Hypo Osmotic Swelling Test (HOST) in Semen Evaluation: Procedure, Principle & Interpretation

Hypo Osmotic Swelling Test (HOST) is a laboratory technique used to assess the functional integrity of the sperm plasma membrane, which is critical for sperm viability and fertility.

The integrity of plasma membrane is a prerequisite 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.

Principle

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.

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.

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

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.

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.

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. The frozen semen samples should have a minimum of 50% reacted sperm.