Aquaculture Research, 2014, 45, 949–960
12037
Replacement of fresh algae with commercial formulas
to enrich rotifers in larval rearing of yellowtail
kingfish Seriola lalandi (Valenciennes, 1833)
Zhenhua Ma & Jian G Qin
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide 5001, Australia
Correspondence: J G Qin, School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide 5001, Australia.
E-mail: Jian.Qin@Flinders.edu.au
Abstract
This study compared the efficacy of four products
that are commonly used in hatchery for nutritional
enhancement of rotifer Brachionus plicatilis as the
starter food for yellowtail kingfish Seriola lalandi
larvae. This experiment consisted of one fresh algae
and three enrichment products: (1) Fresh algae
were a mixture of Nannochloropsis and Isochrysis at
2:1 on a cell concentration basis; (2) S.presso,
(Selco S.presso ®, INVE Aquaculture); (3) Algamac
3050® (Aquafauna, USA); (4) Nutrokol ® (Nutra-
Kol, Australia). Survival rates of the fish fed rotifers
enriched with fresh microalgae (40.69%) and
S.presso (31.21%) were higher than those fed
Algamac 3050 (10.31%). On 3 day post hatch
(DPH), fish feeding incidence in the fresh algae treat-
ment was significantly higher than that in other
treatments. On 6 DPH, fish showed the lowest feed-
ing incidence in the Algamac 3050 treatment. The
methods of enrichment did not affect total lipid
levels in either rotifer or fish larvae, but Algamac
3050 enrichment achieved the highest DHA/EPA
ratio and lowest EPA/ARA ratio in both rotifers and
fish larvae. This study indicates that fresh algae can
be replaced by S.presso, but Algamac 3050 is not as
good as other formula for rotifer enrichment in
rearing yellowtail kingfish larvae in this system.
Keywords: growth, survival, deformity, lipid,
fatty acids
Introduction
Efficacy of nutritional supplement of live food for
fish larvae has been a challenge in marine fish
rearing (Palmtag, Faulk & Holt 2006). The
© 2012 John Wiley & Sons Ltd
doi:10.1111/are.
adequate nutritional supply is critical to fish
embryonic development and metamorphosis (Wa-
tanabe & Kiron 1994). Before first feeding, yolk
sac reserve is the major source of nutrition for fish
larvae (Sarasquete, Gonzalez de Canales, Arellano,
Munoz-Cueto, Ribeiro & Dinis 1996), and all these
nutrient reserves are derived from broodstock
(Hilton, Poortenaar & Sewell 2008). In the early
stages of fish development, first feeding is a critical
period for fish survival because larvae are poten-
tially at a risk of starvation when food is not avail-
able (Watanabe & Kiron 1994). Thus, quality and
quantity of the food supply to fish at first feeding
govern the success of larval rearing.
Requirements of essential fatty acids (EFA) in fish
larvae have been extensively studied in the past
(Watanabe, Tamiya, Oka, Hirata, Kitajima & Fujita
1983; Izquierdo, Arakawa, Takeuchi, Haroun &
Watanabe 1992; Kjørsvik, Olsen, Wold, Hoehne-
Reitan, Cahu, Rainuzzo, Olsen, Øie & Olsen 2009).
Especially, the requirements of marine fish for eico-
sapentaenoic acid 20:5n-3 (EPA), docosahexaenoic
acid 22:6n-3 (DHA) and arachidonic acid 20:4n-6
(ARA) have been extensively quantified (Hamre,
Opstad, Espe, Solbakken, Hemre & Pittman 2002;
Bell & Sargent 2003; Faulk, Holt & Davis 2005).
The long-chain polyunsaturated fatty acids (PUFA)
such as EPA, DHA, and ARA play important roles
in growth, survival and stress resistance in most
marine fish larvae (Watanabe 1993; Bell & Sargent
2003; Faulk & Holt 2003).
DHA is high in neutral tissue and plays an
important role in neutral membrane structure and
functions (Sargent, Bell, McEvoy, Tocher & Estevez
1999a; Copeman, Parrish, Brown & Harel 2002),
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Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
but its requirement varies among fish species.
Cold-water fish species such as yellowtail flounder
Limanda ferruginea and Atlantic halibut Hippoglossus
hippoglossus require high dietary DHA (McEvoy,
Naess, Bell & Lie 1998; Copeman et al. 2002). In
contrast, turbot larvae Scophthalmus maximus
require low DHA to sustain growth and survival
(Planas & Cunha 1999). Besides DHA, more and
more evidence suggests that the DHA/EPA ratio
should be used as an index to determine the opti-
mal levels of both fatty acids for growth and devel-
opment in fish larvae (Koven, Tandler, Sklan &
Kissil 1993; Tocher, Mourente & Sargent 1997;
Rodriguez, Perez, Badia, Izquierdo, Fernandez-Pala-
cios & Lorenzo-Hernandez 1998). The abundance
of DHA and EPA in cell membranes serve as a
major source of energy to absorb fat-soluble vita-
mins (A, D, E and K) and as precursors for prosta-
glandin hormones (Sargent, McEvoy, Estevez, Bell,
Bell, Henderson & Tocher 1999b; Sargent et al.
1999a; Rezek, Watanabe, Harel & Seaton 2010).
A shortage or excess of EPA and DHA in diet can
affect survival and malformation of fish larvae
(Sargent et al. 1999b). Due to low activity of
essential enzymes in fish larvae (Ghioni, Tocher,
Bell, Dick & Sargent 1999; Bell & Sargent 2003),
most larval fish lack the ability to convert short
chain fatty acids into long chain fatty acids to
meet normal physiological activities (Kanazawa,
Teshima & Ono 1979; Sargent, Bell, Bell, Henderson
& Tocher 1995; Sargent et al. 1999b; Sargent,
Tocher & Bell 2002). Therefore, it is necessary to
provide these essential fatty acids through diet
(Palmtag et al. 2006).
Yellowtail kingfish (Seriola lalandi, YTK),
belonging to the Carangidae family, is widely dis-
tributed throughout the warm-temperate waters
of the world (Fowler, Ham & Jennings 2003;
Hutson, Ernst, Mooney & Whittington 2007;
Benetti 2008). This species has been identified as
an aquaculture candidate due to its fast growth,
high flesh quality and suitability for cage culture,
and expands rapidly in Australia (Fowler et al.
2003). Up to date, survival rate from hatching
to fingerlings has been below 10% and deformity
of fish juveniles is over 40%, which significantly
impedes fingerling production of this species in
South Australia. The state of the art in yellow-
tail kingfish culture in the larval phase is based
on the usage of rotifers and Artemia nauplii as
live feeds. However, rotifers are low in PUFA
(Conceicao, Yufera, Makridis, Morais & Dinis
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Aquaculture Research, 2014, 45, 949–960
2010) and nutrient deficiency in rotifers has
resulted in slow growth, mass mortality, mal-pig-
mentation and deformity in larvae of various
marine fish species (Watanabe, Kitajima, Araka-
wa, Fukusho & Fujita 1978; Miki, Taniguchi,
Hamakawa, Yamada & Sakurai 1990; Takeuchi,
Dedi, Haga, Seikai & Watanabe 1998; Olivotto,
Rollo, Sulpizio, Avella, Tosti & Carnevali 2006;
Avella, Olivotto, Gioacchini, Maradonna & Carnevali
2007).
As rotifer nutrition can be improved through
food source manipulation (Watanabe et al. 1983;
Koven, Kissil & Tandler 1989), the method of roti-
fer enrichment is critical to growth and survival of
fish larvae (Rainuzzo, Reitan & Olsen 1997).
Although microalgae have been considered an
excellent diet for enrichment (Dhert, Rombaut,
Suantika & Sorgeloos 2001), there is a trend to
replace fresh algae with commercial formulas to
feed rotifer because of the costly operation to
culture algae in hatchery (Borowitzka 1997). Over
the past two decades, enrichment formulas have
replaced fresh algae as food for rotifers in many
species of marine fish larvae (Rodriguez, Olsen
& Rosenlund 1989; Kissil & Koven 1990; Lie, Haa-
land, Hemre, Maage, Lied, Rosenlund, Sandnes &
Olsen 1997; Tocher et al. 1997; Harel, Koven,
Lein, Bar, Behrens, Stubblefield, Zohar & Place
2002; Castell, Blair, Neil, Howes, Mercer, Reid,
Young-Lai, Gullison, Dhert & Sorgeloos 2003; Liu,
Kelly, Cook, Black, Orr, Zhu & Dong 2007; Estudil-
lo-del, Gapasin & Leaño 2009; Kotani, Genka,
Tanabe, Miyashima, Fushimi & Hayashi 2010).
However, the efficacy of replacing fresh algae with
enrichment formulas for rotifers varies greatly in
the culture of different fish species (Nordoy,
Aakvaag & Larsen 1993; Wedemeyer 1996;
Garcia, Parrish, Brown, Johnson & Leadbeater
2008b). For instance, Garcia, Parrish and Brown
(2008a) found that a combination enrichment of
Pavlova sp. and Algamac 2000® resulted in the
best growth and survival during the rotifer phase
in Atlantic cod Gadus morhua when compared with
a series of enrichment formulas including Algamac
2000®, AquaGrow® Advantage, Pavlova sp.+ Alga-
mac 2000® and DC DHA Selco® + Algamac
2000®. However, when compared the larval
performance of haddock (Melanogrammus aeglefi-
nus) using Algamac 2000®, AquaGrow® Advan-
tage, Pavlova sp. and Pavlova sp. + Algamac
2000® for rotifer enrichment, fish growth was not
significantly affected by the enrichment formula,
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960

Aquaculture Research, 2014, 45, 949–960 Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
but a better survival was achieved using the
combination of Pavlova sp. + Algamac 2000®
while Algamac 2000® treatment showed the poor-
est fish survival (Garcia et al. 2008b). In yellowtail
snapper (Ocyurus chrysurus) larvae, fish grow
faster using the formulas of Isochrysis galbana,
AquaGrow® Advantage, AquaGrow® Advantage +
AquaGrow® Arachidonic acid or Algamac 2000®
than using Nannochloris oculata for rotifer enrich-
ment (Faulk et al. 2005). These studies suggest
that the response of fish larvae to the enrich
formula is species-specific. In YTK larval culture, the
use of fresh algae has been proposed to be replaced
with enrichment formulas for rotifers, but up to date,
it still remains unclear what the most suitable
enrichment formula is for rotifer enrichment.
The objective of this study was (1) to quantify the
growth and survival rate of YTK larvae fed live
feeds enriched with different products; (2) to quan-
tify the nutritional content of rotifers and YTK
larvae under the experimental condition; (3) to
examine the impact of enrichment formulas on the
jaw deformities of fish larvae; 4) to test feeding
incidence of fish larvae fed live feed enriched with
different products. The understanding of these key
issues will help improve the success and efficiency
of larval fish rearing and choose the optimal
protocols to deliver nutrition to fish larvae at first
feeding.
Materials and methods
Eggs and larval fish rearing
Fertilized eggs of yellowtail kingfish were obtained
from Arno Bay, South Australia, and were trans-
ported to the South Australian Research and
Development Institute Aquatic Science Centre,
Adelaide. Upon arrival, all eggs hatched in 200-L
fibreglass incubators at 21.5°C. After hatching,
fish larvae were stocked into 172-L fibreglass
rearing tanks at a density of 60 larvae L 1. All
fish tanks were supplied with filtered seawater
through a 5-lm filter in a flow-through system
with a daily water exchange rate of 400% tank
volume. One air stone was used in each tank to
maintain dissolved oxygen at saturation and to
homogenize live food distribution. Light intensities
were maintained at 2100–2500 lux and a photo-
period of 14-h light and 10-h dark was used.
Salinity was maintained at 38& throughout the
experiment. Water temperature was controlled at
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960
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22.1 ± 0.4°C. This experiment was finished on
12 DPH.
Experiment design
This experiment included four dietary treatments
with five replicates each. Three commercial enrich-
ment products and one fresh microalgae mix were
used, including (1) Fresh algae mix of Nannochlor-
opsis sp. and T-Isochrysis = 2:1 by algal cell
number; (2) S.presso (Selco S.presso ®, INVE Aqua-
culture); (3) Algamac 3050® (Aquafauna, USA);
and (4) Nutrokol ® (Nutra-Kol, Australia). In the
rotifer culture, Brachionus plicatilis (L-type) were fed
with S.parkle ® (INVE Aquaculture) at a density of
500 rotifers mL 1. The condition for rotifer culture
was set at 24.3 ± 0.7°C, >5.8 mg dissolved oxygen
L 1, pH 7.95–8.11 and 37.5 g L 1 salinity. Prior
to enrichment, the rotifers collected from the cul-
ture tank were rinsed with filtered seawater on a
100-lm mesh screen. During enrichment, the den-
sity of rotifers increased to 1000 mL 1. Rotifers
were then separately enriched by four enrichment
products, i.e. S.presso at 350 mg L 1, Nutrokol at
600 mg L 1, Algamac 3050 at 200 mg L 1 and a
fresh algal mix of 12 9 106 cell mL 1 Nannochlor-
opsis and 6 9 106 cell mL 1 T-Isochrysis in each
enrichment tank at a density of 1000 rotifers
mL 1. After enrichment for 12 h, the rotifers were
harvested and fed to fish larvae. The fresh microal-
gae used in this study were cultured in a continu-
ous culture system with a series of 200-L plastic
bags using the AlgaBoost (2000x) f/2 culture med-
ium (AusAqua, Australia). The algal culture condi-
tion was maintained at 20–22°C, 23 g L 1
salinity, 5000 lux light intensity and 16 L:8 D
photoperiod. Starting from 2 DPH, the density of
rotifers was kept at 15 mL 1 in the larval fish rear-
ing tanking. The instant microalgae (Nanno 3600
paste, Reed Mariculture, USA) were also added into
all the larval fish tanks to create a green colour
background.
Every 2 days, the standard length and fish feed-
ing incidence were examined by sampling 10 fish
larvae from each rearing tank and measured on a
dissect microscope. The standard length was mea-
sured from the upper jaw to the end of the noto-
chord. The feeding incidence was calculated as:
feeding incidence = 100 9 N1/N0, where N0 is the
total number of larvae, and N1is the number of
larvae with live food in the gut (Lein, Holmefjord
& Rye 1997). Larval growth was determined by
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Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
specific growth rate (SGR) as% day 1 (Hopkins
1992): SGR = 100(Ln(SLf) – Ln(SLi))/Dt, where
SLf and SLi are the final and initial fish standard
length (mm), respectively, and Dt is the time
interval (days) between samples. At the end of the
experiment, 50 larvae from each tank were
sampled for wet weight, standard length and jaw
deformity assessments.
Jaw deformity
Jaw malformation was assessed on a stereo micro-
scope (Lieca MZ6, Leica Microsystems, Germany)
using the criteria described by Cobcroft and Bat-
taglene (2009). The appearance of the jaws of
each larvae was rated on a scale of 0–3 according
to the jaw malformation index (Cobcroft, Pank-
hurst, Poortenaar, Hickman & Tait 2004) modified
for yellowtail kingfish larvae. A score of 0 indi-
cated a normal jaw while a score of 0.5 indicated
very minor malformation that would not be con-
sidered malformation from a commercial perspec-
tive. Larvae were defined as malformed when the
jaw score reached 1 (minor), 2 or 3 (major).
Lipids and fatty acids analysis
The nutritional content of rotifers was assessed on
4, 8 and 12 DPH. After enrichment, four million
rotifers from each treatment in two replicates were
collected and preserved in liquid nitrogen until
analysis. On 2 DPH and 12 DPH, 0.5 g fish larvae
(wet weight) in five replicates were sampled for
lipid and fatty acid analyses. All rotifer and fish
samples were pre-washed using an ammonium
formate solution (0.5 M) to remove salt, and paper
tower were used to remove extra water before
preservation in liquid nitrogen.
The lipids and fatty acids were analysed at
National Collaborative Research Infrastructure
Strategy, Adelaide, Australia. Total lipids were
extracted as described by Folch, Lees and Stanley
(1957). The fat content was measured and then
samples were methylated in 5 mL of 1% H2SO4 in
methanol at 70°C for 3 h. The fatty acid methyl
esters were extracted by adding 750 lL distilled
water and 2 mL of n-heptane. The heptane layer
was transferred to a 2-mL vial for analysis using
gas chromatography (GC, PerkinElmer gas chro-
matograph Clarus 500). Fatty acid methyl esters
(FAMEs) were separated and measured on the GC
equipped with a 30-m capillary column (0.32-mm
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Aquaculture Research, 2014, 45, 949–960
internal diameter, Zebron ZB-FFAP). Helium was the
carrier gas (1.5 mL min 1). The injector tempera-
ture was set at 250°C and the detector temperature
at 300°C. The initial oven temperature was 140°C
for 3 min, then ramped at 1:10.0 min 1–160°C for
5 min followed by 10.0 min 1–230°C for 10 min.
FAMEs were identified on the GC using software of
TotalChrom Navigator (version 6.3.2 0646, Perkin
Elmer). The level of the internal standard 17:0 was
used to calculate the FAME concentration in each
sample.
Statistical analysis
The data in this article were expressed as mean ±
SD, and tested using one-way ANOVA (PASW Statis-
tics 18.0, IBM, Chicago, IL, USA). When signifi-
cant treatment effect was found, Tukey’s test was
performed for multiple range comparisons
(P < 0.05). All the data were tested for normality,
homogeneity and independence to satisfy the
assumptions for ANOVA.
Results
Larval fish growth and survival
The specific growth rate was not affected by
the enrichment formulas (P > 0.05, Fig. 1). Fish
specific growth rates were 1.0–1.6% day 1 among
all the enrichment treatments. However, at the
end of this experiment, significant differences were
observed in fish survival. The final survival rate of
YTK larvae fed rotifers enriched in Algamac 3050
was significantly lower than that in other treat-
ments (P < 0.05), except in Nutrokol (P > 0.05).
No significant differences were found in final fish
survival rates between S.presso and Nutrokol
treatments (P > 0.05, Fig. 1). In all the treat-
ments, fresh microalgae achieved the highest
survival rate (40.69 ± 11.65%), although there
was no difference between the fresh microalgae
and the S.presso treatments (P > 0.05).
Fish feeding incidence
Fish feeding incidence increased with age (Fig. 2).
Enrichment formulas significantly affected the feed-
ing incidence rates at first feeding. On 3 DPH, the
average feeding incidence was relatively low, but
fish in the fresh microalgae treatment showed
higher feeding incidence rates than in other
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960

Aquaculture Research, 2014, 45, 949–960 Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
Figure 1 Specific growth rate and final survival rate
of yellowtail kingfish larvae in four enrichment treat-
ments on different days post hatch (DPH).
Figure 2 Feeding incidence rates of yellowtail kingfish
larvae in four enrichment treatments on different days
post hatch (DPH).
treatments (P < 0.05, Fig. 2). The feeding incidence
of fish fed rotifers enriched with fresh microalgae
was 66 ± 19.49%, but the feeding incidence of fish
fed rotifers enriched with S.presso, Algamac 3050,
and Nutrokol was <30%. On 6 DPH, the feeding
incidence increased in all treatments compared
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960
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with that on 3 DPH. However, the feeding
incidence in the Algamac 3050 treatment was sig-
nificantly lower than in other treatments (P <
0.05), but there were no significant differences
between other treatments (P > 0.05). On 9 DPH,
the average feeding incidence reached 94% in all
treatments, but the feeding incidence of fish in the
Algamac 3050 treatment was significantly lower
than that in other treatments (P < 0.05).
Jaw deformities
By 12 DPH, normal jaws accounted for over 95%
in all fish and jaw malformation was not signifi-
cantly affected by the type of enrichment formulas
(P > 0.05). Considering the distribution of jaw
malformation, only categories 0 and 1 deformity
were observed. Fish in the Algamac 3050 treat-
ment showed a higher rate of category 1 jaw
malformation than in other treatments, whereas
fish in the S.presso treatment showed the lowest
category 1 jaw malformation.
Fatty acid composition in rotifers and fish larvae
The total lipid in rotifers was not affected by the
enrichment formulas in this experiment (P > 0.05,
Table 1). However, some specific fatty acids signifi-
cantly varied between treatments. The amount of
EPA (20:5n-3) in the rotifers enriched with fresh
microalgae (9.18%) or Nutrokol (10.45%) was
nearly twice as much as in other treatments
(P < 0.05), but no significant differences were
found between un-enriched rotifers (5.56%) and
S.presso (4.08%) or between un-enriched rotifers
and Algamac 3050 (3.22%) (P > 0.05). After
enrichment, the amount of DHA (22:6n-3) in roti-
fers enriched with Algamac 3050 was significantly
higher (34.53%) than that in other treatments
(P < 0.05), while no significant differences were
found between the treatments of S.presso
(19.65%) and Nutrokol (13.29%), or fresh algae
(4.04%) and Nutrokol (13.29%, P > 0.05). The
ratio of DHA/EPA in the rotifers was highest in
the Algamac 3050 enrichment (10.81), followed
by the S.presso enrichment (4.82) (P < 0.05), but
the Nutrokol and fresh microalgae did not change
the DHA/EPA ratio compared with un-enriched
rotifers. The EPA/ARA ratio in the rotifers
enriched with Nutrokol was highest (7.36)
(P < 0.05) and there was no significant difference
between algal enrichment and un-enrichment
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Enrichment formulas for rotifers in larval fish rearing Z Ma et al. Aquaculture Research, 2014, 45, 949–960

Table 1 Fatty acid composition (% of total fatty acids) of enriched and un-enriched rotifers

Un-enrich Microalgae S.presso Algamac Nutrokol

20:4n-6 (ARA) 1.40 ± 0.81a

2.10 ± 0.07 a
1.66 ± 0.04 a
2.35 ± 0.14a
1.42 ± 0.05a
20:5n-3 (EPA) 5.56 ± 2.18a 9.18 ± 0.65b 4.08 ± 0.12a 3.22 ± 0.48a 10.45 ± 0.47b
22:6n-3 (DHA) 11.42 ± 7.71a,b 4.04 ± 0.12a 19.65 ± 0.78b 34.53 ± 2.14c 13.29 ± 0.74a,b
DHA/EPA 1.83 ± 0.87a 0.44 ± 0.03a 4.82 ± 0.24b 10.81 ± 1.05c 1.27 ± 0.02a
EPA/ARA 4.63 ± 1.68c 4.37 ± 0.22b,c 2.46 ± 0.02a,b 1.38 ± 0.24a 7.36 ± 0.15d
Total Sats 20.65 ± 3.00a 23.36 ± 1.05a 24.43 ± 0.82a 21.99 ± 1.70a 23.74 ± 0.79a
Total Monos 42.29 ± 16.08b 37.80 ± 1.55b 27.34 ± 1.41a,b 17.02 ± 3.09a 36.87 ± 1.26a,b
Total n-9 7.15 ± 1.28b 6.95 ± 0.50b 5.03 ± 0.24a 3.53 ± 0.61a 7.40 ± 0.37a
Total n-7 35.14 ± 14.81b 30.85 ± 1.80a,b 22.31 ± 1.19a,b 13.49 ± 2.49a 29.46 ± 0.99a,b
Total Poly Unsats 28.91 ± 12.39a 33.87 ± 1.07a,b 44.55 ± 1.38b,c 57.10 ± 3.05c 34.31 ± 1.60a,b
Total n-6 9.01 ± 2.12b,c 9.68 ± 0.80b,c 12.13 ± 0.30c 4.35 ± 0.43a 7.81 ± 0.33b
Total n-4 0.68 ± 0.29a 1.20 ± 0.08b 0.49 ± 0.01a 0.40 ± 0.02a 0.48 ± 0.02a
Total n-3 19.22 ± 10.56a 22.99 ± 1.43a 31.93 ± 1.08a 52.35 ± 3.42b 26.03 ± 1.33a
Total lipids (mg g 1) 16.37 ± 1.42a 8.37 ± 2.48a 15.77 ± 1.61a 18.50 ± 7.93a 13.80 ± 3.25a

Different letters represent significant differences at P < 0.05.

(P > 0.05). However, the EPA/ARA ratio in roti-
fers was reduced in the Algamac 3050 and
S.presso treatments compared with the un-enrich-
ment control.
Total lipids in fish larvae were not significantly
affected by the methods of rotifer enrichment
(P > 0.05, Table 2). However, by 12 DPH at the
end of the experiment, ARA (20:4n-6) in all fish
larvae was significantly higher than that in the
2-DPH larvae (P < 0.05). The reason of choosing
the 2-DPH larvae for comparison was because no
enriched food was applied to larvae before 2 DPH.
In the fresh microalgae treatment, ARA in larval
fish was highest in all treatments, followed by that
in the Algamac 3050 treatment (P < 0.05), but
no significant differences were found between
the treatments of S.presso and Nutrokol. EPA
(20:5n-3) of the 12-DPH fish in the treatment
of S.presso and Algamac 3050 was significantly
lower than that in the un-enriched 2-DPH lar-
vae (P < 0.05). In contrast, EPA content of the
12-DPH fish in the treatment of fresh microalgae
was significantly higher than that in the un-
enriched 2-DPH larvae. DHA of the larvae in the
Algamac 3050 treatment was significantly higher
than that in other treatments and in the 2-DPH

Table 2 Fatty acid composition (%) in unfed 2-DPH fish and 12-DPH fish fed rotifers enriched with microalgae,
S.presso, Algmac and Nutrokol.

2-DPH larvae 12-DPH larvae

Treatment Microalgae S.presso Algamac Nutrakol

20:4n-6 (ARA) 1.76 ± 0.01a 4.78 ± 0.21d 3.70 ± 0.23b 4.27 ± 0.12c 3.39 ± 0.13b
20:5n-3 (EPA) 5.19 ± 0.02c 8.04 ± 0.45d 2.63 ± 0.53b 1.73 ± 0.13a 5.60 ± 0.34c
22:6n-3 (DHA) 26.16 ± 0.19b,c 16.61 ± 0.78a 28.16 ± 0.96c 30.46 ± 0.94d 25.79 ± 0.67b
DHA/EPA 5.16 ± 0.19b 2.07 ± 0.19a 11.01 ± 1.98c 17.71 ± 1.17d 4.62 ± 0.26a,b
EPA/ARA 2.83 ± 0.18c 1.69 ± 0.16b 0.72 ± 0.20a 0.40 ± 0.04a 1.65 ± 0.13b
Total Sats 25.58 ± 0.24a 33.61 ± 0.60c 31.84 ± 0.53b,c 32.30 ± 1.43b,c 30.85 ± 0.83b
Total Monos 20.79 ± 1.66c 20.13 ± 0.63c 17.02 ± 1.00b 14.91 ± 0.19a 20.22 ± 0.49c
Total n-9 4.59 ± 0.02a,b,c 5.06 ± 0.35c 4.12 ± 0.39a 4.20 ± 0.19a 4.92 ± 0.31b,c
Total n-7 16.20 ± 1.68d 15.07 ± 0.76c,d 12.89 ± 0.97b 10.71 ± 0.20a 15.31 ± 0.58c,d
Total Poly Unsats 40.35 ± 0.21a 38.38 ± 0.81a 41.61 ± 0.73b 39.95 ± 0.97a,b 41.69 ± 0.75b
Total n-6 3.52 ± 0.02a 9.34 ± 0.13e 8.35 ± 0.13d 5.63 ± 0.07b 6.78 ± 0.11c
Total n-4 0.43 ± 0.01a 0.51 ± 0.14a 0.52 ± 0.24a 0.60 ± 0.20a 0.42 ± 0.00a
Total n-3 36.40 ± 0.19e 28.53 ± 0.68a 32.73 ± 0.74b,c 33.72 ± 1.03c,d 34.49 ± 0.67d,e
Total lipids (mg g 1) 29.31 ± 6.38a 28.11 ± 4.75a 32.71 ± 23.47a 43.26 ± 18.47a 35.81 ± 18.30a

Different letters represent significant differences at P < 0.05.

954 © 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960

Aquaculture Research, 2014, 45, 949–960
larvae, but the DHA content in the 12-DPH fish
enriched with S.presso or Nutrokol was similar to
that in the 2-DPH larvae. Nevertheless, the DHA
of the larvae in the fresh microalgae treatment
was significantly lower than that in other treat-
ments and in the 2-DPH larvae. The DHA/EPA
ratio was highest in the 12-DPH fish fed rotifers
enriched with Algamac 3050, followed by the
S.presso enrichment (P < 0.05), but there were no
differences in DHA/EPA ratios between fish fed rot-
ifers enriched with fresh microalgae and Nutrokol,
or between the 2-DPH larvae and the 12-DPH
larvae in the Nutrokol treatment (P > 0.05). In
contrast, the EPA/ARA ratio in all 12-DPH fish
was lower than that in the 2-DPH larvae. Among
all treatments, the EPA/ARA ratios of the fish fed
rotifers enriched with fresh microalgae or Nutrokol
were higher than those in the Algamac 3050 and
S.presso treatments (P < 0.05), but there was no
significant difference between fish fed rotifers
enriched with fresh microalgae or Nutrokol, or
between the Algamac and S.presso treatments
(P > 0.05).
Discussion
In this study, enrichment formulas did not change
the total lipid of rotifers. Similar results have been
also found in other studies (Frolov, Pankov,
Geradze, Pankova & Spektorova 1991; Caric,
Sanko-Njire & Skaramuca 1993; Fernández-Reiriz,
Labarta & Ferreiro 1993; Palmtag et al. 2006).
However, the specific fatty acid composition in
rotifers was significantly different between treat-
ments. Rotifers enriched with Algamac 3050
delivered higher concentration of DHA (34.53%
total fatty acids, TFA) and the DHA/EPA (10.81:1)
ratio, but this treatment did not lead to fast fish
growth and high survival. On the contrary, a bet-
ter survival was obtained in the fresh microalgae
treatment where the DHA/EPA ratio was only
0.44:1. Low fish survival in the Algamac 3050
treatment supports the claim in a previous study
that a high DHA content and a high DHA/EPA
ratio may reduce larval fish survival (Planas &
Cunha 1999), because unbalance in lipid class
composition affects fatty acids’ digestion and
absorption (Diaz, Guyot, Vigier & Connes 1997;
Salhi, Izquierdo, Hernandez-Cruz, Socorro & Fer-
nandez-Palacios 1997; Salhi, Hernández-Cruz,
Bessonart, Izquierdo & Fernández-Palacios 1999).
For instance, DHA and EPA utilize same enzymes
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960
13652109, 2014, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/are.12037 by INIDEP-Instituto Nacional de Investigacion y Desarrollo Pesq, Wiley Online Library on [05/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
to esterify fatty acids into the phospholipid struc-
ture (Sargent et al. 1999b), and an excess of EPA
in cell membranes may negatively affect larval
vitality (Watanabe 1993).
The ideal diet for fish larvae should contain
similar lipids to those in the egg yolk (Sargent
et al. 1999a). Sargent et al. (1995) recommend
that the optimal DHA/EPA ratio be around 2:1.
However, increasing evidence suggests that the
optimum DHA/EPA ratio for fish larvae varies
between species (Rodriguez, Perez, Diaz, Izquierdo,
Fernandez-Palacios & Lorenzo 1997; Copeman
et al. 2002; Harel et al. 2002). In this study, fish
at 2 DPH were used as a reference for the nutri-
tional status of fish embryo because fish had not
been fed at this stage. The ratio of DHA/EPA in
the 2-DPH larvae was 5.16:1, which was higher
than the DHA/EPA ratio in the rotifers enriched
with microalgae (0.44:1) or Nutrokol (1.27:1), but
was lower than the DHA/EPA ratio in the roti-
fers enriched with Algamac 3050 (10.81:1). By
comparing the DHA/EPA ratios in rotifers and in
the fish embryo, survival was higher in fish fed
the rotifers with the DHA/EPA ratio of 0.44~
4.82:1. The upper DHA/EPA ratio in the diet is
closed to the DHA/EPA ratio in the unfed YTK on
2 DPH, and is also close to the DHA/EPA ratios
(4.2:1–4.7:1) in YTK eggs (Battaglene & Cobcroft
2007). The low survival of fish fed rotifers
enriched with Algamac 3050 at an extreme high
DHA/EPA ratio (10.81:1) suggests that rotifers
should be enriched with a formula close to the
nutrition composition in fish eggs or embryos.
In marine fish, both EPA and DHA are essential
to fish growth (Rezek et al. 2010). Rodriguez et al.
(1997) reported that a higher DHA/EPA ratio
(1.4:1–0.3:1) during the rotifer feeding stage can
improve the growth of gilthead sea bream Sparus
aurata L. Copeman et al. (2002) found that yellow-
tail flounder Limanda ferruginea fed a high DHA/
EPA ratio (8:1) grew faster than those fed a DHA/
EPA ratio of 1.9:1. In this study, the DHA/EPA
ratios varied at 0.44–10.81 in enriched rotifers.
However, fish growth was not affected by the
methods of enrichments. Similar results have also
been reported in Japanese flounder Paralichthys
olivaceus, and turbot Scophthalmus maximus when
live food was enriched with formulas of different
DHA/EPA ratios (Estevez, McEvoy, Bell & Sargent
1999; Furuita, Konishi & Takeuchi 1999).
Compared with EPA and DHA, the importance of
ARA in fish nutrition has been overlooked (Bell &
955

Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
Sargent 2003). Despite the relatively small amount
of ARA in fish tissue, it is essential to most marine
carnivorous fish (Koven, Barr, Lutzky, Ben-Atia,
Weiss, Harel, Behrens & Tandler 2001). Gilthead
sea bream Sparus aurata L. larvae fed rotifers
enriched with ARA show high survival (Koven
et al. 2001). The addition of dietary ARA is also
beneficial to salmonids in undergoing parr-smolt
transformation (Tocher, Bell, Dick, Henderson,
McGhee, Mitchell & Morris 2000). In this study,
fish growth was not affected by the dietary ARA
levels from 1.40 ± 0.16 to 2.35 ± 0.14% in total
fatty acids, which is supported by the results from
gilthead sea bream Sparus aurata, Japanese flounder
Paralichthys olivaceus and white bass Morone chrys-
ops (Estevez, Kaneko, Seikai, Tagawa & Tanaka
2001; Koven et al. 2001). In our study, the lowest
EPA/ARA ratio (1.38 ± 0.24) was found in the
rotifer enriched with Algamac 3050. Coincidently,
the lowest EPA/ARA ratio (0.40 ± 0.04) in fish
larvae was also found in the Algamac 3050 treat-
ment on 12 DPH, which may contribute to the
lowest fish survival rate in the Algamac 3050
treatment. In addition, the low survival of fish fed
rotifers enriched with Algamac 3050 may also be
attributed to the low feeding incidence of fish in
this treatment.
Jaw malformation not only affects fish growth
and survival but also reduces the market value of
marine fish (Barahona-Fernandes 1982; Cobcroft
et al. 2004). Izquierdo, Socorro and Roo (2010)
suggest that PUFA is important in bone formation,
and several other studies also indicate that dietary
lipids can affect the fatty acid composition in bone
and cartilage (Kokkinos, Shaye, Alam & Alam
1993; Watkins, Shen, Memurtry, Xu & Bain
1997; Liu, Veit & Denbow 2004). As the source of
dietary lipids is primarily from rotifers, enrichment
formula on rotifer may possibly affect jaw malfor-
mation. In this study, however, the jaw deformities
rates were less than 5% and no significant differ-
ences were found between treatments. This may
suggests that the current enrichment formula may
suffice the nutritional requirement for jaw develop-
ment at this stage.
Microalgae are on the bottom of the food web
and support nutrition for majority of aquatic
animals and particularly for the production of live
food for marine fish larvae (Ferreira, Coutinho,
Seixas, Fábregas & Otero 2009). Green-water tech-
nology has been widely used in the culture of
marine fish larvae (Reitan, Rainuzzo, Øie & Olsen
956
13652109, 2014, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/are.12037 by INIDEP-Instituto Nacional de Investigacion y Desarrollo Pesq, Wiley Online Library on [05/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Aquaculture Research, 2014, 45, 949–960
1993) and microalgae serve as a key food compo-
nent for zooplankton and also provide a contrast
background for fish feeding (Reitan, Rainuzzo,
Oeie & Olsen 1997). Therefore, algae have been
considered an indispensable component in larval
fish rearing (Reitan et al. 1993; Stottrup, Gravnin-
gen & Norsker 1995; Ferreira, Maseda, Fábregas &
Otero 2008; Ludwig & Rawles 2008; Ferreira et al.
2009). In this study, we explored the possible
replacement of fresh algae with enrichment formulas
and found that compatible fish survival was
achieved between YTK larvae fed rotifers enriched
with fresh microalgae and with an enrichment for-
mula S.presso. However, the use of fresh algae for
rotifer enrichment increased fish feeding incidence
at the first feeding. The colour of rotifers enriched
with fresh microalgae, S.presso, Algamac 3050
and Nutrokol was brown, pink, pale white and
bright white respectively. The possible reason for
the discrepancy in feeding incidence is that rotifers
enriched with fresh algae are more visible to fish
as suggested by (Blaxter 1986). It seems that
brown food particles are more attractive to the
first feeding YTK larvae than by other coloured
food particles. In this study, however, we did not
investigate other nutritional components such as
protein, micronutrients in rotifers. In a previous
study, Øie, Makridis, Reitan and Olsen (1997)
reported that protein and carbon utilization in
rotifers B. plicatilis could impact the survival of
turbot larvae. Therefore, besides fatty acids, the
impact of other nutritional components in rotifers
on larval fish performance warrants further study
in future.
In summary, total lipid of rotifers after enrich-
ment was not significantly affected by enrichment
formulas, but the specific fatty acid in rotifers was
affected by enrichment formulas. Rotifers enriched
with Algamac 3050 increased DHA and DHA/EPA
ratio, but did not increase fish growth and survival.
Enrichment formulas did not affect fish growth and
jaw deformity, but best fish survivals were
achieved in rotifers enriched with fresh algae and
S.presso. This study suggests that ratios of DHA/
EPA and EPA/ARA affect fish survival more than
their absolute values. Although fresh microalgae
and S.presso resulted in similar fish survival, roti-
fers enriched with fresh microalgae improved fish
feeding incidence at first feeding. S.presso enrich-
ment formula achieved similar fish survival, but
Algamac 3050 may not be suitable to enrich roti-
fers for rearing YTK larvae.
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960

Aquaculture Research, 2014, 45, 949–960 Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
Acknowledgments
This research was funded by the Australian
Seafood CRC. Clean Seas Tuna kindly provided
fish eggs for this study. Paul Skordas, Bennan
Chen and Wayne Hutchinson provided technical
support to this project. Margaret Stopa and Bar-
bara Rone-Clarke from National Collaborative
Research Infrastructure Strategy, Adelaide, Austra-
lia provided support for fatty acids analysis. South
Australian Research and Development Institute
provided research facilities.
References
Avella M.A., Olivotto I., Gioacchini G., Maradonna F. &
Carnevali O. (2007) The role of fatty acids enrich-
ments in the larviculture of false percula clownfish
Amphiprion ocellaris. Aquaculture 273, 87–95.
Barahona-Fernandes M.H (1982) Body deformation in
hatchery reared European sea bass Dicentrarchus labrax
(L).Types, prevalence and effect on fish survival. Jour-
nal of Fish Biology 21, 239–249.
Battaglene S.C. & Cobcroft J.M. (2007) Yellowtail kingfish
juvenile quality: Identify timing and nature of jaw
deformities in yellowtail kingfish and scope the likely
causes of this condition. Final report of project 2007/
718, the Australian Seafood CRC, pp. 150.
Bell J.G. & Sargent J.R. (2003) Arachidonic acid in aqua-
culture feeds: current status and future opportunities.
Aquaculture 218, 491–499.
Benetti D.D. (2008) Review of Seriola spp Aquaculture.
In: Proceedings of International Workshop: Developing a
Sustainable Aquaculture Industry in the Azores. (ed. by
C.K. Pham, R.M. Higgins, M. de Girolamo & E. Isidro),
pp. 83–85. Universidade dos Acores, Arquipélago.
Blaxter J.H.S. (1986) Development of sense organs and
behaviour of teleost larvae with special reference to
feeding and predator avoidance. Transactions of the
American Fisheries Society 115, 98–114.
Borowitzka M.A. (1997) Microalgae for aquaculture: oppor-
tunities and constraints. Journal of Applied Phycology 9,
393–401.
Caric M., Sanko-Njire J. & Skaramuca B. (1993) Dietary
effects of different feeds on the biochemical composition
of the rotifer (Brachionus plicatilis). Aquaculture 110,
141–150.
Castell J., Blair T., Neil S., Howes K., Mercer S., Reid J.,
Young-Lai W., Gullison B., Dhert P. & Sorgeloos P.
(2003) The effect of different HUFA enrichment emul-
sions on the nutritional value of rotifers (Brachionus
plicatilis) fed to larval haddock (Melanogrammus aeglefi-
nus). Aquaculture International 11, 109–117.
Cobcroft J.M. & Battaglene S.C. (2009) Jaw malformation
in striped trumpeter Latris lineata larvae linked to
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960
13652109, 2014, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/are.12037 by INIDEP-Instituto Nacional de Investigacion y Desarrollo Pesq, Wiley Online Library on [05/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
walling behaviour and tank colour. Aquaculture 289,
274–282.
Cobcroft J.M., Pankhurst P.M., Poortenaar C., Hickman
B. & Tait M. (2004) Jaw malformation in cultured
yellowtail kingfish (Seriola lalandi) larvae. New
Zealand Journal of Marine and Freshwater Research 38,
67–71.
Conceicao L.E.C., Yufera M., Makridis P., Morais S. &
Dinis M.T. (2010) Live feeds for early stages of fish
rearing. Aquaculture Research 41, 613–640.
Copeman L.A., Parrish C.C., Brown J.A. & Harel M. (2002)
Effects of docosahexaenoic, eicosapentaenoic, and
arachidonic acids on the early growth, survival, lipid
composition and pigmentation of yellowtail flounder
(Limanda ferruginea): a live food enrichment experiment.
Aquaculture 210, 285–304.
Dhert P., Rombaut G., Suantika G. & Sorgeloos P. (2001)
Advancement of rotifer culture and manipulation tech-
niques in Europe. Aquaculture 200, 129–146.
Diaz J.P., Guyot E., Vigier S. & Connes R. (1997) First
events in lipid absorption during post-embryonic devel-
opment of the anterior intestine in gilt-head sea bream.
Journal of Fish Biology 51, 180–192.
Estevez A., McEvoy L.A., Bell J.G. & Sargent J.R. (1999)
Growth, survival, lipid composition and pigmentation
of turbot (Scophthalmus maximus) larvae fed live-prey
enriched in arachidpnic and eicosapentaenoic acids.
Aquaculture 180, 321–343.
Estevez A., Kaneko T., Seikai T., Tagawa M. & Tanaka
M. (2001) ACTH and MSH production in Japanese
flounder Paralichthys olivaceus larvae fed arachidonic
acid enriched live prey. Aquaculture 192, 309–319.
Estudillo-del Castillo C., Gapasin R.S. & Leaño E.M.
(2009) Enrichment potential of HUFA-rich thrausto-
chytrid Schizochytrium mangrovei for the rotifer Brachi-
onus plicatilis. Aquaculture 293, 57–61.
Faulk C.K. & Holt G.J. (2003) Lipid nutrition and feeding
of Cobia Rachycentron canadum larvae. Journal of the
World Aquaculture Society 34, 368–378.
Faulk C.K., Holt G.J. & Davis D.A. (2005) Evaluation of
fatty acid enrichment of live food for yellowtail snapper
Ocyurus chrysurus larvae. Journal of the World Aquacul-
ture Society 36, 271–281.
Fernández-Reiriz M.J., Labarta U. & Ferreiro M.J. (1993)
Effects of commercial enrichment diets on the nutri-
tional value of the rotifer (Brachionus plicatilis). Aqua-
culture 112, 195–206.
Ferreira M., Maseda A., Fábregas J. & Otero A. (2008)
Enriching rotifers with “premium” microalgae. Isochry-
sis aff. galbana clone T-ISO. Aquaculture 279, 126–130.
Ferreira M., Coutinho P., Seixas P., Fábregas J. & Otero
A. (2009) Enriching rotifers with “premium” microal-
gae Nannochloropsis gaditana. Marine Biotechnology 11,
585–595.
Folch J., Lees M. & Sloane Stanley G.H. (1957) A simple
method for the isolation and purification of total lipides
957

Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
from animal tissues. The Journal of Biological Chemistry
226, 497–509.
Fowler A.J., Ham J.M. & Jennings P.R. (2003) Discrimi-
nating between Cultured and Wild Yellowtail Kingfish
(Seriola Lalandi) in South Australia. Sardi Aquatic
Sciences Publication No. Rd03/0159, 96pp. Australian,
Adelaide.
Frolov A.V., Pankov S.L., Geradze K.N., Pankova S.A. &
Spektorova L.V. (1991) Influence of the biochemical
composition of food on the biochemical composition of
the rotifer Brachionus plicatilis. Aquaculture 97, 181–202.
Furuita H., Konishi K. & Takeuchi T. (1999) Effect of
different levels of eicosapentaenoic acid and docosa-
hexaenoic acid in Artemia nauplii on growth, survival
and salinity tolerance of larvae of the Japanese floun-
der Paralychthys olivaceus. Aquaculture 170, 59–69.
Garcia A.S., Parrish C.C. & Brown J.A. (2008a) Use of
enriched rotifers and Artemia during larviculture of
Atlantic cod (Gadus morhua Linnaeus, 1758): effects on
early growth, survival and lipid composition. Aquacul-
ture Research 39, 406–419.
Garcia A.S., Parrish C.C., Brown J.A., Johnson S.C. &
Leadbeater S. (2008b) Use of differently enriched
rotifers, Brachionus plicatilis, during larviculture of
haddock, Melanogrammus aeglefinus: effects on early
growth, survival and body lipid composition. Aquacul-
ture Nutrition 14, 431–444.
Ghioni C., Tocher D.R., Bell M.V., Dick J.R. & Sargent
J.R. (1999) Low C18 to C20 fatty acid elongase activity
and limited conversion of stearidonic acid, 18:4(n-3),
to eicosapentaenoic acid, 20:5(n-3), in a cell line
from the turbot, Scophthalmus maximus. Biochimica et
Biophysica Acta 1437, 170–181.
Hamre K., Opstad I., Espe M., Solbakken J., Hemre G.-I. &
Pittman K. (2002) Nutrient composition and metamor-
phosis success of Atlantic halibut (Hippoglossus hippog-
lossus, L.) larvae fed natural zooplankton or Artemia.
Aquaculture Nutrition 8, 139–148.
Harel M., Koven W., Lein I., Bar Y., Behrens P., Stubble-
field J., Zohar Y. & Place A. (2002) Advanced DHA,
EPA, And ArA enrichment materials for marine aqua-
culture using single cell heterotrophs. Aquaculture
213, 347–362.
Hilton Z., Poortenaar C.W. & Sewell M.A. (2008) Lipid
and protein utilisation during early development of
yellowtail kingfish (Seriola lalandi). Marine Biology 154,
855–865.
Hopkins K.D. (1992) Reporting fish growth: a review of
the basics. Journal of the World Aquaculture Society 23,
173–179.
Hutson K.S., Ernst I., Mooney A.J. & Whittington I.D.
(2007) Metazoan parasite assemblages of wild Seriola
lalandi (Carangidae) from eastern and southern Austra-
lia. Parasitology International 56, 95–105.
Izquierdo M.S., Arakawa T., Takeuchi T., Haroun R. &
Watanabe T. (1992) Effect of n-3 HUFA levels in
958
13652109, 2014, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/are.12037 by INIDEP-Instituto Nacional de Investigacion y Desarrollo Pesq, Wiley Online Library on [05/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Aquaculture Research, 2014, 45, 949–960
Artemia on growth of larval Japanese flounder (Para-
lichthys olivaceus). Aquaculture 105, 73–82.
Izquierdo M.S., Socorro J. & Roo J. (2010) Studies on the
appearance of skeletal anomalies in red porgy: effect of
culture intensiveness, feeding habits and nutritional
quality of live preys. Journal of Applied Ichthyology 26,
320–326.
Kanazawa A., Teshima S. & Ono K. (1979) Relationship
between essential fatty acid requirements of aquatic
animals and the capacity for bioconversion of linolenic
acid to highly unsaturated fatty acids. Comparative
Biochemistry and Physiology 63B, 295–298.
Kissil G.W. & Koven W.M. (1990) Preparation of oils,
enhanced in highly unsaturated fatty acid (HUFA) con-
tent, by low temperature crystallization separation, for
rotifer (Brachionus plicatilis) enrichment. Aquaculture
88, 69–74.
Kjørsvik E., Olsen C., Wold P.-A., Hoehne-Reitan K.,
Cahu C.L., Rainuzzo J., Olsen A.I., Øie G. & Olsen Y.
(2009) Comparison of dietary phospholipids and
neutral lipids on skeletal development and fatty acid
composition in Atlantic cod (Gadus morhua). Aquacul-
ture 294, 246–255.
Kokkinos P.P., Shaye R., Alam B.S. & Alam S.Q. (1993)
Dietary lipids, prostaglandin E2 levels, and tooth move-
ment in alveolar none of rats. Calcified Tissue Interna-
tional 53, 333–337.
Kotani T., Genka T., Tanabe M., Miyashima A., Fushimi
H. & Hayashi M. (2010) Effect of Nutritional Enrich-
ment Method on Fatty Acid Contents of Rotifer Brachi-
onus plicatilis. Journal of the World Aquaculture Society
41, 884–892.
Koven W.M., Kissil G.W. & Tandler A. (1989) Lipid and
n-3 requirement of Sparus aurata larvae during starva-
tion and feeding. Aquaculture 79, 185–191.
Koven W.M., Tandler A., Sklan D. & Kissil G.W. (1993)
The association of eicosapentaenoic and docosahexae-
noic acids in the main phospholipids of different-age Spa-
rus aurata larvae with growth. Aquaculture 116, 71–82.
Koven W., Barr Y., Lutzky S., Ben-Atia I., Weiss R.,
Harel M., Behrens P. & Tandler A. (2001) The effect of
dietary arachidonic acid (20:4n–6) on growth, survival
and resistance to handling stress in gilthead seabream
(Sparus aurata) larvae. Aquaculture 193, 107–122.
Lein I., Holmefjord I. & Rye M. (1997) Effects of tempera-
ture on yolk sac larvae of Atlantic halibut (Hippoglos-
sus hippoglossus L.). Aquaculture 157, 123–135.
Lie O., Haaland H., Hemre G.-I., Maage A., Lied E.,
Rosenlund G., Sandnes K. & Olsen Y. (1997) Nutri-
tional composition of rotifers following a change in diet
from yeast and emulsified oil to microalgae. Aquacul-
ture International 5, 427–438.
Liu D., Veit H.P. & Denbow D.M. (2004) Effects of long-
term dietary lipids on mature bone mineral content,
collagen, crosslinks, and prostaglandin E2 production
in Japanese quail. Poultry Science 83, 1876–1883.
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960

Aquaculture Research, 2014, 45, 949–960 Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
Liu H., Kelly M.S., Cook E.J., Black K., Orr H., Zhu J.X. &
Dong S.L. (2007) The effect of diet type on growth and
fatty-acid composition of sea urchin larvae, I. Paracen-
trotus lividus (Lamarck, 1816) (Echinodermata). Aqua-
culture 264, 247–262.
Ludwig G.M. & Rawles S.D. (2008) Effect of rotifer
enrichment on Sunshine bass Morone chrysops x M.
saxatilis larvae growth and survival and fatty acid
composition. Jounral of the World Aquaculture Society
39, 158–173.
McEvoy L.A., Naess T., Bell J.G. & Lie O. (1998) Lipid
and fatty acid composition of normal and malpigment-
ed Atlantic halibut (Hippoglossus hippoglossus) fed
enriched Artemia: a comparison with fry fed wild cope-
pods. Aquaculture 163, 237–250.
Miki N., Taniguchi T., Hamakawa H., Yamada Y. &
Sakurai N. (1990) Reduction of albinism in hatchery-
reared flounder “Hirame”, Paralichthys olivaceus by
feeding on rotifer enriched with vitamin-A. Suisanzos-
hoku 38, 147–155.
Nordoy E.S., Aakvaag A. & Larsen T.S. (1993) Metabolic
adptations to fasting in harp seal pups. Physiological
Zoology 66, 926–945.
Øie G., Makridis P., Reitan K.I. & Olsen Y. (1997) Protein
and carbon utilization of rotifers (Brachionus plicatilis) in
first feeding of turbot larvae (Scophthalmus maximus L.).
Aquaculture 153, 103–122.
Olivotto I., Rollo A., Sulpizio R., Avella M., Tosti L. &
Carnevali O. (2006) Breeding and rearing the Sunrise
Dottyback Pseudochromis flavivertex: the importance of
live prey enrichment during larval development. Aqua-
culture 255, 480–487.
Palmtag M.R., Faulk C.K. & Holt G.J. (2006) Highly
unsaturated fatty cid composition of rotifers (Brachi-
onus plicatilis) and Artemia fed various enrichments.
Jounral of the World Aquaculture Society 37, 126–131.
Planas M. & Cunha I. (1999) Larviculture of marine fish:
problems and perspectives. Aquaculture 177, 171–190.
Rainuzzo J.R., Reitan K.I. & Olsen Y. (1997) The signifi-
cance of lipids at early stages of marine fish: a review.
Aquaculture 155, 103–115.
Reitan K.I., Rainuzzo J.R., Øie G. & Olsen Y. (1993)
Nutritional effects of algal addition in first-feeding of
turbot (Scophthalmus maximus L.) larvae. Aquaculture
118, 257–275.
Reitan K.I., Rainuzzo J.R., Oeie G. & Olsen Y. (1997)
A review of the nutritional effects of algae in marine
fish larvae. Aquaculture 155, 211–225.
Rezek T.C., Watanabe W.O., Harel M. & Seaton P.J. (2010)
Effects of dietary docosahexaenoic acid (22:6n–3) and
arachidonic acid (20:4n–6) on the growth, survival,
stress resistance and fatty acid composition in black
sea bass Centropristis striata (Linnaeus 1758) larvae.
Aquaculture Research 41, 1302–1314.
Rodriguez Rainuzzo J., Olsen Y. & Rosenlund G. (1989)
The effect of enrichment diets on the fatty acid compo-
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960
13652109, 2014, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/are.12037 by INIDEP-Instituto Nacional de Investigacion y Desarrollo Pesq, Wiley Online Library on [05/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
sition of the rotifer Brachionus plicatilis. Aquaculture 79,
157–161.
Rodriguez C., Perez J.A., Diaz M., Izquierdo M.S., Fernan-
dez-Palacios H. & Lorenzo A. (1997) Influece of the
EPA/DHA ratio in rotifers on gilthead seabream (Sparus
aurata) larval development. Aquaculture 150, 77–89.
Rodriguez C., Perez J.A., Badia P., Izquierdo M.S.,
Fernandez-Palacios H. & Lorenzo-Hernandez A. (1998)
The n - 3 highly unsaturated fatty acids requirements
of gilthead seabream (Sparus aurata L.) larvae when
using an appropriate DHA/EPA ratio in the diet.
Aquaculture 169, 9–23.
Salhi M., Izquierdo M.S., Hernandez-Cruz C.M., Socorro J.
& Fernandez-Palacios H. (1997) The improved incorpo-
ration of polyunsaturated fatty acids and changes in
liver structure in larval gilthead seabream fed on mic-
rodiets. Journal of Fish Biology 51, 869–879.
Salhi M., Hernández-Cruz C.M., Bessonart M., Izquierdo
M.S. & Fernández-Palacios H. (1999) Effect of different
dietary polar lipid levels and different n-3 HUFA
content in polar lipids on gut and liver histological
structure of gilthead seabream (Sparus aurata) larvae.
Aquaculture 179, 253–263.
Sarasquete C., Gonzalez de Canales M.L., Arellano J.M.,
Munoz-Cueto J.A., Ribeiro L. & Dinis M.T. (1996)
Histochemical aspects of the yolk-sac and digestive
tract of larvae of the Senegal sole, Solea senegalensis
(Kaup, 1858). Histology and Histopathology 11,
881–888.
Sargent J.R., Bell J.G., Bell M.V., Henderson R.J. & Tocher
D.R. (1995) Requirement criteria for essential fatty
acids. Symposium of European Inland Fisheries Advi-
sory Commission. Journal of Applied Ichthyology 11,
183–198.
Sargent J., Bell G., McEvoy L., Tocher D. & Estevez A.
(1999a) Recent developments in the essential fatty
acid nutrition of fish. Aquaculture 177, 191–199.
Sargent J., McEvoy L., Estevez A., Bell G., Bell M.,
Henderson J. & Tocher D. (1999b) Lipid nutrition of
marine fish during early development: current status
and future directions. Aquaculture 179, 217–229.
Sargent J.R., Tocher D.R. & Bell J.G. (2002) The lipids.
In: Fish Nutrition, (3rd edn.) (ed. by J.E. Halver),
pp. 181–257. Academic Press, San Diego.
Stottrup J.G., Gravningen K. & Norsker N.H. (1995) The
role of different algae in the growth and survival of
turbot larvae (Scophthalmus maximus L.) in intensive
rearing system. ICES. Marine Science Symposia 201,
173–186.
Takeuchi T., Dedi J., Haga Y., Seikai T. & Watanabe T.
(1998) Effect of vitamin A compounds on bone defor-
mity in larval Japanese flounder (Paralichthys olivac-
eus). Aquaculture 169, 155–161.
Tocher D.R., Mourente G. & Sargent J.R. (1997) The use
of silages prepared from fish neural tissues as enrichers
for rotifers (Brachionus plicatilis) and Artemia in
959

Enrichment formulas for rotifers in larval fish rearing Z Ma et al.
the nutrition of larval marine fish. Aquaculture 148,
213–231.
Tocher D.R., Bell J.G., Dick J.R., Henderson R.J., McGhee
F., Mitchell D. & Morris P.C. (2000) Polyunsaturated
fatty acid metabolism in Atantic salmon (Salmo salar)
undergoing parr-smolt transformation and the effects
of dietary linseed and rapeseed oils. Fish Physiology and
Biochemistry 23, 59–73.
Watanabe T. (1993) Importance of docosahexaenoic acid
in marine larval fish. Journal of the World Aquaculture
Society 24, 152–161.
Watanabe T. & Kiron V. (1994) Prospects in larval fish
dietetics. Aquaculture 124, 223–251.
Watanabe T., Kitajima C., Arakawa T., Fukusho K. &
Fujita S. (1978) Nutritional quality of rotifer Brachi-
onus plicatilis as a living feed from the viewpoint of
960
13652109, 2014, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/are.12037 by INIDEP-Instituto Nacional de Investigacion y Desarrollo Pesq, Wiley Online Library on [05/07/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Aquaculture Research, 2014, 45, 949–960
essential fatty acids for fish. Bulletin of the Japanese
Society of Scientific Fisheries 44, 1109–1114.
Watanabe T., Tamiya T., Oka A., Hirata M., Kitajima C.
& Fujita S. (1983) Improvement of dietary value of live
foods for fish larvae by feeding them on n-3 highly
unsaturated fatty acids and fat-soluble vitamins. Bulle-
tin of the Japanese Society of Scientific Fisheries 49,
471–479.
Watkins B.A., Shen C.L., Memurtry J.P., Xu H. & Bain
S.D. (1997) Dietary lipids modulate bone prostaglandin
E2 production, insulin-like growth factor-I concentra-
tion and formation rate in chicks. The Journal of Nutri-
tion 127, 1084–1091.
Wedemeyer G.A. (1996) Physiology of Fish in Intensive
Culture Systems. Chapman & Hall, International
Thompson Publsihing, New York, USA.
© 2012 John Wiley & Sons Ltd, Aquaculture Research, 45, 949–960