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Nov 2019
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A Reproducible Protocol to Measure the Critical Swimming Speed of Adult Zebrafish
一种可重复性测定成年斑马鱼临界游泳速度的方法   

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Abstract

The quantitative measurement of water flow-induced swimming of fish species using a swimmill is a powerful method to evaluate motor ability of individual fish. Zebrafish is a commonly used vertebrate that enables the study of morphological, physiological and behavioral characteristics associated with genes. We here established a reproducible method that allows to measure the body length and the critical swimming speed of adult zebrafish using a swimmill.

Keywords: Motor (运动), Speed (速度), Swimmill (Swimmill), Swimming (游泳), Zebrafish (斑马鱼)

Background

To evaluate motor ability of fish, swimmill systems have been used for measurement of critical swimming speed (Ucrit), which is the maximum water velocity in which fish can keep swimming. Many different swimmill equipments and protocols have been used for zebrafish studies (Table 1) and thus, it was difficult to compare results of different experiments and different protocols due to the lack of detailed calibration methods and protocols. We applied commercially available swimmill equipment Swim Tunnel Respirometer, which is the major and standard one among zebrafish researchers, established a reproducible protocol for obtaining basic swimming characteristics of adult zebrafish, such as adequate water temperature and adequate length of caudal fin among various wild-type strains (Wakamatsu et al., 2019). We here describe the detailed methods to monitor dissolved oxygen for the estimation of oxygen consumption in swimming, to measure body size of adult zebrafish, to conduct calibration of water velocity, to acclimate zebrafish to the narrow swimming chamber and to measure swimming speed. This protocol would be useful for researchers who use swimmill.

Table 1. Swimmill equipments. The absolute value of Ucrit measured in adult wild-type zebrafish (3-12 months old) appears to vary by the difference of the swimmill equipments. This is probably due to the size difference of swimming chamber that generates uneven water flow between center and peripheral. Therefore, the comparison of Ucrit should be done in the assays that use the same equipment.


Materials and Reagents

  1. 50 ml conical tube (TrueLine, catalog number: TR2005 )
  2. Zebrafish wild-type strain 3-9 months old adult (Neos Pet Shop, catalog number: a2-1020v3 )
  3. Green fluorescent PE micro spheres (Loligo Systems, catalog number: AC10555 )
  4. Oxygen can 5 L (Pinole, catalog number: 4530896201169 )
  5. Sodium sulfite (Wako, catalog number: 192-03415 )

Equipment

  1. Swim Tunnel Respirometer model 170 ml, 230 V, 50 Hz (Loligo Systems, model: SW10000 )
  2. High-speed video camera (Imaging Development Systems, model: UI-3240CP-C-GL )
  3. Green laser pointer 300 mW, 532 nm (Loligo Systems, model: AC10551 )
  4. Eheim pump 5 L/min, 230 V, 50 Hz (Loligo Systems, model: PU10050 )
  5. Aquarium heater 300 W (Nisso, model: R300W )
  6. 10 ml measurement cylinder (Iwaki, model: 3022CYL10S )
  7. Electric balance 0.01 g resolution (A&D, model: FZ-3000i )

Software

  1. AutoRespTM version 2 (Loligo Systems #AR12600)
  2. uEye Cockpit (Imaging Development Systems # UI-3240CP-C-GL )
  3. Digital Particle Tracking Velocimetry (DPTV) (Loligo Systems #AC10550)
  4. ImageJ (https://imagej.nih.gov/ij/)
  5. Microsoft Excel (Microsoft)

Procedure

  1. Calibration of water velocity
    This system consists of an outside tank and a double cylinder (Figure 1A). The outside tank contributes to maintain water temperature of the double cylinder, which is composed of an external cylinder and an inner swimming chamber (Figure 1B). A propeller in the swimming chamber generates water flow in the double cylinder by controlling the propeller rotation depending on an input voltage controlled by the AutoResp software. It is necessary to calibrate the input voltage and water velocity. This calibration can be done by flowing green fluorescent micro beads in the swimming chamber using high-speed camera controlling software uEye Cockpit and analyzing velocity of moving beads using DPTV.


    Figure 1. Structure of the swimmill. A. The schematic diagram of the Swim Tunnel Respirometer. C: high speed video camera; D: dissolved oxygen densitometer, T: thermometer. B. Size of the double cylinder. C. An enlarged view of the double cylinder. The propeller rotation generates a directional water flow in the double cylinder. Fluorescent beads flow in the double cylinder and glitter by receiving the excitation light from the green laser pointer. M: mesh; P: propeller. D. In the presence of water flow, zebrafish keep swimming by the same speed with water velocity to stay at the same position. Two plastic meshes were put in the swimming chamber to avoid the fatigued fish hit the propeller.

    1. Set up the Swim Tunnel Respirometer with 18 L of water in the outside tank and inside double cylinder along with a heater that maintain the outside water temperature at 26 ± 0.2 °C (Video 1). Fish is not necessary for the calibration of water velocity.

      Video 1. Set up the Swim Tunnel Respirometer

    2. Replace the dissolved oxygen densitometer, which is inserted into the inner swimming chamber, with fluorescent laser pointer for visualization of fluorescent beads (Video 2).

      Video 2. Calibration of water velocity

    3. Suspend 1 mg of fluorescent beads in 2 ml of water and then apply it to the double cylinder through a water inlet at the top of the side cover of the double cylinder.
    4. Start the AutoResp software and set the input voltage at 0.5 V, initiating rotation of the propeller to generate water flow in the inner swimming chamber (Figure 1C) (https://loligosystems.com/FileCatalog/GetCatalogItemFileUpload?catalogItemId=82).
    5. Start the uEye Cockpit software for video capturing and record the movement of fluorescent beads in the inner swimming chamber (https://en.ids-imaging.com/ueye-cockpit.html).
    6. Repeat Steps A4 and A5 by changing input voltage from 0.5 to 4.5 V in a stepwise manner with a 0.25 V increment.
    7. Analyze the speed of ~10,000 fluorescent beads in movies using the DPTV software and calculate the water velocity at each input voltage. The user manual of the DPTV software is available here.
    8. Make a graph of input voltage and water velocity with a best-fit approximate line using Microsoft Excel (Figure 2).
    9. Input the graph parameters in the AutoResp software so as to automatically calibrate the input voltage and water velocity.


      Figure 2. Voltage-Water velocity plot. The approximate line becomes straight because water velocity is proportional to the input voltage.

  2. Calibration of dissolved oxygen concentration
    The probe of dissolved oxygen densitometer, which can be inserted into the inner swimming chamber during swimmill assay, records the dissolved oxygen (%) per saturated dissolvable oxygen (constant) at the water temperature. This calibration of dissolved oxygen densitometer can be carried out in the AutoResp software using oxygen-saturated water (100%) and sodium sulfite solution (0%).
    1. Place the probe about 5 cm depth in 40 ml of water in a 50 ml conical tubes and check the dissolved oxygen in the AutoResp software (Video 3).

      Video 3. Calibration of dissolved oxygen concentration

    2. Bubble ~5 L of oxygen from an oxygen can into water through a nozzle (inner diameter 1 mm) until the value of dissolved oxygen no longer increase.
    3. Calibrate the probe for 100% by setting “Lock Hi” in the AutoResp software.
    4. Dissolve 2 g of sodium sulfite, which is an oxygen scavenger agent, in 40 ml of water to make oxygen-free water.
    5. Clean the probe and place it in the oxygen-free water then calibrate the probe for 0% by setting “Lock Lo” in the AutoResp software.

  3. Measurement of swimming speed
    1. Zebrafish (Danio rerio) are reared and maintained at 26-28 °C under a 14 h light and 10 h dark photoperiod and fed twice a day in the regular care. Zebrafish used for swimming assay are kept unfed for 20~24 h prior to the measurement, because swimming speed of zebrafish decreases after eating food. This measurement is done between AM 10:00 and PM 3:00.
    2. Put an adult zebrafish into a 10 ml measurement cylinder (Φ 11 mm) that contains 5 ml of water and measure the volume (increase of water height) and the mass of the fish by an electric balance (Figure 3).


      Figure 3. Measurement of the volume and mass of adult fish. The increment of the water height by putting a zebrafish in the measurement cylinder is the volume of the fish. The weight of the fish is simultaneously measurable using an electric balance.

    3. Transfer the zebrafish into a 50 ml plastic tube filled with water and put it in the outside tank for 10 min to acclimate the fish to the narrow cylinder and water temperature (26 ± 0.2 °C).
    4. Pre-mark 3 cm distance lines on the inner swimming chamber using an oil-based marker. This helps to measure total length and standard length of fish in the analysis of swimming movies (see Data analysis B).
    5. Add a single zebrafish in the inner swimming chamber of the double cylinder (Video 4).

      Video 4. Loading zebrafish into Swim Tunnel Respirometer

    6. Set the double cylinder in the Swim Tunnel Respirometer.
    7. Start the AutoResp and uEye Cockpit software to be ready for the control of input voltage and video-recording, respectively.
    8. Initiate propeller rotation at 10 cm/s water flow and video-recording simultaneously (Video 5).

      Video 5. A zebrafish could keep swimming in 10 cm/s water velocity

    9. After 1 min of warming-up at 10 cm/s water flow, the water velocity is set at 15 cm/s to start automatic step protocol in the AutoResp software (Figure 4).


      Figure 4. A protocol of the swimming time and water velocity. The water velocity is initially set at 10 cm/s for 1 min and eventually increased to 15 cm/s for 1 min. Thereafter step protocol increases the water velocity 1 cm/s every 1 min.

    10. After the fish get fatigued and failed to continue swimming (Video 6), stop the propeller rotation and video-recording.

      Video 6. A zebrafish failed to keep swimming in 33 cm/s water velocity

    11. Record the water temperature and dissolved oxygen concentration as well as water velocity and time when the fish failed to keep swimming.
      Note: If you want to change the water temperature, you can increase the temperature by altering the setting of thermal heater or decrease it by putting an ice bag in the outside water tank.

Data analysis

  1. Calculation of the critical swimming speed
    The critical swimming speed (Ucrit) is the maximum water velocity in which fish can keep swimming. The Ucrit of fish is calculated according to the established formula (Brett, 1964). Ucrit = Umax + T/60. Umax (cm/s): the highest water velocity when zebrafish continued to swim for whole 1 min. T (s): time elapsed when fish failed to keep swimming in 1 min.

  2. Measurement of the body size
    Three different definitions of body size have been established; total length, standard length and caudal fin length (Figure 5) (Plaut, 2000). The total length and standard length are measurable in comparison with pre-marked 3 cm distance lines in a frame of swimming movies (dorsal view). In ImageJ software, draw a line of 3 cm distance and measure the length by pixel using the Analyze/Measure function. Similarly, draw a line along the midline of the fish from the head to the tail and measure the length by pixel and then calculate the actual length by hands. The standard length is calculated by the line from the head to the root of caudal fin. The difference of total and standard lengths is the caudal fin length. The motor ability of zebrafish varies by the length of caudal fin (Wakamatsu et al., 2019).


    Figure 5. The definition of body size of zebrafish. Three different parameters of body size are established in fish studies.

Notes

We could obtain reproducible results using this protocol (Wakamatsu et al., 2019).

Acknowledgments

We thank Hirata Lab members for helpful comments. This work was supported by a Grant-in-Aid for Scientific Research B (16H04657, 19H03329) and Scientific Research on Innovative Areas (17H05578) from the MEXT, Japan, the Naito Foundation and the Japan Epilepsy Research Foundation.

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments described in this manuscript and guidelines for use of zebrafish have been approved by Animal Care and Use Committee at Aoyama Gakuin University (A9: valid until March 2021).

References

  1. Brett, J. (1964). The respiratory metabolism and swimming performance of young sockeye salmon. J Fish Research Board of Can 21: 1183-1226.
  2. Conradsen, C. and McGuigan, K. (2015). Sexually dimorphic morphology and swimming performance relationships in wild-type zebrafish Danio rerio. J Fish Biol 87(5): 1219-1233.
  3. Conradsen, C., Walker, J. A., Perna, C. and McGuigan, K. (2016). Repeatability of locomotor performance and morphology-locomotor performance relationships. J Exp Biol 219(Pt 18): 2888-2897.
  4. Gemberling, M., Karra, R., Dickson, A. L. and Poss, K. D. (2015). Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish. Elife 4: e05871.
  5. Gilbert, M. J. H., Zerulla, T. C. and Tierney, K. B. (2014). Zebrafish (Danio rerio) as a model for the study of aging and exercise: physical ability and trainability decrease with age. Experimental Gerontology 50: 106-113.
  6. Leris, I., Sfakianakis, D. G. and Kentouri, M. (2013). Are zebrafish Danio rerio males better swimmers than females? J Fish Biol 83(5): 1381-1386.
  7. Lucas, J., Percelay, I., Larcher, T. and Lefrancois, C. (2016). Effects of pyrolytic and petrogenic polycyclic aromatic hydrocarbons on swimming and metabolic performance of zebrafish contaminated by ingestion. Ecotoxicol Environ Saf 132: 145-152.
  8. Massé, A. J., Thomas, J. K. and Janz, D. M. (2013). Reduced swim performance and aerobic capacity in adult zebrafish exposed to waterborne selenite. Comp Biochem Physiol C Toxicol Pharmacol 157(3): 266-271.
  9. Messerli, M., Aaldijk, D., Haberthur, D., Ross, H., Garcia-Poyatos, C., Sande-Melon, M., Khoma, O. Z., Wieland, F. A. M., Fark, S. and Djonov, V. (2020). Adaptation mechanism of the adult zebrafish respiratory organ to endurance training. PLoS One 15(2): e0228333.
  10. Mokalled, M. H., Patra, C., Dickson, A. L., Endo, T., Stainier, D. Y. and Poss, K. D. (2016). Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish. Science 354(6312): 630-634.
  11. Palstra, A. P., Tudorache, C., Rovira, M., Brittijn, S. A., Burgerhout, E., van den Thillart, G. E., Spaink, H. P. and Planas, J. V. (2010). Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression. PLoS One 5(12): e14483.
  12. Parisi, A., Blattmann, P., Lizzo, G., Stutz, V., Strohm, L., Richard, J., Civiletto, G., Charpagne, A., Raymond, F., Gobet, C., et al. (2018). PGC1a and Exercise Adaptations in Zebrafish. BioRxiv 483784.
  13. Plaut, I. (2000). Effects of fin size on swimming performance, swimming behaviour and routine activity of zebrafish Danio rerio. J Exp Biol 203(Pt 4): 813-820.
  14. Thomas, J. K., Wiseman, S., Giesy, J. P. and Janz, D. M. (2013). Effects of chronic dietary selenomethionine exposure on repeat swimming performance, aerobic metabolism and methionine catabolism in adult zebrafish (Danio rerio). Aquatic Toxicology 130: 112-122.
  15. Wakamatsu, Y., Ogino, K. and Hirata, H. (2019). Swimming capability of zebrafish is governed by water temperature, caudal fin length and genetic background. Sci Rep 9(1): 16307.
  16. Widrick, J. J., Gibbs, D. E., Sanchez, B., Gupta, V. A., Pakula, A., Lawrence, C., Beggs, A. H. and Kunkel, L. M. (2018). An open source microcontroller based flume for evaluating swimming performance of larval, juvenile, and adult zebrafish. PLoS One 13(6): e0199712.

简介

[摘要 ] 的使用swimmill鱼类的水流产生的游泳的定量测量是评估个体的鱼的运动能力的强大方法。斑马鱼是一种常用的脊椎动物,可以研究与基因相关的形态,生理和行为特征。在这里,我们建立了一个可重复的方法,使测量体长,并用成年斑马鱼的临界游泳速度swimmill 。

[背景 ] 至E 计价马达鱼的能力,swimmill系统已被用于关键游泳速度(测量ü 爆击),这是最大的水的流速,其中鱼可以保持游泳。许多不同的游泳设备和协议已用于斑马鱼的研究(表1 ),因此,由于缺乏详细的校准方法和协议,很难比较不同实验和不同协议的结果。我们使用了市售的游泳设备Swim Tunnel呼吸计,这是斑马鱼研究人员的主要标准装备,它为获得成年斑马鱼的基本游泳特征(例如各种野生型中适当的水温和足够的尾鳍长度)建立了可重现的协议。菌株(Wakamatsu 等人,2019)。我们在这里描述详细的方法,以监测溶解氧以估计游泳中的氧气消耗,测量成年斑马鱼的体型,进行水速校准,使斑马鱼适应狭窄的游泳室并测量游泳速度。该协议对于使用游泳机的研究人员将非常有用。



表1.游泳设备。在成年野生型斑马鱼(3-12个月大)中测得的U crit 绝对值似乎因游泳设备的不同而有所不同。这可能是由于游泳室的大小差异所致,从而在中心和外围之间产生了不均匀的水流。因此,U crit 的比较应在使用相同设备的测定中进行。

游泳场

û 爆击(厘米/秒)

参考文献

游泳隧道呼吸器型号170毫升

230 V,50赫兹

Loligo系统#SW10000

25 - 40

Massé 等。,2013

托马斯等。,2013

卢卡斯等。,2016

Parisi 等。,2018

若松等。,2019

游泳隧道呼吸器型号5 L

230 V,50赫兹

Loligo系统#SW10050

70 至90

Gemberling 等。,2015

Mokalled 等。,2016

游泳隧道呼吸仪型号10 L

230 V,50赫兹

Loligo系统#SW10100

40 - 80

吉尔伯特等。,2014

康拉德森和麦圭根,2015年

Conradsen 等。,2016

Messerli 等。,2020

作者制作的游泳系统

50 - 65

普劳特,2000年

作者制作的游泳系统

55

Palstra 等。,2010

作者制作的游泳系统

25 - 30

Leris 等。,2013

水槽

35 - 45

Widrick 等。,2018

关键字:运动, 速度, Swimmill, 游泳, 斑马鱼

材料和试剂


 


50 ml锥形管(True Line ,目录号:TR2005)
斑马鱼野生型3至9个月大的成年病毒(Neos Pet Shop ,目录号:a2-1020v3)
绿色荧光PE微球(Loligo Systems,目录号:AC10555)
氧气罐5 L(Pinole ,货号:4530896201169)
亚硫酸钠(和光,目录号:192-03415)
 


设备


 


型号170 ml,230 V,50 Hz的游泳隧道呼吸计(Loligo Systems ,型号:SW10000)
高速摄像机(影像开发系统,型号:UI-3240CP-C-GL)
绿色激光笔300 mW,532 nm (Loligo Systems,型号:AC10551)
Eheim 泵5 L / min,230 V,50 Hz(Loligo Systems ,型号:PU10050)
水族馆加热器300瓦(日产,型号:R300W)
10 ml量筒(Iwaki,型号:3022CYL10S)
电子天平0.01 g分辨率(A&D ,型号:FZ-3000i)
 


软件


 


AutoResp TM 版本2(Loligo Systems#AR12600)
uEye座舱(影像开发系统#UI-3240CP-C-GL)
数字粒子跟踪测速(DPTV)(Loligo Systems#AC10550)
图片J (https://imagej.nih.gov/ij/)
Microsoft Excel(Microsoft)
 


程序


 


水速校准
该系统由一个外部水箱和一个双缸组成(图1A)。外部水箱有助于维持双缸水温,双缸由外缸和内部游泳室组成(图1B)。在游泳室A螺旋桨通过控制产生所述双缸水流螺旋桨ř 浮选取决于由AutoResp软件控制的输入电压。必须校准输入电压和水速。可以通过使用高速相机控制软件uEye Cockpit使绿色荧光微珠在游泳室中流动并使用DPTV分析运动的珠子的速度来进行此校准。


 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ Figs jpg \ Figure1.jpg


图1 。游泳馆的结构。一。游泳隧道呼吸计的示意图。C:高速摄像机;D:溶解氧密度计,T:温度计。乙。双缸尺寸。Ç 。双缸的放大图。该螺旋桨旋转产生的双缸在定向水流。荧光珠在双圆柱体中流动并通过接收来自绿色激光指示器的激发光而闪烁。M:网眼;警:螺旋桨。d 。在有水流的情况下,斑马鱼以相同的速度与水速保持相同的位置游泳。在游泳室里放了两个塑料丝网,以免疲劳的鱼撞击螺旋桨。


 


设置一个游泳隧道呼吸计,在外部水箱和内部双气瓶中装有18升水,以及一个将外部水温保持在26±0.2的加热器 °C(视频1)。鱼不是校准水速所必需的。
 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ video1.jpg


视频1.设置游泳隧道呼吸计


 


用荧光激光指示器更换荧光珠指示器(视频2),将溶解氧密度仪插入内部游泳室内。
 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ video2.jpg


视频2.水速校准


 


将1 mg荧光珠悬浮在2 ml水中,然后通过双筒侧面盖顶部的进水口将其施加到双筒中。
启动AutoResp软件并将输入电压设置为0.5 V,启动螺旋桨旋转以在内部游泳室内产生水流(图1C)(https://loligosystems.com/FileCatalog/GetCatalogItemFileUpload?catalogItemId=82)。
启动uEye Cockpit软件以进行视频捕获,并记录荧光珠在内部游泳室内的运动(https://en.ids-imaging.com/ueye-cockpit.html)。
重复小号吨EP 小号甲4和甲5b的与为0.25V增量ý变化的输入0.5至4.5 V的电压以逐步方式。
使用DPTV软件分析电影中约10,000个荧光珠的速度,并计算每个输入电压下的水流速度。可在此处获得DPTV软件的用户手册。
使用Microsoft Excel使用最合适的近似线来绘制输入电压和水流速度的图形(图2)。
在AutoResp软件中输入图形参数,以便自动校准输入电压和水速。
 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ Figs jpg \ Figure2.jpg


图2 。电压-水速度图。由于水流速度与输入电压成比例,因此近似线变为直线。


 


溶解氧浓度校准
溶氧密度计的探头可以在泳车分析过程中插入内部游泳室,记录水温下每饱和d 可溶氧(常数)的溶氧(%)。可以在AutoResp软件中使用氧饱和水(100%)和亚硫酸钠溶液(0%)进行溶解氧浓度计的校准。


将探针放在50毫升锥形管中的40毫升水中约5厘米深,并在AutoResp软件中检查溶解的氧气(视频3)。
 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ video3.jpg


视频3.溶解氧浓度的校准


气泡〜5升从氧气罐成水的氧通过一个喷嘴(内径1mm)的,直到溶解的氧的值不再增加。
通过在AutoResp软件中设置“ Lock Hi”,将探头校准为100%。
将2 g亚硫酸钠(一种除氧剂)溶于40 ml水中,制成无氧水。
清洁探头并将其放在无氧水中,然后通过在AutoResp软件中设置“ Lock Lo”将探头校准为0%。
 


MEA 游泳速度的surement
斑马鱼(斑马鱼)饲养,并保持在26 - 28 ℃下在14小时光照和10小时黑暗的光周期下,每天两次在常规护理供给。在测量之前,用于游泳分析的斑马鱼要保持20至24小时不进食,因为斑马鱼进食后会降低游泳速度。这种测量之间进行AM 10 :00 和下午3 :00 。
把一个成年斑马鱼到10ml测量圆筒(Φ 11毫米),其含有5ml水,并测量体积(水高度的增加)和鱼的由质量ELECTRI 碳平衡(图3)。
 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ Figs jpg \ Figure3.jpg


图3 。成年鱼体积和质量的测量。通过将斑马鱼放入量筒中,水高的增量即为鱼的体积。鱼的重量可同时使用电子天平测量。


 


将斑马鱼转移到装满水的50毫升塑料管中,然后将其放在室外水箱中10分钟,使鱼适应狭窄的圆柱体和水温(26±0.2 °C )。
使用油性标记笔在内部游泳室上预先标记3厘米的距离线。这有助于在游泳影片分析中测量鱼的总长度和标准长度(请参阅数据分析B)。
在双缸的内部游泳室内添加单个斑马鱼(视频4)。
 


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视频4.将斑马鱼加载到游泳隧道呼吸计中


 


将双气瓶放在游泳隧道呼吸计中。
启动AutoResp和uEye Cockpit软件,分别准备控制输入电压和视频记录。
以10 cm / s的水流量启动螺旋桨旋转并同时进行视频录制(视频5)。
 


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视频5. 斑马鱼可以以10 cm / s的水速继续游泳


 


在以10 cm / s的水温预热1分钟后,将水速设置为15 cm / s,以在AutoResp软件中启动自动步进方案(图4)。
 


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图4 。游泳时间和水速的协议。水速最初设置为10 cm / s,持续1分钟,最终提高到15 cm / s,持续1分钟。此后,分步方案每1分钟将水速提高1 cm / s。


 


鱼变得疲劳且无法继续游泳(视频6)之后,停止螺旋桨旋转和视频录制。
 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ video6.jpg


视频6.斑马鱼未能以33 cm / s的水速继续游泳


 


记录鱼未能继续游泳时的水温和溶解氧浓度以及水速和时间。
注:我F你想改变水的温度,你可以通过改变热加热器的设置来增加温度或通过将一个冰袋在外面水箱减少它。






数据分析


 


临界游泳速度的计算
临界游泳速度(U crit )是鱼可以保持游泳的最大水流速度。根据确定的公式计算鱼的U 临界值(Brett,1964)。U crit = U max + T / 60。U max (cm / s):斑马鱼继续游动整个1分钟时的最高水流速度。T(s):鱼在1分钟内没有继续游泳的时间。


 


身体尺寸的测量
已经建立了三种不同的体型定义;总长度,标准长度和尾鳍长度(图5)(Plaut,2000年)。与游泳电影画面(背面视图)中预先标记的3 cm距离线相比,可测量总长度和标准长度。在ImageJ软件中,画一条3厘米距离的线,并使用分析/测量功能按像素测量长度。同样,沿着鱼的中线从头到尾画一条线,以像素为单位测量长度,然后用手计算实际长度。标准长度由从尾鳍的头部到根部的线计算。总长度与标准长度之差为尾鳍长度。斑马鱼的运动能力随尾鳍的长度而变化(Wakamatsu et al。,2019)。


 


D:\ Reformatting \ 2020-6-1 \ 2003132--1480 Hiromi平田854726 \ Figs jpg \ Figure5.jpg


图5 。斑马鱼体型的定义。在鱼类研究中建立了三种不同的体型参数。


 


笔记


 


我们可以使用该方案获得可重复的结果(Wakamatsu 等人,2019)。


 


致谢


 


我们感谢平田实验室成员的有用评论。这项工作得到了MEXT,日本,内藤基金会和日本癫痫研究基金会的B项科研补助金(16H04657、19H03329)和创新领域的科学研究(17H05578)的支持。


 


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


本手稿和斑马鱼使用指南中描述的所有动物实验均已获得青山学院大学动物保护和使用委员会的批准(A9:有效期至2021年3月)。


 


参考文献


 


Brett,J。(1964)。红鲑幼鱼的呼吸代谢和游泳性能。 Can的J Fish研究委员会21:1183-1226。             
Conradsen ,C.和McGuigan,K.(2015)。野生斑马鱼Danio rerio的性二态形态与游泳成绩的关系。鱼生物学杂志87(5):1219-1233。
Conradsen ,C.,Walker,JA,Perna ,C.和McGuigan,K.(2016)。运动性能的可重复性与形态运动性能的关系。J Exp Biol 219(Pt 18):2888-2897。
Gemberling ,M.,Karra ,R.,Dickson,AL和Poss ,KD(2015)。Nrg1是斑马鱼中内源性心脏再生程序的损伤诱导心肌有丝分裂原。Elife 4:e05871。
吉尔伯特(MJH),泽鲁拉(Zerulla),技术委员会和蒂尔尼(KB)(2014)。斑马鱼(Danio rerio )作为研究衰老和运动的模型:身体能力和可训练性随年龄的增长而降低。 实验老年学50:106-113。
Leris,I.,Sfakianakis ,DG和Kentouri ,M.(2013)。斑马鱼Danio rerio 男性的游泳者是否比女性更好?鱼生物学杂志83(5):1381-1386。              
Lucas,J.,Percelay ,I.,Larcher ,T.和Lefrancois ,C.(2016)。热解和成岩多环芳烃对被摄入污染的斑马鱼游泳和代谢性能的影响。Ecotoxicol Environ Saf 132:145-152。
Massé ,AJ,Thomas,JK和Janz ,DM(2013)。暴露于水性亚硒酸盐的成年斑马鱼的游泳表现和有氧运动能力下降。Comp Biochem Physiol C Toxicol Pharmacol 157(3):266-271。
梅塞利(Messerli,M.),奥尔迪克(Aaldijk ),哈伯特(Haberthur ),罗斯(Ross),加西亚· 波亚托斯(Garcia - Poyatos ),桑德·梅隆(Sande-Melon),科马(Khoma),奥兹(OZ),维兰(Wieland),FAM,法克(Fark),S。和德约诺夫(Djonov ) (2020)。成年斑马鱼呼吸器官对耐力训练的适应机制。PLoS One 15(2):e0228333。
Mokalled,MH,Patra,C.,Dickson,AL,Endo,T.,Stainier ,DY and Poss ,KD(2016)。损伤引起的ctgfa指导斑马鱼的神经胶质桥接和脊髓再生。科学354(6312):630-634。
Palstra,AP,Tudorache ,C.,Rovira ,M.,Brittijn ,SA,Burgerhout ,E.,van den Thillart ,GE,Spaink ,HP和Planas ,JV(2010)。建立斑马鱼作为一种新颖的运动模型:游泳经济,游泳增强的生长和肌肉生长标记基因的表达。PLoS One 5(12):e14483。
Parisi ,A.,Blattmann ,P.,Lizzo,G.,Stutz,V.,Strohm ,L.,Richard,J.,Civiletto ,G.,Charpagne ,A.,Raymond,F.,Gobet ,C.,等。(2018)。PGC1a与斑马鱼的运动适应。 BioRxiv 483784。
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Thomas JK,Wiseman S.,Giesy JP和DM Janz (2013)。长期饮食中硒代蛋氨酸的暴露对成年斑马鱼(Danio rerio )重复游泳表现,有氧代谢和蛋氨酸分解代谢的影响。水生毒理学130:112-122。
Y.Wakamatsu,K.Ogino和Hirata,H。(2019)。斑马鱼的游泳能力受水温,尾鳍长度和遗传背景的影响。 科学代表9(1):16307。
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引用:Wakamatsu, Y., Kashima, M. and Hirata, H. (2020). A Reproducible Protocol to Measure the Critical Swimming Speed of Adult Zebrafish. Bio-protocol 10(16): e3712. DOI: 10.21769/BioProtoc.3712.
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Ian Hunger Guldner
Is there any volume to the videos? If so, it does not seem to be working.
2021/2/4 20:13:41 回复
Fanglian He
Bio-protocol

No audio in those orginal videos.

2021/2/8 18:00:59 回复