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.