16244
Aquaculture
Vlaams Instituut voor de Zee
ELSEV IER
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w w w .elsevier.nl/lo c a te /a q u a -o n lin e
Fatty acid changes in enriched and subsequently
starved Artem ia franciscana nauplii enriched with
different essential fatty acids
Kyungm in H a n a’*, Inge G eu rd en ab, Patrick S orgeloosa
a Laboratory o f Aquaculture and A rtem ia R eference Center, Ghent University, R ozier 44,
9000 Ghent, B elgium
b Fish Nutrition L aboratory INRA-IFREM ER, B.P. 3, 64310 St. Pée, France
Received 14 A ugust 2000; received in revised form 27 N ovem ber 2000; accepted 27 N ovem ber 2000
A bstract
T he p resen t study aim s to ev alu ate d ifferences in th e incorporation efficiency and th e possible
interactions am ong highly u n sa tu rated fatty acids (H U F A ) du rin g enrichm ent and starvation o f
A rte m ia nauplii. A rtem ia fra n c is c a n a nauplii w ere e n rich ed w ith em ulsions containing docosahex aen o ic acid (D H A , 2 2 :6 n — 3), eico sapentaenoic a c id (E PA , 2 0 :5 « — 3) o r arachidonic acid
(A A , 2 0 :4 « - 6) as sole H U F A o r w ith differen t ratios o f th ese H U F A during 24 h at 28°C and
su bsequently starved fo r 24 h at th e sam e tem perature.
T he com parison o f H U F A in co rp o ratio n efficiency w h e n supplying the three H U FA separately
show ed a less efficient e n rich m en t o f D H A as c om pared to A A o r E PA . D H A incorporation w as
alw ays accom panied by an E P A in crease, in d icatin g the m etab o lic conversion o f D H A to E P A by
th e nauplii during the e n rich m en t process. W h en o fferin g th e H U FA together, w e found no
com p etitiv e interaction o f E P A o r o f A A on D H A incorporation. O nly in the case o f the 97% (%
total fatty acids) n — 3 H U F A em ulsion, som e negative in te rfere n c e m ight have occurred betw een
th e H U F A , as it gave a low er incorporation o f 2 2 :6 « - 3 and 2 0 :5 « - 3 than the em ulsions w ith
lo w e r n - 3 H U FA content.
D uring the su b seq u en t starvation o f E P A - o r D H A -enriched A rte m ia , relative E PA and D H A
lo sse s w ere sim ilarly high in b o th treatm ents. In contrast, th e presence o f D H A in n aupliar lipids
in creased the E P A retention, w h ich m ight h o w ev er be re la te d to D H A retroconversion. © 2 0 0 1
E lsev ier Science B.V. A ll rights reserved.
Keywords: Fatty acid; EPA; D HA
Corresponding author. Tel.: +32-9-264-3754; fax: +32-9-264-4193.
E-m ail address: kyungm in.han@ rug.ac.be (K. Han).
0 0 4 4 - 8 4 8 6 /0 1 /$ - see front m atter © 2001 E lsevier Science B.V. A ll rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 ( 0 0 ) 0 0 5 9 6 - 2
R e prin ted w ith kind p e rm is s io n fro m Elsevier S cie n ce
94
K. Han et al. / A quaculture 199 (2001) 93—105
1. Introduction
T he dietary requirem ents o f n — 3 highly u n saturated fatty acids (H U F A ), p articu­
larly docosahexaenoic acid (D H A , 2 2 :6n — 3) and eicosapentaenoic acid (E P A , 2 0:5n —
3), h ave been docum ented fo r various species o f m arin e fish (Izquierdo et al., 1992;
W atanabe, 1993; Sargent et al., 1997). D H A , as w ell as its ratio to E PA , appears to be
critical during the early larval stages as it affects gro w th and survival o f m arine fish
(K anazaw a, 1993; W atanabe, 1993; R eitan et al., 1994; Furuita et al., 1996). R elated to
its role as a precursor o f the eicosanoids, arachidonic acid (A A , 2 0 :4 n — 6), w as later
added to the list o f dietary essential fatty acids for m a rin e fish (C astell e t al., 1994;
E stév ez et al., 1999; S argent e t al., 1999). T he discrepancy betw een the essential fatty
acid requirem ents o f m arine fish larvae and the fatty acid com position o f their
com m only used live prey has resulted in the develo p m en t o f enrichm ent protocols
im proving the H U FA content o f rotifers and A rte m ia nauplii (L éger et al., 1987;
T akeuchi et al., 1992; W atanabe, 1993; R ainuzzo et al., 1994). L iterature data clearly
do cum ent that D H A , E PA and A A can b e incorporated in A rtem ia nauplii, but also
show their catabolism during subsequent starvation (T riantaphyllidis et al., 1995;
E vjem o e t al., 1997; E stevez et al., 1998, H an et al., 2000b). It rem ains unclear how ever
if and how the different H U FA interact am ong each oth er during the enrichm ent and
starvation processes.
T h e aim o f the present study w as to evaluate the differences in fatty acid incorpora­
tion in A rtem ia nauplii w hen using em ulsions containing each o f the essential fatty
acids (i.e. D H A , EPA o r A A ) and to exam ine p ossib le com petitive interactions during
enrichm ent and subsequent starvation w hen using em ulsions containing varying propor­
tions o f these fatty acids (i.e. D H A /E P A and D H A /E P A /O A (oleic acid); D H A /A A
and D H A /A A /O A ).
2. Materials and methods
2.1. D iets
P ure oils o f D H A, E PA , O A ( > 95% purity) and A A (40% purity) w ere m ixed in
v arious ratios (Table 1) before being em ulsified. E ach lipid em ulsion contained 60%
lip id (W .W .), 30% w ater, 0.02% antioxidants (ethoxyquin, Sigm a, B elgium ), 10%
em ulsifier (IN V E A quaculture, B aasrode, B elgium ) and 0.1% vitam in E. T he fatty acid
com position o f th e various em ulsions w as analyzed by gas-chrom atography and is given
in T able 2.
2.2. C yst hatching, enrichm ent a n d su b seq u en t starvation
A rtem ia fra nciscana cysts (A R C . N o: 1320) from G reat S alt L ake (U T , U SA ) w ere
u sed in the experim ents. T h e cysts (4 g D 1) w ere disinfected in a 200-m g I -1
h ypochlorite solution for 20 m in before hatching. A fter w ashing w ith tap w ater to
rem o v e the rem aining hypochlorite, th e cysts w ere incubated at a density o f 2 g I -1 in
K. Han et a l/A q u a c u ltu r e 1 9 9 (2001) 9 3 -1 0 5
95
T able 1
Com binations o f the oils used for preparing the enrichm ent em ulsions for A. franciscana nauplii
Em ulsion
R a tio o f oils in
em u lsio n s (% )“
D H A /O A
E P A /O A
A A /O A
D H A /E P A
D H A /E P A /O A
D H A /A A
D H A /A A /O A
50:50
50:50
50:50
50:50
50:25:25
50:50
50:25:25
aDHA, docosahexaenoic acid ethyl ester (95% purity). Itochu Techno-Chem ical.
E PA , eicosapentaenoic acid ethyl ester (95% purity). Itochu T echno-C hem ical, Japan.
O A , oleic acid ethyl ester (98% purity). Sigma.
A A , arachidonic acid triacylglycerol (40% purity). M artek, U S A .
filtered seaw ater at 28°C under continuous aeratio n and light. A fter hatching, nauplii
w ere separated from the cy st shells and tran sferred to 2-1 glass tubes (cylindroconical
shape) in a w ater bath at 28°C w ith continuous aeration. T h e aeration consisted o f an
open tube at th e bottom o f the eone and an ad d itio n al airstone to keep oxygen levels
above 4 m g I - 1 . Freshly hatched A rtem ia n au p lii w ere enriched w ith the respective
enrichm ent em ulsions at a dose o f 0.2 g f 1 at th e beginning o f enrichm ent ( t = 0 h) and
after 1 2 h ( f = 1 2 h ) . A fter 24-h enrichm ent ( t = 24 h), a random sam ple o f 100 nauplii
w as taken to verify their m olting stage (i.e. in sta r I vs. instars II and III). A t t 24 the
surviving nauplii w ere transferred into a new g lass tube at a density o f 125 + 10 ind
m l - 1 and kept in the w ater bath at 28°C for a subsequent starvation o f 24 h ( i 48). Each
treatm ent w as conducted in triplicate and sam ples w ere taken at t0, t l2, t24, t36, and Z48
T ab le 2
M ajor fatty acid com position (% o f total fatty acids) o f the various enrichm ent em ulsions3
14:0
16:0
16:1« —7
18:0
18:1« - 9
18:2« —6
18:3« —3
2 0 :4 « - 6
2 0 :5 « - 3
2 2 :6 « - 3
« - 3 HUFAC
« - 6 PUFA
D H A /O A
E P A /O A
A A /O A
D H A /E P A /
OA
D H A /E P A
D H A /A A /
OA
D H A /A A
0.1 (0.0)
0.3 (0.0)
0.2 (0.0)
0.1 (0.0)
50.4 (0.9)
0.1 (0.0)
nd
nd
2.2(0.1)
4 5 .8 (1 .7 )
4 8 .0 (1 .1 )
0.2 (0.0)
0.1 (0.0)
ndb
0.6 (0.0)
0.6 (0.1)
49.1(0.7)
nd
nd
nd
4 4 .3 (1 .2 )
0.7 (0.2)
45.0 (0.9)
0.2 (0.0)
0.2 (0.0)
2.6 (0.3)
0.2 (0.0)
3.4 (0.6)
66.0 (0.5)
2.6 (0.6)
0.1 (0.0)
19.5 (1.1)
nd
nd
nd
2 4 .2 (1 .9 )
0.1 (0.0)
0.2 (0.0)
0.2 (0.0)
0.3 (0.1)
26.1(0.8)
0.2 (0.0)
nd
nd
25.4 (0.7)
45.6 (0.9)
71 .1(1.5)
0.4 (0.1)
0.1 (0.0)
0.2 (0.0)
0.2 (0.0)
0.7 (0.2)
0.1 (0.0)
0.1 (0.0)
nd
nd
5 0 .0 (1 .5 )
4 7 .0 (1 .3 )
97.0 (2.4)
0.4 (0.2)
0.1 (0.0)
1.3 (0.0)
nd
1.7 (0.3)
32.8 (0.6)
1.2 (0.1)
nd
10.2 (0.7)
2.8 (0.5)
4 8 .4 (1 .3 )
5 1 .2 (1 .9 )
12.3(1.3)
0.1 (0.0)
2.4 (0.1)
nd
3.3 (0.2)
17.3 (0.4)
2.4 (0.3)
nd
20.0 (0.9)
2.4 (0.6)
4 7 .2 (1 .7 )
4 9 .6 (2 .1 )
2 4 .6(1.6)
aM inor fatty acids ( < 0.1% ) not m entioned in table.
bnd: N ot detected.
c > 20:3« —3.
K. Han et al. / Aquaculture 199 (2001) 9 3 -1 0 5
96
after hatching for fatty acid analysis. A ll sam ples w ere stored u n d er nitrogen at —30°C
until further analysis.
2.3. F atly a cid analysis
T h e fatty acid com position o f th e A rtem ia n au p lii w as analyzed by a direct
transm ethylation m ethod according to L epage and R o y (1984). T h e internal standard
w as 2 0 :2 n — 6. T he resulting fatty acid m ethyl esters (FA M E ) w ere separated and
identified on a C hrom pack CP 9001 gas chrom atograp h equipped w ith an autosam pler
and a tem perature program m able on-colum n injector (T P O C I). S am ples w ere injected on
a po lar 50 m capillary colum n, B P X 70 (SG E , A ustralia), w ith a diam eter o f 0.32 mm
an d a layer thickness o f 25 |xm connected to a 2 .5-m m ethyl deactivated pre-colum n.
T h e carrier gas was H 2, at a pressure o f 100 kP a and the detection m ode flam e
ionization detector (FID ). T he oven tem perature w as set to increase from the initial
tem perature o f 85°C to 150°C at a rate o f 3 0 ° C /m in , from 150°C to 152°C at
0 .1 ° C /m in , from 152°C to 172°C at 0 .6 5 °C /m in , fro m 172°C to 187°C at 2 5 ° C /m in
and to stay at 187°C for 7 min. T he injector w as h eated from 85°C to 190°C at 5 ° C /s
and stayed at 190°C for 30 min. Identification w as based on standard reference m ixtures
(N u-C hek-P rep, USA). Integration and calculations w ere done w ith a softw are program
(M aestro, Chrom pack).
2.4. Statistical analysis
D ata are expressed in absolute am ounts (m g g -1 D W ) or as relative changes (m g g ~ 1
D W ~ 1) calculated over 12-h intervals as [(m g g ~ 1 D W at tim e x — m g g" 1 D W at tim e
X — 1 2 ) /1 2 h] in T able 4. D ata represent m eans o f trip licate analysis and w ere further
80
80
80
60
60
60
40
40
40
20
20
20
0
0
12
24
T im e (h)
36
48
0
12
24
T im e (h)
36
48
12
24
36
T im e (h)
Fig. 1. C hanges in the HU FA and OA contents (m g g ~ 1 D W ) in A. franciscana nauplii (SD ) enriched for 24
h w ith fatty acid m ixtures D H A /O A (A ), E P A /O A ( B ) and A A /O A (C) and during a subsequent 24-h
starvation.
K. Han et a l / Aquaculture 1 9 9 (2001) 9 3 -1 0 5
97
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Fig. 2. C hanges in the HUFA and OA contents (m g g 1 DW ) in A. franciscana nauplii (SD) enriched for 24
h with fatty acid m ixtures D H A /E P A /O A (A ) and D H A /E P A (B ) and during a subsequent 24-h starvation.
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K. Han et al. / Aquaculture 199 (2001) 9 3 -1 0 5
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Sums include minor fatty acids not shown in table.
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K. Han et al. / Aquaculture 199 (2 0 0 1 ) 9 3 -1 0 5
3. Results
T he fatty acid com position o f A. fra n c isc a n a n a u p lii enriched w ith the fatty acid
m ixtures D H A /O A , E P A /O A and A A /O A is sh o w n in Fig. 1. W ith the D H A /O A
em ulsion, the content o f 2 2 :6 « — 3 and 20:5« — 3 in c re a se d from 0 to 26.3 m g g -1 D W
and from 8.5 to 14.6 m g g _l DW , respectively. In th e E P A /O A em ulsion, the content
o f 2 0 :5 « — 3 increased from 8.5 to 44.4 m g g _l D W and in the A A /O A em ulsion,
2 0 :4 « — 6 increased from 1.7 to 13.6 m g g ~ 1 D W . D u rin g the starvation period ( ri4_48),
several changes w ere noticed in the treatm ents. In the D H A /O A treatm ent, the level o f
2 2 :6 n — 3 decreased rapidly from 26.3 to 8.9 m g g _1 D W , w hereas the content o f
2 0:5« — 3 w as only slightly reduced from 14.6 to 11.5 m g g ~ ‘ D W . In the E P A /O A
treatm ent, the content o f 2 0 :5 « - 3 decreased from 4 4 .4 to 17.6 m g g ~ 1 D W and in the
A A /O A treatm ent, levels o f 20:4« — 6 and E P A d ec reased from 13.6 to 8.5 m g g _l
D W and 8.5 to 2.5 m g g -1 D W , respectively.
T he results o f the A n e m ia enrichm ent w ith com b in atio n s o f 2 2 :6 n — 3 and 2 0:5n - 3
are show n in Fig. 2. In the D H A /E P A /O A em ulsio n , the content o f 22 :6 n — 3 and
2 0:5« — 3 increased during the enrichm ent period (¿ 0 - 2 4 ) fr° m 0 t0 26.1 and 46.9 mg
g ~ ' D W , respectively. In the D H A /E P A em ulsio n , the content o f 2 2 :6 « — 3 and
2 0 :5 « — 3 increased to 19.9 and 37.8 mg g ~ 1 D W , respectively. In the D H A /E P A /O A
and D H A /E P A em ulsion, the content o f 22 :6 « — 3 rapidly decreased during the
starvation period ( i 24_48) from 26.1 to 5.0 m g g _l D W and from 19.9 to 3.8 m g g -1
D W , respectively. T he content o f 20:5« — 3 d ecreased fro m 46.9 to 34.4 m g g _1 D W
and from 37.8 to 18.1 m g g -1 D W in the D H A / E P A /O A and D H A /E P A em ulsion,
respectively.
Fig. 3 show s th e changes in fatty acid com p o sitio n o f A rtem ia enriched w ith
com binations o f 2 2 :6 « — 3 and 20 :4 « — 6. In the D H A / A A /O A and D H A /A A
em ulsions, the content o f 2 2 :6 « - 3 increased to 26.8 and 25.9 m g g _ l D W , accom pa­
n ied by a slight increase o f EPA to 17.7 and 15.6 m g g _1 D W , respectively. In the
D H A /A A em ulsion, the content o f 20 :4 « — 6 rapid ly increased fro m 1.7 to 16 m g g " 1
D W during the first 12-h enrichm ent period ( i 0- 1 2 ^ co m p ared to 8.0 m g g -1 D W in the
D H A / A A /O A em ulsion. In the D H A /A A and D H A / A A /O A em ulsions, the 2 0:4«
— 6 con ten t further increased to 18.2 and 12.6 m g g -1 D W after 24-h enrichm ent,
respectively. D uring the starvation period ( f 24_48), th e content o f 2 2 :6 « — 3 decreased
m o re strongly than that o f 20 :4 « — 6 and 20:5« — 3 in b oth em ulsions. T he E P A /A A
ratio rem ained stable during the 24-h starvation perio d (T able 3).
T able 4 show s the relative increase and decrease in m g g _1 D W h -1 o f 2 2 :6 « — 3,
2 0:5« — 3 and 2 0 :4 « — 6 during the enrichm ent and subsequent starvation period for all
the em ulsions. D uring the first enrichm ent period 0 0_ l2), the increase o f 2 2 :6 « — 3 in
the D H A /E P A em ulsion w as significantly ( P < 0.0 5 ) low er than in the other treat­
m ents, w hich contained th e sam e 2 2 :6 « — 3 concentration ( D H A /E P A /O A em ulsion,
50% o f the em ulsion). In the second enrichm ent p eriod ( t n _24), no difference in
2 2 :6 « — 3 uptake w as noticed. T he 22 :6 « — 3 level decreased m ore rapidly during the
first starvation period ( i 24_36) than during the second starvation period ( t 36 _48). The
increase o f 20:5« — 3 w ith the E P A /O A em ulsion during the first enrichm ent period
( /o- 1 2 ) w as significantly ( P < 0.05) higher than w ith th e D H A /E P A em ulsion and even
K. H an et al. / Aquaculture 1 9 9 (2001) 9 3 -1 0 5
105
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