Name: a swimming-induced zebrafish exercise apparatus for versatile training approaches
Uploaded: 2024-10-18T12:50:00.000+00:00
Duration: 10 min 34 s
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-built. For details, see Figure 1, Supplementary Table 1, Supplementary File 1, and Supplementary File 2.
<ol>
	<li>Place a submersible pump (N) inside a water tank (O) (&#8805;30 L). Ensure that the water meets the following conditions: pH of 7.2 &#177; 0.5 and 400 &#177; 50 &#181;S, 28 &#177; 1 &#176;C.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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&#39;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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
2. Apparatus operation
<ol>
	<li>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.</li>
	<li>Use the globe valve to control flow speed, diverting water back to the reservoir when needed.</li>
	<li>Utilize the gate valve (F2) for precise flow adjustment and to manage fish access.</li>
	<li>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&#176; relative to the axis of the acrylic tube; this will facilitate the drainage of water carrying the exhausted fish along with it.</li>
	<li>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 k&#937;, 0.25 W through-hole resistor, and a 10 k&#937; 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).
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Endurance test
NOTE: This step outlines the procedure for the Endurance test to determine the maximum swimming speed (Umax) of zebrafish.
<ol>
	<li>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.</li>
	<li>Place the zebrafish individually in the apparatus.</li>
	<li>Testing conditions: Position the fish against a water flow with an initial speed of 0.06 m/s for 10 min.</li>
	<li>Velocity Increments: Increase the water flow in discrete stages, with velocity increments of 0.02 m/s occurring every minute for 40-50 min.</li>
	<li>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.</li>
	<li>Close the valve (E) upon observing exhaustion criteria. Then, open valve F and rotate it 180&#176; relative to the axis of the acrylic tube. This will facilitate the drainage of water, carrying the exhausted fish.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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 H&#250;ngaro et al.18. During the first 10 min, the fish was acclimated to the same speed as the sedentary group (0.05 m/s).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#215; time), and these durations should remain consistent between the exercised groups.</li>
	<li>Adjust the swimming time in response to the increased water flow, thereby standardizing the training load across the exercised groups.</li>
</ol>
5. Body measurements
<ol>
	<li>Anesthetize the fish with 0.0075% tricaine (w/v) by immersion for conducting body measurements (weight and size)20.</li>
	<li>Photograph and weigh the fish to determine body dimensions using ImageJ software.</li>
	<li>Express the data in terms of Body Condition Indices (weight [g]/standard length [mm]2; BMI) and Body Condition Scoring (BCS)20.</li>
	<li>To eliminate size and weight variations caused by egg formation, subject the fish to standard breeding20, followed by measurements and weighing.</li>
</ol>

<ol>
	<li>Seo, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+animal+exercise+model+for+clinical+implication.">Humanized animal exercise model for clinical implication.</a> Pflugers Arch Eur. J Physiol. 466 (9), 1673-1687 (2014).</li><li>Nyl&#233;n, E. S., Gandhi, S. M., Lakshman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiorespiratory+fitness,+physical+activity,+and+metabolic+syndrome.">Cardiorespiratory fitness, physical activity, and metabolic syndrome.</a> Cardiorespiratory Fitness in Cardiometabolic Diseases: Prev. &amp; Manag. in Clin. Pract. , 207-215 (2019).</li><li>Cholewa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+models+modeling+resistance+training:+an+update+for+basic+scientists+interested+in+study+skeletal+muscle+hypertrophy.">Basic models modeling resistance training: an update for basic scientists interested in study skeletal muscle hypertrophy.</a> J Cell Physiol. 229 (9), 1148-1156 (2014).</li><li>Martin, B., Ji, S., Maudsley, S., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Control&amp;#34;+laboratory+rodents+are+metabolically+morbid:+Why+it+matters.">34;Control&amp;#34; laboratory rodents are metabolically morbid: Why it matters.</a> Proc Natl Acad Sci USA. 107 (14), 6127-6133 (2010).</li><li>Palstra, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming-induced+exercise+promotes+hypertrophy+and+vascularization+of+fast+skeletal+muscle+fibres+and+activation+of+myogenic+and+angiogenic+transcriptional+programs+in+adult+zebrafish.">Swimming-induced exercise promotes hypertrophy and vascularization of fast skeletal muscle fibres and activation of myogenic and angiogenic transcriptional programs in adult zebrafish.</a> BMC Genomics. 15 (1), 1-20 (2014).</li><li>Blazina, A. R., Vianna, M. R., Lara, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spinning+task:+A+new+protocol+to+easily+assess+motor+coordination+and+resistance+in+zebrafish.">The spinning task: A new protocol to easily assess motor coordination and resistance in zebrafish.</a> Zebrafish. 10 (4), 480-485 (2013).</li><li>Gilbert, M. J. H., Zerulla, T. C., Tierney, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+as+a+model+for+the+study+of+aging+and+exercise:+Physical+ability+and+trainability+decrease+with+age.">Zebrafish (Danio rerio) as a model for the study of aging and exercise: Physical ability and trainability decrease with age.</a> Exp Gerontol. 50, 106-113 (2014).</li><li>Usui, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+French+press:+A+repeatable+and+high-throughput+approach+to+exercising+zebrafish+(Danio+rerio).">The French press: A repeatable and high-throughput approach to exercising zebrafish (Danio rerio).</a> Peer J. 2018 (1), 1-12 (2018).</li><li>Tierney, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+performance+assessment+in+fishes.">Swimming performance assessment in fishes.</a> J. Vis. Exp. (51), e2572 (2011).</li><li>Palstra, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+zebrafish+as+a+novel+exercise+model:+Swimming+economy,+swimming-enhanced+growth+and+muscle+growth+marker+gene+expression.">Establishing zebrafish as a novel exercise model: Swimming economy, swimming-enhanced growth and muscle growth marker gene expression.</a> PLoS One. 5 (12), e0014483 (2010).</li><li>Arnold, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheotropism+in+fishes.">Rheotropism in fishes.</a> Biol Rev Camb Philos Soc. 49 (4), 515-576 (1974).</li><li>Messerli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+mechanism+of+the+adult+zebrafish+respiratory+organ+to+endurance+training.">Adaptation mechanism of the adult zebrafish respiratory organ to endurance training.</a> PLoS One. 15 (2), 1-20 (2020).</li><li>Bek, J. W., De Clercq, A., Coucke, P. J., Willaert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ZE-tunnel:+An+affordable,+easy-to-assemble,+and+user-friendly+benchtop+zebrafish+swim+tunnel.">The ZE-tunnel: An affordable, easy-to-assemble, and user-friendly benchtop zebrafish swim tunnel.</a> Zebrafish. 18 (1), 29-41 (2021).</li><li>Lucon-Xiccato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+low-cost+swim+tunnel+for+measuring+swimming+performance+in+fish.">An automated low-cost swim tunnel for measuring swimming performance in fish.</a> Zebrafish. 18 (3), 231-234 (2021).</li><li>Blazina, A. R., Vianna, M. R., Lara, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spinning+task:+A+new+protocol+to+easily+assess+motor+coordination+and+resistance+in+zebrafish.">The spinning task: A new protocol to easily assess motor coordination and resistance in zebrafish.</a> Zebrafish. 10 (4), 480-485 (2013).</li><li>Depasquale, C., Leri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+exercise+on+anxiety-like+behavior+in+zebrafish+(Danio+rerio).">The influence of exercise on anxiety-like behavior in zebrafish (Danio rerio).</a> Behav Processes. 157, 638-644 (2018).</li><li>Karsenty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+review+of+integrated+hall+effects+in+macro-,+micro-,+nanoscales,+and+quantum+devices.">A comprehensive review of integrated hall effects in macro-, micro-, nanoscales, and quantum devices.</a> Sensors. 20 (15), 4163 (2020).</li><li>H&#250;ngaro, T. G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+exercise+exacerbates+acute+kidney+injury+induced+by+LPS+via+toll-like+receptor+4.">Physical exercise exacerbates acute kidney injury induced by LPS via toll-like receptor 4.</a> Front Physiol. 11, 1-13 (2020).</li><li>Marcinko, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+intensity+interval+training+improves+liver+and+adipose+tissue+insulin+sensitivity.">High intensity interval training improves liver and adipose tissue insulin sensitivity.</a> Mol Metab. 4 (12), 903-915 (2015).</li><li>Chen, W., Ge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonad+differentiation+and+puberty+onset+in+the+zebrafish:+Evidence+for+the+dependence+of+puberty+onset+on+body+growth+but+not+age+in+females.">Gonad differentiation and puberty onset in the zebrafish: Evidence for the dependence of puberty onset on body growth but not age in females.</a> Mol Reprod Develop. 80 (5), 384-392 (2013).</li><li>Conradsen, C., Walker, J. A., Perna, C., McGuigan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeatability+of+locomotor+performance+and+morphology-locomotor+performance+relationships.">Repeatability of locomotor performance and morphology-locomotor performance relationships.</a> J Exp Biol. 219 (18), 2888-2897 (2016).</li><li>Widrick, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+source+microcontroller+based+flume+for+evaluating+swimming+performance+of+larval,+juvenile,+and+adult+zebrafish.">An open source microcontroller based flume for evaluating swimming performance of larval, juvenile, and adult zebrafish.</a> PLoS ONE. 13 (6), 1-14 (2018).</li><li>Gilbert, M. J. H., Zerulla, T. C., Tierney, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+(Danio+rerio)+as+a+model+for+the+study+of+aging+and+exercise:+Physical+ability+and+trainability+decrease+with+age.">Zebrafish (Danio rerio) as a model for the study of aging and exercise: Physical ability and trainability decrease with age.</a> Exp Gerontol. 50 (1), 106-113 (2013).</li><li>Hammer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatigue+and+exercise+tests+with+fish.">Fatigue and exercise tests with fish.</a> Exp Gerontol. 112 (1), 1-20 (1995).</li><li>Takahiro Hasumura, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise+quantity-dependent+muscle+hypertrophy+in+adult+zebrafish+(Danio+rerio).">Exercise quantity-dependent muscle hypertrophy in adult zebrafish (Danio rerio).</a> J Comp Physiol B. 186, 603-614 (2016).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aerobic+exercise+as+a+treatment+on+type+2+diabetes+mellitus+with+depression-like+behavior+zebrafish.">Effect of aerobic exercise as a treatment on type 2 diabetes mellitus with depression-like behavior zebrafish.</a> Life Sciences. 300, 120578 (2022).</li><li>Conradsen, C., McGuigan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexually+dimorphic+morphology+and+swimming+performance+relationships+in+wild-type+zebrafish+Danio+rerio.">Sexually dimorphic morphology and swimming performance relationships in wild-type zebrafish Danio rerio.</a> J Fish Bio. 87 (5), 1219-1233 (2015).</li><li>Hughes, D. C., Ellefsen, S., Baar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptations+to+endurance+and+strength+training.">Adaptations to endurance and strength training.</a> Cold Spring Harb Perspect Med. 8 (6), 1-18 (2018).</li></ol>

It is essential to clarify that there are no competing financial interests associated with the research presented in this manuscript. No financial partnerships or affiliations with organizations or entities have been established that could potentially influence or bias the outcomes of this work. This statement serves as an assurance that the research process was straightforward and honest, with no financial conflicts influencing the results. The presentation of this work is motivated by a genuine passion for the subject matter, driven solely by a love for academia and the pursuit of scientific knowledge.

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.

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

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 ...

a-swimming-induced-zebrafish-exercise-apparatus-for-versatile

Research

JoVE Journal

Biology

1.1K Views.  Federal University of São Paulo. 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.

Video: A Swimming-Induced Zebrafish Exercise Apparatus for Versatile Training Approaches

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Developmental Biology

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

Behavior

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Neuroscience

Zebrafish Maintenance and Husbandry

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to their small size, large numbers of zebrafish can be housed in the laboratory at low cost. Although zebrafish are relatively easy to maintain, special consideration must be given to both diet and water quality to in order to optimize fish health and reproductive success.
This video will provide an overview of zebrafish husbandry and maintenance in the lab. After a brief review of the natural zebrafish habitat, techniques essential to recreating this environment in the lab will be discussed, including key elements of fish facility water recirculation systems and the preparation of brine shrimp as part of the zebrafish diet. Additionally, the presentation will include information on how specific zebrafish strains are tracked in a laboratory setting, with specific reference to the collection of tail fin samples for DNA extraction and genotyping. Finally, experimental modifications of the zebrafish environment will be discussed as a means to further our understanding of these fish, and in turn, ourselves.

Maintenance and Husbandry of Danio rerio Zebrafish

The zebrafish (Danio rerio) is a powerful vertebrate model system for studying development, modeling disease, and screening for novel therapeutics. Due to ...

Education

JoVE Science Education

Basic Biology

Biology II: Mouse, Zebrafish, and Chick

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

Source: Cresci, A., et al. <a href="https://www.jove.com/v/59229/" target="_blank">Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish</a>.&#160;J. Vis. Exp.&#160;(2019)
This video describes the rheotaxis assay in zebrafish. The method involves the measurement of orientation behavior of zebrafish in response to different water flow rates under the influence of different magnetic fields.

Source: Cresci, A., et al. Assessing the Influence of Personality on Sensitivity to Magnetic Fields in Zebrafish.&#160;J. Vis. Exp.&#160;(2019)
This video ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Adult

Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

Source: Richendrfer, H., et al. <a href="https://www.jove.com/v/50622/" target="_blank">Automated High-throughput Behavioral Analyses in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2013)
The video describes the avoidance and thigmotaxis assay of zebrafish larvae. The video examines the behavior of zebrafish larvae in response to visual aversive stimulus.

Source: Richendrfer, H., et al. Automated High-throughput Behavioral Analyses in Zebrafish Larvae. &#160;J. Vis. Exp.&#160;(2013)
The video describes the ...