Veterinary Andrology and Reproductive Techniques

MATERIALS REQUIRED AND PROCEDURE FOR ESTIMATION OF FRUCTOSE

Materials required

• Phosphate buffered saline (0.25 M) with pH 7.4. (Prepare the phosphate buffer by mixing 9.6 ml of 3.4% potassium dihydrogen phosphate and 40.4 ml of 3.55% sodium hydrogen phosphate solutions)
  • Zinc sulphate (0.2%)
  • Sodium hydroxide solution (N/10)
  • Resorcinol (0.1%) in ethanol
  • Hydrochloric acid (30%)
  • Electric calorimeter

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Procedure for estimation of fructose

• Modified method on Mann (1964) for fructose estimation in semen sample is as follows.
• Take 0.4 ml of fresh semen in a tube containing 0.6 ml of 0.25 M phosphate buffer (pH 7.4).
• 0.1 ml of above mixture semen buffer is added to 1.9 ml of distilled water.
• To the above mixture add 1 ml of 2% zinc sulphate solution and 1 ml of N/10 NaOH solution for deproteinisation (zero hour sample).
• 0.9 ml of semen buffer mixture (from Sr.No. a) is incubated at 37⁰C for one hour.
• After incubation deproteinise it as in step (b) & (c) [one hour sample].
• Heat both the deproteinised samples in boiling water for one minute.
• Cool and filter the samples.
• Take 0.5 ml of above filtrate in two separate tubes and make the volume to 2 ml with distilled water.
• Add 2 ml of 0.1% resorcinol in ethanol and add 6 ml of 30% HCl to each sample.
• Mix the contents properly and maintain the sample in water bath at 80^oC for 10 minutes.
• Immediately cool the contents to room temperature in running water. Reddish brown colour develops in the solution.
• Compare the intensity of colour with the help of a visual electric calorimeter (Dubosco Hellige) with a standard fructose* solution of equal volume run as a blank**.
• *Standard fructose solution is prepared by dissolving 400 mg of fructose in 100 ml of saturated benzoic acid. 0.02% and 0.1% fructose solutions are prepared out of 0.4% stock solution for use of comparision with one hour and zero hour samples respectively.
• **Prepare the blank solution by adding 0.5 ml of 2% zinc sulphate and 0.5 ml sodium hydroxide solutions to 1.0 ml of standard fructose solution.
• Then follow the steps described from step No. 10 onwards.

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