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ANIMAL GENETICS |
* Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany; and
Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: longissimus dorsi • myosin heavy chain • pig • real-time polymerase chain reaction
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Accordingly, muscle fibers may be differentiated by their metabolic, contractile, or both, properties as oxidative, oxido-glycolytic, and glycolytic fibers or as slow-twitch oxidative, fast-twitch oxidative, and fast-twitch glycolytic fibers (Ashmore et al., 1972; Picard et al., 2002). The different fibers contain different myosin heavy chain (MyHC) isoforms. Four out of 8 isoforms known in mammals have been identified in porcine muscle (Chang and Fernandes, 1997).
By real-time PCR, a large proportion of fast isoform IIb contributing to cross-sectional fiber area of pig longissimus dorsi (LD) was uncovered (da Costa et al., 2002; Lefaucheur et al., 2004). Here, a question arises whether the differences in MyHC composition in pigs with small and large loin eye muscle area are due to differences in IIb fibers. We aimed to address the hypothesis that MyHC IIb is reflective for muscle growth-hypertrophy.
Therefore, relative proportions of MyHC isoform transcripts were determined by real-time PCR in animals from 3 pure breeds known to differ in muscularity (Pietrain, Duroc, and Mongcai) and 2 crosses [Duroc x Pietrain (DUPI) and Duroc x Berlin Miniature pigs (DUMI)] for comparisons between populations. Moreover, within the 2 crosses (DUPI and DUMI), sib pairs discordant for the trait longissimus area were examined to provide comparisons of extreme phenotypes within populations. Results of relative MyHC isoform transcript quantification were compared with results from histological profiling to assess the correlation between these 2 methods of determining the composition of skeletal muscle. We intended to evaluate the application of relative quantification of MyHC isoform transcripts by real-time PCR as a new tool to reflect muscle fiber types for further research toward understanding the significance of muscle type in modulating muscle growth.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All handling of the animals was done in accordance with German law for the protection of animals and was approved by the institutional animal welfare protection commission.
In this study, 3 pig breeds (Mongcai, Pietrain, and Duroc) and 2 crosses (DUPI and DUMI) were examined. All animals were free of the RYR1 mutation responsible for malignant hyperthermia syndrome. From the pure-bred Mongcai raised on a state farm in central Vietnam, 6 finishing pigs with BW 33.8 ± 2.4 kg at the age of 215 ± 7 d were sampled. Fresh LD muscle samples taken at the 13th to 14th ribs were stored in liquid N and further processed for RNA and cDNA synthesis at the advanced laboratory of Cantho University, Vietnam, as detailed below. All other animals were kept at the research farm Frankenforst, University of Bonn, Germany, where they were performance-tested according to the German performance test directives (ZDS, 2003). From the commercial breeds (Pietrain and Duroc), 9 unrelated animals were sampled (Pietrain and Duroc: carcass weight 83.5 ± 1.1 and 89.1 ± 3.1 kg, and age at slaughter 173 ± 6 and 197 ± 5 d, respectively). In addition, 2 F2 populations were generated and used in this study: a population based on reciprocal crossing of Duroc and Pietrain (DUPI) and a cross of Duroc and Berlin Miniature pigs (DUMI), which resulted from crosses of Vietnamese pot belly pigs, saddle back pigs, and German Landrace (Hardge et al., 1999). From the population of 598 F2 DUPI pigs, 30 animals of 15 full-sib families were used to compare the results of relative MyHC isoform transcript quantification by real-time PCR with the results from histological profiling. An additional 6 DUPI animals were selected that were discordant sibs of animals of the set of 30 animals, to establish a set of 6 discordant sib pairs, balanced for sex, representing extremes for traits related to muscularity and carcass composition. Thus, in total, 36 DUPI animals were used. Of 420 DUMI F2 animals, 6 discordant sib pairs, balanced for sex, were also selected. In both DUPI and DUMI resource populations, ranking of the animals was based on LM area, measured from digital images of a slice of LM taken between the 13th and 14th ribs.
Isolation of RNA and cDNA Synthesis
Tissue samples of all animals were taken from the LD between the 13th and 14th ribs. All samples were processed for RNA isolation and cDNA synthesis by the same person applying the same protocols either in the advance laboratory of Cantho University, Vietnam, or in the laboratory of the Institute of Animal Science, University of Bonn, Germany. Total RNA was isolated from LD muscle using Trizol Reagent (Invitrogen, Karlsruhe, Germany) according to the protocol of the manufacturer and treated with DNase I (Roche, Mannheim, Germany) to decontaminate trace genomic DNA. The RNA was then purified using the Qiagen RNeasy kit (Qiagen, Hilden, Germany). The RNA was quantified by photospectrometry, and its integrity was evaluated on 1% agarose gels containing formaldehyde and ethidium bromide.
The RNA and corresponding cDNA were used as templates in the PCR reactions using intron-spanning primers of the GAPDH gene to confirm the absence of genomic DNA. First-strand cDNA were synthesized from individual RNA using Superscript II enzyme (Invitrogen). In brief, the reaction was initiated by adding oligo (dT)15 primer and random primer to total RNA and incubating at 68°C for 5 min followed by cooling on ice for 5 min. A transcription mixture including first-strand 5X buffer, DTT, deoxynucleoside triphosphate, SuperScript II reverse transcriptase, and RNasin ribo-nuclease inhibitor was prepared to make a final volume of 20 µL. The reaction was incubated at 25°C for 5 min followed by 42°C for 1 h and stopped by heating at 70°C for 15 min. The cDNA were tested for their suitability to amplify the housekeeping gene, 18S rRNA, and kept at –20°C until further use.
Primer Design
Primer Express Software (version 2.0, Applied Biosystems, Darmstadt, Germany) was used to design primers for amplification of the MyHC isoforms and the 18S rRNA gene. The 4 MyHC isoforms share similar sequences. To obtain specific PCR products, these sequences were aligned, and primers were selected in specific regions showing low similarity. Primer sequences and PCR conditions are listed in Table 1.
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Reverse transcription and PCR were performed on samples from individuals from each group. Real-time reverse transcription-PCR was performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) in the laboratory of the Institute of Animal Science, University of Bonn, Germany. The reaction mixture consisted of cDNA, 0.5 µM of upstream and downstream primers, and SYBR Green Universal PCR Mastermix (Applied Biosystems) containing SYBR Green I Dye, AmpliTaq Gold DNA Polymerase, deoxynucleoside triphosphate with deoxyuridine triphosphate, passive reference, and buffer components. Thermal parameters used to amplify the template began with initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s denaturation, and 60°C for 1 min annealing and extension. A dissociation curve was generated at the end of the last cycle by collecting the fluorescence data at 60°C and taking measurements every 7 s until the temperature reached 95°C. Final quantification analysis was done by amplifying serial dilutions of target plasmid DNA. Using the ABI Prism 7000 SDS software (Applied Biosystems), the concentration of unknown cDNA was calculated according to the standard curve, and expression levels of transcripts were described relative to the transcript of the 18S gene, which was found to be stable between the samples containing equal amounts of analyzed cDNA. Based on these data, the relative abundance of the 4 adult MyHC isoforms was calculated as the ratio of the normalized expression level of each MyHC isoform to the total expression of MyHC.
Histological Analysis
To differentiate the 3 main fiber types, samples were stained using the myosin ATPase (mATPase) method (Brooke and Kaiser, 1970). In brief, cross-sectional samples of 10 µm were cut in a cryostat at –20°C from pieces of the center of a sample of the LD muscle taken between the 13th and 14th ribs. These sections were stained for mATPase activity after both acid (pH = 4.6 and 4.3) and alkaline (pH = 9.4) preincubation. The samples were examined to differentiate type I (slow, red muscle, oxidative), type IIa (fast, red muscle, oxidative), and type IIb/IIx (fast, white muscle, glycolytic) fibers. In total, 30 samples taken from F2 animals of the DUPI population were used. The determination of fiber type frequency was done on images captured with a CCD camera (Photometrix Sensys, Kew, Australia) and processed with IP-Lab Spectrum acquisition software (Scanalytics, Rockville, MD). Per sample, 400 fiber cross-sections were independently scored by 2 investigators, and fiber type frequencies were obtained from the ratio of the number of each fiber type to the total number of fibers counted (Wimmers et al., 2006).
Statistical Analysis
The SAS package (SAS Inst. Inc., Cary, NC) was used for statistical analyses. For each isoform, least squares means of the relative expression within the individual populations were estimated using PROC GLM of SAS and compared pairwise between populations using a t-test that was adjusted for multiple comparisons using the Tukey-Kramer correction.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). We aimed to evaluate the relationship between muscle fiber type composition as examined by histochemistry, LM area measured at the 13th/14th rib (suspected to be related to differences in muscle fiber type proportions), and relative abundance of MyHC isotype transcripts. Relative abundance of transcripts of MyHC isoforms determined by real-time PCR may be a new phenotype suitable as an alternative phenotype to muscle fiber typing based on histological methods in attempts to elucidate the relationship of functional and structural muscle properties with meat and carcass traits.
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), whereas ATPase staining after preincubation at pH 4.3 and pH 9.4 only allowed discrimination of type I and type II fibers. In the same samples, by real-time reverse transcription-PCR with specific primers for 4 adult MyHC isoforms, the relative abundance of the transcripts was determined. Correlations between these phenotypes ranged from 0.53 to 0.72 and were highly significant for the corresponding pairs of muscle samples characterized by histochemistry and relative MyHC isoform transcript abundance, respectively. Both methods showed that MyHC II fibers were predominant (Table 2).
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) between the 2 groups within both the DUPI and DUMI populations. Owing to genetic characteristics, larger LM areas were observed in DUPI than in DUMI pigs. However, this does not affect the shift of direction of fiber type composition, as seen in Figures 3 and 4. Discordant sib pairs in the DUMI and the DUPI populations differed significantly in the relative abundance of MyHC type IIb transcripts.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The growth and weight of muscle is mainly determined by the number and size of the muscle fibers and prolificacy of satellite cells. The proportion of different muscle fibers, their structure, and functional properties affect the growth performance of the animal and are endogenous factors for postmortem meat quality traits (Lengerken et al., 1994). The dramatic improvement of growth performance and lean content of pigs is suspected to coincide with altered meat quality. Inconsistency of results regarding the relationship of muscle fiber traits and meat quality and muscularity demonstrates the difficulty in determining the most advantageous muscle fiber types (Larzul et al., 1997; Fiedler et al., 1999, 2003; Lefaucheur et al., 2004). The fact that some muscle fibers cannot be assigned to a particular type due to intermediate properties, and the recent findings of 4 isotypes of adult MyHC protein in porcine muscle, whereas conventional muscle fiber typing only classifies 3 types of fibers, demonstrates the need to define new phenotypes to understand the relationship of muscle structural and functional properties with meat quality and quantity and genetic control mechanisms.
Breed probably accounts for most of the genetic factors affecting the muscle fiber composition of a certain muscle. Pietrain is well known for its muscularity and leanness, whereas Duroc has desirable meat quality in terms of marbling, tenderness, and juiciness. The third breed, Mongcai, is a popular local breed in the Central coastline, the Red River Delta, and other northern provinces of Vietnam with favored characteristics of high prolificacy, good adaptation to poor-quality feed, and disease resistance but low performance with small body size and low growth rate. Genetically, Pietrain, Duroc, and Mongcai are ostensibly distinct breeds in growth and muscularity. Enormous genetic variation among these breeds could account for the variation in MyHC relative expression across breeds.
The relative number of white fibers (IIx and IIb types) was indicated to contribute up to 85.2% of total fiber number in Pietrain (Müller et al., 2002), whereas only 8.5% of muscle fibers were type I fibers. Moreover, in Duroc muscle, the composition of muscle fiber types was slightly different (i.e., 14.5% for type I and 81.5% for type IIx and IIb) from that of Pietrain (Chang et al., 2003). Results from the current study are comparable (86.3 and 77.8% for IIb + IIx in Pietrain and Duroc, respectively) to those findings and, therefore, support the statement that, in modern pig breeds, fiber composition is directed to a greater proportion of larger type IIb fibers (Ruusunen and Puolanne, 2004). Similar pro-files of muscle fiber types were reported in other commercial pure breeds, including Large White (Lefaucheur et al., 2002, 2004; Chang et al., 2003) and Landrace and Yorkshire (Ruusunen and Puolanne, 1997). In addition to the identification of muscle fiber composition in commercial pigs, the proportions of these fibers were also determined for the native Mongcai breed, in which a dramatic difference in relative expression profile was found. Indeed, the proportion of IIx and IIb fibers in Mongcai pigs was lower (47.3%) compared with other conventional breeds, as analyzed here and in other studies involving breeds such as Berkshire (85.7%), Hampshire (75.3%), and wild boar (84.2%) (Ruusunen and Puolanne, 1997; Müller et al., 2002; Chang et al., 2003). When taking only IIb fiber into consideration, the proportion in Meishan LD muscle (Lefaucheur et al., 2004) was nearly in line with our findings in Mongcai (17.1 and 11.4%, respectively), but in Meishan, IIx fibers were shown to be most prominent (61.1%; Lefaucheur et al., 2004), whereas in Mongcai, type I, IIa, and IIx were equally frequent.
The percentage of MyHC IIb is an important feature, because it contributes to increases in muscle mass, which is desired by animal breeders. Moreover, as shown in this and other studies, the IIb isoform is the predominant form in LD muscle and is most likely responsible for the formation of large muscle fiber types. Kristensen et al. (2002) reported that large muscle fibers and high growth rate are also associated with greater protein turnover, which may increase the synthesis of proteolytic enzymes and thereby have a positive effect on meat tenderness. Nevertheless, this conclusion is not always in agreement with results of other experiments. For instance, Chang et al. (2003) concluded that color characteristics, better water-holding capacity, and better tenderness were positively related to the presence of oxidative fibers, and, hence, the main favorable fiber types were IIa and IIx. Sensory meat quality is closely related to the content of i.m. fat, but various studies showed different outcomes regarding the location of lipids in fiber types. Whereas Henckel et al. (1997) reported that the frequency of IIb fiber and i.m. fat content were positively correlated, Essen-Gustavsson et al. (1994) found lipids present mainly in type I/slow and some IIa fibers. Mongcai is known to have high levels of i.m. fat and thereby shows highly preferred meat quality but unsatisfactory muscularity in comparison with commercial breeds with high muscularity but inferior meat quality. Although the lipid content was not determined in the current study, the high percentage of MyHC IIa isoform from Mongcai muscle suggests an important role in lipid storage. That high values of i.m. fat were found in a cross of Duroc x Berlin Miniature pigs additionally supports this view (Fiedler et al., 2003).
Muscle Fiber Composition of Large and Small Muscle Within Breed
Muscle fiber type distribution in commercial cross-bred pigs is similar to that of pure breeds. As an example, Ryu et al. (2004) showed that the greatest percentage of myofibers was white fiber IIx and IIb with a value of 80.2% in a Duroc x (Yorkshire x Landrace) cross. A slightly lower proportion was previously reported by Fiedler et al. (1999) for a Pietrain x Landrace cross (75%). Results from our study indicated the trend that the contributions of IIx and IIb fibers in DUPI were lower compared with the pure breeds (Duroc and Pietrain) and vice versa for type I fibers, which were elevated by up to 15% in the DUPI. However, differences were not statistically different at P < 0.05. In the DUMI resource population, there was evidence that the genetic background of Berlin Miniature pigs led to decreased IIb fiber proportion (66.7%), whereas the relative expression of type I increased to 25.9%. Correspondingly, muscle fibers detected by histochemical differentiation had contributions of 71% (type IIb and IIx) and 15.3% (type I; Fiedler et al., 2003).
In this study, we examined relative MyHC isoform transcript levels as an alternative phenotype to assess muscle fiber composition of the same muscle (LD) between 2 pig groups within 2 populations (DUPI and DUMI). Several factors potentially affect fiber proportion, including birth weight, growth rate, and slaughter weight. Although birth weight was indicated to have an association with enlarged muscle fiber area (Gondret et al., 2006), its influence on muscle fiber composition of LD muscle and other muscles such as longissimus lumborum or rhomboideus was not found (Bee, 2004). Growth rate also has an effect on the cross-sectional area of fibers, especially on IIa, which was shown to increase with increasing growth rate (Ruusunen and Puolanne, 2004). However, the IIa fibers are a minor fiber with low numbers shown in most studies; therefore, they may not have strong effects on muscle size. Ryu et al. (2004) discovered that muscle fiber composition was not significantly associated with carcass weight or loin eye area. Increasing BW and age at slaughter significantly affected cross-sectional area but did not change the numerical percentage of any fibers (
andek-Potokar et al., 1999). Thus, combining available findings with the controlled pig selection in our study, these weight and growth traits can be excluded as factors altering muscle fiber proportions.
Muscle Fiber Typing by mATPase Staining and the Consistency Between Real-Time PCR and Histochemistry Methods
Various methods of muscle fiber differentiation describe different phenotypes of the muscle. Muscle fiber types are characterized by the content of different MyHC isoforms, enzymes, structural proteins, and organelles that warrant specific biochemical and biophysical properties of the fibers. A combination of different methods of examining the fiber types may yield the most comprehensive characterization of muscles and further analysis of the genetic mechanisms of muscle growth. Conventionally, histochemistry by mATPase stability has been used in most studies for many years. Using this methodology, porcine fibers can be classified into type I, IIa, and IIb (Lefaucheur et al., 1991; Fazarinc et al., 1995). For more accurate muscle typing to distinguish pure IIx and IIb, and the hybrid of these 2 fibers, immunocytochemistry with specific antibodies and quantitative real-time PCR have been applied more recently (Lefaucheur et al., 2004; Toniolo et al., 2004). The fiber proportions in our research were similar to the findings reported by Chang et al. (2003) and Lefaucheur et al. (2004), despite slight differences that were likely due to breed-specific effects.
In summary, we have compared histological and quantitative real-time assays to estimate the proportion of fiber types in LD porcine skeletal muscle. The highly significant correlations between the corresponding MyHC isoforms evidenced the consistency of results between the 2 methods. The real-time PCR quantification of MyHC isoform transcripts reveals an average value of the contribution of the different MyHC isoforms to the muscle phenotypes. The formal classification into muscle fibers is artificial as shown by the slightly different results depending on histological method used and the existence of hybrid fiber types. The real-time PCR assay represents a new phenotype close to the effect of genes, which is probably more suitable to unravel the genetic background in variation of traits related to muscle and meat properties depending on muscle fiber distribution. However, this assay does not provide information on fiber size and fiber number.
Moreover, we also observed that differences in fiber composition between large and small muscle groups were due to type IIb, which is not surprising, because in most cases, it contributes greatly to the composition of muscle fiber. By analyzing well-selected sib pairs of the experimental populations DUPI and DUMI, which were specifically discordant in the trait loin eye muscle area but similar in other traits that are possible factors altering the fiber proportion (Table 4
), we can, for the first time, demonstrate that IIb is the determining fiber contributing to the differentiation of large and small loin eye muscle area in the pig. The comparison among different breeds confirmed the trend of high MyHC IIb transcript abundance going together with high muscularity. Further studies are encouraged to discover the genetic backgrounds or which genes are involved in this phenomenon.
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Footnotes |
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2 Corresponding author: s.wimmers{at}fbn-dummerstorf.de
Received for publication August 1, 2006. Accepted for publication December 13, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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andek-Potokar, M., L. Lefaucheur, B. 
lender, and M. Bonneau. 1999. Effect of slaughter weight and/or age on histological characteristics of pig longissimus dorsi muscle as related to meat quality. Meat Sci. 52:195–203.[CrossRef]This article has been cited by other articles:
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  Y. J. Xu, M. L. Jin, L. J. Wang, A. D. Zhang, B. Zuo, D. Q. Xu, Z. Q. Ren, M. G. Lei, X. Y. Mo, F. E Li, et al. Differential proteome analysis of porcine skeletal muscles between Meishan and Large White J Anim Sci, August 1, 2009; 87(8): 2519 - 2527. [Abstract] [Full Text] [PDF]
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