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ANIMAL PRODUCTS |
* Department of Food Science, Royal Veterinary and Agricultural University, DK–1958 Frederiksberg C, Denmark;
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Department of Food Science, Danish Institute of Agricultural Sciences, DK–8830 Tjele, Denmark; and
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Danish Meat Research Institute, Maglegaardsvej 2, DK–4000 Roskilde, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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0.001) fractional growth rate in the second feeding period than those in the ALA group. Growth rate was correlated to the activity of m-calpain (r = 0.37; P 0.01), ß-glucuronidase (r = 0.48; P 0.001), and cathepsins B (r = 0.47; P 0.001) and B+L (r = 0.31; P 0.04). The LM of RA80-gilts received higher tenderness scores than the LM of ALA gilts, but tenderness scores were similar among barrows regardless of treatment (gender x treatment; P = 0.02). Conversely, tenderness scores were higher for the biceps femoris of ALA barrows than either ALA gilts or RA90 barrows (gender x treatment; P = 0.02). Desmin and troponin-T degradation, as well as myofibrillar fragmentation index, of the LM were not (P 
0.24) affected by treatment. No dietary treatment effects were observed on the activities of µ-calpain (P = 0.15), m-calpain (P = 0.74), or calpastatin (P = 0.91) at slaughter. The cathepsin inhibitors, cystatins, tended to be increased (P = 0.06) in RA80 and RA90 pigs. Sarcomere length was longer (P = 0.003) in the LM of gilts than barrows. Barrows in the RA80 group had lower i.m. fat concentrations than ALA; however, no differences were found in the LM of gilts (gender x treatment; P = 0.03). The underlying hypothesis that compensatory growth leads to an increased proteolytic potential at the time of slaughter could not be verified in this study.
Key Words: Calpain • Cathepsin • Desmin • Intramuscular Fat • Muscle Growth • Proteolysis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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; Kristensen et al., 2002). Compensatory growth is a phenomenon that is observed in pigs given ad libitum access to feed following a period of restricted feeding, which leads to an increased growth rate compared with pigs given ad libitum access to feed during the whole period of growth (McMeekan, 1940; Prince et al., 1983; Oksbjerg et al., 2002). Postnatal muscle growth reflects the difference between the rate of protein synthesis and the rate of protein degradation, and it has been suggested that both the rates of protein synthesis and degradation are elevated during periods of the compensatory growth response (Millward et al., 1975; Jones et al., 1990).
In a recent study, we demonstrated that gilts expressing compensatory growth tended to have increased proteolysis at the time of slaughter and a faster rate of tenderization than pigs with ad libitum access to feed and pigs restrictively fed during the entire period of growth (Kristensen et al., 2002). Simultaneously, it was reported that the length of compensatory growth influenced the level of in vivo protein turnover and postmortem proteolysis (Therkildsen et al., 2002). From the latter study, it was suggested that a compensatory growth period beyond 42 d was required for an increase in postmortem proteolysis (Therkildsen et al., 2002). Therefore, the objective of the current study was to examine the effects of compensatory growth on meat tenderness, postmortem proteolysis, and the activities of the calpain and lysosomal enzymes systems in gilts and barrows.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Nine litters of three gilts and three barrows (Duroc x Landrace x Large White) were allocated within litter and gender to three dietary groups. Two ad libitum feeding periods beyond 42 d were used as suggested by Therkildsen et al. (2002). Pigs had either ad libitum access to the diet from weaning at d 28 to slaughter at d 140 (ALA) or were restricted to 69% of voluntary feed intake from d 28 to either d 80 (RA80) or d 90 (RA90), and then given ad libitum access to the diet until slaughter (d 140). All pigs were penned individually and fed diets according to their age. Composition of diets is shown in Table 1. Pigs were fed a four-phase diet program: a starter diet from weaning to 56 d, a transition diet from Starter to Grower-1 and Grower-1 to Grower-2 at d 71 and 112, respectively, and a finisher diet from d 113 to slaughter (d 140). The restricted feeding scale started at 0.15 kg/d at weaning, and increased weekly to 1.1 kg/d at d 70 and 1.7 kg/d (as-fed basis) at d 90 of age. Pigs were slaughtered according to procedures described by Therkildsen et al. (2004).
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At 24 h postmortem, pH was measured as previously described, except that the electrode was calibrated in buffers equilibrated to 3°C. Biceps femoris (BF) from left sides was removed and sampled for determination of total and soluble collagen, desmin degradation, and myofibrillar fragmentation index (MFI) at 1, 2, and 4 d postmortem. Samples collected 1 d postmortem were snap frozen in liquid N and stored at –80°C, whereas 2- and 4-d samples were aged for 1 or 3 d at 3°C before sampling, and then treated similarly to 1 d samples. The right side BF was removed, vacuum packaged, and aged for an additional day at 3°C before storage at –20°C until sensory analysis. Additionally, a portion of LM was removed from the last lumbar vertebra of right sides, and, from the cranial direction, 25 cm was used for sensory analysis, 1 cm for sarcomere length, desmin and tropinin-T degradation, and MFI, and the last 21 cm was cut into three 7-cm sections for Warner-Bratzler shear force (WBSF) determinations on d 1, 2, and 4 postmortem. Samples for desmin and troponin-T degradation and MFI were treated as described previously for BF samples. The LM section designated for sensory panel evaluations was vacuum packaged, aged for an additional day at 3°C, and stored at –20°C until evaluation. Samples for WBSF were vacuum-packaged and either stored at –20°C (d-1 sample) or aged for an additional 1 or 3 d before storage at –20°C. An additional LM section was removed between the 10th and 12th thoracic vertebra for determination of drip loss. Drip loss, calpastatin, m-calpain, µ-calpain, WBSF, total and soluble collagen, and MFI were determined as described in Kristensen et al. (2002).
Sensory Analysis
The sensory panel consisted of nine panelists (seven females and two males) between the ages of 46 and 64 yr, who were familiar with pork and descriptive sensory analysis. Before the analysis, the panel was trained during two sessions using samples represented in the experiment. Pork was cooked in roasting bags in a convection oven preheated to 90°C. Temperature was measured in the center of each roast with a handheld probe (Testo 926; Testoterm, Buhl and Bundsoe, Virum, Den-mark). Roasts were removed from the oven at an internal temperature of 70°C, wrapped in foil, and held at room temperature for at least 20 min. Samples from each pig were cut into 15-mm-thick slices. Slices were then cut into 5 cm x 5 cm pieces, and one piece was served warm to each panelist. Tenderness was rated on a continuous scale from 0 (extremely tough) to 15 (extremely tender).
Sarcomere Length
Longissimus muscle samples (approximately 5 mm x 5 mm x 5 mm) were fixed in a borate solution (0.25 M KCl, 0.29 M Boric acid) containing 2.5% glutaraldehyde and stored at 4°C until analysis. Then, samples were moved to another tube and homogenized in a 0.25 M sucrose solution for 30 s with an Ultra Turrax T25 mixer (Ika Laboratechnik, Staufen, Germany) at 16,000 rpm. A few drops of the homogenate were placed on a glass slide and examined by phase-contrast microscopy. Lengths of 20 sarcomeres from 10 myofibrils were measured using the image-analyzing system TEMA (Scan Beam APS, Hadsund, Denmark).
Lysosomal Enzyme Activity
Sample preparation and assays were conducted as described by Kirschke et al. (1983) with some modifications, and, unless otherwise stated, all chemicals were of analytical grade (Sigma Chemicals Co., St. Louis, MO). For total enzyme activity, frozen meat samples were finely chopped with a handheld knife and 1 g was homogenized (Ultra Turrax mixer T25, Ika Laboratechnik) for 30 s at 8,000 rpm and then for another 30 s at 24,000 rpm in 4.0 mL of buffer (100 mM sodium acetate, 0.2% Triton X-100; pH 5.5). The samples were then centrifuged for 15 min at 32,000 x g at 4°C. For the measurement of cathepsin B+L and cathepsin B total activity, 30 µL of supernatant was added to 270 µL of activation buffer-1 (340 mM sodium acetate, 60 mM acetic acid, 4 mM EDTA, 0.1 % Brij 35, and 0.8 mM freshly added dithiothreitol; pH 5.5) and left for 10 min in a 40°C water bath for temperature equilibration. Substrate solution was added (200 µL of 12.5 µM Z-Phe-Arg-NMec for cathepsin B+L or 12.5 µM Z-Arg-Arg-NMec for cathepsin B), and, after 10 min, the reaction was stopped by adding 2.0 mL of stop buffer-1 (100 mM sodium hydroxide, 30 mM sodium acetate, 70 mM acetic acid, 100 mM monochloroacetic acid; pH 4.3). Standard curve-1 was produced using 25 to 200 nM 4-amino-7-methyl coumarin dissolved in stop buffer-1. Fluorescence was determined (LS 50 B fluorescence spectrophotometer, Perkin Elmer, Boston, MA) with excitation and emission wavelengths set at 360 and 440 nm, respectively, using stop buffer-1 as the blank. One unit (U) equals the release of 1 µmol of Nmec/min. For total activity of ß-glucuronidase, the same procedure was used except that 50 µL of the supernatant was added to 250 µL of activation buffer-2 (100 mM sodium citrate, 250 mM sucrose, 0.02% Triton X-100; pH 5.0). Other reagents included 500 µM substrate (4-methyli-umbelliferyl-ß-D-glucuronidide) and stop buffer-2 (1.0 M sodium carbonate), and the standard curve-2 was produced using 4-methyliumbelliferyl.
Cystatin Activity
Frozen muscle samples were prepared as described previously. One milliliter of supernatant was heated at 100°C for 3 min to remove all protease activity, and subsequently centrifuged at 20,000 x g for 3 min. Activation buffer-1 was added to 5, 10, or 15 µL of heat-treated supernatant to a final volume of 500 µL. Papain solution (100 µL; 0.25 mg/mL in 0.1 % Brij 35) was added and left for 10 min in a 40°C water bath for temperature equilibration. Substrate solution was added (400 µL of 12.5 µM Z-Phe-ARG-NMec), and after 10 min, the reaction was stopped by adding 4.0 mL of stop buffer-1. Using linear regression, the slope (asample) between the fluorescent signal and added volume of supernatant in the assay was calculated, and one unit of cystatin activity was defined as [–(asample/astandard)/t], where astandard is the slope of standard curve-1 and t is the reaction time.
Western Blotting
Sample preparation was carried out according to Wheeler and Koohmaraie (1999), and the protein concentrations were determined using a microbicinchoni-nic acid assay (Pierce, Chemical Co., Rockford IL). Samples were diluted to 2.0 mg of protein/mL using treatment buffer (125 mM Tris, 4% SDS and 20% glycerol; pH 6.8) containing 100 mM 1,4-dithioerythritol and 0.08 mg/mL of bromophenol blue. Samples were then mixed and heated for 10 min in a 50°C heating block and subsequently stored at –80°C. For desmin, samples were run on 10.0%, 18-well Criterion gels (Biorad Laboratories, Hercules, CA), whereas for troponin-T, samples were run on 12.5%, 26-well Criterion gels (Biorad Laboratories). Thawed samples were heated for 10 min in a 50°C heating block before loading a volume equal to 10 µg of protein in each lane. Samples from three pigs (same litter and gender) and four aging times (0, 1, 2, and 4 d postmortem) were loaded on one gel together with an internal standard. The internal standard consisted of a randomly selected d-0 sample, which was used for all samples.
Gels were run at 200 V for 45 min at room temperature, and the proteins were transferred from the gels to polyvinylidene fluoride membranes as described by Towbin et al. (1979). The transfer was done at 200 mA for 2.5 h at 4°C with gentle stirring. Membranes were rinsed with deionized water and blocked in Tween-Tris-buffered saline (TTBS) (20 mM Trisma-base, 137 mM NaCl, 5 mM KCl, 0.05% Tween 20; pH 7.4) with 5% nonfat dry milk (Biorad, Laboratories) overnight at 4°C. Membranes were washed twice for 10 min in TTBS, and incubated for 90 min with primary antibody diluted with TTBS. Monoclonal antidesmin clone de-u-10 (Sigma d-1033; Sigma Chemical Co.) and monoclonal antitroponin-T clone jlt-12 (Sigma t-6277; Sigma Chemical Co.) were used in dilutions of 1:10,000. Then, membranes were washed six times for 5 min in TTBS and incubated in secondary antibody (Rabbit-anti-mouse AP conjugated, Dako d-0314; Dako A/S Denmark) diluted 1:1.000 in TTBS for 1 h, and washed twice for 10 min and four times for 5 min in TBBS before the final rinse with water. Blots were developed with a Biorad amplified alkaline phosphatase conjugate substrate kit (170–6412; Biorad Laboratories) according to the instruction manual. Membranes were scanned with the use of Image Master software (Amersham Phamacia Biotech, Uppsala, Sweden), and the intensity of the desmin and troponin-T bands at d 0, 1, 2, and 4 postmortem were evaluated relative to intact desmin (55 kDa) and troponin-T (39 kDa) bands of the internal standard, respectively.
Intramuscular Fat
The i.m. fat content was determined by gravimetric analysis according to NMKL (1989). Briefly, the sample was treated with 8 M HCl and, after the addition of ethanol, the liberated fat is extracted with a mixture of diethyl ether and petroleum ether. The solvent is then evaporated and the fat is weighted.
Statistical Analyses
Data were tested for normal distribution using the Shapiro-Wilkes test within the univariate procedure of SAS (SAS Inst., Inc., Cary, NC). Variables not satisfying the requirement of a normal distribution were transformed. Data for cathepsin B and B+L, desmin and collagen in the BF, and i.m. fat were not normally distributed, and were therefore transformed using 1/x (cathepsins, i.m. fat, and soluble collagen), x2 (desmin in the BF), or 1/x4 (total collagen in the BF). Data were then analyzed for treatment and gender effects in a complete randomized block design using the mixed model procedure of SAS, with the fixed effects of treatment, gender, and their interaction. Litter origin (blocking factor) was included in the model as a random effect, and weaning weight was used as a covariate. Least squares mean were separated using the PDIFF option. Analysis of WBSF, desmin, troponin-T, and MFI data included days postmortem as a repeated measurement, pig as the subject, and an unstructured covariance structure with the covariance between days set to zero. This model also included the random effects of litter and the pig x treatment interaction. Analysis of the sensory data included panelists as a repeated measurement with pig as subject, and an unstructured covariance structure with the covariance between panelists set to zero. The model for sensory data also included the random effects of litter and animal x treatment. All results are presented as least squares means. Correlation coefficients were calculated using the GLM procedure of SAS after removing the effects of treatment, gender, and the interaction.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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). Additional performance results from the current study have been published in detail elsewhere (Therkildsen et al., 2004). In brief, results showed that during compensatory growth, the ADG was increased by 7.8% due to a 5.6% improvement in feed conversion ratio without any change in feed intake. This resulted in similar slaughter weights, meat yields and fat thickness among treatments indicating complete compensation (Therkildsen et al., 2004). No (P > 0.10) effect of treatment or gender on drip loss was observed (results not shown).
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) or by treatment with porcine growth hormone (Sève et al., 1993). It was suggested that the calpain system and the lysosomal enzymes are involved in controlling the rate of protein degradation in living animals (Goll et al., 1998). This may explain the significant positive correlation coefficients observed between the fractional rate of growth and the proteolytic potential of muscles, especially correlations to m-calpain, cathepsin B+L, cathepsin B, and glucoronidase (Table 3). Brooks et al. (1983), Spencer et al. (1997), and Kristensen et al. (2002) also observed a positive relation between m-calpain activity and muscle growth. However, correlations between lysosomal enzyme activities and muscle growth (Table 3) have, to our knowledge, not been reported previously.
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and Figure 1. A gender x treatment interaction on subjective pork tenderness was detected for both the LM and BF (P = 0.02). The LM from ALA barrows and RA80 gilts received higher (P < 0.05) tenderness scores than did the LM from ALA gilts. Moreover, the BF from ALA barrows was rated more tender (P < 0.05) than the BF from RA90 barrows and ALA gilts. Results obtained on gilts supports the observation by Kristensen et al. (2002), who also found a positive effect of compensatory growth in gilts on meat tenderness using a feeding strategy comparable to the one used in this study. Conversely, Therkildsen et al. (2002) did not observe an effect of compensatory growth on pork tenderness of either gilts or barrows; however, they suggested that the lack of treatment effects was the response to a too-short period of ad libitum access to feed following the restricted feeding period. Restrictive feeding results in a lower protein turnover compared with a feeding strategy with ad libitum access to the diet (Therkildsen et al., 2004). The increased growth rate observed during compensatory growth is due to a gradual increase in both the rate of protein synthesis and the rate of protein degradation, with a relatively larger increase in the rate of protein synthesis (Millward et al., 1975; Jones et al., 1990). As hypothesized by Kristensen et al. (2002), this might increase postmortem proteolysis and thereby increase meat tenderness. In the study by Therkildsen et al. (2002), the length of the ad libitum period until slaughter was found to have a positive relationship with in vivo protein turnover and postmortem proteolysis. It was suggested that an ad libitum feeding period beyond 42 d was needed before the rate of protein degradation in compensating pigs exceeded that of pigs with ad libitum access to feed throughout the experiment, and consequently increased the tenderness (Therkildsen et al., 2002).
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; Taylor et al., 1995; Huff-Lonergan et al., 1996). In addition, degradation of troponin-T in beef has been observed to be strongly related to the tenderization process (Penny and Dransfield, 1979). The levels of intact desmin in the LM and BF, as well as troponin-T in the LM, during storage are presented in Table 4. As expected, an effect (P < 0.001) of postmortem storage time on desmin and troponin-T degradation was observed; however, no treatment (P 0.24) or interactive (P 0.22) effects were observed on either desmin or troponin-T degradation. The fragmentation of myofibrils has been associated with postmortem proteolysis and meat tenderization (Davey and Gilbert, 1969; Møller et al., 1973), and the method to measure fragmentation has been named MFI (Olson and Parrish, 1977). However, neither compensatory growth (P 0.08) nor gender (P 0.55) affected the MFI values of either the BF or LM (Table 4); however, as expected, MFI values increased (P < 0.001) with increasing duration of storage.
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. Only the activity of the lysosomal enzymes cathepsin B and ß-glucuronidase was affected by an interaction between gender and treatment (P = 0.03 and 0.006, respectively). However, the interaction was due to a higher activity of both enzymes in RA80 barrows, and this interaction cannot explain the observed gender x treatment interaction on sensory panel-evaluated tenderness (Figure 1).
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). Cystatins are a superfamily of cysteine proteinase inhibitors. They form tight complexes with cysteine proteases, such as cathepsin B and L, thereby inhibiting their activity. Cystatins are located in the sarcoplasm, where they are believed to protect the cell from the activity of cathepsins unleashed from the lysosomes (Bond and Butler, 1987). Cystatin levels tended to increase (P = 0.06) in pigs showing compensatory growth (RA80 and RA90; Table 5), suggesting that cystatin activity is upregulated in pigs showing compensatory growth, regardless of gender.
Kristensen et al. (2002) and Therkildsen et al. (2002) observed a tendency for increased activity of µ-calpain in pigs showing compensatory growth; however, such a tendency was not (P = 0.15) observed in this study in either barrows or gilts (Table 5). Kristensen et al. (2002) suggested that gilts showing compensatory growth had a higher proteolytic potential at slaughter, as evidenced by increased µ-calpain activity, higher MFI values, and lower shear force values compared with the control group. As explained by Therkildsen et al. (2002), the response of the in vivo proteolytic system going from restricted feeding to ad libitum feeding is a rather slow process, which requires at least 42 d before an effect can be expected on meat tenderness. The lack of treatment effects on proteolysis in this study may be due to the shorter period of ad libitum access to feed. Another explanation for the lack of an effect on the calpain system is presented by Ilian and Forsberg (1994), who found that fasting rabbits for 8 d increased protein degradation twofold and increased the concentrations of mRNA encoding µ-calpain, m-calpain, and calpastatin, indicating an upregulation of the calpain system during fasting. However, the upregulation was not followed by increased µ- or m-calpain activity. This contradiction may be caused by increased turnover rate of calpains in fasted animals. The calpain system might be upregulated during compensatory growth, but because of faster autolysis, no effect of treatment could be found on the at-slaughter extractable activity (Table 5).
Besides proteolysis, meat tenderness is influenced by a wide range of factors, including sarcomere length (Marsh and Leet, 1966; Møller and Vestergaard, 1986, 1987), i.m. fat (Bejerholm and Barton-Gade, 1986; van Laack et al., 2001), and the quantity and quality of connective tissue (Dransfield, 1977; Møller, 1981). The percentage of total collagen in the BF was higher (P = 0.01) and sarcomere length was longer (P = 0.003) in gilts than in barrows (Table 6). The i.m. fat levels were affected by an interaction between gender and treatment (P = 0.03), with no difference in i.m. fat in gilts among the treatment groups; however, i.m. fat was decreased in RA80 and RA90 barrows compared with ALA barrows (Figure 1). Neither of these results could explain the gender x treatment interaction on tenderness.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Footnotes |
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2 Correspondence: Rolighedsvej 30 (phone: +4535283246; fax: +4535283341; e-mail: lak{at}kvl.dk).
Received for publication December 19, 2003. Accepted for publication August 20, 2004.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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