Page 553 - Equine Clinical Medicine, Surgery and Reproduction, 2nd Edition

528 CHAPTER 2

VetBooks.ir Total and progressive motility Sperm concentration
Sperm concentration of the non-gel fraction can
A good quality microscope with phase contrast optics
and a heated stage greatly improves the quality and
simeter. Densimeters measure the amount of light
repeatability of semen evaluations. Total and progres- be determined using a haemocytometer or a den-
sive motility can be evaluated, prior to dilution with transmitted through a sample (photometry) and
extender, by placing a small drop of raw semen on a correlate this with the sperm concentration of the
pre-warmed (37°C [98.6°F]) microscope slide with a sample. Examples of commercially available densim-
coverslip. Several fields are evaluated with a magni- eters include the SpermaCue (Minitube of America,
fication of ×200 to ×40 with phase contrast, and the Wisconsin, USA) and the ARS System (Animal
average motility of the fields recorded. Computer- Reproduction Systems, Chino, USA). Photometric
assisted sperm motion analysis systems are available instruments must be calibrated properly and are
that reduce the subjective nature of motility analy- less accurate at extremely high or low concentra-
sis (Fig. 2.136); however, their considerable expense tions. Contaminated samples will give spurious
makes them of little value in the field situation. results. Photometric instruments rely on the den-
The motility evaluation is repeated with semen sity of the sample and therefore cannot be used to
extended in a 1:1 ratio or to a concentration of 30–50 determine the concentration of sperm in frozen-
million sperm cells per ml in order to permit evalu- thawed samples, due to egg-yolk and other extender
ation of the motion characteristics of individual components. An automated cytometry method, the
sperm. Total motility is defined as the percentage of Nucleocounter SP-100 (CHemoMetec A/S, Allerod,
sperm cells moving, in any manner, in the sample. Denmark) is available for rapid determination of
Progressive motility is defined as the percentage of equine sperm cell concentration in both fresh and
sperm cells moving forward in a progressive manner frozen-thawed semen samples. The Nucleocounter
in the sample. An additional description of sperm SP-100 uses fluorescent staining of the cells and does
velocity or vigour may also be given. One example not rely on the density of the sample.
is a scale of 0 to 4, with 0 being non-motile, 1 being Originally designed for counting blood cells, the
very sluggish motility, and a score of 4 describing haemocytometer remains a useful method for deter-
fast or highly vigorous motility. Retrograde or a mination of sperm concentration despite the unavail-
tightly circular motion is abnormal. ability of the Unopette diluting system. To use this
simple and inexpensive method, 20 µl of raw semen
are diluted with 1.98 ml of formal saline (or 10%

2.136 buffered formalin; sodium citrate can also be used).
The diluted sample (a 1:100 dilution) is mixed well,
and then a small volume (10–20 µl) is drawn into a
pipette. This sample is loaded by capillary action
onto both sides of the haemocytometer chamber
with a coverglass already in place, being careful not
to overfill it. After allowing the sample to settle for
a short time, the haemocytometer is viewed under
the microscope at ×20, and the central 5 × 5 grid
located. The number of sperm within all 25 squares
within the central large grid are counted. The count
is repeated on the other side, and the average of the
two counts is calculated. This is the number of sperm
Fig. 2.136 Total and progressive motility can be in 0.1 µl of the sample, since this is the volume of the
evaluated by computer-assisted sperm motion analysis haemocytometer chamber. Taking into account the
systems, which reduce the subjective nature of dilution of 1:100, this means that the concentration
motility analysis.