Histochemical identification of fiber types in canine skeletal muscle.

Proximal and distal skeletal muscles from pectoral and pelvic limbs were histochemically examined in 18 neuromuscular disease-free dogs. On the basis of the human system of classification and nomenclature and results of standard adenosine triphosphatase (ATPase) and glycine-formaldehyde preincubation procedures, the fiber types identified in immature and mature canine skeletal muscles were I, IIA, and IIC.     

Histochemical skeletal muscle fibre types in the sheep

In this study, the differentiation of adult and postnatal muscle fibres in sheep longissimus thoracis muscle has been characterized. By using a variety of histochemical methods, we have investigated the m-ATPase and metabolic activities of skeletal muscle fibres in adult sheep and lambs aged between 1 day and 3 months. Types I, IIA, IIB and IIC fibres were identified. The results showed that the interpretation of the fibre type composition depends on the methods used. The findings also revealed that the fibre types IIA and IIB can be separated histochemically in sheep by using the correct m-ATPase technique, even at early stages of postnatal development, and that the origin of the four different fibres of the adult can be traced back to early postnatal stages.     

Simplified technique for histochemical determination of three fiber types in equine skeletal muscle

For determination of 3 muscle fiber types in equine skeletal muscle, a comparison of 2 preincubation buffers, each followed by myosin adenosine triphosphatase staining, was made. Serial sections of the muscle samples (n = 75) were preincubated in an acid buffer (pH 4.6) or a formaldehyde-glycine buffer (pH 7.25) and then were stained for myosin adenosine triphosphatase. Differentiation of muscle fibers into type I, IIA, and IIB was identical with both techniques; however, in the samples prepared at pH 4.6, type I fibers were black; type IIA, light gray; and type IIB, dark gray. In the samples prepared at pH 7.25, types I, IIA, and IIB fibers were white, light gray, and dark gray respectively. The formaldehyde-glycine preincubation buffer (at pH 7.25) gave more consistent results, was easier to prepare, and retained cytoarchitecture better, compared with the samples prepared at pH 4.6.     

Histochemical staining characteristics of normal horse skeletal muscle

The histologic and histochemical staining characteristics of the triceps brachii (long head), extensor carpi radialis, gluteus medius, vastus lateralis, biceps femoris, semimembranosus, semitendinosus, and extensor digitorum longus muscles of 8 Thoroughbreds, 2 Quarter Horses, 1 Arabian, 1 Paso Fino, and 1 Shetland Pony are described. Muscle fiber morphology, staining distribution and intensity, amount of IM connective tissue, number of IM blood vessels and IM nerves, calcium-activated adenosine triphosphatase activity (CaATPase), percentage of fibertype population, percentage of relative fibertype area, mean fiber diameter, nonspecific esterase activity, alkaline phosphatase activity, and acid phosphatase activity were evaluated, using 10 common histochemical and histologic stains. Two fiber types (I, II) and 3 subtypes (IIA, IIB, IIC) were observed, using CaATPase-, nicotinamide-adenine dinucleotide-tetrazolium reductase-, periodic acid-Schiff hematoxylin-, and nonspecific esterase-stained frozen serial muscle sections. Type I muscle fibers in general had low CaATPase activity, high oxidative capacity, low glycogen capacity, and low esterase activity. Type IIA muscle fibers had high CaATPase activity, intermediate oxidative capacity, high glycogen concentration, and high esterase activity. Type IIB fibers had high CaATPase activity, low oxidative capacity, high glycogen concentration, and a high esterase activity. Type IIC muscle fibers had high CaATPase activity, high oxidative capacity, variable glycogen concentration, and high esterase activity. Type II (IIA and IIB) muscle fibers predominated in the muscles. The percentage of muscle fiber population, mean minimal muscle fiber diameter, and percentage of relative muscle fiber area were determined for each sampled muscle. Type IIA and IIB muscle fibers predominated in the percentage of muscle fiber population and percentage of relative muscle fiber area. Type IIB muscle fibers had the greatest minimal fiber diameter, type IIA muscle fibers had intermediate minimal fiber diameter, and type I muscle fibers had the smallest minimal fiber diameter. The percentage of relative muscle fiber area was less variable (P less than or equal to 0.05) than the percentage of muscle fiber population. Mean muscle fiber diameter did not significantly differ between breeds. Alkaline and acid phosphatase activities were at low levels in all muscles biopsied and were limited to the IM connective tissue fibrocytes, macrophages, and capillaries.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histochemical properties of muscle fibres types and enzyme activities in skeletal muscles of Standardbred trotters of different ages

Fibre characteristics and enzyme activities were determined for the gluteus, semitendinosus, vastus lateralis and triceps brachii muscles of 55 Standardbred trotters of different ages. Four fibre types (I, IIA, IIB, IIC) were demonstrated by histochemical staining of myofibrillar adenosine triphosphatase after preincubation at different pH values. Type II fibres predominated in all the muscles and the type IIA/IIB ratio was higher in horses over 5 years than in younger horses, except in the vastus in which the IIA/IIB ratio did not change with age. The vastus had the highest proportion of type IIA fibres and the semitendinosus the highest proportion of type IIB fibres. Histochemical demonstration of NADH dehydrogenase disclosed that almost 100 per cent of the type IIA and many of the type I and IIB fibres were medium-stained; the remaining type I fibres were darkly stained and the type IIB fibres lightly stained. In older horses more fibres were stained for NADH dehydrogenase. The activity of triosephosphate dehydrogenase decreased that that of 3-hydroxy-acyl-coA dehydrogenase and citrate synthase increased in all the muscles except the vastus with increasing age. The greatest increase in oxidative capacity occurred in the gluteus and triceps. Training, rather than age, was regarded as the factor inducing these changes. The results emphasise that histochemical data are only semiquantitative, and there are apparent discrepancies in the intensities of histochemical staining and the biochemical evaluation of various enzymes.     

Technique for physiological examination of canine skeletal muscle in vivo

A technique is described for studying the physiological function of canine skeletal muscle in vivo. The contractile properties of the tarsal flexor muscles were examined in three beagle dogs under general anaesthesia. The force responses to electrical stimulation of the common peroneal nerve were measured at various frequencies to determine the frequency:force relationship for this muscle group. Fatigue characteristics were also examined during intermittent stimulated activity delivered in a set pattern of frequencies. The results provide quantitative characterisation of muscle function which is repeatable. The technique described could be applied to other animals and is a potentially powerful tool for evaluating the effects of drugs on muscle performance.     

Effect of dietary protein on calpastatin in canine skeletal muscle

The cysteine proteinases, mu- and m-calpain, along with their inhibitor, calpastatin, have been hypothesized to play a role in skeletal muscle protein degradation. Because nutrition has previously been shown to influence the expression of calpastatin, the working hypothesis of this study was that the quantity and source of dietary protein could influence regulation of the calpain system in muscle. The objectives to support this hypothesis were to determine the effects of dietary protein (amount and source) on the expression of calpastatin in canine skeletal muscle. This study comprised eight diets with seven dogs per diet. A biopsy was taken from the biceps femoris of all 56 dogs before and after 10 wk on their respective diets. This experimental design allowed examination of change within individual dogs. Diets 1 to 4 contained 12% total protein derived from chicken and/or corn gluten meal in ratios of 100:0, 67:33, 33:67, and 0:100%, respectively. Diets 5 to 8 contained 28% total protein with protein sources and ratios identical to Diets 1 to 4. Differences in calpastatin were examined qualitatively using SDS-PAGE and immunoblotting, and semiquantitatively with densitometric analyses. The majority of the calpastatin blots showed three distinct calpastatin bands, the uppermost appearing at approximately 110 kDa. Diet 5 (28% CP, 100% chicken) resulted in an increase in the expression of the 110-kDa calpastatin band compared with the other two lower molecular weight bands in the same samples. Muscle from dogs fed Diet 5 showed greater increase in (P < 0.05) calpastatin intensity of the topmost band than those fed Diet 8 (0:100; chicken:corn gluten meal). Diet 5 (100:0; chicken:corn gluten meal) showed greater total calpastatin intensity than Diet 8 (0:100; chicken:corn gluten meal). These data suggest that dogs fed a diet containing a higher total percentage of chicken protein may have a greater potential to regulate calpain-mediated degradation of muscle protein than dogs fed diets containing corn gluten meal.     

Simultaneous histochemical determination of three fiber types in single sections of ovine, bovine and porcine skeletal muscle

Classification of muscle fiber types involves tedious comparisons of serial muscle tissue sections. Procedures that would incorporate pertinent histochemical properties into one simplified assay for the successful differentiation and determination of fiber types were evaluated using muscle samples from three different species (ovine, bovine and porcine). For ovine and porcine muscle tissue, the best staining procedure involved initial preincubation of the tissue section at 4.35 (for ovine) and 4.30 (for porcine) followed by incubating for succinate dehydrogenase (SDH) activity, then staining for myofibrillar adenosine triphosphatase (ATPase) activity after an acid preincubation and, finally, counterstaining with hematoxylin. For bovine muscle tissue, the procedure involves first incubating for SDH activity, after which the section is incubated at pH 4.15 and then stained for myofibrillar ATPase activity after an acid preincubation, with a concluding counterstaining using hematoxylin. These procedures permit successful differentiation and identification of three fiber types (beta R, alpha R and alpha W) from a single section of muscle tissue with approximately a 65% saving in time and materials. The results of histochemically typing muscle fibers in a single tissue section stained with these techniques were similar to those obtained with the traditional methods comparing serial sections.     

Fiber types and size in equine skeletal muscle.

Frozen sections of equine musculus semitendinosus were examined for myosin adenosine triphosphatase (ATPase) and reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), using standard histochemical procedures, and the proportions of the various fiber types and average fiber sectional size were determined. With ATPase staining, approximately 70% of the fibers were classified as alpha fibers (ATPase positive), and 30%, as beta fibers (ATPase negative). In addition, 2 populations of alpha fibers could be readily distinguished on the basis of the intensity of the ATPase reaction, and these were designated alpha positive and alpha intermediate. The relationship of this difference in ATPase reaction to contraction speed of the fibers is not known. With NADH-TR staining, fibers were classified as either red fibers (positive) having aerobic metabolism or white fibers (negative) having primarily anaerobic metabolism. All beta fibers were red by NADH-TR; thus, they conformed to the criteria for beta R fibers. All alpha positive fibers were white by NADH-TR, as were most of the alpha intermediate fibers, and would be classified alpha W. Some of the alpha intermediate fibers gave an intermediate reaction with NADH-TR and could be classified as alpha R fibers which have not transformed to alpha W fibers. The alpha positive fibers were 7 to 10 mum larger in diameter than either beta or alpha intermediate fibers.     

Identification and characterization of proteoglycans in bovine neonatal skeletal muscle

In order to provide background for understanding biological roles of proteoglycans (PG) in developing skeletal muscle, we have isolated and characterized PG in bovine neonatal skeletal muscle. Two types of PG were isolated from skeletal muscle by density gradient ultracentrifugation and ion‐exchange chromatography. One was a small PG (PG‐S) with a molecular size of 100–130 kDa, another was a large PG (PG‐L) with a molecular size of 300–500 kDa. The glycosaminoglycan chains of PG‐S and PG‐L were dermatan sulfate and chondroitin sulfate, respectively, judged by cellulose acetate membrane electrophoresis. Immunoblot assays revealed that both PG bound to type I, II, III and IV collagen, laminin and fibronectin. Unlike PG‐S, PG‐L bound to type V collagen and hyaluronic acid. Small proteoglycans had a core protein of 45 kDa, which reacted with the antibody against the decorin core protein. The N‐terminal amino acid sequence of the PG‐S core protein was consistent with that of decorin from bovine bone and tendon. Thus, PG‐S from neonatal skeletal muscle was identified as decorin in bovines. Immunohistochemical analysis with antibodies against PG‐L and PG‐S demonstrated that PG‐L was located both in the perimysium and endomysium, but PG‐S was localized exclusively in the perimysium. These findings suggest that the characterized PG may have distinct roles in the ECM construction of developing skeletal muscle.