Nutrient evaluation of rotifers and zooplankton: feed for marine fish larvae

1 2 1

1 Norwegian college of fishery science, Faculty of biosciences, Fisheries and economics (BFE), University of Tromsø, Tromsø,

Norway; 2 National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway

The objective of this study was to examine the biochemical

composition of intensively reared rotifers after enrichment

with three commonly used enrichment media, Multigain,

Ori-Green and DHA-enriched freshwater algae Chlorella,

using standard enrichment protocols at a local cod larvae

producer and compare it with that of natural zooplankton

from Lofilab AS, a cod larvae producer using semi-inten-

sive rearing techniques. Unenriched rotifers were analysed

to examine whether the enrichment procedures were suc-

cessful in increasing the content of essential nutrients to

level requirements for marine fish larvae. Neither total lip-

ids nor proteins were affected by enrichment. Unenriched

rotifers were significantly lower in highly unsaturated fatty

acids (HUFAs) and significantly higher in linoleic acid

(LA, C18:2, n-6), than were zooplankton. Enrichment with

Chlorella and Multigain increased the HUFAs significantly,

while they were slightly reduced after enrichment with Ori-

Green. Total amino acids and mineral content were unaf-

fected by enrichment. Zooplankton was rich in taurine and

selenium, whereas rotifers were devoid of it, both prior to

and after enrichment. Using zooplankton as a reference for

the nutritional requirements of marine fish larvae, results

from this study demonstrate that enrichment media cur-

rently in use are not effective for enhancing the nutritional

quality of rotifers.

KEY WORDS: fatty acids, lipids, live feed enrichment, sele-

nium, taurine, zooplankton

Received 6 July 2011; accepted 8 March 2012

Correspondence: Hanne K. Mæhre, Norwegian college of fishery science,

BFE, University of Tromsø, N-9037 Tromsø, Norway. E-mail: hanne.

[email protected]

Farming of cod (Gadus morhua L.) and other marine spe-

cies faces several challenges regarding growth, survival and

physical development of the fish at early life stages when

compared with salmon (Salmo salar) farming. Owing to a

poorly developed digestive system in marine fish larvae, the

uptake and utilization of essential nutrients is difficult.

Although there have been made attempts to start-feed mar-

ine fish larvae with formulated feeds, the common percep-

tion is that they depend on easily digestible live feed during

this developmental stage. In Norway, most of the produc-

tion of marine fish larvae is based on intensive rearing sys-

tems using rotifers (Brachionus sp.) as prey organism.

However, some hatcheries, as for instance Lofilab AS in

Lofoten, Norway, produce their larvae in closed seawater

ponds using the natural prey, zooplankton as feeding

source. Growth studies performed at Lofilab AS have

shown that cod larvae fed zooplankton can achieve a daily

growth rate, twice as high as larvae fed rotifers (Busch et al.

2009, 2010). Although live feeds are used for only 20–

25 days post hatch, long-term studies from Lofilab AS have

shown that the difference in growth has already been mani-

fested and increases further on (D. Hansen, unpublished

data). These data correspond well with the study of Imsland

et al. (2006), where the difference in growth was maintained

for several weeks after weaning onto formulated feed.

Furthermore, zooplankton-fed marine fish larvae have a

higher survival rate (Rajkumar & Vasagam 2006; Busch

et al. 2010) and a lower frequency of skeletal deformities

(Imsland et al. 2006). Atlantic halibut larvae fed zooplank-

ton also had less pigmentation and eye migration errors

(Hamre et al. 2002) than those fed Artemia.

Difference in nutritional quality is one explanatory factor

for the differences in rearing success of fish larvae fed the

different live prey (Cahu et al. 2003; Hamre et al. 2008;

van der Meeren et al. 2008). Rotifers are deficient in

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ª 2012 Blackwell Publishing Ltd

2012 doi: 10.1111/j.1365-2095.2012.00960.x. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition

important nutritional components, such as the highly

unsaturated fatty acids (HUFAs), eicosapentaenoic acid

(EPA, C20:5, n-3) and docosahexaenoic acid (DHA, C22:6,

n-3). Other limiting nutrients are some amino acids (Ara-

gao et al. 2004a,b), vitamins and minerals (Hamre et al.

2008). Enrichment of these organisms to enhance their

nutritional value before feeding them to the larvae is there-

fore a necessity, and the main focus has traditionally been

to optimize the fatty acid profile. Numerous studies have

been performed and published on this research field, with

focus on growth, survival rate and fatty acid profile in the

fish (Park et al. 2006; Garcia et al. 2008a,b,c), but because

of the high specific growth rate in the larval period, the

composition of amino acids in the enriched rotifers may be

just as important (Aragao et al. 2004a).

Based on the growth studies performed at Lofilab AS,

the objective of this study was to compare the biochemical

composition of rotifers after enrichment with three com-

monly used enrichment media using standard enrichment

protocols to that of zooplankton from Lofilab AS. Unen-

riched rotifers were also analysed to examine whether the

enrichment procedures were successful in increasing the

content of essential nutrients to level requirements for mar-

ine fish larvae. Owing to lack of direct requirement mea-

sures on cold water marine fish larvae, National Research

Council (NRC 1993), requirements of fish were used as ref-

erence in this study.

Zooplankton/Copepods As a result of light and tempera-

ture conditions in North Norway, pond production of zoo-

plankton is limited to the summer season. The

zooplankton/copepods used in this experiment were col-

lected at Lofilab AS (Steine, Norway), in September 2008.

A characterization of the copepods performed in the spring

of 2008 revealed that the most numerous of the adult cope-

pods were Eurythemora affinis, Calanus finmarchicus and

Microsetella norvegica. There were also a large number of

nauplii and copepodites present, but these could not be

characterized because of their very general appearance.

The production of zooplankton/copepods followed a

standard procedure at Lofilab AS and the experimental

conditions described in this study are common for this pro-

duction site. This included fertilization of closed seawater

ponds, monitoring of temperature, salinity and oxygen sat-

uration of the water. The aim of fertilization is to improve

the production of phytoplankton which in turn gives rise

to an increased amount of zooplankton/copepods. Fertil-

ization also helps to keep the oxygen saturation in the

water at a little over 100%, and the typical frequency of

fertilization is 4–5 times per season. In 2008 the fertilizer

used was NPK 21-4-10 and 18-3-15 (Felleskjøpet, Norway).

Oxygen saturation and water temperature on the collection

day were 121% and 10.6 °C respectively. Day length on

the collection day was 14 h.

Seawater from the closed pond was filtrated through sev-

eral filters with pore sizes ranging from 50 to 1000 lm,

after which the different fractions of zooplankton/copepods

were fed continuously to the rearing tanks containing the

cod larvae. (Nora A. Rist & Espen Vang, personal commu-

nication). During a feeding period of total 20–25 days,

the fraction size >250 lm is used between 13 and 25 DPH.

The smaller fractions are used for shorter periods, fraction

50–150 lm between 2 and 8 DPH, and fraction 150–250 lm

between 9 and 12 DPH (Busch et al. 2010).

Zooplankton samples (n = 3) were collected during one

feeding cycle and from one pond. At the time of sampling,

mainly zooplankton from the largest fraction (>250 lm)

were available and they were collected from the outlet of

the filtering unit, rinsed with fresh seawater and frozen

immediately. The samples were 50–80 g wet weight.

Rotifers Rotifers were collected from a local producer of

cod larvae in October 2008. According to the producer’s

standard procedures, rotifers were kept in large tanks and

given 0.3 g of DHA-enriched Chlorella (Chlorella Industry

Co. Ltd., Tokyo, Japan) per million rotifers per day as

maintenance feed. The enrichments were carried out in

600 L tanks filled with seawater, salinity being

34–35 g L�1. The water was aerated during enrichment

and oxygen saturation was 100–120%. Time and tempera-

ture were 2 h and 20 °C respectively. Prior to enrichment,

the rotifers were rinsed and transferred to enrichment

tanks where they were enriched with either Multigain

from Dana Feed AS (Horsens, Denmark), Ori-Green from

Trouw France SA (Fontaine les Vervins, France) or the

DHA-enriched freshwater algae Chlorella sp. Multigain

and Ori-Green were given at a dose of 0.2 g per million

rotifers while Chlorella was given at a dose of 0.5 mL per

million rotifers. After enrichment the rotifers were rinsed

again (Thor Arne Hangstad & Kristin Skar, personal

communication). Owing to the short duration of enrich-

ments, a substantial loss of rotifers was not expected and

hence the need of registration of rotifer conditions was

not considered necessary.

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Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd

Rotifer samples (n = 3 in each group) were collected

prior to enrichment and after each of the three enrichment

treatments. Sample sizes were approximately 100 g wet

weight and all samples were filtered to remove seawater

and subsequently frozen. Samples of the enrichment media

were also collected. As not all enrichment procedures are

performed every day, the samples used in these experiments

were collected over several days in October 2008.

Samples were subjected to proximate analysis (water, lip-

ids and ash), fatty acid composition, phospholipid analysis,

mineral analysis and analyses of total amino acids (TAA)/

protein. For comparison purposes, all raw materials were

freeze-dried prior to chemical analyses using a Vir-Tis Gen-

esis 35EL freeze dryer (SP Industries, Gardiner, NY,

USA). Freeze-dried samples were stored in the dark at

room temperature and all analyses were performed within

1 month of freeze-drying.

All reagents used in these analyses were of analytical

grade. Chloroform and methanol were purchased from

BDH (Poole, Dorset, UK). All other solvents and chemi-

cals were purchased from Merck (Darmstadt, Germany),

unless otherwise stated.

Water Water content was determined using a modified

version of AOAC method 925.04 (Horwitz 2004), where

10 g of sample was dried at 105 °C until constant weight

and water content was determined gravimetrically. Analy-

ses were performed in triplicate.

Ash Ash content was determined using a modified version

of AOAC method 938.08 (Horwitz 2004). The water free

sample was combusted at 500 °C for 12 h, and ash content

was determined gravimetrically. Analyses were performed

in triplicate.

Extraction of lipids Lipids were extracted according to

Folch et al. (1957) and fat content was determined gravi-

metrically. Analyses were performed in triplicate. The

extracted lipids were subjected to analysis of fatty acid

composition.

Fatty acid composition Each lipid extract was redissolved

to a concentration of approximately 10 mg mL�1 in chloro-

form:methanol (2:1, (v/v)). The samples were trans-methy-

lated, according to Stoffel et al. (1959), using 20 g L�1

H2SO4 instead of 50 g L�1 HCl for the trans-methylation

and heptane instead of petroleum ether for extraction of the

methyl esters. Gas chromatography was performed using an

Agilent 6890N equipped with a 7683 B auto injector and a

flame ionization detector (FID) (Agilent Technologies Inc.,

Santa Clara, CA, USA), with He as the carrier gas. A Var-

ian CP7419 capillary column (50 m 9 250 lm 9 0.25 lm

nominal) (Varian Inc., Middelburg, the Netherlands) was

used. Injector and detector temperatures were 240 and

250 °C respectively. A predefined temperature programme

was used to ensure the best possible separation of the fatty

acids (50 °C for 2 min, then 10 °C per min to 150 °C, fol-

lowed by 2 °C per min to 205 °C and finally 15 °C per min

until 255 °C and stabilization for 10 min). The fatty acids

were identified by comparison with the fatty acid standards

1895, 1893, 1891, PUFA no 1 and PUFA no 3 from Sigma

(Sigma Chemicals Co, St. Louis, MO, USA) and fatty acid

standard 68D from NuChek (NuChec Prep. Inc., Elysian,

MN, USA).

Amino acids Analysis of total amino acids, except trypto-

phan, was performed after acid hydrolysis according to

Moore & Stein (1963). An alkaline hydrolysis was per-

formed for determination of tryptophan, according to

Levine (1982). Analyses were performed in triplicate.

All amino acid samples were analysed by chromato-

graphic separation on an ion exchange column, using lith-

ium citrate buffers of different pH and ionic strength and a

pre-defined temperature programme, as described in Spack-

man et al. (1958). The analysis was performed using a Bio-

chrom 30 amino acid analyser (Biochrom Co, Cambridge,

UK) and the UV signals were analysed by Chromeleon

software (Dionex, Sunnyvale, CA, USA) and compared

with A9906 physiological amino acids (Sigma Chemicals

Co, St. Louis, MO, USA). Protein content of the samples

are given as the sums of individual amino acid residues

(the molecular weight of each amino acid less the molecular

weight of water), according to recommendations by FAO

(2003).

Minerals Calcium (Ca), iron (Fe), magnesium (Mg), phos-

phorus (P), manganese (Mn), copper (Cu), zinc (Zn) and

selenium (Se) were analysed. Analyses were performed

according to method 186 of the Nordic Committee on

Food Analysis (2007). Samples were added to concen-

trated HNO3 and hydrogenperoxide (300 g L�1, (w/v)) and

digested in a microwave oven. Quantitative ICP-MS was

used for determination of the elements and calculation of

the concentrations was based on individual standard

curves. Scandium was used as an internal standard for Ca,

Fe, Mg and P, whereas Rhodium was used as an internal

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


standard for Mn, Cu, Zn and Se. Analyses were performed

in duplicate. Criteria for acceptance of the analytical result

is that the difference between two parallel determinations

should be <10% when the mean is above 10 times the limit

of quantification (LOQ) and <25% when the mean is below

this level.

One-way analysis of variance (ANOVA) was performed using

SPSS 15.0 (SPSS Inc., Chicago, IL, USA). As the sample

size of each collection was rather small a Levene’s test of

homogeneity of variance was performed and in most cases

this test revealed that the variances were not equal. Hence,

the Dunnet T3 test was chosen as post hoc test for evalua-

tion of statistics. Means were considered significantly dif-

ferent at P < 0.05.

As shown in Table 1, zooplankton had a proximate compo-

sition of 32 g kg�1 lipids, 350 g kg�1 proteins and

320 g kg�1 ash, whereas the rotifers had a proximate com-

position of 107 g kg�1 lipids, 400 g kg�1 proteins and

200 g kg�1 ash, prior to enrichment. There were no signifi-

cant changes in lipid content after enrichment with either

media, but rotifers enriched with Chlorella were significantly

lower in fat compared with rotifers enriched with Multigain

and Ori-Green. Protein content was not significantly

affected by enrichment. Ash content was significantly lower

after enrichment with Multigain and Chlorella, whereas it

remained unchanged after enrichment with Ori-Green.

In Table 2, the fatty acid composition of zooplankton and

rotifers is presented. There were significant differences in

several important fatty acids, including EPA (140 and

41 g kg�1 lipid respectively) and DHA (250 and 66 g kg�1

lipid respectively) between zooplankton and rotifers, prior

to enrichment. The sums of polyunsaturated fatty acids

(PUFAs) and n-3 fatty acids were also significantly higher

in zooplankton than in rotifers (569 vs. 392 g kg�1 lipid

and 557 vs. 192 g kg�1 lipid respectively), whereas the ratio

between n-6 and n-3 fatty acids was lower (0.02 vs. 1.04

respectively). Linoleic acid (LA, C18:2, n-6), however, was

significantly higher in rotifers than in zooplankton (200 Table

1Proxim

ate

compositionofrotifers

priorto

andafter

enrichment,thecorrespondingenrichmentmedia

andzooplankton

Enrich

mentmedia

Rotifers

Zooplankton

Multigain

Ori-G

reen

Chlorella

Unenrich

ed

rotifers

Rotifers

enrich

ed

withMultigain

Rotifers

enrich

ed

withOri-G

reen

Rotifers

enrich

ed

withChlorella

Waterco

ntent

before

freeze

drying

(gkg�1WW

1)

89±1B

52±2A

883±3C

871±2b

861±3a

873±1b

858±1a

906±2c

Waterco

ntent

afterfreeze

drying

(gkg�1DW

2)

78±6B

18±3A

12±1A

108±8b

26±1a

110±9b

22±1a

17±1a

Lipids3

(gkg�1DW

4)

389±30C

269±6B

192±16A

107±15ab

133±10b

121±14b

87±3a

83±4a

Protein

5(g

kg�1DW

3)

113±5A

352±17B

412±3C

393±10

348±23

373±11

349±10

348±30

Ash

(gkg�1DW

3)

98±4B

114±5C

82±3A

199±5c

157±3b

199±6c

135±6a

320±4d

Differentcapitalletters

ineach

row

indicate

significantdifferences(P

<0.05)betw

eenthethreedifferentenrich

mentmedia.Differentsm

allletters

ineach

row

indicate

signifi-

cantdifferencesbetw

eenrotifers

andzo

oplankton.

1Weightexp

ressedasgkg�1oftheoriginalwetweight(W

W)samples.

2Weightexp

ressedasgkg�1ofthefreeze

-driedmaterial.

3Lipid

extractionperform

edbytheFo

lchmethod.

4Calculatedweight,

withrespect

totheresidualwaterin

thesample

afterfreeze

-drying.

5Protein

basedontotalaminoacidresidues.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Table

2Main

fattyacidsin

rotifers

priorto

andafter


andzooplankton

Enrich

mentmedia

Rotifers

Zooplankton

Multigain

Ori-G

reen

Chlorella

Unenrich

ed

rotifers

Rotifers

enrich

ed

withMultigain

Rotifers

enrich

ed

withOri-G

reen

Rotifers

enrich

ed

withChlorella

C14:0

56.2

±1.4

C17.2

±0.1

BbdlA

14.9

±0.4

ab

27.2

±1.9

c17.5

±0.8

b13.4

±0.9

a34.3

±1.6

d

C16:0

371.3

±0.6

C172.0

±0.4

B137.6

±7.6

A248.3

±2.7

bc

324.3

±39.3

d280.3

±2.8

cd219.3

±1.7

ab

184.2

±0.9

a

C16:1,n-7

bdl

bdl

bdl

15.5

±3.0

b12.1

±1.4

ab

12.5

±0.9

ab

13.4

±0.9

b7.5

±0.3

a

C18:0

15.0

±6.3

A50.9

±0.3

B15.0

±1.0

A68.8

±5.3

c48.1

±6.0

b71.5

±10.8

c51.4

±0.6

b28.2

±1.0

a

C18:1,n-9

28.8

±3.9

B116.0

±0.5

C20.6

±1.9

A35.2

±5.5

bc

55.9

±15.8

c81.1

±4.4

d32.5

±2.2

ab

12.9

±1.0

a

C18:1,n-7

bdlA

16.2

±0.0

C11.7

±1.6

B12.1

±0.8

a16.1

±7.4

ab

16.3

±0.9

ab

9.5

±0.3

a21.4

±0.5

b

C18:2,n-6

38.3

±0.7

A133.0

±0.2

B218.4

±2.2

C200.0

±8.7

c152.3

±14.4

b177.9

±15.4

bc

205.3

±4.1

c12.1

±0.3

a

C18:3,n-3

6.2

±0.1

A29.5

±0.0

B79.3

±0.6

C42.5

±1.5

b38.4

±3.2

ab

33.4

±2.1

a53.0

±1.1

c50.0

±0.3

c

C18:4,n-3

bdl

bdl

bdl

17.1

±2.1

b9.3

±0.6

a13.9

±3.2

ab

10.6

±0.4

ab

108.8

±0.8

c

C20:0

bdl

bdl

bdl

18.9

±0.6

b15.3

±1.4

ab

19.1

±0.8

b14.5

±0.2

bbdla

C20:5,n-3

15.7

±0.1

A47.9

±0.0

B81.2

±2.9

C40.6

±0.8

b61.7

±3.9

c31.7

±1.1

a61.4

±1.5

c139.7

±0.9

d

C24:0

bdl

bdl

bdl

8.9

±0.4

b10.5

±0.4

a5.9

±0.5

a15.1

±0.5

ab

4.9

±0.2

a

C24:1

bdl

bdl

bdl

7.8

±0.4

cbdla

8.3

±0.9

c5.5

±0.4

b16.2

±0.8

d

C22:5,n-3

bdlA

15.1

±0.0

B24.7

±1.1

C25.8

±0.7

c32.5

±2.4

d18.8

±0.9

b39.6

±0.7

e8.5

±0.2

a

C22:6,n-3

305.8

±6.4

B292.2

±0.9

B181.7

±9.8

A65.8

±1.2

a97.0

±6.8

b61.2

±2.1

a94.9

±0.4

b249.7

±3.9

c

ΣsaturatedFA

s442.4

±5.6

C240.1

±0.6

B152.6

±8.6

A359.8

±7.2

c416.7

±32.4

d394.2

±12.0

cd313.7

±0.9

b250.0

±2.3

a

ΣmonounsaturatedFA

s28.8

±3.8

A156.7

±0.1

B32.3

±1.5

A70.6

±3.5

a74.7

±31.5

a118.1

±5.4

b60.9

±3.6

a55.5

±4.0

a

ΣpolyunsaturatedFA

s366.0

±6.9

A517.6

±0.7

B585.2

±12.2

C391.9

±8.3

b388.1

±25.2

b336.8

±14.2

a464.7

±4.5

c568.9

±3.7

d

-where

ofn-3

327.7

±6.4

A384.6

±0.9

B366.9

±14.3

B191.9

±0.6

b235.7

±12.5

c158.9

±2.2

a259.4

±0.6

d556.8

±3.4

e

n-6/n-3

ratio

0.12±0.00A

0.35±0.00B

0.60±0.03C

1.04±0.05c

0.65±0.04b

1.12±0.11c

0.80±0.01b

0.02±0.00a

Valuesare

exp

ressedasmean±SD

(n=3)andin

gkg�1lipid.Differentcapitalletters

ineach

row

indicate


<0.05)betw


-


allletters

ineach

row

indicate

significantdifferencesbetw

eenrotifers

andzo

oplankton.bdl,below

detectionlimit.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


and 12 g kg�1 lipid respectively). The fatty acid composi-

tion of the enrichment media was quite different and as a

general trend, the changes in fatty acid composition of roti-

fers after enrichment were related to their respective med-

ium. After enrichment with Multigain and Chlorella, EPA

and DHA in rotifers were significantly increased to 62 and

97 g kg�1 lipid and 61 and 95 g kg�1 lipid respectively.

Enrichment with Ori-Green resulted in a significant reduc-

tion in the EPA level, whereas the DHA level remained

unchanged. The increased EPA and DHA levels, after

enrichment with Multigain and Chlorella also gave an

increased amount of n-3 fatty acids and hence lowered the

ratio between n-6 and n-3 fatty acids. Enrichment with

Chlorella also increased the sum of PUFAs. Both the sum

of PUFAs and the amount of n-3 fatty acids was signifi-

cantly reduced after enrichment with Ori-Green, whereas

the ratio between n-6 and n-3 remained unchanged. The

amount of LA was significantly reduced after enrichment

with Multigain.

There were no significant difference in TAA content

between rotifers and zooplankton, but the content of essen-

tial amino acids were significantly lower in zooplankton

than in unenriched rotifers. The most abundant amino

acids in rotifers were Glu, Lys, Asp and Leu, whereas Glu,

Gly, Pro and Lys were dominant in zooplankton. Taurine

content in zooplankton was approximately 8 g kg�1,

whereas rotifers were devoid of it. Rotifers enriched with

Multigain was significantly lower in Lys, Pro, Ser, Ala and

Tyr compared with unenriched rotifers, whereas rotifers

enriched with Chlorella was significantly lower in Lys and

Pro compared with unenriched rotifers.

As shown in Table 4, rotifers prior to enrichment were

2–10 times lower in all of the minerals analysed than were

zooplankton, the only exception being the level of phos-

phorus, which was slightly higher in rotifers. Selenium was

not detected in rotifers, neither prior to nor after enrich-

ment, whereas zooplankton contained 1.53 mg kg�1 dry

weight (DW). The mineral composition of the enrichment

media was quite heterogeneous, but all of them were

mainly within the requirement ranges defined by the NRC

(1993). All of the media were lower in Ca than the require-

ments and in addition, Multigain was low in P and Chlo-

rella was low in Zn and Se. The differences in the mineral

content of the enrichment media were not reflected in the

rotifers, as enrichment had little, if any, effect on the min-

eral content of the rotifers.

The exact nutritional requirements of most cold water mar-

ine fish larvae have yet to be defined, but the National

Research Council (NRC 1993) has given an overview of

the nutritional requirements of adult fish and juveniles. The

species listed are, however, mainly tropical and subtropical

species, and some deviations should be expected for cold

water species. In addition, this list presents the require-

ments for adult and juvenile fish. Several studies (Izquierdo

et al. 1989; Mourente & Tocher 1992) have shown that lar-

vae often have quantitatively higher requirements of, for

instance n-3 HUFAs. Zooplankton, or copepods, is the

main nutrient source for wild marine fish larvae. These

organisms contain large amounts of essential nutrients,

such as HUFAs, good quality proteins and minerals, and

their biochemical composition is probably close to the true

requirements of the larvae, but their biochemical composi-

tion will differ substantially with geographical location,

season and size. There is, however, a possibility that the

nutrient levels of zooplankton/copepods are much higher

than the actual requirements of cod larvae (Hamre et al.

2008). Dietary requirements are most often determined by

manipulation of diets and this is difficult to achieve in live

feeds. Until formulated feed suitable for cold water marine

fish larvae has been developed and actual requirements can

be tested, the biochemical composition of zooplankton/co-

pepods should be used as reference.

Rotifers are deficient or low in several essential nutrients,

such as HUFAs, some amino acids, vitamins and minerals

(reviewed by Conceicao et al. (2010)). To make rotifers

suitable as feed for marine fish larvae, their biochemical

composition has to be improved and this is accomplished

through different enrichment procedures. Most enrichment

procedures focus on increasing the content of HUFAs and

this goal is reflected in the composition of the enrichment

media, which normally are rich in HUFAs, especially

DHA. Along with the composition of the media, the result

of the enrichment is also affected by physical factors, such

as duration of enrichment, water temperature and light

conditions. The duration of the rotifer enrichment used in

these experiments was 2 h, which was according to the

standard procedure at the local producer and also similar

to the experiment described by Busch et al. (2010). Rotifers

have been shown to have a rapid gut filling rate, with a

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


maximum of 35 min (Baer et al. 2008). This indicates that

a short enrichment period could be sufficient for a ‘boost’

in nutritional value. However, the gut is only a small part

of the rotifers’ total body volume and a longer-term enrich-

ment may be necessary to allow an uptake of nutrients into

the rotifers’ tissues. Focusing on lipids, there has been

described that 24 h of enrichment improved the effect com-

pared with 8 and 16 h (Park et al. 2006), and 24 h seems

to be a standard duration for rotifer enrichment (Ferreira

et al. 2008, 2009; Garcia et al. 2008b). Hence, there is a

possibility that the effects of enrichment could be improved

by increasing the duration of enrichment.

The main objective of most enrichment procedures has tra-

ditionally been to increase the content of HUFAs to cover

the dietary requirements for marine larvae. In this study,

the EPA + DHA in rotifers prior to enrichment were

107 g kg�1 total lipid, corresponding to 11 g kg�1 diet.

After enrichment with Multigain and Chlorella, the

EPA + DHA levels were 155 g kg�1 and 151 g kg�1

respectively, corresponding to 21 g kg�1 and 14 g kg�1

diet. Enrichment with Ori-Green, however, did not have an

impact on the EPA + DHA level in the diet. The sum of

EPA + DHA in zooplankton was 389 g kg�1 total lipid,

corresponding to 32 g kg�1 diet. The requirements of

EPA + DHA for the marine species listed by the NRC

(1993) range from 5 g kg�1 to 20 g kg�1 diet for adult and

juvenile fish. Owing to these fatty acids’ important role in

early development, larvae often have higher requirements

than adults and the optimum dietary levels of different

marine larvae range from 0.3 g kg�1 to 39 g kg�1 (Iz-

quierdo 1996). Environmental factors, such as temperature,

salinity and light have been shown to affect lipid composi-

tion of fish tissue, and requirements could also be affected

by these factors (Izquierdo & Koven 2011). As the content

of HUFAs is important, for instance in the regulation of

the viscosity of membranes, the requirements for cold-

water larvae probably are in the upper range, and hence

the levels of these fatty acids seem not to be adequate in

either of the enriched rotifers.

The fatty acid composition of the feeding sources are

reflected in the larvae and several studies have shown that

larvae fed rotifers contain more LA along with less EPA

and DHA, compared with newly hatched larvae and larvae

fed zooplankton (Busch et al. 2009; Copeman & Laurel

2010). In this study, the LA content in rotifers was 12–17

folds higher than in zooplankton. The regulation of meta-

bolic processes is fine-tuned and hence an altered fatty acid

composition in early life could affect several factors, such

as growth, survival and general development.

The specific growth rate during the larval stage is high and

hence a sufficient intake of amino acids, both essential and

non-essential, is crucial and the balance between them is

important for a proper protein synthesis. However, protein

level, amino acid composition and relative amount of free

amino acids in rotifers have been shown to be rather unaf-

fected by the enrichment media (Srivastava et al. 2006) and

this was to some degree confirmed in this study. None of

the enrichments managed to increase the amino acid con-

tent in the rotifers, but enrichment with Multigain, which

was the media with the lowest protein content, resulted in

lower amounts of Lys, Pro, Ser, Ala and Tyr compared

with unenriched rotifers. As shown in Table 3, there were

no significant differences between zooplankton and the rot-

ifers in the amount of TAAs, whereas the amount of essen-

tial amino acids were lower in zooplankton than in rotifers.

Compared with the requirements given by the NRC (1993),

the relative amount of Met was insufficient, whereas Trp

was just barely sufficient both in rotifers and zooplankton

and this could have impact on protein synthesis. All of the

other essential amino acids were within the requirements.

There was, however, one major difference in the amino

acid composition of zooplankton and rotifers, taurine.

Zooplankton contained approximately 8 g kg�1 DW,

whereas rotifers were devoid of it. Neither of the enrich-

ment media contained taurine. Taurine (2-aminoethanesulf-

onic acid) is an exclusively free amino acid, as it is not

bound to proteins. Taurine is one of the most abundant

FAAs in animal tissues, and apart from some algae, it is

absent in plants. Seafood is normally rich in taurine, but

some studies have shown that the synthesis rate is low dur-

ing the first stages of development even in fish and shellfish

and that supplementation via the feed for larvae and juve-

niles may be necessary (Litaay et al. 2001; Takeuchi 2001;

Kim et al. 2005; Pinto et al. 2010). Japanese flounder and

red sea bream larvae fed on rotifers enriched with taurine

showed increased growth compared with unenriched roti-

fers (Chen et al. 2004, 2005). Supplementation of taurine in

fish feed has been reported to give increased specific growth

rate, increased feed intake and to lower the feed conversion

factor, which may be as a result of its feeding stimulatory

effects in fish (Carr 1982). In addition, the bile-salt acti-

vated lipase (BAL) activity is increased by the presence of

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Table

3Totalaminoacidsin

rotifers

priorto

andafter


andzooplankton

Enrich

mentmedia

Rotifers

Zooplankton

Multigain

Ori-G

reen

Chlorella

Unenrich

ed

rotifers

Rotifers

enrich

ed

withMultigain

Rotifers

enrich

ed

withOri-G

reen

Rotifers

enrich

ed

withChlorella

Essentialaminoacids(g

kg�1DW)

Thr

5.4

±0.2

A15.2

±0.7

B21.0

±0.3

C20.2

±0.5

b17.7

±1.3

ab

19.3

±0.9

ab

18.0

±0.4

ab

17.4

±1.4

a

Val

7.2

±0.5

A23.0

±1.0

B30.7

±0.4

C27.1

±0.9

b24.5

±1.6

ab

26.3

±0.7

b25.2

±1.1

ab

21.7

±2.0

a

Met

2.0

±0.2

A5.8

±0.4

B7.8

±0.1

C8.9

±0.3

8.1

±0.5

8.3

±0.2

8.1

±0.4

9.2

±0.8

Ile

5.3

±0.4

A20.5

±1.2

B20.4

±0.1

B25.9

±1.0

b23.5

±1.4

b24.6

±0.5

b23.5

±0.9

b18.3

±1.6

a

Leu

8.6

±0.5

A35.3

±2.1

B44.9

±0.3

C38.8

±1.0

b35.4

±2.1

b36.3

±0.7

b35.9

±1.0

b29.0

±2.5

a

Phe

5.4

±0.3

A23.9

±1.1

B25.4

±0.3

B25.0

±0.7

b22.9

±1.2

b23.9

±0.9

b23.0

±0.7

b17.1

±1.6

a

Lys

6.5

±0.2

A30.6

±1.4

B44.2

±0.3

C41.2

±0.9

b35.0

±2.3

a37.3

±1.4

ab

35.6

±1.0

a32.6

±2.8

a

His

2.4

±0.2

A9.5

±0.6

B10.0

±0.1

B9.6

±0.1

b8.7

±0.5

ab

9.4

±0.3

ab

9.1

±0.3

ab

8.4

±0.7

a

Arg

12.6

±0.7

A35.1

±1.7

B34.3

±0.3

B29.4

±0.9

26.3

±1.9

28.0

±0.7

26.7

±0.8

29.5

±3.0

Trp

1.8

±0.1

A5.4

±0.2

B8.4

±0.2

C6.2

±0.7

4.4

±0.3

5.8

±0.4

5.2

±0.5

4.9

±0.3

Conditionallyessentialaminoacids(g

kg�1DW)1

Tau

bdl

bdl

bdl

bdla

bdla

bdla

bdla

7.7

±0.6

b

Pro

6.4

±0.5

A21.7

±0.8

B29.0

±0.5

C33.4

±1.1

b26.6

±1.2

a31.3

±1.3

b26.6

±0.6

a33.0

±1.4

b

Cys

bdlA

2.7

±0.2

C1.2

±0.1

B3.8

±0.3

ab

3.5

±0.3

a4.1

±0.1

b3.4

±0.2

a4.0

±0.3

b

Non-essentialaminoacids(g

kg�1DW)

Asp

10.6

±0.7

A37.7

±1.7

C34.3

±0.2

B39.2

±0.9

b35.5

±2.4

ab

37.0

±1.2

b35.2

±0.9

ab

31.5

±3.0

a

Ser

5.3

±0.3

A18.8

±0.9

B17.6

±0.3

B21.9

±0.6

c18.8

±1.5

ab

21.8

±0.6

c19.3

±0.6

bc

16.3

±1.6

a

Glu

32.2

±1.3

A69.9

±3.7

C58.3

±0.6

B62.0

±1.4

56.6

±3.5

59.8

±1.4

56.8

±1.4

57.5

±4.8

Gly

7.3

±0.3

A19.1

±0.9

B31.4

±0.3

C22.7

±0.6

a20.3

±1.4

a21.6

±0.8

a20.6

±0.8

a33.1

±3.1

b

Ala

9.1

±0.5

A20.4

±1.1

B46.6

±0.6

C25.1

±0.7

bc

21.0

±1.6

a23.5

±0.8

ab

21.5

±0.5

ab

28.9

±2.8

c

Tyr

3.3

±0.1

A13.8

±0.7

B15.5

±0.2

C17.7

±0.8

b14.6

±0.9

a17.4

±0.8

ab

15.1

±0.4

ab

14.7

±1.8

a

Orn

0.6

±0.0

A0.2

±0.0

BbdlC

bdla

bdla

bdla

0.4

±0.1

c0.1

±0.0

b

Sum

AA

132.8

±5.5

A409.0

±19.9

B481.0

±3.3

C458.2

±11.1

402.0

±25.0

435.7

±13.3

407.5

±11.9

417.1

±35.9

Sum

essentialAA

56.7

±2.4

A204.4

±10.2

B247.1

±1.7

B232.3

±5.2

b206.3

±12.5

ab

219.3

±6.5

b208.6

±9.0

ab

188.1

±16.6

a

Valuesare

exp

ressedasmean±SD

(n=3)andin

gkg�1DW.Differentcapitalletters

ineach

row

indicate


<0.05)betw


-


allletters

ineach

row

indicate

significantdifferencesbetw

eenrotifers

andzo

oplankton.bdl,below

detectionlimit.

1Theaminoacidscysteine,glutamine,hyd

roxyproline,prolineandtaurineare

classifiedasco

nditionallyessentialforfish

inLi

etal.(2009).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


taurine, indicating that a high level of this amino acid in

the feed could improve lipid metabolism by increasing the

utilization of TAGs (Chatzifotis et al. 2008).

Minerals are involved in a wide variety of functions in fish,

and as marine fish have an uptake of several minerals

through the gills, skin and mouth; it is difficult to define

specific dietary requirements for the different minerals. As

shown in Table 4, all minerals reported, except P, were

higher in zooplankton than in rotifers, both prior to and

after enrichment. None of the minerals analysed was

affected by the enrichment procedures. According to NRC

(1993), rotifers contained sufficient amounts of all of the

minerals analysed, except Se, which could not be detected

in either of the rotifer samples. Selenium content in zoo-

plankton was sufficient, according to the NRC (1993). Sele-

nium is an important part of several enzymes with

antioxidant activity, such as glutathione peroxidases (GPx).

Cod larvae fed Se-enriched rotifers have been shown to

exhibit a higher GPx activity and mRNA expression than

larvae fed control rotifers (Penglase et al. 2010). A low

level of these enzymes could affect the defence against oxi-

dative stress (Sies 1986), and these findings indicate that

rotifers are unable to deliver sufficient amounts of Se to

support an optimal antioxidant status. Other effects of Se

deficiency have been shown to include growth depression

(Gatlin & Wilson 1984), pathological changes in nerve cells

and anaemia (Bell et al. 1986, 1987).

This study has shown that the enrichment media cur-

rently in use do not enhance the nutritional quality of roti-

fers to a level sufficient for marine fish larvae. Using

zooplankton as a reference on adequate nutrition for these,

there is a need for improvement of enrichment procedures.

Previous studies have shown that an increased uptake of

nutrients is correlated with increased duration of enrich-

ment procedures but also improvement of the composition

of enrichment media could be advantageous. Enhancing

the rotifers’ content of important nutrients, such as HU-

FAs, taurine and Se, one at a time, has been shown to

improve growth and survival of marine fish larvae, but as

the overall performance of larvae fed zooplankton is better

than for larvae fed rotifers, an enrichment medium with a

total biochemical composition more close to zooplankton

would probably be beneficial. Utilization of organisms on a

lower trophic level, such as krill and calanus, could be an

option. Small scale commercial catch of these species has

been initiated, with extraction of oils as food supplements

for humans being the main target. Krill and calanus are, as

most lower trophic organisms, rich in HUFAs, taurine and

Se, which are all limiting factors in enrichment media cur-

rently in use. As a conclusion, the differences in fatty acid

composition, taurine and Se content between rotifers and

zooplankton observed in this study, can either separately

or in combination, be a part of an explanation to why mar-

ine fish larvae fed zooplankton have an overall better per-

formance than the ones fed rotifers.

Aragao, C., Conceicao, L.E.C., Dinis, M.T. & Fyhn, H.J. (2004a)

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tions: nutritional implications for fish larvae. Aquaculture, 234,

429–445.Aragao, C., Conceicao, L.E.C., Fyhn, H.J. & Dinis, M.T.

(2004b) Estimated amino acid requirements during early ontog-

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242, 589–605.

Table 4 Macro- and micro-minerals in rotifers prior to and after enrichment, the corresponding enrichment media and zooplankton

Enrichment media Rotifers

ZooplanktonMultigain Ori-Green Chlorella

Unenriched

rotifers

Rotifers enriched

with Multigain

Rotifers enriched

with Ori-Green

Rotifers enriched

with Chlorella

Macrominerals (mg kg�1 DW)

Ca 1324 754 617 2628 2495 2807 1973 17355

Mg 3526 1161 3370 6178 5257 6115 4703 10468

P 4138 6191 14939 11260 10637 10794 11074 9023

Fe 122 295 243 152 144 152 153 1577

Microminerals (mg kg�1 DW)

Mn 42 11 32 10 10 9 10 63

Cu 21 3 4 4 5 4 4 15

Zn 96 37 10 50 48 50 45 214

Se 0.49 0.15 <0.1 <0.1 <0.1 <0.1 <0.1 1.53

Values are expressed as mean of two parallels and in mg kg�1 DW.

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Nutrient evaluation of rotifers and zooplankton: feed for marine fish larvae