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 mix the sample

manually by swirling the container several times. Thorough

mixing is essential for accurate counting. Calibrated

automatic pipettes are used to prepare a dilution. Because

of the viscosity of semen, the semen should be added to

the diluent using a positive pressure pipettor.

The dilution often used for routine sperm counts is 1:20

but the actual dilution factor will vary depending on the

402 Concise Book of Medical Laboratory Technology: Methods and Interpretations total sperm count. For high concentration specimens, a

greater dilution will be necessary. For low concentrations,

an undiluted or minimally diluted specimen may be

required. The appropriate dilution is determined by

estimating the concentration needed to do a count of at

least 100 cells per side of the loaded hemacytometer.

The diluent that may be used for sperm counts on a

hemacytometer can be as follows: 5 g of sodium bicarbonate

in 100 mL of distilled water, plus 1 mL of formalin (neutral).

Other Counting Chambers

Some professionals believe that sperm counts done by

hemacytometer are not accurate because of the need

to dilute the viscous semen prior to counting. There are

several other counting methods available to assess sperm

concentration.

The advantages of the following methods are:

¾ The specimen does not have to be diluted

¾ Motile and non-motile sperm can both be counted

avoiding the need for wet mount evaluation of motile

cells.

Note that counting moving sperm can be difficult and

takes significant practice to avoid error.

For each of these methods accurate counts are best

obtained when at least 100 sperm per replicate are

counted.

¾ Makler (Zygotek Systems, Inc.): An undiluted sample is

placed on the chamber and covered with the coverglass.

Ten squares on the grid contain 0.000001 mL

¾ CellVu (Millennium Sciences, Inc.): Two sides of a

special slide are loaded with a drop of undiluted semen.

Coverslips with special grids are placed on top of the

sperm according to manufacturer’s directions. Sperm

on both sides are counted

¾ MicroCell (Conception technologies) has two

chambers on a single, disposable slide. A special

eyepiece with a grid is needed for counting.

Loading and Counting Using a Hemacytometer

Fill both sides of the hemacytometer. Focus on the large

center square with the 20X objective. The counting area

consists of five small squares in the large center square.

The squares usually counted are the four corner squares

and the center square, all of which are marked R. A

minimum of 100 sperm should be counted in the five

small “R” squares. If the number of sperm is low then 10

squares or all squares may be counted to obtain the 100

per side. Count both sides of the hemacytometer and take

the average of the two counts to calculate the actual count

per mL.

Neubauer Hemacytometer

The picture on the right shows the counting chamber of

the Neubauer hemacytometer. This counting method is

used to count many types of cells. To use this chamber

for counting sperm the specimen will usually need to

be diluted. Proper loading of the hemacytometer is also

important for accurate sperm counts to be obtained.

When Count are Towards Lower Limit Use the Method

Given as Under

Counting can be done with WBC pipette. Following

liquefaction draw semen till ‘0.5’ mark, dilute with the

diluting fluid till 11 mark. Mix properly. Charge the

chamber, let stand for 2 minutes (sperms settle down). The

spermatozoa in 4 sq mm (four large squares) are counted.

Multiply this number by 50,000 to get the number of

sperms per milliliter of semen. When seminal viscosity

is markedly increased, a mucolytic agent prior to pipette

dilution can be added in equal volume (1:1 dilution) and

then the final count be multiplied by 2. Normal sperm

count range is 60 to 150 million/mL with an average of

100 million/mL. Counts of less than 20 million/mL are

considered distinctly abnormal.

Motility

Active motility is a must for normal spermatozoa as they

have to migrate from cervix to the fallopian tubes where

fertilization of the ovum occurs. A drop of liquefied semen

should be kept on a prewarmed slide and coverslipped.

The coverslip should be ringed by petrolatum. Count at

least 200 spermatozoa, the whole depth of the fluid should

be screened and the nonmotile sperms settled at the

bottom be included also to assess motility. The percentage

of sperms showing actual progressive motion should be

recorded. Normal semen contains more than 70% motile

sperms. Semen should be considered abnormal if fewer

than 60% of spermatozoa show progressive motion in

specimens examined within 3 hours of collection.

Sperm Morphology (Fig. 13.1)

This is assessed by performing differential count of

morphologically normal and abnormal spermatozoa types

on stained smears (Fig. 13.1). Smears are made on slides as

for blood smears. Place the smear immediately in a fixative

95% ethanol (v/v) or 50% (v/v) ethanol ether before drying

has occurred. The most suitable stain is Papanicolaou. Air

dried smears can be stained by Mayer’s hematoxylin [air

dried smears in 10% (v/v) formalin for 1 minute; water

rinse; Mayer’s hematoxylin 2 minutes; water rinse; air dry],

but this is not a very satisfactory method. Other staining

Semen Analysis 403

techniques include Giemsa, basic fuchsin and crystal

violet. Basic fuchsin and crystal violet need heat fixing.

At least 200 spermatozoa should be examined under oil

immersion and the percentage of abnormal forms noted.

Normal semen has fewer than 30% abnormal forms. In

addition to sperm morphology, the presence of RBCs,

WBCs and epithelial cells should be noted. Differentiate

immature germinal cells from macrophages or leukocytes.

Numerous granules and globules are normally present in

the semen.

Chemical Examination

Fructose: This is the main sugar of semen and reduced

levels of seminal fructose correspond well with diminished

androgen deficiency. There is an inverse relationship

between fructose level and sperm count. A low fructose

concentration is the result of a low testosterone level or

seminal vesical insufficiency. Resorcinol method is quite

simple and inexpensive.

This principle of the test being that fructose heated with

resorcinol in an acidic medium produces a red precipitate.

The reaction involves the conversion of fructose to

hydroxymethyl furfural that condenses with resercinol to

form the real precipitate.

Reagent

Resorcinol 50 mg

Concentrate hydrochloric acid 33 mL.

Dissolve and make to 100 mL with distilled water.

Method

¾ Take 5 mL of resorcinol reagent in a test tube (15 mL)

¾ Add 0.5 mL of seminal fluid

¾ Bring the solution to the boil (take care—the test tube

opening should not be facing you)

¾ Examine the solution and report as mentioned below.

Reporting

Negative: No change in color seen.

Positive: Red colored precipitate forms within 30 seconds.

Check the positive reaction with 0.5% aqueous fructose

solution. 2% glucose can evoke similar test result, but such

quantities of glucose are not seen in semen.

Other Tests

Postcoital (Sims-Huhner) test: Optimum time ovulatory

phase at midcycle. The woman reports within 8 hours after

coitus.