Examination of temperature dependence and caffeine affect on live Gallus Domesticus in the Stage of Organogenesis Aimee Hilton Lissette Herrera, Sean Kerr Rachelle Christie Section 902 February 24, 2009 Introduction The fertilization and development of an egg is a remarkable beginning to the rise of a complex organism. Although characteristics are very different as mature organisms, chick embryos and human embryos share many of the same structures. Since human embryos are more difficult to look at in a human placenta, especially in a biology laboratory, chick embryos can be used to note key steps in the development of an embryo. Chick embryos develop in a disk shape on top of a very abundant yolk sac. This thick yolk sac is one of the primary reasons chicks have meroblastic cleavage; only the embryo will divide. This early division takes place during the cleavage and gastrulation stages of development. This experiment focuses on the next stage of organogenesis, which is the formation of organs and the three tissue layers. The ectoderm (outer region), the mesoderm (middle region), and the endoderm (inner region) make up the different tissue layers that form during organogenesis. Organogenesis is the major stage in development when cells will migrate; cells signal between different tissues, and the cells begin to take their shape. The chick embryo has almost identical development during organogenesis to the human embryo, allowing the lab to develop basic understanding of human development. This lab focuses on observing the development of two systems, the nervous system and the circulatory system. By looking at different embryos that were incubated at different periods of time and different temperatures we can observe the changes. By 18 hours into the development the very first organ system is visible by the appearance of the neural tube, the start of the nervous system. The neural tube is made up of ectoderm tissue that will develop into the brain and spinal cords. Somites, made of mesoderm tissue will also be visible at this time. Somites form at the cranial end of the embryo, giving rise to the skeletal system; rib cartilage, and the muscular system; musculatures on back and limbs. The most noticeable landmark in 24 hours is the notochord, which is the supportive rod that is found done the center of the embryo. QuickTime™ and a decompressor are need ed to see this picture. Figure 1: A chick embryo at 24 hours of development pointing out the major landmarks for reference. The figure was used from the Vertebrate Development: Organogenesis in the Chick Embryo By around 33 hours into development the heart and blood vessels should be visible in the embryo. By 48 hours, the embryo is showing more distinct landmarks. The brain is now visible and three divided sections may be seen. The notochords will be migrating towards the posterior, or tail, region in the embryo, which is starting its development into the spinal cord. Somites will be more visible and pairs of somites may even be countable. QuickTime™ an d a decompressor are need ed to see this p icture . Figure 2: A chick embryo at 48 hours of development pointing out the major landmarks for reference. The figure was used from the Vertebrate Development: Organogenesis in the Chick Embryo The second portion of the lab looked at cardiovascular physiology of a chick embryo by testing different concentrations of caffeine on the heart rate. By testing concentrations of caffeine on a live chick embryo, an observation can be made on the percent difference of the basal heart rate to the heart rate after caffeine is applied. This data can be used to show the effect of caffeine on the cardiovascular system. The goal of the chick lab is to observe the stages of the chick embryo development using live chick embryos at different hours of development and at different incubation temperatures. By observing different landmark events we can conclude a relative incubation temperature and the effects of a higher incubation temperature. This lab will also look at the effects of caffeine on the cardio system by collecting data on the heartbeat of the embryo at different caffeine concentrations. With the basic knowledge from the lab manual, the group hypothesized that the chick embryo incubated at a higher temperature would be further developed than the chick embryo incubated at the lower temperature. We also hypothesized that caffeine would have a considerable increase on heart rate, showing negative effects on the cardiovascular system. Materials and Methods The first part of the lab focuses on the observation of chick embryos incubated at 37C for 48 hours and 72 hours. Using whole mount slides in the lab classroom, chick embryos can be looked at under the microscope to observe landmark stages in the development of the chick embryo. The next part of the lab is to observe the effect of incubation temperature on live chick embryos. Each group receives two Petri dishes each containing a yolk sac with an embryo on top saturated with warm saline solution. The eggs previously have been cracked for this first procedure. It is very important to keep the embryo warm and moist so that the chick embryo does not die. This can be done by keeping the embryo under a close light source and bathing the embryo with a small amount of warm saline solution from time to time. These two embryos were incubated for the same amount of time, however one embryo was incubated at 37C and one was incubated at 38C. Under the microscope look at each embryo on top of the yolk sac and record the observations in table 1. After all observations are completed move on to taking the embryo off of the yolk sac, which is useful to see developmental structures better. Taking the embryo off of the yolk sac was a difficult process that required a steady hand. First cut a piece of filter paper into a circle that is smaller then the yolk sac but bigger then the embryo. By first placing a slit in the center of the paper, an inner circle can be cut in the filter paper that is about the size of the embryo. Next place the filter paper over the embryo so that the embryo is inside the hole of the filter paper. Carefully, with steady hands, cut around the outside of the filter paper cutting the vitelline membrane with quick snapping cuts. Using forceps, tweezers, hold the filter paper and embryo together so that the embryo does not slide outside of the filter paper and accidentally get cut. Once the whole oval if the filter paper is cut, grasp both the vitelline membrane and the filter paper; this should be easily done if the vitelline membrane stuck to the filter paper. Finally place the embryo in a Petri dish with warm saline solution. Keep the embryo on the filter paper because if the filter paper is removed the embryo will curl and will not be viewable for observation. The process of taking the embryo off the yolk may have to be repeated several times if the embryo dies before the end of the last experiment on the observations of caffeine. Once both chick embryos are off the yolk record observations of the landmark structures in the bottom of table 1. By using the lab manual and observations in table one predict the temperature of incubation for each embryo. The final part of the lab is to test the effects of caffeine on the cardiovascular system of the embryo. If the embryos from the previous experiment are still alive they can be used for this experiment. First record the basal heart beat by counting the number of heart beats for 15 seconds. Two people on the same embryo, one using the microscope and one observing the heart beat with the naked eye should do this. Once the heartbeat is counted for 15 seconds, times that number by four to know the resting heart rate per minute. Once the basal heart rate is determined with a transfer pipet remove some of the buffer solution and add a small amount of the lower concentration of caffeine. The lower concentration must be used first so that the embryo can easily return back to its basal heart rate before the next concentration is added. After adding the lower concentration of caffeine count the number of heart beats for 15 seconds, then multiple that number by four. Using a transfer pipet remove the drug solution from the Petri dish and bathe the embryo with warm saline solution. Wait a couple of minutes to allow the embryo to return to basal heart rate. Calculate the basal heart rate again by counting the number of heart beats for 15 seconds and multiplying that number by four. Using the same technique as the lower concentration, now add a small amount of the higher concentration of caffeine. Calculate the heart rate by counting the number of heartbeats for 15 seconds and multiplying that number by four. If at any time the embryo dies, a new embryo has to be obtained, removed from the yolk, and everything recalculated. The effect of caffeine should be looked at on two different embryos. Results By looking at the lab manual and comparing the different developmental stages shown to the embryos under the microscope, the group was able to observe different structures that are noted in table 1. Table 1 shows the observations from the group on both an embryo incubated at 37C and an embryo incubated at 38C, although when making the observation which temperature the embryo was incubated at is unknown. The table includes observations first taken while the embryo was on the yolk; this was incase the embryo died while it was removed from the yolk, and observations of the embryo off of the yolk. The embryo was easier to look at off the yolk because the microscope light created a better image. Everything that was seen on the embryo with the yolk was seen without the yolk, just is not listed again for repetitive sake. Heart beat Posterior limb bud Circulatory system Heart beat Blood vessels Somites Notochord QuickTime™ and a decompressor are neede d to see this picture. Clearer somites Mesencephalon 4 countable pairs Myelencephalon of somites High Temp. Low Temp Table 1: Observations of two chick embryos incubated at different temperatures for the same period of time. The tabled was used from the Vertebrate Development: Organogenesis in the Chick Embryo After observing both of the chick embryos and keeping both embryos alive, the same embryos were used for the next part of the experiment. The basal heart rate and the heart rate with different concentrations of caffeine we calculated. The group also took the heart rate a step further and transferred a small amount of Red Bull Energy Drink onto the fetus to see the effects of the energy drink on the chick embryo. Red Bull is a drink millions of humans, consume on a regular bases, without really thinking about the effect. Effect of Caffeine on Chick Embryos Chick Embryo 2 Heart Chick Embryo 1 Heart Rate Rate 15 Seconds (x4) 1 min. 15 seconds (x4) 1 min. Basal 21 84 15 60 1% Concentration 23 92 20 80 Basal 19 76 17 68 5% Concentration 27 108 24 96 Basal 18 72 17 68 Red Bull 16 64 20 80 Table 2: The effect of caffeine on chick embryos by calculating there number of heartbeats for 15 seconds, then multiplied by four to get the number of beats per minute. Once the table of the different concentrations of caffeine was created, the percent difference in the heart rate with the different treatments was calculated. Difference in Heart Rate with Different Concentrations of Caffeine Chick Embryo 1 Chick Embryo 2 1% Concentration 8 20 5% Concentration 32 28 Table 3: The difference from the basal heart rate to the heart rate when the caffeine concentration was added Diff erence in Heart Rate Vs. Caff eine Concentration 35 30 Heart Rate (per min) 25 20 C hic k E mbryo 1 15 C hic k E mbryo 2 10 5 0 Caf f eine Concentrat on Graph 1: Shows the increase in heart rate for Caffeine concentration for each chick embryo Discussion When first receiving the two chick embryos in the Petri dish, a hypothesis was made that embryo two was the chick that was incubated at the higher temperature. This educational guess was made because chick embryo two was visibly a bigger embryo by the size that the blood vessels had branched out. There was a notable size difference in the embryo of chick two compared to chick one; chick two being a little smaller then a half dollar coin, chick one bring a little smaller then a quarter. Once the chicks were observed under the microscope, the first hypotheses proved to be very wrong. Chick embryo one was much further developed then chick embryo two. By looking at the observation table 1, a conclusion could be made that more structural landmarks were evident in chick embryo one, which means it, must have been developed at a high temperature. Emory University Health Science Center has looked at the temperature dependence in chick embryos, by conducting experiment of cardiac gap junctions. Their studies including comparing embryos at room temperature, “At room temperature, embryonic cardiac gap junctions contain channels with conductance states near 240, 200, 160, 120, 80 and 40 pS” (Chen, YH and RL, DeHaan), to embryos that were either placed in a cooler temperature or placed in a higher temperature. Their findings showed that when embryos were placed at a cooled temperature the gap junctions were almost non- active and the ion channels would no be open. This study and the experiment performed in the biology laboratory show that chick embryos need a stable temperature to develop at, and development can be greatly effected by a change in temperature. Caffeine is a drug that humans consume on a daily basis, whether it is their fill of cups and cups of coffee, the flourishing energy drinks, or the newly invented five-hour energy shot. Each of these drinks can be loaded with sugar, but also contains an addictive caffeine overdose. Although there is a noticeable size difference, by testing the effects of caffeine on the heart rate of a chick embryo, the picture of caffeine, in much lager amounts, can easily be painted in the human head. When different concentrations of a small amount of caffeine were transferred onto the chick embryo, the initial results were not seen. After waiting one minute, with the naked eye an increase in the heartbeat could be observed. After calculating the heartbeat and the difference in heartbeat from the different concentrations of caffeine, a significant raise in the chick embryos heartbeat was observed. The percent difference is the amount of increase from the basal heart rate to the heart rate when the caffeine was added. This increase shows that caffeine has an overall negative effect on the cardiac system. An experiment conducted on the effects of cardioteratogenic doses of caffeine on cardiac function in a chick embryo, shows that it’s not the amount of caffeine that affects the embryo, but the concentration of caffeine does indeed affect not just the heart rate of a chick embryo; “However, the increase in cardiac rate was not related to dose. At 20 hours after treatment, caffeine increased stroke volume, ejection fraction and cardiac output relative to the controls.” (Bruyere Jr.) This experiment demonstrates the damaging effects caffeine can have. The most shocking experiment was one that was not even directed in this lab, but was something the group felt would be interesting to look at and took the experiment a step further. A small amount of red bull was transferred onto the chick embryo. The data collected for this part of the experiment is not accurate at all because the true effects of red bull were not noticed until the second chick embryo. For the first embryo, the embryo was bathed with a little under 1ml of red bull. An immediately observation was noticed that the heart became a lot smaller and the chick seemed to be dieing. The heartbeat for 15 seconds was quickly counted with only a total of 16 beats, which is seen in table 2. There was no way! About two minutes after counting the heartbeat, the chick embryo was not recovering from this over load of caffeine. The red bull was showing signs of killing the embryo. Chick embryo two would hopefully work better. All the necessary experiments for the lab were conducted and then the red bull experiment was performed on this chick embryo. This embryo was bathed with a small amount of red bull and again the heart became very small. After very close observation of this embryo, the heart was in-fact beating, but at a pace that was considerably hard to count. The heart was pumping fast, but filling only a tiny amount so that the change was very difficult to see. The red bull was an overload for the tiny chick embryo, again nearly killing it. This experiment can be used as a substitute to examine the development of a human embryo and the effect of caffeine. The findings from this experiment and experiments similar can one day be used in the research of embryonic stem cells. The research in embryonic stem cells will be vital in the development for the treatment and cure of life threatening diseases. References Bruyere Jr., HJ, BJ Michaud, EF Gilbert, and JD Folts. "The Effects of Cardioteratogenic Doses of Caffeine on Cardiac Function in the 3-day Chick Embryo." (1987). Pub Med. 17 Feb. 2009 <http://www.ncbi.nlm.nih.gov/pubmed/3624779?ordinalpos=24&itool=EntrezSystem 2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RV DocSum>. Chen, YH and RL, DeHaan. "Temperature dependence of embryonic cardiac gap junction conductance and channel kinetics." (1993). PubMed. Atlanta. 17 Feb. 2009 <http://www.ncbi.nlm.nih.gov/sites/entrez> Nelson, Kimberlyn. "Biology 240W." The Pennsylvania State University, University Park, PA Vertebrate Development: Organogenesis In The Chick Embryo. 2009. Department of Biology, The Pennsylvania State University, University Park, PA.
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