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* Department of Food Science and
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Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, Tjele, Denmark, and
and
Department of Anatomy and Physiology, the Royal Veterinaryand Agricultural University, Copenhagen, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Growth • Maternal Nutrition • Meat Quality • Muscle Fiber • Pig
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Rehfeldt et al., 1999; Pedersen et al., 2001). Doubling the feed intake of sows in restricted periods of gestation results in a tendency toward an increased number of muscle fibers in the offspring with a positive effect on postnatal growth (Dwyer et al., 1994). Others have shown that feeding strategies involving either reduced protein diets or feed level below 2 kg/d during gestation severely reduce postnatal growth of the offspring in pigs (Buitrago et al., 1974; Schoknecht et al., 1993). It therefore seems that maternal nutrition may have an effect on myogenesis and subsequent postnatal growth of the offspring.
Muscle fibers develop in the fetal stage, and the number is fixed at birth in pigs (Wigmore and Stickland, 1983). Two populations of fibers are formed: the first population to be formed in pigs (d 25 to 50) are primary fibers (P-fibers) followed by a secondary fiber population (S-fibers) (d 45 to 80), using P-fibers as templates (Swatland and Cassens, 1973; Ashmore et al., 1973). It is believed that the S-fiber population is more susceptible to environmental factors than is the P-fiber population (Wigmore and Stickland, 1983; Dwyer and Stickland, 1991).
Studies have shown that fast-growing pigs with a high number of muscle fibers with a small cross-sectional area produce meat with a better quality than pigs with a low number of muscle fibers with a large cross-sectional area, thus indicating that muscle fiber number and area have an impact on meat quality (Oksbjerg et al., 2000; Rehfeldt et al., 2000).
The purpose of the present study was to examine how increased feed intake of the sow during early- to mid-gestation affects sow performance and muscle fiber number, performance, and meat quality of the offspring.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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).
Slaughter Procedure and Muscle Sampling
The pigs were slaughtered litterwise at an average live body weight of 104 ± 14 kg. The carcass weight was measured right after slaughter. Also just after slaughter, the right semitendinosus muscle (ST) was dissected and weighed, and a cross-section from the mid-belly of the muscle was taken for measurement of muscle cross-sectional area. The cross-sectional area was photographed 2 to 5 h postmortem. Five muscle samples (200 mg/sample) were taken at fixed points within the muscle cross-sectional area for histochemical analysis. One sample was taken in the red part of ST, whereas the other four samples were taken in the white part. The average of the five samples was used for data analysis. The samples were mounted in embedding medium (catalog no. 4583; O.C.T. Compound, Proshop, Vaerlose, Denmark) and frozen in isopenthane (-160°C) cooled in liquid nitrogen. Another sample (200 mg) was taken at the center for DNA and RNA analysis and frozen in liquid nitrogen. The samples were kept at -80°C until analysis was performed.
Pig Performance
Pig body weight changes from 30 kg until slaughter at 104 ± 14 kg were used for calculation of daily gain. Percentage of meat was measured with a Fat-O-Meater 24 h postmortem (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.
Meat Quality Measures
At 24 h postmortem, the pH (pH24) was measured in longissimus dorsi (LD) muscle at the last rib in the left side. Two samples were removed from LD at the last rib in 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 content was analyzed using the method described by Oksbjerg et al. (2000). Drip loss was measured over 48 h on approximately 100 g of LD muscle using the plastic bag method described by Honikel (1998).
Histochemistry
The muscle samples from the heaviest (HW), middle (MW), and lightest weight (LW) pigs of each sex selected by carcass weight were analyzed per litter. In some litters, the sex distribution was not even; thus, for these litters, less than six pigs per litter were analyzed. Transverse serial sections (10 µm) were cut in a cryostat at -20°C, picked up on cover slips, and allowed to thaw and dry at room temperature overnight. The sections were immunohistochemically stained for slow myosin heavy-chain (MHC) isoform (CRL-2043; American Type Culture Collection, Rockville, MD) to identify type I fibers. Fibers not staining for slow MHC isoform are referred to as type II 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. The methods used for immunohistochemistry are described by Pedersen et al. (2001).
The percentage and the mean area of fiber type I and II and the mean area of all fibers (MFA) were calculated on the basis of 200 to 300 fibers per muscle sample (five samples/animal), using a computer-assisted image analysis system (TEMA; CheckVision Aps, 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 numbers of P- and S-fibers were estimated on the basis of type I fiber clusters. During gestation, the P-fibers develop into type I fibers, whereas the S-fibers develop into type II fibers. As the muscle matures, some of the type II fibers nearest to type I fibers convert into type I fibers. Thus, postnatally there will be clusters of type I fibers surrounded by type II fibers. According to Handel and Stickland (1987) only one fiber from each type I cluster 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 Picogreen dsDNA quantification reagent kit (P-7589, Molecular Probes, Leiden, Netherlands), and RNA concentrations were measured using 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
For analyzing sow and litter performance, ANOVA was performed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC), with boar and treatment groups as class variables. Litter size at birth was included as a covariate for weights of piglets at birth and weaning when significant. Milk yield estimation was analyzed by a split-plot model using the MIXED procedure of SAS, with treatment, week of lactation, and their interactions as fixed effects, and with boar and sow nested within boar as random effects. All pigs within litter were included for analysis of litter performance.
The LW, MW, and HW pigs of each sex within litter were used for analysis of pig performance, meat quality, histochemistry, and DNA and RNA content. Data were analyzed using the MIXED procedure of SAS, with treatment, sex, pig weight, and interactions between these as fixed effects. Sow, boar, and interactions between sow or boar and the fixed effects were used as random effects. The ratio between female pigs and barrows and litter size at birth was used as covariate when significant. Also, carcass weight was used as a covariate in the analyses of meat quality aspects and percentage of meat, and days at slaughter was used as a covariate in analysis for muscle mass, muscle deposition rate, weight, and cross-sectional area of ST, fiber area, and DNA and RNA content when significant. The five muscle samples analyzed per animal for histochemical properties were used as repeated measurements with animal as subject.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). The extra feed was 116 and 181 kg, and the total extra NE was 864 MJ and 1,351 MJ, respectively, for A25–50 and A25–70. The body weight gain of sows was increased by 35 and 54 kg, respectively. In lactation, there was no significant difference in feed intake among treatments, although there was a tendency for a higher weight loss of sows in the two treatment groups (P = 0.06). Litter size at birth and weaning and also birth and weaning weights were similar across treatments.
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Pig Performance and Meat Quality
There was a negative effect of treatment A25–50 on muscle ST weight (P = 0.019) compared with control and A25–70 pigs (Table 3). Although not significant, average daily gain (P = 0.149), carcass weight (P = 0.162), and muscle deposition rate (P = 0.07) were also numerically lower for pigs from treatment A25–50. Interactions between treatment and pig weight within litter were found for muscle mass (P = 0.012), muscle deposition rate (P = 0.006, Figure 1), and carcass weight (P = 0.104). The LW pigs from A25–50 had a lower muscle deposition rate than control pigs, whereas MW and HW pigs from A25–50 were not significantly different from control pigs. Although not significantly different, MW and HW pigs from treatment A25–70 had higher deposition rates than control and A25–50 pigs. The same trends were evident for muscle mass and carcass weight. There was no effect of maternal nutrition on any of the measured meat quality traits (Table 4). The within-litter variation in all measured characteristics is addressed by Nissen et al. (2003).
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). The S/P ratio and the percentage of type I fibers are presented in Figure 2, but neither was influenced by treatment. Also, the areas of type I and type II fibers, and, consequently, the mean fiber area (MFA), were not different across treatments (Figure 2c).
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DNA and RNA Content
There were no effects of maternal nutrition on measured concentrations or calculated amounts of DNA and RNA in ST (Table 5).
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. The P-fiber number was found to be higher in barrows than in female pigs (P = 0.034), but there was no effect of sex on total- or S-fiber number. There was also no difference in fiber area between barrows and female pigs. No significant differences were found between sexes on any measures of DNA and RNA content of ST (Table 5
), although DNA concentration tended to be higher in female pigs than barrows (P = 0.091) and RNA/DNA ratio therefore tended to be lower in female pigs (P = 0.082).
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, barrows had a higher average daily gain (P < 0.0001) and carcass weight (P < 0.0001) than female pigs. Also, calculated muscle mass tended to be higher in barrows than in female pigs (P = 0.12). On the other hand, female pigs had a higher meat percentage (P < 0.0001) than barrows. No significant differences were found on any measured meat quality traits (Table 4). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. Others have found that increased feed intake throughout or in parts of gestation have a negative effect on feed intake during lactation (Dourmad, 1991; Weldon et al., 1994; Close and Cole, 2000), which might influence milk yield. We did find a tendency toward an interaction between treatment and week of lactation on milk yield, where sows on treatment A25–50 and A25–70 had lower milk yield in the first week of lactation compared with control sows, but this was compensated for in the second and third week of lactation. The similar lactation feed intake among treatments and slightly increased milk yield is in accordance with the larger weight loss in A25–50 and A25–70 sows. Thus, increasing gestation feed intake in early to mid-gestation does not seem to influence lactation feed intake and average milk yield significantly. Litter size at birth and at weaning and average piglet birth weight in this study were not affected by different feeding strategies during gestation, which is in agreement with previous studies (Pond et al., 1987; Dwyer et al., 1994).
The postnatal growth of muscle tissue is dependent on muscle fiber number and muscle fiber area. Muscle fibers are formed prenatally and might be influenced by maternal treatments during gestation, thereby affecting postnatal muscle growth of the offspring. In this experiment, the estimated number of total muscle fibers of the offspring was not significantly affected by maternal nutrition, and the S/P ratio was also similar across treatments, which is in contrast to results obtained by Dwyer et al. (1994). Some studies with guinea pigs and rats have shown that nutritional restriction compared with ad libitum feeding during gestation causes a reduction in the number of muscle fibers of the offspring (Dwyer et al., 1995; Wilson et al., 1988), whereas other studies with rats and lambs do not show any difference in muscle fiber number between offspring from ad libitum or restrictively fed dams or single contra-twin fetal lambs (Beermann, 1983; Nordby et al., 1987; McCoard et al., 2000). Hormonal treatment of pregnant sows in early gestation with pGH causes an increase in the number of muscle fibers in low- and middle-weight pigs (Rehfeldt et al., 1993; Rehfeldt et al., 2001).
Thus, there are contradictory results on how different maternal feeding strategies affect myogenesis in the offspring. In experiments where restrictive and ad libitum maternal feeding are compared, the grade of restriction seems to affect the traits observed. Thus, restrictive feeding under requirements probably affects myogenesis, whereas feeding at or above requirements has less or no effect on muscle fiber development in the fetus. The time period of treatment during gestation, the nutrients, and amounts involved in the treatment and the muscle investigated also seem to be important (Ward and Stickland, 1991; Dwyer and Stickland, 1994; Dwyer et al., 1994; Dwyer et al., 1995). There may also be species differences, especially in relation to number of fetuses per parity, as fetuses with littermates probably are more prone to undernutrition in utero due to competition for nutrients than singleton fetuses. Also, variation in litter size within species may influence the variation in muscle fiber number. Thus, we found a negative correlation between number of pigs born per litter and number of muscle fibers (r = -0.50; P = 0.002). In this study, the maternal feed intake was very high during the period of ad libitum feeding compared with control. Whether such a high feed intake can cause hormonal changes in the dam, so that nutrients are directed toward maternal tissues instead of fetal tissues, is not known, but the sows did put on a lot of weight with no effect on offspring birth weights.
The estimate for muscle fiber number is dependent on the measured cross-sectional area of ST, and any factors affecting the cross-sectional area will affect the estimation of muscle fiber number. Thus, whether the muscle was in pre- or postrigor at the time of measure will be of importance to the estimated number of muscle fibers. The muscle cross-sectional area was measured 2 to 5 h postmortem, and although we did not measure whether the muscle had reached rigor, the muscle slice was cold and no spontaneous contractions could be seen, when the muscle cross-sectional area was photographed, implying that the muscles was in postrigor.
Postnatally, the growth of the individual muscle fiber is of great importance to the overall muscle growth, and the MFA can be used as a measurement of postnatal muscle fiber growth rate, if the data studied are from animals of the same age. The muscle fiber growth rate is dependent on satellite cell proliferation and protein synthesis and degradation. Accumulation of DNA postnatally reflects satellite cell proliferation, and the concentration of RNA is related to the protein synthesis capacity.
We found no effect of maternal nutrition, neither on the area of type I and II fibers nor, consequently, on MFA at the same age. In fetal sheep at 140 d of gestation, a significant difference in muscle fiber area was found between single and twin lambs in parts of the adductor femoris muscle (11 to 24% slow-twitch fibers) (McCoard et al., 2000). These results show that there might be muscle variation in the response to intrauterine growth restriction on fiber area and that the period of restriction during gestation is of importance because the difference between single and twin lambs can be compared to restriction throughout gestation.
Maternal feeding did not have a significant influence on any DNA and RNA measurements in this experiment, indicating that neither satellite cell proliferation nor protein synthesis capacity had been affected. Other studies in pigs and lambs have shown that nutritional deprivation and hormonal treatment during gestation does not affect the concentrations of DNA and RNA in muscle (Pond et al., 1990; Pond et al., 1991; Rehfeldt et al., 1993; Schoknecht et al., 1993; Greenwood et al., 1999; McCoard et al., 2000), whereas studies in lambs have shown that nutritional deprivation decreases the muscle weight, the total amount of DNA and DNA per fiber in muscle (Greenwood et al., 1999; McCoard et al., 2000). As intrauterine growth restriction had no effect on muscle fiber number in lambs (Nordby et al., 1987; McCoard et al., 2000), the difference in muscle weights and amounts of DNA might be explained by a reduced satellite cell proliferation, which decreases the amount of DNA and thereby reduces the protein synthesis. Thus, the total amount of DNA and probably DNA per muscle fiber seems to follow the size of the muscle or fiber, showing that increased muscle growth is dependent on increases in the amount of DNA.
The percentage of type I fibers was not different across treatments in this study, which is consistent with our other results, showing no effect of maternal feeding on muscle fiber characteristics and meat quality traits. Experiments with lambs equally show no effect of intrauterine growth restriction on the percentage of fiber types in various muscles (Nordby et al., 1987; Greenwood et al., 1999; McCoard et al., 2000).
Pigs from treatment A25–50 had a reduced weight of ST, and the significant interaction between treatment and pig weight for muscle mass and muscle deposition rate suggests that muscle growth was compromised in LW pigs compared to MW and HW pigs, which were not different from control. Although not significant, MW and HW pigs from treatment A25–70 also had higher muscle deposition rates than control and A25–50 pigs. These results could not be explained by the data on muscle fiber characteristics. Thus, it seems that increased maternal feeding from d 25 to 50 is a disadvantage for the offspring in relation to muscle growth, but prolonging the period of increased maternal feeding from d 25 to 70 overrules this disadvantage. This suggests that there must be a kind of compensation in relation to growth in the fetuses from d 50 to 70. One could then speculate whether the possibility of increased maternal feeding only from d 50 to 70 might be advantageous to the offspring in relation to muscle growth compared with control animals.
No significant effects of treatments were found on any meat quality traits. This is to be expected, as we did not find any significant effects of treatments on fiber number, fiber area, and fiber type distribution.
Sex Differences
Differences between sexes in this experiment were seen on the number of P-fibers, with barrows having a higher number than female pigs, whereas there was no significant effect of sex on total- and S-fiber number, S/P-ratio, or the percentage of type I fibers. Other studies in pigs and rats did not show any effect of sex on muscle fiber number (Beermann, 1983; Dwyer et al., 1994), whereas studies in lambs show that wether lambs have higher muscle fiber number than ewes (Nordby et al., 1987; Shackelford et al., 1995).
The MFA and area of type I and II fibers were not influenced by sex in this study when compared at the same age. Other studies in pigs and lambs have shown that male animals have a smaller cross-sectional area of ST than female animals, but in these studies the comparison has been at the same body weight (Petersen et al., 1998; Nordby et al., 1987). In contrast, Shackelford et al. (1995) did not find any effect of sex on fiber area in LD and psoas major.
No significant differences were found between sexes on any DNA and RNA measurements in ST, which is consistent with our findings on muscle mass, where we also found no difference between sexes. In rats, Beermann (1983) found significant differences between sexes on muscle weights, total DNA, and RNA but not on concentrations of DNA or RNA in various muscles. Theses studies were comparing intact males with females, making a large difference in postnatal growth characteristics compared with castrated males.
As expected, barrows had significantly higher average daily gain and carcass weight than female pigs, and also muscle mass tended to be higher in barrows than female pigs. Percentage of meat, on the other hand, was higher in female pigs than in barrows. As we found no effect of sex on total DNA or the weight of ST, and because the meat percentage was higher in female pigs than in barrows, some of the difference in daily gain and carcass weight between sexes must be due to a higher degree of fat deposition in barrows. If the male pigs in this experiment had been intact males, we might have found significant differences in the weight of ST, muscle mass, and muscle deposition rate between sexes. No significant effects of sex were found on any meat quality traits.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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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 April 25, 2003. Accepted for publication August 11, 2003.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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—), or fed ad libitum from d 25 to 50 (A25–50, —
—) or from d 25 to 70 (A25–70, —
—) of gestation and as control in the remaining periods. Within pig weight group, values with different letters differ (P < 0.05).