Essentiality of dietary phospholipids for carp (Cyprinus carpio L.) larvae

Aquaculture Aquaculture 131 (1995) 303-314

ELSEVIER

Essentiality of dietary phospholipids for carp (Cyprinus carpio L.) larvae Inge Geurdena**, Jo20 Radiinz-Netob, Pierre Bergotb “Laboratory of Aquaculture & Artemia Reference Center, University of Ghent, Rozier 44, 9000 Ghent, Belgium “UnitP Mixte INCA-IFREMERde Nutrition de Poissons, Station d’Hydrobiologie, BP 3. 64310 Saint-Pie-surNivelle, France

Accepted 28 October 1994

Abstract Two experiments were carried out in order to evaluate the essentiality of phospholipid (PL) addition to semi-purified diets for first-feeding carp larvae. In Experiment I (25 days), a casein-based diet was supplemented with 0,2 or 4% of a purified PL source (PL level in source: 98%) and with 0 or 4% of peanut oil (PO). One casein-based diet without PL was supplemented with choline chloride. In Experiment 11 (2 1 days), the same casein-based diet was supplemented with 7 commercial or experimental PL sources (PL content in sources: 55-81%). Sources included soybean PL, lyso soybean PL, obtained by treatment with phospholipase A,, and soybean PL with Ca2+ added in order to decrease the oil/water (01 W) emulsifying properties. Diets were made isolipidic by adding PO. One diet was formulated to be PL-free.In both experiments, all semi-purified diets supplemented with PL significantly improved survival and growth of carp larvae in comparison with PL-deficient diets. Results indicate that the beneficial effect of PL was not due to the correction of a choline deficiency or to the correction of an essential fatty acid deficiency and thus supported the idea of the essentiality of the PL entity for the early nutrition of larvae. The effect of PL seemed unrelated to their emulsifying properties as larval performances were not affected by Ca*+ enrichment while they were decreased by lyso-PL supplementation. It is presumed that the PL requirement for membrane building and renewal could be especially high during the fast growing larval stages and could exceed the endogenous ability of PL synthesis. Attention should be paid to an adequate dietary PL supply when larvae are offered artificial diets instead of PL-rich live food. Keywords: C.yprinus carpio; Feeding and nutrition-fish,

* Corresponding

larval rearing; Semi-purified

author.

0044.8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDlOO44-8486(94)00344-O

diet; Phospholipid

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1. Introduction Recent reviews concerning lipid nutrition of fish suggest that certain larval stages require phospholipids (PL) in their diets (Kanazawa, 1993; Sargent et al., 1994). As animals in general, including juvenile and adult fish, are known to be able to synthesize PL (Sargent, 1976)) the nature of the requirement (the PL entity or only parts of them) requires further investigations. There are only a limited number of experimental data available on the larval response to dietary PL manipulation, which contrasts with the large number of nutritional studies conducted to examine the essential fatty acid requirements of fish larvae. This is partly due to the difficulty in modifying the PL content of live prey (Artemiu) by the classical enrichment techniques (Tackaert et al., 1991). Moreover, since most start-feeding fish do not accept artificial diets, studies on the effect of PL inclusion in artificial diets concern mainly larvae which were initially fed live food (Kanazawa et al., 1981, 1983a, 1985; Koven et al., 1993). In this context, carp larvae offer some advantages since they can be fed exclusively on semi-purified diets and strongly react to PL supplementation (RadiinzNeto et al., 1994). In the first experiment we examined: (a) whether an increased addition of PL to the diet (4% instead of 2%) improved larval performance; (b) whether the PL effect was linked to the supplementation of triglycerides in the diet, as proposed in studies with crustacean and fish larvae where PL improved the absorption of neutral lipids; and (c) whether addition of choline could replicate the effect of PL. In the second experiment, we compared the efficacy of diets containing different PL sources of plant and animal origin to that of a PLdeficient diet, while the effect of modifying the emulsifying properties of a given PL source was also evaluated.

2. Materials and methods Three days after hatching, carp (Cyprinus carpio) larvae were randomly distributed in 4.5 dm3 tanks (300 ind. per tank) of the rearing system described by Charlon and Bergot ( 1984). Starting from the following day (day 0 of the experiment) food was delivered in excess by automatic feeders throughout the 16 h light period. During the first week, the temperature was increased from 19°C (day 0) to 24”C, whereafter it was kept constant at 24 + 1°C. The first experiment lasted 25 days and the second 21 days. Mortality was recorded daily. At the end of the experiment the number of missing larvae (never more than 5%) was calculated and distributed over the experiment as a proportion of actually observed mortality. Twice a week, length measurements were performed on 10 larvae from each replicate with a semi-automatic image analyser (VIDS, Systemes Analytiques, France). At the end of the experiment the remaining populations were anaesthetized with phenoxy ethanol and weighed in order to determine the final individual wet weight. All experimental diets were fed in duplicate and were based on the same casein-based diet (Table 1) . Lipids were added as an emulsion to the mixed dry ingredients, whereafter the moist blend was pelletized with a meat grinder. Diets were dried for 48 h in a ventilated oven at 40°C. A group fed a yeast-based diet (diet C, Expt. I) and a starved group (S,

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Table 1 Lipid supplementation Experiment I Diet Casein-based’ Yeast-based2 POr3 PL4 Choline’ Experiment II Diet Casein-based’ PO”6 PL’

to the semi-purified

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diets used in Experiments

305

I and II (8 dry weight)

A0

B,

A,

B,

A4

B,

BC

C

100 _

96 _

94

96

92

93.6 _

100

_ _

4 _

98 _ 2 -

4 2

_ 4

4 4

4 _

MAR

ovo

LYS

CA-S

SOY

SUN

94 3.6 2.4

94 3.2 2.8

94 3.3 2.7

94 3.3 2.1

94 3.3 2.1

94 3.1 2.9

PO 94 6

2.4

RAP 94 3.2 2.8

‘Casein-based diet: 42.5% casein Prolabo no. 22544292; 12.5% casein sodium salt Sigma C8654; 5% casein hydrolysate, Sigma C0626; 5% mineral premix, Luquet (1971); 25% dextrin, Sigma D2256; 10% vitamin premix, containing 5% inositol and 10% choline (50% pure), EIFAC (1971). *Control yeast-based diet: 80% yeast powder, Protibel, Bel, France; 5% casein hydrolysate; 5% mineral premix; 10% vitamin premix. ‘Peanut oil, Lesieur, France. ?-a-Phosphatidylcholine (PC), type XI-E, Sigma P9671 (from fresh-frozen egg yolk). ‘Choline chloride salt, Sigma C7017, approximate choline content: 60%. 6Peanut oil, Vandemoortele N.V., Belgium. ‘MAR = marine experimental phospholipids (CTPP, France) ; OVO = egg yolk PL, Ovothin 160, (Lucas Meyer, Germany) ; SOY, LYS, CA-S, SUN and RAP = respectively, de-oiled soybean lecithin (Vamothin F), de-oiled lyso-soybean lecithin, de-oiled Ca*’ -enriched soybean lecithin, de-oiled sunflower and de-oiled rapeseed lecithin (prepared by VAMO MILLS Protein & Lecithin Division, Belgium).

Expts. I and II) served as control groups. Table 1 gives an overview of the lipid composition of the semi-purified test diets. In Experiment I, the diets of the A-series (A,,, AZ, A4) contained 0% peanut oil (PO) and 0,2 and 4% PL (Sigma, from dried egg yolk) respectively, while diets of the B-series (B,, B2, B4) contained 4% PO and PL inclusion levels corresponding to those in the diets of the A-series. In the B-series a supplemental diet (B,) was formulated in which PL was replaced by choline chloride (2.4%).In the diets of Experiment II, the lipid level was kept constant by supplementing the PL sources with PO up to 6%. The PL sources of animal origin were a marine phospholipid extract (prepared by CTPP, France) and an egg yolk extract (Ovothin 160, Lucas Meyer, Germany). Vegetable PL sources were de-oiled lecithin from soybean, sunflower and rapeseed, an enzymatically ( phospholipase A,, hydrolysed soybean lecithin and soybean lecithin enriched with Ca*+ (equimolarrel a t’ive to the phospholipid content). They were prepared by VAMO MILLS Protein & Lecithin Division, Belgium. All PL sources of plant origin contained 100% acetone insolubles. Marine PL and Ovothin 160 contained 20 and 27% triglycerides, respectively. Lipids were extracted according to the procedure of Folch et al. ( 1957) and transesterified using a methanol/acetylchloride mixture (20: 1, volumetric). Fatty acid methyl ester determination was performed on a gas chromatograph (Carlo Erba Mega 5 160 HRGC) equipped

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with a fused silica capillary column (25 m X 0.32 mm, i.d.) with hydrogen as carrier gas. Details on the fatty acid analysis procedure are given in Coutteau and Sorgeloos ( 1995). Phospholipid class composition of the egg yolk lecithin, the basal casein diet and the control yeast diet was quantified with the Iatroscan thin-layer chromatography flame ionization detection system (Mark V, TLC-FID). Approximately 20 kg of total lipid was spotted (Hamilton syringe, 0.4 ~1) at the origin of each Chromarod (S-III type) and polar lipids were developed in chloroform/methanol/water (70:35:3.5, volumetric), whereafter they were dried at 100°C for 6 min. The FID was operated at an air flow of 0.2 l/min and a Hz pressure of 3 X lo5 Pa. For the identification of individual PL classes, a purified standard mixture of known composition and concentration was spotted on 4 rods accompanying the samples. Each sample was spotted on 3 rods. The determination of PL classes of the PL sources in Experiment II, except from the marine PL source (analysed by Iatroscan), was performed by high-performance thin-layer chromatography (HPTLC) followed by densitometry (Vandemoortele Coordination Center, R & D Department). One ~1 of a 20 mg/ ml solution of chloroform/methanol (2: 1, volumetric) of each sample was spotted together with a calibration mixture (the “ILPS mixed soy phospholipid standard”) on Whatman HP-K plates ( 10 X 10 cm) _After development of the plates in chloroform/acetone/methanol/acetic acid/water (50:20:5: 15:5.5, volumetric), they were dried for 30 min at 60°C. PL-spots were revealed by dipping the plates for 1 min in an aqueous solution containing 0.1% ANS ( 8-anilino- 1-naphthalene sulfonic acid, ammonium salt), whereafter they were dried again for 25 min at 40°C and scanned with a Desaga CD60 densitometer (remission/ fluorescence, wavelength: 367 nm) . Table 2 shows the composition in PL classes of the basal diet, the control yeast diet and the PL sources used. Total lipid content of the basal casein diet was 1.4% (dry diet basis) from which 12% consisted of polar lipids or approximately 0.15% on diet dry matter basis. Table 2 Selected phospholipid of total lipids) PL class*

class composition

Experiment

of total lipid from basal and control diet and dietary PL sources (weight%

Experiment

I

II

Casein based* *

Yeast based* *

PL

MAR

OVO

LYS

CA-S

SOY

SUN

RAP

31 30 n.d. n.d.

68 28 n.d. _ 21

44 3 n.d. 17 172

60 6 n.d. 31

3 2 10 2 33 13

20 19 14 9

22 21 14 6

22 12 15 4

24 13 16 4

Lyso Others* * *

5 6 n.d. n.d. _ 1

SUM PL

12

69

98

81

69

51

65

PC PE PI PA

2

33

21 65

n.d. = not detected. *See text for abbreviatons and specifications on lyso-PL. **Total lipid in casein-based diet: 1.4% and in yeast-based control diet: 7.5% (% dry matter). ***Unknown, unless marked with superscript: ‘sphingomyelin (SM), ‘6% phosphatidylserine SM. ‘N-acyl-PE (APE).

23 55

23 59

(PS) and 11%

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The control yeast diet contained 7.5% of lipids (dry diet basis). More than two thirds of the lipid included in the control yeast diet consisted of polar lipids with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as most important PL classes. PL sources in Experiment II were less pure (5 l-80%) than the egg yolk PL in Experiment I (98%). The latter contained 68% PC and 28% PE. The marine PL source (MAR) had 80% PL, with more than 60% contained in the PC (44%) and lyso-PC ( 17%) fraction and 11% sphingomyelin (SM). The predominant PL class in the OVO egg yolk was PC (60%). Apart from the higher PE content in the soybean PL, proportions of PL classes in vegetable PL sources showed a close resemblance to each other. The enzymatically prepared lyso-soybean (LYS) contained 14% lyso-PC, 12% lyso-PE and 6% lyso-phosphatidylinositol (lyso-PI) . The fatty acid (FA) composition of total lipid from the basal diet (&), the control diet (C) and the dietary lipid sources (PO and PL) are shown in Table 3. Both the basal diet and the yeast-based diet had very low levels of highly unsaturated fatty acids. Both egg yolk PL from Experient I and OVO in Experiment II had similar FA patterns, except for a slightly higher content of DHA (22:6n-3) in OVO. The FA composition of the peanut oil in Experiments I and II ( POL and POv) differed in percentages of oleic ( 18: ln-9) and linoleic ( 18:2n-6) acid. Marine PL had the highest levels of EPA (20:5n-3) and DHA. The major FA in the plant PL sources were palmitic ( 16:0), oleic and linoleic acids. Rapeseed lecithin (RAP) had the lowest level of linoleic acid and sunflower lecithin (SUN) had the lowest level of linolenic acid ( 18:3n-3). The percentage linoleic acid in the lyso-soybean PL (LYS) was lower than that of the original soybean PL (SOY), whereas the percentage of palmitic acid was increased. Statistical analyses were carried out with the help of a computer-assisted program (ITCF, 1988). Data were compared by the Neumann-Keuls test, after transformation log( weight), arcsin( survival) and length, without transformation, at the 0.05 significance level. Table 3 Selected fatty acid composition Experiment Basal

I

Control

14:o

9.8

1.0

16:O 18:O 20:o

30.8 9.3 -

14.3

16:ln-7

of basal and control diets and of dietary lipid sources (% total fatty acids)

1.7 _

Experiment POL 11.2 3.3 1.2

PL

PO”

II

MAR

ovo

LYS

CA-S

SOY

0.1

0.1

2.9

2.0

-

-

_

31.0 16.0 -

9.8 2.2 I.0

21.9 17.1 -

25.9 14.2 _

30.3 5.7 0.1

19.5 3.7 -

19.3 3.9 0.1

SUN

RAP

16.1 5.6 0.4

10.9 0.8 0.1

_

1.4

1.6

0.1

1.0

0.1

3.5

1.3

-

_

18:ln-9 20: 1n-9

25.3 0.6

48.6 0.3

59.2 1.0

26.4 0.2

40.0 1.6

0.9 6.1

29.0 0.2

10.3 -

9.3

8.7

10.5 _

45.1

18:2n-6 20:4n-6 22:5n-6

6.8 0.6 0.1

16.5 -

20.2 _ _

15.2 5.3 1.4

39.4 0.1 -

0.9 _

17.1 4.7

46.2 -

59.5

58.9

35.0 _

-

-

65.6 _ _

18:3n-3 20:5n-3 22:6n-3

0.8 1.2 1.5

1.8 0.1 -

0.2 _ -

0.1

0.3

8.2 _

0.3

6.7 _

-

_ 3.5

6.9

1.4

10.0 10.5

-

5.9 _ -

_

_

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3. Results In Experiment I, larvae fed the yeast-based control diet had the best final survival (95%), whereas total mortality in the starved groups had occurred by day 9 (Fig. 1). According to Table 4 Growth results’ for Experiment

I

Diet

s.d.’

Total length (mm) D7 Dll D14 D18 D21 D25

9.7ak 11.s* 12.9’ 15.2abc 17.2b 20.1bc

10.0” 11.3” 12.7” 15.6ab 16.5& 21.8b

9.7ab 11.6& 13.1& 16.2ab 18.6” 20.7be

10.la 12.08 13.6’ 16.7” 19.5” 24.2”

9.2’” lO.lb 10.9b 12.2d 13.1’ 15.0d

9.1’ 10.3b 11.3b 13.5&* 14.2e’ 16.5”

9.1bc 10.lb 10.9b 12.8=’ 15.1de 16.9’

47”

71C

83be

121ab

136=b

142”b

181’

2d

4=

3”

10”

13ab

12ab

16’

9.w 10.7b 11.P 14.3ab”d 15.P 18.0*

0.20 0.26 0.33 0.79 0.47 1.06

Wet weight (mg) D25

83be

12

8b

18

Final biomass, WXS D25

(8)

‘Means of two replicate groups. Within rows, means not sharing a common superscript letter are significantly different (P < 0.05). *s.d. = residual standard deviation between replicates within groups expressed in mm for length and % for logtransformed weight and biomass.

0

1111111:II.11111111II.~I~( 0

5

IO

IS

20

25

days of the experiment

Fig. 1. Daily survival in Experiment

I of carp larvae fed the experimental

diets and of the starved group

( S)

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Table 5 Growth results’ for Experiment Diet

309

131 (1995) 303-314

II

PO

MAR

ovo

LYS

CA-S

SOY

SUN

RAP

8.1’ 7.Sb 11.1”

9.2b 10.0a 12.6ab

9.4b 9.8” 15.1”

9.1b 9.5” 12.Sab

9.4b 10.3a 14.7a

10.la 1o.78 15.1s

9.1 10.8 15.7

9.2b 9.8” 14.08

27b

42ab

80”

46ab

67a

75”

80

62a

s.d.’

Total length (mm) DlO D14 D21

0.19 0.61 0.75

Wet weight (mg) D21

20

Final biomass, WXS D21

(9) 1.4b

0.5’

1.6b

2.9ab

5.5”

5.9”

5.7

4.2”b

1.6

‘Means of two replicate groups. Within rows, means not sharing a common superscript letter are significantly different (P < 0.05). Since for diet SUN one replicate was lost, data for SUN represent one replicate and were therefore not included in the statistical analyses. However, in a subsequent experiment, the same diets SUN and SOY as in Experiment II were fed to duplicate groups of carp larvae and supportedhigh survival and growth, without significant differences between the two diets. ‘s.d. = residual standard deviation between replicates within groups expressed in mm for length and % for logtransformed weight and biomass.

the overall survival throughout the 25day Experiment I, larvae fed the semi-purified diets can be clearly divided into two major groups, related to PL supplementation (diets A,, B,, A4 and B4) or PL deficiency (diets A,, B, and B,) . Final survival of groups fed the former diets varied between 81 and 92% as against final survival of 31-57% in the latter groups. The beneficial effect of PL addition on survival became significant from day 11 onwards, whereas the addition of 4% PL compared to 2% PL did not further improve survival (Fig. 1). Mortality was low from day 14 onwards. Growth of fish fed the control diet C was loo

20

PO

0 0

7

14

21

daysoftbeexpriment

Fig. 2. Daily survival in Experiment

II of carp larvae fed the experimental

diets and of the starved group (S)

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303-314

inferior to those which received the PL-supplemented diets. The growth-promoting effect of PL supplementation was significant on day 7 (Table 4). The addition of 2% PL to the PL-deficient diets of the A and B series increased the final weight by a factor of almost 3 and 2, respectively. The product of survival by weight (W X S or theoretical biomass) was 3-5 times higher for groups fed PL-enriched diets than of those fed the PL-deficient diets (Table 4). Results of Experiment II also confirmed the beneficial effect of adding PL to the purified larval diets (Table 5 and Fig. 2). Final survival of larvae fed the PL of plant origin (6882%) was significantly higher, except for the lyso-soybean PL, than for those fed the PLdeficient diet PO ( 19%) and the diets enriched with PL of animal origin (33-36%). After 14 days, total length was significantly affected by PL supplementation, whereas no differences were detected between groups fed the different PL-supplemented diets. In contrast to their final survival rate, which was similar to final survival of those fed PO and LYS, fish fed the OVO diet showed good growth performances as expressed in length and weight data. In the latter group, skeletal deformities also appeared by the end of the experiment. The final biomass was 5.5-5.9 g for fish fed diets SOY and Ca-enriched soy (CA-S), while the final biomass of the group fed the LYS diet was approximately 4 times lower. The final biomass of fish fed the PL-free diet (PO) was more than lo-fold lower than that of larvae fed diet SOY.

4. Discussion

Mortality of the yeast-fed and starved control groups were in accordance with previous results obtained with carp larvae under similar experimental conditions (Radiinz-Neto et al., 1994, 1995). The good match between daily mortality counting and final number of surviving larvae from all groups excluded cannibalism as a possible source of nutrients to the larvae. Experiment I clearly confirmed that addition of PL to semi-purified diets based on casein and dextrin was important to obtain good initial survival and growth of first-feeding carp larvae, as also observed by Radtinz-Neto et al. (1994) in carp and by Szlaminska et al. (1993) in goldfish. In a previous experiment, a diet with 2% PL, identical to diet A,, provided better larval performance than a diet with only 1% PL (Radiinz-Neto et al., 1995). Further increasing of the PL level to 4% improved final weight after 25 days, but did not significantly increase survival or growth during the first 10-14 days. With larvae of rockbream (Oplegnathus fasciatus) fed casein-based diets with graded PL levels, Kanazawa ( 1993) also found that the addition of 7% of soya lecithin (containing 53% PL) did not provide better rearing results than 5% PL supplementation, which was considered the optimum level. Experiment II showed that several PL sources, more particularly of plant origin, also promoted better survival and growth than a PL-deficient diet. The beneficial effect of diet supplementation with soybean lecithin had been reported earlier for sturgeon juveniles (Hung et al., 1987). However, for the latter species, the initial hypothesis of a requirement for dietary lecithin was subsequently rejected because the initial experimental diets were

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311

deficient in choline and since addition of choline duplicated the lecithin effect (Hung and Lutes, 1988). In the present case, the observed beneficial effect of PL did not seem to be due to the correction of a choline deficiency, as the experimental test diets contained an appreciable amount of choline ( 10 g choline chloride or 5 g choline per kg diet dry weight, Table 1), which was more than 3 times as high as the estimated requirement for juvenile carp (Ogino et al., 1970; NRC, 1993). In Experiment I, a further increase in the choline chloride level up to about 3.4% on a dry weight basis (diet B,) did not improve survival or growth of the larvae compared to the 1% choline chloride diet (diet B,) and, consequently, did not replace the observed PL effect (diet B, and diet B4). Likewise, the inositol level in the larval diets was relatively high (0.5% on a diet dry weight basis, Table 1) and therefore it seemed unlikely that the beneficial effect of the plant PL sources (Expt. II) was related to the provision of inositol as such. In the present study, no relationship was observed between the essential fatty acid (EFA) content of the added PL sources and their efficiency in promoting larval performances. Previous experiments have shown that carp larvae, in contrast to marine fish larvae, are able to survive and grow well on diets with a n-3 FA as low as 0.05-O. 1% on a dry diet basis, without significant improvement by supplementing the diets with methyl linolenate or cod liver oil (Radiinz-Neto et al., 1995). Experiment II of the present study confirmed this observation as the SUN diet, which had an even smaller amount of n-3 FA than the abovementioned level, allowed good larval performance, contrary to the MAR diet, which is relatively rich in n-3 HUFA. Kanazawa et al. ( 1981) similarly found that soybean lecithin, poor in n-3 HUFA, gave results that were slightly superior to chicken egg lecithin in ayu larvae. Also, in the case of marine fish larvae, the effect of PL (PC and PE) did not appear to be related to provision of EFA (Takeuchi et al., 1992). With regard to n-6 FA, the requirement was estimated at approximately 1% linoleic acid on total dry weight of diets for carp juveniles (Takeuchi and Watanabe, 1977) and for first-feeding carp larvae (Radtinz-Neto et al., 1995). Since almost all presently tested diets (B-series Expt. I and diets Expt. II) contained a high amount of 18:2n-6, derived from the added peanut oil, there was no n-6-FA deficiency to be expected. The observation that the PL effect was not related to a specific nutritional constituent leads to the idea of a specific role of the PL entity itself for fish larvae. PL are known to act as emulsifiers in an aquaeous environment such as the intestinal lumen. According to Kanazawa ( 1993) they could be essential in allowing the absorption of dietary neutral lipids such as cholesterol and triglycerides, as found in crustacean larvae. The possible function of dietary lecithin as emulsifier has also been suggested by Koven et al. ( 1993) in seabream larvae to explain a higher incorporation of labelled FA in larval tissue. The explanation of the PL effect by their emulsifying function was not supported by the present results (diets of the A-series). In Experiment I, PL proved to have a beneficial effect both in the diets containing peanut oil (B-diets) and in those without oil supplementation with a very low triglyceride content (A-diets). Moreover, in Experiment II, the emulsifying properties of the PL sources were not in relation to fish performance: Ca2+ enrichment of the soybean PL, which decreases the emulsifying properties (diet CA-S), gave similar results as diet SOY, whereas diet LYS, containing a better oil/water emulsifier than diet SOY, gave poorer results. Possibly, inferior rearing results of carp fed the marine PL source

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could be related to its relatively high content of lyso-PC. On the one hand, it could be expected that lyso-soybean PL, obtained by phospholipase A,, would be more effective than non-hydrolyzed soybean lecithin, since sn-1-acyl-lyso-PL is thought to be the most abundant natural hydrolytic product of PL in the fish intestinal lumen. On the other hand, the existence of a phospholipase A2 in fish has been questioned (Sargent et al., 1994). Whether the strong effect of PL supplementation in the present study could be directly attributed to improvement of the physicochemical properties of the food particles, by reducing the leaching of water-soluble nutrients or by preventing oxidation, is doubtful. The food delivery was continuous and the risk of oxidation was low since the experimental feed had a very low HUFA content and high levels of vitamins, including vitamin C and vitamin E, and since they were prepared only 2 days before the start of the experiment whereafter they were kept at 4°C prior to feeding. So far, another hypothesis remains to be considered, more particularly, the limited ability of fish larvae to synthesize PL at a sufficient rate to fulfil the demand for building and renewal of cellular membranes (Kanazawa, 1993). Potential specific growth rates and protein synthesis rates are much higher in larvae than in juvenile or adult carp (Fauconneau, 1984). It may be speculated that formation of lipoproteic assemblages such as membranes and circulating lipoproteins have a high requirement for rapid PL synthesis, which could possibly exceed the larval capabilities in the absence of dietary supply. The observation of mortality in the present study suggested that larval sensitivity to PL deficiency is restricted to the first 2-3 weeks of feeding, in accordance with previous results (Radiinz-Neto et al., 1994). The temporary aspect of the need for dietary supply of PL is also supported by the observations that PL supplementation had a more marked effect in lo-day larval ayu than in lOO-day-old ayu (Kanazawa et al., 198 1) and that PL were shown to be non-essential in juvenile ( 10-30 g) white sturgeon (Hung and Lutes, 1988). It is noteworthy that fish larvae, during embryonic development and subsequent stages in their natural feeding habitat, always have different types of PL classes at their disposal. Those PL originate from the egg reserves (Fraser et al., 1988) and later from the live food organisms (Teshima et al., 1987). Under artificial conditions, problems arise when larvae are offered formulated diets with a PL content that might be inadequate in quantity and/or quality. The present work has shown that several PL sources in casein-based diets can improve initial survival and growth, but precise requirement and optimal supply, in terms of PL classes and possibly fatty acid composition, remain unclear. The use of purified PL and PLclasses in further studies could help to solve some problems. For instance, it is not clear from the present results whether the provision of a single PL class is sufficient to meet the requirements or whether the supply of a mixture of PL classes, similar to the PL class composition of fish larvae, is more effective.

Acknowledgements We wish to thank Ir. J. De Kock (Vandemoortele N.V., Group R&D Center, Izegem, Belgium) for the preparation and analysis of the phospholipid sources of plant origin; P. Bouchez (CTPP, Boulogne-sur-Mer, France) for the preparation of the marine phospholipid

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extract and B. Gaeremynck (Lucas Meyer, Oudenaarde, Belgium) for providing Ovothin 160. This work was partially supported by the Conselho National de Desenvolvimento Cientifico et Tecnologico, Brazil (Grant 200069/89 VT/DF), the RCgion Aquitaine, France, and the Belgian National Science Foundation.

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