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Methylene Blue Reduction Test (MBRT) for Semen Quality Evaluation in Animals
Methylene blue dye reduction test (MBRT) is a very simple test used to measure the metabolic activity of the spermatozoa for semen quality evaluation.
This test is based on the principle that the hydrogen ions are liberated during sperm metabolism which will reduce the blue coloured methylene blue into colourless leuco-methylene blue.
The hydrogen ions are released due to the dehydrogenase enzyme present in active sperm.
The time taken to change the colour is directly related with the motility and concentration of the spermatozoa. More the motility, concentration will leads to more the hydrogen ion release which will cause less time taken by the methylene blue to colourless leuco-methylene blue.
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 percent 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 colour from blue to colourless.
Result
Based on the time taken to change the colour, the semen sample is classified as follows:
| Time Interval | Classification |
|---|---|
| 3-5 minutes | Good |
| 9 minute | Average |
| >9 minutes | Poor |
