Aquaculture Intemationall1: 109-117,2003.@ 2003 Kluwer Academic Publishers. Printed in the Netherlands.

98277

The effect of different HUFA enrichment emulsions onthe nutritional value of rotifers (Brachionus plicatilis)fed to larval haddock (Melanogrammus aeglefinus)

JOHN CASTELL1.*,TAMMYBLAIR1,STEVENNEIL', KENNETHHOWESI, SARAHMERCERI, JOHN REIDI, WILFREDYOUNG-LA!I,BRANDIGULLISON1,PHILIPPEDHERT2and PATRICKSORGELOOS2lFisheries and Oceans Canada. Biological Station. 531 Brandy Cove Rd. St. Andrews. E5B 2L9. NB,Canada; 2Laboratory of Aquaculture & Artemia Reference Center, Ghent University. Rozier 44, 9000Gent. Belgium; *Author for correspondence (e-mail: CasteIU@mar.dfo-mpo.gc.ca; phone: (506) 529-5904; fax: (506) 529-5862)

Received 12 December 2001; accepled 6 March 2002

Key words: Arachidonic acid, Docosahexaenoic acid, Eicosapenlaenoic acid, Enrichment, Lipid emul-sions, Marine fish larvae, Microalgae, Rotifer

Abstract. A feeding study was conducled in the winter 2001 to determine the effects of feeding rotifers(Brachionus plicatilis) enriched with various levels of essential fatty acids on the growth and survival ofhaddock larvae (Melanogrammus aeglefinus). Rotifer enrichment treatments were: I) mixed algae, 2)high DHA (docosahexaenoic acid, 22:6n-3), 3) high DHA and EPA (eicosapenlaenoic acid, 20:5n-3),and 4) DHA, EPA, and AA (arachidonic acid, 20:4n-6). Larvae were fed rotifers enriched with the dif-ferent treatments from days I to 16 post-hatch. From day 17 until 25 all treatment groups were fedrotifers reared on mixed algae and then weaned onto the International Council for Exploration of theSEA (ICES) Slandard Reference Weaning diet (http://al1serv.rug.ac.tIaquaculturelrendlrend.htm) over afive day period. The experiment was lerminated on day 41 post-hatch. The enrichment treatments af-feeled the fatty acid composition of the rotifers and correlated with the accumulation of these fatty acidsin the haddock larvae. However, no significant differences in larval growth or survival to 40 days posthatch were deteeled, suggesting that all treatments provided the minimal essential fatty acid require-ments for haddock.

Abbreviations: EPA - eicosapentaenoic acid, DHA - docosahexaenoic acid, AA - arachidonic acid,

dph - days post hatch,EFA- essentialfatty acid, HUFA- highly unsaturated fatty acids, ICES - In-ternationalCouncilfor the Explorationof the Sea, ppt -parts per thousand, sem -slandarderror of themean

Introduction

Haddock (Melanogrammus aeglefinus) has been identified as a candidate speciesfor commercial culture in Atlantic Canada (Litvak 1998; Henry 1997). The princi-pal obstacle to haddock culture is poor survival (average -2%, range 0-33%)through the first-feeding larval stage, which requires live food organisms, to wean-ing and metamorphosis. The essential fatty acid (EFA) requirements of first-feed-

VLlZ (VZW)

VLAAMSINSTlTUUTVOOR DE ZEE

FLANDERS MARINE INSTITUTEOostende - Belgium

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ing haddock larvae have not been identified, however, two other gadoids closelyrelated to haddock do require live food organisms enriched with docosahexaenoicacid (DHA) (Gadus macrocephalus Takeuchi et al. (1994); Gadus morhua, Gallo-way et al. (1998)). Rotifers (Brachionus plicatilis), particularly when reared onyeast-based diets, have been found deficient in n-3 highly unsaturated fatty acids(HUFA; particularly 20:5n-3 or eicosapentaenoic acid (EPA) and 22:6n-3 or DHA),if used as the live food for cool-water marine fish larvae (Rodrfguez et al. 1996;Rainuzzo et al. 1997). Recent research has shown that the n-6 HUFA, arachidonicacid (20:4n-6 or AA) is also important for growth, survival and stress resistance(Koven et al. 2001). An experiment was designed to determine the effect of differ-ent rotifer enrichments during the first l5d of larval haddock feeding on subsequentgrowth, survival and fatty acid composition.

Materials and methods

Rotifers (Brachionus plicatilis, Aquafarms FL, USA) were batch cultured in 1.4 m3fiberglass tanks and fed a mixed algal diet of lsochrysis galbana, Tetraselmis sue-cia, Pavlova lutheria and Nannochloropsis sp. in seawater (24 0c) at 200-250rotifers.ml-' under constant light. Harvested rotifers were enriched in 25 L glasscarboys using the following ICES emulsions (ICES 1997): ICES 30/4/C (DHA);ICES 30/0.6/C (DHA:EPA); ICES DHAlEPNAA; and a mixed-algae control of thefour species mentioned above. The fatty acid composition of algal lipids and en-richment emulsions are presented in Table 1. More information on the productionand composition of the ICES enrichment emulsions can be found on the Universityof Ghent web site (http://allserv.rug.ac.bef"jdhont/rendlrend.htm). Rotifers, with en-richment media or mixed algae, were acclimated to larval culture temperature (60c) over a period of approximately 16 h prior to being fed to the larvae. The lipidcomposition giving the main fatty acids of enriched rotifers are shown in Table 2.

A single batch of eggs, spawned by captive broodstock, was incubated at 6 °Cin 250 L upwelling conical-bottom incubators. The broodstock haddock had beenmaintained at the Department of Fisheries and Oceans Biological Station, St. An-drews, New Brunswick on a diet of herring, squid and shrimp supplemented withvitamins, Corey Feed Mills dry marine feed and moist feed. Hatched larvae weretransferred to 20x50-1 black plastic tanks (5 tanks/treatment) and stocked at 20larvae.L -I. Larval tanks were supplied with 1 p,m filtered seawater (0.2 L.min-I,salinity 31 ppt), 24 h light (240 lux), and 500 ml algae 2 x/d to "green" the waterfrom day 1 to day 30. Water temperature was 6 °C initially and increased graduallyto 12 °C over the course of the experiment. Larvae utilized their yolksac reservesand began exogenous feeding on day-l post hatching. The enriched rotifer treat-ments were fed to the larvae 3 times per day (9, 15, 20 h, to maintain a concen-tration of 5 rotifers per ml) from 1 dph (days post hatch) to 16 dph, then all larvaewere fed mixed-algae rotifers until weaning onto ICES Standard Reference Wean-ing Diet (Coutteau et al. 1995) at 25 dph. Larvae samples at 16 and 41 dph, en-

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Table 1. Fatty acid composition (percentage of total fatty acids) of principal fatty acids present in en-richment emulsionsand algal species.(Valuesare meansfor three replicatesampleanalysis).

T-Iso Tetraselmis Nanno. Pavlovalutheri 30/4/C 30/0.6/C DHAlEPAlAA

Fatty acid! % % % % (%) (%) (%)12:0 0.0 0.0 0.0 0.0 11.2 0.1 0.014:0 17.7 0.3 3.8 8.9 5.5 6.8 0.516:0 12.6 29.2 15.8 12.3 22.9 20.1 8.118:0 0.3 0.3 0.2 0.4 4.1 2.6 4.8

16:ln-11 1.0 3.7 0.0 0.0 0.0 0.0 0.016:ln-9 0.0 0.7 2.4 0.4 0.1 9.5 0.016:ln-7 4.1 0.3 26.9 20.3 2.8 0.1 0.418:ln-9 9.9 16.4 2.0 0.3 18.0 13.5 12.218:1n-7 0.8 3.0 0.2 1.8 1.0 3.6 1.518:2n-6 13.5 16.4 1.6 0.9 4.6 3.5 7.818:3n-6 1.2 0.9 0.2 0.6 0.2 0.1 0.020:4n-6 0.1 1.3 3.2 0.7 0.7 0.5 17.522:5n-6 1.1 0.0 0.1 0.9 0.8 0.2 0.316:4n-3 0.1 6.5 0.1 0.0 0.0 1.0 0.018:3n-3 7.2 7.0 0.1 1.5 0.4 0.6 0.318:4n-3 15.8 2.9 0.0 2.4 0.3 1.4 0.320:5n-3 0.5 2.9 26.5 30.7 2.6 17.8 18.5

22:5n-3 0.0 0.0 0.0 0.0 0.45 1.3 2.122:6n-3 11.1 0.1 0.1 11.3 17.7 10.6 17.9Sum satsII 30.9 30.1 20.3 22.2 45.9 30.5 14.5Sum mono'" 17.1 26.0 32.2 24.2 23.4 28.6 17.3Sum n-6lv 16.1 18.6 5.7 3.8 6.7 4.8 27.8Sum n_3V 34.8 23.6 27.1 45.9 22.5 33.3 40.2n-3/n-6 2.16 1.28 4.75 12.11 3.38 7.04 1.44DHAlEPA 20.7 0.04 0.00 0.37 6.93 0.59 0.97EPAlAA 10.5 2.32 8.37 42.79 3.43 35.25 1.06

% Dry matter 13.7 33.1 27.8 34.2

% Lipid 17.5 13.7 20.8 17.0-I Fatty acids present in small quantities and not reported here included 16:2n-4, 16:3n-4 and 18:2n-4iI Saturated fatty acids might also include small quantities of 12:0, 15:0. 20:0. 22:0 and 24:0OHMonoenoic fatty acids also include 14:ln-5. 16:1n-9. 16:1n-5, 20:1n-11, 20:1n-9. 2-:1n-7, 22:1n-11and 22: 1n-9

Iv Sum of n-6 PUFA also included small amounts of 16:2n-6. 18:3n-6. 20:2n-6, 20:3n-6, 22:3n-6and22:4n-6

vSum of n-3 PUFA also included small amounts of 16:2n-3, 20:3n-3, 20:4n-3, 22:3n-3, 22:4n-4 and22:5n-3

richment samples and enriched rotifers were analyzed for dry matter and fatty acid

; Fatty acids present in small quantities and not reported here included 16:2n-4, 16:3n-4 and 18:2n-4ii Saturated fatty acids might also include small quantities of 12:0, 15:0, 20:0, 22:0 and 24:0iii Monoenoic fatty acids also include 14:1n-5, 16:1n-9, 16:1n-5, 20:1n-ll, 20:1n-9, 2-:1n-7, 22:1n-ll,22: In-9ivSum of n-6 PUFA also included small amounts of 16:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 22:3n-6, 22:4n-6and 22:5n-6

VSumof n-3 PUPA also included small amounts of 16:2n-3, 16:4n-3, 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3,22:3n-3, 22:4n-4 and 22:5n-3

composition. Dry matter was determined gravimetrically, as the percentage of ma-terial remaining after drying in an oven at 105°C overnight. Lipids were extractedwith chlorofonnlmethanol (2:1. v/v) by the method of Folch et al. (1957). Samplesof lipid (approximately 1 mg) were transmethylated by incubation with 1 ml oftoluene and 2 ml 7% BF3 in methanol at 50°C in a TeflonTM-linedscrew cap 10ml glass test tube for 16 hours (a modification of the method of Morrison and Smith(1964)). The fatty acid methyl esters were separated and analysed using a Varian3400 model gas liquid chromatograph as described by Nanton and Castell (1998),after purification of the fatty acid methyl esters on Silica Gel G high performancethin layer plates (HPTLC) using hexane:diethyl ether:acetic acid (90:10:1 v/v/v)solvent. Percent survival of the larvae was determined at 41 dph. Statistical analy-sis was performed using SYSTAT 10 (ANOVA and Tukeys HSD).

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Table 2. Fatty acid composition (percentage of total fatty acids) in rotifers cold enriched for 16 hr with

each enrichment emulsion or mixed algae. (Values are means:!: standard deviations for three replicatesample analysis).-Fatty Acid; Mixed algae DHAlEPAlAA ICES 30/0.6/C ICES 30/4/C

14:0 1.3 :!:2.2 1.4 :!:0.5 4.5 :!: 1.9 4.8 :!: 1.1

16:0 14.8 :!:2.1 12.4 :!:0.7 20.3 :!: 3.0 23.9 :!:4.9

18:0 3.9 :!: 1.6 3.7:!: 1.3 3.0:!: 0.6 4.3:!: 0.9

16:ln-7 3.1 :!: 1.5 2.5 :!: 1.5 8.5 :!:0.3 4.4 :!: 1.1

18:ln-9 16.2:!: 4.2 10.3 :!: 1.2 12.6:!: 1.1 17.2:!: 1.4

18:ln-7 2.4:!: 0.8 2.1:!: 0.9 4.4:!: 0.1 1.6 :!: 1.2

18:2n-6 19.0:!: 1.9 9.8 :!: 1.9 6.1 :!:0.8 7.3 :!:2.1

20:4n-6 2.4 :!:0.1 9.9:!: 4.4 1.0 :!:0.3 1.4 :!:0.4

20:5n-3 4.9:!: 0.9 11.5:!: 0.5 13.3 :!: 1.1 3.9:!: 0.8

22:6n-3 3.8:!: 0.8 17.9:!: 0.5 11.7:!: 2.5 17.0:!: 2.0

Sum SatU 21.0 :!:2.2 18.5 :!: 1.9 28.6 :!:5.6 34.7 :!:8.1

Sum mono'u 30.8 :!: 3.7 19.3:!: 2.1 28.6 :!: 1.3 24.5 :!: 1.4

Sum n-6iv 24.0 :!:2.4 23.7 :!: 1.8 9.1 :!: 1.7 10.8 :!:2.7Sumn-3v 20.8 :!: 3.6 35.8 :!: 1.5 30.4 :!:5.3 23.8 :!:3.5

n-3/n-6 0.9:!: 0.2 1.5 :!:0.2 3.3 :!:0.0 2.3 :!:0.4

DHAlEPA 0.8:!: 0.2 1.6 :!:0.1 0.9 :!:0.1 4.5:!: 0.6

EPAlAA 2.1 :!:0.5 1.3 :!:0.5 13.1 :!:2.1 2.7:!: 0.2

1.5

0.0o 10

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...~Aigaem_DHA-0-- DHA:8"A-!r- DHA:8"A:AA!

20Days after Hatch

30 40

Figure 1. Mean:!: sem of dry weights of fish larvae fed rotifers enriched with different lipid emulsions

or mixed algae. Inset shows growth from day 0 to 16.

Results

Growth

At 16 dph, larvae fed the mixed-algae rotifers had the greatest dry weight (mean :I:sd; 0.22 :I:0.08 mg); followed by larvae fed DHA:EPA:AA rotifers (0.21 :I:0.07),larvae fed DHArotifersand larvaefed DHA:EPArotifers (both0.20 :I:0.08), how-ever, none of the mean dry weights after 16 or 41 d (Figure 1) were significantlydifferent (p < 0.05).

Survival

One larval tank fed mixed-algae rotifers, and two tanks fed DHA:EPA:AA rotifersexperienced 100% mortality by 41 dph. Survival in the other tanks ranged from 0.4to 11.9%. Because of the large differences in survival among replicates, there wereno significant differences in survival among the treatments. The survival rates(mean:l: sem) of larvae by treatment were: ICES 30/0.6/C (DHA:EPA) 5.9 :I:2.4%;mixed-algae, 4.7:1: 1.8%; ICES 30/4/C (DHA) 3.1:1: 1.1; and DHA:EPA:AA 3.1 :I:1.9%.

1.00>.s

0 16C

0.5

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A) 20"0'5. 15:::i

]j 10oI- 5'0?fl 0

B) 20"0'5. 15:::i

]j 10oI- 5'0?fl 0

C) 40"0'5. 30:::i

m 20'0

!: 10o

?fl 0

A, 0 Enrichment

'-1IIIRotifers ;---.-.-------.--..----iIs:JLarvae :

H.. _m...0______----

b b b

DHA DHA:EPAEnrichment Treatment

Figure2. % EFA(mean:!: sem) in enrichments. rotifers, and larvae (16 dph): A) AA, B) EPA and C)DHA. Different letters indicate significant differences among treatments. Neither the enrichments northe rotifers treated with DHA:EPA had enough replicates for statistical analysis.

Algae DHA:EPA:AA

Fatty acid composition

Although the enrichments did not significantly affect survival and growth of had-dock larvae, they did significantly affect the fatty acid composition of the rotifersand the larvae. No statistical analyses were done with the DHA:EPA enriched ro-tifers because only two replicate samples were available for analysis. The AA con-tent of the rotifers enriched with DHA:EPA:AA (9.9 :I: 4.4%) was significantlyhigher (p < 0.01) than the algae (2.4 :I:0.1), DHA:EPA (1.0 :I:0.3) or DHA (l.4 :I:0.4) enriched rotifers. The AA content of rotifers fed the low AA enrichments wasprobably retained from the algae they were fed prior to enrichment. All larvae hadAA levels higher than the rotifers they were fed, except those enriched withDHA:EPA:AA (Figure 2A).

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The mean percent of EPA in the rotifers enriched with DHA:EPA:AA was sig-nificantly higher (p < 0.001) than those enriched with mixed algae or DHA. Themean value for the EPA of DHA:EPA enriched rotifers was even higher than that ofthe DHA:EPA:AA enriched rotifers but statistical analysis was not done becauseonly two DHA:EPA enriched rotifer samples were analyzed. Larvae fed mixed-al-gae or DHA enriched rotifers were significantly lower (p < 0.005) in EPA than theDHA:EPA treatment (Figure 2B). Except for the DHA treatment, the percentagesof EPA decreased from enrichment to rotifer to larvae, suggesting that in 3 treat-ments, the dietary levels of EPA were in excess of the requirement.

The DHA content of the rotifer lipids in all treatments reflected that of the en-richment (Figure 2C). The mixed-algae rotifers had significantly lower levels ofDHA than those enriched with DHA or DHA:EPA:AA. The content of DHA in all

larvae remained high (greater than 35%) after 15 days of feeding and there was nosignificant effect of treatment.

By day 41, after larvae had been weaned to the ICES Standard Reference Wean-ing Diet, there were no significant differences (p > 0.05) in AA, EPA or DHA amongtreatment groups. There were interesting developmental stage differences in larvalfatty acid compositions which are reflected in the data for the larvae fed the mixedalgae enriched rotifers (Figure 3). Initially the larvae were high in EPA, which de-creased significantly by day 16 and remained low at 41 dph. The DHA was initiallyhigh and remained high after 16 days but decreased to about 25% by 41 dph. TheAA was initially low but increased after 16 days and remained high at 41 dph.

Discussion

Though the mean survival rates for the haddock larvae in this experiment may seenlow, they are consistent with the mean values ranging from 2.8 to 6.3% obtainedby Hamlin and Kling (2001) for haddock weaned unto micorparticulate diets from14 to 35 dph. In their second experiment Hamlin and Kling (2001) were able toimprove survival when haddock larvae were fed live food until 35 or 42 dph. Simi-larly their mean dry weights after 42 dph ranged from 1.4 to 2.2 mg, similar to therange of values found in the present experiment. None of the emulsion enrichmenttreated rotifer diets resulted in any significant advantage in growth or survivalcompared to the larvae fed rotifers reared only on mixed algae.

The n-3/n-6 ranged from 19.6 in newly hatched larvae to 3.7 and 3.9 in larvae at16 dph fed the mixed algae and DHA:EPA:AA rotifers, respectively. However, by41 dph, after the larvae had been weaned to the ICES Standard Reference WeaningDiet, the n-3/n-6 ratio had decreased to about 2.5 for larvae from all treatmentgroups. DHA dominated the HUFA of the newly hatched and 16 dph larvae rang-ing from 35 to 39% of the total fatty acids in spite of the fact that it was only 10 to17% of the dietary lipid fatty acids. Either there is a strong specific retention ofdietary DHA, or the larvae highly conserve the maternal DHA that was provided inthe egg yolk. The content of AA (0.9 :!:0.3%), EPA (10.0 :!:0.1%) and DHA (37.4

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Mixed Algae Rotifer Treatment

5

IiIlnitial :1J15 Day:.!=L4J. I?ay

40

35

11I30'C'uc(25~.f20ca(515....

~10

a

b

o20:4n-6 20:5n-3

Fatty Acid22:6n-3

Figure 3. Mean percentages of AA, EPA and DHA in mixed-algae rotifer fed larvae I, 16 and 41 dayspost hatch. Vertical bars are sem. Significant differences (p < 0.001) for each fatty acid are indicated bydifferent letters above the histogram.

::t:1.3%) in newly hatched larvae, as expected, were almost the same as levels inhaddock eggs (1.1 ::t:0.1%, 10.6 ::t:1.2%, and 35.3 ::t:2.0%, respectively). These eggvalues are the mean and standard deviations from analyses of 20 egg samples takenfrom the same haddock broodstock that supplied the eggs used for the present study(unpublished results). EPA levels decreased in all treatment groups during the first16 days of feeding, while the DHA remained the same or increased slightly. ThoughDHA dominated as the major lillFA, the levels of AA in all treatment groups in-creased compared with the initial percentage in newly hatched larval lipids (0.9 ::t:0.3). Since the AA levels in the larvae were higher than the level in rotifer lipids(except for larvae fed the DHA:EPA:AA enriched rotifers), and AA was higher thanin newly hatched larvae, it is probable that this fatty acid is also important in earlylarval development. We speculate that a level between 3 and 5% of the total fattyacids in the diet will prove to be optimal, provided that requirements for DHA andEPA are satisfied. Sargent et al. (1999) have stated that both the amount and pro-portions of DHA, EPA and AA are important in marine fish nutrition and suggestedthat the optimum ratios may vary with specific species but would be in the range of10:5:1 for DHA:EPA:AA. We speculate that an increase or decrease in the propor-tion of one of these lillFA in the larvae relative to the live food would indicate a

deficiency or excess of that particular lillFA, respectively. Based upon that hypo-thesis, from our results, we suggest that the optimal ratios of DHA:EPA:AA in thediet would be 10:1:1 for larval haddock. However, since the larvae can obviously

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specifically retain DHA and AA at higher concentrations than provided in the diet,the required levels for these fatty acids in the diet might be lower. In the case ofour study it appears that rotifers grown on a mixed algae diet of lsochrysis gal-hana, Tetraselmis suecia, Nannochloropsis sp. and Pavlova lutheria were able tosatisfy the EFA requirements of haddock larvae, since none of the enrichment treat-ments provided any significant improvement in growth or survival. The specificdietary requirements by larval haddock for each of these HUFA will only be deter-mined by starting with yeast fed rotifers that are deficient in these fatty acids andenriching these with various levels and ratios of the HUFA before feeding to had-dock larvae.

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