Muscle Fiber Population and Biochemical Properties of Whole Body Muscles in Thoroughbred Horses
Article first published online: 2 SEP 2009
Copyright © 2009 Wiley-Liss, Inc.
The Anatomical Record
Volume 292, Issue 10, pages 1663–1669, October 2009
How to Cite
Kawai, M., Minami, Y., Sayama, Y., Kuwano, A., Hiraga, A. and Miyata, H. (2009), Muscle Fiber Population and Biochemical Properties of Whole Body Muscles in Thoroughbred Horses. Anat Rec, 292: 1663–1669. doi: 10.1002/ar.20961
- Issue published online: 18 SEP 2009
- Article first published online: 2 SEP 2009
- Manuscript Revised: 15 MAY 2009
- Manuscript Accepted: 15 MAY 2009
- Manuscript Received: 22 JAN 2009
- The Japanese Ministry of Education, Science and Culture. Grant Number: 21300251
- muscle fiber type;
- metabolic enzyme
We examine the muscle fiber population and metabolic properties of skeletal muscles from the whole body in Thoroughbred horses. Postmortem samples were taken from 46 sites in six Thoroughbred horses aged between 3 and 6 years. Fiber type population was determined on muscle fibers stained with monoclonal antibody to each myosin heavy chain isoform and metabolic enzyme activities were determined spectrophotometrically. Histochemical analysis demonstrated that most of the muscles had a high percentage of Type IIa fibers. In terms of the muscle characteristic in several parts of the horse body, the forelimb muscles had a higher percentage of Type IIa fiber and a significantly lower percentage of Type IIx fiber than the hindlimb muscles. The muscle fiber type populations in the thoracic and trunk portion were similar to those in the hindlimb portion. Biochemical analysis indicated high succinate dehydrogenase activity in respiratory-related muscle and high phosphofructokinase activity in hindlimbs. We suggested that the higher percentage of Type IIa fibers in Thoroughbred racehorses is attributed to training effects. To consider further the physiological significance of each part of the body, data for the recruitment pattern of each muscle fiber type during exercise are needed. The muscle fiber properties in this study combined with the recruitment data would provide fundamental information for physiological and pathological studies in Thoroughbred horses. Anat Rec, 2009. © 2009 Wiley-Liss, Inc.
Scientific investigation of Thoroughbred racehorse running ability could provide significant insight into exercise physiology, not only for racehorses but also for humans. It is well known that muscles of the Thoroughbred horse are composed of Type I, Type IIa, and Type IIx fibers (Billeter et al.,1988; Sosnicki et al.,1989; Rivero et al.,1996; Serrano et al.,1996). Furthermore, the development of immunohistochemical techniques has permitted identification of hybrid muscle fibers expressing two types of myosin heavy chain (MHC) isoforms (Linnane et al.,1999; Rivero et al.,1999; Quiroz-Rothe and Rivero,2001). Although some previous studies, including our study, demonstrated that hybrid fibers (I/IIa and IIa/x) were detected in the gluteus medius muscle in Thoroughbred horses, the percentages of these fibers decreased to less than 4.9% until they were 24 months old (Yamano et al.,2005). On the basis of the result we speculate that the contractile properties of the muscle can be roughly determined by the populations of three main fiber types in mature Thoroughbred race horses, as mentioned in a previous study (Snow and Guy,1980).
Although studies of skeletal muscle in horses have greatly advanced since Lindholm and Piehl (1974) developed a muscle biopsy procedure, most physiological studies have been performed in the gluteus medius muscle. Recently, many articles dealing with horses suggest that the forelimb muscles may also play an important role in running. In fact, most of the damage during running occurs in the forelimbs (60% for bone, 40% for other tissue) in Thoroughbred horses (Peloso et al.,1994; Cohen et al.,1999). It has been hypothesized that the hindlimb produces a net propulsive force, whereas the forelimb produces a net braking force in horse trotting at a constant speed (Merkens et al.,1993; Dutto et al.,2004). The previous studies demonstrated, using a combination of kinetic and kinematic analysis, that the major functions of forelimb and hindlimb muscles were antigravity and propulsive force production, respectively (Niki et al.,1984; Dutto et al.,2006). In addition, the horse muscles of the shoulder girdle, the suspending forelimb from the trunk anatomically, have a suitable structure for generating antigravity force (Payne et al.,2004). Furthermore, the flexion-extension movements in the back of horses were recently examined using EMG and 3D biomechanical analysis (Audigié et al.,1999; Robert et al.,2001). They concluded that the primary role of trunk muscles is to control the stiffness of the back rather than to induce movements.
In addition to the physiological aspect, it has been demonstrated that the clinical and pathological features in some disorders of equine motor system are closely related to the muscle fiber population (Valentine et al.,1994). Therefore, as basic knowledge, to examine the muscle fiber type population of each muscle sampled from whole body in Thoroughbred horses is important for both physiological and pathological studies.
In this study, we examined the muscle fiber type population and metabolic properties (oxidative and glycolytic enzyme activities) of 46 muscle samples from the whole body in six Thoroughbred horses.
MATERIALS AND METHODS
Clinically healthy Thoroughbred horses with no neuromuscular dysfunction (six males; mean age = 4.5 ± 1.1 years; mean bodyweight = 482 ± 9 kg) were examined. All horses received pasture exercise and conventional training from birth, and water and hay were available ad libitum. The horses were euthanized using muscle relaxants after being anesthetized with 2 g of thiopental sodium. Then, the blood was removed from the carotid, and the muscles on the left side were dissected within 2 hr after the horses were euthanized. Each whole muscle was isolated, and then a ∼1 cm3 block was taken from the center of the superficial part, basically the intersection of the long axis and a short axis. These blocks were frozen in liquid nitrogen and stored at −80°C until analysis. The name of the muscle and location are shown in Tables 1–5.
|Muscle name||Type I||Type IIa||Type IIx||SDH||PFK||SDH/PFK|
|Soleus||86.0 (15.0)||14.0 (15.0)||0.0 (0.0)||4.27 (1.92)||9.84 (2.83)||0.43|
|Gastrocnemius (caput laterale)||9.8 (2.2)||73.3 (5.8)||17.0 (5.5)||4.52 (1.56)||35.07 (14.18)||0.13|
|Gastrocnemius (caput mediale)||11.4 (3.5)||70.3 (10.9)||18.3 (9.2)||4.55 (1.46)||30.75 (11.21)||0.15|
|Extensor Digitorum longus||10.7 (5.9)||67.7 (13.4)||21.7 (15.8)||3.84 (1.87)||35.84 (4.62)||0.11|
|Gluteus medius||5.2 (5.7)||52.6 (9.1)||42.2 (6.4)||4.87 (1.01)||57.25 (25.71)||0.09|
|Gluteus medius (middle part)||13.9 (7.5)||55.4 (5.5)||30.7 (2.7)||4.05 (0.57)||40.28 (24.73)||0.10|
|Semitendinosus||8.5 (11.2)||65.8 (18.8)||25.7 (22.7)||5.04 (1.47)||45.87 (11.12)||0.11|
|Semimembranosus||9.3 (6.1)||63.9 (6.0)||26.8 (8.3)||4.74 (1.52)||45.98 (13.30)||0.10|
|Biceps femoris||16.0 (3.8)||62.8 (11.4)||21.2 (11.7)||4.77 (1.25)||39.86 (20.71)||0.12|
|Tensor fasciae latae||49.1 (7.7)||43.7 (6.9)||7.1 (5.8)||4.17 (1.21)||18.00 (8.79)||0.23|
|Gracilis||24.9 (13.7)||66.9 (13.3)||8.2 (10.8)||5.31 (1.30)||23.59 (10.87)||0.22|
|Vastus lateralis||2.5 (2.1)||82.3 (12.0)||15.2 (10.8)||2.07 (1.04)||38.02 (12.41)||0.05|
|Iliacus||46.2 (12.8)||38.5 (6.8)||15.3 (11.0)||4.39 (1.78)||30.02 (21.37)||0.15|
|Psoas major||36.3 (7.3)||40.1 (6.7)||23.6 (7.3)||5.03 (0.51)||39.12 (12.73)||0.13|
|Average||23.6 (22.5)||56.9 (17.2)||19.5 (10.2)||4.40 (0.76)||34.96 (11.67)|
|Muscle name||Type I||Type IIa||Type IIx||SDH||PFK||SDH/PFK|
|Latissimus dorsi||21.5 (8.2)||59.4 (1.41)||19.1 (11.9)||5.17 (1.58)||32.77 (12.83)||0.16|
|Pectoralis descendens||31.9 (14.9)||53.2 (12.2)||14.8 (4.1)||5.56 (1.67)||30.13 (11.30)||0.18|
|Pectoralis transverses||17.5 (6.8)||62.2 (11.7)||20.3 (14.1)||5.56 (1.16)||28.36 (9.38)||0.20|
|Trapezius (pars thoracica)||32.6 (9.8)||65.7 (10.4)||1.8 (2.1)||5.80 (1.15)||20.41 (8.97)||0.28|
|Rhomboideus thoracis||40.2 (11.4)||59.4 (11.0)||0.3 (0.8)||6.53 (1.04)||23.50 (9.81)||0.28|
|Longissimus thoracis||22.8 (3.5)||53.1 (8.7)||24.2 (6.4)||6.19 (1.23)||45.32 (17.98)||0.14|
|Longissimus lumborum||9.8 (3.6)||54.4 (6.6)||35.8 (8.9)||4.59 (1.32)||59.34 (41.87)||0.08|
|Pectoralis profundus||26.6 (13.5)||49.7 (9.0)||23.7 (11.3)||6.67 (1.34)||46.32 (10.94)||0.14|
|Subclavius||23.3 (5.3)||72.3 (5.9)||4.5 (1.5)||5.57 (0.38)||31.26 (8.86)||0.18|
|Average||25.1 (8.5)||58.8 (6.7)||16.0 (11.2)||5.70 (0.60)||35.30 (11.80)|
|Muscle name||Type I||Type IIa||Type IIx||SDH||PFK||SDH/PFK|
|Triceps brachii (caput longum)||16.2 (17.2)||68.1 (15.3)||15.7 (13.4)||4.47 (0.95)||33.70 (12.79)||0.13|
|Triceps brachii (caput laterale)||18.8 (5.9)||76.7 (5.7)||4.5 (3.3)||4.90 (2.46)||38.26 (11.60)||0.13|
|Biceps brachii||29.4 (9.9)||67.6 (10.2)||2.9 (2.7)||4.50 (1.04)||20.21 (8.60)||0.22|
|Deltoideus||21.9 (7.1)||54.1 (12.0)||24.0 (13.9)||3.48 (1.18)||30.48 (9.84)||0.11|
|Supraspinatus||12.2 (5.1)||83.4 (5.8)||4.4 (2.6)||4.89 (1.62)||26.85 (5.90)||0.18|
|Infraspinatus||26.2 (11.1)||68.1 (11.6)||5.7 (2.6)||5.43 (1.47)||29.89 (9.43)||0.18|
|Teres minor||34.8 (17.7)||54.7 (8.6)||10.5 (13.0)||4.57 (1.78)||24.14 (7.04)||0.19|
|Brachialis||48.5 (8.7)||51.5 (8.7)||0.0 (0.0)||4.65 (1.96)||20.46 (2.51)||0.23|
|Extensor carpi radialis||30.8 (11.2)||63.9 (7.7)||5.3 (4.1)||3.68 (0.85)||34.54 (12.13)||0.11|
|Flexor carpi radialis||36.3 (4.4)||59.9 (0.6)||3.8 (3.8)||5.27 (1.99)||31.16 (11.01)||0.17|
|Extensor carpi ulnaris||28.3 (1.9)||58.8 (19.0)||13.0 (18.4)||5.80 (0.08)||13.79 (2.84)||0.42|
|Flexor carpi ulnaris (caput ulnare)||30.7 (1.9)||65.5 (5.2)||3.8 (5.4)||4.46 (0.33)||12.52 (2.48)||0.36|
|Extensor digitorum communis||25.2 (3.0)||63.1 (16.2)||11.7 (13.1)||4.21 (1.72)||20.14 (5.39)||0.21|
|Flexor digitorum superficialis||43.1 (5.6)||56.9 (5.6)||0.0 (0.0)||4.25 (2.46)||11.04 (3.52)||0.39|
|Flexor digitorum profundus||24.2 (11.0)||75.8 (11.0)||0.0 (0.0)||3.74 (0.63)||11.79 (5.02)||0.32|
|Average||28.4 (9.4)||64.5 (8.8)||7.0 (6.5)||4.60 (0.60)||23.90 (8.70)|
|Muscle name||Type I||Type IIa||Type IIx||SDH||PFK||SDH/PFK|
|Masseter||77.8 (18.2)||22.0 (18.0)||0.3 (0.6)||3.94 (1.86)||12.96 (8.94)||0.30|
|Omotransversarius||23.4 (6.7)||70.5 (7.2)||6.2 (5.0)||3.55 (0.52)||35.10 (9.30)||0.10|
|Cleidocephalicus (Pars mastoidea)||17.0 (7.8)||80.4 (6.4)||2.6 (3.3)||4.04 (1.19)||31.16 (12.33)||0.13|
|Sternocephalicus (Pars mandibularis)||23.4 (11.8)||72.4 (6.6)||4.2 (6.6)||4.04 (1.41)||28.93 (5.98)||0.14|
|Splenius||49.0 (12.3)||45.8 (8.1)||5.2 (5.0)||6.41 (1.52)||16.61 (0.75)||0.39|
|Average||38.1 (22.7)||58.2 (21.5)||3.7 (2.1)||4.37 (0.74)||20.98 (8.25)|
|Muscle name||Type I||Type IIa||Type IIx||SDH||PFK||SDH/PFK|
|Diaphragm||59.3 (9.9)||39.8 (9.3)||1.0 (0.9)||9.64 (1.62)||22.61 (11.03)||0.43|
|Serratus vertralis thoracis||42.4 (5.8)||56.4 (4.8)||1.2 (1.7)||5.73 (2.17)||18.72 (4.02)||0.31|
|Obliquus internus abdominis||41.5 (6.9)||53.1 (6.5)||5.5 (4.3)||6.59 (2.74)||28.20 (12.17)||0.23|
|Obliquus externus abdominis||36.7 (4.4)||60.4 (6.0)||2.9 (3.7)||5.07 (0.89)||25.44 (8.40)||0.20|
|Rectus abdominis||26.9 (4.8)||69.8 (5.2)||3.3 (3.0)||5.16 (0.87)||31.33 (13.07)||0.16|
|Average||41.3 (10.5)||55.9 (9.8)||2.8 (1.6)||5.32 (1.68)||24.73 (6.62)|
All experimental procedures were reviewed and approved by the Animal Welfare and Ethics Committee of the Equine Research Institute, Tochigi Branch of the Japan Racing Association.
Several cross sections of 10 μm thickness were obtained from each block of frozen muscle on cryostat (Leica, Nussloch, Germany) at −20°C. The sections were allowed to warm to room temperature and then preincubated in goat normal serum in 0.2 M phosphate buffer (pH 7.6) at 25°C for 10 min. Primary monoclonal antibody was then applied: (1) Fast myosin, which specifically reacts with MHC-IIa and -IIx; (2) BA-D5, which specifically reacts with MHC-I; and (3) SC-71, which specifically reacts with MHC-IIa. The sections were incubated at 25°C for 180 min, then washed with phosphate buffer and reacted with a secondary antibody conjugated with horseradish peroxidase at 25°C for 180 min, and then washed with phosphate buffer again. Diaminobenzidine tetrahydrochloride was used as a chromogen to localize peroxidase in all primary antibodies (Fig. 1).
After the staining was performed, microscopic images of muscle fibers were obtained by use of a computer and image-processing system (Nikon, Tokyo, Japan). Based on the immunohistochemical staining images, the fibers were classified as Type I, IIa, and IIx fibers, and the population of each muscle fiber type (percentage of number) in about 800 fibers (200 × 4 sites) were calculated.
The activities of two enzymes, succinic dehydrogenase (SDH) and phosphofructokinase (PFK), were measured to determine the oxidative and glycolytic capacities of muscle samples, respectively. All enzyme assays were performed in triplicate at 25°C spectrophotometrically (Japan Spectroscopic, Tokyo, Japan).
The SDH activity was determined on the basis of the technique by Cooperstein et al. (1950). The muscle sample was homogenized in ice-cold 33.3 mM phosphate buffer (pH 7.4). The electronic transfer system was blocked by the addition of cyanogens, and the extent of reduction in cytochrome C was determined based on the change in absorbance measured at 550 nm.
Another piece of muscle was homogenized in ice-cold homogenization medium containing 150 mM KCl, 50 mM KHCO3, and 6 mM EDTA. PFK activity was evaluated on the basis of a technique of Shonk and Boxer (1964). Both enzyme activities were represented in μmol/g wet weight.
All values were reported as mean ± standard deviation. To determine significant differences among the five portions of the body, one-way ANOVA and post hoc analyses (t test with Bonferroni adjustment) were used. To determine significance differences between fiber type population and metabolic enzyme activity, a linear regression analysis was performed in individual values of each horse and mean values of six horses. Significance was set at P < 0.05.
Population of Muscle Fiber Type
Group 1: (hindlimb muscles, Table 1).
The mean percentages of Type I, IIa, and IIx fibers were 23.0%, 57.5%, and 19.5%, respectively. The soleus muscle had the highest percentage of Type I fiber in the hindlimb muscles (77.7%). The tensor fascia latae, iliacus, and psoas major muscles had relatively high percentages of Type I fiber (49.1%, 46.2%, and 36.3%, respectively). The mean percentage of Type IIx fiber in the gluteus medius muscle was 42.2%. The other muscles had a relatively higher percentage of Type IIa fiber (>60%), especially, the gastrocnemius (caput laterale and caput mediale) and the vastus lateralis had higher percentages of Type IIa fiber (70.3%, 73.3%, and 82.3%, respectively).
Group 2: (thoracic and trunk muscles, Table 2).
The mean percentages of Type I, IIa, and IIx fibers were 25.1%, 58.8%, and 16.0%. All muscles were composed of 53%–72% of Type IIa fiber. In the pectoralis, the descendens muscle had a higher percentage of Type I fiber and a lower percentage of Type IIx fiber than those in the transverses muscle. Almost all of the muscle fiber in the trapezius (pars thoracica) and rhomboideus thoracis were Type I and IIa fibers. The longissimus lumborum muscle had 35.8% Type IIx fiber. This value was 11% higher than that of the same muscle at the longissimus thoracis muscle.
Group 3: (forelimb muscles, Table 3).
The mean percentages of Type I, IIa, and IIx fibers were 28.4%, 64.5%, and 7.0%. Six muscles of the 16 obtained forelimb muscles had over 30% of Type I fiber, and most of the muscle had less than 10% of Type IIx fiber. However, only the deltoideus muscle had a high percentage of Type IIx fiber (24.0%) and a relatively lower percentage of Type IIa fiber (54.1%).
Group 4: (head and neck muscles, Table 4).
The mean percentages of Type I, IIa, and IIx fibers were 38.1%, 58.2%, and 3.7%, respectively. The masseter muscle had 77.8% Type I fiber. The percentages of Type I and IIa fibers in the splenius muscle were approximately equivalent. The other three muscles had similar fiber type percentages, around 20% for Type I and 75% for Type IIa fiber.
Group 5: (respiratory-related muscles, Table 5).
The mean percentages of Type I, IIa, and IIx fibers were 41.3%, 55.9%, and 2.8%, respectively. All muscle had more than 90% of combination of Type I and IIa fibers. Especially, the diaphragm muscle contained a large amount of oxidative fiber, 59.3% of Type I and 39.8% of Type IIa fibers.
Comparison of the Muscle Fiber Type Population Among Muscle Groups
As shown in Fig. 2, Group 5 (respiratory-related) showed the highest percentage of oxidative muscle fibers (Type I + IIa) among the five groups, and followed by rank order of Group 4 (head and neck) > Group 3 (forelimb) > Group 2 (thoracic and trunk) > Group 1 (hindlimb). Significant differences were found in percentage of Type IIx fibers between Group 1 and Groups 3, 4, and 5. There were no significant differences among the other muscle groups.
Correlation Between the Muscle Fiber Type Population and Enzyme Activities
The oxidative (SDH), glycolytic (PFK) enzyme activities and the ratio (SDH/PFK) in mean values of 46 skeletal muscles are presented in Tables 1–5. The correlations between percentages of Type I fiber and enzyme activities are presented in Figs. 3 (SDH activity), 4 (PFK activity), and 5 (SDH/PFK activities). The correlation coefficients were statistically significant in Figs. 4 and 5, but not in Fig. 3. The correlation coefficient between percentage of oxidative fiber (Type I + IIa fibers) and SDH activity was not significant (r = 0.11).
The same linear regression analysis was performed in individual horses. The correlation coefficients between percentages of Type I fiber and PFK activities were significant (r = −0.38 to −0.62) in five of six horses. Significant correlations between percentages of Type I fiber and SDH/PFK activities (r = 0.40 to 0.68) were found in all horses. Significant correlation between percentages of Type I fiber and SDH activity was found in only one horse (r = 0.33).
Group 2 (thoracic and trunk) and Group 5 (respiratory-related muscles) had relatively high SDH activities, especially diaphragm muscle (Fig. 3). The soleus and masseter muscles showed low SDH activities, despite having higher percentages of Type I fiber.
As shown in Fig. 4, Group 1 (hindlimb), except for soleus muscle, and Group 2 (thoracic and trunk) had relatively high PFK activities and Group 4 (head and neck) had relatively low PFK activities.
For the SDH/PFK activities ratio (Fig. 5), most Group 1 (hindlimb) muscles had lower values than expected for the percentage of Type I fibers. Group 3 muscles had higher values than expected for the percentage of Type I fibers.
In this study, we investigated the muscle fiber populations and metabolic properties of skeletal muscles obtained from the whole body in six Thoroughbred racehorses. In total, 276 (46 muscles × 6) samples were investigated including 13 from hindlimb, 9 from thoracic and trunk, 8 from the upper arm in forelimb, 7 from the forearm in forelimb, 5 from the head and neck, and 5 from respiratory-related muscles.
Characteristic Features in Fiber Type Population
As shown in Table 1 and a previous study (van den Hoven et al,1985), some differences in muscle fiber populations were observed within large and thick muscles (i.e., superficial and middle portions of gluteus medius). Furthermore, as shown in Fig. 1, an antibody that specifically reacts with MHC-IIx was not used to identify Type IIX fibers. Therefore, we have to mention the methodological limitations of our study concerning with the lack of consideration of the hybrid fiber (Type IIa/x). We speculate an overestimation of Type IIa fibers with a concomitant underestimation of Type IIx fibers. However, we determined that the functional properties could be represented by the three muscle fiber type populations of superficial portions in most muscles in this study.
The remarkable characteristic of Thoroughbred skeletal muscles was that many muscles had a low percentage of Type I fiber and a high percentage of Type IIa fiber. This tendency is largely in agreement with the results of previous studies that performed muscle fiber typing for eight Thoroughbred horses (Andrews and Spurgeon,1986). However, in some muscles, there are substantial differences in the percentage of fiber types (i.e., 7.3% vs. 30.8% Type I fiber in extensor carpi radialis muscle). Although the muscle sampling site is basically identical in both studies, the horses in the previous study (Andrews and Spurgeon,1986) were about 2 years younger than our horses. Furthermore, it is unknown whether the training state of their horses was similar to that of our horses. Therefore, we speculate that there are some age and/or training-related differences in fiber type composition between previous studies and this study. In general, it is suggested that larger mammals have a higher percentage of Type I fiber to sustain their heavy body weight. In fact, most of the limb muscles in small animals such as rat had more than 60% Type IIb (Ariano et al.,1973). Many studies with Thoroughbred racehorses have reported that the percentage of Type IIa fiber is increased with conventional training (Yamano et al.,2006; Rivero et al.,2007). Therefore, we speculate that the higher percentage of Type IIa fibers in Thoroughbred racehorses is attributed to the training effect.
In terms of the muscle characteristic in several parts of the horse body, first we noticed a remarkable difference between the forelimb and hindlimb muscles. The forelimb muscle had a higher percentage of Type IIa fiber and a significantly lower percentage of Type IIx fiber (Fig. 2). This result supports the hypothesis that the forelimb muscles play a less propulsive role than hind limbs during daily activities (Niki et al.,1984; Merkens et al.,1993; Payne et al.,2004; Dutto et al.,2004,2006).
Second, we found that the muscle fiber type populations in the thoracic and trunk portion were similar to those in the hindlimb portion. The muscle fiber type population of the hindlimb was significantly higher in Type IIx fiber than the other parts (Fig. 2). It is speculated that the rich Type IIx muscle fiber in hindlimb muscles could play an important role to produce propulsive forces during horse running. Although the thoracic and trunk muscles are not likely involved in dynamic action with fast contraction (Audigié et al.,1999; Robert et al.,2001), more recruitment of Type IIx fibers may be needed for faster movement of thoracic and trunk in Thoroughbred horses. Further studies are needed to confirm the recruitment patterns of thoracic and trunk muscles during high-speed running.
There was a large variability in the muscle fiber type population in Group 1 (hindlimb) and Group 4 (head and neck) muscles. For example, soleus, tensor fascia latae, iliacus, and psoas major muscles in Group 1 (Table 1), and masseter and splenius muscles in Group 4 (Table 4) had a high percentage of Type I. Soleus muscle in other animals, especially humans, is a typical posture muscle and composed of more than 80% Type I fiber (Johnson et al.,1973). The data of soleus muscle obtained in this study were consistent with many previous studies in the other animals. However, we doubt the function of soleus muscles because the muscle is rather diminutive with respect to the overall size of muscles in the hindlimb of horses (Meyers and Hermanson,2006). Meyers and Hermanson (2006) suggested that the soleus muscle could have a significant role in proprioceptive function instead of a significant force during locomotion, because the muscle had many sensory organs (muscle spindles). The iliacus and psoas major muscles in Thoroughbred horses might play an important role for antigravity activity in hindlimb muscles. A high percentage of Type I fibers in masseter and splenius muscles seems to be reasonable for continued activity for mastication and sustaining the heavy head, respectively.
The tensor fascia latae muscle is a flexor of the hip joint, so we expected that this muscle would have many Type IIx fibers. In practice, ∼50% of the muscle fiber in this muscle were Type I fibers (Table 1). Tokuriki and Aoki (1995) reported that the EMG signal of the tensor fascia latae was not apparent in the swing phase when the hip joint is flexed. However, the muscle was active in the stance phase when this joint was extended. Therefore, they suggested that this muscle might act as an extensor or stabilizer of the stifle joint during locomotion. We speculate that the higher percentage of Type I fibers in tensor fascia latae may support the suggestion based on EMG analysis.
Metabolic Enzyme Properties and Fiber Type Population
Because the muscle samples from the whole body were taken 2 hr after the horses were euthanized, the SDH and PFK activities of gluteus medius muscle (4.05 ± 0.57 and 40.28 ± 24.73 μmol/g wet weight) decreased substantially, when compared with our unpublished data (5.26 ± 1.08 and 44.25 ± 4.96 μmol/g wet weight) in gluteus medius muscle (5 cm depth from surface) of same age Thoroughbred horses (N = 8) obtained by needle biopsy. Therefore, we estimate that about a 25% and 10% decreases in oxidative and glycolytic enzyme activities, respectively, exists in postmortem samples in this study.
Although the fiber type population was identical between Group 1 (hindlimb) and Group 2 (thoracic and trunk), the SDH activities in Group 1 were lower than those of Group 2 (Fig. 3). The hindlimb muscles are required to produce a propulsive force in anaerobic conditions and the thoracic and trunk muscles function to stabilize the trunk, which is the heaviest part of a horse (Audigié et al.,1999; Robert et al.,2001). These functional demands may cause dynamic contraction in hindlimb muscles and isometric contraction in the trunk muscle. Therefore, although the fiber type population is basically important for muscle enzymatic properties, the activity pattern of these muscles has significant impacts on enzymatic properties. The main respiratory muscle (diaphragm) had the highest SDH activity, indicating that mitochondrial enzyme activities depend on the functional demand of the muscle.
As shown in Fig. 4, the PFK activities were highly correlated with the fiber type population (r = 0.67). This relationship suggests that glycolytic enzyme activities strongly depend on the muscle fiber type population, not like SDH activities. Furthermore, as shown in Fig. 5, the SDH/PFK activities were more closely correlated to fiber type population (r = 0.72). The ratio of oxidative to glycolytic enzyme is considered to represent metabolic properties of the muscle. On the basis of this concept, it is concluded that the Group 1 (hindlimb) and Group 3 (forelimb) muscles are typical glycolytic and oxidative muscle groups, respectively.
To consider further physiological properties of each muscle group, data for the recruitment pattern of each muscle fiber type during exercise are needed. The muscle fiber properties in this study combined with the recruitment data would provide valuable information to determine optimal training intensities for Thoroughbred horses. In addition to the physiological aspect, these data would be expected to facilitate understanding for some disorders of the equine motor system.
The authors thank Dr. Sugiura in Yamaguchi University for supplying all antibodies for myosin heavy chains, and the Management Section of the Equine Research Institute for their help throughout all the experiments.
- 1986. Histochemical staining characteristics of normal horse skeletal muscle. Am J Vet Res 47: 1843–1852. , .
- 1973. Hindlimb muscle fiber populations of five mammals. J Histochem Cytochem 21: 51–55. , , .
- 1999. Kinematics of the equine back: flexion-extension movements in sound trotting horses. Equine Vet J Suppl 30: 210–213. , , , , .
- 1988. Gel electrophoresis of proteins from single equine muscle fibers. In: GillespieJR, RobinsonNE, editors. Equine exercise physiology 2. Davis, CA: ICEEP Publications. p 359–366. , , , .
- 1999. Frequency of musculoskeletal injuries and risk factors associated with injuries incurred in quarter horses dueing races. J Am Vet Med Assoc 215: 662–669. , , , , .
- 1950. A microspectrophotometric method for the determination of succinic dehydrogenase. J Biol Chem 186: 230–243. , , .
- 2006. Joint work and power for both the forelimb and hindlimb during trotting in the horse. J Exp Biol 209: 3990–3999. , , , , .
- 2004. Ground reaction forces in horses trotting up an incline and on the level over a range of speeds. J Exp Biol 207: 3507–3514. , , , .
- 1973. Data on the distribution of fibre types in thirty-six human muscles. An autopsy study. J Neurol Sci 18: 111–129. , , , .
- 1974. Fibre composition, enzyme activity and concentrations of metabolites and electrolytes in muscles of standardbred horses. Acta Vet Scand 15: 287–309. , .
- 1999. Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants. J Anat 194: 363–372. , , .
- 1993. Ground reaction force patterns of Dutch Warmblood horses at normal trot. Equine Vet J 25: 134–137. , , , .
- 2006. Horse soleus muscle: postural sensor or vestigial structure? Anat Rec A Discov Mol Cell Evol Biol 288: 1068–1076. , .
- 1984. A force plate study in equine biomechanics 2. The vertical and fore-aft components of floor reaction forces and motion of equine limbs at walk and trot. Bull Equine Res Inst 21: 8–18. , , .
- 2004. The role of the extrinsic thoracic limb muscle in equine locomotion. J Anat 205: 479–490. , , .
- 1994. Prevalence of, and factors associated with, musculoskeletal racing injuries of Thoroughbreds. J Am Vet Med Assoc 204: 620–626. , , .
- 2001. Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains. Histochem Cell Biol 116: 299–312. , .
- 2007. Effects of intensity and duration of exercise on muscular responses to training of Thoroughbred racehorses. J Appl Physiol 102: 1871–1882. , , , , , , , .
- 1999. Analysis of myosin heavy chains at the protein level in horse skeletal muscle. J Muscle Res Cell Motil 20: 211–221. , , , , .
- 1996. Myosin heavy chain isoforms in adult equine skeletal muscle: an immunohistochemical and electrophoretic study. Anat Rec 246: 185–194. , , .
- 2001. Effects of treadmill speed on the mechanics of the back in the trotting saddlehorse. Equine Vet J Suppl 33: 154–159. , , , , .
- 1996. Myosin isoforms and muscle fiber characteristics in equine gluteus medius muscle. Anat Rec 244: 444–451. , , , .
- 1964. Enzyme patterns in human tissues. I. Methods for the determination of glycolytic enzymes. Cancer Res 24: 709–724. , .
- 1980. Muscle fibre type composition of a number of limb muscles in different types of horse. Res Vet Sci 28: 137–144. , .
- 1989. Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers. J Histochem Cytochem 37: 1731–1738. , , , .
- 1995. Electromyographic activity of the hindlimb muscles during the walk, trot and canter. Equine Vet J Suppl 18: 152–155. , .
- 1994. Acquired equine motor neuron disease. Vet Pathol 31: 130–138. , , , , , , .
- 1985. Variation of fiber types in the triceps brachii, longissimus dorsi, gluteus medius, and biceps femoris of horses. Am J Vet Res 46: 939–941. , , , , .
- 2006. Recruitment pattern of muscle fibre type during high intensity exercise (60–100% VO2max) in thoroughbred horses. Res Vet Sci 80: 109–115. , , , .
- 2005. Evaluation of developmental changes in the coexpression of myosin heavy chains and metabolic properties of equine skeletal muscle fibers. Am J Vet Res 66: 401–405. , , , , , , .