For performing the myomere cryoinjury on zebrafish, use a custom manufactured stainless steel cryoprobe. To avoid frostbite while handling the probe during the procedure, insert the probe handle into a pipette tip and secure it by wrapping it with tape. Before beginning with the cryoinjury procedure, prepare a beaker for the anesthesia working solution, a spoon for handling the fish, a moist sponge, and a tank with system water for post-procedure recovery of the fish.
Prepare the anesthesia stock solution following the steps shown on the screen. Then add four milliliters of the prepared stock solution to 100 milliliters of system water in a beaker to prepare the tricaine working solution. To begin the cryoinjury procedure, immerse the probe in liquid nitrogen for a minimum of three minutes.
Wet the sponge in system water and place it on a flat surface. Then one to two minutes after transferring a single adult zebrafish into the tricaine working solution, confirm its unresponsiveness by touching the fish gently with the spoon. Place the anesthetized fish onto the wet sponge.
Locate the caudal peduncle posterior to the anal fin and anterior to the caudal fin. At this stage, remove the cryoprobe from the liquid nitrogen. Shake the probe gently to ensure no residual liquid nitrogen remains on the tip.
Place the edge of the spatula perpendicular to the body on the caudal peduncle. Keep the probe in this position for six seconds without applying pressure, before releasing the cryoprobe from the tissue. Then transfer the fish into the system water in the recovery tank.
Monitor the fish while it resumes breathing and swimming after waking up from the anesthesia. If opercular movements do not occur after 30 seconds, stimulate the fish by pipetting system water into the gills until the animal initiates respiration by itself. Video recording of control and cryoinjured fish at one day post-injury or DPCI revealed that although the cryoinjured fish swam less actively, they did not display any abnormal movements, such as swirling, convolution, or reduced equilibrium.
In the husbandry system, their position in the tank and food intake was similar to those of the uninjured fish. As observed at 5 DPCI, normal behavior was still persistent indicating the cryoinjury procedure did not severely affect the wellbeing of the animals.
