Effect of fast ph decline during the early postmortem period on calpain activity and cytoskeletal protein degradation of broiler M.

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1 Effect of fast ph decline during the early postmortem period on calpain activity and cytoskeletal protein degradation of broiler M. pectoralis major J. C. Huang,, J. Yang, F. Huang, M. Huang, K. J. Chen, X. L. Xu,,1 and G. H. Zhou Department of Engineering, Nanjing Agricultural University, Nanjing , China; Key Laboratory of Meat Processing and Quality Control, Ministry of Education China, College of Food Science and Technology, Nanjing Agricultural University, Nanjing , China; and Institute of Agro-Products Processing Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing , China ABSTRACT The objective of this study was to determine the effects of fast ph decline during the early postmortem period on calpain activity and the degradation of cytoskeletal proteins in broilers. Eighty broilers were randomly categorized into two groups: physical restraint (PR) and free struggle (FS). M. pectoralis major (PM) was used for determination of calpain activity, shear value, ultrastructure of myofibrils, and the degradation of desmin, titin, nebulin, and troponin-t. The ph (6.05) of FS group is significantly low than PR group (6.38) at 0.3 h postmortem. Fast ph decline during the early postmortem period led to a decrease of μ/m-calpain activities at 0.3 and 3 h postmortem (P < 0.05), but did not affect the ultimate μ/m-calpain activity. An initial fast decrease in ph increased the degradation of desmin, titin, nebulin, and increased the 30 kda degradation fragments of troponin-t. Therefore, the fast ph decline during the early postmortem period decreased the μ/m-calpain activity and increased the degradation of cytoskeletal proteins in broiler muscle. It is possible that the fast ph decline experienced an earlier activation of calpains that resulted in earlier protein degradation and ultimately lower shear force. Key words: autolysis, broiler, calpain, casein zymography, tenderness 2016 Poultry Science 95: INTRODUCTION Tenderness of meat is considered to be an important trait of broilers (An et al., 2010). Many researchers have reported that postmortem hydrolysis of some key cytoskeletal proteins is essential for meat tenderization (Koohmaraie, 1996; Koohmaraie and Geesink, 2006). It is currently believed that calpains are the most important endogenous enzymes involved in postmortem cytoskeletal protein hydrolysis (Taylor et al., 1995; Lee et al., 2008). In mammals, the calpain system consists of μ-calpain, m-calpain, and calpastain (Huff Lonergan et al., 2010). The calpain system of birds is not exactly the same as in mammals. There exists a distinct calpain which is defined as μ/m-calpain (Sorimachi et al., 1995). There are also reports that the activity of μ/m-calpain can be affected by fasting and electrical stimulation (Northcutt et al., 1998; Wang et al., 2012). C 2016 Poultry Science Association Inc. Received January 15, Accepted May 11, Corresponding author: xlxu@njau.edu.cn Postmortem meat tenderization is affected by the rate of ph decline during the early postmortem period (O Halloran et al., 1997). Some researchers have found that a fast ph decline during the early postmortem period can improve the tenderization of postmortem meat (Alvarado and Sams 2000; Pomponio et al., 2010; Kahraman et al., 2011). Other researchers have shown the opposite, whereby a fast ph decline during the early postmortem period delays the tenderization of postmortem meat (Yu and Lee, 1986; Northcutt et al., 1998; Molette et al., 2005). Most of the previous studies have been done in mammals, and to our knowledge, there is little information available regarding the rate of ph decline during the early postmortem period as it affects the calpain system and protein hydrolysis in broilers. Further, the precise mechanism of the interaction between postmortem ph changes and tenderness in broiler is presently unknown. Because tenderness is affected by cytoskeletal degradation, and μ/m-calpain can affect cytoskeletal degradation, the μ/m-calpain may be a link factor between ph decline and broiler meat tenderness. Therefore, the present study was designed to investigate the effect of fast ph decline during the early postmortem period on μ/m-calpain activities and cytoskeletal protein degradation in broiler PM. 2455

2 2456 HUANG ET AL. MATERIALS AND METHODS Samples and Treatment All procedures were approved by the Animal Care and Use Committee of the College of Food Science and Technology of Nanjing Agricultural University. Eighty Arbor Acres broilers (45 d old, 2.0 to 2.5 kg, mixed sex), were obtained from the holding area of a commercial plant and randomly divided into two groups [Free struggle (FS) and physical restraint (PR)]. They were then transported from a holding area to the commercial slaughter house within about 30 min. After resting for one hour, one group of the broilers were physically restrained with a nylon fabric with hook-and-loop straps to secure the wings to the body as described by Papinaho et al. (1995) and the other group (FS) was without treatment (free struggle). The live broilers were hung upside down on metal shackles (according to commercial practice). All the broilers were hung for about 30 s (induced wing flapping in the FS group) and then slaughtered directly. Slaughter was achieved by severing the jugular vein and carotid artery using handheld knife on one side of the neck to allow bleeding for 150 s. The broilers were then scalded at 54 C for 120 s and plucked for 30 s. After evisceration, both of the M. pectoralis major (PM) were immediately removed and placed on ice. At 0.3, 3, 6, and 24 h postmortem, samples for calpain activity, purification of myofibrils, and transmission electron microscopy were collected on the left PM site of ten carcasses for each treatment. Determination of lactate were collected (frozen in liquid nitrogen), ph was measured on the left PM, and the shear value was measured on the right PM site of each carcass. The samples for transmission electron microscopy and shear force were taken from the cranial end of the PM and the samples for calpain activity and Sarcoplasmic protein extraction were taken from middle of the PM. Muscle ph and Lactate Concentration The ph of PM at 0.3, 3, 6, and 24 h postmortem was measured using a calibrated ph meter (FE-20, Mettler-Toledo Instruments Co., Ltd., Zurich, Switzerland). Each muscle sample was measured in triplicate with a spear-type electrode at the thickest part of the PM muscle. Muscle lactate concentration was measured on homogenized frozen samples using a lactic acid kit (Nanjing Jiancheng Bioengineering Institute, China). Each muscle sample was measured in triplicate according to the manufacturers instructions. Sarcoplasmic Protein Extraction Sarcoplasmic proteins were extracted according to the process described by Huang et al. (2012) with slight modifications. Three minced broiler samples (0.5 g) of each PM were homogenized in 2 ml of extraction buffer which contained 0.1% (v/v) β-mercaptoethanol (MCE), 10 mm EDTA, 100 mm Tris-HCl, ph 8.3, and protease/phosphatase inhibitor cocktail (#5872, Cell Signaling Technology, Denver, America). The samples were homogenized twice using a polytron at a speed of 15,000 rpm for 15 s each with a cooling break between. The homogenate was centrifuged at 16,000 g for 30 min at 3 C, and then the supernatant was collected. Protein content of each sample was determined using the Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, Massachusetts, America), according to the instructions of the manufacturer. The collected supernatant samples were used for determination of the relative calpain activity using casein zymography. Casein Zymography The method for casein zymography described by Raser et al. (Raser et al., 1995) was used with slight modification and each sample was measured in triplicate to quantify the activities of the calpains. Samples from the sarcoplasmic protein extraction were thawed and immediately loaded onto non-denaturing 12.5% polyacrylamide gel electrophoresis (PAGE) casein gels (separating gel = acrylamide:n, N -bis-methylene acrylamide = 70:1 [w/w], 0.05% [v/v] N N N N - tetramethylethylenediamine [TEMED], 0.05% [w/v] ammonium persulfate [APS], casein [2.1 mg/ml], and 375 mm Tris-HCl, ph 8.8; stacking gel = acrylamide: N, N -bis-methylene acrylamide = 37.5:1 [w/w], 0.125% [v/v] TEMED, 0.075% [w/v] APS, and 125 mm Tris- HCl, ph 6.8). Each well of the gels was loaded with 35 μg protein. Gels (8 cm tall and 9 cm wide) were prerun at 100 V for 15 min in running buffer (1 mm EDTA, 192 mm glycine, 0.05% [v/v] MCE, and 25 mm Tris, ph 8.3) on mini slab electrophoresis units (Bio-Rad Laboratories, Hercules, CA) and then run for approximately 4 h at a constant voltage of 100 V. After electrophoresis the gels were incubated in three changes of 20 min each using incubating buffer (5 mm CaCl 2, 0.1% [v/v] MCE, and 50 mm Tris-HCl, ph 7.5). The gels were incubated for 12 h in incubating buffer. Then, the gels were stained with staining solution (7% [v/v] glacial acetic acid, 0.1% [w/v] Coomassie brilliant blue R-250, and 40% [v/v] methanol) for 1 h, and subsequently destained using an excess of destain solution containing 7% [v/v] glacial acetic acid and 40% [v/v] methanol. Calpain activity was indicated by the visual density of the clear zones in the stained gels which scanned with Imagescaner III (GE) and analyzed with Quantity One software (Bio-Rad, Philadelphia, America). Quantification of calpain activity was done by expressing the density of the bands from the samples relative to the mean density of standards within each gel. The standard on each gel comprised equivalent volumes of all samples.

3 FAST ph DECLINE AND PROTEIN DEGRADATION 2457 Myofibril Preparation Myofibrils from 0.3, 3, 6 and 24 h postmortem broiler PM were purified at 4 C according to the method of Huang et al. (2009). Three minced broiler samples (0.5 g) of each PM were homogenized using a blade type homogenizer (Vir-Tis) at a speed of 15,000 rpm for 30 s with 4 mlof pyrophosphate relaxing butter containing 2 mm MgCl 2, 1 mm DTT, 100 mm KCl, 1mMNaN 3, 2 mm EDTA, 10 mm Tris-maleate, ph 6.8, 2 mm Na 4 P 2 O 7 and then the homogenate was centrifuged at 1000 g for 15 min. The sediment was washed with 5 ml low salt buffer (2 mm MgCl 2, 1 mm DTT, 100 mm KCl, 1 mm NaN 3, 2 mm EDTA, 10 mm Trismaleate, ph 6.8) and the process was repeated eight times. In the final step, the myofibrils were homogenized in Tris EDTA buffer including 5 mm EDTA, 10 mm Tris, ph 8.0, and then immediately removed and mixed with treatment buffer (20% glycerol, 125 mm Tris, 4% SDS); the samples were boiled in a 100 C water bath for 10 min, and centrifuged at a speed of 16,000 g (30 min). Protein content of each supernatant was determined using the Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, America), according to the manufacturer instructions, and diluted to 3.5 μg/μl using treatment buffer (0.001% bromophenol blue, 10% MCE). Samples were mixed and heated at 50 C for 20 min, and then stored at 80 C for subsequent SDS PAGE and western blotting. SDS PAGE Gels (9 cm wide and 8 cm tall) for analysis of desmin and troponin-t degradation were run on AE-6530 M mini slab electrophoresis units (Bio-Rad Laboratories, Hercules, CA). The separating gels of 10% and 12.5% polyacrylamide (acrylamide:n, N -bis-methylene acrylamide = 37.5:1 [w/w], 0.1% [w/v] SDS, 0.05% [v/v] TEMED, 0.05% [w/v] APS, and 0.5 M Tris-HCl, ph 8.8), and stacking gel (acrylamide: N, N -bis-methylene acrylamide = 37.5:1 [w/w], 0.125% [v/v] TEMED, 0.075% [w/v] APS, and 125 mm Tris-HCl, ph 6.8) were used to detect the degradation of myofibrillar proteins desmin and troponin-t. The running buffer contained 192 mm glycine, 25 mm Tris, and 0.1% [w/v] SDS. Gels were loaded with 35 μg per well of protein for desmin and troponin-t, and run at a constant voltage of 120 V for approximately 2 h. A 5% polyacrylamide continuous gel (acrylamide: N, N -bis-methylene acrylamide = 100:1 [w/w], 0.1% [w/v] SDS, 0.067% TEMED, 0.1% [w/v] APS, 2 mm EDTA, and 200 mm Tris HCl, ph 8.0) was used for determination of titin and nebulin. Each well was loaded with samples containing 70 μg myofibrillar protein. Gels (8 cm wide, 8 cm tall and 1.5 mm thick) were run on the Bio-Rad Mini-Protean II system (Bio-Rad Laboratories, Hercules, CA). The gels were run at a constant current of 4 ma per gel for 20 h. For examination of all protein bands, gels were stained for 6 h in an excess of staining solution (0.1%, [w/v] Coomassie brilliant blue R-250, 40% [v/v] ethanol, and 7% [v/v] glacial acetic acid), and then the gels were destained in an excess of the same solution without the Coomassie brilliant blue R-250. The gels were scanned with Imagescaner III (GE) and analyzed with Quantity One software (Bio-Rad Laboratories). Quantification of titin and nebulin protein was done by expressing the density of the bands from the samples relative to the mean density of standards within each gel. The standard on each gel comprised equivalent volumes of all samples. Western Blotting Western blotting and chemiluminescent detection were performed according to the methods of Huff- Lonergan et al. (1996). Gels for desmin and troponin- T were immediately transferred to nitrocellulose membranes (Bio-Rad Laboratories) in transfer buffer (192 mm glycine, 25 mm Tris, and 10% [v/v] methanol) using a Mini-Protean II system (Bio-Rad Laboratories), and run at a constant voltage of 90 V (90 min) and 60 V (60 min), respectively. After transfer, the membrane was blocked using blocking buffer which consisted of 5% skimmed milk powder and Tween tris buffer solution (TTBS) (137 mm NaCl, 20 mm Tris, 0.05% Tween 20, 5 mm KCl) at room temperature for 2 h. Primary antibodies included mouse anti-desmin (RD301; abcam; diluted: 1:500 Massachusetts, America) and mouse anti-troponin-t (T6277; sigma; diluted: 1:500 Missouri, America) were used to incubate membrane for 12 h at 2 C. After washing five times with TTBS for 6 min each, the membrane was incubated with a secondary antibody, goat anti-mouse IgG HRP (BS12478; Bioworld technology CO; diluted: 1:5,000) for 1.5 h at room temperature. After five further washes, the immunoreactive protein bands were detected by enhanced chemiluminescence (Thermo). Quantification of desmin and troponin-t protein was done by expressing the density of the bands from the samples relative to the mean density of standards within each nitrocellulose membrane. The standard on each nitrocellulose membranes comprised equivalent volumes of all samples. Transmission Electron Microscope Transmission electron microscopy of muscle samples at 24 h postmortem was determined as described by Huang et al. (2011). Warner-Bratzler Shear Force Shear value was measured as described by Huang et al. (2014b). The right PM site of each carcass individually placed inside polyethylene bags, and cooked in a water bath at 85 C until an internal temperature of 70 C had been reached. During cooking, the internal temperature was tracked using a portable needletipped thermometer. The cooked samples were cooled in

4 2458 HUANG ET AL. Figure 1. Effects of physical restraint and free struggle on ph and lactate contents of broiler M. pectoralis major. PR: physical restraint, FS: free struggle. All measurements were expressed as the mean ± SEM (n = 40). Means with different letters were significantly different (P < 0.05). running water to ambient temperature and cooled at 4 C for 24 h, then removed from the bags and dried with filter paper. The dried samples were trimmed into strips (1 1 cm) along the direction of the myofibrils. Each strip was subjected to 6 shear measurements across the long axis of the muscle fibers using a texture analysis machine with a Warner-Bratzler single-blade type (model TA-XT2i; Stable Micro Systems, England). Test speed was 3 mm/s and the results were expressed in newtons. Statistical Analyses The effect of treatments on shear value data were analyzed using a 1-way ANOVA. The main and interactive effects of treatments and postmortem time on other variables were evaluated using a mixed-model ANOVA, where the measured variables were set as dependent variables, time, treatment, and time treatment as fixed effects, and animal as random effect. The number of replications for shear value, ph, and lactate contents was 40 and for others was 10. The pairwise differences between means were evaluated by Bonferroni s method. Effects and differences were considered significant at P < Analyses of variance were conducted using the SAS program (version 8; SAS Inst. Inc., Cary, NC). RESULTS Muscle ph and Lactate Concentrations The ph decline and lactate accumulation of broiler PM during postmortem aging is shown in Figure 1. The ph declined with time postmortem and the ph of FS group was significantly lower (P < 0.01) than the PR group at 0.3 and 3 h postmortem (Figure 1a). In the FS group, the muscle had declined to the ultimate ph at 3 h postmortem, but the PR group did not reach its final ph until 6 h postmortem. There were no significant differences (P > 0.05) in ph between the FS and PR groups at 6 and 24 h postmortem. The lactate concentration of the FS group was significantly higher (P < 0.01) than PR group at 0.3 and 3 h postmortem and it was not significantly different (P > 0.05) between treatments after 6 h postmortem (Figure 1b). Calpain Activity Fast ph decline during the early postmortem period and the time postmortem both had an influence on the densities of μ/m-calpain bands (Figure 2). The densities of μ/m-calpain in the FS group were significantly lower (P < 0.05) than in the PR group at 0.3 and 3 h postmortem. In the PR group, the band density of μ/mcalpain was not significantly different (P > 0.05) between 0.3 and 3 h postmortem, but the density of μ/mcalpain at 0.3 and 3 h postmortem was significantly higher (P < 0.05) than those at 6 and 24 h postmortem. In the FS group, there were no significant differences (P > 0.05) between postmortem times for μ/m-calpain density, except for 0.3 h postmortem which was significantly higher than at other times. The intensities were not different (P > 0.05) between the PR and FS groups for μ/m-calpain at 6 and 24 h postmortem. Protein Degradation The effects of the early postmortem fast ph decline on the degradation of desmin, titin, nebulin, and troponin-t during postmortem storage of broiler PM is shown in Figures 3, 4, and5. Desmin Figure 3 shows the effect of the fast ph decline during the early postmortem period on the relative contents of desmin. The desmin band continued to deminish in all samples and was storage-time dependent (Figure 3a). The relative density of the desmin band in the FS group was significantly lower (P < 0.05) than in the PR group at 0.3, 3, 6, and 24 h postmortem. Titin and Nebulin Figure 4 shows the effects of the fast ph decline during the early postmortem period on the relative

5 FAST ph DECLINE AND PROTEIN DEGRADATION 2459 Figure 2. Representative casein zymography gel and changes in relative densities of μ /m-calpain at 0.3, 3, 6, and 24 h postmortem in M. pectoralis major from physically restrained and free struggle broilers. PR: physical restraint, FS: free struggle. Physical restrained (A, C, E, G), Free struggle (B, D, F, H), 0.3 h (A, B), 3 h (C, D), 6 h (E, F), 24 h (G, H), Standard (Std). 35 μ g of proteins were loaded per lane. All measurements were expressed as the mean ± SD (n = 10). Means with different letters were significantly different (P < 0.05). Figure 3. Representative Western blot patterns (a) and changes in relative densities of desmin at 0.3, 3, 6, and 24 h postmortem in M. pectoralis major from physically restrained and free struggle broilers. PR: physical restraint, FS: free struggle. Physical restrained (A, C, E, G), Free struggle (B, D, F, H), 0.3 h (A, B), 3 h (C, D), 6 h (E, F), 24 h (G, H), Standard (Std). 35 μ g of proteins were loaded per lane. All measurements were expressed as the mean ± SD (n = 10). Means with different letters were significantly different (P < 0.05). contents of titin and nebulin. Titin is a mega myofibrillar protein which has a molecular weight of approximately 3,000 kda. It is the largest and third most abundant protein found in mammalian muscle tissues (Clark et al., 2002). Titin migrated as a doublet at early times postmortem; the upper band has been identified as in tact T1 and the lower band as T2 (migrates only slightly faster) as a degradation product of T1 (Wang et al., 1979). In the current experiment, results showed that the degradation of titin increased progressively with time in all lanes. The relative density of T1 bands in the FS group was significantly lower (P < 0.05) than for the PR group at each measured time (Figure 4b). In the PR group, the relative density of T1 was not significantly different (P > 0.05) between 0.3, 3 and 6 h postmortem, but it was significantly lower (P < 0.01) at 24 h postmortem than at other times. In the FS group, the relative density of T1 was significantly higher at 0.3 h postmortem than at other times. The T1 band at 24 h postmortem was rapidly degraded and had almost vanished, and the relative density was significantly lower (P < 0.01) than at other times. There was no significant difference (P > 0.05) between 3 and 6 h postmortem. The relative density of T2 increased with postmortem time and the relative density of T2 bands in FS group was significantly (P < 0.05) higher than in the PR group at each time measured (Figure 4c). The degradation of the nebulin bands were detected by continuous SDS PAGE gels (Figure 4d). Nebulin is also a mega myofibrillar protein which has a molecular weight of approximately 800 kda. As found for titin, there were large differences in the rates of degradation observed between the PR and FS groups (P < 0.05). The density of nebulin in the FS group was significantly lower than in the PR group and the degradation of nebulin in the FS group was fast and had almost disappeared at 24 h postmortem. Troponin-T The appearance of bands having molecular weights ranging from 28 to 32 kda from the postmortem

6 2460 HUANG ET AL. Figure 4. Representative electrophoresis patterns of polyacrylamide gel (a) showing the degradation of titin and nebulin at 0.3, 3, 6 and 24 h postmortem in M. pectoralis major from physical restrained (PR) and free struggle (FS) broilers. 70 μ g of proteins were loaded per lane. Physical restrained (A, C, E, G), Free struggle (B, D, F, H), 0.3 h (A, B), 3 h (C, D), 6 h (E, F), 24 h (G, H), Standard (Std). Figure 4b to d show the relative densities of T1 (intact titan), T2 (large degradation product) and nebulin at 0.3, 3, 6 and 24 h postmortem, respectively. All measurements were expressed as the mean ± SD (n = 10). Means with different letters were significantly different (P < 0.05). degradation of troponin-t has been frequently reported (Ho et al., 1994; Taylor et al., 1995; Huff-Lonergan et al., 1996). Even though troponin-t is not considered as an important structural element in maintaining protein organization, the appearance of the 30 kda degradation fragment of the intact troponin-t has been demonstrated to be an indicator of muscle tenderization and aging (Harris et al., 2001; Prates et al., 2001). In the present research, the Western blot membrane showed the presence of 30 and 32 kda degradation fragments of troponin-t (Figure 5a). The current result is con sistent with previous research of Huang et al. (2009) who also found the 30 and 32 kda degradation fragments of troponin-t. The 30 and 32 kda degradation bands accumulated with postmortem time and there were differences between the PR and FS group samples with regards to their rates of appearance (Figure 5a). The 32 kda degradation bands appeared until 3 h postmortem in the FS group. The 30 kda degradation fragments appeared after 6 h postmortem, but the relative density was not significantly different (P > 0.05) between the PR and FS groups at 6 h postmortem

7 FAST ph DECLINE AND PROTEIN DEGRADATION Figure 5. Representative Western blot patterns (a) and relative densities (b) showing changes in troponin-t at 0.3, 3, 6, and 24 h postmortem in M pectoralis major from physically restrained and free struggle broilers. PR: physical restraint, FS: free struggle. Physical restrained (A, C, E, G), Free struggle (B, D, F, H), 0.3 h (A, B), 3 h (C, D), 6 h (E, F), 24 h (G, H), Standard (Std). 35 μ g of proteins were loaded per lane. All measurements were expressed as the mean ± SD (n = 10). Means with different letters were significantly different (P < 0.05). (Figure 5b). At 24 h postmortem, the relative density of the 30 kda degradation fragments of FS group was significantly higher (P < 0.05) than the PR group. Myofibrillar Ultrastructure and Shear Force Figure 6 shows transmission electron micrographs and shear value of broiler PM muscle at 24 h postmortem. Treatments affected the major ultrastructural components of myofibrils. Electron microscopy of PR samples revealed good preservation of normal structures, having distinct I, A bands and Z, M lines. Compared with PR, samples from FS group showed serious disruption of meat structures. The thick and thin filaments were markedly degraded, and certain areas of the Z line and A bands had become pierced. The shear value of FS group was significantly lower than PR group (P < 0.01). DISCUSSION Anaerobic glycolysis is a very important energy production pathway after slaughter of broilers. This leads to a ph decline in postmortem muscle due to the accumulation of lactate. The ph in the FS group was lower than in the PR group during the early postmortem pe 2461 riod, which is consistent with previous studies that preslaughter and during bleeding struggle leads to a fast ph decline during the postmortem period (Berri et al., 2005; Huang et al., 2014a). Many researchers have observed that a fast ph decline during the early postmortem period can reduce the activity of μ-calpain in livestock muscles (Claeys et al., 2001; Hwang and Thompson, 2001). This possibly results from the earlier activation of calpain in muscles and increased autolysis of calpain as a result of fast ph decline (Pomponio et al., 2010). Our results are consistent with previous studies that the μ/m-calpain activity in PM of the FS group was lower (P < 0.05) than in the PR group during the early postmortem period. Previous research with pigs has shown that the sarcoplasmic calcium concentration was higher in the muscle which had a fast ph decline during the early postmortem period (Cheah et al., 1984). This was likely due to the early release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells in those muscles having a fast ph decline, resulting in earlier activation of μcalpain and enhanced autolysis (Wilhelm et al., 2010). In our work, the μ/m-calpain activity at early postmortem was lower in the fast ph decline group, thus supporting the notion that a fast ph decline induces early release of Ca2+ ions, leading to early activation of μ/m-calpain and thus enhanced autolysis. During the initial post mortem period, the muscle of an animal carcass is converted to meat and this process is mediated by complex biochemical interactions (Herrera-Mendez et al., 2006). Although these reactions influence the ultimate texture and tenderness of the meat, they are not completely understood. However, fragmentation by proteolysis of myofibrils and some of key skeletal cytoskeletal proteins, including desmin, titin, nebulin, and troponin-t, is generally believed to be responsible for meat tenderization (Koohmaraie, 1996). The molecular weight of desmin is approximately 53 kda and it is essential for maintaining the integrity of muscle cyto-architecture through forming a threedimensional scaffold around the myofibrillar Z-disk. Many researchers believe that the tenderization of postmortem meat is significantly impacted by desmin degradation (Chang and Chou, 2010; Huff Lonergan et al., 2010). Nebulin could be the link between actin and myosin where it may have a regulatory function in skeletal muscle contraction (Bang et al., 2006; Bang et al., 2009). Postmortem degradation of nebulin may have a function in weakening the thin filament which connects with the Z-line (Taylor et al., 1995). It has been considered that the tenderness of postmortem meat can be influenced by the degradation of titin and nebulin, which are likely to affect the integrity of the myofibrillar structures (Huff-Lonergan et al., 1995). The research of Wu et al. (2014) found that the degradation of titin and nebulin in high ultimate ph meat was more serious than in low ultimate ph meat and the rapid degradation of intact titin and nebulin resulted in more tender meat.

8 2462 HUANG ET AL. Figure 6. Effects of physical restraint and free struggle on ultrastructure of myofibrils (n = 10 each treatment) ane shear value (n = 40 each treatment) of broiler M. pectoralis major at 24 h postmortem. PR: physical restraint, FS: free struggle. All measurements were expressed as the mean ± SD. Means with different letters differ (P < 0.05). Troponin-T is a tropomyosin-binding component of the troponin complex. Even though it is not considered to significantly contribute to the maintenance of myofibrillar structures, it is believed that troponin-t has an effect on regulation of the actin myosin interaction and it is possible that the degradation of troponin-t causes weakening of the strong acitin myosin crossbridge bonds (Herrera-Mendez et al., 2006; Huff Lonergan et al., 2010). Previously it has been found that fast ph decline during early postmortem can boost desmin and titin degradation in porcine muscles, and earlier appearances of the 30 kda fragment have been shown in beef muscle (O Halloran et al., 1997; Melody et al., 2004). Our findings are in agreement with previous results in that fast ph decline during early postmortem increased desmin, titin, nebulin, and troponin-t degradation in broiler muscle. The present study shows that fast ph decline during the early postmortem period can influence the rate of protein degradation in broiler breast muscle. Fast ph decline during the early postmortem period can reduce the activity of μ/m-calpain during the early postmortem period possibly by earlier activation, but it did not influence the ultimate μ/m-calpain activity. Fast ph decline during the early postmortem period increased the degradation rate of desmin, titin, nebulin, and troponin-t. This may be due to the earlier postmortem activation of μ/m-calpain leading to an earlier degradation of cytoskeletal proteins. ACKNOWLEDGMENTS This research was funded by National Natural Science Foundation of China (Grant No.: ), China Agricultural Research System (CARS-42), Special Fund for Agro-scientific Research in the Public Interest ( ). The authors thank Dr. Ron Tume (CSIRO Food and Nutrition Flagship, QLD, Brisbane, Australia) for his careful revision of this paper. REFERENCES Alvarado, C. Z., and A. R. Sams The influence of postmortem electrical stimulation on rigor mortis development, calpastatin activity, and tenderness in broiler and duck pectoralis. Poult. Sci. 79: An, J. Y., J. X. Zheng, J. Y. Li, D. Zeng, L. J. Qu, G. Y. Xu, and N. Yang Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens. Poult. Sci. 89:

9 FAST ph DECLINE AND PROTEIN DEGRADATION 2463 Bang, M. L., M. Caremani, E. Brunello, R. Littlefield, R. L. Lieber, J. Chen, V. Lombardi, and M. Linari Nebulin plays a direct role in promoting strong actin-myosin interactions. Faseb J. 23: Bang, M. L., X. D. Li, R. Littlefield, S. Bremner, A. Thor, K. U. Knowlton, R. L. Lieber, and J. Chen Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle. J. Cell Biol. 173: Berri, C., M. Debut, V. Santé-Lhoutellier, C. Arnould, B. Boutten, N. Sellier, E. Baéza, N. Jehl, Y. Jégo, and M. J. Duclos Variations in chicken breast meat quality: implications of struggle and muscle glycogen content at death. British Poult. Sci. 46: Chang, Y. S., and R. G. Chou Postmortem degradation of desmin and calpain in breast and leg and thigh muscles from Taiwan black-feathered country chickens. Journal of the science of food and agriculture. 90: Cheah, K. S., A. M. Cheah, A. R. Crosland, J. C. Casey, and A. J. Webb Relationship between Ca(2+) release, sarcoplasmic Ca(2+), glycolysis and meat quality in halothane-sensitive and halothane-insensitive pigs. Meat Sci. 10: Claeys,E.,S.DeSmet,D.Demeyer,R.Geers,andN.Buys Effect of rate of ph decline on muscle enzyme activities in two pig lines. Meat Sci. 57: Clark, K. A., A. S. Mcelhinny, M. C. Beckerle, and C. C. Gregorio Striated muscle cytoarchitecture: An intricate web of form and function. In: R. Schekman (ed.) Annual Review of Cell and Developmental Biology. Volume 18. USA. Harris, S. E., E. Huff-Lonergan, S. M. Lonergan, W. R. Jones, and D. Rankins Antioxidant status affects color stability and tenderness of calcium chloride-injected beef. J. Anim. Sci. 79: Herrera-Mendez, C. H., S. Becila, A. Boudjellal, and A. Ouali Meat ageing: Reconsideration of the current concept. Trends in Food Science and Technology. 17: Ho, C., M. Stromer, and R. Robson Identification of the 30 kda polypeptide in post mortem skeletal muscle as a degradation product of troponin-t. Biochimie. 76: Huang, J. C., M. Huang, P. Wang, L. Zhao, X. L Xu, G. H. Zhou, and J. X. Sun. 2014b. Effects of physical restraint and electrical stunning on plasma corticosterone, postmortem metabolism, and quality of broiler breast muscle. J. Anim. Sci. 92: Huang, J. C., M. Huang, J. Yang, P. Wang, L. Zhao, X. L Xu, and G. H. Zhou. 2014a. The effects of electrical stunning methods on broiler meat quality: Effect on stress, glycolysis, water distribution, and myofibrillar ultrastructures. Poult. Sci. 93: Huang, M., F. Huang, M. Xue, X. Xu, and G. Zhou The effect of active caspase-3 on degradation of chicken myofibrillar proteins and structure of myofibrils. Food Chem. 128: Huang, M., F. Huang, H. Ma, X. Xu, and G. Zhou Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle. Meat Sci. 90: Huang, M., F. Huang, X. L. Xu, and G. Zhou Influence of caspase3 selective inhibitor on proteolysis of chicken skeletal muscle proteins during post mortem aging. Food Chem. 115: Huff-Lonergan, E., T. Mitsuhashi, D. D. Beekman, F. Parrish, D. G. Olson, and R. M. Robson Proteolysis of specific muscle structural proteins by mu-calpain at low ph and temperature is similar to degradation in postmortem bovine muscle. J. Anim. Sci. 74: Huff-Lonergan, E., F. Parrish, and R. M. Robson Effects of postmortem aging time, animal age, and sex on degradation of titin and nebulin in bovine longissimus muscle. J. Anim. Sci. 73: Huff Lonergan, E., W. Zhang, and S. M. Lonergan Biochemistry of postmortem muscle Lessons on mechanisms of meat tenderization. Meat Sci. 86: Hwang, I., and J. Thompson The interaction between ph and temperature decline early postmortem on the calpain system and objective tenderness in electrically stimulated beef longissimus dorsi muscle. Meat Sci. 58: Kahraman, T., A. G. Bayraktaroglu, A. Vural, G. Issa, and E. Ergun Electron microscopy of contractile bands and quality characteristics in high-voltage electrical stimulation broiler breast meat. Poult. Sci. 90: Koohmaraie, M Biochemical factors regulating the toughening and tenderization processes of meat. Meat Sci. 43: Koohmaraie, M., and G. H. Geesink Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system. Meat Sci. 74: Lee, H. L., V. Sante-Lhoutellier, S. Vigouroux, Y. Briand, and M. Briand Role of calpains in postmortem proteolysis in chicken muscle. Poult. Sci. 87: Melody, J., S. Lonergan, L. Rowe, T. Huiatt, M. Mayes, and E. Huff- Lonergan Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles. J. Anim. Sci. 82: Molette, C., H. Remignon, and R. Babile Modification of glycolyzing enzymes lowers meat quality of turkey. Poult. Sci. 84: Northcutt, J., R. Buhr, and L. Young Influence of preslaughter stunning on turkey breast muscle quality. Poult. Sci. 77: O Halloran, G., D. Troy, D. Buckley, and W. Reville The role of endogenous proteases in the tenderisation of fast glycolysing muscle. Meat Sci. 47: Papinaho, P., D. Fletcher, and R. Buhr Effect of electrical stunning amperage and peri-mortem struggle on broiler breast rigor development and meat quality. Poult. Sci. 74: Pomponio, L., P. Ertbjerg, A. H. Karlsson, L. N. Costa, and R. Lametsch Influence of early ph decline on calpain activity in porcine muscle. Meat Sci. 85: Prates, J. A. M., A. M. R. Ribeiro, and A. A. D. Correia Role of cysteine endopeptidases (EC ) in rabbit meat tenderisation and some related changes. Meat Sci. 57: Raser, K. J., A. Posner, and K. K. Wang Casein zymography: a method to study μ-calpain, m-calpain, and their inhibitory agents. Archives of Biochemistry and Biophysics. 319: Sorimachi, H., T. Tsukahara, M. Okada-Ban, H. Sugita, S. Ishiura, and K. Suzuki Identification of a third ubiquitous calpain species chicken muscle expresses four distinct calpains. Biochimicaet Biophysica Acta (BBA)-Gene Structure and Expression. 1261: Taylor, R. G., G. Geesink, V. Thompson, M. Koohmaraie, and D. Goll Is Z-disk degradation responsible for postmortem tenderization? J. Animal sci. 73: Wang, K., J. McClure, and A. Tu Titin: major myofibrillar components of striated muscle. Proceedings of the National Academy of Sciences. 76: Wang, S., C. Li, X. L Xu, and G. Zhou Effect of fasting on energy metabolism and tenderizing enzymes in chicken breast muscle early postmortem. Meat Sci. 93: Wilhelm, A. E., M. B. Maganhini, F. J. Hernández-Blazquez, E. I. Ida, and M. Shimokomaki Protease activity and the ultrastructure of broiler chicken PSE (pale, soft, exudative) meat. Food Chem. 119: Wu, G., M. M. Farouk, S. Clerens, and K. Rosenvold Effect of beef ultimate ph and large structural protein changes with aging on meat tenderness. 98: Yu,L.P.,andY.B.Lee.1986.EffectsofpostmortempHandtemperature on bovine muscle structure and meat tenderness. J. food Sci. 51: