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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- challenge in all birds, but the peak PAP was lower in
ate the effects of arginine, vitamin E (VE), and vitamin the AEC group than in all the other groups, whereas
C (VC) on cardiopulmonary performance and ascites birds in the AE and AC groups had lower PAP peaks
parameters of broilers reared under a cold environmen- than did the control group. After 120 s of challenge, the
tal temperature. One-day-old male broilers were fed a PAP was lower in AEC birds compared with the other
basal corn-soybean meal diet (control, 1.2% arginine birds. The PAP returned to pre-Epi amounts within
and 40 IU of VE), or the basal diet supplemented with 300 s in all groups. The PAP was increased (P < 0.05)
1% arginine and either 200 IU vitamin E (AE), 500 within 60 s after the aminoguanidine hemisulfate and
mg of vitamin C (AC), or a combination of VE and N-nitro-l-arginine methyl ester challenges in all groups,
VC at the same amounts (AEC) per kilogram of feed. but no differences were found among groups. The mean
Pulmonary arterial pressure (PAP) and mean arterial arterial pressure responses did not differ among groups.
pressure were recorded in clinically healthy, anesthe- Plasma NO was greater in the AEC group than in all
tized birds (28 to 42 d old) before and after an epineph- the other groups before and after the Epi challenge.
rine (Epi) challenge (0.5 mg/kg of BW, i.v.), an amin- These results showed that Epi elicited lower amplitude
oguanidine hemisulfate challenge (100 mg/kg of BW, PAP and less prolonged increases in PAP in birds from
i.v.), and an N-nitro-l-arginine methyl ester challenge the AEC group, and this may have been related to the
(50 mg/kg of BW, i.v.) at 20-min intervals. Data were increased vasodilation attributable to NO production.
analyzed by repeated measures ANOVA, and the Stu- The AEC may have had complementary effects against
dent Newman-Keuls test was used to separate means oxidative stress, protecting the endothelium and pre-
within groups. The PAP increased 30 s after the Epi serving NO function.
Key words: arginine, pulmonary hypertension, vitamin E, vitamin C
2009 Poultry Science 88:526–535

INTRODUCTION output must be propelled through the noncompliant
pulmonary vasculature, which requires increases in pul-
Pulmonary hypertension syndrome (PHS, ascites) monary arterial pressure (PAP) and right ventricle
is a common metabolic disorder of modern, fast-grow- (RV) work (Wideman, 1988). Sustained increases in
ing strains of broilers. Ascites mortality peaks close to PAP cause hypertrophy and subsequent dilation of the
market age when the birds are 5 to 6 wk old, which RV (Peacock et al., 1990; Wideman, 1999).
represents a significant loss to the industry, given the By reducing the pulmonary vascular resistance, it
cost of production up to this point (Maxwell and Rob- is possible to reduce the PAP needed to propel the
ertson, 1998). Ascites can be initiated by an increase in cardiac output required to match the metabolic de-
metabolic rate, which implies a greater oxygen demand mands of the broilers (Wideman et al., 1995) and delay
(Scheele et al., 1991; Buys et al., 1999; Wideman and the pathophysiological progression leading to ascites.
Tackett, 2000). In a gas exchange system working close Nitric oxide is an important endogenous vasodilator
to its physiological limit, this elevation in metabolic produced by endothelial cells through an oxygen-de-
demand can lead to cellular hypoxia (Scheele et al., pendent 5-electron oxidation of a terminal guanidine
1991). To avoid cellular hypoxia, an increased cardiac 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
©2009 Poultry Science Association Inc.
Received September 18, 2008.
reported that ARG amounts supporting the maximal
Accepted October 21, 2008. growth rate are not adequate for maximal NO produc-
Corresponding author: tion (Taylor et al., 1992; Dietert and Austic, 1994).

Supplemental ARG reduced the incidence of ascites in increased the incidence of ascites in broilers (Walton et
broilers exposed to a cool temperature (Wideman et al., al., 2001).
1995; Tan et al., 2005), improved pulmonary vasodila- The combined effects of ARG, VE, and VC on car-
tion (Wideman et al., 1996; Lorenzoni and Ruiz-Feria, diopulmonary performance and ascites parameters have
2006), and facilitated vasorelaxation in fowl (Martinez- not been studied. We hypothesized that ARG and an-
Lemus et al., 1999). However, the effects of ARG on tioxidant vitamins may have complementary effects on
ascites incidence have not been consistent (Ruiz-Feria cardiovascular performance, with ARG providing extra
et al., 2001). substrate for NO production, and vitamins E and C
Oxidative stress is also involved in the etiology of providing protection against oxidative stress. In this
PHS (Peacock et al., 1990; Maxwell et al., 1992; En- study, we evaluated the effects of ARG and VE or VC,
kvetchakul et al., 1993; Bottje and Wideman, 1995; or a combination of ARG, VE, and VC on cardiopul-
Grobe et al., 2006). A sustained high PAP is associated monary responses after an acute Epi challenge and
with hypoxia, endothelial damage, and reactive oxy- subsequent intravenous injections of aminoguanidine
gen species production. Chronic hypoxia increases ROS hemisulfate (AG) and N-nitro-l-arginine methyl ester
concentrations and impairs endothelial NO-dependent (l-NAME) on plasma concentrations of NO, and on
relaxation in mice pulmonary arteries (Fresquet et the hematocrit values of broiler chickens reared under
al., 2006). The superoxide anion causes a loss of NO cold exposure.
bioavailability by shortening its half-life (Jung et al.,
2003), thereby reducing the potential for endothelial MATERIALS AND METHODS
vasodilation (Lopez-Lopez et al., 2001). Furthermore,
the reaction of superoxide anion with NO leads to pro- A total of 96 one-day-old male Cobb 500 broiler
duction of peroxynitrite, a potent oxidant agent respon- chicks were used in each of 2 experiments. Chicks were
sible for direct tissue damage (Beckman et al., 1990; wing-banded and reared in light-, temperature-, and
Szabo, 1996). ventilation-controlled environmental chambers. All
Ascitic broilers have been reported to have low con- broilers were brooded at the recommended brooding
centrations of vitamin E (VE; Bottje et al., 1995) and temperatures until 16 d of age. Thereafter, all broilers
ascorbic acid (Enkvetchakul et al., 1993) in the lungs were subjected to a cool temperature (16°C) until 6
and liver. Vitamin E supplementation reduced the inci- wk of age to amplify the incidence of PHS. The data
dence of ascites when administered as a subcutaneous obtained from the 2 experiments were pooled before
implant, but not when provided as a dietary supple- statistical analysis.
ment (Bottje et al., 1995, 1997). We previously report- All chicks were fed a basal corn-soybean meal diet to
ed that VE at 400 IU/kg of feed, when supplemented in meet or exceed the NRC (1994) requirements, includ-
combination with ARG, was associated with less effec- ing 22.5% CP and 3,150 kcal/kg of ME (1 to 21 d), or
tive pulmonary vasodilation after an epinephrine (Epi) 19% CP and 3,250 kcal/kg of ME (22 to 42 d). The
challenge, compared with birds receiving only ARG basal diet included ARG at 1.2% (wt/wt) and VE at
(Lorenzoni and Ruiz-Feria, 2006). In addition, birds 40 IU/kg of feed for both growing periods; the details
receiving ARG and large amounts of VE had greater of the diet composition, along with micronutrients, are
concentrations of TBA-reactive substances than birds described elsewhere (Baurhoo et al., 2007). Birds were
fed normal diets, suggesting that large amounts of VE divided into 4 groups: a control group (CTL, basal
increased oxidative stress. Vitamin E reacts with or- diet), a high-ARG and high-VE group [AE; basal diet
ganic peroxides, interrupting the chain reaction of lipid plus 1% supplemental l-arginine monohydrochloride
peroxidation involving the formation of tocopheroxy (Sigma, Oakville, Ontario, Canada), and 200 IU of dl-
radicals, which can act as prooxidative agents and ini- α-tocopheryl acetate (Rovimix E 50 SD, DSM nutri-
tiate oxidative processes (Schneider, 2005). tional products, Ayr, Ontario, Canada)], a high-ARG
Ascorbate has been shown to prevent the prooxidant and VC group (AC; basal diet plus 1% ARG, and 500
activity of α-tocopherol by reducing the α-tocopheroxyl mg/kg of ascorbic acid; Sigma), and a high-ARG, high-
radical to α-tocopherol, and then acting as a coan- VE, and VC group (AEC; basal diet plus 1% ARG,
tioxidant (Carr et al., 2000). Through this mechanism, 200 IU of α-tocopherol acetate, and 500 mg/kg of VC).
vitamin C (VC) supplementation has been reported The diets were not pelletized and were mixed in small
to increase or restore plasma and tissue VE (Liu and batches to prevent inactivation of the ascorbic acid.
Lee, 1998; Keller et al., 2004). Furthermore, ascor-
bate has been reported to have beneficial effects on Surgery
endothelium-dependent vasodilation by decreasing the
concentrations of superoxide radicals, which inactivate From d 28 to 42, clinically healthy birds were select-
NO (Dudgeon et al., 1998). Vitamin C, when supple- ed for the evaluation of cardiopulmonary performance
mented in feed at 500 mg/kg, reduced the incidence of (n = 7 and n = 16 per group for experiments 1 and
PHS in broilers (Ladmakhi et al., 1997; Xiang et al., 2, respectively). Birds were anesthetized to a surgi-
2002). However, diets containing flax oil, VE, and VC cal plane with allobarbital (5,5-diallyl-barbituric acid,
528 Ruiz-Feria

Dial mixture, Sigma, St. Louis, MO; 50 mg/kg of BW manely killed and the heart was dissected to determine
i.m.) and lidocaine hydrochloride (Xylocaine, Astra- the RV to total ventricle ratio (RV:TV) as an indica-
Zeneca Canada Inc., Mississauga, Ontario, Canada; 2% tor of PHS (Burton et al., 1968; Cueva et al., 1974).
s.c.) as a supplemental local anesthetic at the incision All animal procedures were approved by the McGill
sites. Birds were fastened in dorsal recumbency, and University animal care committee.
the wings and legs were extended on a heated surgical
board regulated to maintain a surface temperature of Determination of NO in blood
30°C and a 20° head-up angle. The left brachial artery
was isolated and cannulated with 30 cm of heparinized Blood samples (2 mL) for NO determination were
polyethylene tubing (PE-50, Becton Dickinson Canada collected in Vacutainer (Becton Dickinson Canada Inc.)
Inc., Oakville, Ontario, Canada). The left brachial vein tubes containing EDTA at 5 min before the Epi chal-
was cannulated by using 30 cm of heparinized Silas- lenge and at 20 min after the l-NAME challenge. The
tic tubing (0.012 i.d. × 0.025 o.d., VWR International, plasma was separated by centrifugation and stored at
Mississauga, Ontario, Canada) and the proximal end −20°C until analysis. Plasma NO concentration was
was advanced through the vein and RV until it reached determined by using a Nitrate/Nitrite Colorimetric As-
the pulmonary artery. Both distal ends of the PE-50 say Kit (Cayman Chemical, Ann Arbor MI). This test
polyethylene tubing and the Silastic tubing were at- is based on a 2-step process that converts nitrate to
tached to blood pressure transducers interfaced with nitrite by using nitrate reductase, followed by the ad-
a Transbridge preamplifier to a Biopac MP100 data dition of Griess reagents, which convert nitrite into a
acquisition system using Acknowledge software (Biopac deep purple azo compound. Analyses were carried out
Systems Inc., Goleta, CA) for the continuous measure- in 96-well microtiter plates, and absorbance of the azo
ment of PAP (mm Hg), mean systemic arterial pressure chromophore was measured at 540 nm.
(MAP, mm Hg), and heart rate (HR, beats/min).
Hematocrit, RV:TV Ratio, and PHS Mortality
Experimental Protocols
Hematocrit values were recorded from wk 3 to 6.
Once birds were cannulated, they were allowed to Blood samples were collected from the wing vein into
stabilize for 10 min. During this period, representative blood capillary tubes from randomly selected broilers
PAP and MAP readings were taken at 300 and 60 s (n = 8 to 9 per group for experiment 1, and n = 18
before an Epi [4-(1-hydroxy-2-[methylamino]ethyl)-1,2- for experiment 2) and centrifuged at 9,000 × g for 3
benzenediol hydrochloride, Sigma, St. Louis, MO] chal- min. Birds that died during the experimental period
lenge (0.5 mg/kg of BW, i.v.), to obtain basal values. were examined for lesions of heart failure and ascitic
Epinephrine exerts a strong vasonstrictive effect (Smith fluid in the abdominal cavity. The heart was removed,
et al., 2000), increasing MAP and PAP. Evaluation of the RV was carefully cut from the left ventricle, and
the vasodilation capacity was estimated by measuring the RV:TV ratio was calculated (Burton et al., 1968;
the increment of the PAP after each challenge and the Cueva et al., 1974). At the end of 6 wk, all birds were
time birds within each dietary treatment took to re- humanely killed and RV:TV ratios were calculated.
turn to the basal measurement. The PAP, MAP, and
HR responses were measured at 30, 60, 120, 300, 600, Statistical Analysis
720, and 1,200 s after the Epi challenge. At 1,200 s
after the Epi challenge, birds were injected with AG Body weight, RV:TV ratio, hematocrit, plasma NO,
(100 mg/kg of BW, i.v.), and the PAP, MAP, and HR and values within sampling points for PAP, MAP, and
were recorded at the same time intervals (30, 60, 120, HR were analyzed by one-way ANOVA, and means
300, 600, 720, and 1,200 s after the AG challenge). A were separated by the Student-Newman-Keuls method
third challenge with l-NAME (50 mg/kg of BW, i.v.) in both experiments. Plasma concentrations of NO be-
was conducted 1,200 s after the AG challenge, and the fore and after challenge within treatment group were
same parameters were recorded at the interval times compared by using a paired t-test, and differences were
described previously. declared at P < 0.05. The PAP, MAP, and HR were
The Biopac MP100 system monitored 2 primary analyzed within treatment group over time by using
channels, including MAP and PAP. Values for these the repeated measures ANOVA of SigmaStat (Jandel
parameters were averaged electronically during 10-s re- Scientific, 1994), and means were separated by the Stu-
cordings at the above-mentioned representative sam- dent-Newman-Keuls method. Sampling times at −300
pling times. The protocol used for data averaging was and −60 s were used as basal values for the Epi chal-
previously demonstrated to compensate accurately for lenge. The sampling time of 1,200 s after the Epi chal-
the influences of pulse pressure and respiratory cycles lenge was considered the basal measurement for the AG
on PAP and MAP (Wideman et al., 1996). Heart rate challenge, and the sampling time of 2,400 s was consid-
was obtained by counting systolic peaks over time in ered the basal measurement for the l-NAME challenge.
the PAP recording coincident with each sampling time Differences were declared when the PAP, MAP, and
interval. At the end of the experiment, birds were hu- HR during the challenges were statistically different (P
< 0.05) from their respective baseline values. Data for on the cardiopulmonary response. The extra plasma
PAP, MAP, HR, and plasma NO were highly consistent concentrations of ARG may become available for enzy-
in both experiments and were pooled before analysis. matic formation of NO, whereas antioxidant vitamins
A value of P < 0.05 was considered statistically signifi- may protect the bioavailability of NO and protect the
cant. endothelial integrity, and in this way, improve the va-
sodilatory capacity of the lungs. In that regard, our
RESULTS AND DISCUSSION results also indicated that VE or VC, in combination
with ARG, provided limited protection against oxida-
The pulmonary arterial relaxation was improved tive stress, but when both antioxidant vitamins were
when ARG was supplemented above the NRC (1994) re- combined, the protective effect was enhanced. For in-
quirements, and this has been attributed to an increase stance, the superoxide anion (·O2−) caused a loss of
in NO production (Wideman et al., 1996; Lorenzoni NO bioavailability by shortening its half-life, causing,
and Ruiz-Feria, 2006). In addition, antioxidants such among other effects, a decrease in endothelial vaso-
as VE (Bottje et al., 1995) and VC (Xiang et al., 2002) dilation (Lopez-Lopez et al., 2001). Furthermore, the
have been shown to reduce PHS mortality. However, reaction of·O2− with NO led to the production of per-
the effects of ARG, VE, or VC on cardiopulmonary oxynitrite, a potent oxidant agent responsible for di-
function and ascites incidence have not been consistent rect tissue damage by oxidation, peroxidation, and ni-
when used alone (Ruiz-Feria et al., 2001; Lorenzoni and tration of lipids, proteins, and DNA (Beckman et al.,
Ruiz-Feria, 2006; Walton et al., 2001). We hypothesized 1990; Szabo, 1996). The additive effects of VE and VC
that ARG and antioxidant vitamins may have comple- on antioxidant capacity have been reported in laying
mentary effects on cardiopulmonary performance, with hens reared at high temperatures (Sahin et al., 2002)
ARG providing extra substrate for NO production, and and in apolipoprotein E-deficient mice (Nespereira et
antioxidant vitamins providing protection against oxi- al., 2003; Rodriguez-Gomez et al., 2005). It has been
dative stress and increasing NO bioavailability. documented that VC ensures α-tocopherol regeneration
The basal PAP (before challenge) was not different from the α-tocopheroxy radical, thereby preventing VE
among clinically healthy broilers in the different treat- prooxidant activity, acting as a “coantioxidant,” and
ment groups (Figure 1; time −300 and −60 s). There inhibiting oxidation (Carr et al., 2000). We previously
was a significant increase in PAP at 30 s after the Epi reported that VE supplementation (400 IU/kg of feed),
challenge within all treatment groups (P < 0.05; time along with ARG, increased TBA-reactive substances
30 s vs. basal PAP). The PAP after the Epi challenge in the plasma of broilers reared in cold environments
remained greater, compared with the basal measure- (Lorenzoni and Ruiz-Feria, 2006). Chickens can syn-
ments, for up to 300 s in the CTL and AE groups, but thesize VC, but under intensive farming conditions and
for less than 300 s in the AC and AEC groups. Further- stressors such as rapid growth, heat, or cold, birds may
more, when comparing the PAP among treatments at be unable to synthesize adequate amounts of VC (Par-
the 30-s time (the peak response in PAP), the PAP was due and Thaxton, 1986). In addition, ARG may be-
lowest (P < 0.05) in the AEC group and greatest in the come a prooxidative agent under some circumstances;
CTL group, whereas the AE and AC groups had an in- for instance, greater concentrations of NO production
termediate (lower than the CTL group, but greater than have been associated with the production of superoxide
the AEC group) and comparable PAP. At the 60-, 120-, radicals (Hishikawa and Lüscher, 1997), and Ruiz-Feria
and 300-s times, the PAP of the CTL group remained et al. (2004) reported that high concentrations of ARG
greater than the PAP of the AEC group, but was not were associated with increased endothelial lesions in
different from the PAP of the AE and AC groups. At the aorta of birds, presumably because of increased oxi-
60 s after the Epi challenge, the PAP of the AEC group dative stress. The results of these experiments strongly
was lower than the PAP of the AC group, but was not suggest that the combination of VE and VC had addi-
different from the PAP of the AE group. At 120 s after tive effects on improving cardiopulmonary performance
the challenge, the PAP of the AEC group was lower and reducing pulmonary hypertension, and these may
than the PAP of both the AE and AC groups, but at have been mediated by reductions in oxidative stress
300 s after the Epi challenge, there were no differences and an increased availability of NO, as discussed be-
among the PAP of the AE, AC, and AEC groups. After low.
the 600-s recording, the PAP was not different among Aminoguanidine is a specific inhibitor of the induc-
treatments. ible form of nitric oxide synthase (NOS). After the AG
Therefore, the birds in the AEC group showed the challenge, the PAP values increased (P < 0.05) in all
best pulmonary vasodilation response after an acute groups within 30 s, compared with basal values (time
Epi challenge, as evidenced by the lower peak in PAP 1,200 s), and returned to the basal measurements after
at 30 s, the lower PAP from 60 to 300 s, and the rapid 120 s in all treatments, except in the AEC group, in
return of the PAP to basal measurements, whereas the which PAP returned to the basal measurement before
birds in the AE and AC groups had a better pulmo- 120 s. The peak of PAP was recorded at 60 s after the
nary vasodilation response than the CTL birds. These AG challenge in all treatments (time 1,260 s; Figure 1);
results suggest an additive effect of ARG, VE, and VC 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
that the PAP did not increase in either ascites-suscep- ferences in PAP discussed above can be attributable to
tible or ascites-resistant lines of broilers within 10 min group differences in the pulmonary vascular tone.
after AG injection. On the other hand, Teshfam et al. The in vivo half-life of NO is only a few seconds
(2006) reported that cold exposure increased endothe- in dissolved biological fluids, where NO and reactive
lial NOS and inducible NOS (iNOS) gene expression, nitrogen species are rapidly converted into the stable
and Schroeder et al. (2000) observed that rats that de- oxidation products nitrite and nitrate (Dweik et al.,
veloped hepatic and pulmonary hypertension presented 2001). Before the Epi challenge, we found that dietary
a 9-fold increase in iNOS activity in the pulmonary supplementation of ARG in combination with antioxi-
endothelium. Therefore, the increase in PAP recorded dant vitamins (AEC group) significantly increased the
after the AG challenge in these experiments may be the plasma concentrations of NO (nitrates and nitrites) in
result of cold temperature exposure and birds develop- the blood, as compared with birds fed ARG and either
ing PHS, further supporting the complementary effects one of the antioxidant vitamins (AE or AC groups) or
of ARG, VE, and VC in reducing the deleterious effects a normal diet (CTL group; Table 1). In addition, before
of cold exposure on pulmonary vasodilation. the Epi challenge, the NO concentrations between the
The PAP increased (P < 0.05) within 30 s after l- AE and AC birds were not different, but were greater
NAME injection in the AC and AEC groups, increased when compared with the NO concentrations of the CTL
within 60 s in the CTL and AE groups, and remained birds. These results strongly suggest that the better
elevated up to 300 s in all groups, but with no differ- pulmonary vasodilatory capacity shown by the AEC
ences among treatments. The l-NAME challenge had a birds after the Epi challenge (Figure 1) could be attrib-
longer lasting effect on PAP than did the AG challenge. uted to a greater availability of NO. Furthermore, birds
When both endothelial NOS and iNOS are inhibited by showing intermediate cardiopulmonary performance af-
l-NAME, the ensuing reduction of NO synthesis leads ter the Epi challenge (AE and AC birds) also had inter-
to pulmonary arterial vasoconstriction (Martinez-Le- mediate concentrations of NO metabolites in the blood,
mus et al., 1999, 2003; Wideman and Chapman, 2004). supporting the idea that VE and VC have complemen-
Wideman et al. (2005) reported that an l-NAME injec- tary effects, protecting and enhancing the availability
tion (40 mg) did not cause an increase in PAP within 5 of NO, probably through reductions in oxidative stress.
min, but effectively amplified the pulmonary vasocon- In addition to their antioxidant effects, VC and VE
striction elicited by a cellulose microparticle injection. have been shown to improve the synthesis of endotheli-
The responsiveness of the birds to l-NAME in these al-derived NO through the regeneration of tetrahydro-
experiments may be explained by the difference in dose biopterin (an essential cofactor for NO synthesis) and
(50 mg/kg of BW), the exposure to a cold temperature the inhibition of protein kinase C activity, respectively
(which could have begun to create endothelial damage), (Carr et al., 2000). As expected, the concentration of
and exposure to the previous challenges, reducing the plasma NO metabolites was reduced after the l-NAME
availability of NO. challenge; however, even after the l-NAME challenge,
The MAP (Figure 2) showed the same tendencies birds in the AEC group had greater concentrations of
as described for PAP. The first peak in MAP occurred NO than birds in the other treatments (experiments 1
within 30 s of the Epi challenge and returned to its and 2, and pooled data), further suggesting the benefi-
basal measurement within 300 s after the Epi challenge. cial effects of the concurrent supplementation of ARG,
The MAP increased 60 s after the AG challenge and re- VE, and VC on NO availability.
turned to its basal measurement within 120 s after the At 3 wk of age (5 d after cold exposure began), the
challenge in all groups. Sixty seconds after the l-NAME hematocrit value was greater in the CTL group than
challenge, the MAP increased and returned to its basal in the AEC group, but at wk 4, there were no dif-
measurement within 600 s in all groups. The MAP was ferences among groups (Figure 3). However, at wk 5
not different among treatment groups at any sampling and 6, hematocrit values were lowest (P < 0.05) in the
time. Heart rate was decreased in all groups after the AEC birds, and greatest in the CTL group. Hematocrit
Epi challenge and returned to its original measurements values for the AE and AC groups were similar, but
within 300 s in all groups (data not shown). Epineph- lower when compared with the CTL birds, and greater
rine triggers immediate increases in total peripheral when compared with the AEC birds (Figure 3). These
resistance and pulmonary vascular resistance, which in- hematocrit values were greater than those previously
crease the MAP and PAP in spite of reductions in HR reported by Maxwell et al. (1992), but were similar to
and cardiac output (Wideman, 1999). A decreased HR the ones we reported previously (Lorenzoni and Ruiz-
was also recorded after the AG and l-NAME challeng- Feria, 2006). A greater hematocrit value is associated
es. Previous studies have shown similar observations with sustained hypoxia (Yersin et al., 1992) and has
for HR and MAP after an Epi or l-NAME challenge been shown to be correlated with ascites susceptibility
(Wideman, 1999; Wideman et al., 2005; Lorenzoni and (Wideman et al., 1998). The hematocrit values in the
Ruiz-Feria, 2006). In these experiments, neither MAP present experiments matched the results of cardiopul-
nor HR presented differences among treatments, and monary performance and plasma NO concentrations,
the time they took to return to basal measurements further supporting the role of ARG and antioxidants
was similar among treatments, suggesting that the dif- 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.
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
Means in the same column with different superscripts are statistically different (P < 0.05).
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).
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 a greater substrate for NOS, and presumably by reduc-
any detrimental effect on the bird, and could be han- ing the losses of NO associated with oxidative stress
dled easily by the mechanisms of the bird to control the and reducing endothelial damage by NO-derived radi-
acid-base balance. cals. Further research is warranted to elucidate these
In summary, the concurrent supplementation of mechanisms.
ARG, VE, and VC significantly improved the pulmo-
nary vasodilation of broiler chickens grown under cold ACKNOWLEDGMENT
environmental conditions after an acute Epi challenge;
the improved pulmonary vasodilation was associated This research was funded by grants from the Fonds
with increased plasma NO concentrations and lower québécois de la recherche sur la nature et les technolo-
hematocrit concentrations. Thus, ARG, VE, and VC gies, and Hatch project H70280. Sunil Kawthekar con-
may have complementary effects on cardiopulmonary ducted the experiments and submitted the results in
performance by increasing NO bioavailability through 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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