Protein and carbon utilization of rotifers (Brachionus plicatilis) in first feeding of turbot larvae (Scophthalmus maximus L.)

ELSEVIER

Aquaculture

153 (1997) 103-122

Protein and carbon utilization of rotifers (Bmchimus pZicatiEis) in first feeding of turbot larvae (Scophthalmus maximus L.) Gunvor f&e *J, Pavlos Makridis *, Kjell Inge Reitan, Yngvar Olsen 2 SINTEF Applied Chemistry, Center of Aquaculture, N-7034 Trondheim, Norway Accepted

28 October

1996

Abstract The effect of three different rotifer enrichments was examined on growth, survival, pigmentation and viability of first feeding turbot larvae. The diets differed in rotifer content of protein, lipid and ratio of protein/lipid. The diets were fed to turbot with or without algae (Zsochrysis galbuna) added to the larval tanks. The turbot larvae were fed rotifers for 10 days and thereafter the same Artemia diet was fed to all treatments for the rest of the experimental period. Growth and survival of fish larvae were higher in tanks containing algae than in tanks where no algae were added. Independent of algal addition, the highest growth rate and survival was obtained by feeding rotifers containing the highest protein content. Larvae reared in greenwater consumed higher numbers of rotifers during the stagnant period than larvae kept in clearwater conditions, while analysis of the larval gut contents showed lower rotifer numbers in the gut of larvae reared in greenwater conditions. This must imply longer residence time of the food in the larval gut, and presumably also higher digestion and assimilation efficiencies of larvae maintained without algae than in larvae maintained with algae. Calculation of protein and carbon conversion efficiency showed higher utilization in larvae maintained without algae (IS-28% for protein, 12-19% for carbon) than in larvae maintained with algae (6-9% for protein, 4-7% for carbon). No significant

* Corresponding author. Tel: +47-73590581; Fax: + 47-735963 11; E-mail: [email protected]. ’ Present address: Brattora Research Centre, Norwegian University of Science and Technology, Dragvoll, Norway. * Present address: Trondhjem Biological Station, Norwegian University of Science and Technology, Trondheim, Norway. 0044-8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(96)01514-l

N-7055 N-7018

104

G. 0ie et al./Aquaculture

I53 (1997) 103-122

differences in pigmentation rate and stress sensitivity were observed among the larvae of the various treatments. 0 1997 Elsevier Science B.V. Keywords: Larvae

Scophthalmus

maximus

L.; Brachionus

plicatilis;

First feeding;

Nutrition:

Protein

utilization;

1. Introduction Established methods for larval rearing of turbot normally involve feeding regimes with rotifers (Bruchionus plicatilis) and Artemia, both with and without supplementation of microalgae to the tanks (Jones et al., 1981; Gatesoupe, 1990; Reitan et al., 1993; Minkoff and Broadhurst, 1994). Addition of microalgae to larval tanks has improved the rearing success of larvae in most cases (Howell, 1979; Scott and Middleton, 1979; Jones et al., 1981; Reitan et al., 1993). A critical factor for the nutritional value of the live food used during first feeding is their content of n-3 HUFA, including 20:5n-3 and 22:6n-3 (Witt et al., 1984; Kanazawa, 1985; Sargent et al., 1989; Koven et al., 19921, and major efforts have been made to optimize the lipid content and fatty acid composition of both rotifers and Artemia (LCger et al., 1986; Olsen et al., 1993; Dhert et al., 1994; Rainuzzo et al., 1994). It is known that the lipid content and fatty acid composition of marine microalgae vary among species and cultural conditions (Pohl, 1982; Reitan et al., 1994a), and algae fed to rotifer cultures or larval tanks will alter the lipid and fatty acid composition of the rotifers (Watanabe et al., 1983; Ben-Amotz et al., 1987; Whyte and Nagata, 1990; Reitan et al., 1994b). The changes in fatty acid composition are rapid, and significant changes may therefore occur during the period when the rotifers reside in the larval tanks before being consumed by the larvae (Olsen et al., 1993; Reitan et al., 1993). The protein level in rotifers has been reported to be in the range of 28 to 67% of dry weight, whereas no difference in the amino acid profile of rotifers fed different diets has been reported (Lubzens et al., 1989). Furthermore, the amino acid profile of rotifer does not deviate significantly from that of natural zooplankton or other cultivated zooplankton species recognized as beneficial for growth of larvae (Lubzens et al., 1989). Information on the importance of protein level and protein/lipid ratio of the live food organisms during first feeding are scarce. Generally, more attention has been paid to the lipid composition than to the protein level of the live food organisms. The aim of the present study was to examine the effect of protein level and protein to lipid ratio of rotifers on growth and survival of turbot larvae during the early stages of first feeding. Rotifers with different protein content and protein/lipid ratio were fed to turbot larvae with and without algae added to the larval tanks. The effect of the different rotifer treatments on larval growth, survival, pigmentation and viability was examined, and we present an evaluation of protein conversion efficiency in turbot larvae.

2. Material

and methods

2.1. Liue food treatment Rotifers (Bruchionus plicutilis, L-strain) were cultured in 250-l conical vessels (20°C 20%0) and fed baker’s yeast and 10% Super Selco (INVE Aquaculture SA, Belgium;

G. 0ie et al./Aquaculture Table 1 Review of cultivation in the fish tanks

and enrichment

Cultivation conditions Volume replaced per day Short-term enrichment ( pg food per rotifer day- ’ ) Abbreviations Tanks with algae added (three replicates) Control tanks without algal addition (two replicates)

treatments

105

153 (1997) 103-122

of the rotifers, and abbreviations

for the different treatments

P-rotifers

L-rotifers

N-rotifers

20%

5%

0.8 cLg Protein Selco

0.4 CLg DHA Selco

5% No short-term enrichment

PA

LA

NA

PC

LC

NC

citation of commercial products is not to be considered an endorsement) once a day (Olsen et al., 1993). The rotifers were grown in two cultures from which 20% or 5% of the culture volume was removed daily and replaced by 20%0 seawater. High dilution rate (20% day- ’ ) will result in rotifers with higher growth rate (0.19 day- ’ ), with higher individual carbon and protein content than in those grown in cultures with a low dilution Three different enrichment procedures were used for the rate (5%, = 0.05 day-‘). rotifers to establish the diets (Table 1). The first diet (P-rotifers) consisted of fast growing rotifers (20% dilution) successively enriched with 0.8 pg Protein Selco per rotifer for 24 h. This treatment resulted in high individual carbon, lipid and protein content of the rotifers. In the second diet (L-rotifers), slow growing rotifers (5% dilution) were short-term enriched with 0.4 pg DHA-Selco per rotifer. The last treatment (N-rotifers) involved slow growing rotifers (5% dilution) without any shortterm enrichment. The rotifers were harvested, rinsed with seawater and then fed to the turbot larvae. Samples of rotifers were taken for chemical analysis at days 1, 7 and 9. The Artemia (EG-cysts, INVE Aquaculture SA) were hatched at 28°C in 10-l conical plastic vessels containing 10%~ seawater. After hatching (24 h) the nauplii were harvested with a plankton net (70 pm mesh size), washed in seawater (25°C) and then short-term enriched (24-36 h, in seawater, 28°C) with High DHA-Super Selco (0.2 g 1-l). The algae Isochrysis galbana, (clone 7’. Zso) was cultivated in 200-l transparent plastic tubes with f/2 medium (Gulliard and Ryther, 1962). The cultures were run semi-continuously with a daily dilution rate corresponding to 50% of the maximum growth rate, in order to produce constant and reproducible nutritional value of the algae (Reitan et al., 1994a). The effect of microalgal addition to larval tanks on the carbon and protein content of the rotifers was simulated in separate experiments without fish larvae at 18°C. The rotifers were cultivated on yeast and Super Selco (N-rotifers), and transferred to water with and without I. gulbunu ( > 2 mg C l- ’ >. Samples of the rotifers were taken from the tanks at different times during the following 3 days for determination of carbon and nitrogen.

106

G. 0ie et al./Aquacubure

153 (1997) 103-122

2.2. Larval treatments The first-feeding experiment was performed in 200-l cylindrical tanks with a flat bottom. The seawater (34%0) was filtered (pore size 0.2 pm), tempered and microbial matured (Skjermo et al., 1997) before being used in the larval tanks. The eggs of turbot, Scophthalmus maximus (obtained from the broodstock at Tinfos Aqua AS, Norway), were transferred to 15 replicate tanks (10 eggs l- ’ ). The tanks were individually illuminated by 40-W bulbs, and slight air bubbling was introduced 1 day after hatching. The temperature was gradually increased from 13S”C at hatching to 18°C 5 days post-hatching, and was than maintained at 17- 18°C. The larvae were maintained in stagnant water during the first 5 days. Between Days 5 and 9, the water was exchanged at a rate of 1.4 day - ’ (daily water volume supplied/water volume in tank). The water exchange rate was increased to 2.9 day-’ after Day 9. Each rotifer diet (see Table 1) was fed to five tanks. Larvae in three tanks also received algae. The algae I. galbana was maintained at concentrations of l-2 mg C 1-l in the fish tanks by daily addition from the first day after hatching until Day 5, and then two additions until Day 12, when Artemia was introduced. The larvae were fed rotifers from Day 2 to Day 12. The rotifer density was adjusted to 3000 rotifers 1-l and 5000 rotifers 1-’ from Days 2 and 4, respectively. The rotifer density and egg ratio (ER) in the fish tanks were counted just before feeding and adjusted to 5000 rotifers 1-l daily. After Day 12 the larvae were fed 500-1500 Artemia 1-l three times daily depending on larval density. 2.3. Analytical

procedures

Larval survival during the first feeding experiment was estimated based on counts of dead larvae removed from the tanks and the final number of fish, corrected for larval sampling. The carbon content of individual larvae was measured by transferring individual larvae into tin capsules (n = 20-30), dried at 60°C for 48 h and analyzed in a Carbon Erba Element analyzer, Model 1106, using acetanilide as the standard. The average carbon and nitrogen content per rotifer was measured on rotifers previously rinsed and concentrated (1000-2000 rotifers ml- ’ > in a beaker. The exact number of rotifer was counted and the respective carbon and nitrogen content was analyzed in lOO-~1 samples (n = 8, Eq. (2)). The background content of carbon was analyzed in 100 ~1 of the same water. The tin capsules were dried and analyzed as described above. Total amino acid composition of the rotifers and larvae was analyzed using reversed phase HPLC technique (see Oie et al., 1994). The protein content of live food was estimated as the average of two different methods. One estimate was obtained by (A): Nitrogen content X 4.2, and the other estimate was calculated as (B): sum of water-free amino acids/0.9. The sum of water-free amino acids has earlier been regarded as a minimum estimate (0ie et al., 1994) because some amino acids may not be detected (i.e. proline, cysteine and tryptophan). In this equation the amount of undetected amino acids was assumed to be 10% of the total amount of amino acids (Roald, 1994).

G. Oie et al./Aqwculture

107

153 (1997) 103-122

The lipid content of live food and turbot larvae was estimated by a modified Bligh and Dyer (1959) method (see Rainuzzo et al., 1994). Larval consumption of rotifers was estimated through an analysis of mass balance based on population dynamics of both rotifers and larvae in the first feeding tanks (see Reitan et al., 1993). The gut content of sampled larvae was analyzed by counting the number of rotifers in the gut at Days 3 and 5. The larvae (10 larvae per tank) were dissected and the number of rotifers (lorica) in the fore- and midgut counted. The numbers of colony-forming units (CFU) attached to the rotifers and the culture water were determined before the rotifers were fed to the larvae (Day 2) and in rotifer samples taken from the larval tanks (Day 8). Sampled rotifers (5 ml) were washed four times with autoclaved 80% seawater (800 ml seawater and 200 ml distilled water), and were then aseptically homogenized with seawater (80%) in a glass homogenizer. All samples were plated on three different media: TCBS Cholera Medium (Oxoid) for enumeration of presumptive Vibrio, Pseudomonas C-F-C agar (Oxoid) which is selective for Pseudomonas and M65seawater agar (0.5 g peptone, 0.5 g tryptone, 0.5 g yeast extract, 15 g agar), for enumeration of the total number of CFU. The pigmentation rate of 40-day-old turbot fry was visually estimated by two persons not aware of the treatment given to the different groups (blind tests). Thirty larvae were randomly sampled from each replicate tank and classified as completely pigmented or as not completed pigmented individuals. The viability of 30-day-old larvae was tested in a salinity test, where 30 larvae were transferred to a beaker with 60%0 salinity water. The mortality of the larvae was monitored for 3 h. 2.4. Calculations Rotifer growth rate ( p) in the larval tanks was calculated based on egg ratio (Reitan et al., 1993):

by the empirical

p = ER x 0.95 - 0.17

equation

(1)

The individual carbon and nitrogen content in the rotifers was calculated based on the numbers of rotifers in the samples and the results of the carbon (C) analysis: C per rotifer = C - C background/no. of rotifers The dry weight content of the rotifers was calculated

(2) based on the equation:

DW = 2.25 X carbon The specific growth rate (SGR) of the larvae was calculated ment of the individual larval carbon content by the equation: SGR = ln( N,/N,)/t

(3) based on the measure-

(4)

where N, and N, denote the individual larval carbon content at time 0 and time t, respectively. The relationship between SGR and daily percent weight increase (DWI) is: DWI = lOO( eSGR - 1)

(5)

108

G. 0ie et al./Aquaculture

The larval utilization equation:

efficiency

153 (1997) 103-122

(%o) of protein

and carbon

was calculated

by the

utilization eff. ( %) = ( G, - G, ) X 100/Z X t (6) where G, and G, denote the larval protein or carbon content at time 0 and time t, respectively, I is the daily average consumption of rotifers (protein or carbon) and t is the period of larval growth. The utilization efficiency was calculated from Day 2 to Day 6 (stagnant period) and Days 5-6 (flow through period). The utilization efficiency was estimated by two calculations, by use of carbon and protein content of the rotifers given to the larvae (I), and the measured nutritional value of the rotifers after transfer to the fish tanks (II), corrected for rotifer addition during the period. New rotifers were added to the fish tanks at Day 2 (2/5 of the total amount) and Day 5 (l/2 of the total amount). The nutritional content of the rotifers in the fish tanks was calculated based on the simulation experiment. N-rotifers were used in the simulation experiment and it was assumed that the nutritional value of P- and L-rotifers would follow the same pattern of variation. The experimental data were tested for statistical significance using Student’s t-test, and the level of statistical significance is given at the 5 or 10% level. The confidence limits of the data are given as standard error of the mean. 3. Results 3.1. Characteristics

of the live food

Rotifer density during the production period was maintained at the same level for all treatments (Table 2), but a significantly higher egg ratio (ER) (P < 0.05) was found for P-rotifers than for the other treatments. Also the individual carbon contents (C, Eq. (2)) Table 2 Rotifer density and egg ratio (ER) during the production period (n = lo), together with individual carbon and dry weight (ng per ind.) content, content of protein (ng per ind. and mg g-’ DW), lipid (ng per ind. and mg ratio in rotifers given to the larval tanks (mean + SEM) g - ’ DW) content and protein/lipid

Ind. ml-’ ER ng C per ind. Total Body ng DW per ind. Total Body ng protein per ind. ng lipid per ind. mg protein g-’ DW mg lipid g-t DW Protein/lipid Estimation Estimation

P-rotifers

L-rotifers

N-rotifers

235rfr13 0.34 + 0.02

193* 10 0.17*0.01

245 + 14 0.14+0.01

226 * 15 174* 14

149+6 132+6

169+9 149+9

502+33 3a7+31 172 + 34 91.3* 18 364+8.2 182& 13 2.0+0.1

331*13 293+ 13 111*10 70.3 * 6.3 336 + 2.4 212k6.2 1.6fO.l

376 + 20 331+20 137+23 54.2 * 8.9 347+11.4 144*0.5 2.4+0.1

of ng protein per individual: Average of nitrogen of mg protein g-’ DW: Total amino acids/0.9.

per individual X 4.2 and total amino acids/0.9.

G. 0ie et al./Aquaculture

109

153 (1997) 103-122

and dry weight (DW, Eq. (3)) were significantly higher (P < 0.05) in P-rotifers as compared to L-rotifers and N-rotifers, and a gradient in individual biomass between P-rotifers, N-rotifers and L-rotifers was obtained. The protein content of the individual rotifers and the protein content expressed per dry weight showed the highest protein content in P-rotifers and the lowest in L-rotifers (P < 0.1 for individual protein content and P < 0.05 for protein content in dry weight). No significant differences in protein content were found, however, between P-rotifers and N-rotifers or L-rotifers and N-rotifers. The individual lipid level was higher in P-rotifers than in L-rotifers, and lowest in N-rotifers. The lipid level expressed per dry matter was significantly higher (P < 0.05) in L-rotifers than in P-rotifer and N-rotifers and was lowest in N-rotifers. The protein/lipid ratio was higher in N-rotifers (P < 0.0% than in P-rotifers and L-rotifers, and lowest in L-rotifers. The total fatty acid content of the rotifers, expressed in dry matter, was significantly higher in L- and P-rotifers (P < 0.05) than in N-rotifers (Table 3). The content of 20:5n-3, 22:6n-3 and n-3 HUFA was highest in L-rotifer and lowest in P-rotifers (P < 0.05). L-rotifers also exhibited a higher DHA/EPA ratio than N-rotifers, which again had a higher ratio than P-rotifers. Artenzia enriched for 24-36 h with High DHA

Table 3 Total fatty acid content (% of total lipid) and individual three replicates + SEM) and short-term enriched Artemia P-rotifers Fatty acids (% of total lipid)

64.3 + 6.0

fatty acid pattern (%I in enriched

L-rotifers 68.4 + 2.2

N-rotifers 53.5 + 1.4

rotifer (mean of Artemia a 72.5

Fatty acid (%) 14:o 16:0 18:O 16:l 18:l 20: 1 22: 1 18:2n-6 20:4n-6 18:3n-3 18:4n-3 20:5n-3 22:5n-3 22:6n-3

2.5 f 0.2 9.2kO.3 3.1+0.1 11.1 kO.3 20.0 + 0.6 5.5 * 0.2 3.4+0.1 6.7 + 0.3 0.8 + 0.02 1.4kO.l 0.7+-0.1 14.5+0.6 4.2kO.2 16.6 * 0.9

1.1+0.02 6.7 + 0.2 2.2kO.l 7.1+0.1 11.1+0.4 3.2kO.l 2.2kO.l 4.3 * 0.2 0.720.1 0.6+0.01 0.6f0.02 18.6f0.2 5.1+0.1 35.9+0.7

1.4kO.l 8.0+0.1 3.1 kO.03 12.OkO.3 15.2+0.1 5.4+0.2 3.5+0.1 3.7kO.l 0.8 k 0.04 0.6+0.01 0.4 + 0.02 14.6 f 0.3 5.8kO.03 25.0 + 0.4

0.5 7.1 4.3 2.0 12.6 0.5 0.9 3.6 1.1 14.9 2.3 22.6 2.15 23.8

G-3 En-6 n-3 HUFA 22:6/20:5 n-3/n-6

37.9 * 1.3 7.4kO.2 35.2 + 1.3 1.1 f0.1 5.1+0.3

61.6k 1.0 4.9+0.1 59.6 * 0.9 1.9+0.02 12.8kO.6

46.9 i 0.4 4.5+0.1 45.4 * 0.4 1.7+0.1 10.5 f 0.2

67.4 4.7 48.5 1.1 14.3

“n=l.

110

G. 0ie et al./Aquaculture

153 (1997) 103-122

Table 4 The content of total water-free amino acids (mg g- ’ DW) and amino acid composition (% weight of total) in rotifer after enrichment (mean + SEM of three replicates) and in short-term enriched Arfemia (n = 1)

Total amino acids (mg g-

’ DW)

Ammo acid (% by weight) Aspartic acid + aspargine Glutamic acid + glutamine Serine Histidine Glycine Threonine Arginine Alanine Tyrosine Methionine Valine Phenylalanine Isoleucine Leucine Lysine

P-rotifers

L-rotifers

N-rotifers

Artemia

328.0 + 7.4

302.6 + 2.2

312.5 + 10.3

298.8

ll.OIkO.4 13.3kO.4 4.1 Ito. 2.2ItO.l 4.2kO.l 4.3fO.l 8.6 +0.4 6.6kO.l 4.9fO.l 1.7kO.2 6.0+0.1 6.1 +O.l 5.9+0.1 8.5 + 0.2 12.8* 1.3

10.3+0.5 13.7*0.5 4.3 +0.1 2.2+0.1 4.4+0.1 4.3*0.1 8.7 + 0.4 7.0fO.l 5.0+0.1 2.0+0.1 6.2 * 0.2 6.2kO.l 6.1 kO.1 8.7+0.1 10.9 + 0.4

11.1+0.2 13.8kO.3 4.2kO.l 2.2kO.l 4.2kO.l 4.3+0.1 8.4 k 0.4 6.6*0.1 4.1+0.8 2.0f0.2 6.1 kO.1 6.2kO.l 6.0+0.1 8.8 * 0.3 12.0 + 0.6

10.2 14.9 3.8 2.2 5.0 4.6 9.1 8.5 4.9 2.1 6.0

Table 5 The content of free amino acids (mg g- ’ DW) and free amino acid composition (mean * SEM of three replicates) and in short-term enriched Artemia (n = 1)

Total free amino acid (mg g Amino acid (% of weight) Aspartic acid Glutamic acid Aspargine Serine Histidine + glutamine Glycine Threonine Arginine Taurine Alanine Tyrosine Tryptophan Methionine Valine Phenylalanine Isoleucine Leucine Lysine

’ DW)

5,3 5.4 7.7 10.5

(% of total) in the rotifers

P-rotifers

L-rotifers

N-rotifers

Artemia

15.7& 1.1

19.Ok5.9

11.4+0.6

6.4.

1.6+0.2 10.3 + 0.5 5.8kO.3 6.0+ 1.0 9.9kl.O 6.4iO.2 2.3 50.4 16.7+ 1.1 2.5 + 0.2 12.3+ 1.4 8.1+0.2 0.2+0.1 0.6kO.l 3.9 + 0.4 1.5rtO.l 3.1 kO.3 2.8zkO.3 6.2+0.2

1.4kO.3 11.2+0.9 3.3 * 0.4 7.4+ 2.8 9.6+ 1.6 4.5 f 0.9 3.4+ 1.5 10.7&0.1 2.9 + 0.7 13.1*4.6 7.1 +2.4 0.2*0.1 0.4*0.1 6.7 + 2.7 3.0+ 1.5 4.5 * 1.9 6.2 f 2.7 4.5 &-1.3

2.0*0.1 12.6+ 1.1 6.2 f 0.7 6.0 + 0.6 13.8 + 2.0 4.2 i 0.5 1.6kO.2 19.3 &-1.6 2.3+ 1.1 12.5 +0.3 7.8 f 0.4 _

2.7 13.1 2.7 5.2 3.6 3.7 4.4 9.9 7.1 11.3 4.7 1.0 2.1 4.4 4.0 3.9 6.4 7.6

0.5*0.1 2.4kO.3 1.6kO.l 2.4*0.2 1.3fO.l 3.6k 1.0

G. 0ie et al./Aquaculture

153 11997) 103-122

111

Super Selco (INVE Aquaculture SA, Belgium) contained a high level of 18:3n-3, 205-i-3 and 22:6n-3. The relative composition of total amino acids (% weight of total, Table 4) showed almost equal profiles for all treatments and the amino acid profile of Artemia was close to the rotifer profiles. The content of free amino acids (mg g -i DW) was of the same magnitude in all

a

L-rot1fer

P-rot1fer

N-rotder

b lE+7

-

1E+6

-

f E 2 lE+5

0

-

lEi4

lE+3

-

1E+2 i P-water

1

L-water

n M-65 q TCBS

Fig. 1. Colony-forming bacteria (CFU) associated cultures fed different diets. The bacterial growth ) and Pseudomonas agar ( q 1.

1

..

N-water

0 Pseudormans

with rotifers (a) and suspended in the culture water (b) in was tested on three different agar types: M-65 agar (W 1,

112

G. C&e et al./Aquaculture

Table 6 Rotifer density and rotifer growth rate (/I, units in rotifers plated on M65 agar

Rotifer ml - ’ p day-’ CFU per rotifer

day-’

153 (1997) 103-122

) in fish tanks between Days

1 and 5 and colony-forming

PA

PC

LA

LC

NA

NC

5.4+ 1.2 0.43 + 0.08 408 + 19.2

4.6+ 1.2 -0.02+0.06 70.0 + 4.2

4.2 + 0.8 0.21 fO.l 124f3.6

3.8 + 0.9 - 0.01 + 0.02 455 + 12

4.1 kO.6 0.20 * 0.08 177t0.1

4.0 * 0.7 - 0.09 * 0.02 428 f 35.0

rotifer diets, but was significantly higher (P < 0.05) in P- and L-rotifers than in N-rotifers (Table 5). Almost 50% of the free amino acids (% weight of sum) was glutamic acid, histidine, glutamine, arginine and alanine for all treatments. The content of free amino acids (mg g -’ DW) in Artemia was half the level found in rotifers, but the profile was comparable to those of the rotifers. The numbers of colony forming units (CFU) per rotifer on M-65, TCBS and Pseudomonas agar showed no systematic differences for the different treatments (Fig. la). No CFU of Pseudomonas were found in N-rotifers. For all agar types, a significantly lower number of CFU was found in the cultivation water of N-rotifers compared to those of L- and P-rotifers (Fig. lb). 3.2. Characteristics

of rotifers in larval tanks

Rotifers were added to the tanks at Days 2, 4 and 5 in order to keep the rotifer density in the larval tanks at five rotifers ml-‘, and no significant difference in rotifer density was observed for the different treatments from Days 1 to 5 (Table 6). The growth rate of rotifers in the larval tanks was high in all tanks with algae, especially in

150

I

40 Time

(hours)

Fig. 2. Change (%) in protein and carbon content in N-rotifer after transfer to fish tanks conditions with and without algae added: W, carbon in water with algae; 0, carbon in water without algae; * , protein in water with algae: l , protein in water without algae.

113

G. 0ie etal./Aquaculture153 (1997) 103-122

100

80

20

LA

OT 0

I

I

/

I

5

10

15

20

0

5

10

15

20

25

Days

Fig. 3. Survival (% of hatched larvae) of turbot larvae from hatching to Day 23. Triplicate tanks for all enrichment with addition of algae (A) and duplicate tanks for enrichment without algae present (B).

the tanks fed with P-rotifers, whereas the population growth rate was negative (i.e. declining density) in all tanks without algae (Table 6). The simulation experiment with N-rotifers showed a rapid decrease in protein and carbon content per individual rotifer in tanks without algal addition, whereas the individual contents increased in tanks where algae were added (Fig. 2). The number of colony forming units (CFU) per rotifer on M-65 agar varied between 70-455 CFXJ per rotifer (Table 6), which was much lower than the values obtained during the cultivation conditions (Fig. 1). The numbers of CFU on TCBS- and Pseudomonas agar were lower than 40 CFW per rotifer. In tanks fed L- and N-rotifers, the numbers of CFU were lower in tanks with algae than without algae.

Table 7 Specific growth rate (SGR, Days 5-12), daily weight increase (DWI, Days 5-12), mean feeding rate (I) of larvae in the period 2-6 days (triplicate tanks with algal addition and two replicate tanks without algal addition), and survival of the larvae at Day 23, with different treatments during first feeding (mean * SEMI. Pigmentation (%) 41 days after hatching (three replicates of 30 larvae) and mortality (%) after a stress test on 30-day-old larvae (two replicates of 30 larvae) PA SGR (day- ’ ) DWI (% day-‘) Ingestion (rotifer day- ’ per larvae) Survival Day 23 (%) Full pigm. (%) Mortality (%) after stress

’ n = 1 (30 larvae).

PC

LA

LC

NA

NC

0.29kO.01 0.241tO.01 0.28+0.01 0.21*0.02 0.27+0.01 0.26kO.02

34 303 + 23 54.4k1.9 99+1 7855

21 130+8 24.1k9.8 94*4 75_+8

32 400* 16 28.8k7.1 93+7 78+1

23 143+7 6.9k2.2 100&O 52 z

31 325 f 24 46.2+3.1 99+1 57+2

30 31s+47 11.1+0.8 ss+1 83* 1

PA,LAandNA:

n=3.PC,LCandNC:

?~=2,~n=l.

40.5 3.3 37.7 4.3 12.4

41.5 2.8 38.9 4.3 14.6

G-3 In-6 n-3 HUFA DHA/EPA n-3/n-6

140.3

4.6 18.62 4.7 3.8 17.1 5.3 2.2 2.1 1.2 1.0 1.0 6.6 2.5 28.6

129.1

5.1 16.8 3.6 4.3 17.3 5.9 2.8 2.2 0.6 0.9 1.1 6.8 3.0 29.2

DW)

Day2a

Fatty acid (%) 14:o 16:O 18:0 16:l IS:1 2O:l 22: 1 18:2n-6 20:4n-6 18:3n-3 18:4n-3 20:5n-3 22:5n-3 22:6n-3

Total fatty acids (mg g-’

DayOa

40.7*0.5 6.5+0.1 34.6+0.5 2.7kO.l 6.3kO.2

4.7+0.1 13.1 fO.l 5.9*0.1 5.2kO.2 17.2kO.2 3.9*0.1 2.8kO.l 5.3kO.l 0.8+0.1 1.9*0.1 3.5 + 0.2 8.5 *0.2 3.3 f 0.2 22.8 f 0.4

106.4+6.3

PA

Day 12

1.8

42.4 f 0. I 6.OkO.l 40.5kO.2 2.7+0.1 7.1+0.1

1.6kO.l 13.0&0.1 7.7*0.1 5.3+0.1 18.2+0.1 4.3*0.1 1.6&O 4.6+0.1 1.4*0.1 0.7*0.1 0.3*0.1 9.8kO.l 5.5kO.l 25.1kO.3

86.4*

PC

46.5 f 0.9 5.3*0.3 40.2 f 1.6 3.5kO.2 8.8 + 0.6

5.0fO.l 14.2 f 0.3 6.9 i 0.3 4.6f0.2 15.2kO.2 3.4rfO.l 2.8 kO.2 4.2 f 0.2 1.1+0.1 1.7f0.2 3.6kO.3 7.2kO.l 7.2 j10.2 25.6f0.4 42.6 * 0.6 5.4+0.1 36.1 +0.5 3.6kO.l 8.0f0.2

48.9 + 3.6 4.8rfr0.1 46.9 f 3.8 3.3fO.l 10.2 f 1.o

91.0f7.6

NA

1.4fO.l 13Sf 1.1 8.3 i 0.5 5.2kO.5 13.0f0.9 3.4 f 0.4 1.5rtO.l 3.4*0.1 1.4fO.l 0.7+0.1 0.3*0.1 10.0 10.6 4.2ctrO.5 32.8zt2.6

89.5 f 8.4

104.5 + 10.0

4.9 *0.4 14.4*0.5 6.6 i 0.5 3.6+0.2 13.0+0.2 3.1 kO.2 2.7+0.5 4.2 f 0.3 1.1 f0.1 1.9*0.3 3.750.6 8.4kO.6 3.0t0.2 28.8kO.8

LC

LA

41.2f3.9 4.4f0.2 38.5 + 4.0 2.9fO.l 9.5+ 1.3

1.6kO.2 14.7 + 1.6 9.5+ 1.1 4.9kO.6 18.0f 1.1 3.8rtO.2 2.0*0.1 3.0*0.1 1.3fO.l 0.4kO.l 1.0+0.1 8.3 i 0.7 5.6kO.7 24.5 rt 2.6

64.4 + 4.0

NC

52.8 + 0.7 6.9f0.4 36.4 f 0.7 1.2fO.l 8.0 f 0.4

0.5fO.l 11.3*0.1 7.110.1 1.8fO.l 17.7 f 0.3 0.8ztO.l l.O&O.l 4.6 f 0.4 2.3f0.1 13.0f0.3 1.8fO.l 15.3 i0.2 3.420.1 17.61tO.7

123.1 k2.5

Day 23

Table 8 Content of total fatty acids (mg g- ’ DW) and fatty acid composition (% of total fatty acids) in turbot larvae at different ages and feeding regimes (mean + SEM of three replicate tanks with algal addition and two replicate tanks without algal addition)

G. Oie et al. /Aquaculture

3.3. Growth and development

115

153 (1997) 103-122

of turbot larvae

All larval groups exhibited highest mortality from Day 5 to Day 12. The larval mortality of all groups was low from Day 12 onwards. Survival of the turbot larvae from hatching until Day 23 was significantly higher throughout in tanks with microalgae added than in tanks without algae (P < 0.05, Fig. 3). PA-larvae showed the highest average survival at Day 23 (54.8 f 1.9, range 5 1.8-59.0%) whereas LA-larvae showed the lowest and most variable survival (28.8 f 7.1, range 15.8-45.2%) in tanks with algae added. The same survival pattern was also observed in tanks mn without algal addition; the highest survival was observed for PC-larvae (16.9 f 5.0, range 9.8-24.2%) and lowest for LC-larvae (6.9 + 2.2, 3.8-10.0%). Specific growth rate (SGR) of the individual larvae during the rotifer period (i.e. Days 5-12) was slightly higher in tanks with algae added (0.27-0.29 day-‘, Table 7) than in tanks without algae present (0.21-0.26 day-’ ). Daily weight increase (DWI) from Day 5 to Day 12 followed the same pattern of variation as SGR (Table 7). Pigmentation of the turbot fry was evaluated at Day 41 after hatching, and no significant differences in pigmentation were observed for the larval groups (94-100% fully pigmented), except in NC-larvae (88% k 1) (Table 7). Salinity tests mn with 30-day-old larvae resulted in high mortality in all larval groups, and no systematic difference in viability among the treatments was observed (Table 7). The total fatty acid content (mg g- ’ DW) of the larvae increased from hatching up to Day 2, and then decreased until Day 12 (Table 8). At the end of the rotifer period (Day Table 9 Amino acid composition replicates).

(% of total amino acids) in turbot larvae at Day 2 and Day 12 (mean f SEM of two

The last column shows the average amino acid composition

Total amino acid

mgg-’

DW

of all treatments

Day 2 Day 12

392

PA

PC

LA

LC

NA

NC

352k24

287+45

354+14

362+32

285+60

367f20

Average

(% of weight) 10.3

lO.lkO.6

10.3k3.2

10.1+0.4

10.3k2.9

lO.Ok2.1

lO.Ok2.8

lO.l+O.l

Glutamic acid + glutamine 12.2

Aspartic acid + aspargine

12.6&0.5

12.91k3.9

12.2+0.4

12.3k3.5

12.1 +2.5

12.1 +3.4

12.4+0.1

Serine

5.0

5.3zkO.l

5.8+1.9

5.4kO.2

5.1+1.4

5.4ztO.l

5.3+1.5

5.4kO.l

Histidine

1.8

1.7+0.1

1.7+0.5

l.lkO.5

1.6+0.4

1.7kO.4

1.5ztO.5

1.5+0.1

Glycine

10.2

12.9kO.3

13.0+4.1

12.8f0.4

11.9+3.4

12.6k2.6

12.7+3.5

12.7+0.2

Threonine

5.4

5.4+0.1

5.9k1.9

5.4zkO.2

5.3+1.5

5.2kl.l

5.1+1.4

5.4kO.l

Arginine

5.4

6.3+0.2

6.6k2.0

6.4+0.2

5.9k1.7

6.0+1.1

6.2k1.7

6.2+0.1

Alanine

9.2

10.2kO.2

10.5k3.3

10.2+0.49.5+2.7

10.0*2.0

10.2k2.8

lO.l*O.l

Tyrosine

3.8

3.3kO.2

3.4kl.O

3.5+0.2

3.2kO.l

3.3+0.7

3.2kO.9

3.3+0.1

Methionine

2.2

2.2+0.3

0.9kO.3

2.3kO.l

2.0k0.6

1.7k0.8

1.3+0.5

1.7kO.2

Valine

7.1

6.9k0.3

7.Ok2.2

6.8kO.2

7.O~t2.0

6.8k1.4

6.71t1.9

6.9kO.l

Phenylalanine

4.4

4.0+0.2

4.1k1.3

3.9+0.1

4.Okl.l

4.0+0.8

3.9fl.l

4.0*0.1

Isoleucine

5.2

5.0+0.3

5.1k1.6

4.91!zO.l

5.11t1.4

5.0*0.1

5.Ok1.2

5.0*0.1

Leucine

8.9

8.9k0.5

9.4k2.8

9.1+0.5

9.1k2.6

9.0+1.8

8.9k2.5

9.1+0.1

Lysine

8.8

5.2+2.5

3.5k1.8

5.8+1.9

7.7k2.2

7.2k1.5

7.9f2.2

6.2+0.6

116

G. 0ie et al./Aquaculture

153 (1997) 103-122

12), all groups cultivated with algae showed significantly (P < 0.05) higher total fatty acid content than the groups cultured without algae, except for the L-groups where the differences were statistical insignificant. The content of total fatty acids was identical in all larval groups at Day 23, and pooled values are shown in Table 8. No significant differences in relative fatty acid composition were found from hatching to Day 2. The main differences at Day 12 for larval groups reared without algae consisted of a significantly higher (P < 0.05) content of DHA and n-3 HUFA in LC-larvae than in the other treatments. Larvae maintained in tanks to which algae were added showed a higher content of 14:0, 18:2n-6, 18:3n-3, 18:4n-3 and 22: I, and a lower content of 18:0, 20:4n-6 and 20:5n-3. The ratio DHA/EPA was quite high for all treatments (2.7-3.6). The total amino acid composition (% of weight) of the larvae remained constant from Day 2 to Day 12 and the amino acid composition was not affected by the diets (Table 9).

a Day 3

PA PC

LA LC

PA PC

LA LC

NA NC

PA PC

LA LC

PA PC

LA LC

NA

NC

i NA NC

Fig. 4. Ingestion observed (a) at Day 3 and Day 5 and calculated

NA NC

(b) at Days 3-4 and at Days 5-6.

G. 0ie et al. /Aquaculture

117

153 (1997) 103-122

3.4. Feeding and food conversion

The number of rotifers found in the gut of the turbot larvae at Day 3 was equal for all treatments (Fig. 4a), whereas higher numbers were found at Day 5 for larvae reared without microalgae than with algae. No significant differences in numbers were, however, found for the different rotifer diets. Larval consumption rate of rotifers, estimated through an analysis of mass balance (Reitan et al., 1993), is shown in Fig. 4b. Contrary to the gut contents, the consumption rate of rotifers was significantly higher for larvae reared in tanks with algae than in tanks without algae, both through Days 3-4 and Days 5-6 (P < O.OS>,except for Days 3-4 in N-tanks. The protein and carbon content per larvae at Day 6, and the corresponding weight increases (Days 2-6), were higher in P- and N-larvae than in L-larvae (Table 10). Moreover, larvae reared in tanks with algae contained more carbon and protein than larvae reared in tanks without algal addition. The larval utilization efficiency (%) of dietary protein in the stagnant period was, in general, higher than for carbon (Table 10). Calculation of the use of the carbon and protein of the enriched live food given to the tanks (Table 2) showed a utilization efficiency for carbon of 5.7-9.6% in tanks where algae were added, and of 8.3- 13.1% in tanks where algae were not added. The protein utilization efficiency was 10.5-15.3% in tanks where algae were added and 15.0-24.5% in tanks without algae (Table 10). The rotifers nutritional content will be modified after

Table 10 Weight ( pg per larva) at Day 6 and weight increase ( pg per larva) from Days 2 to 6. Utilization efficiency (%) of carbon and protein in the period from Days 2-6 and after water flow was introduced (Days 5-6). I: Utilization efficiency calculated with initial nutritional value of the rotifers. II: Utilization efficiency calculated from average nutritional value of the rotifers after addition to the fish tanks, corrected for rotifer addition at Day 2 and Day 5 PA

PC

LA

LC

NA

NC

Carbon W, Day 6 AW, Days 2-6

35.2+0.3 20.2+ 1.0

30.5 f 0.4 15.5 + 1.4

28.6 f 0.3 13.6+ 1.1

25.3 +0.4 10.3 + 1.4

36+0.3 21 kO.9

32.9 f 0.5 17.9+ 1.5

Utilization efficiency Days 2-6 (I) Days 2-6, (II) Days 5-6 (I)

7.4+0.8 5.3 *0.7 4.9* 1.0

13.1+ 1.7 18.6*2.3 6.5 f 2.6

5.7+0.6 4.1+0.5 4.5* 1.1

12.0+ 1.8 17.2+4.1 5.5+3.0

9.6+ 1.0 6.9 f 0.8 5.5kO.9

8.3f 1.5 11.9k2.1 6.5+ 1.5

Protein W, Day 6 AW, Days 2-6

42.6* 1.6 26.6 + 2.3

38.1+2.1 22.1+ 2.2

34.7 * 1.4 18.7k2.1

30.9+ 1.8 14.9 + 2.4

43.2* 1.3 27.2 + 2.1

42.1+ 2.7 26.1k3.1

Utilization efficiency Days 2-6 (I) Days 2-6, (II) Days 5-6 (I)

12.8k2.7 7.2+ 1.7 7.4+ 1.9

24.5 + 5.5 28.Ok4.7 10.4*4.6

10.5 + 1.2 5.9*0.7 7.2+ 1.7

23.3 f 2.2 26.3 k9.5 9.1 f 8.3

15.3 + 0.5 9.0* 1.9 8.2+ 1.7

15.0+0.1 17.8 + 3.7 10.4+2.9

Protein per larva: nitrogen per larva X 5.1.

118

G. 0ie et al./Aquaculture

153 (1997) 103-122

being transferred to the fish tanks (see Fig. 2). An estimation of the utilization efficiency in larvae (Days 2-6), which considered the change in nutritional content of the rotifers in the larval tanks, resulted in carbon utilization efficiencies of 4.1-6.9% in tanks with algae present and 11.9- 18.6% in tanks without algae. The comparable protein utilization efficiency values were 5.9-9.0% and 17.G28.0% for tanks with and without algae, respectively (Table 10). No significant differences in utilization efficiency values were obtained between treatments at Days 5-6 when the water flow was initiated. The values for protein still remained higher than those for carbon.

4. Discussion The larval survival was affected by the nutritional value of the rotifers and the survival was systematically higher when algae were added along with the rotifers. The positive effect of algal addition on survival was far stronger than the effect of the variable protein and lipid enrichments of the rotifer diets. Irrespective of algal addition or not, the survival was highest for larvae fed protein-enriched rotifers (PA and PC larvae) and lowest for larvae fed lipid-enriched rotifers (LA and LC larvae>. This suggests that P-rotifers, with their high and balanced content of protein and lipid, were beneficial compared to the other rotifer diets used. It is notable that L-rotifers, which were normal short-term enriched rotifers widely used in marine aquaculture, gave the poorest survival in this experiment. Algal addition resulted in enhanced larval growth rate between Days 5 and 12 (P < 0.05) compared to tanks without algal addition. However, no significant difference in growth rate for the rotifer diets was observed. The enhanced initial growth rate of larvae cultivated with algae in the water corresponded well with the enhanced feeding rates of rotifers found for the early larval stages (stagnant phase, Days 2-61, and with earlier reports (Naas et al., 1992; Reitan et al., 1993). The quantitative and relative content of essential n-3 fatty acids of the larvae were high and quite close for all treatments, suggesting satisfactory fatty acid content of the diets (Le Milinaire et al., 1983; Rainuzzo et al., 1994). Pigmentation was adequate in all groups, in good agreement with the high DHA/EPA ratio found in 12-day-old larvae (Reitan et al., 1994b). Larval composition of essential amino acids was in agreement with earlier reports (Watanabe et al., 1983; Cowey, 1994). The results showed a higher content of glycine, alanine, valine and leucine and a lower content of glutamic acid + glutamine, histidine, methionine and lysine in turbot larvae, than in whole body tissue of rainbow trout, Atlantic salmon and channel catfish (Wilson, 1989). The microbial conditions of the larvae, and the viability of the larvae during the salinity test, were not systematically related to the experimental treatments. To define an integrated unified description of the nutritional value of the three diets, we assume that high protein per rotifer, high lipid per rotifer, and high protein to lipid ratio are beneficial nutritional characteristics of rotifers used in first feeding of turbot larvae. If, additionally, these factors are given the same weight, we can rank the diets on a relative scale based on the figures given in Table 2. The relative score estimate (Table 11) are proportional to the absolute values of Table 2, with the lowest value fixed at 1,

119

G. Bie et al./Aqwculture 153 (1997) 103-122 Table I1 Relative values treatments

of protein

per ind., lipid per ind. and the ratio of protein/lipid

in the different

Relative values

P-rotifers

L-rotifers

N-rotifers

Protein per ind. Lipid per ind. Protein/lipid Sum

1.5 1.7 1.3 4.5

1.0 1.3 1.0 3.3

1.2 1.0 1.5 3.7

feeding

and their sum expresses the integrated nutritional value of the rotifers, based on the assumptions made above. Table 11 shows that P-rotifers gave the highest relative score for protein per rotifer, lipid per rotifer and integrated nutritional value, whereas N-rotifers showed the highest score for the ratio of protein to lipid. L-rotifers showed the lowest score for both protein per rotifer and protein per lipids, and a total score close to the minimum of 3. It is notable that percent survival through the larval stage, both with and without algae added, corresponded exactly with the integrated nutritional score of Table 11. Assuming that the content of essential n-3 fatty acids in the diets was satisfactory, this may suggest that the protein and energy contents of the rotifers affect the survival of turbot larvae during early larval stages. The pronounced differences in survival with and without algae added may be explained as a factor of enhanced rotifer energy and protein content. The simulations made for protein stability in fed and starved rotifers (Fig. 2) and earlier simulations made for fatty acids and lipids (Olsen et al., 1993; Reitan et al., 1994b) clearly show that the lipid and protein content was maintained or enhanced in tanks with algae present, while without algal addition both lipid and protein were lost quite rapidly. The changes in biochemical composition which may be expected to take place in the larval tanks before consumption by the larvae may be more pronounced than the differences in the rotifer diets (i.e. Fig. 2). This may be attributed to the lipid content (Reitan et al., 1993) but also to the improved protein and energy level of rotifers maintained in larval tanks together with algae contribute to the higher survival of the larvae. The supply of protein and energy in the individual rotifers seemed to play an important role, directly and indirectly, and the importance of protein and the protein/lipid ratio during early larval stages presumably has been underestimated so far. The present experiment allows a closer evaluation of the effect that algae have on protein availability and conversion in early stages of turbot larvae. The resolution of the data was not good enough to judge between the diets. It was demonstrated that larvae maintained with algae consumed a higher numbers of rotifers and these rotifers contained higher levels of protein and energy during the very early stages. The estimated conversion efficiencies (Table 10) indicate that only a minor fraction of the protein (lo-25%) and energy (6-13%) of the rotifer diets was actually converted into larval protein or biomass to sustain the growth rate measured. The results for P- and L-larvae indicate that a lower efficiency was required when algae were added, whereas N-larvae showed approximately equal figures with and without added algae (estimate I, Table

120

G. Oie et al./Aquaculture

153 (1997) 103-122

10). If the changes in rotifer nutritional value in the larval tanks (Fig. 2) and the feeding of new rotifers to the tanks during the period are considered (estimate II, Table lo), the lower protein and energy requirements to sustain actual growth becomes obvious for all groups of larvae maintained with algae. In fact, use of algae may reduce the required efficiency by about 50% compared to the initial situation, whereas no use of algae may give a slight increase of the required efficiency needed to sustain the growth rate measured in these tanks. Although we cannot exactly quantify how much the rotifers change in the larval tanks, we conclude that use of algae will reduce the required conversion efficiency of protein and carbon through the initial 2-6 days by more than 50% when the larvae are maintained under stagnant conditions. The highest protein conversion efficiencies of 26-28% found for larvae maintained without algae may be close to the maximum conversion possible. This cannot be concluded for sure, but the fact that the average initial specific growth rate of the larvae maintained without algae was significantly lower (P < 0.05) than the average rate with algae, suggests that protein supply was limiting, or close to limiting, in the groups maintained without algae. The conversion efficiencies of protein and energy for Days 5-6, the first day of water exchange, was of the same magnitude for all treatments. We expect the nutritional effects of algae on the rotifers in larval tanks to be of minor importance when the rotifers residence time is as short as a few hours. The confidence limits of both the protein and energy conversion efficiencies are too broad to see diet-dependent variations. The values estimated for Days 5-6 may also act as an internal control of accuracy of methods, including the estimation of rotifer ingestion rates, measurements of larval growth and chemical composition, and measurements of the rotifer biochemical composition. The results of the analysis of the larval gut contents support the above evaluation of digestion efficiency. Larvae maintained without algae showed systematically higher rotifer numbers in their gut compared to larvae maintained with algae, although their respective ingestion rate was lower (Fig. 4). This may imply a longer residence time of the food in the larval gut, and presumably also higher digestion and assimilation efficiencies, of larvae maintained without algae than in larvae maintained with algae. Others have shown that high food rations may result in an overloading of the intestinal digestive capacity and thereby a reduced absorption efficiency of food (Werner and Blaxter, 1980; Boehlert and Yoklavich, 1984) and a high evacuation rate in marine larvae (Chitty, 1981). We must emphasize, however, that we cannot explain why the larvae ingested the food much faster when algae were added to the tanks. This observation was also made in earlier experiments (Naas et al., 1992; Reitan et al., 1993). The food concentration in terms of rotifer concentrations was equal for all treatments. The higher feeding rates of turbot larvae maintained with algae in the very first period of feeding may be related to the different microbial colonization of the larval intestine during the first days. It has been shown that algal addition or thorough maturation of the bacterial community of the rearing water, may both reduce the time lag in initial growth response and make survival less variable and on average higher (Skjermo and Vadstein, 1993; Bergh et al., 1994; Skjermo et al., 1997).

G. 0ie et al./Aqwculture

153 (1997) 103-122

121

We conclude that the protein and energy content of the rotifers, as well as their balance between protein and lipid, is important for obtaining high growth rate and survival during the early stages of turbot cultivation. We also suggest that the positive effect on larval survival obtained by supplying algae during the early stages of larval cultivation, may partly originate in enhanced energy and protein supplementation to the larvae. There may, however, also likely be an element of improved microbial conditions involved when algae are added, and this effect is assumed to be important during the very first days.

Acknowledgements We thank Marte Schei and Merethe Selnes for technical assistance during the experiments. This study was supported by the Norwegian Research Council (NFR) and the EU project ‘Effect of broodstock management and broodstock nutrition on quality of turbot Scophthalmus maximus production’.

References Ben-Amotz, A., Fishler, R. and Schneller, A., 1987. Chemical composition of dietary species of marine unicellular algae and rotifers with emphasis on fatty acids. Mar. Biol., 95: 31-36. Bergh, 0.. Naas, K. and Harboe, T., 1994. Shift in intestinal microflora of Atlantic halibut (Hippoglassus hippogloppus) larvae during feeding. Can. J. Fish. Aquat. Sci., 51: 1899-1903. Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. B&hem. Physiol., 37: 911-917. Boehlert, G.W. and Yoklavich, M.M., 1984. Carbon assimilation as a function of ingestion rate in larval pacific herring Clupea harengus pullusi Valenciennes. J. Exp. Mar. Biol. Ecol., 79: 251-262. Chitty, N., 1981. Behavioral observations of feeding larvae of bay anchovy, Anchoa mitchilli, and bigeye anchovy, Anchoa lumprotaeniu. Rapp. P.-V. Reun. Cons. hit. Explor. Mer., 178: 320-321. Cowey, C.B., 1994. Amino acid requirements of fish: a critical appraisal of percent values. Aquaculture, 124: l-11. Dhert, P., Lavens, P., Dehasque, M. and Sorgeloos, P., 1994. Improvements in the larviculture of turbot Scophthalmus maximus: zootechnical and nutritional aspects, possibility for disease control. In: P. Lavens and R.A.M. Remmerswaal (Editors), Turbot Culture: Problems and Prospects. European Aquaculture Society, Special Publication No. 22, Gent, Belgium. pp. 32-46. Gatesoupe, F.J., 1990. The continuous feeding of turbot larvae, Scophthalmus marimus, and control of the bacterial environment of rotifers. Aquaculture, 89: 139-148. Gulliard, R.R.L. and Ryther, J.H., 1962. Studies on marine planktonic diatoms. I. Cyclotella nann Hudtedt, and Detonulu conferuacea (Cleve) Gran. Can. J. Microbial., 8: 229-239. Howell, B.R., 1979. Experiments on the rearing of larval turbot Scophthalmus maximus L. Aquaculture, 18: 215-225. Jones, A., Prickett, R.A. and Douglas, M.T., 1981. Recent developments in techniques for rearing marine flatfish larvae, particularly turbot (Scophthalmus muximus), on a pilot commercial scale. Rapp. P.-V. R&n. Cons. Int. Explor. Mer., 178: 522-526. Kanazawa, A., 1985. Essential fatty acid and lipid requirement of fish. In: C.B. Cowey, A.M. Mackie and J.G. Bell (Editors), Nutrition and Feeding in Fish. Academic Press, London. pp. 281-298. Koven, W.M., Tandler, A., Kissil, G.W. and Sklan, D., 1992. The importance of n-3 highly unsaturated fatty acids for growth in larval Sparus aurora and their effect on survival, lipid composition and size distribution, Aquaculture, 104: 91-104.

122

G. 0ie et al./Aquaculture

153 (19971 103-122

Ltger, P., Bengtson, D.A., Simpson, K.L. and Sorgeloos, P., 1986. The use and nutritional value of Artemia as food source. Oceanogr. Mar. Biol. Ann”. Rev., 24: 521-623. Le Milinaire, C., Gatesoupe, F.J. and Stephan, G., 1983. Approche du besoin quantitatif en acides gras longs polyinsaturatts de la serie n-3 chez la larve de turbot (Scophthalmus maximus). C.R. Acad. Sci. Paris, 269: 917-920. Lubzens, E., Tandler, A. and Minkoff, G., 1989. Rotifers as food in aquaculture. Hydrobiologia, 186/187: 387-400. Minkoff, G. and Broadhurst, A.P., 1994. Intensive production of turbot, Scophthalmus maximus, fry. In: P. Lavens and R.A.M. Remmerswaal (Editors), Turbot Culture: Problems and Prospects. European Aquaculture Society, Special Publication No. 22, Gent, Belgium, pp. 14-29. Naas, K.E., Nress, T. and Harboe, T., 1992. Enhanced first feeding of halibut larvae (Hippoglossus hippoglossus) in green water. Aquaculture, 105: 143-156. Olsen, Y., Reitan, K.I. and Vadstein, 0. 1993. Dependence of temperature on loss rates of rotifers, lipids, and 03 fatty acids in starved Brachionus plicatilis cultures. Hydrobiologia, 255/256: 13-20. Pohl, P., 1982. Lipids and fatty acids of microalgae. In O.R. Zaborsky (Editor), CRC Handbook of Biosolar Resources, Vol, I, Part I. CRC Press, Boca Raton, FL, pp. 383-404. Rainuzzo, J.R., Reitan, K.I. and Olsen, Y., 1994. Effect of short- and long term lipid enrichment on total lipids, lipid class and fatty acid composition in rotifers. Aquacult. Int., 2: 19-32. Reitan, K.I., Rainuzzo, J.R., 0ie, G. and Olsen, Y., 1993. Nutritional effects of algal addition in first feeding of turbot (Scophthdmus manimus L.) larvae. Aquaculture, 118: 257-275. Reitan, K.I., Rainuzzo, J.R. and Olsen, Y., 1994a. Effect of nutrient limitation on fatty acid and lipid content of marine microalgae. J. Phycol., 30: 972-979. Reitan, K.I., Rainuzzo, J.R. and Olsen, Y., 1994b. Influence of lipid composition of live feed on growth, survival and pigmentation of turbot larvae. Aquacult. Int., 2: 33-48. Roald, H., 1994, Studier av amino syre/protein behov hos marin yngel-analysegrunnlaget. (In Norwegian), Diploma-thesis, Laboratory of Biotechnology, Institute of Technology, University of Trondheim, pp. 46-52. Sargent, J.R., Henderson, R.J. and Tocher, D.R.,1989. The lipids. In: J. Halver (Editor), Fish Nutrition, 2nd ed., Academic Press, San Diego, pp. 153-217. Scott, A.P. and Middleton, C., 1979. Unicellular algae as a food of turbot (Scophthalmus maximus L.) larvae -the importance of dietary long-chain polyunsaturated fatty acids. Aquaculture, 18: 227-240. Skjermo, J. and Vadstein, O., 1993. The effect of microalgae on skin and gut bacterial flora of halibut larvae. In: H. Reinertsen, L.A. Dahle, L. Jorgensen and K. Tvinnereim (Editors), Proceedings from International Conference on Fish Farming Technology, August 1993, Trondheim, Norway. Balkema, Rotterdam, pp. 61-67. Skjermo, J., Salvesen, I., 0ie, G., Olsen, Y. and Vadstein, O., 1997. Microbially matured water: a technique for selection of a nonopportunistic bacterial flora in water that may improve performance of marine larvae. Aquacult. Int., in press. Watanabe, T., Kitajima, C. and Fujita, S., 1983. Nutritional values of live food organisms used in Japan for mass propagation of fish: a review. Aquaculture, 34: 115-143. Werner, R.G. and Blaxter, J.H.S., 1980. Growth and survival of larval herring (Clupea harmgus) in relation to pray density. Can. J. Fish. Aquat. Sci., 37: 35-46. Whyte, J.N.C. and Nagata, W.D., 1990. Carbohydrate and fatty acid composition of the rotifer, Brachionus plicatilis, fed monospecific diets of yeast or phytoplankton. Aquaculture, 89: 263-272. Wilson, R.P., 1989. Amino acids and proteins. In: J.E. Halver (Editor), Fish Nutrition. Academic Press, Inc., San Diego, pp. 111-152. Witt, Il., Quantz, G., Kuhlmann, D. and Kattner, G., 1984. Survival and growth of turbot larvae Scophthalmus maximus L. reared on different food organisms with special regard to long-chain polyunsaturated fatty acids. Aquacult. Eng., 3: 177-190. 0ie, G., Reitan, K.I. and Olsen, Y., 1994. Comparison of rotifer quality with yeast and algal-based diets. Aquacult. Int., 2: 225-238.