1. The Effects of Caffeine on the Heart Rate of the Six Day In Vitro Chicken Embryo Alyssa Miller, Krista Pummer, & Dr. Jacqueline McLaughlin Dr. Jacqueline McLaughlin Biology 240W March 25, 2004

2. Chicken Heart Development Embryonic heart has 4 regions: Embryonic heart has 4 regions: conotruncus conotruncus atrium atrium ventricle ventricle sinus venosus sinus venosus 33 hours: heart bends to form an “S” shape. 33 hours: heart bends to form an “S” shape. Sinus Venosus conotruncus ventricle atrium Figure 1. 33 hour chick embryo.

3. Chicken Heart Development 48 hours: heart folds upon itself forming a single loop. 48 hours: heart folds upon itself forming a single loop. atrium ventricle conotruncus Sinus Venosus Figure 2. Right side of 48 hour chick embryo.

4. Chicken Heart Development 72 hours: heart begins preparation for the division of the atrium. 72 hours: heart begins preparation for the division of the atrium. Time of heart completion varies. Time of heart completion varies. conotruncus atrium ventricle Sinus Venosus Figure 3. Right side of 72 hour chicken embryo.

5. Caffeine Caffeine is a cardiac stimulant. (Rxlist 2003) Caffeine is a cardiac stimulant. (Rxlist 2003) Caffeine increases heart rate, respiration, and metabolic rate. (Erowid 1995) Caffeine increases heart rate, respiration, and metabolic rate. (Erowid 1995) Caffeine relaxes smooth muscles. (Erowid 1995) Caffeine relaxes smooth muscles. (Erowid 1995) Caffeine may cause birth defects and cardiac arrhythmias. (Erowid 1995) Caffeine may cause birth defects and cardiac arrhythmias. (Erowid 1995) Caffeine decreases in effectiveness over time. (Rxlist 2003) Caffeine decreases in effectiveness over time. (Rxlist 2003)

6. Purpose To test the effects of two different concentrations of caffeine (0.1 mg/ml and 0.5 mg/ml) on the six day chicken embryo. To test the effects of two different concentrations of caffeine (0.1 mg/ml and 0.5 mg/ml) on the six day chicken embryo. To evaluate the effects of caffeine over time. To evaluate the effects of caffeine over time.

7. Hypothesis Caffeine added exogenously to the 6-day (120 hour) chicken embryo will initially increase heart rate. Caffeine added exogenously to the 6-day (120 hour) chicken embryo will initially increase heart rate. Over time, heart rate will decrease as the embryo becomes accustomed to the amount of caffeine in its system. Over time, heart rate will decrease as the embryo becomes accustomed to the amount of caffeine in its system.

8. Methods Prepare “stock” solution of 2% caffeine. Then serially diluted stock solution with chick saline to create 0.1 mg/ml and 0.5 mg/ml caffeine solutions. Prepare “stock” solution of 2% caffeine. Then serially diluted stock solution with chick saline to create 0.1 mg/ml and 0.5 mg/ml caffeine solutions. Window egg according to the of Cruz et al. 1993. Window egg according to the of Cruz et al. 1993.

9. Methods Explant the embryo according to the method of Cruz et al. 1993: Explant the embryo according to the method of Cruz et al. 1993: Place a filtering paper doughnut around the embryo. Place a filtering paper doughnut around the embryo. Cut around the doughnut, through the vitelline layer, to free the embryo. Cut around the doughnut, through the vitelline layer, to free the embryo. Lift the doughnut with the attached embryo and place into a warm saline filled Syracuse dish. Lift the doughnut with the attached embryo and place into a warm saline filled Syracuse dish. Remove the doughnut and place the dish under a stereoscopic microscope heated by a warming lamp. Remove the doughnut and place the dish under a stereoscopic microscope heated by a warming lamp. Obtain in vitro HR five times. Obtain in vitro HR five times. Six day chick embryo in vivo.

10. Methods Application of Caffeine: Application of Caffeine: Using a 4 quadrant Petridish, fill quadrant 1 with saline solution and quadrant 2 with 0.1% of caffeine solution (see figure 1). Using a 4 quadrant Petridish, fill quadrant 1 with saline solution and quadrant 2 with 0.1% of caffeine solution (see figure 1). Place embryo into quadrant 1 and allow thirty seconds of adjustment time before taking the HR. Place embryo into quadrant 1 and allow thirty seconds of adjustment time before taking the HR. Observe the in vitro HR and count the beats for fifteen seconds, then multiply by four. Record and repeat step four more times and obtain an average for the five counts. After one minute check HR again using the same method and record. Observe the in vitro HR and count the beats for fifteen seconds, then multiply by four. Record and repeat step four more times and obtain an average for the five counts. After one minute check HR again using the same method and record. Gently move the embryo into the quadrant with the 0.1mg/ml caffeine solution and repeat steps. Gently move the embryo into the quadrant with the 0.1mg/ml caffeine solution and repeat steps. Repeat steps using 0.5 mg/ml caffeine sloution. Repeat steps using 0.5 mg/ml caffeine sloution. Repeat steps on six different embryos. Repeat steps on six different embryos. Quadrant 1: Saline solution Quadrant 2: Caffeine solution Figure 1: Four quadrant Petridish.

11. Control The in vivo and in vitro heart rates for a 6-day chicken embryo will be recorded before exposure to caffeine. The in vivo and in vitro heart rates for a 6-day chicken embryo will be recorded before exposure to caffeine. Six day chick embryo in vitro.

12. Heart Beat (bpm) in 0.1% Caffeine Solution EmbryoTrial12345AVERAGE 1 In vivo144184 144136158.4 In vitro6013614472136109.6 18860 686468 272 64325258.4 3404840 41.6 2 In vivo104 108104108105.6 In vitro7668128446476 1808880 8482.4 2844852845664.8 3404852 4046.4 3 In vivo172164152 144156.8 In vitro1216 202417.6 1323640363235.2 2524448 5248.8 3524852 51.2

13. Heart Beat (bpm) in 0.5% Caffeine Solution EmbryoTrial12345AVERAGE 1 In vivo10410096889696.8 In vitro201612 813.6 1889256404868.4 2728076564465.6 3282016 20 2 In vivo156 128148140145.6 In vitro20 24 22.4 140485260 52 296326044 55.2 3 3 In vivo608868808876.8 In vitro323632566444 1767268524863.2 28836721628 316 2016.8 = cardiac arrest (0 bpm)

14. Heart Rate vs. Caffeine Concentration Heart Rate (bpm) Caffeine Concentration (%)

15. Heart Rate with 0.1% Caffeine Solution Heart Rate (Beats per 15 seconds) Seconds

16. Heart Rate with 0.5% Caffeine Solution Heart Rate (Beats per 15 seconds) Seconds

17. Data Interpretation In vivo heart rate is dramatically higher than our in vitro heart rate. In vivo heart rate is dramatically higher than our in vitro heart rate. This may be due to colder temperatures of the saline solution. This may be due to colder temperatures of the saline solution. This may be due to trauma after disrupting its natural environment. This may be due to trauma after disrupting its natural environment. Data from 5 out of our 6 embryos supported our hypothesis that heart rate will increase after exposure to caffeine. Data from 5 out of our 6 embryos supported our hypothesis that heart rate will increase after exposure to caffeine. The 0.5% solution increased heart rate more than the 0.1% solution. The 0.5% solution increased heart rate more than the 0.1% solution. Data from 5 out of 6 supported our hypothesis that over time heart rate would decrease from exposure to caffeine. Data from 5 out of 6 supported our hypothesis that over time heart rate would decrease from exposure to caffeine. The 0.5% solution decreased heart rate more than the 0.1% solution. The 0.5% solution decreased heart rate more than the 0.1% solution. Both solutions caused sporadic heart rates leading to bradycardia and tachycardia. Both solutions caused sporadic heart rates leading to bradycardia and tachycardia.

18. Conclusion Our results support our hypothesis. Our results support our hypothesis. Caffeine at 0.5 mm/ml concentration increased the heart rate more than the 0.1 mm/ml caffeine solution. Caffeine at 0.5 mm/ml concentration increased the heart rate more than the 0.1 mm/ml caffeine solution. Over time as the embryo became accustomed to the amount of caffeine in its system, the heart rate decreased. Over time as the embryo became accustomed to the amount of caffeine in its system, the heart rate decreased.

19. Future Experiments Since temperature of the chicken embryos environment is critical, temperature must be regulated throughout the experiment. Since temperature of the chicken embryos environment is critical, temperature must be regulated throughout the experiment. Use more varieties of caffeine solutions to test the effects of different concentrations of caffeine over time. Use more varieties of caffeine solutions to test the effects of different concentrations of caffeine over time. Use different media solutions containing critical nutrients and electrolytes for prolonged life of the chicken embryo. Use different media solutions containing critical nutrients and electrolytes for prolonged life of the chicken embryo.

20. References Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic Press, San Diego, California, 241 pages. [ISBN 0-12-198390-0] [book]. Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic Press, San Diego, California, 241 pages. [ISBN 0-12-198390-0] [book]. McLaughlin, Jacqueline S., and Elizabeth R. McCain. In Vitro and In Vivo Development of the McLaughlin, Jacqueline S., and Elizabeth R. McCain. In Vitro and In Vivo Development of the Chicken Heart. 1998. Pennsylvania State University of Lehigh Valley Department of Biology, Muhlenberg College Biology Department. 21 Mar. 2004. http://www.zoo.utoronto.ca/able/volumes/vol-19/mini/01-mccain/01-mccain.htm Park, Joyce. The Effect of Caffeine on the Heart Rate of the Three Day Chick Embryos. 02 Aug. 2001. Swarthmore College. 13 Feb. 2004.. Park, Joyce. The Effect of Caffeine on the Heart Rate of the Three Day Chick Embryos. 02 Aug. 2001. Swarthmore College. 13 Feb. 2004..http://www.swarthmore.edu/N Rxlist. 31 Dec. 2003. 13 Feb. 2004. Rxlist. 31 Dec. 2003. 13 Feb. 2004. http://www.rxlist.com/cgi/generic3/caffeinecp.htm Sherwood, Lauralee. Human Physiology: From Cells to Systems. California: Brooks/Cole, 2001. Sherwood, Lauralee. Human Physiology: From Cells to Systems. California: Brooks/Cole, 2001. The Vaults of Erowid: Caffeine. 1995. Erowid. 20 Mar. 2004. The Vaults of Erowid: Caffeine. 1995. Erowid. 20 Mar. 2004.http://www.erowid.org/chemicals/caffeine/caffeine.shtml

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