Estrus, its detection and artificial insemination

The Estrus cycle can be divided into 4 stages:

 

Proestrus Phase:

     About 16 to 18 days after a heat period under the influence of GnRH the pituitary produce FSH this is released into the blood stream. FSH causes development of follicle of ovary. The follicle in turn over a 3 to 5 days period produces estrogen as it grows in size. Estrogen level peaks at 19 to 20 days. Estrogen causes following changes in cow.

 

Physical:        

•  Increase of blood lining of the uterus
• Thickening of wall of vagina
• Increase growth of cell and cilia in oviduct
• Relaxation of cervix
• Mucus production by cells of cervix
• Mucus is usually thick and milky in color
• Decline of previous CL on ovary

Behavioral:   

• Late in this period, the female animal usually exhibits interest in the male

 

Hormonal:

• High estrogen level cause production of LH about day 20.

 

Estrus Phase:

     FSH and LH affect the ovary to stimulate follicle rupture and release of the egg contained within. Physically this period is characterized by sexual desire and acceptance of male. Estrogen continues to dominate the reproductive tract and produce the following changes.

 

Physical:

•  Engorgement of the uterus wall
•  Contraction of oviduct
•  Relaxation of cervix
•  Mucus secretion is clear, transparent and stringy
• Vulva is swollen and relaxed
• Ovulation occurs after 12 hours after the end of this period

Behavioral:

• Effects of estrogen of CNS produce the characteristic behavioral pattern of reception of male

 

Met estrus phase:

         This stage last for 3 to 4 days, the same length of time it takes the ova to reach the uterus. The egg is picked up by the oviduct, and the follicle collapses. LH causes the production of a new cell type, the luteal cell in the ruptured cell, in the ruptured follicle. This gives rise 1^st to the Corpus hemorrhagicum where the blood invades the follicular space and then the Corpus luteum which produces progesterone which changes the state of reproductive tract.

Progesterone prevents the FSH secretion and therefore prevents the further development of follicles.

Physical Change:

• Blood discharge occurs about 2 days after estrous in 50 to 80% of all cows. This blood does not indicate ovulation or success or failure of the breeding; but, it indicates some hemorrhage of the uterus lining
•  Mucus secretions decrease
• The uterus becomes soft, pliable and relaxed
• Uterus wall grows thicker in preparation for pregnancy
•  Cervix begins to close

 Diestrus Phase:

     This is the longest period in the estrus cycle, generally lasting from day 4 to 18

The Corpus luteum matures and produces progesterone with the influence of reproductive tract.

Physical Change:

• Thickening of uterine lining
• Cervix closes
• Vaginal mucus is scant and sticky
•  Uterus muscles are relaxed

        If the egg is not fertilized in the oviduct, the CL grows for 10 to 12 days. If no embryo is present, high progesterone level causes production of PGF 2a. Prostaglandin influences the ovary and causes degeneration of CL and therefore, a decrease in progesterone. This leads to FSH production and re entry into the proestrus phase with the development of a new follicle. This normal cyclic activity repeats itself

        If the egg is fertilized, the CL body continues to produce progesterone. The recognition of an embryo by the dam blocks the release of PGF2a. Progesterone relaxes the uterine muscle and encourages growth of uterine lining to nourish the fertilized egg. Progesterone production by the CL is important for maintainace of pregnancy during the 1^st 2/3 of gestation. After that progesterone is produced by the cells of the placenta and adrenal glands.

  

STAGES LEADING TO FERTILIZATION:

1.  Ovulation and insemination
2. Transport of ova and sperm
3. Capacitation of sperm
4. Activation of egg
5. Formation of zygote

1: Ovulation and Insemination:

       Towards the end of the estrus period, the cow becomes receptive to the male. The follicle ruptures and the ova or egg travels into the oviduct or fallopian tube. In natural services sperm is deposited in the anterior vagina next to the cervix. In artificial insemination, the sperm is deposited within the cervix or just inside the uterus.

 

2: Transport of Ova and Sperm:

    The hormonal changes of the estrus cycle are very important in changing of the reproductive environment to facilitate ova and sperm transport. The oviduct experience contractions. Hair like cilia on the inside the oviduct creates waves in the oviduct fluid and assist in ova transport.

The sperm moves rapidly through the uterus to the upper 1/3 of the oviduct where fertilization occurs

The egg is fertile for approximately 10 to 12 hours. If fertilization occurs after this time, an abnormal embryo results. This may lead to and early embryonic death or early abortion

 3: Capacitation of Sperm:

For fertilization to occur, a process known as capacitation must 1^st occurs. Cpacitation refers to the functional change in the head of the sperm which then allows it to penetrate the cells of the ova

4: Activation of Egg:

Once the sperm has penetrated the egg, a reaction occurs preventing penetration of further sperm. Male and female pro nuclei form, a mixing of the chromosomes occurs and the union forms the zygote

5: Fertilization:

Once the fertilized ova has formed the cells within divide. The ova goes through 2/4/8/16/32 cell stages. Fertilized ova usually enter the uterus from the oviduct, about 4 days after estrus.

 Important times to remember:

• Fertile life of egg is 10 to 12 hour after ovulation
• Time required for capacitation of sperm is 4 to 6 hours
• Life time of sperm is 24 hours
• Transit of fertilized ova from oviduct to uterus is day 4
• If insemination occurs too early, the sperm may have died prior to ovulation
• If insemination occurs too late the ova has passed its fertile life period before the sperm gains the ability to fertile the egg
• In general cows noticed in heat in the morning should be bred later the same day. Cows noticed in heat in the after noon should be bred next morning

 

 Physiology of female reproductive Organs:

 

1: Reproduction, Sexual maturity and breed maturity:

    Puberty is the period of a cow’s life when it is able to release ova and to manifest complete sexual behavior sequence. Puberty is basically the result of a gradual adjustment between increasing sexual hormonal activities from the pituitary and the ovaries.

    At puberty in heifers there is an increase in ovarian size due the presence of follicle. This is associated with the ability of follicles to ovulate as a consequence of LH peak, a hormone secreted by pituitary for ovulation. The time of onset of this activity appears to be a function more of the weight than age of the animal. Restricted nutrition delays puberty in cattle.

    Dairy cattle reach puberty when its body weight is 30 to 40% that of their adult weight whereas in beef cattle this percentage is higher. Generally speaking in normal conditions, puberty occurs between 300 to 360 days of age in dairy cattle and between 320-460 days of age for beef breeds.

2: Sexual or Heat cycle:

The estrus cycle is divided into four arbitrary stages:

• Proestrus
• Estrus
• Metestrus
• Diestrus

Each stage is controlled by reproductive hormones produced by the nervous system and by ovaries and uterus.

3: Cycles of Ovary

    Oogenesis is defined as formation of primordial follicle. The ovary of a newborn calf contains several hundred and thousands oocytes. Only a small percentage is ovulated during the life. The primordial follicle forms preovulatory Graffialn follicle. The mature follicle extends through thickness and protrudes above the ovarian surface like a blister.

The active follicle produces the hormones called estrogen mainly responsible for estrus signs

    Ovulation is the rupture of the Graffian follicle and release of mature oocyst. The oocyst is then called ovum. Present evidence shows that high level of LH stimulates the plasminogin and collaginase in the follicular wall that will participate in the digestion of intercellular connective tissue. This will result in release of the ovum.

After ovulation the follicular cavity fills with blood forming a corpus hemorrhagicum. The granuloseand thical cells enlarge to fill the follicular cavity. The corpus luteum is highly vascularized. This process is called lutinization. CL produces progesterone that inhibits estrus behavior.

4: Cycles of the Uterus:

During the follicular phase of estrus cycle, the vascularity of the uterus is increased due to estrogenic stimulation. The uterus becomes engorged with blood, especially in the carouncular area. It is firm and turgid

At this stage the mucus cells of the cervix are stimulated and thin watery mucus is discharged from the cervix and expelled from the vagina. The glands in the vagina are also stimulated to secrete mucus. The increase in blood circulation in vaginal wall will induce slight edema.

 5: The Oestrual Cycle:

In cows the estrus cycle lasts 20 to 21 days. An open cow will continue to start a new reproductive cycle until it is bred and become pregnant. Cycle can range in length from 18 to 24 days. The cycle can be divided into 2 phases

• Follicular
• Luteal

The 1^st day a cow is in heat is called day zero of the cycle. The follicular phase is ended by the ovulation of a mature oocyst. After ovulation follicular cell are transformed into the CL. This is the beginning of the luteal phase. The end of the luteal phase begins with luteolysis, the destruction of CL to allow rapid growth of the new ovulatory follicle

 6: Hormones of Reproduction:

    Hormones are chemical agent synthesized and secreted by specialized glands and carried by the blood to other parts of the body where they act on specific tissues or organs

In cow reproduction we are primarily concerned with three different types of hormones.

• Releasing hormones
• Gonadotropic hormones
• Sex steroid hormones

     Releasing hormones originate from the hypothalamus and control the synthesis and release of hormone from the anterior portion of the pituitary gland. The hypothalamus is part of the brain.

    The gonadotropic hormones produced in the pituitary are directly involved in maturation and release of ova. They also stimulate the secretion of sex steroids hormones from the ovary. The pituitary gland is located in a pocket beneath the brain to which it is connected by the pedicle of nervous and blood vessels.

    The sex steroid hormones are involved in the behavioral aspects of reproduction, in development and maintainace of secondary sex characteristics in maintainace of reproductive organs and in regulation of reproductive cycle and pregnancy.

HORMONE	ORIGIN	FUNCTION
GnRH	Hypothalamus	Stimulation and release of FSH and LH
FSH	Pituitary gland	Follicular growth and production
LH	Pituitary gland	Final maturation of follicle, ovulation, CL formation
Estrogen	Ovary (Follicle)	Growth of uterus , estrus behavior, cervical mucus secretion, release of LH for ovulation
Progesterone	Ovary (CL)	Maintains pregnancy, keeps cow from cycling
Oxytocin	Ovary (CL), Pituitary	Milk excretion and prostaglandin synthesis
PGF2A	Uterus	Lysis of CL

 

7: Control of Sexual Cycle:

     GnRH from hypothalamus cuase release of FSH from the pituitary gland. The FSH reaches the ovaries by blood circulation and stimulates the development of follicles.

    The follicles in the ovaries produce esterdiol which brings the cow in estrus. One follicle produces more esterdiol than the others on the ovaries and grows more rapidly.

T    he level of esterdiol in the blood rises causing a greater discharge of GnRH by the hypothalamus. The pituitary then release less FSH. The level of esterdiol causes the pituitary gland to secrete a massive discharge of LH in response to frequent stimulation by GnRH. This provokes around 36 hours after the LH spike, the final maturation of the follicle which ruptures to expel the ova. Under the stimulates of LH the follicle cell transform into luteal cells to form CL. The CL progressively produces progesterone. CL reaches at its maximum size at day 11-13.

If pregnancy does not occur at day 16-17 CL begins to regress due to production of oxytocin produced by the CL cells and the luteolytic PGF2a secreted by the uterus.

If pregnancy occurs the CL remains and continues to produce progesterone until the birth of the calf.

 

PRIMARY EXAMINATIONS

1: Pre-Insemination checks:

• Restrain the cow or heifer but do not tie down or over excite
• Identify the cow to be inseminated.
• Approach the cow slowly, talk to her, do not rush, do nothing to excite her

Check for previous services and in case of repeats, note observations such as late repeats over 23 days, early and quick repeats less than 17 days and watch for possible pregnancy

 

 2: Heat Signs:

	Coming into heat	Standing heat	Going out of heat
Appetite	Noticeable decrease	Sharp decrease	Slowly returning
Nervous behavior	Very noticeable	Very noticeable	Noticeable
Fence walking	Very noticeable	Very noticeable	Noticeable
Bawling	Frequent	Less frequent	Frequent
Persistent trailing of other animal	Yes	Yes	Very little
Temperament towards other animal	Very antagonistic	Less antagonistic	Back to normal
Head butting	Quite frequent	less frequent	Frequent
Licking other animal	Yes	Yes	Yes
Mounting behavior	Will jump others but will not stand to be ridden	Will jump on others and will stand to be ridden	May jump others but not stand to be ridden
Response to observe	Cautions	Interested	Shy, avoid
Mucus present	Occasionally	May occur frequent	More frequent
Mucus consistency	Watery	Cohesive strand	Cohesive strands
Vulva	Red and puffy	Red and puffy	Less swollen
Bloody discharge	Not usual	Not usual	May present

 

3: General conditions of the Animal:

• Cow should be in good physical condition. Cows gaining body weight conceive better than cows in a weight loss condition
• Observe for abnormal discharge from vulva. They are signs of infections and need to be treated before breeding
• Observe for bloody discharge. It may be to late to bred       

 

4: Insemination Procedure:

All insemination equipments and supplies must be clean and sanitary. Use disposable equipments only once. Be extremely careful when handling the insemination gun, sheath and straw of semen to keep them clean and free from contamination

Summary in points form describing the step by step procedure eh inseminating the cow:

1. Have a thawing thermos prepared with the proper temperature of water, 37C
2. Place insemination kit close to liquid nitrogen semen tanks.
3. In case of repeats, note if the animal in question is a late repeat or is she early or quick repeat. Watch for possible signs of pregnancy when examining the cow prior to insemination.
4. Remove the lid and place it in a clean area
5. Prior to lifting any conister from your liquid nitrogen tank, assure yourself of the semen location
6. Lift conister high enough to reach the cane containing the dose of semen. Be assure not to lift the consiter above the frost line in the neck of your tank
7. Identify and remove dose as quickly as possible from the tank. The recommended time is between 15 to 1 minutes
8. In order to reduce the chance of straw exploding after they were removed from the nitrogen tank, immediately and without hesitation shake the straw in a short swing movement similar to that of nurse shaking thermometer. This will remove small drops of nitrogen which may be trapped at one end of the straw. The plug is needed to expel the semen form the straw
9. After this now load the gun properly
10. Approach the cow slowly and gently and talk to her
11. Lubricate your gloved hand with mineral oil; rub gently the anal area of the cow. This will relax the animal and make the entry of your hand into the rectum easier
12. With your four finger and thumb, a wedge is formed which in turn slowly and firmly pushed into the rectum of the cow
13. Gently massage the rectum and examine the reproductive organ. Note the abnormalities
14. Pull your hand back towards vulva. Now you are in position to clean the vulva with the clean, dry paper towel. Do this 2 to 3 times if necessary. Use a new towel each time. Remove as much manure as you can.
15. Now gently aside the vulvular lips for clean entry. The inseminator should not touch the outer lips of vulva while he is carefully inserting the insemination device into the vagina
16. Be sure to insert the insemination device into the vagina at a 45 angle to avoid entry into urethral opening
17. After the insemination device has been manipulated into the body of uterus the semen may now expelled from the insemination device. Push the plunger very slowly. It is advisable to count backwards from 10 to zero.
18. In order to prevent injury to the reproductive tract remove the insemination instrument very slowly
19. Remove arm form rectum slowly
20. Dispose the dirty glove and sheath
21. Before going to next case make sure your equipment is clean and free from manure
22. It is a good practice to remove all of your equipments from your kit and clean it thoroughly once a week

Thawing Frozen Semen:

Since the discovery of frozen semen technique by Dr. Chris Polge in 1949, thawing procedure for frozen semen has changed dramatically since that time.

The volume of semen dose for each insemination has also been changed from origin 1 ml ampoule to the present 0.5 and 0.25 ml straws. The volume and surface relationship of each packaging system influences the thawing rate of spermatozoa.

Following are the most commonly used semen packages system throughout the world.

1. 1 ml ampoule
2. 0.5 ml
3. 0.25 ml ampoule

Insemination Technique:

• One should pay special attention that 1/3 of the insemination device which is entering the animal be clean.
• Injuring the animal with the insemination pipette can cause failure of conception
• An injury to mucus membrane can cause bleeding. This blood is harmful to semen, and fertilization may not occur, because the sperm cells agglutinate. Even if the fertilization has taken place the fertilized embryo may be unable to implant in the lining of uterus causing an early embryonic death.
• In order to prevent the injury to the rectum of the animal the inseminator should have great care to always trim the nail.

 

Most common error during insemination

Fundamentals when using semen stored in the liquid nitrogen. The development and use of frozen semen stored in the liquid nitrogen and especially the reduction of semen packages to 0.5 to 0.25 ml respectively requires special training and careful handling of these packages

The operator must e precise and gentle as well as technically competent to introduce the semen into the animal. The insemination equipments for the straw should be thin and rigid. The insemination should not be performed roughly.

Errors when animal are not restrained:

A high degree of injury to the rectum of the reproductive tract can occur

And exact, careful and smooth introduction of the insemination equipments is difficult

Inseminator is at great risk of injury

Deficient Hygiene:

 

1: Rough and Uncontrolled Insemination technique:

Injuring the animal with the insemination pipette can cause failure of conception

An injury to mucus membrane can cause bleeding. This blood is harmful to semen, and fertilization may not occur, because the sperm cells agglutinate. Even if the fertilization has taken place the fertilized embryo may be unable to implant in the lining of uterus causing an early embryonic death.

2: Intra cervical insemination:

This method should be used only when a cow is not showing clear and distant signs of estrus.  It is also advisable to use intracervical semen deposition if a chance of pregnancy exists. If the tip of the insemination rod is passed through the cervix into the uterus the animal may abort.

3: Deep intrauterine Deposit:

This type of insemination is to be avoided. This can cause injury to delicate uterine membrane with instruments. It is seen that deposition in the uterine body is considerably superior that other depositions