Refereed

THE EFFECT OF EXERCISE AND CONDITIONING ON EQUINE RED BLOOD CELL CHARACTERISTICS

Carolyn L. Stull, PhD1 and Laurie M. Lawrence, PhD2

SUMMARY

The effect of exercise and conditioning on 2,3- diphosphoglycerate levels was studied in nine mature horses. During a 12 minute exercise bout producing heart rates of 165 bpm, 2,3-DPG was significantly increased (p<.05). In addition, exercising levels of 2,3-DPG were increased (19<.05) approximately 8% after a six-week submaximal conditioning program. These increases could not be entirely attributed to changes in erythrocyte number. Mean corpuscular volume was also increased during exercise (p<.05) but was not altered by conditioning.

INTRODUCTION

The glycolytic intermediate, 2,3-diphosphoglycerate (2,3- DPG), is present in high concentrations only in red blood cells (RBC). The 2,3-DPG molecule has been shown to combine reversibly with deoxyhemoglobin, thus shifting the oxygen dissociation curve to the right and promoting the unloading of oxygen at higher tissue oxygen tensions1. As a result, 2,3-DPG content may influence the capacity of an individual for aerobic exercise. In humans, 2,3-DPG levels have been shown to increase with conditioningL The effect of conditioning on 2,3-DPG levels in the horse has not been well studied. Several researchers observed a decrease in levels in trained horses at rest 3,4, or post exercise 4, but there are no reports on exercising values.

IAuthor's address: Department of Animal Science, University of California-Davis, Davts, CA 95616. 2Departrnent of Animal Science, university of Illinois, Urban=, IL 61801

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An increase in 2,3-DPG levels would be expected when RBC are released from the spleen of the horse during an exercise bout. Some researchers5 have reported an increase in packed cell volume (PCV), hemoglobin (Hb) and RBC number with conditioning, thus a concomitant increase in 2,3-DPG could also be expected. This study was conducted tO determine whether venous 2,3-DPG levels increase with exercise and conditioning in the horse, and whether any changes in 2,3-DPG are proportional to changes in Hb or RBC number.

MATERIALS AND METHODS

Nine mature Quarter Horse mares were divided into 2 groups for use in a cross-over design. All mares were accustomed to working on a motorized treadmill, but had no regular exercise for at least 8 weeks prior to the study. The mares in the conditioned group were exercised 4 times per week for 6 weeks on the treadmill, which was located indoors. Environmental temperature was kept constant between 15 to 19°C and humidity varied between 40 to 76 percent. Each exercise bout consisted of a 5 minute warm- up and a 5 minute cool-out session at the walk, with a trot being maintained between walking sessions. The trotting period progressed up to 24 minutes in duration by the 5th week. The trotting speed was 3.6 m/sec, while the speed of the warm-up and cool-out walk was 1.4 rn/sec and 1.0 m/sec, respectively. The treadmill was set at a 9% grade t o insure that heart rates (HR) at the trot were in the range of 150 to 180 beats per minute (bpm). The horses in the nonconditioned group were worked on the treadmill 2 times per week for 2 minutes at the walk and 1 minute at the trot.

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TABLE 1

The Effect of the SET on Heart Rate (bpm) In Conditioned and Nonconditioned Horses (mean; n=g)

Activity

Conditioned

Nonconditioned

*significant difference (p<.05) between groups within an activity. +significant differeace (p<.05) with ~ t .

tsignificant difference (p<.05) between trot and recovery.

Rest Trot Recovery S.E.

39.4* 167.2+ 81.7+t 4.2

55.2 164.1+ 85.7*t

Heart rate was monitored by a hardwire electrocardiogram at rest while standing on the treadmill and during each minute of exercise, once weekly in both groups.

A submaximal standardized exercise test (SET) was given to all mares at the conclusion of the conditioning program. The SET consisted of 2 minutes of rest, 2 minutes of walking, followed by 9 minutes of trotting and 9 minutes of standing recovery. This protocol subjectively provided a strenuous test, but was within the capabilities of the unfit horse to finish the entire trotting phase. Heart rates were monitored every minute during rest, exercise and recovery. Resting and recovery heart rates were recorded while the horse stood quietly on the treadmill. Indwelling catheters were inserted into the left jugular vein at least 15 minutes before the SET was started. Blood samples were drawn during the second minute of rest and walk, and the 1st, 3rd, 5th, 7th and 9th minute of both trotting and recovery sessions. The parameters measured included 2,3-DPG levels, RBC number, lactic acid, Hb and PCV.

At the conclusion of the SET, all mares were maintained in a drylot for 8 weeks with no forced exercise. The treatments were then reversed and the procedure repeated. There were no differences due to period, so the data from both parts of the cross-over were combined for statistical analysis.

Analysis for 2,3-DPG was performed withing 24 hours on the supernatant obtained from whole blood which had been deproteinized by immediate acidification and subsequently centrifuged. A commercially available assay a for 2,3-DPG which colorimetrically measured the phosphorous resulting from enzymatic degradation of 2,3- DPG was used. Red blood cell number was determined using a Coulter Counted,. Hemoglobin determinations were made using the cyanomethemoglobin technique. The quantification of venous lactic acid was peformed by a spectrophotometric enzyme assay kit~. Values for mean

aSigma Chemical Co. Kit No. 665, SL Louis, MO 63178 bCoulter Diagnostic Co., Model ZBI, Miami Springs, FL 33166 CSigma Chemical Co. Kit No. 665, St. Louis, MO 63178 Volume 6, Number 4

cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC) were calculated4.

A split-split-plot analysis of variance was performed on the data to obtain appropriate estimates of experimental error. An F protected least significant difference was computed to compare means between the conditioned and control groups within an activity and also to compare means between activities within a group.

RESULTS

Lactic acid levels and heart rates (HR) were monitored in order to evaluate the intensity of the exercise bout and the effectiveness of the conditioning program. Heart rates averaged 165 bpm during the exercise bout, which is 69% of an estimated HRma ~ of 240 bpm. Although conditioning did not affect exercising HR, the conditioned horses had a lower resting heart rate than the nonconditioned horses (Table 1). The effectiveness of the conditioning program

3 5 . 0

3 0 . (

~ 25 . (

~20.o .'g

~ 15.o.

10.0

5.{

Coc¢l i t ioned Group . . . . . . . . Non Conditioned G r o u p

/

/ "--,...,, ! /

/

I. I I I I I I I I ! I I I I I I I I I I I I I

2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 I I I I

Rest W a l k Tro t Recove ry

11me (minutes)

Figure 1. The effect of SET on venous lactic acid levels in exercising horses.

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TABLE 2

The Effect of the SET on PCV, Hb and RBC Number In Conditioned and Nonconditloned Horses (mean; n=9)

RBC (IOI2/L) Conditioned

Nonconditioned Hb (g/100ml)

Conditioned

Nonconditioned PCV (%)

Conditioned

Nonconditioned

*significant difference (p<.05) between groups within an activity. +significant difference (p<.05) with resL Isignificant difference (p<.05) between trot and recovery.

Activity

Rest Trot Recovery S.E

9.23 10.22 + 10.12 + .27

9.50 10.38 + 10.22 +

12.22" 16.52+ 15.51+1 .32

13.53 16.79 + 15.74+t

36.22 48.32 + 45.69 + 1.61

36.11 49.20 + 46.33 +

was further evidenced by the serum lactic acid concenu'ations (Figure 1). In the nonconditioned state, lactic acid increased 244% during the SET. However, after conditioning, the lactic acid increased only 79%. The SET produced elevations in PCV, Hb and RBC numbers (p<.05) in both groups (Table 2).

The levels of 2,3-DPG were also increased by exercise (p<.05) and were higher (p<.05) in the conditioned state than in the nonconditioned state, during the trot (Figure 2). When 2,3-DPG levels were related to RBC number (DPG- RBC), these effects remained (Table 3). When 2,3-DPG levels were related to Hb levels (DPG-Hb) the effect of conditioning was observed at rest as well as during the trot

but the effect of the exercise bout was no longer observed (Table 3). The response of the RBC indices to exercise and conditioning is shown in Table 4. The MCV and MCH were greater at the trot than during rest, but there was no effect (p>.05) due to conditioning at the trot for either index.

DISCUSSION

Lactic acid levels in venous blood are traditionally indicative of conditioning effects of submaximal exercise in both humans and horses7,S, 9. The effectiveness of this

TABLE 3

The Effect of the SET on 2,3-DPG Levels as Related to Hb and RBC Number in Conditioned and Nonconditioned Horses (mean; n=9)

Activity

DPG-Hb (gM DPG/g Hb) Conditioned

Nonconditioned DPG-RBC (raM DPG/1012RBC)

Conditioned

Nonconditioned *significant difference (p<.05) between groups within an activity. +signif'w.ant difference (p<.05) with rest.

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Rest Trot Recovery S.E

17.7" 17.4" 17.3 .06

15.8 16.0 16.5 +

.236 .282*+ .266+ .01

.225 .259+ .255+

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TABLE 4

The Effect of the SET on RBC Indices In Conditioned and Noncondilioned Horses (mean; n=9)

Activity

MCV ~3) Conditioned

Nonconditioned MCHC (g/lO0 ml)

Conditioned

Noncondidoned MCH (pg)

Conditioned

Nonconditioned

Rest Trot Recovery S.E

*significant difference (p<.05) between groups within an activity. +significant deference (p<.05) with rest. "~significant difference (p<.05) between trot and recovery.

39.6 47.4 + 45.3* 1.2

41.6 47.7+ 45.5+

33.8* 34.2 34.0 .5

34.3 34.2 33.9

13.4" 16.2 + 15.4+t .4

14.3 16.2 + 15.5 +

conditioning program was evidenced by lower lactate levels (Figure 1) during the SET in the conditioned group. The lower lactate levels may reflect an adaptation by the muscle to derive a larger percentage of its energy through aerobic metabolism and/or faster degradation of the lactate. The conditioning effect of resting bradycardia was observed in the conditioned group (Table 1), and has been previously reported in Standardbred horses 10.

The exercise bout in this study caused PCV to increase approximately 33%. This increase can be attributed primarily to splenic release of red blood cells into the circulation. The MCV, MCH and DPG-RBC values during exercise suggest that these cells differ from those found in the circulation during rest. It has been suggested that there is a decrease in mean cell volume during endurance exercise in horses z]. An earlier study reported a decrease in trained horses between resting and post-exercise 2,3-DPG concentrations; untrained horses showed no difference 4. The results of this study contradict both of these findings. In this study, MCV was greater during exercise than at rest in both groups. In addition, there was an increase in the amount of 2,3-DPG found per RBC. levels of 2,3-DPG are known to decrease with aging]2, thus the cells released by the spleen during exercise may represent a population of less mature cells. The present findings may differ from those previously reported because of the different type of exercise bout imposed.

Conditioning has been reported to cause a decrease in resting3,4 and post exercise 4 levels of 2,3-DPG , but no values have been reported during an exercise bout in horses. In this investigation, conditioning resulted in an 8% Volume 6, Number 4

increment in 2,3-DPG concentration during exercise which is consistent with previous human studies13,14 that found a 5% and 12% increase in 2,3-DPG, respectively. In humans, conditioning increased 2,3-DPG levels independently of Hb levels 2. By relating the 2,3-DPG levels to RBC number (DPG-RBC) or Hb level (DPG-Hb) it is apparent in this study that conditioning produced an effect on 2,3-DPG which was independent of either RBC number of Hb level.

3.0

2[ : ~2 .6 (9

2,= o~

2[ 2[

I Rest Walk

roup - - - - - - - N o n Conditioned Group

I I I I I I I I I I I I I ! I I I I I I I I 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

I I I I Trot Recovery

Time (minutes)

Figure 2. The effect of SET on venous 2,3-dlphosphoglycerate levels in exercising horses.

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Conditioning may increase 2,3-DPG levels by stimulating erythropoiesis and increasing the number of younger cells that have greater levels of 2,3-DPG2. These younger cells also possess a steeper oxygen dissociation curve 15. A change in the slope of the oxygen dissociation curve and an increase in 2,3-DPG could augment the uptzke of oxygen by muscle and complement other aerobic conditioning effects.

Changes in 2,3-DPG concentrations of .5 to .7 mM were observed from rest through trotting. A lmM change in total concentration of 2,3-DPG causes a shift in the oxygen dis- sociation curve in whole blood oxygen affinity of 1.5 to 4.0 mm Hg at half saturatio# 6. The change in 2,3-DPG during the exercise bout would correspond to a .75-2.8 mm Hg change to the right in the oxygen dissociation curve. The difference in plasma 2,3-DPG due to conditioning is only .2 mM which would result in a much smaller shift. It is possible that longer or more strenuous exercise or condi- tioning programs would produce larger changes in 2,3-DPG levels. However, any shift in the dissociation curve due to increased 2,3-DPG content would be far outweighed by pH and temperature changes. At this exercise intensity it seems unlikely that the increased 2,3-DPG makes a comparably large contribution to conditioning effects in the horse.

MCH and MCHC were lower at rest in the conditioned state but were not affected by conditioning during exercise. Similarly, conditioning state did not alter MCV, so these indices do not appear to confirm a population shift with conditioning. Consequently, it appears that in horses, submaximal conditioning does not result in a shift in erythrocyte population as has been speculated for humans.

REFERENCES 1. Benesch R and Benesch RE: Intracellular o~anie phosphates as

regulator of oxygen release by hemoglobin, lVature 221:618-664,

1969. 2. Braurnan KM, Boring B, Tro~ F: Bohr effect and slope of

oxygen dissociation curve after physical training. J Appl Physioi:Respirat Environ Exercise Physiol 52:1524-1529, 1982.

3. Lewis IM and McLean JG: Physiological variations in levels of 2,3-diphosphoglycerate in horse erythrocytes. Res Vet Sci 18:186- 189, I975.

4. Lykkeboe G, Schougaard H and Johansen K: Training and exercise change respiratory properties of blood in race horses. Resp Physiology 29:315-325, 1977e

5. Allen BV, Powell 13(3: Effects of training and time of day of blood sampling on the variation of some common haematological parameters in normal Thoroughbred racehorses. In Equine Exercise Physiology, DH Snow, SGB Persson, RJ Rose, ed. Cambridge, Granla Press, 1983, pp 328-335.

6. Wintrobe MM: Clinical Hematology, 6th Ed Philadelphia; Lea and Febiger, 1967, pp 436437.

7. Cobb LA and Johnson WP: Hemodynamic relationships of anaerobic metabolism and plasma free fatty acids during prolonged strenuous exercise in ttmned and untrained subjects. J Clin Invest. 42:800-810, 1963.

8. Asheim A, Knudsen O, Lindholm A, Ruleker C and Saltin B: Heart rates and blood lactate concentrations of Standardbred horses during training and racing. JAVMA 157:304-312, 1970.

9. Engelhardt W: Cardiovascular effects of exercise and training in horses. Adv Vet Sci and Comp Med 21:173-205, 1977.

10. Marsland WP: Heart rate response to submaximal exercise in the Standardbred horse. J Appl Physiol 24:98-101, 1968

11. Boneher JH, Ferguson EW, Wilhehnsen LL, Statham N and McMeekin RW. Erythrocyte alterations during endurance exercise in horses. J Appl Physiol:Respirat Environ Exercise Physiol 5:131-134, 1981.

12. Hejlm M: Erythrocyte glycolytic intermediates and cofactors with hemoglobin concentrations in human neonate and adults. In Adv in Exp Med and Biology. G. Brewer, ed. New York, Plenum Press, 1970, pp 81-94.

13. Shappell SD, Murray JA, Bellingham AJ, Woodson RD, Detten JC and Lenfact C: Adaptation to exercise. Role of hemoglobin affinity for oxygen and 2,3-dlphosphoglyeerate. J Appl Physiol 30: 827-832, 1971.

14. Brotherhood J, Brozovic B and Pugh LGC: Hematological status of middle- and long-distance runners. Clin Scl and Mol Med 48:139-145, 1975.

15. Neville, JR. Erythrocyte age and slope of the oxygen dissociation curve (abs) Proc lnt Congr Physiol Sci 27th Paris 23:548,1977.

16. de Verdier C: The erythrocyte as a vehicle for oxygen with self- regulating adjustment of unloading. Scand d Clin Lab Invest 26(3):193- 198, 1970.

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