Semen Concentration Estimation: Methods and Haemocytometer Procedure Explained

Semen concentration estimation is the process of determining the number of sperm cells per milliliter (million/mL) of semen.

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.

Methods of Concentration Estimation

Concentration estimation of semen can be done by:

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

Haemocytometer

Haemocytometer 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
• Useful when only a few bulls require estimation

Disadvantages

• Requires skill
• The procedure is time-consuming, so it is not suitable for situations where large numbers of samples are processed

Materials Required

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

Procedure

• 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 up to 0.5 mark.
• Clean the tip of the pipette with blotting paper.
• Draw the dilution fluid in the same RBC pipette up to 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 cover slip 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.
• Count the number of sperms in left top, right top, right bottom and left bottom and center squares of RBC chamber and calculate the concentration.

Calculation

Normal Sperm Concentration in Different Species

Nomenclature