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The page below is a sample from the LabCE course Semen Analysis. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Sperm viability (vitality) should be assessed if a low percentage of sperm are progressively motile, eg., 30 - 40%. Since motile cells are inherently viable, a viability assessment may not be necessary when motility is high. This test is important to determine if the non-motile spermatozoa are alive or dead.
The percentage of live spermatozoa is determined by identifying sperm with an intact cell membrane. This is usually done by using a dye exclusion method where dye enters a non-vital (dead) cell due to the damaged plasma membrane. Therefore, viable cells will not appear stained, but non-viable cells will take up the stain.
Viability testing should be performed as soon as possible after liquefaction. To assess viability, place a drop of semen on a slide. Add an equal volume of a vital stain such as trypan blue. Cover with a coverslip. Allow color to develop for several minutes, but not more than 5 minutes. Count 100 cells (both motile and non-motile cells) on each of 2 slides. During the count, differentiate between the nonstained cells (living) and stained cells (non-living).
Another commonly used staining method is eosin-nigrosin. The advantages to this stain are that permanent slides can be made and the nigrosin provides a dark background for easier recognition of the non-stained, viable cells. Non-viable sperm have red or dark-pink heads and viable sperm have white or faintly-pink heads,as shown in the image on the right.
If viability testing is not performed in your laboratory and a low percent motility is reported (eg, less than 30 - 40%), a comment should be added to the the report that the decreased motility may be the result of non-viable or non-motile sperm.