Principles of Genetics and Breeding

Counting of fish sperm and checking the motility

Sperm count and motility are the most commonly used parameters to evaluate sperm quality, since sperm count must be more and motile to achieve fertilization. Unlike mammal, the teleost spermatozoa are found immotile in the testis as well as in seminal fluid. Healthy fish spermatozoa on activation shows a vigorous movement. The motility of such activated spermatozoa is evaluated by six point (++++++) scale after adding 100 times of water to a drop of milt.

Motility percentage Score Condition

0 --10 + All dead

11--30 ++ Slightly active

31--50 +++ Oscillating

51--70 ++++ Moving

71--90 +++++ Active

91--100 ++++++ Excellent

Procedure

1. Collect spermatozoa directly from the testis (for catfish and murrels). Otherwise strip the milt by pressing the abdomen gently (for carps).

2. Take 10 μl of sperm sample and add 490 μl of diluent (HBSS) in a microcentrifuge.

3. Draw 10 μl of the above diluted sample and add 90 μl of diluent (1:50 and 1: 10 dilution).

4. Transfer a drop of the milt content to a clean haemocytometer. Each square is divided into16 smaller cells (4x4). Each of these smaller cells has a volume of 1/4000 mm ³

5. Count the sperm heads in each small cells from the 5 squares marked “x”. It gives a total of 80 cells (4x4x5). This is the number of sperm per 1/4000 mm ³

6. Calculate the concentration of sperm as given below:

Con. (sperm cells /ml) = Average mean x 4000 x 1000 x dilution factor

7. Check the motility under a microscope after adding a drop of tap water