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Tuesday, 14 July 2015

There are essentially 4 stages of pregnancy in cattle; Fertilization, Cleavage, Implantation and Gestation. These processes are the most important factor in relation to failure of a cow or heifer becoming pregnant, therefore it is important to understand these processes in order to minimise embryonic loss and improve the total yearly calving percentage.

Stage 1 – FERTILIZATION

The entire process of sexual reproduction is centred on the act of fertilization; essentially this act is the fusion of two cells (the male sperm cell and the female egg), to form one single cell, known as the zygote.


Cow sex cells

Cow body cell
Each sperm cell or egg cell contains half the normal number of chromosomes (chromosomes are the structures of DNA, genetic material, that are held within the nucleus of cells). All body cells contain a full set of chromosomes and cattle have 30 pairs (so 60 individual chromosomes altogether) in a normal adult cell; sex cells contain half this number, so cattle sex cells have 30 individual chromosomes. The main reason for this is so when the egg and sperm fuse, the resulting zygote contains a full, and not double, set of chromosomes. Cells that contain a full set are known as diploid and those that contain half are called haploid.


For example, using the diagram above, the orange and green cells (sex cells) are haploid cells – they contain half the genetic number of chromosomes. If they fuse together to create a zygote, the zygote will contain the correct number of chromosomes (30 pairs) and can go on to form an embryo.

In cows, fertilization takes place at the ampullaristhmic junction of the oviduct (this is the stretch of fallopian tube between the ampulla and the isthmus).


The female egg, which was released from the ovarian follicle surrounded by cumulus cells at the time of ovulation, arrives at the junction 1 -2 days after standing heat. The egg is denuded of the cumulus cells and ready for fusion with the sperm. Cumulus cells are follicular cells that protect the egg up until it is ready to be met with a sperm. The fertile life span of the ovum is very short (8 – 12 hours).
Sperm are deposited in the vagina during natural service (mating with a bull) and into the uterus during artificial insemination. Although the total number of sperm deposited into the female tract measures in the thousands of millions, the number travelling as far as the ampulla is probably not much more than 1000 in any mammal. Some sperm reach the site of fertilization very quickly (15 minutes) but in order to fertilize the egg, sperm must go through changes termed capacitation. In cattle, sperm capacitation takes about 4 hours. The fertile life span of sperm is in the range of 30 – 48 hours.

The sperm must penetrate two membranes on the egg in order to complete fertilization. The first outer membrane that the sperm must penetrate is called the zona pellucida. Passage of the sperm into the zona pellucida is facilitated by proteolytic enzymes (chemicals which break down the components of the membrane) which are released from the sperm head when the acrosome is lost.



The zona pellucida undergoes some sort of change after the passage of a sperm, which makes it less easy for subsequent sperm to enter. This is called the zona reaction and is one measure of protection against polyspermy.


The last stage of sperm penetration into the egg involves the attachment of the sperm head to the surface of the vitelline membrane. This is a vital period in the fertilization process since it is at this time that activation occurs. Stimulated by the close proximity of the sperm, the egg awakens from its dormancy and development begins. The other defence mechanism against polyspermy (more than one sperm fertilizing the egg) is shown by the vitelline membrane and is termed vitelline block.
The fertilizing sperm is actively engulfed by the vitelline; but subsequently, the vitelline surface becomes unresponsive to sperm contact and no further sperm are engulfed.

The reason for the changes to the membranes of the egg, which only allow one sperm to reach the egg, is that polyspermy leads to an abnormal number of chromosomes in the embryo, which is a fatal condition.

Once the male and female membranes fuse, the genetic material from each parent is able to join in a process called syngamy to form one new individual. The process of fertilization is now complete and the fertilized ovum undergoes its first cleavage to produce a two celled embryo. Each daughter cell now contains the normal diploid number of chromosomes, half of which have been derived from the egg and half from the sperm. The duration of fertilization from the time of penetration of the sperm to the first cleavage is estimated to be around 20 – 24 hours in cows.



Male sex cell (sperm) with 30 chromosomes

Zygote with 60 chromosomes

Female sex cell (ovum) with 30 chromosomes





STAGE 2 – CLEAVAGE

Cleavage of the newly fertilized embryo is simply mitotic division of one cell into two cells, two cells into four cells, four into eight, 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.


The embryo moves from the oviduct into the uterus at about the 8-cell stage, usually 3 – 3 ½ days after ovulation. At this point, the embryo is free-floating within the uterus, and depends upon uterine secretions for nourishment.
By the 16-32 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. Fluid begins to collect between the cells and an inner cavity or blastocyst appears. Once the cavity begins to expand, the embryo is known as a blastocyst.
Some embryonic loss occurs during the time of blastocyst formation and may be due to embryonic chromosome defects. In cattle, the blastocyst stage occurs between days 7-8 after ovulation. This is the time when embryos are flushed from a donor cow and implanted into recipient cows during embryo transfer.

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. The knob of cells, or inner cell mass, will give rise to the adult organism while the cells of the trophoblast will from the placenta embryonic membranes. In the cow, the zona pellucida sheds at about day 8 and blastocyst elongation begins a few days later.

Maternal recognition of pregnancy must take place about day 16 to 17 or the uterus will produce prostaglandin F2A (PG) which will cause the corpus luteum to regress, leading to a decline in progesterone concentration. A lack of progesterone is fatal for pregnancy – the embryo will not be able to implant (which is the next stage).

Therefore in the case of a pregnancy, the embryo must somehow signal to the mother that she is pregnant, so that the regression of the CL can be inhibited. It has been demonstrated that bovine and ovine (cattle and sheep) embryos produce and release a specific pregnancy protein – this protein is known as Interferon-T (INF-T). The mechanism of luteolysis (regression of CL) inhibition is now well established…
·         Oxytocin receptors on the uterine luminal epithelium are inhibited.
·        


Usually PG is stimulated to be produced by a PG synthesis hormone; in the case of a pregnancy, an 
inhibitor is produced which blocks this synthesising hormone.


When the female cow is not pregnant

When the female cow is pregnant

Significant embryo losses can occur around the time of maternal recognition of pregnancy due to either failure of the embryo to produce signals or failure of the mother to recognize the signal from the embryo. If the embryo died, or if the cow fails to recognize pregnancy, the corpus luteum will regress normally and the cow will return to estrus at about 21 days after mating.

STAGE 3 – IMPLANTATION

While the embryo is undergoing cleavage and blastocyst formation, the uterus is also undergoing changes which prepare the way for implantation. The embryo is said to be implanted when it becomes fixed in position and physical contact with the mother is established. Implantation in ruminants (including cattle) is non-invasive and some people prefer to use the term attachment – as there is close attachment between embryonic membranes (i.e. membranes of the foetus) and the endometrium overlying caruncles (i.e. membranes of the maternal uterus). Shortly thereafter, the placenta is established.

Ruminants have a cotyledonary placenta – instead of having a single large area of contact between maternal and foetal vascular systems (points where blood/serum can be exchanged), these animals have numerous smaller placentae. The terminology used to describe ruminant placentation is:
·         Cotyledon: the foetal side of the placenta         

Caruncle: the maternal side of the placenta
·        Placentome: a cotyledon and caruncle together



Caruncles are oval or round thickenings in the uterine mucosa resulting from proliferation of sub epithelial connective tissue. As shown in the image below, caruncles are readily visible in the non-pregnant uterus. Further, they are the only site in the uterus to form attachments with the foetal membranes. Patches of chorioallantoic membrane become cotyledons by developing villi that extend into crypts in the caruncular epithelium.

The image below shows caruncles in an incised non-pregnant sheep uterus (left) and cross sections through placentomes from a mid-gestation sheep (right). Bovine placentomes look similar, but have a convex appearance rather than the concave shape seen in sheep…



Pregnant sheep, goats and cattle have been 75 and 125 placentomes. The image below shows an incised uterus from a pregnant sheep roughly 50 days of gestation. The numerous button-like structures are placentomes and the surfaces in view are actually cotyledons. The slightly milky-looking membrane covering and between placentomes is the chorioallantois. The foetus is clearly visible inside the amnion.



The image below shows a bovine conceptus dissected away from the uterus. The size of the chorioallantois relative to the amnion and foetus is evident. The cotyledons are readily observed; caruncles have been left behind with the uterus. Cattle foetus are located in one uterine horn, the large chorioallantois fills both uterine horns and placentomes are present throughout the uterus.



During parturition, there is substantial loosening of the cotyledonary villi from caruncles, and the placentomes expand laterally. After expulsion of the foetus and loss of foetal circulation to the cotyledons, capillaries within the villi collapse, leading to a decrease in their size. The uterus contracts and the caruncular stock shrinks, further enhancing the separation of cotyledons from caruncles. In the normal case, foetal membranes with cotyledons are delivered within 12 hours from birth. However, in many cases which are still considered normal, the membranes will not be passed up until the 10th day after calving, at which time almost all cows will expel the membranes.
Failure to do so can lead to detrimental infections and cycling problems.
The images below show what the expulsion of membranes should look like…





Has your cow still not passed her foetal membranes? Does your cow have a temperate post calving? These problems are more often than not associated with membrane retention - For more information on post pregnancy processes and care, follow this link to my specific blog post… *insert link*

Structure of the Placenta…

A prominent feature of the ruminant placenta is the presence of large numbers of binucleate cells (cells with two nuclei). These cells arise early as part of the foetal trophoblast from cells that fail to undergo cytokinesis (the separating of one dividing cell, into two separate cells) following nuclear division. They invade and fuse with caruncular epithelial cells to from small syncytia (multi-nucleated cells forming specialised tissues). Binucleate cells secrete placental lactogen which can stimulate mammary cell growth and milk secretion.

Ruminants basically have an epitheliochorial placenta, but because the uterine epithelium is modified by invasion and fusion of binucleate cells, its structure if generally referred to as synepitheliochorial.

“Epitheliochorial placenta:
The uterine epithelium of the uterus and the chorion are in contact in this placentation, and there is no erosion of the epithelium. It is characteristic of cows, sows and mares. Also called adeciduate placenta.”

General aspects of placental transport are similar to that seen in other species. Immunoglobulins (antibodies) are not transported across the placenta from mother to foetus, and therefore, apart from foetal infections should they occur, the young ruminant is born without circulating antibodies. This is why it is extremely important for calves to receive sufficient and continuous colostrum within the first few days of life.

The major hormones of ruminant placentae are progesterone and other progestins, oestrogens and placental lactogen.

Generally speaking, most cattle and goats require a corpus luteum throughout pregnancy in order to maintain sufficient levels of progesterone. This is in contrast to sheep, which at around day 70 can maintain pregnancy even without a corpus luteum.



In cows, the foetal placenta starts to produce placental lactogen around 4 months of gestation and remains low throughout parturition. This hormone stimulates mammary growth and prepares the mammary gland for the impending period of lactation – it does not stimulate milk production, it is has more of a preparatory action than an active role.
http://www.partners-in-reproduction.com/reproduction-cattle/pregnancy-conception-gestation.asp
In cattle, the embryo remains in the uterine cavity and whatever attachment it forms with the wall of the uterus before the formation of the placenta is of an extremely loose nature. Progesterone secreted by the corpus luteum of the ovary acts to decrease muscular activity of the uterus. In addition, progesterone increases by supply to the uterus and stimulates proliferation of the uterine epithelium and an increase in uterine milk secretions.
By 33 days post-mating, the foetal chorionic membrane has formed a fragile attachment with two to four of the cotyledons surrounding the foetus; within a few days, maternal caruncles and foetal cotyledons have become so intimately interdigitated that the embryo is being completely nourished through the cotyledons. Growth of the cotyledons is also stimulated by progesterone. The other significant loss of embryos occurs at the implantation stage. Cows losing embryos at this time will return to oestrus approximately 40 – 42 days post mating.

STAGE 4 – GESTATION
The length of gestation extends from the time of fertilization until birth of the offspring. On average, gestation lasts for 283 days in cattle (ranging from 279 – 285 days).
Changes occur in the female’s reproductive tract as pregnancy progresses. The foetal and maternal hormone systems interact throughout pregnancy such that pregnancy not only is maintained but that continued development and growth of the foetus is assured. Perhaps the most marked example 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 synchronized with parturition (giving birth).
The foetal placenta is the major unit producing hormones during pregnancy, and the foetus plus placenta contribute to hormonal changes in the mother’s circulation in late pregnancy. Hormone production by the foetus may also affect its own growth and regulate maternal function to assure adequate environmental conditions (I.e. oxygen, nutrients, water and minerals).
Swelling (oedema) and an increase in blood supply are the major reactions of the vulva to pregnancy. The oedema increasing with the progress of pregnancy. These vulval changes occur around the fifth month in heifers and the seventh month in cows. The lining of the vagina is pale and dry during most of gestation but becomes swollen and pliable toward the end of pregnancy.
During pregnancy, cervical secretions increase to produce very viscid mucus, serving to seal the cervical canal by the so called ‘mucous plug’ of pregnancy. Before parturition, this seal breaks down and is discharged as strings. During pregnancy, the external orifice of the cervix remains tightly closed. A few days before the onset of labour, relaxin is released by the CL of pregnancy; along with the increasing levels of oestrogen, acting to relax the cervix and pelvis.
As pregnancy progresses, the uterus undergoes gradual enlargement to permit expansion of the foetus, but its muscular walls remain quiet to prevent premature expulsion. Three phases can be identified in the adaption of the uterus to accommodate pregnancy – proliferation, growth and stretching.
Uterine proliferation occurs before blastocyst attachment and is promoted by high levels of progesterone. Characteristic changes of the inside lining of the uterus initiated by progesterone are increased blood supply, growth and coiling of the uterine glands and leucocyte infiltration.
Uterine growth starts after implantation. Uterine growth includes muscular hypertrophy and an extensive increase in connective tissue. Modification of connective tissue is important both during uterine adaption to the growing foetus and during involution after calving. The structural changes which take place in the pregnant uterus are reversible but are restored at different rates after parturition.
During the period of uterine stretching (last trimester) uterine growth diminishes while its contents are growing at an accelerating rate.
When the mother recognizes the presence of a viable foetus in the uterus, the CL persists as the ‘CL of pregnancy’ (CLoP) and subsequent oestrous cycles are suspended. During early pregnancy this suspension may not be complete as considerable follicular development occurs in the ovaries and some may even reach preovulatory size; however, these follicles eventually become atretic (i.e. the follicle degenerates before reaching maturity). The CLoP in the cow persists at a maximal size and continues to produce progesterone throughout pregnancy. However, from about day 150 – 250 of gestation, the foetal-placental unit is capable of sustaining progesterone production and pregnancy should the CL abnormally/prematurely regress.
Usually, animals gain weight during pregnancy due to the growth of the foetus as well as increases in maternal body weight. In heifers, nutrient retention due to growth may mask actual weight gain due to pregnancy. Therefore, heifers must receive adequate diets so that both the heifer and the foetus grow during pregnancy.

 Considerable alternation in the distribution of water occurs in pregnancy. Some of this is mechanical and is related to the increase in venous pressure due to the weight of the enlarging uterus. Oedema extending from the udder to the umbilicus is frequently observed during late gestation in cows.



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