Influence of size and nutritional value of Artemia franciscana on growth and quality of halibut larvae (Hippoglossus hippoglossus) during the live feed period

Ž .Aquaculture 179 1999 475–487

Influence of size and nutritional value of Artemiafranciscana on growth and quality of halibut larvaež /Hippoglossus hippoglossus during the live feed

period

Atle Ivar Olsen a,), Yngve Attramadal b,1, Arne Jensen c,Yngvar Olsen c

a SINTEF Fisheries and AquaculturerN-7465 Trondheim, Norwayb Stolt Sea Farm Øye, N-4484 Øyestranda, Norway

c Norwegian UniÕersity of Science and Technology, N-7491 Trondheim, Norway

Abstract

Ž .In a first feeding experiment, halibut larvae were offered either short term enriched ST 1-dayŽold Artemia franciscana or A. franciscana of successively increasing size ST, 2-, 3- and 4-day

. Žold juveniles , from day 0 to day 60. No differences, either in growth approximately 6.5% daily. Ž .weight increase, DWI or in survival ca. 25% between the two treatments were observed, but

both were satisfactory. However, feeding increasing sizes of A. franciscana increased the numberof completely pigmented and metamorphosed larvae from 4 to 20%. Proteinrlipid ratio in the feedseems to be important for pigmentation and metamorphosis, provided the levels of essential fatty

Ž .acids esp. DHA are sufficient. Juvenile A. franciscana may therefore be a better live feed thanST A. franciscana for halibut larvae. The fact that a relatively high load of bacteria wasintroduced by 4-day old A. franciscana, may have had a negative influence on the growth of thelarvae. Tests indicated that a great part of the bacteria exhibited hemolytic activity, and many ofthe bacteria were apparently Vibrios. Strict microbial control in the production of live feed is verylikely just as important as control of the nutritional composition of the larval feed. This is achallenge for further work. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Artemia franciscana; Halibut larvae; Proteinrlipid ratio; Larval quality

) Corresponding author. Tel.: q47-7359-6387; Fax: q47-7359-6363; E-mail: [email protected] Present address: NMC Norwegian Halibut, Ryumsjøen, 7900 Rørvik, Norway.

0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0044-8486 99 00181-7

( )A.I. Olsen et al.rAquaculture 179 1999 475–487476

1. Introduction

In Norway, halibut fry have been produced either with harvested zooplankton,Ž .cultivated live feed such as rotifers Brachionus plicatilis and brine shrimp Artemia

Žsp., or on mixtures of harvested and cultivated live feed Olsen, 1997; van der Meeren.and Naas, 1997; Olsen et al., 1999 . Cultivation of live feed can provide sufficient

quantities at all times of the year and allows better control of nutritional and microbialŽ .quality bacteria, parasites, etc. . This is essential for a continuous industrial production

of fry. However, there are developmental problems associated with the use of cultivatedŽ .live feed, especially with regard to pigmentation and metamorphosis Næss et al., 1995 .

Ž .An earlier report by Holmefjord et al. 1989 also linked Artemia sp. with poor growth.This may be attributed to inferior quality of the feed because the experiment was

Ž .performed before proper attention was paid to the quality of the live feed Olsen, 1997 .In more recent experiments quite similar growth rates have been obtained when halibut

Žlarvae were offered either Artemia franciscana or collected zooplankton Reitan et al.,.1993a; Næss et al., 1995 .

The growth rate of larvae may also be effected by the size of the feed. The sizedistribution of prey organisms appears to be more important than the total number of

Žprey per unit of volume, for some marine larvae. This applies both for growth Frankand Leggett, 1986; Crowder et al., 1987; Mills et al., 1989a,b; Miller et al., 1990;

. ŽBremigan and Stein, 1994 as well as for survival Ware, 1975, 1977; Frank and.Leggett, 1986; Bremigan and Stein, 1994 . Experiments have shown that the halibut

larvae have no significant preference for B. plicatilis over 1-day old Artemia sp. naupliiŽthe first two days after first feeding is initiated Gulbrandsen, 1993; own unpublished

.results . After the first two days they prefer small A. franciscana when offered a widerange of size classes of A. franciscana, until they reach the size of about 3 mg dry

Ž .weight about 20 days after first feeding . The larvae then select for longer A.Ž .franciscana individuals )1.20 mm , although they consume a wide range of sizes all

Ž .through the live feed period own unpublished results . When wild zooplankton is used,halibut larvae are normally offered a wider size range giving the larva more to choosefrom. This may give better growth relative to larvae feeding on small A. franciscananauplii throughout the live feed period. Increasing the sizes of A. franciscana offeredduring this period would most likely give better growth.

In order to provide the larvae with the required amounts of essential fatty acidsŽ .Watanabe, 1982 , Artemia sp. nauplii are normally enriched with lipids before beingused as feed. This inevitably leads to a high fat content and a concomitant lowproteinrlipid ratio, a very different composition from that of zooplankton believed to be

Ž .natural food for halibut larvae Evjemo and Olsen, 1997 . This may be the reason forsome of the problems encountered in the use of cultivated live feed for halibut larvae.

The present study was conducted to evaluate the effect of feeding increasing sizeclasses of A. franciscana during the live feed period of halibut fry, thereby increasingboth prey size and proteinrlipid ratio of the diet. Growth, survival and quality werecompared with the performance of a control group which received short term enrichedŽ .ST 1-day old A. franciscana nauplii during the whole live feed period.

( )A.I. Olsen et al.rAquaculture 179 1999 475–487 477

2. Materials and methods

2.1. Experimental design

The experiment was conducted in four identical tanks, and the larvae in the twotreatments were fed according to the following feeding regimes:

Two tanks: day 1–day 60 1-day old ST A. franciscanaTwo tanks: day 1–day 14 1-day old ST A. franciscana

day 14–day 20 2-day old A. franciscanaday 20–day 25 3-day old A. franciscanaday 25–day 60 4-day old A. franciscana

y1 Ž .Larval density at the start was 1–2 larvae l , and the temperature was 128C 11–13y1 Ž y1 .throughout the experiment. The flow rate was 0.7 l min dilution rate 0.7 day at

y1 Ž y1 .start, increasing to about 1.5 l min dilution rate 1.4 day at day 7, and 3.0–3.6 ly1 Ž y1 .min dilution rate 2.9–3.5 day from day 28 and onwards. Algae were added

during the experimental period once a day to give a density of 1–1.5 mg C ly1 just afterthe addition, mostly Tetraselmis sp. and sometimes Isochrysis galbana, but always thesame algae to all tanks.

Careful aeration was applied in the centre of the tank to keep A. franciscana evenlyŽ 3.distributed throughout the tank volume. The tank volume 1.5 m had a diameter of 144

Ž .cm at the top, and had a cover with a circular opening Ds104 cm in the middle. Thiswas necessary to ensure an even distribution of the larvae and to avoid wall-nosingbecause of light reflections.

The density of ST A. franciscana was 100–300 animals ly1 in the morning. Thetanks that were given juvenile A. franciscana from day 14 were stocked with at least

y1 Ž .100 A. franciscana l . The larvae were fed three times a day, morning 0800 h ,Ž . Ž .afternoon 1500 h and in the evening 2100 h .

The tanks were cleaned every two days and dead larvae removed and counted, exceptfor a period between day 23–30 when the tanks were cleaned every day. After samplingon day 60, the larvae were pooled in one tank per treatment and subsequently weaned toa dry feed diet.

2.2. Production of different size classes of A. franciscana

ŽThe A. franciscana EG quality, Great Salt Lake, Utah, USA, INVE Aquaculture,.Belgium were enriched for 12 h after hatching to produce ST A. franciscana. The

y1 Žfollowing procedure was used: 10–12 h with 0.15 g l DHA Selco INVE Aquacul-.ture, Belgium at 26–288C, followed by rinsing and used as feed 24 h after hatching.

The cysts were not decapsulated before hatching.ŽJuvenile A. franciscana were fed micronized fish meal Norwegian Herring Oil and

.Meal Industry Research Institute, Bergen, Norway and lipids mixed with water before


Žfeeding. The lipid source was either DHA Selco or fish oil from Pronova EPAX 1040.TG, Pronova Biocare, Sandefjord, Norway . Pronova oil was used only for the produc-

tion of 3-day old A. franciscana. Juvenile A. franciscana were produced under thefollowing conditions: 27–288C, ca. 20 ind. mly1 and two feedings a day up to a feedinglevel of 0.09 g fish meal ly1 and either 0.036 g DHA Selco ly1 or 0.054 g Pronova oilly1. To maintain the feeding level in the culture, the optical density of the culture waterwas measured at 750 nm after the first addition of feed. At later feedings, the opticaldensity of the culture water in the tanks was brought back to the previously measuredlevel by addition of feed. Before used as live feed, juvenile A. franciscana were fed

Ž .algae Tetraselmis sp. for 4 h according to a procedure which was developed for 2-dayŽ y1old A. franciscana 100 ind. ml , 208C, initial concentration of alga was about 7 mg C

y1 .l .

2.3. Bacteriological samples

Samples for determination of the number and composition of bacteria in the gut ofthe halibut larvae were taken on days 16 and 45. For A. franciscana bacterial samples

Ž .were taken from the outlet of the tanks on days 17 ST and 2-day old A. franciscanaŽ .and 44 ST and 4-day old A. franciscana . Bacteria associated with the larvae and the

Ž .A. franciscana were characterised and quantified as colony forming units CFU usingthree different types of agar. The total number of culturable bacteria was estimated by

Žplating on M65 seawater agar 0.5 g peptone, 0.5 g tryptone, 0.5 g yeast extract, 15 g.agar, 200 ml distilled water, and 800 ml sterile seawater, Skjermo and Vadstein, 1993 .

Ž .To estimate the number of Vibrio spp., TCBS Cholera Agar Oxoid was used, and theŽ .number of bacteria with hemolytic activity was estimated using blood agar base Merck

Ž y1 . Ž y1 .added calf blood 50 ml l medium and NaCl 5 g l . The larvae were rinsed inŽ . Ž .sterile seawater SSW , surface disinfected with benzalconiumchloride 0.1% for 60 s,

Žthen rinsed twice for 2=20 s in SSW and finally homogenized in SSW Skjermo and.Vadstein, 1993 . The homogenates were serially diluted in SSW and plated out in

duplicate. Thus, the samples from the larvae represented bacterial content and composi-tion in the gut, whereas the samples of A. franciscana collected from the outlet of thefish tanks were expected to characterise the feed the larvae had ingested. The A.

Ž .franciscana were rinsed in SSW in a plankton net, a known number 20–30 individualswere homogenized and then serially diluted and plated out in duplicate as describedabove. The plates were incubated in darkness at 208C and counted after 2 and 7 days.

2.4. Chemical analyses

Larval samples for lipid and fatty acid analysis were taken on days 0, 13, 20, 27 and60. Samples were taken of the different size classes of A. franciscana used during theexperiment; four samples of ST A. franciscana, five samples of 2-day old juvenile A.franciscana, and two samples for 3- and 4-day old A. franciscana, respectively. Theanimals were rinsed in fresh water, stored in either liquid nitrogen or at y808C in anitrogen atmosphere. The analyses were carried out as described by Rainuzzo et al.Ž .1992 .


2.5. Samples and measurements of A. franciscana

Ž .The dry weight DW of A. franciscana was determined by rinsing the animals infresh water to remove seawater and then placing 3=10 animals in pre-weighed tincapsules and drying them at 608C for 48 h before weighing. The length was measured at25 = magnification in a stereomicroscope. For each different size class of A. francis-cana, 12 individuals were measured at 1–4 different times during the experimental

Ž . Ž .period, and all length data were pooled. The carbon C and nitrogen N contents of theŽ .A. franciscana were obtained by CN analysis ns4 of aliquots of the freeze-dried

samples taken for lipid analysis, using a Carlo Erba Element Analyzer model 1106 withŽ .acetanilide C H NO as standard.8 9

2.6. Measurement of DW of larÕae and estimation of surÕiÕal

Ž y1Larval dry weights were determined by anaesthetising metomidate, 5 mg l. Žseawater , rinsing in freshwater and placing individual larva a total of 12 from each

.tank at each sampling time in pre-weighed tin capsules, drying for 48 h at 608C andthen weighing. This was done for at days 0, 6, 13, 20, 27, 34 and 41. An established

Ž .ratio between DW and wet weight WW was used to estimate the DW on days 51 and60. Some 23–29 larvae from each of the four tanks were sampled on day 51, and62–102 larvae on day 60. To obtain the ratio between DW and WW, 15 larvae were

Ž .sampled on day 45, anaesthetised, dried carefully in a net by placing on a paper towel ,weighed, placed in a pre-weighed tin capsule and treated as previously described.

Survival were estimated on basis of the number of survivors on day 60 corrected forlarvae removed for DW determination, lipid analysis and bacterial samples, and deadlarva removed from the bottom of the tanks during cleaning. Larvae with jaw deformi-ties, which comprised 30% of the total larvae on day 0, were excluded from thecalculations.

2.7. Quality eÕaluation of the halibut fry

Ž .A top quality larva is described as having: a completely pigmented upper ocularside, no pigmentation on the lower side, complete eye migration and no visibledeformities. On day 71, some of the biggest larvae were sorted out, and 64 from thegroup fed juvenile A. franciscana and 59 from the group fed ST A. franciscana wereexamined. On day 89, the remaining larvae were evaluated by checking 100 larvae fromeach treatment.

2.8. Statistics

The statistical test applied was a Student’s t-test, after ensuring normality of data byŽ .plotting residuals on a normal probability plot Minitab 11.2 . The differences in quality


between the two treatments were tested for significance with a chi-square test usingMinitab 11.2. The results are given as mean"S.D.

3. Results

3.1. Characterisation of the feed

Biometric data and elemental composition for the different types of A. franciscanaused in the experiment are given in Table 1. The CrN ratio was lower for all juvenile

Ž .A. franciscana than for the ST A. franciscana nauplii P-0.05 , reflecting the lowerŽ . Žproteinrlipid ratio of the latter Table 2 . The sums of lipid and protein calculated as

. Ž .N=6.25 of the animals Table 2 were almost equal in all size classes.The fatty acid contents presented in Fig. 1 showed that ST and 3-day old A.

Ž .franciscana had similar P)0.05 and the highest DHA content. Juvenile 2- and 4-dayold A. franciscana, which were given DHA Selco as lipid source, had a much lower

Ž .DHA content P-0.05 , and there were no difference in the DHA content betweenŽ .them P)0.05 . The content of DHA in the live feed was 18.8, 3.8, 17.5 and 2.6 mg g

DWy1, for ST, 2-, 3- and 4-day old A. franciscana, respectively. Fig. 1a also revealsŽ .that ST A. franciscana had much higher content of lipids 26.7% than juvenile A.

franciscana which showed 15.6, 18.7 and 10.8% for 2-, 3- and 4-day old A. francis-Ž .cana, respectively. The lipid contents were significantly different P-0.05 between

the four different size classes of A. franciscana. The percentage of DHA of the totalfatty acids in the live feed was 11.4, 3.8, 17.5 and 4.8%, for ST, 2-, 3- and 4-day old A.franciscana, respectively. The DHArEPA ratios for the same animals were 1.07, 0.27,1.08 and 0.31, respectively.

3.2. Growth and surÕiÕal of larÕae

Growth and survival of the halibut larvae are shown in Fig. 2. The growth of thelarvae from both treatments was similar through the whole period. The weight increased

Ž .exponentially throughout the period day 6 to day 60, the daily weight increase DWIŽ .was 6.5%, with no significant difference in growth between the two treatments P)0.5 .

Table 1Ž . Ž . ŽLength mm, "S.D. and range, ns12–48 dry weight mgrindividual, "S.D., ns3 , and content % of

.DW, "S.D., ns4 and ratio of C and N in A. franciscana used in the experiment

A. franciscana Length Range DW % C % N CrN

ST nauplii 0.84"0.07 0.71–0.98 2.41"0.17 49.7"0.2 8.9"0.1 5.6"0.082-day old 1.09"0.07 0.98–1.22 3.36"0.41 45.6"0.2 10.5"0.2 4.3"0.063-day old 1.30"0.11 1.14–1.56 5.01"0.41 46.9"0.2 10.2"0.3 4.6"0.124-day old 1.42"0.16 1.18–1.88 6.68"0.44 43.6"0.6 11.0"0.2 3.9"0.09

The differences between each of the different A. franciscana in length, DW, % C, % N and CrN ratio wereŽ .all significant P -0.05 .


Table 2Ž . Ž .Protein P s N =6.25, ns4 and lipid L, ns2–5 content of the different size classes of A. franciscana

A. franciscana % Lipid % Protein Sum Lq P Ratio PrL

ST nauplii 26.7"1.4 55.9"0.9 82.6"1.7 2.09"0.122-day old 15.6"1.4 65.9"1.2 81.5"1.8 4.23"0.393-day old 18.6"0.8 63.6"1.6 82.2"1.8 3.41"0.174-day old 10.8"0.9 69.0"1.2 79.8"1.5 6.38"0.52

The differences between each of the different A. franciscana in % lipid and % protein were significantŽ .P -0.05 .

Identical survival values of 25% for the two treatments were observed on day 60Ž .Ps0.94 . The relationship between DW and WW on day 45 is shown in Fig. 3. The

Ž .coefficient of correlation was high 0.99 .Fig. 4a shows the lipid and fatty acid contents in the larvae throughout the

Žexperiment, and Fig. 4b shows the fatty acid profiles. The content of DHA mg g

Ž . Ž y1 . Ž . Ž .Fig. 1. a Lipid and fatty acid content mg g DW and b the fatty acid composition % of total fatty acidsin the ST-, 2-, 3-, and 4-day old A. franciscana used as feed in the experiment. DHA S means that DHA Selco

Ž .was used as lipid source, while Pronova means that Pronova oil EPAX 1040 TG was used as the lipid source.


Ž . Ž . Ž .Fig. 2. Growth a and survival b of halibut larvae from day 0 to day 60 "S.D. The larvae were fed ST A.franciscana and gradually increasing size classes of A. franciscana, respectively.

y1 .DW in the larvae was 28.6 on day 0 and 15.1 on day 13. It was higher in larvae fedŽST A. franciscana than in the larvae fed juvenile A. franciscana on day 20 11.1 vs.

. Ž . Ž . Ž8.6 and 60 11.1 vs. 5.9 , but not on day 27 7.1 vs. 7.8 The coefficient of variation.for the GC analysis of DHA is normally 0.045, range 0.015–0.12, Reitan et al., 1993b .

On day 60, the larvae fed increasing sizes of A. franciscana showed a lipid content of12.5% of DW, the same as on day 0, whereas the other group had 15.9%. Throughoutthe live feed period, the lipid content varied between 14.2 and 16.7%, but the group fedST A. franciscana had 1.2–1.3% higher lipid content at all sampling times, and the

ŽFig. 3. Relationship between DW and WW of halibut larvae on day 45 after initial first feeding valid between.2.5 and 35 mg DW, ns15 .


Ž . Ž y1 . Ž . Ž .Fig. 4. a Lipid and fatty acid content mg g DW and b the fatty acid composition % of total fatty acidsof the halibut larvae in the period day 0 to day 60. ST: Fed short term enriched A. franciscana nauplii. JUV:Fed juvenile A. franciscana after day 14.

Ž .difference was significant P-0.05 on days 27 and 60. The proportion of DHAŽ .decreased Fig. 4b from 44.3% of the total fatty acids on day 0 to 19.6% on day 13. In

larvae fed ST A. franciscana nauplii, the level of DHA was still quite high on day 20with 17.1% compared to 12.1% in the larvae fed juvenile A. franciscana for 6–7 days.At day 60, the numbers were 13.2 and 8.5%, respectively. At day 27, however, thediscrepancy had narrowed to 11.4 and 12.5%, respectively. The total content of DHAshowed the same pattern. The DHArEPA ratio in the larvae showed a similar patternwith 3.3 and 1.6 on days 0 and 13, respectively, 1.23 vs. 0.85 on day 20, 0.79 vs. 0.76on day 27 and 0.85 vs. 0.42 on day 60.


Table 3Changes in total CFU, and numbers of bacteria classified as Vibrios and hemolytic bacteria in larvae and on

Ž .A. franciscana in the tanks ns2–6

Feed-type Days after Total"S.D. Hemolytic"S.D. Vibrios"S.D.first-feeding

3 y1 a a a10 Larvae ST 16 49.3"33.3 9.4"2.7 8.9"0.33 y1 a b b10 Larvae Juveniles 16 37.5"5.9 21.7"3.6 45.7"16.33 y1 b c a,b10 Larvae ST 45 81.0"16.6 57.5"5.9 38.3"26.03 y1 c c b10 Larvae Juveniles 45 200"89.6 119"77.4 70.0"22.5

y1 a a aA. franciscana ST 17 300"141 50"71 150"49y1 a a aA. franciscana ST 44 191"73 20"14 35"28y1 a a,b aA. franciscana 2-day old 17 385"0 165"0 173"43y1 b b bA. franciscana 4-day old 44 1440"191 1144"561 808"243

Ž .Different subscript letters within a column indicate significant differences P -0.05 .Statistical tests are either run between A. franciscana or between larvae.

3.3. Microbiology

Ž . ŽGut contents of bacteria in larvae days 16 and 45 and on A. franciscana days 17.and 44 are given in Table 3. The differences observed on day 16 were small, with total

Ž . y1numbers averaging ca. 40 000 colony forming units CFU larvae . However, thepercentages of hemolytic bacteria and Vibrios in the gut of the larvae fed 2-day old A.

Ž .franciscana, were higher P-0.05 than in those fed only ST A. franciscana nauplii. Inthe larvae fed 2-day old A. franciscana, 58% of the bacteria exhibited hemolytic activitycompared to 19% in the group fed only ST A. franciscana. For Vibrios the numberswere 100 and 18% of the total numbers, respectively. The absolute differences weregreater between the two treatments on day 45. The larvae which were offered juvenile

Ž .A. franciscana had significantly higher total number of bacteria P-0.05 , around200 000 compared to 81 000 CFU larvaey1 on those fed only ST A. franciscana. Also,the number of hemolytic bacteria and the number of probable Vibrio bacteria were

Ž .higher not significant, Ps0.21 and Ps0.12, respectively in the gut of larvae fedjuvenile A. franciscana, than in the group fed only ST A. franciscana. The 4-day old A.

Table 4Quality evaluation on day 71 of the largest larvae, and on the remaining smaller larvae on day 89

Ž .Larvae Number Perfect % Complete Complete CompletercorrectŽ .evaluated pigmentation pigmentation metamorphosis %

Ž . Ž .% upper side %a a a aSmall fed juvenile 100 17 39 71 33

b b b aSmall fed ST 100 4.0 6.0 23 23a a c bLarge fed juvenile 64 28 33 42 75

b b b cLarge fed ST 59 3.4 8.5 15 47

Ž .Different subscript letters within a column indicate significant differences P -0.05 .Perfect implies complete pigmentation on upper side, no pigmentation on lower side and complete metamor-phosis.


Žfranciscana also contained more bacteria per unit weight calculated from Tables 1 and. Ž y1 .3 than ST A. franciscana nauplii 215 vs. 124 CFU mg DW , especially with regard

Ž y1 .to bacteria with hemolytic activity 171 vs. 21 CFU mgDW .

3.4. EÕaluation of pigmentation and metamorphosis

The results of the quality evaluation of the largest juvenile halibut larvae from eachŽ . Ž .group day 71 and of the remaining, small larvae day 89 are shown in Table 4. More

than 20% of the fish in the group fed juvenile A. franciscana were of top quality,compared to around 4% in the group fed ST A. franciscana nauplii.

4. Discussion

The brine shrimp A. franciscana has been instrumental in the many attempts toproduce an ideal live feed for larvae of marine fish. In this connection, the requirementfor highly unsaturated fatty acids typical of many cold water marine species has beenmet through enrichment techniques. The resulting high fat content has rendered A.

Ž .franciscana sub-optimal as food for fish larvae Conceicao, 1997 by reducing the˜relative content of protein. One of the goals of producing live feed, has been to mimicthe natural zooplankton believed to be the normal diet for the marine larvae in nature.Zooplankton typically have relatively low total lipid content, 9–12% of dry weight, ahigh amount of DHA, often as much as 40% of total fatty acids in cold water speciesŽ .Evjemo and Olsen, 1997 , and a high proteinrlipid ratio compared to cultivated livefeed.

The juvenile A. franciscana used in this experiment seemed to be more optimal thanthe ST A. franciscana with respect to the proteinrlipid ratio and also to the relativeDHA content, although the lipid and DHA content varied between the different sizeclasses of juvenile A. franciscana. When DHA Selco was used as a lipid source, thetotal amount of lipid fed to A. franciscana was reduced. This could be the explanationfor the lower DHA level in 2-day old juvenile A. franciscana as well as the low level in4-day old juvenile A. franciscana. In addition, other experiments have shown that theDHA level in juvenile A. franciscana is decreasing dramatically after 3 days of

Žcultivation, regardless of lipid source and feed level and concentration in the feed own.unpublished results .

In the present study, an increase in proteinrlipid ratio in A. franciscana used as firstfeed to halibut larvae had no measurable effect on growth and survival of larvae. Onepossible explanation is that the positive effects of a better live feed and protein supplywere counteracted by a simultaneous increase in bacterial load. On day 45 larvae that

Žwere fed juvenile A. franciscana had a higher number of bacteria 2.5 times totalnumber, four times the number of possible Vibrio bacteria, and two times the number of

.bacteria with hemolytic activity than the larvae fed ST A. franciscana. This differencemay be attributed to differences between the flora on 4-day old A. franciscana and onST A. franciscana nauplii. Introduction of algae to fish tanks seems to reduce the

Ž .number of bacteria colonising halibut larvae Skjermo and Vadstein, 1993 , and aŽ .method to improve the microbial quality numbers and composition of 2-day old A.

franciscana by incubation with algae prior to the use as feed has shown promising


Ž .results own unpublished results . We used this method in the production of all juvenileA. franciscana in this experiment, but the method apparently has to be improved for useon 3- and 4-day old juvenile A. franciscana.

It was established that the quality of the larvae, expressed as percentage of top qualityindividuals and number of larvae with complete pigmentation, was significantly in-creased by feeding juvenile A. franciscana with higher protein to lipid ratios. In thepresent case, an improved protein supply seemed to be more important to larvalperformance than the higher bacterial loads.

Ž y1 .The rather low DHA content of the 4-day old A. franciscana 2.6 mg g DW , didapparently not have any impact on the pigmentation and metamorphosis of the larvaeoffered this feed, even though the larvae had a DHA content of only 5.9 mg g DWy1 onday 60, compared to 11.1 mg g DWy1 in the other group. It is possible that this DHAlevel still was high enough to satisfy the demands, or the feeding with 4-day old A.franciscana started after the most critical period for the larvae. The larvae had a quitegood growth rate and started metamorphosis around day 20, eye migration after day 30,and they received 4-day old A. franciscana from day 25.

In fact, the level of DHA on day 27 was highest in the larvae that were fed juvenileA. franciscana. This was most probably because day 27 was just after the period when

Ž .the larvae were fed the DHA rich 3-day old A. franciscana. Næss et al. 1995 havesuggested a critical stage for the normal pigmentation and metamorphosis sometimeafter day 19 and before the eye migration started on day 35. The present data seem tosupport this idea.

At any rate, the introduction of juvenile A. franciscana represents an advancement inlive feed technology in the sense that both pigmentation and metamorphosis areaccomplished more easily. Larval growth was exponential and survival was good,indicating that the developed methodology very well can serve as a basis for furtherresearch in the field.

We can conclude that 3-day old juvenile A. franciscana seems to represent the bestdiet; they reach an acceptable size in 3 days, and they have a satisfactory DHA level andDHArEPA ratio, and a rather low lipid content. The challenge is, however, to improvethe microbiological quality of the live food.

5. Conclusions

Halibut larvae fed gradually increasing size classes of A. franciscana up to day 60showed the same satisfactory growth and survival as larvae fed ST A. franciscananauplii, despite the higher bacterial loads of the former. The quality of the larvae,however, improved significantly, with 20% top-quality larvae in the group offeredincreasing prey sizes compared to about 4% in the control group.

Acknowledgements

This study was supported by Stolt Sea Farm and the Research Council of Norway.The experiment were conducted at SSF Øye, and we thank the staff at SSF Øye forskillful assistance.


References

Bremigan, M.T., Stein, R.A., 1994. Gape-dependent larval foraging and zooplankton size: implications for fishrecruitment across systems. Can. J. Fish Aquat. Sci. 51, 913–922.

Conceicao, L.E.C., 1997. Growth in early life stages of fishes. An explanatory model. PhD. Thesis.˜Wageningen Agricultural University, The Netherlands.

Crowder, L.B., McDonald, M.E., Rice, J.A., 1987. Understanding recruitment of Lake Michigan fishes: theimportance of size-based interactions between fish and zooplankton. Can. J. Fish. Aquat. Sci. 44, 141–147.

Evjemo, J.O., Olsen, Y., 1997. Lipid and fatty acid content in cultivated live feed organisms compared toŽ .marine copepods. In: Hagiwara, A., Snell, T.W., Lubzens, E., Tamaru, S. Eds. , Live Food in Aquacul-

ture. Hydrobiologia, Vol. 358, pp. 159–162.Frank, K.T., Leggett, W.C., 1986. Effect of prey abundance and size on the growth and survival of larval fish:

an experimental study employing large volume enclosures. Marine Ecology Progress Series 34, 11–22.Ž .Gulbrandsen, J., 1993. Prey consumption of Atlantic halibut Hippoglossus hippoglossus L. larvae: selectivity

or availability?. Aquaculture 116, 159–170.Ž .Holmefjord, I., Bolla, S., Reitan, K.I., 1989. Start feeding of Atlantic halibut Hippoglossus hippoglossus L.

on enriched rotifers and Artemia compared with collected plankton. Rapp. P-V. Reun. Cons. Int. Explor.Mer. 191, 474–483.

Miller, T., Crowder, L.B., Binkowski, F.P., 1990. Effects of changes in the zooplankton assemblage on growthof bloater and implications for recruitment success. Trans. Am. Fish. Soc. 119, 483–491.

Mills, E.L., Pol, M.V., Sherman, R.E., Culver, T.B., 1989a. Interrelationships between prey body size andgrowth of age-0 yellow perch. Trans. Am. Fish. Soc. 118, 1–10.

Mills, E.L., Sherman, R.E., Robson, D.S., 1989b. Effect of zooplankton abundance and body size on growth ofŽ .age-0 yellow perch Perca flaÕescens in Oneida Lake, New York, 1975–86. Can. J. Fish Aquat. Sci. 46,

880–886.Ž .Næss, T., Germain-Henry, M., Naas, K.E., 1995. First feeding of Atlantic halibut Hippoglossus hippoglossus

using different combinations of Artemia and wild zooplankton. Aquaculture 130, 235–250.Olsen, Y., 1997. Larval-rearing technology of marine species in Norway. In: Hagiwara, A., Snell, T.W.,

Ž .Lubzens, E., Tamaru, S. Eds. , Live Food in Aquaculture. Hydrobiologia, Vol. 358, pp. 27–36.Olsen, Y., Evjemo, J.O., Olsen, A.I., 1999. Status of the cultivation technology for production of Atlantic

Ž .halibut Hippoglossus hippoglossus juveniles in NorwayrEurope. Aquaculture, in press.Rainuzzo, J.R., Reitan, K.I., Jørgensen, L., 1992. Comparative study on the fatty acid and lipid composition of

four marine fish larvae. Comp. Biochem. Physiol. 103B, 21–26.ŽReitan, K.I., Evjemo, J.O., Olsen, Y., Øie, G., 1993. Biomass growth in Atlantic halibut Hippoglossus

.hippoglossus L. from eggs to metamorphosis. In: Reinertsen, H., Dahle, L.A., Jørgensen, L., Tvinnereim,Ž .K. Eds. , Proceedings of the First International Conference on Fish Farming Technology. A.A. Balkema,

Rotterdam, pp. 417–420.Reitan, K.I., Rainuzzo, J.R., Øie, G., Olsen, Y., 1993b. Nutritional effects of algal addition in first feeding of

Ž .turbot Scophthalmus maximus L. larvae. Aquaculture 118, 257–275.Skjermo, J., Vadstein, O., 1993. The effect of microalgae on skin and gut bacterial flora of halibut larvae. In:

Ž .Reinertsen, H., Dahle, L.A., Jørgensen, L., Tvinnereim, K. Eds. , Proceedings of the First InternationalConference on Fish Farming Technology. A.A. Balkema, Rotterdam, pp. 61–67.

van der Meeren, T., Naas, K.E., 1997. Development of rearing techniques using large enclosed ecosystems inŽ .the mass production of marine fish fry. Rev. Fish. Sci. 5 4 , 367–390.

Ware, D.M., 1975. Relation between egg size, growth, and natural mortality of larval fish. J. Fish. Res. BoardCan. 32, 2503–2512.

Ware, D.M., 1977. Spawning time and egg size of Atlantic mackerel, Scomber scombrus, in relation to theplankton. J. Fish. Res. Board Can. 34, 2308–2315.

Watanabe, T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol. 73B, 3–15.

Documents

Influence of size and nutritional value of Artemia franciscana on growth and quality of halibut larvae (Hippoglossus hippoglossus) during the live feed period