Оптимизированный<em> Экс-ово</em> Культивирование куриных эмбрионов в продвинутых стадиях разработки

The overall goal of this method is to present an optimized X ovo culture technique for chicken embryos, which enables the observation of embryonic development from hamburger and Hamilton Stage 19 into late stages of development up to stage 40. This method is useful for students in a developmental biology laboratory setting, as well as researchers who want to use the chicken embryo as a model organism to set up this X ovo culture method. First, the eggs are prepped.

The eggs have been incubated at 37 degrees Celsius with 40%humidity for approximately three and a half days, which roughly correlates to hamburger and Hamilton stage 19. The eggs are prepared by spraying them with 70%ethanol so that when the shell is cracked, the embryo is not contaminated. After the eggs have been sprayed, they’re left momentarily to dry.

While the humidity chamber is prepped, the humidity chamber is made out of a Ziploc container. The first step in building the humidity chamber is to add a one to two centimeter layer of absorbent padding. In this case, Kim wipe, although layers of cotton padding can be used as well.

This layer of absorbent padding fits into the base of the Ziploc container shown here. Once The absorbent layer is in place, approximately 150 milliliters of distilled water is poured into the base of the container. The majority of this water should be absorbed by the Padding.

The humidity chamber is now complete and can be placed to the side. A small, sterile way boat is used to cradle the embryo when it is removed from the shell. The way boat measures 8.8 centimeters squared due to the orientation of the eggs in the incubator, the embryo is located on the top surface of the egg, and therefore the shell should be cracked on the opposite side.

The shell is then gently cracked and the egg is placed very close to the way boat. In one smooth motion, the egg is pulled apart, releasing the embryo gently into the way Boat. The embryo Is then inspected to ensure the yoke is still intact and that the embryo is viable, as shown here with a visible heartbeat and no obvious abnormalities.

Next, we add an antibiotic to the embryo to help fight infection. Penicillin streptomycin at a concentration of 5, 000 units of penicillin and five milligrams of streptomycin per mil is allotted to minimize the frequency of freeze thaw.Events. 40 microliters is taken from the aliquot and added to the surrounding albumin using a pipette.

Now the embryo is ready to be carefully placed into the humidity chamber. Once in the humidity chamber, the embryo is given a lid to help prevent contamination. A square Petri dish lid measuring nine and a half centimeters squared is placed over the way boat.

It is important to ensure that there is no moisture on the rim of the way boat so that a seal is not formed between the Petri dish and the way boat, which would restrict oxygen to the embryo. Then to complete the humidity chamber, a lid is placed on the Ziploc container. However, only two corners of the lid are pressed into place, leaving the other corners open and allowing airflow to reach the embryo.

Once the humidity chamber is complete, it is placed in a 37 degree incubator so the embryo can continue developing after four and a half days. The humidity chamber can be removed from the incubator and the embryo can be observed at approximately stage 34. At this point, students Can easily observe the embryo, including the vasculature, the large eye, and the limb buds.

The embryo can be further observed under a dissecting microscope, and more advanced research can be performed such as embryonic manipulations. After observing the embryo at this stage, it can be returned to a 37 degree incubator and would continue to develop until at least stage 40, which is 14 days of incubation. In conclusion, This method shows the optimization of culturing chick embryos outside of the eggshell.

It provides an opportunity for students to observe chick embryo development in a busy lab environment, and in addition, provides researchers access to embryos if they’re doing Manipulations.