The effects of ethanol concentrations
on developing chicken embryos
Background Information:
Fetal Alcohol Syndrome (FAS) results from prenatal alcohol exposure.
Those affected by the disease are often born with birth defects and
developmental disorders, such as, mental retardation (Welch-Carre,
2005). FAS is noticed within 1% of all live births and unlike other diseases
it is 100% preventable (Burd, et al, 2004).
Ethanol is one of the components found within alcohol. Although,
ethanol is a relatively simple organic compound, it is a known teratogen. A
teratogen is an agent that can interfere with the normal development of a
fetus. Ethanol disrupts the normal development of the Central Nervous
System (CNS) (Costa, et al, 2002). Primarily, the substance inhibits glial
cell proliferation (Costa, et al, 2002). Glial cells, the main cells that make
up the nervous system, provide support and nutrition to the neurons.
Specifically with FAS, the timing of ethanol exposure often determines the
severity of the damage (Costa, et al, 2002). The most common
manifestation of ethanol exposure within the CNS is characteristic growth
suppression (Figure 1).
Figure 1: CNS growth suppression
www.stpetershealthcare.org
Prior research has shown that the use of chicken embryos can aid in
understanding the complexity of FAS. The results of past investigations
show that ethanol induces growth suppression within developing chicken
embryos (Bupp & Shibley, et al, 1998). There are several advantages in
using a chicken model for examining FAS (Hartl & Shibley, et al, 2002).
When dealing with chicken eggs, concern about maternal malnutrition or
drug use can be eliminated. (Bupp & Shibley, et al, 1998). Also, the
incubator offers a self-contained environment in which the eggs can be
monitored at all times during development (Bupp & Shibley, et al, 1998).
The 21 day gestation period is also an advantage when using the chicken
model. Unlike human embryos that can take up to nine months to
develop, it only takes 21 days for a chicken embryo to fully develop.
Additionally, the direct effects of ethanol can be studied by manipulating
ethanol dosages as well as the number of eggs within the experiment
(Bupp & Shibley, et al, 1998).
In other experiments concerning this topic, the focus was on the effects
of a high versus a low dosage of ethanol (Bupp & Shibley, et al, 1998).
Assessments comparing the torso versus head weights of the chickens
were then evaluated. These measurements were used to determine if the
ethanol caused an overall embryonic growth suppression or if the ethanol
affected the head and torso independently (Bupp & Shibley, et al, 1998).
Moreover, various chicken strains have been used within these
experiments; among those are layers and broilers (Bupp & Shibley, et al,
1998). Broiler chickens are known for their high meat yield and rapid
development, layers are known for their slow growth, yet early sexual
maturity (Bupp & Shibley, et al, 1998).
My senior research will examine the effects of high versus low dosages of
ethanol on embryonic chickens. I will only use layer chickens, specifically,
White Leghorns. To determine the effects of ethanol, I will measure the
total weight suppression of the embryos.
Dianna M. Jarvis
Marietta College
Hypothesis:
The effects of low/high dose ethanol concentrations will
suppress the growth of embryonic chickens. High ethanol
concentrations will cause greater growth suppression of
embryonic chickens, than low ethanol concentrations.
Procedures:
Two dozen fertile, White Leghorn chicken eggs were ordered from
Florida. When the eggs arrived, they were kept at room temperature for
no longer than two days. In preparation for ethanol injections, the small
ends of each egg were cleaned with 70% ethanol. Once cleaned, each
egg was then labeled with a pencil according to the designated injection
group. Next, a puncture site was made into the air cell of each egg and
milliliter of solution was injected into the air cell (Figure 2).
Figure 2: Air Cell
The composition of each milliliter of solution was based on the
designated dosage group. The low dose groups solution was composed
of two milliliters of 200 proof ethanol and 48 milliliters of distilled water or
saline solution. The high dose groups solution was composed of eight
milliliters of 200 proof ethanol and 42 milliliters of distilled water or saline
solution.
After ethanol was placed inside the air cells of each egg, the injection
sites were sealed with paraffin wax. The eggs were then placed within a
25.3 watt Hova- Bator incubator (model 1602N) with an automatic egg
turner. The incubator was kept at 90 degrees Fahrenheit and the eggs
were candled periodically throughout development.
On day nine of development, the eggs were opened and the chicken
embryos were removed. The total weight of each embryo was recorded.
The length and width of each embryos head was collected using a
caliber. Observations were also made of any noticeable, morphological
abnormalities.
Acknowledgements:
MC Biology Department
Dr. Peter Hogan, Capstone/MC advisor
Sarah Zumbro, lab assistant
Brandon Coughenour, lab assistant
Results:
Results of previous studies have shown a reduction in embryonic weights.
Higher ethanol concentrations have shown an increase in growth
suppression in both broiler and layer chicken strains (Bupp & Shibley, et al,
2002). However, within this investigation, growth suppression could not be
statistically verified. Also, differences in growth suppression between high
and low ethanol dosage groups could not be concluded. As seen in Figure
3, the mean values of the groups did not decrease linearly with increased
ethanol concentrations as hypothesized. A student t-test of the recorded
means confirmed no statistical significance.
Figure 3: Embryonic Weights
Even though growth suppression could not be concluded statistically, visually
tissue necrosis, decrease size, and abnormal growths were observed (Figure 4).
Figure 4: Abnormal Growths
Conclusions:
Past research demonstrates that chicken embryos can be used to study
FAS. My research does not statistically support that embryonic growth
suppression is due to ethanol exposure. The graphs show some decrease
in embryonic weights due to ethanol exposure, but there is not enough data
to support my hypothesis. A problem likely exists in the number of chicken
embryos used within the experiment. A larger sample size might have
demonstrated, statistically, embryonic growth suppression. Additionally, the
measurements taken of the width and length of the embryos heads were
inconclusive, as well as variable. Again, the head caliber data might have
been significant if a larger sample size was available.
Future Research:
Some anatomical effects were observed due to ethanol injections.
Investigations on the cellular level are needed to determine the specific
interactions between ethanol and the developing embryo. Through cellular
testing, an embryo exposed to ethanol versus a normal embryo could be
compared. Multiple injections of ethanol could be given, instead of one
injection at the beginning of incubation. The ethanol injection amounts
could be smaller by splitting up the volume over nine days. Development
could also be evaluated past day nine.
Works Cited:
Bupp Becker SR, Shibley IA. 1998. Teratogenicity of ethanol in different chicken strains.
Alcohol & Alcoholism (33): pp. 457-464.
Burd L, Wilson H. 2004. Fetal, infant, and child mortality in the context of alcohol abuse.
American Journal of Medical Genetics (127). pp. 51-58.
Costa LG, Guizzetti M. 2002. Inhibition of Muscarinic Receptor-induced proliferation of
Astroglial cells by Ethanol: Mechanisms and Implications for Fetal Alcohol Syndrome.
Neurotoxicology (23): pp. 685-691.
Hartl MW, Shibley IA. 2002 Supraphysiological acetaldehyde levels suppress growth of
chicken embryos. Alcohol (28): pp. 111-115.
Welch-Carre E. 2005. The Neurodevelopment consequences in prenatal alcohol exposure.
Advances in Neonatal care (5): pp. 217-229.