Live and Dead Sperm Estimation: Procedure, Staining, and Interpretation in Semen

The estimation of live and dead sperm is an important quality control test in all semen banks. The live sperm concentration is directly related with the fertility.

The live sperm percentage is also directly related with motility. More the number of motile sperms higher will be the live sperms. But the live sperm percentage will not give indication about progressively motile sperms.

Sperms with other motilities like circular, oscillatory and bunting movements also will be live. So semen samples with lower motility will also have normal live sperm percentage.

So the results should always be correlated with motility and the interpretation should be done critically.

Principle

• The spermatozoa will have a plasma membrane as its outer covering.
• This plasma membrane is intact in live sperms and its integrity is lost in dead sperms.
• The live and dead sperm is assessed by a vital stain-eosin-nigrosin which was found by Hancock during 1951.
• The eosin is a vital stain which can only pass through the loosely integrated plasma membrane of dead sperm and stains it as pink colour.
• The stain will not pass through the live sperm and it appears as white in colour. The nigrosin is a negative stain which gives background colour.
• Initially the live and dead sperm was assessed by Mayer et al. (1947) by using eosin (vital stain) and opal blue (background stain).
• Later Hancock (1951) used eosin (vital stain) and nigrosin (background stain) and it is most commonly followed.

Materials Required

• Semen sample (fresh/frozen)
• Glass slides
• Eosin stain (5%)
• Nigrosin stain (10%)
• Immersion oil
• Phase contrast microscope

Staining Procedure

• A drop of eosin, four drops of nigrosine and a small drop of semen are placed on a clean, grease free slide.
• Mix the semen first with eosin and then immediately with nigrosin stain.
• The mixture is taken on the edge of a slide and pulled across the top of another slide leaving a smear.
• Allow it to dry in air.
• 200 spermatozoa are counted under oil immersion at a magnification of 1000X in different areas of smear and the live sperm percent is calculated.

Calculation

Interpretation

• Unstained sperms are considered as LIVE
• Sperms stained pink by eosin are considered as DEAD
• Partially stained sperms are considered as DEAD
• The fresh semen sample should have 80% live sperms
• The frozen semen samples should have 50% live sperms
• If a semen sample will have more dead sperms, it is called as necrozoospermia