Fatty acid changes in enriched and subsequently starved Artemia franciscana nauplii enriched with different essential fatty acids

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ELSEVIER Vlaams Instituut voor de Zee f7" rfsa l ^ ^ W ( 2 1 ) 9 3-1 5 16244 Aquaculture www.elsevier.

Sorgeloosa a Laboratory o f Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9 Ghent, Belgium b Fish Nutrition Laboratory INRA-IFREMER, B.P. 3, 6431 St.

possible interactions am ong highly unsaturated fatty acids (H U FA ) during enrichm ent and starvation o f A rtem ia nauplii.

1 ELSEVIER Vlaams Instituut voor de Zee f7" rfsa l ^ ^ W ( 2 1 ) Aquaculture cate/aqua-online Fatty acid changes in enriched and subsequently starved Artemia franciscana nauplii enriched with different essential fatty acids Kyungmin H an a *, Inge G eurdenab, Patrick Sorgeloosa a Laboratory o f Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9 Ghent, Belgium b Fish Nutrition Laboratory INRA-IFREMER, B.P. 3, 6431 St. Pée, France Received 14 August 2; received in revised form 27 Novem ber 2; accepted 27 November 2 Abstract T he present study aim s to evaluate differences in the incorporation efficiency and the possible interactions am ong highly unsaturated fatty acids (H U FA ) during enrichm ent and starvation o f A rtem ia nauplii. A rtem ia francisca n a nauplii w ere enriched w ith em ulsions containing docosahexaenoic acid (D H A, 22:6 n 3), eicosapentaenoic acid (EPA, 2:5«3) o r arachidonic acid (AA, 2 :4 «- 6) as sole H U FA o r w ith different ratios o f these H U FA during 24 h at 28 C and subsequently starved for 24 h at the sam e tem perature. The com parison o f H U FA incorporation efficiency w hen supplying the three HU FA separately show ed a less efficient enrichm ent o f D H A as com pared to A A or EPA. DHA incorporation was alw ays accom panied by an E PA increase, indicating the m etabolic conversion o f DHA to E PA by the nauplii during the enrichm ent process. W hen offering the H U FA together, w e found no com petitive interaction o f EPA or 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 FA em ulsion, som e negative interference m ight have occurred betw een the H U FA, as it gave a low er incorporation o f 2 2:6«- 3 and 2:5«- 3 than the em ulsions w ith low er n - 3 HU FA content. D uring the subsequent starvation o f E PA - o r D H A -enriched A rtem ia, relative EPA and D H A losses w ere sim ilarly high in both treatm ents. In contrast, the presence o f DHA in naupliar lipids increased the E PA retention, w hich m ight how ever be related to D H A retroconversion. 2 1 Elsevier Science B.V. A ll rights reserved. Keywords: Fatty acid; EPA; DHA Corresponding author. Tel.: ; fax: address: kyungmin.han@ rug.ac.be (K. Han) / 1 /$ - see front m atter 21 Elsevier Science B.V. All rights reserved. PII: S ( ) Reprinted w ith kind perm ission fro m Elsevier Science

94 K. Han et al. / Aquaculture 199 (21) 93 15 1.

for various species of m arine fish (Izquierdo et al., 1992; W atanabe, 1993; Sargent et al., 1997).

2 94 K. Han et al. / Aquaculture 199 (21) Introduction The dietary requirem ents o f n 3 highly unsaturated fatty acids (H U FA), particularly docosahexaenoic acid (DHA, 22:6n 3) and eicosapentaenoic acid (EPA, 2:5n 3), have been docum ented for various species of m arine fish (Izquierdo et al., 1992; W atanabe, 1993; Sargent et al., 1997). DHA, as well as its ratio to EPA, appears to be critical during the early larval stages as it affects grow th and survival o f m arine fish (Kanazawa, 1993; W atanabe, 1993; R eitan et al., 1994; Furuita et al., 1996). Related to its role as a precursor o f the eicosanoids, arachidonic acid (AA, 2:4n 6), was later added to the list of dietary essential fatty acids for m arine fish (Castell et al., 1994; Estévez et al., 1999; Sargent et al., 1999). The discrepancy between the essential fatty acid requirem ents of m arine fish larvae and the fatty acid com position of their com m only used live prey has resulted in the developm ent o f enrichm ent protocols im proving the HUFA content o f rotifers and Artem ia nauplii (Léger et al., 1987; Takeuchi et al., 1992; W atanabe, 1993; Rainuzzo et al., 1994). Literature data clearly docum ent that DHA, EPA and AA can be incorporated in Artem ia nauplii, but also show their catabolism during subsequent starvation (Triantaphyllidis et al., 1995; Evjem o et al., 1997; Estevez et al., 1998, Han et al., 2b). It remains unclear however if and how the different H U FA interact am ong each other during the enrichm ent and starvation processes. T he aim o f the present study was to evaluate the differences in fatty acid incorporation in Artem ia nauplii when using em ulsions containing each o f the essential fatty acids (i.e. D HA, EPA or A A ) and to exam ine possible com petitive interactions during enrichm ent and subsequent starvation w hen using em ulsions containing varying proportions of 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. Diets Pure oils o f DHA, EPA, OA ( > 95% purity) and AA (4% purity) w ere m ixed in various ratios (Table 1) before being em ulsified. Each lipid em ulsion contained 6% lipid (W.W.), 3% water,.2% antioxidants (ethoxyquin, Sigma, Belgium ), 1% em ulsifier (IN V E Aquaculture, Baasrode, Belgium ) and.1% vitam in E. The fatty acid com position of the various em ulsions w as analyzed by gas-chrom atography and is given in Table C yst hatching, enrichm ent and subsequent starvation Artem ia franciscana cysts (ARC. No: 132) from G reat Salt Lake (UT, U SA ) were used in the experiments. T he cysts (4 g D 1) w ere disinfected in a 2-m g I -1 hypochlorite solution for 2 m in before hatching. A fter washing with tap w ater to rem ove the rem aining hypochlorite, the cysts w ere incubated at a density of 2 g I -1 in

K. Han et a l/a q u a c u ltu r e 1 9 9 (21) 9 3-1 5 95 Table 1 Combinations of the oils used for preparing the enrichment emulsions for A.

docosahexaenoic acid ethyl ester (95% purity). Itochu Techno-Chemical. EPA, eicosapentaenoic acid ethyl ester (95% purity). Itochu Techno-Chemical, Japan. OA, oleic acid ethyl ester (98% purity).

3 K. Han et a l/a q u a c u ltu r e (21) Table 1 Combinations of the oils used for preparing the enrichment emulsions for A. franciscana nauplii Emulsion R atio of oils in em ulsions (%) D H A /O A 5:5 E P A /O A 5:5 A A /O A 5:5 D H A /E P A 5:5 D H A /E P A /O A 5:25:25 D H A /A A 5:5 D H A /A A /O A 5:25:25 adha, docosahexaenoic acid ethyl ester (95% purity). Itochu Techno-Chemical. EPA, eicosapentaenoic acid ethyl ester (95% purity). Itochu Techno-Chemical, Japan. OA, oleic acid ethyl ester (98% purity). Sigma. AA, arachidonic acid triacylglycerol (4% purity). Martek, USA. filtered seaw ater at 28 C under continuous aeration and light. A fter hatching, nauplii w ere separated from the cyst shells and transferred to 2-1 glass tubes (cylindroconical shape) in a w ater bath at 28 C w ith continuous aeration. The aeration consisted of an open tube at the bottom of the eone and an additional airstone to keep oxygen levels above 4 mg I - 1. Freshly hatched A rtem ia nauplii w ere enriched with the respective enrichm ent emulsions at a dose of.2 g f 1 at the beginning o f enrichm ent ( t = h) and after 1 2 h ( f = 1 2 h ). After 24-h enrichm ent ( t = 24 h), a random sam ple of 1 nauplii w as taken to verify their m olting stage (i.e. instar I vs. instars II and III). A t t24 the surviving nauplii were transferred into a new glass tube at a density of ind m l - 1 and kept in the w ater bath at 28 C for a subsequent starvation of 24 h ( i48). Each treatm ent was conducted in triplicate and sam ples w ere taken at t, t l2, t24, t36, and Z48 Table 2 M ajor fatty acid composition (% of total fatty acids) of the various enrichment emulsions3 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 14:.1 (.).1 (.).2 (.).1 (.).1 (.).1 (.).1 (.) 16:.3 (.) ndb 2.6 (.3).2 (.).2 (.) 1.3 (.) 2.4 (.1) 16:1«7.2 (.).6 (.).2 (.).2 (.).2 (.) nd nd 18:.1 (.).6 (.1) 3.4 (.6).3 (.1).7 (.2) 1.7 (.3) 3.3 (.2) 18:1« (.9) 49.1(.7) 66. (.5) 26.1(.8).1 (.) 32.8 (.6) 17.3 (.4) 18:2«6.1 (.) nd 2.6 (.6).2 (.).1 (.) 1.2 (.1) 2.4 (.3) 18:3«3 nd nd.1 (.) nd nd nd nd 2 :4 «- 6 nd nd 19.5 (1.1) nd nd 1.2 (.7) 2. (.9) 2 :5 « (.1) 44.3(1.2) nd 25.4 (.7) 5.(1.5) 2.8 (.5) 2.4 (.6) 2 2 :6 « (1.7).7 (.2) nd 45.6 (.9) 47.(1.3) 48.4(1.3) 47.2(1.7) «- 3 HUFAC 48.(1.1) 45. (.9) nd 71.1(1.5) 97. (2.4) 51.2(1.9) 49.6(2.1) «- 6 PUFA.2 (.).2 (.) 24.2(1.9).4 (.1).4 (.2) 12.3(1.3) 24.6(1.6) am inor fatty acids ( <.1%) not mentioned in table. bnd: Not detected. c > 2:3«3.

96 K. Han et al. / Aquaculture 199 (21) 93-

C until further analysis. 2.3. Fatly acid analysis The fatty acid com position o f the Artem ia nauplii was analyzed by a direct transm ethylation m ethod according to Lepage and R oy (1984).

The resulting fatty acid m ethyl esters (FAM E) w ere separated and identified on a Chrom pack CP 91 gas chrom atograph equipped w ith an autosam pler and a tem perature program m able on-colum n

4 96 K. Han et al. / Aquaculture 199 (21) after hatching for fatty acid analysis. A ll sam ples w ere stored under nitrogen at 3 C until further analysis Fatly acid analysis The fatty acid com position o f the Artem ia nauplii was analyzed by a direct transm ethylation m ethod according to Lepage and R oy (1984). T he internal standard w as 2 :2n 6. The resulting fatty acid m ethyl esters (FAM E) w ere separated and identified on a Chrom pack CP 91 gas chrom atograph equipped w ith an autosam pler and a tem perature program m able on-colum n injector (TPO CI). Sam ples w ere injected on a polar 5 m capillary column, BPX 7 (SGE, A ustralia), with a diam eter of.32 mm and a layer thickness of 25 xm connected to a 2.5-m methyl deactivated pre-column. T he carrier gas was H 2, at a pressure of 1 kpa and the detection m ode flame ionization detector (FID). The oven tem perature was set to increase from the initial tem perature o f 85 C to 15 C at a rate of 3 C /m in, from 15 C to 152 C at.1 C /m in, from 152 C to 172 C at.6 5 C /m in, from 172 C to 187 C at 2 5 C /m in and to stay at 187 C for 7 min. The injector was heated from 85 C to 19 C at 5 C /s and stayed at 19 C for 3 min. Identification was based on standard reference mixtures (Nu-Chek-Prep, USA). Integration and calculations w ere done with a softw are program (M aestro, Chrompack) 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 [(mg g ~ 1 D W at time x mg g" 1 DW at time X 1 2 )/1 2 h] in Table 4. D ata represent m eans of triplicate analysis and w ere further Tim e (h) T im e (h) T im e (h) Fig. 1. Changes in the HUFA and OA contents (m g g ~ 1 DW ) in A. franciscana nauplii (SD) enriched for 24 h with fatty acid mixtures 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.

$5 o o> 1 O) E 4 3 2 1 / * \ t B _ 6-5 - - / / 4 - / / ^ - 3 V / _ 1 12 24 36 48 Time (h) 2 : : AA s EPA Ár-- DHA -T- OA V ^ ---- # ------ ------ ------ 12 24 36 48 Time (h) Fig. 2. Changes in the HUFA and OA contents (mg g 1 DW) in A.$ franciscana nauplii (SD) enriched for 24 h with fatty acid mixtures D H A /E P A /O A (A) and D H A /E P A (B ) and during a subsequent 24-h starvation.

franciscana nauplii (SD) enriched for 24 h with fatty acid mixtures D H A /E P A /O A (A) and D H A /E P A (B ) and during a subsequent 24-h starvation.

5 K. Han et a l / Aquaculture 199 (21) CT5 6? 5 o o> 1 O) E / * \ t B _ / / 4 - / / ^ - 3 V / _ Time (h) 2 : : AA s EPA Ár-- DHA -T- OA V ^ ---- # Time (h) Fig. 2. Changes in the HUFA and OA contents (mg g 1 DW) in A. franciscana nauplii (SD) enriched for 24 h with fatty acid mixtures D H A /E P A /O A (A) and D H A /E P A (B ) and during a subsequent 24-h starvation. analyzed by one-way A NOVA follow ed by T ukey s H onest Significant Difference test ( P <.5) (Sokal and Rohlf, 1981) "O 2 O) CT> 1 : * 7 / Time (h) Time (h) 48 A A E P A D HA O A Fig. 3. Changes in the HUFA and OA contents (mg g 1 DW) in A. franciscana nauplii (SD) enriched for 24 h with fatty acid mixtures D H A /A A /O A (A) and D H A /A A (B) and during subsequent 24-h starvation.

98 K. Han et al. / Aquaculture 199 (21) 93-15 Q O' Q / s es io IO oo oo 'ici s? p o Ö O CM O X es Ö p o p O p s o O o es O P es o Ö O ' - O.

cn VO ) q' q voq o cd r 1 y * o cn CN ö i o q o d O d q s d CN Ö Ö O S Ö Ö o o d d Ö oo q rt in- q q GO m CN p ) <N d d q -o cn oo en o (N q d ^ -o -a ^ VO d en 1-4» «*-* ON vo ON c en c n Ö d d o c

6 98 K. Han et al. / Aquaculture 199 (21) Q O' Q / s es io IO oo oo 'ici s? p o Ö O CM O X es Ö p o p O p s o O o es O P es o Ö O ' - O. O Ö o Ö 3 Ö cd s*-- > ' CO N ' p P et- s e M-e es p p oo es p es p st 2 S3 es r-~ es O O «o ici i -o S3 Ml- ici cd es o s c ^ ^ 1-1 ci sf O s C es (S Ö C) 1 O 1-1 O c C o es es io es r- o /-N s? cn VO ) q' q voq o cd r 1 y * o cn CN ö i o q o d O d q s d CN Ö Ö O S Ö Ö o o d d Ö oo q rt in- q q GO m CN p ) <N d d q -o cn oo en o (N q d ^ -o -a ^ VO d en 1-4» «*-* ON vo ON c en c n Ö d d o c c o CN en m s- I o o ö vo oo vo p es o o o es Ö O Ö Ö Ö vo G CG es es O O ^ => Ö <=>. D o o ^ o fn -s vc CN en * < 9 N e o o m O; -î corho\ p t o\ o\ m - ioei lri -< OHNifl-'ONON /^s 'q ^*NIO'S S /-N S ON en S o d q d CN q 3 q O o q q q o o d d d d d d O o o o d d d o_ d - p w ON < CN W q q no q CN r- *o CN en q d q CN q q q vo q -o -o ^ vo o en d VO O n o en en d 3 O r-hd o c c: o CN en ON 1. S q q S S s q q q i d T 1 d pf- S o d y * o y 1, J q» 1> i 1 _\ i/-) d d d d d d d d d d w d d d d d V-' en q vo q q q d q q d q en ON N" q en q q q q q q q oo d en o * d oo r- oo d en en d d d d d c o c o CN ON CN S3 2 Q q S q ^^ q S y * m d d vo o CN en O en o ' <CN o O CN v: d S_c d d d d d d d d d d V-/ d d d NO vo o VO s*»/ w en en q d q * «d d q < d vo CN vo q q m d q T3 -o <1 "O d d q r < IO r- vo d en CN d ci d» H d O C G O G CN ON en q q q /^ v S q oo S CN q q «n q d q q d q O o o q d o q o d CN d '- e1 d, d d d d d d d d 3 d d d q 'w' q oo q q w v / w q q q q ON oo en q d VO q q ON vo en oo q q q d q "ô -o q vo CN Os q d. ^ d d VOr- o CN CN d d o f-h d o G G O CN CN ON r-»-hr-> P? D -Ç -Ö es sf o cd Ö ' " Ç ^ ^ T S O'O O co O C d 3 p p p p u d s -i h B N iii io S i E- tt, r- î6 r- r ON 1 ī I vo en en ON vo VO en en en vo en en i i 1 1 i I i I 1 1 I 1 i i i I 1 1 I c: s: C ss c C s: SC C - s: c s: C s: q O H q < i (Nq en q q y * en q «n m q q q q d d vb oo oo oo ÓÑoc oô d d q q d o * H CN d CN CN * CN CN CN CN CN CN CN cn CN CN CN co < < Ü &. vo en I il

K. Han et al./a quaculture 199 (21) 93-15 99 /Ä v a CO 5 ON ON p P <N P CN 1 p p o o d o o o 'n ^ Ö Ö s> ' 3 d d 3 3 d 3, i s' ' cn ON cn v ' os P cn 5 CN O.

H ^ >n ^ d d o»i c h o c c o (N «h t O --i P p CN /-N o o Q > 1 5 r - d P 5 1 CN CN o * > CN CN r < o CN d d d d d d d d d d d d o P O n CN CN O y, «< CN v q VO cn C"i p «p CN p 1-H oo n - P - o T3 P.

7 K. Han et al./a quaculture 199 (21) /Ä v a CO 5 ON ON p P <N P CN 1 p p o o d o o o 'n ^ Ö Ö s> ' 3 d d 3 3 d 3, i s' ' cn ON cn v ' os P cn 5 CN O. Ö p p OO cn P CN d cn Ö r-h cn 5 b CN * 1 Ö o o *-* d d c a o N N co a p cn p 3 *-< 1 d 3 o d d d p w io p IO oo 5 CN p CN ON o p d y 1 * 1 io c CN c n o t~~ m oo co so co T3 -a et r-. H ^ >n ^ d d o»i c h o c c o (N «h t O --i P p CN /-N o o Q > 1 5 r - d P 5 1 CN CN o * > CN CN r < o CN d d d d d d d d d d d d o P O n CN CN O y, «< CN v q VO cn C"i p «p CN p 1-H oo n - P - o T3 P. i-h CN NO d CN y ' d i «d d d O C C O cn io co ^ co <5 co ) sd d- t~~ tj- o cn P 'P I O NO Q cn / N o d IO CN d CN r- o CN CN o * ' ' i i oj io OO IO d o d d d d 3 o o o Ö p s_ d ON N; p r-h w OS p p p p p cn p ON p p Tf p <N T3 ^ CO. 13 " O cr! p io CN p 5 p p r-< IO oo N- P O CN Ö Ö C i Ö CO t: C C= O io CN r- 5 C O 1 o 'r o m os o o. oo co Ö co o o o o o CO N IO «IO -3 Ö Ö Ö Ö OO c co co Nt Ö c c e o CO O ) O S CN S ci S O OO CN T f o o OO O S CO CN ^ s t M CN / s N- NO r-h r -i o P P P o d d d d ^ s> ^ o o w ' r - VO o O M N H3 -et O i * <O q o, s s w s r-^ooo c o -<o e c h - OO 5 ^ o vc s o c o i~~ CN o CN» < p cn * 1 p - O y ' < P P O 'S d d d d d ' ' d d d o d d ' ' 3 d 3 - -er ^ cd o. o P c I v / w ' o o P d p T < cn ON P p P P p en r i P T3 -o P p d cn IO d CN r-h O M N } d d d d o c c o oo p r** r- os NO en co OS SO so co CO co SO cn en I 1 c c c C s: p O p p P xt; K K K K O s 1 i i C S CO d- 't en in n C o C s: P p NO NO OC OC oc oo ON T~H d Ö (N Ö (M Ö CN Ö Ol Ö CN Ö CN CN CN CN P CN CN CN O io CN w N- SO CN '. oo i >o io co o < < S o- o- SD CO B s e c 'tr: <.1 mgg 1 DW. Sums include minor fatty acids not shown in table.

1 K. Han et al. / Aquaculture 199 (21) 93-15 3. Results The fatty acid com position of A.

W ith the D H A /O A em ulsion, the content o f 22:6«3 and 2:5«3 increased from to 26.3 m g g -1 DW and from 8.5 to 14.6 mg g _l DW, respectively.

8 1 K. Han et al. / Aquaculture 199 (21) Results The fatty acid com position of A. franciscana nauplii enriched with the fatty acid m ixtures D H A /O A, E P A /O A and A A /O A is show n in Fig. 1. W ith the D H A /O A em ulsion, the content o f 22:6«3 and 2:5«3 increased from to 26.3 m g g -1 DW and from 8.5 to 14.6 mg g _l DW, respectively. In th e E P A /O A em ulsion, the content o f 2:5«3 increased from 8.5 to 44.4 mg g _l D W and in the A A /O A emulsion, 2:4«6 increased from 1.7 to 13.6 mg g ~ 1 DW. D uring the starvation period (ri4_48), several changes w ere noticed in the treatments. In the D H A /O A treatm ent, the level of 22:6n 3 decreased rapidly from 26.3 to 8.9 mg g _1 D W, w hereas the content of 2:5«3 was only slightly reduced from 14.6 to 11.5 mg g ~ DW. In the E P A /O A treatm ent, the content o f 2:5«- 3 decreased from to 17.6 mg g ~ 1 DW and in the A A /O A treatm ent, levels of 2:4«6 and EPA decreased from 13.6 to 8.5 m g g _l D W and 8.5 to 2.5 m g g -1 DW, respectively. The results o f the A n em ia enrichm ent with com binations o f 22:6 n 3 and 2:5n - 3 are show n in Fig. 2. In the D H A /E P A /O A em ulsion, the content o f 22:6n 3 and 2:5«3 increased during the enrichm ent period ( - 24) fr m t 26.1 and 46.9 mg g ~ ' D W, respectively. In the D H A /E P A em ulsion, the content o f 22:6«3 and 2:5«3 increased to 19.9 and 37.8 mg g ~ 1 DW, respectively. In the D H A /E P A /O A and D H A /E P A em ulsion, the content of 22:6«3 rapidly decreased during the starvation period ( i24_48) from 26.1 to 5. m g g _l D W and from 19.9 to 3.8 mg g -1 DW, respectively. The content o f 2:5«3 decreased from 46.9 to 34.4 mg g _1 DW and from 37.8 to 18.1 mg g -1 DW in the D H A /E P A /O A and D H A /E P A emulsion, respectively. Fig. 3 shows the changes in fatty acid com position of Artem ia enriched with com binations of 22:6«3 and 2:4«6. In the D H A /A A /O A and D H A /A A em ulsions, the content of 22:6«- 3 increased to 26.8 and 25.9 mg g _l DW, accom panied by a slight increase o f EPA to 17.7 and 15.6 m g g _1 DW, respectively. In the D H A /A A em ulsion, the content of 2:4«6 rapidly increased from 1.7 to 16 m g g " 1 D W during the first 12-h enrichm ent period ( i- 12^ com pared to 8. mg 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 2:4«6 content further increased to 18.2 and 12.6 m g g -1 DW after 24-h enrichment, respectively. D uring the starvation period (f24_48), the content of 2 2 :6 «3 decreased m ore strongly than that o f 2:4«6 and 2:5«3 in both emulsions. The E P A /A A ratio rem ained stable during the 24-h starvation period (Table 3). Table 4 shows the relative increase and decrease in mg g _1 D W h -1 o f 22:6«3, 2:5«3 and 2:4«6 during the enrichm ent and subsequent starvation period for all the em ulsions. D uring the first enrichm ent period _ l2), the increase o f 22:6«3 in the D H A /E P A em ulsion w as significantly ( P <.5) low er than in the other treatm ents, w hich contained the same 22:6«3 concentration (D H A /E P A /O A emulsion, 5% o f the em ulsion). In the second enrichm ent period ( t n _24), no difference in 22:6«3 uptake was noticed. The 22:6«3 level decreased more rapidly during the first starvation period ( i24_36) than during the second starvation period ( t36 _48). The increase o f 2:5«3 with the E P A /O A em ulsion during the first enrichm ent period ( /o- 12) was significantly (P <.5) higher than with the D H A /E P A em ulsion and even

K. Han et al. / Aquaculture 199 (21) 93-15 15 Rainuzzo, J.R., Reitan, K.I., Olsen, Y., 1994.

Influence o f lip id composition o f live feed on growth, survival and pigmentation of turbot larvae. Aquacult. Int. 2, 33 48. Sargent, J.R., Bell, J.G., McEvoy, L.A., 1997.

9 K. Han et al. / Aquaculture 199 (21) Rainuzzo, J.R., Reitan, K.I., Olsen, Y., Effect of sh o rt and long-term lipid enrichment on total lipids, lipid class and fatty acid composition in rotifers. A quacult. Int. 2, Reitan, K.I., Rainuzzo, J.R., Olsen, Y., Influence o f lip id composition o f live feed on growth, survival and pigmentation of turbot larvae. Aquacult. Int. 2, Sargent, J.R., Bell, J.G., McEvoy, L.A., Requirem ents, presentation and sources of polyunsaturated fatty acids in marine fish larval feeds. Aquaculture 155, Sargent, J.R., McEvoy, L., Estévez, A., Bell, G., Bell, M., Henderson, J., Tocher, D., Lipid nutrition of marine fish during early development: current status an d future directions. Aquaculture 179, Sokal, R.R., Rohlf, F.J., Biometry. Freeman, New Y ork, USA, 859 pp. Takeuchi, T., Toyota, M., W atanabe, T., C om parison of lipid and n 3 highly unsaturated fatty acid incorporation between Artem ia enriched with various types of oil by direct method. Nippon Suisan Gakkaishi 58, Triantaphyllidis, G.V., Coutteau, P., Sorgeloos, P., T h e stability of n 3 highly unsaturated fatty acids in various Artem ia populations following enrichment an d subsequent starvation. In: Lavens, P., Jasper, E., Roelants, I. (Eds.), Larvi 95 Fish and Shellfish Sym posium. European Aquaculture Society, Special Publication, vol. 24, pp W atanabe, T., Importance of docosahexaenoic acid in marine larval fish. J. W orld Aquacult. Soc. 24, Watanabe, T., Oowa, F., Kitajima, C., Fujita, S., N utritional quality o f brine shrimp. Artemia salina, as a living feed from the viewpoint of essential fatty acids for fish. Bull. Jpn. Soc. Sei. Fish. 44,

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Live Feeds for Marine Fish Larvae. Aquaculture Center for Training, Education and Demonstration

Live Feeds for Marine Fish Larvae Aquaculture Center for Training, Education and Demonstration Why live feeds? Important Criteria For Selecting Live Feeds Is it the appropriate size for target species?