PhD
Thesis
[ ee
o
eeding
gil hcad
scab cam
(Spa us
au a a)
wi h di c cn Ic cls
u
n-3
and
n-6 lipids om cgc ablc oils on ish
hcal h
and
esis ancc o s ess
n
ｊｾＡＱ＠
Anexo I
Da
BEGOÑA ACOSTA HERNÁNDEZ, SECRETARIA DEL
INSTITUTO UNIVERSITARIO
DE
SANIDAD ANIMAL Y
SEGURIDAD ALIMENTARIA
DE
LA
UNIVERSIDAD
DE
LAS
PALMAS
DE
GRAN CANARIA,
CERTIFICA,
Que
el
Consejo de Doc o es del Depa amen o en
su
sesión de
echa 26 de no iemb e de 2009 omó
el
acue do de da
el
consen imien o pa a
su
ami ación, a
la
esis doc o al i ulada
"E ec o eeding gil head seab eam (Spa us au a a) wi h di e en
le els o n-3 and n-6 lipids om ege able oils
on
ish heal h and
esis ance o s ess
",
p esen ada po
el
doc o ando D Rachid Ganga
y di igida po
la
Doc o a
Da
Ma isol Izquie do López y el Doc o D.
Go don Bell.
y pa a que así cons e, y a e ec os de
lo
p e is o en
el
A O
73.2 del
Reglamen o de Es udios de Doc o ado de es a Uni e sidad, i mo
la
p esen e en Las Palmas de G an Cana ia, a ein iseis de no iemb e
de dos mil nue e.
Anexo
11
UNIVERSIDAD
DE
LAS PALMAS DE GRAN CANARIA
Depa amen o: Ins i u o Uni e si a io de Sanidad
An
imal y Segu idad
Alimen a ia
P og ama de Doc o ado: Acuicul u a
Tí ulo de
la
Tesis
"E ec o eeding gil head seab eam (Spa us a u a a ) wi h di e en
le els o n-3 and n-6 lipids om ege able oils
on
ish heal h and
esis ance o s ess",
Tesis Doc o al p esen ada po O Rachid Ganga
Di igida po
la
Ooc o a
oa
Ma isol Izquie do López y
el
Ooc o
O.
Go don
Bell.
El/la Ooc o ando/a,
de G an Cana ia, a 26 de no iemb e de 2009
 
E ec o eeding gil head seab eam
(Spa us au a a) wi h di e en le els o
n-3 and n-6 lipids om ege able oils on
ish heal h and esis ance o s ess
Rachid Ganga
G upo de In es igación en Acuicul u a,
Ins i u o Cana io de Ciencias Ma inas
Uni e sidad de Las Palmas de G an Cana ia (IUSA)
Being a hesis submi ed o he deg ee o
Doc o o Phylosophy
In he Uni e si y o Las Palmas de G an Cana ia, 2009
Di ec o s:
P o . Ma isol Izquie do López & P o . Go don Bell






“ En el nomb e de Alá, el Compasi o, el Mise ico dioso, Él es
Quien ha suje ado el ma pa a que comáis de él ca ne esca y
ob engáis de él ado nos que pone os. Y es que las na es lo
su can. Pa a que busquéis Su a o . Quizás, así, seáis
ag adecidos”
16 - Las abejas (Al nahl), e so 14. Re elado an es de Hig ah
(Sag ado CORAN)

CONTENTS
Lis o ables
Lis o igu es
Lis o abb e ia ions
Acknowledgmen s
1. Gene al In oduc ion 1
1- Lipids as a cons i uen o ish die s………………………………………………1
1.1- Lipids and a y acids……………………………………….....………….…1
1.2- Lipids unc ions in ish..…..……………………………………………...…2
1.3- Lipids sou ces in aqua eeds………………………………………..……….5
1.4- Vege able oils and ish heal h………………………………….…………...8
1.5- Lipid nu i ion and ish esis ance o s ess…….………………………..11
2- S ess in Fish….……………………………………….................……………...12
2.1- S ess esponses…………………………………………......……….……..12
2.2- Head kidney as a key o gan in s ess esponse…………………………...15
2.3- Co isol: as a s ess ho mone indica o in ish……………………......….17
2.4- Co icos e oids biosyn hesis by in e enal cells…...………….……….....19
2.5- Co isol oles in ish…………………………………………………..……20
3- Gil head Seab eam as a model species o his hesis.….…………………….20
4- Objec i es……..…………………………………………………………………22
2. Gene al ma e ials and me hods 39
2.1. Expe imen al condi ions………..………….……………………………….…39
2.2. S udied species…………………… …......……………………………………41
2.3 - T ial I: pa o RAFOA P ojec ....…………………………...……...……….42
2.3.1- Animals and die s……………………………………...…………………42
2.3.2- Sampling p ocedu e……………………………………...……………....45
2.3.3- Lipids ex ac ion and analysis……………………………..……………45
2.3.4. Ex ac ion, sepa a ion and enzyme immunoassay o PGE isome s..…46
2.3.5. Lep in immunoassay……………………………………………………..49
2.4. T ial II: Pa o LINOSALUD p ojec ..……………………………………..51
2.4.1. Animals and Die s……………………......………………………….…...51
2.4.2. Sampling p ocedu e……………………………………………………...54
2.4.3. P epa a ion and s imula ion o head kidney issue…………………….54
2.4.4. S ess Panels……………………………………………………………....55
2.4.5. Biochemical analysis…………………………………………………..….56
2.4.6. Co isol measu emen s…………………………………………………...56
2.5. S a is ical analysis…….……………………………..………………..…….57
3. E ec o eeding seab eam (Spa us au a a) wi h a blend o soybean and linseed
oil on g ow h, eed u iliza ion and body a y acids composi ion…………………..59
4. S ess esponse in seab eam (Spa us au a a) held unde c owded condi ions and
ed die s wi h di e en le els o inclusion o linseed and/o soybean oil…………..91
5. E ec o die a y lipids on plasma a y acid p o iles and p os aglandina and
lep in p oduc ion in gil head seab eam (Spa us au a a)…………………………..119
6. Modula ion o ACTH-induced co isol elease by polyunsa u a ed a y acids in
in e enal cells om gil head seab eam, Spa us au a a…………………………...129
7. ACTH-s imula ed co isol- elease by he head-kidney-in e enal issue om
seab eam (Spa us au a a) ed wi h linseed and soybean oil………………………137
8. Gene al conclusions…………………………………………………………....….163
9. Summa y………………………………………………….………………….……165
Resumen…………………………………………………………………………...167
A abic summa y…………………………………………………………………..171
10. Resumen ampliado…………….…..……...……………………………………..173
Lis o Publica ion……………………………………………………………………227
Cu iculum i ae ……………………………………………………………………..229
Lis o Tables
Table 2.1: The ypes and % o oils in he expe imen al die s expe imen
I………………………………………………………………………………………....43
Table 2.2: Main a y acids o he di e en expe imen al die s (g/100 g a y acid)
(5mm) ………………………………………………………………………………….44
Table 2.3: Main ing edien s con en s o he expe imen al die s (in %) expe imen II....51
Table 2.4: Vi amins and mine als con en s o he expe imen al die s………………….52
Table 2.5: The ypes and % o oils in he expe imen al die s…………………………..52
Table 2.6: Fa y acids p o ile o he expe imen al die s………………………………..53
Table 3.1: Main ing edien s con en s o he expe imen al die s………………………..63
Table 3.2: P oxima e composi ion (% d y weigh ) o expe imen al die s and p opo ions
e en lipid sou ces (% o oil inclusion) con ained in he expe imen al die s...63 o he di
Table 3.3: Fa y acid composi ions o he expe imen al die s (g/100 g o al a y
acids)……………………………………………………………………………………64
Table 3.4: G ow h and e iciency in u iliza ion o eed by gil head seab eam ed he
expe imen al die s……......……………………………………………………………..69
Table 3.5: Fa y acid p o ile o  muscle om gil head sea b eam ed he
expe imen aldie s…………………………………………………………………………………………...70
Table 3.6: Fa y acid p o ile o li e om gil head sea b eam ed he expe imen al
die s……………………………………………………………………………………..71
Table 3.7: Fa y acid p o ile o gills om gil head sea b eam ed he expe imen al
die s……………………………………………………………………………………..72
Table 4.1: Main ing edien s o he expe imen al die s used……...……………………95
Table 4.2: Fa y acid composi ion o he expe imen al die s (g a y acid/ 100g o o al
a y acids)…………………………………………………………………………......98
Table 4.3: E ec o eeding ege able oils on HK a y acids p o ile (g a y acid/100g
o al a y acids)……………………………………………………………………….99
Table 4.4: Co isol in plasma o ish om he c owding ial (ng/ml o plasma)……100
Table 4.5: Co ela ion coe icien s be ween co isol le els and di e en head kidney
a y acids ……………………………………………………………………………102
Table 5.1: Fa y acid composi ions o he 5 mm expe imen al die s…………………121
Table 5.2: Plasma pola lipid a y acid composi ions……………………………......122
Table 5.3: Plasma neu al lipid a y acid composi ions……………………………...123
Table 5.4: The a io be ween a y acid pe cen in plasma lipid classes and die a y
pe cen o he same a y acid (% Fa y acid in plasma/% a y acid in die )…………..123
Table 5.5: Concen a ion o PGE2 and PGE3 in plasma om seab eam ed expe imen al
die s……………………………………………………………………………………123
Table 6.1: E ec o wo a y acid concen a ions (50 and 150 mM) and wo incuba ion
imes (1 h and 3 h) o h ee polyunsa u a ed a y acids on co isol sec e ion s imula ion
ac o ……………………………………………………………………………….….130
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Acknowledgmen s
The ollowing PhD hesis was pa o he EU F amewo k V p ojec , Resea ching
Al e na i es o Fish Oil in Aquacul u e (RAFOA), Q5RS-200-30058 and Spanish
p ojec “E ec o de la sus i ución pa cial del acei e de pescado po acei e de lino en la
die a sob e la salud y esis encia al es és” (AGL2004-08151-CO302). Du ing my PhD
wo k, I was g an ed om di e en ins i u ions, I am e y g a e ul o Di ección Gene al
de Relaciones con A ica del Gobie no de Cana ias, Agencia Española de Coope ación
In e nacional (AECI) and CIHEAM o gi ing me his g ea oppo uni y. I was also
suppo ed, in pa , h ough an awa d o GR om he Eu opean Union Access o
Resea ch In as uc u es (ARI) Ac ion o he Imp o ing Human Po en ial P og amme
(con ac HPRI-CT-2001-00180) du ing his s age in Ins i u e o Aquacul u e, Uni e si y
o S i ling.
I wish o exp ess my since e g a i ude o P o Ma isol Izquie do o leading me
o he ascina ing wo ld o ish nu i ion, o he g ea scien i ic, mo al, p o essional
suppo and o opening me he way o be a iend. I am e y g a e ul and I can only say
THANK YOU.
Pa o he expe imen s o he hesis was done a he depa men o nu i ion,
Ins i u e o Aquacul u e, Uni e si y o S i ling. I am g a e ul o P o Go don Bell, o
he oppo uni y o wo k a his labo a o y and his ad ices ha e been in aluable. I am
g a e ul o him o accep ing o be a co-supe iso o he p esen hesis and his
a ailabili y all hese yea s. Fiona McGhie is also acknowledged o he echnical
suppo wi h he p os aglandins and lep ins measu emen s. I wish o exp ess my
g a i ude o all he s a o he depa men o nu i ion o hei help and hei assis ance,
especially o D . James Hende son, P o . D. Toche , D . J. Good, Vasilis, Jo ge and
Yannis o sha ing a wonde ul days.
A pa o he in i o expe imen and co isol analysis was done a he
depa men o Cell Biology, Physiology and Immunology, Uni e si a Au onòma de
Ba celona. I wish o hank P o Lluis To o his assis ance and supe ision du ing he
in i o expe imen and co isol analysis. My hanks also, o Sheila Ons, C is ina
Ma quez, Lau a Ace e e, Ad iana, C is ina and Ca men Doña e o hei since e
iendship and use ul help.

I wish o hank P o William Powell, Meakins Ch is ies Labo a o ies, McGill
Uni e si y, Mon eal Canada, o opening me he doo s o his labo a o y o lea n HPLC
echniques o sepa a es Lipoxygenases me aboli es, and his own house o sha e a ew
momen s wi h his amily. I spend a e y wonde ul 3 mon hs he e. Thanks o Syl ie,
Chan al, Gail and F ancois o making my aining e y p oduc i e and amazing a he
same ime.
I wish o hank D . Daniel Mon e o, o his ad ices and his special conside a ion
o ou ela ionship. Many hanks o D . Lidia Robaina, D , Juan Manuel A onso, D .
Ra ael Ginés, D . Ma ia Jose Caballe o, D . Hipóli o Fe nandez, D . Juan Soco o, D .
Rica do Ha oun and D . Ja ie Roo o hei use ul help and ad ices. I did no ha e
enough ime o know A. Valencia, bu I am su e he was a g ea man God me cy o him.
I am e y g a e ul o P o Albe Tacon o his ad ices, cons uc i e c i icism and
h ough e iew o some pa s o he hesis manusc ip . I also hank him o his
con idence and his iendship.
I wish o hank also, all my o me and p esen colleagues a G upo de
In esi gación en Acuicul u a. Especially I wish o hank my b o he in li e Eyad and his
amily o all he kindness and lo e du ing all hese yea s, Eneko and O es es o
sha ing e e y hing wi h me and showing me he igh way o clam down and don’
loose he pa h in di icul momen s. Amaia, Edua do, Men o , Vale ia, Yo yia, Tibiabin,
Mapi, Ge cende, Ta iana, Mohamed, Sil ia, Alex, F an, Jezabel, Fe i, Da inia, Ca men,
Dominique, Lahoussine, Reda, Fa ima-Zah a, Ada, Moneiba and Manolo o many
help ul ad ices and good con e sa ions du ing all hese yea s, and o unde s anding
me. We ha e gone h ough so many hings oge he . Wi h you I can be jus me…
My wa ms hanks I owe o my pa en s and b o he s, I am a esul o all he lo e
and sac i ices you showed me du ing all hese yea s. The wo ds a e no able o exp ess
my deep hanks and espec . I wish ha my a he would be p esen , bu I know ha he
was always p oud o us. I plead God o co e him wi h his me cy.
Chap e 1
Gene al In oduc ion
Chap e 1 Gene al In oduc ion
1
1- Lipids as a cons i uen o ish die s
1.1- Lipids and a y acids
Lipids a e a la ge and di e se g oup o na u ally occu ing o ganic compounds
ha ha e in common a eady solubili y in o ganic sol en s (hyd oca bons, chlo o o m,
benzene, e he s and alcohols) and gene al insolubili y in wa e . The e is a g ea
s uc u al a ie y among he lipids, ha ing as a basic o m a hyd oca bon chain wi h a
ca boxylic g oup a one end and a e minal me hyl g oup a he o he (n o w ca bon).
The acyl chain could be sa u a ed o unsa u a ed and can be es e i ied o o he
molecules; hen, lipids include a di e se ange o compounds, like a y acids and hei
de i a i es, ca o enoids, e penes, s e oids and bile acids.
Lipids cons i u e a he e ogeneous g oup o compounds, and di e en
classi ica ions a e used o dis inguish hem. Fo ch oma og aphy pu poses, lipids a e
di ided in wo b oad classes including complex lipids and simple lipids. The la e
include nea ly all he comme cially impo an a s and oils o animal and plan o igin
such as, iacylglyce ols, diacylglyce ols, 2-monoacyl-sn-glyce ols, s e ols, waxes,
ocophe ols and ee a y acids. Complex lipids including glyce ophospholipids,
glycoglyce olipids, sphingomyelin, glycosphingolipids and eicosanoids. O he wise,
acco ding o hei deg ee o pola i y, lipids a e sepa a ed in o pola o neu al lipids.
Pola lipids including glyce ophospholipids (cu en ly named phospholipids),
glucoglyce olipids, sphingomyelin and glycosphingolipids, whe eas neu al lipids
con ain a y acids and hei de i a i e glyce olipids, s e ols, waxes and ocophe ols,
among o he s.
Fa y acids a e he basic building block o he lipids, ce ain a e essen ial
componen s o he die , as hey canno be syn hesised by animals and ha e speci ic
physiological and s uc u al unc ions. Polyunsa u a ed a y acids (PUFA) a e a y
acids con aining mo e han 16 ca bon a oms wi h wo o mo e double bonds. The PUFA
mos equen ly ound in na u e a e: docosahexaenoic acid (DHA; 22:6n-3),
docosapen aenoic acid (DPA; 22:5n-3 o n-6), eicosapen aenoic acid (EPA; 20:5n-3),
a achidonic acid (ARA; 20:4n-6), γ-linolenic acid (GLA; 18:3n-6), α-linolenic acid
(LNA; 18:3n-3) and linoleic acid (LA; 18:2n-6). These a e impo an die a y nu ien s
o mammals including humans (Simopoulos, 2000). Highly unsa u a ed a y acids
(HUFA), such as DHA, DPA, EPA and ARA a e hose PUFA o 20 and mo e ca bon
a oms in hei alipha ic chain wi h 3 o mo e unsa u a ed bonds, and ish a e he majo
Chap e 1 Gene al In oduc ion
2
die a y sou ce o n-3 HUFA (Ackman, 1980; Sa gen and Tacon, 1999). ARA gene ally
accoun s o only 1-2% o he o al a y acids in ish phosphoglyce ides, wi h he
no able excep ion o he phospha idylinosi ols whe e ARA can be he majo PUFA
(Izquie do, 1996; Sa gen e al., 1993), hus, ish ha e high nu i ional equi emen s o
ARA, EPA and DHA (Sa gen e al., 1997).
Fish na u ally consume die s ich in (n-3) PUFA, howe e , he issue a y acid
composi ions o cul i a ed ish is gene ally de e mined bo h by he ype o die a y lipid
inges ed and he abili y o he indi idual ish species o modi y ha die a y inpu ia
bo h pa hways o ca abolism and con e sion including desa u a ion and elonga ion
(Hende son and Toche , 1987; Sa gen e al., 1989; To s ensen e al., 2000; Bell e al.,
2001, 2002). I is gene ally unde s ood ha ma ine ish a e incapable o hese
con e sions (Toche and Ghioni, 1999) and equi e p e o med EPA, ARA and DHA in
hei die s.
1.2- Lipid unc ions in ish
In common wi h o he e eb a e so a s udied including humans, ish ha e
obliga o y die a y equi emen s o bo h (n-6) and (n-3) PUFA. In o de o p o ide he
ish wi h adequa e equilib a ed die s, i is necessa y o know he physiological and
me abolic unc ions o he di e en die a y nu ien s. Fu he mo e, lipids ha e mul iple
s uc u al and physiological unc ions in ish, since a y acids a e he main cell
memb ane cons i uen s.
Fu he mo e, some a y acids a e also impo an p ecu so s o a ange o highly
biologically ac i e media o s o ish me abolism and physiology. Thus, a y acids can
ac as second messenge s equi ed o ansla ion o ex e nal signals, as hey a e
p oduced apidly as a consequence o he binding o speci ic agonis s o plasma
memb ane ecep o s. Wi hin cells, a y acids can ac o ampli y o o he wise modi y
signals o in luence he ac i i ies o such enzymes as p o ein kinases, phospholipases,
and many mo e. They a e in ol ed in egula ing gene exp ession, mainly a ge ing
genes ha encode p o eins wi h oles in a y acid anspo o me abolism ia e ec s on
ansc ip ion ac o s, i.e. pe oxisome p oli e a o s-ac i a ed ecep o s (PPARs) in he
nuclei o cells, such e ec s can be highly speci ic o pa icula a y acids. Indeed,
phospholipids play mul iple oles in cells o he han es ablishing pe meabili y ba ie s,
hey p o ide a ma ix o he assembly and unc ion o a wide a ie y o enzymes, hey
pa icipa e in he syn hesis o mac omolecules, and hey ac as molecula signals o

Chap e 1 Gene al In oduc ion
3
in luence me abolic e en s.
The ins abili y o biomemb anes, including mechanical agili y and osmo ic
leakiness is a ec ed by i s a y acids composi ion. The e o e, en i onmen may change
memb ane luidi y, which ac s as a sensi i e ecep o ini ia ing cellula egula ion
(Beney and Ge ais, 2001). I is impo an ha memb ane luidi y is also a ec ed by he
con en o HUFA in cons i uen phospholipids, which due o hei h ee dimensional
s uc u e and lowe hyd ogen-hyd ogen in e ac ions, inc ease p o ein inse ion. Fo
example, he a y acid composi ion o phospholipids om memb anes de e mines he
le el o i s luidi y (Bell e al., 1986), and conside ing ha ish is a poikilo he m
o ganism, i s memb ane a y acid con en is c ucial o adap o he en i onmen al
empe a u e changes. Fu he mo e, changes in he a y acid composi ion o he cell
memb anes could di ec ly a ec he ac i i ies o memb ane-bound enzymes and
ecep o s, pe u bing consequen ly di e en physiological p ocesses (g ow h,
ep oduc ion, immune sys em…e c).
The essen ial a y acid (EFA) equi emen s o eshwa e and ma ine ish
species ha e been ex ensi ely s udied o e he pas 20 yea s and a e known o a y bo h
quan i a i ely and quali a i ely (Sa gen e al., 1989, 1995, 2002). In eshwa e ish,
EFA equi emen s can be me by supplying LA and/o LNA, al hough be e
pe o mances can be achie ed by supplying he “bioac i e” o ms o he n-3 HUFA,
mainly EPA and DHA (Kanazawa, 1985). Die a y lipids p o ide essen ial PUFA
necessa y o g ow h and de elopmen o cells and issues (Sa gen e al., 1995) and a e
also a majo sou ce o ene gy (Sa gen e al., 1989) yielding 9 Kcal/g , p omo ing hei
use o pa ially spa e p o ein in aquacul u e eeds and enhancing die p o i abili y
(Ve ga a e al., 1996; Mo ow e al., 2004). In addi ion, ish phosphoglyce ides a e
na u ally ich in DHA and EPA and i can be assumed ha hese a y acids ul il he
same s uc u al ole in biomemb ane phosphoglyce ides ha ARA does in highe
e es ial mammals (Sa gen , 1995).
In ish, some epo s show posi i e e ec s o n-3 FA on immune esponse, as he
inc eased ac i i y o head kidney mac ophages has been associa ed wi h highe le els o
die a y n-3 a y acids in ca ish (Blaze , 1991; Sheldon and Blaze , 1991). Ash on e al.
(1994) ha e ound ha head kidney supe na an s de i ed om ainbow ou ed a die
en iched wi h n-3 a y acids had g ea e mig a ion s imula ing abili y han supe na an s
om ish ed an n-6 a y acid en iched die . The eby, adequa e amoun o n-3 a y
acids was ound o be essen ial o he co ec unc ion o immune sys em.
Chap e 1 Gene al In oduc ion
4
While long-chain PUFAs possess a wide ange o cellula unc ions, one o hei
mos impo an is o supply p ecu so s o he p oduc ion o eicosanoids, which a e
bioac i e a y acid me aboli es ha can modula e many immune unc ions (Ge shwin e
al., 1985; Uhing e al., 1990). Eicosanoids a e p oduced om C20 PUFA, especially
dihomo-gamma-linolenic-acid (20:3n-6; DHGLA), ARA and EPA, by he ac ion o
cyclooxygenase (COX) and lipoxygenase (LOX) enzymes o yield a wide ange o
bioac i e compounds, including p os aglandins (PG) and h omboxanes (TX) p oduced
by he ac ion o COX. LOX yields a ange o monohyd oxy a y acids (e.g. 5 (S)-
hyd oxy-eicosa e aenoic acid; 5-HETE de i ed om ARA), while di- and i-hyd oxy
a y acids, such as leuko ienes (LT), lipoxins (LX), a e also o med ia epoxy
in e media es (Samuelson, 1983).
ARA is he majo eicosanoid p ecu so in mammalian cells, gi ing ise o,
among o he p oduc s, 2-se ies p os aglandins (Ho obin, 1983). O he C20 PUFAs, such
DHGLA and EPA a e also subs a es o eicosanoid p oduc ion and yield
p os aglandins o he 1- and 3-se ies, espec i ely. Al hough DHGLA and EPA a e
gene ally poo e subs a es o p os aglandin syn he ase han ARA (C aw o d, 1983),
hey bo h compe e o he enzyme binding si e and can educe he p oduc ion o ARA-
de i ed p os aglandins (Willis, 1981; Bell e al., 1994)
Eicosanoids ha e di e se pa hophysiological ac ions including immune esponse
and in lamma o y p ocesses. Thus, hey a e in ol ed in he egula ion o he immune
sys em by hei di ec e ec s on cells such as mac ophages and lymphocy es o hei
indi ec e ec s ia cy okines (Rowley, 1995). PG, especially PGE2 a e impo an in
modula ing he unc ion o immune cells and while PGE2 is equi ed o no mal
immune unc ion, o e p oduc ion can be immunosupp essi e (Kinsella e al., 1990).
Fu he mo e, he lipoxygenase p oduc LTB4 is a powe ul modula o o immune cell
unc ion, augmen ing p oli e a ion o T and B cells, s imula ing he elease o cy okines
om monocy es and T cells, ac ing as a po en chemoa ac an and inducing na u al
kille (NK) cell ac i i y (Kinsella e al., 1990; Claesson e al., 1992). LOX de i a i es
a e also shown o egula e and media e he in e ac ions be ween inna e and adap i e
immuni y o he hos de ence (Hedi and No be , 2004). Recen s udies indica e ha
some pa hogens disable in e eb a e immuni y by inhibi ing eicosanoid ac ions in
cellula immuni y, indica ing he key ole o eicosanoids on immune de ence (Dean e
al., 2002; S anley and Mille , 2006). Die a y n-3HUFA le els in luence pa e ns o
eicosanoid p oduc ion in ish issues, possibly as a consequence o changes in he
Chap e 1 Gene al In oduc ion
5
EPA/ARA a io in issues (Bell e al., 1993).
1.3- Lipids sou ces in aqua eeds
Aquacul u e has adi ionally used p oduc s om indus ial ishe ies, namely
ish meal (FM) and ish oil (FO), o con e ela i ely cheap p o ein and oil in o high
alue p oduc s, a p ac ice ha is bo h scien i ically and comme cially sound (Bell e al.,
2002). Howe e , he apid de elopmen o aquacul u e, which is expanding a o e 10%
pe yea (Tidwell and Allan, 2002), oge he wi h he FO uses o domes ic animals
eeds and pha macological uses ha e g ea ly inc eased i s cos and a ec ed i s
a ailabili y. Consequen ly, demand o hese p oduc s is apidly inc easing and cu en
es ima es sugges ha aquacul u e eeds will consume app oxima ely 90% and 60-70%
o he wo ld FO and FM supplies espec i ely by 2010 (Figu e 1.1) (Allodi, 2007) and
i is expec ed ha by 2012 he FO needs o aquacul u e will exceed he o al
p oduc ion. The e o e, a ailabili y o FM and FO o aqua eeds is limi ed (Anon, 2002)
du ing s abilised pe iods o wild ishe ies ca ches, and endange ed when wild ca ches
dec ease as a consequence o na u al phenomena such as “El Niño” (Anon, 2002;
Naylo , 2004). Mo eo e , he con inuous use o FM and FO may place added p essu e
on na u al esou ces and con e aquacul u e o a ne use a he han in a ne
con ibu o o wo ld ish supply, wi h disas ous consequences o he ecosys em
(S ani o d, 2002; Milewski, 2002; New, 2002; Allan, 2004). Elsewhe e, one o he long
con inued deba es in aquacul u e is he use o FM and FO in eeds and he amoun o
wild ish i akes o p oduce a med ish, called Fish In-Fish Ou (FIFO) a ios anging
om 3:1 o 10:1 (In e na ional Fishmeal and Fish Oil O ganisa ion: IFFO, 2008); and
he mos ecen e iew published by Tacon & Me ian (2008) ga e he FIFO o salmon
as 4.9:1, meaning ha i akes 4.9 onnes o wild ish o p oduce 1 onne o salmon.
Mo eo e , he FO p ices doubled du ing he las 5 yea s (Figu e 1.1), and hey con inue
o ise making i mo e cos -e ec i e o di e inc easing p opo ions o hese
ing edien s o human consump ion. The e o e, he es ic ions in cos and a ailabili y
imposed on FM and FO ha e lead, du ing he las 15 yea s, o he g adual subs i u ion
o hese ing edien s by al e na i e sus ainable lipid and p o ein sou ces
Chap e 1 Gene al In oduc ion
6
Figu e 1.1: The e olu ion and expec a ion o Fish Oil uses and needs om 1995 o 2015. Based
on da a om Allodi 2007.
This g aph shows clea ly he signi ican inc ease o FO use o aquacul u e
du ing he las 15 yea s and i s p ojec ion o inc ease in he u u e 5 yea s. The
no iceable d op in FO p oduc ion in 1998 was due o “El Niño” phenomena. Thus, FO
uses o aqua eeds inc eased om less han 30% o mo e han 80% o o al FO
p oduc ion annually and i is expec ed ha he all FO needs o aqua eeds could exceed
he o al FO p oduc ion by 2011. This p essu e on he limi ed wild esou ces caused, FO
p ices o double du ing he las decade (Figu e 1.1) as he p ices ose om 400 US
$/ onne in 2000 o mo e 750 US $/ onne oday, and i is expec ed ha i will con inue o
inc ease up o 900 US $/ onne in 2015 (Tacon and Me ian, 2008). In consequence,
al e na i es o FO a e u gen ly equi ed, wi h ege able oils as he p ime candida es
(Bell and Waagbø, 2008)
Ma ine ish a e mos ly adap ed o a ca ni o ous/pisci o ous die ha is na u ally
ich in n-3 HUFAs, de eloping a ela i e de iciency in he enzymes (desa u ases and
elongases) esponsible o he con e sion pa hway om LA and LNA o hei HUFA
end p oduc s (Toche and Ghioni, 1999; Ghioni e al., 1999; Zheng e al., 2009). As
wi h e eb a es, PUFA a e essen ial in he die o ish, bu he e a e special
equi emen s o HUFAs such EPA and DHA, a he han sho e chain PUFA ound in
VO (Toche , 2003a,b).
A p esen , mo e han 100 million onnes o ege able oils a e p oduced pe yea
including palm, soybean, linseed, apeseed, oli e and sun lowe oils, wi h palm and
Chap e 1 Gene al In oduc ion
13
such as g ading, anspo a ion and accina ion.
S ess ul condi ions ha e nega i e consequences on ish pe o mance in
aquacul u e, leading o g ow h educ ion, immune-supp ession and inc eased
suscep ibili y o in ec ious diseases, esul ing in majo economic losses o ish a me s.
The esponse o he ish o such s esso s in ol es all le els o o ganiza ion om he
cell (High owe , 1991) o he indi idual o ganism (Ba on and Iwama, 1991; Mommsen
e al., 1999) and o he s uc u e o he popula ion (Adams, 1990).
Unde s ess si ua ions, ish unde goes a se ies o physiological eac ions in an
a emp o eco e he homeos asis which could lead o an adap i e esponse o o a
maladap i e esponse which comp omises ish pe o mances. These esponses a e
di e en ia ed in h ee di e en ypes acco ding o he so-called gene al adap a ion
synd ome (GAS; Selye, 1975).
a) A p ima y esponse, he ala m eac ion, which includes he o ganism’s
pe cep ion o he new al e ed s a e and he elease o s ess ho mones and
neu o ansmi e s. The e o e, he ca echolamines a e eleased om he ch oma in issue
si ua ed in he head kidney o eleos s and he endings o ad ene gic ne es (Randall and
Pe y, 1992). Co isol is eleased om he in e enal issue, loca ed in he head kidney,
in esponse o se e al pi ui a y ho mones, pa icula ly he ad enoco ico ophic
ho mone (ACTH) ha in u n is libe a ed om he hypo halamus by he co ico opic
eleasing ho mone (CRH) (Donaldson, 1981; Vijayan e al., 2005; Alsop and Vijayan
2008).
b) A seconda y esponse o s age o esis ance, du ing which he o ganism
adjus s i s me abolism o cope wi h he dis u bance. Thus, he eleased s ess ho mones
ac i a e a numbe o me abolic pa hways including hose implying he mobiliza ion and
ealloca ion o ene gy, osmo ic dis u bance and inc ease in ca diac ou pu , oxygen
up ake and ans e , esul ing in al e a ions in egula blood chemis y and haema ology
(Ba on and Iwama, 1991, Iwama e al., 2006).
c) A e ia y esponse o s age o exhaus ion may occu when he o ganism is
unable o adap o o e come he changes caused by he s esso . The e o e, se e e s ess
can cause massi e mo ali y, and suble hal s ess may comp omise se e al beha iou al
and physiological unc ions (Campbell e al., 1992; To e al., 1996; Iwama e al.,
1997; Wendelaa Bonga, 1997; O uño e al., 2002; Vijayan e al., 2005). Thus, ish
g ow h and ep oduc ion pe o mances a e educed in s ess si ua ions (Ba on e al.,

Chap e 1 Gene al In oduc ion
14
1986; Mesa, 1994; Sch eck e al., 2001), supp esses cellula immuni y by a ec ing
in lamma o y signaling pa hways (O uño e al., 2002; Holland e al., 2003; MacKenzie
e al., 2006; Alu u and Vijayan, in p ess), and also i encou ages in ec ious diseases and
can e en cause dea h (Pankhu s and Van de K aak, 1997).
The neu o-endoc ine egula ion o he s ess esponse in ish is simila o ha o
highe e eb a es and in ol es wo egula o y axes (Figu e 1.3) (Wendelaa Bonga,
1997; Ba on, 2002):
 Hypo halamus-Sympa he ic ne es-Ch oma in (HSC) cell
axis, equi alen o he Sympa he ic-Ad enal-Medulla (SAM) sys em in
e eb a es. As a esul o he ac i a ion o he sympa he ic ne e ib es,
which inne a e he ch oma in cells, s imula ing he elease o
ca echolamines (epineph ine and no epineph ine) ia choline gic
ecep o s (Reid e al., 1996). Ca echolamines, p edominan ly epineph ine
in eleos ean ishes, a e eleased apidly o he ci cula ion sys em in
esponse o s ess s imulus (Randall and Pe y, 1992) and a e anspo ed
o he di e en o gans, al e ing he no mal unc ions o he di e en
physiological p ocesses ( espi a ion, ep oduc ion, immune
sys em…e c.) (Wendelaa Bonga, 1997).
 Hipo halamus-Pi ui a y-In e enal Cell (HPI) Axis: Is
equi alen o he Hipo halamus-Pi ui a y-Ad enal Axis, HPA in high
e eb a es. The HPI axis is he egula o o s ess esponse in ish, once
ac i a ed in esponse o almos all o ms o s ess (Wendelaa Bonga,
1997). B ie ly, in esponse o a s ess si ua ion, he hypo halamus
eleases he Co ico opin Releasing Ho mone (CRH) ha ac s on he
Pa s dis alis o he adenohypophysis, close o he oo o he mou h,
igge ing he elease o Ad enoco ico opin (ACTH), and, o a lesse
ex en , α-melanocy e-s imula ing ho mone (g-MSH) and lipo opic
ho mone (b-LPH), which a e in e media e p oduc s o ACTH syn hesis.
A e wa ds, ACTH induces he p oduc ion and elease o co isol, om
he in e enal cells loca ed in he head kidney, o he ci cula ion sys em
(Figu e 1.3). Co isol elease is con olled h ough a nega i e eedback a
di e en le els o he HPI axis (Donaldson, 1981; B ad o d e al., 1992;
Wendelaa Bonga, 1997).
Chap e 1 Gene al In oduc ion
15
Figu e 1.3: A simpli ied ep esen a ion o he cen al and pe iphe al componen s o he s ess
esponse in ish. Co ico opic eleasing ho mone (CRH), ad enoco ico ophic ho mone (ACTH),
ca echolamine ecep o (CR), glucoco icoid ecep o (GR).
Acco ding o he in ensi y and du a ion o he s ess challenge, se e al ypes o
s ess si ua ions a e ecognized: acu e, ch onic, epe i i e, i egula epe i i e, e c. Acu e
s ess is p oduced by an in ense s esso o sho du a ion, whe eas a ch onic s ess
si ua ion implies a long- e m dis u bance ha ac s as an in ense o mild s esso .
2.2- Head kidney as a key o gan in s ess esponse
Ana omically, ish don’ posses an ad enal gland as in mammals. Teleos ean
kidney consis s o a head and body kidney, de i ing om p oneph os and mesoneph os
issues, espec i ely. The ex e nal o m o he ish kidney a ies acco ding o species.
The head kidney is an o gan encased in bone, i mus ha e aken up i s posi ion in he
head, and ha e pene a ed bo h he ai -bladde and he scapula a ch, whe eas many
neph ons and in e s i ial lymphoid issue cons i u e he body kidney. The head kidney
con ains he in e enal gland (homologous o he ad enal co ex in mammals)
esponsible o co isol elease and he ch oma in cells (homologous o he ad enal
medulla), su ounding he pos ca dinal ein and i s b anches (Milano e al., 1997).
Ch oma in cells a e loca ed singly o in clus e s in he walls o he pos ca dinal ein
Chap e 1 Gene al In oduc ion
16
su ounded by he in e enal cells (Imagawa e al., 1996). The loca ion o he in e enal
and ch oma in cells nea he pos ca dinal ein acili a es hei egula ion by he
endoc ine sys em ia he bloods eam. The in e enal issue exhibi s conside able
mo phological a ia ion among axonomic g oups (Nandi, 1962), and i is conside ed as
he majo endoc ine, haema opoie ic and lympha ic issue in ish (Takashima and
Hibiya, 1995). Thus, head kidney is conside ed as a key issue in s ess esponse.
The e is a li le a ailable in o ma ion on he unc ions o he head kidney issue
in ish, and ecen s udies a e in e es ed in using his o gan in di e en expe imen al
p epa a ions o s udy i s unc ions. Va iabili y in he size and numbe o cells o he
head kidney be ween indi idual ish (Po inge e al., 1995) and he ela i ely la ge
numbe s o ish equi ed p eclude he ou ine use o whole o agmen ed head kidneys
in s a ic o pe usion sys ems (Pa iño e al., 1986; Bengui a and Hon ela, 2000, Ro llan
e al., 2000a,b) o in i o sc eening es s o ad eno oxican s (example, igu e 1.4).
Figu e 1.4: C oss sec ions o pa a in embedded head kidney issue o eleos ean ish consis ing
o haema opoe ic issue (HT), blood essels (BV) and in e enal issue (IT) con aining he s e oidogenic
cells (SC).
Chap e 1 Gene al In oduc ion
17
2.3- Co isol: as a s ess ho mone indica o in ish
The blood ci cula ion le el o co isol is commonly used as an indica o o
deg ee o s ess expe ienced by ish (Ba on and Iwama, 1991; Wendelaa Bonga, 1997)
since, 1) co isol is he majo glucoco icoid in eleos ish and i s plasma le els a e
known o inc ease in esponse o a a ie y o s esso s (Ba on and Iwama, 1991), 2) i
is easily and eliably measu ed using adioimmunoassay (RIA) and enzyme-linked
immunoso ben assays and 3) i plays a c i ical egula o y ole in many impo an
physiological p ocesses (Mommsen e al., 1999). The pa hway o co isol elease
begins in he HPI axis wi h he elease o CRH by he hypo halamic neu osec e o y
cells, which in u n s imula es he co ico ophic cells o he adenohypophysis o sec e e
ACTH. Ci cula ing ACTH, in u n, s imula es he in e enal cells embedded in he head
kidney o syn hesize and elease co icos e oids in o he ci cula ion o dis ibu ion o
a ge issues (Figu e 1.3).
The p oduc ion o co isol is unde he con ol o he HPI (Wendalaa Bonga,
1997; Mommsen e al., 1999). Fo ins ance, co isol eleasing ac o (CRF) sec e ed
om hypo halamic neu ons s imula es he elease o ACTH om he pi ui a y. This
pi ui a y pep ide binds o melanoco in 2 ecep o (MC2R) on he s e oidogenic cells in
he in e enal issue leading o co icos e oidogenesis (Alu u and Vijayan, 2009). I is
widely belie ed in mammals ha ACTH s imula es co isol sec e ion h ough adenosine
3’,5’-cyclic monophospha e (cAMP), which p o ides subs a e choles e ol by ac i a ing
choles e ol es e hyd olase and acili a ing anspo o choles e ol o he side-chain
clea age (scc) enzyme. Dempshe e al. (1984) ha e es ablished a model o co isol
elease (Figu e 1.5), hus ACTH binds o one o mo e speci ic ecep o s in he
ad enoco ical cell memb ane (MC2R, ep esen ed by A and B in he igu e below)
which has he e ec o ac i a ing adenyla e cyclase (ACase). The cy osolic
concen a ion o cAMP inc eases, in u n a) ac i a ing choles e ol es e (CE) hyd olase,
which ca alyses he con e sion o choles e ol es e in o ee choles e ol, and b)
acili a ing he ans e o choles e ol wi hin he mi ochond ion o a si e accessible o he
scc enzyme. Fu he mo e, low densi y lipop o ein ecep o -media ed up ake o plasma
choles e ol oge he wi h cy osolic choles e ol syn hesis p o ide subs a e o s e oid
syn hesis, and bo h hese p ocesses can be con olled by an in acellula pool o
choles e ol.
Fu he mo e, o he s udies ha e poin ed ou ha he main pa hway leading o
Chap e 1 Gene al In oduc ion
18
co icos e oid syn hesis by ACTH s imula ion in ol es a signalling cascade in eg a ing
G-p o eins, adenyl cyclase, cAMP and p o ein kinase A (PKA) (Mille 1988, Schimme
1995). E idence sugges s ha co isol exe s a nega i e eedback e ec on he ACTH
sec e ion in he pi ui a y and also supp esses CRH syn hesis in he hypo halamus. In
addi ion, he e is also a nega i e eedback e ec o ACTH on he sec e ion o CRH.
The e o e, he en i e p ocess can be hough o as a sel - egula ing sys em, as
summa ised in he igu e below. In addi ion, choles e ol modula es i s own syn hesis by
inhibi ing be a-hyd oxy-be a-me hylglu a yl (HMG)-CoA educ ase in he
ad enoco ical cell (Dempshe e al., 1984).
Se e al bio ic and abio ic s esso s a e known o modula e co isol biosyn hesis
in ish by al e ing he exp ession pa e n o genes encoding p o eins in ol ed in he
unc ioning o he HPI axis (Alsop and Vijayan, 2009). In eleos s, mul iple
glucoco icoid ecep o s (GRs) and one mine aloco icoid ecep o (MR) a e in ol ed in
co isol signalling (Vijayan e al., 2005; P une e al., 2006; Alsop and Vijayan, 2008).
11-deocyco icos e one was iden i ied as a ligand o ou MR using in i o epo e
assays, a physiological ole o his ligand is lacking in i o (S u m e al., 2005;
McCo mick e al., 2008). A dis inc physiological ole o mul iple GR iso o ms in
co isol signalling has no been es ablished in i o (Bu y e al., 2003).
Figu e 1.5: A mechanis ic model o ACTH-s imula ed co isol sec e ion in mammals (Dempshe
e al., 1984). Adenyla e cyclase (ACase), choles e ol es e (CE), adenosine 3’,5’-cyclic monophospha e
(camp), Adenosine iphospha e (ATP).

Chap e 1 Gene al In oduc ion
19
2.4- Co icos e oids biosyn hesis by in e enal cells
S e oid ho mones a e impo an egula o s o nume ous physiological p ocesses,
including glucoco icoids, mine aloco icoids and sex s e oids, such as oes ogens and
and ogens (Mille , 1988). Howe e , only low le els o aldos e one ha e been ound in
eleos s and he unc ion o mine aloco icoids seems o be ca ied ou by he
glucoco icoids (Wendelaa Bonga, 1997).
The s e oid ho mones a e s uc u ally simila and a e syn hesized om
choles e ol in he s e oidogenic cells o he head kidney. Thus, co isol is o med om
choles e ol ia he in e media es p egnenolone, p oges e one, 17-hyd oxyp oges e one
and 11-deoxyco isol. 17-hyd oxyp oges e one can be con e ed o ei he
co icos e oids by 21-hyd oxylase o o sex s e oids by 17-hyd oxylase which hus is a
b anch s ep in he s e oid biosyn hesis pa hway. The 11-deoxyco isol o med by he
mic osomal 17-hyd oxyp oges e one 21-hyd oxylase is e en ually me abolised o
co isol by he mi ochond ial 11-hyd oxylase (Figu e 1.6). In ish he enzymes
esponsible o co isol p oduc ion ha e been poo ly examined. In mammals he 21-
hyd oxyla ion o 17-hyd oxyp oges e one is ca alysed by mic osomal cy och ome
P450c21 in co isol p oducing ad enoco ical cells (Mille e al., 1997). P oduc ion o
co isol in ex a in e enal issues o ish is poo ly examined. In mammals, he enzyme
esponsible o con e sion o 17-hyd oxy oges e one o p oges e one is belie ed o be
ano he o m o P450 han he P45021c ha is esponsible o he con e sion o 17α-
hyd oxyp oges e one o 11-deoxyco isol.
Figu e 1.6: Rep esen a ion o he s e oidogenic pa hways in eleos ish and he impo an ole o
P450c17 in he o ma ion o glucoco icoids and and ogens/oes ogens. Enzymes implica ed a e 1)
P450scc, 2) 3β-HSD, 3) P450c17 (hyd oxylase), 4) P450c17 (lyase), 5) P450c21 (Ruane, 2002).
Chap e 1 Gene al In oduc ion
20
2.5- Co isol oles in ish
While ca echolamines induce apid, sho - e m ele a ion in blood glucose
p ima ily h ough he glycogenoly ic pa hway (Vijayan and Moon, 1992), co isol has a
key ole in egula ing many impo an physiological unc ions in eleos s. Thus, i is
in ol ed in longe - e m mobiliza ion o non-ca bohyd a e ene gy s o es (Wendelaa
Bonga, 1997) such as issues p o ein and lipids, implying inc eases in plasma a y acids
(Mazeaud e al., 1977). Besides i s eedback ac ions on he co ico opic axis,
ch onically ele a ed co isol le els a e esponsible o he de imen al e ec s o s ess
on i al physiological unc ions o ish, such as ep oduc ion (Foo and Lam, 1993,
Small, 2004), osmo egula ion (Redding e al., 1991; Mance a e al., 1994), g ow h
(Ba on e al., 1987; Small, 2004) and immune sys em (Ro lan e al., 1997; Wey s e
al., 1998, MacKenzi e al., 2006; Alu u and Vijayan, in p ess). In eshwa e ish, he
esponse o ch onic s ess may also include: loss o elec oly es, dec eases in hema oc i
and hemodilu ion om ca echolamine-induced inc eases in gill pe meabili y (Randall
and Pe y, 1992) o inc eased loss o ions h ough he u ine (McDonald and Milligan,
1997).
Co isol alue le els a y and should se e as gene al guidelines since,
indi idual condi ions, including species di e ences, s ain gene ic cha ac e is ics, p io
ea ing his o y, and local en i onmen will modi y he plasma alues o con ol and
s essed s a es (Ba on e al., 2002). Fo ins ance, esul s o con inemen s udies in
ainbow ou , Onco hynchus mykiss, (Po inge e al., 1992) and A lan ic salmon,
Salmo sala (Fe olden e al., 1991) ha e indica ed ha he co isol esponse o s ess in
eleos ish is a highly indi idualized ai . Some indi iduals display a consis en ly high
co isol s ess esponse while o he s ha e a consis en ly low co isol esponse. The
easons o hese di e ences a e p esen ly unclea . Fu he mo e, selec i e b eeding
p og ams ha e demons a ed ha s ess esponsi eness is he i able and ha indi idual
esponsi eness is s able o e ime (Fe olden e al., 1991; A onso e al., 1998).
3- Gil head Seab eam as a model species o his hesis
Gil head seab eam is a ma ine ish species ha has a long his o y in he
Medi e anean egion, wi h e idence o ish cap u e and a ening da ing back mo e han
2000 yea s. Bu , om 1980, he p oduc ion s a ed o g ow apidly, expanding om
1100 onnes in 1985 o 8400 MT in 1990. This inc ease was suppo ed by he
Chap e 1 Gene al In oduc ion
21
de elopmen o new echnologies and he a ailabili y o na ional and EU inancial aids.
Nowadays, gil head seab eam cons i u es one o he mos impo an ma ine ish species
cul u ed in he Medi e anean, and he o al Eu opean aquacul u e p oduc ion o
seab eam a ained app oxima ely 140.000 onnes in 2007, abou 40.000 onnes mo e
han 2005 (APROMAR, 2008) (Figu e 1.7). In Spain, seab eam is a much app ecia ed
ish species and has a high economic impo ance. Gil head seab eam p oduc ion
echniques a e he e o e well de eloped and i cons i u es an excellen model o
physiological s udies in ma ine wa m wa e species. Howe e , since i s cul u e is
ela i ely new in compa ison o salmonids and o he cold wa e species, many
physiological and nu i ional aspec s emain uns udied.
Figu e 1.7: Gil head seab eam global p oduc ion e olu ion in Eu ope du ing he las decade.
APROMAR (2008)
Chap e 1 Gene al In oduc ion
22
4- Objec i es
The o e all aim o his hesis is o p omo e he subs i u ion o ish oil by
ege able oils in on-g owing die s o gil head seab eam wi hou comp omising ish
wel a e by imp o ing ou knowledge on he mechanisms in ol ed in he egula ion o
s ess esis ance by die a y lipids.
Fo ha pu pose se e al objec i es we e add essed:
1- To de e mine he e ec o high le els o ish oil subs i u ion by n-3 o n-6
a y acids ich ege able oils on cul u e pe o mance and a y acid composi ion o
di e en issues in gil head seab eam un il comme cial size.
2- To s udy he e ec o ish oil subs i u ion le els and n-3/n-6 a y acid a ios
on seab eam wel a e and hei esis ance o se e al ypes o s ess.
3- To in es iga e he e ec s o di e en le els o subs i u ion o ish oil by
ege able oils blends on gil head seab eam heal h and wel a e in e ms o plasma a y
acid composi ions and p os aglandin and lep in p oduc ion.
4- To be e unde s and he mechanisms in ol ed in he egula ion o s ess
esis ance by polyunsa u a ed a y acids by s udying he p oduc ion and elease o
co isol in gil head seab eam in e enal cells incuba ed wi h ad enoco ico opic
ho mone.
5- To cla i y he e ec o eeding ege able oils along gil head seab eam on-
g owing on wel a e in e ms o co isol p oduc ion and elease by head kidney and he
physiological pa hways in ol ed.
To achie e hese goals, ow eeding expe imen s along he whole on-g owing
pe iod and se e al “in i o” s udies we e conduc ed. The esul s we e o ganized in i e
scien i ic s udies ha ha e been al eady published o submi ed o publica ion. Some o
hose s udies we e included in wo esea ch p ojec s: RAFOA (2001-2005) unded by
he EU (Q5RS-2000-30058) and LINOSALUD (2005-2007) unded by he Spanish
Go e nmen (AGL2004-08151-CO302).
Chap e 1 Gene al In oduc ion
29
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le els and DHA/EPA a ios on g ow h, su i al and osmo ic s ess ole ance o
Chinese mi en c ab E iochei sinensis la ae. Aquacul u e, 273:139-150.
Tacon, A.G.J. and Me ian, M. (2008) Aquacul u e eed and ood sa e y: he ole o FAO
and Codex Alimen a ius. Annals o New Yo k Academy o Science, 1140:50-59.
Takashima, F. and Hibiya, T. (1995) An A las O Fish His ology: No mal And
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342-364.
Thompson, K.D., Ta ne , M.F. and Hende son, R.J. (1996) E ec s o die a y (n-3) and
(n-6) polyunsa u a ed a y acid a io on he immune esponse o A lan ic
salmon, Salmo sala L. Aquac Nu , 2:21-31.
Tidwell, J.H. and Allan, G.L. (2002) Fish as ood: aquacul u e’s con ibu ion. Wo ld
Aquacul Soc, 33:44-48.
Toche D.R. and Ghioni, C. (1999) Fa y acid me abolism in ma ine ish: low ac i i y o
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Chap e 1 Gene al In oduc ion
37
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Chap e 2 Ma e ials & Me hods
44
Table 2.2: Main a y acids o he di e en expe imen al die s (g/100 g a y acid) (5mm)
FO
60LO
60RO
100LO
% Lipids (d.w)
20.24
21.36
22.79
25.14
14:0
5.92
3.05
2.93
0.79
15:0
0.50
0.28
0.24
0.08
16:0
19.30
14.99
15.95
15.92
16:1n-7
7.21
3.93
3.23
1.23
16:2n-6
-
-
-
-
17:0
0.26
0.15
0.13
0.02
16:4n-3
0.17
0.04
0.03
0.02
18:0
3.37
3.21
3.36
3.40
18:1n-9
11.71
24.61
29.89
32.98
18:2n-6
5.84
11.87
12.50
13.67
18:3n-6
0.07
0.10
0.08
-
18:4n-6
0.17
0.03
0.03
-
18:3n-3
1.62
14.36
12.25
23.02
18:4n-3
2.18
1.28
0.87
0.17
20:0
0.21
0.24
0.32
0.23
20:1n-9
2.38
2.05
1.98
2.23
20:1n-7
-
-
-
-
20:2n-6
0.15
0.10
0.08
0.03
20:4n-6
0.66
0.34
0.28
0.06
20:4n-3
0.54
0.27
0.21
0.05
20:3n-3
0.08
0.05
0.04
0.02
20:5n-3
11.90
6.10
4.86
1.06
22:0
0.08
0.10
0.24
0.13
22:1n-11
2.98
2.35
2.20
2.41
22:1n-7
-
0.02
-
-
22:4n-6
0.18
-
0.03
0.01
22:5n-6
0.24
0.12
0.11
-
22:4n-3
-
-
0.03
0.02
22:5n-3
1.17
0.56
0.47
0.08
22:6n-3
14.14
7.36
3.21
2.10
Sa u a ed
30.01
22.22
23.33
20.63
Monoenoics
27.70
33.12
37.44
38.85
Σ n-3
32.23
30.27
25.22
26.57
Σ n-6
7.37
12.59
13.13
13.78
Σ n-9
14.56
26.81
31.91
35.22
Σ n-3 HUFA
27.84
14.34
11.82
3.33
n-3/n-6
4.37
2.40
1.92
1.93

Chap e 2 Ma e ials & Me hods
45
2.3.2. Sampling p ocedu e
A he end o he eeding ial, day 281, ish we e indi idually sampled om
each ank. Blood was collec ed om caudal eins in hepa inised sy inges om 6 ish
pe each ank (18 ish pe die ) and ans e ed o an eppendo ube coa ed wi h li hium
hepa in as an an icoagulan . The blood was cen i uged immedia ely a 3000 pm o 10
min o sedimen he cells. One millili e o plasma was emo ed, 50 µl/ml o 2 M o mic
acid was added and he acidi ied samples we e ozen in liquid ni ogen (-80 °C) be o e
he eicosanoids analysis. 250 µl o plasma we e emo ed and s o ed a -80 °C o lep in
analysis and he emainde plasma was pooled pe each ank and s o ed a -80 °C o
a y acid analysis.
Blood leukocy es sepa a ion was conduc ed on only 3 die a y ea men s as
su icien ish ed 100 LO die we e no a ailable. Se en millili es o blood was
collec ed om he caudal ein in hepa inised sy inges om 9 ish pe die and
ans e ed o clean glass ubes kep on ice. Blood was hen cen i uged a 500 g o 10
min a 4 ºC. A e elimina ion o supe na an cells we e esuspended in 10 ml o HBSS
Ca–Mg- ee and hen cen i uged a 500 g o 10 min a 4 ºC. Cells we e sepa a ed in o
2 sub-samples which we e esuspended in 6 ml o HBSS Ca–Mg- ee, and ca e ully
laye ed o e 6 ml o 46% Pe coll and cen i uged a 450 g o 40 min a 4 ºC. The
leukocy es (whi e in e media e laye ) we e collec ed and washed wi h 10 ml o HBSS
Ca–Mg ee. The leukocy es ob ained om 3 ish we e pooled and esuspended in 4 ml
o HBSS, 2 ml o chlo o o m we e added and he sample was s o ed a -80 ºC p io o
lipid ex ac ion.
2.3.3. Lipids ex ac ion and analysis
Ex ac ion o o al lipid om plasma samples, die s and leukocy es was
pe o med by he me hod o Folch e al (1957) using a mix u e o chlo o o m: me hanol
(2:1)( : ) con aining 0.01% BHT, as an an ioxidan , and KCl (0.88 %); 300 µl o
pooled plasma we e used. Vigo ous o ex mixe ollowed by cen i uga ion o assis
sepa a ion o chlo o o m and aqueous laye s ex ac ed he lipids om plasma, die and
leukocy es samples. The lowe laye s is il e ed h ough Wha man il e pape and d ied
unde a low o ni ogen, o al lipids we e weigh ed. Neu al and pola ac ions we e
sepa a ed by adso p ion ch oma og aphy on silica ca idges Sep-pak, (Wa e s, Mil o d,
MA) as desc ibed by Juaneda and Rocquelin (1985).
Chap e 2 Ma e ials & Me hods
46
Fa y acid me hyl es e s we e p oduced om aliquo s o o al lipids ex ac ed
om die and plasma samples by acid-ca alyzed ansme hyla ion pe o med o e nigh
a 50°C as desc ibed by Ch is ie (1982). Fa y acids me hyl es e s we e sepa a ed and
quan i ied by Gas-ch oma og aphy (Shimadzu C-R5A, 30m*0,32mm Silice column
wi h Supleco-10) acco ding o condi ions desc ibed by (Izquie do e al., 1990).
Indi idual me hyl es e s we e iden i ied by compa ison wi h known s anda ds and
published da a.
2.3.4. Ex ac ion, sepa a ion and enzyme immunoassay o PGE isome s
a. Pu i ica ion o eicosanoids
The ozen plasma o eicosanoids analysis was hawed and cen i uged a 1000
x g o 5min o p ecipi a e he deb is. The supe na an s we e ex ac ed using oc adecyl
silyl (C18) “Sep-Pak” mini-columns (Millipo (UK), Wa o d) as desc ibed in de ail by
Bell e al (1994). 200 µl o supe na an s was applied o he column, which had been p e-
washed wi h 5 ml me hanol and 10 ml dis illed wa e . The column was washed
successi ely wi h 10 ml dis illed wa e , 5 ml o 15% ( / ) e hanol and 5 ml
hexane/chlo o o m (65:35, / ) be o e elu ion o p os anoids wi h 10 ml o e hyl
ace a e. This ex ac was d ied unde ni ogen and esuspended in 100 µl me hanol and
s o ed in a small glass ial in he deep- eeze (- 4 ºC) be o e analyzing by
immunoassay.
b. Sepa a ion o PGE3 by HPLC
PGE3 was sepa a ed by e e se-phase HPLC using a Sphe iso b 5 µm C18
(ODS2) column. The ch oma og aphic sys em was equipped wi h Wa e s Model M-45
pumps and Wa e s 680 moni o ed a 196 nm using a Pye-Unicam LC-UV de ec o o
de e mine elu ion o p os aglandin s anda ds. An isoc a ic sol en sys em was used
con aining 17 mM phospho ic acid/ace oni ile (70:30, / ) a a low o 0,75ml/min.
The emaining 50 µl o pu i ied eicosanoids om plasma ex ac was injec ed o he
column and 2.25 ml ac ions we e collec ed using an LKB 2112 “Redi ac”. F ac ions
co esponding o PGE3 we e applied o a C18 “Sep-Pak” which had been-p ewashed as
desc ibed abo e, and he p os aglandin elu ed in 5 ml e hyl ace a e. Samples we e d ied
unde ni ogen and edissol ed in 100 µl o immunoassay bu e . Measu emen o PGE3
was pe o med using enzyme immunoassay (EIA) ki s o PGE2 acco ding o he same
p o ocol desc ibed abo e (SPI-bio, Gi su Y e e, F ance). The speci ici y o he ki
Chap e 2 Ma e ials & Me hods
47
an ibodies used in his immunoassay wi h PGE2 is 100% bu i is only 43% wi h PGE3,
which is coun ed when calcula ing esul s.
c. P os aglandins E2 and E3 immunoassay
Fo p os aglandins E2: 50 µl o he me hanol ex ac we e aken, d ied unde
ni ogen and e-dissol ed in 500 µl o EIA bu e and s o ed in he idge ( 4°C). This
assay is based on he compe i ion be ween PGE2 and a PGE2-ace ylcholines e ase
(AchE) conjuga ed PGE2 ( ace ) o a limi ed amoun o PGE2 monoclonal an ibody.
Because he concen a ion o PGE2 ace is cons an while he concen a ion o PGE2
a ies depending on he sample, he amoun o PGE2 ace ha is able o bind o PGE2
monoclonal an ibody is in e sely-p opo ional o he concen a ion o PGE2 in he well.
This an ibody PGE2 complex binds o goa polyclonal an i-mouse IgG ha has been
p e iously a ached o he well. The pla e is washed o emo e any unbound eagen s
and hen Ellman’s Reagen (which con ains he subs a e o AchE) is added o he well.
The p oduc o his enzyma ic eac ion has dis inc yellow colo and abso bs s ongly a
412 nm. The in ensi y o his colo , de e mined spec ome ically, is p opo ional o he
amoun o PGE2 ace bound o he well, which is in e sely p opo ional o he amoun
o ee PGE2 p esen in well du ing he incuba ion. All assay-speci ic eagen s we e
p epa ed be o e s a ing he assay.
• EIA Bu e : Dilu ing he con en s o he ial o EIA Bu e Concen a e wi h 90
ml o Ul aPu e wa e .
• Wash Bu e : Dilu ing he con en o Wash Bu e Concen a e 1:400 wi h
Ul aPu e wa e and adding Tween (0.5ml/li e o Wash Bu e ).
• P os aglandin E2 s anda d: The concen a e o The PGE2 s anda d (10 ng) is
econs i u ed wi h 1ml o EIA Bu e . Eigh clean ubes (#1-8) we e p epa ed;
360 µl EIA Bu e o ube #1 and 200 µl EIA Bu e o ubes # 2-8. 40 µl o he
bulk s anda d (10 ng/ml) is ans e ed o he ube # 1 and mix ho oughly.
Se ially, he s anda d was dilu ed by emo ing 200 µl om # 1 and placing in
ube # 2, mixing. Nex , emo ing 200 µl om ube 2 o ube # 3, mixing. The
p ocess was epea ed o ubes # 3-8. These dilu ed s anda ds should no be
s o ed o mo e han 24 hou s.
• P os aglandin E2 AchE: The concen a e is dilu ed wi h 6 ml EIA Bu e , The
ace dye is added a a inal dilu ion o 1:100.
Chap e 2 Ma e ials & Me hods
48
• P os aglandin E2 Monoclonal An ibody: The concen a e is dilu ed wi h 6 ml
EIA, he dye o he econs i u ed an ise um is added a a inal dilu ion o 1:100.
Once he immunoassay ki s was opened, each pla con ained eigh Blanks (Blk), one
non-speci ic bindings wells (NSB), one maximum binding wells (B0), and eigh poin
s anda d cu e (S1-S8). A es wi h di e en samples dilu ions was un o choose he
bes (80 µl).
Pipe ing he eagen s: di e en ips a e used o pipe he bu e , s anda d, sample,
ace , and an ibody.
1. EIA Bu e : 100 µl EIA bu e we e added o non-speci ic Binding (NSB) wells
and 50 µl o maximum binding (B0) wells.
2. P os aglandin E2 S anda d: 50 µl om ube # 8 o he lowes s anda well (S8).
50 µl om ube # 7 we e added o he nex s anda d well (S7). The same
p ocedu e was used un il all s anda ds we e aliquo ed.
3. Samples: 80 µl o sample we e added pe well.
4. P os aglandin E2 AchE T ace : 50 µl we e added o each well excep he Blank
(Blk) wells.
5. P os aglandin E2 Monoclonal An ibody: 50 µl we e added o each well excep
he Non Speci ic Binding (NSB), and he Blank (Blk) wells.
6. The pla e was co e ed wi h plas ic ilm and incuba ed o 18 hou s a 4°C which
inc ease he sensi i i y o he assay.
7. De eloping he pla e: Be o e de eloping he pla e, Ellman’s Reagen is
econs i u ed wi h 20 ml o ul apu e wa e . This should be p epa ed and used
he same day i is p epa ed, and p o ec ed om ligh when no in use. The wells
we e emp ied and insed i e imes wi h Wash Bu e . 200 µl o Ellman’s
Reagen we e added o each well and 5 µl o ace o he To al Ac i i y wells.
The pla e is co e ed wi h plas ic ilm and de eloped by using an o bi al shake
and da k du ing 75 min.
8. Reading he pla e: Is accomplished a a wa eleng h be ween 405.
9. Calcula ing he esul s: The esul s we e calcula ed manualy as ollows:
• A e age he abso bace eadings om Blk wells.
• Subs ac a e age Blk om all he o he wells.
• Subs ac NSB om all h o he wells.
• Calcula e %B/B0 (% Sample s anda d Bound/Maximum Bound) o he
emaining wells.
Chap e 2 Ma e ials & Me hods
49
• The s anda d cu e is aced (%B/B0 wi h s anda d concen a ion on PGE2 en
pg/ml).
• The concen a ion o each sample in PGE2 is calcula ed.
2.3.5. Lep in immunoassay
a. P inciple o he assay
This assay uses a cuan i a i e sandwich enzyme immunoassay echnique. A
monoclonic an ibody o lep in is applied o he pla es. A monoclonal an ibody (Human
lep in an ibody) speci ic o lep in had been p e-coa ed on o a mic opla e. S anda ds and
samples a e pipe ed in o he wells and any lep in p esen is bound by he immobilized
an ibody. A e washing away any unbound subs ances, an enzyme-linked monoclonal
an ibody speci ic o lep in is added o he wells. Following a wash o emo e any
unbound an ibody-enzyme eagen , a subs a e solu ion is added o he wells and colo
de elops in p opo ion o he amoun o lep in bound in he ini ial s ep. The colo
de elopmen is s opped and he in ensi y o he colo is measu ed.
b. Reagen s
• Lep in mic opla es: 96- well. Mi copla e o polys y ene (12 columns o 8 well)
wi h monoclínic an ibody lep in.
• Lep in conjuga e (Pa 890574): 21 ml o an ibody monoclonic agains Lep in o
a e wi h p ese a i .
• Lep in es anda d (Pa 890575): 10 ng o ecombinan lep in human in bue e d
p o eins wi h p ese a i .
• Diluen s Analysis RD1-19 (Pa 895467): 11ml o bu e ed p o ein base wi h
p ese a i .
• Calib a o diluen e RD5P (5x) Concen a e (Pa 895151): 21ml o solu ion
concen a e o bu e ed p o ein base wi h p ese a i .
• Concen a e wash bu e (Pa 895003): 21 ml o 25- old concen a e o solu ion
bu e ed su ac an wi h p ese a i .
• Dye eac i i A (pa 895000): 12.5 ml o es abilized hy dogen p eoxidase.
• Dye eac i B (Pa 895002): 12.5 ml de es abilized ch omogen
( e ame hylbenzidine).
• S op solu ion: 6 ml o 2 N Sul i ic acid.
c. Reagen s p epa a ion
All eagen s a e b ough o he oom empe a u e be o e he assay.

Chap e 2 Ma e ials & Me hods
50
• Wash Bu e : The concen a e ial is mixed gen ly and dilu ed in o 1:25 wi h
deionized wa e . 500 ml o wash bu e we e p epa ed.
• Subs a e solu ion: Colo eagen A and B we e mixed oge he in equal olumes
and pu in da k wi hin 15 minu es be o e using.
• Calib a o Diluen RD5P (1X): he concen a is dilu ed o 1:5. 100 ml we e
p epa ed.
• Lep in s anda d: I is p epa ed 15 minu es be o e use. The concen a is mixed
gen ly and dilu ed wi h 1 ml o deionized wa e . This econs i u ion p oduces a
s ock solu ion o 10.000 pg/ml. 8 ubes s anda d a e p epa ed as expained abo e
(P os aglandin s anda d).
d. Assay p ocedu e
The ozen plasma we e hawed and cen i uged a 1000 X g o 5 min o
p ecipi a e he deb is.
• 100 µl o assay diluen RD1-19 we e added o each well.
• 100 µl o S anda d a e added o each s adan d well.
• 100 µl o con ol we e added o each well ollowed by 200 µl o Plasma sample.
Mul i-channel pipe e was used.
• The pla e is co e ed and incuba ed o 2 hou s a oom empe a u e.
• All he wells we e aspi a ed and washed wi h 400 µl o Wash bu e . This
p ocess is epea ed 4 imes. A he las wash, he wells we e bol ed agains clean
pape o emo e any emaining wash bu e
• 200 µl o Lep in Conjuga e we e added o each well, he pla e was co e ed wi h
new adhesi e s ip and incuba ed o 1 hou a oom empe a u e.
• The wells we e aspi a ed and washed as in s ep 5.
• 200 µl o Subs a e Solu ion we e added o each well and he pla e was
incuba ed o 30 minu es a oom empe a u e and p o ec ed om ligh .
• 50 µl o S op Solu ion we e added o each well.
• The pla e is de elopped wi hin 30 minu es in shake and he op ical densi y o
each well was de e mined using a mic opla e eade se o 450 nm.
• Calcula ion o esul s: The s anda d cu e is d awn by plo ing op ical densi y o
he s anda ds e sus he concen a ion o he s anda ds. The da a is linea ized
using a loga i mic ans o ma ion. The equa ion o s anda d cu e is de e mined
and he inal Lep in concen a ion in plasma is de e mined by simple applica ion
o hei abso bance in he s anda d cu e equa ion.
Chap e 2 Ma e ials & Me hods
51
2.4. T ial II: Pa o LINOSALUD p ojec
2.4.1. Animals and Die s
Gil head seab eam (Spa us au a a) jnu eniles (45 g ini ial body weigh ) we e
dis ibu ed in 45 anks o 500l (50 ish/ ank, each die assayed in iplica e) supplied
wi h seawa e a a empe a u e anging om 20 ºC a he beginning o he expe imen o
24.2 ºC a he end, and ae a ion. Tow housand ow hund ed i y animals we e used.
Eigh iso-ene ge ic and isoni ogenous die s wi h lipids con en abou 18 % we e
o mula ed. Ancho y oil was he only added lipid sou ce in Die FO ( ish oil). All he
o he die s con ained a blend o ege able oils o subs i u e FO wi h di e en le els o
subs i u ion as men ioned in he ables (2.3, 2.4, 2.5). The a y acids composi ion o he
expe imen al die s o al lipids is showed in Table 2.6. Fish we e ed he expe imen al
die s un il appa en sa ia ion (3 imes/day, 6 days/ week), un il hey eached he
comme cial size a e 26 weeks.
Feed in ake was de e mined daily and all ish we e indi idually weighed
mon hly. CI and SGR we e calcula ed ollowing he o mulas desc ibed abo e.
Mo ali ies we e daily eco ded and su i al was mon hly de e mined.
Table 2.3: Main ing edien s con en s o he expe imen al die s (in %)
% o d y weigh
Oils (Fish oila/linseed/soybean)
Sou h-ame ican ish meal
Whea
Soybean meal 47% b
Sun lowe meal
Vi amins p emixc
Mine als p emixc
16.32
47.26
7.00
25.00
3.67
0.27
0.48
a Sou h-ame ican, ancho y oil.
b Soybean meal wi h 47% as a b u p o ein, “no GMO”
c Vi amin and mine al p emixes p epa ed acco ding o P oaqua A/S comme cial s anda ds
Chap e 2 Ma e ials & Me hods
52
Table 2.4: Vi amins and mine als con en s o he expe imen al die s
Vi amins/ Mine als
Uni s
1-Vi amins
A-Re inol
D3- Cholecalci e ol
E- Tocophe ol
C (S ay C)- Asco bic acid
B1- Thiamin
B2- Ribo la in
B3- Nico inic acid/Niacin
B5- Pan ho henic acid
B6- Py idoxin
B8- Bio in
B9- Folic acid
B12- Cyanocobalamin
K- Menadion
Inosi ol
2- Mine als
I
Zn
Fe
Cu
Mn
Mg
Co
Se
11200.0 IU/kg
112.0 IU/kg
280.0 mg/kg
336.0 mg/kg
9.0 mg/kg
15.7 mg/kg
179.2 mg/kg
31.4 mg/kg
13.4 mg/kg
0.5 mg/kg
4.5 mg/kg
0.036 mg/kg
6.7 mg/kg
44.8 mg/kg
4.5 mg/kg
44.8 mg/kg
67.2 mg/kg
3.6 mg/kg
14.6 mg/kg
136.1 mg/kg
0.2 mg/kg
0.06 mg/kg
Table 2.5: The ypes and % o oils in he expe imen al die s
FO
70L
100L
70S
100S
20L50S
50L20S
50L50S
FO
100
30
-
30
-
30
30
LO
-
70
100
-
-
20
50
50
SO
-
-
-
70
100
50
20
50
Chap e 2 Ma e ials & Me hods
53
Table 2.6: Fa y acids p o ile o he expe imen al die s (% o al iden i ied a y acids)
Fa y acids
FO
70L
100L
70S
100S
20L50S
50L20S
50L50S
14:0
9.23
3.11
1.57
3.32
1.59
2.67
2.60
1.37
15:0
0.26
0.10
0.06
0.11
0.06
0.32
0.23
0.06
16:0ISO
0.11
0.04
0.03
0.05
0.03
0.01
0.06
0.03
16:0
22.21
12.19
10.21
15.84
13.69
15.46
12.16
11.75
16:1n-7
11.25
3.91
1.98
4.06
2.01
3.07
3.24
1.73
16:1n-5
0.38
0.14
0.09
0.16
0.09
0.07
0.10
0.08
16:2n-4
1.83
0.61
0.28
0.61
0.29
0.11
0.51
0.24
17:0
0.85
0.38
0.25
0.41
0.28
0.52
0.31
0.25
16:3n-4
2.00
0.66
0.29
0.67
0.31
0.59
0.54
0.24
16:3n-3
0.15
0.07
0.05
0.07
0.05
0.57
0.03
0.05
16:3n-1
0.12
0.05
0.04
0.06
0.04
0.07
0.07
0.03
16:4n-3
0.72
0.26
0.13
0.24
0.11
0.04
0.05
0.09
16:4n-1
-
-
-
-
-
0.25
0.29
-
18:0
3.85
3.96
3.99
3.43
3.20
10.20
3.49
3.74
18:1n-9
9.10
13.75
15.31
18.60
21.48
15.30
18.48
19.04
18:1n-7
3.16
1.57
1.36
2.15
1.77
1.15
1.37
1.36
18:1n-5
0.14
0.08
-
0.12
0.11
0.09
0.10
0.10
18:2n-6
4.06
12.38
16.21
29.93
38.51
22.25
18.50
27.25
18:2n-4
0.38
0.13
0.06
0.12
0.06
0.18
0.12
0.05
18:3n-6
0.36
0.13
-
-
0.07
0.13
0.12
0.09
18:3n-4
0.04
-
0.04
0.18
0.09
0.70
0.12
0.05
18:3n-3
0.48
31.94
37.63
5.63
6.01
11.38
20.67
23.03
18:4n-3
1.94
0.76
0.40
0.73
0.40
0.06
0.75
0.36
18:4n-1
-
-
0.03
-
-
-
0.08
-
20:0
0.28
0.21
0.19
0.28
0.28
0.72
0.26
0.24
20:1n-9
2.59
1.94
1.84
2.08
1.89
1.74
2.35
1.91
20:1n-7
0.26
0.13
0.09
0.13
0.10
0.11
0.15
0.09
20:2n-9
-
0.04
-
0.04
0.01
0.02
0.03
0.01
20:2n-6
0.19
0.11
0.08
0.11
0.09
0.10
0.04
0.08
20:3n-9
-
-
-
-
-
0.08
0.15
-
20:3n-6
0.25
0.09
0.04
0.06
0.03
0.08
0.08
0.03
20:4n-6
1.11
0.43
0.24
0.43
0.24
0.40
0.39
0.21
20:3n-3
-
-
0.07
-
-
0.06
0.08
-
20:4n-3
0.96
0.35
0.16
0.33
0.16
0.32
0.38
0.13
20:5n-3
10.05
4.00
2.07
3.77
2.06
3.85
3.99
1.73
22:1n-11
1.79
1.52
1.48
1.67
1.61
1.45
1.91
1.64
22:1n-9
-
-
0.42
-
-
0.33
0.52
-
22:4n-6
0.34
0.15
0.09
0.14
0.09
0.15
0.14
0.08
22:5n-6
-
-
-
-
-
0.64
0.75
-
22:5n-3
1.74
0.66
0.29
0.60
0.29
0.39
0.11
0.24
22:6n-3
7.82
4.16
2.92
3.87
2.91
4.35
4.69
2.63
Sa u a es
36.68
19.95
16.28
23.40
19.10
29.88
19.06
17.41
Monoenic
28.79
23.10
22.62
29.03
29.09
23.33
28.57
25.98
n-3
23.42
42.19
43.72
15.23
12.00
21.02
30.74
28.26
n-6
6.27
13.26
16.66
30.67
39.02
23.76
19.99
27.74
n-9
22.99
19.66
19.55
24.78
25.40
17.48
24.79
22.69
n-3 HUFA
20.57
9.17
5.50
8.57
5.42
8.97
9.24
4.73
n-3/n-6
3.74
3.18
2.62
0.50
0.31
0.88
1.54
1.02
Chap e 3 G ow h, eed u iliza ion and body a y acids
59
E ec o eeding seab eam (Spa us au a a) wi h a blend o soybean and linseed oil
on g ow h, eed u iliza ion and body a y acids composi ion
Rachid Ganga, Bell, J.G.1, Mon e o, D., Fe nández-Vaque o, A.2, A alah, E. and
Izquie do M.S.
G upo de In es igación en Acuicul u a (ULPGC & ICCM) P.O. Box 56. 35200. Telde,
Las Palmas. Cana y Islands, Spain.
1Ins i u e o Aquacul u e, Uni e si y o S i ling FK9 4LA, Sco land, UK.
2Bioma Ibe ia/ P oAqua Nu i ion, S.A., A-62, Km, 99, ES-34210 Dueñas, Spain.
Abs ac
T iplica e g oups o gil head seab eam we e ed eigh p ac ical- ype die s in
which he added lipid was subs i u ed wi h a blend o linseed oil (LO) and soybean oil
(SO) a di e en le els o a pe iod o 26 weeks. Ancho y oil was he only lipid sou ce
in he FO ( ish oil) die , whe eas he o he die s con ained di e en le els o ege able
oils, 70L (70% LO and 30 FO), 70S (70% SO and 30FO), 100L (100% as LO), 100S
(100% as SO), 50S20L (50% as SO, 20% as LO and 30% as FO), 20S50L (20% wi h
SO, 50% wi h LO and 30% wi h FO) and 50S50L (50% as SO and 50% wi h LO).
Resul s showed ha subs i u ion up o 70% o mo e o FO by ege able oils in die s o
gil head seab eam, signi ican ly educed g ow h and a ec ed eed u iliza ion. Fa y acid
composi ions o muscle lipid co ela ed wi h SO o LO inclusion in he die , hus he
p opo ions o 18:2n-6, 18:3n-3 and 18:1n-9 all inc eased wi h inc easing die a y VO
%. The concen a ions o eicosapen aenoic acid (20:5n-3), docosahexaenoic acid
(22:6n-3) and a achidonic acid (20:4n-6) in muscle lipid we e signi ican ly educed
(P<0.05), along wi h o al sa u a ed a y acids, wi h inc easing die a y VOs, while he
la e 2 a y acids we e less educed in he muscle han in he die , indica ing hei
selec i e e en ion. The elongase and Δ6 des au ase p oduc s om linoleic acid (18:2n-
6) and linolenic acid (18:3n-3) we e also inc eased wi h inc easing VOs. Die -induced
changes in li e and gill a y acid composi ions we e b oadly simila o hose in
muscle, wi h some excep ions. Limi ed supplies o ma ine ish oils equi e ha
subs i u es be ound wi hou comp omising ish wel a e. Thus, LO and SO can be used
success ully in he cul u e o gil head seab eam a le els less han 70% o die a y lipid
wi hou comp omising g ow h, bu subs an ial educ ions occu in muscle 20:5n-3,
22:6n-3 and he n-3/n-6 polyunsa u a ed a y acid (PUFA) a io, which will esul in
educed p oduc quali y o he consume .
Key wo ds: Gil head seab eam, Fish Oil, Linseed oil, Soybean oil, g ow h, EPA, DHA, n-3 HUFA, n-
3/n-6.

Chap e 3 G ow h, eed u iliza ion and body a y acids
60
In oduc ion
Ma ine ish oils (FO), na u ally ich in omega 3 highly unsa u a ed a y acids
(HUFA) (>30%) which play e y impo an oles in aqua ic o ganisms, ha e been
adi ionally used as a p e e ed lipid sou ce in aqua eeds, due o i s high diges ibili y
and con en o essen ial a y acids. Since hose a y acids also ha e bene icial e ec s
on human heal h (C aig-Schmid , 2001), global consume demand o sea ood has
g own emendously and aquacul u e is playing an inc easingly impo an ole in
ul illing he inc eased demand (Tacon, 2004; FAO, 2007a). The e o e, global demand
o FO o aquacul u e has been inc easing wi h ish p oduc ion. Indeed, FO supply is
becoming a challenge o he aqua eeds indus y, since i s p oduc ion elies on ishe ies;
an o e exploi ed sou ce o HUFA (FAO, 2007b) and esea ch on al e na i e lipid
sou ces has become a majo goal o sus ainable aquacul u e de elopmen .
Vege able oils (VO), mo e sus ainable, eliable and, equen ly, wi h mo e
compe i i e p ices han FO, a e seen as good al e na i e sou ces. VOs assayed in ish
eeds include linseed (LO) (Bell e al., 2003; Izquie do e al., 2003, 2005; Benedi o-
Palos e al., 2007; Jobling e al., 2008), apeseed (Bell e al., 2001, Mon e o e al.,
2005), palm (Bell e al., 2002), soybean (SO) (G isdale-Hellan e al., 2002) and
sun lowe oils (To s ensen e al., 2000; B ansden e al., 2003). In gil head seab eam
(Spa us au a a), an impo an ish in Medi e anean aquacul u e, i is possible o
pa ially eplace FO by VO wi hou comp omising g ow h o eed u iliza ion (Caballe o
e al., 2002; Izquie do e al., 2003, 2005; Mon e o e al., 2005). Howe e , comple e
subs i u ion migh be mo e di icul as he VO lack he n-3 HUFA essen ial o his
species (Ibeas e al., 1994). This means ha , as in o he ma ine ish (Wa anabe e al.,
1983; Sa gen e al., 1989; Cas ell e al., 1994), seab eam has a e y low Δ5 desa u ase
and elongase ac i i ies o allow syn hesis o eicosapen aenoic (20:5n-3; EPA) and
docosahexaenoic (22:6n-3; DHA) acids om hei p ecu so linolenic (18:3n-3; LNA)
acid (Seiliez e al., 2003; Izquie do e al., 2005, 2008). The e o e, seab eam minimum
equi emen s o n-3 HUFA, pa icula ly EPA and DHA should be included in die in
o de o main ain ish g ow h, heal h and wel a e (Izquie do e al., 2003, 2005; Mon e o
e al., 2003; Caballe o e al., 2004), as well as ile quali y (Kaloge opoulos e al., 1992;
Izquie do e al., 2003, 2005). Mo eo e , ce ain VOs such as soybean oil, ha e a high
con en in n-6 a y acids, pa icula ly linoleic acid (18:2n-6, LA) which a e no p esen
in high amoun s in ma ine en i onmen s.
Chap e 3 G ow h, eed u iliza ion and body a y acids
61
Inclusion o hose n-6 ich ege able oils, wi h a low n-3/n-6 a y acids a io
(0.3-1) will p oduce die s wi h a n-3/n-6 a y acids p o ile e y di e en om FO (n-
3/n-6 o 9-6:1) (Robaina e al., 1998; B ansden e al., 2003) and he na u al ood o
ma ine ish. Indeed, eeding high lipid die s wi h a 60% FO eplacemen by ce ain
VOs, pa icula ly soybean oil, inc eased lipid deposi ion in hepa ocy es (Caballe o e
al., 2002) and a ec ed some immune pa ame e s (Mon e o e al., 2003, 2008)
sugges ing undesi able e ec s on ish heal h. A well balanced n-3/n-6 a y acid a io is
de e minan o good human heal h (Simopoulos, 2008), since he unc ions and
e iciency o hese a y acids di e in many aspec s o lipid me abolism such as
diges ion, abso p ion and anspo in ish (Izquie do e al., 2000, 2001; Caballe o e al.,
2006). In addi ion, ene gy p oduc ion, cell memb anes s uc u al componen s and
signaling molecules and egula o s o gene exp ession can all be a ec ed by changes in
die a y a y acids (Calde and Bu dge, 2004; Yaqoob and Calde , 2007). Elsewhe e, n-
6 and n-3 PUFA can in luence mechanisms conce ned wi h di e en physiological and
in lamma o y p ocesses in humans (Sande s, 1993), some imes in opposing ways, so
high die a y n-6 PUFA and n-6/n-3 PUFA a ios ha e been sugges ed o play a ole in
many ch onic diseases endemic in Wes e n popula ions (Simopoulos, 2008), hus
al e ed balance be ween he n-6 and n-3 se ies in aquacul u e p oduc s may educe he
bene icial e ec s o FO o human heal h. P oducing ish ich in LA wi h consequen ly
highe n-6/n-3 a io when eeding soybean oil (Caballe o e al., 2002; Mon e o e al.,
2003, Izquie do e al., 2005), no only educes he heal h p omo ing p ope ies o he
ish bu wo sens an al eady imbalanced human die . The e o e, he challenge when
subs i u ing FO wi h di e en VOs is o main ain high quali y ish p oduc s by assu ing
a high n-3/n-6 a io in he aquacul u e p oduc s a ailable o he consume .
The p esen s udy was conduc ed o be e unde s and he consequences o
eeding comme cial die s con aining n-3 and/o n-6 a y acid ich oils, and blends o
hese oils gi ing di e en n-3/n-6 a y acids a ios, on cul u e pe o mance o gil head
seab eam, pa icula ly in ela ion o eed u iliza ion, ish g ow h, and lipid me abolism
in di e en issues.
Chap e 3 G ow h, eed u iliza ion and body a y acids
62
Ma e ials and me hods
Fish husband y
Gil head seab eam (Spa us au a a) ju eniles (45 g ini ial body weigh ) we e
dis ibu ed in 39 anks o 500 l (50 ish/ ank wi h each die assayed in iplica e).
Seawa e was supplied in an open sys em (10 % enewal/h) a a empe a u e o 20-24.2
ºC. Fish we e ed he expe imen al die s (Table 3.1) un il appa en sa ia ion (3
imes/day, 6 days/week) o 26 weeks. Eigh iso-ene ge ic and isoni ogenous die s wi h
a lipid con en o ~18 % we e o mula ed and p o ided by P oaqua, S.A. (Dueñas,
Spain). Ancho y oil was he only added lipid sou ce in he FO die , whe eas in he o he
die s he FO was eplaced by ei he LO, SO o blends o bo h oils (Table 3.2).
Feed in ake (FI) was de e mined daily and all ish we e weighed indi idually
e e y mon h. Feed con e sion a io (FCR = eed in ake/weigh gain) and speci ic
g ow h a e (SGR= ((Ln inal weigh - Ln ini ial weigh )/ )*100, =expe imen al pe iod
(days)) we e calcula ed mon hly. Mo ali ies we e eco ded daily and su i al was
de e mined mon hly.
A e 26 weeks o eeding, samples o muscle, li e and gills om 18 ish ed
each expe imen al die we e collec ed, immedia ely ozen and s o ed a -80 ºC un il
hey we e analysed. Due o he complexi y and he high numbe o samples p oduced,
and acco dingly o he e ec on ish g ow h, we chose only he 6 ex eme die s o a y
acid analysis (100FO, 70L, 100L, 70S, 100S, 50S50L). Lipids om die s, li e , gills
and muscle we e ex ac ed wi h chlo o o m: me hanol (2:1 : ) (Folch e al., 1957). The
a y acids me hyl es e s we e ob ained by anses e i ica ion wi h 1% sulphu ic acid in
me hanol (Ch is ie, 1982), pu i ied by abso p ion ch oma og aphy on NH2 Sep-pak
ca idges (Wa e , S.A., Mil o d, Massachuse s) and sepa a ed, iden i ied and
quan i ied by gas-liquid ch oma og aphy unde he condi ions p e iously desc ibed
(Izquie do e al., 1990). Fa y acid me hyl es e s we e iden i ied by compa ison o
ex e nal s anda ds.
Chap e 3 G ow h, eed u iliza ion and body a y acids
63
Table 3.1: Main ing edien s con en s o he expe imen al die s
% o d y weigh
Oils (Fish oila/linseed/soybean)
16.32
Sou h-Ame ican ish meal
47.26
Whea
7.00
Soybean meal 47%b
25.00
Sun lowe meal
3.67
Vi amins p emixc
0.27
Mine als p emixc
0.48
a Sou h-Ame ican, ancho y oil.
b Soybean meal wi h 47% g oss p o ein, “no GMO”
cVi amin and mine al p emixes p epa ed acco ding o P oaqua A/S comme cial s anda ds.
Table 3.2: P oxima e composi ion (% d y weigh ) o expe imen al die s and p opo ions o he di e en
lipid sou ces (% o oil inclusion) con ained in he expe imen al die s
Lipid sou ce
FO
70L
100L
70S
100S
50S20L
20S50L
50S50L
FO (Ancho y oil)
100
30
-
30
-
30
30
-
LO
-
70
100
-
-
50
50
50
SO
-
-
-
70
100
20
20
50
Composi ion
D y Weigh (%)
Lipid
17.97
17.97
17.51
16.22
17.59
15.51
17
17.67
P o ein
44.35
46.97
45.83
47.42
47.51
47.62
44.83
46.35
Ash
4.06
5.22
4.61
5.05
5.42
5.5
4.55
4.83
Chap e 3 G ow h, eed u iliza ion and body a y acids
64
Table 3.3: Fa y acid composi ions o he expe imen al die s (g/100 g o al a y acids)
Fa y acids
FO
70L
100L
70S
100S
50S20L
20S50L
50S50L
14:0
9.23
3.11
1.57
3.32
1.59
2.67
2.60
1.37
15:0
0.26
0.10
0.06
0.11
0.06
0.32
0.23
0.06
16:0ISO
0.11
0.04
0.03
0.05
0.03
0.01
0.06
0.03
16:0
22.21
12.19
10.21
15.84
13.69
15.46
12.16
11.75
16:1n-7
11.25
3.91
1.98
4.06
2.01
3.07
3.24
1.73
16:1n-5
0.38
0.14
0.09
0.16
0.09
0.07
0.10
0.08
16:2n-4
1.83
0.61
0.28
0.61
0.29
0.11
0.51
0.24
17:0
0.85
0.38
0.25
0.41
0.28
0.52
0.31
0.25
16:3n-4
2.00
0.66
0.29
0.67
0.31
0.59
0.54
0.24
16:3n-3
0.15
0.07
0.05
0.07
0.05
0.57
0.03
0.05
16:3n-1
0.12
0.05
0.04
0.06
0.04
0.07
0.07
0.03
16:4n-3
0.72
0.26
0.13
0.24
0.11
0.04
0.05
0.09
16:4n-1
-
-
-
-
-
0.25
0.29
-
18:0
3.85
3.96
3.99
3.43
3.20
10.20
3.49
3.74
18:1n-9
9.10
13.75
15.31
18.60
21.48
15.30
18.48
19.04
18:1n-7
3.16
1.57
1.36
2.15
1.77
1.15
1.37
1.36
18:1n-5
0.14
0.08
-
0.12
0.11
0.09
0.10
0.10
18:2n-9
0.04
0.02
-
-
-
-
0.03
-
18:2n-6
4.02
12.36
16.21
29.93
38.51
22.25
18.47
27.25
18:2n-4
0.38
0.13
0.06
0.12
0.06
0.18
0.12
0.05
18:3n-6
0.36
0.13
-
-
0.07
0.13
0.12
0.09
18:3n-4
0.04
-
0.04
0.18
0.09
0.70
0.12
0.05
18:3n-3
0.48
31.94
37.63
5.63
6.01
11.38
20.67
23.03
18:4n-3
1.94
0.76
0.40
0.73
0.40
0.06
0.75
0.36
18:4n-1
-
-
0.03
-
-
-
0.08
-
20:0
0.28
0.21
0.19
0.28
0.28
0.72
0.26
0.24
20:1n-9
2.59
1.94
1.84
2.08
1.89
1.74
2.35
1.91
20:1n-7
0.26
0.13
0.09
0.13
0.10
0.11
0.15
0.09
20:2n-9
-
0.04
-
0.04
0.01
0.02
0.03
0.01
20:2n-6
0.19
0.11
0.08
0.11
0.09
0.10
0.04
0.08
20:3n-9
-
-
-
-
-
0.08
0.15
-
20:3n-6
0.25
0.09
0.04
0.06
0.03
0.08
0.08
0.03
20:4n-6
1.11
0.43
0.24
0.43
0.24
0.40
0.39
0.21
20:3n-3
-
-
0.07
-
-
0.06
0.08
-
20:4n-3
0.96
0.35
0.16
0.33
0.16
0.32
0.38
0.13
20:5n-3
10.05
4.00
2.07
3.77
2.06
3.85
3.99
1.73
22:1n-11
1.79
1.52
1.48
1.67
1.61
1.45
1.91
1.64
22:1n-9
-
-
0.42
-
-
0.33
0.52
-
22:4n-6
0.34
0.15
0.09
0.14
0.09
0.15
0.14
0.08
22:5n-6
-
-
-
-
-
0.64
0.75
-
22:5n-3
1.74
0.66
0.29
0.60
0.29
0.39
0.11
0.24
22:6n-3
7.82
4.16
2.92
3.87
2.91
4.35
4.69
2.63
Sa u a es
36.68
19.95
16.28
23.40
19.10
29.88
19.06
17.41
Monoenoics
28.79
23.10
22.62
29.03
29.09
23.33
28.57
25.98
n-3
23.42
42.19
43.72
15.23
12.00
21.02
30.74
28.26
n-6
6.27
13.26
16.66
30.67
39.02
23.76
-
27.74
n-9
22.99
19.66
19.55
24.78
25.40
17.48
24.79
22.69
n-3 HUFA
20.57
9.17
5.50
8.57
5.42
8.97
9.24
4.73
n-3/n-6
3.74
3.18
2.62
0.50
0.31
0.88
1.54
1.02

Chap e 3 G ow h, eed u iliza ion and body a y acids
65
S a is ical analysis
All he da a we e s a is ically ea ed using ANOVA and Tukey’s es a P<0.05 was
applied as a mul iple sample compa ison analysis using a SPSS S a is ical So wa e
Sys em 10.0 (SPSS Inc., Chicago, Illinois) (Sokal and Rol . 1995).
Resul s
Lipid pe oxida ion p oduc s we e de e mined as hioba bi u ic acid eac i e
subs ances (TBARS) and hey showed no signi ican di e ences be ween he die s,
anging be ween 8.56 and 3.85 µmole o malonaldehyde (MDA)/kg o we die . No
die a y e ec was obse ed on TBARS concen a ion (P<0.05). The a y acid p o ile o
he di e en die s e lec ed he inclusion le el o di e en VOs (Table 3.3). Sa u a ed
a y acids dec eased wi h highe FO eplacemen in he die s, anging om 36.68 in FO
die o 17.41 % in 50SO50LO die . To al monounsa u a es (MUFA; mainly oleic acid
(18:1n-9, OA) end o be educed wi h he inclusion o LO and o inc ease wi h he
inclusion o SO, anging om 22.62 % in 100LO die o 29.09 in 100S. LA in die
inc eased wi h inc easing VO inclusion, pa icula ly SO, anging om 4.02% in FO die
o 38.51% in 100S die , while LNA was also inc eased, pa icula ly by LO inclusion,
anging om 0.48% in FO die o 37.63% in 100L die . The pe cen ages o ARA, EPA
and DHA we e p opo ionally educed by he eplacemen inc ease, anging om 0.21,
1.73 and 2.63 %, espec i ely, in 50S50L die o 1.11, 10.05 and 7.82%, espec i ely, in
FO die . Thus, in die s wi h 70% FO subs i u ion by plan oils he con en s o ARA,
EPA and DHA we e educed by 56%, 62% and 46% espec i ely, while when
subs i u ing up o 100% o FO, he educ ion o hese a y acids was 74%, 83% and
62% compa ed o hei espec i e alues in he con ol die .
All ish accep ed all he expe imen al die s and no ish died du ing he eeding
pe iod. All ea men s showed good g ow h bu , a e 4 mon hs o eeding ish ed FO
die showed he highes inal body weigh (Figu e 3.1). Mon hly SGR inc eased wi h
empe a u e along he expe imen being slowe a he beginning and highe a he end o
he eeding pe iod. A e age SGRs we e signi ican ly lowe in ish ed 70S20L, 20S50L
and 50S50L compa ed o he con ol die (Table 3.4). CI anged om 1.23 o ish ed
FO o 1.64 o ish ed 20L50S a he end o he ial (Table 3.4). Thus, ish ed he
con ol die showed a signi ican ly highe inal weigh , wi h 277.1 g, compa ed o he
o he ea men s. The weigh gain ollowed a simila pa e n wi h a signi ican ly highe
Chap e 3 G ow h, eed u iliza ion and body a y acids
66
alue o ish ed he con ol die and he lowes alues o ish ed 100S and 50S50L
die s.
Figu e 3.1: E ec o di e en die a y oils on gil head seab eam body weigh (expe imen I)
(n = 150). * Deno es signi ican di e ences (P < 0.05) be ween 100F and he es o he expe imen al
g oups.
Fa y acid composi ions o ille o al lipid o sea b eam a e 26 weeks o
eeding he expe imen al die s a e shown in Table 3.5. To al sa u a es (mainly 16:0) in
gil head sea b eam ille s we e p opo ionally educed by he inclusion o die a y plan
oils, bu o a lowe ex en han in he die s. To al MUFA con en s we e simila in all
ille s bu oleic acid was signi ican ly highe in ille s om ish ed he VO die s
compa ed o he con ol ea men . Muscle con en s o LA we e also highes in ish ed
die s wi h pa ial subs i u ion o die a y FO, due o he high le els o LA in SO and o a
lesse ex en in LO die s. The same pa e n was ue o o al n-6 PUFA since LA
cons i u ed he main n-6 componen . In con as , ARA was signi ican ly lowe in ish
ed 70L, 70S, 100S and 50S50L compa ed o he FO. The con en on LNA in lesh o al
lipids was highes in ish ed 100L ollowed by ish ed 70L, hen hose ed 100S and
50S50L, and he lowes con en was ound in ish ed 70S and FO. EPA, DHA and o al
n-3 HUFA we e signi ican ly highes in ish ed FO due o i s highes con en o hese
Chap e 3 G ow h, eed u iliza ion and body a y acids
67
a y acids and dec eased wi h inc eased subs i u ion by VO. The end p oduc s o Δ6-
desa u ase and elongase om LA, including 18:3n-6, 20:2n-6 and 20:3n-6, we e
inc eased in muscle om ish ed SO. In addi ion he dead end p oduc o C18/C20
elongase om LNA, 20:3n-3, was inc eased in ish ed LO.
The e ec s o he expe imen al die s on a y acid composi ions o li e we e
quali a i ely he same as desc ibed o lesh. Howe e , a numbe o no able quan i a i e
di e ences occu ed, pa icula ly wi h espec o MUFA. Li e o al lipid a y acid
composi ion om sea beam ed he expe imen al die s is shown in Table 3.6. A e he
eeding pe iod, o al sa u a es we e signi ican ly highe in ish ed FO. To al MUFA,
p ima ily OA, we e signi ican ly highe in li e o al lipids om ish ed 100L, 70S,
100S and 50S50L compa ed o he FO die , due o he highe inclusion o his a y acid
in he blends o VO. LA was signi ican ly highe in all li e o al lipids om ish ed
VO and he highes con en was egis e ed in ish ed 100S. To al n-6 was signi ican ly
highe in ish ed SO and 50S50L e lec ing i s highe con en o LA. The li e o al
lipid con en o LNA was also a ec ed by he eeding ial, as ish ed LO showed
signi ican ly he highes con en s on his a y acid, ollowed by 50S50L. Li e o al
lipid pe cen age o EPA, DHA and o al n-3 HUFA was signi ican ly highe in ish ed
FO as a consequence o he die a y abundance o hese a y acids. Li e con en o
LNA also e lec ed he die a y inpu o his a y acid, such ha ish ed he LO die
showed signi ican ly he highes le els, ollowed by 50S50L and 100S. The Δ6-
desa u ase and elongase p oduc s om LA and LNA, such as 18:3n-6, 20:2n-6, 20:3n-6
and 20:3n-3, we e signi ican ly inc eased in ish ed VO compa ing o FO and
acco ding o he amoun o he p ecu so a y acid in he die .
Fa y acid composi ion om he gills showed a simila pa e n (Table 3.7), hus
o al sa u a es we e signi ican ly educed when eeding seab eam LO and SO compa ed
o he FO die . This can be explained mainly by he high con en in FO o 16:0. OA was
signi ican ly inc eased in gills om ish ed all he expe imen al VO die s compa ed o
FO. LA was signi ican ly inc eased in all ish ed he VO die s wi h he maximum
obse ed o he 100S die . LNA was also inc eased signi ican ly in ish ed LO
acco ding o he le el o inclusion and consequen ly o al n-3 con en was highe . ARA
was signi ican ly educed in gills om ish ed VO compa ed o FO, and he le els o
his FA we e highe in his o gan compa ed o he li e o muscle up o almos 2 old.
EPA, DHA and o al n-3HUFA we e signi ican ly dec eased in ish ed LO and SO
Chap e 3 G ow h, eed u iliza ion and body a y acids
68
compa ed o he con ol die . The amoun s o 18:3n-6, 20:2n-6 and 20:3n-6 we e
inc eased in SO ed ish, while 20:3n-3 we e inc eased in LO ed ish compa ed o FO.
The n-3/n-6 a io was clea ly a ec ed by bo h he ype and he le el o inclusion o each
oil and highe n-3/n-6 a ios we e obse ed in all he issues o ish ed FO. Howe e ,
inc easing die a y VO concen a ions led o a p og essi e dec ease in he n-3/n-6 a io.
Die induced changes on sa u a ed and MUFA concen a ions o all he issues.
Chap e 3 G ow h, eed u iliza ion and body a y acids
75
g ow h pe o mances. Al hough, Mon e o and colleagues (2008) ha e demons a ed ha
ish ed VOs in he p esen expe imen ha e dec eased phagocy ic ac i i y, educed
se um al e na i e complemen pa hway ac i i y and ele a ed basal cons i u i e le els o
Mx ansc ip exp ession in he li e .
P e ious s udies in seab eam ha e demons a ed ha a y acid composi ions
e lec he die a y a y acid composi ion (Mon e o e al., 1996; 2003; 2005, 2008;
Izquie do e al., 2000; 2003, 2005). This was clea ly shown in he p esen s udy, in
muscle, li e and gills. Speci ically, in muscle and li e , posi i e co ela ions be ween
die a y and issue FA concen a ion we e shown o 18:2n-6, 18:3n-3, 18:1n-9, 20:4n-6,
20:5n-3 and 22:6n-3 (Figu e 3.2). In ag eemen wi h hese s udies, he linea co ela ion
ob ained in he p esen ial e ealed di e ences be ween he ela ionships o die a y
and issue FA o each indi idual FA. In pa icula , EPA and DHA we e p esen in
highe concen a ions in muscle compa ed o die in all he ea men s. In line wi h
p e ious s udies, hese sugges ha when hese FA we e p o ided o he ish in low
concen a ions hey we e selec i ely e ained in he issues (Bell e al., 2004; Mon e o e
al., 2005). The di e ences in e en ion and u ilisa ion o speci ic FA in muscle and li e
a e ela ed o he di e en unc ions o FA in he wo issues. Li e is he p ima y lipid
s o age o gan in ma ine ish and his is la gely FA s o ed as iacylglyce ols while he
muscle issue has mos o i s FA con ained in memb ane phospholipids. Since HUFA,
especially EPA and DHA, a e i al o he unc ion o cell memb anes he e en ion o
hese HUFA in phospholipids is gene ally mo e e icien han in iacylglyce ols
(Izquie do e al., 2000; Sa gen e al., 2002).
HUFA le els signi ican ly dec eased in lesh, li e and gills om ish ed VO
compa ed o he con ol ish. Fa y acids deposi ed in he lesh e lec ed hose inges ed
in he die . Howe e , in pa icula , DHA and ARA le els in lesh we e highe han in
die , indica ing a selec i e deposi ion and e en ion. DHA is known o be mo e abundan
in pola lipid o lesh (Izquie do e al., 2005) wi h he con en being double ha o EPA.
The possible mechanisms unde lying his selec i e deposi ion include he high
speci ici y o a y acyl ans e ases o DHA and he ela i e esis ance o DHA o β-
oxida ion s emming om he complex ca abolic pa hway o his HUFA (F øyland e
al., 2000). In spi e o his selec i e e en ion o DHA, he con ibu ion o he desa u ase
and/o elongase ac i i y upon i s p ecu so (LNA) is e y limi ed and, in consequence,
DHA and also EPA a e essen ial and mus be p o ided by he die (Wa anabe e al.,
1989; Wa anabe, 1993; Ibeas e al., 1994; Izquie do, 1996, e al., 2000; Mon e o e al.,

Chap e 3 G ow h, eed u iliza ion and body a y acids
76
1998). Indeed, ish ed VOs showed a lowe EPA in ille han in he die s p o ided,
indica ing ha his a y acid is no subjec o speci ic e en ion. The same pa e n was
ound in p e ious s udies and he au ho s sugges ed a p e e en ial oxida ion o EPA
o e DHA in he muscle (Izquie do e al., 1989a,b, 2005), since he mi ochond ial
oxida ion is p edominan in he muscle (F oyland e al., 2000) and EPA had a high
a ini y wi h his ype o oxida ion (mi ochond ial be a-oxida ion) (Madsen e al., 1998).
In addi ion, i has been demons a ed ha DHA inhibi s EPA inco po a ion in o
phospha idylcholine and phospha idyle hanolamine in sea b eam la ae (Izquie do e
al., 2000). Consequen ly, he ille nu i ional quali y could be a ec ed by high le els o
VO inclusion, due o he dec ease o n-3 HUFAs, ha a e essen ial nu ien s o human
heal h (Simopoulos, 1999; Sa gen e al., 2001).
Humans a e no e y e icien a syn hesizing e y-long-chain PUFAs and i has
been pos ula ed ha his is because hey ha e e ol ed wi h die s abundan in ish and
game mea s, which a e qui e ich in hese ypes o a s (Co dain e al., 2000, 2005). I is
hough ha a de iciency o n-3 HUFA, especially EPA and DHA ound in FO, can be
linked o many in lamma o y diseases o he de eloped wo ld, such as ca dio ascula
disease and in lamma o y diso de s such as a h i is (Hibbeln e al., 2006). Fu he mo e,
DHA has been ecognized as being i ally impo an o neu al unc ion, and a
de iciency o his HUFA has been linked o dep ession, cogni i e diso de s, and men al
illness (Conklin e al., 2007).
In u n, C18 a y acids a e selec i ely deposi ed in lesh, when p esen a high
concen a ions in he die (Bell e al., 2001, 2002, 2003; Caballe o e al., 2002;
Izquie do e al., 2003, 2005, 2008). This sugges s ha he monoenes, mainly 18:1n-9, as
well as LA and LNA, a e eadily oxidized when p esen a high concen a ions
(Mou en e and Bell, 2006). In muscle, li e and gills, 18:2n-6 and 18:3n-3 we e ound
in much lowe concen a ions han in he die when he ish we e ed he VO die s,
indica ing ha when hese FA we e abundan in he die hey we e selec i ely u ilized
o me abolism, p obably o ene gy p oduc ion (Hende sen and Sa gen , 1985;
S ubhaug e al., 2006). In e es ingly, he concen a ion o 18:1n-9 in li e and gills was
highe han in he die , sugges ing a p e e en ial e en ion, whe eas in muscle p esen ed
a highe con en han in he die only o ish ed 70S and 100S. The selec i e e en ion
o his FA in seab eam, a he han mobiliza ion o ca abolism, may e lec i s
s uc u al unc ion in memb ane phospholipids whe e i is o en ound in he sn-1
Chap e 3 G ow h, eed u iliza ion and body a y acids
77
posi ion o phospholipids wi h HUFA being a ou ed in he sn-2 posi ion (Bell and
Dick, 1991).
LO is ich in 18:3n-3 and p e ious s udies ha e shown an inc eased issue
con en o his a y acid and o al n-3 when i is included in he die (Izquie do e al.,
2003, 2005; Menoyo e al., 2007). In he p esen s udy, eeding seab eam wi h die s
con aining inc easing le els o LO signi ican ly inc eased hei con en o 18:3n-3 in
muscle, li e and gills in a pa allel manne . This a y acid has been demons a ed o be
bene icial o human nu i ion by imp o ing many physiological unc ions (Fe e i and
Flanagan, 1996; Newcome e al. 2001; Mailla d e al., 2002; B ouwe e al., 2004),
including ca diop o ec i e e ec s, modula ion o in lamma o y esponse, and a posi i e
impac on bo h cen al ne ous sys em unc ion and beha iou (S a k e al., 2008). In
addi ion, i is shown ha his a y acid educed he numbe o ca diac dea hs by 70% in
Medi e anean consume s (De Lo ge il e al., 1994, 1999) and i is also used in
li es ock eeding (Sande son e al., 2002). While SO con ains high le els o LA, and is
eadily a ailable a a a ou able p ice, se e al au ho s claim ha highe le els o n-6
a y acids p esen in some VOs can unbalance he die a y n-3/n-6 a io and induce
a hy hmias which subsequen ly can lead o ca diac dys unc ions (Jou en e al., 2001;
Lea , 2001); Thus, i s inclusion in die s o ish should be limi ed due o he g ea e
educ ion o he n-3/n-6 a io ha a ises when using he p oduc . In he p esen s udy,
di e en issues om seab eam ed SO showed a linea deposi ion o n-6, mainly LA,
wi h inc easing SO inclusion le el wi h a high posi i e co ela ion (Figu e 3.2). Thus,
ailo ing he a y acid composi ion o a med ish o sui consume p e e ence,
pa icula ly om he poin o iew o supplying heal hy ood, was conside ed when
subs i u ing FO wi h VOs in die o ma ine ish. P e ious s udies ha e shown ha
eeding seab eam wi h die s con aining a highe con en o LA o LNA ich oils,
signi ican ly inc eased he con en s o hese a y acids in he lesh and consequen ly
educed i s quali y o he consume (Bell e al., 2001, 2002, 2003; Caballe o e al.,
2002; Mon e o e al., 2003, 2005; Izquie do e al., 2003, 2005).
In ish ed SO and LO, he muscle, li e and gills p esen ed highe le els o
p oduc s o Δ6 desa u a ion and elonga ion om hei C18 p ecu so s, including 20:2n-6
and 20:3n-6, in SO ed ish and 20:3n-3 in LO ed ish, sugges ing a mo e e icien
elongase han desa u ase ac i i y, specially o n-3 se ies. This was also ound in
ano he s udy (no published da a) showing highe del a 6 desa u ase ac i i y in ish ed
LO compa ing o apeseed oil o FO die in se e al issues such as in es ine, li e bu
Chap e 3 G ow h, eed u iliza ion and body a y acids
78
wi h lowe le els in he muscle, deno ing he impo an oles o he i s 2 o gans in
lipid me abolism. 20:3n-3 was 9 old highe in ish ed LO compa ed o he con ol,
while 20:2n-6 was only inc eased 5 old, while 20:3n-6 was only inc eased 2 old,
which is in acco dance wi h p e ious s udies (Toche e al., 2000; Menoyo e al., 2005).
Nume ous mammalian s udies ha e es ablished ha a a y acid can compe e and
in luence he me abolism o ano he a y acid (Ga cia and Holman, 1965). In addi ion,
he n-3 PUFA a e known o be usually mo e e ec i e in inhibi ing he me abolism o n-
6 PUFA han ice e sa (Ho obin, 1991). The same e ec s we e ound in p e ious
s udies analysing di e en issues om he same species (Mon e o e al., 2005; Ganga
e al., 2005; Izquie do e al., 2003, 2005) deno ing he s imula ion e ec o VOs in
inc easing he a y acyl desa u a ion/elonga ion pa hway (Bell e al., 2002; Toche e
al., 2002, 2003). The e o e, in e ms o p oduc nu i ional quali y, he e is a ma ked
e ec o eeding VOs on seab eam lesh quali y exp essed by i s dec eased con en o n-
3 HUFAs.
Li e PUFA composi ion was a ec ed by VO die s ollowing a simila bu mo e
ex eme pa e n han in he ish ille (Mon e o e al., 2003; Izquie do e al., 2003).
The e was a selec i e e en ion o 16:0 and 18:1n-9 in VO ea men s, which could be
conside ed as a lipid s o age depo associa ed wi h ese es o me abolic ene gy a he
han an immedia e syn hesis o cell memb ane phospholipids (Sa gen e al., 2002).
Indeed, 16:0 and 18:1n-9 a e he p e e ed subs a es o β-oxida ion in ish (Hende son
and Sa gen , 1985, To s ensen e al., 2000). In addi ion, 18:1n-9, LA and LNA ha e
always shown lowe concen a ions in li e han in he die (Bell e al., 1994, 2001,
2003; Caballe o e al., 2002; Izquie do e al., 2003, 2005) deno ing hei eady
oxida ion when p esen a highe concen a ions. As in lesh he e is an inc easing
amoun o 20:2n-6, 20:3n-6 and 20:3n-3 in li e s om ish ed VOs, deno ing again he
p esence o he desa u ase and elongase ac i i ies in his me abolic issue. The
dec eased con en o EPA and ARA when eeding VO, e en when he le els o 20:3n-3
and 20:3n-3 a e inc eased, may indica e ha he Δ5 desa u ase ac i i y is e y low i no
absen in ma ine ish (Ghioni e al., 1999; Toche and Ghioni, 1999; Seiliez e al., 2003;
Izquie do e al., 2008).
Gill a y acids p o iles showed he same pa allel e ec s caused by he die a y
inpu s wi h some excep ions, e.g. ARA con en was 2 old highe compa ed o he o he
issues. This o gan has many i al physiological unc ions such as espi a ion and
osmo egula ion. Thus, he a y acid composi ion o his o gan is de e minan o i s
Chap e 3 G ow h, eed u iliza ion and body a y acids
79
op imal unc ion. ARA is known o be he main eicosanoid p ecu so o highly ac i e
ho mones wi h many physiological unc ions (Ho obin e al., 1991; Bell e al., 1994).
Sa u a es we e dec eased in gills om ish ed di e en VOs compa ed o FO, while
MUFA we e no signi ican ly a ec ed. Fish gill is a mul i unc ional o gan ha , in
addi ion o p o iding aqua ic gas exchange, plays dominan oles in osmo ic and ionic
egula ion, acid-base egula ion and exc e ion o ni ogenous was es (E ans e al., 2005)
Thus all hese unc ions need ene gy, la gely om a y acids, and especially sa u a es
and MUFA a e conside ed as p e e able o oxida ion and ene gy p oduc ion, as 16:0,
16:1 and 18:1n-9 ha e been demons a ed o be p e e ed subs a es o β-oxida ion
(Keissling, 1993; Hende son, 1996; F øyland e al., 2000). In his expe imen , MUFA
and sa u a es we e mo e deposi ed in gills in compa ison o he o he issues, sugges ing
he ole o hese a y acids migh be o cope wi h he high ene ge ic demands o his
o gan. I is ai ly well es ablished ha he abili y o salmon o osmo egula e is di ec ly
ela ed o hei die and his may be media ed h ough changes in gill pola lipid
composi ions, in pa icula ARA and EPA le els and he esul an e ec s on
p os aglandin p oduc ion (Toche e al., 2000).
Some scien is s sugges ed ha he e en ion e iciencies o he n-6 EFAs a e
lowe han n-3 a y acids, and i is di ec ly ela ed o he quan i ies o n-6 EFAs p esen
in he die . This sugges s ha he ca abolic deg ada ion o n-6 EFAs was ela i ely
lowe when hey we e supplied in la ge amoun s ia ege able oils (Bendiksen and
Jobling, 2003) and his is in acco dance wi h ou esul s whe e we obse ed ha he
inco po a ion o LA in muscle was sligh ly lowe in 100SO compa ed o 70SO die s
(Table 3.5). The C18 a y acids we e inco po a ed mo e in he muscle compa ed o he
o he issues. Feeding seab eam wi h VOs in p e ious s udies has demons a ed ha he
lesh con en o n-3HUFA could be e-es ablished by a inishing die wi h FO o a
pe iod o 3 mon hs be o e ha es ing (Izquie do e al., 2005). Ne e heless, he lowe
e en ion o n-6 a y acids in compa ison wi h n-3 EFAs may be indica i e o highe
a es o oxida ion o he n-6 a y acids ela i e o n-3 a y acids. The oxida ion o DHA
in ish issues is low, whe eas LA is oxidized mo e eadily (Hende son, 1996). In
addi ion, lipids ha con ain HUFAs a e gene ally conside ed o be mo e easily diges ed
and abso bed han hose con aining less-unsa u a ed a y acids (Hende son and Toche ,
1987; Higgs and Dong, 2000; Johnsen e al., 2000). As such, i is also possible ha he
lowe e en ion e iciency o he n-6 EFAs could also esul om educed diges ion and
abso p ion in compa ison wi h n-3 EFAs (Bendiksen and Jobling, 2003).
Chap e 3 G ow h, eed u iliza ion and body a y acids
80
Tissue lipids o seab eam a e eadily in luenced by he a y acid composi ion o
he die . The p esen s udy clea ly es ablishes linea co ela ions be ween he a y acid
composi ions o die s, and hei inco po a ion in lesh and li e (Figu e 3.2). These
co ela ions a e o p ac ical use o p edic ing ou comes o eeding di e en blends o a
gi en subs i u ing oil in place o ish oil. They also e eal how di e en a y acids in
die a y lipid a e selec ed o o agains e en ion ela i e o issue lipids. Thus, when
subs i u ing FO wi h VOs in die s o gil head seab eam, ca e should be aken o choose
he co ec blend o VOs in o de o main ain he adequa e n-3/n-6 le els and gua an ee
he inal nu i ional quali y o he lesh. Gi en p esen conce ns abou imbalance o n-6
and n-3 PUFA in he die s o de eloped na ions and he encou agemen o consume oily
ish, such as macke el, sa dines, salmon and ou , i is impo an ha cul u ed seab eam
main ain a high le el o essen ial (n-3) HUFA in he edible lesh.
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Izquie do, M.S. (1996) Re iew a icle: essen ial a y acid equi emen s o cul u ed
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A onso, J.M. (2008) Regula ion o g ow h, a y acid composi ion and del a 6
desa u ase exp ession by die a y lipids in gil head seab eam la ae Spa us
au a a. Fish Physiol Biochem, 34:117-127.
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Op imum EFA le els in A emia o mee he EFA equi emen s o ed seab eam
(Pag us majo ). In: Takeda, M., Wa anabe, T. (Eds.), The cu en S a us o Fish
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la as de peces ma inos. Modi icación de la composición lipídica de las p esas.
Thesis Doc o al, La Laguna Uni e si y, Spain.
Chap e 4
S ess esponse in seab eam (Spa us au a a) held unde
c owded condi ions and ed die s wi h di e en le els o
inclusion o linseed and/o soybean oil
Rachid Ganga, Bell, J.G., Mon e o, D., Fe nández-Vaque o, A.,
To , L., Ace e e, L., Beni ez San ana, T. and Izquie do, M.S.
To be submi ed

Chap e 4 S ess panels and head kidney a y acids
91
S ess esponse in seab eam (Spa us au a a) held unde c owded condi ions and
ed die s wi h di e en le els o inclusion o linseed and/o soybean oil
Rachid Ganga, Bell, J.G.1, Mon e o, D., A alah, E., Fe nández-Vaque o, A.2, To , L.,
Ace e e, L.2, Beni ez San ana, T. and Izquie do, M.S.
G upo de In es igación en Acuicul u a (ULPGC & ICCM) P.O. Box 56. 35200. Telde,
Las Palmas. Cana y Islands, Spain
1Ins i u e o Aquacul u e, Uni e si y o S i ling FK9 4LA, Sco land, UK.
2Bioma Ibe ia/ P oAqua Nu i ion, S.A., A-62, Km, 99, ES-34210 Dueñas, Spain.
Abs ac
The physiological esponse o s esso s in ish, including ho monal p o iles and
associa ed issue esponsi eness, a e less documen ed. The aim o his s udy was o
e alua e he e ec o eeding gil head seab eam (Spa us au a a) wi h die s con aining
linseed oil (LO) and soybean oil (SO) as a subs i u e o ish oil (FO) and hei e ec on
a y acid p o ile o head kidney and he consequen e ec on s ess esponse o a
c owding es . Fish we e ed wi h di e en die s wi h di e en le els o subs i u ion 0%
(FO), 70% (70LO, 70SO, 20LO50SO and 50LO20SO) and 100% (100LO, 100SO and
50LO50SO) o e a pe iod o 8 mon hs. A he end o he eeding ial, samples o head
kidney biochemical analysis we e collec ed and he ish we e challenged by a c owding
es . Samples o plasma o co isol analysis we e collec ed a di e en imes du ing he
es , 0h, 2h, 5h, 24h, 48 and 1 week. Basal co isol le els we e signi ican ly inc eased in
ish ed 70LO, 100LO and 50LO50SO. The physiological esponse o c owding was
signi ican ly a ec ed by he die . A e 2 h o c owding, all he ea men s showed
highe co isol, wi h ish ed 100LO signi ican ly di e en wi h he maximum esponse
o 131.38 pg/ml compa ed o 50LO20SO which had he lowes plasma co isol esponse
wi h only 18.73 pg/ml. A e 5h and 24h, plasma co isol was educed in all ea men s
excep o 50LO20SO. A e 48 h o c owding, he plasma co isol was inc eased in all
ea men s wi h he maximum alue seen in ish ed 100LO (72.12 pg/ml). These alues
we e dec eased a e 1 week in ish ed FO, 70LO, 100LO and 50L050SO a e 1 week
o c owding, bu emained highe in ish ed 70SO, 100SO, 20LO50SO and
50LO20SO. In gene al, ish ed wi h LO we e cha ac e ized by a as e and s onge
s ess esponse and apid ecupe a ion, while ish ed SO had a slow esponse and a
longe ecupe a ion.
Keywo ds: Seab eam, s ess, co isol, a y acids, head kidney, linseed oil, soybean oil.
Chap e 4 S ess panels and head kidney a y acids
92
In oduc ion
Fin ish aquacul u e has adi ionally used die s con aining la ge amoun s o ish
meal (FM) and ish oil (FO), p ima ily as a cos -e ec i e sou ce o highly diges ible
animal p o ein and lipids. Mo eo e , FO is an impo an die a y sou ce o omega-3
a y acids, and in pa icula he long chain polyuns au a ed a y acids (PUFA),
eicosapen aenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3)
(Ackman, 1989; Hende son and Toche , 1987). In pa icula , FO is impo an in ish
nu i ion as i is used as he main sou ce o hese essen ial a y acids (EFA) equi ed
o ish o ensu e op imal g ow h, de elopmen and ep oduc ion (Izquie do e al.,
1989; Sa gen e al., 1999). The s eady inc ease in global p oduc ion olume in
aquacul u e o 8-10% a yea (Tacon, 2004, 2006) has esul ed in inc easing p ices and
limi ed a ailabili y o hese ing edien s in he ma ke . Consequen ly, i is expec ed ha
he demand o FO by aquacul u e will p obably exceed a ailable esou ces o e he
nex decade (Pike and Ba low, 2003; Tacon, 2004, 2005). The e o e, ecen ly much
in e es has ocused on esea ching al e na i es o FO and FM in aqua eeds, and many
s udies ha e epo ed he success ul use o ege able oils (VO) and ege able p o eins
as good subs i u es wi hou comp omising ish g ow h o eed u iliza ion (Bell e al.,
2001, 2002; To s ensen e al., 2000; Caballe o e al., 2002; Mon e o e al., 2003, 2005;
Izquie do e al., 2003, 2005).
VOs a e de oid o C20 n-3 PUFA, including EPA and DHA while he le els o
C18 PUFA, linoleic acid (LA; 18:2n-6), linolenic acid (LNA; 18:3n-3) and
monounsa u a ed a y acids (MUFA; mainly oleic acid OA, 18:1n-9) a e high in VO.
Thus, including VOs in die s o ma ine ish ha e been ound o lead o inc eased C18
PUFAs and educed n-3 HUFA in he lesh o he ish, po en ially comp omising he
nu i ional quali y o consume s (Bell e al., 2001, 2002; To s ensen e al., 2000;
Mon e o e al., 2003, 2005; Izquie do e al., 2003, 2005). In addi ion, modi ying he
a y acid p o iles o ish issue by educing i s HUFA con en could ha e many
physiological and me abolic impac s in ish; since hese componen s ha e impo an
oles in me abolism, canno be syn hesized de no o and, he e o e, mus be ob ained
om he die (Ghioni e al., 1999). Al e a ion o he phospholipid composi ion may
induce s uc u al modi ica ions ha in u n modula e he unc ional p ope ies o he
memb anes (Wills, 1985; Ch is on e al., 1992), indica ing he undamen al ela ionship
be ween nu i ion and animal's esponses o i s en i onmen .
PUFA/HUFA a e memb ane componen s, sou ces o ene gy and cell media o s
Chap e 4 S ess panels and head kidney a y acids
93
ha a e essen ial o cell s uc u e and unc ion. I is well documen ed ha die a y a
can in luence he a y acid composi ions o di e en ish issues, including hose o
cell memb anes (Bell e al., 2001, 2002; Izquie do e al., 2003). They may al e a ious
p ope ies o he memb anes such as in eg i y, luidi y, pe meabili y, dis ibu ion and
numbe s o ecep o si es (Yaqoob, 1998). HUFAs a e also p ecu so s o a ange o
highly ac i e C20 compounds called eicosanoids, ha a e o med in small o e en ace
amoun s by i ually e e y issue o he body and in ol ed in a wide a ie y o
physiological unc ions (Bell e al., 1991). In b oad e ms hey a e p oduced in
esponse o s ess ul si ua ions, bo h a cellula and whole body le el. In ish, HUFA
a e epo ed o modula e s ess esponse (Mon e o e al., 1998, 2003; Tago e al.,
1999). Whe eas, he mechanisms by which HUFA could modula e ish esis ance o
s ess a e s ill unknown, some ecen s udies ha e sugges ed ha hese e ec s could be
media ed pa ly by eicosanoids (Ko en e al., 2001a,b; Van Anhol e al., 2004a,b;
Ganga e al., 2006). Thus, incuba ing head kidney issue wi h di e en HUFA in an in
i o supe usion sys em a ec ed signi ican ly hei ACTH-s imula ed co isol elease,
and his e ec was pa ly media ed by cycloxygenase (COX) and lipoxygenase (LOX)
me aboli es (Ganga e al., 2006).
The e is a end owa ds inc eased conce n in animal wel a e and, ecen ly, his
conce n has expanded o include he wel a e o ish. High densi ies a e gene ally used
in in ensi e aquacul u e compa ed o he wild ishe ies (Tu nbull e al., 2005) o
imp o e business p o i abili y. Gene ally his p ac ice is conside ed as a po en ial
sou ce o s ess, wi h a nega i e e ec on ish g ow h a e and wel a e (Le ançois e
al., 2001; Ellis e al., 2002; Van de Nieuwegiessen e al., 2008, 2009), su i al and
eeding a e (Rowland e al., 2006). In eleos s, he s ess esponse comp ises o a
numbe o physiological p ocesses, which a e la gely egula ed by he hypo halamus-
pi ui a y-in e enal (HPI) axis. Following exposu e o a s esso , he hypo halamus
sec e es co ico opin- eleasing ho mone (CRH), which s imula es he pi ui a y o
sec e e ad enoco ico opin ho mone (ACTH). Binding o ACTH o memb ane
ecep o s o he s e oidogenic cells ac i a es a se ies o enzyma ic eac ions using
choles e ol as a subs a e o he syn hesis o co isol (Pa iño e al., 1986; Lac oix and
Hon ela, 2001). Co isol p oduc ion is unde he con ol o he HPI axis ac i a ion
(Wendelaa Bonga, 1997; Mommsen e al., 1999) and is conside ed a key esponse o
s ess, in o de o cope wi h he physiological changes and o eco e homeos asis.
Se e al s udies ha e epo ed a ela ionship be ween he s ess esponse in ish unde
Chap e 4 S ess panels and head kidney a y acids
94
c owded condi ions and inc eased plasma-co isol le els (Mon e o e al., 1999; Ruane
and Komen, 2003; T enzado e al., 2006; Van de Nieuwegiessen e al., 2008, 2009).
Al hough plasma co isol esponse o s esso s is well es ablished in eleos s, he
magni ude and du a ion o his s e oid esponse is dependen upon he ype, in ensi y
and du a ion o he s esso as well as he his o y o he animal (Wendelaa Bonga,
1997; Iwama e al., 2006) as well as he eeding his o y being c ucial in de e mining
he s ess esponse. Unde condi ions o acu e s ess, co isol ele a ion usually las s
hou s, bu wi h ch onic s ess, such as p olonged con inemen , alues may emain high
o days o weeks (Picke ing and Po inge , 1989; Iwama e al., 2006), only e u ning o
basal alues o e conside able ime (Picke ing and S ewa , 1984).
The aim o he p esen s udy was o con ibu e o ou unde s anding o how
eeding lipid om ege able sou ces (LO and SO), ich in C18 a y acids (mainly LA,
LNA and OA), could a ec a y acid me abolism and hei inco po a ion in head
kidney issue and hei consequen e ec on s ess esis ance caused by c owding. This
was s udied by measu ing he plasma basal and pos -s ess co isol le els.
Ma e ials and Me hods
Fish and Die s
Two housand wo hund ed and i y ju eniles o gil head seab eam (Spa us
au a a) (45 g ini ial body weigh ) we e dis ibu ed in 45 anks o 500 l (50 ish/ ank,
each die assayed in iplica e) supplied wi h seawa e a a empe a u e anging om
20-24.2 ºC and cons an ae a ion. Fi e iso-ene ge ic and isoni ogenous die s wi h lipid
con en ~22 % we e o mula ed. Ancho y oil was he only added lipid sou ce in he
FO die ( ish oil). In he o he die s, ish oil was eplaced by linseed (LO) o soybean
oil (SO) oils a 70% (70LO and 70SO, 20LO50SO, 50LO20SO) o 100% (100LO,
100SO and 50LO50SO). Fish we e ed he expe imen al die s con aining di e en
ing edien s (Table 4.1) un il appa en sa ia ion (3 imes/day, 6 days/ week) o 240
days, when hey eached comme cial ma ke size.
Chap e 4 S ess panels and head kidney a y acids
95
Table 4.1: Ing edien s o he expe imen al die s used
% o d y weigh
Oils (Fish oila/linseed/soybean)
16.32
Sou h-Ame ican ish meal
47.26
Whea
7.00
Soybean meal 47%b
25.00
Sun lowe meal
3.67
Vi amins p emixc
0.27
Mine als p emixc
0.48
a Sou h-ame ican, ancho y oil.
b Soybean meal wi h 47% as a b u p o ein, “no GMO”
c Vi amin and mine al p emixes p epa ed acco ding o P oaqua A/S comme cial s anda ds.
Biochemical analysis
Ex ac ion o o al lipid om die s and ish head kidney was pe o med by he
me hod o Folch e al. (1957) using a mix u e o chlo o o m: me hanol (2:1)( : )
con aining 0.01% BHT, as an an ioxidan , ollowed by phase pa i ion wi h KCl (0.88
%). Vigo ous o ex mixing ollowed by cen i uga ion o assis sepa a ion o
chlo o o m and aqueous laye s ex ac ed he lipids om die and head kidney samples.
The lowe laye was il e ed h ough Wha man il e pape and d ied unde ni ogen,
o al lipids we e weighed ollowing desicca ion.
Fa y acid me hyl es e s we e p oduced om aliquo s o o al lipids ex ac ed
om di e en samples by acid-ca alyzed ansme hyla ion pe o med o e nigh a 50°C
as desc ibed by Ch is ie (1982). Fa y acids me hyl es e s we e sepa a ed and quan i ied
by gas-ch oma og aphy (GC; The mo Finnigan) wi h He as a ca ie gas using a used
silica, ca bowax 20M, 30m * 0.32 mm i.d. (d =0.27 m) column (Supleco, Belle on e,
USA). The ini ial empe a u e o he column was se o 170ºC o 10min, and hen i
was aised o 220ºC a 2.5 ºC/min and inally main ained a 215ºC o a u he 5min.
The empe a u e o he injec ion po was 250ºC. The peaks in he ch oma og am we e
iden i ied by compa ison o a well-cha ac e ised ex e nal s anda d (Sigma). Indi idual
me hyl es e s we e iden i ied by compa ison wi h known s anda ds and published da a.
S ess Panels
A e he eeding pe iod, 20 ish pe ank we e exposed o con inemen s ess.
Thus, ish we e dis ibu ed in small loa ing cages (50x30x15cm) in 5 g oups o 4 ish,

Chap e 4 S ess panels and head kidney a y acids
96
ano he 4 ish pe ank we e used as con ol ish o basal plasma co isol de e mina ion
jus be o e con inemen . Subsequen ly, all ish om di e en cages we e sampled a 5
di e en ime in e als (2h, 5h, 24h, 48h and 1 week) a e he beginning o con inmen
o encompass “ea ly” and “la e” esponse o he s esso . A each sampling ime one
cage was ca e ully emo ed om each ank, opened and ish blood was sampled in less
han 2 min o handling ime, blood was ob ained by caudal sinus punc u e wi h a 1 ml
plas ic sy inge. Aliquo s o blood samples we e immedia ely ans e ed o an
Eppendo ube coa ed wi h li hium hepa in as an icoagulan . The plasma was ob ained
by cen i uga ion a 3000 pm o 10 min and s o ed a -80º C p io o co isol
de e mina ion and hen he ish we e libe a ed. The same p ocedu e was epea ed a e
5h, 24h, 48h and 1 week. This app oach was aken o keep any acu e dis u bance o he
emaining ish, om ish emo al, as low as possible; bo h densi y g oups we e ea ed
in he same manne . Thus, o al cap u e ime was less han 8 min pe ank o minimize
cap u e s ess e ec s on analyzed pa ame e s (Sump e , 1997).
Co isol measu emen s
Co isol concen a ion in he pe used luid was de e mined by
adioimmunoassay (RIA) (Ro llan e al., 2001). The an ibody, Biolink, S.L. (Cos a
Mesa, Cali o nia), was used a a inal dilu ion o 1:6000. This an ibody c oss eac i i y
is 100% wi h co isol, 11.40% wi h 21-desoxyco icos e one, 8.90% wi h 11-
desoxyco isol and 1.60% wi h 17a-hyd oxyp oges e one. The adioac i i y was
quan i ied using a liquid scin illa ion coun e . Co isol le els a e gi en as ng/ml o
plasma.
S a is ical analysis
Signi icance o di e ence (P<0.05) be ween die a y ea men s was de e mined
by one-way analysis o a iance (ANOVA) ollowed by Duncan’s mul iple compa ison
es s (Sokal and Rol 1995). Analyses we e pe o med using SPSS so wa e (SPSS o
windows 13).
Resul s
Lipid pe oxida ion p oduc s we e de e mined as hioba bi u ic acid eac i e
subs ances (TBARS) and hey showed no signi ican di e ence be ween he die s,
anging be ween 8.56 and 3.85 mmole o malonaldehyde (MDA)/kg o we die . No
die a y e ec was obse ed on TBARS concen a ion (P<0.05).
Chap e 4 S ess panels and head kidney a y acids
97
The con ol die (FO), o mula ed wi h 100% FO, con ained app oxima ely 37%
o al sa u a es, mainly 16:0, almos 29% o al monounsa u a ed a y acids wi h
app oxima ely one- hi d as 18:1n-9, 6 % n-6 a y acids, p edominan ly 18:2n-6, and
29% n-3 a y acids p edominan ly n-3HUFA, mainly EPA (Table 4.2). Inclusion o
di e en VO le els esul ed in inc eased pe cen ages o 18:1n-9, 18:2n-6 and 18:3n-3
wi h concomi an dec eased p opo ions o n-3 HUFA, o al PUFA and long chain
monoenes. Thus, he le els o 18:1n-9, 18:2n-6 and 18:3n-3 inc eased o 15, 16 and
38% o o al a y acids, espec i ely, in he die o mula ed wi h 100% LO, and o 21,
38 and 6% o o al a y acids, espec i ely, in he die wi h 100% SO wi h an a e age
alue o 5.5%. n-3HUFA in bo h die s. The 20LO50SO and pa icula ly 50LO20SO die
showed highe con en s o n-3, n-6 and n-9 a y acids. The die 50LO50SO had
18.97%, 27.15% and 22.95% o 18:1n-9, 18:2n-6 and 18:3n-3 espec i ely, and
egis e ed he lowes con en on n-3 HUFA, wi h only 4.71%.
Fish HK a y acid p o ile o o al lipids e lec ed he die a y lipid composi ion
(Table 4.3). N-3 a y acids we e signi ican ly inc eased in ish ed he LO die , while n-
6 con en was signi ican ly inc eased in SO ed die . N-9 a y acids we e also inc eased
in ish ed he LO and VO die s compa ed o con ol die , due mainly o an inc ease in
OA. Howe e , he con en o n-3 HUFA was signi ican ly (P<0.05) educed in ish ed
he wo ege able oils sepa a ely, o as a blend, compa ed o con ol die ed ish oil.
DHA was educed by 45 % in HK o ish ed 70LO, 55 % in ish ed 100LO, 58 % in
ish ed 70SO, 61 % in ish ed 100SO, 35,5% in ish ed 20LO50SO, 19% in ish ed
50LO20SO and 64% in ish ed 50LO50SO. Simila ly EPA was educed by 64 % in
HK o ish ed 70LO, 76 % in ish ed 100LO, 73 % in ish ed 70SO, 80 % in ish ed
100SO, 57% in ish ed 20LO50SO, 42% in ish ed 50LO20SO and 82% in ish ed
50LO50SO. Rega ding ARA, a 47 % educ ion was ound in ish ed 70LO, 16% in ish
ed 100LO, 88% in ish ed 70SO, 86% in ish ed 100SO, 45% in ish ed 20Lo50SO,
40% in ish ed 50LO20SO and 52% in ish ed 50LO50SO. Die a y inclusion o
ege able oils educed DHA/EPA a ios in ish HK, ega dless o he ype o oil used. In
ish ed LO and/o SO he end p oduc s o Δ6 desa u ase and elongase om 18:2n-6 and
18:3n-3 (mainly 20:2n-6, 20:3n-6 and 20:3n-3) we e inc eased in ish ed SO and LO
espec i ely compa ed o he con ol ish. Figu e 1 shows a highposi i e co ela ion
be ween die a y LA, LNA, EPA and hei con en in he head kidney wi h a 2=0.99,
0.93 and 0.91, espec i ely, and o a lesse ex en die a y DHA was also posi i ely
co ela ed wi h die a y DHA con en in he head kidney wi h 2=0.71.
Chap e 4 S ess panels and head kidney a y acids
98
Table 4.2: Fa y acid composi ion o he expe imen al die s (g a y acid/ 100g o o al a y acids)
Fa y acids
FO
70L
100L
70S
100S
20L50S
50L20S
50L50S
14:0
9.23
3.11
1.57
3.32
1.59
2.99
2.6
1.36
15:0
0.26
0.10
0.06
0.11
0.06
0.45
0.27
0.09
16:0ISO
0.11
0.04
0.3
0.05
0.03
0.04
0.08
0.09
16:00
22.21
12.19
10.21
15.84
13.69
27.42
12.21
11.87
16:1n-7
11.25
3.91
1.98
4.06
2.01
3.44
3.23
1.79
16:1n-5
0.38
0.14
0.09
0.16
0.09
0.21
0.10
0.08
16:2n-4
1.83
0.61
0.28
0.61
0.29
0.57
0.64
0.29
17:00
0.85
0.38
0.25
0.41
0.28
0.56
0.31
0.25
16:3n-4
2.00
0.66
0.29
0.67
0.31
0.62
0.53
0.24
16:3n-3
0.15
0.07
0.05
0.07
0.05
0.03
0.03
0.05
16:3n-1
0.12
0.05
0.04
0.06
0.04
0.07
0.07
0.03
16:4n-3
0.72
0.26
0.13
0.24
0.11
0.04
0.05
0.09
16:4n-1
-
-
-
-
-
0.26
0.28
-
18:00
3.85
3.96
3.99
3.43
3.20
7.69
3.48
3.73
18:1n-9
9.10
13.75
15.31
18.60
21.48
12.49
18.42
18.97
18:1n-7
3.16
1.57
1.36
2.15
1.77
1.23
1.37
1.35
18:1n-5
0.14
0.08
-
0.12
0.11
0.09
0.10
0.10
18:2n-9
0.04
0.02
-
-
-
-
0.03
-
18:2n-6
4.02
12.36
16.21
29.93
38.51
16.40
18.42
27.15
18:2n-4
0.38
0.13
0.06
0.12
0.06
0.12
0.12
0.05
18:3n-6
0.36
0.13
-
-
0.07
0.14
0.12
0.09
18:3n-4
0.04
-
0.04
0.18
0.09
0.18
0.12
0.05
18:3n-3
0.48
31.94
37.63
5.63
6.01
11.36
20.61
22.95
18:4n-3
1.94
0.76
0.40
0.73
0.40
0.75
0.75
0.36
18:4n-1
-
-
0.03
-
-
0.07
0.08
-
20:00
0.28
0.21
0.19
0.28
0.28
0.51
0.26
0.24
20:1n-9
2.59
1.94
1.84
2.08
1.89
1.65
2.34
1.90
20:1n-7
0.26
0.13
0.09
0.13
0.10
0.11
0.15
0.09
20:2n-9
-
0.04
-
0.04
0.01
0.02
0.03
0.01
20:2n-6
0.19
0.11
0.08
0.11
0.09
0.04
0.04
0.08
20:3n-9
-
-
-
-
-
0.10
0.15
-
20:3n-6
0.25
0.09
0.04
0.06
0.03
0.09
0.08
0.03
20:4n-6
1.11
0.43
0.24
0.43
0.24
0.43
0.39
0.20
20:3n-3
-
-
0.07
-
-
0.06
0.08
-
20:4n-3
0.96
0.35
0.16
0.33
0.16
0.34
0.38
0.13
20:5n-3
10.05
4.00
2.07
3.77
2.06
4.15
3.98
1.72
22:1n-11
1.79
1.52
1.48
1.67
1.61
1.33
1.91
1.63
22:1n-9
-
-
0.42
-
-
0.31
0.52
-
22:4n-6
0.34
0.15
0.09
0.14
0.09
0.15
0.14
0.08
22:5n-6
-
-
-
-
-
0.70
0.74
-
22:5n-3
1.74
0.66
0.29
0.60
0.29
0.24
0.11
0.23
22:6n-3
7.82
4.16
2.92
3.87
2.91
2.59
4.68
2.62
Sa u a es
36.68
19.95
16.28
23.40
19.10
39.17
18.86
17.45
Monoenoics
28.79
23.10
22.62
29.03
29.09
20.86
28.13
25.92
n-3
23.42
42.19
43.72
15.23
12.00
19.55
30.65
28.15
n-6
6.27
13.26
16.66
30.67
39.02
17.98
19.95
27.60
n-9
22.99
19.66
19.55
24.78
25.40
14.57
21.49
20.88
n-3 HUFA
20.57
9.17
5.50
8.57
5.42
7.38
9.22
4.71
n-3/n-6
3.74
3.18
2.62
0.50
0.31
1.09
1.54
1.02
Chap e 4 S ess panels and head kidney a y acids
99
Table 4.3: E ec o eeding ege able oils on HK a y acids p o ile (g a y acid/100g o al a y acids)
FO
70LO
100LO
70SO
100SO
20LO50SO
50LO20SO
50LO50SO
14:00
7.85±0.17
3.98±0.16
2.79±0.13
3.86±0.12
2.34±0.03
3.11±0.04
4.58±0.53
2.34±0.07
15:00
0.25
0.15±0.01
0.12±0.02
0.14
0.08±0.01
0.52±0.09
0.69±0.36
0.09±0.01
15:1n-5
0.49±0.33
0.45±0.03
0.33±0.03
0.54±0.06
0.33±0.05
0.07±0.05
0.03±0.02
0.35±0.06
16:0 ISO
0.13
0.23±0.03
0.29±0.09
0.10±0.03
0.04±0.03
0.11±0.03
0.11±0.03
0.08±0.01
Me16:0
0.16±0.03
0.14±0.01
0.10±0.01
0.12±0.01
0.09±0.01
-
-
0.1
16:00
22.72±0.12
18.90±0.51
17.13±0.52
20.65±0.58
17.95±0.47
16.75±0.4
18.16±1.27
17.7±0.48
16:1n-9
0.07
0.06
0.28±0.01
0.05±0.03
0.07
-
-
0.15±0.08
16:1n-7
12.33±0.2
5.79±0.15
3.69±0.07
5.76±0.12
3.35±0.01
5.18±0.1
7.11±0.44
3.26±0.06
16:1n-5
0.28
0.17±0.01
0.13±0.01
0.13±0.02
0.09±0.02
0.13±0.01
0.16±0.01
0.1±0.01
16:2n-6
0.34
0.19
0.15
0.20±0.01
0.18
0.21
0.21±0.02
0.16
16:2n-4
1.31±0.02
0.52±0.01
0.35
0.50
0.30
0.52
0.68±0.03
0.28
17:00
0.60
0.41±0.01
0.33±0.01
0.39
0.28
0.36±0.01
0.42±0.08
0.3±0.01
16:3n-4
1.21±0.02
0.49
0.35
0.49±0.01
0.34
0.08±0.01
0.11±0.02
0.32
16:3n-3
0.16
0.37±0.05
0.09±0.01
0.09
0.07
0.51±0.02
0.67±0.04
0.07
16:3n-1
0.35±0.09
-
0.39±0.06
0.28±0.03
0.25
0.03±0.03
0.04
0.33±0.04
16:4n-3
0.30±0.09
0.13±0.06
0.20±0.02
0.12±0.08
0.19
0.16±0.14
0.21±0.12
0.17±0.02
16:4n-1
0.02±0.02
0.05±0.01
0.02
0.02±0.02
0.02±0.02
0.31±0.1
0.26±0.09
0.02±0.02
18:00
4.57±0.12
5.94±0.1
5.91±0.23
4.94±0.07
4.60±0.17
5.77±1.35
4.31±0.76
5.5±0.13
18:1n-9
14.06±0.1e
19.94±0.4d
21.57±0.18c
23.50±0.31b
25.04±0.4a
20.39±0.9bc
20.72±1.2bc
24.64±0.35a
18:1n-7
3.98±0.01
2.52±0.05
2.05±0.06
2.94±0.07
2.51±0.03
1.62±0.45
2.38±0.28
2.33±0.05
18:1n-5
0.19±0.02
0.05±0.07
0.12
0.10±0.07
0.14
0.13±0.02
0.16±0.01
0.13
18:2n-9
0.06
0.04±0.01
0.03±0.01
0.02±0.02
0.05
0.04±0.04
0.02±0.01
0.03±0.02
18:2n-6
3.47±0.04
9.50±0.17e
11.69d
20.62±0.46b
27.94±0.3a
20.52±0.4b
12.85±1.02c
19.63±0.27b
18:2n-4
0.35
0.17
0.11±0.03
0.12±0.01
0.07
0.14±0
0.14±0.08
0.07±0.01
18:3n-6
0.28
0.18
0.15±0.78
0.22±0.01
0.30±0.01
0.25±0.13
0.18±0.05
0.23±0.02
18:3n-4
0.55±0.02
0.14±0.1
0.07±0.02
0.13±0.09
-
0.17±0.15
0.53±0.12
-
18:3n-3
1.16±0.02
16.39±0.55b
20.42±0.02a
2.79±0.14e
3.19±0.13e
7.71±0.1d
6.3±1.13d
11.53±0.31c
18:3n-1
0.95±0.02
0.39±0.03
0.03
-
-
0.01±0.01
-
-
18:4n-3
0.15
0.06
0.31±0.01
0.28±0.02
0.22±0.02
0.44±0.02
0.14±0.25
0.26±0.02
18:4n-1
-
0.09±0.13
0.04±0.03
0.03±0.02
0.02±0.02
0.06
0.1±0.02
0.01±0.01
20:00
0.23
0.15±0.11
0.24
0.24
0.23
0.23±0.01
0.23±0.04
0.24
20:1n-9
1.80±0.04
1.54±0.02
1.47±0.04
1.84±0.02
1.66±0.01
2.03±0.01
3.05±0.28
1.74±0.03
20:1n-7
0.08±0.12
-
0.13±0.01
0.16
0.12
0.15±0
0.2±0.03
0.13
20:2n-9
0.10±0.01
0.09±0.01
0.05
0.08±0.07
0.05±0.04
0.1±0.05
0.03±0.05
0.04±0.03
20:2n-6
0.17
0.21±0.03
0.28±0.01
0.44±0.01
0.65±0.01
0.1±0.02
0.15±0.12
0.49±0.03
20:3n-9
-
-
0.08±0.04
0.14±0.02
0.23±0.02
0.46±0.02
0.31±0.01
0.14±0.02
20:3n-6
0.18±0.01d
0.08±0.01e
0.34±0.01b
0.39±0.01a
0.29±0.01c
0.2±0.01b
0.13±0.04d
0.26±0.02c
20:4n-6
0.96±0.08a
0.81±0.03c
0.51±0.04b
0.11±0.01c
0.13±0.01c
0.53±0.06b
0.58±0.11b
0.46±0.03b
20:3n-3
0.11b
0.31±0.02b
0.51±0.13a
0.26±0.01b
0.19±0.01b
0.31±0.01
0.22b
0.23±0.02
20:4n-3
0.79±0.01
0.37±0.02
0.12±0.09
-
0.07±0.05
0.41±0.01
0.53±0.12
0.04±0.03
20:5n-3
5.07±0.08a
1.83±0.18cd
1.22±0.06de
1.38±0.09cde
1.03±0.1de
2.18±0.16bc
2.93±0.78b
0.92±0.07e
22:1n-11
1.12±0.03
0.99±0.02
0.93±0.02
1.19±0.02
1.06±0.02
1.23±0.06
1.96±0.19
1.14±0.01
22:1n-9
0.53
0.67±0.04
0.65±0.04
0.57±0.02
0.62±0.07
0.59±0.07
0.68±0.05
0.71±0.05
22:4n-6
0.25
0.12±0.01
0.09
0.07±0.05
0.03±0.04
0.14±0.01
0.16±0.04
0.05±0.03
22:5n-6
0.27±0.01
0.12±0.01
0.28±0.27
0.09±0.01
0.07±0.01
1.19±0.02
1.52±0.4
0.07±0.01
22:5n-3
2.40±0.04
0.94±0.07
0.41±0.29
0.75±0.05
0.59±0.06
0.08±0.01
0.07±0.03
0.5±0.04
22:6n-3
7.40±0.19a
4.07±0.28cd
3.35±0.09cd
3.08±0.23d
2.89±0.23d
4.77±0.33bc
5.98±1.34ab
2.69±0.19d
Sa u a es
36.6±0.18ª
29.8±0.76bc
26..7±0.9bcd
30.49±0.74b
25.6±0.66d
26.7±1.05cd
28.4±2.99bcd
26.16±0.7cd
Monoenes
34.8±0.51a
32.03±0.5bc
31.2±0.14c
36.54±0.34a
34.8±0.37a
31.58±1.1c
36.55±2.07a
34.68±0.5ab
n-3
17.7±0.3b
24.73±1.15a
26.48±0.72a
8.82±0.57c
8.48±0.59c
16.56±0.3b
17.05±3.12b
16.41±0.69b
n-6
5.97±0.05
11.02±0.23e
13.87±0.51d
22.33±0.46b
29.75±0.3a
23.06±0.4b
15.77±1.36c
21.33±0.38b
n-9
16.79±0.2e
22.55±0.43d
24.28±0.1cd
26.42±0.2ab
27.85±0.3ª
23.62±0.9cd
24.81±1.47bc
27.38±0.26a
n-3 HUFA
15.91±0.3ª
7.89±0.57bc
5.34±0.28cd
5.51±0.37cd
4.79±0.45c
7.74±0.51bc
9.72±2.21b
4.38±0.36d
n-3/n-6
2.96±0.02
2.24±0.06
1.91±0.04
0.4±0.02
0.28±0.02
0.72±0.02
1.07±0.11
0.77±0.02
DHA/EPA
1.46±0.02
2.23±0.07
2.76±0.05
2.24±0.09
2.80±0.07
2.18±0.02
2.06±0.16
2.94±0.05
Chap e 4 S ess panels and head kidney a y acids
106
expec ed (Mon e o e al., 2005). Ne e heless a e 1 week o c owding, he plasma
co isol le els dec eased in FO, 70LO and 100LO ed ish compa ed o hei alues a
48 hou s, bu emained a he same le els in he o he ea men s wi h 100SO
egis e ing he highes concen a ion. This die a y e ec on s ess esponse could be
explained by he ype and he quan i y o di e en die a y a y acids (Ganga e al.,
2006). While he mechanism o he s ess esponse is s ill con o e sial in ish and i
depends on many ac o s. C owding o b own ou caused inc eased co isol le els o
he i s 2 days, howe e by 6 days, plasma co isol le els e u ned o alues seen p io
o c owding (Picke ing and Po inge , 1987). In addi ion, ecen obse a ions indica ed
ha GR mRNA le els we e lowe ed in sea bass (Dicen a chus lab ax) li e in
esponse o ch onic c owding s ess (Te o a e al., 2005) sugges ing ha he s ess
e ec on he GR message may be ei he species-speci ic and/o dependen on he ype,
in ensi y and du a ion o he s esso (Wiseman e al., 2007) I may be ha he e was a
speci ic esponse due o he speci ic a y acids p esen in each VO (LO o SO) in he
p esen s udy. . Howe e , i is hough ha he adap i e signi icance o GR u no e is
au o egula ed by co isol (Sa hiya and Vijayan, 2003) sugges ing ha GR esponse may
be an impo an pa o he adap i e s ess esponse ha is egula ed by plasma co isol
le el. Inc eased clea ance a e o co icos e oids om he blood has been sugges ed as
one o he possible mechanisms o explain acclima ion o ch onic s ess in salmonids
(Redding e al., 1984). Al e na i ely, he lack o inc ease in co isol in long- e m
s essed indi iduals may be due o a nega i e eedback o his daily inc ease in co isol
on he HPI (Picke ing, 1992; Picke ing and Po inge , 1987).
In he p esen s udy, he co isol esponse o c owding was di e en acco ding o
he die a y ea men wi h ish ed he LO die egis e ing he highes co isol alue.
This was suppo ed by i s highes co ela ion wi h he head kidney LNA con en ( 2=
0.61), and his in acco dance wi h he esul s om a p e ious s udy showing inc eased
plasma co isol le els when eeding seab eam wi h die s ich in his a y acid (Mon e o
e al., 2005).
The conse ed s ess esponse has adap i e alue and is hough o allow
animals o egain homeos asis a e a s esso insul (Mommsen e al., 1999; Iwama e

Chap e 4 S ess panels and head kidney a y acids
107
al., 2006). Some epo s ha e sugges ed ha p olonged exposu e o a s esso can lead
o allos asis, which is he abili y o he body o e u n o physiological le els seen p io
o s ess challenge (McEwen, 1998, Sch eck, 2000). Howe e , e en hough animals
ch onically s essed seem o compensa e physiologically o he s esso , hei abili y o
pe o m impo an unc ions a he whole o ganism le el may be a ec ed (Sch eck,
2000). The e o e, many s udies ha e iden i ied e ec s o co isol on di e en
physiologic p ocesses, including osmo egula ion, immune unc ion, neu oendoc ine
unc ion and beha iou . Thus, co isol sup esses cellula immuni y by a ec ing
in lamma o y signaling pa hways (Holland e al., 2003; MacKenzie e al., 2006; Alu u
and Vijayan, 2009). Mo eo e , 1h pos -handling dis u bance was ound o inc ease he
ac i i ies o glycoly ic enzymes ha may be c i ical o cope wi h he inc eased li e
ene gy demand (Iwama e al., 2006; Wiseman e al., 2007). I is also demons a ed ha
a 24 h es ain s ess was shown o modula e Il-1β and i s ecep o exp ession in he
head kidney and b ain o common ca p, leading o he hypo hesis ha IL- 1β plays a
key ole in he s ess-media ed pe iphe al immune esponse as well as cen ally in he
ac i a ion o he HPI axis (Me z e al., 2006).
I was also demons a ed ha when subjec ing seab eam o con inemen , he
a y acid composi ion o he head kidney was a ec ed and PUFA we e dec eased (Van
Anhol e al., 2004a). In he p esen s udy, we demons a ed ha eeding ish wi h LO
and/o SO se iously a ec ed he head kidney a y acid composi ion by inc easing C18
and dec easing n-3 HUFA, indica ing he e ec o eeding highe le els o LO and/o
SO in inc easing plasma co isol concen a ion o e a long pe iod (Mon e o e al.,
2005). Consequen ly he co isol elease a e c owding s ess was signi ican ly
di e en be ween ea men s. n-3 PUFA, in addi ion o a ec ing gene al p ope ies o
cells as memb ane componen s, play a ole in modula ing he p oduc ion o bo h lipid
(eicosanoids) and p o ein (cy okines) media o s. Feeding ish wi h dec easing le els o
n-3 HUFA, by including LO in he die s, dec eased signi ican ly he plasma le els o
PGE3 (Ganga e al., 2005) known as an an i-in lamma o y local ho mone. Mo eo e , a
numbe o au ho s ha e a ibu ed a DHA e ec on s ess esis ance (Kanazawa, 1997;
Tago e al., 1999; Ha el e al., 2001).
Chap e 4 S ess panels and head kidney a y acids
108
These esul s indica e clea ly ha co isol elease is di ec ly co ela ed o he
head kidney composi ion o di e en a y acids, pa icula ly n-3 HUFA (EPA and
DHA) wi h 2 =0.82 and 0.8, espec i ely a e 5 h o c owding. This is in acco dance
wi h p e ious esul s showing he s imula o y e ec o EPA and DHA on co isol
elease in head kidney (Ganga e al., 2006). In ano he s udy, Welke and Congle on
(2004) ound ha eeding salmon ju eniles wi h LO and SO in di e en p opo ions
showed ha he highes co isol le els we e egis e ed in ish ed SO. They sugges ed
ha he co isol esponse may ha e been in luenced by he a io o p os aglandin 1 and
2 se ies o p os aglandin 3-se ies p ecu so a y acids p o ided by di e en die s
(Welke and Congle on, 2004) concluding ha he e is a ela ionship be ween die a y
lipid sou ce and s ess in chinook salmon (Onco hynchus shawy scha).
Many o he s udies ha e epo ed ha die a y ARA imp o es ish su i al and
esi ance o s ess (Cas ell e al., 1994, Bessona e al., 1999; Ko en e al., 2001a,b,
Van Anhol e al., 2004a,b). Ne e hless, he mechanisms by which hese HUFAs could
modula e he s ess esponse a e s ill unknown, bu many ecen s udies ha e sugges ed
he implica ion o COX and LOX me aboli es in his esponse (Ko en e al., 2001a,b,
2003; Van Anhol e al., 2004a). P os aglandin and PGE2, in pa icula , ha e been
shown o modula e he sensi i i y o he mammalian HPA axis and consequen ly
change he s ess esponse (Di Luigi e al., 2001). In a p e ious s udy, we demons a ed
ha eeding seab eam wi h highe le els o LO, inc eased signi ican ly plasma PGE3
concen a ion (Ganga e al., 2005). Al hough less s udied in ish, he p os aglandins
likely modula e he elease o hypo halamic CRH and/o pi ui a y ACTH, as shown in
mammals (Abou-Sam a e al., 1986). Fu he mo e, Ko en e al., (2001a, b, 2003) ha e
demons a ed he impo ance o eeding die s ich in ARA o seab eam la ae in
imp o ing i s esponse o s ess and inc easing hei su i al, and hey sugges ed ha
his esponse is media ed by COX me aboli es de i ed om ARA.
In conclusion, high s ocking densi ies could be conside ed as a ch onic s ess
ac o ha a ec s ish wel a e in aquacul u e (Wedemeye , 1997, Van de
Nieuwegiessen e al., 2008, 2009), and good p ac ices in aquacul u e business a e o
g ea impo ance o imp o e ish wel a e and can minimize s ess on a med ish and
Chap e 4 S ess panels and head kidney a y acids
109
main ain high s anda ds o ish wel a e (Te o a e al., 2005). This s udy showed clea ly
ha eeding seab eam wi h LO and SO as subs i u es o FO esul ed in a p o ound
al e a ion o head kidney a y acids p o iles. N-3 HUFA we e dec eased and OA, LA
and LNA we e dec eased. Sho - e m o e c owding (2 h) in seab eam esul ed in sho
e m inc eases in plasma co isol. These al e a ions, a ec ed he s ess esponse o
c owding o all he die a y ea men s wi h ish ed wi h highe con en s o LO and/o
SO showing signi ican ly highe plasma basal and pos -s ess co isol le els. Thus,
when subs i u ing ish oil wi h ege able oils in die s o seab eam, ca e should be
aken o gua an ee ha su icien con en o EFAs necessa y o no mal s ess esponse
a e p o ided o sa is y ish physiological equi emen s.
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coa ed wi h li hium hepa in as an an icoagulan . The blood was
cen i uged immedia ely a 600g o 10 min 4 -C o sedimen
he cells. One millili e o plasma was emo ed, 50 AL/mL o
2M o mic acid was added and he acidi ied samples we e
ozen in liquid ni ogen (80 -C) p io o eicosanoid analysis.
250 AL o plasma was emo ed and s o ed a 80 -C o lep in
analysis and he emaining plasma was pooled pe each ank
and s o ed a 80 -C o a y acid analysis.
2.4. Blood leukocy e sepa a ion
This assay was conduc ed on only 3 die a y ea men s as
su icien ish ed 100 LO die we e no a ailable. Se en
millili e s o blood was collec ed om he caudal ein in
hepa inised sy inges om 9 ish pe die and ans e ed o clean
glass ubes kep on ice. Blood was hen cen i uged a 500 g o
10 min a 4 -C. A e elimina ion o supe na an cells we e
esuspended in 10 mL o HBSS Ca–Mg- ee and hen
cen i uged a 500 g o 10 min a 4 -C. Cells we e sepa a ed
in o 2 sub-samples which we e esuspended in 6 mL o HBSS
Ca–Mg- ee, and ca e ully laye ed o e 6 mL o 46% Pe coll
and cen i uged a 450 g o 40 min a 4 -C. Cells collec ed om
he whi e in e media e laye we e esuspended in 10 mL o
HBSS Ca–Mg- ee and cen i uged a 500 g o 10 min a 4 -C.
Collec ed cells we e esuspended in 6 mL HBSS, Ca–Mg- ee,
ca e ully laye ed o e 6 mL o 46% Pe coll and cen i uged a
450 g o 40 min a 4 -C. The leukocy es (whi e in e media e
laye ) we e collec ed and washed wi h 10 mL o HBSS Ca–Mg-
ee. The leukocy es ob ained om 3 ish we e pooled and e-
suspended in 4 mL o HBSS, 2 mL o chlo o o m we e added
and he sample was s o ed a 80 -C p io o lipid ex ac ion.
2.5. Lipid analysis
Ex ac ion o o al lipid om die s, plasma samples and
blood leukocy es was pe o med by he me hod o Folch e al.
(1957). Neu al and pola ac ions we e sepa a ed by
adso p ion ch oma og aphy on silica Sep-Pak ca idges
(Wa e s, Mil o d, MA) as desc ibed by Juaneda and Rocquelin
(1985). Fa y acid me hyl es e s we e p oduced om aliquo s o
o al lipids ex ac ed om die , plasma samples and blood
leukocy es by acid-ca alyzed ansme hyla ion pe o med
o e nigh a 50 -C as desc ibed by Ch is ie (1982).
Fa y acid me hyl es e s we e sepa a ed and quan i ied by
gas–liquid ch oma og aphy (Shimadzu C-R5A gas ch oma o-
g aph, 30 m0.32 mm ID capilla y column (Suplecowax-10,
Sigma-Ald ich, Mad id, Spain) acco ding o condi ions de-
sc ibed by Izquie do e al. (1990). Indi idual me hyl es e s
we e iden i ied by compa ison wi h known s anda ds and
published da a.
2.6. Ex ac ion, sepa a ion and enzyme immunoassay o
eicosanoids
The ozen acidi ied plasma samples we e hawed and
cen i uged (1000 g, 5 min) o p ecipi a e cell deb is. The
supe na an s we e ex ac ed using oc adecyl silyl (ODS, C18)
FSep-Pak_mini-columns (Millipo e (UK), Wa o d) by he
me hod o Powell (1982) and as desc ibed in de ail by Bell e
al. (1994b). The inal ex ac was edissol ed in 100 ALo
me hanol p io o HPLC. PGE
3
was sepa a ed by e e se phase
HPLC using he me hodology simila o ha desc ibed in Bell
e al. (1994a). An isoc a ic sol en sys em was employed
con aining 17 mM phospho ic acid/ace oni ile (70/30, / ) a a
low a e o 0.75 mL/min. Fi y mic oli e s o he eicosanoid
plasma ex ac s was injec ed on o he column and 2.25 mL
ac ions was collec ed using an Wa e s F ac ion Collec o
(Wa e s L d., Wa o d, UK). F ac ions co esponding o he
PGE
3
elu ion ime we e pooled and ex ac ed as ollows. The
pooled ac ions we e applied o a C18 FSep-Pak,_which had
been p e-washed wi h 5 mL me hanol and 10 mL dis illed
wa e . The column was hen washed wi h u he 10 mL
dis illed wa e and he PGE
3
elu ed in 5 mL o e hyl ace a e.
Samples we e d ied unde ni ogen and edissol ed in
immunoassay bu e . Measu emen o PGE
3
was pe o med
using an enzyme immunoassay (EIA) ki o PGE
2
acco ding o
he manu ac u e s p o ocol (SPI-Bio, Gi su Y e e, F ance).
The c oss- eac i i y o he PGE
2
an ibody wi h PGE
3
was 43%.
The emaining 50 Al o he eicosanoid plasma ex ac s was
used o measu e p os aglandin PGE
2
concen a ion.
Table 1
Fa y acid composi ions o he 5 mm expe imen al die s (g/100 g a y acid)
FO 60 LO 60 RO 100 LO
%Lipids (dw) 20.24 21.36 22.79 25.14
14:0 5.92 3.05 2.93 0.79
16:0 19.30 14.99 15.95 15.92
16:1n-7 7.21 3.93 3.23 1.23
16:4n-3 0.17 0.04 0.03 0.02
18:0 3.37 3.21 3.36 3.40
18:1n-9 11.71 24.61 29.89 32.98
18:2n-6 5.84 11.87 12.50 13.67
18:3n-3 1.62 14.36 12.25 23.02
18:4n-3 2.18 1.28 0.87 0.17
20:1n-9 2.38 2.05 1.98 2.23
20:2n-6 0.15 0.10 0.08 0.03
20:3n-6 n.d. n.d. n.d. n.d.
20:4n-6 0.66 0.34 0.28 0.06
20:3n-3 n.d. n.d. n.d. n.d.
20:4n-3 0.54 0.27 0.21 0.05
20:5n-3 11.90 6.10 4.86 1.06
22:1n-11 2.98 2.35 2.20 2.41
22:5n-6 0.24 0.12 0.11 –
22:5n-3 1.17 0.56 0.47 0.08
22:6n-3 14.14 7.36 6.21 2.10
Sa u a ed
a
30.01 22.22 23.33 20.63
Monounsa u a es
b
27.70 33.12 37.44 38.85
~n-3
c
32.23 30.27 25.22 26.57
~n-6
d
7.37 12.59 13.13 13.78
~n-9 14.56 26.81 31.91 35.22
~n-3 HUFA 27.84 14.34 11.82 3.33
n-3/n-6 4.37 2.40 1.92 1.93
dw—d y weigh .
n=3. Values in he same ow wi h di e en supe sc ip le e s a e signi ican ly
di e en ( P<0.05).
a
Includes 15:0, 17:0, 20:0 and 22:0.
b
Includes 22:1n-7.
c
Includes 20:3n-3 and 22:4n-3.
d
Includes 18:3n-6 and 22:4n-6.
R. Ganga e al. / Compa a i e Biochemis y and Physiology, Pa B 142 (2005) 410– 418412

2.7. Lep in immunoassay
Plasma lep in concen a ion was measu ed wi h an enzyme
immunoassay ki using a monoclonal an ibody o human lep in
acco ding o he manu ac u e s p o ocol (SPI-bio, Gi su
Y e e, F ance).
2.8. S a is ical analysis
Signi icance o di e ence ( P<0.05) be ween die a y ea -
men s was de e mined by one-way analysis o a iance
(ANOVA) ollowed by Duncan mul iple compa ison es .
Analyses we e pe o med using SPSS so wa e (SPSS o
windows 11.0).
3. Resul s
3.1. Fa y acid composi ion o plasma
The a y acid composi ions o plasma pola and neu al
lipids a he end o he ial a e summa ized in Tables 2 and 3.
In pola lipids, DHA was he mos abundan a y acid in ish
ed 100 FO, 60 RO and 60 LO, ollowed by 16:0 and 18:1n-9.
Howe e in ish ed 100 LO, 18:1n-9 and 16:0 we e he mos
abundan a y acids ollowed by DHA. 18:2n-6, 20:2n-6,
20:3n-6 and o al n-6 a y acids we e all inc eased wi h
inc easing die a y 18:2n-6. 18:3n-3 was also signi ican ly
inc eased in ish ed ege able oils. The elongase p oduc o
18:3n-3, 20:3n-3, was also highe in ish ed 60 RO, 60 LO and
100 LO die s han in ish ed FO die , despi e he low die a y
con en . 20:4n-3 was also high, bu no signi ican ly, in ish ed
VO. DHGLA was signi ican ly highe and ARA lowe
(P<0.05) in ish ed he die wi h he highes le el o 18:2n-
6 (100 LO) when compa ed wi h FO ed ish. Besides, EPA
con en showed a signi ican educ ion ( P<0.05) in ish ed
ei he 60 RO o 100 LO, compa ed o ish ed FO. The ARA/
EPA a io was ela i ely simila in he ou ea men s.
Howe e , he DHGLA/ARA a io was signi ican ly lowe in
ish ed FO (0.09) and inc eased wi h inc easing die a y LA
and gammalinolenic acid (18: 3n-6) (GLA), being highes in
ish ed 100 LO (0.49). DHA and o al n-3 PUFA con en s
dec eased, hough no signi ican ly, acco ding o hei espec-
i e die a y le els, leading o a lowe n-3/n-6PUFA a io.
Fa y acid composi ion o neu al lipids was gene ally highe
in LA and GLA con en , han ha o pola lipids, LA being
g ea e in ish ed VO. Fish ed VO also exhibi ed highe
amoun s o GLA, while hose ed FO we e signi ican ly highe
in EPA and DHA. DHA/EPA was inc eased wi h eeding VO,
and ARA/EPA was also sligh ly bu no signi ican ly inc eased.
Table 4 shows he ela ionship be ween he plasma con en o
some a y acids and i s le el in he die . Oleic acid was
pa icula ly inco po a ed in o plasma neu al lipids compa ed
wi h pola lipids. The a io o inco po a ion dec eased wi h
inc easing die a y con en o his a y acid, he highes a io
being obse ed in ish ed FO (1.41) and he lowes in ish ed
100 LO (0.75). The a io o inco po a ion o palmi ic acid was
no di e en among he ea men s, being sligh ly highe in pola
lipids. By con as , ARA EPA and DHA we e p e e en ially
inco po a ed in o pola lipid, inc easing wi h VO inclusion.
The ARA, EPA and EPA/ARA composi ion o o al lipids
om blood leukocy es is shown in Fig. 1. Bo h palmi ic and
oleic acids, ollowed by DHA and EPA we e he mos abundan
a y acids in hese cells (da a no shown). Fish oil subs i u ion
wi h VO ma kedly a ec ed a y acid composi ion o blood
leukocy es by signi ican ly educing he EPA con en s, and
sligh ly ARA ones. Inco po a ion o linolenic acid was always
lowe han o linoleic acid. ARA/EPA alues we e only
sligh ly lowe han hose ound in plasma pola lipids and
sligh ly highe han hose ound in plasma neu al lipids.
3.2. Plasma p os aglandin analysis.
Plasma PGE
2
and PGE
3
concen a ions a e shown in Table
5. The concen a ion o PGE
2
was no signi ican ly di e en
among he ou ea men s wi h he lowes alue in ish ed FO
(33.72 pg/mL) and he highes in ish ed 60 RO (44.74 pg/
mL). Howe e , he concen a ion o PGE
3
dec eased acco d-
Table 2
Plasma pola lipid a y acid composi ions (g/100 g o o al a y acids)
(meanTSD)
Fa y acids FO 60 LO 60 RO 100 LO
12:0 1.59T0.36 1.61T0.72 1.44T0.51 1.38T0.85
14:0 2.15T0.16 1.32T0.74 1.01T0.19 0.90T0.34
16:0 22.91T0.93 18.76T4.33 23.30T1.29 21.97T1.78
16:1n-7 2.99T0.88 1.27T0.64 1.43T0.66 1.05T0.69
18:0 6.34T0.13 6.12T2.42 7.16T1.34 7.24T0.49
18:1n-9+18:1n-7 10.47T0.96 16.15T4.81 14.28T0.30 15.73T1.12
18:2n-6 6.04T2.64
b
8.47T2.78
a,b
8.07T1.89
a,b
11.75T1.63
a
18:3n-6 0.09T0.00 0.07T0.02 0.05T0.02 0.10T0.03
18:3n-3 0.59T0.23
c
3.05T0.95
a,b
2.19T0.06
b,c
4.98T1.54
a
18:4n-3 0.10T0.00 0.11T0.08 0.04T0.05 0.06T0.06
20:0 0.21T0.09 0.20T0.13 0.19T0.14 0.12T0.01
20:1n-9 0.53T0.04 0.70T0.13 0.67T0.01 0.81T0.25
20:2n-6 0.16T0.01 0.32T0.01 0.36T0.06 0.46T0.09
20:3n-6 0.12T0.02
b
0.26T0.07
a,b
0.31T0.12
a
0.31T0.06
a
20:4n-6 1.28T0.15
a
1.02T0.15
a,b
1.05T0.32
a,b
0.65T0.12
b
20:3n-3 0.09T0.00
b
0.34T0.02
a
0.38T0.16
a
0.32T0.2
a
20:4n-3 0.26T0.04 0.39T0.06 0.38T0.13 0.39T0.08
20:5n-3 9.02T0.90
a
7.53T1.16
a,b
6.87T0.82
b
4.52T1.04
b
22:1n-11 0.32T0.04 0.29T0.14 0.31T0.01 0.39T0.20
22:5n-6 0.47T0.02 0.64T0.28 0.51T0.30 0.29T0.04
22:5n-3 1.89T0.22 2.04T0.16 1.96T0.34 1.47T0.07
22:6n-3 28.63T3.56 26.28T3.20 26.03T4.86 22.95T1.77
To al sa u a es
1
34.11T0.06 28.56T5.07 33.62T3.37 31.92T2.06
To al monoenes
2
15.24T2.09 19.22T5.93 17.12T1.19 18.58T0.76
n-3
3
41.01T4.47 40.09T3.47 37.97T6.08 34.87T1.21
n-6
4
8.44T2.41 10.98T2.23 10.42T1.26 13.67T1.75
n-9
5
11.43T1.01 17.24T5.08 15.12T0.52 16.97T1.03
n-3 HUFA 39.99T4.71 36.68T4.53 35.64T6.28 29.69T2.65
n-3/n-6 4.86T1.37
a
3.65T1.08
a,b
3.64T1.03
a,b
2.58T0.40
b
DHA/EPA 3.22T0.01 3.45T0.01 3.85T0.02 5.01T0.03
ARA/EPA 0.14T0.00 0.14T0.00 0.15T0.03 0.14T0.01
DHGLA/ARA 0.09T0.004
c
0.26T0.09
b
0.28T0.02
b
0.49T0.15
a
1
Includes 15:0, 17:0, and 22:0.
2
Includes 16:1n-5, 18:1n-5, 20:1n-5, 20:1n-7,
22:1n-9, and 22:1n-7.
3
Includes 22:4n-3.
4
Includes 22:4n-6.
5
Includes 18:2n-9,
18:3n-9, 20:2n-9, 20:3n-9 and 22:1n-9. Includes 16:2n-3, 16:2n-4, 16:4n-3 and
16:4n-1.
n=36. Values in he same ow wi h di e en supe sc ip le e s a e
signi ican ly di e en ( P<0.05).
R. Ganga e al. / Compa a i e Biochemis y and Physiology, Pa B 142 (2005) 410– 418 413
ingly o he EPA con en in he die , being signi ican ly lowe
(P<0.05) in ish ed 100 LO (Fig. 2).
The co ela ion be ween PGE
3
and i s p ecu so (EPA) in
plasma, is shown in Fig. 3. A high co ela ion (
2
=0.97) was
ound be ween plasma PGE
3
and EPA concen a ion wi h a
posi i e ela ionship ( Y=3.26X+18.61).
3.3. Plasma lep in analysis
The p oduc ion o he ho mone lep in in plasma is shown in
Fig. 4. No signi ican di e ences we e seen among he ou
die s.
Howe e , high nega i e co ela ion was also ound be ween
plasma lep in and PGE
2
(
2
=0.85; Fig. 5).
4. Discussion
Fa y acid composi ion o plasma lipids was ma kedly
a ec ed by he inclusion o VO. LA and LNA, main
componen s o ege able oils, signi ican ly inc eased in plasma
o ish ed VO, being p e e en ially inco po a ed in o NL, as i
has been ound in o he cells and issues o his specie
(Izquie do e al., 2003, in p ess) and o he species (Waagbo e
al., 1995). In e es ingly, DHGLA also inc eased in ish ed VO,
wi h maximum alues o he 100 LO g oup. DHGLA is he
p edominan p oduc o desa u a ion and elonga ion in salmo-
nids, eeding ish wi h die s con aining high le el o LA and
LNA (Bell e al., 1991, 2002; Toche e al., 2000).
Plasma DHA con en was e en highe han in he die s,
being pa icula ly inco po a ed in PL. Simila esul s we e
ound in cod (Gadus mo hua)(
Waagbo e al., 1995), Eu opean
sea bass (Dicen a chus lab ax)(
Fa ndale e al., 1999) and
seab eam (Caballe o, 2002). O he p e ious s udies ha e also
demons a ed ha DHA was p e e en ially e ained unde
die a y essen ial a y acids de iciency (Izquie do, 1996;
Izquie do e al., 2001; Mon e o e al., 2001), showing he
Table 3
Plasma neu al lipid a y acid composi ions (g/100g o o al a y acids)
(meanTSD)
Fa y acids FO 60 LO 60 RO 100 LO
12:0 4.70T2.09 3.14T1.09 3.34T0.69 2.97T0.90
14:0 5.52T3.28 2.42T0.55 2.31T0.46 2.43T0.40
16:0 22.36T0.53 16.96T0.36 18.15T1.11 18.77T1.07
16:1n-7 4.13T1.11 2.55T0.40 1.99T0.15 2.09T1.13
18:0 4.10T0.97 5.70T0.23 4.89T0.55 5.07T0.64
18:1n-9 + 18:1n-7 16.46T2.34 22.78T0.72 27.31T0.60 24.65T3.37
18:2n-6 6.34T2.35
b
13.37T3.18
a
11.09T3.26
a,b
10.61T0.52
a,b
18:3n-6 0.14T0.06 0.07T0.00 0.16T0.03 0.23T0.5
18:3n-3 1.06T0.09
c
7.09T0.44
a,b
6.39T0.33
b
9.11T3.71
a
18:4n-3 0.40T0.03 0.50T0.05 0.36T0.13 0.46T0.09
20:0 0.18T0.08 0.40T0.11 0.28T0.10 0.28T0.16
20:1n-9 1.51T0.18 1.46T0.04 1.54T0.13 1.33T0.06
20:2n-9 0.09T0.01 0.18T0.05 0.18T0.01 0.37T0.04
20:2n-6 0.18T0.02 0.32T0.16 0.27T0.02 0.28T0.03
20:3n-6 0.09T0.03 0.10T0.03 0.17T0.11 0.16T0.05
20:4n-6 0.52T0.14 0.30T0.01 0.34T0.10 0.49T0.23
20:3n-3 0.38T0.02 0.38T0.11 0.35T0.07 0.36T0.10
20:4n-3 0.42T0.22 0.33T0.05 0.30T0.08 0.25T0.03
20:5n-3 6.19T1.73
a
3.27T0.17
b
2.85T0.59
b
2.68T1.64
b
22:1n-11 1.12T0.04 0.89T0.13 0.83T0.04 0.70T0.05
22:5n-6 0.37T0.08 0.21T0.01 0.22T0.01 0.22T0.01
22:5n-3 2.37T0.65 1.36T0.05 1.32T0.20 1.02T0.44
22:6n-3 14.85T4.02
a
10.67T1.00
b
10.54T2.07
b
10.15T2.09
b
To al sa u a es
1
38.08T6.91 29.45T0.70 29.68T1.68 29.84T1.42
To al monoenes
2
25.28T3.47 29.36T0.69 33.22T0.62 29.81T1.96
n-3
3
26.32T6.13 24.05T1.40 22.52T3.01 24.92T1.54
n-6
4
8.19T1.90
b
14.68T3.05
a
12.43T3.14
a,b
12.93T1.30
a,b
n-9
5
18.88T2.71 25.35T1.02 29.84T0.77 26.99T2.99
n-3 HUFA 24.42T6.54 16.13T1.04 15.49T2.96 14.58T4.26
n-3/n-6 3.39T1.53
a
1.68T0.44
b
1.81T0.73
b
1.93T0.08
b
DHA/EPA 2.38T0.00 3.23T0.04 3.70T0.02 4T0.11
ARA/EPA 0.08 0.09 0.12 0.18
1
Includes 15:0, 17:0, and 22:0.
2
Includes 16:1n-5, 18:1n-5, 20:1n-5, 20:1n-7,
22:1n-9, and 22:1n-7.
3
Includes 22:4n-3.
4
Includes 22:4n-6.
5
Includes 18:2n-9,
18:3n-9, 20:3n-9 and 22:1n-9. Includes 16:2n-3, 16:2n-4, 16:4n-3 and 16:4n-1.
n=36. Values in he same ow wi h di e en supe sc ip le e s a e
signi ican ly di e en ( P<0.05).
Table 4
The a io be ween a y acid pe cen in plasma lipid classes and die a y pe cen
o he same a y acid (% Fa y acid in plasma/% a y acid in die )
Fa y acid in plasma lipid classes FO 60 LO 60 RO 100 LO
Oleic acid (OA) Pola 0.89 0.66 0.48 0.48
Neu al 1.41 0.93 0.91 0.75
Palmi ic acid Pola 1.19 1.25 1.46 1.38
Neu al 1.16 1.13 1.14 1.18
A achidonic acid (ARA) Pola 1.94 3.00 3.75 10.83
Neu al 0.79 0.88 1.21 8.17
EPA Pola 0.76 1.23 1.41 4.26
Neu al 0.52 0.54 0.59 2.53
DHA Pola 2.02 3.57 4.19 10.93
Neu al 1.05 1.45 1.7 4.83
0
1
2
3
4
5
6
7
8
9
10
ARA EPA EPA/ARA
g/100g o al F.A.
FO
60LO
60RO a
bb
Fig. 1. P opo ion (g/100 g o al a y acids) o blood leukocy es EPA, ARA and
EPA/ARA. Columns assigned a di e en le e a e signi ican ly di e en
(P<0.05).
Table 5
Concen a ion o PGE
2
and PGE
3
in plasma om seab eam ed expe imen al
die s (meanTSD)
P os aglandins FO 60 LO 60 RO 100 LO
PGE
2
(pg/mL) 33.72T6.52 41.87T7.18 44.74T16.47 35.15T4.58
PGE
3
/PGE
2
1.45T0.33 1.00T0.22 0.91T0.46 0.96T0.12
N=36. Values in he same ow wi h di e en supe sc ip le e s a e
signi ican ly di e en ( P<0.05). The speci ici y o he an ibody was 100%
o PGE
2
and 43% o PGE
3
.
R. Ganga e al. / Compa a i e Biochemis y and Physiology, Pa B 142 (2005) 410– 418414
impo ance o his a y acid as a majo s uc u al componen o
ish cell memb anes (Wa anabe, 1993; Sa gen e al., 1995).
Selec i e inco po a ion o DHA in o ish lipids is di ec ly
ela ed wi h he speci ici y o some PL syn hesis enzyme
complexes such as 1-lysophospha idylacylCoA ans e ase
(Gu and Ha wood, 1991; Caballe o, 2002) and he a ini y
o a y acid binding p o eins (FABP) (Si e and Ve nie , 1981).
ARA, EPA and DHA we e p e e en ially inco po a ed in o
PL lipids o plasma, and DHGLA accumula ed wi h he
inc eased VO inclusion. The highe con en o EPA in plasma
PL sugges he selec i e inco po a ion o his a y acid in o
memb ane lipids in ag eemen wi h he esul s ound in sea
bass leukocy es (Fa ndale e al., 1999) and seab eam an e io
kidney mac ophages (Mon e o e al., 2003). A selec i e
deposi ion o ARA in PL was ound a low die a y concen a-
ions in ag eemen wi h p e ious esul s ound in o he cellula
ypes o his specie (Mon e o e al., 2003; Foun oulaki e al.,
2003) o o he species such as cod (Waagbo e al., 1995) o sea
bass (Fa ndale e al., 1999), and deno ing he impo ance o his
a y acid o op imal cell unc ion. Besides, his a y acid is
e ained in essen ial a y acid de icien ish (Izquie do, 1996)
and is inco po a ed and e ained in PI o seab eam (Mou en e
and Toche , 1993) and u bo (Scoph almus maximus)(Lina es
and Hende son, 1991).
The die a y ea men s employed in he p esen s udy ha e
esul ed in p o ound al e a ions o DHGLA/ARA a ios ( a io
in able), bu no in ARA/EPA (Table 2). ARA, EPA and
DHGLA a e all p ecu so s o eicosanoid p oduc ion (Bell e
al., 1994a) and changes in he a ios o hese HUFA had
impo an consequences o he quan i y and spec um o
eicosanoids p oduced by u bo (Bell e al., 1998). ARA-
de i ed PGE
2
p oduc ion in plasma was no a ec ed by VO, in
ag eemen wi h simila eicosanoid p ecu so a io (ARA/EPA)
in leukocy es o al lipid and plasma phospholipids among ish
ed he di e en die a y ea men s. Howe e , educ ion in
die a y ARA/EPA lead o a signi ican educ ion in PGE
2
concen a ion in hea , b ain and kidney in u bo (Bell e al.,
1995). This con o e sy may be ela ed o die a y, issue o
species di e ences be ween bo h s udies.
In e es ingly, he p oduc ion o PGE
3
was signi ican ly
di e en among ish ed he ou expe imen al die s and
s ongly co ela ed wi h plasma pola lipid concen a ions o i s
p ecu so , EPA. Mo eo e , PGE
3
was he majo p os aglandin
p oduced in plasma o ish ed FO, in con as wi h p e ious
s udies conduc ed ‘‘in i o’’ in b ain as oglial u bo cells and
pineal o gan o A lan ic salmon (Salmo sala )(
Bell e al.,
0
10
20
30
40
50
60
FO
60LO
60RO
100LO
Die
Plasma PGE3 (pg/ml
aab ab
b
Fig. 2. Plasma concen a ion o PGE
3
(pg/mL) om seab eam ed he
expe imen al die s. Columns assigned a di e en le e a e signi ican ly
di e en ( P<0.05).
y = 3,26x + 18,61
R2 = 0,97
30
45678910
32
34
36
38
40
42
44
46
48
50
EPA in
p
lasma
(
% in
p
ola li
p
ids
)
Plasma PGE3 in pg/ml
PGE3
Lineal (PGE3)
Fig. 3. The ela ionship be ween PGE
3
(pg/mL) and EPA (pe cen in pola
lipids) concen a ion in plasma om seab eam ed he expe imen al die s wi h
pink do s
2
=0.97 and line Y=3.26x+18.61.
20,5
21,0
21,5
22,0
22,5
23,0
23,5
FO
60LO
60RO
100LO
Die
Plasma lep in (pg/ml)
Fig. 4. Lep in concen a ion in plasma om seab eam ed he ou expe imen al
die s.
y = -0,08x + 25,02
R2 = 0,85
21,20
0102030405
0
21,60
21,40
21,80
22,00
22,20
22,40
22,60
PGE2
Lep in
Fig. 5. Rela ion be ween plasma lep in and PGE
2
in seab eam ed he ou
expe imen al die s wi h pink do s
2
=0.85.
R. Ganga e al. / Compa a i e Biochemis y and Physiology, Pa B 142 (2005) 410– 418 415
1994a; Hende son e al., 1996). Such indings ema ks he
impo ance o EPA as a p ecu so o PG in ma ine ish, a leas
o he co ec unc ion o hei blood cells, and co ela es well
wi h he p edominan ole o his a y acid in immune
egula ion in his specie (Mon e o e al., 1998). Ne e heless,
PG p oduc ion ma kedly di e s among issues o he same
species. Fo ins ance, PGE
2
p oduc ion was highe in u bo
kidney mac ophages han in blood leukocy es (Ta alla e al.,
1999). In he p esen s udy, eeding VO lead o a dec ease in
plasma EPA which in u n educed plasma PGE
3
concen a ion.
PL a y acid composi ion de e mines he physical p ope ies o
cell memb anes, in luencing he ac i i ies o memb ane-
associa ed p o eins and enzymes (Spec o and Yo ek, 1985)
and he ca aly ic ac i i y o he phospholipase A
2
enzymes
(Bell e al., 1996). Since a p e ious s udy conduc ed in ainbow
ou (Salmo gai dne i) has demons a ed ha blood e y h o-
cy es a e no capable o eicosanoid syn hesis (Pe i e al.,
1989), plasma eicosanoids e lec p incipally leukocy e o
ascula p oduc s. Despi e ha he plasma PGE
2
le els we e
no being signi ican ly di e en , he lowe p oduc ion in ish
ed FO may be explained by he highe con en o EPA in he
PL o hese ish, since his a y acid is a po en compe i o o
ARA. Hence, hese esul s show ha subs i u ion o FO wi h
VO in die s o seab eam modi ies plasma PUFA composi ion
p o oundly, a ec ing he 3-se ies p os aglandin p oduc ion.
Inc eased in DHGLA in pola lipid a y acid composi ion
could be ela ed wi h he esul s ob ained in PG p oduc ion. Fo
ins ance, supplemen a ion wi h bo h DHGLA and EPA signi -
ican ly educed ARA-de i ed p os aglandin p oduc ion in
salmon (Bell e al., 1993) sugges ing a compe i i e inhibi ion
by hose a y acids a he cyclooxygenase ac i e si e (Bell e al.,
1996). Also, phospholipase A
2
ac i i y, which is key o elease
o eicosanoid p ecu so a y acids om cell memb anes, is
a ec ed by die a y a y acids. In salmon ed a die wi h educed
n-3/n-6 PUFA a io, he phospholipase A ac i i y was inc eased
in ca diac issue compa ed o hose ed ish oil (Bell e al., 1993).
The abili y o DHGLA and EPA o a enua e he p oduc ion o
ARA-de i ed eicosanoids is undamen al in he con ol o
pa hophysiological p ocesses in nume ous in lamma o y condi-
ions occu ing in humans (Ho obin, 1992).
Recen ly, he in e ac ion be ween lep in and p os aglandin
elease has been desc ibed by Ze ani e al. (2005) who
sugges ed ha ci cula ing le els o lep in may ac as a
me abolic signal modula ing PG elease h ough media ion o
he ni ic oxide syn hase/ni ic oxide sys em. In he p esen
s udy a nega i e co ela ion was ound be ween plasma PGE2
and lep in plasma concen a ion in ag eemen wi h he esul s
ound by Ze ani e al. (2005). Mo eo e , Campbell e al. (1998)
demons a ed ha lep in elec opho e ic mobili y depends
ma kedly on he ee a y acids in plasma, inc eases o oleic
acid inducing a dec ease o lep in mobili y. This may esul in a
change o con o ma ion o lep in and consequen ly may a ec
he anspo /binding o his p o ein o a ge issues. In
ag eemen wi h hese indings, oleic acid con en s in plasma
neu al lipids we e nega i ely co ela ed o lep in le els, which
in u n we e nega i ely ela ed wi h PGE
2
. Besides, an e ec o
a achidonic acid inc easing he elease o lep in has been also
desc ibed (Fain e al., 2001), in ag eemen wi h he esul s
ob ained in he p esen s udy, when ish wi h highe le els o
ARA in plasma neu al lipids showed he lowe le els o
PGE2. Howe e , u he expe imen s a e equi ed o cla i y he
in e ac ion be ween plasma a y acids and lep in le els.
In summa y, he p esen s udy shown ha inc eased
inclusion o VO in die s o seab eam may p o oundly a ec
he a y acid composi ion o plasma and leukocy es, especially
HUFA, and consequen ly he p oduc ion o PGE
3
, which can
be a majo PG in plasma. Al e a ion in he amoun and ype o
PG p oduced can be a leas pa ially esponsible o he
changes in he immune sys em and heal h pa ame e s o ish
ed die s wi h high inclusion o VO (Blaze , 1992; Thompson
e al., 1996; Mon e o e al., 2003). Fu he esea ch is needed o
cla i y he e ec s o die a y a y acids on plasma lep in le els
and i s ela ion wi h p os aglandins p oduc ion.
Acknowledgemen
This expe imen was pa o he EU F amewo k V p ojec ,
Resea ching Al e na i es o Fish Oil in Aquacul u e (RAFOA),
Q5RS-200-30058. This s udy was also suppo ed, in pa ,
h ough an awa d o GR om he Eu opean Union Access o
Resea ch In as uc u es (ARI) Ac ion o he Imp o ing
Human Po en ial P og amme (con ac HPRI-CT-2001-00180).
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Chap e 6
Modula ion o ACTH-induced co isol elease by
polyunsa u a ed a y acids in in e enal cells om
gil head seab eam, Spa us au a a
Ganga, R., To , L., Ace e e, L., Mon e o, D. and Izquie do, M.S.
Jou nal o Endoc inology, 190:39-45
Modula ion o ACTH-induced co isol elease by polyunsa u a ed a y
acids in in e enal cells om gil head seab eam, Spa us au a a
R Ganga, L To
1
, L Ace e e
1
, D Mon e o and M S Izquie do
G upo de In es igacio
´n en Acuicul u a, Ins i u o Cana io de Ciencias Ma inas, PO Box 56, 35200-Telde, Las Palmas, Cana y Islands, Spain
1
Depa men o Cell Biology, Physiology and Immunology, Uni e si a Au ono
`ma de Ba celona, Bella e a 08193, Spain
(Reques s o o p in s should be add essed o R Ganga; Email: gaqua2003@yahoo. )
Abs ac
Highly unsa u a ed a y acids a e essen ial componen s o
cellula memb anes o e eb a es and can modula e
physiological p ocesses, including memb ane anspo ,
ecep o unc ion and enzyma ic ac i i ies. In gil head sea
b eam, die a y de iciencies o essen ial a y acids o ma ine
ish aise he basal co isol le els and al e he pa e n o
co isol elease a e s ess. The aim o he p esen s udy was o
cla i y he e ec o di e en essen ial a y acids on
ad enoco ico opic ho mone (ACTH)-induced co isol
p oduc ion and elease in ish, h ough in i o s udies o sea
b eam in e enal cells main ained in supe usion and
incuba ed wi h di e en ypes o a y acids and eicosanoid
p oduc ion inhibi o s. Resul s showed he i s e idence o
he e ec o ce ain a y acids on co isol p oduc ion by
ACTH-s imula ed in e enal cells in ish. Bo h a achidonic
acid (ARA) and pa icula ly eicosapen aenoic acid (EPA)
p omo ed co isol p oduc ion in sea b eam in e enal cells.
Mo eo e , incuba ion wi h indome acin (INDO) educed
he inc eased co isol p oduc ion induced by EPA and ARA,
sugges ing media ion by hei cyclooxygenase-de i ed
p oduc s. Docosahexaenoic acid s imula ed co isol p o-
duc ion o a lesse ex en han ha caused by EPA o ARA,
bu he inhibi o y e ec o INDO was no as ma ked as i was
o he o he a y acids. In con as , supplemen a ion wi h
dihomogammalinoleic acid educed co isol p oduc ion,
deno ing he inhibi o e ec o his a y acid in co isol
sec e ion.
Jou nal o Endoc inology (2006) 190, 39–45
In oduc ion
Highly unsa u a ed a y acids wi h 20 o mo e ca bon a oms
and h ee o mo e double bonds (HUFA) a e essen ial
componen s o cellula memb anes and can modula e
physiological p ocesses, including memb ane anspo ,
ecep o unc ion and enzyma ic ac i i ies. Hence, die a y
a y acids ha e been shown o ha e ma ked e ec s on a a ie y
o immunological and haemos a ic pa ame e s (Bal y e al.
2001, Mon e o e al. 2001). HUFA possess a wide ange o
cellula unc ions. One o he mos impo an unc ions is o
supply p ecu so s o he syn hesis o eicosanoids, which a e
p oduced in esponse o a ious ex acellula s imuli by wo
main ypes o dioxygenase enzymes: cyclooxygenases (COX)
and lipoxygenases (Ho obin 1983). Following cell s imu-
la ion, bo h a achidonic acid (ARA; 20:4n-6) and eicosapen-
aenoic acid (EPA; 20:5n-3) a e eleased om he memb ane
by he ac ion o phospholipase A
2
. La e hese a y acids a e
ans o med by a ange o lipoxygenases and cyclooxygenases
o yield p os aglandins (PG), leuko ienes, lipoxins and o he
compounds, which can modula e se e al immune unc ions
(Uhing e al. 1990).
Eicosanoids ha e been ound in a la ge ange o eshwa e
and ma ine ish (Ma sumo o e al. 1989, Mus a a & S i as a a
1989) and in many issues (Hende son & Toche 1987, Bell
e al. 1994a, Toche 1995). In ish, a p e e ed eicosanoid
p ecu so o cyclooxygenase seems o be ARA (20:4n-6)
(Toche & Sa gen 1987, Bell e al. 1994a, 1994b, 1998), bu
EPA (20:5n-3) and dihomo-g-linolenic acid (DHGLA;
20:3n-6) a e also impo an eicosanoid p ecu so s which
can modula e p oduc ion and biological e icacy o ARA-
de i ed eicosanoids (Ho obin 1983, Bell e al. 1994a, Ganga
e al. 2005). In addi ion, he high con en o docosahexaenoic
acid (DHA; 22:6n-3) in cellula memb anes a ec s eicosanoid
p oduc ion (Nablone e al. 1990). This a y acid is also
ecognised as a p ecu so o ce ain biologically ac i e
ioxila ed de i a i es (Ge man e al. 1983, Hong e al.
2005). The e o e, he supply o p ecu so polyunsa u a ed
a y acids wi h 18 o mo e ca bon a oms and wo o mo e
double bonds (PUFA) o eicosanoid syn hesis is di ec ly
ela ed o he a y acid composi ion o memb ane
phospholipids, which in u n is in luenced by die a y PUFA
in ake and me abolism (Lands 1989).
In gil head sea b eam, die a y de iciencies on n-3 HUFA,
essen ial a y acids o ma ine ish (Izquie do 1996), aised
he basal plasma co isol le els and al e ed he pa e n o
co isol elease a e s ess (Mon e o e al. 1998). Co isol is a
key co icos e oid ho mone o homeos a ic esponse o s ess
39
Jou nal o Endoc inology (2006) 190, 39–45 DOI: 10.1677/joe.1.06770
0022–0795/06/0190–039 q2006 Socie y o Endoc inology P in ed in G ea B i ain Online e sion ia h p://www.endoc inology-jou nals.o g
in all e eb a es, h ough i s e ec s on me abolism and
immune unc ion (Hon ela 1997, Wendelaa Bonga 1997) as
well as he osmo egula ion p ocess (Wendelaa Bonga 1997).
Thus, he inc ease in plasma co isol le els is ega ded as he
mos eliable me hod o di e en ia ing be ween s essed and
non-s essed ish (Thompson e al. 1993, Yin e al. 1995,
Ro llan & To 1997). Mo eo e , eeding ela i ely low le els
o n-3 HUFA, al hough no a ec ing g ow h and eed
e iciency, signi ican ly aised plasma co isol le els (Mon e o
e al. 2003).
Howe e , he physiological mechanisms by which hese
HUFA egula e he ho mone-induced plasma co isol le els
a e no clea . In ish, se e al s udies ha e sugges ed ha ARA is
in ol ed in he elease o co isol, al hough he ac ual
mechanisms ha e no been in es iga ed (Gup a e al. 1985,
Bessona e al. 1999, Ha el e al. 2001, Ko en e al. 2003, Van
Anhol e al. 2004). In mammals, ce ain s udies sugges ha
PG play an impo an ole in media ing he co icos e oido-
genic ac ion o ad enoco ico opic ho mone (ACTH) (Kocsis
e al. 1999), and hus he ole o a y acids in s ess esponse
seems o be media ed by he p oduc ion o eicosanoids.
The p esen s udy aims o cla i y he e ec o di e en
HUFA on ACTH-induced co isol p oduc ion and elease by
gil head sea b eam in e enal cells.
Ma e ial and Me hods
Animals
Sexually imma u e gil head sea b eam (Spa us au a a) o body
weigh 54$7G11$2 g supplied by a Spanish ish a m
(Masnou, Ba celona, Spain) we e kep o 2 weeks in wo
ib eglass anks o 1000 l held in a semi-closed seawa e
ci cula ion sys em equipped wi h physical and biological
il e s. Wa e empe a u e was main ained a 16–18 8C, he
salini y a 35–40% and pho ope iod a 12 h ligh :12 h
da kness. Fish we e ed once a day wi h a comme cial eed
un il 24 h be o e he in i o ials o a oid eed in e e ence.
A o al numbe o 30 ish we e employed in he expe imen s.
Supe usion ials
A e 2 weeks o acclima isa ion, ish we e andomly aken
om he anks in less han 1 min, immedia ely anaes he ised
wi h 2-phenoxye hanol (1:1000 / ) and blood collec ed
wi h a hypode mic sy inge om he caudal ein o minimise
he haemo hage. Head kidney issue was emo ed om wo
ish in each supe usion ial and cu in o e y small agmen s
in Hepes Ringe medium, which was used as he supe usion
medium. A e wa ds, head kidney homogena es we e pooled
and dis ibu ed in eigh supe usion chambe s ( olume:
0$2 ml) in o de o ob ain a homogeneous aliquo om
each o hem. Tissues we e supe used wi h a Hepes (pH 7$4)
Ringe ’s solu ion con aining 171 mM NaCl, 2 mM KCl,
2 mM CaCl
2
H
2
O, 0$25% (w/ ) glucose and 0$03% (w/ )
bo ine se um albumin (Ro llan e al. 2001). The sys em was
empe a u e-con olled a 15 8C and supe usion medium was
pumped h ough he chambe a a a e o 75 ml/min by a
Mas e plex L/S
R
mul ichannel pe is al ic pump (Cole
Pa me Ins umen Co. Ve non Hills, IL, USA).
T ials we e s a ed a e 3 h o supe usion when co isol
eached a s able baseline le el (Ro llan e al.2000a,2000b)due
o se e al ac o s such as he di e en dispe sion o in e enal
cells in he pe usion p epa a ion, indi idual di e ences and he
p e-s ess le el o each ish. A e he s abilisa ion pe iod o 3 h,
issues we e subsequen ly incuba ed wi h di e en a y acids. A
se ies o p elimina y es s we e pe o med in quad uplica e, o
de e mine he adequa e a y acid concen a ion (50, 150 o
300 mM) and incuba ion ime (1 o 3 h) o any o he h ee a y
acids assayed (ARA, EPA and DHA). Bes co isol s imula ion
was ound wi h a y acid concen a ions o 50 mMandan
incuba ion ime o 1 h (Table 1) and hese condi ions we e used
a e wa ds in all he esea ch expe imen s. Bo h in hese
p elimina y es s and in he esea ch expe imen s, pe usion
medium was supplemen ed wi h he co esponding concen-
a ion o di e en a y acids ARA, EPA, DHA and DHGLA
(dilu ed in less han 0$5% o e hanol/medium / ) p io o
issue incuba ion. In a second se ies o expe imen s o cla i y he
ac ion mechanisms o hese a y acids, issues we e incuba ed
wi h a COX inhibi o indome acin (INDO) o 20 min a a
concen a ion o 25 mM dilu ed in supe usion medium. A e
incuba ion wi h he a y acids, he pe used issues we e
s imula ed wi h ACTH a a concen a ion o 5 nM hACTH
1–39
(Sigma) o 20 min. Subsequen ly, pe usion was main ained o
ano he 170 min, ac ion samples being collec ed e e y 20 min
du ing his pe iod. Co isol s imula ion ac o was calcula ed by
he compa ison o maximum co isol eleased a e ACTH
s imula ion wi h baseline co isol eleased (maximum eleaseK
baseline elease)/(baseline elease) (Ro llan e al. 2001). In all
he se ies o expe imen s, each ea men was assayed in
quad uplica e.
Co isol measu emen s
Co isol concen a ion in he pe used luid was de e mined
by RIA (Ro llan e al. 2001). The an ibody used o he assay
was pu chased om Biolink, S.L. (Cos a Mesa, CA, USA) in a
Table 1 E ec o wo a y acid concen a ions (50 and 150 mM) and
wo incuba ion imes (1 h and 3 h) o h ee polyunsa u a ed a y
acids on co isol sec e ion s imula ion ac o
1h 3h
T ea men
Con ol 14$71G2$41 13$28
EPA 50 mM29$63G2$59 7$79G3$29
150 mM7$79G3$29 –
ARA 50 mM22$26C6$29 11$75G4$16
150 mM12$25G1$86 –
DHA 50 mM35$72G9$28 2$60G1$16
150 mM4$47G0$28 –
R GANGA and o he s $Modula ion o ACTH-induced co isol elease40
Jou nal o Endoc inology (2006) 190, 39–45 www.endoc inology-jou nals.o g
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