Tests on the semen system

 

Boar health

Ejaculation details

Routine records

Colorimeter

Semen morphology

Daily checks

Daily checks

AI storage controls

 

Inspections:

Manager

Veterinarian

 

 

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

 

Read the operating instructions that came with the colorimeter

 

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 Newton's rings are clearly visible on the contact surfaces.

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.