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The Relationship Between Respiratory Exchange Ratio, Plasma Lactate and Muscle Lactate Concentrations in Exercising Horses Using a Valved Gas Collection System Gail M. Gauvreau, Simon S. Young, Henry Staempfli, L. Jill McCutcheon, Brian A. Wilson, and Wayne N. McDonell
ABSTRACT A valved gas collection system for horses was validated, then used to examine the relationship between the respiratory exchange ratio (RER), and plasma and muscle lactate in exercising horses. Four healthy Standardbred horses were trained to breathe through the apparatus while exercising on a treadmill. Comparisons of arterial blood gas tensions were made at 3 work levels for each horse, without (control), and with the gas collection system present. At the highest work level, the arterial oxygen tension (PaO2) was significantly lower (P < 0.05), and the arterial carbon dioxide tension (PaCO2) was significantly higher (P < 0.05), than control levels when the apparatus was present; however arterial oxygen content remained unchanged. The horses completed a standardized incremental treadmill test on 4 occasions to determine the repeatability of measurements of oxygen consumption (102), carbon dioxide production (VCO2), inspired minute ventilation (VI), respiratory exchange ratio (RER), yentilatory equivalent for oxygen (V1/VO2), tidal volume (VT), and ventilatory frequency (VF). All gas exchange and respiratory measurements showed good reproducibility with the mean coefficient of variation of the 4 horses ranging from 3.8 to 12%. We examined the relationship between 3 indices of energy metabolism in horses performing treadmill exercise: respiratory exchange ratio
(RER), central venous plasma and muscle lactate concentrations. A relationship between RER and plasma lactate concentration was established. To compare muscle and plasma lactate concentrations, the horses completed a discontinuous exercise test without the gas collection apparatus present. Significant relationships (P < 0.05), between plasma lactate concentration and RER, and between plasma and muscle lactate concentration, were described for each horse. The valved gas collection system produced a measurable but tolerable degree of interference to respiration, and provided reproducible measurements of gas exchange and ventilatory measurements. It was concluded that measurements of both gas exchange and blood lactate may be used to indicate increased glycolytic activity within exercising skeletal muscle. RESUME Un systeme, muni de valves, de recolte des gazs respiratoires pour le cheval a ete valide afin d'etre utilise pour etudier la relation entre le rapport d'echange respiratoire (RER) et les concentrations des lactates plasmatiques et musculaires chez le cheval a l'exercice. Quatre chevaux Strandardbred en bonne sante ont ete entraines a respirer au travers de cet appareil tout en etant a l'exercice sur un tapis roulant. Au niveau de travail le plus eleve, la tension arterielle en oxygene
(PaO2) etait plus basse (P < 0,05) et la tension arterielle en dioxyde de carbone (PaCO2) etait plus elevee (P < 0,05) par rapport au niveau de controle quand l'appareil etait present, mais le contenu arteriel en oxygene restait inchange. Les chevaux ont accompli un test croissant sur le tapis roulant "a quatre occasions pour determiner la repetabilite des mesures de consommation en oxygene (VO2), de production en dioxyde de carbone (VCO2), de ventilation inspir;ee minute (V1), de RER, d'equivalent de la ventilation pour l'oxygene (V1/VO2), du volume courant (VT) et de la frequence respiratoire (VF). Toutes les mesures respiratoires et d'echange gazeux ont montre une bonne reproductibilite avec un coefficient de variation chez ces quatre chevaux variant de 3,8 a 12 %. La relation entre trois indices du metabolisme energe'tique chez les chevaux exerces sur le tapis roulant a t examinee : RER, concentrations plasmatiques veineuses centrales et musculaires de lactates. Une relation entre RER et la concentration plasmatique de lactates fut etablie. Pour comparer les concentrations plasmatiques et musculaires de lactates, les chevaux ont accompli un test d'exercice intermittent sans le systeme de collection des gaz respiratoires. Une relation significative (P < 0,05) entre la concentration plasmatique de lactate et le RER, et entre les concentrations plasmatiques et musculaires de lactate a ete montree chez chaque cheval. Le systeme de collection des
Department of Clinical Studies (Gauvreau, Young, Staempfli, McDonell), Department of Pathology (McCutcheon), Department of Human Biology (Wilson), University of Guelph, Guelph, Ontario NIG 2W1. Correspondence should be sent to Gail M. Gauvreau, Health Sciences Center - 3U1, Department of Medical Sciences, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5. Requests for reprints should be sent to Dr. H. Staempfli. Support for this project was received from the Dynasty Equine Trust, the Ontario Ministry of Agriculture, Food and Rural Affairs and the Equine Research Centre at the University of Guelph. Submitted February 22, 1995.
Can J Vet Res 1996; 60: 161-171
161
Figure 1. Schematic of valved gas collection system.
gaz respiratoires provoque un degre d'interference mesurable mais tolerable a la respiration et fournit des mesures de ventilation et d'echanges gazeux qui sont reproductibles. II est conclu que les mesures d'echanges gazeux et de lactates sanguins peuvent etre utilisees pour indiquer une augmentation d'activite glycolytique dans le muscle A l'exercice. (Traduit par docteure Sophie Cuvelliez)
INTRODUCTION Gas exchange and ventilatory measurements have become useful for assessing the exercise potential of performance horses (1,2,3). Both open flow and valved gas collection systems have been developed to measure oxygen consumption (VO2) and carbon dioxide production (VCO2) in exercising horses (4-7); however, simultaneous measurements of minute ventilation (V,) and gas exchange require the use of valves or breath-bybreath measurements with a mass spectrometer. Although valved systems have been associated with rebreathing and resistance, these problems can be minimized through careful design. It has been reported that measurements of gas exchange are very reproducible
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in the horse when using open flow (4) or valved (5) gas collection systems, although neither study reported the repeatability of ventilatory measurements for a given horse at various work levels. Only 2 replicates were carried out in the open flow study, while the study using the valved collection system used 3 replicates. Horses with higher aerobic capacities may rely less on glycolyticallyderived energy, and achieve a lower respiratory exchange ratio (RER) and plasma lactate concentrations at the same absolute work intensity than do less fit horses. Measurements of gas exchange have been used to estimate aerobic capacity in exercising horses (1,8,9). Until recently, plasma lactate concentrations were commonly used for the same purpose. High circulating lactate concentrations have been associated with a low aerobic capacity in the horse (8,9,10,11); however, measurements of blood and plasma lactate concentration have a large re-test variability (4) and may not consistently reflect aerobic capacity and the accumulation of lactic acid in the
performance testing. We describe a custom-designed low resistance valved gas collection system that permits simultaneous measurement of gas exchange and ventilatory variables in horses without the need for a mass spectrometer, and we report the effects of this gas collection system on arterial blood gas tensions during exercise. Repeatability of blood lactate determination and of simultaneous gas exchange measurements of VO 2' VCO2 and RER, as well as ventilatory measurements of X8, tidal volume (VT) and respiratory frequency (VF), were determined during a standardized exercise test. In a subsequent investigation, the relationship between RER, central venous plasma lactate, and muscle lactate concentrations was measured. These relationships have not previously been investigated in the same horses during exercise.
MATERIALS AND METHODS
The study was conducted in 3 parts using the same animals. Part 1 of the study was designed to assess the influence of the facemask and valved gas collection system on arterial blood gas tensions during strenuous exercise on the treadmill. In Part 2 of the study, 4 replicate measurements of gas exchange and ventilatory variables as well as plasma lactate determinations were carried out during a standardized treadmill exercise protocol on each of the 4 horses. Part 3 of the study examined the relationship between RER and central venous plasma lactate and muscle lactate concentrations. All aspects of the experimental protocol were approved by the institutional Animal Care Committee, and the care and handling of the horses during and between experiments followed the recommendations of the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, Ontario). Four healthy, trained Standardbred horses were used. All horses were 45 y old with a mean (+SEM) bodymuscle. The present study was carried out weight of 463.5 ± 17.1 kg. The horses to provide additional insight into the were normal on clinical examination, utility, repeatability and interrelation- arterial blood gas analysis and a nitroship of a number of indices that are gen washout test (12). Three of the currently utilized in equine exercise horses were maintained on a regular
exercise schedule 2 mo before and throughout the test period. The 4th horse was exercised regularly for 3 wk before, and maintained at the same training level during the testing period. All horses had participated in previous treadmill experiments and were accustomed to breathing through the gas collection apparatus while exercising at speeds up to 8.2 m-s-' at a 7% incline on a high speed treadmill (Sato, Sweden).
Model S-3A/1, Pittsburgh, Pennsylvania, USA) which measured the fractional, mixed, expired carbon dioxide (FECO2) and oxygen (FEO2), respectively. Analyzers were calibrated daily using certified gas mixtures (Canox-Canadian Oxygen Limited, Toronto, Ontario). Inspiratory flow was measured by 2 ultrasonic flow metres (NOVEX UF202, Kou & Assoc Consultant Co, Redmond, Washington, USA), each with a cross-sectional area of 20.3 cm2 APPARATUS and a length of 18 cm. The flow A valved gas collection system was metres were calibrated using a volume designed to simultaneously measure integration method (15) and a 10 L syringe (Houston Atlas Inc, V02, VCO2, V9, VT, and VF. A light precision Houston, Texas, USA). Analog signals weight, airtight respiratory mask (13) were converted to a digital signal, was secured around the muzzle of the averaged digitally using a scientific horse, and the port of the mask was software package (Superscope, G.W. attached to a 15 cm diameter Instruments MasSomerville, lightweight PVC T connector, to which two 1.5 m lengths of 15 cm sachusetts, USA) and stored in a comdiameter flexible tubing were also puter (Macintosh Classic, Apple fastened (Fig. 1). While the mask was Canada Inc). Measured flows were in use, the mandibular groove of the integrated digitally to give V,. The volume of the system between horse was packed with cotton wool the flow metres and mixed expired and the air bladder within the mask line was 101.8 L, as meagas sample was inflated around the horse's muzsured water by displacement. Approxzle with a hand held pump to a pres3 were required imately tidal volumes of mm sure 60 Hg to create an airtight to clear from the mask expired gas seal. In this manner, the dead space in into the chamber submixing during the mask was reduced to approxiexercise. maximal Peak-to-peak presmately 1.5 L. The mask did not interin the port of the mask fere with nostril flare during exercise, sures measured maximal exercise were less during as confirmed by visual inspection of 7 than cm smallest H20. Increasing nostril function while the horse ran on cross-sectional area on thethe inspiratory the treadmill. Unidirectional flow side of the apparatus from 40.5 cm2 through the tubing was maintained by (2 flow metres) (4 flow custom made 1-way valves positioned metres) did not to 81.1 cm2 reduce the measurably at each end of the tubing and secured pressure differences measured in the to the sides of the treadmill. The 1-way of the mask during mild or maxivalves, each with a cross-sectional port mal The compliance of the exercise. area of 133 cm2, were constructed flexible tubing in the apparatus was from transparent plexi-glass using a measured at 60 mL-cm H20-1. modification of a previously published 2-way design (14). Expired gas was channelled from the expiratory PART 1 - EFFECTS OF A VALVED GAS valve, through 0.5 m of 15 cm diame- COLLECTION SYSTEM ON ARTERIAL ter flexible tubing, to a 25 L chamber. BLOOD The expired gas was mixed with a fan, To permit serial arterial blood gas then exhausted to the atmosphere sampling, a 20 G, 5 cm catheter through a flexible 15 cm diameter, (Insyte-W, Becton Dickinson, Sandy, 30 cm length of tubing. A sample of Utah, USA) was inserted into the mixed expired gas was drawn through transverse facial artery, and an 8.5 F a drying column of anhydrous calcium percutaneous introducer (Arrow Intersulfate (Drierite, Xenia, Ohio, USA) national Inc, Reading, Pennsylvania, into the CO2 analyzer (Ametek, Model USA) was inserted into the right juguCD-3A, Pittsburgh, Pennsylvania, lar vein for placement of a temperaUSA) and 02 analyzer (Ametek, ture probe. The catheters were inserted
percutaneously using an aseptic technique and local anesthesia (1 mL of 2% lidocaine hydrochloride), and they were kept patent by periodic flushing with a dilute heparin/saline solution. A sterile temperature probe (Yellow Springs Instruments, Yellow Springs, Ohio, USA) that had been calibrated with a precision thermometer (Fisher Scientific, Ottawa, Ontario) was passed approximately 25 cm through the inlet of the introducer into the right jugular vein. The temperature probe was found to be linear over the range 36.0 to 45.0°C, with an r value of 1.0. The calibration factor did not drift after sterilization or after several experiments. The time required to reach 95% of the actual temperature was calculated to be 9 s. Each horse was weighed then fitted with a safety harness that was connected to an emergency stop switch above the treadmill. Heart rate (HR) electrodes were placed beneath the harness on the sternum and to the left of the withers, and an HR metre (Equistat Model HR-8A, EQB Inc, Unionville, Pennsylvania, USA) was used to determine HR. The HR metre was calibrated by recording raw ECG traces with another set of electrodes. An airtight mask was placed over the muzzle of the horse and held in place by nylon straps joined with velcro tape.
Experimental Design - Each of the 4 horses performed a standardized exercise test, both with and without the gas collection apparatus on the same day, in a random order. The horses were maintained for 4 min at each of 3 work levels; standing quietly on the treadmill, at a 1.6 ms-' walk at a 0% incline, and trotting or pacing at each horse's predetermined maximal HR, 6.4 to 7.6 m-s-' at 8.7% incline, which was an estimate of the exercise intensity needed to produce V02 max. Over the last minute of each work level, HR was recorded, and four 5 mL arterial blood samples were collected anaerobically into heparinized 5 mL plastic syringes simultaneous with measurements of venous temperature. The blood gas analyzer (Radiometer Model ABL3, Copenhagen, Denmark) was maintained at 37°C and was calibrated every 2 h using laboratory standards (Radiometer, Copenhagen,
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TABLE I. Arterial blood gas and hematological values (mean ± SEM) from 4 Standardbred horses wearing a face mask attached to a valved gas collection apparatus (Mask) and not wearing a face mask (Control) at rest, while walking and at an exercise intensity estimated to produce maximal oxygen uptake (C02 max). Values from 4 sampling periods during the 4th min under each exercise condition were averaged for each horse prior to calculating SEM
Variablea
Control
VO, Max
Walk
Rest
Mask
Control
Mask
Control
Mask
51.6 ± 1.7** 35.0 ± 1.2 40.5 ± 1.3* 37.4 ± 0.7 41.9 ± 1.2 39.3 ± 1.2 61.5 ± 1.2* 73.8 ± 2.2 95.6 ± 3.3 95.9 ± 1.7 98.9 ± 1.8 96.1 ± 2.8 22.2±0.5 24.3±0.7 18.0± 1.5 18.0± 1.0 16.3± 1.2 16.0± 1.4 CaO,(mL.O00mL-') 7.22 ± 0.02 7.38 ± 0.04 7.48 ± 0.01 7.50 ± 0.01 7.46 ± 0.01 7.47 ± 0.01 pH -7.54 ± 1.59 -3.60 ± 2.2 6.36 ± 0.56 5.96 ± 0.50 5.31 ± 0.69 4.93 ± 0.55 ABE (mM) 52.5 ± 1.6 51.9 ± 1.9 38.0 ± 2.3 36.9 ± 2.1 34.6 ± 1.5 35.4 ± 1.3 PCV (L-L-') 59.6 ± 2.4 62.6 ± 1.9 52.1 ± 1.3 53.4 ± 0.8 51.5 ± 0.8 TP (g-L-') 52.9 ± 1.6 94.7 ± 1.0 86.4 ± 1.1 98.3 ± 0.2 97.9 ± 0.4 98.0 ± 0.2 98.0 ± 0.2 Hb saturation (%) 38.5 ± 0.3 36.9 ± 0.2 36.6 ± 0.2 35.9 ± 0.2 36.9 ± 0.2 36.4 ± 0.7 TV (OC) a PaCO : arterial carbon dioxide tension; PaO,: arterial oxygen tension; CaO,: arterial oxygen content; ABE: actual base excess; PCV: packed cell volume; TP: total serum protein; Hb saturation: hemoglobin saturation; TV: venous temperature * Statistically different between control and mask at P < 0.05 ** Statistically different between control and mask at P < 0.025
PaCO, (mmHg) PaO2 (mmHg)
Denmark). All blood samples were placed in an ice/water bath and analyzed during the same 2 h calibration period within 90 min of collection; values were corrected to the venous temperature measured at the time of blood collection. The following measurements were taken; arterial 02 and CO2 tensions (PaO2 and PaCO2), hemoglobin saturation, venous temperature, pH, actual base excess (ABE), packed cell volume (PCV), and total serum protein (TP). Arterial 02 content (CaO2) was calculated using a hemoglobin saturation curve for exercising horses (16). Comparisons between exercise in the control state (no mask, no breathing apparatus) and while wearing the apparatus were made using all 4 samples collected during the last minute of the 3 exercise levels. The data were analyzed with a statistics computer program (SAS Institute Inc, Cary, North Carolina, USA) using an analysis of variance procedure for repeated measures, where the between-mean squares sources of variations were corrected to account for the correlation resulting from repeated measurements on the same horses in successive trials. Maximal HR were compared using a two-tailed Student's paired t-test. PART 2- REPEATABILITY OF MEASUREMENTS
An intravenous catheter (57 mm, 14 G; Critikon, Tampa, Florida, USA)
164
and percutaneous sheath introducer (8.5 F; Arrow International Inc, Reading, Pennsylvania, USA) were inserted aseptically into the jugular vein under local analgesia (2% lidocaine hydrochloride). A saline-filled polyethylene tube (120 cm long, 0.2 cm i.d./0.26 cm o.d.) with multiple holes at the distal end was advanced through the introducer into the pulmonary artery. Correct placement of the catheter was ensured by observing characteristic pressure traces with an oscilloscope monitor (Tektronics 401, Spacelabs Medical Products Ltd., Mississauga, Ontario) and pressure transducer (DTX Model T36AD-R, Viggo-Spectromed, Oxnard, California, USA). After the catheter tip had passed through the pulmonary valve, it was advanced another 5.0 cm and secured in place. Extension lines were fastened to each of the catheters; total dead space volumes were 2.0 mL and 5.0 mL in the jugular vein and pulmonary artery catheters, respectively. Each horse was then fitted with a safety harness, HR metre, and gas collection apparatus as described earlier.
Experimental Design - Each of the 4 horses performed a standardized incremental treadmill test 4 times, 35 d apart. All tests were completed within 17 d. For each test, the treadmill protocol consisted of a 2 min warmup at 1.6 m-s-1 with no incline and 1 min at 3.2 m-s-1 on a 7% incline. The horses then exercised at a constant gait (trot or pace) for 7
speeds on a 7% incline. The treadmill velocity ranged from 3.2 to 8.2 m-s-' and was increased by 0.91 m-s-' at the beginning of each minute. Inspired airflow, FEO2, FECO2, HR, jugular and pulmonary artery blood were simultaneously measured at rest and during the last 15 s of each minute. The test was stopped if the horse could not keep up with the treadmill or if HR was over 220 min-'. Jugular and mixed venous blood samples for lactate determination were simultaneously collected into 5.0 mL disposable syringes, transferred immediately into chilled sodium fluoride tubes, and submerged in an ice bath. The blood samples were centrifuged for 10 min at 3500 rpm within 20 min of collection. Plasma samples were frozen at -70°C and analyzed in duplicate (23 L Lactate Analyzer, YSI Company Inc, Yellow Springs, Ohio, USA) within 7 d of collection. Earlier experiments confirmed that lactate concentrations did not change in plasma frozen for up to 3 mo. The lactate analyzer was calibrated daily with 1.0 mmol-L-' and 15.0 mmol L- laboratory standards supplied by the manufacturer, and rechecked frequently during use with a serum based 1.5 mmol-L-' laboratory standard. Simultaneous measurements of inspiratory airflow, FEO2 and FECO2 were sampled at a frequency of 20 Hz. The analog signals were digitized and processed with a Macintosh Classic computer (Apple Canada Inc) using a
TABLE II. Mean (±SEM) coefficients of variation of metabolic and ventilatory parameters measured in 4 Standardbred horses at 7 work levels of a standardized exercise test. The coefficient of variation for each individual horse was based on 4 replicate tests Variablesa
VCO, (mL-kg-'-min-') VO2 (mL-kg-' min-') RER J Lac- (mmol L-') PA Lac- (mmol.L-')
VT (L BTPS) V, (L-min- IBTPS) YF(min -')
VI/VO2(LBTPS/LSTPD)
3.2
3.7
9.7 ± 1.7 7.2 ± 1.8 4.0 ± 2.2 22.0 ± 2.7 21.6 ± 4.7 7.2 ± 1.0 10.6 ± 0.9*
11.2 ± 1.7 9.2 ± 2.1 4.5 ± 1.8 22.1 ± 5.0 22.4 ± 6.2* 7.2 ± 1.6* 9.8 ± 1.3
Speed (m.s ') 4.6
5.5
6.4
7.3
8.2
12.0 ± 2.4 8.2 ± 1.4 5.3 ± 1.0 21.9 ± 5.8 24.7 ± 7.8 8.0 ± 1.8 11.5 ± 2.5 9.3 ±2.3
8.8 ± 1.9 5.3 ± 0.7 4.0 ± 0.7 27.7 ± 5.2 28.2 + 5.9 7.7 ± 1.0 8.9 ± 2.7
8.4 ± 2.7 4.8 ± 2.1 4.8 ± 1.1 25.1 ± 6.5 23.7 ± 4.8 5.5 ± 0.8 9.3 ± 3.1
7.7 ± 2.7 5.9 ± 1.4* 3.8 ± 1.0 19.3 3.1 19.9 2.4 6.0 ± 1.0 9.0 ± 2.7*
8.9±2.0
12.0±2.6
7.5±2.2
5.8±1.8
6.0±2.0
9.6±2.2
8.8±2.0
11.6 ± 2.2 9.8 ± 1.7 4.2 ± 1.2 24.2 ± 3.4 19.6 ± 5.6 9.3 ± 2.3 11.1 ± 0.9 8.2± 1.5
7.7±2.0
8.8±2.4
8.3±3.1
7.0±3.1
5.0+0.8
Carbon dioxide production (VCO2); oxygen consumption (VO,); respiratory exchange ratio (RER); jugular lactate concentration (J Lac-); pulmonary artery lactate concentration (PA Lac-); tidal volume (VT); inspiratory minute ventilation (V); ventilatory frequency (VF); ventilatory equivalent for
a
oxygen *
(V,/V02)
Statistically significant differences between tests at P < 0.05
scientific software package (Superscope, GW Instruments, Somerville Massachusetts, USA). Subsequently, VI, VO2, VCO2 and RER were calculated using standard equations and averaged over the sampling period. Algorithms were developed to determine the VF and VT during each data collection period. Measurements of V02 and VCO2 were corrected to standard temperature and pressure dry (STPD), VT and VI were corrected to BTPS, and the ventilatory equivalent for oxygen (V1/V02) was corrected to BTPS/STPD. A 2-way analysis of variance was used to assess the test variability on values of gas exchange, ventilation and plasma lactate concentration, after accounting for between horse variations. The error term was the horse by test interaction because this was a non-replicated experiment. Individual and group coefficients of variation (CV) for each of the parameters were calculated at each speed level. PART 3 - RELATIONSHIP BETWEEN RER AND LACTATE CONCENTRATION
A discontinuous treadmill test was performed once on each of the 4 horses. As previously described, a polyethylene catheter was advanced into the pulmonary artery to collect mixed venous blood samples. Four sites on the skin overlying the right gluteal muscle were shaved and aseptically prepared for muscle biopsies. Sensation in a small area of the skin and subcutaneous tissue was blocked
120F 100L
a
62 0
G140
so40 C,/
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o
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E
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E
1~~~~60
-
7
120
>~~~~~~~~40
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4040
35
3
4
5
Treadmill Velocity
6
7
(M.S
1);
8 7% Incline
Figure 2. Mean (± SEM) oxygen consumption (VO2), carbon dioxide production (VCO2) and ventilatory equivalent for oxygen (V,/VO2) of each of 4 Standardbred horses measured at 7 work loads of a standardized incremental treadmill test. Means were calculated for each horse using data from 4 repeated exercise tests.
last 15 s of the workload. The treadmill was temporarily stopped (30-45 s) upon termination of the workload, and a muscle biopsy was rapidly obtained percutaneously using a custom built biopsy needle. Muscle biopsies weighing 0.2 to 0.5 g were obtained at a depth of 5 cm. The muscle sample was immediately frozen in liquid nitrogen and stored at -80°C until analyzed. Immediately after the muscle biopsy was completed the horse commenced exercise at the next workload. The wet to dry ratio was calculated by weighing each muscle sample before and after freeze drying. Muscle lactate concentration was determined by extraction and spectrophotometric analysis, as described elsewhere (9), and expressed as a dry weight value. Central venous plasma lactate concentrations were determined as previously described. The RER at each work intensity was predicted using the measured central venous plasma lactate concentration and the 2nd order polynomial relationship that had been determined between plasma lactate and RER for each horse in Part 2.
with 2% lidocaine hydrochloride before making a 1.5 cm skin incision with a scalpel. RESULTS Blood and muscle samples were obtained pre-exercise and at speeds shown to elicit 50, 75 then 100% of PART 1 - EFFECTS OF A VALVED GAS the horse's peak V02 (for horses not COLLECTION SYSTEM ON ARTERIAL demonstrating a plateau of V02) which BLOOD had been determined using a 7% A comparison of the hematological treadmill incline in Part 2. Each speed and blood gas values for control and was maintained trotting or pacing for apparatus exercise tests is shown in 2 min and blood was drawn during the Table I. Individual resting PaCO2 165
means (±SEM) of artery. The average RER coefficients 206.6 ± 2.1 and 211.8 ± 2.2 for control of variation ranging from 3.8 to 5.3% were lower than the 19.3 to 28.2% and apparatus present, respectively. range of CV for measurements of plasma lactate in the pulmonary artery PART 2- REPEATABILITY OF and jugular vein (Table II). Test-to-test MEASUREMENTS measurements were repeatable for Mean V02 and VCO2 values for each RER, and only significantly different horse are shown in Fig. 2 and the (P < 0.05) for jugular vein and pulpooled CV of each measurement are monary artery lactate concentrations shown in Table II. The V02 was sig- at the lowest speed level. There was a nificantly different (P < 0.05) between significant (P < 0.01) 2nd order polyhorses at all work intensities, with the nomial relationship between RER and exception of the 3.7 m-s-' speed level. pulmonary artery plasma lactate conIndividual horse CV for V02 mea- centration for each of the 4 horses. sured during the exercise test ranged Plasma lactate concentrations in between 1.0 and 14.7%. Inter-test dif- samples collected from the jugular ferences for 02 were only significant vein during submaximal exercise (P < 0.05) at the highest speed level. remained lower and increased at a Mean VCO2 values of each horse lower rate than that in the pulmonary (Fig. 2) were significantly different artery during submaximal exercise (P < 0.05) at 5 of the 7 speed levels of (Fig. 4). The differences between lacthe standardized exercise test. Indi- tate concentration in the jugular vein vidual horse CV for VCO2 across all and pulmonary artery increased as the speed levels ranged from 1.7 to work intensity rose. Variability in 17.3%. Test-to-test variability was not lactate concentration was similar significant (P < 0.05) at any speed between the 2 sites of measurement. level of the exercise test. Mean V,, VT and VF values for the 4 horses are shown in Fig. 3. Significant PART 3 RELATIONSHIP BETWEEN differences (P < 0.05) in VT were RER AND LACTATE CONCENTRATION Muscle lactate and central venous observed among horses at all work loads, whereas VX and VF were signifi- lactate concentrations increased as the cantly different among horses at all exercise intensified (Fig. 5). Respiraspeeds except 7.3 m-s-'. Only 1 horse tory exchange ratios were calculated reached maximal ventilation at the on the basis of the measured pulhighest speed level, as assessed by a monary artery lactate concentration and previously established polynolevelling of both VT and VF. Test-to-test measurements of VI, VT mial relationships between RER and and VF were repeatable at most of the pulmonary artery plasma lactate conspeed levels. Individual horse CV at centration for each horse (P < 0.01, each speed level ranged from 3.2 to r > 0.93; Part 2) are shown in Fig. 5. 15.1%, 2.0 to 15.1% and 3.1 to 17.6% Three horses demonstrated similar for VT, VI and VF, respectively. Mean plasma lactate concentrations and CV for VI and VT of the 4 horses were RER at the highest work load. Mean higher at slower speed levels (Table II). maximal values for RER, plasma, and Mean V1/V02 are shown for each of muscle lactate concentrations were the horses in Fig. 2. Significant differ- 1.03 ± 0.03, 5.88 ± 1.04 mmol-L-', ences (P < 0.05) among horses were and 96.95 ± 14.05 mmol.kg'1 dm, found at all but 2 speed levels. Indi- respectively: Horse 2 responded to vidual horse CV ranged from 1.3 to 100% peak V02 work intensity with 15.4% across all speed levels, and lower RER and plasma lactate contest-to-test measurements were not centrations than the other horses, significantly different (P > 0.05) over despite higher muscle lactate concenthe 4 repeated exercise tests. trations, indicating either higher proMean RER and plasma lactate con- duction of lactic acid in muscle, centrations rose as the work intensity and/or poor diffusion into the blood. increased (Fig. 4). The slope of the An inefficient gait or pushing against RER relationship versus speed was the front of the treadmill during the steeper at the higher speed levels, as test can lead to a plateau of VO2 at low work loads (Fig. 3). Significant were the plasma lactate concentrations in both jugular vein and pulmonary linear relationships (P < 0.05), were
different, with
Un a-
1600 1400
1200 .-
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25
1000
800 .>7
-1 20
600 /
U)
m Ji
15 90
I.
/l
;
8070
i
60-
3
4
5
6
Treadmill Velocity (m.
7
I);
8 7% incline
Figure 3. Mean (± SEM) minute ventilation (Vd), tidal volume (VT) and ventilatory frequency (VF) of each of 4 Standardbred horses measured at 7 work loads of a standardized incremental treadmill test. Means were calculated for each horse using data from 4 repeated exercise tests.
values measured without the gas collection system ranged from 34.7 to 45.6 mmHg. As the exercise intensity increased, all horses in the control state became progressively more hypocapnic. There were significantly higher (P < 0.05) PaCO2 levels when the gas collection system was present, ranging from 37.3 to 46.8 mmHg while walking and 46.0 to 58.3 mmHg while exercising at the estimated V02 max. With the gas collection apparatus in place, arterial hypoxemia was significantly worse (P < 0.05) than control levels; ranging between 57.9 and 64.6 mmHg at the maximal HR. Jugular temperatures during exercise tended to be higher with the mask applied, but the difference was not significant. The calculated CaO2 during maximal exercise with the mask applied ranged from 19.9 to 23.1 mL per 100 mL blood. These were not significantly different (P > 0.05) from the control concentrations which ranged between 22.0 and 25.9 mL per 100 mL blood. Furthermore, there were no significant differences between hemoglobin saturation, venous temperatures, pH, ABE, PCV, or TP at any speed when control tests were compared to tests in which the gas collection apparatus was worn. The maximum HR achieved under the 2 conditions was not significantly 166
o Horse y--0.5476+0.3280x R=0.98 * Horse 2 y= 0.6417+0.0713x R= 1.0 S7 Horse 3 y= 0.1750+0.424Hx R=0.96 *v Hose 4 y=-0.1308+0.1737x R=0.89
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Figure 4. Mean (+ SEM) respiratory exchange ratio (RER), pulmonary artery and jugular plasma lactate concentration of each of 4 Standardbred horses measured at 7 work loads of a standardized incremental treadmill test. Means were calculated for each horse using data from 4 repeated exercise tests.
observed between plasma lactate and muscle lactate concentrations for each of the horses (Fig. 6); however the slopes of the relationships were different between horses, ranging between 0.07 and 0.42.
DISCUSSION The results of this study demonstrate good repeatability of selected gas exchange and ventilatory indices in horses using a standardized treadmill exercise protocol and a valved gas collection system The mean CV
for V02, VCO2, RER, VI, VT and VNV2 were under 10% at the highest speed, and measurements of RER were especially repeatable with mean CV between 3.8 ± 1.0% (highest speed) and 5.3 ± 1.0%. In contrast, the measurement of jugular vein or pulmonary artery plasma lactate levels was much more variable with CV ranging from 19.3 ± 3.1% (highest speed) to 28.2 ± 5.9%. These results are in general agreement with earlier studies done using both open (4,9), and closed (5,6), gas collection systems, and somewhat different exercise protocols.
Figure 5. The relationship between respiratory exchange ratio (top), pulmonary artery plasma (middle) and middle gluteal muscle (bottom) lactate concentration measured in 4 Standardbred horses at rest and while exercising at 50,75 and 100% peak V02.
The decision to utilize a valved gas collection system rather than an open system (4) in the present study was based on our desire to measure ventilatory variables along with V02 max and RER determinations, and the fact that the response rate of our oxygen and carbon dioxide analyzers would not permit continuous breath-by-breath determinations. The latter analysis requires a mass spectrometer (17,18), which was not available. Since others have demonstrated that the use of valved collection systems (of other designs) significantly interferes with gas exchange in the submaximal and maximally exercising horse (19,20), our choice of measurement system warrants some additional comment. In a subsequent study reported elsewhere, we used the same apparatus to measure V02 max in 2 groups of healthy race fit Standardbreds with a mean racing time difference of 4.4 s (2). The V02 max values determined (164.9 ± 4.3 and 147.8 ± 2.9 mL-min-'.kg-1, or the faster and slower groups, respectively) were similar to those reported for racing Thoroughbreds (161.9 ± 12.9 (sd) mL*min-'*kg-') measured with an open system (21), and to those measured using breath-by-breath uptake studies with a mass spectrometer (3). We were thus assured that the valved collection system used in the present study provides realistic V02 max determinations, although it obviously does produce altered blood gas levels under
Figure 6. The correlation between pulmonary artery plasma lactate concentration and middle gluteal muscle lactate concentration measured in 4 Standardbred horses at rest and-while exercising at 50, 75 and 100% peak V02.
maximal exercise conditions. There are several advantages of the apparatus described here over other valved systems; the flow metres and valves are firmly anchored away from the horse and thus are less affected by movement, the 1-way valves are light weight and have a large cross-sectional area, especially compared to masked systems employed in previous studies (19), and the mask is tight fitting and light with mninimal dead space. The primary purpose of the present study was to critically examine the repeatability of gas exchange, ventilatory and lactate measurements, and to examine the relationship of RER alterations to muscle and lactate values. The consistency and accuracy of the measurement system employed permitted these determinations, even if it might be argued that the absolute value of W2max or the onset time and intensity of anaerobic glycolysis was different on the valved system than might have been observed using an open system. PART 1 - EFFECTS OF A VALVED GAS COLLECTION SYSTEM ON ARTERIAL BLOOD
Horses exercising without the gas collection apparatus (control state) showed no change in PaO2 during submaximal exercise, but they developed hypoxemia in response to maximal exercise as expected (22). The mean PaO2 value of 73.8 ± 2.2 mmHg measured in our maximally exercising horses during control tests were similar to published PaO2 values of 70.6 ± 167
2.7 mmHg for Standardbreds (20), and 69.5 ± 3.0 mmHg and 68.0 ± 3.3 mmHg, respectively, for Thoroughbreds exercising maximally at the highest speed of an incremental test (19), and Thoroughbreds exercising at 91% V02 max for 4 min (23). With the collection system in place, PaO2 values were 12.3 mmHg lower during maximum exercise, a change that is statistically significant and would seem, intuitively, to be of biological importance. The added hypoxemia introduced by the mask and collection system was of a very similar magnitude to that observed by others (PaO2 of 60.9 ± 1.2 mmHg) in strenuously exercising Standardbreds when they employed a system with 1.5 L dead space and inspiratory and expiratory valve cross-sectional areas of 115 and 125 cm2 respectively (20). Others have reported greater falls in PaO2 to as low as 52.3 ± 0.4 mmHg (19,24); however the valved collection systems they employed either used valves with a comparatively low cross-sectional area (48 cm2) or relatively thick rubber flap valves which appeared to perform poorly. Although there was a tendency for hemoglobin saturation levels to be lower at maximum exercise levels when the horses breathed through the collection apparatus, this was not reflected in a significant change in CaO2, in contrast to the observations of Evans and coworkers (20). This may have been a reflection of our sample size since CaO2 did tend to be lower with the apparatus in place than in the control state (22.2 ± 0.5 versus 24.3 ± 0.7 mL 02 per 100 mL blood). In this, and previous studies (17,25), CaO2 values (control and mask) increased with moderate and strenuous exercise, reflecting the increase in hematocrit produced by the splenic release of erythrocytes (26). It has been stated that arterial hypoxemia is unlikely to limit performance at or below V02 max because CaO2 is maintained and actually increases (17). Oxygen extraction by muscle is increased during exercise (20), and the unloading of oxygen into the tissues is assisted by a rightward shift of the hemoglobin saturation curve resulting from tissue acidosis, hypercarbia and hyperthermia. Presumably there is a lower blood to tissue concentration gradient during strenuous exercise 168
with the mask and collection system since PaO2 is 12 mmHg lower. The biological significance of this is open to question, but it would seem that the difference is more likely to be related to the treadmill speed at which V02 max develops or to the time to fatigue rather than V02 max determinations, or the relationship of RER changes to alterations in muscle and blood lactate levels. In a subsequent study where we directly compared blood gas levels and V02 max determinations using the present valved system and an open system on eight racing pacers, fatigue appeared to occur at a lower treadmill speed using the valved system (27). The PaCO2 values measured during maximal exercise without a mask in the present study (35.0 ± 1.2 mmHg) were lower than the values of 45.2 ± 0.8 mmHg measured in Standardbreds during the last minute of an incremental test (20), and of 43.9 ± 1.7 mmHg in Thoroughbreds after 4 min of exercise at 91% V02 max (23). The PaCO2 values measured during maximal exercise in our horses with the mask and apparatus applied (51.6 ± 1.7 mmHg), were lower than the values of 55.0 ± 0.4 mmHg measured in Standardbreds (20), or values of 57.6 ± 1.3 mmHg measured in Thoroughbreds performing a maximal 1 min work increment connected to a valved system (24), but higher than the PaCO2 values of 46.6 ± 1.3 mmHg measured in Thoroughbreds performing maximally using a flow through system (19). The nature of the exercise protocols used in these studies may have a large impact on the CO2 levels measured in the blood. Increasing the exercise duration can change the exercise response from hypercapnic to hypocapnic, thus the studies are not entirely comparable. Values of PaO2 and PaCO2 in the present study may be higher than those reported in other studies (19,20,23) because they were corrected to a measured jugular temperature, rather than to rectal temperatures as in earlier studies. Rectal temperatures are not an accurate reflection of blood temperature during exercise (23,28). Venous pH values are also affected by the temperature correction, and may cause estimates of blood pH at V02 max to be biased if they are corrected to rectal temperature rather than to the temperature of the blood (25). In the present study, the jugular temperature with the
apparatus in place was higher, although not significantly higher than control, likely due to ineffective cooling of the head. Correction of arterial blood gas values to erroneously high jugular temperatures during the tests with the mask applied may have underestimated the degree of hypoxemia present and reduced apparent differences between mask and control tests. On the other hand, the same temperature correction of arterial blood, if erroneous, would have increased the apparent level of CO2 in the blood with the mask applied, thus overemphasizing the apparent hypercapnic effect. PART 2- REPEATABILITY OF MEASUREMENTS
Although an attempt was made to ensure that the 4 horses in the study were all fit and at a reasonably similar level of training, the primary objective in the repeatability study was to ensure that the level of training did not change appreciably during the course of the repeatability trials. The horses were not trained to a competitive level. Measurements of V02 were significantly different (P < 0.05) among the horses at most speeds and reflected the individual running efficiencies of the animals. The range of V02 max values achieved by the 4 horses was similar to that reported by others for 6 Standardbred horses using a similar exercise protocol (5), and the CV for repeat measurements in the same horse was similar to that reported using an open flow system (9). Intertest differences in V02 measurement for each horse were only significantly different (P < 0.05) at the highest speed level of 8.2 m-s-'. Although training effects of the exercise test itself may influence subsequent peak V02 values of each of the horses, none of these horses showed a significant (P < 0.05) linear effect of time on oxygen consumption at the highest speed level. Thus, re-test differences at 8.2 m-s-' were most likely due to the fatiguing effects of the exercise test on the 2 horses that reached their V02 max at 7.3 m-s-1, and the sensitivity of the ANOVA to these outlying data points. Increasing the work load beyond V02 max of the horse frequently resulted in a small decrease in V02, as has been described in earlier investigations (29). This decrease
may have been caused by exhaustion which would prevent the horse from maintaining its position on the treadmill without being pulled by the safety harness. The CV of gas exchange, ventilatory and blood lactate measurements are shown in Table II. Larger CV for V02 that were present at the slower speeds were most likely a result of the lower accuracy of the flow measuring devices at low flow rates. Mean CV for VCO2 were slightly higher than values measured for V02. The signal output from the CO2 analyzer fluctuated over a small range (based on observations of the visual display) and this might have contributed to the slightly greater variability of CO2 measurements. Just as with the measurements of VO2, higher VCO2 coefficients of variation at the lower speeds were likely a reflection of a higher variability of the flow metres at low flow rates. Since inter-test variability was not found to be significant at any speed level, VCO2 proved to be a very repeatable measurement. The ventilatory measurements of VI, VT and VF, were similar to reported values for pacing and trotting Standardbred horses (29,30). A small error was introduced into our volume measurements when correcting to BTPS due to the assumption of a 38°C body temperature at all exercise levels, because body temperatures may have reached 42°C during maximal exercise. The maximum error in correcting the volume to BTPS caused by assuming a body temperature of 38°C is only 2.9%. Maximal ventilation may have been limited by the 1-way valves imposing extra resistance, thereby preventing further increases in VT. However, the large cross-sectional areas of valves utilized in the study were designed to minimize resistance within the gas collection system. Ventilatory measurements of horses working at similar loads using masks with 1way valves (32) were found to be similar to measurements using masks without valves (29). A less variable VI at high speed levels may be the cause of lower CV of VCO2 and VO2 found at the higher speeds. The flow metres used introduced some error into measurements of VT, VI, and subsequently M)2 and VCO2, due to the intrinsic + 1% full scale accuracy of the flow metres (and
noise and zero drift on the output of the flow metres). These inaccuracies introduced a constant small error into the measured flow, causing a comparatively large variablity at the lowest flow rates and a much smaller variability at the highest flow rates. The V/VO2 measured in the Standardbred horses in the present study was higher than values previously reported for Standardbred horses (30). This difference could be due to a lesser degree of fitness in our horses, as V,/VO2 has been shown to decrease significantly after training ( 1 8). The low variability of RER measured with our valved gas collection system was similar to that reported using a flow-through system (4). Calculation of RER allows V1 to be removed from the equation, thus eliminating error introduced by the flow meters. During incremental exercise, RER rose in a similar fashion to what has been reported in Standardbreds (30), and Thoroughbreds (4,18,21,33). The significantly different RER values measured between horses at absolute work loads are a reflection of the fact that a given workload (i.e. treadmill speed) probably constituted a different level of difficulty for each horse. Although the horses had been trained in a similar manner, and in the case of 3 horses for the same duration, none of the horses were racefit, and their individual levels of fitness were not similar. Training has been shown to significantly decrease RER at a given workload (18,33,34). As observed by others (21,34), plasma lactate concentration increased exponentially as the work intensity increased. Blood lactate concentrations have been used to estimate the aerobic capacity (8,10), as well as the contribution of anaerobic glycolysis to meet the energy requirements in exercising horses (21,35). As with the RER, training and the level of fitness influences blood lactate accumulation in horses (34,36), and this may be reponsible for the variable plasma lactate concentrations present in our horses at the same absolute work loads. Lactate concentrations measured in samples collected from the pulmonary artery, jugular vein and carotid artery are reported to be similar in the horse at rest and during submaximal exercise (37). However, at higher levels of exercise, the pulmonary artery lactate
concentration should exceed that of the jugular vein which contains blood returning from the head where the metabolic rate is lower, assuming there is no uptake or addition of lactate to jugular blood from the brain and other cranial tissues. The present study demonstrates that pulmonary artery and jugular vein plasma lactate concentrations differ consistently at higher exercise intensities. Test-to-test measurements of gas exchange were more repeatable than plasma lactate concentrations at all speed levels tested, thus confirming earlier work reported by Seeherman and Morris (4). The ventilatory measurements demonstrated low test-totest variability, confirming the use of these measures in evaluating ventilatory responses to exercise in the horse. The between horse variability in measurements of VO2, VCO2, V, VT and VF illustrates the sensitivity of the valved gas collection system in identifying different exercise responses between individual horses. This may be of value in assessing respiratory function in horses with poor performance syndrome (1). When the metabolic response to exercise was examined, our results clearly demonstrated that the RER is related to circulating lactate concentration as a function of work intensity (34). The rate of CO2 generated from the combination of H+ with HCO3-, however, depends on the rate of lactate increase rather than on the absolute value of lactate (38). As the rate of CO2 generation increases, greater ventilation is required to offload CO2 at the lung, which subsequently drives up the VCO2 and increases the RER. Since VCO2 depends on the rate of lactate increase, and RER is proportional to VCO2, it may be more valid to establish a relationship between RER and the rate of lactate increase rather than RER and circulating lactate concentration. PART 3 - RELATIONSHIP BETWEEN RER AND LACTATE CONCENTRATION
Higher muscle lactate concentrations were measured after exercise at 50, 75 and 100% peak V02 than at rest, and the absolute values were similar to those measured in untrained Standardbreds after maximal treadmill exercise (39). Between horse differences in muscle lactate concentra169
tion measured after exercise at the same relative work intensities could be due to differences in aerobic capacity and training state of the horse. Horses I and 3 had the highest peak VO2's and the lowest muscle lactate concentrations at the same relative work intensities. Endurance training in humans has been shown to lower muscle lactate concentrations during submaximal exercise due to the increased use of fat, reducing muscle and liver glycogen utilization (40). Standardbred horses trained for high intensity exercise also have been shown to have lower resting muscle lactate concentrations than untrained Standardbred horses (41). Lower muscle lactate concentrations and increased fat oxidation by active skeletal muscle may be accomplished by training-induced increases in oxidative enzymes (41), thus permitting greater aerobic contribution to the total energy production. In the present study, muscle lactate accumulation was positively correlated with an increased plasma lactate concentration. The rate of lactate accumulation in the circulation should stoichiometrically account for the increased rate of ATP production by glycolysis; however, the rate of increase of muscle lactate concentration measured in the horses was greater than the rate of increase of plasma lactate. This suggests a delay in transport of lactate out of the muscle. The linear relationship between muscle lactate and plasma lactate concentration in our discontinuous treadmill test was similar to the relationship observed during progressive bicycle ergometer exercise in humans (42). Determination of circulating lactate concentration in exercising horses offers a less invasive method to evaluate the occurrence of glycolysis in the muscle. Trained horses are capable of a higher VO2 than are untrained horses, require less glycolytically-derived energy, and consequently have lower muscle and circulating lactate concentrations when exercising at the same absolute work load. The 4 Standardbred horses in the present study demonstrated similar relationships between muscle versus central venous plasma lactate concentrations, and central venous plasma 170
lactate concentration versus RER. The strength of these relationships indicates that both circulating lactate concentrations and RER mirror an accumulation of lactate in muscle, and may be used qualitatively as an indication of the occurrence of glycolysis during exercise. Due to the variability between horses, it does not appear that measures of RER or plasma lactate could be used to quantify muscle lactate concentration. When profiles of aerobic capaclty (peak V02) were compared amongst the horses in the present study, glycolytic activity appeared to be lower in horses with the highest peak V02. We concluded that aerobic capacity and training state of the horse may be evaluated by indices of glycolytic activity during maximal exercise, and expressed by invasive measures of muscle or plasma lactate concentration, or by non-invasive measures of RER.
ACKNOWLEDGMENTS The authors gratefully acknowledge Dan Schnurr, Ginger Howell, Cassandra Leicht, Chris Lewis and Sean McIntosh for their excellent technical assistance, and Dr. M. Shoukri and Dr. M. Lindinger for advice. The study would not have been possible without the generous help of the Equine Research Centre which provided horses and technical assistance.
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