Tests on the
semen system
|
||
Inspections: |
Use of a Colorimeter
Colorimeters are only of
serious use on studs of more than 10 boars, below this it is better to just to
collect from additional boars.
This equates to serving up to
80 sows a week, this is for more than a 1800 sow farm
weaning weekly
Note even with the best
colorimeters, spectomphotometers and haemocytomer checking they are only ±15% accurate. The major reason is variation in semen fluid
opacity
1 |
Switch on and allow a minimum of
15 minutes warm-up time before taking readings. |
2 |
Fill a clean colorimeter tube
with 9.9 ml of 2.9% sodium citrate
buffer solution (prepared by dissolving
36.0 g sodium citrate in pure water to volume of 1000 ml) for each boar being
collected. Wipe the outside of the
tube and place in the colorimeter, ensuring the mark on the tube is aligned
with the mark on the tube holder. |
3 |
A dirty colorimeter tube or liquid
on the outside of the tube will affect the passage of light and result in an
inaccurate reading. |
4 |
Check the tube containing the
blank solution is correctly aligned, set 'course' switch to left hand
position and set the pointer to zero using the 'fine' control. |
5 |
In order to obtain accurate
readings on the left hand side of the meter scale it is necessary to dilute
samples of boar semen in sodium citrate buffer solution at the rate of 1:100
(semen:buffer).
Ensure that the ejaculate is
well mixed. Remove a 0.1 ml sample of
raw semen from the collection bottle using a 0.1 ml automatic pipetter and disposable tip. Wipe off excess semen from the tip of the
pipette and run into the buffer solution.
Fill and rinse the pipette tip once with solution from the tube,
remembering to blow out the last drop (eject to second position on the
pipette). |
6 |
Remove and invert the tube
containing the semen sample twice and wipe the outside with a clean
tissue. Place sample tube in
colorimeter ensuring it is correctly aligned and note the reading. |
7 |
Re-check the zero with the blank
and repeat procedure with the sample tube to confirm reading (within one
point on meter scale). |
8 |
When certain of the reading, take
the ready-reckoner chart and read off the sperm
concentration, semen volume per bottle and dilution rate. These figures should be recorded on the
individual boar data card, together with motility and ejaculate volume. The number of bottles can then be calculated
at the required dose rate. |
9 |
Ensure tubes are put into
soak/wash immediately after use. When
finished with the colorimeter switch off at mains and cover with polythene
cover. |
10 |
Should the colorimeter appear to
be faulty or the readings erratic, consult the maintenance and
trouble-shooting section in the instrument instruction manual. |
11 |
Theoretically the calibration
curve will remain constant for a given instrument and set of conditions. However, it is advisable to confirm the validity
of the calibration at regular intervals (every 6 months) and whenever the
bulb is replaced. |
12 |
Calculate the amount of diluent
required (i.e. number of bottles x 75 ml minus semen volume) using the ready reckoner and write this volume together with the dose
rate and number of bottles on the collection bottle label. |
Colorimeter Calibration
Introduction |
|
|
This
instrument contains a photoelectric cell which reacts to light passing through
the glass colorimeter tubes filled with diluted semen samples of different
optical densities, shown by the deflection of the pointer across the
logarithmic meter scale. The optical
density of the semen sample is proportional to the concentration of sperm
present. Once accurately calibrated,
the colorimeter provides a rapid method of determining semen
concentration. The machine should be
re-calibrated every 6 months. |
Calibration
procedure |
|
1 |
Obtain
50 ml of raw semen and allow it to settle out for 4 hours. Gently pour off the clear fluid. |
2 |
Pour
1 ml of concentrated semen into a colorimeter tube, number tube 1. |
3 |
To 1 ml
of concentrated semen add 1 ml of standard diluent and gently mix well,
number the tube 2. |
4 |
Remove
0.5 ml from tube 2 and add to colorimeter tube 3. |
5 |
Add
0.5 ml of diluent to tube 3 and gently mix well. |
6 |
Repeat
steps 2 to 5 until you have 8 serial dilution's of
the concentrated semen sample. |
7 |
Record
the colorimeter reading at each of the dilution's. |
8 |
Determine
the semen concentrations at each of the dilutions with a haemocytometer
(described below). |
9 |
Draw
a best-fit (regression) line on graph paper and construct a ready reckoner chart for the lab giving the colorimeter reading
at regular intervals and the corresponding sperm concentration x 106/ml plot the maximum and minimum dilution rates per 100 ml
of raw semen. |
The
Haemocytometer |
|
The
procedure for using the improved Neubauer double
counting chamber model (BS478) is as follows: |
|
1 |
Make
a 1:100 dilution of the semen sample by accurately pipetting
0.1 ml of semen sample into 9.9 ml of 3.6% sodium citrate buffer solution (in
a glass colorimeter tube for convenience when calibrating colorimeter). |
2 |
Add
one drop of formalin to the tube to immobilise the
spermatozoa. Note COSHH requirements of
formalin |
3 |
Clean
the glass haemocytometer and special coverslip
thoroughly with a soft tissue. Press
the coverslip onto the slide so that |
4 |
Ensure
that the diluted semen sample is thoroughly mixed. A drop is then expelled gently into the
chamber from a fine pipette so that the entire cavity is filled with diluted
semen. The process is repeated for the
second counting chamber. Excess semen
should not be allowed to flow into the grooves bounding the two chambers. |
Loading the haemocytometer |
|
|
The
counting chamber is a flat rectangular, glass block with a central recess,
i.e. an area slightly lower than those on either side from which it is separated
by grooves. When in use, this recess
is bridged over by cover glass. On the
recessed area are engraved fine rulings to mark out the small areas in which
the sperm cells are to be counted. It
is usual for two identical ruled areas to be engraved on the same counting
chamber, separated by a longitudinal groove.
|
1. |
Upper
surface, showing the cover glass in position on the ruled areas |
2. |
Method
of filling the counter chamber with a Pasteur pipette. The tip of the pipette (held at 45o
to the vertical) is applied to the gap between the cover glass and the
underlying recessed area, and a small amount of semen is then discharged. |
|
|
Counting the
sperm numbers |
|
|
Haemocytometer chamber viewed under the
microscope |
1 |
Allow
the cells to settle for 5 minutes then examine the slide with the microscope
(x100). Locate the block of 25 squares
and count the number of spermatozoa in 5 of these large squares, e.g. one at
each corner of the block and the centre one.
Each large square is divided into 16 smaller squares. Count the heads of the spermatozoa using a
hand tally counter. Some of the cells
will lie across the lines at the edges of the square; to avoid counting the same cell twice,
count any spermatozoa on the top and right lines and ignore those on bottom
and left lines. Repeat the count on
the second chamber and use the mean of the two counts for the calculation of
the concentration. One large square of
the counting grid of a haemocytometer chamber is shown below. The arrow indicates the direction to follow
when counting spermatozoa in each of the 16 small squares; only those heads shown in black would be
counted in square 1. |
2 |
Calculation
of the number of spermatozoa per ml; - where 5 large squares (each divided
into 16 small squares) have been counted.
One
small square has an area of 1/400 sq mm and a depth of 0.1 mm. Thus
the volume of one small square is 1/4000 cu mm. Where
N is the mean count from the two chambers. |
|
Sperm
concentration = (N/80) x 4000 x 1000 x 100 (dil
rate) i.e,(original semen) = (per small sq) x
(per cu mm) x (per ml) x (per ml original). = 5N x
106 sperm per ml. |
3 |
For greater
accuracy, count the sperm in all 25 large squares. The formula will then be: Sperm
concentration = N x 106 sperm per ml. |