Name: a swimming-induced zebrafish exercise apparatus for versatile training approaches
Uploaded: 2024-10-18T12:50:00
Description: To comprehensively investigate the effects of exercise on health and disease, animal models play a pivotal role. Zebrafish, a widely utilized vertebrate ...

Gratitude is extended to Dr. Omar Mertins for generously providing access to the laboratory for the maintenance of fish and execution of tests. Furthermore, acknowledgments are given to FAPESP (process numbers 2020/12084-8 and 2021/14313-7), CNPq (process number 151756/2022-8), and CAPES for awarding fellowships to support this research.

The procedures received prior approval from the Animal Use Ethics Committee of the Federal University of S&#227;o Paulo (CEUA/UNIFESP no. 9206260521). Only adult female wild-type Danio rerio, aged 6 months and weighing 2.5-3 g, were employed in this study. The equipment and reagents needed for the study are listed in the Table of Materials.
1. Custom-made Zebrafish exercise apparatus
NOTE: The exercise apparatus was custom-bu...

<ol>
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In this study, an innovative, cost-effective exercise system was developed inspired by the swim tunnel respirometer from Loligo Systems21 and the flume system22 for the comprehensive examination of zebrafish swimming performance. The Umax was determined by systematically increasing water flow in discrete stages, with speed increments occurring over short intervals (20-30 min) until the fish reached exhaustion, which was characterized by three consecutive fatigues or the ina...

Physical exercise encompasses any bodily movement performed by skeletal muscles that result in increased energy expenditure, with exercise being a structured and repetitive subset of physical activities1. Exercise, a multifactorial and cost-effective activity involving the entire body, yields numerous health benefits, such as preventing metabolic syndrome and sarcopenia2. Consequently, the field of exercise physiology holds significant interest as it seeks to elucidate how the body adapts to the acute stress of exercise, the chronic stress of physical training, and the overall impact of exercise on health1.
Conducting exercise physiology studies in humans can be both expensive and time-consuming due to challenges in experimental design and participant monitoring3. Therefore, the use of animal models in laboratory settings has been highly recommended due to their genetic and physiological uniformity. Moreover, under controlled laboratory conditions, animals typically have sedentary lifestyles and regulated food intake4. Among animal models, rodents have been the most widely employed in research involving physical exercise1. However, zebrafish (Danio rerio; Hamilton, 1822) is a complementary model to murine and other species for exercise studies5,6,7,8.
In zebrafish research, physical exercise can be conducted using commercially available or custom-built swim tunnels. Among the commercially available options, the Blazka-type tunnel, developed by the Loligo System, is the most frequently utilized7,9,10. This system induces forced swimming through a propeller coupled to an electric motor, generating a continuous water flow within the tunnel. This swimming capability is rooted in the principle of rheotaxis, an innate behavior in fish that drives them to swim against water currents and maintain their position11. Rheotaxis enables the measurement of critical swimming speed (Ucrit), representing the maximum velocity a fish can sustain for a specific duration. However, it is worth noting that this equipment, while valuable for assessing swimming behavior and oxygen consumption, comes with a significant cost12.
Researchers have developed alternative apparatus for exercising zebrafish, often based on the Blazka-type mechanism10,13,14 or simpler mechanisms8,15,16. Nonetheless, these methods may be constrained by the technical demands of the protocol, including extended durations, substantial equipment expenses, and limitations in throughput and precision. Consequently, the primary objective of the study was to design an affordable and user-friendly zebrafish exercise system using readily available materials, providing a new alternative apparatus for physical exercise in fish. A secondary goal was to implement both aerobic and anaerobic exercise regimes in zebrafish, further advancing the utilization of the zebrafish model as an intervention strategy in exercise research.

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			<td>BXV</td>
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			<td>10 k&#937;, 0.25W t</td>
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			<td>Lab Support Stand With Clamp with 30 inch rod&#160;</td>
			<td>Masiye Labs</td>
			<td>RSC0001</td>
			<td>Support the horizontal pipes</td>
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			<td>LCD screen&#160;</td>
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			<td>Perforated Circuit Board single sided</td>
			<td>KY WIN ROBOT</td>
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			<td>5 x 10 cm</td>
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			<td>Potentiometer</td>
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			<td>DL-ALPSA01</td>
			<td>10k&#937;</td>
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			<td>One World</td>
			<td>5603131000</td>
			<td>To avoid leaks</td>
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			<td>Silicone hose</td>
			<td>Tigre</td>
			<td>14211250</td>
			<td>2 cm inner</td>
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			<td>QHTITEC</td>
			<td>EU/US PLUG</td>
			<td>Arduine system welding&#160;</td>
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			<td>Solder Wire Spool</td>
			<td>BEEYIHF</td>
			<td>I001-A001-Set</td>
			<td>Arduine system welding&#160;</td>
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			<td>Threaded Male Socket and Unthreaded Female Socket CPVC Pipe Fitting</td>
			<td>TIgre</td>
			<td>35447849</td>
			<td>3/4-inch&#160;</td>
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			<td>Tricaine (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Anesthetic</td>
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			<td>UNO-R3 board UNO R3 CH340G+MEGA328P Chip 16Mhz&#160;</td>
			<td>FSXSEMI</td>
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			<td>For Arduino UNO R3 Development board</td>
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			<td>Unthreaded CPVC Tee Pipe Fitting, Female</td>
			<td>Tigre</td>
			<td>22200267</td>
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			<td>Unthreaded Female CPVC Socket Pipe Fitting</td>
			<td>Tigre</td>
			<td>22170260</td>
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			<td>Water Flow Sensor&#160; model YF-B5&#160;</td>
			<td>Siqma Robotics</td>
			<td>SQ8659</td>
			<td>1-25 L/min</td>
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			<td>Water Pump&#160;</td>
			<td>Sunsun</td>
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			<td>Model HJ-2041, 3000L/h, 65W</td>
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			<td>Water reservoir</td>
			<td>Custom</td>
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a swimming-induced zebrafish exercise apparatus for versatile training approaches

To comprehensively investigate the effects of exercise on health and disease, animal models play a pivotal role. Zebrafish, a widely utilized vertebrate model organism, offers a unique platform for such studies. This study introduced the development of a cost-effective apparatus tailored for zebrafish exercise studies utilizing readily available materials. The device is founded on the principles of a swim tunnel and encompasses a network of pipes and valves linked to a submersible pump. Water flow is meticulously monitored by a sensor and regulated via valves. To assess the apparatus&#39;s effectiveness, two training protocols were implemented: moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT). Fish were trained collectively, and their swimming performance was assessed through an endurance test. Both training protocols led to improvements in swimming performance following 30 days of training and induced alterations in the molecular response to exercise compared to a sedentary control group. Notably, HIIT demonstrated superior efficiency over MICT. The zebrafish training system proved to be a valuable tool for investigations in exercise physiology and further advances the utility of the zebrafish model in this field.

The exercise apparatus demonstrated remarkable efficiency in regulating flow velocity. To gradually enhance swimming speed, water flow was incrementally increased weekly for all groups, except for the SED group, which was maintained at a constant flow velocity of 0.06 m/s. Notably, the apparatus allowed for a remarkable level of precision, achieving flow velocity adjustments as fine as 0.001 m/s. However, the error rate was 30% at low velocities, such as 0.06 m/s. At high velocities, such as 0.3 m/s and 0.5 m/s, the erro...

The exercise apparatus designed for less fortunate fish facilitates the implementation of diverse exercise protocols with varying intensities by manipulating water flow speed, achievable through rheotaxis.

A Swimming-Induced Zebrafish Exercise Apparatus for Versatile Training Approaches

To comprehensively investigate the effects of exercise on health and disease, animal models play a pivotal role. Zebrafish, a widely utilized vertebrate ...

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.

Research

JoVE Journal

Biology

Experimental Approaches to Tissue Engineering

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...