The Facts of Life
- Dec 1, 2003
The fertilization of an egg and subsequent growth of one tiny cell into the perfect foal is a very complex process. Understanding the various stages of development can help you understand how pregnancies can be challenged and what's going on in the event of a problem.
Pregnancy in the mare can be divided into four main events--fertilization, early embryo development, placentation (formation of the placenta), and organ growth.
The ovum, or egg, released by the follicle is directed through the infundibulum, then down the fallopian tube (also called the oviduct) where it waits in the ampulla region for the arrival of the sperm. It is unable to pass through the uterotubal junction until it has been fertilized.
The sperm, having been ejaculated into the lumen (cavity) of the uterus, make their way up through the uterus to the uterotubal junction using contractions of the tract and the driving actions of their own tails. The sperm are attracted to the ovum by chemical stimulants the ovum produces while awaiting fertilization. On arrival at the uterotubal junction, they pass through to the fallopian tube and if the timing is correct, meet a waiting ovum in the ampulla.
As the sperm pass up through the female tract, they come in contact with uterine secretions, which induce a capacitation response in the acrosome region at the top of the sperm head. Capacitation activates enzymes in the acrosome region, which are essential to allowing penetration of the ovum and thus fertilization. Once in the vicinity of the ovum, sperm stick to the outer gelatinous layer.
Sperm force themselves through this outer gelatinous layer to the zona pellucida (the next layer in) by whipping their tails. They then digest a pathway through the zona pellucida using the enzyme acrosin released during capacitation.
As the sperm head meets the vitelline membrane of the ovum, the two fuse. The nuclei of the sperm and the ovum can then unite, joining their haploid complements of chromosomes (32) to give the full diploid set (64) of the new individual. This newly combined genetic material now dictates all the characteristics of the new individual.
Five to six days after fertilization, the embryo actively controls its passage through the uterotubal junction to the uterine horn, possibly via the localized secretion of estrogens or prostaglandin E2.
Early Embryo Development
Twenty-four hours after mating, the fertilized ovum--now termed a zygote--divides by mitosis (growth by cell division) into two cells. At this stage the outer gelatinous layer is lost and the embryo, still within the zona pellucida, continues to divide into four cells, eight cells, 16 cells, etc. At four days old, it is a bundle of cells and is termed a morula.
At this stage, the total volume and external size of the bundle of cells has not changed from the zygote stage. The cytoplasm of the original ovum has either been divided up between the cells in the morula or been used for energy. Nevertheless, the amount of genetic material has dramatically increased, giving a full identical complement to all cells of the morula.
As the cells continue to divide, the morula makes its way toward the uterotubal junction by counter-clockwise rotation. It passes through this junction and arrives in the uterus at Day 5-6.
It is very important that the uterus is in a fit condition to accept the embryo. This is a potential problem in mares which suffer from prolonged or acute post-mating endometritis (inflammation of the uterine wall; see "Make Room for Baby" on page 45). If the infection and/or inflammation has not cleared up prior to the embryo arriving in the uterus, the embryo will not survive in such a hostile environment.
At Day 5, a thin acellular glycoprotein layer--termed the capsule--appears in the space between the trophoblast (layer of cells around the morula that becomes the placenta) and the zona pellucida. This capsule is relatively unique to the mare, and its exact function is unclear. It might help prevent adhesion of the embryo to the uterine endometrium (wall), hence allowing the prolonged mobility (free-living) phase characteristic of equine conceptuses (embryos and their membranes).
From Day 6 the total size of the embryo starts to increase, which helps to force the thinning of the zona pellucida that will eventually break. The embryo then hatches through this break and is left surrounded by its capsule. At this time, the conceptus starts to derive nutrients for its growth and cell division from the surrounding uterine secretions, as by this stage it has used up all of its own reserves. These nutrients are passed through the capsule to the embryonic cells. The provision of such additional nutrients allows a further increase in size.
The morula is now in its mobility phase, free and floating within the uterus and deriving all its nutritional requirements from uterine histotroph (the part of the nutrition of the embryo derived from cellular sources other than blood). This secretion is designed to exactly match the requirements of the developing conceptus. This period of mobility is unusual; such a long period of free movement within the uterus is not seen in other mammals. In the mare, it lasts for at least 25 days, during which the conceptus moves from horn to horn and throughout the uterine body. Indeed this movement appears to be essential for informing the maternal system of the presence of the conceptus and maintaining the pregnancy. If the mobility is restricted--for example, by numerous uterine cysts or uterine adhesions--then the pregnancy will fail.
At Day 8, the cells of the morula become differentiated (organized) into three distinct areas--the embryonic disc, the blastocoel (fluid-filled cavity), and the trophoblast (see above). The morula is now termed a blastocyst.
These three distinct areas go to form the embryo proper (embryonic disc), the yolk sac (blastocoel), and the placenta (trophoblast). This cell differentiation marks the beginning of the switching on and off of various genes, by which cells become destined to pursue set lines of development. Prior to this differentiation, all cells in theory are capable, if extracted from the morula, of each developing into a new individual as none of their genes have been switched off. After differentiation, this is no longer possible as certain cells have been given the message to only pursue set lines of development. The mechanism behind this switching on and off of genes and its trigger are unknown in the horse. It is important to note that, at this differentiation stage, the conceptus is very susceptible to external physical effects such as drugs, other chemicals, disease, radiation, etc. These can disrupt the differentiation process, resulting in deformities, abnormalities, and a high risk of abortion or resorption. It is also notable that after this stage of development, embryo transfer is largely unsuccessful.
From this stage on, further differentiation takes place. At Day 9, two germ layers (cell layers)--the ectoderm (trophoblast), consisting of the outer blastocyst cell layers, and the endoderm, consisting of the inner cell lining--become evident.
The endoderm grows and develops, working its way around the inside of the ectoderm to give a complete inner layer. Together they form the yolk sac wall and provide the means by which the embryonic disc receives its nourishment from the uterine secretions. The blastocoel, or fluid-filled center, is now termed the yolk sac and acts as a temporary nutrient store (see above). This remains the major source of nutrients to the embryo until implantation or fixation to the uterus occurs.
At this stage the embryo is a clear sphere in shape--this means that like the human embryo, but unlike the cow and ewe, the embryo can be easily identified at an early stage using ultrasound (see early embryo sizes on page 38).
At Day 14, when the embryo has reached 0.5 inches (1.3 cm) in diameter, the mesoderm or third germ cell layer begins to develop. It becomes progressively evident between the ectoderm and endoderm, in the center of the yolk sac wall, again working its way down from the embryonic disc to enclose the whole blastocyst. These three germ cell layers are the cell layers from which all placental and embryonic tissue development originates. In the case of the placenta, the ectoderm forms the outer cell layers nearest the uterus, the mesoderm forms the blood vessels and nutrient transport system, and the endoderm forms the inner cell lining that will become the allantoic or waste sac (see above).
At Day 16, folds appear in the outer cell layers and the beginnings of the protective layers--which will surround the embryo--become evident. The ectoderm folds over the top of the embryonic disc, taking the mesoderm with it. These two folds fuse, producing a fluid-filled protective space for the embryonic disc; this is the amniotic sac containing the amniotic fluid (see above). At this stage, the first fixation of the embryo to the uterine wall is reported to occur, although this attachment might only be temporary (more on this later).
Initially, the amnion is visible as a clear fluid-filled bubble surrounding the embryo (see Day 20 illustration on previous page). As pregnancy progresses, it tends to collapse and lie close to the embryo. Throughout its life in utero, the amniotic sac provides a clean and protective environment in which the embryo can develop. The volume of amniotic fluid surrounding the fetus is about 0.1 gallons (0.4 liters) at 100 days post-fertilization and increases to 0.9 gallons (3.5 liters) at full term.
From this stage on, embryology can be dealt with in two sections--placentation and organ development.
The placenta has two major functions--the first is protection, and the second is regulation of the fetal environment in the form of nutrient intake and waste output. The placenta develops from the extra embryonic membranes--the trophoblast of the blastocyst. The first source of nutrients, and therefore a form of primitive placenta, is the yolk sac or blastocoel. This provides both a temporary store and a transport system for nutrients from uterine secretions to the embryo.
Day 14 sees the first evidence of blood vessels developing in the center of the yolk sac wall--the mesoderm. This will become the blood system of the placenta. By Day 18, the vitelline arteries (which carry blood from the embryo to the mare) and the vitelline vein (carrying blood from the mare to the embryo) are identifiable.
On Day 20, an out-pushing of the embryonic hindgut can be seen right below the placenta. This is termed the allantois, and it continues to grow with the embryo. This sac fills with allantoic fluid and is encompassed by the allantochorionic membrane (allantois) or placenta. Secretions of the allantochorion plus urinary fluid from the fetal bladder via the urachus (cord of fibrous tissue) within the umbilical cord form the allantoic fluid (see Day 20 illustration).
The volume of the allantois at Day 45 is approximately 3.7 ounces (110 mL), increasing to 2.2 gallons (8.5 liters) by Day 310, a considerably larger volume than is seen in the amniotic sac. The allantoic fluid increases in volume as the fetus grows.
During the first trimester (three to four months), the allantoic fluid is clear yellow and changes to brown/yellow as the pregnancy progresses. It is largely allantoic fluid that is evident at parturition when the waters break. The developing allantoic sac moves over the top of the fetus as its contents increase, forcing the fetus down to the bottom of the blastocyst, reducing, as it goes, the extent of the yolk sac, until the yolk sac is hardly visible (see Day 40 illustration).
As the allantoic sac increases in size, the umbilical cord becomes evident. The umbilicus consists of two vitelline arteries, one vitelline vein, the urachus that transfers waste products to the allantois, and supporting and connective tissue.
As the fetus develops, its nutrient demand increases. The nutrients provided via the yolk sac soon do not meet this demand; thus a more intimate relationship needs to develop between the mother and the fetus, and hence its period of mobility ceases and it begins to implant. This occurs as a gradual process from Day 25 onward. At this stage, the capsule begins to degenerate so the embryo starts to lose its clear spherical shape, and the lack of a capsule allows the fetus to adhere to the uterine wall.
The first identifiable attachment between mother and fetus around Day 25 is the chorionic girdle. This is a temporary attachment and normally implants the conceptus at the junction between the uterine body and the uterine horn. The chorionic girdle is a band of shallow folds encircling the allantochorionic membrane. Cells within this girdle invade the uterine endometrium. This attachment, though only tenuous, does provide a significant nutrient and gaseous exchange unit with the mother.
At Day 38, the chorionic girdle attachment separates and is replaced by the second form of attachment, the endometrial cups. In this, fetal cells invade the endometrium, forming a band around the inside of the uterus at the junction of the body and horn. These endometrial cups secrete equine chorionic gonadotropin (eCG), previously referred to as pregnant mare serum gonadotropin (PMSG), which is essential for the maintenance of early pregnancy and is often used in blood sample pregnancy tests.
Around Day 90, the endometrial cups begin to degenerate and slough away from the uterine endometrium. The reason for this seeming rejection is not fully understood, but might be a maternal rejection of the "foreign" fetal tissue. The remains of these sloughed off endometrial cups might be resorbed by the fetus during the remainder of the pregnancy, or they might be seen in the placenta at birth as pouches in the allantochorion.
Gradually over time as the endometrial cup attachment is lost, the rest of the fetal placenta begins to attach to the uterine epithelium. This attachment, which is independent of regression of the endometrial cups, begins between Day 45 and Day 70 and gradually becomes firmer over the next 100 days, becoming a solid attachment by Day 150.
At Day 45-70, the placenta takes on a velvety appearance created by fine microvilli (tiny finger-like projections) over its entire surface; hence the equine placenta is termed diffuse (widely distributed). These microvilli organize themselves into discrete microscopic bundles, or tufts, that invade into receiving invaginations in the uterine epithelium. These bundles of microvilli are termed microcotyledons, and their attachment develops over a period of time, becoming fully complete and functional by Day 150 (see illustration on page 32).
The placenta forms a strong attachment between fetus and mother, and is relatively thick with six cell layers--three cell layers on the fetal side and three on the maternal side--and four basement membranes. The equine placenta is termed epitheliochorial (the chorion is in contact with the endometrium) and covers the whole surface of the uterus, except the cervix (the cervical star) and the two uterotubular junctions.
The presence of the microcotyledons increases the surface area of the placenta and, therefore, the area for nutrient and gas exchange. Within each microcotyledon, the maternal and fetal blood supply system come into close proximity, allowing quite efficient diffusion despite the thickness of the placenta. However, the thickness of the placental attachment prevents the diffusion of any large protein molecules.
As immunoglobulins (antibodies) are large protein molecules, their diffusion across the placenta, and thus the foal's passive immunity, are limited. Passage of immunoglobulins via colostrum (first milk after birth) is, therefore, of utmost importance for the foal.
The thickness of the placenta varies in mammals, but in general, the thicker the placenta, the less efficient the transfer of passive immunity in utero and hence the increased reliance upon colostrum. Primates, with their relatively thin placentas, do not rely as heavily upon colostrum. However, a thicker placenta as seen in the mare has the advantage of providing extra protection to the fetus from harmful maternal blood-borne factors.
As pregnancy progresses and the fetus grows, demands upon the placenta increase. In the mare, the placenta is fully formed at Day 150 and cannot develop further after this time. However, as pregnancy progresses, the maternal epithelium stretches as the uterus increases in size. As a result, the placenta also stretches and becomes thinner and hence the resistance to gaseous and nutrient exchange decreases. The placenta then becomes more efficient as the demands of the fetus increase.
By full term, the placenta in a 15- to 16-hand horse weighs about 8.8 pounds (4 kg). Its surface area is approximately 15.1 square feet (14,000 cm2) and it is about 0.04 inches (1 mm) thick. The foal's birth weight is directly proportional to the surface area of the placenta, as this is the limiting factor controlling nutrient and gas exchange and hence their availability to the developing fetus. The surface area of a placenta can be restricted for several reasons, including the presence of twins.
Twinning presents an increasing problem in broodmare management, especially in intensively bred horses (i.e., Thoroughbreds). The incidence of twin ovulations, which have the potential to result in twin conceptuses in the Thoroughbred, is 20-25%. Of this potential number of twins, significant natural reduction does occur with one twin dying (70% of twins are unilateral, or found in one horn, of which 85% naturally reduce; 30% are bilateral, or found in both horns, none of which naturally reduce).
If twins do develop to the placentation stage, the area of the uterus available for each placenta is restricted by the presence of the other fetus. If the division of uterine surface area available to each twin is equal, then both twins have an equal chance of survival, but their birth weights will be reduced. If the division is unequal, then the smaller one might cause the whole pregnancy to abort or, if the pregnancy is not well advanced, it might die and become mummified.
If mummification occurs, the pregnancy can continue, but if beyond Day 150, the placenta of the larger surviving fetus cannot expand into the uterine surface originally occupied by the now dead fetus. At term, therefore, a single foal will be born, but with a reduced birth weight due to placental restriction (left).
Organ development arises from the reorganization of cell populations within the embryonic disc. This organization is related to that which occurs in placentation previously discussed. This can be divided into two basic sections--gastrulation and neurulation.
Gastrulation is defined as the organization of the embryo into three germ layers--ectoderm, mesoderm, and endoderm. First, the central blastomeres (cells of the embryonic disc) organize themselves into smaller outer and larger inner blastomeres.
The larger blastomeres collect underneath the disc and migrate in two directions. First, they line the remaining ectoderm of the blastocyst, forming the endoderm. Second, they move within the embryonic disc, creating at Day 11 the first asymmetry, a thicker area at the caudal (tail) end and a thinner area at the cranial (head) end.
At Day 14, a change in the embryonic disc (neural plate) becomes evident with the beginnings of the primitive streak appearing following the movement of some epiblast cells (see Day 14 illustration on page 30). The primitive streak is the longitudinal axis of the embryo, and at this stage it is about 0.4 inches (10 mm) in length.
The epiblast cells now move inward and back to the center of the caudal (rearward) end of the disc (see Day 14 illustration). At this stage, three types of cells--ectoderm, mesoderm, and endoderm--are evident within the embryonic disc, the same as these seen in the extra-embryonic tissue. These three cell layers will form all the main body structures.
The moving epiblast cells reappear as mesoderm between the ectoderm and the endoderm, or hypoblast. As the cells move through to the lower level, they leave a depression in the upper surface. These migrating epiblast cells move in greater concentrations at the caudal end of the primitive streak, making it wider.
At Day 15, epiblast cell movement slows down, the slight indentation along the longitudinal axis of the primitive streak becomes deeper as cells continue to move out from underneath and are not replaced by migrating cells above. This deep groove is now termed the primitive groove. The cells associated with the primitive groove are termed node cells to differentiate them from the cells of the remainder of the embryo.
At Day 15 these node cells can be identified as precursors of future body organs. The ectoderm node cells form the neural plate running the length of the top of the primitive groove, the cranial end of which goes to form the head. The spreading mesoderm in the immediate vicinity of the neural plate goes to form the somites, or body trunk, and the mesoderm immediately below the primitive groove goes to form the spine and central nervous system. Finally, the wide caudal end forms the tail end of the embryo.
The next stage, neurulation, involves the development of the central nervous system (CNS), gut, and heart. Day 16 sees three major changes. First, the ectoderm near the neural plate thickens and two neural folds develop on either side. The neural plate becomes depressed and these folds fold over, join, then fuse to enclose a hollow tube, the spine and CNS-to-be.
Second, the mesoderm on either side of the neural plate organizes into 14 somites (body muscle blocks). Third, increased cell growth is apparent above the surface at the cranial end, with an increase in the length of the neural plate. This cell growth folds over to form the head process, heart, and pharynx.
By Day 18, lateral folds are beginning to develop on either side of the head process, which lifts it away from the underlying tissue (see Day 18-19 illustrations).
Lateral folds also move down from the cranial end to the caudal end, lifting the whole body away from the underlying tissue, leaving just one central attachment point, the first evidence of the umbilical cord. The embryo continues to lift away from the underlying tissue, and the head and tail processes fold back down to give the embryo its characteristic "C" shape configuration.
The gut tube also now begins to develop from the pharynx fold by closure of the endoderm folds, in a similar way to the neural tube formation. The hindgut of the fetus now extends out into the blastocoel to form the allantois (see Days 20 and 40 illustrations) and blood is evident in the lumen of the tubular heart.
The embryo now lies away from the underlying placental tissue and is connected directly to the mother only by the umbilical cord. The embryo now has identifiable CNS, head, and brain areas. Its pharynx and gut tube are present, as are the somites. Therefore, by Day 23, all the basic bodily structures are evident, though only in a rudimentary form.
At Day 23, the basic plan for the whole body is laid out. What is required from this time on is growth and the fine differentiation of the basic organ structures. By Day 40, all of the main body features are evident--e.g., limbs, tail, nostrils, pigmented eyes, ears, elbow and stifle regions, eyelids--and the embryo is now termed a fetus. Days 39-45 herald sexual differentiation and evidence of external genitalia.
At Day 60, eyelids close and finer eye development occurs, teats are present on females, and the oral palate is fused. Day 160 sees the first evidence of hair around the eyes and muzzle, and by Day 180 hair has begun to develop at the tip of the tail and the beginnings of a mane is evident. By Day 270 hair covers the whole body surface.
Internally, the weight of the fetal gonads reaches a maximum at Day 180-200. This increase in gonad size is unusual and might correspond to the period of masculinarization or feminization of the fetus.
After Day 320 the testes in the male fetus might descend through the inguinal canal (passage in the abdominal wall); however, this does not occur in all colt fetuses as some don't drop until after birth.
The size of the embryonic vesicle in early pregnancy is given at left, and the main milestones in equine fetal development are summarized on page 36.
At full term, the fetus is very well developed, as is typical of a plains-dwelling animal. At birth foals are capable of all basic bodily functions, including walking within 30-60 minutes, thus allowing them to escape predators soon after birth.
The development of a foal is a complicated, marvelous series of changes. There are many times when things can go wrong and the fetus can be lost or deformed. However, having a more intricate knowledge of the development of your foal can help you understand how to best care for the mare, and the developing fetus.
About the Author
Mina Davies Morel, PhD, is head of the equine group at the Institute of Biological, Environmental and Rural Sciences at Aberystwyth University in the United Kingdom. She has particular interest in equine reproductive physiology and its application to stud management, and she is the author of a number of scientific papers and text books on the subject. She is a leisure rider and owner of Welsh Cob Section Ds.
POLL: University Equine Hospitals