Chick Lab by N815QKpB

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									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.




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  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.
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    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

37C 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 37C and one was incubated at 38C. 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 37C and an embryo incubated at 38C, 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




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                         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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