Comparative Biochemistry and Physiology, Part C 141 (2005) 69 75 www.elsevier.

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Influence of organic selenium on hsp70 response of heat-stressed and enteropathogenic Escherichia coli-challenged broiler chickens (Gallus gallus )
Kamel Z. Mahmouda,*, F.W. Edensb
a

Department of Animal Production, Jordan University of Science and Technology, Box 3030, Irbid 22110, Jordan b Department of Poultry Science, North Carolina State University, Raleigh, NC 27695-7635, USA Received 28 December 2004; received in revised form 5 May 2005; accepted 6 May 2005 Available online 16 June 2005

Abstract The effect of dietary selenium yeast, a source of organic selenium, on heat shock protein 70 (hsp70) responses, redox status, growth and feed utilization were evaluated either in enteropathogenic Escherichia coli -challenged (EPEC) or in heat-stressed (HS) male broiler chickens grown to 42 days of age. One day-old chicks in experiment 1 were challenged orally with EPEC (106 cfu/chicken on day 1 and boosted by water application on days 2, 3, and 4) and fed diets with or without selenium yeast. Body weight (BW), feed conversion ratio (FCR), and total mortality were determined at 42 days of age, and this was followed by collection of ileal tissue for the quantification of total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70 in randomly selected chickens from each treatment. In experiment 2, male broiler chickens were fed diets with or without selenium yeast under a thermoneutral rearing condition. At four weeks of age, blood and hepatic tissue were collected from chickens maintained in the thermoneutral environment and from chickens subjected to HS (40 -C for 1 h) and analyzed for TGSH, GSH, GSSG, and hsp70. Selenium yeast improved BW, FCR, and decreased mortality in both control and EPEC-challenged chicks. Selenium yeast significantly attenuated hsp70 expression in EPEC-challenged chickens and in those subjected to HS. The EPEC challenge increased TGSH and GSSG levels and decreased GSH / GSSG ratio. However, GSSG level accumulated in chickens fed diets without selenium supplementation resulting in a lower GSH / GSSG ratio in the selenium yeast-fed group. Heat stress increased GSSG level and decreased GSH / GSSG ratio. Selenium yeast-fed groups maintained higher levels of GSSG before and after HS with a resultant lower GSH / GSSG ratio. The hsp70 response was significantly less in those chickens fed selenium yeast and challenged with either EPEC or HS than in those chickens given no supplemental selenium. The results of this study suggest that selenium yeast supplementation had imparted resistance to oxidative stress associated with enteric bacteria infection and to high temperature exposure. It is believed that the resistance to the stressors was due to an improved redox status of the selenium yeast-fed chickens. D 2005 Elsevier Inc. All rights reserved.
Keywords: Selenium; Chicken; Escherichia coli ; Heat stress; Hsp70; Glutathione

1. Introduction
Abbreviations: GPx, Glutathione Peroxidase; TGSH, total glutathione; GSH, Reduced Glutathione; GSSG, Oxidized Glutathione; Organic selenium/selenium yeast, Sel-Plex\; hsp, heat shock protein; hsp70, heat shock protein 70; EPEC, enteropathogenic Escherichi coli ; TR, thioredoxin; CAT, catalase; SOD, superoxide dismutase; ROS, reactive oxygen species; BW, body weight; FCR, feed conversion ratio; ME, metabolizable energy; CP, crude protein; HS, heat distress. * Corresponding author. Tel.: +962 2 720 1000x22212; fax: +962 2 709 5069. E-mail address: kmahmoud@just.edu.jo (K.Z. Mahmoud). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2005.05.005

Poultry diets deficient in selenium result in poor growth and development, increased mortality, reduced egg production, decreased hatchability, pancreatic fibrosis, and muscle myopathies (Walter and Jensen, 1963; Scott et al., 1967). In light of the faster growth rates of current commercial broiler stocks compared to those grown two decades ago, dietary selenium level and/or source need to be reconsidered. Supplemental selenium as inorganic sodium selenite in feed or drinking water of animals can range between 0.1 and 0.3

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ppm (NRC, 1994). In June 2000, selenium yeast was approved by the US-FDA (Federal Register, 2000) as a source for feed-supplemented organic selenium for chickens. Organic selenium from yeast (Sel-Plex\, Alltech, Inc.) is a highly available form of selenium for chickens and other livestock and provides antioxidant protection at a level greater than inorganic selenium (Mahan, 1999; Mahmoud and Edens, 2003). Organic selenium from selenium yeast has been shown to enhance meat quality (Edens, 1996; Mahan et al., 1999), growth of feathers (Edens, 1996), and positively influence thyroxine conversion to tri-iodothyronine and passive immunity of newborn lambs (Rock et al., 2001). Reactive oxygen species (ROS) are generated as byproducts of normal biochemical processes of oxygen metabolism in all aerobic organisms (Halliwell and Gutteridge, 1989; Pahl and Baeuerle, 1994). The antioxidant defense system in most cells is composed of two components: (1) the antioxidant enzyme component that includes enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx, primarily selenium-dependent), selenium-dependent thioredoxin reductase, and (2) the low molecular weight antioxidant components that includeamong othersglutathione (GSH) and thioredoxin (TR). Oxidative stress occurs when the production of ROS exceeds the bodys natural antioxidant defense mechanisms, causing damage to biomolecules such as lipids, proteins, and nucleic acids (Halliwell and Gutteridge, 1989). Selenium is an essential trace element that is involved in the regulation and control of the bodys antioxidant glutathione and GPx system, which plays a major role in the control of ROS (Palmer and Paulson, 1997). Exposure of animals to thermal extremes and bacterial infections are stressors that increase metabolic activity with a resultant increase in ROS production (Palmer and Paulson, 1997). It has been reported that inorganic selenium supplementation as sodium selenite to chickens improved their resistance to the stress caused by E . coli infection and by exposure to cold stress (Larsen et al., 1997). Heat shock proteins (hsps) are expressed in both prokaryotic and eukaryotic cells in response to a variety of stressors (for reviews, see Lindquist and Craig, 1988; Welch, 1992; Parsell and Lindquist, 1994; Mahmoud, 2000). Furthermore, the ubiquitous hsp70 apparently is involved in mechanisms protecting the body from the deleterious effects of ROS (Favatier et al., 1997; Polla, 1998). Interestingly, cellular GSH was found to be elevated under thermal stress conditions (Mitchell et al., 1983). Exposure of poultry species to mild stressors over a period of time enhances hsp70 expression, but eventually, the birds become acclimated and no further increase in cellular hsp70 can be demonstrated (Wang and Edens, 1998). There are reports that suggest a strong relationship between thiol oxidation and hsp70 synthesis in stressed cells (SierraRivera et al., 1988; Mahmoud and Edens, 2003). Mahmoud

and Edens (2003) have clearly shown that chickens provided organic selenium in yeast are more resistant to thermal stress than chickens fed inorganic sodium selenite, and this was demonstrated by a lower expression of hsp70. Thus, it was important to continue the study of the influence of organic selenium in yeast supplements on the induction of hsp70 and glutathione responses to a variety of other stressors in chickens. In this report, enteropathogenic E. coli (EPEC) challenge and acute heat distress were examined.

2. Materials and methods 2.1. Broiler chickens and husbandry This project was approved and conducted under the supervision of the North Carolina State University Animal Care and Use Committee that has adopted Animal Care and Use Guidelines governing all animal use in experimental procedures. Newly hatched male broiler chickens (Gallus gallus f. domesticus ) (Arbor Acres Arbor Acres) were obtained from the North Carolina Agricultural Research Service Poultry Field Laboratory hatchery, and were reared in an environmentally controlled isolation facility. The day-old chicks were identified with plastic transdermal neck tags and placed in heated brood-grow batteries with raised wire floors. Brooding temperature was 33 -C and this temperature was decreased incrementally to 22 -C by the time the birds were 21 days old. The experimental chickens were provided continuous lighting from incandescent lamps in the ceilings of each room, but each brood-grow battery provided an area of subdued light for sleeping and resting. The experimental diets consisted of a starter diet3177 kcal/kg ME, 22.5% CP (1 16 days of age), grower diet 3168 kcal/kg ME, 19.5% CP (16 35 days of age), and finisher diet3160 kcal/kg ME and 17.5% CP (35 42 days of age), and the vitamin E content in all of the broiler diets was 33 IU/kg of feed as reported by Edens et al. (2001). Other dietary nutrient components of the diet were equal to or greater than levels recommended by the National Research Council (1994). The diets were supplemented with selenium yeast (Sel-Plex\, Alltech, Inc., Nicholasville, KY, USA) as a source for organic selenium at an elemental selenium level of 0.2 mg/kg of feed. The background levels of selenium were 0.28, 0.28, and 0.24 mg/kg, in the starter, grower and finisher diets, respectively. Body weights (BW) and feed conversion ratios (FCR) were determined at 42 days of age, and mortality was recorded on a daily basis. 2.2. Experiment 1 (enteropathogenic Escherichia coli) A total of 128 day-old chicks were allocated among 16 pens, from which four randomly designated pens were assigned to each of the following four dietary treatments: (1) no supplemental selenium, sterile trypticase soy broth, (2)

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organic selenium (Sel-Plex\), sterile trypticase soy broth, (3) no selenium, enteropathogenic E . coli (serotype O1; EPEC), and (4) Sel-Plex\, EPEC. The EPEC was grown overnight at 37 -C in trypticase soy broth. The EPEC challenge dose (106 CFU/mL) was administered in a volume of 100 Al into the external nares of 1 day-old chicks. The EPEC challenge was boosted on days 2, 3, and 4-post hatch by adding 15 mL of the culture (10 106 CFU/mL) to the drinking water. At four weeks of age, 16 chicks from each treatment group were killed by carbon dioxide asphyxiation. To minimize the probability of a handling effect on hsp70 induction and GSH reduction, only two chicks in a group were processed at any time. A 1 g sample of the ileum from each chick was dissected and placed into ice-cold protein buffer (0.05 M Tris HCl, pH = 7.5, 0.15 M NaCl, 2 mM DTT, 5 mM EDTA, and 0.2% Tween-20). The tissues were washed three times to rinse away blood and were then homogenized. The measured variables were total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70. 2.3. Experiment 2 (heat stress) A total of 128 day-old chicks were allocated among 16 different pens, and each of two dietary treatments was assigned randomly to eight different pens. The first dietary treatment represented a control group without organic selenium supplementation (control). The second one supplemented with 0.20 mg/kg feed of organic selenium (SelPlex\). At four weeks of age, blood samples were collected by cardiac puncture from 20 chicks per treatment group before they were killed by carbon dioxide asphyxiation. The remaining chickens were subjected to acute heat distress for 1 h (40 -C) in a temperature-controlled chamber. Mortality was monitored, but there were no deaths during the short exposure period. The heat distressed chickens (20 per group) were then bled via cardiac puncture before they were killed by carbon dioxide asphyxiation. The liver was dissected and placed in an ice-cold protein buffer before being processed for quantifying the amount of hsp70, and the blood was used to quantify the amounts of TGSH, GSH, and GSSG. 2.4. Determination of TGSH, GSH, and GSSG A quantification of TGSH, GSH, and GSSG in the ileum (EPEC challenge study) and blood (heat distress study) was made using a colorimetric kit (GSH / GSSG-412; Oxis International, Inc., Portland, OR, USA). In the EPEC challenge study, 1 g ileum samples were dissected and placed into ice-cold protein buffer. The tissues were washed three times to rinse away blood and were then homogenized. Supernatants were collected after 1 h of centrifugation at 100,000 g and then total protein content was determined for each sample. The assays in the kit were adapted for a microplate reader with modifications of existing methods

(Tietze, 1969; Griffith, 1980). The GSH values were obtained by subtracting GSSG from TGSH. 2.5. Determination of hsp70 One gram tissue samples were washed three times to rinse away residual blood before the sample was minced in 4 mL of ice-cold protein buffer and subsequently homogenized with a Polytron Homogenizer (Heat System Ultrasonics, Plainview, NY, USA) for protein quantification. The homogenates were centrifuged at 10,000 g for 15 min, the supernatants were removed and further centrifuged at 100,000 g for 1 h (Beckman L5-50; Beckman Instruments, Palo Alto, CA, USA). Total protein content of the supernatant was measured using a Protein Assay Kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as a standard following the vendors instructions. Coefficients of variation, among replicates of the same sample, were less than 3%. Enzyme linked immunosorbent assay (ELISA) for liver and ileum hsp70 was performed as described by Anderson et al. (1993) with some modifications as

A
2.5 2.3

a bc c b

No EPEC EPEC

Bwt (Kg)

2.1 1.9 1.7 1.5

No Sel Plex

Sel Plex

2.25 2.15

a b c b

FCR

2.05 1.95 1.85 1.75 1.65

No Sel Plex

Sel Plex

C
Mortality (%)

40 35 30 25 20 15 10 5 0

b c
No Sel Plex

Sel Plex

Fig. 1. The effect of enteropathogenic E . coli (EPEC) challenge on Bwt (Panel A), FCR (Panel B), and mortality (Panel C) of broiler chicken with or without organic selenium (Sel-Plex\) supplementation. a,b,cHistogram columns with unlike superscripts differ significantly ( P  0.05).

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K.Z. Mahmoud, F.W. Edens / Comparative Biochemistry and Physiology, Part C 141 (2005) 69 75

HSP70 (ng/mg total protein)

A
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

a a

No EPEC EPEC

2.6. Statistical analysis A 2 2 factorially arranged completely randomized design was used for these experiments. The data were analyzed using by the General Linear Model procedure of SAS (SAS Institute, 1996) utilizing the following statistical model: Yijk = a + a i + b j + ab ij + e ijk . The effects of EPEC (a ), Sel-Plex\ (b ), and their interaction (ab ) were tested against the residual error (e ). The same model was used to test the effect of Sel-Plex\, heat distress, and their interaction. Statements of statistical significance were based on P  0.05 unless stated otherwise.

No Sel Plex

Sel Plex Pre-heat

HSP70 (ng/mg total protein)

B
a
6 5 4 3 No Sel Plex Sel Plex

ab b b

Post-heat

3. Results There were significant effects due to Sel-Plex\ and EPEC challenge (Fig. 1) on BW, FCR, and mortality. Broilers fed Sel-Plex\ were significantly ( P  0.05) heavier, had improved FCR, and they had lower mortality rates than birds without selenium supplementation. Additionally, there was a Sel-Plex\ EPEC interaction in which, SelPlex\-supplemented broilers with EPEC challenge were significantly ( P  0.05) heavier, had improved FCR, and lower mortality than EPEC-challenged broilers without the selenium supplementation. The hsp70 responses in the ileum of broiler chicks were elevated ( P  0.001) due to EPEC-challenge (Fig. 2A). Heat-distress increased significantly ( P  0.05) the hsp70 expression in the liver of the chickens (Fig. 2B). Similarly, the increase in hsp70 levels was higher ( P  0.001) between

Fig. 2. The effect of enteropathogenic E . coli (EPEC; Panel A) and heat distress (Panel B) on hsp70 expression in intestinal ileum (Panel A) or liver (Panel B), respectively, in broiler chickens with or without organic selenium (Sel-Plex\) supplementation. a,bHistogram columns with unlike superscripts differ significantly ( P  0.05).

previously described by Mahmoud and Edens (2003). Dividing the optical density results of each sample by its initial total protein (TP) concentrations facilitated the standardization of the hsp70 expression.

Table 1 Influence of selenium yeast (Sel-Plex\) on intestinal total glutathione (TGSH), oxidized glutathione (GSSG), reduced glutathione (GSH), and reduced : oxidized glutathione ratios (GSH / GSSG) in broiler chickens given a dietary supplement of organic selenium and challenged with an enteropathogenic E . coli (EPEC) Variable Selenium EPEC TGSH (AM/g) Control Sel-Plex Pooled SEM Control Sel-Plex Pooled SEM Control Sel-Plex Pooled SEM Control Sel-Plex Pooled SEM 18.95a,y 17.04a,y 1.65 1.60b,y 2.24a,y 0.14 13.35a,y 13.32a,y 1.36 11.35a,x 6.34b,y 1.12 + 21.90b,x 22.50b,x 1.65 3.05a,x 3.31a,x 0.14 19.10a,x 18.91a,x 1.36 6.30b,y 5.78b,y 1.12 P -value Selenium NS EPEC * Interaction NS

GSSG (AM/g)

**

NS

GSH (AM/g)

NS

**

NS

GSH / GSSG

0.08

a,b Means in the same column within a variable with unlike superscripts differ significantly ( P  0.05); x,ymeans in the same row within a variable with unlike superscripts differ significantly ( P  0.05). NSnot significant. * P  0.05. ** P  0.001.

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Table 2 Influence of selenium yeast (Sel-Plex\) on intestinal total glutathione (TGSH), oxidized glutathione (GSSG), reduced glutathione (GSH) and reduced : oxidized glutathione ratios (GSH / GSSG) in broiler chickens given a dietary supplement of organic selenium and subjected to acute heat distress Variable Selenium Temperature Pre-stress TGSH (AM/L) Control Sel-Plex Pooled SEM Control Sel-Plex Pooled SEM Control Sel-Plex Pooled SEM Control Sel-Plex Pooled SEM 1715 1776a,x 45 49a,y 60a,y 6 1617a,x 1646a,x 47 33a,x 27a,x 3
a,x

P -value Post-stress 1817 1821a,x 45 69b,x 91a,x 6 1619a,x 1639a,x 47 23a,y 18a,y 3
a,x

Selenium NS

Temperature NS

Interaction NS

GSSG (AM/L)

***

0.09

GSH (AM/L)

NS

NS

NS

GSH / GSSG

NS

**

NS

a,b Means in the same column within a variable with unlike superscripts differ significantly ( P  0.05); x,ymeans in the same row within a variable with unlike superscripts differ significantly ( P  0.05). NSnot significant. * P  0.05. ** P  0.01. *** P  0.001.

the two groups that were not supplemented with Sel-Plex\ compared to the supplemented group ( P = 0.056). With heat distress, Sel-Plex\ supplementation was associated with significantly lower constitutive (4.50 vs. 5.15 ng/mg total protein) and HS inducible (4.60 vs. 5.54 ng/mg total protein) liver hsp70 (Fig. 2B). EPEC challenge increased ileum TGSH content, but SelPlex\ treatment did not have a significant influence (Table 1). In broilers without EPEC challenge, the Sel-Plex\ treatment elevated significantly the GSSG level in the ileum, but both GSSG and GSH in the ileum were elevated significantly by EPEC challenge, which was not modified by Sel-Plex\ treatment. The Sel-Plex\ EPEC interactions for ileum GSSG and GSH were not significant ( P > 0.05) (Table 1). The ileum GSH / GSSG ratios were elevated significantly in birds given diets without supplemental selenium in comparison to the ratios for birds in the SelPlex\ groups. Neither heat distress nor Sel-Plex\ had any significant influence on either blood TGSH or GSH concentrations (Table 2) despite the numerical increases in both variables. However, Sel-Plex\ supplementation in non-heat distressed as well as heat distressed broilers elevated significantly ( P  0.05 and P  0.001, respectively) blood GSSG, which resulted in lower ( P  0.01) GSH / GSSG ratios in SelPlex\-treated broilers.

4. Discussion The National Research Council established the minimum level of supplemental selenium to sustain growth and performance in broiler chickens to be 0.1 ppm (NRC, 1994). The background selenium levels in the feeds used in these studies reported herein were 0.24 to 0.28 ppm and

ostensibly were adequate. However, those background levels were not sufficient to allow broilers to attain the same BW performance as those given Sel-Plex\, a newly approved source of organic selenium in yeast (Federal Register, 2000, 2002). The FCR and mortality rates were also significantly elevated when birds were given diets with less than adequate selenium, and those birds ultimately suffered from the development of a stress reaction characterized by elevated tissue concentrations of hsp70. Based upon the hsp70 response, one could conclude that consumption of inadequate levels of dietary selenium conferred the ability to resist some stressors, but that may be an inappropriate conclusion. The expression of hsp70 is a classical sign of stress in animals because it is the physical manifestation of specific genes that are induced to combat stressors (Edens et al., 1992; Wang and Edens, 1994; Polla, 1998; Wang and Edens, 1998; Mahmoud, 2000; Mahmoud and Edens, 2003). There is a physiological and metabolic cost for this response, and it is paid in reduced growth due to a lower rate of synthesis of structural proteins in chronically stressed animals. In the studies reported herein, broilers given diets without supplemental selenium had higher constitutive levels of hsp70 than those given organic selenium as SelPlex\. Additionally, when those animals were subjected either to an EPEC challenge or to an acute heat distress, the inducible hsp70 concentrations were significantly greater in those birds given diets without supplemental selenium. These results force the conclusion that inadequate dietary selenium is a stressor in its own right and increases the level of stress proteins leading to reduced growth. Sies (1993) reported that the physiological strategies for antioxidant defense are organized in three categories: prevention, interception, and repair. In this context, selenium and the glutathione GPx system work mostly at the level of

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interception. The main point of intercepting a damaging species, once formed, is to exclude it from further activity and lead to transfer of the pro-oxidant away from more sensitive compartments of the cell. The glutathione redox cycle must be considered very important in the process because hsp70 expression is responsive to increased thiol oxidation (GSSG production) and loss of cellular GSH as indicated by a lower GSH / GSSG ratio (Sierra-Rivera et al., 1988), which was illustrated in the current report and previously reported by Mahmoud and Edens (2003). Normally, stress will induce an increase in blood and tissue concentrations of GSH (Cantor et al., 1982; Mitchell et al., 1983; Mahmoud and Edens, 2003), which were observations consistent with those reported herein. The increased oxidation of GSH can then allow increased ROS formation leading to elevated hsp70 expression. Both EPEC challenge and heat distress have the potential to increase ROS formation in association with elevation of hsp70 in birds without supplemental selenium. However, with Sel-Plex\, a lower level of hsp70 expression indicated improved tolerance to the stress of EPEC infection. Katoh et al. (1991) reported that the hepatic content of GSH decreased dramatically after ethylene oxide treatment and that both hsp32 mRNA increased (approximately 40-fold) and hsp90 mRNA increased (approximately 3-fold) after high dose exposure to ethylene oxide. However, hsp70 mRNA levels did not change under these conditions (Katoh et al., 1991). Recently, Mahmoud and Edens (2003) reported that heat stress depletion of hepatic GSH was the greatest among broiler chicks without supplemental selenium followed by those supplemented with sodium selenite, but broilers given organic selenium showed the least depletion during heat stress. Hepatic hsp70 levels in heat distressed broilers in this study were less than the levels found in broilers without supplemental selenium and coincidentally those broilers given supplemental organic selenium had the least GSH mobilization. Although not measured in these studies, GPx activity has been reported to be elevated significantly with the addition of organic selenium to poultry diets (Cantor et al., 1982; Mahmoud and Edens, 2003). Reduction of organic peroxides by glutathione is catalyzed by GPx and leads to increased oxidation of GSH to GSSG (Burk et al., 1978). Normally, within a few days after chickens begin to consume Sel-Plex\, maximal GPx activity can be achieved. Mahmoud and Edens (2003) reported that higher GPx activity in Sel-Plex\-fed broilers will catalyze a more rapid oxidation of GSH to GSSH consistent with observations in these studies. The GSSG is reduced normally back to GSH by GSH reductase activity, which can be limited by the glutathione reductase activity if GSSG accumulates rapidly. In fact, there was an accumulation of GSSG in the SelPlex\-fed broilers indicating a more efficient clearing of ROS in those birds. If GSSG accumulation increases hsp70 expression (Sierra-Rivera et al., 1988), then tissues from stressed chickens should have shown increased hsp70

concentrations, but that was not the situation in Sel-Plex\fed broilers. Induction of hsp70 is an acute phase response that cannot be tolerated for long periods of time without resulting in the slowing of growth and performance in animals. Previously, Wang and Edens (1998) have shown that chronic exposure to heat distress decreases hsp70 expression as the birds become acclimated. By the end of the 6-week growing period for those Sel-Plex\-fed broilers, their resistance to stressors was improved as indicated by superior performance traits, lower hsp70 production, and lower GSH / GSSG ratios.

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