Summary

Geoptimaliseerd<em> Ex-ovo</em> Het kweken van kippenembryo's tot geavanceerde stadia van ontwikkeling

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

Opmerking: Alle leveringen zijn opgenomen in tabel 1. 1. Het opslaan van de kippenembryo's Incubeer kippeneieren van de stam Gallus gallus horizontaal bij 37 ° C met ongeveer 40% vochtigheid en draai eieren of tweemaal daags. Het draaien van de eieren is belangrijk om te voorkomen dat het embryo uit het naleven van de eierschaal. Niet om het ei draaien in de 24 uur voorafgaand aan het opzetten van de kweek anders de embryo zich ventraal van de do…

Representative Results

Deze ex ovo methode maakt het mogelijk voor de observatie van embryo's uit de vroege stadia van ontwikkeling (HH 19/20) tot de late stadia van ontwikkeling (HH 40-41) (Figuur 1A en 1B). Het opzetten van de cultuur bij HH 19-20 verhoogt de overlevingskansen van de embryo's in de cultuur. Voorafgaand aan de kop te draaien (vóór 53 HPF) overlevingskansen is zeer laag in de cultuur en na etappe 21, het embryo heeft de neiging om meer te houden aan de shell op verwijdering…

Discussion

Ex ovo kweken en vensteropdrachten beide hebben voordelen en uitdagingen. Hier vergelijken we de voordelen en uitdagingen van het plastic bekertje ex ovo-methode en de window-methode om onze geoptimaliseerde ex ovo methode hier getoond. Met onze methode kunnen manipulatie en gemakkelijke observatie van het kippenembryo in de late stadia van de ontwikkeling en onze verfijningen aan de traditionele ex ovo methode 1, 2, 3 maken het bovendien zeer makkelijk te gebruiken in unde…

Disclosures

The authors have nothing to disclose.

Acknowledgements

We willen graag Paul Poirier, de Media Producer, bij Mount Saint Vincent University bedanken voor zijn werk in het filmen en bewerken van de video deel van dit manuscript. Wij erkennen de Natural Science and Engineering Research Council van Canada voor financiering.

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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