No copper supplementation in a corn-soybean basal diet has no adverse effects on late-phase laying hens under normal and cyclic high temperatures

No copper supplementation in a corn-soybean basal diet has no adverse effects on late-phase... Abstract Over supplementation of copper (Cu) in animal diets may cause serious pollution in soil, water and harvested crops. To minimize the potential pollution, the effects of corn-soybean basal diet with or without supplementation of 8 mg Cu/kg on laying performance, plasma biochemical metabolic indices, and antioxidant status in laying hens were evaluated under normal and cyclic high temperatures. A total of 240 Hy-Line Brown laying hens were randomly allotted to 4 treatments with 6 replicates of 10 hens per replicate according to factorial design involved in 2 temperatures [normal temperature (NT) vs. cyclic high temperature (CHT)] and 2 dietary Cu addition amount [Cu0 (0 mg/kg) vs. Cu8 (8 mg/kg in the form of CuSO4·5H2O)]. The experimental period included 1-week adaptation, 2-week heat stress and 2-week convalescence. The temperatures of NT groups in the same period or any groups during other periods were kept at 26 ± 2°C except that of CHT groups were 26 ± 2°C∼33 ± 2°C cyclically during heat stress period. CHT groups increased (P < 0.05) the rectal temperature and plasma glucose content under heat stress, but decreased (P < 0.01) the egg yield at the second week of heat stress and the first week of convalescence, and the plasma triglyceride, uric acid, and triiodothyronine levels under heat stress. Cu8 groups increased (P < 0.05) egg weight at the first week of convalescence, and plasma thyroxin level during the whole convalescence. Interactions between temperature and Cu content existed (P < 0.05) in the laying rate at the first week of convalescence, and the plasma lactic dehydrogenase level under heat stress. Conclusively, the CHT impaired laying performance. The Cu content (10.3 mg/kg) in corn-soybean basal diet might be sufficient for meeting the maintenance and production requirements of late-phase laying hens, and no Cu supplementation had no adverse effects on egg production and antioxidant indices under cyclic high (26 ± 2°C∼33 ± 2°C) or normal (26 ± 2°C) temperatures. INTRODUCTION Heat stress is of great concern in all types of poultry operations. High environmental temperature can cause a significant reduction in egg production and eggshell thickness of commercial laying hens (Emery et al., 1984; Star et al., 2008a). High environmental temperature also induces a series of physiological and metabolic changes such as elevated rectal temperature (Mashaly et al., 2004) and plasma creatine kinase activity and decreased plasma triiodothyronine levels in laying hens (Star et al., 2008b). Moreover, heat stress from high environmental temperature can result in oxidative stress and lipid peroxidation with an elevated level of malondialdehyde (MDA) in the plasma of laying hens (Sahin and Kucuk, 2001; Lin et al., 2008). Copper (Cu) is an essential element in the diet of poultry due to its biological activity. It is a cofactor of many enzymes such as superoxide dismutase (SOD), cytochrome oxidase, and ceruloplasmin (Prohaska et al., 1983). Therefore, the importance of Cu in animal nutrition is associated with its roles in many biological processes including antioxidant activity, immune function, and neuropeptide synthesis (Bonham et al., 2002). According to the National Research Council (NRC), the requirement for Cu is 4.0–5.0 mg/kg diet in laying hens (0–18 wk). However, there is still uncertainty about the need for Cu in laying hens after 18 wk (NRC, 1994). Basal concentration in a corn-soybean meal diet without supplemented Cu containing 9.2 mg Cu/kg DM seems to be enough for maintaining egg production and shell quality of laying hens (ISA Brown, 45–53 wk) (Skrivan and Skrivanova, 2006). When dietary Cu concentration was decreased from 12 to 4 mg/kg, no effect was observed in growth rate, feed conversion and mortality in Ross 308 broilers at the age of 0–17 d (Dozier et al., 2003). Farmers in China believe that the supplementation of Cu in diets of laying hens (from 4 to 25 mg/kg feed) ought to alleviate the negative effects of heat stress in hens and the recommended supplementation in diet is 8 mg Cu/kg feed for laying hens in China's Feeding Standard of Chicken (Ministry of Agriculture of People's Republic of China, 2004). Hubert et al. (1989) studied the effect of diet supplemented with 250 mg Cu/kg feed on pigs under different temperatures, demonstrating that there was no interaction between environmental temperature and Cu supplementation in daily gain, feed consumption, and feed per unit of the gain of pigs. Moreover, dietary 110 mg Cu/kg DM (Yang et al., 2017) or 600 mg Cu/kg feed (Chiou et al., 1997) may induce acute or chronic toxicity because the oxidative damage and cell death is observed in vivo with a high level of Cu (10 μmol/L Cu2+) in Leibovitz L15 medium (Nawaz et al., 2005). Result from previous study also indicated that only 20% of the supplemented Cu could be digested and absorbed. The rest is excreted with feces that may result in pollution in plant, soil, and aquatic environments, subsequently impacting human health (Zhao et al., 2010). Therefore, it is not clear whether Cu should be supplemented in the diet of the laying hens after 18 wk. In order to understand the optimal application of Cu in laying hens especially under heat stress, and to minimize the potential pollution in the environment, this study was conducted to assess the effects of diets with or without supplementation of Cu on laying performance, plasma biochemical, and antioxidant indices of laying hens under normal and cyclic high temperatures. MATERIALS AND METHODS Experimental Design and Treatments A completely randomized design with a 2 temperatures (normal 26 ± 2°C (NT) vs. cyclic high temperature (CHT, 26 ± 2°C∼33 ± 2°C) × 2 dietary Cu supplementation levels (0 mg Cu (Cu0) vs. 8 mg Cu/kg feed (Cu8) in the form of CuSO4·5H2O) factorial arrangement was used. Thus, there were a total of 4 treatments (NT-Cu0, NT-Cu8, CHT-Cu0, and CHT-Cu8) in this experiment. Animals and Diets The protocol was reviewed and approved by the Animal Care and Use Committee of China Agricultural University. All procedures were performed strictly in accordance with the guidelines of recommendations in the Guide for Experimental Animals of the Ministry of Science and Technology (Beijing, China), and all efforts were made to minimize suffering. Two hundred and forty 47-week-old Hy-Line Brown laying hens with a similarity in laying rate (89.3 ± 0.93%) and body weight (BW, 1.68 ± 0.05 kg) were allotted randomly to one of 4 treatments with 6 replicates of 10 hens per replicate. Ten hens in each replicate were kept in five neighboring ladder stainless steel cages with 2 hens per cage. The cage size was 0.45 m wide × 0.45 m deep × 0.45 m height. Laying hens were maintained on a 16-h light schedule and allowed ad libitum access to experimental diets and water. The experimental period was 5 weeks including 1 week of adaptation (47 wk), 2 weeks of heat stress (48–49 wk) and 2 weeks of convalescence (50–51 wk). During the adaptation period, all laying hens were fed with the corn-soybean basal diet (Table 1) with no Cu addition. During the heat stress period, the room temperature for hens in treatments of NT-Cu0, NT-Cu8 was maintained at 26 ± 2°C, whereas the room temperature for hens in treatments of CHT-Cu0, CHT-Cu8 was increased step-wise from 26 ± 2°C to 33 ± 2°C in 2 h (between 07:00 and 09:00), and maintained at 33 ± 2°C for 8 h (between 09:00 and 17:00), and then decreased step-wise from 33 ± 2°C to 26 ± 2°C (between 17:00 and 19:00), and maintained at 26 ± 2°C until 07:00 of the next day. During the convalescent period, both of the two rooms were maintained at 26 ± 2°C. The temperature program is referred to the method of Mashaly et al. (2004). Relative humidity was kept at 50 ± 10% for the two rooms during the experimental period of 5 weeks (47–51 wk of age). Table 1. Composition and nutrient levels of basal diet (as-fed basis) Ingredient  Inclusion (%, unless noted)  Nutrient3  Nutrient composition (%, unless noted)  Corn  63.68  AME (MJ/kg)  11.70  Soybean meal, 44% CP  24.80  CP  15.40  Limestone (CaCO3)  9.00  Ca  3.64  Dicalcium phosphate  1.60  Available P  0.35  Salt  0.30  Methionine  0.36  DL-Methionine  0.11  Methionine + cysteine  0.58  L-Lysine HCL (98.5%)  0.08  Lysine  0.81  Threonine  0.02  Threonine  0.61  Tryptophan  0.02  Tryptophan  0.18  Vitamin premix1  0.04  Cu (mg/kg)  10.27  Mineral premix2  0.35      Total  100.00      Ingredient  Inclusion (%, unless noted)  Nutrient3  Nutrient composition (%, unless noted)  Corn  63.68  AME (MJ/kg)  11.70  Soybean meal, 44% CP  24.80  CP  15.40  Limestone (CaCO3)  9.00  Ca  3.64  Dicalcium phosphate  1.60  Available P  0.35  Salt  0.30  Methionine  0.36  DL-Methionine  0.11  Methionine + cysteine  0.58  L-Lysine HCL (98.5%)  0.08  Lysine  0.81  Threonine  0.02  Threonine  0.61  Tryptophan  0.02  Tryptophan  0.18  Vitamin premix1  0.04  Cu (mg/kg)  10.27  Mineral premix2  0.35      Total  100.00      1Provided per kilogram of diet: 8500 IU of vitamin A (retinol acetate); 3600 IU of vitamin D3; 21 IU of vitamin E (DL-α-tocopherolacetate); 4.2 mg of vitamin K3 (menadione dimethpyrimidinol); 3.0 mg of vitamin B1 (thiamin mononitrate); 10.2 mg of vitamin B2 (riboflavin); 45 mg of niacin; 15 mg of calcium pantothenate; 5.4 mg of vitamin B6 (pyridoxine); 0.15 mg of biotin; 0.9 mg of folic acid and 0.024 mg of vitamin B12. 2Provided per kilogram of diet: 60 mg of Fe (FeSO4·7H2O), 80 mg of Zn (ZnSO4·7H2O), 60 mg of Mn (MnSO4·H2O), 0.35 mg of I (KI), and 0.3 mg of Se (Na2SeO3). Cu additive was added to diets by replacing an equal weight of corn starch. 3CP, Ca, and Cu levels were analyzed. Each value was based on triplicate determinations, but all other nutrient levels were calculated. View Large Both BW and rectal temperature (RT) of laying hens were measured at 12:00 on the last day of each week. The RT was monitored using a thermo-code electric gauge (JM222, JinMing, Tianjin, China) with an accuracy of 0.1°C. Eggs were collected daily at 15:00 and the number of eggs and egg weight in each replicate were recorded. The feed intake of laying hens in each replicate was recorded at 20:00 on the last day of each week of the experimental period. The basal corn-soybean basal diet (Table 1) was formulated to meet or exceed the nutrient requirements for laying hens (NRC, 1994), except for Cu, which was added to the basal diet according to the experimental design. Copper sulfate pentahydrate was reagent grade (Beijing Chemical Company, Beijing, China), and contained 25.5% Cu on a basis of analysis (purity > 99%). A single batch of basal diet was mixed and then divided into two aliquots according to the experimental treatments. The content of Cu in tap wat0er was undetectable. The analyzed content of Cu in diets of Cu0 and Cu8 were 10.3, 17.6 mg/kg, respectively. Sample Collections and Preparations The feed ingredients and diet samples from all the treatments were collected and analyzed for CP, Ca, Cu before the initiation of the experiment to confirm the correct preparation of diets. Blood samples were collected via a bronchial vein from 2 fasted laying hens with average BW in each replicate at 12:00 on the last day of each week for the heat stress and convalescent periods. Plasma samples were obtained by centrifuging blood samples at 3000 × g for 20 min at 4°C and stored in −20°C for further analyses. All samples from 2 hens in each replicate were pooled into one sample in equal volume before analysis. Sample Analyses CP, Ca, Cu Contents. Concentrations of CP in feed ingredients and diet samples were determined using the Association of Official Analytical Chemists methods (AOAC, 1990). The concentrations of Cu in water and diets, and Ca in feed ingredients and diet samples, were measured using an inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA, USA) after wet digestions using HNO3 and HCLO4 as described by Zhao et al. (1994). Plasma Biochemical Parameters. Plasma glucose (GLU), cholesterol (CHO), triglyceride (TG), uric acid (UA) contents and activities of alkaline phosphatase (ALP), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH), and creatine kinase (CK) were measured using a HITACHI 7600 automatic biochemical analyzer (Hitachi Co., Ltd, Tokyo, Japan) with the detection kits (Prodia diagnostics, Boetzingen, Germany), respectively. Plasma triiodothyronine (T3), thyroxin (T4), and corticosterone (CORT) levels were measured using a Sn-69,513 RIA counter (Shanghai nuclear power Co., Ltd, Shanghai, China) with the detection kits (Jiuding medical biological engineering Co., Ltd, Tianjin, China). Plasma total antioxidant capacity (T-AOC) content was measured using the specific detection kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) with a microplate spectrophotometer (Epoch, BioTek Instruments Inc., Winooski, VT). Plasma Cu and zinc superoxide dismutase (CuZnSOD) activity was measured using the nitrite method as described by Kobayashi et al. (1978). Plasma malondialdehyde (MDA) content was determined using the thiobarbituric acid colorimetric method as described by Mak et al. (1983). Statistical Analyses Data were analyzed by 3-way ANOVA using the PROC GLM procedure of SAS (2010, release 9.2, SAS Institute Inc., Cary, NC, USA), and the model included temperature, dietary Cu, period and the interactions between these factors. The period is referred to the 48–49 wk (2-week heat stress), and the 50–51 wk (2-week convalescence), respectively. The replicate served as the experimental unit. No interactions (P > 0.05) between period and temperature or dietary Cu were observed for RT, daily feed intake, feed: egg ratio, BW, morality, and plasma biochemical parameters. Therefore, the main effects of temperature, dietary Cu and their interaction on the above indices were presented for the heat stress period (48–49 wk) and the convalescent period (50–51wk). Because the significant interactions (P < 0.05) between period and temperature were observed for laying rate, egg weight, and egg yield, the main effects of temperature, dietary Cu and their interaction on these three indices were reported for each week during the heat stress and convalescent period. Data of hen mortality was transformed to arc sine values before statistical analysis. Differences among means were tested using the LSD method, and statistical significance was set at P ≤ 0.05. RESULTS Rectal Temperature Dietary Cu did not affect (P > 0.05) RT during the experimental period (47–51 wk). The temperature impacted (P < 0.01) RT during the heat stress period (48–49 wk) but not during the adaptation (47 wk) and convalescent periods (50–51 wk). Compared with NT, CHT increased (P < 0.01) the RT during 48–49 wk of age. No interactions between dietary Cu and temperature were detected (Table 2). Table 2. Effects of environmental temperature and dietary Cu on rectal temperature (RT) of hens at 47 to 51 wk of age Item  RT (°C)    47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  39.8  39.9  40.4    Cu82  40.0  39.8  40.3  CHT1,3  Cu0  39.9  40.7  40.6    Cu8  39.9  40.8  40.3    Pooled SEM  0.08  0.10  0.08  TEMP1,4  NT  39.9  39.8B  40.3    CHT  39.9  40.8A  40.5    Pooled SEM  0.06  0.07  0.05  Dietary Cu4  Cu0  39.9  40.3  40.5    Cu8  40.0  40.3  40.3    Pooled SEM  0.06  0.07  0.05  P-value5  TEMP  0.73  <0.0001  0.082    Cu  0.26  0.97  0.064    TEMP × Cu  0.18  0.50  0.22  Item  RT (°C)    47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  39.8  39.9  40.4    Cu82  40.0  39.8  40.3  CHT1,3  Cu0  39.9  40.7  40.6    Cu8  39.9  40.8  40.3    Pooled SEM  0.08  0.10  0.08  TEMP1,4  NT  39.9  39.8B  40.3    CHT  39.9  40.8A  40.5    Pooled SEM  0.06  0.07  0.05  Dietary Cu4  Cu0  39.9  40.3  40.5    Cu8  40.0  40.3  40.3    Pooled SEM  0.06  0.07  0.05  P-value5  TEMP  0.73  <0.0001  0.082    Cu  0.26  0.97  0.064    TEMP × Cu  0.18  0.50  0.22  a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Laying Performance All data are shown in Tables 3 and 4. No difference (P > 0.05) was found on laying performance at the beginning of the study (47 wk of age). Compared to NT, CHT decreased (P < 0.01) the laying rate at 49 wk of age, egg yield at 49 and 50 wk of age, daily feed intake and feed: egg ratio during the stage of 48–49 wk. The BW (P < 0.04) was also decreased during the stages of 48–49 wk, and 50–51 wk, but the feed: egg ratio was increased (P < 0.03) during 50–51 wk of age in hens under CHT as compared to NT. Compared to Cu0, hens fed with Cu8 diet had a higher (P < 0.04) egg weight at 50 wk of age. No interactions between temperature and dietary Cu were observed in any of the above-mentioned indices except for the laying rate (P < 0.04) at 50 wk of age, and egg weight (P < 0.03) during the heat stress period. No difference between dietary Cu treatments was observed in the laying rate of the hens under NT at 50 wk of age. However, hens in CHT-Cu8 had a decreased (P < 0.01) laying rate compared to NT-Cu8 with no difference (P > 0.30) between CHT-Cu0 and NT-Cu0. Moreover, hens in CHT-Cu0 had decreased (P < 0.03) egg weight compare to CHT-Cu8 with no difference (P > 0.35) between NT-Cu0 and NT-Cu8 during 48 and 49 wk of age. Table 3. Effects of environmental temperature and dietary Cu on laying rate, egg weigh, and egg yield of hens at 47 to 51 wk of age Item  Laying rate (%)  Egg weight (g)  Egg yield (g/hen·d)    47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  NT1,3  Cu02  88.3  88.6  88.0  84.3a,b  87.7  61.3  61.4a  62.0a  60.9  60.7  54.2  54.4  54.5  51.3  53.2    Cu82  90.5  89.3  88.7  88.4a  83.3  60.9  60.7a  61.7a  61.3  60.9  55.1  54.3  54.7  54.2  50.7  CHT1,3  Cu0  88.9  85.4  81.0  82.1b,c  85.7  61.0  58.7b  57.6c  59.2  59.7  54.2  50.1  46.7  48.6  51.2    Cu8  89.4  86.9  79.6  78.0c  84.8  61.1  60.6a  59.6b  61.1  61.6  54.6  52.5  47.5  47.7  52.1    Pooled SEM  1.72  2.57  2.53  1.74  2.30  0.53  0.54  0.44  0.52  0.54  1.11  1.59  1.56  0.97  1.41  TEMP1,4  NT  89.4  88.9  88.3A  86.3  85.5  61.1  61.1  61.8  61.1  60.8  54.7  54.3  54.6A  52.7A  52.0    CHT  89.2  86.1  80.3B  80.0  85.2  61.1  59.6  58.6  60.2  60.6  54.4  51.3  47.1B  48.1B  51.6    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  Dietary Cu4  Cu0  88.6  87.0  84.5  83.2  86.7  61.2  60.0  59.8  60.0b  60.2  54.2  52.2  50.6  49.9  52.2    Cu8  90.0  88.1  84.1  83.2  84.0  61.0  60.6  60.6  61.2a  61.2  54.9  53.4  51.1  50.9  51.4    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  P-value5  TEMP  0.88  0.29  0.005  0.002  0.91  0.84  0.013  <0.0001  0.093  0.72  0.82  0.071  0.0001  0.0001  0.82    Cu  0.46  0.68  0.89  0.99  0.26  0.83  0.27  0.079  0.035  0.068  0.54  0.47  0.77  0.33  0.60    TEMP × Cu  0.62  0.89  0.67  0.030  0.46  0.64  0.028  0.019  0.18  0.14  0.81  0.43  0.87  0.063  0.24  Item  Laying rate (%)  Egg weight (g)  Egg yield (g/hen·d)    47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  NT1,3  Cu02  88.3  88.6  88.0  84.3a,b  87.7  61.3  61.4a  62.0a  60.9  60.7  54.2  54.4  54.5  51.3  53.2    Cu82  90.5  89.3  88.7  88.4a  83.3  60.9  60.7a  61.7a  61.3  60.9  55.1  54.3  54.7  54.2  50.7  CHT1,3  Cu0  88.9  85.4  81.0  82.1b,c  85.7  61.0  58.7b  57.6c  59.2  59.7  54.2  50.1  46.7  48.6  51.2    Cu8  89.4  86.9  79.6  78.0c  84.8  61.1  60.6a  59.6b  61.1  61.6  54.6  52.5  47.5  47.7  52.1    Pooled SEM  1.72  2.57  2.53  1.74  2.30  0.53  0.54  0.44  0.52  0.54  1.11  1.59  1.56  0.97  1.41  TEMP1,4  NT  89.4  88.9  88.3A  86.3  85.5  61.1  61.1  61.8  61.1  60.8  54.7  54.3  54.6A  52.7A  52.0    CHT  89.2  86.1  80.3B  80.0  85.2  61.1  59.6  58.6  60.2  60.6  54.4  51.3  47.1B  48.1B  51.6    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  Dietary Cu4  Cu0  88.6  87.0  84.5  83.2  86.7  61.2  60.0  59.8  60.0b  60.2  54.2  52.2  50.6  49.9  52.2    Cu8  90.0  88.1  84.1  83.2  84.0  61.0  60.6  60.6  61.2a  61.2  54.9  53.4  51.1  50.9  51.4    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  P-value5  TEMP  0.88  0.29  0.005  0.002  0.91  0.84  0.013  <0.0001  0.093  0.72  0.82  0.071  0.0001  0.0001  0.82    Cu  0.46  0.68  0.89  0.99  0.26  0.83  0.27  0.079  0.035  0.068  0.54  0.47  0.77  0.33  0.60    TEMP × Cu  0.62  0.89  0.67  0.030  0.46  0.64  0.028  0.019  0.18  0.14  0.81  0.43  0.87  0.063  0.24  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Table 4. Effects of environmental temperature and dietary Cu on daily feed intake, feed: egg ratio, body weight (BW), and mortality of hens at 47 to 51 wk of age Item  Daily feed intake (g/hen·d)  Feed:egg ratio (g/g)  BW (kg)  Mortality (%)    47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  98.0  95.3  102  1.85  1.75  2.05  1.64  1.69  1.66  1.67  1.67  0.00    Cu82  105  101  106  1.93  1.85  2.09  1.71  1.74  1.72  0.00  0.00  0.00  CHT1,3  Cu0  105  75.1  105  1.96  1.57  2.16  1.66  1.62  1.65  0.00  1.67  1.85    Cu8  105  74.2  108  1.95  1.48  2.26  1.66  1.65  1.65  0.00  5.00  0.00    Pooled SEM  2.09  2.37  2.05  0.05  0.05  0.06  0.02  0.03  0.02  0.83  2.07  0.93  TEMP1,4  NT  102  98.0A  104  1.89  1.80A  2.07b  1.68  1.72A  1.69a  0.84  0.83  0.00    CHT  105  74.7B  106  1.96  1.53B  2.21a  1.66  1.64B  1.65b  0.00  3.33  0.93    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  Dietary Cu4  Cu0  102  85.2  103  1.91  1.66  2.11  1.65  1.66  1.66  0.84  1.67  0.93    Cu8  105  87.4  107  1.94  1.67  2.18  1.69  1.70  1.69  0.00  2.50  0.00    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  P-value5  TEMP  0.11  <0.0001  0.30  0.23  <0.0001  0.020  0.39  0.007  0.038  0.33  0.26  0.33    Cu  0.091  0.37  0.085  0.52  0.87  0.26  0.065  0.17  0.19  0.33  0.84  0.33    TEMP × Cu  0.14  0.21  0.74  0.43  0.10  0.58  0.071  0.65  0.11  0.33  0.26  0.33  Item  Daily feed intake (g/hen·d)  Feed:egg ratio (g/g)  BW (kg)  Mortality (%)    47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  98.0  95.3  102  1.85  1.75  2.05  1.64  1.69  1.66  1.67  1.67  0.00    Cu82  105  101  106  1.93  1.85  2.09  1.71  1.74  1.72  0.00  0.00  0.00  CHT1,3  Cu0  105  75.1  105  1.96  1.57  2.16  1.66  1.62  1.65  0.00  1.67  1.85    Cu8  105  74.2  108  1.95  1.48  2.26  1.66  1.65  1.65  0.00  5.00  0.00    Pooled SEM  2.09  2.37  2.05  0.05  0.05  0.06  0.02  0.03  0.02  0.83  2.07  0.93  TEMP1,4  NT  102  98.0A  104  1.89  1.80A  2.07b  1.68  1.72A  1.69a  0.84  0.83  0.00    CHT  105  74.7B  106  1.96  1.53B  2.21a  1.66  1.64B  1.65b  0.00  3.33  0.93    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  Dietary Cu4  Cu0  102  85.2  103  1.91  1.66  2.11  1.65  1.66  1.66  0.84  1.67  0.93    Cu8  105  87.4  107  1.94  1.67  2.18  1.69  1.70  1.69  0.00  2.50  0.00    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  P-value5  TEMP  0.11  <0.0001  0.30  0.23  <0.0001  0.020  0.39  0.007  0.038  0.33  0.26  0.33    Cu  0.091  0.37  0.085  0.52  0.87  0.26  0.065  0.17  0.19  0.33  0.84  0.33    TEMP × Cu  0.14  0.21  0.74  0.43  0.10  0.58  0.071  0.65  0.11  0.33  0.26  0.33  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Plasma Biochemical Parameters The results on plasma GLU, CHO, TG, UA, T3, T4 levels, and ALP, GOT, GPT, LDH, CK activities, and CORT level of hens are given in Tables 5 and 6. CHT decreased plasma TG (P < 0.04), UA (P < 0.01), and T3 (P < 0.03) levels, but increased plasma GLU (P < 0.03) level compared to NT during the stage of 48–49 wk. Compared to hens with Cu0, hens fed with Cu8 diet had a lower (P < 0.01) plasma UA concentration during the 48–49 wk of age, but a higher (P < 0.04) T4 level during the stage of 50–51 wk. There were interactions between temperature and dietary Cu in plasma LDH (P < 0.05) activity during the stage of 48–49 wk, and CK (P < 0.01) activity during the stage of 50–51 wk. No interactions between temperature and dietary Cu were observed in any other plasma biochemical indices. No difference was observed in plasma LDH activity between dietary Cu treatments under NT during the stage of 48–49 wk. However, the hens in CHT-Cu0 had a higher (P < 0.01) LDH activity compared to CHT-Cu8 with no difference (P > 0.90) between CHT-Cu8 and NT-Cu8. No difference was observed also in plasma CK activity between dietary Cu treatments under NT during the stage of 50–51 wk, but the hens in CHT-Cu8 had a higher (P < 0.01) CK activity compared to CHT-Cu0 with no difference (P > 0.05) between CHT-Cu0 and NT-Cu0. Table 5. Effects of environmental temperature and dietary Cu on plasma glucose (GLU), cholesterol (CHO), triglyceride (TG), uric acid (UA) triiodothyronine (T3), and thyroxin (T4) levels of hens at 48 to 51 wk of age Item  GLU (mmol/L)  CHO (mmol/L)  TG (mmol/L)  UA (μmol/L)  T3 (ng/mL)  T4 (ng/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  12.6  13.3  2.38  3.34  11.3  15.4  194  232  1.95  2.12  46.3  46.4    Cu82  12.9  13.1  2.57  3.05  12.3  13.7  177  211  1.94  2.07  45.0  61.4  CHT1,3  Cu0  13.3  13.0  2.41  3.11  10.6  15.4  174  210  1.81  2.10  47.0  46.4    Cu8  13.1  13.3  2.25  2.74  9.32  12.2  129  235  1.62  2.00  45.8  47.8    Pooled SEM  0.18  0.19  0.12  0.29  0.81  1.56  8.42  22.1  0.10  0.18  1.97  3.59  TEMP1,4  NT  12.8b  13.2  2.48  3.20  11.8a  14.6  186A  222  1.95a  2.10  45.7  53.9    CHT  13.2a  13.2  2.33  2.93  10.0b  13.8  152B  223  1.72b  2.05  46.4  47.1    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  Dietary Cu4  Cu0  13.0  13.2  2.40  3.23  11.0  15.4  184A  221  1.88  2.11  46.7  46.4b    Cu8  13.0  13.2  2.41  2.90  10.8  13.0  153B  223  1.78  2.04  45.4  54.6a    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  P-value5  TEMP  0.027  0.70  0.25  0.36  0.036  0.62  0.0006  0.98  0.028  0.79  0.73  0.074    Cu  1.00  0.70  0.91  0.27  0.88  0.12  0.002  0.93  0.33  0.70  0.54  0.033    TEMP × Cu  0.25  0.16  0.18  0.89  0.16  0.63  0.11  0.32  0.34  0.88  0.95  0.074  Item  GLU (mmol/L)  CHO (mmol/L)  TG (mmol/L)  UA (μmol/L)  T3 (ng/mL)  T4 (ng/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  12.6  13.3  2.38  3.34  11.3  15.4  194  232  1.95  2.12  46.3  46.4    Cu82  12.9  13.1  2.57  3.05  12.3  13.7  177  211  1.94  2.07  45.0  61.4  CHT1,3  Cu0  13.3  13.0  2.41  3.11  10.6  15.4  174  210  1.81  2.10  47.0  46.4    Cu8  13.1  13.3  2.25  2.74  9.32  12.2  129  235  1.62  2.00  45.8  47.8    Pooled SEM  0.18  0.19  0.12  0.29  0.81  1.56  8.42  22.1  0.10  0.18  1.97  3.59  TEMP1,4  NT  12.8b  13.2  2.48  3.20  11.8a  14.6  186A  222  1.95a  2.10  45.7  53.9    CHT  13.2a  13.2  2.33  2.93  10.0b  13.8  152B  223  1.72b  2.05  46.4  47.1    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  Dietary Cu4  Cu0  13.0  13.2  2.40  3.23  11.0  15.4  184A  221  1.88  2.11  46.7  46.4b    Cu8  13.0  13.2  2.41  2.90  10.8  13.0  153B  223  1.78  2.04  45.4  54.6a    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  P-value5  TEMP  0.027  0.70  0.25  0.36  0.036  0.62  0.0006  0.98  0.028  0.79  0.73  0.074    Cu  1.00  0.70  0.91  0.27  0.88  0.12  0.002  0.93  0.33  0.70  0.54  0.033    TEMP × Cu  0.25  0.16  0.18  0.89  0.16  0.63  0.11  0.32  0.34  0.88  0.95  0.074  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Table 6. Effects of environmental temperature and dietary Cu on plasma alkaline phosphatase (ALP), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH), creatine kinase (CK) activities, and corticosterone (CORT) level of hens at 48 to 51 wk of age Item  ALP (U/L)  GOT (U/L)  GPT (U/L)  LDH (U/L)  CK (U/L)  CORT (nmol/L)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  1148  2152  185  156  6.97  8.08  100b  106  18,734  19,985a,b  68.6  61.9    Cu82  1119  1750  177  160  6.72  7.58  94.9b  109  19,266  18,180b  62.5  62.3  CHT1,3  Cu0  1361  2184  186  152  6.61  7.67  138a  118  19,963  18,237b  66.7  61.6    Cu8  1164  1980  192  153  6.61  6.92  93.4b  101  19,303  21,895a  66.8  65.0    Pooled SEM  183  257  8.58  8.12  0.29  0.38  9.42  12.0  545  656  2.36  3.11  TEMP1,4  NT  1134  1951  181  158  6.85  7.83  97.5  108  19,000  19,083  65.6  62.1    CHT  1263  2082  189  153  6.61  7.30  116  110  19,633  20,066  66.8  63.3    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48  385  464  1.67  2.20  Dietary Cu4  Cu0  1255  2168  186  154  6.79  7.88  119  112  19,349  19,111  67.7  61.8    Cu8  1142  1865  185  157  6.67  7.25  94.2  105  19,285  20,038  64.7  63.7    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48.,  385  464  1.67  2.20  P-value5  TEMP  0.49  0.62  0.37  0.49  0.43  0.17  0.068  0.87  0.26  0.15  0.61  0.70    Cu  0.54  0.25  0.91  0.71  0.68  0.12  0.014  0.56  0.91  0.17  0.21  0.53    TEMP × Cu  0.65  0.71  0.42  0.83  0.67  0.75  0.049  0.42  0.29  0.0005  0.20  0.64  Item  ALP (U/L)  GOT (U/L)  GPT (U/L)  LDH (U/L)  CK (U/L)  CORT (nmol/L)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  1148  2152  185  156  6.97  8.08  100b  106  18,734  19,985a,b  68.6  61.9    Cu82  1119  1750  177  160  6.72  7.58  94.9b  109  19,266  18,180b  62.5  62.3  CHT1,3  Cu0  1361  2184  186  152  6.61  7.67  138a  118  19,963  18,237b  66.7  61.6    Cu8  1164  1980  192  153  6.61  6.92  93.4b  101  19,303  21,895a  66.8  65.0    Pooled SEM  183  257  8.58  8.12  0.29  0.38  9.42  12.0  545  656  2.36  3.11  TEMP1,4  NT  1134  1951  181  158  6.85  7.83  97.5  108  19,000  19,083  65.6  62.1    CHT  1263  2082  189  153  6.61  7.30  116  110  19,633  20,066  66.8  63.3    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48  385  464  1.67  2.20  Dietary Cu4  Cu0  1255  2168  186  154  6.79  7.88  119  112  19,349  19,111  67.7  61.8    Cu8  1142  1865  185  157  6.67  7.25  94.2  105  19,285  20,038  64.7  63.7    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48.,  385  464  1.67  2.20  P-value5  TEMP  0.49  0.62  0.37  0.49  0.43  0.17  0.068  0.87  0.26  0.15  0.61  0.70    Cu  0.54  0.25  0.91  0.71  0.68  0.12  0.014  0.56  0.91  0.17  0.21  0.53    TEMP × Cu  0.65  0.71  0.42  0.83  0.67  0.75  0.049  0.42  0.29  0.0005  0.20  0.64  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Both temperature and dietary Cu had no effect (P > 0.05) on the plasma contents of T-AOC and MDA and plasma CuZnSOD activity. No interactions between temperature and dietary Cu were observed in the above-mentioned three indices during the stages of 48–49, and 50–51 wk (Table 7). Table 7. Effects of environmental temperature and dietary Cu on plasma total antioxidant capacity (T-AOC), Cu and zinc superoxide dismutase (CuZnSOD) activity, and malondialdehyde (MDA) level of hens at 48 to 51 wk of age Item  T-AOC (U/mL)  CuZnSOD (NU/mL)  MDA (nmol/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  5.97  6.08  190  262  3.17  4.02    Cu82  6.36  6.11  190  271  3.67  4.64  CHT1,3  Cu0  5.69  6.72  188  264  3.46  4.07    Cu8  5.64  5.98  179  251  2.97  3.50    Pooled SEM  0.25  0.36  5.28  7.20  0.25  0.29  TEMP1,4  NT  6.17  6.10  190  267  3.42  4.33    CHT  5.67  6.35  184  258  3.22  3.79    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  Dietary Cu4  Cu0  5.83  6.40  189  263  3.32  4.05    Cu8  6.00  6.05  185  261  3.32  4.07    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  P-value5  TEMP  0.073  0.49  0.24  0.21  0.43  0.076    Cu  0.50  0.34  0.45  0.81  0.99  0.93    TEMP × Cu  0.39  0.31  0.40  0.14  0.064  0.065  Item  T-AOC (U/mL)  CuZnSOD (NU/mL)  MDA (nmol/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  5.97  6.08  190  262  3.17  4.02    Cu82  6.36  6.11  190  271  3.67  4.64  CHT1,3  Cu0  5.69  6.72  188  264  3.46  4.07    Cu8  5.64  5.98  179  251  2.97  3.50    Pooled SEM  0.25  0.36  5.28  7.20  0.25  0.29  TEMP1,4  NT  6.17  6.10  190  267  3.42  4.33    CHT  5.67  6.35  184  258  3.22  3.79    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  Dietary Cu4  Cu0  5.83  6.40  189  263  3.32  4.05    Cu8  6.00  6.05  185  261  3.32  4.07    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  P-value5  TEMP  0.073  0.49  0.24  0.21  0.43  0.076    Cu  0.50  0.34  0.45  0.81  0.99  0.93    TEMP × Cu  0.39  0.31  0.40  0.14  0.064  0.065  1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effect. View Large DISCUSSION In the current study, the RT was significantly increased by approximately 1°C when laying hens were exposed to CHT, indicating that all hens under CHT were in a heat stress status throughout heat stress period. However, the temperature did not affect RT in hens during the stage of 50–51 wk. This was probably because the temperature of 2 henhouses was maintained at normal temperature (26 ± 2°C) during the convalescent period. Hyperthermia severely depresses laying performance, feed intake of commercial laying hens (Emery et al., 1984; Star et al., 2008a). Therefore, the egg production and feed intake were significantly decreased in hens exposed to the CHT in the current study. Similar results were observed also in the previous studies with laying hens (Mashaly et al., 2004; Star et al., 2008b). Moreover, results from a recent study (Mack et al., 2013) showed that hens subjected to heat stress conditions spend less time eating, but more time drinking and panting. Meanwhile, heat stress could prolong the time of egg formation (Nordstrom, 1973), and disrupt the normal status of reproductive hormones in the hypothalamus (Donoghue et al., 1989) and ovary (Rozenboim et al., 2007). The lack of feed intake is very likely the starting point of the most detrimental effects of heat stress on production, leading to a decreased BW, feed efficiency, egg production and quality (Mashaly et al., 2004). Thus, the decrease in the feed: egg ratio and BW of the heat stressed hens observed in this study probably was associated with the decreased feed intake and this adverse effect actually lasted into the convalescent period. The heat stress could alter some plasma endocrine and biochemical indices involved in various aspects of reproductive performance and nutrient metabolism in commercial layers (Star et al., 2008a,b). The plasma GLU, CHO, and TG levels have been used as indicators of stress in fowls (Odihambo Mumma et al., 2006). Usually the glucocorticoids is increased in circulating system of hens under stressed status, which could induce gluconeogenesis and then lead to an elevation of plasma GLU (Nagra and Meyer, 1963). Moreover, the glucocorticoids also inhibit GLU uptake into peripheral tissues by antagonizing the effect of insulin (Buren et al., 2002). In consistent with the previous reports (Sahin and Kucuk, 2001; Star et al., 2008a), we found that the plasma GLU level significantly increased in hens under CHT, but TG level decreased. Geraert et al. (1996) reported that endocrinological changes caused by heat stress stimulate lipid accumulation via increasing in de novo lipogenesis, reducing lipolysis, and enhancing amino acid catabolism. We noticed that the plasma TG and UA levels were decreased in hens under CHT, which appeared to be consistent with the results obtained by Geraert et al. (1996) and Lin et al. (2008) and Song et al. (2012). The laying hens could not maintain their performance after 7 d of heat exposure even by the mobilization of energy and nitrogen stores in this study. This consequence might be caused by insufficient protein deposits, as indicated by the lower UA detected in the plasma of heat-exposed hens (Song et al., 2012). Body temperature and metabolic activity are regulated by T3, T4 and their balance. The decreased plasma T3 level is indicative of the physiological adaptation to heat exposure (Lara and Rostagno, 2013). In this study, CHT decreased plasma T3 level but had no effect on plasma T4 level, which was in agreement with the result of Mack et al. (2013). A number of enzymes are used in the clinical biochemistry as tools for differential diagnosis, such as ALP, GOT, GPT, LDH, and CK. Since they are located in different tissues, their abnormal appearance in plasma can indicate specific muscle or organ damages (Pech-Waffenschmidt, 1992). Significant increases in activities of GOT, GPT, LDH, and CK were observed in plasma of chickens exposed to high temperature (Melesse et al., 2011). CHT had no effect on plasma ALP, GOT, GPT, LDH and CK activities, and CORT level in the present study. According to the results obtained by Pech-Waffenschmidt et al. (1995), heat exposure did not significantly change the enzyme activities in the laying hens’ serum. This was also supported by previous findings of Ward and Peterson (1973), who reported that the activities of GOT and CK were not influenced even by acute heat exposure. Clearly, plasma GLU, CHO, and TG levels are stress indicators in fowls, however, CORT is not accepted by all as a stress response in laying hens (Odihambo Mumma et al., 2006). The magnitude and duration of heat stress imposed on birds may affect their metabolic response of the stressed hens, resulting in changes in stress indicators in plasma. However, the possible mechanisms which might have caused such conflicting responses in enzyme activities in heat stressed birds remain unclear. The adverse effect of CHT on plasma biochemical indices did not last into the convalescent period in this study, probably due to normal ambient temperature during 50–51 wk, but the possible mechanisms need to be further studied. Chiou et al. (1997) reported that White Leghorn layers fed diet supplemented with 200 mg Cu/kg feed in the form of CuSO4·5H2O had higher egg production than diet with 0, 400, 600, 800 mg Cu/kg feed. Similar results were also reported by Pesti and Bakalli (1998). However, the supplementation of Cu over 200 mg/kg in the diet reduced the feed intake of Hy-Line Brown laying hens (Kim et al., 2016) and egg production of white light hybrid hens (Pearce et al., 1983). These results suggest that there are negative impacts on productive performance of hens with Cu supplementation over 200 mg/kg feed. Further studies indicated that dietary supplementation of Cu in the range of 0–300 mg/kg feed had no effect on feed intake and egg production of Hisex-Brown hens (Balevi and Coskun, 2004). A possible explanation for such contradictory reports might be the different breeds of laying hens and added amount of Cu additives. Indeed, there was no difference in laying rate between Cu0 and Cu8 under NT in the current study. However, there was a decreasing trend in the laying rate of hens in CHT-Cu8 in the second week of heat stress (49 wk), and the laying rate of CHT-Cu8 was significantly decreased in the first week of convalescence (50 wk). Mooyoung et al. (1970) found that the isthmus of the oviduct was rich in Cu. It is speculated that laying hens have lower tolerance of the Cu toxicity under heat stress, which has an adverse effect on the normal function of the isthmus. However, the exact reasons for the decreased laying rate of hens in CHT-Cu8 remain unclear. In the present study, there was a significant decrease in egg weight in Cu0, which was in agreement with the result of Pekel and Alp (2011). The effect of dietary Cu and the interaction between temperature and Cu had no influence on other laying performance except for laying rate and egg weight in the current study, which might mostly be related to the low added level of Cu compared to other studies (Chiou et al., 1997; Balevi and Coskun, 2004; Kim et al., 2016), but the possible mechanisms need to be further elucidated. Diet supplemented with 8 mg of Cu (Cu8) significantly decreased the plasma UA level compared to the diet with no supplementation of Cu (Cu0) during the heat stress period, indicating that laying hens are very sensitive to the dietary Cu level under heat stress. UA has been proposed to be a potent scavenger of free radicals in human and poultry (Becker, 1993) and its formation is considered as one of the mechanisms leading to a longer lifespan of birds (Simoyi et al., 2003). The elevated plasma T4 of hens in Cu8 group during the convalescent period might be the result of a decreased peripheral deiodination in the hens after heat exposure as reported previously by Kühn et al. (1987). ALP, GOT, GPT, and LDH are widely distributed in liver, kidney, heart and muscle cells, and CK is the specific enzyme in heart and muscle tissue as shown by Mitchell and Sandercock (1995). Chiou et al. (1997) reported that serum LDH and CK activities of hens with Cu supplementation over 500 mg/kg feed were significantly increased. In the present study, the plasma LDH activity of hens in CHT-Cu0 and the plasma CK activity of hens in CHT-Cu8 were increased during the heat stress period and convalescent period, respectively. This implies that kidney, heart, and muscle damage has occurred and leads to the release of the enzymes from cells to the blood stream when plasma LDH and CK activities increase. Heat stress can enhance the formation of reactive oxygen species (ROS) that cause oxidative injury such as lipid peroxidation (Flanagan et al., 1998). The antioxidative enzyme system (comprising SOD, glutathione peroxidase, and catalase) acts as the first line of antioxidant defense. Modification of these enzymes activities can alter the balance between the ROS production and the antioxidant system. Cu is the cofactor of the CuZnSOD, which could scavenge free radicals. The integrative index of T-AOC reflects the activity of scavenging free radicals (Lewis et al., 1995). Previous studies demonstrated a significant increase in free radicals production together with an increase in the expression of antioxidant enzymes under heat stress (Sahin and Kucuk, 2001). These increases in antioxidant enzyme activities have been considered to be a protective response against oxidative stress. Thus, it is implied that the balance has already been disturbed by heat stress. MDA is the biomarker of lipid peroxidation (Lin et al., 2008). Moreover, the relationship in biological systems between lipid peroxidation and high temperature has been discussed previously (Borisiuk and Zinchuk, 1995). In the present study, both temperature and dietary Cu had no effect on plasma T-AOC, CuZnSOD, and MDA levels, and no interactions between temperature and Cu were observed in the three plasma indices. A possible explanation for such contradictory results might be the short duration of heat stress and all hens have 12 h normal temperature each day during heat stress period. Meanwhile, age and genotype are crucial factors for resilience to heat stress in laying hens (Mignon-Grasteau et al., 2015), which imply that the hens have robust adaptive ability at the 48–51 wk of age. Moreover, the added amount Cu in the present study is lower than the previous studies (Pesti and Bakalli, 1998; Kim et al., 2016; Yang et al., 2017). Considering the UA has been proposed to be a potent scavenger of free radicals, and thyroid hormones could accelerate the oxidative metabolism via an increase in the mitochondria mass, mitochondria cytochrome content, and respiratory rate (Asayama et al., 1987; Lin et al., 2008). It might suggest that the scavengers of free radicals induced by oxidative injury contain many substances except for the antioxidative enzyme system. CONCLUSION Cyclic high temperature (26 ± 2°C∼33 ± 2°C) impaired laying performance. 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No copper supplementation in a corn-soybean basal diet has no adverse effects on late-phase laying hens under normal and cyclic high temperatures

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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/pex447
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Abstract

Abstract Over supplementation of copper (Cu) in animal diets may cause serious pollution in soil, water and harvested crops. To minimize the potential pollution, the effects of corn-soybean basal diet with or without supplementation of 8 mg Cu/kg on laying performance, plasma biochemical metabolic indices, and antioxidant status in laying hens were evaluated under normal and cyclic high temperatures. A total of 240 Hy-Line Brown laying hens were randomly allotted to 4 treatments with 6 replicates of 10 hens per replicate according to factorial design involved in 2 temperatures [normal temperature (NT) vs. cyclic high temperature (CHT)] and 2 dietary Cu addition amount [Cu0 (0 mg/kg) vs. Cu8 (8 mg/kg in the form of CuSO4·5H2O)]. The experimental period included 1-week adaptation, 2-week heat stress and 2-week convalescence. The temperatures of NT groups in the same period or any groups during other periods were kept at 26 ± 2°C except that of CHT groups were 26 ± 2°C∼33 ± 2°C cyclically during heat stress period. CHT groups increased (P < 0.05) the rectal temperature and plasma glucose content under heat stress, but decreased (P < 0.01) the egg yield at the second week of heat stress and the first week of convalescence, and the plasma triglyceride, uric acid, and triiodothyronine levels under heat stress. Cu8 groups increased (P < 0.05) egg weight at the first week of convalescence, and plasma thyroxin level during the whole convalescence. Interactions between temperature and Cu content existed (P < 0.05) in the laying rate at the first week of convalescence, and the plasma lactic dehydrogenase level under heat stress. Conclusively, the CHT impaired laying performance. The Cu content (10.3 mg/kg) in corn-soybean basal diet might be sufficient for meeting the maintenance and production requirements of late-phase laying hens, and no Cu supplementation had no adverse effects on egg production and antioxidant indices under cyclic high (26 ± 2°C∼33 ± 2°C) or normal (26 ± 2°C) temperatures. INTRODUCTION Heat stress is of great concern in all types of poultry operations. High environmental temperature can cause a significant reduction in egg production and eggshell thickness of commercial laying hens (Emery et al., 1984; Star et al., 2008a). High environmental temperature also induces a series of physiological and metabolic changes such as elevated rectal temperature (Mashaly et al., 2004) and plasma creatine kinase activity and decreased plasma triiodothyronine levels in laying hens (Star et al., 2008b). Moreover, heat stress from high environmental temperature can result in oxidative stress and lipid peroxidation with an elevated level of malondialdehyde (MDA) in the plasma of laying hens (Sahin and Kucuk, 2001; Lin et al., 2008). Copper (Cu) is an essential element in the diet of poultry due to its biological activity. It is a cofactor of many enzymes such as superoxide dismutase (SOD), cytochrome oxidase, and ceruloplasmin (Prohaska et al., 1983). Therefore, the importance of Cu in animal nutrition is associated with its roles in many biological processes including antioxidant activity, immune function, and neuropeptide synthesis (Bonham et al., 2002). According to the National Research Council (NRC), the requirement for Cu is 4.0–5.0 mg/kg diet in laying hens (0–18 wk). However, there is still uncertainty about the need for Cu in laying hens after 18 wk (NRC, 1994). Basal concentration in a corn-soybean meal diet without supplemented Cu containing 9.2 mg Cu/kg DM seems to be enough for maintaining egg production and shell quality of laying hens (ISA Brown, 45–53 wk) (Skrivan and Skrivanova, 2006). When dietary Cu concentration was decreased from 12 to 4 mg/kg, no effect was observed in growth rate, feed conversion and mortality in Ross 308 broilers at the age of 0–17 d (Dozier et al., 2003). Farmers in China believe that the supplementation of Cu in diets of laying hens (from 4 to 25 mg/kg feed) ought to alleviate the negative effects of heat stress in hens and the recommended supplementation in diet is 8 mg Cu/kg feed for laying hens in China's Feeding Standard of Chicken (Ministry of Agriculture of People's Republic of China, 2004). Hubert et al. (1989) studied the effect of diet supplemented with 250 mg Cu/kg feed on pigs under different temperatures, demonstrating that there was no interaction between environmental temperature and Cu supplementation in daily gain, feed consumption, and feed per unit of the gain of pigs. Moreover, dietary 110 mg Cu/kg DM (Yang et al., 2017) or 600 mg Cu/kg feed (Chiou et al., 1997) may induce acute or chronic toxicity because the oxidative damage and cell death is observed in vivo with a high level of Cu (10 μmol/L Cu2+) in Leibovitz L15 medium (Nawaz et al., 2005). Result from previous study also indicated that only 20% of the supplemented Cu could be digested and absorbed. The rest is excreted with feces that may result in pollution in plant, soil, and aquatic environments, subsequently impacting human health (Zhao et al., 2010). Therefore, it is not clear whether Cu should be supplemented in the diet of the laying hens after 18 wk. In order to understand the optimal application of Cu in laying hens especially under heat stress, and to minimize the potential pollution in the environment, this study was conducted to assess the effects of diets with or without supplementation of Cu on laying performance, plasma biochemical, and antioxidant indices of laying hens under normal and cyclic high temperatures. MATERIALS AND METHODS Experimental Design and Treatments A completely randomized design with a 2 temperatures (normal 26 ± 2°C (NT) vs. cyclic high temperature (CHT, 26 ± 2°C∼33 ± 2°C) × 2 dietary Cu supplementation levels (0 mg Cu (Cu0) vs. 8 mg Cu/kg feed (Cu8) in the form of CuSO4·5H2O) factorial arrangement was used. Thus, there were a total of 4 treatments (NT-Cu0, NT-Cu8, CHT-Cu0, and CHT-Cu8) in this experiment. Animals and Diets The protocol was reviewed and approved by the Animal Care and Use Committee of China Agricultural University. All procedures were performed strictly in accordance with the guidelines of recommendations in the Guide for Experimental Animals of the Ministry of Science and Technology (Beijing, China), and all efforts were made to minimize suffering. Two hundred and forty 47-week-old Hy-Line Brown laying hens with a similarity in laying rate (89.3 ± 0.93%) and body weight (BW, 1.68 ± 0.05 kg) were allotted randomly to one of 4 treatments with 6 replicates of 10 hens per replicate. Ten hens in each replicate were kept in five neighboring ladder stainless steel cages with 2 hens per cage. The cage size was 0.45 m wide × 0.45 m deep × 0.45 m height. Laying hens were maintained on a 16-h light schedule and allowed ad libitum access to experimental diets and water. The experimental period was 5 weeks including 1 week of adaptation (47 wk), 2 weeks of heat stress (48–49 wk) and 2 weeks of convalescence (50–51 wk). During the adaptation period, all laying hens were fed with the corn-soybean basal diet (Table 1) with no Cu addition. During the heat stress period, the room temperature for hens in treatments of NT-Cu0, NT-Cu8 was maintained at 26 ± 2°C, whereas the room temperature for hens in treatments of CHT-Cu0, CHT-Cu8 was increased step-wise from 26 ± 2°C to 33 ± 2°C in 2 h (between 07:00 and 09:00), and maintained at 33 ± 2°C for 8 h (between 09:00 and 17:00), and then decreased step-wise from 33 ± 2°C to 26 ± 2°C (between 17:00 and 19:00), and maintained at 26 ± 2°C until 07:00 of the next day. During the convalescent period, both of the two rooms were maintained at 26 ± 2°C. The temperature program is referred to the method of Mashaly et al. (2004). Relative humidity was kept at 50 ± 10% for the two rooms during the experimental period of 5 weeks (47–51 wk of age). Table 1. Composition and nutrient levels of basal diet (as-fed basis) Ingredient  Inclusion (%, unless noted)  Nutrient3  Nutrient composition (%, unless noted)  Corn  63.68  AME (MJ/kg)  11.70  Soybean meal, 44% CP  24.80  CP  15.40  Limestone (CaCO3)  9.00  Ca  3.64  Dicalcium phosphate  1.60  Available P  0.35  Salt  0.30  Methionine  0.36  DL-Methionine  0.11  Methionine + cysteine  0.58  L-Lysine HCL (98.5%)  0.08  Lysine  0.81  Threonine  0.02  Threonine  0.61  Tryptophan  0.02  Tryptophan  0.18  Vitamin premix1  0.04  Cu (mg/kg)  10.27  Mineral premix2  0.35      Total  100.00      Ingredient  Inclusion (%, unless noted)  Nutrient3  Nutrient composition (%, unless noted)  Corn  63.68  AME (MJ/kg)  11.70  Soybean meal, 44% CP  24.80  CP  15.40  Limestone (CaCO3)  9.00  Ca  3.64  Dicalcium phosphate  1.60  Available P  0.35  Salt  0.30  Methionine  0.36  DL-Methionine  0.11  Methionine + cysteine  0.58  L-Lysine HCL (98.5%)  0.08  Lysine  0.81  Threonine  0.02  Threonine  0.61  Tryptophan  0.02  Tryptophan  0.18  Vitamin premix1  0.04  Cu (mg/kg)  10.27  Mineral premix2  0.35      Total  100.00      1Provided per kilogram of diet: 8500 IU of vitamin A (retinol acetate); 3600 IU of vitamin D3; 21 IU of vitamin E (DL-α-tocopherolacetate); 4.2 mg of vitamin K3 (menadione dimethpyrimidinol); 3.0 mg of vitamin B1 (thiamin mononitrate); 10.2 mg of vitamin B2 (riboflavin); 45 mg of niacin; 15 mg of calcium pantothenate; 5.4 mg of vitamin B6 (pyridoxine); 0.15 mg of biotin; 0.9 mg of folic acid and 0.024 mg of vitamin B12. 2Provided per kilogram of diet: 60 mg of Fe (FeSO4·7H2O), 80 mg of Zn (ZnSO4·7H2O), 60 mg of Mn (MnSO4·H2O), 0.35 mg of I (KI), and 0.3 mg of Se (Na2SeO3). Cu additive was added to diets by replacing an equal weight of corn starch. 3CP, Ca, and Cu levels were analyzed. Each value was based on triplicate determinations, but all other nutrient levels were calculated. View Large Both BW and rectal temperature (RT) of laying hens were measured at 12:00 on the last day of each week. The RT was monitored using a thermo-code electric gauge (JM222, JinMing, Tianjin, China) with an accuracy of 0.1°C. Eggs were collected daily at 15:00 and the number of eggs and egg weight in each replicate were recorded. The feed intake of laying hens in each replicate was recorded at 20:00 on the last day of each week of the experimental period. The basal corn-soybean basal diet (Table 1) was formulated to meet or exceed the nutrient requirements for laying hens (NRC, 1994), except for Cu, which was added to the basal diet according to the experimental design. Copper sulfate pentahydrate was reagent grade (Beijing Chemical Company, Beijing, China), and contained 25.5% Cu on a basis of analysis (purity > 99%). A single batch of basal diet was mixed and then divided into two aliquots according to the experimental treatments. The content of Cu in tap wat0er was undetectable. The analyzed content of Cu in diets of Cu0 and Cu8 were 10.3, 17.6 mg/kg, respectively. Sample Collections and Preparations The feed ingredients and diet samples from all the treatments were collected and analyzed for CP, Ca, Cu before the initiation of the experiment to confirm the correct preparation of diets. Blood samples were collected via a bronchial vein from 2 fasted laying hens with average BW in each replicate at 12:00 on the last day of each week for the heat stress and convalescent periods. Plasma samples were obtained by centrifuging blood samples at 3000 × g for 20 min at 4°C and stored in −20°C for further analyses. All samples from 2 hens in each replicate were pooled into one sample in equal volume before analysis. Sample Analyses CP, Ca, Cu Contents. Concentrations of CP in feed ingredients and diet samples were determined using the Association of Official Analytical Chemists methods (AOAC, 1990). The concentrations of Cu in water and diets, and Ca in feed ingredients and diet samples, were measured using an inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA, USA) after wet digestions using HNO3 and HCLO4 as described by Zhao et al. (1994). Plasma Biochemical Parameters. Plasma glucose (GLU), cholesterol (CHO), triglyceride (TG), uric acid (UA) contents and activities of alkaline phosphatase (ALP), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH), and creatine kinase (CK) were measured using a HITACHI 7600 automatic biochemical analyzer (Hitachi Co., Ltd, Tokyo, Japan) with the detection kits (Prodia diagnostics, Boetzingen, Germany), respectively. Plasma triiodothyronine (T3), thyroxin (T4), and corticosterone (CORT) levels were measured using a Sn-69,513 RIA counter (Shanghai nuclear power Co., Ltd, Shanghai, China) with the detection kits (Jiuding medical biological engineering Co., Ltd, Tianjin, China). Plasma total antioxidant capacity (T-AOC) content was measured using the specific detection kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) with a microplate spectrophotometer (Epoch, BioTek Instruments Inc., Winooski, VT). Plasma Cu and zinc superoxide dismutase (CuZnSOD) activity was measured using the nitrite method as described by Kobayashi et al. (1978). Plasma malondialdehyde (MDA) content was determined using the thiobarbituric acid colorimetric method as described by Mak et al. (1983). Statistical Analyses Data were analyzed by 3-way ANOVA using the PROC GLM procedure of SAS (2010, release 9.2, SAS Institute Inc., Cary, NC, USA), and the model included temperature, dietary Cu, period and the interactions between these factors. The period is referred to the 48–49 wk (2-week heat stress), and the 50–51 wk (2-week convalescence), respectively. The replicate served as the experimental unit. No interactions (P > 0.05) between period and temperature or dietary Cu were observed for RT, daily feed intake, feed: egg ratio, BW, morality, and plasma biochemical parameters. Therefore, the main effects of temperature, dietary Cu and their interaction on the above indices were presented for the heat stress period (48–49 wk) and the convalescent period (50–51wk). Because the significant interactions (P < 0.05) between period and temperature were observed for laying rate, egg weight, and egg yield, the main effects of temperature, dietary Cu and their interaction on these three indices were reported for each week during the heat stress and convalescent period. Data of hen mortality was transformed to arc sine values before statistical analysis. Differences among means were tested using the LSD method, and statistical significance was set at P ≤ 0.05. RESULTS Rectal Temperature Dietary Cu did not affect (P > 0.05) RT during the experimental period (47–51 wk). The temperature impacted (P < 0.01) RT during the heat stress period (48–49 wk) but not during the adaptation (47 wk) and convalescent periods (50–51 wk). Compared with NT, CHT increased (P < 0.01) the RT during 48–49 wk of age. No interactions between dietary Cu and temperature were detected (Table 2). Table 2. Effects of environmental temperature and dietary Cu on rectal temperature (RT) of hens at 47 to 51 wk of age Item  RT (°C)    47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  39.8  39.9  40.4    Cu82  40.0  39.8  40.3  CHT1,3  Cu0  39.9  40.7  40.6    Cu8  39.9  40.8  40.3    Pooled SEM  0.08  0.10  0.08  TEMP1,4  NT  39.9  39.8B  40.3    CHT  39.9  40.8A  40.5    Pooled SEM  0.06  0.07  0.05  Dietary Cu4  Cu0  39.9  40.3  40.5    Cu8  40.0  40.3  40.3    Pooled SEM  0.06  0.07  0.05  P-value5  TEMP  0.73  <0.0001  0.082    Cu  0.26  0.97  0.064    TEMP × Cu  0.18  0.50  0.22  Item  RT (°C)    47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  39.8  39.9  40.4    Cu82  40.0  39.8  40.3  CHT1,3  Cu0  39.9  40.7  40.6    Cu8  39.9  40.8  40.3    Pooled SEM  0.08  0.10  0.08  TEMP1,4  NT  39.9  39.8B  40.3    CHT  39.9  40.8A  40.5    Pooled SEM  0.06  0.07  0.05  Dietary Cu4  Cu0  39.9  40.3  40.5    Cu8  40.0  40.3  40.3    Pooled SEM  0.06  0.07  0.05  P-value5  TEMP  0.73  <0.0001  0.082    Cu  0.26  0.97  0.064    TEMP × Cu  0.18  0.50  0.22  a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Laying Performance All data are shown in Tables 3 and 4. No difference (P > 0.05) was found on laying performance at the beginning of the study (47 wk of age). Compared to NT, CHT decreased (P < 0.01) the laying rate at 49 wk of age, egg yield at 49 and 50 wk of age, daily feed intake and feed: egg ratio during the stage of 48–49 wk. The BW (P < 0.04) was also decreased during the stages of 48–49 wk, and 50–51 wk, but the feed: egg ratio was increased (P < 0.03) during 50–51 wk of age in hens under CHT as compared to NT. Compared to Cu0, hens fed with Cu8 diet had a higher (P < 0.04) egg weight at 50 wk of age. No interactions between temperature and dietary Cu were observed in any of the above-mentioned indices except for the laying rate (P < 0.04) at 50 wk of age, and egg weight (P < 0.03) during the heat stress period. No difference between dietary Cu treatments was observed in the laying rate of the hens under NT at 50 wk of age. However, hens in CHT-Cu8 had a decreased (P < 0.01) laying rate compared to NT-Cu8 with no difference (P > 0.30) between CHT-Cu0 and NT-Cu0. Moreover, hens in CHT-Cu0 had decreased (P < 0.03) egg weight compare to CHT-Cu8 with no difference (P > 0.35) between NT-Cu0 and NT-Cu8 during 48 and 49 wk of age. Table 3. Effects of environmental temperature and dietary Cu on laying rate, egg weigh, and egg yield of hens at 47 to 51 wk of age Item  Laying rate (%)  Egg weight (g)  Egg yield (g/hen·d)    47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  NT1,3  Cu02  88.3  88.6  88.0  84.3a,b  87.7  61.3  61.4a  62.0a  60.9  60.7  54.2  54.4  54.5  51.3  53.2    Cu82  90.5  89.3  88.7  88.4a  83.3  60.9  60.7a  61.7a  61.3  60.9  55.1  54.3  54.7  54.2  50.7  CHT1,3  Cu0  88.9  85.4  81.0  82.1b,c  85.7  61.0  58.7b  57.6c  59.2  59.7  54.2  50.1  46.7  48.6  51.2    Cu8  89.4  86.9  79.6  78.0c  84.8  61.1  60.6a  59.6b  61.1  61.6  54.6  52.5  47.5  47.7  52.1    Pooled SEM  1.72  2.57  2.53  1.74  2.30  0.53  0.54  0.44  0.52  0.54  1.11  1.59  1.56  0.97  1.41  TEMP1,4  NT  89.4  88.9  88.3A  86.3  85.5  61.1  61.1  61.8  61.1  60.8  54.7  54.3  54.6A  52.7A  52.0    CHT  89.2  86.1  80.3B  80.0  85.2  61.1  59.6  58.6  60.2  60.6  54.4  51.3  47.1B  48.1B  51.6    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  Dietary Cu4  Cu0  88.6  87.0  84.5  83.2  86.7  61.2  60.0  59.8  60.0b  60.2  54.2  52.2  50.6  49.9  52.2    Cu8  90.0  88.1  84.1  83.2  84.0  61.0  60.6  60.6  61.2a  61.2  54.9  53.4  51.1  50.9  51.4    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  P-value5  TEMP  0.88  0.29  0.005  0.002  0.91  0.84  0.013  <0.0001  0.093  0.72  0.82  0.071  0.0001  0.0001  0.82    Cu  0.46  0.68  0.89  0.99  0.26  0.83  0.27  0.079  0.035  0.068  0.54  0.47  0.77  0.33  0.60    TEMP × Cu  0.62  0.89  0.67  0.030  0.46  0.64  0.028  0.019  0.18  0.14  0.81  0.43  0.87  0.063  0.24  Item  Laying rate (%)  Egg weight (g)  Egg yield (g/hen·d)    47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  47 wk  48 wk  49 wk  50 wk  51 wk  NT1,3  Cu02  88.3  88.6  88.0  84.3a,b  87.7  61.3  61.4a  62.0a  60.9  60.7  54.2  54.4  54.5  51.3  53.2    Cu82  90.5  89.3  88.7  88.4a  83.3  60.9  60.7a  61.7a  61.3  60.9  55.1  54.3  54.7  54.2  50.7  CHT1,3  Cu0  88.9  85.4  81.0  82.1b,c  85.7  61.0  58.7b  57.6c  59.2  59.7  54.2  50.1  46.7  48.6  51.2    Cu8  89.4  86.9  79.6  78.0c  84.8  61.1  60.6a  59.6b  61.1  61.6  54.6  52.5  47.5  47.7  52.1    Pooled SEM  1.72  2.57  2.53  1.74  2.30  0.53  0.54  0.44  0.52  0.54  1.11  1.59  1.56  0.97  1.41  TEMP1,4  NT  89.4  88.9  88.3A  86.3  85.5  61.1  61.1  61.8  61.1  60.8  54.7  54.3  54.6A  52.7A  52.0    CHT  89.2  86.1  80.3B  80.0  85.2  61.1  59.6  58.6  60.2  60.6  54.4  51.3  47.1B  48.1B  51.6    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  Dietary Cu4  Cu0  88.6  87.0  84.5  83.2  86.7  61.2  60.0  59.8  60.0b  60.2  54.2  52.2  50.6  49.9  52.2    Cu8  90.0  88.1  84.1  83.2  84.0  61.0  60.6  60.6  61.2a  61.2  54.9  53.4  51.1  50.9  51.4    Pooled SEM  1.21  1.82  1.79  1.23  1.63  0.36  0.38  0.31  0.37  0.38  0.78  1.12  1.11  0.68  1.00  P-value5  TEMP  0.88  0.29  0.005  0.002  0.91  0.84  0.013  <0.0001  0.093  0.72  0.82  0.071  0.0001  0.0001  0.82    Cu  0.46  0.68  0.89  0.99  0.26  0.83  0.27  0.079  0.035  0.068  0.54  0.47  0.77  0.33  0.60    TEMP × Cu  0.62  0.89  0.67  0.030  0.46  0.64  0.028  0.019  0.18  0.14  0.81  0.43  0.87  0.063  0.24  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Table 4. Effects of environmental temperature and dietary Cu on daily feed intake, feed: egg ratio, body weight (BW), and mortality of hens at 47 to 51 wk of age Item  Daily feed intake (g/hen·d)  Feed:egg ratio (g/g)  BW (kg)  Mortality (%)    47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  98.0  95.3  102  1.85  1.75  2.05  1.64  1.69  1.66  1.67  1.67  0.00    Cu82  105  101  106  1.93  1.85  2.09  1.71  1.74  1.72  0.00  0.00  0.00  CHT1,3  Cu0  105  75.1  105  1.96  1.57  2.16  1.66  1.62  1.65  0.00  1.67  1.85    Cu8  105  74.2  108  1.95  1.48  2.26  1.66  1.65  1.65  0.00  5.00  0.00    Pooled SEM  2.09  2.37  2.05  0.05  0.05  0.06  0.02  0.03  0.02  0.83  2.07  0.93  TEMP1,4  NT  102  98.0A  104  1.89  1.80A  2.07b  1.68  1.72A  1.69a  0.84  0.83  0.00    CHT  105  74.7B  106  1.96  1.53B  2.21a  1.66  1.64B  1.65b  0.00  3.33  0.93    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  Dietary Cu4  Cu0  102  85.2  103  1.91  1.66  2.11  1.65  1.66  1.66  0.84  1.67  0.93    Cu8  105  87.4  107  1.94  1.67  2.18  1.69  1.70  1.69  0.00  2.50  0.00    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  P-value5  TEMP  0.11  <0.0001  0.30  0.23  <0.0001  0.020  0.39  0.007  0.038  0.33  0.26  0.33    Cu  0.091  0.37  0.085  0.52  0.87  0.26  0.065  0.17  0.19  0.33  0.84  0.33    TEMP × Cu  0.14  0.21  0.74  0.43  0.10  0.58  0.071  0.65  0.11  0.33  0.26  0.33  Item  Daily feed intake (g/hen·d)  Feed:egg ratio (g/g)  BW (kg)  Mortality (%)    47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  47 wk  48–49 wk  50–51 wk  NT1,3  Cu02  98.0  95.3  102  1.85  1.75  2.05  1.64  1.69  1.66  1.67  1.67  0.00    Cu82  105  101  106  1.93  1.85  2.09  1.71  1.74  1.72  0.00  0.00  0.00  CHT1,3  Cu0  105  75.1  105  1.96  1.57  2.16  1.66  1.62  1.65  0.00  1.67  1.85    Cu8  105  74.2  108  1.95  1.48  2.26  1.66  1.65  1.65  0.00  5.00  0.00    Pooled SEM  2.09  2.37  2.05  0.05  0.05  0.06  0.02  0.03  0.02  0.83  2.07  0.93  TEMP1,4  NT  102  98.0A  104  1.89  1.80A  2.07b  1.68  1.72A  1.69a  0.84  0.83  0.00    CHT  105  74.7B  106  1.96  1.53B  2.21a  1.66  1.64B  1.65b  0.00  3.33  0.93    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  Dietary Cu4  Cu0  102  85.2  103  1.91  1.66  2.11  1.65  1.66  1.66  0.84  1.67  0.93    Cu8  105  87.4  107  1.94  1.67  2.18  1.69  1.70  1.69  0.00  2.50  0.00    Pooled SEM  1.48  1.72  1.45  0.04  0.04  0.04  0.01  0.02  0.01  0.59  1.47  0.65  P-value5  TEMP  0.11  <0.0001  0.30  0.23  <0.0001  0.020  0.39  0.007  0.038  0.33  0.26  0.33    Cu  0.091  0.37  0.085  0.52  0.87  0.26  0.065  0.17  0.19  0.33  0.84  0.33    TEMP × Cu  0.14  0.21  0.74  0.43  0.10  0.58  0.071  0.65  0.11  0.33  0.26  0.33  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Plasma Biochemical Parameters The results on plasma GLU, CHO, TG, UA, T3, T4 levels, and ALP, GOT, GPT, LDH, CK activities, and CORT level of hens are given in Tables 5 and 6. CHT decreased plasma TG (P < 0.04), UA (P < 0.01), and T3 (P < 0.03) levels, but increased plasma GLU (P < 0.03) level compared to NT during the stage of 48–49 wk. Compared to hens with Cu0, hens fed with Cu8 diet had a lower (P < 0.01) plasma UA concentration during the 48–49 wk of age, but a higher (P < 0.04) T4 level during the stage of 50–51 wk. There were interactions between temperature and dietary Cu in plasma LDH (P < 0.05) activity during the stage of 48–49 wk, and CK (P < 0.01) activity during the stage of 50–51 wk. No interactions between temperature and dietary Cu were observed in any other plasma biochemical indices. No difference was observed in plasma LDH activity between dietary Cu treatments under NT during the stage of 48–49 wk. However, the hens in CHT-Cu0 had a higher (P < 0.01) LDH activity compared to CHT-Cu8 with no difference (P > 0.90) between CHT-Cu8 and NT-Cu8. No difference was observed also in plasma CK activity between dietary Cu treatments under NT during the stage of 50–51 wk, but the hens in CHT-Cu8 had a higher (P < 0.01) CK activity compared to CHT-Cu0 with no difference (P > 0.05) between CHT-Cu0 and NT-Cu0. Table 5. Effects of environmental temperature and dietary Cu on plasma glucose (GLU), cholesterol (CHO), triglyceride (TG), uric acid (UA) triiodothyronine (T3), and thyroxin (T4) levels of hens at 48 to 51 wk of age Item  GLU (mmol/L)  CHO (mmol/L)  TG (mmol/L)  UA (μmol/L)  T3 (ng/mL)  T4 (ng/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  12.6  13.3  2.38  3.34  11.3  15.4  194  232  1.95  2.12  46.3  46.4    Cu82  12.9  13.1  2.57  3.05  12.3  13.7  177  211  1.94  2.07  45.0  61.4  CHT1,3  Cu0  13.3  13.0  2.41  3.11  10.6  15.4  174  210  1.81  2.10  47.0  46.4    Cu8  13.1  13.3  2.25  2.74  9.32  12.2  129  235  1.62  2.00  45.8  47.8    Pooled SEM  0.18  0.19  0.12  0.29  0.81  1.56  8.42  22.1  0.10  0.18  1.97  3.59  TEMP1,4  NT  12.8b  13.2  2.48  3.20  11.8a  14.6  186A  222  1.95a  2.10  45.7  53.9    CHT  13.2a  13.2  2.33  2.93  10.0b  13.8  152B  223  1.72b  2.05  46.4  47.1    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  Dietary Cu4  Cu0  13.0  13.2  2.40  3.23  11.0  15.4  184A  221  1.88  2.11  46.7  46.4b    Cu8  13.0  13.2  2.41  2.90  10.8  13.0  153B  223  1.78  2.04  45.4  54.6a    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  P-value5  TEMP  0.027  0.70  0.25  0.36  0.036  0.62  0.0006  0.98  0.028  0.79  0.73  0.074    Cu  1.00  0.70  0.91  0.27  0.88  0.12  0.002  0.93  0.33  0.70  0.54  0.033    TEMP × Cu  0.25  0.16  0.18  0.89  0.16  0.63  0.11  0.32  0.34  0.88  0.95  0.074  Item  GLU (mmol/L)  CHO (mmol/L)  TG (mmol/L)  UA (μmol/L)  T3 (ng/mL)  T4 (ng/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  12.6  13.3  2.38  3.34  11.3  15.4  194  232  1.95  2.12  46.3  46.4    Cu82  12.9  13.1  2.57  3.05  12.3  13.7  177  211  1.94  2.07  45.0  61.4  CHT1,3  Cu0  13.3  13.0  2.41  3.11  10.6  15.4  174  210  1.81  2.10  47.0  46.4    Cu8  13.1  13.3  2.25  2.74  9.32  12.2  129  235  1.62  2.00  45.8  47.8    Pooled SEM  0.18  0.19  0.12  0.29  0.81  1.56  8.42  22.1  0.10  0.18  1.97  3.59  TEMP1,4  NT  12.8b  13.2  2.48  3.20  11.8a  14.6  186A  222  1.95a  2.10  45.7  53.9    CHT  13.2a  13.2  2.33  2.93  10.0b  13.8  152B  223  1.72b  2.05  46.4  47.1    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  Dietary Cu4  Cu0  13.0  13.2  2.40  3.23  11.0  15.4  184A  221  1.88  2.11  46.7  46.4b    Cu8  13.0  13.2  2.41  2.90  10.8  13.0  153B  223  1.78  2.04  45.4  54.6a    Pooled SEM  0.13  0.13  0.09  0.20  0.57  1.10  5.96  16.0  0.07  0.13  1.39  2.54  P-value5  TEMP  0.027  0.70  0.25  0.36  0.036  0.62  0.0006  0.98  0.028  0.79  0.73  0.074    Cu  1.00  0.70  0.91  0.27  0.88  0.12  0.002  0.93  0.33  0.70  0.54  0.033    TEMP × Cu  0.25  0.16  0.18  0.89  0.16  0.63  0.11  0.32  0.34  0.88  0.95  0.074  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). a,bMeans within a row with no common superscripts differ significantly (P < 0.01). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Table 6. Effects of environmental temperature and dietary Cu on plasma alkaline phosphatase (ALP), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), lactic dehydrogenase (LDH), creatine kinase (CK) activities, and corticosterone (CORT) level of hens at 48 to 51 wk of age Item  ALP (U/L)  GOT (U/L)  GPT (U/L)  LDH (U/L)  CK (U/L)  CORT (nmol/L)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  1148  2152  185  156  6.97  8.08  100b  106  18,734  19,985a,b  68.6  61.9    Cu82  1119  1750  177  160  6.72  7.58  94.9b  109  19,266  18,180b  62.5  62.3  CHT1,3  Cu0  1361  2184  186  152  6.61  7.67  138a  118  19,963  18,237b  66.7  61.6    Cu8  1164  1980  192  153  6.61  6.92  93.4b  101  19,303  21,895a  66.8  65.0    Pooled SEM  183  257  8.58  8.12  0.29  0.38  9.42  12.0  545  656  2.36  3.11  TEMP1,4  NT  1134  1951  181  158  6.85  7.83  97.5  108  19,000  19,083  65.6  62.1    CHT  1263  2082  189  153  6.61  7.30  116  110  19,633  20,066  66.8  63.3    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48  385  464  1.67  2.20  Dietary Cu4  Cu0  1255  2168  186  154  6.79  7.88  119  112  19,349  19,111  67.7  61.8    Cu8  1142  1865  185  157  6.67  7.25  94.2  105  19,285  20,038  64.7  63.7    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48.,  385  464  1.67  2.20  P-value5  TEMP  0.49  0.62  0.37  0.49  0.43  0.17  0.068  0.87  0.26  0.15  0.61  0.70    Cu  0.54  0.25  0.91  0.71  0.68  0.12  0.014  0.56  0.91  0.17  0.21  0.53    TEMP × Cu  0.65  0.71  0.42  0.83  0.67  0.75  0.049  0.42  0.29  0.0005  0.20  0.64  Item  ALP (U/L)  GOT (U/L)  GPT (U/L)  LDH (U/L)  CK (U/L)  CORT (nmol/L)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  1148  2152  185  156  6.97  8.08  100b  106  18,734  19,985a,b  68.6  61.9    Cu82  1119  1750  177  160  6.72  7.58  94.9b  109  19,266  18,180b  62.5  62.3  CHT1,3  Cu0  1361  2184  186  152  6.61  7.67  138a  118  19,963  18,237b  66.7  61.6    Cu8  1164  1980  192  153  6.61  6.92  93.4b  101  19,303  21,895a  66.8  65.0    Pooled SEM  183  257  8.58  8.12  0.29  0.38  9.42  12.0  545  656  2.36  3.11  TEMP1,4  NT  1134  1951  181  158  6.85  7.83  97.5  108  19,000  19,083  65.6  62.1    CHT  1263  2082  189  153  6.61  7.30  116  110  19,633  20,066  66.8  63.3    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48  385  464  1.67  2.20  Dietary Cu4  Cu0  1255  2168  186  154  6.79  7.88  119  112  19,349  19,111  67.7  61.8    Cu8  1142  1865  185  157  6.67  7.25  94.2  105  19,285  20,038  64.7  63.7    Pooled SEM  129  182  6.07  5.74  0.21  0.27  6.67  8.48.,  385  464  1.67  2.20  P-value5  TEMP  0.49  0.62  0.37  0.49  0.43  0.17  0.068  0.87  0.26  0.15  0.61  0.70    Cu  0.54  0.25  0.91  0.71  0.68  0.12  0.014  0.56  0.91  0.17  0.21  0.53    TEMP × Cu  0.65  0.71  0.42  0.83  0.67  0.75  0.049  0.42  0.29  0.0005  0.20  0.64  a,bMeans within a row with no common superscripts differ significantly (P < 0.05). 1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effects. View Large Both temperature and dietary Cu had no effect (P > 0.05) on the plasma contents of T-AOC and MDA and plasma CuZnSOD activity. No interactions between temperature and dietary Cu were observed in the above-mentioned three indices during the stages of 48–49, and 50–51 wk (Table 7). Table 7. Effects of environmental temperature and dietary Cu on plasma total antioxidant capacity (T-AOC), Cu and zinc superoxide dismutase (CuZnSOD) activity, and malondialdehyde (MDA) level of hens at 48 to 51 wk of age Item  T-AOC (U/mL)  CuZnSOD (NU/mL)  MDA (nmol/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  5.97  6.08  190  262  3.17  4.02    Cu82  6.36  6.11  190  271  3.67  4.64  CHT1,3  Cu0  5.69  6.72  188  264  3.46  4.07    Cu8  5.64  5.98  179  251  2.97  3.50    Pooled SEM  0.25  0.36  5.28  7.20  0.25  0.29  TEMP1,4  NT  6.17  6.10  190  267  3.42  4.33    CHT  5.67  6.35  184  258  3.22  3.79    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  Dietary Cu4  Cu0  5.83  6.40  189  263  3.32  4.05    Cu8  6.00  6.05  185  261  3.32  4.07    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  P-value5  TEMP  0.073  0.49  0.24  0.21  0.43  0.076    Cu  0.50  0.34  0.45  0.81  0.99  0.93    TEMP × Cu  0.39  0.31  0.40  0.14  0.064  0.065  Item  T-AOC (U/mL)  CuZnSOD (NU/mL)  MDA (nmol/mL)    48–49 wk  50–51 wk  48–49 wk  50–51 wk  48–49 wk  50–51 wk  NT1,3  Cu02  5.97  6.08  190  262  3.17  4.02    Cu82  6.36  6.11  190  271  3.67  4.64  CHT1,3  Cu0  5.69  6.72  188  264  3.46  4.07    Cu8  5.64  5.98  179  251  2.97  3.50    Pooled SEM  0.25  0.36  5.28  7.20  0.25  0.29  TEMP1,4  NT  6.17  6.10  190  267  3.42  4.33    CHT  5.67  6.35  184  258  3.22  3.79    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  Dietary Cu4  Cu0  5.83  6.40  189  263  3.32  4.05    Cu8  6.00  6.05  185  261  3.32  4.07    Pooled SEM  0.18  0.26  3.74  5.09  0.18  0.21  P-value5  TEMP  0.073  0.49  0.24  0.21  0.43  0.076    Cu  0.50  0.34  0.45  0.81  0.99  0.93    TEMP × Cu  0.39  0.31  0.40  0.14  0.064  0.065  1NT = normal temperature; CHT = cyclic high temperature; TEMP = environmental temperature. 2Cu0 = the diet supplemented with 0 mg/kg Cu from CuSO4·5H2O; Cu8 = the diet supplemented with 8 mg/kg Cu from CuSO4·5H2O. 3Each value represented the means of 6 replicates (n = 6). 4Each value represented the means of 12 replicates (n = 12). 5Probability values for main effect. View Large DISCUSSION In the current study, the RT was significantly increased by approximately 1°C when laying hens were exposed to CHT, indicating that all hens under CHT were in a heat stress status throughout heat stress period. However, the temperature did not affect RT in hens during the stage of 50–51 wk. This was probably because the temperature of 2 henhouses was maintained at normal temperature (26 ± 2°C) during the convalescent period. Hyperthermia severely depresses laying performance, feed intake of commercial laying hens (Emery et al., 1984; Star et al., 2008a). Therefore, the egg production and feed intake were significantly decreased in hens exposed to the CHT in the current study. Similar results were observed also in the previous studies with laying hens (Mashaly et al., 2004; Star et al., 2008b). Moreover, results from a recent study (Mack et al., 2013) showed that hens subjected to heat stress conditions spend less time eating, but more time drinking and panting. Meanwhile, heat stress could prolong the time of egg formation (Nordstrom, 1973), and disrupt the normal status of reproductive hormones in the hypothalamus (Donoghue et al., 1989) and ovary (Rozenboim et al., 2007). The lack of feed intake is very likely the starting point of the most detrimental effects of heat stress on production, leading to a decreased BW, feed efficiency, egg production and quality (Mashaly et al., 2004). Thus, the decrease in the feed: egg ratio and BW of the heat stressed hens observed in this study probably was associated with the decreased feed intake and this adverse effect actually lasted into the convalescent period. The heat stress could alter some plasma endocrine and biochemical indices involved in various aspects of reproductive performance and nutrient metabolism in commercial layers (Star et al., 2008a,b). The plasma GLU, CHO, and TG levels have been used as indicators of stress in fowls (Odihambo Mumma et al., 2006). Usually the glucocorticoids is increased in circulating system of hens under stressed status, which could induce gluconeogenesis and then lead to an elevation of plasma GLU (Nagra and Meyer, 1963). Moreover, the glucocorticoids also inhibit GLU uptake into peripheral tissues by antagonizing the effect of insulin (Buren et al., 2002). In consistent with the previous reports (Sahin and Kucuk, 2001; Star et al., 2008a), we found that the plasma GLU level significantly increased in hens under CHT, but TG level decreased. Geraert et al. (1996) reported that endocrinological changes caused by heat stress stimulate lipid accumulation via increasing in de novo lipogenesis, reducing lipolysis, and enhancing amino acid catabolism. We noticed that the plasma TG and UA levels were decreased in hens under CHT, which appeared to be consistent with the results obtained by Geraert et al. (1996) and Lin et al. (2008) and Song et al. (2012). The laying hens could not maintain their performance after 7 d of heat exposure even by the mobilization of energy and nitrogen stores in this study. This consequence might be caused by insufficient protein deposits, as indicated by the lower UA detected in the plasma of heat-exposed hens (Song et al., 2012). Body temperature and metabolic activity are regulated by T3, T4 and their balance. The decreased plasma T3 level is indicative of the physiological adaptation to heat exposure (Lara and Rostagno, 2013). In this study, CHT decreased plasma T3 level but had no effect on plasma T4 level, which was in agreement with the result of Mack et al. (2013). A number of enzymes are used in the clinical biochemistry as tools for differential diagnosis, such as ALP, GOT, GPT, LDH, and CK. Since they are located in different tissues, their abnormal appearance in plasma can indicate specific muscle or organ damages (Pech-Waffenschmidt, 1992). Significant increases in activities of GOT, GPT, LDH, and CK were observed in plasma of chickens exposed to high temperature (Melesse et al., 2011). CHT had no effect on plasma ALP, GOT, GPT, LDH and CK activities, and CORT level in the present study. According to the results obtained by Pech-Waffenschmidt et al. (1995), heat exposure did not significantly change the enzyme activities in the laying hens’ serum. This was also supported by previous findings of Ward and Peterson (1973), who reported that the activities of GOT and CK were not influenced even by acute heat exposure. Clearly, plasma GLU, CHO, and TG levels are stress indicators in fowls, however, CORT is not accepted by all as a stress response in laying hens (Odihambo Mumma et al., 2006). The magnitude and duration of heat stress imposed on birds may affect their metabolic response of the stressed hens, resulting in changes in stress indicators in plasma. However, the possible mechanisms which might have caused such conflicting responses in enzyme activities in heat stressed birds remain unclear. The adverse effect of CHT on plasma biochemical indices did not last into the convalescent period in this study, probably due to normal ambient temperature during 50–51 wk, but the possible mechanisms need to be further studied. Chiou et al. (1997) reported that White Leghorn layers fed diet supplemented with 200 mg Cu/kg feed in the form of CuSO4·5H2O had higher egg production than diet with 0, 400, 600, 800 mg Cu/kg feed. Similar results were also reported by Pesti and Bakalli (1998). However, the supplementation of Cu over 200 mg/kg in the diet reduced the feed intake of Hy-Line Brown laying hens (Kim et al., 2016) and egg production of white light hybrid hens (Pearce et al., 1983). These results suggest that there are negative impacts on productive performance of hens with Cu supplementation over 200 mg/kg feed. Further studies indicated that dietary supplementation of Cu in the range of 0–300 mg/kg feed had no effect on feed intake and egg production of Hisex-Brown hens (Balevi and Coskun, 2004). A possible explanation for such contradictory reports might be the different breeds of laying hens and added amount of Cu additives. Indeed, there was no difference in laying rate between Cu0 and Cu8 under NT in the current study. However, there was a decreasing trend in the laying rate of hens in CHT-Cu8 in the second week of heat stress (49 wk), and the laying rate of CHT-Cu8 was significantly decreased in the first week of convalescence (50 wk). Mooyoung et al. (1970) found that the isthmus of the oviduct was rich in Cu. It is speculated that laying hens have lower tolerance of the Cu toxicity under heat stress, which has an adverse effect on the normal function of the isthmus. However, the exact reasons for the decreased laying rate of hens in CHT-Cu8 remain unclear. In the present study, there was a significant decrease in egg weight in Cu0, which was in agreement with the result of Pekel and Alp (2011). The effect of dietary Cu and the interaction between temperature and Cu had no influence on other laying performance except for laying rate and egg weight in the current study, which might mostly be related to the low added level of Cu compared to other studies (Chiou et al., 1997; Balevi and Coskun, 2004; Kim et al., 2016), but the possible mechanisms need to be further elucidated. Diet supplemented with 8 mg of Cu (Cu8) significantly decreased the plasma UA level compared to the diet with no supplementation of Cu (Cu0) during the heat stress period, indicating that laying hens are very sensitive to the dietary Cu level under heat stress. UA has been proposed to be a potent scavenger of free radicals in human and poultry (Becker, 1993) and its formation is considered as one of the mechanisms leading to a longer lifespan of birds (Simoyi et al., 2003). The elevated plasma T4 of hens in Cu8 group during the convalescent period might be the result of a decreased peripheral deiodination in the hens after heat exposure as reported previously by Kühn et al. (1987). ALP, GOT, GPT, and LDH are widely distributed in liver, kidney, heart and muscle cells, and CK is the specific enzyme in heart and muscle tissue as shown by Mitchell and Sandercock (1995). Chiou et al. (1997) reported that serum LDH and CK activities of hens with Cu supplementation over 500 mg/kg feed were significantly increased. In the present study, the plasma LDH activity of hens in CHT-Cu0 and the plasma CK activity of hens in CHT-Cu8 were increased during the heat stress period and convalescent period, respectively. This implies that kidney, heart, and muscle damage has occurred and leads to the release of the enzymes from cells to the blood stream when plasma LDH and CK activities increase. Heat stress can enhance the formation of reactive oxygen species (ROS) that cause oxidative injury such as lipid peroxidation (Flanagan et al., 1998). The antioxidative enzyme system (comprising SOD, glutathione peroxidase, and catalase) acts as the first line of antioxidant defense. Modification of these enzymes activities can alter the balance between the ROS production and the antioxidant system. Cu is the cofactor of the CuZnSOD, which could scavenge free radicals. The integrative index of T-AOC reflects the activity of scavenging free radicals (Lewis et al., 1995). Previous studies demonstrated a significant increase in free radicals production together with an increase in the expression of antioxidant enzymes under heat stress (Sahin and Kucuk, 2001). These increases in antioxidant enzyme activities have been considered to be a protective response against oxidative stress. Thus, it is implied that the balance has already been disturbed by heat stress. MDA is the biomarker of lipid peroxidation (Lin et al., 2008). Moreover, the relationship in biological systems between lipid peroxidation and high temperature has been discussed previously (Borisiuk and Zinchuk, 1995). In the present study, both temperature and dietary Cu had no effect on plasma T-AOC, CuZnSOD, and MDA levels, and no interactions between temperature and Cu were observed in the three plasma indices. A possible explanation for such contradictory results might be the short duration of heat stress and all hens have 12 h normal temperature each day during heat stress period. Meanwhile, age and genotype are crucial factors for resilience to heat stress in laying hens (Mignon-Grasteau et al., 2015), which imply that the hens have robust adaptive ability at the 48–51 wk of age. Moreover, the added amount Cu in the present study is lower than the previous studies (Pesti and Bakalli, 1998; Kim et al., 2016; Yang et al., 2017). Considering the UA has been proposed to be a potent scavenger of free radicals, and thyroid hormones could accelerate the oxidative metabolism via an increase in the mitochondria mass, mitochondria cytochrome content, and respiratory rate (Asayama et al., 1987; Lin et al., 2008). It might suggest that the scavengers of free radicals induced by oxidative injury contain many substances except for the antioxidative enzyme system. CONCLUSION Cyclic high temperature (26 ± 2°C∼33 ± 2°C) impaired laying performance. The Cu content (10.3 mg/kg) in corn-soybean basal diet might be sufficient for meeting the maintenance and production requirements of late-phase laying hens, and no Cu supplementation had no adverse effects on egg production and antioxidant indices under normal (26 ± 2°C) or cyclic high temperatures. Acknowledgements We greatly appreciate the supports of the China Agriculture Research System (CARS-40-K08), National Natural Science Foundation of China (Grant No. 31772621), Public Sector (Agriculture) Scientific Research of China (Grant No.201403047), and China Program for New Century Excellent Talents in University (NCET-13–0558). We would also like to express our gratitude and our thanks to Dr. Rainer Hubertus Mosenthin (University of Hohenheim) for his dedication in improving the language and syntax of the manuscript. REFERENCES Asayama K., Dobashi K., Hayashibe H., Megata Y., Kato K.. 1987. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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