Aquaculture nutrition, tập 18, số 5, 2012

Aquaculture Nutrition 2012 18; 465492 1 doi: 10.1111/j.1365-2095.2012.00943.x 3,4 Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal, India; Aquaculture Laboratory, Department of Zoology, University of Burdwan, Burdwan, West Bengal, India; Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsứ, Tromsứ, Norway; Aquaculture Protein Centre (a CoE), Department of Aquatic Medicine and Nutrition, Norwegian School of Veterinary Medicine, Oslo, Norway Digestion of food depends on three main factors: (i) the ingested food and the extent to which the food is susceptible to the effects of digestive enzymes, (ii) the activity of the digestive enzymes and (iii) the length of time the food is exposed to the action of the digestive enzymes Each of these factors is affected by a multitude of secondary factors The present review highlights the experimental results on the secondary factor, enzymatic activity and possible contribution of the fish gut microbiota in nutrition It has been suggested that fish gut microbiota might have positive effects to the digestive processes of fish, and these studies have isolated and identified the enzyme-producing microbiota In addition to Bacillus genera, Enterobacteriaceae and Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus, unidentified anaerobes and yeast are also suggested to be possible contributors However, in contrast to endothermic animals, it is difficult to conclude the exact contribution of the gastrointestinal microbiota because of the complexity and variable ecology of the digestive tract of different fish species, the presence of stomach and pyloric caeca and the relative intestinal length The present review will critically evaluate the results to establish whether or not intestinal microbiota contribute to fish nutrition KEY WORDS: contribution, digestive tract, enzyme-producing bacteria, fish, nutrition, review Received 16 June 2011, accepted January 2012 Correspondence: Arun Kumar Ray, Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan-731 235, West Bengal, India E-mail: aray51@yahoo.com, arun_ray1@rediffmail.com ê 2012 Blackwell Publishing Ltd Traditionally, digestion is described as the process by which food in the gastrointestinal (GI) tract is split into simpler absorbable compounds performed primarily by the digestive enzymes However, what happens in the alimentary tract is only one part of a continuous process that also includes factors outside the GI tract The traditional aspects involved in digestion and absorption have been comprehensively reviewed by several authors (Kapoor et al 1975; Faănge et al 1979; Ash et al 1985; Sheridan 1988; Smith & Halver 1989; Sire & Vernier 1992; Olsen & Ringứ 1997; Bakke et al 2010) However, these reviews have not focused on the gut microbiota and their possible influence on digestibility of nutrients An understanding of the contribution of endosymbionts to digestion requires information on the relative importance of exogenous (produced by the GI endosymbionts) and endogenous (produced by the host) digestive enzymes (Clements et al 1997) Prior to the discussion of the contribution of the gut microbiota in production of digestive enzymes, a brief introduction regarding endogenous enzyme activities in fish seems pertinent The endogenous digestive enzymes, which are secreted to the lumen of the alimentary canal, originate from the oesophageal, gastric, pyloric caeca and intestinal mucosa and from the pancreas (De Silva & Anderson 1995) The presence of endogenous digestive enzymes in fish has been reported in numerous studies (e.g Dhage 1968; Kawai & Ikeda 1972; Shcherbina et al 1976; Fagbenro 1990; Das & Tripathi 1991; Fagbenro et al 2000) All fish species investigated per se possess the enzymatic apparatus for hydrolysis and absorption of simple and complex carbohydrates (Krogdahl et al 2005) Digestive a-amylase has been localized throughout the entire GI tract of numerous fish species (Dhage 1968; Kawai & Ikeda 1972; Chiu & Benitez 1981; Fagbenro 1990; Sabapathy & Teo 1993; Chakrabarti et al 1995; Kuzmina 1996; Peres et al 1997; Hidalgo et al 1999; de Seixas et al 1999; Fagbenro et al 2000; Tengjaroenkul et al 2000; Alarcon et al 2001; Fernandez et al 2001) In general, amylase activity in the digestive tract of omnivorous fish is higher than that of carnivorous fish (Kitamikado & Tachino 1960; Shimeno et al 1977; Cowey et al 1989; German et al 2004, 2010), but the activity is also affected by dietary manipulation (German et al 2004, 2010; Skea et al 2005, 2007) Moreover, it is likely that the activity differs with the structure of the digestive tract, developmental stages and ambient temperatures of fish (Kitamikado & Tachino 1960; Kawai et al 1975; Takeuchi 1991; Cahu & Zambonino Infante 2001; Kamaci et al 2010; Miegel et al 2010) Chitinolytic activity is reported to be present throughout the GI tract, and high activity is localized in stomach and pyloric tissue, indicating that these organs or the diet are the main sources of the enzymes (e.g Micha et al 1973; Faănge et al 1979; Danulat & Kausch 1984; Lindsay 1984, 1986; Danulat 1986; Krogdahl et al 2005; Ringứ et al 2012) Endogenous cellulase activity has been reported in the digestive tract of several fish species indicating that these fish species may be able to utilize cellulose and similar fibrous carbohydrates (Fagbenro 1990; Das & Tripathi 1991; Szlaminska et al 1991; Chakrabarti et al 1995; Saha & Ray 1998; Salnur et al 2009) Saha & Ray (1998) observed a diet-dependent cellulase activity both in intestine and hepatopancreas of rohu (Labeo rohita) fingerlings However, a sharp decline in the level of cellulase activity was observed in fish fed diets containing the antibiotic tetracycline (active against Streptococcus, Mycoplasma etc.), indicating that cellulase activity in rohu is contributed largely by the microorganisms present in the digestive tract The early study of Shcherbina & Kazlauskiene (1971) proposed that an endogenous cellulase is secreted into the anterior portion of the digestive tract of carp (Cyprinus carpio), while the remaining cellulose absorption takes place in the posterior portion of the digestive tract, indicating the presence of microbial cellulase in this region Lipase activity has been reported in the gut or gut contents of most fish species studied, and it seems like a general rule that most of the intestinal lipase activity if present is located in the pyloric caeca and the proximal intestine (Olsen & Ringứ 1997) The principal sites for secretion of endogenous proteases in teleosts are stomach, pancreas and intestine (De Silva & Anderson 1995) In fish, adaptive changes in the activity of proteolytic enzymes have been reported in relation to diet (Kawai & Ikeda 1972; Shcherbina et al 1976; Dabrowski & Glogowski 1977; Clements et al 2006; German et al 2010) Although intestinal phytase activity has been detected in several fish species, it was insufficient for any significant improvement in phytate hydrolysis in most teleosts (Ellestad et al 2003) Numerous studies have reported diverse microbial communities in the GI tract of carnivorous, herbivorous and omnivorous fish species (e.g Fishelson et al 1985; Rimmer & Wiebe 1987; Clements et al 1989; Cahill 1990; Sakata & Lesel 1990; Clements 1991; Rahmatullah & Beveridge 1993; Luczkovich & Stellwag 1993; Ringứ et al 1995; Ringứ & Gatesoupe 1998; Ringứ & Birkbeck 1999; Bairagi et al 2002a; Ramirez & Dixon 2003; Fidopiastis et al 2006; Izvekova et al 2007; Sun et al 2009; Li et al 2009; Merrifield et al 2010a; Nayak 2010a) However, surprisingly, the endosymbiotic community and its role in digestion, of the dominant aquatic vertebrate herbivore fish, are poorly investigated In the reviews of Stone (2003), Krogdahl et al (2005) and Rowland (2009), the topic is either neglected or only hinted Cahill (1990), Ringứ et al (1995), Austin (2006) and Nayak (2010a) presented some information on studies of exogenous enzyme activity in fish, but a more comprehensive review is needed as the GI microbiota of fish have been reported to produce a wide range of enzymes; amylase, cellulase, lipase, proteases, chitinase and phytase (Tables 16) Furthermore, the role of enzyme-producing fish gut bacteria as probiotics in enhancement of food digestibility and their effect on gut enzyme activity has been evaluated through several investigations (Table 7) In the present review, we addressed the issue to provide an overview of the information available on the enzymeproducing microbiota isolated from the GI tracts of fish together with a critical evaluation of the results obtained so far The results cited include works published in wellknown as well as minimally circulated journals This is performed to indicate that there are numerous interesting investigations published on the topic enzyme-producing microorganisms isolated from the digestive tracts of fish The gut microbiota of fish is classified as autochthonous or indigenous when they are able to adhere and colonize the hosts gut epithelial surface or allochthonous, when they are incidental visitors in the GI tract and are rejected after some time without colonizing (Ringứ & Birkbeck 1999; Ringứ et al 2003; Kim et al 2007; Merrifield et al 2011) However, one study has hinted that the allochthonous microbiota might be able to colonize the area between the Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd Table Amylase-producing bacteria isolated from the digestive tract of fish Microorganism Isolated from References Strict anaerobes and Aeromonas hydrophila N.i N.i* Vibrio spp Aeromonas spp.; Bacteroidaceae; Clostridium spp Grass carp Grass carp Grass carp Sea bass larvae Ayu, carp, channel catfish, Japanese eel and tilapia Grass carp, carp and tilapia Rohu Rohu and murrel Roach Trust et al (1979) Lesel et al (1986) Das & Tripathi (1991) Gatesoupe et al (1997) Sugita et al (1997) Seven freshwater teleosts Bata Three species of Indian major carps Mondal et al (2008) Mondal et al (2010) Ray et al (2010) Atlantic salmon Askarian et al (2012a) Atlantic cod Askarian et al (2012b) N.i Bacillus circulans; B pumilus; B cereus N.i Aeromonas spp.; Enterobacteriaceae; Pseudomonas spp.; Flavobacterium spp N.i Bacillus licheniformis; Bacillus subtilis Citrobacter sp.; Enterobacter sp.; Bacillus coagulans and an uncultured bacterium clone isolated from the PI of Catla catla Bacillus cereus isolated from the DI of C catla Bacillus sp isolated from the PI of Cirrhinus mrigala Bacillus cereus, Citrobacter freundii and an uncultured bacterium clone isolated from the DI of C mrigala Bacillus sp isolated from the DI of Labeo rohita Bacillus thuringiensis, B cereus, Bacillus sp isolated from the GI tract of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp Isolated from the GI tract of S salar fed 5% chitin supplemented diet Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal Bairagi et al (2002a) Ghosh et al (2002) Kar & Ghosh (2008) Skrodenyte-Arbaciauskiene (2007) N.i no information was given; N.i* indicates the presence of microbial amylase; PI proximal intestine; DI distal intestine; Channel catfish Ictalurus punctatus; Grass carp Ctenopharyngodon idella; Rohu Labeo rohita; Murrel Channa punctatus; Roach Rutilus rutilus; Bata Labeo bata; Ayu Plecoglossus altivelis; Tilapia Oreochromis niloticus; Carp Cyprinus carpio; Japanese eel Anguilla japonica; Sea bass Dicentrarchus labrax; Atlantic salmon Salmo salar; Atlantic cod Gadus morhua microvilli under special conditions such as stress, when a peal of effect of mucus occurs (Olsen et al 2005) Based on the criteria for testing autochthony of microorganisms reported in the GI tracts of endothermic animals, Ringứ & Birkbeck (1999) proposed some criteria for testing indigenous microorganisms in fish: (i) the microorganisms should be detected in healthy individuals, (ii) colonize early stages and persist throughout the life cycles, (iii) demonstrated in both free-living and hatchery-cultured fish, (iv) able to grow anaerobically and (v) be detected associated with the epithelial mucosa in the stomach, proximal or distal intestine In addition, several factors such as (i) gastric acidity, (ii) bile salts, (iii) peristalsis, (iv) digestive enzymes, (v) immune response and (vi) indigenous bacteria and the antibacterial compounds that they produce are suggested to influence adhesion and colonization of the microbiota within the digestive tract (Ringứ et al 2003) The historical data stem from culturing methods of the fish digestive tracts reported that aerobes or facultative Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd anaerobes are dominant in the digestive tract of fish (e.g Trust & Sparrow 1974; Cahill 1990; Sakata & Lesel 1990; Ringứ et al 1995; Ringứ & Birkbeck 1999; Bairagi et al 2002a; Saha et al 2006) However, these results are based on culture methods and mainly evaluated aerobes and facultative anaerobes with a subsequent underestimation of the obligate anaerobic microbiota and the un-culturable microbiota This is clearly demonstrated in numerous recent publications evaluating the fish gut microbiota by using molecular methods (e.g Moran et al 2005; Pond et al 2006; Clements et al 2007; Hovda et al 2007; Liu et al 2008; Navarrete et al 2009; Ferguson et al 2010; He et al 2010; Zhou et al.2011) In addition, several authors have suggested that electron microscopic (EM) examinations of the GI tract should be included as an important tool for investigating the microbial ecology of the gut ecosystem and determining the presence of autochthonous or allochthonous microbiota (e.g Fishelson et al 1985; Clements 1991; Andlid et al 1995; Ringứ et al 2003; Table Cellulase-producing bacteria isolated from the digestive tract of fish Microorganism Isolated from References N.i* N.i* N.i N.i* N.i was given about the CMCase-producing bacteria Anaerobic CMCase-producing bacteria N.i Bacillus circulans; B pumilus; B Cereus N.i N.i Bacillus circulans, B megaterium N.i N.i N.i Bacillus licheniformis; B Subtilis Citrobacter sp.; Enterobacter sp.; Bacillus coagulans and an uncultured bacterium clone isolated from the PI of Catla catla Bacillus cereus isolated from the DI of C catla Bacillus sp isolated from the PI of Cirrhinus mrigala Bacillus cereus, Citrobacter freundii and an uncultured bacterium clone isolated from the DI of C mrigala Bacillus sp isolated from the DI of Labeo rohita Aeromonas sp Bacillus subtilis, B velesensis Bacillus thuringiensis, B cereus, Bacillus sp isolated from the GI tract of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp Isolated from the GI tract of S salar fed 5% chitin supplemented diet Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal Carp Different fish species Grass carp Grass carp Pinfish Pinfish Rohu Rohu Rohu and murrel Grass carp, carp and silver carp Tilapia, Grass carp Grass carp Grass carp Seven freshwater teleosts Bata Three species of Indian major carps Shcherbina & Kazlauskiene (1971) Stickney & Shumway (1974) Lesel et al (1986) Das & Tripathi (1991) Luczkovich & Stellwag (1993) Stellwag et al (1995) Saha & Ray (1998) Ghosh et al (2002) Kar & Ghosh (2008) Bairagi et al (2002a) Saha et al (2006) Li et al (2009) He et al (2009) Mondal et al (2008) Mondal et al (2010) Ray et al (2010) Grass carp Pacu, Piaucom-pinata Atlantic salmon Jiang et al (2011) Peixoto et al (2011) Askarian et al (2012a) Atlantic cod Askarian et al (2012b) N.i* indicates the presence of microbial cellulase; N.i no information was given; Pinfish Lagodon rhomboids; Silver carp Hypophthalmichthys molitrix; Tilapia Oreochromis mossambica; Pacu Piaractus esoiptamicus; Piaucom-pinata Leporinus friderici Fidopiastis et al 2006; German 2009; Ghosh et al 2010; Merrifield et al 2010b, 2011; Harper et al 2011) However, to the authors knowledge, only one recent study has used EM examination related to the gut enzyme-producing microbiota of fish (Ghosh et al 2010) Scanning electron microscopy (SEM) evaluation revealed that bacteria present in the GI tract of rohu were rod shaped, probably bacilli, attached to the intestinal fold associated with mucous As this topic is underestimated, we recommend that the topic merits further investigations It has only been during the last decade that there has been an improved understanding of the importance of commensal intestinal microbiota in fish intestine Nevertheless, the first studies on enzyme production by the fish gut bacteria, to the authors knowledge, were reported in 1979 (Hamid et al 1979 and Trust et al.1979) Since then, numerous studies have been carried out, and an overview of these studies is presented in Tables 16 Microbial amylase activity in the fish gut has been documented in several studies (Table 1) To the authors knowledge, occurrence of amylolytic bacteria (strict anaerobes and Aeromonas hydrophila) in the gut of grass carp (Ctenopharyngodon idella) was first reported by Trust et al (1979) Later, Lesel et al (1986) demonstrated amylolytic bacteria in the digestive tract of grass carp, but the bacteria were not characterized and identified In their study on grass carp, Das & Tripathi (1991) suggested the presence of amylase-producing bacteria, but no specific information was given Gatesoupe et al (1997) reported amylase-producing Vibrio spp isolated from sea bass (Dicentrarchus labrax) larvae, but the activity of the gut bacteria was affected by diet formulation Sugita et al (1997) detected Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd Table Protease-producing bacteria isolated from the digestive tract of fish Microorganism Isolated from References Enterobacter spp.; Vibrio spp.; Pseudomonas spp.; Acinetobacter spp.; Aeromonas spp Strict anaerobes and Aeromonas hydrophila N.i* Vibrio spp Pseudomonas sp Flavobacterium balustinum Bacillus cereus N.i Gray mullet Hamid et al (1979) Grass carp Grass carp Sea bass larvae Arabesque greenling Salmon Gray mullet Nine different freshwater teleosts Rohu Roach Hake Roach Trust et al (1979) Das & Tripathi (1991) Gatesoupe et al (1997) Hoshino et al (1997) Morita et al (1998) Esakkiraj et al (2009) Bairagi et al (2002a) Rohu and murrel Seven freshwater teleosts Three species of Indian major carps Kar & Ghosh (2008) Mondal et al (2008) Ray et al (2010) Bata Atlantic salmon Mondal et al (2010) Askarian et al (2012a) Atlantic cod Askarian et al (2012b) Bacillus cereus; B circulans; B pumilus N.i Pseudoalteromonas sp Aeromonas spp.; Enterobacteriaceae; Pseudomonas spp.; Flavobacterium spp.; Micrococcus sp N.i N.i Citrobacter sp.; Enterobacter sp.; Bacillus coagulans and an uncultured bacterium clone isolated from the PI of Catla catla Bacillus cereus isolated from the DI of C catla Bacillus sp isolated from the PI of Cirrhinus mrigala Bacillus cereus, Citrobacter freundii and an uncultured bacterium clone isolated from the DI of C mrigala Bacillus sp isolated from the DI of Labeo rohita Bacillus licheniformis; B subtilis Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from the GI tract of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp Isolated from the GI tract of S salar fed 5% chitin supplemented diet Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal Ghosh et al (2002) Skrodenyte-Arbaciauskiene (2000) Belchior & Vacca (2006) Skrodenyte-Arbaciauskiene (2007) N.i* indicates the presence of microbial protease; N.i no information was given; Gray mullet Mugil cephalus; Salmon Oncorhynchus keta; Hake Merluccius hubbsi; Arabesque greenling Pleurogrammus azonus amylase production by the intestinal microbiota in cultured ayu (Plecoglossus altivelis), common carp (C carpio), channel catfish (Ictalurus punctatus), Japanese eel (Anguilla japonica) and tilapia (Oreochromis niloticus) Of the 206 isolates examined, 65 (31.6%) produced ! 0ã01 U amylase mL1, and they were identified as Aeromonas spp., Bacterioidaceae and Clostridium spp In a more recent study, enumerating the specific enzyme-producing bacterial community in the gut of nine species of adult freshwater teleosts, Bairagi et al (2002a) observed higher densities of amylolytic strains in herbivorous grass carp, common carp and tilapia (Oreochromis mossambica), but these bacteria were not characterized and identified Furthermore, the authors could not detect amylolytic bacterial strains in the GI tract of carnivorous catfish (Clarias batrachus) and murrel (Channa punctatus) Amylolytic bacteria (Bacillus circulans, Bacillus pumilus and Bacillus cereus) have been documented in the gut of rohu (Ghosh et al 2002) indicating its possible link with feeding habit Skrodenyte- Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd Arbaciauskiene (2007) examined in vitro amylolytic activities of bacteria isolated from the intestinal tract of adult roach (Rutilus rutilus) that feed mainly on mollusks and macrophytes Of total 60 bacterial strains isolated from the intestinal contents, amylolytically active isolates comprised 50%, 65% and 55% of all bacteria isolated from the foregut, midgut and hindgut, respectively Of the 34 bacteria isolated displaying in vitro amylolytic activity, 29 isolates belonged to Aeromonas spp However, amylolytic activity was only detected in bacteria belonging to Enterobacteriaceae, Pseudomonas and Flavobacterium isolated from the foregut Kar & Ghosh (2008) reported amylase-producing bacteria in the digestive tracts of rohu and murrel, but no information was given about their identification Protease and cellulase activities were exhibited by all bacterial strains isolated from rohu and murrel, but amylase production was poorly detected in strains isolated from murrel Mondal et al (2008) documented higher densities of amylolytic strains in the foregut region of two carps species Table Lipase-producing bacteria isolated from the digestive tract of fish Microorganism Isolated from References Strict anaerobes; Aeromonas hydrophila N.i* N.i Agrobacterium; Pseudomonas; Brevibacterium; Microbacterium; Staphylococcus Vibrio spp., Acinetobacter spp Enterobacteriaceae, Pseodomonas spp Vibrio sp N.i Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from the GI tract of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp Isolated from the GI tract of S salar fed 5% chitin supplemented diet Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal Grass carp Grass carp Turbot, rainbow trout and piranha Arctic charr Trust et al (1979) Das & Tripathi (1991) Mckenzie (1994) Ringứ et al (1995) Sea bass larvae Gatesoupe et al (1997) Arctic charr Nine different freshwater teleosts Atlantic salmon Henderson & Millar (1998) Bairagi et al (2002a) Askarian et al (2012a) Atlantic cod Askarian et al (2012b) N.i* indicates the presence of microbial lipase; N.i no information was given; Rainbow trout Oncorhynchus mykiss; Arctic charr Salvelinus alpinus; Sea bass Dicentrarchus labrax; Turbot Scophthalmus maximus; Piranha Serrasalmus nattereri Table Phytase- and tannase-producing microorganisms isolated from the digestive tract of fish Enzyme Microorganisms Isolated from References Phytase Two strains of Bacillus licheniformis and 20 not identified strains Bacillus subtilis; Bacillus atrophaeus Rhodococcus sp Several marine yeasts Ten fresh water teleosts Roy et al (2009) Freshwater teleosts Catla Sea cucumber and marine fish species Atlantic salmon Khan & Ghosh (2011) Khan et al (2011) Li et al (2008a,b) Atlantic cod Askarian et al (2012b) Fresh water teleosts Mandal & Ghosh (2010) Tannase Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from the GI tract of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp Isolated from the GI tract of S salar fed 5% chitin supplemented diet Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal Yeasts; Pichia spp.; Candida spp Askarian et al (2012a) Catla Catla catla; Sea cucumber Holothuria scabra; Marine fish species Hexagrammos otakii and Synecogobius hasts (Labeo calbasu and Labeo bata) [12.2 103 colony-forming units (CFU) g1 gut tissue and 11.5 103 CFU g1 gut tissue, respectively] in comparison with the hindgut region In a more recent study, Mondal et al (2010) isolated amylase-producing Bacillus licheniformis and Bacillus subtilis from the digestive tract of bata (L bata) Ray et al (2010) detected a huge population of amylase-producing bacteria in the GI tract of three Indian major carps, catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (L rohita), where amylase production was considerably higher by the strains isolated from the proximal intestine of catla and mrigal, except the strain CF4, isolated from the proximal intestine of catla A description of the identified bacteria in the study of Ray et al (2010) is given in Table Cellulose consists of a b-1,4-glycosidic linkages and is estimated as the most abundant biomass (1015 metric tons; Wilson et al 1999) in the world Complete cellulose hydrolysis to glucose demands the action of exoglucanases (also called cellobiohydrolyses), endoglucanases and b-glucosidases Exoglucanases (1,4-b-D-glucan cellobio-hydrolase, EC 3.2.1.91) are usually active on crystalline cellulose and are Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd Table Chitinase-producing bacteria isolated from the digestive tract of fish Microorganism Isolated from References Enterobacter spp., Vibrio spp., Pseudomonas spp Aeromonas spp and Vibrio spp Acinetobacter sp., Enterobacteriaceae, Flavobacterium sp., Photobacterium spp., Vibrio spp and a unidentified Gram-negative rod Plesiomonas shigelloides and Aeromonas hydrophila Aeromonas caviae, A hydrophila, A jandaei, A sobria and A veroni Gray mullet Tilapia Dover sole Hamid et al (1979) Sakata et al (1980) MacDonald et al (1986) Tilapia Carp, crucian carp and gray mullet Various Japanese costal fishes Sakata & Koreeda (1986) Sugita et al (1999) Marinobacter lutaoensis, Ferrimonas balearica, Pseudoalteromonas piscicida, Enterovibrio norvegicus, Grimontia hollisae, Photobacterium damselae spp damselae, P leiognathi, P lipolyticum, P phosphoreum, P rosenbergii, Vibrio campbelli, V chagasii, V fischeri, V fortis, V gallicus, V harveyi, V natrigenes, V nigripulchritudo, V ordalii, V parahaemolyticus, V pomeroyi, V ponticus, V proteolyticus, V rumoiensis, V shilonii, V tasmaniensis and V tubiashii Vibrio fischeri, V harveyi, V scophthalmi V iscthyoenteri group type 1, V scophthalmi V iscthyoenteri group type and V scophthalmi V iscthyoenteri group type Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from the GI tract of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp Isolated from the GI tract of S salar fed 5% chitin supplemented diet Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal Itoi et al (2006) Japanese flounder Sugita & Ito (2006) Atlantic salmon Askarian et al (2012a) Atlantic cod Askarian et al (2012b) Dover sole Solea solea; Japanese flounder Paralichthys olivaceus; Crucian carp Carassius carassius lacking from incomplete cellulose systems Endogluconases (1,4-b-D-glucan-4-glucanohydrolase, EC 3.2.1.4) are more active against the amorphous regions of cellulose, and they can also hydrolyze substituted celluloses, such as carboxymethylcellulose (CMC) and hydroxyethyl-cellulose (HEC) Cellobiohydrolases cleave disaccharide (cellobiose) units either from non-reducing or reducing ends, whereas endoglucanases hydrolyze the cellulose chain internally b-glucosidases (EC 3.2.1.21) are needed to cleave cellobiose and other soluble oligosaccharides to glucose (Beguin 1990) Cellulose is completely hydrolyzed to its constituent oligomers by the cellulase (endogluconase,1,4-b-D-glucan-4-glucanohydrolase, EC 3.2.1.4) Thus, many cellulose-eating animals require the aid of symbiotic microorganisms in their GI tract to digest cellulose and make the energy in this compound available to the host (Bergman 1990; Mo et al 2004; Karasov & Martinez del Rio 2007) Reports on the existence of cellulase activity in the digestive system of fish are rare with contradictory result In early studies on fish, Fish (1951), Barrington & Brown (1957) and Yokoi & Yasumasu (1964) believed that fish not posses endogenous cellulase However, cellulase activity has been reported in several fish species, indicating that fish may be able to utilize cellulose and similar fibrous carbohydrates (Chakrabarti et al 1995) To the authors knowledge, the first study indicating the presence of microbial cellulase in the GI tract of fish was Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd reported in the distal intestine of common carp by Shcherbina & Kazlauskiene (1971) Later, Stickney & Shumway (1974) investigated cellulase activity in the stomachs of 62 species of elasmobranches and teleost fish Of the 62 species studied, 17 showed cellulase activity One species of freshwater catfish (channel catfish, I punctatus) demonstrated cellulase activity Channel catfish exposed to streptomycin (Gram-positive bacteria are more susceptible than Gramnegatives) for 24 h showed no cellulase activity while control fish, not exposed to the antibiotic, continued to demonstrate cellulase activity Based on their results, the authors hinted that the cellulase activity, at least in I punctatus, was derived from alimentary tract microbiota rather than from cellulase secreting cells within the fish Stickney (1975) evaluated cellulase activity in a number of freshwater species and concluded that herbivores are unlikely to have the enzyme, while omnivores and carnivores might pick up cellulolytic bacteria from the invertebrates that harbour the bacteria, which might explain the presence of the cellulolytic bacteria within the GI tract of carnivore fishes Lindsay & Harris (1980) displayed cellulase activity in the digestive tract of 138 fish representing 42 species and suggested that the source of cellulase activity originates from the microbial population, although the authors discarded the hypothesis of a stable cellulolytic microbiota in fish In a study on catfish (Clarias isheriensis) fed an omnivorous diet, mainly the pond plankton Cyanophycea, Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd Arabesque greenling Common carp and grass carp Common carp Cellulase and amylase Protease Cellulase Cellulase and amylase Cellulase Mozambique tilapia Common carp Optimization of cellulase production by gut bacteria Evaluated protease production at low temperature Water hyacinth leaf meal inoculated with fish intestinal bacteria Pretreatment of de-oiled soybean white flake Optimization of cellulase production by gut bacteria Leaf meal inoculated with fish intestinal bacteria Supplemented to the diet Supplemented to the diet Rohu fingerlings N.i N.i Rohu fingerlings Atlantic salmon N.i Rohu spawns Rohu spawns Rohu fingerlings Rohu fingerlings Rohu fingerlings Rohu fingerlings Rohu fingerlings Fish species Bairagi et al (2002b) Pretreatment ANFs (tannins and phytic acid), total FFA and AA in diet growth of nutritive value of meal ANFs (tannins, phytic acid and b-ODAP) in diet CFC and ANFs (tannins and phytic acid) in diet growth, FCR and PER Production of novel halophilic thermo stabile protease capable of converting fish waste proteins for protease production growth and survival growth and endogenous enzyme activities (a-amylase and protease) FAA and FA in leaf meal CF, cellulose, hemi cellulose and ANFs (tannin, phytic acid and mimosine) in leaf meal growth, FCR, PER and ANPU Fermentation CP and FAA and CFC in diet growth and survival were noticed in spawns fed fermented diet Fermentation feed allergen(s), soy factor(s) Solid-state fermentation was suitable for increased cellulase production FAA and FA in leaf meal CF, cellulose, hemi cellulose and ANFs (tannin, phytic acid and mimosine) in leaf meal growth, FCR, PER and ANPU Solid-state fermentation was suitable for increased cellulase production protease production at low temperature FAA and FA in leaf meal CF, cellulose, hemi cellulose and ANFs (tannin and phytic acid) in leaf meal Hoshino et al (1997) Saha & Ray (2011) Ray et al (2007) Bairagi et al (2004) Ray et al (2007) Refstie et al (2005) Ghosh et al (2004) Bairagi et al (2004) Ghosh et al (2002) Ghosh et al (2003) Ramachandran & Ray (2007) Esakkiraj et al (2009) Ramachandran et al (2005) References Results N.i no information was given; ANF antinutritional factors; FFA free fatty acids; AA amino acids; FCR feed conversion ratio; PER protein efficiency ratio; CP crude protein; FAA free amino acids; CFC crude fibre content of the diet; ANPU apparent net protein utilization; Duckweed Lema polyhiza; Rohu Labeo rohita; Common carp Cyprinus carpio; Black gram Phaseolus mungo; Grass pea Lathyrus sativus; Grey mullet Mugil cephalus; Leaf meal Leucaena leucocephala; Water hyacinth Eichhornia crassipes; Mozambique tilapia Oreochromis mossambicus; Arabesque greenling Pleurogrammus azonus; Atlantic salmon Salmo salar Symbols represent an increase () or decrease () Pseudomonas sp Bacillus subtilis, B megaterium Bacillus subtilis N.i N.i Atlantic salmon Pretreatment of diet Cellulase and amylase Mozambique tilapia Bacillus circulans Lactic acid bacteria Leaf meal inoculated with fish intestinal bacteria N.i N.i Rohu Rohu Bacillus cereus Diet formulation Incorporating fermented black gram seed meal Evaluated protease production during processing of tuna waste Amylase, cellulase, lipase and protease Protease Pretreatment of grass pea seed meal Pretreatment of duckweed leaf meal Method of application Adult common carp Gray mullet N.i Enzyme N.i Isolated from Adult common carp Bacillus sp Microorganism Table Overviews of research towards application of enzyme-producing fish gut bacteria in aquaculture high cellulase activities were detected in both the stomach and in the proximal and distal parts of the mid intestine (Fagbenro 1990) Several studies have reported cellulase-producing bacteria isolated from the GI tract of fish (Table 2) Lesel et al (1986) reported cellulolytic gut bacteria in grass carp, but the bacteria were not characterized and identified In a study on digestive enzymes in grass carp, cellulase activity was reported both in hepatopancreas and intestine, and dietary cellulose level significantly affected the cellulase activity (Das & Tripathi 1991) The fact that cellulase activity was reduced to approximately one-third when tetracycline (effective against Vibrio, Mycoplasma and Streptococcus) was supplemented to the diet indicates that the gut microbiota may contribute to the cellulolytic activity in the intestinal tract of grass carp In a study evaluating cellulase activity in rohu, Saha & Ray (1998) reported cellulase-producing bacteria, but they were not characterized and identified Abundance of cellulolytic bacteria has further been documented in the GI tract of grass carp (Bairagi et al 2002a; Saha et al 2006; Li et al 2009), common carp and silver carp (Hypophthalmichthys molitrix) (Bairagi et al 2002a), rohu (Saha & Ray 1998; Ghosh et al 2002; Kar & Ghosh 2008; Ray et al 2010), catla and mrigal (Ray et al 2010), bata (Mondal et al.2008, 2010), tilapia (Saha et al 2006), murrel (Kar & Ghosh 2008) and wood-eating catfishes of genus Panaque (Nelson et al 1999) Bairagi et al (2002a), however, failed to isolate cellulolytic bacteria in the GI tract of carnivorous catfish and murrels In contrast to these results, Kar & Ghosh (2008) reported the presence of cellulolytic bacteria in murrel Nelson et al (1999) isolated several aerobic bacteria from the guts of wood-eating catfishes that showed the ability to grow on cellulose and to produce cellulases Nelson and colleagues also measured cellulases in the fish guts Based on their results, they concluded that wood-eating catfishes digested cellulose in their guts with the aid of aerobic endosymbiotic microbes Mondal et al (2008) evaluated enzyme-producing bacteria in the foregut and hindgut regions of seven freshwater teleosts and quantitatively assayed the cellulase activity However, the authors did not identify the isolated strains Ray et al (2010) isolated and enumerated cellulase-producing autochthonous bacteria in the proximal and distal intestine of three species of Indian major carps and identified the most promising strains by 16S rRNA gene sequence analysis Recently, Jiang et al (2011) investigated the bacterial community in the gut of grass carp using genomic DNA-based 16S rRNA gene library The analysis revealed 28 different bacteria species Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd belonging to seven genera; Vibrio, Acinetobacter, Providencia, Yersinia, Pseudomonas, Morganella and Aeromonas, respectively All cellulase-producing bacteria isolated from the intestine of grass carp belonged to Aeromonas Peixoto et al (2011) evaluated the cellulolytic potential of B subtilis P6 and Bacillus velesensis P11 originally isolated from the midgut of the South American warm water teleosts, pacu (Piaractus esoiptamicus) and piaucom-pinata (Leporinus friderici), respectively The authors reported bacterial growth and cellulase production (mainly endoglucanases), and the highest residual cellulase activity was reported at pH values between 7.0 and 9.0 Luczkovich & Stellwag (1993) and Stellwag et al (1995) reported carboxymethylcellulase (CMCase)-producing microbes from the intestinal tract of the omnivorous pinfish (Lagodon rhomboids) Stellwag et al (1995) isolated a total of 550 anaerobic bacterial strains, 200 from environmental samples and 350 isolates from the intestinal tract contents of seven different pinfish and screened them for CMCase activity The 200 environmental strains revealed no detectable CMCase activity, whereas 36 of the 350 (10.3%) obligate anaerobes recovered from the intestinal tract contents of seagrass-consuming pinfish expressed CMCase activity To understand the taxonomic relationships among CMCase-producing strains, the authors conducted morphological, physiological and biochemical characterization of 36 strains but did not identify them In a study on common carp, Kihara & Sakata (2002) showed that intestinal bacteria isolated from the fish was able to metabolize oligosaccharides commonly found in soy and other beans with the liberation of short-chain fatty acids, carbon dioxide and methane gas Diaz & Espana (2002) reported that the hindgut chamber of the king angelfish (Holacanthus passer) contained a high population level of microorganisms able to hydrolyze complex carbohydrates The hindgut of this fish species is highly vascularized indicating absorption in this gut segment Populations of symbiotic organisms in the gut of most terrestrial vertebrate herbivores play a key role in digestion by breaking down plant cell walls (cellulose and hemicelluloses) to simple compounds such as short-chain fatty acids (SCFAs) that are taken by the host and are used for energy generation and biosynthesis (Stevens & Hume 1995; Seeto et al 1996) The SCFAs produced are rapidly absorbed from the gut lumen The major SCFA is normally acetate with minor amounts of propionate and butyrate (Stevens & Hume 1995) Acetate produced by microbial fermentation constitutes an important source of energy to the host (Mountfort et al 2002) Besides their contribution to energy metabolism, SCFAs perform various physiological functions SCFAs stimulate cell proliferation in the intestinal epithelium in vivo, while in in vitro, they inhibit cell proliferation, but they are potent enhancers of gene expression in cultured cells (Von Engelhardt et al 1989) Propionate is converted to glucose in the liver and may modify hepatic metabolism Butyrate is the preferred fuel for the colony epithelial cells (Roediger 1980; Von Engelhardt et al 1989) It has also been shown that butyrate protects these cells against agents that lead to cellular differentiation and may even inhibit tumour growth (Young et al 1994) Many marine herbivorous fishes contain SCFA (predominantly acetate) in their hindgut, which indicate microbial activity (Clements et al 1994; Clements & Choat 1995; Mountfort et al 2002) Such information is essential to understand the contribution of gut microorganisms to contribute to the energy needs of the fish Mountfort et al (2002) estimated the rates of acetate production in the gut of three species of temperate marine herbivorous fish from north-eastern New Zealand, viz., Kyphosus sydneyanus, Odax pullus and Aplodactylus arctidens The rates of turnover of acetate were in the same order of magnitude as those values detected in the intestinal tracts of herbivorous reptiles and mammals, even though the ectothermic fishes were held at much lower temperatures (1723 C) However, this result does not support the previous hypothesis that high temperatures are a prerequisite for efficient fermentation systems to operate in marine herbivores (Kandel et al 1995) The importance of SCFAs to overall energy supply and metabolism has not yet been quantified for any of these herbivores, but it may be substantial (Mountfort et al 2002) Titus & Ahearn (1988, 1991) reported the concentration of SCFAs along the gut of tilapia, O mossambicus, and characterized a specific transport system for acetate However, in this study, the authors did not determine the role of SCFA metabolism in the investigated species Algae consumed by marine fishes contain much more complex and different carbohydrates than vascular plants with mainly cellulose and hemicellulose-based structural components (Clements et al 2009) In addition to different sets of secondary metabolites, digestion is achieved in a differing ionic environment Neither has attracted much attention by researchers Cellulase yields appear to depend on a complex relationship involving a variety of factors, like inoculums size (carbon source and cellulose quality), pH, temperature, presence of inducers, medium additives, aeration and growth time (Immanuel et al 2006) Ray et al (2007) investigated the optimum environmental and nutritional conditions required to enhance cellulase production by B subtilis CY5 and B circulans TP3, originally isolated from the gut of common carp and Mozambique tilapia (O mossambicus), respectively The authors concluded that solid-state fermentation was suitable for increased cellulase production by the bacterial strains The strains could readily utilize the substrate at 40 C in in vitro culture at pH 7.5, and organic nitrogen sources were reported to be more suitable for optimum cellulase production Proteases are hydrolytic enzymes that catalyse the total hydrolysis of proteins in to amino acids Although proteases are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth, the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for their various applications (Chu 2007) Bacteria belonging to Bacillus sp are by far the most important source of several commercial microbial enzymes (Ferrero et al 1996; Kumar et al 1999; Sookkheo et al 2000; Singh et al 2001; Gupta et al 2002; Beg & Gupta 2003; Shafee et al 2005; Chu 2007; da Silva et al 2007) Some information is available regarding production of proteases by fish gut bacteria (Table 3) To our knowledge, the first studies on protease-producing bacteria isolated from the digestive tract of fish, gray mullet and grass carp were carried out by Hamid et al (1979) and Trust et al (1979), respectively Gatesoupe et al (1997) displayed that protease activity of gut bacteria isolated from sea bass larvae was affected by diet formulation In this study, all bacteria (Vibrio spp.) isolated from larvae fed the compound diet showed amylase activity, while larvae fed Artemia only, 40% of the gut bacteria displayed protease activity This finding could be related to the fact that gut microbiota was more diverse when the larvae were fed Artemia In a study isolating bacteria from intestinal contents of Arabesque greenling (Pleurogrammus azonus), one of the isolates showed strong proteolytic activity (Hoshino et al 1997) The isolate was identified to genus Pseudomonas and displayed highest protease production at 10 C, but the activity decreased with increasing cultivation temperature Morita et al (1998) detected protease activity in the culture medium of Flavobacterium balustinum isolated from salmon (Oncorhynchus keta) intestine The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5, and maximal Aquaculture Nutrition 18; 465492 ê 2012 Blackwell Publishing Ltd The activity of GPx was higher with the supplementation of Se Thyroid hormone levels were higher in the Se group as well, possibly due to lower oxidative stress in the thyroid follicles However, further work must be done to fully understand the eects of Se on TH production and also test its interaction with iodine We are indebted to the highly skilled technicians at CCMAR and NIFES This work was supported by the DIGFISH project, POCI/ CVT/58790/2004 (FCT, Portugal) Ribeiro A.R.A and Ribeiro L benet from grants SFRH/BD/24803/2005 and SFRH/BPD/7148/2001 (FCT, Portugal), respectively Abdel-Tawwab, M., Mousa, M.A.A & Abbass, F.E (2007) Growth performance and physiological response of African catsh, Clarias gariepinus (B.) fed organic selenium prior to the exposure to environmental copper toxicity Aquaculture, 272, 335345 Arthur, J.R., Nicol, F & Beckett, G.J (1993) Selenium deciency, thyroid hormone metabolism, and thyroid hormone deiodinases Am J Clin Nutr., 53, 236239 Beckett, G.J & Arthur, J.R (2005) Selenium and endocrine systems J Endocrinol., 184, 455465 Beckett, G.J., MacDougall, D.A., Nicol, F & Arthur, R (1989) Inhibition of type I and type II iodothyronine deiodinase activity in rat liver, kidney and brain produced by selenium deciency Biochem J., 259, 887892 Beckett, G.J., Nicol, F., Rae, P.W., Beech, S., Guo, Y & Arthur, J.R (1993) Eects of combined iodine and selenium deciency on thyroid hormone metabolism in rats Am J Clin Nutr., 57, 240S 243S Bell, J.G., Pirie, B.J., Adron, J.W & Cowey, C.B (1986) Some eects of selenium deciency on glutathione peroxidase (EC 1.11.1.9) activity and tissue pathology in rainbow trout (Salmo gairdneri) Br J Nutr., 55, 305311 Canavate, J.P & Fernandez-Diaz, C (1999) Inuence of co-feeding larvae with live and inert diets on weaning the sole Solea senegalensis onto commercial dry feeds Aquaculture, 174, 255263 Conceicáao, L.E.C., Ribeiro, L., Engrola, S., Aragao, C., Morais, S., Lacuisse, M., Soares, F & Dinis, M.T (2007) Nutritional physiology during development of Senegalese sole (Solea senegalensis) Aquaculture, 268, 6481 Cotter, P.A., Craig, S.R & McLean, E (2008) Hyperaccumulation of selenium in hybrid striped bass: a functional food for aquaculture? 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172179 Wang, Y., Han, J., Li, W & Xu, Z (2007) Eect of dierent selenium source on growth performances, glutathione peroxidase activities, muscle composition and selenium concentration of allogynogenetic crucian carp (Carassius auratus gibelio) Anim Feed Sci Technol., 134, 243251 Yufera, M., Parra, G., Santiago, R & Carrascosa, M (1999) Growth, carbon, nitrogen and caloric content of Solea senegalensis (Pisces: Soleidae) from egg fertilization to metamorphosis Mar Biol., 134, 4349 Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00933.x 2012 18; 568580 1 1 Department of Biochemistry and Molecular Biology I, Faculty of Sciences, University of Granada, Granada, Spain; Biochemistry and Molecular Biology Section, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaen, Jaen, Spain; Department of Animal Biology, Faculty of Sciences, University of Granada, Granada, Spain white-muscle protein-turnover rates and tissue hyperplasia in gilthead sea bream Maslinic acid (MA) is a pentacyclic triterpene used, at experimental level, as an additive to stimulate growth, protein-turnover rates and hyperplasia in fish In this experiment we have studied the effects of feeding with MA at daily fixed ration of 10 g kg1 of body mass, on growth, protein-turnover rates and nucleic acid concentration in white muscle of gilthead sea bream grown in a local fish farm The experimental groups contained (control), 50 (MA50) and 100 (MA100) mg MA per kg of diet Two groups were provided feed ad libitum (control AL and MA100AL), while three were given a fixed ration (control R, MA50R, and MA100R) We have determined the following: growth rate; nucleic acid and protein levels; relative and absolute protein-accumulation (KG, AG), synthesis (KS, AS) and degradation (KD, AD) rate, protein-synthesis capacity (CS), protein-synthesis efficiency (KRNA), proteinsynthesis rate per DNA (KDNA) and protein-retention efficiency At the end of the experiment, higher body weights and muscle growth rates were found in both groups of fish fed with 100 mg MA per kg of diet Feed-efficiency rate, protein-efficiency ratio and protein productive value, were higher in the MA100AL group than in control AL Fractional and absolute protein-synthesis and degradation rates in white muscle of MA100AL and MA100R fish were higher than in the control, resulting in a higher protein-accumulation rate and tissue growth Total DNA content, indicated cell hyperplasia, was higher in MA100AL and MA100R than its respective controls Studies of optical and electronic microscopy corroborated these results These findings indicate that MA added to the diet can stimulate the growth, KEY WORDS: growth, hyperplasia, maslinic acid, proteinturnover rates, white muscle Received July 2011; accepted 14 December 2011 Correspondence: Jose A Lupianez, Department of Biochemistry and Molecular Biology I, Faculty of Sciences, University of Granada, Granada, Spain E-mail: jlcara@ugr.es Triterpenic acids are a large family of natural products biosynthetically derived from squalene and widely found in nature They have been used for centuries in folk medicine as anti-inflammatory, hepatoprotective, gastroprotective, anti-ulcer, anti-diabetic, hypolipidemic, anti-atherosclerotic, immunoregulatory (Dzubak et al 2006) and hepatoprotective agents (Liu et al 1995) Recently, they have attracted scientific interest because of their anti-carcinogenic properties (He & Liu 2007; Struch et al 2008), which make them attractive for use in cosmetics and healthcare products as functional compounds Maslinic acid (MA) is one type of these triterpenes widely distributed throughout the plant kingdom, being especially abundant in olive fruits and leaves (Vazquez & Janer 1969) MA is concentrated on the surface of olive leaves to form a physical barrier that prevents microbes from penetrating the leaf (Kubo et al 1985) MA is also present in high concentrations in the epicarp of the fruit as part of the waxes that cover it (Romero et al 2010) ê 2012 Blackwell Publishing Ltd MA is isolated solid olive waste during the olive oil production Maslinic acid has been attributed with antioxidant (Tsai & Yin 2008), anti-hyperglycaemic (Liu et al 2007; Sato et al 2007), anti-inflammatory (Aladedunye et al 2008) and anti-cancer activities (Reyes-Zurita et al 2009) Moreover, it inhibits the spread of the HIV virus (Garc a-Granados 1998b) and influences certain enzymatic activities For example, it has been described as an inhibitor of glycogen phosphorylase (Wen et al 2005, 2006), elastase (Sultana & Lee 2007), acyl cholesterol transferase (Kim et al 2005), DNA topoisomerase and RNA polymerase in muscle of rabbit (Pungitore et al 2007) Furthermore, MA has been studied as inhibitor of serin proteases in microorganisms of genera Criptosporidium (Garc a-Granados 1998a) and human immunodeficiency virus, HIV (Garc a-Granados 1998b; Vlietinck et al 1998) This property was an approach to study the growth in certain animals, so animals were healthier and had an optimum development and growth For this, MA has been used as a food additive in some fish In rainbow trout (Onchorhynchus mykiss), the amount of MA used was 0, 1, 5, 25 and 250 mg kg1 of diet, and the result indicated that the greater maslinic amount, the higher growth rate in whole animal, liver and white muscle, increasing the protein-accumulation rate mainly as a result of the regulation of protein synthesis rates (Fernandez-Navarro et al 2006, 2008) On the other hand, in Dentex dentex, 0, 20, 40 and 80 mg kg1 of MA added to the diet had no effect on whole-body specific growth rate (Hidalgo et al 2006) Results found by our research group in the gilthead sea bream (Sparus aurata) fed 0, 50 and 100 mg kg1 of MA in the diet were increased liver and whole-body growth as well as higher protein-synthesis rates in these tissues (Rufino-Palomares et al 2011) The aim of the present study was to clarify whether MA addition to the diet is related to protein accretion and growth rate in the fast-growing S aurata The present work investigates whether white-muscle growth can be regulated and with it the whole-body growth rate This experiment was performed in white muscle of gilthead sea bream for being the most abundant tissue in this fish, and its growth rate is very similar to that of the whole body (Weatherley & Gill 1989) Growth has a close relationship with protein accretion (Carter & Houlihan 2001) and is the result of the positive balance between protein synthesis and degradation rates Hence, there is a need to combine growth studies with protein turnover On the other hand, most research has been performed with fishes provided feed ad libitum (De la Higuera et al 1998) Some authors rec- Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd ommend using low-fat diets when the animals are fed to satiety or close to it (Vergara et al 1996) or, alternatively, reducing the amount of feed to below satiety levels (Company et al 1999) Similar studies have been made in cows, chickens and pigs (Daza et al 2006; Heyer & Lebret 2007) In this sense, moreover, our experimental conditions involve ad libitum diet effects and diet daily rations at 10 g kg1 by animal weight Currently, restricted food application in farmed fish is being studied to elucidate the selection criteria, such as fat accumulation, compensatory growth (sparing effect; Summers et al 1990), food efficiency (Robinson et al 1992), reduction in locomotor complications and the control of metabolic diseases (Arce & Keen 1992) 6-3H] Phenylalanine (37 MBq mL1) was supplied by Amershan Biosciences, Buckinghamshire, UK L-Phenylalanine, L-tyrosine decarboxylase, b-phenylethylamine leucylalanine, pyridoxal phosphate and Hoăechst 33258 came from Sigma, St (Louis, MO, USA) All other chemical compounds were bought from Fluka (Buchs SG, Switzerland) and were of analytical grade MA was provided by Biomaslinic S.L., University of Granada (Granada, Spain) L-[2, The experiments were conducted at a local fish farm The gilthead sea bream coming from industrial production tanks were transferred to 15 experimental polyester tanks of m3 The mean initial weight of the fish was 12 g The experiments were always made in triplicate, and the experimental time was of 210 days Continuous open-circuit water maintained a flow of L min1 The water came from filtered seawater with 36 g L1 salinity The light photoperiod was natural, whereas the water temperature ranged between 15 and 21 C The mean oxygen concentration was 6.9 mg L1 Three different experimental diets were used in the experiment (Table 1) The amount of MA was mg (control diet), 50 mg (MA50 diet) and 100 mg (MA100 diet) per kg of diet Crude protein, total lipids, ash and moisture were analysed using an Association Official Analytical Chemist Table Composition and biochemical analysis of the experimental diets Control MA50 MA100 Ingredients (g kg ) Fish meal 570 Soy meal 130 Wheat meal 205 Fish oil 85 Vitamin premixture Mineral premixe Maslinic acid Analysis of diets (g kg1 dry matter) Moisture 92.4 Protein 498.4 Lipid 139.6 Ash 110.9 Nitrogen-free extract 251.1 Maslinic acid (mgkg1diet) Energy content Gross energy (kJg1) 19.6 570 130 205 85 5 0.05 570 130 205 85 5 0.1 91.0 502.8 146.7 111.9 238.6 48.1 1.1 91.7 515.1 148.4 109.5 227.0 97.3 2.0 19.7 19.8 Control, MA50 and MA100 correspond to the diets without maslinic acid and with 50 and 100 mg kg1 diet, respectively The vitamin supplement contained the following (g kg1): thiamine, 2; riboflavin, 3; pyridoxine, 1.5; calcium pantothenate, 7.5; nicotinic acid, 12.5; folic acid, 0.75; inositol, 50; choline, 250; biotin, 0.15; cyanocobalamin, 0.33; ascorbic sulphate, 50; vitamin A, 0.0075; vitamin D, 3.75; vitamin E, 12.5; vitamin K, 1.25; and sucrose to 1000 g of mixture The mineral supplement contained the following (g kg1 mixture): calcium phosphate monobasic, 600; calcium carbonate, 175; potassium chloride, 50; sodium chloride, 80; magnesium sulphate, 4; ferric sulphate, 30; magnesium chloride, 92; potassium iodide, 0.4; copper sulphate, 1; zinc sulphate, 4; cobalt sulphate, 1; sodium selenite, 0.0435; and sucrose aluminium sulphate to 1000 g of mixture MA content was expressed as mean SEM of 12 samples It was assumed that the energy value of protein, fat and carbohydrates were 19.6, 39.5 and 17.2 kJ g1, respectively The following nutritional indexes were determined: feedefficiency rate, (FER), protein-efficiency ratio (PER), protein productive value (PPV) and thermal growth coefficient (TGC), calculated as [(W1)1/3 (W0)1/3] (days Temp.C)1, where W is the weight of the sampled fish in grams, and W0 and W1 are the initial and the final fish mean weights in grams At the beginning of the experiment, all the fish were weighed individually and separated into different tanks, 150 fish per tank In addition, eight fish (different from those used for the experiment) were initially sampled, after which their muscles were weighed and the muscles protein content was measured The protein concentration was determined following the methods of Lowry et al (1951) and Bradford (1976); we were using two methods to corroborate the protein content The initial mean protein values were used as reference to determine the whole-body and white-muscle growth rate (GR) as well as the whitemuscle protein-accumulation rate (KG) throughout the experiment Nine fish in each treatment were used for this measurement White-muscle KG was calculated as a percentage increase in muscle protein per day, using the following equation: KG % day1 ị ẳ 100LnP2 LnP1 ị=t2 t1 ị method, (AOAC 2000) MA was analysed using the method described elsewhere (Romero et al 2010) Fish were distributed into five experimental groups as follows: two groups were fed to satiety (ad libitum, AL, control and MA100), and three groups were fed with a ration equivalent to 10 g kg1 of fish weight (fixed ration, R, control, MA50 and MA100) We include two experimental groups in the situation of daily fixed ration (MA50R and MA100R) to know how the compound could respond in this nutritional condition The fish were fed by hand twice daily, days a week, and the food ration was prepared and checked daily also Food intake was recorded daily, and the overall weight increase in the fish was determined by weighing the individual at the beginning and the end of the experimental period The relative daily intake was calculated by dividing the absolute daily diet intake by the mean body weight plotted where P1 and P2 represent the total tissue-protein content at times t1 and t2, respectively The fractional white-muscle protein-synthesis rate (KS) was determined as described by Fernandez-Navarro et al (2008) at 195 days after the beginning of the experiment, in which the fish were fed and 100 mg MA per kg diets Both in ad libitum (AL) and in restriction food (R), MA100 was the MA concentration at which the greatest effects on growth were detected and thus in these situations, proteinturnover rates and nucleic acid concentrations were determined The rest of the experimental conditions were similar to use by Rufino-Palomares et al (2011) The white-muscles protein increase was calculated in each fish by subtracting the average initial values (of a reference group) from the final protein content Muscle samples were extracted in the cold and then freeze-clamped in liquid nitrogen Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd All protein-turnover parameters (KS, AS, KD, KG, AD, AG and PRE) were calculated as Rufino-Palomares et al (2011) 812 (Burke & Geiselman 1971) Ultrathin sections of 700 A thickness, cut with a Reichert-Jung ultramicrotome, were contrasted with uranyl acetate and lead citrate (Reynolds 1963) The RNA concentration was determined by the method described by Munro & Fleck (1966) and modified by Fernandez-Navarro et al (2008).The DNA concentration was determined fluorometrically using the method described by Labarca & Paigen (1980) The method is based on the enhancement of fluorescence seen when bisbenzimidazole (Hoăechst 33258) binds to DNA The RNA and DNA concentrations were expressed as milligram per grams of tissue The protein/DNA and RNA/DNA ratios were calculated together with the protein, RNA, and DNA contents and expressed per 100 g of white muscle Because values of the protein-synthesis rate are too largely proportional to RNA concentrations, protein-synthesis capacity (CS) can also be defined as RNA/protein ratio and expressed as mg g1 Protein-synthesis efficiency (KRNA) was defined as the amount (g) of protein synthesized per day, and the RNA unit (g) was calculated as [(KS/CS) 10] The protein-synthesis rate/DNA unit (KDNA) was defined as the amount (g) of protein synthesized per day and DNA unit and was calculated as [(KS/100) protein/DNA] (Sugden & Fuller 1991) Data are shown as mean the standard error of the mean (SEM) The effects of the experimental diets on the different parameters were examined using the two-way analysis of variance (ANOVA) The statistical significance of differential findings between experimental groups and controls was determined by Students t-test using SPSS (IBM Corporation, Somer, NY, USA) version 15.0 for Windows software package P values smaller than 0.05 were considered statistically significant After 195 days of experimental time, muscles from four fish per experimental group were dissected out, cut into 1- to 2mm blocks and fixed for 48 h in Bouins fluid) Afterwards, the tissue samples were embedded in paraffin and cut into serial sections mm thick These sections were stained with Harris haematoxylin and 1% aqueous eosin solution, by Alcian Blue, pH 2.5 (AB), by periodic acid-Schiff (PAS; Lillie 1954) and by its combination (PAS + AB) These stained samples were used to determine the nature of the filling material of the lumen of the organelles For the TEM ultrastructural study, white-muscle sections were fixed with a solution of g L1 glutaraldehyde in 0.1 mol L1 cacodylate buffer, pH 7.4 (Watson 1958) Next, a new section was made and was again fixed in a solution of 1.5 g L1 osmium tetraoxide in cacodylate buffer Then, it was dehydrated with acetone and finally embedded in Epon Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd Whole-body weight, final white-muscle weight, relative daily intake and feed-conversion parameters were monitored for 210 days in five groups of gilthead sea bream fed with 0, 50 and 100 mg of MA per kg of diet The results are shown in Table The fish fed with MA increased significantly in body weight with respect to control Moreover, a significant increase in total white-muscle weight was found in fish fed with the MA100 diet Fish fed MA100AL increased some 27% compared to control AL and 16% in the MA100R group with respect to its control R (Table 2) These same groups showed a greater white-muscle growth rate (GR) The GR values for the MA100AL group increased 15% compared to control, and the MA100R group raised a 10% respect to control R Differences were found in the miosomatic relationship (RMS) among the groups fed with fixed diet and ad libitum, the difference being 13% higher in the AL Thermal growth coefficient values were higher in MA100AL than Control AL, indicated that MA better growth in these fishes The rest of the nutritional indexes values are shown in Rufino-Palomares et al (2011) In gilthead sea bream, the whole-body composition fed with different experimental diets are show in Table At the final time, protein values expressed in g kg-1 dry matter Table Whole-body, white muscle growth, food intake and main nutritional indexes of the different experimental diets (n) (450) (450) (450) (n) (144) (156) (138) (n) (297) (303) (294) (296) (303) (n) (8) (8) (8) (8) (8) (7) (n) (n) (7) (7) (450) (143) (450) (157) (7) (7) (7) (7) (7) (7) (7) (7) (7) (7) (n) (7) (n) (297) (303) (294) (n) (297) (303) (294) (n) (297) (303) (294) (303) (294) (296) (294) (296) (303) (296) (303) (n) (297) (n) (297) (n) (297) (7) (303) (303) (294) (296) (296) (296) (303) (303) (303) (303) MA50, MA100, group of fish fed with 50, 100 mg MA kg diet, respectively Results are means SEM of n data The results were analysed by a two-way ANOVA followed by Students t-test Probabilities of P < 0.05 or less were considered statistically significant For each parameter, for comparison between different feeding ration (AL versus R), the data followed by different superscript (a, b) are statistically different For comparison between different feeding diets (Control versus MA100), the data followed by different subscript (x, y) are statistically different GR, whole-body growth rate; FER, feed-efficiency ratio, wet weight gain (g) dry diet intake (g)1; PER, proteinefficiency ratio, wet weight gain (g) dry protein intake (g)1; PRE, protein retention efficiency, retained protein (g) in whole fish of consumed protein (g) and its calculated by 100 grams of fish; TGC, thermal-unit growth coefficient growth rate of the MA100AL group were approximately 10% higher than the rest experimental groups The body-lipid content at half the experimental time (105 days) decreased significantly in the groups fed with MA, whereas this decrease was not found at the end of the experiment The fat data of the control AL was 30% higher than in the MA100AL group while the control R value was 13.5% and 22.4% higher than for MA50R and MA100R, respectively The values ash showed no significant changes at any time during the experiment between the different groups The moisture data did not differ between the fishes fed MA, but registered significant differences at the end of the experiment, with an overall decline in all treatments after 210 days The concentration of total protein and total RNA in the white muscle of gilthead sea bream of the different experimental groups are shown in Fig The total-protein values showed significant changes only between the groups fed ad libitum This parameter increased 27% in the group MA100AL compared with control AL group MA added to the diet boosted the total RNA content (mg), this rise being a 74% in fish fed the MA100AL diet and a 56% in the MA100R group compared with its respective control groups Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd Table Whole-body composition of gilthead sea bream fed with maslinic acid Ad Libitum feeding (AL) Time (days) Protein (g kg1 dm) Fat (g kg1 dm) Ash (g kg1dm) Moisture (g kg1) 105 210 105 210 105 210 105 210 Fixed ration (R) Control MA100 Control MA50 MA100 577 ặ 18ax 552 ặ 32ax 573 ặ 23ad x 309 ặ 23ax 318 ặ 28ax 315 ặ 13ax 112 ặ 3ax 128 ặ 2ax 127 ặ 2ax 686 ặ 8ax 647 ặ 41ax 637 ặ 4ay 577 ặ 18ax 627 ặ 17axy 637 ặ 7by 309 ặ 23ax 244 ặ 7by 273 ặ 9az 112 ặ 3ax 118 ặ 13ax 120 ặ 4ax 686 ặ 8ax 704 ặ 23ax 641 ặ 2ay 577 ặ 18ax 528 ặ 6ay 557 ặ 5ax 309 ặ 23ax 337 ặ 9ax 312 ặ 33ax 112 ặ 3ax 123 ặ 2ax 117 ặ 1ax 686 ặ 8ax 641 ặ 16ay 617 ặ 17ay 577 ặ 18ax 590 ặ 14ax 596 ặ 7cx 309 ặ 23ax 297 ặ 4cx 284 ặ 12ax 112 ặ 3ax 126 ặ 3ax 116 ặ 4ax 686 ặ 8ax 635 ặ 20ay 656 ặ 3ay 577 ặ 18ax 528 ặ 10ax 571 ặ 10cd x 309 ặ 23ax 276 ặ 10cx 277 ặ 12ax 112 ặ 3ax 117 ặ 4ax 116 ặ 3ax 686 ặ 8ax 696 ặ 24ax 645 ặ 1ay The results in g kg-1 dry matter (dm) are expressed as the mean SEM of eight data The experimental times in all cases were initial, 105 and 210 days Different superscripts in same line indicate significant differences between diets, and different superscripts in same column indicate significant differences over the experimental time Data were treated with two-way ANOVA followed by Students t-test P < 0.05 was considered to be statistically different White-muscle growth is plotted in Fig After 195 days of experiment, the total DNA content, which indicates the number of cells and thus cell hyperplasia, increased by 50% and 33% for gilthead sea bream fed with MA100AL and MA100R, respectively, compared to its control groups The protein/DNA ratio, indicating that cell size and thus hypertrophy, declined in the groups of animal fed with MA added to the diet, under both conditions, AL and R, although no significant changes appeared between experimental groups The result of both parameters indicated that MA in white-muscle stimulated DNA synthesis and therefore the formation of new cells The effects of the different experimental conditions on protein-turnover rates in the white muscle of gilthead sea bream are shown in Table MA increased KS values in the group MA100AL at the end of experiment The rise was 40% with its respective control group KD values showed the same behaviour for KS, being MA100AL values 32% higher than control AL values Moreover, the value of control AL was the lowest of all the groups The food restriction did not show changes between groups On the other hand, KG values were higher in the MA100AL group with respect its control, the increase being 39%, while these values in restriction groups did not significantly change The other parameters related with protein turnover, such as, CS, KRNA, KDNA and protein-retention efficiency Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd (PRE), are shown in Table CS values were significantly higher by the addition of MA to diet The increase was 37% for the MA100AL and 46.5% for MA100R compared with their respective controls MA100AL KDNA values were 35% higher than control AL The rest of parameters did not significantly change between groups, either in the case of fish fed the AL diet or the fish fed the fixed diet (R) In addition, absolute protein-turnover (AS, AD, AG) rates were also determined in the white muscle of these fish These parameters are shown in Fig The value of AS for the MA100AL group was a 55.2% higher than control AL In animals fed the fixed diet, no changes were found The values of AD showed the same trend as the AS values, observing a higher protein-degradation rate for the group fed MA100AL with its respective control AL Furthermore, this same behaviour was observed in values of AG The fish fed with MA100AL diet had values higher than did control AL, with a rise of 33.8% compared with control AL The importance of muscle tissue as reservoir proteins and its involvement in the processes of growth led us to study the structure of this tissue The white-muscle structure of gilthead sea bream from different experimental groups was analysed using both optical and TEM The results are shown in Fig In a and b panel of Fig presents a white-muscle cross-section under optical microscopy for two different treatments: one control AL (panel a) and another with addition of MA (MA100AL, panel b) No significant 0.6 140 120 100 1 0.4 80 60 0.2 40 20 0.0 500 5.0 450 400 4.0 350 300 3.0 250 200 2.0 150 100 50 1.0 Control AL 0.0 100 100 Figure Maslinic acid (MA) and fixed ration effect on white-muscle protein and RNA levels Data are presented as mg protein and mg RNA per 100 g of muscle in gilthead sea bream fed different doses of MA and ration The results are expressed as the means SEM of eight observations Data were treated with a two-way ANOVA followed by a Students t-test Probabilities of P < 0.05 or less were considered statistically significant Letters a, b above the bars indicate statistical differences between different feeding ration (ad libitum versus fixed ration), and letters x, y above the bars indicate statistical differences between treatments (control versus MA100) changes were observed between the two, so that MA showed no signs of tissue damage Muscle structure in these images corresponds to a typical structure of this tissue in fish The muscle cells or myocytes were different sizes and highly vascular The nucleus cell of them was well stained and myofibrils appear in their interiors These filaments, arranged in alternate and characteristic form, appeared in striated muscle under a microscopic observation The connective tissue surrounding individual cells or endomysium was apparent, composed of basal lamina and MA 100 AL Control R MA 100 R Figure Maslinic acid (MA) and fixed ration effect on whitemuscle DNA level and protein/RNA ratio Data are presented as mg DNA per 100 g of muscle and protein/RNA rate in gilthead sea bream fed with different dosages of MA and ration The results are expressed as the mean SEM of eight observations The data were treated with a two-way ANOVA followed by a Students t-test Probabilities of P < 0.05 or less were considered statistically significant Letters a, b above the bars indicate statistical differences between different feeding ration (ad libitum versus fixed ration), and letters x, y above the bars indicate statistical differences between treatments (control versus MA100) reticular fibres Moreover, a blood vessel was visible with an interior surrounded by endothelium, as well as flattened nuclei of these endothelial cells Sarcoplasm was distinguished for a large number of myofibrils and the nucleus Furthermore, we studied the muscle ultrastructure for TEM under control AL and MA100AL conditions The results are shown in panels c, d, e and f of Fig In these appeared the typical structure of the anatomic, functional and repetitive units or sarcomeres that compound the muscle In both experimental situations, the sarcomere was well Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd Table White-muscle changes in protein-turnover rates and protein-retention efficiency in Sparus aurata Ad Libitum feeding (AL) KS KD KG CS KRNA KDNA PRE Fixed ration (R) Control MA100 Control MA100 1:32 ặ 0:10ax 0:31 ặ 0:04ax 1:01 ặ 0:12ax 27:94 ặ 3:11ax 0:47 ặ 0:06ax 4:28 ặ 0:36ax 76:52 ặ 6:30ax 1:85 ặ 0:21ay 0:45 ặ 0:04ay 1:40 ặ 0:12ay 38:32 ặ 2:62ay 0:48 ặ 0:04ax 5:80 ặ 0:34ay 76:68 ặ 5:91ax 1:81 ặ 0:14bx 0:63 ặ 0:06bx 1:18 ặ 0:14bx 30:98 ặ 1:55ax 0:58 ặ 0:04ax 5:61 ặ 0:25bx 65:19 ặ 7:49bx 1:95 ặ 0:12ax 0:73 ặ 0:09bx 1:22 ặ 0:21bx 45:41 ặ 4:41ay 0:43 ặ 0:03ay 5:18 ặ 0:35ax 62:56 ặ 6:83bx MA100, group of fish fed with 100 mg MA kg1 diet Results are means SEM of seven data The results were analyzed by a twoway ANOVA followed by Students t-test Probabilities of P < 0.05 or less were considered statistically significant For each parameter, for comparison between different feeding ration (AL versus R), the data in each row followed by different superscript (a, b) are statistically different For comparison between different feeding diets (Control versus MA100), the data in each column followed by different subscript (x, y) are statistically different KG, protein accumulation rate (% day1) KS, protein synthesis rate (%ãday1), KD, protein-degradation rate (% day1) CS, proteinsynthesis capacity (mg RNAãg1 protein), KDNA, protein synthesis rate/DNA unit (KS/100)ã(protein/DNA), g protein synthesizedãday1ãg DNA1), KRNA, protein-synthesis efficiency ((KS/CS) 10), g protein synthesizedãday1 PRE, protein-retention efficiency (KG/KS) 100, % synthesized protein retained as growth defined, all bands and lines composing it being visible A sarcomere unit was composed of two lines, A and I, where Z was zone A (anisotropic) and zone I (isotropic) In the I band, actin filaments were seen, and in the A band, myosin filaments responsible for muscle contraction were visible Moreover, intracellular triads of three components were seen: the sarcolemma invagination, called the T tubule between two cisterns of sarcoplasmic reticulum The TEM resolution, higher OM, revealed glycogen granules around all the sarcoplasm In the present work, we study how MA as a natural compound affects growth and protein-turnover rates of white muscle in S aurata, a saltwater species Moreover, in this study, ad libitum feeding (AL) and a restricted daily ration (10 g kg1 of body weight) were used to investigate the effect of feeding dosage as well as its combination with MA Gilthead sea bream were fed for 210 days with the different experimental diets (control AL, MA100AL, control R, MA50R, and MA100R) This study was conducted at a local fish farm; where all the fish were subjected to the same environmental farming conditions Elsewhere, the Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd effects of food ration, feeding frequency on fish growth, feed-conversion efficiency, and different nutrients in the diet have been studied for several fish species, including (Jarboe & Grant 1997), rainbow trout (Grayton & Beamish 1977), yellowtail flounder (Whalen et al 1998), flounder (Lee et al 2000), Atlantic and short-nose sturgeon (Giberson & Litvak 2003), and gilthead sea bream (SanchezMuros et al 2003) The results of these studies vary widely because of differences in feeding frequencies applied in different fish species, and within a fish species over time, at different temperatures, and with water qualities, nutrients qualities/quantities, culture systems, fish sizes and feed rations In the present study, the experimental results show that dietary MA concentrations were well accepted and ingested by the fish MA caused an increase in whole-body and white-muscle growth Similar results were found in trout fed with MA in experimental tanks (Fernandez-Navarro et al 2006, 2008) In the present work, MA did not change the daily relative intake, although it did modify the intake, water temperature and restriction feeding Velazquez et al 2006b observed that when gilthead sea bream of 130 g to size, fed by hand or in a automatic/free or modulated way, intake values were 1.2 (hand) and 1.1 (automatic) at 25 C of temperature and 12 : 12; L : D of photoperiod When winter (17 C, 9L : 15D) and summer (26 C, 12L : 12D) conditions are simulated, the average voluntary intake was 0.6 and 1.4 g per 100 g fish per day, respectively Temperature affects all fish species in the sense that when temperatures rise within a normal range, feed intake and growth increase (Jobling 1988; Velazquez et al 2006a) independently of diet composition (Sierra 1995) The TGC expresses growth rates for changing temperatures and is used in growth models of intensive aquaculture (Querellou 1984) The group fed with MA100 and AL diet presented the major value of this parameter, indicating that MA is beneficial for the animal growth Our results demonstrate that the nutritional indexes calculated, such as FER, PER and PPV, were higher in animals fed on diets containing MA and fed AL These differences were also observed in the restricted groups for FER and PPV (Rufino-Palomares et al 2011) MA seems to compensate for any shortcomings of food intake with respect to ad libitum groups These results are consistent with those found in rainbow trout (Fernandez-Navarro et al 2008), but in our case the differences appeared at a lower MA dosage (100 versus 250 mg MA per kg of diet) The values of nutritional indexes indicated that protein was better used for growth and that diet utilization for growth 250 200 150 S 100 50 80 70 60 50 D 40 30 20 10 160 140 120 100 G 80 60 40 20 100 100 Figure White-muscle absolute protein-turnover rates (AS, protein synthesis; AD, protein degradation; and AG, protein-accumulation rate) in gilthead sea bream fed different doses of MA and ration The results are expressed as the mean SEM of seven observations Data were treated with a two-way ANOVA followed by a Students t-test Probabilities of P < 0.05 or less were considered statistically significant Letters a, b above the bars indicate statistical differences between different feeding ration (ad libitum versus fixed ration), and letters x, y above the bars indicate statistical differences between treatments (control versus MA100) MA, maslinic acid improved when MA was added to the diet regardless of the feeding regimen (AL or R) A possible mechanism of MA to improve growth will be discussed below in relation with muscle protein turnover The whole-body composition was notably higher in protein content in animals fed with MA, with only a slight decrease in lipid content The average protein and lipid values were within the normal range for individuals of the same age/weight (Grayton & Beamish 1977; Riche et al 2004) With age, gilthead sea bream increased in fat and protein content from 160 g kg-1 of protein and 70 g kg-1 of lipid content in animals of 10 g body weight (Marcouli et al 2006) to 185200 g kg-1 protein and 90160 g kg-1 fat content in animals weighing 200450 g (Grigorakis & Alexis 2005; Mart nez-Llorens et al 2009) Other studies have reported that the feeding system does no effect fishbody composition (Azzaydi et al 1998; Valente et al 2006) Growth is considered a consequence of protein accretion (Carter & Houlihan 2001), and muscle is the most representative tissue of growth, and therefore we studied the nature of white-muscle growth and protein-turnover rates The results presented here show that MA intake increased white-muscle weight of gilthead sea bream with ad libitum feeding and with fixed rations Furthermore, total protein, DNA and RNA content increased The nature of liver growth can be interpreted in terms of increases in cell number or size (Peragon et al 1998), but in white-muscle tissue, where the possibility of polynucleate cells arises, changes in total DNA and the protein/DNA ratio cannot be so easily explained in the same terms (Peragon et al 2001) Nevertheless, when the DNA unit is considered to be the cytoplasmic volume controlled by a single nucleus, the terms hypertrophy and hyperplasia can be used to describe changes in the size and number of muscle DNA units (Waterlow et al 1978) The parameters with regard to growth nature have been determined, and the results indicate a greater total DNA content (cell number) in the gilthead sea bream fed MA without changes in the protein/ DNA ratio (cell size) Previously, studies in trout demonstrated the same (Fernandez-Navarro et al 2008) In short, under our experimental conditions, MA stimulates whitemuscle growth of these fishes by inducing those processes of generation of new cells or muscle fibres Weatherley & Gill (1984) studying trout white muscle observed that somaticgrowth differences are intimately related to hypertrophy processes and new fibre generation, so that for every fish size, there is an exact distribution of muscle-fibre diameters Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd (a) (c) (b) (d) (e) (f) Figure Effect of maslinic acid (MA) and fixed food on white-muscle structure in gilthead sea bream fed control AL and MA100AL diets In a (control AL) and b (MaA100AL) panels show images to OM of white-muscle cross-section indicating different myocyte size M, myofibrils; N, nucleus; E, endomysium In c and d (control AL) and in e and f (MA100AL) panels shown ultrastructure of muscle to transmission electron microscopy Rs, sarcoplasmatic reticule; T, T tubules; M, myofibrils; A and I bands, Z lines and the zone indicated for the arrow: glycogen granules Muscle-cell hypertrophy processes predominate during phases of rapid animal growth, specifically during the first developmental phases, although in large fish the generation of new muscle fibres is most important (Weatherley 1990; Kiessling et al 1991; Peragon et al 2001) The effects of MA in gilthead sea bream match the second growth pattern usually observed in larger fish In fish, white muscle is the largest tissue and the main protein-accumulation site Amino acids derived from it are used to synthesize around 30% of the total body protein (Houlihan et al 1988) Although its fractional protein rate is the lowest of all the tissues, it is the one that most efficiently accumulates the newly synthesized protein Studies in codfish (Gadus morhua) have described the relation between protein-synthesis rate (KS) and growth rate when these fish are fed different diet rations (Houlihan et al 1988) Additionally, other studies (Weatherley & Gill 1989; Peragon et al 1998) demonstrate that white-muscle growth rate is very similar to the full-body rate, and the body-mass time course determines the growth and final size of the fish White-muscle KS values described in gilthead sea bream fed AL with a control diet was 1.32% day1 These agree with findings reported elsewhere (Sierra 1995; Peragon et al 2001; Fernandez-Navarro et al 2008), while other related parameters, such as CS, KRNA, KDNA and PRE are Aquaculture Nutrition 18; 568580 ê 2012 Blackwell Publishing Ltd significantly higher than those found in rainbow trout fed with different MA quantities (Fernandez-Navarro et al 2008) Absolute protein-turnover rates follow behaviour similar to that described for the fractional protein-turnover rates Therefore, the fixed ration boosted the absolute protein-degradation rate Fish fed with MA100AL diet increased in relative and absolute synthesis and degradation protein-turnover rates as well as absolute accumulation protein-turnover rates Hence, as protein growth is the result of the difference between protein synthesis and degradation, it has been hypothesized that protein-degradation rates play a key role in regulating protein growth (Dolby et al 2004) The results here suggest that MA, in white muscle, encouraged protein accumulation or reserve, translating as major body growth Thus, understanding the role of MA in protein metabolism may be important for aquaculture, as they reveal the mechanisms that determine the efficiency by which feed is converted into growth (Hidalgo et al 2006) When nutrients and the energy supply are restricted by the ration effect, MA has less influence because the stimulation is used to compensate for the food restriction In rainbow trout, a study of the parameters related to protein turnover and results showed a higher protein-synthesis rate in the tissue without changes in protein degradation, this being a mechanism that results to a greater protein accumulation and weight gains of the organism (Fernandez-Navarro et al 2008) Therefore, we also studied white-muscle structure in gilthead sea bream by optical and TEM The results revealed irregular and varied myocyte morphology, high quantities of myofibrils No apparent differences were found between experimental groups, and only the myofibrils in MA100AL group were slightly more dilated than in the control AL group, due probably to the techniques of tissue fixation Flattened nuclei endothelial cells were clearly observed in all cases In addition, TEM showed considerable quantities of glycogen granules, mitochondria with a normal size and structure, as well as the sarcomere, the functional unit to the muscle cell, being perfectly structured The size and number of muscle cells in either treatment were not significantly different Thus, MA and/or food restriction caused no structural differences in the gilthead sea bream muscle with respect to the control, leading to the conclusion that there was no muscle damage attributable to these two experimental conditions Fernandez-Navarro et al (2006) reported that MA leads to a high accumulation of glycogen in rainbow trout and can also act as a new type of glycogen phosphorylase inhibitor (Wen et al 2005, 2006), the enzyme responsible for glycogen degradation in liver and whitemuscle tissues The anabolic effect of this molecule can contribute to glycogen accumulation in the tissue and a good general state of the fish (Fernandez-Navarro et al 2008) In conclusion, the results presented in this work indicate that MA can be used as a feed additive to stimulate whitemuscle growth during initial juvenile stages gilthead sea bream In these phases, the white muscle is involved in overall active metabolism, where the growth rate of the whole body and its nutritional requirements are stimulated and absolute protein-accumulation rates in with MA100AL groups indicate that proteins are accumulated in reservoirs, producing higher growth in these fish The results concerning the food-restriction group suggest that the increase in the protein-synthesis rate, without changes in 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