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

HYPO OSMOTIC SWELLING TEST (HOST)

Introduction and Principle of the test

• The integrity of plasma membrane is a pre-requisite for maintaining fertility.
• The spermatozoa undergo important events in the female reproductive tract like capacitation and acrosome reaction to attain fertilizing ability.
• For these events to take place the spermatozoa must maintain the functional integrity of cell membrane.
• Further the process of fertilization involves complex biochemical and physiological events that are not measured by the gross physical indicators used in routine semen evaluation.
• Hence there is a need for a test that is inexpensive, technically simple and reasonably accurate that could be used as an adjunct to the standard semen evaluation methods.
• Fluid transport occurs in an intact cell membrane under hypo osmotic conditions until equilibration is reached between inside and outside the cell.
• Due to influx of fluid there will be bulging or bending of the tail fibre occurs.
• This phenomenon is known as "tail curling" or the sperm with curled tail is known as "swollen sperm".
• Spermatozoa with chemically and physically intact membrane will show tail curling under hypo osmotic conditions whereas spermatozoa with an inactive membrane will not.
• During cryopreservation, spermatozoa are subjected to stress that can alter membrane integrity.

Hypo osmotic swelling test (HOST) is found to be useful as a measure of cryosurvival of spermatozoa.

• Various media are used to provide hypo osmotic conditions to spermatozoa to test the functional integrity of cell membrane.
• Distilled water can be used as hypo osmotic medium in HOST.
• The results of the studies on HOST and fertility of frozen semen in bulls indicate that with higher HOS spermatozoa give higher fertility rate.
• HOS also has positive correlation with freezability of semen.

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Pre requisites

Materials required

• Fresh/frozen semen
• Sugar tube
• Distilled water/ HOS media
• Water bath
• Pipette and tips
• Preparation of HOS media

For 75 mosm media

• Sodium citrate - 0.367 gm
• Fructose - 0.675 gm
• Distilled water - 100 ml

The osmolality of water is '0'. This can be used as hypo osmotic media. Otherwise hypo osmotic media (with different osmolalities like 75, 100, 125, 150 & 250 mOsm) can be prepared with sodium citrate and fructose in certain combination.

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Procedure and interpretion of result

(Click here for video demonstration Part I & Video demonstration part II )

Procedure

• Take 0.9 ml of HOS media /distilled water in a sugar tube
• Keep the tube in 37 ° C water bath for 5 minutes to bring the temperature of media to 37 ° C
• Then add 0.1 ml of semen
• Incubate the mixture at 37 ° C water bath for 30 minutes
• Place a drop of semen in a clean, grease free glass slide and put a cover slip over it.

Examine under phase contrast microscope to see the tail curling

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If the clarity is not there, do the following procedure

• Make a thin smear of the incubated mixture in a clean slide and dry it.
• Stain the slide with 3% Rose Bengal stain for 20 minutes
• Wash the slide under running tap water and dry it
• Examine the slide under oil immersion microscope for tail curling
• Count at least 200 spermatozoa
  • Calculate the percentage of spermatozoa with showing tail curling.
  • Samples showing higher percentage of sperms with tail curling indicate good samples

Calculation

The fresh semen samples should have a minimum of 70% and above HOS reacted sperms and the frozen semen samples should have a minimum of 50% reacted sperm.

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