The living embryo and making of whole mounts 72-and 96- hour chick

embryo

Joemark T. Narsico
Natural Sciences and Mathematics Division
University of the Philippines Visayas Cebu College
joemark_narsico@yahoo.com.ph

Abstract

This experiment investigates the development of Gallus domesticus chick

embryo. Gallus domesticus eggs were incubated and were opened to be
examined anatomically. Whole amounts were examined using a
microscope. It was observed that the 72 hour chick embryo has evidently
distinct regions of the brain, which was bent ventrally. In this stage
unpigmented optic vesicles, lemon-shaped otic vesicles, and asymmetrical
limb buds were seen to develop in this stage. The 96-hour embryo has
pigmented eyes, a beating heart, an elongated heart, and developing tail
buds. Further observation of the chick on its twelfth day of incubation
showed that embryos developed scaly hind limbs hardened beaks and
feathered tracts. The eggs were incubated until its 21st day however the
eggs did not hatched.

Introduction

From three weeks of incubation a chick emerges from the shell. During the three weeks
duration the cell division occurs as long as the 80 degrees Fahrenheit is maintained in
the environment. On the third day of incubation the developing wing and limb buds begin
to appear. Torsion and flexion continue on the fourth day. The chicks body rotates 90
degrees and the head and tail come close together to form a “C” Shaped embryo. The
heart at this stage continues to enlarge. Many complex physiological processes take
place during the development of the chick. They include the use of its yolk for the chick’s
nutrition, respiratory functions and the removal of waste and carbon dioxide and the
uptake of oxygen.

After 21 days of incubation the chick finally escapes from its shell. The allantois which
serves as its lungs during incubation dries out and the chick uses its own lungs after its
escape. After hatching the chick can survive without food for 72 hours. The yolk is
deposited in the chick’s body. The yolk is deposited just before the chick’s hatching. The
yolk contains fats, proteins and minerals.

The living chick embryo can be used as a model for determining the possible ill-effects of
environmental conditions and substances. Relationships can be drawn from the human
and chick embryo.
Mckenna (2006) demonstrated that the application of alcohol to the embryonic-5 day old
chicken heart in vitro had very harmful effects to the heart rates and to the embryo as a
whole. Albert (2006) also demonstrated the adverse effects of caffeine in the 4-day old
chick embryo. In both experiments presented there was a rapid increase in the heart
rates when alcohol and caffeine were applied to the chick embryo.

Objectives

To observe the morphological developments of the 72-and 92-hour chick embryo

To determine the effects of caffeine and light to the living chick embryo

Materials and Methods

The egg incubator was assembled. The egg incubator was made from the Styrofoam ice
chest and a electric light bulb was attached to the egg incubator. Water was placed in
the containers and placed inside the incubator to maintain the 57% relative humidity.
The eggs were placed in a box with a piece of cloth. A thermometer was placed inside
the incubator to monitor the temperature of the incubator. The incubator must maintain a
37.5-38 degrees centigrade temperature for the chick to develop. The blunt end of the
egg faced upward. This was to ensure that it would be accessible to be observed during
dissection.

After three days an egg was opened and dissected. The heartbeat was observed and
morphological characteristics were observed. The developmental stage was noted using
the Hamburger-Hamilton Index (1953). The response of the embryo to touch was also
observed. The effects of the lamp were also recorded.

On the seventh day of incubation another egg was also dissected and observed. The
stage of the chick embryo dissected was noted. Morphological changes and
development were observed and recorded.

On the twelfth day of incubation another egg was dissected and observed. The chick
embryo dissected was staged using the Hamburger-Hamnilton Index (1953). Drops of
coffee were applied to the chick embryo. Its effects were observed. The responses of the
embryo to touch and light were also tested.

When the chick embryo was removed the extra embryonic membranes were cut using
forceps and scissors. The embryo was then placed in a petri dish containing the Ringer’s
solution. The staging of the embryo was done using a microscope.

The length of the third toe and the beak was measured using a ruler. Uncertainties of the
measurements were also recorded. The number of beats per minute was also recorded.
The remaining eggs were allowed to be hatched until the 21st day of incubation was
reached. The incubator was cleaned once in a while to insure that no ants could grow.

Results and Discussion

On the 3rd day of incubation the dissected egg we observed that there was an atrial and
ventricular beating in the chick embryo. We also found that there was no skeletal
muscular movement. We observed that there was an increase in the number of heart
beats per minute when the chick embryo was exposed to the lamp. The lamp has
intense light and temperature. The normal heart beat of the chick was 121 beats per
minute while the heart beat exposed to the lamp was 130 beats per minute.

Figure 1. Chick embryo on the third day of incubation. Eyes are visible. Chick has
relatively large head and eyes. The beak inits mandible is distinct.

We observed that the eyes have already formed in the chick embryo. There were
already toes and defined wings. The head is relatively larger than its body and the eyes
are occupying much of the head. The ventral region of the embryo is not yet fully formed.
The beak was observed to be defined. The eyes were colored black. There was still no
feather. The skin was colored white.

On the 7th day of incubation, the dissected embryo exhibited a lot of changes compared
to the chick embryo dissected on the third day of incubation. The chick embryo exhibited
characteristics described in stages 29-36. There was a rudiment in the 5th toe and
feather germs were also visible. A beak was already seen at the anterior tip of the
chick’s mandible. There were already phalanges in the toes observed. The eyes were
more defined compared to the embryo observed on the third day. The embryo is also
larger compared to the 7th day embryo. There were already three digits in the hind limb.
Tail was also observed to be distinct.
Figure 2. Chick embryo on 7th day of incubation. Eyes are relatively smaller compared to
the 3rd day. Feather tract are now visible. Scales on the hind limbs are seen. The Wings
can already be distinguished.

The embryo on the 9th day of incubation was sorted to be in the 38th stage according to
the Hamburger Hamilton Staging Index (1953). We observed that the measured value
for third toe is 84.00±0.30 centimeters. The beak measured 3.1±0.30 centimeters. The
four digits of the chick’s limbs were already visible. Feather tracts were already exhibited
in the embryo during this stage. The eyes have slits and the wings of the embryo are
already defined. The texture o the beak was relatively softer. The head is relatively
larger.

Figure 3. Chick embryo on ninth day of incubation. Feather tracts are very distinct. Eyes
relatively reduced in size compared to the seventh embryo. Phalanges was
visible. Ventral region of the embryo was not yet completely sealed.

When the embryo was exposed to the Ringer’s solution had less than 15 heart beats per
minute. The heart beat of the embryo when exposed to the lamp was irregular and
faster. There were 35 heart beats per minute when exposed to the lamp. The heart beat
was also irregular when the embryo was exposed to caffeine.

The first functional organ of the chick embryo is the heart. The heart functions
independently from the external environment. The heart muscle cell secretes signals,
which depend on the sodium-calcium pump. Other signaling molecules such as
acetylcholine and norinephrine can also affect the embryonic heart (Gilbert 2006).
Caffeine can induce morphological changes which may cause irreversible harm on a
developing embryo.

We tried allow the remaining eggs to undergo hatching and leaved these eggs for twenty
one days in the incubator, however, the eggs were not able to hatch.

Caffeine is a xanthine alkaloid commonly used as a psychoactive stimulant drug. It is

found in beans, leaves and fruits, where it acts as a insect repellant for the plant.
Caffeine is stimulant to the central nervous system in humans. It is a major component in
teas, softdrinks and coffee.

Bruyere et al (1986) demonstrated the cardioteratogenic effects of caffeine on three day

chick embryo. They reported that there is a decreases cardiac output of the heart
suggesting decreased flow through the embryonic heart, followed by an increase in
ejection fraction, which suggest increased cardiac workload. This finding is related to the
observation we gathered in this study. The increase in heart beats per minute manifest
the increase of workload in the embryonic heart.

Lee and his colleagues demonstrated the toxic and terratogenic effects on the chick
embryo. They found out that embryos in earlier stages were more susceptible to the
terratogenic and toxic effects of caffeine. Caffeine at concentrations is sufficient to inhibit
the uplifting of neural folds, the closure of the neural tube, and the thinning of
microfilament bundles. They found that caffeine causes neural tube defects through
inhibitory action of contractile activity of the apical microfilament bundles in developing
neuroepithelial cells.

In mouse embryo cultures, failure of neural tube closures, excessive proliferation of

neuroepithelial cells, and premature evagination of the telencephalon were related to
high dosage intake of caffeine (Marret et al 1997). Marret et al also added that when
caffeine reaches the embryonic neural tube before migration, caffeine regionally
modifies the schedule and or rate of neural cell proliferation.

Gimeno et al (1966) reported that under certain conditions of visible light exposure to
chick embryo accelerates the rate of heart beat. She further demonstrated that activity of
cells in tissue culture is greatly affected by the exposure of light. There have been
reports of increased metabolic activity and of accelerated growth rate and hatching of
the cultures in visible light.

Niiyama and Miyakawa (1986) studied the effects of the temperature on the heart beat
rate of the chick embryo. The rhythmic recurrence of heartbeats is found to be
decreased in frequency as the temperature decreases. This behavior is found to be
described as ν∝exp (-W/kT), where T is the absolute temperature, k is the Boltzmann's
constant and ν is the frequency of heartbeats. The variable W can be interpreted as the
minimum activation energy necessary for one heartbeat, the magnitude of which
increases with the developmental stage. Their findings support the result of our
experiment on the increase of heartbeat when chick embryo is exposed to light. Light is
correlated with temperature.

Ultraviolet light could also induce the increase of heart beat and termination of beating in
chick embryo (Nathan, Dehaan, Pooler 1976). This is attributed due to the depolarization
of the membrane and a decline in the fast sodium conductance of the heart in chick
embryos.

Conclusion

This study demonstrated the development of live embryo cultures for whole mounts. We
have recorded the morphological changes that the chick embryo has undergone from
the third, seventh and the twelfth day of incubation. These changes correspond to the
stages presented by Hamburger-Hamilton Index (1953).

Caffeine and light induces great number of heart beats per minute. The presented works
of other researchers suggest that caffeine could be toxic to the developing chick embryo
because it induces morphological and biochemical changes within the developing heart
of the chick.

For future experiments it is advised that researchers employ replicates and use
statistical analysis to test the reliability of the data when conducting a study on the effets
of light and caffeine on the chick embryo.
 References

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Lee H, RH Nagele, JF Pietrolungo. 1981. Toxic and teratologic effects of caffeine on

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