Aquaculture Research, 2009, 1^12 doi:10.1111/j.1365-2109.2009.02223.

REVIEW ARTICLE
Water-soluble vitamins in fish ontogeny

Rune Waagb
National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway

Correspondence: Dr R Waagb, NIFES, P.O. Box 2029 Nordnes, N-5817 Bergen, Norway. E-mail: rune.waagbo@nifes.no

Abstract 1993;Woodward1994; Dabrowski 2001; Halver 2002;

Webster & Lim 2002; Koshio 2007). Among this
Studies on vitamin requirement at early stages are
group of eight vitamins, vitamin C or ascorbic acid is
di⁄cult and vary in quality, both due to the scienti¢c
by far the most studied (Dabrowski 2001). This is due
approach and vitamin analysis. Focus has been on
to the rapid and dramatic consequences at de¢ciency,
water-soluble vitamins that cause dramatic losses of the
and the high risk for de¢ciency using traditional and
o¡spring in practical farming situations or in wild life,
practical ingredients in ¢sh feed production (Sandnes
like vitamin C and thiamine de¢ciencies respectively.
1991). Further, the essentiality of ascorbic acid is ver-
Practical solutions including vitamin administration
i¢ed in several farmed ¢sh species, demonstrated by
through brood stock and larvae diets have con¢rmed
the lack of the enzyme gulonolactone oxidase,
and corrected the vitamin de¢ciencies. For the other
needed for ascorbic acid biosynthesis (Mland &
water-soluble vitamins, the situation is not so obvious.
Waagb 1998; Moreau & Dabrowski 2001). Finally,
Descriptive studies of folate and vitamin B6 during ¢sh
the need for a stable and bioavailable ascorbic acid
ontogeny have shown a net loss of vitamin during
derivative in feed for all commercial farmed ¢sh spe-
endogenous feeding and a steady transfer of vitamin
cies has been a driving force in vitamin C research for
from the yolk sac into the body compartment, and
a long period.
¢nally, dramatic increases in body vitamin levels after
For other water-soluble vitamins far less informa-
the start of feeding. The kinetics of mass transfer with
tion is available, and we like to think that live feed,
ontogeny appears, however, to di¡er between vita-
practical feed ingredients and safety supplementa-
mins. Start of feeding of ¢sh larvae with live or formu-
tions are su⁄cient supplies for these vitamins to pre-
lated feeds includes several challenges with respect to
vent suboptimal conditions in all farmed species at all
water-soluble vitamins, including aspects of live feed
stages of development. For salmonids, Woodward
enrichment and stability, micro-diet leaching, variable
(1994) suggested that the listed minimum require-
feed intakes, immature gastrointestinal tract, variable
ments at that time were extremely high compared
bioavailability of vitamins and larvae vitamin storage
with that for other species, probably re£ecting practi-
capacity. Consequently, the exact minimum require-
cal recommendations rather than minimum require-
ments are di⁄cult to estimate and vitamin recom-
ments for optimal growth and health. The latter is
mendations need to consider such conditions.
indeed a safer approach for a rapidly growing aqua-
culture industry (Hardy 2001). The focus of the mar-
Keywords: ¢sh, vitamin, ontogeny, endogenous
ine ¢sh farming research has also been severely
feeding, start feeding, live feed
in£uenced by the commercial interests and pro-
gresses that have been made side by side. Practical
and basic researches on larvae nutrition have faced
Introduction
challenges with feed processing and storage stability,
The traditionally de¢ned water-soluble vitamins live feed enrichment, leaching, bioavailability, nutri-
seem to be essential to all examined farmed ¢sh spe- ent interactions and a situation-dependent increase
cies so far. The basic roles and de¢ciency symptoms of in requirements. Such conditions may lead to subop-
the single vitamins are detailed in the literature (NRC timal vitamin nutrition with potential consequences

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Journal Compilation r 2009 Blackwell Publishing Ltd 1
Water soluble vitamins in ¢sh ontogeny R Waagb
for the larvae quality, and later growth and welfare of
the ¢sh (Cahu, Infante & Takeuchi 2003).
The aim of the present communication is to dis-
cuss the requirement for water-soluble vitamins for
¢sh species during early ontogeny in light of updated
literature. However, the technical approaches and ex-
periments vary in scienti¢c strength, depending on
the experimental design, ¢sh species, developmental
stage of the ¢sh and not the least the feed type.
Experimental approaches and analytical
methods
Experimental designs and methods used in vitamin
studies on ¢sh larvae and juveniles have mostly been
traditional, including start of feeding experiments
with live feed and dose^response studies with formu-
lated diets (Boonyaratpalin 1989; Halver 1995). The
technical nature of the diets to ¢sh larvae does not al-
ways allow exact determination of requirements in the
same manner as formulated puri¢ed diets for vitamin
studies in larger ¢sh, in terms of feed levels of vitamins
and intakes (Boonyaratpalin 1989; Woodward 1994).
Firstly, there were uncertainties in the suitability of
the live feed organism for the ¢sh species and the
given developmental stage. Further, the overall live
pray production procedure is a compromise for both
macro- and micro-nutrient enrichments, with a
minor focus on the chemical form and the technical
quality of the water-soluble vitamin. Finally, own
metabolism of the live prey in the period from feeding
to being ingested by the ¢sh larvae may result in re-
duced live pray quality, as illustrated for essential fatty
acids (Evjemo, Cotteau, Olsen & Sorgeloos 1997).
Some studies include selective vitamin feeding of
brood stocks and subsequent evaluation of the o¡-
spring quality and performance (Fig. 1), like a study
on vitamin C in rainbow trout (Blom & Dabrowski
1995). More descriptive studies on the mass transfer
of vitamins between the yolk sac and the larval body
compartments during endogenous feeding have been
used as an estimate of the requirement of vitamins
for growth until exogenous feeding (Rnnestad, Lie
& Waagb 1997; Rnnestad, Hamre, Lie & Waagb
1999; Mland, Rnnestad & Waagb 2003;Fig.1). Im-
portantly, the larvae examined in these studies origi-
nated from high-quality o¡spring.
At the start of feeding (Fig. 1), minimum require-
ments are determined on the basis of selected
markers at di¡erent biological organization levels in
the ¢sh, like mortality, growth, body/tissue vitamin
saturation (maintenance of steady-state tissue con-
2 Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 1^12
Aquaculture Research, 2009, 1^12
centration), retention of nutrients, speci¢c biochem-
ical measures like enzyme activities, histopathology
and lately also vitamin speci¢c molecular markers.
Using more speci¢c markers, vitamin recommenda-
tion levels have generally been reduced.
Gouillou-Coustans, Bergot and Kaushik (1998) de-
termined the ascorbic acid requirement of common
carp (Cyprinio carpio) larvae with semi-puri¢ed diets
added with graded levels of a stable and bioavailable
ascorbyl phosphate (AP) form, and demonstrated that
the level for tissue saturation was six times higher than
that of maximum growth (45 mg AA equiv. kg  1). This
shows that body vitamin saturation may not necessa-
rily be a useful requirement indicator in ¢sh larvae,
due to the limited storage capacity for water-soluble
vitamins and thereby low retention e⁄cacies. In juve-
nile Atlantic salmon fed graded levels of AP, tissue vita-
min saturation varied both by dose and by time of
feeding, illustrating the importance of both feed level
and feed intake (Waagb, Glette, Raa-Nilsen & Sandnes
1993). For ¢sh larvae, the e⁄cacy of intestinal phos-
phate hydrolysis of AP and subsequently AA uptake
may increase the insecurity of the requirement esti-
mates (Dabrowski, Moreau & El-Saidy 1996).
The speci¢city, reliability and accuracy of analyti-
cal methods for determination of water-soluble vita-
mins comprise a part of the discussion of vitamin
requirements. Normally, one would expect con¢rm-
ing feed and tissue vitamin analyses re£ecting the
dietary input. For ascorbic acid, the discussion has
focused on assay techniques and vitamer forms of
AA in the tissues (AA, dehydroAA and ascorbate-sul-
phate) (Dabrowski, Matusiewicz & Blom 1994; Halver
& Felton 2001). Feed levels and chemical forms (AA,
coated AA, polymere AA, AA-palmitate, AA-sulphate
and AA-phosphate salts) have varied, as well as their
respective stability and bioavailability. In enrichment
procedures to boost live feed with vitamin C for ¢sh
larvae, microalgae (Lie, Haaland, Hemre, Maage,
Lied, Rosenlund, Sandnes & Olsen 1997), AA-palmi-
tate (Merchie, Lavens, Dhert, Garcia Ulloa Go¤mez,
Nelis, De Leenheer & Sorgeloos 1996) and AA-phos-
phate have been used successfully.
Although HPLC analytical techniques are increas-
ingly used for vitamin analysis, many of the B vita-
mins are analysed using standardized microbiological
assays (Mland, Rnnestad, Fyhn, Berg & Waagb
2000). These methods include growth of vitamin-sen-
sitive microorganisms in tissue or ¢sh larvae homoge-
nates. Because the microbial growth and thereby
analytical results can be in£uenced by growth factors
within the sample homogenates, the results need to be
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Aquaculture Research, 2009, 1^12 Water soluble vitamins in ¢sh ontogeny R Waagb

Brood stock feeding Endogenous feeding Live & formulated

experiments studies feed experiments

Oocyte Egg Yolk sac larvae Fed larvae

Final maturation
Spawning Hatching First feeding
fertilisation

Vitamin accumulation Vitamin consumption Vitamin feeding

5 -7 mm 100 days Atlantic salmon

3-3.5 mm 50 days Atlantic halibut

1-1.2 mm 10 days Turbot

Figure 1 Vitamin research in ¢sh ontogeny includes brood stock feeding experiments, studies on endogenous feeding,
and start of feeding experiments. These re£ect vitamin status at start of the new life, early consumption and external
vitamin supply, respectively. The ¢gure illustrates that farmed ¢sh species have di¡erent starting points and development
(modi¢ed from Tonheim 2004).

carefully evaluated by the technicians. As for ascorbic
acid, most di⁄culties in vitamin analytical work were
in proper homogenization and extraction of live feed
and larvae, but also measurement of di¡erent vitamer
forms versus the total vitamin concentration. Some
species, like carp and herring, contain various activ-
ities of thiaminase (Wistbacka & Bylund 2008), which
may rapidly reduce thiamine in homogenized samples
from these species, before a potential heat-treatment
step in the analysis inactivates the enzymes (own un-
published data). In such cases, heat treatment before
homogenization will be necessary.
post-translatory hydroxylation of proline and lysine
moieties in collagen, covered by 10^20 mg AA kg  1
in several ¢sh species (Sandnes 1991), it has been
shown to interfere with mineral metabolism
(Sandnes 1991), the stress response (Fletcher 1997),
immunity (Sealy & Gatlin 2001; Waagb 2006),
wound repair (Wahli, Verlhac, Girling, Gabaudan &
Aebischer 2003) and detoxi¢cation reactions (Norrg-
ren, B˛rjeson, F˛rlin & —kerblom 2001) in ¢sh species
at considerably higher concentrations than the mini-
mum requirement for good growth and survival.

Discussion
The water-soluble vitamins may be roughly classi¢ed
according to their main biochemical roles into those
taking part in molecular chemical modi¢cations,
coenzyme function in energy metabolism and in
redox reactions. However, novel roles of vitamins
and more sensitive markers for requirements are
continuously being discovered in human and animal
nutrition research and these aspects should also be
considered in future evaluation of requirements for
¢sh. Suggested ‘beyond de¢ciency requirements’ may
di¡er considerably from the estimates arising from
classical studies. Again, ascorbic acid (AA) serves as
a good example. Besides its classical essential role in
Ascorbic acid
The importance of vitamin C during ontogeny can
easily be demonstrated by high mortalities in o¡-
spring from vitamin C-de¢cient brood ¢sh (Sandnes
1984, 1991; Mangor-Jensen, Holm, Rosenlund, Lie &
Sandnes 1994; Blom & Dabrowski 1995). This is
mainly related to its well-de¢ned role in collagen
synthesis in the formation of connective tissues like
cartilage and bone in the developing ¢sh larvae
(Terova, Saroglia, Papp & Cecchini 1998), but also to
other essential functions related to the role of AA in
the integrated antioxidant system, constituting an
important part of the cellular water-soluble antioxi-
dant capacity together with GSH. Recently, Fontagne,
Lataillade, Breque and Kaushik (2008) demonstrated

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Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 1^12 3
Water soluble vitamins in ¢sh ontogeny R Waagb
an active antioxidant system in the developing rain-
bow trout until swim-up fry, recorded as changes in
gene expression of major antioxidant enzymes. Since
the mRNA expressions could be in£uenced by
oxidized lipids, it would not be surprising if the status
of antioxidants could impact the enzyme activities
and thereby the overall antioxidant capacity.
In the brood ¢sh, ascorbic acid has been investigated
for its roles in steroid synthesis and vitellogenesis
(Waagb, Thorsen & Sandnes 1989). Similarly, egg
AA deposition levels may easily be tailored by feeding
brood ¢sh elevated AA before and during vitellogen-
esis (Waagb et al. 1989; Mangor-Jensen et al. 1994;
Nortvedt, Mangor-Jensen, Waagb & Norberg 2001,
2003). The fate of AA during embryogenesis has been
studied in several ¢sh species and the weight concen-
tration has been shown to decline until start of feed-
ing. Maternally caused AA de¢ciency results in high
mortalities and severely reduced quality of the o¡-
spring. Surprisingly, there is not much detailed docu-
mentation on AA de¢ciency symptoms in marine ¢sh
larvae during endogenous feeding, except for low
gross performance and survival. The dynamics of col-
lagen synthesis, as evaluated by egg and larva hydro-
xyl-proline was, however, studied in sea bass and
gilthead sea bream o¡spring originating from brood
¢sh fed normal or excess levels of AA. The respective
egg and larvae re£ected the maternal AA feeding,
and higher and earlier collagen synthesis were
observed in the o¡spring fed excess AA compared
with the control (Terova et al.1998). The mass transfer
and loss of vitamin C from the yolk sac to the larval
body during development was studied in a population
of well-performing Atlantic halibut larvae (Rnnes-
tad et al. 1999). A constant AA content in the larvae
from hatching and until ¢rst feeding indicated minor
loss of vitamin in this period, in contrast to losses
found in other species like white¢sh (Dabrowski
1990). Depending on the overall antioxidant and
energy status, oxidized AA (dehydro AA) may be
regenerated to AA and thereby recycled. A start of
feeding study on juvenile Atlantic salmon (0.2 g)
showed that vitamins C and E interact and the
requirement levels of both growth, mortality and
collagen synthesis are in£uenced in vivo (Hamre,
Waagb, Berge & Lie 1997). After start of feeding
the halibut larvae with zooplankton containing
600^1000 mg AA g  1 dw, the whole bodyAA content
increased rapidly along with the growth, while a con-
stant concentration of bodyAA in larvae 410 mg in-
dicated a state of regulation of excess AA (Rnnestad
et al. 1999). A similar observation was also made in
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Aquaculture Research, 2009, 1^12
striped trumpeter, Latris lineata (Brown, Battaglene,
Morehead & Brock 2005).
The minimum requirement established in salmo-
nids that were fed on formulated feed with graded
levels of the stable and bioavailable AP derivative is
10^20 mg kg  1 (Sandnes,Torrissen & Waagb 1992),
which also seems to be adequate for European
sea bass and turbot (Merchie, Lavens, Dhert, Mai
Soni, Nelis, Ollevier, De Leenheer & Sorgeloos
1995). Updated knowledge has shown that common
carp (Cyprinus carpio) also need AA through the
diet (Gouillou-Coustans et al. 1998), despite earlier
published evidences for the opposite (Sato,Yoshinaka,
Yamamoto & Ikeda 1978; Yamamoto, Sato & Ikeda
1978). Interestingly, Atlantic salmon fry fed diets
devoid of vitamin C from the start of feeding did
not show mortality or develop skeletal deformities
(classical scoliosis and lordosis) until as late as 11
weeks of feeding, in accordance with no detectable
liver AA levels (Sandnes et al. 1992; Hamre et al.
1997). On the other hand, Gapasin, Bombeo, Lavens,
Sorgeloos and Nelis (1998) demonstrated bene¢cial
e¡ects on the occurrence of opercular malformations
in milk¢sk larvae fed rotifers or Artemia enriched
with AA and highly unsaturated fatty acids, as com-
pared with the same live feed enriched with the fatty
acids alone. Cat¢sh (Clarias gariepinus) larvae fed
AA enriched Artemia as high as 2300 mg g  1 showed
higher growth and stress resistance than larvae
fed lower AA levels (Merchie et al. 1995). The same
observations were made for sea bass, although
at lower Artemia AA concentrations (Terova, Cecchi-
ni, Saroglia, Caricato & Jeney 2001). The docu-
mented e¡ects of elevated AA on growth, collagen
synthesis and resistance to stress are mainly
observed in larvae fed AA far above any assessed
minimum requirements. Tissue or whole larvae
re£ect the live feed AA concentrations, demonstrat-
ing the bioavailability of this form of AA, but the
retention e⁄cacy is di⁄cult to estimate. Weaning
of Atlantic halibut with formulated feed with
increasing doses of AA in the form of ascorbic phos-
phate suggested that AA from Artemia (control)
retained more e⁄ciently than AP from the formulated
feeds, but this changed during the course of the
experiment (Mland, Rosenlund, Stoss & Waagb
1999). Similar observations with available AA from
Artemia were made for white¢sh (Dabrowski 1990;
Merchie, Lavens,Verreth, Ollevier, Nelis, De Leenheer,
Storch & Sorgeloos 1997). The seemingly elevated
requirements in for example carp and milk¢sh larvae
may therefore re£ect immaturity with respect to
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Aquaculture Research, 2009, 1^12 Water soluble vitamins in ¢sh ontogeny R Waagb

intestinal development, vitamin absorption e⁄cacy
and tissue vitamin storage.
B vitamins
B vitamins participate in biochemical reactions in the
intermediate energy metabolism (Table 1), and high
concentrations of the vitamins are found in metaboli-
cally active ¢sh tissues. The roles of the single B vita-
mins in the ¢sh body are not fully known, and there
is a large gap between historical ¢sh requirement
experiments (NRC 1993; Halver 2002; Webster & Lim
2002) and experiments with other species conducted
under modern intensive incubation and farming
conditions. Uncertainties in water-soluble vitamin
requirement estimates are illustrated by the wide
ranges reported in the literature. Cold water carnivor-
ous ¢sh may not have a regular intestinal micro-£ora
that supplies additional vitamins. The exogenous sup-
ply of B vitamins are therefore the natural content of
the live feed, feed ingredients and additional enrich-
ment or supplementation of the diet through vitamin
premixes. While vitamin C de¢ciency causes drastic
changes, lack of B vitamins is less obvious to reveal,
especially at larvae and juvenile stages of farmed
species that normally experience high mortalities
early in the larvae production chain.
Fish seem to require B vitamins according to their
growth and metabolic rate. Accordingly, the require-
ments have been suggested to be higher at larval
stages compared with juvenile and adult stages
(Dabrowski 1986). Analysis of vitamins in ¢sh eggs
and natural live pray supports this assumption
(Brkkan 1959; De Roeck-Holtzhauer, Quere & Claire
1991; Mland et al. 2000). The developing ¢sh larvae
seem to oxidize the energy-yielding substrates in the
sequence glycogen, amino acids and lipids (Finn
1994), which may in£uence the need for selected B
vitamins di¡erently during development.
Descriptive studies of vitamin B6 (Rnnestad et al.
1997) and folate (Mland et al. 2003) in developing
Atlantic halibut larvae showed a net loss of vitamin
during endogenous feeding and a steady transfer of
vitamin from the yolk sac into the body compart-
ment. The requirement of folate is related to growth
rates and cellular proliferation, which may be espe-
cially high during rapid maternal and larvae growth.
Indeed, for humans, increased preconceptional folic
acid intake is recommended in several countries
(Scott, Weir & Kirke 1995). In the hatched halibut
egg, there was a 50% net loss of folate from the yolk
compartment during endogenous feeding, of which
half was retained in the larval body (Mland et al.
2003). The data suggest a need for folate for metabolic
and growth purposes during embryogenesis of
2 mg g  1 weight gain, which is in agreement
with the requirement suggested by (NRC 1993) for
cold-water ¢sh (calculations based on a feed conver-
sion factor of 1g feed g  1 weight). According to this
requirement, the authors expressed concerns regard-
ing the critical low folate status observed in selected
batches of egg from Atlantic halibut brood ¢sh
(Mland et al. 2003). Studies on commercially
halibut farming in Norway and Iceland during
1998^2000 con¢rmed a large variation in egg folate
concentration (Table 3). A trend towards increased
egg folate concentration with time was observed, in-

Table 1 Trivial names, importance in metabolism, sensitive organ at de¢ciency in ¢sh and main biochemical function of the
single B vitamins

Vitamin Familiar name Essential for Sensitive organs Biochemical function

Vitamin B1 Thiamin Carbohydrates Nerve system, muscle tissue Carboxylation,

decarboxylation
Vitamin B2 Riboflavin Lipid, protein, carbohydrate, Skin, epithelium, nerves, Redox reactions
energy eye tissues
Niacin Nicotinic acid, Lipid, protein, carbohydrate, Skin, epithelium, nerves, Redox reactions
Vitamin B3 energy muscle cell
Vitamin B6 Pyridoxine Protein, amino acids, lipid Muscle cell, nerves, blood cell Transaminations/deaminations
Biotin Vitamin H Lipid, carbohydrates Skin, gill, liver tissues Carboxylation, decarboxylations
Pantothenic acid Vitamin B5 Carbohydrates, proteins, Gills, skin, nerve system Acetylations, acylations
fatty acids
Folate Folic acid Amino acids, nucleic acids Blood cells, nerves, skin, 1C transfers
cell division
Vitamin B12 Cyano-cobalamin Amino acids, nucleic acids, Blood cells, nerves, cell division Methylations
fatty acids

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Water soluble vitamins in ¢sh ontogeny R Waagb
£uenced by the increasing focus on overall maternal
vitamin nutrition. Feeding halibut brood stocks with
graded levels of folate, however, did not a¡ect fertili-
zation, egg survival or hatchability in eggs ranging
between 0.18 and 0.71 mg g  1 wet weight (ww) (Nort-
vedt et al. 2001, 2003). Unfortunately, the larvae were
not followed during further development, so later
performance of the o¡spring was not assessed. Criti-
cal low levels of folate may constitute a potential pro-
blem until start of feeding, after which the
requirement can be met by folate from several live
prey alternatives (Mland et al. 2000; Hamre, Srivas-
tava, Rnnestad, Mangor-Jensen & Stoss 2008).
The whole halibut larvae content of vitamin B6
started to decline 10 days post hatch and resulted in a
net 25% reduction until ¢rst feeding (Rnnestad et al.
1997). This expenditure of whole larvae vitamin B6
was attributed to as a need for vitamin B6 for protein
synthesis and growth (15 nmol vitamin B6 g  1 ww
gain), according to the hypothesis of Woodward
(1994). Similarly, Albrektsen, Sandnes, Lie and Waagb
(1994) showed a stable egg vitamin B6 of approxi-
mately 1 mg B6 g  1 until hatching at 500 day degrees
in Atlantic salmon, with a subsequent decline in the
body compartment from 1.0 to 0.6 mg B6 g  1 until the
start if feeding 6 weeks post hatch. The kinetics of
vitamin B6 transfer from the yolk to the body was
slower than the main bulk dry matter. This may
indicate need for vitamin B6 according to protein
degradation and increasing transaminase acitivity
(Sato, Yoshinaka, Kuroshima, Morimoto & Ikeda 1987;
Rnnestad, Groot & Fyhn 1993) or simply that the yolk
has excess vitamin relative to the need of the develop-
ing larvae, and where the vitamin is mediated by a
speci¢c transporter mechanism.
Thiamine requirements are estimated to range
between 0.5 and 1mg kg  1 in several ¢sh species
based on weight gain, as summarized by Woodward
(1994), and enzyme saturation (erythrocyte transke-
tolase) was achieved at higher concentrations.While
there are few recent studies on thiamine in farmed
species, there is a substantial body of recent literature
on thiamine de¢ciency in yolk-sac fry of wild salmo-
nid species su¡ering from the M74 syndrome (M74)
in the Baltic sea, early mortality syndrome (EMS) in
the Great Lakes in North America and the Cayuga
syndrome (CS) in the New York State Finger Lakes
(reviewed byAmco¡ 2000). Mortality rates of M74 yolk
sac fry may be as high as 40^95%. The development
of the disorders has been related to environmental
pollution, change in prey species (to species expres-
sing thiaminase; Wistbacka, Heinonen & Bylund
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Aquaculture Research, 2009, 1^12
2002; Wistbacka & Bylund 2008) or oxidative stress
(Pickova, Kiessling, Petterson & Dutta 1998; Vuori &
Nikinmaa 2007). Such maternally transmitted thia-
mine de¢ciency manifests in the fry as abnormal
swimming behaviour, general weakness and poor re-
sponse to physical stimuli, darker skin pigmentation,
whitened liver, pale gills, reduced tissue glycogen
levels, extremely low thiamine levels (6% of healthy
fry values) and necrotic changes in the brain areas
(Amco¡, B˛rjeson, Norrgren & Pesonen 1998b). At
the terminal stages, the yolk-sac fry are lethargic
and have convulsions and bradycardia (Lundstrom,
B˛rjeson & Norrgren 1998). The development of the
M74 disease occurs normally midway in the yolk-
sac phase and has been categorized in severity into
preclinical, clinical and terminal stages, where the
stages depend on the egg and hatched larvae thia-
mine status. Recently, the development of the M74
syndrome was studied using a cDNA micro-array
and real-time PCR to explore the transcriptional
responses behind the typical neurological, cardio-
vascular and pathological symptoms. Most of the
gene expressions could be related to reactions asso-
ciated with environmental stress, and terminally
to hypoxia (Vuori, Koskinen, Krasnov, Koivumaki,
Afanasyev,Vuorinen & Nikinmaa 2006).
The M74 syndrome serves as a good example of a
classical vitamin de¢ciency that can be completely
prevented by thiamine injection of the brood ¢sh or by
bathing the egg and fry in thiamine (4500 mg L  1)
solutions (Amco¡, B˛rjeson, Eriksson & Norrgren
1998a). The thiamine-dependent enzymes transketo-
lase and a-ketoglutarate dehydrogenase were lower in
M74 diseased yolk-sac fry compared with healthy con-
trols and may be part of the M74 pathogenesis (Amco¡
2000; Amco¡, —kerman, B˛rjeson,Tjarnlund, Norrgren
& Balk 2000). The enzyme activities were restored
with thiamine treatment, with activities correlating
with the thiamine status obtained (Amco¡, Akerman,
Tjarnlund, Borjeson, Norrgren & Balk 2002a). Finally,
yolk-sac fry exposed to the thiamine antagonists pyr-
ithiamine and oxythiamine developed M74-like symp-
toms, although not the complete array of pathology
(Amco¡, Lundstr˛m, Teimert, B˛rjeson & Norrgren
1999; Amco¡, —kerman, Tjrnlund, B˛rjeson, Norrg-
ren & Balk 2002b). Eyed eggs with a thiamine status
below a certain threshold (0.4^0.8 nmol g  1) had a
higher risk of developing M74, while healthy feral
and farmed eggs had thiamine concentrations
41nmol g  1 (Amco¡ et al. 1998b). The respective
low thiamine threshold for yolk-sac fry was 0.3^
0.5 nmol g  1 (Amco¡ et al. 1998a). With a relatively
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Aquaculture Research, 2009, 1^12
short half-life of thiamine during development, a nor-
mal reduction in thiamine during endogenous feeding
may be as high as 75% (Sato et al.1987).The occurrence
of the discussed thiamine de¢ciency-related syn-
dromes shows the importance of a single vitamin
during ontogeny, with interacting environmental and
dietary factors contributing to the state of de¢ciency.
As for most of the B vitamins, the minimum require-
ment of ribo£avin is relatively low, estimated to be
relatively equal, between 3 and11mg kg  1 for di¡erent
species. De¢ciency symptoms of ribo£avin are
observed in feeding trials with unsupplemented diets
with a low natural content of ribo£avin. Besides show-
ing weight reduction as the ¢rst sign observed, de¢-
ciency symptoms include eye disorders (corneal
lesions and cataracts), nervousness and abnormal skin
coloration. Poorly vascularized tissues such as the cor-
nea and eye lens are often a¡ected (Tacon 1992; NRC
1993). Little is known about ribo£avin during onto-
geny. According to historical data, the ovaries showed
the highest values of ribo£avin concentration among
selected organs of several wild-caught ¢sh species
(Brkkan 1959), indicating importance in embryogen-
esis and for the developing larvae. Live feed ribo£avin
concentrations seem to be in several fold excess of
known requirements for ¢sh species, which indicate
no problems with ribo£avin after start of feeding
(Brown, Je¡rey, Volkman & Dunstan 1997; Hamre
et al. 2008; Van der Meeren, Olsen, Hamre & Fyhn
2008). Pantothenic acid is a part of coenzyme A that is
required in energy-yielding reactions of glucose, fatty
acids and amino acids. Few requirement studies have
been conducted in ¢sh. Sandnes, Rosenlund, Mangor-
Jensen and Lie (1998) showed, however, pantothenic
acid accumulation in the ovaries of turbot at a much
lower rate than for example vitamin B6. Sato et al.
(1987) showed that the pantothenic acid concentration
declined by 50% in developing rainbow trout eggs,
from fertilization to start of feeding ready fry. In the
same study, biotin showed a similar decline with devel-
opment (Sato et al.1987).
Vitamin requirement estimates from the start
of feeding trials
Start of feeding of altrical ¢sh larvae is mostly per-
formed with live feed organisms, but often enriched
to complement the requirement of the larvae. Natural
marine zooplankton (copepods) has been regarded as
the gold standard feed organisms because they
support successfully the growth and normal develop-
ment of marine ¢sh larvae (Hamre, Rnnestad, Rain-
r 2009 The Authors
Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 1^12
Water soluble vitamins in ¢sh ontogeny R Waagb
uzzo, Barr & Harboe 2007). Recently, the nutritional
composition of rotifers was examined with respect
the ful¢llment of the nutritional requirements of
marine larvae like Atlantic cod, Gadus morhua
(Hamre et al. 2008). The choice of feed organism (size
and composition) and the enrichment procedure is
always a compromise in optimizing macro and
micro-nutrient composition, and for use of enriched
Artemia and rotifers to ¢sh larvae, suboptimal levels
of vitamin C, thiamine and vitamin B6 have been in
focus among the water-soluble vitamins.
There are several challenges with respect to water-
soluble vitamins in live or formulated feeds, besides
aspects of live feed enrichment, like vitamin stability
and leaching. Micro bound and encapsulated diets
may undergo severe leaching of up to 90% of water-
soluble vitamins during feeding (nal & Langdon
2005; Hamre et al. 2007). They reported on a new gen-
eration of encapsulated complex diets, with use of
marine phospholipids or polymerization agents that
seem to cope better with the leaching problems. Bio-
logical aspects of requirement assessment from start
of feeding include variable individual feed intakes,
maturation of the gastrointestinal tract, bioavailabil-
ity of inherent and added vitamins and the vitamin
storage capacity in the developing larvae (Dabrowski
1986; Segner, R˛sch, Verreth & Witt 1993). Conse-
quently, exact minimum requirements are di⁄cult
to estimate, and established and future recommenda-
tions need to consider such imperfect conditions.
Some newer studies on water-soluble vitamin re-
quirements in ¢sh species have recently been con-
ducted, that adds to and are in accordance with the
existing literature (Table 2).
External interfering conditions
Growth and metabolic rate of ¢sh larvae are highly
in£uenced by water temperature, and other external
rearing conditions (Lein 1996). Generally, elevated
water temperatures lead to increased growth and
intermediary metabolism, and possibly vitamin
requirement. Elevated water temperature or genetic
di¡erences in the growth rate did not, however, a¡ect
the requirement of ribo£avin, and this has been used
as a support for the fairly equal requirement among
¢sh species (Woodward 1985; NRC 1993). Uncon-
trolled temperature conditions, however, like criti-
cally elevated or £uctuating temperatures during
egg development, cause heat shock and developmen-
tal errors in some studied ¢sh species, and an asso-
7
Water soluble vitamins in ¢sh ontogeny R Waagb Aquaculture Research, 2009, 1^12

Table 2 Recently published requirement studies for the single B vitamins mostly con¢rm existing requirements (NRC 1993;
Halver 2002; Webster & Lim 2002) for similar species

Requirement
Vitamin (mg kg  1) Species, weight Analysed markers Reference

Vitamin B1 0.5–1 Woodward (1994)

Vitamin B2 4.1–5.0 Sunshine bass, 2 g Growth, pathology, cataract inspection, hepatic Deng & Wilson (2003)
D-amino acid oxidase activity
6 Atlantic salmon, 17.5 g Growth performance, pathology, eye lens histology; Brønstad, Bjerkås &
body composition; tissue riboflavin Waagbø (2002)
Niacin 25 Indian catfish, 9.4 g Growth performance, pathology, body composition; Mohamed &
tissue niacin Ibrahim (2001)
33 African catfish, 17 g Growth performance, pathology, body composition Morris, Baker &
Davies (1998)
7.4 Channel catfish, 5.6 g Growth performance, pathology, body composition; Ng, Serrini, Zhang &
liver NAD; haematology Wilson (1997)
Vitamin B6 28% protein: Tilapia, 0.73 g Growth performance, feed efficiency; liver tissue Shiau & Hsieh (1997)
1.7–9.5 enzyme activity; haematology
36% protein:
15–16.5
1.75–2.05 Grouper, 14.8 g Growth performance, pathology, body composition; Huang, Tian, Du,
tissue enzyme activity; haematology Yang & Liu (2005)
Biotin 0.25 Indian catfish, 3.6 g Maximal weight gain, body biotin content, liver Mohamed (2001)
pyruvate carboxylase and acetyl CoA carboxylase
0.06 Juvenile hybrid tilapia, Maximal weight gain; body composition; Shiau & Chin (1999)
0.98 g body biotin; hepatic pyruvate carboxylase
and acetyl CoA carboxylase activities
Pantothenic 10 Blue tilapia, 0.7 g Maximal weight gain; prevention of deficiency Soliman &
acid signs Wilson (1992)
Folate 0.3–0.6 Rainbow trout 1.4 and Growth indices; tissue folate; hematology Cowey &
2.8 g Woodward (1993)
1.1 Channel catfish Weight gain, hematocrit, erythrocyte and leukocyte Duncan & Lovell
numbers, and plasma and liver folate (1994)
Vitamin B12 0.014 Atlantic salmon, 0.2 g Prevention of anaemia with immature erythrocytes (Sandnes, Mæland &
Waagbø 1993,
unpublished data)

Table 3 Selected range (minimum and maximum concentrations) of water-soluble vitamins (wet weight) in egg batches
from Atlantic halibut (Hippoglossus hippoglossus) brood stocks during the period 1997^2001

Dry matter (g kg  1) Thiamine (lg g  1) Folate (lg g  1) Ascorbic acid (lg g  1)

Range min–max 44–139 o0.1–4.4 0.02–0.71 5–55

1998 0.6–2.2 0.05–0.09 1–20
1999 2.1–2.3 0.05–0.06 24–29
2000 1.4–3.0 0.03–0.13 31–40

The vitamin concentration increased during the period 1998^2000, re£ecting a general increase in vitamin levels in the brood stock
diets (data from Nortvedt et al. 2003).

ciation with selected vitamins has been suggested
(Waagb 2008).
With respect to salinity, there seems to be minor
di¡erences in requirement between fresh water and
marine species in for example AA requirement
(Gouillou-Coustans & Kaushik 2001).
Changes in external farming conditions, use of
novel feed ingredients and exposure to contaminants
may increase the relative requirement for selected
vitamins in brood ¢sh, and start fed larvae and fry.
The discussed thiamine de¢ciency syndromes in sal-
monid species have been related to environmental
pollution, resulting in a low thiamine status in brood
¢sh and o¡spring (Vuorinen, Paasivirta, Keinnen,
Koistinen, Rantio, Hy˛tylinen & Welling 1997).
Whether this is entirely due to the low maternal input

r 2009 The Authors

8 Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 1^12
Aquaculture Research, 2009, 1^12
of thiamine to the eggs during vitellogenesis or also
increased consumption during yolk-sac fry develop-
ment is not completely clear. Low maternal status
has, however, been associated with environmentally
induced changes in the relative biomasses of salmon
prey species towards more thiaminase-rich pelagic
¢sh species (Wistbacka & Bylund 2008).
Conclusions
Evaluating the progress in vitamin research in ¢sh
during the last decade is easy, relative to the substan-
tial body of published literature within other areas of
aquaculture nutrition research. However, any pro-
gress in other ¢elds is likely to in£uence the vitamin
nutrition of ¢sh, and especially in the sensitive juve-
nile stages. Regular reevaluations of vitamin require-
ments at di¡erent stages of ¢sh development are
therefore needed, including brood stock and larvae
nutrition. Strict requirements for water soluble vita-
mins are probably relatively similar among ¢sh spe-
cies and in metabolically active tissues. There are,
however, elevated needs during ontogeny due to lar-
vae immaturity and imperfect diets. Because ¢sh lar-
vae are more susceptible to de¢ciency, larvae diets
should contain safe and surplus vitamin levels to
compensate for varying biological and technical con-
ditions.
Acknowledgments
I wish to thank Dr. Konrad Dabrowski (The OHIO
State University, USA) for arranging this Special
session on Basic and applied aspects of Aquaculture
Nutrition: Healthy ¢sh for healthy consumers at Aqua-
culture Europe 0 08 conference in Krakow, Polen, and
the OECD for the economical support.
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