Histochemical and morphometric aspects of the lateral musculature of different species of teleost marine fish of the Percomorphi order

Cross-sections of the lateral musculature of 19 species of teleost fish of the Percomorphi order were studied histochemically and morphometrically. The red, pink and white muscles were observed in all the species except in Seriola dumerilii and Scomber japonicus, in which no pink muscle was found. The red fibres were the smallest due to their minimal diameters. Histochemically and according to their location at least five types were found in the same species: typical red, in rosette, of the border, infiltrated pink and in mosaic. The pink fibres were larger than the red but smaller than the white. The form and disposition of the pink muscle varied according to the species. This muscle is made up by fibres of medium size, moderate m-ATPase and intermediate oxidative activities. Their histochemical properties change near red and white muscles, where they acquire the histochemical characteristics of the typical red fibres or the low m-ATPase activity of white fibres, respectively. The white muscle is the widest muscular layer. All of the species, except Thalassoma pavo are made up by three fibre types (small with high m-ATPase activity, medium with moderate m-ATPase activity, and large with low m-ATPase activity). They are mixed and show a histochemical and morphometric mosaic form. The extent of this mosaic depends on the species. In most of the analysed species it was observed in areas close to the pink muscle, but in three of them (Liza aurata, Seriola dumerilii and Coris julis) it occupied all of the myotome.     

Characterization of the swimming muscle of the anchovy Engraulis anchoita (Hubbs and Martini 1935)

The lateral muscle characteristics of individual female anchovies (Engraulis anchoita) were studied by histochemical and ultrastructural techniques. Red, pink and white fibres were discerned on the basis of the activity of the metabolic enzymes succinic dehydrogenase, phosphorylase and myofibrilar ATPase, and the usage of Sudan and periodic-acid Schiff staining techniques. Red fibres were located at the surface, white fibres deep inside and pink fibres in between. The red fibres appeared flattened or ribbon-shaped in transverse section and they were located in rows separated by myosepta of connective tissue. The red and pink fibres were multi-innervated whereas the white fibres were terminally innervated. The relative proportion of the red muscle increased toward the caudal region, the white muscle diminished toward that region, whereas the proportion of pink muscle did not vary according to region, being scarce throughout. The distribution of capillaries was also investigated, and as a result, the vascular supply proved to be scarce in both white and pink muscle.     

The Post-larval Development of Lateral Musculature in Gilthead Sea Bream Sparus aurata (L.) and Sea Bass Dicentrarchus labrax (L.)

Fibre-type differentiation of lateral musculature has been studied in gilthead sea bream Sparus aurata (L.) and sea bass Dicentrarchus labrax (L.) during post-larval development using ultrastructural, histochemical and morphometric techniques. The study showed three muscle layers: red, intermediate (or pink) and white. Initially, most of the red muscle showed low myosin ATPase (m-ATPase) activity fibres, whereas near the transverse septum some small high m-ATPase activity fibres appeared and later acquired a rosette aspect. Afterwards, during adult growth the red muscle showed a histochemical mosaic appearance. The pink muscle in sea bass was observed at the beginning of juvenile development by the oxidative technique (NADH-RT reaction) whereas in gilthead sea bream it was also observed at the end of larval development. The pink layer consists of high m-ATPase activity fibres. However, along the muscle development other low and moderate m-ATPase activity fibres were observed close to the red and white muscles, respectively. The white muscle of juvenile fish showed a histochemical mosaic appearance near the pink muscle. In adult specimens the mosaic white muscle spread out occupying the whole of the myotome. Morphometric analysis shows a significant increase in mean fibre diameter during post-larval development, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibres from the myosatellite cells (hyperplastic growth).     

Characterization of Lateral Musculature in the Striped Weakfish (Cynoscion striatus Cuvier)

Histochemical and ultrastructural studies were performed on lateral musculature from individual female weakfish (Cynoscion striatus Cuvier). Based upon SDH, PhR, Sudan and myosin-ATPase determinations, different kinds of red, pink and white fibres were discerned at the anterior, medium and posterior regions. Levels of glycolytic activity increased from red to white fibres, being intermediate in the pink ones. Contrarily, oxidative activity decreased from red to pink and white fibres. Histochemical stain showed a complex distribution of fibre types in each layer. A considerable variation in size and distribution was also found among fibres.     

Morphological, histochemical and morphometric study of the myotomal muscle tissue of the pacu (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei)

Histochemical, ultrastructural and morphometric methods were used to study growth patterns of red, pink and white muscle fibres and their relation to body weight and total length in the fast-growing freshwater fish Piaractus mesopotamicus Holmberg. The correlations amongst body weight, body length and diameter of red, pink and white fibres were low. From 10-15 to 40-50 cm, body weight increased 102.7 times, while the diameter of each type of fibre increased by factors of 0.94, 0.74 and 0.70, respectively. Muscle fibres revealed different morphological and histochemical stages of maturation. The frequencies of < 20 microns fibres of red, pink and white muscle tissue in the youngest and oldest classes were 64.5 and 11.0, 38.2 and 7.7 and 24.0 and 1.4%, respectively. In 30-40 cm fish, the frequency of < 20 microns fibres in the red and pink tissue was 24.5 and 25.5%, while in the white tissue it was 11.5%. During sexual maturity (40-50 cm), the recruitment of < 20 microns fibres in white muscle was 1.4%. Muscle fibres of this species showed continuous growth by both hyperplastic and hypertrophic mechanisms, and hyperplasia was particularly active in the juvenile phase.     

Suspected mitochondrial myopathy in a Jack Russell terrier

A Jack Russell terrier with a history of progressive exercise intolerance was examined at the age of four months and again 10 months later. Clinical examination revealed a stunted, thin dog with a stilted gait. The dog had raised lactate levels before and after feeding and a raised lactate/pyruvate ratio after feeding, indicating a metabolic abnormality. Histochemical evaluation of muscle biopsies revealed subsarcolemmal accumulation of oxidative activity when stained with nicotinamide adenine dinucleotide tetrazolium reductase and ragged red fibres when stained with modified Gomori trichrome; all fibre types were involved. Ultrastructural examination of the muscle confirmed the presence of subsarcolemmal accumulations of mitochondria. Histochemical staining for the activity of enzymes of the Krebs cycle, oxidative phosphorylation and other metabolic cytosolic enzymes failed to demonstrate an abnormality. In view of the clinical picture and the biochemical and histological findings, a tentative diagnosis of mitochondrial myopathy was made. The difficulties associated with diagnosing mitochondrial disorders are discussed.     

Muscle Development in Gilthead Sea Bream (Sparus aurata, L.) and Sea Bass (Dicentrarchus labrax, L.): Further Histochemical and Ultrastructural Aspects

The histochemical profiles - mATPase and NADH-TR reactions - of the red and white muscle fibres of gilthead sea bream and sea bass were determined from the first week after hatching. Modifications of the mATPase technique by combinations of pH/time/molarity were carried out in order to compare the sensitivity of the myosin ATPase of each muscle fibre type of the lateral muscle. Results showed that the staining of muscle fibres was independent of small modifications in the technique. The intermediate ‘pink’ muscle was histochemically defined towards the end of the larval life and is considered to be implicated in the growth of the myotome. A layer of external cells was observed, by electron microscopical examination, between the connective tissue of the skin and the superficial red muscle fibres of larvae and postlarvae. It is suggested that the external cells are unlikely to be a source of red muscle fibres and implicated on the growth of the myotome, but rather a part of the dermatome. The timing, areas and mechanisms of hyperplastic growth of the myotome were defined and discussed.     

Red muscle development of gilthead sea bream Sparus aurata (L.): structural and ultrastructural morphometry

The transverse red and white muscle area, the superficial red muscle fibres area and their percentages of mitochondria (%mit), myofibrils (%myof) and sarcoplasm (%sarc) were determined in the Mediterranean teleost gilthead sea bream, Sparus aurata (L.). Fish aged from hatching to 78 days were studied. The proportional growth of the red and white muscles was higher for the red muscle in the first half of the larval stage (1-35 days). Then the opposite relationship was observed. The hypertrophic growth of the superficial muscle fibres was continuous except in the first week after hatching. The percentage of mitochondria and percentage of myofibrils showed a significant change just after the half of the larval stage. Whereas the %mit of the superficial muscle fibres was higher than %myof from the first week after hatching to 35 days (average 66.64%), then the %mit decreased significantly and at 73-78 days both parameters were close to an average value of 50%. The meaning of these morphological changes is discussed in relation to the functional role of the red muscle of larvae and the onset of the gills respiration.     

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.     

Morphometry and histochemistry of the semitendinosus muscle of tufted capuchin monkeys (Cebus apella Linnaeus, 1758)

Considering monkeys are animals closely related to the human, and semitendinosus muscle has been used in many postural research experiments, we have decided to study its histochemical characteristics. Samples were removed from the proximal, middle and distal regions of the semitendinosus muscles of five adult male tufted capuchin monkeys and observed for reaction with m-ATPase (with alkaline and acid pre-incubation), nicotinamide adenine dinucleotide tetrazolium reductase, and haematoxylin and eosin. The muscle fibres were classified as fast glycolytic (FG), fast oxidative glycolytic (FOG) and slow oxidative (SO) and quantified in terms of frequency and area. The three regions of the semitendinosus muscle showed no significant differences in frequency or area of the respective fibre types, and therefore the muscle can be considered histoenzymologically homogeneous. FG fibres presented higher frequencies and larger areas. The sum of FOG and SO fibres was 57.5%, suggesting that the semitendinosus muscle of the tufted capuchin monkey is adapted to an action involved in posture maintenance and in long arboreal dislocation. The present data agrees with the notion of differentiated quadrupedalism in some primates, which support a lower percentage of their weight on the fore limbs.     

Relation between myoglobin levels and succinate dehydrogenase activity in muscle fibers

We compared the histochemical activity of succinate dehydrogenase (SDH) and the intensity of reaction to the presence of myoglobin (Mb) in the same muscle fibres m. longissimus thoracis (MLT), m. semimembranosus (MSM), m. rectus femoris (MRF) and caput longum m. tricipitis brachii (MTB). At the same time we also ascertained the myoglobin content of one gram of fresh muscle. We found that the muscle fibre in all the muscles studied in which there is the most intensive reaction in the test for SDH are also the most intensively coloured in the test for Mb. There is also the same relationship in intermediary and white fibres. The reaction in testing for SDH on a cross section is greatest in the subsarcolemmic area. Similar tendencies appear in the distribution of Mb, although they are less marked and only in fibres with intensive reaction. Different muscles have different myoglobin levels. Muscles with a higher proportion of red and intermediary fibres contain more myoglobin than muscles with a high proportion of white fibres. The quantitative values of the levels of Mb (g/fresh muscle) are not, however, proportional to the share of individual types of muscle fibres in the muscles studied.     

Caracteristicas Histoquimicas y Morfometricas de Algunos Musculos Extraoculares del Perro

Histochemical and Morphometric Aspects of some Extraocular Muscles of the Dog
This investigation was carried out on retractor bulbi, lateral and medial rectus muscles of six adult dogs. Tissues were collected from near the center of individual muscle bellies. These were stained for m-ATPase at varying pHs during preincubation, NADH-TR, Alpha-GPDH, Modified Mason trichrome and Hematoxyline and Eosin. Muscle fibers were classified as type I and type II, based on their reaction for m-ATPase. The retractor bulbi muscle was composed entirely of type II, oxidative, muscle fibers, with no glycolytic fibers. The rectus muscles presented a stratified composition, with superficial muscle fiber bundles containing a mixture of type II fibers oxidative and glycolytic, and central bundles of type I, mixed with type II. It was observed that there was less interstitial tissue in the center of the muscle bellies. Large diameter nerve fibers were also observed in the central layers.     

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.     

Energy metabolism in relation to skeletal muscle fibre properties during treadmill exercise

The skeletal muscle metabolism of horses with a wide range of cardiocirculatory capacities was studied during a standardised near-maximal exercise test in relation to muscle fibre composition. Although the same amount of work was performed by all horses the amount of triglycerides and glycogen utilised and lactate accumulated differed widely. Both blood and muscle lactate accumulation were positively related to the amount of glycogen utilised. These factors were in turn positively associated with the percentage of Type IIB fibres and consequently negatively associated with the percentage of Type IIA fibres. The percentage of Type IIB fibres was negatively correlated to the activities of the oxidative enzymes citrate synthase and 3-hydroxyacyl-CoA-dehydrogenase and positively associated with the lactate dehydrogenase activity. The results indicate that the horses with a high proportion of Type IIB fibres had a lower muscle oxidative capacity and were, therefore, for energy production, more dependent upon glycolysis with subsequent lactate accumulation than horses which had a high proportion of Type IIA fibres. The different metabolic properties of the muscle fibre types indicate that the pattern of muscle fibre recruitment could have a significant effect on intramuscular substrate utilisation.     

The leucine metabolite 3-hydroxy-3-methylbutyrate (HMB) modifies protein turnover in muscles of laboratory rats and domestic chickens in vitro

This study was conducted to assess the in vitro effects of leucine and 3-hydroxy-3-methylbutyrate (HMB) on protein synthesis and degradation in isolated rat and chick muscles. Isolated muscles — extensor digitorum longus (EDL) and soleus (S) in rats and EDL and tibialis anterior (TA) in chicks — were taken out intact and incubated in Krebs—Henseleit buffer with [¹⁴C]phenylalanine. Protein synthesis was then determined by measuring [¹⁴C]phenylalanine incorporation into protein and protein breakdown was estimated by net release of tyrosine from muscle proteins. In all muscles investigated leucine stimulated protein synthesis (average 20%; p < 0.05 versus control) but only slightly reduced proteolysis (average 8%, NS). In contrast, HMB had no significant effect on protein synthesis but consistently decreased protein degradation in the range from −29% (p < 0.01 versus control) in rat extensor digitorus longus to −5% (NS) in rat soleus muscles. HMB appeared to suppress protein degradation more in white muscle fibres than in red muscle fibres (at least in the rat). These data support observations in humans indicating that HMB decreases muscle proteolysis. It is suggested that metabolism of leucine to HMB could be responsible for the reported suppressive effects of leucine on protein breakdown.     

The effect of testosterone on gastrocnemius muscle fibres in growing and adult male and female rats: a histochemical,morphometric and ultrastructural study

In this study, the effect of testosterone on gastrocnemius muscle fibres in growing and adult rats (male and female) was examined using histochemical, morphometric and ultrastructural techniques. After physiological saline (PS), olive oil (OvO) or olive oil + testosterone (OvOT) injections on 72 rats (growing and mature, 36 male and 36 female), the sample tissues of fibre types of the gastrocnemius muscle taken were examined by histochemical [alkaline adenosine triphosphatase (alk-ATPase), acid ATPase (ac-ATPase)], morphometric and ultrastructural techniques. In PS-injected control groups, the gastrocnemius muscle of both sexes contained all the fibre types studied [slow-oxidative muscle fibres (type I), fast-oxidative glycolytic muscle fibres (type IIA) and fast-glycolytic muscle fibres (type IIB)]. The type I fibres had the smallest diameter, type IIA had a medium diameter and type IIB fibres had the largest diameter. In OvO-injected groups, it was observed that the OvO had little effect on the gastrocnemius muscles of either sex, although there was significant enlargement of type IIB fibres. After the injection of OvOT, hypertrophy of muscle fibres was determined by morphometric study. The biggest increase in diameter was on type I fibres. In addition, degenerations on some mitochondria, accumulation of lipid droplets on type I and type II fibres, an increase in glycogen particles, bifurcation of myofibrils, an increase in the number and diameter of units resembling T tubules and an increase in ribosomal content were also observed in the same group by transmission electron microscope. Consequently, it was determined that testosterone can induce protein synthesis in gastrocnemius muscle fibres, and induces changes in shape and size, and also can change the appearance and the number of fibres.     

Factors Responsible for Smaller Pelvic Muscle Mass in Dysplastic Dogs

Pectineus muscles of 23 German shephard dogs were studied. 10 dogs had normal hips, 13 dogs had hip dysplasia. The relative weight of each pectineus muscle was determined, when the dogs were 24 months old. Cryostat sections were stained for demonstration of ATPase, thereby differentiating muscle fibres into type I (slow twitch), type II (fast twitch) and intermediate fibres. The results (Tables 1–3) shows, that the relative pectineus muscle weight was smaller in dysplastic dogs and there were significantly fewer type I muscle fibres in the pectineus muscles of dysplastic dogs.     

Swine foetal myogenesis in different gestation periods

This experiment was conducted to evaluate the effects of sex and uterus position on swine foetal myogenesis at different gestational ages. Fifteen primiparous sows were divided into three groups according to gestational age: 50, 80, and 106 days. The experiment was a block randomized factorial design with two sexes (male and female) and three uterine regions (apex, middle, and base). After slaughter, each uterus horn was divided into three segments of equal length: apex region near the ovary; base region near the uterine body; and the middle region, lying between the apex and base regions. The foetuses were weighed, identified, and longitudinally opened to harvest the semitendinosus muscle for later morphological analysis. After 50 days of pregnancy, male foetuses had greater (p  < .05) weight than females. The number of primary fibres at 50 days of gestation was negatively correlated (r  = ?.29, p  = .04) with the number of foetuses in utero. After 80 days, foetuses in the base region had less (p  < .05) secondary area of muscle fibres compared to the apex region, which was accompanied by differences in the weight of the foetuses, the lowest weight were for foetuses located in the base region (p  < .05). In the same period, the ratio of secondary to primary fibres had a positive correlation with weight. In conclusion, sex did not influence myogenesis in the gestational ages studied and the development of secondary muscle fibres of the foetuses at 80 days of gestation was affected by their uterine position with foetuses at the base of the uterine horn being less developed.     

Temperature effect on muscle growth of the axial musculature of the sea bass (Dicentrarchus labrax L.)

In order to determine the temperature effect on the axial muscle growth of sea bass, a stock of larvae was subjected to the following incubation and cultivation temperatures, respectively: 15 degrees C/ambient, 15/17 degrees C, 17 degrees C/ambient and 17/17 degrees C. In all groups the cross-sectional area of white and red muscles and the number and average area of the white and red muscle fibres were quantified. Results showed that the embryonic period, pre-larval phase and the end of metamorphosis were accelerated at higher temperatures. During the endogenous feeding period, muscle growth took place by fibrillar hypertrophy, and was not influenced by the temperature. Thereafter (external feeding) muscular hyperplasia began, and growth of all the muscular parameters was favoured by the effect of high incubation and cultivation temperatures, with the latter having higher influence. High incubation temperature had an slight effect on muscle growth and body length, which was only observed from 15 days. Metamorphosis finished at 3 +/- 0.4 cm in all the larvae, but this length was earlier reached at higher temperatures. At 120 days, the largest growth was obtained in the larvae maintained at a higher temperature.     

Cytochemistry for lectins, actin, nucleotide tetrazolium reductases and several phosphatases in the porcine semitendinosus muscle: vascular development in young pigs

An ontogeny study of the porcine semitendinosus muscle was conducted to meet two objectives: 1) to evaluate enzyme histochemistry, lectin cytochemistry and actin staining for usefulness as quantitative markers of muscle capillaries and 2) to describe the ontogeny of capillary density changes in developing porcine muscle. Muscle samples were obtained from three to eight crossbred pigs at each of the following ages: newborn, 1 to 2 d and 1, 2, 3, 4.5 and 24 wk. Cryostat sections were stained or reacted for alkaline and neutral phosphatase, dehydrogenase, actin, a panel of nine plant lectins (fluorescein isothiocyanate conjugated) and routine cytochemical tests for muscle fiber typing. Capillaries were quantitatively marked by reactions for dehydrogenase activity in the young pigs but not in the oldest animals. The lectins, soybean agglutinin, Bauhinia purpuria agglutinin, Ulex europeus agglutinin-I and Griffonia simplicifolia agglutinin-I (GS-I) quantitatively stained capillaries at all ages. Histological observations of thin sections of epon-embedded tissues served to validate the lectin and cytochemical capillary staining. Micrographs of sections stained with the lectin GS-I were used to count capillaries and muscle fibers so the capillary:fiber ratio (C/F) could be calculated. Deep (red) and superficial (white) aspects of muscle sections had different C/F at birth, 2, 4 and 24 wk. The deep aspects (DA) had higher C/F than superficial (SA) aspects (at all four ages), and there were age-dependent increases (P less than .001) in C/F of DA and SA. This study demonstrates the usefulness of lectin staining for determining C/F in porcine muscle.