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*
Virginia Polytechnic Institute and State University, Blacksburg, VA 24601-0606 and
Hebrew University, Rehovot, Israel
1To whom correspondence should be addressed at Campus Box 219, Virginia Intermont College, Bristol, VA 24201. E-mail: thiers{at}3wave.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • protein • acid base • fat • horses
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Increased
needs for protein during exercise include maintenance of new muscle
mass, repair of existing muscle mass and replacement of any nitrogen
that would be lost in sweat (Meyer 1987
). The upper
limit of dietary protein for exercising horses should minimize
thermogenic, ureogenic and acidogenic effects, but the lower limit must
avoid protein deficiency.
The current dietary protein recommendations are 9.8, 10.4 and
11.4% crude protein (CP)2
for horses
in light, moderate and intense exercise, respectively (National Research Council 1989
). Surveys in Michigan (Gallagher et al. 1992a and 1992b
) and Australia (Southwood et al. 1993
) revealed that thoroughbreds and standardbreds in training
were fed an average of 14% CP. Another study in central North Carolina
(Honore and Uhlinger 1994
) found that 70% of the horses
were being fed protein in excess of National Research Council
recommendations.
Heat production from higher levels of protein may interfere with
exercise by attenuating fatigue (Kronfeld 1996
) and an
increased acidity will interfere with glycolysis and muscle fiber
contraction (Mainwood and Renaud 1985
). Restriction of
dietary protein may be beneficial during strenuous exercise by
diminishing production of heat and acid. A restricted protein diet has
been shown to reduce the acidity of exercise in
Arabian horses performing repeated sprints resulting in higher pH and
bicarbonate levels (Graham-Thiers et al. 1999b
).
Fat adaptation refers to the combined effect of training and prolonged
consumption of a high fat (HF) diet (Kronfeld and Downey 1981
). Fat adaptation has influenced acid-base responses to
repeated sprints, mainly by reducing the increase in
PCO2 in venous blood (Kronfeld et al. 1998
). An increase in
PCO2 in venous blood has resulted in
an acidosis but a concurrent decrease in
PCO2 in arterial blood has resulted
in an alkalosis (Taylor and Kronfeld 1995
). Other
influences on acid-base balance include strong ion difference
(SID), which is the difference between strong cations and strong anions
in the body, and total weak acids (Atot; Stewart 1981
).
These factors may be influenced by a reduction in dietary protein that
may reduce anions. Separating the effects of
PCO2, SID and total weak acids on
hydrogen ion concentration revealed that restricted dietary protein
reduced hydrogen ion concentration mainly due to an increase in SID.
Restricted dietary protein reduced the level of anions thus improving
SID and lowering hydrogen ion concentration (Graham-Thiers et al. 1999c
).
Fat supplementation may spare protein during energy demanding states,
such as food deprivation or during energy restriction (McCargar et al. 1989
). This study tested the hypothesis that fat
adaptation may reduce the risk of protein deficiency when dietary
protein is restricted and energy demand increased, such as during
strenuous exercise. Addition of limiting amino acids may also reduce
the risk of protein deficiency. This study also tested the hypothesis
that acid-base responses to repeated sprints will be reduced by the
combination of dietary protein restriction and fat adaptation. In
another paper, we reported that several indices of protein status were
not diminished by the restricted level of crude protein (fortified with
lysine and threonine) in this experiment (Graham-Thiers et al. 1999a
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twelve previously unconditioned Arabian horses (6 mares, 6 geldings,
ages 5–11 y) were housed in a dry lot with free access to water.
General health and feed intake were observed daily, and body weight,
body condition and coat condition were monitored every 2 wk. Body
condition was evaluated by a standardized system (Henneke et al. 1983
). Coat was evaluated on shine and smoothness. The protocol
was approved by the Institutional Animal Care and Use Committee.
Diets.
Horses were randomly assigned to four complete feeds (HPHF, LPHF, HPLF,
LPLF) which were formulated to provide 12.5 MJ of digestible
energy/kg of dry matter (Table 1
). Protein was also formulated at two levels: high protein (HP; 14.5
g/100 g CP) and low protein (LP; 7.5 g/100 g CP) supplemented with
0.5% L-lysine and 0.3% L-threonine (Heartland
Lysine, Chicago, IL) to match the amino acids in HP. The National
Research Council currently recommends a dietary CP level of 11.4% for
horses in intense exercise (National Research Council 1989
). Fat was also at two levels: either 13 g/100 g (10 g/100
g added corn oil; HF) or 3 g/100 g (no fat supplementation, low fat
[LF]). A 4-wk adaptation period began the experiment. Feed intake was
adjusted to maintain body condition.
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All horses underwent 11 wk of conditioning using a high speed treadmill
(Mustang 2000; Kagra, Fahrwangen, Switzerland). The protocol consisted
of interval training divided into three phases (Table 2
). Horses were interval trained twice a week and were walked at 1.5 m/s
for 30 min on a mechanical walker on rest days.
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Before conditioning, but after the dietary accommodation period, all
horses performed a standard exercise test (SET-U). The SET consisted of
6 min at the walk (1.5 m/s; 3 min on no slope and 3 min on a 6%
slope), a 5-min warm-up at the trot (3.5 m/s) followed by six
1-minute sprints at 7 m/s on a 6% slope separated by 4-min walks (1.5
m/s), and a 30-min recovery period at the walk with no slope. After the
conditioning period, two SET were conducted 7 d apart (Fig. 1
). These two SET were similar to SET-U except that the sprints were
at 10 m/s. This exercise testing was designed to simulate a typical
sport horse’s events, often being repetitive in a short period.
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50%. A
14-guage, 150-cm polyethelene catheter (Intramedic polyethylene tubing;
Becton Dickinson, Sparks, MD) was fed down the left jugular vein into
the pulmonary artery and a 18-guage catheter was placed in the right
carotid artery (Angiocath Deseret Medical Inc., Sandy, UT) 60 min
before the SET (Taylor and Kronfeld 1995
). Placement of
the end of the tubing in the pulmonary artery was facilitated by using
an oscilloscope attached to a pressure transducer (Propaq 140; Protocol
Systems, Beaverton, OR). A thermocouple (Model IT-18; Physitemp
Instruments, Clifton, NJ) was inserted to the catheter tip in the
pulmonary artery. Sampling and analysis.
During the SET, 10 mL of arterial and 30 mL of mixed venous blood were
taken at rest, during the last 15 s of sprint 1, 2 and 6 as well
as at 5, 10, 20 and 30 min of recovery. Blood samples were taken
anaerobically into heparinized syringes (Sherwood Medical, St. Louis,
MO) for analysis of PO2,
PCO2, pH, [Na+] and
[K+] (STAT Profile 2; Nova Biomedical, Waltham, MA).
Bicarbonate was calculated from pH and PCO2.
Additional heparinized blood was centrifuged to obtain plasma. An
aliquot of plasma was immediately deproteinized in ice-cold
perchlorate for lactate (La-) assay (Proc. No. 826-UV;
Sigma Diagnostics, St. Louis, MO). The remaining plasma was frozen for
later analysis of total protein (TP; Proc. No. 541; Sigma Diagnostics),
Cl- (Proc. No. 461; Sigma Diagnostics) and cortisol
(Coat-A-Count Cortisol, St. Louis, MO). Atot were estimated from TP
using a conversion factor of 0.21 mEq/L TP (Stämpfli et al. 1999
). SID+ was calculated as the algebraic sum
of [Na+], [K+], [La-] and
[Cl-]. Samples were analyzed in tripicate, with
duplicates being selected that varied by <1%.
Statistics.
Data were summarized as least squares means and standard errors.
Analysis of variance with repeated measures was used to evaluate the
effects of time (exercise and/or recovery), fat level, protein level
and interactions in the SET (SAS 1991
). Data that were
not significantly different between SET-2 and SET-3 in conditioned
horses were pooled and reported as SET-C. Significance of
difference was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rest.
Before SET-1, arterial [La-] was higher in the LP groups (P = 0.0022) but lower in the HF groups (P = 0.011). There were no differences in PO2, PCO2, SID, Atot, [Na+], [K+], [Cl-], pH or [HCO3-]. Venous [La-] was also higher for the LP groups (P = 0.0076) but was not affected by fat level. No differences were found in PO2, PCO2, SID, Atot, [Na+], [K+], [Cl-], pH or [HCO3-].
Before SET-2, arterial [K+] was higher in the LP groups (P = 0.039) as was [HCO3-] (P = 0.032). No differences were found in pH, PO2, PCO2, lactate, SID, Atot, [Na+], [K+] or [Cl-]. Venous [HCO3-] was higher (P = 0.024) in the LP groups as well as [K+] (P = 0.025). Plasma cortisol was lower in the HF groups (P = 0.0008) as well as in the LP groups (P = 0.0043). There were no differences for pH, PO2, PCO2, SID, Atot, [La-], [Na+] or [Cl-].
Exercise.
During SET-1, arterial PCO2 was decreased (P = 0.0001). Increases were observed in [La-] and [K+] (P = 0.0001), PO2 (P = 0.001), [Na+] (P = 0.0009) and [Cl-] (P = 0.0007) and pH (P = 0.0001). Exercise had no effect on arterial [HCO3-] and SID. In venous plasma, decreases were observed in pH, [Cl-] and PO2 during the SET (P = 0.0001). Increases were observed in PCO2, [Na+], [K+], [HCO3-] and [La-] (P = 0.0001). Venous SID was unaffected by exercise.
During SET-2, arterial [HCO3-] and PCO2 decreased (P = 0.0001), along with pH (P = 0.0086) but [La-] increased (P = 0.0001). Plasma [Cl-] increased (P = 0.0003) along with [K+] (P = 0.07), [Na+] (P = 0.0048) and PO2 (P = 0.0007). Arterial SID was unaffected by exercise. In venous plasma, decreases were observed in pH and PO2 during the SET (P = 0.0001). Increases were observed in PCO2, [HCO3-], [La-] and [K+] (P = 0.0001). Plasma [Na+] also increased (P = 0.0002) along with SID (P = 0.0038) and plasma cortisol (P = 0.0004).
Diet and exercise.
During SET-1, venous pH was higher (P = 0.0056) in the
LP groups (Fig. 2
) but was not affected by fat level. Venous
[HCO3-] was not affected by
fat or protein level.
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Venous SID was affected by a time x protein interaction
(P = 0.022) being higher in the LP groups during the
sprints (Fig. 3
). Venous [Na+], [K+]
and [Cl-] were not affected by protein and
there was not an effect of fat. Venous [La-]
was higher (P = 0.023) in the LF groups but was not
affected by protein level (Fig. 4
).
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During SET-2, venous pH was affected by a fat x protein
interaction (P = 0.022) being higher in the LPHF group
and lower in the LPLF group (Fig. 5
). Venous [HCO3-] also had a
fat x protein interaction (P = 0.043) during
recovery, remaining elevated in the LPHF group (Fig. 6
).
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Venous SID had a time x protein interaction (P = 0.038) with higher SID in the LP groups during the later stages of the
sprints (Fig. 7
). Venous [Na+] was higher (P = 0.007) in the LP groups but was not affected by fat. Venous
[K+] was higher (P = 0.026) for
the LP groups, especially during the sprints, but again, was not
affected by fat. Venous [Cl-] was higher
(P = 0.046) during the sprints in the HF groups. Venous
[La-] was higher (P = 0.05) in
the LF groups, especially during the sprints, but was not affected by
protein.
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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the comprehensive physico-chemical model of acid-base status
by Stewart (Stewart 1981
), the dependent variables,
hydrogen ion and bicarbonate ion, are influenced by changes in the
three independent variables, PCO2,
SID and Atot. During SET-U, venous pH was higher for horses in the
LP group. This difference in pH was attributable mainly to SID because
PCO2 and Atot were not affected by
diet. During SET-C, the lower venous pH in exercising horses fed
the two low fat diets could be explained by the higher
PCO2. In studies of intensely
exercising horses, PCO2 was the main
determinant of changes in venous or arterial plasma pH (Kronfeld 1996
). In contrast, the higher venous pH found in horses fed
the LPHF diet could be explained by a combination of reduced
PCO2, due to fat adaptation, and
increased SID, due to protein restriction.
The effect of protein restriction on venous plasma pH observed here
confirms previous observations (Graham-Thiers et al. 1999b
). This earlier study also revealed that the higher pH was
influenced through changes in SID (Graham-Thiers et al. 1999c
). The lack of effect on pH and
PCO2 in arterial blood is consistent
with arterial blood reflecting pulmonary ventilation more than
peripheral metabolism (Carlson 1995
)
Dietary protein contains sulfur and phosphorus that become oxidized to
sulfate and phosphate. The amino acids that are oxidized during
exercise (leu, iso, val) do not contain sulfur or phosphorus so dietary
protein is not expected to directly affect the acidogenic effect of
sprint exercise. Prolonged restriction of dietary protein is expected
to exert a chronic anti-acidogenic effect. Oxidation of
sulfur-containing amino acids increases the net acid load in the
body by 2 mol for every mole of sulfur-containing amino acid
oxidized. Endogenous acid is also increased by dibasic and
phosphorylated amino acids (Patience 1990
). In the
present study, venous SID was higher in the LP group over the course of
the SET, compared with the HP group. Restricting dietary protein may be
an alternative to replacing NaCl with sodium bicarbonate for reducing
exercise acidosis, especially in horses consuming a HF diet. This would
be beneficial for horses working hard in the heat because HP is
thermogenic and will affect water balance with increased urination and
possibly higher sweat loss (Meyer 1987
).
In a previous study, sodium bicarbonate administration had an additive
or, perhaps, synergistic effect with fat adaptation in moderating the
blood lactate increase during repeated sprints (Ferrante et al. 1994
). Dietary protein restriction had a similar interaction
with fat adaptation in moderating the acidogenic effect of repeated
sprints in the present experiment.
Dietary fat has also been observed to spare protein, especially under
conditions of high energy demand, such as exercise. The present results
suggest that protein deficiency may have been avoided partly because of
fat adaptation (in addition to added lys and thr) in exercising horses
fed a restricted protein diet. Fat adaptation has previously been
suggested to improve metabolic regulation as indicated by plasma
lactate responses in repeated sprints and incremental excercise.
Moreover, fat adaptation increases fatty acid oxidation, which is more
efficient than amino acid oxidation (Kronfeld 1996
).
Lower plasma cortisol levels in the HF group may provide evidence of
less reliance on glucose as fuel and more reliance on fat metabolism,
with a diminished need for gluconeogenisis. With less amino acid
oxidation, less endogenous acid would be produced in the body
(McCargar et al. 1989
). This would partly explain the
higher pH and [HCO3-] in
venous blood of horses in the LPHF group during SET-C.
In conclusion, the acidogenic effect of sprinting exercise was diminished by both LP diets, regardless of fat level before conditioning, but only by the LPHF diet after conditioning. Thus, the combination of protein restriction and fat adaptation was needed for horses in training to reduce the acidogenic effect of intense exercise.
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FOOTNOTES |
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Manuscript received June 30, 2000. Initial review completed January 12, 2001. Revision accepted March 26, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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