Ø    Pregnancy or gestation begins when the ovum is fertilised and it ends at birth.

Ø    It includes the unique event of fertilisation by which the genetic material of the parents is brought together in the zygote, the single cell destined to contribute to a cell life which continues until death. The length of gestation extends from the time of fertilisation until birth of the offspring and averages 335-340 days in the mare.

Ø    It is a period of foetal development and growth to a state at which the new individual is sufficiently mature to survive in the extrauterine environment.

 

The zygote, by virtue of its small size can survive as a free-living entity in the oviduct. Similarly, the early stage embryo can also survive in the oviduct and uterus. As the embryo increases in size, simple diffusion of nutrients from the endometrium is not adequate to meet the nutritional requirements for growth and development. Consequently, later stages of the embryo and all stages of foetal growth derive their nutrients from the mare via the placenta. The placenta may be conveniently defined as the organ of exchange between the dam and the embryo. It provides nutrients and oxygen for the foetus and removes wastes. The placenta serves other important roles, in addition to providing nutrients. For instance, the placenta is an important source of hormones in the mare (It acts as an endocrine organ). These hormones serve to maintain pregnancy in the mare. Indeed one of the most remarkable examples of the ability of the foetus to regulate the mother's system is its ability to program development of the udder so that milk production is synchronised with parturition. The placenta also serves a number of protective roles, cushioning the foetus in a number of fluid compartments, and acting as a barrier to prevent bacteria or viruses from reaching the foetus. As we shall see, this barrier is not always effective and some disease causing micro-organisms and some toxins can cross the placenta to damage the foetus.

Ø   Failure or dysfunction of development of the foetus presents problems of abortion, neonatal illness and errors in foetal development.  

Diagnosing these problems presents a problem to the horse owner and veterinarian since the foetus is remote, hidden from direct view and usual methods of clinical investigation.

            
In the next few lectures, we will consider the gestational points of particular interest.  These include foetal development and endocrinology, an account of management and husbandry of the pregnant mare, pregnancy diagnosis, the foetal environment and abortion.

 

Fertilisation and Early Development

Fertilisation occurs in the proximal end of the fallopian tube, in the region termed the ampulla. 

 

Fertilized human egg (Zygote).
 Male and female genetic material (DNA) is contained in the 2 pronuclei seen in the center. The second polar body can be seen at one o’clock.

The oocyte and spermatazoan unite to form a single-cell entity called the zygote. Once the male and female membranes fuse, the genetic material from each parent is able to join to form one new individual. Only the zygote descends into the uterus. The zygote, undergoes its first cleavage to produce a two celled embryo. The interval from the time of penetration of sperm to the first cleavage is estimated to be 20 to 24 hours in the mare. So the zygote is a very short lived, albeit important structure.

Unfertilised ova remain in the mare’s fallopian tube degenerating over seven to eight months. Developing embryos influence their own transport to the uterus, because they can overtake old, unfertilised, eggs en route. Development of the zygote beyond at least the two- to four-cell stage is necessary for normal transport through the fallopian tube to the uterus.

A 2-cell embryo. Note that there are still some cumulus cells attached at 7-9 o’clock. The zona is still in place and you can see some unsuccessful spermatozoa stuck in the zona.

 

Cleavage

Cleavage is the process by which a zygote or fertilized ovum subdivides into smaller cells called blastomeres. Cleavage of the newly fertilised embryo simply involves mitotic divisions of one cell into two cells, two cells into four cells, four cells into eight cells, and so on. In each mitotic division, the genetic information is duplicated such that each daughter cell arising from the original cell contains exactly the same chromosomes.

            Each resulting daughter cell thus contains the normal diploid number of chromosomes, half of which have been derived from the egg and half from the sperm.

The embryo moves from the oviduct into the uterus in the mare at about the 16-32 cell stage, usually 5-6 days after ovulation. At this point, the embryo is free-floating within the uterus, and depends upon uterine secretions for nourishment.

            

By the 16- to 32-celled stage, the cells of the embryo are crowded together into a compact group still within the zona pellucida.  The embryo is now known as a morula, (this term refers to the mulberry shape of the dividing cells). Because the divisions are not accompanied by cell growth, the morula remains the same size as the zygote from which it was formed.

 

The fertilized ovum arrives in the uterus about 6 days after ovulation.

 

 

 

 

EMBRYOLOGY

            Embryology is the study of the development of the individual from the single celled zygote.  Three types of activity - growth, cell division and differentiation. All are necessary for embryonic development. 

            Organogenesis is the process whereby the various organs and organ systems develop from the totipotent zygote. Organogenesis involves, first, the formation of germ layers.

A.        Cells differentiate from discrete germ layers

a.         Endoderm

            i.          Gut

            ii.          Lungs

            iii.         Liver

 

b.         Mesoderm

            i.          Muscle

            ii.          Skeleton

            iii.         Cardiovascular System

            iv.         Reproductive System

c.         Ectoderm

            i.          Nervous System

            ii.          Skin

            iii.         Hair

            iv.         Some Reproductive System                   (External Genitalia)

Let’s take a look at the developmental processes in a little more detail.

 

 

 

 

 

 

 

 

 

 

Fluid begins to collect between the cells, and an inner cavity or blastocyst appears. The morula starts to form a new animal from a clump cells called the inner call mass (A).

Then the morula forms itself into a layer of cells called the trophoblast (B) surrounding a fluid filled space - the blastocoele (C) . Once the cavity begins to expand, the embryo is known as a blastocyst. The zona pellucida covering the surface of the blastocyst now breaks down and disappears.

 

 

LOSS OF TOTIPOTENCY

Prior to the blastocyst stage, individual cells of the embryo are totipotent; that is, each cell contains the full range of developmental capabilities necessary to form a complete individual. That means that each cell can form any differentiated tissue. If all other cells in the embryo were destroyed or separated, the remaining individual cell would be capable of developing into a complete individual. With formation of the blastocyst and allocation of cells to either the trophoblast or the inner cell mass, the cells in these two entities become changed biochemically and developmentally. Under normal circumstances, the cells can no longer give rise to a complete individual. The recent cloning of an adult demonstrates that, under the right conditions, a mammary cell nucleus can give rise to all tissues of the body when injected into a fertilized egg. It is likely that other mature cells can also de-differentiate and give rise to an adult.

After formation of the blastocyst, gastrulation occurs. This allows the formation of the three primary germ layers. The ectoderm, mesoderm, and endoderm form and give rise to all of the adult tissues and organs in the body.

 

 

 

  

Blastocyst Stage: A=Inner Cell Mass; B=Trophoblast and C=Blastocoele.

In cattle and horses, the blastocyst stage occurs between day seven to eight days after ovulation and is the time when embryos are flushed from the donor and implanted into recipient during embryo transfer.

With the formation of the blastocoele, the embryo is termed a blastocyst or blastula.  

             The outer single layer of cells within the blastocyst are the trophoblast cells and will contribute to the outer layers of the placenta. Within the trophoblast layer is a small group of cells referred to as the inner cell mass. Proteases are released from the blastocyst and they breakdown the zona pellucida so the blastocyst is said to "hatch". Once freed of the zona pellucida, it grows and elongates quickly

·      A single layer of large flattened cells, the trophoblast layer, surrounds a knob of smaller cells which lie to one side of the central cavity. This knob of cells called the inner cell mass, will give rise to the adult organism while the cells of the trophoblast form the placenta embryonic membranes. The trophoblast contributes only to the placenta and is lost at birth.

 

Gastrulation

The next step is the formation of a double-walled sac or cup, the gastrula. A third layer of cells next develops between the other two walls. These three layers differentiate into organs

·      The embryo developing from the inner cell mass becomes roofed-over by amniotic folds (D) that later fuse to form a complete layer - the amnion.

·      Endoderm cells (F; which eventually form the gut and its associated organs like the liver) spread from the inner cell mass over the inner surface of the trophoblast and, at this stage, the blastocyst is said to be bilaminar or two- layered.

·      The blastocyst becomes trilaminar or three-layered when mesoderm cells (E; which eventually will form muscles, bones, and fat) migrate from the inner cell mass spreads between the outer trophoblast layer and the inner endoderm layer.

·      The mesoderm layer then splits internally, and a cavity expands within the mesoderm to become the extra-embryonic coelom(G).

The process of gastrulation converts the simple structure of the blastula into several layers that are structurally and functionally specialised.

 

Foetal Membranes:

Although the very young embryo can exist for a short time by absorbing nutrients from the uterine fluids, its size soon becomes a limiting factor. The increase in size prevents diffusion of nutrients and waste into and from the embryo. A more effective source of nutrition and waste removal must be established. To accomplish this, membranes that support, protect, and nourish the embryo are all formed around the embryo. These membranes are collectively known as the extraembryonic membranes.  

 

 

A=Developing Amnion; B=Chorion; C=Yolk Sac; D=Allantoic Cavity; E=Trophoblast

By 21 days the equine blastocyst is pear-shaped, measures 6 X 7 cm and contains the embryo at the broad rounded end. The outer surface of the blastocyst is the trophoblast consisting of columnar epithelial cells which probably 'absorb' uterine secretions and so provide the foetus with nourishment.

 
All vertebrate foetuses possess 4 distinct foetal membranes. These are the yolk sac, the chorion, the allantois and amnion. These membranes are not part of the foetus proper and are discarded at birth. The foetal membranes function:

1.    to protect the developing embryo and foetus,

2.    to provide the developing embryo and foetus with a means of obtaining oxygen and nutrients and

3.    to remove metabolic waste products.

 

1.The yolk sac. Although the equine ovum does not contain any significant amount of yolk, a yolk sac is formed early during embryonic development. The yolk sac in the foal is small and functions only in the first 20 days of embryonic growth. 

The yolk sac is formed by the embryo very early in development, is part of the primitive gut, but is not included with the body when the edges of the embryo fold in to form the gut. Instead, as the embryo folds, the yolk stalk extends below it to the yolk sac. During this time the yolk sac supplies nutrients to the embryo. Blood vessels develop in the walls of the yolk sac and carry materials absorbed from the uterine fluids of the mother to the embryo. The yolk sac only remains functional for a short time and its function is soon taken over by the allantois. It becomes vestigial and is not found in more mature fetal-placental units.

 

2.The amnion. From each side of the embryo, a fold made up of one layer of mesoderm and one layer of ectoderm grows up and over the embryo. The layers fuse at the top and enclose the embryo in a double layered sac known as the amnion. The amnion becomes filled with a clear fluid that holds the embryo suspended. It forms a protective cushion against external shock and prevents the surface of the embryo from adhering to the surrounding membranes.

The amnion is the innermost membrane and forms the second water bag or amnionic cavity.

 

3.The chorion. The outer cell layer of the blastocyst is initially referred to as the trophoblast. When the three germ layers are formed, this becomes known as the chorion. The primary role of the chorion is initially to absorb nutrients. Later it fuses with the allantois to form the allanto-chorion. The chorion is the layer of the placenta that makes contact with the uterine wall and is critical in the exchange of nutrients and waste products between the embryo and maternal vascular system. The allantois is a membrane that lines the inside of the chorion. It forms the first water bag or allantoic cavity. The allantoic cavity is continous with the foetal bladder by way of a connection called the urachus[1]. This is a duct that passes through the umbilical cord.

 

 

 

 

As can be seen from this figure, the early equine embryo is a very small structure. Even by day 10, most equine embryos have a diameter of only 3-5 mm. Despite this small size, it is now possible to detect pregnancies with very good accuracy, even as early as day 11. This is possible because of the developments in ultrasound technology. Some of the events and changes that can be detected by ultrasound examination are shown in the figure overleaf.