins growth and development that ultimately will become a live, healthy foal.
The information comes from many sources. We are indebted, for example, to the likes of W.R. (Twink) Allen, BVSc, PhD, ScD, DESM, MRCVS, of the University of Cambridge in the United Kingdom, who gave the keynote presentation "The Physiology of Early Pregnancy in the Mare" at the 2000 San Antonio AAEP meeting in November of 2000; O.J. Ginther, DVM, PhD, University of Wisconsin, who enthralled his audience during the Frank Milne Lecture on Reproduction at the AAEP gathering in 1998; the highly informative book Equine Reproductive Physiology, Breeding and Stud Management by M.C.G. Davies Morel, DVM, BSc, PhD, of the University of Wales; a treatise by Dale L. Paccamonti, DVM, MS, Dipl. ACT, who wrote a chapter called "Early Embryonic Death" in the textbook The Horse: Diseases and Clinical Management; Mats Troedsson, DVM, PhD, Dipl. ACT, from the University of Minnesota; researchers at Colorado State University, Texas A&M, University of Minnesota, University of Florida...the list is almost endless.
Let The Story Begin
It all starts when the egg or ovum is released by the ovarian follicle and begins its passage down through the fallopian tube. It arrives at the uterotubal junction, that spot where the fallopian tube links up with the uterus at a uterine horn. There the egg must wait, because it is unable to pass through this junction until it has been fertilized.
In a normal breeding by natural cover, the sperm will have been ejaculated into the cervix. This is the beginning of a journey for the sperm that will take them through the uterus and uterine horns until they, too, reach the uterotubal junction. The sperm move along on this journey via the driving action of their own tails, along with contractions of the female reproductive tract. Modern technological advances now make it possible in artificial insemination to place sperm at the uterotubal junction, thus bypassing the somewhat hostile environment of the uterus.
Once the sperm reach the uterotubal junction, they are able to slip right through, unlike the egg that must await fertilization. As the sperm have traveled along, they have been "capacitated" by uterine secretions. Being capacitated means that enzymes in the sperm head are activated in such a way that the sperm now are capable of penetrating the egg.
There might be many sperm still alive when the egg is reached, or there could be only a few, but one thing is certain--only a single member of the survivors will penetrate the egg. The moment the membrane of the egg is penetrated, there is an instantaneous response within the membrane. This response results in an impenetrable barrier that no other sperm are able to penetrate.
There is now a uniting of chromosomes within the fertilized egg, and the blueprint for that new life is "set in stone." Immediately, the development of what will be an embryo begins.
The first five days of this development occur in the fallopian tube, where the fertilization took place. This length of time in the fallopian tube is unique to the horse. By comparison, Allen says, a pig embryo will remain in the fallopian tube for only 48 hours, and a ruminant embryo will remain in the fallopian tube for about 72 hours.
About 24 hours after a single spermatozoa penetrates the equine egg, the fertilized ovum, now called a zygote, has divided by mitosis (growth by cell division) into two cells. The cell division continues until, by Day 4, there are a bundle of cells and our embryo-to-be is now called a morula.
As the cells continue to divide, the morula makes its way to the uterus, arriving on about Day 6. Up to that point, the new little bundle of life has remained about the same size. Now in the uterus, that changes as the morula grows.
At this stage, the morula has used up all of its own nutritional reserves and begins "feeding" on uterine secretions. The morula remains free and is floating about the uterus.
As Day 8 arrives, the cells of the morula become organized or differentiated, and three distinct areas can be identified--the embryo mass (shield); the blastocoel, which is a fluid-filled cavity that will become the yolk sac; and the trophoblast, the outer layer that ultimately will become the placenta (the vascular organ that maintains metabolic exchanges between the mare and fetus). Time for another new name--the morula is now called a blastocyst.
Once the cells have differentiated or organized, they begin to take on specific functions. Prior to this differentiation, all cells were theoretically capable of developing into a new individual if extracted from the morula. Once differentiation has occurred, this is no longer possible as each of the cells is given a message to pursue only a particular line of development.
It is important to note, Davies Morel of the University of Wales tells us, that at this differentiation stage, the conceptus is very susceptible to external physical effects, such as those induced by drugs, illness, chemicals, and so on. These, she reports, easily can disrupt the differentiation process, resulting in deformities, abnormalities, and a high risk of abortion.
Thus, we learn that even at this early stage of pregnancy, early embryonic death can occur as the result of outside forces that are capable of disrupting the delicate developmental balance.
Differentiation continues, and by Day 9 there are two cell layers surrounding the con-ceptus or embryo. The outer layer is called the ectoderm and the inner layer or lining is called the endoderm.
At this point the fluid-filled center is officially termed the yolk sac.The ectoderm and endoderm together form the yolk sac wall.
By Day 14, the embryo has grown to about 1.3 centimeters in diameter. It is at this point that a third cell layer begins to develop. It will be located between the endoderm and ectoderm, and will be called the mesoderm. The mesoderm, like the endoderm and ectoderm, eventually will encase the entire blastocyst or em-bryo. From these three cell layers, all placental and embryonic tissue will originate and develop.
At Day 16, the ectoderm folds over the top of the embryonic shield, taking the mesoderm with it. These two folds fuse, Davies Morel tells us, producing a fluid-filled protective space for the embryonic shield or mass. This is the amniotic sac (or amnion) containing the amniotic fluid, and it will protect the embryo for the rest of its prenatal (before birth) life.
The outer layer of the ectoderm and mesoderm folds also form a protective outer lining called the chorion.
During these first 16 days, the equine embryo has been bopping about the uterus like a nomad. It floats hither, thither, and yon, but is never still. At this point in its fragile life, its existence has not yet been recognized by the mare's reproductive system.
Ironically, during this period of its existence, the roaming conceptus is secreting a substance that can cause its demise--prostaglandin. It is assumed that this prostaglandin is used to stimulate the peristaltic contractions and relaxations of the myometrium (muscular uterine wall). These contractions and relaxations are what propel the conceptus through the uterine space during those first 16 days when it is awaiting recognition by the mare, according to Allen.
It is important to note at this point that prostaglandin can shut down the production of progesterone by the corpus luteum (the follicle left after ovulation of the egg). Progesterone is essential in maintaining pregnancy from very early on, all the way through gestation.
"The situation seems ironic," Allen says, "and no doubt reflects a finely balanced mechanism of action and interaction that, in order to distribute its all-important recognition message through the uterus, the equine conceptus must secrete the very hormone (prostaglandin) which it is striving to prevent the neighboring maternal endometrium from releasing to ensure its survival in a progesterone-dominated uterus."
When the conceptus or embryo secretes too much prostaglandin, the result can be early embryonic death.
"One cannot help the suspicion," Allen says, "that at least some of the relatively high proportion of total pregnancy losses in the mare which occur between Days 12 and 30 after ovulation (32%) stem not from any failure of release of sufficient maternal recognition of pregnancy factor from the conceptus to suppress the normal cyclical luteolytic pathway, but more from the secretion of too much prostaglandin by the wandering conceptus, which then gains untoward access to the peripheral circulation and thereby accidentally induces luteolysis (a shutdown or destruction of the progesterone-producing corpus luteum) of the ultra-sensitive corpus luteum."
In the above scenario, the conceptus might be developing normally, but if it secretes too much prostaglandin, it causes the destruction of the corpus luteum, which produces the progesterone that maintains a favorable uterine environment. Thus, the conceptus has provided itself with a death sentence.
When such a compromised conceptus is examined with ultrasound between Days 14 and 18, Allen says, it will appear to be "well developed and apparently normal."
However, he added, the ultrasound image also reveals that the mare's endometrium is edematous (containing an abnormal amount of fluid). This condition, Allen says, "is heralding the imminent onset of true estrus and the resulting relaxation of the cervix, leading to the expulsion of the conceptus from the uterus."
Day 15 Is "D-Day"
Let us back up a couple of days in the development process. If all has gone well during the first 14 days of pregnancy, the tiny embryo now faces another challenge. Davies Morel describes Day 15 of pregnancy as D-Day. It is at this point that, if the pregnancy is to be sustained, the mare's reproductive system must receive a message telling it that there is a life within the uterus that needs to be supported.
Just how this information is conveyed hasn't been totally defined, but it is believed that estrogen, which is also being secreted by the embryo, is the message bearer.
Once the mare's reproductive system acknowledges the pregnancy message, her body instantly goes to work to support the new life. From Day 15 onward, progesteroneproduced by the mare and estrogens secreted by the fetus are the dominant hormones in the system. Among other duties, they stimulate the production of "uterine milk" containing the necessary nutrients for survival of the conceptus, which survives as a free-living form for about 38 days. During that time, its sustenance comes from the uterine secretions it absorbs.
Again, we are faced with knowledge of the delicate balance that exists. At Day 15, if insufficient estrogens have been secreted by the conceptus, the re-productive system of the mare might fail to receive the pregnancy message, which means early embryonic death would be the result.
Once the message that a pregnancy exists is received by the mare's reproductive system and a normal response en-sues, and progesterone becomes the most important hormone. This hormone inhibits the production of prostaglandin and enables the pregnancy to continue.
Between Days 6 and 14, Davies Morel says, the concentration of progesterone (in the mare's bloodstream) is 8 to 15 nanograms per milliliter (ng/mL). However, when D-Day--Day 15--arrives, there is a marked increase in progesterone within the pregnant mare. Day 15 is the high point for progesterone concentration, and it declines somewhat after that. However, the levels continue to be significantly higher than in non-pregnant mares.
By Day 30, progesterone levels in the pregnant mare will have dropped to approximately 6 ng/mL. By Days 45-55, progesterone levels will have risen to 8 to 10 ng/mL and will remain at that level until Day 150.
Once more, we are faced with the fact that if this delicate balance is disrupted, early embryonic death can occur. If insufficient levels of progesterone are secreted by the mare's corpus luteum, the embryo will not survive.
We now add another phenomenon that occurs at Day 17. The free-roaming embryo ceases movement. Allen describes this stage in the life of the embryo in these rather colorful terms:
"A free-living, fully encapsulated equine embryo rattles around the maternal uterus for 10 days, liberating significant quantities of estrogens and prostaglandins through the capsule in an outward direction to maintain progesterone dominance of the uterus for its very existence, while at the same time imbibing quantities of protein-rich uterine milk though its capsule in the opposite or inward direction to sustain the growth and development it must undergo during this period.
"Movement stops abruptly around Day 17 with the sudden increase in myometrial tone. The precise underlying cause of this has yet to be determined, although it is, quite reasonably, considered widely to be the result of an interaction between the longer than normal period of progesterone dominance from the now-prolonged corpus luteum and the increasing quantity of estrogens secreted by the enlarging conceptus."
For at least the first 75-80 days of its life, the conceptus is dependent on progesterone produced by the corpus luteum and secondary corpus luteum for survival.
A quick refresher concerning the corpus luteum. In the non-pregnant mare, ovarian activity is stimulated by follicle stimulating hormone (FSH). This hormone causes a dominant ovarian follicle to develop and mature, eventually resulting in the ovulation of a mature egg. The eggless follicle then collapses and becomes the corpus luteum, which produces progesterone. If pregnant, the mare's uterus will later secrete the hormone pregnant mare serum gonadotropin (PMSG), which is high in follicle stimulating hormone (FSH) and luteinizing hormone (LH). This new hormonal activity between Days 40-60 of gestation results in another dominant follicle being developed. Once again there is ovulation, and in its wake a secondary corpus luteum, which also produces progesterone, is formed.
It is believed that PMSG plays a secondary role in maintaining a pregnancy. It very well might serve as a blocking agent to prevent the mare's body from expelling the fetus as a foreign body, writes Davies Morel. The uterus, she explains, is a privileged site--the only place in the body that under the influences of the hormonal changes associated with pregnancy, will tolerate a foreign body--the embryo. The "foreign" aspect stems from the fact that only half of the genetic makeup of the embryo comes from the mare. The other half is from the stallion--thus the "foreign" aspect.
As time goes on, the secondary corpus luteum takes over in the production of progesterone, ultimately relieving the primary corpus luteum of the lion's share of this duty. The prime source for progesterone remains the corpus luteum and secondary corpus luteum until the pregnancy reaches about 75 to 80 days of age. At that point the placenta begins to produce progesterone.
If at any point during those first 75-80 days the corpus luteal function is compromised and progesterone production shut down, the mare will abort unless she is supplemented with an outside source of progesterone.
Between Days 35 and 40, when the secondary corpus luteum is being formed, there will be a sharp increase in the amount of another hormone--maternal estrogen. Between Days 0-35, estrogen levels remain very similar to those observed during the non-pregnant diestrus period. However, between Days 35-40, estrogen levels rise sharply. It is believed, Davies Morel tells us, that estrogen is having the same effect on follicle stimulation in the pregnant mare that it does in the non-pregnant mare. However, for some reason this estrogen activity does not cause the mare to exhibit estrual behavior as it does in the non-pregnant mare, even though she develops a mature follicle and ovulates.
It's another of nature's little balancing acts and mysteries in equine reproduction.
Knowledge about the role and function of progesterone has grown a great deal in the past decade or so. That knowledge has enabled researchers to find ways to use the hormone in synthetic form to prevent early embryonic death from a lack of normally produced progesterone, and thus to aid in maintaining a pregnancy all the way to term.
More about that a bit later. For now, let us return to our examination of the developing conceptus during its first 35-40 days.
Growth Of The Placenta
On Day 16, reports Davies Morel, the first evidence of blood vessels developing in the center of the yolk sac wall can be seen. This network of blood vessels will become the blood system of the placenta. By Day 18, the artery carrying blood toward the mother and the vein carrying blood away from the mother can be identified.
On Day 20, according to Davies Morel, a pouch of the embryonic hindgut can be seen immediately below the placenta. This is known as the allantois, and it continues to grow in pace with the embryo. As the fluid-filled allantoic sac increases in size, the umbilical cord becomes evident.
As the conceptus continues to grow, its nutrient needs expand, and at this point the fetus must call upon the mare for additional nourishment. In order to receive nourishment from the mare, the fetus must have a lifeline or attachment--the umbilical cord.
The attaching or implanting begins at about Day 20. The first identifiable attaching between mother and fetus occurs around Day 25. This is a temporary attachment, with a permanent attachment being made at about Day 38.
A great deal has happened in 38 days. The minuscule conceptus has time after time faced hazards that could terminate its existence. If it has survived for 35 days and is developing normally, its chances of being carried to term increase.
In discussing the early days in the life of a conceptus or embryo, we have paused to take a look at potential causes of EED. Time now to take a look at some of these suspected causes in more detail and, at the same time, examine some preventive measures.
Possible Cause: Hormone Deficiency
As already mentioned, one of the prime suspects in early embryonic death is the lack of progesterone.
Researchers have reasoned that if a lack of progesterone can cause early embryonic death, then providing the mare with an outside source of this essential hormone could prevent EED. One of the leaders in progesterone supplementation research has been E.L. Squires, PhD, of Colorado State University (CSU). In one study, the CSU researchers removed the ovaries of pregnant mares very early in the pregnancy. Without ovaries, of course, the progesterone-producing corpus luteum was gone. Thus, at that stage of pregnancy, the mares would be unable to generate any progesterone.
The researchers then provided an outside source of progesterone to the mares. The outside source of progesterone was able to make up for the loss from natural sources and most of the mares remained pregnant. Without the progesterone, the embryos died.
Many pregnant mares which do not produce an adequate supply of progesterone have been successfully supplemented with outside sources of progesterone, such as Regumate, and have carried foals to term.
Supplemental progesterone normally is required until the placenta begins producing sufficient quantities of progesterone, usually sometime after Days 75-80. In some mares, supplementation might be required even beyond the 75- to 80-day mark, but that is a subject for another article.
While outside sources of progesterone have taken on the status of embryonic life-savers, there can be pitfalls. Troedsson from the University of Minnesota told a gathering of theriogenologists (reproduction specialists) in San Antonio in December 2000 that administration of progesterone can have unwanted effects in the case of fetal death. He showed slides of a mummified fetus that was delivered months after it had died in the uterus. However, the mare was being supplemented with an outside source of progesterone, which did not permit her body to react normally and expel the non-living fetus. It is highly important, he emphasized, that when progesterone is supplemented, the fetus be monitored closely to determine its health.
Age Of The Mare
Unfortunately, an inadequate supply of progesterone is only one of a myriad of potential causes of early embryonic death. Even the age of the mare can be factor.
It has been found, for example, that very young (less than three years) and very old mares (more than 18 years) are more apt to lose a pregnancy in the early stages than are mares in the in-between years.
The high rate of early embryonic death in yearling mares, Paccamonti tells us, has been attributed to immaturity, inadequate nutrition, and/or physical stress. Older mares, he reports, were found to have a lower pregnancy rate, higher loss rate, and lower live foaling rate compared with younger mares.
Of course, when discussing older mares, it often is difficult to differentiate whether the problem arises because of age only or because the mare might be sub-fertile due to multiple pregnancies that have taken their toll on uterine well-being, infections that might have compromised uterine health, and/or various forms of trauma that could have left irreparable uterine damage in its wake.
It also appears that the problem in sub-fertile mares is not just an inability to carry a foal to term. Embryos collected from the oviducts of sub-fertile donors four days post-ovulation and transferred to normal recipients resulted in lower pregnancy rates at Day 14 than embryos from normal mares, according to Paccamonti. Also, Paccamonti reported that abnormal embryos have been collected more often from sub-fertile mares than from maiden mares.
Of course, sometimes the mare isn't at fault when early embryonic death results from an abnormal embryo--the problem could be the stallion. It has been reported that some stallions appear to be involved in more cases of EED than others.
However, Paccamonti tells us, most chromosomal abnormalities leading to EED probably are not inherited, but arise during the formation and the aging of the gametes (cells that make up sperm and ovum). These abnormalities might result in an inability of the resulting zygote to develop into a viable embryo.
It has been found, Paccamonti says, that EED caused by aged gametes might be increased when mating is not closely timed to ovulation. Although insemination in the wake of ovulation can result in a pregnancy, he says, the incidence of EED is higher than when insemination occurs before ovulation.
Fallopian tube roblems can cause EED. Post-mortem examinations, for example, have revealed signs of inflammation in this location in several mares which lost pregnancies early on.
Something else often implicated in EED is endometritis. Simply put, this is an inflammation of the endometrium, which is the inner lining of the uterus. The death of the conceptus might result from a direct toxic effect of invading bacteria or something more complicated, such as the release of prostaglandins as a result of the inflammation.
The use of anabolic steroids in the mare also has been implicated in EED. Long-term anabolic steroid treatment has negative effects on subsequent reproductive performance, Paccamonti reports. He said that a trend toward higher EED was observed in mares treated for the previous year with anabolic steroids.
The mare's nutritional state can be another factor in early embryonic death. It is generally believed that mares in a poor nutritional state will be more apt to lose a conceptus during those early days of pregnancy than will mares in good physical condition.
While it's important to know what can cause early embryonic death, it's also important to realize that some presumed causes don't stand the test of scientific scrutiny.
For example, it was thought for some years that transporting a mare while she was in various stages of the estrous cycle and early days of pregnancy could first of all affect the cycle, and secondly decrease the embryo's chance of survival. Researchers at Colorado State University took a hard look at the first part of this supposition in one of their studies. Their conclusion was that transportation, even for as long as 12 hours during the pre-ovulatory stages of estrus, did not affect the estrous cycle.
The same was true in other studies involving pregnant mares, according to Paccamonti. One study found that early pregnancy losses in mares which were hauled for nine hours were no different than for mares which were not transported.
Many have been concerned that repeated palpation or ultrasound examinations might increase the likelihood of early embryonic death. Yet, no scientific evidence states this to be true.
Some owners think EED is caused by breeding on the foal heat. Paccamonti states that while pregnancy rates might be lower for mares bred on foal heat, there are no greater losses due to EED than in mares which were bred 30 days or more after foaling.
Now that we know some of the causes of EED, we should turn our attention to prevention.
Managing To Prevent EED
We have already discussed the use of progesterone, which is its own success story. However, automatically putting mares on Regumate isn't necessarily the end-all answer for the breeder. The proper approach for each at-risk mare must be left to the veterinarian. It will be for him or her to determine if the mare should be supplemented, how much should be administered, and within what timeframe.
Cleanliness is a strong ally when battling EED. Everything possible should be done to prevent any form of contamination during breeding.
Mares which suffer from endometritis should be appropriately treated so that when the conceptus arrives in the uterus, it will have a clean and healthy environment in which to develop.
When poor nutrition is involved, the treatment method is obvious--make certain the mare is on the appropriate nutritional plane long before breeding time, and keep her there all the way through the pregnancy and into the lactation phase.
The final question before researchers is this: Will mares which have experienced EED once be more likely to experience it again?
There is no direct answer to that question. Much depends on what caused EED in the first place. However, if a hostile uterine environment is the culprit and the uterus remains in that state, one can expect that EED would occur again. The same would be true if the mare is unable to produce enough progesterone and is not supplemented.
Thus, we end our tale on a bad news, good news note. The bad news is that early embryonic death will continue to take its toll. The good news is that scientific knowledge and technology enable us to cut into the toll at present, with the hope that more advances through research will lower it even more in the future.
Schweizer, Christine M. Understanding Breeding Management. Lexington: Eclipse Press, 1999.
Schweizer, Christine M. Understanding the Broodmare. Lexington: Eclipse Press, 1998.
About the Author
Les Sellnow is a free-lance writer based near Riverton, Wyo. He specializes in articles on equine research, and operates a ranch where he raises horses and livestock. He has authored several fiction and non-fiction books, including Understanding Equine Lameness and Understanding The Young Horse, published by Eclipse Press and available at www.exclusivelyequine.com or by calling 800/582-5604.
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