27
EQUINE VETERINARY JOURNAL Vol. 7. No. 1. January 1975
The Effect of Exercise on Blood Metabolite LeveIs in the Horse MARION G. ANDERSON Department of Veterinary Pharmacology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow Present address: University of Glasgow, Wellcome Surgical Research Institute, Garscube Estate, Bearsden Road, Bearsden, Glasgow
MATERIALS AND METHODS THE variations in blood levels of glucose, pyruvate and lactate during muscular activity in man have been Animals extensively studied (Christensen and Hansen, 1939; A group of nine clinically normal mares and geldings Huckabee, 1958b; Johnson, Walton, Krebs and William- of both hunter and Thoroughbred types (aged 4 to son, 1969). High concentrations of blood lactate and 15 years) were used in this study. The animals were pyruvate have been observed following exercise, housed in loose-boxes and maintained on a diet conparticularly in untrained individuals. Changes in blood sisting of 16 kg hay, 0.5 kg oats, 1.5 kg bran, 2 kg pony glucose levels are less consistent. The effects of exercise cubes and 20 ml treacle per day. Daily maintenance on lipid metabolism in man are also well documented exercise was rigidly controlled and consisted of walking (Carlson and Pernow, 1959; Havel, Naimark and and trotting for 20-30 minutes over a distance of 3-5 km. Two experimental exercise programmes were used : Borchgrevink, I963 ; Johnson, et al., 1969). Increased mobilisation of free fatty acids (FFA) and glycerol Exercise A4 km trot 2 km canter 4 km gallop=lO km from adipose tissue has been observed during exercise. In contrast comparatively little is known of carbo- Exercise B4 km trot 6 km canter =10 km hydrate and lipid metabolism during exercise in other species although some studies have been made in the rat For comparative purposes, the paces trot, canter and (Gollnick, 1967) and in the dog (Issekutz, Miller, Paul gallop were characterised by the following speeds. and Rodahl, 1965; Issekutz and Paul, 1966). Few During trotting, the speed ranged from 3-5 mjsec studies have been made on changes in blood metab- depending on the horse. Similarly, during cantering olites, i.e. glucose, lactate, pyruvate, FFA, glycerol and galloping, speeds ranged from 5.2-6.7 mjsec and and ketone bodies in the horse-a species selectively 11.0-13.3 mjsec respectively again depending on the adapted for exercise. Of particular interest is the horse. In each case, however, the horse galloped at its relative importance of lipid and carbohydrate metab- maximum speed. Throughout the tests, all the horses olism during exercise in this species. Carlson, Froberg were ridden by the same person to avoid any effects due and Persson (1969, studied the concentration of glucose to different weights or styles of riding. Experimental exercises were carried out in a large and the concentration and turnover of FFA in the plasma of horses during trotting and cantering. Asheim, field once a week between 10.00 a.m. and 12.00 p.m. and, Knudsen, Lindholm, Rulcker and Saltin (1970) and on days when the animals were subjected to one of these Tagaki and Sakurai (1971) have measured blood lactate exercises, the daily maintenance exercise was omitted. Horses were “trained” by repeated exposure at weekly levels in the horse during exercise. In the present study, the effects of exercise of different intervals to exercise A. Alternatively, with one horse, intensities on blood concentrations of glucose, lactate, an experimental exercise consisting of 13 km trotting 9 km canter) was repeated pyruvate, lactate/pyruvate ratio, FFA and glycerol and cantering (4 km trot were studied together with the effects of training. In daily for 3 weeks as a training regime. To determine the effect of diet on blood metabolite addition the blood acid-base status of two horses was determined before and after exercise. In the past, levels two meals, one consisting of 2 kg pony cubes the relationship between blood lactate and pyruvate (composition-3 per cent oil, 10 per cent protein and levels has been used in man as an index of the oxygen 14 per cent fibre), 0.75 kg bran and 8 kg hay and the deficiency of tissues (Huckabee, 1958a and b). In the other consisting of 8 kg hay alone were compared. present study this relationship was used to distinguish Throughout the experiment the animals were kept in between aerobic and anaerobic metabolism during loose-boxes and were not exercised. The appropriate meal was provided at 9.30 a.m. and blood samples were exercise in the horse.
+ +
+
+
EQUINE VETERINARY JOURNAL
28
taken before feeding and then at 30-45 minute intervals for 3-4 hours. Preparation of blood for metabolite estimations Blood was prepared immediately on sampling since blood levels of metabolites alter very rapidly. 20 ml of blood withdrawn from the jugular vein was divided as follows : For glucose estimations, approximately I ml was added to a tube containing sodium fluoride and sodium citrate and mixed. For lactate estimations, 10 ml of blood were deproteinised immediately by pipetting into a 50 ml centrifuge tube containing 10 ml of 0.7M ice-cold perchloric acid, mixing the contents and centrifuging at 2,000g for 10 minutes. The supernatant fluid was then stored at -15°C until estimations could be made. For free fatty acid and glycerol determinations, the remainder of the blood was collected in a plain polystyrene tube and serum prepared. For blood pC0, and bicarbonate measurements, 2-3 ml of blood were collected in a heparinised syringe and the needle tip sealed with a rubber stopper to prevent loss of CO, to the atmosphere. The syringe was kept in ice until determinations were made (within 3-4 hours of sampling). Estimation of blood metabolites All measurements were made in duplicate and blanks and standards were included with each batch of samples. Blood lactate levels were estimated using the Biochemica Test Combination Cat. No. I5972 (Boehringer, 1969) which is based on the enzymatic method of Hohorst (1965). This method was found to be linear up to a lactate concentration of at least 160 mg/ml blood and the presence of a high concentration of pyruvate (16 mg per cent) did not interfere with the estimation. If the extinction change exceeded 1.5 units the sample was diluted with 0.5M glycine buffer, pH 9.0 and the assay repeated. Blood glucose levels were measured colorimetrically by the Nelson-Somogyi method (Nelson, 1944). Blood pyruvate levels were assayed using a method for the simultaneous enzymatic determination of acetoacetate and pyruvate (Bergmeyer, 1965). The method was found to be linear up to a pyruvate concentration of at least 4 mg per cent. If the extinction change exceeded 1 unit, however, the sample was diluted with O.IM phosphate buffer, pH 7.0-7.4 and the assay repeated. Serum glycerol levels were estimated using the Biochemica Test Combination Cat. No. 15989 (Boehringer, 1969) which is based on the enzymatic method of Eggstein and Kreutz (1966). This method was found
-~
~
GLUCOSE
T
PY RUVATE
---;--b 0.6
1
FFA
-
L
.
L
I.
1 GLYCEROL
10
m 0 ' 0 5 / ~ ~ -- - -1 ~ - -- . - - I~ ]
I
~
HOURS AFTER FEEDING - - Fig. 1. Effect of different diets on blood glucose, lactate, pyruvate, free fatty acid and glycerol concentrations. Each point on the graph represents the mean of at least two results f standard deviation. hay alone, __ haylnutslbran.
-----
to be linear up to a glycerol concentration of at least 10 mg/100 ml serum. Free fatty acids (FFA) in serum were estimated using the colorimetric method of Itaya and Ui (1965) with one modification-the last traces of cupric aqueous layer were removed by passing through Whatman phase-separating paper, thus avoiding contamination of the chloroform layer. The method was shown to be linear up to a FFA concentration of at least 3.0 mEq/l. When applying this method to horse
TABLE I NORMAL RANGE OF BLOOD METABOLITES IN THE RESTING, FASTING HORSE (9 ANIMALS) Metabolite
No. of Samples
Mean
S.D.
44 43 41 39 32 30
13 5.5 0.50 11.4 0.23 0.55
f 8.35 f 1.76 f 0.19 f 3.01 f 0.14 f 0.25
~
Glucose (mg %) Lactate (rng %) Pyruvate (mg %) Lactate/Pyruvate Ratio FFA (mEq/l) Glycerol (mg %)
* Hawk's Physiological Chemistry (1965). t Laudahn (1959). Landon, Fawcett and Wynn (1962).
Normal Values for Human
Range .
_
(54-86) (2.8--.I1 .O) (0.24-1.28) (4.7-17.8) (0.03-0.49) (0.25-1.43)
_
65-1 10.
9-16? 0.36-0.591 0.09-0.6§ 0.5-1.7T
5 Howorth, Gibbard and Marks (1966).
7 Eggstein (1966).
29
EQUINE VETERINARY JOURNAL
I
5t-
I
I
I
I
I PYRUVATE
1
was observed. Blood pyruvate, lactate, FFA and glycerol levels showed slight insignificant variations throughout the morning irrespective of which meal was provided. On the basis of these findings, all subsequent experiments were carried out on horses which had access only to hay. In the present studies, such animals are referred to as “fasting”. Normal levels of blood glucose, lactate, pyruvate, FFA and glycerol were measured in nine different horses on several different occasions. In each case samples were taken in the early morning before the animals were exercised or fed (as close as possible to basal conditions). Table 1 lists the findings together with data obtained from the literature for man. In general, the normal values are similar for both species.
Effect of exercise Blood glucose, lactate and pyruvate levels were monitored before, during and immediately after exercise in seven horses subjected to exercise A (each horse being tested two or three times). In all cases, blood LACTATE lactate and pyruvate levels rose significantly during PY RUVATE 60 * O L exercise, particularly during galloping, and began to fall immediately the exercise ceased (fig. 2). The lactate] pyruvate ratio also increased during exercise. Changes in blood glucose levels were less consistent with some 0 -- -horses showing an increase while others remained constant. In most of those animals which showed an 25 50 75 100 125 increase, a slight initial decrease in blood glucose was observed, followed by the increase. Fig. 3 shows the effect MINUTES AFTER EXERCISE BEGINS of exercise A on serum FFA and glycerol for five horses. Fig. 2. Eflect of exercise A on blood lactate, pyruvate and Glycerol levels increased during exercise and remained lactatelpyruvate ratio. Each point on the graph represents elevated for some time after the exercise had ceased. the mean of at least six results f standard deviation. _ _ _ - trotlcanter, _ -__ gallop. FFA levels, on the other hand, increased initially and then decreased slightly as the intensity of the exercise serum some interference arose from the presence in increased. When the exercise ceased, FFA levels rose horse serum of high concentrations of bilirubin and again. Since it appeared that the pace or intensity of the similar pigments which are extracted into the chloroform exercise was the major factor determining blood layer and which absorb at the same wavelengths as the metabolite levels, four horses were subjected to exercise copper-fatty acid salts. This was compensated for by A (trot/canter/gallop) and also to exercise B (trot/canter). making up a chloroform blank for such sera. Blood A fifth horse cantered for 4.2 km (6.7 mjsec) and, on a pH and pC0, measurements were made using the different day, covered the same distance at the gallop Blood Micro System Type BMS3 and Digital Acid-Base (1 1.8 mjsec). Table 11 compares the effect on the various metabolite levels of these two paces. In each case, Analyser PHM72.’ blood lactate, pyruvate and lactate/pyruvate ratio increased dramatically during the gallop while, during RESULTS cantering, there was little change in these parameters. Egect of diet A severe metabolic acidosis, indicated by a large negative Prior to any exercise studies, the effect of diet on Base Excess and pH change (horses No. 2 and No. 4) blood metabolite levels was investigated to determine also developed during galloping. Body lipids were whether feeding the animal before exercise would mobilised at both paces, indicated by increased blood interfere with or mask the metabolic effect of the exercise. glycerol levels, but in the absence of isotopic lahelling The effect of a meal containing 2 kg pony cubes, studies the degree of mobilisation and utilisation cannot 0.75 kg bran and 8 kg hay and one consisting of 8 kg be estimated. hay alone were compared. At the beginning of each To investigate how quickly changes in blood lactate experiment the animals were in a fasting condition, the etc. occurred during galloping, two horses were subjected last meal having been taken the previous evening (1 7- to gallops of increasing length, i.e. 200, 400, 800 and 18 hours before). Blood samples were taken before 1,600 m at two or three day intervals. Blood lactates feeding and then at 30-45 minute intervals for three to and pyruvates were measured before and immediately four hours. Glucose, lactate, pyruvate, FFA and after each exercise and the results are shown in Table 111. glycerol estimations were carried out on blood from It can be seen that, for both animals, even after a 200 m three horses (fig. 1). Blood glucose levels remained gallop, blood lactate levels were very significantly fairly constant when the animal had access to hay alone elevated as were the lactate/pyruvate ratios. Blood but, when the complete meal was provided, a glucose peak lactate levels reached a plateau after 400-800 m at which stage lactate was presumably being removed from the circulation as quickly as it was formed. 1. Radiometer Ltd., Copenhagen.
w : ,; T
1
30
E,QUINE VETERINARY JOURNAL
Eflect of training When the horses were exposed repeatedly (at weekly intervals) to exercise A and blood metabolite levels measured before, during and after exercise on each occasion, no significant effect was observed on the changes in these metabolites resulting from such exercise, i.e. no training effect was seen. An alternative form of exercise, namely daily trotting and cantering for 13 km over a three week period, was performed by one horse. Blood samples for metabolite assays were taken before and immediately after exercise on the Ist, 8th and 16th day of the programme. In this case, the size of the increases in lactate, pyruvate and lactate/ pyruvate ratio decreased over the three week period. The animal was, therefore, showing a training effect when subjected to exercise daily rather than once weekly., Further tests on the effect of training, carried out on more animals, are necessary.
qualitative conclusions can be drawn regarding the metabolism of fats and carbohydrate in the horse during exercise. The influence of diet in the horse cannot readily be measured since some of the dietary carbohydrate is converted to volatile fatty acids in the caecum of this species. These short chain fatty acids are readily absorbed and converted to long-chain fatty acids. Whether this results in an increase in the importance of free fatty acids as an energy source for muscle in the horse, in analogy with the dietary influence in man, is not known. The influence of diet in blood metabolites is reported here. A peak in blood glucose levels is seen about two hours after the provision of concentrates plus hay but not after hay alone (fig. 1). This peak is probably due to the presence i n concentrates of easily absorbable mono- or disaccharides. The effect of exercise on blood glucose levels in the horse is variable with some animals showing an increase and some a decrease (Table 11). The blood glucose DISCUSSION There are several possible energy sources readily level itself does not provide much information on available for use by muscles. These include glucose, carbohydrate metabolism during exercise since this free fatty acids, glycerol, amino acids and lactate. In level reflects both the uptake of glucose by working tissues the absence of severe malnutrition, amino acids can be and its replenishment by hepatic glycogenolysis and discounted as a likely fuel during short term exercise, depends, therefore, on the balance between these two while lactate can only be used as a fuel in tissues such as processes. In this respect, it is interesting to note that, cardiac muscle which have an adequate supply of during studies in man, Johnson, et al. (1969) observed increases in blood glucose levels during exercise in oxygen. It is now known that, in the human at least, the trained athletes while untrained individuals showed a relative importance of lipids and carbohydrate in muscle slight decrease. Also, in a recent publication, Lindholm metabolism can be influenced to some extent by the and Saltin (1974) reported significant increases in blood diet. Christensen and Hansen (1939) showed that glucose in highly trained horses subjected to a 2100 m subjects living on a high-fat diet were exhausted during race. The product of glucose metabolism in working muscle moderate cycling exercises more quickly and had a lower respiratory quotient than subjects living on a is pyruvate which, in the presence of oxygen, is comnormal or high-carbohydrate diet. It was also shown pletely oxidised to carbon dioxide and water via the that the closer the subject worked to his maximum tricarboxylic acid cycle. In the absence of sufficient capacity, the higher the percentage of energy supplied oxygen, however, excess lactate is formed from pyruvate and the lactate/pyruvate ratio increases. In the present by carbohydrate. From the results obtained in the present studies, some studies, blood lactate and pyruvate levels increased during TABLE 11 EFFECT O F EXERCISE 1NTENSITY ON BLOOD METABOLITE LEVELS IN THE HORSE
I
Increase in Horse No.
Lactate me%
1
Canter Gallop
Pyruvate
Lactate/ Pyruvate
0.03 4.00
3
Canter Gallop
4
Canter Gallop -~
5
3.5 107.7
Canter Gallop
I 1
5.1 61.9
12;:; 4.5 146.4
0.06 1.20 0.48 2.36 0.31 2.07
-
19.7 17.8
Glucose
-
+I5 - t 33
t-0.41 -t0.03
-9 -4
0.49 -0.14
Glycerol
mg
mg % i 0.54
+0.81
~~
0.27 4.53
I 1
~-
Base Excess mEd1
~~
~-
Canter Gallop
Blood pH
mg%
____
9.8 136.5
~
2
Change in
-~
Pace
2.6 11.5
I I
8.2 25.8 0.9 35.8
i 0.005 -0. I35
I I
-
_ -
-
+
10.25 -1- 0.03
i0.009 -0.106
_
1.8 49.0
-3.9 -9.1
-t0.05 ~
+
0.09 -0.03
-t 1.99 11.52 j
0.96 -
+ 1.01
+ 1.23 $0.21 t 0.90
EQUINE VETERINARY JOURNAL
31
exercise (fig. 2 and Table It) indicating increased utilisation of glucose by the working muscle. The relative dependence on anaerobic metabolism during the gallop is indicated by the greatly increased lactate/pyruvate ratio. Neither lactate, pyruvate nor lactate/pyruvate ratio changed to the same degree at the canter. When the pace is restricted to a canter, therefore, the cardiovascular system can presumably carry enough oxygen to the working tissues to meet the demand. In a recent study, Lindholm and Saltin (1974) also found that blood and muscle lactate levels only increased dramatically when horses were running at or near their maximal speeds ( 1 1.4-12.5 misec). TABLE Lu EFFECT OF DISTANCE ON LACTATE PRODUCTION DURING GALLOPING
I
Horse 6
I
~
~
(m)
Lactate (mg%)
Lip
0 200
3.7 79.8 116.7 119.5 145.2
Distance
-
_
~ _ _ .
400 800 1600
11.6 55.4 109.1 123.2 115.2
It has been suggested that part of the 0, debt incurred during exercise results from the circulatory lag at the beginning of exercise (Knuttgen, 1962). During this time excess lactate is not produced, indicating either that work during this time is performed aerobically using O L stored in myoglobin and haemoglobin, or alternatively, that available muscle stores of highenergy compounds such as phosphocreatine are being used to regenerate ATP anaerobically. Astrand, Astrand, Christensen and Hedman (1960) and Christensen, Hedman and Saltin (1960) have shown that even very high work loads can be carried out for long periods without the production of excess lactate if the work is performed in periods of
EQUINE VETERINARY JOURNAL Vol. 7. No. 1. January 1975
The Effect of Exercise on Blood Metabolite LeveIs in the Horse MARION G. ANDERSON Department of Veterinary Pharmacology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow Present address: University of Glasgow, Wellcome Surgical Research Institute, Garscube Estate, Bearsden Road, Bearsden, Glasgow
MATERIALS AND METHODS THE variations in blood levels of glucose, pyruvate and lactate during muscular activity in man have been Animals extensively studied (Christensen and Hansen, 1939; A group of nine clinically normal mares and geldings Huckabee, 1958b; Johnson, Walton, Krebs and William- of both hunter and Thoroughbred types (aged 4 to son, 1969). High concentrations of blood lactate and 15 years) were used in this study. The animals were pyruvate have been observed following exercise, housed in loose-boxes and maintained on a diet conparticularly in untrained individuals. Changes in blood sisting of 16 kg hay, 0.5 kg oats, 1.5 kg bran, 2 kg pony glucose levels are less consistent. The effects of exercise cubes and 20 ml treacle per day. Daily maintenance on lipid metabolism in man are also well documented exercise was rigidly controlled and consisted of walking (Carlson and Pernow, 1959; Havel, Naimark and and trotting for 20-30 minutes over a distance of 3-5 km. Two experimental exercise programmes were used : Borchgrevink, I963 ; Johnson, et al., 1969). Increased mobilisation of free fatty acids (FFA) and glycerol Exercise A4 km trot 2 km canter 4 km gallop=lO km from adipose tissue has been observed during exercise. In contrast comparatively little is known of carbo- Exercise B4 km trot 6 km canter =10 km hydrate and lipid metabolism during exercise in other species although some studies have been made in the rat For comparative purposes, the paces trot, canter and (Gollnick, 1967) and in the dog (Issekutz, Miller, Paul gallop were characterised by the following speeds. and Rodahl, 1965; Issekutz and Paul, 1966). Few During trotting, the speed ranged from 3-5 mjsec studies have been made on changes in blood metab- depending on the horse. Similarly, during cantering olites, i.e. glucose, lactate, pyruvate, FFA, glycerol and galloping, speeds ranged from 5.2-6.7 mjsec and and ketone bodies in the horse-a species selectively 11.0-13.3 mjsec respectively again depending on the adapted for exercise. Of particular interest is the horse. In each case, however, the horse galloped at its relative importance of lipid and carbohydrate metab- maximum speed. Throughout the tests, all the horses olism during exercise in this species. Carlson, Froberg were ridden by the same person to avoid any effects due and Persson (1969, studied the concentration of glucose to different weights or styles of riding. Experimental exercises were carried out in a large and the concentration and turnover of FFA in the plasma of horses during trotting and cantering. Asheim, field once a week between 10.00 a.m. and 12.00 p.m. and, Knudsen, Lindholm, Rulcker and Saltin (1970) and on days when the animals were subjected to one of these Tagaki and Sakurai (1971) have measured blood lactate exercises, the daily maintenance exercise was omitted. Horses were “trained” by repeated exposure at weekly levels in the horse during exercise. In the present study, the effects of exercise of different intervals to exercise A. Alternatively, with one horse, intensities on blood concentrations of glucose, lactate, an experimental exercise consisting of 13 km trotting 9 km canter) was repeated pyruvate, lactate/pyruvate ratio, FFA and glycerol and cantering (4 km trot were studied together with the effects of training. In daily for 3 weeks as a training regime. To determine the effect of diet on blood metabolite addition the blood acid-base status of two horses was determined before and after exercise. In the past, levels two meals, one consisting of 2 kg pony cubes the relationship between blood lactate and pyruvate (composition-3 per cent oil, 10 per cent protein and levels has been used in man as an index of the oxygen 14 per cent fibre), 0.75 kg bran and 8 kg hay and the deficiency of tissues (Huckabee, 1958a and b). In the other consisting of 8 kg hay alone were compared. present study this relationship was used to distinguish Throughout the experiment the animals were kept in between aerobic and anaerobic metabolism during loose-boxes and were not exercised. The appropriate meal was provided at 9.30 a.m. and blood samples were exercise in the horse.
+ +
+
+
EQUINE VETERINARY JOURNAL
28
taken before feeding and then at 30-45 minute intervals for 3-4 hours. Preparation of blood for metabolite estimations Blood was prepared immediately on sampling since blood levels of metabolites alter very rapidly. 20 ml of blood withdrawn from the jugular vein was divided as follows : For glucose estimations, approximately I ml was added to a tube containing sodium fluoride and sodium citrate and mixed. For lactate estimations, 10 ml of blood were deproteinised immediately by pipetting into a 50 ml centrifuge tube containing 10 ml of 0.7M ice-cold perchloric acid, mixing the contents and centrifuging at 2,000g for 10 minutes. The supernatant fluid was then stored at -15°C until estimations could be made. For free fatty acid and glycerol determinations, the remainder of the blood was collected in a plain polystyrene tube and serum prepared. For blood pC0, and bicarbonate measurements, 2-3 ml of blood were collected in a heparinised syringe and the needle tip sealed with a rubber stopper to prevent loss of CO, to the atmosphere. The syringe was kept in ice until determinations were made (within 3-4 hours of sampling). Estimation of blood metabolites All measurements were made in duplicate and blanks and standards were included with each batch of samples. Blood lactate levels were estimated using the Biochemica Test Combination Cat. No. I5972 (Boehringer, 1969) which is based on the enzymatic method of Hohorst (1965). This method was found to be linear up to a lactate concentration of at least 160 mg/ml blood and the presence of a high concentration of pyruvate (16 mg per cent) did not interfere with the estimation. If the extinction change exceeded 1.5 units the sample was diluted with 0.5M glycine buffer, pH 9.0 and the assay repeated. Blood glucose levels were measured colorimetrically by the Nelson-Somogyi method (Nelson, 1944). Blood pyruvate levels were assayed using a method for the simultaneous enzymatic determination of acetoacetate and pyruvate (Bergmeyer, 1965). The method was found to be linear up to a pyruvate concentration of at least 4 mg per cent. If the extinction change exceeded 1 unit, however, the sample was diluted with O.IM phosphate buffer, pH 7.0-7.4 and the assay repeated. Serum glycerol levels were estimated using the Biochemica Test Combination Cat. No. 15989 (Boehringer, 1969) which is based on the enzymatic method of Eggstein and Kreutz (1966). This method was found
-~
~
GLUCOSE
T
PY RUVATE
---;--b 0.6
1
FFA
-
L
.
L
I.
1 GLYCEROL
10
m 0 ' 0 5 / ~ ~ -- - -1 ~ - -- . - - I~ ]
I
~
HOURS AFTER FEEDING - - Fig. 1. Effect of different diets on blood glucose, lactate, pyruvate, free fatty acid and glycerol concentrations. Each point on the graph represents the mean of at least two results f standard deviation. hay alone, __ haylnutslbran.
-----
to be linear up to a glycerol concentration of at least 10 mg/100 ml serum. Free fatty acids (FFA) in serum were estimated using the colorimetric method of Itaya and Ui (1965) with one modification-the last traces of cupric aqueous layer were removed by passing through Whatman phase-separating paper, thus avoiding contamination of the chloroform layer. The method was shown to be linear up to a FFA concentration of at least 3.0 mEq/l. When applying this method to horse
TABLE I NORMAL RANGE OF BLOOD METABOLITES IN THE RESTING, FASTING HORSE (9 ANIMALS) Metabolite
No. of Samples
Mean
S.D.
44 43 41 39 32 30
13 5.5 0.50 11.4 0.23 0.55
f 8.35 f 1.76 f 0.19 f 3.01 f 0.14 f 0.25
~
Glucose (mg %) Lactate (rng %) Pyruvate (mg %) Lactate/Pyruvate Ratio FFA (mEq/l) Glycerol (mg %)
* Hawk's Physiological Chemistry (1965). t Laudahn (1959). Landon, Fawcett and Wynn (1962).
Normal Values for Human
Range .
_
(54-86) (2.8--.I1 .O) (0.24-1.28) (4.7-17.8) (0.03-0.49) (0.25-1.43)
_
65-1 10.
9-16? 0.36-0.591 0.09-0.6§ 0.5-1.7T
5 Howorth, Gibbard and Marks (1966).
7 Eggstein (1966).
29
EQUINE VETERINARY JOURNAL
I
5t-
I
I
I
I
I PYRUVATE
1
was observed. Blood pyruvate, lactate, FFA and glycerol levels showed slight insignificant variations throughout the morning irrespective of which meal was provided. On the basis of these findings, all subsequent experiments were carried out on horses which had access only to hay. In the present studies, such animals are referred to as “fasting”. Normal levels of blood glucose, lactate, pyruvate, FFA and glycerol were measured in nine different horses on several different occasions. In each case samples were taken in the early morning before the animals were exercised or fed (as close as possible to basal conditions). Table 1 lists the findings together with data obtained from the literature for man. In general, the normal values are similar for both species.
Effect of exercise Blood glucose, lactate and pyruvate levels were monitored before, during and immediately after exercise in seven horses subjected to exercise A (each horse being tested two or three times). In all cases, blood LACTATE lactate and pyruvate levels rose significantly during PY RUVATE 60 * O L exercise, particularly during galloping, and began to fall immediately the exercise ceased (fig. 2). The lactate] pyruvate ratio also increased during exercise. Changes in blood glucose levels were less consistent with some 0 -- -horses showing an increase while others remained constant. In most of those animals which showed an 25 50 75 100 125 increase, a slight initial decrease in blood glucose was observed, followed by the increase. Fig. 3 shows the effect MINUTES AFTER EXERCISE BEGINS of exercise A on serum FFA and glycerol for five horses. Fig. 2. Eflect of exercise A on blood lactate, pyruvate and Glycerol levels increased during exercise and remained lactatelpyruvate ratio. Each point on the graph represents elevated for some time after the exercise had ceased. the mean of at least six results f standard deviation. _ _ _ - trotlcanter, _ -__ gallop. FFA levels, on the other hand, increased initially and then decreased slightly as the intensity of the exercise serum some interference arose from the presence in increased. When the exercise ceased, FFA levels rose horse serum of high concentrations of bilirubin and again. Since it appeared that the pace or intensity of the similar pigments which are extracted into the chloroform exercise was the major factor determining blood layer and which absorb at the same wavelengths as the metabolite levels, four horses were subjected to exercise copper-fatty acid salts. This was compensated for by A (trot/canter/gallop) and also to exercise B (trot/canter). making up a chloroform blank for such sera. Blood A fifth horse cantered for 4.2 km (6.7 mjsec) and, on a pH and pC0, measurements were made using the different day, covered the same distance at the gallop Blood Micro System Type BMS3 and Digital Acid-Base (1 1.8 mjsec). Table 11 compares the effect on the various metabolite levels of these two paces. In each case, Analyser PHM72.’ blood lactate, pyruvate and lactate/pyruvate ratio increased dramatically during the gallop while, during RESULTS cantering, there was little change in these parameters. Egect of diet A severe metabolic acidosis, indicated by a large negative Prior to any exercise studies, the effect of diet on Base Excess and pH change (horses No. 2 and No. 4) blood metabolite levels was investigated to determine also developed during galloping. Body lipids were whether feeding the animal before exercise would mobilised at both paces, indicated by increased blood interfere with or mask the metabolic effect of the exercise. glycerol levels, but in the absence of isotopic lahelling The effect of a meal containing 2 kg pony cubes, studies the degree of mobilisation and utilisation cannot 0.75 kg bran and 8 kg hay and one consisting of 8 kg be estimated. hay alone were compared. At the beginning of each To investigate how quickly changes in blood lactate experiment the animals were in a fasting condition, the etc. occurred during galloping, two horses were subjected last meal having been taken the previous evening (1 7- to gallops of increasing length, i.e. 200, 400, 800 and 18 hours before). Blood samples were taken before 1,600 m at two or three day intervals. Blood lactates feeding and then at 30-45 minute intervals for three to and pyruvates were measured before and immediately four hours. Glucose, lactate, pyruvate, FFA and after each exercise and the results are shown in Table 111. glycerol estimations were carried out on blood from It can be seen that, for both animals, even after a 200 m three horses (fig. 1). Blood glucose levels remained gallop, blood lactate levels were very significantly fairly constant when the animal had access to hay alone elevated as were the lactate/pyruvate ratios. Blood but, when the complete meal was provided, a glucose peak lactate levels reached a plateau after 400-800 m at which stage lactate was presumably being removed from the circulation as quickly as it was formed. 1. Radiometer Ltd., Copenhagen.
w : ,; T
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E,QUINE VETERINARY JOURNAL
Eflect of training When the horses were exposed repeatedly (at weekly intervals) to exercise A and blood metabolite levels measured before, during and after exercise on each occasion, no significant effect was observed on the changes in these metabolites resulting from such exercise, i.e. no training effect was seen. An alternative form of exercise, namely daily trotting and cantering for 13 km over a three week period, was performed by one horse. Blood samples for metabolite assays were taken before and immediately after exercise on the Ist, 8th and 16th day of the programme. In this case, the size of the increases in lactate, pyruvate and lactate/ pyruvate ratio decreased over the three week period. The animal was, therefore, showing a training effect when subjected to exercise daily rather than once weekly., Further tests on the effect of training, carried out on more animals, are necessary.
qualitative conclusions can be drawn regarding the metabolism of fats and carbohydrate in the horse during exercise. The influence of diet in the horse cannot readily be measured since some of the dietary carbohydrate is converted to volatile fatty acids in the caecum of this species. These short chain fatty acids are readily absorbed and converted to long-chain fatty acids. Whether this results in an increase in the importance of free fatty acids as an energy source for muscle in the horse, in analogy with the dietary influence in man, is not known. The influence of diet in blood metabolites is reported here. A peak in blood glucose levels is seen about two hours after the provision of concentrates plus hay but not after hay alone (fig. 1). This peak is probably due to the presence i n concentrates of easily absorbable mono- or disaccharides. The effect of exercise on blood glucose levels in the horse is variable with some animals showing an increase and some a decrease (Table 11). The blood glucose DISCUSSION There are several possible energy sources readily level itself does not provide much information on available for use by muscles. These include glucose, carbohydrate metabolism during exercise since this free fatty acids, glycerol, amino acids and lactate. In level reflects both the uptake of glucose by working tissues the absence of severe malnutrition, amino acids can be and its replenishment by hepatic glycogenolysis and discounted as a likely fuel during short term exercise, depends, therefore, on the balance between these two while lactate can only be used as a fuel in tissues such as processes. In this respect, it is interesting to note that, cardiac muscle which have an adequate supply of during studies in man, Johnson, et al. (1969) observed increases in blood glucose levels during exercise in oxygen. It is now known that, in the human at least, the trained athletes while untrained individuals showed a relative importance of lipids and carbohydrate in muscle slight decrease. Also, in a recent publication, Lindholm metabolism can be influenced to some extent by the and Saltin (1974) reported significant increases in blood diet. Christensen and Hansen (1939) showed that glucose in highly trained horses subjected to a 2100 m subjects living on a high-fat diet were exhausted during race. The product of glucose metabolism in working muscle moderate cycling exercises more quickly and had a lower respiratory quotient than subjects living on a is pyruvate which, in the presence of oxygen, is comnormal or high-carbohydrate diet. It was also shown pletely oxidised to carbon dioxide and water via the that the closer the subject worked to his maximum tricarboxylic acid cycle. In the absence of sufficient capacity, the higher the percentage of energy supplied oxygen, however, excess lactate is formed from pyruvate and the lactate/pyruvate ratio increases. In the present by carbohydrate. From the results obtained in the present studies, some studies, blood lactate and pyruvate levels increased during TABLE 11 EFFECT O F EXERCISE 1NTENSITY ON BLOOD METABOLITE LEVELS IN THE HORSE
I
Increase in Horse No.
Lactate me%
1
Canter Gallop
Pyruvate
Lactate/ Pyruvate
0.03 4.00
3
Canter Gallop
4
Canter Gallop -~
5
3.5 107.7
Canter Gallop
I 1
5.1 61.9
12;:; 4.5 146.4
0.06 1.20 0.48 2.36 0.31 2.07
-
19.7 17.8
Glucose
-
+I5 - t 33
t-0.41 -t0.03
-9 -4
0.49 -0.14
Glycerol
mg
mg % i 0.54
+0.81
~~
0.27 4.53
I 1
~-
Base Excess mEd1
~~
~-
Canter Gallop
Blood pH
mg%
____
9.8 136.5
~
2
Change in
-~
Pace
2.6 11.5
I I
8.2 25.8 0.9 35.8
i 0.005 -0. I35
I I
-
_ -
-
+
10.25 -1- 0.03
i0.009 -0.106
_
1.8 49.0
-3.9 -9.1
-t0.05 ~
+
0.09 -0.03
-t 1.99 11.52 j
0.96 -
+ 1.01
+ 1.23 $0.21 t 0.90
EQUINE VETERINARY JOURNAL
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exercise (fig. 2 and Table It) indicating increased utilisation of glucose by the working muscle. The relative dependence on anaerobic metabolism during the gallop is indicated by the greatly increased lactate/pyruvate ratio. Neither lactate, pyruvate nor lactate/pyruvate ratio changed to the same degree at the canter. When the pace is restricted to a canter, therefore, the cardiovascular system can presumably carry enough oxygen to the working tissues to meet the demand. In a recent study, Lindholm and Saltin (1974) also found that blood and muscle lactate levels only increased dramatically when horses were running at or near their maximal speeds ( 1 1.4-12.5 misec). TABLE Lu EFFECT OF DISTANCE ON LACTATE PRODUCTION DURING GALLOPING
I
Horse 6
I
~
~
(m)
Lactate (mg%)
Lip
0 200
3.7 79.8 116.7 119.5 145.2
Distance
-
_
~ _ _ .
400 800 1600
11.6 55.4 109.1 123.2 115.2
It has been suggested that part of the 0, debt incurred during exercise results from the circulatory lag at the beginning of exercise (Knuttgen, 1962). During this time excess lactate is not produced, indicating either that work during this time is performed aerobically using O L stored in myoglobin and haemoglobin, or alternatively, that available muscle stores of highenergy compounds such as phosphocreatine are being used to regenerate ATP anaerobically. Astrand, Astrand, Christensen and Hedman (1960) and Christensen, Hedman and Saltin (1960) have shown that even very high work loads can be carried out for long periods without the production of excess lactate if the work is performed in periods of
