Aquaculture Research, 2006, 37, 1662^1670 doi:10.1111/j.1365-2109.2006.01617.

Effect of Artemia enrichment on the growth and

survival of Pacific bluefin tuna Thunnus orientalis
(Temminck & Schlegel) larvae

Amal Kumar Biswas, Jun Nozaki, Michio Kurata, Kenji Takii, Hidemi Kumai & Manabu Seoka
Fisheries Laboratory, Kinki University, Uragami,Wakayama, Japan

Correspondence: A Kumar Biswas, Fisheries Laboratory, Kinki University, Uragami, Nachikatsuura,Wakayama 649-5145, Japan. E-mail:
ns -akb@nara.kindai.ac.jp

Abstract to meet the consumer demand. The growing demand

for raw tuna in the local market led scientists to de-
This study was carried out to investigate the suitabil-
velop techniques for egg production and rearing of
ity of Artemia enriched with docosahexaenoic acid
larvae in captivity to make up the shortfall between
(DHA) and choline as live food on the growth and
the ¢shery supply and demand. Beginning in 1970,
survival rate of the Paci¢c blue¢n tuna (PBT;Thunnus
scientists at the Kinki University, Japan, achieved the
orientalis) larvae. The PBT larvae were fed eitherArte-
successful completion of the PBT life cycle under
mia enriched with oleic acid (Diet 1), DHA (Diet 2),
aquaculture conditions for the ¢rst time in 2002
DHA1choline 1.0 mg L 1 (Diet 3) and DHA1choline
(Sawada, Miyashita, Murata & Kumai 2004). The
2.0 mg L 1 (Diet 4) or striped knifejaw larvae (Diet 5,
spawning of this broodstock resulted in several studies
reference diet), in duplicate for12 days. Enrichment of
of their early life history, including the behaviour and
Artemia with DHA signi¢cantly increased the DHA
morphological development of larvae and early juve-
levels to 13.9, 13.8 and 12.5 mg g 1 on a dry matter
niles, food selectivity of larvae and the growth perfor-
basis in Diets 2, 3 and 4 respectively; however, the
mance (Sawada & Kumai 2000; Miyashita 2002).
levels were signi¢cantly lower than the reference
For the larval stage of PBT, a combination of di¡er-
diet (26.9 mg g 1 dry matter basis; Diet 5). Although
ent feeds such as rotifers, Artemia and newly hatched
growth and survival rate were signi¢cantly improved
larvae of the striped knifejaw Oplegnathus fasciatus
by the enrichment of Artemia with DHA and choline,
(Temminck & Schlegel) has been used to obtain opti-
the improvement was negligible compared with the
mum growth and survival rates (Sawada et al. 2004).
enhanced growth and survival rate of the ¢sh lar-
However, producing this live feed combination re-
vae-fed group (Po0.05). The results demonstrated
quires time and e¡ort to produce su⁄cient amounts
that enriched Artemia does not seem to be the right
for mass culture of tuna larvae. Therefore, providing
choice to feed the PBT larvae perhaps because of the
a suitable feed for larval culture remains an unre-
di⁄culties in achieving the correct balance of fatty
solved problem. The failure of an earlier attempt to
acid with higher DHA/EPA from Artemia nauplii.
use Artemia as the sole feed for tuna larvae was
assumed to be attributed to the lack of n-3 highly
Keywords: Thunnus orientalis, Artemia enrichment,
unsaturated fatty acids (HUFAs), especially docosa-
growth, survival, fatty acids
hexaenoic acid (DHA) in Artemia (Dobbeleir, Adam,
Bossuyt, Bruggman & Sorgeloos 1985). The e¡ective-
ness of Artemia enrichment has been demonstrated
Introduction
in a number of marine species (Watanabe 1993; Fur-
The Paci¢c blue¢n tuna (PBT; Thunnus orientalis) is uita, Takeuchi & Uematsu 1998; Czesny, Kolkovski,
one of the most popular ¢sh in Japan because of its Dabrowski & Culver 1999; Furuita, Konishi & Takeu-
high meat quality and popularity in ‘sashimi’ and chi 1999; Gapasin & Duray 2001; Koueta, Boucaud-
‘sushi’ markets. Every year, Japan needs to import Camou & Noel 2002) although, in some cases, the ef-
thousands of metric tones of raw tuna from abroad fect of this enrichment on growth enhancement was

r 2006 The Authors

1662 Journal Compilation r 2006 Blackwell Publishing Ltd
Aquaculture Research, 2006, 37, 1662^1670
not conclusive (Hanley, Shashar, Smolowitz, Bullis,
Mebane, Gabr & Hanlon 1998). However, there is no
information on how the enrichment of Artemia nau-
plii in£uences the growth and survival rate of PBT
larvae.
Choline has several important metabolic func-
tions. It is required: (1) for the synthesis of acetylcho-
line; (2) for the synthesis of phosphatidylcholine (PC)
(lecithin) and other complex choline-containing
phospholipids (PL); and (3) as a source of methyl
groups, via betaine, for the synthesis of various
methylated metabolites (Zeisel 1990; NRC 1993). Diet-
ary choline requirements have been estimated for
several species of ¢sh (Hung1989; Rumsey1991; Grif-
¢n,Wilson,White & Brown 1994; Craig & Gatlin 1996;
Zhang & Wilson 1999). It has been demonstrated that
PC, which is the most abundant PL class, has a major
role in ¢sh larvae performance (Takeuchi, Arakawa,
Satoh & Watanabe 1992), and membrane function,
lipoprotein synthesis in the liver and the transport
of lipids from the enterocytes to the larval tissues
(Hadas, Koven, Sklan & Tandler 2003). Previous ob-
servations in this laboratory revealed a lower choline
and PC content in Artemia compared with the striped
knifejaw larvae. In addition, there is no information
on the e¡ect of choline on the growth performance
of PBT larvae. The aim of this study was to investigate
the e¡ect of Artemia enrichment with DHA and cho-
line on the growth and survival rate of PBT larvae.
Materials and methods
Artemia hatching and enrichment
In this study, Artemia franciscana cysts (INVE Aqua-
culture, Dendermonde, Belgium) from Great Salt
Lake (UT, USA) were used. The cysts (4 g L 1) were
disinfected in a 5 mg L 1 hypochlorite solution for
30 min before hatching. After washing with tap
water to remove the remaining hypochlorite, the
cysts were incubated at a density of 2 g L 1 in ¢ltered
seawater at 28 1C under continuous aeration and
light. After hatching, the nauplii were separated from
the cyst shells and unhatched cysts and transferred
to a 100-L-tank (cylindroconical shape) in a water
bath at 28 1C with continuous aeration. The aeration
consisted of several airstones to maintain oxygen
near 100% saturation levels.
For enrichment, instar-II stage Artemia nauplii
were introduced into 10-L buckets at a density of
100^150-individuals-mL 1. Artemia were enriched
with four types of emulsions: (1) 0.5 mg L 1 oleic acid
r 2006 The Authors
Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1662^1670
Towards a suitable feed for tuna larvae A K Biswas et al.
(OA); (2) 0.5 mg L 1 DHA; (3) 0.5 mg L 1 DHA1
1.0 mg L 1 choline; and (4) 0.5 mg L 1 DHA1
2.0 mg L 1 choline. The emulsion OA has been used
in this laboratory to maintain lipid content in Arte-
mia. It has been demonstrated earlier that Artemia
nauplii enriched with OA showed better performance
compared with Artemia nauplii without OA enrich-
ment (personal communication). Polyoxyethylene
sorbitan mono-oleate  2% was added to each emul-
sion to ensure a stable and uniform emulsion. Enrich-
ment lipids and choline were mixed with distilled
water and homogenized for 5 min. Particle size distri-
bution and stability were checked according to Harel,
Ozkizilcik, Lund, Behrens and Place (1999). Two equal
portions of each emulsion were mixed with 2 L sea-
water and added to the Artemia nauplii containing
bucket at 8 and 20 h posthatch. Artemia were en-
riched for 24 h at 25 1C in duplicate for each emul-
sion. The purity of the OA (Wako, Osaka, Japan) ethyl
ester was 490%, and that of DHA (Harima Chemi-
cals, Osaka, Japan) was 70% and both were esters.
Fish, experimental design and data collection
Nineteen-day-old PBT larvae were obtained from the
Fish Nursery Center of Kinki University, Ohshima, Ja-
pan, where they were fed with Artemia, rotifers and
the striped knifejaw larvae. The experiment was de-
signed in duplicate for each of ¢ve treatments. One
thousand larvae (the initial total length and weight
were 12.2  1.0 mm and 28.5  7.4 mg, n 5 20) were
randomly divided into 10 groups and stocked into
each of 500 L tanks. The PBT were fed either Artemia
enriched with OA (Diet 1), DHA (Diet 2), DHA1
choline 1.0 mg L 1 (Diet 3) and DHA1choline
2.0 mg L 1 (Diet 4) or stripped knifejaw larvae (Diet
5, reference diet). The larvae were fed four to ¢ve
times daily at 5^10  104 nauplii per tank at each
time for 12 days. Seawater was supplied at a rate of
350^700 mL min 1 and continuously aerated with
air stones. The water temperature was maintained
at 28  1 1C. At the termination of the experiment,
the survival rate and growth performance were
recorded.
Lipid analysis
Lipids from Artemia and ¢sh larvae were extracted
with a mixture of chloroform and methanol (2:1, v/v;
Folch, Lees & Sloane 1957). Polar and neutral lipids
were separated with silica cartridges (Sep-pak; Japan
1663
Towards a suitable feed for tuna larvae A K Biswas et al.
Waters, Tokyo, Japan) as described by Juaneda and
Rocquelin (1985). The fatty acid methyl esters were
analysed using 2 N NaOH^methanol and 2 N HCl^
methanol according to Yoshinaka and Satoh (1989)
with a gas chromatograph (G-3000; Hitachi, Tokyo,
s
Japan) equipped with an Ultra Alloy capillary
column (30 m  0.25 mm ID; Frontier Laboratories,
Fukushima, Japan). The column temperature was
increased from 180 to 280 1C at a rate of 4 1C min 1.
and the carrier gas was nitrogen with source and col-
umn head pressure at 5 and 1kgf cm 2 respectively.
The ¢nal temperatures for the injector and detector
were 260 and 290 1C, respectively, and the detection
mode was a £ame ionization detector (FID). Peak
quanti¢cation was performed with an integrator (D-
2500; Hitachi). Choline content was determined by a
kit (Wako) according to the manufacturer’s instruc-
tions with some modi¢cation. In brief, total choline
was extracted directly from the samples with a mix-
ture of ethanol and diethylether (3:1 v/v). For PC-
bound choline, lipids were ¢rst extracted from the
samples with a mixture of chloroform and methanol
(2:1 v/v) according to Folch et al. (1957). Phosphatidyl-
choline-bound choline was then extracted from li-
pids with a mixture of ethanol and diethylether
(3:1 v/v). Phosphatidylcholine-bound choline was
analysed to investigate whether choline enrichment
increased total choline or PC-bound choline in ¢sh.
Statistical analysis
All statistical analyses were carried out using SPSS
for Windows (v. 10.0). Data were expressed as the
mean  SD of two replicates, and the means within
each treatment and among treatments were com-
pared using Tukey’s test of multiple comparison with
a 95% signi¢cant level.
Results
Fatty acid composition and choline content of
Artemia and knifejaw ¢sh larvae
The fatty acid contents of Artemia and knifejaw ¢sh
larvae are shown in Table 1. Enrichment of Artemia
with DHA signi¢cantly increased the DHA levels
to 13.9, 13.8 and 12.5 mg g 1 on a dry matter basis
in Diets 2, 3 and 4 respectively; however, the levels
were signi¢cantly lower than the reference diet
(26.9 mg g 1 dry matter basis; Diet 5). The level of
OA was signi¢cantly higher in Diet 1 than that of
1664 Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1662^1670
Aquaculture Research, 2006, 37, 1662^1670
other diets. The DHA/EPA ratio in Diet 5 was around
three times higher than that of other diets. However,
the OA/DHA ratio in Diets 2, 3 and 4 was around
three times higher than that of Diet 5.
Figure 1 shows the levels of total choline and
PC-bound choline in di¡erent diets. The choline en-
richment in Artemia signi¢cantly elevated only total
choline levels in Diet 4; however, there was no signif-
icant di¡erence in PC-bound choline levels among
Diets 1, 2, 3 and 4. Both total choline and PC-bound
choline levels were signi¢cantly higher in the refer-
ence diet than in other diets (Po0.05).
Growth and survival rate of tuna larvae
The PBT larvae fed DHA-de¢cient Artemia had the
lowest growth (Table 2) and survival rate (Fig. 2).
Growth and survival rate were signi¢cantly im-
proved by the enrichment of Artemia with DHA com-
pared with the DHA-de¢cient Artemia; however, the
improvement was negligible compared with the
growth and survival rate of the striped knifejaw lar-
vae-fed group (Po0.05).
Fatty acid composition and choline content in
the PBT larvae
The whole-body fatty acid composition of polar and
neutral lipid fractions of 31-day-old tuna larvae is
presented in Tables 3 and 4. Owing to poor survival
and insu⁄cient samples for Diet 1, the whole-body
fatty acid composition could not be analysed. The le-
vel of DHA in the striped knifejaw-fed group was sig-
ni¢cantly higher than the levels of other ¢sh groups
in both polar and neutral lipid fractions (Po0.05). In
the polar lipid fraction, the striped knifejaw-fed group
had a signi¢cantly lower OA level than that of other
¢sh groups; however, there were no signi¢cant di¡er-
ences in OA levels among the treatments in neutral
lipid fraction. Docosahexaenoic acid was the most
abundant fatty acid in both polar and neutral lipid
fractions in the striped knifejaw-fed group. The
DHA/EPA ratio in the striped knifejaw-fed group was
around two times higher than that of the other ¢sh
groups in both lipid fractions. However, the OA/DHA
ratio in DHA- and DHA1choline-enriched Artemia-
fed groups was about three times higher than that of
the striped knifejaw-fed group in both polar and neu-
tral lipid fractions.
Fish fed the diet without choline enrichment (Diets
1 and 2) showed levels of whole-body total choline
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Aquaculture Research, 2006, 37, 1662^1670 Towards a suitable feed for tuna larvae A K Biswas et al.

1
Table 1 Fatty acid contents (mg g DW) of enriched Artemia (Diets 1^4) and striped knifejaw larvae (Diet 5)

Fatty acid Diet 1 Diet 2 Diet 3 Diet 4 Diet 5

14:0 0.3  0.5 ND ND ND 2.3  2.0

16:0 15.4  2.2a 10.7  0.6b 10.7  0.7b 12.5  0.7ab 33.0  0.8c
16:1 14.0  1.6a 5.0  0.4b 5.0  0.6b 5.9  0.3b 6.8  0.1b
17:0 0.5  0.5 0.6  0.1 0.6  0.0 0.6  0.1 0.2  0.3
17:1 3.0  0.3 1.2  0.1 1.2  0.1 1.3  0.2 0.2  0.3
18:0 6.0  0.7 5.4  0.3 5.2  0.1 6.1  0.2 8.2  0.2
18:1n-9 128.5  13.5a 30.9  2.2b 29.8  1.6b 34.3  1.7b 19.6  0.4b
18:1n-7 2.9  1.1a 8.2  0.4b 8.2  0.7b 9.2  0.4b 5.5  0.4c
18:2n-6 10.9  1.0a 4.4  0.3b 4.3  0.2b 5.0  0.3bc 6.2  0.2c
18:3n-6 1.2  0.2 0.5  0.4 0.7  0.1 0.8  0.1 ND
18:3n-3 18.7  2.2a 18.8  1.3a 18.9  0.9a 21.8  1.0a 0.2  0.3b
20:1n-9 0.8  0.1 0.7  0.1 0.7  0.1 0.8  0.1 1.7  0.1
20:4n-6 1.4  0.2 1.2  0.1 1.1  0.1 1.3  0.1 1.9  0.1
20:5n-3 3.6  0.3a 13.7  1.3b 13.6  1.1b 14.7  1.0b 7.7  0.2c
22:5n-3 ND 2.3  0.3 2.2  0.3 2.2  0.2 3.7  0.1
22:6n-3 ND 13.9  1.7a 13.8  1.5a 12.5  0.9a 26.9  0.8b
DHA/EPA 1.0 1.0 0.9 3.5
18:1n-9/DHA 2.2 2.2 2.7 0.7

Values in a row with di¡erent letters are signi¢cantly di¡erent (Tukey’s Test, Po0.05).
ND, not detected; DHA, docosahexaenoic acid.

Total choline Discussion

15 PC-bound choline
a x The results demonstrated that growth and survival
a
were improved in the DHA- and DHA1choline-en-
12 riched groups compared with the ¢sh fed with DHA
y and choline-de¢cient diets. However, this improve-
ab y ab y y
9
ment was negligible compared with the stimulated
b growth and survival rate of the ¢sh larvae-fed group.
The lack of success of enriched Artemia may be attrib-
6 uted to a number of factors that are discussed below.
The signi¢cantly lower levels of PC-bound choline
3
in DHA- and DHA1choline-enriched Artemia com-
pared with the striped knifejaw larvae fed ¢sh may
be the dominant reason for the reduced growth. This
0 may be explained by the bene¢cial e¡ects of PL on
Diet 1 Diet 2 Diet 3 Diet 4 Diet 5
growth and survival that have been demonstrated in
Figure 1 Total choline and phosphatidylcholine (PC)- the larval and juvenile stages of various marine spe-
bound choline content in di¡erent diets. Data are pre- cies (Kanazawa,Teshima, Inamori & Matsubara 1983;
sented as the mean  SD of two replicates of each treat- Poston 1991; Hadas et al. 2003). It has been suggested
ment. Bars for total choline and PC-bound choline with that PC exerts the greatest in£uence on ¢sh larvae
di¡erent letters are signi¢cantly di¡erent (Po0.05). performance over other PL classes (Takeuchi et al.
1992). Owing to the important role of PC in mem-
brane function, lipoprotein synthesis in the liver and
similar to those observed in ¢sh fed diets containing the transport of lipids from the enterocytes to the lar-
di¡erent choline levels (Fig. 3). Although statistical val tissues (Hadas et al. 2003), the reduced concentra-
analysis could not be performed because of a single tions of PC-bound choline in enriched Artemia and
value for Diet 1, ¢sh fed the striped knifejaw larvae Artemia-fed tuna larvae would likely have contribu-
showed higher PC-bound choline than other ¢sh ted to their reduced weight gain. The lack of signi¢-
groups. cant di¡erence in PC-bound choline among Artemia

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Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1662^1670 1665
Towards a suitable feed for tuna larvae A K Biswas et al. Aquaculture Research, 2006, 37, 1662^1670

Table 2 Average body length and weight of tuna larvae fed for 12 days either on enriched Artemia or striped knifejaw larvae

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5

Average body length (mm)

Initial (n 5 20) 12.2  1.0 12.2  1.0 12.2  1.0 12.2  1.0 12.2  1.0
Final (n 5 30) 17.7  1.8b 18.5  1.4b 19.1  1.3b 19.1  0.8b 43.3  3.4a
Average body weight (mg)
Initial (n 5 20) 28.5  7.4 28.5  7.4 28.5  7.4 28.5  7.4 28.5  7.4
Final (n 5 30) 65.1  17c 88.2  16.9b 96.9  18.9b 93.8  12.9b 1175.5  244.3a

n 512.
Values in a row with di¡erent letters are signi¢cantly di¡erent (Tukey’s test, Po0.05).

120
Survival rate (%)
100
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10
Rearing period (day)
Figure 2 Variation in survival rate among the treat-
ments. Statistical analysis was carried out only to ¢nal va-
lues. The values for Diets 2, 3 and 4 are indicated by the
similar letter as there were no signi¢cant di¡erences
among the groups.
groups enriched with or without DHA and choline
suggests that Artemia does not appear to be a suitable
diet for tuna larvae. Similar results were observed in
postlarval Penaeid shrimp by Tackaert, Camara and
Sorgeloos (1991). Moreover, in studies with carp lar-
vae Geurden, Radunz-Neto and Bergot (1995) demon-
strated that the growth-stimulating e¡ects of PL
could not be mimicked by providing choline.
An overall balance between energy and meeting
essential fatty acid requirements is needed in lipid
nutrition, i.e. an overall balance is required between
saturated, monounsaturated and polyunsaturated
fatty acids, especially in rapidly growing and develop-
ing animals such as ¢sh larvae (Sargent, McEvoy, Es-
te¤vez, Bell, Bell, Henderson & Tocher 1999). However,
the results from this study suggested that the balance
Diet 1
Diet 2 between energy and essentiality was not maintained
Diet 3
Diet 4
in enriched Artemia as the level of OA (18:1n-9) was
Diet 5 higher in enriched Artemia groups, whereas the
DHA content was lower than the striped knifejaw lar-
vae. Similarly, the balance in palmitic acid (16:0),
which is involved in energy storage and is also asso-
ciated with HUFAs in diacyl glycerophospholipids of
a ¢sh (Bell & Dick 1991), was also poor in enriched Ar-
temia compared with striped knifejaw larvae. The
abundance of 18:3n-3 in enriched Artemia may also
exacerbate the problem. Precisely how much DHA
b and EPA is required is not known but it is suggested
that su⁄cient DHA and EPA must be available in the
c feed ingested by the larvae to outcompete and replace
the 18:3n-3 (Sargent et al. 1999). However, the levels
11 12 of DHA in enriched Artemia nauplii were lower than
that of 18:3n-3 in this study. The higher growth per-
formance in the knifejaw larvae-fed group (Diet 5)
also suggests that the PBT larvae require substan-
tially higher DHA levels in diet than the levels avail-
able in enriched Artemia. Therefore, the DHA
requirement of PBT larvae may be di¡erent than that
of other species where the larvae are able to grow and
survive either fed on Artemia nauplii absent in DHA
(Villalta, Este¤vez, Bransden & Bell 2005) or a small
amount (0.1^0.5%) of this fatty acid (Takeuchi,Toyo-
ta, Satoh & Watanabe 1990; Dickey-Collas & Ge¡en,
1992; Izquierdo, Arakawa,Takeuchi, Haroun & Wata-
nabe 1992; Morais, Narciso, Dores & Pousao-Ferreira
2004).
A major problem in determining the e¡ects of var-
ious HUFAs in ¢sh is that the requirement for any gi-
ven HUFA is determined not only by its absolute
amount in the diet but also by the absolute amounts
of other HUFA in the diet due to competitive inhibi-
tion (Rainuzzo, Reitan & Olsen 1997; Sargent et al.
1999; Corraze 2001). Recently, the DHA/EPA ratio
has been the focus of study in di¡erent ¢sh species
(Izquierdo 1996; Rainuzzo et al. 1997; Rodr|¤ guez,

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1666 Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1662^1670
Aquaculture Research, 2006, 37, 1662^1670 Towards a suitable feed for tuna larvae A K Biswas et al.

Table 3 Fatty acid composition (% of total fatty acid) in polar lipid of tuna larvae

Fatty acid Diet 2 Diet 3 Diet 4 Diet 5

14:0 ND 0.3  0.0 ND ND

16:0 18.2  0.6 18.2  2.3 18.6  0.8 24.2  2.3
16:1 2.0  0.1 1.9  0.1 2.1  0.1 1.6  0.3
17:0 0.7  0.0 0.7  0.0 0.8  0.1 ND
17:1 0.6  0.0 0.7  0.1 0.6  0.0 ND
18:0 10.9  0.4 11.4  0.3 11.4  0.1 10.0  0.2
18:1n-9 16.9  0.6a 16.9  0.8a 17.6  0.3a 9.4  0.7b
18:1n-7 5.4  0.1a 5.5  0.1a 5.5  0.1a 3.0  0.1b
18:2n-6 2.3  0.1 2.2  0.1 2.3  0.0 2.7  0.1
18:3n-6 0.3  0.0 0.3  0.0 0.3  0.0 ND
18:3n-3 3.3  0.1 3.3  0.1 3.4  0.1 ND
20:1n-9 0.6  0.1 0.7  0.0 0.7  0.0 ND
20:4n-6 3.0  0.0 2.9  0.2 3.0  0.1 3.4  0.1
20:5n-3 7.9  0.1a 7.4  0.5ab 7.6  0.1a 6.5  0.0b
22:5n-3 3.0  0.1 3.0  0.4 3.0  0.2 2.5  0.1
22:6n-3 20.9  0.8a 20.5  1.8a 18.8  0.9a 35.9  0.3b
DHA/EPA 2.7 2.8 2.5 5.5
18:1n-9/DHA 0.8 0.8 0.9 0.3

Values in a row with di¡erent letters are signi¢cantly di¡erent (Tukey’s Test, Po0.05).
ND, not detected; DHA, docosahexaenoic acid.

Table 4 Fatty acid composition (% of total fatty acid) in neutral lipid of tuna larvae

Fatty acid Diet 2 Diet 3 Diet 4 Diet 5

14:0 ND ND ND ND
16:0 14.0  2.4a 16.1  2.9ab 10.9  0.6a 23.2  0.8b
16:1 4.6  0.7 4.6  1.0 3.9  0.4 5.4  0.6
17:0 ND ND ND 0.3  0.2
17:1 ND ND ND 0.3  0.2
18:0 10.1  0.1 9.9  2.4 8.1  0.1 6.4  0.1
18:1n-9 21.2  0.9 18.0  3.3 21.2  0.8 17.3  0.6
18:1n-7 6.5  0.6 6.0  1.1 6.9  0.2 4.6  0.1
18:2n-6 2.5  0.3a 2.3  0.8a 3.2  0.4a 4.7  0.1b
18:3n-6 ND 1.1  0.6 ND ND
18:3n-3 6.3  1.2ab 4.8  0.5a 8.9  0.2b 0.2  0.2c
20:1n-9 ND ND ND 1.6  0.2
20:4n-6 4.0  0.3a 3.8  0.2a 4.3  0.4a 1.6  0.1b
20:5n-3 6.9  0.4a 4.9  0.4b 7.4  0.6a 6.7  0.1a
22:5n-3 ND ND ND 4.0  0.1
22:6n-3 9.9  0.9a 6.6  2.2a 8.5  2.2a 23.0  0.4b
DHA/EPA 1.4 1.4 1.2 3.4
18:1n-9/DHA 2.1 2.7 2.5 0.8

Values in a row with di¡erent letters are signi¢cantly di¡erent (Tukey’s Test, Po0.05).
ND, not detected; DHA, docosahexaenoic acid.

Pe¤rez, D|¤ az, Izquierdo, FernaŁndez-Palacios & Lorenzo-
Hernandez 1997; Sargent, McEvoy & Bell 1997). Var-
ious investigators have used DHA/EPA ratios as an
index of the optimal level required for normal growth
and development in ¢sh larvae (Koven,Tandler, Sklan
& Kissil 1993; Reitan, Rainuzzo & Olsen 1994; Tocher,
Mourente & Sargent 1997; Rodr|¤ guez, Pe¤rez, Badia,
Izquierdo, FernaŁndez-Palacios & Lorenzo-Hernandez
1998). Di¡erent ¢sh species require di¡erent levels of
DHA/EPA ratios for their growth and survival. Gapa-
sin and Duray (2001) suggested that for milk¢sh
(Chanos chanos), a DHA/EPA ratio of at least  1.0
in the live food would be appropriate for normal
growth and good survival in an intensive larvicul-
ture system. Rodr|¤ guez et al. (1997) observed signi¢-
cantly higher growth rates in sea bream larvae fed

r 2006 The Authors

Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1662^1670 1667
Towards a suitable feed for tuna larvae A K Biswas et al.
Total choline
PC-bound choline
1400
1200
1000
800
600
400 *
200
0
Diet 1 Diet 2 Diet 3 Diet 4 Diet 5
Figure 3 Total choline and phosphatidylcholine (PC)-
bound choline content in tuna larvae fed with di¡erent
diets. Data are presented as the mean  SD of two repli-
cates of each treatment. No replicates.
rotifers with a DHA/EPA ratio of 1.5 compared with
those fed DHA/EPA ratios o0.6. In seahorse (syng-
nathid ¢sh), Woods (2003) observed better growth
performance with a DHA/EPA ratio as high as 1.21
in Algamac-3050-enriched Artemia and as low as
0.31 in Eyre Peninsula Aquaculture brine shrimp
food (90% EPABSF)/spirulina-enriched Artemia.
However, this study demonstrated that the DHA/
EPA ratio of around 1.0 in enriched Artemia would
not be su⁄cient to stimulate the desired growth and
survival rate of the PBT larvae, suggesting that the
DHA/EPA ratio in the diet should be more than 1.0
for PBT larvae. Another essential fatty acid ratio eval-
uated for marine ¢n¢sh nutrition is OA (18:1n-9) to
DHA (Takeuchi, Masuda, Ishizaki, Watanabe, Kane-
matsu, Imaizumi & Tsukamoto 1996; Furuita et al.
1999). The results from striped knifejaw larvae sug-
gested that an OA/DHA ratio o1 would be satisfac-
tory for the growth and survival of PBT larvae.
Similar results were observed in the Japanese £oun-
der where the larvae showed higher total length and
stress resistance when the OA/DHA ratio in the diet
was o1 (Furuita et al. 1999).
In conclusion, enriched Artemia does not provide
for optimum growth and survival in PBT larvae. The
di⁄culties in obtaining a balanced fatty acid compo-
sition with higher DHA/EPA and lower OA/DHA ra-
tios from Artemia nauplii are a major drawback to
their suitability as live feed for the PBT larvae be-
cause the fatty acid composition of the reference diet
(striped knifejaw larvae) suggests that the PBT larvae
1668 Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 1662^1670
Aquaculture Research, 2006, 37, 1662^1670
require a balance diet with higher DHA/EPA and
lower OA/DHA ratios. Further studies are required
using one of the many PL-rich enrichment emulsions
and/or dry diets that are now available as an alterna-
tive to Artemia and knifejaw larvae. In addition, the
results obtained in this study will also help consider-
ably in furthering the understanding of dietary com-
position of arti¢cial microdiet, which would be our
further investigation, for the PBT larvae.
Acknowledgments
This study was supported by the 21st Century COE
program of the Ministry of Education, Culture, Sport,
Science and Technology, Japan. The expenses were
also defrayed in part by a Grant-in-Aid for Scienti¢c
Research (No. S 14104007) from the Ministry of Edu-
cation, Culture, Sport, Science and Technology.
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