THE ANATOMICAL RECORD PART A 281A:1363–1371 (2004) Skeletal Muscle Development in Normal and Double-Muscled Cattle JULIE K. MARTYN, JOHN J. BASS, AND JENNY M. OLDHAM* Growth Physiology, Animal Genomics, AgResearch, Hamilton, New Zealand ABSTRACT This study examined the effect of genotype on prenatal muscle development in both normal-muscled (NM) animals and in double-muscled (DM) animals harboring a mutation in the gene for myostatin that results in the production of a functionally inactive protein. The following muscle development parameters were analyzed at four gestational ages: muscle weight, ﬁber type, by both enzyme histochemistry and myosin heavy-chain (MHC) immunocytochemistry, and average ﬁber area. The weights of both M. vastus lateralis and M. vastus medialis were greater throughout prenatal development in the DM animals compared to NM. The percentage of type 1 muscle ﬁbers initially declined with gestational age and subsequently increased in both NM and DM. The percentage of type 1 ﬁbers was consistently lower in DM than in NM. A pattern of MHC isoform localization was shown in DM muscle that is indicative of a delay in muscle development relative to NM. Muscle ﬁber size was differentially regulated in NM and DM, depending on ﬁber type. Type 1 ﬁbers were smaller in DM than NM in late gestation, while type 2 ﬁbers were smaller throughout gestation. This study suggests that the inactivating myostatin mutation in DM animals may be associated with changes in both skeletal muscle ﬁber type and ﬁber size during bovine muscle development. © 2004 Wiley-Liss, Inc. Key words: bovine; muscle; development; ﬁber type; ﬁber size; myosin heavy chain; myostatin The major determinants of skeletal muscle mass are muscle ﬁber number and muscle ﬁber size. During development, these factors are controlled by a series of events, including myoblast proliferation, myotube formation, and myoﬁber maturation. A number of regulatory factors can inﬂuence each of these stages of muscle development, including the genetic background and breed of an animal. Within double-muscled (DM) cattle breeds, there is an overall increase in muscle mass (Boccard, 1981). The genetic basis of the DM condition in Belgian Blue animals arises from an 11 bp deletion mutation in the gene for myostatin in these animals, resulting in a severely truncated myostatin protein (Grobet et al., 1997; Kambadur et al., 1997; McPherron and Lee, 1997). Myostatin is a negative regulator of muscle mass (McPherron et al., 1997), which has been shown to inhibit both myoblast proliferation (Thomas et al., 2000; Joulia et al., 2003) and differentiation (Langley et al., 2002; Rios et al., 2003). Each of these effects is mediated through differential regulation of a key component of cell cycle progression, the cyclin-dependent kinase inhibitor, p21. In addition to regulation of cell cycle progression, myostatin also has a direct effect on myogenesis through its action in regulating levels of the © 2004 WILEY-LISS, INC. myogenic regulatory factors (Langley et al., 2002; Joulia et al., 2003). This dysregulation of myogenic proliferation and differentiation leads to muscle hyperplasia and hypertrophy as seen in myostatin-null animals (Langley et al., 2002). The extent of the muscular hypertrophy exhibited by DM animals has been reported to vary among different muscles in a number of DM breeds (Butterﬁeld, 1966; Rollins et al., 1969; Boccard, 1981). Muscles with a large surface area tend to be the most enlarged, while deeper muscles tend to be reduced in size relative to NM. As all of Grant sponsor: the New Zealand Foundation for Research, Science and Technology. *Correspondence to: Jenny M. Oldham, Animal Genomics, AgResearch, PB3123, Hamilton, New Zealand, Fax: 64-7-8385536. E-mail: email@example.com Received 18 September 2003; Accepted 22 March 2004 DOI 10.1002/ar.a.20140 Published online 5 November 2004 in Wiley InterScience (www.interscience.wiley.com). 1364 MARTYN ET AL. these muscles have increased muscle ﬁber numbers, it follows that the reduced muscles must have a smaller ﬁber size (Ouhayon and Beaumont, 1968). No studies have been carried out to determine if these differences in muscle mass and ﬁber size are present in prenatal animals, so we examined the development of the M. vastus lateralis, which is 15% heavier in adult DM than NM, and M. vastus medialis, which is 38% lighter in mature animals (Boccard, 1981). Double-muscled cattle have a different composition of histochemical ﬁber types compared with NM, both during prenatal development (Ashmore et al., 1974) and in the postnatal animal (Holmes and Ashmore, 1972), where there are increased proportions of type 2 muscle ﬁbers and fewer type 1 ﬁbers. In addition to histochemical staining, myosin heavy-chain (MHC) immunohistochemistry has been widely used in the investigation of muscle development in DM animals. The sequence of MHC isoform transitions which myotubes undergo as they mature is an important determinant of skeletal muscle ﬁber type. The relationship between the patterns of MHC isoforms expressed by primary myotubes and the ﬁber type composition of mature muscles is not yet fully understood (Whalen et al., 1984; Dhoot, 1986; Zhang and McLennan, 1998). Immunohistochemistry using antibodies against developmental MHC isoforms has enabled changes in MHC isoform expression to be examined during normal bovine development (Robelin et al., 1993; Picard et al., 1994, 1995b) and in DM animals (Picard et al., 1995a). This latter study reported developmental differences in MHC expression between NM and DM, with DM tending to express more immature isoforms at the same gestational age during the ﬁrst two-thirds of fetal life (Picard et al., 1995a). In normal bovine muscle, ﬁber size varies according to ﬁber type. Type 2B ﬁbers have the largest cross-sectional area, type 2A ﬁbers are intermediate, and type 1 ﬁbers are the smallest. In DM cattle, muscle ﬁber size is altered relative to NM, with both increases and decreases in ﬁber size being reported. These variations in ﬁber size are related to differences in ﬁber type composition and animal age (Holmes and Ashmore, 1972; Ashmore, 1974). In postnatal animals, there is an increase in the size and frequency of type 2B ﬁbers in DM animals (Holmes and Ashmore, 1972) and this contributes to the overall increase in the whiteness of meat from these animals. In muscles from adult DM animals that are decreased in size relative to normal animals, average muscle ﬁber size is smaller (Ouhayon and Beaumont, 1968). The aims of this study were to test the hypothesis that the relative difference in size between M. vastus lateralis and M. vastus medialis that occurs in adult NM and DM animals was present during prenatal development, and that differences in ﬁber type composition and ﬁber size during prenatal development contribute to overall differences in skeletal muscle mass between NM and DM fetuses. of one hind limb from each animal and weighed. A 5 mm slice was taken through the mid belly of the muscle, at right angles to the direction of the muscle ﬁbers. These samples were stored at ⫺80°C for ﬁber typing and immunohistochemistry. There were ﬁve fetuses in each of the age groups for the normal animals and in the 120- and 160-day groups for the DM and three in the 210- and 260-day groups for the DM. These gestational ages were selected to cover the late stages of primary myotube formation, which is nearing completion by 120 days (Stickland, 1978; Robelin et al., 1991), a period of active secondary ﬁber formation at around 160 days (Stickland, 1978) and a period when all ﬁbers were undergoing hypertrophic growth (210 –260 days). This study was carried out with the approval of the Animal Ethics Committee of Ruakura Research Center. MATERIALS AND METHODS Animal Data Statistical analysis of muscle weight, ﬁber type, and ﬁber size was carried out using analysis of variance with age and breed as the main effects. Data were log-transformed before analysis as required and back-transformed for presentation of results. Values are presented as means; errors are pooled or individual standard errors of the means (SEMs). Covariate analysis was carried out within groups using sex ratio and body weight and estab- Normal-muscled (NM) and double-muscled calves were generated as previously described (Oldham et al., 2001). Fetuses at 120, 160, 210, and 260 days of gestation were collected after slaughter of the recipient cows. The M. vastus lateralis and M. vastus medialis were dissected out Fiber Typing Muscle ﬁber typing was carried out on the M. vastus lateralis according to a modiﬁcation of the myosin ATPase method of Guth and Samaha (1969). After ﬁxation, slides were incubated in 0.1 M potassium acetate buffer at pH 5.0 for 10 min. The remainder of the procedure was identical to that of Guth and Samaha (1969). This procedure results in a staining pattern identical to that of ﬁxed sections preincubated at pH 9.4, with type 1 ﬁbers showing lighter staining and type 2 ﬁbers darker, but with improved histology. Immunohistochemistry Immunohistochemistry was carried out on serial cryostat sections of M. vastus lateralis. Slides were ﬁxed in neutral buffered formaldehyde, blocked in dilute normal serum, and incubated using primary antibodies speciﬁc for fast MHC (MY32), slow MHC (NOQ.4.D; Sigma, St. Louis, MO), and embryonic MHC (2B6; gift of Dr. D. A. chman). Detection of the primary antibody was carried out using a biotinylated secondary antibody followed by streptavidin-biotin complex and diaminobenzidine substrate. Image Analysis Three sections stained for mATPase activity were analyzed from each group for ﬁber type proportions and average area for each ﬁber type. All ﬁbers within each of ﬁve fascicles were analyzed for each animal, giving a total of 200 –300 ﬁbers per muscle. The rationale behind sampling entire fascicles to enable a more accurate assessment of ﬁber type proportions was based on reports suggesting that the ﬁber type composition of fascicles recapitulates that of entire muscles (Maier et al., 1992). Quantitative image analysis was carried out using the NIH image system for the Macintosh. Digital images were captured using the ScionCorp CMS-700 image analysis system. Statistical Analysis 1365 BOVINE SKELETAL MUSCLE DEVELOPMENT TABLE 1. Muscle weights and sex ratio of normal and double muscled fetuses at four gestational ages* Vastus lateralis Vastus medialis NM Days 120 160 210 260 wt (g) 2.7 14.5 47.7 123.6 DM SEM 0.1 0.5 1.5 4.1 wt (g) a 3.48 19.8a 66.4a 177.9a NM SEM 0.1 0.6 2.6 7.7 wt (g) 0.9 4.8 17.1 33.3 Sex ratio (M:F) DM SEM 0.1 0.4 1.2 2.6 wt (g) b 1.3 5.9c 23.2d 61.2a SEM NM DM 0.1 0.4 2.2 6.2 1:4 2:3 3:2 3:2 2:3 3:2 1:2 1:2 *Values are least-squares means, adjusted within groups for body weight and sex ratio, ⫾ SEM (n ⫽ 3–5 per group). Data are back-transformed after log transformation for analysis. a P ⱕ 0.001. b P ⱕ 0.01. c P ⱕ 0.05. d Not signiﬁcant. lished that these factors did not contribute to the breed effects. RESULTS Fetal Data Both M. vastus lateralis and M. vastus medialis weights showed a highly signiﬁcant increase with increasing gestational age (P ⱕ 0.001) and both muscles were signiﬁcantly larger in the DM animals relative to NM (54% for VL, 30% for VM; P ⱕ 0.001; Table 1). Fiber Typing and Morphological Analysis Muscle ﬁber type. Qualitative analysis of sections stained using mATPase histochemistry showed similar ﬁber morphology between NM and DM at all gestational ages. In both breeds, however, there was a noticeable difference between 120 and 160 days gestation in the morphology of the presumptive primary myotubes, from being large and vacuolated to more closely resembling mature myoﬁbers. The smaller size of the type 1 ﬁbers in the DM muscles was readily seen at 260-day gestation (Fig. 1H). The percentage of type 1 ﬁbers in both DM and NM showed a biphasic pattern of change throughout development (Fig. 2), with numbers decreasing between 120 and 160 days and then increasing again between 210 and 260 days (P ⱕ 0.001). There were consistently fewer type 1 muscle ﬁbers in DM than in NM (P ⱕ 0.001; Fig. 2). Immunohistochemistry. This study has shown a similar pattern of MHC expression in both DM and NM at 120-day gestation, with both presumptive primary and presumptive secondary ﬁbers staining positively for embryonic MHC (Fig. 3). All primary ﬁbers were also positive for slow MHC and all secondary ﬁbers were also positive for fast MHC. At 160-day gestation, the pattern was similar, but there were a number of ﬁbers in NM that were negative for embryonic MHC (Fig. 3G). At 210 days, a number of presumptive secondary ﬁbers in the DM were negative for embryonic MHC and others were positive for slow MHC (Fig. 4). Some presumptive secondary ﬁbers in NM and DM were positive for all MHC isoforms (Fig. 4A–F). At 260 days, all ﬁbers were negative for embryonic MHC in NM (Fig. 4G), while immunostaining remained quite strong in smaller presumptive secondary ﬁbers in DM (Fig. 4J). Muscle ﬁber size. The average area of type 1 ﬁbers in both DM and NM decreased from 120 to 160 days of gestation (Fig. 5a), as their morphology changed from that of primary myotubes with a central region devoid of myoﬁbrils to more mature muscle ﬁbers surrounded by developing secondary ﬁbers. By 210 days, type 1 ﬁbers of NM muscles markedly increased in size as they continued to mature, while DM ﬁbers had only very small increases in size, failing to regain the size they had previously been at 120 days. By main-effect analysis, type 1 ﬁbers were signiﬁcantly smaller overall in DM than in NM (P ⱕ 0.001) due to the reduced size at 210 and 260 days (Fig. 5a). The average area of type 2 muscle ﬁbers increased with age (P ⱕ 0.001) and was less in DM than in NM (P ⱕ 0.05). This effect was consistent across age groups from 160 to 260 days (Fig. 5b). One of the most sensitive measures of overall ﬁber type composition is total % area of a speciﬁc ﬁber type, which is the product of average ﬁber area and average numerical percentage of each ﬁber type. The total % area of type 1 ﬁbers declined between 120 and 160 days of gestation in both NM and DM, then remained relatively constant throughout the remainder of the period studied. The net effect of the changes in size and proportions of type 1 ﬁbers in the DM animals was that the muscle overall had a signiﬁcantly lower proportion of total area given over to type 1 ﬁbers at all gestational ages (P ⱕ 0.001; Fig. 6). DISCUSSION This study describes the quantitative and qualitative analysis of bovine muscle development in normal animals and in animals harboring a mutation in the gene for myostatin. Skeletal muscle mass was increased in DM fetuses relative to NM in both the M. vastus medialis and the M. vastus lateralis. This contrasts with observations from postnatal DM animals in which the M. vastus medialis is 38% smaller in size relative to NM animals, while the M. vastus lateralis is 15% larger (Boccard, 1981). This suggests that the difference in muscle mass between DM and NM is not due to a direct effect of the myostatin mutation on muscle development. The atrophy may occur as a result of an effect that is not expressed until the postnatal period, or it may also be a consequence of postnatal environmental effects. In humans, injury to or diseases of the knee joint may be associated with atrophy of the M. vastus medialis and hypertrophy of the M. vastus 1366 MARTYN ET AL. Fig. 1. Myosin ATPase staining in M. vastus lateralis at four gestational ages: 120 days (A and B), 160 days (C and D), 210 days (E and F), and 260 days (G and H) of gestation. NM in panels on left (A, C, E, and G); DM in panels on right (B, D, F, and H). A and B stained after preincubation at pH 4.2; C-G stained after preincubation at pH 5.0 according to modiﬁed histochemical staining procedure. Dotted lines indicate primary myotubes; open arrows indicate type 1 ﬁbers. Scale bar ⫽ 50 m. BOVINE SKELETAL MUSCLE DEVELOPMENT Fig. 2. Average percentages of type 1 muscle ﬁbers in M. vastus lateralis from NM (ﬁlled circle) and DM (open circle) fetuses at four gestational ages (n ⫽ 3 per group). Values are mean ⫾ pooled SEM. Triple asterisk, P ⱕ 0.001; asterisk, P ⱕ 0.05. lateralis (Speakman and Weisberg, 1977). Animals exhibiting extreme muscular hypertrophy have some abnormalities in stance and in the anatomy of the front limbs and hocks (Kieffer et al., 1972). This postural abnormality may therefore result in atrophy of the M. vastus medialis in DM animals during postnatal life. Quantitative analysis of histochemical ﬁber type showed the percentage of type 1 muscle ﬁbers initially declines with gestational age and then increases again. In developing human muscle, a proportion of primary myotubes degenerate between 16 and 20 weeks of gestation (Fidzianska and Goebel, 1991), a time period that coincides with the decrease in the percentage of type 1 ﬁbers seen in the current study. This would have the net effect of decreasing the percentage of type 1 ﬁbers, as seen in this study. An alternative mechanism for the reduction in the percentage of type 1 ﬁbers may be that a proportion of ﬁbers underwent transformation from type 1 to type 2 ﬁbers (Whalen et al., 1984). This possibility was not directly tested in this study, as individual ﬁbers could not be followed throughout gestation. During late gestation, the percentage of type 1 ﬁbers increases in both NM and DM. A similar observation to this has been previously made in developing ovine muscle (Maier et al., 1992). Maier et al. (1992) suggested that the majority of the ﬁbers transforming to slow MHC initially expressed an adult fast MHC isoform. This was not, however, supported by other investigators, who suggested that in predominantly fast twitch muscles, all the slow ﬁbers originated from primary generation myotubes (Picard et al., 1994). Although the current study did not investigate temporal changes in MHC isoforms in individual ﬁbers, this remains an area for future investigation in order to determine whether those ﬁbers that express slow MHC isoforms in late gestation are indeed the original population of primary myoﬁbers. The pattern of change in ﬁber type proportions described in this study was similar in both NM and DM, although in the DM the percentage of type 1 ﬁbers was consistently lower than in the NM. This result had been 1367 previously reported both during prenatal development (Ashmore et al., 1974) and in adult animals (Holmes and Ashmore, 1972). On that basis, it appears that the initial decrease in type 1 ﬁbers and the subsequent increase again are unrelated to the DM condition. The overall ﬁber type composition is, however, affected by the mutation. Previous studies have generally shown a positive association between myostatin expression and fast MHC isoforms. Myostatin mRNA was undetectable in the slow MHC expressing soleus muscle in mice (Carlson et al., 1999), and in vitro myostatin was associated with myotubes expressing the MHC type II isoform (Artaza et al., 2002). In rats, in which muscle atrophy was induced following dexamethazone treatment, myostatin mRNA was elevated, and there was an increase in type 2 muscle ﬁbers (Ma et al., 2003). The mechanism through which myostatin mediates effects on MHC expression remains unknown. In the earliest periods of gestation covered by this study, differences in MHC isoform expression between NM and DM were restricted to a population of ﬁbers in NM that were negative for embryonic MHC at 160 days. As this is a developmental isoform, expression of which is lost as ﬁbers mature, this suggests a relatively more advanced stage of development in the NM at this gestational age. Ninety and 130 days of gestation have been identiﬁed as the ages during which developmental differences were most marked between NM and DM (Picard et al., 1995a). The current study extended the period of gestation a further 50 days beyond that of Picard et al. (1995a) and has demonstrated that at 260 days of gestation NM no longer expresses embryonic MHC, although it is still relatively abundant in DM. These results suggest that later muscle development in DM fetuses is delayed relative to NM with respect to the expression of MHC isoforms. An alternative explanation for the prolonged period of expression of immature MHC isoforms is that the period of secondary ﬁber formation is extended in DM. This is consistent with the observation of an overall increase in muscle ﬁber number in DM animals. An elevation in satellite cell numbers in muscle ﬁbers from mstn⫺/⫺ mice also suggests that myostatin deﬁciency may be a mechanism through which muscle ﬁber number may be increased in DM animals (McCroskery et al., 2003). A number of in vitro studies have identiﬁed a role for myostatin in blocking cell cycle progression and differentiation (Langley et al., 2002; Rios et al., 2003), with inhibition of myostatin synthesis leading to enhanced cell cycle withdrawal and the stimulation of myoblast differentiation (Joulia et al., 2003). One proposed mechanism for the mediation of cell cycle progression may be a heightened response to MyoD in DM animals (Oldham et al., 2001; Spiller et al., 2002). A C313Y mutation in the myostatin gene results in hyperplasia but not hypertrophy in both cattle (Berry et al., 2002) and mice (Nishi et al., 2002), suggesting that different dominant negative mutations in the myostatin gene can have different effects on muscle ﬁber number and ﬁber size. The average size of primary myotubes initially decreased from 120 to 160 days of gestation as they developed into mature myoﬁbers. A similar result has been previously reported in bovine muscle (Stickland, 1978). After the initial decrease in ﬁber size that occurred in both breeds, type 1 ﬁbers in muscles from NM fetuses began to enlarge, but ﬁbers from DM fetuses did not. This result 1368 MARTYN ET AL. Fig. 3. Photomicrographs showing MHC immunohistochemistry in M. vastus lateralis at 120 (A–F) and 160 days (G–L) of gestation. NM in rows A–C and G–I, and DM in rows D–F and J–L. Embryonic MHC is shown in left column, slow MHC in central column, and fast MHC in right column. Dotted lines indicate type 1 ﬁbers. Scale bar ⫽ 50 m. suggests that during late gestation, some hypertrophic stimulus induces growth in type 1 ﬁbers of NM only, while in DM, either this stimulus is not present or the muscle ﬁbers are unable to respond. The time period during which a difference develops in the average area of type 1 ﬁbers between NM and DM is from 160 to 210 days of gestation. Type 2 ﬁbers grew at a relatively constant rate throughout the time period studied and were consistently smaller in DM than NM. The smaller ﬁber size in DM may be explained by the apparent developmental delay in these animals with myoﬁber hypertrophy lagging behind that of NM, in the same way as expression of more mature MHC BOVINE SKELETAL MUSCLE DEVELOPMENT 1369 Fig. 4. Photomicrographs showing MHC immunohistochemistry in M. vastus lateralis at 210 (A–F) and 260 days (G–L) of gestation. NM in rows A–C and G–I, and DM in rows D–F and J–L. Embryonic MHC is shown in left column, slow MHC in central column, and fast MHC in right column. Solid arrows indicate type 1 ﬁbers, open arrows indicate ﬁbers positive for all MHC isoforms. Scale bar ⫽ 50 m. isoforms was delayed. In postnatal animals, type 2 ﬁbers have been shown to be larger in myostatin-deﬁcient DM animals than in NM (Holmes and Ashmore, 1972). This is consistent with studies in humans, in which chronic disuse atrophy of the vastus muscles, resulting in a reduction in the area of type 2A and 2B muscle ﬁbers, was associated with an increase in myostatin expression (Reardon et al., 2001). It is well established that ﬁber type composition between DM and NM changes from prenatal to postnatal life (Holmes and Ashmore, 1972; Ashmore et al., 1974). Fiber size differences between DM and NM also appear to vary with developmental age. The total area of muscle classiﬁed as type 1 or type 2 ﬁbers is a more accurate measure of overall ﬁber type 1370 MARTYN ET AL. Fig. 6. Total percentage of area of type 1 ﬁbers calculated from average ﬁber number per fascicle multiplied by average ﬁber area in M. vastus lateralis from NM (ﬁlled circle) and DM (open circle) fetuses at four gestational ages (n ⫽ 3 per group). Values are least-squares mean ⫾ SEM. Data was log-transformed for analysis. Triple asterisk, P ⱕ 0.001; double asterisk, P ⱕ 0.01. Fig. 5. Average cross-sectional area of (a) type 1 and (b) type 2 muscle ﬁbers in M. vastus lateralis NM (ﬁlled circle) and DM (open circle) fetuses at four gestational ages (n ⫽ 3 per group). Values are mean ⫾ pooled SEM. A t-test was used for the calculation of signiﬁcant differences within time periods. Triple asterisk, P ⱕ 0.001; asterisk, P ⱕ 0.05. composition than measurement of either ﬁber type percentage or average ﬁber area in isolation (Holmes and Ashmore, 1972; West, 1974). The total area of type 1 muscle ﬁbers in NM and DM declines between 120 and 160 days, largely because of the remodeling of the vacuolated primary myotubes, then remains essentially level. The increase in the percentage of type 1 ﬁbers in the DM at 210 and 260 days of gestation is able to compensate fully for the smaller average ﬁber size, with no overall decrease in the total area occupied by type 1 ﬁbers. Although myosin ATPase activity in developing muscle is not necessarily a reliable indicator of contraction speed or metabolic activity (Guth and Samaha, 1972), this decline may represent a change in the metabolic pathway being employed by the muscles at this stage of development. Previous researchers proposed that a transformation toward more glycolytic ﬁber types in DM animals may reﬂect an inability of the cardiovascular system to supply the excess musculature, resulting in a compensatory shift toward more anaerobic metabolism (Ashmore, 1974). In summary, in contrast to postnatal animals, the M. vastus medialis in DM animals is not reduced in mass relative to NM during prenatal growth. In M. vastus lateralis, type 1 muscle ﬁbers in both NM and DM exhibited a biphasic change in proportions with gestational age, and proportions of type 1 ﬁbers were consistently lower in DM, suggesting that differences in ﬁber type composition are associated with the myostatin mutation. There are patterns of MHC isoform localization in DM muscle that are indicative of a delay in development relative to NM. There is an increase in the proportion of type 2 muscle ﬁbers that may contribute to the increase in muscle mass seen in DM animals during postnatal growth. Finally, type 1 ﬁbers are smaller in DM than NM in late gestation only and type 2 ﬁbers are smaller throughout gestation. 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