# Relationship between dietary phospholipid classes and neutral lipid absorption in newly-weaned turbot, shape Scophthalmus maximus

Inge I. Geurden, Pierre Bergot, L. Schwarz, P. Sorgeloos

► To cite this version:

Inge I. Geurden, Pierre Bergot, L. Schwarz, P. Sorgeloos. Relationship between dietary phospholipid classes and neutral lipid absorption in newly-weaned turbot, shape Scophthalmus maximus. Fish Physiology and Biochemistry, 1998, 19 (3), pp.217-228. 10.1023/A:1007723515204. hal-02690818

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# Relationship between dietary phospholipid classes and neutral lipid absorption in newly-weaned turbot, *Scophthalmus maximus*

I. Geurden¹, P. Bergot², L. Schwarz¹ and P. Sorgeloos¹

¹Laboratory for Aquaculture & Artemia Reference Center, University of Ghent, Rozier 44, B-9000 Gent, Belgium
(Fax: +32-9 2644193; E-mail: inge.geurden@rug.ac.be); ²Unité mixte INRA-IFREMER de Nutrition des Poissons, Station d'Hydrobiologie, F-64310 Saint-Pée-sur-Nivelle, France

**Key words:** phospholipid classes, phosphatidylcholine, HUFA, DHA, marine fish, turbot

## Abstract

A 28-day feeding trial was conducted for comparing the effect of different dietary phospholipid (PL) classes on the growth of post-larval turbot and on the incorporation of dietary neutral lipid fatty acids into their body lipids. Prior to the experiment the turbot were weaned for one week on a PL-free diet. The nine experimental diets were isolipidic and contained an equal amount of highly unsaturated fatty acids in the form of ethyl esters. They differed by their PL content (0, 1 or 2%) and by the PL class composition of the added soybean PL fractions.

Compared to the PL-free diet, diets enriched with phosphatidylcholine (PC) resulted in a better growth, a higher triglyceride content (% body dry matter) and increased levels of docosahexaenoic acid (% total fatty acids) in each of the examined body lipid classes (neutral lipid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol). The effects of the other soybean PL fractions were less explicit than those noted for soybean PC.

The results support the idea that dietary PC plays a role in the intestinal absorption of neutral lipid fatty acids. This might, at least partially, explain the superiority of PC for enhancing growth.

**Abbreviations:** DHA – docosahexaenoic acid (22:6n-3); EPA – eicosapentaenoic acid (20:5n-3); HUFA – highly unsaturated fatty acid; PA – phosphatidic acid; PC – phosphatidylcholine; PE – phosphatidylethanolamine; PI – phosphatidylinositol; PL – phospholipid; PS – phosphatidylserine; PUFA – polyunsaturated fatty acid.

## Introduction

Considerable attention has been paid to n-3 highly unsaturated fatty acids (HUFA) in nutritional studies on marine fish larvae (Watanabe et al. 1983; Izquierdo et al. 1989; Watanabe 1993). It is usually accepted that marine fish have a poor elongation-desaturation capacity and that the provision of EPA (20:5n-3) and DHA (22:6n-3), instead of shorter chain n-3 fatty acids, is necessary to meet the requirement (Cowey et al. 1976; Sargent et al. 1993). The same applies for the fatty acids of the n-6 series, as 18:2n-6 can not be transformed in 20:4n-6 (Bell et al. 1994; Castell et al. 1994).

The provision of n-3 HUFA to marine fish larvae is mostly performed by enriching the live preys (*Brachionus* and *Artemia*) with neutral lipid rich in these HUFA (Léger et al. 1987; Dhert et al. 1990; McEvoy et al. 1996). Also the weaning diets of marine fish are supplemented with n-3 HUFA-rich lipid sources. A concentrated form consists of fatty acid ethyl esters prepared from fish oil (Coutteau et al. 1995), which were demonstrated to result in a DHA level in the fish proportional to that in the diet (Coutteau et al. 1996). Recently, we established that for a similar supply of DHA ethyl esters by the diet, the DHA level in the fish was increased by supplementing the weaning diet with phospholipids (PL), either of vegetable or of animal origin (Geurden et al. 1997b, c).
The aim of the present study was to compare the
efficiency of different PL classes for enhancing the
incorporation of HUFA presented in the diet as neu-
tral lipid. For this purpose, diets having the same
amount of HUFA ethyl esters were supplemented with
PL fractions with very different lipid class compo-
sition and fed to newly-weaned turbot. These PL
fractions were prepared from soybean PL and were
consequently free of HUFA. Assuming the absence
of significant elongation-desaturation activities, the
capacity of each dietary PL fraction to increase the
incorporation of the dietary HUFA into the fish was
directly assessed by the level of these HUFA in fish
lipid. Several body lipid classes (NL, PC, PE, PI)
were examined since they respond differently to di-
etary fatty acids, as shown *in vitro* and *in vivo* (Tocher
and Mackinlay 1990; Bell et al. 1994). Furthermore,
the growth-promoting effect of the various experimen-
tal PL fractions was compared and eventual changes in
the lipid class composition of the turbot in relation to
that of the diet were examined.

**Materials and methods**

Fish and experimental design

Unweaned 36-day old turbot, *Scophthalmus maximus*,
obtained from Ferme Marine de Douhet (France),
were stocked for one week in two 1 m³ tanks for ac-
climation to the laboratory conditions. During the first
4 days the larvae were weaned by gradually dimin-
ishing the number of freshly-hatched *Artemia* nauplii
(EG grade, INVE Aquaculture N.V., Belgium) and in-
creasing the amount of weaning diet, which was fed in
excess for the remaining acclimation period.

At the age of 43 days, the larvae were randomly
stocked in the recirculating rearing system (Coutteau
et al. 1995) at a density of 67 ind. per 30 l tank. From
the first day of stocking, each experimental diet was
given to triplicate groups by means of automatic feed-
ers (Charlon and Bergot 1984) delivering food every
30 min throughout the artificial 12-h light period.
The feeding experiment lasted 28 days and general
rearing conditions were as previously given (Geurden
et al. 1997b). Temperature, salinity and ammonia-
N averaged around 19 °C, 32 g l⁻¹ and 0.3 mg l⁻¹,
respectively.

Experimental diets

The nine experimental diets were prepared accord-
ing to Coutteau et al. (1995). The common extruded
basal diet (92.5% diet) has been described previously
(Geurden et al. 1997b) and was coated with different
lipid fractions (7.5% diet). These fractions contained
equal amounts of the fish oil ethyl ester concentrate
providing the HUFA, but differed by their PL compo-
nents (Table 1). Hydrogenated coconut oil was used
for rendering the diets isolipidic. All PL-sources were
derived from de-oiled soybean PL, containing glycol-
ipids as major impurity, and their PL content varied
between 52 and 100% (% PL-source). The negative
control diet D1 was not supplemented with PL. The
other diets contained 2% total PL, except diets D2 and
D7 which contained 1% PL. According to the PC-
content (diet weight%), 3 categories of diets can be
distinguished: PC-free (D1), PC-poor (D2-D5, <0.5%
PC) and PC-rich (D6-D9, > 0.5% PC) diets. The PC-
poor diet D2 contained PI-enriched PL (100% PL),
diets D3, D4, D5 and D8 were supplemented with PL-
fractions enriched up to about 50% of their PL-content
in PA, PI, PE and PC, respectively. Diet D6 contained
the original soybean PL blend. Diets D7 and D9 con-
tained respectively 1 and 2% highly purified soybean
PC.

The diet particle size was 500-800 µm. A smaller-
sized (300-500 µm) diet with identical lipid compo-
sition as diet D1 served as weaning diet. The PL
composition of the diets shown in Table 1 is calculated
from analytical data of the PL-source (Vandemoortele
N.V., Belgium; Lucas Meyer GmbH, Germany; INRA
Laboratoire de Biochimie et Technologie des Pro-
téines, France) and does not take into account the 0.2%
PL included in the basal diet (Geurden et al. 1997b).

Sampling

The final survival was calculated from daily mortal-
ity percentages based on the actual number of fish in
each tank. For the initial wet weight determination 60
fish were sampled. The intermediate growth data were
based on a weekly sample of 5 fish taken randomly
in each of the 3 replicate dietary treatment groups and
final weight on a sample of 25 fish from each group.
The fish were starved for 16 h prior to sampling, dried
on a paper towel, weighed individually and frozen
at -40 °C. Dry weight was determined on freeze-
dried samples, corrected for residual water content
obtained by placing subsamples (100 mg) of homoge-
nized freeze-dried fish in an oven (60 °C) for 24 h. The
<table><caption>**Table 1.** Formulation of the variable part of the coated lipid fractions<fnref n="1" /> and the phospholipid (PL) composition of the experimental diets²</caption><thead><tr><th>Diet</th><th>D1</th><th>D2</th><th>D3</th><th>D4</th><th>D5</th><th>D6</th><th>D7</th><th>D8</th><th>D9</th></tr><tr><th>Major diet PL<sup>3</sup></th><th>-</th><th>PI</th><th>PA</th><th>PI</th><th>PE</th><th>SL</th><th>PC</th><th>PC</th><th>PC</th></tr></thead><tbody><tr><td colspan="10">Coated lipid fraction (% diet)</td></tr><tr><td>PL-source<sup>4</sup></td><td>-</td><td>1.0</td><td>3.7</td><td>3.5</td><td>2.5</td><td>3.2</td><td>1.0</td><td>3.1</td><td>2.1</td></tr><tr><td>Hydrogenated oil<sup>5</sup></td><td>4.4</td><td>3.4</td><td>0.7</td><td>1.0</td><td>1.9</td><td>1.3</td><td>3.4</td><td>1.4</td><td>2.4</td></tr><tr><td>Fish oil EE<sup>6</sup></td><td>2.6</td><td>2.6</td><td>2.6</td><td>2.6</td><td>2.6</td><td>2.6</td><td>2.6</td><td>2.6</td><td>2.6</td></tr><tr><td colspan="10">PL content (mg g<sup>-1</sup> diet)</td></tr><tr><td>PC</td><td>-</td><td>0.3</td><td>1.1</td><td>1.7</td><td>4.9</td><td>6.6</td><td>9.8</td><td>12.0</td><td>19.6</td></tr><tr><td>PE</td><td>-</td><td>2.0</td><td>4.4</td><td>5.0</td><td>9.6</td><td>6.3</td><td>0.1</td><td>6.5</td><td>0.2</td></tr><tr><td>PI</td><td>-</td><td>6.4</td><td>4.1</td><td>11.2</td><td>2.9</td><td>4.5</td><td>-</td><td>0.6</td><td>-</td></tr><tr><td>PA</td><td>-</td><td>0.8</td><td>10.2</td><td>1.8</td><td>0.8</td><td>2.0</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Total PL<sup>7</sup></td><td>-</td><td>10</td><td>20</td><td>20</td><td>20</td><td>20</td><td>10</td><td>20</td><td>20</td></tr></tbody></table>

homogenized fish powder was pooled according to the dietary treatment groups and stored under vacuum at -40 °C for further analysis.

## Lipid analysis

The fatty acid composition of the diets was determined by a direct esterification following Lepage and Roy (1984), using 23:0 as internal standard. Turbot whole body lipid was extracted from triplicate subsamples according to Folch et al. (1957). The lipid extracts were dried under nitrogen, weighed, dissolved at a concentration of 10 mg ml⁻<fnref n="1" /> chloroform / methanol (2:1, v/v plus 0.01%, w/v butyl hydroxytoluene) and stored at -20 °C.

The lipid composition of the tissues was analyzed using unidimensional, double development, high-performance thin-layer chromatography (HPTLC) according to Olsen and Henderson (1989). The 10 × 10 cm silica gel 60 plates were predeveloped in hexane: diethylether (1:1) for the removal of impurities whereafter they were dried for 30 min at 110 °C. Approximately 30 µg of lipid extract was spotted on

the 2 mm spot sites. The polar development system consisted of methyl acetate: isopropanol: chloroform: methanol: 0.25% aqueous KCl (25:25:25:10:9) and the subsequent neutral system of hexane: diethylether: acetic acid (85:15:1.5). After development, the plates were dried under vacuum, sprayed with 3% (w/v) cupric acetate in 8% (v/v) phosphoric acid and charred for 20 min at 160 °C. The plates were scanned by a Sharp JX-325 color scanner (reflection mode). The optical density reading of the scanner was calibrated according to a grey-value reference standard (Kodak). The scanner was connected to image analysis software (ImageMaster, Pharmacia Biotech, the Netherlands) which performed the peak integration (surface × optical density). Identification of peaks was based on standards (Avanti Polar Lipids, Inc., USA).

For the preparation of the fatty acid methyl esters of fish PC, PE, PI and NL, approximately 2.5 mg of lipid was applied on 20 × 20 cm glass plates coated with silica gel 60. The classes were separated in the same polar solvent mixture as for HPTLC. The spots were visualized under UV light after spraying the plates with 0.1% 2'-7' dichlorofluorescein in 97%

<footnote n="1">¹ The total coated fraction comprised 7.5% of the diet weight. Additional components were: 0.5% emulsifier (INVE Aquaculture N.V., Belgium), 0.02% Vit E (dl-alfa-tocopherol acetate, Roche N.V., Belgium) and 0.01% ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinolin, Sigma, E-8260); ² Theoretical values, calculated from weight% composition PL-source; ³ PI: phosphatidylinositol, PA: phosphatidic acid, PE: phosphatidylethanolamine, SL: soybean lecithin with PC and PE as major PL classes, PC: phosphatidylcholine; ⁴ All PL-sources are derived from de-oiled soybean PL. D2: purified PI-enriched soybean PL (100% PL) (INRA, Laboratoire de Biochimie et Technologie des Protéines, Nantes, France); D3, D4, D5, D8: de-oiled soybean lecithin enriched in PA (54% PL), PI (57% PL), PE (79% PL) and PC (66% PL), respectively, and D6: de-oiled soybean lecithin (63% PL, Vamothin F1) (Vandemoortele N.V., Belgium), D7, D9: highly purified soybean PC (Epikuron 200, 97% PC) (Lucas Meyer GmbH, Germany); ⁵ Hydrogenated coconut oil, Cocos 32/43 (Vandemoortele N.V., Belgium); ⁶ Concentrate of ethyl esters containing approximately 50% n-3 HUFA (INVE Aquaculture N.V., Belgium); ⁷ Total includes lyso PC and N-acyl PE.</footnote>
<table><caption>Table 2. Fatty acid composition of the experimental diets (mg g⁻<fnref n="1" /> dry diet) <fnref n="1" /></caption><thead><tr><th>Diet<br>Major diet PL</th><th>D1<br>-</th><th>D2<br>PI</th><th>D3<br>PA</th><th>D4<br>PI</th><th>D5<br>PE</th><th>D6<br>SL</th><th>D7<br>PC</th><th>D8<br>PC</th><th>D9<br>PC</th></tr></thead><tbody><tr><td>14:0</td><td>14.7</td><td>12.7</td><td>9.1</td><td>9.5</td><td>10.5</td><td>9.8</td><td>12.8</td><td>9.5</td><td>10.8</td></tr><tr><td>16:0</td><td>14.5</td><td>13.8</td><td>15.7</td><td>13.5</td><td>13.3</td><td>14.8</td><td>12.6</td><td>13.2</td><td>15.9</td></tr><tr><td>16:1n-7</td><td>0.6</td><td>0.5</td><td>0.6</td><td>0.6</td><td>0.5</td><td>0.6</td><td>0.5</td><td>0.6</td><td>0.7</td></tr><tr><td>18:0</td><td>11.2</td><td>10.2</td><td>8.6</td><td>9.0</td><td>8.4</td><td>9.2</td><td>10.5</td><td>8.3</td><td>10.4</td></tr><tr><td>18:1n-9</td><td>6.4</td><td>6.1</td><td>7.8</td><td>8.0</td><td>7.7</td><td>8.1</td><td>6.1</td><td>7.9</td><td>7.8</td></tr><tr><td>18:1n-7</td><td>0.6</td><td>0.6</td><td>0.9</td><td>1.0</td><td>1.0</td><td>0.9</td><td>0.9</td><td>1.0</td><td>0.8</td></tr><tr><td>18:2n-6</td><td>4.2</td><td>6.9</td><td>15.5</td><td>12.3</td><td>12.5</td><td>14.6</td><td>8.5</td><td>15.6</td><td>13.0</td></tr><tr><td>18:3n-3</td><td>0.4</td><td>0.6</td><td>1.8</td><td>1.3</td><td>1.2</td><td>1.8</td><td>0.9</td><td>1.5</td><td>2.0</td></tr><tr><td>20:0</td><td>0.2</td><td>0.2</td><td>0.3</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.2</td></tr><tr><td>20:1n-9</td><td>0.6</td><td>0.5</td><td>0.5</td><td>0.6</td><td>0.6</td><td>0.6</td><td>0.6</td><td>0.6</td><td>0.5</td></tr><tr><td>20:4n-6</td><td>0.3</td><td>0.3</td><td>0.3</td><td>0.4</td><td>0.4</td><td>0.4</td><td>0.3</td><td>0.4</td><td>0.3</td></tr><tr><td>20:5n-3</td><td>5.5</td><td>5.3</td><td>5.4</td><td>5.3</td><td>5.4</td><td>5.5</td><td>5.1</td><td>5.2</td><td>5.3</td></tr><tr><td>22:5n-6</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.3</td><td>0.2</td><td>0.3</td><td>0.2</td><td>0.3</td><td>0.2</td></tr><tr><td>22:5n-3</td><td>1.2</td><td>1.2</td><td>1.2</td><td>1.1</td><td>1.2</td><td>1.3</td><td>1.1</td><td>1.2</td><td>1.2</td></tr><tr><td>22:6n-3</td><td>12.4</td><td>12.2</td><td>11.9</td><td>11.4</td><td>12.0</td><td>12.4</td><td>11.5</td><td>11.6</td><td>11.9</td></tr><tr><td>Saturates</td><td>40.6</td><td>37.0</td><td>33.8</td><td>32.4</td><td>32.6</td><td>34.1</td><td>36.3</td><td>31.4</td><td>37.4</td></tr><tr><td>Monoenes</td><td>8.4</td><td>8.0</td><td>10.1</td><td>10.3</td><td>9.9</td><td>10.5</td><td>8.3</td><td>10.2</td><td>10.2</td></tr><tr><td>n-6 PUFA</td><td>4.9</td><td>7.5</td><td>16.0</td><td>12.8</td><td>13.2</td><td>15.2</td><td>9.1</td><td>16.3</td><td>13.5</td></tr><tr><td>n-3 PUFA</td><td>20.1</td><td>19.9</td><td>21.2</td><td>20.1</td><td>20.4</td><td>21.8</td><td>19.3</td><td>20.2</td><td>20.4</td></tr><tr><td>n-3 / n-6</td><td>4.1</td><td>2.7</td><td>1.3</td><td>1.6</td><td>1.6</td><td>1.4</td><td>2.1</td><td>1.2</td><td>1.5</td></tr></tbody></table>

ethanol. The classes were scraped off and transmethylated overnight at 50 °C (Christie 1982). The resulting methyl esters were analyzed by on column injection in a Chrompack CP 9001 gas chromatograph (GC) with H₂ as carrier gas. The temperature was programmed to raise from 85 (injection) to 150 °C at 30 °C per min, from 150 to 152 °C at 0.1 °C per min, from 152 to 172 °C at 0.65 °C per min and further from 172 to 187 °C at 25 °C per min. The final temperature of 187 °C was maintained for 7 min. The GC was equipped with a 2.5 m methyl deactivated precolumn connected to a 50 m × 0.32 mm i.d. polar capillary column (BPX70, SGE, Australia). Peak identification was based on reference standards (Nu-Check Prep. Inc, USA).

## Statistical analysis

Survival, weight, fish total lipid content and fish lipid class and HUFA composition were subjected to a one-way analysis of variance (ANOVA) for evaluating the effect of the dietary PL composition. Only the HUFA were considered in the ANOVA as these fatty acids were supplied in equal amounts by the different diets.

For testing the effects of diet, lipid class and sampling period a three-way ANOVA was performed on the HUFA (log transformed) detected in NL, PC and PE for both day 7 and day 28. HUFA levels in PI were available only for day 28 and were compared in a separate one-way ANOVA. When a significant (p<0.05) difference was evidenced by the ANOVA, data were further analyzed by Duncan's multiple range test (Statistica, StatSoft Inc.).

## Results

### Dietary fatty acid composition

Table 2 summarizes the fatty acid composition of the diets. They all contained similar amounts of essential HUFA, related to the equal amount of fish oil ethyl esters. DHA accounted for 1.1-1.2%, EPA for 0.5% and 20:4n-6 for 0.3-0.4% of diet dry weight. Variations between the diets were noted for 18:2n-6 and 18:3n-3, depending on the soybean PL added to the diets. The amount of saturated fatty acids varied according to the level of hydrogenated coconut oil. The

<footnote n="1">¹ Data represent means of duplicate esterifications and sums include minor fatty acids not listed in the table.</footnote>
<table><caption>Table 3. Survival (%), weight (mg), lipid content and lipid class composition of turbot at the start (initial) and at the end of the 28-day feeding trial<fnref n="1" /></caption><thead><tr><th>Diet<br>Major diet PL</th><th>Initial</th><th>D1</th><th>D2<br>PI</th><th>D3<br>PA</th><th>D4<br>PI</th><th>D5<br>PE</th><th>D6<br>SL</th><th>D7<br>PC</th><th>D8<br>PC</th><th>D9<br>PC</th></tr></thead><tbody><tr><td>Survival (%)</td><td></td><td>87</td><td>90</td><td>93</td><td>87</td><td>91</td><td>90</td><td>85</td><td>92</td><td>99</td></tr><tr><td>Wet weight (day 7)</td><td>210</td><td>364</td><td>404</td><td>374</td><td>420</td><td>391</td><td>448</td><td>397</td><td>440</td><td>443</td></tr><tr><td>Wet weight (day 28)</td><td>210</td><td>930<sup>d</sup></td><td>110<sup>cd</sup></td><td>1030<sup>cd</sup></td><td>1230<sup>bc</sup></td><td>1210<sup>bc</sup></td><td>1240<sup>bc</sup></td><td>1410<sup>ab</sup></td><td>1310<sup>abc</sup></td><td>1460<sup>a</sup></td></tr><tr><td>Dry weight, DW (day 28)</td><td>36.4</td><td>161<sup>d</sup></td><td>188<sup>cd</sup></td><td>176<sup>cd</sup></td><td>214<sup>bc</sup></td><td>215<sup>bc</sup></td><td>223<sup>b</sup></td><td>254<sup>a</sup></td><td>242<sup>ab</sup></td><td>268<sup>a</sup></td></tr><tr><td>Total lipid (% DW)</td><td>10.3</td><td>10.7<sup>d</sup></td><td>12.4<sup>b</sup></td><td>12.6<sup>b</sup></td><td>13.4<sup>ab</sup></td><td>11.7<sup>c</sup></td><td>13.6<sup>a</sup></td><td>13.8<sup>a</sup></td><td>13.8<sup>a</sup></td><td>14.2<sup>a</sup></td></tr><tr><td colspan="11">Total lipid composition of turbot whole body (% total lipid)</td></tr><tr><td>Total neutral lipid</td><td>47.9</td><td>48.0<sup>c</sup></td><td>52.7<sup>a</sup></td><td>51.0<sup>b</sup></td><td>49.3<sup>b</sup></td><td>49.5<sup>b</sup></td><td>52.5<sup>a</sup></td><td>51.4<sup>b</sup></td><td>53.1<sup>a</sup></td><td>53.4<sup>a</sup></td></tr><tr><td>Triglyceride</td><td>20.9</td><td>22.0<sup>d</sup></td><td>27.0<sup>ab</sup></td><td>22.9<sup>c</sup></td><td>26.3<sup>b</sup></td><td>24.8<sup>b</sup></td><td>27.5<sup>a</sup></td><td>25.5<sup>b</sup></td><td>27.9<sup>a</sup></td><td>28.1<sup>a</sup></td></tr><tr><td>Cholesterol</td><td>14.6</td><td>13.2<sup>b</sup></td><td>13.1<sup>b</sup></td><td>14.4<sup>a</sup></td><td>11.9<sup>c</sup></td><td>12.2<sup>c</sup></td><td>11.9<sup>c</sup></td><td>12.2<sup>c</sup></td><td>12.2<sup>c</sup></td><td>11.8<sup>c</sup></td></tr><tr><td>Free fatty acid</td><td>2.2</td><td>2.1</td><td>2.1</td><td>2.3</td><td>1.9</td><td>3.0</td><td>2.9</td><td>3.0</td><td>3.0</td><td>2.5</td></tr><tr><td>Sterol ester</td><td>10.2</td><td>10.7</td><td>10.6</td><td>11.4</td><td>9.3</td><td>9.5</td><td>10.2</td><td>10.7</td><td>10.0</td><td>11.0</td></tr><tr><td>Total polar lipid<sup>2</sup></td><td>52.1</td><td>52.0<sup>a</sup></td><td>47.3<sup>c</sup></td><td>49.0<sup>b</sup></td><td>50.7<sup>b</sup></td><td>50.5<sup>b</sup></td><td>47.5<sup>c</sup></td><td>48.6<sup>b</sup></td><td>46.9<sup>c</sup></td><td>46.5<sup>c</sup></td></tr><tr><td colspan="11">Polar lipid composition of turbot whole body (% total identified PL)</td></tr><tr><td>Phosphatidylcholine<sup>3</sup></td><td>50.9</td><td>50.3</td><td>49.2</td><td>51.6</td><td>49.8</td><td>49.0</td><td>51.0</td><td>48.9</td><td>49.9</td><td>50.2</td></tr><tr><td>Phosphatidylethanolamine</td><td>26.3</td><td>26.0</td><td>26.0</td><td>26.2</td><td>25.1</td><td>26.3</td><td>25.8</td><td>25.7</td><td>27.5</td><td>27.6</td></tr><tr><td>Phosphatidylserine</td><td>9.4</td><td>10.6</td><td>11.2</td><td>9.9</td><td>10.4</td><td>10.8</td><td>10.2</td><td>11.1</td><td>9.5</td><td>9.4</td></tr><tr><td>Phosphatidyinositol</td><td>7.2</td><td>7.0</td><td>7.7</td><td>6.9</td><td>8.4</td><td>7.8</td><td>6.9</td><td>8.1</td><td>7.0</td><td>7.6</td></tr><tr><td>Phosphatidic acid<sup>4</sup></td><td>6.2</td><td>6.1</td><td>5.9</td><td>5.5</td><td>6.3</td><td>6.1</td><td>6.1</td><td>6.2</td><td>6.2</td><td>5.2</td></tr></tbody></table>

total lipid content of the diets was 12.5 ± 0.3% of diet dry weight.

## Growth, lipid content and lipid class composition of turbot

The principal rearing characteristics of the turbot are shown in Table 3. Final survival was high (85-99%), without significant difference among the various treatment groups. The wet body weight of the turbot increased by a factor 1.7-2.1 after 7 days. After 28 days this factor was comprised between 4.4 and 6.9, depending on the diet. As compared to the PL-free diet D1, all PL-supplemented diets, except diets D2 and D3, resulted in a significantly higher final weight. The best growth was obtained with the diets containing the highest levels of PC (D7, D8, D9), without difference between a 1 (D7) or 2% (D9) dietary PC supply. Turbot fed the PC-rich phospholipid fractions also contained more total lipid per unit of dry matter

and more triglycerides in their body lipids than those fed the PL-free diet. When calculating the amount of triglycerides per individual fish, it was found that fish fed the PC-rich diet D9 had almost three-fold the amount of triglycerides found in fish fed the PL-free diet D1 (11 mg vs. 4 mg per fish). Regarding the fish polar lipid components, PC and PE were the major PL classes together accounting for about 75% of total body PL. The polar lipid class composition of the fish, expressed as a percentage of total body PL, was very uniform in all dietary groups and did not reflect the differences in polar lipid composition of the diets.

## Fatty acid composition of initial turbot

Table 4 details the fatty acid (FA) profiles in neutral lipid (NL), PC, PE and PI of whole turbot at the start of the experiment. Initial PC and PI were the most saturated with 16:0 and 18:0 as most abundant saturated FA, respectively. PE was the less saturated and con-

<footnote n="1">¹ Mean values (n=3 for rearing results and total lipid, n=2 for lipid class compositions) in a same row sharing a common superscript are not significantly different (p>0.05). The coefficient of variation for each mean value never exceeded 5%; ² Total polar lipids include unidentified glycolipids and pigments; ³ PC includes a small amount of lyso PC and sphingomyelin (< 2% of total PL); ⁴ PA includes cardiolipin.</footnote>
<table><caption>**Table 4.** Fatty acid composition of major lipid classes of turbot whole body at the beginning of the feeding trial (% fatty acids)</caption><thead><tr><th rowspan="2"></th><th colspan="4">PL class initial turbot</th></tr><tr><th>NL</th><th>PC</th><th>PE</th><th>PI</th></tr></thead><tbody><tr><td>14:0</td><td>4.1</td><td>3.5</td><td>0.5</td><td>1.4</td></tr><tr><td>16:0</td><td>11.4</td><td>27.4</td><td>8.1</td><td>6.2</td></tr><tr><td>16:1n-7</td><td>4.7</td><td>1.1</td><td>0.6</td><td>1.1</td></tr><tr><td>18:0</td><td>4.3</td><td>5.0</td><td>10.3</td><td>29.6</td></tr><tr><td>18:1n-9</td><td>19.3</td><td>18.6</td><td>12.2</td><td>8.2</td></tr><tr><td>18:1n-7</td><td>6.1</td><td>6.6</td><td>7.6</td><td>3.5</td></tr><tr><td>18:2n-6</td><td>5.5</td><td>5.8</td><td>3.2</td><td>1.9</td></tr><tr><td>18:3n-3</td><td>15.1</td><td>6.4</td><td>3.5</td><td>2.2</td></tr><tr><td>18:4n-3</td><td>2.0</td><td>0.5</td><td>0.5</td><td>0.2</td></tr><tr><td>20:0</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.1</td></tr><tr><td>20:1n-9</td><td>0.8</td><td>0.5</td><td>0.7</td><td>0.4</td></tr><tr><td>20:1n-7</td><td>0.2</td><td>0.1</td><td>0.2</td><td>0.1</td></tr><tr><td>20:2n-6</td><td>0.5</td><td>0.7</td><td>0.6</td><td>0.5</td></tr><tr><td>20:3n-6</td><td>0.2</td><td>0.2</td><td>0.2</td><td>0.3</td></tr><tr><td>20:4n-6</td><td>1.4</td><td>1.7</td><td>3.6</td><td>16.8</td></tr><tr><td>20:3n-3</td><td>1.8</td><td>1.1</td><td>1.8</td><td>0.6</td></tr><tr><td>20:4n-3</td><td>0.9</td><td>0.7</td><td>0.7</td><td>0.4</td></tr><tr><td>22:0</td><td>0.2</td><td>0.2</td><td>tr</td><td>0.1</td></tr><tr><td>20:5n-3</td><td>8.6</td><td>7.7</td><td>10.0</td><td>9.3</td></tr><tr><td>22:1n-9</td><td>0.1</td><td>0.1</td><td>-</td><td>0.3</td></tr><tr><td>22:1n-7</td><td>0.7</td><td>0.1</td><td>-</td><td>0.4</td></tr><tr><td>22:4n-6</td><td>0.1</td><td>0.1</td><td>0.4</td><td>0.1</td></tr><tr><td>22:3n-3</td><td>0.1</td><td>tr</td><td>0.1</td><td>-</td></tr><tr><td>22:5n-6</td><td>0.2</td><td>0.2</td><td>0.8</td><td>0.2</td></tr><tr><td>22:4n-3</td><td>0.1</td><td>-</td><td>-</td><td>-</td></tr><tr><td>24:0</td><td>0.1</td><td>-</td><td>-</td><td>0.1</td></tr><tr><td>22:5n-3</td><td>2.3</td><td>1.9</td><td>6.0</td><td>3.7</td></tr><tr><td>24:1n-9</td><td>0.1</td><td>0.4</td><td>-</td><td>0.1</td></tr><tr><td>22:6n-3</td><td>4.8</td><td>6.2</td><td>25.6</td><td>10.0</td></tr><tr><td>Saturates</td><td>21.9</td><td>37.7</td><td>21.0</td><td>38.9</td></tr><tr><td>Monoenes</td><td>34.3</td><td>29.1</td><td>22.4</td><td>14.8</td></tr><tr><td>n-6 PUFA</td><td>8.1</td><td>8.8</td><td>8.6</td><td>19.9</td></tr><tr><td>n-3 PUFA</td><td>35.7</td><td>24.4</td><td>47.9</td><td>26.3</td></tr><tr><td>n-3 / n-6</td><td>4.4</td><td>2.8</td><td>5.5</td><td>1.3</td></tr></tbody></table>

tained 18:0 as major saturated FA. The highest level of monoenoic FA was found in NL and PC, the lowest in PI. NL and PI had similar proportions of total PUFA, but with a very distinct n-3/n-6 ratio of 4.4 and 1.3, respectively. This ratio was lowered in the turbot PI fraction due to the important accumulation of n-6 HUFA, more specifically 20:4n-6. The high amount of

n-3 PUFA in the NL of the initial fish was mainly due to 18:3n-3, accounting for 15% in NL vs 2.2-6.4% in the polar lipid classes and was characterized by a very low DHA/EPA ratio (0.6). The DHA/EPA ratio was the highest in PE (2.6) where DHA accounted for 26% of total FA.

### Major changes in the fatty acid profiles of turbot due to the feeding

The fatty acid profiles of the body lipid classes of turbot, fed the diets for 7 and 28 days, are summarized in Tables 5 and 6, respectively. The NL fraction showed the strongest dietary response. In NL, saturated fatty acids increased from 22 to 31-36% after 7 days and to 32-42% after 28 days. The amounts of monoenes dropped from 34 to 25-31% after 7 days, down to 21-28% in final fish. Total n-6 PUFA in NL varied between 6 and 21% according to the diet. Despite the doubling of DHA, the percentage of total n-3 PUFA decreased due to the important decline of 18:3n-3 and, to a lesser extent, of 20:5n-3. The fatty acid groups in whole body PC reacted similarly as in NL, but not as drastically. In PE and PI the proportions of the major fatty acid groups remained more constant.

The PL-deprived turbot stored in all body lipid classes a higher amount of monoenoic and of saturated FA and lower amounts of n-6 and n-3 PUFA than the PL-fed fish (Tables 5 and 6). The levels of monoenoic FA and of n-3 PUFA in the fish were not in accordance with the dietary levels, as they were similar in the PL-free and the PL-supplemented diets. The higher level of 18:2n-6 in the PL-fed fish reflected the higher dietary level. However, when comparing the 18:2n-6 levels among the PL-supplemented groups, an effect of the PL class composition of the diet was noted. For instance, in spite of the similar amounts of 18:2n-6 in the PA-rich diet D3 and in the PC-rich diet D8, fish receiving diet D8 had incorporated a higher amount of 18:2n-6. In all body classes, increased levels of 18:2n-6 were accompanied by increased levels of 20:2n-6. In the NL and PC fractions of the final fish, the dead-end elongation product 20:2n-6 accounted often for more than 4 times the level of 20:4n-6.

### Effect of dietary PL class composition on HUFA incorporation in turbot

Tables 7 and 8 give the results of the 3-way analysis of variance for evaluating the effect of the diet (D), the sampling period (P) and the lipid class (C) on the retention of the dietary HUFA in the fish. The most

<footnote n="1">¹ Values represent means of the duplicate lipid extracts. Sums include minor fatty acids not listed in the table, tr <0.1, -: not detected.</footnote>
<table><caption>Table 5. Selected fatty acid composition of major lipid classes of whole turbot fed the experimental diets for 7 days. The HUFA values in each lipid class were subjected to a one-way analysis of variance<fnref n="1" /></caption><thead><tr><th>Diet<br>Major diet PL</th><th>D1<br>-</th><th>D2<br>PI</th><th>D3<br>PA</th><th>D4<br>PI</th><th>D5<br>PE</th><th>D6<br>SL</th><th>D7<br>PC</th><th>D8<br>PC</th><th>D9<br>PC</th></tr></thead><tbody><tr><td colspan="10">Turbot neutral lipid (% fatty acids NL)</td></tr><tr><td>Saturates</td><td>36.3</td><td>31.6</td><td>32.0</td><td>33.1</td><td>31.5</td><td>31.5</td><td>30.9</td><td>30.8</td><td>30.6</td></tr><tr><td>Monoenes</td><td>31.4</td><td>29.6</td><td>29.5</td><td>27.6</td><td>29.6</td><td>26.8</td><td>28.7</td><td>26.0</td><td>25.1</td></tr><tr><td>n-6 PUFA</td><td>8.6</td><td>9.8</td><td>10.9</td><td>12.0</td><td>12.1</td><td>12.5</td><td>12.0</td><td>16.0</td><td>16.4</td></tr><tr><td>18:2n-6</td><td>6.4</td><td>7.4</td><td>8.4</td><td>9.6</td><td>9.7</td><td>10.1</td><td>9.2</td><td>12.5</td><td>12.9</td></tr><tr><td>20:2n-6</td><td>0.6</td><td>0.6</td><td>0.8</td><td>0.9</td><td>0.9</td><td>0.9</td><td>0.9</td><td>1.2</td><td>1.3</td></tr><tr><td>20:4n-6</td><td>1.0</td><td>1.1</td><td>1.1</td><td>0.9</td><td>0.9</td><td>0.9</td><td>1.1</td><td>0.9</td><td>0.9</td></tr><tr><td>n-3 PUFA</td><td>23.8</td><td>28.9</td><td>27.6</td><td>27.3</td><td>26.8</td><td>29.3</td><td>28.5</td><td>27.2</td><td>28.0</td></tr><tr><td>18:3n-3</td><td>7.2</td><td>7.6</td><td>7.8</td><td>6.5</td><td>7.7</td><td>7.4</td><td>8.1</td><td>5.8</td><td>6.7</td></tr><tr><td>20:5n-3</td><td>5.3<sup>c</sup></td><td>6.6<sup>a</sup></td><td>6.0<sup>b</sup></td><td>6.3<sup>b</sup></td><td>6.1<sup>b</sup></td><td>6.7<sup>a</sup></td><td>6.8<sup>a</sup></td><td>6.3<sup>ab</sup></td><td>6.5<sup>ab</sup></td></tr><tr><td>22:5n-3</td><td>2.2<sup>b</sup></td><td>3.0<sup>a</sup></td><td>2.4<sup>b</sup></td><td>2.7<sup>a</sup></td><td>2.5<sup>b</sup></td><td>2.8<sup>a</sup></td><td>3.0<sup>a</sup></td><td>2.7<sup>a</sup></td><td>2.7<sup>a</sup></td></tr><tr><td>22:6n-3</td><td>6.4<sup>d</sup></td><td>8.8<sup>ab</sup></td><td>8.5<sup>b</sup></td><td>9.3<sup>a</sup></td><td>7.7<sup>c</sup></td><td>9.5<sup>a</sup></td><td>8.4<sup>b</sup></td><td>9.6<sup>a</sup></td><td>9.7<sup>a</sup></td></tr><tr><td colspan="10">Turbot phosphatidylcholine (% fatty acids PC)</td></tr><tr><td>Saturates</td><td>41.8</td><td>38.9</td><td>40.0</td><td>39.0</td><td>39.1</td><td>38.8</td><td>38.2</td><td>38.1</td><td>37.9</td></tr><tr><td>Monoenes</td><td>28.0</td><td>25.1</td><td>26.0</td><td>23.7</td><td>23.5</td><td>22.7</td><td>24.7</td><td>21.6</td><td>22.0</td></tr><tr><td>n-6 PUFA</td><td>9.7</td><td>10.8</td><td>12.2</td><td>13.7</td><td>14.5</td><td>15.2</td><td>12.6</td><td>17.2</td><td>16.6</td></tr><tr><td>18:2n-6</td><td>7.2</td><td>8.1</td><td>9.6</td><td>10.9</td><td>11.6</td><td>12.0</td><td>9.0</td><td>14.8</td><td>13.2</td></tr><tr><td>20:2n-6</td><td>1.1</td><td>1.0</td><td>1.2</td><td>1.3</td><td>1.6</td><td>1.5</td><td>1.3</td><td>1.7</td><td>1.5</td></tr><tr><td>20:4n-6</td><td>0.9</td><td>1.0</td><td>0.9</td><td>0.9</td><td>1.0</td><td>1.1</td><td>0.8</td><td>0.8</td><td>0.9</td></tr><tr><td>n-3 PUFA</td><td>20.0</td><td>25.2</td><td>21.8</td><td>23.6</td><td>22.9</td><td>23.3</td><td>24.5</td><td>23.1</td><td>23.5</td></tr><tr><td>18:3n-3</td><td>2.9</td><td>2.6</td><td>2.5</td><td>2.4</td><td>2.4</td><td>2.8</td><td>2.6</td><td>2.2</td><td>2.3</td></tr><tr><td>20:5n-3</td><td>6.1<sup>b</sup></td><td>6.9<sup>a</sup></td><td>6.2<sup>b</sup></td><td>6.7<sup>a</sup></td><td>6.2<sup>b</sup></td><td>6.8<sup>a</sup></td><td>6.8<sup>a</sup></td><td>6.6<sup>a</sup></td><td>6.4<sup>ab</sup></td></tr><tr><td>22:5n-3</td><td>1.9<sup>c</sup></td><td>2.5<sup>a</sup></td><td>2.2<sup>b</sup></td><td>2.3<sup>b</sup></td><td>2.2<sup>b</sup></td><td>2.3<sup>b</sup></td><td>2.2<sup>b</sup></td><td>2.2<sup>b</sup></td><td>2.3<sup>b</sup></td></tr><tr><td>22:6n-3</td><td>8.0<sup>d</sup></td><td>11.1<sup>a</sup></td><td>8.8<sup>c</sup></td><td>10.9<sup>a</sup></td><td>10.0<sup>b</sup></td><td>10.9<sup>a</sup></td><td>11.4<sup>a</sup></td><td>11.0<sup>a</sup></td><td>11.1<sup>a</sup></td></tr><tr><td colspan="10">Turbot phosphatidylethanolamine (% fatty acids PE)</td></tr><tr><td>Saturates</td><td>26.2</td><td>22.8</td><td>23.8</td><td>22.3</td><td>26.1</td><td>23.6</td><td>22.4</td><td>24.5</td><td>23.2</td></tr><tr><td>Monoenes</td><td>19.8</td><td>19.8</td><td>18.1</td><td>18.6</td><td>17.2</td><td>17.0</td><td>16.8</td><td>15.7</td><td>16.0</td></tr><tr><td>n-6 PUFA</td><td>8.8</td><td>9.2</td><td>11.4</td><td>12.9</td><td>11.8</td><td>12.1</td><td>11.5</td><td>14.0</td><td>15.0</td></tr><tr><td>18:2n-6</td><td>4.5</td><td>5.3</td><td>6.2</td><td>8.7</td><td>7.6</td><td>7.2</td><td>6.8</td><td>9.6</td><td>9.7</td></tr><tr><td>20:2n-6</td><td>0.6</td><td>0.8</td><td>0.8</td><td>1.1</td><td>1.0</td><td>1.0</td><td>1.0</td><td>1.2</td><td>1.3</td></tr><tr><td>20:4n-6</td><td>2.3</td><td>2.1</td><td>2.5</td><td>2.1</td><td>2.3</td><td>2.1</td><td>2.3</td><td>2.1</td><td>2.2</td></tr><tr><td>n-3 PUFA</td><td>45.2</td><td>48.1</td><td>46.6</td><td>46.2</td><td>45.0</td><td>47.3</td><td>49.3</td><td>45.7</td><td>45.8</td></tr><tr><td>18:3n-3</td><td>2.4</td><td>2.8</td><td>2.1</td><td>2.6</td><td>1.9</td><td>2.1</td><td>2.9</td><td>2.0</td><td>2.2</td></tr><tr><td>20:5n-3</td><td>9.3<sup>a</sup></td><td>9.0<sup>a</sup></td><td>9.1<sup>a</sup></td><td>7.8<sup>b</sup></td><td>8.5<sup>ab</sup></td><td>7.9<sup>b</sup></td><td>9.0<sup>a</sup></td><td>7.9<sup>b</sup></td><td>7.8<sup>b</sup></td></tr><tr><td>22:5n-3</td><td>5.5</td><td>5.1</td><td>5.7</td><td>5.0</td><td>5.0</td><td>5.1</td><td>5.5</td><td>5.0</td><td>5.0</td></tr><tr><td>22:6n-3</td><td>26.4<sup>c</sup></td><td>28.2<sup>b</sup></td><td>28.4<sup>b</sup></td><td>28.5<sup>b</sup></td><td>27.9<sup>b</sup></td><td>30.2<sup>a</sup></td><td>29.7<sup>a</sup></td><td>29.4<sup>a</sup></td><td>30.0<sup>a</sup></td></tr></tbody></table>

<footnote n="1">¹ Footnote as given in Table 4. Only HUFA are considered by the ANOVA as they are supplied in equal amounts by the different diets. Mean HUFA values sharing a common letter are not significantly different (p>0.05).</footnote>
<table><caption>Table 6. Selected fatty acid composition of major lipid classes of whole turbot at the end of the 28-day feeding trial. The HUFA values in each lipid class were subjected to a one-way analysis of variance <fnref n="1" /></caption><thead><tr><th>Diet<br>Major diet PL</th><th>D1<br>-</th><th>D2<br>PI</th><th>D3<br>PA</th><th>D4<br>PI</th><th>D5<br>PE</th><th>D6<br>SL</th><th>D7<br>PC</th><th>D8<br>PC</th><th>D9<br>PC</th></tr></thead><tbody><tr><td colspan="10">Turbot neutral lipid (% fatty acids NL)</td></tr><tr><td>Saturates</td><td>41.5</td><td>37.6</td><td>35.3</td><td>35.0</td><td>34.8</td><td>33.0</td><td>36.4</td><td>32.0</td><td>35.0</td></tr><tr><td>Monoenes</td><td>28.2</td><td>26.8</td><td>25.6</td><td>24.3</td><td>23.2</td><td>22.4</td><td>24.1</td><td>21.0</td><td>20.8</td></tr><tr><td>n-6 PUFA</td><td>6.2</td><td>11.3</td><td>14.9</td><td>16.9</td><td>17.6</td><td>17.9</td><td>14.8</td><td>21.3</td><td>19.3</td></tr><tr><td>18:2n-6</td><td>3.8</td><td>8.6</td><td>12.2</td><td>13.9</td><td>14.7</td><td>15.0</td><td>11.7</td><td>18.0</td><td>16.4</td></tr><tr><td>20:2n-6</td><td>0.9</td><td>1.0</td><td>1.6</td><td>1.9</td><td>1.9</td><td>1.8</td><td>1.1</td><td>2.3</td><td>1.9</td></tr><tr><td>20:4n-6</td><td>0.6</td><td>0.5</td><td>0.5</td><td>0.5</td><td>0.3</td><td>0.5</td><td>0.4</td><td>0.5</td><td>0.4</td></tr><tr><td>n-3 PUFA</td><td>24.0</td><td>24.3</td><td>24.2</td><td>23.8</td><td>24.4</td><td>26.7</td><td>24.7</td><td>25.8</td><td>24.9</td></tr><tr><td>18:3n-3</td><td>6.2</td><td>3.2</td><td>4.3</td><td>3.1</td><td>3.5</td><td>3.9</td><td>3.4</td><td>3.3</td><td>3.7</td></tr><tr><td>20:5n-3</td><td>4.9</td><td>5.1</td><td>4.9</td><td>5.1</td><td>5.0</td><td>5.3</td><td>5.0</td><td>5.4</td><td>5.1</td></tr><tr><td>22:5n-3</td><td>3.1<sup>b</sup></td><td>3.5<sup>a</sup></td><td>3.3<sup>ab</sup></td><td>3.8<sup>a</sup></td><td>3.5<sup>a</sup></td><td>3.6<sup>a</sup></td><td>3.6<sup>a</sup></td><td>3.5<sup>a</sup></td><td>3.4<sup>a</sup></td></tr><tr><td>22:6n-3</td><td>8.2<sup>d</sup></td><td>11.4<sup>b</sup></td><td>10.0<sup>c</sup></td><td>10.9<sup>b</sup></td><td>11.4<sup>b</sup></td><td>12.4<sup>a</sup></td><td>11.6<sup>b</sup></td><td>12.3<sup>a</sup></td><td>11.8<sup>ab</sup></td></tr><tr><td colspan="10">Turbot phosphatidylcholine (% fatty acids PC)</td></tr><tr><td>Saturates</td><td>40.8</td><td>39.1</td><td>39.9</td><td>38.1</td><td>37.8</td><td>36.9</td><td>36.6</td><td>35.3</td><td>36.0</td></tr><tr><td>Monoenes</td><td>31.8</td><td>28.6</td><td>28.5</td><td>27.3</td><td>25.1</td><td>24.4</td><td>27.5</td><td>21.9</td><td>22.2</td></tr><tr><td>n-6 PUFA</td><td>10.0</td><td>11.6</td><td>13.7</td><td>15.0</td><td>17.9</td><td>17.1</td><td>15.7</td><td>20.0</td><td>19.7</td></tr><tr><td>18:2n-6</td><td>7.5</td><td>8.5</td><td>10.7</td><td>11.9</td><td>14.5</td><td>13.8</td><td>12.5</td><td>16.2</td><td>16.3</td></tr><tr><td>20:2n-6</td><td>1.0</td><td>1.3</td><td>1.6</td><td>1.8</td><td>2.2</td><td>1.9</td><td>1.5</td><td>2.6</td><td>2.0</td></tr><tr><td>20:4n-6</td><td>0.5</td><td>0.5</td><td>0.4</td><td>0.5</td><td>0.5</td><td>0.6</td><td>0.6</td><td>0.5</td><td>0.5</td></tr><tr><td>n-3 PUFA</td><td>17.0</td><td>20.7</td><td>16.9</td><td>19.5</td><td>19.4</td><td>21.6</td><td>20.2</td><td>22.7</td><td>22.1</td></tr><tr><td>18:3n-3</td><td>1.0</td><td>1.0</td><td>1.1</td><td>1.0</td><td>1.1</td><td>1.6</td><td>1.1</td><td>1.3</td><td>1.3</td></tr><tr><td>20:5n-3</td><td>4.0<sup>b</sup></td><td>4.9<sup>a</sup></td><td>4.0<sup>b</sup></td><td>4.3<sup>b</sup></td><td>4.6<sup>ab</sup></td><td>4.8<sup>a</sup></td><td>4.7<sup>a</sup></td><td>5.1<sup>a</sup></td><td>5.0<sup>a</sup></td></tr><tr><td>22:5n-3</td><td>2.0</td><td>2.3</td><td>2.0</td><td>2.4</td><td>2.2</td><td>2.4</td><td>2.2</td><td>2.3</td><td>2.1</td></tr><tr><td>22:6n-3</td><td>9.5</td><td>11.7</td><td>9.4</td><td>11.3</td><td>11.1</td><td>11.9</td><td>11.9</td><td>13.4</td><td>12.9</td></tr><tr><td colspan="10">Turbot phosphatidylethanolamine (% fatty acids PE)</td></tr><tr><td>Saturates</td><td>25.1</td><td>26.7</td><td>25.8</td><td>24.6</td><td>24.3</td><td>25.3</td><td>24.8</td><td>24.0</td><td>25.0</td></tr><tr><td>Monoenes</td><td>20.8</td><td>16.6</td><td>16.9</td><td>15.4</td><td>15.6</td><td>14.4</td><td>15.9</td><td>14.7</td><td>14.9</td></tr><tr><td>n-6 PUFA</td><td>10.8</td><td>9.1</td><td>10.0</td><td>11.1</td><td>10.4</td><td>11.7</td><td>10.3</td><td>12.0</td><td>12.5</td></tr><tr><td>18:2n-6</td><td>5.8</td><td>5.6</td><td>6.7</td><td>7.1</td><td>6.6</td><td>7.7</td><td>6.8</td><td>8.7</td><td>8.7</td></tr><tr><td>20:2n-6</td><td>0.9</td><td>1.0</td><td>1.1</td><td>1.5</td><td>1.3</td><td>1.4</td><td>1.2</td><td>1.6</td><td>1.6</td></tr><tr><td>20:4n-6</td><td>1.4</td><td>1.3</td><td>1.4</td><td>1.5</td><td>1.6</td><td>1.5</td><td>1.5</td><td>1.5</td><td>1.4</td></tr><tr><td>n-3 PUFA</td><td>43.3</td><td>47.6</td><td>47.3</td><td>49.0</td><td>49.8</td><td>48.6</td><td>49.0</td><td>49.3</td><td>47.4</td></tr><tr><td>18:3n-3</td><td>1.1</td><td>0.7</td><td>0.8</td><td>0.7</td><td>0.6</td><td>0.6</td><td>0.9</td><td>0.6</td><td>1.0</td></tr><tr><td>20:5n-3</td><td>7.1<sup>a</sup></td><td>6.9<sup>a</sup></td><td>7.1<sup>a</sup></td><td>6.5<sup>b</sup></td><td>6.9<sup>a</sup></td><td>6.2<sup>b</sup></td><td>6.1<sup>b</sup></td><td>6.0<sup>b</sup></td><td>6.0<sup>b</sup></td></tr><tr><td>22:5n-3</td><td>4.8<sup>b</sup></td><td>5.1<sup>a</sup></td><td>5.3<sup>a</sup></td><td>5.3<sup>a</sup></td><td>5.4<sup>a</sup></td><td>5.1<sup>a</sup></td><td>4.9<sup>ab</sup></td><td>5.3<sup>a</sup></td><td>5.0<sup>a</sup></td></tr><tr><td>22:6n-3</td><td>28.4<sup>e</sup></td><td>33.0<sup>c</sup></td><td>32.3<sup>d</sup></td><td>34.9<sup>a</sup></td><td>34.8<sup>a</sup></td><td>34.8<sup>a</sup></td><td>34.0<sup>b</sup></td><td>35.2<sup>a</sup></td><td>33.9<sup>b</sup></td></tr><tr><td colspan="10">Turbot phosphatidylinositol (% fatty acids PI)</td></tr><tr><td>Saturates</td><td>45.4</td><td>42.2</td><td>40.6</td><td>42.1</td><td>40.0</td><td>41.5</td><td>40.8</td><td>39.3</td><td>40.3</td></tr><tr><td>Monoenes</td><td>19.2</td><td>16.0</td><td>16.7</td><td>14.9</td><td>16.4</td><td>12.1</td><td>15.7</td><td>14.7</td><td>13.4</td></tr><tr><td>n-6 PUFA</td><td>10.3</td><td>12.8</td><td>13.4</td><td>14.0</td><td>14.5</td><td>16.1</td><td>14.1</td><td>15.4</td><td>15.5</td></tr><tr><td>18:2n-6</td><td>2.0</td><td>2.7</td><td>3.9</td><td>4.0</td><td>4.3</td><td>4.7</td><td>3.5</td><td>4.6</td><td>4.4</td></tr><tr><td>20:2n-6</td><td>0.3</td><td>0.4</td><td>0.9</td><td>0.9</td><td>1.0</td><td>1.2</td><td>0.6</td><td>1.1</td><td>1.0</td></tr><tr><td>20:4n-6</td><td>7.2<sup>c</sup></td><td>8.1<sup>b</sup></td><td>7.8<sup>b</sup></td><td>8.4<sup>ab</sup></td><td>8.6<sup>a</sup></td><td>8.8<sup>a</sup></td><td>8.6<sup>a</sup></td><td>9.1<sup>a</sup></td><td>8.7<sup>a</sup></td></tr><tr><td>n-3 PUFA</td><td>25.1</td><td>29.0</td><td>28.3</td><td>29.0</td><td>29.1</td><td>30.4</td><td>29.4</td><td>30.6</td><td>30.8</td></tr><tr><td>18:3n-3</td><td>0.4</td><td>0.4</td><td>0.7</td><td>0.5</td><td>0.5</td><td>0.8</td><td>0.4</td><td>0.6</td><td>0.5</td></tr><tr><td>20:5n-3</td><td>6.4<sup>b</sup></td><td>7.1<sup>a</sup></td><td>6.7<sup>ab</sup></td><td>7.1<sup>a</sup></td><td>7.2<sup>a</sup></td><td>7.4<sup>a</sup></td><td>7.3<sup>a</sup></td><td>7.5<sup>a</sup></td><td>7.6<sup>a</sup></td></tr><tr><td>22:5n-3</td><td>3.7<sup>b</sup></td><td>4.2<sup>a</sup></td><td>4.0<sup>a</sup></td><td>4.1<sup>a</sup></td><td>4.2<sup>a</sup></td><td>4.2<sup>a</sup></td><td>4.2<sup>a</sup></td><td>4.5<sup>a</sup></td><td>4.5<sup>a</sup></td></tr><tr><td>22:6n-3</td><td>13.7<sup>c</sup></td><td>15.8<sup>b</sup></td><td>16.0<sup>b</sup></td><td>16.5<sup>b</sup></td><td>16.4<sup>b</sup></td><td>17.3<sup>a</sup></td><td>16.1<sup>b</sup></td><td>17.6<sup>a</sup></td><td>17.1<sup>a</sup></td></tr></tbody></table>

<footnote n="1">¹ Footnotes as given in Table 5.</footnote>
<table><caption>Table 7. Mean squares<fnref n="1" /> for main effects and interactions given by the 3-way ANOVA on HUFA levels in turbot lipid classes (NL, PC and PE) sampled at two different periods (day 7 and day 28 of the trial)</caption><thead><tr><th rowspan="2">HUFA<br>(df =8)</th><th colspan="3">Main effects</th><th colspan="3">Interactions</th><th rowspan="2">Error<br>df = 54</th></tr><tr><th>Diet (D)<br>(df =1)</th><th>Period (P)<br>(df =2)</th><th>Class (C)<br>(df =8)</th><th>D × P<br>(df =16)</th><th>D × C<br>(df =2)</th><th>P × C</th></tr></thead><tbody><tr><td>20:4n-6</td><td>0.23</td><td>166.9***</td><td>217.0***</td><td>0.39</td><td>0.50</td><td>4.62***</td><td>0.212</td></tr><tr><td>20:5n-3</td><td>0.69</td><td>201.2***</td><td>105.5***</td><td>0.45</td><td>1.81**</td><td>4.47**</td><td>0.019</td></tr><tr><td>22:5n-3</td><td>0.38**</td><td>3.1***</td><td>123.8***</td><td>0.12</td><td>0.17</td><td>4.42***</td><td>0.010</td></tr><tr><td>22:6n-3</td><td>10.8***</td><td>76.5***</td><td>1475.0***</td><td>0.39</td><td>1.07*</td><td>5.22***</td><td>0.046</td></tr></tbody></table>

<table><caption>Table 8. Duncan's multiple range test (p<0.05) for the main effects in the 3-way ANOVA from Table 7. Data represent mean HUFA values (% total fatty acids) averaged over all the observations (n = number of observations) <fnref n="1" /></caption><thead><tr><th rowspan="2"></th><th colspan="9">Diet<br>(n=12)</th><th colspan="3">Lipid class<br>(n=36)</th><th colspan="2">Sampling<br>(n=54)</th></tr><tr><th>D1</th><th>D2</th><th>D3</th><th>D4</th><th>D5</th><th>D6</th><th>D7</th><th>D8</th><th>D9</th><th>NL</th><th>PC</th><th>PE</th><th>day7</th><th>day28</th></tr></thead><tbody><tr><td>20:4n-6</td><td>1.1</td><td>1.1</td><td>1.1</td><td>1.1</td><td>1.1</td><td>1.1</td><td>1.1</td><td>1.0</td><td>0.7<sup>b</sup></td><td>0.7<sup>b</sup></td><td>1.8<sup>a</sup></td><td>1.4<sup>a</sup></td><td>0.8<sup>b</sup></td></tr><tr><td>20:5n-3</td><td>6.1</td><td>6.5</td><td>6.2</td><td>6.1</td><td>6.2</td><td>6.3</td><td>6.4</td><td>6.2</td><td>5.7<sup>b</sup></td><td>5.6<sup>b</sup></td><td>7.5<sup>a</sup></td><td>7.1<sup>a</sup></td><td>5.4<sup>b</sup></td></tr><tr><td>22:5n-3</td><td>2.3<sup>b</sup></td><td>3.6<sup>a</sup></td><td>3.5<sup>a</sup></td><td>3.6<sup>a</sup></td><td>3.5<sup>a</sup></td><td>3.6<sup>a</sup></td><td>3.5<sup>a</sup></td><td>3.5<sup>a</sup></td><td>3.1<sup>b</sup></td><td>2.2<sup>c</sup></td><td>5.2<sup>a</sup></td><td>3.4<sup>b</sup></td><td>3.6<sup>a</sup></td></tr><tr><td>22:6n-3</td><td>14.5<sup>e</sup></td><td>17.3<sup>c</sup></td><td>16.2<sup>d</sup></td><td>17.6<sup>b</sup></td><td>17.1<sup>c</sup></td><td>18.3<sup>a</sup></td><td>17.8<sup>b</sup></td><td>18.5<sup>a</sup></td><td>9.9<sup>c</sup></td><td>10.9<sup>b</sup></td><td>31.1<sup>a</sup></td><td>15.9<sup>b</sup></td><td>18.7<sup>a</sup></td></tr></tbody></table>

significant dietary effect (D) on HUFA levels was detected for DHA (Table 7). The DHA values, averaged over the 3 lipid classes at the 2 periods, were 12 to 30% higher in PL-fed fish than in PL-deprived fish (Table 8). In addition, significant differences were seen between the PL-supplemented diets with as ranking order: D3 < (D2, D5) < (D4, D7) < (D6, D8, D9), denoting a higher DHA incorporation in fish fed the PC-rich diets. The other HUFA (22:5n-3, 20:5n-3 and 20:4n-6) did not distinguish the diets as explicitly as did DHA. The level of 22:5n-3 was significantly lower in fish fed the PL-free diet, without any further distinction between the dietary PL-fractions. Based on the EPA levels, the diet effect was not significant, whereas an important diet x class (D x C) interaction (p<0.01) was observed, indicating different responses of the lipid classes in turbot to the dietary EPA supply. Indeed, in PC and NL the EPA levels were higher in most groups fed the PL-supplemented diets than in fish fed the PL-free diet, in agreement with findings for DHA, whereas a reverse pattern was found in the fish PE. Based on the accumulation of 20:4n-6 no diet effect was noted in the 3 considered body lipid classes.

In contrast, in body PI, higher levels of 20:4n-6 were observed in fish fed the PC-rich diets (Table 6).

Aside from the variations of the HUFA levels related to the dietary effects, HUFA levels also varied according to the sampling period and the body lipid class (Table 7). The whole body PE fraction contained a significantly higher level of each considered HUFA than PC or NL. Regarding the sampling time, the average levels of DHA and 22:5n-3 increased, whereas EPA and 20:4n-6 decreased between day 7 and day 28 (Table 8).

## Discussion

This study confirms the positive effect of phospholipids (PL) on growth observed in foregoing studies on larvae and post larvae of different fish species (Kanazawa et al. 1981, 1983a, b; Geurden et al. 1997a, b). Clear differences according to the polar head group of the dietary PL were also evident, with a superiority of PC over the other PL classes. Some of the PC fractions were highly purified as compared to the other PL fractions, which contained noticeable amounts of gly-

<footnote n="1">¹Mean squares are based on log-transformed values and are multiplied by a common factor 100. The F value is based on the residual variance DxPxC (df = 16). Error: analytical error between duplicate analyses. Total degrees of freedom (df) = 108. *, **, *** denotes a significance at p<0.05, p<0.01 and p<0.001, respectively.</footnote>

<footnote n="1">¹ For each effect, data in a row sharing a common superscript are not significantly different.</footnote>
colipids. It seems however unlikely that the observed
growth differences could be explained by a negative
effect of glycolipids. The superiority of PC over PI
was also noticed when comparing the groups fed di-
ets D2 (PI-rich) and D7 (PC-rich), which were both
supplemented with purified PL fractions.

The superiority of PC over the other dietary PL
classes is in general accordance with previous data on
larvae or post-larvae of other fish species (Takeuchi
et al. 1992; Kanazawa 1993). In contrast, PI has been
reported to be as efficient as PC in a study on lar-
val ayu *Plecoglossus altivelis* (Kanazawa et al. 1985).
The larval response of common carp *Cyprinus car-
pio* to PC and PI was distinct, i.e. PC stimulated the
initial growth, but induced deformities and a subse-
quent mortality, while PI prevented the deformities
and ensured a high survival (Geurden et al. 1997a,
1998). The growth differences presently observed be-
tween the turbot in relation to the dietary PL-supply
are smaller than in carp larvae. This discrepancy in
response, as well as the absence of a PL effect on the
survival of the turbot, is probably explained by the
more advanced developmental stage of weaned turbot
as compared to first-feeding carp (210 mg vs. 2 mg
initial body weight, respectively).

Another distinctive characteristic of the PL-fed fish
is the significantly higher deposition of triglycerides
in their whole body lipid. Based on the high recovery
of labelled fatty acids, Linares and Henderson (1991)
suggested a very active synthesis of triglycerides in
turbot. The increased ratio of 'storage to membrane
lipid' following a dietary PL supplementation was also
noticed in newly-weaned European sea bass (Geurden
et al. 1997c). But, as shown in the present study, the
PL class profile of the fish, expressed as a percent-
age of total polar lipid, remained unaffected by the PL
class composition of the diet.

The specific distribution of the various fatty acids
in each of the examined lipid classes agrees with data
reported for the whole body (Linares and Henderson
1991) and for separate tissues of the same species
(Bell et al. 1985, 1994, 1995). The high n-3/n-6 and
EPA/DHA ratios at the beginning of the experiment
are typical of newly-weaned fish and reflect the pre-
vious *Artemia* feeding (Coutteau et al. 1996; Geurden
et al. 1997b, c). Whereas some specific dietary fatty
acid responses were noted in the separate fish lipid
classes, general tendencies affecting all classes were
seen during the course of the experiment, such as the
increase of 18:2n-6 relative to 18:3n-3 and the increase
of DHA relative to EPA. The levels of 18:2n-6 in the

fish were related to the dietary levels, being higher in
the PL-supplemented diets than in the PL-free diet.
However, the example of diet D3 (PA-rich) which
provided the same amount of 18:2n-6 as diet D8 (PC-
rich), but which in the fish resulted in a considerably
lower 18:2n-6 level than the PC-rich diet, indicates a
stimulating influence of dietary PC on the fatty acid
incorporation. The increasing deposition of 20:2n-6, a
‘dead-end’ product, with increasing 18:2n-6 levels in
diet and fish agrees with the data of Bell et al. (1994)
in juvenile turbot and indicates an inhibition of the
delta-6-desaturase activity (Tocher 1993), possibly by
the high HUFA level in the diet. In contrast, when
culturing turbot cells in a medium poor in n-3 HUFA,
18:2n-6 was more desaturated than elongated (Tocher
and Mackinlay 1990).

Following our introductory hypothesis, the com-
parison of HUFA levels in fish fed diets containing
an equal amount of HUFA, presented as neutral lipid,
should indicate the capacity of the different dietary PL
classes for enhancing the absorption of neutral lipid
fatty acids. In fact, the outcome varied according to
the HUFA, i.e. DHA allowed the best discrimination
and separated the PL-free, the PC-poor and the PC-
rich diets; 22:5n-3 distinguished the PL-free diet from
the other diets; EPA gave results dependent on the fish
class and 20:4n-6 was discriminant only in fish PI. The
utilization of the body level of a specific fatty acid as a
quantitative indicator of absorption implies a minimal
degradation or synthesis of this fatty acid. These con-
ditions seem valid for DHA. Bell et al. (1995) showed
a DHA increase proportional to dietary levels in tur-
bot PC and PE, without simultaneous EPA increase.
Low retroconversion of DHA to EPA in turbot was
also reported by Tocher and Mackinlay (1990) and
Mourente et al. (1991). Furthermore, the synthesis of
DHA from EPA, via 24:5 and 24:6n-3 (Mourente and
Tocher 1994), seems unlikely when considering the
inhibition of the delta-6 desaturase, suggested before
by the accumulation of 20:2n-6 in the fish and the high
DHA/EPA ratio in the diets. In freshwater fish, n-3
HUFA addition to the diet completely retro-inhibited
the conversion of 18:3n-3 into DHA (Léger et al. 1981;
Olsen et al. 1990) and a similar situation might be as-
sumed in present turbot fed diets which are relatively
rich in n-3 HUFA.

The higher level of 22:5n-3 in fish fed PL could
be due either to an increased EPA elongation in PL-
fed fish, or, more likely, to an enhanced absorption of
this fatty acid from the diet, in analogy with the above
findings for DHA. The validity of EPA as a ‘marker’ of
fatty acid uptake is dubious. The general decrease of
this fatty acid throughout the experiment might have
hindered an improved accumulation by PL. However,
in NL and PC, EPA had decreased less in the PL-fed
fish, in agreement with the expected PL effect. But,
this was not the case in PE where EPA levels appeared
inversely related to DHA levels. Antagonistic relations
between DHA and EPA levels in marine fish PE have
been reported before by Watanabe (1993). The low
response of fish 20:4n-6 to the PL supplementation
is possibly explained by the low analytical precision
linked to the small amount in the fish. But, in fish
PI, where 20:4n-6 reached high levels, there was an
increased incorporation of this fatty acid related to the
dietary PC supply. Moreover the latter increase was
not associated to an inverse variation of EPA in fish
PI, as found by Bell et al. (1995) in turbot fed diets
with different 20:4n-6 contents, but was accompanied
by an increase of all n-3 HUFA.

The observed relationship between the amount of
PC in the diet, fish growth and fish DHA content could
lead to several interpretations. For instance, the higher
DHA in fish fed PC-rich diets might be looked at
as a trivial consequence of their higher final weight.
This is however contradicted by the higher amount of
DHA in the 400-450 mg fish, fed the PC-diets for one
week, compared to the 950 mg PL-deprived fish at
the end of the trial. The other way around, the better
growth of the PL-fed fish is unlikely the consequence
of a better absorption of DHA preventing an essential
fatty acid deficiency, taking into account the important
amount of DHA in all diets (1.2% diet dry weight),
which should be sufficient to satisfy the requirement
of postlarval turbot (Léger et al. 1979; Le Millinaire
et al. 1983). Moreover, the considerable increase of
DHA in the body neutral lipids in all groups during
the course of the experiment suggests the fulfillment of
the DHA requirement for proper membrane functions.
The presently proposed interpretation is that DHA can
be considered as a marker of the neutral fatty acid ab-
sorption. Hence, the above findings are consistent with
the idea that PC stimulates the absorption of dietary
neutral lipid, with as direct consequences more energy
for growth, an increased triglyceride synthesis and an
increased incorporation of diet specific FA.

This conclusion is consistent with previous results
indicating an impaired lipid export from the intestinal
absorptive cells in carp larvae fed PL-free or PI-rich
diets instead of PC-rich diets, resulting in smaller
liver and hepatocyte size (Fontagné et al. 1998). The
link between the latter histological observations and

the growth of the PC-fed carp (Geurden et al. 1998)
coincides with the link between the increased DHA
and triglyceride level and the better growth of PC-fed
turbot in present study. An increased absorption of di-
etary fatty acids is also in accordance with the specific
enhancement of the secretion of intestinal lipoproteins
by PC, as reported in rat cell lines (Field and Mathur
1995).

Acknowledgements

This study was supported by the Flemish Fund for
Scientific Research (I.G. is Research Assistant of
the FWO), the Belgian Ministry of Science Policy
and INVE Aquaculture N.V (Belgium). We wish to
thank Vandemoortele N.V., Coordination Center R&D
(Belgium) and Lucas Meyer GmbH (Germany) for
the provision of the phospholipids and Bühler Ltd
(Switzerland) for providing the facilities and technical
assistance for the extrusion of the experimental diets.
The help of Badia Moustafa and Karla Van Ryck-
eghem is very much appreciated. The authors would
also like to thank Dr. Peter Coutteau for the comments
during the preparation of the experiment.

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