Effect of feed at different times priorto exercise and chelated chromiumsupplementation on the athleticperformance of MangalargaMarchador mares

Lilian de Rezende Jordao1, Jose Aurelio G Bergmann1,Raquel S Moura1, Marılia M Melo2, Maria LL Costa1,Patrıcia CB Moss1, Helio M Aquino Neto3

and Adalgiza Souza Carneiro de Rezende1,*1Department of Animal Science, School of Veterinary Medicine, Universidade Federalde Minas Gerais, Belo Horizonte, MG, Brazil2Department of Veterinary Surgery, School of Veterinary Medicine, UniversidadeFederal de Minas Gerais, Belo Horizonte, MG, Brazil3Arapiraca Campus – Vicosa, College of Veterinary Medicine, Universidade Federalde Alagoas, Maceio, AL, Brazil*Corresponding author: adalgizavetufmg@gmail.com

Submitted 4 July 2010: Accepted 23 January 2011 – First published online 14 March 2011 Research Paper

AbstractNutritional management studies to improve the performance of Mangalarga Marchador (MM) horses during themarcha test are limited. This study was designed to test the hypothesis that chelated trivalent chromium (CR)feed supplementation may reduce the suitability of the length of the interval between concentrate feeding andthe marcha test among MM horses. A total of 12 healthy mares (4.25 ^ 0.62 years) were randomly assignedto one of six dietary treatments (0 or 10 mg Cr by concentrate, fed 0.5, 2 or 4 h before exercise), according toa completely randomized design, with a split-plot arrangement. The diet was Cynodon pasture and concentrate(50:50 ratio). The first 29 days of the trial were for diet, Cr and exercise adaptation; during the next 15 days,horses were submitted to three 50-min field marcha tests, once a week. Heart rate (HR) was measured before,during and until 25 min after the exercise. Respiratory rate and rectal temperature were measured; blood sampleswere collected before, at the end and 25 min after the test. There was no effect of Cr by concentrate feeding strat-egy on any physiological variables (P . 0.05). Supplementation of Cr increased glycaemia before and soon afterthe second marcha test (P , 0.01). In addition, Cr reduced HR during the second marcha test and decreasedthe time to first post-exercise HR recovery (P , 0.05). Insulinaemia was greater when the concentrate wasprovided 2 h prior to the test (P , 0.05). Concentrate provided 0.5 and 2 h before the test reduced plasma tria-cylglycerol in the first and second tests, respectively. The interval between concentrate feeding and marcha testsshould not be decreased in horses supplemented with Cr. Horses should be fed more than 2 h before that test. Crsupplementation during training may improve the cardiac performance of MM mares during the marcha test.

Keywords: carbohydrate feeding; horse; insulin; marcha test; micromineral

Introduction

The Mangalarga Marchador (MM) horse is the most

important Brazilian horse breed, and its natural gait is

marcha, rather than trot. The marcha is a comfortable

four-beat lateral and diagonal gait with moments of

triple support and no suspension. The MM horse has

been used for a specific functional trial, called the

marcha test. This test was characterized as an eques-

trian submaximal intensity competition1 of approxi-

mately 50 min with no rest and an average speed of

3.3 m s21. It is a standard evaluation procedure for

Comparative Exercise Physiology 7(3); 133–140 doi:10.1017/S1755254011000043

q Cambridge University Press 2011

most of the official breed championships. The training

and nutritional management carried out in Brazilian

farms are still quite empirical. It is necessary to inves-

tigate procedures that have currently been used for

marcha tests to propose protocols for animal training.

Moreover, the proportion of different types of musclefibres in the MM horses has not been defined.

Ralston2 contraindicated soluble carbohydrate

supplies to horses with less than 4 h before the

prolonged exercise. The resulting hyperinsulinaemia

may negatively interfere with lipid mobilization and

lead to fatigue3. However, a Cr-enriched diet of MM

horses submitted to the marcha test may facilitate

the use of alternative energy sources and therebyreduce carbohydrate oxidation. As part of the chromo-

dulin, Cr may potentiate the effects of insulin (INS)4.

Therefore, the purpose of this study was to measure

the effect of three different intervals between concen-

trate feeding and the beginning of the marcha test and

Cr feed supplementation on biochemical and clinical

responses in MM horses during and after exercise.

Materials and methods

HorsesThe experiment was carried out at Haras Catuni

(latitude, 16841016100S; longitude, 43831021000W; alti-

tude, 784 m), Montes Claros, Minas Gerais, Brazil,from January to February 2008. A total of 12 healthy

MM mares, offspring of two stallions (mean ^ SD),

4.25 ^ 0.62 years old, 339.40 ^ 23.10 kg body

weight (BWT) and with body condition scoring5

(CS) 2–3 (range 1–5), were used. Prior to the exper-

iment the animals had received no training related

to the marcha test. They were kept in an 8.3 ha

paddock, continuously grazing Coast cross (Cynodon

dactylon £ Cynodon nlemfluensis) with water and

mineral supplements ad libitum. In addition, concen-

trate (1.25% BWT day21) was offered subsequent to

moderate work6 at 06.00 and 18.00 hours (Table 1).

BWT and CS were measured weekly. All mares were

treated at the beginning of the pre-experimental

phase for internal and external parasites with 2%

ivermectin (Altec; Tortuga Cia. Zootecnica Agraria,

Sao Paulo, Brazil) and deltamethrin (Butoxw P CE 25;

Intervet/Shering-Plough Brasil, Brazil), respectively.

The Ethics Committee in Animal Experimentation

(CETEA/UFMG) approved the experimental protocol.

Experimental designEach of the 12 mares was assigned to one of two

groups: Cr (10 mg chelated trivalent Cr capsules;

Tortuga Cia. Zootecnica Agraria) and CON (no sup-

plemental Cr), in a completely randomized design,

with a split-plot arrangement. The capsules wereorally administered daily, at 18.00 hours just before

concentrate feeding. The distribution of the animals

within the two experimental groups was based on

the following criteria: each group included two

mares, daughters of the same sire, of similar age,

BWT, body CS, mean heart rate for marcha (HRM)

and HR recovery index (D%). Each pair was assigned

for CON or Cr supplementation. Within each group,two mares were randomly assigned to feed concen-

trate (0.63 kg 100 kg21 BWT) 0.5, 2 or 4 h before the

marcha test. HRM and D% were observed after a

period of 13 min in a continuous session divided into

the following: 4 min walking, 4 min marching, 2 min

walking and 3 min standing at rest, the latter two

being the recovery period. D% was calculated as

½ðHRM 2 HRR5Þ=HRM� £ 100, where HRR5 is the HR atthe end of the recovery period. The first 29 days

(pre-experimental phase) were used for diet, Cr and

exercise adaptation, and the last 15 days were the

experimental phase. During the experimental phase,

the animals were submitted to three 50 min field

marcha tests once a week, in the 1st, 8th and 15th

days of this phase, according to the XXVI Mangalarga

Marchador National Exhibition regulations7.The adaptation of the animals to simulate the

marcha competition1,8 was carried out on an oval

track (0% slope; approximately 20 m £ 50 m). During

the experimental phase, the ridden animals were sub-

mitted to a 5 min walk for warm-up (average speed

1.6 m s21), followed by a 50 min cadenced marcha

(25 min in each direction and average speed

Table 1 Nutritional composition of feed ingredients on a DM basis

Feed DM (%)Digestible energy

(Mcal kg21) CP (%) Lysine (%) NDF (%)

Forage 34.93 2.25 8.06 0.22iii 68.32Concentratei 90.49 3.29ii 15.32 0.78iii 26.06

ADF (%) Ca (%) P (%) Ca:P Cr (mg kg21)

Forage 33.61 0.21 0.23 0.91:1 18.24Concentratei 6.47 1.73 0.63 2.75:1 9.24

i Corn (61%), soyabean meal (19%), wheat bran (15%), limestone (3%), dicalcium phosphate (1%) and mineral salt without Cr

(1%) (Coequi Plus; Tortuga Cia. Zootecnica Agraria) (basic composition per kg: Na, 120 g; Ca, 185 g; P, 60 g; K, 20 g). ii In addition

to the 0.368 Mcal provided by 50 ml of soyabean oil added to the concentrate daily. iii From Valadares Filho32. DM – dry matter;

CP – crude protein; NDF – neutral detergent fibre; ADF – acid detergent fibre; Ca – calcium; P – phosphorus; Cr – chromium.

LR Jordao et al.134

3.33 m s21). The marcha tests were performed at

10.00 and 16.00 hours each day, with six animals in

each period. In each period, there was a mare from

each treatment group (Cr by concentrate feeding).

Thus, two mares were used for each concentrate feed-

ing schedule, one supplemented with Cr and the otherwith placebo.

The mares remained in the paddock without any

athletic activity 1 day before and 1 day after the

marcha test. In the remaining days of this phase,

they were trained every other day, as in the protocol

adapted in the pre-experimental phase.

Diet analysesFeed samples were collected (forage being hand-

plucked9) to determine the chemical composition:

dry matter (DM), crude protein (CP), gross energy

(GE), Ca, P, Cr (measured by atomic absorption spec-

troscopy10), neutral detergent fibre (NDF) and acid

detergent fibre11 (ADF). Faeces were analysed for DM

and GE concentrations10.

Environmental analysesThe room temperature (T), relative air humidity (RH)

(thermo hygrometer MT-242; Minipa Industria e

Comercio, Sao Paulo, Brazil), temperature–humidity

index (THI) and wet bulb globe temperature (WBGT)

index12 were measured during the days and periods

of the marcha tests. A value of THI or WBGT #70

indicated a normal condition, not stressful; values

between 71 and 78 indicated a critical condition;values between 79 and 83 indicated a danger con-

dition; and values .83 indicated an emergency

condition.

Clinical and biochemical analysesPolar RS400SD heart rate monitors (Polar Electro Oy,

Kempele, Finland) were used to assess HR immediately

before the test (BT), during the test, soon after thetest (AT), and 5, 10, 15, 20 and 25 min after. Respi-

ratory rate (RR), rectal temperature (RT) and blood

INS (BET Laboratories Endocrinologia Veterinaria, Rio

de Janeiro, Brazil), cortisol (CORT; BET Laboratories),

glucose (GLU; Accutrend GCT, Roche Diagnostics,

Mannheim, Germany) and lactate (LACT; Accutrend

Lactate, Roche Diagnostics), and plasma glycerol

(GLY; Bioclin Quibasa Quımica Basica, Minas Gerais,

Brazil), and triacylglycerol (TG; triglicerides lıquido

estavel kit, K055, Bioclin Quibasa Quımica Basica)

levels were measured BT, AT and 25 min AT (Table 2).

In addition, the INS:GLU ratio was calculated. The

blood samples were collected via the jugular vein invacuum tubes using no anticoagulant or heparin.

After the analyses of blood GLU and LACT, samples

were maintained in a cooler with ice and centrifuged

on the same day at 3000 rpm for 20 min. The super-

natants were frozen at 2808C with liquid nitrogen

until processing.

StatisticsThe effects of treatments were analysed by ANOVA

and the means compared by F-test using SAEG version

9.1 software (Universidade Federal de Vicosa, Minas

Gerais, Brazil). Data subsets were created to

analyse the effect of the treatments on each marcha

test day. Post hoc comparisons were made, where

appropriate, using the Fisher least significant differ-

ence test for RR and the Newman–Keuls test forthe other variables. Significance was accepted when

P , 0.05. A Lilliefors test for normality was conducted.

Logarithmic transformations to approach normality

were applied to RR and INS:GLU variables. A regres-

sion adjustment model was used to assess the effect

of time on HR. Correlations between the biochemical

and clinical variables were calculated using Pearson’s

correlation analysis.

Results

Environmental parametersThe WBGT index was (mean ^ SD) 76.62 ^ 3.56

during the marcha tests (critical condition12). T (8C),

RH (%) and THI were high at the time of the tests:

30.32 ^ 2.708C, 47.83 ^ 10.30% and 75.79 ^ 2.74

(critical condition12), respectively.

Biochemical parametersConsidering all three marcha tests, there was no effect

of Cr by the time of concentrate feeding prior to

exercise (FEEDING) and the moment of evaluation

(MOMENT; BT, AT or 25 min AT) on the biochemical

variables analysed (P . 0.05). However, for the three

Table 2 Methods used to determine concentrations of the analytesinvestigated in this study

Analyte Sample Method

CORT Serum Radioimmunoassay, solid phaseGLU Whole blood Amperometry, GLU dehydrogenaseGLY Heparinized plasma Enzymatic colourimetric assayINS Serum Radioimmunoassay, solid phaseLACT Whole blood Amperometry, LACT oxidaseTG Heparinized plasma Enzymatic colourimetric assay

Feed prior to exercise and Cr supplementation 135

marcha tests, the effects of MOMENT on CORT, GLY

(Fig. 1) and LACT were significant (P , 0.01). Blood

LACT levels of AT (2.09 mmol l21) and 25 min AT

(2.29 mmol l21) were similar; however, these values

were higher (P , 0.01) than BT (1.84 mmol l21); co-

efficient of variance (CV) 24.63%. Nevertheless, there

was no effect of either Cr or FEEDING on the variables

GLU and TG (P . 0.05).

Table 3 presents a concentrate feeding strategy by

MOMENT interaction effects on INS (P , 0.05) and

INS:GLU (P , 0.001). The 4 h group showed the least

severe insulinaemia BT, AT and 25 min AT. In this

group, INS was lower AT and 25 min AT than BT. Inter-

estingly, INS was higher when the concentrate was fed

2 h rather than 0.5 h prior to exercise BT, AT and

25 min AT. In both FEEDING groups, INS was higher

BT, followed by AT, reducing further 25 min AT. Also,

concentrate feeding , 2 h before the marcha test

increased INS:GLU AT and 25 min AT. INS:GLU for

the 0.5 h group remained high during the three time

points of analysis.

In the first test, the 0.5 h group showed the lowest

TG (P , 0.001; Table 4). In contrast, the 2 h group

showed the lowest TG on the second test (P , 0.05;

Table 4).

There was a Cr by MOMENT effect interaction on

GLU in the second test (P , 0.01; Table 5). Surpris-

ingly, Cr increased glycaemia BT and AT and main-

tained almost constant GLU at these moments. In

contrast, the CON group showed a decline in GLU at

the end of the exercise.

In the third test, there was a FEEDING by MOMENT

effect interaction on GLU (P , 0.01; Table 4). The

group that consumed concentrate 4 h prior to the

test showed GLU levels that were almost constant

over the three moments of analysis. The 2 h group

ended the test with lower GLU that returned to

a value close to pre-exercise values by 25 min AT.

The 0.5 h group began the exercise with reduced

GLU in relation to the two moments AT. During the

test, the 0.5 h group showed a higher GLU than the

other groups.

Finally, there was a weak negative correlation between

INS and CORT after exercise (r ¼ 20.42; P , 0.02),

and the level of INS showed a weak positive correlation

with GLU only 25 min AT (r ¼ 0.44; P , 0.01).

Heart rateThe main results of the HR analysis are shown in

Figs 2a and 2b and Tables 3 and 5. The result of the

HRM test was 145 ^ 32 beats min21 (bpm). According

to the regression prediction model (Fig. 2a), the

peak HR (HRpeak) of the marcha test was 153 bpm

at 17 min (P , 0.0001). HR reached stability after2 min, remaining almost constant until the end of the

activity period.

In accordance with the regression prediction model

of post-exercise HR recovery (Fig. 2b), there was a rapid

reduction in HR over the first 2 min, with 50% reduction

in the HRpeak 25 min after exercise (P , 0.01). However,

recovery values were higher (52 ^ 10 bpm), even at

the end of post-exercise recovery (77 bpm).

0

50

100

150

200

250

300

BT AT 25 min AT

CORT (ng ml–1)GLY (mg dl–1)

RR (bpm)

RT (°C)

ab

c

a

a

a

b

c bcb

b

FIG. 1 Blood CORT and plasma GLY levels (P , 0.01), RR and RT (P , 0.0001) of MM mares BT, AT and 25 min AT. Mean ^ SD CV,34.67% (CORT); 55.56% (GLY); 10.72% (RR); 1.56% (RT). bpm, breaths per min a, b, c: Means followed by different letters vary by theNewman–Keuls test (CORT, GLY and RT) or Fisher’s least significant difference test (RR).

Table 3 Blood INS levels, INS:GLU ratio and post-exercise HRrecovery of MM mares fed at different times before the marchatestsi

Feeding (h:min)

Moment 4:00 2:00 0:30

INS (pmol l21)*BT 151.89Ac 222.21Aa 186.84Ab

AT 88.32Bc 186.89Ba 159.43Bb

25 min AT 42.48Bc 171.70Ca 116.67Cb

INS:GLU (pmol mg l21 dl21)***BT 1.15Ab 1.94Aa 0.72Ab

AT 0.50Bb 1.22Ba 1.36Aa

25 min AT 0.29Bb 0.79Ba 1.22Aa

Post-exercise HR recovery*Feeding (h:min) HR (bpm)

4:00 87B

2:00 85B

0:30 97A

* P , (0.05). *** P , (0.001). i Only the results that were statistically differ-

ent are shown in the table. Feeding, time of concentrate feeding prior to

exercise. Means in rows followed by different lower case letters and in

columns by different upper case letters differ by the Newman–Keuls test.

CV, 64.79; 35.37 and 30.26%, respectively.

LR Jordao et al.136

There was no Cr by FEEDING by MOMENT effect

interaction on HR (test and post-exercise recovery;

P . 0.05). Also, there was no effect of the test day

(TEST) on these variables (P . 0.05). However, therewas an interaction of Cr by TEST effect on HR of

test and post-exercise recovery values (P , 0.05;

Table 5). Cr was responsible for the lowest HR in the

second test and the lowest post-exercise HR recovery

in the first test. In addition, the 0.5 h group showed

the highest level of post-exercise HR recovery

(P , 0.05; Table 3).

Respiratory rate and rectal temperatureThere was no effect of Cr or FEEDING on RR and RT

(P . 0.05). Also, there was no effect of TEST on

these variables (P . 0.05). However, Fig. 1 presents

higher values of RR and RT AT, followed by 25 min

AT (P , 0.0001). There was a strong positive corre-

lation between RR and RT (r ¼ 0.80; P , 0.0001).

Discussion

The HRpeak for the marcha test was lower than

the maximum HR (HRmax)13 (220–260 bpm). When

associated with the HRpeak value, the stability of the

HR values was used to characterize this exercise as

submaximal intensity (as in the study by Prates

et al.1). In addition, it may be deduced that the

marcha test is essentially aerobic, as the HR remained

close to 150 bpm and blood LACT remained close to

2 mmol l21 during exercise14.

From the HRM, the average VO2 of animals was

estimated at 66.4 ml kg21 min21 15. This value allowed

us to estimate the mean energy expenditure during

the test, assuming that each horse metabolizes

approximately 4.86 kcal for each litre of oxygen

utilized6. Additionally, from the HRpeak of marcha and

considering the HRmax as 220 bpm13, the oxygen utili-

zation reached 55.3% of maximum VO2 (VO2max)16.

This value suggests that the marcha test is a moderate-

intensity exercise (range 35–55% VO2max).

Dietary Cr supplementation has attracted attention

because this micromineral improved both the perfor-

mance of exercising horses1,17 and GLU tolerance in

humans18,19, horses20 and other farm animals21,22.

It was therefore hypothesized that, in this study, Cr

would reduce the interval between the marcha test

and pre-exercise carbohydrate feeding (according to

Ralston2, 4 h). However, Cr did not interact with

FEEDING and did not prevent the adverse effects of

this nutritional management.

Unexpectedly, Cr was responsible for increased

glycaemia before and after the second test. A possible

explanation could be a blood GLU-sparing effect,

mediated by increased reliance on fat substrate

stores, based on evidence of its lipolytic effect23.

Another possibility is that Cr stimulated hepatic glyco-

genolysis and gluconeogenesis.

In this study, 10 mg of Cr did not reduce the RR or

RT at the end of the exercise (in contrast to the results

published by Prates24). However, Cr reduced HR

during the test and accelerated the speed of decline

in HR recovery, improving cardiac performance (as

in the study by Prates et al.1). Furthermore, Cr made

the marcha more energy efficient, because there is a

strong relationship between HR and oxygen utilization

at submaximal exercise intensities15.

Horses fed grain and subjected to prolonged

aerobic exercise may have a higher degree of Cr

Table 5 Cr effects on glycaemia and HRof MM mares during each marcha test andsubsequent post-exercise recoveryi

Blood GLU (mg dl21) on the second dayof testing**

Moment Cr CON

BT 132.12Aa 120.14Ab

AT 142.50Aa 98.50Bb

25 min AT 125.50Aa 134.33Aa

HR during tests (bpm)*Cr CON

First 157Aa 147Aa

Second 139Bb 159Aa

Third 145ABa 150Aa

Post-exercise HR recovery (bpm)*Cr CON

First 77Bb 98Aa

Second 98Aa 91Aa

Third 89Aba 83Aa

* P , (0.05). ** P , (0.01). i Only the results that

were statistically different are shown in the table.

Means in rows followed by different lower case

letters and in columns by different upper case

letters differ by the Newman–Keuls test. CV, 29.68;

29.75 and 38.36%, respectively.

Table 4 Plasma TG and blood GLU levels of MMmares from three different marcha testsi

Feeding (h:min) TG (mg dl21)***

GLU (mg dl21)* and TG (mg dl21)* by test day

First test4:00 41.33A

2:00 40.56A

0:30 17.48B

Second test4:00 40.87A

2:00 24.02B

0:30 39.24A

Third testGLU (mg dl21)**

Feeding (h:min) BT AT 25 min AT4:00 131.09Aa 130.50Ba 126.00Aa

2:00 141.63Aa 119.50Bb 142.25Aa

0:30 115.54Ab 153.50Aa 136.75Aa

*P , (0.05). **P , (0.01). ***P , (0.001). i Only the results that

were statistically different are shown in the table. Feeding, time

of concentrate feeding prior to exercise. Means in rows followed

by different lower case letters and in columns by different upper

case letters differ by the Newman–Keuls test. CV, 32.71; 49.83

and 17.80%, respectively.

Feed prior to exercise and Cr supplementation 137

deficiency due to increased excretion in the urine,

especially in situations of high stress25,26. Therefore,

the favourable effect of Cr on the marcha test can

be explained partially by the intense thermal discom-

fort and high thermoregulatory demand suffered

during the exercise.

Cr did not interfere with INS, CORT, lipolysis (which

would be evidenced by higher GLY and TG) or LACT.

These results are different from those published by

Pagan et al.17, in which exercising horses sup-

plemented with 5 mg Cr yeast had higher plasma TG

levels after a standardized exercise test (SET) and

lower plasma CORT before and at the beginning of

the exercise. There was still a trend towards a decrease

in plasma INS (P , 0.10) 1 h after grain feeding and a

trend towards a decrease in plasma LACT (P ¼ 0.08) in

the final stages of the SET.

In competition, MM horses perform various tests for

about 50 min and are provided with short periods of

rest between, often under intense heat and high RH.

As the animals progress through the various compe-

tition stages, they may be allowed to compete for

two to three consecutive days. Thus, a rapid recovery

provides obvious advantages in marcha competitions.

Based on the results of this study, Cr supplementation

in the diet of MM horses submitted to the marcha test

may be considered an effective nutritional ergogenic

aid. However, Cr is neither a panacea nor a drug;

rather, it is a nutrient. The complexity of neuroendo-

crine regulation during exercise (inherent in the type

of exercise and food status involved) and the different

methodological and environmental conditions could

explain the variation in results recorded in the

literature.

It is possible that the intravenous administration of

GLU before exercise increases the time to fatigue

during moderate-intensity exercise27. However, in

long-duration and low-intensity exercise (.3 h) such

as endurance racing, the concentrate intake may

increase the level of insulinaemia during exercise.

This would decrease the degree of fat utilization with

a concomitant increase in carbohydrate oxidation

during exercise and acceleration of fatigue3. The

marcha test is a moderate-intensity exercise, and it

has intermediate duration between explosion exercises

(e.g. Thoroughbred racing) and endurance. Thus, its

duration is not extensive enough for hypoglycaemia

to occur, and a marked increase in lactataemia

occurs even if concentrated meals are offered less

than 2 h before exercise. Additionally, regular exercise

may improve glycaemic CON28 and prevent hypo-

glycaemia caused by concentrate feeding.

Contrary to expectations, animals in the 0.5 h group

did not show an increase in INS as high as that

observed in the 2 h group. When the mares were fed

concentrate 30 min BT, there was probably insufficient

time for digestion of the ingested concentrate, result-

ing in the recorded blood INS levels. According to

Frape29, ingesta remain in the stomach for 15 min on

average, and their passage through the small intestine

occurs 30–90 min after food intake. Another expla-

nation for this is that there is reduced blood flow to

the splanchnic vascular bed during exercise30, which

may impair the digestion and absorption of nutrients.

The 4 h group experienced minimal fluctuations

in GLU on the third test, which may reflect improved

homeostatic regulation on GLU in this approach to

nutritional management. In addition, the lower INS

and INS:GLU recorded in this group are in agreement

with Ralston2, who maintained that concentrate

should be offered at least 4 h before the long-duration

exercise to avoid hyperinsulinaemia. On the other

hand, the slower return of HR recovery to resting

values in the 0.5 h group is an indication of a clinical

HR = 155.75 – (44.01/T ); R2 = 91.85%

0

20

40

60

80

100

120

140

160

180a) b)

0 10 20 30 40 50Time (min)

HR

(bp

m)

HR = 137.44 – (6.90 T + 0.18 T 2);R2 = 87.71%

0

20

40

60

80

100

120

140

160

0 10 20 30Time (min)

HR

(bp

m)

HR_estHR_obs

HR_estHR_obs

FIG. 2 (a) HR of Cr and CON experimental groups during the 50 min of all three marcha tests (P , 0.0001). (b) HR during the 25 min ofall three post-exercise recoveries (P , 0.01). HR_obs – observed HR; HR_est – estimated HR. T – time (min)

LR Jordao et al.138

abnormality such as severe dehydration, and is associ-

ated with fatigue13. Therefore, it is likely that this

group experienced haemodynamic changes related to

the displacement of fluid into the gastrointestinal tract

associated with excessive sweating (subjectively ana-

lysed) during the marcha test.

As the concentrate supply 0.5 or 2 h before the

marcha test increased INS and INS:GLU and had anegative influence on lipid mobilization, we recommend

that researchers should not provide a concentrated

meal within 2 h before the test. The marcha test can

reach 2 h of duration in extraordinary conditions, and

so higher blood INS levels during the exercise may

lead to early fatigue.

Finally, CORT is considered a stress hormone and a

factor for immunosuppression. It has an antagonisticeffect on INS31, and a characteristic response was

noted in this experiment (shown by the negative cor-

relation between INS and CORT at the end of the

test). CORT levels are increased to avoid the deleter-

ious effects of hypoglycaemia, which can occur

during an exercise of prolonged duration.

In conclusion, this study showed that dietary Cr

did not reduce the appropriate interval between con-centrate feeding and the marcha test in MM mares.

Cr supplementation during training may improve

cardiac performance during the test, and MM mares

should be fed more than 2 h BT. The HR values

characterized the marcha test as a predominantly

aerobic and moderate-intensity submaximal exercise.

Acknowledgements

We thank the FAPEMIG, CAPES, CNPq, Escola de Veter-inaria da UFMG, Pro-Reitoria de Pesquisa da UFMG and

Tortuga Cia. Zootecnica Agraria for funding this

project. We would also like to thank Mr Joao Carlos

Moreira’s family for providing animals and logistics.

In addition, we are grateful to Neimar Severo (ABS Pec-

Plan), BET Laboratories, ABCCMangalarga Marchador

and Leonardo de Freitas (Bioclin Quibasa Quımica

Basica) for their support. Dr Angela Lana and DrDanusa Soares contributed invaluable advice with

respect to the experimental design.

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