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J. Anim. Sci. 2002. 80:2862-2871
© 2002 American Society of Animal Science

Dietary-induced changes of muscle growth rate in pigs: Effects on in vivo and postmortem muscle proteolysis and meat quality1

L. Kristensen*,2, M. Therkildsen{dagger}, B. Riis{dagger}, M. T. Sørensen{ddagger}, N. Oksbjerg{dagger}, P. P. Purslow*,3 and P. Ertbjerg*

* Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark , and and {dagger} Department of Animal Product Quality and and {ddagger} Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Foulum, DK-8830 Tjele, Denmark

2 Correspondence:
E-mail:
lak{at}kvl.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The effects of various growth rates in pigs induced by four different feeding strategies on the activity of the calpain system and on postmortem (PM) muscle proteolysis and tenderness development were studied. An increased growth rate may be caused by an increased protein turnover, which results in up-regulated levels of proteolytic enzymes in vivo that, in turn, possibly will affect PM tenderness development. It can be hypothesized that increased proteolytic activity preslaughter will increase the PM tenderization rate. From postnatal d 28 to d 90 (phase 1) the pigs were divided into two groups, given either ad libitum (A) or restricted (R, 60% of ad libitum) access to feed. The two groups were then divided into two subgroups, given either restricted or ad libitum access to feed from d 91 to slaughter at d 165 (phase 2). Measurements of the activity of µ-calpain, m-calpain, and calpastatin; concentrations of total collagen and the percent of soluble collagen; and RNA, DNA, and elongation factor-2 where made at slaugther. Myofibrillar fragmentation index (MFI) was determined at slaughter and 24 h PM. Warner-Braztler shear force was determined 1 d and 4 d PM. Pigs fed restricted diets in phase 1 and fed ad libitum in phase 2 (RA pigs) had increased growth rates in the last phase compared to pigs fed ad libitum during both phase 1 and phase 2 (AA pigs). The increased growth rate (compensatory growth) was followed by an increased proteolytic potential (µ-calpain:calpastatin ratio), increased MFI values, and higher tenderization rates. There was a positive correlation between the activities of m-calpain and growth rates (r = 0.35, P = 0.03), and between RNA levels and growth rates (r = 0.43, P = 0.006). The proposed hypothesis is largely supported by the results. The activities of both µ- and m-calpain at slaughter were highest in fast-growing pigs. The calpain activity was highest in RA pigs, which in turn also had the fastest growth rates prior to slaughter among the four groups. This implies that the synthesis of these enzymes was up-regulated during the second feeding period to a larger extent in RA pigs. The proteolytic potential and the MFI values indicate that the up-regulated in vivo calpain activity had an effect on PM protein degradation, which also is supported by the higher tenderization rate in RA pigs.

Key Words: Calpain • Growth • Pork • Proteolysis • RNA • Tenderness


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
It has been known for decades that accelerated growth rates occur in pigs having free access to feed following a period of restricted feeding (McMeekan, 1940; Prince et al., 1983; Mersmann et al., 1987). The accelerated growth rate is termed "compensatory growth," and may be related to the severity of the feed restriction and the length of the restriction period and the following ad libitum feeding period. The compensatory growth response is interesting from a meat quality aspect, because an increased in vivo protein turnover may be a result of upregulated levels of proteolytic enzymes that, in turn, possibly will affect the postmortem (PM) tenderness development.

The growth rate is principally determined by the ratio between the rates of protein synthesis and degradation, as outlined in Figure 1Go. Subsequent to slaughter, protein synthesis stops, but some protein degradation processes will continue. It can be hypothesized that an increased proteolytic activity preslaughter will affect the PM tenderization rate (i.e., high proteolytic activity preslaughter leads to a fast tenderization rate (Figure 1Go). If this hypothesis is correct, then compensatory growth could be a way to optimize the tenderness development in meat-producing animals.



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  		Figure 1. Hypothesis for the relation between feeding intensity, in vivo protein turnover, and postmortem tenderization rate.

 
The calpain system has been proposed as the primary proteolytic enzyme system responsible for the tenderization process (Koohmaraie et al., 1986; Huff-Lonergan et al., 1996). In live animals, calpains are suggested to be involved in muscle growth by initiating myofibrillar protein degradation (Dayton et al., 1975; Goll et al., 1998). It is therefore likely that an increased muscle growth rate in vivo may affect the tenderization rate by increasing proteolytic potential, as hypothesized in Figure 1Go.

In this study, our objective was to evaluate the variation in muscle growth rate on the calpain system and the tenderness development due to varying feeding strategies during growing and finishing phases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals and Diets

Ten litters of four female pigs (Danish Landrace x Danish Yorkshire) were within litter, allocated to four treatment groups ensuring that the average weight of the animals was the same across treatments. From weaning at postnatal d 28 to d 90 (phase 1) the pigs were given either ad libitum access to feed or restrictive access (60% of ad libitum feeding) as described by Oksbjerg et al. (2002). From d 91 to slaughter at d 165 (phase 2), the pigs were divided into two subgroups given either ad libitum (AA, RA) or restricted (AR, RR) (60% of ad libitum feeding) access to feed. From d 28 (start of experiment) to d 56, the pigs were offered a weaning diet containing 14.19 MJ of metabolic energy and 242 g of crude protein per kilogram of feed; from d 57 to d 84, a grower diet (Grower-1) containing 13.98 MJ of metabolic energy and 230 g of crude protein per kilogram of feed was given; from d 85 to d 112, a grower diet (Grower-2) containing 13.65 MJ of metabolic energy and 199 g of crude protein per kilogram of feed was provided; and from d 113 to slaughter, the pigs had access to a finishing diet containing 13.79 MJ of metabolic energy and 181 g of crude protein per kilogram of feed. The compositions of the diets are shown in Table 1Go. The pigs were raised individually in pens with partly slatted floors. Chopped straw was used as bedding. Feed intake was recorded individually.


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  		Table 1. Composition of diets used in the experiment
 
At d 90, the cross-sectional area of the longissimus dorsi (LD) muscle and the fat thickness were measured ultrasonically. The pigs were slaughtered by litter group at Research Center Foulum’s experimental slaughterhouse with CO2 stunning. Samples for collagen, RNA, DNA, m-calpain, µ-calpain, calpastatin, elongation factor-2 (eEF-2), and myofibrillar fragmentation index (MFI) were taken 15 min after slaughter from the left-side LD at first lumbar vertebra. This was immediately frozen in liquid nitrogen and afterwards stored at -80°C until analysis. The left-side semitendinosus muscle was dissected 30 min PM, weighed, and sampled for DNA and RNA. Twenty-four hours PM, the pH24 was measured in the LD at the last rib with a PHM201 pH meter (Radiometer, Copenhagen, Denmark) equipped with a Metrohm probe-type glass electrode WOC (Metrohm Ag, Herisau, Switzerland). The electrode was calibrated in pH 4.01 and 7.00 IUPAC buffers (Radiometer) equilibrated to 3°C. A section of LD (12th thoracic to 5th lumbar vertebra) from the right carcass side was removed 24 h PM and split in three parts. From the cranial end, the first part was cut in three chops and used for MFI at d 1, color measurement after 1 h of blooming, and drip-loss determination, respectively. Drip loss was measured on approximately 75 g of LD muscle using the plastic bag method described by Honikel (1998). Twenty-four hours PM, the meat color of bloomed (1 h at 3°C) pork chops was measured using a Minolta Chroma Meter CR-300 (Osaka, Japan), with a D65 light source calibrated against a white tile. Tristimulus parameters L*, a*, and b* (representing lightness, redness, and yellowness, respectively) were measured on five fixed sites on each chop surface. Parts 2 and 3, each 7 cm, were vacuum packed and used for Warner-Bratzler shear force (WBSF) determination. One chop was immediately stored at -20°C and the other was aged for additionally 3 d and afterwards stored. Total RNA and DNA were quantified according to Oksbjerg et al. (2000), with small modifications as described in Therkildsen et al. (2002b); MFI was measured according to Culler et al. (1978), with small modifications as described in Therkildsen et al. (2002a); protein content was measured according to Hansen (1989) and Sweency (1989).

Elongation Factor-2

The muscle eEF-2 was concentrated by binding to an ion-exchange matrix. Two milliliters of muscle tissue homogenate was mixed with 0.25 mL of DEAE Sephacel (Amersham Pharmacia Biotech, Uppsala, Sweden) previously equilibrated in buffer X (20 mM Tris/HCl, pH 7.6, 70 mM KCl, 14.4 mM 2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol). The column material was centrifuged (5 min, 1,500 x g) and the supernate discarded. The column material was washed twice with 10 mL of buffer X and the supernate discharged. The DEAE-bound eEF-2 was eluted by adding 0.3 mL of buffer Y (20 mM Tris/HCl, pH 7.6, 0.2 M KCl, 14.4 mM 2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol). The mixture was centrifuged (2 min, 1,500 x g) and the supernate collected for eEF-2 assay. The eEF-2 assay is described in detail elsewhere (Riis et al., 1989; Christophersen et al., 2002).

Thaw Loss, Cooking Loss, and Shear Force

Samples were thawed in vacuum bags for 24 h at 4°C using forced-air circulation and the thaw loss was determined by weighing meat and exudates. The meat was trimmed for fat and connective tissue, vacuum packed, and heat-treated for 60 min at 80°C in circulating water. Afterwards, the meat was cooled in ice water for 20 min and the cooking loss was determined by weighing the meat and exudates. Samples were kept overnight at 4°C, after which the WBSF was determined according to Møller (1981), except that a square blade was used (12 mm wide, 1.1 mm thick). An Instron testing machine (Model 4301, Instron Ltd., Buckinghamshire, England) mounted with a 1-kN load cell was used. Twelve single deformations were performed on each sample using a cross-head speed of 50 mm/min, and the mean maximum force was recorded.

Collagen Determination

The meat was analyzed for total and soluble collagen. Unless stated otherwise, all chemicals were of analytical grade and were obtained from Sigma (Sigma Chemicals Co., St. Louis, MO). Samples were partly thawed and finely chopped using a food processor. The meat (6.0 g) was mixed with 20 mL of water in a 50-mL glass centrifuge tube and heat-treated for 2 h in circulating water at 90°C. The tube was cooled to 40°C and homogenized for 1 min using an Ultra Turrax Mixer T25 at 9,500 rpm (IKA-WERKE, Stanfen, Germany), which afterwards was flushed with 10.0 mL of water (40°C). The homogenate was centrifuged for 15 min at 4,000 x g, and the supernate was filtered through paper into a second glass centrifuge tube. The filter was added to the pellet, and 30 mL of 6.0 M HCl was added to the supernate and 50 mL of 6.0 M HCl was added to the pellet. Both were then hydrolyzed overnight in a sand bath (160°C; Harry Gestigkeit GmbH type ST32, Düsseldorf). The concentration of hydroxyproline was determined according to the NMKL method described by Kolar (1990). The amount of soluble collagen was calculated from the hydroxyproline concentration in the supernatant, and the total collagen was calculated from the sum of the hydroxyproline concentration in the pellet and in the supernatant (Kolar, 1990).

Calpain Determination

The activities of µ- and m-calpain were determined by modification of the method described by Ertbjerg et al. (1999). Samples were finely chopped and 10 g was homogenized (Ultra Turrax Mixer T25, 2 x 30 s at 9,500 rpm and 2 x 30 s at 13,500 rpm, 30 s of cooling between bursts) in 60 mL of buffer (50 mM Trizma-base, 5 mM EDTA; Fluka Chemie AG, CH-9471, Buchs, Switzerland), 10 mM monothioglycerol, 1 µM leupeptin, pH 8.00). The homogenate was centrifuged for 20 min at 25,000 x g, 4°C, and the supernate was filtered through a 0.2-µm pore-size filter (Sartorius, Göttingen, Germany). One milliliter of filtrate was heated at 100°C for 3 min and stored at -80°C until analysis for total calpastatin activity. The two calpains were separated using an ion-exchange column: 20 mL of the filtrate were loaded on a Resource-Q 6-mL column (Amersham Phamacia Biotech, Uppsala, Sweden) and eluted with an NaCl gradient from 0 to 0.6 M NaCl in 20 mM Trizma-base, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5, using a run time of 20 min. Fractions eluted between 0.29 and 0.35 M NaCl were pooled and analyzed for m-calpain activity using the assay described by Ertbjerg et al. (1999). Fractions eluted between 0.08 and 0.23 M NaCl were pooled and analyzed for µ-calpain activity using the method described by Geesink and Koohmaraie (1999b). Explained briefly, the calpastatin activity in the pooled fractions was measured before and after heat inactivation of µ-calpain. The difference in calpastatin activity equals the activity of µ-calpain. Calpastatin activity was determined by the method described by Ertbjerg et al. (1999) using partially purified µ-calpain (see below). Determination of total calpastatin activity was achieved using partially purified m-calpain.

Partial Purification of µ- and m-Calpain

Calpastatin assays require the use of partially purified calpain. A pig LD was obtained 2 h PM from a local slaughterhouse. The muscle was trimmed from fat and connective tissue and 150 g was homogenized (Ultra Turrax Mixer T25, 2 x 30 s at 9,500 rpm and 2 x 30 s at 13,500 rpm, 30 s of cooling between burst) in 900 mL of buffer (50 mM Trizma-base, 5 mM EDTA, 10 mM monothioglycerol, 1 µM leupeptin, pH 8.00). The homogenate was centrifuged for 20 min at 25,000 x g, 4°C, and the supernate was filtered through a cheese cloth. The supernate was slowly added (NH4)2SO4 to a final saturation of 50% and was then kept at 0°C while slowly stirring for 30 min. The solution was centrifuged at 20,000 x g for 20 min, the supernate discharged, and the pellet was dissolved in 60 mL of buffer A (20 mM Trizma-base, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5) and afterwards dialyzed (cut-off: 12,000 to 14,000 kDa) overnight in 3 L of buffer A. The dialysat was centrifuged for 10 min at 20,000 x g and the supernate was adjusted to 0.80 M (NH4)2SO4, which was loaded on a butyl sepharose 4 fast-flow column (26 x 40 cm; Pharmacia Biotech) previously equilibrated with buffer B (0.80 M (NH4)2SO4, 20 mM Trizma-base, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5). Calpastatin and calpains were separated using a linear gradient from 100% buffer B to 100% buffer A in 90 min, and 4-mL fractions were collected and screened for calpain activity using the method described by Ertbjerg et al. (1999). Fractions containing calpain activity were pooled and ionic strength was adjusted to 5 mS by dilution with buffer A. The adjusted solution was loaded on a Resource-Q 6-mL column (Phamacia Biotech), and eluted with a NaCl gradient from 0 to 0.6 M NaCl in 20 mM Trizma-base, 1 mM EDTA, 10 mM monothioglycerol, pH 7.5, using a run time of 20 min. Fractions containing µ- or m-calpain activity were pooled and glycerol was added to a final concentration of 30% and stored at 4°C until use.

Statistical Analysis

Data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC), by means of a model including the fixed effect of treatment in phase 1 and 2 and the interaction. The experiment is a typical 2 x 2 factorial design, and is analyzed as such, with the pig being the experimental unit. The 2 x 2 factorial design is composed of two types of feeding (ad libitum and restricted) and two phases (phase 1: d 28 to d 90; phase 2: d 91 to d 165). The effect of litter type was included in the model as a random effect to remove the variation caused by the different litters. All results are presented as least squares means. In the calculation of correlations between different traits, the effect of treatment and day of analysis was removed before calculation, and thus the correlations were calculated using the residuals from a GLM procedure. As WBSF, collagen content, and calpain/calpastatin activity of pigs from one litter was analyzed in 1 d, the random effect of day of analysis was confounded with litter in the case of these traits.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
One pig was excluded from the analysis due to illness. At d 90, RA and RR pigs reached an average weight of 41.2 kg, which was lower (P < 0.001) than that in AA and AR pigs (47.8 kg) in the same phase (Table 2Go). This difference was achieved by a 5% higher fractional rate of growth (FRG) in pigs given ad libitum access to feed compared to restrictively fed pigs. The thickness of the subcutaneous fat layer and the area of LD also were lower (P < 0.001) in restrictively fed pigs at d 90. At slaughter there was no difference in live weight, meat percentage, carcass weight 24 h PM, muscle mass, subcutaneous fat, or weight of semitendinosus between AA pigs and RA pigs or between RR pigs and AR pigs. However, the differences between AA and RA pigs or AR and RR pigs in the second phase were highly significant (P < 0.001). The FRG2 for RA pigs was significantly higher than in the other groups and 12% higher than AA pigs, which also were given free access to feed in the second feeding phase.


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  		Table 2. Least square means of animal weight, fractional rate of growth (FRG), meat percentage, muscle mass, protein content, subcutaneous fat, and muscle size at d 28, d 90 and at slaughter, as affected by treatment
 
The drip loss and the cooking loss were not affected by feeding; however, the thaw loss of meat was significantly higher in RR pigs (Table 3Go). There was a difference (P = 0.02) in ultimate pH between AA pigs and AR pigs, the former being lowest. Pigs fed restrictively in phase 2 had a higher (P = 0.02) percent of soluble collagen. Neither total collagen nor the color measurements L* and b* were affected by treatment. The a* value was lower (P = 0.04) in pigs fed restrictively in the first feeding phase.


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  		Table 3. Least square means of drip loss, thaw loss, cooking loss, pH24h, total collagen, soluble collagen, and color as affected by treatment
 
The concentrations of RNA and DNA (Table 4Go) were not affected by treatment, except for the DNA level in the LD between AA pigs and RR pigs measured at slaughter, the latter being lowest (P = 0.04). The total RNA content in the ST was significantly higher in AA and RA pigs. There was a tendency to a higher (P = 0.1) ratio between RNA and DNA in the LD in pigs that had been restrictively fed in the first feeding phase. This tendency was not found in the ST.


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  		Table 4. Least square means of RNA, DNA, and elongation factor-2 (eEF-2) measured at slaughter and at d 105 as affected by treatment
 
The activity at slaughter of both µ- and m-calpain was higher (P = 0.02, 0.03) in AA and RA pigs, (Table 5Go), whereas the calpastatin activity was unaffected by treatment. The numerical values of the activity of µ- and m-calpain were highest in RA pigs. The ratio between µ-calpain and calpastatin activity was higher (P = 0.05) in RA pigs than in RR pigs. At d 0, no differences in MFI values were observed between treatment groups. The development in MFI values from d 0 to d 1 was lower (P < 0.001) in AR and RR pigs. Pigs in group RA had higher MFI values at d 1 than pigs in group AR and RR (P = 0.003 and P < 0.001 respectively). The P-value for differences between AA pigs and RA pigs was 0.14, the former being lowest. There was a highly significant difference in WBSF after 1 d of storage between AA and RA pigs and AR and RR pigs, the latter having the highest values (P < 0.001). Pigs in group RA had the lowest shear force results, with P-values for differences from AA pigs, AR pigs, and RR pigs at 0.07, 0.004, and < 0.001, respectively. After 4 d storage, AA and RA pigs had a lower (P = 0.03) shear force compared to AR and RR pigs. The P-values for differences between RA pigs and AR and RR pigs were 0.04 and 0.09, respectively. No differences in shear force were observed between AA and RA pigs after 4 d storage.


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  		Table 5. Least square means for activity of the calpain system, myofibrillar fragmentation index (MFI), and Warner-Braztler shear force (WBSF) as affected by treatment
 
The RNA level and the m-calpain activity in the LD measured at slaughter were correlated (P = 0.006 and 0.03) to FRG2, and also significantly intercorrelated (Table 6Go). No correlations were detected between µ-calpain and FRG2 or RNA.


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  		Table 6. Adjusted correlations between fractional rate of growth in the second feeding phase (FRG2), RNA concentrations, and calpain activity
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
The results presented in Table 2Go clearly show the effects of the four different feeding strategies, with a highly significant compensatory growth response in RA pigs as indicated by the FRG2. This is in agreement with several other studies showing compensatory growth in pigs following a period of restricted feeding (McMeekan, 1940; Gädeken et al., 1983; Prince et al., 1983; Mersmann et al., 1987; Critser et al., 1995; Oksbjerg et al., 2002). Pigs in treatment group AA were given ad libitum access to feed during the whole experiment, according to traditional feeding strategy in Danish pig production, and were therefore used as a control treatment. Pigs showing compensatory growth (RA pigs) did not differ significantly from control pigs (group AA) in any of the commercially important parameters, such as live weight, meat percentage, or subcutaneous fat thickness. However, there were clear differences between AA, RA pigs and AR, RR pigs in the second phase in terms of these parameters.

The physiological role of the calpain system both in muscle and in nonmuscle cells remains unclear (Goll and Thompson, 1999). However, there is evidence that supports a possible role in the initiation of the myofibrillar protein turnover, as first suggested by Dayton et al. (1975) and summarized by Goll et al. (1989, 1992, 1998). Brooks et al. (1983) found increased calpain activity after refeeding rats that had been starved up to 8 d. The calpain activity decreased upon refasting. Spencer et al. (1997) found a 90% increase in m-calpain concentration in rat muscles that had been mechanically stimulated in order to initiate muscle growth after a period of muscle inactivity. No effect was found on µ-calpain concentration. These results suggest that the remodeling of myofibrils that occurs during muscle growth requires m-calpain activity. The results presented in Table 6Go support this, since m-calpain activity in the LD at slaughter correlates positively with FRG2, and no correlation is observed for µ-calpain activity. The results in Table 6Go also suggest that the protein synthesis rate is increased in fast-growing muscles, as indicated by the positive correlation between RNA concentration in the LD and the FRG2.

To calculate the tenderization rate in a precise manner, the tenderness value at slaughter is required. However, as discussed elsewhere (Devine, 1998; Dransfield, 1998; Koohmaraie and Goll, 1998), it is highly questionable to measure tenderness before the muscle has entered rigor. The tenderization process in pork is believed to begin very early PM, and a significant part of the process takes place in the first 24 h PM (Dransfield, 1994a). In this study, the WBSF value 24 h PM is therefore used as a measure of the tenderization rate. This choice is debatable because differences in collagen content, sarcomere length, and intramuscular fat (IMF) content also could influence the WBSF 24 h PM. Pigs in group RR have a higher content of soluble collagen and a numerically lower content of total collagen compared to pigs in group AA and RA (Table 3Go). Increased collagen solubility and decreased total collagen have been associated with a decrease in the connective tissue component of meat toughness (Møller, 1981; Dransfield, 1994b; Berge et al., 2001). The decreased meat toughness in our study observed for meat from RA pigs in relation to RR pigs therefore cannot be explained by the observed changes in collagen. Rather, it is likely to arise from changes in the myofibrillar component of toughness. The sarcomere length and the content of IMF have not been measured in this study, so these factors cannot be ruled out in explaining the differences in tenderization rate between treatment groups. Drip loss has been shown to be linearly related to the sarcomere length at normal-to-short muscle lengths (Honikel et al., 1986), and as no differences were observed in drip loss in this study (Table 3Go), it indicates that no major differences in sarcomere length exist between groups that can explain the observed differences in WBSF between treatment groups. As discussed later in this paper, the restrictively fed pigs had a higher meat percentage, which indicates a lower IMF content. van Laack et al. (2001) found a significant but very small correlation (r = -0.11) between IMF and WBSF after 2 d storage of pork. This implies that differences in IMF may explain only a minor part of the observed differences in WBSF in this study.

The proteolytic potential indicates how fast a muscle may tenderize PM, and it has been argued that the ratio between the µ-calpain activity and the calpastatin activity at slaughter is a suitable way to measure proteolytic potential in muscles (O’Halloran et al., 1997; Ertbjerg et al., 1999; Geesink and Koohmaraie, 1999a). The rational behind this is that µ-calpain is suggested to be the primary proteolytic enzyme that causes tenderness development and that calpastatin is an inhibitor of µ-calpain (Koohmaraie et al., 1986; Huff-Lonergan et al., 1996; Koohmaraie, 1996). Although only significantly different to treatment group RR, RA pigs had the highest proteolytic potential of the four groups when expressed as µ-calpain/calpastatin activity (Table 5Go). The development in MFI values is believed to reflect the proteolytic degradation that occurs during the first 24 h PM. The MFI results presented in Table 5Go show the same tendency as those for µ-calpain/calpastatin, with the highest value in RA pigs and the lowest values in RR pigs 24 h PM. These results indicate an increased tenderization rate in RA pigs, which the results from the WBSF measurements after 1 d storage also confirm. It is well known that tenderness of meat increases during aging to a point described as "background toughness" whereafter only minor changes in tenderness occur (Dransfield, 1994a; Wheeler and Koohmaraie, 1994). After 4 d of aging, the differences in shear force between group RA and groups AR and RR are still significant, but the difference between group AA and RA has vanished, which is probably due to an identical background toughness in pigs from the two groups (i.e., RA pigs tenderize faster than AA pigs, but in time the two groups end up at the same shear force values). Whether groups AR and RR will arrive at the same background toughness as groups AA and RA at prolonged aging is not clear; however, the difference in shear force between pigs on restricted or ad libitum diets at d 4 is less than at d 1.

Due to the lower feed consumption in restrictively fed pigs (60% of ad libitum), their live weights are lower at both d 90 and at slaughter than those of pigs given free access to feed. A restricted energy intake will have a negative influence on the deposition of fat during growth (i.e., there is less energy that can be stored in lipid tissue). The lower feed consumption can therefore explain the thinner layer of subcutaneous fat in restrictively fed pigs at both d 90 and at slaughter. The restrictively fed pigs have a higher meat percentage, which is probably caused by a lower content of IMF. This content has not been measured in this study; however, earlier work found a positive correlation between IMF content and subcutaneous fat thickness (Wood, 1990).

Muscle growth is dependent on at least two mechanisms—satellite cell proliferation and protein accretion rate (Allen et al., 1979)—the latter being the difference between the protein synthesis rate and the protein degradation rate. Since satellite cell proliferation increases the total amount of DNA in a muscle (Oksbjerg et al., 2002), and the protein synthesis capacity is reflected in the RNA levels (Millward et al., 1973), the two nucleotides were measured in addition to eEF-2, which also is related to protein synthesis in vivo. The FRG2 values clearly indicate that RA pigs have the highest growth rate in the second feeding period. However, this was not reflected in the levels of DNA, RNA, or eEF-2 at slaughter in group RA in either the LD or ST muscles. In another experiment (Therkildsen et al., unpublished data) designed to clarify how the length of the restrictive phase before the phase with free-access feeding affects protein synthesis and protein degradation, results indicated that muscle protein synthesis increases at a faster rate following changes to free-access feeding compared with increases in muscle protein degradation. In order to utilize compensatory growth to increase the tenderization rate, animals should be slaughtered at a time with maximum muscle protein degradation. This is probably not at a time when they could show superior growth compared with control pigs, but could be at later stages when both synthesis and degradation have increased (Therkildsen et al., unpublished data). This explains why there seems to be an effect of the compensatory growth response on the calpain system and not on the levels of nucleotides and eEF-2 when comparing AA pigs and RA pigs (i.e., the protein synthesis rate in group RA had caught up with the synthesis rate in group AA long before the animals were slaughtered). [The levels of nucleotides measured at d 105 in fact point toward increased protein synthesis rates in RA pigs compared to AA pigs with P-values of 0.25 and 0.14 for RNA and DNA, respectively.]

The hypothesis presented in Figure 1Go is largely supported by the results presented in this paper. The activities of both µ- and m-calpain at slaughter were highest in AA and RA pigs, which also showed the highest growth rates expressed as FRG2. Although no significant differences were observed between AA and RA pigs, the numerical values for the calpain activity were highest in RA pigs, which in turn also had the fastest growth rates among the two groups. This implies that the synthesis of these enzymes were up-regulated during the second feeding phase to a larger extent in group RA than in group AA. The proteolytic potential and the MFI values also were found to be highest in RA pigs, which indicates that the up-regulated in vivo calpain activity had an effect on the PM protein degradation. This is furthermore supported by the higher tenderization rate in RA pigs.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
The results obtained from this study demonstrate that manipulation with animal growth rate using a compensatory growth strategy might be an easy way to increase meat quality and at the same time decrease production costs by reducing feed quantity. The results also imply that m-calpain is involved in muscle growth in pigs and that µ-calpain and calpastatin activity at slaughter is connected to the postmortem tenderization process in pork. Pigs are known to have a faster tenderization rate than cattle for instance, therefore no direct conclusion on how restricted feeding and compensatory growth could affect other species can be drawn.


   Footnotes
 
1 The authors would like to thank the Ministry of Food, Agriculture, and Fisheries, and the Danish Slaughterhouses for their financial support of this project, and Hans Busk for carrying out ultrasound measurements. Ahmad Abdal-kader Kabel, Marianne Rasmussen, Anna Halborg-Madsen, and Anne-Grete Dyrvig Petersen are gratefully acknowledged for their technical assistance in this study. Back

3 Current address: Centre for Extracellular Matrix Biology, Department of Biological Sciences, University of Stirling, Stirling FK9 4LA, Scotland, UK. Back

Received for publication March 5, 2002. Accepted for publication June 26, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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