Okajimas Folia Anat. Jpn., 70(6): 273-284, March, 1994

Differentiation of Histochemical Properties of Masticatory Muscles in Bovidae and Cervidae (Artiodactyla)

By

Iwao SATO, Kazuyuki SHIMADA, Gen MURAKAMI, Narumi SAGARA and Tooru SATO

Department of Anatomy, School of Dentistry at Tokyo, Nippon Dental University, 2)Department of Anatomy, School of Medicine, Showa University, 3)Department of Anatomy, School of Medicine, Nihon University

- Received for Publication, September 11, 1993-

Key Words: Artiodactyla, Histochemistry, masticatory muscle

Summary: We analyzed the masticatory Muscles (masseter, temporal, medial pterygoid and lateral pterygoid muscles) of Bovidae and Cervidae (Artiodactyla) for the histochemical characteristics of their fiber types. Analysis of muscle fiber types in the present study was based on the staining reaction for SDH, Sudan black B, α-GPDH, and myosin-A TPase after alkaline preincubations. Histochemical properties were found to contribute to masticatory function, including a slow-twitch fatigue resistant activity derived from the high percentage of red fibers, in spite of the differences in the distributions of fiber types in three portions (superficial, medial and profound portions) of each masticatory muscle. These results indicate a correlation between the histochemical profiles of individual masticatory muscles in these species and their functions during jaw movements.

Histochemical profiles of fiber types in masticatory et al., 1961), glycolytic associated capacity (Wat- muscles of mammals (masseter muscle of cattle, tenberg and Leong, 1960) and respiratory capacity sheep, dog, guinea, pig, and rat, Suzuki, 1977; lateral (Gauthier and Padykula, 1966). pterygoid Muscle of cat, Taylor et al. , 1973; masseter The purpose of this study was to describe the and temporal muscles of Rhesus monkey, maxwell histochemical properties of enzymes in the masticatory et al. , 1979; masseter, temporal and lateral pterygoid muscles of Artiodactyla (Cervidae and Bovidae) muscles of macaques, Clark and Luschei, 1981; and to clarify differences in the functions of these masseter and temporal muscles of cat and dog, masticatory muscles during jaw movements. Rowlerson et al., 1983; masseter, temporal and lateral pterygoid muscles of hedgehog, Lindman et al. , 1986; human masseter, temporal, medial Materials and Methods pterygoid muscles, Eriksson and Thornell, 1983; human lateral pterygoid muscle, Eriksson et al., Masticatory muscles of adults, juvenile and infant 1981) have provided information about contraction Artiodactyla (Cervidae, 5 species; Bovidae, 7 species) speed and metabolic properties. In Bovidae and were used in this study (Table 1). Each muscle was Cervidae, however, histochemical analysis of the cut transversely, rapidly embedded into OCT com- fiber types in the masticatory muscles has not been pound (Tissue-Tek) and frozen in liquid nitrogen. performed. Such analysis would provide information Serial cross-sections (about 10 rim) were cut in on the role of each jaw muscle during a feeding a cryostat at —20°C and air dried. These sections were cycle, and a correlation with jaw movement. Previous incubated for Ca-activated adenosine triphosphatase histochemical analyses of Bovidae and Cervidae at pH 9.4 (Padykula and Herman, 1955) after prein- (Artiodactyla) masticatory muscles have utilized cubation at various pH levels (pH 10.4, 9.4, 5.0, 4.7. reactions and staining techniques for myosin-ATPase, 4.5 or 4.3) (Brooke and Kaiser, 1970), and succinic SDH, Sudan black B, and a-GPDH to characterize dehydrogenase (SDH) activity (Novikoff et al., muscle fiber types for contraction speed (Padykula 961), Sudan black B stain (Gauthier and Pady- and Herman, 1955), oxidative capacity (Novikoff kula , 1966) and glycerophosphate dehydrogenase

Address correspondence to Dr. Shimada Kazuyuki, D.D.Sc., Ph 1 .D., Department of Anatomy. School of Medicine . Showa University. -5-8 Hatanodia , Shinagawa-ku, Tokyo 142

273 274 L Sato et al.

Table 1. Tabulation of Artiodactyla for this study

* Nec , Necro-number of Zoological society of San Diego

(ot-GPDH) (Pearse, 1972), were used to demonstrate Table 2. Staining reactions of fiber types of masticatory muscles the types of muscle fibers. An ocular micrometer in Bovidae and Cervidae (Artiodactyla). marked with lines forming a grid of squares of 10 [Am on a side was used for calculating the percentages of types of muscle fibers (Fig. 1). According to their Sudan black B and SDH activities, fibers were classified into three types, red, intermediate and white. Muscle fibers were scanned continuously along each line of the micrometer grid by moving the section with a mechanical stage. For each grid square, cells partially outside of the left and upper lines of the grid were excluded from the total count. In contrast, cells partially outside of the right and lower For glycolysin-associatedcapacities fibers are classifiedas fast lines of the grid were counted. The percentage of twitchglycolytic (FG), fastoxidative glycolytic (FOG), and slow muscle fiber types located in 10 grid squares randomly oxidative(SO) fibers, according to systemof Putnamet al. (1980). selected throughout each section was determined. Fibertypes are classified as Red. Intermediate, and White according to systemof Novikoffet al. (1961)and Gauthierand Padykula (1966). Results

Histochemical analysis various pH levels (pH 9.4, 5.0, 4.7, 4.5 or 4.3), In Artiodactyla; Cervidae {Cervus d. duvauceli succinic dehydrogenase (SDH) activity, Sudan black (CDD), Muntiacus r. reevesi (MRR), Hydropotes i. B stain, and glycerophosphate dehydrogenase inermis (H11), Muntiacus reevesi micrirus (MRM), (a-GPDH) showed various reactions in each fiber and Cervus elaphus sibiricus (CES)} , and Bovidae type of the masticatory muscles (Fig. 2). The oxidative {Capra ibex nubiana (CIN), Ovis orientalis musimou and respiratory capacities of masticatory muscles (00M), Saiga t. tatarica (SIT), Oryx gazella beisa were well demonstrated by SDH activity and Sudan (OGB), Addax nasomaculatus (AN), Kobus 1. lenche black B staining, which showed extensive reactions (KLL), and GazeIla t. thmosoni (GIT)), masticatory in red fibers of masticatory muscles. The reaction muscles were used in this study. products were present in the region near the outline of muscle fibers, and appeared as small spots. Two Histochemical observations (Table 2) types of red fibers were identified in all cells stained Staining methods including Ca-activated adenosine by these two techniques. The red fibers were classified triphosphatase at pH 9.4 after preincubation at into two types, large and small. Small red fibers were Histochemical Properties in Artiodactyla Masticatory Muscle 275 stained more heavily than the large fibers by the two CIN (NEC 27055: 124, NEC 26895: 191) and MRM techniques. Reactions for SDH activity and Sudan (130). The high NMF in medial pterygoid muscle black B staining were moderate in the intermediate was found in MRR (2,179) and GTT (2,140), in fibers. These were also of two sizes; small and large. contrast to a low NMF in CIN (NEC 27055) (80), The reaction for SDH activity was very weak in the SIT (NEC 26908) (168), and MRM (179). A high white fibers, which were likewise divided into large NMF in lateral pterygoid muscle was found in 0GB and small types (Fig. 3). (1,228), in contrast to a low NMF in CIN (NEC 27055) (76) and MRM (190) (Fig. 4). Proportions of three muscle fiber types The highest percentage of red fibers was found in the profound portion of the masseter muscle in our Discussion examined species. A higher percentage of white fibers was found in the superficial portion of the There are many functional elements of the ana- masseter muscle (ca. 23-24%) in CDD and HII tomical relationships in the feeding appratus: the than in MRM, AN, and CIN which (ca. 3%). White oral condition, soft plate and oropharyngeal struc- fibers constituted a low percentage of fibers in each ture, occlusion, movement of the tongue, movement species. White fibers were only 2 to 3 percent of the of the lower jaw, movement of the upper jaw, masti- total in the masseter muscle of MRM, KLL, and catory muscles, and central nervous system (Hiiemae CIN. and Crompton, 1985). The relationship between the The highest percentage of red and intermediate feeding mechanism and masticatory muscles is, fibers was found in whole portions of temporal however, unknown because of the many elements in muscle. White fibers in the superficial and profound the feeding apparatus that are involved during jaw portions of the temporal muscle were about 12-14% movements. EMG analysis of masticatory muscles in CDD (NEC 2754 see Table 1) and MRR. White demonstrates the mechanism of chewing (human, fibers constituted a low percentage in whole portions Ahlgren and Owall, 1970; albino rat, Weijs and of the temporal muscle in MRM and AN (ca. Dantuma, 1975, rabbit, Weijs and Dantuma, 1981; 2-3%). monkey, Hylander and Johnson, 1989). However, The highest percentage of red and intermediate there are technical difficulties in measuring bite force fibers was found in whole portions of the medial with time and surface electrodes. Sampling muscle pterygoid muscle in our species. The highest per- for histochemical fiber types provides a means to centage of white fibers in the medial portion of the obtain an accurate estimate of the fiber population medial pterygoid muscle was about 43% in 0GB. (Goslow, 1985). Brooke and Kaiser (1970) have White fibers constituted the lowest percentage in the classified muscle fibers as type I 11A, 11B with respect medial pterygoid muscle in MRM (ca. 0.9%). to myosin-ATPase activity. Peter et al. (1972) dis- The highest percentage of red and intermediate tinguished SO, FG and FOG fibers with respect to fibers was found in three portions of the lateral the metabolic activities of whole masticatory muscles pterygoid muscle in our species. The highest per- of various mammals. We have simply referred to centage of white fibers in the superficial portion of these fibers as white, intermediate and red muscle the lateral pterygoid muscle was about 32.4% in fiber types on the basis of their reactivity to SDH, MRR. White fibers constituted the lowest percentage Sudan black B, a-GPDH and myosin-ATPase at in the whole region of the lateral pterygoid muscle in pH 9.4. These results indicate that there are major CES and OOM (ca. 1.5%) (Fig. 4). differences in the functioal characteristics of mam- malian masticatory muscles. Differentiation in muscle Number of muscle fibers per sq mm (NMF) fiber type compostion among the masticatory muscles In Bovidae, NMF was high in the masseter and reflects the different functions they play during temporal muscles in comparison with Cervidae. the feeding cycle. Therefore, movement of the jaw Number of muscle fibers per sq mm was low in during the feeding cycle is relatively complex. the temporal muscle of Cervidae and the medial In Bovidae and Cervidae muscle fibers showed pterygoid muscle of Bovidae. either no or a weak reaction fof myosin-ATPase at A high NMF in masseter muscle was found in pH 9.4, in contrast to other reactions for SDH. 0GB (2,348), MRR (1,630), and CDD (NEC 26888) Sudan black B, and a-GPDH. In the cattle and (1,038), in contrast to a low NMF in MRM (169), sheep, whole muscle fibers of masseter muscles CIN (NEC 27055) (131), and STT (NEC 26908) also showed no reaction to myosin-ATPase after (190). A high NMF in temporal muscle was found in preincubatiion at pH 10.7 (Suzuki, 1)77). Suzuki 0GB (2,856), MRR (1,810), GTT (1,491) and CDD (1977) showed that the masseter muscle of cattle (NEC 26888) (1,048), in contrast to a low NMF in and sheep contributed to the feeding function as 276 I. Sato et al. slow and long-term exercise properties during jaw 4) Eriksson P-0, Eriksson A, Ringqvist M and Thrornell L-E. movements. In general, slow, long-term exercise Special Histochemical-muscle fiber characteristics of the requires a high oxidative capacity and glycolytic lateral pterygoid. Arch Oral Biol 1981;26:495-507. activity. In our results, whole masticatory muscle 5) Eriksson P-0 and Thrornell L-E. Histochemical and mor- phologicalmuscle fiber characteristicsof the human masseter, fibers showed high activity in the three portions the medial pterygoid and temporal muscles. Arch oral Biol except for the medial pterygoid muscle of Oryx 1983;28:781-795. gazella beisa. This characteristic property suggests 6) Gauthier GF and Padykula HA. Cytological studies of that the muscle contributes to slow-twitch fatigue- fiber types in skeletal muscle: a comparative study of the resistant activity because of the distribution of fiber mammalian diaphragm. Cel Biol 1966;28:333-354. 7) Goslow GE. Neural control of locomotion, In: Bramble types. Differences in the distribution of muscles fiber DM, Liem KF, Wake DB (eds) Functional vertebrate mor- types (superficial, medial, and profound portions) phology, The Belknap Press of Harvard University Press, may show different parts of the functional movement England, 1985;338-365. that occurs during the feeding cycle. There are clearly 8) Hiiemae KM, Cromton AW. Mastication, food transport three types of fibers in the three portions; however, and swallowing. In: Bramble DM, Liem KF, Wake DB (eds) Functional vertebrate morphology, The Belknap Press the ratios of composition of the three muscle fiber of Harvard University Press, England, 1985;262-265. types are different. The superficial and medial 9) Hylandder WL and KR Johnson. The relationship between portions of this muscle have the highest percentage masseter force and masseter electromyogram during masti- of white fibers, in contrast to the high percentage cation in the monkey. Macaca Fascicularis. Arch Oral Biol 1989;34:713-722. and large size of the red fibers in the deeper portions. 10) Lindman R, Eridksson P-0 and Thornell L-E. Histochemical However, the histochemical properties and numer enzyme profile of the masseter, temporal and lateral of muscle fibers per sq mm in CDD in CIN change pterygoid muscles of the European hedgehog (Erinaceus with aging. In general, in the infant and juvenile europeaus). Arch Oral Biol 1986;31:51-55. stages, the low percentage of oxidative capacity and 11) Maxwell LC, Carlson DS, Mcnamara JA and Faulkner JA. Histochemical characteristicus of the masseter and high number of muscle fibers per sq mm are mainly temopralis muscles of the rhesus monkey (Macaca mulatta). found in Cervidae rather than Bovidae (Fig. 5). This Anta Rec 1979;193:389-402. suggests that the functional properties of masticatory 12) Novikoff AB, Shin W and Drucker J. Mitochondria' locali- muscles are reflected in muscle fiber size. There are zation of oxidative enzymes: staining results with two various important elements (e.g., histochemical terazolium salts. J Biophys Biochem Cytol 1961;9:47-61. 13) Padykula HA and Herman E. The specificity of the his- properties, muscle fiber size, muscle weight, and tochemical method adenosine triphosphatase. J Histochem other morphological properties) in jaw movement, Cytochem 1955;3:170-185. and our histochemical data indicate that each muscle 14) Pearse AGE. Oxcidoredictase II. In: Pearse AGE (ed) plays an important role in jaw movement. Histochemstry. Vol. 2, Churchill Livingstone, London, 1972;929-930. 15) Peter JB, Barnard RJ, Edgerton VR, Gillespie CA and Steple KE. Metabolic profiles of three fiber types of skeletal Acknowledgments muscle in guinea pigs and rabbits. Biochem 1972;11:2627-2633. We would like to thank Dr. V. A. Lance 16) Rowlerson A, Mascara() F, Vegatti A and Carpane E. The fiber type composiition of the first brachial arch muscles (C.R.E.S., San Diego Zoo, U.S.A.), Dr. A. T. in Carnivoram and Primates. J Muscle Res Cell Motil Kumamoto (C.R.E.S., San Diego Zoo, U.S.A.), 1983;4:443-472. and the staffs of San Diego Zoological Society, San 17) Suzuki A. A comparative histochemical study of the masseter Diego, California, U.S.A., for their help with this muscle of the cattle, sheep, swine, dog, guinea pig, and rat. research. Histochem 1977;51:121-131. 18) Taylor A, Cody FWJ and Bosley MA. Histochemical and mechanical properties of jaw muscles of the cat. Exp Neurol 1973;38:99-109. References 19) Wattenberg LW and Leong JL. Effects of coenzyme Q and menadione on succinate dehydrogenase activity as measured 1) AhlgrenJ and Owall B. Muscularactivity and chewing by tetrazoluim salt reaction. J Histochem Cytochem force,a polygraphicstudy of humanmandibular movements. 1960;8:296-303. Arch OralBio 1970;15:271-280. 20) Weijs WA and Dantuma R. Electromyographyand mechanics 2) BrookeMH and Kaiser KK. Muscle fiber types: How many of mastication of the albino rat. J Morph 1975;146:1-34. andWhat kind?Arch Neurol1970;23:369-379. 21) Weijs WA and Dantuma R. Functional anatomy of the 3) Clark WRS and LuscheiHS. Histochemicalcharacter- masticatory muscle apparatus in the rabbit (Oryctolagus istics of mandibularmuscles of monkey. Exp Neurol cuniculus). Nether J Zool 1981;31:99-147. 1987;74:654 — 672 . Histochemical Properties in Artiodactyla Masticator) Muscle 277 Plate I

Explanation of Figures

Plate I

Fig. 1 Schematic explanation of measurement methods and sampling portions (a—c, superficial portion; medial portion, d—f; PRO, profound portion, g—k). a: Lateral view of masticatory muscles: b: Drawing of ocular grid (vertical line, 1-38; horizontal line, a—k) superimposed on a cross-section of each masticatory muscle. Square areas (9-e) are sampled for muscle fiber types and the number of muscle fibers per sq mm is counted. c: Enlargement of grid space, showing distribution of red (R), intermediate (I) and white (W) fiber types. Abbreviations: LP, lateral pterygoid muscle; MID, medial portion; MM, masseter muscle; MP, medial pterygoid muscle; PRO, profound portion, SUP, superficial portion; TM, temporal muscle. 278 I . Sato et al.

Plate II

Plate II Fig. 2 Serial transverse sections of the masseter muscle stained for (a) SDH, (b) a-GPDH, (c) Sudan black B and myosin-ATPase at pH 9.4 (d) and pH 4.7 (e); red (R), intermediate (I) and white (W) fiber types. Bar = 100 Histochemical Properties in Artiodactyla Masticatory Muscle 279 Plate Ill

Plate III Fig. 3a Serial transverse sections of the temporal muscles in Cervidae stained for reaction to Sudan black B. (a) Cervus d. duvauceli, (b) Muntiacus r. reevesi, (c) Hydropotes i. inermis, (d) Muntiacus reevesi micrirus, (e) Cervus elaphus sibiricus. Small muscle fibers size were in each masticatory muscle of Cervus d. duvauceli and Muntiacus r. reevesi. Bar = 250 tan 280 I. Saw et al.

Plate IV Fig. 3b Serial transverse sections of the temporal muscles in Bovidae stained for reaction to Sudan black B. (f) Capra ibex nubiana, (g) Ovis orientalis musimou), (h) Saiga t. tatarica, (1) Oryx gazella beida, (j) Addax nasomaculatus, (k) Kobus 1. lenche, (1) Gazella t. thmosoni. Very small muscle fibers were found in each masticatory muscle of Oryx gazella beisa and Gazella t. thmosoni. Bar = 250 pi.M Histochemical Properties in Artiodactyla Masticatory Muscle 281 Plate IV 282 I. Sato et al .

Plate V

Plate V Fig. 4 Graph showing oxidative capacity according to the composition of the three fiber types and number of muscle fibers 100 squares of 10ium per side in the three portions (superficial, medial, and profound) in each masticatory muscle (see Fig. 1). Fig. 4a masseter muscle, Fig. 4b temporal muscle, Fig. 4c medial pterygoid muscle, Fig. 4d lateral pterygoid muscle, Bar length shows the number of muscle fibers. Abbreviations: MID, medial portion; MEAN, the mean of three portions; PRO, profound portion, SUP, superficial portion; CDD, Cervus d. duvauceli; MRR, Muntiacus r. reevesi (MRR); HII, Hydropotes i. inermis; MRM, Muntiacus reevesi micrirus; CES, Cervus elaphus sibiricus; CIN, Capra ibex nubiana; 00M, Ovis orientalis musimou; Saiga t. tatarica; 0GB, Oryx gzella beisa; AN, Addax nasomaculatus; KLL, Kobus I. lenche; G'IT, Gazellat t. thmosoni. Histochemical Properties in Artiodactyla Masticatory Muscle 283 Plate V

Oxidative capacity 284 L Saw et al .

Plate VI

Plate VI Fig. 5 Relationship between oxidative capacity according to the composition of the three fiber types and number of muscle fibers in 100 squares of 101AMper side in the masticatory muscles (see Fig. 1). Bar length shows the number of muscle fibers. Abbreviations are as in Fig. 4.