Printer Friendly

Proteomic assessment of the relevant factors affecting pork meat quality associated with longissimus dorsi muscles in Duroc pigs.

INTRODUCTION

Variation in meat quality traits is a well-known problem. Meat quality traits are closely related to biological traits of live animal. Hence, biological sciences including genetics, physiology, cell biology, and biochemistry have been widely employed for decades to characterize the biological mechanisms behind major variability of meat quality traits (Bendixen, 2005). Basic knowledge of these mechanisms is essential to reduce the variation in meat quality traits such as tenderness, water-holding capacity, and color. They are also important to understand the physiology of meat animals, especially on muscle growth and development (Lametsch et al., 2002; Hwang et al., 2005). Understanding and changes related to physiochemical factors, genotypes, and many other factors influence postmortem metabolism (Monin et al., 1995; Brocks et al., 1998; Wheeler et al., 2005). Some previous studies have indicated that meat quality is determined by postmortem muscle metabolism (Pette, 2002; Spangenburg and Booth, 2003). At slaughter, muscles become deprived of oxygen as the circulatory system shuts down. This lack of oxygen results in a shift to glycolytic (anaerobic) metabolism and a buildup of lactic acid, causing a drop in muscle pH (Frisby et al., 2005). Accelerated postmortem glycolysis reduces pH and increases temperature within muscle, resulting in excessive protein denaturation and inferior meat quality (Julve et al., 2000). Although extensively researched, the underlying mechanisms of many different meat quality traits are far from well understood due to many factors affecting the quality of meat (Mullen et al., 2006; Hollung et al., 2007). The proteome expressed from the genome is influenced by environmental conditions. Proteome is the molecular link between the genome and the functional quality characteristics of the meat. Therefore, proteomics is a promising and powerful tool in meat science (Lametsch and Bendixen, 2001; Morzel et al., 2004; Jia et al., 2006; Sayd et al., 2006). However proteomics has been, and still are, used in numerous studies on skeletal muscle (Picard et al., 2010).

In this study, we focus on its use in the study of livestock muscle development and meat quality with a focus on the differential expression patterns of proteins and their interactions for the development of meat quality traits.

MATERIALS AND METHODS

Animals and sample collection

The meat quality characteristics were assessed from 200 randomly selected great grandparent Duroc pigs raised from October 2011 to March 2012 for one production cycle. The live weight ranged from 100 to 120 kg. The carcasses were kept in a freezer (0[degrees]C) for 24 h after slaughtering. The frozen carcasses were thawed, deboned, and trimmed. The left side loin was transferred to the laboratory and placed in a deep-freezer (-45[degrees]C) for analysis

Gel electrophoresis and silver staining

High quality longissimus dorsi muscles (HQLD) and low quality longissimus dorsi muscles (LQLD) tissues were collected from Duroc pigs. Total protein isolation was performed using PRO-PREP protein extraction solution (iNtRON biotechnology, Sungnam, Korea) according to the manufacturer's instructions. Concentrations of eluted proteins were measured using Pierce BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). Equal amounts of protein samples were precipitated with cold acetone. Protein pellets dissolved in 1 x sodium dodecyl sulfate (SDS) sample buffer were separated by 8% and 12% SDS-polyacrylamide gel electrophoresis (PAGE). Following SDS-PAGE, protein spots were visualized using protocols described in PlusOne Silver staining kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). The complete protocol was followed to analyze gels. To prepare gels, the protocol was modified so that glutaraldehyde was omitted from the sensitization step and formaldehyde was omitted from the silver reaction step (Yan et al., 2000). Silver-stained gels were scanned (UMAX PowerLook 2100KL Imaging system, UMAX, Taiwan) and protein profiles were compared.

Liquid chromatography-tandem mass spectrometry

The resulting tryptic peptides were separated and analyzed using reversed-phase capillary high-performance liquid chromatography directly coupled to a Thermo LTQ Orbitrap mass spectrometer using published procedure described by Zuo et al. (2001) with slight modifications. Briefly, a 0.075 x 20 mm trapping column and a 0.075 x 120 mm resolving column were packed with C18AQ 218MS low formic acid C18 beads (5 pm in size, 200[Angstrom] pore size; C18AQ, Michrom BioResources, Auburn, CA, USA) and placed in-line. Peptides were bound to the trapping column for 10 min with 2% (vol/vol) aqueous acetonitrile containing 0.1% (vol/vol) formic acid. The bound peptides were then eluted with a 67 min gradient of 2% to 90% (vol/vol) acetonitrile containing 0.1% (vol/vol) formic acid at a flow rate of 0.2 [micro]L/min. For tandem mass spectrometry, the full mass scan range mode was set at m/z = 50 to 2,000 Da. After determining the charge states of the ion zoom scans, product ion spectra were acquired in MS/MS mode with relative collision energy of 55%. The individual spectra from MS/MS were processed using Protein discoverer 2.1 software (Thermo scientific, USA). The generated peak list files were used to query either the MSDB or the NCBI database using the MASCOT program (http://www.matrixscience.com). We considered modifications of methionine and cysteine, peptide mass tolerance at 2 Da, MS/MS ion mass tolerance at 0.8 Da, allowance of missed cleavage at 2, and charge states (namely, +1, +2, and +3). Only significant hits as defined by MASCOT probability analysis were initially considered.

Cell culture

Mitotic C2C12 mouse myoblasts were obtained from Chonbuk University (Jeonju, Korea). C2C12 were passaged as subconfluent monolayers in growth medium (GM) using Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY, USA) supplemented with 20% fetal bovine serum, 200 mM L-glutamine, 10 units/mL penicillin, and 10 [micro]g/mL streptomycin. Confluent (90%) myoblasts were differentiated into myotubes by culturing the cells in differentiation medium (DM) with Dulbecco's modified Eagle's medium supplemented with 2% horse serum.

3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay

The effects of [H.sub.2][O.sub.2] on cell viability were estimated using MTS Assay Kit (Promega, Madison, WI, USA). C2C12 cells were seeded into a 96-well plate for 24 h and treated with [H.sub.2][O.sub.2] (12.5 [micro]M to 1 mM) for 24 h or 48 h. MTS solution was added to the plates and incubated at 37[degrees]C with 5% C[O.sub.2] for 2 h. Absorbance at 490 nm was recorded using a GloMax-Multi Microplate Multimode Reader (Promega, USA).

Immunocytochemistry

To detect myosin heavy chain (MYH), myogenic differentiation (MyoD), and myogenin (Myog), cells were blocked with 1% bovine serum albumin and incubated with monoclonal anti-MYH (B-5), anti-MyoD (E-1), or anti-Myog (M-225) antibody at 4[degrees]C overnight (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Probed cells were reacted with a 488-conjugated anti-mouse or 594-conjugated anti-rabbit secondary antibody. For nucleus staining, cells were treated with mounting medium with 4'6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc. Burlingame, CA, USA). The cells were visualized using a FluoView confocal laser microscope (Fluoview FV10i, Olympus Corporation, Tokyo, Japan).

Reverse transcription polymerase chain reaction and real-time polymerase chain reaction analysis

Total RNA isolation was performed using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Briefly, total RNA levels were quantified by absorbance at 260 nm. RNA integrity was evaluated by 1.2% (w/v) agarose gel. Total RNA (2 [micro]g amounts) was reverse-transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. Real-time polymerase chain reaction analysis (PCR) was performed with SYBR green Premix Ex Taq II (Takara, Dalian, China) using Applied Biosystems StepOne Plus Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA). Relative quantification analysis was performed using the comparative Ct (2 [sup.(-[DELTA][DELTA]CT)]) method. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and [beta]-actin was used as endogenous control for the detection of mRNA expression levels. Primers used in the study were listed in Table 4.

Kinetic determination of LDH activity

Commercially available kits for lactate dehydrogenase (DLDH-100, QuantiChrom Lactate Dehydrogenase Kit, Gentaur Molecular, Hayward, CA, USA) were used according to the manufacturers' instructions (Stentz et al., 2010).

RESULTS AND DISCUSSION

Animals and phenotypes

The average weight before and after slaughtering of the GGP pigs were 98.23 kg and 89.98 kg, respectively. Difference of 10 kg before and after slaughtering was recorded. Important economic traits such as lean percent and eye muscle area showed an average value of 54.74% and 26.03 [cm.sup.2], respectively (Table 1).

Protein profiles in HQLD and LQLD from Duroc pigs

Meat qualities were evaluated by Korea Institute for Animal Products Quality Evaluation (KAPE) authorized by South Korean government to perform animal products grading service. The normality test was applied to show normal distribution of the traits. The highest and lowest meat grades for pH and water holding capacity were identified from the sample which accounted for 10% (20 heads) of the total population (Table 2). To obtain a comprehensive overview of protein components in HQLD and LQLD from 12 individuals, protein profiles of whole lysate of HQLD and LQLD separated by 8% and 12% SDS-PAGE were assessed by silver-stained image analysis (Figure 1A). Patterns of total protein components in whole lysate of HQLD and LQLD were similar among the individual six groups. However, significantly fewer proteins (i.e. band a and b) were expressed in HQLD compared to in LQLD. Two different protein spots were identified by a mass spectrometric analysis. Myosin binding protein C (MYBPC2) expressed during skeletal muscle development (Gurnett et al., 2010) had higher expression in HQLD than in LQLD. However, lactate dehydrogenase A (LDHA) catalyzing the conversion of pyruvate to lactate during glycolysis (Fan et al., 2011) had higher expression in LQLD than in HQLD (Figure 1B).

Protein identification and gene ontological classification by LC-MS/MS-based proteomic analysis

Next, we performed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis to elucidate the proteins involved in longissimus dorsi muscle (i.e. HQLD and LQLD) properties involved in meat quality. Among the total 24 proteins identified, 10 and 14 proteins were confirmed to be highly expressed in HQLD and LQLD, respectively. All identified proteins were clustered into the following seven categories (Figure 2A) based on information obtained from DAVID gene ontology (GO) database (http://david.abcc.ncifcrf.gov) and UniProt (http://www.uniprot.org): Catalytic activity (31%), ATPase activity (19%), oxidoreductase activity (13%), cytoskeletal protein binding (13%), actin binding (12%), calcium ion binding (6%) and structural constituent of muscle (6%) (Supplementary Table S1). The GO analysis was performed using DAVID Bioinformatics Resources 6.7 categories for both molecular function (MF) and biological process (BP). Depending on the MF in which the proteins were involved, they were categorized into the following three groups (Figure 2B): Cytoskeletal protein binding (40%), actin binding (40%), and structural constituent of muscle (20%) (Supplementary Table S2). Depending on the BP in which the proteins were involved, they were categorized into the following five groups (Figure 2C): Primary metabolic process (26%), cellular metabolic process (26%), catabolic process (18%), nitrogen compound metabolic process (19%), and oxidation reduction (11%) (Supplementary Table S3). The expression changes of the up- and down-regulated proteins in HQLD and LQLD of Duroc pigs were summarized in Table 3. LDHA was selected and subjected to further analysis by LDH activity assay and in vitro study of myogenesis under oxidative stress conditions.

Gross changes in C2C12 myoblasts in response to myogenic differentiation

C2C12 myoblasts serve as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development (Kislinger et al., 2005). A temporally well-defined myogenic differentiation program can be selectively triggered in cultured C2C12 myoblasts upon withdrawal of GM and mitogens (Gramolini and Jasmin, 1999). When switched to DM, mitotic C2C12 myoblasts rapidly cease proliferation and initiate a synchronously terminal differentiation program (Figure 3A). To investigate the patterns of protein expression and efficiency of myotube formation during myogenesis under oxidative stress condition, undifferentiation C2C12 cells were treated with various concentrations of [H.sub.2][O.sub.2] (12.5 [micro]M to 1 mM). Cytotoxicity was negligible with 200 gM H2O2. However, up to 1 mM H2O2 did reduce viability which was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay (Figure 3B). Control cells exhibited striking morphological changes over the course of 3 to 7 days, eventually fusing into mature multinucleated myotubes (i.e. by day 5). However, [H.sub.2][O.sub.2]-treated cells exhibited thread-like shape without fusing into mature multinucleated myotubes (Figure 3C). Myotubes were identified by immunocytochemistry with anti-myosin heavy chain (MYH) antibody (Figure 3D).

In vitro model of myogenesis under oxidative stress condition

The expression of myogenic regulatory factors consisting of MyoD and Myog characterizes various phases of skeletal muscle development, including myoblast proliferation, cell-cycle exit, cell fusion, and the maturation of myotubes to form myofibers (Lee et al., 2014). MyoD, the chief regulatory molecule of myogenic differentiation (Langen et al., 2004), plays an important role in cell cycle exit of differentiating myoblasts (Guo et al., 1995; Halevy et al., 1995) Terminal differentiation of myoblast, driven by expression of Myog, is essential for the formation of functional multinucleated myofibers (Liu et al., 2012). In order to study the patterns of MyoD and Myog expression (gene and protein levels) depending on oxidative stress condition during myogenesis, we used western blot and RT-PCR analyses. Our data revealed that, among various genes subjected to comparison, MyoD had significant (p<0.0001) higher expression under oxidative stress condition than under normal condition on day 3. The expression of MyoD on day 3 was not significantly different from that on day 2 under oxidative stress condition. Myog had significant (p<0.0001) higher expression under normal condition than under oxidative stress condition at day 6. Under oxidative stress condition, the expression of Myog was significantly (p<0.05) decreased at day 6 compared to that at day 5 (Figure 3E). The mRNA expression levels for selected genes were analyzed by quantitative real-time PCR with specific primer (Table 4). Western blot analysis data revealed that, of different proteins subjected to comparison, MyoD had significantly (p<0.0001) higher expression under oxidative stress condition than under normal condition at day 2. Under oxidative stress condition, the expression of MyoD at 12 h was not significantly different from that at 6 h. Moreover, Myog had significantly (p<0.0001) higher expression under normal condition than under oxidative stress condition at day 4. Under oxidative stress condition, the expression of Myog was significantly more decreased (p = 0.0016) at day 4 than at day 3 (Figure 3F). These results indicate that [H.sub.2][O.sub.2]-induced oxidative stress inhibits myogenesis through the down-regulation of Myog and the continuous up-regulation of MyoD.

Relationships between LDHA gene expression and myogenesis

Previous studies reported that porcine myogenic differentiation 1 (MyoD1) gene has been mapped to swine chromosome 2p14-p17 which is involved in the regulation of the proliferation and differentiation of skeletal muscle cells. LDHA genes mapped close to MyoD are involved in energy metabolism and protein transport processes. LDHA genes might play important roles in muscle development (Qiu et al., 2010). However, little is known about porcine LDHA genes. Therefore, we determined the relationships between LDHA gene expression and myogenesis under normal and oxidative stress condition. To investigate whether oxidative stress regulated LDHA expression at genetic levels, we used quantitative real-time PCR. The mRNA expression levels of selected genes were subjected to quantitative real-time PCR with specific primers (Table 4). Figure 4A showed that LDHA genes were increased up to day 5 during myogenesis under normal and oxidative stress condition. LDHA expression was significantly (p = 0.0088) higher under oxidative stress condition. These results indicate that up-regulated LDHA genes induced by oxidative stress might play dysfunctional roles in myogenesis.

Antioxidant properties of resveratrol

Resveratrol (RSV), a well-known phytocompound and food component, has antioxidative and multifunctional bioactivities (Wu et al., 2013). Previous studies have reported that RSV in skeletal muscle acts on protein catabolism and muscle function and confers resistance against oxidative stress, injury, and cell death. However, its action mechanisms and protein targets in myogenesis process are not completely understood (Montesano et al., 2013). Therefore, we determined the effect of RSV on LDHA gene expression in myogenesis under oxidative stress condition. C2C12 cells were treated with various concentrations of RSV (6.25 [micro]M to 100 [micro]M). Cytotoxicity was negligible with RSV (25 [micro]M) under normal and oxidative stress conditions. However, up to 100 [micro]M RSV did reduce viability which was confirmed by MTS assay (Figure 4B). However, after RSV treatment, there was no significant difference in mRNA expression of LDHA between normal condition and oxidative stress condition (Figure 4C).

Confirmation of lactate dehydrogenase activity

Elevation of plasmatic LDH levels are characteristic responses to strenuous exercise which are often used as indicators of muscle damage (Kanter et al., 1988; Bouzid et al., 2014). However, difference of LDH activity between HQLD and LQLD of Duroc pigs is not well determined. Our data showed that LDH activity was significantly (p = 0.0003) higher in LQLD than in HQLD of Duroc pigs (Figure 5A). Moreover, higher LDH activity was positively correlated with in vitro model of myogenesis under oxidative stress condition. In addition, LDH activity was significantly reduced by RSV treatment (Figure 5B). We also confirmed the patterns of MyoD and Myog expression under oxidative stress condition and RSV treatment during myogenesis by immunocytochemistry. Oxidative stress induced down-regulation of Myog and continuous upregulation of MyoD. The down-regulation induced by oxidative stress was recovered by RSV treatment. The upregulation of MyoD induced by the oxidative stress was reduced by the treatment of RSV in myogenesis (Figure 5C). In addition, there was a significant correlation between MyoD and Myog expression (gene and protein levels) under oxidative stress condition during myogenesis (Figure 5D). These results indicate that high activity of LDH by oxidative stress will result in dysfunction of myogenesis and that RSV treatment will result in its functional recovery.

In conclusion, our data demonstrated that up- or down regulation of genes and proteins are involved in muscle development, muscle function, actin organization, oxidative stress, cell proliferation, cell differentiation, and cell growth. In this paper, differential expression patterns of genes and their interaction are found to be important for the development of meat quality traits. Our proteome data provided valuable information on differentially expressed genes (LDHA) and activity of LDH in HQLD and LQLD from Duroc pigs, which may aid in the regulation of muscle development. Our study provided experimental evidence for RSV as an important regulator to improve meat quality grades in porcine. However, further study is required to determine the relationship between differential expression of genes or proteins and their direct effects on meat quality.

http://dx.doi.org/ 10.5713/ajas.16.0050

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

ACKNOWLEDGMENTS

This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011682)" Rural Development Administration, Republic of Korea.

REFERENCES

Bendixen, E. 2005. The use of proteomics in meat science. Meat Sci. 71:138-149.

Bouzid, M. A., O. Hammouda, R. Matran, S. Robin, and C. Fabre. 2014. Changes in oxidative stress markers and biological markers of muscle injury with aging at rest and in response to an exhaustive exercise. PLoS One 9:e90420.

Brocks, L., B. Hulsegge, and G. Merkus. 1998. Histochemical characteristics in relation to meat quality properties in the Longissimus Lumborum of fast and lean growing lines of Large White pigs. Meat Sci. 50:411-420.

Fan, J., T. Hitosugi, T. W. Chung, J. Xie, Q. Ge, T. L. Gu, R. D. Polakiewicz, G. Z. Chen, T. J. Boggon, S. Lonial, F. R. Khuri, S. Kang, and J. Chen. 2011. Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells. Mol. Cell. Biol. 31:4938-4950.

Frisby, J., D. Raftery, J. P. Kerry, and D. Diamond. 2005. Development of an autonomous, wireless pH and temperature sensing system for monitoring pig meat quality. Meat Sci. 70:329-336.

Gramolini, A. O. and B. J. Jasmin. 1999. Expression of the utrophin gene during myogenic differentiation. Nucleic Acids Res. 27:3603-3609.

Guo, K., J. Wang, V. Andres, R. C. Smith, and K. Walsh. 1995. MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation. Mol. Cell. Biol. 15:3823-3829.

Gurnett, C. A., D. M. Desruisseau, K. McCall, R. Choi, Z. I. Meyer, M. Talerico, S. E. Miller, J. S. Ju, A. Pestronk, A. M. Connolly, T. E. Druley, C. C. Weihl, and M. B. Dobbs. 2010. Myosin binding protein C1: a novel gene for autosomal dominant distal arthrogryposis type 1. Hum. Mol. Genet. 19:1165-1173.

Halevy, O., B. G. Novitch, D. B. Spicer, S. X. Skapek, J. Rhee, G. J. Hannon, D. Beach, and A. B. Lassar. 1995. Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. Science 267:1018-1021.

Hollung, K., E. Veiseth, X. Jia, E. M. Faergestad, and K. I. Hildrum. 2007. Application of proteomics to understand the molecular mechanisms behind meat quality. Meat Sci. 77:97-104.

Hwang, I. H., B. Y. Park, J. H. Kim, S. H. Cho, and J. M. Lee. 2005. Assessment of postmortem proteolysis by gel-based proteome analysis and its relationship to meat quality traits in pig longissimus. Meat Sci. 69:79-91.

Jia, X., K. Hollung, M. Therkildsen, K. I. Hildrum, and E. Bendixen. 2006. Proteome analysis of early post-mortem changes in two bovine muscle types: M. longissimus dorsi and M. semitendinosis. Proteomics 6:936-944.

Julve, J., J. C. Escola-Gil, A. Marzal-Casacuberta, J. Ordonez-Llanos, F. Gonzalez-Sastre, and F. Blanco-Vaca. 2000. Increased production of very-low-density lipoproteins in transgenic mice overexpressing human apolipoprotein A-II and fed with a high-fat diet. Biochim. Biophys. Acta 1488:233-244.

Kanter, M. M., G. R. Lesmes, L. A. Kaminsky, J. La Ham-Saeger, and N. D. Nequin. 1988. Serum creatine kinase and lactate dehydrogenase changes following an eighty kilometer race. Relationship to lipid peroxidation. Eur. J. Appl. Physiol. Occup. Physiol. 57:60-63.

Kislinger, T., A. O. Gramolini, Y. Pan, K. Rahman, D. H. MacLennan, and A. Emili. 2005. Proteome dynamics during C2C12 myoblast differentiation. Mol. Cell. Proteomics 4:887-901.

Lametsch, R. and E. Bendixen. 2001. Proteome analysis applied to meat science: characterizing postmortem changes in porcine muscle. J. Agric. Food Chem. 49:4531-4537.

Lametsch, R., P. Roepstorff, and E. Bendixen. 2002. Identification of protein degradation during post-mortem storage of pig meat. J. Agric. Food Chem. 50:5508-5512.

Langen, R. C., J. L. Van Der Velden, A. M. Schols, M. C. Kelders, E. F. Wouters, and Y. M. Janssen-Heininger. 2004. Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization. FASEB J. 18:227-237.

Lee, E. J., A. Malik, S. Pokharel, S. Ahmad, B. A. Mir, K. H. Cho, J. Kim, J. C. Kong, D. M. Lee, K. Y. Chung, S. H. Kim, and I. Choi. 2014. Identification of genes differentially expressed in myogenin knock-down bovine muscle satellite cells during differentiation through RNA sequencing analysis. PLoS ONE 9:e92447.

Liu, Q. C., X. H. Zha, H. Faralli, H. Yin, C. Louis-Jeune, E. Perdiguero, E. Pranckeviciene, P. Munoz-Canoves, M. A. Rudnicki, M. Brand, C. Perez-Iratxeta, and F. J. Dilworth. 2012. Comparative expression profiling identifies differential roles for Myogenin and p38alpha MAPK signaling in myogenesis. J. Mol. Cell Biol. 4:386-397.

Monin, G., E. Lambooy, and R. Klont. 1995. Influence of temperature variation on the metabolism of pig muscle in situ and after excision. Meat Sci. 40:149-158.

Montesano, A., L. Luzi, P. Senesi, N. Mazzocchi, and I. Terruzzi. 2013. Resveratrol promotes myogenesis and hypertrophy in murine myoblasts. J. Transl. Med. 11:310.

Morzel, M., C. Chambon, M. Hamelin, V. Sante-Lhoutellier, T. Sayd, and G. Monin. 2004. Proteome changes during pork meat ageing following use of two different pre-slaughter handling procedures. Meat Sci. 67:689-696.

Mullen, A. M., P. C. Stapleton, D. Corcoran, R. M. Hamill, and A. White. 2006. Understanding meat quality through the application of genomic and proteomic approaches. Meat Sci. 74:3-16.

Pette, D. 2002. The adaptive potential of skeletal muscle fibers. Can. J. Appl. Physiol. 27:423-448.

Picard, B., C. Berri, L. Lefaucheur, C. Molette, T. Sayd, and C. Terlouw. 2010. Skeletal muscle proteomics in livestock production. Brief. Funct. Genomics 9:259-278.

Qiu, H., X. Xu, B. Fan, M. F. Rothschild, Y. Martin, and B. Liu. 2010. Investigation of LDHA and COPB1 as candidate genes for muscle development in the MYOD1 region of pig chromosome 2. Mol. Biol. Rep. 37:629-636.

Sayd, T., M. Morzel, C. Chambon, M. Franck, P. Figwer, C. Larzul, P. Le Roy, G. Monin, P. Cherel, and E. Laville. 2006. Proteome analysis of the sarcoplasmic fraction of pig semimembranosus muscle: implications on meat color development. J. Agric. Food Chem. 54:2732-2737.

Spangenburg, E. E. and F. W. Booth. 2003. Molecular regulation of individual skeletal muscle fibre types. Acta Physiol. Scand. 178:413-424.

Stentz, R., R. J. Bongaerts, A. P. Gunning, M. Gasson, and C. Shearman. 2010. Controlled release of protein from viable Lactococcus lactis cells. Appl. Environ. Microbiol. 76:3026-3031.

Wheeler, T. L., L. V. Cundiff, S. D. Shackelford, and M. Koohmaraie. 2005. Characterization of biological types of cattle (Cycle VII): Carcass, yield, and longissimus palatability traits. J. Anim. Sci. 83:196-207.

Wu, R. E., W. C. Huang, C. C. Liao, Y. K. Chang, N. W. Kan, and C. C. Huang. 2013. Resveratrol protects against physical fatigue and improves exercise performance in mice. Molecules 18:4689-4702.

Yan, J. X., R. Wait, T. Berkelman, R. A. Harry, J. A. Westbrook, C. H. Wheeler, and M. J. Dunn. 2000. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666-3672.

Zuo, X., L. Echan, P. Hembach, H. Y. Tang, K. D. Speicher, D. Santoli, and D. W. Speicher. 2001. Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins. Electrophoresis 22:1603-1615.

Jin Hyoung Cho (1,a), Ra Ham Lee (1,a), Young-Joo Jeon (1,2), Seon-Min Park (3), Jae-Cheon Shin (3), Seok-Ho Kim (4), Jin Young Jeong (5), Hyun-sung Kang (6), Nag-Jin Choi (7), Kang Seok Seo (6), Young Sik Cho (8), MinSeok S. Kim (9), Sungho Ko (10), Jae-Min Seo (11), Seung-Youp Lee (12), Jung-Hyun Shim (13,14), *, and Jung-Il Chae (1), *

(1) Department of Dental Pharmacology, School of Dentistry and Institute of Dental Bioscience, BK21 plus, Chonbuk National University, Jeonju 651-756, Korea

* Corresponding Authors: Jung-Hyun Shim. Tel: +82-61-450-2684, Fax: +82-61-450-2684, E-mail: s1004jh@gmail.com / Jung-Il Chae. Tel: +82-63-270-4024, Fax: +82-63-270-4037, E-mail: jichae@jbnu.ac.kr

(2) National Marine Biodiversity Institute of Korea, Seocheon 33662, Korea.

(3) Pohang Center for Evaluation of Biomaterials, Pohang 37668, Korea.

(4) Aging Research Institute, Korea Research Institute of Bioscience & BioTechnology, Daejeon 34141, Korea.

(5) Division of Animal Genomics and Bioinformatics, National Institute of Animal science, Rural Development Administration, Suwon, 441-706, Korea.

(6) Department of Animal Science and Technology, Sunchon National University, Suncheon 540-742, Korea.

(7) Department of Animal Science, College of Agricultural and Life Science, Chonbuk National University, Jeonju 651-756, Korea.

(8) Department of Pharmacy, Keimyung University, Daegu 704-701, Korea.

(9) Department of Biomedical Engineering, Konyang University, Daejeon 35365, Korea.

(10) Department of Applied Bioscience, CHA University, Seongnam 463-836, Korea.

(11) Department of Prosthodontics, School of Dentistry and Institute of Oral Bio-Science and Research Institute of Clinical Medicine, Chonbuk National University, Jeonju 561-756, Korea.

(12) Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju 561-756, Korea.

(13) Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Mokpo 534-729, Korea.

(14) The China-US (Henan) Hormel Cancer Institute, Zhengzhou, Henan 127, China.

(a) These authors contributed equally to this work.

Submitted Jan. 19, 2016; Revised Feb. 21, 2016; Accepted Mar. 29, 2016

Table 1. Phenotypic record of meat quality traits in Duroc pigs

Traits                            N     Mean     Min      Max

Eye muscle area ([cm.sup.2])     199   28.13    20.99    33.00
Age at 90 kg (d)                 199   142.97    123      171
Average daily gain (g)           199   643.34   493.30   780.00
Backfat thickness (mm)           199   14.78    10.26    24.23
Loin depth (mm)                  199   55.61    50.40    60.40
Weight at end of test day (kg)   199   99.18    75.00    120.00
Meat quality characteristics
  pH 24h                         199    5.71     5.43     6.03
  Brightness                     199   54.50    46.71    62.25
  Redness                        199   16.98     9.75    21.10
  Yellowness                     199   11.31     5.45    16.29
  Water holding capacity         199   73.55    56.99    89.04
  Cooking loss                   199   14.53     6.65    22.95
  Moisture                       199   72.76    63.67    79.33
  Color, sensory test            199    5.37     2.50     7.50
  Flavor, sensory test           199    5.28     1.25     8.00
  Tenderness, sensory test       199    5.28     1.25     8.50
  Juiciness, sensory test        199    5.10     1.25     9.75
  Palatability, sensory test     199    5.13     1.25     8.50
  Shear force                    199    6.24     2.46    12.94
Biochemical measures
  Palmitic acid                  199   25.58    22.74    28.08
  Oleic acid                     199   35.89    10.89    44.91
  Stearic acid                   199   17.19     9.72    43.71
  Linolenic acid                 199    8.42     5.31    13.06

Traits                           Median    Std

Eye muscle area ([cm.sup.2])     28.15    0.218
Age at 90 kg (d)                  142     0.960
Average daily gain (g)           641.75   5.806
Backfat thickness (mm)           14.48    0.184
Loin depth (mm)                  55.80    0.189
Weight at end of test day (kg)   100.00   0.861
Meat quality characteristics
  pH 24h                          5.71    0.109
  Brightness                     54.40    2.981
  Redness                        17.27    1.984
  Yellowness                     11.52    2.076
  Water holding capacity         72.80    6.874
  Cooking loss                   14.64    2.998
  Moisture                       73.00    1.922
  Color, sensory test             5.42    0.873
  Flavor, sensory test            5.33    1.187
  Tenderness, sensory test        5.33    1.450
  Juiciness, sensory test         5.25    1.506
  Palatability, sensory test      5.25    1.491
  Shear force                     5.63    2.215
Biochemical measures
  Palmitic acid                  25.68    1.044
  Oleic acid                     40.00    10.118
  Stearic acid                   13.17    9.799
  Linolenic acid                  8.06    1.743

Table 2. High-or low-meat quality traits of longissimus dorsi muscles
in Duroc pigs

Traits                                      N    Mean     Min

High quality of longissimus dorsi muscles
  pH 24h                                    20   5.82    5.73
  Water holding capacity                    20   73.96   65.60
  Cooking loss                              20   14.40   7.30
  Moisture                                  20   72.95   70.17
  Shear force                               20   6.44    3.83

Low quality of longissimus dorsi muscles
  pH 24h                                    20   5.60    5.55
  Water holding capacity                    20   73.76   62.33
  Cooking loss                              20   15.39   9.27
  Moisture                                  20   72.74   68.00
  Shear force                               20   6.14    2.46

Traits                                       Max    Median    Std

High quality of longissimus dorsi muscles
  pH 24h                                    6.03     5.80    0.083
  Water holding capacity                    85.75   73.42    5.699
  Cooking loss                              21.45   13.82    3.457
  Moisture                                  75.33   72.67    1.369
  Shear force                               10.55    6.02    2.229

Low quality of longissimus dorsi muscles
  pH 24h                                    5.64     5.60    0.031
  Water holding capacity                    87.35   72.60    7.135
  Cooking loss                              22.95   15.41    2.850
  Moisture                                  75.00   73.00    1.713
  Shear force                               10.29    5.60    2.036

Table 3. List of differentially expressed genes on high-and low
quality of longissimus dorsi muscles in duroc pigs

No   UniProt (1)    UniGene (2)     Protein identified

Highly expressed genes in HQLD
1    Q5S1S4         Ssc.10960       Carbonic anhydrase 3
2    F1RKI3         Ssc:100518898   histidine triad
                                      nucleotide-binding protein 1
3    Q9TSX9         Ssc.2979        Peroxiredoxin-6
4    D3GGC9         Ssc.3835        Actinin-associated LIM
                                      protein 3
5    F1RGK5         Ssc.51787       Tropomyosin alpha-3 chain
6    P34930         Ssc.5145        Heat shock 70 kDa protein 1A
7    Q04967         Ssc.114         Heat shock 70 kDa protein 6
8    F1SEN8         Ssc.97236       LIM domain-binding protein 3
9    F1RH20         Ssc.83876       Myosin-binding protein C,
                                      fast-type
10   F1SMN5         Ssc.46794       Filamin-C

Highly expressed genes in LQLD
11   P00355         Ssc.16135       Glyceraldehyde-3-phosphate
                                      dehydrogenase
12   P00339         Ssc.50275       L-lactate dehydrogenase A
13   F1SKJ8         Ssc.26154       Parvalbumin 1
14   F1SLA0         Ssc.279         ATP synthase subunit beta
15   Q9GJT2         Ssc.217         S-formylglutathione hydrolase
16   F1RFH9         Ssc.55270       Sarcoplasmic/endoplasmic
                                      reticulum calcium ATPase 1
17   Q06AA5         Ssc.26272       Tetraspanin-9
18   F1S6Q7         Ssc:100523423   ATP synthase subunit delta,
                                      mitochondrial -like
19   A1X898         Ssc.97027       Procollagen-proline
                                      2-oxoglutarate-4-dioxygenase
20   F1RFU5         Ssc.3588        Aspartate aminotransferase
21   E7EBY5         Ssc.26469       MACRO domain containing
                                      protein 1
22   Q2XQV4         Ssc.11147       Aldehyde dehydrogenase,
                                      mitochondrial
23   I3LL15         Ssc.16302       Uricase
24   F1SLR6         Ssc.5041        Putative ribosomal RNA
                                      methyltransferase NOP2

No   UniProt (1)    UniGene (2)      Gene name      pI    MW (kDa)

Highly expressed genes in HQLD
1    Q5S1S4         Ssc.10960           CA3        7.85     29.4
2    F1RKI3         Ssc:100518898   LOC100518898   6.87     13.7

3    Q9TSX9         Ssc.2979           PRDX6       6.01      25
4    D3GGC9         Ssc.3835           PDLIM3      8.12     30.5

5    F1RGK5         Ssc.51787           TPM3       4.75     28.9
6    P34930         Ssc.5145           HSPA1A      5.73      70
7    Q04967         Ssc.114            HSPA6       6.06     71.1
8    F1SEN8         Ssc.97236           LDB3       7.78     75.8
9    F1RH20         Ssc.83876          MYBPC2      6.55    127.6

10   F1SMN5         Ssc.46794           FLNC       5.96    290.2

Highly expressed genes in LQLD
11   P00355         Ssc.16135          GAPDH       8.35     35.8

12   P00339         Ssc.50275           LDHA       8.07     36.6
13   F1SKJ8         Ssc.26154          PVALB1      5.07     12.1
14   F1SLA0         Ssc.279            ATP5B       5.27     56.3
15   Q9GJT2         Ssc.217             ESD        7.02     31.5
16   F1RFH9         Ssc.55270          ATP2A1      5.29    109.1

17   Q06AA5         Ssc.26272          TSPAN9      7.44     26.8
18   F1S6Q7         Ssc:100523423   LOC100523423   5.25     17.5

19   A1X898         Ssc.97027          P4HA1       6.01     60.9

20   F1RFU5         Ssc.3588            GOT2       8.73     24.1
21   E7EBY5         Ssc.26469         MACROD1      9.22     35.1

22   Q2XQV4         Ssc.11147          ALDH2       6.87     56.9

23   I3LL15         Ssc.16302           UOX        8.32     34.9
24   F1SLR6         Ssc.5041            NOP2       9.06     90.2

                                               Individual ion
                                                  score (3)

No   UniProt (1)    UniGene (2)      Seq.      HQLD     LQLD
                                    Cov (%)
Highly expressed genes in HQLD
1    Q5S1S4         Ssc.10960        53.46    150.33   82.96
2    F1RKI3         Ssc:100518898    11.11     3.48      0

3    Q9TSX9         Ssc.2979         7.59      2.67      0
4    D3GGC9         Ssc.3835         5.42      5.96     2.24

5    F1RGK5         Ssc.51787        5.24      7.13      0
6    P34930         Ssc.5145         3.12     12.17     4.39
7    Q04967         Ssc.114          2.95      9.69     3.98
8    F1SEN8         Ssc.97236        1.68      2.83     2.13
9    F1RH20         Ssc.83876        1.58      2.57      0

10   F1SMN5         Ssc.46794        0.44      4.59      0

Highly expressed genes in LQLD
11   P00355         Ssc.16135        46.85    81.05    447.46

12   P00339         Ssc.50275        39.76    129.85   230.24
13   F1SKJ8         Ssc.26154        20.91    15.89    22.65
14   F1SLA0         Ssc.279          8.71       0       4.89
15   Q9GJT2         Ssc.217          7.45       0       2.32
16   F1RFH9         Ssc.55270        6.45      1.96    21.11

17   Q06AA5         Ssc.26272        6.28       0       2.32
18   F1S6Q7         Ssc:100523423    5.36       0       4.15

19   A1X898         Ssc.97027        4.49       0       2.47

20   F1RFU5         Ssc.3588         3.72       0       4.7
21   E7EBY5         Ssc.26469        3.69      2.39     4.3

22   Q2XQV4         Ssc.11147        3.26      3.59     4.95

23   I3LL15         Ssc.16302        2.63      1.78     4.37
24   F1SLR6         Ssc.5041         1.95       0       6.07

pI, isoelectric point of the protein; MW, molecular weight of the
protein; Seq. Cov, percentage of sequence coverage; HQLD, high
quality of longissimus dorsi muscles; LQLD, low quality of
longissimus dorsi muscles.

(1) UniProt, Accession number in the UniProt database.

(2) UniGene, UniGene number from NCBI (National Center for
Biotechnology Information) database.

(3) Individual ion score, TurboSEQUEST or gMASCOT score.

Table 4. Primer sequences used to generate templates for reverse
transcription polymerase chain reaction

Gene name                    Symbol    GenBank
                                         ID

Myogenic                      MyoD    NM_010866
differentiation

Myogenin                      Myog    NM 031189

Glyceraldehyde-3-phosphate   GAPDH    NM 008084
dehydrogenase

Gene name                    Primer sequence                   Size
                             (5' [right arrow] 3')             (bp)

Myogenic                     F: GAT GGC ATG ATG GAT TAC AGC    528
differentiation              R: GAC TAT GTC CTT TCT TTG GGG

Myogenin                     F: GCT CAG CTC CCT CAA CCA G      424
                             R: ATG TGAATG GGG AGT GGG GA
Glyceraldehyde-3-phosphate   F: ACC ACA GTC CAT GCC ATC AC     452
dehydrogenase                R: TAC AGC AAC AGG GTG GTG GA

Figure 1. Protein profiles of high quality longissimus dorsi muscles
(HQLD) and low quality longissimus dorsi muscles (LQLD) from
Duroc pigs by image analysis. (A) The overall patterns of total
protein bands from individuals. All gels were visualized by sliver
staining. (B) Two different protein spots were identified by a mass
spectrometric analysis. a *: MYBPC2, myosin binding protein C; b #:
LDHA, lactate dehydrogenase A.

B

Band   Accession   UniGene     Gene     Protein        MW      Score
       no.         ID          name     name           (kDa)

a *    F1rH20      Ssc.83876   MYBPC2   Myosin         127.6   120.82
                                        binding
                                        protein C

b #    P00339      Ssc.50275   LDHA     Lactate         36.6    58.91
                                        dehydrogenase
                                        A

Figure 2. Ontological classifications of differentially regulated
proteins in high quality longissimus dorsi muscles (HQLD) and low
quality longissimus dorsi muscles (LQLD) from Duroc pigs. Of the total
24 identified proteins, 10 and 14 proteins were highly expressed
in HQLD and LQLD, respectively. (A) The identified proteins were
clustered into 7 categories based on information obtained from
DAVID gene ontology (GO) database; (B) The identified proteins
were clustered into 3 categories based on their molecular function;
(C) The identified proteins were clustered into 5 categories based on
their biological processes.

A

ATPase activity                       19%
oxidoreductase activity               13%
cytoskeletal protein binding          13%
actin binding                         12%
Structural consttuent of muscle        6%
calcium ion binding                    6%
catalytic activity                    31%

B

Actin binding                         40%
Structurral consituent of muscle      20%
Cytoskeletal protein binding          40%

C

Cellublar metabolic process           26%
Primary metabolic process             26%
Catabolic process                     18%
Nitrogen compound metabolic process   19%
Oxidation reduction                   11%

Note: Table made from pie chart.


----------

Please note: Some tables or figures were omitted from this article.
COPYRIGHT 2016 Asian - Australasian Association of Animal Production Societies
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2016 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Cho, Jin Hyoung; Lee, Ra Ham; Jeon, Young-Joo; Park, Seon-Min; Shin, Jae-Cheon; Kim, Seok-Ho; Jeong,
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Date:Nov 1, 2016
Words:6362
Previous Article:Rapamycin inhibits expression of elongation of very-long-chain fatty acids 1 and synthesis of docosahexaenoic acid in bovine mammary epithelial cells.
Next Article:Effects of [beta]-glucan on the release of nitric oxide by macrophages stimulated with lipopolysaccharide.
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters