The Increase in Embryonic Size of Zebrafish ( Danio rerio ) after Embryonic Vitamin D Exposure. Cindy Zheng

Transcription

1 The Increase in Embryonic Size of Zebrafish ( Danio rerio ) after Embryonic Vitamin D Exposure Cindy Zheng WInSTEP Research Paper 2018 AP Biology, Kohler High School, Kohler, Wisconsin [1]

2 The Increase in Embryonic Size of Zebrafish (Danio rerio) after Embryonic Vitamin D Exposure WInSTEP Research Paper Presented to The Faculty of Wisconsin Inquiry-based Scientist-Teacher Education Partnership (WInSTEP) Program University of Wisconsin-Milwaukee As part of the Science Education Partnership Award (SEPA) Program sponsored by the National Institutes of Health (NIH) Cindy Zheng Kohler, Wisconsin Advisor: Dr. Mary Eldridge [2]

3 CONTENTS Abstract Introduction Materials and Methods Results Discussion Conclusion Literature Cited Acknowledgments.. 18 [3]

4 Abstract Maternal complications involving the impairment of insulin may result in gestational diabetes. Like other types of diabetes, gestational diabetes affects how your cells use sugar, and affected women may have the risk of high blood pressure, preeclampsia, and Type 2 diabetes later in life (Mayo Foundation, 2017). Nevertheless, most doctors have prescribed Vitamin D to diabetics to combat insulin resistance, with no conclusive scientific data connecting Vitamin D and health (Moonjur Healthcare Inc., 2017). So, recent studies on gestational diabetes have utilized animal models, such as zebrafish, Danio rerio, to display the morphological and behavioral effects of Vitamin D. I exposed zebrafish embryos to high doses of glucose (14 mm) and then treated the zebrafish embryos with the addition of Vitamin D (300 nm). I tested the hypothesis that Vitamin D exposure affect the developmental stages of zebrafish, and thereby the addition of Vitamin D has an effect on overall mortality rates. The results show that embryonic exposure to a high concentration of glucose and then treatment of Vitamin D does indeed affect the morphology and behavior of larval zebrafish. Embryos in Vitamin D exposure treatments exhibited differences in morphology as compared to embryos in the control treatments. Findings from one treatment group even suggest that Vitamin D exposure can lead to an increase in embryonic size double the standard amount. As a result, Vitamin D plays a key role in maintaining healthy development of an embryo. To conclude, the statistics suggest there is a benefit for women to take Vitamin D during pregnancy in regards to the baby s health. Keywords : Vitamin D, gestational diabetes, glucose [4]

5 Introduction Broad Question Based on an initial observation, I determined that pregnant women take a multitude of prenatal vitamins, one of which being Vitamin D. Does the consumption of Vitamin D truly make a difference in affecting embryonic health? Background Information Gestational diabetes is a form of diabetes in which the body does not produce adequate amounts of insulin to regulate sugar during pregnancy (American Pregnancy Association, 2017). While there is no specific cause of gestational diabetes, there are several theories. To begin with, the placenta supports the baby and provides the essential hormones needed for growth and development. However, these hormones also block the mother s insulin from functioning in her body, more commonly known as insulin resistance. When the insulin levels become dangerously low, glucose cannot leave the blood and be changed to energy. So, glucose levels being to build up in the blood. This condition is called hyperglycemia (American Diabetes Association, 2016). Testable Question Do zebrafish embryos have a greater mortality when placed in the wells with the highest dose of glucose over the span of 96 hours as compared to when placed in the wells with the highest dose of glucose in addition to a treatment dose of Vitamin D over a span of 96 hours? Biological Rationale According to a 2014 analysis by the Centers for Disease Control and Prevention, the prevalence of gestational diabetes is as high as 9.2% (4). As a result of gestational diabetes affecting the mother in late pregnancy, the baby is still growing and has the potential of being affected. For instance, when a mother has gestational diabetes, extra blood glucose molecules may pass through the placenta and give the baby high blood glucose levels. This, in turn, may cause the baby s pancreas to make extra insulin to [5]

6 get rid of the blood glucose. Especially dangerous because the baby is getting more energy than needed, the extra energy is stored as fat and possibly lead to macrosomia, or a fat baby. Babies with macrosomia face health problems of their own, including damage to their shoulders during birth, higher risk for breathing problems, and a higher risk for type 2 diabetes (4). Hypothesis If elevated glucose exposure causes adverse effects on the cardiovascular system of a developing embryo and we compare the heart rate of zebrafish embryos exposed to concentrations ranging from 4 mmol to 14 mmol for 96 hours post-fertilization (hpf), then there will be a statistically significant increase in mortality in the group that is exposed to 14 mmol glucose rather than in the zebrafish embryos exposed to 14 mmol glucose and treated with Vitamin D. Experimental Approach This will be a manipulative experiment. Zebrafish placed in wells with the high concentration of glucose and zebrafish placed in wells with the high concentration of glucose and addition of Vitamin D will be analyzed to see their respective rates of mortality and morphological and behavioral differences. The dependent variable will be the size (from head to tail) after 96 hours post fertilization. The independent variable will be the addition of Vitamin D. The constants will be the size of the well, amount of hours between treatments, ml of solution in each well, incubator temperature, and others. The control will be the size of embryos in the embryo media (modeling salt water). We expect the data to yield data that highlights Vitamin D to make an effect on embryonic health. [6]

7 Materials and Methods Ethics Statement All animal experimental procedures were conducted in accordance with local and international regulations. In raising of the larvae, fertilized eggs were kept in an incubator maintained at 28 C. In addition, larvae were kept in embryo medium or other treatment solution with 50% or more of the solution changed on a daily basis. At these points, the removal of any dead zebrafish was conducted. Throughout the entire experiment, zebrafish were checked regularly to ensure their health and wellbeing. Safety Glucose must be handled with care and stored in a well-sealed container that is separated from strong oxidants. It must be kept away from flames and other electrical equipment. If placed in a slightly ajar container during the course of an experiment, the chemical must be ventilated consistently. I will not directly smell or inhale the substance as safety concerns and health issues will arise. Moreover, safety goggles must be worn at all times when handling the chemical and experimenting with it. General Materials The following is a brief summary of the materials needed for my experiment. 66 zebrafish embryos from the University of Wisconsin-Milwaukee were placed in 18 wells each. Three glucose solutions (concentrations of 4 mmol, 8 mmol, and 14 mmol) and one Vitamin D solution were added to the wells accordingly to experimental design. When solutions were reapplied to the zebrafish, glass beakers (50 ml or larger), graduated cylinders (50 ml or larger), plastic microsyringes (2-20 microliters), and plastic pipettes were utilized. Furthermore, when documenting data, a digital device was needed along with a functioning light microscope. Finally, safety goggles, gloves, and aprons (if applicable) were needed at all times. [7]

8 General Procedures I used zebrafish from a breeding facility maintained at the University of Wisconsin-Milwaukee. As illustrated in the flow diagram in Figure 1, zebrafish embryos were first exposed to glucose according to assigned treatment groups. Then, only the zebrafish embryos that were exposed to the high concentration of glucose were treated with the addition of Vitamin D. I took digital images of the embryos every 24 hours post fertilization. All embryos were placed in wells and stored in an incubator maintained at 28 C. I completed size analysis to estimate the morphological size of each zebrafish compared to the control zebrafish. Figure 1. The general procedure of the experiment. [8]

9 Statistical Analysis of Morphologies To determine differences in morphology of the embryos in different treatments, I used a two-sample t-test. I used a t-test because this type of statistical test allows you to determine if there is a statistically signficant difference between two treatments. In addition, the two-sample t-test is a hypothesis test for answering questions about the mean when the data are collected from two random samples of independent observations, each from an underlying normal distribution. I completed these tests on GraphPad, and the p-value is identified as being less than Furthermore, the t-test was applied because of small sample size in the treatment used. [9]

10 Results Using the experimentally determined standard curve, the morphological size of the embryos in the treatment group with high glucose concentration was significantly less than the morphological size of the embryos in the treatment group with high glucose concentration and treated with Vitamin D. The t-value of the two-sample t-test was with degrees of freedom being 22 and a standard error of difference being High Concentration of Glucose Morphology The average length of embryos exposed to a high concentration of glucose was significantly lower than those of the control hatchings and those treated with Vitamin D. Regarding the statistical summary information, the mean was μm and standard deviation μm. In addition, the standard error of the mean was 4.69 μm with a sample size of High Concentration of Glucose and Treatment of Vitamin D Morphology The average length of embryos exposed to a high concentration of glucose and treated with Vitamin D was significantly higher than those treated without Vitamin D. Regarding the statistical summary information, the mean was μm and standard deviation μm. In addition, the standard error of the mean was 7.11 μm with a sample size of [10]

11 Group Specific Well Size (µm) Stats Group 3: High Glucose Concentration Group 6: High Glucose Concentration with Vitamin D 3.1 n = µm 3.2 n = µm 3.3 n = µm 6.1 n = µm 6.2 n = µm 6.3 n = µm Max = 133 Median = 100 Mean = 111 Min = 100 Max = 300 Median = 300 Mean = 283 Min = 250 Results for Groups 1, 2, 4, and 5 can be found by scanning QR Figure 2. General table of statistical results of the experiment. Figure 3. Boxplot of the distribution of average sizes in the experiment. [11]

12 Figure 4 : Above is the 400X microscopic view of an unhatched zebrafish embryo exposed to a high amount of glucose solution, modeling pregnant women with elevated blood glucose levels. Figure 5 : Above is the 400X microscopic view of a zebrafish embryo exposed to high amount of glucose solution and treated with Vitamin D solution, modeling pregnant women with elevated blood glucose levels taking Vitamin D supplements. [12]

13 Figure 6 : Above is the 100X microscopic view of an unhatched zebrafish embryo exposed to a high amount of glucose solution, modeling pregnant women with elevated blood glucose levels. Figure 7 : Above is the 100X microscopic view of a hatched zebrafish embryo exposed to a high amount of glucose solution and treated with Vitamin D solution, modeling pregnant women with elevated blood glucose levels taking Vitamin D supplements. [13]

14 Discussion The results of the experiment provide evidence that the addition of Vitamin D demonstrated drastic changes in morphology. When the zebrafish embryos were exposed to the Vitamin D, the morphological size (from head to tail) increased in size while the zebrafish embryos not exposed to Vitamin D did not follow the same pattern. Broad Study Question Do zebrafish embryos have a greater mortality when placed in the wells with the highest dose of glucose over the span of 96 hours as compared to when placed in the wells with the highest dose of glucose in addition to a treatment dose of Vitamin D over a span of 96 hours? Support or Reject Hypothesis Based on the results of the experiment, I rejected the null hypothesis. The null hypothesis was that the addition of Vitamin D has no effect on embryonic development. The alternative hypothesis was that the addition of Vitamin D does have an effect on embryonic development. I reject the null hypothesis because there was a significantly higher percentage of surviving zebrafish after the four day period in the wells with the high concentration of glucose and treated with the addition of Vitamin D. Interpret Data The p-value told us that there was a % chance of finding a difference as large as than the one in my study given that the null hypothesis is true. So, I reject the null hypothesis and support the alternative hypothesis: the addition of Vitamin D does have an effect on embryonic development. Generating New Knowledge Furthermore, there was a larger increase in embryonic yolk sac for the zebrafish treated with Vitamin D. From a scientific approach, this could suggest Vitamin D plays a key role in the functioning and development of a healthy embryo. [14]

15 Evaluating Design and Results Potential sources of error in our experiment could be adding the correct dosages of Vitamin D. Although I did the daily dosage recommended by doctors, the levels may differ to other individuals. On another note, we could repeat the experiment again but with a larger sample size to see if the results are confirmed. New Questions and Future Studies On the topic of varying the dosages of Vitamin D, I would like to repeat the experiment again but this time treat a high concentration of glucose with varying concentrations of Vitamin D. In this case, I want to see, on a statistical standpoint, if one concentration is more beneficial than others. Furthermore, I would like to test if a certain brand of Vitamin D has an effect on total effectiveness. [15]

16 Conclusion Overall, the results of this study indicate that Vitamin D is indeed a factor in embryonic growth. Specifically, we found that the inclusion of Vitamin D more than doubled the embryonic size. As a result, the morphology and growth of the zebrafish are impacted. Nevertheless, a repeat of this experiment would be beneficial with a larger sample size. This would make our results more conclusive and accurate. [16]

17 Literature Cited American Diabetes Association. (2016, November 21). What is Gestational Diabetes? Retrieved March 23, 2018, from American Pregnancy Association. (2017, April 12). Gestational Diabetes: Testing and Treatment. Retrieved March 23, 2018, from Mayo Foundation. (2017, April 28). Gestational diabetes. Retrieved March 23, 2018, from Moonjur Healthcare Inc. (2017, September 16). Vitamin D and its Connection with Diabetes. Retrieved March 23, 2018, from [17]

18 Acknowledgments I am deeply grateful for the help and guidance of my advisor, Dr. Mary Eldridge. Her encouragement and innovative approach to modern science enabled the completion of this research paper. This report could not have been completed without the hard work of Rene Hasselback, Outreach Specialist of the UWM-WinSTEP SEPA Program. Her commitment to making this program run as efficiently as possible was greatly appreciated. Many thanks to research partners, Kate O Leary and Nicole Konecke, for their help with daily treatments, zebrafish maintenance, and hours spent after class to complete this project. This project was supported by the Wisconsin Inquiry-based Scientist-Teacher Education Partnership (WInSTEP) Program, which is part of the NIH Science Education Partnership Award (SEPA) Program. [18]