Antioxidant Defenses in Lung Lining Fluid of Broilers: Impact of Poor Ventilation Conditions W. G. BOTTJE,*,1 S. WANG,* F. J. KELLY,† C. DUNSTER,† A. WILLIAMS,† and I. MUDWAY† *Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville Arkansas 72701, and †Cardiovascular Research, The Rayne Institute, St. Thomas Hospital, London, SE1 7EH, United Kingdom in Controls. An increase in the GSSG to total glutathione (GSx) ratio, an indicator of oxidative stress, was also observed in birds maintained in the House environment. Ascorbic acid was not detected in House-reared birds and detected in only 4 of 12 Controls. Regression analysis revealed positive correlations between lung lining fluid protein and uric acid (r = 0.71; P < 0.01), protein and GSSG (r = 0.73; P < 0.01), and uric acid and GSSG concentrations (r = 0.69, P < 0.01). Additionally, GSSG was positively correlated (r = 0.66; P < 0.01) with the right ventricular weight ratio, an index commonly used in identifying the development of pulmonary hypertension syndrome in broilers. These data, the first to document lung lining fluid antioxidants in avian species, indicate an oxidative stress can be detected in fluid of broilers exposed to high levels of dust and ammonia in a simulated poultry house environment.
ABSTRACT Lung lining fluid antioxidants represent a potentially important protective barrier of lung epithelial cells to damaging effects of air pollutants, yet no information is apparently available concerning lung lining fluid antioxidants in broilers. Therefore, goals of this study were to establish uric acid, ascorbic acid, reduced (GSH) and oxidized (GSSG) glutathione, and protein concentrations in lung lining fluid obtained from male broiler chickens maintained for 6 to 7 wk within environmentally controlled rooms (Control) or chronically exposed to high levels of dust and ammonia within a broiler rearing house (House). The entire respiratory tract was carefully removed following an overdose of anesthetic and lavage fluid was collected after flushing the lungs with heparin-saline (10 mL per lung). There was no difference in GSH, but GSSG, uric acid, and protein concentrations were higher in House birds than
(Key words: antioxidants, lung lining fluid, glutathione, uric acid, ascorbic acid) 1998 Poultry Science 77:516–522
is no information to our knowledge on antioxidant composition of lung lining fluid in avian species, a primary objective of this study was to obtain baseline information on antioxidant concentrations in lung lining fluid in broiler chickens maintained under clean housing conditions. In commercial broiler production, poultry houses are ventilated primarily to remove moisture from the house or to cool the birds during warm weather (Carr and Carter, 1985). The rate of ventilation is generally sufficient to remove house air pollutants such as dust and gases (e.g., ammonia and carbon monoxide). However, during periods of cool weather, poultry producers often decrease ventilation to reduce heating, thereby allowing dust and gaseous pollutants to accumulate in the air.
INTRODUCTION The epithelial surface of the mammalian lung is covered by a thin layer of epithelial lining fluid that is derived from plasma exudate and secretions from underlying lung and resident immune cells (Reynolds, 1987). Protection of lung epithelial cells from inhaled pollutants has been hypothesized to be facilitated by antioxidants present in the lung lining fluid (Cantin et al., 1990; Slade et al., 1993; Cross et al., 1994; Kelly et al., 1995). Typically, epithelial lining fluid contains reduced glutathione (GSH) (Cantin et al., 1987), ascorbic acid (Skoza et al., 1983; Bui et al., 1992), uric acid (Crissman et al., 1989) and a-tocopherol (Mustafa, 1990), antioxidant enzymes (Cantin et al., 1987; Avissar et al., 1996), and metal binding proteins (Pacht and Davis, 1988). As there
Received for publication March 21, 1997. Accepted for publication November 11, 1997. 1To whom correspondence should be
[email protected]
Abbreviation Key: GSH = reduced glutathione; GSSG = oxidized glutathione; Gsx = total glutathione; PHS = pulmonary hypertension syndrome; RV:TV = right ventricular weight ratio.
addressed:
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ANTIOXIDANTS IN LUNG FLUID OF BROILERS TABLE 1. Starter and grower diet composition
Component
Starter diet
MATERIALS AND METHODS Grower diet
(%) Corn Soybean meal Poultry by-product Animal fat Dicalcium phosphate Calcium carbonate Salt Alimet 88% Mineral premix1 Vitamin premix2
58.10 30.23 5.00 3.58 1.10 1.03 0.40 0.26 0.10 0.20
63.64 27.85 2.50 3.00 0.98 1.38 0.32 0.03 0.10 0.20
1Supplied per kilogram of diet: Mn (MnSO ·H O), 100 mg; Zn 4 2 (ZnSO4·7H2O), 100 mg; Fe (FeSO4·7H2O), 50 mg; Cu (CuSO4·7H2O), 10 mg; I [CaI(O3)2·H2O], 1 mg. 2Supplied per kilogram of diet; vitamin A (retinyl acetate), 7,710 IU; cholecalciferol, 2,203 IU; vitamin E, 17 IU; vitamin B12, 0.013 mg; riboflavin, 6.6 mg; niacin 38.5 mg; pantothenic acid, 16 mg; menadione, 1.5 mg; folic acid, 0.881 mg; pyridoxine, 3.3 mg; thiamine, 1.5 mg; dbiotin, 0.07 mg.
A costly metabolic disease in poultry, termed pulmonary hypertension syndrome (PHS, ascites), has been associated with cold temperatures and poor ventilation of poultry houses (Lopez-Coello et al., 1985; Julian, 1993). Low ventilation and cool temperatures have been used experimentally to increase the incidence of PHS mortality (Enkvetchakul et al., 1993; Bottje et al., 1995a; Wideman et al., 1995). An underlying factor predisposing poultry to PHS is an insufficiency of the pulmonary vasculature to cope with increases in cardiac output that occur as a consequence of hypoxic-mediated systemic vasodilation and/or increased metabolic rate (Powell et al., 1985; Timmwood et al., 1987; Vidyadaran et al., 1990; Wideman and Bottje, 1993). This inability to cope with even relatively small increases in cardiac output can quickly lead to increases in pulmonary arterial pressure in broilers (Wideman et al., 1996). Increased pulmonary arterial pressure is followed by exudation of fluids from the liver, vascular congestion, and death from congestive heart failure. Recently, we reported that antioxidant levels were depressed in lung and liver of broilers in advanced stages of PHS (Bottje et al., 1995a) and hypothesized a role of oxidative stress in the pathophysiology of this syndrome (Bottje and Wideman, 1995). Lung lining fluid antioxidants could represent an important protective barrier against inhaled pollutants in the broiler lung and might be overwhelmed by high levels of dust, ammonia, and other pollutants that accumulate in poorly ventilated poultry houses. Thus, a second objective of this study was to determine the effect of low ventilation and cool temperatures on lung lining fluid antioxidants.
2Randall Road Hatchery, Tyson Foods, Inc., Springdale, 3Dra ¨ ger No. 1005, Ro¨hrchen Dra¨gerwerk, AG Lubeck,
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AR 72762. Germany.
Animals Male broiler chicks (Cobb 500) were obtained from a local hatchery2 at 1 d of age. The birds were fed a commercial starter (Weeks 0 to 3) and grower (Weeks 3 to 7) diet (Table 1) formulated to meet or exceed all requirements of the bird (NRC, 1995) with ad libitum access to feed and water. All procedures used were approved by the University of Arkansas Institutional Animal Care and Use Committee (protocols 96-288 and 96-302). The chicks were randomly assigned to Control or House environments. Control birds were maintained in battery cages (approximately 32 cm2 per bird) within environmentally controlled rooms under clean (Control) conditions. Ammonia levels were kept to a minimum (< 5 ppm), as was dust in the air, by adequate ventilation of the room and regular removal of feces, down, and dander material from cage trays. Control birds were kept thermoneutral by maintaining ambient temperatures at 32, 29, 27, and 24 C for Weeks 1, 2, 3, and 4 to 6, respectively. In contrast to Controls, birds in the House environment were placed at 1 d of age in floor pens (approximately 30 cm2 per bird). A layer of fresh wood shavings covered old litter in the pens that had been previously used for four flock cycles, a practice commonly used in the U.S. poultry industry. House birds were kept at thermoneutral temperatures of 32 and 29 C for Weeks 1 and 2, but air temperature was lowered from 29 to 18 C during Week 3, and kept between 10 and 15 C for Weeks 4 to 7 by decreasing supplemental heat and adjusting thermostatically regulated ventilation fans. Ammonia concentrations at bird level, detected using a Dra¨ger tube,3 was 18 ppm on Day 1 and 36 ppm on Day 42, but particulate content was not quantified. High ammonia levels were easily detected when working in the house and corresponded to an obvious visual accumulation of dust. The House conditions described above were used to simulate conditions known to induce PHS in the field (Enkvetchakul et al., 1993; Bottje et al., 1995a; Wideman et al., 1995).
Sampling of Lung Lining Fluid Lung lining fluid was obtained in the following manner. The birds were quickly overdosed with sodium pentobarbital (100 mg/kg BW i.v.). The entire respiratory tract (trachea and both lungs) was carefully removed from the body cavity and placed in a beaker containing ice-cold heparin-saline (200 units heparin per milliliter 0.85% saline). Blood was removed from the outside of the lungs by gentle rinsing in the heparin-saline solution. Lung lining fluid was obtained by infusing either 20 mL of heparin-saline into both lungs through a tube inserted in the trachea or 10 mL of heparin-saline into one lung through a tube advanced to the lung through a primary
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BOTTJE ET AL.
bronchus. The tubes were secured with suture to prevent fluid back leak. Due to ease of handling, data presented below were derived mainly from fluid obtained from one lung (n = 22) as opposed to both lungs (n = 5). No differences in antioxidant concentrations in lung lining fluid were observed following lavage of one or both lungs (data not shown). The introduction of heparin-saline over a 10 to 20 s period resulted in an immediate expansion of the lungs followed by percolation of fluid from the lungs via ostia that connect the air sacs to mediodorsal secondary bronchi, neopulmonic parabronchi, intrapulmonary primary bronchus, and lateroventral secondary bronchi (Dunker, 1972). The volume of effluent fluid was recorded to determine percent recovery of the fluid, aliquoted into microcentrifuge tubes, immediately frozen in liquid N2, and stored at –80 C. All antioxidant determinations were made within 6 wk after collection. Samples with a pink color, presumably from erythrocyte contamination, were not analyzed. Twelve broilers were studied in the Control and House groups, respectively. The average body weights of these sampled birds were 2,703 ± 75 g and 2,659 ± 147 g and average wet weight (± SEM) of lungs prior to lavage were 6.9 ± 0.3 and 5.9 ± 0.6 g for Control and House birds, respectively. After completing the lavage procedure, the weights of the right ventricle and total ventricle were determined as an index of pulmonary arterial pressure (Burton et al., 1968).
Measurement of Glutathione Total glutathione (GSx) concentration was determined using the enzymatic recycling method described by Tietze (1969) adapted for use on a microplate reader (Baker et al., 1990). In this method, standards containing 0 to 165 pmol/ 50 mL oxidized glutathione (GSSG), equivalent to 0 to 330 pmol/50 m: GSx, were prepared in 150 mmol/L NaCl, 1 mmol/L EDTA (pH 7.5). Fifty milliliters of standard or sample were transferred into micro-titer plate wells and 100 mL of reaction mixture was added to each well consisting of 0.15 mmol/L dithionitro benzoic acid, 0.2 mmol/L NADPH, and 1 unit of GSH-reductase final concentration. Immediately after addition of this reaction mixture, the micro-titer plate was placed on a micro-titer plate reader4 for analysis. The rate of thionitrobenzoic acid formation was followed by the rate of change in absorbance at 405 nm over a 2-min period at 30 C. The amount of GSSG in samples was determined by adding 5 mL of undiluted 2-vinyl pyridine to 130 mL of sample or standard. The conjugation of the vinyl pyridine was facilitated by vortexing and incubation for 1 h at room temperature. Standards and samples were then plated and assayed as outlined above. Reduced glutathione
4Model EL 340, Biokinetics Reader, Winooski, VT 05404. 5Model 231, Gilson Medical Electronics, Middleton, WI 53562. 6Jones Chromatography, Hengoed, Wales, UK. 7 Kit No. BCA-1, Sigma Chemical Co., St. Louis, MO
63178-9916.
(GSH) concentration was determined by subtraction of GSSG from GSx concentration.
Measurement of Uric Acid, Ascorbic Acid, and Protein Determination of uric acid and ascorbic acid concentrations in lung lining fluid was based on the HPLC method of Iriyama et al. (1984). Lung fluid effluent samples (450 mL) were acidified with 50 mL of 16% metaphosphoric acid followed by extraction of lipid components with 100 mL of heptane. The mixture was vortexed vigorously for 40 s and centrifuged at 13,000 × g for 5 min at 4 C. The lower aqueous layer was carefully removed and transferred to a 1-mL HPLC. vial. Aliquots of 20 mL were injected for analysis using a Gilson Autosampler.5 A 10 × 300 mm, 5-mm C18 column was eluted with a 0.2 mol/L K2HPO4H3PO4 mobile phase running buffer (pH 2.1, containing 0.25 mmol/L octanesulfonic acid) at a flow rate of 1.8 mL/ min. An EG & G electrochemical detector6 was used for detection with E set at 810 mV, a time constant of 5 s, and the cathodic output and sensitivity of 100 nA. Protein concentrations in lung lining fluid were determined using a bicinchoninic acid assay.7
Statistical Analysis Data were analyzed by Student’s t test to determine differences between treatments. However, as one parameter (GSSG) failed the test for normality of distribution, Wilcoxon’s nonparametric signed rank test for paired observations was used here to determine differences in lung lining antioxidant concentrations between Control vs House conditions. A value of P < 0.05 was considered significant. Additionally, relationships between lung lining constituents with each other or with the right ventricular weight ratio (RV:TV) were assessed using Spearman’s correlation coefficient and significance determined by linear regression analysis using the least squares means method. Heterogeneity of linear regression of Control and House data were tested separately for significance. Data are shown for those relationships in which the regressions for House and Control data were significant for both groups and in which the slope and intercept did not differ (P > 0.05) from each other.
RESULTS Concentrations of antioxidants (in micromoles per liter) and protein (milligrams per milliliter) in lung lavage fluid from birds maintained under Control and House conditions are presented in Table 2A. Although there were no differences in GSH between groups, House birds exhibited higher GSSG, uric acid, and protein concentrations, and a higher GSSG:GSx ratio than Controls. Ascorbic acid was detected in only 4 of 12 Control birds and was not detected in any bird maintained in the House environment. As differences in
ANTIOXIDANTS IN LUNG FLUID OF BROILERS
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TABLE 2. Concentrations of reduced (GSH) and oxidized (GSSG) glutathione, uric acid, ascorbic acid, and protein expressed as either concentration per liter of lavage fluid, concentration per gram of lung lavaged, or concentration per milligrams of protein in lavage fluid in male broilers maintained under Control and House conditions1 Variable
Control
House
mmol/L GSH GSSG GSSG/GSx Uric acid Ascorbic acid2 Protein, mg/mL nmol/g lung3 GSH GSSG GSSG/Gx Uric acid Ascorbic acid Protein, mg/g lung nmol/mg Protein GSH GSSG GSSG/GSx Uric acid Ascorbic acid
(n = 12) 2.51 ± 0.23 0.20 ± 0.06 0.07 ± 0.01 2.92 ± 0.44 1.46 ± 0.39 0.18 ± 0.02 (n = 11) 4.57 ± 0.55 0.34 ± 0.08 0.07 ± 0.01 4.94 ± 0.38 0.27 ± 0.39 0.32 ± 0.03 (n = 12) 14.9 ± 1.47 1.05 ± 0.21 0.07 ± 0.01 17.0 ± 1.86 11.9 ± 4.85
(n = 12) 3.67 ± 0.60 0.74 ± 0.09* 0.16 ± 0.03* 6.79 ± 1.10* ND 0.35 ± 0.05* (n = 12) 4.78 ± 0.60 0.95 ± 0.09* 0.19 ± 0.03* 9.01 ± 1.10* ND 0.40 ± 0.03* (n = 12) 11.0 ± 1.26 2.29 ± 0.25* 0.20 ± 0.04* 21.5 ± 1.80* ND
1Values represent the mean ± SEM with the number (n) of observations shown in parentheses. 2Values for ascorbic acid represent the mean ± SEM of four observations; ascorbic acid values in the remaining birds in the Control group were below the level of detection. 3Values were determined by dividing the total amount of antioxidant obtained in lavage fluid (corrected for percent lavage fluid recovery) by the weight of lung tissue lavaged. *House means are different from Control (P < 0.05).
lung lining fluid antioxidants might be attributed to the amount of lung tissue lavaged or lavage fluid recovered, concentrations of GSH, GSSG, and uric acid were also calculated as the total amount of compound corrected for lavage fluid recovery per gram of lung (Table 2B). The percent of lavage fluid recovered was 86.3 ± 3.5 and 79.8 ± 2.4 for Control and House groups, respectively, and were not different (P > 0.05). As higher levels of GSSG, the GSSG:GSx ratio, uric acid, and protein in lavage fluid were also observed in House birds when lung lining components were corrected for recovery and amount of lung tissue lavaged (Table 2B) differences between groups (Table 2A) were apparently not due to a collection artifact. Expression of lung lining components as concentration per milligrams of protein according to Slade et al. (1993) revealed similar results (Table 2C). Regression analysis revealed significant and positive correlations between protein and uric acid (Figure 1A), between protein and GSSG (Figure 1B), and between uric acid and GSSG (Figure 1C). Significant relationships between lavage fluid protein concentration and GSH or ascorbic acid were not observed (data not shown). The RV:TV was higher in birds maintained in House (0.37 ± 0.06) than in Control (0.19 ± 0.02) conditions. The RV:TV ratio was positively correlated with lung lining GSSG (Figure 2).
FIGURE 1. Relationships of between (A) protein and uric acid concentration (B) protein and oxidized glutathione (GSSG), and (C) uric acid and GSSG in lung lining lavage fluid. The regression equation, r value, and significance level are shown on the inset of the graph. Closed circles and open triangles represent values for birds maintained under Control and House conditions, respectively.
DISCUSSION Antioxidants in the lung lining fluid form a first line of defense against inhaled toxins such as the air pollutants, ozone and nitrogen dioxide (Cantin et al., 1990; Slade et al., 1993; Cross et al., 1994; Kelly et al., 1995; 1996a,b). In the present study, lung lining antioxidant concentrations were determined in lavage fluid obtained from birds maintained in a clean, control environment and from birds following chronic exposure to a dirty, ostensibly air polluted environment. Whereas the presence of surfactant-like material has been demon-
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FIGURE 2. Relationships of the right ventricular weight ratio (RV: TV) to oxidized glutathione (GSSG) in lung lining lavage fluid. The regression equation, r value, and significance level are shown on the inset of the graph. Closed circles and open triangles represent values for birds maintained under Control and House conditions, respectively.
strated in avian lung (Pattle and Hopkinson, 1963; Fujiwara et al., 1970), to our knowledge, this is the first time antioxidant concentrations have been determined in lung lining fluid in any avian species. The method of obtaining lung lining fluid in the present study differs from that used to examine pulmonary surfactant phospholipids in turkeys (Fujiwara et al., 1970). In their study, Fujiwara et al. (1970) flushed 500 mL of saline in and out through the trachea of turkeys five times (7 to 10 kg BW) followed by final flush of 400 mL. This procedure, unlike that used in the present study, would result in flushing of air sac lining fluid in addition to lung epithelial lining fluid. Birds in this study were lavaged with 10 mL of saline per lung, an amount ranging between 7 and 10 mL/kg body weight. This volume of lavage fluid is 3.5 to 5 times lower than the 35 mL of lavage fluid/kg body weight used by Slade et al. (1993). Slade et al. (1993) reported uric acid and GSH concentrations in lavage fluid of 2.7 and 11.2 nmol/mg protein in rats, and 4.3 and 12.1 nmol/mg protein in guinea pigs. If antioxidant values (Table 1C) were divided by 4 to account for differences in lavage fluid volume between studies outlined above, lavage fluid uric acid concentration in Control broilers would be approximately the same as that found for guinea pigs and rats, and GSH would be 10 times less than values in these species reported by Slade et al. (1993). Uric acid and GSH concentrations in human lung lavage fluid were 0.2 to 0.3 mmol/L and 0.4 to 0.5 mmol/L, respectively (Kelly et al., 1996b). If an average body weight of 70 kg is assumed, the amount of saline used for lavage (180 mL per person) would be 2.6 mL/kg body weight, or 25 to 30% of the volume used in the present study. Multiplication of Control values (Table 1A) by 0.25 and 0.30 yields GSH (0.8 to 1.2 mmol/ L) and uric acid (1 to 2 mmol/L) values that are in a similar range as concentrations of these compounds reported in human lavage fluid (Kelly et al., 1996b). It should be pointed out that these comparisons may not account for differences in lung surface area between
species, but does allow a cursory species comparison to be presented. Pulmonary hypertension syndrome is a metabolic disease estimated to cost the world-wide poultry industry up to $500 million annually in mortality losses (Bottje et al., 1995b). An underlying factor predisposing broilers to PHS is a basic insufficiency of the pulmonary vasculature to cope with increases in cardiac output that occur as a consequence of hypoxic-mediated systemic vasodilation or increased metabolic rate (Powell et al., 1985; Timmwood et al., 1987; Wideman et al., 1996). It has been known for many years that PHS mortality is elevated during cold weather when ventilation of houses is decreased to reduce fuel expenditure. The effects of low ventilation and cool temperatures on PHS mortality have also been well documented experimentally (Enkvetchakul et al., 1993; Bottje et al., 1995a; Wideman et al., 1995). Results of recent studies (Enkvetchakul et al., 1993; Bottje et al., 1995a) have lead to a hypothesis that oxidative stress may play a role in the etiology of this syndrome (Bottje and Wideman, 1995). During oxidative stress, when reducing equivalents becoming limiting for GSH reductase, an enzyme that reduces GSSG to GSH, GSSG is actively transported out of cells into extracellular fluid to maintain cell redox status and to avoid oxidation of critical cellular components (Srivasta and Beutler, 1969; Sies et al., 1972; Ishikawa and Sies, 1984; Deleve and Kaplowitz, 1991). Elevations in GSSG and GSSG:GSx ratio in lung lining fluid obtained from broilers chronically exposed to poor air quality indicate that these conditions exerted an oxidative stress in lung tissue of these birds in comparison to broilers maintained under clean conditions. These findings lend further support to the hypothesized role of oxidative stress in the pathophysiology of PHS (Bottje and Wideman, 1995). Birds maintained in the House environment also exhibited elevated levels of protein in lavage fluid. This increased protein is likely of vascular origin, having crossed the blood/air barrier due to increased vascular permeability or may also reflect damage to epithelial cells. Several researchers have indicated the presence of antioxidant enzymes and metal binding proteins in lung lining fluid (Pacht et al., 1988; Cantin et al., 1990; Avissar et al., 1996). Although not measured, if levels of these metal binding proteins or antioxidant enzyme activity had increased in lung lining fluid of birds chronically exposed to air pollutants, this increase would represent enhanced antioxidant protection to the lung. In addition, a certain amount of nonspecific antioxidant protection to the lung would be afforded simply by the increase in protein level (Yu, 1994). A protective role of uric acid against gaseous pollutants has been proposed as levels of uric acid in bronchioalveolar lavage fluid decrease immediately following nitrogen dioxide exposure in humans (Kelly et al., 1996b). Additionally, Mudway (1996) demonstrated that uric acid was consumed at a faster rate than either
ANTIOXIDANTS IN LUNG FLUID OF BROILERS
ascorbic acid or GSH following exposure to ozone in vitro, implicating uric acid as a primary sacrificial antioxidant in lung lining fluid. Thus, higher levels of uric acid in lung lining fluid of birds maintained in the House environment may represent an adaptive response to help protect the lung from high levels of air pollutants (dust and ammonia) present in the House environment. Based on the results of this study, it is difficult to assess the role ascorbic acid plays in lung lining fluid of broilers. The lack of ascorbic acid in lung lining fluid in House-maintained birds might imply that ascorbic acid functions as an important sacrificial antioxidant in birds subjected to chronic air pollution, but this would not explain the lack of ascorbic acid in lung lining fluid in 8 of 12 Controls. Ascorbic acid was detected in lung lining fluid in 3 of 3 additional broilers that had been subjected to anesthesia and abdominal surgery (unpublished observations). Thus, further research is required to delineate a role of ascorbic acid in lung lining fluid in broilers. It should be noted that broilers raised in the House environment were exposed to cool temperatures in addition to low ventilation conditions. Consequently, differences between Control and House antioxidant concentrations in lung lining fluid could be partially due to a temperature effect and not strictly result from poor air quality. However, if House temperatures had been maintained similar to that of the Control environment, it is likely that even higher amounts of ammonia would have been present in the House air, which might have augmented differences in lung lining variables between treatments. In summary, lung lining fluid in male broiler chickens contains significant concentrations of the low molecular weight, water-soluble antioxidants GSH and uric acid, whereas ascorbic acid was detected in only 4 of 24 birds in this study. Birds maintained in a broiler house environment (House), in which low ventilation allowed dust and ammonia to accumulate, exhibited elevated levels of GSSG, protein, and uric acid in lung lining fluid in comparison to birds maintained in a clean environment (Control). These results indicate that the poor air quality in House air exerted an oxidative stress on lung lining fluid antioxidants in birds. Positive correlations between lung lining GSSG and the RV:TV ratio indicate that oxidative stress in lung lining fluid may be related to the subsequent development of PHS in broiler chickens.
ACKNOWLEDGMENTS Support for a professional leave of absence for W. G. Bottje to analyze lung lining fluid in Frank J. Kelly’s laboratory was provided by the Director of the Agriculture Research Experiment Station, University of Arkansas who has also approved publication of these data. The authors would like to thank Cory Evenson and Robert Moore for technical assistance.
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