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Unité Mixte INRA-IFREMER de Nutrition des Poissons, 64310 Saint-Pée-sur-Nivelle, France
2To 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: • fish larvae • carp • dietary lipid • medium-chain fatty acids • tricaprylin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Dabrowski et al. 1984
, Szlaminska et al. 1991
), for marine
larvae, this task is more difficult and the first positive results are
quite recent (Cahu et al. 1998
). Knowledge of the
nutrition of fish larvae, which are independent zooplankton-eaters,
appears to be far from those of young suckling mammals. However, some
common features can be considered, such as the high energy demand
related to a high growth rate and the limited absorptive capacity of
the poorly differentiated digestive tract (Zambonino Infante et al. 1997
). In seabass larvae pre-fed live food for 8 d, the lipid enrichment (phospholipids and fish oil) of the dry diet
fed afterward improves survival and growth rates (Zambonino Infante and Cahu 1999
). A phospholipid requirement has been
shown in fish larvae (Geurden et al. 1997
,
Kanazawa 1985
). Besides the specific role of
phosphatidylcholine for intestinal absorption of neutral lipids
(Fontagné et al. 1998
), a beneficial effect of a
high energy supply in diets for fish larvae can be hypothesized.
In mammals, medium-chain fatty acids
(MCFA),3
with chain length
between 6 and 12 carbon atoms, are readily utilized (Bach and Babayan 1982
, Kennedy 1991
) and, due to their
specific properties, MCFA are widely employed in enteral and parenteral
nutrition as energy sources (Bistrian 1997
,
Lasekan et al. 1992
, Wolfram 1998
). By
analogy with mammals, it could be speculated that in larval fish also,
MCFA could be easily absorbed and enhance the energy supply without (or
with limited) competition with long-chain fatty acids. High levels
of MCFA are found only in a small number of terrestrial lipid
sources such as coconut oil or milk fat (Gurr and Harwood 1991
). MCFA are absent or of minor importance in the natural
aquatic food chain of fish. However, some studies have shown that fish
can utilize dietary MCFA, presented as coconut oil or as
medium-chain triacylglycerols (MCT). MCT were found to lower body
fat deposition without impairing body growth in ayu (Nematipour et al. 1990
). In juvenile red drum, coconut oil can replace
efficiently other dietary fats such as tallow or fish oil (Craig and Gatlin 1995
). In contrast to coconut oil, tricaprylin
reduces growth in juvenile red drum (Craig and Gatlin 1995
) and in larval carp (Fontagné et al. 1999
), suggesting that fish response depends on the dietary
MCFA.
The present study was initiated to evaluate the use of MCFA with different chain length provided by purified MCT as dietary lipid for the larval stage of carp.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Common carp (Cyprinus carpio L.) larvae were obtained by
induced spawning from one female and two males (trial 1) or two females
and four males (trial 2) of the captive broodstock maintained in our
experimental fish farm (INRA, Donzacq, Landes, France). Rearing
conditions were the same as previously reported (Fontagné et al. 1999
). All animal procedures and handling were conducted
in compliance with guidelines for laboratory animals (The Council of Europe 1986
). Trial 1 lasted 21 d and trial 2,
22 d. One day after hatching, larvae were randomly distributed
into 17 (trial 1) or 8 tanks (trial 2) in a semi-recirculating
system as described by Charlon and Bergot (1984)
with
400 larvae per 6 L tank and 300 larvae for the second part of trial 2.
The water temperature was monitored and risen from 21 to 24°C within
3 d whereupon it remained at 24°C. First-feeding (d 0)
started 2 d after hatching. Automatic feed dispensers delivered
food in excess throughout the 16-h light period. Food particle size and
water flow rate in each tank were increased progressively every week.
In trial 1, five diets were fed to triplicate groups of
first-feeding larvae. Two tanks were food-deprived and served
as a negative control in order to assess the possible availability of
unwanted food in the recirculated water system and for comparison with
the other experiments performed in similar conditions. In trial 2, two
diets were tested with three replicates on first-feeding larvae (d
0–d 11) or on larvae fed previously for 11 d with the control
diet (d 11–d 22).
The dry diets were based on casein and soluble fish protein concentrate
(Table 1
). In trial 1, the lipid level was 23% including 10% of supplemented,
synthetic triacylglycerols. In trial 2, the lipid level was 18%
including 10% supplied as triacylglycerols. The difference in the
lipid level in the two trials might be due to the difference in sources
of soluble fish protein concentrate. The requirement for phospholipids
of carp larvae was satisfied by soybean lecithin (Geurden et al. 1997
). Essential fatty acid requirements
(Radünz-Neto et al. 1996
) were met by (n-3) and
(n-6) fatty acids from the soluble fish protein concentrate and soybean
lecithin, respectively. Triolein (Prolabo, Fontenay-sous-Bois, France)
was determined by gas chromatographic analysis to be in fact a blend of
64 g/100 g oleic acid and of 13% linoleic acid with some saturates and
monoenes from 12:0 to 18:0 (Fontagné et al. 1999
).
In trial 1, the control diet TOL contained 10% triolein. The four
other diets contained 5% triolein and 5% medium-chain
triacylglycerols (MCT) which were either tricaproin (TC6), tricaprylin
(TC8), tricaprin (TC10) or trilaurin (TC12). MCT were provided as
purified sources with one MCFA amounting to 98% of total fatty acids.
In trial 2, two diets were used: a control diet with 10% triolein and
another diet with 10% tricaprylin instead of triolein. Chemical
composition analysis of the diets was made using the following
procedures: dry matter after drying at 105°C for 24 h, protein
(N · 6.25) by the Kjeldahl method after acid hydrolysis, ash by
incineration at 550°C for 16 h and gross energy in an adiabatic
bomb calorimeter (IKA, Heitersheim, Germany). The fatty acid
composition of the diets, determined as the fatty acid composition of
larvae, is shown in Table 2
.
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The final survival was calculated from daily mortality and from the final record of surviving fish in each tank. Three samples of 100 larvae on d 1 and all larvae from each tank at the end of the trial were collected for wet weight determination. The larvae were food-deprived for 1 d, killed by anesthesia in 2-phenoxyethanol (Prolabo, Fontenay-sous-Bois, France), water-rinsed and blotted on absorbent tissue before wet weight determination and storage at -80°C for lipid analyses. In trial 1, 10 larvae were sampled and killed by anesthesia on d 0 and from each tank on d 7, 11, 15 and 21 for length measurements performed with a semi-automatic image analyzer (VIDS Version IV; Systèmes Analytiques, Les Clayes-sous-Bois, France).
Lipid extraction and analysis.
Total lipids of diets and larvae were extracted and measured
gravimetrically according to Folch et al. (1957)
using
dichloromethane instead of chloroform, the former being less toxic than
the latter. In trial 2, total lipids were separated into neutral and
polar fractions according to Juaneda and Rocquelin (1985)
. Fatty acid methyl esters were prepared by
acid-catalyzed transmethylation of total lipids (trial 1) or of
both neutral and polar lipids (trial 2) according to Shantha and Ackman (1990)
and were analyzed using a Varian 3400 gas
chromatograph (Les Ulis, France). The chromatograph was equipped with a
DB Wax fused silica capillary column (30 m · 0.25 mm i.d., film
thickness: 0.25 µm; J&W Scientific, Folsom, CA). Helium was used as
carrier gas (1.4 mL/min). The thermal gradient was 100 to 180°C at
8°C/min, 180 to 220°C at 4°C/min and a constant temperature of
220°C for 20 min. Injector and flame-ionization detector
temperatures were 260 and 250°C, respectively. Fatty acid methyl
esters were identified by comparison with known standards and
quantified using a Spectra Physics 4270 integrator (San Jose, CA).
Statistical analysis.
Diet-related differences were analyzed using one-way ANOVA. The
Newman-Keuls multiple range test was used to compare means when a
significant difference was found. Percentage data were arc-sin
transformed and weight data were log-transformed before analysis.
The theoretical biomass of each group, calculated as the product of
survival by the mean weight of 100 larvae, was also log-transformed
before analysis. In both trials, survival, weight and biomass results
were analyzed using the tank (three replicates per diet) as the
statistical unit. In trial 1, lipid and fatty acid data obtained from
two analyses per tank (three tanks per dietary treatment and one tank
in TC8 group) were compared by a nested ANOVA. The tank effect tested
in the analytical variance was never significant. In trial 2, lipid and
fatty acid data were obtained from two analyses of pooled larvae from
three tanks in all dietary treatments. All the statistical analyses
were performed according to Snedecor and Cochran (1968)
and with the computing program STAT-ITCF (ITCF 1988
). Differences were considered significant when
P values were <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). On d 7, the mean survival rate of the unfed larvae became significantly
lower than that of the fed groups. Mortality of the food-deprived
group was almost complete by d 10 (>99%). On d 11, carp larvae fed
TC8 had a significantly lower survival rate (90 ± 3%) compared
with other fed groups (97 ± 1%). At the end of the trial, final
survival of the TC8 group was 56 ± 12% and 92 ± 7% for
the other groups TOL, TC6, TC10 and TC12. Among larvae with the best
survival rates, larvae fed TOL and TC10 exhibited significantly greater
survival (96 ± 2%) than the group fed TC12 (82 ± 8%). In
trial 2, mortality of the food-deprived group began on d 8 and was
complete on d 11. Survival of larvae fed TOL was significantly lower on
d 11 of the trial 2 than on d 11 of the trial 1 (71 ± 16% vs. 98
± 1%, respectively) due to a reduced survival in one tank of
larvae fed TOL in trial 2 (53% vs. 80 ± 2% for the two other
tanks). The difference in survival between TC8 and TOL groups (55
± 11% vs. 71 ± 16%) was not significant between d 0 and d
11 (P = 0.21) but was significant for the
period d 11–d 22 (67 ± 4% vs. 78 ± 5%, respectively).
|
and Table 4
). In trial 1, larvae fed TC8 were significantly smaller (Fig. 2
) than larvae fed TC6 and TOL on d 11 (larval total length: 10.3 ± 0.5 mm vs. 12.1 ± 0.7 mm, respectively) and than larvae from all
other dietary groups on d 21 (11.6 ± 0.1 mm vs. 15.3 ± 1.4
mm, respectively). The combined index biomass (final weight of
surviving larvae for 100 initial larvae) of larvae fed TC8 was
significantly lower than that of larvae fed TOL, TC6, TC10 and TC12. In
trial 2, total lipid contents of larvae fed TC8 were significantly
lower than the TOL fed control due to a significantly reduction of
neutral lipid contents of larvae fed TC8, whereas no significant
differences in polar lipid contents were found. A tendency for lower
total lipid contents was also noted for larvae fed TC8 in trial 1
(P = 0.30).
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) except for polar lipids from larvae fed TC8 in the first part of trial
2 which were smaller and exhibited lower (n-6) and higher (n-3) fatty
acids levels than in the control (Table 6
). In trial 1, the (n-3) and (n-6) fatty acid composition of larvae fed
TC8 reflected more the initial fatty acid profile of 1-d larvae with
greater amounts of polyunsaturated fatty acids and lower amounts of
18:3 (n-3) and 18:2 (n-6) compared to other dietary groups. Total
lipids from larvae fed the control diet TOL contained the most of
monoenes followed by larvae fed TC6 and larvae fed the other
MCT-diets (53 vs. 48 and 42 g/100 g total fatty acids,
respectively). Compared to larvae fed TOL, larvae fed MCT contained
more saturates (16:0 and 18:0). Levels of the tested MCFA found in
larval total lipids were significantly higher for 10:0 (7.3 g/100 g of
total fatty acids) and 12:0 (8.1 g/100 g) than for 6:0 (not detected)
and for 8:0 (1.3 g/100 g). Separate analyses of larval neutral and
polar lipid in trial 2 indicated that fatty acids shorter than 14:0
were found only in neutral lipid and that the level of 10:0 in neutral
lipid of larvae fed TC8 reached 25 g/100 g of total fatty acids on d
11.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
In carp larvae, in contrast to rats, tricaprylin significantly reduced
survival and growth rates compared to other MCT and triolein. This
peculiar effect of tricaprylin is in agreement with previous comparison
of tricaprylin against coconut oil in larval carp
(Fontagné et al. 1999
) and in juvenile red drum
(Craig and Gatlin 1995
). The latter authors also found
higher levels of ketone bodies in the blood of red drum fed
tricaprylin. However, these effects of tricaprylin cannot be
extrapolated to tricaproin, which yielded high survival and growth in
the present study. Moreover, in a further experiment with carp larvae,
tricaproin was found to be less ketogenic than tricaprylin and resulted
also in better survival and growth rates (Fontagné et al, unpublished results).
Whole body lipid contents of larvae were higher in the present study
than in previous studies (Geurden et al. 1999
), likely
due to the higher lipid content of the diets used here (18–24%
instead of 6%). The dietary effect on fatty acid profile was more
marked in the neutral lipid than in the polar lipid fraction of larval
lipids. The fact that no fatty acid shorter than 14:0 was incorporated
into polar lipid is consistent with previous observations in juvenile
red drum (Davis et al. 1999
) and rabbits fed MCT
(Linseisen and Wolfram 1993
). The stability of the
saturated fatty acid level in phosphatidylcholine, the major
phospholipid class, has already been underlined in carp larvae
(Geurden et al. 1999
). Compared to triolein, dietary
MCFA enhanced saturated fatty acid level at the expense of monoenes,
whereas polyunsaturated (n-3) and (n-6) fatty acid levels remained
globally unaffected, as already found in carp larvae fed coconut oil
vs. triolein (Fontagné et al. 1999
).
The part of each MCFA fed recovered in larval total lipids rose
with the fatty acid chain length. This finding was consistent with the
gradient of MCFA reported in body lipid of rats fed tricaproin,
tricaprylin, tricaprin or trilaurin (Clifford et al. 1983
).
In the present study, the MCFA deposit of 8:0, 10:0 and 12:0 in
larval lipids was associated with higher concentration of 10:0, 12:0
and 14:0, respectively, compared to the TOL group. This phenomenon was
less clear in plasma lipids of rats, except for trilaurin, with 3%
14:0 vs. 9% 12:0 (Clifford et al. 1983
). A distinct
feature found in larval carp for tricaprylin was the levels of 10:0
higher than those of 8:0, which was not the case for 12:0 and 14:0
after feeding with tricaprin and trilaurin, respectively. The 10:0
formation after 8:0 feeding was still more prominent in trial 2 with
larvae fed TC8 during 11 d after first-feeding as the 10:0
level reached 25% of total fatty acids in the neutral lipid fraction.
This unexpected level of 10:0 raises the question of the nature of the
elongase responsible. The fatty acid synthase, present in vertebrates
is known as a regulated multi-enzyme system which produces 16:0 by
successively adding 2-carbon units to an acyl chain (Gurr and Harwood 1991
). Intermediate-length products are not usually
released, with a noticeable exception for the mammary gland. The high
levels of 10:0 thus suggest a disorganization or an abnormal
functioning of the fatty acid synthase if the latter enzyme is implied.
Another possible explanation is that another elongating system is
responsible. Besides the fatty acid synthase, the elongases allow the
formation of polyunsaturated fatty acids by successive
elongation-desaturation (Gurr and Harwood 1991
).
Horie et al. (1989)
have described an activity of rat
liver peroxisomes to elongate medium-chain acyl-CoA with
octanoyl-CoA as the preferred substrate. According to these
authors, the peroxisomal elongation system may consist of the reverse
reaction of the ß-oxidation system except for the last step, which is
catalyzed by enoyl-CoA reductase.
Irrespective of the elongating system involved, the unusual level of
10:0 suggests that a large part of 8:0 is not directed toward energy
production nor normal fat deposition. In contrast, 6:0 appears to
follow the classical way of complete degradation into acetyl-CoA
and resynthesis via fatty acid synthase. This pathway has been
described in HepG-2 cells incubated with 8:0 (Pakula et al. 1997
) or in rats fed 8:0, which exhibited a body fatty acid
profile similar to that of control rats fed a fat-free diet
(Demarne et al. 1978
).
Deposition of 10:0 was lowered in larvae fed TOL for 11 d then TC8
for 11 other days. In the latter case, growth was impaired, and body
fat content was reduced compared to the TOL-fed group. This
suggests that 10:0 formation probably depends on energy balance in the
fish. In food-deprived rats, MCFA are mainly ß-oxidized, whereas
in refed rats, MCFA can be partly retailored to long-chain fatty
acids by peroxisomal ß-oxidation followed by synthesis of palmitic
acid (Christensen et al. 1989
). In pre-ruminant
calves and lambs, addition of 8:0 or 10:0 to the diet may yield
different protein accretion or fat deposition, according to the supply
of other nutrients especially carbohydrates and amino acids and to
other factors related to the age and the growth potential of the animal
(Aurousseau et al. 1984
, Aurousseau et al. 1989
). Similar situation may exist in fish, but in the
present study, dietary amino acid supply was not limiting. Besides,
utilization of carbohydrates as energy source is rather limited in
fish.
In conclusion, this study pointed out the peculiarity of tricaprylin among other MCT which seem well utilized by carp larvae up to 20–30 g/100 g of total dietary fatty acids. More information is needed before introducing MCT in practical diets of larvae. The exception of tricaprylin raises the question of the metabolic pathways followed by this MCT, especially for the suggested direct elongation of caprylic acid into capric acid.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: MCFA, medium-chain fatty acids; MCT medium-chain triacylglycerols; TC6, TC8, TC10, TC12, diets supplemented with 5% of tricaproin, tricaprylin, tricaprin and trilaurin, respectively; TOL, diet supplemented with 10% of triolein.
Manuscript received December 16, 1999. Initial review completed February 7, 2000. Revision accepted April 12, 2000.
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