Sperm Preparation
Sperm Preparation for Insemination of Oocytes
While motility is being evaluated, other members of the lab group will need to determine the concentration of sperm in the sperm pellet and prepare a sample at the correct concentration for insemination of oocytes.
Use a hemocytometer to determine sperm concentration. Follow the instructions below but view the hemocytometer page for general instructions and associated calculations.
- Recommended dilution to count on hemocytometer - to 5 µl of the percoll separated sperm pellet add 95 µl of water (dilution factor = 20).
- Counting 5 large squares on each side of hemocytometer as explained in instructions.
- Average count = (count in 5 squares side A) * (count in 5 squares side B) / 2
- Concentration
in sperm/ml = (Average Count) * (Dilution Factor) *
(50,000)
- for ease of calculations express as 106 sperm/ml
- Make
100 µl
of semen at 25 X 106 sperm/ml from the
sperm pellet using BGM-3
medium
for the dilution.
- 100 µl = 0.1 ml
- for
the following calculations
- sperm pellet concentration = y sperm/ml
- eventual sperm pellet volume used = z ml
- sperm required = (0.1 ml) * (25 X 106 sperm/ml) = 2.5 X 106 sperm
- volume
of semen from sperm pellet = (2.5 X 106sperm)
/ (y sperm/ml)
= z ml
- note the answer is in ml, so convert it to µl by multiplying by 1000
- volume of BGM3 to add = 100 µl - z µl
- make the dilution of sperm as you have just determined and use 2 µl to inseminate the 50 µl drop of oocytes in fertlization medium. Thus there will be 1 X 106 sperm/ml in the fertilization drop.