Metabolic characteristics and oxidative damage to skeletal muscle in broiler chickens exposed to chronic heat stress

Emerging evidence has shown that acute heat exposure affects metabolic characteristics and causes oxidative damage to skeletal muscle in birds. Little is known, however, about such phenomena under chronic heat stress conditions. To address this, we designed the present study to determine the influence of cyclic (32 to 24 to 32 °C: 32 °C for 8 h/d, 32–24–32HS ), and constant (32 and 34 °C, 32HS and 34HS, respectively) heat exposure on the metabolic and peroxide status in skeletal muscle of 4-wk-old male broiler chickens. Heat stress, particularly in the 32HS and 34HS groups, depressed feed intake and growth, while cyclic high temperature gave rise to a less severe stress response in performance terms. Malondialdehyde (MDA) levels in skeletal muscle were enhanced (P < 0.05) by constant heat treatment; the degree of enhancement was not as large as the changes observed in our previous ‘acute’ heat stress model. The 3HADH (3-hydroxyacyl CoA dehydrogenase related to fatty acid oxidation) and CS (citrate synthase) enzyme activities were lowered (P < 0.05) by both the cyclic and constant 34HS treatments, and constant 34HS group, respectively. These results suggest that chronic heat exposure decreases metabolic oxidation capacity in skeletal muscle of broiler chickens. On exposure to chronic heat stress, GPx activity remained relatively constant, though a temperature-dependent elevation in Cu/Zn-SOD activity was observed, implying that anti-oxidation ability was disturbed by the chronic stress condition. From these results it can be concluded that chronic heat stress did not induce oxidative damage to a major extent. This may probably be due to a decrease in metabolic oxidation capacity or due to a self-propagating scavenging system, though the system was not fully activated.     

Accelerated tissue aging and increased oxidative stress in broiler chickens fed allopurinol

Uric acid has been hypothesized as being one of the more important antioxidants in limiting the accumulation of glycosylated endproducts in birds. Study 1 was designed to quantitatively manipulate the plasma concentrations of uric acid using hemin and allopurinol while study 2 determined their effects on skin pentosidine, the shear force value of Pectoralismajor muscle, plasma glucose, body weight and chemiluminescence monitored oxidative stress in broiler chickens. Hemin was hypothesized to raise uric acid concentrations thereby lowering oxidative stress whereas allopurinol was hypothesized to lower uric acid concentrations and raise measures of oxidative stress. In study 1 feeding allopurinol (10 mg/kg body weight) to 8-week-old broiler chicks (n=50) for 10 days decreased plasma uric acid by 57%. However, hemin (10 mg/kg body weight) increased uric acid concentrations 20%. In study 2, 12-week-old broiler chicks (n=90) were randomly assigned to either an ad libitum (AL) diet or a diet restricted (DR) group. Each group was further divided into three treatments (control, allopurinol or hemin fed). Unexpectedly, hemin did not significantly effect uric acid concentrations but increased (P<0.05) measures of chemiluminescence dependent oxidative stress in both the DR and AL birds probably due to the ability of iron to generate oxygen radicals. Allopurinol lowered concentrations of uric acid and increased (P<0.05) the oxidative stress in the AL birds at week 22, reduced (P<0.05) body weight in both the AL and DR fed birds at 16 and 22 weeks of age, and markedly increased (P<0.001) shear force values of the pectoralismajor muscle. Skin pentosidine levels increased (P<0.05) in AL birds fed allopurinol or hemin fed birds, but not in the diet restricted birds at 22 weeks. The significance of these studies is that concentrations of plasma uric acid can be related to measures of oxidative stress, which can be linked to tissue aging.     

Effect of indomethacin on hyperthermia induced by heat stress in broiler chickens

 An investigation was carried out to verify whether the heat stress hyperthermia response of broilers is prostaglandin-dependent. Male broiler chickens of the Hubbard-Petterson strain, aged 35–49 days, were used. Chickens were injected with indomethacin (1 mg/kg intraperitoneally ) 15 min before or 2 h after heat exposure (at 35°C for 4 h), and rectal temperature was measured before injection and up to 4 h thereafter. Birds were separated into two groups with and without access to water during heat stress. The increase in rectal temperature was lower (P<0.05) in birds with access to drinking water during heat exposure. All birds injected with indomethacin exhibited an increase in rectal temperature, irrespective of whether indomethacin was administered before or in the course of the rise in temperature. The results revealed that the increase in rectal temperature during heat exposure is not prostaglandin-dependent, and that the use of cyclooxigenase inhibitors is not recommended to attenuate heat stress hyperthermia in broiler chickens. Received: 15 December 1997 / Revised: 29 June 1998 / Accepted: 31 July 1998     

Supplemental zinc and vitamin A can alleviate negative effects of heat stress in broiler chickens

This experiment was conducted to evaluate the effects of zinc (ZnSO₄H₂O) and vitamin A (retinol) supplementation on performance, carcass characteristics, and serum concentrations of glucose, cholesterol, total protein, and malondialdehyde (MDA) as an indicator of lipid peroxidation in broiler chickens (Ross) reared at a high temperature (34°C). One hundred twenty 10-d-old male broilers were randomly assigned to 4 treatment groups, 3 replicates of 10 birds each. The birds were fed either a basal diet or the basal diet supplemented with either 30 mg Zn/kg diet, 4.5 mg (15,000 IU) retinol/kg diet, or 30 mg Zn+4.5 mg retinol/kg diet. Supplemental zinc and vitamin A significantly increased live weight gain and improved feed efficiency (p<0.05). However, a combination of zinc and vitamin A, rather than each separately, provided a greater performance. Hot and chilled carcass weights and yields and the weights of internal organs with the exception of abdominal fat were greater for each supplement (p<0.05) compared to the control group. Abdominal fat decreased (p<0.05) upon dietary zinc and vitamin A supplementation. Supplemental treatments resulted in an increased total serum protein but decreased glucose, cholesterol, and MDA concentrations. The results of the study show that, separtely or as a combination, zinc and vitamin A supplementation resulted in an improved live weight gain, feed efficiency, and carcass traits, as well as a decrease in serum MDA concentrations. The results of the present study also suggest that zinc and vitamin A have similar effects and that a combination of zinc and vitamin A may offer a potential protective management practice in preventing heat-stress-related depression in performance of broiler chickens.     

Exploring the molecular mechanism of acute heat stress exposure in broiler chickens using gene expression profiling

The process of heat regulation is complex and its exact molecular mechanism is not fully understood. In this study, to investigate the global gene regulation response to acute heat exposure, gene microarrays were exploited to analyze the effects of heat stress on three tissues (brain, liver, leg muscle) of the yellow broiler chicken (Gallus gallus). We detected 166 differentially expressed genes (DEGs) in the brain, 219 in the leg muscle and 317 in the liver. Six of these genes were differentially expressed in all three tissues and were validated by qRT-PCR, and included heat shock protein genes (HSPH1, HSP25), apoptosis-related genes (RB1CC1, BAG3), a cell proliferation and differentiation-related gene (ID1) and the hunger and energy metabolism related gene (PDK). All these genes might be important factors in chickens suffering from heat stress. We constructed gene co-expression networks using the DEGs of the brain, leg muscle and liver and two, four and two gene co-expression modules were identified in these tissues, respectively. Functional enrichment of these gene modules revealed that various functional clusters were related to the effects of heat stress, including those for cytoskeleton, extracellular space, ion binding and energy metabolism. We concluded that these genes and functional clusters might be important factors in chickens under acute heat stress. Further in-depth research on the newly discovered heat-related genes and functional clusters is required to fully understand their molecular functions in thermoregulation.     

Breast muscle and plasma metabolomics profile of broiler chickens exposed to chronic heat stress conditions

Understanding the variations of muscle and plasma metabolites in response to high environmental temperature can provide important information on the molecular mechanisms related to body energy homeostasis in heat-stressed broiler chickens. In this study, we investigated the effect of chronic heat stress conditions on the breast muscle (Pectoralis major) and plasma metabolomics profile of broiler chickens by means of an innovative, high-throughput analytical approach such as the proton nuclear magnetic resonance (¹H NMR) spectrometry. A total of 300 Ross 308 male chicks were split into two experimental groups and raised in either thermoneutral conditions for the entire rearing cycle (0–41 days) (TNT group; six replicates of 25 birds/each) or exposed to chronic heat stress conditions (30 °C for 24 h/day) from 35 to 41 days (CHS group; six replicates of 25 birds/each). At processing (41 days), plasma and breast muscle samples were obtained from 12 birds/experimental group and then subjected to ¹H NMR analysis. The reduction of BW and feed intake as well as the increase in rectal temperature and heterophil: lymphocyte ratio confirmed that our experimental model was able to stimulate a thermal stress response without significantly affecting mortality. The ¹H NMR analysis revealed that a total of 26 and 19 molecules, mostly related to energy and protein metabolism as well as antioxidant response, showed significantly different concentrations respectively in the breast muscle and plasma in response to the thermal challenge. In conclusion, the results obtained in this study indicated that chronic heat stress significantly modulates the breast muscle and plasma metabolome in fast-growing broiler chickens, allowing to delineate potential metabolic changes that can have important implications in terms of body energy homeostasis, growth performance and product quality.     

Effects of allopurinol on uric acid concentrations,xanthine oxidoreductase activity and oxidative stress in broiler chickens

The purpose of this study was to determine the effects of allopurinol (AL) on xanthine oxidoreductase (XOR) activity and uric acid (UA) levels in chickens. Thirty 5-week-old broilers were divided into three groups and fed 0 (control), 25 (AL25) or 50 (AL50) mg AL per kg of body mass for 5 weeks. Chicks were weighed twice weekly and leukocyte oxidative activity (LOA) and plasma purine levels were determined weekly in five birds per group. Chicks were sacrificed after 2 or 5 weeks, and samples from tissues were taken for analysis of XOR activity. Plasma UA concentrations were lower (P < 0.001) and xanthine and hypoxanthine concentrations were greater (P < 0.001) in AL25 and AL50 birds compared to controls, whereas no differences (P = 0.904) were detected in allantoin concentrations. By week 5, body mass was reduced (P < 0.001) to 84.0 and 65.1% of that in controls for AL25 and AL50 broilers, respectively, and LOA was 4.1 times greater (P < 0.05) in AL25 compared to control birds. Liver XOR activity was increased by 1.1 and 1.2 times in AL25 and AL50 birds, but there was no change (P > 0.05) in XOR activity in the pancreas and intestine. These results suggest that AL effect on XOR activity is tissue dependent.     

Effect of chronic heat stress on gastrointestinal histology and expression of feed intake-regulatory hormones in broiler chickens

Heat stress (HS) dramatically impairs the growth performance of broiler chickens, mainly as a consequence of reduced feed intake due to the loss of appetite. This study was aimed at evaluating the alterations induced by chronic HS conditions on the morphological and morphometric features of the gastrointestinal (GI) tract and on the expression of some enteroendocrine cells (EECs) involved in the regulation of feed intake in chickens. Three hundred male chickens (Ross 308) were divided into two experimental groups and raised either in thermoneutral environment for the whole fattening period (0–41 days) (TNT group) or subjected to chronic HS conditions (30 °C for 24 h/day) from 35 to 41 days (HS group). Samples of proventriculus, duodenum, jejunum and cecum were collected from 24 broilers (12/group). Haematoxylin-eosin was used for the morphometric evaluations, while immunohistochemistry was applied for the evaluation of EECs expressing ghrelin (GHR), cholecystokinin (CCK), neuropeptide Y (NPY), glucagon-like peptide-1 (GLP-1), and serotonin (5-HT). In the proventriculus, HS reduced total wall thickness and mucous layer height (P ≤ 0.01) as well as mean diameter, circumference, and area of the compound tubular glands (P ≤ 0.001) with respect to TNT. The small intestine of HS birds was characterised by decreased villous height and total thickness (duodenum, P ≤ 0.01; jejunum, P ≤ 0.001), whereas crypt depth and width were reduced only in the jejunum (P ≤ 0.01). HS had negligible effects on the morphological aspects of the cecum. In the proventriculus, an increase in GHR and NPY EECs was observed in response to HS (P ≤ 0.001). Similarly, the small intestine villi of the HS group showed greater GLP-1 (P ≤ 0.05), 5-HT (P ≤ 0.001) and CCK (P ≤ 0.01) EECs. Moreover, the expression of 5-HT EECs was higher in the duodenal (P ≤ 0.01) and jejunal (P ≤ 0.01) crypts of HS birds, whereas GLP-1 and CCK EECs increased only in jejunal crypts (P ≤ 0.05). Finally, 5-HT EEC expression was increased in the cecum of HS group (P ≤ 0.01). In conclusion, these outcomes demonstrate that chronic HS induces morphometric alterations not only in the small intestine but also in a key organ such as the proventriculus. Furthermore, HS conditions affect the presence and distribution of EECs, suggesting that some GI peptides and biogenic amine may be implicated in the regulation of appetite and voluntary feed intake in heat-stressed broiler chickens.     

Tryptophan loading induces oxidative stress

In previous studies tryptophan loads have been administered to human subjects in order to raise central levels of 5-hydroxytryptamine (5HT) and assess the effects of 5HT on behaviour and mood. However, tryptophan is metabolised primarily along the oxidative kynurenine pathway. In this study a 6 g oral tryptophan load was administered to 15 healthy volunteers and the levels of kynurenines and lipid peroxidation products (indicative of oxidative stress) were measured. The results demonstrate that tryptophan loading produces a highly significant increase in lipid peroxidation products in parallel with increased kynurenines. The oxidative stress may result from the generation of quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are known to have the ability to generate free radicals. The results may have implications for the use of tryptophan loading in psychiatric practice, and for the chronic use of diets high in tryptophan.     

Effect of heat stress and drinking water salt supplements on plasma electrolytes and aldosterone concentration in broiler chickens

An experiment was conducted to evaluate the effects of supplementing drinking water with isomolar (0.067 mol/l) KCl or NaCl on mass gain, food and water consumption, rectal temperature, and plasma concentrations of aldosterone, Na⁺, and K⁺ in broiler chickens reared in thermoneutral and cycling heat stressing environments. Heat stress decreased (P0.05) mass gain, food consumption, and plasma concentrations of Na⁺ and K⁺, while increases (P0.05) in plasma concentrations of aldosterone, rectal temperature, and water consumption were observed. Drinking water supplemented with either KCl or NaCl increased (P0.05) broiler mass gain and water consumption, but had no effect (P>0.1) on the other variables evaluated. The results of this study indicate that broiler chickens in a heat stress environment are under osmotic stress and supplementing drinking water with 0.067 mol/1 KCl or NaCl does not lessen this stress.     

Drought induces oxidative stress in pea plants

Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (leaf water potential of -1.3 MPa) showed major reductions in photosynthesis (78), transpiration (83), and glycolate oxidase (EC 1.1.3.1) activity (44), and minor reductions (18) in the contents of chlorophyll a, carotenoids, and soluble protein. Water stress also led to pronounced decreases (72–85) in the activities of catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), but resulted in the increase (32–42) of non-specific peroxidase (EC 1.11.1.7) and superoxide dismutase (EC 1.15.1.1). Ascorbate peroxidase (EC 1.11.1.11) and monodehydroascorbate reductase (EC 1.6.5.4) activities decreased only by 15 and the two enzymes acted in a cyclic manner to remove H₂O₂, which did not accumulate in stressed leaves. Drought had no effect on the levels of ascorbate and oxidized glutathione in leaves, but caused a 25 decrease in the content of reduced glutathione and a 67 increase in that of vitamin E. In leaves, average concentrations of catalytic Fe, i.e. Fe capable of catalyzing free-radical generation by redox cycling, were estimated as 0.7 to 7 M (well-watered plants, depending on age) and 16 M (water-stressed plants); those of catalytic Cu were 4.5 M and 18 M, respectively. Oxidation of lipids and proteins from leaves was enhanced two- to threefold under stress conditions and both processes were highly correlated. Fenton systems composed of the purported concentrations of ascorbate, H₂O₂, and catalytic metal ions in leaves produced hydroxyl radicals, peroxidized membrane lipids, and oxidized leaf proteins. It is proposed that augmented levels and decompartmentation of catalytic metals occurring during water stress are responsible for the oxidative damage observed in vivo.Abbreviations and Symbol ASC ascorbate - DW dry weight - DHA dehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - MDHA monodehydroascorbate (ascorbate free radical) - SOD Superoxide dismutase - _wa water potential We thank Dr. R. Picorel (E.E. de Aula Dei, CSIC) for allowing us access to HPLC equipment. J.F.M., 1.1., and S.F. were the recipients of predoctoral fellowships from the Comunidades Autónomas de Aragon, Pais Vasco, and Navarra, respectively. R.V.K. thanks the U.S. Department of Agriculture (grant 91-37305-6705) for travel support. This work was financed by grants from the Comisión Interministerial de Ciencia y Tecnología (AGR-91-0857-C02 to P.A. and M.B.) and the Dirección General de Investigación Científica y Técnica (PB92-0058 to M.B) of Spain.     

Acrolein induces oxidative stress in brain mitochondria

Acrolein, a byproduct of lipid peroxidation, has been shown to inflict significant structural and functional damage to isolated guinea pig spinal cord. Reactive oxygen species (ROS) are thought to mediate such detrimental effects. The current study demonstrates that acrolein can directly stimulate mitochondrial oxidative stress. Specifically, exposure of purified brain mitochondria to acrolein resulted in a dose-dependent increase of ROS and decreases in glutathione content and aconitase activity. This effect was not accompanied by significant intramitochondrial calcium influx or mitochondrial permeability transition, but rather by impaired function of the mitochondrial electron transport system. As well, we detected a significant inhibition of mitochondrial adenine nucleotide translocase (ANT) in the presence of acrolein. This inhibition of ANT likely contributes to acrolein-induced ROS elevation since application of atractyloside, a specific ANT inhibitor, induced significant increase of ROS. We hypothesize that inhibition of ANT may mediate, in part, the acrolein-induced ROS increase in mitochondria.     

Circadian rhythms of biomarkers of oxidative stress and their characteristics in broiler chickens reared under natural light/dark cycle

For the purpose of studying circadian rhythms of biomarkers of oxidative stress in broiler chickens maintained under natural photoperiod, we collected blood from the wing veins of 10 matured broiler chickens every three hours for a period of 24 h. The blood samples were analyzed for superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA). The fitting in of single cosinor procedure showed that CAT and MDA exhibited clear circadian rhythms, while SOD did not. The amplitude of daily rhythms for the parameters was small, while the acrophases were all restricted to the light phase of the light:dark cycle. In conclusion, to our knowledge, this is the first time investigation of the circadian rhythms of antioxidants in broilers chickens, reared under natural photoperiod in the tropics. The result may be exploited for a more precise targeting of reactive oxygen species, thereby offering better protection for the cells to combat oxidative stress.     

Probiotic bacteria maintain normal growth mechanisms of heat stressed broiler chickens

Probiotics have growth promoting effects even under periods of heat stress challenge. More information is needed to understand the mechanisms by which probiotics maintain the growth performance. The aim of this study was to evaluate the effect of a probiotic based on Bacillus subtilis bacteria on growth related mechanisms of broilers under heat stress conditions. Specifically, growth performance, skeletal bone characteristics, skeletal muscles size, intestinal villus-crypt structure, intestinal bacteria, growth hormone (GH), insulin-like growth factor-1 (IGF-1), cholesterol, and glucose. A total of 1200 one day old Ross 308 male broilers were randomly distributed into 4 treatments, with 12 replicates per treatment and 25 birds per replicate. A 2 × 2 factorial arrangement was used; the main factors were environmental temperature (thermoneutral or heat stress) and diet (control or control + B. subtilis; 3 × 10⁷ cfu/kg of feed). From d 22 to 35 of age, birds were either exposed to thermoneutral conditions (21 °C) or chronic heat stress (30 °C). During the same period, each group was divided into 2 subgroups and fed either the control diet or the B. subtilis supplemented diet. The results demonstrated that B. subtilis had positive effects (P < 0.05) on the body weight gain, feed conversion ratio, villus height, crypt depth, villus surface area, absorptive epithelial cell area and viable counts of intestinal beneficial bacteria. B. subtilis increased (P < 0.05) serum GH, IGF-1 and maintain normal levels of cholesterol and glucose under heat stress conditions. In addition, broilers fed B. subtilis under heat stress conditions exhibited higher (P < 0.05) skeletal muscles size and improved (P < 0.05) tibia traits and lower (P < 0.05) abdominal fat pads deposition compared with the controls. B. subtilis had no effect on rectal temperature under thermoneutral or heat stress conditions. It is concluded that B. subtilis can be used as growth promoters in broilers, particularly during the periods of heat stress conditions.     

Aminonaphthoquinone induces oxidative stress in Staphylococcus aureus.

The biological activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) on Staphylococcus aureus was investigated in comparison with the unsubstituted 1,4-naphthoquinone (NQ). Complete inhibition of microbial growth was observed with ANQ and NQ at 50 and 10 microg/mL, respectively. The antibacterial effect of naphthoquinones decreased in the presence of sodium ascorbate, but the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) was able to protect S. aureus only from the harmful effect of ANQ. Naphthoquinones blocked oxygen uptake and induced cyanide-insensitive oxygen consumption. When combining rotenone or salicylhydroxamic acid with ANQ or NQ, a slight decrease in respiratory activity was observed. Assays in the presence of naphthoquinones induced an increase of lipid peroxidation in S. aureus, as determined by thiobarbituric acid reactive substances. These results showed that 1,4-naphthoquinones effectively act as electron acceptors and induce an increase in reactive oxygen species that are toxic to S. aureus cells.     

Hypobaric hypoxia induces oxidative stress in rat brain

High altitude exposure results in decreased partial pressure of oxygen and an increased formation of reactive oxygen and nitrogen species (RONS), which causes oxidative damage to lipids, proteins and DNA. Exposure to high altitude appears to decrease the activity and effectiveness of antioxidant enzyme system. The antioxidant system is very less in brain tissue and is very much susceptible to hypoxic stress. The aim of the present study was to investigate the time dependent and region specific changes in cortex, hippocampus and striatum on oxidative stress markers on chronic exposure to hypobaric hypoxia. The rats were exposed to simulated high altitude equivalent to 6100 m in animal decompression chamber for 3 and 7 days. Results indicate an increase in oxidative stress as seen by increase in free radical production, nitric oxide level, lipid peroxidation and lactate dehydrogenase levels. The magnitude of increase in oxidative stress was more in 7 days exposure group as compared to 3 days exposure group. The antioxidant defence system such as reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and reduced/oxidized glutathione (GSH/GSSG) levels were significantly decreased in all the three regions. The observation suggests that the hippocampus is more susceptible to hypoxia than the cortex and striatum. It may be concluded that hypoxia differentially affects the antioxidant status in the cortex, hippocampus and striatum.     

Aortic coarctation induces oxidative stress in rat tissues

The purpose of this study was to evaluate the induction of oxidative stress in heart and erythrocytes from rats with abdominal aorta coarctation (Coa) compared with sham-operated normotensive controls (Sham). The group of Coa animals developed myocardial hypertrophy, showing heart homogenates markedly increased levels of reduced glutathione (48%), lipid peroxidation (148%) and activation of superoxide dismutase and glutathione peroxidase (189% and 37%, respectively), compared with controls. Other oxidative stress indicators were also altered in erythrocytes from Coa rats: increased protein carbonyl content (141%) and total glutathione level (349%) were determined. Inactivation of the antioxidant enzymes catalase (27%), superoxide dismutase (58%) and glutathione peroxidase (25%) was observed in erythrocytes from the Coa group. Taken jointly our results provide strong evidence for the production of oxidative stress in heart and erythrocytes from aortic coarcted rats.