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Revista chilena de anatomía

Print version ISSN 0716-9868

Rev. chil. anat. vol.17 n.1 Temuco  1999 



* Antonio de Castro Rodrigues
** Maeli Dal Pai - Silva
* Maria Lúcia Eleutério
* Progresso José Garcia
*** Carlos Roberto Padovani
SUMMARY: A description is provided of the different fiber-type composition of two flexors and two extensor muscles of the pig. In addition, cross-section areas of each fiber type and an estimation of the relative myonuclei by the muscle fibers in the tested muscles are also provided. Four muscles were selected for study, based in their suitability for future neurophysiological analysis as components of segmental motor system, and on their homologies with muscles in other vertebrates. The tested muscles were soleus, flexor digitorum superficialis (FDS), extensor digitorum longus (EDL) and extensor halux longus (EDHL). Serial sections of these muscles were stained for NADH-diaphorase and Haematoxilin and Eosin (HE). The relationships between the present results and previous findings on homologous muscles of the mammalian hindlimb are discussed.

KEY WORDS: 1. NADH; 2. Diaforase; 3. Muscle fiber; 4. Pig.


When the physiological, biochemical and histochemical properties of single fibers are considered together, fibers can be classified into various types (STEIN & PadIkula, 1962; ROMANUL, 1964; NISHIYAMA, 1965; GAUTHIER, 1969; CLOSE, 1972; DUSTERHOFTETAL et al., 1990).

The locomotive muscles of various classes of vertebrates, e.g., mammals (PETER et al., 1972; ARMSTRONG et al., 1982; ARMSTRONG & PHELPS, 1984; RODRIGUES et al., 1990, 1994); amphibians (LANNERGREN & SMITH, 1966; SMITH & OVALLE, 1973), birds (TALESARA & GOLDSPINK, 1978; ROSIER & GEORGE, 1986), fishes (DAL PAI-SILVA et al., 1995) and reptiles (GOTH, 1981), have been shown to contain at least three histochemicaly identifiable fiber types.

Several generalizations, based on a survey of the motor control (BURKE, 1981; HENNEMAN & MENDEL, 1981; SKETELJ et al., 1991) can be made regarding the usage of the three fiber types: 1) slow-twitch oxidative (SO) fibers are recruited during activities requiring slow and sustained force development; 2) fast-twitch-oxidative-glycolytic (FOG) fibers are involved in activities requiring rapid and sustained force development; and 3) fast-twitch fibers with low oxidative capacities (FG) are recruited during activities requiring rapid and brief force development. Thus, knowledge of a muscle's "histochemical profile" can provide important insights into its functional capabilities.

Over the past ten years, the use of the pig as a model for the study of issues central to our understanding of the function of the muscle system has increased dramatically.

The purpose of this study was to stablish a histochemical profile for four muscles of the hindlimb in the pig (Sus scrofa) in order to observe the possible variations between flexor and extensor muscles.


Muscle selection. Four muscles were selected for study, based our suitability for future neurophysiological analysis as components of segmental motor system, and on their homologies with muscles in other vertebrates.

Table I Attachments and presumed actions of the four test muscles selected for analysis.


Table I. Attachments and presumed actions of the four-hindlimb muscle selected for analysis.

Muscle (Abbr.) origin insertion actions

Soleus epicondyle of the femur tuber calcanei Plantar flexes foot
FDS medial part of posterior surface of tibia tuber calcanei digitis flexes
EDL extensor groove of femur distal phalanges digitis extends
EDHL proximal third of anterior surface of fibula medial phalange

great toe extends

Tissue processing. The biopsy of the muscle was carefully obtained from five pigs (Sus scrofa) (weighing about 11.8 Kg and 77cm in length). The animals were first sedated with hydrochlorate and then anesthetized with sodium pentobarbitone I.V. (1ml/Kg). Immediately afterwards the soleus, FDS, EDL and EDHL muscles were assessed and a small fragment of these muscles were removed from right leg. The samples were combined in a single block and frozen in liquid nitrogen (DUBOWITZ & BROOKE, 1973). Serial cryostat sections were stained with HE - Haematoxilin-eosin and NADH-TR - nicotinamide adenine dinucleotide tetrazolium reductase (PEARSE, 1972) to demonstrate the metabolic activity of the fibers.

Fiber-type composition. Some pig muscles (DAVIES, 1972) and many mammalian muscles (BOTTERMAN et al., 1978; ENGLISH & LETBETTER, 1982; WINDHORST et al., 1989; DALL PAI et al., 1991) exhibit significant compartmentalization.

Muscle fiber-type proportions were assessed, using a sample of fibers representing 10-30% of each muscle's cross-section area. This sampling range reflects important consideration about fiber-type compartmentalization.

Cross-section area. The area of individual fibers was measured by tracing around their perimeters with a digitizer. Care was taken to ensure that measurements were made only on fibers that were free of ice-artifact and appeared to be sectioned perpendicular to their long axis. However, given the shape of some pig muscles it is recognized that all fibers in a muscle cross section cannot be sectioned normal to their long axis. Based on maximum pinnation angles (up to 20o ) that have been reported for some cat limb muscles (SACKS & ROY, 1982), we estimate this error to approach 7% for a fiber sectioned at 20o perpendicular to its long axis.

Statistical analysis. Morphometric analysis were made by using a software image analyzer (OPTMAS 4.10 - Image Corporation USA). The obtained data were submitted to the statistical tests (MORRISON, 1976). A significance level of p<0.05 was used in all statistical tests.


The histological organization of the studied muscles was similar to the other muscles in the hindlimb mammalian muscles. Large numbers of parallel muscle fibers were grouped into fascicles. The muscle fibers, the fascicles, and the whole muscle were each invested by connective tissue that forms a continuous stroma. The muscle as a whole was includes by a connective tissue layer called the epimysium. The soleus and FDS muscle presented small fibers inside some fascicles. In the soleus muscle these fibers were agrouped in "metabolic group". This situation was observed only in the soleus muscle. In the FDS these small fibers were scattered inside the fascicles and in the other muscles was not observed "metabolic groups" (Fig. 1).


Fig. 1. Transversal section of tested muscles: a)m. soleus; b)m.FDS; c)m.EDL and d)m. EDLH. The perimysium (p), endomysium (e) and myonuclei (n) are showed. The metabolic group is indicated (arrow). HE. ( X120).

As revealed by the NADH-diaforase the soleus, FDS, EDL and EDLH muscles presented a heterogeneous population of fibers. Based on the intensity of reaction and the cytological distribution pattern of the formazan, three main kinds of muscle fibers were individualized: a small fiber, presenting a high degree of NADH-diaforase activity especially at the subsarcolemmal level called SO. Large fiber, presenting a low degree of enzyme activity, was the formazan is uniformly distributed, called FG fiber, and a fiber with intermediate characteristics called FOG. This reaction confirms the presence of the called "metabolic group" only in the soleus muscle (Fig. 2).

Fig. 2. Transversal section of different fibers of the studied muscle. a) m. soleus; b) m. FDS; c) m. EDL and d) m.EDLH. The "metabolic group" is showed (between the brackets). NADH - diaforase reaction. ( X120).

Mean cross-sectional areas of soleus muscle fibers was 500.5 mm2; in FDS muscle was 486.8 mm2; in EDL muscle was 476.7 mm2 and in EDLH muscle was 431.6 mm2. The mean number of myonuclei around each fiber was: in the soleus muscle, 1.54; in FDS muscle, 1.38; in EDL muscle, 0.49 and in EDLH muscle, 0.52. These findings are summarized in the Table II.

Mean area between the flexor muscle (soleus and FDS) fibers had not significant difference. Otherwise, the mean areas between the extensor muscle (EDL and EDLH) fibers were different.

The flexor muscles (soleus and FDS) presented mean area of their fibers larger than the extensor muscle (EDL and EDLH) (Table III).

The number of myonuclei around each fiber was larger in the soleus muscle. There was not significant difference between extensor muscles (Table III).

Mean area of the soleus, FDS and EDL muscles had not significant difference. The fibers of the flexor muscles were larger if compared to the extensor muscles. Otherwise the mean area of the extensor muscles (EDL and EDLH) presented significant difference.

The number of myonuclei present in the extensor muscles (EDL and EDLH) was similar. It also was observed that the number of myonuclei in the flexor muscles was larger if compared to the extensor muscles (Table III).

The frequency of the SO and FOG fiber was similar in the soleus and FDS muscles (15.03% - 15.11% and 30.43% - 27.73%, respectively). In the EDL and EDLH muscle the frequency of these fibers was similar too (11.40% - 10.35% and 35.81% - 34.67%, respectively). The S0 fibers were more frequent in the flexor muscles than extensor muscles. Otherwise the FOG fibers were more frequent in the extensor muscles than in the flexor muscles (Table IV).

Table II. Mean area and myonuclei values of fibers in the tested muscles; n=5.

Muscle (Abbr.)
area (µm2)
Soleus 500.5 ± 60.1 1.54 ± 0.05
FDS 486.8 ± 53.5 1.38 ±  0.11
EDL 476.7 ±  61.9 0.49 ± 0.05
EDHL 431.6 ± 64.7 0.52 ±  0.04

Values are means ± standard errors; n=100 fibers per muscle in each animal.

Table III. Statistical results about the compared areas and myonuclei number in the studed muscles.


Muscle (Abbr.) area (µm) myonuclei

Soleus 500.50 b 1.54 c
FDS 486.80 c 1.38 b
EDL 476.72 b 0.49 a
EDHL 431.64 a 0.52 

Flexors X Extensors                78.94 (p<0.05)              1.91 (p<0.05)

DMS (5%) = 31.01 for Tukey test of the muscle area
DMS (5%) = 0.11 for Tukey test of the average myonuclei number
S (5%) = 47.74 for Scheffé test of the muscle area
S (5%) = 0.17 or Scheffé test of the average fibers number
AREA: (m.soleus = FDS = m. EDL) > m. EDLH and Flexors > extensors
MYONUCLEI: m. soleus > m. FDS > (m.EDL = m.EDLH) and Flexors > Extensors
Table IV. Statistical results about the frequency of fiber-type composition (%) in the test muscles.

Muscle (Abbr.) SO FOG FG

Soleus 15.03 b 30.43 a 54.54 ab
FDS 15.11 b 27.73 a 56.16 b
EDL 11.40 a 35.81 b 52.79 a
EDLH 10.35 a 34.67 b 54.98 ab

Results of the global test 0 = 0.92 

Results of test by variable F = 33.85 
F = 20.07 
F = 4.50 

SO = (m. soleus = m. FDS) > (m. EDL = m. EDLH)
FOG = (m. soleus = m. FDS) < (m. EDL = m. EDLH)
FG = m. FDS > m. EDL


These data clearly demonstrate predominance of the FG fiber type in the studied muscle of the pig. The dominance of this fiber type may reflect to a certain extent the sedentary existence of the animals. There are a number of physiological correlates as reviewed by BURKE (198l). For example, there appears to be a typical recruitment pattern of the three fiber types within muscles for most locomotive movements with SO fibers being activated under conditions of low force production and FOG and FG fibers being progressively recruited with increasing levels of force.

LAUGHLIN & ARMSTRONG (1983) have observed that patterns of blood-flow distribution within and among the muscles before and during locomotive exercise are closely related to the fiber-type populations and, presumably, in turn, to the patterns of muscle-fiber recruitment. Thus, when the pig is simply standing on the treadmill, the blood flow is primarily directed to the deep postural muscles in the extensor, antigravity groups that are mainly composed of slow-twitch fibers. When the animal walks or runs on the treadmill, however, the elevations in blood flow over pre-exercise are primarily in muscles and muscle parts with significant numbers of FOG fibers. Thus, the fiber type, as classified by mitochondria enzyme activities, has functional correlates that have been identified.

The existence of these correlates would seem to support the practicality and validity of classifying fibers by the method (PETER et al.) we have employed. However, it should be emphasized that several major disadvantages exist with this method. As mentioned above, using a mitochondria marker enzyme to distinguish between FOG and FG fibers requires arbitrary delineation, since a spectrum of oxidative potentials exists within the fast-twitch population. Recently some authors (BEGO et al., 1996) claim for a new classification to the muscle fibers based on their different diameters. Thus, the muscle fiber should be classified in MFUs (morphofunctional units).

The relative sizes of the fibers of each type vary both within and among the different muscles in the hindlimb. For example, the SO and FOG fiber located in the flexor muscles were generally larger than those in the extensor muscles were. Similarly, the FG fiber had largest area in the extensor muscles than those in the flexor muscles.

In mammalian muscles, fiber cross-section area in each of the three fiber types is generally in the order FG > FOG > SO (BURKE). As a result, contraction of FG fibers accounts for a greater proportion of total muscle force than their fiber-type proportion alone would suggest, whereas contraction of SO fibers with their relatively smaller cross-section areas will produce correspondingly lower whole muscle forces. The FG fibers in the studied muscles were larger in extensor muscles than flexor muscles. Otherwise the SO fibers were larger in flexor muscles than extensor muscles. It is conceivable that the flexor muscles might be preferentially involved in slow movements that require relatively high sustained force outputs. Such possibilities deserve experimental testing (SKETELJ et al.), because they help to stablish the boundary conditions needed to propose theories on the "rules" used by the CNS in motor control.

Normally, the different kind of fibers is scattered in the whole muscle in mosaic pattern. It was observed, only in the soleus muscle, the called "metabolic group", i.e., group of SO fibers surrounded by FOG fibers. Similar results were observed in the longuissimos dorsis, trapezius, semitendinosus and gracilis muscles of the pig (MODDY & CASSENS, 1968; STICKLAND & HANDEL, 1986; HORAK, 1988).

The functional significance of these "metabolic groups" within the muscles remains to be determined. However, this arrangement appear to be more evenly frequent in red muscles, i.e., postural muscles, than in the white muscles.


The authors thank Mr. Gelson Rodrigues for his technical assistance. CNPq (Proc.300062/93-1) provided financial support.

RESUMEN: Se describen diversos tipos de fibras musculares de dos músculos flexores y de dos músculos extensores del cerdo (Sus scrofa). Además, en áreas seccionadas transversalmente, se efectuó una estimación relativa del número de mionúcleos por fibras de los músculos examinados. Los músculos fueron seleccionados de acuerdo a la conveniencia de realizar un futuro análisis neurofisiológico como componente del sistema motor segmentario y por su homología con músculos de otros vertebrados. Los músculos seleccionados fueron: sóleo, flexor superficial de los dedos, extensor largo de los dedos y extensor largo del hálux. Las secciones seriadas de estos músculos fueron teñidas con NADH-diaforasa y hematoxilina-eosina. Se discuten las relaciones entre los resultados obtenidos y hallazgos previos en músculos homólogos del miembro posterior de otros mamíferos.

PALABRAS CLAVE: 1. NADH; 2. Diaforasa; 3. Fibra muscular; 4. Cerdo.

* Department of Anatomy, IB, UNESP, Botucatu, SP, Brazil
** Department of Morphology, IB, UNESP, Botucatu, SP, Brazil
*** Department of Biostatiscs, IB, UNESP, Botucatu, SP, Brazil
CNPq (Proc.300062/93-1)
 Correspondence to:
Prof. Dr. Antonio de Castro Rodrigues
Department of Anatomy
IB, UNESP, Botucatu
18618-000 Rubião Júnior,
São Paulo
Telephone: +55 014 821 6040
FAX : +55 014 821 3744

Recibido : 04-03-1999
Aceptado: 30-05-1999


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