The Effects of Temperature on Organogenesis and Caffeine Concentration on

Heart Rate in Chicken Embryos

Greg Kanski

Biology 240W

TA: Gaurav Bhardwaj

Section 16
Introduction

The process by which an embryo matures and develops is referred to as embryogenesis.

This process involves the formation of all body tissues and structures from a single celled zygote.

In nearly all vertebrates, the early stages of embryogenesis are very similar. To study the

processes and features of embryogenesis, chickens are the model organism. In a chick embryo,

structures of the nervous system are the first to develop. After twenty-four hours, the neural

fold, the neural groove, and notochord develop. These structures will give rise to the spinal cord.

Somites, which become part of the muscular and skeletal systems, also begin to appear. After

forty-eight hours, the brain and neural tube can be identified, and more somites will be present.

In addition, the heart will now be visible. An embryo after seventy-two hours of development

will be larger and will be surrounded by blood vessels that supply nutrients to developing

structures. The brain is larger and more developed. Along with growth of the brain, the head

will have grown, and the eyes will be visible. Again there will be an increased number of

somites (approximately 36 pairs at this point). Further development of the heart allows one to

see an embryo’s heart beating, even with the naked eye (Burpee, 2008).

The structures that appear during the early stages of embryogenesis are markers for the

stages of development. One can estimate the age of an embryo based on this knowledge.

However, temperature can increase or decrease the rate at which the landmark structures form.

One of the goals of this experiment is to show how temperature of incubation influences

development. To a certain point an increase in temperature will speed up development and

landmark structures will form before they would at a lower temperature. Too much heat can

denature enzymes that play crucial roles in development. Denaturation prevents enzymes from

functioning and halts development, killing the embryo.

 The heart develops fairly early in chicken embryos and after approximately seventy-two

hours a heart beat can be detected and heart rate can be measured. Caffeine is a stimulant drug

found in many food items, including coffee, soda, and chocolate, that is known to have an effect

on the nervous systems of many animals and leads to an increase in heart rate. This experiment

tested the effects of caffeine, at two different concentrations, on the heart rate of developing

chicken embryos. The addition of caffeine to the embryos will increase the heart rate, and the

higher caffeine concentration will cause a greater increase in heart rate.

Materials and Methods

This experiment began with the preparation of eggs that were incubated at two different

temperatures. Two eggs, one from each incubation group, were cracked into separate display

dishes containing saline solution. Each embryo, found resting on top of the yolk, was examined

under a dissecting microscope. The embryos were left on the yolks during examination. At any

given time, the embryo under examination was kept warm with a microscope light, and the other

embryo was kept under a desk lamp. Each embryo was thoroughly examined, and landmark

structures were visually identified using a dissection microscope, recorded, and sketched. All

data was obtained visually and results were qualitative. From the results, it was determined

which embryo was incubated at a higher temperature and which was incubated at a lower

temperature. Then using the more mature embryo (the one incubated at a higher temperature),

the effects of caffeine on heart were tested. First, a basal heart rate (in bpm) was taken and

recorded for the embryo. The heart rate was obtained by observing the heart with the naked eye

and counting the number beats per minute. Then, five drops of a 0.1% caffeine solution was

added on top of the embryo using a thin stem pipet. The heart rate was taken again and recorded.

The embryo was rinsed using saline solution. After a period of about two minutes to allow the
heart rate to reach a normal level, a second basal heart rate was taken. Finally, five drops of a

0.5% caffeine solution was added on top of the embryo and the heart rate was taken again and

recorded.

Throughout the lab process there were a few deviations from the lab manual. First of all,

the embryos were not removed from the yolk, as the manual instructed, but were viewed resting

on the yolk. As a result, there was no comparison done between the visibility of structures while

the embryo was on the yolk and when was removed from the yolk. In addition, several groups

were able to test the effects of caffeine on the embryo heart rate for both of their embryos (one of

which was more mature than the other).

Results

Table 1: Embryos incubated at different temperatures

Embryo A Embryo B

Sketch
  Heart  Heart (well developed)

Major  Blood vessels  Blood vessels

landmark
 Notochord  Notochord
structures
observed  Optic cup  Optic cup and lens

 Somites (vague)  Mesancephalon (brain)

Table 1 shows the information obtained from examining the two embryos that were incubated at

two different temperatures. A sketch of each is provided along with the landmark structures

observed. Once this information was obtained, the caffeine experiment was carried out on the

more mature embryo or on both if a heart beat could be detected in both. The data from that

experiment is shown in Table 2 below.

Table 2: Caffeine Concentrations and Heart Rate (HR)

Group Embryo Basal HR 1 (bpm) 0.1% caffeine HR (bpm) Basal HR 2 (bpm) 0.5% caffeine HR (bpm)
1 A 53 64 54 72
B 51 52 56 60
2 A 35 40 36 40
B 31 34 30 52
3 A 60 52 59 56
B 33 44 x x
4 A 52 55 50 56
B no trial no trial no trail no trial
5 A 64 92 72 92
B no trial no trial no trial no trial
Average HR 47.4 54.1 50 61.1
Standard
12.7 17.9 14.2 16.6
Deviation

Table 2 shows the data obtained from the entire class. Groups 4 and 5 did not perform the

experiment on a second embryo; therefore no data was recorded. This is denoted “no trial” in the
table. Embryo B for group 3 died after the 0.1% caffeine heart rate was taken so no results were

recorded for the second basal rate or 0.5% caffeine rate. This is denoted with an “x”. The

average heart rates from each category were calculated and are shown in Figure 1.

Figure 1: Average Heart Rates

Figure 1 also illustrates the standard deviations, denoted with error bars, of each category. The

average heart rates were used to calculate the percent increase from the first basal heart rate to

the 0.1% caffeine solution heart rate and from the second basal heart rate to the 0.5% caffeine

solution heart rate. The percent increases are shown in Table 3 and in Figure 2.

Table 3: Percent Increase in Heart Rate

Caffeine Concentration % Increase of Heart Rate from Basal

0.1% caffeine solution 14.1

0.5% caffeine solution 19.8

Figure 2: Percent Increase in Heart Rate

Trends in the data can be interpreted from the results of both experiments. The results from the

first experiment show that Embryo B had more landmark structures, specifically the lens of the

eye and the mesancephalon. These are both indicators of maturity. Embryo B was also larger

and was further in its development than Emryo A. The second experiment shows that on average

the heart rates were higher for 0.1% caffeine solution than for the first basal rate, and the rates

were higher for the 0.5% caffeine solution than for the second basal rate. Also, the 0.5%

caffeine solution rate was higher than the 0.1% caffeine solution rate and the second basal rate

was higher then the first basal rate. Results also show that there was an average percent increase

in heart rate from the two caffeine solutions. The 0.5% caffeine solution produced a greater

percent increase than the 0.1% caffeine solution. The main deviation from these average trends

comes from the group 3 data. Embryo A data shows a decrease in heart rate with the addition of

caffeine and Embryo B did not survivie past the testing with the 0.1% caffeine solution.
Discussion

Data obtained from the first experiment shows that Embryo B was more developed than

Embryo A. This is a clear indication that it was incubated at a higher temperature than Embryo

A. This supports the hypothesis that increased temperature (to a point) increases the rate of

embryonic development. This information is important because it provides insight into

development of other organisms, including humans. Development of a human embryo can be

affected by temperature, and therefore this information is important to doctors and researchers in

embryological development. Monitoring temperature during development of human embryos

could help prevent certain birth defects. Further experiments can be performed to to obtain more

information about the effects of temperature on development. Testing chicken embryos at many

different temperatures will reveal the temperature at which development occurs ideally. Also,

other organisms can be used to test the effects of different temperatures on embyological

development.

Results of the second experiment show that caffeine does in fact speed up heart rate in

chicken embryos and that a higher concentration of caffeine will produce a greater change. This

conclusion is consistent with the findings of professional researchers. Swedish scientists Nils-

Gunnar Ilbäck, Max Siller and Torbjörn Stålhandske, performed experiments on rats to test the

cardiovascular effects of caffeine. Wistar female rats were given three different concentrations of

caffeine and the effects were recorded. The research concludes that the effects of caffeine range

from a moderate increase in heart rate to more severe cardiac symptoms that can lead to death

(Ilbäck etal, 2007). The deviaitoins from this trend in this experiment performed on chicken

embryos could be due to some experimental error. In group 3, there could have been some error

in the addition of caffeine solutions that resulted in it having no effect. For example, the caffeine
may have never come into direct contact with the embryo but rather was diluted by the saline

solution. The counting of heart beats could have been inconsistent, which would lead to results

differing from the trends. It is also possible that Embyo A from this group had some

developmental complications that resulted in caffeine having effects opposite of what is normal.

There are other sources of error that could affected the outcome of the experiment. The chick

embryos could have been kept in conditions that were less than ideal. Although lamps were used

to warm the embryos, the heat from each lamp and the postioning of the lamp were most likely

inconsistent throughout the groups. This could have affected the embryo heart rates.

This experiment supports the claim that caffeine increases animal heart rates. These

findings are significant because caffeine is a drug found in very common foods and drinks, that

people consume everyday. It is important that people understand what effects certain substances

have on their bodies and the potential harm these substances can do. The results of this

experiment and others like it can also provide insight into how caffeine molecules interact with

target cells and tissues in the body, and how signals are sent to the rest of the body. To further

explore the effects of caffeine on cardiovascular systems, other experiments can be performed.

The same concentrations of caffeine can be tested on more mature chickens or on a different

organism. Different concentrations of caffeine can be used on chicken embryos. Also, the

caffeine can be delivered to the organism in a more direct way. If caffeine is delivered through a

syringe, the results may be different. Further experiments like these could confirm or refute the

conclusion of this experiment and perhaps allow for a better understanding of embryogenesis.
Works Consulted

Burpee, D. 2008. Vertebrate Development: Organogenesis in the Chick Embryo. Department

of Biology, The PennsylvaniaStateUniversity, University Park, PA. Adapted from Price, M. and
D. Burpee (eds). 2004. A Laboratory Manual for Biology 240W: Function and Development of
Organisms. Department of Biology, The PennsylvaniaStateUniversity, University Park, PA

Ilbäck, Nils-Gunnar, Max Siller, and Torbjörn Tålhandske. 2007. "Evaluation of Cardiovascular
Effects of Caffeine Using Telemetric Monitoring in the Conscious Rat." Food and Chemical
Toxicology 834-842.

Patten, B. M., and B. M. Carlson. 1974. Foundations of Embryology, Third Edition. McGraw-
Hill Book Company, New York, NY.

Raven, P. H., and G. B. Johnson. 2005. Biology. Seventh Edition. McGraw-Hill, a business unit
of The McGraw-Hill Companies, Inc., New York, NY.