Effects of alcohol on the development of the cardiovascular system in Pekin Ducks (Anas platyrhynchos): An assessment of current empirical findings and the development of a research protocol utilizing Pekin Ducks A project completed in partial fulfillment of the requirements for the Honors Program By Josephine McKean May 8, 2021 Department of Biological and Environmental Sciences Capital University Approved by Nancy J Swails Name, Advisor __________John Mersfelder______________________________________ Name, Department Chair Accepted by ________________________________________________ Name, Director, Capital University Honors Program 1 Copyrighted by Josephine McKean 2021 2 Abstract Fetal alcohol syndrome is a serious condition that affects the development of fetuses with irreversible effects that can impact individuals throughout their lives. The cardiovascular system is one example of an organ system in which abnormalities caused by alcohol can occur. The heart is one of the first structures to be formed, and heart development is highly conserved among amniotes. There are difficulties studying the effects of ethanol on human embryos due to ethical concerns; as a result, the use of animal models, particularly avian models, is widely used. The effects of ethanol have not been widely studied on Pekin ducks, Anas platyrhynchos, and ducks offer advantages compared to other model organisms, such as their larger size and durability. The purpose of this study was to develop a method for testing the effects of ethanol on the development of the heart and cardiovascular system in ducks. The development of the cardiovascular system occurs over several stages of development, and treatment of ethanol at different stages leads to various potential abnormalities of heart structure and function. The developed protocol determines which stages of heart development are most sensitive to ethanol effects, and what anomalies are expected to form after exposure to ethanol. ii Acknowledgments I express gratitude to my advisor, Dr. Nancy Swails, for all her continued advice and support throughout this project. I greatly appreciate all her dedication and guidance during the course of this research. I would also like to thank the Capital University Honors Program for providing encouragement for and the opportunity to pursue undergraduate research. iii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments .............................................................................................................. iii List of Figures ..................................................................................................................... v Chapter 1. Introduction ....................................................................................................... 1 Chapter 2. Methods ............................................................................................................. 5 Chapter 3. Use of Avian Models ......................................................................................... 6 Chapter 4. Development of the Cardiovascular System ..................................................... 7 Chapter 5. Ethics Statement .............................................................................................. 13 Chapter 6. Proposed Methods ........................................................................................... 14 Chapter 7. Expected Results ............................................................................................. 19 Avian Models ................................................................................................................ 19 Mammalian Models ...................................................................................................... 20 Physiological Findings .................................................................................................. 21 Chapter 8. Discussion and Conclusions ............................................................................ 22 Figures............................................................................................................................... 25 References ......................................................................................................................... 26 iv List of Figures Figure 1 ............................................................................................................................. 25 v Chapter 1. Introduction Alcohol use can cause negative impacts on people other than the person consuming it, such as physical violence, car accidents, relationship problems, or financial difficulties (Laslett et al., 2011). In addition to these, alcohol consumption during pregnancy is another common example of alcohol use that drastically impacts another individual’s life. Ethanol is a teratogen, meaning that it is a substance that can cross the placenta and cause damage to the organs of the developing embryo (Popova et al., 2017). Ethanol consumption during pregnancy has been linked to various risk factors and adverse pregnancy outcomes, such as stillbirth due to fetoplacental dysfunction (Kensmodel et al., 2002), spontaneous abortion (Abertsen et al., 2004; Patra et al., 2011), low birthweight (Patra et al., 2011; O'Callaghan, 2003), and preterm birth (Albertsen et al., 2004; Patra et al., 2011). Ethanol consumption has also been found to cause congenital abnormalities, such as growth deficiencies, microcephaly, cleft palate, joint abnormalities, anomalous external genitalia, epicanthal folds, and maxillary hypoplasia (Jones & Smith, 1973). Additionally, alcohol use can lead to developmental, cognitive, or learning disabilities in children (Mesa et al., 2017; Popova et al., 2017). Collectively, all impacts of ethanol consumption during pregnancy are referred to as Fetal Alcohol Spectrum Disorders (FASD), and an estimated 119,000 children are born with FAS every year (Popova et al., 2017). 1 Over 40 years ago, distinct patterns of altered growth and morphogenesis, including growth deficiencies, craniofacial abnormalities, joint abnormalities, cardiac abnormalities, and fine-motor function difficulties were observed in infants born to alcoholic mothers. This unique pattern of abnormalities was described as Fetal Alcohol Syndrome (FAS) (Jones & Smith, 1973; Jones et al., 1973), and further research has expanded upon the original presentation to provide further evidence of FAS (Hanson et al., 1978; Clarren & Smith, 1978). Since then, additional effects of ethanol intake during pregnancy have been described as Fetal Alcohol Spectrum Disorders (FASD), which include a wide range of physical, mental, behavioral, and learning disabilities seen in individuals whose mother consumed alcohol during pregnancy, and symptoms can present in utero through early childhood (Benz et al., 2009; Cook et al., 2016; Caputo et al., 2016). Common behavioral or cognitive disabilities associated with FASD include mental retardation, developmental delays, or attention deficit hyperactivity disorder- ADHD (Caputo et al., 2016). Many studies find a positive correlation between increased alcohol consumption and adverse embryological effects (Hanson et al., 1978; Kensmodel et al., 2002; Albertsen et al., 2004; Patra et al., 2011; O’Callaghan et al., 2003). Some of the most severe birth defects occur in the brain and neurologic systems (Jones & Smith, 1973; Caputo et al., 2016), but other severe birth defects are found in the heart and cardiovascular system (Caputo et al., 2016). One significant effect of ethanol consumption is defects in the development of the cardiovascular system. Prior research has shown that FAS and FASD do affect the cardiac systems with cardiac abnormalities (Jones et al, 1973). Babies born to mothers 2 who consumed alcohol during pregnancy were more likely to have cardiac murmurs (Hanson et al, 1978). Up to one-third of individuals affected by FASD have congenital cardiac diseases (Krasemann & Klingebiel, 2007). The heart is one of the first structures to develop because the cardiovascular system is critical in distributing nutrients to the developing embryo. Prior research has found several defects in cardiac function due to ethanol exposure, including abnormal flow, excitation, contraction, and calcium transients, and can cause cellular, molecular, functional, and structural abnormalities (Karunamuni et al, 2014). Heart defects associated with FASD are often some of the most life-threatening and require surgical correction in newborns (Karunamuni et al, 2014). In fact, embryo death during organogenesis is most commonly associated with cardiovascular impairment (Flentke & Smith, 2017). Additionally, the cardiac orienting response in human infants has been evaluated as a possibility to detect neurological defects that usually appear later in life (Mesa et al, 2017). Experimental studies to evaluate the timing and amount of ethanol exposure on embryonic development have been completed in animal models. Previous experiments have studied the effects of ethanol on cardiac abnormalities on many model organisms, including Zebrafish larva (Li et al, 2016; Dlugos & Raban, 2010), quail embryos (Ma et al, 2016; Karunamuni et al 2014; Karunamuni et al 2015; Serrano et al, 2010; Twal & Zile, 1997; Peterson et al., 2017), white leghorn chick (Serrano et al, 2010), mice (Serrano et a, 2010), and baboons (Seleverstov et al, 2017). Prior research