The research presented in this article was supported by previous grants from National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR) R01DE020884 to J.K.E. and National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA) F32AA021320 to C.B.L. and by the current grant from National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 to C.B.L. We thank Rueben Gonzales for providing and assisting with gas chromatograph analysis. We thank Tiahna Ontiveros and Dr. Gina Nobles writing assistance.

All zebrafish embryos used in this procedure were raised and bred following established IACUC protocols27. These protocols were approved by the University of Texas at Austin and the University of Louisville.
NOTE: The zebrafish line Tg(fli1:EGFP)y1 was used in this study28. All water used in this procedure is sterile reverse osmosis water. All statistical analyses were performed using Graphpad Prism v8.2.1.
1. Making embryo media
<ol>
	<li>To make a 20x stock of embryo media, dissolve 17.5 g of NaCl, 0.75 g of KCl, 2.9 g of CaCl2, 0.41 g of K2HPO4, 0.142 g of NA2HPO4, and 4.9 g of MgSO4&#183;7H2O in 1 L of water. Ignore the white precipitate that forms; this will not impact the media. Filter sterilize the stock solution and store at 4 &#176;C.</li>
	<li>To create the working embryo media solution, dissolve 1.2 g of NaHCO3 in 1 L of 20x embryo media stock and add 19 L of water. Maintain the working embryo media solution at 28 &#176;C.</li>
</ol>
2. Measuring the embryonic volume using water displacement
NOTE: In this protocol, 24 h postfertilization (hpf) embryos (Figure 1) are used. The embryos used in the volume measurements are not used in the ethanol analysis.
<ol>
	<li>Place 10 embryos and extraembryonic fluid in 1.5 mL microcentrifuge tube marked at a volume of 250 &#956;L (Figure 2A). Add water to the 250 &#956;L fill line (see sample with dyed water in Figure 2B).</li>
	<li>Repeat step 2.1 to set up receptacles for embryos that have had their chorions removed.
	<ol>
		<li>To remove the chorion, place the embryos in their chorions in a 100 mm Petri dish with 2 mg/mL of protease cocktail in embryo media at room temperature (RT) for 10 min. Every few minutes, gently swirl the embryos to break the chorion.</li>
		<li>Once all the embryos are free of their chorion, remove the dechorionated embryos from protease cocktail/embryo media and place in a new 100 mm Petri dish with fresh embryo media to wash the embryos. Repeat this wash step 1 more time (for a total of 2 washes). Transfer the embryos to a fresh 100 mm Petri dish.</li>
	</ol>
	</li>
	<li>Using the smallest tip possible and without damaging the embryos, carefully remove all liquid from around the embryos using a p200 micropipettor (Figure 2C) and weigh the water with a scale with &#60;0.1 mg precision. To determine the volume of the sample of 10 embryos, subtract the weight/volume of the water (1 mL of water = 1 g of water.) removed from 250 &#956;L. To determine the volume of a single embryo, divide the difference between 250 &#956;L and the weight of the water removed by 10.</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/60766/60766equ01.jpg" />
<img alt="Equation 2" src="/files/ftp_upload/60766/60766equ02.jpg" />
3. Treating embryos with ethanol
<ol>
	<li>Gather embryos from mating tanks and place in a standard 100 mm Petri dish. Count out no more than 100 embryos per single Petri dish and incubate at 28.5 &#176;C. 
	NOTE: If the chorion is to be removed (step 2.2), it needs to be removed before adding ethanol.</li>
	<li>At 6 hpf, add up to 100 embryos to a new standard 100 mm Petri dish either with embryo media or embryo media + 1% ethanol (v/v). Cover, but DO NOT seal the Petri dish. Place the embryos in a low temp incubator set at 28.5 &#176;C for 18 h, or until the embryos reach the developmental time point of 24 hpf.</li>
</ol>
4. Preparing workflow before processing the embryos for head space gas chromatography
<ol>
	<li>Make a solution of 5 M NaCl in water (450 &#956;L will be needed per sample tube) to denature all proteins and prevent ethanol metabolism. Make the protease cocktail (Table of Materials) at a concentration of 2 mg/mL in embryo media.</li>
	<li>Label a 1.5 mL microcentrifuge tube and a 2 mL gas chromatograph vial for each sample. Label additional 2 mL gas chromatograph vials for the ethanol standards described below as well as air, water, and the 5 M NaCl/protease cocktail blanks.</li>
	<li>Set up three p200 micropipettors two set to 50 &#956;L, and the third set to 200 &#956;L; a p1000 micropipettor set to 450 &#956;L and a p2 micropipettor set to 2 &#956;L. For the glass pipettes used to transfer embryos from the Petri dishes to the 1.5 mL microcentrifuge tubes, quickly pass the pipette tip through a flame to smooth the edges to not damage the embryos when drawing them into the pipette.</li>
</ol>
5. Processing embryos for head space gas chromatography
NOTE: Both embryos in their chorions and those previously removed from their chorions are treated the same for consistency in the calculation of dilution factors.
<ol>
	<li>Using the two p200 micropipettors set to 50 &#956;L, draw 50 &#956;L of the protease cocktail solution into one and draw 50 &#956;L of water into the second.</li>
	<li>Using the glass pipette and micropipettor, quickly place 10 embryos (from step 3.2) in a 1.5 mL microcentrifuge tube and close the cap (as described in Figure 2A). Repeat for all samples to be tested, controls, and ethanol treated embryos.</li>
	<li>Using the p200 micropipettor set to 200 &#956;L, quickly open the cap and remove all residual embryo media (as described in Figure 2C). Quickly place the pipette containing 50 &#956;L of water in the tube and rapidly but gently (to not damage the embryos) add, then remove the water. Quickly add 50 &#956;L of the protease cocktail solution from the waiting pipettor and close the tube cap (Figure 2D). 
	NOTE: Perform this process one sample at a time.</li>
	<li>Let the sample sit at RT for 10 min to allow the protease cocktail to degrade the chorion. Then, quickly add 450 &#956;L of 5 M NaCl and close the tube cap (Figure 2E). Vortex the samples for 10 min. To speed up the process, set up the mixer to vortex multiple samples at the same time. 
	NOTE: Add a small amount (~100 &#956;L) of a silica bead mixture (2 sizes, 0.5 mm and 1 mm bead) to any tube with embryos older than 24 hpf. In older embryos, the notochord will remain intact regardless of how long they are homogenized.</li>
	<li>After homogenizing for 10 min, quickly remove 2 &#956;L of homogenized embryo supernatant and add to a gas chromatograph vial. Quickly seal the vial with the polytetrafluoroethylene cap.</li>
</ol>
6. Preparing media and ethanol standards
<ol>
	<li>To prepare the media standards, dilute the media by a factor of 10 with a 5 M NaCl/protease cocktail solution. Add 2 &#956;L of each sample to a gas chromatograph vial and seal with a polytetrafluoroethylene cap.</li>
	<li>To prepare the ethanol standards, create a serial dilution of 100% ethanol in 5 M NaCl/protease cocktail solution to the following concentrations: 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 mM. Add 2 &#956;L of each standard to a gas chromatograph vial and seal with a polytetrafluoroethylene cap.</li>
</ol>
7. Preparing the head space gas chromatograph
NOTE: This setup and protocol may need to be changed depending on the gas chromatograph used. Head space gas chromatography is used to quantify ethanol levels, not for separation.
<ol>
	<li>Set the heater for the autosampler to 58 &#176;C and turn on. Allow the heater to reach 58 &#176;C, and turn on the air and hydrogen gas lines feeding the gas chromatograph (for flame ionization used to quantify the ethanol). 
	NOTE: The psi should be set to properly operate the chromatograph according to the manufacturer&#39;s specifications. Make sure the helium line is on and set to the proper psi.</li>
	<li>For the septum, make sure the number of samples injected is not &#62;100. For the injection fiber, make sure the number of samples injected is not &#62;500. 
	NOTE: If either is over the amount of injections, they will need to be changed before running the test samples.</li>
	<li>Turn on the analysis software and make sure the workstation is set up properly. Store all data according to lab/department standards. Create a new sample list for the samples to be run.</li>
	<li>Initiate the startup method and wait for the flame ionization detector to stabilize before running the samples. Once stable, run the software startup method to clean the fiber.</li>
</ol>
8. Sample measurements using head space gas chromatography
<ol>
	<li>Once the startup method is complete, create 1 new line per sample in the sample list. From the methods menu in the analysis software, load 436 Current spme ethanol 2013 3min absorb 2_5min rg run.METH for each sample on the sample list.
	<ol>
		<li>Fill out the sample list, starting with the air, water, 5 M NaCl/protease cocktail blanks. Then enter in the standards in order, from 0.3125 to 40 mM. Follow the standards with a second round of the air, water, and 5 M NaCl/protease cocktail blanks.</li>
		<li>Enter all homogenized embryo supernatant samples to be tested, from lowest to highest predicted ethanol concentration. End by entering a third and final round of the air, water, and 5 M NaCl/protease cocktail blanks.</li>
	</ol>
	</li>
	<li>Add the gas chromatograph vials to the autosampler in the order in which the samples were entered. Allow samples to warm for 10&#8211;15 min. Start the sample runs in the software.</li>
	<li>After all the samples and the final blanks have run, activate the shutdown method in the software by adding a final sample in the sample list and running standby.METH. Back up all data acquired during the sample runs. Turn off the equipment in the following order: autosampler heater, hydrogen tank and, only after the chromatograph temperature reaches 30 &#176;C, the air tank. Leave the helium tank on to preserve the wax column.</li>
</ol>
9. Sample ethanol peak integration and sample concentration analysis
NOTE: All values from 9.3 on were calculated in an excel file that all equations prefilled.
<ol>
	<li>Once the shutdown method is complete, click on Open chromatogram. Open the folder containing the results. Samples are automatically integrated in this program.</li>
	<li>In the results, make sure the correct peaks have been integrated (ethanol peaks between 2 and 2.5). Once all samples have been confirmed, print or export the results.</li>
	<li>Plot the peak height of the ethanol standards on a graph. Calculate the slope, Y intercept and R2 values for the ethanol standards (R2 should be &#62;0.99). 
	NOTE: These values will be used to determine the ethanol concentration from the sample peak height.</li>
	<li>For each sample, subtract the peak height of the sample from the Y intercept of the ethanol standards. Divide this value by the slope of the ethanol standards to obtain the ethanol value for each sample in the GC vials.</li>
</ol>
<img alt="Equation 3" src="/files/ftp_upload/60766/60766equ03.jpg" />
<ol start="5">
	<li>To calculate the ethanol concentration in the embryos, first calculate the dilution factor for each sample. Take the volume measure calculated in step 2.3 of this protocol and divide it by the volume measure plus 500 &#956;L. This represents the 5 M NaCl/protease cocktail solution added to each sample during embryo processing (steps 5.3 and 5.4).</li>
</ol>
<img alt="Equation 4" src="/files/ftp_upload/60766/60766equ04.jpg" />
<ol start="6">
	<li>Using this sample dilution factor, multiply by the sample ethanol value for each sample. The results will be in mM concentration. Calculate media reference samples by multiplying the media ethanol value by the dilution factor of 10.</li>
</ol>
<img alt="Equation 5" src="/files/ftp_upload/60766/60766equ05.jpg" />
<img alt="Equation 6" src="/files/ftp_upload/60766/60766equ06.jpg" />

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Validation+of+a+Method+for+Alcohol+Analysis+in+Brain+Tissue+by+Headspace+Gas+Chromatography+with+Flame+Ionization+Detector.">Development and Validation of a Method for Alcohol Analysis in Brain Tissue by Headspace Gas Chromatography with Flame Ionization Detector.</a> Journal of Analytical Toxicology. 40 (8), 653-658 (2016).</li><li>Schlatter, J., Chiadmi, F., Gandon, V., Chariot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+methanol,+acetaldehyde,+acetone,+and+ethanol+in+human+blood+by+gas+chromatography+with+flame+ionization+detection.">Simultaneous determination of methanol, acetaldehyde, acetone, and ethanol in human blood by gas chromatography with flame ionization detection.</a> Human and Experimental Toxicology. 33 (1), 74-80 (2013).</li><li>Schier, C. J., Mangieri, R. A., Dilly, G. A., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+ethanol+during+operant+ethanol+self-administration+and+ethanol+determination+by+gas+chromatography.">Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography.</a> Journal of Visualized Experiments. (67), e4142 (2012).</li><li>Adalsteinsson, E., Sullivan, E. V., Mayer, D., Pfefferbaum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+quantification+of+ethanol+kinetics+in+rat+brain.">In vivo quantification of ethanol kinetics in rat brain.</a> Neuropsychopharmacology. 31 (12), 2683-2691 (2006).</li><li>Quertemont, E., Green, H. L., Grant, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+ethanol+concentrations+and+ethanol+discrimination+in+rats:+effects+of+dose+and+time.">Brain ethanol concentrations and ethanol discrimination in rats: effects of dose and time.</a> Psychopharmacology. 168 (3), 262-270 (2003).</li><li>Flentke, G. R., Klinger, R. H., Tanguay, R. L., Carvan, M. J., Smith, 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=An+evolutionarily-conserved+mechanism+of+calcium-dependent+neurotoxicity.">An evolutionarily-conserved mechanism of calcium-dependent neurotoxicity.</a> Alcoholism: Clinical and Experimental Research. 38 (5), 1255-1265 (2014).</li><li>Reimers, M. J., Flockton, A. R., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol-+and+acetaldehyde-mediated+developmental+toxicity+in+zebrafish.">Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish.</a> Neurotoxicology and Teratology. 26 (6), 769-781 (2004).</li><li>Zhang, C., Ojiaku, P., Cole, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forebrain+and+hindbrain+development+in+zebrafish+is+sensitive+to+ethanol+exposure+involving+agrin,+Fgf,+and+sonic+hedgehog+function.">Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function.</a> Birth Defects Research Part A: Clinical and Molecular Teratology. 97 (1), 8-27 (2013).</li></ol>

The authors have nothing to disclose.

As a developmental model system, zebrafish are ideally suited to study the impact of environmental factors on development. They produce large numbers of externally fertilized embryos, which allows for precise timing and dosage paradigms in ethanol studies. This, combined with the live imaging capabilities and the genetic and developmental conservation with humans, make zebrafish a powerful model system for teratology studies. Described is a protocol for measuring embryonic ethanol concentrations in developing zebrafish embryos using head space gas chromatography.
Determining embryonic ethanol concentration is critical to understanding the impact of ethanol to the developing embryo. In rodent models, blood ethanol concentrations correlate to tissue concentrations26. However, it is not possible to assess blood ethanol levels in early embryonic development in a zebrafish. Head space gas chromatography has been used to measure ethanol levels in various experimental paradigms, including zebrafish25,30,31,32,33,34,35. However, this protocol can be used to measure many different environmental factors, even though the volatility of these various factors needs to be high enough to measure quantitatively and a gas chromatograph column needs to be chosen based on the polarity of the compounds in question. In addition, zebrafish are best suited to study the impact of water-soluble factors, because exposure to hydrophobic factors is extremely difficult to analyze in fish.
This protocol will allow researchers to directly assess ethanol concentrations in zebrafish embryos. However, several factors have to be addressed to ensure accurate measurements of embryonic ethanol concentrations. Because ethanol levels in total embryonic tissues were measured, embryonic volume had to be considered. After 10 hpf the embryo begins somitogenesis (somite formation) and is no longer a sphere (Figure 1). To assess embryonic volume, water displacement was used on 10 pooled embryos per sample. Each sample consisted of 10 embryos for two reasons: first, to reduce error from pipetting small volumes, and second, to average out small fluctuations in embryo volumes. When measuring embryonic volume, it is important to consider the precision of pipetting and weighing. Even the use of 10 embryos per sample is at the lower limit of precision for both the pipettes and the scale used to weigh the embryos. With the equipment available, using fewer than 10 embryos would have impacted the analyses. This does not limit researchers from using fewer embryos per sample as long as equipment precision is considered.
Embryonic volume is one factor impacting sample analysis. A second key factor is embryonic processing speed, because ethanol is volatile. In addition, ethanol quickly equilibrates in multiple animal studies25,36,37. Our wash protocol is designed to quickly remove any ethanol adhering to the exterior surface of the chorion. In this study this wash protocol did not impact the embryonic ethanol levels, though longer or multiple washes could impact embryonic ethanol concentrations25,38. Yet a third factor to take into account is the dilution of the embryonic volume during processing. This protocol uses a protease cocktail to degrade the chorion as well as 5 M NaCl to denature all proteins, including alcohol processing enzymes.
Finally, a range of analyses and reporting methods have been described in a wide variety of zebrafish studies. Due to variation within different samples of a study or comparing different studies, proper reporting of ethanol concentration is critical25,38. Methods using indirect (usually enzymatic measures) rely on analysis of a secondary metabolite. In that case, the rate of enzymatic activity can vary and impede precise analysis of ethanol concentration. This method directly measures ethanol levels, and ethanol concentrations are reported as a ratio of embryo to media concentrations. Our results are in line with a growing consensus of a 24 hpf embryo containing ~30% of media concentrations (Table 3)25,38,39,40. Surprisingly, this 30% embryonic ethanol concentration is independent of media concentration, and it is not well understood what creates this equilibrium25,38. Embryos older than 24 hpf show decreases in ethanol concentrations, with 48 hpf embryos containing nearly half of the ethanol content of a 24 hpf embryo25. However, our results do not determine the ethanol-water equilibrium. Given the speed at which ethanol equilibrates, as well as the volatility of ethanol, it is incredibly difficult to assess water volume changes in the embryo itself due to ethanol exposure. Trying to measure the volume of an ethanol treated embryo sample using water displacement will result in loss of ethanol from the embryo during the measurement. Using more precise microscopy and spectroscopy methods than those described above, it may be possible to determine the ethanol-water equilibrium in an embryo by directly measuring both at the same time.
Overall, the protocol described here is a powerful tool with which to assess embryonic ethanol concentrations in developing zebrafish embryos. This information is critical in standardizing FASD studies using zebrafish. The ability to precisely control ethanol treatment paradigms and directly quantify embryonic ethanol concentrations strengthens the functionality of the zebrafish model for human FASD studies. Ultimately, this work will greatly improve our understanding of FASD.

Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of neurological impairments and craniofacial dysmorphologies associated with embryonic ethanol exposure1. Multiple factors, including timing and dosage of ethanol exposure and genetic background, contribute to the variation of FASD2,3. In humans, the complex relationship of these variables makes studying and understanding the etiology of FASD challenging. Animal models have proven crucial in developing our understanding of the mechanistic basis of ethanol teratogenicity. A wide variety of animal model systems has been used to study multiple aspects of FASD and results have been remarkably consistent with what is found in exposure in humans4. Rodent model systems are used to examine many aspects of FASD, with mice being the most common5,6,7. The majority of this work has focused on developmental defects to early ethanol exposure8, though later exposure to ethanol has been shown to cause developmental anomalies as well9. Moreover, the genetic capabilities of mice have greatly aided in our ability to probe the genetic underpinnings of FASD10,11. These studies in mice strongly suggest that there are gene-ethanol interactions with the sonic hedgehog pathway, retinoic acid signaling, Superoxide dismutase, Nitric oxide synthase I, Aldh2 and Fancd28,10,11,12,13,14,15,16,17,18,19,20,21. These studies show that animal models are critical to advancing our understanding of FASD and its underlying mechanisms.
The zebrafish has emerged as a powerful model system to examine many aspects of ethanol teratogenesis22,23. Due to their external fertilization, high fecundity, genetic tractability, and live imaging capabilities, zebrafish are ideally suited to study factors such as timing, dosage, and genetics of ethanol teratogenesis. Ethanol can be administered to precisely staged embryos and the embryos can then be imaged to examine the direct impact of ethanol during developmental processes. This work can be related directly to humans, because the genetic programs of development are highly conserved between zebrafish and humans and can therefore help guide FASD human studies24. While zebrafish have been used to examine ethanol teratogenesis, a lack of consensus in reporting embryonic ethanol concentrations makes comparison to humans difficult25. In mammalian systems, blood-alcohol levels correlate directly to tissue ethanol levels26. Many of the zebrafish studies treat embryos before complete formation of their circulatory system. With no maternal sample to examine, a process to assess ethanol concentrations is required to quantify ethanol levels within the embryo. Here we describe a process to quantify ethanol concentrations in a developing zebrafish embryo using head space gas chromatography.

<table><tbody><tr><td>Air</td><td></td><td></td><td>Provided by contract to the university</td></tr><tr><td>Analytical Balance</td><td>VWR</td><td>10204-962</td><td></td></tr><tr><td>AutoSampler, CP-8400</td><td>Varian</td><td></td><td>Gas Chromatograph Autosampler</td></tr><tr><td>Calcium Chloride</td><td>VWR</td><td>97062-590</td><td></td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>2701</td><td></td></tr><tr><td>Gas chromatograph vial with polytetrafluoroethylene/silicone septum and plastic cap 2 mL</td><td>Agilent</td><td>8010-0198</td><td>Can reuse the vials after cleaning, but not the caps/septa</td></tr><tr><td>Gas Chromatograph, CP-3800</td><td>Varian</td><td></td><td></td></tr><tr><td>Helium</td><td></td><td></td><td>Provided by contract to the university</td></tr><tr><td>HP Innowax capillary column</td><td>Agilent</td><td>19095N-123I</td><td>30 m x 0.53 mm x 1.0 &amp;mu;m film thick</td></tr><tr><td>Hyrdogen</td><td></td><td></td><td>Provided by contract to the university</td></tr><tr><td>Magnesium Sulfate (Heptahydrate)</td><td>Fisher Scientific</td><td>M63-500</td><td></td></tr><tr><td>Microcentrifuge tube 1.5 mL</td><td>Fisher Scientific</td><td>2682002</td><td></td></tr><tr><td>Micropipette tips 10 &amp;mu;L</td><td>Fisher Scientific</td><td>13611106</td><td></td></tr><tr><td>Micropipette tips 1000 &amp;mu;L</td><td>Fisher Scientific</td><td>13611127</td><td></td></tr><tr><td>Micropipette tips 200 &amp;mu;L</td><td>Fisher Scientific</td><td>13611112</td><td></td></tr><tr><td>Petri dishes 100 mm</td><td>Fisher Scientific</td><td>FB012924</td><td></td></tr><tr><td>Pipetman L p1000L Micropipette</td><td>Gilson</td><td>FA10006M</td><td></td></tr><tr><td>Pipetman L p200L Micropipette</td><td>Gilson</td><td>FA10005M</td><td></td></tr><tr><td>Pipetman L p2L Micropipette</td><td>Gilson</td><td>FA10001M</td><td></td></tr><tr><td>Polytetrafluoroethylene/silicone septum and plastic cap</td><td>Agilent</td><td>5190-7021</td><td>Replacement caps/septa for gas chromatograph vials</td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>P217-500</td><td></td></tr><tr><td>Potassium Phosphate (Dibasic)</td><td>VWR</td><td>BDH9266-500G</td><td></td></tr><tr><td>Pronase</td><td>VWR</td><td>97062-916</td><td></td></tr><tr><td>Silica Beads .5 mm</td><td>Biospec Products</td><td>11079105z</td><td></td></tr><tr><td>Silica Beads 1.0 mm</td><td>Biospec Products</td><td>11079110z</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>VWR</td><td>BDH9280-500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Sodium Phosphate (Dibasic)</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>Solid-phase microextraction fiber assembly Carboxen/Polydimethylsiloxane</td><td>Millipore Sigma</td><td>57343-U</td><td>Replacement fibers</td></tr><tr><td>Star Chromatography Workstation</td><td>Varian</td><td></td><td>Chromatography software</td></tr><tr><td>Thermogreen Low Bleed (LB-2) Septa</td><td>Millipore Sigma</td><td>23154</td><td>Replacement inlet septa</td></tr></tbody></table>

quantification of ethanol levels in zebrafish embryos using head space gas chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

Blood ethanol levels cannot be determined in early embryonic zebrafish, because they lack a fully formed circulatory system. To determine the level of ethanol concentration in the zebrafish embryos, the ethanol levels are measured directly from homogenized embryonic tissue. To properly measure the embryonic ethanol concentrations, the embryonic volume has to be taken into account. The embryo (yolk attached) sits inside the chorion (eggshell) surrounded by extraembryonic fluid (Figure 1). Any volume measure of the embryo has to be made on embryos at the time of exposure, because embryonic volume will change over developmental time as the embryo increases in size. In addition, how the embryo is processed has to be taken into account as well as the embryonic volume, because both of these elements create dilution factors used to calculate embryonic ethanol levels.
Because the embryo is not a perfect sphere, especially after 10 hpf, water displacement was used to assess embryonic volume. This study used embryos at 24 hpf. The volume of embryos both in their chorion and those removed from their chorion were measured. These volumes were 1.97 (&#177; 0.4 SD) &#956;L for the embryo with extraembryonic fluid, and 1.1 (&#177; 0.22 SD) &#956;L for the embryo alone (Table 1). This difference between the embryo with extraembryonic fluid and the embryo alone is the volume of the extraembryonic fluid, which surrounds the embryo inside the chorion (Figure 1). This difference is critical in determining the ethanol concentration of the embryo alone in the samples. From the analysis, the embryo comprised 56% of the total of the volume of the embryo with extraembryonic fluid inside the chorion (Table 1). As the embryo grew, the embryonic volume increased over time, resulting in a decrease in the volume of the extraembryonic fluid25. When measuring ethanol concentrations from embryos still in their chorion, the water content in both the embryo and the extraembryonic fluid must be taken into account.
Previously published work has shown that the water fraction in the embryo alone is 73.6%29. This result was obtained using a combination of volume measures, electron spin resonance spectroscopy, and magnetic resonance microscopy. To confirm this result, embryos were weighed alone before and after baking at 70 &#176;C, as previously described25. This process removes all water from the embryo. The change in weight represents the amount of water in the embryo. From this analysis, a 24 hpf embryo contained ~72% water while a 48 hpf embryo contained ~76%. Both of these values are extremely similar to the 73.6% embryonic water volume previously calculated29. This suggests that the water volume of the embryo changes little throughout early development.
Based on the published work using multiple approaches to calculate water volume in the embryo, 73.6% was used as the embryonic water content for all of the presented analyses. This resulted in water comprising 0.82 &#956;L of the 1.1 &#956;L of embryonic volume (Table 1). From this, the volume of the extraembryonic fluid was calculated as 0.86 &#956;L. Of the total water volume of the embryo with extraembryonic fluid, 49% is contained in the embryo and 51% is in the extraembryonic fluid (Table 1).
For the gas chromatography analysis, only 24 hpf embryos still in their chorion were used (Figure 1). In this ethanol regimen, embryos were treated with 1% (171 mM) ethanol media concentration from 6&#8211;24 hpf, though different ethanol concentrations and exposure time windows can be used depending on experimental design. Two groups of 15 samples (10 embryos per sample) and the media with which they were treated over 2 different experimental days (Table 2) were analyzed. Media levels can vary from experiment to experiment. To account for this, media ethanol levels were measured 5x per experimental group. In this analysis, media levels were 143.6 (&#177; 2.3 SD) mM for group 1 and 133.6 (&#177; 2.7 SD) mM for group 2. These values are significantly different using an unpaired t test (p = 0.0002). The embryos with extraembryonic fluid averaged 63.5 (&#177; 17.1 SD) mM and 53.1 (&#177; 12.6 SD) mM for groups 1 and 2, respectively. However, the concentration was not significantly different between the 2 groups of embryos with extraembryonic fluid (unpaired t test, p = 0.07). Untreated control embryos were measured at 0 mM. Due to the variation in media levels, a ratio of embryonic ethanol concentration/media levels of ethanol for each sample was calculated. Group 1 had 44% of the media ethanol levels, while group 2 had 40% of the media ethanol levels (Figure 3 and Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60766/60766fig01.jpg" /> 
Figure 1: An image of an embryo at 24 h postfertilization (hpf) inside the chorion. The embryo and the yolk are surrounded by the extraembryonic fluid, all located inside the chorion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60766/60766fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60766/60766fig02.jpg" /> 
Figure 2: Protocol to measure embryonic volume and process the embryos for analysis. (A) Ten 24 hpf embryos transferred to a 1.5 mL microcentrifuge tube marked at a 250 &#956;L volume. (B) The microcentrifuge tube with embryos filled with water (water is dyed so it is easier to see at the 250 &#956;L mark). (C) All of the water is removed from the tube and weighed. (D) Ten embryos transferred to a 1.5 mL microcentrifuge tube. The water was removed as in (C) and replaced with 50 &#956;L of protease cocktail. (E) After 10 min, 450 &#956;L of 5 M NaCl was added to the tube from (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60766/60766fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60766/60766fig3a.jpg" /> 
Figure 3: Box and whisker plot of the ratio of ethanol concentration of samples to media. Ethanol concentration for each group was divided by the average of the media samples of that group. Group 1 contained 44% of media levels, while group 2 contained 40%. In both groups, n = 15; 10 embryos per sample. Groups were compared using a one-way ANOVA with a Tukey&#39;s post hoc analysis (****p &#60; 0.0001). The whiskers represent minimum to maximum. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60766/60766fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="7">Embryo and Embryo + Extraembryo Water Volumes (&#181;L)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td colspan="4">Watera</td>
		</tr>
		<tr>
			<td></td>
			<td>Total Volumeb</td>
			<td>% of Total Volume (V)</td>
			<td>Embryonic Volume (W)</td>
			<td>Extraembryonic Volume (X)</td>
			<td>% Water in Embryo (Y)</td>
			<td>% Water in Extraembryonic (Z)</td>
		</tr>
		<tr>
			<td>Embryo + Extraembryonic (EE)c</td>
			<td>1.97 &#177; 0.4</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embryo 24 hpf (E)c</td>
			<td>1.11 &#177; 0.22</td>
			<td>56%</td>
			<td>0.82</td>
			<td>0.86</td>
			<td>49%</td>
			<td>51%</td>
		</tr>
		<tr>
			<td colspan="7">Calculations</td>
		</tr>
		<tr>
			<td colspan="7">VEmbryo % of Total volume = Total Volume of E &#247; Total Volume of EE</td>
		</tr>
		<tr>
			<td colspan="7">WEmbryonic Water Volume = V &#215; 73.6%</td>
		</tr>
		<tr>
			<td colspan="7">XExtraembryonic Water Volume = W &#8211; Total Volume of EE</td>
		</tr>
		<tr>
			<td colspan="7">Y% Water in Embryo = (W + X)/W &#215; 100</td>
		</tr>
		<tr>
			<td colspan="7">Z% Water in Extraembryonic = 100 &#8211; Y</td>
		</tr>
		<tr>
			<td colspan="7">aEmbryonic water volume was calculated from Hagedorn et al.29.</td>
		</tr>
		<tr>
			<td colspan="7">bValues are reported as &#177; standard deviation (SD).</td>
		</tr>
		<tr>
			<td colspan="7">cn = 13; 10 embryos per sample</td>
		</tr>
	</tbody>
</table>
Table 1: Calculation of water volume for the embryos with extraembryonic fluid. The volume was calculated from 13 samples (10 embryos per sample) from 24 hpf embryos with extraembryonic fluid still in the chorion and embryos removed from their chorion. Values are reported as the mean &#177; SD.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Ethanol Concentration of embryos and media (mM)a</td>
		</tr>
		<tr>
			<td></td>
			<td>Group 1</td>
			<td>Group 2</td>
			<td>Combined</td>
		</tr>
		<tr>
			<td>Media (M)b</td>
			<td>143.6 &#177; 2.3</td>
			<td>133.6 &#177; 2.7</td>
			<td>138.6 &#177; 5.8</td>
		</tr>
		<tr>
			<td>Embryo + Extraembryonic (EE)c</td>
			<td>63.5 &#177; 17.1</td>
			<td>53.1 &#177; 12.6</td>
			<td>58.3 &#177; 15.7</td>
		</tr>
		<tr>
			<td>Ratio of ethanol - embryo to media (1)</td>
			<td>0.44 &#177; 0.12</td>
			<td>0.40 &#177; 0.09</td>
			<td>0.42 &#177; 0.11</td>
		</tr>
		<tr>
			<td colspan="4">Calculations</td>
		</tr>
		<tr>
			<td colspan="4">1Ratio of ethanol = EE &#247; M</td>
		</tr>
		<tr>
			<td colspan="4">aValues are reported as &#177; SD.</td>
		</tr>
		<tr>
			<td colspan="4">bn = 5</td>
		</tr>
		<tr>
			<td colspan="4">cn = 15; 10 embryos per sample</td>
		</tr>
		<tr>
			<td colspan="4">dValues are the average of groups 1 and 2.</td>
		</tr>
	</tbody>
</table>
Table 2: Calculations of ethanol concentration in embryos with extraembryonic fluid and media (mM). Media samples were direct measures from the media (n = 5 per group). Embryonic ethanol concentration was calculated from 15 samples (10 embryos per sample) from 24 hpf embryos with extraembryonic fluid. The ratio of the ethanol concentrations of the embryos with extraembryonic fluid to media were calculated. Values are reported as the mean &#177; SD.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Calculation of ethanol in the embryoa,b</td>
		</tr>
		<tr>
			<td>Embryo (Y)</td>
			<td>Ratio to media (Z)</td>
		</tr>
		<tr>
			<td>32.7 &#177; 8.8 mM</td>
			<td>0.24 &#177; 0.06</td>
		</tr>
		<tr>
			<td colspan="2">Calculations</td>
		</tr>
		<tr>
			<td colspan="2">YEmbryonic Ethanol = 58.3 mM (Table 2) &#215; 56% (Table 1)</td>
		</tr>
		<tr>
			<td colspan="2">ZRatio of ethanol = Y &#247; 138.6 mM (Table 2)</td>
		</tr>
		<tr>
			<td colspan="2">aValues are the average of groups 1 and 2.</td>
		</tr>
		<tr>
			<td colspan="2">bValues are reported as &#177; SD.</td>
		</tr>
	</tbody>
</table>
Table 3: Calculation of ethanol in the embryo. Embryonic ethanol concentration was calculated as 56% of the total ethanol concentration of the embryo with extraembryonic fluid. This was then reported as a ratio of embryo to media. Values are reported as the mean &#177; SD.

Watch this Scientific Journal Video about Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography at JoVE.com

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

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Developmental Biology

5.6K Views.  University of Louisville. This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Video: Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genetics

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Medicine

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Immunology and Infection

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Neuroscience

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Behavior

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Summary

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Chapters in this video

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Metabolic Profile Analysis of Zebrafish Embryos