Research Article Received: 17 February 2014

Revised: 9 May 2014

Accepted article published: 28 May 2014

Published online in Wiley Online Library: 4 July 2014

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6749

Constant heat stress reduces skeletal muscle protein deposition in broilers Jianjun Zuo,a† Mei Xu,a† Yusuf Auwalu Abdullahi,a,b Limei Ma,a Zhongyue Zhanga and Dingyuan Fenga* Abstract BACKGROUND: This experiment was conducted to evaluate the effects of constant heat stress on growth performance and protein metabolism in skeletal muscle of Arbor Acres broilers. RESULTS: Two hundred and seventy 21-day-old Arbor Acres broilers with similar body weight (1298 ± 28 g) were selected for a 3-week trial (29–49 days of age). The broilers were randomly assigned to three groups including the control group, constant heat stress group and pair-fed group. Up-regulation of the rectal temperature and the mRNA expression of heat shock protein 70 in liver indicate that the model for constant heat stress was success. The average daily gain, feed conversion ratio, breast and thigh muscle weight, percentage of breast muscle, crude protein content in breast and thigh muscle in constant heat stress group were significantly lower than in control group and pair-fed group. Serum uric acid content and the glutamic-oxaloacetic transaminase activity were significantly higher, while protein content and glutamic-pyruvate transaminase activity were significantly lower in liver of heat stress group than of the control and pair-fed groups. The expression of insulin-like growth factor 1, phosphatidylinositol 3-kinase and p70S6 kinase associated with protein synthesis were lower in breast muscle but higher in thigh muscle in heat stress group compared to the control or fed-pair groups. In thigh muscles, the expression of muscle ring-finger protein-1 and MAFbx associated with protein degradation were higher in the heat stress group than in the control and pair-fed groups. CONCLUSION: Poor performance of the birds under heat stress may be due to lower synthesis and increased degradation of proteins. © 2014 Society of Chemical Industry Keywords: heat stress; protein; broilers; muscle

INTRODUCTION

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AKT1. Once activated, AKT1 can phosphorylate a series of protein substrates to activate its downstream mTOR/p70S6K passage. p70S6K is a protein with an important role in mediating ribosomal protein mRNA translation, thus affecting total protein synthesis.10 The ubiquitin–proteasome pathway is the main pathway of intracellular protein degradation.11 – 14 Protein breakdown is primarily associated with increased expression of two muscle-specific ubiquitin ligases (E3s): muscle atrophy F-box (MAFbx, also called atrogin-1) and muscle ring finger 1 (MuRF1).15 In muscle-loss models, mRNA expression of two genes is up-regulated, such as paralysis, starvation, diabetes, renal failure, sepsis, and glucocorticoid.16 – 18 Furthermore, mice lacking either of the two genes show partial resistance to muscle atrophy caused by denervation.17,19 – 21 Koyama et al.22 reported that when wild-type mice and MuRF1 knockout mice were fed a diet lacking amino



Correspondence to:D.Y. Feng, No. 483, Wushan Road, Tianhe district, Guangzhou 510642, China. E-mail: [email protected]

† J.J. Zuo and M. Xu contributed equally to this study. a College of Animal Science of South China Agricultural University, Guangzhou 510642, China b Department of Animal Science of Kano University of Science and Technology, Wudil P. M. B, 3244, Kano, Nigeria

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The impact of heat stress on livestock and poultry production performance is a common phenomenon in tropical and sub-tropical climatic conditions. Heat stress can lead to heat burden, including high mortality, poor feed consumption, and low body weight (BW) gain in broilers.1,2 A range of reasons for poor performance has been proposed from reduced feed intake (accounted for 63%)3 and decreased nutrient digestibility.4 Yunianto et al.5 also reported that heat stress severely inhibited poultry protein metabolism. Protein metabolism involves protein synthesis and degradation where protein deposition is the combined effect of the synthesis and degradation. In mammals, protein synthesis is mainly affected by insulin-like growth factor 1 (IGF1), phosphatidylinositol 3-kinase (PI3K/AKT), mammalian target of rapamycin (mTOR) and the p70S6 kinase (p70S6K) signalling pathway, but in poultry the signalling pathway is shorter and cellular protein degradation occurs mainly through the ubiquitin–protein enzyme (proteasome) process.6 In birds, IGF1 firstly binds to the IGF1 receptor (IGFR) and phosphorylates the IGFR. Phosphorylated IGFR collects insulin receptor substrate-1 (IRS1) and activates its downstream signalling pathway PI3K/AKT.7 Previous studies have shown that the activation of PI3K can induce muscle hypertrophy.8,9 As a lipid kinase, PI3K can phosphorylate diphosphate phosphatide inositol to generate triphosphate phosphatide inositol. The latter provides a membrane-binding site for protein kinase B (AKT1) and activates

www.soci.org acids, MuRF1 knockout mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Bodine et al.16 found that MAFbx was a muscle-specific F-box protein highly expressed during muscle atrophy. In the past decade, various studies have reported important roles of MAFbx and MuRF1 in the degradation of skeletal muscle protein.17,18,23 – 25 In southern China, the frequent high temperatures in summer cause serious impact on broilers. Therefore, studying the mechanism of how heat stress reduces the performance of broilers is necessary. This study was conducted to evaluate the effect of constant heat stress on growth performance, carcass and muscle composition, blood biochemical indexes, transaminase activities and gene expression related to protein metabolism in Arbor Acres broilers, exploring the mechanism of the decrease in protein deposition in skeletal muscle of broilers under heat stress conditions.

MATERIALS AND METHODS Experimental design and diets Two hundred and seventy 21-day-old Arbor Acres broilers with similar body weights (1298 g ± 28 g) were selected for the study. The birds were adapted for 7 days before the experiment, which lasted for 3 weeks (from the age of 29 to 49 days). The broilers were randomly assigned to three groups with six replicates (pens) with 15 birds of each pen in each group. The three groups were: (1) the control group (house temperature was 23 ∘ C), (2) the constant heat stress group (house temperature was 34 ∘ C), and (3) the pair-fed group (temperature was 23 ∘ C and pair-fed to the constant heat stress group), respectively. The experiment was conducted in a laboratory belonging to Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition. The laboratory was equipped for controlling ambient conditions where temperature and humidity were monitored and maintained with an automatic control system. The experimental diets and nutrient composition were designed according to the NRC Nutrient Requirements of Poultry.26 Feed composition and nutrient levels are shown in Table 1. Growth performance At the end of the experiment, broiler performance was assessed through feed intake, BW gain, and feed conversion ratio (FCR). The FCR was calculated based on the BW gain/feed intake for each replicate pen. Characteristic response to constant heat stress On day 49, immediately after weighing, 10 birds from each pen were randomly taken for rectal temperature measurement. Another three birds from each pen, whose BWs were near the average, were chosen to collect liver samples for analysis of HSP70 mRNA expression. The livers were placed in liquid nitrogen immediately after collection and then stored at −80 ∘ C. Serum biochemical indexes Before collecting the tissue samples, 2 mL jugular vein blood was collected from three birds from each pen. Serum was isolated and mixed to measure uric acid and total protein concentration by the phospho-tungstic acid deoxidising method and bicinchinonic acid (BCA) assay.27,28

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Liver transaminase variables A 0.1 g sample of liver was taken to produce a homogenate. The liquid were separated by centrifugation at 2810xg for 15 min.

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Table 1. Diet composition and nutrient levels (air dry basis, %) Ingredient Corn Wheat Soybean meal Corn gluten meal Soybean oil Wheat bran Lysine (98.5%) Methionine (98%) Limestone CaHPO4 NaCl Choline chloride Antioxidant Premix* Total Nutrient levels† ME, MJ kg−1 Crude protein Ca Lysine Methionine Methionine + cystine

22 to 49 days 12.00 55.00 18.00 4.00 3.50 4.29 0.45 0.18 1.13 0.85 0.30 0.06 0.02 0.22 100.00 13.14 19.70 0.86 1.10 0.49 0.88

* The premix provides the following per kg of diet: Mn 100 mg, Zn 95 mg, Fe 80 mg, Cu 8 mg, Se 150 μg, I 350 μg, vitamin A 12 500 IU, vitamin D3 2500 IU, vitamin K3 2.65 mg, vitamin B1 2.0 mg, vitamin B2 6.0 mg, vitamin B6 3.0 mg, vitamin B3 12.0 mg, vitamin B12 0.025 mg, biotin 32.5 μg, vitamin E 30 IU, niacin 5 mg, folic acid 1.25 mg. † Nutrient levels were calculated values.

Eight hundred microlitres of supernatant was collected and used to determine glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activity using the method of Reitman and Frankel.29 Carcass traits and muscle composition After collection of jugular blood, the breast and thigh muscles were immediately isolated and weighed. The muscle samples were sliced and freeze dried, then weighted to calculate the moisture content. The crude protein content in muscles was determined by the Kjeldahl method.30 Analysis of mRNA expression of genes Liver, breast and thigh muscles were collected and placed immediately in liquid nitrogen and then stored at −80 ∘ C for analysis. These samples were later used for real-time polymerase chain reaction (PCR). The mRNA expressions of IGF1, PI3K, p70S6K, MuRF1 and MAFbx in liver, breast and thigh muscles were determined by RT-PCR. Total RNA was extracted from 100 mg tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and genomic DNA was eliminated using RNase-free DNase (TaKaRa, Watagawashi, Gunmaken, Japan) according to the manufacturer’s instructions. RNA concentrations and purity were determined by measuring the absorbance at 260 nm and by the ratio of the absorbances at 260 nm and 280 nm. The cDNA was synthesised by M-MLV reverse transcriptase (TaKaRa) at 42 ∘ C for 60 min with oligo-dT–adaptor primer, following the manufacturer’s protocol.

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Heat Stress Reduces Protein Deposition

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Table 2. Sequences and parameters of primers for real-time polymerase chain reaction Item 𝛽-Actin IGF-1 HSP70 PI3K p70S6K MAFbx MuRF1

Primer sequence (5′ to 3′ )

T m (∘ C)

Size (bp)

F: FCCCCAGCCATGTATGTAGCC R: FTCTGTCAGGATCTTCATGAGGTAG F: FAGCCTACAGGGTATGGATCCAGCAG R: FCAGTGTGGCGCTGAGCACGTA F: FCAAGCCCAAGGTGCAGGTGG R: FGGCGCTGGGAGTCGTTGAAG F: FCCGTGAGGCCACACTGCTAAC R: FCCGCAGGTCACAAAGTCTCCG F: FAAGAGGTGCTTCTGCCAGCG R: FTCATGCGCAAGTGCTCTGGTC F: FCCAACAACCCAGAGACCTGT R: FGGAGCTTCACACGAACATGA F: FACCCCCAACCCCATGATCCAG R: FTACACTGCTGTGGCCCCCAT

Accession No.

154

59.0

NM_205518

182

61.5

NM_001004384

150

63.8

FJ217667.1

170

61.7

AF001076

200

62.1

NM_001030721

180

56.5

NM_001039309

160

59.2

NM_204639

F, forward primer; R, reverse primer. IGF-1, insulin-like growth factor 1; HSP70, heat shock protein 70; PI3K, phosphatidylinositol 3-kinase; MuRF1, muscle ring-finger protein-1.

a

44.0 43.5 Rectal temperature (°C)

Genes were analysed by real-time quantitative PCR (RT-qPCR). The RT-qPCR was performed in triplicate using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA, USA) and the 7500 real-time PCR system (Applied Biosystems). Primers were designed using Primer software 5.0 and synthesised by Shanghai Sangon Co., Ltd (Shanghai, China). Conditions for RT-qPCR were as follows: 95 ∘ C for 60 s, 40 cycles of 95 ∘ C for 15 s, 72 ∘ C for 15 s, then followed by a hold at 4 ∘ C. Primer sequences and parameters are shown in Table 2.

43.0

b

b

pair-fed group

control group

42.5 42.0 41.5 41.0 40.5

RESULTS Characteristic response to constant heat stress The rectal temperature (Fig. 1) and the mRNA expression of HSP70 (Fig. 2) in the livers of birds under heat stress were significantly higher than those in the control group and the pair-fed group. There were no differences between the pair-fed and the control groups. The results indicated that the model can be used to study the effects of heat stress.

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heat stress group

Figure 1. Rectal temperature of broilers (n = 6). Bars that share a common superscript do not differ (P > 0.05).

1.6

a

1.4 1.2

b

b

pair-fed group

control group

1.0 0.8 0.6 0.4 0.2 0.0

heat stress group

Figure 2. Relative mRNA expression of HSP70 in livers of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of HSP70 mRNA were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units.

Effects of constant heat stress on carcass and muscle composition Constant heat stress significantly decreased the breast and thigh muscle weight by 35.38% and 18.74% compared to the control group, 28.25% and 12.29% to the pair-fed group, respectively.

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Effects of constant heat stress on growth performance Constant heat stress decreases the growth performance of broiler chicken (Table 3). The average daily gain (ADG), feed intake and feed conversion ratio (FCR) of heat stress group were significantly lower than of control by 39.48%, 15.72% and 28.47%, respectively. Compared with pair-fed groups, the ADG and FCR of heat stress group were significantly lower by 28.01% and 28.14% based on the same feed intake. Although the ADG of the control group was higher than that of the pair-fed group (P < 0.05), there was no significant difference in FCR between them.

40.0

The relative expression of HSP70 in liver

Statistical analysis Data were analysed by using Statistical Product and Service Solutions 18.0 software (IBM Corporation, Armonk, NY, USA), and presented as means ± SE. The difference between means was assessed by ANOVA and the Tukey test was then used to compare parametric data among treatments. Statistical significance between treatments was based on P < 0.05.

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Table 5. Serum uric acid and total protein content in broilers (n = 6)

Growth parameter

Constant heat stress group

Pair-fed group

Control group

Initial body weight (g) Final body weight (g) Average daily gain (g) Average daily feed intake (g d−1 ) Feed/gain ratio

1294.8 ± 10.2

1298.3 ± 12.6

1302.1 ± 24.7

2540.5 ± 29.2c

3029.3 ± 32.5b

3361.7 ± 45.2a

59.33 ± 1.10c

82.41 ± 1.90b

98.03 ± 1.24a

175.10 ± 2.10b

175.52 ± 1.80b

207.76 ± 2.06a

2.95 ± 0.04b

2.12 ± 0.05a

2.11 ± 0.04a

Results are given as means ± SE. a,b,c Means within a row with different superscripts differ at P < 0.05.

Table 4. Carcass and muscle composition of broilers (n = 6)

Body weight (g) Thigh weight (g) Percentage of breast (g kg−1 ) Percentage of thigh (g kg−1 ) Breast muscle Moisture content (g kg−1 ) Crude protein (g kg−1 ) Thigh muscle Moisture content (g kg−1 ) Crude protein(g kg−1 )

Constant heat stress group

Pair-fed group

Control group

424.0 ± 12.0c 404.1 ± 6.0b 166.9 ± 0.51b

590.9 ± 20.1b 460.7 ± 9.2b 194.8 ± 0.54a

656.1 ± 7.1a 497.3 ± 5.0a 195.1 ± 0.30a

159.0 ± 0.19

151.8 ± 0.51

188.0 ± 0.21

744.2 ± 0.46

733.1 ± 0.53

743.0 ± 0.34

882.3 ± 0.02b

901.7 ± 0.05a

907.4 ± 0.01a

724.8 ± 0.64

720.4 ± 0.40

727.3 ± 0.44

661.4 ± 0.91b

698.3 ± 0.68a

696.2 ± 0.32a

Results are given as the means ± SE. a,b,c Means within a row with different superscripts differ at P < 0.05.

The breast muscle ratio of heat stress group was significantly lower than the control and pair-fed groups by 14.45% and 14.32%, respectively. However, no differences were observed in thigh muscle ratio among the three groups (Table 4). There were no differences in moisture contents in breast and thigh muscle among the three groups. Compared to the control group and pair-fed group, the crude protein concentration in breast and thigh muscle of heat stress group was decreased significantly, but no differences were observed between the two.

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Effects of constant heat stress on serum biochemical indexes and transaminase activities in liver The serum uric acid content and glutamic-oxaloacetic transaminase activity in liver of heat stress group were significantly higher than those of the control group by 17.05% and 20.55%, pair-fed group by 17.88% and 28.26%, respectively, but the serum total protein content and glutamic-pyruvic transaminase activity in the livers of the heat stress group were significantly lower than those of the control group by 13.44% and 48.94%, pair-fed group by

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Constant heat stress group

Component Serum uric acid (mg L−1 ) Serum total protein (mg mL−1 )

Pair-fed group

Control group

106.73 ± 2.91a

90.54 ± 3.22b

91.18 ± 2.77b

25.38 ± 1.24b

28.92 ± 0.93a

29.32 ± 1.01a

Results are given as the means ± SE. a,b Means within a row with different superscripts differ at P < 0.05.

Table 6. Glutamic-oxaloacetic transaminase (GOT) glutamic-pyruvic transaminase (GPT) in livers of broilers (n = 6) Constant heat Transaminase stress group

Pair-fed group

and

Control group

GOT (IU L−1 ) 6321.43 ± 349.67a 4928.76 ± 359.63b 5243.87 ± 270.92b GPT (IU L−1 ) 176.85 ± 67.42b 352.37 ± 93.64a 346.35 ± 97.71a Results are given as the means ± SE. a,b Means within a row with different superscripts differ at P < 0.05.

3.00 IGF1/β-actin mRNA(×10-3) of liver

Table 3. Growth performance of broilers (n = 6)

Parameter

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a

a

pair-fed group

control group

2.50 2.00 1.50

b

1.00 0.50 0.00

heat stress group

Figure 3. Relative mRNA expression of IGF1 in livers of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of IGF1 mRNA were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units.

12.24% and 49.81%, respectively. Moreover, there were no differences between the control group and pair-fed group for the indexes above (Table 5 and Table 6). Effects of constant heat stress on insulin-like growth factor 1 mRNA expression The IGF1 mRNA expression in liver (Fig. 3) and breast muscle (Fig. 4) of broilers in the heat stress group was significantly lower than that in the control group and pair-fed group, but the IGF1 mRNA expression was similar in thigh muscle. There was no significant difference between the pair-fed and control groups. Expression of genes associated with protein synthesis PI3K (Fig. 5) and p70S6K (Fig. 6) were expressed differently in the breast and thigh muscles. In the breast muscle, PI3K mRNA expression in the heat stress group was significantly lower than that in the pair-fed group, and the pair-fed group was significantly

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5.0

a

pair-fed group b

control group

4.0 3.0 2.0 1.0 0.0

breast

PI3K/β-actin mRNA(×10-3)

20.0 a

A B C

5.0

breast

p70S6k/β-actin mRNA(×10-3)

heat stress group

7.0

pair-fed group

6.0

control group

5.0 4.0

6.0 4.0 2.0

a b

b

pair-fed group

thigh control group

Figure 7. Relative mRNA expression of MuRF1 in muscles of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of MuRF1 were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units. 14.0 12.0 10.0

heat stress group

A

pair-fed group control group

8.0 6.0 B 4.0

B

2.0

thigh

Figure 5. Relative mRNA expression of PI3K in muscles of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of PI3K mRNA were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units.

8.0

8.0

0.0

0.0

B

10.0

breast heat stress group

heat stress group pair-fed group control group

c

B

12.0

0.0

b

10.0

A

14.0

thigh

Figure 4. Relative mRNA expression of IGF1 in muscles of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of IGF1 mRNA were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units.

15.0

16.0

heat stress group

MAFbx/β-actin mRNA(×10-3

IGF-1/β-actin mRNA(×10-3)

a

MURF1/β-actin mRNA(×10-3)

6.0

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breast

thigh

Figure 8. Relative mRNA expression of MAFbx in muscles of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of MAFbx were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units.

Expression of genes associated with protein degradation MuRF1 (Fig. 7) and MAFbx (Fig. 8) mRNA expression had the same changes in the breast and thigh muscles. mRNA expression of both genes was higher in the heat stress group than in the pair-fed group and the control group. The increase is more significant in thigh muscle, but there were no differences between the pair-fed group and the control group.

A

B B

3.0

DISCUSSION

1.0

It has been reported that heat-exposed chickens exhibit a lower growth rate and a depressed protein retention, which may result from an alteration in protein metabolism.31 It also decreases feed consumption in order to reduce metabolic heat production and maintain homeo-thermic conditions leading to a lower BW gain.32 – 35 Compared with pair-fed birds exposed to thermo-neutrality, heat-exposed chickens still exhibit slower growth and decreased feed efficiency.34,36,37 The present study was in agreement with the above authors, in the sense that the ADG, feed intake, feed conversion ratio, the percentage of breast muscle and thigh muscle of broilers in the heat stress group were significantly lower than in the control group and the pair-fed group. Although the ADG of the pair-fed group was significantly lower than in the control group, there were no significant differences in the FCR, the percentage of breast muscle and thigh muscle between the pair-fed group and the control group.

0.0

breast

thigh

Figure 6. Relative mRNA expression of p70S6K in muscles of broilers. All samples were normalised using 𝛽-actin expression as an internal control in each real-time PCR. Relative level of p70S6K were analysed by the 2(−Delta Ct) method. Bars that share a common superscript do not differ (P > 0.05). Data are presented as means ± SE (n = 6), in arbitrary units.

lower than in the control group. The p70S6K mRNA expression in the heat stress group and the pair-fed group showed no significant difference, but both were significantly lower than in the control group. In thigh muscle, PI3K and p70S6K mRNA expression was significantly higher in the heat stress group than in the pair-fed group and control group, but the pair-fed group and the control group had no significant difference. J Sci Food Agric 2015; 95: 429–436

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2.0

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Zhang38 studied the impact of heat stress on broilers’ hypothalamic appetite regulatory gene expression and found that, although the heat stress group and the pair-fed group had the same feed intake, the regulation of gene expression is different. In the heat stress group, the expression of suppression foraging genes POMC and CRH was significantly increased. Zhang concluded that the up-regulation of suppression foraging genes was the main reason of decreasing feed intake, and the high temperature directly contribute to the suppression of the up-regulation of the foraging genes. Zhang’s findings were in agreement with Li et al.39 where rat given Pro-opiomelanocortin (POMC) were consistently gained less BW, and the difference in body mass between the POMC group and control group gradually increased. High expression of POMC led to the degradation of brown adipose tissue and the likely mechanisms underlying the lower BW gain.40 The result is consistent with the work of Temim et al.37 who highlighted that chronic heat exposure appears to have a depressive effect independent of the decrease in feed intake. A similar conclusion has been drawn by comparing chickens reared at 22 ∘ C and 32 ∘ C and receiving the same feed intake.34,36 Conversely, in rats and piglets an improvement in feed efficiency was observed at high temperatures.41,42 Temim et al.37 added that heat stress reduced nitrogen retention but the effect is independent of the feed intake. Geraert et al.36 reported that chronic exposure to 32 ∘ C also decreases protein gain (−54%) and protein retention efficiency (−46%). Therefore, we suggest that, compared with heat stress, the reduced feed intake had less effect on protein synthesis and degradation. In addition to reduced feed intake, heat stress leads to lower digestibility,4 impairs performance parameters and induces mild intestinal enteritis,1 causes oxidative stress and lipid peroxidation in broilers.43 It can be concluded that in Arbor Acres broiler chickens, apart from the impact of feed intake, constant heat stress greatly reduces growth performance and protein deposition of broilers. Heat stress reduces protein synthesis in breast muscle while induces protein degradation in thigh muscle. The expression of IGF1, PI3K and p70S6K, which promote protein synthesis, changed differently in the breast and thigh muscles. In the breast muscle, heat stress reduced the gene expression, but in the thigh muscle it was reversed. This is similar to the finding by Temim et al.37 where tissue samples performed in 5to 6-week-old chicks showed varying effects to heat according to the muscles studied: at 32 ∘ C, the proportion of pectoralis major muscle (as a percentage of BW) appeared slightly reduced (reduction lower than 10%), whereas the proportion of two leg muscles were increased (+10 to +15 S % for the sartorius muscle; +5% for the gastrocnemius muscle). Howlider and Rose35 similarly found a reduced yield of breast meat. These results are also in agreement with those of Ain Baziz et al.,44 who observed a decreased proportion of breast muscle (−12%) and a slight but significant increase in the proportion of leg muscles, i.e. thigh plus drumstick proportion (+6%). IGF1 stimulates muscle growth and protein synthesis,45,46 and Dupont et al.47 reported that IGF1 mainly promotes amino acid transport and protein synthesis and inhibits protein degradation, resulting in skeletal muscle cell hypertrophy, which is in agreement with the results obtained by Musarò et al.24 Additionally, Onagbesan et al.48 found that IGF1 stimulates DNA synthesis and cell proliferation in thecal and granulosa cells derived from chicken follicles. Sacheck et al.15 emphasised that the over-expression of IGF1 in mouse by transgenic technology caused a two-fold increase in muscle size by suppressing protein breakdown and expression of

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atrophy-related ubiquitin ligases, MAFbx and MuRF1. Goldspink and Yang23 previously came to the same conclusion. In the current study, the relative expression of IGF1 mRNA of liver and breast muscle in the heat stress group broiler was significantly lower than in the air-fed group and the control group, which might indicate that heat stress inhibits protein synthesis in the broiler breast muscle, while there were no significant changes in the thigh muscle. There have been many studies on the mTOR signalling pathway in mammals, but few can be found in the literature relating to poultry.49 Previous studies have shown that the mTOR signalling pathway is critical to the regulation of protein synthesis.50,51 Tesseraud et al.52 have reported that, in chicken muscle, insulin and re-feeding can activate the PI3K/mTOR/p70S6K pathway, and this pathway plays an important role in the nutritional regulation of mRNA transcription and protein synthesis. The downstream p70S6K, a cytoplasmic serine/threonine kinase, is critical for translational regulation of genes that encode essential components of the protein synthesis apparatus.53 Bigot et al.54 proved that p70S6K is expressed in chicken skeletal muscle and plays a key role in the control of insulin-dependent protein synthesis; this is also consistent with Tawa et al.12 In this study, the PI3K and p70S6K mRNA expression were reduced in the breast muscle while increased in the thigh muscle under the heat stress group, which might demonstrate that protein synthesis was inhibited in the breast muscle while promoted in the thigh muscle. Muscle loss has been linked to increased expression of two ubiquitin ligases termed muscle atrophy F-box (MAFbx) and muscle ring finger 1 (MuRF1).18 Sacheck et al.15 found a highly significant linear relationships between MAFbx expression (up to 200% of control) and protein breakdown (70–110% of control; R2 = 0.79, P < 0.001). The two kinases are regulated by nutritional status and insulin in vivo despite the particularities of the insulin signalling pathway in chicken muscle.54 In the present study, both MAFbx and MuRF1 were highly expressed in the breast and thigh muscle under the heat stress group, which might indicate that heat stress greatly promoted the protein degradation. The above results showed that the mechanisms behind the heat stress reductions in broiler breast and thigh muscle protein depositions were different. In breast muscle, it mainly reduces protein synthesis, while in thigh muscle, it mainly increases protein degradation. The increased mRNA of PI3K and p70S6K were not a result of IGF1, as the IGF1 showed no significant increase in the thigh muscle under heat stress. There are many studies on negative feedback regulation about PI3K55,56 and p70S6K.57,58 The increase of PI3K and p70S6K in the thigh muscle under heat stress might be the result of negative feedback regulation of highly increased MAFbx, for balancing the great loss of protein and ensuring survival. The result in the present study showing that heat stress had different impacts protein metabolism in broiler breast and thigh muscles was similar to the work of Chen.59 Temim et al.31 measured protein synthesis in vivo in the pectoralis major, sartorius and gastrocnemius muscles and determined protein breakdown in the same muscles. Their result showed that chronic heat stress markedly reduces protein synthesis, irrespective of muscle type that was related to the lower capacity for protein synthesis (muscle RNA/protein). They further emphasised that chronic heat exposure also decreased protein breakdown in the pectoralis major and sartorius muscles, which was not observed in the gastrocnemius muscle. Protein synthesis was more affected than protein breakdown, leading to reduced protein deposition, at least in the pectoralis major and gastrocnemius muscles. It was concluded that chronic heat exposure

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J Sci Food Agric 2015; 95: 429–436

Heat Stress Reduces Protein Deposition

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decreased muscle protein deposition, mainly by reducing protein synthesis.

ACKNOWLEDGEMENTS This research was funded by Guangdong Province Natural Science Foundation of China (Project No. S2013010013215). The authors greatly appreciate the assistance of the postgraduate students of the Feed Biotechnology Laboratory.

REFERENCES

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1 Quinteiro-Filho WM, Rodrigues MV, Ribeiro A, Ferraz-de-Paula V, Pinheiro ML, Sá LR, et al, Acute heat stress impairs performance parameters and induces mild intestinal enteritis in broiler chickens: Role of acute hypothalamic–pituitary–adrenal axis activation. J Anim Sci 90:1986–1994 (2012). 2 Sohail MU, Hume ME, Byrd JA, Nisbet DJ, Ijaz A, Sohail A, et al, Effect of supplementation of prebiotic mannan-oligosaccharides and probiotic mixture on growth performance of broilers subjected to chronic heat stress. Poult Sci 91:2235–2240 (2012). 3 Dale NM and Fuller HL, Effect of diet composition on feed intake and growth of chicks under heart stress. Poult Sci 59:1434–1441 (1980). 4 Bonnet S, Geraert PA, Lessire M, Carre B and Guillaumin S, Effect of high ambient temperature on feed digestibility in broilers. Poult Sci 76:857–863 (1997). 5 Yunianto VD, Hayashi K, Kaneda S, Ohtsuka A and Tomita Y, Effect of environmental temperature on muscle protein turnover and heat production in tube-fed broiler chickens. Br J Nutr 77:897–909 (1997). 6 Glass D J, Skeletal muscle hypertrophy and atrophy signaling pathways. Int J Biochem Cell Biol 37:1974–1984 (2005). 7 Coleman ME, DeMayo F, Yin KC, Lee HM, Geske R, Montgomery C, et al, Myogenic vector expression of insulin-like growth factor stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J Biol Chem 270:12109–12116 (1995). 8 Shioi T, McMullen JR, Kang PM, Douglas PS, Obata T, Franke TF, et al, Akt/protein kinase B promotes organ growth in transgenic mice. Mol Cell Biol 22:2799–2809 (2002). 9 Lai KM, Gonzalez M, Poueymirou WT, Kline WO, Na E, Zlotchenko E, et al, Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy. Mol Cell Biol 24:9295–9304 (2004). 10 Gingras AC, Raught B and Sonenberg N, Regulation of translation initiation by FRAP/mTOR. Genes Dev 15:807–826 (2001). 11 Solomon V and Goldberg AL, Importance of the ATP–ubiquitin–proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts. J Biol Chem 271:26690–26697 (1996). 12 Tawa NE Jr, Odessey R and Goldberg AL, Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles. J Clin Invest 100:197–203 (1997). 13 Mitch WE, Bailey JL, Wang X, Jurkovitz C, Newby D and Price SR, Evaluation of signals activating ubiquitin–proteasome proteolysis in a model of muscle wasting. Am J Physiol 276:C1132–C1138 (1999). 14 Smith IJ and Dodd SL, Calpain activation causes a proteasome-dependent increase in protein degradation and inhibits the Akt signalling pathway in rat diaphragm muscle. Exp Physiol 92:561–573 (2007). 15 Sacheck JM, Ohtsuka A, McLary SC and Goldberg AL, IGF-I stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1. Am J Physiol Endocrinol Metab 287:591–601 (2004). 16 Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, et al, Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294:1704–1708 (2001). 17 Gomes MD, Lecker SH, Jagoe RT, Navon A and Goldberg AL, Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. PNAS B 98:14440–14445 (2001). 18 Zhao W, Wu Y, Zhao J, Guo S, Bauman WA and Cardozo CP, Structure and function of the upstream promotor of the human Mafbx gene: The proximal upstream promotor modulates tissue-specificity. J Cell Biochem 96:209–219 (2005).

19 Dehoux MJ, van Beneden RP, Fernández-Celemin L, Lause PL and Thissen JP, Induction of MafBx and Murf ubiquitin ligase mRNAs in rat skeletal muscle after LPS injection. FEBS Lett 544:214–217 (2003). 20 Wray CJ, Mammen JM, Hershko DD and Hasselgren PO, Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle. Int J Biochem Cell Biol 35:698–705 (2003). 21 Nikawa T, Ishidoh K, Hirasaka K, Ishihara I, Ikemoto M, Kano M, et al, Skeletal muscle gene expression in space-flown rats. FASEB J 18:522–524 (2004). 22 Koyama S, Hata S, Witt CC, Ono Y, Lerche S, Ojima K, et al, Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis. J Mol Biol 376:1224–1236 (2008). 23 Goldspink G and Yang SY, Gene expression associated with muscle adaptation in response to physical signals. Chapter 7, in Cell and Molecular Response to Stress Cell, ed. by Storey KB and Storey JM. Elsevier press, Amsterdam, Netherlands, pp. 87–96 (2001). 24 Musarò A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, et al, Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 27:195–200 (2001). 25 Cong H, Sun L, Liu C and Tien P, Inhibition of atrogin-1/MAFbx expression by adenovirus-delivered small hairpin RNAs attenuates muscle atrophy in fasting mice. Hum Gene Ther 22:313–324 (2010). 26 National Research Council (NRC), Nutrient Requirements of Poultry, 9th revised edition. National Academy Press, Washington DC (1994). 27 Henry JB, Clinical Diagnosis and Management by Laboratory Methods. WB Saunders Co, Philadelphia PA (1979). 28 Smith PK, Krohn RL, Hermanon GT, Mallia AK, Gartner FH, Provenzano MD, et al, Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85 (1985). 29 Reitman S and Frankel S, A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 28:56–63 (1957). 30 Kjeldahl JZ, A new method for the determination of nitrogen in organic bodies. Anal Chem 22:366 (1883). 31 Temim S, Chagneau AM, Peresson R and Tesseraud S, Chronic heat exposure alters protein turnover of three different skeletal muscles in finishing broiler chickens fed 20 or 25% protein diets. J Nutr 130:813–819 (2000). 32 Austic RE, Feeding poultry in hot and cold climates, in Stress Physiology in Livestock, ed. by Youssef MK. CRC Press, Boca Raton, pp. 123–136 (1985). 33 Charles DR, Temperature for broilers. World Poult Sci J 42:249–258 (1986). 34 Geraert PA, Métabolisme énergétique du poulet de chair en climat chaud. INRA Prod Anim 4:257–267 (1991). (In French) 35 Howlider MAR and Rose SP, Rearing temperature and the meat yield of broilers. Br J Nutr 30:61–67 (1989). 36 Geraert PA, Padilha JC and Guillaumin S, 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 (1996). 37 Temim S, Chagneau AM, Guillaumin S, Michel J, Peresson R, Geraert PA, et al, Effects of chronic heat exposure and protein intake on growth performance, nitrogen retention and muscle development in broiler chickens. Reprod Nutr Dev 39:145–156 (1999). 38 Zhang ZY, The mechanism of chronic heat stress inducing growth performance and lipid peroxidation of tissues in AA broilers. PhD dissertation. College of Animal Science, South China Agricultural University, Guangzhou (2012). (In Chinese) 39 Li G, Mobbs CV and Scarpace PJ, Central pro-opiomelanocortin gene delivery results in hypophagia, reduced visceral adiposity, and improved insulin sensitivity in genetically obese Zucker rats. Diabetes 52:1951–1957 (2003). 40 Pritchard LE, Turnbull AV and White A, Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signalling and obesity. J Endocrinol 172:411–421 (2002). 41 Christon R, Le Dividich J, Seve B and Aumaitre A, Effect of ambient temperature on the metabolic use of dietary energy and nitrogen in growing rat. Reprod Nutr Dev 24:327–341 (1984). 42 Rinaldo D and Le Dividich J, Effects of warm exposure on adipose tissue and muscle metabolism in growing pigs. Comp Biochem Physiol A Comp Physiol 100:995–1002 (1991).

www.soci.org ˘ A, Altan A, Konyalioglu ˘ S and Bayraktar H, Effect 43 Altan O, Pabuccuoglu of heat stress on oxidative stress, lipid peroxidation and some stress parameters in broilers. Br Poul Sci 44:545–550 (2003). 44 Ain Baziz H, Geraert PA, Padilha JC and Guillaumin S, Chronic heat exposure enhances fat deposition and modifies muscle and fat partition in broiler carcasses. Poult Sci 75:505–513 (1996). 45 DeVol DL, Rotwein P, Sadow JL, Novakofski J and Bechtel PJ, Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. Am J Physiol 259:89–95 (1990). 46 McMurtry JP, Francis GL and Upton Z, Insulin-like growth factors in poultry. Domestic Anim Endocrinol 14:199–229 (1997). 47 Dupont J, Tesseraud S and Simon J, Insulin signaling in chicken liver and muscle. Gen Comp Endocrinol 163:52–57 (2009). 48 Onagbesan OM, Peddie MJ and Williams J, Regulation of cell proliferation and estrogen synthesis by ovine LH, IGF-I, and EGF in theca interstitial cells of the domestic hen cultured in defined media. Gen Comp Endocrinol 94:261–272 (1994). 49 Lee MY, Jo SD, Lee JH and Han HJ, L-leucine increases [H-3]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways. J Cell Biochem 14:1410–1419 (2008). 50 Martin KA and Blwnis J, Coordinate regulation of translation by the PI3-kinase and mTOR. Adv Cancer Res 86:1–39(2002). 51 Kimball SR and Jefferson LS, Molecular mechanisms through which amino acids mediate signaling through the mammalian target of rapamycin. Curr Opin Clin Nutr Metab Care 7:39–44 (2004).

JJ Zuo et al.

52 Tesseraud S, Abbas M, Duchene S, Bigot K, Vaudin P and Dupont J, Mechanisms involved in the nutritional regulation of mRNA translation: features of the avian model. Nutr Res Rev 19:104–116 (2006). 53 Dufner A and Thomas G, Ribosomal S6 kinase signaling and the control of translation. Exp Cell Res 253:100–109 (1999). 54 Bigot K, Taouis M and Tesseraud S, Refeeding and insulin regulate S6K1 activity in chicken skeletal muscles. J Nutr 133:369–373 (2003). 55 Fukao T, Tanabe M, Terauchi Y, Ota T, Matsuda S, Asano T, et al, PI3K-mediated negative feedback regulation of IL-12 production in DCs. Nat Immunol 3:875–881 (2002). 56 Kozma SC and Thomas G, Regulation of cell size in growth, development and human disease: PI3K, PKB and S6K. Bioessays 24:65–71(2002). 57 Klionsky DJ, Meijer AJ, Codogno P, Neufeld TP and Scott RC, Autophagy and p70S6 kinase. Autophagy 1:59–61 (2005). 58 Park S, Zhao D, Hatanpaa KJ, Mickey BE, Saha D, Boothman DA, et al, RIP1 activates PI3K-Akt via a dual mechanism involving NF-kB-mediated inhibition of the mTOR-S6K-IRS1 negative feedback loop and down-regulation of PTEN. Cancer Res 69:4107–4111 (2009). 59 Chen B, Effect of oligopeptide on digestive physiology and protein synthesization regulation for broilers. PhD dissertation. Feed Research Institute of CAAS, Beijing (2005). (In Chinese)

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J Sci Food Agric 2015; 95: 429–436

Constant heat stress reduces skeletal muscle protein deposition in broilers.

This experiment was conducted to evaluate the effects of constant heat stress on growth performance and protein metabolism in skeletal muscle of Arbor...
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