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1. Gently add 5 microliters of thawed sperm to each egg. Do this by softly touching the tip of the pipette to the jelly without piercing the jelly or the egg. If there is not enough sperm to fertilize all eggs, remove those that aren’t fertilized.
2. Allow sperm and eggs to sit exposed to air for 10 minutes
3. Fill fertilization glass container with 50% Axolotl rearing water (ARW) diluted 1:10 with DI water so that the eggs are fully submerged. Pour the ARW off to the side of the eggs slowly as opposed to directly on top.
4. Change 50% ARW daily.
5. Assess embryo development after 24 and 72 hrs, and after hatching.

Egg collection and fertilization

https://youtu.be/AUGr0hfTF3g

Recipe for 50% Axolotl rearing water (ARW):

• Deionized (DI) water
• NaCl (0.69 g/L)
• CaCl2.2H2O (0.015 g/L)
• KCl (0.02 g/L)
• MgSO4.7H2O (0.08 g/L)
• NaHCO3 (0.04 g/L)

1. Combine ingredients one at a time and increase to the final volume with DI water.

• It may help to mix a stock solution (5-10X concentration) and dilute accordingly with DI water.