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The effect of different times of ethanol exposure on the embryonic development of Danio rerio

Anna Doutt
Department of Biology
Randolph-Macon College
Ashland, Virginia 23005

Abstract
Fetal Alcohol Syndrome is becoming more common in pregnant women who drink not
knowing they are pregnant or who don’t realize the dangers of drinking while pregnant
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The purpose of this experiment is to define the critical periods for
embryonic development in Danio rerio
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Danio rerio embryos were divided into three groups and then exposed to 0% and
6% ethanol at different time periods for four and a half hours at a time
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We discovered that there is a critical period for
embryonic development in Danio rerio embryos, which is 24-28
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During this time, the embryos developed spinal deformities, a depressed heart rate, short body
length and a decreased percent that reached prim-11 stage
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Introduction
Fetal Alcohol Syndrome (FAS) is becoming more and more common in pregnant women
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Drinking even weeks into a woman’s pregnancy can affect the development of
embryos
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2010)
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2011)
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2004)
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Danio rerio, more commonly known as
Zebrafish work as a good model organism for this experiment because they are a vertebrate
animal, making them similar to humans, they reproduce prolifically, providing many to work
with and they reach adulthood within three months, which speeds up the process of evaluating
them (Bilotta et al
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For this experiment, we hypothesized that Danio rerio embryos exposed to ethanol
early in development would have more of an effect than embryos exposed later in
development
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Methods and Materials
We collected 9-15 Danio rerio embryos and then randomly assigned each one to one of
three groups
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The embryos in the control group were exposed to
zero percent ethanol and our two treatment level groups were exposed to six percent ethanol
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All of
the embryos were placed in wells containing 3ml of spring water
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Then, at 32-hours, the other group
was dosed with six percent ethanol for four and a half hours
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Every day, for five days, the embryos were checked on and staged
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Results
The Danio Rerio embryos exposed to no ethanol had the highest average heart rate
(104
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93 and the embryos exposed to ethanol between 24-28
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25) with the SEM being 9
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The embryos exposed
between 32-36
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63) with the SEM being
5
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This shows that the critical period for
heart development is between 24-28
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The p-value for this was calculated using and
ANOVA test and was 0
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Figure 2 shows that the embryos
not exposed to ethanol had the longest average body length which was 3
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15,
while the embryos exposed to ethanol between the hours of 32-36
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9 with the SEM being 0
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The average body length of the embryos
exposed to ethanol between 24-28
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3 and the SEM was 0
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This shows that the
critical period for fry length is between 32-36
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The p-value was calculated using the
ANOVA test and was 0
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Figure 3 shows that embryos exposed to no
ethanol had the highest average number of live fry, which was 2
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5 while
the groups exposed between 24-28
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5 hours both had an average of 1
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The SEM for 24-28
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5 and the SEM for 32-36
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6
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Using the ANOVA test, the p-value was calculated to be 0
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Figure 4 shows that embryos exposed to no ethanol resulted in having the least
amount of deformities with an average of 0
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2, while the embryos exposed at
24-28
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7 and a SEM of 0
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The average
number of embryos with deformities at 32-36
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8 with a SEM of 0
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This shows
that 24-28
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The p-value for
this was 0
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Figure 5
shows the effect of ethanol on how many Danio rerio embryos are alive at 0, 1, 2 and 5 days
post fertilization
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The highest percent of Danio rerio embryos reaching the prim-11 stage or beyond
were the embryos not exposed to ethanol at 38
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The embryos not exposed to ethanol
had 38
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The embryos exposed at 24-28
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22
percent reach prim-11 stage or beyond and the embryos exposed at 32-36
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66

percent reach prim-11 stage or beyond
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5 hours
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000

Average Hert Rate (bpm)

100
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000
60
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000
20
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000
NO ethanol

24-28
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5 hpf

Exposure Time to Ethanol (6 %)

Figure 1
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The
graph displays the means and SEM for each group
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5 and the standard error of mean was 8
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(n=10)
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5 was 91
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99
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For hours 32-36
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63 and the standard error of mean was 5
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(n=11)
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498
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5

Average Fry Length (mm)

4
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5
3
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5
2
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5
1
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5
0
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5 hpf

32-36
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The effect of 6% ethanol exposure on the fry length at different time periods
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For the group with no ethanol exposure,
the mean was 3
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15
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For the group exposed to
ethanol for 24-28
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3 and the standard error of mean was 0
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(n=4)
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5 hours, the mean was 2
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29
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Using the ANOVA test, the p-value was 0
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3
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5
2
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5
1
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5
0
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5 hpf

32-36
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The effect of 6% ethanol exposure on the average number of live fry at different time
periods
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The mean for the group with no
ethanol is 2
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5
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For the hours 24-28
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8 and the standard error of mean was 0
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(n=11)
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5, the mean was
1
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6
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Using the ANOVA test, the p-value was
0
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Number of Fry With Deformities

2
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0

1
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0

0
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0
No ethanol

24-28
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5 hpf

Exposure Time to Ethanol (6%)

Figure 4
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The graph displays the means and the SEM
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3 and the standard error of mean was 0
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(n=2)
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5, the mean was 1
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4
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For hours 32-36
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8 and the standard error of mean was 0
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(n=5)
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1046
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5 hpf
40

32-36
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The effect of 6% ethanol exposure on the number of fries alive at X days post
fertilization
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Number of Fries at or Beyond Prim-11 Stage
(Percent)

50
45
40
35
30
25
20
15
10
5
0
No Ethanol

24-28
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5 hpf

Exposure Time to Ethanol (6%)

Figure 6
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The graph displays the percent of fries at or beyond the prim-11
stage
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46
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22 for
the group exposed between 24-28
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5, the percent
was 46
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Discussion
As a result of this experiment, it can be concluded that there is a critical period for different
parts of embryonic development of Danio rerio
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Although there were some
tests with results that did not support our hypothesis, they were not significant
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It could also be
repeated using different times of exposure, the time exposed to ethanol, the percent of ethanol

exposed to and comparing different physical deformities
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We discovered that for the most part the critical
period for Danio rerio embryonic development is between 24-28
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2010)
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2004)
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Pregnant women need to be more informed and aware of how
damaging ethanol exposure is to embryonic development
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Literature Cited
Ali, D
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, Brewster, D
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, and Sylvain, N
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2011
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Neurotoxicology and Teratology 33:
313-321
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W
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L
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J
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Zebrafish embryos exposed to alcohol undergo
abnormal development of motor neurons and muscle fibers
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Barnett, J
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, Bilotta, J
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, and Saszik, S
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Ethanol exposure alters zebrafish
development: A novel model of fetal alcohol syndrome
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