The Effects of Caffeine on the Heart Rate of the Six Day In Vitro Chicken Embryo

1
The Effects of Caffeine on the Heart Rate of the
Six Day In Vitro Chicken Embryo
Alyssa Miller, Krista Pummer, Dr. Jacqueline
McLaughlin Biology 240W March 25, 2004
2
Chicken Heart Development

• Embryonic heart has 4 regions
• conotruncus
• atrium
• ventricle
• sinus venosus
• 33 hours heart bends to form an S shape.

conotruncus
ventricle
Sinus Venosus
atrium
Figure 1. 33 hour chick embryo.
3
Chicken Heart Development

• 48 hours heart folds upon itself forming a
  single loop.

conotruncus
atrium
Sinus Venosus
ventricle
Figure 2. Right side of 48 hour chick embryo.
4
Chicken Heart Development

• 72 hours heart begins preparation for the
  division of the atrium.
• 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 increases heart rate, respiration, and
  metabolic rate. (Erowid 1995)
• Caffeine relaxes smooth muscles. (Erowid 1995)
• Caffeine may cause birth defects and cardiac
  arrhythmias. (Erowid 1995)
• 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 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.
• 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.
• Window egg according to the of Cruz et al. 1993.

9
Methods

• Explant the embryo according to the method of
  Cruz et al. 1993
• Place a filtering paper doughnut around 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.
• Remove the doughnut and place the dish under a
  stereoscopic microscope heated by a warming lamp.
• Obtain in vitro HR five times.

Six day chick embryo in vivo.
10
Methods

• 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).
• 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.
• 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 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.

Six day chick embryo in vitro.
12
Heart Beat (bpm) in 0.1 Caffeine Solution
Embryo Trial 1 2 3 4 5 AVERAGE
1 In vivo 144 184 184 144 136 158.4
1 In vitro 60 136 144 72 136 109.6
1 1 88 60 60 68 64 68
1 2 72 72 64 32 52 58.4
1 3 40 48 40 40 40 41.6
2 In vivo 104 104 108 104 108 105.6
2 In vitro 76 68 128 44 64 76
2 1 80 88 80 80 84 82.4
2 2 84 48 52 84 56 64.8
2 3 40 48 52 52 40 46.4
3 In vivo 172 164 152 152 144 156.8
3 In vitro 12 16 16 20 24 17.6
3 1 32 36 40 36 32 35.2
3 2 52 44 48 48 52 48.8
3 3 52 48 52 52 52 51.2
13
Heart Beat (bpm) in 0.5 Caffeine Solution
Embryo Trial 1 2 3 4 5 AVERAGE
1 In vivo 104 100 96 88 96 96.8
1 In vitro 20 16 12 12 8 13.6
1 1 88 92 56 40 48 68.4
1 2 72 80 76 56 44 65.6
1 3 28 20 16 16 20
2 In vivo 156 156 128 148 140 145.6
2 In vitro 20 20 24 24 24 22.4
2 1 40 48 52 60 60 52
2 2 96 32 60 44 44 55.2
2 3
3 In vivo 60 88 68 80 88 76.8
3 In vitro 32 36 32 56 64 44
3 1 76 72 68 52 48 63.2
3 2 8 8 36 72 16 28
3 3 16 16 16 16 20 16.8
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.
• This may be due to colder temperatures of the
  saline solution.
• 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.
• 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.
• The 0.5 solution decreased heart rate more than
  the 0.1 solution.
• Both solutions caused sporadic heart rates
  leading to bradycardia and tachycardia.

18
Conclusion

• 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.
• 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.
• 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.

20
References

• 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
• Chicken Heart. 1998. Pennsylvania State
  University of Lehigh Valley Department of
  Biology, Muhlenberg College Biology Department.
  21 Mar. 2004 lthttp//www.zoo.utoronto.ca/able/vol
  umes/vol-19/mini/01-mccain/01-mccain.htmgt.
• Park, Joyce. The Effect of Caffeine on the Heart
  Rate of the Three Day Chick Embryos. 02 Aug.
  2001. Swarthmore College. 13 Feb. 2004.
  lthttp//www.swarthmore.edu/NatSci/sgilber1?DBlab/
  Chick/chickadv.htmlgt.
• Rxlist. 31 Dec. 2003. 13 Feb. 2004.
  lthttp//www.rxlist.com/cgi/generic3/caffeinecp.htm
  gt
• Sherwood, Lauralee. Human Physiology From Cells
  to Systems. California Brooks/Cole, 2001.
• The Vaults of Erowid Caffeine. 1995. Erowid. 20
  Mar. 2004 lthttp//www.erowid.org/chemicals/caffei
  ne/caffeine.shtmlgt.

21
Thank you for listening to our presentation.

• Any Questions?