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Fish Nutrition Laboratory, Unité mixte INRA-IFREMER,
*
B.P 70, 29280 Plouzané,
Station d’Hydrobiologie, INRA 64310 St. Pée-sur-Nivelle, France
1To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • ascorbic acid • requirement • hepatic saturation • hydroxyproline • Dicentrarchus labrax
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Soliman et al. 1985
) and some acipenserids (Moreau et al. 1996
), which appear to possess an active
L-gulonolactone oxidase (GLO), an enzyme involved in AA
synthesis. Regarding marine finfish, Maeland and Waagbo (1998)
did not
detect GLO activity in several species.
Quantitative data on vitamin C requirements of marine finfish are
scarce (Boonyaratpalin et al. 1992
, NRC 1993
, Saroglia and Scarano 1992
, Teshima et al. 1993
). Recommendations on vitamin requirements for these
species are generally based on data obtained for freshwater species as
reported by the NRC (1993)
. The dietary AA requirement for optimal
growth and normal development (tissue HyPro saturation levels) would be
in the range of 10–20 mg AA/kg for most freshwater fish such as
rainbow trout, Oncorhynchus mykiss (Cho and Cowey 1993
); channel catfish, Ictalurus punctatus
(El Naggar and Lovell 1991
); Atlantic salmon,
Salmo salar (Sandnes et al. 1992
) and hybrid
tilapia, Oreochromis niloticus x O. aureus (Shiau and Hsu 1995
). Vitamin C requirement could also be based on body
vitamin storage status such as the hepatic AA concentration. Thus, in
European sea bass, (Dicentrarchus labrax), the requirement
for normal growth and hepatic saturation was estimated to be 200 mg of
AA/kg when a crystalline form of AA was included in the diets
(Saroglia and Scarano 1992
).
Since the form of dietary AA can greatly influence the estimates of the
requirement for this labile water-soluble vitamin
(Boonyaratpalin et al. 1989
, 1992
), the
aim of this study was to estimate vitamin C requirements of European
sea bass using a stable AA phosphate form incorporated into practical
diets using different physiological response criteria: skin and whole
fish HyPro concentration and hepatic AA concentration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was conducted in the experimental flow-through facilities
of IFREMER, Brest, France. Seawater was filtered with a high-
pressure sand filter and thermoregulated (salinity: 35
;
water temperature: 20 ± 1°C; photoperiod: 12 h light/12 h
dark). Groups of 100 juvenile sea bass originating from our own stock
(IFREMER), with an initial body weight (IBW) of 0.7 g were reared
in 24 different tanks of 50-L capacity for 15 wk. Three tanks were
allotted at random to each diet.
Diets.
Eight practical diets were formulated to contain graded levels of AA
(0, 5, 10, 20, 40, 80, 160, 320 mg of AA/kg). The raw materials
including fish meal Norseamink® and soluble fish protein
concentrate CPSP® (Lorientaise des produits de la
pêche, Lorient, France), corn gluten meal (Roquette, Lille,
France), soybean meal (Drogou, St. Renan, France), whey (ICI-Proseca,
Epinay sur Orge, France) and wheat middlings (Moulin du Buis, Brest,
France) were ground, sifted to 600 µm and well blended with the other
ingredients (Table 1
). The minimal vitamin C (AA and dehydroascorbic acid) concentration
measured by fluorimetric detection (Bourgeois et al. 1989
) in the mixture before ascorbyl polyphosphate (AP)
incorporation was 2 mg/kg. The moist diets were pelleted and dried for
1 h in a ventilated drier at 40°C. The dried diets were then
ground and sieved in three diameters: 1–1.6, 1.6–2 and 2–2.5 mm. The
diets were distributed to visual satiety by hand four times/day
according to fish size over a 15-wk period (5% body weight/day at the
beginning of the trial, decreasing to 3% body weight/day at the end of
the trial).
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At the end of the experiment, fish were weighed and examined for
external deficiency signs. All the fish captured for analysis were
anesthetized with phenoxy-2 ethanol and killed by cervical section. A
pool of 30 fish from each tank was collected for analysis of HyPro
concentration in the whole fish. Five other fish were sampled from each
tank for individual analysis of AA concentration in the liver and HyPro
concentration in the skin. Analysis of vitamin C (ascorbic and
dehydroascorbic acid) in hepatic tissues was performed by fluorimetric
detection (Bourgeois et al. 1989
). Concentrations of
HyPro in skin and whole body were measured using a colorimetric
detection after acid hydrolysis with 72% perchloric acid
(Bonnet and Kopp 1984
).
Data analysis.
Growth data were analyzed by one-way ANOVA, followed by
Newman-Keuls test, using the computing program STAT-ITCF (1991)
. Differences were considered significant at P
< 0.05.
Relationships between dietary AA supplementation and HyPro
concentrations in the skin or in the whole body or liver AA
concentration were computed using the four-parameter saturation
kinetics model as proposed by Mercer et al. (1986)
for describing the
nutrient-response curves. Since the responses did not decline at
high intake levels in our study, the initial four-parameter model
was judged more pertinent than the inhibited nutrient-response
curve (Mercer et al. 1989
). Based on this model, other
parameters representing, respectively, the level at which the organism
is the most sensitive to changes in intake (Ims, intake at maximum
slope) and the intake levels at which the organism operates most
efficiently (Ime, intake level at maximum efficiency) were also
calculated. Subsequently, considering that an intake level leading to a
95% decrease in slope would represent the requirement level
(Mercer et al. 1986
), the minimum requirement levels for
each type of response (maximum concentration of HyPro in skin and whole
body, maximum AA concentration in liver) were computed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The nutrient response curves (Mercer et al. 1986
) for
HyPro in skin and whole body are shown in Figure 1
and Figure 2
, respectively. The skin HyPro concentration was low in AA-deficient
sea bass (217 nmol/g skin), compared to AA-supplemented diet fed
fish (Rmax = 297 nmol/g skin). The whole
body Hypro concentrations were low in fish fed 0, 5, 10 mg of AA/kg (in
the range of 15–18 nmol/g whole body). Whole body HyPro levels
increased with increasing dietary levels, reaching a maximum
concentration of 45 nmol/g whole body.
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. Hepatic AA concentration increased with increasing dietary AA levels,
reaching a maximum storage of 474 pmol/g liver
(Rmax). Values of Ims (intake at maximum
slope) and Ime (intake at maximum efficiency) were 21 mg AA/kg and 35
mg AA/kg, respectively.
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led to
values of 5, 31 and 121 mg of AA/kg based on skin HyPro concentration,
whole body HyPro concentration and liver AA concentration,
respectively. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, El Naggar and Lovell 1991
,
Matusiewicz et al. 1995
) including European sea bass
(Amerio et al. 1998
). However, although the latter study
showed that ascorbyl-2 polyphosphate was more bioavailable than
ascorbyl-2 sulfate in sea bass, the AA requirement was not determined.
Over the past decade, development of these stable AA forms has led to a
re-evaluation of vitamin C requirements of several teleosts. Data
obtained with stable phosphate forms show that the minimum dietary
requirement is in the range of 10–20 mg of AA/kg for freshwater fish
(Cho and Cowey 1993
, El-Naggar and Lovell 1991
, Sandnes et al. 1992
) and 12.6–47 mg of
AA/kg for some marine fish (Boonyaratpalin et al. 1992
,
Kanazawa et al. 1992
, Teshima et al. 1993
). In addition to growth, other response criteria such as
HyPro concentration in skin or in backbone or in whole body fish or AA
liver concentration have been tested independently but never used
simultaneously as valid criteria within the same study.
For juvenile European sea bass, the minimum dietary AA requirement
needed to maintain normal skin collagen concentration and maximal
growth (5 mg of AA/kg) is below the requirement of other fish. A
plateau in the whole body HyPro concentration was reached at a dietary
level of about 31 mg of AA/kg. This would therefore appear to be the
minimal AA requirement to maintain normal collagen formation in the
whole fish. HyPro content in the whole fish seems to be a more
sensitive biochemical criterion of AA deficiency than HyPro
concentration in the skin. In Atlantic salmon, the minimum dietary
requirement to maintain normal collagen formation was estimated to be
10 and 20 mg of AA/kg, respectively, for normal HyPro concentration in
the backbone and in the skin (Sandnes et al. 1992
).
These authors concluded that the skin HyPro concentration was a more
sensitive indicator of AA status.
Up to a dietary level of 80 mg AA/kg, there was a good correlation
between dietary AA level and liver vitamin C concentration. Based on
the value of Ims (21 mg AA/kg), we can deduce that below this level,
fish would develop deficiency symptoms due to depletion of body stores
of AA and above this value, fish would store AA. At a dietary AA level
of 35 mg of AA/kg (Ime), seabass are most efficient in terms of hepatic
AA storage. The hepatic AA concentration reaches a plateau (474 pmol/g
liver) at an intake of at least 121 mg of AA/kg. Similarly, hepatic
saturation of AA was only observed in yellowtail fed 14–28 mg of AA/kg
(Kanazawa et al. 1992
). Although a dose-response was
observed in Salmonids fed graded levels of vitamin C, no hepatic
saturation of AA was detected by Cho and Cowey (1993)
and Sandnes et al. (1992)
.
If the optimal dietary concentration of vitamin C is equivalent to that
allowing for maintenance of steady-state tissue concentration as
suggested by Dabrowski (1990)
, a dietary vitamin C level at least 121
mg AA/kg is required for juvenile European sea bass, leading to a
maximum liver storage of 474 pmol/g of tissue. This result is 2.5-fold
higher than the recommended dietary levels by NRC (1993)
, based on
survival and growth of young salmonids. In the same manner, Blom and Dabrowski (1995)
propose that the saturation level in mature ova
represents the requirement level in the rainbow trout brood-stock
diet supplemented with 357 mg of AA/kg.
To conclude, depending upon physiological response criteria, dietary AA requirement of juvenile European sea bass would vary: 5 mg of AA/kg to maintain maximum growth and for maximal HyPro concentration in the skin, 31 mg of AA/kg for maximal HyPro concentration in whole body and 121 mg of AA/kg for maximum hepatic storage of AA. Finally, hepatic AA saturation appears to be the most stringent response criterion for the determination of vitamin C requirement of European sea bass.
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FOOTNOTES |
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Manuscript received July 1, 1999. Revision accepted November 4, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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