This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.


Thawing and Using Frozen Sperm for In Vitro Fertilization

(Source: C. Walker and G. Streisinger)

1. Obtain eggs as described in the section Embryo Production By In Vitro Fertilization.
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 min, then add 0.75 ml fish water and let it sit another 2 min.
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:

[10 ul (capillary vol.) x mm sm] / 90 mm (capillary length) = vol (ul)

c. Multiply the calculated volume x 10 and use that much 100% Hank's saline for thawing.

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