Concurrent supplementation of arginine, vitamin E, and vitamin C improve
cardiopulmonary performance in broilers chickens
C. A. Ruiz-Feria1
Department of Poultry Science, Texas A&M University, College Station 77843-2472
ABSTRACT Two experiments were conducted to evalu-
ate the effects of arginine, vitamin E (VE), and vitamin
C (VC) on cardiopulmonary performance and ascites
parameters of broilers reared under a cold environmen-
tal temperature. One-day-old male broilers were fed a
basal corn-soybean meal diet (control, 1.2% arginine
and 40 IU of VE), or the basal diet supplemented with
1% arginine and either 200 IU vitamin E (AE), 500
mg of vitamin C (AC), or a combination of VE and
VC at the same amounts (AEC) per kilogram of feed.
Pulmonary arterial pressure (PAP) and mean arterial
pressure were recorded in clinically healthy, anesthe-
tized birds (28 to 42 d old) before and after an epineph-
rine (Epi) challenge (0.5 mg/kg of BW, i.v.), an amin-
oguanidine hemisulfate challenge (100 mg/kg of BW,
i.v.), and an N-nitro-l-arginine methyl ester challenge
(50 mg/kg of BW, i.v.) at 20-min intervals. Data were
analyzed by repeated measures ANOVA, and the Stu-
dent Newman-Keuls test was used to separate means
within groups. The PAP increased 30 s after the Epi
Key words: arginine, pulmonary hypertension, vitamin E, vitamin C
INTRODUCTION
Pulmonary hypertension syndrome (PHS, ascites)
is a common metabolic disorder of modern, fast-grow-
ing strains of broilers. Ascites mortality peaks close to
market age when the birds are 5 to 6 wk old, which
represents a significant loss to the industry, given the
cost of production up to this point (Maxwell and Rob-
ertson, 1998). Ascites can be initiated by an increase in
metabolic rate, which implies a greater oxygen demand
(Scheele et al., 1991; Buys et al., 1999; Wideman and
Tackett, 2000). In a gas exchange system working close
to its physiological limit, this elevation in metabolic
demand can lead to cellular hypoxia (Scheele et al.,
1991). To avoid cellular hypoxia, an increased cardiac
©2009 Poultry Science Association Inc.
Received September 18, 2008.
Accepted October 21, 2008.
1
Corresponding author: ciro.ruiz@poultry.tamu.edu
526
challenge in all birds, but the peak PAP was lower in
the AEC group than in all the other groups, whereas
birds in the AE and AC groups had lower PAP peaks
than did the control group. After 120 s of challenge, the
PAP was lower in AEC birds compared with the other
birds. The PAP returned to pre-Epi amounts within
300 s in all groups. The PAP was increased (P < 0.05)
within 60 s after the aminoguanidine hemisulfate and
N-nitro-l-arginine methyl ester challenges in all groups,
but no differences were found among groups. The mean
arterial pressure responses did not differ among groups.
Plasma NO was greater in the AEC group than in all
the other groups before and after the Epi challenge.
These results showed that Epi elicited lower amplitude
PAP and less prolonged increases in PAP in birds from
the AEC group, and this may have been related to the
increased vasodilation attributable to NO production.
The AEC may have had complementary effects against
oxidative stress, protecting the endothelium and pre-
serving NO function.
2009 Poultry Science 88:526–535
doi:10.3382/ps.2008-00401
output must be propelled through the noncompliant
pulmonary vasculature, which requires increases in pul-
monary arterial pressure (PAP) and right ventricle
(RV) work (Wideman, 1988). Sustained increases in
PAP cause hypertrophy and subsequent dilation of the
RV (Peacock et al., 1990; Wideman, 1999).
By reducing the pulmonary vascular resistance, it
is possible to reduce the PAP needed to propel the
cardiac output required to match the metabolic de-
mands of the broilers (Wideman et al., 1995) and delay
the pathophysiological progression leading to ascites.
Nitric oxide is an important endogenous vasodilator
produced by endothelial cells through an oxygen-de-
pendent 5-electron oxidation of a terminal guanidine
nitrogen of l-arginine (ARG; McQueston et al., 1993;
Palmer et al., 1998). l-Arginine is an essential amino
acid in birds (Tamir and Ratner, 1963), and it has been
reported that ARG amounts supporting the maximal
growth rate are not adequate for maximal NO produc-
tion (Taylor et al., 1992; Dietert and Austic, 1994).
 ARGININE, ANTIOXIDANT VITAMINS, AND ASCITES
Supplemental ARG reduced the incidence of ascites in
broilers exposed to a cool temperature (Wideman et al.,
1995; Tan et al., 2005), improved pulmonary vasodila-
tion (Wideman et al., 1996; Lorenzoni and Ruiz-Feria,
2006), and facilitated vasorelaxation in fowl (Martinez-
Lemus et al., 1999). However, the effects of ARG on
ascites incidence have not been consistent (Ruiz-Feria
et al., 2001).
Oxidative stress is also involved in the etiology of
PHS (Peacock et al., 1990; Maxwell et al., 1992; En-
kvetchakul et al., 1993; Bottje and Wideman, 1995;
Grobe et al., 2006). A sustained high PAP is associated
with hypoxia, endothelial damage, and reactive oxy-
gen species production. Chronic hypoxia increases ROS
concentrations and impairs endothelial NO-dependent
relaxation in mice pulmonary arteries (Fresquet et
al., 2006). The superoxide anion causes a loss of NO
bioavailability by shortening its half-life (Jung et al.,
2003), thereby reducing the potential for endothelial
vasodilation (Lopez-Lopez et al., 2001). Furthermore,
the reaction of superoxide anion with NO leads to pro-
duction of peroxynitrite, a potent oxidant agent respon-
sible for direct tissue damage (Beckman et al., 1990;
Szabo, 1996).
Ascitic broilers have been reported to have low con-
centrations of vitamin E (VE; Bottje et al., 1995) and
ascorbic acid (Enkvetchakul et al., 1993) in the lungs
and liver. Vitamin E supplementation reduced the inci-
dence of ascites when administered as a subcutaneous
implant, but not when provided as a dietary supple-
ment (Bottje et al., 1995, 1997). We previously report-
ed that VE at 400 IU/kg of feed, when supplemented in
combination with ARG, was associated with less effec-
tive pulmonary vasodilation after an epinephrine (Epi)
challenge, compared with birds receiving only ARG
(Lorenzoni and Ruiz-Feria, 2006). In addition, birds
receiving ARG and large amounts of VE had greater
concentrations of TBA-reactive substances than birds
fed normal diets, suggesting that large amounts of VE
increased oxidative stress. Vitamin E reacts with or-
ganic peroxides, interrupting the chain reaction of lipid
peroxidation involving the formation of tocopheroxy
radicals, which can act as prooxidative agents and ini-
tiate oxidative processes (Schneider, 2005).
Ascorbate has been shown to prevent the prooxidant
activity of α-tocopherol by reducing the α-tocopheroxyl
radical to α-tocopherol, and then acting as a coan-
tioxidant (Carr et al., 2000). Through this mechanism,
vitamin C (VC) supplementation has been reported
to increase or restore plasma and tissue VE (Liu and
Lee, 1998; Keller et al., 2004). Furthermore, ascor-
bate has been reported to have beneficial effects on
endothelium-dependent vasodilation by decreasing the
concentrations of superoxide radicals, which inactivate
NO (Dudgeon et al., 1998). Vitamin C, when supple-
mented in feed at 500 mg/kg, reduced the incidence of
PHS in broilers (Ladmakhi et al., 1997; Xiang et al.,
2002). However, diets containing flax oil, VE, and VC
527
increased the incidence of ascites in broilers (Walton et
al., 2001).
The combined effects of ARG, VE, and VC on car-
diopulmonary performance and ascites parameters have
not been studied. We hypothesized that ARG and an-
tioxidant vitamins may have complementary effects on
cardiovascular performance, with ARG providing extra
substrate for NO production, and vitamins E and C
providing protection against oxidative stress. In this
study, we evaluated the effects of ARG and VE or VC,
or a combination of ARG, VE, and VC on cardiopul-
monary responses after an acute Epi challenge and
subsequent intravenous injections of aminoguanidine
hemisulfate (AG) and N-nitro-l-arginine methyl ester
(l-NAME) on plasma concentrations of NO, and on
the hematocrit values of broiler chickens reared under
cold exposure.
MATERIALS AND METHODS
A total of 96 one-day-old male Cobb 500 broiler
chicks were used in each of 2 experiments. Chicks were
wing-banded and reared in light-, temperature-, and
ventilation-controlled environmental chambers. All
broilers were brooded at the recommended brooding
temperatures until 16 d of age. Thereafter, all broilers
were subjected to a cool temperature (16°C) until 6
wk of age to amplify the incidence of PHS. The data
obtained from the 2 experiments were pooled before
statistical analysis.
All chicks were fed a basal corn-soybean meal diet to
meet or exceed the NRC (1994) requirements, includ-
ing 22.5% CP and 3,150 kcal/kg of ME (1 to 21 d), or
19% CP and 3,250 kcal/kg of ME (22 to 42 d). The
basal diet included ARG at 1.2% (wt/wt) and VE at
40 IU/kg of feed for both growing periods; the details
of the diet composition, along with micronutrients, are
described elsewhere (Baurhoo et al., 2007). Birds were
divided into 4 groups: a control group (CTL, basal
diet), a high-ARG and high-VE group [AE; basal diet
plus 1% supplemental l-arginine monohydrochloride
(Sigma, Oakville, Ontario, Canada), and 200 IU of dl-
α-tocopheryl acetate (Rovimix E 50 SD, DSM nutri-
tional products, Ayr, Ontario, Canada)], a high-ARG
and VC group (AC; basal diet plus 1% ARG, and 500
mg/kg of ascorbic acid; Sigma), and a high-ARG, high-
VE, and VC group (AEC; basal diet plus 1% ARG,
200 IU of α-tocopherol acetate, and 500 mg/kg of VC).
The diets were not pelletized and were mixed in small
batches to prevent inactivation of the ascorbic acid.
Surgery
From d 28 to 42, clinically healthy birds were select-
ed for the evaluation of cardiopulmonary performance
(n = 7 and n = 16 per group for experiments 1 and
2, respectively). Birds were anesthetized to a surgi-
cal plane with allobarbital (5,5-diallyl-barbituric acid,
528 Ruiz-Feria
Dial mixture, Sigma, St. Louis, MO; 50 mg/kg of BW
i.m.) and lidocaine hydrochloride (Xylocaine, Astra-
Zeneca Canada Inc., Mississauga, Ontario, Canada; 2%
s.c.) as a supplemental local anesthetic at the incision
sites. Birds were fastened in dorsal recumbency, and
the wings and legs were extended on a heated surgical
board regulated to maintain a surface temperature of
30°C and a 20° head-up angle. The left brachial artery
was isolated and cannulated with 30 cm of heparinized
polyethylene tubing (PE-50, Becton Dickinson Canada
Inc., Oakville, Ontario, Canada). The left brachial vein
was cannulated by using 30 cm of heparinized Silas-
tic tubing (0.012 i.d. × 0.025 o.d., VWR International,
Mississauga, Ontario, Canada) and the proximal end
was advanced through the vein and RV until it reached
the pulmonary artery. Both distal ends of the PE-50
polyethylene tubing and the Silastic tubing were at-
tached to blood pressure transducers interfaced with
a Transbridge preamplifier to a Biopac MP100 data
acquisition system using Acknowledge software (Biopac
Systems Inc., Goleta, CA) for the continuous measure-
ment of PAP (mm Hg), mean systemic arterial pressure
(MAP, mm Hg), and heart rate (HR, beats/min).
Experimental Protocols
Once birds were cannulated, they were allowed to
stabilize for 10 min. During this period, representative
PAP and MAP readings were taken at 300 and 60 s
before an Epi [4-(1-hydroxy-2-[methylamino]ethyl)-1,2-
benzenediol hydrochloride, Sigma, St. Louis, MO] chal-
lenge (0.5 mg/kg of BW, i.v.), to obtain basal values.
Epinephrine exerts a strong vasonstrictive effect (Smith
et al., 2000), increasing MAP and PAP. Evaluation of
the vasodilation capacity was estimated by measuring
the increment of the PAP after each challenge and the
time birds within each dietary treatment took to re-
turn to the basal measurement. The PAP, MAP, and
HR responses were measured at 30, 60, 120, 300, 600,
720, and 1,200 s after the Epi challenge. At 1,200 s
after the Epi challenge, birds were injected with AG
(100 mg/kg of BW, i.v.), and the PAP, MAP, and HR
were recorded at the same time intervals (30, 60, 120,
300, 600, 720, and 1,200 s after the AG challenge). A
third challenge with l-NAME (50 mg/kg of BW, i.v.)
was conducted 1,200 s after the AG challenge, and the
same parameters were recorded at the interval times
described previously.
The Biopac MP100 system monitored 2 primary
channels, including MAP and PAP. Values for these
parameters were averaged electronically during 10-s re-
cordings at the above-mentioned representative sam-
pling times. The protocol used for data averaging was
previously demonstrated to compensate accurately for
the influences of pulse pressure and respiratory cycles
on PAP and MAP (Wideman et al., 1996). Heart rate
was obtained by counting systolic peaks over time in
the PAP recording coincident with each sampling time
interval. At the end of the experiment, birds were hu-
manely killed and the heart was dissected to determine
the RV to total ventricle ratio (RV:TV) as an indica-
tor of PHS (Burton et al., 1968; Cueva et al., 1974).
All animal procedures were approved by the McGill
University animal care committee.
Determination of NO in blood
Blood samples (2 mL) for NO determination were
collected in Vacutainer (Becton Dickinson Canada Inc.)
tubes containing EDTA at 5 min before the Epi chal-
lenge and at 20 min after the l-NAME challenge. The
plasma was separated by centrifugation and stored at
−20°C until analysis. Plasma NO concentration was
determined by using a Nitrate/Nitrite Colorimetric As-
say Kit (Cayman Chemical, Ann Arbor MI). This test
is based on a 2-step process that converts nitrate to
nitrite by using nitrate reductase, followed by the ad-
dition of Griess reagents, which convert nitrite into a
deep purple azo compound. Analyses were carried out
in 96-well microtiter plates, and absorbance of the azo
chromophore was measured at 540 nm.
Hematocrit, RV:TV Ratio, and PHS Mortality
Hematocrit values were recorded from wk 3 to 6.
Blood samples were collected from the wing vein into
blood capillary tubes from randomly selected broilers
(n = 8 to 9 per group for experiment 1, and n = 18
for experiment 2) and centrifuged at 9,000 × g for 3
min. Birds that died during the experimental period
were examined for lesions of heart failure and ascitic
fluid in the abdominal cavity. The heart was removed,
the RV was carefully cut from the left ventricle, and
the RV:TV ratio was calculated (Burton et al., 1968;
Cueva et al., 1974). At the end of 6 wk, all birds were
humanely killed and RV:TV ratios were calculated.
Statistical Analysis
Body weight, RV:TV ratio, hematocrit, plasma NO,
and values within sampling points for PAP, MAP, and
HR were analyzed by one-way ANOVA, and means
were separated by the Student-Newman-Keuls method
in both experiments. Plasma concentrations of NO be-
fore and after challenge within treatment group were
compared by using a paired t-test, and differences were
declared at P < 0.05. The PAP, MAP, and HR were
analyzed within treatment group over time by using
the repeated measures ANOVA of SigmaStat (Jandel
Scientific, 1994), and means were separated by the Stu-
dent-Newman-Keuls method. Sampling times at −300
and −60 s were used as basal values for the Epi chal-
lenge. The sampling time of 1,200 s after the Epi chal-
lenge was considered the basal measurement for the AG
challenge, and the sampling time of 2,400 s was consid-
ered the basal measurement for the l-NAME challenge.
Differences were declared when the PAP, MAP, and
HR during the challenges were statistically different (P
 ARGININE, ANTIOXIDANT VITAMINS, AND ASCITES
< 0.05) from their respective baseline values. Data for
PAP, MAP, HR, and plasma NO were highly consistent
in both experiments and were pooled before analysis.
A value of P < 0.05 was considered statistically signifi-
cant.
RESULTS AND DISCUSSION
The pulmonary arterial relaxation was improved
when ARG was supplemented above the NRC (1994) re-
quirements, and this has been attributed to an increase
in NO production (Wideman et al., 1996; Lorenzoni
and Ruiz-Feria, 2006). In addition, antioxidants such
as VE (Bottje et al., 1995) and VC (Xiang et al., 2002)
have been shown to reduce PHS mortality. However,
the effects of ARG, VE, or VC on cardiopulmonary
function and ascites incidence have not been consistent
when used alone (Ruiz-Feria et al., 2001; Lorenzoni and
Ruiz-Feria, 2006; Walton et al., 2001). We hypothesized
that ARG and antioxidant vitamins may have comple-
mentary effects on cardiopulmonary performance, with
ARG providing extra substrate for NO production, and
antioxidant vitamins providing protection against oxi-
dative stress and increasing NO bioavailability.
The basal PAP (before challenge) was not different
among clinically healthy broilers in the different treat-
ment groups (Figure 1; time −300 and −60 s). There
was a significant increase in PAP at 30 s after the Epi
challenge within all treatment groups (P < 0.05; time
30 s vs. basal PAP). The PAP after the Epi challenge
remained greater, compared with the basal measure-
ments, for up to 300 s in the CTL and AE groups, but
for less than 300 s in the AC and AEC groups. Further-
more, when comparing the PAP among treatments at
the 30-s time (the peak response in PAP), the PAP was
lowest (P < 0.05) in the AEC group and greatest in the
CTL group, whereas the AE and AC groups had an in-
termediate (lower than the CTL group, but greater than
the AEC group) and comparable PAP. At the 60-, 120-,
and 300-s times, the PAP of the CTL group remained
greater than the PAP of the AEC group, but was not
different from the PAP of the AE and AC groups. At
60 s after the Epi challenge, the PAP of the AEC group
was lower than the PAP of the AC group, but was not
different from the PAP of the AE group. At 120 s after
the challenge, the PAP of the AEC group was lower
than the PAP of both the AE and AC groups, but at
300 s after the Epi challenge, there were no differences
among the PAP of the AE, AC, and AEC groups. After
the 600-s recording, the PAP was not different among
treatments.
Therefore, the birds in the AEC group showed the
best pulmonary vasodilation response after an acute
Epi challenge, as evidenced by the lower peak in PAP
at 30 s, the lower PAP from 60 to 300 s, and the rapid
return of the PAP to basal measurements, whereas the
birds in the AE and AC groups had a better pulmo-
nary vasodilation response than the CTL birds. These
results suggest an additive effect of ARG, VE, and VC
529
on the cardiopulmonary response. The extra plasma
concentrations of ARG may become available for enzy-
matic formation of NO, whereas antioxidant vitamins
may protect the bioavailability of NO and protect the
endothelial integrity, and in this way, improve the va-
sodilatory capacity of the lungs. In that regard, our
results also indicated that VE or VC, in combination
with ARG, provided limited protection against oxida-
tive stress, but when both antioxidant vitamins were
combined, the protective effect was enhanced. For in-
stance, the superoxide anion (·O2−) caused a loss of
NO bioavailability by shortening its half-life, causing,
among other effects, a decrease in endothelial vaso-
dilation (Lopez-Lopez et al., 2001). Furthermore, the
reaction of·O2− with NO led to the production of per-
oxynitrite, a potent oxidant agent responsible for di-
rect tissue damage by oxidation, peroxidation, and ni-
tration of lipids, proteins, and DNA (Beckman et al.,
1990; Szabo, 1996). The additive effects of VE and VC
on antioxidant capacity have been reported in laying
hens reared at high temperatures (Sahin et al., 2002)
and in apolipoprotein E-deficient mice (Nespereira et
al., 2003; Rodriguez-Gomez et al., 2005). It has been
documented that VC ensures α-tocopherol regeneration
from the α-tocopheroxy radical, thereby preventing VE
prooxidant activity, acting as a “coantioxidant,” and
inhibiting oxidation (Carr et al., 2000). We previously
reported that VE supplementation (400 IU/kg of feed),
along with ARG, increased TBA-reactive substances
in the plasma of broilers reared in cold environments
(Lorenzoni and Ruiz-Feria, 2006). Chickens can syn-
thesize VC, but under intensive farming conditions and
stressors such as rapid growth, heat, or cold, birds may
be unable to synthesize adequate amounts of VC (Par-
due and Thaxton, 1986). In addition, ARG may be-
come a prooxidative agent under some circumstances;
for instance, greater concentrations of NO production
have been associated with the production of superoxide
radicals (Hishikawa and Lüscher, 1997), and Ruiz-Feria
et al. (2004) reported that high concentrations of ARG
were associated with increased endothelial lesions in
the aorta of birds, presumably because of increased oxi-
dative stress. The results of these experiments strongly
suggest that the combination of VE and VC had addi-
tive effects on improving cardiopulmonary performance
and reducing pulmonary hypertension, and these may
have been mediated by reductions in oxidative stress
and an increased availability of NO, as discussed be-
low.
Aminoguanidine is a specific inhibitor of the induc-
ible form of nitric oxide synthase (NOS). After the AG
challenge, the PAP values increased (P < 0.05) in all
groups within 30 s, compared with basal values (time
1,200 s), and returned to the basal measurements after
120 s in all treatments, except in the AEC group, in
which PAP returned to the basal measurement before
120 s. The peak of PAP was recorded at 60 s after the
AG challenge in all treatments (time 1,260 s; Figure 1);
however, the peak was greater in the CTL group than in
530 Ruiz-Feria

Figure 1. Pulmonary arterial pressure (PAP) after an epinephrine (Epi) challenge (0.5 mg/kg of BW), an aminoguanidine hemisulfate (AG)
challenge (100 mg/kg of BW), and an N-nitro-l-arginine methyl ester (l-NAME) challenge (50 mg/kg of BW) in male broilers (n = 23 per treat-
ment) raised at 16°C and fed a standard corn-soybean meal diet (control, CTL), or the CTL diet supplemented with either 1% arginine and 200
IU of α-tocopherol/kg of feed (AE); 1% arginine and 500 mg of vitamin C (AC); or 1% arginine, 200 IU of α-tocopherol, and 500 mg of vitamin
C (AEC) per kilogram of feed. Data were averaged during representative sample intervals at 300 and 60 s before the Epi challenge (basal values)
and at 30, 60, 120, 300, 600, 720, and 1,200 s after the Epi injection. The AG challenge was administered 1,200 s after the Epi challenge, and this
time served as the basal value for the AG challenge; the same sampling intervals as in the first challenge were used. The l-NAME was administered
1,200 s after the AG challenge (time 2,400 s), and this value served as the basal value for the l-NAME challenge. Each point represents the mean
± SE. Asterisks represent the values that are different (P < 0.05) from all their respective basal values within the same treatment. Letters (a, b)
represent values that are different (P < 0.05) among treatments at the same sampling time.

the AEC group, whereas the AE and AC groups had an al. (2006) reported that AG stabilized, but did not in-
intermediate PAP peak, which was not different from crease, the PAP during the period preceding micropar-
the peak PAP of the CTL or AEC group. Wideman et ticle injection; similarly, Bowen et al. (2006) reported
 ARGININE, ANTIOXIDANT VITAMINS, AND ASCITES
that the PAP did not increase in either ascites-suscep-
tible or ascites-resistant lines of broilers within 10 min
after AG injection. On the other hand, Teshfam et al.
(2006) reported that cold exposure increased endothe-
lial NOS and inducible NOS (iNOS) gene expression,
and Schroeder et al. (2000) observed that rats that de-
veloped hepatic and pulmonary hypertension presented
a 9-fold increase in iNOS activity in the pulmonary
endothelium. Therefore, the increase in PAP recorded
after the AG challenge in these experiments may be the
result of cold temperature exposure and birds develop-
ing PHS, further supporting the complementary effects
of ARG, VE, and VC in reducing the deleterious effects
of cold exposure on pulmonary vasodilation.
The PAP increased (P < 0.05) within 30 s after l-
NAME injection in the AC and AEC groups, increased
within 60 s in the CTL and AE groups, and remained
elevated up to 300 s in all groups, but with no differ-
ences among treatments. The l-NAME challenge had a
longer lasting effect on PAP than did the AG challenge.
When both endothelial NOS and iNOS are inhibited by
l-NAME, the ensuing reduction of NO synthesis leads
to pulmonary arterial vasoconstriction (Martinez-Le-
mus et al., 1999, 2003; Wideman and Chapman, 2004).
Wideman et al. (2005) reported that an l-NAME injec-
tion (40 mg) did not cause an increase in PAP within 5
min, but effectively amplified the pulmonary vasocon-
striction elicited by a cellulose microparticle injection.
The responsiveness of the birds to l-NAME in these
experiments may be explained by the difference in dose
(50 mg/kg of BW), the exposure to a cold temperature
(which could have begun to create endothelial damage),
and exposure to the previous challenges, reducing the
availability of NO.
The MAP (Figure 2) showed the same tendencies
as described for PAP. The first peak in MAP occurred
within 30 s of the Epi challenge and returned to its
basal measurement within 300 s after the Epi challenge.
The MAP increased 60 s after the AG challenge and re-
turned to its basal measurement within 120 s after the
challenge in all groups. Sixty seconds after the l-NAME
challenge, the MAP increased and returned to its basal
measurement within 600 s in all groups. The MAP was
not different among treatment groups at any sampling
time. Heart rate was decreased in all groups after the
Epi challenge and returned to its original measurements
within 300 s in all groups (data not shown). Epineph-
rine triggers immediate increases in total peripheral
resistance and pulmonary vascular resistance, which in-
crease the MAP and PAP in spite of reductions in HR
and cardiac output (Wideman, 1999). A decreased HR
was also recorded after the AG and l-NAME challeng-
es. Previous studies have shown similar observations
for HR and MAP after an Epi or l-NAME challenge
(Wideman, 1999; Wideman et al., 2005; Lorenzoni and
Ruiz-Feria, 2006). In these experiments, neither MAP
nor HR presented differences among treatments, and
the time they took to return to basal measurements
was similar among treatments, suggesting that the dif-
531
ferences in PAP discussed above can be attributable to
group differences in the pulmonary vascular tone.
The in vivo half-life of NO is only a few seconds
in dissolved biological fluids, where NO and reactive
nitrogen species are rapidly converted into the stable
oxidation products nitrite and nitrate (Dweik et al.,
2001). Before the Epi challenge, we found that dietary
supplementation of ARG in combination with antioxi-
dant vitamins (AEC group) significantly increased the
plasma concentrations of NO (nitrates and nitrites) in
the blood, as compared with birds fed ARG and either
one of the antioxidant vitamins (AE or AC groups) or
a normal diet (CTL group; Table 1). In addition, before
the Epi challenge, the NO concentrations between the
AE and AC birds were not different, but were greater
when compared with the NO concentrations of the CTL
birds. These results strongly suggest that the better
pulmonary vasodilatory capacity shown by the AEC
birds after the Epi challenge (Figure 1) could be attrib-
uted to a greater availability of NO. Furthermore, birds
showing intermediate cardiopulmonary performance af-
ter the Epi challenge (AE and AC birds) also had inter-
mediate concentrations of NO metabolites in the blood,
supporting the idea that VE and VC have complemen-
tary effects, protecting and enhancing the availability
of NO, probably through reductions in oxidative stress.
In addition to their antioxidant effects, VC and VE
have been shown to improve the synthesis of endotheli-
al-derived NO through the regeneration of tetrahydro-
biopterin (an essential cofactor for NO synthesis) and
the inhibition of protein kinase C activity, respectively
(Carr et al., 2000). As expected, the concentration of
plasma NO metabolites was reduced after the l-NAME
challenge; however, even after the l-NAME challenge,
birds in the AEC group had greater concentrations of
NO than birds in the other treatments (experiments 1
and 2, and pooled data), further suggesting the benefi-
cial effects of the concurrent supplementation of ARG,
VE, and VC on NO availability.
At 3 wk of age (5 d after cold exposure began), the
hematocrit value was greater in the CTL group than
in the AEC group, but at wk 4, there were no dif-
ferences among groups (Figure 3). However, at wk 5
and 6, hematocrit values were lowest (P < 0.05) in the
AEC birds, and greatest in the CTL group. Hematocrit
values for the AE and AC groups were similar, but
lower when compared with the CTL birds, and greater
when compared with the AEC birds (Figure 3). These
hematocrit values were greater than those previously
reported by Maxwell et al. (1992), but were similar to
the ones we reported previously (Lorenzoni and Ruiz-
Feria, 2006). A greater hematocrit value is associated
with sustained hypoxia (Yersin et al., 1992) and has
been shown to be correlated with ascites susceptibility
(Wideman et al., 1998). The hematocrit values in the
present experiments matched the results of cardiopul-
monary performance and plasma NO concentrations,
further supporting the role of ARG and antioxidants
on the gas exchange rate and overall cardiopulmonary
532 Ruiz-Feria

function in the AEC birds, followed by birds fed ARG and the AEC group (4/48). The BW and RV:TV ratio
and either VE or VC. The mortality attributed to as- were not different among treatments (data not shown).
cites was numerically greater in the CTL group (9/48), The results of the present experiments also indicated
followed by the AC group (8/48), the AE group (6/48), that the increase in H+ ions when arginine HCl was

Figure 2. Mean arterial pressure (MAP) after an epinephrine (Epi) challenge (0.5 mg/kg of BW), an aminoguanidine hemisulfate (AG) chal-
lenge (100 mg/kg of BW), and an N-nitro-l-arginine methyl ester (l-NAME) challenge (50 mg/kg of BW) in male broilers (n = 23 per treatment)
raised at 16°C and fed a standard corn-soybean meal diet (control, CTL), or the CTL diet supplemented with either 1% arginine and 200 IU of
α-tocopherol/kg of feed (AE); 1% arginine and 500 mg of vitamin C (AC); or 1% arginine, 200 IU of α-tocopherol, and 500 mg of vitamin C (AEC)
per kilogram of feed. Data were averaged during representative sample intervals at 300 and 60 s before the Epi challenge (basal values) and 30, 60,
120, 300, 600, 720, and 1,200 s after the Epi injection. The AG challenge was administered 1,200 s after the Epi challenge, and this time served
as the basal value for the AG challenge; the same sampling intervals as in the first challenge were used. The l-NAME was administered 1,200 s
after the AG challenge (time 2,400 s), and this value served as the basal value for the l-NAME challenge. Each point represents the mean ± SE.
Asterisks represent the values that are different (P < 0.05) from all their respective basal values within the same treatment.
 ARGININE, ANTIOXIDANT VITAMINS, AND ASCITES 533
Table 1. Plasma NO concentrations in anesthetized broilers chickens reared in a cold environment and fed different levels of arginine,
vitamin E, and vitamin C, before an epinephrine challenge, and after an epinephrine, aminoguanidine, and N-nitro-l-arginine methyl
ester (l-NAME) challenge at 20-min intervals
Experiment 1 Experiment 2 Pooled data
1 2 2
Treatment n Before After n Before After n Before After2

CTL 9 6.6 ± 0.4c 3.9 ± 0.3c 14 6.8 ± 0.5c 4.7 ± 0.5b 23 6.7 ± 0.4c 4.4 ± 0.3b
AE 7 9.9 ± 1.1b 5.7 ± 0.7b 15 10.4 ± 0.2b 6.2 ± 0.8b 22 10.2 ± 0.4b 6.0 ± 0.6b
AC 5 9.9 ± 0.8b 6.7 ± 0.4b 14 10.7 ± 0.3b 6.6 ± 0.9b 19 10.5 ± 0.3b 6.6 ± 0.7b
AEC 4 24.6 ± 1.8a 12.5 ± 1.2a 14 16.9 ± 1.5a 11.5 ± 1.6a 18 18.7 ± 1.4a 11.7 ± 1.3a
a–c
Means in the same column with different superscripts are statistically different (P < 0.05).
1
The basal diet included arginine at 1.2% (wt/wt) and vitamin E at 40 IU/kg of feed. Treatment groups: control group (CTL, basal diet); a high-
arginine and high-vitamin E group (AE, basal diet plus 1% supplemental l-arginine monohydrochloride, and 200 IU of vitamin E); a high-arginine and
vitamin C group (AC, basal diet plus 1% l-arginine and 500 mg/kg of vitamin C); and a high-arginine, high-vitamin E, and vitamin C group (AEC,
basal diet plus 1% l-arginine, 200 IU of α-tocopherol acetate, and 500 mg/kg of vitamin C).
2
Plasma NO levels were lower after the l-NAME injection (paired t-test, P < 0.05).

supplemented at amounts of 1% (wt/wt) did not have
any detrimental effect on the bird, and could be han-
dled easily by the mechanisms of the bird to control the
acid-base balance.
In summary, the concurrent supplementation of
ARG, VE, and VC significantly improved the pulmo-
nary vasodilation of broiler chickens grown under cold
environmental conditions after an acute Epi challenge;
the improved pulmonary vasodilation was associated
with increased plasma NO concentrations and lower
hematocrit concentrations. Thus, ARG, VE, and VC
may have complementary effects on cardiopulmonary
performance by increasing NO bioavailability through
a greater substrate for NOS, and presumably by reduc-
ing the losses of NO associated with oxidative stress
and reducing endothelial damage by NO-derived radi-
cals. Further research is warranted to elucidate these
mechanisms.
ACKNOWLEDGMENT
This research was funded by grants from the Fonds
québécois de la recherche sur la nature et les technolo-
gies, and Hatch project H70280. Sunil Kawthekar con-
ducted the experiments and submitted the results in
partial fulfillment of his master’s degree.

Figure 3. Mean hematocrit values of male broilers raised at 16°C and fed a standard corn-soybean meal diet (control, CTL) or the CTL diet
supplemented with either 1% arginine and 200 IU of α-tocopherol/kg of feed (AE); 1% arginine and 500 mg of vitamin C (AC); or 1% arginine,
200 IU of α-tocopherol, and 500 mg of vitamin C (AEC) per kilogram of feed. Each bar is the mean ± SE of 26 to 27 observations. Bars with
different letters (a–c) within the same age group are statistically different (P < 0.05).
534 Ruiz-Feria
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