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Poult Sci 2006. 85:2241-2250
© 2006 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Effects of Vitamin E and L-Arginine on Cardiopulmonary Function and Ascites Parameters in Broiler Chickens Reared Under Subnormal Temperatures

A. G. Lorenzoni1 and C. A. Ruiz-Feria2

Department of Animal Science, McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9

2 Corresponding author: ciro.ruiz-feria{at}mcgill.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Two experiments were conducted to evaluate the effects of arginine (Arg) and vitamin E (VE) on ascites (pulmonary hypertension syndrome) parameters, nitric oxide synthase (NOS) activity, and cardiopulmonary performance after an acute challenge with epinephrine (Epi). One-day-old male broilers (n = 100) were fed a commercial corn-soybean meal–based diet meeting NRC (1994) requirements, including 1.2% Arg and 40 IU of VE/kg. In experiment 1, birds were provided tap water (control), water with 0.3% Arg (HArg), water with 400 IU of VE/L (HVE), or a combination of both compounds (Arg-VE). In experiment 2, the treatment groups were similar but the VE was incorporated in the diet (400 IU/ kg of feed). At d 18, temperature was reduced to amplify the incidence of pulmonary hypertension. Body weight and hematocrit were recorded weekly. From d 28 to 42, cardiopulmonary performance was evaluated in clinically healthy, anesthetized birds (n = 7 to 8/treatment). A pulmonary artery and a systemic artery were cannulated, the birds were allowed to stabilize for 10 min (basal), an i.v. injection of Epi was applied (1 or 0.5 mg/kg of BW, experiment 1 and 2, respectively), and a second dose was applied 20 min later. Pulmonary arterial pressure (PAP), mean arterial pressure (MAP), and heart rate (HR) were recorded continuously and data were analyzed by repeated measures ANOVA. The NOS activity was estimated through the conversion of 14C-Arginine to 14C-citrulline in isolated pulmonary arteries. Right/total ventricular weight ratio (RV/TV) was recorded at the end of the experiment. Body weight, RV/TV, and hematocrit values were not significantly affected by the dietary treatments. The PAP increased (P < 0.01) within 30 s after Epi in all treatments, except the HArg treatment in experiment 2. Overall, the time taken for PAP to return to basal levels was longer in the Arg-VE birds and shorter in the HArg birds, particularly after the second challenge. However, although NOS activity was highly variable, birds fed HArg tended to have the lowest NOS activity of all groups. The levels of VE supplementation used in these experiments did not improve cardiopulmonary performance or NOS activity in isolated pulmonary arteries.

Key Words: arginine • vitamin E • pulmonary hypertension • nitric oxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Modern strains of broilers are able to achieve market weight in 60% less time than broilers from 40 yr ago (Card, 1961). Nevertheless, pulmonary and cardiac capacity of modern broilers is very similar compared with the old broiler strains, which forces the cardiopulmonary system to work very close to its physiological limit. Consequently, pulmonary hypertension syndrome (PHS, ascites) is a common problem that causes considerable mortality (Julian, 1993).

Pulmonary hypertension syndrome can be initiated by an increase in metabolic rate, which implies higher oxygen demands (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 output must be propelled through the noncompliant pulmonary vasculature, which requires increases in pulmonary arterial pressure (PAP) and right ventricle work (Wideman, 1988). Sustained increases in PAP cause hypertrophy and subsequent dilation of the right ventricle. An elevated right to total ventricular weight ratio (RV/TV) is thus an anatomical symptom of PHS and has been used as a sensitive index to determine the presence of pulmonary hypertension in broilers (Cueva et al., 1974; Peacock et al., 1990; Wideman, 1999). Broilers with PHS die due to right-sided congestive heart failure, which is associated with central venous congestion, pressure-induced liver cirrhosis, and transudation of ascitic fluid into the abdominal cavity (Julian, 1993).

By reducing the pulmonary vascular resistance, it is possible to reduce the PAP needed to propel the cardiac output required to match broilers’ metabolic demands (Wideman et al., 1995) and delay the pathophysiological progression leading to ascites. Nitric oxide (NO) is a potent endogenous pulmonary vasodilator that acts by increasing the levels of cyclic guanosine monophosphate (cGMP) and thereby reducing intracellular Ca2+ levels in vascular smooth muscle cells (Forstermann et al., 1986). Nitric oxide is produced in the pulmonary endothelium where the enzyme NO synthase (NOS) converts L-argi-nine to L-citrulline (Jorens et al., 1993; McQueston et al., 1993). Arginine is an essential amino acid in birds, and must be supplemented in the diet (Tamil and Ratner, 1963). There is evidence showing that the Arg levels supporting maximal growth rate are not adequate to support maximal NO production (Dietert and Austic, 1994). Experimental supplementation of Arg has been used successfully to reduce ascites mortality induced by cool temperatures; nevertheless, its effects have not been consistent (Wideman et al., 1995; Ruiz-Feria et al., 2001; Tan et al., 2005).

Oxidative stress is also involved in the pathophysiological progression leading to ascites (Maxwell et al., 1986). Oxygen-derived free radicals play an important role in the genesis of tissue damage. The superoxide anion causes a loss of NO bioavailability by shortening its half-life and thereby reducing the potential for endothelial vasodilation (Lopez-Lopez et al., 2001). Furthermore, the reaction of the superoxide anion with NO leads to the production of peroxynitrite, a potent oxidant agent responsible for direct tissue damage by oxidation, peroxidation and nitration of lipids, proteins, and DNA (Beckman et al., 1990; Szabo, 1996). Excessive production of free radicals could cause damage of the pulmonary vascular endothelium causing destruction of the cells and thus a reduction in the endothelial NOS available for the production of NO and maintenance of blood pressure homeostasis. Vitamin E (VE) is known to be a powerful lipid-soluble antioxidant that scavenges lipid radicals. It has the ability to react with fatty acid peroxyl radicals, the primary products of lipid peroxidation, and intercepts the chain reaction preventing further radical reactions. During the antioxidant reaction, {alpha}-tocopherol is turned into a stable radical (Burton and Ingold, 1981). Vitamin E could thus help in reducing the oxidative stress in lung vessels and, in this way, reduce endothelial damage. It has been reported that birds developing ascites have low levels of {alpha}-tocopherol in lung and liver, providing evidence that a compromised antioxidant status is involved in the etiology of PHS (Enkvetchakul et al., 1993). Nevertheless, studies supporting the use of antioxidants to reduce mortality for ascites have been successful only when the VE is administered as a subcutaneous implant, and showing no effects in ascites mortality when VE was given as a feed additive (Bottje et al., 1995, 1997).

The combined effects of Arg and VE on the pathophysiology of pulmonary hypertension syndrome have not been studied. We hypothesize that Arg and VE may have complementary effects in reducing the incidence of ascites, with Arg providing extra substrate for NO synthesis, and VE protecting the endothelium from oxidative stress damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Two experiments were conducted. In both experiments 1-d-old Cobb-500 male broilers (n = 100) from a commercial hatchery were randomly distributed and placed on fresh wood shavings litter in 4 environmentally controlled chambers (controlled for temperature, ventilation, and light). Recommended brooding temperatures were used during wk 1, wk 2, and the beginning of wk 3 (32, 30, and 27°C). At d 18 or 15 (experiment 1 or 2, respectively) the temperature was reduced to 16°C to amplify the incidence of PHS. The daily photoperiod was 23L:1D throughout the experiment. Feed and water were provided ad libitum.

Treatments
In experiment 1 we evaluated 2 levels of L-arginine monohydrochloride (Sigma Canada, Oakville, Ontario, Canada) and 2 levels of VE (DL-{alpha}-tocopherol acetate, A.P.A., Vetoquinol Canada, Lavaltrie, Quebec, Canada). Birds were provided tap water (control), water with 0.3% Arg (HArg), water with 400 IU of VE/L (HVE), or water containing both compounds at the same levels (Arg-VE). Birds were fed a starter diet (21.6% CP; 3,120 kcal of ME/ kg) from 0 to 3 wk; and a grower corn-soybean meal–based diet (19.1% CP; 3,200 kcal of ME/kg) from 3 to 7 wk. In experiment 2 the birds were fed a corn-soybean meal–based diet (23% CP; 3,200 kcal of ME/kg) for the complete 7-wk period with 2 levels of VE inclusion (40 and 400 IU of VE/kg), and tap water or water containing 0.3% Arg with the following arrangement of treatments: tap water and feed with 40 IU of VE/kg (control), water with 0.3% Arg and feed with 40 IU of VE/kg (HArg), tap water and feed with 400 IU/kg (HVE), or water containing 0.3% Arg and feed with 400 IU of VE/kg (Arg-VE). The diets were formulated to meet or exceeded NRC (1994) requirements. Birds in the second experiment were exposed to low temperatures at a younger age and received a diet with a higher nutrient density compared with birds in the first experiment as a measure to further increase the incidence of PH. Body weight was recorded weekly throughout the experiment; hematocrit was also determined weekly starting at wk 3.

Surgery
From d 28 to 42, clinically healthy birds were selected for the evaluation of cardiopulmonary performance (n = 7 to 8/treatment). Birds were anesthetized by injecting allobarbital (Dial mixture, 5,5 diallyl-barbituric acid; 50 mg/kg of BW i.m., Sigma, St. Louis, MO) with lidocaine hydrochloride (2% s.c., Xylocaine, Astra-Zeneca Canada Inc., Mississauga, Ontario, Canada) injected as a supplemental local anesthetic at incision sites. Birds were fastened in dorsal recumbency, with wings and legs 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 PE-50 tubing (PE-50 polyethylene, Becton Dickinson Canada, Inc., Oakville, Ontario, Canada). The left brachial vein was cannulated using 30 cm of heparinized Silastic tubing (0.012 internal diameter x 0.025 outside diameter, VWR International, Mississauga, Ontario, Canada) and the proximal end was advanced through the vein and right ventricle until it reached the pulmonary artery. Both distal ends of the PE-50 polyethylene tubing and the Silastic tubing were attached to blood pressure transducers interfaced with a Trasbridge preamplifier to a Biopac MP100 data acquisition system using Acknowledge software (Biopac Systems Inc., Goleta, CA) for the continuous measurement of mean systemic arterial pressure (MAP, mmHg), PAP (mmHg), 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 180, 120, and 60 s before an epinephrine (Epi; 4-(1-hydroxy-2-[methylamino]ethyl)-1,2-benzenediol hydrochloride, Sigma) challenge (1.0 and 0.5 mg/kg of BW in experiments 1 and 2, respectively) to obtain basal values. Epinephrine acts on the adrenoreceptors of vascular smooth muscle and exerts a strong vasoconstrictive effect (Smith et al., 2000), thereby increasing MAP and PAP. Evaluation of the vasodilation capacity was estimated by measuring the time that birds within each dietary treatment took to return to basal levels. The PAP, MAP, and HR responses were measured at 30, 60, 120, 180, 300, 600, and 1,200 s after Epi. After the first 1,200-s recovery period, a second Epi challenge was applied and the same points described for the first challenge were used to sample the resulting curve.

The Biopac MP100 system monitored 2 primary channels, including MAP and PAP. Values for these parameters were averaged electronically during 10-s recordings at the representative sampling times described above. The protocol used for data averaging was previously demonstrated to accurately compensate for the influences of pulse pressure and respiratory cycles on pulmonary and systemic arterial pressures (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 humanely killed and the heart was dissected to determine RV/TV as an indicator of PHS (Burton et al., 1968; Cueva et al., 1974).

Determination of NOS Activity
The activity of the NOS enzyme was estimated by the conversion of L-[14C]arginine to L-[14C]citrulline in vitro using isolated lung arteries (NOS assay kit, Cayman Chemical Co., Ann Arbor, MI). Immediately after the bird was killed the left lung was removed and placed on ice-cold physiologic saline solution. The pulmonary artery was carefully isolated, frozen in liquid nitrogen, and stored at –80°C until analysis.

The arteries were weighed, thawed, and homogenized in a previously chilled manual tissue grinder, containing 10 mL of cold homogenization buffer per gram of tissue (25 mM Tris-HCl, pH 7.4; 1 mM EDTA; 1 mM ethylenglycol-bis (ß-aminoethylether)-N,N, N',N' tetraacetic acid). The homogenate was centrifuged at 10,000 x g for 15 min at 4°C and the supernatant was transferred to microcentrifuge tubes and kept on ice.

The reaction buffer containing 1 µL of L-[14C]arginine monohydrochloride (50 µCi/mL; Amersham Biosciences, GE Healthcare Canada, Montreal, Quebec, Canada), 5 µL of 10 mM nicotinamide adenine dinucleotide phosphate (freshly prepared in 10 mM Tris-HCl, pH 7.4), 5 µL of 6 mM CaCl2 (Calbiochem, La Jolla, CA), and 4 µL of deionized H2O was combined with 10 µL of tissue homogenate. The reaction was removed from ice and incubated for 45 min at room temperature. After incubation, the reactions were stopped with 400 µL of stop buffer [50 mM N-2-hydroxymethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 5.5, and 5 mM EDTA]. To process the samples, 100 µL of equilibrated resin was added to each microtube to bind unreacted arginine, and each reaction mix was transferred to a microcentrifuge tube equipped with a filter to retain the resin, allowing the citrulline to flow. Samples were centrifuged in a microcentrifuge at 17,000 x g for 30 s and the filter removed.

The resulting eluate was transferred to scintillation vials and 3 mL of liquid scintillation cocktail (Universol, ICN Biomedicals, Costa Mesa, CA) was added. Radioactivity corresponding to L-[14C] citrulline was measured (cpm) in a liquid scintillation counter (LKB Wallak, Turku, Finland). The conversion of arginine to citrulline was calculated with the following equation: Percentage conversion = (cpm of the eluate – cpm blank)/total cpm) x 100. The blank corresponded to a reaction mixture incubated with N{omega}-nitro-L-arginine methyl ester, and the total count corresponded to a reaction mixture not filtered through the resin. The percentage conversion and the specific activity of the L-[14C]arginine (305 mCi/mmol) were used to estimate the NOS activity as arginine converted to citrulline (pg/min per g of artery).

Determination of Thiobarbituric Acid Reactive Substances
Vacutainers containing EDTA were used to collect 2 mL of blood from 7 healthy birds in each group. The blood was centrifuged at 1,500 x g for 5 min; plasma was collected in labeled tubes and stored at –80°C until analysis. After thawing, 100 µL of plasma was placed in a labeled glass tube and mixed with the reagents of a commercial kit (Oxitek, Buffalo, NY) for the measurement of thiobarbituric acid reactive substances (TBARS), and each tube was covered with a glass marble and incubated at 95°C for 60 min. The tubes were removed from incubation and allowed to cool in an ice bath for 10 min. Once cooled, the tubes were centrifuged at 1,100 x g for 15 min and the supernatant carefully removed from the tubes for analysis. The absorbance of the supernatants was measured at 532 nm using a UV/VIS spectrophotometer (Gildford Instrument Laboratories, Inc., Oberlin, OH) and the results were compared against a standard curve made with 100, 50, 25, 12.5, and 0 nmol/mL of malondialdehyde dimethyl acetal.

Statistical Analysis
Body weight, hematocrit, RV/TV, and values within sampling points for PAP, MAP, and HR were analyzed using a 1-way ANOVA and means were separated by the Student-Newman-Keuls method in both experiments, using the GLM procedure of SAS (SAS Institute, 1998). The PAP, MAP, and HR values were analyzed within group over time using the SigmaStat (Jandel Scientific, 1994) repeated-measures ANOVA, and means were separated with the Student-Newman-Keuls method. Sampling times of –180, –120, and –60 s were considered as baseline values for the first Epi challenge, and the sampling time of 1,200 s was considered as baseline for the second Epi challenge. Differences were declared when the PAP, MAP, and HR parameters during the Epi challenges were statistically different (P < 0.05) from all respective baselines values.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Previous experiments have reported the use of Arg to reduce cold-induced ascites mortality in broilers, with positive results when this amino acid is supplemented above the NRC recommendation, probably due to improvement in NO production; however, these observations have not been consistent (Wideman et al., 1995; Ruiz-Feria et al., 2001; Tan et al., 2005). In the same way, VE provided to chickens at levels above the NRC (1994) recommendations has been shown to reduce the incidence of ascites when administered as a subcutaneous implant, but without any effect on ascites mortality when provided as a dietary supplement (Bottje et al., 1995, 1997). Oxygen-derived free radicals cause a loss of NO bioavailability leading to a reduction in endothelial vasodilation (Lopez-Lopez et al., 2001). Subsequently, we hypothesize that an increment of antioxidant levels in the diet of broiler chickens may contribute to protecting the NO bioavailability, as well as the endothelium integrity, thus increasing the pulmonary vasodilation capacity. The objectives of these experiments were to study the effects of Arg in pulmonary circulation and parameters associated with PH, and to investigate if these effects were enhanced by the combination of VE and Arg.

In experiment 1, PAP increased (P < 0.01) within 30 s after Epi challenge in all treatments compared with the prechallenge values (Figure 1Go). There were no differences among treatments in the PAP values within the same sampling period. However, the time required for each experimental group to return to basal levels did differ. After the first Epi challenge, PAP in the control group returned to basal levels within 60 s, the HArg group recovered within 120 s, the HVE group recovered within 180 s, and it took more than 600 s for the Arg-VE group to return to basal levels. After the second Epi challenge, PAP in the control, HVE, and Arg-VE groups returned to basal values within 300 s, but in the HArg group, PAP returned to prechallenge values 120 s earlier (within 180 s). Accordingly, the best vasodilatory response in experiment 1 (estimated by the time taken for PAP to return to prechallenge values) was achieved after the first challenge by the control group followed by the HArg group. After the second challenge, the vasodilator capacity of the HArg group was better than that of the other treatments, possibly because the first challenge depleted the existing pool of Arg readily available as NOS substrate in the control, HVE, and VE-Arg groups. In this context the additional availability of Arg in the HArg group may have supported increased NO production, representing a comparative advantage in terms of vasodilatory capacity. Because increases in PAP represent a key step in the pathogenesis of ascites (Wideman, 1988), improvements in pulmonary vasorelaxation should help reduce the mortality caused by ascites (Wideman et al., 1995). Although plasma concentrations of Arg were not measured in these experiments, we previously demonstrated that supplementation with 0.3% Arg in the drinking water increased plasma Arg levels from 480 to 540 nmol/mL within 3 h of Arg ingestion (Ruiz-Feria et al., 2001).


Figure 1
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  		Figure 1. Pulmonary arterial pressure (PAP, experiment 1), after 2 epinephrine (Epi) challenges (1 mg/kg of BW i.v.) in male broilers consuming a commercial corn-soybean meal–based diet (1.2% Arg and 40 IU of VE/kg). Birds were provided tap water (control, n = 7), water with 0.3% Arg (HArg, n = 7), water with 400 IU of VE/L (HVE, n = 7), or a combination of both compounds at the same levels (Arg-VE, n = 7). Data were averaged during representative samples intervals at 180, 120, and 60 s before the first Epi challenge (basal values) and 30, 60, 120, 180, 300, 600, and 1,200 s post-Epi injection. The second Epi challenge was administered at the time 1,200 s, the value at time 1,200 s before the challenge served as the basal value, and the same sampling intervals as in the first challenge were used. Asterisks represent values that are different (P < 0.05) from all the respective basal values within a group.

 
Birds in experiment 2 were injected with a lower dose of Epi than those in experiment 1 (0.5 vs. 1.0 mg/kg of BW). We decided to reduce the dose of Epi in the second experiment because the PAP response in the first experiment was very strong for all the treatment groups. In experiment 2, PAP also increased (P < 0.01) within 30 s after the first Epi challenge in all treatments, except in the HArg treatment (Figure 2Go). The PAP of the control, HVE and Arg-VE groups returned to pre-Epi values within 600 s after the Epi challenge. After the second challenge, PAP in the HArg group did not show significant increases compared with the basal levels, whereas birds in the other treatments showed a significant increase in PAP 30 s after the Epi challenge. The PAP of birds in the control and Arg-VE groups returned to basal levels within 120 and 300 s, respectively, but in the HVE group, PAP returned to basal levels after 600 s. All the treatments groups had PAP values lower than basal levels in the last sampling point (1,200 s), except for the Arg-VE treatment group. There were no differences in PAP among treatments within the same sampling period.


Figure 2
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  		Figure 2. Pulmonary arterial pressure (PAP, experiment 2), after 2 epinephrine (Epi) challenges (0.5 mg/kg of BW i.v.) in male broilers fed a commercial corn-soybean meal–based diet (1.2% Arg). Birds were provided tap water and a diet containing 40 IU/kg of feed of vitamin E (control, n = 7), water with 0.3% Arg and a diet with 40 IU/kg of feed of vitamin E (HArg, n = 8), tap water and a diet with 400 IU/kg of feed of vitamin E (HVE, n = 8), or water with 0.3% Arg and a diet with 400 IU/kg of feed of vitamin E (Arg-VE, n = 8). Data were averaged during representative samples intervals 180, 120, and 60 s before Epi (basal values), and 30, 60, 120, 180, 300, 600, and 1,200 s post-Epi injection. The second Epi challenge was administered at the time 1,200 s, the value at time 1,200 s before the challenge served as the basal value, and the same sampling intervals as in the first challenge were used. Asterisks represent values that are different (P < 0.05) from all the respective basal values within a group.

 
In general, birds receiving supplemental Arg showed a better pulmonary vasodilation response in both experiments, as reflected by the rapid return to basal levels in experiment 1, and by the lack of a significant response of the HArg group to the Epi challenge in experiment 2. These results support the hypothesis that supplemental Arg improves pulmonary arterial relaxation, which in turn could reflect improved NO synthesis because of higher substrate availability (Taylor et al., 1992; Wideman et al., 1995, 1996). On the other hand we found that VE supplementation did not improve or augment the effects of Arg on vasodilation, and it appears that VE supplementation at the levels used in this experiment negated the ability of Arg to improve pulmonary vasodilation under acute challenges. Catecholamines stimulate the production of NO (Chi et al., 2003; Lin et al., 2005), for this reason we have attributed the return of PAP to basal levels after Epi challenge solely to the effects of NO, although other mechanisms may also be involved.

It was anticipated that vitamin E might improve vasodilation by protecting the endothelium from free radical damage, and a healthier endothelium might have more functional cells with higher constitutive levels of NOS, which could contribute to a higher NO production. We did not find significant differences in NOS activity among dietary treatments in either experiment (Table 1Go), which in part may be attributable to the large variability within treatments. However, we found a consistent tendency for the HArg group to present the lowest values for NOS activity in both experiments; in this group, we had more samples with undetectable levels of citrulline. This observation contrasts with the performance of the HArg group, which presented the best pulmonary relaxation response after the Epi challenge. The NOS could be down-regulated in the HArg group because of the better vasodilation response elicited by a higher substrate for NO production and a resulting lower shear stress. Schroeder et al. (2000), in a rat model designed to study hepatic hypertension, observed that rats that developed hepatic and pulmonary hypertension presented a 9-fold increase in inducible NOS activity in the pulmonary endothelium. Thus, it may be possible that birds developing PHS also present an increased activity of inducible NOS, explaining the tendency of the treatments other than HArg to have higher NOS activity.


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  		Table 1. Conversion of arginine to citrulline (expressed as a percentage of added 14C-arginine) and citrulline produced (pg/min) in a homogenate of pulmonary arteries to estimate nitric oxide synthase activity of male broilers consuming a corn-soybean meal based diet and different levels of arginine and vitamin E (VE; means ± SE)
 
We further anticipated that vitamin E might improve vasodilation by protecting NO from direct free radical attack, increasing its life span, and avoiding the formation of peroxynitrite (Beckman and Koppenol, 1996). The levels of TBARS in peripheral blood were measured as an indicator of lipid peroxidation and oxidative stress before and after exposure to low temperature. Levels of TBARS did not differ among treatments before cold exposure at d 14 (Table 2Go). However, there was a tendency for the birds in the HArg, HVE, and Arg-VE groups to have higher TBARS values than birds in the control group (P = 0.06) after cold exposure at d 35. It has been documented that both Arg and VE may become prooxidative agents under some circumstances. For instance, higher levels 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 levels of Arg were associated with increased endothelial lesions in the aorta of birds, presumably due to increased oxidative stress. On the other hand, excessive VE intake, when combined with salmon oil in the diet, lowers the antioxidant activity of superoxide dismutase, glutathione peroxidase, and glutathione in rat erythrocytes (Eder et al., 2002). Vitamin E can react with organic peroxides interrupting the chain reaction of lipid peroxidation. This reaction involves the formation of tocopheroxyl radicals, which are regenerated by means of hydrogen donors. If this reduction is incomplete, the tocopheroxyl radicals can initiate oxidative processes (Mukay, 1993; Thomas et al., 1996; Schneider, 2005).


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  		Table 2. Plasma levels of thiobarbituric acid reactive substances (TBARS, malondialdehyde equivalents, nmol/mL) before (d 14) and 19 d after cold exposure (d 35) in male broilers consuming a corn-soybean meal–based diet and different levels of arginine and vitamin E (VE; means ± SE)
 
In these experiments VE did not improve the vasorelaxation effects of Arg; moreover, VE combination with Arg in both experiments decreased the pulmonary vasodilation performance. The fact that groups provided high levels of VE had the longest recovery period after Epi challenge suggests that VE at the levels used in this experiment may have detrimental effects on pulmonary relaxation. These results agree with recent epidemiological studies in humans in which the use of VE in patients to prevent cardiovascular diseases has had null or even negative effects in high-risk patients (Kris-Etherton et al., 2004). There is evidence of chemical differences between the isomers of VE that could be important to consider for future research. Although {gamma}-tocopherol is considered a less potent chain-breaking antioxidant compared with {alpha}-tocopherol, its molecular arrangement appears to make it better to trap lipophilic electrophiles such as reactive nitrogen oxide species (Kamal-Eldin and Appelqvist, 1996). This feature may be important to rapidly decrease molecules like peroxynitrite, a potent oxidant believed to cause direct tissue injury (Beckman and Koppenol, 1996). Furthermore, {gamma}-tocopherol has antiinflammatory properties and a less hydrophobic condition than {alpha}-tocopherol (Jiang et al., 2000, 2001).

The MAP presented the same tendency in all treatments, increasing markedly within 30 s after the Epi injection in both experiments and returning to basal levels within 1,200 s in experiment 1 and within 600 s in experiment 2, due to the different Epi doses used in the 2 experiments (data from experiment 1 shown in Table 3Go). After the Epi challenge, HR decreased in all groups and it returned to basal levels 600 s after the Epi injection in both experiments (data from experiment 1 shown in Table 3Go). Epinephrine triggers immediate increases in total peripheral resistance and pulmonary vascular resistance, which increase the MAP and PAP in spite of reductions in HR and cardiac output (Wideman, 1999). In these experiments, neither MAP nor HR presented differences among treatments, and the time that they took to return to basal levels was similar among treatments, suggesting that the differences in PAP discussed above can be attributable to group differences in the pulmonary vascular tone.


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  		Table 3. Mean arterial pressure (MAP, mmHg), and heart rate (beats/min) after 2 epinephrine (Epi) challenges (1.0 mg/kg of BW i.v.) in male broilers fed a corn-soybean meal–based diet and different levels of arginine and vitamin E (VE)1
 
Hematocrit values were not different among treatments in experiment 1; however, in experiment 2, the HVE and HArg groups had lower hematocrit values than the control and Arg-VE groups at wk 6 (Table 4Go). A higher hematocrit is associated with sustained hypoxia (Yersin et al., 1992) and is correlated with ascites susceptibility (Wideman et al., 1998), suggesting some advantage in the gas exchange rate in the HVE and HArg groups. However, the RV/TV (a better indicator of sustained hypertension) was not different among treatments. Hematological changes in response to systemic hypoxemia may occur more rapidly than cardiac hypertrophic responses, explaining the differences found in hematocrit values but not in RV/TV (Table 4Go). As expected there was no difference among the treatments in BW in both experiments, showing that the different levels of VE and Arg among the treatments did not affect feed consumption, and neither presented detectable toxicity evidence.


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  		Table 4. Hematocrit (H) from wk 3 to 6 and right ventricle/total ventricle ratio (RV/TV) in male broilers consuming a corn-soybean meal based diet and different levels of arginine and vitamin E (VE)
 
In summary, Arg supplementation improved pulmonary arterial relaxation, probably by increasing NO levels due to the extra substrate availability for NOS. The supplementation of VE (water soluble or in the feed) alone or in combination with Arg did not improve pulmonary relaxation capacity after an acute Epi challenge. High levels of VE were associated with longer recovery periods of PAP after challenge, suggesting that VE at the levels used in these experiments may have detrimental effects on pulmonary relaxation. Neither Arg nor VE supplementation at the levels used in these experiments improved the bioavailability of the enzyme NOS, measured as the in vitro capacity of this enzyme to transform arginine to citrulline.


   ACKNOWLEDGMENTS
 
This research was supported by a grant from the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQNRT).


   FOOTNOTES
 
1 Present address: Department of Poultry Science, University of Arkansas, Fayetteville 72701. Back

Received for publication May 12, 2006. Accepted for publication July 26, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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