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Proteome analysis using iTRAQ reveals the alterations in stress-induced dysfunctional chicken muscle Tong Xing, Chong Wang, Xue Zhao, Chen Dai, Guanghong Zhou, and Xinglian Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05835 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017
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Proteome analysis using iTRAQ reveals the alterations in
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stress-induced dysfunctional chicken muscle
3 Tong Xing,† Chong Wang,† Xue Zhao,† Chen Dai,‡ Guanghong Zhou† and Xinglian Xu*,†
4 5 †
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Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Synergetic
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Innovation Center of Food Safety and Nutrition, College of Food Science and Technology,
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Nanjing Agricultural University, Nanjing 210095, China
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‡
Experimental Teaching Center of Life Science, Nanjing Agricultural University, Nanjing 210095,
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People’s Republic of China
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* Corresponding author: Xinglian Xu. Email:
[email protected]; Tel.:+86-25-84395939; Fax:
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+86-25-84395939.
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ABSTRACT: The current study was designed to investigate the changes in the protein profiles of
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pale, soft and exudative (PSE)-like muscles of broilers subjected to transportation under high
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temperature conditions, using isobaric tags for relative and absolute analysis quantitation (iTRAQ).
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Arbor Acres chickens (n = 112) were randomly divided to two treatments: unstressed control
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(CON) and 0.5 h of transport (T). Birds were transported based on a designed protocol. Pectoralis
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major (PM) muscle samples in the T group were collected and classified as normal (T-NOR) or
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PSE-like (T-PSE). Plasma activities of stress indicators, muscle microstructure and proteome were
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measured. Results indicated that broilers in the T-PSE group exhibited higher activities of plasma
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stress indicators. The microstructure of T-PSE group showed a looser network and larger
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intercellular spaces in comparison to the other groups. Proteomic analysis, based on iTRAQ
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revealed 29 differentially expressed proteins in the T-NOR and T-PSE groups that were involved
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in protein turnover, signal transduction, stress and defense, calcium handling, cell structure and
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metabolism. In particular, proteins relating to the glycolysis pathway, calcium signaling, and
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molecular chaperones exhibited significant differences that may contribute to the inferior
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postmortem meat quality. Overall, the proteomic results provide a further understanding about the
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mechanism of meat quality changes in response to stress.
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KEYWORDS: Broiler; Stress; Proteome; Glycolysis; Calcium signaling; Molecular chaperone
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INTRODUCTION
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During the past few decades, the selection of broilers for augmented growth rate and breast
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yield has been accompanied with severe muscle dysfunction and pectoral myopathy, among which
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pale, soft and exudative (PSE)-like syndrome is the most serious 1. Due to the abnormal color,
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lower water holding capacity (WHC), and impaired functional properties, it has limited value in
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the meat-processing sector, thereby causing huge losses to the modern poultry industry 2. Previous
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studies indicated that pre-slaughter stressors, such as transportation conditions and environmental
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heat, have deleterious effects on broiler welfare, and may increase the incidence of PSE-like meat
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3, 4
.
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The emergence of novel proteomic techniques in recent years has greatly aided with
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developed understanding of biological mechanisms and the discovery of different meat quality
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biomarkers
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stress and early postmortem chilling processes, which are attributed to increased postmortem
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glycolysis and a rapid decrease of pH 7. The use of two-dimensional electrophoresis, combined
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with MALDI-TOF-MS, indicated that myosin-light chain 1, troponin T, and alpha-crystalline were
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less proteolyzed and HSP27 was undetectable in the PSE zones of pig semimembranosus muscle,
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indicating a probable difference in the underlying physiological mechanism derived from stress 8.
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Di Luca et al.
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and found proteins differ in metabolism, oxidative stress response, muscle structure, and signal
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transduction. The decreased expression of stress-related proteins was correlated with an impaired
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WHC when compared with the normal, illustrating the significance of the stress response in the
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improvement of WHC.
5, 6
. Factors that directly cause the incidence of PSE-like meat are genetic, slaughter
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compared the protein profiles of pork with high and low drip loss phenotypes,
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Research on alterations in proteins in pectoralis major (PM) muscle of broilers in response to
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stress and the mechanism triggering the development of PSE-like meat is limited. In addition, the
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reason for the variation in meat quality caused by intra-broiler variability in response to stress
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remains unclear. To depict the potential mechanism of meat quality development, isobaric tags for
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relative and absolute quantitation (iTRAQ)-labeling combined with LC-MS/MS were used for
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identifying the changes in protein profiles in the PM muscle of broilers, based on categorized
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normal and PSE-like muscles collected after subjecting broilers to acute transport under high
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ambient temperature.
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MATERIALS AND METHODS
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Experimental design. Arbor Acres broiler chickens (n = 112) at 42 day of age, with an
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average weight of 2.75 kg, were divided to two treatments: control (CON) and 0.5 h transport (T).
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All procedures were consistent as recommended by the Commercial Broiler Management Guide.
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Feed and water were withdrawn 8 h prior to slaughter. Birds were held in crates (760mm ×
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480mm × 380mm) containing seven birds each with 8 replicates, totaling 56 birds that were then
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distributed at the rear of a truck. The birds in the T group were transported at an average speed of
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45 km/h on a flat road. The ambient temperature was about 33°C, and the temperature inside the
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crate was about 41°C during transportation. All birds were slaughtered in accordance with the
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commercial slaughter process, using electrical stunning, then exsanguination via the carotid
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arteries and jugular veins, followed by scalding and evisceration. Blood was collected in
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heparinized tubes and mixed gently. The tubes were centrifuged at 2,000 g at 4°C for 10 min and
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the supernatant was kept at -20°C for stress indicator determinations. Immediately after
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exsanguination, ten grams of the right PM muscle was taken quickly, frozen in liquid nitrogen and 4
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stored at -80°C for proteome measurements. The other muscles were used for assessment of
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muscle pH at 0.5 h, meat quality and histological analysis at 24 h postmortem.
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Sample collection. The criteria for categorization of muscles in the T group as normal (46
1.5 or
