Ll Xin, XlA An-qi, CHEN Li-juan, DU Man-ting, CHEN Li, KANG Ning, ZHANG De-quan

Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing 100193, P.R.China

Abstract The objective of this study was to investigate the effect of lairage after transport on post mortem muscle glycolysis, protein phosphorylation and lamb meat quality. Two preslaughter animal treatments, transport for 3 h and lairage for 0 h (T3L0) and transport for 3 h and then lairage for 12 h (T3L12), were compared with a control treatment of 0 h transport and 0 h lairage.Data obtained showed that preslaughter transport had a significant effect on lamb meat quality. Loins from lambs of the T3L0 treatment showed higher (P=0.026) pH24 h and higher (P=0.021) pH48 h values, but lower (P＜0.001) drip loss and lower(P＜0.05) glycolytic potential at 0 h post mortem than those of the T3L12 and control groups. Muscle samples of the T3L0 group showed higher (P=0.046) shear force and lower (P=0.005) b＊ value than those of the T3L12 group. Muscle glycogen concentration at 0, 2, 4 h post mortem were lower (P＜0.05) in the T3L0 group than in control. No significant difference(P＞0.05) in most meat quality parameters was determined between the T3L12 group and control, showing lairage for 12 h allowed lambs to recover from the effects of transport for 3 h and resulted in similar meat quality characteristics compared to no transport. Lairage after transport did not affect most meat quality indices in comparison with control, but increased the meat drip loss and b＊ value of lambs possibly through decreasing glycogen concentration and glycolytic potential.

Keywords: lamb, transport, lairage, meat quality, glycolytic metabolite, protein phosphorylation

1. lntroduction

Most preslaughter handling leads to stress reactions that subsequently decrease meat quality (Hall and Bradshaw 1998; Knowles 1998; Ferguson and Warner 2008). Animals can be exposed to a series of stressors during the preslaughter period including human contact,physical exercise, confinement, transportation, unfamiliar environments, food and water deprivation, changes in socialstructure and extreme climatic conditions. On the other hand, some preslaughter operations, such as appropriate rest after transportation, may relieve stress and hence improve meat quality (Ekiz et al. 2012).

After slaughter, anaerobic glycolysis continues to utilise the glycogen reserves in muscle to supply ATP with the production of lactic acid and thereby reduce the muscle pH.Depletion of muscle glycogen before slaughter results in low lactate content in muscle and higher ultimate muscle pH postmortem (Ferguson and Warner 2008). The rate and extent of pH decline post mortem are related to muscle glycolytic potential which is an estimate of muscle glycogen in vivo and a stress-induced increase in glycolysis (Hambrecht et al. 2005). Both the rate of pH decline and the ultimate pH of post mortem muscle are important for meat quality formation, which influence meat tenderness, water holding capacity and colour (Díaz et al. 2014).

Protein phosphorylation regulates many physiological and biochemical processes, such as signal transduction and consequential protein kinase activity (Graves and Krebs 1999; Hunter 2000; Huang et al. 2011) and plays an important role in post mortem meat quality development(Chen et al. 2016; Li M et al. 2017). Protein phosphorylation influences meat quality development through regulation of actomyosin dissociation, protein degradation and µ-calpain activity post mortem (Du et al. 2017; Gao et al. 2017; Li Z et al. 2017). Preslaughter stress has been revealed to regulate pork quality post mortem through protein phosphorylation and glycolysis (Shen et al. 2006).

Transportation of animals from farm to abattoir is usually unavoidable and stresses animals (Thompson et al. 1987;Schwartzkopf-Genswein et al. 2012). A study showed that preslaughter transportation resulted in higher stressed animals and lairage allowed lambs to recover from stress(Ekiz et al. 2012). Other studies confirmed the reverse effects of transportation (Knowles et al. 1996; Fisher et al. 2010) and lariage on lamb meat quality (Jacob et al.2005a, b, 2006; Díaz et al. 2014), however, the detailed mechanisms how preslaughter handling regulate lamb meat quality are not well understood. The objective of this study was to compare the effect of two lairage periods on glycolysis and protein phosphorylation with a control treatment of 0 h transport and 0 h lairage in relation to meat quality and provide some new insights in the biochemistry of post mortem of meat quality development.

2. Materials and methods

2.1. Animals and treatments

Thirty crossbred lambs of Chinese Fat-tail Han and Smalltail Han sheep breed were randomly selected from the same flock. The animals (six months old) were intensively housed and raised under one feeding strategy without significant variations in terms of age and pre-transport weight of animals. They were randomly assigned to one of the following treatments (n=10): (1) control: transport for 0 h and lairage for 0 h; (2) transport for 3 h and lairage for 0 h(T3L0); (3) transport for 3 h and lairage for 12 h (T3L12).The transportation and lairage treatments were done outside with the outside temperature ranged from 4 to 10°C and a relative humidity of 35.5%. The stocking density was 0.25 m2per animal. The animals were denied access to feed and water during transport. The road surface was flat and the speed was constant 50 km h–1. The animals were loaded and unloaded using driving board. The animals were given access to water ad libitum but no food during lairage.

2.2. Slaughter and sampling

All of the animals used in the present study were slaughtered in the morning in a commercial abattoir according to their standard halal slaughter procedures. This involved exsanguination without electrical stunning. There was no electrical stimulation at the end of carcase dressing. The mean hot carcass weight was 20.08 kg (standard deviation 1.71). The longissimus thoracis et lumborum (LTL) muscles from the 1st rib to the last lumbar vertebrae were removed from both sides of carcasses 2 h after slaughter (n=10).The LTL muscles were chilled at 4°C for 24 h on a tray wrapped with oxygen permeable polyvinyl chloride film in the chilling room.

The LTL muscles from the left side were used to measure pH, meat colour, drip loss, cooking loss, glycolysis and protein phosphorylation analysis. The samples for glycolysis and protein phosphorylation analysis were quickly frozen in liquid nitrogen until analysis. The LTL muscles from the right side were vacuum-packed and frozen (–20°C) for Warner-Bratzler shear force measurement and sensory evaluation.

In this experiment, all procedures were undertaken following the guidelines set out by the Animal Care and Ethics Committee for animal experiments, produced by the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences.

2.3. Meat quality measurements

The pH was measured manually at 0, 24 and 48 h postmortem at the same location of LTL muscle sample from the left side carcass using a portable pH meter (Testo 205,Lenzkirch, Germany).

Meat colour (CIE-L＊a＊b＊) was measured at 24 h postmortem on a fresh cut surface of LTL muscle after blooming in air for 30 min using a Chroma Meter (CR-400, Konica Minolta Sensing Inc., Osaka, Japan) with illuminant C and 11 mm measurement diameter. The instrument was calibrated against a standard white plate and four measurements were made on each sample. The parameters L＊ (lightness), a＊(redness) and b＊ (yellowness) were recorded.

Drip loss and cooking loss were measured using the method described by Honikel (1998). The LTL muscles(1st–2nd lumbar vertebrae) used for drip loss measurement were weighed, placed in a plastic bag without contacting with the bag and hung at 4°C for 24 h. The samples were weighed again after gently wiping the sample surface. Drip loss was expressed as the percentage of weight loss after suspension for 24 h. For cooking loss measurement, the LTL muscles (2nd–3rd lumbar vertebrae) were weighed,cooked in a water bath at 80°C for 45 min, chilled at 4°C overnight and reweighed. Cooking loss was expressed as the percentage of weight loss during cooking.

Warner-Bratzler shear force was measured using a Texture Analyser (TA.XT2, Stable Micro Systems, UK).Frozen samples from the right side LTL muscles (8 cm length, approximately 60 g) were thawed at 4°C overnight,packed in vacuum, cooked at 75°C in water bath until the core temperature reached 70°C and then cooled in cold water. For each animal, 12 muscle strips of 2 cm×1 cm×1 cm(length×width×height) were cut out with the longitudinal axis paralleled to the muscle fibre direction. The shear force was recorded as the peak force (kg) to cut the strips in the direction perpendicular to the muscle fibres.

Sensory analysis was carried out using the method described by Fernández and Vieira (2012). An eight-member sensory panel was trained according to the International Standard Method (ISO 8586: 2012 2012). The LTL samples from the right side carcass were thawed overnight at 4°C and cut into 5 cm×5 cm×2 cm (length×width×height) steaks.The steaks were roasted in an oven setting at 160°C and 50% humidity for 15 min to reach the internal temperature of 70°C. The cooked steaks were cut into pieces of 1 cm×1 cm×1 cm in size, placed on white plastic trays covered with aluminium foil and stored in an oven (about 10 min) at 70°C until tasting. Scores for lamb odour (1=very slight; 9=verystrong), tenderness (1=very tough; 9=very tender), juiciness(1=very dry; 9=very juicy) and overall acceptability (1=very unacceptable; 9=very acceptable) were recorded. Each member tasted sample from each treatment independently.Three sessions were applied for sensory evaluation and the panellists were served with three samples in each session.The sensory evaluation was carried out in a test room which was kept constant at 20°C and odour free by air conditioner.The colour differences were masked by red light when the trial was being carried on.

2.4. Glycolytic metabolite measurements

Muscle glycogen and lactate concentrations at 0, 2, 4 and 24 h post mortem were determined using the methods described by Zhang et al. (2009) with some modification.Muscle samples of 500 mg were cut and homogenized(T10, IKA, Germany) for 30 s in 4.5 mL of 0.85 mol L–1HClO4and the homogenates were centrifuged at 2 500×g,4°C for 10 min. The supernatant fraction was neutralized with 10 mol L–1KOH and stored at -80°C for subsequent analysis. Lactate in the supernatant fraction was determined spectrophotometrically using a commercial Lactic Acid Kit (A019-2, Nanjing Jiancheng Bioengineering Institute,Nanjing, China). The glycogen in the supernatant fraction was enzymatically hydrolyzed by amyloglucosidase (A7420,Sigma-Aldrich Inc., St. Louis, MO, USA) in sodium acetate buffer (pH 4.8) at 55°C for 2 h and then neutralized with 10 mol L–1KOH. Glucose concentration was measured using spectrophotometric method according to the manual of the Glucose Oxidase Kit (Shanghai Rongsheng Biotech Co., Ltd., Shanghai, China). The concentration of glucose-6-phosphate and glucose was not determined separately but was included in the glycogen determination. The glycolytic potential was calculated as the sum of 2×[glycogen]+[lactate](Monin and Sellier 1985; Hambrecht et al. 2005).

2.5. Protein phosphorylation

The global protein phosphorylation levels in muscle at 0,4 and 24 h post mortem were determined as described by Chen et al. (2016). Briefly, a total of 1 g of frozen muscle was homogenized on ice in 6 mL ice cold buffer containing 100 mmol L–1Tris (pH 8.3), 10 mmol L–1DTT, complete protease inhibitor (one tablet for each 50 mL; Roche,Mannheim, Germany) and phosphatase inhibitor (two tablets for each 50 mL; PhosSTOP, Roche, Mannheim, Germany).The homogenates were centrifuged at 25 000×g, 4°C for 20 min to separate sarcoplasmic proteins and myofibrillar proteins. The pellets of myofibrillar proteins were further homogenized for 30 s in 25 mL SDS buffer, incubated at 80°C for 20 min. Both prepared sarcoplasmic and myofibrillar proteins were stored at -80°C for SDS-PAGE analysis. Protein concentration was determined by the BCA assay (Pierce Chemical Company, Rockford, USA).

One-dimensional gel electrophoresis and staining were carried out using the methods described by Lametsch et al.(2006) and Huang et al. (2012). The sample buffer (2×,reducibility) containing the following media: 40 mL 10% SDS,2 mL glycerin, 2 mL 0.5 mol L–1Tris·HCl (pH 6.8), 2.0 mL 1 mol L–1DTT, 0.01 g bromophenol blue was added. Mixture was heated in boiling water for 10 min and centrifuged at 12 000×g for 2 min. An aliquot of 10 µL supernatant with the same amount of total protein was loaded on SDS-PAGE gels. The gels were run in electrophoretic running buffer at 70 V for stacking gel (4%) and 120 V for separation gel(12%) until bromophenol blue reached the front edge of gels.

The Pro-Q Diamond (Invitrogen, USA) and SYPRO Ruby dye (Invitrogen, USA) were used to stain the phosphoproteins and total proteins. Fluorescently-labelled proteins were visualised using the Typhoon Trio Variable Mode Imager System (GE, USA). Phosphoproteins images were scanned under Cy3 channel with 200-mm resolution and excitation and emission wavelength of 532 and 580 nm respectively. The excitation and emission wavelength were 532 and 610 nm respectively for total proteins. The Quantity One 4.6.2 software (Bio-Rad, USA) was used for analysis of the protein band intensities. Protein phosphorylation level was calculated as the ratio of the intensity of phosphorylated proteins (P) in each band in the Pro-Q Diamond image to the intensity of total protein (T) in SYPRO Ruby image(P/T ratio). Global phosphorylation level was calculated by measuring all protein bands in an entire lane for P/T ratios.

2.6. Statistical analysis

The statistical analysis was carried out using the SPSS Statistics (Version 17.0, SPSS Inc., Chicago, USA). The one-way ANOVA was used with preslaughter handling treatment as the fixed factor and individual animal as the experimental unit. The model used for data from the sensory evaluation also included assessor as random factor with data averaged over the sensory triplicates. Multiple comparisons were performed using the Duncan’s multiple rang test. Reported P-values were evaluated at the 5%significance level.

3. Results

3.1. Meat quality

Treatment had significant effects on pH, drip loss, shear force and b＊ value (Table 1). The T3L0 treatment resulted in higher (P＜0.05) pH values than those of T3L12 and control treatments at 24 and 48 h post mortem. The drip loss in T3L12 treatment was higher (P＜0.05) than that in T3L0 and control treatments and it was also higher (P＜0.05) in control group than in T3L0 group. Warner-Bratzler shear force was increased (P＜0.05) in the T3L0 group than in the T3L12,indicating increased toughness. However, meat from both T3L0 and T3L12 groups were not different from control in tenderness. The b＊ value in T3L12 group was higher(P＜0.05) than in T3L0 and control groups. No significant effects (Table 1, P＞0.05) of lairage after transport on muscle pH0h, L＊, a＊, cooking loss or sensory characteristics were observed in this study.

3.2. Glycolytic metabolite concentrations

In the present study, glycogen, glucose and glucose-6-phosphate were spectrophotometrically measured together and referred to as glycogen. The glycogen concentration in muscle of the control group was significantly higher (Fig. 1-A,P＜0.05) than that of the T3L0 group at 0, 2 and 4 h postmortem. The control group also showed higher (P＜0.05)glycogen content than the T3L12 group at 4 h post mortem,showing preslaughter transport increased usage of glycogen in muscle. Lairage after transport had no significant effects(Fig. 1-B, P＞0.05) on ovine muscle lactate concentration.The calculated glycolytic potential was higher (Fig. 1-C,P＜0.05) in the T3L12 groups than in the T3L0 group at 0 and 24 h post mortem and it was also higher (P＜0.05) in the control group than that in the T3L0 group at 0 h post mortem.

3.3. Global protein phosphorylation

The lairage after transport treatments did not affect(Figs. 2–4, P＞0.05) global protein phosphorylation during 24 h post mortem. The global protein phosphorylation level(P/T ratio) of both sarcoplasmic proteins and myofibrillar proteins was not significantly different (P＞0.05) between T3L0, T3L12 and control groups at 0, 4, and 24 h postmortem.

Table 1 Effects of lairage after transport on ovine meat quality and sensory characteristics

Fig. 1 Changes in the concentrations of glycogen (A), lactate(B) and glycolytic potential (C) in ovine muscle during the first24 h post mortem after different preslaughter handling treatments. Control, transport for 0 h and lairage for 0 h; T3L0,transport for 3 h and lairage for 0 h; T3L12, transport for 3 h and then lairage for 12 h. Data are mean±SD (n=10). Different letters at the same post mortem time indicated significant difference between different treatments (P＜0.05).

Fig. 2 Gel images (SDS-PAGE) of phosphorylated sarcoplasmic proteins (A) and myofibrillar proteins (B) of ovine muscle within 24 h post mortem after different preslaughter handling treatments. M, marker; A0–A24, control group (transport for 0 h and lairage for 0 h) at 0, 4 and 24 h post mortem, respectively; B0–B24, T3L0 group (transport for 3 h and lairage for 0 h) at 0, 4 and 24 h postmortem, respectively; C0–C24, T3L12 group (transport for 3 h and then lairage for 12 h) at 0, 4 and 24 h post mortem, respectively.

4. Discussion

Fig. 3 Gel images (SDS-PAGE) of total sarcoplasmic proteins (A) and myofibrillar proteins (B) of ovine muscle within 24 h postmortem after different preslaughter treatments. M, marker; A0–A24, control group (transport for 0 h and lairage for 0 h) at 0, 4 and 24 h post mortem, respectively; B0–B24, T3L0 group (transport for 3 h and lairage for 0 h) at 0, 4 and 24 h post mortem, respectively;C0–C24, T3L12 group (transport for 3 h and then lairage for 12 h) at 0, 4 and 24 h post mortem, respectively.

Fig. 4 Comparison of global phosphorylation of ovine muscle sarcoplasmic proteins (A) and myofibrillar proteins (B) at post mortem after different preslaughter handling treatments. Control, transport for 0 h and lairage for 0 h; T3L0, transport for 3 h and lairage for 0 h; T3L12, transport for 3 h and then lairage for 12 h. P/T ratio is protein phosphorylation level which is calculated as the ratio of the intensity of phosphorylated proteins (P) in each band in the Pro-Q Diamond image to the intensity of total proteins (T) in SYPRO Ruby image. Data are mean±SD (n=10).

Many factors related to preslaughter handling affect meat quality such as transportation and lairage duration (Ruizde-la-Torre et al. 2001; Kadim et al. 2009). Transport for 3 h without lairage resulted in increased pH at 24 and 48 h postmortem compared to the control in the present study, which was in agreement with the findings of other studies (Jacob et al. 2005a; Zhong et al. 2011).

The increased shear force and decreased drip loss of LTL muscle of T3L0 lambs indicated that transport without lairage resulted in increased toughness of meat. These results were in consistent with earlier studies showed that the meat from animals that were transported before slaughter had higher ultimate pH and shear force than non-transported animals(Kadim et al. 2009; Ekiz et al. 2012). In general, the shear force values obtained in this study were a little bit higher compared with other reports. It might be due to the feedingstrategy or perhaps the chilling procedure of the samples had effects on meat tenderness, which needs to be clarified in further study.

The results of studies on meat colour are inconsistent.Kadim et al. (2009) and Ekiz et al. (2012) reported no significant effect of transportation and lairage time on meat L＊, a＊ and b＊ values. Others found higher a＊ values and total heme pigment content in muscles of animals allowed for longer time of recovery (Kannan et al. 2000; Zhong et al.2011). Meat colour analysis in the current study showed that b＊ values of T3L12 group was higher than those of the T3L0 and control groups, but there was no significant difference in L＊ and a＊ values. The variation of b＊ value between treatments may be correlated with the structure change of muscle and the water holding capacity. In this study, similar trend was also observed on drip loss between treatments.

Glycogen content in muscle affects lactate concentration and ultimate pH of post mortem muscle and subsequently meat tenderness, colour, drip loss and juiciness (Rosenvold et al. 2001; Hamilton et al. 2003; Bee et al. 2006).Preslaughter stress could lead to depletion of muscle glycogen reserves of animal before slaughter, insufficient meat acidification at post mortem and increased incidence of DFD (dark, firm, dry) meat (Lawrie and Ledward 2006).Depletion of glycogen during transportation could be restored during preslaughter lairage (Warriss et al. 1984; Del Campo et al. 2010). However, Daly et al. (2006) reported that time-off feed prior to slaughter had no effect on post-slaughter muscle glycogen concentrations, pHu, or rate of pH decline. In this study, the glycogen concentration in control group was significantly higher than that in T3L0 group at early post mortem, but there was no difference on glycogen concentrations between the control and the T3L12 groups,which indicated that lairage for 12 h after 3 h transport can allow the recovery of muscle from stress. Richter and Galbo(1986) suggested that higher initial glycogen concentrations could result in higher glycogen breakdown. The rate of pH decline achieved stability when glycogen concentration reached approximately 56 mmol kg–1muscles. Below this level, lower muscle glycogen resulted in slower rates of pH decline, whereas above this level there was no effect of increased glycogen on pH decline rate. This might be the reason that no difference was obtained on lactate concentration between treatments.

The rate of pH decline post mortem is regulated by the activity of glycolytic enzymes (Clarke et al. 1980). Protein phosphorylation regulates muscle metabolism and indirectly affected meat quality (Sola-Penna et al. 2010; Li M et al.2017). Glycolytic enzymes function is regulated by protein phosphorylation (Randle 1981; Cohen 1983; Singh et al.2004). Huang et al. (2012) reported the changes of protein phosphorylation of porcine myofibrillar proteins within 24 h post mortem. Their results showed that the change of protein phosphorylation in myofibrillar proteins within 24 h postmortem influenced meat quality development. However,few data were available on the protein phosphorylation of sarcoplasmic and myofibrillar under different preslaughter handlings which probably has a relationship to meat quality.However, the global P/T ratio of both sarcoplasmic and myofibrillar proteins had no difference between treatments within 24 h post mortem in this study. As the global P/T ratio demonstrates the general protein phosphorylation level of all proteins, phosphorylation level of individual protein may have opposite variation leading to insignificant global phosphorylation level between treatments (Li et al.2017a). Identification of individual phosphorylated protein may provide further understanding about the mechanism of meat quality regulation.

5. Conclusion

Lairage for 12 h allowed lambs to recover from stress induced by preslaughter transport for 3 h and resulted in similar meat quality characteristics compared to control except increased drip loss and b＊ value of lambs meat which was possibly caused by the depletion of glycogen and decreased glycolytic potential in muscle. Analysis of protein phosphorylation showed no difference in the phosphorylation levels of total proteins between treatments.Identification of individual phopho-proteins are needed to clarify the role of protein phosphorylation in post mortem lamb quality formation in response to preslaughter handling.

Acknowledgements

The authors thank the financial support from the National Agricultural Science and Technology Innovation Program in China.