To collect data, the cross-sectional area of the heart and body were determined by mounting and examining each D. magna under the EVOS microscope. For the purposes of this experiment, the term “cross-sectional area” was defined as the widest outlined area visible from a top view of the organism (equivalent to the area of a two-dimensional slice through the widest part of the object in question, whether that be the heart or the organism itself).
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The images were analyzed through ImageJ, a computer software program used for image analysis that allows the operator to analyze scale measurements based on pixel ratio. For each image captured by the EVOS microscope, there was also a corresponding scale that related to the real-life size of the object being observed. ImageJ was used to measure the pixel length of this scale on the computer and create a conversion factor based on the known distance provided by the scale. Once the scale had been set and globalized, the outside of the heart was traced using the “freeform” tool and the “measure” button from the top menu. These actions produced the value of the cross sectional area within the previously drawn outline.
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The data was organized into a spreadsheet where water contents, body area, and heart area were recorded for each organism. Google Sheets functions were then used to calculate the ratio of heart cross-sectional area to body cross-sectional area as a percentage. The average areas and ratios were quantified for each group, as well as the average standard deviations from the mean for both.
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The average heart-body ratio for the experimental D. magna was 2.62%, while the average ratio for control D. magna was 1.88%. We observed a... 39.36% increasein heart size after estrogen exposure, as calculated by dividing the difference between the two average ratios by 1.88%.
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Mean heart area was 26507.4 μm2 for EE2 exposed D. magna and 16930.6 μm2 for control D. magna, demonstrating a significant increase in size after exposure to EE2. A comparison of the average body areas between the two groups revealed a difference of only 52,789.5μm2 – only 5.80% when compared to the body area of an average juvenile control D. magna. The average standard deviation for ratios in experimental D. magna was 0.18%, but in the control group, this value was only .09%.
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control (no EE2)
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EE2 (.0003 g/L)
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