9. Differential Staining of Live and Dead Spermatozoa. Determination of the Resistance of Spermatozoa

What is the principle of the differential staining method for live and dead spermatozoa?

The principle of the differential staining method is based on the ability of the cytoplasmic membrane of dead spermatozoa to allow the diffusion of eosin dye into the cytoplasm, while the membrane of live spermatozoa does not. Dead spermatozoa stain pink or red due to eosin, while live spermatozoa remain colourless. Nigrosine is used to provide a blue-black background.

How is the differential staining method performed?

The procedure for the differential staining method involves placing a small drop of semen on a watch glass and adding eosin dye. After 30 seconds, nigrosine dye is added and gently mixed. One or more smears are prepared using the stained semen and dried rapidly. The resulting smear should appear slightly pink or almost white, with live sperm remaining unstained and dead or drying sperm stained pink or pinkish.

What is the resistance test of spermatozoa?

The resistance test is a biological test that determines the resistance of spermatozoa to the detrimental effects of 1% NaCl (salt solution). The test assesses the ability of sperm cells to withstand the penetration of chloride ions into the cytoplasm, which can result in sperm death. The test evaluates the protective function of the lipoprotein complex coating the sperm cells.

How is the resistance test performed?

The resistance test is performed by adding varying amounts of 1% NaCl to test tubes containing semen samples. The motility of the spermatozoa is evaluated at specific time intervals after each addition of NaCl. If progressive movement is still present, more NaCl is added, and the motility is reassessed. The resistance of the sperm cells is expressed by the amount of NaCl required to destroy sperm structure. The minimal resistance value is typically set at 20,000.