Effect of epigallocatechin gallate on growth performance and serum biochemical metabolites in heat-stressed broilers

Effect of epigallocatechin gallate on growth performance and serum biochemical metabolites in heat-stressed broilers Jingxian Luo,∗ Jiao Song,†,1 Longzhou Liu,∗ Bo Xue,∗ Guangming Tian,∗ and Ye Yang∗,2 ∗

College of Animal Science, Yangtze University, Jingzhou, P.R. China; and † State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, P.R. China

ABSTRACT This study aims to investigate the effects of epigallocatechin gallate (EGCG) on the growth performance and serum metabolic characteristics of heat-stressed broilers. A total of 192 14-day-old Arbor Acres broilers were divided into 4 groups with 6 replicates per group (8 chickens/cage). Thermoneutral group (Group TN) was fed the basal diet and maintained at 28◦ C for 24 h/d. The heat-stressed groups were housed at 35◦ C for 12 h/d and 28◦ C for 12 h/d and fed the basal diet supplemented with EGCG at 0, 300, and 600 mg/kg diet (Groups HS0, HS300, and HS600, respectively). The production performance and serum metabolic characteristics were analyzed at d 21, 28, and 35, respectively. At d 35 of age, heat stress reduced (P < 0.05) the body weight (BW), feed intake (FI) and the contents of serum total protein (TP) and glucose (GLU); inhibited (P < 0.05) alkaline phosphatase (ALP) activity; But increased (P < 0.05) the contents of uric acid (UA), cholesterol (CHOL),

triglyceride (TG), and the activities of creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST). Heat-stressed chickens fed EGCG exhibited a linear increase (P < 0.05) in BW, FI, the levels of serum TP, GLU, and ALP activity; and linear decrease (P < 0.05) in the contents of serum UA, CHOL, and TG, as well as the activities of LDH, CK, and AST. Heat stress also reduced (P < 0.05) the activities of serum glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) on d 35 and increased (P < 0.05) the GSH-Px and SOD activity on d 21 and malondialdehyde (MDA) contents. There was a linear increase (P < 0.05) in activities of GSH-Px, SOD and CAT at 35 d of age, and linear decreased (P < 0.05) in MDA contents. In conclusion, EGCG can improve the growth performance of broilers by enhancing antioxidant property and alleviating oxidant damage caused by heat stress.

Key words: broiler, heat stress, epigallocatechin gallate, growth performance, serum biochemical metabolite 2017 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pex353

INTRODUCTION

(Mujahid et al., 2007; Yang et al., 2010). Therefore, mitochondrial damage and energy distribution following heat stress result in significant economic losses and healthy problem (Tomanek and Zuzow, 2010). As global temperatures increase, the negative effects will become more and more apparent in the future. Many phytochemicals, such as resveratrol (Liu et al., 2014; Zhang et al., 2017), epigallocatechin-3-gallate (Sahin et al., 2010), lycopene (Sahin et al., 2016), essential oil (Akbarian et al., 2014), plant extracts (Varmuzova et al., 2015) were used to improve antioxidation property and alleviate heat stress in poultry. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, is considered to be the most bioactive component of green tea and has a strong antioxidant activity (Rains et al., 2011; Wang et al., 2014). Tea polyphenols and EGCG play an important role in regulating meat quality (Zhang et al., 2012; Zhang et al., 2017), improving growth performance and egg quality (Liu et al., 2014; Yuan et al., 2016), modulating gut health (Mar´ın et al., 2015). More important, EGCG exhibits a strong antioxidant capacity to scavenge free

Heat stress is the main environmental factor affecting the production performance and economic benefit of livestock and poultry. Heat stress affects poultry performance mainly by causing oxidative stress (Yang et al., 2010; Gu et al., 2012; Zhang et al., 2012). Oxidative stress is presented by the imbalance between the production of ROS and the activity of antioxidant systems. Studies also have shown that heat stress-induced oxidant stress activated the in vivo antioxidant system to eliminate free radicals or ROS (Yang et al., 2010), and the increase in antioxidant system activation required more energy consumption (Hofmann and Todgham, 2010). At the same time, oxidative stress leads to lipid peroxidation and oxidative damage to proteins, resulting in impaired cellular and mitochondrial functions  C 2017 Poultry Science Association Inc. Received January 31, 2017. Accepted October 28, 2017. 1 joint first authors. 2 Corresponding author: [email protected]

1 Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

2

LUO ET AL.

radicals and reduce the formation of H2 O2 and reactive oxygen species (Dembinska-Kiec et al., 2008), to restore the cellular redox balance and mitochondrial functions (Scalbert et al., 2005; Kim et al., 2014; Oliveira et al., 2016), and eventually, to reduce the oxidative stressinduced cell damage and alleviate the impact of oxidative stress on poultry (Sahin et al., 2010; Bogdanski et al., 2012; Orhan et al., 2013). Changes in heat stress–induced serum metabolic characteristics appear to reflect the status of metabolism and energetics (Baumgard and Rhoads, 2013), oxidative damage (Xie et al., 2015; Marchini et al., 2016), and immune system (Renaudeau et al., 2012). Much of the literature highlights the heat stress– induced serum metabolic characteristics (Habibian et al., 2014; Xie et al., 2015) and phytochemicals’ regulating response (Akbarian et al., 2014; Liu et al., 2014; Sahin et al., 2016; Zhang et al., 2017). But we still know little about the effects of time-course and dosage-course of EGCG on serum metabolic characteristics in heatstressed broilers. This study aims to investigate the effects of EGCG on serum metabolic characteristics and the serum antioxidant capacity of broilers under heat stress.

MATERIALS AND METHODS This study was carried out in the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (CAAS, Beijing, China) and conducted in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology (Beijing, China). The protocols were approved by the Science Research Department (in charge of animal welfare issues) of the Institute of Animal Sciences, CAAS (Beijing, China).

Experimental Design After 1-day-old male Arbor Acres broilers (purchased from Huadu Broiler Company, Beijing, China) were pre-fed for 2 wk, 192 healthy broiler chicks of similar weight were randomly divided into 4 groups. Each group had 6 replicate cages with 8 chickens per replicate. Among them, one group (thermoneutral group) was housed in an environmentally controlled chamber at 28◦ C (Group TN) and was fed with the basal diet, a commercial diet provided by Huadu Feed Company (Beijing, China). The other 3 groups (heat stress [HS] groups) were housed in a cyclic high-temperature environmentally controlled chamber (35◦ C from 07:00 to 19:00 h; 28◦ C from 19:00 to 7:00 h). The basal diets for the HS group were supplemented with EGCG at the following doses: 0 mg/kg (Group HS0); 300 mg/kg (Group HS300); and 600 mg/kg (Group HS600). The diets were based on corn-soybean meal (Table 1). The chickens were raised in cages and supplied diet and

Table 1. Composition and nutrient levels of basal diets (air-dry basis). Item Ingredient, % Corn Soybean meal fish meal Corn oil Dicalcium phosphate Limestone Sodium chloride D,L-Methionine L-Lysine Vitamin-mineral-premix1 Total Nutrient level2 ME, MJ/kg CP, % Lysine, % Methionine, % Calcium, % Available phosphorus, %

0 ∼ 21 d

22 ∼ 35d

61.2 29.0 3.0 2.5 1.60 1.10 0.32 0.18 0.10 1.0 100

65.2 25.7 3.0 2.30 1.40 1.00 0.30 0.10 1.00 100

12.48 20.5 1.05 0.45 0.99 0.51

12.85 19.0 0.95 0.43 0.89 0.46

1 Mineral-vitamin premix provided the following per kilogram of diet: vitamin A, 12,000 IU; vitamin D3, 3,500 IU; vitamin E, 25 IU; nicotinic acid, thiamine 60 mg; vitamin B12, 0.014 mg; calcium pantothenate, 20 mg; vitaminK3,2.0 mg; thiamine, 2.0 mg; riboflavin, 8.0 mg; vitamin B6, 7.0 mg; folic acid, 0.8 mg; biotin, 0.2 mg; Fe, 100 mg; Cu, 8 mg; Mn, 120 mg; Zn, 100 mg; I, 0.7 mg; Se, 0.3 mg; 2 The nutritional level was a calculated value.

water ad libitum. The lighting program was a period of 23 h of light and 1 h of darkness.

Growth Performance Record and Serum Sample Collections After 12 h fasting, feed intake (FI) and body weight (BW) of chickens were recorded with the replicate cage as a unit at 21, 28, and 35 d of age respectively, and feed/gain (F/G) ratios were calculated. And then multiple blood samples were collected from one broiler per cage (24 broilers; 6 broilers from each of 4 groups) on d 7, d 14, and d 21 of heat stress. The serum was separated at 1,000 g/min for 15 min and stored at −20◦ C until to measure serum metabolic characteristics and antioxidant-related indicators.

Serum Metabolic Characteristics Analysis Serum biochemical metabolic characteristics were determined by a Toshiba 120 automated biochemical analyzer (Toshiba, Tokyo, Japan). The levels of serum total protein (TP), glucose (GLU), uric acid (UA), serum cholesterol (CHOL), and blood triglycerides (TG) were measured by the method of biuret reaction, glucose oxidase, uricase, cholesterol oxidase, and glycerophosphate oxidase, respectively (Xie et al., 2015).

Analysis of Serum Enzymes Related to Tissue Damage The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

3

EPIGALLOCATECHIN GALLATE AND HEAT-STRESSED CHICKEN Table 2. Effects of heat stress and EGCG on the growth performance.1 Effects of EGCG under HS Treatment Item BW, kg/bird 14 d 21 d 28 d 35 d FI3 , g/bird per d 14–21 d 22–28 d 29–35 d 14–35 d F/G3 14–21 d 22–28 d 29–35 d 14–35 d

2

TN vs.HS0

P-value

TN

HS0

HS300

HS600

SEM

P-value

Main effect

Linear

Quadratic

0.38 0.81 1.24 1.68

0.37 0.76 1.16 1.51

0.38 0.79 1.18 1.53

0.38 0.81 1.26 1.73

0.01 0.03 0.04 0.09

0.129 0.003 0.007 0.013

0.045 0.001 0.005

0.015 0.001 0.003

0.639 0.077 0.122

0.73 0.82 0.93 0.83

0.62 0.77 0.85 0.75

0.68 0.76 0.86 0.77

0.73 0.79 0.95 0.82

0.04 0.05 0.08 0.03

0.001 0.147 0.103 0.002

0.007 0.766 0.119 0.013

0.002 0.681 0.066 0.005

0.721 0.554 0.333 0.363

1.69 1.91 2.12 1.91

1.61 1.90 2.59 1.98

1.68 1.97 2.49 2.01

1.72 1.71 2.06 1.83

0.09 0.11 0.24 0.11

0.306 0.982 0.074 0.310

0.295 0.013 0.083 0.059

0.129 0.026 0.037 0.054

0.785 0.033 0.425 0.133

1

Results are the mean of 6 replicate cages, 6–8 birds per cage (as a result of slaughter). TN: thermoneutral (28◦ C for 24 h/d); HS0: heat-stressed (35◦ C for 12 h/d followed by 28◦ C for 12 h/d) broilers without EGCG; HS300: heat-stressed broilers with EGCG 300 mg/kg diet; HS600: heat-stressed broilers with 600 EGCG mg/kg diet. 3 feed intake; F/G: feed/gain ratio. 2

(LDH), and creatine kinase (CK) were measured by the method of ultraviolet-malate dehydrogenase, 2amino-2-methyl-1-propanol (AMP) buffer, lactic acid substrate, and N-acetylcysteine, respectively, with a Toshiba 120 automated biochemical analyzer (Toshiba, Tokyo, Japan).

evaluate the linear and quadratic effects of EGCG levels on broilers in the HS environment. Statistical significance was considered at P < 0.05.

RESULTS Growth Performance

Measurement of the Activity of Antioxidant Enzymes and Lipid Peroxidation The activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), and the contents of malondialdehyde (MDA) were determined according to the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). SOD activity was determined using nitrite coloration method with absorbance at 550 nm. CAT activity was measured using ammonium molybdate method with spectrophotometric determination of absorbance at 405 nm. GSH-Px activity was measured at 412 nm by quantifying the rate of oxidation of reduced GSH to oxidized glutathione. MDA content was determined using thiobarbital (TBA) method with absorbance at 532 nm (Yang et al., 2010; Xie et al., 2015).

The effects of heat stress and EGCG on the growth performance of broilers are given in Table 2. Compared with Groups TN, the BW at d 21, d 28, and d 35 of age and FI during d 14 to 35 was decreased (P < 0.05) in the Groups HS0. There was a linear increase in BW on d 21 (P = 0.015), d 28 (P = 0.001), d 35 (P = 0.003) and FI during during d 14 to 21 (P = 0.002) and the overall experimental period (P = 0.005) to dietary EGCG supplementation for the heat-stressed broilers. There was no difference (P = 0.310) in F/G between Groups TN and Groups HS0 during d 14 to 35. But there was a linear decrease in F/G during d 22 to 28 (P = 0.026), d 29 to 35 (P = 0.037), and the overall experimental period (P = 0.054) to dietary EGCG supplementation for the heat-stressed broilers.

Serum Metabolic Characteristics Statistical Analysis The data were analyzed by using the one-way analysis of variance (ANOVA) procedure (SAS Version 8). Data on growth performance parameters (BW, FI, and F/G) were analyzed on a cage basis, whereas data on serum biochemical parameters and antioxidant capacity were based on individual broilers. A single degree of freedom contrast was used to evaluate the effect of HS versus TN for broilers on the basal diet without EGCG. Orthogonal polynomial contrasts were also used to

The analyzed serum metabolic characteristics are presented in Table 3. Compared with the Group TN, heat stress lowered (P < 0.05) the contents of serum TP on d 35, GLU on d 28, d 35 d and serum ALP activity on d 21, d 28 and d 35. On the other hand, an increase was observed in the contents of UA on d 21 and d 35, TG on d 21, d 28, and d 35, and CHOL on d 28 and d 35, as well as the activities of LDH, CK, and AST on d 28 and 35 d (P < 0.05). There was a linear increase (P < 0.05) in contents of serum TP on d 28

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

4

LUO ET AL. Table 3. Effects of heat stress and EGCG on the serum metabolites parameters.1 Effects of EGCG under HS Treatment Item

2

TN vs.HS0

P-value

TN

HS0

HS300

HS600

SEM

P-value

Main effect

Linear

Quadratic

18.5 30.2 30.2

18.1 29.2 28.0

18.6 31.3 29.8

19.2 31.5 30.7

0.86 0.93 1.02

0.401 0.139 0.002

0.254 0.001 0.002

0.104 0.001 0.001

0.986 0.053 0.351

0.53 0.78 0.65

0.413 0.037 0.021

0.207 0.002 0.161

0.084 0.001 0.061

0.766 0.383 0.883

10.08 17.58 13.80

0.039 0.071 0.014

0.004 0.026 0.157

0.002 0.008 0.061

0.249 0.952 0.726

3

TP , g/L 21 d 28 d 35 d GLU3 , mol/L 21 d 28 d 35 d UA3 , μ mol/L 21 d 28 d 35 d CHOL3 , mmol/L 21 d 28 d 35 d TG3 , mmol/L 21 d 28 d 35 d

9.61 7.20 10.05 213 182 138

9.33 6.16 8.71 229 201 160

9.51 7.45 9.05 213 184 154

9.84 8.06 9.32 209 167 143

4.09 4.08 3.97

4.01 4.43 4.53

3.72 4.17 3.9

3.62 3.83 3.6

0.18 0.24 0.22

0.407 0.046 0.001

0.005 0.003 < 0.001

0.002 0.001 < 0.001

0.280 0.745 0.155

0.48 0.47 0.37

0.57 0.54 0.47

0.36 0.46 0.39

0.41 0.44 0.37

0.04 0.05 0.04

< 0.001 0.066 0.002

< 0.001 0.021 0.004

< 0.001 0.010 0.002

< 0.001 0.239 0.261

1

Results are the mean of 6 replicate cages, 6 birds per cage. TN, thermoneutral (28◦ C for 24 h/d). HS0, heat-stressed (35◦ C for 12 h/d followed by 28◦ C for 12 h/d) broilers without EGCG; HS300, heat-stressed broilers with EGCG 300 mg/kg diet; HS600: heat-stressed broilers with 600 EGCG mg/kg diet. 3 TP, total protein; GLU, glucose; UA, uric acid; CHOL, cholesterol; TG, triglycerides. 2

and d 35, and GLU on d 28, as well as ALP activity on d 21, d 28, d 35, and a decrease (P < 0.05) in the contents of serum UA on d 21 and d 28, CHOL and TG on d 21, d 28, d 35, as well as the activities of LDH on d 21, d 28, d 35, and CK on d 21, d 35, and AST on d 35 to dietary EGCG supplementation for the heat-stressed broilers.

Serum Enzyme Related to Tissue Damage Compared with the Group TN, heat stress reduced (P < 0.05) the serum ALP activity on d 21, d 28, and d 35, but increased (P < 0.05) the activities of LDH, CK and AST on d 28 and 35 d. A linear increase was observed (P < 0.05) in ALP activity on d 21, d 28, and d 35, and a linear decrease was observed (P < 0.05) in activities of LDH on d 21, d 28, and d 35; CK on d 21 and d 35; and AST on d 35 when dietary EGCG was added to the heat-stressed broilers (Table 4).

Serum Antioxidant Capacity and Lipid Peroxidation Compared with the Group TN, heat stress increased the GSH-Px (P < 0.05) and SOD (P < 0.05) activity on d 21, but decreased (P < 0.05) the activities of serum GSH-Px on d 35, SOD on d 28, d 35 and CAT on d 21, d 28, d 35. In contrast, the MDA contents were increased (P < 0.05) in Group HS0 compared with Group TN on d 21, d 28 and d 35 (Table 5). The diets supplemented with different levels of EGCG exhibited a linear increase (P < 0.05) in the activities of GSH-Px and SOD at 21,

28, 35 d of age and CAT at 28, 35 d of age and decrease (P < 0.05) in MDA contents. There was a quadratic increase (P < 0.05) in the activities of GSH-Px on d 28 and d 35 and SOD on d 28 (Table 5).

DISCUSSION Heat stress is a major factor affecting the production performance of broilers. Numerous studies have shown that heat stress can significantly reduce the production performance of broilers (Liu et al., 2014; Sahin et al., 2016). The results of this study also show that the daily weight gain and feed intake of the broilers were decreased significantly under heat stress. The rapid decline in feed intake is a distinct feature of heat stress. The feed intake was decreased by 2.2% per degree centigrade increase in room temperature above the thermal comfort zone (Oliveira et al., 2016). Traditional studies have suggested that decreases in poultry production performance are caused by a heat stress–induced decline in nutrient uptake. Therefore, early studies have focused on increasing the feed intake of heat-stressed poultry through various methods to improve production performance. However, the recent report (Rhoads et al., 2013) found that the decline in production performance was caused not by a lack of nutrient intake but by changes in the physiological metabolism of livestock and poultry under heat-stressed conditions. Oxidant stress induced by heat stress is the main cause of decreasing growth production. Therefore many antioxidant additives such as vitamins, trace elements, or plant extracts can alleviate heat stress and improve growth production (Sahin et al., 2010; Renaudeau et al.,

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

5

EPIGALLOCATECHIN GALLATE AND HEAT-STRESSED CHICKEN Table 4. Effects of heat stress and EGCG on the activities of enzyme related to tissue damage.1 Effects of EGCG under HS Treatment Item

2

TN vs.HS0

TN

HS0

HS300

HS600

SEM

P-value

3412 3370 2756

3043 2961 2388

3377 3483 2869

3645 3653 3131

252.6 277.1 264.0

0.049 0.018 0.016

2942 2710 2766

3086 2982 3034

2796 2814 2622

2659 2636 2445

171.9 223.2 182.6

4415 4120 4090

4547 4479 4419

4184 4311 4317

4055 4245 4084

159.2 251.4 186.5

297 284 280

298 302 316

299 311 268

307 294 248

P-value Main effect

Linear

Quadratic

0.001 0.002 0.001

< 0.001 0.001 < 0.001

0.761 0.241 0.443

0.101 0.048 0.013

0.003 0.093 0.001

0.001 0.032 < 0.001

0.421 0.969 0.234

0.120 0.043 0.025

0.001 0.303 0.011

< 0.001 0.140 0.004

0.197 0.697 0.451

0.849 0.044 0.020

0.522 0.270 < 0.001

0.324 0.425 < 0.001

0.580 0.159 0.077

3

ALP , U/L 21 d 28 d 35 d LDH3 , U/L 21 d 28 d 35 d CK3 , U/L 21 d 28 d 35 d AST3 , U/L 21 d 28 d 35 d

15.01 14.76 16.52

1

Results are the mean of 6 replicate cages, 6 birds per cage. TN, thermoneutral (28◦ C for 24 h/d). HS0, heat-stressed (35◦ C for 12 h/d followed by 28◦ C for 12 h/d) broilers without EGCG; HS300, heat-stressed broilers with EGCG 300 mg/kg diet; HS600: heat-stressed broilers with 600 EGCG mg/kg diet. 3 ALP, alkaline phosphatase; LDH, lactate dehydrogenase; CK, creatine kinase; AST, aspartate aminotransferase. 2

Table 5. Effects of heat stress and EGCG on serum antioxidant capacity.1 Effects of EGCG under HS Treatment Item

TN

GSH-Px , μ mol/L 21 28 35 SOD3 , U/mL 21 28 35 CAT3 , U/mL 21 28 35 MDA3 , nmol/L 21 28 35

HS0

2

TN vs.HS0

P-value

HS300

HS600

SEM

P-value

Main effect

Linear

Quadratic

3

596 477 806

648 459 748

666 606 875

690 615 826

30.78 48.27 49.38

0.001 0.502 0.008

0.022 < 0.001 0.007

0.006 < 0.001 0.036

0.825 0.022 0.009

141 158 170

151 138 160

156 157 172

162 160 173

6.42 6.26 5.89

0.011 0.001 0.017

0.099 < 0.001 0.003

0.035 < 0.001 0.002

0.856 0.004 0.086

4.55 4.70 8.59

3.67 3.75 6.34

3.84 4.06 7.15

3.91 4.73 7.48

0.46 0.51 0.61

0.001 0.001 0.001

0.352 0.014 0.028

0.167 0.005 0.010

0.740 0.510 0.477

4.32 4.39 4.33

5.04 6.34 5.49

4.39 5.15 5.32

4.12 4.49 4.73

0.48 0.51 0.52

0.003 0.001 0.016

0.018 0.001 0.009

< 0.001 0.001 0.004

0.472 0.425 0.282

1

Results are the mean of 6 replicate cages, 6 birds per cage. TN: thermoneutral (28◦ C for 24 h/d); HS0: heat-stressed (35◦ C for 12 h/d followed by 28◦ C for 12 h/d) broilers without EGCG; HS300: heat-stressed broilers with EGCG 300 mg/kg diet; HS600: heat-stressed broilers with 600 EGCG mg/kg diet. 3 GSH-Px, GSH peroxidase; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde. 2

2012). In this study, the broilers fed diet supplement EGCG exhibited higher BW and FI, which also indicated that the effective measure of alleviating heatstress response is to inhibit oxidant stress (Sahin et al., 2010; Liu et al., 2014). The main effects of heat stress on poultry largely come from damage to tissues and cells by heat stressinduced oxidative stress (Yang et al., 2010; Gu et al., 2012; Zhang et al., 2012) and are mainly reflected by increasing in the MDA content of the tissues and cells during heat stress. MDA levels indicate the extent of lipid peroxidation mediated by oxygen free radicals (Tan et al., 2010). EGCG could reduce

both the formation of H2 O2 and ROS and the MDA content (Dembinska-Kiec et al., 2008). Furthermore, it could enhance the antioxidant capacity of the liver in poultry and reduce the effect of oxidative stress on poultry by inhibiting the inflammatory signaling pathways (Sahin et al., 2010; Bogdanski et al., 2012; Orhan et al., 2013). The results of this study also show that EGCG could significantly decrease the serum MDA content and increase the activities of the antioxidant enzymes SOD and GSH-Px in the heat-stressed broiler chickens, indicating that EGCG can alleviate the heat-stress effect by enhancing the activities of antioxidant enzymes (Franco et al., 2013).

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

6

LUO ET AL.

EGCG may up-regulate phospho-AMP-activated protein kinase (p-AMPK) (Hwang et al., 2005; Reiter et al., 2010; Park et al., 2012; Kim et al., 2013) and Nrf2 (Han and Loa, 2007; Sahin et al., 2010) expression to exert their antioxidant effects. Ye et al. (2012) also reported that EGCG increases the cell’s antioxidant capability by enhancing the gene transcription and protein expression of PGC-1α, Sirt1, SOD1, and GPX1; in addition, EGCG increases the activities of antioxidant enzymes and promotes the scavenging of ROS via the PGC-1α/Sirt1 signal pathway. Therefore, the effects of heat stress and EGCG on antioxidant enzymes activities may associate with the function and structural integrity of mitochondria. In this study, the results indicate that heat stress significantly increases SOD and GSH-Px activities at 21 d, but dramatically reduced the SOD activity at d 28 and d 35, and CAT activity at d 21, 28, and 35. Differences in antioxidant enzyme activities might largely depend on the intensity and duration of heat stress (GonzalezEsquerra and Leeson, 2006; Akbarian et al., 2014). The studies showed that acute or chronic short-term heat stress may increase SOD, CAT, and GSH-Px activities of serum and liver in poultry (Ramnath et al., 2008; Pamok et al., 2009; Tan et al., 2010; Yang et al., 2010), but long-term heat stress reduced these antioxidant enzyme activities (Seven et al., 2009; Sahin et al., 2010; Liu et al., 2014). On the other hand, the mild heat stress induces mitochondrial biogenesis and upregulated AMPK and PGC-1α expression, which activated the expression of Nrf2, SOD, and GPX genes (Liu et al., 2012; Wenz, 2013). But the mitochondrial biogenesis could be impaired under long-term heat stress which reduced the AMPK and PGC-1α expression, and decreased the antioxidant enzyme activities (Yang et al., 2015). Recent studies have suggested that mitochondrial function is improved by antioxidative action of EGCG (Oliveira et al., 2016). Thus EGCG enhanced PGC-1α expression and up-regulated the biosynthesis of mitochondria proteins and alleviated mitochondria damage induced by heat stress (Lagouge et al., 2006; Park et al., 2012). Changes to the serum metabolites also reflect the degree of oxidative damage in poultry tissues caused by heat stress. The serum ALP, CK, AST, and LDH are used as indicators of liver damage (Ozaki et al., 1995; Xie et al., 2015). Sustained heat stress exacerbates the oxidative stress in tissues and significantly increases activities of serum CK, LDH, and AST (Xie et al., 2015), which is consistent with the results of this study. Heat stress also significantly increases the contents of serum UA, CHLO, and TG and significantly reduces GLU and TP content in present study, which indicates changes in fat, carbohydrate, and protein metabolism during heat stress. Studies have shown that chickens increase fat deposition during heat stress, especially in the abdominal region (Rhoads et al., 2013). Heat stress reduces the rate of lipolysis and the activity of lipolytic enzymes, increasing the levels of serum CHOL and TG (Geraert

et al., 1996). Change in the GLU content is an important index for measuring the energy metabolism of heat stress. Skeletal muscle is the main site for glucose utilization, and glycolysis and lactic acid content increase during heat stress, indicating that heat stress increases the glucose utilization by skeletal muscle, which results a decrease in blood glucose levels (DeFronzo, 1992). Rhoads et al. (2013) also reported that animals in heat stress could only use carbohydrates via glycolysis as energy, which was consistent with the increase of LDH activity under the condition of heat stress. The effect of EGCG on regulating heat stress in broilers was also reflected by changes in the serum metabolites. EGCG significantly decreased the levels of serum UA, CHOL, and TG and the activities of serum enzymes (CK, LDH, and AST) in heat-stressed broiler chickens. Firstly, EGCG alleviated the oxidative stress and protect protein from degradation and damage (Inoue et al., 2013). Secondly, EGCG could alleviate fat deposition in broilers through inhibiting fat anabolism and stimulating lipid catabolism in broilers (Huang et al., 2015). The regulating mechanism of EGCG on lipid metabolism may also associate with AMPK signal pathway (Kim et al, 2014). Our previous study also showed that AMPK expression negatively related to intramuscular lipid in chickens (Yang et al., 2015). Therefore, EGCG inhibited lipogenesis and increased the lipid oxidation by the activation of p-AMPK (Suliburska et al., 2012; Kim et al., 2013; Wang et al., 2014).

ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (Grant No.: 31474117). In addition, we sincerely thank the LetPub for languageediting service.

REFERENCES Akbarian, A., J. Michiels, A. Golian, J. Buyse, Y. Wang, and S. De Smet. 2014. Gene expression of heat shock protein 70 and antioxidant enzymes, oxidative status, and meat oxidative stability of cyclically heat-challenged finishing broilers fed Origanum compactum and Curcuma xanthorrhiza essential oils. Poult. Sci. 93:1930–1941. Baumgard, L. H., and R. P. Rhoads. 2013. Effects of heat stress on postabsorptive metabolism and energetics. Annu. Rev. Anim. Biosci. 1:311–337. Bogdanski, P., J. Suliburska, M. Szulinska, M. Stepien, D. PupekMusialik, and A. Jablecka. 2012. Green tea extract reduces blood pressure, inflammatory biomarkers, and oxidative stress and improves parameters associated with insulin resistance in obese, hypertensive patients. Nutr. Res. 32:421–427. DeFronzo, R. A. 1992. Pathogenesis of type 2 (non-insulin dependent) diabetes mellitus: A balanced overview. Diabetologia. 35:389–397. Dembinska-Kiec, A., O. Mykk¨ anen, B. Kiec-Wilk, and H. Mykk¨ anen. 2008. Antioxidant phytochemicals against type 2 diabetes. Br. J. Nutr. 99:ES109–ES117. Franco, J. G., P. C. Lisboa, N. S. Lima, T. A. S. Amaral, N. PeixotoSilva, A. C. Resende, E. Oliveira, M. C. F. Passos, and E. G. Moura. 2013. Resveratrol attenuates oxidative stress and prevents steatosis and hypertension in obese rats programmed by early weaning. J. Nutr. Biochem. 24:960–966.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

EPIGALLOCATECHIN GALLATE AND HEAT-STRESSED CHICKEN Geraert, P. A., J. C. Padilha, and S. Guillaumin. 1996. Metabolic and endocrine changes induced by chronic heat exposure in broiler chickens: Growth performance, body composition and energy retention. Br. J. Nutr. 75:195–204. Gonzalez-Esquerra, R., and S. Leeson. 2006. Physiological and metabolic responses of broilers to heat stress-implications for protein and amino acid nutrition. World. Poult. Sci. J. 62:282–295. Gu, X. H., Y. Hao, and X. L. Wang. 2012. Overexpression of heat shock protein 70 and its relationship to intestine under acute heat stress in broilers: 2. Intestinal oxidative stress. Poult. Sci. 91:790–799. Habibian, M., S. Ghazi, M. M. Moeini, and A. Abdolmohammadi. 2014. Effects of dietary selenium and vitamin E on immune response and biological blood parameters of broilers reared under thermoneutral or heat stress conditions. Int. J. Biometeorol. 58:741–752. Han, X., and T. Loa. 2007. Dietary polyphenols and their biological significance. Int. J. Mol. Sci. 8:950–988. Hofmann, G. E., and A. E. Todgham. 2010. Living in the now: physiological mechanisms to tolerate a rapidly changing environment. Annu. Rev. Physiol. 72:127–145. Huang, J. B., Y. Zhang, Y. B. Zhou, X. C. Wan, and J. S. Zhang. 2015. Effects of epigallocatechin gallate on lipid metabolism and its underlying molecular mechanism in broiler chickens. J. Anim. Physiol. Anim. Nutr. 99:719–727. Hwang, J. T., I. J. Park, J. I. Shin, Y. K. Lee, S. K. Lee, H. W. Baik, J. Ha, and O. J. Park. 2005. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase. Biochem. Biophys. Res. Commun. 338:694–699. Inoue, H., M. Maeda-Yamamoto, A. Nesumi, T. Tanaka, and A. Murakami. 2013. Low and medium but not high doses of green tea polyphenols ameliorated dextran sodium sulfate-induced hepatoxicity and nephrotoxicity. Biosci. Biootechnol. Biotechem. 77:1223–1228. Kim, H. S., M. J. Quon, and J. A. Kim. 2014. New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. Redox Biol. 2:187–195. Kim, J. J., Y. Tan, L. Xiao, Y. L. Sun, and X. Q. Qu. 2013. Green tea polyphenol epigallocatechin-3-gallate enhance glycogen synthesis and inhibit lipogenesis in hepatocytes. BioMed Res. Int. Article ID 920128, 8 pages, http://dx.doi.org/10.1155/2013/920128. Lagouge, M., C. Argmann, Z. Gerhart-Hines, H. Meziane, C. Lerin, F. Daussin, N. Messadeq, J. Milne, P. Lambert, P. Elliott, B. Geny, M. Laakso, P. Puigserver, and J. Auwerx. 2006. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 127:1109– 1122. Liu, C. T., A. George, and G. A. Brooks. 2012. Mild heat stress induces mitochondrial biogenesis in C2C12 myotubes. J. Appl. Physiol. 112:354–361. Liu, L. L., J. H. He, H. B. Xie, Y. S. Yang, J. C. Li, and Y. Zou. 2014. Resveratrol induces antioxidant and heat shock protein mRNA expression in response to heat stress in black-boned chickens. Poult. Sci. 93:54–62 Marchini, C. F. P., M. B. Caf´e, E. G. Ara` ujo, and M. R. B. M. Nascimento. 2016. Physiology, cell dynamics of small intestinal mucosa, and performance of broiler chickens under heat stress: a review. Rev. Colomb. Cienc. Pecu. 29:159–168. Mar´ın, L., E. M. Migu´elez, C. J. Villar, and F. Lomb´ o. 2015. Bioavailability of dietary polyphenols and gut microbiota metabolism: antimicrobial properties. BioMed. Res. Int. Article ID 905215, 18 pages. http://dx.doi.org/10.1155/2015/905215. Mujahid, A., N. R. Pumford, W. Bottje, K. Nakagawa, T. Miyazawa, Y. Akiba, and M. Toyomizu. 2007. Mitochondrial oxidative damage in chicken skeletal muscle induced by acute heat stress. J. Poult. Sci. 44:439–445. Oliveira, M. R., S. F. Nabavi, M. Daglia, L. Rastrelli, and S. M. Nabavi. 2016. Epigallocatechin gallate and mitochondria—A story of life and death. Pharmacol. Res. 104:70–85. Orhan, C., M. Tuzcu, H. Gencoglu, N. Sahin, A. Hayirli, and K. Sahin. 2013. Epigallocatechin-3-gallate exerts protective effects against heat stress through modulating stress-responsive transcription factors in poultry. Br. Poult. Sci. 54:447–453.

7

Ozaki, M., S. Fuchinoue, S. Teraoda, and K. Ota. 1995. The in vivo cytoprotection of ascorbic acid against ischemia/reoxygenation injury of rat liver. Arch. Biochem. Biophys. 318:439–445. Pamok, S., W. Aengwanich, and T. Komutrin. 2009. Adaptation to oxidative stress and impact of chronic oxidative stress on immunity in heat-stressed broilers. J. Thermal. Biol. 34:353–357. Park, S. J., F. Ahmad, A. Philp, K. Baar, T. Williams, H. Luo, H. Ke, H. Rehmann, R. Taussig, A. L. Brown, M. K. Kim, M. A. Beaven, A. B. Burgin, V. Manganiello, and J. H. Chung. 2012. Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases. Cell. 148:421–433. Rains, T. M., S. Agarwal, and K. C. Maki. 2011. Antiobesity effects of green tea catechins: a mechanistic review. J. Nutr. Biochem. 22:1–7. Ramnath, V., P. S. Rekha, and K. S. Sujatha. 2008. Amelioration of heat stress induced disturbances of antioxidant defense system in chicken by Brahma Rasayana. Evid. Based Compl. Alt. Med. 5:77–84. Reiter, C. E., J. A. Kim, and M. J. Quon. 2010. Green tea polyphenol epigallocatechin gallate reduces endothelin-1 expression and secretion in vascular endothelial cells: roles for AMP-activated protein kinase, Akt, and FOXO1. Endocrinology. 151:103–114. Renaudeau, D., A. Collin, S. Yahav, V. de Basilio, J. L. Gourdine, and R. J. Collier. 2012. Adaptation to hot climate and strategies to alleviate heat stress in livestock production. Animal 6:707–728. Rhoads, R. P., L. H. Baumgard, and J. K. Suagee. 2013. 2011 and 2012 Early Careers Achievement Awards:Metabolic priorities during heat stress with an emphasis on skeletal muscle. J. Anim. Sci. 91:2492–2503. Sahin, K., C. Orhan, M. Tuzcu, S. Ali, N. Sahin, and A. Hayirli. 2010. Epigallocatechin-3-gallate prevents lipid peroxidation and enhances antioxidant defense system via modulating hepatic nuclear transcription factors in heat-stressed quails. Poult. Sci. 89: 2251–2258. Sahin, K., C. Orhan, M. Tuzcu, N. Sahin, A. Hayirli, S. Bilgili, and O. Kucuk. 2016. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers. Poult. Sci. 95:1088–1095. Scalbert, A., I. T. Johnson, and M. Saltmarsh. 2005. Polyphenols: antioxidants and beyond. Am. J. Clin. Nutr. 81:215S–217S. Seven, P. T., S. Yilmaz, I. Seven, I. H. Cerci, M. A. Azman, and M. Yilmaz. 2009. Effects of propolis on selected blood indicators and antioxidant enzyme activities in broilers under heat stress. Acta Vet. Brno. 78:75–83. Suliburska, J., P. Bogdanski, M. Szulinska, M. Stepien, D. PupekMusialik, and A. Jablecka. 2012. Effects of green tea supplementation on elements, total antioxidants, lipids, and glucose values in the serum of obese patients. Biol. Trace Elem. Res. 149:315–322 Tan, G. Y., L. Yang, Y. Q. Fu, J. H. Feng, and M. H. Zhang. 2010. Effects of different acute high ambient temperatures on function of hepatic mitochondrial respiration, antioxidative enzymes, and oxidative injury in broiler chickens. Poult. Sci. 89:115–122. Tomanek, L., and M. J. Zuzow. 2010. The proteomic response of the mussel congeners Mytilus galloprovincialis and M. trossulus to acute heat stress: implications for thermal tolerance limits and metabolic costs of thermal stress. J. Exp. Biol. 213:3559–3574. Varmuzova, K., M. E. Matulova, L. Gerzova, D. Cejkova, D. GardanSalmon, M. Panh´eleux, F. Robert, F. Sisak, H. Havlickova, and I. Rychlik. 2015. Curcuma and Scutellaria plant extracts protect chickens against inflammation and Salmonella Enteritidis infection. Poult. Sci. 94:2049–2058. Wang, S., N. Moustaid-Moussa, L. X. Chen, H. B. Mo, A. Shastri, R. Su, P. Bapat, I. S. Kwun, and C. L. Shen. 2014. Novel insights of dietary polyphenols and obesity. J. Nutr. Biochem. 25:1–18. Wenz, T. 2013. Regulation of mitochondrial biogenesis and PGC-1α under cellular stress. Mitochondrion. 13:134–142. Xie, J. J., L. Tang, L. Lu, L. Y. Zhang, X. Lin, H. C. Liu, L. Odle, and X. G. Luo. 2015. Effects of acute and chronic heat stress on plasma metabolites, hormones and oxidant status in restrictedly fed broiler breeders. Poult. Sci. 94:1635–1644. Yang, L., G. Y. Tan, Y. Q. Fu, J. H. Feng, and M. H. Zhang. 2010. Effects of acute heat stress and subsequent stress removal on function of hepatic mitochondrial respiration, ROS production and lipid peroxidation in broiler chickens. Comp. Biochem. Phys. Part C. 151:204–208.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018

8

LUO ET AL.

Yang, Y., J. Song, R. Q. Fu, Y. F. Sun, and J. Wen. 2015. Expression of adenosine monophosphate-activated protein kinase subunit related to the rate of intramuscular lipogenesis in fast and slow-growing chicken strains. Avian Biol. Res. 8: 138–144. Ye, Q. Y., L. F. Ye, X. J. Xu, B. X. Huang, X. D. Zhang, Y. G. Zhu, and X. C. Chen. 2012. Epigallocatechin-3-gallate suppresses 1-methyl-4-phenyl-pyridine-induced oxidative stress in PC12 cells via the SIRT1/PGC-1αsignaling pathway. BMC Complem. Alt. Med. 12:82. Yuan, Z. H., K. Y. Zhang, X. M. Ding, Y. H. Luo, S. P. Bai, Q. F. Zeng, and J. P. Wang. 2016. Effect of tea polyphenols on

production performance, egg quality, and hepatic antioxidant status of laying hens in vanadium-containing diets. Poult. Sci. 95:1709–1717. Zhang, Z. Y., G. Q. Jia, J. J. Zuo, Y. Zhang, J. Lei, L. Ren, and D. Y. Feng. 2012. Effects of constant and cyclic heat stress on muscle metabolism and meat quality of broiler breast fillet and thigh meat. Poult. Sci. 91:2931–2937. Zhang, C., L. Wang, X. H. Zhao, X. Y. Chen, L. Yang, and Z. Y. Geng. 2017. Dietary resveratrol supplementation prevents transport-stress-impaired meat quality of broilers through maintaining muscle energy metabolism and antioxidant status. Poult. Sci. 96:2219–2225.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pex353/4739538 by University of Tasmania Library user on 04 January 2018