The Role of Caffeine on Zebrafish Embryogenesis
Jack Steed
MaST Community Charter School
Question:
If a zebrafish embryo is dose with caffeine during embryonic development, will it have more
teratogenic effects than a zebrafish embryo that has not been dosed with caffeine?
Hypothesis:
Caffeine is something that is part of a pregnant women’s everyday life, whether it be coffee or
supplementation for sleep deprivation. Although there is little known about the teratogenic
effects of caffeine consumption on the fetus during pregnancy, clinical studies have found that
the teratogenic effects of caffeine depend on dose, with higher doses producing the more visible
effects.
These effects have ranged between low birth weight, preterm birth, and low
testosterone levels. There is little known if the time or dose of caffeine consumption during
pregnancy can limit or eliminate the teratogenic effects of caffeine. However, given that caffeine
has teratogenic effects on the outcomes of pregnancies in the forms of low birth weight and
preterm birth, a delay in the consumption of caffeine during pregnancy will minimize caffeine’s
teratogenic effects.
Experimentation:
Two separate experiments, using Zebrafish embryos as an experimental model, one to
determine the dose of caffeine needed to produce a substantial effect and the other to
implement this dose at different points during development the effects of caffeine on embryonic
development will be unravelled. The first experiment will use doses of caffeine ranging from
10mg to 300mg will be dissolved into the embryonic medium at the beginning of development
they will be viewed at each stage to quantifying growth deformities or anomalies, such as using
NIH Image J Software. Anomalies during each embryonic period will be quantified such as:
During the different periods, observe different aspects of the embryos: zygote period (0-.075
hours)- the size of yolk sacks; cleavage period (1-2 hours) and the blastula period (2-5 hours)size of yolk sacks and visual amnion; gastrula period (5-10 hours)- visual activity of yolk sack;
segmentation period (10-24 hours)- size of gut sac; hatching period (48-72 hours)- time of
hatching; early larval period (72 hours)-the curvature of spine. Throughout the experiment,
different set-ups constants such as: nitrites, nitrate, ammonia, and pH will be kept constant.
The effective teratogenic dose found from the first experiment will be used for the second
experiment. In the second experiment this effective dose will be applied at each developmental
stage to see if there is any difference in the deformities or anomalies based upon when caffeine
is consumed. During both experiments key points in the development of the embryo will be
measured for including heart rate, stroke volume, cardiac output, growth changes, and mortality
using Image J (NIH) software. To observe the growth changes, measure zebrafish growth by
the perimeters of notochord length and eye growth diameter and yolk sac growth, where
appropriate. To measure the cardiovascular changes, (heart rate, cardiovascular output, and
stroke volume) the heart on each organism will be located under a microscope and measured.
Heart rate will be measure using a digital timer to determine the number of heart beats in 30
seconds. Using the same software, NIH Image J, the stroke volume will be analyzed by making
measurements of the end-diastolic and end-systolic volumes of the ventricle. After attaining
these measurements, the cardiac output will be calculated by multiplying the stroke volume and
the heart rate. To measure the survival rate, the dead embryos and each subsequent day will
be counted and sorted; calculating the fraction of surviving larvae at each stage of development.
Before using the Image J (NIH) software, pictures will be taken of the embryos at each stage to
do these measurements. The controls for both experiments will be embryos that have not
received any caffeine doses. Using the different acquired data, and taking a translational
approach, these results will then help determine the ultimate question as to whether consuming
caffeine increases a mother’s risk of birth defects and miscarries.
References
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"Care and Mainainance of Zebrafish." Zebrafish. 11 Sept. 2001. 21 Dec. 2008
<http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Fish/fish_animals.html>.
2.
The Journal of Experimental Biology 204, 4335-4343 (2001)B. Bagatto1,*, B. Pelster2 and
W. W. Burggren1
3.
"Stages of Embryonic Development of the Zebrafish." Developmental Dynamics (1995):
253-310.
4.
"Translational Medicine and the Patient." BioBank Central. 2 Dec. 2008
<http://www.biobankcentral.org/translational/whatis.php>.
5.
"Translational Medicine Research." Translational Medicine Research. 2 Dec. 2008
<http://www.wyeth.co.uk/Home/About--Wyeth/Research---Development/Translational-Med>.
6.
The Journal of Experimental Biology 204, 4335-4343 (2001)B. Bagatto1,*, B. Pelster2 and
W. W. Burggren1