Counting Spermatozoa - Part 1

By B. W. Pickett, Ph.D.
Professor Emeritus, Colorado State University

A hemacytometer, originally designed to count blood cells, has been used for many years to count other
types of cells. Toward the end of World War II, with the advent of artificial insemination in cattle, it became
painfully obvious that other methods of counting spermatozoa had to be developed, because of the length of
time it required for an accurate count to be obtained with the
hemacytometer.

Since the late 1930's numerous scientific papers have been published
describing how to use the hemacytometer to calibrate other instruments
to estimate the number of spermatozoa more rapidly in a sample of
semen than is possible with the hemacytometer. Consequently, today
(2004), the number of spermatozoa are estimated in bull, boar, stallion,
chicken, turkey and dog semen using electronic methods. However, the
initial calibration of these instruments is done with a hemacytometer. In
general, for an accurate calibration it requires 70 normal, clean semen
samples ranging in count, per milliliter of semen, from 50 million to 1
billion, depending upon the species. Regardless of species, the
important point is that the sperm concentration of the 70 ejaculates must
be equally distributed over the range of expected counts regardless of frequency of collection of semen.
Unfortunately, it requires 8 to 10 counts of each of the 70 samples to obtain an average count that is highly
accurate. Of course a "quick and dirty" estimate can be made with fewer counts, but is not satisfactory for
scientific purposes or any other purpose when accuracy is essential.

During the calibration procedure the same samples used for the hemacytometer counts are placed in the
electronic instrument to be calibrated. Once the 70 samples have been through the process, a regression
equation is "fitted", then the electronic instrument is used to estimate the number of spermatozoa per
milliliter of semen. The electronic method provides a quick, simple, repeatable method, provided the semen
sample is "clean". The presence of gel in the ejaculates of stallion or boar semen will cause the electronic
instrument to estimate a larger number of spermatozoa than are actually present. The same is true of my
sample containing extraneous material such as dirt, hair or cells other than spermatozoa. Consequently, it is
not uncommon for an appropriately calibrated electronic instrument to overestimate the number of
spermatozoa in a series of unselected semen samples, particularly if the electronic instrument in question
was calibrated with a hemacytometer with too few samples selected over a narrow range, or if there were a
majority of samples within a narrow range. To further complicate matters, the individual counting
spermatozoa must have a good, appropriately adjusted phase-contrast microscope and be skilled in utilizing
the counting procedure, as well as the instrument.

The method of counting spermatozoa with a hemacytometer is based upon the principle that the
hemacytometer is loaded with a constant volume of semen between the cover slip and counting grid, or
chamber. Accurate counts can be obtained with this method when used properly.

It is highly unlikely that a breeding farm would consider using a hemacytometer on a routine basis due to:
a) the number of chambers that must be counted for reasonable
accuracy,
b) the time required to prepare the sample for counting with the
hemacytometer,
c) the time required to count the number of spermatozoa in the appropriate
number of squares,
d) procedure for counting the spermatozoa,
e) preparing and counting a sample of semen requires considerable
technical skill and equipment.

However, the hemacytometer is very useful in counting semen samples that have been placed in an
extender and those that are contaminated with debris.
Counting Spermatozoa - Part 2 Return to to part 1
By B. W. Pickett, Ph.D. About the
Professor Emeritus, Colorado State University
Author...
B. W. Pickett, Ph.D is
In Part 1 five factors were listed that significantly influence the use of a hemacytometer to Professor Emeritus,
count the number of spermatozoa in a semen sample. They are as follows: Colorado State University.

a) The number of chambers that must be counted for reasonable accuracy.

Considering all the variables associated with

hemacytometer counts, it was statistically
determined that a minimum of eight chambers
should be counted to determine a mean accurate
count for a single sample of semen. In the event any
one of the eight counts is 10% or more above or
below the mean, that count should be eliminated and
another count made. Under these circumstances,
the accuracy of the hemacytometer is superior, in Figure 1. Hemacytometer (black arrow), dilution
general, to an electronic method, but the precision media (Unopette, white arrow) and high quality,
phase-contrast microscope used to determine
(or deviation) is less with the electronic counter, concentration of spermatozoa.
because there are fewer variables in preparing the
semen sample for counting. Further, the human (technician) error involved in preparing a
sample for electronically counting is relatively minimal compared to preparing a sample
for counting in the hemacytometer.

2b) The time required to prepare a sample for counting with the hemacytometer.

In recent years a system for counting cells with a

hemacytometer (Unopette) has been developed (Becton-
Dickinson, Rutherford, NJ) that has certainly simplified the
older methods, which utilized a white or red blood cell
pipette.

Presented in Figure 1 is the

equipment necessary for
Figure 2. Puncturing Unopette reservoir utilizing this newer method
using the pointed shield.
of counting spermatozoa.
Once the semen sample has been collected and
thoroughly mixed, a sub-sample is taken for ease of
sampling. The Unopette is used to puncture the plug of
the reservoir with the pointed end of the plastic shield
(Figure 2). With the shield in place, and serving as a cap
for the reservoir, remove the capillary tube (Figure 3)
Submerge the capillary tube in a thoroughly mixed sample Figure 3. Removing capillary tube
from the pointed shield, which will
of raw semen (Figure 4). Allow semen to fill the capillary be used to load an appropriate
tube, then remove it from the sample. Wipe excess semen volume of raw semen for an
from the outside of the capillary tube, being careful not to accurate hemacytometer count.
remove any semen from within the capillary tube. Remove cap and lightly squeeze the
reservoir between thumb and forefinger. Place pipette into the reservoir and release the
pressure (Figure 5), this allows semen to be siphoned into the reservoir. Rinse the
capillary tube by gently squeezing the reservoir several times without forcing fluid out of
the top. Remove and invert the pipette (Figures 6 and 7).
 Figure 4. Loading an appropriate volume
of raw semen by capillary action for
counting in a hemacytometer. Figure 5. Adding raw semen to diluent
reservoir in order to provide proper
dilution for an accurate hemacytometer
count.

Figure 7. Inverting pipette and placing it

on the reservoir to be used as a dispenser
Figure 6. Removing pipette from in loading the hemacytometer.
diluent reservoir

Presented in Figure 8 is a schematic drawing of the top and side view of the
hemacytometer. There is a grid on each of the two shorter, wider portions, (chambers) of
the hemacytometer. The two elongated portions (one on each side of the chambers) are
slightly higher than the chambers. Consequently, when the weighted cover slip is placed
over the chambers, there is a very specific volume of diluted semen over the grid, which
must be used as one of the multiplication factors to calculate the number of spermatozoa
per milliliter of semen.

Figure 8. Drawing of the top and side view of a

hemacytometer.

c) The time required to count the number of spermatozoa in the appropriate

number of squares.

Obviously, the length of time required to prepare and count the sample (including the
calculations) will vary from 10 to 20 minutes for the two chambers (one on each side)
depending upon the experience of the technician. It must be emphasized that a phase-
transparent cells can be seen in rich contrast to their background. Consequently, without
a phase-contrast microscope it is difficult, if not impossible to differentiate some cellular
debris from spermatozoa.

d) Procedure for counting the spermatozoa.

Place the hemacytometer on a flat surface, such as a

counter top. Position the special weighted cover slip so
that it is centered and cover both counting grids (Figure 9).
Load the diluted sample onto the hemacytometer by
allowing a single drop of diluted semen to be drawn across
the counting grid by capillary action (Figure 10). To assure
an accurate count, do not overfill the space between the
cover slip and the counting grid. If overfilling occurs, the
chamber must be dismantled, cleaned and the procedure Figure 9. Placing weighted cover slip
repeated. Load both counting chambers on the over both etched counting surfaces of
hemacytometer. Place the loaded hemacytometer on the the hemacytometer.

microscope stage (Figure 11). Locate the counting grid on the monitor. Adjust
magnification so that the central block of 25 squares can be observed (Figure 12). Count
the spermatozoa in the 25

squares on both sides of the hemacytometer. Be sure to

count all spermatozoa within a square and only those
spermatozoa on or across two of the four borders. This will
prevent counting the same spermatozoon twice on two
adjoining squares. At a 1:100 dilution, this total number
represents millions of spermatozoa ml in this sample of
gel-free semen. For a reasonably accurate count, two
chambers (25 squares/ chamber) should be counted on
four hemacytometers, thus duplicate counts made on each
Figure 10. Loading the hemacytometer
by placing a single drop of diluted sample of semen. Since utilization of a hemacytometer for
semen in the loading slot of the estimating sperm numbers can be very time consuming,
hemacytometer.
more rapid procedures would save time, and thus,
eventually money.

Figure 11. Placing loaded hemacytometer on the

stage of a phase-contrast microscope so that the
counting grid is visible on the monitor.

Figure 12. A drawing of the 25 squares to be

counted on a grid of a loaded hemacytometer..

Counting is further complicated when semen samples are very dilute or extremely thick,
which changes the number of large squares that must be counted for sufficient accuracy.

e) Preparing and counting a sample of semen requires considerable

technical skill and equipment. Therefore, very few farms are prepared
to make accurate counts with a hemacytometer.

It should be noted that the hemacytometer requires some special care. A dirty or
1. Wash the hemacytometer chamber and cover slip individually with a
mild detergent (Ivory). Be careful not to scratch the chamber. Rub the
surface gently with a facial tissue, lens paper or fingertip.

2. Rinse well in warm water.

3. Rinse in distilled or deionized water.

4. Rinse in alcohol or acetone to dry or dry with lens paper.

5. Store in a dust-free location with the cover slip and hemacytometer

wrapped separately in tissue.