(Source: C. Walker and G. Streisinger from Zebrafish Book 5th Edition)
1. Obtain eggs as described in the section Embryo Production By In Vitro Fertilization (see page 2.14).
2. Place a 70 μl droplet of ice-cold 100% Hank's saline next to the eggs on the Petri dish.
3. Remove the capillary from the liquid nitrogen and thaw for a few seconds in air.
4. Expel the sperm into the droplet of 100% Hank's saline with gentle pressure, mix gently, and then mix with the eggs.
5. Let sit 0.5-1 minute, then add 0.75 ml fish water and let it sit another 2 minutes.
6. Add more water to bring the solution level up to about half the volume of the Petri dish.
The above recipe assumes an average of 10-11 mm of sperm. To calculate the amount of thawing medium needed for different amounts of sperm:
Sperm Thawing Medium:
a. Measure the mm of sperm + freezing medium (sm) in the capillary.
b. Convert to volume:
10μl (cap. vol.) x mm sm = vol. (μl) 90 mm (cap. length) 1
c. Multiply the calculated volume x 10 and use that much 100% Hank's saline for thawing.