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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Food Science, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark and
and
Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark
Abstract
The objective of this study was to examine the intralitter variation in postnatal growth performance, meat quality, and muscle fiber characteristics when littermates were categorized by carcass weight. Thirty-nine litters were weaned at 4 wk of age and had free access to feed from 2 wk of age until slaughter. They were slaughtered by litter at an average BW of 104 ± 14 kg, and six pigs per litter were selected for analysis: the heaviest- (HW), middle- (MW), and lightest-weight (LW) pig of each sex. Categorizing littermates in LW, MW, and HW pigs at the same age reflected the differences in postnatal growth rate within a litter; thus ADG, muscle mass, and muscle deposition rate differed across pig weight groups (P < 0.001). Also, the total DNA content was different among pig weight groups (P < 0.001) and reflected differences in muscle growth rate. The difference in muscle growth rate between LW and MW pigs could be explained by a larger (P < 0.05) mean fiber area (MFA) in MW pigs, whereas the number of muscle fibers was similar. Growth rate differences between MW and HW pigs could in part be explained by a higher number (P < 0.01) of equal-sized muscle fibers in HW pigs. The difference in MFA was due to a higher estimated DNA and RNA content per muscle fiber in MW and HW compared with LW pigs (P < 0.05). Pigment content was higher in MW and HW compared with LW pigs (P < 0.01), but no other measured meat quality traits were significantly different across pig weight groups. These results indicate that both the number and the growth rate of muscle fibers contribute to intralitter variation in postnatal growth performance.
Key Words: Meat Quality • Muscle Fiber Characteristics • Performance • Pigs • Within-Litter Variation
Introduction
The within-litter variation in muscle growth, and consequently in performance, is large. Reducing this variation will benefit production economy. Postnatal muscle growth rate is positively correlated with the number of muscle fibers and the growth rate of the individual muscle fiber (Dwyer et al., 1993
; Rehfeldt et al., 2000). Muscle fibers develop in the fetal stage, and the number in most mammals is fixed prenatal.
Categorizing littermates by birth weight, Handel and Stickland (1987a) and Dwyer and Stickland (1991) found no relationship between pig weight and muscle fiber number. Categorizing littermates by weight at 5 wk of age, Dwyer and Stickland (1991) found that low-weight pigs have a reduced number of muscle fibers compared with their largest littermates. Some low-birth-weight pigs show catch-up growth, and these pigs have a muscle fiber number not unlike their largest littermates (Handel and Stickland, 1988). Categorizing littermates by birth or weaning weight does not enable an evaluation of the contribution of the muscle fiber area to postnatal muscle growth because growth of the individual muscle fiber is an ongoing process in the growing animal. Thus, categorizing littermates by carcass weight will enable an examination of the intralitter variation in muscle fiber characteristics of animals with very different postnatal growth potential.
Fiber number and fiber cross-sectional area might influence final meat quality (Oksbjerg et al., 2000; Rehfeldt et al., 2000); thus, variation in fiber number and muscle fiber area within litter might reveal differences in final meat quality. Therefore, the purpose of the present study was to examine the contribution of muscle fiber characteristics to the intralitter variation in postnatal growth performance and meat quality traits.
Materials and Methods
Animals and Feed
The experiment was conducted at the Danish Institute of Agricultural Sciences, Research Centre Foulum (Tjele, Denmark) from late 1997 to early 2001. Thirty-nine litters from Danish Landrace x Large White sows and Danish Landrace or Large White boars were used in this experiment. The sows were in their fourth parity. They were assigned randomly to one of three treatments in which the sows were either: 1) fed restrictively (15 MJ of NE/d from d 1 to 90, then 24 MJ NE/d from d 91 to 112, and again 15 MJ of NE/d from d 113 to 115 of gestation; control); 2) fed ad libitum from d 25 to 50 (A25–50); or 3) fed ad libitum from d 25 to 70 (A25–70) and fed the same as control sows in the remaining periods. In this study, the within-litter aspects are analyzed without respect to maternal treatment during gestation, which had no significant effect on muscle fiber characteristics (Nissen et al., 2003). The piglets were weaned at 4 wk of age, and penned by litter thereafter. They were offered feed ad libitum from 2 wk of age until slaughter. The composition of the diets for piglets and growing-finishing pigs is shown in Table 1. At slaughter, a maximum of six pigs per litter was selected for analysis: the heaviest- (HW), middle- (MW), and lightest-weight (LW) pigs of each sex, based on carcass weight. In litters with an uneven distribution in relation to sex, fewer than six pigs were selected for analysis.
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Pig Performance and Meat Quality Measures
Body weight changes were recorded until slaughter for calculation of daily gain from 30 to 100 kg of BW. Percentage of meat was measured 24 h postmortem with a Fat-O-Meater (SFK Ltd., Hvidovre, Denmark; Kempster et al., 1985). The muscle mass of the carcass was calculated as the meat percentage multiplied by cold carcass weight (24 h postmortem), and the muscle deposition rate was calculated as the muscle mass divided by days at slaughter.
At 24 h postmortem, the pH (pH24) of LD was measured at the last rib of the left side. Two samples were removed from LD at the last rib on the right side: one for color and pigment determination and the other for determination of water-holding capacity measured as drip loss. Color was measured using a Minolta Chroma Meter CR-300 (Osaka, Japan) calibrated against a white tile (lightness = 92.30, redness = 0.32, and yellowness = 0.33). The samples were allowed to bloom for 1 h at 4°C before measurements. The three lightness and color parameters were measured on five fixed sites of each sample surface. Muscle pigment concentration was analyzed using the method described by Oksbjerg et al. (2000). Drip loss was measured on approximately 100 g of LD muscle using the plastic bag method described by Honikel (1998).
Histochemistry Analysis
Transverse serial sections (10 µm) were cut in a cryostat at -20°C, picked up on cover slips, and then allowed to thaw and dry at room temperature overnight. The sections were immunohistochemically stained for slow myosin heavy chain isoform (catalog No. CRL-2043, American Type Culture Collection, Rockville, MD) to identify type-I fibers. Other sections were stained for collagen IV (catalog No. M0785, DAKO, Glostrup, Denmark) to outline cell membranes for quantification of muscle fiber number and area. Methods used for immunohistochemistry were described by Pedersen et al. (2001).
The percentage and the mean area of fiber types I and II and the mean area of all fibers (MFA) were calculated on the basis of 200 to 300 fibers per sample (five samples per animal), using a computer-assisted image analysis system (TEMA, CheckVision, Hadsund, Denmark) developed and validated as described by Henckel et al. (1998). These data were used to estimate the total number of fibers (cross-sectional area of ST divided by the MFA). The number of primary (P)- and secondary (S)-fibers was estimated on the basis of type-I fiber clusters. Initially, the P-fibers develop into type-I fibers, whereas the surrounding fibers develop into type-II fibers. As the muscle matures, some of the type-II fibers closest to type-I fibers change fiber type characteristics, thereby becoming type-I fibers. Thus, postpartum, there will be clusters of type-I fibers surrounded by type-II fibers. According to Handel and Stickland (1987a), only one fiber from each type-I cluster was originally formed as a P-fiber, thus the estimated number of type-I clusters is the same as the originally developed number of P-fibers. The total number of fibers minus the number of P-fibers is then the estimate for the number of S-fibers.
Determination of DNA and RNA
Concentrations of DNA and RNA in ST were measured fluorometrically. Concentrations of DNA were quantified using a Picogreen double-stranded DNA quantification reagent kit (catalog No. P-7589, Molecular Probes, Leiden, The Netherlands), and RNA concentrations were measured using a SyrbGreen II RNA gel-staining kit (S-7568, Molecular Probes, Leiden, The Netherlands). The methods are described by Oksbjerg et al. (2000) and modified by Therkildsen et al. (2002).
Data Analysis
To analyze pig performance, meat quality, histochemistry, and DNA and RNA content, ANOVA was performed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) with sex, pig weight within litter, and interactions between these as fixed effects. Sow, boar, their interactions, and the fixed effects were used as random effects. The within-litter ratio between female pigs and barrows and litter size at birth was used as covariate when significant. Also, meat percentage and age at slaughter (days) was used as a covariate in the analysis for muscle mass, muscle deposition rate, weight and cross-sectional area of ST, fiber areas, and DNA and RNA content when significant.
Results
Pig Performance
As expected, birth weight differed within litter (P < 0.001; Table 2). Thus, the HW pigs had a higher birth weight than LW and MW pigs, which had similar birth weights. At weaning, there was a difference between weights of LW, MW, and HW pigs (P < 0.001). Due to selection of the pig weight groups, carcass weight and ADG were different across pig weight groups (P < 0.001). Correlations between BW at birth and weaning, carcass weight, and daily gain varied between 0.24 and 0.60 (P < 0.01; Table 2). Meat percentage was not significantly different among pig weight groups within litter. Also, calculated muscle mass and muscle deposition rate were lowest for the LW pigs and highest for the HW pigs, with MW pigs intermediate (P < 0.001). Meat deposition rate was correlated to birth weight, weaning weight, carcass weight, and daily gain (P < 0.001; Table 3), with the highest correlation between meat deposition rate, and carcass weight and daily gain.
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), although there was a tendency for a higher yellowness in HW pigs compared with both LW and MW pigs (P = 0.053). Pigment content was found to be higher in MW and HW pigs compared with LW pigs (P < 0.01).
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and 2). Total and S-fiber number was correlated to birth, weaning, and carcass weight (P < 0.05), but not to daily gain or meat deposition rate (Table 2
). The P-fiber number was correlated to BW, daily gain, and meat deposition rate (P < 0.05). There was no difference in the percentage of type-I fibers across pig weight groups (Table 5). The area of both type-I and -II fibers, and thus MFA (Figure 1), was lower for LW pigs compared with MW and HW pigs (P < 0.05). The cross-sectional area and the weight of ST followed the weight of the pig and were thus different across weight groups (P < 0.001) (Table 2).
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). The total amount of DNA and RNA in ST followed the weight of the pig; thus, the LW pigs had the lowest amount and the HW pigs had the highest amount (P < 0.001). The LW pigs had a lower amount of DNA and RNA per fiber compared with MW and HW pigs (P < 0.05), which were similar.
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The present study demonstrated a large intralitter variation in growth performance, which can be explained by variation in both the number of muscle fibers and the growth rate of the fibers. Thus, HW pigs had a significantly higher number of total muscle fibers than MW and LW pigs, which did not differ from each other. This is in agreement with studies of large and small fetal pigs (Wigmore and Stickland, 1983), but in contrast to comparisons of large and small pigs at birth (Handel and Stickland, 1987a; Dwyer and Stickland, 1991; Dwyer et al., 1993). When comparing the fiber number of small and large littermates based on weight at 5 wk instead of weight at birth, a significantly lower number of total muscle fibers in small littermates was found (Dwyer and Stickland, 1991). These results are in agreement with our study and show that there is a variation in the total number of muscle fibers within a litter of pigs, and that the pigs with the lowest postnatal growth rate have a lower number of total muscle fibers than their largest littermates.
Both P- and S-fiber numbers were higher in HW pigs in this study. Even though the numbers of P- and S-fibers were similar in LW and MW pigs, the S:P ratio was significantly lower for MW pigs compared with LW pigs. This can be explained by a numerically higher number of P-fibers and a lower number of S-fibers in MW pigs. Others have found that small littermates have a lower S:P ratio than their large littermates due to a higher number of S-fibers with no difference in P-fiber numbers (Wigmore and Stickland, 1983; Dwyer and Stickland, 1991). In contrast, Handel and Stickland (1987a) found no difference in total fiber number or primary fiber number between small and large littermates at birth, and thus no difference in S:P ratio. We found a positive correlation between P-fiber number and pig carcass weight within litter (r = 0.24; P < 0.01). We also found that the S:P ratio was higher in low-weight pigs at slaughter compared with larger littermates. This indicates that more S-fibers were developed around each P-fiber in LW vs. MW and HW pigs, which is in contrast to findings by Wigmore and Stickland (1983) and Dwyer and Stickland (1991).
The estimation of P- and S-fiber numbers in this study was based on the method developed by Handel and Stickland (1987a). However, there is increasing evidence that some P-fibers might develop into type-II fibers, particularly in the white part of ST (Dunglison et al., 1999). If this were the case, the estimated P-fiber percentage (type I clusters) in this study would be underestimated. However, in this study, the estimated type-I cluster percentage was determined in each of the five muscle samples per animal on only half the number of animals (data not shown). Data analysis on these animals showed no interaction between the site of sampling within the muscle cross-sectional area (white or red part of ST) and the weight of the pig (LW, MW, and HW) for type-I cluster percentage. Thus, even though the estimated number of P-fibers may not be fully correct, the within-litter comparison of P- and S-fibers does not seem to be affected by this, and differences between weight groups therefore represents real differences.
Daily gain and meat deposition rate had a very low and nonsignificant correlation to total and S-fiber numbers in our study, which is in agreement with studies by Larzul et al. (1997). In contrast, Dwyer et al. (1993), Rehfeldt et al. (2000), and Herfort et al. (2001) found a significant correlation between daily gain and total muscle fiber number (r = 0.42; r = 0.44; r = 0.46, respectively). The lack of correlation between total fiber number and postnatal growth in this study may be explained by a significant difference in growth rate between LW and MW pigs, with no significant difference in total muscle fiber number.
Also, the growth of the individual muscle fiber is of overall importance to postnatal muscle growth. The MFA at slaughter at the same age reflects the growth potential of the muscle fibers. In contrast to our results, Handel and Stickland (1987b) found no difference in mean area of muscle fibers between large and small pig littermates selected by birth weight when pigs were slaughtered at the same age between d 0 and 128 postpartum. This indicates that the rate of muscle fiber growth was the same within litter.
The underlying cause of variation in size at birth has been suggested to be undernutrition in utero in low-birth-weight pigs, indicating a reduced number of muscle fibers developed prenatally and thus resulted in a lower postnatal growth rate (Dwyer and Stickland, 1991). In our study, pigs with a low to middle carcass weight had a lower number of both P- and S-fibers than did pigs with a high carcass weight, indicating a variation in the formation of muscle fibers prenatal. It is believed that variation in P-fiber number is more genetically controlled than the variation in S-fiber number (Wigmore and Stickland, 1983; Dwyer and Stickland, 1991). Because the P-fibers develop very early on in fetal development (approximately d 25 to 50 of gestation), it may be suggested that nutrients are not limiting to development at this stage. Thus, the variation in P-fiber number seen within litter is probably a normal variation in genetic potential within a litter of pigs, whereas the variation in S-fiber number (develops approximately between d 45 to 80 of gestation) is more variable and is affected by both genetic and environmental factors, such as nutrient supply.
Differences in MFA between littermates at the same age at slaughter reflect a difference in satellite cell proliferation and protein turnover. Thus, the higher MFA in HW and MW pigs in our study is in agreement with a higher amount of estimated DNA per fiber compared with LW pigs. The higher amount of DNA per fiber may have occurred due either to a higher rate of satellite cell proliferation postpartum or the fact that the individual muscle fibers had more satellite cells associated during prenatal development. The higher amount of RNA per fiber in MW and HW pigs is the basis for increased protein turnover as both synthesis of muscle specific proteins and proteolytic enzymes might be enhanced. Because fetal growth is highest during the last trimester, it might be that nutrient supply becomes limited to some fetuses at this stage, and that this has an impact on satellite cell proliferation, and thus on postnatal growth potential.
The intralitter variation in total DNA and DNA per muscle fiber may not be due solely to differences in muscle DNA. Also, differences in connective, epithelial, and nervous tissue may occur. Next to muscle tissue, connective tissue takes up the largest proportion of the entire muscle. Thus, minor differences in the amount of connective tissue between littermates might occur, but it is not possible to distinguish between muscle DNA (myonuclei and satellite cells) and other sources of DNA in this study.
Fiber number and fiber area are believed to have an impact on meat quality (Oksbjerg et al., 2000; Rehfeldt et al., 2000). In our study, MW and HW pigs had higher pigment content than LW pigs. Muscle pigment content increases with age and is thus related to muscle fiber size in the pig (Oksbjerg et al., 2000). This is in agreement with our results, where MW and HW pigs had a larger MFA than LW pigs. Pigment content is also positively related to redness of the meat (Oksbjerg et al., 2000). Although a significant variation was found in pigment content among pig weight groups, this variation was not large enough to cause variation in redness in the present study. Pigs with large muscle fibers and a high growth rate, such as the MW and HW pigs in this study, are also associated with a higher protein turnover than pigs with smaller muscle fibers and lower growth rate. A higher protein turnover may increase the synthesis of proteolytic enzymes and thereby have a positive effect on meat tenderness (Kristensen et al., 2002).
Implications
This study showed that both the number of muscle fibers and the growth rate of the individual muscle fiber could explain the large variation in postnatal growth performance within a litter of pigs. Thus, within-litter variation in muscle fiber formation during fetal development occurs, showing that pigs with the highest postnatal growth rate form more fibers prenatally than pigs with the lowest or middle growth rate. Also, a variation in muscle fiber growth rate postnatally shows that satellite cell proliferation and protein turnover vary within litter, with pigs with the lowest postnatal growth rate having the lowest muscle fiber growth. The reason for these findings is not clear.
Footnotes
1 The authors gratefully acknowledge the technical assistance of I. L. Sorensen, A. H. Madsen, L. Kjeldsen, S. G. Handberg, S. V. Blom, and C. Sorensen. 
2 Correspondence: Research Centre Foulum, P.O. Box 50, 8830 Tjele (phone: +45 89991279; fax: +45 89991564; e-mail: piam.nissen{at}agrsci.dk).
Received for publication July 7, 2003. Accepted for publication September 26, 2003.
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