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Unité Mixte de Nutrition des Poissons IFREMER-INRA, 29280 Plouzané, France
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Dicentrarchus labrax • dietary lipid • pancreatic enzymes • intestinal enzymes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For years, it was hypothesized that young fish larvae did not have the
enzymes needed to digest a compound diet (Lauff and Hofer 1984
); live prey digestion was explained by an autolysis of
these organisms in gut larvae. Recent studies using experimental
compound diets have shown some specifics of larval protein and
carbohydrate digestion (Péres et al. 1998
,
Zambonino Infante et al. 1997
) and have provided the
first data on qualitative and quantitative protein requirements.
Moreover, these studies showed that the nature and concentration of
dietary components affect the maturation of digestive function in
seabass larvae (Cahu and Zambonino Infante 1995
) as has
been extensively reported in mammals (Henning 1987
).
Unlike protein, lipid requirements in fish larvae have been extensively
studied using live prey enriched by different oils (for review, see
Watanabe and Kiron 1994
). This experimental approach led
to an incomplete determination of lipid requirements; the accurate
determination of larvae lipid needs will be reached only by the use of
a compound diet.
The aims of this study were to determine the optimal lipid level in a compound diet for larvae and to understand the effects of dietary fat concentration on maturation of the digestive tract in seabass larvae.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eggs of European sea bass (Dicentrarchus labrax) were
obtained from Aquanord. Larval rearing was conducted at the
Ifremer-Station de Brest and lasted 38 d, which corresponds to
the end of larval development. Newly hatched larvae were transferred
from incubators to 25 conical fiber glass tanks (35 L) with black walls
(initial stocking density, 80 larvae/L, i.e., 2800
larvae/tank). They were supplied with running sea water that had been
filtered through a sand filter, then passed successively through a
tungsten heater and a degassing column packed with plastic rings.
Throughout the experiment, the water temperature and salinity were
18–19°C and 35 g/L, respectively. The oxygen level was maintained
above 6 mg/L by setting the water exchange to 30%/h (flow rate, 0.18
L/min). The light intensity was 9 W/m2 maximum at the
surface. All animal procedures and handling were conducted in
compliance with NIH guidelines (NRC 1985
).
The larvae were fed live prey from mouth opening until d 14 with the
following sequence expressed per larvae and per day: d 6–9, 50–200
rotifers (Brachionus plicatilis); d 10–14, 200 rotifers
plus 30–100 Artemia nauplii. Then the larvae were
divided into five groups (4 tanks per group) and fed for 23 d five
formulated diets of equal protein level with increasing fat level
(10–30%) and decreasing carbohydrate level (20–0%). The diets were
designated as
L10,2
L15, L20, L25 and L30 (Table 1). In added lipids, the soy lecithin/cod liver oil ratio was
maintained at 0.8. Diet formulation took into account the estimated
requirements of marine fish larvae in phospholipids and in the two main
highly unsaturated fatty acids, eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) (Table 2). The size of the microparticulate diets was 125–200
µm during the first 5 d, then 200–400
µm. Fish were fed continuously in large excess for 18
h/d using a belt feeder. Food ingestion was monitored by observing the
larvae digestive tract under a binocular microscope because dietary
microparticles are visible by transparency.
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To monitor growth, 10 larvae per tank (n = 4 tanks for each dietary group) were taken once a week from each tank. For this, water volume was lowered and 10 larvae were collected at one time using an appropriate net. These 10 larvae were thus representative of the tank population. At the end of the experiment, larval survival rates were determined by counting individuals.
At d 15, 28 and 38, 50 larvae were collected for enzymatic studies from each tank before morning food distribution; larvae were immediately stored at -80°C pending dissection and assays. Larvae (n = 50) were collected for mRNA studies from only three tanks; they were dissected and RNA extraction was immediately performed.
Dissection under microscope was conducted on a glass maintained at
0°C; individuals were cut into four parts as described by Cahu and Zambonino Infante (1994)
: head, pancreatic segment,
intestinal segment and tail, in order to limit the assay of enzymes to
specific segments. This dissection inevitably produced a crude mixture
of organs in each segment. The pancreatic segment also contained liver,
heart, muscle and spine. The intestinal segment contained intestine,
muscle and spine.
Analytical methods.
The pancreatic segments were homogenized into 5 volumes (v/wt) of
ice-cold distilled water. Trypsin (EC 3.4.21.4) and amylase (EC
3.2.1.1) activities were assayed according to Holm et al. (1988)
and Métais and Bieth (1968)
,
respectively. Phospholipase A2 (PLA2; EC
3.1.1.4) was assayed by the reverse-phase HPLC method of
Tojo et al. (1993)
. This chromatographic method was
adapted for the assay of lipase (EC 3.1.1.3) using triolein as
substrate (Tietz et al. 1989
). Purified brush border
membranes (BBM) from the intestinal segment homogenate were obtained
according to a method developed for intestinal scraping (Crane et al. 1979
). The degree of purification of BBM, taking
alkaline phosphatase and aminopeptidase N as markers of cell membrane
fraction, was close to that reported by Crane et al. (1979)
, i.e., 13.5- and 10-fold, respectively. Enzymes of the
BBM, alkaline phosphatase (EC 3.1.3.1), aminopeptidase N (EC 3.4.11.2)
and 
-glutamyl transpeptidase (GT; EC 2.3.2.2) were assayed
according to Bessey et al. (1946)
, Maroux et al. (1973)
and Meister et al. (1981)
, respectively.
Assay of a cytosolic peptidase, leucine-alanine (Leu-Ala) peptidase
was performed using the method of Nicholson and Kim (1975)
. Enzyme activities were expressed as specific
activities, i.e., mU/mg protein. Ratios of enzyme activities of BBM
related to Leu-Ala peptidase activity were calculated using the
segmental activities, i.e., the total activity of each enzyme per
larvae in the intestinal segment. Protein was determined by the
Bradford procedure (Bradford 1976
).
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Total RNA was extracted using the TRIzol reagent kit procedure (Life Technologies, Grand Island, NY). For synthesis of cDNA, 5 µg of total RNA was treated with FPLCpure Moloney-Murine Leukemia virus reverse transcriptase using the Ready-To-Go-T-Primed First Strand Kit (Pharmacia Biotech, Uppsala, Sweden). PCR was carried out by an initial denaturation at 94°C for 1 min, followed by 35 cycles at a temperature of 94°C for 30 s, specific annealing temperatures for 1.5 min, and 72°C for 1 min; the final extension was conducted at 72°C for 7 min. The PCR mixture contained 0.8 µL of the cDNA, 0.2 units Taq Polymerase (Appligene, Gaithersburg, MD), 100 µmol/L dNTP, 50 µmol of each primer and 1X Reaction Buffer (Appligene), in a final volume of 50 µL. Sequences and annealing temperatures for the sense and antisense oligonucleotides were as follows: 5'-CAggTgTCTCTgAAC-3' and 5'-CCCARGACACAACACCCTg-3' (60°C) for trypsin, 5'-gCCATCAATgACCCCTT-3' and 5'-ggTgCAggATgCATTgC-3' (50°C) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ACTACggYTgCTACTg-3' and 5'-CggTCACAgTTRCAgA-3' (48°C) for PLA2.
These primers were selected after alignment of different species sequences of selected mRNA, obtained using the Sequence Retrieval System WWW server at EMBL-EBI (Cambridge, UK). Alignments of RNA sequence of species from different phyla were necessary because fish RNA sequences were scarce. The resulting PCR products were cloned using the TOPO-TA Cloning kit (Invitrogen, Leek, The Netherlands) and sequenced by Cybergene (St Malo, France); the trypsin, GAPDH and PLA2 sequences obtained have been registered by EMBL under the accession numbers AJ006882, AJ006883 and AJ006339, respectively.
Trypsin, GAPDH and PLA2 were amplified using the same cDNA
sample; 1 to 10 µL of each PCR product were applied on
a 1.2% agarose/1 mg/L ethidium bromide gel. cDNA spots were quantified
with a Fluor-SMultilmager and its MultiAnalyst Software (Bio-Rad,
Hercules, CA), using appropriate calibration. We generated a standard
curve by plotting the UV absorbance of the spots (resulting after a
35-cycle PCR) against the input concentration of the studied cDNA. The
limits of the exponential phase and the beginning of the saturation
phase of the amplification reaction were determined for each gene; this
ensured a linear relationship between input RNA and final RT-PCR
product or the ability to maintain it by an appropriate dilution of the
input cDNA. The values obtained for trypsin and PLA2 mRNA
were normalized relative to the GAPDH mRNA by calculating the
PLA2/GAPDH mRNA ratio and the trypsin/GAPDH mRNA ratio.
Indeed, Sölch and Arnold (1996)
showed that this
normalization relative to GAPDH provides a widely applicable value for
comparative studies of gene expression at the mRNA level.
Statistical analyses.
Results are given as means ± SEM (n
= 4; n = 3 for mRNA studies). Survival rates
and ratios of segmental enzymatic activities were
arcsin(x1/2) transformed. The variance homogeneity of the
data was checked using Bartlett's test (Dagnelie 1975
).
Weight, survival rate and ratios of enzymatic segmental activity data
were compared by a one-way ANOVA followed by Newman-Keuls
multiple range test (Dagnelie 1975
) when significant
differences were found at the 0.05 level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)although the weight of larvae fed the L10 diet did not differ from
that of those fed the L15 diet.
|
), even though no significant difference in survival (P
< 0.05) was noted between the groups fed the L15 and L20 diets.
|
). Amylase, trypsin, lipase and PLA2 were
active as early as d 15.
|
) and PLA2 (Fig. 3A
) were affected by the dietary lipid level. Lipase specific
activity generally increased with dietary lipid level, but significant
differences were found only between the group fed the L10 diet and
groups fed diets with lipid levels >20% (P < 0.05).
In response to high lipid diets, PLA2 activity
was markedly increased and was 2.75 times higher in the group fed the
L30 diet than in the group fed L10. PLA2 mRNA
levels were lower in groups fed L10 and L15 diets than in those fed
diets with greater lipid concentrations (Fig. 3
B).
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were affected by the dietary lipid level at d 38. On
the contrary, amylase specific activity (Table 3)
was lower in groups
fed L25 or L30 than in those fed L10 or L15; This lower amylase
activity resulted from the lower dietary starch level.
Secretion of pancreatic enzymes was calculated as follows: enzyme
assayed in the intestinal segment divided by enzyme assayed in
pancreatic plus intestinal segments. Trypsin secretion was
significantly greater in groups fed L20, L25 and L30 than in groups fed
low lipid levels (Table 3)
. Amylase secretion generally increased with
the dietary lipid level (Table 3)
.
The specific activity of a BBM enzyme, AP (Table 3
), was
maximally stimulated when dietary fat reached 20% dry matter and was
significantly lower when dietary fat was 10 or 15% dry matter.
Segmental activity ratios of two BBM enzymes to Leu-Ala peptidase
are reported in Table 5. Groups fed L25 and L30 diets exhibited a higher
aminopeptidase/Leu-Ala peptidase ratio than the other groups as early
as d 28, whereas no difference among groups was observed for the
GT/Leu-Ala peptidase ratio. At d 38, the ratio between
aminopeptidase and Leu-Ala peptidase was greater in larvae fed L25
and L30 diets than in those fed L10, L15 or L20 diets. The ratio in
larvae fed L30 was almost three times that of the group fed the L10
diet. The GT/Leu-Ala peptidase ratios obtained in the three groups
fed 20% or more lipid did not differ and were significantly greater
than those obtained in groups fed the L10 and L15 diets.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Moreover,
data on compound diet feeding as early as the d 15 of fish larval
development are scarce (Brinkmeyer and Holt 1998
,
Cahu and Zambonino Infante 1994
). Indeed, at this
developmental stage, larvae are generally fed live prey, rotifers and
Artemia. A compound diet formulated to meet the specific
nutritional requirements of fish larvae could replace live prey in
larvae feeding sequence and would lower the juvenile production cost in
hatcheries. The lack of a compound diet available for the early larval
stages explains the poor knowledge in larvae nutritional requirements.
We have chosen to balance the variation in lipid concentration with
carbohydrates instead of nonnutritive filler. Indeed, our previous
experiments suggested that the incorporation of nonnutritive filler
such as cellulose strongly reduces larvae growth and in that case,
conclusive results cannot be drawn in a nutritional study.
In our experiment, the five diets were able to sustain some growth and
survival in seabass larvae. The lowest final weight observed in this
study (group L10) was close to the best weight obtained by
Péres et al. (1996)
who fed seabass larvae a
compound diet containing 50% protein and 15% lipid. The increase in
dietary lipid concentration from 10 to 30% led to 100% growth gain. A
maximal 10-fold increase was observed in the weight of fish larvae
between d 15 and 38. We can assume that such a growth rate requires
high energy diets.
In the same way, the increase in dietary lipid level positively
affected the larva survival rate. The best survival obtained in this
study, 48% in the L30 group, was close to the optimal survival
obtained by feeding larvae live prey (Person-Le Ruyet et al. 1993
). These results suggest that the optimal lipid
concentration in diet for larvae would be ~30%, higher than the
10–20% lipid level generally adopted in the experimental larvae diets
(Brinkmeyer and Holt 1998
, Fernandez-Diaz and Yufera 1997
).
Activities of lipolytic enzymes, lipase and PLA2,
were revealed in very young larvae (15 d). Until now, several authors
argued that young fish larvae could not thrive on a compound diet
because of an insufficient digestive capacity (Kolkovski et al. 1997
). In particular, the attendance of lipolytic enzymes was
debated (Cousin and Baudin-Laurencin 1985
,
Koven et al. 1993
); only in recent studies have
PLA2 activities in young fish larvae been
reported (Evans et al. 1998
, Ozkizilcik et al. 1996
). The results obtained in our study complete the data
recently acquired on protein and carbohydrate digestion in early fish
larvae stages (Cahu and Zambonino Infante 1994
,
Péres et al. 1998
) and support the idea
that young larvae have functional digestive enzymes.
The lipolytic enzymes, lipase and PLA2, were
stimulated in 38-d-old larvae by the increase in their respective
substrates, triglycerides and phospholipids in the diet. This enzymatic
response has been extensively reported in mammals, especially for
lipase (Sheele 1993
) but, to our knowledge, has never
been described in fish. The PLA2 specific
activity in the different experimental groups generally increased with
the mRNA levels. Nevertheless, post-transcriptional regulation also
appeared to be involved because PLA2 activity in
groups fed diets containing <4.5% phospholipids was not consistent
with the mRNA levels. Ying et al. (1993)
reported that
changes in PLA2 synthesis occurred, in spite of
similar mRNA levels, after a stimulation of pancreas by caerulein, a
cholecystokinin (CCK)-like peptide. It can be assumed that the
efficiency of PLA2 mRNA translation would be
modulated by hormonal mechanisms in seabass larvae.
The plateau observed in the activity of lipase and PLA2 suggests that maximal capacity of lipolytic enzyme synthesis was reached at 15% triglycerides and 4.5% phospholipids in the diet, respectively (group L20). This assertion was backed up by similar PLA2 mRNA levels in larvae fed 20% or more dietary lipid.
As expected, amylase activity at d 38 was affected by dietary starch
concentration (Péres et al. 1996
). Trypsin
activity and trypsin mRNA levels did not differ in the five groups
because the diets contained the same kind and concentration of protein.
It must be pointed out that the secretion of amylase and trypsin
generally increased with the dietary lipid level. Pancreatic secretion
is mediated mainly by an intestinal hormone, cholecystokinin. Liddle (1995)
showed that the CCK secretion in rats was stimulated by the
ingestion of protein but also fat, through a CCK-releasing factor.
We suggested previously the existence of a CCK-releasing factor in
seabass larvae fed different levels of casein hydrolysate (Cahu and Zambonino Infante 1995
). High pancreatic secretion levels
related to high dietary lipid levels reinforce the hypothesis of the
existence of a CCK-releasing factor in seabass.
Seabass larvae normally achieve the development of their digestive
tract around wk 4 of life by the onset of BBM digestion of enterocytes,
concurrently with the decline of cytosolic digestion. This maturational
process has been well examined in mammals (Henning 1987
)
and has been described also in fish (Zambonino Infante et al. 1997
). The developmental stage of intestinal digestion can be
evaluated by considering the segmental activity ratio of brush border
enzymes vs. a cytosolic enzyme, Leu-Ala peptidase. This ratio
reflects the relative importance of BBM digestion compared with
intracellular digestion at a given developmental stage of the larvae
(Zambonino Infante et al. 1997
). At d 28, the higher
aminopeptidase/Leu-Ala peptidase ratio in the L25 and L30 groups
indicated an earlier enterocyte maturation compared with the other
groups. At d 40, the positive effect of high dietary fat level on the
enterocyte differentiation was also illustrated by the high
GT/Leu-Ala peptidase ratio in the L20, L25 and L30 groups.
Intestinal maturation and mucosal adaptation are nutrient-sensitive
processes; Vanderhoof (1993)
pointed out that highly
unsaturated long-chain fats are especially efficient in stimulating
these processes. Alessandri et al. (1993)
showed that
the enterocyte differentiation in rats involves an increasing
incorporation of (n-6) fatty acids whose availability is controlled by
diet. A similar incorporation would occur in fish larvae during the
maturation process of enterocytes, but this incorporation might involve
(n-3) fatty acids, the major constituents of cell membranes in marine
organisms. Enterocyte differentiation in seabass larvae would be
favored therefore by diets containing >20% lipids, i.e., >2.7% EPA
+ DHA. These data are consistent with the (n-3) highly unsaturated
fatty acid requirements that were determined by Izquierdo et al. (1989)
to be ~3.6% in red seabream fed live prey. On the
other hand, it must be pointed out that diets containing >20% lipids
also incorporated >4.5% phospholipids, which is higher than the
requirement of 3% usually accepted for fish larvae (Coutteau et al. 1997
).
The maximal specific activities of alkaline phosphatase that were found when dietary fat reached 20% reinforced the idea of better development of the BBM of enterocytes in larvae fed high dietary fat levels. Nevertheless, alkaline phosphatase activity generally increased when larvae were fed dietary phospholipids ranging from 2.3 to 4.5%, indicating a stimulation of this enzyme by one of its known substrates.
In previous studies, we noted that the early maturation of enterocytes
led to an improvement in survival (Cahu and Zambonino Infante 1995
). In this experiment, higher survival rates were also
found in the two groups, L25 and L30, in which enterocyte
differentiation had begun earlier than in the other experimental
groups.
In conclusion, this study shows that a high dietary fat level favored seabass larvae development. It remains to be clarified whether the beneficial effect is attributable mainly to dietary energy or more particularly to some lipid components, i.e., essential fatty acids or phospholipids.
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FOOTNOTES |
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GT,
-glutamyl transpeptidase; L10, 15, 20, 25, 30, diets containing
10,15, 20, 25 and 30% lipid, respectively; Leu-Ala,
leucine-alanine peptidase; PLA2, phospholipase
A2; RT-PCR, reverse transcriptase-polymerase chain
reaction. 
Manuscript received December 18, 1998. Initial review completed January 21, 1999. Revision accepted March 2, 1999.
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