Zebrafish Rheotaxis Assay: A Method for Measuring Orientation Behavior in Response to Different Water Flow Rates

- First, carefully transfer the fish to the swimming tunnel and switch on the water pump. Let the fish acclimate for one hour to reduce its stress level. Fish tend to display positive rheotaxis or orienting upstream in flowing water. Using video analysis and a modeling software program, track the time and fish movement by marking its eye position at each upstream to downstream turn with a dot, and at each downstream to upstream turn with dot of a different color. Increase the water flow rate and repeat the trial. We can calculate a rheotactic index by comparing the time spent facing upstream to the total, thereby, quantifying the fish's orientation behavior. The example protocol is part of an experiment to separate the influences of water flow and magnetic fields on the orientation behavior of fish.

- To set up a magnetic field with one dimensional magnetic field manipulation, keep the tunnel open and switch on power unit and magnetometer. Place a magnetic probe connected to a gas Tesla meter inside the tunnel and verify the voltage necessary to obtain the chosen magnetic field value along the major axis of the tunnel. Then disconnect the probe and connect the flow tunnel to the swimming apparatus. To set up a magnetic field with three dimensional magnetic field manipulation, turn on the computer, digital to analog converter, and coil drivers and set the chosen magnetic field on the xy and z-axis, then place the tunnel in the center of the Helmholtz pairs set. And the magnetic field of choice has been set up, select one to five fish for the rheotactic test. Transfer the fish in the tunnel, set the flow rate in the tunnel to 4.5 liters per minute and turn on the pump.

- Be sure to close the outlet valve before filling the tunnel with water, and transfer the fish and open it before setting the flow rate.

- After the animals have acclimated in the swimming tunnel for an hour, start recording the behavior of the fish within the tunnel and begin the stepwise increase of the flow rate according to the chosen experimental protocol.