# 5.4.3. Sperm count protocol (unstained sperm)

This protocol is covered in some detail in
Human *et al.,* 2013. Despite that, we
include an expanded version of the technique here as we list details specific
to counting sperm.

- Dissect out the spermatheca of a mated queen or obtain the sperm sample from a desired drone (queen: section 5.4.1., drone: section 5.4.2.). *NOTE: Always use glassware when dealing with sperm, because they can stick to plastic.
- Add 10.0 ml of diluent or water to a glass petri dish. *NOTE: water causes live sperm to contract and contort their shape, so only use water with frozen or dead sperm.
- Break open the spermatheca using forceps, taking care to remove all sperm, and remove the remaining tissue.
- Mix well with a clean glass pipette about 40 times until all sperm are dispersed. Use caution to prevent air bubbles or excess splattering.
- Immediately add a drop of the diluent/sperm mixture to both sides of a haemocytometer on which a cover slip has been placed. Capillary action will fill each chamber (the area between the cover slip and slide) with solution. View under 250× magnification. Start counts on the gridded section after the sperm have settled (~ 20 seconds).
- The sperm appear headless, translucent, and filamentous. They are usually about 0.25 mm long, but are often coiled or looped. It is best to bring the focus slightly upwards from the grid and to keep the light somewhat dim in order to best see them. Rapidly change the fine-tuning focus on the microscope to observe those that are not laying on the bottom of the haemocytometer.
- Count the number
of sperm in five of the large 1.00 mm
^{2}squares in the grid, preferably the large squares in the four corners and the one in the centre (5 bold, black squares seen in Fig. 26). The centre square will contain a smaller grid (used to count red blood cells at a greater magnification), while the remaining squares will be divided into 16 squares (4 × 4 grids). *NOTE: There are different types of haemocytometers so it is important to follow manufacturer’s directions when using one as calculations and haemocytometer volume may differ between types. Our calculations are done using a Bright-Line haemocytometer (Hausser Scientific). - Since sperm often
overlap the boundaries of the squares, only count those sperm which are
entirely within a square or are
*only*on the top and left boundaries (or bottom and right, if you prefer). This procedure will prevent double counting of sperm and give a more accurate count.

Calculate the total number of sperm in 5 large squares in *both* chambers of the haemocytometer.
Divide the total number of sperm in the 10 large squares (5 large squares per 2
chambers) by 100 to estimate the number of millions of sperm. (The volume of
each large square is 1.0 mm × 1.0 mm × 0.1 mm = 100 nl, making the total volume
counted for ten squares = 1000 nl, Table 5. Thus the number of sperm, for an
initial dilution of 10.0 ml, is to the order of 10^{4} which is
equivalent to 10^{-2} million).

Alternative method for counting sperm:

In this method, the sperm are induced to coil in the solution, which facilitate their counting (Woyke 1979).

- Add a drop of saline solution into a small porcelain evaporator, preferably one having blue bottom.
- Break open the spermatheca, remove all sperm, and remove the remaining tissue.
- Stir with the dissecting needle and add more solution up to a total of 1 ml.
- Add 9 ml of tap water, which will result in a total of 10 mL of solution, and mix well. The tap water causes the sperm to coil.
- Mix well with a clean glass pipette until all sperm are dispersed. Use caution to prevent air bubbles or excess splattering.
- The Fuchs-Rosenthal count chamber is used to count the sperm (Fig. 27). There are two counting grids and the depth of the counting chamber is 0.2 mm.
- Add a drop of the diluent with sperm on both grids and cover with a cover slip.
- Wait for the sperm to settle.
- Use 250× magnification for counting the sperm.
- Count the number
of sperm in 5 large 1 mm
^{2}squares on both grids (totalling 10 squares). The sperm will be easy to count because they will be coiled. Some sperm will be located over the square boundary. To avoid double counting, count only those that are over the top and left boundaries. - Calculate the
total number of sperm as follows: the total volume of the solution over the 10
large squares (5 squares in both grids) in which the sperm are counted is: 1 ×
1 × 0.2 × 10 = 2 mm
^{3}. However, the total volume of the solution in which the sperm were dispersed is 10 cm^{3}= 10,000 mm^{3}. Thus, the volume of the dispersion solution is 10,000:2 = 5,000 times higher. To get the total number of spermatozoa, multiply the number of spermatozoa counted over the 10 large squares by 5,000.

** Fig. 26.** Haemocytometer grid. Red square (and each of the 5
large squares with bold, black lines added) = 1 mm

^{2}(100.00 nl); green square = 0.0625 mm

^{2 }(6.250 nl); yellow square = 0.040 mm

^{2}(4.00 nl); blue square = 0.0025 mm

^{2}(0.25 nl). All squares are at a depth of 0.1 mm. For area and volume calculations per certain grid dimensions on the haemocytometer, see Table 5. Information is for Bright-Line Haemocytometer (Hausser Scientific). Figure from Wikipedia.

** Fig. 27.** Diagram of a Fuchs-Rosenthal grid that can be used for counting sperm.