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

Joemark T. NarsicoNatural Sciences and Mathematics Division

University of the Philippines Visayas Cebu Collegejoemark_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 5 th 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 38 th 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

Albert M. 2006. The effects of caffeine on the four day old chick embryonic heart rate. Journal of Chicken Embryology.

Bruyere H, B Michaud, E Gilbert, J Folts. 1986. The effects of cardioteratogenic doses of caffeine on cardiac function in the 3-day chick embryo. Journal of applied toxicology

Gimeno M, CM Roberts, JL Webb. 1966. Acceleration of rate of the early chick embryo heart by visible light. Nature. 214: 1014-1016

Lee H, RH Nagele, JF Pietrolungo. 1981. Toxic and teratologic effects of caffeine on explanted embryos. Teratology. 25(1):19-25

Marret, S, P Gressens, GVM Fabry, J Picaerd, P Evrard. 1998. Caffeine-induced disturbances of early neurogenesis in whole mouse embryo cultures. Brain Research. 773(1-2): 213-216

Mckenna C. 2006. Determining the effects of alcohol on the 5 day chicken embryo heart rate. Unpublished

. Niiyama W, K Miyakawa. 1986. The effects of temperature on the rhythm heartbeats of

chick embryos. J. Phys. Soc. Japan. 55:3745-3748

Nathan RD, JP Pooler, R DeHaan. 1976. Ultraviolet-induced alterations of beat rate and electrical properties of embryonic chick heart cell aggregates. The Journal of Genral Physiology. 67: 27-44