The effects of ethanol concentrations on developing chicken embryos

1. Dianna M. Jarvis Marietta College The effects of ethanol concentrations on developing chicken embryos Results: Results of previous studies have shown a reduction in embryonic weights. Higher ethanol concentrations have shown an increase in growth suppression in both broiler and layer chicken strains (Bupp & Shibley, et al, 2002). However, within this investigation, growth suppression could not be statistically verified. Also, differences in growth suppression between high and low ethanol dosage groups could not be concluded. As seen in Figure 3, the mean values of the groups did not decrease linearly with increased ethanol concentrations as hypothesized. A student t-test of the recorded means confirmed no statistical significance. Background Information: Fetal Alcohol Syndrome (FAS) results from prenatal alcohol exposure. Those affected by the disease are often born with birth defects and developmental disorders, such as, mental retardation (Welch-Carre, 2005). FAS is noticed within 1% of all live births and unlike other diseases it is 100% preventable (Burd, et al, 2004). Ethanol is one of the components found within alcohol. Although, ethanol is a relatively simple organic compound, it is a known teratogen. A teratogen is an agent that can interfere with the normal development of a fetus. Ethanol disrupts the normal development of the Central Nervous System (CNS) (Costa, et al, 2002). Primarily, the substance inhibits glial cell proliferation (Costa, et al, 2002). Glial cells, the main cells that make up the nervous system, provide support and nutrition to the neurons. Specifically with FAS, the timing of ethanol exposure often determines the severity of the damage (Costa, et al, 2002). The most common manifestation of ethanol exposure within the CNS is characteristic growth suppression (Figure 1). Figure 3: Embryonic Weights Hypothesis: The effects of low/high dose ethanol concentrations will suppress the growth of embryonic chickens. High ethanol concentrations will cause greater growth suppression of embryonic chickens, than low ethanol concentrations. Figure 1: CNS growth suppression Even though growth suppression could not be concluded statistically, visually tissue necrosis, decrease size, and abnormal growths were observed (Figure 4). Procedures: Two dozen fertile, White Leghorn chicken eggs were ordered from Florida. When the eggs arrived, they were kept at room temperature for no longer than two days. In preparation for ethanol injections, the small ends of each egg were cleaned with 70% ethanol. Once cleaned, each egg was then labeled with a pencil according to the designated injection group. Next, a puncture site was made into the air cell of each egg and ½ milliliter of solution was injected into the air cell (Figure 2). Figure 4: Abnormal Growths www.stpetershealthcare.org Prior research has shown that the use of chicken embryos can aid in understanding the complexity of FAS. The results of past investigations show that ethanol induces growth suppression within developing chicken embryos (Bupp & Shibley, et al, 1998). There are several advantages in using a chicken model for examining FAS (Hartl & Shibley, et al, 2002). When dealing with chicken eggs, concern about maternal malnutrition or drug use can be eliminated. (Bupp & Shibley, et al, 1998). Also, the incubator offers a self-contained environment in which the eggs can be monitored at all times during development (Bupp & Shibley, et al, 1998). The 21 day gestation period is also an advantage when using the chicken model. Unlike human embryos that can take up to nine months to develop, it only takes 21 days for a chicken embryo to fully develop. Additionally, the direct effects of ethanol can be studied by manipulating ethanol dosages as well as the number of eggs within the experiment (Bupp & Shibley, et al, 1998). In other experiments concerning this topic, the focus was on the effects of a high versus a low dosage of ethanol (Bupp & Shibley, et al, 1998). Assessments comparing the torso versus head weights of the chickens were then evaluated. These measurements were used to determine if the ethanol caused an overall embryonic growth suppression or if the ethanol affected the head and torso independently (Bupp & Shibley, et al, 1998). Moreover, various chicken strains have been used within these experiments; among those are layers and broilers (Bupp & Shibley, et al, 1998). Broiler chickens are known for their high meat yield and rapid development, layers are known for their slow growth, yet early sexual maturity (Bupp & Shibley, et al, 1998). My senior research will examine the effects of high versus low dosages of ethanol on embryonic chickens. I will only use layer chickens, specifically, White Leghorns. To determine the effects of ethanol, I will measure the total weight suppression of the embryos. Figure 2: Air Cell Conclusions: Past research demonstrates that chicken embryos can be used to study FAS. My research does not statistically support that embryonic growth suppression is due to ethanol exposure. The graphs show some decrease in embryonic weights due to ethanol exposure, but there is not enough data to support my hypothesis. A problem likely exists in the number of chicken embryos used within the experiment. A larger sample size might have demonstrated, statistically, embryonic growth suppression. Additionally, the measurements taken of the width and length of the embryos’ heads were inconclusive, as well as variable. Again, the head caliber data might have been significant if a larger sample size was available. Future Research: Some anatomical effects were observed due to ethanol injections. Investigations on the cellular level are needed to determine the specific interactions between ethanol and the developing embryo. Through cellular testing, an embryo exposed to ethanol versus a normal embryo could be compared. Multiple injections of ethanol could be given, instead of one injection at the beginning of incubation. The ethanol injection amounts could be smaller by splitting up the volume over nine days. Development could also be evaluated past day nine. The composition of each ½ milliliter of solution was based on the designated dosage group. The low dose group’s solution was composed of two milliliters of 200 proof ethanol and 48 milliliters of distilled water or saline solution. The high dose group’s solution was composed of eight milliliters of 200 proof ethanol and 42 milliliters of distilled water or saline solution. After ethanol was placed inside the air cells of each egg, the injection sites were sealed with paraffin wax. The eggs were then placed within a 25.3 watt Hova- Bator incubator (model 1602N) with an automatic egg turner. The incubator was kept at 90 degrees Fahrenheit and the eggs were candled periodically throughout development. On day nine of development, the eggs were opened and the chicken embryos were removed. The total weight of each embryo was recorded. The length and width of each embryo’s head was collected using a caliber. Observations were also made of any noticeable, morphological abnormalities. Works Cited: Bupp Becker SR, Shibley IA. 1998. Teratogenicity of ethanol in different chicken strains. Alcohol & Alcoholism (33): pp. 457-464. Burd L, Wilson H. 2004. Fetal, infant, and child mortality in the context of alcohol abuse. American Journal of Medical Genetics (127). pp. 51-58. Costa LG, Guizzetti M. 2002. Inhibition of Muscarinic Receptor-induced proliferation of Astroglial cells by Ethanol: Mechanisms and Implications for Fetal Alcohol Syndrome. Neurotoxicology (23): pp. 685-691. Hartl MW, Shibley IA. 2002 Supraphysiological acetaldehyde levels suppress growth of chicken embryos. Alcohol (28): pp. 111-115. Welch-Carre E. 2005. The Neurodevelopment consequences in prenatal alcohol exposure. Advances in Neonatal care (5): pp. 217-229. Acknowledgements: MC Biology Department Dr. Peter Hogan, Capstone/MC advisor Sarah Zumbro, lab assistant Brandon Coughenour, lab assistant