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

FRUCTOLYSIS INDEX

Introduction

• Fructose, a glycolysable sugar, present in the semen, which is produced by the accessory sex glands, is utilized as a source of energy by active spermatozoa.
• Its level in semen is regulated by the male sex hormone testosterone.

Fructolytic index is defined as the amount of fructose utilized by 10⁹ spermatozoa in one hour at 37⁰C.

• Greater the metabolic activity of spermatozoa more will be the amount of fructose metabolized in any semen sample.
• The quality of the semen can be assessed by measuring the rate of utilization of fructose.
• Fructolysis in the semen is assessed by measuring the disappearance of sugars and accumulation of lactic acid by a constant number of spermatozoa in a specific time and under specified conditions.
• Gassner and Hill (1952), Bonadonna and Pozzi (1954), Probine et al. (1958) reported a relationship between fructolysis and fertility.
• A highly significant relationship was found between fructolysis and concentration of live sperm.
• In buffalo semen fructose level is low as compared to cattle semen. Fructolysis was lowest during summer and superior during spring season.
• Mann (1948) for the first time proposed fructolysis as an index for evaluating the activity of semen.

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Materials required and procedure for estiation 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)
  • Resorsinol (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 filterate 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 comparison 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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Calculation

• A standard curve is prepared by taking 0.02 – 0.1% standard fructose solution in satrated benzoic acid.
• The amount of fructose is calculated from this curve.
• Blank is prepared by adding all the reagents and in place of semen water is added.
• The amount of fructose present in semen immediately after collection and present after one hour’s incubation are worked out.
• The difference between the two concentrations of fructose is on account of fructolysis in one hour.
• Fructolysis may also be worked out in respect of 10⁹ live spermatozoa.
• This method is useful as compared to 10⁹ sperm concentration and provides a true comparison of metabolic activities of semen samples.

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