Aquaculture nutrition, tập 17, số 6, 2011

Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00904.x 2011 17; 585–594 1 2 Department of Biology, University of Bergen, Bergen, Norway; (NIFES), Bergen, Norway Vitamin K belongs to the lipid soluble vitamins, and occurs naturally as phylloquinone (vitamin K1) and menaquinone (vitamin K2) In addition, there is a synthetic provitamin, menadione (vitamin K3), primarily used as a vitamin K source in animal feed Menadione is unstable during feed processing and storage and the dietary content may reach critically low levels Recent publications also question the availability of menadione in feed for salmonids Vitamin K plays vital roles in blood coagulation and bone mineralization in fish, but the suggested minimum requirement varies considerably depending on the vitamin K source used Vitamin K deficiency is characterized by mortality, anaemia, increased blood clotting time and histopathological changes in liver and gills However, one should assess both inherent and supplemented forms of vitamin K in feeds for exact determinations, as relevant novel feed ingredients of plant origin may be sufficient to meet the requirement for vitamin K The current review gives an overview of the biochemical role of vitamin K, and discusses vitamin K requirement in fish in light of updated literature, with special emphasis on salmonids key words: fish, menadione, menaquinone, phylloquinone, requirement, vitamin K Received 31 January 2011; accepted 28 July 2011 Correspondence: Christel Krossøy, Department of Biology, University of Bergen, PBox 7803, N-5020 Bergen, Norway E-mail: Christel.Krossoy@ bio.uib.no Fish, like all other animals, need a certain amount of vitamins for optimal growth and proper health that vary Ó 2011 Blackwell Publishing Ltd National Institute of Nutrition and Seafood Research according to factors like nutritional status, external stressors, age and health status Vitamin requirements published by the NRC (1993) usually designate minimum requirements as the vitamin level required to avoid clinical deficiency signs and support normal growth (Woodward 1994) There is a distinction between minimum requirement and requirement for optimal growth or optimal health, which could lead to the definition of higher requirement or recommendation levels adapted to a specific function or to certain conditions In intensive commercial fish farming, the last decade has brought with it changes in genetics, husbandry and diet composition leading to increased growth rates and subsequently changes in the minimal requirement of micronutrients (Waagbø 2008) However, detailed evaluations of the nutrient requirements for fish have not kept pace with the changes as most of the vitamin requirements of salmonids were determined more than 30 years ago It is thus unclear if the given requirements are appropriate for modern diet formulations The earliest requirement studies on fish were performed in an effort to increase the survival of the stock in juvenile stages Test diets and growth rates were not comparable to commercial rearing, and the response criteria used were mostly survival, weight gain, absence of deficiency signs and maximum tissue storage The latter resulted in relatively high requirement estimates, but the cost of adding too high levels of vitamins were lower than the cost of suffering high mortalities As commercial farming became more efficient, more sensitive response criteria for vitamins were used, some measuring metabolically active forms and specific enzyme activity This lowered the recommendations for most vitamins (Woodward 1994) Historically, vitamin K is best known for its essential role in blood coagulation (Olson 1999), being responsible for the posttranslational modification and activation of the vitamin K-dependent (VKD) proteins (Knapen et al 1993; Luo et al 1997; Boskey et al 1998; Lee et al 2007), and the first VKD proteins identified were those involved in vitamin K haemostasis (Nelsestuen et al 1974; Stenflo et al 1974; Ferland 1998) In the past few decades, it has become clear that vitamin K plays an important role in other biological processes, such as bone metabolism and growth control (Price 1988; Manfioletti et al 1993) The diverse range of functions of VKD proteins implicates a broad biological impact of vitamin K (Berkner 2008), but the exact roles of vitamin K and VKD proteins have been difficult to assess, and the physiological consequences of non-carboxylated and undercarboxylated proteins are unknown Estimates of dietary vitamin K requirement differ widely among fish species, and the quantitative requirement of vitamin K for most fish is still unknown (NRC 1993) In the current review, we will give an overview of the biochemical role of vitamin K, and discuss vitamin K requirement in fish in light of updated literature with special emphasis on salmonids However, differences in experimental design, fish species, developmental stage, biomarkers, as well as inclusion level and forms makes the published studies challenging to compare Overall, the minimum requirement of vitamin K has been difficult to estimate owing to natural occurrence in feed ingredients, feed processing and storage stability of inherent and added vitamin K, vitamin leaching, variable feed intakes and variable bioavailability of the different K vitamers Lipid soluble vitamin K was first discovered by the Danish scientist Henrik Dam in 1929 as an antihaemorrhagic factor in chicks (Olson 1999) The factor was later shown to be related to the absence of prothrombin activity in plasma For decades, it was believed that the only function of vitamin K was in the coagulation cascade, but several vitamin K dependent proteins have now been isolated from bone, dentin, cartilage, kidney, atherosclerotic plaque and numerous soft tissues (Vermeer et al 1995, 1996; Shearer et al 1996; Booth 1997; Ferland 1998) Vitamin K refers to a family of compounds derived from quinone, that share a common 2-methyl-1,4-naphthoquinone ring, but differ in the side chain at the C3-position (Lambert & De Leenher 1992) All vitamers K are insoluble in water, slightly soluble in alcohol and readily soluble in non-polar organic solvents (Koivu-Tikkanen 2001) They have a relatively high thermostability (Lambert & De Leenher 1992), but are sensitive to light and alkaline conditions (KoivuTikkanen 2001) There are at least two naturally occurring forms of vitamin K, designated vitamin K1 and K2 Vitamin K1 (phylloquinone; 2-methyl-3-phytyl-1,4-naphthoquinone, (a) (b) (c) Figure The chemical structures of (a) vitamin K1 (phylloquinone): 2-methyl-3-phytyl-1,4-naphthoquinone; (b) vitamin K2 (menaquinones): 2-methyl-3-(prenyl)n-1,4-naphthoquinone; and (c) vitamin K3 (menadione): 2-methyl-1,4-naphthoquinone Fig 1a) is synthesized by plants, and is mainly found in green leafy vegetables (Booth & Suttie 1998) Phylloquinone has a phytyl group with one double bond in the side chain Vitamin K2 (menaquinones; MK; 2-methyl-3-(prenyl)n-1,4-naphthoquinone, Fig 1b), on the other hand, is primarily of microbial origin, and is found in fermented products and in foods of animal origin (Booth & Suttie 1998) Menaquinones include a range of vitamin K forms, named according to the number (n) of prenyl groups in the unsaturated side chain, thus designated MK-n, with n ranging from to 14 (Lambert & De Leenher 1992) Menaquinone-4 (MK-4) and MK-7 are the most relevant nutritional menaquinones (Fodor et al 2010) Of these, MK-4 is unique as it is the product of certain tissue-specific conversions directly from dietary phylloquinone (Thijssen & Drittij-Reijnders 1994; Ronden et al 1998; Okano et al 2008) Menaquinones may be synthesized by bacteria in the gut (Conly & Stein 1993), and the requirement of vitamin K in mammals is met by a combination of dietary intake and intestinal bacterial synthesis Both diet composition and the use of antibiotics are known to affect intestinal production (Mathers et al 1990) The quantitative significance and role of menaquinones produced by the intestinal microflora in maintaining vitamin K status is still unknown (Conly & Stein 1993; Suttie 1995; Vermeer et al 1995), but bacterially derived long-chain menaquinones have been found in human liver (Usui et al 1989; Thijssen & DrittijReijnders 1996) However, the importance of intestinal production of vitamin K or the effect of antibiotics has not been established in fishes or crustaceans (Tan & Mai 2001) Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone, Fig 1c) are chemically synthesized vitamin K compounds used in commercial feeds for domestic animals It is a vitamin K derivate in the form of water soluble salts, like menadione Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd sodium bisulphite (MSB) and menadione nicotinamide bisulphite (MNB) Menadione has no side chain, and is chemically unstable compared to the naturally occurring vitamin K forms (Marchetti et al 1995, 1999) It is not itself biologically active and is easily excreted, but can at least be partly alkylated enzymatically to MK-4 in tissues when present in animal feeds (Dialameh et al 1971; Udagawa 2000; Graff et al 2002, 2010; Okano et al 2008; Krossøy et al 2009a) Most of the work within vitamin K research has been conducted on humans and laboratory animals It was long thought that the role of vitamin K was limited to the synthesis of factors within the coagulation system, but the discovery of vitamin K as a cofactor and the identification of additional VKD proteins, significantly expanded the understanding of its physiological roles (Stenflo et al 1974; Suttie 1992; Ferland 1998; Vermeer et al 1998) Key VKD proteins include coagulation proteins, anticoagulation proteins and bone proteins, in addition to the VKD growth factor growtharrest-specific-6 (Gas6, Table 1; Suttie 1992; Ferland 1998) Calcium binding is essential for the activation of the seven VKD proteins that mediate blood coagulation and anticoagulation Coagulation factors II (prothrombin), VII, IX and X make up core actors of the coagulation cascade, while proteins C, S and Z belong to the anticoagulation proteins With the exception of protein S, which is also synthesized by osteoblasts, these proteins are produced exclusively in the liver (Ferland 1998) Blood clotting follows the same fundamental pattern in both mammals and teleosts, generating thrombin by pathways involving VKD factors (see Hanumanthaiah et al 2002 and Jiang & Doolittle 2003; and references cited therein) In addition to protein S, the VKD proteins found in bone are bone Gla-protein (BGP; synonym for osteocalcin) and matrix Gla-protein, MGP (Vermeer Table Vitamin K-dependent (VKD) proteins Coagulation proteins Anticoagulation proteins Bone proteins Other proteins Prothrombin (Factor II) Factor VII Factor IX Factor X Protein C Protein S Protein Z Bone Gla Protein Matrix Gla Protein Growth-arrest-specific-6 Gla-rich Protein Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd et al 1995; Ferland 1998) Although the exact role of BGP is not clear, it is suggested to function as a regulator of bone formation and bone mineral maturation (Ducy et al 1996; Boskey et al 1998) BGP is produced by osteoblasts and odontoblasts only (Dimuzio et al 1983) The protein was originally isolated from bovine bone where it was shown to inhibit the formation of hydroxyapatite (Price et al 1976) Vitamin K is involved in the posttranslational modification of VKD proteins and acts as a cofactor for the enzyme c-glutamylcarboxylase (GGCX) GGCX catalyses the carboxylation of glutamic acid (Glu) residues in VKD proteins resulting in its conversion to c-carboxyglutamic acid (Gla) residues (Stenflo et al 1974) Although VKD c-carboxylation occurs only on specific Glu-residues in a small number of proteins, it is critical for the functionality of these proteins (Suttie 1992) Both phylloquinone and menaquinones act as co-factors in the GGCX mediated carboxylation (Buitenhuis et al 1990), where the naphthoquinone ring is the active site for the carboxylation reaction (Shea & Booth 2008) As a first step, vitamin K is reduced to vitamin K hydroquinone (KH2; Fig 2) The KH2 provides the energy to drive the carboxylation reaction, leading to formation of Gla residues and vitamin K epoxide (KO) KO is subsequently reduced by KO-reductase to vitamin K, in a process commonly called the vitamin K cycle (Ferland 1998; Berkner 2000; Stafford 2005) which conserves the available vitamin K very efficiently The resulting Gla domain formed from the carboxylation is a calcium-binding amino acid moiety required for the function of VKD proteins In the presence of calcium ions, these proteins undergo a structural transition leading to the exposure of a phospholipid (membrane) binding site Vitamin K deficiency leads to the occurrence of undercarboxylated proteins with Glu-residues, and are most often biologically inactive Lower VKD enzymatic activities or degree of VKD protein carboxylation can be used as markers for suboptimal vitamin K nutrition (Ferland 1998; Vermeer et al 1998; Furie et al 1999) Bone Gla-protein missing one or more Gla residues is termed under-carboxylated osteocalcin (ucOC), and the ratio between fully carboxylated and ucOC has been suggested as a sensitive marker for vitamin K deficiency (Vermeer et al 1995; Ferland 1998) In humans, a correlation between osteoporosis and ucOC has been found (Szulc et al 1994, 1996) When supplemented with vitamin K, the level of ucOC, bone resorption and urinary calcium secretion is reduced, while bone formation increases (Braam et al 2003) MGP, originally purified from mammalian bone (Price & Williamson 1985), is a small VKD protein synthesized by osteoblasts and a wide variety of other cells, like in turbot, Scophthalmus maximus (Roberto et al 2009) In Atlantic salmon (Salmo salar L.), BGP and MGP are expressed in vertebrae, as well as in fin, gills and scales, confirming the presence of vitamin K in bone, and suggesting involvement of vitamin K in bone metabolism of Atlantic salmon (Krossøy et al 2009b) The latest addition to the VKD family, is Gla-rich protein isolated from sturgeon (Acipenser nacarii) cartilage This VKD protein is highly expressed in chondroblasts, chondrocytes, osteoblasts and osteocytes, and is suggested to regulate calcium in the extracellular environment (Viegas et al 2008) Figure The vitamin K cycle: The vitamin K-dependent (VKD) c-carboxylation system consists of the vitamin K-dependent enzyme c-glutamylcarboxylase (GGCX) which requires the reduced vitamin K form, vitamin K hydroquinone (KH2), as a cofactor and the enzyme vitamin K 2,3-epoxide reductase (KO-reductase) Vitamin K is reduced to KH2 by KO-reductase The GGCX converts glutamic acid (Glu) residues in VKD proteins to c-carboxyglutamic acid (Gla) residues by adding CO2 to newly synthesized proteins, using KH2 as a cofactor for the posttranslational reaction The conversion of KH2 to vitamin K 2,3-epoxide (KO) coincide with the c-carboxylation The epoxide is subsequently reduced back to vitamin K by KOreductase, ready to enter another cycle (Enzyme nomenclature adapted from http://www.chem.qmul.ac.uk/iupac/iupac.html) chondrocytes and vascular smooth muscle cells It contains five Glu-residues that need modification to Gla for its activation (Schurgers et al 2007) Animal studies suggest that MGP is a physiological inhibitor of tissue calcification (Luo et al 1997; Lee et al 2007; Schurgers et al 2007), and its gene structure, amino acid sequence and tissue distribution are similar among examined animal species (Laize´ et al 2005) MGP is also important in chondrocyte differentiation and maturation, regulating endochondral and intramembranous ossification (Luo et al 1997; Newman et al 2001) As in mammals, studies have shown that MGP expression and function is associated with regulation of mineralization Bone and spinal deformities represent a recurring problem for commercial fish farming, and have raised ethical concerns in animal welfare issues in recent years Suggested risk factors are nutrition, genetics, environment, vaccination and fast growth (Waagbø et al 2005; Waagbø 2008) The importance of vitamin K in bone health has been established in mammals (Vermeer et al 1995, 1996; Shearer et al 1996; Booth 1997; Ferland 1998), and the interest in vitamin K requirement for normal bone development in fish has recognized that the vitamin K supply may be suboptimal for bone but sufficient to maintain normal growth and prevent mortality (Udagawa 2000) To date, there is no information on the form or the levels of vitamin K required to achieve optimal bone health neither in humans nor in fish Only a few reports have dealt with the impact of vitamin K deficiency on fish bone health (Udagawa 2001, 2004; Graff et al 2002; Roy & Lall 2007; Krossøy et al 2009a) Studies on mummichog (Fundulus heteroclitus) larvae have shown that diets without vitamin K supplementation caused a higher incidence of deformities in the vertebrae and caudal skeleton (Udagawa 2001) Further, the effect of parental vitamin K deficiency on bone structure was examined in the developing mummichog larvae (Udagawa 2004) The author concluded that the offspring from fish fed a vitamin K deficient diet had abnormal vertebral formation days posthatching compared to larvae from fish fed a vitamin K rich diet with significantly lower incidences of malformations More specifically, vitamin K deficiency caused the formation of thin and weak bone, and induces bone structure abnormalities such as vertebral fusion and row irregularity, both in early development and during later growth in mummichog (Udagawa 2001, 2004) Radiological and histological findings in haddock (Melanogrammus aeglefinus L.), however, showed that vitamin K deficiency decreased bone mineralization and increased the occurrence of bone deformities, without affecting the number of Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd osteoblasts (measured by histomorphometry) in the vertebrae This indicates that vitamin K is necessary for bone mineralization in haddock (Roy & Lall 2007) Investigations of bone health, performed by mechanical testing and radiological and/or visual examination, revealed no signs of vertebral deformities in juvenile Atlantic salmon (Krossøy et al 2009a) and Atlantic salmon smolts (Graff et al 2002) given an un-supplemented diet Moreover, neither phylloquinone nor MK-4 were detected in samples of vertebrae (Graff et al 2010), but both bgp and mgp were expressed in vertebrae, gills and pectoral fin as analysed by in situ hybridization and qPCR (Krossøy et al 2009a,b) Furthermore, gene expression of ggcx was found in vertebrae, scales, operculum and fin of adult Atlantic salmon, indicating GGCX activity in bony tissues of Atlantic salmon (Krossøy et al 2010) Although the exact role of the VKD bone proteins BGP and MGP remains unknown, they may be important in regulation of bone growth (Dimuzio et al 1983; Boskey et al 1998) Together these latter results suggested the involvement of vitamin K in bone metabolism of Atlantic salmon (Krossøy et al 2009b) Lately, studies in mammals have proposed multiple roles of vitamin K beyond coagulation that are both dependent and independent of its classical role as an enzyme cofactor, as reviewed by Booth (2009) A novel mechanism of vitamin K function in transcriptional regulation of osteoblastic cells was demonstrated by Tabb et al (2003), showing that menaquinone is a transcriptional regulator of bone markers, such as alkaline phosphatase and MGP in osteoblastic cells It has been shown that menaquinone is a ligand for the nuclear pregnance X receptor (PXR; also known as steroid xenobiotic receptor or SXR), suggesting a role of menaquinone in regulation of bone homoeostasis (Tabb et al 2003; Zhou et al 2009) and collagen formation (Ichikawa et al 2006) Menaquinones potentially contribute to improved bone quality by gene regulation (Ichikawa et al 2006; Horie-Inoue & Inoue 2008) in addition to its role as an enzymatic co-factor Gas6 is involved in regulating cell survival and proliferation, and protecting against cellular apoptosis (see review by Hafizi & Dahlba¨ck 2006) Gas6 is found throughout the nervous system, as well in the heart, lungs, stomach, kidneys and cartilage (Ferland 1998; Hafizi & Dahlba¨ck 2006) It affects vascular smooth muscle cell movement and apoptosis (Danziger 2008), and appears to play important physiological roles in inflammation, energy metabolism, renal disease, sepsis and neoplasia (Manfioletti et al 1993; Arai et al 2008; Booth 2009) Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd Lastly, a role of vitamin K in prevention of oxidative damage of the brain and sphingolipid synthesis has been suggested, as reviewed by Shearer & Newman (2008) The minimum requirements given by NRC (1993) are primarily determined for small fish and the studies are performed under optimal experimental conditions, using purified, synthetic or semi-synthetic diets produced under conditions causing minimal losses These studies and requirements are obviously not valid for commercial conditions using practical diets Normally, most vitamins are supplemented at levels above the NRC minimum requirements to compensate for factors influencing the vitamin level Thus, practical vitamin allowances correct for losses under feed production and storage (Marchetti et al 1999), and should take into consideration the bioavailability of vitamin forms, challenging rearing conditions and the developmental stage of the fish (Hamre et al 2010) Practical dietary vitamin K recommendations given for optimum health and productivity of farmed fish are therefore often several folds above the minimum requirement In fish, typical vitamin K deficiency signs include increased blood coagulation time, reduced growth, anaemia, haemorrhages, loss of fin tissue, weak bones, and occurrence of spinal curvature, short tails and increased mortality (Taveekijakarn et al 1996; Udagawa 2004; Lall & Lewis-McCrea 2007) The earliest vitamin K requirement studies in fin fish were based on increased blood coagulation time and mortality as the primary criteria (Kitamura et al 1967; Poston 1976; Murai & Andrews 1977) Studies with vitamin K deficient feed caused no detectable deficiency symptoms in rainbow trout (Kitamura et al 1967) and channel catfish, Ictalurus punctatus (Murai & Andrews 1977), but reduced growth and increased mortality in amago salmon, Oncorhynchus rhodurus (Taveekijakarn et al 1996), and increased mortality in mummichog (Udagawa & Hirose 1998) Lately, more sensitive biomarkers have been used As the major function of vitamin K is to act as co-factor for GGCX, the activity of this enzyme may provide a biomarker for deficiency Results from recent studies in juvenile Atlantic salmon confirmed that GGCX activity is a sensitive marker for evaluating vitamin K status and intake (Krossøy et al 2009a, 2010) However, altered enzyme activity does not necessarily represent a deficiency state and because there were no indications of deficiency in any of the other parameters measured, Krossøy et al (2009a) concluded that the minimum requirement in salmon juveniles was at, or less Table Overview over published vitamin K requirement and recommendations in fish (1970–2011) Fish species Response criteria Diet K vitamer Lake trout Salmonids Atlantic cod Haematology, coagulatin time Growth Mortality, haematology, coagulatin time Growth, mortality Growth, mortality Growth Growth, bone health Growth, coagulation time, bone health Semisynthetic Purified Practical n.g K1 n.g Semipurified/Practical Semipurified/Practical K3 K3 n.g K3 K1 Salmonids European seabass Salmonids Haddock Atlantic salmon Semipurified Practical Recommendations/ Requirement (mg kg)1 feed) 0.5–1 0.45 0.2 1.5 1.5 10* 20 0.1 Reference Poston (1976) Woodward (1994) Grahl-Madsen & Lie (1997) Kaushik et al (1998) Kaushik et al (1998) Halver (2002) Roy & Lall (2007) Krossøy et al (2009b) n.g.,not given; *, vitamin recommendation for growth than, the basal level of phylloquinone found in the diets (0.1 mg kg)1 feed) This is comparable to the study of Graff et al (2002) where the basal level of vitamin K in the diet for Atlantic salmon was 0.06 mg phylloquinone kg)1 feed, and where no signs of deficiency were recorded Current estimates of dietary vitamin K requirement differ in what is considered adequate levels in the feeds for fish (Table 2) In NRC (1993) recommendation, the minimum requirement for growing lake trout (Salvelinus namaycush) is 0.5–1 mg vitamin K kg)1 diet (based on Poston 1976), while in Halver (2002) the vitamin K recommendation for growth in trout and salmon is 10 mg kg)1 diet A previous comprehensive review of vitamin requirement studies in fish suggested that vitamin K concentrations equivalent to 0.45 mg phylloquinone kg)1 feed might be sufficient for salmonid fish (Woodward 1994) In addition, Kaushik et al (1998) showed that supplementation of practical diets with 1.5 mg menadione kg)1 was sufficient to maintain growth and prevent deficiency signs in juvenile rainbow trout (Oncorhynchus mykiss), Chinook salmon (Oncorhynchus tschawytscha) and European seabass (Dicentrachus labrax) In the same period, Grahl-Madsen & Lie (1997) suggested that 300 g kg)1) tended to have negative effects on the intestinal morphology, although the most significant effect was observed in the jejunum part In addition, a regression equation generated the predicted model of the effects of RWS on intestinal morphology These negative effects by RWS on the intestinal morphometry were related to growth performance traits The present study demonstrated that RWS can be incorporated in a practical diet and partially replace FM (on isonitrogenous and isocaloric basis) without any significant, adverse effects on the growth performance, carcass 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Bendiksen, E.A˚., Campbell, P.J & Bell, J.G (2008) The role of phospholipids in nutrition and metabolism of teleost fish Aquaculture, 280, 21–34 Uran, P.A., Schrama, W.J., Rombout, J.H.W.M., Obach, A., Jensen, L., Koppe, W & Verreth, J.A.J (2008) Soybean meal-induced enteritis in Atlantic salmon (Salmo salar L.) at different temperatures Aquacult Nutr., 14, 324–330 Whittington, R., Lim, C & Klesius, P.H (2005) Effect of dietary b-glucan levels on the growth response and efficacy of Streptococcus iniae vaccine in Nile tilapia, Oreochromis niloticus Aquaculture, 248, 217–225 Aquaculture Nutrition 17; 685–694 Ó 2011 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2011.00873.x 2011 17; 695–700 1 Department of Fish Diseases and Biology, University of Life Sciences, Lublin, Poland; Engineering, University of Life Sciences, Lublin, Poland The effects of Echinacea purpurea supplementation on growth performance, nutrient utilization, body composition and disease resistance were studied in the ornamental fish Poecilia reticulata Five diets were used, each differing in Echinacea content (0 g kg)1 diet – control, g kg)1 diet – group 1, 10 g kg)1 diet – group 2, 20 g kg)1 diet – group 3, 30 g kg)1 diet – group and 60 g kg)1 diet – group 5), and the fish were fed twice daily at a rate of 30 g kg)1 body weight per day for 67 days The gain in the body weight and the condition factor were significantly increased in groups 2-5, whereas specific growth rate and feed conversion ratio were significantly increased in the supplemented groups (P < 0.05) Cumulative mortalities after challenge infection with the fish pathogen Aeromonas bestiarum were the lowest in the groups supplemented with Echinacea Log-rank tests showed significant differences between the supplemented groups 1, 2, 3, and and the control group (P = 0.0074, P = 0.0075, P = 0.00507, P = 0.00001 and P = 0.00001, respectively) The results of this study indicate that Echinacea improves body weight gain and resistance against challenge infection in fish key words: diet, Echinacea purpurea, fish, growth, health, supplementation Received 14 September 2010, accepted April 2011 Correspondence: Leszek Guz, Department of Fish Diseases and Biology, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 13, 20-950 Lublin, Poland E-mail: leszek.guz@up.lublin.pl Echinacea (E.) purpurea herb, commonly known as the purple coneflower, red sunflower and rudbeckia, has a long and Department of Food Process well-established tradition of human medicinal use in North America and Europe (Percival 2000) It is believed to stimulate the immune system, preventing or reducing the severity of bacterial and viral infections Echinacea has been shown to have non-specific immunostimulatory properties in vitro (Bauer & Wagner 1991), including increased phagocytosis (Roesler et al 1991a), increased cytokine production (Burger et al 1997), natural killer cell activity (See et al 1997), chemotaxis and oxidative burst of either neutrophils (Wagner et al 1988; Graisbauer et al 1990) or macrophages (Stimpel et al 1984; Luettig et al 1989) Rehman et al (1999) have shown an increase in primary and secondary immunoglobulin G response in rats treated with Echinacea Echinacea can enhance the production of cytokines, including TNFa, IL-1, IL-6 and IL-10, by macrophages (Burger et al 1997) Moreover, peritoneal macrophages have been shown to be able to kill tumour cells (WEHI 162 cells) and cells infected either with the parasite Leishmania enriettii or with yeast cells of Candida albicans (Roesler et al 1991b; Steinmu¨ller et al 1993; Percival 2000) The glycoproteins, polysaccharides, caffeic acid derivatives and alkylamides present in Echinacea have all been reported to have immunostimulating activity (Burger et al 1997; See et al 1997; Bauer 1998; Rehman et al 1999) Among the numerous plants used in traditional medicine for the treatment and control of diseases, a few have been tested in fish Dietary intakes of aloe (Aloe vera) (Kim et al 1999), tulsi (Ocimum sanctum) (Logambal et al 2000), heartleaf moonseed (Tinospora cordifolia) (Binuramesh et al 2006; Sudhakaran et al 2006), purple coneflower (E purpurea) and garlic (Allium sativum) (Aly & Mohamed 2010), mistletoe (Viscum album), nettle (Urtica dioica) and ginger (Zingiber officinale) (Du¨genci et al 2003) and ashwagandha (Withania somnifera) (Sharma et al 2010) have been reported to enhance the immune response of fish against disease In Nile tilapia (Oreochromis niloticus), the immunostimulatory effects of Echinacea have been determined from differences Ó 2011 This article is a U.S Government work and is in the public domain in the USA between treatment and control groups in body weight gain, specific growth rates (SGR), survival rate, resistance to challenge infection, total and differential leucocyte counts, lysozyme activity, haematocrit values and nitroblue tetrazolium values (Aly et al 2007, 2008; Aly & Mohamed 2010) To the best of our knowledge, there is no information in the literature on the role of E purpurea, added as a supplement to fish feed, in the prevention of infectious diseases in ornamental fish The aim of the present study was to investigate the effect of E purpurea on the growth and survival of guppies (P reticulata) after experimental infection with A bestiarum Six diets were formulated to contain different levels of Echinacea The composition of the six experimental diets and the composition of the Echinacea plant meal used are shown in Table The Echinacea meal was incorporated into the diets as follows: no Echinacea meal (control), g Echinacea meal kg)1 diet (group 1), 10 g Echinacea meal kg)1 diet (group 2), 20 g Echinacea meal kg)1 diet (group 3), 30 g Echinacea meal kg)1 diet (group 4) and 60 g Echinacea meal kg)1 diet (group 5) The Echinacea plant meal used in this study was provided by Herbal Farm – Waldemar Lupa, Kolonia Dobrynio´w, Poland Extruded feeds were produced at the Department of Food Process Engineering, University of Life Sciences in Lublin, Poland Extruder parameters are specified in Table Extrusion was carried out using a single-screw laboratory extruder TS-45 (modified to L/D = 16/1) (Metalchem, Gliwice, Poland) Live-bearing guppies, P reticulata, were obtained from a private fish breeder in Lublin The experiments were carried out in the fish laboratory of the Department of Fish Diseases and Biology, Faculty of Veterinary Medicine in Lublin, Poland The fish had not been vaccinated or exposed to fish diseases and were determined to be pathogen-free using standard microbiological techniques Before the start of the experiments, the fish were acclimated for week in aquaria and were fed a standard Echinacea (g kg)1 diet) Ingredients (g kg)1 diet) Wheat sharps Yellow lupine1 Soybean sharps1 Fodder yeast1 Soybean oil1 II – calcium phosphate1 Chalk fodder1 Vitamin C3 Fish meal1,4 Vit/min mix2,5 Echinacea Proximate composition Crude protein Crude fibre Crude fat Total Ca Total P Estimated metabolic energy (MJ kg)1)6 10 20 30 60 209 100 320 50 90 13 200 10 – 210 100 314 50 90 13 200 10 210 100 309 50 90 13 200 10 10 200 100 309 50 90 13 200 10 20 190 100 309 50 90 13 200 10 30 160 100 309 50 90 13 200 10 60 386.7 66.7 95.2 14.9 7.2 15.16 389.1 60.8 96.6 13.8 7.4 15.34 385.5 62.1 94.8 13.9 7.0 15.26 379.6 73.3 93.8 14.0 7.1 14.71 379.8 75.4 98.2 14.2 7.0 14.72 388.5 75.1 93.5 14.5 6.9 14.93 Table Formulation (g kg)1), chemical composition (g kg)1) and metabolic energy (MJ kg)1) of the tests diets The source is Animex Grupa Drobiarska SA, Zamosc, Poland The source is BASF Polska, Kutno, Poland The source is POCH, Gliwice, Poland Crude protein, 720 g kg)1 Vit/min mix (IU or g kg)1 diet): vitamin A, 4400.00 IU; vitamin D3, 680.00 IU; vitamin E, 0.006 g; thiamin, 0.0006 g; riboflavin, 0.0012 g; pyridoxine, 0.0008 g; vitamin B12, 0.006 g; folic acid, 0.00016 g; biotin, 0.00004 g; niacin, 0.02 g; Ca, 0.004 g; Mn, 0.016 g; Zn, 0.02 g The estimated dietary metabolic energy was calculated in accordance with the Polish Committee for Standardization, PKN, PN-A-79011-6 (1998) Aquaculture Nutrition 17; 695–700 Ó 2011 This article is a U.S Government work and is in the public domain in the USA Table Extruder parameters during feed processing Parameters Planned extrusion temperature (°C) Mass temperature (°C) Feeding rate (kg h)1) Fat addition (%) Screw speed (rpm) Mass pressure (MPa) SME1 (kWh kg)1) 140.0 137.0–140.0 21.25 ± 1.32 10.0 100.0 0.08 0.147 ± 0.012 Values are given as (mean ± SD, n = 6) Specific mechanical energy commercial feed (TetraGuppy, Melle, Germany; proximate composition: 475.0 g kg)1 crude protein, 105.0 g kg)1 crude ash, 80.0 g kg)1 crude fat, 20.0 g kg)1 crude fibre, 60.0 g kg)1 water) at a rate of 30.0 g kg)1 of biomass per day After the acclimation period, the average weight of the fish was 0.177 ± 0.02 g, and the fish were randomly distributed into 20 L aquariums, each containing 40 fish/ replicate The fish were kept for 67 days at 26 ± °C under natural photoperiod The experimental diets were hand-fed to triplicate tanks two times daily (at 09.00 and 15.00 h) The fish were weighed every weeks starting from the beginning of the experiment Before weighing, the fish were starved for 24 h, allowing the gut to be emptied During handling, the fish were anaesthetized using a solution of tricaine methanesulphonate (MS-222; Sandoz LTD, Basle, Switzerland) at a concentration of 0.1 g L)1 At the end of the experiment, fish from each tank were individually weighed and measured The fish were stored at )20 °C to measure whole-body amino acids and protein composition At the termination of the 67-day feeding trial, the fish in each tank were individually weighed 24 h after the last feeding The survival percentage, mean individual weight and length increment were estimated Moreover, the SGR, feed conversion ratio (FCR) and condition factor (CF) were used as indicators for growth performance and feed utilization (Mohseni et al 2008) Feed conversion ratio ðFCRÞ ¼ feed intake/weight gain Specific growth rate ðSGRÞ ¼ 100  ½ðln Wf À ln Wi Þ=days], where Wf is mean final weight and Wi is mean initial weight Condition factor (CF) ¼ 100  ðfinal body weight/final body length): A challenge test was carried out in three replicates where 10 fish from each aquarium of both control and Echinacea treatment groups were transferred to aquaria (10-L) The challenge was performed by immersing the fish in a washed A bestiarum suspension (107 CFU mL)1) for h The temperature during the immersion challenges varied from 22 to 24 °C The challenged fish from each aquarium were observed for 14 days to record mortality The pathogenic strain of A bestiarum used in the present study was isolated from an ulcer on the skin of a diseased carp and was kindly provided by Dr Kozin´ska (Department of Fish Diseases, National Veterinary Research Institute, Puawy, Poland) Concentrations of amino acids were determined by ionexchange chromatography in freeze-dried whole-body samples using an INGOS AAA 400 amino acid analyzer (Ingos, Prague, Czech Republic) (Davis & Thomas 1973) Protein (N · 6.25) was analysed by the Kjeldahl method using the Kjel-Tec system (FOSS, Warsaw, Poland) All data were subjected to one-way analysis of variance (ANOVA) Significant differences among treatments were evaluated by TukeyÕs test at P < 0.05 Results are given as mean ± SD Mortality curves obtained for each group in the disease challenge experiments were analysed using the logrank test All statistical tests were performed using the Statistica 8.0 package (StatSoft, Cracow, Poland) The effects of Echinacea diets on growth performance and feed utilization of P reticulata are shown in Tables and Mortalities during the 67 days of the experiment were rare (from 5.8% to 9.2%) and unrelated to treatment (Table 3) The mean individual weight increment and the mean individual length increment were significantly different between the control and the experimental groups 2, 3, and (Table 4) Significant differences in FCR and SGR between control and Echinacea-treated groups were also observed (Table 3) FCR was significantly affected by dietary treatments (P < 0.05) and was the highest (3.74 ± 0.07) in group SGR increased significantly with increasing levels of Aquaculture Nutrition 17; 695–700 Ó 2011 This article is a U.S Government work and is in the public domain in the USA Experiment Survival (%) FCR Control Group Group Group Group Group 90.8 92.5 92.5 94.2 94.2 93.8 3.92 3.74 3.71 3.68 3.50 3.55 ± ± ± ± ± ± 1.4 2.5 2.5 1.4 3.8 1.4 SGR ± ± ± ± ± ± 0.07a 0.07b 0.07b 0.04b 0.05bc 0.04bc 0.74 0.84 0.97 0.98 1.11 1.03 CF ± ± ± ± ± ± 0.08a 0.06a 0.07b 0.06b 0.09b 0.07b 0.95 0.94 1.03 1.05 1.06 1.09 ± ± ± ± ± ± 0.08a 0.08a 0.06b 0.06b 0.09b 0.08b Table Survival, food conversion ratio and specific growth rate of live-bearing fish fed diets containing different levels of E purpurea for 67 days Each data represents the mean ± SD of triplicate tanks Mean values with same superscripts in each column are not significant (P > 0.05) Experiment Initial length (mm) Initial weight (g) Mean individual length increment (mm) Control Group Group Group Group Group 26.0 25.8 26.3 26.7 25.9 26.6 0.176 0.170 0.177 0.182 0.171 0.188 5.50 6.2 6.70 7.02 7.77 7.57 ± ± ± ± ± ± 1.0 1.4 1.2 1.4 1.3 1.5 ± ± ± ± ± ± 0.021 0.016 0.018 0.022 0.018 0.020 ± ± ± ± ± ± 0.74a 0.73a 0.63b 0.47b 0.82b 0.98b Mean individual weight increment (g) 0.115 0.130 0.163 0.171 0.188 0.187 ± ± ± ± ± ± Table Growth performance of livebearing fish fed diets containing different levels of E purpurea for 67 days 0.015a 0.014a 0.014b 0.019b 0.015b 0.019b Each data represents the mean ± SD of triplicate tanks Mean values with same superscripts in each column are not significant (P > 0.05) dietary Echinacea meal and ranged from 0.69 to 0.74 The best SGR was obtained in group which showed better growth than the other groups and had the best FCR (Table 3) Aly et al (2008) noted that, in Nile tilapia, body weight gain and SGRs were significantly higher in a group supplemented with Echinacea (at a rate of 0.25 · 10)9 g kg)1 on a dry weight basis for months) than in a control group, although there was no significant change in the CF between the experimental and the control groups Variations in condition coefficients may change with environment, age and feeding conditions The CF is a useful index for the monitoring of feeding intensity, age and growth rates in fish (Oni et al 1983) The mean CFs for the groups studied in our experiment are shown in Table The results indicate that there were significant differences between the CFs of the control and the experimental groups 2, 3, and Contrary to our findings, Aly et al (2008) did not observe any changes in CFs in Echinacea-treated Nile tilapia (Oreochromis niloticus) Nutrient requirements of fish are summarized by the National Research Council (NRC 1993) and these generally fall within the range of 350.0 ± 45.0 g kg)1 crude protein Therefore, in our experiment, diets containing 384.8 ± 4.2 g kg)1 protein were used Whole-body crude protein content tended to increase in the experimental groups (1–5) when compared with the control group (Table 5) Amino acids regulate key metabolic pathways that are crucial for maintenance, growth and health of fish (Li et al 2009) To the best of our knowledge, there is no published information regarding the amino acid composition of wholebody proteins in fish fed diets supplemented with Echinacea In our study, the whole-body concentration of valine (Val) was significantly higher in groups and 5, whereas the concentrations of alanine (Ala), arginine (Arg), asparagine (Asn), glutamine (Gln), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), proline (Pro), threonine (Tre) and tyrosine (Tyr) were significantly higher in group Increased contents of glycine (Gly), cysteine (Cys), histidine (His) and serine (Ser) were found in whole bodies of fish following an increase in Echinacea concentration in the diet Methionine (Met), on the other hand, showed a somewhat irregular behaviour (Table 5) The major amino acids were Asp, Glu, Gly, Leu and Lys, and their contents ranged from 11.1 to 19.5 g kg)1 In general, amino acid contents seemed to be higher in whole bodies of fish fed higher Echinacea diets As there are many different active components found in Echinacea (Burger et al 1997; See et al 1997; Bauer 1998; Rehman et al 1999), it is most likely that some of these constituents play an important role in enriching tissues with amino acids The exact mechanism of this effect requires further and more advanced studies Although many natural and synthetic substances have been reported to potentiate fish immunity and increase disease resistance, plant extracts have recently received particular attention as they are easily incorporated into the diet and have a low impact on the environment Echinacea is best Aquaculture Nutrition 17; 695–700 Ó 2011 This article is a U.S Government work and is in the public domain in the USA Table Amino acid composition (g kg)1) and total protein (g kg)1) of the fish P reticulata fed the experimental diets Control Group Amino acids (AA) Mean ± SD (n = 3) Mean ± SD (n = 3) P-value1 Mean ± SD (n = 3) P-value1 Asp Ser Glu Pro Gly Ala Tyr Cys Val Ile Leu Tre Phe His Lys Arg Met Total AA Total protein 11.4 6.4 17.2 6.4 9.7 7.8 3.8 1.7 5.6 4.9 9.7 5.6 5.5 3.6 10.0 7.8 3.9 121.1 136.0 11.7 6.3 17.7 6.7 10.1 8.2 3.9 1.6 6.9 5.1 9.7 5.6 5.5 3.6 10.1 8.2 3.6 130.0 138.0 NS NS NS NS NS NS NS NS 0.006 NS NS NS NS NS NS NS NS 0.005 NS 13.5 7.0 19.5 7.3 10.5 8.7 4.3 2.0 6.2 5.5 11.1 6.3 5.8 3.7 11.2 8.5 4.5 135.4 140.0 0.020 NS 0.001 0.020 NS 0.004 0.028 NS 0.015 0.013 0.008 0.020 0.034 NS 0.015 0.025 NS 0.006 NS ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.9 0.2 0.4 0.4 0.6 0.2 0.2 0.1 0.1 0.2 0.2 0.1 0.1 0.3 0.4 0.2 0.3 1.3 14.0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.6 0.7 1.7 0.5 0.6 0.7 0.3 0.4 2.1 0.4 1.0 0.7 0.4 3.3 1.1 0.6 0.4 1.8 8.0 Group ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 0.3 0.3 0.3 0.6 0.2 0.2 0.5 0.3 0.2 0.2 0.2 0.2 0.3 0.3 0.3 1.1 4.6 9.0 NS, not significant (P > 0.05) P-value: group compared to control by Tukey’s test known for its effects on the immune system (Bauer & Wagner 1991; Sun et al 2001) The compounds responsible for its immunomodulatory effects are polysaccharides, glycoproteins, caffeic acid conjugates and alkamides The immunostimulatory properties of Echinacea appear to target both non-specific and specific immune function (Bauer & Wagner 1991; Burger et al 1997; Rehman et al 1999) In this report, we demonstrated that E purpurea, a fish diet supplement, can confer resistance to challenge with A bestiarum The fish pathogen, A bestiarum, was used to test the disease resistance of the experimental groups of fish by immersion assay The cumulative mortalities of experimental and control fish challenged with A bestiarum are given in Table The cumulative mortalities of control fish were higher than those observed in the supplemented fish groups Survivorship deTable Cumulative mortality (%) of guppies (P reticulata) challenged with A bestiarum and fed diets containing graded levels of E purpurea Group Mortality1 P-value2 Group Group Group Group Group Control 31.1 29.9 33.3 6.5 6.5 57.8 0.00740 0.00750 0.00507 0.00001 0.00001 ± ± ± ± ± ± 1.9 6.5 7.6 3.4 3.4 6.9 Values are mean ± SD of triplicate tanks P-value: group compared to control by log-rank test creased with time in all of the supplemented groups and even more dramatically in the control group Log-rank tests showed that the supplemented groups 1, 2, 3, and differed from the control group (P < 0.001–0.007) Earlier studies revealed that 3-month dietary supplementation with Echinacea enhanced disease resistance against A hydrophila in Nile tilapia (Aly & Mohamed 2010) Probably, the enhancement of non-specific immune parameters by an Echinacea supplemented diet is possibly an important factor in protecting fish against bacterial challenge and in reducing their percentage mortality Aly et al (2008) reported that Echinacea acts as a disease control agent in fish In the present study, Echinacea meal was an excellent dietary substitute in guppy at a substitution level of 30 g kg)1 It is apparent that the use of dietary Echinacea plays a role in the control of diseases of ornamental fish This work provides a new perspective for the use of medicinal plants, which can be added to fish feed as adjuvant therapy for the prevention of fish diseases However, before this new method of feed supplementation is recommended for application in ornamental fish, a full commercial cost analysis is necessary This study was supported by the University of Life Sciences in Lublin Project number DS-8 Aquaculture Nutrition 17; 695–700 Ó 2011 This article is a U.S Government work and is in the public domain in the USA Aly, 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is a U.S Government work and is in the public domain in the USA [...]... of channel catsh (Ictalurus punctatus) fed diets containing various percentages of canola meal Aquaculture, 150, 103112 Aquaculture Nutrition 17; 605612 ể 2011 Blackwell Publishing Ltd Aquaculture Nutrition 2011 17; 613626 1,2,3 1 doi: 10.1111/j.1365-2095 .2011. 00861.x 3 Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Nubeena Crescent, Taroona, Tas., Australia;... carp (Cyprinus carpio var Jian) Aquacult Nutr., 14, 381386 Aquaculture Nutrition 17; 595604 ể 2011 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095 .2011. 00857.x 2011 17; 605612 1,2 3 3 4 4 1 1,2 1 Gesellschaft fuăr Marine Aquakultur mbH, Hafentoărn, Buăsum; 2 Department of Marine Aquaculture, Christian-AlbrechtsUniversitaăt zu Kiel, Kiel; 3 Pilot Panzenoăltechnologie... X.-Q (2011) Eects of pyridoxine on antioxidative parameters in juvenile Jian carp (Cyprinus carpio var Jian) Aquacult Nutr., 17, e226e232 Huang, H.-H., Feng, L., Liu, Y., Jiang, J., Hu, K., Jiang, W.-D., Li, S.H & Zhou, X.-Q (2011) Eects of dietary thiamin supplement on growth, body composition and intestinal enzymes activities of juvenile Jian carp (Cyprinus carpio var Jian) Aquacult Nutr., 17, e233e240... bass (Morone chrysops ã M saxatilis) Aquaculture, 287, 414418 Aquaculture Nutrition 17; 595604 ể 2011 Blackwell Publishing Ltd Li, W., Zhou, X.-Q., Feng, L., Jiang, J & Liu, Y (2009b) Eect of dietary riboavin on growth, feed utilization, body composition and intestinal enzyme activities of juvenile Jian carp (Cyprinus carpio var Jian) Aquacult Nutr., 16, 137143 Lin, Y & Zhou, X.-Q (2006)... accepted 15 February 2011 Correspondence: Cedric J Simon, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Nubeena Crescent, Taroona, Tas 7053, Australia E-mail: cedricjsimon@gmail.com ể 2011 Blackwell Publishing Ltd The development of a cost-eective and nutritionally adequate formulated diet is fundamental to the future viability of spiny lobster aquaculture (Williams... Comp Endocrinol., 124, 343358 Caruso, D & Schlumberger, O (2002) Haematological baseline values of juvenile Silurus glanis (Siluridae) reared in intensive conditions Cybium, 26, 6570 Aquaculture Nutrition 17; 605612 ể 2011 Blackwell Publishing Ltd Chabanon, G., Chevalot, I., Framboisier, X., Chenu, S & Marc, I (2007) Hydrolysis of rapeseed protein isolates: kinetics, characterization and... function of intestine of juvenile Jian carp (Cyprinus carpio var Jian) Aquaculture, 2 56, 389394 Ling, J, Feng, L., Liu, Y., Jiang, J., Hu, K., Jiang, W.-D., Li, S.H & Zhou, X.-Q (2010) Eect of dietary iron levels on growth, body composition and intestinal enzymes activities of juvenile Jian carp (Cyprinus carpio var Jian) Aquacult Nutr., 16, 616624 Livingstone, D.R., Garcia, M.P., Michel, X., Narbonne, J.F.,.. .Aquaculture Nutrition doi: 10.1111/j.1365-2095 .2011. 00853.x 2011 17; 595604 1,2 1 1,2 1,2 1,2 1,2 1 1,2 1 Animal Nutrition Institute, Sichuan Agricultural University, Yaếan, China; Nutrition of China Ministry of Education,... that MHA was converted into L-methionine for eective utilization in chicken liver (Dibner & Knight 1984) and small intestine (Mart n-Venegas et al 2006) Through converting Aquaculture Nutrition 17; 595604 ể 2011 Blackwell Publishing Ltd into L-methionine, MHA may participate or regulate protein synthesis, and thus inuence antioxidant enzymes activities in intestine and hepatopancreas Meanwhile,... Proc Natl Acad Sci USA., 102, 1257812583 Elia, A.C., Anastasi, V & Drr, A.J.M (2006) Hepatic antioxidant enzymes and total glutathione of Cyprinus carpio exposed to three Aquaculture Nutrition 17; 595604 ể 2011 Blackwell Publishing Ltd disinfectants, chlorine dioxide, sodium hypochlorite and peracetic acid for supercial water potabilization Chemosphere, 64, 1633 1641 Feng, L (2009) Eect