Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Summary

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Abstract

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Introduction

Ex ovo culturing has played an important role in the study of development of the chicken^{1, 2}. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes ^{3, 4, 5}.

There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup^6,7,8 or a glass bowl⁵. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate hum....

Protocol

Note: All supplies are listed in Table 1.

1. Storing the Chicken Embryos

1. Incubate chicken eggs of the strain Gallus gallus horizontally at 37^oC with approximately 40% humidity and turn eggs once or twice daily. Turning eggs is important to prevent the embryo from adhering to the eggshell.
2. Do not to turn the egg in the 24 hr prior to setting up the culture as otherwise the embryo will be located ventral to the yolk mass and will be damaged on opening the egg in step 3. In addition, keep the eggs at 4^oC degrees for no more than one week before incubation to “halt” developme....

Results

This ex ovo method allows for the observation of embryos from early stages of development (HH 19/20) to late stages of development (HH 40-41) (Figure 1A and 1B). Setting up the culture at HH 19-20 increases survivability of the embryos in the culture. Prior to the head turning (before 53 hpf) survivability is very low in culture and after stage 21, the embryo tends to stick more to the shell on removal so fewer intact embryos are obtained. In general, survivability of the embryo.......

Discussion

Ex ovo culturing and windowing both have advantages and challenges. Here we compare the advantages and challenges of the Styrofoam cup ex ovo method and the windowing method to our optimized ex ovo method shown here. Our method enables manipulation and easy observation of the chick embryo at late stages of development and our refinements to the traditional ex ovo method^{1, 2, 3} make it additionally very easy to use in undergraduate teaching laboratory classes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Penicillin/Streptomycin	Sigma	P4458	Make small aliquots to avoid freeze/thaw events
Square Petri Dish			9.5 cm x 9.5 cm
Weigh Boat	Fischer Scientific	8732113	88 x 88 x 23 mm
Ziplock container	Ziplock	N/A	12 cm x 12 cm x 6 cm