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

Procedure and result interpretation

PROCEDURE AND RESULT

(Materials required, Preparation of eosin, Preparation of nigrosin, Staining procedure, Calculation and 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 4⁰C.

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 4⁰C.

Staining procedure (Click here for video demonstration )

• 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 (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