The Effects of Caffeine on the The Effects of Caffeine on the Heart Rate of the Six Day Heart Rate of the Six Day In In

VitroVitro Chicken Embryo Chicken Embryo

Alyssa Miller, Krista Alyssa Miller, Krista Pummer, &Pummer, &

Dr. Jacqueline McLaughlinDr. Jacqueline McLaughlin

Biology 240WBiology 240W

March 25, 2004March 25, 2004

Chicken Heart DevelopmentChicken Heart Development Embryonic heart has 4 Embryonic heart has 4

regions:regions:

conotruncusconotruncus

atriumatrium

ventricleventricle

sinus venosussinus venosus 33 hours: heart bends to 33 hours: heart bends to

form an “S” shape.form an “S” shape.

Sinus Venosus

conotruncusventricleatrium

Figure 1. 33 hour chick embryo.

Chicken Heart DevelopmentChicken Heart Development

48 hours: heart 48 hours: heart folds upon itself folds upon itself forming a single forming a single loop.loop. atriu

mventricle

conotruncus

Sinus Venosus

Figure 2. Right side of 48 hour chick embryo.

Chicken Heart Chicken Heart DevelopmentDevelopment

72 hours: heart 72 hours: heart begins preparation begins preparation for the division of for the division of the atrium.the atrium.

Time of heart Time of heart completion varies.completion varies.

conotruncus

atrium ventricle

Sinus Venosus

Figure 3. Right side of 72 hour chicken embryo.

CaffeineCaffeine Caffeine is a cardiac Caffeine is a cardiac

stimulant. stimulant. (Rxlist 2003)(Rxlist 2003) Caffeine increases Caffeine increases

heart rate, respiration, heart rate, respiration, and metabolic rate. and metabolic rate. (Erowid 1995)(Erowid 1995)

Caffeine relaxes Caffeine relaxes smooth muscles. smooth muscles. (Erowid (Erowid 1995)1995)

Caffeine may cause Caffeine may cause birth defects and birth defects and cardiac arrhythmiascardiac arrhythmias. . (Erowid 1995)(Erowid 1995)

Caffeine decreases in Caffeine decreases in effectiveness over effectiveness over time. time. (Rxlist 2003)(Rxlist 2003)

PurposePurpose

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

To evaluate the effects of caffeine To evaluate the effects of caffeine over time.over time.

HypothesisHypothesis

Caffeine added exogenously to the 6-Caffeine added exogenously to the 6-day (120 hour) chicken embryo will day (120 hour) chicken embryo will initially increase heart rate.initially increase heart rate.

Over time, heart rate will decrease Over time, heart rate will decrease as the embryo becomes accustomed as the embryo becomes accustomed to the amount of caffeine in its to the amount of caffeine in its system. system.

MethodsMethods Prepare “stock” solution of 2% caffeine. Then serially Prepare “stock” solution of 2% caffeine. Then serially

diluted stock solution with chick saline to create 0.1 diluted stock solution with chick saline to create 0.1 mg/ml and 0.5 mg/ml caffeine solutions.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.

MethodsMethods Explant the embryo according to the method of Explant the embryo according to the method of

Cruz et al. 1993: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 Cut around the doughnut, through the vitelline layer, to free the

embryo. embryo. Lift the doughnut with the attached embryo and place into a Lift the doughnut with the attached embryo and place into a

warm saline filled Syracuse dish. warm saline filled Syracuse dish. Remove the doughnut and place the dish under a stereoscopic Remove the doughnut and place the dish under a stereoscopic

microscope heated by a warmingmicroscope heated by a warming lamp.lamp. Obtain Obtain in vitroin vitro HR five times HR five times..

Six day chick embryo in vivo.

MethodsMethods Application of Caffeine:Application of Caffeine:

Using a 4 quadrant Petridish, fill quadrant 1 with saline solution and quadrant Using a 4 quadrant Petridish, fill quadrant 1 with saline solution and quadrant 2 with 0.1% of caffeine solution (see figure 1). 2 with 0.1% of caffeine solution (see figure 1).

Place embryo into quadrant 1 and allow thirty seconds of adjustment time Place embryo into quadrant 1 and allow thirty seconds of adjustment time before taking the HR. before taking the HR.

Observe the Observe the in vitroin vitro HR and count the beats for fifteen seconds, then multiply HR and count the beats for fifteen seconds, then multiply by four. Record and repeat step four more times and obtain an average for 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 the five counts. After one minute check HR again using the same method and record. record.

Gently move the embryo into the quadrant with the 0.1mg/ml caffeine Gently move the embryo into the quadrant with the 0.1mg/ml caffeine solution and repeat steps.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.

ControlControl

The The in vivoin vivo and and in vitroin vitro heart rates for a 6-day heart rates for a 6-day chicken embryo will be chicken embryo will be recorded before recorded before exposure to caffeineexposure to caffeine..

Six day chick embryo in vitro.

Heart Beat (bpm) in 0.1% Caffeine Heart Beat (bpm) in 0.1% Caffeine SolutionSolution

Embryo Trial 1 2 3 4 5 AVERAGE

1

In vivo 144 184 184 144 136 158.4

In vitro 60 136 144 72 136 109.6

1 88 60 60 68 64 68

2 72 72 64 32 52 58.4

3 40 48 40 40 40 41.6

2

In vivo 104 104 108 104 108 105.6

In vitro 76 68 128 44 64 76

1 80 88 80 80 84 82.4

2 84 48 52 84 56 64.8

3 40 48 52 52 40 46.4

3

In vivo 172 164 152 152 144 156.8

In vitro 12 16 16 20 24 17.6

1 32 36 40 36 32 35.2

2 52 44 48 48 52 48.8

3 52 48 52 52 52 51.2

Heart Beat (bpm) in 0.5% Caffeine Heart Beat (bpm) in 0.5% Caffeine SolutionSolution

Embryo Trial 1 2 3 4 5 AVERAGE

1

In vivo 104 100 96 88 96 96.8

In vitro 20 16 12 12 8 13.6

1 88 92 56 40 48 68.4

2 72 80 76 56 44 65.6

3 28 20 16 16 20

2

In vivo 156 156 128 148 140 145.6

In vitro 20 20 24 24 24 22.4

1 40 48 52 60 60 52

2 96 32 60 44 44 55.2

3

3

In vivo 60 88 68 80 88 76.8

In vitro 32 36 32 56 64 44

1 76 72 68 52 48 63.2

2 8 8 36 72 16 28

3 16 16 16 16 20 16.8

= cardiac arrest (0 bpm)

Heart Rate vs. Caffeine Heart Rate vs. Caffeine ConcentrationConcentration

Heart

Rate

(b

pm

)

Caffeine Concentration (%)

0

20

40

60

80

100

120

140

In vivo In vitroControl

In vitro .01% caffeine

In vitro .05% Caffeine

Embryo 1Embryo 2Embryo 3

Heart Rate with 0.1% Caffeine Heart Rate with 0.1% Caffeine SolutionSolution

0

5

10

15

20

25

30

35

40

0 100 200 300 400

Seconds

Hea

rt R

ate

(BP

S)

Embryo 1 Embryo 2 Embryo 3

Heart

Rate

(B

eats

per

15

secon

ds)

Seconds

Heart Rate with 0.5% Caffeine Heart Rate with 0.5% Caffeine SolutionSolution

0

5

10

15

20

25

30

0 100 200 300 400

Seconds

Hea

rt R

ate

(BP

S)

Embryo 1 Embryo 2 Embryo 3

Heart

Rate

(B

eats

per

15

secon

ds)

Seconds

Data InterpretationData Interpretation In vivoIn vivo heart rate is dramatically higher than our heart rate is dramatically higher than our in vitroin vitro heart rate. 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 Data from 5 out of our 6 embryos supported our hypothesis that heart rate will increase after exposure to caffeine. 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 Data from 5 out of 6 supported our hypothesis that over time heart rate would decrease from exposure to caffeine.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.

ConclusionConclusion

Our results support our hypothesis.Our results support our hypothesis. Caffeine at 0.5 mm/ml concentration Caffeine at 0.5 mm/ml concentration

increased the heart rate more than the 0.1 increased the heart rate more than the 0.1 mm/ml caffeine solution.mm/ml caffeine solution.

Over time as the embryo became Over time as the embryo became accustomed to the amount of caffeine in its accustomed to the amount of caffeine in its system, the heart rate decreased.system, the heart rate decreased.

Future ExperimentsFuture Experiments Since temperature of the chicken embryos environment is Since temperature of the chicken embryos environment is

critical, temperature must be regulated throughout the critical, temperature must be regulated throughout the experiment.experiment.

Use more varieties of caffeine solutions to test the effects of Use more varieties of caffeine solutions to test the effects of different concentrations of caffeine over time.different concentrations of caffeine over time.

Use different media solutions containing critical nutrients and Use different media solutions containing critical nutrients and electrolytes for prolonged life of the chicken embryo.electrolytes for prolonged life of the chicken embryo.

ReferencesReferences Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic Cruz, Y.P. 1993. Laboratory Exercises in Developmental Biology. Academic

Press, San Diego, Press, San Diego, California, 241 pages. [ISBN 0-12-198390-0] California, 241 pages. [ISBN 0-12-198390-0] [book]. [book].

McLaughlin, Jacqueline S., and Elizabeth R. McCain. McLaughlin, Jacqueline S., and Elizabeth R. McCain. In VitroIn Vitro and and In VivoIn Vivo Development of the Development of the

Chicken Heart. 1998. Pennsylvania State University of Lehigh Chicken Heart. 1998. Pennsylvania State University of Lehigh Valley Department of Biology, Muhlenberg College Biology Department. 21 Valley Department of Biology, Muhlenberg College Biology Department. 21 Mar. 2004 Mar. 2004 <http://www.zoo.utoronto.ca/able/volumes/vol-19/mini/01-<http://www.zoo.utoronto.ca/able/volumes/vol-19/mini/01-mccain/01-mccain.htm>. mccain/01-mccain.htm>.

Park, Joyce. The Effect of Caffeine on the Heart Rate of the Three Day Chick Park, Joyce. The Effect of Caffeine on the Heart Rate of the Three Day Chick Embryos. 02 Aug. 2001. Embryos. 02 Aug. 2001. Swarthmore College. 13 Feb. 2004. Swarthmore College. 13 Feb. 2004. <<http://www.swarthmore.edu/NatSci/sgilber1?DBlab/Chick/chickadv.htmlhttp://www.swarthmore.edu/NatSci/sgilber1?DBlab/Chick/chickadv.html>. >.

Rxlist. 31 Dec. 2003. 13 Feb. 2004. Rxlist. 31 Dec. 2003. 13 Feb. 2004. <http://www.rxlist.com/cgi/generic3/caffeinecp.htm> <http://www.rxlist.com/cgi/generic3/caffeinecp.htm>

Sherwood, Lauralee. Sherwood, Lauralee. Human Physiology: From Cells to SystemsHuman Physiology: From Cells to Systems. California: . California: Brooks/Cole, 2001. 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>. <http://www.erowid.org/chemicals/caffeine/caffeine.shtml>.

Thank you for listening to Thank you for listening to our presentation.our presentation.

Any Questions?Any Questions?