Fructolytic Index in Semen: Definition, Procedure, and Calculation for Assessing Sperm Metabolic Activity

The Fructolytic Index in semen is defined as the amount of fructose utilized by 10⁹ spermatozoa in one hour at 37°C. Fructose, secreted by the accessory sex glands, serves as an essential energy source for spermatozoa.

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. It’s 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 37°C. 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.

Mann (1948) for the first time proposed fructolysis as an index for evaluating the activity of semen. It has been described as the milligram of fructose utilized by 10⁹ spermatozoa in one hour at 37°C.

Greater is the metabolic activity of spermatozoa more will be the amount of fructose metabolized per unit of time in any semen sample.

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

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).
• Take 0.1 ml of above mixture and add 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 is incubated at 37°C for one hour.
• After incubation deproteinise it as above [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°C 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.

Calculation

• A standard curve is prepared by taking 0.02-0.1% standard fructose solution in saturated 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.