CHAPTER 7 - GENETIC METHODS
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.
The Zebrafish Book
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