- Email: [email protected]
Anaerobic threshold in rats
Camp. Biochem. Physiol. Vol. 106A, No. 2, pp. 285-289,
0300-9629/93 $6.00 + 0.00 Q 1993 Pergamon Press Ltd
1993
Printed in Great Britain
ANAEROBIC WIE~EAWPnts,*t
THRESHOLD
IN RATS
RYSZARDZARZECZNY,$ J~ZEF LANGFORT&HANNA KACIUBA-USCIEKO,$ KRYSTYNANAZAR§ and JANUSZWOJTYNA~
*Department of Physiology, Academy of Physical Education, 72 A Mikoiowska str., 40-065 Katowice, Poland; fUniversity of Pedagogigs, 4/8 Waszyngtona str., 42-200 Czestochowa, Poland; $Department of Applied Physiology, Medical Research Centre, Polish Academy of Sciences, 17 Jazgarzewska str., 00-730 Warsaw, Poland (Received
23 November 1992; accepted 15 January
1993)
Abstract-l. The aim of this study was to find out whether the anaerobic threshold (AT) can be estimated in rats running at increasing speed and if so what is the reproducibility of the measurements. 2. Lactate (LA) concentrations in blood taken from 11 rats were determined during a discontinued, multistage treadmill exercise test repeated four times in each animal. 3. It was found that blood LA changes vs speed have an exponential pattern with a distinct, rapid rise at the speed above 25 m/min which corresponds to blood LA of approx. 4 mmol/l. 4. The variation coefficient of the speed at which AT occurred in individual animals ranged between 10 and 20%. 5. These results offer a potential application of AT determination in the animal studies concerning mechanisms controlling exercise metabolism.
INTRODUCTION It is well known that in human subjects lactate (LA) concentration in blood increases exponentially with exercise intensity. The break point on the curve of blood LA vs exercise load has been commonly termed the anaerobic threshold (AT) although anaerobiosis as a cause of the accelerated LA production has been questioned (Brooks, 1986; Connett et al., 1990; Hughson et al., 1987). Anaerobic threshold occurs within the range of submaximal exercise intensities, usually between 50 and 80% of the maximal load and at blood LA concentration approximating 4 mmol/l. Despite many attempts (Wasserman et al., 1973; Brooks 1986; Connett et al., 1990; Beaver et al., 1986; Conconi et al., 1982) the physiological basis of AT has not been yet fully explained. Nevertheless, the threshold estimations, i.e. determinations of exercise intensity at which LA starts to accumulate in blood during incremental exercise, were found useful in the assessment of the subjects’ endurance exercise ability. It has become increasingly popular for an evaluation of training effectiveness in athletes, assuming that the higher the threshold the greater is the endurance capacity (Kinderman et al., 1979; Reinhard et al., 1979; Kumagai et al., 1982; Smith et al., 1984; Weber et al., 1984). Measurements of AT also proved to be of a marked value in clinical studies with cardiac patients (Wasserman and McIlroy, 1964; Marcus et al., 1971; Wasserman et al., 1973; Matsumura et al., 1983). Since there are obvious limitations in the investigations with human subjects an animal tTo whom all correspondence should be addressed. cm.4
106,*--1
model would be important for a deeper insight into the physiological mechanisms underlying AT. Surprisingly, according to the authors’ knowledge, the anaerobic threshold has not been determined in animals. Thus, the main purpose of this study was to analyse blood LA changes during a multistage treadmill exercise in rats in order to find out whether AT can be systematically detected in these animals.
MATERIALSANDMETHODS Experimental
procedure
Eleven untrained male Wistar rats, weighing 250 f SD (15 g) were used for this study. They were housed in groups in the wire mesh cages at 2&22”C with the light on from 6 a.m. to 6 p.m. The animals had free access to water and the commercial laboratory chow (Murigram, Poland). After being well familiarized with handling and the treadmill running each animal participated in seven experimental series with 1 week intervals between them. The first four series of identical protocol were designed to follow up changes in blood lactate (LA) concentrations during the multistage running test, and if possible to estimate the anaerobic threshold as well as to check its reproducibility. In each experiment the rats were transferred individually to special, small wire cages allowing them to reduce their motion. After a period of 10min rest the end of animal’s tail was cut and heparinized. Five min later the first (resting) capillary blood sample (0.05 ml) was taken from the tail for blood LA determination. Then, the animals were put on the treadmill and started to run at a speed of
286
WIFSZAW PILIS et a/.
13 m/min at 10% of the tradmill inclination for 3 min. Afterwards, the animals performed several exercise bouts with increasing treadmill velocity. At each bout the speed was increased by 4m/min than in the previous one until 37 m/min was attained. Each exercise load lasted 3 min and was followed by 3 min rest during which blood samples were taken. For this purpose the rats were transferred from the treadmill to the small cages to avoid struggling. After achieving a speed of 37 m/min the treadmill velocity was further increased until the individual maximum speed was reached, and then the last blood sample was taken. The aim of the fifth series was to evaluate a relationship between blood LA, blood bicarbonate (HCOr-) and the base excess (BE) concentrations using the protocol basically similar to the one described above, except that more blood (0.1 ml) was taken after each exercise load. The sixth and seventh series were carried out to follow-up blood LA concentrations during repeated bouts of identical exercise of the intensity either below or above the anaerobic threshold. For this purpose the rats performed six bouts of 3 min exercise at 17 m/min (sixth series) and six exercise bouts at 33 m/min (seventh series). During the 3min rest intervals between separate bouts blood samples were taken (as described above) for LA determinations. Analytical methods Blood LA concentration was determined enzymatitally using commercial kits (Boehringer Diagnostica, Mannheim, F.R.G.). Blood HCO,and BE levels were measured using the 168 pH/Blood-Gas Analyzer (CibaCorning, Basel, Switzerland). Calculations The anaerobic threshold was estimated from individual plots of blood LA vs the treadmill speed. In addition, blood LA concentration at AT was calculated from the individual plots by interpolation.
Rat’s No.
Table 1. Maximal
running
Maximal
running (mimin)
speed and maximal
Reproducibility of the threshold evaluated for each animal in four experimental series was checked by means of the two-way analysis of variance, followed by Duncan’s multiple range test (Armitage, 1971). Besides, the linear regressions were calculated using conventional methods. The null hypothesis was rejected when P < 0.05. The data presented throughout the paper are means f SD. RESULTS
The maximal speed achieved by the rats of the first 4 series was on the average 52.82 + 5.61 m/min (Table 1) and blood LA concentrations at that running speed reached 15.14 + 4.75 mmol/ x 1. The mean values of blood LA concentration at the consecutive work loads (running speed) in all rats exercising four times (N = 44) are presented in Fig. 1. As it can be seen, the course of changes in blood LA in relation to the speed of running was exponential rather than linear, with a distinct, rapid increase in blood LA at the speed of running above 25m/min. An analysis of individual curves enabled us to determine the anaerobic threshold (AT). The mean AT, expressed as the running speed at which LA started to accumulate in blood in all the rats from four series (N = 44), was 26.4 k 3.75 m/min, while the mean interpolated blood LA level at this speed was 4.12 f 1.36 mmol/l. There were no significant differences between the mean values of either the running speed or blood LA level at AT obtained in the four experimental series. The individual values of running speed and blood LA levels at AT are given in Table 2. The mean AT values obtained in the same rats in four experimental series showed relatively high reproducibility. It should be noted, however, that in five rats the variation coefficient of the speed at which AT was achieved exceeded 10% but in none it reached 20%. The values of blood LA at AT showed greater variation than those of speed in the repeated tests (the variation coefficients was between 11.2 and 43.5%.
blood lactate concentration
of individual
rats obtained
in four repetitions
Maximal blood lactate concentration (mmol/l)
speed
I
II
III
IV
Mean
SD
CV%*
I
II
III
IV
Mean
SD
CV%
I 2 3 4 5 6 7 8 9 10 I1
45 55 50 37 55 37 52 55 55 50 55
50 48 55 50 60 50 55 55 55 45 55
50 55 55 55 60 55 60 55 55 37 55
so 55 55 55 55 60 60 58 55 SO 55
48.75 53.25 53.75 49.25 57.50 50.50 56.75 55.75 55.00 45.50 55.00
2.50 3.50 2.50 8.50 2.87 9.88 3.95 1.50 0.00 6.14 0.00
5.13 6.57 4.65 17.26 4.99 19.56 6.96 2.69 0.00 13.49 0.00
8.37 16.89 20.40 21.46 12.86 22.50 12.79 10.24 15.03 12.71 20.38
IO.40 20.85 20.32 23.28 15.29 25.70 15.49 12.14 12.61 12.90 20.61
18.19 13.60 21.08 24.89 9.32 21.52 10.40 10.27 14.31 9.75 14.36
II.27 10.74 10.32 13.75 14.70 14.19 14.94 Il.05 10.83 9.73 13.70
12.06 15.52 18.03 20.85 13.04 20.98 13.41 10.93 13.20 11.27 17.26
4.27 4.35 5.15 4.93 2.69 4.86 2.32 0.89 I .87 I .77 3.74
35.41 28.03 28.56 23.65 20.63 23.16 17.30 8.14 14.17 15.71 21.67
mean SD CV%
49.64 7.00 14.10
52.5s 4.27 8.13
53.82 6.19 11.50
55.27 3.29 5.95
15.78 4.84 30.67
17.24 5.12 29.70
15.24 5.41 35.50
12.29 1.95 15.87
*CV% indicates
the coefficient of variation
in percentage
of the mean values.
Anaerobic threshold in rats
SPEED
OF RUNNING
287
lm . mid)
Fig. 1. The mean (k SD) values of blood LA concentrations in relation to the increased speed of running in rats.
The results of the fifth series of experiments showed that blood concentrations of HCO,- and BE decreased with the intensity of exercise, in opposition to blood LA level (Fig. 2). The inverse correlations were ascertained between blood LA and HCO,- concentrations (y = 13.53 -0.45x, r = - 0.73, N = 71, P < 0.001) as well as between blood LA and BE concentrations (y = 1.32 - 0.49x, r = - 0.75, N=71, P
Tabk
2. Running
speed and blood lactate concentration Running
Rat’s No. 1 2 3 4 5 6 7 8 9 10 11 mCan
DISCUSSION
Our study clearly demonstrated that in rats, running on the treadmill with increasing intensity, changes in blood lactate show a pattern similar to that described in human subjects. This enabled us to determine the level of exercise intensity at which blood lactate concentration starts to increase rapidly. This level, corresponding to the anaerobic threshold (AT), was achieved during running on the treadmill set at a 10” slope and the speed of approximately 25 m/min. Taking into account the reported data concerning maximal oxygen uptake (% YOz max) in rats (Bedford et al., 1979; Sonne and Galbo, 1980; Brooks and White, 1978; Glesson and Baldwin, 1981), it may be concluded that AT in rats corresponds to 5565% of VOz max. This is within the range of exercise intensities at which AT is achieved in human subjects. The mean value of LA measured after the maximal exercise as well as that at the anaerobic threshold, were in rats 15.14 f 4.75 and 4.12 f 1.36,
at the anaerobic tests
speed at AT (m/min)
threshold
of individual
rats obtained
in four multistage
exercise
Blood lactate concentration at AT (mmol/l)
I
II
III
IV
Mean
SD
CV%
I
II
III
IV
Mean
SD
CV%
29 25
25 25
25 29
:: 29 25 25 25 25 29 25
:: 33 29 29 29 2s 29 29
:: 29 29 21 29 29 29 29
25 33 21 21 25 33 25 29 25 29 29
26.00 28.00 20.00 22.00 29.00 29.00 25.00 28.00 26.00 29.00 28.00
2.00 3.83 3.83 2.00 3.27 3.27 3.26 2.00 2.00 0.00 2.00
7.7 14.0 19.1 9.1 11.3 11.3 13.1 7.1 7.7 0.0 7.1
3.2 3.0 5.0 3.1 4.0 6.8 4.4 2.5 2.2 6.8 2.0
2.6 4.3 4.0 7.2 5.6 6.8 4.2 3.9 2.4 4.2 4.4
3.1 4.1 4.0 4.6 3.0 5.3 1.6 3.9 4.1 4.2 4.0
2.6 5.2 5.8 4.6 2.8 6.4 2.4 3.9 4.0 3.6 4.0
3.04 4.30 4.70 4.88 3.85 6.33 3.15 3.55 3.18 4.70 3.60
0.48 0.94 0.87 1.70 1.28 0.71 1.37 0.70 0.77 I .43 I .08
15.8 21.9 18.5 34.9 33.2 11.2 43.5 19.7 24.2 30.4 30.0
25.70 2.41 9.38
26.80 4.14 15.45
26.09 4.42 16.94
26.80 4.14 15.45
3.91 I .69 43.22
4.51 1.50 33.26
4.19
4.12 1.29 31.31
1.26 30.07
288
WIESZAW
PlLIS
et al.
HCOj o-----Q
BE
-
LA
-26
--3
-20
s-6
% . 169
- -9 d
g
g
- 10
4!i WEED
OF RUNNING
4Q
53
r
- -12
67
(m emid 1
Fig. 2. Blood concentration of LA, HCO,- and BE during the increased speed of running in rats. Results are presented as mean k SD. thus being close to those found in men (Heck et al., 1985; Green et al., 1983). Four repetitions of the multistage exercise in the same rats showed fairly good reproducibility of AT, expressed as the speed of running. It should be noted that those AT levels were more reproducible than blood LA concentrations at a given speed (values of blood LA measured after maximal exercise and the values corresponding to AT). The concept of AT assumes that exercise of the intensity below the threshold can be performed for a long time without accumulation of lactate in blood, whilst during exercise exceeding the threshold blood LA concentration progressively increases with time of effort. This concept was tested in the present study on the animal model, using several bouts of intermittent exercise of identical intensity. Continuous exercise could not be applied in this case, respectively,
20 %
L
18 16lL-
iz g
12-
a
lo
SPEED OF RUNNING: 0
17
0 33m,
mid I T
1
P
P
2
3
I!l
OL
T
m -mid
1
NUMBER OF SEauENllAL
P
9
P
1
5
6
EXERCISE REPETITION
Fig. 3. Blood LA concentrations after six consecutive runs at the speed below (17 m/min) and above (33 m/min) AT. Results are presented as mean f SD.
since breaks were necessary for blood sampling. Despite this the data confirmed the assumption mentioned above indicating practically no change in blood LA level at the running speed of 17 m/min (below AT) and its progressive increase at the speed of 33 m/min. In human subjects in addition to blood lactate concentration changes in several physiological or biochemical variables e.g. pulmonary ventilation, CO, output, heart rate, blood bicarbonate (HCO,-), base excess (BE) and hydrogen ion concentrations are measured during incremental exercise test and used for determining the anaerobic threshold. All of them, except HR, are related to lactic acid accumulation in blood and show a close correlation with blood LA level (Beaver et al., 1986; Heck et al., 1985; Kumagai et al., 1982; Yoshida et al., 1981). To check whether such relationships can also be found in rats in one of the experimental series blood HCO,- and BE concentrations were followed together with blood LA during the multistage exercise. The data showed a similar pattern of changes in those variables to that in men. Moreover, close inverse correlations were ascertained between blood LA, and HC03- or BE levels (r = -0.73,P c 0.001 and r = -0.75, P c 0.001, respectively). In conclusion the present study demonstrated that in rats changes in capillary blood lactate concentration during a discontinuous multistage exercise of increasing intensity allow us to determine the anaerobic threshold, which is fairly reproducible. These results offer a potential application of AT determinations for serial evaluations of animal working ability under various experimental conditions, in the course of physical training etc. The animal model can be of value for further investiaations directed to
Anaerobic threshold in rats explain physiological
mechanisms
responsible
for AT
phenomenon.
289
function in progressive exercise. J. appl. Physiol. 62, 197>1981.
Kinderman W., Simon G. and Keul J. (1979) The significance of the aerobic-anaerobic threshold transition for the determination of work load intensities during endurance training. Eur. J. appl. Physiol. Occup. Physiol. 42,
REFERENCES
25-34.
Armitage P. (1971) Statistical Methoak in Medical Research. John Wiley & Sons, New York. Beaver W. L., Wasserman K., and Whipp B. J. (1986) A new method for detecting anaerobic threshold by gas exchange. J. appl. Physiol. 60, 202&2027. Bedford T. G., Tipton C. M., Wilson N. C., Oppliger R. A. and Gisolfi C. V. (1979) Maximum oxygen consumption of rats and its changes with various experimental procedures. J. appl. Physiol. 41, 1278-1283. Brooks G. A. (1986) Lactate production under fully aerobic conditions: the lactate shuttle during rest and exercise. Fed. Proc. 45, 2924-2929.
Brooks G. A. and White T. P. (1978) Determination of metabolic and heart rate responses of rats to treadmill exercise. J. appl. Physiol. 45,-1009-1015. Conconi F.. Ferrari M.. Zialio P. G.. Droahetti P. and Codeca L. (1982) Detekination ‘of the anaerobic threshold by a noninvasive field test in runners. J. appl. Physiol. 52, 869-873.
Connett R. J., Honig C. R., Gayeski T. E. J. and Brooks G. A. (1990) Defining hypoxia: a systems view of VO,, glycolysis, energetics, and intracellular PO,. J. appl. Physiol. 68, 833-842.
Glesson T. T. and Boldwin K. M. (1981) Cardiovascular response to treadmill exercise in untrained rats. J. appl. Physiol. 50, 120&1211.
Green H. J., Hughson R. L., Orr G. W. and Ranney D. A. (1983) Anaerobic threshold, blood lactate, and muscle metabolites in progressive exercise. J. appl. Physiol. (Respir. Em. E.&&e
Physiol.) 54, 1032-lij8.
_
Heck H., Mader A.. Hess G., Mucke S.. Muller R. and Hollmann W. (1985) JustiBcation of 4mmol/l lactate threshold. Int. J. Sports Med. 6, 117-130. Hughson R. L., Weisiger K. H. and Swanson G. D. (1987) Blood lactate concentration increases as a continuous
Kumagai S., Tanaka K., Matsumura Y., Matsuzaka A., Hirakoba K. and Asana K. (1982). Relationships of the anaerobic threshold with 5 km, 10 km, and 10 mile races. Eur. J. appl. Physiol. 49, 13-23.
Matsumura N., Nishijima H., Kojima S., Hashimoto F., Minami M. and Yasuda H. (1983) Determination of anaerobic threshold for assessment of functional state in patiens with chronic heart failure. Circulation 68, 36&367.
Reinhard U. P., Muller P. H. and Schmulhng R. M. (1979) Determination of anaerobic threshold by ventilation equivalent in normal individuals. Respiration 38, 3&42. Sonne B. and Galbo H. (1980) Simultaneous determinations of metabolic and hormonal responses, heart rate, temperature, and oxygen uptake in running rats. Acta Physiol. &and. 109, 201-209. Smith D. A. and O’Donnell T. V. (1984) The time course during 36 weeks’ endurance training of changes in VO, max and anaerobic threshold as determined with a new computerized method. Clin. Sci. Land. 67, 229-236. Yoshida T., Nagata A., Muro M., Takeuchi N. and Suda Y. (1981) The validity of anaerobic threshold determination by a Douglas bag method compared with arterial blood lactate concentration. Eur. J. appl. Physiol. 46,423430.
Wasserman K. and McIlroy M. (1964) Detecting the threshold of anaerobic metabolism in cardiac patents during exercise. Am. J. Cardiol. 14, 844852. Wasserman K., Whipp B. J., Koyal S. N. and Beaver W. L. (1973) Anaerobic threshold and respiratory gas exchange during exercise. J. appl. Physiol. 35, 236243. Weber K. T., Wilson J. S., Janicki J. S. and Likoff M. J. (1984) Exercise testing in the evaluation of the patient with chronic cardiac failure. Am. Rev. Respir. Dis. 129. S60-S62.
0300-9629/93 $6.00 + 0.00 Q 1993 Pergamon Press Ltd
1993
Printed in Great Britain
ANAEROBIC WIE~EAWPnts,*t
THRESHOLD
IN RATS
RYSZARDZARZECZNY,$ J~ZEF LANGFORT&HANNA KACIUBA-USCIEKO,$ KRYSTYNANAZAR§ and JANUSZWOJTYNA~
*Department of Physiology, Academy of Physical Education, 72 A Mikoiowska str., 40-065 Katowice, Poland; fUniversity of Pedagogigs, 4/8 Waszyngtona str., 42-200 Czestochowa, Poland; $Department of Applied Physiology, Medical Research Centre, Polish Academy of Sciences, 17 Jazgarzewska str., 00-730 Warsaw, Poland (Received
23 November 1992; accepted 15 January
1993)
Abstract-l. The aim of this study was to find out whether the anaerobic threshold (AT) can be estimated in rats running at increasing speed and if so what is the reproducibility of the measurements. 2. Lactate (LA) concentrations in blood taken from 11 rats were determined during a discontinued, multistage treadmill exercise test repeated four times in each animal. 3. It was found that blood LA changes vs speed have an exponential pattern with a distinct, rapid rise at the speed above 25 m/min which corresponds to blood LA of approx. 4 mmol/l. 4. The variation coefficient of the speed at which AT occurred in individual animals ranged between 10 and 20%. 5. These results offer a potential application of AT determination in the animal studies concerning mechanisms controlling exercise metabolism.
INTRODUCTION It is well known that in human subjects lactate (LA) concentration in blood increases exponentially with exercise intensity. The break point on the curve of blood LA vs exercise load has been commonly termed the anaerobic threshold (AT) although anaerobiosis as a cause of the accelerated LA production has been questioned (Brooks, 1986; Connett et al., 1990; Hughson et al., 1987). Anaerobic threshold occurs within the range of submaximal exercise intensities, usually between 50 and 80% of the maximal load and at blood LA concentration approximating 4 mmol/l. Despite many attempts (Wasserman et al., 1973; Brooks 1986; Connett et al., 1990; Beaver et al., 1986; Conconi et al., 1982) the physiological basis of AT has not been yet fully explained. Nevertheless, the threshold estimations, i.e. determinations of exercise intensity at which LA starts to accumulate in blood during incremental exercise, were found useful in the assessment of the subjects’ endurance exercise ability. It has become increasingly popular for an evaluation of training effectiveness in athletes, assuming that the higher the threshold the greater is the endurance capacity (Kinderman et al., 1979; Reinhard et al., 1979; Kumagai et al., 1982; Smith et al., 1984; Weber et al., 1984). Measurements of AT also proved to be of a marked value in clinical studies with cardiac patients (Wasserman and McIlroy, 1964; Marcus et al., 1971; Wasserman et al., 1973; Matsumura et al., 1983). Since there are obvious limitations in the investigations with human subjects an animal tTo whom all correspondence should be addressed. cm.4
106,*--1
model would be important for a deeper insight into the physiological mechanisms underlying AT. Surprisingly, according to the authors’ knowledge, the anaerobic threshold has not been determined in animals. Thus, the main purpose of this study was to analyse blood LA changes during a multistage treadmill exercise in rats in order to find out whether AT can be systematically detected in these animals.
MATERIALSANDMETHODS Experimental
procedure
Eleven untrained male Wistar rats, weighing 250 f SD (15 g) were used for this study. They were housed in groups in the wire mesh cages at 2&22”C with the light on from 6 a.m. to 6 p.m. The animals had free access to water and the commercial laboratory chow (Murigram, Poland). After being well familiarized with handling and the treadmill running each animal participated in seven experimental series with 1 week intervals between them. The first four series of identical protocol were designed to follow up changes in blood lactate (LA) concentrations during the multistage running test, and if possible to estimate the anaerobic threshold as well as to check its reproducibility. In each experiment the rats were transferred individually to special, small wire cages allowing them to reduce their motion. After a period of 10min rest the end of animal’s tail was cut and heparinized. Five min later the first (resting) capillary blood sample (0.05 ml) was taken from the tail for blood LA determination. Then, the animals were put on the treadmill and started to run at a speed of
286
WIFSZAW PILIS et a/.
13 m/min at 10% of the tradmill inclination for 3 min. Afterwards, the animals performed several exercise bouts with increasing treadmill velocity. At each bout the speed was increased by 4m/min than in the previous one until 37 m/min was attained. Each exercise load lasted 3 min and was followed by 3 min rest during which blood samples were taken. For this purpose the rats were transferred from the treadmill to the small cages to avoid struggling. After achieving a speed of 37 m/min the treadmill velocity was further increased until the individual maximum speed was reached, and then the last blood sample was taken. The aim of the fifth series was to evaluate a relationship between blood LA, blood bicarbonate (HCOr-) and the base excess (BE) concentrations using the protocol basically similar to the one described above, except that more blood (0.1 ml) was taken after each exercise load. The sixth and seventh series were carried out to follow-up blood LA concentrations during repeated bouts of identical exercise of the intensity either below or above the anaerobic threshold. For this purpose the rats performed six bouts of 3 min exercise at 17 m/min (sixth series) and six exercise bouts at 33 m/min (seventh series). During the 3min rest intervals between separate bouts blood samples were taken (as described above) for LA determinations. Analytical methods Blood LA concentration was determined enzymatitally using commercial kits (Boehringer Diagnostica, Mannheim, F.R.G.). Blood HCO,and BE levels were measured using the 168 pH/Blood-Gas Analyzer (CibaCorning, Basel, Switzerland). Calculations The anaerobic threshold was estimated from individual plots of blood LA vs the treadmill speed. In addition, blood LA concentration at AT was calculated from the individual plots by interpolation.
Rat’s No.
Table 1. Maximal
running
Maximal
running (mimin)
speed and maximal
Reproducibility of the threshold evaluated for each animal in four experimental series was checked by means of the two-way analysis of variance, followed by Duncan’s multiple range test (Armitage, 1971). Besides, the linear regressions were calculated using conventional methods. The null hypothesis was rejected when P < 0.05. The data presented throughout the paper are means f SD. RESULTS
The maximal speed achieved by the rats of the first 4 series was on the average 52.82 + 5.61 m/min (Table 1) and blood LA concentrations at that running speed reached 15.14 + 4.75 mmol/ x 1. The mean values of blood LA concentration at the consecutive work loads (running speed) in all rats exercising four times (N = 44) are presented in Fig. 1. As it can be seen, the course of changes in blood LA in relation to the speed of running was exponential rather than linear, with a distinct, rapid increase in blood LA at the speed of running above 25m/min. An analysis of individual curves enabled us to determine the anaerobic threshold (AT). The mean AT, expressed as the running speed at which LA started to accumulate in blood in all the rats from four series (N = 44), was 26.4 k 3.75 m/min, while the mean interpolated blood LA level at this speed was 4.12 f 1.36 mmol/l. There were no significant differences between the mean values of either the running speed or blood LA level at AT obtained in the four experimental series. The individual values of running speed and blood LA levels at AT are given in Table 2. The mean AT values obtained in the same rats in four experimental series showed relatively high reproducibility. It should be noted, however, that in five rats the variation coefficient of the speed at which AT was achieved exceeded 10% but in none it reached 20%. The values of blood LA at AT showed greater variation than those of speed in the repeated tests (the variation coefficients was between 11.2 and 43.5%.
blood lactate concentration
of individual
rats obtained
in four repetitions
Maximal blood lactate concentration (mmol/l)
speed
I
II
III
IV
Mean
SD
CV%*
I
II
III
IV
Mean
SD
CV%
I 2 3 4 5 6 7 8 9 10 I1
45 55 50 37 55 37 52 55 55 50 55
50 48 55 50 60 50 55 55 55 45 55
50 55 55 55 60 55 60 55 55 37 55
so 55 55 55 55 60 60 58 55 SO 55
48.75 53.25 53.75 49.25 57.50 50.50 56.75 55.75 55.00 45.50 55.00
2.50 3.50 2.50 8.50 2.87 9.88 3.95 1.50 0.00 6.14 0.00
5.13 6.57 4.65 17.26 4.99 19.56 6.96 2.69 0.00 13.49 0.00
8.37 16.89 20.40 21.46 12.86 22.50 12.79 10.24 15.03 12.71 20.38
IO.40 20.85 20.32 23.28 15.29 25.70 15.49 12.14 12.61 12.90 20.61
18.19 13.60 21.08 24.89 9.32 21.52 10.40 10.27 14.31 9.75 14.36
II.27 10.74 10.32 13.75 14.70 14.19 14.94 Il.05 10.83 9.73 13.70
12.06 15.52 18.03 20.85 13.04 20.98 13.41 10.93 13.20 11.27 17.26
4.27 4.35 5.15 4.93 2.69 4.86 2.32 0.89 I .87 I .77 3.74
35.41 28.03 28.56 23.65 20.63 23.16 17.30 8.14 14.17 15.71 21.67
mean SD CV%
49.64 7.00 14.10
52.5s 4.27 8.13
53.82 6.19 11.50
55.27 3.29 5.95
15.78 4.84 30.67
17.24 5.12 29.70
15.24 5.41 35.50
12.29 1.95 15.87
*CV% indicates
the coefficient of variation
in percentage
of the mean values.
Anaerobic threshold in rats
SPEED
OF RUNNING
287
lm . mid)
Fig. 1. The mean (k SD) values of blood LA concentrations in relation to the increased speed of running in rats.
The results of the fifth series of experiments showed that blood concentrations of HCO,- and BE decreased with the intensity of exercise, in opposition to blood LA level (Fig. 2). The inverse correlations were ascertained between blood LA and HCO,- concentrations (y = 13.53 -0.45x, r = - 0.73, N = 71, P < 0.001) as well as between blood LA and BE concentrations (y = 1.32 - 0.49x, r = - 0.75, N=71, P
Tabk
2. Running
speed and blood lactate concentration Running
Rat’s No. 1 2 3 4 5 6 7 8 9 10 11 mCan
DISCUSSION
Our study clearly demonstrated that in rats, running on the treadmill with increasing intensity, changes in blood lactate show a pattern similar to that described in human subjects. This enabled us to determine the level of exercise intensity at which blood lactate concentration starts to increase rapidly. This level, corresponding to the anaerobic threshold (AT), was achieved during running on the treadmill set at a 10” slope and the speed of approximately 25 m/min. Taking into account the reported data concerning maximal oxygen uptake (% YOz max) in rats (Bedford et al., 1979; Sonne and Galbo, 1980; Brooks and White, 1978; Glesson and Baldwin, 1981), it may be concluded that AT in rats corresponds to 5565% of VOz max. This is within the range of exercise intensities at which AT is achieved in human subjects. The mean value of LA measured after the maximal exercise as well as that at the anaerobic threshold, were in rats 15.14 f 4.75 and 4.12 f 1.36,
at the anaerobic tests
speed at AT (m/min)
threshold
of individual
rats obtained
in four multistage
exercise
Blood lactate concentration at AT (mmol/l)
I
II
III
IV
Mean
SD
CV%
I
II
III
IV
Mean
SD
CV%
29 25
25 25
25 29
:: 29 25 25 25 25 29 25
:: 33 29 29 29 2s 29 29
:: 29 29 21 29 29 29 29
25 33 21 21 25 33 25 29 25 29 29
26.00 28.00 20.00 22.00 29.00 29.00 25.00 28.00 26.00 29.00 28.00
2.00 3.83 3.83 2.00 3.27 3.27 3.26 2.00 2.00 0.00 2.00
7.7 14.0 19.1 9.1 11.3 11.3 13.1 7.1 7.7 0.0 7.1
3.2 3.0 5.0 3.1 4.0 6.8 4.4 2.5 2.2 6.8 2.0
2.6 4.3 4.0 7.2 5.6 6.8 4.2 3.9 2.4 4.2 4.4
3.1 4.1 4.0 4.6 3.0 5.3 1.6 3.9 4.1 4.2 4.0
2.6 5.2 5.8 4.6 2.8 6.4 2.4 3.9 4.0 3.6 4.0
3.04 4.30 4.70 4.88 3.85 6.33 3.15 3.55 3.18 4.70 3.60
0.48 0.94 0.87 1.70 1.28 0.71 1.37 0.70 0.77 I .43 I .08
15.8 21.9 18.5 34.9 33.2 11.2 43.5 19.7 24.2 30.4 30.0
25.70 2.41 9.38
26.80 4.14 15.45
26.09 4.42 16.94
26.80 4.14 15.45
3.91 I .69 43.22
4.51 1.50 33.26
4.19
4.12 1.29 31.31
1.26 30.07
288
WIESZAW
PlLIS
et al.
HCOj o-----Q
BE
-
LA
-26
--3
-20
s-6
% . 169
- -9 d
g
g
- 10
4!i WEED
OF RUNNING
4Q
53
r
- -12
67
(m emid 1
Fig. 2. Blood concentration of LA, HCO,- and BE during the increased speed of running in rats. Results are presented as mean k SD. thus being close to those found in men (Heck et al., 1985; Green et al., 1983). Four repetitions of the multistage exercise in the same rats showed fairly good reproducibility of AT, expressed as the speed of running. It should be noted that those AT levels were more reproducible than blood LA concentrations at a given speed (values of blood LA measured after maximal exercise and the values corresponding to AT). The concept of AT assumes that exercise of the intensity below the threshold can be performed for a long time without accumulation of lactate in blood, whilst during exercise exceeding the threshold blood LA concentration progressively increases with time of effort. This concept was tested in the present study on the animal model, using several bouts of intermittent exercise of identical intensity. Continuous exercise could not be applied in this case, respectively,
20 %
L
18 16lL-
iz g
12-
a
lo
SPEED OF RUNNING: 0
17
0 33m,
mid I T
1
P
P
2
3
I!l
OL
T
m -mid
1
NUMBER OF SEauENllAL
P
9
P
1
5
6
EXERCISE REPETITION
Fig. 3. Blood LA concentrations after six consecutive runs at the speed below (17 m/min) and above (33 m/min) AT. Results are presented as mean f SD.
since breaks were necessary for blood sampling. Despite this the data confirmed the assumption mentioned above indicating practically no change in blood LA level at the running speed of 17 m/min (below AT) and its progressive increase at the speed of 33 m/min. In human subjects in addition to blood lactate concentration changes in several physiological or biochemical variables e.g. pulmonary ventilation, CO, output, heart rate, blood bicarbonate (HCO,-), base excess (BE) and hydrogen ion concentrations are measured during incremental exercise test and used for determining the anaerobic threshold. All of them, except HR, are related to lactic acid accumulation in blood and show a close correlation with blood LA level (Beaver et al., 1986; Heck et al., 1985; Kumagai et al., 1982; Yoshida et al., 1981). To check whether such relationships can also be found in rats in one of the experimental series blood HCO,- and BE concentrations were followed together with blood LA during the multistage exercise. The data showed a similar pattern of changes in those variables to that in men. Moreover, close inverse correlations were ascertained between blood LA, and HC03- or BE levels (r = -0.73,P c 0.001 and r = -0.75, P c 0.001, respectively). In conclusion the present study demonstrated that in rats changes in capillary blood lactate concentration during a discontinuous multistage exercise of increasing intensity allow us to determine the anaerobic threshold, which is fairly reproducible. These results offer a potential application of AT determinations for serial evaluations of animal working ability under various experimental conditions, in the course of physical training etc. The animal model can be of value for further investiaations directed to
Anaerobic threshold in rats explain physiological
mechanisms
responsible
for AT
phenomenon.
289
function in progressive exercise. J. appl. Physiol. 62, 197>1981.
Kinderman W., Simon G. and Keul J. (1979) The significance of the aerobic-anaerobic threshold transition for the determination of work load intensities during endurance training. Eur. J. appl. Physiol. Occup. Physiol. 42,
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