Aquaculture nutrition, tập 18, số 2, 2012

Aquaculture Nutrition 2012 18; 117131 doi: 10.1111/j.1365-2095.2011.00919.x 1 Faculty of Biosciences, Fisheries and Economics, Norwegian College of Fishery Science, University of Tromsứ, Tromsứ, Norway; Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China; Institute of Marine Research, Matre Research Station, Matredal, Norway; School of BioScience, Handong University, Pohang, South Korea Chitin consists of b-1,4-linked N-acetylglucosamine residues and is estimated as the second most abundant biomass in the world after cellulose However, relatively little chitin is utilized as a material for industrial, agricultural and medical applications and aquacultural purposes Chitin may be useful as a constitutive material in formulated fish feed, and the interesting effects in fish merit further evaluation There is evidence that fish and aquatic animals harbour a gut bacterial community that is distinctly different from that reported in the surrounding habitat or in the diet Thus, the gut environment provides a specific niche, and bacterial activity in the gut is not merely a continuum of that observed in the environment Today, it is well accepted that the gut microbiota in fish are modulated by dietary manipulations But to what extent can dietary chitin and krill (chitin-rich) modulate the intestinal microbiota of fish and how these dietary components affect the immune system? These questions will be discussed in the present review KEY WORDS: dietary chitin and krill, digestion, fish, growth, gut microbiota, immunology Received 18 March 2011; accepted December 2011 Correspondence: E Ringứ, Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsứ, Tromsứ, Norway E-mail: Einar.Ringo@uit.no Chitin was first investigated in 1811 by Professor M Henri Braconnot, who discovered it in the cell walls of mush- ể 2012 Blackwell Publishing Ltd rooms, but it was first named chitin by Odier (1823) Chitin (C8H13O5N)n consists of b-1,4-linked N-acetylglucosamine residues (Fig 1), is estimated as the second most abundant biomass in the world after cellulose and forms a major structural component of many organisms, including fungi, crustaceans, molluscs, coelenterates, protozoan and green algae (Rinaudo 2006; Khoushab & Yamabhai 2010) Chitin occurs in two major forms: (i) a-chitin, which has a very rigid crystalline structure because of intersheet and intrasheet hydrogen bonding, and (ii) b-chitin, which has a relatively weak intermolecular force because of intrasheet interaction (Jang et al 2004) The degree of acetylation also varies from 0% to 100% (chitosan) Fungi contain only a-chitin, and a-chitin is the most abundant form found in animals (Ruiz-Herrera 1992) The annual biosynthesis of chitin has been estimated at more than 100 billion tons (Li & Roseman 2004) The chitin content of various copepods that are food organisms for wild juvenile fish has been reported to range from 21 to 95 mg g1 (average of 46 mg g1) by dry weight (for review, see Bamstedt 1986) The chitin content of Artemia urminanca cyst shells range between 29.3% and 34.5% of the shells dry weight (Tajik et al 2008) Furthermore, chitin has been reported to make up 3.6% (wet weight) of the stomach contents of 50-day-old juvenile black sea bream (Acanthopagrus schlegeli) (Om et al 2003), but this value decreases with growth The ability to degrade chitin in vivo is thought to involve the action of the enzymes chitinase (EC 3.2.1.14) and chitobiase (EC 3.2.1.30) (Jeuniaux 1961) Chitinase degrades chitin to di- and trisaccharides and chitobiases (N-acetylglucosaminidases), which further degrades these to the sugar monomer beta-N-acetyl-D-glucosamine (Danulat Figure Chemical structure of chitin 1987) In Atlantic cod (Gadus morhua L.), strong chitinolytic activities have been measured in the stomach (Danulat & Kausch 1984; Lindsay & Gooday 1985; Danulat 1986) In addition, substantial chitinase activity has been reported in the pyloric caecae and the intestine of the cod (Danulat 1986) Chitinases in the guts of cod hydrolysed chitin both in vivo and in vitro (Danulat 1987), and only up to 9% of ingested chitin was recovered from intestinal contents and the faeces of the fish Based on this finding, the authors concluded that Atlantic cod is able to digest chitin to a large extent Feeding Atlantic salmon (Salmo salar L.) diets where the fishmeal was replaced with chitin-containing krill meals showed no influence of diet on the apparent digestibility coefficients (ADC) of dry matter and protein, while chitin was not utilized to any major extent (Olsen et al 2006) They also reported the ADC for chitin ranging from 13% to 40% and that the chitin digestibility tended to decrease with increasing krill inclusion (Olsen et al 2006) Moreover, oligomers of N-acetyl-D-glucosamine produced by chitinases in the digestive tract of fish might function as a bioactive substance (Tamai et al 2003), although beneficial effects on fish have not yet been defined Readers with special interest in chitinases in fish are referred to the comprehensive review of Krogdahl et al (2005) and Ray et al (2012) and the research paper of German & Bittong (2009) Although numerous investigations have been conducted to determine the effects of diet on the intestinal microbiota (for review, see Ringứ et al 2011), little information is available about the effect of chitin and krill (chitin-rich) on the gut microbiota (Sera & Kimata 1972; Kono et al 1987a; Kumar et al 2006; Ringứ et al 2006; Askarian et al 2012; Z Zhou, S He, R E Olsen, B Yao & E Ringứ, unpublished data) From a microbial point of view, one of the most important goals has been to obtain a stabile indigenous gut microbiota in fish The practical effect of this activity is the exclusion of invading populations of non-indigenous microorganisms, including pathogens that attempt to colonize the gastrointestinal (GI) tract (Ringứ et al 2005, 2010; Merrifield et al 2010) This fundamental topic arises as numerous papers published during the last 25 years have suggested that the alimentary tract is involved in Aeromonas and Vibrio infections (e.g Groff & LaPatra 2000; Birkbeck & Ringứ 2005; Harikrishnan & Balasundaram 2005; Jutfelt et al 2006; Ringứ et al 2007, 2010; Sugita et al 2008) Therefore, one can hypothesize that beneficial bacteria colonizing the digestive tract by producing, for example, bacterocins may offer protection against invading fish pathogens (Ringứ et al 2005; Desriac et al 2010) The effects of chitin on the fish immune system have been relatively well studied One of the major purposes of these studies were to investigate the possibility that chitin could be used as an immunostimulatory additive for fish feed and that chitin-fed fish might be protected against fish pathogenic bacteria In order to determine the optimized conditions of the chitin additive, multiple conditions have been investigated: the mode of chitin administration (Esteban et al 2001), the size of chitin additive (Cuesta et al 2003) and the dietary chitin supplementation To our knowledge, a broad range of studies have been conducted, including various fish species and shellfish Although some differences exist depending on the conditions of chitin treatment and fish species, generally dietary chitin activates the innate immune system of the fish and shellfish tested Information is also available that chitin-fed organisms were protected against pathogens in challenge experiments Readers with special interest in the applications of chitin are referred to the comprehensive review of Kumar (2000) The objectives of the present review were to evaluate the available information in aquatic animals with regard to the effect of chitin and krill on growth and feed utilization, bacterial capacity to degrade chitin, modulation of the gut microbiota and the effect on the innate immune system and disease resistance The results cited in the present review might be of importance for future aquaculture, and further directions will be discussed In order for fish to utilize chitin, fish must be able to digest and utilize it If not, chitin will become an energy sink lowering the potential energy availability of the fish Furthermore, undigested chitin may also affect nutrient utilization For example, chitin is known to absorb lipid and bile in Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd the GI tract, thus lowering lipid digestion and absorption (Tharanathan & Kittur 2003) High nutrient utilization is particularly important in intensive aquaculture practices with high growth rates as reported for Atlantic salmon and Atlantic cod Most fish examined to date seem to possess some form of chitin-degrading enzymes such as such as chitinases and/or chitobiases in their GI tract (Danulat & Kausch 1984; Lindsay 1984, 1985, 1987; Lindsay & Gooday 1985; Danulat 1986; Rehbein et al 1986; Kono et al 1987b, 1990; Sabapathy & Teo 1993; Moe & Place 1999; Gutowska et al 2004; Karasuda et al 2004; Fines & Holt 2010) The location of the enzymes seems to vary between studies and species, with some finding most activity in the stomach, while others have more in the intestine There are also some data indicating that fish feeding on chitin-rich prey have higher chitinase activity than other fish (Gutowska et al 2004) and that feeding chitin-rich diets will increase enzyme activity (Danulat 1986) However, current data are rather incomplete and to some extent contradictory However, possessing chitinase activity does not guarantee that there will be a complete digestion of chitin At present, such utilization can only be carried out in real-time experiments In cyprinids, inclusions of small amounts of chitin will not affect growth rates and feed utilization to any major extent In golden mahseer (Tor putitora), feeding up to 2% of diet as chitin has no effect on growth rate, indicating a not-well-developed system for utilization of chitin (Mohan et al 2009) In other cyprinids, such as snow trout Schizothorax richardsonii, 2% chitin will actually enhance growth most likely due to factors other than energy consumption (Mohan et al 2009) In common carp Cyprinus carpio, chitin has no effect on growth rate as such, but chitosan, the deacetylated form of chitin, will enhance growth and feed conversion when included at 12% levels (Victor et al 2004; Gopalakannan & Arul 2006) In hybrid tilapia (Oreochromis niloticus O aureus), on the other hand, growth and feed utilization is reduced in a dosedependent manner up to at least 10% inclusion (Shiau & Yu 1999) The same appears to be true for many other cichlids (Spataru 1978; Spataru & Zorn 1978; Buddington 1980), suggesting a generalized poor ability to utilize prey chitin In their study with juvenile cobia (Rachycentron canadum), Fines & Holt (2010) reported that chitinase activity was only detected in the stomach As antibiotic treatment did not reveal differences in chitinase activity, the authors suggested that the bacterial contribution from chitinolytic bacteria was not significant Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd Salmonids also seem to have a low ability to utilize chitin In practical experiments, a high rate of replacement of fish meal with krill meal containing chitin affects fish performance In Atlantic salmon, chitin is not digested to any significant extent, and complete replacement of fishmeal with krill meal will reduce growth and lipid utilization (Olsen et al 2006) Growth reductions have also been seen in chum salmon (Oncorhynchus keta) fingerlings (Murai et al 1980) and rainbow trout (Oncorhynchus mykiss) fed high levels of krill meal (Wojno & Dabrowska 1984) It has also been shown that rainbow trout are unable to digest chitin (Buddington 1980) However, 6% chitin supplementation has been reported to enhance growth in rainbow trout juveniles (Lellis & Barrows 2000) and may thus indicate that chitin under some circumstances may be digested and indirectly enhance fish performance There are few data on the performance of marine fish fed chitin But so far, most data point to good abilities with respect to dietary utilization At 10% inclusion level, growth is enhanced in red sea bream (Pagrus major) and to a lesser extent in Japanese eel (Anguilla japonica) and yellowtail (Seriola quinqueradiata) (Kono et al 1987c) Likewise, chitin is well digested in Atlantic cod (Danulat 1987), and growth studies have shown good growth rates with high inclusion levels of chitin-rich krill meal (Moren et al 2006) or up to 5% purified chitin (ỉ Karlsen, H Amlund, E Ringứ, A Berg & R E Olsen, unpublished data), indicating that chitin is well utilized under intensive conditions Chitin is completely hydrolysed to its constituent oligomers and monomer by the binary chitinase enzyme system: chitinase (EC 3.2.1.14) and b-N-acetylglucosaminidase (EC 3.2.1.30) It can also directly inhibit the growth of a wide range of fungi and bacteria (for review, see Tsai et al 2002) The observation that chitin does not accumulate in nature has prompted numerous researchers to investigate the prevalence of chitin-degrading bacteria and the rates of chitin degradation in the environment To our knowledge, the first study reporting chitin destruction by bacteria was conducted by Benecke (1905), who reported the isolation of Bacillus chitinovorus from the polluted waters of Kiel Harbour Three years later, Stoărmer (1908) reported that actinomycetes in soil were able to degrade chitin The bacterial capacity to degrade chitin is widespread among taxonomic groups of prokaryotes, including gliding bacteria, vibrios, Acinetobacter, Achromobacter, Agarbacterium, Chromobacterium, Cytophaga, Photobacterium, Plesiomonas, Pseudomonas, Flavobacterium, Marinobacter, Micrococcus, Sulfitobacter, Streptomyces, Listeria, Enterococcus faecalis, enteric bacteria, actinomycetes, bacilli, carnobacteria, clostridia, marine bacteria and archaea An overview of bacteria able to digest chitin is presented in Table The general method to evaluate chitinolytic bacteria is using caseinchitinagar with the following composition: 0.2% casein, pancreatic digest, 0.8% colloidal chitin and 1.5% technical-grade agar, made up with 75% aged filtered sea water and pH adjusted to 6.7 Hydrolysis of chitin can also be measured on ZA agar plates containing 0.5% chitin and phenolphthalein diphosphate sodium salt (0.01%) according to the method of Cowan (1974) or using 1/20 PYBG agar plates containing 0.2% colloidal chitin (Itoi et al 2006) In the study of Sugita & Ito (2006), the authors reported that 98.8% of the bacterial strains isolated from the GI tract of Japanese flounder were chitinolytic, and they were identified as Vibrio fischeri, Vibrio harveyi and Vibrio scophthalmiVibrio ichthyoenteri group The authors also analysed partial sequences of the chiA gene from V harveyi and V scophthalmiV ichthyoenteri isolates Sugita & Ito (2006) did not detect the chiA gene in chitinolytic isolates of V fischeri, but Ramaiah et al (2000) detected and sequenced the chiA gene in V fischeri Based on these results, one can suggest that V fischeri isolates produce various chitinases, and Sugita & Ito (2006) suggested that the PCR amplification technique for the chiA gene might be useful in detecting chitinolytic bacteria associated with fish GI tract Studies on fish-associated microorganism carried out in the 1970s, 1980s and 1990s used culture-dependent techniques of dubious sensitivity, and mostly aerobic and facultative heterotrophic bacteria were investigated (Cahill 1990; Ringứ et al 1995; Montes et al 1999) At that time, anaerobic bacteria and unculturable clones were neglected (Kamei et al 1985; Lee & Lee 1995; Gonzalez et al 1999), possibly through the shortage of techniques and the expensive nature of the culture-independent techniques available However, today there is increasing evidence that microorganisms occur in large numbers in the alimentary tract of fresh and marine fish In line with other studies on microbial biodiversity, emphasis has been focussed on molecularbased culture-independent techniques, which are generating interesting data and revealed the presence of anaerobes and uncultured organisms (e.g Moran et al 2005; Clements et al 2007; He et al 2010; Zhou et al 2011) Furthermore, there are some data available that the gut microbiota of fish is more closely related to the food than to the rearing water (Han et al 2010) Thus, the gut environment provides a specific niche, and bacterial activity in the guts is not merely a continuum of that observed in the environment This has consequences for the ecosystem as a whole, and the activities and abilities of these distinct bacterial communities may contribute uniquely to the nutrient cycling in the system Today, it is well accepted that the gut microbiota in fish are modulated by dietary manipulations; to our knowledge, the first study to evaluate the effect of dietary chitin on gut microbiota was carried out in 1972 (Sera & Kimata 1972) Since then, five studies have been carried out investigating the effect of dietary krill and chitin on the culturable heterotrophic gut microbiota of aquatic organisms, and an overview of these studies is presented in Table One of the studies listed in Table evaluate the gut microbiota by using denaturing gradient gel electrophoresis Atlantic salmon Ringứ et al (2006) evaluated the effect of dietary krill supplementation on epithelium-associated bacteria in the distal intestine (DI) of Atlantic salmon (Salmo salar L.) In this study, both microbial and electron microscopical investigations were included The adherent DI microbiota was modulated by dietary krill meal as the Gram-positive bacteria Carnobacterium maltaromaticum, Microbacterium oxydans, Microbacterium luteolum and Staphylococcus equorum ssp linens and the Gram-negatives Psychrobacter spp and Psychrobacter glacincola were not detected in the DI of fish fed krill meal but were reported to be present in non-krill-fed fish Transmission electron microscopy (TEM) revealed bacteria-like profiles between the microvilli in both rearing groups, indicating an autochthonous microbiota TEM evaluations also showed that by feeding fish a krill meal diet, the DI enterocytes were replete with numerous irregular vacuoles not detected when the fish were fed the control diet Whether the changes in gut microbiota and histology influence fish health was not evaluated and should be investigated in future studies Red sea bream snapper and crimson sea bream To the authors knowledge, the first study reporting the effect of chitin on fish gut microbiota was carried out by Sera & Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd Table Bacteria isolated from aquatic organisms and aquatic environment able to digest chitin Bacteria species (no or % in parentheses if available) Isolated from References Achromobacter labrum (1), A ureasophorum (1), A lipophagum (1), A hyperopticum (1), Flavobacterium indoltheticum (1), Pseudomonas cryothasia (1), P subruba (1) and Micrococcus colpogenes Agarbacterium spp (3), Beneckea lipophaga (1), Beneckea indolthetica (1) and Pseudomonas sp (1) No information was given Marine mud Campbell & Williams (1951) Sea bottom deposits of Kisarazu Coast in Tokyo Bay Stomach and intestinal contents of octopus and squid and intestinal contents of swell fish Intestinal contents of red sea bream Stomach and intestinal contents of red sea bream Gastrointestinal (GI) tract of red sea bream GI tract of yellow tail Gut microbiota of different fish species GI tract of tilapia GI tract of Dover sole Kihara & Morooka (1962) Sediment of an estuarine environment GI tract of tilapia Water Intestinal digesta of red sea bream and Japanese eel River and marine water of Tokushima Pel & Gottschal (1986) Different gut bacteria but no specific identification was given Vibrio spp Vibrio spp Vibrio spp Vibrio spp Aeromonas spp and Vibrio spp Acinetobacter sp (1), Enterobacteriaceae (4), Flavobacterium sp (1), Photobacterium spp (6), Vibrio spp (48) and an unidentified Gram-negative rod Rod-shaped, Gram-negative, terminal spherical spores that swelled the sporangium (8) Plesiomonas shigelloides and Aeromonas hydrophila Cytophaga Different gut bacteria but no specific identification was given Vibrio parahaemolyticus (4), V alginolyticus (21), V fluviales (9), V mimicus (1), Listonella anguillarum (1) and Aeromonas hydrophila (12) Vibrio anguillarum and V parahaemolyticus Aeromonas caviae, A hydrophila, A jandaei, A sobria and A veroni Bacteroides spp., Bacteroidaceae, Streptococcus spp Aeromonas spp., A hydrophila and A salmonicida Achromobacter spp., Bacillus spp and Enterobacteriaceae No information was given Cytophaga/Flavobacteria (1), Marinibacter sp (1) and Sulfitobacter pontiacus (1) Enterobacter aerogenes (28%), Aeromonas sp (25%) and Chromobacterium sp (16%) Aeromonas hydrophila (46%) and Aeromonas sp (15%) Marinobacter lutaoensis (1), Ferrimonas balearica (1), Pseudoalteromonas piscicida (1), Enterovibrio norvegicus (13), Grimontia hollisae (10), Photobacterium damselae spp damselae (20), P leiognathi (28), P lipolyticum (1), P phosphoreum (39), P rosenbergii (9), Vibrio campbelli (5), V chagasii (5), V fischeri (6), V fortis (4), V gallicus (1), V harveyi (77), V natrigenes (15), V nigripulchritudo (3), V ordalii (18), V parahaemolyticus (33), V pomeroyi (26), V ponticus (12), V proteolyticus (4), V rumoiensis (1), V shilonii (2), V tasmaniensis (2) and V tubiashii (24) Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd Prawn Intestinal digesta of common carp, crucian carp, grey mullet, surface water and sediment samples from the Hikiji River Minke whale Lake Jeziorak Surface water, water above the sediment and bottom sediments of lake Jeziorak, Jeziorak Maly and Tynwald Foregut of Eriocheir sinensis Organic particles of the upper water column of the Equatorial Atlantic Oligo-mesotrophic lake Eutrophic lake Intestinal contents from various Japanese costal fishes Seki & Taga (1963) Sera & Ishida (1972a) Sera & Ishida (1972b) Sera et al (1974) Sakata et al (1978a) Sakata et al (1978b) Sakata et al (1980) MacDonald et al (1986) Sakata & Koreeda (1986) Dempsey & Kitting (1987) Kono et al (1987a) Osawa & Koga (1995) Rivonker et al (1999) Sugita et al (1999) Olsen et al (2000) Brzezinska & Donderski (2001) Donderski & Brzezinska (2001) Xue & Wu (2003) Berkenheger & Fischer (2004) Brzezinska & Donderski (2006) Itoi et al (2006) Table (Continued) Bacteria species (no or % in parentheses if available) Isolated from References Vibrio fischeri (39), V harveyi (2), V scophthalmi V iscthyoenteri group type (1), V scophthalmi V iscthyoenteri group type (4) and V scophthalmi V iscthyoenteri group type (36) Carnobacterium divergens and C maltaromaticum Bacillus, Acinetobacter sp Japanese flounder intestine Sugita & Ito (2006) Different sources Proximal and distal intestine of Atlantic salmon fed 5% chitin Leisner et al (2007, 2008) Askarian et al (2012) Kimata (1972) In this study, red sea bream and crimson sea bream (Evynnis japonica) were fed diets supplemented with or without 15% chitin The population level of allochthonous heterotrophic and chitin-decomposing bacteria in the stomach and intestine was investigated after 5, 15 and 25 days of feeding, but the authors did not distinguish between the different fish species Some variation in the allochthonous bacterial population level was observed between the different dietary groups The heterotrophic population level was generally higher at all sampling points when fish were fed the chitin-supplemented diet, and the frequency of gut bacteria able to utilize chitin seemed higher when the fish were fed chitin compared to those fed the fish meat paste Red sea bream and Japanese eel In a study with red sea bream and Japanese eel (Anguilla japonica), Kono et al (1987a) compared the effect of a 20% chitin-supplemented diet with that of fish fed a control diet Red sea bream were fed the experimental diets for 30 days, and total viable counts and chitin-decomposing bacteria counts were estimated in the stomach and intestinal contents The population level of chitin-decomposing bacteria in stomach and intestinal contents of fish fed the control diet was generally tenfold less than the total viable counts, and this trend was not changed when chitin was supplemented to the diet Based on their findings, the authors suggested that supplementation of chitin had no effect Crustaceans In a 45-day feeding trial of giant freshwater prawn (Macrobrachium rosenbergii), Kumar et al (2006) evaluated the effect of dietary chitin, at 5% and 10% inclusion, on total viable counts and chitinoclastic bacteria The total viable bacteria count was gradually reduced as the chitin level increased, but the rate of reduction was less in natural chitin-fed groups in comparison with the purified chitin groups The authors suggested that this may be due to the antimicrobial properties of chitin, but no evidence was presented No chitinoclastic bacteria were observed in the gut of any of the treatment groups, which is in agreement with the findings of Fox (1993), who reported few chitinoclasts and concluded that the synthesis of endogenous chitinase in the digestive gland of shrimp (Penaeus monodon) occurs at a slow rate and hence that juvenile shrimps are able to digest only small amounts of dietary chitin in the absence of bacterially produced chitinase Atlantic salmon Askarian et al (2012) identified 139 autochthonous bacterial strains (based on biochemical and physiological properties) isolated from proximal intestine (PI) and DI of Atlantic salmon fed control or 5% chitin diet Seventy-four of these isolates were further identified by 16S rRNA gene sequencing The predominant autochthonous bacteria in the PI and DI of fish fed the control diet belonged to Staphylococcus, Bacillus and Agrococcus, while Staphylococcus, lactobacilli, bacilli and Acinetobacter were dominant in the intestine of fish fed the chitin diet A further description of the dietary effect of chitin on the autochthonous microbiota is presented in Table The most promising gut bacteria isolated from the control group, displaying chitinase activity, belonged to the Bacillus genera, while gut bacteria showing highest chitinase activity when the fish were fed the 5% chitin diet belonged to bacilli and Acinetobacter One surprising observation noticed in this study was that the lactic acid bacteria (LAB) (only isolated from the GI tract of fish fed the chitin-enriched diet) displayed no chitinase activity; however, these LAB were able to inhibit the growth of fish pathogenic bacteria in vitro Atlantic cod In their study with Atlantic cod (Gadus morhua L.), Z Zhou, S He, R E Olsen, B Yao & E Ringứ, unpublished data, investigated the autochthonous gut microbiota of fish fed diets with or without 5% chitin supplementation by using denaturing gradient gel electrophoresis (DGGE) DGGE was used as this method is reliable, rapid, sensitive and easy to operate From each of the two Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd Atlantic salmon (Salmo salar L.) Red sea bream (Pagrus major) and crimson sea bream (Evynnis japonica) Red sea bream northern krill (32%) chitin (15%) 92 days 115 days 45 days 60 days 30 days 5, 15 and 25 days 46 days Length of the study Not investigated In this study, only DGGE analysis was carried out Dietary chitin had no effect on the TVC of adherent microbiota in proximal and distal intestine Dietary chitin had no significant effect on TVC Dietary chitin had no significant effect on TVC in stomach and intestine TVC was reduced by increasing chitin level Dietary chitin increased TVC of allochthonous microbiota in stomach and intestine Dietary northern krill increase TVC in distal intestine Effect on total viable counts (TVC) Chitin modulates the autochthonous gut microbiota Psychrobacter ikaite, Nesterenkonia sp., Acinetobacter spp., Lactobacillus sakei, Leuconostoc citreum, Macrococcus equipercicus and Staphylococcus equorum were not isolated when fish were fed the control diet Aeromonas sp., Agrococcus baldri, Bacillus cereus and Micrococcus luteus were not isolated from fish fed the chitin diet Chitin modulates the autochthonous gut microbiota Proximal intestine: Escherichia coli HPC119-like, Erwinia chrysanthemi strain 1015-1-like, uncultured bacterium clone wom-12-like and Thermus sp TC4-like were not identified when fish were fed the control diet Uncultured bacteria clone GA61-like, uncultured proteobacterium clone MS037A1_F05-like, Vibrio salmonicida isolate PB3-7 rrnA-like, Escherichia coli strain G6F1162-like and uncultured bacterium isolate DGGE gel band 15-like were not identified when fish were fed the chitin diet Distal intestine: Swine faecal bacterium SD-Xy16-like, Lactococcus lactis strain C9-6, Aliivibrio sp HM3-18, Allivibrio wodanis isolate AV11/2007-like and uncultured bacterium clone nbt36h02-like were not identified in the control diet Uncultured bacterium clone GA61-like was not identified in the chitin diet No information was given, and no chitinoclastic bacteria were observed in any of the treatment groups No information was given, and dietary chitin had no effect on chitin-decomposing microbiota in stomach and intestine No information was given, and dietary chitin had no effect on chitin-decomposing bacteria Carnobacterium maltaromaticum, Microbacterium oxydans, Microbacterium luteolum, Staphylococcus equorum spp linens, Psychrobacter spp and Psychrobacter glacincola were not isolated when fish were fed the krill diet, while Pseudomonas fulgida, Pseudomonas reactans and Stenotrophomonas maltophila were not isolated from fish fed fish meal (control diet) No information was given, but frequency of chitin utilizing bacteria increased by dietary chitin Effect on gut microbiota TVC, total viable counts of culturable heterotrophic gut bacteria; DGGE, denaturing gradient gel electrophoresis chitin (5%) Atlantic cod (Gadus morhua L.) Giant fresh water prawn (Macrobrachium rosengergii) Atlantic salmon chitin (5% and 10%) chitin (5%) Japanese eel (Anguilla japonica) chitin (20%) chitin (20%) Species Supplement Table Effects of krill and chitin on gut microbiota in fish, eel and prawn Z Zhou, S He, R E Olsen, B Yao & E Ringứ, unpublished data Askarian et al (2012) Kumar et al (2006) Kono et al (1987a) Kono et al (1987a) Sera & Kimata (1972) Ringứ et al (2006) References dietary groups (control and chitin-supplemented diet), samples were taken from the PI and DI of three individual fish The results clearly displayed that dietary chitin modulated the intestinal microbiota (Table 2) and that the gut microbiota in the dietary chitin treatment was more diverse than that of the control group Furthermore, the presence of Escherichia coli-like bacteria, Anaerorhabdus furcosa and an uncultured bacterium was depressed by chitin in the PI, while Aliivibrio wodanis was stimulated in the PI by chitin Analysis of the DI microbiota revealed that uncultured Spirochete-like bacterium, a swine faecal bacterium and an uncultured bacterium were significantly stimulated by dietary chitin The immunological effects of chitin have been studied in many different fish and shellfish species However, very few, if any, reports have been published about the effects of chitin on the adaptive immune system of fish This may be due to the inadequate resources to understand the fish adaptive immune system In mouse systems, however, chitin activities have been studied in much more detail (for review, see Lee et al 2008) Chitin activates the mouse lung alveolar macrophages to express cytokines such as IL-12, tumour necrosis factor-a and IL-18, leading to INF-c production mainly by NK cells (Shibata et al 1997) Although chitin promotes the type I immune responses, it down-regulates type II immune responses of mice (Shibata et al 2001) Chitin induces an immune response characterized by the infiltration of cells that express IL-4, IL-13, eosinophils and basophiles, a response that typically has been associated with allergic and parasitic worm immune responses (Reese et al 2007) Recently, chitin receptor molecules on target cells have been reported A chitin-binding protein was discovered in rainbow trout (Russell et al 2008) The protein was homologous to human and murine plasma interlectins and localized in the gill, spleen, hepatic sinus, renal interstitium, intestine, skin, swim bladder and within the leucocytes of the rainbow trout Another type of chitin receptor was identified in mice (Schlosser et al 2009) The chitin receptor is a novel homotetrameric transmembrane protein encoded by the FIBCD1 gene The receptor protein was conserved among major vertebrates including fish and was highly expressed in the GI tract The receptor is involved in endocytosis through binding acetylated components of chitin and chitin fragments in a calcium-dependent manner with a high affinity The innate immune system is a fundamental defence mechanism in fish (Magnadottir 2006) Innate immunity is the initial response to microbes that prevent, control or eliminate infections of the fish Innate immunity to microbes stimulates adaptive immune responses and can also influence the nature of the adaptive responses to make them optimally effective against different types of microbes Components of the innate immune system consist of epithelial barriers, phagocytes (neutrals, monocytes, macrophages and dendritic cells) and circulating proteins (the complement system, pentraxins, collectins and ficolins) (Abbas et al 2010) The scales of fish, the mucous surfaces of skin and gills, and the epidermis act as the primary barrier against infection (Ellis 2001) Fish mucus contains immune components such as lectins, pentraxins, lysozyme, complement proteins, antibacterial peptides and IgM (Alexander & Ingram 1992; Rombout et al 1993; Aranishi & Nakane 1997) Innate immune cells recognize non-self molecules through germline-encoded pattern recognition receptors (PRR) or pattern recognition proteins (PRP) that are present in a relatively low numbers and are vertically transmitted (Kaisho & Akira 2001) The molecules that are recognized by PRR or PRP of the innate immune cells contain a highly conserved molecular pattern Two categories of the molecular patterns can induce immune responses by interacting with PRR or PRP: foreign or pathogen-associated molecular patterns (PAMP) and danger signals that are released from the damaged host cells and tissues owing to infection, necrosis and natural cell death PAMP is the collective term used for highly conserved molecules in microbes and is not generally expressed in multicellular organisms These molecules include polysaccharides (LPS) in bacterial cell walls, peptidoglycans, bacterial DNA and double-stranded viral RNA In contrast, danger signals include the molecules such as the hosts DNA, RNA, heatshock proteins and other chaperons, and oligomannose of presecreted glycoproteins (Matzinger 1998; Elward & Gasque 2003) Among the parameters of the innate immune system, the phagocytic, the lysozyme and spontaneous haemolytic activities and, in some cases, pentraxins have been used to determine the effects of the inherent or external factors on the immune system and the disease resistance of fish Such factors include immunostimulants, fish diets and feed additives in addition to genetic traits, seasonal factors, environmental temperature, pollution, handling and crowding Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd stress, probiotics, and the effects of diseases and vaccination (Magnadottir 2006) The activities of phagocytic cells of the fish treated with immunostimulants such as chitin can be detected by phagocytosis, killing of pathogens and cytokine production The killing activity of phagocytic cells is most important in the elimination of pathogens and can be determined by measuring the direct cell killing of macrophages or the respiratory burst activity, which indicates the production level of reactive oxygen species, a mediator of oxygen-dependent killing (Sakai 1999) In this section, the effects of chitin on the immune system of various species of fish and shellfish are discussed As several studies have been carried out on the effects of chitin on the immune system, growth and survival of various species of fish and shellfish, an overview of these studies is presented in Table Crayfish Zhu et al (2010) reported the effect of chitin on the survival and immune reactivity of crayfish (Procambarus clarkia) In general, crayfish fed chitin displayed elevated total hemocyte counts (THCs), prophenol oxidase and superoxide dismutase activities However, cumulative mortality of P clarkia challenged with white spot syndrome virus was not significantly affected by chitin Gilthead seabream In a study with gilthead seabream (Sparus aurata L.), Esteban et al (2000) injected chitin (0.1 mg of chitin particles < 10 lm in 100 lL PBS) intravenously (i.v.) or with mg of chitin intraperitoneally (i.p.) and assessed humoral and cellular immune responses at 3, or 10 days postinjection Seabream injected with chitin i v remained unaffected in their innate immune responses However, fish i.p injected showed an increase in humoral and cellular immune responses such as natural haemolytic complement activity, headkidney leucocyte respiratory burst activity, and phagocytic and cytotoxic activities An assumption as to why chitin i.p injection solely showed an increase in the fishes immune response was suggested to be of the immunomodulatory activity of chitin on the macrophages In fish, similar to mammals, a lectin-type receptor may be involved in the recognition and attachment of chitin particles to the macrophage membrane, and the production of gamma-interferon may trigger the activation of the macrophage Esteban et al (2001) evaluated the effects of dietary chitin on the innate immune response of gilthead seabream Dietary feeding is non-stressful and can act as a vector to provide the immunostimulant to a larger number of fish, minimizing cost and effort Seabream were fed 0, 25, 50 or 100 mg chitin kg1 for 2, and weeks and were analysed for the non-specific modulation of haemolytic Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd complement activity, leucocyte respiratory burst activity and cytotoxicity Fish fed 25 and 50 mg chitin kg1 showed increased activity of the seabream innate immune system Natural haemolytic complement activity and cytotoxic activity increased after weeks of chitin feeding The respiratory burst activity and phagocytic activity showed a delayed response with increased activity observed after and weeks, respectively However, lysozyme activity and growth performance were unaffected by the chitin supplementation These studies indicate that the high-chitin diet may not be beneficial to the innate immunity of seabream and that the immunological parameters are enhanced sequentially at different time points of post-chitin dietary provision Not only the concentration but also the size of chitin particles has been reported to be important for the activation of leucocytes (Cuesta et al 2003) Three different sizes of chitin particles (unfiltered, 10 lm) were prepared and incubated with gilthead seabream head kidney leucocytes for 1, 4, 24 or 48 h The leucocytes were able to phagocytose only the chitin particles that were smaller than 10 lm Leucocyte phagocytosis of bacteria was increased after chitin incubation for or h, while the respiratory burst activity was unaffected Preincubation of leucocytes with chitin particles enhanced phagocytosis, tumour cell cytotoxicity and respiratory burst Yellowtail Yellowtail (Seriola quinqueradiata) injected only with chitin showed an increase in protection against the P piscicida challenge until 45 days after the treatment (Kawakami et al 1998) Although chitin increased the nonspecific protective immunity in yellowtail, chitin did not show adjuvant effects, as had been demonstrated in mice and guinea pigs Rainbow trout In an early study, Sakai et al (1992) reported that rainbow trout injected with chitin showed stimulated macrophage activities and increased resistance to V anguillarum infection Based on their results, the authors suggested that chitin has immunostimulating effects in rainbow trout Carp Gopalakannan & Arul (2006) fed and 10 g chitin kg1 to common carp (Cyprinus carpio) under field conditions During 90 days of feeding, body weights of ten randomly selected fish from each pond were measured every 15 days The growth of chitin-fed fish was not significantly improved Lysozyme activity and neutrophil activity of the treated carp were also significantly stimulated When the treated fish were intraperitoneally injected with Aquaculture Nutrition 18; 117131 ể 2012 Blackwell Publishing Ltd Gilthead seabream (Sparus aurata L.) Gilthead seabream Chitin injection i.p (0.1 mg) 3, and 10 days Chitin (25, 50, 100 mg kg1) 2, 4, weeks Chitin (different sizes of particles) 1, 4, 24 and 48 h Chitin injection i.p 45 days White shrimp (Litopenaeus vannamei) Artemia franciscana Tilapia (Oreochromis niloticus ì O aureus) Shore crab (Carcinus maenas) Yellow tail (Seriola quinqueradiata) Rainbow trout (Oncorhynchus mykiss) Common carp (Cyprinus carpio) Rohu (Labeo rohita) survival rate in the natural state, number of hyaline haemocytes and survival in the challenge experiment ? phagocytic activity 5% inclusion level number of cultivable bacteria in the hepato-pancreas lg chitin g1 immune ability and resistance to V alginolyticus infection ? resistance in the challenge experiment lysozyme activity and neutrophil activity and resistance ? survival ? respiratory burst activity, body weight gain, survival rate and resistance in the challenge study growth rate total hemocyte count, prophenol oxidase and superoxide dismutase activities No significant effect on cumulative mortality when challenged with white spot syndrome virus natural haemolytic complement activity, head kidney leucocyte reparatory burst activity and phagocytic and cytotoxic activities haemolytic complement activity, cytotoxicity (2 weeks), respiratory burst and phagocytosis (4, weeks) ? lysozyme acidity and growth rate phagocytosis of bacteria only in leucocytes treated with the chitin particles smaller than 10 lm non-specific immunity and resistance ? adjuvant effects macrophage activities and resistance Effect on the immune system, growth and survival Symbols represent an increase (), no effect (?) or decrease () on the specific response Chitin injection (4, and lg g1) days Chitin (20 g kg1) days Chitin (0, 20, 50 or 100 g kg1) weeks Chitin (0, 50100 g kg1 in feed) 11 weeks (50 and 100 g kg1) and 10 weeks (control) Chitin (25 mg kg1) 15 days Chitin (10 g kg1) 90 days Chitin (100 mg kg1) 14 days Crayfish (Procambarus clarkia) Chitin (5, 10 and 15 mg g1) weeks Gilthead seabream Species Dose and length of administration Table Effects of chitin on the immune system, growth and survival of various fish and shellfish species Wang & Chen (2005) Soltanian et al (2007) V campbellii Powell & Rowley (2007) Shiau & Yu (1999) Gopalakannan & Arul (2006) Choudhury et al (2005) Sakai et al (1992) Kawakami et al (1998) Cuesta et al (2003) Esteban et al (2001) Esteban et al (2000) Zhu et al (2010) References V alginolyticus V alginolyticus A hydrophila A hydrophila V anguillarum P piscicida Challenge with a pathogen We thank to Scientic and Technological Research Council of Turkey (TUBITAK), project nr 109O413 and National Academy of Sciences of Belarus (NASB) for their supports to this project Abele, D & Puntarulo, S (2004) Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and sh Comp Biochem Physiol., 138C, 405415 Barroso, J.B., Peragon, J., Garc a-Salguero, L., de 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44, 10151018 Peragon, J., Barroso, J.B., de la Higuera, M & Lupianez, J.A (1998) Relationship between growth and protein turnover rates and nucleic acids in the liver of rainbow trout (Oncorhynchus mykiss) during development Can J Fish Aquat Sci., 55, 649 657 Reznick, A.Z., Packer, L & Sen, C.K (1998) Strategies to assess oxidative stress In: Oxidative Stress in Skeletal Muscle (Reznick, A.Z., Packer, L., Sen, C.K., Holloszy, J.O & Jackson, M.J eds), pp 4358 Birkhauser Verlag, Basel, Switzerland Riley, J.C.N & Behrman, H.R (1991) Minireview: oxygen radicals and reactive oxygen species in reproduction Proc Soc Exp Biol Med., 198, 781791 Rudeva, I.I (1997) Blood antioxidant system of Black Sea elasmobranch and teleosts Comp Biochem Physiol., 118C, 255260 Aquaculture Nutrition 18; 211219 ể 2011 Blackwell Publishing Ltd Satoh, S., Takeuchi, T., Narabe, Y & Watanabe, T (1983a) Eects of deletion of several trace elements from sh meal diets on growth and mineral composition of rainbow trout 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a-tocopherol requirement of rainbow trout Bull Jpn Soc Sci Fish., 47, 1463 1471 Watanabe, T., Kiron, V & Satoh, S (1997) Trace minerals in sh nutrition Aquaculture, 151, 185207 Woodward, B (1994) Dietary vitamin requirements of cultured young sh with emphasis on quantitative estimates for salmonids Aquaculture, 124, 133168 Aquaculture Nutrition 2012 18; 220232 doi: 10.1111/j.1365-2095.2011.00898.x 1,2,3 1,2 1,3 1,3 1,4 1,4 1,3 1,2 1,2,3 Animal Nutrition Institute, Sichuan Agricultural University, Sichuan, Yaếan, China; Key Laboratory for Animal DiseaseResistance Nutrition of China, Ministry of Education, Sichuan Agricultural University, Sichuan, Yaếan, China; Key Laboratory of Sichuan Province, Fish Nutrition and Safety Production University, Yaếan, China; Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu, China Correspondence: Xiao-Qiu Zhou, Animal Nutrition Institute, Sichuan Agricultural University, Yaếan 625014, China E-mail: zhouxq@sicau edu.cn; xqzhouqq@tom.com A total of 1200 juvenile Jian carp (Cyprinus carpio var Jian) (8.76 0.02 g) were fed diets containing graded levels of histidine at 2.3 (unsupplemented control), 4.4, 6.3, 8.6, 10.8 and 12.7 g kg)1 diet for 60 days to investigate the eects of histidine levels on growth performance, body composition, intestinal enzymes activities and microora Specic growth rate (SGR), feed eciency, protein eciency ratio, protein productive value, body protein content and lipid content of sh were lowest in sh fed the basal diet (P < 0.05) Activities of glutamate-pyruvate transaminase in muscle and hepatopancreas, trypsin, chymotrypsin, amylase, lipase activities in intestine and hepatopancreas, and Na+, K+ATPase, creatine kinase, alkaline phosphatase, c-glutamyl transpeptidase activities in three intestinal segments were improved by dietary histidine (P < 0.05), whereas glutamateoxaloacetate transaminase activities and plasma ammonia content followed an opposite trend The amounts of Lactobacillus, Escherichia coli and Aeromonas were signicantly aected by dietary histidine levels (P < 0.05) These results suggested that histidine could improve growth and enhance intestinal enzymes activities of juvenile Jian carp The dietary histidine requirement of juvenile Jian carp (8.7668.02 g) based on SGR was 7.8 g kg)1 diet or 2.38 g 100 g)1 protein by quadratic regression analysis key words: var Jian, growth, histidine, intestinal enzymes activity, protein deposition Received April 2011, accepted 20 June 2011 Histidine is known as an indispensable amino acid for common carp (Cyprinus carpio L.) (Nose 1979) Lack of dietary histidine has been shown to cause poor growth performance and feed utilization in Indian major carp (Labeo rohita) (Murthy & Varghese 1995) and Atlantic salmon (Salmo salar L.) (Breck et al 2005), as well as negative eect on protein eciency ratio (PER) of Indian major carp (Cirrhinus mrigala) (Ahmed & Khan 2005) and African catsh (Clarias gariepinus) (Khan & Abidi 2009) Growth of sh is largely a process of protein deposition (Cowey 1994) The deciency of dietary histidine resulted in lowered body protein deposition in Indian major carp (Ci mrigala) (Ahmed & Khan 2005) The protein accretion is a balance between protein anabolism and catabolism (Kaushik & Seiliez 2010) Glutamate-oxaloacetate transaminase (GOT) and glutamatepyruvate transaminase (GPT) are the most important transamination enzymes in sh, which are involved in protein and amino acid metabolism (Ramaswamy et al 1999) In teleosts, ammonia is the major component of nitrogen excretion, and its production rate is directly related to the rate of protein catabolism (Waarde 1983) A recent study from our laboratory has indicated that GOT and GPT activities and plasma ammonia content could be aected by dietary methionine hydroxy analogue in juvenile Jian carp (C carpio var Jian) (Xiao et al 2011) However, little was known about the eect of histidine on the GOT and GPT activities and plasma ammonia content in sh ể 2011 Blackwell Publishing Ltd The digestion ability and absorption function, which were dependent mostly on the activities of digestive enzymes and brush border enzymes in the intestine, play a crucial role in the growth of sh (Fountoulaki et al 2005; Hakim et al 2006) The information about the relationship between dietary histidine and the activities of intestinal enzymes in sh is not available Studies on mammals showed that histidine serves as an essential component of the active centre in many enzymes, such as serine peptidases, amylase, creatine kinase (CK) and alkaline phosphatase (AKP) (Schneider 1978; Ishikawa et al 1992; Polgar 2005) Serine peptidase family is constituted of a group of peptidases including trypsin, chymotrypsin and carboxypeptidase, which play a very important role in protein digestion (Polgar 2005) Intestinal AKP in carp is considered to take part in the absorption of nutrients, such as lipid and glucose (Villanueva et al 1997) It has been noted that histidine can increase uptake or act to redistribute zinc to body tissues of rainbow trout (Glover et al 2003) Tan et al (2011) reported that dietary zinc could improve the activities of intestinal trypsin, chymotrypsin, lipase, amylase, AKP, Na+, K+-ATPase and c-glutamyl transpeptidase (c-GT) in juvenile Jian carp These ndings indicated that the activities of intestinal enzymes in sh may be aected by dietary histidine Furthermore, digestive system developmental changes are associated with food assimilation of sh (Gisbert et al 2004) The development of pancreas and intestine rely on the structural integrity of cells (Hamlin et al 2000) The changes in enterocyte structure are characterized by morphological alterations (Bonaldo et al 2008) Recodo (1991) noted extensive necrosis in the epithelial cells of hepatopancreas of shrimp fed histidine-excess diets Histological changes in the hepatopancreas and midgut gland were also observed in prawns with sub-optimal or super-optimal levels of histidine in the diet (Recodo et al 2000) The structural integrity of intestinal epithelial cells in sh was partly related to the antioxidant ability of cells (Chen et al 2009) It was reported that histidine could inhibit oxidative stress-induced inammation in human intestinal epithelial cells (Son et al 2005) Therefore, histidine may be related to the growth and development of pancreas and intestine of sh The digestive tract of sh carries a heavy bacterial load that plays an important role in host health, both as a source of infection and conversely in protection against disease (Cahill 1990) Ringứ & Strứm (1994) suggested that the changes in intestinal microora might have been caused by changes in either the gut epithelium or attachment sites for the microora Peterson et al (1998) reported that histidine protected the intestinal tissue of mouse from Salmonella typhimuriuminduced damage Histidine-rich polypeptides (HRPs) isolated Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd from human salivary gland exerted antifungal activity against Candida albicans (Pollock et al 1984) and Streptococcus mutans (MacKay et al 1984) However, no study has been conducted to investigate the eect of dietary histidine on intestinal microora of sh, which warrants investigation The main purpose of this study was to investigate the eect of dietary histidine levels on growth, body composition and intestinal enzymes activities of juvenile Jian carp, which could provide partial theoretical evidence for the eect of histidine on sh growth The dietary histidine requirement of juvenile Jian carp was also estimated based on growth performance Formulation of the basal diet is presented in Table Fish meal (Pesquera Lota Protein Ltd., Villagram, Chile) and gelatin (Rousselot Gelatin Co., Ltd., Guangdong, China) were used as dietary protein sources Crystalline amino acids (Jiangsu Nantong Eastern Amino Acid Co Ltd., Nantong, China) were used to simulate the amino acid prole of diets to that of 340 g kg)1 whole chicken egg protein, except for histidine The histidine concentrations in sh meal and gelatin were measured, respectively, before the formulation by the method of Llames & Fontaine (1994) The experimental diets were supplemented with L-histidine hydrochloride monohydrate to provide histidine levels at 2.5 (unsupplemented control), 4.5, 6.5, 8.5, 10.5 and 12.5 g kg)1 diet All diets were made isonitrogenous with the addition of appropriate amounts of glycine Zinc, ferrum, pyridoxine, pantothenic acid, inositol, thiamin and riboavin were formulated to meet the nutrient requirements of Jian carp according to our laboratoryếs studies (He et al 2009; Jiang et al 2009; Wen et al 2009; Li et al 2010; Ling et al 2010; Huang et al 2011; Tan et al 2011) The levels of other nutrients met the requirements of common carp according to NRC (1993) The pH of each diet was adjusted to 7.0 by the addition of 6.0 N NaOH (Nose et al 1974) The pellets were produced and stored at )20 C (Akiyama et al 1985) The histidine concentration in the experimental diets was measured to be 2.3 (unsupplemented control), 4.4, 6.3, 8.6, 10.8 and 12.7 g kg)1 diet as described by Llames & Fontaine (1994) Juvenile Jian carp were obtained from Tong Wei Hatchery (Sichuan, China) Fish were adapted to the experimental environment for weeks A total of 1200 sh with an average initial weight of 8.76 0.02 g were randomly assigned to 24 Ingredients g kg)1 Nutrients content1 g kg)1 Fish meal Gelatin Amino acid mix2 Amino acid premix3 Fish oil Soybean oil a-starch Corn starch Vitamin premix4 Trace mineral premix5 Ca (H2PO4)2 Choline chloride (500 g kg)1) Cellulose Ethoxyquin (300 g kg)1) 153.0 70.0 187.0 50.0 16.3 18.9 230.0 208.3 10.0 10.0 24.7 1.3 20.0 0.5 Crude protein Crude lipid n-3 n-6 Available phosphorus 328.0 61.1 10.0 10.0 6.0 Table Composition and nutrient content of the basal diet Crude protein and crude fat were measured value Available phosphorus, n-3 and n-6 contents were calculated according to NRC (1993) and Bell (1984) Amino acid mix (g kg)1): arginine, 11.01 g; isoleucine, 13.60 g; leucine, 22.29 g; lysine, 17.69 g; methionine, 8.42 g; cystine, 0.50 g; phenylalanine, 14.90 g; tyrosine, 12.30 g; threonine, 12.80 g; tryptophan, 4.11 g; valine, 16.75 g; glycine, 52.63 g L-histidine hydrochloride monohydrate was added to obtain graded level of histidine Each mixture was made isonitrogenous with the addition of reduced amounts of glycine and compensated with appropriate amounts of corn starch Per kilogram of amino acid premix composition from diet to was as follows (g kg)1): L-histidine hydrochloride monohydrate 0.00, 41.44, 82.88, 124.31, 165.75 and 207.19 g; glycine 298.63, 238.90, 179.18, 119.45, 59.73 and 0.00 g; and corn starch 701.37, 719.66, 737.94, 756.24, 774.52 and 792.81 g, respectively Per kilogram of vitamin premix (g kg)1): retinyl acetate (500 000 IU g)1), 0.80 g; cholecalciferol (500 000 IU g)1), 0.48 g; DL-a-tocopherol acetate (500 g kg)1), 20.00 g; menadione (500 g kg)1), 0.20 g; cyanocobalamin (100 g kg)1), 0.01 g; D-biotin (200 g kg)1), 0.50 g; folic acid (960 g kg)1), 0.52 g; thiamin nitrate (980 g kg)1), 0.10 g; ascorhyl acetate (920 g kg)1), 7.24 g; niacin (980 g kg)1), 2.85 g; meso-inositol (980 g kg)1), 52.86 g; calcium-D-pantothenate (980 g kg)1) 2.51 g; riboflavine (800 g kg)1), 0.63 g; pyridoxine hydrochloride (980 g kg)1), 0.76 g All ingredients were diluted with corn starch to kg Per kilogram of mineral premix (g kg)1): FeSO4ặ7H2O (197 g kg)1 Fe), 69.70 g; CuSO4ặ5H2O (250 g kg)1 Cu), 1.20 g; ZnSO4ặ7H2O (225 g kg)1 Zn), 21.64 g; MnSO4ặH2O (318 g kg)1 Mn), 4.09 g; KI (38 g kg)1 I), 2.90 g; NaSeO3 (10 g kg)1 Se), 2.50 g All ingredients were diluted with CaCO3 to kg experimental aquaria (90 L ã 30 W ã 40 H cm), each of which was connected to a closed recirculating water system with continuous aeration Feeding management was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Animal Nutritional Institute, Sichuan Agricultural University Water change rates in each aquarium were maintained at 1.2 L min)1, and the water was drained through biolters to decrease micro-organism, reduce ammonia concentration and remove solid substances in the water Dissolved oxygen was higher than mg L)1, and water temperature and pH were maintained at 26 C and 7.0 0.3, respectively The experimental units were under natural light and dark cycle For the feeding trial, each of six experimental diets was fed to quadruplicate of sh six times daily from to 30 days and four times daily from 31 to 60 days Fish were fed to satiation, and uneaten feed was removed by siphoning after each meal Fish in each aquarium were weighed at the initiation and termination of the feeding trial Thirty sh from the same population were randomly selected to determine initial carcass proximate composition After the feeding trial, four sh from each aquarium were frozen for subsequent body composition analysis (AOAC 1998) Another 15 sh from each aquarium were anaesthetized in benzocaine bath (50 mg L)1) 12 h after the last feeding and sampled for the determination of hepatopancreas protein content (HPC), intestine protein content (IPC), activities of hepatopancreatic, intestinal and muscle enzymes The hepatopancreas, intestine and muscle were removed, weighed and frozen in liquid nitrogen and then stored at )70 C until analysed Intestines of another four sh from each aquarium were sampled to measure the height of intestinal folds according to Lin & Zhou (2006) In Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd specic growth rate (SGR), feed eciency (FE), PER and protein productive value (PPV) Hepatopancreas and intestine weight (IW), and hepatopancreas and intestine protein were used to calculate hepatosomatic index (HSI), intestosomatic index (ISI), HPC and IPC Besides, relative gut length (RGL) was calculated addition, three sh were collected from each aquarium and then extruded the intestinal content for measuring the amount of Lactobacillus, Escherichia coli and Aeromonas All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Agricultural University Hepatopancreas, intestine and muscle samples were homogenized in 10 volumes (w/v) of ice-cold physiological saline and centrifuged at 6000 g at C for 20 min, and then the supernatant was conserved for enzymes activities analysis Trypsin and chymotrypsin activities were determined according to Hummel (1959), lipase and amylase activities were assayed according to the method described by Furne et al (2005), and CK, AKP, Na+, K+-ATPase and c-GT activities were determined according the procedure described by Tanzer & Gilvarg (1959), Bessey et al (1946), Weng et al (2002) and Bauermeister et al (1983), respectively Hepatopancreas and IPC were determined by the method of Bradford (1976) GOT and GPT activities in muscle and hepatopancreas were, respectively, determined according to the method described by Bergmeyer & Bernt (1974a,b) Six hours after the last feeding, blood of ve sh collected from each aquarium was drawn from the caudal vein into heparinized syringes Prior to sampling, sh in each aquarium were fed solely and blood was drawn in order The blood samples were centrifuged at 4000 g for 15 (Liu et al 2009) Plasma was collected for the determination of ammonia concentration by the method of Tantikitti & Chimsung (2001) The amounts of intestinal microora were measured using the method of Refstie et al (2006) SGR ẳfẵln mean FWị ln mean initial weightị= number of daysg 100 FE ẳ wet weight gain gị=FI gị PER ẳ weight gain gị=protein intake gị PPV ẳ ẵfish protein gain gị=protein intake gị 100 HSI ẳ ẵwet hepatopancreas weight gị= wet body weight gị 100 ISI ẳ ẵwet IW gị=wet body weight gị 100 HPC ẳ ẵhepatopancreas protein gị= wet hepatopancreas weight gị 100 IPC ẳ ẵintestine protein gị=wet IW gị 100 RGL ẳ ẵintestine length cmị=total body length cmị 100 Results were presented as means SD All data were subjected to one-way analysis of variance (ANOVA) followed by the Duncanếs multiple-range test to determine signicant dierences among treatment means at the level of P < 0.05 through SPSS 13.0 (SPSS Inc., Chicago, IL, USA) The parameters with signicant dierences were subjected to a second-degree polynomial regression analysis Dietary histidine requirement based on SGR was estimated by quadratic regression analysis (Robbins et al 1979) Data on initial weight, nal weight (FW), feed intake (FI) and proximate composition of carcass were used to calculate Table Initial body weight (IBW, g sh)1), nal body weight (FBW, g sh)1), feed intake (FI, g sh)1), feed eciency (FE), specic growth rate (SGR) and protein eciency ratio (PER) of juvenile Jian carp (Cyprinus carpio var Jian) fed diets with graded levels of histidine for 60 days1 Dietary histidine levels (g kg)1 diet) 2.3 IBW FBW SGR FI FE PER 8.77 53.6 3.02 66.5 0.68 2.12 4.4 a 0.01 1.0a 0.04a 0.1a 0.01a 0.05a 8.75 59.2 3.19 70.9 0.71 2.24 6.3 a 0.02 1.6c 0.04c 0.2c 0.03b 0.08b 8.77 64.9 3.34 74.6 0.76 2.37 8.6 a 0.01 0.9d 0.03d 0.2e 0.01c 0.04cd 8.75 68.0 3.42 76.0 0.78 2.46 10.8 a 0.02 1.6e 0.04e 0.2f 0.02c 0.07d 8.76 60.7 3.23 71.4 0.73 2.29 12.7 a 0.02 1.6c 0.04c 0.3d 0.03b 0.08bc 8.76 56.1 3.09 67.7 0.70 2.20 0.02a 0.9b 0.03b 0.2b 0.01ab 0.05ab All data were expressed as means SD (n = 4) Mean values within the same row with different superscripts are significantly different (P < 0.05) Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd 0.85 y = 0.003x2 + 0.047x + 0.5763 R2 = 0.7204 Body composition and PPV of juvenile Jian carp fed diets containing graded levels of histidine are presented in Table 3.50 y = 0.0121x2 + 0.1892x + 2.6269 R2 = 0.8962 3.40 SGR 3.30 3.20 X = 7.8 3.10 0.75 0.70 0.65 X = 7.8 0.60 0.55 0.50 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Dietary histidine levels (g kg1 diet) Figure Quadratic regression analysis of feed efciency for juvenile Jian carp (Cyprinus carpio var Jian) fed diets containing graded levels of histidine for 60 days Protein efficiency ratio Eects of graded concentrations of dietary histidine on growth parameters are given in Table Signicant dierences were observed in SGR, FI, FE and PER Fish receiving diet with 8.6 g kg)1 histidine obtained a maximum SGR that is signicantly higher than other dietary treatments (P < 0.05) As shown in Fig 1, on subjecting the SGR data and dietary histidine levels to second-degree polynomial regression analysis, optimum histidine level was found at 7.8 g kg)1 diet or 2.38 g 100 g)1 protein The relationship was described by the following equation: YSGR = )0.0121x2 + 0.1892x + 2.6269, R2 = 0.8962, P < 0.01 FI was signicantly enhanced with increasing dietary histidine levels up to 8.6 g kg)1 diet and decreased thereafter (P < 0.05) The FI to dietary concentrations of histidine relationship was estimated by the following quadratic regression equation: YFI = )0.3078x2 + 4.7535x + 56.788, R2 = 0.9576, P < 0.01 FE and PER were improved with the increase in dietary histidine levels and were signicantly higher in sh fed diets with 6.3 and 8.6 g kg)1 histidine compared with other groups (P < 0.05) The FE and PER to dietary levels of histidine relationship were described by quadratic regression analysis, respectively (YFE = )0.003x2 + 0.047x + 0.5763, R2 = 0.7204, P < 0.01; YPER = )0.0093x + 0.1479x + 1.8103, R2 = 0.7287, P < 0.01) (Figs and 3) Based on the above equations, the best FE and PER occurred at histidine levels of 7.8 and 8.0 g kg)1 diet, respectively Feed efficiency 0.80 2.70 y = 0.0093x2 + 0.1479x + 1.8103 R2 = 0.7287 2.50 2.30 2.10 X = 8.0 1.90 1.70 1.50 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Dietary histidine levels (g kg1 diet) Figure Quadratic regression analysis of protein efciency ratio for juvenile Jian carp (Cyprinus carpio var Jian) fed diets containing graded levels of histidine for 60 days The whole body lipid was lower in sh fed the diet with histidine concentration at 2.3 g kg)1 (unsupplemented control) than any other groups (P < 0.05) The protein content of sh carcasses also increased signicantly with dietary histidine levels higher than 2.3 g kg)1 (P < 0.05), except for the group receiving dietary histidine at 10.8 g kg)1 However, no signicant dierences in body moisture and ash content among the groups were evident (P > 0.05) The PPV was signicantly improved with increasing dietary histidine levels up to 8.6 g kg)1 diet and thereafter decreased (P < 0.05) The relationship between PPV and dietary concentrations of histidine was described by the following second-degree polynomial regression equation: YPPV = )0.1454x2 +2.3971x + 21.449, R2 = 0.7933, P < 0.01 3.00 2.90 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 Dietary histidine levels (g kg1 diet) Figure Quadratic regression analysis of specic growth rate for juvenile Jian carp (Cyprinus carpio var Jian) fed diets containing graded levels of histidine for 60 days As shown in Table 4, the activities of GOT and GPT and plasma ammonia content were signicantly aected by dietary histidine The activities of GOT in muscle and Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd Table Body composition (g kg)1) and protein productive value (PPV) of juvenile Jian carp fed diets with graded levels of histidine for 60 days1 Dietary histidine levels (g kg)1 diet) 2.3 Moisture Protein Lipid Ash PPV 735 126 104 25.7 26.3 4.4 a 21 3a 11a 1.1a 0.7a 732 130 115 24.6 28.9 6.3 a 4b 6b 1.5a 1.0b 726 131 118 25.2 30.8 8.6 a 12 3b 4b 0.6a 0.9c 726 132 119 25.2 32.2 10.8 a 1b 6b 1.0a 0.2d 727 130 120 25.1 29.3 12.7 a 1ab 6b 1.5a 0.4b 727 132 119 25.6 28.8 20a 2b 9b 2.0a 0.6b Values are means SD of four replicate groups, with four fish in each group Mean values with the different superscripts in the same row are significantly different (P < 0.05) Table The activities of glutamate-oxaloacetate transaminase (GOT, U g)1 tissue) and glutamate-pyruvate transaminase (GPT, U g)1 tissue) in muscle and hepatopancreas and plasma ammonia concentration (PAC, lmol L)1) of juvenile Jian carp fed diets with diets with graded levels of histidine for 60 days Dietary histidine levels (g kg)1 diet) GOT1 Muscle Hepatopancreas GPT1 Muscle Hepatopancreas PAC2 2.3 4.4 6.3 8.6 10.8 12.7 7151 373d 4112 162d 6688 437c 3121 125c 6135 308b 3067 187bc 5618 442a 2354 224a 6278 381bc 2871 171b 7169 345d 3032 146bc 772 48a 2355 130a 217 23c 1040 72b 2704 190b 207 10bc 1674 143d 2713 131b 150 20a 1892 108e 4087 262c 183 22b 1451 52c 4077 304c 181 17b 1112 57b 3922 505c 203 12bc Mean values of four replicate groups, with six fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) Mean values of four replicate groups, with five fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) hepatopancreas decreased signicantly with increasing histidine levels up to 8.6 g kg)1 diet and thereafter increased (P < 0.05) Regression analysis showed that GOT activities were signicantly quadratic response to graded levels of dietary histidine [YGOT (muscle) = 48.507x2 ) 753.15x + 8802.6,R2 = 0.6315, P < 0.01; YGOT (hepatopancreas) = 34.221x2 ) 609.28x + 5299.9, R2 = 0.8216, P < 0.01] The trend of muscle GPT activity was properly opposite to that in GOT[YGPT (muscle) = )30.692x2 + 504.51x ) 339.62, R2 = 0.8418, P < 0.01] The hepatopancreas GPT activity was signicantly improved with increasing dietary histidine levels up to 8.6 g kg)1 diet (P < 0.05), where the response reached a plateau The second-degree polynomial regression equation about the relationship between hepatopancreas GPT activity and dietary histidine levels was presented as following: YGPT (hepatopancreas) = )10.936x2 + 347.67x + 1455.1, R2 = 0.7392, P < 0.01 The concentration of plasma ammonia decreased signicantly with the increase in histidine levels up to 6.3 g kg)1 diet and thereafter increased (P < 0.01) Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd Hepatopancreas weight (HW), HSI, HPC, intestine length (IL), RGL, IW, ISI and IPC of juvenile Jian carp fed diets containing graded levels of histidine are given in Table HW and IW were signicantly enhanced with the increase in dietary histidine levels up to 10.8 and 8.6 g kg)1, respectively, and then decreased (P < 0.05) The relationship between HW and IW and dietary histidine levels were described by the following quadratic equation, respectively: YHW = )0.022x2 + 0.3475x + 1.1697, R2 = 0.8523, P < 0.01; YIW = )0.0134x2 + 0.2108x + 1.3772, R2 = 0.5874, P < 0.01 The IL was the highest in sh fed diet with dietary histidine at 8.6 g kg)1, and no signicant dierences were found among the other groups (P > 0.05) The HPC and IPC also showed similar trend HSI, RGL and ISI were not inuenced by dietary histidine levels (P > 0.05) The intestinal folds heights in all intestinal segments are presented in Table The folds Table Hepatopancreas weight (HW, g sh)1), intestine weight (IW, g sh)1), intestine length (IL, cm sh)1), intestosomatic index (ISI), hepatosomatic index (HSI), relative gut length (RGL), hepatopancreas protein content (HPC) and intestine protein content (IPC) of juvenile Jian carp fed diets with graded levels of histidine for 60 days Dietary histidine levels (g kg)1 diet) 2.3 Hepatopancreas HW1 1.88 HSI1 3.50 HPC2 3.30 Intestine IL1 21.8 RGL1 170 IW1 1.86 ISI1 3.46 IPC2 2.35 0.19a 0.23a 0.11a 1.3a 11a 0.18a 0.32a 0.20a 4.4 6.3 8.6 10.8 12.7 2.21 0.20bc 3.74 0.18a 3.39 0.17a 2.51 0.26d 3.88 0.25a 3.43 0.32ab 2.55 0.30d 3.75 0.33a 3.72 0.27b 2.36 0.24cd 3.90 0.16a 3.21 0.27a 2.03 0.17ab 3.62 0.27a 3.25 0.16a 21.6 165 1.95 3.30 2.37 1.5a 9a 0.15ab 0.25a 0.13a 22.1 165 2.08 3.21 2.49 1.9a 8a 0.25b 0.34a 0.06a 24.0 170 2.38 3.50 2.70 1.1b 14a 0.18c 0.42a 0.07b 22.0 169 2.05 3.37 2.48 1.5a 9a 0.20b 0.33a 0.19a 21.8 173 1.87 3.34 2.45 1.4a 9a 0.23a 0.41a 0.18a Mean values of four replicate groups, with 15 fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) Mean values of four replicate groups, with six fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) Table Folds height (lm) in proximal intestine (PI), midintestine (MI) and distal intestine (DI) of juvenile Jian carp fed diets with graded levels of histidine for 60 days1 Dietary histidine levels (g kg)1 diet) 2.3 PI MI DI 4.4 a 777 53 425 35a 435 18a 6.3 b 830 41 451 40b 439 25a 8.6 cd 888 30 528 22d 493 24b 10.8 d 908 33 492 17c 512 28c 12.7 c 883 27 426 17a 434 33a 847 38b 434 17ab 423 22a Mean values of four replicate groups, with four fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) heights of proximal intestine (PI), midintestine (MI) and distal intestine (DI) were signicantly improved with dietary histidine levels up to 8.6, 6.3 and 8.6 g kg)1 diet, respectively, and thereafter declined (P < 0.05) Regression analysis suggested that intestinal folds height in PI extreme signicantly quadratically responded to the increase in dietary histidine levels (Yintestinal folds height in PI = )3.2414x2 + 55.946x + 659.93, R2 = 0.9124, P < 0.01) Marked dierences were observed in the activities of digestive enzymes in sh fed diets with graded levels of histidine (Table 7) The activities of trypsin, chymotrypsin, lipase and amylase in both hepatopancreas and intestine signicantly increased with dietary histidine levels up to 8.6 g kg)1 diet and thereafter declined (P < 0.05) Digestive enzymes activities in hepatopancreas were positive correlated with that in intestine (rtrypsin = +0.873, P < 0.05; rchymotrypsin = +0.887, P < 0.05; rlipase = +0.803, P = 0.05; ramylase = +0.832, P < 0.05) The activities of digestive enzymes in intestine to dietary concentrations of histidine relationships were estimated by the following quadratic regression equations: Yintestinal trypsin = )0.0221x2 + 0.3858x ) 0.2417, R2 = 0.6718, P < 0.01; Yintestinal chymotrypsin = )0.0517x2 + 0.7905x + 1.0816, R2 = 0.7277, P < 0.01; Yintestinal lipase = )75.914x2 + 1302.4x ) 589.08, R2 = 0.7283, P < 0.01; Yintestinal amylase = )6.1724x2 + 102.04x + 904.03, R2 = 0.6652, P < 0.01 Based on the above equations, the maximum activities of intestinal trypsin, chymotrypsin, lipase and amylase occurred at dietary histidine concentration of 8.7, 7.6, 8.5 and 8.3 g kg)1 diet, respectively Eects of graded levels of dietary histidine on the activities of brush border enzymes in PI, MI and DI are presented in Table The activity of AKP in PI of the sh fed diet containing 2.3 g kg)1 (unsupplemented control) histidine was found to be signicantly lower than those fed other dietary levels (P < 0.05) AKP activities in mid- and DI signicantly increased with increasing histidine levels up to 8.6 g kg)1 diet and thereafter decreased (P < 0.05) The activities of Na+, Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd Table The activities of trypsin (U g)1 tissue), chymotrypsin (U g)1 tissue), lipase (U g)1 tissue) and amylase (U g)1 tissue) in hepatopancreas and whole intestine of juvenile Jian carp fed diets with graded levels of histidine for 60 days1 Dietary histidine levels (g kg)1 diet) 2.3 Hepatopancreas Trypsin Chymotrypsin Lipase Amylase Intestine Trypsin Chymotrypsin Lipase Amylase 4.4 6.3 8.6 10.8 12.7 0.45 2.36 2162 1003 0.04a 0.13a 167b 45a 0.79 2.92 2595 1044 0.06b 0.09b 205cd 37a 0.94 3.83 2756 1184 0.08c 0.26d 178d 44c 1.23 4.92 3783 1198 0.09d 0.17e 335e 43c 0.73 3.50 2378 1116 0.03b 0.11c 167bc 32b 0.72 2.83 1838 1026 0.04b 0.28b 167a 40a 0.68 2.72 2208 1101 0.06a 0.23a 187a 43a 0.78 3.56 3426 1241 0.06a 0.29b 250b 35bc 1.19 3.64 4112 1290 0.07c 0.34b 409c 44c 1.79 4.33 6015 1372 0.13d 0.32c 344d 39d 1.26 3.75 4036 1222 0.12c 0.33b 344c 52b 1.06 2.64 3807 1232 0.10b 0.19a 236c 40b Mean values of four replicate groups, with five fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) Table The activities of alkaline phosphatase (AKP, mmol of nitrophenol released g)1 tissue h)1), Na+, K+-ATPase (lmol of phosphorus released g)1 tissue h)1), c-glutamyl transpeptidase (c-GT, mmol of 5-amino-2-nitrobenzoate released g)1 tissue min)1) and creatine kinase (CK, lmol of phosphorus released g)1 tissue h)1) in proximal intestine (PI), midintestine (MI) and distal intestine (DI) of juvenile Jian carp fed diets with graded levels of histidine for 60 days1 Dietary histidine levels (g kg)1 diet) 2.3 AKP PI 761 MI 445 DI 78.3 Na+, K+-ATPase PI 163 MI 303 DI 163 c-GT PI 32.8 MI 39.3 DI 48.7 CK PI 275 MI 149 DI 113 4.4 6.3 8.6 10.8 12.7 72a 38a 4.5a 1182 88b 723 70b 89.1 3.0ab 1139 83b 801 78b 114 9c 1153 98b 912 45c 217 12e 1068 60b 725 59b 166 15d 1067 97b 729 52b 96.1 9.4b 9a 15b 11a 320 18d 296 20b 161 11a 411 25e 320 25b 277 17c 282 21c 297 15b 266 20c 229 16b 299 24b 261 14c 215 13b 269 15a 216 15b 2.7a 2.2a 4.0bc 38.1 2.6b 40.6 3.2ab 49.7 3.7bc 39.3 2.4b 43.9 2.9b 53.2 4.8c 57.1 36d 49.4 3.3c 69.1 5.1d 58.9 5.5d 51.5 3.9c 48.0 3.7b 45.6 3.3c 39.6 1.5a 32.5 2.0a 19b 8b 9c 320 11c 145 10b 121 7c 314 21c 149 13b 114 9c 325 20c 160 13c 140 6d 167 14a 122 5a 92 6b 178 13a 118 8a 61 5a Mean values of four replicate groups, with five fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) K+-ATPase in proximal and DI were signicantly improved with dietary histidine levels from 2.3 (control) up to 6.3 and 10.8 g kg)1 diet, respectively, and thereafter declined (P < 0.05) Na+, K+-ATPase in MI did not dier among the treatments, excepting at 12.7 g kg)1 dietary histidine level where a signicantly lower activity was observed (P < 0.05) The activities of c-GT in proximal, mid- and DI signicantly increased with dietary histidine levels up to 10.8, 10.8 and 8.6 g kg)1, respectively, and decreased with levels further increasing (P < 0.05) The activities of CK at 10.8 and 12.7 g kg)1 dietary histidine levels in all intestinal segments Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd were signicantly lower than other dietary treatments (P < 0.05) The amounts of Lactobacillus, E coli and Aeromonas in the intestine are presented in Table Fish fed diets with 2.3 and 4.4 g kg)1 histidine had signicantly lower amounts of Lactobacillus than those fed other dietary levels (P < 0.05) The amount of E coli was highest at 12.7 g kg)1 dietary histidine followed by those receiving diet containing Table Effect of diets containing graded levels of histidine on Lactobacillus (LB, log CFU g)1 intestine content), Escherichia coli (EC, log CFU g)1 intestine content) and Aeromonas (log CFU g)1 intestine content) in whole intestine of juvenile Jian carp1 Dietary histidine levels (g kg)1 diet) 2.3 LB EC Aeromonas 4.4 a 6.99 0.08 6.90 0.13ab 9.32 0.03c 6.3 a 7.11 0.06 6.85 0.06ab 9.08 0.04b 8.6 b 7.34 0.10 6.77 0.03a 9.04 0.01b 10.8 c 7.49 0.03 6.75 0.10a 8.92 0.03a 12.7 bc 7.41 0.09 6.98 0.11b 9.28 0.14c 7.39 0.04bc 7.33 0.11c 9.68 0.05d Mean values of four replicate groups, with three fish in each group Mean values within the same row with different superscripts are significantly different (P < 0.05) 10.8 g kg)1 histidine (P < 0.05) The amount of Aeromonas decreased signicantly with the increase in dietary histidine levels up to 8.6 g kg)1 diet and thereafter increased (P < 0.05) The present study showed that growth performance was signicantly inuenced by dietary histidine levels Juvenile Jian carp fed the histidine-decient diet obtain poor weight gain and SGR, which was also found in common carp (Nose 1979) and Indian major carp (L rohita) (Murthy & Varghese 1995) In these studies, the growth-depressing eect of feeding higher amounts of histidine than optimum has also been noted, which may be partly attributed to that excess levels of essential amino acid (EAA) in diets results in higher levels of ammonia-N excretion (Yang et al 2002) Based on the quadratic regression analysis for SGR, the requirement of histidine for juvenile Jian carp (8.7668.02 g) was estimated to be 7.8 g kg)1 diet or 2.38 g 100 g)1 protein, which was higher than that for common carp (Nose 1979) This may be attributed to the fast-growing characteristic of Jian carp compared to common carp (Sun et al 1995) It is consistent with the ndings of lysine (Zhou et al 2008) and methionine (Tang et al 2009) requirement for juvenile Jian carp Correlation analysis showed that SGR was positive correlated to FI (r = +0.997, P < 0.01) and FE (r = +0.990, P < 0.01) The enhancement of sh growth in this experiment may be related to the fact that FI and FE were improved with optimum dietary histidine level, which was in agreement with the results in chum salmon (Akiyama et al 1985) and Atlantic salmon (S salar L.) (Breck et al 2005) Kasaoka et al (2004) reported that food intake of rats decreased with increased dietary histidine and suggested that dietary histidine suppresses food intake possibly by activated histamine neurons Quadratic regression analyses for FE and PER data indicated the optimum dietary histidine requirement at 7.8 and 8.0 g kg)1 diet, respectively, which were close to that analysed for SGR In the present study, both body protein and lipid content of sh were improved with histidine supplementation The body moisture and ash content were not aected by dietary histidine levels, which were also reported in Indian major carp (L rohita) (Abidi & Khan 2004) The major part of the weight increase is related to the deposition of protein (Cowey 1994), which is an important tool in studying sh FE (Belal 2005) The PPV in the present study increased with increasing dietary histidine levels up to 8.6 g kg)1 diet, just supporting the highest FE in this group and were in accordance with the results in Indian major carp (Ci mrigala) (Ahmed & Khan 2005) and African catsh (Cl gariepinus) (Khan & Abidi 2009) Protein deposition is the sum of two continuous processes, synthesis and degradation (Kaushik & Seiliez 2010) The improvement of protein deposition in our study might be related to protein and amino acid metabolism GOT and GPT exist in hepatic and muscle cells widely and play an important role in protein metabolism (Ramaswamy et al 1999) The activity of these transaminases could be good indices of EAA utilization (Balogun & Fetuga 1980) The present results showed that GOT activity in hepatopancreas and muscle exhibited negative quadratic relationship, respectively, with dietary histidine levels Ramaswamy et al (1999) suggested that sh adaptively elevate the activity levels of transaminases, particularly in liver and muscle, thereby probably aiding gluconeogenesis through transamination of glucogenic amino acids to meet the energy demand The catabolism of protein for energy and for synthesis of glucose reduces protein retention and increases the nitrogen release (Lee et al 2003) The production of ammonia is directly related to the rate of protein catabolism (Waarde 1983) Amino acid imbalance of diets had a signicant eect on plasma ammonia levels (Fournier et al 2003) In the present study, plasma ammonia concentration was lowest at the optimum dietary histidine level, indicating that the diet contained more sucient or balanced amino acids Correlation analysis showed that PPV was negative correlated to GOT activities in hepatopancreas (r = )0.915, P < 0.05), GOT activities in muscle (r = )0.891, P < 0.05) and plasma ammonia content Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd (r = )0.712, P = 0.11), respectively This nding suggested that sh fed with optimum dietary histidine may utilize much more amino acid for protein retention but not for energy source Interestingly, the GPT activities both in hepatopancreas and muscle were enhanced with dietary histidine up to an optimum level A possible explanation could be that GPT is related to the synthesis of carnosine (b-alanine-L-histidine) Studies on mammals showed that carnosine is an important metabolism product of histidine and is mainly synthesized from its constituent amino acids in muscle and liver (Stifel & Herman 1971), whereas the transamination mediated by GPT can produce alanine (Goldstein & Newsholme 1976) The enhanced growth of sh obtained from live food has been partially attributed to the digestive enzymes activity (Kolkovski et al 1993) Trypsin and chymotrypsin, which belong to the serine peptidase family with a catalytic triad (serine, histidine and aspartic) in their catalytic sites, are important enzymes in protein digestion (Polgar 2005) Histidine residue serves for transferring the proton from serine to aspartic, which is a necessary step in catalytic reaction (Polgar 2005) Functional roles of histidine residues at the active site in human pancreatic a-amylase were examined (Ishikawa et al 1992) Little is known about the relationship between dietary histidine and the activities of trypsin, chymotrypsin and amylase In our study, trypsin, chymotrypsin and amylase activities in intestine were improved by dietary histidine Intestinal lipase also followed the same trend The interactions of histidine with transition metal ions have been established (Sundberg & Martin 1974) Positive action of histidine on the uptake of zinc in rainbow trout was reported by Glover et al (2003), whereas dietary zinc supplementation could improve the activities of intestinal digestive enzymes in juvenile Jian carp (Tan et al 2011) Thus, digestive enzymes activities may be aected by dietary histidine through the interaction with zinc The dietary histidine requirements estimated by quadratic regression analysis for the activities of intestinal trypsin, lipase and amylase (8.7, 8.5 and 8.3 g kg)1 diet, respectively) were higher than those analysed for SGR (7.8 g kg)1 diet), whereas those analysed for intestinal chymotrypsin activity (7.6 g kg)1 diet) were a little lower The exocrine cells of pancreas secrete enzymes into the digestive tract (Slack 1995) The increase in intestine digestive enzymes activities by histidine may partly because of the promotion of pancreatic digestive enzymes activities Our results showed that there is a positive correlation between digestive enzymes activities in hepatopancreas and that in intestine The ontogenetic development of hepatopancreas was correlated with the digestion ability of sh (Gisbert et al 2004) In our study, HW and protein content were improved by histidine Aquaculture Nutrition 18; 220232 ể 2011 Blackwell Publishing Ltd supplementation, which suggested that histidine could enhance the growth of hepatopancreas The integrity of hepatopancreas is necessary for its growth and development (Hamlin et al 2000) The histological changes of liver are important indicators of the nutritional and physiological status of sh (Gatta et al 2011) It has been noted that an optimum level of dietary histidine has a benet to keep the histological structure of hepatopancreas epithelial cells in shrimp and prawns (Recodo 1991; Recodo et al 2000) Histidine has also been identied as a unique factor to control the integrity of rat liver mitochondria (Connelly & Myron 1971) The positive eects of dietary histidine on the activities of pancreatic digestive enzymes may partially be attributed to the enhanced growth of hepatopancreas The enzymes located in the brush border section of the intestine are responsible for the nal stages of degradation and assimilation of the food (Klein et al 1998) AKP is considered to be involved in absorption of lipid, glucose, calcium and inorganic phosphate (Villanueva et al 1997) CK is involved in energy metabolism and the couple of ATP and kinase (Wallimann & Hemmer 1994) Histidine serves as an essential component of the active centre in AKP and CK (Schneider 1978) However, no study has been conducted to investigate the eect of dietary histidine on the activities of AKP and CK In the present study, histidine increased the activities of AKP and CK Na+, K+-ATPase is associated with a number of sodium-coupled transport events across the apical domain of the cell (Sweeney & Klip 1998) c-GT is also an enzyme localized at intestinal brush border and involved in amino acid transport via the c-glutamyl cycle (Bell et al 1987) Our results suggested that the activities of Na+, K+ATPase and c-GT were improved by dietary histidine The benets of dietary histidine on sh brush border enzyme activities were probably due to the improvement of intestine growth and development In the present study, intestinal length, weight and protein content increased with increasing dietary histidine levels up to the optimum level Moreover, intestinal folds height in PI, MI and DI was improved by dietary histidine Caballero et al (2003) suggested that maintaining integrity of the intestine should be a major concern to optimize feed utilization Histological damages in the midgut gland of prawns were observed with sub-optimal or super-optimal levels of histidine in the diet (Recodo et al 2000) The structural integrity of sh intestinal epithelial cells was partly related to the antioxidant ability of cells (Chen et al 2009) Son et al (2005) reported histidine could inhibit the inammation induced by oxidative stress in human intestinal epithelial cells Unfortunately, little is known about the eect of dietary histidine on the intestinal antioxidant status, which awaits investigation The ability of lactobacilli to adhere to the gastrointestinal mucosa has been suggested to inuence their interaction with the host and the other bacteria present, by aecting the local microbial composition and/or by stimulating the hostếs immune system (Deepika & Charalampopoulos 2010) Escherichia coli in intestine were pathogens with the particular potential of causing enteric infection (Rio-Rodriguez et al 1997) Aeromonas species are regarded as enteropathogens and in some cases are linked to gastroenteritis (Merino et al 1995) The current study showed that histidine could inuence the balance of intestinal microora by promoting the growth of Lactobacillus and depressing the growth of E coli and Aeromonas A similar tendency was reported in a previous study from our laboratory, in which the eect of methionine on sh intestinal microora has been considered (Tang et al 2009) However, the mechanism about eects of histidine on intestinal microora of sh is still unknown In summary, the present results demonstrated that dietary histidine improved the growth performance and protein deposition, enhanced intestinal enzymes activities and optimized the intestinal microora of Jian carp Meanwhile, the dietary histidine requirement for maximum growth of juvenile Jian carp (8.7668.02 g) was determined to be 7.8 g kg)1 diet or 2.38 g 100 g)1 protein by quadratic regression analysis However, further studies should be carried out to reveal the mechanisms underlying eects of dietary histidine on sh intestinal enzymes activities and intestinal microora This research was nancially supported by the National Department Public Benet Research Foundation (Agriculture) of China (201003020) and Program for New Century Excellent Talentsin University (NCET-08-0905) The authors would like to thank the personnel of these teams for their kind assistance Abidi, S.F & Khan, M.A (2004) Dietary histidine requirement of ngerling Indian major carp, Labeo rohita (Hamilton) Isr J Aquac Bamidgeh, 56, 200208 Ahmed, I & Khan, M.A (2005) Dietary histidine requirement of ngerling Indian major carp, Cirrhinus mrigala (Hamilton) Aquacult Nutr., 11, 359366 Akiyama, T., Arai, S., Murai, T & Nose, T (1985) Threonine, histidine and lysine requirements of chum salmon fry Bull Jpn Soc Sci Fish., 51, 635639 AOAC (Association of Ocial Analytical Chemists) (1998) Ocial 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