CLINICAL TECHNIQUES
Counting Spermatozoa with a Hemacytometer
Dirk K. Vanderwall, DVM, PhD, Dipl. ACT
Determining the concentration of spermatozoa in the
gel-free fraction of an ejaculate of semen is a fundamental
procedure when performing a complete breeding sound-
ness evaluation or preparing semen for artificial insemina-
tion. In a clinical setting, there are two primary methods
of determining the concentration of spermatozoa, spectro-
photometrically using a commercially available sperm
countera or by direct counting of spermatozoa using a he-
macytometer.1,2 The primary advantages of the spectro-
photometric method are rapidity and ease of use;
however, these instruments are moderately expensive
($1,000$3,500), and spectrophotometric methods can-
not be used to count a semen sample that has been mixed
with semen extender (eg, cooled semen that has been
received for insemination), because the opacity of the ex-
tender interferes with the analysis. Similarly, spectrophoto-
metric methods cannot be used to accurately count
a semen sample that is contaminated with cellular debris,
blood, urine, purulent material, or premature germ cells.3
Advantages of the direct counting method using a hemacy-
tometer are that most clinical laboratories have the neces-
sary equipment (microscope and hemacytometer), and
the direct method can be used to count a semen sample
that has been mixed with semen extender (eg, Kenney-
type) or is contaminated with cellular debris. Therefore,
even in clinical settings in which spectrophotometric
methods are routinely used for counting spermatozoa, sit-
uations that require direct enumeration of spermatozoa us-
ing a hemacytometer are likely to arise. This ‘‘Clinical
Tips’’ will review the methods for counting spermatozoa
with a standard Neubauer-type hemacytometer.
For an accurate count, precise dilution of a well-mixed
semen sample is essential. A simple method of accurately
preparing a 1:100 dilution of semen is the BD Unopette
System #365855 for white blood cell/platelet determina-
tion that consists of a 20-mL capillary pipette and prefilled
diluting reservoir containing 1.98 mL buffered ammo-
nium oxalate (Fig. 1).b Use of the Unopette System for
counting spermatozoa is essentially the same as for hema-
tology procedures and is summarized in Table 1. Once
the 1:100 dilution of semen sample is prepared in the Un-
opette reservoir, both sides of the hemacytometer (with
From the Northwest Equine Reproduction Laboratory, Department of Animal and
Veterinary Science and Center for Reproductive Biology, University of Idaho, Moscow,
ID.
Reprint requests: Dirk K. Vanderwall, DVM, PhD, Dipl. ACT, PO Box 442201,
University of Idaho, Moscow, ID 83844.
0737-0806/$ - see front matter
Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.jevs.2008.02.003
244
REFEREED
Figure 1. Three Unopettes are shown in this image.
The one on the left shows the 20-mL capillary pipette
covered by its protective shield next to the prefilled
dilution chamber; the one in the middle shows the
capillary pipette inserted into the dilution chamber for
filling/dilution; and the one on the right shows the
capillary pipette seated in the neck of the dilution
chamber, forming a ‘‘dropper assembly’’ for loading the
diluted sample into a hemacytometer. See text and
Table 1 for further details on use of the Unopette
System.
cover slip in place) are filled with the diluted sample, and
the hemacytometer is left undisturbed for approximately
3 to 5 minutes to allow the spermatozoa to settle to a
uniform focal plane. Then with a microscope at 200 to
400 magnification, the number of spermatozoa (intact
or separated heads) present in the 25 small squares within
the large central square is determined on both sides of the
hemacytometer (Figs. 2 and 3). As for hematology cell
counts, spermatozoa touching the top/left lines of an indi-
vidual square are counted, and spermatozoa touching the
right/bottom lines of an individual square are not counted
to ensure spermatozoa are counted only once. The number
a
Spectrophotometric instruments for counting spermatozoa are available from:
Animal Reproduction Systems, Chino, CA (590a or 591B Equine Densimeter)
Hamilton Research, Hamilton, MA (Model 10 Sperm Counter)
IMV USA, Maple Grove, MN (Accuread Equine Photometer)
Minitube of America, Inc., Verona, WI (SpermaCue)
b
BD UnopetteÔ System #365855 is available from the following distributors:
Butler Animal Health Supply, Dublin, OH (catalog #000255)
Henry Schein Animal Health, Melville, NY (catalog #9877817)
MWI Veterinary Supply, Meridian, ID (catalog #006584)
Webster Veterinary, Sterling, MA (catalog #07-803-2090)
Journal of Equine Veterinary Science  Vol 28, No 4 (2008)
DK Vanderwall  Vol 28, No 4 (2008) 245

Table 1. Procedures for diluting a semen sample with the BD Unopette System*
Step Procedure
1. Using the protective plastic shield covering the capillary pipette, puncture the diaphragm of the reservoir
chamber.
2. Remove the shield from the capillary pipette, and while holding the pipette nearly horizontally, place the
tip into the sample to be analyzed and allow the pipette to fill completely via capillary action.
3. Wipe any excess sample from the outside of the capillary pipette, being careful to avoid drawing sample out
of the pipette.
4. Squeeze the reservoir chamber slightly to force out a small amount of air (do not expel any diluent), and
while maintaining pressure on the reservoir, insert the tip of the pipette into the fluid within the reservoir
chamber and seat the base of the pipette securely in the reservoir neck.
5. Relieve pressure on the reservoir chamber, which will draw the sample out of the pipette into the diluent
within reservoir chamber, and then gently squeeze the reservoir chamber two or three times to rinse the
pipette bore with diluent, being careful not to allow diluent/sample to overflow from the open top of the
pipette.
6. Gently mix the contents of the reservoir chamber, and then convert it to a ‘‘dropper assembly’’ by removing
the pipette from the reservoir chamber and reseating the base of the pipette in the neck of the reservoir
chamber in the reverse position (ie, with the tip of the capillary pipette exposed for dispensing).
7. Carefully squeeze the reservoir chamber to discard the first 3 or 4 drops of fluid, and then use the same
procedure to carefully fill both sides of the hemacytometer.
* Adapted from BD Product Circular for the Unopette System, Rutherford, NJ (6/84).

Figure 2. Schematic drawing of the markings on
a Neubauer-type hemacytometer. The black circle
outlines the large central square that is subdivided into
25 small squares. As described in the text, with a 1:100
dilution, the number of spermatozoa present in all 25
small squares represents the concentration of
spermatozoa in millions/mL.
Figure 3. Photomicrograph of 1 of the 25 small squares
of the hemacytometer containing 14 spermatozoa; as
shown, the small square is further subdivided into 16
squares. As described in the text, with a 1:100 dilution,
the number of spermatozoa counted in all 25 small
squares represents the concentration of spermatozoa in
millions/mL. The small square is 0.2  0.2 mm. Image
magnification ¼ 400.
246
of spermatozoa counted on each side of the hemacytome-
ter should be within 10% of one another, which indicates
that both sides are representative (ie, no loading errors,
and so forth). With a 1:100 dilution, the number of sper-
matozoa present in the 25 small squares represents the
sperm concentration in the original sample in millions/
mL. For example, if the number of spermatozoa counted
in the 25 small squares on one side of the hemacytometer
is 150 and on the other it is 160, the average is 155, which
indicates the concentration of spermatozoa in the original
sample is 155 million/mL. Although other dilution ratios
can be used when counting spermatozoa with a hemacy-
tometer, using a 1:100 dilution greatly simplifies the calcu-
lations involved when performing the procedure. If the
original semen sample was undiluted (ie, raw/neat) semen,
the concentration of spermatozoa is multiplied by the vol-
ume of gel-free semen to determine the total number of
spermatozoa in the ejaculate. Similarly, if the original se-
men sample was mixed with extender before counting, as
in a cooled-transported insemination dose, the concentra-
tion of spermatozoa is multiplied by the volume of the
entire extended sample to determine the total number of
spermatozoa in the sample (ie, in the insemination dose).
DK Vanderwall  Vol 28, No 4 (2008)
In summary, although spectrophotometric methods of
counting spermatozoa are rapid and easy to use, their inabil-
ity to accurately count extended or contaminated semen
samples may necessitate the use of direct counting of sper-
matozoa with a hemacytometer; therefore, it is prudent
for all breeding facilities to maintain the supplies and equip-
ment needed to count spermatozoa with a hemacytometer
and be familiar with their use as described in this article.
REFERENCES
1. Rigby SL, Varner DD, Thompson JA, Love CC, Brinsko SP,
Blanchard TL. Measurement of sperm concentration in stallion ejacu-
lates using photometric or direct sperm enumeration techniques. Pro-
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Practitioners 2001;47:236–238.
2. Macpherson ML. How to evaluate semen in the field. Proceedings An-
nual Convention American Association of Equine Practitioners 2001;
47:412–416.
3. Varner DD, Schumacher J, Blanchard TL, Johnson L. Breeding
soundness examination. In: Pratt PW, ed. Diseases and management
of breeding stallions. Goleta, CA: American Veterinary Publications;
1991:61–96.