Effect of Creatine On the Pulsation and Regeneration Rates of Lumbriculus

variegatus

Abstract

In this experiment the pulsation and regeneration rates of the California

blackworm, Lumbriculus variegatus, were tested using Creatine, a supplement used
mainly by body builders and athletes to increase strength and muscle appearance.
The worms were placed in a control group of spring water, 0.4mg/mL Creatine
solution, and 0.6mg/mL Creatine solution to test their pulsation rates. They were
then placed under the microscope and their pulsation rates were tested. The
regeneration rates of Lumbriculus regeneration rates they were placed in a control
group of spring water, 0.2mg/mL Creatine solution, and 0.4mg/mL Creatine solution.
After one week in the solutions the worms were put under the microscope and the
additional segments regenerated were counted. The results showed that the
worms’ pulsation rates were increased by the Creatine solution, but only after 20
minutes in the solution were the pulsation rates significant. The regeneration rate
results also showed an increase with the addition of the Creatine solution, but it was
concluded that the results were insignificant.

Introduction/Background

This was a biological study of the effects of Creatine on the pulsation and
regeneration rates the California blackworm, Lumbriculus variegatus. Creatine
(C4H9N3O2) is a supplement widely used by athletes and body builders to build and
sustain muscle mass more effectively than the normal body processes would allow.
This is done by the body storing an amount Creatine in a muscle cell, known a
muscle fiber, as Creatine phosphate. The Creatine attracts water to the cell,
thereby enlarging it. This super hydration of the cells causes increased strength,
and fuller appearance of the muscle. It has also been hypothesized that water
retention caused by the Creatine may also trigger protein synthesis and minimize
catabolism. Creatine provides faster muscle recovery, as well as better tolerance
toward lactic acid. It does this by increasing the amount of Adenosine Triphosphate
(ATP). Cells use ATP as their first source of energy with Adenosine Diphosphate
(ADP) as its only by product. With enough ADP left over it can be used to create
more ATP, and such the cycle continues until the ADP supply is close to depletion.
Depletion only takes 10 seconds, because there is a lack of ADP to produce more
ATP. The cell would then move on to its second source, glycogen. Glycolysis (the
process of converting glycogen into energy) produces a byproduct of lactic acid.
When too much lactic acid is produced it causes muscle contractions to stop. What
Creatine phosphate does is provide more phosphate to turn ADP into ATP, thus
increasing the amount of ATP the cell has stored to use. That adds more time
before glycolysis must begin, delaying the production of lactic acid, decreasing
recovery time, and increasing overall muscle performance.i

Lumbriculus variegatus was tested on its pulsation and regeneration rates

with various amounts of Creatine solution. The pulsation and regeneration rates are
the dependent variables, meaning that their outcome depends on the independent
variable. The different Creatine solutions are the independent variables, which are
what is not affected by any other varibles. Our hypothesis is that Creatine will
increase both the pulsation and regeneration rates of Lumbriculus variegatus. We
will test the pulsation rate by placing the worms in two different concentrations of
solutions for two different periods of time and then compare those results to that of
our control group. This information could help in determining whether or not
Creatine causes a potentially harmful increase in heart rate of a human. For the
regeneration rates we will be putting the worms in two different concentrations of
Creatine for a week in complete darkness. After the elapsed time we will separately
count the new segments the worms have regenerated. That information would
provide extra evidence that Creatine is a good tool for regeneration of muscle. 1

Hypothesis

If Lumbriculus variegatus is exposed to a Creatine solution, then its pulsation and

regeneration rates will both increase.

Methodology

The first experiment we will be tested the effect of Creatine on the California
blackworms’ pulsation rate. Six experiments were conducted, each with a different
amount of solution, with a total of 36 worms. There were 6 worms per experiment
in 3 different solution concentrations. The first was simply a control group, the
worm’s natural environment, of spring water. The second contained 0.4mg of
Creatine for every 100mL of water, 3.1X10-3M C4H9N3O2. The third contained 0.6mg
of Creatine for every 100mL of water, 4.6X10-3M C4H9N3O2. The worms were tested
twice in each solution. In the first test they were placed in 1mL of the liquid for 10
minutes, and in the second they were placed in 1mL the liquid for 20 minutes. After
the time was elapsed the worms were placed under the microscope and the
pulsation rate was measured. This was done by finding the dorsal vessel of the
worm (because it is the easiest to see pulsation from), choosing a segment of the
worm to focus on, and then counting how many times the vessel pumps past that
segment for a period of 10 seconds at a time.ii

The second experiment was the testing of Creatine’s effect on the

regeneration of Lumbriculus variegatus. Three experiments were conducted using
12 worms per solution, a total of 36 worms in all. The first solution only had a
control group of spring water. The second solution had a 0.2mg of Creatine per
100mL of water, 1.5X10-3M C4H9N3O2. The third solution had a 0.4mg of Creatine per
100mL of water, 3.1X10-3M C4H9N3O2. Small pieces of brown paper bag were placed
in each well (the container that will be housing the worm while it regenerates) to
replicate its natural environment of sand and other sediment. This was done in
hopes that it may encourage the worm in some way to grow at a greater pace. The
worms were be cut in half, and then a 5-8 segment long piece were cut from the
anterior end (front; head) and the posterior end (back; rear). Both pieces were
placed in separate wells adjacent to each other containing 2mL of the solution. This
was done because the anterior end tends to regenerate at a more rapid pace than
the posterior end, because the anterior end is the worms’ control center and
contains the aortic arches (worm’s hearts) that help pump the blood. The worms
were then covered with a plastic film and a lid so to keep them from working their
way out of the wells. They were then put them in the dark to help simulate its
natural environment of dark murky water. After a week had elapsed the worms
were taken out individually and the new segments were counted of the anterior and
posterior ends that appeared lighter than the rest of the worm. Those results were
then compared to the results of the control group.

Results

The results of the pulsation rate experiment showed a general increase of the
worm’s pulsation with the addition of time and dosage (Figure 1). There was no
correlation between the dosages. The time, however, after 20 minutes showed a
significant increase in the pulsation rates. That being true, the p-values obtained
from the T-test using the ANOVA (JMP v.7 SAS) program show that the all results,
except the pulsation rate after 20 minutes, were not significant, so the hypothesis
must be rejected.

Figure 1
 The results of the regeneration rate showed an increase in the worm’s
regeneration rate, given the factors of time and dosage (Figure 2). There was no
trend with dosage and body part. Once again, the p-values obtained from the T-test
using ANOVA (JNP v.7 SAS) program shows that all the results were not significant,
and the hypothesis must be rejected.

Figure 2

Conclusion

In conclusion, the data shows that Creatine may have a significant effect on
both the pulsation and regeneration rates of Lumbriculus variegatus, but the overall
experiment was too small of scale to make significant results. Also, how the worms
were handled may have affected the pulsation rates by exciting the worms and
increasing their pulsation rates prior to examination. The manner in which the
worms were cut in the regeneration experiment may have determined whether the
worms lived throughout the week . In future experiments consideration should be
taken towards the number of test subjects. Without enough test subjects the
results acquired may not be significant and would then be rejected. This is the
reason why the hypotheses for the pulsation and regeneration experiments were
rejected. If there had been more worms the results would have been accepted and
a proper conclusion could have been prepared.

Bibliography
i
Andres, Robert H., Angélique D. Ducray, Uwe Schlattner, Theo Wallimann, and Hans
Rudolf Widmer. Functions and effects of creatine in the central nervous system. Brain
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922bd93ba4f3%40sessionmgr109 Accessed 2008 July 2.

ii
Sardo, A. M., A. M. V. M. Soares, and A. Gerhardt. Behavior, growth, and reproduction of
Lumbriculus variegatus (oligochaetae) in different sediment types. Human & Ecological
Risk Assessment 2007;13 519-527. Available from: Ebscohost [online database].
http://web.ebscohost.com/ehost/detail?vid=4&hid=109&sid=e026fada-b9d0-4e43-8ed3-
bde9e25c047e%40sessionmgr106 Accessed 2008 July 1.