This protocol is significant because it helps in understanding the pharmacokinetics of ethanol in developing zebrafish embryos and this will help standardize ethanol exposure regimes in zebrafish studies. The main advantage of our technique is that it allows researchers to rapidly and reproducibly measure ethanol levels during embryonic development, particularly in embryos where blood supply is not available. This method can be easily applied to other model systems as well as other environmental factors assuming proper technique and equipment setup.
I would expect individuals performing this protocol for the first time to struggle with the speed necessary to prevent ethanol evaporation during embryo processing. Begin by transferring 10 embryos with extra embryonic fluid into 1.5 milliliter microcentrifuge tubes marked with a line at a volume of 250 microliters. Add water to the fill line.
Prepare additional microcentrifuge tubes for dechorionated samples. To remove the chorion, incubate the embryos in a 100 millimeter Petri dish with two milligrams per milliliter of protease cocktail in embryo media for 10 minutes. Gently swirl the dish every few minutes to break the chorion.
Once all the embryos are free of the chorion, remove them from the protease cocktail and wash them twice with fresh media. Then transfer the embryos to a fresh 100 millimeter dish. When all samples are ready for volume measurement, use a P200 micropipette with the smallest tip possible to carefully remove all liquid from around the embryos.
Weigh the water with a scale with less than 0.1 milligram precision, then determine the volume of the 10 embryos by subtracting the weight of the removed water from 250 microliters. To determine the volume of a single embryo, divide the difference by 10. Gather the embryos from the mating tanks and place them in a standard 100 millimeter Petri dish then incubate the dish at 28.5 degrees Celsius.
At six hours post fertilization, transfer up to 100 embryos to a new dish with embryo media and 1%ethanol or media alone. Cover but to not seal the Petri dishes and place them in a 28.5 degree Celsius incubator for 18 hours or until they reach 24 hours post fertilization. Use two P200 micropipettes set to 50 microliters and aspirate the protease cocktail solution into one and water into the second.
Then quickly place 10 embryos in a 1.5 milliliter microcentrifuge tube using a glass pipette and close the cap. Repeat this process for all samples to be tested, controls and ethanol treated embryos. Working with one sample at a time, quickly open the cap and aspirate all residual embryo media with a P200 micropipette set to 200 microliters.
Rapidly but gently add and remove the 50 microliters of water, then add the 50 microliters of protease cocktail and close the tube cap. Let the sample sit for 10 minutes at room temperature then quickly add 450 microliters of five molar sodium chloride and close the tube cap. Homogenize the sample by vortexing for 10 minutes and transfer two microliters of supernatant to a gas chromatography vial.
Seal the vial with a polytetrafluoroethylene cap. After completing the startup method, load 436 current SPME ethanol two to five-minute RG run. meth from the methods menu in the analysis software for each sample on the sample list.
Fill out the sample list starting with the air, water, and five molar sodium chloride protease cocktail blanks. Then enter in the ethanol standards in order from 0.3125 to 40 millimolar. Follow the standards with the second round of blanks.
Enter all homogenized embryo supernatant samples to be tested from lowest to highest predicted ethanol concentration and finish by entering a third round of blanks. Add the gas chromatography vials to the autosampler in the order in which the samples were entered and allow them to warm for 10 to 15 minutes. Then start the sample runs using the software.
After all runs are completed, activate the shutdown method by adding a final sample in the sample list and running standby.meth. Back up all data acquired during the sample run. Once the shutdown method is complete, click on open chromatogram and open the folder with the results.
Samples are automatically integrated in this program. In the results, make sure the correct peaks have been integrated. Once all samples have been confirmed, print or export the results.
Plot the peak height of the ethanol standards on a graph, then calculate the slope, y-intercept, and r squared values. Obtain the ethanol value for each sample by subtracting the peak height of the sample from the y-intercept of the standards and dividing this value by the slope of the standards. To calculate the ethanol concentration in the embryos, calculate the dilution factor for each sample and multiply the sample ethanol value by this dilution factor.
Finally, calculate media reference samples by multiplying the media ethanol value by a dilution factor of 10. To properly calculate the embryonic ethanol concentrations, the volumes of embryos both in their chorion and removed from their chorion were measured. In this analysis, the embryo comprised 56%of the total volume inside the chorion.
Based on previously published work, 73.6%was used as the embryonic water content for all the presented analyses. This resulted in water comprising 0.82 microliters of the 1.1 microliters of embryonic volume. Of the total water volume, 49%is contained in the embryo and 51%is in the extraembryonic fluid.
For the gas chromatography analysis, two groups of 15 samples and the media with which they were treated measured five times per group were analyzed. Media ethanol levels were 143.6 millimolar for group one and 133.6 millimolar for group two. The ethanol concentrations of the embryos averaged 63.5 millimolar and 53.1 millimolar for groups one and two respectively.
A ratio of embryonic ethanol concentration to media levels of ethanol for each sample was calculated. Group one had 44%of the media ethanol levels while group had 40%of the media ethanol levels. Untreated control embryos were measured at zero millimolar media ethanol concentration.
The most important thing to remember when attempting this procedure is that processing the embryos for GC analysis properly is critical as ethanol is volatile and will evaporate quickly. The best way to perform this step is described, but it needs to be practiced to develop proper muscle memory. This procedure will help standardize ethanol treatment regimes in zebrafish and allow future work to dissect the genetic and cellular mechanisms of ethanal teratogenicity.
