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.     

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.     

Lateral musculature in the whitemouth croaker (Micropogonias furnieri): its characterization with respect to different gonadal conditions

Histochemical and ultrastructural studies were performed on the lateral musculature from individual female whitemouth croaker, Micropogonias furnieri, at the anterior, medium and posterior regions. Based upon histochemical myosin-ATPase (m-ATPase) determination, diverse types of red, pink and white fibres were discerned. Red muscle had abundant mitochondria and stained intensely for aerobic enzymes, white muscle scarcely stained for the same enzymes and pink muscle responded in an intermediate manner. Both white and pink muscle had few mitochondria. The relative proportion of red muscle increased towards the caudal region; pink muscle diminished towards this region and white muscle modified its proportion only in the anterior region. m-ATPase activity showed differences in relation to the gonadal condition along the body, particularly in the white fibres at the anterior and medium regions.     

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.     

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.     

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.     

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.     

Muscle cellularity at cranial and caudal levels of the trunk musculature of commercial size sea bass, Dicentrarchus labrax (Linnaeus, 1758)

In eight specimens of Atlantic sea bass of commercial size (congruent with 350 g) muscle cellularity was studied at two selected sampling levels of the trunk axial musculature: caudal (anal opening) and cranial (fourth radius of the dorsal fin). The following parameters were quantified at both sampling levels: white muscle cross-sectional area, white muscle fibre diameter (900-1200 fibres), muscle fibre number and muscle fibre density. Results showed a higher total cross-sectional area at cranial than at caudal level (P < 0.05), what is related with their different gross morphology. However, the white muscle fibre size distribution, as well as the muscle fibre number and density did not show significant differences between them. This study contributes to typify muscle fibre sampling in sea bass of commercial size what is of great interest for morphometric studies where white muscle cellularity is commonly correlated with textural or organoleptic parameters.     

Gill morphology in two Mediterranean Sea fishes of similar feeding preferences: sea bream (Sparus aurata L) and sea bass (Dicentrarchus labrax)

The aim of the present study was to describe the gill morphology of the gilthead sea bream and sea bass fishes that showed the same feeding habits. Our study focused on the gross morphological, scanning and light microscopical features of the gills, where a many differences in the gill arches and gill rakers surface in both species were explained. Our results marked that; there was an interbranchial median elevation appeared in sea bream. The gill arches length and the gaps between decreased medial wards in both species. The gill rakers had similar arrangement and varied number in both species. The first lateral rakers row was the longest one in sea bass. However the gill filaments appeared in double rows. Gill filaments were long at middle and short at extremities of gill arch in both species. Scanning electron microscopy revealed the surfaces of gill arch covered by a mosaic of pavement cells, varied sized pores of chloride cells and many pointed spines on the rakers ventral border in both species. Two types of taste buds marked on the gill arches and rakers in sea bass. Many lamellae appeared on filaments in both species.     

Virulence and enterobacterial repetitive intergenic consensus PCR of Vibrio alginolyticus strains isolated from Tunisian cultured gilthead sea bream and sea bass outbreaks

Vibrio alginolyticus was isolated from the internal organs of diseased gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) cultured in two fish farms located on the Tunisian Mediterranean coast, from 2003 to 2005. After phenotypic characterisation, a selection of 34 isolates from gilthead sea bream and sea bass were molecularly typed by repetitive intergenic consensus PCR (ERIC-PCR) showing a high polymorphism among the isolated strains (19 genotypes). Most of the isolates were resistant to atleast two antimicrobial agents. All the tested strains were resistant to ampicillin. However, 91.17% were resistant to nitrofurantoin and 35.29% to tetracycline. Several strains isolated from diseased gilthead sea bream and sea bass were tested for virulence in both fish species by intraperitoneal injection. The selected isolates (n=7) were pathogenic for gilthead sea bream and sea bass. LD(50) values ranged from 5.01 x 10(4) to 6.20 x 10(7)CFU/fish. This is the first report on characterisation and virulence of V. alginolyticus for sea bass and sea bream in Tunisia.     

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.     

Insulin,IGF-I,and muscle MAPK pathway responses after sustained exercise and their contribution to growth and lipid metabolism regulation in gilthead sea bream

Herein, we studied whether sustained exercise positively affects growth of gilthead sea bream by alterations in a) plasma concentrations of insulin and IGF-I, b) signaling pathways in muscle, or c) regulation of lipid metabolism. Specifically, we evaluated the effects of moderated swimming (1.5 body lengths per second; BL/s) on the circulating concentrations of insulin and IGF-I, morphometric parameters, and expression of genes related to lipid metabolism in gilthead sea bream (80–90 g BW). Exercise increased the specific growth rate (P < 0.05) and reduced the hepatosomatic index (P = 0.006). Plasma IGF-I concentrations increased in exercised fish (P = 0.037), suggesting a role for this endocrine factor in the control of muscular growth and metabolic homeostasis during swimming. The observed decrease in plasma insulin concentrations (P = 0.016) could favor the mobilization of tissue reserves in exercised fish. In this sense, the increase in liver fatty acid content (P = 0.041) and the changes in expression of peroxisome proliferator-activated receptors PPARα (P = 0.017) and PPARγ (P = 0.033) indicated a hepatic lipid mobilization. Concentration of glycogen in both white and red muscles was decreased (P = 0.021 and P = 0.017, respectively) in exercised (n = 12) relative to control (n = 12) gilthead sea bream, whereas concentrations of glucose (P = 0.016) and lactate (P = 0.0007) were decreased only in red muscle, indicating the use of these substrates. No changes in the glucose transporter and in lipoprotein lipase mRNA expression were found in any of the tissues studied. Exercised sea bream had decreased content of PPARβ mRNA in white and red muscle relative to control sea bream expression (P = 0.001 and P = 0.049, respectively). Mitogen-activated protein kinase phosphorylation was significantly down-regulated in both white and red muscles of exercised sea bream (P = 0.0374 and P = 0.0371, respectively). Tumor necrosis factor-α expression of white muscle was down-regulated in exercised gilthead sea bream (P = 0.045). Collectively, these results contribute to the knowledge base about hormonal regulation of growth and lipid metabolism in exercised gilthead sea bream.     

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.     

Growth Dynamics of White Muscle Fibres in Relation to Somatic Growth of Larvae of European Sea Bass (Dicentrarchus labrax, L.)

The body length and diameter of muscle fibres from the right hypaxial tail myomeres were measured in 100 sea bass larvae (Dicentrarchus labrax, L.) at ages of 40 to 90 days, from a fish farm in Croatia. The larvae were kept at a temperature of 19-20 degrees C and fed with standard feed for their respective ages. Body lengths of larvae 40-90 days old varied from 8 to 42 mm, increasing significantly in 10-day intervals (p < 0.001), except between days 50 and 60 when there was no increase in the body length. The measured diameters of muscle fibres ranged from 10 to 80 microm, the mean value increasing except between days 50 and 60. The fibre diameter in larvae aged up to 60 days was 10-40 microm, while that in larvae older than 70 days was 51-80 microm. In 90-day-old larvae there were no fibres of 10-20 microm in diameter; however, the number of fibers of 61-80 microm diameter increased. A statistically significant concordance was determined between the body length and fibre diameter of larvae 70 to 90 days old. Between days 50 and 60, arrested growth of the larval body and corresponding decrease in fibre diameter was observed. From day 45 until days 59, larvae were fed with Artemia nauplii and microparticles of formulated diets. From the day 60, minced fish was added to formulated diets. This change in diet probably resulted in the observed growth arrest.     

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.     

Effect of two thermal regimes on the muscle growth dynamics of sea bass larvae,Dicentrarchus labrax L

Muscle growth was studied in larvae of sea bass, Dicentrarchus labrax L., reared at two temperatures: real ambient temperature ( congruent with 15 degrees C during vitelline phase and increased gradually) and 19 degrees C from fertilization until the end of larval development. Muscle cellularity, body length and body weight were measured. Early temperature influenced larval development and so, pre-larval phase finished earlier at 19 degrees C than at ambient temperature (4 and 6 days, respectively). Temperature also affected muscle growth such that at hatching and at mouth opening hypertrophy of muscle fibres was greater at 19 degrees C (P < 0.05), whereas hyperplasia was similar in both groups. After 25 days, the cross-sectional area of the white muscle was greater at 19 degrees C (P < 0.05), which was mainly associated with a higher proliferation of new white muscle fibres. At this stage the body length was also higher at 19 degrees C. Metamorphosis finished earlier in fish reared at 19 degrees C (52 days) than at natural temperature (82 days). At this developmental stage body length and cross-sectional area of the myotome were similar in both groups. However, muscle cellularity differed between groups. Thus, hypertrophy of muscle fibres was higher in fish reared at ambient temperature (P < 0.05), whereas proliferation of new muscle fibres was higher at 19 degrees C (P > 0.05).     

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.     

Ozone Disinfection of Eggs from Gilthead Seabream Sparus aurata,Sea Bass Dicentrarchus labrax,Red Porgy,and Common Dentex Dentex dentex

Abstract

The risk of fish pathogen transmission via eggs can be reduced by disinfection in ozonated seawater. The aim of this study was to determine the suitable conditions for ozone disinfection of the eggs of gilthead seabream Sparus aurata, sea bass Dicentrarchus labrax, red porgy Pagrus pagrus, and common dentex Dentex dentex. The eggs were disinfected with a concentration (C) of 0.5 mg of ozone/L of water at four different exposure times (T = 2, 4, 8, and 16 min). The hatching rate was determined in triplicate for each treatment. Bacterial colonies were counted on tryptic soy agar and thiosulfate–citrate–bile salts–sucrose agar. At the end of the experiment, bacterial load and hatching rate were assessed together to determine the optimal ozone treatment values, which were estimated in CT units (i.e., C [= 0.5 mg/L] × T [min]). Optimal values were CT 2–4 (T = 4–8 min; 18°C) for gilthead seabream and red porgy, CT 2 (T = 4 min; 18°C) for common dentex, and CT 4 (T = 8 min; 15°C) for sea bass.

Received April 12, 2011; accepted February 2, 2012     

Estudio Histoquímico y Morfométrico de la Población Fibrilar del Músculo Diafragma. Parte I: Caballo y Perro

Histochemical and morphometric study of fibrilar population in diaphragm muscle. Part I: Horse and Dog By using m-ATPase techniques, under alkaline and acid pre-incubations, as well as metabolic techniques (α-MGPDH and NADH-TR), sections from the lumbar and costal portions of diaphragm muscle in horse and dog were analysed. Fibre samples were exposed to image-analysis observations in order to determine the lowest fibre diameter and obtain estadistical data. The findings revealed that the lumbar portion of horse diaphragm consists of fibre types I, IIA and IIB; in the costal portion, the fibre types I and IIA were present. In the dog, the lumbar and costal portions consisted of fibre types I, IIA and IIC, which showed some specific histochemical characteristics. Some data concerning both morphometrical and statistical fibre-type analysis are presented.     

Oxidative capacity of skeletal muscle fibres in racehorses: histochemical versus biochemical analysis

The oxidative capacity of skeletal muscle fibre types was evaluated histochemically using the nicotinamide dinucleotide diaphorase (NADH-D) staining, and biochemically by measuring the activity of citrate synthase (CS) in both whole muscle samples and in pools of fibres of identified type. Duplicate determinations of the NADH-D staining pattern resulted in standard deviations (sd) between duplicates of 6 and 11 per cent for two observers. The NADH-D pattern was found to differ between observers. Duplicate determinations of CS activity in the same fibre pools resulted in an sd value of 2.9 mumol/g/min. Measurements of whole muscle CS activity did not provide information about the distribution of oxidative capacity among fibre types. The NADH-D stain and CS activity in fibre pools both showed that, in general, type I and IIA fibres had a higher oxidative capacity than type IIB fibres. Biochemical techniques also showed, however, that the CS activity in type I and IIA fibres of different horses could vary as much as twofold, whereas the NADH-D rating showed a high intensity staining for most type I and IIA fibres in all horses. Furthermore, type IIB fibres received a lower NADH-D rating than the other fibre types even when the CS activities were quite similar. For purposes of research, biochemical measurement of oxidative capacity in individual muscle fibre types provides valuable quantitative and comparative information. The ease of histochemical NADH-D staining in comparison to fibre dissections makes this technique more practical for routine estimates of the distribution of oxidative capacity among muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)