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Title: Effect of Alcohol as a Teratogen in Explant Cultures of Chick Embryos Abstract: Studies have been done to show that

a developing organism is susceptible to external and internal factors during certain stage of the organism development. This study was designed to investigate the effect of ethanol exposure, simulating the Fetal Alcohol Syndrome (FAS), on a chick embryo model system. Chick embryos were subjected to varying concentrations of ethanol injections, and then measured with respect to age for developmental phenotypes such as vascular, yolk sac sizes, and body length. The result of ethanol treatments on the embryos does not compare up to the standard effects of FAS, which suggests that at low concentrations, ethanol may serve as a positive factor in certain development features. Introduction: The consequences of ethanol are The effects of alcohol (or ethanol) dependent on the time of administration, on the developing fetus are a variety of concentration of treatment, and duration characteristic abnormalities term Fetal of treatment. Alcohol Syndrome (FAS). Ethanol exposure to the fetus causes Materials and Methods: developmental defects that lead to The following materials were used mental retardation. during this experiment: 3-3.5 day old fertilized chick eggs Chick embryos serve as an 1 ml syringes acceptable model for observing the 3 vials of ethanol/saline solution of effects of ethanol at developmental stages involving neurogenesis, neuronal concentrations 0%, 0.2%, and 2% migration and differentiation, cell to cell ethanol Plastic boats in square Petri dishes connectivity, and synaptic function. Embryos of 1-3 days old, when treated with ethanol, show a decrease in normal Following the protocol provided by neuronal phenotypes in later Armstrong, et al, 2007, fertilized eggs developments. Embryos of 1 day of were wiped with 70% ethanol to embryos are most vulnerable to the disinfect, air dried, then cracked to effects of ethanol because day 1 in chick transfer the intact yolks from the eggs to embryos is when neurogenesis and the Petri dishes. The eggs were labeled neuro-organization are actively with an X to indicate that the embryo processed. The neural tube elements are would be directly under the X, so the also made during this early embryonic cracking would be done to the opposite period. Exposure to elements such as of the X. that way when the shell was ethanol during this dynamic pried open, the yolk would flow out with neuroembryonic period may interfere the embryo on top. with important parameters of neuronal The embryos with the best looking growth like cell connectivity, neuronal 3-3.5 day features were selected and migration and phenotypic expression. grouped into experimental groups;

control (A), 0.2% ethanol (B), and 2% ethanol (C). With the syringes, 1 ml of the ethanol/saline solutions with different concentrations was injected to embryos of corresponding groups. The embryos were then incubated at 37 degrees Celsius. Daily observations were made of the embryo. Traits chosen to be observed for our experiment were vascular area

(mm of the longest diameter), yolk sac width (mm of the longest diameter), and body length (mm). Time zero measurement was made on all embryos, then followed up till the chick embryos were 10 days old. The collected data were then pooled from 3 lab tables fro analysis. There were 7 embryos total, 3 in control group, 2 in 0.2% ethanol group, and 2 in 2% ethanol group.

Result: The raw data sheets are included at the end of the lab report. Below are tables averaging the measurements taken

for the 3 experimental embryo groups for traits vascular area (VAS, mm), yolk sac width (YOLK, mm), and body length (BODY, mm).

Table 1: Group A (0% ethanol, control) Chick Culture Traits Measurement Averages Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 (-1)* VAS 24.67 43 48.5 70.5 73 75 76.5 79 BODY 6 7 10 16 20 21.5 22 24 YOLK 49 58.66 62 72 74 74 75 75.5 * indicates how many embryos died at this point, thus less measurement calculated in the final average Table 2: Group B (0.2% ethanol, control) Chick Culture Traits Measurement Averages Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 VAS 26 46.5 55 71.5 73 74.5 78.5 79 BODY 7.5 7.5 10 15.5 21 22.5 23.5 26 YOLK 49.5 54.5 61 73 74 74 73 69.5 * indicates how many embryos died at this point, thus less measurement calculated in the final average Table 3: Group C (2% ethanol, control) Chick Culture Traits Measurement Averages Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 (-1)* VAS 23 42.5 48.5 47 55 75 76 79 BODY 7.5 7.5 11.5 16 20 23 23 23 YOLK 48 52.5 55.5 59.5 74 75 76 74 * indicates how many embryos died at this point, thus less measurement calculated in the final average.

90 80 70 60 50 40 30 20 10 0

Tr ends i n Vascul ar Ar ea Changes

D am i eter i n m m

VAS ( A) VAS ( B) VAS ( C)

There appears to be an upwards trend of vascular area enlargement for embryos treated with low concentration of ethanol and control group, such as group A and B. They also appear to have a similar increasing rate. For group C, which was treated with high concentration of ethanol, the embryos

D 3D 4D 5 D 6D 7 D 8D 9 D ay ay ay ay ay ay ay ay 10 D ays

also show an upwards trend of vascular area enlargement, but the growing rate of vascular area is slightly slower than the control group at day 4 to day 7. At day 7, group C has a sudden jump and result in equal vascular area as the control and low concentration ethanol group.

Trends i n B ody Lengt h changes Body l engt h i n m m 30 25 20 15 10 5 0 D 3 D 4 D 5 D 6 D 7 D 8 D 9 ay ay ay ay ay ay ay

All three groups show a similar rate of increasing in body length.

BO Y ( A D ) BO Y ( B D ) BO Y( C D )

D ays

D ay 10

Trends i n Yol k Sac Wdt h Changes i Yol k Sac di am er i n m et m 80 70 60 50 40 30 20 10 0

YO ( A) LK YO ( B) LK YO ( C) LK

D 3D 4D 5 D 6D 7 D 8D 9 D ay ay ay ay ay ay ay ay 10 D ays All three groups show a similar rate of increase in yolk sac width.
Discussion: Looking at the vascular area trends across the experimental groups, it is curious to see that the embryos treated with some extent of ethanol were able to develop a large area of vascular structures as the control embryos. It is also important to point out that out of 3 groups, only group B, which was treated with low concentration of ethanol, has a 100% survival rate. The control group A has a 33% mortality rate, whereas the high concentration ethanol group C has a 50% mortality rate before 6 days of age. The data thus may be skewed at the later dates since there were fewer samples of embryos to take the averages of for control group A and high concentration ethanol group C. Looking at group B and C, we can say that despite the presence of ethanol, the embryos were able to maintain the vascular structures. In fact, all three groups have the same size of vascular area by the end. It is not surprising to see a same size of vascular area across the groups because the vascular expanded to the maximum area permitted, and that is the area of the Petri dish. There is a similar trend in yolk sac widths across the groups. This result tells us that ethanol does not seem to affect the ability of embryo to exploit the yolk sacs, perhaps because that process is a different subject than development. The areas of the yolk sacs become bigger as the chick embryos get older. As the chick embryos get to day 10, it shows a slightly decreasing in yolk sac areas for all these groups, perhaps because the older chick required more nutrients, which are stored in the yolk sacs. The chick embryos have a similar body length for both control group and treated groups. Perhaps the ethanol is not playing a role in growing of body length. It is very hard to measure the exact length of the chick body because the chick body is cured up. Therefore the measurement for thee chick body length

might varied. Looking at all these observed traits across the 3 groups, you can see that for the most part, an increase in vascular area corresponding to an increasing yolk sac width and an increasing body length. That is probably because as the yolk sac expands, there is more space for the vascular structures to grow on. Also as the chick gets older with an increasing vascular area, the chick body length gets longer. Regarding initial hypothesis that FAS displays abnormality in chick embryo developments, the result of a higher survival ratio of low ethanol-

treated embryos seems to contradict the expected, which is a higher survival rate of control embryos. This outcome could be due to various factors, human errors. The embryos selected for control group may have a non-intact yolk, or some other damages that cause their deaths. In order to do a conclusive investigation on the effect of ethanol on chick embryo developments, more traits need to be observed such as number of somites and heart rate. Also more embryos are needed for each group.

References: Brodie, C. and Vernadakis, r (1990) Critical Periods to Ethanol Exposure during Early Neuroembryogenesis in the Chick Embryo: Cholinergic Neurons. Developmental Brain Research. Volume 56:223-228 Gribert, S. F., Developmental Biology, 7th ed., Massachusetts: Sinauer Associates, 2003. Armsrong, P., Embryology Laboratory (MCB150L) Class Syllabus. 2007

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