view - Department of Reproduction, Obstetrics and Herd Health

3.2.3.3. The different chambers and loading technique
CHAPTER 3.2
The different types of counting chambers with their specific characteristics used in this study
are presented in Table 3. Disposable chambers are marked with a “d” following the depth in µm
while reusable chambers are marked with an “r”. The ISAS20r chamber was filled both with
capillarity (C) as well as using the drop filled cover slide technique (M). A detailed description of the
loading technique per chamber is presented in addendum II. Briefly, chambers filled with capillarity
(Leja10d, Leja12d, Leja20d, ISAS12d, ISAS16d, ISAS20d and ISAS20rC) were loaded by placing an
abundance of semen in the loading area. When the semen reached the opposite air valve, the excess
of semen was carefully removed by means of a Kimtech Science ® tissue (Kimberly-Clark, Surrey, UK).
Drop filled chambers (ISAS20rM, Makler10r and WHO) were filled by placing the exact volume on
the designated place, followed by immediate application of the coverslide. All chambers were left to
rest (at 37°C) prior to analysis until drifting of the cells ceased (approximately 30s).
Table 3. Characteristics of the different counting chambers used to analyze equine semen with
CASA.
Name
Chambers/
slide
Depth
(µm)
Volume/
chamber (µL)
Way of
filling
Volume
loaded (µL)
Reusable -
Disposable
Leja10d 4 10 1 capillarity 2.5 disposable
Leja12d 2 12 3 capillarity 5 disposable
Leja20d 4 20 2 capillarity 5 disposable
ISAS12d 4 12 - capillarity 5 disposable
ISAS16d 4 16 - capillarity 5 disposable
ISAS20d 4 20 - capillarity 5 disposable
ISAS20rM 2 20 - drop filled 5 reusable
ISAS20rC 2 20 - capillarity 5 reusable
Makler10r 1 10 - drop filled 5 reusable
WHO 1 20.6 - drop filled 10 reusable
3.2.3.4. Experimental design
The fifty straws were thawed individually, concentration was determined using the
NucleoCounter and the semen was diluted within the above mentioned range and loaded in a
chamber. A random rotation with the 10 viewing chambers was done to avoid time effects. After
every scan with the CASA system, the playback function was used to ensure the correct reading of
the sample. If a given sample loaded in a certain chamber yielded a concentration out of range for
that chamber, an additional dilution was prepared based on the later results to ensure a motility
analysis within the designed concentration range.
91