Thawing And Using Frozen Sperm For In-Vitro Fertilization

(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.