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This works best with two people. One person squeezes eggs from females while the other person thaws the sperm.
1. Set a water bath to 33ºC.
2. Remove cryovial from liquid nitrogen freezer and transfer to liquid nitrogen-containing Dewar flask until ready to thaw.
3. Squeeze eggs from females into 35 mm plastic Petri dish. If possible, try to obtain 3 clutches of eggs and combine into one dish. If you cannot get three good clutches within 1 min of your first clutch, then proceed to step 4 (one good clutch is sufficient). Definition of good clutch: >150 uniformly nice looking eggs (i.e. yellowish, with no white debris indicative of degradation). Keep dish covered while sperm is thawed.
4. Remove vial from liquid nitrogen and remove cap. Quickly immerse vial ~1/2 way into 33ºC water bath for 8-10 sec.
5. Quickly add 70 µl room temperature Hanks to vial and mix by pipetting up and down. Immediately add to eggs and mix gently with pipette tip.
6. Without delay, activate sperm and eggs by adding 750 µl fish water. Swirl to mix. Incubate 5 min at room temperature.
7. After 5 min, fill dish with fish water and incubate dish at 28ºC. After 2-3 hrs, count and transfer fertile embryos to 100 mm dishes (50/dish). Count infertile eggs so that fertilization frequency can be determined.
8. Take good care of the larvae! For the first 5 days, change water daily and remove dead embryos. Put only the larvae that have swim bladders into nursery on day 5.
References
Ginsburg, A. S. (1963) Sperm-egg association and its relationship to activation of the egg in salmonid fishes. J. Embryol. Exp. Morphol. 11: 13-33.
Harvey, B., R. N. Kelley and M. J. Ashwood-Smith (1982) Cryopreservation of zebra fish spermatozoa using methanol. Can. J. Zoology. 60: 1867-1870.