Anas Platyrhynchos, and Ducks Offer Advantages Compared to Other Model Organisms, Such As Their Larger Size and Durability

Home , Anterior cardinal vein, Aorticopulmonary septum, Aortic sac, Blood islands, Bulbus cordis, Chorion

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

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

Copyrighted by

Josephine McKean

2021

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.

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

List of Figures

Figure 1 ...... 25

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 has used animal models and injected ethanol into developing embryos. Dlugos and Rabin (2010) found that their treated group of zebrafish had pericardial edema, heart enlargement,

3 misshapen atria and ventricles, and the appearance of small chambers spread out over serial sections. Additionally, ventricle wall thickness was thinner in treatment groups than controls. Li et al. (2016) also reported pericardial edema, as well as blood vessels that were narrower or fewer in number in zebrafish in treatment groups compared to controls. Samples treated with higher doses of ethanol experienced more significant defects.

Studying the effects of ethanol on embryonic development using animal models is necessary to understand the implications on human development and FASD. The purpose of this research is to develop a method for evaluating what effect, if any, ethanol has on the development of the cardiovascular system of Pekin ducks (Anas platyrhynchos). A literature review was completed to compare the development of Pekin ducks to the more widely studied chick and quail embryos to understand how to modify prior methods to duck embryos. In addition, several possible stages of development were identified as potential timing of ethanol injections to cause anomalies in the cardiovascular system.

Chapter 2. Methods

I performed a literature review using several different databases, including

GoogleScholar, BIOSIS, and Web of Science. Common search phrases used throughout this review include “fetal alcohol syndrome,” “fetal alcohol spectrum disorders,”

“cardiovascular and heart development,” “avian model with ethanol exposure,” “ethanol and cardiovascular development,” and many others. I chose to include studies that used avian models or animal models that evaluated ethanol effects on the cardiovascular system.

Chapter 3. Use of Avian Models

Avian embryos are often used to study development for several reasons. First, avian embryos are enclosed in a calcium carbonate shell that can easily be windowed for observation. Second, development occurs under conditions (37-38 degrees Celsius and

50-55% humidity) that are easily obtained in incubators. Third, the development of avians and mammals is largely conserved, which is evidenced by the fact that avians and mammals are both amniotes, meaning they possess an amnion that surrounds the embryo, and many developmental processes at the molecular and cellular levels between avian and mammalian development are similar (Flentke & Smith, 2017). In particular, heart valve formation in embryos has been largely conserved in vertebrates, including between chickens and humans (Combs & Yutzey, 2009). Because of these reasons, researchers concluded the development of avians and mammals, including humans, is essentially equivalent. This has allowed researchers to better understand the development of avians and mammals.

Chapter 4. Development of the Cardiovascular System

In amniotes, the cardiovascular system is the first system to function in the developing embryo. Cardiac cords form during the third week of development in humans, and they canalize to form the endocardial heart tubes that fuse cranially to caudally to form a single tube that will eventually develop into the heart. Mesenchyme surrounds the heart tube and thickens to form the myoepicardial mantel. The endocardial tube eventually develops into the endocardium, the endothelial lining of the heart. The myoepicardial mantel becomes the myocardium and the epicardium. The tubular heart begins to elongate and develop dilations and constrictions. The dilations become the bulbus cordis, ventricles, atria, the truncus arteriosus, and sinus venosus. The truncus arteriosus continues to grow caudally with the bulbus cordis and grow cranially with the aortic sac. Next, the heart folds into a U-shaped loop known as the bulboventricular loop, and the atrium and sinus venosus move to lie dorsal to the bulbus cordis, truncus arteriosus, and ventricle. Then, the sinus venosus develops left and right horns. The left horn will become the coronary sinus, while the right horn becomes the sinus venarum.

The heart invaginates into the pericardial cavity as it bends. Initially, the heart is suspended by a dorsal mesentery. The central portion of the mesentery degenerates, and the transverse pericardial sinus is formed between the right and left sides of the

7 pericardial cavity. At this point, the heart is suspended only by the vessels at the cranial and caudal ends.

Heart contraction begins early in embryonic development, by day 22 of human development. At this point, the ventricle and atrium muscle layers are continuous.

Contractions begin at the sinus venosus and move through the heart in peristaltic-like waves. Initially, there is an ebb and flow circulation. By the end of the fourth week in humans, there are coordinated contractions. When the heart contracts, blood flows through the heart. Blood circulation in the early embryonic heart is different from blood flow in later hearts. Blood flows through the sinus venosus to the sinoatrial valve. From the sinoatrial valve, blood flows to the atrioventricular valve to the primitive ventricle to the bulbus cordis to the truncus arteriosus into the aortic sac.

The heart begins to separate into four chambers around the fourth week of development in humans, and this partitioning is essentially complete by the fifth week.

Endocardial cushions form on the dorsal and ventral walls of the atrioventricular canal.

The cushions enlarge and fuse to divide into the left and right halves. Two different septa form from endothelial cells in the atrium: the septum primum that grows down from the roof of the atrium towards the endothelial cushions and the septum secundum that grows from the roof of the atrium to the right of the septum primum towards the endocardial cushions. The septum secundum grows to the right of the septum primum. As the septum primum fuses with the endocardial cushions, the foramen primum is obliterated, but the foramen secundum remains patent. The septum secundum does not fully reach the endocardial cushions, resulting in a hole, the foramen ovale. This remains open but is

8 overlapped by the septum primum, which forms a valve. In the fetal heart, blood can flow directly from the right atrium through the foramen ovale into the left atrium. At birth, the septum primum becomes pressed against the other septum due to increased pressure.

Eventually, the two septa will fuse, causing blood to no longer flow from the right atria to the left atria. The sinus venosus forms the right atrium dorsal wall.

The left atrium forms primarily from the primitive pulmonary veins, which forms as an out-growth of the dorsal atrial wall. The pulmonary veins become incorporated into the atrial wall, resulting in separate openings for veins. The primitive ventricle separates into separate ventricles. This process begins with the appearance of a muscular ridge near the ventricular apex. This ridge elevates as the ventricles dilate on each side. Myoblasts form the thick muscular part of the interventricular septum. An interventricular foramen is present until the seventh week of development in humans. This allows communication between the left and right ventricle.

Bulbar ridges form in the walls of the bulbus cordis. Truncal ridges form in the truncus arteriosus and are continuous with the bulbar ridges. The bulbar and truncal ridges fuse to form the spiral aorticopulmonary septum that creates two separate channels: the aorta and the pulmonary trunk, which twist around each other. The interventricular septum fuses with the aorticopulmonary septum. This causes the pulmonary trunk to communicate with only the right ventricle and the aorta to communicate with only the left ventricle. The bulbus cordis is incorporated into the walls of the ventricles and forms the aortic vestibule in the left ventricle.

Angiogenesis is the formation of blood vessels. The process begins when the mesenchymal cells, also known as angioblasts, aggregate to form isolated masses and cords called blood islands. Cavities form in these islands, and angioblasts form the endothelium. Vessels fuse to form endothelial channels that extend by budding and fusing. Early plasma and blood cells develop initially in the yolk sac and allantois. Actual blood formation does not form in the embryo until the fifth week in humans in the embryonic mesenchyme of the liver, spleen, marrow, and lymph nodes.

Three paired veins drain the heart: the vitelline veins, the common cardinal veins, and the umbilical veins. The vitelline veins follow the yolk sac into the embryo and enter the sinus venosus after passing through the septum transversum. Hepatic veins form from the right vitelline vein; the anastomotic network formed around the duodenum by the vitelline veins forms the hepatic portal vein. The right umbilical vein degenerates, but the left umbilical vein remains and carries blood from the placenta to the fetus. The ductus venosus develops within the liver as a shunt between the umbilical vein and the inferior vena cava. After birth, the umbilical vein becomes the ligamentum teres, and the ductus venosus becomes the ligamentum venosum. There are three sets of cardinal veins: the anterior cardinal veins, the posterior cardinal veins, and the common cardinal veins. The anterior cardinal veins drain the cranial portion of the embryo, and the posterior cardinal veins drain the caudal portions of the embryo. They empty into the sinus venosus through the right and left common cardinal veins. The anterior cardinals become connected by an anastomosis and shunts the blood from left to right. The caudal portion of the left anterior cardinal vein becomes the left brachiocephalic vein. The right anterior cardinal vein and

10 the right common cardinal vein become the superior vena cava. The posterior cardinal veins are associated with the mesonephros kidneys and degenerate when the mesonephros kidneys are replaced with the metanephros kidneys. The portions that remain become the root of the azygous vein and the common iliac veins.

There are two major tributaries of the posterior cardinal veins: the subcardinal veins and the supracardinal veins. The subcardinal veins replace and supplement the posterior cardinal veins. The subcardinal veins form the stem of the left renal vein, the suprarenal veins, the gonadal veins, and a segment of the inferior vena cava. The supracardinal veins are the last to develop and parts of the azygous vein and hemiazygous vein and the inferior part of the inferior vena cava. The inferior vena cava is formed from the hepatic segment, the prerenal segment, the renal segment, and the postrenal segment.

The hepatic segment is derived from the hepatic veins and the hepatic sinusoids. The prerenal segment is derived from the right subcardinal. The renal segment is derived from the subcardinal and supracardinal anastomosis. The postrenal segment is derived from the right supracardinal vein.

The branchial arches form during the fourth and fifth week. The aortic arches grow into the branchial arches and come from the aortic sac. The dorsal aortae fuse to form a single dorsal aorta that is caudal to the branchial arches. Approximately 30 segmental arteries branch off of the dorsal aorta and carry blood to somites and their derivatives. The persisting segmental arteries in adult humans are the pulmonary arteries, the intercostal arteries, the lumbar arteries, the common iliac arteries, the suprarenal arteries, the renal arteries, and the gonadal arteries. The caudal end of the dorsal aorta

11 becomes the median sacral artery. The vitelline arteries are unpaired branches from the dorsal aorta in the ventral midline that pass to the yolk sac and later the gut. The celiac artery goes to the foregut, the superior mesenteric artery to the midgut, and the inferior mesenteric artery goes to the hindgut. The paired umbilical arteries go to the chorion and carry deoxygenated blood. The proximal portions become the internal iliac arteries and superior vesical arteries. The distal parts degenerate and become the median umbilical ligaments.

Six pairs of aortic arches normally develop in an embryo, but not all six are present at the same time. The primitive aortic arch pattern is transformed into the adult pattern during the sixth to eighth weeks of development. The first aortic arches mostly disappear, but some parts remain to form the maxillary arteries and may contribute to the external carotid arteries. The second aortic arches become the stapedial arteries. The third aortic arches become the common carotid arteries and the internal carotid arteries. the fourth aortic arch becomes the aortic arch and the subclavian arteries. The fifth aortic arch degenerates and leaves no derivatives. The sixth aortic arch becomes the pulmonary arteries and the ductus arteriosus.

Chapter 5. Ethics Statement

Institutional Animal Care and Use Committee (IACUC) approval is not required for this experiment because the embryos will be euthanized prior to three days before hatching. Pekin ducks typically have an incubation period of 28 days, and the experiment will not be carried out this far.

Chapter 6. Proposed Methods

The purpose of this experiment is to determine what effect, if any, ethanol has on the development of Pekin ducks (Anas platyrhynchos). Although the effects of FAS using avian models have been widely studied, most experiments use quail or chick embryos, and the effects of ethanol on the development of Pekin ducks (Anas platyrhynchos) have not been widely studied. Pekin duck embryos offer many advantages over other avian models, including proving to be hardier with a fertility rate of 95% based on data obtained from Ridgeway Hatchery. Duck embryos are larger than chick embryos, and as a result, they are easier to manipulate and work with. Many studies involving chicks or quail require the use of imaging to evaluate cardiovascular defects. Since ducks are bigger in size, a large research lab with access to this imaging equipment is not required.

Furthermore, the development of Pekin ducks and chicks is highly conserved, so there are very few differences in their development. Most of the differences in development occur in late stages of organogenesis and growth, and most likely as a result of differences in the timing of particular events and/or growth trajectories of a specific structure, also known as heterochrony. Additionally, chicks and ducks develop at different rates and have differences in periods of gestation: chicks have a gestation length of 21 days while ducks have a period of 28 days. Li et al. (2019) compared the embryonic development of ducks (Anas platyrhynchos) to the more widely studied chicken (Gallus gallus) and goose

(Anser cygnoides). They compared the development of these three animals and placed them all into the common stages of development. The timing in hours of when each organism is expected to enter each stage was recorded. The major anatomical developments of each stage were also recorded.

In this experiment, duck eggs will randomly be divided into three groups: one control group receiving no injection, one control group receiving an injection of 300 microliters of sterile water, and one group receiving an injection of 300 microliters of

45% alcohol. The injection procedure will be performed as previously studied (Pinkard &

Swails, 2017; Taylor & Koenig, 2018; Taylor & Koenig, 2019). The alcohol dosage for use in ducks was determined based on previous experimentation (Pinkard & Swails,

2017). Taylor and Koening (2018) compared the percentage of duck embryos that had arrested development with varying ethanol doses: 30%, 40%, and 45%. The 45% alcohol dose was determined to be the best dose to use in this research. In one study, 10% alcohol revealed little to no adverse effects on chick and quail egg development, but 25%/35% ethanol caused cardiac valve abnormalities in over two-thirds of embryos (Serrano et al.,

2010). Karunamuni et al. (2014, 2015) and Peterson et al. (2017) injected 40 micrograms of 50% ethanol into quail eggs, and this dosage was equated to a single binge drinking event in humans. Twal and Zile (1997) injected a solution composed of 0.5%-2% ethanol into quail embryos. The ethanol dosage for this experiment is higher than prior studies since duck embryos are larger than those of chicks or quail. Larger organisms require a larger dose to mimic the effect of a human binge drinking episode.

In the proposed method, eggs are injected with 300 microliters of their solution, either ethanol or sterile water, and treatment groups will be sealed with parafilm.

Previous research has used sterile water or saline as a control injection into avian eggs or mammalian embryos. Taylor and Koenig (2019) found no statistically significant difference in degree of development between controls injected with water compared to saline. As a result of these findings, eggs in this experiment will be injected with sterile water. A control injection is used to ensure that any changes in development are due to ethanol injections, not just the injection process itself (Pinkard & Swails, 2017; Taylor &

Koenig, 2018; Taylor & Koenig, 2019). Prior studies have shown physiological changes with control embryos injected with saline compared to non-injected embryos, but these changes were associated with the placenta, not the heart (Serrano et al., 2010). Pekin ducks are not mammals, so they do not have a placenta, so this is not expected to be an issue. All eggs will be incubated at 38 degrees Celsius with 65% humidity, and all eggs will be removed from the incubator for the same amount of time when injections are being performed and when embryonic development is being studied (Pinkard & Swails,

2017; Taylor & Koenig, 2018; Taylor & Koenig, 2019).

Injections could be made at several critical times in development. An initial injection could be made at HH stage 4-5, gastrulation (Hamburger & Hamilton, 1951; Li et al, 2019). When compared to humans, at this point in a pregnancy, a woman might not know she was pregnant (Karunamuni et al., 2014). Previous research has shown that embryos are particularly vulnerable to alcohol-induced congenital heart defects (CHD) when exposed to alcohol during gastrulation (Serrano et al, 2010; Karunamuni et al.,

2014; Karunamuni et al., 2015). Another possible stage for the initial injections could be

HH stage 8-9 because this is when the paired primordia begin to fuse (Hamburger &

Hamilton, 1951; Li et al, 2019; Ma et al., 2016; Twal & Zile, 1997). Another possible stage for ethanol exposure could be HH stage 19 because this is the stage when cardiac cushions are actively forming (Combs and Yutzey, 2009; Karunamuni et al., 2014; Ma et al., 2016).

The embryos will continue to develop until HH stage 34-35 because, at this stage, the heart is expected to have completely separated into four chambers, vessels should have achieved their mature branching pattern, and left atrioventricular valve leaflets should be distinct. At this time, the heart and major vessels are expected to be fully formed (Hamburger & Hamilton, 1951; Li et al., 2019). When the control embryos have reached HH stage 34-35, all embryos will be dissected to determine the degree of development they reached. Prior research has allowed avian embryos to develop to this stage to evaluate for cardiac defects (Serrano et al., 2010; Karunamuni et al., 2014; Ma et al., 2016). All embryos will be staged based on limb size, visceral arches, and other features to determine how far they developed (Hamburger & Hamilton, 1951; Li et al,

2019). Another physical aspect that can be measured is the degree of cervical flexure.

Prior research compared cardiac defects with structural morphology, and they found that most embryos exposed to ethanol experienced differences in morphology and embryo twisting, and they related this to the heart looping mechanism that occurs in development

(Karunamuni et al, 2014).

In addition to using body features to stage the embryos, the degree of cardiac development will be observed. Cardiac development will be compared between controls and treatment groups to see if there is a significant difference in the development of the heart and/or vessels. Any abnormalities, delays, or differences in folding structure in heart structure between the controls and the treatment groups will be recorded.

Chapter 7. Expected Results

It is expected that ethanol will have an effect on cardiac and overall development of Pekin duck embryos. Research using avian embryos as models have found various cardiac defects associated with ethanol exposure during development: thinner interventricular septae, malformed aortic valves, and ventricular wall thinning

(Karunamuni et al, 2014), bilateral heart fields not fusing, cardiac tissue situated anterior to the head, and left-looping hearts (Serrano et al, 2010), ventricular septal defects, missing or misaligned great vessels, double outlet right ventricle, and hypoplastic or abnormally rotated ventricles (Karunamuni et al, 2015), and ballooning or tube-like unlooped hearts and failure to connect the dorsal aorta and the extraembryonic circulation

(Twal & Zile, 1997). It is also expected that embryos exposed to ethanol will be less developed in terms of limbs and visceral arches as well as experience greater amounts of death than control embryos.

Avian Models

Fang et al. (1987) report three main types of cardiovascular defects in chicks when exposed to ethanol: intracardiac abnormalities, including ventricular septum defects and right ventricle or common aorticopulmonary trunk double outlets; aortic arch abnormalities; and subclavian artery anomalies. Intracardiac anomalies were reported in 19

64.8% of samples, and 11.2–89.1% of samples had subclavian artery abnormalities.

Serrano et al. (2010) found several cardiac abnormalities that would most likely be lethal in quail embryos, including the bilateral heart fields not fusing, cardiac tissue that was located anterior to the head, signifying a truncation of the neural tube, and left looping hearts. Karunamuni et al. (2014) recorded smaller atrioventricular valves in treatment quail embryos compared to controls, thinner interventricular septae, malformed aortic valves, and ventricular wall thinning. Karunamuni et al. (2015) found a wide array of cardiovascular defects in quail embryos, including perimembranous and muscular ventricular septal defects, missing or misaligned great vessels, double outlet right ventricle, and hypoplastic or abnormally rotated ventricles.

Mammalian Models

Seleverston et al. (2017) studied the effect of ethanol exposure on baboons. They reported significantly decreased peak systolic velocity of the middle cerebral artery 120 minutes after alcohol exposure when compared to pre-exposure values. The cerebral arteries provide vital nutrients to the developing brain and neurological system. This decreased peak systolic velocity following alcohol exposure is consistent with dilation of the fetal middle cerebral artery. This could lead to a drop in perfusion pressure, which could result in cognitive deficiency and skull malformation.

Physiological Findings

Heart function was also affected by ethanol treatments in prior research. 44% of individuals suffering from FASD have electrocardiograms that revealed shortened QRS intervals (Krasemann & Klingebiel, 2007). Zebrafish treated with ethanol had a significant increase in heart volume and a significant decrease in heart rate when compared to controls (Dlugos & Rabin, 2010; Li et al., 2016). Serrano et al. (2010) report several findings on electrocardiograms of mice embryos treated with ethanol, such as atrioventricular valve or semilunar valve regurgitation. Other research has shown that ethanol-treated embryos have significantly higher levels of retrograde flow compared to control groups (Karunamuni et al., 2014; Peterson et al., 2017). This likely relates to the development of the endocardial cushions.

Chapter 8. Discussion and Conclusions

Despite the fact that FASD and FAS are well-documented, women continue to use alcohol throughout their pregnancies. Floyd and Sidhu (2004) reported that approximately 40% of American women drink alcohol during pregnancy, and 3-5% drink heavily during pregnancy. 10% of women consume alcohol during pregnancy, and approximately 1.5% of babies delivered to these mothers develop FAS. The risk is far greater in heavy drinkers; approximately 1 in 23 babies born to mothers who drink two or more alcoholic drinks per day or five to six drinks per occasion will develop FAS. FASD is estimated to be nine to ten times as common as FAS (Popova, 2017). Worldwide, an average of 9.8% of women report any amount of alcohol use during pregnancy with an average of 14.6/10,000 cases of FAS. The highest percentage of alcohol use during pregnancy was in Europe, with 25.2% and 37.4/10,000 cases of FAS. The lowest percentage of alcohol use during pregnancy was in the Eastern Mediterranean region, with 0.2% and 0.2/10,000 cases of FAS (Popova et al., 2017).

When first described in 1973, the physical impairments of FAS included craniofacial, limb, cardiovascular defects, growth deficiency, and developmental delay

(Jones & Smith, 1973; Jones et al, 1973). The exact mechanism of how ethanol affects development to cause birth defects is unknown since studies find such a wide range of results. It is difficult to directly study the effects of ethanol exposure to human

22 development due to ethical concerns, so many studies rely on observation of abnormalities or autopsy findings (Jones & Smith, 1973; Jones et al., 1973) or surveys of pregnant women and new mothers (Albertsen et al., 2004). Survey methods present challenges because participants may not provide the whole truth, either due to forgetfulness or social stigma (Benz et al., 2009). It is also impossible to conduct a controlled experiment studying the effects of ethanol on human development. For this reason, experiments that control the amount and timing of alcohol exposure can be valuable in studying the mechanism of anatomical and physiological defects on embryos due to alcohol. The proposed methods could determine if cardiovascular defects are visible after exposure to ethanol, and this would confirm the usefulness of controlled experiments involving ethanol and embryonic development. Ducks in particular are useful for comparison to humans because both are amniotes with highly conserved embryonic development.

If access to a full research lab was available, additional imaging and testing could be performed to further determine which defects are present in the embryos. With the proposed method, I can only identify if there are anatomical abnormalities present; it is not possible to diagnose which anomalies are present. Karunamuni et al. (2014 and 2015) and Ma et al. (2016) used optical coherence tomography, a nondestructive imaging technique that maps cardiovascular structure and hemodynamics in real-time. If access to this imaging was available, this would allow us to determine if there are physiological changes to the cardiovascular system associated with ethanol exposure as well as diagnose various heart conditions and abnormalities.

Further research could expand on this method to determine the effect, if any, variation in the timing of ethanol exposure has on development. For example, does early exposure to ethanol lead to more obvious defects compared to late exposure? In addition, several injections over time could be compared to one single injection. This is useful because some women continuously drink alcohol throughout pregnancy (Kensmodel et al., 2002) and do not have only one binge drinking event, as modeled by several experiments (Karunamuni et al., 2014; Karunamuni et al., 2015; Twal & Zile, 1997;

Peterson et al., 2017).

Further controlled experiments involving ethanol and embryonic development must be performed to understand the implications of FAS and FASD on humans. Pekin ducks offer an advantage over other highly studied avian models, and more research should be completed using ducks as model organisms. In addition, further efforts should be made to educate the general public about the dangers of FAS and FASD and the long- term effects they have on children and adults. FASD is an entirely preventable disorder, so further understanding of it, and potentially how to prevent or treat it, is a major obstacle in modern science.

Figures

Series 1 3 Series 2 Series 3 2

0 Category 1 Category 2 Category 3 Category 4

Figure 1. Pekin duck (Anas platyrhynchos) at approximately HH Stage 20. The heart of the embryo is visible. This embryo was not injected with ethanol. Photo Credit: Taylor &

Koenig 2018

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