Effects of a diet lacking HUFA on lipid and fatty acid content of intestine and gills of male...

Effects of a diet lacking HUFA on lipid and fatty acidcontent of intestine and gills of male gilthead seabream(Sparus aurata L.) broodstock at different stagesof the reproductive cycle

M. V. Martın • E. Almansa • J. R. Cejas •

A. Bolanos • S. Jerez • A. Lorenzo

Received: 4 March 2011 / Accepted: 22 April 2011 / Published online: 7 May 2011

� Springer Science+Business Media B.V. 2011

Abstract A feeding experiment was carried out to

determine the effect of a diet lacking n-3 and n-6

highly unsaturated fatty acids (HUFA) on lipid and

fatty acid content in intestine and gills of male

gilthead seabream (Sparus aurata L.) broodstock at

different stages of the reproductive cycle: November

(pre-spermatogenesis), March (spermatogenesis), and

June (post-spermatogenesis). For this purpose, gilt-

head seabream broodstock were fed either a control

diet (C) or an n-3 and n-6 HUFA-deficient diet (D).

The results showed no changes in fatty acid content

of polar lipids of intestine and gills from fish fed diet

C at different stages of the reproductive cycle.

However, significant changes were observed in the

fatty acid content of neutral lipids in intestine but not

in gills in this group. Thus, between November and

March, saturates and n-3 HUFA decreased while

monoenes increased. In June, the contents of these

fatty acids had returned to their initial values

(November). Moreover, in fish fed diet D, the fatty

acid content of neutral lipid changed in both intestine

and gills. In intestine NL, a decrease in saturates and

n-3 HUFA and an increment in monoenes were

observed from November to June. In gills, a decrease

was also observed in n-3 HUFA from NL along the

cycle. Nevertheless, n-6 HUFA content remained

unchanged. These results show both tissue specificity

in seasonal mobilization of fatty acids linked to

reproductive processes and the influence of dietary

fatty acids on body composition.

Keywords Broodstock � Dietary fatty acids �Gilthead seabream � HUFA deficiencies � Males

Introduction

Numerous studies have shown that improvement of

broodstock nutrition and feeding greatly enhances the

quality of both sperm and eggs (Harel et al. 1994;

Fernandez-Palacios et al. 1995; Labbe et al. 1995;

Navas et al. 1997; Rodrıguez et al. 1998; Almansa

et al. 1999; Bruce et al. 1999; Asturiano et al. 2001;

Bromage et al. 2001; Mazorra et al. 2003; Jerez et al.

2006; Alavi et al. 2009; Martın et al. 2009; Tocher

2010). As in other teleosts, the maturation cycle in

gilthead seabream broodstock requires large quanti-

ties of macronutrients such as lipids and proteins to

M. V. Martın (&) � E. Almansa � J. R. Cejas � S. Jerez

Instituto Espanol de Oceanografıa, Centro Oceanografico

de Canarias, Gral. Gutierrez N�4, C.P. 38003 Santa

Cruz de Tenerife, Spain

e-mail: [email protected]

A. Bolanos � A. Lorenzo

Laboratorio de Fisiologıa Animal, Departamento de

Biologıa Animal, Facultad de Biologıa, Universidad de La

Laguna, Av. Astrofısico Fco. Sanchez s/n, 38206 Santa

Cruz de Tenerife, Spain

123

Fish Physiol Biochem (2011) 37:935–949

DOI 10.1007/s10695-011-9491-2

be made available for transfer to the developing of

oocytes and spermatozoids. Gilthead seabream (Spa-

rus aurata) continue eating during sexual maturation

and through the spawning season, which in Canary

Islands takes place from the end of December to the

beginning of June (Cejas et al. 1992; Jerez et al.

2006). In these circumstances, the necessary energy

for gonadal development must come from both

dietary of the broodstock and/or from its body

reserves. Teleost gonads are particularly rich in

DHA and EPA (Sargent et al. 2002, Bobe and Labbe

2010). Specifically, spermatozoa membrane contain

extremely high levels of DHA (Labbe et al. 1995;

Asturiano et al. 2001; Bobe and Labbe 2010) Thus,

these fatty acids derived directly from the dietary

input of broodstock as well as from body reserves are

crucial to gonadogenesis in both male and female and

also to fecundity and to embryo and early larval

development (Wade et al. 1994; Mercure and Van

Der Kraak 1995; Asturiano et al. 2001). Previous

studies conducted by our research group have shown

that both ovary and testicle of gilthead seabream

exhibited a dramatic increase in DHA and EPA

contents before the beginning of spawning, being it

partly due to mobilization from the liver and muscle

(Almansa et al. 2001; Martın et al. 2009). Consider-

ing the current trend of replacement of fish meal and

fish oils with plant-derived products in the existing

commercial diets, it is important to know to what

extent these seasonal lipid mobilizations can also

change the fatty acid composition of tissues, espe-

cially if the diet is inadequate to meet the broodstock

requirements. Most studies of lipid mobilization

associated with reproduction have focused on tissues

like liver and muscle, however, no information is

available about seasonal changes in lipid composition

linked to reproductive cycle in other organs like

intestine and gills, whose participation in these

processes could compromise its functionality, espe-

cially if the diet is not adequate to meet the

requirements of the species. The aim of the present

study was to determine the lipid content and fatty

acid composition of intestine and gills of male

gilthead seabream at different stages of the repro-

ductive cycle, when fish were fed with a diet rich in

HUFA (control) or a diet lacking in these fatty acids,

in an attempt to achieve a better understanding of the

patterns of mobilization and retention of lipids and

essential fatty acids during reproduction.

Materials and methods

Animals and diets

Gilthead seabream (S. aurata L.) broodstock were

selected and randomly sorted into six groups (three

groups/diet) of 12 fish each, six females (1.5 kg mean

mass) and six males (0.7 kg mean mass). Each group

of fish was stocked in an 8-m3 tank with a seawater

supply of 30 l/min, assuring suitable water conditions

and an oxygen level close to saturation. The fish were

exposed to natural photoperiod, and the water tem-

perature throughout the experimental period ranged

from 19.0 to 22.8�C.

In the Canary Islands and under captivity, the

spawning season of S. aurata takes place from

January until May to early June, although males

reach maturity in December. Therefore, the experi-

mental period began 1 month before spermatogenesis

and 2 months before spawning (November) and

ended once spermatogenesis and spawning had

concluded (June). The fish had been fed a commercial

diet (Trouw S.A., Spain) containing 12% lipid and

48% protein until the beginning of the experimental

period. At this point, three groups of fish were fed for

7 months a control diet (diet C) containing fish oil as

sole lipid source and therefore, rich in n-3 and n-6

HUFA. The other three groups were fed a deficient

diet (diet D) containing linseed oil and olive oil as

lipid sources and lacking n-3 and n-6 HUFA but rich

in 18:1n-9, 18:3n-3, and 18:2n-6. Fish were fed once

a day 1.5% of their biomass.

In order to ensure that the diets differed only in

terms of fatty acid composition, ingredients used to

formulate the two experimental diets were exactly the

same, with the exception of the lipid source. In this

sense, two diets containing 12% lipids and 46%

protein (dry mass basis, DMB) but with different

fatty acid profiles were manufactured (Table 1).

Taking into account the total amount of lipid supplied

by the fish meal (Canagrosa, Canary Islands, Spain),

65 g kg-1 of fish oil was added to diet C. Therefore,

the amount of n-3 HUFA in the control diet was

approximately 1.8% DMB and EPA/DHA ratio 2:1,

values adequate to meet the n-3 HUFA requirements

of this species broodstock (Fernandez-Palacios et al.

1995). To formulate the n-3 and n-6 HUFA-deficient

diet (diet D), the fat content from the fish meal was

removed using trichloromethane. Then, 42 g kg-1 of

936 Fish Physiol Biochem (2011) 37:935–949

123

linseed oil (ICN, Barcelona, Spain) and 70 g kg-1 of

a commercially available olive oil were added to this

diet. Finally, 10 g kg-1 of vitamin E (Sigma, St.

Louis, MO) was added to both diets. Furthermore, a

mineral and vitamin mixture (Premix) supplied by

Dibaq (Madrid, Spain) was added to both experi-

mental diets according to NRC (1993) recommenda-

tions for fish. After adding water and mixing all the

ingredients in an electric blender, diets were pelleted

by machine and dried at room temperature. After-

ward, pellets were stored at -20�C prior to use.

The tissue samplings were made in November

(before the start of feeding the experimental diets),

March (after 4 months of feeding), and June (after

7 months of feeding). Prior to sampling, the fish were

starved for 48 h to clear the intestinal tract. At each

sampling, three males from each tank were killed and

anterior intestine and gills samples were taken for

lipid analysis. The entire experiment was conducted

in accordance with Spanish law 223/1988 (B.O.E.

18th March) for protection of experimental animals,

in agreement with European law 89/609/CE.

Biochemical analysis

Moisture content analyses were performed using stan-

dard methods (Horwitz 1980). Total lipid from original

tissue was extracted with chloroform/methanol (2:1 by

vol) according to a modification of the method described

by Folch et al. (1957), with the lipid content being

determined by gravimetry (Christie 1982).

Neutral (NL) and polar (PL) lipid fractions were

also obtained from the crude lipids by means of silica

Sep-Pak cartridges (Millipore, Milford, MA), using

chloroform and methanol as solvents according to

Juaneda and Rockelin (1985). NL and PL were

subjected to acid-catalyzed transmethylation with

1% sulphuric acid (v/v) in methanol. The resultant

fatty acid methyl esters (FAME) were purified by

using thin-layer chromatography (TLC) plates (Chris-

tie 1982). FAME were separated and quantified by

using a Shimadzu GC-14A gas chromatograph

equipped with a flame ionization detector (250�C)

and a fused silica capillary column SupelcowaxTM 10

(30 m 9 0.32 mm I.D.). Helium was used as carrier

gas, and the oven initial temperature was 180�C for

10 min, followed by an increase at a rate of 2.5�C/min

to a final temperature of 215�C. Individual FAME was

identified by reference to authentic standards and to

well-characterized fish oil. Prior to transmethylation,

heneicosanoic acid (21:0) was added to the lipid

fractions as an internal standard. The results were

expressed as milligrams of each fatty acid per gram of

tissue dry mass (mg g-1 dry mass).

TLC (20 9 20 cm 9 0.25 mm) plates, precoated

with silica gel (without fluorescent indicator) were

purchased from Macherey–Nagel (Duren, Germany).

Table 1 Proximate composition (mg g-1) and fatty acid

composition (percent of total fatty acids) of the experimental

diets. (Diet C, control diet; Diet D, HUFA-deficient diet)

Diet C Diet D

Crude protein 460.0 460.0

Total lipid 123.9 121.7

Moisture 405.0 390.1

Fatty acids (%)

14 : 0 8.5 ± 1.2 0.7 ± 0.3

15 : 0 0.5 ± 0.1 0.0

16 : 0 21.5 ± 1.6 11.4 ± 1.1

16 : 1# 8.0 ± 0.9 1.0 ± 0.2

16 : 3 0.9 ± 0.1 0.0

16 : 4 1.4 ± 0.1 0.0

18 : 0 3.9 ± 0.2 4.2 ± 0.1

18 : 1 n-9 18.4 ± 0.7 57.1 ± 1.4

18 : 1 n-7 3.5 ± 0.1 1.5 ± 0.1

18 : 2 n-6 5.1 ± 0.1 7.5 ± 0.2

18 : 3 n-3 2.2 ± 0.2 13.8 ± 0.6

18 : 4 n-3 1.6 ± 0.1 0.0

20 : 1? 2.3 ± 0.3 tr

20 : 4 n-6 0.8 ± 0.1 tr

20 : 5 n-3 8.8 ± 1.1 tr

22 : 1? 1.8 ± 0.4 0.0

22 : 5 n-3 1.0 ± 0.2 0.0

22 : 6 n-3 4.3 ± 0.8 0.0

Unknown 2.2 ± 0.2 0.8 ± 0.3

Totals

Saturates 35.0 ± 2.6 16.8 ± 1.3

n-9 18.4 ± 0.7 57.1 ± 1.4

n-3 18.7 ± 2.4 14.4 ± 0.4

n-6 6.5 ± 0.3 7.8 ± 0.2

n-3 HUFA 14.9 ± 2.2 0.6 ± 0.3

n-6 HUFA 1.2 ± 0.3 tr

Results represent means ± SD of three replicates. Totals

include some minor components not shown# Contains n-9 and n-7 isomers? Contains n-11 and n-9 isomers. tr, values 0.3–0.5%; 0.0

values B 0.3%

Fish Physiol Biochem (2011) 37:935–949 937

123

All organic solvents used were of reagent grade and

were purchased from Panreac (Barcelona, Spain).

Statistical analysis

Results are presented as means ± standard deviation

(S.D.) after the analysis of triplicate samples The data

were checked for normal distribution with the one-

sample Kolmogorov–Smirnoff test as well as for

homogeneity of the variances with the Levene test

and, when necessary, arcsine transformation was

performed. The effects of three different spawning

periods on intestine and gills fatty acid composition

for each specific diet were analyzed by one-way

ANOVA followed by Student–Newman–Keuls

(SNK) multiple comparison tests. When normal

distribution and/or homogeneity of the variances were

not achieved, data were subjected to the Kruskall–

Wallis nonparametric test, followed by a nonpara-

metric multiple comparison test (Zar 1984). Differ-

ences between the two dietary treatments were

established by treating the pairs of means with a

Student’s t test. In all statistical tests used, P values of

less than 0.05 were considered statistically different.

The values of each fatty acid were chemometrically

analyzed by principal components analysis (PCA).

Factor scores were subsequently analyzed by one-way

ANOVA followed by SNK multiple comparison tests.

The statistical analysis was performed using the SPSS

package version 15.0 (SPSS Inc, Chicago, USA).

Results

Total lipid content on DMB in both broodstock diets

were almost identical, being 12.39 and 12.17% for

diets C and D, respectively (Table 1). However, they

differed in their fatty acid contents. Diet D was

characterized by having increased proportions of

monounsaturated (18:1n-9) and polyunsaturated

(18:3n-3) fatty acids compared to diet C that had a

higher content of saturated (16:0, 14:0) and highly

unsaturated n-3 and n-6 fatty acids (20:4n-6, 20:5n-3,

22:6n-3).

The total neutral and polar lipid and fatty acid

compositions of neutral and polar lipid from gilthead

seabream (S. aurata L.) intestine fed with C and D

diets are shown in Tables 2 and 3. Neither dietary

lipid nor stages of reproductive cycle had any

significant effect on total neutral and polar lipid

contents of intestine (Tables 2, 3). However, differ-

ences in the fatty acid compositions of the diets and

stages of reproductive cycle were reflected in the

fatty acid compositions of intestine. Major NL fatty

acids in the intestine were 16:0, 18:0, 18:1, 18:2n-6,

20:5n-3, and 22:6n-3. Principal variations were

detected in saturates, monoenes, and n-3 HUFA fatty

acids. From November to March, saturates and n-3

HUFA displayed diminished content in both dietary

groups of fish. The decrease in n-3 HUFA was higher

in fish fed diet D (73%) than in fish fed diet C (55%)

and was due to decreased 22:6n-3, 22:5n-3, 20:5n-3,

and 20:4n-3. By contrast, monoenes showed an

increase of 184 and 191% in diet C and D fish,

respectively, in this period, due to increased 18:1.

Between March and June, in fish fed diet C, total

monoenes, n-6 HUFA and n-3 HUFA recovered the

levels observed in November. However, in fish fed

diet D, total monoenes continued to increase and n-3

HUFA continued to diminish. Therefore, in June, the

levels of monoenes and n-6 were higher in diet D fish

compared to diet C fish, while the n-3 HUFA level

was 3-fold lower in diet D fish than in diet C fish,

reflecting clearly the diet compositions. Thus, prin-

cipal components analysis (PCA), used to examine

the multivariate structure of the data set, confirmed

these trends. The two components of PCA accounted

for the 82% of variation of this data set, although

55% of variation was explained by principal compo-

nent 1 (PC1) itself (Fig. 1a). The PC1 clearly

separated fatty acids that predominate in diet D,

18:1, 18:3n-3 (on the left) from those characteristic of

diet C, 22:5n-3, 22:6n-3 (on the right) reflecting the

differences in diet profiles. Four groups were signif-

icantly separated (SNK, P \ 0.05) and identified as

diet D June, diet D March, diet C March and

November ? diet C June in the first factor score

(Fig. 1b). The principal component 2 (PC2)

accounted for 26.9% of variation, and separated fatty

acids that diminished along the cycle (above the zero

line) from those that did not undergo changes (below

the zero line) (Fig. 1a). Therefore, the fatty acid

profile was also affected, in PC2, by the differences

among the three stages of the reproductive cycle.

Thus, three major clusters were significantly sepa-

rated (SNK, P \ 0.05) as a) November and March

groups, b) June groups, and c) a group corresponding

to March and June diet D (Fig. 1c).


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Fatty acid composition of intestine PL from fish

diet C (Table 3) showed no significant differences

throughout the study period. However, after 7 months

of feeding (June), a decrease in n-3 HUFA content,

particularly 20:5n-3 and 22:6n-3, was observed in fish

fed diet D reflecting clearly the deficient diet. When

data of PL were analyzed by PCA, the two principal

components accounted for 54.3% of variation

(Fig. 2a). PC1 explained 34.2% of variation and

separated several fatty acids representative of diet D

from those characteristic of diet C grouping three

clusters: November and diet C groups, diet D March

group, and diet D June group (SNK, P \ 0.05)

(Fig. 2b). However, the fatty acid profile was also

affected in PC2 (20.1%) by the differences among the

three stages of the reproductive cycle separating

November and March stages from the June stage

(SNK, P \ 0.05) (Fig. 2c).

The NL and PL fatty acid compositions of the gills

in fish fed with diet C and D, in the three stages of the

reproductive cycle, are shown in Tables 4 and 5.

Major fatty acids in the NL gills were 16:0, 18:1n-9,

and 22:6n-3. In diet C fish, no changes were observed

during the three periods studied. However, the fatty

Table 2 Total neutral lipid and fatty acid content (mg g-1 dry mass) of neutral lipids of gilthead seabream intestine from fish fed

with two experimental diets C and D at different stages of the reproductive cycle

November Diet C Diet D

March June March June

Neutral lipid 74.7 ± 6.0 83.1 ± 16.1 67.1 ± 8.6 72.4 ± 13.1 80.1 ± 16.3

14 : 0 1.9 ± 1.01 1.7 ± 0.6 0.3 ± 0.3 0.8 ± 0.32 0.3 ± 0.03

16 : 0 11.0 ± 1.5a1 8.3 ± 0.3b 9.4 ± 0.8ab 5.7 ± 0.42* 6.7 ± 0.92*

18 : 0 3.7 ± 0.3a1 2.7 ± 0.4b 3.6 ± 0.2a 2.6 ± 0.42 3.5 ± 0.512

Saturates 17.2 ± 2.0a1 13.2 ± 0.5b 14.3 ± 1.0ab 9.5 ± 0.52* 10.9 ± 1.42*

16 : 1# 2.8 ± 1.21 2.5 ± 0.6 1.4 ± 0.6 1.2 ± 0.312* 0.6 ± 0.02

18 : 1# 11.3 ± 2.5b3 28.0 ± 7.3a 9.8 ± 1.4b 32.4 ± 4.62 48.3 ± 7.11*

Monoenes 18.3 ± 5.1b3 33.6 ± 6.1a 12.4 ± 2.4b 35.0 ± 4.22 49.6 ± 7.31*

18 : 2 n-6 3.9 ± 0.9a 4.2 ± 0.6a 1.9 ± 0.3b 4.1 ± 0.7 5.2 ± 0.5 *

20 : 4 n-6 1.0 ± 0.2ab1 0.5 ± 0.1b 1.7 ± 0.7a 0.5 ± 0.22 0.4 ± 0.02*

22 : 4 n-6 0.1 ± 0.1 0.0 0.2 ± 0.1 0.0 0.1 ± 0.0

22 : 5 n-6 0.2 ± 0.0b1 0.1 ± 0.0b 0.3 ± 0.1a 0.1 ± 0.112 0.02

Total n-6 5.5 ± 0.8 5.0 ± 0.6 3.7 ± 0.8 5.0 ± 0.5 5.9 ± 0.5*

18 : 3 n-3 0.6 ± 0.3b3 3.7 ± 1.4a 0.5 ± 0.1b 5.5 ± 1.52 7.7 ± 0.51*

18 : 4 n-3 0.6 ± 0.3a 0.4 ± 0.1a 0.1 ± 0.1b 0.2 ± 0.2 0.0

20 : 4 n-3 0.6 ± 0.21 0.3 ± 0.0 0.3 ± 0.0 0.1 ± 0.12 0.0c3*

20 : 5 n-3 3.4 ± 0.5a1 2.4 ± 0.7ab 1.7 ± 0.1b 0.9 ± 0.32* 0.2 ± 0.12*

22 : 5 n-3 1.3 ± 0.31 0.7 ± 0.2 1.4 ± 0.4 0.4 ± 0.22 0.2 ± 0.02*

22 : 6 n-3 8.2 ± 1.81 2.7 ± 0.5 7.9 ± 3.7 2.1 ± 1.12 0.9 ± 0.12*

Total n-3 14.9 ± 1.9 10.3 ± 0.3 11.1 ± 3.5 9.4 ± 0.4* 9.3 ± 0.3

n-6 HUFA 1.3 ± 0.3ab1 0.6 ± 0.1b 2.3 ± 0.9a 0.7 ± 0.22 0.5 ± 0.12*

n-3 HUFA 13.7 ± 2.5a1 6.2 ± 1.3b 10.9 ± 3.8ab 3.7 ± 1.62 1.5 ± 0.23*

18:1/n-3 HD 0.8 ± 0.3b2 4.7 ± 2.1a 1.0 ± 0.5b 10.0 ± 4.42 31.8 ± 5.01*

Results represent means ± SD of three replicates. Values in the same row bearing different superscript symbols are significantly

different (P \ 0.05); letters correspond to the comparison made in diet C group and numbers correspond to the comparison made in

diet D group. Totals include some minor components not shown

* Pairs of means corresponding to the C and D diet values at each spawning period that were significantly different (P \ 0.05)# Contains n-9 and n-7 isomersD 18:1n-9/n-3 HUFA. 0.0, values \0.05 mg g-1


123

acid profile of diet D fish clearly reflected those of

diets. Specifically, total n-3 HUFA, particularly

20:5n-3, 22:5n-3, and 22:6n-3, were lower after

7 months of feeding (June) compared to November.

However, 20:4n-6 remained unchanged despite its

low concentration in the diet. Also, in June, the levels

of 18:1n-9, 18:2n-6, and 18:3n-3 were higher and

20:5n-3 and 22:5n-3 lower in diet D fish compared to

diet C fish. The PCA confirmed these differences and

disclosed that the first two factors account for over

69.4% of the variability within the data (Fig. 3a).

PC1 was positively related with saturated and mono-

ene fatty acids, and no grouping was recognized on

the basis of the first factor score. However, PC2

clearly separated the most abundant fatty acids in

diets C and D. Plots of factor 2 demonstrate a

significant separation of diet D June group from other

groups (P \ 0.05) (Fig. 3b).

In the PL of gills, no changes were observed in fish

fed diet C (Table 5). In fish fed diet D, monoenes,

specifically 16:1 and 22:1, increased from November

to March. In June, 18:2n-6, 18:3n-3, and 18:1n-9/n-3

Table 3 Total polar lipid and fatty acid content (mg g-1 dry mass) of polar lipids of gilthead seabream intestine from fish fed with

two experimental diets C and D at different stages of the reproductive cycle



Polar lipid 27.0 ± 5.6 26.1 ± 8.3 25.6 ± 3.3 28.6 ± 7.7 36.8 ± 10.8

14 : 0 0.4 ± 0.1a1 0.3 ± 0.0ab 0.1 ± 0.0b 0.2 ± 0.12 0.1 ± 0.12

16 : 0 4.7 ± 0.9 3.7 ± 0.4 4.6 ± 1.3 3.1 ± 0.5 4.9 ± 1.7

18 : 0 2.7 ± 0.6 1.8 ± 0.6 2.5 ± 0.8 2.3 ± 0.5 3.6 ± 1.3

Saturates 8.1 ± 1.6 6.0 ± 0.9 7.5 ± 2.2 5.6 ± 1.1 8.9 ± 3.1

16 : 1# 0.4 ± 0.1 0.4 ± 0.2 0.2 ± 0.0 0.2 ± 0.1 0.1 ± 0.1

18 : 1# 2.0 ± 0.5 4.2 ± 1.3 1.8 ± 0.1 5.2 ± 1.8 7.7 ± 3.8

20 : 1? 0.2 ± 0.0a 0.2 ± 0.0a 0.1 ± 0.0b 0.2 ± 0.1 0.2 ± 0.1

Monoenes 3.2 ± 0.6 5.2 ± 1.3 2.2 ± 0.3 5.9 ± 1.9 8.2 ± 3.9

18 : 2 n-6 0.9 ± 0.3 1.0 ± 0.4 0.5 ± 0.1 1.3 ± 0.6 1.6 ± 0.7

20 : 2 n-6 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.1 0.1 ± 0.0 0.1 ± 0.0

20 : 4 n-6 0.3 ± 0.02 0.4 ± 0.1 0.4 ± 0.0 0.5 ± 0.11 0.6 ± 0.21

22 : 5 n-6 0.1 ± 0.0 0.2 ± 0.1 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0

Total n-6 1.4 ± 0.3 1.8 ± 0.4 1.3 ± 0.1 2.1 ± 0.7 2.6 ± 0.9

18 : 3 n-3 0.1 ± 0.0 0.4 ± 0.3 0.1 ± 0.0 0.8 ± 0.4 1.1 ± 0.6

20 : 4 n-3 0.1 ± 0.01 0.1 ± 0.0 0.1 ± 0.0 0.012 0.02*

20 : 5 n-3 0.9 ± 0.1b1 1.3 ± 0.3a 0.4 ± 0.1b 0.6 ± 0.21* 0.3 ± 0.12

22 : 5 n-3 0.3 ± 0.0 0.5 ± 0.1 0.4 ± 0.1 0.3 ± 0.0* 0.3 ± 0.1

22 : 6 n-3 4.6 ± 0.41 3.7 ± 0.4 4.1 ± 0.3 3.0 ± 0.62 2.9 ± 0.72*

Total n-3 6.1 ± 0.5 6.2 ± 0.8 5.1 ± 0.3 5.0 ± 1.2 4.7 ± 1.6

n-6 HUFA 0.4 ± 0.0 0.7 ± 0.2 0.7 ± 0.1 0.6 ± 0.1 0.9 ± 0.2

n-3 HUFA 5.9 ± 0.61 5.7 ± 0.6 5.0 ± 0.2 4.1 ± 0.812 3.6 ± 0.92

18:1/n-3 HD 0.3 ± 0.1b2 0.7 ± 0.2a 0.3 ± 0.0b 1.2 ± 0.312 2.0 ± 0.71*


different (P\0.05); letters correspond to the comparison made in diet C group and numbers correspond to the comparison made in


* Pairs of means corresponding to the C and D diet values at each spawning period that were significantly different (P \ 0.05)# Contains n-9 and n-7 isomers? Contains n-11 and n-9 isomersD 18:1n-9/n-3 HUFA. 0.0, values \ 0.05 mg g-1


123

HUFA were higher in fish fed diet D compared to diet

C, reflecting the diet profile. However, no changes

were observed in n-3 and n-6 HUFA. From principal

components analysis, two principal components

accounted for 67.4% of variation (Fig. 4a). PC1

explained 37.4% of variation and separated fatty

acids representative of diet C from those character-

istic of diet D. Thus, two groups were significantly

separated (P \ 0.05) in the first factor score

(Fig. 4b), whereas no grouping was recognized on

the basis of second factor score.

Discussion

Designing diets for broodstock fish requires a thor-

ough understanding of the changes in lipid and fatty

acid deposition over the seasonal reproductive cycle

Fig. 1 Component plot (a) and factor score plots (b, c) of the PCA for the intestine fatty acid profile of neutral lipids in fish at

different stages of the reproductive cycle. Circles stand for different clusters in the factor score 1 (b) and factor score 2 (c)


123

in order to adjust diet composition to the species-

specific requirements. Several studies have shown

that lipids are mobilized from muscle and liver to

support reproductive development and spawning

(Harel et al. 1994; Almansa et al. 2001; Cejas et al.

2004; Rodrıguez et al. 2004; Martın et al. 2009).

However, lipid composition of other organs such as

intestine and gills may also be affected by seasonal

changes associated with reproductive cycle, espe-

cially if the diet is not adequate to meet the lipid

requirements of the species.

The results of the present study showed that in

March, at the stage of spermatogenesis, an important

loss of n-3 HUFA fatty acids together with an

increase in monoene fatty acids was detected in

neutral lipid of anterior intestine not only in fish fed

diet D but also in diet C fish. The decrease in these

fatty acids from intestinal tissue in fish fed diet C may

be related with the important demand of n-3 HUFA,

specifically 22:6n-3, in this stage, possibly linked to

reproductive processes. It is known that both gonads

and sperm have a high content of DHA (Labbe et al.

Fig. 2 Component plot (a) and factor score plots (b, c) of the PCA for the intestine fatty acid profile of polar lipids in fish at different

stages of the reproductive cycle. Circles stand for different clusters in the factor score 1 (b) and factor score 2 (c)


123

1995; Asturiano et al. 2001; Martın et al. 2009, Bobe

and Labbe 2010). In fish fed diet D, this reduction

was even more pronounced due to dietary deficiency

in these fatty acids. Moreover, an accumulation of

18:1n-9 in this stage was observed in both groups of

fish, with this increase being more pronounced in fish

fed diet D due to its higher content in the diet

supplied to this group. However, the accumulation of

18:1n-9 also in diet C fish suggests a metabolic

strategy in this tissue to maintain the unsaturation

index (Farkas et al. 2001; Jobling and Bendiksen

2003; Ruyter et al. 2006).

In June (post-spermatogenesis), when the higher

demand for DHA has concluded, fish fed with diet C

were able to recover the original intestine fatty acid

composition of NL by increasing the DHA content.

These metabolic changes observed in fish fed control

diet throughout the reproductive cycle, confirmed by

PCA analysis, suggest seasonal lipid changes in

intestine associated with reproductive condition.

Similar findings were reported by Huynh et al.

(2007) in herring (Clupea harengus pallasi), in which

differences between fatty acid composition of intes-

tine from spawning and non-spawning fish were

Table 4 Total neutral lipid and fatty acid content (mg g-1 dry mass) of neutral lipids of gilthead seabream gills from fish fed with

two experimental diets C and D at different stages of the reproductive cycle



Neutral lipid 27.7 ± 2.6 22.4 ± 6.3 25.0 ± 3.6 23.1 ± 3.9 25.2 ± 2.9

14 : 0 0.5 ± 0.2 0.5 ± 0.1 0.4 ± 0.0 0.6 ± 0.2 0.3 ± 0.1

16 : 0 3.3 ± 0.5 3.2 ± 0.9 3.1 ± 0.4 3.4 ± 0.9 3.4 ± 0.3

18 : 0 0.9 ± 0.1 0.8 ± 0.2 1.0 ± 0.3 0.8 ± 0.2 1.1 ± 0.1

Saturates 4.9 ± 0.6 4.5 ± 1.2 4.6 ± 0.8 4.9 ± 1.3 5.0 ± 0.4

16 : 1# 1.3 ± 0.3 1.3 ± 0.5 1.0 ± 0.1 1.3 ± 0.3 0.9 ± 0.1

18 : 1 n-9 3.8 ± 0.7 3.8 ± 1.4 3.5 ± 0.2 4.4 ± 1.1 5.9 ± 1.1*

18 : 1 n-7 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.4 ± 0.0

20 : 1? 0.5 ± 0.1 0.4 ± 0.2 0.3 ± 0.0 0.5 ± 0.1 0.4 ± 0.1

Monoenes 6.6 ± 1.3 6.3 ± 2.4 5.8 ± 0.4 7.1 ± 1.7 7.8 ± 1.1*

18 : 2 n-6 0.8 ± 0.212 0.6 ± 0.2 0.6 ± 0.0 0.6 ± 0.12 1.0 ± 0.11*

20 : 4 n-6 0.4 ± 0.112 0.2 ± 0.0 0.5 ± 0.2 0.2 ± 0.12 0.4 ± 0.11

n-6 1.4 ± 0.21 1.0 ± 0.3 1.3 ± 0.3 1.0 ± 0.22 1.7 ± 0.11

18 : 3 n-3 0.1 ± 0.12 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.02 0.4 ± 0.11*

20 : 5 n-3 0.8 ± 0.11 0.5 ± 0.2 0.6 ± 0.2 0.3 ± 0.12 0.2 ± 0.12*

22 : 5 n-3 0.4 ± 0.01 0.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.02 0.1 ± 0.12*

22 : 6 n-3 2.9 ± 0.41 1.8 ± 0.7 2.2 ± 0.4 1.4 ± 0.612 1.3 ± 0.82

n-3 4.5 ± 0.41 2.9 ± 1.1 3.3 ± 0.7 2.2 ± 0.82 2.2 ± 1.12

n-6 HUFA 0.5 ± 0.112 0.3 ± 0.1 0.6 ± 0.3 0.3 ± 0.12 0.6 ± 0.11

n-3 HUFA 4.3 ± 0.41 2.6 ± 1.0 3.2 ± 0.7 2.0 ± 0.82 1.7 ± 1.02

18:1/n-3 HD 0.9 ± 0.3b 1.4 ± 0.0a 1.1 ± 0.2ab 2.5 ± 1.2 4.2 ± 2.3




* Pairs of means corresponding to the C and D diet values at each spawning period that were significantly different (P\0.05)# Contains n-9 and n-7 isomers? Contains n-11 and n-9 isomersD 18:1n-9/n-3 HUFA. 0.0, values \ 0.05 mg g-1


123

found. Likewise, in Esox lucius, intestinal lipids are

mobilized before any change in lipid from other

tissues is apparent (Navarro and Gutierrez 1995).

In fish fed with diet D, the deficiencies in HUFA

were accentuated in June, while monoene content was

increased, reflecting clearly the influence not only

seasonal changes linked to reproductive process but

also the fatty acid profile of the deficient diet supplied

to this group. This notion is supported by PCA analysis

that revealed that the 55% of variation in the intestine

fatty acid profile of NL is explained by dietary

components. In addition, NL of November group and

fish fed diet C groups (March and June) appeared as a

highly homogeneous group, whereas fish fed diet D

groups deficient in HUFA appeared as outlier groups

in the PCA analysis. Likewise, numerous studies have

shown the critical influence of diet on fatty acid

composition of intestine and enterocytes (Olsen et al.

1999, 2000; Cahu et al. 2000; Caballero et al. 2003;

Tocher et al. 2002, 2004; Ruyter et al. 2006).

On the other hand, the fatty acid composition of

PL was less affected by diet than the fatty acid

Table 5 Total polar lipid and fatty acid content (mg g-1 dry mass) of polar lipids of gilthead seabream gills from fish fed with two

experimental diets C and D at different stages of the reproductive cycle



Polar lipid 11.0 ± 0.8 11.1 ± 2.9 12.6 ± 3.5 11.2 ± 1.7 11.0 ± 0.7

14 : 0 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.3 ± 0.1 0.2 ± 0.0*

16 : 0 2.2 ± 0.4 2.7 ± 0.8 3.2 ± 0.7 2.4 ± 0.4 2.5 ± 0.3

18 : 0 0.9 ± 0.1 0.9 ± 0.3 1.2 ± 0.2 0.8 ± 0.2 1.1 ± 0.1

Saturates 3.5 ± 0.5 3.9 ± 1.2 4.9 ± 1.1 3.5 ± 0.6 3.9 ± 0.4

16 : 1# 0.2 ± 0.02 0.4 ± 0.1 0.3 ± 0.1 0.4 ± 0.11 0.2 ± 0.02

18 : 1 n-9 1.0 ± 0.1 1.3 ± 0.3 1.2 ± 0.4 1.7 ± 0.3 1.5 ± 0.3

18 : 1 n-7 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.1 0.2 ± 0.0 0.1 ± 0.0

20 : 1? 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.0 0.1 ± 0.0

22 : 1? 0.1 ± 0.02 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.01 0.1 ± 0.012

Monoenes 1.6 ± 0.22 2.2 ± 0.5 2.0 ± 0.6 2.8 ± 0.51 2.2 ± 0.212

18 : 2 n-6 0.2 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.0*

20 : 2 n-6 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.1 0.1 ± 0.0

20 : 4 n-6 0.1 ± 0.01 0.0 0.1 ± 0.1 0.02 0.1 ± 0.012

Total n-6 0.4 ± 0.012 0.3 ± 0.0 0.3 ± 0.2 0.3 ± 0.02 0.4 ± 0.01

18 : 3 n-3 0.0 0.0 0.0 0.0 0.1 ± 0.0*

20 : 5 n-3 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.1 0.0 0.0

22 : 5 n-3 0.1 ± 0.0 0.0 0.1 ± 0.0 0.0 0.0

22 : 6 n-3 0.7 ± 0.3 0.4 ± 0.1 0.4 ± 0.2 0.3 ± 0.1 0.2 ± 0.1

Total n-3 1.0 ± 0.4 0.5 ± 0.1 0.6 ± 0.3 0.4 ± 0.2 0.4 ± 0.1

n-6 HUFA 0.1 ± 0.01 0.1 ± 0.0 0.1 ± 0.1 0.02 0.1 ± 0.012

n-3 HUFA 1.0 ± 0.4 0.5 ± 0.1 0.6 ± 0.3 0.3 ± 0.2 0.3 ± 0.1

18:1/n-3 HD 1.1 ± 0.5b 2.6 ± 0.7a 2.2 ± 0.5ab 6.0 ± 2.7 5.0 ± 1.7*




* Pairs of means corresponding to the C and D diet values at each spawning period that were significantly different (P\0.05)# Contains n-9 and n-7 isomers? Contains n-11 and n-9 isomersD 18:1n-9/n-3 HUFA. 0.0, values \ 0.05 mg g-1


123

composition of NL. Despite this, the n-3 HUFA

levels in the intestine PL of diet D fish diminished

significantly after 7 months of feeding with a HUFA-

deficient diet. Former studies have also reported that

a period of feeding a HUFA-deficient diet can

generate deficiency in the proportion of these fatty

acids in the polar lipids of intestine in species such as

rainbow trout (O. mykiss W.) (Di Costanzo et al.

1983), turbot (Scophthalmus maximus L.) (Bell et al.

1985), and gilthead seabream (Sparus aurata L.)

Fig. 3 Component plot

(a) and factor score plot

(b) of the PCA for the gills

fatty acid profile of neutral

lipids in fish at different

stages of the reproductive

cycle. Circles stand for

different clusters in the

factor score 2


123

(Fountoulaki et al. 2003). Also, it has been seen that

the enterocyte membranes are influenced by the

dietary lipid source (Olsen et al. 1999, 2000, 2003;

Cahu et al. 2000; Mourente et al. 2005; Almaida-

Pagan et al. 2007).

The gills from fish fed diet C, unlike the intestine,

did not experience significant changes in their neutral

and polar lipid content, nor in the fatty acid content

along the reproductive cycle. These findings show

tissue specificity in seasonal variations of certain

Fig. 4 Component plot

(a) and factor score plot

(b) of the PCA for the gills

fatty acid profile of polar

lipids in fish at different

stages of the reproductive

cycle. Circles stand for

different clusters in the

factor score 1


123

fatty acids. However, the diet lacking in HUFA was

reflected in the fatty acid composition of gills from

fish fed diet D after 7 months of feeding. At this time,

a decrease was observed in n-3 HUFA fatty acids that

affected the NL. The factor score plot showed two

major clusters corresponding to the diet D June group

and the other groups (November, March diet C and D,

and diet C June) which appeared as a homogeneous

group, evidence of robustness of the NL fatty acid

profile of this tissue along the reproductive cycle

when requirements are covered. Moreover, the n-3

HUFA content of gills PL in fish fed diet D did not

diminish along the cycle. The decrease in n-3 HUFA

in the NL compared with their conservation in PL,

when these fatty acids are absent from the diet,

appears to be a strategy to conserve these fatty acids

in the PL to maintain membrane structure and

function.

In fish, 20:4n-6 plays an important role despite

not presenting a high percentage, as it is the most

important precursor of eicosanoids in fish (Bell and

Sargent 2003; Evans et al. 2005). The present results

showed preferential conservation of this fatty acid in

both intestine and gills PL after 7 months of feeding

a diet lacking in HUFA. This retention may be

necessary to maintain eicosanoid synthesis in these

tissues, which guarantees the integrity and function-

ality of the branchial and intestinal epithelia.

Supplementation of 20:4n-6 in commercial gilthead

seabream broodstock diets is quite low despite the

fact that this species in the wild contains 10 times

the amount present in cultured seabream (Grigorakis

et al. 2002). Previous studies have suggested the use

of arachidonic-supplemented diets for several mar-

ine teleosts (Bell et al. 1996; Cejas et al. 2003,

2004; Rodrıguez et al. 2004; Perez et al. 2007;

Martın et al. 2009). The retention of this fatty acid

observed in the present study in both groups of fish

leads us to suggest a similar recommendation for

gilthead seabream males, particularly at the repro-

duction stage.

In summary, the results of the present study

showed changes in the fatty acid composition of

both intestine and gills of male gilthead seabream

broodstock fed either a control diet or an n-3 and

n-6 HUFA-deficient diet, these changes being more

accentuated in intestine than in gills. These tissue-

specific variations can be attributed not only to the

influence of diet fatty acid profile but also probably

to seasonal changes associated with reproduction.

Acknowledgments Dr. Antonio Lorenzo is member from the

Instituto de Tecnologıas Biomedicas (ITB) of La Laguna

University (Spain).

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