A Swimming-Induced Zebrafish Exercise Apparatus for Versatile Training Approaches

Summary

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.

Abstract

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'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.

Introduction

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 studies^5,6,7,8.

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 utilized^7,9,10. 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 mechanism^10,13,14 or simpler mechanisms^8,15,16. 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.

Protocol

The procedures received prior approval from the Animal Use Ethics Committee of the Federal University of São 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-built. For details, see Figure 1, Supplementary Table 1, Supplementary File 1, and Supplementary File 2.

1. Place a submersible pump (N) inside a water tank (O) (≥30 L). Ensure that the water meets the following conditions: pH of 7.2 ± 0.5 and 400 ± 50 µS, 28 ± 1 °C.
2. Flowing Supplementary Table 1 and Figure 1, connect Pipe (I) to point Tee Pipe (B), and attach a small pipe (G) to the side of B. From G, establish connections to Globe Valve (F), then to another G, and in sequence to Pipe Elbow (A) and I, thereby completing the segment responsible for regulating water pressure within the system. This regulation is achieved through a return flow to the water tank (O).
3. In the alternate section of B, link it to a Pipe (J), followed by connections to A and G. Utilize Socket Pipe Fitting (D) to connect the Gate Water Valve (E) to G.
4. Integrate a fish entry port into the system by connecting B to G at one end and attaching another G at the opposite end. Subsequently, connect Socket Pipe Fitting (C) to this second G, establishing a sequence that connects to the Acrylic Pipe (K), which is crucial for visualizing swimming behavior.
   1. To link K to the Water Flow Sensor (M), utilize pipes C, G, and D. Proceed to connect M to G using D, and then integrate A, G, and H to facilitate the water's return to the reservoir.
      NOTE: Insert a mosquito screen in the short pipe segment between the gate and globe valve (F2) to prevent fish from accessing other sections of the apparatus. The globe valve (F) serves a dual purpose. The first globe valve (F1) controls the water flow returning to the reservoir before entering the remainder of the apparatus, acting as a system pressure control valve. The globe valve (F2) is an entry and exit point for the zebrafish within the system.
5. Attach a water flow sensor downstream of the acrylic pipe.
   NOTE: The flow sensor should be connected to an LCD display and programmed using an Arduino (Figure 1). Details of the Arduino setup are provided in Supplementary File 2.

2. Apparatus operation

1. To safely introduce the fish into the system, it is essential to interrupt the water flow. To achieve this, close the Gate Valve (E) while keeping the Globe Valve (F1) open. Subsequently, open the Globe Valve (F2), which serves as the entrance to the system, gently introduce the fish, and promptly close the F2 valve. Finally, open the E valve to fill the exercise area with water.
2. Use the globe valve to control flow speed, diverting water back to the reservoir when needed.
3. Utilize the gate valve (F2) for precise flow adjustment and to manage fish access.
4. To remove the fish at the end of the test, close valve (E) upon observing exhaustion criteria. Then, open valve F and rotate it 180° relative to the axis of the acrylic tube; this will facilitate the drainage of water carrying the exhausted fish along with it.
5. Perform the flow monitoring.
   NOTE: It is necessary to monitor the water flow speed through a system that incorporates an Arduino Nano, a 16 x 2 LCD screen, a 10 kΩ, 0.25 W through-hole resistor, and a 10 kΩ potentiometer. The flow sensor continuously monitors water flow speed based on Hall Effect technology¹⁷. Each pulse of current corresponds to one revolution of the sensor flopper, resulting in a frequency (Hz) of 6.6 x Q (flow rate in L/min).
   1. Connect the appropriate wires of the flow sensor to the 5 V, GND, and D2 pins of the Arduino Nano (Supplementary Table 1). Load the provided sketch (Supplemental File 1) into the Arduino using the Arduino IDE. Power the system through the Arduino USB port.
      NOTE: The flow measurements are displayed on the 16 x 2 LCD screen. The calibration of the water flow sensor is depicted in Figure 2. The schematics of the Arduino connections to the LCD are illustrated in Figure 3.

3. Endurance test

NOTE: This step outlines the procedure for the Endurance test to determine the maximum swimming speed (Umax) of zebrafish.

1. First, allow the fish to adapt for 60 min per day to a low water flow speed (0.06 m/s) inside the swimming tunnel for two weeks.
   NOTE: After a 24 h preconditioning period, individual zebrafish will undergo the sustained swimming performance test. The purpose of this test is to establish the Umax of each fish.
2. Place the zebrafish individually in the apparatus.
3. Testing conditions: Position the fish against a water flow with an initial speed of 0.06 m/s for 10 min.
4. Velocity Increments: Increase the water flow in discrete stages, with velocity increments of 0.02 m/s occurring every minute for 40-50 min.
5. Umax determination: Recpod the maximum swimming speed (Umax) when the fish meet the exhaustion criteria.
   NOTE: Exhaustion is defined when the first of the following situations is observed: (1) Inability to maintain its position against the water flow for more than three instances, or (2) Inability to sustain its position for longer than 5 s.
6. Close the valve (E) upon observing exhaustion criteria. Then, open valve F and rotate it 180° relative to the axis of the acrylic tube. This will facilitate the drainage of water, carrying the exhausted fish.

4. Exercise groups and procedure

NOTE: To establish distinct exercise protocols, it is essential to include a sedentary group exposed to identical experimental conditions to compare the effects of exercise protocols, albeit without undergoing high-intensity exercise. It is also essential to establish the Umax because the fractions of the Umax value are necessary to determine the intensity of exercise protocols.

1. Sedentary (SED) group: Subject the fish to forced swimming against the water flow at 0.06 m/s for 60 min.
   NOTE: The apparatus generates a continuous water flow, compelling the fish to swim against this current based on the principle of rheotaxis¹¹.
2. Moderate-Intensity Continuous Training (MICT) group: Subject the fish to forced swimming against the water flow at 60% of Umax, as determined in the maximal capacity test, for 35 min.
   NOTE: This protocol was adapted from Húngaro et al.¹⁸. During the first 10 min, the fish was acclimated to the same speed as the sedentary group (0.05 m/s).
3. High-Intensity Interval Training (HIIT) group: Subject the fish to forced swimming alternating the swimming speeds: 2 min at 90% of Umax followed by 2 min at 30% of Umax, repeated for 18 min (9 cycles). This protocol was adapted from Marcinko et al.¹⁹.
   NOTE: During the first 10 min of the exercise period, it is necessary to acclimate the fish to the same speed as the sedentary group (0.06 m/s).
4. Implement all exercise protocols for 5 days a week over a four-week period.
   NOTE: Fish should be housed in aquariums that provide suitable conditions, and they should only be introduced into the exercise apparatus during designated exercise periods. Fish should be provided with tropical fish-flaked food three times daily, and the water in maintenance aquariums should undergo a partial change every 2 days.
5. Repeat the endurance test at the end of each week, with latency and velocity data at the point of fatigue as indicators of physical conditioning parameters.
6. To induce overtraining effects, increase the water flow velocity weekly based on the results of the endurance test conducted after each 4-day cycle of training. Training durations must be adjusted to account for the distance traveled (velocity × time), and these durations should remain consistent between the exercised groups.
7. Adjust the swimming time in response to the increased water flow, thereby standardizing the training load across the exercised groups.

5. Body measurements

1. Anesthetize the fish with 0.0075% tricaine (w/v) by immersion for conducting body measurements (weight and size)²⁰.
2. Photograph and weigh the fish to determine body dimensions using ImageJ software.
3. Express the data in terms of Body Condition Indices (weight [g]/standard length [mm]2; BMI) and Body Condition Scoring (BCS)²⁰.
4. To eliminate size and weight variations caused by egg formation, subject the fish to standard breeding²⁰, followed by measurements and weighing.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
CPVC Female 90-Degree Elbow for Plumbing	Tigre	22150260	3/4-inch
24AWG Wire	Sky Cablo Store		Connection between components in the Perforated Circuit Board (1m)
Acrylic pipe	The Clear Plastic Shop	41138408	3/4-inch
Aquarium Submersible Fish Tank	Aqua Tank		300w
CPVC Pipe	Tigre	10121787	3/4-inch
Female Threaded Gate Water Valve	Tigre	27950310	3/4-inch
Female Threaded Globe Water Valve	Tigre	27940510	3/4-inch
hrough-hole resistor	BXV		10 kΩ, 0.25W t
Lab Support Stand With Clamp with 30 inch rod	Masiye Labs	RSC0001	Support the horizontal pipes
LCD screen	Eichip		16 x 2, model JHD162A
Male x Male Dupont Jumpers	Chyan		Connection between arduino and flow sensor (30 cm)
Perforated Circuit Board single sided	KY WIN ROBOT		5 x 10 cm
Potentiometer	LUSYA	DL-ALPSA01	10kΩ
Roll of Water Blocking Tape	One World	5603131000	To avoid leaks
Silicone hose	Tigre	14211250	2 cm inner
Solder Station	QHTITEC	EU/US PLUG	Arduine system welding
Solder Wire Spool	BEEYIHF	I001-A001-Set	Arduine system welding
Threaded Male Socket and Unthreaded Female Socket CPVC Pipe Fitting	TIgre	35447849	3/4-inch
Tricaine (MS-222)	Sigma-Aldrich	E10521	Anesthetic
UNO-R3 board UNO R3 CH340G+MEGA328P Chip 16Mhz	FSXSEMI		For Arduino UNO R3 Development board
Unthreaded CPVC Tee Pipe Fitting, Female	Tigre	22200267	3/4-inch
Unthreaded Female CPVC Socket Pipe Fitting	Tigre	22170260	3/4-inch
Water Flow Sensor  model YF-B5	Siqma Robotics	SQ8659	1-25 L/min
Water Pump	Sunsun		Model HJ-2041, 3000L/h, 65W
Water reservoir	Custom		30 L