Collection of Semen 

Recovery method

It is the oldest method. In this method bull is allowed to jump on cow and semen is collected from vagina by spoon, aspiration or sponge.

Drawbacks:

• Bacteria are mixed

•  Vaginal fluid is mixed

• Later it was improved by placing bag in vagina but still mixing of urine

Advantage:

• Maximum semen can be collected.

Massage Method

Gently insert lubricated hand and forearm into bull rectum. Remove feaces and gently massage the seminal vesicle  with fingers by backward and downward strokes toward urethra. An assistant with receptacle will collect seminal fluid and semen as drips from penis. Ampule is massaged similarly in a slow rhythmic way. Sometimes semen is retained in sigmoid flexture of penis or in the proximal portion of the prepuce. Therefore after massage the ampule, sigmoid curvature should be straightened up.

Precaution:
Carefully wash, rinse and dry the prepuce and prepucial hairs with warm normal saline. To avoid the inflammation, massage should be done gently.

Collection by Electroejaculation

Apparatus:
Electroejaculator, salt solution, semen receptacle, K jelly, tranquilizers.

Principle:
Ejaculation of semen is brought about by inserting the probe or electrode into bull rectum and stimulating the nerves to the reproductive system by gradually increasing the voltage with rhythmic fashion.

Procedure:
Rectum of bull is flushed with normal salt solution. The bipolar electrode is held in contact with floor of rectum. The nerves are stimulated by gradually increasing the voltage from 0 upto 10-15 volts. 2 volts increase at the interval of 5-10 seconds and then returning to zero after each increase. This brings about erection and ejaculation. Electroejeculation may be added by giving bull tranquilizers prior to collection as tranquilizer reduces straining. This method is normally used in those valuable bulls that due to one or other reason are not able to mount.

Collection by Artificial Vagina

It has been proved to be the best method and is now most commonly used. For cattle and buffalo artificial vagina consists of:

• A heavy hard outer cylinder open at both ends with a nozzle for air and water in and outlet.

• Inner sleeve of rubber or rubber liner.

• The semen receiving cone or rubber cone.

• Semen collection tube

Before using for semen collection all the parts are washed thoroughly and sterilized properly, and assembled as artificial vagina. The rubber liner is inserted into the hose; inverting both ends back by folding back from either side

opening, and fastening with rubber bands. Now the space between the hard rubber hose and inner rubber liner is filled with hot water to provide. The graduated semen collection tube is fixed to the narrow end of the artificial vagina hose, and fastened by a rubber band. The inner side of the rubber liner on the anterior side of the artificial vagina is lubricated with sterile jelly to a length of 3 to 4 inches. Air is blown through the nozzle into the water jacket, to create pressure.

The temperature of the artificial vagina is more important and should always be checked before making the collection. Too high temperature may cause injury to the penis and the bull may refuse to serve in future, whereas at lower temperature, there may not be complete ejaculation.

Prior to collection, bulls are usually allowed to become excited by bringing to cow or dummy. It will smell and also will make attempts to ride. During collection, the artificial vagina is held at an angle of 45^o with the horizontal plane of the cow or dummy. It is so because the penis of the bull enters the vagina of the cow at that angle. When the bull mounts the penis is quickly guided into the artificial vagina with the operator’s hand. Care is taken not to touch the exposed part of the penis, as this stimulates the bull to ejaculate. After the bull dismounts, the artificial vagina is taken off the penis and is kept in an upright condition after releasing the pressure by draining water and air out of it. This allows the ejaculate to flow from the cone to the vial, which is then detached from the cone and taken to the laboratory for examination.

  Evaluation of Semen 

Just after collection the ejaculate should be placed in a water bath maintain at 35 ^oC. It is important that semen examination should be carried out rapidly so that dilution and cooling can be initiated as soon as possible after semen collection.

Gross Semen Evaluation

Volume:
Volume of semen is constant for each species. For bull it is 3-5 ml per ejaculation. Volume of semen depends upon sexual activity of bull. Small ejaculation may be in young bull which is due to excessively incomplete ejaculation, failure of ejaculation, or bilateral seminal vesiculitis. Volume of ejaculation increases with age of bull, body size, and reproductive health.

General Appearance:
Normally semen of bull is white or creamy and its degree of opaqueness depends upon concentration of sperms. Sometimes color is yellow due to riboflavin. Often abnormal semen has pus, plaques, clamps of leukocytes, urine or water that is leaked from artificial vagina. It should be discarded.
 

Presence of Pseudomonas aerogenosa changes the normal color to yellow green color. Clumps, clots, large plaques in semen indicate presence of varying amount of blood. Light brown color due to presence of feces.

pH of Semen:
It is measured by pH meter or pH paper. pH of bull semen is 6.5-7.2

Microscopic Evaluation

Mass activity

Apparatus:
Glass slide, glass rod, microscope, tissue paper.

Procedure:
Place a drop of semen with the help of glass rod on clean slide and observe under microscope.

Precaution:

• Temperature of apparatus should be 37 ^oC

• Mass activity should be observed immediately after collection.

• The drop of semen should be thick and observe under low power and reduced light.

Detection of Individual Motility of Spermatozoa

Individual motility is observed in order to estimate the total percentage of motile sperms in the ejaculate.

Apparatus:
Microscope, glass slide, glass rod, cover slip, 2.9 % sodium citrate, hot plate and tissue paper.

Procedure:
Take a glass slide and clean it with tissue paper. Place a drop of whole semen with the help of glass rod in the centre of the slide and near to this place a drop of sodium citrate solution with the help of other glass rod. Then gently mix these two drops, place cover slip and observe under low power microscope. Estimate whether 50 % of sperms are motile or less than 50 %. Then observe under high power and select an area on the slide where there are about 10 sperms. Count the number of dead or live sperms. If 8 out of 10 are dead, 80 % is answer.

All live sperms are motile sperms. Those which have abnormal movement will not be able to fertilize the ovum and hence are considered as dead sperm. The motility of sperm is categorized in following category:

•  Progressive rectilinear motility: the sperm move in straight forward direction.

• Circling motility: the sperms move in circle because of mid piece or tail defect indicating cold shock to sperms.

• Reverse circling motility: the sperms move in a circular backward direction. This type is observed in post thaw semen and also in bulls severely affected by FMD.

• Penetration motility: the spermatozoa exhibit jerky serpentine motility without showing marked progress. This type of motility is observed in the aged semen sample.

Progressive rectilinear is normal others are dead.

Semen samples are classified according to rate of motility as follows:

80-100 %         very good

60-80 %           good

40-60 %           fair

20-40 %           poor

0-20 %             very poor

Concentration of Spermatozoa

Concentration is variable from ejaculate to ejaculate of the same bull, from bull to bull and from species to species. It is mostly used for research purposes, determining the rate at which semen should be diluted and for determining the bull output.

Apparatus:
RBC counting pipette and hemocytometer chamber, 3 % solution of NaCl, methylene blue solution and microscope.

Procedure:
Clean and dry the RBC counting pipette. Semen is diluted at 1:100 with 3% solution of NaCl. 0.1-0.2 ml of well mixed semen is poured in a small clean vial and few drops of methylene blue solution is added to give semen a distinctive blue color which will aid in an accurate sampling. Suck the semen upto chosen mark slowly and carefully and wipe outside the pipette either with clean cloth or lens paper to remove the adhering semen. Then dilute 100 times by drawing into pipette, the diluting fluid is 3 % NaCl. Shake the pipette for at least 1-2 minutes. The hemocytometer chamber and its cover slip are thoroughly wash and dry. Place the diluted semen under cover slip of counting chamber. The hemocytometer is left for 5 minute so that all spermatozoa settle down. The counting chamber is then placed under microscope first at low power then at high power. Spermatozoa are counted in five of the large squares, four at corners and one in centre.

Formula:   

Where 

X = total cells counted in squares

80 = squares where sperms were counted

400 = total squares

100 = dilution factor

10 = depth of counting chamber

In modern semen production units semen evaluation is carried out through computerized system.

  Storage of Semen 

After evaluation semen is diluted in an 'extender'. This provides an appropriate concentration of spermatozoa, allowing more inseminations from each sample. A dilution of around 50 times is usual. The extender also nourishes and protects the spermatozoa during storage and distribution. Typically, the extender contains:

• Milk or egg yolk to protect against cold shock (the initial cooling below body temperature);

• Glycerol as a cryoprotectant (to protect damage due to the formation otherwise of ice crystals during freezing);

• A buffer (usually citrate) to prevent pH changes due to, for example, lactic acid produced during sperm metabolism;

• Glucose (and/or other sugars) to provide an energy source for the spermatozoa, as well as the correct overall water potential for their survival;

• Antibiotics, to kill pathogens.

Semen is packed into the plastic straws and stored in liquid nitrogen at -196° C. Each straw contains around 20 million spermatozoa. There is slow deterioration of the effectiveness of semen with time.

For use, the straws are thawed in water bath at 37 ° C for a few seconds before insemination to reactivate the spermatozoa.

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