VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

METHYLENE BLUE DYE REDUCTION TEST

Principle

Methylene blue dye reduction test (MBRT) is a very simple test used to measure the metabolic activity of the spermatozoa.

• This test is based on the principle that the hydrogen ions are liberated during sperm metabolism which will reduce the blue colored methylene blue into colorless leucomethylene blue.
• The hydrogen ions are released due to the dehydrogenase enzyme present in active sperm.
• The time taken to change the color is directly related with the motility and concentration of the spermatozoa.
• More the motility and concentration will lead to more the hydrogen ion release which will cause less time taken by the methylene blue to colourless leucomethylene blue.

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Procedure and interpretaion of reuslt

Materials required

• Fresh semen
• Egg yolk citrate diluent
• 5 ml test tube
• Incubator
• Methylene blue solution (50 mg of methylene blue in 100 ml of 2.9 per cent sodium citrate buffer)
• Liquid paraffin
• Water bath

Procedure

• Take 0.2 ml of fresh semen and 0.8 ml of egg yolk citrate diluent in a sterile 5 ml test tube
• Add 0.1 ml methylene blue solution and mix the contents
• Place 1 cm layer of liquid paraffin
• Keep the test tube in a water bath of 46.5° C
• Observe the time taken to change in color from blue to colorless

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Result

Based on the time taken to change the color, the semen sample is classified as follows.

S.No.	Time interval	Classification
1	3 - 5 min	Good
2	9 min	Average
3	> 9 min	Poor

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