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

DILUENT PREPARATION

(Diluent preparation steps, Points to be considered before preparation of diluent, Preparation of buffer, Sterilization of buffer, Preparation of diluent)

The diluent preparation involves two steps

• Preparation of buffer
• Preparation of diluents

Points to be considered before preparation of diluent

• The chemicals should be purchased from reliable source.

The chemicals of only highest purity (GR or AR grade) should be purchased to prepare buffer and diluent.

• The compounds used should be chemically pure and weighing should be accurate.
• The triple glass distilled water or Millipore water should be used for dissolving the salts.
• Completely dissolve one salt before adding another salt.
• The buffer solution should be sterilized by autoclaving before use.

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Preparation of buffer (Click here for video demonstration)

Materials required

• Triple distillation water/Millipore water
• Chemicals (Tris, Citric acid monohydrate, Fructose)
• Glycerol
• Weigh the chemicals as per the requirement given above for preparation of 1 liter diluent

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Buffer preparation (for 1 litre diluent)

• Take the triple distilled water in a sterile beaker according to the requirement
• Weigh the chemicals accurately
• Add the chemicals one by one and dissolve it by magnetic stirrer by required speed
• Finally add the glycerol at 7% level ie. 70 ml
• Make the volume up to 800 ml by adding distilled water
• Completely mix the glycerol by magnetic stirrer for at least 20 minutes
• Care should be taken while stirring that air bubbles should not be formed by over speed of the stirrer
• The pH of buffer should be 6.8-6.9.

• Glycerol is added @ 7%.
• pH of buffer - 6.8-6.9.

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Sterilization of buffer

• The prepared buffer is sterilized by autoclaving at 100⁰C for 15 min at 5 psi.
• Cool the buffer to room temperature
• Generally the buffer is prepared on previous day evening, sterilized and kept under laminar air flow over night.
• The diluent is prepared by adding egg yolk in the morning immediately before semen collection.

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Preparation of diluent (Click here for video demonstration)

• The egg yolk is prepared as per the standard procedure
• For 1 liter of diluent, 200 ml of egg yolk is required
• The egg yolk is added and mixed by using magnetic stirrer
• The antibiotics are also added while stirring with egg yolk
• Care should be taken while stirring that air bubbles should not be formed by over speed of the stirrer
• The pH of the diluents should be 6.7-6.8.

• Egg yolk is added @ 20%.
• pH of buffer - 6.7-6.8.

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