Aquaculture nutrition, tập 17, số 6, 2011

Aquaculture Nutrition
doi: 10.1111/j.1365-2095.2011.00904.x

2011 17; 585–594

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1
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2

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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

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Ó 2011 Blackwell Publishing Ltd

2

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 1 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 2 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

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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 1 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

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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 2 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 />
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 5 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

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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).

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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 2 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 <0.2 mg vitamin
K kg)1 feed as the minimum dietary requirement for Atlantic
cod (Gadus morhua). Recently, Krossøy et al. (2009a) found
that 0.1 mg phylloquinone kg)1 feed was sufficient to meet

the minimum requirement for normal growth, health and
bone strength in juvenile Atlantic salmon fed a diet without
vitamin K supplementation from start feeding. Confounding
factors in this study may have been the transfer of vitamin K
to offspring from the broodfish, as analyses show that salmon
eggs may contain approximately 0.0032 ± 0.0002 lg K1 and
0.016 ± 0.005 lg MK-4 per egg (R. Ørnsrud, unpublished
data). Considering that the vitamin K cycle efficiently recycles vitamin K, this endogenous source may cover the minimum requirement for start feeding fry.

There may be several reasons for the variation between fish
species in vitamin K requirement research. First, differences
in biological efficacy among the chemical vitamin K forms
(phylloquinone, menaquinones and menadione) have to be
taken into consideration. In addition, different outcomes
may arise from differences in absorption, metabolism and
excretion, feed intakes, and choice of biomarkers. Lastly, the
use of the very labile forms of menadione as a vitamin K
source in the feed, and challenges with the vitamin K analysis, might have led to an overestimated requirement (Graff
et al. 2010).
Menadione has been reported to cause toxicity symptoms
like abnormalities in liver, kidney and lungs, as well as
haemorrhage and haemolytic anaemia in mammals (Smith
et al. 1943; Rebhun et al. 1984), while no symptoms of toxicity have been reported for phylloquinone and menaquinones. Available data for fish is contradictory. As an
example, 30 mg MSB kg)1 feed reduced growth in rainbow
trout after 20 weeks of feeding (Grisdale-Helland et al.
1991), while 20 mg MSB kg)1 feed reduced growth in
Atlantic cod after 23 weeks of feeding (Grahl-Madsen & Lie
1997). In a study by Udagawa (2001), the upper tolerance
levels of phylloquinone was 100 mg kg)1 feed while the
tolerance for MSB was 2500 mg kg)1. For rainbow trout fed

up to 2000 mg MNB kg)1 feed (Marchetti et al. 1995) and
Atlantic salmon fed diets with up to 1000 mg MNB kg)1 feed
(Krossøy et al. 2009a; Graff et al. 2010), no signs of toxicity
in growth, mortality or health measures were recorded,
showing high dietary tolerance of MNB. However, safe
upper levels of vitamin K for fish have not been established.

Currently, the most common menadione forms added to
feeds for farmed fish are MSB and MNB. Only a limited

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number of the papers published on vitamin K requirement in
fish include analysis of menadione in the feed (Grahl-Madsen
& Lie 1997; Roy & Lall 2007; Graff et al. 2010), and thereby
validate if the levels of menadione in the feed are in accordance with the targeted amounts. Graff et al. (2010) reported
that the analysed levels of menadione in the experimental
feeds were very low compared to the target levels (added
0–1000, analysed 0–46.5 mg menadione kg)1). This agrees
with findings of Tavcˇar-Kalcher & Vengusˇ t (2007) showing
that up to 90% of the menadione in premixes may be lost
during 12 months of storage. The instability of menadione
may be increased by addition of choline in the vitamin premix (Marchetti et al. 1995, 1999; Tavcˇar-Kalcher & Vengusˇ t
2007). When 0–20 mg menadione kg)1 feed in the form of
MSB was added to Atlantic cod diets, Grahl-Madsen & Lie
(1997) were able to analyse the same amount added in the
feed. Roy & Lall (2007), on the other hand, reported a 50%

reduction from added to analysed dietary menadione content
using levels of MSB corresponding to 0–40 mg menadione
kg)1 in feeds for haddock. The differences in recovery
between the works of Grahl-Madsen & Lie (1997) and Roy &
Lall (2007) compared to Graff et al. (2010) may be related to
several factors. First, the feeds may have been produced by
different processing methods (not described in the papers).
Secondly, feeds for cod and haddock contain less fat
(100 g kg)1) than feeds for salmon (300 g kg)1) and may
thus be easier to analyse. Thirdly, the stability of MSB and
MNB may differ.
Knowledge about the availability of inherent and synthetic
K vitamers in fish feed is scarce (Lall 2005). Menadione must
be alkylated enzymatically to MK-4 in animal tissues to
become biologically active (Udagawa 2000), and is thought
to have lower bioavailability than the naturally occurring K
vitamers, as shown in mummichog (Udagawa 2001; Udagawa & Murai 2001). Results from Krossøy et al. (2010) confirmed that menadione did not act directly as a co-factor for
GGCX in Atlantic salmon, despite its structural similarities
with the inherent vitamin K forms (Lambert & De Leenher
1992). This is also in line with earlier studies by Sadowski
et al. (1976) and Buitenhuis et al. (1990), demonstrating that
only phylloquinone and menaquinones can function as a
co-factor for GGCX. However, menadione is known to be
converted to MK-4 in the tissues of several fish species
including sardine, Sardinops melanosticus (Udagawa et al.
1993), Atlantic cod (Grahl-Madsen & Lie 1997), mummichog
(Udagawa & Hirose 1998), ayu (Plecoglossus altivelis)
(Udagawa et al. 1999) and Atlantic salmon (Graff et al. 2002,
2010; Krossøy et al. 2009a), and can thus be used as a
vitamin K source in aquaculture feeds. A dose–response

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Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd

relationship has been found between menadione intake and
the liver MK-4 concentration in Atlantic salmon (Krossøy
et al. 2009a; Graff et al. 2010), despite extremely low conversion and retention of MK-4. It has to be clarified if the
conversion of menadione to MK-4 is severely rate limited, or
if the low liver MK-4 levels are solely related to extremely
low stability and intake from feed. In the study by Graff et al.
(2010), a comparison between MNB and phylloquinone fed
salmon showed a considerably higher retention of phylloquinone compared to menadione, necessitating validation of
actual levels of menadione in the feeds after feed production.
Additional knowledge about retention of K vitamers in fish
fillets is also an important step to tailor fish fillets towards
improved nutritional value (Bell & Waagbø 2008).

Recommendations for vitamin requirements must be seen in
relation to factors like species, life stage, overall feed composition and farming conditions (Waagbø 2008). Welfare
and quality of farmed fish is a serious issue of debate, and
more scientific investigations should be directed towards
providing properly formulated feeds, securing optimal
nutrient content throughout all production stages. The
increased global production of fish through farming and
the worldwide shortage of marine resources, has led to
replacement of fish meal and fish oil by novel protein and
lipid sources of plant origin and from marine by-products.
Some relevant alternative oils in fish feed, like soybean oil
(2.7 mg kg)1) and canola oil (1.1 mg kg)1), may contain

higher levels of natural vitamin K forms compared to
marine ingredients (0.01–1.0 mg kg)1 wet weight) (Ostermeyer & Schmidt 2001, Wollard et al. 2002; Suttie 2006).
To reduce unnecessary vitamin supplementation and feed
costs, information is needed on the concentration and bioavailability of naturally occurring vitamers in the feed
ingredients used.
Based on the points mentioned, it is necessary to define a
more precise dietary requirement for vitamin K. The stability
of vitamin K in processing and storage should be further
investigated, and a focus on the bioavailability of inherent
and synthetic forms, is needed. Traditionally, estimation of
vitamin requirements is mostly performed on the basis of one
vitamin, but it has become clear that it is necessary to have a
more integrated approach with multiple variables. This is
related to the fact that different vitamins show interactions
with each other. As an example, some of the non-infectious
diseases and deformities causing trouble for the aquaculture
industry have been related to suboptimal production


conditions and nutrition, and nutritional factors like the lipidsoluble vitamins A, D and K have been proposed as important for development and mineralization of bone (Waagbø
et al. 2005). The formation, deposition and break-down of
bone proteins and minerals are controlled by bone cells. Bone
cells react on external stimuli, such as the hormone like
compounds vitamins A and D. Vitamin A may accumulate in
fish and lead to hypervitaminosis (Ørnsrud et al. 2002) and an
increase in the occurrence of both craniofacial and spinal
deformities (Ørnsrud et al. 2008), while vitamin D is shown to
be an important regulator of mineral homoeostasis (Fraser
1995; Lock et al. 2010). Both vitamins A and D influence
expression and synthesis of VKD proteins (Lian et al. 1989;

Fu et al. 2008; Darias et al. 2010) and have shown strong
regulatory effects on the activity of bone cells (Lall &
Lewis-McCrea 2007), while vitamin K is responsible for the
posttranslational modification and activation of the VKD
proteins (Knapen et al. 1993; Luo et al. 1997; Boskey et al.
1998; Lee et al. 2007). Intensive rearing, the use of novel feed
ingredients and exposure to diseases and contaminants may
alter the relative requirement of some vitamins, especially in
the juvenile stages. Therefore, a re-evaluation of the vitamin
K requirement in farmed fish species is needed.

This project is part of the research programme ÔRoles of fat
soluble vitamins in bone development and mineral metabolismÕ, funded by the Research Council of Norway (project
number 153472).

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Aquaculture Nutrition
doi: 10.1111/j.1365-2095.2011.00853.x

2011 17; 595–604

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1,2

1

1,2

1,2

1,2

1,2

1

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1

Animal Nutrition Institute, Sichuan Agricultural University, YaÕan, China;
Nutrition of China Ministry of Education, YaÕan, China

Oxidative damage and antioxidant status of intestine and
hepatopancreas for juvenile Jian carp (Cyprinus carpio var.
Jian) fed graded levels of methionine hydroxy analogue
(MHA: 0, 5.1, 7.6, 10.2, 12.7, 15.3 g kg)1 diet) for 60 days
were studied. Radical scavenging ability, antioxidant
enzymes activities such as superoxide dismutase (SOD),
catalase (CAT), glutathione-S-transferase (GST), glutathione
peroxidase (GPX) and glutathione reducase (GR), as well as
glutathione (GSH), protein carbonyl (PC) and malondialdehyde (MDA) contents were assayed in these tissues.
Results indicated that anti-superoxide anion capacity in
intestine and anti-hydroxyl radical capacity in hepatopancreas significantly improved with dietary MHA levels

up to 7.6 and 10.2 g kg)1 diet respectively, whereupon they
decreased (P < 0.05). SOD, CAT, GST, GPX, GR activities
in intestine and hepatopancreas, as well as GSH content in
hepatopancreas significantly increased with optimal MHA
levels which were in the range of 5.1–10.2 g kg)1 diet, and
thereafter decreased (P < 0.05). Meanwhile, MDA and PC
contents in these tissues together with GOT and GPT
activities in plasma significantly decreased with optimal
MHA levels which were in the range of 5.1–7.6 g kg)1 diet,
and thereafter increased (P < 0.05). These results suggested
that MHA improved antioxidant status and depressed lipid
and protein oxidation in intestine and hepatopancreas.
antioxidant enzymes, Cyprinus carpio var. Jian,
lipid peroxidation, MHA, oxygen radicals, protein oxidation
KEY WORDS:
WORDS:

Received 3 July 2010, accepted 12 January 2011
Correspondence: Xia-Qiu Zhou, Animal Nutrition Institute, Sichuan
Agricultural University, YaÕan 625014, China. E-mail:

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Ó 2011 Blackwell Publishing Ltd

2

Key Laboratory for Animal Disease-resistance

Methionine has been demonstrated to be a dietary essential

amino acid for normal growth of juvenile Jian carp (Cyprinus
carpio var. Jian) (Tang et al. 2009) and Indian major carp
(Cirrhinus mrigala) (Ahmed et al. 2003). As a common synthetic methionine source, supplement methionine hydroxy
analogue (MHA) to methionine-deficient diets can partly
substitute DL-methionine to promote the growth of rainbow
trout (Oncorhynchus mykiss) (Poston 1986), juvenile sunshine
bass (Morone chrysops $ · M. saxatilis #) (Keembiyehetty &
Gatlin 1995) and juvenile red drum (Sciaenops ocellatus)
(Goff & Gatlin 2004). Our previous study also indicated that
supplementation of MHA and DL-methionine on equalsulphur basis made no significant difference in growth
performance of Jian carp (Xiao et al. 2010a). By other hand,
it has been reported that animal growth is dependant on
digestion and absorption ability (Hakim et al. 2006). Our
previous research found that MHA promoted digestion and
absorption ability and thus growth of Jian carp by improving
intestine and hepatopancreas function (Xiao et al. 2010b).
Shoveller et al. (2005) reviewed that intestinal growth and
function are usually involved in its antioxidant status. Previous studies with Jian carp also indicated that improved
growth and function of intestine and hepatopancreas were
positively related to antioxidant status in these organs by
nutrients such as glutamine (Lin & Zhou 2006; Chen et al.
2009), pyridoxine (He et al. 2009; Hu et al. 2011) and myoinositol (Jiang et al. 2009b, 2010). Reactive oxygen species
(ROS) such as the superoxide anion (O2•–) and hydrogen
peroxide (H2O2) are generated as part of the normal aerobic
cellular metabolism, and these reactive species in turn may
promote the production of many other reactive molecules
such as hydroxyl radical (OH•), hypochlorous acid (HClO)


and peroxynitrite (HOONO) (Storz & Imlayt 1999). Like all

aerobic organisms, antioxidant defence systems in fish also
consist of low-molecular-weight antioxidants and antioxidant enzymes (Martınez-Alvarez et al. 2005). When the ROS
generation rate exceeds that of their removal, oxidative stress
occurs which may produce deleterious effects including protein oxidation, DNA strand-break damage and peroxidation
of unsaturated lipids (Martınez-Alvarez et al. 2005). Peroxidation of membrane lipid often initiates the loss of membrane integrity, which may induce injury of tissues so as to
the leak of enzymes or ions (Veena et al. 2006). According to
this, serum glutamate-oxaloacetate transaminase (GOT) and
glutamate-pyruvate transaminase (GPT) act often as markers
of liver injury in terrestrial animals (Yoshikawa et al. 2002).
Humtsoe et al. (2007) also implied that reduced GOT and
GPT in muscle and liver of rohu carp (Labeo rohita) when
exposed to oxidative stress might be related to the leak of
them into serum. Therefore, GOT and GPT activities in
serum warrants investigation to further reflect the antioxidant status of tissues influenced by MHA.
Methionine as an important sulphydryl-containing compound is particularly susceptible to oxidation by ROS and
then converted to methionine sulphoxide (MeSOX) by biochemical assays in vitro (Levine et al. 1996). However, studies
with yeast (Moskovitz et al. 1997), bovine (Moskovitz et al.
1996) and human tissue (Kuschel et al. 1999) implied that the
enzyme MeSOX reductase could reduce MeSOX back to
methionine if the MeSOX was not further oxidation. It
appears that the cycle of methionine oxidation and reduction
represents a natural scavenging system for ROS. Furthermore, studies had demonstrated that MHA was converted
into L-methionine for effective utilization in chicken liver
(Dibner & Knight 1984) and chicken small intestine (Martı´ nVenegas et al. 2006). By other hand, methionine is not only a
substrate for protein synthesis (Me´tayer et al. 2008), but also
a regulator that can modulate phosphorylation and protein
synthesis in avian myoblast cell line (Tesseraud et al. 2003),
and simultaneously can affect DNA methylation and further
influence gene expression in mouse as an important source of
methyl group (Tremolizzo et al. 2002; Dong et al. 2005).

Based on these observations, methionine may influence
antioxidant enzymes activities in vivo. Studies with rats
indicated that methionine improved SOD, GPX and CAT
activities in the heart of male Sprague–Dawley rat (Seneviratne et al. 1999), as well as GR and GST activities in tertbutylhydroperoxide induced brain synaptosomes of albino
rat (Slyshenkov et al. 2002). A few research have been
conducted to study the antioxidant defence of methionine
source in fish, showing that both DL-methionine and MHA

increased glutathione (GSH) in juvenile sunshine bass liver
(Keembiyehetty & Gatlin 1995) and declined thiobarbituric
acid reactive substances (TARS) in hybrid striped bass
(Morone chrysops $ · M. saxatilis #) liver (Li et al. 2009a).
However, few studies have evaluated effects of methionine
sources on free radical generation and antioxidant enzymes
in fish, which needs further experimental investigation.
This study was a part of a larger study that involved in the
determination of the effects of MHA on digestive and
absorptive capacity in Jian carp (Xiao et al. 2010b), and
provides a first insight into the possible effects of MHA on
free radical generation and antioxidant enzymes response in
fish. The results can provide partial theoretical evidence for
the effect of MHA on digestive and absorptive capacity in
fish.

Formulation of the basal diet is presented in Table 1. Except
methionine, dietary components (amino acids, vitamins and
minerals) were supplemented to meet the requirements of
juvenile Jian carp according to our previous studies (Zhou
et al. 2008; Feng 2009; He et al. 2009; Jiang et al. 2009b; Li
et al. 2009b; Wen et al. 2009; Huang et al. 2011; Ling et al.

2010; Tan et al. 2010) and reported nutritional requirements
for common carp (NRC 1993). Six experimental diets were
formulated according to MHA supplementation: 0 (control),
5.1, 7.6, 10.2, 12.7 and 15.3 g MHA kg)1 diet. Liquid MHA
product (measured with 879 g kg)1 active substance)
(Sumitomo-chemical, Tokyo, Japan) was added to the test
diets to provide different concentrations, and the amount of
corn starch was reduced to compensate final amount. The
methionine concentration in the basal (control) diet was 6.9 g
kg)1 diet, which was determined by the method of Spindler
et al. (1984). Experimental diets and the procedures for diet
preparation and storage ()20 °C) were the same as was
previously reported (Xiao et al. 2010b).

Hatchery-reared juvenile Jian carp were obtained from the
TongWei Hatchery (Sichuan, China). Before starting the
experiment, fish were acclimatized to the aquaria system and
fed six times daily (08:00, 10:30, 13:00, 15:30, 18:00, 20:30)
for 4 weeks with a diet containing 320 g crude protein kg)1
diet, which was similar to that of basal experimental diet. A
total of 900 fish with an initial weight of 8.24 ± 0.03 g were

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Aquaculture Nutrition 17; 595–604 Ó 2011 Blackwell Publishing Ltd


Table 1 Ingredients and composition of the basal diet1
)1

Ingredients

g kg

Fish meal
Soybean meal
Rice gluten meal
Cottonseed meal
Rapeseed meal
Wheat flour
Fish oil
Soybean oil
Vitamin premix2
Trace mineral premix3
Ca (H2PO4)2
Choline chloride (50%)
Carboxymethyl cellulose
Ethoxyquin (30%)
Threonine (98.5%)
Lysine (78.8%)
MHA premix4
Proximate composition
Moisture (g kg)1)
Crude protein (g kg)1 dm)
Crude fat (g kg)1 dm)
Crude ash (g kg)1 dm)
Cysteine (g kg)1)
Methionine (g kg)1)

75.0

75.0
100.0
147.8
295.6
165.7
22.5
7.2
10.0
10.0
26.7
1.3
20.0
0.5
5.9
6.8
–
117
321.4
54.4
87.9
7.9
6.9

1
Fish meal, soybean meal, rapeseed meal, cottonseed meal and
rice gluten meal were used as dietary protein sources. Fish oil,
soybean oil and wheat flour were used as dietary lipid and carbohydrate source respectively. Lysine, threonine, available phosphorus, n-3 and n-6 calculated contents were 20.0, 17.0, 6.0, 10.0
and 10.0 g kg)1 diet respectively according to NRC (1993) and Bell
(1984).
2

Per kilogram of vitamin premix (g kg)1 diet): retinyl acetate, 0.80
(500 000 IU g)1); cholecalciferol, 0.48 (500 000 IU g)1); DL-atocopherol acetate, 20.00 (50%); menadione, 0.20 (50%); cyanocobalamin, 0.01 (10%); D-biotin, 0.50 (20%); folic acid, 0.52 (96%);
thiamin nitrate, 0.10 (98%); ascorhyl acetate, 7.23 (92%); niacin,
2.86 (98%); meso-inositol, 52.86 (98%); calcium-D-pantothenate,
2.51 (98%); riboflavine, 0.63 (80%); pyridoxine hydrochloride, 0.76
(98%). All ingredients were diluted with corn starch to 1 kg.
3
Per kilogram of mineral premix (g kg)1 diet): FeSO4Æ7H2O, 69.70
(19.7% Fe); CuSO4Æ5H2O, 1.20 (25.0% Cu); ZnSO4Æ7H2O, 21.64
(22.5% Zn); MnSO4ÆH2O, 4.09 (31.8% Mn); KI, 2.90 (3.8% I); NaSeO3,
2.50 (1.0% Se). All ingredients were diluted with CaCO3 to 1 kg.
4
MHA premix (30 g kg)1 diet) was added to obtain graded level of
MHA, and the amount of corn starch was reduced to compensate.
Six MHA premix were elaborated according to different proportion
liquid MHA (g) and corn starch (g): 0/1000.0, 193.3/806.7, 290.0/
710.0, 386.7/613.3, 483.3/516.7 and 580.0/420.0.

randomly distributed to each of 18 experimental aquaria
(90 L · 30 W · 40 H, cm). Each experimental diet was randomly assigned to triplicate aquaria. The aquaria system,
culture system, and water quality were the same as our previous study (Xiao et al. 2010b). The experimental fish were
hand-fed with the respective diet to apparent satiation for
60 days six times daily (08:00, 10:30, 13:00, 15:30, 18:00,
20:30) from day 1 to 30 and four times daily (08:00, 12:00,

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Aquaculture Nutrition 17; 595–604 Ó 2011 Blackwell Publishing Ltd

16:00, 20:00) from day 31 to 60. Thirty minutes after the

feeding, uneaten feed were removed by siphoning and then
air dried. The experimental units were under a natural light
and dark cycle and water temperature was 25 ± 1°C.

At the end of the feeding trial, fish were anaesthetized with
benzocaine (50 mg L)1) 12 h after the last meal. Immediately, blood of 15 fish from each aquarium was drawn from
the caudal vein, stored at 4 °C overnight, and then centrifuged at 3000 g at 4 °C for 10 min. The plasma was stored at
)20 °C for further glutamate-oxaloacetate transaminase
(GOT) and glutamate-pyruvate transaminase (GPT) activity
assays. Intestine and hepatopancreas of the same 15 fish
were quickly removed, weighed and frozen in liquid nitrogen
and stored at )70 °C until analysed. Tissue samples of six
fish per tank were homogenized on ice in 10 volumes (w/v)
of ice-cold physiological saline (0.7 g mL)1) and centrifuged
at 6000 g for 20 min at 4 °C respectively, and then supernatants were stored at )20 °C for antioxidant parameters
analysis.
The anti-superoxide anion capacity (O2•)-scavenging
ability) and anti-hydroxy radical capacity (OH•-scavenging
ability) were determined by the method described by (Zhang
et al. 2005; Jiang et al. 2009a). Anti-superoxide anion (ASA)
capacity was determined by using the superoxide anion free
radical detection Kit (Nanjing Jiancheng Bioengineer Institute) Superoxide radicals (O2•)) were generated by the action
of xanthine and xanthine oxidase. With the electron acceptor
added, a coloration reaction is developed by using the Griess
reagent. The coloration degree is directly proportional to the
quantity of superoxide anion in the reaction. Tissue ASA
capacity was expressed in mU mg)1 protein. One mU was
defined as the amount that scavenged superoxide anion free
radical in 40 min per milligram of tissue protein which
equalled to per microgram of vitamin C scavenging at the

same condition. Anti-hydroxy radical (AHR) capacity was
determined by using the hydroxyl free radical detection Kit
(Nanjing Jiancheng Bioengineer Institute). It was on the basis
of Fenton reaction (Fe2++H2O2 ) Fe3++OH)+OH•).
Hydroxyl radicals (OH•) are generated in the Fenton reaction. With the electron acceptor added, a coloration reaction
is developed by using the Griess reagent. The coloration
degree is directly proportional to the quantity of hydroxyl
radicals in the reaction. Tissue AHR capacity was expressed
in U mg)1 protein. One U was defined as the amount that
decreased 1 mmol L)1 H2O2 in 1 min per milligram of tissue
protein.


bance at 370 nm, using an absorption coefficient of
21 000 M)1 cm)1 and expressed in nmol mg)1 protein. The
protein concentration of samples was determined by the
method of Bradford (1976).

Superoxide dismutase (SOD) and glutathione peroxidase
(GPX) activities were assayed as described by Zhang et al.
(2008). Tissues SOD activity was expressed in U mg)1 protein.
One U means 50% of inhibition by SOD of nitric ion production. Tissues GPX activity was expressed in U mg)1
protein. One U was defined as the amount that reduced
1 lmol L)1 GSH in 1 min per milligram of tissue protein.
Catalase (CAT) activity was determined by the decomposition of hydrogen peroxide (Aebi 1984). The result was
expressed in U mg)1 protein. One U was defined as the
amount that decreased 1 lmol L)1 H2O2 in 1 s per milligram
of tissue protein. Glutathione-S-transferase (GST) activity
was measured by monitoring the formation of an adduct
between GSH and 1-chloro-2,4-dinitrobenzene (Lushchak

et al. 2001). The result was expressed in U mg)1 protein. One
U was defined as the amount that decreased 1 lmol L)1 GSH
in 1 min per milligram of tissue protein. Glutathione reductase (GR) activity was measured according to described by
Lora et al. (2004) and given as mU mg)1 protein. One mU was
defined as the amount that decreased 1 mmol L)1 NADPH in
1 min per milligram of tissue protein. GSH content was
determined by the formation of 5-thio-2-nitrobenzoate followed spectrophoto-metrically at 412 nm (Vardi et al. 2008).
The amount of GSH in the extract was expressed as mg g)1
protein and commercial GSH was used as standard.
GOT and GPT activities in serum were determined by the
method of Bergmeyer & Bernt (1974a) and Bergmeyer &
Bernt (1974b) respectively. Both GOT and GPT activities
were expressed in U L)1 plasma. One U was defined as the
amount that produced 1 lmol L)1 pyruvic acid in 1 min. The
malondialdehyde (MDA) content was assayed as described
by Livingstone et al. (1990) using the thiobarbituric acid
reaction. The results were expressed in nmol mg)1 protein.
The protein carbonyl content was determined according to
the method described by Armenteros et al. (2009). The protein carbonyl content was calculated from the peak absor-

All data were subjected to one-way analysis of variance
(ANOVA) followed by the Duncan method to determine significant differences among treatment groups. All results were
expressed as mean ± SEM. The parameters with significant
differences were subjected to quadratic regression analysis
with dietary MHA level.

SOD, CAT, GST, GPX, GR activities and GSH content in
intestine are displayed in Table 2. SOD activity significantly
increased as dietary MHA levels was up to 7.6 g kg)1 diet, and
then the data significantly decreased maintaining a plateau.

CAT activity also significantly increased as dietary MHA
levels was up to 7.6 g kg)1 diet, after that it manifested a
significant gradual decrease. When MHA level was up to 5.1 g
kg)1 diet, GSH content significantly improved, and then
plateaued. GST activity in fish fed diet 1 and 6 were significantly lower than that of fish fed the other four diets. GPX
activity was significantly increased with higher MHA levels
showing the highest values with 7.6 g kg)1 diet MHA levels,
but a significant decreased activity was manifested in fish fed
diets containing MHA ‡ 10.2 g kg)1 diet. GR activity was the
lowest in fish fed the MHA-unsupplemented diet, and the
highest in fish fed diet containing 7.6 g MHA kg)1 diet, and
then significantly decreased with MHA levels up to 15.3 g kg)1
diet. Intestinal CAT, GST and GR activities manifested
quadratic responses to the increasing dietary MHA levels
(YCAT = 1.1833 + 0.1846X ) 0.0132X2, R2 = 0.772,

Table 2 Superoxide dismutase (SOD, U mg)1 protein), catalase (CAT, U mg)1 protein), glutathione-S-transferase (GST, U mg)1 protein),
glutathione peroxidase (GPX, U mg)1 protein), glutathione reducase (GR, mU mg)1 protein) activities and glutathione (GSH, mg g)1 protein)
content in intestine of juvenile Jian carp fed diets containing graded levels of methionine hydroxy analogue (MHA g kg)1 diet) for 60 days1
Dietary MHA level
0
5.1
7.6
10.2
12.7
15.3

SOD
54.29
58.98

61.59
54.74
52.98
53.29

CAT
±
±
±
±
±
±

a

4.00
5.53ab
5.93b
5.40a
3.98a
3.73a

1.11
1.90
2.05
1.38
1.29
1.05

GST

±
±
±
±
±
±

a

0.04
0.12c
0.14d
0.14b
0.12b
0.09a

36.95
41.77
42.25
44.26
43.28
37.57

GPX
±
±
±
±
±
±

a

3.37
1.08b
3.82b
4.63b
3.06b
2.38a

50.86
54.24
65.94
47.18
50.18
49.71

GR
±
±
±
±
±
±

ab

4.30
3.96b
4.92c

2.61a
4.47ab
4.43ab

55.73
61.08
68.65
65.10
65.72
61.95

GSH
±
±
±
±
±
±

a

3.59
4.52ab
5.58c
5.14bc
3.28bc
4.74b

12.31
15.07

14.70
14.76
15.18
14.27

±
±
±
±
±
±

1.09a
1.04b
1.38b
0.90b
0.72b
1.35b

1

Values are mean of three groups of fish (n = 3), with six fish per group. Mean values within the same column with different superscripts
are significantly different (P < 0.05).

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Aquaculture Nutrition 17; 595–604 Ó 2011 Blackwell Publishing Ltd


P = 0.109; YGST = 36.4970 + 1.6786X ) 0.1000X2, R2 =

0.843, P = 0.062; YGR = 55.1850 + 2.2907X ) 0.1192X2,
R2 = 0.824, P = 0.074).
SOD, CAT, GST, GPX, GR activities and GSH content in
hepatopancreas are presented in Table 3. SOD activity of fish
fed MHA-unsupplemented diet and diets containing MHA ‡
10.2 g kg)1 diet were significantly lower than that of fish fed
diet containing 5.1 g MHA kg)1. CAT activity enhanced with
increasing dietary MHA levels up to 10.2 g kg)1 diet, after
that it significantly decreased. GSH content was the highest in
fish fed diet containing 5.1 g MHA kg)1 and then decreased,
but no significant difference was obtained with dietary MHA
level from 10.2 to 15.3 g kg)1 diet. GST activity gradually
increased with dietary MHA levels up to 10.2 g kg)1 diet,
whereupon it significantly decreased. GPX activity significantly improved with MHA level up to 7.6 g kg)1 diet, and
then a decreased activity was sustained for the remaining
groups. GR activity of fish fed diets containing MHA from
5.1 to 10.2 g kg)1 diet was significantly higher. Hepatopancreas CAT, GST and GR activities were quadratic responses
to the increasing dietary MHA levels (YCAT = 24.2770 +
1.5826X ) 0.0570X2, R2 = 0.771, P = 0.110; YGST = 60.3440
+ 3.3125X ) 0.1718X2, R2 = 0.723, P = 0.146; YGR =
77.4710 + 1.7579X ) 0.1231X2, R2 = 0.804, P = 0.087).
MDA, protein carbonyl content, anti-superoxide anion
and anti-hydroxy radical capacities in intestine of juvenile
Jian carp fed graded levels of MHA are presented in Table 4.
MDA content, which was the highest in fish fed the MHAunsupplemented diet, decreased with dietary MHA levels up
to 7.6 g kg)1 diet, and then significantly increased with dietary MHA concentration further up to 12.7 and 15.3 g kg)1
diet. Protein carbonyl content was decreased as dietary MHA
level was up to 5.1 g kg)1 diet, and significantly increased
with MHA level ‡ 12.7 g kg)1 diet. Anti-superoxide anion

Table 4 Malondialdehyde content (MDA, nmol mg)1 protein),
protein carbonyl content (PC, nmol mg)1 protein), anti-superoxide
anion (ASA, mU mg)1 protein) and anti-hydroxy radical (AHR, U
mg)1 protein) capacities in intestine of juvenile Jian carp fed diets
containing graded levels of methionine hydroxy analogue (MHA g
kg)1 diet) for 60 days1
Dietary
MHA
level
0
5.1
7.6
10.2
12.7
15.3

MDA
1.45
1.30
0.85
0.87
1.29
1.36

±
±
±
±
±
±

PC
c

0.14
0.04b
0.07a
0.08a
0.11b
0.14bc

2.10
1.30
1.40
1.31
1.58
1.73

ASA
±
±
±
±
±
±

c

0.17
0.13a

0.15a
0.11a
0.12b
0.18b

103.8
105.5
119.7
94.7
94.9
92.5

AHR
±
±
±
±
±
±

b

6.3
8.2b
10.1c
5.6a
2.6a
4.5a

274.5

289.0
277.0
287.2
273.1
284.2

±
±
±
±
±
±

19.4a
12.9a
24.3a
24.1a
17.5a
19.2a

1

Values are mean of three groups of fish (n = 3), with six fish per
group. Mean values within the same column with different
superscripts are significantly different (P < 0.05).

capacity significantly increased as dietary MHA levels was up
to 7.6 g kg)1 diet and then the data significantly decreased
maintaining a plateau. No significant difference was manifested for anti-hydroxy radical capacity among six diets.
MDA, protein carbonyl content, anti-superoxide anion

and anti-hydroxy radical capacities in hepatopancreas of
juvenile Jian carp fed graded levels of MHA are displayed in
Table 5. MDA content was the highest in fish fed the MHAunsupplemented diet, and decreased with increasing dietary
MHA levels up to 7.6 g kg)1 diet, and then significantly
increased with dietary MHA concentration further up to 12.7
and 15.3 g kg)1 diet. Protein carbonyl content significantly
decreased with dietary MHA level up to 7.6 g kg)1 diet, and
then gradually increased. Fish fed on six experimental diets
showed no significant difference for anti-superoxide anion
capacity. Anti-hydroxy radical capacity gradually improved

Table 3 Superoxide dismutase (SOD, U mg)1 protein), catalase (CAT, U mg)1 protein), glutathione-S-transferase (GST, U mg)1 protein),
glutathione peroxidase (GPX, U mg)1 protein), glutathione reducase (GR, mU mg)1 protein) activities and glutathione (GSH, mg g)1 protein)
content in hepatopancreas of juvenile Jian carp fed diets containing graded levels of methionine hydroxy analogue (MHA g kg)1 diet) for
60 days1
Dietary
MHA
level
0
5.1
7.6
10.2
12.7
15.3

SOD
125.8
138.0
131.7
125.9

122.2
126.7

CAT
±
±
±
±
±
±

a

8.4
6.5b
10.1ab
8.2a
6.6a
5.0a

24.63
30.52
30.60
38.63
33.46
35.11

GST
±
±

±
±
±
±

a

2.18
2.56b
1.67b
2.86d
2.44c
1.60c

61.38
68.38
77.53
81.18
70.42
71.57

GPX
±
±
±
±
±
±

a

4.44
3.18b
2.66c
4.46c
4.94b
3.20b

159.7
161.8
178.1
165.8
153.5
154.8

GR
±
±
±
±
±
±

a

15.8
10.0a
8.7b
6.7a
7.0a

11.5a

77.17
84.39
82.30
84.53
77.58
76.53

GSH
±
±
±
±
±
±

a

7.04
5.23b
4.71ab
5.18b
5.14a
4.74a

8.29
9.91
8.16
6.90

6.69
7.29

±
±
±
±
±
±

0.50b
0.52c
0.67b
0.55a
0.32a
0.58a

1
Values are mean of three groups of fish (n = 3), with six fish per group. Mean values within the same column with different superscripts
are significantly different (P < 0.05).

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Aquaculture Nutrition 17; 595–604 Ó 2011 Blackwell Publishing Ltd


0
5.1
7.6
10.2

12.7
15.3

MDA
2.56
2.35
1.87
1.92
2.37
2.41

±
±
±
±
±
±

PC
b

0.25
0.13b
0.16a
0.19a
0.17b
0.18b

2.12
1.61

1.48
1.89
2.11
2.49

ASA
±
±
±
±
±
±

c

0.14
0.13a
0.15a
0.07b
0.20c
0.24d

151.5
147.2
146.5
146.4
151.4
149.2

AHR

±
±
±
±
±
±

a

3.8
8.2a
6.6a
2.6a
3.8a
2.2a

150.2
218.5
234.9
253.4
216.4
199.9

±
±
±
±
±
±

15.3
19.0bc
15.9cd
23.8d
21.5bc
14.7b

1

Values are mean of three groups of fish (n = 3), with six fish per
group. Mean values within the same column with different
superscripts are significantly different (P < 0.05).

with increasing dietary MHA levels up to 10.2 g kg)1 diet,
after that significantly decreased. Furthermore, anti-hydroxy
radical capacity showed quadratic response with dietary
MHA supplementation (YAHR = 148.7700 + 20.1630X )
1.1071X2, R2 = 0.947, P = 0.012).
GOT and GPT activities in serum of juvenile Jian carp fed
graded levels of MHA are shown in Table 6. GOT activity
was the highest for fish fed the MHA-unsupplemented diet,
and decreased with increasing dietary MHA levels up to 7.6 g
kg)1 diet, and then gradually increased. The same trend was
obtained for serum GPT activity. Both GOT and GPT
activities showed quadratic response with dietary MHA
supplementation (YGOT = 34.1800 ) 1.7490X+0.1000X2,
R2 = 0.942, P = 0.014; YGPT = 5.4035 ) 0.1294X+0.0111
X2, R2 = 0.895, P = 0.034)

Table 6 The activities of glutamate-oxaloacetate transaminase

(GOT, U L)1) and glutamate-pyruvate transaminase (GPT, U L)1)
in serum of juvenile Jian carp fed diets with graded supplemental
levels of methionine hydroxy analogue (MHA g kg)1 diet) for
60 days1
Dietary
MHA level
0
5.1
7.6
10.2
12.7
15.3
1

GOT
33.89
29.07
25.68
26.75
27.82
31.03

2.5
2.0
1.5
1.0
0.5
0.0
0.0

a

y = 0.0105x2 – 0.1782x + 2.0645
R2 = 0.922
3.0
6.0
9.0
12.0
15.0
MHA supplemental levels (g kg–1 diet)

18.0

(b)
Hepatopancreas protein carbonyl
(nmol mg–1 protein)

Dietary
MHA
level

(a)
Intestine protein carbonyl
(nmol mg–1 protein)

Table 5 Malondialdehyde content (MDA, nmol mg)1 protein),
protein carbonyl content (PC, nmol mg)1 protein), anti-superoxide
anion (ASA, mU mg)1 protein) and anti-hydroxy radical (AHR, U
mg)1 protein) capacities in hepatopancreas of juvenile Jian carp fed
diets containing graded levels of methionine hydroxy analogue

(MHA g kg)1 diet) for 60 days1

3.0
2.5
2.0
1.5
1.0

y = 0.0121x2 – 0.1565x + 2.0993
R2 = 0.945

0.5
0.0
0.0

3.0
6.0
9.0
12.0
15.0
MHA supplemental levels (g kg–1 diet)

18.0

Figure 1 Quadratic regression analysis of protein carbonyl content
in intestine (a) and hepatopancreas (b) for juvenile Jian carp fed diets
with graded levels of MHA for 60 days [Each point represents the
mean of three groups (n = 3), with six fish per group. Optimal
supplemental levels of MHA for protein carbonyl content in intestine
and hepatopancreas was 8.5 and 6.5 g kg)1 diet, respectively.].

The optimal supplemental level of MHA for protein carbonyl content was estimated by quadratic regression analysis
(Fig. 1). Based on protein carbonyl content in intestine, the
optimal supplemental level of MHA was estimated to be
8.5 g kg)1 diet. The regression equation was as follows:
Y = 2.0645 ) 0.1782X+0.0105X2 (R2 = 0.922, P = 0.022)
(Fig. 1a). Based on protein carbonyl content in hepatopancreas, the optimal supplemental level of MHA was estimated
to be 6.5 g kg)1 diet. The regression equation was as follows:
Y = 2.0993 ) 0.1565X+0.0121X2 (R2 = 0.945, P = 0.013)
(Fig. 1b).

GPT
±
±
±
±
±
±

d

1.75
1.25bc
1.79a
1.35a
2.53ab
2.03c

5.44
5.00

4.92
5.30
5.74
5.89

±
±
±
±
±
±

0.23cd
0.23ab
0.24a
0.37bc
0.23df
0.23f

Values are mean of three groups of fish (n = 3), with six fish per
group. Mean values within the same column with different
superscripts are significantly different (P < 0.05).

In general, antioxidant defence systems in fish consist of lowmolecular-weight antioxidants and antioxidant enzymes
(Martınez-Alvarez et al. 2005). When ROS generation rate
exceeds that of their removal in cell, ROS can oxidize cell
constituents such as lipids, proteins and DNA, and thus pose
a threat to cell integrity (Scherz-Shouval & Elazar 2007).
Therefore, the main purpose of this study was to investigate
the effects of methionine hydroxy analogue (MHA) on

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Aquaculture Nutrition 17; 595–604 Ó 2011 Blackwell Publishing Ltd


antioxidant response and oxidative damage in intestine and
hepatopancreas by evaluating lipid peroxidation and protein
oxidation, to determine whether MHA can protect their
structure integrity.
The scavenging ability of MHA against superoxide radical
(O2•)) and hydroxy radical (OH•) are strongly involved in
oxidative damage. Superoxide radical (O2•)) formed whenever molecular oxygen chemically oxidizes electron carrier
can further produce H2O2 that through the Fenton reaction
to generate reactive molecules such OH• causing a wide range
of oxidative damage within the cell (Hoshi & Heinemann
2001). In this study, intestine superoxide radical-scavenging
ability was improved by dietary optimal MHA supplementation, whereas intestine hydroxyl radical-scavenging ability
showed no alterations. Nevertheless, the trend of superoxide
and hydroxyl radical-scavenging ability in hepatopancreas
was opposite displaying a curious different behaviour
depending on tissue assayed. The reason for these interesting
results was not clear. However, these data implied that MHA
would have an antioxidant role in enhancing the superoxide
or hydroxyl radical-scavenging ability.
In the present study, antioxidant enzymes and GSH content response were also measured to further determine the
mechanism of MHA-induced inhibition of radical generation
in intestine and hepatopancreas. SOD is the first enzyme
involved in antioxidant defence systems to clear superoxide
radical (Visner et al. 1990), while CAT is an essential defence

against the potential toxicity of free radical like hydroxyl
radical by catalyzing the degradation of H2O2 (David et al.
2008). In our study, the data displayed that SOD and CAT
activities in intestine and hepatopancreas significantly
improved with increasing dietary MHA level up to 5.1, 7.6 or
10.2 g kg)1 diet respectively, showing that optimal level of
MHA can reduce the superoxide and hydroxyl radical in
tissues. GPX, GST and GR are three important enzymes
dependent on GSH (Rudneva 1997). Among them, GPX can
catalyse the reduction of hydroperoxides and H2O2 (Lackner
1998), and GST is able to detoxify compounds containing
reactive electrophilic centres to facilitate their excretion from
cells (Elia et al. 2006). The results in present study indicated
that a significant enhancement of GPX and GST activities
were obtained in intestine and hepatopancreas with dietary
MHA levels up to 5.1–10.6 g kg)1 diet respectively, further
supporting that MHA can increase enzymatic antioxidant
capacity in fish intestine and hepatopancreas to prevent
oxidative damage in these tissues. Studies had demonstrated
that MHA was converted into L-methionine for effective
utilization in chicken liver (Dibner & Knight 1984) and small
intestine (Martı´ n-Venegas et al. 2006). Through converting

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Aquaculture Nutrition 17; 595–604 Ó 2011 Blackwell Publishing Ltd

into L-methionine, MHA may participate or regulate protein
synthesis, and thus influence antioxidant enzymes activities in
intestine and hepatopancreas. Meanwhile, a few studies had

implied that excess methionine intake might make DNA
hypermethylated, which appeared to down-regulate some
genes expression in mouse (Waterland 2006). This discovery
may interpret the depression of all antioxidant enzymes
activities in intestine and hepatopancreas when dietary MHA
level was high.
GSH is a major low-molecular-weight antioxidant in vivo
that acts as a substrate for GPX and GST, enzymes that
catalyse the reactions for detoxification of xenobiotics and
ROS (Atmaca 2004). The present study indicated that GSH
content in intestine and hepatopancreas all significantly increased as dietary MHA level was at 5.1 g kg)1 diet. Similar
result was found in liver of juvenile sunshine bass fed on diet
with MHA supplementation (Keembiyehetty & Gatlin 1995).
GSH homeostasis is maintained through de novo synthesis
from precursor methionine and cysteine or regeneration from
its oxidized form GSSG (Shoveller et al. 2005). GR catalyses
the reduction of GSSG back to GSH by the expense of the
NADPH (Elia et al. 2006). In the present study, GR activities
in intestine and hepatopancreas increased with 5.1 or
7.6 g kg)1 diet MHA supplementation respectively. As was
previously commented, MHA could convert into L-methionine in male Ross chicken small intestine, after that diverting
to the transsulphuration pathway to the synthesis cysteine,
which is the precursor for GSH synthesis (Martı´ n-Venegas
et al. 2006). Accordingly, the improvement of GSH content
in digestive organs by MHA may be due to the increment of
precursor for GSH synthesis, and the elevated GR activity
for GSH regeneration. Furthermore, studies with human
colon epithelial cells indicated that cysteine availability and
local GSH concentration have a direct influence on epithelial
cell proliferation and survival (Shoveller et al. 2005). In our

previous study, MHA improved the intestine and hepatopancreas weight and intestine length (Xiao et al. 2010b),
which may be related to the enhancement of GSH content in
intestine and hepatopancreas cells. However, this hypothesis
needs further investigation.
Most components of cellular structure and function are
likely to be the potential targets of ROS, polyunsaturated
fatty acids in the biomembrane being the most susceptible
substrates for oxidation, which undergo peroxidation rapidly
(Zhang et al. 2004). MDA is one of the most readily assayed
end-products of both enzymatic and non-enzymatic lipid
peroxidation reactions (Requena et al. 1996). The present
study showed that optimal level of MHA significantly
declined MDA content in intestine and hepatopancreas,


suggesting that lipid peroxidation in these organs was depressed by MHA. According to this, Li et al. (2009a,b)
indicated that MHA level was negatively correlated with
thiobarbituric acid reactive substances (TBARS) in hybrid
striped bass liver. Peroxidation of membrane lipid often initiates the loss of membrane integrity, which may lead to the
leak of ions or enzymes from tissue cells (Veena et al. 2006).
Therefore, GOT and GPT activities in serum were further
investigated to study tissue damages associated to lipid peroxidation. With the increasing of MHA levels, GOT and
GPT activities in serum decreased to a point, and then
increased in this study. The trend was opposite with GOT
and GPT activities in muscle and hepatopancreas of Jian
crap in our previous study (Xiao et al. 2010b). Humtsoe et al.
(2007) implied that GOT and GPT in muscle and liver of
rohu carp might release into serum when exposed to oxidative stress. Therefore, the reduced GOT and GPT activities in
serum further demonstrated that lipid peroxidation was
depressed by MHA in tissues. Also, protein oxidation damage can be directly induced by ROS (Berlett & Stadtman

1997), and can also be led by lipid peroxidation end products
such as MDA and 4-hydroxynonenal (Negre-Salvayre et al.
2008). Protein carbonyl content is the most widely used
biomarker for oxidative damage to proteins, and reflects
cellular damage induced by ROS (Baltacıog˘lu et al. 2008). In
our study, we observed that optimal level of MHA significantly declined protein carbonyl content in intestine and
hepatopancreas, suggesting that protein oxidation was also
depressed in these organs by MHA. The optimal MHA
supplemental level for protein carbonyl content in intestine
and hepatopancreas by the quadratic regression analysis was
estimated to be 8.5 (R2 = 0.922, P = 0.022) and 6.5
(R2 = 0.945, P = 0.013) g kg)1 diet, respectively. In our
results, we also found that MDA and protein carbonyl
content enhanced, while all antioxidant enzymes activities
decreased in intestine and hepatopancreas when dietary
MHA levels were high. It seems that excess levels of dietary
MHA have some bad effects on antioxidant status in fish
intestine and hepatopancreas.
In conclusion, MHA could promote the antioxidant defence in fish intestine and hepatopancreas by increasing
enzymatic antioxidant capacity, GSH content and clearance
of oxygen radicals, thus protecting the structure and function
of these organs. Therefore, the result of this study can provide some theoretical evidence for our previous research
about the improvement of digestive and absorptive capacity
by MHA in Jian carp (Xiao et al. 2010b). Nevertheless, the
specific molecule mechanism that MHA mediates antioxidant
defence in fish needs further investigation.

This study was financially supported by Sumitomo-chemical
(Japan), National Science Foundation of China (30771671
and 30871926), Programme for New Century Excellent Talentsin University (NCET-08-0905) and the Key Project of

Chinese Ministry of Education (208120). The authors would
like to thank the Sumitomo-chemical for providing financial
assistance, and thank the personnel of these teams for their
kind assistance.

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Aquaculture Nutrition
doi: 10.1111/j.1365-2095.2011.00857.x

2011 17; 605–612

..........................................................................................

1,2

3

3

4

4

1

1,2
1

Gesellschaft fu¨r Marine Aquakultur mbH, Hafento¨rn, Bu¨sum; 2 Department of Marine Aquaculture, Christian-AlbrechtsUniversita¨t zu Kiel, Kiel; 3 Pilot Pflanzeno¨ltechnologie Magdeburg e.V., Berliner Chaussee, Magdeburg; 4 Johann Heinrich
von Thu¨nen-Institut, Federal Research Institute of Rural Areas, Forestry and Fisheries; Institute of Fisheries Ecology,
Wulfsdorfer Weg, Ahrensburg, Germany

The potential of rapeseed protein concentrate as fish meal
alternative in diets for wels catfish (initial average weight
86.5 ± 1.9 g) was evaluated. Sixteen fish were stocked into
each of 12 experimental tanks being part of a freshwater
recirculation system. Fish were organized in triplicate groups
and received isonitrogenous (603 ± 3 g CP kg)1) and isocaloric (23.0 ± 0.3 kJ g)1) experimental diets with 0%, 25%,
50% and 75% of fish meal replaced with rapeseed protein
concentrate (710 g CP kg)1). At the end of the 63-day feeding
period, weight gain, standard growth rate, feed intake, feed
conversion ratio and protein efficiency showed no significant
difference between control group and fish fed on diets with
25% reduced fish meal content by inclusion of rapeseed protein
concentrate. Higher dietary fish meal replacement negatively
affected diet quality and palatability resulting in reduced feed
intake, feed efficiencies and fish performance. However, blood
serum values of triglycerides, glucose and protein were not
significantly different between treatment groups, still indicating a favourable nutrient supply from all experimental diets.
KEY WORDS: feed evaluation, fish meal, growth trial, rapeseed,
rapeseed protein concentrate, wels catfish

Received 24 September 2010, accepted 4 February 2011
Correspondence: Hanno Slawski, Institute of Fisheries Ecology, Wulfsdorfer Weg 204, 22926 Ahrensburg, Germany. E-mail: hanno.slawski@
vti.bund.de

In 2009, worldwide production of rapeseed (including canola) was 61.6 Mio t. Thus, rapeseed, commonly produced in

..............................................................................................

Ó 2011 Blackwell Publishing Ltd

temperate regions, ranked as number three oilseed worldwide, only surpassed by soybean (222.2 Mio t) and cotton
seed (64.0 Mio t) (FAO 2010). For soybean, a crop mainly
cultivated in warm regions, efforts and research have been
undertaken to make it a commonly accepted fish feed
ingredient and fish meal alternative (Gatlin et al. 2007). The
usage of rapeseed products as fish feed ingredients, however,
is limited. Either simple oilcakes or rapeseed meals with
increased protein content produced from oilcakes that were
de-oiled with organic solvents have been tested as protein
sources in feeding trials with several fish species, among them
Oncorhynchus mykiss (Burel et al. 2000a,c, 2001; Thiessen
et al. 2003, 2004; Shafaeipour et al. 2008), Oreochromis
mossambicus (Davies et al. 1990), Ictalurus punctatus (Webster et al. 1997), Cyprinus carpio (Dabrowski & Kozlowska
1981), Pagrus auratus (Glencross et al. 2004) and Psetta
maxima (Burel et al. 2000a,b). In general, experimental
results showed that the nutritional quality of simple rapeseed
products is below that of fish meal although they contained a
well-balanced amino acid profile. Particularly, antinutritional
factors (ANF) determine the quality of rapeseed products for
fish nutrition. Prominent ANF in rapeseed products are
glucosinolates, phytic acid, phenolic constituents (e.g. tannins) and indigestible carbohydrates (Mawson et al. 1995;
Francis et al. 2001). Several processing techniques can be
adapted to reduce the level of antinutrients in rapeseed
products and improve their value for fish nutrition. Dehulling of seeds and utilization of high temperatures and organic
solvents (hexane) during oil extraction as well as sieving of
meal decrease content of glucosinolates, phytate, fibre, cellulose, hemicellulose, sinapin and tannins (Fenwick et al.
1986; Anderson-Hafermann et al. 1993; Tripathi et al. 2000)
and increase protein level in meals (Mwachireya et al. 1999).
Protein extraction from meals by methanol–ammonia

treatment or ethanol treatment will increase protein level and
effectively remove glucosinolates, phenolic compounds, soluble sugars, such as sucrose, and some oligosaccharides (e.g.
raffinose and stachyose) (Naczk & Shahidi 1990; Chabanon
et al. 2007). Last but not least water treatment appears to be
a cost-effective method for removing glucosinolates from
rapeseed meals (Tyagi 2002). Sporadically, rapeseed protein
products of high quality are being produced in different
countries for application in animal nutrition. However, these
products are produced for test purposes in small volumes
until their potential as protein source in animal nutrition is
clarified. Besides nutritive quality, their costs of production
will have to become low enough to make rapeseed protein
products available at a competitive price compared to other
protein sources, especially fish meal. In this study, a highquality rapeseed protein concentrate containing 710 g CP
kg)1 was tested as fish meal replacement in fish diets. Diets
were fed to wels catfish (Silurus glanis L.), a carnivorous
species that is believed to have potential for indoor recirculation farming in Europe as a high-value product for local
markets (Mazurkiewicz et al. 2008). Fish performance and
blood serum parameters were investigated to evaluate rapeseed protein concentrate as fish meal alternative in diets for
wels catfish.

Four experimental diets were formulated in which fish meal
was replaced with rapeseed protein concentrate (RPC) at
0%, 25%, 50% and 75% level (designated as R0, R25, R50
or R75, respectively). Solvent extracted RPC was obtained
from the Pilot Pflanzeno¨ltechnologie Magdeburg e.V.,
Magdeburg, Germany. For the production of RPC, a batch
of rapeseed (variety Lorenz; Norddeutsche Pflanzenzucht,

Hohenlieth, Germany) was conditioned in a vacuum dryer
for 15 min at 60–70 °C to inactivate the enzyme myrosinase.
Then, rapeseed was cold-pressed. To remove residual oil
from the oilcake (129 g kg)1 oil, 313 g kg)1 protein), it was
crushed into 1- to 5-mm particle size followed by a hexane
treatment. The treatment lasted for 2 h, and the incubation
temperature was 60 °C. Hexane-treated rapeseed meal
extract was desolventized under pressure to remove hexane
(<300 ppm), then rapeseed meal extract was further crushed
to a particle size of 0.2–0.1 mm. A four-step treatment using
a 75% ethanol solution (35 min at 60 °C) aimed to remove
glucosinolates from rapeseed meal extract. This resulted in a
residual oil content of the nearly glucosinolate-free rapeseed

meal extract of 11 g kg)1 and a protein content of
398 g kg)1. In the following, protein was gained through liquid water extraction (rapeseed meal extract 1 : 15 water).
For this, the suspension was heated to 40–45 °C followed by
2 h of constant agitation. Afterwards, the suspension was
decanted. Following decantation, the solvent was collected,
and residue material was secondly extracted (residue 1 : 10
water, 5% NaCl) at 40–45 °C and 1-h contact time under
constant agitation. Following extraction, the suspension was
decanted. Solvent was collected, and residue prepared for a
third extraction. Then, solvents of extraction 1, 2 and 3 were
collected to remove low-molecular compounds and to concentrate dissolved proteins by dia- and ultrafiltration. During
filtration, conductivity was checked. Protein washing ended,
when conductivity was 5–6 mS cm)1, corresponding to a
protein content of 600 g kg)1. The gained material was
spray-dried at 70–80 °C, which led to a rapeseed protein
concentrate with 710 g kg)1 protein content (Table 1).

Vitamins and minerals were added to diets to meet the
dietary requirements of freshwater fish [NRC (National
Research Council) 1993]. The diets were formulated to be
isonitrogenous (603 ± 3 g CP kg)1) and isocaloric (23.0 ±
Table 1 Nutrient composition (g kg)1 dry matter) and essential
amino acid profiles (g kg)1 protein) of fish meal (FM) and rapeseed
protein concentrate (RPC) and concentration of antinutritional
factors detected in RPC

Dry matter
Crude protein
Crude fat
Ash
Phosphorus
Crude fibre
NfE1
Gross energy2 (kJ g)1)
Essential amino acids
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophane
Valine
Glucosinolates (lmol g)1)
Phytic acid (g kg)1)

Tannins (g 100 g)1)

FM

RPC

916
690
70
207
29
5
28
19.9

942
710
22
92
21
48
128
25.2

58.4
20.0
36.3
64.6
65.5
23.7

35.2
39.0
8.4
44.5

74.9
29.9
42.9
78.1
57.0
20.3
42.8
44.4
14.2
54.3
0.2
<0.5
<0.005

Nitrogen-free extract (g kg)1) = 1000 ) (crude protein + crude
fat + ash + fibre).
2
Calculated by: crude protein = 23.9 MJ kg)1; crude fat =
39.8 MJ kg)1; NfE, fibre: 17.6 MJ kg)1.
1

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Aquaculture Nutrition 17; 605–612 Ó 2011 Blackwell Publishing Ltd

0.3 MJ kg)1). Essential amino acid concentrations did
not differ considerably between experimental diets. The
diets were manufactured to give pellets of 4 mm in diameter
(L 14-175; AMANDUS KAHL, Reinbek, Germany). Diet
formulations, proximate compositions and amino acid
profiles are given in Table 2.
The growth trial was conducted at the Johann Heinrich
von Thu¨nen Institute of Fisheries Ecology, Ahrensburg,
Germany. Juvenile wels catfish (Silurus glanis L.) were
obtained from the Ahrenhorster Edelfisch GmbH & CO KG
(Ahrenhorst, Germany). Two weeks before the experiment
started, 17 fish were stocked in each of nine experimental
tanks (96 L; bottom surface 480 cm2), being part of a recir-

Table 2 Formulation (g kg)1), essential amino acids composition
(g kg)1 crude protein) and proximate composition (g kg)1 dry matter) of experimental diets
Ingredients
1

Herring meal
Rapeseed protein concentrate
Soyprotein concentrate2
Blood meal3
Wheat gluten4
Fish oil1
Dextrose3
Vit/MinMix5
Essential amino acids
Arginine

Histidine
Isoleucine
Leucine
Lysine
Methionine + cysteine
Phenylalanine
Threonine
Valine
Proximate composition
Dry matter (g kg)1)
Crude protein
Crude fat
Ash
Phosphorus
NfE + fibre6
Calculated glucosinolates
(lmol g)1)
Gross energy7 (MJ kg)1)

R0

R25

R50

R75

500
0
110

105
65
120
80
20

375
122
110
105
65
126
77
20

250
243
110
105
65
132
75
20

125
362
110
105
65
138

75
20

52.9
30.9
30.2
75.9
59.3
27.5
42.9
34.1
49.9

55.6
33.0
31.7
79.1
59.2
28.9
44.9
35.6
52.0

58.3
33.9
33.2
81.3
57.7
30.4
46.4

36.6
53.5

59.1
34.6
32.1
81.7
54.7
31.1
47.1
37.1
52.6

922
607
158
131
14.8
104
0

931
603
165
114
14.1
118
0.02

939

600
158
99
13.0
143
0.05

944
600
148
83
12.7
169
0.07

22.6

23.1

23.1

23.2

1

VFC GmbH, Cuxhaven, Germany.
IMCOSOY 60 Piglet, IMCOPA, Araucaria, Brasil.
3
Euroduna-Technologies GmbH, Barmstedt, Germany.
4

Cargill Deutschland GmbH, Krefeld, Germany.
5
AA-Mix 517158 & 508240, Vitfoss, Gra˚sten, Denmark.
6
Nitrogen-free extract (g kg)1) = 1000 ) (crude protein + crude
fat + ash).
7
Calculated by: crude protein = 23.9 MJ kg)1; crude fat = 39.8 MJ
kg)1; NfE, fibre: 17.6 MJ kg)1.
Tryptophane was not analysed.
2

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Aquaculture Nutrition 17; 605–612 Ó 2011 Blackwell Publishing Ltd

culation system. Tanks were provided with freshwater at
2 L min)1 (temperature: 26.9 ± 0.7 °C; O2: 6.8 ± 0.5 mg
)1
À
L)1; pH: 7.3 ± 0.5; NHþ
4 : <0.6 mg L ; NO2 : <0.2 mg
)1
L ). Photoperiod was in accordance with natural rhythmic.
In respect of the fishesÕ light sensitivity, tanks were halfcovered with translucent plastic lids. For a 2-week adaptation period, fish were fed the control diet in four daily meals
until apparent satiation. After the adaptation period, initial
average fish weight was determined (86.5 ± 1.9 g). For an
experimental period of 63 days, triplicate groups of fish were
fed the experimental diets in four daily meals (8:00 and 11:00
a.m., 2:00 and 5:00 p.m.) until apparent satiation. At the

beginning and at end of the experiment, two fish per tank
were removed and stored at )23 °C for the determination of
initial and final body composition.

At the end of the feeding period, blood samples from the
caudal vein and artery of eight fish per experimental treatment were taken with a heparinized syringe (1 mL). Blood
haematocrit percentage was determined after centrifugation
(10 000 g, 6 min) of glass tubes filled with fresh blood in a
haematocrit centrifuge (Haematokrit 210; Andreas Hettich
GmbH & Co. KG, Tuttlingen, Germany). Remaining fresh
blood was filled in Eppendorf tubes and centrifuged
(1000 g, 5 min). Supernatant blood plasma was separated
into Eppendorf tubes and stored at )84 °C in a freezer.

Diets and homogenized fish bodies were analysed in duplicate for proximate composition. Dry matter was calculated
from weight loss after drying in an oven at 105 °C until
constant weight. Fat content was determined after HCl
hydrolysis (Soxtec HT6; Tecator, Ho¨gana¨s, Sweden) and
total nitrogen content by the Kjeldahl technique (protein = N · 6.25, Kjeltec Auto System; Tecator, Ho¨gana¨s,
Sweden). Ash content was calculated from weight loss after
incineration of samples in a muffle furnace for 2 h at 550 °C.
Dietary amino acid concentrations were analysed by nearinfrared reflectance spectroscopy according to van Kempen
& Bodin (1998). Blood plasma concentrations of triglycerides, glucose and protein were determined using a microplate reader (Infinite 200Ò; TECAN Group Ltd, Ma¨nnedorf,
Switzerland, CH) and commercial kits (Triglycerides GPO
and Glucose GOD-PAP; Greiner Diagnostic GmbH, Bahlingen, Germany; RotiÒ-Quant; CARL ROTH GmbH +
Co.KG, Karlsruhe, Germany).


Fish performance was determined, using the following formulae:
Specific growth rate (SGR, % day)1) = (ln final body

weight ) ln initial body weight) · 100/days fed.
Feed intake as % body weight day)1 = the mean feed
consumption per fish per day as a percentage of the daily fish
body weight for the experimental period. The daily fish body
weight was calculated using daily SGR values equal to the
final SGR of each tank.
Feed conversion ratio (FCR) = g dry feed intake/g wet
body weight gain.
Protein efficiency ratio (PER) = g wet body weight gain/
g protein intake.
Condition factor (CF) = g body weight/cm total
length3 · 100.
Survival (%) = (initial fish count ) dead fish count)/
initial fish count · 100.
All diets were assigned by a completely randomized design.
Biological and analytical data were checked for normal distribution using the Kolmogoroff–Smirnov test and eventually
subjected to transformation. Data were subjected to regression and one-way analysis of variance (ANOVA) using SPSS
17.0 for Windows (SPSS Inc., Chicago, IL, USA). When
differences among groups were identified, multiple comparisons among means were made using TukeyÕs HSD test.
Statistical significance was determined by setting the aggregate type I error at 5% (P < 0.05) for each set of comparisons.

No significant differences in growth performance parameters
and feed efficiencies were detected between control diet and
R0
Initial weight (g)
Final weight (g)
SGR (%)
Feed intake (g DM)
Feed intake as %
body weight day)1

FCR
PER
GEI (MJ)
CF

R25

R50

R25 diet-fed fish. Compared to the control group, fish
growth performance, voluntary feed intake and feed efficiencies declined at fish meal replacement levels above 25%.
Feed intake as per cent body weight was not affected up to
50% fish meal replacement level (Table 3). While fish growth
performance, voluntary feed intake and feed efficiencies significantly correlated with the dietary inclusion level of RPC
(Table 3), no correlation was found between feed intake as
per cent body weight and dietary inclusion of RPC. No significant differences in whole-body composition were detected
between fish fed on the control diet and fish receiving RPC
diets (Table 4). Significant correlations were found between
dietary RPC and phosphorus level and whole-body moisture
(R2 = 0.50 and 0.48, P < 0.05), fat (R2 = 0.54 and 0.53,
P < 0.01) and ash (R2 = 0.56 and 0.58, P < 0.01) content.
Haematocrit values as well as blood serum values determined
showed no significant difference between treatment groups
and were not correlated to the dietary inclusion level of RPC
(Table 5).

While usability and limitations of simple rapeseed products
as fish feed ingredients have been widely investigated
(Dabrowski & Kozlowska 1981; Davies et al. 1990; Webster
Table 4 Proximate whole body composition (g kg)1 wet weight) of

wels catfish fed the experimental diets
Parameter

R0

Moisture
Crude protein
Crude fat
Ash

740
153
85
24

R25
±
±
±
±

12
6
5
2

746
152
82
23

R50
±
±
±
±

10
7
7
3

755
150
76
21

R75
±
±
±
±

10
8
6
1

763
144

72
20

±
±
±
±

11
5
6
2

Initial body composition: moisture 823 g kg)1, crude protein
131 g kg)1, crude fat 28 g kg)1 and ash 18 g kg)1.

R75

R2

P

87.8
279.8a
1.84a
102.9a
0.96a

±
±

±
±
±

2.7
9.1
0.10
3.8
0.04

85.5
264.0a
1.79a
97.1a
1.02a

±
±
±
±
±

1.2
7.7
0.06
3.2
0.03

86.1
218.4b

1.48b
85.7b
0.97a

±
±
±
±
±

1.5
15.7
0.10
5.3
0.02

86.7
159.4c
0.97c
60.9c
0.81b

±
±
±
±
±

2.0
4.4

0.04
3.1
0.01

0.92
0.86
0.88
0.36

<0.01
<0.01
<0.01
ns

0.53a
1.13a
2.33a
0.57

±
±
±
±

0.03
0.06
0.09
0.04

0.56a

1.11a
2.24a
0.62

±
±
±
±

0.02
0.02
0.07
0.04

0.68b
0.92b
1.98b
0.61

±
±
±
±

0.04
0.05
0.12
0.05

0.86c

0.72c
1.41c
0.61

±
±
±
±

0.04
0.02
0.07
0.04

0.84
0.88
0.85
0.08

<0.01
<0.01
<0.01
ns

Table 3 Growth response, feed intake,
feed efficiencies, condition factor (CF)
and survival of wels catfish fed experimental diets

R2: parameter values are regressed to the dietary level of rapeseed protein concentrate.
Values are given as mean ± standard deviation. Values in the same row with common superscript

letters are not significantly different (P < 0.05).
FCR, Feed conversion ratio; PER, Protein efficiency ratio; SGR, Specific growth rate.

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Aquaculture Nutrition 17; 605–612 Ó 2011 Blackwell Publishing Ltd


Table 5 Blood haematocrit content and blood serum values of wels
catfish fed experimental diets
Parameter

R0

Haematocrit
0.23 ± 0.04
(Proportion of 1)
Triglycerides
4.72 ± 2.21
(mM)
Glucose (mM)
6.54 ± 1.33
Protein (g L)1)
36.0 ± 2.8

R25

R50

R75

0.24 ± 0.04 0.25 ± 0.02 0.24 ± 0.02
6.55 ± 4.89 7.20 ± 4.52 8.47 ± 1.46
7.10 ± 1.27 6.66 ± 1.11 6.43 ± 1.27
35.5 ± 2.1 36.1 ± 2.8 37.1 ± 1.9

et al. 1997; Burel et al. 2000a,b,c, 2001; Thiessen et al. 2003,
2004; Glencross et al. 2004; Shafaeipour et al. 2008), lack of
information exists about the benefits of high-quality products
originating from rapeseed oilcakes with protein contents
comparable to or above that of fish meal. Higgs et al. (1982)
successfully replaced 25% of dietary protein from a fish meal
control diet for juvenile Oncorhynchus tshawytscha with
rapeseed protein concentrate (613 g CP kg)1) without
reducing growth rate and food (protein) utilization. In the
study, however, higher fish meal replacement levels with
rapeseed protein concentrate were not evaluated.
The results of our study demonstrate that 25% of dietary
fish meal can be replaced with RPC in diets fed to wels
catfish without negative effects on feed efficiencies and fish
growth. When 50% of dietary fish meal was replaced with
RPC, the feed intake as per cent of fish body weight was not
significantly different from the control group but feed efficiencies and fish growth were reduced. At 75% fish meal
replacement level, fish showed reduced diet acceptance and
reluctant feed intake as a result of unfavourable diet taste. It
appears, therefore, that the level of blood meal incorporated
into diets as feed attractant did not effectively counteract the
negative effects on diet taste resulting from rapeseed protein
concentrate. It is known that the bitter taste exuded by
glucosinolate metabolites, such as isothiocyanates and vinyloxazolidinethiones, present in rapeseed meals can potentially retard diet acceptance by fish. This was found in

O. mykiss and P. maxima at dietary glucosinolate levels of
7.3 or 18.7 lmol g)1, respectively (Burel et al. 2000b,c).
Because the RPC used in our study contained 0.2 lmol
glucosinolates g)1 (Table 1), the highest calculated dietary
glucosinolate concentration was 0.07 lmol g)1 in diet R75
(Table 2). This value is far below the level when glucosinolates become detrimental on food intake of O. mykiss and
P. maxima (Burel et al. 2000b,c) but to our observation, the
typical mustard smell of glucosinolates was still noticeable in
diets R50 and R75. It seems, therefore, that wels catfish is
more sensitive towards a bitter diet taste than other carnivorous fish species. This goes together with the fishÕs

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Aquaculture Nutrition 17; 605–612 Ó 2011 Blackwell Publishing Ltd

excellent developed olfactory organ (Jakubowski & Kunysz
1979). Reduced feed intake in fish fed on diets R50 and R75
resulted in lower growth rates and reduced feed conversion
compared to the control group (Table 3). For prospective
feeding trials with rapeseed protein products in wels catfish,
it appears recommendable to use other feed attractants than
blood meal. Fish protein hydrolysate, squid hydrolysate,
stick water or krill meal at dietary levels from 30 to
50 g kg)1 have shown to be effective feed attractants and
sources of amino acids and minerals when diets low in fish
meal were fed to carnivorous fish (Espe et al. 2006, 2007;
Torstensen et al. 2008; Kousoulaki et al. 2009). As fish behaved calm in all treatment groups, increased energy
expenditure because of feed-searching activity in high RPC
groups did not deplete feed conversion. Thus, lower feed
efficiency might be a result of reduced diet digestibility because of RPC inclusion.

In this study, we did not determine the digestibility of
nutrients and minerals from RPC in wels catfish. We discovered that faeces collection from wels catfish to determine
nutrient and mineral digestibility appears hardly possible. On
the one hand, faeces of wels catfish are slimy and rapidly
dilute in water. This precludes faeces collection with an
automatic collector. On the other hand, faeces stripping, even
when fish are anaesthetized, will stress the sensitive fish. As a
result, wels catfish will stop feed intake for days. Killing fish,
as a last alternative to gain faeces, requires a high number of
individuals to collect enough faeces for laboratory analysis.
In this study, fish count was not sufficient to gain required
amounts of faeces for laboratory analysis. Therefore,
assumptions regarding nutrient and mineral digestibility of
RPC in wels catfish are based on studies conducted with
rapeseed protein products in other fish species. Mwachireya
et al. (1999) stated that fibre levels, either alone or together
with phytate, can have greatest adverse effects on the
digestibility of canola protein products for O. mykiss. The
authors reported that among different canola products
tested, only canola protein isolate (908 g CP kg)1) met
nutrient digestibility coefficients corresponding to fish meal.
In our study, the applied processing techniques to produce
RPC from rapeseed oilcake led to relatively low levels of
phytic acid in the final RPC (0.5 g kg)1). Accordingly, calculated dietary phytic acid concentrations originating from
RPC were 0.1 and 0.2 g kg)1 in diets R50 and R75, respectively. In fish nutrition studies, phytic acid concentrations
that negatively influence mineral and nutrient availability are
commonly higher. Spinelli et al. (1983) observed decreased
growth rates in rainbow trout fed a diet containing 5 g kg)1
synthetic phytic acid. Synthetic phytic acid at concentrations