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Effects of stress and high voltage electrical stimulation on tenderness of lamb m. longissimus
Meat Science 57 (2001) 265±271
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Eects of stress and high voltage electrical stimulation on tenderness of lamb m. longissimus G.H. Geesink *, M.H.D. Mareko, J.D. Morton, R. Bickerstae Molecular Biotechnology Group, Animal and Food Sciences Division, Lincoln University, PO Box 84, Canterbury, New Zealand Received 19 May 2000; received in revised form 9 August 2000; accepted 9 August 2000
Abstract This study was conducted to investigate the reported eect of pre-slaughter stress on meat tenderness independent from its eect on ultimate pH, and its interaction with electrical stimulation. From a group of 80 Coopworth lamb, 40 were stressed by subjecting the animals to a swim wash 3 h before slaughter and the use of dogs to assemble the animals to the access ramp of the abbatoir. Half of the carcasses of each group was electrically stimulated within 30 min post mortem. Temperature and pH decline of the longissimus was monitored and shear force of the cooked muscle was determined at 2 days post mortem and after 6 weeks vacuum storage at 1 C. To investigate an eect of stress independent of ultimate pH, 10 muscles with an ultimate pH below 5.8 were selected from each group for detailed analysis. This analysis consisted of determination of calpastatin activity and sarcomere length, and immunoblotting of m-calpain and calpain substrates. The stress treatment led to an increase in the number of muscles with an ultimate pH above 5.8 (32.5 vs 15%), and muscles with an ultimate pH above 5.8 were signi®cantly tougher than muscles with an ultimate pH below 5.8 at 2 days post mortem. Electrical stimulation improved tenderness at two days post mortem. This eect could be attributed to an eect on muscle contraction, but not on post mortem proteolysis of calpain substrates. A large variation in tenderness at 2 days post mortem was observed and this was not reduced by electrical stimulation. Six weeks of vacuum storage resulted in a 6 kgF drop in mean shear force and a uniformly tender product. Despite the fact that the stress treatment was similar to those in earlier studies, we failed to observe an eect of stress independent of ultimate pH on tenderness. The reason for this is unclear, but dierences in the response to stress between breeds may be responsible. The results of the present study underscore the importance of minimizing pre-slaughter stress and adequate post mortem storage for meat quality. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Stress; Electrical stimulation; Lamb; Tenderness
1. Introduction Stressing meat animals before slaughter may aect meat quality in several ways. Pre-slaughter stress may lead to increased bruising and depletion of muscle glycogen stores. The latter results in meat with an elevated ultimate pH which negatively aects meat colour, ¯avour and keeping quality (Lister, Gregory & Warris, 1981). In addition, an ultimate pH of meat between 5.8 and 6.2 is associated with increased toughnes (Purchas, 1990). Another quality aspect related to treatment of animals before slaughter stems from animal welfare concerns. Many consumers are aware of the ethics of meat * Corresponding author. Tel.: +64-3-325-2811, ext. 8172; fax: +64-3-325-3851. E-mail address: [email protected] (G.H. Geesink).
production and want their meat to be produced in a way which takes animal welfare into account (Warris, 1995). However, developments in the meat industry may have increased the incidence of pre-slaughter stress. The meat industry has become centralised into fewer, larger plants. Consequently, the distance animals have to be transported has increased. The large plants often operate at higher speeds compared to smaller plants. The need to move and process animals rapidly may increase stress in the period immediately before slaughter. Apart from a toughening eect of stress through an increase in ultimate pH, a toughening eect independent of ultimate pH has been observed (Bickerstae, Morton, Daly & Keeley, 1996; Daly, Simmons & Devine, 1995; Morton, Bickerstae, Le Couteur & Keeley, 1997). In addition, this eect was more pronounced when low voltage electrical stimulation was used to immobilize the
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00101-7
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animals, suggesting an interaction between stress and electrical stimulation. In both studies, high voltage electrical stimulation (HVES) was applied to all carcasses within 30 min after stunning, but it was not determined whether there was an interaction of stress and HVES on meat tenderness. Also, in both studies it was not determined whether the dierences in tenderness were due to dierences in contraction, post mortem proteolysis, or a combination of these factors. The purpose of the present study was to further investigate the possibility of a toughening eect of pre-slaughter stress independent of ultimate pH and its possible interaction with HVES. 2. Material and methods 2.1. Reagents All chemicals were analytical grade obtained from BDH Laboratory Supplies, Poole, Dorset, UK and BioRad Laboratories, Hercules, CA, USA. Anti-m-calpain (9A4H8D3) was obtained from Anity Bioreagents, Inc., Golden, CO, USA. Anti-titin (9D10) was developed by M. Greaser, anti-troponin-T (CT3) was developed by J.J.C. Lin and anti-desmin (D3) was developed by D.A. Fishman. These antibodies were obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract N01-HD-7-3263 from the NICHD. Alkaline phosphatase conjugated anti-mouse IgG was obtained from Sigma (St Louis, MO). 2.2. Animals, carcass measurements and sampling From a group of 80 Coopworth lambs (12 months of age, pasture fed), 40 were stressed by subjecting the animals to a swim-wash through a 15 m cold water bath 3 h before slaughter and using dogs during the 15 min needed to assemble the animals to the access ramp. The remaining 40 were not subjected to a swim wash, and the animals were assembled without the use of dogs. The lambs were electrically stunned, immobilised by spinal discharge, exsanguinated, and dressed in accordance with standard New Zealand industry practices (carcass weight 16.31.4 kg). Within 30 min post mortem half of the carcasses within each group was subjected to high voltage electrical stimulation (ES: 1130 V, 14.3 Hz, 90 s). At about 1 h post mortem the longissimus of the right carcass side was exposed at the 12th rib by knife dorsal surface cuts. The pH and temperature of the muscles were measured through this cut at several occasions over a 24 h period. The pH and temperature were measured using an Orion 8163 glass electrode and a temperature probe attached to a Hanna HI 9025 portable
pH meter. The carcasses were held at 8 C for 1 h and then transferred to a 0 C chiller. At 2 days post mortem, the carcasses were transferred to the cutting room and the longissimus was cut from the carcass. The muscle from the right carcass side was used to evaluate tenderness and related attributes at 2 days post mortem. The muscle from the left carcass side was sampled after 6 weeks of vacuum storage at 1 C. 2.3. Shear force measurements Samples for shear force measurements were stored frozen at ÿ30 C and cooked after thawing overnight at 2 C. Samples were cooked individually in plastic bags immersed in a water bath at 80 C until they reached an internal temperature of 75 C as measured using Fluke type K temperature probes attached to Fluke 52 meters. The cooked meat was cooled on ice and six to eight pieces of meat were removed parallel to the muscle ®bres. Test pieces were placed separately in the MIRINZ tenderometer (Chrystall & Devine, 1991) and the shear force (kgF) to cut across the ®bres was determined. 2.4. Calpastatin activity and sarcomere length Samples for calpastatin activity determinations were prepared according to Shackelford, Koohmaraie, Cundi, Gregory, Rohrer and Savell (1994) with minor modi®cations. Calpastatin activity was determined according to Koohmaraie (1990). Minced muscle was homogenised in three volumes extraction buer (100 mM Tris/HCl, pH 8.3, 5 mM EDTA, 0.05% 2-mercaptoethanol, 4 C) using a polytron. The homogenate was centrifuged at 1500 gmax and 5 ml of the supernatant was collected for calpastatin activity determination. The remaining supernatant was discarded and the pellet was resuspended and centrifuged twice with extraction buer pH 7.5. Washed myo®brils were used to determine the sarcomere length using the ®lar micrometer method as described by Cross, West and Dutson (1980±1981). 2.5. SDS-PAGE and Western blotting Samples were minced and homogenised in three volumes of extraction buer (100 mM Tris/HCl, pH 8.3, 5 mM EDTA, 4 C) using a polytron. The homogenate was centrifuged at 1500 g for 15 min. Five volumes of the supernatant were mixed with one volume 6SDS-PAGE sample buer (0.35 M Tris/HCl, pH 6.8, 10% SDS, 5% MCE, 0.01% bromophenol blue) and heated in a boiling waterbath for 3 min. The pellet was washed three times with three volumes extraction buer (pH 7.5), and ®ve volumes of the myo®brillar suspension were mixed with one volume 6SDS-PAGE sample buer and heated in a boiling waterbath for 3 min. Protein concentration was determined according to Karlsson, Ostwald, Kabjorn
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result indicates that the stress treatment caused some depletion of the muscle glycogen stores ante mortem resulting in a decreased lactic acid production post mortem.
and Andersson (1994) and adjusted to 3 mg/ml. To evaluate the dierences between the treatment groups equal volumes of the samples representing the groups were pooled. SDS-PAGE (25 mg protein per lane) was performed according to Laemmli (1970) on 0.75 mm thick 12.5% (troponin-T, desmin and titin degradation products) or 7.5% (m-calpain) (37.5:1 ratio of acrylamide to N,N0 -methylene-bis-acrylamide) separating gels. Western blotting was performed as described by Geesink and Koohmaraie (1999) using mouse anti-titin (9D10), mouse anti-troponin-T (CT3), mouse anti-desmin (D3), and mouse anti-m-calpain (9A4H8D3).
3.2. Tenderness Shear force was determined at 2 days and 6 weeks post mortem because this re¯ects the time the meat may enter the domestic and overseas markets, respectively. At 2 days post mortem a large variation in shear force was observed ranging from 4.1 to 19.4 kgF. This variation was observed regardless of the treatments with dierences between the lowest and highest shear forces ranging between 11.3 and 14.4 kgF. Clearly, marketing this meat at 2 days post mortem would have resulted in a product with a large inconsistency in tenderness. Six weeks of post mortem storage resulted in a uniformly tender product with shear forces ranging from 1.5 to 5.3 kgF. Electrical stimulation improved tenderness at 2 days post mortem, but not after 6 weeks of post mortem storage (Table 2). The stress treatment did not result in dierences in tenderness (Table 2). In agreement with the results of Purchas (1990) and Devine, Graafhuis, Muir and Chrystall (1993), muscles with an ultimate pH between 5.8 and 6.1 exhibited the highest shear force at 2 days post mortem (Table 3). However, after 6 weeks of storage this situation was reversed. This observation supports the conclusion of Watanabe, Daly and Devine (1996) that the increased toughness of meat with an ultimate pH between 5.8 and 6.2 is due to a slow rate of tenderisation. Although the stress treatment did not result in a signi®cant dierence in tenderness at 2 days post mortem, 32.5% of muscles from the stressed animals had a pH between 5.8 and 6.1 versus 15% of the control muscles. Thus, the stress treatment increased the percentage of muscles with an ultimate pH associated with undesirable tenderness and colour characteristics (Purchas, 1990; Warris, 1995).
2.6. Analysis of data Statistical analysis was by a Minitab version 10 computer package. Mean comparison was performed using Students t-test. 3. Results 3.1. pH and temperature Electrical stimulation accelerated the pH-decline to the extent that the pH of stimulated muscles was signi®cantly lower than the non-stimulated muscle up to 24 h post mortem (Table 1). According to Bendall (1972), cold-shortening, and thus toughening, of muscles may occur when the muscle pH is above 6.0 when the temperature drops below 10±12 C. At 6 h post mortem, the muscle temperature was 10.9 C and the pH of the nonstimulated muscles was about 6.4 (Table 1), indicating that the non-stimulated muscles where at risk of coldshortening. The stress treatment resulted in a signi®cant increase in pH at several times post mortem which persisted as a trend (P< 0.10) at 48 h post mortem (Table 1). This
Table 1 Temperature decline and the eect of electrical stimulation and pre-slaughter stress on pH decline of lamb m. longissimus (n=80)a Hours post mortem
1 2 3 6 10 13 24 48 a
Temperature ( C)
30.2 25.1 21.4 10.9 2.9 1.4 1.0 0.4
SEM
0.11 0.10 0.09 0.11 0.06 0.04 0.01 0.02
pH
SEM
Stimulated
Non-stimulated
6.20b 6.00b 5.87b 5.74b 5.74b 5.72b 5.80b 5.73
6.74a 6.75a 6.58a 6.39a 6.26a 6.07a 5.88a 5.78
Means within a main eect with diering letters are signi®cantly dierent (P< 0.05). NS, not signi®cant (P>0.05). *Signi®cant (P<0.05).
b
0.04 0.02 0.03 0.03 0.03 0.02 0.02 0.02
pH Stressed
Relaxed
6.54 6.43a 6.27 6.10 6.05a 5.93a 5.88a 5.78
6.40 6.32b 6.18 6.04 5.95b 5.86b 5.79b 5.72
SEM
Interaction
0.06 0.06 0.06 0.06 0.05 0.03 0.02 0.02
* NSb NS NS NS NS NS NS
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Table 2 The eect of electrical stimulation and pre-slaughter stress on shear force lamb m. longissimus (n=80) Item
Days post mortem
Shear force (kgF) a b
2 42
Stimulation
SEM
Stimulated
Non-stimulated
9.0ba 3.2a
11.4a 2.8b
0.5 0.1
Stress Stressed
Relaxed
9.8 3.0
10.4 3.1
SEM
Interaction
0.6 0.1
NSb NS
Means within a main eect with diering letters are signi®cantly dierent (P<0.05). NS, not signi®cant (P>0.05).
Table 3 The eect of ultimate pH on shear force of lamb m. longissimus pH range
5.8±6.1 5.7±5.8 5.6±5.7
na
20 34 25
Shear force (kgF) day 2
SEM
day 42
SEM
12.22ab 9.79b 9.12b
0.68 0.50 0.83
2.66b 3.00ab 3.22a
0.16 0.18 0.15
a A sample with an ultimate pH of 6.5 was not included in this table. b Means with diering letters are signi®cantly dierent (P<0.05).
3.3. Tenderness and related attributes of selected lamb m. longissimus with an ultimate pH < 5.8 The mean ultimate pH of the selected muscles in the treatment groups varied from 5.70 to 5.75 for the stimulated-stressed, and non-stimulated-stressed groups, respectively. As indicated earlier, the cooling conditions were such that the non-stimulated muscles were at risk of cold-shortening. This is supported by the lower sarcomere lengths in the non-stimulated muscles (Table 4). Sarcomere length was signi®cantly correlated with shear force at 2 days post mortem (r=ÿ0.42; P< 0.05). The increased shortening in the non-stimulated muscles may thus explain their increased toughness at 2 days post mortem. Calpastatin activity at 2 days post mortem was not aected by stimulation or pre-slaughter stress (Table 4).
However, in agreement with the results reviewed by Koohmaraie, Killefer, Bishop, Shackelford, Wheeler and Arbona (1995), the variation in calpastatin activity explained a signi®cant part of the variation in shear force at 2 days post mortem (R2=0.31, P< 0.001). Degradation of calpastatin by m-calpain is probably responsible for the reduction in calpastatin activity during post mortem storage of muscle (Doumit & Koohmaraie, 1999). The calpastatin activity results, therefore, indicate that neither of the treatments signi®cantly aected the activity of m-calpain. This conclusion is supported by Western blots of calpain substrates and m-calpain (Figs. 1 and 2). At 2 days post mortem no obvious dierences were observed in degradation of titin, troponin-T and desmin. Autolysis of m-calpain had progressed to a similar level in all treatments. This result is in agreement with the results on degradation of calpain substrates if the extent of mcalpain autolysis is interpreted as a measure of its activation. A more detailed analysis of m-calpain autolysis was performed on the samples with shear forces closest to the mean of the group (Fig. 2). The autolysis patterns con®rmed that, although there were dierences in the extent of autolysis of individual samples, no obvious dierence was observed between the treatment groups. Taken together, the calpastatin activity and Western blotting results indicate that m-calpain activity was not aected by the treatments and that the dierence in shear force at 2 days post mortem between stimulated and non-stimulated muscles was not due to a dierence in the extent of post mortem proteolysis.
Table 4 The eect of electrical stimulation and pre-slaughter stress on tenderness and related attributes of selected lamb m. longissimus with an ultimate pH below 5.8 (n=40)c Item
Shear force (kgF) Calpastatin activity Sarcomere length (mm) a
Days post mortem
2 42 2 2
Stimulation
SEM
Stimulated
Non-stimulated
8.0ba 3.0 4.3 1.78a
9.8a 2.9 4.5 1.69b
Means within a main eect with diering letters are signi®cantly dierent (P<0.05). NS, not signi®cant (P>0.05). *Signi®cant (P<0.05).
b
0.6 0.2 0.1 0.01
Stress Stressed
Relaxed
8.7 2.9 4.3 1.73
9.3 3.0 4.5 1.75
SEM
Interaction
0.6 0.2 0.1 0.02
NSb NS NS NS
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Fig. 1. Western blot analysis of titin degradation products in the soluble muscle fraction (A), desmin (B), troponin-T (C), and autolysis of m-calpain (D) after 2 and 42 days post mortem storage of lamb m. longissimus. Lanes 1±4: pooled samples from relaxed/stimulated, relaxed/non-stimulated, stressed/non-stimulated, and stressed/stimulated groups, respectively. A protein that probably cross-reacted with the primary antibody.
At 42 days post mortem, no dierences in shear force were observed (Table 4), and post mortem proteolysis was extensive in all groups (Fig. 1). A large number of titin degradation products were detected in the soluble muscle fraction, desmin was completely degraded, and troponin-T was largely degraded. In agreement with the results of Geesink and Koohmaraie (1999), autolysis of the large subunit of m-calpain did not appear to proceed beyond the 76 kDa autolysis product. The reason for this is that partly autolysed m-calpain is unstable under
post mortem muscle conditions and, as a result, loses its activity at this stage of autolysis (Geesink & Koohmaraie, in press). The uniform tenderness after 42 days of post mortom storage (Table 4) indicates that the extent of post mortem proteolysis was sucient to overcome the toughening eect of shortening in the non-stimulated muscles. Additionally, the similar extent of post mortem proteolysis at 42 post mortem supports the conclusion that neither of the treatments aected m-calpain activity.
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Fig. 2. Western blot analysis of m-calpain autolysis in lamb longissimus after 2 days of post mortem storage. Samples represent muscles with shear forces closest to the mean of the groups. Blots A±D: relaxed/non-stimulated, relaxed/stimulated, stressed/non-stimulated, and stressed/stimulated, respectively.
3.4. General discussion The present results apparently contradict the results of earlier studies which used the same stress treatment (Bickerstae et al., 1996; Morton et al., 1997) in the sense that there was no eect of pre-slaughter stress on tenderness independent of ultimate pH. However, in the earlier studies three levels of stress were used. In the non-stressed animals the customary swim wash upon arrival to the plant the day before slaughter was omitted. In the present experiment all animals were swim washed the day before slaughter. It is unclear why omission of swim washing the day before slaughter would result in an improved tenderness, but it is doubtful that this eect justi®es reduction of hygiene requirements. Low stress and moderate stress treatments in the cited studies were similar to the treatments in the present experiment. Nevertheless, in both of the cited studies a small, but signi®cant, dierence in tenderness of the longissimus between the low and moderately stressed animals was observed independent of ultimate pH. The reason for the discrepancy between the present and cited results is unclear, but dierences in the response to stress between breeds may have been a factor. Similar to the present study, Bray, Graafhuis and Chrystall (1989) used Coopworth lambs to investigate the eect of dierent stress factors on tenderness. Although the dierent factors, or combinations thereof, did have an eect on ultimate pH of the muscles, tenderness was not aected. Regarding the eect of electrical stimulation on post mortem proteolysis, either no eect (Geesink, Smulders, van Laack, van der Kolk, Wensing & Breukink, 1993; Ho, Stromer, Rouse & Robson, 1997), or accelerated
proteolysis (Geesink, van Laack, Barnier & Smulders, 1994; Ho, Stromer & Robson, 1996; Uytterhaegen, Claeys & Demeyer, 1992) has been reported. This discrepancy in results may be explained by dierences in stimulation intensity and subsequent chilling conditions. Activation of m-calpain in post mortem muscle as a result of the rise in intracellular Ca2+ has been estimated to occur when the muscle pH drops to about 6.2 (Drans®eld, Etherington & Taylor, 1992), and tenderisation of bovine sternomandibularis has been estimated to increase 2.4-fold with a 10 C rise in temperature (Davey & Gilbert, 1976). This information implies that accelerated proteolysis as a result of electrical stimulation is most pronounced when there is a relatively large dierence in the rate of pH fall between stimulated and non-stimulated muscles and when carcasses are chilled slowly. No temperature and pH data were reported by Ho et al. (1996, 1997), but in the present and previous studies (Geesink et al., 1993, 1994; Uyterhaegen et al., 1992) carcasses were eectively stimulated. However, the studies that found an accelerating eect of stimulation on post mortem proteolysis used slow chilling, whereas the ones that did not observe this eect used rapid chilling. Despite the fact that the present study does not shed more light on the reported eect of stress, independent of ultimate pH, on tenderness and its interaction with electrical stimulation, some conclusions can be drawn. The present results con®rm that an intermediate ultimate muscle pH (5.8±6.1) is associated with increased toughness after limited post mortem storage. Graafhuis and Devine (1994) found that 30% of lamb in New Zealand exhibit an ultimate muscle pH above 5.8 and that 50% of the variation in the ultimate pH can be explained by a number of stress factors. These ®ndings indicate that minimizing pre-slaughter stress potentially has considerable bene®ts for meat quality. Regardless of the use of electrical stimulation, a large variation in tenderness was observed after 2 days of post mortem storage, but not after extended storage. This observation stresses the importance of ageing in the production of uniformly tender meat.
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Eects of stress and high voltage electrical stimulation on tenderness of lamb m. longissimus G.H. Geesink *, M.H.D. Mareko, J.D. Morton, R. Bickerstae Molecular Biotechnology Group, Animal and Food Sciences Division, Lincoln University, PO Box 84, Canterbury, New Zealand Received 19 May 2000; received in revised form 9 August 2000; accepted 9 August 2000
Abstract This study was conducted to investigate the reported eect of pre-slaughter stress on meat tenderness independent from its eect on ultimate pH, and its interaction with electrical stimulation. From a group of 80 Coopworth lamb, 40 were stressed by subjecting the animals to a swim wash 3 h before slaughter and the use of dogs to assemble the animals to the access ramp of the abbatoir. Half of the carcasses of each group was electrically stimulated within 30 min post mortem. Temperature and pH decline of the longissimus was monitored and shear force of the cooked muscle was determined at 2 days post mortem and after 6 weeks vacuum storage at 1 C. To investigate an eect of stress independent of ultimate pH, 10 muscles with an ultimate pH below 5.8 were selected from each group for detailed analysis. This analysis consisted of determination of calpastatin activity and sarcomere length, and immunoblotting of m-calpain and calpain substrates. The stress treatment led to an increase in the number of muscles with an ultimate pH above 5.8 (32.5 vs 15%), and muscles with an ultimate pH above 5.8 were signi®cantly tougher than muscles with an ultimate pH below 5.8 at 2 days post mortem. Electrical stimulation improved tenderness at two days post mortem. This eect could be attributed to an eect on muscle contraction, but not on post mortem proteolysis of calpain substrates. A large variation in tenderness at 2 days post mortem was observed and this was not reduced by electrical stimulation. Six weeks of vacuum storage resulted in a 6 kgF drop in mean shear force and a uniformly tender product. Despite the fact that the stress treatment was similar to those in earlier studies, we failed to observe an eect of stress independent of ultimate pH on tenderness. The reason for this is unclear, but dierences in the response to stress between breeds may be responsible. The results of the present study underscore the importance of minimizing pre-slaughter stress and adequate post mortem storage for meat quality. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Stress; Electrical stimulation; Lamb; Tenderness
1. Introduction Stressing meat animals before slaughter may aect meat quality in several ways. Pre-slaughter stress may lead to increased bruising and depletion of muscle glycogen stores. The latter results in meat with an elevated ultimate pH which negatively aects meat colour, ¯avour and keeping quality (Lister, Gregory & Warris, 1981). In addition, an ultimate pH of meat between 5.8 and 6.2 is associated with increased toughnes (Purchas, 1990). Another quality aspect related to treatment of animals before slaughter stems from animal welfare concerns. Many consumers are aware of the ethics of meat * Corresponding author. Tel.: +64-3-325-2811, ext. 8172; fax: +64-3-325-3851. E-mail address: [email protected] (G.H. Geesink).
production and want their meat to be produced in a way which takes animal welfare into account (Warris, 1995). However, developments in the meat industry may have increased the incidence of pre-slaughter stress. The meat industry has become centralised into fewer, larger plants. Consequently, the distance animals have to be transported has increased. The large plants often operate at higher speeds compared to smaller plants. The need to move and process animals rapidly may increase stress in the period immediately before slaughter. Apart from a toughening eect of stress through an increase in ultimate pH, a toughening eect independent of ultimate pH has been observed (Bickerstae, Morton, Daly & Keeley, 1996; Daly, Simmons & Devine, 1995; Morton, Bickerstae, Le Couteur & Keeley, 1997). In addition, this eect was more pronounced when low voltage electrical stimulation was used to immobilize the
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00101-7
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animals, suggesting an interaction between stress and electrical stimulation. In both studies, high voltage electrical stimulation (HVES) was applied to all carcasses within 30 min after stunning, but it was not determined whether there was an interaction of stress and HVES on meat tenderness. Also, in both studies it was not determined whether the dierences in tenderness were due to dierences in contraction, post mortem proteolysis, or a combination of these factors. The purpose of the present study was to further investigate the possibility of a toughening eect of pre-slaughter stress independent of ultimate pH and its possible interaction with HVES. 2. Material and methods 2.1. Reagents All chemicals were analytical grade obtained from BDH Laboratory Supplies, Poole, Dorset, UK and BioRad Laboratories, Hercules, CA, USA. Anti-m-calpain (9A4H8D3) was obtained from Anity Bioreagents, Inc., Golden, CO, USA. Anti-titin (9D10) was developed by M. Greaser, anti-troponin-T (CT3) was developed by J.J.C. Lin and anti-desmin (D3) was developed by D.A. Fishman. These antibodies were obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract N01-HD-7-3263 from the NICHD. Alkaline phosphatase conjugated anti-mouse IgG was obtained from Sigma (St Louis, MO). 2.2. Animals, carcass measurements and sampling From a group of 80 Coopworth lambs (12 months of age, pasture fed), 40 were stressed by subjecting the animals to a swim-wash through a 15 m cold water bath 3 h before slaughter and using dogs during the 15 min needed to assemble the animals to the access ramp. The remaining 40 were not subjected to a swim wash, and the animals were assembled without the use of dogs. The lambs were electrically stunned, immobilised by spinal discharge, exsanguinated, and dressed in accordance with standard New Zealand industry practices (carcass weight 16.31.4 kg). Within 30 min post mortem half of the carcasses within each group was subjected to high voltage electrical stimulation (ES: 1130 V, 14.3 Hz, 90 s). At about 1 h post mortem the longissimus of the right carcass side was exposed at the 12th rib by knife dorsal surface cuts. The pH and temperature of the muscles were measured through this cut at several occasions over a 24 h period. The pH and temperature were measured using an Orion 8163 glass electrode and a temperature probe attached to a Hanna HI 9025 portable
pH meter. The carcasses were held at 8 C for 1 h and then transferred to a 0 C chiller. At 2 days post mortem, the carcasses were transferred to the cutting room and the longissimus was cut from the carcass. The muscle from the right carcass side was used to evaluate tenderness and related attributes at 2 days post mortem. The muscle from the left carcass side was sampled after 6 weeks of vacuum storage at 1 C. 2.3. Shear force measurements Samples for shear force measurements were stored frozen at ÿ30 C and cooked after thawing overnight at 2 C. Samples were cooked individually in plastic bags immersed in a water bath at 80 C until they reached an internal temperature of 75 C as measured using Fluke type K temperature probes attached to Fluke 52 meters. The cooked meat was cooled on ice and six to eight pieces of meat were removed parallel to the muscle ®bres. Test pieces were placed separately in the MIRINZ tenderometer (Chrystall & Devine, 1991) and the shear force (kgF) to cut across the ®bres was determined. 2.4. Calpastatin activity and sarcomere length Samples for calpastatin activity determinations were prepared according to Shackelford, Koohmaraie, Cundi, Gregory, Rohrer and Savell (1994) with minor modi®cations. Calpastatin activity was determined according to Koohmaraie (1990). Minced muscle was homogenised in three volumes extraction buer (100 mM Tris/HCl, pH 8.3, 5 mM EDTA, 0.05% 2-mercaptoethanol, 4 C) using a polytron. The homogenate was centrifuged at 1500 gmax and 5 ml of the supernatant was collected for calpastatin activity determination. The remaining supernatant was discarded and the pellet was resuspended and centrifuged twice with extraction buer pH 7.5. Washed myo®brils were used to determine the sarcomere length using the ®lar micrometer method as described by Cross, West and Dutson (1980±1981). 2.5. SDS-PAGE and Western blotting Samples were minced and homogenised in three volumes of extraction buer (100 mM Tris/HCl, pH 8.3, 5 mM EDTA, 4 C) using a polytron. The homogenate was centrifuged at 1500 g for 15 min. Five volumes of the supernatant were mixed with one volume 6SDS-PAGE sample buer (0.35 M Tris/HCl, pH 6.8, 10% SDS, 5% MCE, 0.01% bromophenol blue) and heated in a boiling waterbath for 3 min. The pellet was washed three times with three volumes extraction buer (pH 7.5), and ®ve volumes of the myo®brillar suspension were mixed with one volume 6SDS-PAGE sample buer and heated in a boiling waterbath for 3 min. Protein concentration was determined according to Karlsson, Ostwald, Kabjorn
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result indicates that the stress treatment caused some depletion of the muscle glycogen stores ante mortem resulting in a decreased lactic acid production post mortem.
and Andersson (1994) and adjusted to 3 mg/ml. To evaluate the dierences between the treatment groups equal volumes of the samples representing the groups were pooled. SDS-PAGE (25 mg protein per lane) was performed according to Laemmli (1970) on 0.75 mm thick 12.5% (troponin-T, desmin and titin degradation products) or 7.5% (m-calpain) (37.5:1 ratio of acrylamide to N,N0 -methylene-bis-acrylamide) separating gels. Western blotting was performed as described by Geesink and Koohmaraie (1999) using mouse anti-titin (9D10), mouse anti-troponin-T (CT3), mouse anti-desmin (D3), and mouse anti-m-calpain (9A4H8D3).
3.2. Tenderness Shear force was determined at 2 days and 6 weeks post mortem because this re¯ects the time the meat may enter the domestic and overseas markets, respectively. At 2 days post mortem a large variation in shear force was observed ranging from 4.1 to 19.4 kgF. This variation was observed regardless of the treatments with dierences between the lowest and highest shear forces ranging between 11.3 and 14.4 kgF. Clearly, marketing this meat at 2 days post mortem would have resulted in a product with a large inconsistency in tenderness. Six weeks of post mortem storage resulted in a uniformly tender product with shear forces ranging from 1.5 to 5.3 kgF. Electrical stimulation improved tenderness at 2 days post mortem, but not after 6 weeks of post mortem storage (Table 2). The stress treatment did not result in dierences in tenderness (Table 2). In agreement with the results of Purchas (1990) and Devine, Graafhuis, Muir and Chrystall (1993), muscles with an ultimate pH between 5.8 and 6.1 exhibited the highest shear force at 2 days post mortem (Table 3). However, after 6 weeks of storage this situation was reversed. This observation supports the conclusion of Watanabe, Daly and Devine (1996) that the increased toughness of meat with an ultimate pH between 5.8 and 6.2 is due to a slow rate of tenderisation. Although the stress treatment did not result in a signi®cant dierence in tenderness at 2 days post mortem, 32.5% of muscles from the stressed animals had a pH between 5.8 and 6.1 versus 15% of the control muscles. Thus, the stress treatment increased the percentage of muscles with an ultimate pH associated with undesirable tenderness and colour characteristics (Purchas, 1990; Warris, 1995).
2.6. Analysis of data Statistical analysis was by a Minitab version 10 computer package. Mean comparison was performed using Students t-test. 3. Results 3.1. pH and temperature Electrical stimulation accelerated the pH-decline to the extent that the pH of stimulated muscles was signi®cantly lower than the non-stimulated muscle up to 24 h post mortem (Table 1). According to Bendall (1972), cold-shortening, and thus toughening, of muscles may occur when the muscle pH is above 6.0 when the temperature drops below 10±12 C. At 6 h post mortem, the muscle temperature was 10.9 C and the pH of the nonstimulated muscles was about 6.4 (Table 1), indicating that the non-stimulated muscles where at risk of coldshortening. The stress treatment resulted in a signi®cant increase in pH at several times post mortem which persisted as a trend (P< 0.10) at 48 h post mortem (Table 1). This
Table 1 Temperature decline and the eect of electrical stimulation and pre-slaughter stress on pH decline of lamb m. longissimus (n=80)a Hours post mortem
1 2 3 6 10 13 24 48 a
Temperature ( C)
30.2 25.1 21.4 10.9 2.9 1.4 1.0 0.4
SEM
0.11 0.10 0.09 0.11 0.06 0.04 0.01 0.02
pH
SEM
Stimulated
Non-stimulated
6.20b 6.00b 5.87b 5.74b 5.74b 5.72b 5.80b 5.73
6.74a 6.75a 6.58a 6.39a 6.26a 6.07a 5.88a 5.78
Means within a main eect with diering letters are signi®cantly dierent (P< 0.05). NS, not signi®cant (P>0.05). *Signi®cant (P<0.05).
b
0.04 0.02 0.03 0.03 0.03 0.02 0.02 0.02
pH Stressed
Relaxed
6.54 6.43a 6.27 6.10 6.05a 5.93a 5.88a 5.78
6.40 6.32b 6.18 6.04 5.95b 5.86b 5.79b 5.72
SEM
Interaction
0.06 0.06 0.06 0.06 0.05 0.03 0.02 0.02
* NSb NS NS NS NS NS NS
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Table 2 The eect of electrical stimulation and pre-slaughter stress on shear force lamb m. longissimus (n=80) Item
Days post mortem
Shear force (kgF) a b
2 42
Stimulation
SEM
Stimulated
Non-stimulated
9.0ba 3.2a
11.4a 2.8b
0.5 0.1
Stress Stressed
Relaxed
9.8 3.0
10.4 3.1
SEM
Interaction
0.6 0.1
NSb NS
Means within a main eect with diering letters are signi®cantly dierent (P<0.05). NS, not signi®cant (P>0.05).
Table 3 The eect of ultimate pH on shear force of lamb m. longissimus pH range
5.8±6.1 5.7±5.8 5.6±5.7
na
20 34 25
Shear force (kgF) day 2
SEM
day 42
SEM
12.22ab 9.79b 9.12b
0.68 0.50 0.83
2.66b 3.00ab 3.22a
0.16 0.18 0.15
a A sample with an ultimate pH of 6.5 was not included in this table. b Means with diering letters are signi®cantly dierent (P<0.05).
3.3. Tenderness and related attributes of selected lamb m. longissimus with an ultimate pH < 5.8 The mean ultimate pH of the selected muscles in the treatment groups varied from 5.70 to 5.75 for the stimulated-stressed, and non-stimulated-stressed groups, respectively. As indicated earlier, the cooling conditions were such that the non-stimulated muscles were at risk of cold-shortening. This is supported by the lower sarcomere lengths in the non-stimulated muscles (Table 4). Sarcomere length was signi®cantly correlated with shear force at 2 days post mortem (r=ÿ0.42; P< 0.05). The increased shortening in the non-stimulated muscles may thus explain their increased toughness at 2 days post mortem. Calpastatin activity at 2 days post mortem was not aected by stimulation or pre-slaughter stress (Table 4).
However, in agreement with the results reviewed by Koohmaraie, Killefer, Bishop, Shackelford, Wheeler and Arbona (1995), the variation in calpastatin activity explained a signi®cant part of the variation in shear force at 2 days post mortem (R2=0.31, P< 0.001). Degradation of calpastatin by m-calpain is probably responsible for the reduction in calpastatin activity during post mortem storage of muscle (Doumit & Koohmaraie, 1999). The calpastatin activity results, therefore, indicate that neither of the treatments signi®cantly aected the activity of m-calpain. This conclusion is supported by Western blots of calpain substrates and m-calpain (Figs. 1 and 2). At 2 days post mortem no obvious dierences were observed in degradation of titin, troponin-T and desmin. Autolysis of m-calpain had progressed to a similar level in all treatments. This result is in agreement with the results on degradation of calpain substrates if the extent of mcalpain autolysis is interpreted as a measure of its activation. A more detailed analysis of m-calpain autolysis was performed on the samples with shear forces closest to the mean of the group (Fig. 2). The autolysis patterns con®rmed that, although there were dierences in the extent of autolysis of individual samples, no obvious dierence was observed between the treatment groups. Taken together, the calpastatin activity and Western blotting results indicate that m-calpain activity was not aected by the treatments and that the dierence in shear force at 2 days post mortem between stimulated and non-stimulated muscles was not due to a dierence in the extent of post mortem proteolysis.
Table 4 The eect of electrical stimulation and pre-slaughter stress on tenderness and related attributes of selected lamb m. longissimus with an ultimate pH below 5.8 (n=40)c Item
Shear force (kgF) Calpastatin activity Sarcomere length (mm) a
Days post mortem
2 42 2 2
Stimulation
SEM
Stimulated
Non-stimulated
8.0ba 3.0 4.3 1.78a
9.8a 2.9 4.5 1.69b
Means within a main eect with diering letters are signi®cantly dierent (P<0.05). NS, not signi®cant (P>0.05). *Signi®cant (P<0.05).
b
0.6 0.2 0.1 0.01
Stress Stressed
Relaxed
8.7 2.9 4.3 1.73
9.3 3.0 4.5 1.75
SEM
Interaction
0.6 0.2 0.1 0.02
NSb NS NS NS
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Fig. 1. Western blot analysis of titin degradation products in the soluble muscle fraction (A), desmin (B), troponin-T (C), and autolysis of m-calpain (D) after 2 and 42 days post mortem storage of lamb m. longissimus. Lanes 1±4: pooled samples from relaxed/stimulated, relaxed/non-stimulated, stressed/non-stimulated, and stressed/stimulated groups, respectively. A protein that probably cross-reacted with the primary antibody.
At 42 days post mortem, no dierences in shear force were observed (Table 4), and post mortem proteolysis was extensive in all groups (Fig. 1). A large number of titin degradation products were detected in the soluble muscle fraction, desmin was completely degraded, and troponin-T was largely degraded. In agreement with the results of Geesink and Koohmaraie (1999), autolysis of the large subunit of m-calpain did not appear to proceed beyond the 76 kDa autolysis product. The reason for this is that partly autolysed m-calpain is unstable under
post mortem muscle conditions and, as a result, loses its activity at this stage of autolysis (Geesink & Koohmaraie, in press). The uniform tenderness after 42 days of post mortom storage (Table 4) indicates that the extent of post mortem proteolysis was sucient to overcome the toughening eect of shortening in the non-stimulated muscles. Additionally, the similar extent of post mortem proteolysis at 42 post mortem supports the conclusion that neither of the treatments aected m-calpain activity.
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Fig. 2. Western blot analysis of m-calpain autolysis in lamb longissimus after 2 days of post mortem storage. Samples represent muscles with shear forces closest to the mean of the groups. Blots A±D: relaxed/non-stimulated, relaxed/stimulated, stressed/non-stimulated, and stressed/stimulated, respectively.
3.4. General discussion The present results apparently contradict the results of earlier studies which used the same stress treatment (Bickerstae et al., 1996; Morton et al., 1997) in the sense that there was no eect of pre-slaughter stress on tenderness independent of ultimate pH. However, in the earlier studies three levels of stress were used. In the non-stressed animals the customary swim wash upon arrival to the plant the day before slaughter was omitted. In the present experiment all animals were swim washed the day before slaughter. It is unclear why omission of swim washing the day before slaughter would result in an improved tenderness, but it is doubtful that this eect justi®es reduction of hygiene requirements. Low stress and moderate stress treatments in the cited studies were similar to the treatments in the present experiment. Nevertheless, in both of the cited studies a small, but signi®cant, dierence in tenderness of the longissimus between the low and moderately stressed animals was observed independent of ultimate pH. The reason for the discrepancy between the present and cited results is unclear, but dierences in the response to stress between breeds may have been a factor. Similar to the present study, Bray, Graafhuis and Chrystall (1989) used Coopworth lambs to investigate the eect of dierent stress factors on tenderness. Although the dierent factors, or combinations thereof, did have an eect on ultimate pH of the muscles, tenderness was not aected. Regarding the eect of electrical stimulation on post mortem proteolysis, either no eect (Geesink, Smulders, van Laack, van der Kolk, Wensing & Breukink, 1993; Ho, Stromer, Rouse & Robson, 1997), or accelerated
proteolysis (Geesink, van Laack, Barnier & Smulders, 1994; Ho, Stromer & Robson, 1996; Uytterhaegen, Claeys & Demeyer, 1992) has been reported. This discrepancy in results may be explained by dierences in stimulation intensity and subsequent chilling conditions. Activation of m-calpain in post mortem muscle as a result of the rise in intracellular Ca2+ has been estimated to occur when the muscle pH drops to about 6.2 (Drans®eld, Etherington & Taylor, 1992), and tenderisation of bovine sternomandibularis has been estimated to increase 2.4-fold with a 10 C rise in temperature (Davey & Gilbert, 1976). This information implies that accelerated proteolysis as a result of electrical stimulation is most pronounced when there is a relatively large dierence in the rate of pH fall between stimulated and non-stimulated muscles and when carcasses are chilled slowly. No temperature and pH data were reported by Ho et al. (1996, 1997), but in the present and previous studies (Geesink et al., 1993, 1994; Uyterhaegen et al., 1992) carcasses were eectively stimulated. However, the studies that found an accelerating eect of stimulation on post mortem proteolysis used slow chilling, whereas the ones that did not observe this eect used rapid chilling. Despite the fact that the present study does not shed more light on the reported eect of stress, independent of ultimate pH, on tenderness and its interaction with electrical stimulation, some conclusions can be drawn. The present results con®rm that an intermediate ultimate muscle pH (5.8±6.1) is associated with increased toughness after limited post mortem storage. Graafhuis and Devine (1994) found that 30% of lamb in New Zealand exhibit an ultimate muscle pH above 5.8 and that 50% of the variation in the ultimate pH can be explained by a number of stress factors. These ®ndings indicate that minimizing pre-slaughter stress potentially has considerable bene®ts for meat quality. Regardless of the use of electrical stimulation, a large variation in tenderness was observed after 2 days of post mortem storage, but not after extended storage. This observation stresses the importance of ageing in the production of uniformly tender meat.
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