The overall goal of this procedure is to treat zebrafish embryos with nitrite and determine its teratogenic effect. This method can help answer some of the key questions in the field of developmental toxicology such as assessing the teratogenic effects of chemicals on embryonic development. The main advantage of this procedure is that it uses zebrafish embryo which develop externally and can be observed under a microscope.
For the experiments demonstrated here maintain wild-type zebrafish strains, such as the Tuebingen strain, at 28.5 degrees Celsius and a light-dark cycle of 14 hours and 10 hours, respectively. The night before harvesting eggs, add fish water and dividers into mating tanks and place male and female fish on separate sides of the divider. To ensure that enough eggs will be produced set up 30 pairs of fish.
The next morning, after the lights turn on, remove the dividers to initiate mating. Check the mating tanks for eggs every 15 minutes. Once the fish lay eggs, use a tea strainer to harvest the embryos and combine them all into a large container with E3 buffer.
Incubate the eggs at 28.5 degrees Celsius. At 1.5 hours post fertilization, or HPF, under a dissecting microscope use a plastic transfer pipette to discard unfertilized eggs that will appear opaque. Transfer 50 embryos each into 100 by 15 millimeter glass petri dishes containing 50 milliliters of E3 buffer in triplicate for each treatment condition.
For example for 11 treatment conditions, prepare 33 dishes of 50 embryos each. To three petri dishes of embryos at 2 HPF remove the E3 buffer and add 50 milliliters of 300 millimolar ethanol diluted in E3 buffer. Cover the dishes with parafilm to minimize evaporation of ethanol.
Incubate the embryos in the ethanol buffer for 22 hours at 28.5 degrees Celsius, then remove the solution and use E3 buffer to wash out the ethanol, swirling the dish several times. Repeat the wash two more times. To 3 petri dishes of embryos at 2 HPF remove the E3 and replace with 50 milliliters of fresh E3.For nine petri dishes at 2 HPF remove the E3 and add 50 milliliters of 1, 000 milligrams per milliliter of sodium nitrite dissolved in E3 and incubate at 28.5 degrees Celsius, replacing the sodium nitrite solution daily.
After 46 hours, remove three of the nine sodium nitrite plates, remove the solution, and use E3 buffer to wash the plates three times. 24 hours later remove three different sodium nitrite plates from the incubater, remove the solution, and use fresh E3 buffer to wash the embryos three times. 24 hours later, perform similar wash steps for the last three plates.
Next, for another set of three petri dishes, replace the E3 buffer with 200 milligrams per milliliter of sodium nitrite, then set up groups of three dishes of 50 embryos each with 400, 600, 800, and 1, 000 milligrams per milliliter of sodium nitrite. Incubate the embryos for 70 hours, replacing the sodium nitrite solution daily. After the incubation, remove the solution from the plates, and use E3 buffer to wash the plates three times.
For another three petri dishes of embryos, replace the E3 buffer with 50 milliliters of 1, 000 milligrams per milliliter of sodium nitrate in E3 buffer. After exposing the embryos for 70 hours, use E3 to wash the embryos as previously demonstrated. During each day of exposure, under a stereo microscope count the number of dead embryos, looking for a lack of heartbeat and blood circulation, or a lack of mobility after one minute of observation.
Remove the dead embryos. At the end of the experiments, to euthanize the larvae, use a transfer pipette to remove the E3 buffer, then add 50 milliliters of 0.2%MS-222 and incubate for 10 minutes. After one E3 wash to remove the MS-222, leaving some volume to properly pipette the embryo, add the larvae to 4%PFA, swirl the dish several times, and use a transfer pipette to place the larvae into a glass vial with enough PFA to fill the vial.
Then fix the larvae in the refrigerator overnight. After fixation, use a stereoscope and a digital camera at 30X magnification to take pictures of the larvae. Orient the larvae so that the anterior is to the left and the dorsal surface is at the top of the field.
Exposure to 300 millimolar ethanol for 22 hours had no effect on survival, consistent with previous results. As shown here, observed phenotypes included pericardial edema and swim bladder non-inflation. Cranial facial defects, and developmental delay were also observed and are not shown here.
Treatment with sodium nitrite resulted in mild to severe effects on survival, depending on the concentration, with higher concentrations of sodium nitrite resulting in lower survival rates. This figure illustrates that at higher concentrations, such as 1, 000 milligrams per milliliter, longer incubation times also have a more severe impact on survival. Finally, as shown here, unlike nitrate treated fish, nitrite treated larvae have developmental defects.
Once mastered, this technique can be done in 3 hours and it's important to remember to remove the embryos daily to minimize the contamination. Following this procedure, other methods, like in situ hybridization, can be performed to assess gene expression. This technique is communion for researchers to explore teratogenic potentials of chemicals on development in zebrafish.
