In zebrafish research, physical exercise can be conducted using commercially-available or custom-built swim tunnels. Here, we developed a high-throughput, user-friendly exercise system for zebrafish, using materials that are easily available. The setup is based on the real access behavior of zebrafish.
To test the efficiency of the apparatus, two exercise protocols were employed. Moderate-intensity continuous training, and high-intensity interval training. To monitor the water flow speed indicated by the flow sensor, we employ an Arduino Nano, a 16-by-2 LCD screen, a 10-kiloohm one-quarter watt through-hole resistor, and a 10-kiloohm potentiometer.
Assemble the components following the provided scheme. Using a perf board, solder wire, solder iron, wire, and three male jumpers. Load the provided Arduino sketch using an appropriate USB cable via the Arduino IDE.
Water flow rate, time since the last reset in minutes, and velocity of water are displayed on the LCD screen. To assemble the water recirculation system, use three-quarter inch PVC pipes and three-quarter inch connectors. It is necessary to create two water outlets.
The first will lead water from the main flow back to the reservoir, and the second will direct the flow to the fish training area. Add a global valve to regulate the return flow. Add an upper water outlet valve to regulate the water flow to the training area.
Attach a T-connection to the upper valve. Note that it is necessary to add a net inside the connection near the valve to prevent fish from entering the bottom part of the apparatus. In the T-connection, add a global valve, which will serve as a gate for the entry and exit of fish from the system.
Attach the acrylic pipe to the T-connector. Next, attach the flow sensor to the acrylic pipe. Note that it is necessary to place a screen inside the pipe to prevent fish from passing through.
Connect the partially-assembled apparatus to the submersible pump inside the water reservoir. To maintain water recirculation, it is necessary to connect the hose to the final portion of the apparatus after the flow sensor. Finally, connect the flow sensor to the monitor for monitoring water flow velocity.
Note that these are connectors that must be connected in the correct order. Now it is ready to use. Before adding the zebrafish to the apparatus, it is necessary to set the flow.
To do this, first open both flow control valves completely to allow bubbles to exit the system, keeping the fish inlet and outlet valve closed. Next, adjust the flow to maximum speed, approximately 0.6 meters per second. This will provide greater precision in flow adjustment using the upper valve.
Capture the fish, and then close the upper valve to stop the water flow. Then fully open the next valve that will serve as the fishes'entry gate. It is important to keep the valve opening facing upwards.
Immediately afterwards, close the inlet valve and open the flow control valve. It is possible to place a small group of up to six fishes, which should be inserted all at once. To remove the fishes, completely close the upper valve, rotate the fish inlet outlet valve, turning its opening downward.
Then fully open the valve. Use a container to collect the fish, along with the water that will flow by gravity from the apparatus. Finally, close the inlet outlet valve to stop the water leakage.
Before exercise protocols are employed, it is necessary to acclimate the animals to a relatively low and constant speed for at least 60 minutes per day for one week. Before each exercise session, it is necessary to acclimate the animals for 10 minutes. After the acclimatization period, Umax needs to be determined by assessing the maximum swimming speed against the flow.
For this, the flow rate will be increased by 0.02 meters per second, every minute, until the fish reaches exhaustion. To increase the speed, simply open the upper valve gently, monitoring the speed through the LCD. Subject the fish to forced swimming against the water flow at 60%of Umax, as determined in the maximal capacity test, for 35 minutes.
Note during the first 10 minutes, the fish is acclimated to the same speed as the sedentary group, 0.06 meters per second. The fish swim against the constant water flow rate without reaching a state of physical exhaustion. Subject the fish to forced swimming, alternating between swimming speeds.
Two minutes at 90%of Umax, followed by two minutes at 30%of Umax, repeated for 18 minutes, nine cycles. The fish swim against an alternate water flow rate without reaching a state of physical exhaustion. The setup was precise, allowing fine adjustments in flow velocity.
However, the error was around 30%when the speed was low, 0.06 meters per second. When the speed was high, around 0.3 meters per second and 0.5 meters per second, the error rate was only between three and four percent. The fastest speeds reached during training was 0.4 meters per second in the sedentary group, 0.44 meters per second in the moderate training group, and 0.49 meters per second in the high-intensity group in the final endurance test.
Both training regimens were designed to cover the same distance, but the high-intensity training protocol resulted in quicker performance improvements, as evidenced by the weekly Umax increase. The fishes exposed to high-intensity training improved their performance by 10%each week, totaling around 30%overall. On the other hand, the fishes exposed to moderate training displayed a slower progress, with a noticeable 10%increase in Umax only in the third week, and no further improvement in the following week.
These results highlight how the choice of training protocols may affect zebrafish physical performance differently. In this study, we created a new cost-effective exercise system inspired by existing swim tunnel and flume systems. This system allows for a thorough examination of zebrafish swimming performance.
We determined Umax by gradually increasing water flow until the fish were exhausted, characterized by three consecutive fatigues or the inability to keep swimming against the current. These findings helped us to design two exercise protocols and confirm the effectiveness of our swim tunnel apparatus for assessing zebrafish physical performance. Additionally, this compact system is versatile, covering a wide range of water flow speeds, and making it easy to customize training protocols.
