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EXERCISE PERFORMANCE AND MUSCLE CONTRACTILE PROPERTIES AFTER CREATINE
MONOHYDRATE SUPPLEMENTATION IN AEROBIC-ANAEROBIC TRAINING RATS
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1Department of Physiology, Medical University of Plovdiv, Bulgaria, 2Department
of Anatomy, Histology and Embryology, Medical University of Plovdiv, Bulgaria.
| Received |
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19 March 2007 |
| Accepted |
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02
July 2007 |
| Published |
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01
December 2007 |
©
Journal of Sports Science and Medicine (2007) 6, 423- 428
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| ABSTRACT |
| The purpose of this study was to investigate the effects of creatine
monohydrate supplementation on exercise performance and contractile
variables in aerobic-anaerobic training rats. Twenty 90-day-old male
Sprague Dawley rats were divided into two groups - creatine (Cr) and
controls (K). The creatine group received creatine monohydrate as
a nutritional supplement, whereas the control group was given placebo.
Both groups were trained 5 days a week on a treadmill for 20 days
in a mixed (aerobic-anaerobic) metabolic working regimen (27 m·min-1,
15% elevation for 40 min). The exercise performance (sprint-test),
contractile properties (m. tibialis anterior), oxidative enzyme activity
(SDH, LDH, NADH2) in m. soleus and blood hematological and chemical
variables were assessed in the groups at the end of the experiment.
It was found out that creatine supplementation improved the exercise
performance after 20 days of administration in a dose of 60 mg per
day on the background of a mixed (aerobic-anaerobic) exercise training.
At the end of the trial the Cr-group demonstrated better values for
the variables which characterize the contractile properties of m.
tibialis anterior containing predominantly types IIA and IIB muscle
fibers. On the other hand, a higher oxidative capacity was found out
in m. soleus (type I muscle fibers) as a result of 20-day creatine
supplementation. No side effects of creatine monohydrate supplementation
were assessed by the hematological and blood biochemical indices measured
in this study.
KEY
WORDS: Exercise,
creatine supplementation, rats, performance, muscle contractile
properties.
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| INTRODUCTION |
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Recently,
there has been a considerable scientific interest (Kamber et al.,
1999)
shown in creatine as an ergogenic aid for improving exercise performance.
Creatine supplementation first gained popular attention in the early
1990s after high profile Olympic athletes competing in sprint and
power events at the Barcelona Olympic Games believed that it was
creatine that had a beneficial impact on their performance. Since
this time creatine has become one of the most widely used nutritional
supplements with an estimated worldwide consumption of 2.7 million
kilograms (Williams et al., 1999).
Creatine is not included in the International Olympic Committee
list of banned substances. Most of the consumers of this nutritional
supplement are power athletes, sportsmen with predominantly anaerobic
working regimen of muscles. Harris et al., 1992
found that phosphocreatine (PCr) content in muscles can increase
up to 50% following daily creatine supplementation. As a result,
an increase in total creatine stores may provide an ergogenic effect
during high intensity exercise by enhancing the rate of ATP synthesis
during contraction and by improving the rate of PCr resynthesis
during recovery, which may be beneficial for repeated power or sprint
activity, for example. Most of the studies investigating the effect
of creatine supplementation are performed on subjects (humans or
experimental rats) that do strength/power/sprint sports and do work
with maximal effort of muscle contractions (Mujika et al., 2000;
Vandenberghe et al., 1997;
Volek et al., 1999).
There have been few studies, however, performed on aerobic-anaerobic
training subjects.
The aim of this study was to examine the effects of creatine monohydrate
supplementation of the diet on exercise performance and contractile
variables in a mixed aerobic- anaerobic training regimen in rats.
| METHOD |
|
We
used 20 Sprague-Dawley rats approximately 90 days old at the
baseline. The rats were housed in individual metabolic cages
and were allocated into a creatine (Cr) group and a control
(K) group. The experimental animals were fed the standard
rat chaw ad libitum. The Cr-group received 60 mg creatine
monohydrate (DSM Fine Chemicals, Austria) each day as a supplement
to the standard diet (an equivalent of the recommended dose
of 20 g for humans) (Ipsiroglu et al., 2001).
The K-group received placebo (60 mg dextrose). Both, creatine
and placebo were incorporated in minced meat balls, which
were completely consumed by rats in the morning. Both groups
were trained incrementally by using mixed aerobic-anaerobic
workloads - five days a week on a treadmill (Columbus Instruments,
Columbus, OH). The duration of the run was progressively increased
every day by five minutes, starting from 25 min (27 m·min-1
speed and 15% slope) until the animals were running for 40
min per day (day 4). This duration was maintained till the
end of the experiment (Lambert, 1990).
The overall duration of the study was 20 days.
At the end of the experiment a sprint-test was performed to
assess the sprinting performance of the rats. The rats ran
on a treadmill (15% elevation) at 27 m/min for 3 min. The
speed was then increased to 45 m/min for 30 seconds and again
by 10 m/min every 30 seconds until the rat was unable to maintain
the pace of the treadmill belt. The highest speed which the
rat could maintain for 15 seconds was defined as the maximal
sprinting speed (Lambert, 1990).
The
contractile characteristic of m. tibialis anterior (containing
predominantly types IIA and IIB muscle fibers) (Delp and Duan,
1996)
of each rat in both groups was evaluated by isolating the
right limb muscle under narcosis (thiopental 10 mg/kg). Each
muscle was used immediately after removal. The muscle was
placed in an organ bath containing Krebs-Henseleit solution,
heated to body temperature and thermostatically controlled.
The solution was bubbled in a carbonic mixture to maintain
muscle feasibility (95% O2 and 5% CO2).
The muscle was fixed in the organ bath by traction straps
in the inferior rod and in the tensiometer in its superior
part. The basic tension was adjusted to 50 gram force (gf)
to each investigated muscle (1gf = 0.009807 N). The muscle
was stimulated directly by using platinum electrodes placed
along the long axis of the muscle. Electrical stimulation
was supplied by Grass S44-type stimulator (monophasic pulses
0.5 ms in duration, 10 Hz). The appropriate stimulating voltage
was determined by increasing the voltage until the measured
force of contraction reached its peak measurements of the
isometric-twitch curves. The force displacement transducer
was connected to a special converting device which in turn
was connected to the PC. The twitch curves were made using
computer based software WaveRunner 1.0 (Sigma Plus, Plovdiv,
Bulgaria). The following variables of the isometric contractile
properties, as defined by Close and Hoh (Close and Hoh, 1968),
were measured: 1. initial twitch tension (F0), 2. maximum
twitch tension (Fmax), 3. contraction time of maximum twitch
tension tFmax, 4. contraction times to Fmax-10%, Fmax-25%,
Fmax-50%. 5. the changes in the strength of muscle contraction
(as percentage of Fmax) during the whole time of contraction
(till the 450th second from the beginning of stimulation)
were also evaluated.
The right m. soleus (containing predominantly type I muscle
fibers) (Delp and Duan, 1996)
of each rat was prepared for histochemical analysis of the
following oxidative enzymes: LDH (Hess et al., 1958),
SDH (Nachlas et al., 1957),
and NADH2-cytochrome-c-reductase (Hess et al., 1958).
The analysis of the enzyme activity was performed using Microphot
microscope (Nikon, Japan) and special software DP Soft (Olympus,
Japan). The intensity of enzyme activity was measured by analysis
of 22080 fixed pixels (1840x12). The mean value for each field
was taken for further calculations.
Blood samples were taken from each rat at the end of the experiment
to determine the blood glucose, urea, creatinine, AST, ALT,
LDH, CPK, and WBC, RBC and PLT count, hemoglobin, hematocrit,
mean cell volume (MCV), mean concentration of hemoglobin using
a hematological analyzer (CONE, Finland).
Statistical analysis: the data were analyzed with the one-way
ANOVA and the significance of difference between the variation
series was calculated (Statistica 6.0, StatSoft Corp.). To
determine statistical significance between groups the orthogonal
contrast matrix method was used (Hicks, 1973).
All analyses were tested at p < 0.05. The data are presented
as mean ± SEM.
|
| RESULTS |
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The
body weight of the Cr-group rats was not significantly different
from that of the control rats throughout the experiment (Table
1). This fact was important for comparison of the contractile
properties of the skeletal muscles.
It is well known that the maximal sprinting test is a good
predictor of the anaerobic-aerobic working capacity of the
rats. At day 21 after starting the creatine supplementation
and the training programme, the rats of the Cr-group demonstrated
better results in their sprinting performance than the rats
of the K-group achieving higher velocity of the treadmill
belt during the test (79.00 ± 4.00 m·min- 1 vs.
67.00 ± 3.36 m·min-1, p < 0.05) (Figure
1).
Contractile
measurements
To assess the contractile characteristics of muscles that
are due to the training and creatine supplementation, m. tibialis
anterior was chosen because this muscles contains predominantly
fast twitch (types IIA and IIB) muscle fibers (Delp and Duan,
1996).
These fibers are mainly ATP and PCr dependent for energy support.
The initial twitch tension (F0) evoked by electrical
stimulation was higher in the Cr-group than that in the controls
at the end of the experiment (49.74 ± 0.83 gf vs. 47.24 ±
0.81 gf p < 0.05). On the other hand, the maximum tension
of the isolated m. tibialis anterior during continuous stimulation
(Fmax) in the Cr-group occurred later than that
in the K-group (55.07 ± 1.03 s vs. 45.54 ± 0.93 s, p <
0.001), and was
greater that that in the controls (68.41 ± 1. 19 gf vs. 63.13
± 1.03 gf, p < 0.01).
The analysis of the m. tibialis anterior contraction curve
following the continuous stimulation showed that the Cr-group
rats delayed the time to fatigue of this muscle when compared
with the controls - it took the Cr-rats longer time to reduce
their Fmax by 10%, 25%, and 50% in comparison with
the K-group (138.02 ± 1.86 s vs. 123.95 ± 1. 26 s, p <
0.001; 240.91 ± 3.21 s vs. 223.21 ± 2.54 s, p < 0.001.
and 457.63 ± 2.39 s vs. 420. 07 ± 4.60 s, p < 0.001, respectively)
(Figure 2).
We also studied the dynamics of the contraction strength of
m. tibialis anterior during continuous stimulation presented
as a function: the percentage of Fmax versus time
(Figure 3).
The decline of the strength contraction was analyzed
every 25 seconds from the beginning of the electrical stimulation
until the 450th second of this stimulation. At 100 seconds,
for example, the strength of the induced contraction was 94.04
± 0.62% of Fmax in the Cr-group, and 91.03 ± 0.56%
in the K-group, p < 0.01; at 200 seconds - 74.74 ± 0.92%
of Fmax in the Cr-group, and 70.37 ± 0.68% in the
K- group, p < 0.01; at 300 seconds - 53.90 ± 0.73% of Fmax
in the Cr-group, and 50.14 ± 0.85% in the K-group, p <
0.01; and at 400 seconds - 32.34 ± 0.86% of Fmax
in the Cr-group, and 26.34 ± 0.58% in the K-group, p <
0.001.
Because we used a mixed anaerobic-aerobic training regimen
of the experimental animals, we were also interested in the
aerobic capacity of the training muscles, which consist of
type I muscle fibers. We assessed the activity of some oxidative
enzymes in m. soleus such as LDH,
SDH, and NADH2-cytochrome-c-reductase. We found
higher activity of the selected oxidative enzymes in the Cr-group
than those in the controls (note that data are presented in
Reverse Intensity Units which means lower value - higher enzyme
activity). The SDH and NADH2-cytochrome-c-reductase
showed higher activity in the Cr-group (p < 0.001) (Figure
4).
No differences in the studied blood hematological and biochemical
variables were found between the groups except in the HCT
values. It was lower in the Cr-group than in the K-group (0.419
± 0.006 l·l-1 vs. 0.440 ± 0. 007 l·l-1, p < 0.05). All
values were within the physiological ranges for laboratory
rats.
|
| DISCUSSION |
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Dietary
supplementation of creatine and nutritional formulations containing
creatine have become the most popular nutritional strategy
employed by resistance-trained athletes to promote gains of
strength (Kreider, 1995).
Creatine supplementation has been reported to increase single
effort and/or repetitive sprint capacity (Dawson et al., 1995;
Earnest et al., 1995;
Kreider et al., 1998).
Muscle cells generate mechanical work from an energy liberating
chemical reaction - ATP is split into ADP and P (phosphate).
ATP can be used by muscle cells very quickly, but there is
only an extremely limited supply - usually only enough for
a few seconds of high intensity work. When the ATP is gone,
work stops. Fortunately, the body has several ways to convert
ADP back to ATP. The fastest method is to move the phosphate
group off of phosphocreatine and onto ADP. This yields ATP,
which is immediately available for muscular work, and creatine.
There is enough phosphocreatine to keep ATP levels up for
several more seconds. So at this point we have moved from
2 - 3 seconds of all-out work (ATP) to almost 10 seconds (ATP
+ creatine). The body can recharge creatine back to phosphocreatine,
but this takes time (approximately 30 - 60 seconds). This
ATP + creatine system makes up the fastest component of the
anaerobic system, and is most used by power athletes.
Aerobic endurance athletes, such as distance runners and triathletes,
represent a much different picture from power athletes. Their
levels of ATP and phosphocreatine do not change during exercise
because ATP is generated at the same rate it is used - a pay
as you go mechanism. Aerobic generation of ATP via oxidation
of glucose (and fats) is slower than by anaerobic systems,
but the fuel supply is enormous. Aerobic athletes train their
muscles differently, and indeed the muscle tissue itself is
different from power athletes. Type I muscle fibers have a
slower speed of contraction than type II fibers. Slow twitch
fibers have less glycolytic capacity, but increased mitochondria,
myoglobin, and aerobic enzyme pathways.
Thus, slow twitch athletes cannot generate the speed and force
of their fast twitch colleagues, but they can do their work
for a long time. So the widely accepted conclusion until now
has been that the ATP-creatine system is not that important
for aerobic or aerobic- anaerobic athletes.
Some recent studies have found that creatine supplementation
improves performance in aerobic-anaerobic trained athletes
(Chwalbinska-Moneta, 2003;
Engelhardt et al., 1998).
Our results are consistent with these findings - better results
in the sprint-test for the Cr-group (the data indicated better
anaerobic-aerobic working capacity of the Cr-group at the
end of the trial as a result of the creatine monohydrate supplementation
- Figure 1), and better values for the variables which characterize
the contractile properties. The m. tibialis anterior of the
rats of Cr-group demonstrated better adaptation and during
continuous stimulation had longer time to the point of reducing
the strength of contraction to Fmax-10%, Fmax-25%, and Fmax-50%
(Figure 2). On the other
hand, the fatigue curve (Figure
3) showed higher values for the strength of contraction
versus time in the Cr-group than in the controls during the
whole time of continuous stimulation.
The better adaptation of the oxidative enzymes in m. soleus
(Figure 4) can be explained
with the fact that creatine reduces the basal rate of lactate
production (Ceddia and Sweeney, 2004)
and this ensures a pH optimum values for SDH and NADH2-cytochrome-c-reductase
activity in this type I muscle.
In addition, blood biochemical and hematological data suggest
that 20 days of creatine supplementation in the doses we specified
above has no side effects on the organism. This is in agreement
with the findings of Poortmans and Francaux, 1999.
|
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| CONCLUSION |
| The
results in the present study allow us to conclude that creatine supplementation
of the diet in doses of 60 mg per day (an equivalent of 20 g for humans)
improves the performance results, contractile properties of the fast
twitch muscles, and increases the oxidative enzyme activity of aerobic-anaerobic
trained rats. |
| KEY
POINTS |
- The
creatine monohydrate supplementation of the rats diet improves
their exercise performance after 20 days administration in a dose
of 60 mg per day on the background of a mixed (aerobic-anaerobic)
exercise training.
- The
creatine supplemented rats demonstrate better contractile properties
of m. tibialis anterior which muscle contains predominantly types
IIA and IIB muscle fibers.
- The
soleus muscle (type I muscle fibers) demonstrates a higher oxidative
capacity as a result of 20-days creatine supplementation.
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| AUTHORS
BIOGRAPHY |
Nickolay P. BOYADJIEV
Employment: Department of Physiology, Medical University
of Plovdiv, Bulgaria.
Degree: PhD.
Research interests: Exercise physiology, sport supplementation,
resistance training, exercise endurance.
E-mail: boyad@plov.net
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|
Dobrin
I. POPOV
Employment: Department of Physiology, Medical University
of Plovdiv, Bulgaria.
Degree: MSc.
Research interests: Exercise physiology, sport supplementation,
resistance training, exercise endurance.
E-mail: drpopov@abv.bg
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Slavi
DELCHEV
Employment: Department of Anatomy, Histology and Embryology,
Medical University of Plovdiv, Bulgaria.
Degree: MSc.
Research interests: Muscle and heart structural changes
in resistance and endurance training.
E-mail: msdel@abv.bg |
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