The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee
1. Zebrafish Breeding and Embryo Maintenance
<ol>
	<li>Prepare E3 embryo medium8. To Make 60x stock solution, dissolve 34.8 g NaCl, 1.6 g&#160;KCl, 5.8g CaCl2&#9679;2H2O, and 9.78 g MgCl2&#9679;6H2O in 1.95 L ddiH2O....

<ol>
	<li>Jao, L. E., Wente, S. R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> PNAS. 110, 13904-13909 (2013).</li><li>Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+knock-in+transgenic+zebrafish+carrying+reporter/driver+genes+by+CRISPR/Cas9-mediated+genome+engineering.">Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.</a> Sci. Rep. 4, 6545 (2014).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Brand, M. D., Couture, P., Else, P. L., Withers, K. W., Hulbert, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+energy+metabolism.+Proton+permeability+of+the+inner+membrane+of+liver+mitochondria+is+greater+in+a+mammal+than+in+a+reptile.">Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile.</a> Biochem. J. 275 (Pt 1), 81-86 (1991).</li><li>Renquist, B. J., Zhang, C., Williams, S. Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+assay+for+high-throughput+energy+expenditure+monitoring+in+the+zebrafish.">Development of an assay for high-throughput energy expenditure monitoring in the zebrafish.</a> Zebrafish. 10, 343-352 (2013).</li><li>Livingston, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+volumetric+respirometer+for+long-term+studies+of+small+aquatic+animals.">A volumetric respirometer for long-term studies of small aquatic animals.</a> J.Mar. Biol. Assoc. U.K. 48, 485-497 (1968).</li><li>Makky, K., Duvnjak, P., Pramanik, K., Ramchandran, R., Mayer, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-animal+microplate+assay+for+metabolic+rate+using+zebrafish.">A whole-animal microplate assay for metabolic rate using zebrafish.</a> J. Biomol. Screen. 13, 960-967 (2008).</li><li>Nasiadka, A., Clark, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+breeding+in+the+laboratory+environment.">Zebrafish breeding in the laboratory environment.</a> ILAR J. 53, 161-168 (2012).</li><li>Brand, M., Granato, M., Nusslein-Volhard, C., Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish, A practical approach. , (2002).</li><li>Fry, F. E. J., Hart, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cruising+speed+of+goldfish+in+relation+to+water+temperature.">Cruising speed of goldfish in relation to water temperature.</a> J. Fish. Res. Board Can. 7, 169-175 (1948).</li><li>Oka, 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=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC physiol. 10 (21), (2010).</li><li>Song, Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+genetic+model+of+obesity+in+a+teleost.">Creation of a genetic model of obesity in a teleost.</a> FASEB J. 21, 2042-2049 (2007).</li><li>Maddison, L. A., Joest, K. E., Kammeyer, R. M., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+insulin+resistance+in+zebrafish+induces+alterations+in+beta-cell+number+and+glucose+tolerance+in+an+age+and+diet+dependent+manner.">Skeletal muscle insulin resistance in zebrafish induces alterations in beta-cell number and glucose tolerance in an age and diet dependent manner.</a> Am. J. physiol. Endocrinol. , (2015).</li><li>Flynn, E. J., Trent , C. M., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+nutritional+control+of+adipogenesis+in+zebrafish+(Danio+rerio).">Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio).</a> J. lipid res. 50, 1641-1652 (2009).</li><li>Shimada, Y., Hirano, M., Nishimura, Y., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+fluorescence-based+assay+system+for+appetite-regulating+gene+and+drug+screening.">A high-throughput fluorescence-based assay system for appetite-regulating gene and drug screening.</a> PloS one. 7, e52549 (2012).</li><li>Tingaud-Sequeira, A., Ouadah, N., Babin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+obesogenic+test:+a+tool+for+screening+molecules+that+target+adiposity.">Zebrafish obesogenic test: a tool for screening molecules that target adiposity.</a> J. lipid res. 52, 1765-1772 (2011).</li></ol>

Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 ...

The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 - 4 months) and surpasses even the mouse in ease of genetic manipulation 1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens 3.
To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, including energy expenditure. As metabolites are processed through &#946;-oxidation and the tricarboxylic acid cycle oxygen is consumed and NADH2 is produced. Thus, NADH2 is a direct indicator of the flux of metabolites through metabolic pathways (metabolite oxidation). In poikilotherms, H+ leak through the inner mitochondrial membrane, which uncouples NADH2 oxidation from ATP synthesis, is 4 - 5 fold lower than in homeotherms 4. Accordingly, in zebrafish NADH2 is very tightly linked to ATP production through oxidative phosphorylation. Herein, we describe an assay that measures NADH2 production in larval zebrafish as a proxy for energy expenditure5.
Oxygen consumption is the gold standard for measuring energy expenditure. Yet, to best take advantage of the high throughput potential of the zebrafish, assays of energy expenditure must be amenable to high-throughput. Oxygen consumption systems that depend on a closed chamber circulating system are limited in throughput by the number of chambers available6. Open air O2 consumption/CO2 production assays have also been applied in the zebrafish5,7. These open air 96-well plate based systems are amenable to high throughput. Unfortunately, gas exchange with the environment limits sensitivity of these assays. We recently published the application of an assay that monitors NADH2 using the redox indicator alamarBlue5. This assay overcomes the limitations in throughput and sensitivity common to analyses of oxygen consumption in the zebrafish.
The zebrafish is becoming an increasingly important model for studies of whole body energy homeostasis. In part, because zebrafish are amenable to use in forward genetic screens and drug screens. Moreover, targeted genetic manipulation, including knockdown and knock-in, can be quickly applied. We have previously shown that this assay can be combined with bath drug application and genetic knockdown or knockout to identify compounds and genes that alter metabolic rate5. Moreover, this assay is designed to exploit the high throughput advantages inherent to the zebrafish.

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">AlamarBlue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">10-230-103</td>
			<td style="width:307px;">Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fine Tip DisposableTransfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-711-26</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fisherbrand Graduated Disposable Transfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-771-9AM</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Black sided clear bottom 96-well plates</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">50-320-785</td>
			<td>Manufactured by E&#38;K Scientific Products Inc.</td>
		</tr>
	</tbody>
</table>

high throughput danio rerio energy expenditure assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P&#60;0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are ...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

The overall goal of this high throughput resazurin-based fluorometric assay is to monitor the effect of genetic or pharmacological manipulations on NADH2 production, which is a measure of metabolic rate. This method can help us answer key questions in the obesity field, such as the affect of a given drug or gene on metabolic rate. Additionally, this assay can be applied to screen for potential drug-drug or drug-gene interactions in a whole animal system.The main advantage of this assay, relative to oxygen consumption assays, is the high throughput and the accumulation of signal across time, which allows the user to detect small differences in metabolic rate. Demonstrating this procedure will be Caroline Foy, a graduate student in my laboratory. After preparing a 60X stock solution of E3 medium according to the text protocol, prepare 1X E3 medium by diluting 16.5 milliliters of stock and one liter of double distilled water.Then add 100 microliters of 1%methylene blue. To make flame polish disposable pipette for egg and fish transfer, use scissors to cut a graduated disposable transfer pipette at the 0.25 milliliter graduation. Then, to remove the rough edges, use a Bunsen burner to flame polish the cut tip.After setting up zebrafish crossings and collecting eggs, allow the eggs to settle to the bottom of the dish, and pour off the water. Use a fine-tip disposable transfer pipette to remove any remaining water. Then add back the E3 medium.Incubate the embryonic fish at 28.5 degrees Celsius to four days post fertilization, or DPF. Twice each day use a flame polished graduated disposable transfer pipette to remove any unfertilized eggs or dead embryos. Then once each day pour off the E3 medium.Use a fine-tip transfer pipette to remove any remaining medium, and replace with fresh E3 pre-warmed to 28.5 degrees Celsius. To carry out the energy expenditure assay, prepare the assay solution, following this table. Use a 0.22 micrometer filter to filter sterilize the solution, and warm to 28.5 degrees Celsius in a water bath for the assay.In a petri dish, combine all viable zebrafish embryos from the clutch. After filter sterilizing 75 milliliters of E3 medium, use a fine-tip disposable transfer pipette to remove as much of the E3 medium from the petri dish as possible. Add back 20 milliliters of sterile E3.Then remove and replace the sterile E3 two more times.Next, using a flame-polished graduated plastic disposable transfer pipette, transfer individual embryonic fish to the wells of a 96-well plate. Working with one column of the plate at a time, remove the E3 medium from the eight wells of the first column, taking care not to touch the embryos with the transfer pipette. Then add 300 microliters of assay solution to each well.Repeat with all 12 columns of the plate. To include a drug treatment, after preparing a 100X solution of the desired compound, add three microliters to each of the treatment wells. Then add three microliters of vehicle control to the control wells of fish.To ensure that the fluorescence response to the compound is a result of activity within the fish, set up drug and vehicle treatments to three blank wells each in a 96-well plate. Now, using a fluorescence plate reader, with excitation and emission wave lengths of 530 nanometers and 590 nanometers respectively, read the wells of the fish plate. Place the plate into a humidified incubator at 28.5 degrees Celsius.Depending on the assay and the stability of the compound being tested, read the fluorescence again at a predetermined time point. To assess the relative change in fluorescence, calculate the average change in fluorescence for the control wells. Then divide the change in fluorescence of each well by the average change in fluorescence of the control wells.Here is an example of using two columns of a 96-well plate. Column A wells include vehicle-treated control zebrafish, while column B wells include insulin-treated zebrafish. In table A, the fluorescence measured at time zero is shown.Table B contains the fluorescence measured 24 hours later. In table C, the fluorescence at time zero has been subtracted from the fluorescence at 24 hours to assess the change in fluorescence. Next, calculate the average change in fluorescence in control wells.Table D is the change in fluorescence of each individual well, divided by the average change in the control wells. You can see here that insulin treatment at 10 micromolar caused a 2.77 fold increase in the signal generated by zebrafish over 24 hours by a P value of less than 0.0001. As seen here, the assay solution does not change color or absolute fluorescence levels in the absence of embryos.However, the assay is highly sensitive to small changes in metabolic rate within the well. This figure represents signals generated by embryonic tilapia after incubating for 24 hours. Pink wells are indicative of fish with a high metabolic rate.Purple wells contain fish with moderate metabolic rates, and the blue wells represent a low metabolic rate. This also shows the versatility of this assay and application across teleost species. Because the NADH2 induced reduction of alamar blue is non-reversible, the signal accumulates with time, which allows for small changes to be amplified.Shown here is the relative change in fluorescence induced by one to five fish at one, two and four hours. With each increase in the number of fish, the relative change in fluorescence increase significantly with time, and the greater the number of fish, the greater the magnitude in fluorescence change with time. Once mastered, this technique can be performed on up to 5, 000 embryonic fish per day.While attempting this procedure, it's important to remember to take care to avoid injuring the embryonic fish. After performing this assay, other procedures like Nile red measurement of neutral lipids, or measurement of phagic drive through intake of fluorassay labeled paramecium can be performed. This allows us to compare the relationship between metabolic rate and lipid metabolism, or metabolic rate and phagic drive.After its development, this technique paved the way for researchers in the field of energy metabolism and metabolic disease to study energy expenditure in zebrafish without limitations in throughput. After watching this video, you should have a good understanding of how to use the fluorometric reagent resazurin to measure NADH2 production in the zebrafish.

high-throughput-danio-rerio-energy-expenditure-assay

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12.5K visualizações.  Vanderbilt University Medical Center. The overall goal of this high throughput resazurin-based fluorometric assay is to monitor the effect of genetic or pharmacological manipulations on NADH2 production, which is a measure of metabolic rate. This method can help us answer key questions in the obesity field, such as the affect of a given drug or gene on metabolic rate. Additionally, this assay can be applied to screen for potential drug-drug or drug-gene interactions in a whole animal system.The main advantage of this assay...