Proteomic application in predicting food quality relating to animal welfare. A review

1 Proteomic application in predicting food quality relating 1 to animal welfare. A review 2 3 Daniel Mouzo1, Raquel Rodriguez-Vázquez1, José M. Lorenzo2, 4 Daniel Franco2, Carlos Zapata1 and María López-Pedrouso1* 5 1Department of Zoology, Genetics and Physical Anthropology, University of 6 Santiago de Compostela, Santiago de Compostela -15872, Spain 7 2Centro Tecnológico de la Carne de Galicia, Rúa Galicia Nº 4, Parque 8 Tecnológico de Galicia, San Cibrao das Viñas, 32900 Ourense, Spain 9 *corresponding author: [email protected] 10 11 2 1. Introduction 12 Consumer attitude towards farm animal are changing, and specially, western 13 society is very considerate of animal husbandry conditions. Indeed, animal 14 welfare and questions of ethical farming practices are becoming increasingly 15 appreciated. This fact could contribute to the decline in the demand of meat and 16 the an increase of vegetarians in the last few years (De Backer & Hudders, 2015). 17 On the other hand, animal stress has adverse consequences in food industry. 18 The absence of a proper animal welfare has negative effects on meat quality, 19 because the of stressful situations could impact on the biochemical processes 20 during the transformation of muscle into meat. Moreover, it was demonstrated 21 that there is a relationship between stress and the immune system. Health 22 problems of animals and outbreaks of disease could be avoided by improvement 23 of hygiene conditions and animal welfare standards. Therefore, it may be 24 asserted that stress is not only negative for animal welfare but also causes loss 25 of meat quality and its derived products. As a result of this increasing awareness, 26 the main intrinsic and extrinsic stressors are being examined by many studies 27 researches in detail (Xing, Gao, Tume, Zhou, & Xu, 2018). Intrinsic factors such 28 as sex, genetics and age as well as extrinsic factors such as pre-slaughter 29 transport and handling activities are the most important. Pre-slaughter stress also 30 causes a negative effect on meat quality in terms of sensory and technological 31 parameters. 32 Proteomics is defined as the large-scale analysis of proteins including their 33 identification, localization and interaction. In this regard, the proteome of a certain 34 tissue type is highly variable in response to environmental stimulation resulting a 35 3 suitable tool for comprehensive understanding of complex biological processes 36 (Pandey & Mann, 2000) as is shown in Figure 1. To date, proteomic studies have 37 not only been very useful to know in depth the physiology and biochemical 38 mechanisms, but also to find quality biomarkers (Jia et al., 2009). Advanced 39 techniques are being used to evaluate the relationship between meat quality and 40 proteomics such as 2-DE coupled to MS (mass spectrometry), iTRAQ (isobaric 41 tag for relative and absolute quantitation), MALDI-TOF (Matrix-assisted laser 42 desorption ionization time-of-flight) and LC-MS (Liquid chromatography–mass 43 spectrometry. Furthermore, bioinformatic analysis has been widely used to shed 44 more light on the subject from proteomic data. 45 This review provides an overview of proteomic applications for food industry in 46 relation to animal welfare. Then, the search of protein biomarkers associated with 47 animal stress as well as a deeper knowledge of biochemical mechanisms 48 involved are reviewed. A meta-proteomics analysis is carried out to investigate 49 the key biological processes involved in animal stress provided by many articles. 50 PLEASE INSERT FIGURE 1 HERE 51 2. Biochemical indicators of stress: cortisol, creatine kinase and lactate 52 The measurement and quantification of animal stress are known to be very 53 difficult due to the complexity of the effect. During rearing, animals mainly suffer 54 stress due to the livestock husbandry conditions producing low meat quality. 55 However, animals react quite differently both behaviourally and physiologically 56 against these stress situations. To date, indirect indicators of stress, such as their 57 effects, have been used to assess it on living organisms. The most common 58 indicators employed are cortisol, creatine kinase and lactate level in animal blood. 59 4 Accordingly, the monitoring of animal stress could be conducted using these 60 parameters and many researchers have been studying the more suitable 61 methodology. 62 Cortisol is a steroid hormone produced by adrenal cortex which is released as 63 response to stress and a low level of glucocorticoids in blood. Cortisol is a 64 biochemical parameter that can be measured both in muscle and blood and non65 invasively through saliva, urine or faeces. The levels of cortisol are clearly 66 increased in the organism as a result of a stress situation; therefore, it is used as 67 stress indicator (Rutherford, Lively, & Arnott, 2019). For instance, the cortisol 68 level was high during industrial handling due to a psychological stress in pigs 69 (Brown, Warriss, Nute, Edwards, & Knowles, 1998) and poor meat quality is 70 related to cortisol release during pre-slaughter stress for rabbits and sheep 71 (Trocino et al., 2018; Zimerman, Domingo, Grigioni, Taddeo, & Willems, 2013). 72 In recent years, there are an increasing literature indicating that cortisol levels 73 have been unequivocally correlated with meat quality (Reiche et al., 2019; 74 Trocino et al., 2018). 75 Creatine Kinase (CK) is an enzyme responsible for generation of the ATP 76 necessary during muscle contraction. In general, the presence of creatine kinase 77 in blood is considered an indirect marker of muscle. However, animal stress does 78 not always produce muscle damage, resulting no effect in CK level (Hunter et al., 79 1998). For instance, it was demonstrated that bulls transported under poor 80 conditions increased the cortisol level, unlike the CK level which was unaffected 81 (Rutherford et al., 2019). In the case of broilers, the stress of pre-slaughter 82 resulting from shackling affected the level of CK and it could be associated with 83 a lower meat quality (Fidan, Türkyılmaz, Nazlıgül, Ünübol Aypak, & Karaarslan, 84 5 2015). In beef, it was also found an association between CK and meat quality, 85 since the CK levels were inversely proportional to glycogen, pH and sensory 86 testing (Loudon et al., 2019). All these evidences suggest that CK could be used 87 as an objective biomarker of meat quality. 88 Blood lactate concentration often increases during a stress situation due to 89 muscle hexoses such as glucose or glycogen are converted into lactate. 90 Conversion to lactate occurs in anaerobic conditions as oxygen amount is not 91 enough and their release into the blood flow causing a decrease in pH. One of 92 the main drawbacks of this parameter is that the concentration causes a peak for 93 a short term of time after the stress situation (Rocha, Dionne, Saucier, Nannoni, 94 & Faucitano, 2014). On the other hand, there is a different predisposition to pre95 slaughter stress, and consequently, the lactate levels and other indicators can 96 vary among individuals (Chauhan, Ponnampalam, Dunshea, & Warner, 2019). 97 These biochemical indicators have the inconvenience of showing the values of 98 stress in a reduced period. Altering parameters often return to normal levels and 99 result impossible to measure in the final product. Conversely, the rapid 100 development of proteomic techniques is leading towards industrial applications, 101 because of the knowledge of protein content is helpful for understanding the 102 nutritional and technological properties of foods. Moreover, proteomics applied to 103 food products allow to obtain the biomarkers in each tissue reflecting 104 environmental and processing damages, representing a powerful tool for food 105 control. 106 3. Proteomic approach to monitor beef and milk quality in response to 107 stress 108 6 In cattle production, there is an increasing interest in stress control to prevent 109 economic losses and to improve animal welfare. Currently, beef industry 110 investigates the animal rearing and slaughter conditions to achieve a higher beef 111 quality (color, intramuscular fat content or marbling and water holding capacity). 112 It is proven that poor animal production conditions may simultaneously affect both 113 animal welfare and beef quality. Stress can be a as result of many factors as 114 intrinsic (physiology, age, sex and genotype) and extrinsic (temperature, handling 115 activities, human presence, lairage time, and feed/water deprivation). Among 116 them, the psychological and social aspects are more difficult to control by 117 industry. In bovine livestock, fear is a common stressor caused by handling. Heat 118 stress is especially relevant and it has been proven that leads to physiological 119 stress, causing increases in muscle pH that negatively influence beef quality 120 (Kadim et al., 2004). Another psychological aspect which triggers the fear is the 121 mixture with unfamiliar animals or being isolated (Grandin, 1997; Terlouw et al., 122 2008). Beyond these common factors, the degree of stress is highly variable 123 depending on the type, the intensity and the period. For all these reasons, stress 124 has is a highly high complex character and difficult to monitor from an industrial 125 viewpoint. However, the study of the proteome could have a great relevance for 126 search of biomarkers associated with stress as several studies have shown 127 (Table 1). 128 Moderate to high cattle stress can lead a defective meat, knowledge as DFD 129 (dark, firm and dry) meats. The main problems of this meat are the tenderness 130 alterations due to higher water holding capacity (WHC), an unappealing darker 131 color and lower microbial stability (Newton & Gill, 1981). In addition, the sensorial 132 quality of DFD meats is below normal ones. The phenomenon occurs because 133 7 there is a large consumption of muscle glycogen reserves, which produces a 134 great accumulation of lactic acid under stress conditions. This fact provokes an 135 important pH decline, altering the normal acidification process during aging meat. 136 For these reasons, the ultimate pH (measured at 24-48 h) is often higher than 6.0 137 provoking a scarce protein denaturation and the bound water remain tightly 138 connected. Moreover, the darker color is due to higher intracellular water content, 139 which reflects less light. The myoglobin is not denatured at high pH facilitating an 140 aerobic metabolism at the surface and iron remains in the ferrous state (Miller, 141 2007). In general, bovine muscle undergo noticeable changes in response to pre142 slaughter stress which lead to DFD meats. Proteomics to study stress condition 143 may analyse this type of meats in the discovery of protein biomarkers (Table 1). 144 According to Franco et al. (2015), changes in the proteome between normal and 145 DFD meats were characterised by structural proteins such as myosin light chain 146 isoforms and troponin C type 2. Mahmood, Turchinsky, Paradis, Dixon, & Bruce, 147 (2018) also found differences in structural proteins of insoluble fraction analysing 148 meat with pH higher than 5.9 vs. normal meats. These authors suggested that an 149 increased pH as well as a reduced glucidic potential in muscle might be due to 150 the upregulation of oxidative myofibril proteins during the physiological demand, 151 producing changes in myosin isoforms. On the other hand, soluble proteins such 152 as isoform alpha-1 actin, glycogenin-1 and isoform beta-2 of protein phosphatase 153 were differentially more abundant in darker meats with pH higher than 5.9. A 154 sarcoplasmatic subproteomic study using liquid isoelectric focusing (OFFGEL) 155 and mass spectrometry also detected five protein bands significantly different 156 between normal and DFD meats. These bands were identified as actin, 157 phosphoglucomutase-1, alpha-crystallin B, heat shock protein beta-6 and heat 158 8 shock protein beta-1 (Fuente-Garcia et al., 2019). Furthermore, substantial 159 phosphoproteome changes were detected, resulting in more abundance of 160 phosphoproteins in DFD beef than in normal meat related to structural161 contractile, metabolism, electron transport chain, actin polymerization and stress 162 response (Mato et al., 2019). 163 Stress can also play a negative effect on milk in terms of yield and composition 164 (Pragna et al., 2017; West, 2003). In general, heat stress is one of the major 165 factors which affects both quality and quantity of milk, causing diseases or 166 reducing growth in small ruminants (Berihulay, Abied, He, Jiang, & Ma, 2019). 167 Within dairy cattle, stress undoubtedly is one of the major concerns around the 168 world. Factors such as temperature and relative humidity may result in milk yield 169 reduction and lower quality in relation to fat, protein, casein and lactose content 170 (Bernabucci et al., 2014), because heat stress lead to reduced triacylglycerol lipid 171 production of short and medium chain fatty acids, meanwhile long chain fatty 172 acids are increased. This entails significant changes in physical properties and 173 nutritional value of milk (Liu et al., 2017). Moreover, cows suffering extreme 174 conditions of temperature and humidity produce milk with lower lactose and 175 protein (Garcia, Angeli, Machado, de Cardoso, & Gonzalez, 2015). In the protein 176 fraction, the caseins are the most important in milk entailing around 78% of total 177 protein. A proteomic approach revealed that differentially expressed proteins by 178 heat stress effect are associated with cell-substrate junction assembly and 179 catabolic and metabolic processes, and particularly, synthesis and secretion of 180 milk including milk protein and fat. Regarding caseins, a reduced content of β181 casein together with fatty acids were observed under heat stress conditions (Li, 182 Wang, Li, & Wang, 2017). Another relevant tissue, which is greatly affected by 183 9 heat stress, is the subcutaneous adipose, hence it is interesting for protein 184 biomarkers searching. In this regard, Nrf2-mediated oxidative stress response 185 turned out to be the most affected pathway by seasonal heat stress on dairy cows 186 (Zachut et al., 2017). As mentioned previously, dairy cows are more susceptible 187 to diseases under heat stress conditions. Indeed, bacterial mastitis is the most 188 relevant disease in dairy cows. The intramammary infections lead to a high value 189 of somatic cells (leukocytes and epithelial cells) in milk with respect to normal 190 milk. For this reason, the somatic cells count is evaluated for the detection of 191 cows infected with significant pathogens. It has been reported that somatic cells 192 count in milk was higher in animals under thermal stress (cold and heat stress) 193 due to immune function depression caused by oxidative stress (Hammami, 194 Bormann, M’hamdi, Montaldo, & Gengler, 2013). From a proteomic viewpoint, 195 plasma tumor necrosis factor-α and interleukin-6, two pro-inflammatory protein, 196 were increased in cow blood plasma by long-term heat conditions (Min et al., 197 2016). Heat stress also has negative consequences on reproductive 198 performance, decreasing the conception rates during summer seasons affecting 199 milk production indirectly (Polsky & von Keyserlingk, 2017). Moreover, cows 200 during early lactation may suffer fatty liver disease provoked by heat stress due 201 to a function liver deterioration. In a proteomic study of liver tissue, the pathways 202 differing between stressed group by cool and heat were identified as oxidative 203 phosphorylation, mitochondrial dysfunction, farnesoid X receptor/retinoid X 204 receptor (FXR/RXR) activation, and the methyl-malonyl pathway (Skibiel, Zachut, 205 do Amaral, Levin, & Dahl, 2018). Thus, the requirements of lactating dairy cattle 206 must be considered in order to increase milk production and quality. 207 16 one of the most important meat quality parameters and at the same time one of 356 the most difficult to homogenate. The second GO term with a high level of 357 significance is “response to unfolded protein” which is comprised mainly of heat 358 shock proteins (HSPs). HSPs are molecular chaperones whose main function is 359 to assist in protein assembly and disassembly, folding and unfolding or refolding 360 damage proteins. This means that these proteins play an important role 361 protecting cells and structures against stress. Indeed, the concentration of 362 HSP90 was strongly correlated with WHC (cooking and drip losses) as well as 363 meat luminosity in pig longissimus dorsi muscle (Zhang et al., 2014). 364 Finally, the third GO term with a high degree of significance is “hydrogen peroxide 365 catabolic process”. Peroxiredoxin isoforms, manganese superoxide dismutase 366 and glutathione peroxidase are included in this category of Table 2. Peroxiredoxin 367 was proposed as a potential protein biomarker for meat tenderness in bovine and 368 goat longissimus thoracis muscle (Jia et al., 2009; Wang et al., 2016). 369 Manganese superoxide dismutase and glutathione peroxidase are endogenous 370 antioxidants occurring in the pig muscle, that have influence on pork quality (Hu 371 et al., 2019; C. Zhang et al., 2015). 372 Overall, the stress proteins of these three GO terms: muscle filament sliding, 373 response to unfolded protein and hydrogen peroxide catabolic process are clearly 374 associated with pork quality parameters. Proteomic technologies may provide a 375 valuable tool to control pork quality at industrial level. 376 PLEASE INSERT FIGURE 3 HERE 377 5. Proteomic approach to monitor quality of chicken in response to stress 378 17 Poultry meat and eggs are especially consumed above other types of animal food 379 for a wide variety of reasons, representing an important source of high-quality 380 protein. Accordingly, there is also an increasing tendency for chicken 381 consumption more noticeable in developing countries. All over the world, there is 382 a growing demand due to the low cost, a good nutritional quality and the 383 sustainability of the process. Thus, the poultry industry is growing rapidly, and the 384 breeding programs are focused on traits such as a rapid growth of the breast and 385 legs (thigh and drumstick). Over the last few years, the increasing demand for 386 further-processed meats together with a preference for breast has led to select 387 birds with high breast development. However, this selection has increased the 388 incidence of meat abnormalities such as PSE meat (Petracci, Mudalal, Soglia, & 389 Cavani, 2015). PSE meat is a serious problem in breast meat reducing consumer 390 perception because of the presence of moisture and lighter color. With this type 391 of meats, the normal value of pH is above 7 in the muscle of a living animal 392 decreasing to 5.8-5.9 at 6 hours post-mortem. The high glycolytic potential of the 393 muscle will alter the meat acidification reaching lower pH values than the normal 394 meat showing a pale colour and a poor WHC (Petracci et al., 2015). The cation 395 regulation in the cells can increase intracellular calcium and free radical 396 production affecting membrane integrity and muscle acidification (Sandercock, 397 Barker, Mitchell, & Hocking, 2009). In fact, the Ca2+ factor enzymes can trigger 398 the PSE symptoms deteriorating breast quality (Freitas et al., 2017). Therefore, 399 the proteins also were altered, and a proteomic study showed that proteins 400 involved in several biological process (glycolytic, muscle contraction, proteolytic, 401 ATP regeneration, energy metabolism and CO2 hydration) were more abundant 402 in PSE-like meat (Desai et al., 2016). 403 18 Indeed, these fast-growing and heavier birds are more susceptible to heat stress 404 due to reduced thermoregulatory capacity. It has been demonstrated that the 405 selection for fast-growing has reduced their capacity to counteract the acute 406 thermal effects with important implications in protein functionality and 407 consequently in meat quality (Sandercock, Hunter, Mitchell, & Hocking, 2006; 408 Zaboli, Huang, Feng, & Ahn, 2018). In this regard, heat stress is considered the 409 most important environmental stressors in the poultry industry. The analysis of 410 the muscle proteome must be considered in the study of molecular mechanisms 411 associated with stress. It will allow us to evaluate what proteins play an important 412 role in response to stress. According to Xing et al. (2017), heat stress produced 413 a change in proteins involved in glycolysis pathway, calcium signalling and 414 molecular chaperones of pectoralis major muscle. As explained above, these 415 proteins indicated an increase of glycolysis in early post-mortem leading to a fast 416 decline of pH. An abnormal Ca+2 influx into the mitochondria can also be inferred 417 from these data increasing the energy conversion potential. In addition, 418 chaperones such as HSPs involved in protein assembly and folding translocation 419 and interaction with damages proteins were affected by heat stress in breast 420 muscle. 421 Some evidences suggest that the heat stress particularly affect the immune 422 response modulated by the central nervous system and hypothalamic-pituitary423 adrenal and the sympathetic-adrenal medullar axes (Lara & Rostagno, 2013). 424 Indeed, organs such as thymus, spleen and liver showed a significant weight 425 reduction in birds under heat conditions (Bartlett & Smith, 2003). The 426 hypothalamus, spleen and liver tissues were studied from a proteomic approach. 427 Hypothalamus as centre for regulating heat retention or dissipation was damaged 428 19 by acute heat stress through oxidative stress, weakening the protein structures, 429 degrading misfolded proteins or remodelling cytoskeletons (Tu et al., 2018). 430 Additionally, serum proinflammatory cytokines and type I interferons, spleen 431 weight and splenocyte were indicating that an innate immunity and cell death 432 pathways can be associated with heat stress (Ma et al., 2019). To investigate the 433 temporal response to heat stress, Tang et al. (2015) employed label-free 434 quantitative analysis (SWATH) in broiler liver, resulting in the inhibition of 435 extracellular signal-regulated proteins kinases, affected lipid and amino acid 436 metabolism and induced liver immune response associated with heat stress. 437 Other pre-slaughter conditions also affect poultry meat quality such as transport 438 and fasting period (Ali, Kang, & Joo, 2008). These pre-slaughter stages affect 439 muscle glycogen stores changing the post-mortem biochemical reactions in the 440 process of transformation from muscle into meat and increasing the incidence of 441 PSE (Shimokomaki et al., 2017). The response to restrain and transport in thigh 442 muscle was evaluated using a global approach (proteomics, transcriptomics and 443 metabolomics) and resulting in activation of hypothalamic-pituitary-adrenal axe. 444 Proteins altered were mainly related with cytoskeleton structure or carbohydrate 445 metabolism (Hazard et al., 2011). Age and time in transit showed proteomic 446 changes in functions as metabolism, cell division and control of apoptosis in 447 pectoralis muscle. However, the age effect proved to be more relevant than the 448 time in transit (Zanetti et al., 2013). Fasting also causes a reduction in the 449 intestinal absorption and consequently in performance in the last moment of life. 450 In this sense, the alteration in the small intestine was studied from a proteomic 451 point of view resulting in the expression of structural proteins, and proteins 452 20 associated with lipid transport, stress response and intestinal defence as the most 453 affected (Simon et al., 2019). 454 PLEASE INSERT TABLE 3 HERE 455 6. Conclusions and future trends 456 Proteomic technology is useful in search of potential biomarkers which can be 457 linked to food quality defects associated with animal welfare. The relationship 458 relationships between selected meat protein biomarkers with muscle filament 459 sliding, muscle contraction and regulation of muscle contraction in beef meat and 460 muscle filament sliding, response to unfolded protein and hydrogen peroxide 461 catabolic process in pork were demonstrated. Consequently, significant 462 differences in structural-contractile skeletal proteins could be used as stress 463 biomarkers in the case of beef meat and pork. In addition, the requirements of 464 dairy cattle in relation to heat stress were analysed to search protein biomarkers 465 in order to minimise or at least control the impact on animal health. In the poultry 466 industry, heat stress was also widely discussed and different organs were 467 analysed from a proteomic point of view resulting in a great variety of biomarkers 468 in each case. 469 Declaration of competing interest 470 The authors declared that there is no conflict of interest 471 Acknowledgements 472 Authors are grateful to RTA 2014-00034-C04-00 (INIA-MINECO) for the financial 473 support. Special thanks for financial support from the Xunta de Galicia and the 474 European Union (ESF) for supporting Raquel Rodríguez-Vázquez pre-doctoral 475 21 scholarship. Daniel Franco and José M. Lorenzo are members of the 476 HealthyMeat network, funded by CYTED (Ref. 119RT0568). 477 478 References 479 Ali, M. S., Kang, G.-H., & Joo, S. T. (2008). A Review: Influences of Pre-slaughter 480 Stress on Poultry Meat Quality. Asian-Australasian Journal of Animal 481 Sciences, 21(6). https://doi.org/10.5713/ajas.2008.r.06 482 Bartlett, J. R., & Smith, M. (2003). 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Enrichment 809 of biological process from proteins using only the muscle tissue studies marked 810 with asterisk (Table 1). 811 Figure 3. Functional enrichment analysis of proteins using FunRich. Enrichment 812 of biological process from stress protein biomarkers (Table 2). 813 814 815 816 37 817 Figure 1. 818 819 38 820 Figure 2. 821 822 823 39 824 825 Figure 3. 826 827 40 828 Table 1. Effect of stress on quality of cattle products and search of protein biomarkers using different proteomic technologies. 829 Stress sources Effects of stress Proteomic sample Proteomic technologies Effects on proteome Protein biomarkers Reference Heat stress Alteration of synthesis and secretion of milk (milk protein and fat) Bovine mammary epithelial cells iTRAQ LC-MS/MS Elisa Western Blotting Effects associated with cell-substrate junction assembly, catabolic processes and metabolic processes - Li et al., 2017 Heat stress (long term) Inflammatory response (tumor necrosis factor-α interleukin-6) Blood plasma 2-DE MALDI-TOF/TOF Western Blotting Alteration of plasma proteins Keratin, type II cytoskeletal 3-like Protein HP-25 homolog 1 and 2 Hepatitis A virus cellular receptor 1 Transthyretin precursor Min et al., 2016 Heat stress Deterioration of function liver during early lactation contributing to fatty liver disease Liver biopsia LC-MS/MS Western Blotting Changes in oxidative phosphorylation, mitochondrial dysfunction, farnesoid X receptor/retinoid X receptor (FXR/RXR) activation, and the methylmalonyl pathway. Cytochrome c oxidase subunit 4 isoform 1 Peroxidoxin-3 Skibiel et al., 2018 Heat stress The stress increased the concentration of malondialdehyde and cortisol in blood plasma Subcutaneous adipose tissue Nano-UPLC-ESI Western Blotting Differential abundance of 107 proteins in adipose of pregnant cows, affecting Nrf2mediated oxidative stress response, acutephase response, and FXR/RXR and LXR/RXR activation Dual-specificity mitogen-activated protein kinase kinase 1 GST Mu 1 Stress-induced-phosphoprotein 1 Zachut et al., 2017 41 Different management systems Hardest conditions in the mountains induce a higher oxidative stress in the animal Serum samples Immunoblotting DIGE MALDI-TOF/TOF Increased carbonyl content in plasma proteins and higher GPx and SOD activity FGG protein Complement C3B Complement component C9 precursor Complement C1s subcomplement Serum albumin Paraoxonase Conglutinin Lactate dehydrogenase B Protein AMBP precursor Alpha-2-HS glycoprotein precursor (AHSG Immunoglobulin J chain Selenium dependent glutathione peroxidase MarcoRamell et al., 2012 Transportation, weaning and commingling Increased incidence of bacterial and viral pneumonia detected in epithelial lining fluid of the lungs Epithelial lining fluid of the lungs 2-DE LC-MS/MS Immunoblotting Different abundance in eleven proteins which could be used as biomarkers for stressassociated disease susceptibility Annexin A1 and A5 Odorant-binding protein Isocitrate dehydrogenase Fibrinogen Heme-binding protein α-2-HS-glycoprotein α-1-antichymotrypsin Albumin Mitchell et al., 2008 Unknown DFD meats Longissimus thoracis 2-DE LC-MS/MS MALDI-TOF/TOF Alteration of structuralcontractile proteins and metabolism enzymes Myosin light chain isoforms Skeletal myosin light chain 2 isoforms Troponin C type 2 Cofilin-2 Triosephosphate isomerase ATP synthase Beta-galactoside alpha-2,6sialyltransferase Franco et al., 2015* Unknown DFD meats Bovine loin samples SDS-PAGE LC-MS/MS Sarcoplasmic subproteome was strongly affected Actin Phosphoglucomutase-1 Alpha-crystallin B Heat shock protein beta-6 Heat shock protein beta-1 FuenteGarcia et al., 2019*