Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The overall goal of this procedure is to produce haplo zebra fish embryos by in vitro fertilization. This is accomplished by first isolating mature sperm from adult male zebra fish and then performing ultraviolet irradiation to deactivate the paternal genomes present in the sperm samples. The second step is to isolate unfertilized eggs from an adult female zebra fish.

Next, the XR fertilized with the UV inactivated sperm sample. The final step is to rear the haploid clutch until developmental analysis such as a morphology assay or gene expression analysis using whole mount C two hybridization. The main advantage of using haplo zebrafish embryos is that this technique enables efficient forward genetic screens that use fewer generations, thus saving time and animal space.

This method can help answer fundamental questions in the development field, such as identifying genes that are essential for kidney formation. Generally, individuals new to this method will struggle because the collection of zebrafish gametes can have technical challenges. Visual demonstration of this method is critical because it will help researchers to learn how to handle adult zebrafish to collect mature game meets.

To begin prepare the Hank Stock Solutions and Hank's pre-mixed solution. Next, select the desired number of adult zebrafish females for haplo clutch collection. Once selected, prepare mating cages by placing each adult zebrafish female with an adult zebrafish male in a chamber of system water so that they are separated by a divider overnight.

First, make fresh Hank stock solution number six, by adding 0.35 grams of sodium bicarbonate to 10 milliliters of distilled water. Mix well to dissolve. Next, make the buffered Hanks final working solution in a tube over ice.

After mixing, prepare 500 microliter aliquots in micro centrifuge tubes and place them on ice to collect enough sperm for fertilization. Select four to five male zebrafish for each sperm harvest. Once euthanized, carefully blot the male dry of excess liquid to prevent triggering the activation of sperm.

After decapitating and clearing the abdominal cavity, locate the testes along the dorsal body wall lateral to the swim bladder. They have a white opaque appearance when viewed under this stereo microscope. Once located, use fine forceps to remove the testes taking care to keep them intact.

Place the testes into the sperm solution and store on ice. Repeat these steps until all the males have been dissected. Once all of the samples are collected, homogenize the testes by gently grinding the tissue with a sterile micro tube pestle.

Transfer the supernatant to a small Petri dish on ice. Avoid transferring tissue debris along with the supernatant solution. Since this will create shadows and compromise the UV activation of sperm.

Rinse the tube with between 300 to 400 microliters of additional hank's spinal working solution and add this supernatant wash to the small Petri dish. Next, expose the dish to UV from a bench lamp for a total of two minutes at a distance of 15 inches from the source. Return the dish to the ice bucket and then transfer the UV inactivated sperm to a fresh micro centrifuge tube stored on ice.

Begin by confirming the depth of anesthesia in females selected for a collection. Once an anesthetized, carefully remove excess solution to avoid triggering egg activation. Before the addition of sperm, place the female on her side in a clean Petri dish and visualize her belly under the stereo microscope while supporting the female's back by placing one or two fingers from one hand behind the dorsal body wall.

Stroke the belly gently for approximately 10 to 20 seconds using one or two fingers from the other hand to squeeze eggs from the abdomen. The eggs should come out very easily upon stroking the female's abdomen. If the eggs do not emerge with ease, it is not advisable to push harder and force the eggs from the female as this may cause the female harm.

Once extracted, use a fine probe or small spatula to scoop the X away from the female. Gently lift the female and return her to an appropriately labeled isolation tank containing fish system water. Next, add 50 microliters of UV inactivated sperm solution to the clutch of eggs and incubate at room temperature for 30 seconds.

During this time, affix a corresponding label to the dish to track it with the number assigned to the female parent. Next, add one milliliter of E three solution to the eggs and incubate at room temperature for one minute. Lastly, add sufficient E three solution to fill the dish midway and place the embryos at 28 degrees Celsius for subsequent incubation.

After four to eight hours, retrieve the dish and remove any unfertilized embryos. Unfertilized embryos can display a white opaque appearance or a transparent appearance with a misshapen single cell. Next, wash the remaining embryos by replacing the E three embryo media with fresh E three solution.

The embryos are then incubated until the desired time, point of interest, and can be used in a variety of research applications. This image shows diploid wild type embryos produced by natural spawning and then incubated until approximately 30 HPF. Here haplo embryos were produced by in vitro fertilization and exhibit a range of abnormalities in development.

Relatively normal haplos had a shortened more stock body analogous to the grading scheme of an A haplo, while haplos with gross abnormalities exhibited a severely truncated body axis corresponding to a grade of B.And finally, grade C represents poor quality. Haplos haplo wild types displayed a normal pattern in embryonic kidney structure compared to diploid mutants. The segmented pattern of discrete cell types are visualized by targeting gene transcripts that are unique to the differentiated nephron cell types.

After watching this video, you should have a good understanding of how to produce haplo zebrafish embryos by in vitro fertilization. While attempting this procedure, it is important to remember to handle the adult zebrafish gently, particularly when collecting eggs from the females. Following this procedure, other methods can be performed such as whole Mountain C two hybridization in order to assess the development of cell types such as the segments of the pro naro.

Don't forget that working with ultraviolet radiation sources can be extremely hazardous, and precautions such as eye protection should always be taken while performing this procedure.