The effects of ethanol concentrations on developing chicken embryos

1
The effects of ethanol concentrations on
developing chicken embryos
Dianna M. Jarvis Marietta College
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.
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 3 Embryonic Weights
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.
Figure 1 CNS growth suppression
Even though growth suppression could not be
concluded statistically, visually tissue
necrosis, decrease size, and abnormal growths
were observed (Figure 4).
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 ½
milliliter of solution was injected into the air
cell (Figure 2).
Figure 4 Abnormal Growths
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.
Figure 2 Air Cell
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.
The composition of each ½ 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.
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.
Acknowledgements MC Biology Department Dr. Peter
Hogan, Capstone/MC advisor Sarah Zumbro, lab
assistant Brandon Coughenour, lab assistant