Effects of low-protein diets on acute phase proteins and heat shock protein 70 responses, and growth performance in broiler chickens under heat stress condition

Effects of low-protein diets on acute phase proteins and heat shock protein 70 responses, and... Abstract A study with a 4 × 2 factorial arrangement was conducted to investigate the effects of 4 dietary protein levels and 2 environmental conditions on acute phase proteins (APP), brain heat shock protein (HSP) 70 density, and growth performance of broiler chickens. Day-old broiler chicks (Cobb 500) were fed isocaloric diets but with various levels of crude protein (CP), namely, (1) 21.0 and 19.0% CP in starter and finisher diets, respectively (control), (2) 19.5 and 17.5% CP in starter and finisher diets, respectively (Diet A), (3) 18.0 and 16.0% CP in starter and finisher diets, respectively (Diet B), and (4) 16.5 and 14.5% CP in starter and finisher diets, respectively (Diet C). Equal numbers of birds from each diet were subjected to either 23±1°C throughout or 33±1°C for 6 h per d from 22 to 35 d of age. From d 1 to 21, feed intake (FI) and weight gain (WG) decreased linearly (P = 0.021 and P = 0.009, respectively), as CP level was reduced. During the heat treatment period (d 22 to 35), there were significant (P = 0.04) diet × heat treatment interactions for FCR. Diet had no effect on FCR among the unheated birds, but the ratio increased linearly (P = 0.007) as dietary CP level decreased. Irrespective of ambient temperature, there was a significant linear decrease in FI (P = 0.032) and WG (P < 0.001) as dietary CP level decreased. Low-CP diets improved the survivability of heat-stressed broilers when compared to those fed control diets. Low-CP diets linearly decreased (P < 0.01) APP (ovotransferrin and alpha-acid glycoprotein) responses. Both APP and HSP 70 reactions were elevated following heat treatment. In conclusion, feeding broilers with low-CP diets adversely affect the growth performance of broilers under heat stress condition. However, low-CP diets were beneficial in improving the survivability. Because APP are involved in the restoration of homeostasis, the adverse effect of low-CP diet on the synthesis of these proteins could be of concern. INTRODUCTION Protein ingestion cause a greater increase in heat production than carbohydrates or fat (Musharaf and Latshaw, 1999). Cheng et al. (1997) reported that providing heat-stressed broilers with dietary crude protein (CP) higher than their requirements is detrimental to weight gain, feed efficiency, and carcass composition. Thus, reduction of dietary protein in heat-stressed chickens with adequate fortification of several essential amino acids (EAA) may improve performance (Waldroup et al., 1976; McNaughton et al., 1978; Rahman et al., 2002; Zaman et al., 2008). On the contrary, other studies did not support the recommendation that low-CP diets are beneficial to poultry raised in a hot environment (Alleman and Leclercq, 1997; Temim et al., 2000). Buyse et al. (1992) reported that low-CP diets (15 vs. 20%) increased heat production and depressed growth performance in male broiler chickens. The authors attributed the heat increment in broilers fed a lower protein diet to elevated plasma triiodothyronine (T3) concentration, which may consequently increase heat production. Previous work on low-CP diets in broiler chickens emphasize on growth performance (Rahman et al., 2002; Zaman et al., 2008; Abudabos and Aljumaah, 2012), carcass characteristics (Temim et al., 2000; Bregendahl et al., 2002), nitrogen excretion (Namroud et al., 2008), and behavior (Wallenbeck et al., 2016). However, there is a dearth of information on the physiological stress response to heat exposure in chickens fed low-CP diets. Houshmand et al. (2012) demonstrated that low-CP diets with prebiotics supplementation had a negligible effect on physiological stress response (as measured by heterophil-to-lymphocyte ratios, and blood levels of corticosterone and glucose) in broiler chickens housed at a high stocking density. Acute phase proteins (APP) are a group of proteins that are primarily synthesized in the hepatocytes and released into the bloodstream by a variety of challenges such as bacterial infection, inflammation, tissue injury, endotoxin exposure, and neoplasia (Murata et al., 2004; O’reilly and Eckersall, 2014). The functions of the APP included protease inhibitors, enzymes, transport proteins, coagulation proteins, and modulators of the immune response. Alpha 1-acid glycoprotein (AGP) is a sialoglycoprotein produced and then secreted typically by hepatocytes. According to Fournier et al. (2000), AGP plays an important role in homeostasis maintenance by reducing any tissue damage related to the inflammatory response in extrahepatic cells. Ceruloplasmin (CPN) is a copper-containing ferroxidase that protects tissues from iron-mediated free radical injury by oxidation of toxic ferrous iron to nontoxic ferric form (Patel et al., 2002). Although ovotransferrin (OVT) is typically specified as a negative APP, there is some evidence that chicken serum OVT concentration increased in inflammation response (Tohjo et al., 1995). OVT may be included in the innate immune system by sequestration of ferric ions to prevent parasites and also pathogens from using nutrients (Law, 2002). Recent work in our laboratory showed that serum levels of APP were modified in chickens subjected to adverse non-inflammatory stimuli such as high temperature (Najafi et al., 2015; Olubodun et al., 2015), overcrowding (Shakeri et al., 2014; Najafi et al., 2015), and feed withdrawal (Najafi et al., 2016). Murata et al. (2004) suggested that APP may play a profound role in the restoration of homeostasis in animals with respect to non-inflammatory, psychophysical stressors. Living organisms respond to thermal and non-thermal stressors by synthesizing a group of highly conserved proteins known as heat shock proteins (HSP) (Soleimani et al., 2012a). It is well documented that HSP plays a profound role in modifying physiological stress response and in the acquisition of stress tolerance (Kregel, 2002). Work in chickens indicated that heat challenge (Liew et al., 2003; Zulkifli et al., 2003; Soleimani et al., 2011), feed restriction (Zulkifli et al., 2002; Soleimani et al., 2012a), road transportation (Al-Aqil and Zulkifli, 2009), crating (Zulkifli et al., 2009), and social isolation (Soleimani et al., 2012b) may elicit brain HSP 70 expression. There is no documented work on the effects of low-CP diets on APP and HSP 70 expression in heat-stressed poultry. Thus, the present work determined the effects of low-CP diets fortified with selected EAA on serum levels of APP, brain HSP 70 density, and growth performance in broiler chickens exposed to cyclic heat stress. MATERIALS AND METHODS Experimental design and bird husbandry The experiment was undertaken following the guidelines of the Research Policy on Animal Ethics of University Putra Malaysia. A total of 392 day-old male broiler chicks (Cobb 500) was obtained from a commercial hatchery and raised in 56 battery cages with wire floors (7 chicks/cage) in environmentally controlled rooms. The experiment was a completely randomized design consisting of 4 dietary groups and 2 temperatures with 7 replicates for each diet-temperature subgroup. For the first 2 d, the chicks were exposed to a brooding temperature of 33°C. The temperature was then gradually reduced to 23°C by d 21. Birds were fed ad libitum, and drinking water was always available. Dietary treatments Equal numbers of birds were subjected to one of the following dietary treatments: (1) 21.0 and 19% CP in starter and finisher diets, respectively (control), (2) 19.5 and 17.5% CP in starter and finisher diets, respectively (Diet A), (3) 18.0 and 16.0% CP in starter and finisher diets, respectively (Diet B), and (4) 16.5 and 14.5% CP in starter and finisher diets, respectively (Diet C). The starter and finisher diets were provided from d 1 to 21, and d 22 to 35, respectively. The diets were formulated to meet or exceed the recommended levels of lysine, methionine + cysteine, threonine, and tryptophan in each starter and finisher diet as suggested by the NRC (1994). The ME of starter and finisher diets were 3,000 and 3,150 kcal/kg, respectively. The ingredient and nutrient compositions of the experimental diets are shown in Table 1 and Table 2, respectively. Table 1. Ingredient composition (as-fed basis) of the experimental starter and finisher diets.   Starter (d 1 to 21)  Finisher (d 22 to 35)  Ingredient (%)  Control  Diet A   Diet B   Diet C   Control  Diet A   Diet B   Diet C     (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Corn  58.94  64.00  66.00  69.00  62.52  68.48  73.00  76.46  Soybean meal  34.00  29.60  25.50  21.15  26.99  22.13  18.20  13.65  Palm oil  3.00  2.00  2.40  2.30  4.92  3.65  2.76  2.56  Dicalcium phosphate  1.70  1.75  1.80  1.80  1.69  1.79  1.80  1.80  Limestone  1.20  1.25  1.24  1.25  1.49  1.26  1.60  1.50  Salt (NaCl)  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Sand  0.00  0.04  1.48  2.67  0.98  0.98  0.75  1.87  Vitamin premix1  0.05  0.05  0.05  0.05  0.05  0.05  0.05  0.05  Mineral premix2  0.25  0.25  0.25  0.25  0.25  0.25  0.25  0.25  L-Lysine.HCl  0.17  0.30  0.38  0.48  0.34  0.48  0.55  0.66  DL-Methionine  0.19  0.23  0.28  0.33  0.20  0.24  0.28  0.33  L-Threonine  0.00  0.03  0.10  0.18  0.07  0.15  0.21  0.29  L-Tryptophan  0.00  0.00  0.02  0.04  0.00  0.04  0.05  0.08    Starter (d 1 to 21)  Finisher (d 22 to 35)  Ingredient (%)  Control  Diet A   Diet B   Diet C   Control  Diet A   Diet B   Diet C     (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Corn  58.94  64.00  66.00  69.00  62.52  68.48  73.00  76.46  Soybean meal  34.00  29.60  25.50  21.15  26.99  22.13  18.20  13.65  Palm oil  3.00  2.00  2.40  2.30  4.92  3.65  2.76  2.56  Dicalcium phosphate  1.70  1.75  1.80  1.80  1.69  1.79  1.80  1.80  Limestone  1.20  1.25  1.24  1.25  1.49  1.26  1.60  1.50  Salt (NaCl)  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Sand  0.00  0.04  1.48  2.67  0.98  0.98  0.75  1.87  Vitamin premix1  0.05  0.05  0.05  0.05  0.05  0.05  0.05  0.05  Mineral premix2  0.25  0.25  0.25  0.25  0.25  0.25  0.25  0.25  L-Lysine.HCl  0.17  0.30  0.38  0.48  0.34  0.48  0.55  0.66  DL-Methionine  0.19  0.23  0.28  0.33  0.20  0.24  0.28  0.33  L-Threonine  0.00  0.03  0.10  0.18  0.07  0.15  0.21  0.29  L-Tryptophan  0.00  0.00  0.02  0.04  0.00  0.04  0.05  0.08  1Supplies per kg of the diet: vitamin A (retinyl acetate), 15,000.000 IU; vitamin D3 (cholecalciferol), 5,000.000 IU; vitamin E (dl-α-tocopherol), 50 g; vitamin K3 (menadione dimethylpyrimidinol), 10 mg; thiamin (thiamine mononitrate), 4 mg; riboflavin, 8 mg; pyridoxine, 5 mg; vitamin B12, 0.025 mg; niacin, 50 mg; D-calcium pantothenate, 20 mg; folic acid, 20 mg; biotin, 0.25 mg; ascorbic acid, 75 mg. 2Supplies per kg of the diet: Mn (manganese oxide), 100 mg; Zn (zinc sulfate monohydrate), 150 mg; Fe (ferrous sulfate monohydrate), 100 mg; Cu (cupric sulfate pentahydrate), 20 mg; I (calcium iodate), 1.5 mg; Co (cobalt carbonate), 0.5 mg; Se (sodium selenite), 0.2 mg; Mo (sodium molybdate), 1 mg; Mg (manganese sulfate monohydrate), 50 mg. View Large Table 2. Nutrient composition (%, unless stated otherwise) of the experimental starter and finisher diets.   Starter (d 1 to 21)  Finisher (d 22 to 35)  Item1  Control  Diet A  Diet B  Diet C  Control  Diet A  Diet B  Diet C    (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Calculated composition  ME (kcal/kg)  3000  3000  3000  3000  3150  3150  3150  3150  CP  20.96  19.56  18.02  16.49  19.02  17.50  16.18  14.55  ME:CP ratio  143  153  166  182  166  180  195  216  Ca  0.94  0.94  0.94  0.94  0.94  0.94  0.94  0.94  Available P  0.45  0.45  0.45  0.45  0.45  0.45  0.45  0.45  Supplemental AA (% of Lys)2  Lys  100  100  100  100  100  100  100  100  Met + Cys  74  74  74  74  68  68  68  68  Thr  67  67  67  67  66  66  66  66  Trp  18  18  18  18  17  17  17  17  Analyzed composition  CP  21.30  20.19  18.20  16.55  19.10  17.96  16.17  14.71  Lys  1.23  1.24  1.19  1.22  1.24  1.18  1.23  1.24  Met  0.45  0.43  0.46  0.45  0.44  0.43  0.45  0.45  Thr  0.81  0.82  0.84  0.83  0.82  0.82  0.81  0.83  Trp  0.24  0.23  0.22  0.21  0.23  0.21  0.19  0.19  Arg  1.44  1.31  1.17  1.03  1.21  1.07  0.94  0.80  Val  1.09  1.00  0.91  0.83  0.93  0.84  0.76  0.67  Ile  0.92  0.84  0.75  0.66  0.77  0.68  0.61  0.53  Leu  1.91  1.82  1.68  1.55  1.69  1.58  1.49  1.36  Phe  1.03  0.94  0.85  0.76  0.87  0.79  0.73  0.64  His  0.59  0.56  0.52  0.46  0.53  0.48  0.43  0.38  Gly  0.92  0.83  0.78  0.70  0.80  0.72  0.65  0.57  Ser  1.04  0.95  0.88  0.79  0.90  0.81  0.75  0.66  Cys  0.37  0.34  0.31  0.29  0.31  0.29  0.26  0.24  Tyr  0.55  0.50  0.44  0.40  0.46  0.42  0.37  0.31  Pro  1.31  1.26  1.18  1.07  1.19  1.11  1.04  0.96  Ala  1.07  1.00  0.91  0.87  0.95  0.89  0.84  0.77  Glu  3.26  3.08  2.85  2.58  2.89  2.65  2.45  2.20  Asp  2.15  1.92  1.72  1.51  1.77  1.56  1.38  1.16    Starter (d 1 to 21)  Finisher (d 22 to 35)  Item1  Control  Diet A  Diet B  Diet C  Control  Diet A  Diet B  Diet C    (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Calculated composition  ME (kcal/kg)  3000  3000  3000  3000  3150  3150  3150  3150  CP  20.96  19.56  18.02  16.49  19.02  17.50  16.18  14.55  ME:CP ratio  143  153  166  182  166  180  195  216  Ca  0.94  0.94  0.94  0.94  0.94  0.94  0.94  0.94  Available P  0.45  0.45  0.45  0.45  0.45  0.45  0.45  0.45  Supplemental AA (% of Lys)2  Lys  100  100  100  100  100  100  100  100  Met + Cys  74  74  74  74  68  68  68  68  Thr  67  67  67  67  66  66  66  66  Trp  18  18  18  18  17  17  17  17  Analyzed composition  CP  21.30  20.19  18.20  16.55  19.10  17.96  16.17  14.71  Lys  1.23  1.24  1.19  1.22  1.24  1.18  1.23  1.24  Met  0.45  0.43  0.46  0.45  0.44  0.43  0.45  0.45  Thr  0.81  0.82  0.84  0.83  0.82  0.82  0.81  0.83  Trp  0.24  0.23  0.22  0.21  0.23  0.21  0.19  0.19  Arg  1.44  1.31  1.17  1.03  1.21  1.07  0.94  0.80  Val  1.09  1.00  0.91  0.83  0.93  0.84  0.76  0.67  Ile  0.92  0.84  0.75  0.66  0.77  0.68  0.61  0.53  Leu  1.91  1.82  1.68  1.55  1.69  1.58  1.49  1.36  Phe  1.03  0.94  0.85  0.76  0.87  0.79  0.73  0.64  His  0.59  0.56  0.52  0.46  0.53  0.48  0.43  0.38  Gly  0.92  0.83  0.78  0.70  0.80  0.72  0.65  0.57  Ser  1.04  0.95  0.88  0.79  0.90  0.81  0.75  0.66  Cys  0.37  0.34  0.31  0.29  0.31  0.29  0.26  0.24  Tyr  0.55  0.50  0.44  0.40  0.46  0.42  0.37  0.31  Pro  1.31  1.26  1.18  1.07  1.19  1.11  1.04  0.96  Ala  1.07  1.00  0.91  0.87  0.95  0.89  0.84  0.77  Glu  3.26  3.08  2.85  2.58  2.89  2.65  2.45  2.20  Asp  2.15  1.92  1.72  1.51  1.77  1.56  1.38  1.16  1CP, crude protein; ME, metabolizable energy; Ca, calcium; P, Phosphorus. 2Ratios based on a total requirement basis (NRC, 1994). View Large Heat treatment To elicit heat stress from 22 to 35 d of age, 7 cages of chicks from each diet were exposed to ambient temperature of 33 ± 1°C for 6 h per day. Relative humidity was not controlled but ranged from 70 to 80%. The time taken for the ambient temperature to rise from 24 to 33°C was about 30 minutes. Feed and water were provided ad libitum throughout heat treatment. Data collection and sampling Body weight and feed intake (FI) were recorded on d1, 21, and 35. Weight gains (WG) and feed conversion ratios (FCR) from d 1 to 21 (starter period), d 22 to 35 (finisher period), and d 1 to 35 (overall period) were calculated accordingly. The mortality was recorded as it occurred. On d 35, 2 birds from each cage (14 birds per diet-temperature subgroup) were randomly selected, weighed, and humanely slaughtered according to halal slaughter procedure as outlined in the Malaysian Standard, MS1500 (Department of Standards Malaysia, 2009), for blood and brain sampling. Blood samples were collected in plain tubes for determination of CPN, OVT, and AGP. Blood samples for APP assay were centrifuged, and the serum was separated and stored at -80°C for further analysis. Immediately after blood sampling, brain samples were collected to measure HSP 70 density. The brain samples were immediately frozen in liquid nitrogen and kept at −80°C for further analysis. Laboratory analyses Crude protein and amino acids. The CP in feed ingredients and experimental diets was determined according to the procedure of AOAC (1990), whereas the AA contents were determined using high-performance liquid chromatography (HPLC) as described previously (Awad et al., 2014b). Acute phase proteins. The OVT concentration was measured as described by Najafi et al. (2015), using a modified method from Mancini et al. (1965) (radial-immunodiffusion method). The CPN was determined by the rate of formation of a colored product from CPN and the substrate, 1,4-phenylenediamine dihydrochloride (Najafi et al., 2015). AGP concentration was measured using a commercial ELISA kit specific to chicken (Life Diagnostics Inc., West Chester, PA). Heat shock protein 70. The level of HSP 70 protein expression was determined as previously described by (Soleimani et al., 2012a), using SDS-PAGE and Western Blotting with some modifications. Briefly, around 0.3 g of brain samples was homogenized with 1.5 mL of protein extraction buffer (20 mM Tris, pH 7.5; 0.75 M sodium chloride) and 10 μL/mL of protease inhibitor cocktail (P8340, Sigma Chemical Co., St. Louis, MO), and centrifuged at 20,000 × g for 30 min at 4°C. The supernatant was separated, and the total protein quantity was measured using bicinchoninic acid protein assay kit (Sigma Chemical Co., St. Louis, MO). The final brain HSP 70 concentration was calculated as an arbitrary unit of band density relative to the total protein concentration of each sample. Statistical analysis Results were evaluated by analysis of variance using SAS software (SAS, 1991). Pens served as the experimental units for FI, WG, and FCR, while individual birds served as the experimental units for APP and HSP 70. Orthogonal polynomial contrasts were applied to determine the linear and quadratic effects of lowering dietary protein level. Data of FI, WG, and FCR (d 22 to 35 and d 1 to 35), APP, and HSP 70 were analyzed using 2-way ANOVA in a 4 × 2 factorial arrangement with diet, heat treatment, and their interactions as the main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. Mortality data were subjected to chi-squared analysis. Statistical significance is considered at P ≤ 0.05. RESULTS Table 3 shows the FI, WG, and FCR results during the starter period (d 1 to 21) as affected by diet. Lowering CP linearly reduced FI (P = 0.021) and WG (P = 0.009) without affecting FCR of birds. Table 4 shows the FI, WG, and FCR results as affected by diet and heat treatment during the finisher (d 22 to 35) and overall (d 1 to 35) periods. There were a significant diet x temperature interactions for both finisher (P = 0.040) and overall (P = 0.049) FCR (Table 4). During the finisher period, diet had neither a linear (P = 0.103) nor a quadratic (P = 0.066) effect on FCR among the unheated birds (Table 5). Under the heated condition, however, FCR was linearly (P = 0.007) increased as dietary CP level decreased. There were no significant differences in overall FCR between the heated and unheated birds fed the control diet, Diet A, or Diet B. However, heated birds provided Diet C had poorer (P = 0.041) FCR compared to their unheated counterparts (Table 5). Irrespective of temperature, lowering dietary CP led to a linear decrease in FI during the finisher period (P = 0.032), FI during the overall period (P = 0.039), WG during the finisher period (P < 0.001), and WG during the overall period (P < 0.001) (Table 4). Irrespective of diet, heated birds had significantly lower FI during the finisher period (P = 0.010), FI during the overall period (P = 0.017), WG during the finisher period (P < 0.001), and WG during the overall period (P < 0.001) (Table 4). Diet had no significant effect on mortality rates among unheated birds. However, under heated condition, the mortality rate of the group fed the control diet was significantly (P < 0.05) higher than the groups fed Diet B and Diet C (Table 5). Table 3. Effect of diet on feed intake, weight gain, and feed conversion ratio (FCR) of broiler chickens at 21 d of age.   Feed intake  Weight gain  FCR  Treatment  (g/bird)  (g/bird)  (feed/gain)    (d 1 to 21)  (d 1 to 21)  (d 1 to 21)  Diet  Control (21.0% CP)  1127  701  1.61  Diet A (19.5% CP)  1129  703  1.61  Diet B (18.0% CP)  1131  675  1.68  Diet C (16.5% CP)  1076  659  1.63  SEM1  49  41  0.08  Contrast, P-values  Linear  0.021  0.009  0.411  Quadratic  0.155  0.718  0.057    Feed intake  Weight gain  FCR  Treatment  (g/bird)  (g/bird)  (feed/gain)    (d 1 to 21)  (d 1 to 21)  (d 1 to 21)  Diet  Control (21.0% CP)  1127  701  1.61  Diet A (19.5% CP)  1129  703  1.61  Diet B (18.0% CP)  1131  675  1.68  Diet C (16.5% CP)  1076  659  1.63  SEM1  49  41  0.08  Contrast, P-values  Linear  0.021  0.009  0.411  Quadratic  0.155  0.718  0.057  1SEM = Standard error of the mean for diet effect (n = 14). View Large Table 4. Effect of diet1 and heat treatment2 on feed intake, weight gain, and feed conversion ratio (FCR) of broiler chickens at 35 d of age.   Feed intake (g/bird)  Weight gain (g/bird)  FCR (feed/gain)    Finisher  Overall  Finisher  Overall  Finisher  Overall  Treatment  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  Diet  Control (21.0/19.0% CP)  2120  3247  1208  1909  1.75  1.71  Diet A (19.5/17.5% CP)  2141  3270  1146  1849  1.87  1.77  Diet B (18.0/16.0% CP)  2022  3153  1022  1697  1.98  1.86  Diet C (16.5/14.5% CP)  1982  3058  1006  1665  1.97  1.84  SEM3  131  186  94  111  0.15  0.11  Contrast, P-values  Linear  0.032  0.039  <.001  <.001  0.077  0.094  Quadratic  0.508  0.880  0.007  0.009  0.013  0.016  Heat treatment  Unheated  2148  3253  1157  1841  1.84  1.77  Heated  1992  3111  1034  1718  1.95  1.81  SEM4  85  97  62  75  0.08  0.07  Source of variation  Diet (D)  0.044  0.048  <.001  <.001  <.001  <.001  Heat treatment (H)  0.010  0.017  <.001  <.001  0.039  0.609  D × H  0.761  0.812  0.196  0.142  0.040  0.049    Feed intake (g/bird)  Weight gain (g/bird)  FCR (feed/gain)    Finisher  Overall  Finisher  Overall  Finisher  Overall  Treatment  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  Diet  Control (21.0/19.0% CP)  2120  3247  1208  1909  1.75  1.71  Diet A (19.5/17.5% CP)  2141  3270  1146  1849  1.87  1.77  Diet B (18.0/16.0% CP)  2022  3153  1022  1697  1.98  1.86  Diet C (16.5/14.5% CP)  1982  3058  1006  1665  1.97  1.84  SEM3  131  186  94  111  0.15  0.11  Contrast, P-values  Linear  0.032  0.039  <.001  <.001  0.077  0.094  Quadratic  0.508  0.880  0.007  0.009  0.013  0.016  Heat treatment  Unheated  2148  3253  1157  1841  1.84  1.77  Heated  1992  3111  1034  1718  1.95  1.81  SEM4  85  97  62  75  0.08  0.07  Source of variation  Diet (D)  0.044  0.048  <.001  <.001  <.001  <.001  Heat treatment (H)  0.010  0.017  <.001  <.001  0.039  0.609  D × H  0.761  0.812  0.196  0.142  0.040  0.049  1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. 3SEM = Standard error of the mean for diet effect (n = 14). 4SEM = Standard error of the mean for heat treatment effect (n = 28). View Large Table 5. Mean feed conversion ratios (FCR) (feed/gain) during the finisher and overall periods where diet1 x heat treatment2 interactions were significant, and mortality rate (%) during the finisher period.     Heat treatment      Item  Diet  Unheated  Heated  SEM  P-value  Finisher FCR (d 22 to 35)  Control (19.0% CP)  1.71  1.79  0.15  0.082    Diet A (17.5% CP)  1.86  1.88  0.16  0.106    Diet B (16.0% CP)  1.93  2.03  0.11  0.153    Diet C (14.5% CP)  1.83  2.11  0.18  0.009    SEM  0.19  0.11        Linear  0.103  0.007        Quadratic  0.066  0.272      Overall FCR (d 1 to 35)  Control (21.0/19.0% CP)  1.67  1.72  0.12  0.380    Diet A (19.5/17.5% CP)  1.80  1.74  0.13  0.442    Diet B (18.0/16.0% CP)  1.85  1.86  0.07  0.804    Diet C (16.5/14.5% CP)  1.76  1.92  0.14  0.041    SEM  0.15  0.08        Linear  0.048  0.015        Quadratic  0.058  0.380      Finisher mortality (d 22 to 35)  Control (19.0% CP)  2.86  8.57a  3.54  >0.05    Diet A (17.5% CP)  0.00  2.86a,b  2.02  >0.05    Diet B (16.0% CP)  0.00  0.00b  0.00  >0.05    Diet C (14.5% CP)  0.00  0.00b  0.00  >0.05    SEM  0.71  1.35        P-value  >0.05  <0.05          Heat treatment      Item  Diet  Unheated  Heated  SEM  P-value  Finisher FCR (d 22 to 35)  Control (19.0% CP)  1.71  1.79  0.15  0.082    Diet A (17.5% CP)  1.86  1.88  0.16  0.106    Diet B (16.0% CP)  1.93  2.03  0.11  0.153    Diet C (14.5% CP)  1.83  2.11  0.18  0.009    SEM  0.19  0.11        Linear  0.103  0.007        Quadratic  0.066  0.272      Overall FCR (d 1 to 35)  Control (21.0/19.0% CP)  1.67  1.72  0.12  0.380    Diet A (19.5/17.5% CP)  1.80  1.74  0.13  0.442    Diet B (18.0/16.0% CP)  1.85  1.86  0.07  0.804    Diet C (16.5/14.5% CP)  1.76  1.92  0.14  0.041    SEM  0.15  0.08        Linear  0.048  0.015        Quadratic  0.058  0.380      Finisher mortality (d 22 to 35)  Control (19.0% CP)  2.86  8.57a  3.54  >0.05    Diet A (17.5% CP)  0.00  2.86a,b  2.02  >0.05    Diet B (16.0% CP)  0.00  0.00b  0.00  >0.05    Diet C (14.5% CP)  0.00  0.00b  0.00  >0.05    SEM  0.71  1.35        P-value  >0.05  <0.05      1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. SEM = Standard error of the mean for diet and heat treatment effect (n = 7). a,bMeans within a column with no common superscripts are significantly different at P < 0.05. View Large Table 6 shows the serum concentrations of AGP, OVT, CPN, and density of HSP 70 in broiler chickens as affected by diet and heat treatment at 35 d of age. Significant diet x heat treatment interactions were noted for serum AGP (P = 0.008) and OVT (P = 0.005) levels. Heat treatment did not affect serum levels of AGP or OVT in birds fed Diet A, Diet C, or control diet. However, for those fed Diet B, the serum concentrations of AGP and OVT were significantly (P < 0.001 and P = 0.002, respectively) higher in heated birds compared to their unheated counterparts (Table 7). Under both heated and unheated conditions, lowering dietary CP level resulted in a significant (P < 0.05) linear reduction in AGP and OVT (Table 7). Both CPN and brain HSP 70 density were not affected by diet (Table 6). However, irrespective of diet, heated birds showed higher CPN (P = 0.001) and HSP 70 density (P = 0.042) than their unheated counterparts. Table 6. Effect of diet1 and heat treatment2 on serum α-1 acid glycoprotein (AGP), ovotransferrin (OVT), ceruloplasmin (CPN), and heat shock protein (HSP) 70 densities of broiler chickens at 35 d of age. Treatment  AGP  OVT  CPN  HSP70    (mg/mL)  (mg/mL)  (mg/mL)  density  Diet  Control (21.0/19.0% CP)  7.649  0.443  2.096  1.588  Diet A (19.5/17.5% CP)  7.712  0.451  1.851  1.549  Diet B (18.0/16.0% CP)  7.272  0.423  1.916  1.478  Diet C (16.5/14.5% CP)  6.476  0.352  2.001  1.639  SEM3  0.714  0.062  0.337    Contrast, P-values  Linear  0.017  0.013  0.385  0.189  Quadratic  0.221  0.189  0.548  0.331  Heat treatment  Unheated  6.640  0.396  1.779  1.501  Heated  7.914  0.439  2.153  1.626  SEM4  0.424  0.033  0.196    Source of variation  Diet (D)  0.008  0.006  0.124  0.289  Heat treatment (H)  0.001  0.016  0.001  0.042  D × H  0.008  0.005  0.177  0.159  Treatment  AGP  OVT  CPN  HSP70    (mg/mL)  (mg/mL)  (mg/mL)  density  Diet  Control (21.0/19.0% CP)  7.649  0.443  2.096  1.588  Diet A (19.5/17.5% CP)  7.712  0.451  1.851  1.549  Diet B (18.0/16.0% CP)  7.272  0.423  1.916  1.478  Diet C (16.5/14.5% CP)  6.476  0.352  2.001  1.639  SEM3  0.714  0.062  0.337    Contrast, P-values  Linear  0.017  0.013  0.385  0.189  Quadratic  0.221  0.189  0.548  0.331  Heat treatment  Unheated  6.640  0.396  1.779  1.501  Heated  7.914  0.439  2.153  1.626  SEM4  0.424  0.033  0.196    Source of variation  Diet (D)  0.008  0.006  0.124  0.289  Heat treatment (H)  0.001  0.016  0.001  0.042  D × H  0.008  0.005  0.177  0.159  1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. 3SEM = Standard error of the mean for diet effect (n = 28). 4SEM = Standard error of the mean for heat treatment effect (n = 56). View Large Table 7. Mean serum α-1 acid glycoprotein (AGP) and ovotransferrin (OVT) of broiler chickens where diet1 x heat treatment2 interactions were significant at 35 d of age.     Heat treatment      Item  Diet  Unheated  Heated  SEM3  P-value  AGP (mg/mL)  Control (21.0/19.0% CP)  7.225  8.043  1.03  0.077    Diet A (19.5/17.5% CP)  7.106  8.319  1.26  0.062    Diet B (18.0/16.0% CP)  5.844  8.700  1.31  <.001    Diet C (16.5/14.5% CP)  6.357  6.595  0.95  0.103    SEM3  1.062  1.211        Linear  0.045  0.009        Quadratic  0.084  0.606      OVT (mg/mL)  Control (21.0/19.0% CP)  0.421  0.466  0.06  0.058    Diet A (19.5/17.5% CP)  0.446  0.456  0.04  0.113    Diet B (18.0/16.0% CP)  0.350  0.496  0.08  0.002    Diet C (16.5/14.5% CP)  0.366  0.337  0.05  0.093    SEM3  0.05  0.07        Linear  0.003  0.010        Quadratic  0.011  0.013          Heat treatment      Item  Diet  Unheated  Heated  SEM3  P-value  AGP (mg/mL)  Control (21.0/19.0% CP)  7.225  8.043  1.03  0.077    Diet A (19.5/17.5% CP)  7.106  8.319  1.26  0.062    Diet B (18.0/16.0% CP)  5.844  8.700  1.31  <.001    Diet C (16.5/14.5% CP)  6.357  6.595  0.95  0.103    SEM3  1.062  1.211        Linear  0.045  0.009        Quadratic  0.084  0.606      OVT (mg/mL)  Control (21.0/19.0% CP)  0.421  0.466  0.06  0.058    Diet A (19.5/17.5% CP)  0.446  0.456  0.04  0.113    Diet B (18.0/16.0% CP)  0.350  0.496  0.08  0.002    Diet C (16.5/14.5% CP)  0.366  0.337  0.05  0.093    SEM3  0.05  0.07        Linear  0.003  0.010        Quadratic  0.011  0.013      1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. 3SEM = Standard error of the mean for diet and heat treatment effect (n = 14). View Large DISCUSSION The present findings suggested that the growth performance of broiler chickens was negatively affected by dietary CP level reduction from 21.0 to 16.5% and from 19.0 to 14.5% in the starter and finisher diets, respectively, despite meeting or exceeding recommended levels of those commercially available feed-grade AA (namely L-lysine HCl, DL-methionine, L-threonine, and L-tryptophan). These findings concur with previous work (Pinchasov et al., 1990; Aletor et al., 2000; Bregendahl et al., 2002; Si et al., 2004; Namroud et al., 2008; Awad et al., 2014a) that growth performance was depressed in broilers fed diets in which CP has been reduced by more than 3%, even when all EAA requirements are met. Lower FI in birds fed low-CP diets also has been reported by Si et al. (2004), Namroud et al. (2008), and Awad et al. (2014a). Indeed, reduction of dietary CP will reduce both EAA and non-essential amino acids (NEAA), which alter the concentration/balance of the AA in such low-CP diets. This change in concentration/balance of AA can negatively affect the FI in broilers (Aftab et al., 2006). In birds fed low-CP diets, the amount of ingested free AA into the blood stream increase and may affect the balance of plasma AA profile (Aftab et al., 2006). An imbalanced AA profile in plasma has been shown to reduce FI (Austic et al., 2000). On the contrary, other workers (Bregendahl et al., 2002; Fatufe and Rodehutscord, 2005) indicated no differences or even higher (Aletor et al., 2000) FI when birds were fed low-CP, AA-supplemented diets. Depressed performance in broilers fed the low-CP diets could be associated with insufficient nitrogen quantity for NEAA synthesis. Chickens fed the standard levels of dietary protein can synthesize NEAA in the body from excess EAA. Thus, when low-CP diets are provided, this excess is reduced, leaving less EAA available for conversion to the NEAA (Waldroup, 2007). Findings on protein requirements of broilers under hot environment have been inconsistent. Work by Temim et al. (2000) and Gonzalez-Esquerra and Leeson (2005) showed that the higher levels of dietary CP may compensate the reduction in feed and protein associated with heat stress. Conversely, Cheng et al. (1997) noted adverse effects of high-protein diets (22 vs. 24% CP) on growth performance in 3- to 6-week-old heat-stressed birds. In the current study, however, it appears that low-CP diets were more detrimental to the performance of heat-stressed broilers. A possible explanation for this might be the insufficiency in EAA, due either to lower ingested quantities or decreased AA digestibility in heat-stressed birds (Bonnet et al., 1997). One of the advantages of the feeding low-CP diet in the current study was increase in survivability of heat-stressed birds. Birds fed Diet B and Diet C had significantly lower mortality rates than their control counterparts during the heat treatment period. Heavier birds were more susceptible to heat stress (Chwalibog and Eggum, 1989; Cahaner and Leenstra, 1992) than lighter ones because they had more internally generated heat to dissipate and consequently they may have difficulty maintaining body temperature at high ambient temperature. However, in the present study, the improved survivability rate among those fed low-CP diets may not be associated with body weight because the mortality rates of broilers fed Diet A and Diet B were lower than controls despite all 3 groups having almost a similar body weight. In the present study, irrespective of diet, heat challenge increased CPN. However, the effect of heat treatment on AGP and OVT varied according to diet. Only heated birds that were fed Diet B had higher AGP and OVT than their unheated counterparts. In general, it appears that under both unheated and heated conditions, AGP and OVT decreased with low-CP diets. Studies in laboratory animals indicated that the ability to elicit a hepatic response to inflammatory stimuli was reduced by low-protein diets (Bell and Hoffman-Goetz, 1983; Jennings and Elia, 1990; Grimble et al., 1992; Jennings et al., 1992). Grimble et al. (1992) suggested that in a malnourished organism, the availability of sulfur amino acids may influence antioxidant defenses and thereby affect the pattern of APP synthesis in an indirect manner. To the best of our knowledge, this is the first work showing a low-protein diet may dampen synthesis of APP in the avian species. APP function as protease inhibitors, enzymes, transport proteins, coagulation proteins, and modulators of the immune response. The acute phase response (APR) is considered as part of the innate immune response and is observed across all animal species (Cray et al., 2009). The APR induce a complex systemic reaction to re-establish homeostasis and promote healing (Cray et al., 2009). An increase in serum APP concentrations may be considered as an indicator of intracellular communication, suggesting an increase in the cellular immune response (Cray et al., 2009). Hence, the possible impairment of APP synthesis associated with low-CP diets is of concern and should be considered before recommending such diets for broilers. It is interesting to note that although the chicken provided with low-CP diets had diminished APP reactions, they had better survivability than those fed diets with adequate levels of CP. Payne et al. (1990) reported that protein deficiency reduced immunocompetence in chickens as assessed by antibody titers, white blood cell counts, and T-cell activity. However, the authors noted that dietary modifications had negligible influence on survival following challenge with Pasteurella multocida. Thus, it appears that impaired immune response may not be associated with a higher mortality rate. Irrespective of diet, the heat treatment increased CPN. However, elevations in AGP and OVT were noted only for those fed Diet B. Elicitation of AGP, OVT, and CPN reactions to the hot environment in broiler chickens have been demonstrated by Najafi et al. (2015) and Olubodun et al. (2015). There is no clear explanation for the lack of heat treatment effect on birds fed control, Diet A, or Diet C. The findings, however, suggested that APP response to heat may vary according to the levels of dietary protein. The present findings confirmed earlier work (Liew et al., 2003; Zulkifli et al., 2003; Soleimani et al., 2011) that heat treatment can increase brain HSP 70 expression in chickens. Olubodun et al. (2015), and Zulkifli et al. (2016) reported that dietary L-glutamine and L-glutamate supplementation augmented HSP 70 density in chicks. The current study suggested that low-CP diets had a negligible effect on brain HSP 70 density in broiler chickens. Work in cattle indicated that providing a low-protein diet for 3 mo downregulated HSP 70 and HSP 90 expression in fat tissue (Eitam et al., 2012). In vitro work suggested that AA deprivation resulted in inactivation of the heat shock factor 1 (Hensen et al., 2012) The activation of heat shock gene transcription during the stress response is mediated by heat shock transcription factor, which binds to heat shock elements in the promoters of heat shock genes (Amin et al., 1988; Abravaya et al., 1991). The lack of low-CP diets effect on HSP 70 expression in the present work could be attributed to the adequate levels of lysine, methionine, threonine, and tryptophan in the diets. In conclusion, despite meeting the requirements of the essential limiting AA, feeding broilers with starter and finisher low-CP diets negatively affected growth performance compared to those provided standard CP diet. Low-CP diets seemed to be beneficial in improving survivability rate but not the performance of heat-stressed broiler chickens. Under both unheated and heated conditions, low-CP diets tended to impair AGP and OVT responses. Because APP contributes to non-specific innate immune response and restoration of homeostasis, the practice of feeding low-CP diets to broilers could be of concern. Low-CP diets, however, had negligible influence on brain HSP 70 density. Acknowledgements This work was supported by the Malaysian Ministry of Higher Education under the Long-term Research Grant Scheme. REFERENCES Abravaya K., Phillips B., Morimoto R. I.. 1991. 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Effects of low-protein diets on acute phase proteins and heat shock protein 70 responses, and growth performance in broiler chickens under heat stress condition

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pex436
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Abstract

Abstract A study with a 4 × 2 factorial arrangement was conducted to investigate the effects of 4 dietary protein levels and 2 environmental conditions on acute phase proteins (APP), brain heat shock protein (HSP) 70 density, and growth performance of broiler chickens. Day-old broiler chicks (Cobb 500) were fed isocaloric diets but with various levels of crude protein (CP), namely, (1) 21.0 and 19.0% CP in starter and finisher diets, respectively (control), (2) 19.5 and 17.5% CP in starter and finisher diets, respectively (Diet A), (3) 18.0 and 16.0% CP in starter and finisher diets, respectively (Diet B), and (4) 16.5 and 14.5% CP in starter and finisher diets, respectively (Diet C). Equal numbers of birds from each diet were subjected to either 23±1°C throughout or 33±1°C for 6 h per d from 22 to 35 d of age. From d 1 to 21, feed intake (FI) and weight gain (WG) decreased linearly (P = 0.021 and P = 0.009, respectively), as CP level was reduced. During the heat treatment period (d 22 to 35), there were significant (P = 0.04) diet × heat treatment interactions for FCR. Diet had no effect on FCR among the unheated birds, but the ratio increased linearly (P = 0.007) as dietary CP level decreased. Irrespective of ambient temperature, there was a significant linear decrease in FI (P = 0.032) and WG (P < 0.001) as dietary CP level decreased. Low-CP diets improved the survivability of heat-stressed broilers when compared to those fed control diets. Low-CP diets linearly decreased (P < 0.01) APP (ovotransferrin and alpha-acid glycoprotein) responses. Both APP and HSP 70 reactions were elevated following heat treatment. In conclusion, feeding broilers with low-CP diets adversely affect the growth performance of broilers under heat stress condition. However, low-CP diets were beneficial in improving the survivability. Because APP are involved in the restoration of homeostasis, the adverse effect of low-CP diet on the synthesis of these proteins could be of concern. INTRODUCTION Protein ingestion cause a greater increase in heat production than carbohydrates or fat (Musharaf and Latshaw, 1999). Cheng et al. (1997) reported that providing heat-stressed broilers with dietary crude protein (CP) higher than their requirements is detrimental to weight gain, feed efficiency, and carcass composition. Thus, reduction of dietary protein in heat-stressed chickens with adequate fortification of several essential amino acids (EAA) may improve performance (Waldroup et al., 1976; McNaughton et al., 1978; Rahman et al., 2002; Zaman et al., 2008). On the contrary, other studies did not support the recommendation that low-CP diets are beneficial to poultry raised in a hot environment (Alleman and Leclercq, 1997; Temim et al., 2000). Buyse et al. (1992) reported that low-CP diets (15 vs. 20%) increased heat production and depressed growth performance in male broiler chickens. The authors attributed the heat increment in broilers fed a lower protein diet to elevated plasma triiodothyronine (T3) concentration, which may consequently increase heat production. Previous work on low-CP diets in broiler chickens emphasize on growth performance (Rahman et al., 2002; Zaman et al., 2008; Abudabos and Aljumaah, 2012), carcass characteristics (Temim et al., 2000; Bregendahl et al., 2002), nitrogen excretion (Namroud et al., 2008), and behavior (Wallenbeck et al., 2016). However, there is a dearth of information on the physiological stress response to heat exposure in chickens fed low-CP diets. Houshmand et al. (2012) demonstrated that low-CP diets with prebiotics supplementation had a negligible effect on physiological stress response (as measured by heterophil-to-lymphocyte ratios, and blood levels of corticosterone and glucose) in broiler chickens housed at a high stocking density. Acute phase proteins (APP) are a group of proteins that are primarily synthesized in the hepatocytes and released into the bloodstream by a variety of challenges such as bacterial infection, inflammation, tissue injury, endotoxin exposure, and neoplasia (Murata et al., 2004; O’reilly and Eckersall, 2014). The functions of the APP included protease inhibitors, enzymes, transport proteins, coagulation proteins, and modulators of the immune response. Alpha 1-acid glycoprotein (AGP) is a sialoglycoprotein produced and then secreted typically by hepatocytes. According to Fournier et al. (2000), AGP plays an important role in homeostasis maintenance by reducing any tissue damage related to the inflammatory response in extrahepatic cells. Ceruloplasmin (CPN) is a copper-containing ferroxidase that protects tissues from iron-mediated free radical injury by oxidation of toxic ferrous iron to nontoxic ferric form (Patel et al., 2002). Although ovotransferrin (OVT) is typically specified as a negative APP, there is some evidence that chicken serum OVT concentration increased in inflammation response (Tohjo et al., 1995). OVT may be included in the innate immune system by sequestration of ferric ions to prevent parasites and also pathogens from using nutrients (Law, 2002). Recent work in our laboratory showed that serum levels of APP were modified in chickens subjected to adverse non-inflammatory stimuli such as high temperature (Najafi et al., 2015; Olubodun et al., 2015), overcrowding (Shakeri et al., 2014; Najafi et al., 2015), and feed withdrawal (Najafi et al., 2016). Murata et al. (2004) suggested that APP may play a profound role in the restoration of homeostasis in animals with respect to non-inflammatory, psychophysical stressors. Living organisms respond to thermal and non-thermal stressors by synthesizing a group of highly conserved proteins known as heat shock proteins (HSP) (Soleimani et al., 2012a). It is well documented that HSP plays a profound role in modifying physiological stress response and in the acquisition of stress tolerance (Kregel, 2002). Work in chickens indicated that heat challenge (Liew et al., 2003; Zulkifli et al., 2003; Soleimani et al., 2011), feed restriction (Zulkifli et al., 2002; Soleimani et al., 2012a), road transportation (Al-Aqil and Zulkifli, 2009), crating (Zulkifli et al., 2009), and social isolation (Soleimani et al., 2012b) may elicit brain HSP 70 expression. There is no documented work on the effects of low-CP diets on APP and HSP 70 expression in heat-stressed poultry. Thus, the present work determined the effects of low-CP diets fortified with selected EAA on serum levels of APP, brain HSP 70 density, and growth performance in broiler chickens exposed to cyclic heat stress. MATERIALS AND METHODS Experimental design and bird husbandry The experiment was undertaken following the guidelines of the Research Policy on Animal Ethics of University Putra Malaysia. A total of 392 day-old male broiler chicks (Cobb 500) was obtained from a commercial hatchery and raised in 56 battery cages with wire floors (7 chicks/cage) in environmentally controlled rooms. The experiment was a completely randomized design consisting of 4 dietary groups and 2 temperatures with 7 replicates for each diet-temperature subgroup. For the first 2 d, the chicks were exposed to a brooding temperature of 33°C. The temperature was then gradually reduced to 23°C by d 21. Birds were fed ad libitum, and drinking water was always available. Dietary treatments Equal numbers of birds were subjected to one of the following dietary treatments: (1) 21.0 and 19% CP in starter and finisher diets, respectively (control), (2) 19.5 and 17.5% CP in starter and finisher diets, respectively (Diet A), (3) 18.0 and 16.0% CP in starter and finisher diets, respectively (Diet B), and (4) 16.5 and 14.5% CP in starter and finisher diets, respectively (Diet C). The starter and finisher diets were provided from d 1 to 21, and d 22 to 35, respectively. The diets were formulated to meet or exceed the recommended levels of lysine, methionine + cysteine, threonine, and tryptophan in each starter and finisher diet as suggested by the NRC (1994). The ME of starter and finisher diets were 3,000 and 3,150 kcal/kg, respectively. The ingredient and nutrient compositions of the experimental diets are shown in Table 1 and Table 2, respectively. Table 1. Ingredient composition (as-fed basis) of the experimental starter and finisher diets.   Starter (d 1 to 21)  Finisher (d 22 to 35)  Ingredient (%)  Control  Diet A   Diet B   Diet C   Control  Diet A   Diet B   Diet C     (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Corn  58.94  64.00  66.00  69.00  62.52  68.48  73.00  76.46  Soybean meal  34.00  29.60  25.50  21.15  26.99  22.13  18.20  13.65  Palm oil  3.00  2.00  2.40  2.30  4.92  3.65  2.76  2.56  Dicalcium phosphate  1.70  1.75  1.80  1.80  1.69  1.79  1.80  1.80  Limestone  1.20  1.25  1.24  1.25  1.49  1.26  1.60  1.50  Salt (NaCl)  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Sand  0.00  0.04  1.48  2.67  0.98  0.98  0.75  1.87  Vitamin premix1  0.05  0.05  0.05  0.05  0.05  0.05  0.05  0.05  Mineral premix2  0.25  0.25  0.25  0.25  0.25  0.25  0.25  0.25  L-Lysine.HCl  0.17  0.30  0.38  0.48  0.34  0.48  0.55  0.66  DL-Methionine  0.19  0.23  0.28  0.33  0.20  0.24  0.28  0.33  L-Threonine  0.00  0.03  0.10  0.18  0.07  0.15  0.21  0.29  L-Tryptophan  0.00  0.00  0.02  0.04  0.00  0.04  0.05  0.08    Starter (d 1 to 21)  Finisher (d 22 to 35)  Ingredient (%)  Control  Diet A   Diet B   Diet C   Control  Diet A   Diet B   Diet C     (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Corn  58.94  64.00  66.00  69.00  62.52  68.48  73.00  76.46  Soybean meal  34.00  29.60  25.50  21.15  26.99  22.13  18.20  13.65  Palm oil  3.00  2.00  2.40  2.30  4.92  3.65  2.76  2.56  Dicalcium phosphate  1.70  1.75  1.80  1.80  1.69  1.79  1.80  1.80  Limestone  1.20  1.25  1.24  1.25  1.49  1.26  1.60  1.50  Salt (NaCl)  0.50  0.50  0.50  0.50  0.50  0.50  0.50  0.50  Sand  0.00  0.04  1.48  2.67  0.98  0.98  0.75  1.87  Vitamin premix1  0.05  0.05  0.05  0.05  0.05  0.05  0.05  0.05  Mineral premix2  0.25  0.25  0.25  0.25  0.25  0.25  0.25  0.25  L-Lysine.HCl  0.17  0.30  0.38  0.48  0.34  0.48  0.55  0.66  DL-Methionine  0.19  0.23  0.28  0.33  0.20  0.24  0.28  0.33  L-Threonine  0.00  0.03  0.10  0.18  0.07  0.15  0.21  0.29  L-Tryptophan  0.00  0.00  0.02  0.04  0.00  0.04  0.05  0.08  1Supplies per kg of the diet: vitamin A (retinyl acetate), 15,000.000 IU; vitamin D3 (cholecalciferol), 5,000.000 IU; vitamin E (dl-α-tocopherol), 50 g; vitamin K3 (menadione dimethylpyrimidinol), 10 mg; thiamin (thiamine mononitrate), 4 mg; riboflavin, 8 mg; pyridoxine, 5 mg; vitamin B12, 0.025 mg; niacin, 50 mg; D-calcium pantothenate, 20 mg; folic acid, 20 mg; biotin, 0.25 mg; ascorbic acid, 75 mg. 2Supplies per kg of the diet: Mn (manganese oxide), 100 mg; Zn (zinc sulfate monohydrate), 150 mg; Fe (ferrous sulfate monohydrate), 100 mg; Cu (cupric sulfate pentahydrate), 20 mg; I (calcium iodate), 1.5 mg; Co (cobalt carbonate), 0.5 mg; Se (sodium selenite), 0.2 mg; Mo (sodium molybdate), 1 mg; Mg (manganese sulfate monohydrate), 50 mg. View Large Table 2. Nutrient composition (%, unless stated otherwise) of the experimental starter and finisher diets.   Starter (d 1 to 21)  Finisher (d 22 to 35)  Item1  Control  Diet A  Diet B  Diet C  Control  Diet A  Diet B  Diet C    (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Calculated composition  ME (kcal/kg)  3000  3000  3000  3000  3150  3150  3150  3150  CP  20.96  19.56  18.02  16.49  19.02  17.50  16.18  14.55  ME:CP ratio  143  153  166  182  166  180  195  216  Ca  0.94  0.94  0.94  0.94  0.94  0.94  0.94  0.94  Available P  0.45  0.45  0.45  0.45  0.45  0.45  0.45  0.45  Supplemental AA (% of Lys)2  Lys  100  100  100  100  100  100  100  100  Met + Cys  74  74  74  74  68  68  68  68  Thr  67  67  67  67  66  66  66  66  Trp  18  18  18  18  17  17  17  17  Analyzed composition  CP  21.30  20.19  18.20  16.55  19.10  17.96  16.17  14.71  Lys  1.23  1.24  1.19  1.22  1.24  1.18  1.23  1.24  Met  0.45  0.43  0.46  0.45  0.44  0.43  0.45  0.45  Thr  0.81  0.82  0.84  0.83  0.82  0.82  0.81  0.83  Trp  0.24  0.23  0.22  0.21  0.23  0.21  0.19  0.19  Arg  1.44  1.31  1.17  1.03  1.21  1.07  0.94  0.80  Val  1.09  1.00  0.91  0.83  0.93  0.84  0.76  0.67  Ile  0.92  0.84  0.75  0.66  0.77  0.68  0.61  0.53  Leu  1.91  1.82  1.68  1.55  1.69  1.58  1.49  1.36  Phe  1.03  0.94  0.85  0.76  0.87  0.79  0.73  0.64  His  0.59  0.56  0.52  0.46  0.53  0.48  0.43  0.38  Gly  0.92  0.83  0.78  0.70  0.80  0.72  0.65  0.57  Ser  1.04  0.95  0.88  0.79  0.90  0.81  0.75  0.66  Cys  0.37  0.34  0.31  0.29  0.31  0.29  0.26  0.24  Tyr  0.55  0.50  0.44  0.40  0.46  0.42  0.37  0.31  Pro  1.31  1.26  1.18  1.07  1.19  1.11  1.04  0.96  Ala  1.07  1.00  0.91  0.87  0.95  0.89  0.84  0.77  Glu  3.26  3.08  2.85  2.58  2.89  2.65  2.45  2.20  Asp  2.15  1.92  1.72  1.51  1.77  1.56  1.38  1.16    Starter (d 1 to 21)  Finisher (d 22 to 35)  Item1  Control  Diet A  Diet B  Diet C  Control  Diet A  Diet B  Diet C    (21.0% CP)  (19.5% CP)  (18.0% CP)  (16.5% CP)  (19.0% CP)  (17.5% CP)  (16.0% CP)  (14.5% CP)  Calculated composition  ME (kcal/kg)  3000  3000  3000  3000  3150  3150  3150  3150  CP  20.96  19.56  18.02  16.49  19.02  17.50  16.18  14.55  ME:CP ratio  143  153  166  182  166  180  195  216  Ca  0.94  0.94  0.94  0.94  0.94  0.94  0.94  0.94  Available P  0.45  0.45  0.45  0.45  0.45  0.45  0.45  0.45  Supplemental AA (% of Lys)2  Lys  100  100  100  100  100  100  100  100  Met + Cys  74  74  74  74  68  68  68  68  Thr  67  67  67  67  66  66  66  66  Trp  18  18  18  18  17  17  17  17  Analyzed composition  CP  21.30  20.19  18.20  16.55  19.10  17.96  16.17  14.71  Lys  1.23  1.24  1.19  1.22  1.24  1.18  1.23  1.24  Met  0.45  0.43  0.46  0.45  0.44  0.43  0.45  0.45  Thr  0.81  0.82  0.84  0.83  0.82  0.82  0.81  0.83  Trp  0.24  0.23  0.22  0.21  0.23  0.21  0.19  0.19  Arg  1.44  1.31  1.17  1.03  1.21  1.07  0.94  0.80  Val  1.09  1.00  0.91  0.83  0.93  0.84  0.76  0.67  Ile  0.92  0.84  0.75  0.66  0.77  0.68  0.61  0.53  Leu  1.91  1.82  1.68  1.55  1.69  1.58  1.49  1.36  Phe  1.03  0.94  0.85  0.76  0.87  0.79  0.73  0.64  His  0.59  0.56  0.52  0.46  0.53  0.48  0.43  0.38  Gly  0.92  0.83  0.78  0.70  0.80  0.72  0.65  0.57  Ser  1.04  0.95  0.88  0.79  0.90  0.81  0.75  0.66  Cys  0.37  0.34  0.31  0.29  0.31  0.29  0.26  0.24  Tyr  0.55  0.50  0.44  0.40  0.46  0.42  0.37  0.31  Pro  1.31  1.26  1.18  1.07  1.19  1.11  1.04  0.96  Ala  1.07  1.00  0.91  0.87  0.95  0.89  0.84  0.77  Glu  3.26  3.08  2.85  2.58  2.89  2.65  2.45  2.20  Asp  2.15  1.92  1.72  1.51  1.77  1.56  1.38  1.16  1CP, crude protein; ME, metabolizable energy; Ca, calcium; P, Phosphorus. 2Ratios based on a total requirement basis (NRC, 1994). View Large Heat treatment To elicit heat stress from 22 to 35 d of age, 7 cages of chicks from each diet were exposed to ambient temperature of 33 ± 1°C for 6 h per day. Relative humidity was not controlled but ranged from 70 to 80%. The time taken for the ambient temperature to rise from 24 to 33°C was about 30 minutes. Feed and water were provided ad libitum throughout heat treatment. Data collection and sampling Body weight and feed intake (FI) were recorded on d1, 21, and 35. Weight gains (WG) and feed conversion ratios (FCR) from d 1 to 21 (starter period), d 22 to 35 (finisher period), and d 1 to 35 (overall period) were calculated accordingly. The mortality was recorded as it occurred. On d 35, 2 birds from each cage (14 birds per diet-temperature subgroup) were randomly selected, weighed, and humanely slaughtered according to halal slaughter procedure as outlined in the Malaysian Standard, MS1500 (Department of Standards Malaysia, 2009), for blood and brain sampling. Blood samples were collected in plain tubes for determination of CPN, OVT, and AGP. Blood samples for APP assay were centrifuged, and the serum was separated and stored at -80°C for further analysis. Immediately after blood sampling, brain samples were collected to measure HSP 70 density. The brain samples were immediately frozen in liquid nitrogen and kept at −80°C for further analysis. Laboratory analyses Crude protein and amino acids. The CP in feed ingredients and experimental diets was determined according to the procedure of AOAC (1990), whereas the AA contents were determined using high-performance liquid chromatography (HPLC) as described previously (Awad et al., 2014b). Acute phase proteins. The OVT concentration was measured as described by Najafi et al. (2015), using a modified method from Mancini et al. (1965) (radial-immunodiffusion method). The CPN was determined by the rate of formation of a colored product from CPN and the substrate, 1,4-phenylenediamine dihydrochloride (Najafi et al., 2015). AGP concentration was measured using a commercial ELISA kit specific to chicken (Life Diagnostics Inc., West Chester, PA). Heat shock protein 70. The level of HSP 70 protein expression was determined as previously described by (Soleimani et al., 2012a), using SDS-PAGE and Western Blotting with some modifications. Briefly, around 0.3 g of brain samples was homogenized with 1.5 mL of protein extraction buffer (20 mM Tris, pH 7.5; 0.75 M sodium chloride) and 10 μL/mL of protease inhibitor cocktail (P8340, Sigma Chemical Co., St. Louis, MO), and centrifuged at 20,000 × g for 30 min at 4°C. The supernatant was separated, and the total protein quantity was measured using bicinchoninic acid protein assay kit (Sigma Chemical Co., St. Louis, MO). The final brain HSP 70 concentration was calculated as an arbitrary unit of band density relative to the total protein concentration of each sample. Statistical analysis Results were evaluated by analysis of variance using SAS software (SAS, 1991). Pens served as the experimental units for FI, WG, and FCR, while individual birds served as the experimental units for APP and HSP 70. Orthogonal polynomial contrasts were applied to determine the linear and quadratic effects of lowering dietary protein level. Data of FI, WG, and FCR (d 22 to 35 and d 1 to 35), APP, and HSP 70 were analyzed using 2-way ANOVA in a 4 × 2 factorial arrangement with diet, heat treatment, and their interactions as the main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. Mortality data were subjected to chi-squared analysis. Statistical significance is considered at P ≤ 0.05. RESULTS Table 3 shows the FI, WG, and FCR results during the starter period (d 1 to 21) as affected by diet. Lowering CP linearly reduced FI (P = 0.021) and WG (P = 0.009) without affecting FCR of birds. Table 4 shows the FI, WG, and FCR results as affected by diet and heat treatment during the finisher (d 22 to 35) and overall (d 1 to 35) periods. There were a significant diet x temperature interactions for both finisher (P = 0.040) and overall (P = 0.049) FCR (Table 4). During the finisher period, diet had neither a linear (P = 0.103) nor a quadratic (P = 0.066) effect on FCR among the unheated birds (Table 5). Under the heated condition, however, FCR was linearly (P = 0.007) increased as dietary CP level decreased. There were no significant differences in overall FCR between the heated and unheated birds fed the control diet, Diet A, or Diet B. However, heated birds provided Diet C had poorer (P = 0.041) FCR compared to their unheated counterparts (Table 5). Irrespective of temperature, lowering dietary CP led to a linear decrease in FI during the finisher period (P = 0.032), FI during the overall period (P = 0.039), WG during the finisher period (P < 0.001), and WG during the overall period (P < 0.001) (Table 4). Irrespective of diet, heated birds had significantly lower FI during the finisher period (P = 0.010), FI during the overall period (P = 0.017), WG during the finisher period (P < 0.001), and WG during the overall period (P < 0.001) (Table 4). Diet had no significant effect on mortality rates among unheated birds. However, under heated condition, the mortality rate of the group fed the control diet was significantly (P < 0.05) higher than the groups fed Diet B and Diet C (Table 5). Table 3. Effect of diet on feed intake, weight gain, and feed conversion ratio (FCR) of broiler chickens at 21 d of age.   Feed intake  Weight gain  FCR  Treatment  (g/bird)  (g/bird)  (feed/gain)    (d 1 to 21)  (d 1 to 21)  (d 1 to 21)  Diet  Control (21.0% CP)  1127  701  1.61  Diet A (19.5% CP)  1129  703  1.61  Diet B (18.0% CP)  1131  675  1.68  Diet C (16.5% CP)  1076  659  1.63  SEM1  49  41  0.08  Contrast, P-values  Linear  0.021  0.009  0.411  Quadratic  0.155  0.718  0.057    Feed intake  Weight gain  FCR  Treatment  (g/bird)  (g/bird)  (feed/gain)    (d 1 to 21)  (d 1 to 21)  (d 1 to 21)  Diet  Control (21.0% CP)  1127  701  1.61  Diet A (19.5% CP)  1129  703  1.61  Diet B (18.0% CP)  1131  675  1.68  Diet C (16.5% CP)  1076  659  1.63  SEM1  49  41  0.08  Contrast, P-values  Linear  0.021  0.009  0.411  Quadratic  0.155  0.718  0.057  1SEM = Standard error of the mean for diet effect (n = 14). View Large Table 4. Effect of diet1 and heat treatment2 on feed intake, weight gain, and feed conversion ratio (FCR) of broiler chickens at 35 d of age.   Feed intake (g/bird)  Weight gain (g/bird)  FCR (feed/gain)    Finisher  Overall  Finisher  Overall  Finisher  Overall  Treatment  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  Diet  Control (21.0/19.0% CP)  2120  3247  1208  1909  1.75  1.71  Diet A (19.5/17.5% CP)  2141  3270  1146  1849  1.87  1.77  Diet B (18.0/16.0% CP)  2022  3153  1022  1697  1.98  1.86  Diet C (16.5/14.5% CP)  1982  3058  1006  1665  1.97  1.84  SEM3  131  186  94  111  0.15  0.11  Contrast, P-values  Linear  0.032  0.039  <.001  <.001  0.077  0.094  Quadratic  0.508  0.880  0.007  0.009  0.013  0.016  Heat treatment  Unheated  2148  3253  1157  1841  1.84  1.77  Heated  1992  3111  1034  1718  1.95  1.81  SEM4  85  97  62  75  0.08  0.07  Source of variation  Diet (D)  0.044  0.048  <.001  <.001  <.001  <.001  Heat treatment (H)  0.010  0.017  <.001  <.001  0.039  0.609  D × H  0.761  0.812  0.196  0.142  0.040  0.049    Feed intake (g/bird)  Weight gain (g/bird)  FCR (feed/gain)    Finisher  Overall  Finisher  Overall  Finisher  Overall  Treatment  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  (d 22 to 35)  (d 1 to 35)  Diet  Control (21.0/19.0% CP)  2120  3247  1208  1909  1.75  1.71  Diet A (19.5/17.5% CP)  2141  3270  1146  1849  1.87  1.77  Diet B (18.0/16.0% CP)  2022  3153  1022  1697  1.98  1.86  Diet C (16.5/14.5% CP)  1982  3058  1006  1665  1.97  1.84  SEM3  131  186  94  111  0.15  0.11  Contrast, P-values  Linear  0.032  0.039  <.001  <.001  0.077  0.094  Quadratic  0.508  0.880  0.007  0.009  0.013  0.016  Heat treatment  Unheated  2148  3253  1157  1841  1.84  1.77  Heated  1992  3111  1034  1718  1.95  1.81  SEM4  85  97  62  75  0.08  0.07  Source of variation  Diet (D)  0.044  0.048  <.001  <.001  <.001  <.001  Heat treatment (H)  0.010  0.017  <.001  <.001  0.039  0.609  D × H  0.761  0.812  0.196  0.142  0.040  0.049  1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. 3SEM = Standard error of the mean for diet effect (n = 14). 4SEM = Standard error of the mean for heat treatment effect (n = 28). View Large Table 5. Mean feed conversion ratios (FCR) (feed/gain) during the finisher and overall periods where diet1 x heat treatment2 interactions were significant, and mortality rate (%) during the finisher period.     Heat treatment      Item  Diet  Unheated  Heated  SEM  P-value  Finisher FCR (d 22 to 35)  Control (19.0% CP)  1.71  1.79  0.15  0.082    Diet A (17.5% CP)  1.86  1.88  0.16  0.106    Diet B (16.0% CP)  1.93  2.03  0.11  0.153    Diet C (14.5% CP)  1.83  2.11  0.18  0.009    SEM  0.19  0.11        Linear  0.103  0.007        Quadratic  0.066  0.272      Overall FCR (d 1 to 35)  Control (21.0/19.0% CP)  1.67  1.72  0.12  0.380    Diet A (19.5/17.5% CP)  1.80  1.74  0.13  0.442    Diet B (18.0/16.0% CP)  1.85  1.86  0.07  0.804    Diet C (16.5/14.5% CP)  1.76  1.92  0.14  0.041    SEM  0.15  0.08        Linear  0.048  0.015        Quadratic  0.058  0.380      Finisher mortality (d 22 to 35)  Control (19.0% CP)  2.86  8.57a  3.54  >0.05    Diet A (17.5% CP)  0.00  2.86a,b  2.02  >0.05    Diet B (16.0% CP)  0.00  0.00b  0.00  >0.05    Diet C (14.5% CP)  0.00  0.00b  0.00  >0.05    SEM  0.71  1.35        P-value  >0.05  <0.05          Heat treatment      Item  Diet  Unheated  Heated  SEM  P-value  Finisher FCR (d 22 to 35)  Control (19.0% CP)  1.71  1.79  0.15  0.082    Diet A (17.5% CP)  1.86  1.88  0.16  0.106    Diet B (16.0% CP)  1.93  2.03  0.11  0.153    Diet C (14.5% CP)  1.83  2.11  0.18  0.009    SEM  0.19  0.11        Linear  0.103  0.007        Quadratic  0.066  0.272      Overall FCR (d 1 to 35)  Control (21.0/19.0% CP)  1.67  1.72  0.12  0.380    Diet A (19.5/17.5% CP)  1.80  1.74  0.13  0.442    Diet B (18.0/16.0% CP)  1.85  1.86  0.07  0.804    Diet C (16.5/14.5% CP)  1.76  1.92  0.14  0.041    SEM  0.15  0.08        Linear  0.048  0.015        Quadratic  0.058  0.380      Finisher mortality (d 22 to 35)  Control (19.0% CP)  2.86  8.57a  3.54  >0.05    Diet A (17.5% CP)  0.00  2.86a,b  2.02  >0.05    Diet B (16.0% CP)  0.00  0.00b  0.00  >0.05    Diet C (14.5% CP)  0.00  0.00b  0.00  >0.05    SEM  0.71  1.35        P-value  >0.05  <0.05      1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. SEM = Standard error of the mean for diet and heat treatment effect (n = 7). a,bMeans within a column with no common superscripts are significantly different at P < 0.05. View Large Table 6 shows the serum concentrations of AGP, OVT, CPN, and density of HSP 70 in broiler chickens as affected by diet and heat treatment at 35 d of age. Significant diet x heat treatment interactions were noted for serum AGP (P = 0.008) and OVT (P = 0.005) levels. Heat treatment did not affect serum levels of AGP or OVT in birds fed Diet A, Diet C, or control diet. However, for those fed Diet B, the serum concentrations of AGP and OVT were significantly (P < 0.001 and P = 0.002, respectively) higher in heated birds compared to their unheated counterparts (Table 7). Under both heated and unheated conditions, lowering dietary CP level resulted in a significant (P < 0.05) linear reduction in AGP and OVT (Table 7). Both CPN and brain HSP 70 density were not affected by diet (Table 6). However, irrespective of diet, heated birds showed higher CPN (P = 0.001) and HSP 70 density (P = 0.042) than their unheated counterparts. Table 6. Effect of diet1 and heat treatment2 on serum α-1 acid glycoprotein (AGP), ovotransferrin (OVT), ceruloplasmin (CPN), and heat shock protein (HSP) 70 densities of broiler chickens at 35 d of age. Treatment  AGP  OVT  CPN  HSP70    (mg/mL)  (mg/mL)  (mg/mL)  density  Diet  Control (21.0/19.0% CP)  7.649  0.443  2.096  1.588  Diet A (19.5/17.5% CP)  7.712  0.451  1.851  1.549  Diet B (18.0/16.0% CP)  7.272  0.423  1.916  1.478  Diet C (16.5/14.5% CP)  6.476  0.352  2.001  1.639  SEM3  0.714  0.062  0.337    Contrast, P-values  Linear  0.017  0.013  0.385  0.189  Quadratic  0.221  0.189  0.548  0.331  Heat treatment  Unheated  6.640  0.396  1.779  1.501  Heated  7.914  0.439  2.153  1.626  SEM4  0.424  0.033  0.196    Source of variation  Diet (D)  0.008  0.006  0.124  0.289  Heat treatment (H)  0.001  0.016  0.001  0.042  D × H  0.008  0.005  0.177  0.159  Treatment  AGP  OVT  CPN  HSP70    (mg/mL)  (mg/mL)  (mg/mL)  density  Diet  Control (21.0/19.0% CP)  7.649  0.443  2.096  1.588  Diet A (19.5/17.5% CP)  7.712  0.451  1.851  1.549  Diet B (18.0/16.0% CP)  7.272  0.423  1.916  1.478  Diet C (16.5/14.5% CP)  6.476  0.352  2.001  1.639  SEM3  0.714  0.062  0.337    Contrast, P-values  Linear  0.017  0.013  0.385  0.189  Quadratic  0.221  0.189  0.548  0.331  Heat treatment  Unheated  6.640  0.396  1.779  1.501  Heated  7.914  0.439  2.153  1.626  SEM4  0.424  0.033  0.196    Source of variation  Diet (D)  0.008  0.006  0.124  0.289  Heat treatment (H)  0.001  0.016  0.001  0.042  D × H  0.008  0.005  0.177  0.159  1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. 3SEM = Standard error of the mean for diet effect (n = 28). 4SEM = Standard error of the mean for heat treatment effect (n = 56). View Large Table 7. Mean serum α-1 acid glycoprotein (AGP) and ovotransferrin (OVT) of broiler chickens where diet1 x heat treatment2 interactions were significant at 35 d of age.     Heat treatment      Item  Diet  Unheated  Heated  SEM3  P-value  AGP (mg/mL)  Control (21.0/19.0% CP)  7.225  8.043  1.03  0.077    Diet A (19.5/17.5% CP)  7.106  8.319  1.26  0.062    Diet B (18.0/16.0% CP)  5.844  8.700  1.31  <.001    Diet C (16.5/14.5% CP)  6.357  6.595  0.95  0.103    SEM3  1.062  1.211        Linear  0.045  0.009        Quadratic  0.084  0.606      OVT (mg/mL)  Control (21.0/19.0% CP)  0.421  0.466  0.06  0.058    Diet A (19.5/17.5% CP)  0.446  0.456  0.04  0.113    Diet B (18.0/16.0% CP)  0.350  0.496  0.08  0.002    Diet C (16.5/14.5% CP)  0.366  0.337  0.05  0.093    SEM3  0.05  0.07        Linear  0.003  0.010        Quadratic  0.011  0.013          Heat treatment      Item  Diet  Unheated  Heated  SEM3  P-value  AGP (mg/mL)  Control (21.0/19.0% CP)  7.225  8.043  1.03  0.077    Diet A (19.5/17.5% CP)  7.106  8.319  1.26  0.062    Diet B (18.0/16.0% CP)  5.844  8.700  1.31  <.001    Diet C (16.5/14.5% CP)  6.357  6.595  0.95  0.103    SEM3  1.062  1.211        Linear  0.045  0.009        Quadratic  0.084  0.606      OVT (mg/mL)  Control (21.0/19.0% CP)  0.421  0.466  0.06  0.058    Diet A (19.5/17.5% CP)  0.446  0.456  0.04  0.113    Diet B (18.0/16.0% CP)  0.350  0.496  0.08  0.002    Diet C (16.5/14.5% CP)  0.366  0.337  0.05  0.093    SEM3  0.05  0.07        Linear  0.003  0.010        Quadratic  0.011  0.013      1Control: 21 and 19% crude protein during starter and finisher periods, respectively; Diet A: 19.5 and 17.5% crude protein during starter and finisher periods, respectively; Diet B: 18 and 16.0% crude protein during starter and finisher periods, respectively; Diet C: 16.5 and 14.5% crude protein during starter and finisher periods, respectively. 2Equal numbers of birds from each diet were subjected to either 23±1°C throughout (unheated) or 33±1°C for 6 h per d (heated) from 22 to 35 d of age. 3SEM = Standard error of the mean for diet and heat treatment effect (n = 14). View Large DISCUSSION The present findings suggested that the growth performance of broiler chickens was negatively affected by dietary CP level reduction from 21.0 to 16.5% and from 19.0 to 14.5% in the starter and finisher diets, respectively, despite meeting or exceeding recommended levels of those commercially available feed-grade AA (namely L-lysine HCl, DL-methionine, L-threonine, and L-tryptophan). These findings concur with previous work (Pinchasov et al., 1990; Aletor et al., 2000; Bregendahl et al., 2002; Si et al., 2004; Namroud et al., 2008; Awad et al., 2014a) that growth performance was depressed in broilers fed diets in which CP has been reduced by more than 3%, even when all EAA requirements are met. Lower FI in birds fed low-CP diets also has been reported by Si et al. (2004), Namroud et al. (2008), and Awad et al. (2014a). Indeed, reduction of dietary CP will reduce both EAA and non-essential amino acids (NEAA), which alter the concentration/balance of the AA in such low-CP diets. This change in concentration/balance of AA can negatively affect the FI in broilers (Aftab et al., 2006). In birds fed low-CP diets, the amount of ingested free AA into the blood stream increase and may affect the balance of plasma AA profile (Aftab et al., 2006). An imbalanced AA profile in plasma has been shown to reduce FI (Austic et al., 2000). On the contrary, other workers (Bregendahl et al., 2002; Fatufe and Rodehutscord, 2005) indicated no differences or even higher (Aletor et al., 2000) FI when birds were fed low-CP, AA-supplemented diets. Depressed performance in broilers fed the low-CP diets could be associated with insufficient nitrogen quantity for NEAA synthesis. Chickens fed the standard levels of dietary protein can synthesize NEAA in the body from excess EAA. Thus, when low-CP diets are provided, this excess is reduced, leaving less EAA available for conversion to the NEAA (Waldroup, 2007). Findings on protein requirements of broilers under hot environment have been inconsistent. Work by Temim et al. (2000) and Gonzalez-Esquerra and Leeson (2005) showed that the higher levels of dietary CP may compensate the reduction in feed and protein associated with heat stress. Conversely, Cheng et al. (1997) noted adverse effects of high-protein diets (22 vs. 24% CP) on growth performance in 3- to 6-week-old heat-stressed birds. In the current study, however, it appears that low-CP diets were more detrimental to the performance of heat-stressed broilers. A possible explanation for this might be the insufficiency in EAA, due either to lower ingested quantities or decreased AA digestibility in heat-stressed birds (Bonnet et al., 1997). One of the advantages of the feeding low-CP diet in the current study was increase in survivability of heat-stressed birds. Birds fed Diet B and Diet C had significantly lower mortality rates than their control counterparts during the heat treatment period. Heavier birds were more susceptible to heat stress (Chwalibog and Eggum, 1989; Cahaner and Leenstra, 1992) than lighter ones because they had more internally generated heat to dissipate and consequently they may have difficulty maintaining body temperature at high ambient temperature. However, in the present study, the improved survivability rate among those fed low-CP diets may not be associated with body weight because the mortality rates of broilers fed Diet A and Diet B were lower than controls despite all 3 groups having almost a similar body weight. In the present study, irrespective of diet, heat challenge increased CPN. However, the effect of heat treatment on AGP and OVT varied according to diet. Only heated birds that were fed Diet B had higher AGP and OVT than their unheated counterparts. In general, it appears that under both unheated and heated conditions, AGP and OVT decreased with low-CP diets. Studies in laboratory animals indicated that the ability to elicit a hepatic response to inflammatory stimuli was reduced by low-protein diets (Bell and Hoffman-Goetz, 1983; Jennings and Elia, 1990; Grimble et al., 1992; Jennings et al., 1992). Grimble et al. (1992) suggested that in a malnourished organism, the availability of sulfur amino acids may influence antioxidant defenses and thereby affect the pattern of APP synthesis in an indirect manner. To the best of our knowledge, this is the first work showing a low-protein diet may dampen synthesis of APP in the avian species. APP function as protease inhibitors, enzymes, transport proteins, coagulation proteins, and modulators of the immune response. The acute phase response (APR) is considered as part of the innate immune response and is observed across all animal species (Cray et al., 2009). The APR induce a complex systemic reaction to re-establish homeostasis and promote healing (Cray et al., 2009). An increase in serum APP concentrations may be considered as an indicator of intracellular communication, suggesting an increase in the cellular immune response (Cray et al., 2009). Hence, the possible impairment of APP synthesis associated with low-CP diets is of concern and should be considered before recommending such diets for broilers. It is interesting to note that although the chicken provided with low-CP diets had diminished APP reactions, they had better survivability than those fed diets with adequate levels of CP. Payne et al. (1990) reported that protein deficiency reduced immunocompetence in chickens as assessed by antibody titers, white blood cell counts, and T-cell activity. However, the authors noted that dietary modifications had negligible influence on survival following challenge with Pasteurella multocida. Thus, it appears that impaired immune response may not be associated with a higher mortality rate. Irrespective of diet, the heat treatment increased CPN. However, elevations in AGP and OVT were noted only for those fed Diet B. Elicitation of AGP, OVT, and CPN reactions to the hot environment in broiler chickens have been demonstrated by Najafi et al. (2015) and Olubodun et al. (2015). There is no clear explanation for the lack of heat treatment effect on birds fed control, Diet A, or Diet C. The findings, however, suggested that APP response to heat may vary according to the levels of dietary protein. The present findings confirmed earlier work (Liew et al., 2003; Zulkifli et al., 2003; Soleimani et al., 2011) that heat treatment can increase brain HSP 70 expression in chickens. Olubodun et al. (2015), and Zulkifli et al. (2016) reported that dietary L-glutamine and L-glutamate supplementation augmented HSP 70 density in chicks. The current study suggested that low-CP diets had a negligible effect on brain HSP 70 density in broiler chickens. Work in cattle indicated that providing a low-protein diet for 3 mo downregulated HSP 70 and HSP 90 expression in fat tissue (Eitam et al., 2012). In vitro work suggested that AA deprivation resulted in inactivation of the heat shock factor 1 (Hensen et al., 2012) The activation of heat shock gene transcription during the stress response is mediated by heat shock transcription factor, which binds to heat shock elements in the promoters of heat shock genes (Amin et al., 1988; Abravaya et al., 1991). The lack of low-CP diets effect on HSP 70 expression in the present work could be attributed to the adequate levels of lysine, methionine, threonine, and tryptophan in the diets. In conclusion, despite meeting the requirements of the essential limiting AA, feeding broilers with starter and finisher low-CP diets negatively affected growth performance compared to those provided standard CP diet. Low-CP diets seemed to be beneficial in improving survivability rate but not the performance of heat-stressed broiler chickens. Under both unheated and heated conditions, low-CP diets tended to impair AGP and OVT responses. Because APP contributes to non-specific innate immune response and restoration of homeostasis, the practice of feeding low-CP diets to broilers could be of concern. Low-CP diets, however, had negligible influence on brain HSP 70 density. Acknowledgements This work was supported by the Malaysian Ministry of Higher Education under the Long-term Research Grant Scheme. REFERENCES Abravaya K., Phillips B., Morimoto R. I.. 1991. 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Poultry ScienceOxford University Press

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