Aquaculture Intemationall1: 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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1 Aquaculture Intemationall1: 2003 Kluwer Academic Publishers. Printed in the Netherlands The effect of different HUFA enrichment emulsions on the nutritional value of rotifers (Brachionus plicatilis) fed to larval haddock (Melanogrammus aeglefinus) JOHN CASTELL1.*,TAMMYBLAIR1,STEVENNEIL', KENNETH HOWESI, SARAHMERCERI, JOHN REIDI, WILFREDYOUNG-LA!I, BRANDI GULLISON1,PHILIPPEDHERT2and PATRICKSORGELOOS2 lfisheries 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, 9000 Gent. Belgium; *Author for correspondence ( CasteIU@mar.dfo-mpo.gc.ca; phone: (506) ; fax: (506) ) Received 12 December 2001; accepled 6 March 2002 Key words: Arachidonic acid, Docosahexaenoic acid, Eicosapenlaenoic acid, Enrichment, Lipid emulsions, 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 of haddock 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 different treatments from days I to 16 post-hatch. From day 17 until 25 all treatment groups were fed rotifers reared on mixed algae and then weaned onto the International Council for Exploration of the SEA (ICES) Slandard Reference Weaning diet ( over a five day period. The experiment was lerminated on day 41 post-hatch. The enrichment treatments affeeled the fatty acid composition of the rotifers and correlated with the accumulation of these fatty acids in the haddock larvae. However, no significant differences in larval growth or survival to 40 days post hatch were deteeled, suggesting that all treatments provided the minimal essential fatty acid requirements 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 - InternationalCouncilfor the Explorationof the Sea, ppt-parts per thousand, sem -slandarderror of the mean Introduction Haddock (Melanogrammus aeglefinus) has been identified as a candidate species for commercial culture in Atlantic Canada (Litvak 1998; Henry 1997). The principal obstacle to haddock culture is poor survival (average -2%, range 0-33%) through the first-feeding larval stage, which requires live food organisms, to weaning and metamorphosis. The essential fatty acid (EFA) requirements of first-feed- VLlZ (VZW) VLAAMSINSTlTUUTVOOR DE ZEE FLANDERS MARINE INSTITUTE Oostende - Belgium ---

2 110 ing haddock larvae have not been identified, however, two other gadoids closely related to haddock do require live food organisms enriched with docosahexaenoic acid (DHA) (Gadus macrocephalus Takeuchi et al. (1994); Gadus morhua, Galloway et al. (1998)). Rotifers (Brachionus plicatilis), particularly when reared on yeast-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, arachidonic acid (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 different rotifer enrichments during the first l5d of larval haddock feeding on subsequent growth, survival and fatty acid composition. Materials and methods Rotifers (Brachionus plicatilis, Aquafarms FL, USA) were batch cultured in 1.4 m3 fiberglass tanks and fed a mixed algal diet of lsochrysis galbana, Tetraselmis suecia, Pavlova lutheria and Nannochloropsis sp. in seawater (24 0c) at rotifers.ml-' under constant light. Harvested rotifers were enriched in 25 L glass carboys 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 the four species mentioned above. The fatty acid composition of algal lipids and enrichment emulsions are presented in Table 1. More information on the production and composition of the ICES enrichment emulsions can be found on the University of Ghent web site ( Rotifers, with enrichment media or mixed algae, were acclimated to larval culture temperature (6 0c) over a period of approximately 16 h prior to being fed to the larvae. The lipid composition 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 C in 250 L upwelling conical-bottom incubators. The broodstock haddock had been maintained at the Department of Fisheries and Oceans Biological Station, St. Andrews, New Brunswick on a diet of herring, squid and shrimp supplemented with vitamins, Corey Feed Mills dry marine feed and moist feed. Hatched larvae were transferred to 20x50-1 black plastic tanks (5 tanks/treatment) and stocked at 20 larvae.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 water from day 1 to day 30. Water temperature was 6 C initially and increased gradually to 12 C over the course of the experiment. Larvae utilized their yolksac reserves and began exogenous feeding on day-l post hatching. The enriched rotifer treatments were fed to the larvae 3 times per day (9, 15, 20 h, to maintain a concentration of 5 rotifers per ml) from 1 dph (days post hatch) to 16 dph, then all larvae were fed mixed-algae rotifers until weaning onto ICES Standard Reference Weaning Diet (Coutteau et al. 1995) at 25 dph. Larvae samples at 16 and 41 dph, en-

3 III Table 1. Fatty acid composition (percentage of total fatty acids) of principal fatty acids present in enrichment emulsionsand algal species.(valuesare meansfor three replicatesampleanalysis). T-Iso Tetraselmis Nanno. Pavlovalutheri 30/4/C 30/0.6/C DHAlEPAlAA Fatty acid! % % % % (%) (%) (%) 12: : : : :ln :ln :ln :ln :1n :2n :3n :4n :5n :4n :3n :4n :5n :5n :6n Sum satsii Sum mono'" Sum n-6lv Sum n_3v n-3/n DHAlEPA EPAlAA % Dry matter % Lipid I Fatty acids present in small quantities and not reported here included 16:2n-4, 16:3n-4 and 18:2n-4 ii Saturated fatty acids might also include small quantities of 12:0, 15:0. 20:0. 22:0 and 24:0 OHMonoenoic fatty acids also include 14:ln-5. 16:1n-9. 16:1n-5, 20:1n-11, 20:1n-9. 2-:1n-7, 22:1n-11 and 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-6and 22:4n-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 and 22:5n-3 richment samples and enriched rotifers were analyzed for dry matter and fatty acid --

4 112 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 replicate sample analysis). - Fatty Acid; Mixed algae DHAlEPAlAA ICES 30/0.6/C ICES 30/4/C 14:0 1.3 :!: :!: :!: :!: : :!: :!: :!: :!:4.9 18:0 3.9 :!: :!: :!: :!: :ln :!: :!: :!: :!: :ln :!: :!: :!: :!: :ln-7 2.4:!: :!: :!: :!: :2n :!: :!: :!: :!:2.1 20:4n :!: :!: :!: :!:0.4 20:5n-3 4.9:!: :!: :!: :!: :6n-3 3.8:!: :!: :!: :!: 2.0 Sum SatU 21.0 :!: :!: :!: :!:8.1 Sum mono'u 30.8 :!: :!: :!: :!: 1.4 Sum n-6iv 24.0 :!: :!: :!: :!:2.7 Sumn-3v 20.8 :!: :!: :!: :!:3.5 n-3/n-6 0.9:!: :!: :!: :!:0.4 DHAlEPA 0.8:!: :!: :!: :!: 0.6 EPAlAA 2.1 :!: :!: :!: :!: 0.2 ; Fatty acids present in small quantities and not reported here included 16:2n-4, 16:3n-4 and 18:2n-4 ii Saturated fatty acids might also include small quantities of 12:0, 15:0, 20:0, 22:0 and 24:0 iii 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-9 ivsum 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-6 and 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 material remaining after drying in an oven at 105 C overnight. Lipids were extracted with chlorofonnlmethanol (2:1. v/v) by the method of Folch et al. (1957). Samples of lipid (approximately 1 mg) were transmethylated by incubation with 1 ml of toluene and 2 ml 7% BF3 in methanol at 50 C in a TeflonTM-lined screw cap 10 ml 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 Varian 3400 model gas liquid chromatograph as described by Nanton and Castell (1998), after purification of the fatty acid methyl esters on Silica Gel G high performance thin 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 analysis was performed using SYSTAT 10 (ANOVA and Tukeys HSD).

5 >.s C ~Aigaem _DHA -0-- DHA:8"A -!r- DHA:8"A:AA! 0.0 o Days after Hatch 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), however, none of the mean dry weights after 16 or 41 d (Figure 1) were significantly different (p < 0.05). Survival One larval tank fed mixed-algae rotifers, and two tanks fed DHA:EPA:AA rotifers experienced 100% mortality by 41 dph. Survival in the other tanks ranged from 0.4 to 11.9%. Because of the large differences in survival among replicates, there were no 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%.

6 114 A) 20 "0 '5. :::i 15 ]j o 10 I- '0 5?fl 0 B) 20 "0 '5. 15 :::i ]j 10 o I- 5 '0?fl 0 A, 0 Enrichment '-1IIIRotifers ; iis:jlarvae : H.. b b b _m... C) 40 "0 '5. 30 :::i m 20 '0!: 10 o?fl 0 Algae DHA DHA:EPA Enrichment Treatment DHA:EPA:AA 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 nor the rotifers treated with DHA:EPA had enough replicates for statistical analysis. Fatty acid composition Although the enrichments did not significantly affect survival and growth of haddock larvae, they did significantly affect the fatty acid composition of the rotifers and the larvae. No statistical analyses were done with the DHA:EPA enriched rotifers because only two replicate samples were available for analysis. The AA content of the rotifers enriched with DHA:EPA:AA (9.9 :I: 4.4%) was significantly higher (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 was probably retained from the algae they were fed prior to enrichment. All larvae had AA levels higher than the rotifers they were fed, except those enriched with DHA:EPA:AA (Figure 2A).

7 115 The mean percent of EPA in the rotifers enriched with DHA:EPA:AA was significantly higher (p < 0.001) than those enriched with mixed algae or DHA. The mean value for the EPA of DHA:EPA enriched rotifers was even higher than that of the DHA:EPA:AA enriched rotifers but statistical analysis was not done because only two DHA:EPA enriched rotifer samples were analyzed. Larvae fed mixed-algae or DHA enriched rotifers were significantly lower (p < 0.005) in EPA than the DHA:EPA treatment (Figure 2B). Except for the DHA treatment, the percentages of EPA decreased from enrichment to rotifer to larvae, suggesting that in 3 treatments, 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 enrichment (Figure 2C). The mixed-algae rotifers had significantly lower levels of DHA 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 no significant effect of treatment. By day 41, after larvae had been weaned to the ICES Standard Reference Weaning Diet, there were no significant differences (p > 0.05) in AA, EPA or DHA among treatment groups. There were interesting developmental stage differences in larval fatty acid compositions which are reflected in the data for the larvae fed the mixed algae enriched rotifers (Figure 3). Initially the larvae were high in EPA, which decreased significantly by day 16 and remained low at 41 dph. The DHA was initially high and remained high after 16 days but decreased to about 25% by 41 dph. The AA 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 seen low, they are consistent with the mean values ranging from 2.8 to 6.3% obtained by Hamlin and Kling (2001) for haddock weaned unto micorparticulate diets from 14 to 35 dph. In their second experiment Hamlin and Kling (2001) were able to improve survival when haddock larvae were fed live food until 35 or 42 dph. Similarly their mean dry weights after 42 dph ranged from 1.4 to 2.2 mg, similar to the range of values found in the present experiment. None of the emulsion enrichment treated rotifer diets resulted in any significant advantage in growth or survival compared 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 at 16 dph fed the mixed algae and DHA:EPA:AA rotifers, respectively. However, by 41 dph, after the larvae had been weaned to the ICES Standard Reference Weaning Diet, the n-3/n-6 ratio had decreased to about 2.5 for larvae from all treatment groups. DHA dominated the HUFA of the newly hatched and 16 dph larvae ranging from 35 to 39% of the total fatty acids in spite of the fact that it was only 10 to 17% of the dietary lipid fatty acids. Either there is a strong specific retention of dietary DHA, or the larvae highly conserve the maternal DHA that was provided in the egg yolk. The content of AA (0.9 :!:0.3%), EPA (10.0 :!:0.1%) and DHA (37.4

8 Mixed Algae Rotifer Treatment 35 11I30 'C 'u c(25 ~.f20 ca ( ~10 5 IiIlnitial : 1J15 Day:.!=L4J. I?ay b a o 20:4n-6 20:5n-3 Fatty Acid 22:6n-3 Figure 3. Mean percentages of AA, EPA and DHA in mixed-algae rotifer fed larvae I, 16 and 41 days post hatch. Vertical bars are sem. Significant differences (p < 0.001) for each fatty acid are indicated by different letters above the histogram. ::t:1.3%) in newly hatched larvae, as expected, were almost the same as levels in haddock eggs (1.1 ::t:0.1%, 10.6 ::t:1.2%, and 35.3 ::t:2.0%, respectively). These egg values are the mean and standard deviations from analyses of 20 egg samples taken from the same haddock broodstock that supplied the eggs used for the present study (unpublished results). EPA levels decreased in all treatment groups during the first 16 days of feeding, while the DHA remained the same or increased slightly. Though DHA dominated as the major lillfa, the levels of AA in all treatment groups increased 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 than in newly hatched larvae, it is probable that this fatty acid is also important in early larval development. We speculate that a level between 3 and 5% of the total fatty acids in the diet will prove to be optimal, provided that requirements for DHA and EPA are satisfied. Sargent et al. (1999) have stated that both the amount and proportions of DHA, EPA and AA are important in marine fish nutrition and suggested that the optimum ratios may vary with specific species but would be in the range of 10:5:1 for DHA:EPA:AA. We speculate that an increase or decrease in the proportion 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 hypothesis, from our results, we suggest that the optimal ratios of DHA:EPA:AA in the diet would be 10:1:1 for larval haddock. However, since the larvae can obviously

9 117 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 of our study it appears that rotifers grown on a mixed algae diet of lsochrysis galhana, Tetraselmis suecia, Nannochloropsis sp. and Pavlova lutheria were able to satisfy the EFA requirements of haddock larvae, since none of the enrichment treatments provided any significant improvement in growth or survival. The specific dietary requirements by larval haddock for each of these HUFA will only be determined by starting with yeast fed rotifers that are deficient in these fatty acids and enriching these with various levels and ratios of the HUFA before feeding to haddock larvae. References Coutteau P.. Van Stappen G.. and Sorgeloos P A standard experimental diet for the study of fatty acid requirements of weaning and first ongrowing stages of the European sea bass (Dicentrarchus labrax L.): selection of the basal diet. ICES Marine Science Symposia 201: Folch J., Lees M. and Sloane-Stanley G.H A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226: Galloway T.E. Kryvi H.. Hoehne K. and Kj!<1rsvik E Muscle growth in cod larvae in response to dietary HUFA ratios. In: VIII International Symposium on Nutrition and Feeding of fish. Recent Advances in Finfish & Crustacean Nutrition. June Las Palmas de Gran Canaria, Spain. p Hamlin H.J. and Kling L.J The culture and early weaning of larval haddock (Melanogrammus aeglefinus) using a microparticulate diet. Aquaculture 201: Henry R Overview of recent developments in haddock culture in New Brunswick. Aquaculture Association of Canada Special Publication 2: ICES Report of the Working Group on Marine Fish Culture for the International Council for the Exploration of the Sea. Murcia, Spain June ICES CM 1997 I F:7. Koven W., Barr Y.. Lutzky S., Ben-Atia I.. Weiss R.. Harel M. et al The effect of dietary arachidonic acid (20:4n-6) on growth. survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae. Aquaculture 193: Litvak M The development of haddock culture in Atlantic Canada. Bulletin of the Aquaculture Association of Canada 98: Morrison W.R. and Smith L.M Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride methanol. Journal of Lipid Research 6: Nanton O.A. and Castell J.O The effects of dietary fatty acids on the fatty acid composition of the harpacticoid copepod, Tisbe sp., for use as a live food for marine fish larvae. Aquaculture 163: Rainuzzo J.R.. Reitan KJ. and Olsen Y The significance of lipids at early stages of marine fish: a review. Aquaculture 155: Rodnguez c.. Perez J.A.. Izquierdo M.S.. Cejas J.R., Bolanos A. and Lorenzo A Improvement of the nutritional value of rotifers by varying the type and concentration of oil and the enrichment period. Aquaculture 147: Sargent J.. McEvoy L., Estevez A.. Bell G., Bell M.. Henderson J. et al Lipid nutrition of marine fish during early development: current status and future directions. Aquaculture 179: Takeuchi T.. Zheng E. Yosheda K.. Hirokawa J. and Watanabe T Nutritive value of OHA-enriched rotifer for larval cod. Nippon Suisan Gakkaishi 60: