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Overview of vitamin D and C

requirements in fish and their
influence on the skeletal system
Giorgos Koumoundouros, José Zambonino

Aquaculture

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 Aquaculture 315 (2011) 49–60

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Review

Overview of vitamin D and C requirements in fish and their influence on the

skeletal system
M.J. Darias a,⁎, D. Mazurais a, G. Koumoundouros b, C.L. Cahu a, J.L. Zambonino-Infante a
a
Ifremer Marine Fish Nutrition Team, Nutrition Aquaculture and Genomics Research Unit, UMR 1067, Ifremer, Technopole Brest-Iroise, BP 70, 29280 Plouzané, France
b
University of Patras, Biology Department, 26500 Patras, Rio, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Vitamins D and C are essential in many physiological functions. Vitamin D, a fat soluble vitamin, is crucial to
Received 6 May 2010 preserve calcium and phosphate homeostasis and to protect the skeletal integrity. This hormone functions
Received in revised form 3 December 2010 through the vitamin D receptor (VDR) inducing the expression of various calcium binding and transport proteins
Accepted 17 December 2010
in the intestine to stimulate active calcium uptake, thus preserving normocalcemia and, indirectly, maintaining
Available online 7 January 2011
bone mineralization. Besides, vitamin D also acts directly on osteoblasts, the resident bone-forming cells of the
Keywords:
skeleton, to inhibit proliferation, modulate differentiation, and regulate mineralization of the extracellular matrix.
Vitamin D Vitamin C, a water soluble vitamin, acts as a co-substrate for hydroxylase and oxygenase enzymes involved in the
Vitamin C biosynthesis of pro-collagen, carnitine and neurotransmitters, among other numerous physiological functions
Gene expression such as antioxidant or pro-oxidant. Both vitamins should be supplied by the diet because fish are unable to
Skeletogenesis synthesize them. However, their wide range of action makes it difficult to adjust the adequate amount of these
Skeletal deformities vitamins to achieve an optimal fish performance. Besides, the dietary vitamin needs of fish depend on several
Fish larvae factors such as developmental stage, physiological, environmental/ecological and genetic conditions. In this
sense, vitamin requirements of flatfish do not necessarily meet those of pelagic fish and depends also on their
feeding habits (carnivorous, planktivorous or detritivorous); the dietary vitamin demands of an adult fish differ
from those of a larva; and even within the same fish species and developmental stage, the environmental
conditions would also influence the vitamin needs (i.e., under stress conditions, high vitamin C levels have been
demonstrated to improve stress resistance and, consequently, growth).
The present paper gives a general overview about the requirements of vitamins D and C in fish and specifically
reviews the role of these vitamins in fish skeletogenesis and their influence in the development of skeletal
deformities. In addition, new insights on the molecular pathways involving these vitamins in the skeletal
ossification process are provided.
© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2. Physiological functions of vitamins D and C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.1. Vitamin D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.2. Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3. Dietary vitamins D and C requirements in adults and juvenile fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.1. Vitamin D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.1.1. Unbalanced vitamin D diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.2. Vitamin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.2.1. Unbalanced vitamin C diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4. Dietary vitamins D and C requirements in fish larvae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5. The influence of vitamins D and C in the development of malformations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6. Molecular mechanisms involving vitamin D and C in fish skeletogenesis: the case of European sea bass . . . . . . . . . . . . . . . . . . . . 55
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

⁎ Corresponding author. Present address: Institut de Recerca i Tecnologia Agroalimentaries (IRTA), Ctra. de Poble Nou s/n, km 5.5, 43540, Sant Carles de la Ràpita, Tarragona, Spain.
Tel.: + 34 977 745 427; fax: + 34 977 743 841.
E-mail address: maria.darias@irta.cat (M.J. Darias).

0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.12.030
50 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
8. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1. Introduction
The high incidence of malformed fish is considered as one of the
main problems for fish producers due to the severe economic loss
that this entails. Most malformations develop during skeletogenesis,
therefore the larval period constitutes an important bottleneck in
aquaculture since a great deal about physiological demands of fish
larva remains to be elucidated. Among the different causative
factors, larval nutrition has been suggested to have a key role in
skeletogenesis (Cahu et al., 2003; Lall and Lewis-McCrea, 2007).
Knowledge of larval nutritional needs in the fish farming industry is
limited due to the fast changing needs of the larval requirements
during ontogeny. Rotifers and artemia, the most commonly used live
preys due to their rearing simplicity and possibilities to be enriched,
have contributed extremely to improve marine fish aquaculture.
However, deformities still develop, usually as a consequence of
suboptimal/sublethal conditions (including nutrition).
The influence of vitamins on the appearance of larval deformities has
been already demonstrated (i.e., Dedi et al., 1997; Takeuchi et al., 1998;
Villeneuve et al., 2005; Fernández et al., 2008; Mazurais et al., 2009). Up
to date, most work on vitamin C and D in fish dealt with their deposition
in several tissues of different fish species (Halver, 1972; Graff et al.,
1999); their requirements for fish growth and development and
reproduction (Dabrowski, 1992; Dabrowski and Ciereszko, 2001;
Merchie et al., 1997; Phromkunthong et al., 1997); their roles in several
biological processes such as immunoactivity and stress response, in the
case of AA (Dabrowski, 1992; Xie et al., 2006; Azad et al., 2007), or skin
pigmentation, in the case of vitamin D (Haga et al., 2004). Besides, both
vitamins have proven to induce skeletal deformities in some fish species
(Halver et al., 1975; Haga et al., 2004). However, the mechanisms
underlying the development of malformations are still unknown.
Recent investigations have been focused on the study of the
molecular mechanisms underlying fish skeletogenesis when fish are
fed with different dietary vitamin levels (Villeneuve et al., 2006;
Fernández et al., 2008; Mazurais et al., 2009; Darias et al., 2010a).
These studies showed that unbalanced dietary vitamins induced
skeletal deformities and were associated with the regulation of the
expression of genes involved in morphogenesis. For instance, low
levels of dietary vitamin mix has been shown to induce skeletal
malformations correlated with the modulation of genes involved in
osteoblast determination and differentiation such as BMP-4 (Bone
Morphogenetic Protein 4), IGF-1 (Insulin Growth Factor-1) and
PPARγ (Peroxisome Proliferator-Activated Receptor γ) in European
sea bass larvae (Mazurais et al., 2008). It has also been reported for
the same species that excess and deficiency of dietary vitamin A led to
the appearance of skeletal deformities, related to the regulation of
RARγ (Retinoic Acid Receptor γ) and Hoxd-9 (Homeobox protein
Hox-D9) gene expressions, respectively (Villeneuve et al., 2006;
Mazurais et al., 2009). Concerning vitamins D and C (ascorbic acid,
AA), their suitable dietary level for an optimum larval development
of European sea bass is in a restricted range and it has been recently
shown that subtle variations could unleash severe physiological
disruptions such as delay of intestinal maturation and ossification
correlated with the regulation of genes involved in intestinal AA and
Ca2+ absorption (SVCT-1, sodium-dependent vitamin C transporter 1
and TRPV-6, Transient Receptor Potential cation channel-subfamily V,
member 6, respectively), skeletogenesis (BMP-4, IGF-1, RARγ) and
bone mineralization (VDR-Vitamin D Receptor-, osteocalcin) (Darias
et al., 2010a; 2011). Such disturbances detected at molecular level led
to the development of skeletal abnormalities.
The present review provides new information about the influence
of vitamins D and C in skeletogenesis, and especially in the ossification
process, and gives new insights on whether imbalanced levels of such
vitamins could alter some molecular pathways of the skeletogenesis
process and then promoting the development of skeletal disorders. In
addition, some general information existent up to now about the
physiological functions of vitamins D and C, as well as the dietary
requirements of these vitamins for fish, are also overviewed.
Concerning the latest, new data about the vitamin D and C
requirements for European sea bass larvae are also provided.
Taking into account the above, the review is structured as follows:
i) the physiological functions of vitamins D and C; ii) dietary vitamins
D and C requirements in fish and especially in fish larvae; iii) the
influence of dietary vitamins D and C in the development of
malformations; and iv) the molecular mechanisms involving vitamins
D and C in skeletogenesis of European sea bass larvae.
2. Physiological functions of vitamins D and C
2.1. Vitamin D
Vitamin D was isolated by Angus et al. (1931). Henry et al. (1974)
demonstrated for the first time the metabolism of vitamin D by fish
tissues. However, the physiological importance of this metabolism
and the mechanisms and sites of action of the metabolites are scarcely
known. In most vertebrates, one of the two major forms of vitamin D,
vitamin D3 (cholecalciferol, VD3) (Fig. 1A), is processed into the most
active molecule, 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), after
two hydroxylation steps. The first one results in the formation of 25-
hydroxyvitamin D3 (25-(OH)D3) in the liver. This molecule is
transported to the kidney where it is hydroxylated at the 1α position
by the renal cytochrome P450 enzyme (25-(OH)D3-1α-hydroxylase)
resulting in the formation of 1α,25-(OH)2D3 (Norman et al., 1982)
(Fig. 2A). However, this seems not to be the route for fish. Yanda and
Ghazarian (1980) stated that kidney microsomers could not metab-
olize either VD3 nor 25-(OH)D3. Takeuchi et al. (1991) showed that
25-(OH)D3-1α-hydroxylase exists in the liver of carp (Cyprinus
carpio) and bastard halibut (Paralichthys olivaceus) and suggested
that the 25-(OH)D3 formed in the liver is immediately metabolized
into 1α,25-(OH)2D3 in the same tissue. Moreover, they concluded that
natural feed is the most probable origin of 1α,25-(OH)2D3 in fish
(Takeuchi et al., 1991). The origin and hydroxylation of vitamin D
metabolites in fishes is provided in an excellent review of the
significance of vitamin D for fish (Lock et al., 2010). The active
metabolite of VD3 (1α,25-(OH)2D3) can enter the cell and bind to the
Fig. 1. Structural formula of vitamin D3 (cholecalciferol) (A) and ascorbic acid (B).
 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
Fig. 2. A. Synthesis of vitamin D3 and its active metabolite, calcitriol. B. Synthesis of ascorbic acid. Note that fish must obtain both vitamins from the diet since they are unable to
synthesize them.
vitamin D-receptor (VDR). This complex forms a heterodimer with
the retinoid receptor (RXR) and binds to a vitamin D responsive
element (VDRE) on a responsive gene promoter region to modulate
transcription. Such molecular regulation may take hours or days (Lips,
2006). In humans, VD3 stimulates Ca2+ absorption, osteoclastic bone
resorption, cell differentiation (including osteoblasts, neural and
immune system cells), and decreases parathyroid hormone (PTH)
secretion as well as the production of collagen type 1 (Lips, 2001).
Contrary to mammals, which are able to photosynthesize vitamin D
in the skin, fish obtain the required VD3 from food. Sundell and
Bjomsson (1990) suggested that 25(OH)D3 and 24,25(OH)2D3 may be
active regulators of Ca2+ transport across the intestine of the Atlantic
cod. However, no effects of 1α,25-(OH)2D3 on the Ca2+ influx across the
intestinal mucosa were detected. Interestingly, a recent study made on
European sea bass larvae showed an effect of VD3 in intestinal
maturation that influenced TRPV-6 gene expression, which is the
most predominant Ca2+ transporter at the intestine, thereby indirectly
influencing the absorption of Ca2+ at the intestine (Darias et al., 2010a).
2.2. Vitamin C
Vitamin C (L-ascorbic acid, AA) (Fig. 1B) was isolated and
demonstrated to have anti-scorbutic properties by King and Waugh
(1932). The active form of AA is the reduced ascorbate ion form
(Maulén et al., 2003); therefore, the physiological actions of AA here
reviewed are referred to this compound. AA is a water soluble vitamin
that acts as a co-substrate for hydroxylase and oxygenase enzymes
involved in the biosynthesis of pro-collagen, carnitine and neuro-
transmitters (Tolbert, 1979). AA is necessary for the formation of
collagen and cartilage, as well as bone formation and remodeling
(Wilson and Poe, 1973; Kraus et al., 2004; Darias et al., 2011). It is
directly involved in collagen metabolism by functioning as a cofactor
of prolyl and lysyl hydroxylases, which catalyze the hydroxylation of
prolyne and lysine in the pro-collagen molecule, contributing to bone
and skin formation and therefore to growth (Barnes and Kodicek,
1972). Proof of this is the detection of AA in skin, caudal fin, head, jaws,
cartilage of gills and collagen-areas of the bone in fish (Halver, 1972).
51
AA also has many non-enzymatic actions. It is a powerful water-
soluble antioxidant which protects low density lipoproteins from
oxidation, reduces harmful oxidants in the stomach and promotes iron
absorption. AA is easily oxidized to the unstable dehydroascorbic acid
which is not normally detectable in plasma but may occur transiently
during oxidant stress (Padayatty and Levine, 2001). AA is transported
into the cell by sodium-dependent vitamin C transporters SVCT-1 and
SVCT-2 (Tsukaguchi et al., 1999), whereas its oxidized form,
dehydroascorbic acid, is transported by glucose transporters GLUT-1
and GLUT-3, and, in insulin-sensitive tissues, also by GLUT-4.
In general, fish are unable to synthesize AA due to the lack of the
last enzyme of the biosynthetic pathway (L-gulonolactone oxidase)
(Chatterjee, 1973; Dabrowski, 1990a) (Fig. 2B). As for vitamin D, AA
must therefore be supplied through the feed.
3. Dietary vitamins D and C requirements in adults and
juvenile fish
3.1. Vitamin D
Vitamin D studies of different nature have been performed in
adults and juveniles of several fish species and dietary requirements
of this vitamin have been recently reviewed by Lock et al. (2010).
Graff et al. (1999) demonstrated the existence of various metabolites
of vitamin D (25OHD3, 24,25(OH)2D3 and 1α,25(OH)2D3) in the liver,
kidney, head kidney, gills, spleen and intestine of adults of the Atlantic
mackerel, Scomber scombrus, Atlantic halibut, Hippoglossus hippoglos-
sus and Atlantic cod, Gadus morhua. They observed that organs
producing great amounts of one metabolite also produced consider-
able amounts of the other possible derivatives, suggesting a lower
degree of specificity in fish organs than in those of human. It was
found that 24,25(OH)2D3 was the quantitatively predominant
metabolite and this may be explained by the need of the species in
calcium rich environment to decrease rather than increase their
calcium absorption. Horvli et al. (1998) also found deposition of
vitamin D3 not only in the livers, but also in the intestine, kidney,
spleen, gills, fillet, skin and in the plasma of Atlantic salmon, Salmo
52 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
salar. Moreover, the salmon responded to the two high dietary levels
of vitamin D3 tested (88.4 and 1147.2 IU/g diet) by accumulating the
vitamin in a dose dependent manner similarly in all of the tissues
investigated. This dietary dose-dependent accumulation of vitamin D3
was also found by Takeuchi et al. (1991) in the livers of the bastard
halibut and carp, C. carpio. Although a nonspecific accumulation of
vitamin D3 was observed in the Atlantic salmon tissues with these
high dietary doses, no apparent physiological effects, in terms of
growth, plasma calcium homeostasis, haematocrit or red blood cell
count were appreciated in this study (Horvli et al., 1998). Neverthe-
less, high dietary VD levels have caused physiological disruptions in
other fish species, as described below.
3.1.1. Unbalanced vitamin D diets
Hypervitaminosis D has been shown to elevate alkaline phospha-
tase in trout and salmon (Halver, 1989). High levels of vitamin D
(3750 IU VD3/g diet) caused impaired growth, lethargy and dark
coloration in brook trout, Salvelinus fontinalis (Poston, 1969). Haga et
al. (2004) reported for the first time an effect of dietary vitamin D3
metabolites on skin pigmentation in flatfish. They demonstrated that
20 IU 1α,25(OH)2D3/g diet induced hypermelanosis on the blind side
of juvenile Japanese flounder, Paralichthys olivaceus. In addition, a
higher frequency of vertebral deformities was observed in groups fed
diets supplemented with VD3 or 1 α,25(OH)2D3 (Haga et al., 2004).
Darias et al. (2010a) also found an influence of high levels of dietary
VD3 (42 and 120 IU VD3/g diet) in the development of skeletal
malformations in early juvenile of European sea bass. By contrast, no
evidences of skeletal deformities induction by VD3 excess have been
found in Oncorhynchus mykiss, Atlantic salmon and brook trout
(Hilton and Ferguson, 1982; Poston, 1969; Graff et al., 2002). Such
contradictory results could be due to differences in the stage of
development of fish when the experiments were performed.
Therefore, the physiological consequences of hypervitaminosis D
would depend on which phase of the skeletogenesis process an excess
of dietary VD3 is provided to the fish.
Hypovitaminosis D has been also shown to induce several anomalies.
Tamago salmon (Oncorhynchus rhodurus) fed diets without VD3
exhibited a thinned epidermis consisting of atrophied and necrotic
epidermal cells over the basal cells, lesions of the caudal peduncle, the
skin and the underlying musculature necrotized, degenerated and
necrotic hepatocytes, increased respiratory epithelium and cardiac
muscle fibres and hypocalcaemia (Taveekijakarn et al., 1996). However,
no pathological changes were found in the bone, eyes, brain and
alimentary tract. Fortunately, these pathologies were reversed after
4 weeks of feeding of VD3 supplemented diet.
The NRC (1993) recommends 2.4 IU vitamin D3/kg diet for an optimal
performance of juvenile fish based on analyses made on young rainbow
trout which is the actual value of reference in diet formulation for fish.
3.2. Vitamin C
Several benefits have been attributed to AA supplementation in
fish such as growth, survival, reduction of skeletal deformities,
immunoactivity and stress response (Dabrowski, 1992). Many studies
showed that dietary AA can enhance resistance to bacterial infection
in channel catfish, Ictalurus punctatus (Durve and Lovell, 1982; Li and
Lovell, 1985), rainbow trout (Navarre and Halver, 1989), Atlantic
salmon (Hardie et al., 1991) and grouper, Epinephelus awoara (Qin et
al., 2000). However, as can be noticed below, fish AA requirements
may depend on species and their physiological conditions.
Studies on AA have been done for several purposes in adults of
several fish species. Cuesta et al. (2002) demonstrated that AA increases
the natural cytotoxic activity of head-kidney leucocytes of gilthead sea
bream, Sparus aurata, against tumour cells when administered both in
vitro and in vivo. The positive effect of AA in the protection of leucocyte
functions has been also reported by Ortuño et al. (2003). They observed
a reduction of stress and of deleterious effects on the immune system in
sea bream specimens fed diets supplemented with high doses of AA
(3000 mg/kg) when they were exposed to stressors commonly found in
aquaculture (i.e., water current, high density, and anesthetic treatment).
AA has been proven to improve growth rate and sperm quality of yellow
perch, Perca falvescens (Lee and Dabrowski, 2004) and has a role in
reproductive functions (for review see Dabrowski and Ciereszko, 2001).
The requirement of AA has been specially studied in juvenile fish and
the optimal amount seems to be species-specific. An amount of 30 mg
AA/kg diet improved survival and growth in sea bass, Lates calcarifer
(Phromkunthong et al., 1997) whereas dietary supplementation of
118 mg AA/kg diet was necessary for maximum growth in Parrot fish,
Oplegnathus fasciatus (Wang et al., 2003). Nevertheless, no differences in
growth or survival and no external signs of AA deficiency were observed
in juveniles of angelfish, Pterophylum scalare, fed diets containing AA
levels ranging from 0 to 1440 mg/kg (Blom and Dabrowski, 2000). This
denotes the high resistance of this species against prolonged AA
deficiency. A dietary AA of 79 mg/kg resulted in the maximum
concentration of soluble collagen in the muscle of Atlantic salmon,
whereas levels as low as 33 mg of AA/kg had no detrimental effect on
growth rate (Li et al., 2007). Ai et al. (2006) found in large yellow
croaker (Pseudosciaena crocea) that dietary AA ranging from 0.1 to
489 mg AA/kg diet did not significantly influence the growth response.
Furthermore, when dietary AA supplementation exceeded 23.8 mg/kg
diet, no significant differences in bone collagen were found among
dietary treatments suggesting that this amount was essential to
maintain normal bone collagen formation. However, the minimum
requirement for maximal liver AA storage was at least 87 mg/kg diet,
higher than that for maximal survival, this being in line with previous
studies performed in Atlantic salmon (Thompson et al., 1993) and
gilthead sea bream (Henrique et al., 1998). Dabrowski (1990b)
suggested that the optimal dietary AA is equivalent to that allowing
the maintenance of a constant tissue concentration of AA in fish. Murai
et al. (1978) established a dose of 50 mg AA/kg diet for maximal
growth in channel catfish. However, lower amount was needed to
prevent spinal abnormalities (b25 mg/kg) and higher than 200 mg/kg
was required for maximal liver and blood accumulation. Similar
tendency of AA requirements was found by Chen et al. (2003) in
golden shiners, Notemigonus crysoleucas. They found a minimum AA
requirement of 19.5 mg AA/kg to prevent deformities and of 40.3 mg
AA/kg diet to maximize survival rate in this species. However, an
amount of AA 10 times higher than that required to prevent deficiency
signs strongly improved disease resistance and increased AA in visceral
tissues. The positive effects of AA in the immune response and disease
resistance have been also demonstrated in large yellow croaker, and
Indian major carp, Labeo rohita (Ai et al., 2006; Nayak et al., 2007).
Kaushik et al. (1998) reported that supplementation of practical
diets with a vitamin mix containing the minimal requirement levels of
the NRC (1993) was sufficient to support rapid and maximal weight
gain in juveniles of rainbow trout, chinook salmon (Oncorhynchus
tschawytscha) and sea bass. Merchie et al. (1996a) studied the effects of
dietary AA on weaned juveniles of European sea bass and concluded that
20 mg AA/kg diet was enough for normal survival and growth. In
general, AA requirements for normal growth of juvenile fish range from
10 to 25 mg (see review Merchie et al., 1997), although the NRC (1993)
recommends 50 mg AA/kg diet for an optimal performance of juvenile
fish based on analyses made on young rainbow trout. Affonso et al.
(2007) detected greater weight gain and survival in juvenile matrinxã
(Brycon amazonicus) fed 800 mg AA/kg supplemented diets. In contrast,
juveniles of milkfish (Chanos chanos) fed diets supplemented with
1500 mg AA/kg produced the best antibody and protective responses as
well as final survival upon challenge but not better growth (Azad et al.,
2007). These results indicate that high levels of AA in fish diets induce
species-specific responses, and although lower levels of this vitamin are
enough to achieve the fish basal requirement, higher doses might
improve fish performance. Indeed, it seems that higher levels of
 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
vitamins are required by fish in tropical aquaculture due to increased
physiological stress (Montero et al., 1999; Azad et al., 2007).
An interesting species is the sturgeon, Acipenser fulvescens, because
of its ability to synthesize L-ascorbic acid as they possess in their
kidney L-gulono-1,4-lactone oxidase, the enzyme catalyzing the last
step of AA biosynthesis. Moreau et al. (1999) estimated by in vitro
kinetics data a theoretically biosynthetic rate of 3 mg AA/kg body
weight per day at 15 °C in this species. They stated that juvenile
sturgeons are able to synthesize their required AA levels and therefore
they do not need additional dietary AA supplementation. Moreover,
authors suggested that sturgeons can respond to increased nutritional
needs by synthesizing more AA (Moreau et al., 1999). Nevertheless, it
has been found that dietary AA may be conditionally necessary for the
Siberian sturgeon (Acipenser baerii) to achieve optimal immune
response, particularly in early developmental stages (Xie et al., 2006).
All together these results show the physiological complexity of the
organisms and therefore the difficulty to establish an optimal dose that
could meet the requirements of the different tissues and organs when
they are considered separately. Comparative physiology studies, in
which the different physiological processes are interconnected, need
to be done to attain this objective. In the same manner, the interaction
between nutrients must be also considered. In this particular case, the
interaction of vitamins should be taken into account in the determi-
nation of the optimal requirements of AA. Hamre et al. (1997) stated
that the amount of AA to incorporate into the diet depends on the level
of dietary vitamin E due to the interaction existent between these two
vitamins. Indeed, AA regenerates vitamin E at the liver and amounts of
AA above the required increase the protection of fish against vitamin E
deficiency in a dose dependent-manner. Similarly, interactions
between AA and vitamin D have also been evidenced in European
sea bass (Darias et al., 2010, in press).
3.2.1. Unbalanced vitamin C diets
AA cytotoxicity by hypervitaminosis has not been demonstrated in
vivo. However, it has been shown that AA deficiency causes
deformation of skeletal and cartilaginous tissues, capillary fragility,
slow wound repair, depressed immunity, reduced growth rate and
increased rate of mortality (Halver et al., 1975). Dietary AA
deficiencies caused reduced weight gain, feed efficiency, survival
rate and high incidence of scoliosis, lordosis and depigmentation on
reared channel catfish (Wilson and Poe, 1973). Scoliosis, distorted/
twisted gill filaments, opercula and snout deformities have been
detected in other scorbutic fish species such as juvenile tilapias,
Oreochromis niloticus (Soliman et al., 1986) Mexican native cichlid,
Cichlasoma urophthalmus (Chávez de Martínez, 1990), or milkfish
(Hilomen-Garcia, 1997). McLaren et al. (1947) observed haemor-
rhages in trout fed rations lower than 10 mg AA/kg diet. Kitamura et
al. (1965) and Halver et al. (1969) demonstrated a critical need of AA
in trout and salmon, respectively. A compromise of 200 mg AA/kg diet
was established for adult salmon and rainbow trout. However, the
needs for vitamin C can be increased under stress and/disease
conditions (Halver, 1989). AA deficiency resulted in the development
of spinal deformities and several abnormalities in the gills tissues of
sea bass, specially a hyperplasia of the respiratory epithelium
(Phromkunthong et al., 1997). Parrot fish, Oplegnathus fasciatus, fed
the AA-free diet showed deficiency signs, such as retarded growth,
darkening, anorexia and high mortality after three weeks of
treatment. After six weeks, livers showed severe signs of atrophy
and all specimens died one week later (Wang et al., 2003).
As shown, numerous factors influence the dietary vitamin
requirements in fish. Trout and salmon are the most studied species
in fish nutrition (NRC, 1993; Halver et al., 1969; Kaushik et al., 1998;
Madsen and Dalsgaard, 1999). Nevertheless, the increasing diversity
of cultured species demands the study of vitamin requirements
specific for each species. In addition, there is little information about
the requirements for younger specimens. Considering that the larval
53
stage is the first link in the rearing chain and a very sensitive phase
during which a correct ontogenesis must take place to obtain healthy
and well developed juveniles, it is imperative to deepen in the
knowledge of the nutritional requirements in fish larvae.
4. Dietary vitamins D and C requirements in fish larvae
There is scarce information concerning VD3 requirements during
the larval stage. A recent study performed in European sea bass showed
that larvae performed well with 27.6 IU VD3/g supplemented diet
(Darias et al., 2010a); that is 8.4 IU VD3/g diet more than that
recommended for fish larvae and 11.5 times the amount recommended
for juveniles (NRC, 1993). Due to the high growth and metabolic rate, it
has been suggested that larval fish could deplete the storage of vitamins
faster than juveniles. Therefore, vitamin requirements for larval fish
might be higher than those for juveniles (Dabrowski, 1992). Contrary to
that observed for juveniles (Taveekijakarn et al., 1996), inadequate
levels of dietary VD3 negatively influenced the intestinal tract matura-
tion of European sea bass larvae confirming the noticeable sensitivity of
specimens during the organogenesis period (Darias et al., 2010a).
Studies on AA has been performed in larvae of some species with
different purposes: European sea bass and gilthead sea bream
(collagen dynamics: Terova et al., 1998), turbot, Scophthalmus
maximus (AA transfer from life prey to larvae, stress response and
pigmentation: Merchie et al., 1996b), milkfish (stress response:
Gapasin et al., 1998) and pikeperch, Sander lucioperca (performance:
Kestemont et al., 2007). Merchie et al. (1995b, 1996a) evaluated the
effects of live prey (rotifers and artemia) enriched with different levels
of AA on European sea bass and African catfish larval growth, survival
and stress resistance. They observed that an AA supplement of
2500 mg AA/kg dry weight significantly increased stress resistance in
both species. Besides, in the case of African catfish, AA supplemen-
tation increased larval growth while non-supplemented live prey was
enough to satisfy growth in European sea bass. This result indicates
that nutritional studies during the larval period are constrained when
using live prey because of the limitation and variability of nutritional
enrichments. For instance, rotifers and artemia contain relatively high
levels of AA, thus preventing the evaluation of the effects of low levels
of AA on larval development (Merchie et al., 1995a). The same authors
concluded that levels of 20–130 mg AA/kg diet are suitable for growth
and survival (Merchie et al., 1997), although this range might be more
restricted for optimal morphogenesis since research performed in
European sea bass larvae showed that AA lower than 30 mg and as
high as 400 mg AA/kg diet induced severe skeletal deformities (Darias
et al., 2011). Gouillou-Coustans et al. (1998) reported a requirement
in common carp larvae of 45 mg/kg diet based on growth. European
sea bass larvae requires a minimum amount of 15 mg AA/kg diet to
survive, of 30 mg/kg diet to attain maximal growth, and of 50 mg/kg
diet for an adequate morphogenesis (Darias et al., 2011), the last
being 8 times lower than that recommended for fish larvae and
corresponds to the amount recommended by the NRC (1993) for most
juveniles. This finding indicates that although larvae have higher
metabolic rate than juveniles, the amount of every nutrient in larval
diets does not necessarily have to be higher than for juveniles to
satisfy the larval physiological demands. Moreover, this could also
depend on the physiological role of the nutrient.
Blom and Dabrowski (1995) reported that saturation of ovarian AA
levels in broodstock rainbow trout promoted maximum egg quantity
and quality and established a dietary level of 8 times the recommended
level for optimum growth of salmonids (NRC, 1993) to optimize
reproduction efficiency in trout. Moreover, the same authors suggested
that diets containing less than 500 mg AA/kg may yield decreased
growth and survival of fish from eggs containing less than 5 μg AA/g.
These findings suggested that the required amount of AA in first feeding
larvae strongly depend on the previous maternal supply. Considering
that only 50 mg AA/kg diet induced the best larval performance in
54 M.J. Darias et al. / Aquaculture 315 (2011) 49–60

European sea bass (Darias et al., 2011), this could indicate that eggs
probably counted with sufficient amount of AA for the suitable growth
and development of the embryos. This is very interesting since it
denotes that during the very early larval stage, when patterning takes
place, the AA content is critical for a normal development.
These results showed that vitamin requirements vary according to
their physiological function, are species-specific and depend on the
environment (i.e., rearing conditions), physiological needs and devel-
opmental stage. Indeed, nutrient requirements change throughout the
larval period in line with ontogenesis (molecular, cellular and tissue
development), this being also reported for vitamins D and C (Merchie
et al., 1997; Darias et al., 2010a). Moreover, in the case of AA, considering
there is a transfer of this vitamin from broodfish to eggs and embryos
(Terova et al., 1998) and that collagen synthesis begins during early
embryogenesis, AA requirements during the larval stage would also
depend on egg quality. This indicates that fish larvae containing enough
amount of AA from parental origin to satisfy early collagen synthesis
would not necessarily need high levels of AA later in development.
5. The influence of vitamins D and C in the development
of malformations
Few studies exist on the role of dietary vitamins D and C on the
development of skeletal deformities in fish. Graff et al. (2002) did not
observe skeletal deformities in juvenile of salmon fed from 8 to
2280 IU VD3/g diet. However, another study found 41.6% deformed
juveniles of Japanese flounder fed a control diet containing 1.84 IU
VD3/g diet and 51.6% of vertebral deformities when fed 20.8 IU VD3/g
diet, mostly consisting in winding of the vertebrae caused by
abnormal calcification and impairment of its rigidity (Haga et al.,
2004). Vitamin C has been more studied and it has been shown that
scorbutic fish exhibited skeletal abnormalities such as shortened
opercula and abnormal support cartilage in the gills of salmonids
(Halver et al., 1975; Halver, 1989); scoliosis and broken backs in
channel catfish (Andrews and Murai, 1975; Lim and Lovell, 1978);
distortion of gill filament cartilages and short opercula in juvenile
tilapias (Soliman et al., 1986) and Mexican native cichlid (Chávez de
Martínez, 1990) or a cleft on the branchiostegal membrane,
commonly associated with a deformity or the partial to total absence
of its supporting branchiostegal rays, and deformed operculum in
milkfish (Hilomen-Garcia, 1997). Many of these vitamin C-deficient
signs can be attributed to the impaired collagen and support cartilage
formation in most tissues (Halver et al., 1975; Terova et al., 1998).
Some studies performed on juvenile fish originated from female brood
stock fed a diet devoid of AA and showed signs of deficiency with
severe spinal deformities, like scoliosis, lordosis and the resultant
growth retardation (Soliman et al., 1986). Gapasin et al. (1998)
showed that HUFA and ascorbate supplementation alleviated inci-
dence of opercular deformity in milkfish, possibly indicating that
the syndrome may be an ascorbate deficiency-related case. Merchie
et al. (1997) detected opercular deformities in gilthead sea bream
suggesting a possible sign of AA deficiency. AA influenced both the
cartilaginous tissue development as well as the ossification process
of bony tissues of European sea bass larvae (Darias et al., 2011).
A disruption of the mineralization process could induce skeletal
deformities. European sea bass larvae exhibited lower deformities
score when diets contained 27.6 IU VD3/g diet and 50 mg AA/kg
diet (20% deformities) while lower and upper levels of such vitamins
induced skeletal malformations (Darias et al., 2010a, 2011).
Similar amount of VD3 (20–30 IU VD3/g diet) was optimal in juveniles
of Japanese flounder to prevent the appearance of color abnormali-
ties in the blind side (Takeuchi, 2001). Besides, the type of deformi-
ties depends on the nature of the vitamin and the imbalance
considered (Fig. 3). For instance, low VD3 level (11.2 IU VD3/g diet)

Fig. 3. Skeletal disorders observed at 45 dph in European sea bass larvae induced by inadequate levels of vitamin D3 (VD3) and ascorbic acid (AA). A. Low VD3 content especially provoked
poor mineralization, pugheadness (*) and kyphosis (arrow head). Kyphosis and scoliosis were maximized at high VD3 levels. B. Low AA doses induced poor mineralization, cartilage damage,
such as cartilaginous vertebrae (arrows head) and unformed haemal arches (circled), pugheadness (*) and deformities of the caudal fin, specially affecting the epurals, urostyle and
specialized neural arch. High AA content generated one extra vertebra and deformities of dentary (arrow) and proximal pterygiophores of the dorsal and anal fins. Ep, epural; Ur, uroneural.
 M.J. Darias et al. / Aquaculture 315 (2011) 49–60 55

and higher than 42 IU VD3/g diet levels induced vertebral and

branchiostegal rays deformities in European sea bass, while pughead-
ness and deformities of the jaws and caudal fin were maximized
at the low VD3 dose (Darias et al., 2010a). AA levels lower than
30 mg/kg diet and as high as 400 mg AA/kg diet generated severe
deformities of the jaws and caudal fin, specially affecting to the
epurals, uroneural and specialized neural arch. Low AA levels also
caused cartilage damage, characterized by unformed haemal arches
and cartilaginous vertebrae, pugheadness and the lost of one vertebra,
whereas 400 mg AA/kg diet induced the formation of one extra
vertebra.
It was also noticed that a deficiency of these vitamins is more
harmful for developing European sea bass than an excess. Indeed, sea
bass larvae are highly sensitive to low vitamin D levels than to high
vitamin C levels throughout skeletogenesis. Nevertheless, skeletal
elements that developed earlier were more resistant to high vitamin
D levels than to high vitamin C content (Darias et al., 2010a, 2011).
It has been also postulated that juveniles of salmonids are resistant
to excess of dietary vitamin D3 (88.4 and 1147.2 IU VD3/g diet;
Horvli et al., 1998). This tolerance of megadoses of vitamin D3 in
salmonids was corroborated by Graff et al. (2002) in fry specimens of
Atlantic salmon fed with three different levels of vitamin D3 (8, 200
and 2280 IU VD3/g diet) from first feeding for 14 weeks. They did
not find any significant difference neither in weight, length, spe-
cific growth rate, mortality, or kidney calcium concentration nor in
skeletal malformations or histopathological changes between the
different dietary groups. They suggested that the low circulating level
of 25OHD3 and the decreasing effect of 24,25(OH)2D3 on calcium
absorption could partly explain the high vitamin D3 tolerance in
teleosts (Graff et al., 1999, 2002). By contrast, Darias et al. (2010a)
found an effect of high dietary VD3 levels (42 and 120 IU VD3/g diet)
in the development of skeletal deformities in European sea bass
larvae. The reasons for the inconsistent results could be related to
species-specific physiological demands. For instance, specimens with
high ingestion rate could be more sensitive to dietary toxicity.
Besides, the possibility that toxic effects may be age-related reflecting
varying nutritional demands in different developmental stages
Fig. 4. Incidence of malformations (%) and ossification degree (total pixel number/
might be also considered. This age-dependent sensitivity was also larvae × 100) at 45 dph in European sea bass fed different dietary vitamin D3 levels
suggested by Takeuchi (see Takeuchi, 2001) who found that juveniles (IU VD3/g diet) (A) and different dietary vitamin C levels (mg AA/kg diet) (B). Note that
of Japanese flounder fed 200 IU VD/g diet were more sensitive to the opposite profile existent between the percentage of malformations and the level of
develop color abnormalities of the blind side at a size of 11 mm TL ossification suggests that a disruption of the mineralization process could induce
skeletal deformities.
compared to those fed at 13 or 17 mm TL. They suggested an amount
of 20–30 IU VD/g diet after reaching 13 mm TL to prevent the
occurrence and progress of such color abnormality, which coincided
with optimal VD3 requirement for European sea bass larvae (Darias 6. Molecular mechanisms involving vitamin D and C in fish
et al., 2010a). skeletogenesis: the case of European sea bass
As previously discussed, vitamins D and C also influence the
ossification degree of European sea bass larvae through the It has been shown for European sea bass that the amount of VD3
regulation of osteoblast determination/differentiation and minerali- and AA differentially affects the skeletal development, the larvae
zation processes. Both delay and acceleration of the ossification being highly sensitive to low VD3 and AA levels throughout
process induced 50–60% malformations (Darias et al., 2010a; 2011). skeletogenesis, whereas skeletal elements that develop earlier are
The fact that only 14–16 IU VD3/g above or below the optimal dose more resistant to high VD3 than to AA content (Darias et al., 2010a,
(27.6 IU VD3/g diet) notably increased the incidence of deformities 2011). Within the range of dietary VD3 (from 11.2 to 120 IU/g diet)
denotes the high sensitivity of European sea bass larvae to this and AA (from 0 to 400 mg/kg diet) levels analyzed, amounts lower
vitamin. Moreover, doses ranging from 42 to 120 IU VD3/g diet did and higher than 27.6 IU/g diet for VD3 and than 50 mg/kg diet for AA,
not proportionally modify the rate of malformations confirming promoted the development of skeletal deformities in this species
that an excess of VD3 not only negatively influence the larval (Darias et al., 2010a, 2011). This suggests that requirements of these
development but suppose an economic squander for the aquacul- vitamins vary along the larval development, associated to their
ture industry. physiological demands. Due to its physiological function and mode of
In addition, an opposite profile between the incidence of action, dietary VD3 especially affected skeletal elements undergoing
malformations and the degree of ossification in European sea bass intramembranous ossification, whereas AA notably influenced the
larvae was found (Darias et al., 2010a; 2011) (Fig. 4). This suggests skeletal structures undergoing chondral ossification (Darias et al.,
that a disruption of the mineralization process could induce skeletal 2010a, 2011). Interestingly, all these findings were associated with
deformities. For that reason, the study of the molecular pathways changes in the expression profile of genes involved in the ontogeny of
affecting the ossification process is essential to understand the origin skeleton, shedding light to the mechanisms underlying skeletogen-
of skeletal malformations (Fig. 5). esis, the influence of dietary VD3 and AA in this process and the
56 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
Fig. 5. Diagrammatic representation of the role of VD3 and AA in the regulation of several genes involved in skeletogenesis and, particularly, in bone mineralization.
localization of molecular disruptions that could eventually cause
malformations (Darias et al., 2010a, 2011).
Independently from their biosynthetic ability, all animal cells are
strictly dependent on the presence of functional vitamin C
transporters, which determine the distribution of this molecule
between extra- and intra-cellular fluids. AA is imported by an active
mechanism, requiring two sodium-dependent vitamin C transpor-
ters, SVCT1 and SVCT2 (Savini et al., 2008). AA transport depends on
the number of SVCT proteins present in the plasma membrane, as
well as their substrate affinity (Liang et al., 2002). Therefore, the
amount of SVCT-1, which is considered the most predominant AA
transporter in the intestine, would determine the level of AA
absorption. Indeed, Darias et al. (2011) observed a high correlation
between the gene expression profiles of SVCT-1 in specimens fed
different AA levels with the amount of cartilaginous tissue and bone
mineralization degree. This result evidenced that this gene could be a
suitable marker for intestinal AA absorption and that its implication
on cartilage and bone formation would be proportional to the
amount of dietary AA levels. Besides, a dose–response effect was
observed in the expression of SVCT-1 during the metamorphosis
period of European sea bass larvae (Darias et al., 2011). Indeed, the
amount of SVCT-1 transcripts decreased with the dietary AA increase
suggesting the existence of a control mechanism for AA absorption
at transcriptional level (Darias et al., 2011). Similar findings were
observed in a human intestinal epithelial cell line in which elevated
levels of AA in the intestinal lumen lead to down-regulation of
SVCT1 mRNA in enterocytes (MacDonald et al., 2002). A feedback
mechanism has been demonstrated also in human lung epithelial
cells, where a loss of intracellular AA is compensated by active
uptake thanks to a marked increase of SCVT2 expression (Karaczyn
et al., 2006). This indicates that the two Na+-dependent transporters
can be subjected to a regulation by their own substrate, although the
mechanisms are still unknown. Saavini et al. (2005) proposed
transcriptional regulation or modification of mRNA stability. How-
ever, apart from transcriptional, translational and post-translational
modifications of SVCTs that could eventually regulate vitamin C
uptake (Liang et al., 2002), other mechanisms could be influencing
this process. Since SVCT-1 transcription requires calcium ions
(McDonald et al., 2002), it has been suggested that Ca2+ deficien-
cy/unavailability could be also responsible for the regulation of
SVCT-1 expression (Darias et al., 2011). This seemed to be the case of
European sea bass larvae fed high AA content, being the hypothesis
supported by the concomitant low amount of SVCT-1 and TRPV-6
transcripts observed (Fig. 6A). The last gene codes for the most
important calcium transporter at intestinal level (Hoenderop et al.,
2005). Low TRPV-6 expression could entail low TRPV-6 transporter
synthesis, thus preventing sufficient intestinal absorption of Ca2+ to
promote high levels of SVCT-1 expression (Darias et al., 2011). These
results suggest that high-dose supplements of AA might not be the
most efficient way of increasing the body pool of AA. This is in
accordance to previous studies showing that AA is well absorbed at low
doses, but absorption decreases as the level increases (Padayatty and
Levine, 2001). Dietary VD3 also affected the expression of TRPV6, the
highest levels being observed in groups fed 27.6 IU/g diet (Fig. 7A)
in concomitance with higher intestinal maturation degree (Darias
et al., 2010a). The direct effect of this vitamin on larval growth and
development indirectly influenced bone mineralization by limiting the
amount of absorbed Ca2+ through the modulation of TRPV6 expres-
sion. This happened at the time of vertebral column ossification
(around 22 dph) which could explain that larvae from this group were
more ossified (Darias et al., 2010a).
The fact that AA could influence the expression of TRPV-6, a gene
modulated by the vitamin D receptor (VDR), is an interesting finding
and evidenced for the first time in fish an interaction between AA
and the vitamin D metabolic pathway. Moreover, Darias et al. (2011)
demonstrated that dietary AA modulates the expression of VDRβ
suggesting that VDRβ is a possible target gene for AA (Fig. 6B). They
have previously shown that a decrease of VDRβ expression together
with an increase of ostecalcin expression during ontogenesis is the
expected pattern in developing European sea bass (Darias et al.,
2010a). Vitamin D stimulates the expression of osteocalcin through
binding of its receptor VDR. However, low VD3 and AA levels did not
induce a VDRβ expression decrease indicating a disruption in the
skeletogenesis process (Darias et al., 2010a, 2011; Fig. 7).
The influence of these vitamins in the gene expression of AA and
Ca2+ intestinal transporters (SVCT-1 and TRPV-6) and VDRβ also
 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
Fig. 6. Relative gene expression of several genes involved in skeletogenesis and bone
mineralization of developing European sea bass larvae fed different dietary VD3 levels.
A. Effects of dietary VD3 during metamorphosis (22 dph). The gene expression pattern
of TRPV-6 was correlated with that of osteocalcin, both genes being induced by VDR.
Larvae fed 27.6 IU VD3/g diet displayed the highest levels of expression of these genes.
B. Effects of dietary VD3 at the end of the larval stage (45 dph). The level of IGF-1
expression was higher in VD-27.6 group. The influence of VDR on osteocalcin
expression was noticed in all dietary groups except for the lowest one, preventing
thus well-ossified specimens at the end of the larval period.
affects the following pathways involving VD3 and AA on the
ossification process. For instance, it is well known that vitamin D
deficiency in humans causes higher secretion of parathyroid
hormone (PTH) due to the low serum 1,25(OH)2D3 and low serum
Ca2+, and this results in high bone turnover and increased bone
resorption. This causes bone loss and may contribute to the appear-
ance of osteoporosis (Lips, 2001). In European sea bass, osteocalcin
gene expression, which is one of the marker genes for the
mineralization of the extracellular matrix and regulated by VDRβ
(Lian and Stein, 1995; Darias et al., 2010b), was lower in larvae fed
low VD3 content whereas high dietary VD3 levels caused a positive
impact on osteoblast differentiation, this being not the case for
higher AA levels than 50 mg/kg diet (Fig. 6B, 7B). Indeed, the
expression profile of osteocalcin among larval groups fed different
dietary AA levels followed the same pattern that SVCT-1 and was
highly correlated with the amount of cartilage and degree of bone
mineralization. Moreover, authors observed that this profile was
inversely correlated with the incidence of skeletal deformities
suggesting that a disruption of the mineralization process could be
57
Fig. 7. Relative gene expression of several genes involved in skeletogenesis and bone
mineralization of developing European sea bass larvae fed different dietary AA levels at
the end of the larval period (45 dph). A. The expression pattern of TRPV-6 was
associated with that of SVCT-1, which expression is Ca2+-dependent. Consequently,
and as the RARg expression profile showed, cartilage synthesis was influenced by AA
availability in skeletal tissues. The lowest AA dose induced higher expression of PPARg,
possibly indicating that bipotent cells preferably differentiated into adipocytes.
B. The highest level of IGF-1 and osteocalcin expression was detected in larvae fed
50 mg AA/kg diet. The high VDR expression level obtained in larvae fed the lowest
amount of AA probably prevented an adequate osteocalcin expression. Besides, 400 mg
AA/kg diet seemed to be excessive since IGF-1 and osteocalcin expression was
prevented.
responsible for the induction of skeletal malformations. This was in
accordance with the double staining results that showed low
mineralization degree in the 400 mg AA/kg diet (Fig.4B). Such
results indicate that European sea bass larvae could eventually admit
levels of vitamin D higher than 27.6 IU/g diet, while 400 mg AA/kg
clearly exceed the optimal level.
Disturbances in skeletogenesis process by dietary VD3 and AA
were also supported by the IGF-1, BMP-4, RARγ and PPARγ gene
expression patterns observed (Darias et al., 2010a, 2011). IGF
increases osteoblasts proliferation and plays a major role in
stimulating mature osteoblast function. European sea bass larvae
fed low and high VD3 and AA content displayed low levels of IGF-1
expression (Figs. 6B and 7B), suggesting a low stimulation of
osteoblast differentiation. These results were supported by the also
low expression of osteocalcin and the low degree of bone ossification
observed in such specimens.
It has been shown that AA induces embryonic stem cells to
differentiate into osteoblasts (Carinci et al., 2005). The mechanism by
which AA sustains pre-osteoblast proliferation and commitment is
58 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
mediated through the synthesis of collagen type I, interaction with
α2- and β1-integrin, activation of the mitogen-activated protein
kinase pathway, and phosphorylation of osteoblast-specific transcrip-
tion factors. However, the multifunctional role of AA is not fully
elucidated. BMP-4 is a potent osteoblast differentiation factor and is
essential for osteoblasts to achieve their mature phenotype, which is
crucial for bone mineralization (Yamaguchi et al., 2000; Mazurais
et al., 2008). The influence of this gene on European sea bass larval
morphogenesis takes place during the early stage of development
and its expression decreases with larval growth (Darias et al.,
2010a). Authors suggested that this decrease of BMP-4 expression
during development could be an indicator of the osteoblast mature
phenotype achievement (Darias et al., 2010a, 2011). Indeed, 10 days
were necessary for larvae fed low and high AA levels to reach the
same low levels of BMP-4 expression detected in larvae fed 50 mg
AA/kg diet. Besides, larval groups fed diets with low VD3 and AA
content showed higher RARγ expression than the other groups in
concomitance with lower levels of osteocalcin expression indicating
a delay in skeletal development (Darias et al., 2011). The high
expression levels of RARγ, which is preferentially localized to the
pre-cartilage condensation and cartilage (Cash et al., 1997), were
also associated with high amounts of PPARγ transcripts, which
promote adipocyte differentiation (Casteilla et al., 2007), suggesting
a prevention of osteoblast formation in larval groups fed low AA
content (Fig. 6A; Darias et al., 2011). This loss of osteoblasts is a
likely cause of skeletal deformities (Mazurais et al., 2008). Similarly,
it has been demonstrated that an excess of vitamin A led to the
appearance of cephalic deformities related to an increase of RARγ
transcription and the loss of one vertebra (Villeneuve et al., 2006;
Mazurais et al., 2009).
7. Conclusions
Vitamin D and C requirements are species-specific and could vary
as a function of environmental, nutritional, genetic and developmen-
tal conditions. Both vitamins should appear in adequate dosages in the
diet, and with regard to other nutrients, for fish to achieve an optimal
performance. The disrupted molecular pathways involved in skeleto-
genesis and ossification observed in fish fed inadequate vitamin D and
AA levels highlight that several factors condition the appearance of
deformities: the bone origin, the velocity of development of the
skeletal structures, the type of vitamin and the amount of vitamin at
each developmental stage.
8. Perspectives
The new information provided by the molecular approach show
the complexity of the nutritional action in the skeletogenesis
process and allows establishing the basis for new molecular studies
in more depth to improve this knowledge. The analysis of the
molecular pathways involved in the different ossification processes
would be interesting to understand the nutritional needs adapted to
different stages/physiological demands of fish larval skeletogenesis.
Recent research has given evidences of neofunctionalization of
duplicated genes in teleosts (Karanth et al., 2009). Such a relevant
finding highlights the importance of considering gene isoforms
in future studies dealing with gene expression patterns analysis in
fish.
Considering the consequences of nutrients interaction on fish
physiology this aspect should be further investigated. There are more
and more frequent evidences that comparative physiology studies are
the most pertinent way of improving the knowledge of nutritional
requirements in fish. The microarray technologies could provide new
insights in this field (Darias et al., 2008).
Acknowledgements
Authors would like to thank C. Huelvan, E. Desbruyères, M.M. Le
Gall, P. Quazuguel and H. Le Delliou for their technical assistance. This
work was, in part, supported by FINEFISH, a Collective Research
Project of the sixth Framework Programme of the European Union
(Contract 012451). M.J. Darias was supported by a postdoctoral
fellowship from the Fundación Ramón Areces (Spain).
References
Affonso, E.G., Silva Eda, C., Tavares-Dias, M., de Menezes, G.C., de Carvalho, C.S., Nunes
Eda, S., Ituassú, D.R., Roubach, R., Ono, E.A., Fim, J.D., Marcon, J.L., 2007. Effect of high
levels of dietary vitamin C on the blood responses of matrinxã (Brycon amazonicus).
Comp. Biochem. Physiol. Mol. Integr. Physiol. 147, 383–388.
Andrews, J.W., Murai, T., 1975. Studies on the vitamin C requirements of channel cat fish
(Ictalurus punctatus). J. Nutr. 105, 557–561.
Ai, Q., Mai, K., Tan, B., Xu, W., Zhang, W., Ma, H., Liufu, Z., 2006. Effects of dietary vitamin C
on survival, growth, and immunity of large yellow croaker, Pseudosciaena crocea.
Aquaculture 261, 327–336.
Angus, T.C., Askew, F.A., Bruce, H.M., Callow, R.K., Philpot, J.St.L., Webster, T.A., 1931.
Crystalline vitamin D. Nature 128, 758. See also: Angus, T.C., Askew, F.A., Bourdillon,
R.B., Bruce, H.M., Callow, R.K., Fischmann, C., Philpot, J.St.L., Webster, T.A. Proc. Roy.
Sot. London, Series B, 108,340.
Azad, I.S., Syama Dayal, J., Poornima, M., Ali, S.A., 2007. Supra dietary levels of vitamins C
and E enhance antibody production and immune memory in juvenile milkfish,
Chanos chanos (Forsskal) to formalin-killed Vibrio vulnificus. Fish Shellfish Immunol.
23, 154–163.
Barnes, M.J., Kodicek, E., 1972. Biological hydroxylations and ascorbic acid with special
regard to collagen metabolism. Vitam. Horm. 30, 1–43.
Blom, J.H., Dabrowski, K., 1995. Reproductive success of female rainbow trout
(Oncorhynchus mykiss) in presponse to graded dietary ascorbyl monophosphate
levels. Biol. Reprod. 52, 1073–1080.
Blom, J., Dabrowski, K., 2000. Vitamin C requirements of the angelfish, Pterophylum
scalare. J. World Aquacult. Soc. 31, 115–118.
Cahu, C.L., Takeuchi, T., Zambonino-Infante, J.L., 2003. Nutritional components affecting
skeletal development in fish larvae. Aquaculture 227, 245–258.
Carinci, F., Pezzetti, F., Spinac, A.M., Palmieri, A., Lainoc, G., De Rosac, A., Farinac, E.,
Illianoc, F., Stabellinie, G., Perrottif, V., Piattelli, A., 2005. Effect of Vitamin C on pre-
osteoblast gene expression. Arch. Oral Biol. 50, 481–496.
Cash, D.E., Bock, C.B., Schughart, K., Linney, E., Underhill, T.M., 1997. Retinoic acid
receptor alpha function in vertebrate limb skeletogenesis: a modulator of
chondrogenesis. J. Cell Biol. 136, 445–457.
Casteilla, L., Cousin, B., Carmona, M., 2007. PPARs and Adipose Cell Plasticity. PPAR Res.
2007, doi:68202.
Chatterjee, L.B., 1973. Evolution and biosynthesis of ascorbic acid. Science 182,
1271–1272.
Chávez de Martínez, M.C., 1990. Vitamin C requirement of the Mexican native cichlid
Cichlasoma urophthalmus (Gunther). Aquaculture 86, 409–416.
Chen, R., Lochmann, R., Goodwin, A., Praveen, K., Dabrowski, K., Lee, K.J., 2003.
Alternative complement activity and resistance to heat stress in golden shiners
(Notemigonus crysoleucas) are increased by dietary vitamin C levels in excess of
requirements for prevention of deficiency signs. J. Nutr. 133, 2281–2286.
Cuesta, A., Esteban, M.A., Meseguer, J., 2002. Natural cytotoxic activity in seabream
(Sparus aurata L.) and its modulation by vitamin C. Fish Shellfish Immunol. 13,
97–109.
Dabrowski, K., 1990a. Gulonolactone oxidase is missing in teleost fish. Biol. Chem.
Hoppe Seyler 371, 207–214.
Dabrowski, K., 1990b. Ascorbic acid status in the early life of whitefish (Coregonus
lavaretus L.). Aquaculture 84, 61–70.
Dabrowski, K., 1992. Ascorbate concentration in fish ontogeny. J. Fish Biol. 40, 273–279.
Dabrowski, K., Ciereszko, A., 2001. Ascorbic acid and reproduction in fish: endocrine
regulation and gamete quality. Aquac. Res. 32, 623–638.
Darias, M.J., Zambonino-Infante, J.L., Hugot, K., Cahu, C.L., Mazurais, D., 2008. Gene
expression patterns during the larval development of European sea bass
(Dicentrarchus labrax) by microarray analysis. Mar. Biotechnol. NY 10, 416–428.
Darias, M.J., Mazurais, D., Koumoundouros, G., Glynatsi, N., Christodoulopoulou, S.,
Huelvan, C., Desbruyeres, E., Le Gall, M.M., Quazuguel, P., Cahu, C.L., Zambonino-
Infante, J.L., 2010a. Dietary vitamin D3 affects digestive system ontogenesis and
ossification in European sea bass (Dicentrachus labrax, Linnaeus, 1758). Aquaculture
298, 300–307.
Darias, M.J., Lan Chow Wing, O., Mazurais, D., Cahu, C., Zambonino-Infante, J.L., 2010b.
Alcian Blue–Alizarin red double staining technique for developing sea bass
(Dicentrachus labrax) larvae. J. Appl. Ichthyol. 26, 280–285.
Darias, M.J., Mazurais, D., Koumoundouros, G., Le Gall, M.M., Huelvan, C., Desbruyeres,
E., Quazuguel, P., Cahu, C.L., Zambonino-Infante, J.L., 2011. Imbalanced dietary
ascorbic acid alter molecular pathways involved in skeletogenesis of developing
European sea bass (Dicentrarchus labrax, Linnaeus, 1758). Comp. Biochem. Physiol.
Part A. doi:10.1016/j.cbpa.2011.01.013.
Dedi, J., Takeuchi, T., Seikai, T., Watanabe, T., Hosoya, K., 1997. Hypervitaminosis A
during vertebral morphogenesis in larval Japanese flounder. Fish. Sci. 63, 466–473.
Durve, V.S., Lovell, R.T., 1982. Vitamin C and disease resistance in channel catfish
(Ictalurus punctatus). Can. J. Fish. Aquat. Sci. 39, 948–951.
 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
Gapasin, R.S.J., Bombeo, R., Lavens, P., Sorgeloos, P., Nelis, H., 1998. Enrichment of live
food with essential fatty acids and vitamin C: effects on milkfish (Chanos chanos)
larval performance. Aquaculture 162, 269–286.
Graff, I., Aksnes, L., Lie, O., 1999. In vitro hydroxylation of vitamin D3 and 25-hydroxy
vitamin D3 in tissues of Atlantic salmon Salmo salar, Atlantic mackerel Scomber
scombrus, Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua.
Aquacult. Nutr. 5, 23–32.
Graff, I.E., Høie, S., Totland, G.K., Lie, Ø., 2002. Three different levels of dietary vitamin D3
fed to first-feeding fry of Atlantic salmon (Salmo salar L.): effect on growth,
mortality, calcium content and bone formation. Aquacult. Nutr. 8, 103–111.
Gouillou-Coustans, M.F., Bergot, P., Kaushik, S.J., 1998. Dietary ascorbic acid needs of
common carp (Cyprinus carpio) larvae. Aquaculture 161, 453–461.
Haga, Y., Takeuchi, T., Murayama, Y., Ohta, K., Fukunaga, T., 2004. Vitamin D3
compounds induce hypermelanosis on the blind side and vertebral deformity in
juvenile Japanese flounder Paralichthys olivaceus. Fisher. Sci. 70, 59–67.
Halver, J.E., Ashley, L.M., Smith, R.R., 1969. Ascorbic acid requirements of coho salmon
and rainbow trout. Trans. Am. Fish. Soc. 98, 762–771.
Halver, J.E., 1972. The role of ascorbic acid in fish disease and tissue repair. Bull. Jpn
Soc. Sci. Fish. 38, 79–92.
Halver, J.E., Smith, R.R., Tolbert, B.M., Baker, E.M., 1975. Utilization of ascorbic acid in
fish. Ann. N.Y. Acad. Sci. 258, 81–102.
Halver, J.E., 1989. The vitamins, In: Halver, J.E. (Ed.), Fish Nutrition, 2nd edn. Academic
Press, San Diego, USA, pp. 31–109.
Hamre, K., Waagbo, R., Berge, R.K., Lie, O., 1997. Vitamin C and E interact in juvenile
Atlantic salmon. Free Radic. Biol. Med. 22, 137–149.
Hardie, L.J., Fletcher, T.C., Secombes, C.J., 1991. The effect of dietary vitamin C on the
immune response of the Atlantic salmon (Salmo salar L.). Aquaculture 95, 201–214.
Henrique, M.M.F., Gomes, E.F., Gouillou-Coustans, M.F., Oliva-Teles, A., Davies, S.J.,
1998. Influence of supplementation of practical diets with vitamin C on growth
and response to hypoxic stress of seabream, Sparus aurata. Aquaculture 161,
415–426.
Henry, H.L., Midgett, R.J., Norman, A.W., 1974. Regulation of 25-hydroxyvitamin D3-1-
hydroxylase in vivo. J. Biol. Chem. 249, 7584–7592.
Hilomen-Garcia, G.H., 1997. Morphological abnormalities in hatchery-bred milkfish.
Chanos chanos Forsskal fry and juveniles. Aquaculture 152, 155–166.
Hilton, J.W., Ferguson, H.W., 1982. Effect of excess vitamin D3 on calcium metabolism in
rainbow trout Salmo gairdneri Richardson. J. Fish Biol. 21, 373–379.
Hoenderop, J.G.J., Nilius, B., Bindels, R.J.M., 2005. Calcium absorption across epithelia.
Physiol. Rev. 85, 373–422.
Horvli, O., Lie, O., Aksnes, L., 1998. Tissue distribution of vitamin D3 in Atlantic salmon
Salmo salar: effect of dietary level. Aquacult. Nutr. 4, 127–131.
Fernández, I., Hontoria, F., Ortiz-Delgado, J.B., Kotzamanis, Y., Estévez, A., Zambonino-
Infante, J.L., Gisbert, E., 2008. Larval performance and skeletal deformities in farmed
gilthead sea bream (Sparus aurata) fed with graded levels of vitamin A enriched
rotifers (Brachionus plicatilis). Aquaculture 283, 102–115.
Karaczyn, A., Ivanov, S., Reynolds, M., Zhitkovich, A., Kasprzak, K.S., Salnikow, K., 2006.
Ascorbate depletion mediates up-regulation of hypoxia-associated proteins by cell
density and nickel. J. Cell Biochem. 97, 1025–1035.
Kaushik, S.J., Gouillou-Coustans, M.F., Cho, C.Y., 1998. Application of the recommenda-
tions on vitamin requirements of finfish by NRC (1993) to salmonids and sea bass
using practical and purified diets. Aquaculture 161, 463–474.
Karanth, S., Lall, S.P., Denovan-Wright, E.M., Wright, J.M., 2009. Differential transcrip-
tional modulation of duplicated fatty acid-binding protein genes by dietary fatty
acids in zebrafish (Danio rerio): evidence for subfunctionalization or neofunctio-
nalization of duplicated genes. BMC Evol. Biol. 9, 219–233.
Kestemont, P., Xueliang, X., Hamza, N., Maboudou, J., Imorou Toko, I., 2007. Effect of
weaning age and diet on pikeperch larviculture. Aquaculture 264, 197–204.
King, C.G., Waugh, W.A., 1932. The chemical nature of vitamin C. Science 75, 357.
Kitamura, S., Ohara, S., Suwa, T., Nakagawa, K., 1965. Studies on vitamin requirements of
rainbow trout (Salmo gairdneri). Bull. Jpn Soc. Sci. Fish. 31, 818–826.
Kraus, V.B., Huebner, J.L., Stabler, T., Flahiff, C.M., Setton, L.A., Fink, C., Vilim, V., Clark, A.G.,
2004. Ascorbic acid increases the severity of spontaneous knee osteoarthritis in a
guinea pig model. Arthritis Rheum. 50, 1822–1831.
Lall, S.P., Lewis-McCrea, L., 2007. Role of nutrients in skeletal metabolism and pathology
in fish, an overview. Aquaculture 267, 3–19.
Lee, K.J., Dabrowski, K., 2004. Long-term effects and interactions of dietary vitamin C
and E on growth and reproduction of yellow perch, Perca flavescens. Aquaculture
230, 377–389.
Li, Y., Lovell, R.T., 1985. Elevated levels of dietary ascorbic acid increase immune
responses in channel catfish. J. Nutr. 115, 123–131.
Li, X., Bickerdike, R., Nickell, D., Campbell, P., Dingwall, A., Johnston, I.A., 2007.
Investigations on the effects of growth rate and dietary vitamin C on skeletal
muscle collagen and hydroxylysyl pyridinoline cross-link concentration in farmed
Atlantic salmon (Salmo salar). J. Agric. Food Chem. 55, 510–515.
Lian, J.B., Stein, G.S., 1995. Development of the osteoblast phenotype, molecular
mechanisms mediating osteoblast growth and differentiation. Iowa Orthop. J. 15,
118–140.
Liang, W.J., Johnson, D., Ma, L.S., Jarvis, S.M., Wei-Jun, L., 2002. Regulation of the human
vitamin C transporters expressed in COS-1 cells by protein kinase C. Am. J. Physiol
Cell Physiol. 283, 1696–1704.
Lim, C., Lovell, R.T., 1978. Pathology of the vitamin C deficiency syndrome in channel
catfish (Ictalurus punctatus). J. Nutr. 108, 1137–1146.
Lips, P., 2001. Vitamin D deficiency and secondary hyperparathyroidism in the elderly:
consequences for bone loss and fractures and therapeutic implications. Endocr.
Rev. 22, 477–501.
Lips, P., 2006. Vitamin D physiology. Prog. Biophys. Mol. Biol. 92, 4–8.
59
Lock, E.J., Waagbø, R., Wendelaar Bonga, S., Flik, G., 2010. The significance of vitamin D
for fish: a review. Aquac. Nutr. 16, 100–116.
Madsen, L., Dalsgaard, I., 1999. Vertebral column deformities in farmed rainbow trout.
Oncorhynchus mykiss. Aquaculture 171, 41–48.
Maulén, N.P., Henríquez, E.A., Kempe, S., Cárcamo, J.G., Schmid-Kotsas, A., Bachem, M.,
Grünert, A., Bustamante, M.E., Nualart, F., Vera, J.C., 2003. Up-regulation and
polarized expression of the sodium-ascorbic acid transporter SVCT1 in post-
confluent differentiated CaCo-2 cells. J. Biol. Chem. 278, 9035–9041.
Mazurais, D., Darias, M.J., Gouillou-Coustans, M.F., Le Gall, M.M., Huelvan, C., Desbruyères,
E., Quazuguel, P., Cahu, C., Zambonino-Infante, J.L., 2008. Dietary vitamin mix levels
influence the ossification process in European sea bass (Dicentrarchus labrax) larvae.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R520–R527.
Mazurais, D., Glynatsi, G., Darias, M.J., Christodoulopoulou, S., Cahu, C.L., Zambonino-
Infante, J.L., Koumoundouros, G., 2009. Optimal levels of dietary vitamin A for
reduced deformity incidence during development of European sea bass larvae
(Dicentrarchus labrax) depend on malformation type. Aquaculture 294, 262–270.
MacDonald, L., Thumser, A.E., Sharp, P., 2002. Decreased expression of the vitamin C
transporter SVCT1 by ascorbic acid in a human intestinal epithelial cell line. Br. J.
Nutr. 87, 97–100.
McLaren, B.A., Keller, E., O'Donnell, D.J., Elvehjem, C.A., 1947. The nutrition of rainbow
trout. I. Studies of vitamin requirements. Arch. Biochem. Biophys. 15, 169–178.
Merchie, Cl., Lavens, P., Dhert, Ph., Dehasque, M., Nelis, H., De Leenheer, A., Sorgeloos, P.,
1995a. Variation of ascorbic acid content in different live food organisms.
Aquaculture 134, 325–337.
Merchie, G., Lavens, P., Dhert, Ph., Pector, R., Mai Soni, A.F., Abbes, M., Nelis, H., Ollevier,
F., De Leenheer, A., Sorgeloos, P., 1995b. Live food mediated vitamin C transfer to
Dicentrarchus labrax and Clarias gariepinus. J. Appl. Ichthyol. 11, 336–341.
Merchie, G., Lavens, P., Starch, V., Abel, U., Nelis, H., De Leenheer, A., Sorgeloos, P., 1996a.
Influence of dietary vitamin C dosage on turbot (Scophthalmus maximus) and
European sea bass (Dicentrarchus labrax) nursery stages. Comp. Biochem. Physiol. A
114, 123–133.
Merchie, G., Lavens, P., Dhert, Ph., Garcia Ulloa Gomez, M., Nelis, H., De Leenheer, A.,
Sorgeloos, P., 1996b. Dietary ascorbic acid requirements during the hatchery
production of turbot larvae. J. Fish Biol. 49, 573–583.
Merchie, G., Lavens, P., Sorgeloos, P., 1997. Optimization of dietary vitamin C in fish and
crustacean larvae: a review. Aquaculture 155, 165–181.
Montero, D., Marrero, M., Izquierdo, M.S., Robaina, L., Vergara, J.M., Tort, L., 1999. Effect
of vitamin E and C dietary supplementation on some immune parameters of
gilthead seabream (Sparus aurata) juveniles subjected to crowding stress.
Aquaculture 171, 269–278.
Moreau, R., Dabrowski, K., Czesny, S., Cihla, F., 1999. Vitamin C–vitamin E interaction in
juvenile lake sturgeon (Acipenser fulvescens R.), a fish able to synthesize ascorbic
acid. J. Appl. Ichthyol. 15, 250–257.
Murai, T., Andrews, J.W., Bauerfeind, J.C., 1978. Use of Lascorbic acid, ethocel coated
ascorbic acid and ascorbate 2-sulfate in diets for channel catfish (Ictalurus
punctatas). J. Nutr. 108, 1761–1766.
National-Research-Council, 1993. Nutrient Requirements of Fish. National Academy
Press, Washington DC.
Navarre, O., Halver, J.E., 1989. Disease resistance and humoral anti-body production in
rainbow trout fed high levels of vitamin C. Aquaculture 79, 207–219.
Nayak, S.K., Swain, P., Mukherjee, S.C., 2007. Effect of dietary supplementation of
probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita
(Ham.). Fish Shellfish Immunol. 3, 892–896.
Norman, A.W., Roth, J., Orci, L., 1982. The vitamin D endocrine system: steroid
metabolism, hormone receptor, and biological response (calcium binding proteins).
Endocr. Rev. 3, 331–366.
Ortuño, J., Esteban, M.A., Meseguer, J., 2003. The effect of dietary intake of vitamins C
and E on the stress response of gilthead seabream (Sparus aurata L.). Fish Shellfish
Immunol. 14, 145–156.
Padayatty, S.J., Levine, M., 2001. New insights into the physiology and pharmacology of
vitamin C. CMAJ 164, 353–355.
Poston, H.A., 1969. Effects of massive doses of vitamin D3 on fingerling brook trout. Fish.
Res. Bull. 32, 48–50.
Phromkunthong, W., Boonyaratpalin, V., Starch, V., 1997. Different concentrations of
ascorbyl-2-monophosphate-magnesium as dietary sources of vitamin C for
seabass, Lates calcarifer. Aquaculture 151, 225–243.
Qin, Q.W., Wu, Z.H., Zhou, Y.C., Pan, J.P., 2000. Non-specific immunomodulatory effects
of dietary vitamin C on grouper, Epinephelus awoara. Trop. Oceanol. 19, 58–63.
Savini, I., Rossi, A., Pierro, C., Avigliano, L., Catani, M.V., 2008. SVCT1 and SVCT2: key
proteins for vitamin C uptake. Amino Acids 34, 347–355.
Soliman, A.K., Jauncey, K., Roberts, R.J., 1986. The effect of varying forms of ascorbic
acid on the nutrition of juvenile tilapia (Oreochromis niloticus). Aquaculture 52,
1–10.
Sundell, K., Bjomsson, B.Th., 1990. Effects of vitamin D3, 25(OH) vitamin D3, 24, 25(OH)
2 vitamin D3, and 1, 25(OH)2 vitamin D3 on the in vitro intestinal absorption in the
marine teleost, Atlantic cod (Gadus morhua). Gen. Comp. Endocrinol. 78, 74–79.
Takeuchi, T., Dedi, J., Haga, Y., Seikai, T., Watanabe, T., 1998. Effect of vitamin A
compounds on bone deformity in larval Japanese flounder (Paralichthys olivaceus).
Aquaculture 169, 155–165.
Takeuchi, A., Okano, T., Kobayashi, T., 1991. The existence of 25-hydroxyvitamin
D3-1-hydroxylase in the liver of carp and bastard halibut. Life Sci. 48, 275–282.
Takeuchi, T., 2001. A review of feed development for early life stages of marine finfish in
Japan. Aquaculture 200, 203–222.
Taveekijakarn, P., Miyazaki, T., Matsumoto, M., Arai, S., 1996. Histopathological and
haematological changes in amago salmon, Oncorhynchus rhodurus (Jordan and
McGregor), fed a vitamin-D-free diet. J. Fish Dis. 19, 289–294.
60 M.J. Darias et al. / Aquaculture 315 (2011) 49–60
Terova, G., Saroglia, M., Papp, Z.G., Cecchini, S., 1998. Dynamics of collagen indicating
amino acids, in embryos and larvae of sea bass (Dicentrarchus labrax) and gilthead
sea bream (Sparus aurata), originated from broodstocks fed with different vitamin
C content in the diet. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 121, 111–118.
Thompson, I., White, A., Fletcher, T.C., Houlihan, D.F., Secombes, C.J., 1993. The effect of
stress on the immune response of Atlantic salmon (Salmo salar L.) fed diets
containing different amounts of vitamin C. Aquaculture 114, 1–18.
Tolbert, B.M., 1979. Ascorbic acid metabolism and physiological function. Int. J. Vitam.
Nutr. Res. Suppl. 19, 127–142.
Tsukaguchi, H., Tokui, T., Mackenzie, B., Berger, U.V., Chen, X.Z., Wang, Y., Brubaker, R.F.,
Hediger, M.A., 1999. A family of mammalian Na+-dependent L-ascorbic acid
transporters. Nature 399, 70–75.
Villeneuve, L., Gisbert, E., Le Delliou, H., Cahu, C.L., Zambonino-Infante, J.L., 2005. Dietary
levels of all-trans retinol affects retinoid nuclear receptor expression and skeletal
development in European sea bass larvae. Brit. J. Nutr. 93, 791–801.
Villeneuve, L., Gisbert, E., Moriceau, J., Cahu, C., Zambonino-Infante, J.L., 2006. Intake of
different levels of vitamin A and polyunsaturated fatty acids during different
developmental periods modifies the expression of morphogenesis genes in
European sea bass (Dicentrarchus labrax). Brit. J. Nutr. 95, 677–687.
Wang, X., Kim, K.W., Bai, S.C., Huh, M.D., Cho, B.Y., 2003. Effects of the different levels of
dietary vitamin C on growth and tissue ascorbic acid changes in parrot fish
(Oplegnathus fasciatus). Aquaculture 215, 203–211.
Wilson, R.P., Poe, W.E., 1973. Impaired collagen formation in the scorbutic channel
catfish. J. Nutr. 103, 1359–1364.
Xie, Z., Niu, C., Zhang, Z., Bao, L., 2006. Dietary ascorbic acid may be necessary
for enhancing the immune response in Siberian sturgeon (Acipenser baerii), a
species capable of ascorbic acid biosynthesis. Comp. Biochem. Physiol. A 145,
152–157.
Yamaguchi, A., Komori, T., Suda, T., 2000. Regulation of osteoblast differentiation
mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 21,
393–411.
Yanda, D.M., Ghazarian, J.G., 1980. Vitamin d and 25-hydroxyvitamin d in rainbow trout
(Salmo gairdneri): cytochrome p-450 and biotransformations of the vitamins.
Comp. Biochem. Physiol. B 69, 183–188.