Aquaculture research, tập 41, số 9, 2010

Aquaculture Research, 2010, 41, e1^e7

doi:10.1111/j.1365-2109.2009.02463.x

Review Article
A review of sex determination and searches for
sex-specific markers in sturgeon
Saeed Keyvanshokooh1 & Ahmad Gharaei2
1

Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology,

Khorramshahr, Khouzestan, Iran
2

Department of Fisheries, Faculty of Natural Resources, University of Zabol, Zabol, Sistan & Blouchestan, Iran

Correspondence: S Keyvanshokooh, Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine
Science and Technology, Khorramshahr, Khouzestan, Iran. E-mail:

Abstract
The availability of monosex populations of caviarproducing female sturgeon would considerably enhance the economic viability of domestic caviar
production systems. However, it is not possible to distinguish males from females by morphological characters at larval, juvenile and even adult stages. The
mechanism of sex determination in sturgeons is
poorly understood, and to date no sex-speci¢c markers in sturgeon have been reported. This review concentrates on the methodologies used to elucidate the
mode of sex determination in sturgeon species and
provides information on the molecular tools used to
determine genetic sex markers.

Keywords: sturgeon, sex determination, sex marker, genomics, proteomics

Introduction
Sturgeon species (FamilyAcipenseridae) belonging to
the order Acipenseriformes, are primitive ¢shes (Bemis, Findies & Grande 1997) that evolved approximately 250 million years ago. Sturgeon populations
are declining worldwide due to over¢shing, pollution
and habitat degradation (Birstein 1993; Billard & Lecointre 2001). Production of sturgeon for meat or caviar increasingly has to rely on aquaculture (Logan,
Johnston & Doroshov 1995). As sturgeon species are
highly prized for their caviar, females are more valuable than males. From an aquacultural perspective, it
is important that the sex of sturgeon be identi¢ed in

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd

the early stage of development, so that males can be
reserved for meat and females for caviar production.
None of the sturgeon species exhibit external sexual dimorphism, and the absence of external markers
for sexing has encouraged the search for a practical
technique for gender identi¢cation. Blood plasma
sex steroid levels in sturgeon remain low until the beginning of gonadal development (Doroshov, Moberg
& Van Eenennaam 1997). Although an examination
of plasma steroids could be used to sex sturgeon
(Webb, Feist, Foster, Schreck & Fitzpatrick 2002),
these steroid indicators are in£uenced by age, husbandry conditions and water temperature (Feist,Van
Eenennaam, Doroshov, Schreck, Schneider & Fitzpatrick 2004). The current diagnostic technique for sex
identi¢cation in these sexually monomorphic species
requires surgical biopsy of sexually di¡erentiated gonads (Doroshov et al. 1997). Although survival rate is
nearly100%, this surgery is invasive (Feist et al. 2004)
and non-invasive procedures are needed for sexing
sturgeon. The di¡erent techniques that have been
used to elucidate the sex-determining system and to

identify sex-speci¢c markers in sturgeon are the subject of this review.
Elucidation of sex-determining
mechanism in sturgeon
Sex determination is the genetic or environmental
process by which the sex (male or female) of an individual is established in a simple binary fate decision
(Penman & Piferrer 2008). Fish use a wide array
of mechanisms for sex determination (reviewed by

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Sex determination in sturgeon S Keyvanshokooh & A Gharaei

Devlin & Nagahama 2002). Many species use genetic
systems that determine sex at fertilization. A number
of ¢sh species use intrinsic clues provided by behavioural interactions among conspeci¢cs, some use
changes in environmental factors such as temperature, season or year class, whereas in some cases,
sex may also be determined by genetic^environmental interactions.
Intersexuality (ovotestis) is a condition whereby an
individual possesses oocytes or di¡erent stages of
spermatogonia, at varying degrees of development,
within the normal gonad of the opposite gender (i.e.
spermatocytes in the ovary or oocytes in the testis)
(Jackson, Hurvitz, Din, Goldberg, Pearlson, Degani &
Levavi-Sivan 2006). Hermaphroditism in sturgeon is
not a normal situation, and the few cases of intersexuality that have been reported in wild sturgeon
have all been from polluted habitats (Chapman, Van
Eenennaam & Doroshov 1996; Van Eenennaam &
Doroshov 1998; Harshbarger, Co¡ey & Young 2000).
In addition, the sex ratio in adult populations of

sturgeon has been 1<: 1, (Chapman et al. 1996).
These observations suggest that sturgeon have a gonochoristic type of sexuality and that sex is genetically determined. Environmental sex determination
would lead to variations in sex ratios due to the systematic £uctuations in the environmental factors
that in£uence sex (Bull 1983).
A wide variety of techniques (e.g. cytogenetic studies, analysis of sex ratios among families, examination of progeny sex ratios from sex-reversed
individuals, analysis of sex ratio among gynogenetic
progeny, etc.) have been used to elucidate sex determination mechanisms in ¢sh species (reviewed by Devlin & Nagahama 2002). To date, several studies have
been carried out to analyse the sex-determining systems in sturgeon.
Cytogenetic studies
One of the most rapid ways that the sex-determining
mechanism of a species can be detected is by determining the karyotype of male and female individuals
(Devlin & Nagahama 2002). Species with sex chromosomes can demonstrate male or female heterogamety. Male and female heterogametic species can
exhibit homomorphic or heteromorphic sex chromosomes. Heteromorphic sex chromosomes appear different in size or shape when viewed under the
microscope, or can be similar in size and shape but
di¡erences be evidenced only when using other techniques such as banding (Penman & Piferrer 2008).

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Aquaculture Research, 2010, 41, e1–e7

Fish exhibit a relatively primitive sex chromosome
evolution (Vol¡ 2005) and many ¢sh species do not
display heteromorphic sex chromosomes. About 10%
(176 species) of the ¢sh species studied have been
found to contain cytogenetically distinct sex chromosomes (Devlin & Nagahama 2002). Karyotypes of
¢sh belonging to the order Acipenseriformes are
characterized by a very large number of chromosomes, about half of which are microchromosomes.
The order may be divided into two groups; the ¢rst
group has a chromosome number of approximately
120 and a mean genome size (1C) of 1.6^2.5 pg, and

the second group has a chromosome number of
240^250 and a genome size of 4.0^4.8 pg (Birstein
1993; Blacklidge & Bidwell 1993). Heteromorphic sex
chromosomes have not been identi¢ed in many sturgeon species (Fontana & Colombo 1974; Holcik 1986;
Van Eenennaam 1997). However, monomorphic sex
chromosomes do not preclude a stable gonochoristic
sex ratio of 1<:1,.
Analysis of sex ratio among gynogenetic
progeny
In the absence of cytologically demonstrable sex
chromosomes in most ¢sh species, genetic approaches have been used to show that sex determination in many of the gonochoristic species can be
explained by a chromosomal mechanism (Van Eenennaam, Van Eenennaam, Medrano & Doroshov 1999).
Gynogenesis, as one of the chromosomal manipulation techniques, is a valuable experimental tool for
investigating sex-determining mechanisms in ¢sh
(Felip, Zanuy, Carrilo & Piferrer 2001; Devlin & Nagahama 2002). In species with female homogamety
(XX), gynogenesis will produce all-female progeny. In
species having female heterogamety (WZ), gynogenesis may produce ZZ males, WW super females and/
or WZ females, depending upon the rate of recombination between the sex-determining locus and the
centromere during the meiotic prophase (Nace, Richards & Asher Jr 1970;Van Eenennaam 1997).
Gynogenesis has been used to infer female heterogamety in three sturgeon species: the white sturgeon, Acipenser transmontanus (Van Eenennaam
et al. 1999), the beluga sturgeon, Huso huso (Omoto,
Maebayashi, Adachi, Arai & Yamauchi 2005) and
the shortnose sturgeon, Acipenser brevirostrum
(Flynn, Matsuoka, Reith, Martin-Robichaud & Benfey
2006). In fact, the strongly female-biased sex ratios
of the gynogenetic diploids in the above-mentioned
studies (82% in white sturgeon; 70^80% in beluga

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Aquaculture Research, 2010, 41, e1^e7

sturgeon; 65% in shortnose sturgeon) provide evidence that female is not the homogametic sex in
these species. If it were, then gynogenetic diploids
would all be female, as demonstrated for salmonids,
which have a typical mammalian system for sex determination (XX female, XY male; Devlin & Nagahama 2002). However, these results do not exclude the
possibility of other chromosomal or polygenic factors
having an e¡ect on sex ratio. Regarding the extensive
genome duplication in the evolutionary history of
sturgeon (Ludwig, Bel¢ore, Pitra, Svirsky & Jenneckens 2001), it is possible that sex may be controlled by
multiple chromosomes with multiple sex-determining loci (Flynn et al. 2006). Because the sex-determination systems between closely related species were
reported to be di¡erent in some cases (Devlin & Nagahama 2002), more research should be carried out to
compare the sex-determining mechanisms in sturgeon species. If a female heterogametic sex-determination system is in operation in other sturgeon
species, gynogenesis would be useful for conservation plans for the many endangered sturgeon populations (Birstein 1993; Bemis & Findeis 1994), because
both female and male are predicted in gynogenetic
progeny. On the other hand, inducing gynogenetic diploids in endangered sturgeon populations (where
normal males cannot be recovered) with genetically
inactivated donor sperm from other sturgeon species
is a useful method for saving them from extinction
(Omoto et al. 2005). Moreover, cryopreservation of
sperm, followed by thawing and dispermic fertilization or induced mitotic diploid androgenesis in genetically inactivated donor ova from a closely related
species has been considered as one tool for ensuring
the survival of endangered ¢sh species (Ihssen,
McKay, McMillan & Phillips 1990; Grunina & Neifakh
1991; Thorgaard & Scheerer 1992). However, this
strategy in species having female heterogamety
would produce only the ZZ males, leading to failure
in population recovery (Van Eenennaam et al. 1999).

Thus, knowledge of the sex-determining system operating in sturgeon on the verge of extinction is of importance when using sperm cryopreservation for
conservation programmes.

Molecular markers for sex
DNA markers
One e¡ective solution to identify the sexes at an early
life stage is to use DNA markers. Such markers will be
present in species where one sex possesses an unique

Sex determination in sturgeon S Keyvanshokooh & A Gharaei

chromosome or DNA sequence (Gri⁄ths & Tiwari
1993), and may not be present in species with XX:XO
(ZO:ZZ), multilocus or environmental sex determination systems. The number of sex-linked sequences
found in the genome of ¢sh species is low in comparison with other vertebrates (Devlin & Nagahama
2002), and may not be applicable to more than one
¢sh species or even strain (Iturra, Medrano, Bagley,
Lam,Vergara & Marin 1997).
Several approaches have been used for the isolation of sex-associated DNA markers in sturgeon (see
Table 1). Techniques in the ¢rst class involve a comparison of male and female DNA using di¡erent types
of DNA pro¢ling/¢ngerprinting. The second general
approach is based on candidate genes, where genes
or sequences that are sex determining or sex linked
in one species are searched for in the target species.
Another approach entails subtractive techniques to
¢nd sequences that are di¡erent between the male
and female genomes.
Interlinking three enterprises and four research laboratories, from three European countries, random ampli¢ed polymorphic DNA (RAPD), ampli¢ed fragment
length polymorphism (AFLP) and inter-simple sequence repeats (ISSR) techniques have been used to
search for sex-speci¢c DNA markers in sturgeon.

Wuertz, Gaillard, Barbisan, Carle, Congiu, Forlani, Aubert, Kirschbaum, Tosi, Zane and Grillasca (2006) focused on the identi¢cation of genomic sex-speci¢c
markers in four sturgeon species (Acipenser baerii; Acipenser naccarii; Acipenser gueldenstaedtii and Acipenser
ruthenus). Although 1100^9230 bands were screened
per species, no sex-speci¢c markers were detected. A similar result has been obtained by searching the genome of beluga sturgeon (H. huso) (Keyvanshokooh,
Pourkazemi & Kalbassi 2007) by using bulked segregant analysis (BSA; separate pooling of DNA from
males and females). A total of 310 randomly ampli¢ed
polymorphic DNA primers were used to screen the
bulks, resulting in 4146 bands that were present in
both sexes. Using the RAPD technique, McCormick,
Bos and DeWoody (2008) also failed to ¢nd a sex-speci¢c marker in lake sturgeon (Acipenser fulvescens).
Based on a candidate gene approach, several studies have been conducted to search for sex-speci¢c
markers by amplifying regions within sturgeon genomes with a high sequence similarity to sex-determination pathway genes of other ¢sh species. Members
of the Sox gene family are known to be involved in
numerous developmental processes and sex determination in vertebrates (Koopman, Gubbay, Vivian,
Goodfellow & Lovell-Badge 1991; Wright, Snopek &

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Aquaculture Research, 2010, 41, e1–e7

Table 1 Summary of studies using ¢ngerprinting/pro¢ling techniques to search for sex-speci¢c sequences in sturgeon
species
Method

Species

Comments

References

RAPD

Acipenser baerii
Acipenser naccarii
Acipenser ruthenus
Huso huso
Acipenser fluvescens
A. baerii
Acipenser gueldenstaedtii
Acipenser naccarii
A. baerii
A. gueldenstaedtii
Acipenser sturio

1100 bands screened
800 bands screened
1100 bands screened
4146 bands screened
50 primers screened; no sex markers
4900 fragments screened
4200 fragments screened
7970 bands screened
300 bands screened
130 bands screened

Sox genes were found; none were
sex specific
No Sox genes were sex-specific
No sex-specific marker was observed
No sex-specific marker was found
No protein spot was directly linked to a
sex-determining gene

Wuertz et al. (2006)
Wuertz et al. (2006)
Wuertz et al. (2006)
Keyvanshokooh et al. (2007)
McCormick et al. (2008)
Wuertz et al. (2006)
Wuertz et al. (2006)
Wuertz et al. (2006)
Wuertz et al. (2006)
Wuertz et al. (2006)
Hett and Ludwig (2005)

AFLP
ISSR

Candidate gene
approach

A. fluvescens
Subtractive techniques Acipenser transmontanus
A. fluvescens
Proteomics

Acipenser persicus

McCormick et al. (2008)
Van Eenennaam (1997)
McCormick et al. (2008)
Keyvanshokooh, Kalbassi et al. (2009)

RAPD, random ampli¢ed polymorphic DNA; AFLP, ampli¢ed fragment length polymorphism; ISSR, inter-simple sequence repeats.

Koopman 1993; Russell, Sanchez-Soriano, Wright &
Ashburner 1996). Sox proteins are characterized by
a conserved high mobility group (HMG)-box domain,
which is responsible for DNA binding and bending
(Sinclair, Berta, Palmer, Hawkins, Gri⁄ths, Smith,
Foster, Frischauf, Lovell-Badge & Goodfellow 1990).
Using highly degenerate primers that ampli¢ed a
broad range of HMG boxes, 22 di¡erent sequences
coding for eight Sox genes (Sox2, Sox3, Sox4, Sox9,
Sox11, Sox17, Sox19 and Sox21) were shown to be present in the genome of European Atlantic sturgeon
(Acipenser sturio) (Hett & Ludwig 2005; Hett, Pitra,
Jenneckens & Ludwig 2005). Similarly, sequences
with homology to Sox gene family (Sox2, Sox4,
Sox17 and Sox21) were detected in lake sturgeon
(A. fulvescens) (McCormick et al. 2008). However,
although Sox genes were found in the genomes of
A. sturio and A. fulvescens, none appeared to be the
primary sex-determination gene in any of these species. These results indicate that Sox genes exist in
sturgeon genome and thus may play a role in sexdetermination pathways, but probably they are not
the primary signal used to control sex in sturgeon
species.

Subtractive techniques have been used for the isolation of sex-associated molecular markers in various
species (Devlin, McNeil, Groves & Donaldson 1991;
Nakayama, Foresti, Tewari, Schartl & Chourrout
1994). The basis of these approaches is that the

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homogametic sex contains all sequences that the heterogametic does, except for those in the vicinity of the
sex-determining locus (Devlin & Nagahama 2002).
Another technique that also has been used for the
isolation of molecular sex markers is representational
di¡erence analysis (RDA) (Lisitsyn, Lisitsyn & Wigler
1993). Representational di¡erence analysis is a modi¢cation of the subtractive hybridization process,
which utilizes PCR ampli¢cation to enrich for sequences uniquely found in tester DNA. Subtractive
hybridization and RDA methodologies have been
used to screen for genetic markers associated with
sex in white sturgeon (Van Eenennaam 1997) and
lake sturgeon (McCormick et al. 2008), but no sexspeci¢c marker was observed.
All of the techniques mentioned herein have been
used to search for a sex-speci¢c marker in various ¢sh
species (May, Johnson & Wright 1989; Devlin et al.
1991; Iturra et al. 1997; Matsuda, Kusama, Oshiro,
Kurihara, Hamaguchi & Sakaizumi 1997; Coughlan,
Schartl, Hornung, Hope & Stewart 1999; Kovacs, Egedi, Bartfai & Orban 2001; Li, Hill,Yue, Chen & Orban
2002; Peichel, Ross, Matson, Dickson, Grimwood,
Schmutz, Myers, Mori, Schluter & Kingsley 2004;
Chen, Li, Deng,Tian,Wang, Zhuang, Sha & Xu 2007),
among which a few failed to ¢nd such DNA markers.
Unfortunately, in spite of extensive screening of
sturgeon genomes, all searches to date have failed to

detect sex-speci¢c sequences in sturgeon.With regard

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Aquaculture Research, 2010, 41, e1^e7

to these failures, McCormick et al. (2008) mentioned
that an environmental sex-determining system may
exist in sturgeon. In theory, the lack of sex-speci¢c
markers in the search could be due to the lack of
genetic sex-determination mechanisms, but there is
evidence of a female heterogametic genetic sex-determination system in sturgeons studied (Van Eenennaam et al. 1999; Omoto et al. 2005; Flynn et al.
2006). Based on data consistent with the ZW system,
the female sturgeon should carry sex-speci¢c DNA
sequences. Moreover, hermaphroditism in sturgeon
is very infrequently observed (Chapman et al. 1996;
Van Eenennaam & Doroshov 1998; Harshbarger et al.
2000) and the sex ratio in adult populations of sturgeon is 1<:1, (Chapman et al. 1996). Environmental
sex determination produces variations in sex ratios
when there are systematic £uctuations in the environmental factors in£uencing sex (Bull 1983). In fact,
the failure in the search for this class of DNA markers
could be due to the size of genome, the number of
markers screened and the proportion of the genome
that is sex speci¢c in the species studied (Keyvanshokooh et al. 2007; Penman & Piferrer 2008).

Gene expression approaches
Regarding the failure of DNA-based techniques in
identifying sex-speci¢c markers, gene expression

pro¢ling has recently been considered as an alternative approach (Wuertz et al. 2006). With recent advances in genomic and proteomic approaches, a
systematic study of global patterns of gene expression
is now possible.
While very potent, approaches to global analysis of
gene expression focused exclusively at the mRNA level nevertheless cannot give a complete picture of the
changes occurring in the cell during a signalling response. Also, there is some concern over the correlation between protein level and mRNA (Parrington &
Coward 2002). Post-translational modi¢cations such
as phosphorylation and glycosylation are often extremely important for the function of many proteins,
but most of these modi¢cations cannot yet be predicted from genomic or mRNA sequences (Salekdeh,
Siopongco, Wade, Ghareyazie & Bennett 2002). Proteomics will signi¢cantly support the post-genomic
analysis of gene expression.
To date, only a few reports have been published on
sturgeon proteomes focusing on reproductive biology
and ecotoxicology (Keyvanshokooh & Vaziri 2008;

Sex determination in sturgeon S Keyvanshokooh & A Gharaei

Keyvanshokooh, Kalbassi, Hosseinkhani & Vaziri.
2009; Keyvanshokooh, Vaziri, Gharaei, Mahboudi,
Esmaili-Sari & Shahriari-Moghadam 2009), among
which Keyvanshokooh, Kalbassi et al. (2009) analysed the di¡erential protein expression between mature male and female Persian sturgeon (Acipenser
persicus) gonads. When comparing protein patterns
of the testis and ovary, 48 unique spots were distinguished in the testis while only two spots were
matchless in ovary. All the proteins identi¢ed were
involved in metabolism and energy production, cell
structure, transcription and translation, cell defence,
signal transduction, transport and cell division. None
of the proteins identi¢ed were directly linked to a sexdetermining gene. Regarding the lack of proteins
linked to a sex-determining sequence, it was concluded that the homologous chromosomes, on which
the sex-determining factor was found, were not extensively di¡erentiated in sturgeon species. They also

did not exclude the probability of the presence of low
abundant sex-linked gene products, which might
have been beyond the sensitivity of the methods utilized. Because the sex-determining genes may function only in the early stages of sex di¡erentiation, it
is recommended to use integrated gene expression
methodologies including proteomics and microarray
in order to focus on proteins and transcripts involved
in sex determination and di¡erentiation at di¡erent
stages of gonadal maturation.

Conclusion and future
Sex seems to be genetically determined in sturgeon
species as suggested by a 1< :1, ratio. Also, it is
known that sturgeon species are gonochoristic and
evidence consistent with female heterogametic genetic sex determination has been presented for some
sturgeon species. Because the sex-determination systems between closely related species were reported to
be di¡erent in some ¢sh species, more investigations
should be carried out to compare sex-determining
mechanisms between sturgeon species. Regarding
the failure of DNA techniques to identifying sexspeci¢c markers, alternative approaches based on
gene expression are required in the future. Microarray strategy, together with proteomics that look at
the proteome of reproductive cells and tissues, are
likely to expand our knowledge on sturgeon sex determination and di¡erentiation over the coming
years.

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Sex determination in sturgeon S Keyvanshokooh & A Gharaei

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Aquaculture Research, 2010, 41, e8^e19

doi:10.1111/j.1365-2109.2010.02505.x

REVIEW ARTICLE
Live prey first feeding regimes for short-snouted
seahorse Hippocampus hippocampus (Linnaeus, 1758)
juveniles

Francisco Otero-Ferrer1, Luc|¤ a Molina1, Juan Socorro1, Rogelio Herrera2,
Hipo¤lito FernaŁndez-Palacios1 & Mar|¤ a Soledad Izquierdo1
1

Grupo de Investigacio¤n en Acuicultura, Universidad de las Palmas de Gran Canaria & Instituto Canario de Ciencias Marinas,

Canary Islands, Spain
2
Direccio¤n General de Ordenacio¤n del Territorio, Consejer|¤ a de Medio Ambiente y Ordenacio¤n Territorial, Edif, Usos Mu¤ltiples
II. Prof. Millares Carlo, Canary Islands, Spain
Correspondence: F Otero-Ferrer, Grupo de Investigacio¤n en Acuicultura, Universidad de las Palmas de Gran Canaria & Instituto Canario
de Ciencias Marinas, PO Box 56, 35200 Telde, Las Palmas, Canary Islands, Spain. E-mail:

Abstract
As with many species of seahorses, Hippocampus
hippocampus wild populations are being subjected
to uncontrolled exploitation in their natural environment. Thus, aquaculture could contribute to satisfy
the commercial demand for animals while promoting the recovery of wild stocks. The present
study was conducted to compare the e¡ect of the
substituting Artemia nauplii with rotifers for
¢rst feeding seahorse juveniles. Survival, growth
and biochemical composition of prey organisms
and ¢sh were studied during the feeding trial. In
addition, to help the biometric study, an anaesthetic
test was also carried out using clove oil. The results
showed excellent survival (average 60%) in juveniles
exclusively fed with Artemia, with better values
than those reported previously obtained by other
authors for this species. By comparison, high
mortality and poor growth were observed during

¢rst feeding with seahorses fed on rotifers. This
could have been related to the lower energy intake and poorer nutritional value of the rotifers.
Furthermore, clove oil concentrations of 25 ppm
were found to work well as an anaesthetic for seahorse juveniles. Overall, ¢rst feeding Artemia alone
was found to be an e⁄cient and simpli¢ed method
for feeding young H. hippocampus fry, building the
principles for their culture for ornamental or restocking purposes.

e8

Keywords: seahorse, ¢rst feeding, Artemia, rotifers, lipids

Introduction
The state of seahorse populations shows the uncontrolled overexploitation of marine resources and the
unsuitable management of wild areas (Vincent
1996). For this reason, all seahorse species have been
included in appendix II of the Convention on International Trade in Endangered Species of Wild Fauna
and Flora (CITES 2002) (GonzaŁlez, Piloto, Chevalier
& Rivero 2006). Reproduction in captivity is considered as one of the solutions to reduce the pressure
on wild stocks by ¢shing and ornamental business
operations. The species studied in this work, Hippocampus hippocampus, is present in the Canary Islands,
and catalogued as data de¢cient by the red book of
the IUCN (The World Conservation Union) and as
‘Vulnerable’ in the Catalogue of Threatened Species
of the Canary Islands (Government of the Canary Islands, D. 151/2001, 23 July). For this reason, references about the life history of this species are scarce.
In the wild, H. hippocampus populations appears to
live close to seagrass areas related with arti¢cial
holdfast, living in low densities and little home ranges
(Wilson & Vincent 1999; Curtis & Vincent 2005). In
the natural environment, the short-snouted seahorse

regime is composed basically on little crustaceans

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Aquaculture Research, 2010, 41, e8^e19

Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

as mysis, amphipods and copepods species (Kitsos,
Tzomos, Anagnostopoulou & Koukouras 2008). Like
other seahorses spp., it is the male that becomes pregnant. After a period between 2 and 4 weeks (Boisseau 1967), depending on water temperature, the
male releases his brood. The standard length of newborn seahorses averages 9 mm (Damerval, De¤tienne,
De¤tienne & Vincent 2003; Lourie, Foster, Cooper &
Vincent 2004). Early life steps of this ¢sh are described with a ¢rst pelagic behaviour of 2^3 weeks
(Vincent 2001; Damerval et al. 2003). After this, juveniles use the prehensile tail to settle. They reach sexual maturity around 16^18 weeks for males and 18
for females (Damerval et al. 2003).
Concerning breeding in captivity of H. hippocampus, although there are more than 100 successful references in‘Aquariology Forums’, most of them are not
applicable for large-scale production operations with
the aim of restocking or for commercial trade activities. Usually, live copepods or other zooplanktonic animals caught in the wild are used in seahorse culture
with high survival rates (Gardner 2004; Bentivegna
2006; Jones & Lin 2007). However, copepods are not
generally used for rearing marine ornamental ¢sh as
reliable large-scale production methods are usually
di⁄cult to attain (Payne & Rippingale 2000). By contrast, rotifers and Artemia are commonly used as live
prey food organisms in aquaculture, and have been
also used in seahorse breeding with di¡erent degrees
of success, depending upon animal size and species.
Thus, the reported survival rates obtained in captivity

for H. hippocampus (Linnaeus, 1758), have ranged between 30% and 45% (Damerval et al. 2003), animals
being ¢rst fed with rotifers until day 4 and then fed
with Artemia. Sometimes rotifers have been used in
seahorse breeding, combined with other live prey
such as Artemia, copepods or both. This is due to their
adequate size and better digestibility for the ¢rst 5
days of culture, which is believed to be the critical bottleneck in seahorse culture (Damerval et al. 2003;
Jones & Lin 2007). Other authors used exclusively
Artemia, copepods, or both, with other seahorse species born in aquaria with varied results (Reyes-Bustamante & Ortega-Salas 1999; Wilson & Vincent 1999;
Payne & Rippingale 2000; Woods 2000a, b; Choo &
Liew 2006; GonzaŁlez et al. 2006; Jones & Lin 2007;
Martinez-Cardenas & Purser 2007; Sheng, Lin, Chen,
Shen & Lu 2007;Truong Si Ky & Hoang Duc Lu 2007).
Sometimes, high survival rates in breeding experiments have been related with the use of relatively
simple larval rearing techniques (Wilson & Vincent
1999) such as the decapsulation of Artemia cysts be-

fore hatching so as to eliminate pathogenic bacteria
sources, the nutritional enrichment of live prey organisms before feeding, the use of proper acclimation
procedures using overlapping feeding protocols, or
the maintenance of scrupulous hygienic controls
within seahorse rearing tanks. Moreover, the success
of larval rearing is greatly in£uenced by ¢rst feeding
regimes and the nutritional quality of starter diets
(Izquierdo, Tandler, Salhi & Kolkovsky 2001). In this
sense, the dietary lipids are recognized as one of the
most important nutritional factors that a¡ect larval
growth and survival (Watanabe, Kitajima & Fujita
1983) and in particular highly unsaturated fatty
acids (HUFA). Numerous studies have shown that

the larvae of marine ¢sh have a dietary requirement
for arachidonic (ARA; 20:4n-6), eicosapentaenoic
(EPA; 20:5n-3) and docosahexaenoic acids (DHA;
22:6n-3) (Webster & Lovell 1990; Lemm & Lemarie
1991; Rimmer, Reed, Levitt & Lisle1994; Sargent, Bell,
McEvoy, Tocher & Estevez 1999) and these must be
supplied in the diet to ensure optimal growth and
survival (Chang & Southgate 2001).
In addition to the nutritional quality of ¢rst feeding
regimes, it is important to understand the morphological development and allometric growth patterns of
¢sh (Koumoundouros, Divanach & Kentouri1999; Gisbert, Merino, Muguet, Bush, Piedrahita & Conklin
2002) for the optimization of production in aquaculture. Biometric data can also provide an insight into
possible functional trends and environmental preferences of di¡erent developmental stages (Fukuhara
1988). In most biometric studies, marine organisms
need to be anaesthetized in order to avoid sacri¢cing
animals (Choo & Liew 2006). Anaesthetic methods
have generally been used to reduce ¢sh stress (Ross &
Ross 1999). Optimal doses must be used in order to
achieve a 100% recovery rate from anaesthetic protocols. Anaesthetic episodes in ¢sh can be divided into
di¡erent periods, beginning with equilibrium loss
(phase I), numb stage with operculum movement
(phase II) and without this movement (phase III) (Garc|¤ a-Go¤mez, de la GaŁndara & Raja 2002). Concerning
the use of anaesthetics with seahorses, some researchers have cited the use of MS-222 (Boisseau 1967; Bull
2002; Jones & Lin 2007) and AQUI-S (Woods 2000a,
b), which has been commonly used with di¡erent species. However, authors seldom detail their protocol.
Natural clove (Syzygium aromaticum; Merr. & Perry) essential oil was found to present a number of advantages over other commonly used anaesthetics: a high
e⁄cacy at low doses, cost-e⁄ciency, non-toxicity,
potential positive side e¡ects and no need for a with-

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Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

drawal time for selling the ¢sh after its use (Garc|¤ aGo¤mez et al. 2002). These characteristics are very important for routine tasks in handling cultured marine
¢sh (Munday & Wilson 1997; Waterstrat 1999; Woody,
Nelson & Ramstad 2002).
This study is part of a coordinated research project ( />devoted to evaluate the wild seahorse resources of
the Spanish Atlantic coasts, from NW Iberian Peninsula to the Canary Islands. The aim of this work was
to evaluate the e¡ect of dietary replacement of liveArtemia nauplii by rotifers during the ¢rst 5 days of
feeding for newborn short-snouted seahorses H. hippocampus (Linnaeus, 1758), by quantifying the e¡ects
on survival rate and growth during their ¢rst weeks
(34 days). In addition, the lipid composition and fatty
acid content of the prey organisms and ¢sh were analysed during the trial so as to see if there was a correlation between animal survival and growth with the
di¡erent food organisms.
Material and methods
One pregnant male seahorse was collected by scuba
diving among seaweed (Cymodocea nodosa) in Melenara bay (27159 0 N, 15122 0 W), Eastern coast of Gran
Canaria island (Canary Islands, Spain), in April 2007
(approximate photoperiod of 10 L:14 D). The male
standard length (Villares 2005) was 11.3 cm and the
wet weight was 3.48 g. The pregnant animal was then
transported in a 10 L acclimatization tank with seawater and oxygen to the laboratory research facilities
at the Instituto Canario de Ciencias Marinas (ICCM)
in Telde (Gran Canaria, Canary Islands, Spain).
The adult seahorse was initially held in an aerated
100 L quarantine square glass aquarium supplied

with £ow-through ambient seawater, puri¢ed previously using a pre¢ltration tank equipped with an
under-gravel ¢lter, 5 mm cartridge ¢lters and ¢nally
UV light (Philips, 25W/G25T8UV-C, Holland, the
Netherlands). The £ow rate was 1L min À 1. The salinity was 37%, the temperature was maintained by a
thermostat (JÌguer, 300 W, Wuestenrot, Germany) at
22^23 1C and the oxygen level ranged from 6.5 to
7 mg L À 1. The aquarium was illuminated10 h day À 1
(Woods 2000b) using broad-spectrum £uorescent
tubes (Sera blue daylight, 36 W, 120001 K, Heinsberg,
Germany). Furthermore, a continual cleaning to prevent hydroids and anemones from invading the tank
was performed every day. The ¢sh was fed twice a day
with live mysids (Leptomysis sp. and Paramysis sp.)
caught in the wild. In addition, two seaweed-like

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Aquaculture Research, 2010, 41, e8–e19

plastic attachment substrata were placed in the aquarium to help the ¢sh to acclimatize.
After 10 days, the male released 468 newborn seahorses. Immediately (day 0), 15 o¡spring were randomly selected and sacri¢ced for biometric studies
(length and dry/wet weight). The remaining seahorses were divided equally into six 35-L square glass
aquaria (one triplicate per treatment). The stocking
density within tanks was 2.3 seahorses L À 1. The
rearing tanks were supplied with £ow-through ambient seawater also ¢ltered with 5 mm cartridge ¢lters
and UV light (Sera, 25 W) at a £ow rate of 2 L min À 1.
This high £ow rate was established to assure that untouched living foods (rotifers and Artemia) were removed after each feeding periods. Aeration to each
tank was provided by an air pump located out of the
aquaria (125 mL min À 1). Measurements of physical
parameters like temperature ( 1C) and oxygen level
(mg L À 1) were carried out every day. Nitrogen compounds than can be lethal for ¢sh, including ammonia and nitrites (Otero-Ferrer 2001), were measured

twice a week (Red Sea Europe, Marine and Freshwater Test Lab, Verneuil Sur Avre, France). Light was
provided by one £uorescent tube (Sera blue sky, 36 W,
120001 K, Germany) for each triplicate and the
photoperiod was 10 L:14 D. During the ¢rst 2 weeks,
we applied lighting from the bottom and also darkened o¡ the top and sides of tanks. Seahorses eat
their food below the surface thanks to the live prey’s
positive phototactic behaviour (Wilson & Vincent
1999; Woods 2000b; Warland 2003; Choo & Liew
2006). This period concerned the seahorse planktonic phase (Damerval et al. 2003; Warland 2003;
Choo & Liew 2006; Sheng et al. 2007). The light intensity just above the water surface ranged between 60
and 100 lx (HT Digital Light Meter, HT170N, HT
Instruments, Barcelona, Spain). After this (days 15^
34), the ¢sh were progressively acclimated to normal
lighting applied from the top of tanks. The light intensity just above the water surface then ranged between 800 and 1000 lx. From the 10th day, two white
plastic mesh (12 cm  5 cm) were added in each
aquarium to provide holdfasts and habitats for the
animals (Villares 2005).
In this study, two experimental protocols were
tried with di¡erent food sequences for seahorse ¢rst
feeding. In the ¢rst treatment (RA), newborn seahorses were exclusively fed until day 5 after birth
(DAB) with 5 rotifers mL À 1 mm (Brachionus plicatilis),
L-strain (240 mm). Rotifers had been grown out in a
batch culture system fed with commercial yeast (Saccharomyces cerevisiae) and enriched with DHA pro-

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Aquaculture Research, 2010, 41, e8^e19

Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

tein-Selco (INVE Aquaculture, Dendermonde, Belgium) following the standard protocols of ICCM
(Roo, HernaŁndez-Cruz, Socorro, FernaŁndez-Palacios,
Montero & Izquierdo 2009a). After which, enriched
(Easy ^ DHA, INVE Aquaculture) Artemia Instar II
(EG type 850 mm, INVE Aquaculture) were progressively included for 3 days in the seahorse diet (0.25,
0.50 and 0.75 Artemia mL À 1), until their concentration reached 1Artemia mL À 1. Meanwhile, rotifers
concentrations were reduced to 0 during feeding episodes. In the second treatment (A), seahorses were fed
only with enriched (Easy ^ DHA, INVE Aquaculture)
Artemia (EG type, INVE Aquaculture) Instar II
(1mL À 1). The seahorses were fed twice per day (at
9:00 and 14:00 hours) after removing the uneaten
food and faeces from the bottom of the tanks. During
feeding, the rearing tanks remained without an in£ow of fresh seawater for 2 h. In this experiment, factors that traditionally had relevance in seahorse
breeding, like type (Damerval et al. 2003) and
position (Woods 2000b; Warland 2003) of lighting,
aeration intensity (Molina, Socorro, Herrera, OteroFerrer, Villares, FernaŁndez-Palacios & Izquierdo
2007) or special tank design (Woods 2000a; Warland
2003; Matsushige & Melechinsky 2004), were incorporated into the experimental design.
Each day, mortality (M) and accumulative survival
(AS) for each treatment’s replicate were determined
as follow:
AS ¼ ððNiÀM ij Þ=Ni Þ Â 100
Ni was the initial number of seahorses placed in
each aquarium at day 1.
Mij was the total seahorses died since day 1 until
the day j.
At 0,5,13, 22 and 34 DAB, three biometric measures
were also taken: snout length (SnL), head length (HL)

and trunk length (TrL) (Villares 2005), using a ‘pro¢le
projector’ (Mitutoyo PJ-A3000, Kawasaki, Japan). At
the same time, an anaesthesia test was carried out,
s
testing natural clove essential oil (Guinama , Valencia, Spain), containing 87% eugenol anaesthetic.
Thirty seahorses (¢ve animals from each tank) of 22
were chosen randomly for the trial. Concentrations
of 25 ppm of natural clove essential oil were prepared
in 100 mL beakers with seawater, from a 0.5% (v/v)
s
solution diluted in 96% ethanol (Panreac , Barcelona, Spain). During the test, the seawater temperature
was constant (20 1C). The ¢ve young seahorses from
each aquarium (15 from RA and15 from A treatment)
were introduced into their respective beaker. Behaviour and possible side e¡ects were observed and

noted. When the animals showed symptoms of
numbness with only operculum movement (phase
II) (Iwama & Ackerman 1994), they were then placed
into a Petri dish for the measurement with the pro¢le
projector. To test the e⁄ciency of the anaesthesia, biometric measurements completed during the ¢rst
5 min (Garc|¤ a-Go¤mez et al. 2002) after clove essential
oil administration were considered as ‘positive’, and if
they took 45 min to arrive to phase II anaesthetic,
they were denominated ‘negative’. After that, the seahorses were immersed in a ‘recovery’aerated tank in
order to recover normal activity and regular breathing, before their return to the rearing aquaria.
Weight (dry and wet) was also evaluated at the end
of the trial. For the dry weight, seahorses were placed
in an oven (Jouan, EU 280 EL TS SN Inox, Saint-Herblain, France) at 105 1C until the weight became constant (AOAC 1995). The measurements were taken
with a precision balance (Mettler Toledo, AG204,
Greifensee, Switzerland). The crude lipids and fatty

acid methyl esters content of seahorses (A and RA
treatments) were determined at DAB 5 and DAB 34.
In order to obtain prey nutritional quality, 3 g samples (wet weight) of rotifers (6 h enriched) and Artemia (24 h enriched) were collected throughout the
trial. A commercial trademark protocol was followed
to enrich live preys. The samples were taken at the
same time that the seahorses were fed. Concerning
seahorses, ¢ve random ¢sh of each treatment replicate were taken at days 5 and 34. Samples were collected with an adequate-sized nylon mesh, measured
with a precision balance and washed twice with distilled water and freeze-dried ( À 80 1C) for subsequent analysis. In order to obtain su⁄cient sample
for lipid analysis in seahorses, three replicates from
each treatment were combined before analysis
(Chang & Southgate 2001).
Crude lipid (% wet weight) of seahorses and live
prey (Artemia and rotifers) were extracted following
the method of Folch, Lees & Stanley (1957). Fatty acid
methyl esters from total lipids were prepared by
transmethylation as described by Christie (1982) and
separated and quanti¢ed by das liquid chromatography under the conditions described by Izquierdo,Watanabe,Takeuchi, Arakawa & Kitajima (1990).
Concerning statistical analysis, normality and
homogeneity of the variable were tested using Kolmogorov^Smirnov and Levene’s test respectively
(Zar 1996). Survival was analysed by a loglineal
model, as follow:
ln fij þ m þ ai þ bj þ abij

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Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

ln fij is the expected frequency in row i, column j of
the two-way contingency table.
m is the mean of the logarithms of the expected frequencies.
ai is the e¡ects of category i of factor A (survival).
bj is the e¡ects of category j of factor B (treatment).
abij is the interaction term that expresses the
dependence of category i of factor A on category j of
factor B.
Variations between RA and A treatment concerning growth (length and weight) and seahorse lipid
composition factors were tested using One-way
ANOVA (Zar 1996). All results were processed and analysed using SPSS statistical software system version
16.0.1. (SPSS, Chicago, IL, USA, 1999).

Results
General observations
Since the ¢rst day, visual inspection of seahorses during feeding periods showed hunting behaviour and a
full orange (A treatment) (Fig. 1a) or white (RA treat(a)

Aquaculture Research, 2010, 41, e8–e19

ment) gut. Until DAB 10, the ¢sh of both treatments
showed pelagic activity. The ¢rst seahorse showing
‘benthic’ behaviour was seen on DAB 15 for A treatment and on DAB 22 for RA treatment. At the end of
the trial, all seahorses from both treatments had developed their prehensile tails and were ¢xed in the
plastic nets. During the experiment, some animals
presented bubble problems in the gut (Fig. 1b) or reduction in swimming activities in both treatments,
with some remaining £oating on the water surface
and ¢nally died. Others appeared to be able to expulse the gas bubble and survive. Finally, no sexual
dimorphism was found.

Survival
The average temperature during the experiment was
21.9 Æ 0.6 1C for all the aquariums. Oxygen levels
were 6.8 Æ 0.2 and 6.7 Æ 0.2 ppm for RA and A treatment respectively. Concerning chemical parameters,
ammonia and nitrites levels were always under 0.2
and 0.02 ppm during the trials.
Cumulative survival average at the end of experiment was signi¢cantly lower in RA treatment
(b)

5 mm

5 mm

Figure 1 Day after birth ¢ve o¡spring’s of Hippocampus hippocampus of treatment A with an orange gut after a feeding
episode (a) and having a bubble gas problem, in RA treatment (b).

Figure 2 Daily average mortality observed in each treatment, RA (D) and A (O), during the experiment.

e12

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Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

Figure 3 Measurements of seahorse juveniles’snout (a), head (b) and trunk (c) lengths. ÃSigni¢cant di¡erences between
RA (D) and A (O) treatments.

(25.93% Æ 0.19) (ZRA,vivos 5 À 6662; Po0.0001) compared with A treatment (59.90% Æ 10.76) (ZA,vivos 5
16662, Po0.0001) (Fig. 2). Beside, RA showed high
mortality peaks between 5 and 9, whereas in A, low
mortality was noted gradually (Fig. 2).
Growth
Biometric studies ¢rst showed signi¢cantly lower values with RA treatment for all parameters (snout,
head and trunk lengths) in seahorses, even at the ¢rst
sampling. Nevertheless, at the end of the study, no
signi¢cant (Po0.05) di¡erence was found between
treatments (Fig. 3).
Both wet and dry weights obtained were signi¢cantly lower (Po0.05) with RA treatment
(48.55 Æ 25.39 and 6.3 Æ 2.72 mg respectively) than
with A treatment (62.64 Æ 31.70 and 12.1 Æ 5.10 mg
respectively).
Lipid composition
The average crude lipid content (mean Æ SD) on a
dry weight basis of the live prey food organisms was
32.36 Æ 1.76% for Artemia and 10.24 Æ 0.07% for rotifers. Concerning seahorse juveniles, the average
crude lipid percentage (mean Æ SD) of wet weight
for treatments RA and A was 2.78 Æ 0.14 and
3.27 Æ 0.23% respectively at 5; and 2.88 Æ 0.14% for
RA and 2.88 Æ 0.08% for A at the end of trial. No signi¢cant di¡erence was found in the lipid composition
between treatments (Po0.05).
Concerning the fatty acid composition of Artemia
and rotifers (Table 2) fed to the seahorses, our results
show that the crustacean was characterized a high
palmitic (16:0), oleic (18:1n-9) and linoleic (18:2n-6)
acids, denoting its geographical origin. Besides, they

also showed a higher content than the rotifers in
ARA (20:4n-6), EPA (20:5n-3) and DHA (22:6n-3),
which are considered as essential fatty acids (EFA) for
marine ¢sh (Tandler,Watanabe, Satoh & Fukusho1989;
Izquierdo 1996; Rodr|¤ guez, Pe¤rez, Izquierdo, Cejas, Bolanìos & Lorenzo 1996; Chang & Southgate 2001).
Rotifers were mainly characterized by 16:0, 16:1n-7
and 18:1n-9 and their content in ARA, EPA and DHA
were very low. They were also characterized by a
higher amount of 18:2n-9 than Artemia.
After only 5 days of feeding on Artemia, seahorses
showed a high content of 18:2n-6 and particularly
18:3n-3 (Table 2). On the contrary, the oleic acid content was lower in rotifer-fed seahorses (RA treatment) but ARA, EPA and DHA were higher with RA
treatment than in Artemia-fed seahorses. Moreover,
these ¢sh had a low content of 16:1n-7 and 18:2n-6
despite the fact that both fatty acids were high in the
rotifers. After 34 days of feeding, saturated
fatty acids, particularly 16:0, 17:0 and 18:0 were
reduced in seahorses in comparison with the younger stages, whereas monoenoic ones were increased
Tables 1 and 2.
Anaesthetic trial
All seahorses reached Phase II during the ¢rst 5 min
after anaesthetic administration. They were correctly measured without signi¢cant di¡erences between treatments (Po0.05). Finally, the animals
recovered normal activity within 30 min after introduction in the ‘recovery’ tank.
Discussion
Since the moment the seahorses are expelled from
the paternal pouch, these animals have been de-

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Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

Table 1 Live preys’ main fatty acid composition (g fatty
acids100 g À 1 total fatty acids) during the trial

14
14:1n-7
14:1n-5
15:0
15:1n-5
16:0ISO
16
16:1n-9
16:1n-7
18
18:1n-9
18:1n-7
18:1n-5
18:2n-9
18:2n-6
18:3n-6
18:3n-3
18:3n-1
18:4n-3
20
20:1n-9
20:1n-7

20:2n-9
20:2n-6
20:3n-9
20:3n-6
ARA (20:4n-6)
20:4n-3
EPA (20:5n-3)
22:4n-6
22:5n-3
DHA (22:6n-3)
P
SaturatedÃ
P
Monoenesw
P
n-3z
P
n-6‰
P
n-9z
P
n-3HUFAk
DHA/EPA
OA/n-3HUFA
EPA/ARA

Table 2 Main fatty acid composition (g fatty acids100 g À 1
total fatty acids) of seahorses during each treatment

Artemia

Rotifers

Treatment

A

RA

A

RA

2.0
0.5
0.2
0.5
0.3
0.1
16.0
0.6
4.2
5.8
25.0
4.9
0.1
0.3
5.6
0.4
9.3

0.1
1.3
0.2
2.4
0.2
0.0
0.3
0.0
0.1
1.3
0.5
5.5
0.4
0.7
6.6
25.5
38.3
24.0
8.8
28.3
13.4
1.2
1.9
4.2

3.0
2.1
0.3
1.6
0.2

0.2
18.0
1.3
15.3
5.3
20.4
4.3
0.7
3.7
5.9
0.2
0.6
0.1
0.1
0.2
2.6
0.6
0.8
0.2
0.6
0.2
0.6
0.3
1.8
0.2
0.7
2.2
29.4
48.0
6.8

8.2
29.5
5.0
1.3
4.1
2.7

Day

5

5

34

34

14:0
16:0
16:1n-9
16:1n-7
Me16:0
17
16:3n-4
16:3n-3
18:0
18:1n-9
18:1n-7
18:1n-5
18:2n-9

18:2n-6
18:3n-6
18:3n-3
18:4n-3
20
20:1n-9
20:1n-7
20:2n-6
20:3n-6
ARA (20:4n-6)
20:4n-3
20:5n-3
22:4n-6
EPA (22:5n-3)
DHA (22:6n-3)
P
SaturatedÃ
P
Monoenesw
P
n-3z
P
n-6‰
P
n-9z
P
n-3HUFAk
DHA/EPA
OA/n-3HUFA
EPA/ARA

1.8
15.8

1.63
17.6
0.62
1.87
0.91
0.75
0.16
2.1
12.2
14
3.05
0.1
0.15
3.04
0.48
1.57
0.49
0.42
0.53
0.22
0.3
0.16
5.01
0.17
5.45
0.86

1.87
20
33.8
21.7
31.9
10.1
15.3
27.5
3.66
0.51
1.09

1.47
13.9
1.52
1.8
0.27
0.72
0.05
1.35
11.3
16.9
3.6
0.19
0.37
13.8
0.42
8.34
1.67
0.63

0.8
0.26
0.26
0.15
2.12
0.44
2.45
0.38
1.07
8.96
29.2
26.8
24.7
17.5
19.6
12.9
3.65
1.31
1.15

1.53
13.4
1.27
3.05
0.14
0.72

ÃIncludes 12:0, 14:0, 15:0, 16:0, 17:0, 18:0 and 20:0.
wIncludes 14:1n-5, 14:1n-7, 15:1n-5, 16:1n-9, 16:1n-7, 18:1n-9, 18:1n7, 18:1n-5, 20:1n-9 and 20:1n-7.
zIncludes 16:3n-3, 16:4n-3, 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3,

22:5n-3 and 22:6n-3.
‰Includes 16:2n-6, 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6 and
22:4n-6.
zIncludes 16:1n-9, 18:1n-9, 18:2n-9, 20:1n-9, 20:2n-9 and 20:3n-9.
kIncludes 20:4n-3, 20:5n-3, 22:5n-3 and 22:6n-3.
ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; HUFA, highly unsaturated fatty acid.

scribed as active and voracious (Herald & Rakowicz
1951; Wilson & Vincent 1999; Woods 2000a) visual
predators (Blaxter1980; Martinez-Cardenas & Purser

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Aquaculture Research, 2010, 41, e8–e19

1.9
1.1
0.6
1.3
12.1
17.4
3.8

14.7
8.9
1.7
0.6
0.5

2.2

0.4
2.6
1
8.7
32.7
24
25
16.9
17.9
12.7
3.35
1.37
1.18

1.15
8.04
19.5
7.06
0.15
0.51
6.68
0.51
11.9
2.36
0.26
0.92
0.16
0.32
0.2
1.88

0.53
4.92
0.32
0.77
6.53
25.1
34
28.2
10.5
22.3
12.7
1.33
1.53
2.61

ÃIncludes 12:0, 14:0, 15:0, 16:0, 17:0, 18:0 and 20:0.
wIncludes 14:1n-5, 14:1n-7, 15:1n-5, 16:1n-9, 16:1n-7, 16:1n-5, 18:1n9, 18:1n-7, 18:1n-5, 20:1n-9, 20:1n-7 and 22:1n-11.
zIncludes 16:3n-3, 16:4n-3, 18:3n-3, 18:4n-3, 20:3n-3, 20:4n-3,
20:5n-3, 22:5n-3 and 22:6n-3.
‰Includes 16:2n-6, 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6 and
22:4n-6.
zIncludes 16:1n-9, 18:1n-9, 18:2n-9, 20:1n-9 and 20:2n-9.
kIncludes 20:4n-3, 20:5n-3, 22:5n-3 and 22:6n-3.
ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; HUFA, highly unsaturated fatty acid.

2007; Villares, Socorro, Herrera, Otero, Izquierdo &
Molina 2007), able to catch di¡erent live preys. This
fact explained the gut colour of juveniles after feeding episodes in both treatments. To date, the e⁄ciency

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Aquaculture Research, 2010, 41, e8^e19

Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

of digestion, absorption and assimilation of proteins,
lipids and carbohydrates from the moment of their
birth are still unclear (Damerval et al. 2003; Warland
2003; Martinez-Cardenas & Purser 2007; Sheng et al.
2007; Villares et al. 2007). Thus, it is possible that
live preys are eaten but their nutrients not completely
assimilated by animals in the ¢rst days (Warland
2003).
The planktonic behaviour and ‘settlement’ agree
well with that described by other authors (Damerval
et al. 2003) for H. hippocampus and other seahorse
species (Warland 2003; Choo & Liew 2006). A nutritional de¢ciency in seahorses fed rotifers compared
with those fed Artemia could be explaining the delay
in the settlement process. Thus, lipid analysis showed
low contents in ARA, EPA and DHA in the rotifers denoting a poor enrichment of these live preys (Rodr|¤ guez et al. 1996). Beside, high values of palmitic
(16:0), palmitoleic (16:1n-7) and oleic (18:1n-9) acids
are principal components of the total lipids commonly found in poorly enriched rotifers (Rodr|¤ guez
et al. 1996; Roo, HernaŁndez-Cruz, Socorro, FernaŁndez-Palacios, Montero & Izquierdo 2009b). The possible HUFA de¢ciency in prey could also explain some
alteration in the behaviour of young seahorses, such
as the reduction in swimming and in feeding activities (Izquierdo 1996; Izquierdo, Socorro, Aranztamendi & HernaŁndez-Cruz 2000). Bubble gas
problems could also be related to a de¢cient swim
bladder in£ation described also by Koven (1991) in
gilthead sea bream fed n-3 HUFA-de¢cient rotifers,

which larvae £oating on the water surface. Alternatively, lipid levels in preys are directly related to enrichment conditions (Izquierdo 1988; Rainuzzo,
Reitan, Jorgensen, & Olsen 1994; Rodr|¤ guez et al.
1996). In this way, Roo et al. (2009b) showed better
levels of HUFA in rotifers enriched in the same circumstances. Therefore, it is hypothesized that there
was a problem with an inadequate enrichment time,
which can degrade HUFA in a lipid emulsion (Rodr|¤ guez et al.1996). Regarding Artemia fatty acid composition, our values were close to those found by Roo
et al. (2009b), with the same type of Artemia sp. in similar enrichment conditions. More adequate lipid
composition in preys could explain the fact that ¢sh
fed Artemia presented more than double the wet
weight than rotifer-fed ones at day 5. The high levels
of linolenic (18:3n-3) acid in Artemia are related to a
cyst origin (Webster & Lovell 1991), and markedly differed from those in rotifers (Roo et al. 2009b),
whereas the high EPA (20:5n-3) denoted the success
in the enrichment process.

During the ¢rst weeks, seahorses juveniles fed Artemia presented better biometric values (length and
dry weight) and survival rates than those fed rotifers.
In fact, the mortality peaks found in the last ones, between 5 and 9 are similar to what was expected for a
fry submitted to a starvation episode until 4 (Villares
2005; Molina et al. 2007; Sheng, et al. 2007). However,
we did observe seahorses ingesting rotifers during
the course of the experiment. This fact was also con¢rmed by the higher survival rate we obtained compared with the total mortality found in starvation
experiments performed in similar temperature conditions (Villares 2005; Molina et al. 2007). Nevertheless, the wet weight at 5 did not increase compared
with DAB 1 for rotifer-fed seahorses. This suggests
that energy ingestion in seahorses fed rotifers could
be suboptimal and unbalanced with the e¡ort of
catching preys. In contrast, the high content in linoleic (18:2n-6), and particularly linolenic (18:3n-3)
fatty acids in seahorses after 5 days of Artemia feeding denoted a very active predation on this crustacean, which presents high contents of these fatty
acids, whereas seahorses are not rich in 18:3n-3 at
early life stages (L. Molina, pers. comm.). These ¢sh

also showed ARA, EPA and DHA contents that were
very similar to those of this species at birth (L. Molina, pers. comm.) and to those of 34-day-old ¢sh. Surprisingly, feeding seahorses with rotifers markedly
increased the content of these EFA; this could be related to a poor feeding activity of these ¢sh, because
ARA, EPA and DHA have been found to be retained
during periods of starvation in other ¢sh species (Koven, Kissil & Tandler 1989; Tandler et al. 1989; Ako,
Kraul & Tamaru 1991; Van der Meeren, Klungsoyr,
Wilhelmsen & Kvensenth 1993; Rainuzzo et al. 1994;
Rodr|¤ guez 1994; Izquierdo 1996; Izquierdo et al.
2000). Moreover, the reduction in 18:1n-9 can be also
related to a depletion of this fatty acid to obtain energy during a starving period (Van der Meeren et al.
1993; Izquierdo et al. 2001), because this fatty acid is
one of the main substrates for b oxidation (Izquierdo
1996; Tandler et al. 1989). This fact, together with the
low content in 16:1n-7 and 18:2n-6, despite the fact
that these fatty acids were high in rotifers, support
the low feeding activity of rotifer-fed seahorses. Besides, these live preys were EFA de¢cient, which also
could explain these results (Izquierdo, Watanabe,
Takeuchi, Arakawa & Kitajima 1989; Rodr|¤ guez,
Pe¤rez, Izquierdo, Mora, Lorenzo & FernaŁndezPalacios 1993, 1994; Izquierdo 1996).
In addition, it is important to note that the mortality rate of rotifers-fed seahorses was reduced and

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Live prey ¢rst feeding regimes for H. hippocampus juveniles F Otero-Ferrer et al.

seahorse growth improved when Artemia was progressively included in the diet. At the end of the trial,

both treatments presented similar measurements, as
seen by Damerval et al. (2003), with the same species.
Probably, the increase in HUFA dietary levels supplied
with adequately enriched Artemia in RA tanks after
the high mortality peaks between 5 and 8 DAB could
explain these results (Izquierdo 1996). In the same
way, other hypothesis as compensatory growth after
starvation episodes or size selective mortality could
be considered. Similarly, the lower growth of A-treated animals could mean that the Artemia concentration was not high enough for our ¢sh. This fact
underlines the major role played by food density in
short-snouted seahorse (H. hippocampus) breeding.
Finally, the good percentage survival rate obtained
(60%) in juveniles exclusively fed Artemia (Treatment
A) was high in comparison with other reports for the
same species. Damerval et al. (2003) obtained a
30^45% survival rate with H. hippocampus, using a
feeding protocol, which combined rotifers (1^3) and
Artemia (4^32). Similar results were found with
others Hippocampus sp. of similar size (ReyesBustamante & Ortega-Salas 1999; Jones & Lin 2007).
Wilson and Vincent (1999) achieved a 100% survival
rate until day 120 with three Indo-Paci¢c seahorse
species (Hippocampus fuscus, Hippocampus barbourii
and Hippocampus kuda), but phylogenetic aspects
(Casey, Hall, Stanley & Vincent 2004) or the use of
copepods (Payne & Rippingale 2000) as ¢rst feeding
food before Artemia, could explain these results
(Gardner 2004).
Concerning the anaesthetic trial with clove oil, the
results of this study have shown for the ¢rst time, the
e¡ectiveness of clove oil at a concentration of 25 ppm

without negative side e¡ects for animals at this temperature and after handling. Clove oil has been also
found useful in other species of marine ¢sh (Soto &
Burhanuddin 1995; Garc|¤ a-Go¤mez et al. 2002) and invertebrates (Seol, Lee, Im & Park 2007). However, it is
usually used at higher concentrations (40 ppm for
amberjack, common dentex, gilthead seabream,
European seabass and sharp-snout seabream;
Garc|¤ a-Go¤mez et al. 2002 at 15^17 1C), denoting the
special sensitivity of seahorses to this anaesthetic.
Similarly, Soto and Burhanuddin (1995) used clove
oil concentrations between 33 and 100 ppm for golden-lined spinefoot (Siganus lineatus), at temperatures
from 27 to 29 1C. These di¡erences noticed could also
be due to animal size, development stage and tank
temperature, as all these factors in£uence anaesthetic e⁄ciency (Ross & Ross 1999).

e16

Aquaculture Research, 2010, 41, e8–e19

Conclusions
The good results obtained, especially in terms of survival, showed the adequate nutritive value of enriched Artemia metanauplii administrated in these
conditions. This e⁄cient ¢rst feeding protocol with
Artemia simpli¢es seahorse production methods and
builds the principles for their culture to ornamental
purposes. Nevertheless, other experiments must be
performed to test di¡erent Artemia concentrations or
other parameters related with rearing environment
(light condition, tank wall colour, green water techniques . . .) during the ¢rst days of seahorse life. Rotifer enrichment procedures should also be revised in
order to clarify the bad results obtained with these
preys. These trials could improve the survival rates
achieved until now with this species. In the same

way, further experiments are being performed to con¢rm the importance of food quality on survival and
growth in seahorse breeding.

Acknowledgments
We acknowledge the Consejer|¤ a de Medioambiente
del Gobierno de Canarias and the Ministerio de Educacio¤n y Ciencia for support to this projet (CGL-200505927-C03-02).

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Aquaculture Research, 2010, 41, e20^e30

doi:10.1111/j.1365-2109.2009.02451.x

Feed intake and growth performance of Senegalese
sole (Solea senegalensis Kaup, 1858) fed diets with
partial replacement of fish meal with plant proteins
Joana M G Silva1,2, Marit Espe2, Lu|¤ s E C Conceic°aìo3, Jorge Dias3, Benjamin Costas3 &
Lu|¤ sa M P Valente1
1

CIMAR/CIIMAR, Centro Interdisciplinar de Investigac°aìo Marinha e Ambiental and ICBAS, Instituto de CieŒncias Biome¤dicas

de Abel Salazar, Universidade do Porto, Porto, Portugal
2

NIFES, National Institute of Nutrition and Seafood Research, Bergen, Norway

3

CIMAR/CCMAR, Centro de CieŒncias do Mar do Algarve, Universidade do Algarve ^ Campus de Gambelas, Faro, Portugal

Correspondence: L Valente, CIMAR/CIIMAR, Centro Interdisciplinar de Investigac°aìo Marinha e Ambiental and ICBAS, Instituto de
CieŒ ncias Biome¤dicas de Abel Salazar, Universidade do Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal. E-mail:

Abstract
To be able to study nutrient requirement and utilization in any species, a diet supporting normal feed intake and growth equally well as a traditional ¢sh
meal-based diet is needed. Additionally the formulation of the diet should allow low levels of the nutrient
under study.When studying the amino acid metabolism and requirements, one cannot rely on the ¢sh
meal-based diets as ¢sh meal are nicely balanced according to requirements. Therefore the current study
aimed to develop a plant protein-based diet (with low
¢sh meal inclusion) to be used in the nutritional studies of Senegalese sole juveniles supporting feed intake and growth close to that obtained in a ¢sh
meal-based control feed. Two experiments were conducted to evaluate whether Senegalese sole juveniles
would accept and utilize diets containing high plant
protein inclusion. For testing the acceptance of high
plant protein inclusion, two diets were formulated: a
reference diet that contained ¢sh meal as the main
protein source (450 g kg À 1 dry matter) whereas in
the test diet, ¢sh meal was substituted by a mixture
of plant ingredients (soybean meal, corn and wheat
gluten) with L -lysine supplementation. In order to
improve the palatability, 50 g kg À 1 squid meal was
added to both diets. The indispensable amino acids
(IAA) pro¢le of the test diet was made similar to the
control diet by adding crystalline amino acids.

Further, automatic feeders were used to improve the
feed intake. Fish (24 g initial body weight) were fed
the diets for a period of 4 weeks. As ¢sh accepted both

e20

diets equally well, a second study was undertaken to
test the growth performance. Fish (6 g initial BW)
were fed the diets for a period of 12 weeks. The use of
automatic feeders to deliver the feed and the addition
of both squid and balancing the indispensable amino
acids resulted in growth performance and accretion
not di¡ering from the ¢sh meal fed control. It can be
concluded that juvenile Senegalese sole are able to
grow and utilize high plant-protein diets when both
diet composition and feeding regime are adequate
for this species.

Keywords: Solea senegalensis, plant protein, test
diet development, feed acceptance, growth performance, amino acids

Introduction
Senegalese sole (Solea senegalensis, Kaup) is a strong
candidate species for intensive aquaculture in Southern Europe. Over the last years, signi¢cant advances
have been achieved in optimizing weaning techniques and larvae feeding (Howell 1997; Dinis, Ribeiro,
Soares & Sarasquete 1999; Dinis, Ribeiro, Conceic°aìo
& Aragaìo 2000; Conceic°aìo, Ribeiro, Engrola, Aragaìo,
Morais, Lacuisse, Soares & Dinis 2007). However, information is lacking on most of the nutritional needs
of this £at¢sh species during the juvenile stage (Rema,
Conceic°aìo, Evers, Castro-Cunha, Dinis & Dias 2008;

Rubio, Navarro, Madrid & SaŁnchez-VaŁsquez 2009).
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Journal Compilation r 2010 Blackwell Publishing Ltd


Aquaculture Research, 2010, 41, e20^e30

Most likely, the theoretical principles of practical feed
development in salmonids can be applied for marine
species (Kaushik & Dias 2001). Fine tuning of such
general principles, however, requires a recognition of
the nutritional requirements and the metabolic potential of each species. Protein requirement for maximum protein accretion in Senegalese sole has been
estimated at 60% dry matter diet (Rema et al. 2008).
The protein component of aquaculture diets is the
single most important and expensive dietary nutrient. Because of its high nutritional value and palatability, ¢sh meal still remains the major dietary
protein source comprising 20^60% of ¢sh feeds (Tacon 1995; Watanabe 2002). E¡orts all over the world
are being directed towards ¢nding high-quality alternative protein sources that, ideally, are less expensive
and readily available as substitutes for the more expensive ¢sh meal (FAO 2006). Among the many protein sources available for animal feeds, plant proteins
appear to be the strongest alternatives to ¢sh meal in
¢sh diets. Many plant feedstu¡s have already been
tested in the diets of freshwater and marine ¢sh. Partial replacement of ¢sh meal by plant protein has
been accomplished in many carnivorous cultured
¢sh such as yellowtail (Seriola quinqueradiata), Japanese £ounder (Paralichthys olivaceus), turbot (Psetta
maxima), European sea bass (Dicentrarchus labrax),
tin foil barb (Barbodes altus), Atlantic salmon (Salmo
salar) and Atlantic cod (Gadus morhua) (Masumoto,
Ruchimat, Ito, Hosokawa & Shimeno 1996; Kikuchi
1999; Regost, Arzel & Kaushik 1999; Robaina, Corraze, Aguirre, Blanc, Melcion & Kaushik 1999; Elangovan & Shim 2000; Kikuchi 2001; Espe, Lemme,
Petri & El-Mowa¢ 2007; Hansen, Rosenlund, Karlsen,
Koppe & Hemre 2007; Espe, Hevroy, Liaset, Lemme &

El-Mowa¢ 2008). However, total replacement has
been met with success only in a few cases with rainbow trout (Oncorhyncus mykiss), European sea bass
and Atlantic salmon (Kaushik, Cravedi, Lalles, Sumpter, Fauconneau & Laroche 1995; Adelizi, Rosati,Warner, Wu, Muench, White & Brown 1998; Kaushik,
Coves, Dutto & Blanc 2004; Espe, Lemme, Petri & ElMowa¢ 2006). Furthermore, the successful replacement of ¢sh meal by a soy protein concentrate in
Senegalese sole postlarvae (Aragaìo, Conceic°aìo, Dias,
Marques, Gomes & Dinis 2003) creates good prospects for ¢sh meal replacement in this species.
The amino acid (AA) pro¢le of raw materials plays
an important role in feed formulation. As plant protein sources are not well balanced in indispensable
amino acids (IAA) according to the requirements of
¢sh, supplementation with crystalline AAs is usually

Development of a plant-protein rich diet for sole J M G Silva et al.

necessary. Knowledge of the dietary IAA requirements of a ¢sh is of great importance in evaluating
the nutritive value of a protein source, and also for
formulating a balanced, cost-e¡ective ¢sh feed allowing optimal protein utilization. Dose^response approaches are widely used in AA requirement
studies. In this regard, two types of diets may be formulated, semi-puri¢ed or puri¢ed test diets (Rodehutscord, Borchert, Gregus, Pack & Pfe¡er 2000), or
practical diets in which animal and/or plant ingredients serve as dietary protein sources (Gomes &
Kaushik 1989; Moyano, Cardenete & De la Higuera
1992;Watanabe, Aoki,Viyakarn, Maita,Yamagata, Satoh & Takeuchi 1995; Regost et al. 1999; Espe et al.
2006, 2007, 2008). Semi-puri¢ed/puri¢ed diets were
used in a large range of requirement studies as they
can ensure a low variability in terms of nutrients as
well as an absence of potential anti-nutritional factors that are usually abundant in plant protein
sources (Rodehutscord, Mandel, Pack, Jacobs & Pfeffer 1995; Rodehutscord, Becker, Pack & Pfe¡er 1997).
Puri¢ed diets, however, present a considerably high
proportion or all of the dietary AA supply by crystalline AA rather than by protein-bound AA. The inability of crystalline AA-rich diets to sustain ¢sh growth
has been known for a long time in salmonids (Espe &
Njaa 1991; Cowey 1995), carps, cat¢sh (Ictalurus puctatus) (Andrews, Page & Murray 1977) and tilapia (Oreochromis niloticus) (El-Sayed 1989). The growth
depression was particularly pronounced in A. salmon and rainbow trout fry fed diets containing all

AA in the crystalline form (Espe & Njaa 1991; Espe,
Haaland & Njaa 1992; Dabrowski & Guderley 2002;
Dabrowski, Lee & Rinchard 2003), whereas this
growth depression was not observed when about half
of the dietary AA supplied by protein-bound AA
(Rollin 1999; Rollin, Mambrini, Abboudi, Larondelle
& Kaushik 2003). Therefore, the aim of the present
study was to develop a plant protein-based diet (with
low ¢sh meal inclusion) to be used in nutritional studies of Senegalese sole juveniles. The diet should support feed intake and growth equally well as do the
traditional ¢sh meal diets containing 450 g kg À 1 ¢sh
meal, thereby contributing to reduce the aquaculture
industry’s dependence on ¢sh meal.

Materials and methods
The feed acceptance trial
Two experimental diets were formulated (crude protein, 550^580 g kg À 1 DM and gross energy, 21^

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Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, e20^e30

e21


Development of a plant-protein rich diet for sole J M G Silva et al.

23 MJ kg À 1 DM). A reference diet containing
450 g kg À 1 ¢sh meal as the main protein source
(control group) and a test diet where ¢sh meal was
substituted by a mixture of soybean meal (SBM), corn
and wheat gluten. The test diet was supplemented

with L -lysine [26 g kg À 1 dry matter (DM) basis] to attain similar lysine levels as the reference diet.Wheat
gluten and corn gluten were chosen as the main protein sources, due to their high protein content and
potential high digestibility in ¢sh, while SBM is
known to have either, high crude protein content
(440 g kg À 1 DM) and a reasonably balanced AA
pro¢le (Gatlin III, Barrows, Brown, Dabrowski, Gaylord, Hardy, Herman, Hu, Krogdahl, Nelson, Overturf,
Rust, Sealey, Skonberg, Souza, Stone, Wilson & Wurtele 2007). In the absence of speci¢c data on vitamin,
mineral and trace element requirements of Senegalese sole, requirement data for other species were applied (NRC 1993; Kaushik 1998). With the aim of
increasing the feed acceptance,50 g kg À 1 squid meal
was added to both the control and the test diets. Additionally, IAA pro¢le of the plant-protein diet was
made similar to the control diet through the addition
of 47 g kg À 1 crystalline AA mixture. Moreover,
mono-Ca phosphate was added to the test diet to ensure similar phosphorus availability, as plant proteins utilization had been known to be limited by
dietary minerals (such as phosphorus) due to high levels of phytic acid in plant ingredients (Gatlin et al.
2007). All dietary ingredients were supplied by Sorgal S.A. (Ovar, Portugal) and were ¢nely ground,
mixed and dry pelleted through a 3.2 mm die at 50
1C (CPM, C-300 model, San Francisco, CA, USA). The
diets were dried at 37 1C for 24 h and stored in a refrigerator ( À 4 Æ 1 1C) until use. Formulation and
the proximate composition of the experimental diets
are presented in Table 1 and the corresponding IAA
pro¢le in Table 2.
The study was performed at the experimental facilities of Ramalhete Station of CCMAR (Algarve, Portugal). After arrival at the experimental unit, ¢sh were
acclimated to the new facilities for 2 weeks. Four
homogenous groups of 10 juveniles with an average
initial body weight of 24 g were randomly distributed
among ¢bre glass tanks (55 L), in a recirculation
water system. Individual weight of the animals was
recorded. Each tank was supplied with ¢ltered,
heated (20 Æ 1 1C) saltwater (30 g L À 1), at a £ow rate
of 2 L min À 1. The most important physical and chemical parameters (temperature, dissolved O2, salinity,

pH and nitrogenous compounds) were monitored
during the entire trial and maintained at levels with-

e22

Aquaculture Research, 2010, 41, e20^e30

Table 1 Dietary composition and chemical analysis
(g kg À 1 or MJ kg À 1 DM) of the diets fed to Senegalese sole
in both the feed acceptance and the growth trial

Fish meal Herring
CPSP G
Squid meal
Soybean meal 48
Corn gluten
Wheat meal
Wheat gluten
Gelatin
Fish oil
L-lysine HCl
Aminoacid mix
Choline chloride
Yttrium oxide
Mono Ca phosphate
Mineral mixz
Vitamin mix‰
Vitamin E50
Vitamin C35
Chemical composition

Dry matter (% DM)
Crude protein (% DM)
Crude fat (% DM)
Ash (% DM)
Gross energy (MJ kg À 1 DM)
NFE

Feed
acceptance
trial

Growth trial

Control Test

Control Test

450
25
50
152
90
163
–
–
67
–
–
1
1

–
1
1
0.5
0.3

50
25
50
20
120
211
332
10
76
26
47Ã
1
1
35
1
1
0.5
0.3

450
25
50
153
100

137
–
–
75
–
–
1
1
–
5
2
0.5
0.3

50
25
50
66
100
203
250
20
103
26
62w
1
1
36
5
2

0.5
0.3

918
545
132
105
21.9
218

935
584
105
63
23.2
247

898
555
132
108
22.0
205

953
566
131
61
23.1
242

ÃAmino acids in % of mixtures: Arg 1.20, His 0.30, Ile 0.50, Leu

0.40, Thr 0.50, Trp 0.10, Val 0.90, Met 0.30, Tyr 0.50.
wAmino acids in % of mixtures: Arg 1.25, His 0.45, Ile 0.65, Leu
0.90, Thr 0.75, Trp 0.15, Val 1.0, Met 0.50, Tyr 0.50.
zMinerals (g or mg kg À 1 diet): Mn (manganese oxyde), 20 mg; I
(potassium iodide), 1.5 mg; Cu (copper sulphate), 5 mg; Co (cobalt
sulphate), 0.1mg; Mg (magnesium sulphate), 500 mg; Zn (zinc
oxide) 30 mg; Se (sodium selenite) 0.3 mg; Fe (iron sulphate),
60 mg; Ca (calcium carbonate), 2.15 g; dibasic calcium phosphate, 5 g; KCl (potassium chloride), 1g; NaCl (sodium chloride),
0.4 g.
‰Vitamins (mg or IU kg À 1 diet): vitamin A (retinyl acetate),
8000 IU; vitamin D3 (DL -cholecalciferol), 1700 IU; vitamin K3
(menadione sodium bisul¢te), 10 mg; vitamin B12 (cyanocobalamin), 0.02 mg; vitamin B1 (thiamine hydrochloride), 8 mg;
vitamin B2 (ribo£avin), 20 mg; vitamin B6 (pyridoxine hydrochloride), 10 mg; folic acid, 6 mg; biotin, 0.7 mg; inositol, 300 mg;
nicotinic acid, 70 mg; pantothenic acid, 30 mg; vitamin E (Lutavit E50), 300 mg; vitamin C (Lutavit C35), 500 mg; betaine (Beta¢n S1), 500 mg.
CPSP G, ¢sh soluble protein concentrate (hydrolysed white ¢sh
meal); ¢sh oil extracted from sardine; DM, dry matter; NFE, nitrogen-free extracts 51000 À (CP1CL1CA).

in limits recommended for Senegalese sole. Fish were
exposed to an arti¢cial photoperiod of 12 h light.

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, e20^e30


Aquaculture Research, 2010, 41, e20^e30

Development of a plant-protein rich diet for sole J M G Silva et al.

Table 2 Indispensable amino acids (IAA, g16 g À 1 N) present in the experimental diets in both the feed acceptance
study and the growth trial
Feed
acceptance
trial

Growth trial

IAA

Control

Test

Control

Test

Sole carcass

His
Arg
His
Ile
Leu
Lys
Thr
Val
Met

Phe
Sum IAA
Sum DAA
IAA/DAA

3.3
7.8
3.3
4.0
8.9
6.4
4.7
4.7
2.4a
4.6
46.8
53.2
0.88

2.7
6.8
2.7
3.9
8.1
5.8
4.2
5.1
1.6b
4.3
42.5

57.5
0.73

1.6
5.4
1.6
3.8
8.1
6.0
3.1
4.3
3.4
4.2
39.9
48.3
0.82

1.3
5.6
1.3
4.2
9.0
6.2
3.5
5.5
3.9
4.3
43.5
52.3
0.84

2.2
5.7
2.2
3.2
6.0
6.2
3.5
3.9
2.3
3.4
36.9
61.7
0.83

Trp was not analysed.
The IAA pro¢le of Senegalese sole carcass is listed for
comparison.

Each of the diets was fed to duplicate tanks during a
4-week period. Fish were fed by automatic feeders,
over 24 h to apparent satiety with the aim of maximizing feed intake. The ration o¡ered was adjusted
daily according to the feed losses in each tank.

The study was performed at the experimental facilities of CIIMAR at Porto, Portugal, with Senegalese
sole (S. senegalensis) juveniles supplied from a commercial ¢sh farm (A. Coelho e Castro, Vila do Conde,
Portugal).
Experimental diets were fed to triplicate groups
containing twenty Senegalese sole juveniles each
with a mean initial body weight of 6 g. The same physical conditions and sampling procedures as described above were applied. This trial lasted for a

period of12 weeks and ¢sh were fed by automatic feeders, over 24 h to apparent satiety with the aim of
maximizing feed intake. The ration o¡ered was adjusted daily based on the feed losses in each tank.

Sampling procedures
All ¢sh were weighted at the start and at the end of
the experiment. A pooled sample of 10 ¢sh was analysed for proximate composition at the beginning of
the experiment. Also at the end, four ¢sh from both,
the feed acceptance and the growth trials, were collected from each tank and used for proximate composition analyses. Further, liver weight was recorded
and the hepatosomatic index was calculated. All
samples were maintained at À 20 1C until analysed.

Chemical analyses
Growth trial
A growth trial was carried out aiming to evaluate how
¢sh would perform on diets from the feed acceptance
study over a 12-week period, which is the generally
recommended time for a growth trial. As in the acceptation trial, two diets were formulated (crude
protein, 560^570 g kg À 1 DM and gross energy, 22^
23 MJ kg À 1 DM), a reference diet with ¢sh meal as
the main protein source (450 g kg À 1) and a test diet
in which ¢sh meal was substituted by a mixture of
plant protein-rich ingredients (SBM, corn and wheat
gluten). Using the IAA pro¢le of sole carcass (Table 2)
as a baseline, IAA of the plant-protein diet was made
similar to the control diet through the addition of crystalline AA (62 g kg À 1). Ingredients and proximal composition of control and test diets were made similar to
the diets used in the feed acceptance study (Tables 1
and 2), with the exception of mineral and vitamin
mixtures that were relatively higher in the growth performance trial to ensure the necessary quantities of
these nutrients over the longer growth period.

All chemical analyses were run in duplicates. Frozen
whole body samples were freeze-dried before being
analysed. Feed and whole body samples were analysed for dry matter in an oven (105 1C for 24 h), ash
by combustion in a mu¥e furnace (550 1C for 12 h),
crude protein (Micro-Kjeldahl, K˛nigswinter, Germany N Â 6.25) after acid digestion, lipid content by
petroleum ether extraction (at Soxhlet 40^60 1C) and
gross energy in an adiabatic bomb calorimeter
(Werke C2000, IKA, Staufen, Germany). All analyses
were performed following AOAC (2006) procedures.
Diets were analysed for total AA content. Samples
were hydrolysed in 6 M HCl at 106 1C over 24 h in nitrogen-£ushed glass vials. Total AAs were analysed
by high-pressure liquid chromatography (HPLC) in a
Pico-Tag AminoAcid Analysis System (Waters, Bedford,
MA, USA), using norleucine as internal standard and
according to the procedures described by Cohen,
Meys and Tarvin (1989). Tryptophan was not determined, as it is destroyed during acid hydrolysis.
Asparagine is converted to aspartate and glutamine
to glutamate during acid hydrolysis, and hence the

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, e20^e30

e23


Development of a plant-protein rich diet for sole J M G Silva et al.

values reported for these AAs (Asx and Glx) represent
the sum of the respective amide and acid. Resulting
peaks were analysed using the BREEZE software

(Waters).

Aquaculture Research, 2010, 41, e20^e30

body composition and nutrient accretion analysis were
used as experimental units for statistical analyses.

Results
Calculations

Diets

Data on initial weight, ¢nal weight, feed intake and
proximate composition of diets and carcass were
used to calculate nitrogen-free extracts (NFE), daily
weight gain (WG), daily growth index (DGI), daily
nutrient gain (NG), daily nutrient o¡ered (NO) and
hepatosomatic index (HSI):

The dietary protein in both trials ranged from 550%
to 580% DM whereas the dietary energy ranged from
22 to 23 MJ kg À 1 DM. To avoid di¡erences in AAs
contents, all the IAA were balanced to the level present in the control diet by adding crystalline AAs. In
the feed acceptance trial, the IAA in the test diet were
present at similar levels as the control diet with the
exception of Met (33% less). To avoid di¡erences in
Met content between diets in the growth trial, Met
was slightly enhanced in the AA mixture added to
the test diet. This strategy resulted in similar concentrations of all IAA within both the control and test
diet in the growth trial and with the exception of

His, all IAA exceeded the values found in the sole carcass IAA pro¢le (Table 2).
The IAA/DAA ratio in the growth trial was close to1
(0.82 vs. 0.84, in control and test diets respectively) and
similar to the IAA/DAA ratio in the sole carcass (0.83).

Nitrogen-free extracts ðNFEÞ
¼ 1000 À ðcrude protein þ crude lipids
þ crude ashÞ
Weight gain ð%IBWÞ
final body weight À initial body weight
¼ 100 Â
initial body weight
Daily growth index ðDGIÞ
¼ 100 Â ððfinal body weightÞ1=3 À
ðinitial body weightÞ1=3 Þ=days
Feed conversion ratio ðFCRÞ
¼ dry feed intake=weight gain
Average body weight ðABWÞ
¼ ðfinal body weight þ Initial body weightÞ=2
Daily nutrient gain in ðg kgÀ1 ABW dayÀ1 Þ ðNGÞ
¼ ðfinal body nutrient content À initial body
nutrient contentÞ=ABW=days
Daily nutrient offered ðg kgÀ1 ABW dayÀ1 ÞðNOÞ
¼ nutrient offered/nutrient gain
Hepatosomatic indexðHISÞ
¼ 100
 ½liver weight ðgÞ=whole body weight ðgފ

Statistical analyses
Statistical analyses followed methods outlined by Zar

(1999). All data were tested for normality and homogeneity of variances by Kolmogorov^Smirnov and
Levene’s tests, and then submitted to a two-way ANOVA
with trial and diet as main e¡ects, using the STATISTICS
8.0 package (Stat Soft, Tulsa, OK, USA, 1999). When
these tests turned out signi¢cant (Po0.05), individual means were compared using unequal N HSD
test. Tank average values for feed intake, growth,

e24

Growth performance
Data on growth performance and apparent feed intake of Senegalese sole juveniles fed the experimental
diets are reported in Table 3. The initial body weights
of ¢sh were similar in both the test group and the
control in both trials, but ¢sh used in the feed acceptance trial were bigger than in the growth trial (24.0
vs. 6.5 g respectively).
The feed acceptance trial was run for a short period of time with the purpose only to check if the test
diet was accepted by the ¢sh. Generally, the recommended duration for a trial that evaluate growth with
¢sh juveniles is around 12 weeks. Thus, growth measurements should be interpreted with caution in this
trial. Daily growth index was similar in ¢sh fed either
of the diets (1.05 vs. 0.76, for test and control respectively, Table 3). Data on feed intake showed that ¢sh
fed the test diet consumed approximately the same
amount of dry feed as those fed the control diet (17.6
vs. 18.4 g kg À 1 ABWday À 1, for test and control respectively, Table 4). Further, IAA intake in this trial
was also similar between diets while lipid intake was
signi¢cantly lower in ¢sh fed the test diet (1.9 vs.
2.4 g kg À 1 ABWday À 1), as a re£ection of the lower li-

r 2010 The Authors
Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, e20^e30