Veterinary Andrology and Reproductive Techniques

METHODS OF CONCENTRATION ESTIMATION

Concentration estimation can be done by

• Visual examination
• Cell volume method
• Haemocytometer method
• Colorimeter
• Photometer
• Opacity tubes
• Computer assisted semen analyzer (CASA)

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Visual examination

• Here the semen sample is seen visually by naked eye and the concentration is estimated approximately.
• The estimation depends on the experience of the person.
  • This method is giving satisfactory result in samples with high concentration in bulls and rams.
  • But it will not be useful in samples with medium or low concentration.
  • This method is not useful in stallion, boar and dogs.
  • Sperm concentration and semen color in bulls and rams

S.No.	Bull (sperms/cmm)	Ram (sperms/cmm)	Color
1	-	2,500,000	Thick creamy
2	2,000,000	2,000,000	Creamy
3	1,000,000	1,000,000	Thick milky
4	500,000	500,000	Milky
5	100,000	100,000	Cloudy, watery, translucent
6	Less than 50,000	Less than 50,000	Clear, transparent, watery

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Cell volume method

• The semen is centrifuged immediately after collection.
• The sperms will deposit at bottom and the seminal plasma will come to top.
• The packed volume of sperms is measured and based on this the concentration is estimated.
• This is not an accurate method because the materials other than the sperms will interfere with the result.

The concentration obtained by cell volume method will not be accurate during disease conditions of male reproductive tract and whenever the semen is mixed with foreign materials.

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Colorimeter

• Here the optical density of the semen sample is measured and from which the concentration of the sample is arrived.
• The colorimeters are designed to measure the percentage of light transmitted through a light absorbing media can be used.
• The percentage of transmission is a function of the concentration of the light absorbing agents in the medium.
• This principle is applied is applied to estimate the sperm concentration in a semen sample.
• The percentage of transmission recorded has to be converted as sperm concentration.
• To standardize this colorimeter initially the percentage transmission has to be recorded for a large number of semen samples of various concentrations.
• The actual sperm concentration of the same samples has to be estimated by haemocytometer method.
• Finally the correlation between the sperm concentration and light transmission has to be found.
• Based on this a working chart has to be prepared and it can be used for routine concentration.
• The instrument has to be calibrated once in a month.

Advantages

• Faster than other methods.
• Results are reliable

Disadvantage

• Initial standardization requires time
• Operation requires time and skill.
• The media used for suspending the semen should dust free otherwise the results will be wrong.

  Photometer

  • It is the advanced form of colorimeter.
  • The principle of the equipment is same as colorimeter.
  • This instrument itself will display the final concentration, dilution rate and number of doses can be made. It is faster than the colorimeter.
  • The instrument has to be calibrated once in two weeks with haemocytometer to ensure better working condition.

  

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  Opacity tubes

  • Brown’s opacity tubes have been used for estimating sperm concentration.
  • It is a faster method and can be used in field conditions.
  • Opacity increases with increase in concentration of semen.
    • Here 0.1 ml of semen is diluted with 9.9 ml of formal saline (dilution rate 1:100). This is matched with the Brown’s opacity tubes.
  • Concentration per cmm = Opacity tube number x dilution rate x 100 x 5
    • Semen samples with more epithelial cells, casts, dusts and lubricants will give poor result. However this method is not used nowadays.

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  Computer assisted semen analyzer (CASA)

  • It is the most advanced method. Here a special instrument CASA is used to assess the sperm concentration.
  • But the process is tedious and the instrument is costly. So it is not in regular practice.

  

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  Haemocytometer

  • It is a very old method used to assess the sperm concentration but is most accurate method.

    Haemocytometer is originally a device made for counting blood cells. Now it is used to count sperms cells also. If the procedure is followed correctly, this is the most accurate method to assess the sperm concentration.

  • The procedure is as same as RBC estimation.

  Advantages

  • Accurate method
  • It is used when few bulls requires estimation

  Disadvantages

  • Requires skill
  • The procedure is time consuming.
  • So it cannot be used in places where large samples are processed.

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