Effects of preslaughter shackling on postmortem glycolysis, meat quality, changes of water distribution, and protein structures of broiler breast meat

Effects of preslaughter shackling on postmortem glycolysis, meat quality, changes of water distribution, and protein structures of broiler breast meat X. B. Sun,∗ J. C. Huang,†,1 T. T. Li,∗ Y. Ang,∗ X. L. Xu,∗ and M. Huang∗,2 Nanjing Innovation Center of Meat Products Processing, Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, and College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China; and † College of Engineering, Nanjing Agricultural University, Nanjing 210095, China ABSTRACT The objective of this study was to investigate the effects of preslaughter shackling on stress, postmortem glycolysis, meat quality, water distribution, and protein structures of pectoralis majors. Before slaughter, Arbor Acres broilers (n = 105, 42 days old, 2.0 to 2.5 kg) were randomly categorized into 3 treatment groups: (I) control group without shackling (NS); (II) 2.5 min shackling (SS); (III) 4.5 min shackling (LS). Each treatment group consisted of 5 replicates with 7 broilers each. Results indicated that preslaughter shackling increased (P < 0.05) plasma corticosterone and adrenocorticotropic hormone concentrations in comparison with the control group. Antemortem shackling increased (P < 0.05) activity of glycogen phos-

phorylase and phosphofructokinase-1 (PFK-1) accompanying with rapid glycolysis and pH decline at early postmortem. LS treatment led to myosin denaturation, decreased (P < 0.05) α-helix content, and increased (P < 0.05) β -sheet structures proportion in the myofibrillar proteins. Furthermore, meat from LS treatment had higher (P < 0.05) lightness, redness, and poorer water-holding capacity. These results indicated that the longer shackling duration (4.5 min) increased stress and the rate of glycolysis, causing myosin denaturation and changes of the secondary structure in the myofibrillar proteins, which aggravated the deterioration of meat quality.

Key words: broiler, shackling, stress, glycolysis, protein structure 2019 Poultry Science 0:1–9 http://dx.doi.org/10.3382/ps/pez175

INTRODUCTION

serves at the time of slaughter and accelerate the initial pH decline rate (Fidan et al., 2015). The early postmortem pH decline rate is a key factor in influencing the ability of meat to retain water (Huff-Lonergan and Lonergan, 2005). Huang et al. (2018) observed that preslaughter shackling accelerated glycolysis and led to a low water-holding capacity of breast meat which is not favored by the modern broiler industry owing to loss of profit. Researchers have also found poor water-holding capacity is associated with protein denaturation, mainly myofibrillar and sarcoplasmic proteins (Joo et al., 1999; Xing et al., 2016). Meanwhile, protein structures play a crucial role in water distribution of meat through affecting the interaction between protein-network and water (Wu et al., 2006). We hypothesized that these factors together were conducive to poor water-holding capacity in breast meat. Though previous studies indicated antemortem shackling had a negative influence on meat quality of broilers (Kannan et al., 1997; Fidan et al., 2015), the mechanism lacked adequate explanation. Therefore, further research is still required to explore how preslaughter shackling influences meat quality of broilers. The objective of this study was to further explore how

Unlike other meat-producing animals, the birds are hung upside down in shackles preslaughter. Shackling before slaughter is unavoidable in poultry commercial slaughter plants (Joseph et al., 2013). Previous studies have indicated that broilers display struggling behaviors such as vocalization, wing flapping, and the straightening up of the body after they were suspended by shackles, suggesting that antemortem shackling caused stress to broilers (Lines et al., 2011; Fidan et al., 2014). Some researchers have found that plasma corticosterone (CORT), which is widely considered as an indicator of preslaughter stress, increased in shackled broilers (McFarlane and Curtis, 1989; Nijdam et al., 2005; Bedanova et al., 2007a). In addition, Ngoka et al. (1982) observed shackling before and during bleeding could affect glycolysis in turkey breast muscle. Therefore, shackling stress can decrease muscle glycogen re-

 C 2019 Poultry Science Association Inc. Received October 30, 2018. Accepted March 15, 2019. 1 Co-first author. 2 Corresponding author: [email protected]

1

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

∗

2

SUN ET AL.

shackling stress affects water-holding capacity of breast meat in broilers by investigating the effects of preslaughter shackling on stress level, meat quality, postmortem metabolism, changes of water distribution, and protein structures in pectoralis majors (PM).

Experimental Design All procedures were approved by the Animal Care and Use Committee of the College of Food Science and Technology of Nanjing Agricultural University. For the trial, a total of 105 live Arbor Acres broilers (42 days old, mixed sex, 2.0 to 2.5 kg) were obtained from a commercial farm located in Jiangsu, China in summer. They were randomly divided into 3 treatment groups: (I) control group without shackling (NS); (II) 2.5 min shackling (SS); (III) 4.5 min shackling (LS). Each treatment group consisted of 5 replicates with 7 birds each. Feed was removed from the broilers at about 12 h prior to slaughter. All broilers were transported in a truck for 30 min from the holding area to the abattoir. After resting for an hour, the broilers were slaughtered according to the commercial slaughter process. SS and LS, 2 shackling treatments were hung upside down for 2.5 and 4.5 min, respectively. The broilers following NS treatment were held by hand to avoid struggling behaviors and then slaughtered directly. All the birds were bled by severing the jugular vein and carotid artery using a hand-held knife on one side of the neck to allow bleeding for 150 s.

Blood Parameter Measurements CORT and ACTH concentrations were determined to analyze the stress condition using a chicken CORT ELISA kit and a chicken ACTH ELISA kit (R&D Systems, Minneapolis, MN, USA), respectively. Blood samples were measured in triplicate in accordance with the manufacturers’ instructions.

Meat Color Measurements At 24 h postmortem, the color of meat was measured on the bone side, medial surface in triplicate by a chromameter (Minolta CR400; Konica Minola Company, Tokyo, Japan). Color was recorded as lightness (L∗ ), redness (a∗ ), and yellowness (b∗ ) values in the CIE Lab trichromatic system.

Muscle pH Value Sampling Procedure Blood Samples Blood samples (5 mL) were collected in tubes containing heparinized anticoagulant (Nanjing Dingguo Bioengineering Institute, Nanjing, China) from 15 broilers selected from each replicate in each treatment during bleeding. The samples were centrifuged for 10 min (2,000× g) at 4◦ C, and then the plasma was removed and stored at –80◦ C until required for CORT and adrenocorticotropic hormone (ACTH) assessments. Muscle Samples After bleeding for 150 s, the birds were inserted by an insertion thermometer under the arm to measure the temperature of breast muscle. Then the broilers experienced scalding at 54◦ C for 120 s, plucking for 30 s and evisceration. Both PM muscles were immediately removed manually. The left breast meat from each of 45 broilers whose blood had been collected was stored at 4◦ C. Muscle pieces were obtained at 0.25, 1, 3, and 24 h, and they were stored in antifreeze tubes to be quickly frozen in liquid nitrogen and then kept at –80◦ C for the measurements of pH, lactate, glycogen, glycogen phosphorylase, and phosphofructokinase-1 activity. The right breast meat from all broilers was stored at 4◦ C until 24 h for meat color, drip loss, and cooking loss measurements and 45 birds whose blood had been collected were selected for

The pH of postmortem muscle was determined according to McGeehin et al. (2001) with a slight modification. A frozen sample (1 g) was homogenized in a tube containing 10 mL of ice cold buffer of sodium iodoacetate (5 mmol/L) and potassium chloride (150 mmol/L) adjusted to pH 7.0 by an Ultra Turrax T25 homogenizer (IKA, Staufen, Germany) for 30 s. The pH of each sample was recorded using a FE-20 Mettler pH meter (Mettler-Toledo Instruments, Zurich, Switzerland).

Drip Loss Duplicate samples (about 10 g) removed from the center of each right PM were trimmed into a specific shape (1 × 1 cm). Each strip was weighed (M1 ) and individually hung with a hook in the air-tight container that was stored at 4◦ C. After 24 h, any moisture on the surface of the sample was dried with filter paper. Subsequently, all samples were weighed (M2 ). Drip loss (%) = (M1 – M2 )/M1 × 100%.

Cooking Loss Two strips (approximately 30 g of the thickest place of the right PM, 4 × 3 × 2 cm) were selected from each broiler at 24 h postmortem. Each strip was weighed

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

MATERIALS AND METHODS

low-field nuclear magnetic resonance (LF-NMR), differential scanning calorimetry (DSC) analysis as well, but the muscle pieces obtained at 1 h postmortem were just used for the determination of LF-NMR. The samples, which had been used for NMR measurement at 24 h postmortem, were immediately frozen in liquid nitrogen and kept at –80◦ C prior to sectioning. Thawing and sectioning were carried out in a cold-chamber (4◦ C), transversely to the fiber direction and soak up any moisture. Pieces (1 × 1 × 0.5 cm) were prepared and laid on a clean glass slide one by one for Raman spectroscopy analysis.

SHACKLING AND POSTMORTEM BROILER MUSCLE

3

lines were corrected and normalized against the phenylalanine band at 1,003 cm−1 using Labspec version 5.0 (Horiba Jobin Yvon). The secondary structure of proteins was determined as percentages of α-helix, β -sheet, β -turn, and unordered conformations using the method of Susi and Byler (1988) using PeakFit 4.12 software (SeaSolve Software Inc., USA).

NMR Measurements

Glycogen Determinations

The NMR relaxation measurement (T2 ) was performed according to the method of Huang et al. (2014a) with a slight modification. At 1 and 24 h postmortem, 3 strip samples (1 × 1 × 2 cm, weighing approximately 2 g) from the PM of each broiler were cut along the direction of myofiber and individually put into cylindrical glass tubes, 10 mm in diameter and 35 mm high, which were warmed at 28◦ C for 8 min in a water bath (TW20, Julabo Co., Ltd., German). Then, the samples were measured by a Niumag Pulsed NMR analyzer (Meso MR23; Niumag Corporation, Shanghai, China) with a resonance frequency of 22 MHz at 32◦ C. T2 was analyzed using the Carr-Purcell-Meiboom-Gill sequence with a τ - value of 150 μs between the 90o pulse and 180o pulse. The scan repetitions were set at 32 with an interval 3,500 ms. A total of 3,200 echoes were acquired and fitted with the program MultiExp Inv Analysis software (Niumag Electric Corporation, Shanghai, China).

Glycogen content was determined using a commercial kit (A043, Nanjing Bioengineering Institute, Nanjing, China). Briefly, 90 mg of each sample was mixed with 270 μL concentrated alkali and heated for 20 min in a boiling water bath. After cooling, the mixture was diluted with 1.44 mL distilled water to obtain a 5% glycogen diluent. Then, 2 mL staining solution was mixed with 0.9 mL distilled water and 0.1 mL glycogen diluent and heated for 5 min in a boiling water bath and cooled with running water. The absorbance value was determined at 620 nm using a Microplate Reader (SpectraMax M2, Molecular Devices, CA, USA). Glycogen content was calculated using a glycogen standard curve.

DSC Analysis Protein denaturation was assessed using a differential scanning calorimeter (DSC8000, PerkinElmer Corporation, USA). About 15 mg sample was accurately weighed using an electronic balance with one hundred thousand precision, sealed in an aluminum pan, and then placed into the DSC together with an empty pan as the reference. The heat rate was 5◦ C/min from 25 to 90◦ C. Data recorded were analyzed by the software (Pyris Manager) attached to the machine.

Raman Spectroscopy Analysis Protein structures were measured using a Raman spectrometer with a Jobin Yvon Labram HR800 spectrometer (Horiba Jobi Yvon S.A.S., Longjumeau, France) referring to the method of Shao et al. (2011) with minor modifications. A microscope equipped with a 50× lens was used for focusing the excitation laser beam (514.5 nm exciting line of a Spectra Physics Arlaser) on the sample, and the Raman signal was collected in the backscattered direction. All spectra were recorded in the range of 400 to 2,000 cm−1 . The specific conditions were as follows: 3 scans, 30 s exposure time, 2 cm−1 resolution, and a sampling speed of 120 cm−1 /min, with data collected every 1 cm−1 . The spectra obtained were smoothed, and their base-

Lactate Determinations Lactate content was determined spectrophotometrically (530 nm) using a commercial kit (A019-2, Nanjing Bioengineering Institute, Nanjing, China). Approximately 500 mg of sample was homogenized in 4.5 mL of normal saline. After centrifugation (2,000× g at 4◦ C), 1 mL of the supernatant was diluted with 4 mL distilled water. Then, 20 μL of the diluent was mixed with 1 mL of enzyme reaction mixture and 0.2 mL of the chromogenic agent and then placed in a 37◦ C warm bath for 10 min. Then 2 mL of terminator solution was added and mixed. Lactate standard curves were developed for each set of samples. Linear regression equations were used to determine the lactate concentrations in the corresponding samples. Sample protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc, Waltham, MA, USA).

Measurements of Enzyme Activity Activity of glycogen phosphorylase was analyzed spectrophotometrically (340 nm) using a glycogen phosphorylase kit (BC3340, Beijing Solarbio Science & Technology Company, Beijing, China). Approximately 100 mg of sample was homogenized in 1 mL extract solution. After centrifugation (8,000× g at 4◦ C, 10 min), 50 μL of the supernatant was mixed with 50 μL distilled water and 900 μL reaction mixture in a 1 mL quartz cuvette. Record absorbance value A1 at 5 min and A2 at 10 min, respectively. Protein concentration (Cpr) was determined using a BCA protein assay kit

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

(M3 ) and individually put into a thinwalled plastic bag with an insertion thermometer and then cooked in a water bath at 85◦ C until the central temperature reached 70◦ C. The bags were chilled in running water for 20 min and then stored at 4◦ C. After 24 h, the samples were dried with filter paper and weighed (M4 ). Cooking loss (%) = (M3 – M4 )/M3 × 100%.

4

SUN ET AL.

Table 1. Effects of shackling on plasma parameters of broilers (mean ± SD; n = 15 each treatment); blood samples were collected during bleed out. NS ACTH, ng/L CORT, ng/mL

SS

122.67 ± 12.91 132.70 ± 14.88a a

Table 2. Effects of shackling on breast meat quality (mean ± SD; n = 35 each treatment). Meat quality parameters

LS

157.81 ± 7.07 152.04 ± 8.64b b

183.79 ± 7.72 194.45 ± 11.25c c

(Thermo Fisher Scientific Inc, Waltham, MA, USA). Activity of glycogen phosphorylase was calculated as 643 × A ÷ Cpr (A = A2 – A1 ). Activity of phosphofructokinase-1 was determined (340 nm) spectrophotometrically with a phosphofructokinase-1 kit (H244, Nanjing Bioengineering Institute, Nanjing, China). Approximately 100 mg of sample was homogenized in 1 mL extract solution. After centrifugation (8,000× g at 4◦ C, 10 min), 30 μL of the supernatant was mixed with 810 μL reaction mixture in a 1 mL quartz cuvette. Record absorbance value A1 at 20 s and A2 at 10 min 20 s, respectively. Protein concentration (Cpr) was determined using a BCA protein assay kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Activity of phosphofructokinase1 was calculated as 450 × A ÷ Cpr (A = A1 – A2 ).

Statistical Analysis All results were analyzed using one-way analysis of variance (ANOVA) with the SAS 9.12 software (SAS Institute Inc., Cary, NC, USA) and the mean value of each broiler was a replicate. Duncan’s multiple range tests were used to analyze any difference among treatments significant at P < 0.05. The differences among postmortem time (0.25, 1, 3, and 24 h) were analyzed for pH, lactate, glycogen in each treatment by Duncan’s multiple range tests and significant at P < 0.05. The results are expressed as mean ± SD.

RESULTS AND DISCUSSTION Plasma Parameters When the birds are under stressful conditions, the function of hypothalamic–pituitary–adrenal axis is enhanced. The increasing plasma ACTH acts on the adrenal cortex to release CORT, leading to the resultant accumulation of plasma CORT (Knowles et al., 1995; Bedanova et al., 2007a). It has been reported that plasma CORT is a feasible indicator to evaluate the stress level in broilers (McFarlane and Curtis, 1989; Haensch et al., 2009). In the present study, the concentrations of ACTH and CORT both presented an increase (P < 0.05) in the shackling groups compared to the control group as shown in Table 1, manifesting that preslaughter shackling increased stress to the

L a∗ b∗ Drip loss (%) Cooking loss (%) pH0.25h pH1h pH3h pH24h Breast muscle temperature (◦ C)

NS 47.44 2.78 8.33 2.40 15.60 6.63 6.40 6.16 5.85 39.65

± ± ± ± ± ± ± ± ± ±

SS

LS

1.05 49.76 ± 1.65 52.91 ± 1.05c 0.46a 3.01 ± 0.58a 3.88 ± 0.64b 1.25a 8.93 ± 1.64a 8.57 ± 0.97a 0.71a 3.28 ± 0.50b 3.76 ± 0.28b 0.75a 17.38 ± 0.80b 21.02 ± 1.27c 0.11a,A 6.11 ± 0.14b,A 5.95 ± 0.15c,A 0.14a,B 5.89 ± 0.08b,B 5.91 ± 0.12b,A 0.11a,C 5.82 ± 0.13b,B,C 5.86 ± 0.13b,A 0.15a,D 5.75 ± 0.14a,b,C 5.70 ± 0.09b,B 41.47 ± 0.39c 0.68a 40.73 ± 0.36b a

b

L∗ : lightness, a∗ : redness, b∗ : yellowness, NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group, breast muscle temperature was measured after bleeding for 150 s. a–c Means in the same row with no common superscript differ significantly (P < 0.05); A,D means about pH in the same column with no common superscript also differ significantly (P < 0.05).

broilers. As reported by Belge et al. (2003), ACTH application could provoke the rise of plasma CORT content in chickens through strengthening the function of pituitary–adrenal axis. When compared, the concentrations of CORT and ACTH between the SS and LS groups were higher (P < 0.05) due to longer shackling duration, which indicated that LS treatment resulted in stronger stress to the broilers. This result was in accordance with previous studies that antemortem shackling aroused more stress to broilers due to increased shackling periods (Bedanova et al., 2007b; Fidan et al., 2014).

Meat Quality Lightness was higher (P < 0.05) in the shackling groups than the control group, and a difference (P < 0.05) was observed between the SS and LS groups (Table 2). The breast meat from LS treatment had higher (P < 0.05) redness, as Huang et al. (2014b) observed that PM muscles were characterized in higher redness as birds experienced preslaughter struggle. However, yellowness was not affected (P > 0.05). Meat following shackling treatments had higher (P < 0.05) drip loss and cooking loss, compared to the SS group, cooking loss was higher (P < 0.05) in the LS group. Furthermore, the shackling groups had higher (P < 0.05) postmortem breast muscle temperature and compared with the SS group, an increase (P < 0.05) occurred in the LS group. Meat pH values following shackling treatments were lower (P < 0.05) than NS treatment at 0.25, 1, and 3 h postmortem; pH values in the LS group were lower (P < 0.05) than the SS group at 0.25 h postmortem. The SS group reached ultimate pH at 3 h postmortem; pH in the LS group had no remarkable change during 3 h postmortem and was lower (P < 0.05) at 24 h postmortem by comparison with the control group. Huang et al. (2018) also found that preslaughter shackling led to an increase in the decline rate of pH at early postmortem stages.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

CORT: corticosterone, ACTH: adrenocorticotropic hormone, NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group. a–c Means in the same line with no common superscript differ significantly (P < 0.05).

∗

5

SHACKLING AND POSTMORTEM BROILER MUSCLE 1800 1600

NS

SS

LS

Amplitude

1400

Table 3. Effects of shackling on water populations of corresponding transversal relaxation times of breast meat at 1 and 24 h postmortem (mean ± SD; n = 15 each treatment). T2 relaxation peak area fraction (%)

1200 1000

NS

800 400 200 0 Relaxtion time (ms)

Figure 1. Representative continuous relaxation time spectra of transverse relaxation (T2 ) data. Three components were detected: T2b , a minor component between 1 and 10 ms; T21 , a major component between 10 and 100 ms; and T22 , a component between 200 and 600 ms (n = 15 each treatment). NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group.

Preslaughter stress (transport, high ambient temperature, or shackling) could result in high lightness, poor water-holding capacity, and low pH of breast meat (Xing et al., 2015; Huang et al., 2018). In our present study, breast meat from the LS group had a trend to be PSE-like meat as described in previous studies (Oba et al., 2009; Souza Langer et al., 2010). As expected, LS treatment led to stronger stress to the broilers because of longer shackling period compared with the other 2 treatments, which led to an imbalance of muscle Ca2+ homeostasis causing rapid postmortem glycolysis (Barbut et al., 2008). A rapid accumulation of lactic acid and the consequent low pH promoted the denaturation of myofibrillar and sarcoplasmic proteins, causing PSE-like turkey breast muscle (Sosnicki, et al., 1997). This was similar with our results.

NMR Transverse Relaxation (T2 ) Measurements The distributed exponential fitting analysis of T2 measured at 24 h postmortem is showed in Figure 1. A total of 3 components were identified in every sample, and these were referred to as T2b , T21 , and T22 , respectively: T2b (1 to 10 ms) represents water tightly bound to protein macromolecules; T21 (10 to 100 ms) is considered corresponding to intra-myofibrillar water which accounts for more than 90% of the total water in muscle (Honikel et al., 1986); T22 (200 to 600 ms) reflects a minor ratio water locating in extramyofibrillar space. There is an evident relationship between water-holding capacity and T21 , T22 populations (Bertram et al., 2001). The results about T2b , T21 , and T22 populations are exhibited in Table 3. At 24 h postmortem, the water population of P21 following LS treatment in samples was the lowest (P < 0.05) among 3 groups, and P22 was the highest (P < 0.05). In addition, the population of P22 following SS treatment was higher (P < 0.05) than NS treatment and P21

1h

24 h

2.31 97.00 0.69 2.11 96.82 1.07

± ± ± ± ± ±

0.34 0.33a 0.13a 0.24 0.21a 0.10a

2.26 96.91 0.83 2.13 96.17 1.70

± ± ± ± ± ±

LS 0.28 0.31a,b 0.10a,b 0.44 0.51b 0.22b

2.36 96.60 1.04 2.21 95.60 2.19

± ± ± ± ± ±

0.39 0.33b 0.43b 0.20 0.55c 0.63c

NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group; P2b , P21 and P22 corresponding to water populations of transversal relaxation times of breast meat (T2b , T21 , and T22 , respectively). a–c Means in the same line with no common superscript differ significantly (P < 0.05).

was lower (P < 0.05). It was speculated that the reason for this phenomenon may be that intra-myofibrillar water partially flowed into extra-myofibrillar space. At 1 h postmortem, the population of P21 following LS treatment was the lowest within 3 groups (P < 0.05) and P22 was the highest (P < 0.05), which revealed poor water-holding capacity had occurred at early postmortem stages. Guignot et al. (1993) observed that myofilament spacing starts to drop dramatically when pH is near 5.9 which also appeared in the shackling treatment groups at 1 h postmortem. Maybe the lower pH contributed to shrinkage of myofibrillar space, leading to differences of water distribution among 3 treatments at early postmortem. We assumed that a combination of low pH and high breast muscle temperature resulted from preslaughter stress; this then reduced space within the myofibrils and made contributions to leakage of intra-myofibrillar water to extra-myofibrillar space. Meanwhile, protein denaturation reduced the amount of protein side chains available for water binding (McDonnell et al., 2013). The decrease of intra-myofibrillar water may be the key factor for increased drip loss in the shackling groups.

DSC Analysis DSC is a generally acknowledged and widely used method to analyze protein denaturation characteristics in muscle. Meat samples were measured at 24 h postmortem and the generated typical DSC thermograms are exhibited in Figure 2. Three typical endothermic transition peaks were identified in all samples, which are divided into Peak1 (54 to 58◦ C), Peak2 (60 to 70◦ C), and Peak3 (71 to 83◦ C). In view of previous studies (Stabursvik et al., 1984; Brunton et al., 2006), Peak1 represents the endothermic transition peak formed by denaturation of myosin in muscle. The formation of Peak2 is generally considered to be due to denaturation of sarcoplasmic proteins and collagen. Actin, the most heat-stable protein in muscle, denatured corresponding to Peak3 . The values for endothermic

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

P2b P21 P22 P2b P21 P22

600

SS

6

SUN ET AL.

Table 4. Effects of shackling on endothermic peaks (Td ) of breast meat (mean ± SD; n = 15 each treatment). Temperature (◦ C) Treatments NS SS LS

Tmax1

Tmax2

Tmax3

56.54 ± 0.77b 56.12 ± 0.84a,b 55.40 ± 0.67a

64.16 ± 0.52 64.09 ± 0.52 63.85 ± 0.41

78.49 ± 0.21 78.32 ± 0.24 78.05 ± 0.32

NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group. a,b Means in the same column with no common superscript differ significantly (P < 0.05).

peaks (Td ) are shown in Table 4. The denaturation temperature of myosin in the LS group was lower (P < 0.05) than the NS and SS groups, manifesting that myosin from LS treatment became more susceptible to thermal denaturation, which was consistent with Stabursvik et al. (1984) who also observed a decrease of Tmax1 in PSE meat. But Tmax2 and Tmax3 were not affected (P > 0.05) by shackling treatments. Offer (1991) indicated that myosin was more prone to denaturation at low pH and especially high temperature, which played an important part in lateral shrinkage of myofibrils resulting in a decreased intra-myofibrillar space. Bertram et al. (2006) also found that a negative correlation existed between the denaturation of myosin and immobilized water. Consequently, this result further explained our NMR data and why breast meat from LS treatment had poor water-holding capacity.

Raman Spectroscopy Analysis Variations of protein structures take important roles in water-holding capacity of meat and meat products through affecting the interaction between proteinnetwork and water. Among bands generated from Raman spectroscopy, amide I (1600−1700 cm−1 ) and amide III (1200−1350 cm−1 ) are the most useful to determine the secondary structure. In general, proteins

Figure 3. Secondary structure fractions estimated from the amide I band (n = 15 each treatment). NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group.

with predominantly α-helical structures display an amide I centered at 1,650 to 1,665 cm−1 ; β -sheet peaks are considered corresponding to 1,670 to 1,680 cm−1 and 1,620 to 1,632 cm−1 region; the 1,640 to 1,645 cm−1 and 1,666 to 1,670 cm−1 region have been assigned as random coil peaks; the 1,635 to 1639 cm−1 and 1,680 to 1690 cm−1 region have been assigned as β -turn peaks (Susi and Byler, 1988). The amide I is mainly used to assess the protein secondary structure percentages because of the fact that β -sheet (1230 to 1245 cm−1 ) and random coil (1240 to 1255 cm−1 ) in amide III overlap to some extent (Herrero, 2008). Secondary structural changes in the myofibrillar proteins are exhibited in Figure 3. The content of α-helical structures in LS treatment was lower (P < 0.05) than NS and SS treatments, and the proportion of β -sheet structures was higher (P < 0.05). These findings indicated that preslaughter shackling affected the nature state of myofibrillar proteins. According to Sano et al. (1994), the α-helix structures are mainly stabilized by hydrogen bonds between the carbonyl oxygen (–CO) and amino hydrogen (NH–) of a polypeptide chain. The pH can affect the detailed characteristics of protein interactions owing to its role changing the protonation state of charged amino acids and α-carboxyl and α-amino terminal groups at the surface of proteins (Dumetz et al., 2008). Liu et al. (2010) found the α-helix content in fish myosin significantly decreased due to deviation of pH from neutral to acidity, which was similar with our research. Heating could induce denaturation of protein followed by a gel formation, generally accompanied by the process of β -sheet content increasing at the expense of α-helix structures (Xu et al., 2011). At death, the breast muscle temperature of birds in the shackling groups was higher compared to the NS group. Therefore, a combination of low pH and high breast muscle temperature caused by preslaughter shackling may play a role in affecting the nature state of myofibrillar proteins. Wang et al. (2017) indicated that the reduction of α-helix content in the

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

Figure 2. Typical differential scanning calorimetry thermograms of broiler pectoralis major muscle samples (n = 15 each treatment). NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group.

7

SHACKLING AND POSTMORTEM BROILER MUSCLE Table 5. Effects of shackling on lactate, glycogen of broiers (mean ± SD; n = 15 each treatment). 0.25 h Glycogen mg/g muscle

3h

3.25 ± 0.34

24 h

NS

3.66 ± 0.29

SS LS NS

2.20 ± 0.27a,B 1.01 ± 0.14a,C 1.20 ± 0.05a,A

1.22 ± 0.28b,B 0.93 ± 0.06a,b,C 1.27 ± 0.20a,A

0.98 ± 0.16c,B 0.92 ± 0.06a,b,B 1.71 ± 0.27b,A

0.83 ± 0.08c,A 0.79 ± 0.14b,A 2.10 ± 0.09c,A

SS LS

1.62 ± 0.10a,B 1.77 ± 0.20a,B

1.83 ± 0.07a,b,B 1.96 ± 0.21a,b,B

1.88 ± 0.12b,A 2.15 ± 0.17b,c,B

2.25 ± 0.11c,A 2.23 ± 0.16c,A

b,A

2.23 ± 0.07

c,A

0.95 ± 0.14d,A

NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group. a–d Means in the same row with no common superscript differ significantly (P < 0.05). A–C Means in the same column with no common superscript also differ significantly (P < 0.05).

meat gels was conducive to myosin unfolding as α-helix structures were mainly formed by myosin. Given that myosin denatured partially in the meat from LS treatment, changes of α-helix and β -sheet structures might contribute to this process. Bertram et al. (2006) observed that there was a positive correlation between the intra-myofibrillar water and α-helix structures, and an increase of aggregated structures led to an increase of extra-myofibrillar water. Maybe the changes of αhelix and β -sheet structures resulted in a negative effect on the ability of myofibrillar protein network retaining water, which was attributed to the outflow of intra-myofibrillar water.

Muscle Lactate, Glycogen, and Enzyme Activity Although the supply of oxygen to tissues is suspended due to bleeding at slaughter, metabolic reactions in muscle cells continue for several hours (Shen et al., 2006a). Because of the absence of oxygen, glucose, glycogen, and energy substance conserved in muscle at the time of death mainly can be metabolized by an anaerobic glycolysis pathway. Postmortem muscle lactate and glycogen are exhibited in Table 5. Lactic acid concentrations increased with postmortem time at the expense of glycogen consumption in every group. The lactate content in the LS group was higher (P < 0.05) than the NS group from 0.25 to 3 h postmortem and samples from the SS group had higher lactate content (P < 0.05) at 0.25 and 1 h postmortem, indicating that faster glycolysis had occurred in LS and SS treatments. Corresponding to the accumulation of lactate, there was a decrease of glycogen content with each of the 3 groups. The contents of glycogen in LS and SS treatment were lower (P < 0.05) than NS treatment at postmortem time except 24 h. And compared to LS treatment, higher glycogen content (P < 0.05) was found in SS treatment at 0.25 and 1 h postmortem. In addition, the samples from LS treatment reached ultimate glycogen content at 1 h postmortem, and that following SS treatment reached a terminal value at 3 h postmortem. This indicated that a significant amount of glycogen reserved had been consumed at preslaughter shackling

Figure 4. Effects of shackling on activity of glycogen phosphorylase of broilers (n = 15 each treatment). NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group.

periods, as demonstrated by Fidan et al. (2015) who found that preslaughter shackling caused rapid depletion of glycogen reserved in the skeletal muscle. This was understandable because preslaughter shackling increased stress to broilers, causing strong struggle behaviors accompanied by muscular contraction, which increased activity of metabolic enzymes and consumption of energy. Schilling et al. (2008) and Liste et al. (2009) observed that the increasing of CORT content and muscular contraction was accompanied by rapid glycolysis. Activity of glycogen phosphorylase and phosphofructokinase-1 are shown in Figures 4 and 5, respectively. When tissues are in the state of hypoxia or energy deficiency, glycogen phosphorylase can be phosphorylated and activated, which then promotes glycogenolysis and catalyzes the production of substrate for glycolysis. Glycogen phosphorylase contains a and b 2 forms, but phosphorylase-b is not an active form. Protein kinase A (PKA) can phosphorylase a specific Ser residue in phosphorylase-b to promote its conversion to phosphorylase-a, a fully active form of phosphorylase. Therefore, in our present study, the activity of phosphorylase-a was measured to analyze glycogen phosphorylase activity. The activity

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

Lactate mmol/g protein

1h a,A

8

SUN ET AL.

strong as LS treatment. Accordingly, these factors might contribute to poorer water-holding capacity that was found in the shackling treatment groups compared to the control group.

ACKNOWLEDGMENTS

REFERENCES of glycogen phosphorylase was higher (P < 0.05) in the shackling groups than the control group, when compared to SS treatment, LS treatment gave a rise to (P < 0.05) activity of glycogen phosphorylase at 0.25 h postmortem. And a higher (P < 0.05) activity of glycogen phosphorylase was also found in LS treatment at 1 h postmortem. However, no difference occurred at later time. A similar trend was observed at the changes of PFK-1 activity, an important rate controlling enzyme in glycolysis. The samples from LS treatment had higher (P < 0.05) PFK-1 activity at 0.25 h postmortem, which was also found higher (P < 0.05) in the shackling groups at 1 h postmortem. This indicated that preslaughter shackling increased glycogen phosphorylase and PFK-1 activity at early postmortem, which accelerated glycogen degradation and the rate of glycolysis (anaerobic). These findings may explain faster pH decline in the shackling groups at early postmortem stages. Du et al. (2005) found that when the mice were forced to swim before slaughter, skeletal muscle behaved higher activity of glycogen phosphorylase at early postmortem compared with no swimming group. Preslaughter exercise could increase AMPK activity in skeletal muscle (Morales-Alamo and Calbet, 2016). When AMPK was activated earlier, glycogen phosphorylase and PFK-1 were activated earlier, which promoted generation of substrate for glycolysis and simultaneously accelerated glycolysis (Shen et al., 2006b). The current study indicated that preslaughter shackling could cause stress to broilers, which increased plasma CORT and adrenocorticotropic hormone concentrations, the rate of glycolysis, and early pH decline. In addition, longer shackling duration (4.5 min) led to variations of the secondary structure of myofibrillar proteins accompanied by denaturation of myosin and decreased immobilized water with an increase of free water in PM. Though SS treatment had a similar influence on muscle, the effects were not as

Barbut, S., A. A. Sosnicki, S. M. Lonergan, T. Knapp, D. C. Ciobanu, L. J. Gatcliffe, E. Huff-Lonergan, and E. W. Wilson. 2008. Progress in reducing the pale, soft and exudative (PSE) problem in pork and poultry meat. Meat Sci. 79:46–63. Bedanova, I., E. Voslarova, P. Chloupek, V. Pistekova, P. Suchy, J. Blahova, R. Dobsikova, and V. Vecerek. 2007a. Stress in broilers resulting from shackling. Poult. Sci. 86:1065–1069. Bedanova, I., E. Voslarova, V. Vecerek, V. Pistekova, and P. Chloupek. 2007b. Haematological profile of broiler chickens under acute stress due to shackling. Acta. Vet. Brno. 76:129–135. Belge, F., A. Cinar, and M. Selcuk. 2003. Effects of stress produced by adrenocorticotropin (ACTH) on lipid peroxidation and some antioxidants in vitamin C treated and nontreated chickens. S. Afr. J. Anim. Sci. 33:201–205. Bertram, H. C., H. J. Andersen, and A. H. Karlsson. 2001. Comparative study of low-field NMR relaxation measurements and two traditional methods in the determination of water holding capacity of pork. Meat Sci. 57:125–132. Bertram, H. C., A. Kohler, U. Bocker, R. Ofstad, and H. J. Andersen. 2006. Heat-Induced changes in Myofibrillar protein structures and Myowater of two pork qualities. A combined FT-IR spectroscopy and low-field NMR relaxometry study. J. Agric. Food Chem. 54:1740–1746. Brunton, N. P., J. G. Lyng, L. Zhang, and J. C. Jacquier. 2006. The use of dielectric properties and other physical analyses for assessing protein denaturation in beef biceps femoris muscle during cooking from 5 to 85◦ C. Meat Sci. 72:236–244. Du, M., Q. W. W. Shen, and M. J. Zhu. 2005. Role of betaAdrenoceptor signaling and AMP-activated protein kinase in glycolysis of postmortem skeletal muscle. J. Agric. Food Chem. 53:3235–3239. Dumetz, A. C., A. M. Chockla, E. W. Kaler, and A. M. Lenhoff. 2008. Effects of pH on protein-protein interactions and implications for protein phase behavior. Biochim. Biophys. Acta. Proteins Proteom. 1784:600–610. Fidan, E. D., M. K. T¨ urkyilmaz, A. Nazlig¨ ul, S. U. Aypak, and S. J. V. I. Z. Karaarslan. 2014. The effects of preslaughter shackling on some stress parameters, fear, and behavioural traits in broilers. Veterinarija Ir Zootechnika. 67:24–28. Fidan, E. D., M. K. Turkyilmaz, A. Nazligul, S. U. Aypak, and S. Karaarslan. 2015. Effect of preslaughter shackling on stress, meat quality traits, and glycolytic potential in broilers. J. Agr. Sci. Tech. 17:1141–1150. Guignot, F., X. Vignon, and G. Monin. 1993. Post mortem evolution of myofilament spacing and extracellular space in veal muscle. Meat Sci. 33:333–347. Haensch, F., B. Nowak, and J. Hartung. 2009. Evaluation of a gas stunning equipment used for turkeys under slaughterhouse conditions. Livest. Sci. 124:248–254.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

Figure 5. Effects of shackling on activity of phosphofructokinase1 of broilers (n = 15 each treatment). NS: no shackling group (as control), SS: 2.5 min shackling group, LS: 4.5 min shackling group.

This research was funded by National Natural Science Foundation of China (31701620), Natural Science Foundation of Jiangsu (BK20170715), Fundamental Research Funds for the Central Universities (KJQN201842) Agricultural Industry of Nanjing Science and Technology Development Project (201716055), China Agricultural Research System (CARS-41-Z06) the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

SHACKLING AND POSTMORTEM BROILER MUSCLE

on the quality characteristics and chemical composition of Turkey breast muscles. Poult. Sci. 61:1996–2003. Nijdam, E., E. Delezie, E. Larnbooij, M. J. A. Nabuurs, E. Decuypere, and J. A. Stegeman. 2005. Comparison of bruises and mortality, stress parameters, and meat quality in manually and mechanically caught broilers. Poult. Sci. 84:467–474. Oba, A., M. de Almeida, J. W. Pinheiro, E. I. Ida, D. F. Marchi, A. L. Soares, and M. Shimokomaki. 2009. The effect of management of transport and lairage conditions on broiler chicken breast meat quality and DOA (Death on Arrival). Braz. Arch. Biol. Technol. 52:205–211. Offer, G. 1991. Modelling of the formation of pale, soft and exudative meat: effects of chilling regime and rate and extent of glycolysis. Meat Sci. 30:157–184. Sano, T., T. Ohno, H. Otsukafuchino, J. J. Matsumoto, and T. Tsuchiya. 1994. Carp natural actomyosin: thermal denaturation mechanism. J. Food Sci. 59:1002–1008. Schilling, M. W., V. Radhakrishnan, Y. V. Thaxton, K. Christensen, J. P. Thaxton, and V. Jackson. 2008. The effects of broiler catching method on breast meat quality. Meat Sci. 79:163–171. Shao, J. H., Y. F. Zou, X. L. Xu, J. Q. Wu, and G. H. Zhou. 2011. Evaluation of structural changes in raw and heated meat batters prepared with different lipids using Raman spectroscopy. Food Res. Int. 44:2955–2961. Shen, Q. W., W. J. Means, S. A. Thompson, K. R. Underwood, M. J. Zhu, R. J. McCormick, S. P. Ford, and M. Du. 2006a. Pre-slaughter transport, AMP-activated protein kinase, glycolysis, and quality of pork loin. Meat Sci. 74:388–395. Shen, Q. W., W. J. Means, K. R. Underwood, S. A. Thompson, M. J. Zhu, R. J. McCormick, S. P. Ford, M. Ellis, and M. Du. 2006b. Early post-mortem AMP-activated protein kinase (AMPK) activation leads to phosphofructokinase-2 and -1 (PFK-2 and PFK-1) phosphorylation and the development of pale, soft, and exudative (PSE) conditions in porcine longissimus muscle. J. Agric. Food Chem. 54:5583–5589. Sosnicki, A. A., M. Pietrzak, and M. L. Greaser. 1997. Effect of rapid rigor mortis processes on protein functionality in pectoralis major muscle of domestic turkeys. J. Anim. Sci. 75:2106–2116. Stabursvik, E., K. Fretheim, and T. Froystein. 1984. Myosin denaturation in pale, soft, and exudative (PSE) porcine muscle tissue as studied by differential scanning calorimetry. J. Sci. Food Agric. 35:240–244. Susi, H., and D. M. Byler. 1988. Fourier deconvolution of the Amide I Raman band of proteins as related to conformation. Appl. Spectrosc. 42:819–826. Wang, M., X. Chen, Y. Zou, H. Chen, S. Xue, C. Qian, P. Wang, X. Xu, and G. Zhou. 2017. High-pressure processing-induced conformational changes during heating affect water holding capacity of myosin gel. Int. J. Food Sci. Technol. 52:724–732. Wu, Z., H. C. Bertram, A. Kohler, U. Bocker, R. Ofstad, and H. J. Andersen. 2006. Influence of aging and salting on protein secondary structures and water distribution in uncooked and cooked pork. A combined FT-IR microspectroscopy and H-1 NMR relaxometry study. J. Agric. Food Chem. 54:8589–8597. Xing, T., Y. H. Li, M. Li, N. N. Jiang, X. L. Xu, and G. H. Zhou. 2016. Influence of transport conditions and pre-slaughter water shower spray during summer on protein characteristics and water distribution of broiler breast meat. Anim. Sci. J. 87:1413– 1420. Xing, T., X. L. Xu, G. H. Zhou, P. Wang, and N. N. Jiang. 2015. The effect of transportation of broilers during summer on the expression of heat shock protein 70, postmortem metabolism and meat quality. J. Anim. Sci. 93:62–70. Xu, X. L., M. Y. Han, Y. Fei, and G. H. Zhou. 2011. Raman spectroscopic study of heat-induced gelation of pork myofibrillar proteins and its relationship with textural characteristic. Meat Sci. 87:159–164.

Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez175/5455233 by Bukkyo University user on 17 April 2019

Herrero, A. M. 2008. Raman spectroscopy a promising technique for quality assessment of meat and fish: a review. Food Chem. 107:1642–1651. Honikel, K. O., C. J. Kim, R. Hamm, and P. Roncales. 1986. Sarcomere shortening of prerigor muscles and its influence on drip loss. Meat Sci. 16:267–282. 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. 92:5749–5756. Huang, J. C., M. Huang, J. Yang, P. Wang, 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. 93:2087–2095. Huang, J. C., J. Yang, M. Huang, Z. S. Zhu, X. B. Sun, B. H. Zhang, X. L. Xu, W. G. Meng, K. J. Chen, and B. C. Xu. 2018. Effect of pre-slaughter shackling and wing flapping on plasma parameters, postmortem metabolism, AMPK, and meat quality of broilers. Poult. Sci. 97:1841–1847. Huff-Lonergan, E., and S. M. Lonergan. 2005. Mechanisms of waterholding capacity of meat: the role of postmortem biochemical and structural changes. Meat Sci. 71:194–204. Joo, S. T., R. G. Kauffman, B. C. Kim, and G. B. Park. 1999. The relationship of sarcoplasmic and myofibrillar protein solubility to colour and water-holding capacity in porcine longissimus muscle. Meat Sci. 52:291–297. Joseph, P., M. W. Schilling, J. B. Williams, V. Radhakrishnan, V. Battula, K. Christensen, Y. Vizzier-Thaxton, and T. B. Schmidt. 2013. Broiler stunning methods and their effects on welfare, rigor mortis, and meat quality. Worlds Poult. Sci. J. 69:99–112. Kannan, G., J. L. Heath, C. J. Wabeck, and J. A. Mench. 1997. Shackling of broilers: effects on stress responses and breast meat quality. Br. Poult. Sci. 38:323–332. Knowles, T. G., S. N. Brown, P. D. Warriss, A. J. Phillips, S. K. Dolan, P. Hunt, J. E. Ford, J. E. Edwards, and P. E. Watkins. 1995. Effects on sheep of transport by road for up to 24 hours. Vet. Rec. 136:431–438. Langer, Souza, R. O., G. S. Simoes, A. L. Soares, A. Oba, A. Rossa, and M. S. Elza Iouko Ida. 2010. Broiler transportation conditions in a brazilian commercial line and the occurrence of breast PSE (Pale, Soft, Exudative) Meat and DFD-like (Dark, Firm, Dry) meat. Braz. Arch. Biol. Technol. 53:1161–1167. Lines, J. A., T. A. Jones, P. S. Berry, P. Cook, J. Spence, and C. P. Schofield. 2011. Evaluation of a breast support conveyor to improve poultry welfare on the shackle line. Vet. Rec. 168:129– 129. Liste, G., M. Villarroel, G. Chacon, C. Sanudo, J. L. Olleta, S. Garcia-Belenguer, S. Alierta, and G. A. Maria. 2009. Effect of lairage duration on rabbit welfare and meat quality. Meat Sci. 82:71–76. Liu, R., S. m. Zhao, Y. m. Liu, H. Yang, S. b. Xiong, B. j. Xie, and L. h. Qin. 2010. Effect of pH on the gel properties and secondary structure of fish myosin. Food Chem. 121:196–202. McDonnell, C. K., P. Allen, E. Duggan, J. M. Arimi, E. Casey, G. Duane, and J. G. Lyng. 2013. The effect of salt and fibre direction on water dynamics, distribution and mobility in pork muscle: a low field NMR study. Meat Sci. 95:51–58. McFarlane, J. M., and S. E. Curtis. 1989. Multiple concurrent stressors in chicks. 3. Effects on plasma - plasma- corticostrone and the heterophil-lymphocyte ratio. Poult. Sci. 68:522–527. McGeehin, B., J. J. Sheridan, and F. Butler. 2001. Factors affecting the pH decline in lamb after slaughter. Meat Sci. 58:79–84. Morales-Alamo, D., and J. A. L. Calbet. 2016. AMPK signaling in skeletal muscle during exercise: role of reactive oxygen and nitrogen species. Free Radic. Biol. Med. 98:68–77. Ngoka, D. A., G. W. Froning, S. R. Lowry, and A. S. Babji. 1982. Effects of sex, age, preslaughter factors, and holding conditions

9