Muscle growth in yolk-sac larvae of the Atlantic halibut as influenced by temperature in the egg and yolk-sac stage

Atlantic halibut eggs and yolk-sac larvae were incubated at 1, 5 and 8° C. Eggs incubated at 8° C gave slightly shorter larvae at hatching with a significantly smaller total cross-sectional area of white muscle fibres than eggs incubated at 5° C. Transport of eggs 2 days prior to hatching gave significantly longer larvae at hatching with a significantly larger red fibre cross-sectional area than when eggs were transported shortly after the blastopore closure. A higher survival until 230 degree days after hatching was also observed in the former group. All eggs incubated at 1° C died before hatching and all larvae incubated at 1° C died before 45 degree days after hatching. From hatching until 230 degree days the total white cross-sectional area increased threefold in all temperature groups. The increase in white cross-sectional area was entirely due to hypertrophy between hatching and 150 degree days (10 mm L _S). Recruitment of new white fibres increased in germinal zones at the dorsal, ventral and lateral borders of the myotome from 150 degree days onwards, but at 230 degree days (12–13 mm L _S) the recruitment fibre zone constituted <10% of the total white cross-sectional area. Larval incubation at 8° C gave slightly longer larvae with a significantly larger cross-sectional area of recruitment fibres at 230 degree days than incubation at 5° C. The larval group incubated at 8° C also had a significantly lower survival until 230 degree days than did the 5° C group. Incubation temperature regimes did not affect the volume density of myofibrils in the axial muscle fibres at 230 degree days. Thus hypertrophy is the predominant mechanism of axial white muscle growth in Atlantic halibut yolk-sac larvae and an increased rearing temperature during the yolk-sac stage increases white muscle fibre hyperplasia.     

Histochemical fibre types in the lateral muscle of fishes in fresh, brackish and salt water

The histochemical pattern of red, pink and white muscle of fish living in fresh, brackish, and salt water is reported. The muscle fibres were stained routinely during the year for lactate dehydrogenase (LDH), menadione α-glycerophosphate dehydrogenase (Mα—GPDH), succinic dehydrogenase (SDH), myosin adenosine triphosphatase (myosin ATPase), phosphorylase, lipids and glycogen. The pink and red muscles contain more glycogen and lipids and have a higher SDH activity, which is in accord with their aerobic metabolism and function in sustained swimming activity. The acid labile myosin ATPase activity characteristic of fast twitch fibres is present in the white fibres of most species, however in the white muscle of Gobius paganellus the enzyme activity is stable to both acid and alkali and, in addition, there is a scattered distribution of different fibre types in red and, especially, pink muscle. A study of seasonal variation patterns of myosin ATPase in white muscle of mugilidae over a period of two years has demonstrated, in late summer, the appearance of new small diameter fibres, with a high acid stable enzyme activity, that develop into the large diameter acid labile fibres.     

Temperature- and developmentally-induced variation in the histochemical profile of myofibrillar ATPase activity in carp

The histochemical profile of calcium activated acid stable myofibrillar ATPase (mATPase) activity in developing larval and juvenile carp was investigated. In the larval fish, differentiation of pink muscle fibres occurred after metamorphosis which was delayed by a week at 17° C compared to larvae grown at 27° C. After metamorphosis the 27° C group exhibited some small myofibres with acid stable mATPase activity in the deep white muscle. This was similar for the juvenile carp which were acclimated for more than a month at 25° C. In contrast, the cold (12° C) acclimated juvenile fish, contained very few small white muscle fibres with acid stable mATPase activity. It was also noted that the cold acclimated fish had lower background acid stable mATPase activity than the warm acclimated fish. Results indicate that after metamorphosis and more evidently in juveniles, temperature can influence the rate of myofibre hyperplasia.     

Temperature and neuromuscular development in the tambaqui

The development of muscle innervation pattern was investigated in larvae of the Amazonian fish, the tambaqui Colossoma macropomum. The time to hatching decreased from 28–29 h at 23.5° C to 11–12 h at 31° C. The larvae hatched after the completion of somitogenesis (38-somite stage) at 23.5° C but only at the 33-somite stage at 28–31° C. Embryos were stained for acetylcholinesterase activity and with an acetylated tubulin antibody in order to visualize neural processes. All muscle fibre types were initially innervated at their myoseptal ends. The development of motor innervation to the trunk muscle was delayed with respect to hatching at higher temperatures. At hatching, muscle fibres were innervated only to somites 16–17 at 28–31° C and somite 23–26 at 23.5–25° C (counting from the head), although the larvae swam vigorously to avoid sinking. In contrast, in newly hatched larvae myofibrils were present right along the trunk at all temperatures in both the superficial and inner muscle fibres. At hatching numerous multi-layered membrane contacts with the ultrastructural characteristics of gap junctions, were found between muscle fibres and at the inter-somite junctions, suggesting the somites were initially electrically coupled. These structures disappeared concomitant with the development of muscle endplates right down the trunk. The larvae started feeding 5 days post-hatch at 28° C. First feeding was associated with a dramatic decrease in the volume density of mitochondria and an increase in the volume density of myofibrils in the inner muscle fibres. The polyneuronal and multi-terminal pattern of innervation characteristic of adult slow-muscle fibres also developed around the time of first feeding.     

Morphological and histochemical characterization of the pectoral fin muscle of the stripped weakfish,Cynoscion guatucupa

The fibres of superficial and deep abductor muscles of the pectoral fins of the stripped weakfish, Cynoscion guatucupa have been studied using histochemical techniques: succinic dehydrogenase (SDH) for mitochondria, periodic acid–Schiff (PAS) for glycogen, myosin‐adenosintriphosphatase (mATPase) to identify different fibre types based on the contraction speed and modified ATPase to identify capillaries. The fibre diameters were measured, and the capillaries of the main fibre types – red, pink and white— were counted. The two muscles showed both macroscopically and microscopically two well‐differentiated zones with predominant white fibres. The area of insertion of muscles into the fin rays had red, pink and white fibres. The origin zone of the muscle into the bone was composed by white fibres only. Both zones of white muscle evidenced a mosaic of small, medium and large polygonal white fibres. Red, pink and white muscles showed a wide histochemical diversity of fibre subtypes. The area per peripheral capillary increased from the red to the white muscles. Due to the predominance of white fibres, the pectoral fins of C. guatucupa were mainly involved in rapid movements to stop/discontinue and stabilize the body during swimming.     

The histochemistry of muscle in juvenile Atlantic salmon, Salmo solar L.

In fry, 1- and 2-year old juvenile Atlantic salmon, relatively small superficial red muscle fibres staining well for glycogen and succinic dehydrogenase (SDH) but with little myofibrillar ATPase, were evident on either side of the lateral line, in June. Well differentiated relatively large white fibres contained lower concentrations of glycogen than the red fibres, higher ATPase and no SDH, except traces in the larger 1- and 2-year-olds. Intermediate size pink fibres, which were also intermediate between red and white fibres in their staining properties, occurred in a thin diffuse layer along the red-white boundary, thickest at the apex near the vertebral column, and most evident in the younger fish.     

Muscle development in cultured blackspot seabream Pagellus bogaraveo: preliminary histochemical and immunohistochemical data on the fibre types

We studied muscle ontogeny and fibre type characteristics in the blackspot seabream, a new species for commercial aquaculture. Myosin ATPase and SDH histochemistry and immunohistochemistry were tested at different ontogenetic stages, using a panel of antibodies to myosin isoforms and parvalbumin. In general, deep white muscle was parvalbumin‐positive, and superficial 'red' muscle was parvalbumin‐negative at all ages examined. At 6 days of age (transition from endogenous to exogenous feeding) three layers of muscle fibres were observed with different antimyosin reactivities: superficial monolayer, presumptive slow red (present only as a small group of fibres adjacent to the lateral line nerve), and presumptive fast‐white (forming the bulk of the muscle). The superficial monolayer and presumptive slow fibres were positive for SDH. At 60 days of age (transition from live to artificial feeding) an additional fibre type was identified: a typical 'pink' or intermediate layer. In juveniles, the axial muscle consisted mainly of fast white fibres covered by a slow‐red layer and between them a pink layer. Surprisingly, the red layer could be resolved into two distinct types by myosin immunostaining. Red fibres were also present along the horizontal septum, near the notochord. Both red and white muscle layers showed a mosaic appearance, which was confirmed by ATPase reaction. The work was financed by British Council, CRUP, and FCT (PhD Grant SFRH‐BD‐14068–2003).     

Characterization of swimming muscle in the Atlantic mackerel, Scomber scombrus L.

Both red and white muscle were removed from juvenile and adult Atlantic mackerel, Scomber scombrus L., for histochemical characterization of the muscle fibre types. Staining of white muscle for myosin ATPase, SDH, NADH diaphorase, GPDH and LDH revealed that these fibres are homogeneous. Red muscle was shown to be heterogeneous, of at least two fibre types recognizable on the basis of myosin ATPase staining with preincubation at a pH of 9·8. These two red types are dispersed throughout the red muscle and are present in both juveniles and adults. Red muscle is located both deep within the myotomes and as a superficial layer of muscle fibres. A third group of muscle fibres, intermediate in nature, was distinguished at the apex of the red muscle 'triangle,' between the epaxial and hypaxial muscle, using NADH diaphorase and myosin ATPase stains. This paper discusses the possibility that functionally different muscle fibres occur in the red swimming muscle of the Atlantic mackerel.     

A histochemical and ultrastructural study of heterogeneous red fibres of pigeon pectoralis muscle

The oxidative fibres of pigeon pectoralis muscle are 'type II red' on the basis of high myofibrillar adenosine triphosphatase (ATPase) and succinate dehydrogenase (SDH). The ATPase and SDH reactions do not characterize the heterogeneity of the red fibres in the normal pigeon pectoralis. Therefore, lipid, glycogen, phosphorylase, glycogen synthetase, SDH and ATPase reactions were studied in transverse sections of the pigeon pectoralis red fibre. This study has revealed the existence of an inherent heterogeneity between these red fibres. Three sub-populations distinguished were : (1) 'red1' fibres showing low fat but high glycogen and enzymes of glycogen metabolism (EGM); (2) 'red2' fibres displaying higher fat and higher amount of glycogen and EGM; and (3) 'red3' fibres possessing higher fat but lower content of glycogen and EGM. Ultrastructurally, one category of fibres (presumably red2 and red3) showed a higher concentration of fat; these fibres presumably possess higher synthetic capacity and lower (or higher?) utilization. Other mitochondria-loaded red fibres (presumably red1) documented here for the first time, showed fewer and smaller fat droplets indicating that they are 'predominantly lipid utilizers' and are incapable of storing large quantities of lipid. Moreover, the differing histochemical-ultrastructural attributes are presumably correlated with differences in levels of energetic metabolism, heat production and motor units of the red fibres.     

Histochemical and immunohistochemical profile of pink muscle fibres in some teleosts

Summary The pink muscle of several Teleosts was examined immunohistochemically using antisera specific for the myosins of red and white muscle, and histochemically using various methods for demonstrating myosin ATPase (in ATPase) activity.In the catfish the pink muscle consists of 2 different layers of fibres. The superficial layer has a low mATPase activity after both acid and alkali pre-incubation, whereas the deeper layer has a high mATPase activity after acid and alkali pre-incubation, being more resistent to these conditions even than is the white muscle.In the trout the pink muscle is composed of fibres with the same mATPase activity as in the superficial pink muscle of the catfish, whereas in the rock goby, goldfish, mullet and guppy the pink muscle is like the deep pink layer of the catfish.Immunohistochemically the fibres of the pink muscle behave like the white muscle fibres except in the guppy and rock goby in which at the level of the lateral line there occurs a transition zone between red and pink fibres. The fibres of this region react with both anti-fast and (to a lesser extent) anti-slow myosin antisera, and have a mATPase activity which, going from the superficial to the deeper fibres, gradually loses the red muscle characteristics to acquire those of the main pink muscle layer.     

Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH

Summary The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles.The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres.In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.     

Changes in ATPase and SDH reactions of the rat extrafusal and intrafusal muscle fibres after preincubations at different pH.

The dependence of adenosine-triphosphatase (ATPase) and succinic dehydrogenase (SDH) histochemical reactions on the pH of the preincubation medium was studied in serial cross sections of 1- to 6-month-old rat extensor digitorum longus (EDL) and soleus (SOL) muscles. The use of a wide spectrum of pH values confirmed the previous results showing that: (1) according to their ATPase and SDH reactions 3 types of extrafusal muscle fibres, i.e., fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG) and slow-twitch oxidative (SO) and 3 types of intrafusal muscle fibres, i.e. typical and intermediate nuclear bag fibres and nuclear chain fibres were observed; (2) only acid preincubation (pH 4.35) is necessary to demonstrate the reversal of the ATPase reaction; while (3) alkali preincubation (pH 10.4) does not provide any new important information as compared with ATPase without preincubation. Furthermore, it was shown that: (4) fast-twitch muscle fibres exhibited high ATPase activity on preincubations at pH 4.9 to 10.4, slow-twitch fibres had very high ATPase activity on preincubation at pH 4.3 and 4.5; (5) after preincubation at pH 4.5 two types of FOG fibres were observed, differing in their ATPase activity; (6) in both muscles there were fibres with intermediate ATPase activity both after acid and/or alkali preincubations; (7) the intrafusal muscle fibres exhibited some specific characteristics when compared with extrafusal fibres. In contrast to the ATPase reactions, SDH activity was decreased equally, in both extra- and intrafusal fibres, with increasing acidity and alkality of the preincubation medium.     

Posthatch development of the axial musculature of the common dentex Dentex dentex, L (Teleostei)

The common dentex is a promising candidate for Mediterranean aquaculture. The present work is aimed at describing the development of the axial musculature from hatching to postlarval life. Transmission electron microscopy, histochemical (NADH-TR and mATPase) and immunohistochemical techniques (S-58 and TUNEL) have been used. At hatching superficial red and deep white muscles can be distinguished. Presumptive dermomyotome (external) cells are initially located over the superficial red muscle but shortly (2 days) tend to concentrate towards the epaxial and hipaxial limits of the myotome. Then, these cells enter the myotome and spread around and within the white muscle thus being apparently responsible for the stratified hyperplasia of the myotome. Mosaic hyperplasia is activated during the second half of the larval period and initially relies on differentiation of a population of atypical premyoblastic cells (APC). APC are mononuclear cells with euchromatic nuclei, cytoplasms full of thin longitudinally projected tubules, occasional mitochondria and scattered ribosomes. By the end of the larval period these cells tend to disappear, partly due to apoptosis, but postlarval mosaic hyperplasia continues by differentiation of presumptive myosatellite cells. APC are an unexpected and singular finding of this study which deserves more research, so as to further characterize their ancestry, developmental programme and fate. In addition to the white and superficial red muscle fibres, intermediate (pink) and tonic fibres appear during larval metamorphosis. Later, during the early postlarval life, a new type of slow twitch red muscle fibre is differentiated (red adult type).     

On the heterogeneity of capillaries of pigeon pectoralis muscle: a histoenzymatic and ultrastructural study

Synopsis Earlier studies had failed to show the presence of capillaries between the white fibres of pigeon pectoralis muscle. In this paper, data are reported for the first time documenting that these capillaries occur in both intra- and inter-fasicular areas of the muscle. Fresh frozen sections of pigeon pectoralis major muscle were incubated for alkaline ATPase reaction following pretreatment with different EDTA solutions (4.3 mM, pH 4.3). The results showed the existence of an inherent heterogeneity of capillaries. The capillaries of white fibres stained intensely for K^–/Mg^2–-EDTA or Mg²⁺-EDTA pre-incubated ATPase; the capillaries of red fibres stained poorly. Both white fibre and red fibre capillaries were examined ultrastructurally in the non-perfused pigeon pectoralis muscle. It is suggested that a possible correlation exists between the distinctive metabolic and mechanical characteristics of the Type II white, glycolytic, fast-twitch fast-fatigue muscle fibres and the high ATPase activity of their capillaries.     

A study of the peripheral innervation and muscle fibre types of Ictalurus nebulosus (Lesueur) and Ictalurus punctatus (Rafinesque)

The innervation pattern and fibre types of the axial musculature of two closely related catfish species with differing lifestyles, Ictalurus nebulosus (Lesueur) and I. punctatus (Rafinesque) were investigated. Both fish displayed the multiple innervation pattern in the red muscle. However, the white muscle of I. nebulosus demonstrated terminal innervation while I. puncrurus displayed multiply innervated white muscle fibres. Fibre typing utilizing histochemical techniques for glyco-gen, lipid, succinic dehydrogenase and glucose-6-phosphate dehydrogenase revealed the typical teleostean distribution of red, intermediate and white muscle fibres in both fish. Staining was greatest in the red muscle fibres and least in the white muscle fibres. The white muscle fibres of I. punctatus stained slightly more for lipid than the white fibres of I. nebulosus which may be correlated with a greater aerobic capacity related to lifestyle and possibly innervation.     

Histochemical heterogeneity of intrafusal muscle fibres in slow and fast skeletal muscles of the rat

Summary Intrafusal muscle fibres of the slow soleus (Sol) and fast vastus lateralis (VL) muscles of the rat were studied histochemically. Serial transverse sections were incubated for the localization of succinate dehydrogenase (SDH), alpha glycerophosphate dehydrogenase (GPD) and adenosine triphosphatase (ATPase). The latter was examined further after preincubation in acidic solution held at either low or room temperature (R_T). The bag₂ intrafusal fibres in both muscles displayed high regular and acid stable ATPase, but low SDH and GPD activities. Bag₁ intrafusal fibres showed low to moderate regular ATPase, a regional heterogeneity after R_T acid preincubation (low activity in juxtaequatorial and high in polar zones), moderate SDH, but low GPD reactions. In both muscles the chain fibres usually exhibited high ATPase for both regular and cold acid preincubated reactions, but usually low activity after R_T acid preincubation; they had high SDH but variable GPD activities. In Sol muscle, however, approximately 25% of spindles contained chain fibres that showed high acid-stable ATPase reaction after both cold and R_T acid preincubation. In contrast, chain fibres in some VL spindles had a characteristically low ATPase reaction even after cold acid preincubation. This study, therefore, has delineated the existence of an inherent heterogeneity among chain fibres (with respect to their histochemical reactions) in muscle spindles located within slow and fast muscles and also between those found within populations of either Sol or VL muscle spindles.     

Comparison of red and white muscles by cytophotometry of their muscle fibre populations

Synopsis Samples from two red muscles (vastus intermedius and vastus medialis) and two white muscles (biceps femoris and gluteus medius) were taken from four pigs. Serial transverse sections were reacted for ATPase and NADH oxidative activity. Sections were mapped with a projection microscope so that the staining intensity of individual fibres for the two reactions could be measured with a simple microscope photometer. Transmission values at 600 nm were converted to units of 0–10 for the range from darkest to lightest staining fibres on each section to cancel variation in staining intensity between sections. The aim of the study was to use simple cytophotometry instead of subjective judgement in the categorization of different histochemical types of muscle fibres. Cytophotometry enabled clear resolution of the major fibre types (types I and II using the ATPase reaction), partial resolution of more variable characteristics (NADH oxidative activity in type I and II fibres) and no resolution of subtle subtypes (IA and IB with the NADH oxidative reaction). However, between the major fibre types, cytophotometry revealed variable numbers of fibres with transitional characteristics. There were more of these fibres in red muscles. With sections reacted for ATPase, transmission values for low magnification fields containing 100 to 200 fibres were correlated (r=–0.91) with the ratio of type I:II fibres.     

The immunohistochemical location of cathepsin L in rabbit skeletal muscle

Summary The immunohistochemical location of cathepsin L in rabbit soleus, plantaris and psoas muscles was investigated using the peroxidase-anti-peroxidase (PAP) technique. The amount of enzyme detected varied according to the fibre type, which were identified by histochemical staining of serial sections for succinate dehydrogenase and alkali-stable myosin ATPase. In the three muscles studied labelling was strongest in the highly oxidative fibres and weaker in the other fibre types with least staining in the fast white fibres. Immunoreactive cathepsin L appeared to be most concentrated at the periphery of muscle fibres, especially near to the nuclei, although some staining was seen throughout the fibres.     

A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

Energy metabolism of carp swimming muscles

Summary Electromyography has been used to study the recruitment of red, pink and white muscle fibres of the Mirror carp at different swimming speeds. Locomotion below 0.3–0.5 L/S (lengths per second) is achieved primarily by fin movements after which the red myotomal muscle becomes active. Pink muscle fibres are the next type to be recruited at speeds around 1.1–1.5 L/S. White muscle is only used for fast cruising in excess of 2–2.5 L/S and during bursts of acceleration.Studies of the myofibrillar ATPase activities of these muscles have shown a ratio of 124 for the red, pink and white fibres respectively. The myosin low molecular weight components, which are characteristic of the myosin phenotype, have been investigated by SDS polyacrylamide electrophoresis. The light chain patterns of the pink and white muscles were identical and characteristic of the fast myosin phenotype. Red muscle myosin had a light chain pattern characteristic of slow muscles. It would appear that there is a relationship between the speed of contraction of the fibre types and the locomotory speed at which they are recruited.The activities of some enzymes of energy metabolism have also been determined in the three muscle types. Enzymes associated with oxidate metabolism have high, intermediate and low activities in the red, pink and white muscles respectively. Pyruvate kinase and lactate dehydrogenase activities were considerably higher in the pink than in either red or white muscles. It is suggested that the high capacity for anaerobic glycolysis of the pink muscle is associated with its recruitment for sustained effort at swimming speeds above which the fish can no longer meet all its energy requirements by gas exchange at the gills.Abbreviations used EDTA ethylenediamine tetraacetic acid - L/S lengths, sec^–1 - LDH Lactate dehydrogenase - PFK phosphofructokinase - SDS sodium dodecyl sulphate - TCA trichloroacetic acid