Sperm concentration

ESTIMATION OF SPERM CONCENTRATION

  • Accurate determination of the number of sperm and volume of the ejaculate determines how many females can be inseminated.

In fresh semen samples the concentration estimation helps to determine the dilution rate and in frozen semen samples it is done to ensure sufficient concentration is packed in the straws

Concentration estimation can be done by

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.

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Calorimeter
  • 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 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.

Calorimeter

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.

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Photometer

  • It is the advanced form of colorimeter. Here the principle of the equipment is same as colorimeter.
  • But the 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.

Photometer

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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.

CASA

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Haemocytometer

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

Materials required

  • Semen sample (fresh/frozen) Semen sample
  • Haemocytometer set Haemocytometer
  • Phase contrast microscope Nikon
  • Watch glass Watch glass
  • Dilution fluid (0.1% formal saline or distilled water or 3% chlorazene solution) Formal saline
  • Eosin powder Eosin
  • Blotting paper Blotting paper

Procedure (Click here to view picture)

(Click here for video demonstrationView video)

  • Mix the semen sample gently to get uniform distribution of sperms.
  • Place the semen in a sterile watch glass.
  • Put a speck of eosin powder and mix it with semen.
  • Aspirate the semen from watch glass into a RBC pipette upto 0.5 mark.
  • Clean the tip of the pipette with blotting paper.
  • Draw the dilution fluid in the same RBC pipette upto 101 mark.
  • Roll the pipette between palm of the hands for 2 minutes to ensure through mixing of the fluid and semen.
  • Discard first few drops.
  • Charge the haemocytometer by releasing the fluid below the coverslip which is placed over the haemocytometer.
  • While charging overflowing and air bubble formation should be avoided.
  • Wait for 1-2 minutes for the sperms to settle.
  • Examine the charged haemocytometer for under low power and then in high power. (Click here to view picture).
  • The sperm counting is done in RBC chamber. (Click here to view picture).
  • Count the number of sperms in left top, right top, right bottom, left bottom and center squares of RBC chamber and calculate the concentration. Counting can be done in other directions also but it should be in unbiased manner(Click here to view picture).
  • While counting sperms in individual chamber it should be done in such a way that biasness should not be there. (Click here to view picture)

Calculation

Length of one small square

= 1/20 mm

Breadth of one small square

= 1/20 mm

Depth of one small square

= 1/10 mm

Volume of one small square

= length x breadth x depth

= 1/20 x 1/20 x 1/10

= 1/4000 mm3

Total number of small squares counted

= 16 x 5 = 80

Volume of semen in 80 squares

= 1/4000 x 80

= 1/50 mm3

Consider the total number of spermatozoa counted is

N

The dilution rate is

1:200

Number of sperms are occupying 1/50 mm3 space

=N

So sperms occupied in 1 mm3 space

= N x 50 x 200 mm3

= N x 10000 mm3

Number of sperms in 1 ml of semen

= N x 10000 x 1000

= N x 107 millions

Normal sperm concentration in different species

S.No.

Species

Concentration

1

Cattle bull

1200 (800-1400 millions/ml)

2

Buffalo bull

800 (600-1200 millions/ml)

3

Stallion

250 (200-600 millions/ml)

4

Ram and buck

3000 (2000-4000 millions/ml)

5

Boar

250 (200-500 millions/ml)

6

Dog

250 (125-500 millions/ml)

7

Man

150 (100-200 millions/ml)

Nomenclature

S.No.

Terminology

Explaination

1

Normozoospermia

Normal sperm concentration

2

Oligozoospermia

Reduced sperm concentraion

3

Polyzoospermia

Increased sperm concentration

4

Azoospermia

Zero sperm concentration

Last modified: Monday, 4 June 2012, 10:04 AM