Forsythia suspensa extract protects broilers against breast muscle oxidative injury induced by corticosterone mimicked pre-slaughter acute stress

Forsythia suspensa extract protects broilers against breast muscle oxidative injury induced by... ABSTRACT Broilers were used to determine the protective effects of Forsythia suspensa extract (FSE) against breast muscle oxidative injury induced by corticosterone (CS) mimicking pre-slaughter acute stress. A total of 144 male Arbor Acre broilers was randomly allotted to one of 4 treatments in a 2 × 2 factorial arrangement that included FSE supplementation (0 or 100 mg/kg) and subcutaneous injection of CS (0 or 4 mg/kg) at 3 h before slaughter. Corticosterone increased live BW loss, and the adverse effect was attenuated by FSE in broilers subjected to CS (P < 0.05). Serum levels of CS, uric acid, and glucose were increased, and postmortem breast muscle pH values at 45 min and 24 h were decreased for CS-challenged broilers (P < 0.05). Corticosterone increased lightness and yellowness values and decreased redness of breast muscle (P < 0.05), and FSE decreased yellowness and increased redness of breast muscle (P < 0.05). Drip loss was increased by CS for birds supplemented without FSE (P < 0.05) and decreased by FSE for birds under CS challenge (P < 0.05). Corticosterone increased monounsaturated fatty acid (FA) and decreased polyunsaturated FA in breast muscle (P < 0.05), and saturated FA was decreased and polyunsaturated FA was increased by FSE (P < 0.05). Malondialdehyde and carbonyl contents in breast muscle were increased by CS and decreased by FSE (P < 0.05). Inhibition of 1,1-diphenyl-2-picryl-hydrazyl was decreased by CS and increased by FSE (P < 0.05). The activities of total-antioxidant capacity, glutathione peroxidase, and superoxide dismutase in breast muscle were lower in birds subjected to CS (P < 0.05) and were greater in birds supplemented with FSE (P < 0.05). Collectively, live BW loss and breast muscle oxidative injury were increased by CS in broilers, and these stress-related adverse effects could be attenuated by FSE supplementation via enhanced scavenging ability of free radicals and antioxidant capacity. Therefore, FSE could protect broilers against breast muscle oxidative injury when acute stress happens. INTRODUCTION Broilers are generally challenged by a variety of stress before slaughter induced by several stressors such as feed withdrawal, environmental temperature, catching, crating, transport, stunning, and struggling, all of which can cause physical, psychological, and physiological stresses for birds (Schwartzkopf-Genswein et al., 2012). Most stress would result in the increase of reactive oxygen species (ROS); oxidative stress occurs once the dramatically accumulated ROS level surpasses the capacity of antioxidant defensive systems (Miller et al., 1993; Nordberg and Arnér, 2001). Oxidation by free radicals is the primary mechanism for quality deterioration in foods, especially in meat products (Archile-Contreras and Purslow, 2011). Pre-slaughter oxidative stress can cause undesirable changes in flavor, color, texture, and nutritive value, and may even induce the production of toxic compounds in meat via influence on the pre- or post-slaughter muscle metabolism or both (Ferguson and Warner, 2008). Accordingly, the oxidative status of poultry at the time of slaughter is critical for meat quality (Jensen et al., 1998). Recently, increasing attention has been paid to explore effective ways to reduce pre-slaughter stress response and to improve post-slaughter meat quality of broilers (Zhang et al., 2014; Wang et al., 2015). Forsythia suspensa extract (FSE) is a popular traditional Chinese medicine (Piao et al., 2008, 2009) that is used as a natural source of antioxidants (Matkowski et al. 2013). In our previous studies, FSE has been reported to effectively attenuate stress in broilers induced by high temperature (Wang et al. 2008), high stocking density (Zhang et al. 2013), or corticosterone (CS) (Zeng et al. 2014), in rats induced by diquat (Lu et al. 2010) or lipopolysaccharide (Zhao et al., 2017a), and in pigs induced by soybean β-conglycinin (Hao et al., 2010) or weaning (Han et al., 2012; Zhao et al., 2017b). Therefore, FSE may have the potential to improve oxidative status of poultry at the time of slaughter. Pre-slaughter stressors can activate the hypothalamic–pituitary–adrenal (HPA) axis and cause release of glucocorticords such as CS in poultry (Zeng et al., 2014). Glucocorticoids, as the final effectors of the HPA axis, participate in the control of whole body homeostasis and the response of organisms to stressors by stimulating the release of energy stores, including glucose mobilization and lipolysis (Harvey et al., 1986). Stimulation of the adrenal cortex by exogenous administration of adrenocorticotropin and steroid moieties, including CS and dexamethasone, has been employed as a model to mimic physiological stress in poultry (Puvadolpirod and Thaxton, 2000a; Virden et al., 2007). As reported, physiological stress responses that occur in broilers following CS challenge include poor performance and meat quality (Lin et al., 2007), increased circulating levels of CS, glucose, and uric acid (Puvadolpirod and Thaxton, 2000b; Yuan et al., 2008), depression of the immune system (Minozzi et al., 2008), and impairment of redox balance (Lin et al., 2004a). When subjected to acute stress or chronic stress, broilers show different performance and biochemical responses (Lin et al., 2004b). However, few studies using the mimic stress models were focused on the oxidative responses of muscle, especially the vulnerable breast muscle in broilers. Our lab has reported the protective effects of FSE against physiological stress induced by dietary CS supplementation (from d 14 to 21, chronic exposure) to mimic the chronic physiological stress during the intensive rearing period (Zeng et al., 2014). The results indicated FSE can alleviate CS-induced growth inhibition, impairment of nutrient digestibility, and immune depression in broiler chickens. Unfortunately, the previous model failed to mimic the pre-slaughter acute stress and also failed to study the protective effects of FSE against post-slaughter breast muscle oxidative injury. Therefore, we hypothesized that the pre-slaughter acute stress can be mimicked by subcutaneous injection of CS at 3 h before slaughter, and the post-slaughter breast muscle oxidative injury caused by the mimicked pre-slaughter short-time stress in broilers could be attenuated, partly or completely, by dietary FSE supplementation. The purpose of this study was to investigate the underlying potentiality of FSE in protecting broilers against breast muscle oxidative injury induced by subcutaneous injection of CS at 3 h before slaughter. MATERIALS AND METHODS Preparation And Composition Of FSE Forsythia suspensa extract is derived from a climbing plant widely distributed in China. The dried fruits of Forsythia suspensa were purchased from Tong Ren Tang Company (Beijing, China), and FSE was prepared using the methods described by Wang et al. (2008). The 3 major active antioxidant constituents isolated from FSE have been identified as forsythoside A, forythialan A, and phillygenin by Lu et al. (2010) in our laboratory. The 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Experimental Birds The experimental protocols used in the experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China). A total of 144 one-day-old male Arbor Acre broiler chickens (weighing 44.5 ± 1.2 g) was purchased from Arbor Acres Poultry Breeding Company (Beijing, China). All birds were raised in wire-floored cages in an environmentally controlled room with continuous light and had ad libitum access to feed and water. The ambient temperature was maintained at 33°C at the start of experiment and decreased as the birds progressed in age to ensure a final temperature of 24°C at 35 d and thereafter. All birds were inoculated with inactivated infectious bursa disease vaccine on d 14 and 21 and Newcastle disease vaccine on d 7 and 28. The trial was conducted in 2 phases, consisting of a starter phase from d 1 to 21 and a finisher phase from d 22 to 42. Experimental Design And Diets The broiler chickens were randomly allotted to one of 4 treatments in a 2 × 2 factorial arrangement that included FSE supplementation (0 or 100 mg/kg of diet) and subcutaneous injection of CS [4 mg/kg of body weight (BW) in corn oil] or corn oil (sham control) at 3 h before slaughter. Six cages per treatment were used, with 6 birds per cage. In detail, from d 1 to 42, 12 cages of birds received diets containing FSE (100 mg/kg of diet), whereas 12 cages did not. The supplementation level of FSE was based on previous work from our laboratory (Zhang et al., 2013; Zeng et al., 2014). At 42 d of age, the chickens in each treatment were subjected to one of the following treatments: single subcutaneous administration of CS (4 mg/kg of BW in corn oil) or corn oil at 3 h before slaughter. During the 3-hour exposure, feed was withdrawn, and all the broiler chickens had free access to tap water. Birds were weighed at the start and end of the 3-hour CS challenge, and live BW loss calculated. All diets were fed in mash form and were based on corn-soybean meal (Table 1). All essential nutrients provided in diets met or slightly exceeded NRC recommendations (NRC, 1994). Table 1. Composition and nutrient levels of the experimental basal diets (%, as-fed basis, antibiotic-free).1 Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  1Forsythia suspense extract (100 mg/kg) was directly mixed in the basal diet. 2L-Lys HCl (78.8%) and DL-Met (98.5%) were products of CJ CheilJedang Co., Inc., Seoul, Korea. 3The premix provided the following per kilogram of diet: zinc, 60 mg; iron, 100 mg; manganese, 80 mg; copper, 10 mg; iodine, 0.35 mg; selenium, 0.3 mg; vitamin A, 10,000 IU; vitamin D3, 2850 IU; vitamin E, 30 IU; vitamin K3, 2 mg; vitamin B12, 1.2 mg; riboflavin, 6 mg; nicotinic acid, 40 mg; pantothenic acid, 12 mg; pyridoxine, 3 mg; biotin, 0.2 mg; and choline chloride, 800 mg. 4These values were calculated from data provided by NRC (1994). View Large Table 1. Composition and nutrient levels of the experimental basal diets (%, as-fed basis, antibiotic-free).1 Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  1Forsythia suspense extract (100 mg/kg) was directly mixed in the basal diet. 2L-Lys HCl (78.8%) and DL-Met (98.5%) were products of CJ CheilJedang Co., Inc., Seoul, Korea. 3The premix provided the following per kilogram of diet: zinc, 60 mg; iron, 100 mg; manganese, 80 mg; copper, 10 mg; iodine, 0.35 mg; selenium, 0.3 mg; vitamin A, 10,000 IU; vitamin D3, 2850 IU; vitamin E, 30 IU; vitamin K3, 2 mg; vitamin B12, 1.2 mg; riboflavin, 6 mg; nicotinic acid, 40 mg; pantothenic acid, 12 mg; pyridoxine, 3 mg; biotin, 0.2 mg; and choline chloride, 800 mg. 4These values were calculated from data provided by NRC (1994). View Large Sample Collection And Handling At 3 h after CS treatment, one bird close to the average BW was selected per replicate (cage) and slaughtered via exsanguination of the left jugular artery. Blood was collected (5 mL) by cardiac puncture into a 10-mL anticoagulant-free vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria) and then centrifuged at 3,000 × g for 10 min at 4°C to obtain serum. The serum samples were stored at −20°C for further analysis. Immediately after death, the left breast muscle was excised from the warm carcasses, cryogenically frozen in liquid nitrogen (−196°C), and stored in a −80°C freezer until use for lipid and protein peroxidation, DPPH radical scavenging activity, and antioxidative enzyme activity analysis. Before analysis, tissues were minced and homogenized (10% w/v) in ice-cold sodium, potassium phosphate buffer (0.01 M, pH 7.4) containing 0.86% NaCl (Lu et al., 2010). The homogenate was centrifuged at 3,000 × g for 10 min at 4°C, and the resultant supernatant was used for analysis. The right breast muscle was taken to measure postmortem meat quality in a 4°C cell. Chemical Analysis Serum metabolite analysis. Serum concentrations of glucose and uric acid were measured using an Automated Biochemistry Analyzer (Hitachi 902 Automatic Analyzer, Hitachi, Tokyo, Japan) using colorimetric methods and following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China). Corticosterone concentration was assessed with a commercial enzyme immunoassay kit with the intra-assay precision less than 8% and inter-assay precision less than 10% (Cusabio Biotech. Co., Ltd., Wuhan, Hubei, China) according to the manufacturer's instructions. Meat quality and fatty acid profile measurement. Meat color, including lightness (L*), redness (a*), and yellowness (b*) values, was measured from 3 orientations (middle, medial, and lateral) using a Chromameter (CR-410, Konica Minota, Tokyo, Japan). The pH values at 45 min and 24 h postmortem also were measured at 3 locations using a glass penetration pH electrode (pH-star, Matthaus, Germany). The pH decline rate within 24 h postmortem was calculated as a percentage: [(pH45 min − pH24 h)/pH45 min] × 100. Each sample was measured 3 times, and their average value was taken as the final result. Drip loss over 24 h was measured using the plastic bag method. The drip loss was calculated as a percentage: [(initial weight-final weight)/initial weight] × 100. The homogenized skinless breast meat tissue was defrosted, and samples of breast (20 g) lyophilized for 60 h using a freeze dryer were used for testing fatty acid (FA) concentrations. The FA profiles of lipid sources were determined by gas chromatography (6890 series, Agilent Technologies, Wilmington, DE) according to the procedures of Sukhija and Palmquist (1988). Lipid samples were converted to FA methyl esters using methanolic HCl solution. Undecanoic acid (C11:0) was used as the internal standard. Aliquots of 11 were injected into a capillary column (60 m × 250 m × 250 nm, DB-23, Agilent) with cyanopropyl methyl silicone as the stationary phase. Column oven temperature was programmed with a 1:20 split. Injector and detector temperatures were maintained at 260 and 270°C, respectively. Nitrogen was the carrier gas at a flow rate of 2 mL/min. Fatty acids were expressed as the proportion of each individual FA to the total of all FA present in the sample. The following FA combinations—total saturated FA (SFA), total monounsaturated FA (MUFA), polyunsaturated FA (PUFA), MUFA/SFA and PUFA/SFA ratio—were calculated. Lipid and protein oxidation analysis. First, protein concentration was determined using a Pierce BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and was expressed as mg/mL. Lipid oxidation in muscle samples was measured as 2-thiobarbituric acid-reactive substances and was expressed as nmol of malondialdehyde (MDA) per mg of protein. Protein oxidation was reflected by carbonyl concentration, oxidation byproducts of amino acid residues. The concentrations of MDA and carbonyl were determined using kits from Cayman Chemical Company (Ann Arbor, MI). Homogenates of breast muscle were measured colorimetrically at 540 nm for MDA and 372 nm for carbonyl. Values of both were expressed in nmol/mg of protein. DPPH free radical scavenging assay The DPPH radical-scavenging activity of the supplements was estimated by the method of Blois (1958) with slight modifications. Briefly, 1 mL of 0.2 mM DPPH prepared in methanol was added to 200 μL supernatant and 800 μL distilled water. The mixture was vortexed and left to stand at room temperature (20 to 22°C) for 30 minutes. A tube containing 1 mL of methanol and 1 mL of DPPH solution was used as control, whereas methanol alone was used as a blank. The absorbance of the solution was measured at 517 nm using a spectrophotometer (SPECTRAmax 340 PC, Molecular Devices, Sunny Vale, CA). The scavenging activity of meat sample against DPPH radical was expressed as percent of control and calculated as: inhibition rate of DPPH (%) = [1- (absorbance of sample/absorbance of control)] × 100. Anti-oxidative enzyme activity measurement The total-antioxidant capacity (T-AOC) and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in breast muscle were determined with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) per the manufacturer's instructions. Statistical Analysis All data were analyzed as a 2 × 2 factorial design using the General Linear Model procedure of SAS (version 9.2, 2008). The cage was the experimental unit for BW loss, and the selected bird from each cage was the experimental unit for other parameters. The main effects included FSE, CS, and their interaction. Least squares means were derived for all treatments and compared using the PDIFF procedure (Tukey Adjustment) and STDERR options of SAS (2008). Results were expressed as least squares means and SEM. Probability values less than 0.05 were used as the criterion for statistical significance and less than 0.10 as the tendency to significance. RESULTS Bw Loss And Serum Metabolites There was significant interaction (P = 0.04) in live BW loss between FSE and CS (Fig. 1). Corticosterone increased live BW loss of broilers (P < 0.05), and the adverse increase was attenuated by FSE for broilers subjected to CS (P < 0.05). Figure 1. View largeDownload slide Effect of Forsythia suspense extract (FSE) on live BW loss of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 1. View largeDownload slide Effect of Forsythia suspense extract (FSE) on live BW loss of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Serum levels of CS, uric acid, and glucose were increased (P < 0.05) for CS-challenged broilers, whereas CS and glucose concentrations in serum were unaffected by FSE supplementation (Table 2). Significant interactions were noted between FSE and CS for uric acid concentration (P < 0.05), in that FSE decreased the uric acid concentration in serum of broilers under CS challenge. Table 2. Effects of Forsythia suspense extract (FSE) on serum metabolite parameters of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  a,bMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large Table 2. Effects of Forsythia suspense extract (FSE) on serum metabolite parameters of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  a,bMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large Meat Quality And Fatty Acid Profile Of Breast Muscle Postmortem breast muscle pH values at 45 min and 24 h were decreased (P < 0.05), and the pH decline rate was increased (P < 0.05) in birds challenged by CS, whereas these parameters were not influenced by FSE supplementation (Table 3). Corticosterone increased (P < 0.05) lightness and yellowness values and decreased (P < 0.05) redness of breast muscle in broilers. Conversely, FSE supplementation decreased (P < 0.05) yellowness and increased (P < 0.05) redness of breast muscle. Drip loss was influenced by the interaction between CS and FSE (P < 0.05) in that drip loss was increased by CS challenge for birds supplemented without FSE (P < 0.05) and decreased by FSE supplementation for birds under CS challenge (P < 0.05). Table 3. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large Table 3. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large There was no interaction between CS and FSE on FA profiles of breast muscle (Table 4). The percentages of C18:0 and SFA in breast muscle were decreased (P < 0.05) by FSE supplementation while not influenced by CS administration. Concentrations of C18:1n9c and MUFA were greater in breast muscle of CS-challenged birds than those of non-challenged birds (P < 0.05), whereas no differences were observed among birds supplemented with or without FSE. Corticosterone decreased (P < 0.05) the concentrations of C20:3n3, C20:3n6, C20:4n6, C22:6n3, and PUFA in breast muscle, whereas FSE increased (P < 0.05) C22:6n3 and PUFA content and tended to increase (P = 0.06) C20:4n6 content. The birds subjected to CS had a greater ratio of MUFA/SFA (P < 0.05) and lower ratio of PUFA/SFA (P < 0.05) in breast muscles compared with the unchallenged birds. The ratio of PUFA/SFA in breast muscles was increased in birds supplemented with FSE (P < 0.05). Table 4. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids. View Large Table 4. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids. View Large Oxidative And Antioxidative Status Of Breast Muscle Administration with CS increased MDA and carbonyl content in breast muscle (P < 0.05), and FSE supplementation decreased (P < 0.05) MDA content or tended to decrease (P = 0.07) carbonyl content in breast muscles of broilers (Fig. 2). The interaction between FSE and CS tended to be significant for carbonyl content but was not for MDA content in breast muscle. Figure 2. View largeDownload slide Effect of Forsythia suspense extract (FSE) on oxidation injury of breast muscle in broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; MDA = malondialdehyde. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 2. View largeDownload slide Effect of Forsythia suspense extract (FSE) on oxidation injury of breast muscle in broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; MDA = malondialdehyde. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Inhibition of DPPH radicals in breast muscle of birds was decreased (P < 0.05) by CS and increased (P < 0.05) by FSE (Fig. 3). The activities of T-AOC, GSH-Px, and SOD in breast muscle were lower (P < 0.05) in birds subjected to CS than those without CS challenge and were greater (P < 0.05) in birds supplemented with FSE than those without FSE supplementation (Fig. 4). Neither CS nor FSE affected CAT activity in breast muscle. There were no significant interactions between FSE and CS for antioxidant activities in breast muscle. Figure 3. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant potential in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; DPPH = 1,1-diphenyl-2-picryl-hydrazyl. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 3. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant potential in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; DPPH = 1,1-diphenyl-2-picryl-hydrazyl. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 4. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant enzyme activities in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; T-AOC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; CAT = catalase. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 4. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant enzyme activities in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; T-AOC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; CAT = catalase. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). DISCUSSION Exogenous CS administration has been extensively used as a reliable method to induce stress for studying physiological response in poultry (Lin et al., 2007; Zhang et al., 2011; Zeng et al., 2014). Among the endocrine and metabolic responses, circulating CS level appears to be an extremely sensitive and reliable indicator of stress in broilers (Puvadolpirod and Thaxton, 2000b). Stressed birds normally exhibit elevated plasma CS level, whether treated with adrenocorticotropin (Puvadolpirod and Thaxton, 2000c) or CS (Zhang et al., 2011; Zeng et al., 2014). In this study, the significantly increased circulating CS level in serum after CS injection demonstrated hyper-glucocorticoid and stress status in these treated broilers. Generally, elevated CS level, in turn, causes the increase in energy levels by acting on intermediary metabolism of carbohydrates, protein, and fats. Therefore, live BW loss and circulating glucose and uric acid concentrations have been demonstrated to be other predictable indicators of stress in broilers (Puvadolpirod and Thaxton, 2000c). Circulating glucose level, as a stress-induced physiological response index, is another early indicator of stress condition (Lin et al., 2007). Stress would cause an increase in glucose production by the catabolism of muscle protein (Gao et al., 2008), which is driven directly by CS-activated gluconeogenesis (Puvadolpirod and Thaxton, 2000c). Uric acid is excreted as a major end product of nitrogen metabolism by birds (Dong et al., 2007). Either adrenocorticotropin (Adams, 1968) or CS (Davison et al., 1985) administration can cause a dramatic increase in uric acid level because of exacerbated gluconeogenesis. Therefore, glucose level increases during gluconeogenesis, and concomitantly there is a significant increase in excretory uric acid and BW loss for the rise in protein oxidation in poultry (Lin et al., 2007). Similarly, the simultaneously elevated concentrations of glucose and uric acid illustrates the enhanced gluconeogenesis and protein catabolism in challenged chickens after short-term CS administration, respectively, which was also indirectly confirmed by the increased live BW loss in this study. All the results were in accordance with the effects of short-term CS injection (Lin et al., 2007; Zhang et al., 2011) or long-term dietary CS supplementation (Lin et al., 2004a; Zeng et al., 2014). Collectively, based on the increase in both stress index and stress-induced response indexes, we conclude that the acute pre-slaughter stress can be successfully mimicked by exogenous CS exposure. Live weight loss due to decreased anabolism and enhanced catabolism during pre-slaughter stress is of particular economic importance in animal and meat production (Virden and Kidd, 2009). It has been demonstrated that FSE generally exhibits beneficial effects on performance of broilers reared under stressful conditions rather than under normal conditions (Zeng et al., 2014). Expectedly, broilers fed diets supplemented with FSE showed a lower BW loss than those fed basal diets when subjected to CS, indicating that FSE had a potential to alleviate live BW loss of birds during pre-slaughter stress. Uric acid may play dual functions as an oxidative stress biomarker and an antioxidant in vivo (Simoyi et al., 2003; Machı΄n et al., 2004; Glantzounis et al., 2005). In this study, a dramatic increase in serum level of uric acid caused by CS injection may be due to exacerbated gluconeogesis by metabolizing muscles and increased non-enzymatic antioxidant capacity for stress-induced injury, suggesting the disadvantage influence of acute pre-slaughter stress. When subjected to CS, the birds supplemented with FSE had a lower serum uric acid level in concert with lower BW loss, which indicates preventive changes to counteract the injury induced by CS stress. Stressful stimuli before slaughter may increase levels of glucocorticoids and adrenaline-induced pathways of aerobic energy production, which induce free radical generation and may cause cellular damage resulting in lipid peroxidation and protein oxidation (Est΄evez, 2015). Lipid is the macromolecule most susceptible to free radical damage, which results in cleavage of the double bonds (Lu et al., 2014). Stress-induced lipid peroxidation can be directly reflected by the changes in muscle MDA concentration because MDA is the end product of lipid peroxidation and can generally be used as a biomarker of radical-induced damage and endogenous lipid peroxidation (Perai et al., 2014). Breast muscle is more vulnerable to meat quality deterioration when birds are subjected to stress (Zhang et al., 2014). Oxidative stress arises by increased MDA concentration in breast muscle after CS administration (Lin et al., 2009). The FSE prevented the increase of MDA levels in breast muscle, suggesting that lipid peroxidation associated with CS administration could be prevented by dietary FSE supplementation. The protective effects of FSE against MDA were consistent with studies conducted by Wang et al. (2008) who stated FSE lowed MDA concentration of muscle in broilers under heat stress. The secondary products of lipid peroxidation will induce protein oxidation as well, which may cause fragmentation and conformational changes in the secondary and tertiary structures of proteins and their function (Lu et al., 2014). Amino acid residues are major targets of free radicals (Fellenberg and Speisky, 2006). Carbonyl derivatives are an important oxidation by-product of amino acid residues, which is indicative of the extent of oxidative damage affecting the amino acid residues (Lu et al., 2014). Under stress condition, breast muscle was sensitive to protein oxidation as measured by carbonyl concentration in this study. Dietary FSE supplementation prevented the increase of carbonyl levels in breast muscle of birds, suggesting that FSE could be beneficial for prevention of protein oxidation in muscle. Determining disappearance of free radicals such as DPPH is a rapid and stable method to assess the antioxidant activity (Samarth et al., 2007). In the current study, CS decreased DPPH radical scavenging capability, and FSE exerted a remarkable DPPH scavenging ability towards normalization in broilers after challenge. Several studies demonstrated the free-radical scavenging effects of FSE by measuring DPPH activity (Yang et al., 2004; Wang et al., 2008; Lu et al., 2010) and concurrently, this was further confirmed by decreased oxidation indices including MDA and carbonyl content and increased antioxidant activities of T-AOC, SOD, and GSH-Px in this study. Interaction of antioxidants with DPPH, either the transfer of an electron or a hydrogen atom to DPPH, neutralizes its free radical character (Naik et al., 2003). Therefore, the high DPPH scavenging ability of the meat extract from broilers supplemented with FSE may be attributed to its high hydrogen donating ability. Under stressful conditions, higher production levels of free radicals are associated with changes in the scavenging capacity of antioxidant systems (Lin et al., 2007). The anti-oxidative status of muscle at the time of slaughter has been shown to be critical to meat quality (Jensen et al., 1998). Generally, excessive oxidative radicals are eliminated by antioxidant systems, including non-enzymatic components and a series of antioxidant enzymes. In the present study, the activities of T-AOC, SOD, and GSH-Px in breast muscle were significantly decreased in broilers subjected to CS administration, which means both the enzymatic and non-enzymatic antioxidant levels are suppressed by short-term CS administration. Therefore, the antioxidant activity of enzymes during postmortem processes can be influenced by pre-slaughter treatments as observed in this study. Antioxidants are an important preventive measure against lipid and protein oxidation in meat and meat products. To produce high-quality poultry meat, a superior anti-oxidative status is required when birds undergo pre-slaughter stress. The improved anti-oxidative capacity could protect against pre-slaughter stress-induced increase in lipid and protein oxidation in skeletal muscle (Young et al., 2003). As hypothesized, dietary supplementation with FSE increased the production of muscle T-AOC and SOD and GSH-Px activity, which indicates FSE could activate both non-enzymatic and enzyme antioxidant systems to protect boilers against transport-induced muscle lipid and protein oxidation by quenching free radicals to maintain the meat quality. One of the most well-known factors causing negative effects on meat quality is pre-slaughter stress (Ferguson and Warner, 2008). In this study, we used CS challenge to imitate the effects of pre-slaughter stress on meat quality. In accordance with the previous reports (Lin et al., 2009; Gao et al., 2010), the result indicated that the susceptibility of breast meat to lipid peroxidation was enhanced by pre-slaughter stress physiological responses. Lipid peroxidation is primarily initiated in the highly unsaturated FA components of membrane phospholipids (Gao et al., 2010). It was postulated that the impaired muscle cell membrane integrity is associated with the reduced meat quality through exudative water loss caused by stress (Sandercock et al., 2001; Archile-Contreras and Purslow, 2011). In this study, the increased drip loss in breast muscle of birds injected with CS may be caused by more exudative loss resulting from oxidative injury of muscle cell membrane, which indicated the pre-slaughter acute stress decreased the water-holding capacity of breast muscle. A disadvantage of pre-slaughter stress on meat drip loss could be alleviated by FES, indicating an increased water-holding capacity. The initial postmortem pH could be affected by antemortem stress (Lin et al., 2007). In the present study, coinciding with the increased drip loss of breast meat, the acute pre-slaughter stress had a significant negative effect on breast muscle pH values, which was similar to the results of Gao et al. (2008). The lower postmortem pH in breast muscle of CS-induced boilers indicated the fast acidification after slaughter (Lin et al., 2007). Color is a sensory characteristic used by consumers as an indicator of freshness and quality. Stress-inducing factors in animals may increase the generation of free radicals and ROS in the organism, which can result in cellular damage and cell membrane lipid oxidation and ultimately negatively impact the color and shelf life of meat (Adenkola and Ayo, 2010). Changes in meat color are generally caused by oxidation of red oxymyoglobin to metmyoglobin, which gives the meat an unattractive brown color (Adzitey and Nurul, 2011). Oxidation of myoglobin would result in decreased redness and increased yellowness (El Rammouz et al., 2004). Just as the present study showed, CS administration significantly increased lightness and yellowness values of breast muscle accompanied with decreased redness value, which indicates the presence of the oxidation of myoglobin. Similarly, pre-slaughter stress reduced the redness and increased lightness and yellowness values in meat (Bianchi et al., 2006; Zhang et al., 2011). Dietary FSE supplementation normalized yellowness value accompanied with increased redness of breast muscle, minimizing the oxidation of myoglobin and maintaining breast meat color of stressed broilers. The FA profiles of muscle tissues are mainly affected by dietary FA composition (Bavelaar and Beynen, 2003), but the degree of FA unsaturation in biomembranes is associated with their sensitivity to lipid peroxidation (Rebole et al., 2006). Gao et al. (2010) reported that FA profiles in muscle lipid could be altered by oxidative stress induced by glucocorticoid. In the present study, the effect of CS stress on SFA composition was insignificant. However, CS increased the content of MUFA and decreased the content of PUFA, indicating the altered FA composition by CS administration, which was consistent with the changes of muscle FA in broilers treated by dexamethasone (Gao et al., 2010). Compared with mammals, the FA double bond of total lipids and phosphatidylcholine, phosphatidylethanolamine, and cardiolipin fractions is intrinsically lower in poultry (Pamplona et al., 1999). The low degree of FA unsaturation in biomembranes of long-lived animals, such as birds, may confer advantage by decreasing their sensitivity to lipid peroxidation and prevent lipoxidation-derived damage to other macromolecules (Pamplona et al., 2002). The result suggested that the CS stress could alter the FA profiles by increasing FA saturation in chickens, which have an intrinsically higher degree of FA saturation. Interestingly, FSE altered the FA composition. Stearic acid, one of the main SFA, was decreased, which mainly contributed to the lower percentage of SFA in total FA. In contrast with the CS-induced effects, FSE improved PUFA percentage in breast muscle. The mechanisms by which FSE regulates FA composition of breast meat in chickens are not fully understood, and therefore further studies are warranted. Collectively, acute pre-slaughter stress mimicked by CS administration accelerated muscle lipid and protein oxidation by increasing MDA and carbonyl production and decreasing radical scavenging activity and antioxidant activities, thus increasing the saturation level of breast muscle fatty acids. Dietary supplementation with FSE elevated protective effects by directly scavenging free radicals and activating antioxidant capacity through both enzymatic and non-enzymatic antioxidant defensive systems. Therefore, FSE could be a promising anti-oxidant to protect the animals from oxidative injury and to improve the quality of meat for human consumption. In view of FSE’s good anti-oxidative characteristic concomitant with free radical scavenging properties, further research is warranted to investigate the effectiveness of dietary FSE supplementation as a potential natural antioxidant under more stressful conditions. ACKNOWLEDGMENTS This work was financed by the National Natural Science Foundation of China (No. 31372316; 31772612). REFERENCES Adams B. M. 1968. Effect of cortisol on growth and uric acid excretion in the chick. J. Endocrinol.  40: 145– 151. Google Scholar CrossRef Search ADS PubMed  Adenkola A. Y., Ayo J. O.. 2010. 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Forsythia suspensa extract protects broilers against breast muscle oxidative injury induced by corticosterone mimicked pre-slaughter acute stress

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© 2018 Poultry Science Association Inc.
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ABSTRACT Broilers were used to determine the protective effects of Forsythia suspensa extract (FSE) against breast muscle oxidative injury induced by corticosterone (CS) mimicking pre-slaughter acute stress. A total of 144 male Arbor Acre broilers was randomly allotted to one of 4 treatments in a 2 × 2 factorial arrangement that included FSE supplementation (0 or 100 mg/kg) and subcutaneous injection of CS (0 or 4 mg/kg) at 3 h before slaughter. Corticosterone increased live BW loss, and the adverse effect was attenuated by FSE in broilers subjected to CS (P < 0.05). Serum levels of CS, uric acid, and glucose were increased, and postmortem breast muscle pH values at 45 min and 24 h were decreased for CS-challenged broilers (P < 0.05). Corticosterone increased lightness and yellowness values and decreased redness of breast muscle (P < 0.05), and FSE decreased yellowness and increased redness of breast muscle (P < 0.05). Drip loss was increased by CS for birds supplemented without FSE (P < 0.05) and decreased by FSE for birds under CS challenge (P < 0.05). Corticosterone increased monounsaturated fatty acid (FA) and decreased polyunsaturated FA in breast muscle (P < 0.05), and saturated FA was decreased and polyunsaturated FA was increased by FSE (P < 0.05). Malondialdehyde and carbonyl contents in breast muscle were increased by CS and decreased by FSE (P < 0.05). Inhibition of 1,1-diphenyl-2-picryl-hydrazyl was decreased by CS and increased by FSE (P < 0.05). The activities of total-antioxidant capacity, glutathione peroxidase, and superoxide dismutase in breast muscle were lower in birds subjected to CS (P < 0.05) and were greater in birds supplemented with FSE (P < 0.05). Collectively, live BW loss and breast muscle oxidative injury were increased by CS in broilers, and these stress-related adverse effects could be attenuated by FSE supplementation via enhanced scavenging ability of free radicals and antioxidant capacity. Therefore, FSE could protect broilers against breast muscle oxidative injury when acute stress happens. INTRODUCTION Broilers are generally challenged by a variety of stress before slaughter induced by several stressors such as feed withdrawal, environmental temperature, catching, crating, transport, stunning, and struggling, all of which can cause physical, psychological, and physiological stresses for birds (Schwartzkopf-Genswein et al., 2012). Most stress would result in the increase of reactive oxygen species (ROS); oxidative stress occurs once the dramatically accumulated ROS level surpasses the capacity of antioxidant defensive systems (Miller et al., 1993; Nordberg and Arnér, 2001). Oxidation by free radicals is the primary mechanism for quality deterioration in foods, especially in meat products (Archile-Contreras and Purslow, 2011). Pre-slaughter oxidative stress can cause undesirable changes in flavor, color, texture, and nutritive value, and may even induce the production of toxic compounds in meat via influence on the pre- or post-slaughter muscle metabolism or both (Ferguson and Warner, 2008). Accordingly, the oxidative status of poultry at the time of slaughter is critical for meat quality (Jensen et al., 1998). Recently, increasing attention has been paid to explore effective ways to reduce pre-slaughter stress response and to improve post-slaughter meat quality of broilers (Zhang et al., 2014; Wang et al., 2015). Forsythia suspensa extract (FSE) is a popular traditional Chinese medicine (Piao et al., 2008, 2009) that is used as a natural source of antioxidants (Matkowski et al. 2013). In our previous studies, FSE has been reported to effectively attenuate stress in broilers induced by high temperature (Wang et al. 2008), high stocking density (Zhang et al. 2013), or corticosterone (CS) (Zeng et al. 2014), in rats induced by diquat (Lu et al. 2010) or lipopolysaccharide (Zhao et al., 2017a), and in pigs induced by soybean β-conglycinin (Hao et al., 2010) or weaning (Han et al., 2012; Zhao et al., 2017b). Therefore, FSE may have the potential to improve oxidative status of poultry at the time of slaughter. Pre-slaughter stressors can activate the hypothalamic–pituitary–adrenal (HPA) axis and cause release of glucocorticords such as CS in poultry (Zeng et al., 2014). Glucocorticoids, as the final effectors of the HPA axis, participate in the control of whole body homeostasis and the response of organisms to stressors by stimulating the release of energy stores, including glucose mobilization and lipolysis (Harvey et al., 1986). Stimulation of the adrenal cortex by exogenous administration of adrenocorticotropin and steroid moieties, including CS and dexamethasone, has been employed as a model to mimic physiological stress in poultry (Puvadolpirod and Thaxton, 2000a; Virden et al., 2007). As reported, physiological stress responses that occur in broilers following CS challenge include poor performance and meat quality (Lin et al., 2007), increased circulating levels of CS, glucose, and uric acid (Puvadolpirod and Thaxton, 2000b; Yuan et al., 2008), depression of the immune system (Minozzi et al., 2008), and impairment of redox balance (Lin et al., 2004a). When subjected to acute stress or chronic stress, broilers show different performance and biochemical responses (Lin et al., 2004b). However, few studies using the mimic stress models were focused on the oxidative responses of muscle, especially the vulnerable breast muscle in broilers. Our lab has reported the protective effects of FSE against physiological stress induced by dietary CS supplementation (from d 14 to 21, chronic exposure) to mimic the chronic physiological stress during the intensive rearing period (Zeng et al., 2014). The results indicated FSE can alleviate CS-induced growth inhibition, impairment of nutrient digestibility, and immune depression in broiler chickens. Unfortunately, the previous model failed to mimic the pre-slaughter acute stress and also failed to study the protective effects of FSE against post-slaughter breast muscle oxidative injury. Therefore, we hypothesized that the pre-slaughter acute stress can be mimicked by subcutaneous injection of CS at 3 h before slaughter, and the post-slaughter breast muscle oxidative injury caused by the mimicked pre-slaughter short-time stress in broilers could be attenuated, partly or completely, by dietary FSE supplementation. The purpose of this study was to investigate the underlying potentiality of FSE in protecting broilers against breast muscle oxidative injury induced by subcutaneous injection of CS at 3 h before slaughter. MATERIALS AND METHODS Preparation And Composition Of FSE Forsythia suspensa extract is derived from a climbing plant widely distributed in China. The dried fruits of Forsythia suspensa were purchased from Tong Ren Tang Company (Beijing, China), and FSE was prepared using the methods described by Wang et al. (2008). The 3 major active antioxidant constituents isolated from FSE have been identified as forsythoside A, forythialan A, and phillygenin by Lu et al. (2010) in our laboratory. The 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Experimental Birds The experimental protocols used in the experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China). A total of 144 one-day-old male Arbor Acre broiler chickens (weighing 44.5 ± 1.2 g) was purchased from Arbor Acres Poultry Breeding Company (Beijing, China). All birds were raised in wire-floored cages in an environmentally controlled room with continuous light and had ad libitum access to feed and water. The ambient temperature was maintained at 33°C at the start of experiment and decreased as the birds progressed in age to ensure a final temperature of 24°C at 35 d and thereafter. All birds were inoculated with inactivated infectious bursa disease vaccine on d 14 and 21 and Newcastle disease vaccine on d 7 and 28. The trial was conducted in 2 phases, consisting of a starter phase from d 1 to 21 and a finisher phase from d 22 to 42. Experimental Design And Diets The broiler chickens were randomly allotted to one of 4 treatments in a 2 × 2 factorial arrangement that included FSE supplementation (0 or 100 mg/kg of diet) and subcutaneous injection of CS [4 mg/kg of body weight (BW) in corn oil] or corn oil (sham control) at 3 h before slaughter. Six cages per treatment were used, with 6 birds per cage. In detail, from d 1 to 42, 12 cages of birds received diets containing FSE (100 mg/kg of diet), whereas 12 cages did not. The supplementation level of FSE was based on previous work from our laboratory (Zhang et al., 2013; Zeng et al., 2014). At 42 d of age, the chickens in each treatment were subjected to one of the following treatments: single subcutaneous administration of CS (4 mg/kg of BW in corn oil) or corn oil at 3 h before slaughter. During the 3-hour exposure, feed was withdrawn, and all the broiler chickens had free access to tap water. Birds were weighed at the start and end of the 3-hour CS challenge, and live BW loss calculated. All diets were fed in mash form and were based on corn-soybean meal (Table 1). All essential nutrients provided in diets met or slightly exceeded NRC recommendations (NRC, 1994). Table 1. Composition and nutrient levels of the experimental basal diets (%, as-fed basis, antibiotic-free).1 Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  1Forsythia suspense extract (100 mg/kg) was directly mixed in the basal diet. 2L-Lys HCl (78.8%) and DL-Met (98.5%) were products of CJ CheilJedang Co., Inc., Seoul, Korea. 3The premix provided the following per kilogram of diet: zinc, 60 mg; iron, 100 mg; manganese, 80 mg; copper, 10 mg; iodine, 0.35 mg; selenium, 0.3 mg; vitamin A, 10,000 IU; vitamin D3, 2850 IU; vitamin E, 30 IU; vitamin K3, 2 mg; vitamin B12, 1.2 mg; riboflavin, 6 mg; nicotinic acid, 40 mg; pantothenic acid, 12 mg; pyridoxine, 3 mg; biotin, 0.2 mg; and choline chloride, 800 mg. 4These values were calculated from data provided by NRC (1994). View Large Table 1. Composition and nutrient levels of the experimental basal diets (%, as-fed basis, antibiotic-free).1 Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  Item  Starter phase  Finisher phase  Ingredients  (d 1 to 21)  (d 22 to 42)  Corn grain  60.60  61.42  Soybean meal  28.64  25.82  Fish meal  4.00  4.00  Soybean oil  2.25  4.56  Dicalcium phosphate  1.36  1.24  Limestone  1.33  1.20  Salt  0.40  0.35  L-Lys HCl (78.8%)2  0.16  0.13  DL-Met (98.5%)2  0.26  0.28  Vitamin-mineral premix3  1.00  1.00  Analyzed nutrient levels      CP  21.4  19.5  Ca  1.05  0.89  Total P  0.61  0.62  Calculated nutrient levels4      Available P  0.45  0.40  Lys  1.28  1.12  Met  0.58  0.48  DE, MJ/kg  12.55  12.89  1Forsythia suspense extract (100 mg/kg) was directly mixed in the basal diet. 2L-Lys HCl (78.8%) and DL-Met (98.5%) were products of CJ CheilJedang Co., Inc., Seoul, Korea. 3The premix provided the following per kilogram of diet: zinc, 60 mg; iron, 100 mg; manganese, 80 mg; copper, 10 mg; iodine, 0.35 mg; selenium, 0.3 mg; vitamin A, 10,000 IU; vitamin D3, 2850 IU; vitamin E, 30 IU; vitamin K3, 2 mg; vitamin B12, 1.2 mg; riboflavin, 6 mg; nicotinic acid, 40 mg; pantothenic acid, 12 mg; pyridoxine, 3 mg; biotin, 0.2 mg; and choline chloride, 800 mg. 4These values were calculated from data provided by NRC (1994). View Large Sample Collection And Handling At 3 h after CS treatment, one bird close to the average BW was selected per replicate (cage) and slaughtered via exsanguination of the left jugular artery. Blood was collected (5 mL) by cardiac puncture into a 10-mL anticoagulant-free vacutainer tube (Greiner Bio-One GmbH, Kremsmunster, Austria) and then centrifuged at 3,000 × g for 10 min at 4°C to obtain serum. The serum samples were stored at −20°C for further analysis. Immediately after death, the left breast muscle was excised from the warm carcasses, cryogenically frozen in liquid nitrogen (−196°C), and stored in a −80°C freezer until use for lipid and protein peroxidation, DPPH radical scavenging activity, and antioxidative enzyme activity analysis. Before analysis, tissues were minced and homogenized (10% w/v) in ice-cold sodium, potassium phosphate buffer (0.01 M, pH 7.4) containing 0.86% NaCl (Lu et al., 2010). The homogenate was centrifuged at 3,000 × g for 10 min at 4°C, and the resultant supernatant was used for analysis. The right breast muscle was taken to measure postmortem meat quality in a 4°C cell. Chemical Analysis Serum metabolite analysis. Serum concentrations of glucose and uric acid were measured using an Automated Biochemistry Analyzer (Hitachi 902 Automatic Analyzer, Hitachi, Tokyo, Japan) using colorimetric methods and following the instructions of the manufacturer of the corresponding reagent kit (Zhongsheng Biochemical Co., Ltd., Beijing, China). Corticosterone concentration was assessed with a commercial enzyme immunoassay kit with the intra-assay precision less than 8% and inter-assay precision less than 10% (Cusabio Biotech. Co., Ltd., Wuhan, Hubei, China) according to the manufacturer's instructions. Meat quality and fatty acid profile measurement. Meat color, including lightness (L*), redness (a*), and yellowness (b*) values, was measured from 3 orientations (middle, medial, and lateral) using a Chromameter (CR-410, Konica Minota, Tokyo, Japan). The pH values at 45 min and 24 h postmortem also were measured at 3 locations using a glass penetration pH electrode (pH-star, Matthaus, Germany). The pH decline rate within 24 h postmortem was calculated as a percentage: [(pH45 min − pH24 h)/pH45 min] × 100. Each sample was measured 3 times, and their average value was taken as the final result. Drip loss over 24 h was measured using the plastic bag method. The drip loss was calculated as a percentage: [(initial weight-final weight)/initial weight] × 100. The homogenized skinless breast meat tissue was defrosted, and samples of breast (20 g) lyophilized for 60 h using a freeze dryer were used for testing fatty acid (FA) concentrations. The FA profiles of lipid sources were determined by gas chromatography (6890 series, Agilent Technologies, Wilmington, DE) according to the procedures of Sukhija and Palmquist (1988). Lipid samples were converted to FA methyl esters using methanolic HCl solution. Undecanoic acid (C11:0) was used as the internal standard. Aliquots of 11 were injected into a capillary column (60 m × 250 m × 250 nm, DB-23, Agilent) with cyanopropyl methyl silicone as the stationary phase. Column oven temperature was programmed with a 1:20 split. Injector and detector temperatures were maintained at 260 and 270°C, respectively. Nitrogen was the carrier gas at a flow rate of 2 mL/min. Fatty acids were expressed as the proportion of each individual FA to the total of all FA present in the sample. The following FA combinations—total saturated FA (SFA), total monounsaturated FA (MUFA), polyunsaturated FA (PUFA), MUFA/SFA and PUFA/SFA ratio—were calculated. Lipid and protein oxidation analysis. First, protein concentration was determined using a Pierce BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and was expressed as mg/mL. Lipid oxidation in muscle samples was measured as 2-thiobarbituric acid-reactive substances and was expressed as nmol of malondialdehyde (MDA) per mg of protein. Protein oxidation was reflected by carbonyl concentration, oxidation byproducts of amino acid residues. The concentrations of MDA and carbonyl were determined using kits from Cayman Chemical Company (Ann Arbor, MI). Homogenates of breast muscle were measured colorimetrically at 540 nm for MDA and 372 nm for carbonyl. Values of both were expressed in nmol/mg of protein. DPPH free radical scavenging assay The DPPH radical-scavenging activity of the supplements was estimated by the method of Blois (1958) with slight modifications. Briefly, 1 mL of 0.2 mM DPPH prepared in methanol was added to 200 μL supernatant and 800 μL distilled water. The mixture was vortexed and left to stand at room temperature (20 to 22°C) for 30 minutes. A tube containing 1 mL of methanol and 1 mL of DPPH solution was used as control, whereas methanol alone was used as a blank. The absorbance of the solution was measured at 517 nm using a spectrophotometer (SPECTRAmax 340 PC, Molecular Devices, Sunny Vale, CA). The scavenging activity of meat sample against DPPH radical was expressed as percent of control and calculated as: inhibition rate of DPPH (%) = [1- (absorbance of sample/absorbance of control)] × 100. Anti-oxidative enzyme activity measurement The total-antioxidant capacity (T-AOC) and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in breast muscle were determined with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) per the manufacturer's instructions. Statistical Analysis All data were analyzed as a 2 × 2 factorial design using the General Linear Model procedure of SAS (version 9.2, 2008). The cage was the experimental unit for BW loss, and the selected bird from each cage was the experimental unit for other parameters. The main effects included FSE, CS, and their interaction. Least squares means were derived for all treatments and compared using the PDIFF procedure (Tukey Adjustment) and STDERR options of SAS (2008). Results were expressed as least squares means and SEM. Probability values less than 0.05 were used as the criterion for statistical significance and less than 0.10 as the tendency to significance. RESULTS Bw Loss And Serum Metabolites There was significant interaction (P = 0.04) in live BW loss between FSE and CS (Fig. 1). Corticosterone increased live BW loss of broilers (P < 0.05), and the adverse increase was attenuated by FSE for broilers subjected to CS (P < 0.05). Figure 1. View largeDownload slide Effect of Forsythia suspense extract (FSE) on live BW loss of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 1. View largeDownload slide Effect of Forsythia suspense extract (FSE) on live BW loss of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Serum levels of CS, uric acid, and glucose were increased (P < 0.05) for CS-challenged broilers, whereas CS and glucose concentrations in serum were unaffected by FSE supplementation (Table 2). Significant interactions were noted between FSE and CS for uric acid concentration (P < 0.05), in that FSE decreased the uric acid concentration in serum of broilers under CS challenge. Table 2. Effects of Forsythia suspense extract (FSE) on serum metabolite parameters of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  a,bMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large Table 2. Effects of Forsythia suspense extract (FSE) on serum metabolite parameters of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  Corticoserone (ng/mL)  31.5b  33.1b  43.1a  39.6a  2.45  <0.01  0.67  0.25  Uric acid (umol/L)  110c  93.5c  189a  135b  13.0  <0.01  <0.01  0.01  Glucose (mmol/L)  5.59b  5.73b  8.76a  7.85a  0.58  <0.01  0.39  0.24  a,bMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large Meat Quality And Fatty Acid Profile Of Breast Muscle Postmortem breast muscle pH values at 45 min and 24 h were decreased (P < 0.05), and the pH decline rate was increased (P < 0.05) in birds challenged by CS, whereas these parameters were not influenced by FSE supplementation (Table 3). Corticosterone increased (P < 0.05) lightness and yellowness values and decreased (P < 0.05) redness of breast muscle in broilers. Conversely, FSE supplementation decreased (P < 0.05) yellowness and increased (P < 0.05) redness of breast muscle. Drip loss was influenced by the interaction between CS and FSE (P < 0.05) in that drip loss was increased by CS challenge for birds supplemented without FSE (P < 0.05) and decreased by FSE supplementation for birds under CS challenge (P < 0.05). Table 3. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large Table 3. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  pH45 min  6.33a  6.32a,b  6.20b  6.21a,b  0.04  <0.01  0.98  0.81  pH24 h  5.85a  5.87a  5.60b  5.66b  0.03  <0.01  0.48  0.96  pH decline (%)  7.61b,c  7.11c  9.80a  8.88a,b  0.45  <0.01  0.12  0.64  Lightness  54.1b  53.6b  59.2a  56.8a,b  1.05  <0.01  0.18  0.35  Redness  2.84a  3.40a  1.98b  2.87a  0.22  <0.01  <0.01  0.46  Yellowness  6.40b  5.75b  8.18a  6.58b  0.41  <0.01  0.01  0.25  Drip loss (%)  2.56b  2.47b  3.99a  2.30b  0.18  <0.01  <0.01  0.02  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation. View Large There was no interaction between CS and FSE on FA profiles of breast muscle (Table 4). The percentages of C18:0 and SFA in breast muscle were decreased (P < 0.05) by FSE supplementation while not influenced by CS administration. Concentrations of C18:1n9c and MUFA were greater in breast muscle of CS-challenged birds than those of non-challenged birds (P < 0.05), whereas no differences were observed among birds supplemented with or without FSE. Corticosterone decreased (P < 0.05) the concentrations of C20:3n3, C20:3n6, C20:4n6, C22:6n3, and PUFA in breast muscle, whereas FSE increased (P < 0.05) C22:6n3 and PUFA content and tended to increase (P = 0.06) C20:4n6 content. The birds subjected to CS had a greater ratio of MUFA/SFA (P < 0.05) and lower ratio of PUFA/SFA (P < 0.05) in breast muscles compared with the unchallenged birds. The ratio of PUFA/SFA in breast muscles was increased in birds supplemented with FSE (P < 0.05). Table 4. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids. View Large Table 4. Effects of Forsythia suspense extract (FSE) on meat quality in breast muscle of broilers challenged with corticosterone (CS).1 Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  Item  CS−  CS+  SEM  P-value    FSE−  FSE+  FSE−  FSE+    CS  FSE  CS × FSE  C6:0  0.12  0.13  0.11  0.11  0.02  0.38  0.76  0.92  C8:0  0.04  0.04  0.03  0.04  0.01  0.19  0.88  0.19  C10:0  0.06  0.06  0.04  0.04  0.01  0.16  0.95  0.86  C12:0  0.06  0.06  0.05  0.06  <0.01  0.22  0.53  0.22  C14:0  0.44  0.41  0.41  0.40  0.02  0.42  0.37  0.68  C15:0  0.09  0.08  0.08  0.08  <0.01  0.13  0.28  0.51  C16:0  20.9  21.0  20.8  20.6  0.31  0.43  0.80  0.66  C17:0  0.17  0.18  0.17  0.16  0.01  0.20  0.72  0.20  C18:0  12.2a  10.7b  12.7a  10.6b  0.37  0.61  <0.01  0.42  C20:0  0.18  0.18  0.18  0.19  0.01  0.44  0.33  0.69  C21:0  0.90  0.92  0.85  0.90  0.07  0.62  0.60  0.90  C22:0  0.18  0.20  0.16  0.17  0.01  0.06  0.52  0.86  C24:0  1.83  1.83  1.75  1.76  0.14  0.62  0.95  0.94  SFA  37.2a  35.8a,b  37.3a  35.1b  0.51  0.61  <0.01  0.42  C14:1  0.10  0.08  0.09  0.09  0.01  0.56  0.21  0.21  C16:1  2.41  2.00  2.77  2.55  0.27  0.11  0.25  0.74  C18:1n9c  23.5b  23.7b  26.9a  26.6a  0.53  <0.01  0.88  0.62  C20:1  0.27  0.29  0.27  0.25  0.01  0.18  0.65  0.11  C22:1n9  0.06  0.06  0.05  0.05  0.01  0.16  0.87  0.62  C24:1  0.27  0.30  0.26  0.24  0.02  0.07  0.96  0.21  MUFA  26.6b  26.4b  30.4a  29.7a  0.71  <0.01  0.57  0.77  C18:2n6c  24.3  23.8  22.7  24.3  0.64  0.40  0.41  0.11  C18:3n3  1.72  1.78  1.70  1.72  0.11  0.71  0.75  0.87  C20:3n3  0.11a  0.11a  0.07b  0.06b  0.01  <0.01  1.00  0.48  C20:3n6  1.22a  1.19a  0.85b  0.82b  0.10  <0.01  0.77  0.95  C20:4n6  5.38a,b  6.18a  4.46b  5.27a,b  0.41  0.03  0.06  0.98  C20:5n3  0.68  0.82  0.63  0.72  0.08  0.34  0.13  0.72  C22:6n3  2.83b  3.95a  1.92b  2.26b  0.31  <0.01  0.02  0.22  PUFA  36.2a,b  37.8a  32.3c  35.1b  0.59  <0.01  <0.01  0.30  MUFA/SFA  0.72c  0.74b,c  0.82a,b  0.85a  0.03  <0.01  0.34  0.78  PUFA/SFA  0.98b  1.06a  0.87c  1.00a,b  0.02  <0.01  <0.01  0.26  a-cMeans in the same row with different superscripts differ significantly (P < 0.05). 1CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated fatty acids. View Large Oxidative And Antioxidative Status Of Breast Muscle Administration with CS increased MDA and carbonyl content in breast muscle (P < 0.05), and FSE supplementation decreased (P < 0.05) MDA content or tended to decrease (P = 0.07) carbonyl content in breast muscles of broilers (Fig. 2). The interaction between FSE and CS tended to be significant for carbonyl content but was not for MDA content in breast muscle. Figure 2. View largeDownload slide Effect of Forsythia suspense extract (FSE) on oxidation injury of breast muscle in broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; MDA = malondialdehyde. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 2. View largeDownload slide Effect of Forsythia suspense extract (FSE) on oxidation injury of breast muscle in broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; MDA = malondialdehyde. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Inhibition of DPPH radicals in breast muscle of birds was decreased (P < 0.05) by CS and increased (P < 0.05) by FSE (Fig. 3). The activities of T-AOC, GSH-Px, and SOD in breast muscle were lower (P < 0.05) in birds subjected to CS than those without CS challenge and were greater (P < 0.05) in birds supplemented with FSE than those without FSE supplementation (Fig. 4). Neither CS nor FSE affected CAT activity in breast muscle. There were no significant interactions between FSE and CS for antioxidant activities in breast muscle. Figure 3. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant potential in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; DPPH = 1,1-diphenyl-2-picryl-hydrazyl. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 3. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant potential in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; DPPH = 1,1-diphenyl-2-picryl-hydrazyl. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 4. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant enzyme activities in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; T-AOC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; CAT = catalase. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). Figure 4. View largeDownload slide Effect of Forsythia suspense extract (FSE) on antioxidant enzyme activities in breast muscle of broilers challenged with corticosterone (CS). CS− = corn oil injection; CS+ = CS injection; FSE− = basal diet without FSE supplementation; FSE+ = basal diet with 100 mg/kg FSE supplementation; T-AOC = total antioxidant capacity; SOD = superoxide dismutase; GSH-Px = glutathione peroxidase; CAT = catalase. Values represent the mean ± SE (n = 6 broilers per treatment). a-cMeans with different superscripts differ significantly (P < 0.05). DISCUSSION Exogenous CS administration has been extensively used as a reliable method to induce stress for studying physiological response in poultry (Lin et al., 2007; Zhang et al., 2011; Zeng et al., 2014). Among the endocrine and metabolic responses, circulating CS level appears to be an extremely sensitive and reliable indicator of stress in broilers (Puvadolpirod and Thaxton, 2000b). Stressed birds normally exhibit elevated plasma CS level, whether treated with adrenocorticotropin (Puvadolpirod and Thaxton, 2000c) or CS (Zhang et al., 2011; Zeng et al., 2014). In this study, the significantly increased circulating CS level in serum after CS injection demonstrated hyper-glucocorticoid and stress status in these treated broilers. Generally, elevated CS level, in turn, causes the increase in energy levels by acting on intermediary metabolism of carbohydrates, protein, and fats. Therefore, live BW loss and circulating glucose and uric acid concentrations have been demonstrated to be other predictable indicators of stress in broilers (Puvadolpirod and Thaxton, 2000c). Circulating glucose level, as a stress-induced physiological response index, is another early indicator of stress condition (Lin et al., 2007). Stress would cause an increase in glucose production by the catabolism of muscle protein (Gao et al., 2008), which is driven directly by CS-activated gluconeogenesis (Puvadolpirod and Thaxton, 2000c). Uric acid is excreted as a major end product of nitrogen metabolism by birds (Dong et al., 2007). Either adrenocorticotropin (Adams, 1968) or CS (Davison et al., 1985) administration can cause a dramatic increase in uric acid level because of exacerbated gluconeogenesis. Therefore, glucose level increases during gluconeogenesis, and concomitantly there is a significant increase in excretory uric acid and BW loss for the rise in protein oxidation in poultry (Lin et al., 2007). Similarly, the simultaneously elevated concentrations of glucose and uric acid illustrates the enhanced gluconeogenesis and protein catabolism in challenged chickens after short-term CS administration, respectively, which was also indirectly confirmed by the increased live BW loss in this study. All the results were in accordance with the effects of short-term CS injection (Lin et al., 2007; Zhang et al., 2011) or long-term dietary CS supplementation (Lin et al., 2004a; Zeng et al., 2014). Collectively, based on the increase in both stress index and stress-induced response indexes, we conclude that the acute pre-slaughter stress can be successfully mimicked by exogenous CS exposure. Live weight loss due to decreased anabolism and enhanced catabolism during pre-slaughter stress is of particular economic importance in animal and meat production (Virden and Kidd, 2009). It has been demonstrated that FSE generally exhibits beneficial effects on performance of broilers reared under stressful conditions rather than under normal conditions (Zeng et al., 2014). Expectedly, broilers fed diets supplemented with FSE showed a lower BW loss than those fed basal diets when subjected to CS, indicating that FSE had a potential to alleviate live BW loss of birds during pre-slaughter stress. Uric acid may play dual functions as an oxidative stress biomarker and an antioxidant in vivo (Simoyi et al., 2003; Machı΄n et al., 2004; Glantzounis et al., 2005). In this study, a dramatic increase in serum level of uric acid caused by CS injection may be due to exacerbated gluconeogesis by metabolizing muscles and increased non-enzymatic antioxidant capacity for stress-induced injury, suggesting the disadvantage influence of acute pre-slaughter stress. When subjected to CS, the birds supplemented with FSE had a lower serum uric acid level in concert with lower BW loss, which indicates preventive changes to counteract the injury induced by CS stress. Stressful stimuli before slaughter may increase levels of glucocorticoids and adrenaline-induced pathways of aerobic energy production, which induce free radical generation and may cause cellular damage resulting in lipid peroxidation and protein oxidation (Est΄evez, 2015). Lipid is the macromolecule most susceptible to free radical damage, which results in cleavage of the double bonds (Lu et al., 2014). Stress-induced lipid peroxidation can be directly reflected by the changes in muscle MDA concentration because MDA is the end product of lipid peroxidation and can generally be used as a biomarker of radical-induced damage and endogenous lipid peroxidation (Perai et al., 2014). Breast muscle is more vulnerable to meat quality deterioration when birds are subjected to stress (Zhang et al., 2014). Oxidative stress arises by increased MDA concentration in breast muscle after CS administration (Lin et al., 2009). The FSE prevented the increase of MDA levels in breast muscle, suggesting that lipid peroxidation associated with CS administration could be prevented by dietary FSE supplementation. The protective effects of FSE against MDA were consistent with studies conducted by Wang et al. (2008) who stated FSE lowed MDA concentration of muscle in broilers under heat stress. The secondary products of lipid peroxidation will induce protein oxidation as well, which may cause fragmentation and conformational changes in the secondary and tertiary structures of proteins and their function (Lu et al., 2014). Amino acid residues are major targets of free radicals (Fellenberg and Speisky, 2006). Carbonyl derivatives are an important oxidation by-product of amino acid residues, which is indicative of the extent of oxidative damage affecting the amino acid residues (Lu et al., 2014). Under stress condition, breast muscle was sensitive to protein oxidation as measured by carbonyl concentration in this study. Dietary FSE supplementation prevented the increase of carbonyl levels in breast muscle of birds, suggesting that FSE could be beneficial for prevention of protein oxidation in muscle. Determining disappearance of free radicals such as DPPH is a rapid and stable method to assess the antioxidant activity (Samarth et al., 2007). In the current study, CS decreased DPPH radical scavenging capability, and FSE exerted a remarkable DPPH scavenging ability towards normalization in broilers after challenge. Several studies demonstrated the free-radical scavenging effects of FSE by measuring DPPH activity (Yang et al., 2004; Wang et al., 2008; Lu et al., 2010) and concurrently, this was further confirmed by decreased oxidation indices including MDA and carbonyl content and increased antioxidant activities of T-AOC, SOD, and GSH-Px in this study. Interaction of antioxidants with DPPH, either the transfer of an electron or a hydrogen atom to DPPH, neutralizes its free radical character (Naik et al., 2003). Therefore, the high DPPH scavenging ability of the meat extract from broilers supplemented with FSE may be attributed to its high hydrogen donating ability. Under stressful conditions, higher production levels of free radicals are associated with changes in the scavenging capacity of antioxidant systems (Lin et al., 2007). The anti-oxidative status of muscle at the time of slaughter has been shown to be critical to meat quality (Jensen et al., 1998). Generally, excessive oxidative radicals are eliminated by antioxidant systems, including non-enzymatic components and a series of antioxidant enzymes. In the present study, the activities of T-AOC, SOD, and GSH-Px in breast muscle were significantly decreased in broilers subjected to CS administration, which means both the enzymatic and non-enzymatic antioxidant levels are suppressed by short-term CS administration. Therefore, the antioxidant activity of enzymes during postmortem processes can be influenced by pre-slaughter treatments as observed in this study. Antioxidants are an important preventive measure against lipid and protein oxidation in meat and meat products. To produce high-quality poultry meat, a superior anti-oxidative status is required when birds undergo pre-slaughter stress. The improved anti-oxidative capacity could protect against pre-slaughter stress-induced increase in lipid and protein oxidation in skeletal muscle (Young et al., 2003). As hypothesized, dietary supplementation with FSE increased the production of muscle T-AOC and SOD and GSH-Px activity, which indicates FSE could activate both non-enzymatic and enzyme antioxidant systems to protect boilers against transport-induced muscle lipid and protein oxidation by quenching free radicals to maintain the meat quality. One of the most well-known factors causing negative effects on meat quality is pre-slaughter stress (Ferguson and Warner, 2008). In this study, we used CS challenge to imitate the effects of pre-slaughter stress on meat quality. In accordance with the previous reports (Lin et al., 2009; Gao et al., 2010), the result indicated that the susceptibility of breast meat to lipid peroxidation was enhanced by pre-slaughter stress physiological responses. Lipid peroxidation is primarily initiated in the highly unsaturated FA components of membrane phospholipids (Gao et al., 2010). It was postulated that the impaired muscle cell membrane integrity is associated with the reduced meat quality through exudative water loss caused by stress (Sandercock et al., 2001; Archile-Contreras and Purslow, 2011). In this study, the increased drip loss in breast muscle of birds injected with CS may be caused by more exudative loss resulting from oxidative injury of muscle cell membrane, which indicated the pre-slaughter acute stress decreased the water-holding capacity of breast muscle. A disadvantage of pre-slaughter stress on meat drip loss could be alleviated by FES, indicating an increased water-holding capacity. The initial postmortem pH could be affected by antemortem stress (Lin et al., 2007). In the present study, coinciding with the increased drip loss of breast meat, the acute pre-slaughter stress had a significant negative effect on breast muscle pH values, which was similar to the results of Gao et al. (2008). The lower postmortem pH in breast muscle of CS-induced boilers indicated the fast acidification after slaughter (Lin et al., 2007). Color is a sensory characteristic used by consumers as an indicator of freshness and quality. Stress-inducing factors in animals may increase the generation of free radicals and ROS in the organism, which can result in cellular damage and cell membrane lipid oxidation and ultimately negatively impact the color and shelf life of meat (Adenkola and Ayo, 2010). Changes in meat color are generally caused by oxidation of red oxymyoglobin to metmyoglobin, which gives the meat an unattractive brown color (Adzitey and Nurul, 2011). Oxidation of myoglobin would result in decreased redness and increased yellowness (El Rammouz et al., 2004). Just as the present study showed, CS administration significantly increased lightness and yellowness values of breast muscle accompanied with decreased redness value, which indicates the presence of the oxidation of myoglobin. Similarly, pre-slaughter stress reduced the redness and increased lightness and yellowness values in meat (Bianchi et al., 2006; Zhang et al., 2011). Dietary FSE supplementation normalized yellowness value accompanied with increased redness of breast muscle, minimizing the oxidation of myoglobin and maintaining breast meat color of stressed broilers. The FA profiles of muscle tissues are mainly affected by dietary FA composition (Bavelaar and Beynen, 2003), but the degree of FA unsaturation in biomembranes is associated with their sensitivity to lipid peroxidation (Rebole et al., 2006). Gao et al. (2010) reported that FA profiles in muscle lipid could be altered by oxidative stress induced by glucocorticoid. In the present study, the effect of CS stress on SFA composition was insignificant. However, CS increased the content of MUFA and decreased the content of PUFA, indicating the altered FA composition by CS administration, which was consistent with the changes of muscle FA in broilers treated by dexamethasone (Gao et al., 2010). Compared with mammals, the FA double bond of total lipids and phosphatidylcholine, phosphatidylethanolamine, and cardiolipin fractions is intrinsically lower in poultry (Pamplona et al., 1999). The low degree of FA unsaturation in biomembranes of long-lived animals, such as birds, may confer advantage by decreasing their sensitivity to lipid peroxidation and prevent lipoxidation-derived damage to other macromolecules (Pamplona et al., 2002). The result suggested that the CS stress could alter the FA profiles by increasing FA saturation in chickens, which have an intrinsically higher degree of FA saturation. Interestingly, FSE altered the FA composition. Stearic acid, one of the main SFA, was decreased, which mainly contributed to the lower percentage of SFA in total FA. In contrast with the CS-induced effects, FSE improved PUFA percentage in breast muscle. The mechanisms by which FSE regulates FA composition of breast meat in chickens are not fully understood, and therefore further studies are warranted. Collectively, acute pre-slaughter stress mimicked by CS administration accelerated muscle lipid and protein oxidation by increasing MDA and carbonyl production and decreasing radical scavenging activity and antioxidant activities, thus increasing the saturation level of breast muscle fatty acids. Dietary supplementation with FSE elevated protective effects by directly scavenging free radicals and activating antioxidant capacity through both enzymatic and non-enzymatic antioxidant defensive systems. Therefore, FSE could be a promising anti-oxidant to protect the animals from oxidative injury and to improve the quality of meat for human consumption. In view of FSE’s good anti-oxidative characteristic concomitant with free radical scavenging properties, further research is warranted to investigate the effectiveness of dietary FSE supplementation as a potential natural antioxidant under more stressful conditions. ACKNOWLEDGMENTS This work was financed by the National Natural Science Foundation of China (No. 31372316; 31772612). REFERENCES Adams B. M. 1968. Effect of cortisol on growth and uric acid excretion in the chick. J. Endocrinol.  40: 145– 151. Google Scholar CrossRef Search ADS PubMed  Adenkola A. Y., Ayo J. O.. 2010. 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Poultry ScienceOxford University Press

Published: Mar 5, 2018

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