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

Live and dead sperms

LIVE AND DEAD SPERM ESTIMATION

Introduction

• 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.
• 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 color.
• The stain will not pass through the live sperm and it appears as white in color. The nigrosin is a negative stain which gives background color.
• 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.

Procedure and result interpretation

Materials required

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

Phase contrast microscope

Preparation of 5% eosin stain

Chemical	Quantity
Eosin yellow powder	5 gm
2.9% sodium citrate	100 ml

• Weigh the eosin powder, put in pestle and mortar.
• Prepare 2.9% sodium citrate solution, boil it.
• Add the boiling solution to stain and grind it well. Finally filter and store it at 4 ⁰ C.

Preparation of 10% nigrosin stain

Chemical	Quantity
Nigrosin water soluble powder	10 gm
2.9% sodium citrate	100 ml

• The preparation of the stain is as per the above method. After filtration the stain is stored in an air tight bottle at 4° C.

Staining procedure (click here to view picture)

(Click here for video demonstration)

• A drop of eosin, four drops of nigrosin 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 (Click here to view picture).
• 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. (Click here to view picture)

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