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Effects of incubation temperature on muscle morphology and growth in the pacu (Piaractus mesopotamicus)
Aquaculture 237 (2004) 251 – 267 www.elsevier.com/locate/aqua-online
Effects of incubation temperature on muscle morphology and growth in the pacu (Piaractus mesopotamicus) Jeane M.F. de Assis a, Robson F. Carvalho a, Luciano Barbosa b, Cla´udio A. Agostinho c, Maeli Dal Pai-Silva d,* a
Programa de Po´s Graduacßa˜o em Biologia Celular e Estrutural, UNICAMP, Campinas, SP, Brazil b Departamento de Bioestatı´stica, Instituto de Biocieˆncias, UNESP, Botucatu, Sa˜o Paulo, Brazil c Departamento de Producßa˜o e Exploracßa˜o Animal, Faculdade de Medicina Veterina´ria e Zootecnia, UNESP, Botucatu, Sa˜o Paulo, Brazil d Departamento de Morfologia, Instituto de Biocieˆncias, UNESP, Botucatu, Sa˜o Paulo 18618-000, Brazil Received 27 December 2003; received in revised form 13 April 2004; accepted 19 April 2004
Abstract This study describes the influence of incubation temperature during initial development phase on the morphology and muscle growth characteristics in the pacu (Piaractus mesopotamicus). Pacu eggs were incubated at 25, 27, and 29 jC until hatching. After day 5, fish from each temperature were transferred to 500 l tanks. At hatching and after 5, 25, and 60 days, muscle samples were collected, some were frozen in liquid nitrogen and others fixed in 4% paraformaldehyde or 2.5% glutaraldehyde. These samples were used for morphological, histochemical, immunohistochemical, and morphometric analysis. At hatching, we observed a superficial monolayer of small diameter fibers, lying just beneath the skin surrounding several round cells. From day 5, we observed two distinct populations of muscle fibers distributed in two layers: (1) red—in a superficial region with aerobic activity, and following acid preincubation, high mATPase activity, and 2) white—with anaerobic activity, and following alkaline preincubation, high mATPase activity. Twenty-five days after hatching, an intermediate layer and cell proliferating zones could be seen in the dorsal fin muscle region, with intermediate characteristics. Throughout the experimental period, there was an increase in muscle mass due to new fiber recruitment in the cell proliferating zones and between the more differentiated fibers in red, intermediate, and white muscles. This was more obvious from day 25, and at 29 jC than at 25 and 27 jC. Fiber hypertrophy occurred from hatching to 60 days and was more evident from 5 to 25 days. The number of proliferating nuclei (PCNA-labelling) increased from hatching to 60 days, and was more obvious in the 29 jC group at 60 days. Our results show that at
* Corresponding author: Tel.: +55-14-3811-6264; fax: +55-14-3811-6264. E-mail address: [email protected] (M. Dal Pai-Silva). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.04.022
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incubation temperatures of 25, 27 and 29 jC, hypertrophy was predominantly from hatching to 25 days, after that muscle growth by hyperplastic mechanism increased. The interaction of muscle hypertrophic and hyperplastic growth processes in the 29 jC group produced the largest fish at the end of the experiment. D 2004 Elsevier B.V. All rights reserved. Keywords: Morphology; Muscle growth; Temperature; Piaractus mesopotamicus
1. Introduction Most muscle mass in fish myotomes is composed of white musculature, formed by glycolytic metabolism fibers; this uses anaerobic glycolysis for energy supply. These fibers are fast contracting and are used in fast swimming (Johnston, 1999). The red musculature forms a thin layer in the subdermal region which is more developed in the lateral line nerve region, but makes up less than 30% of total musculature (Greer-Walker and Pull, 1975; Hoyle et al., 1986; Luther et al., 1995). Red fibers show aerobic metabolism and slow contraction; they are associated with slow cruise swimming, such as migration and foraging (Johnston, 1999). There is an intermediate layer between the red and white musculature with intermediate characteristics (Sanger and Stoiber, 2001). Muscle is a post-mitotic tissue (Johnston, 1999) and during fish growth, the myotomal muscle fibers show distinct morphological and functional characteristics. In the first developmental phases of most teleosts, the myotomes are composed of presumptive white immature fibers surrounded by a monolayer of small embryonic red fibers; these can be more differentiated in some species (Talessara and Urfi, 1987; Valente et al., 1999). The intermediate musculature develops later (Nag and Nursall, 1972). Post-embryonic growth is associated with both hypertrophy (as shown by the increase in muscle fiber diameters) and by hyperplasia (recruitment of new muscle fibers) from undifferentiated myoblasts or myosatellite cells. At a particular moment, these cells proliferate and differentiate to form new fibers or are incorporated to pre-existing fibers and this process is regulated by several myogenic regulatory factors (Molkentin and Olson, 1996; Johnston, 1999). This causes a mosaic of different diameter fibers in the muscle, which is either permanent or seasonal (Carpene` and Veggetti, 1981; Rowlerson et al., 1985). Some studies have shown that temperature can influence fish food ingestion and growth rate. The temperature at which the eggs are incubated may influence muscle cell differentiation, the number and size of muscle fibers at hatching, muscle growth, and adult size (Moksness et al., 1995; Vieira and Johnston, 1992; Johnston et al., 1995; Valente et al., 1999). Following this line of investigation, Stickland et al. (1988) observed that incubating salmon eggs at around 10 jC caused more intense hypertrophic growth in muscle fibers immediately after hatching compared to temperatures below 5 jC. However, Alami-Durante et al. (1997) observed that incubating carp eggs at 15, 19, and 23 jC did not have any effect on the total number of muscle fibers after hatching. Vieira and Johnston (1992) analyzed the embryonic development of Clupea harengus at 5, 10, and 15 jC (this normally occurs around 5 to 10 jC); at 15 jC they observed a higher number of small diameter white muscle
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fibers characterizing hyperplasia, this correlates to higher myoblast recruitment for new muscle fiber formation. According to Vieira and Johnston (1992), Veggetti et al. (1993), and Nathanailides et al. (1996), as skeletal striated muscle shows plasticity, differences in egg incubation temperature can affect muscle cellularity, composition of myofibrillar proteins, and the morphophysiological characteristics of muscle fibers. Johnston (1993b) showed that seasonal temperature change may lead to alterations in muscle phenotype, such as changes in contractile properties and metabolic characteristics. The neotropical characid, pacu (Piaractus mesopotamicus) have been extensively used in Brazilian aquaculture programs (Hernandez, 1989). It is one of the most important food species exploited in the Pantanal wetlands area of the Parana´-Paraguai basin. It is an autochthon species with great economic importance in South American commercial fishing (Goulding, 1981). Reproduction is between October and March when temperatures are hot and rain is frequent (Romagosa et al., 1990). This study analyzed the morphological, histochemical, and growth characteristics of skeletal muscle fibers in the pacu (P. mesopotamicus) during the initial developmental phases at different incubation temperatures.
2. Materials and methods 2.1. Fish Fish were obtained after fertilization of eggs from two females with the sperm of three males, at the Aquaculture Sector, FMVZ, UNESP, Botucatu, SP. The fertilized eggs (573 g) were equality divided and transferred to incubators at 25, 27 and 29 jC, with three repetitions each. Temperatures were maintained with 0.5 jC variation throughout the experiment using thermostats. During the experiment, pH (6.65 F 0.2), oxygen (6.7 mg Hg F 0.9), and water temperature were periodically monitored. After hatching, the fish were kept in this environment for 5 days until mouth opening and swim bladder filling. Fish from each incubator were transferred to 500 l tanks (150 fish/m3). All tanks were equally filled with running water with a daily temperature variation between 25 and 28 jC. After mouth opening, the fish were fed newborn brine shrimp (Artemia sp. INVE) up to the 10th day of life, and then ground fish pellets with 40% raw protein (RP). 2.2. Histochemistry and immunohistochemistry Specimens were collected at the following live stages: hatching, and 5, 25, and 60 days after hatching. After length measurement, the fish were sacrificed by MS-222 (3-aminobenzoic acid ethyl ester—Sigma) anesthesia. Five fish per incubation temperature were fixed in 4% paraformadehylde as per Bancroft and Steven (1990), processed, and embedded in Historesin (Kit Historesin Leica) and Paraplast. Histological sections (2 to 4 Am thick) from the dorsal fin region were stained with Haematoxylin – Eosin. Histological sections of the Paraplast-embedded material were submitted to immunohistochemical PCNA reaction to evaluate striated muscle cell proliferation levels for each incubation temperature. The histological slides were incubated with primary antibody (NCL-PCNA—NOVOCAS-
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TRA), secondary antibody (anti-mouse IgG—VECTOR), and then ABC solution (avidin – biotin-peroxidase—VECTOR). After developing with Diaminobenzidine (DAB—SIGMA), the slides were mounted in Permount (Hsu et al., 1981; Foley et al., 1993; Veggetti et al., 1999). Other specimens were frozen in n-hexane, previously cooled in liquid nitrogen ( 196 jC). Small fish were combined with mouse liver or muscle before freezing to facilitate microtomy. Frozen transverse sections (7 to 10 Am) were stained with Haematoxylin –Eosin. Further sections were submitted to NADH-TR reaction for metabolic pattern evaluation (glycolytic or oxidative) and myofibrillar ATPase (mATPase) after acid (pH 4.4– 4.8) and alkaline preincubation (pH 10.4– 10.6) for myosin ATPase characteristics (Dubowitz and Brooke, 1973). NADH is an oxidative enzyme, present in both the sarcoplasmic reticulum and the mitochondria, which takes up hydrogen reversibly to a tetrazolium salt and forms a dark compost, the formazan at the site of enzyme activity. This then reacts intensely for oxidative fibers and mildly for glycolytic fibers, but also identifies fibers of mixed metabolic characteristics, usually referred to as oxidative and glycolytic. Myofibrillar ATPase identifies myosin ATPase enzyme characteristics by the breakdown of ATP and subsequent formation of calcium phosphate. After treatment with cobalt chloride and ammonium sulphide, cobaltous sulphide is produced, which is dark. This reaction is considered alkali-stable for fast fibers, and acid-stable for slow fibers (Loughlin, 1993). 2.3. Electron microscopy Small muscle fragments from three specimens at each incubation temperature were taken at hatching and 5, 25, and 60 days and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), washed in phosphate buffer and post-fixed in 1% osmium tetroxide, dehydrated and embedded in Epon resin. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined and photographed using a Philips EM 301 transmission electron microscope. 2.4. Morphometry Five fish per incubation temperature were taken at 5, 25, and 60 days and embedded in Historesin to determine the number of muscle fibers in one complete epaxial quadrant of the lateral musculature (Fig. 2B). In this quadrant, the number of proliferation nuclei were counted using PCNA immunohistochemical reaction. The smallest diameter per fiber of approximately 500 red and white muscle fibers from each incubation temperature was measured. Smallest fiber diameter was used to avoid any errors caused by cross sections not being completely true (Brooke and Engel, 1969; Dubowitz, 1985). Red and white muscle fibers were grouped into five diameter classes. Morphometric analyses were performed using Qwin (Leica, Germany). 2.5. Statistical analysis Data are expressed as mean F S.D. Fish length, proliferation nuclei data, and muscle fiber distribution frequency were analyzed using one way analysis of variance (ANOVA,
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p < 0.05). Square root values of fiber number data were analyzed using two way analysis of variance (ANOVA, p < 0.05) with incubation temperature and age as factors (SAS, 8.02). The Tukey multiple comparison test was used to locate differences when appropriate.
3. Results Hatching times for pacu were different for each incubation temperature with 50% of eggs hatching after 20 h 20 min at 25 jC, 16 h 30 min at 27 jC, and 14 h at 29 jC postfertilization. There was no significant difference for mortality index between treatments. On day 5, there were no significant differences in fish length at all studied temperatures. On day 25, all groups showed significant increases in length but the 25 jC group was significantly lower than the others. On day 60, length increase in the 29 jC group was significantly higher than the others (Fig. 1). 3.1. Morphological and histochemical analysis In all temperatures, HE stain showed lateral musculature composed of two distinct cell populations at hatching. A thin layer of columnar fibers was seen in the most peripheral region below the skin, involving several layers of polygonal shaped fibers in the inner region (Fig. 2A). On day 5, there was a thickening in muscle mass at all temperatures. Two distinct layers were identified: a superficial, formed by small columnar fibers in cross section; and a deep, forming most of the muscle mass, composed of round fibers separated by a thin layer of loose connective tissue. Most fibers had central nuclei, however, for some, the nuclei were in the fiber periphery (Fig. 2B). The NADH-TR reaction showed small fibers in the superficial region with intense to moderate reaction. In the deep musculature, fiber reaction was weak to negative (Fig. 2C). After acid and alkaline preincubation, mATPase reaction was intense in the small superficial fibers after acid
Fig. 1. Total length of pacu (P. mesopotamicus) at 25, 27 and 29 jC throughout the experiment. #Temperature where fish total length was significant. Values represent means F S.D., five fish per temperature.
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Fig. 2. Transverse section of lateral musculature in pacu (P. mesopotamicus). (A) At hatching, at 29 jC. Superficial columnar muscle fibers (s). Deep round muscle fibers (d). Nerve tissue (nt). HE. Scale bar: 20 Am. (B, C and D) Five days after hatching at 27, 25 and 29 jC, respectively. Scale bars: 30, 40 and 30 Am, respectively. (B) Continuos lines showing the entire epaxial quadrant. HE. (C) Superficial layer fibers with intense NADH-TR reaction. Deep layer fibers with weak enzymatic reaction. NADH-TR. (D) Superficial layer fibers dark for mATPase reaction. Deep layer fibers with weak enzymatic reaction. Myosin ATPase, pH 4.4. Fibers with central nuclei (arrow). Nerve tissue (nt). Notochord (n). Superficial muscle fibers (s); deep muscle fibers (d); horizontal septum region with triangular arrangement of fibers (arrowhead).
preincubation (Fig. 2D); most fibers reacted intensely after alkaline preincubation. In the horizontal septum region that divides the musculature into epaxial and hypoaxial, the columnar fiber layer penetrated the musculature towards the medial plane, assuming a triangular shape (Fig. 2C and D). On days 25 and 60, increased epaxial muscle mass was seen; this was more marked in the 29 jC group (Fig. 3A). In the deep layer, there were many small fibers in between the larger fibers (Fig. 3B and C). An intermediate layer was seen between the superficial and deep layers (Fig. 3B); on day 60, the 29 jC group showed more large areas of small fiber in both the deep layer and the dorsal fin muscle region (Fig. 3C). Also in these periods, the superficial and deep layer fibers showed standard NADH-TR and mATPase similar to those at day 5. In the intermediate layer fibers, NADH-TR reaction was weak, and mATPase ranged from moderate to strong regardless of preincubation (Fig. 3D1 and E). On day 60, the small fibers in between the larger deep layer fibers, reacted moderately to mATPase after both acid and alkaline preincubation (Fig. 3E). In the dorsal fin muscle, the fibers closest to the median plane reacted intensely and the more peripheral fibers reacted weakly to NADH-TR and m-ATPase in both acid and alkaline (Fig. 3D2).
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Fig. 3. Transverse section of lateral musculature in pacu (P. mesopotamicus). (A) Twenty-five days after hatching, at 27 jC. Dorsal fin muscle fibers (*). HE. Scale bar: 40 Am. (B) Sixty days after hatching, at 27 jC. Superficial muscle fiber (s). Intermediate muscle fibers (i). Small fibers (*) between larger fibers in the deep layer. HE. Scale bar: 40 Am. (C) Sixty days after hatching, at 29 jC. Cluster of small fibers (*) between some larger fibers in the deep layer. HE. Scale bar: 30 Am. (D) Twenty-five days after hatching, at 29 jC. Scale bar: 30 Am. (D1) Small fibers between the superficial and deep compartments, moderate intensity NADH-TR reaction (arrowhead). NADH-TR. (D2) Dorsal fin muscle fibers with intense NADH-TR reaction (*). Deep layer fibers with weak NADH-TR reaction (d). NADH-TR. (E) Sixty days after hatching, at 29 jC. Superficial (s) and intermediate (i) muscle fibers with negative and moderate mATPase reaction, respectively. Small fibers (arrowhead) in deep layer with weak to moderate mATPase reaction, between larger fibers with marked reaction. Myosin ATPase, pH 10.4. Scale bar: 30 Am. (F) Nuclei with positive PCNA reaction (arrows). Counterstained with Haematoxylin. Scale bar: 20 Am.
3.2. Muscle ultrastructure Pacu striated musculature was similar at all the incubation temperatures. At hatching, undifferentiated columnar muscle fibers were seen in the superficial region; they were
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more rounded in the deep region. There were few myofibrils and mitochondria, and many ribosomes. Sarcoplasmic reticulum was seen between the myofibrils. The nucleus showed loose chromatin and evident nucleolus and was located in the fiber innermost region.
Fig. 4. Ultrastructure of lateral musculature in pacu (P. mesopotamicus) at all experimental temperatures. (A) At hatching. Columnar muscle fibers (*) in the superficial region. Myofibrils (mi). Mitochondria (M). Ribosomes (arrowhead). Sarcoplasmatic reticulum (arrow). Undifferentiated cell with loose nucleus (N) and ribosomes. Scale bar: 1 Am. (B) At hatching. Muscle fibers from the deepest region. Myofibrils in the fiber peripheral region (mi). Mitochondria (M). Ribosomes (arrowhead). Loose nucleus with evident nucleolus (N). Scale bar: 1 Am. (C) Five days. Superficial muscle fibers with myofibrils (mi), mitochondria (M). Undifferentiated cell with loose nucleus (N). Dermis (D). Scale bar: 1 Am. (D) Five days. Deep layer muscle fibers. Sarcoplasm with radial myofibrils in the periphery (*) and polygonal in the centre (arrow). Mitochondria (M). Sarcoplasmatic reticulum (arrowhead). Scale bar 2 Am. (E) Twenty-five days. Superficial layer fibers with myofibrils (mi) and mitochondria (M) in the subsarcolemmal and central regions. Intermediate layer small fiber (f). Undifferentiated cell with relatively dense nucleus (uc). Dermis (D). Scale bar: 2 Am. (F) Sixty days. Deep compartment fibers with radially oriented myofibrils (*). Sarcoplasmatic reticulum (arrow). Fiber nucleus in peripheral position, with evident nucleolus (N). Small fiber (arrow) between larger fibers. Undifferentiated cell with relatively dense nucleus (uc). Scale bar: 1 Am.
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Table 1 Total number of red (R) and white (W) fibers in one epaxial quadrant of the lateral muscle in pacu (P. mesopotamicus) at 25, 27 and 29 jC, throughout the experiment Total number of fibers Day 5 25 jC 27 jC 29 jC
W R W R W R
Day 25 1AB
64.2 F 7.2 46.4 F 3.51a 58.0 F 7.11A 48.8 F 7.71a 79.4 F 16.31B 47.2 F 4.11a
Day 60 1A
80.5 F 8.7 44.5 F 1.71a 226.0 F 33.81B 78.2 F 6.12b 270.2 F 127.31B 92.2 F 40.21b
834.2 F 383.32A 192.4 F 44.42ab 1111.6 F 237.82AB 183.6 F 42.53a 1961.8 F 906.82B 246.8 F 28.22b
Values represent means F S.D., five fish per each temperature. Significant differences when P < 0.05. Values with the same number in the rows are not statistically significant. Values with the same letters in the columns (capitals for white fibers; small letters for red fibers) are not statistically significant.
Undifferentiated cells with cytoplasm showing many ribosomes, mitochondria, and with loose nucleus were seen between the muscle fibers (Fig. 4A and B). On day 5 post-hatching, muscle fiber showed more myofibrils in the sarcoplasm. The superficial layer fibers were small with a few myofibrils in the sarcoplasm. Most fibers showed areas of sarcoplasm without myofibrils, with many mitochondria, ribosomes, and sarcoplasmic reticulum. In deep musculature, the fibers were larger than the superficial layer. Several groups of myofibrils were seen filling the sarcoplasm. The myofibrils were arranged radially in the periphery and polygonally in the centre. Some mitochondria and sarcoplasmic reticulum cisternae were seen between the myofibrils (Fig. 4C and D). On day 25 post-hatching, large and small fibers were seen, especially in the deep musculature. In the superficial layer fibers, there were well-organized myofibrils and mitochondria, especially in the subsarcolemmal and central regions. In the deep musculature, small fibers were seen with areas of sarcoplasma without myofibrils adjacent to Table 2 Diameter of red (R) and white (W) fibers in one epaxial quadrant of the lateral muscle in pacu (P. mesopotamicus) at 25, 27 and 29 jC, throughout the experiment Diameter of fibers Day 5 25 jC 27 jC 29 jC
W R W R W R
Day 25 1A
9.2 F 3.4 2.2 F 0.51a 9.1 F 3.41A 2.4 F 0.61b 8.9 F 3.61A 2.4 F 0.51b
Day 60 2A
11.5 F 4.3 2.34 F 0.51a 16.77 F 7.52B 3.5 F 0.92b 16.8 F 8.02B 3.8 F 1.22c
15.6 F 13.03A 6.8 F 3.52a 14.77 F 11.03A 6.6 F 2.93b 17.2 F 16.42B 8.7 F 4.12c
Values represent means F S.D., five fish per each temperature. Significant differences when P < 0.05. Values with the same number in the rows are not statistically significant. Values with the same letters in the columns (capitals for white fibers; small letters for red fibers) are not statistically significant.
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large fibers of more differentiated shape, with loose nuclei in the peripheral region, few mitochondria, and well-developed sarcoplasm. Between muscle fibers in the connective tissue, there were cells with little cytoplasm and loose or relatively dense nuclei (Fig. 4E). On day 60 post-hatching, the organization of the muscle fibers was similar to 25-dayold specimens. However, there were frequent small fibers with areas of sarcoplasma without myofilaments, especially in the deep musculature (Fig. 4F). 3.3. Morphometric analysis In the superficial layer, between days 5 and 25 post-hatching, significant increase in number of fibers was only seen in the epaxial quadrant of the 27 jC group; from day 25 onwards, increase was significant at all temperatures. At day 5, there were no significant differences between temperatures for number of fibers. At day 25, increase in fiber
Fig. 5. Muscle fiber diameter distribution in pacu (P. mesopotamicus) at 25, 27, and 29 jC throughout the experiment. Columns represent number of fibers (means F S.D) in each size class, five fish per temperature. (letters in the column show size classes with significant variation, p < 0.05).
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numbers was significantly lower at 25 jC; after 60 days, 29 jC showed a higher number of fibers in the superficial compartment compared to 27 jC; this was not significant compared to 25 jC fish. In the deep layer, there was significant increase in number of fibers in the epaxial quadrant from day 25 for all temperatures. On day 5, number of fibers at 29 jC was significantly higher than 27 jC, but not significant against 25 jC; on day 25, the number of fibers was significantly higher for both 27 and 29 jC groups; on day 60, fish incubated at 29 jC presented a significantly higher number of fibers than at 25 jC, but not significant against 27 jC (Table 1). In relation to mean fiber diameter in the superficial layer, there was increase at 27 jC throughout the study period. From day 25 post-hatching, there was significant increase at 25 jC, but not at 29 jC. At day 5, fiber diameter was significantly higher at 27 and 29 jC; on days 25 and 60, it was significantly higher proportional to increasing temperature. In the deep layer, there was significant increase in mean fiber diameter at 25 jC throughout the study period. At 27 and 29 jC, this increase was significant between days 5 and 25 posthatching; at 27 jC, there was a reduction in average fiber diameter at day 60. On day 5, there was no temperature-related significant difference in fiber diameter; on day 25, this was higher at 27 and 29 jC but at day 60 post-hatching, only in the 29 jC group (Table 2). Throughout the experiment, there was both fiber diameter increase and new muscle fiber recruitment in superficial red and deep white muscle layers. In the superficial layer, the majority of fiber diameters after 5, 25 and 60 days were V 4; V 8; and V 16 Am, respectively. There was temperature-related significant difference in muscle fibers V 8 Am only at 25 days. In the deep layer, most fiber diameters were V 20, V 30, and V 20 Am after 5, 25 and 60 days, respectively. There was temperature-related significant difference in all size classes at 25 days, and in classes V 10 and V 20 Am at 60 days ( p < 0.05) (Fig. 5). At the studied temperatures, proliferating nuclei in the epaxial quadrant were shown by PCNA immunohistochemical reaction (Fig. 3F). The number of nuclei increased during the experiment; on day 5, there was no significant difference between the studied
Fig. 6. Number of proliferating nuclei in one epaxial quadrant of the lateral muscle in pacu (P. mesopotamicus) at 25, 27 and 29 jC throughout the experiment. #Temperature with significant difference. Values represent means F S.D., five fish per temperature.
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temperatures; on day 25 the 25 jC group, and on day 60 the 29 jC group were significantly higher than the others (Fig. 6).
4. Discussion In our study, the time between egg fertilization and hatching was inversely proportional to the incubation temperature. Several researchers have reported that incubation temperature can influence fish development and growth, and egg incubation at higher temperatures can accelerate hatching time (Calvo and Johnston, 1992; Vieira and Johnston, 1996; Johnston et al., 1998; Galloway et al., 1999b). In our experiment, the mortality index was low. This is probably related to the narrow temperature range used which was close to the ideal reproduction temperature for this species (Romagosa et al., 1990). Some studies have shown that incubation temperature can have various effects on muscle tissue growth and development (Vieira and Johnston, 1992; Johnston et al., 1998). This may be related to egg quality, including yolk sac nutrient quantity and growth factor concentration (Johnston et al., 1998). Evidence shows that the chorion can form a barrier to gas exchange between the embryo and the environment, and temperature might influence this exchange and oxygen diffusion through the chorion and perivitellinic fluid (Matschack et al., 1995, 1997). In our study, the intensity of NADH reaction in the red and white fibers was similar in all rearing temperatures from day 5. Red fibers in the superficial layer and dorsal fin muscle region showed aerobic metabolism, and the white fibers that made up the majority of the musculature showed more anaerobic metabolism; these characteristics are similar to other fish (Fernandez et al., 2000). White fibers are used in fast burst swimming, for example, escaping predators and catching prey, and red fibers for low intensity sustained swimming (Bone, 1975; Johnston, 1999). In pacu, the precursor cells of these two muscle fiber populations were present since hatching, as demonstrated by light and electron microscopy. Although some authors have shown the influence of temperature on mitochondria volume density (Vieira and Johnston, 1992; Brooks and Johnston, 1993), our study showed muscle fibers with similar oxidative activity throughout the experiment at all incubation temperatures. At the different temperatures used in our experiment, intermediate muscle fibers could be seen from day 25 post-hatching with characteristics of both red and white fibers. During postembryonic myotomal muscle growth, intermediate fibers commonly appear later than red and white fibers (Matsuoka and Iwai, 1984; Scapolo et al., 1988), and the distribution and frequency of these fibers differ between species and developmental stages (Zhang et al., 1996). Based on histochemical and physiological characteristics, these fibers are recruited at faster cruising speeds (Akster et al., 1995; Johnston and Mclay, 1997; Johnston, 1999). The mATPase reaction showed more variability in the intensities, mainly in the intermediate and white muscle and dorsal fin muscle region. Several histochemical studies have demonstrated that the variation in the m-ATPase staining intensities after both alkali and acid preincubation can be related to the change or transition in the myosin heavy chain isoform composition, or co-expression of the different myosin isoforms (Staron and Pette,
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1986; Scapolo et al., 1988). These can be related to the phenotypic plasticity in muscle contractile functions as the fish grows (Scapolo et al., 1988; Brooks and Johnston, 1993; Veggetti et al., 1993; Mascarello et al., 1995). In our study, these phenotypic variations were more evident 25 and 60 days after hatching at all temperatures. In pacu, from hatching until day 60 at all temperatures, muscle mass thickening was from muscle tissue growth. It has been suggested that post-embryonic growth of fish muscle tissue involves both hypertrophy and hyperplasia (Alami-Durante et al., 1997; Galloway et al., 1999a,b; Rowlerson and Veggetti, 2001). In mammals, muscle tissue growth by fiber hyperplasia is restricted to the pre- and perinatal periods (Schultz, 1974; Stickland, 1983). In our study, muscle fiber hypertrophy was observed from hatching until day 60 at all temperatures. From days 5 to 25, hypertrophy was the main growth process characterized by the significant increase in fiber diameter except for the red fibers at 25 jC. In one epaxial quadrant, fiber number increase was not significant, except for the red fibers at 27 jC. Hypertrophy occurred at all studied temperatures between 25 and 60 days; however at 29 jC, fiber diameter increase was not significant although higher than at 25 and 27 jC. In this period, a significant increase in fiber number for both red and white muscle layers was seen, characterizing new fiber recruitment, as seen by the high number of small diameter fibers ( V 20 Am); this was also seen in the intermediate layer and dorsal fin muscle region. As muscle is a post-mitotic tissue, fiber hyperplasia and hypertrophy involve the activation and proliferation of undifferentiated myoblasts or myosatellite cells (Koumans et al., 1993; Johnston, 1999). In mammals and in the juvenile and adult phases of some fish, these cells are located between the sarcolemma and basal lamina of muscle fibers (Mauro, 1961; Campion, 1984; Veggetti et al., 1990; Koumans and Akster, 1995). In initial developmental stages, myogenic precursor cells are widely scattered throughout the myotomal muscle fibers (Veggetti et al., 1990; Stoiber and Sange¨r, 1996; Johnston et al., 1998, 2000). Ultrastructural analysis of pacu muscle showed nuclei of undifferentiated cells in between the more differentiated fibers in the connective tissue. According to Johnston (1993a) and Johnston et al. (1998), these cells could be undifferentiated or persistent myoblasts involved in muscular growth. These cells can proliferate and their nuclei may enlarge existing fibers by fusion, or they may add new fibers to the musculature (Johnston et al., 1998; Johnston, 2001). In our study, the increase in proliferating nuclei suggests that they participate in hyperplastic and hypertrophic muscle fiber growth. Although on day 25, 25 jC fish were smaller, on day 60 there was no significant difference in proliferating nuclei between 25 and 27 jC groups. This could be related to the higher number of proliferating nuclei in 25 jC on day 25. More intense cell proliferation may have accounted for the higher level of hyperplasia in this period, as shown by the high number of small diameter fibers V 20 Am. On day 60 at 25, 27 and 29 jC, there was an increase in fiber diameter, but there were also many muscle fibers with diameters < 20 Am suggesting highly active new fiber recruitment. The higher frequency of PCNA-positive nuclei at 29 jC suggests they contributed to the higher number of fibers at this temperature. Pacu is a fast growing fish which grows to a large size; the mechanisms of muscle fiber growth by hyperplasia and hypertrophy occur over a long period; hyperplasia is
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predominant in the juvenile phase (Dal Pai et al., 2000); this is different to small species, where fiber recruitment ceases in an early phase (Weatherley and Gill, 1984). In our study, we found that hyperplasia and hypertrophy was more intense between days 25 and 60. Several authors have shown that differences in rearing temperature can modulate the rates of muscle fiber recruitment and hypertrophy in different phases of muscle growth. The mechanism is unknown, but some authors have suggested that variation in temperature can alter the proliferation and differentiation of myogenic stem cells; these events are regulated by myogenic regulatory factors, which play a major role in cell proliferation and terminal muscle fiber differentiation (Olson, 1992; Rudnicki and Jaenisch, 1995). At the end of our experiment, 29 jC incubation temperature fish showed higher fiber diameter, intense hyperplasia, and high cell proliferation rate; this may be linked to their greater length. Although muscular growth by hypertrophy and hyperplasia occurred at all studied temperatures, 29 jC fish could possibly attain a larger size; also an acceleration may occur in the reproductive phase, which requires further studies. This study demonstrates the viability of pacu egg incubation at 29 jC to obtain larger larvae and alevins with higher growth potential.
Acknowledgements This study was supported by grants from FAPESP, Proc. No. 00/06177-0 and FUNDUNESP, Proc. No. 557-01-DCP. The authors wish to thank Jarbas do Amaral, Sueli Cruz Michelin, and Cristina Audi for their skillful assistance.
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Effects of incubation temperature on muscle morphology and growth in the pacu (Piaractus mesopotamicus) Jeane M.F. de Assis a, Robson F. Carvalho a, Luciano Barbosa b, Cla´udio A. Agostinho c, Maeli Dal Pai-Silva d,* a
Programa de Po´s Graduacßa˜o em Biologia Celular e Estrutural, UNICAMP, Campinas, SP, Brazil b Departamento de Bioestatı´stica, Instituto de Biocieˆncias, UNESP, Botucatu, Sa˜o Paulo, Brazil c Departamento de Producßa˜o e Exploracßa˜o Animal, Faculdade de Medicina Veterina´ria e Zootecnia, UNESP, Botucatu, Sa˜o Paulo, Brazil d Departamento de Morfologia, Instituto de Biocieˆncias, UNESP, Botucatu, Sa˜o Paulo 18618-000, Brazil Received 27 December 2003; received in revised form 13 April 2004; accepted 19 April 2004
Abstract This study describes the influence of incubation temperature during initial development phase on the morphology and muscle growth characteristics in the pacu (Piaractus mesopotamicus). Pacu eggs were incubated at 25, 27, and 29 jC until hatching. After day 5, fish from each temperature were transferred to 500 l tanks. At hatching and after 5, 25, and 60 days, muscle samples were collected, some were frozen in liquid nitrogen and others fixed in 4% paraformaldehyde or 2.5% glutaraldehyde. These samples were used for morphological, histochemical, immunohistochemical, and morphometric analysis. At hatching, we observed a superficial monolayer of small diameter fibers, lying just beneath the skin surrounding several round cells. From day 5, we observed two distinct populations of muscle fibers distributed in two layers: (1) red—in a superficial region with aerobic activity, and following acid preincubation, high mATPase activity, and 2) white—with anaerobic activity, and following alkaline preincubation, high mATPase activity. Twenty-five days after hatching, an intermediate layer and cell proliferating zones could be seen in the dorsal fin muscle region, with intermediate characteristics. Throughout the experimental period, there was an increase in muscle mass due to new fiber recruitment in the cell proliferating zones and between the more differentiated fibers in red, intermediate, and white muscles. This was more obvious from day 25, and at 29 jC than at 25 and 27 jC. Fiber hypertrophy occurred from hatching to 60 days and was more evident from 5 to 25 days. The number of proliferating nuclei (PCNA-labelling) increased from hatching to 60 days, and was more obvious in the 29 jC group at 60 days. Our results show that at
* Corresponding author: Tel.: +55-14-3811-6264; fax: +55-14-3811-6264. E-mail address: [email protected] (M. Dal Pai-Silva). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.04.022
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incubation temperatures of 25, 27 and 29 jC, hypertrophy was predominantly from hatching to 25 days, after that muscle growth by hyperplastic mechanism increased. The interaction of muscle hypertrophic and hyperplastic growth processes in the 29 jC group produced the largest fish at the end of the experiment. D 2004 Elsevier B.V. All rights reserved. Keywords: Morphology; Muscle growth; Temperature; Piaractus mesopotamicus
1. Introduction Most muscle mass in fish myotomes is composed of white musculature, formed by glycolytic metabolism fibers; this uses anaerobic glycolysis for energy supply. These fibers are fast contracting and are used in fast swimming (Johnston, 1999). The red musculature forms a thin layer in the subdermal region which is more developed in the lateral line nerve region, but makes up less than 30% of total musculature (Greer-Walker and Pull, 1975; Hoyle et al., 1986; Luther et al., 1995). Red fibers show aerobic metabolism and slow contraction; they are associated with slow cruise swimming, such as migration and foraging (Johnston, 1999). There is an intermediate layer between the red and white musculature with intermediate characteristics (Sanger and Stoiber, 2001). Muscle is a post-mitotic tissue (Johnston, 1999) and during fish growth, the myotomal muscle fibers show distinct morphological and functional characteristics. In the first developmental phases of most teleosts, the myotomes are composed of presumptive white immature fibers surrounded by a monolayer of small embryonic red fibers; these can be more differentiated in some species (Talessara and Urfi, 1987; Valente et al., 1999). The intermediate musculature develops later (Nag and Nursall, 1972). Post-embryonic growth is associated with both hypertrophy (as shown by the increase in muscle fiber diameters) and by hyperplasia (recruitment of new muscle fibers) from undifferentiated myoblasts or myosatellite cells. At a particular moment, these cells proliferate and differentiate to form new fibers or are incorporated to pre-existing fibers and this process is regulated by several myogenic regulatory factors (Molkentin and Olson, 1996; Johnston, 1999). This causes a mosaic of different diameter fibers in the muscle, which is either permanent or seasonal (Carpene` and Veggetti, 1981; Rowlerson et al., 1985). Some studies have shown that temperature can influence fish food ingestion and growth rate. The temperature at which the eggs are incubated may influence muscle cell differentiation, the number and size of muscle fibers at hatching, muscle growth, and adult size (Moksness et al., 1995; Vieira and Johnston, 1992; Johnston et al., 1995; Valente et al., 1999). Following this line of investigation, Stickland et al. (1988) observed that incubating salmon eggs at around 10 jC caused more intense hypertrophic growth in muscle fibers immediately after hatching compared to temperatures below 5 jC. However, Alami-Durante et al. (1997) observed that incubating carp eggs at 15, 19, and 23 jC did not have any effect on the total number of muscle fibers after hatching. Vieira and Johnston (1992) analyzed the embryonic development of Clupea harengus at 5, 10, and 15 jC (this normally occurs around 5 to 10 jC); at 15 jC they observed a higher number of small diameter white muscle
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fibers characterizing hyperplasia, this correlates to higher myoblast recruitment for new muscle fiber formation. According to Vieira and Johnston (1992), Veggetti et al. (1993), and Nathanailides et al. (1996), as skeletal striated muscle shows plasticity, differences in egg incubation temperature can affect muscle cellularity, composition of myofibrillar proteins, and the morphophysiological characteristics of muscle fibers. Johnston (1993b) showed that seasonal temperature change may lead to alterations in muscle phenotype, such as changes in contractile properties and metabolic characteristics. The neotropical characid, pacu (Piaractus mesopotamicus) have been extensively used in Brazilian aquaculture programs (Hernandez, 1989). It is one of the most important food species exploited in the Pantanal wetlands area of the Parana´-Paraguai basin. It is an autochthon species with great economic importance in South American commercial fishing (Goulding, 1981). Reproduction is between October and March when temperatures are hot and rain is frequent (Romagosa et al., 1990). This study analyzed the morphological, histochemical, and growth characteristics of skeletal muscle fibers in the pacu (P. mesopotamicus) during the initial developmental phases at different incubation temperatures.
2. Materials and methods 2.1. Fish Fish were obtained after fertilization of eggs from two females with the sperm of three males, at the Aquaculture Sector, FMVZ, UNESP, Botucatu, SP. The fertilized eggs (573 g) were equality divided and transferred to incubators at 25, 27 and 29 jC, with three repetitions each. Temperatures were maintained with 0.5 jC variation throughout the experiment using thermostats. During the experiment, pH (6.65 F 0.2), oxygen (6.7 mg Hg F 0.9), and water temperature were periodically monitored. After hatching, the fish were kept in this environment for 5 days until mouth opening and swim bladder filling. Fish from each incubator were transferred to 500 l tanks (150 fish/m3). All tanks were equally filled with running water with a daily temperature variation between 25 and 28 jC. After mouth opening, the fish were fed newborn brine shrimp (Artemia sp. INVE) up to the 10th day of life, and then ground fish pellets with 40% raw protein (RP). 2.2. Histochemistry and immunohistochemistry Specimens were collected at the following live stages: hatching, and 5, 25, and 60 days after hatching. After length measurement, the fish were sacrificed by MS-222 (3-aminobenzoic acid ethyl ester—Sigma) anesthesia. Five fish per incubation temperature were fixed in 4% paraformadehylde as per Bancroft and Steven (1990), processed, and embedded in Historesin (Kit Historesin Leica) and Paraplast. Histological sections (2 to 4 Am thick) from the dorsal fin region were stained with Haematoxylin – Eosin. Histological sections of the Paraplast-embedded material were submitted to immunohistochemical PCNA reaction to evaluate striated muscle cell proliferation levels for each incubation temperature. The histological slides were incubated with primary antibody (NCL-PCNA—NOVOCAS-
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TRA), secondary antibody (anti-mouse IgG—VECTOR), and then ABC solution (avidin – biotin-peroxidase—VECTOR). After developing with Diaminobenzidine (DAB—SIGMA), the slides were mounted in Permount (Hsu et al., 1981; Foley et al., 1993; Veggetti et al., 1999). Other specimens were frozen in n-hexane, previously cooled in liquid nitrogen ( 196 jC). Small fish were combined with mouse liver or muscle before freezing to facilitate microtomy. Frozen transverse sections (7 to 10 Am) were stained with Haematoxylin –Eosin. Further sections were submitted to NADH-TR reaction for metabolic pattern evaluation (glycolytic or oxidative) and myofibrillar ATPase (mATPase) after acid (pH 4.4– 4.8) and alkaline preincubation (pH 10.4– 10.6) for myosin ATPase characteristics (Dubowitz and Brooke, 1973). NADH is an oxidative enzyme, present in both the sarcoplasmic reticulum and the mitochondria, which takes up hydrogen reversibly to a tetrazolium salt and forms a dark compost, the formazan at the site of enzyme activity. This then reacts intensely for oxidative fibers and mildly for glycolytic fibers, but also identifies fibers of mixed metabolic characteristics, usually referred to as oxidative and glycolytic. Myofibrillar ATPase identifies myosin ATPase enzyme characteristics by the breakdown of ATP and subsequent formation of calcium phosphate. After treatment with cobalt chloride and ammonium sulphide, cobaltous sulphide is produced, which is dark. This reaction is considered alkali-stable for fast fibers, and acid-stable for slow fibers (Loughlin, 1993). 2.3. Electron microscopy Small muscle fragments from three specimens at each incubation temperature were taken at hatching and 5, 25, and 60 days and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), washed in phosphate buffer and post-fixed in 1% osmium tetroxide, dehydrated and embedded in Epon resin. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined and photographed using a Philips EM 301 transmission electron microscope. 2.4. Morphometry Five fish per incubation temperature were taken at 5, 25, and 60 days and embedded in Historesin to determine the number of muscle fibers in one complete epaxial quadrant of the lateral musculature (Fig. 2B). In this quadrant, the number of proliferation nuclei were counted using PCNA immunohistochemical reaction. The smallest diameter per fiber of approximately 500 red and white muscle fibers from each incubation temperature was measured. Smallest fiber diameter was used to avoid any errors caused by cross sections not being completely true (Brooke and Engel, 1969; Dubowitz, 1985). Red and white muscle fibers were grouped into five diameter classes. Morphometric analyses were performed using Qwin (Leica, Germany). 2.5. Statistical analysis Data are expressed as mean F S.D. Fish length, proliferation nuclei data, and muscle fiber distribution frequency were analyzed using one way analysis of variance (ANOVA,
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p < 0.05). Square root values of fiber number data were analyzed using two way analysis of variance (ANOVA, p < 0.05) with incubation temperature and age as factors (SAS, 8.02). The Tukey multiple comparison test was used to locate differences when appropriate.
3. Results Hatching times for pacu were different for each incubation temperature with 50% of eggs hatching after 20 h 20 min at 25 jC, 16 h 30 min at 27 jC, and 14 h at 29 jC postfertilization. There was no significant difference for mortality index between treatments. On day 5, there were no significant differences in fish length at all studied temperatures. On day 25, all groups showed significant increases in length but the 25 jC group was significantly lower than the others. On day 60, length increase in the 29 jC group was significantly higher than the others (Fig. 1). 3.1. Morphological and histochemical analysis In all temperatures, HE stain showed lateral musculature composed of two distinct cell populations at hatching. A thin layer of columnar fibers was seen in the most peripheral region below the skin, involving several layers of polygonal shaped fibers in the inner region (Fig. 2A). On day 5, there was a thickening in muscle mass at all temperatures. Two distinct layers were identified: a superficial, formed by small columnar fibers in cross section; and a deep, forming most of the muscle mass, composed of round fibers separated by a thin layer of loose connective tissue. Most fibers had central nuclei, however, for some, the nuclei were in the fiber periphery (Fig. 2B). The NADH-TR reaction showed small fibers in the superficial region with intense to moderate reaction. In the deep musculature, fiber reaction was weak to negative (Fig. 2C). After acid and alkaline preincubation, mATPase reaction was intense in the small superficial fibers after acid
Fig. 1. Total length of pacu (P. mesopotamicus) at 25, 27 and 29 jC throughout the experiment. #Temperature where fish total length was significant. Values represent means F S.D., five fish per temperature.
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Fig. 2. Transverse section of lateral musculature in pacu (P. mesopotamicus). (A) At hatching, at 29 jC. Superficial columnar muscle fibers (s). Deep round muscle fibers (d). Nerve tissue (nt). HE. Scale bar: 20 Am. (B, C and D) Five days after hatching at 27, 25 and 29 jC, respectively. Scale bars: 30, 40 and 30 Am, respectively. (B) Continuos lines showing the entire epaxial quadrant. HE. (C) Superficial layer fibers with intense NADH-TR reaction. Deep layer fibers with weak enzymatic reaction. NADH-TR. (D) Superficial layer fibers dark for mATPase reaction. Deep layer fibers with weak enzymatic reaction. Myosin ATPase, pH 4.4. Fibers with central nuclei (arrow). Nerve tissue (nt). Notochord (n). Superficial muscle fibers (s); deep muscle fibers (d); horizontal septum region with triangular arrangement of fibers (arrowhead).
preincubation (Fig. 2D); most fibers reacted intensely after alkaline preincubation. In the horizontal septum region that divides the musculature into epaxial and hypoaxial, the columnar fiber layer penetrated the musculature towards the medial plane, assuming a triangular shape (Fig. 2C and D). On days 25 and 60, increased epaxial muscle mass was seen; this was more marked in the 29 jC group (Fig. 3A). In the deep layer, there were many small fibers in between the larger fibers (Fig. 3B and C). An intermediate layer was seen between the superficial and deep layers (Fig. 3B); on day 60, the 29 jC group showed more large areas of small fiber in both the deep layer and the dorsal fin muscle region (Fig. 3C). Also in these periods, the superficial and deep layer fibers showed standard NADH-TR and mATPase similar to those at day 5. In the intermediate layer fibers, NADH-TR reaction was weak, and mATPase ranged from moderate to strong regardless of preincubation (Fig. 3D1 and E). On day 60, the small fibers in between the larger deep layer fibers, reacted moderately to mATPase after both acid and alkaline preincubation (Fig. 3E). In the dorsal fin muscle, the fibers closest to the median plane reacted intensely and the more peripheral fibers reacted weakly to NADH-TR and m-ATPase in both acid and alkaline (Fig. 3D2).
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Fig. 3. Transverse section of lateral musculature in pacu (P. mesopotamicus). (A) Twenty-five days after hatching, at 27 jC. Dorsal fin muscle fibers (*). HE. Scale bar: 40 Am. (B) Sixty days after hatching, at 27 jC. Superficial muscle fiber (s). Intermediate muscle fibers (i). Small fibers (*) between larger fibers in the deep layer. HE. Scale bar: 40 Am. (C) Sixty days after hatching, at 29 jC. Cluster of small fibers (*) between some larger fibers in the deep layer. HE. Scale bar: 30 Am. (D) Twenty-five days after hatching, at 29 jC. Scale bar: 30 Am. (D1) Small fibers between the superficial and deep compartments, moderate intensity NADH-TR reaction (arrowhead). NADH-TR. (D2) Dorsal fin muscle fibers with intense NADH-TR reaction (*). Deep layer fibers with weak NADH-TR reaction (d). NADH-TR. (E) Sixty days after hatching, at 29 jC. Superficial (s) and intermediate (i) muscle fibers with negative and moderate mATPase reaction, respectively. Small fibers (arrowhead) in deep layer with weak to moderate mATPase reaction, between larger fibers with marked reaction. Myosin ATPase, pH 10.4. Scale bar: 30 Am. (F) Nuclei with positive PCNA reaction (arrows). Counterstained with Haematoxylin. Scale bar: 20 Am.
3.2. Muscle ultrastructure Pacu striated musculature was similar at all the incubation temperatures. At hatching, undifferentiated columnar muscle fibers were seen in the superficial region; they were
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more rounded in the deep region. There were few myofibrils and mitochondria, and many ribosomes. Sarcoplasmic reticulum was seen between the myofibrils. The nucleus showed loose chromatin and evident nucleolus and was located in the fiber innermost region.
Fig. 4. Ultrastructure of lateral musculature in pacu (P. mesopotamicus) at all experimental temperatures. (A) At hatching. Columnar muscle fibers (*) in the superficial region. Myofibrils (mi). Mitochondria (M). Ribosomes (arrowhead). Sarcoplasmatic reticulum (arrow). Undifferentiated cell with loose nucleus (N) and ribosomes. Scale bar: 1 Am. (B) At hatching. Muscle fibers from the deepest region. Myofibrils in the fiber peripheral region (mi). Mitochondria (M). Ribosomes (arrowhead). Loose nucleus with evident nucleolus (N). Scale bar: 1 Am. (C) Five days. Superficial muscle fibers with myofibrils (mi), mitochondria (M). Undifferentiated cell with loose nucleus (N). Dermis (D). Scale bar: 1 Am. (D) Five days. Deep layer muscle fibers. Sarcoplasm with radial myofibrils in the periphery (*) and polygonal in the centre (arrow). Mitochondria (M). Sarcoplasmatic reticulum (arrowhead). Scale bar 2 Am. (E) Twenty-five days. Superficial layer fibers with myofibrils (mi) and mitochondria (M) in the subsarcolemmal and central regions. Intermediate layer small fiber (f). Undifferentiated cell with relatively dense nucleus (uc). Dermis (D). Scale bar: 2 Am. (F) Sixty days. Deep compartment fibers with radially oriented myofibrils (*). Sarcoplasmatic reticulum (arrow). Fiber nucleus in peripheral position, with evident nucleolus (N). Small fiber (arrow) between larger fibers. Undifferentiated cell with relatively dense nucleus (uc). Scale bar: 1 Am.
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Table 1 Total number of red (R) and white (W) fibers in one epaxial quadrant of the lateral muscle in pacu (P. mesopotamicus) at 25, 27 and 29 jC, throughout the experiment Total number of fibers Day 5 25 jC 27 jC 29 jC
W R W R W R
Day 25 1AB
64.2 F 7.2 46.4 F 3.51a 58.0 F 7.11A 48.8 F 7.71a 79.4 F 16.31B 47.2 F 4.11a
Day 60 1A
80.5 F 8.7 44.5 F 1.71a 226.0 F 33.81B 78.2 F 6.12b 270.2 F 127.31B 92.2 F 40.21b
834.2 F 383.32A 192.4 F 44.42ab 1111.6 F 237.82AB 183.6 F 42.53a 1961.8 F 906.82B 246.8 F 28.22b
Values represent means F S.D., five fish per each temperature. Significant differences when P < 0.05. Values with the same number in the rows are not statistically significant. Values with the same letters in the columns (capitals for white fibers; small letters for red fibers) are not statistically significant.
Undifferentiated cells with cytoplasm showing many ribosomes, mitochondria, and with loose nucleus were seen between the muscle fibers (Fig. 4A and B). On day 5 post-hatching, muscle fiber showed more myofibrils in the sarcoplasm. The superficial layer fibers were small with a few myofibrils in the sarcoplasm. Most fibers showed areas of sarcoplasm without myofibrils, with many mitochondria, ribosomes, and sarcoplasmic reticulum. In deep musculature, the fibers were larger than the superficial layer. Several groups of myofibrils were seen filling the sarcoplasm. The myofibrils were arranged radially in the periphery and polygonally in the centre. Some mitochondria and sarcoplasmic reticulum cisternae were seen between the myofibrils (Fig. 4C and D). On day 25 post-hatching, large and small fibers were seen, especially in the deep musculature. In the superficial layer fibers, there were well-organized myofibrils and mitochondria, especially in the subsarcolemmal and central regions. In the deep musculature, small fibers were seen with areas of sarcoplasma without myofibrils adjacent to Table 2 Diameter of red (R) and white (W) fibers in one epaxial quadrant of the lateral muscle in pacu (P. mesopotamicus) at 25, 27 and 29 jC, throughout the experiment Diameter of fibers Day 5 25 jC 27 jC 29 jC
W R W R W R
Day 25 1A
9.2 F 3.4 2.2 F 0.51a 9.1 F 3.41A 2.4 F 0.61b 8.9 F 3.61A 2.4 F 0.51b
Day 60 2A
11.5 F 4.3 2.34 F 0.51a 16.77 F 7.52B 3.5 F 0.92b 16.8 F 8.02B 3.8 F 1.22c
15.6 F 13.03A 6.8 F 3.52a 14.77 F 11.03A 6.6 F 2.93b 17.2 F 16.42B 8.7 F 4.12c
Values represent means F S.D., five fish per each temperature. Significant differences when P < 0.05. Values with the same number in the rows are not statistically significant. Values with the same letters in the columns (capitals for white fibers; small letters for red fibers) are not statistically significant.
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large fibers of more differentiated shape, with loose nuclei in the peripheral region, few mitochondria, and well-developed sarcoplasm. Between muscle fibers in the connective tissue, there were cells with little cytoplasm and loose or relatively dense nuclei (Fig. 4E). On day 60 post-hatching, the organization of the muscle fibers was similar to 25-dayold specimens. However, there were frequent small fibers with areas of sarcoplasma without myofilaments, especially in the deep musculature (Fig. 4F). 3.3. Morphometric analysis In the superficial layer, between days 5 and 25 post-hatching, significant increase in number of fibers was only seen in the epaxial quadrant of the 27 jC group; from day 25 onwards, increase was significant at all temperatures. At day 5, there were no significant differences between temperatures for number of fibers. At day 25, increase in fiber
Fig. 5. Muscle fiber diameter distribution in pacu (P. mesopotamicus) at 25, 27, and 29 jC throughout the experiment. Columns represent number of fibers (means F S.D) in each size class, five fish per temperature. (letters in the column show size classes with significant variation, p < 0.05).
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numbers was significantly lower at 25 jC; after 60 days, 29 jC showed a higher number of fibers in the superficial compartment compared to 27 jC; this was not significant compared to 25 jC fish. In the deep layer, there was significant increase in number of fibers in the epaxial quadrant from day 25 for all temperatures. On day 5, number of fibers at 29 jC was significantly higher than 27 jC, but not significant against 25 jC; on day 25, the number of fibers was significantly higher for both 27 and 29 jC groups; on day 60, fish incubated at 29 jC presented a significantly higher number of fibers than at 25 jC, but not significant against 27 jC (Table 1). In relation to mean fiber diameter in the superficial layer, there was increase at 27 jC throughout the study period. From day 25 post-hatching, there was significant increase at 25 jC, but not at 29 jC. At day 5, fiber diameter was significantly higher at 27 and 29 jC; on days 25 and 60, it was significantly higher proportional to increasing temperature. In the deep layer, there was significant increase in mean fiber diameter at 25 jC throughout the study period. At 27 and 29 jC, this increase was significant between days 5 and 25 posthatching; at 27 jC, there was a reduction in average fiber diameter at day 60. On day 5, there was no temperature-related significant difference in fiber diameter; on day 25, this was higher at 27 and 29 jC but at day 60 post-hatching, only in the 29 jC group (Table 2). Throughout the experiment, there was both fiber diameter increase and new muscle fiber recruitment in superficial red and deep white muscle layers. In the superficial layer, the majority of fiber diameters after 5, 25 and 60 days were V 4; V 8; and V 16 Am, respectively. There was temperature-related significant difference in muscle fibers V 8 Am only at 25 days. In the deep layer, most fiber diameters were V 20, V 30, and V 20 Am after 5, 25 and 60 days, respectively. There was temperature-related significant difference in all size classes at 25 days, and in classes V 10 and V 20 Am at 60 days ( p < 0.05) (Fig. 5). At the studied temperatures, proliferating nuclei in the epaxial quadrant were shown by PCNA immunohistochemical reaction (Fig. 3F). The number of nuclei increased during the experiment; on day 5, there was no significant difference between the studied
Fig. 6. Number of proliferating nuclei in one epaxial quadrant of the lateral muscle in pacu (P. mesopotamicus) at 25, 27 and 29 jC throughout the experiment. #Temperature with significant difference. Values represent means F S.D., five fish per temperature.
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temperatures; on day 25 the 25 jC group, and on day 60 the 29 jC group were significantly higher than the others (Fig. 6).
4. Discussion In our study, the time between egg fertilization and hatching was inversely proportional to the incubation temperature. Several researchers have reported that incubation temperature can influence fish development and growth, and egg incubation at higher temperatures can accelerate hatching time (Calvo and Johnston, 1992; Vieira and Johnston, 1996; Johnston et al., 1998; Galloway et al., 1999b). In our experiment, the mortality index was low. This is probably related to the narrow temperature range used which was close to the ideal reproduction temperature for this species (Romagosa et al., 1990). Some studies have shown that incubation temperature can have various effects on muscle tissue growth and development (Vieira and Johnston, 1992; Johnston et al., 1998). This may be related to egg quality, including yolk sac nutrient quantity and growth factor concentration (Johnston et al., 1998). Evidence shows that the chorion can form a barrier to gas exchange between the embryo and the environment, and temperature might influence this exchange and oxygen diffusion through the chorion and perivitellinic fluid (Matschack et al., 1995, 1997). In our study, the intensity of NADH reaction in the red and white fibers was similar in all rearing temperatures from day 5. Red fibers in the superficial layer and dorsal fin muscle region showed aerobic metabolism, and the white fibers that made up the majority of the musculature showed more anaerobic metabolism; these characteristics are similar to other fish (Fernandez et al., 2000). White fibers are used in fast burst swimming, for example, escaping predators and catching prey, and red fibers for low intensity sustained swimming (Bone, 1975; Johnston, 1999). In pacu, the precursor cells of these two muscle fiber populations were present since hatching, as demonstrated by light and electron microscopy. Although some authors have shown the influence of temperature on mitochondria volume density (Vieira and Johnston, 1992; Brooks and Johnston, 1993), our study showed muscle fibers with similar oxidative activity throughout the experiment at all incubation temperatures. At the different temperatures used in our experiment, intermediate muscle fibers could be seen from day 25 post-hatching with characteristics of both red and white fibers. During postembryonic myotomal muscle growth, intermediate fibers commonly appear later than red and white fibers (Matsuoka and Iwai, 1984; Scapolo et al., 1988), and the distribution and frequency of these fibers differ between species and developmental stages (Zhang et al., 1996). Based on histochemical and physiological characteristics, these fibers are recruited at faster cruising speeds (Akster et al., 1995; Johnston and Mclay, 1997; Johnston, 1999). The mATPase reaction showed more variability in the intensities, mainly in the intermediate and white muscle and dorsal fin muscle region. Several histochemical studies have demonstrated that the variation in the m-ATPase staining intensities after both alkali and acid preincubation can be related to the change or transition in the myosin heavy chain isoform composition, or co-expression of the different myosin isoforms (Staron and Pette,
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1986; Scapolo et al., 1988). These can be related to the phenotypic plasticity in muscle contractile functions as the fish grows (Scapolo et al., 1988; Brooks and Johnston, 1993; Veggetti et al., 1993; Mascarello et al., 1995). In our study, these phenotypic variations were more evident 25 and 60 days after hatching at all temperatures. In pacu, from hatching until day 60 at all temperatures, muscle mass thickening was from muscle tissue growth. It has been suggested that post-embryonic growth of fish muscle tissue involves both hypertrophy and hyperplasia (Alami-Durante et al., 1997; Galloway et al., 1999a,b; Rowlerson and Veggetti, 2001). In mammals, muscle tissue growth by fiber hyperplasia is restricted to the pre- and perinatal periods (Schultz, 1974; Stickland, 1983). In our study, muscle fiber hypertrophy was observed from hatching until day 60 at all temperatures. From days 5 to 25, hypertrophy was the main growth process characterized by the significant increase in fiber diameter except for the red fibers at 25 jC. In one epaxial quadrant, fiber number increase was not significant, except for the red fibers at 27 jC. Hypertrophy occurred at all studied temperatures between 25 and 60 days; however at 29 jC, fiber diameter increase was not significant although higher than at 25 and 27 jC. In this period, a significant increase in fiber number for both red and white muscle layers was seen, characterizing new fiber recruitment, as seen by the high number of small diameter fibers ( V 20 Am); this was also seen in the intermediate layer and dorsal fin muscle region. As muscle is a post-mitotic tissue, fiber hyperplasia and hypertrophy involve the activation and proliferation of undifferentiated myoblasts or myosatellite cells (Koumans et al., 1993; Johnston, 1999). In mammals and in the juvenile and adult phases of some fish, these cells are located between the sarcolemma and basal lamina of muscle fibers (Mauro, 1961; Campion, 1984; Veggetti et al., 1990; Koumans and Akster, 1995). In initial developmental stages, myogenic precursor cells are widely scattered throughout the myotomal muscle fibers (Veggetti et al., 1990; Stoiber and Sange¨r, 1996; Johnston et al., 1998, 2000). Ultrastructural analysis of pacu muscle showed nuclei of undifferentiated cells in between the more differentiated fibers in the connective tissue. According to Johnston (1993a) and Johnston et al. (1998), these cells could be undifferentiated or persistent myoblasts involved in muscular growth. These cells can proliferate and their nuclei may enlarge existing fibers by fusion, or they may add new fibers to the musculature (Johnston et al., 1998; Johnston, 2001). In our study, the increase in proliferating nuclei suggests that they participate in hyperplastic and hypertrophic muscle fiber growth. Although on day 25, 25 jC fish were smaller, on day 60 there was no significant difference in proliferating nuclei between 25 and 27 jC groups. This could be related to the higher number of proliferating nuclei in 25 jC on day 25. More intense cell proliferation may have accounted for the higher level of hyperplasia in this period, as shown by the high number of small diameter fibers V 20 Am. On day 60 at 25, 27 and 29 jC, there was an increase in fiber diameter, but there were also many muscle fibers with diameters < 20 Am suggesting highly active new fiber recruitment. The higher frequency of PCNA-positive nuclei at 29 jC suggests they contributed to the higher number of fibers at this temperature. Pacu is a fast growing fish which grows to a large size; the mechanisms of muscle fiber growth by hyperplasia and hypertrophy occur over a long period; hyperplasia is
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predominant in the juvenile phase (Dal Pai et al., 2000); this is different to small species, where fiber recruitment ceases in an early phase (Weatherley and Gill, 1984). In our study, we found that hyperplasia and hypertrophy was more intense between days 25 and 60. Several authors have shown that differences in rearing temperature can modulate the rates of muscle fiber recruitment and hypertrophy in different phases of muscle growth. The mechanism is unknown, but some authors have suggested that variation in temperature can alter the proliferation and differentiation of myogenic stem cells; these events are regulated by myogenic regulatory factors, which play a major role in cell proliferation and terminal muscle fiber differentiation (Olson, 1992; Rudnicki and Jaenisch, 1995). At the end of our experiment, 29 jC incubation temperature fish showed higher fiber diameter, intense hyperplasia, and high cell proliferation rate; this may be linked to their greater length. Although muscular growth by hypertrophy and hyperplasia occurred at all studied temperatures, 29 jC fish could possibly attain a larger size; also an acceleration may occur in the reproductive phase, which requires further studies. This study demonstrates the viability of pacu egg incubation at 29 jC to obtain larger larvae and alevins with higher growth potential.
Acknowledgements This study was supported by grants from FAPESP, Proc. No. 00/06177-0 and FUNDUNESP, Proc. No. 557-01-DCP. The authors wish to thank Jarbas do Amaral, Sueli Cruz Michelin, and Cristina Audi for their skillful assistance.
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