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A shell-less chick embryo culturing technique, reproduced successfully under

local circumstances

Article  in  Journal of the College of Physicians and Surgeons--Pakistan: JCPSP · October 2008

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SHORT COMMUNICATION
A Shell-Less Chick Embryo
Culturing Technique,
Reproduced Successfully Under
Local Circumstances
Nusrat Zareen and Muhammad Yunus Khan
Animal models have long being playing essential roles
in the development of preventive, diagnostic and
therapeutic procedures. Studies relating to fetal
malformations require invasive investigations in
pregnant animals that may implicate tissue damage and
ethical considerations. It would, therefore, be interesting
to investigate such malformations by observing
developing embryos without invasive interventions. A
shell-less embryo culturing technique was successfully
reproduced at the laboratory of Anatomy Department,
Regional Centre CPSP, Islamabad, to cultivate chick
embryos outside their egg shells.
Shell-less culture is an embryo culture model, where the
intact in-vivo relationship between the embryo and the
yolk sac/albumen is preserved outside the egg-shell and
shell membranes, i.e. in an artificial experimental culture
container. Thus, it allows day-to-day direct observation
of the developing vertebrate embryo and also permits
experimental manipulations. It has the potential to
enhance knowledge on molecular and developmental
anatomy at both basic and clinical science level. More
so, shell-less culture of the chick embryo is a cost
effective, simple, efficient and dependable reproducible
model system to work with.
In this communication, the technique of shell-less
culture model system of the chick embryo is described.
These cultures were prepared using the technique
described by Hamamichi and Nishigori.1 The goal of this
project was to demonstrate shell-less chick embryo
culturing as a potential experimental model in the field of
developmental anatomy.
This project was undertaken at Regional Centre CPSP,
Islamabad from March to April 2005. Freshly laid,
fertilized chicken eggs of “Egyptian Fayoumi” breed
were obtained from Poultry Research Institute Punjab,
Department of Anatomy, CPSP Regional Centre, Islamabad.
Correspondence: Dr. Nusrat Zareen, 211 (Upper Portion),
Street No. 74, I-8/3, Islamabad.
E-mail: nusrat.zareen@gmail.com
Received March 28, 2008; accepted July 7, 2008.
Journal of The College of Physicians and Surgeons Pakistan 2008, Vol. 18 (9): 595-596
Rawalpindi. The fertilized chicken eggs were pre-
incubated for 33 hours under standard conditions of
37.5°C and 65-75% humidity, to bring them to stage 9
(29-33 hours embryo, 7 somites) of Hamburger and
Hamilton staging system.2 After this period, the eggs
were taken out of the incubator, placed horizontally,
wiped with 70% ethanol and permitted to air-dry for 10
minutes to reduce contamination from the egg surface
and also to ensure that the embryo was properly
positioned. The eggs’ contents were then transferred
into the “culture containers” by cracking the undersides
against an edge.
The culture containers consisted of thin, clear, semi-
permeable polyethylene sheet secured with elastic
rubber bands on the mouth of a cylindrical plastic or
paper cup. Before transferring the egg’s content into the
culture container, the polyethylene sheet was sagged
down a little by gloved fingers for comfortable
accommodation of the contents. The brim of the cup
was covered with a sterile Petri dish lid.
Only cultures with the blastodisc positioned to
uppermost side of the yolk were used in the experiments
(Figure 1-A). Each shell-less culture was then again
transferred to the incubator under the above
temperature and humidity conditions. This model
system of shell-less chick embryo culture was then
observed periodically with unaided eyes to watch the
organ development and extra-embryonic membrane
formation and never kept outside the incubator for more
than 5 minutes.
The formation and growth of the embryonic membranes,
the central nervous system – beginning from the vesicle
stage, the circulatory system – including the heart, the
eyes, beak, limbs, skin, feathers, wings and folding of
the body were directly observed (Figure 1-B, 1-C and
1D).
Repeated successful culturing was attempted, tracing
the developmental process of the embryo upto the 15th
day of embryonic life at least after which the survivability
period varied in different embryo cultures. The most
advanced age reached in this project was day 19 of the
embryonic life (Figure 1-D), which in researchers’
understanding is the latest developmental stage in shell-
less environment described as yet. The normal hatching
time of this breed is 21-22 days. The size of these
embryos was smaller as compared to the embryos of
the same age that carried out their development inside
their shells.
This model system has the advantage of providing easy
access to the embryo and extra-embryonic membranes
for the observation of morphogenesis and growth as well
as for the application of different agents under study.
595
Nusrat Zareen and Muhammad Yunus Khan
Figure 1: In-vitro development of a chick embryo in a shell-less culture
system. A: 48-hour chick embryo (arrow) lying in the centre of the blastodisc.
B: Ten-day old embryo, with developed head, eye balls (black spots), ears
and limbs (not visible in picture), and a network of vessels. C: Seventeen-
day old embryo with eyelids, skin with feathers (not visible clearly due to
opacity of the culture) and the noticeable growth of the embryo taking a
definite body form (dotted line). D: Nineteen-day old embryo after the
removal of the membranes. The yolk sac has regressed into the abdominal
cavity, the body is covered with feathers, beak and wings are fully formed
and there are no gross developmental abnormalities.
Recently, the same maneuver has been adopted to
study glucose-induced malformations in the developing
embryos during their early periods of life.3
Shell-less cultures also provide an opportunity for
directly observing the age-specific teratological effects
of various agents. Giles and Boehm utilized the same
technique to evaluate the stage-specific effects of
ethanol on neurodevelopment of chick embryos.4
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596
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Studying the real-time developmental process has
always remained a challenge, which has now been met
with the construction and establishment of this living
in-vitro model system allowing moment to moment
observation of the developing embryo. Recently,
computer-based video imaging system has been
established for analyzing critical phase of cardiac
looping in live chick embryos cultivated in shell-less
cultures.5
This technique is easily reproducible under laboratory
environment and may be utilized for local research
projects enhancing the knowledge of developmental
anatomy.
REFERENCES
1. Hamamichi S, Nishigori H. Establishment of a chick embryo
shell-less culture system and its use to observe change in
behaviour caused by nicotine and substances from cigarette
smoke. Toxicol Lett 2001; 119:95-102.
2. Hill M. Chicken development stages. (On line) 2006 (cited 2008
June). Available from:http://embryology.med.unsw.edu.au/
otheremb/chick1.htm#hhtable
3. Datar S, Bhonde RR. Shell-less chick embryo culture as an
alternative in vitro model to investigate glucose-induced
malformations in mammalian embryos. Rev Diabet Stud 2005; 2: 221-7.
4. Giles S, Boehm P, Brogan C, Bannigan J. The effects of ethanol
on CNS development in the chick embryo. Reprod Toxicol 2008;
25: 224-30.
5. Orhan G, Baron S, Norozi K, Männer J, Hornung O, Blume H,
et al. Construction and establishment of a new environmental
chamber to study real-time cardiac development. Microsc
Microanal 2007; 13:204-10.
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Journal of the College of Physicians and Surgeons Pakistan 2008, Vol. 18 (9): 595-596