Summary

Optimizado<em> Ex-ovo</em> El cultivo de embriones del polluelo a etapas avanzadas de desarrollo

Published: January 24, 2015
doi:

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 chicken1, 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 cup6,7,8 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 humidity6. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses.

The above challenges in the ex ovo method has meant that researchers turn to the windowing method 9,10 to view embryonic chick development. In this technique, a hole or “window” is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax9 to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 – HH 27), viewing the entire embryo at late stages is not easily accomplished.

Here, we describe an improved and simple ex ovo culturing technique11 that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

Protocol

Nota: Todos los suministros se enumeran en la Tabla 1. 1. Almacenamiento del pollo embriones Incubar huevos de gallina de la cepa de la especie Gallus gallus horizontalmente a 37 ° C, con aproximadamente 40% de humedad y girar los huevos una vez o dos veces al día. Volviendo huevos es importante para evitar que el embrión se adhiera a la cáscara del huevo. No girar el huevo en la 24 horas antes de la creación de la cultura de lo contrario el embr…

Representative Results

Este ex ovo método permite la observación de los embriones desde las primeras etapas de desarrollo (HH 19/20) a las etapas finales de desarrollo (HH 40-41) (Figura 1A y 1B). La creación de la cultura en HH 19-20 aumenta la supervivencia de los embriones en la cultura. Antes de la cabeza de giro (antes de 53 hpf) de supervivencia es muy baja en cultivo y después de la etapa 21, el embrión tiende a pegarse más a la cáscara en la eliminación de por lo menos se obtienen emb…

Discussion

Ex ovo cultivo y de ventanas ambos tienen ventajas y desafíos. Aquí se comparan las ventajas y desafíos de la taza de espuma de poliestireno ex ovo método y el método de ventanas a nuestro ex optimizado ovo método que se muestra aquí. Nuestro método permite la manipulación y la fácil observación del embrión de pollo en las etapas finales de desarrollo y nuestros refinamientos al ex ovo tradicional método 1, 2, 3 lo hacen, además, muy fácil de utiliza…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Nos gustaría dar las gracias a Paul Poirier, el Productor de Medios, en la Universidad Mount Saint Vincent por su trabajo en la filmación y la edición de la parte de vídeo de este manuscrito. Reconocemos el de Ciencias Naturales e Ingeniería de Investigación de Canadá para su financiación.

Materials

Penicillin/Streptomycin Sigma P4458 Make small aliquots to avoid freeze/thaw events
Square Petri Dish N/A N/A 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

References

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Cite This Article
Cloney, K., Franz-Odendaal, T. A. Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development. J. Vis. Exp. (95), e52129, doi:10.3791/52129 (2015).

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