Aquaculture nutrition, tập 19, số 1s, 2013

Another striking difference between wild and farmed fish lies in the fatty acid profile of the eggs.. The comparison of farmed and wild eels revealed differ-ences in egg composition, pre

1 2 2 3 4

1

National Institute of Sciences and

To sustain eel aquaculture, development of reproduction in

captivity is vital The aim of this review is to assess our

cur-rent knowledge on the nutrition of broodstock eels in order

to improve the quality of broodstock under farming

condi-tions, drawing information from wild adult eels and other

marine pelagic spawners Freshwater eels spawn marine

pelagic eggs with an oil droplet (type II), and with a large

perivitelline space Compared with other marine fish eggs,

eel eggs are at the extreme end of the spectrum in terms of

egg composition, even within this type II group Eel eggs

contain a large amount of total lipids, and a shortage of

neu-tral lipids has been implied a cause for reduced survival of

larvae Eel eggs have higher ARA but lower EPA and DHA

levels than in other fish Too high levels of ARA negatively

affected reproduction in the Japanese eel, although high

lev-els of 18:2n-6 in the eggs of farmed elev-els were not

detrimen-tal The total free amino acid amount and profile of eel eggs

appears much different from other marine pelagic spawners

Nutritional intervention to influence egg composition seems

feasible, but responsiveness of farmed eels to induced

matu-ration might also require environmental manipulation The

challenge remains to succeed in raising European eel

brood-stock with formulated feeds and to enable the procurement

of viable eggs and larvae, once adequate protocols for

induced maturation have been developed

nutri-tion, fatty acids, feed, minerals, nutrients, vitamins

Received 26 August 2012; accepted 1 May 2013 Correspondence: L Heinsbroek, Aquaculture and Fisheries Group, Wageningen University, Wageningen 6700 AH, The Netherlands E-mail: leon.heinsbroek@wur.nl

Recruitment and wild stock of European eel (Anguillaanguilla, L.) have declined drastically over the last decades.Habitat reduction and over-fishing, climate change, pollu-tion and infections with the swim bladder parasite (Anguill-icoloides crassus) and/or eel viruses have been implicated ascauses for the current decline of the eel population (vanGinneken & Maes 2005) The major part of eel productionnow comes from aquaculture, but this is still capture based,relying on wild caught glass eels To sustain eel aquacul-ture, development of reproduction in captivity is vital.Research on eel reproduction is complicated, becausebroodstock eels stop feeding when silvering in nature.Although silvering is reversible and feeding can be resumedwhen migration is not initiated (Sved€ang & Wickstr€om1997; Durif & Elie 2008), it has been shown for A japonicathat eels caught in the spawning area had not been eating inthe marine phase of the migration (Chow et al 2010) Also

in captivity, feeding is terminated after transfer to saltwaterprior to induction of maturation Thus, for eels, all thequalitative and quantitative requirements for reproductionhave to be met from their body reserves highlighting the

importance of prespawning nutrition For eel embryos and

larvae, the expression ‘you are what you eat’ might be

extended to ‘you are what your parents ate a long time ago’

Furthermore, the life history of A anguilla, being the

lat-est in the anguillid evolution (Aoyama 2009), is in a

num-ber of respects also the most extreme They have the

longest migration distance, the longest larval duration and

the highest body lipid levels and are least mature even in

the silver stage Especially in A anguilla, silver females

puberty, defined as the onset of vitellogenesis (Taranger

2011a), A rostrata (Cottrill et al 2001), and A australis

and A dieffenbachii (Todd 1981; Lokman et al 1998),

seem to be more advanced

To close the life cycle of the European eel, information

on larval, juvenile and adult (broodstock) nutrition is

required The aim of this review is to assess our current

knowledge on broodstock nutrition or on nutritional

influ-ences on reproduction of A anguilla, in order to improve

the quality of broodstock under farming conditions

In nature, a large part of the reproductive investment of

an-guillid eels is spent during migration The energy

require-ments during migration also consist of a ‘fixed’ part

(standard metabolic rate) and a ‘variable’ part (active

metabolism above standard) Therefore, the total costs of

distance to the spawning area and the swimming speed

(Pal-stra & van den Thillart 2010) At optimal swimming speeds

, both females and males eels have a COT

esti-mated from the body energy losses (Van Ginneken et al

2005a; Palstra et al 2008; Burgerhout et al 2010) Based on

this, and depending on the initial body energy content,

pro-vided by body lipids, the remainders mostly by body protein

(Bo€etius & Bo€etius 1980, 1985; Van Ginneken et al 2005a)

Energy (and nutrients) invested during gonad

develop-ment is either deposited in the gonads or used to ‘fuel’ this

deposition When artificially matured, A anguilla males

reach gonado-somatic indices (GSI) of 6–14% (Bo€etius &

Bo€etius 1967; Amin 1991; Van Ginneken et al 2005b;

Mazzeo et al 2010) For A japonica males, GSI of up to40% have been reported (Lau 1987; Tsukamoto et al

2011), but it is not clear whether this is a true difference orthe result of a further advanced emaciation of these eel’s

to variable degree of hydration (Fig 5), 22–28% on a dry

tes-A anguilla, but total energy deposited in the testes seems

initial body mass

1991)

Lipid levels in the immature and early stage ovaries of

1980; Amin 1991; Kokhnenko et al 1977; Mazzeo et al

2008)) In later stages, ovary lipid levels are comparable

dm(Bo€etius & Bo€etius 1980; Ozaki et al 2008) Still, also egglipid levels in A anguilla seem to be somewhat higher,

in the ovaries and the eggs differ between species tion of total lipid and (crude) protein in the ovaries of

Initially more lipid is deposited, but in mature ovaries,

mass Palstra et al (2006) reported a lipid deposition of

of the initial body energy, which again seems somewhat

higher than for A japonica

Gonad development, or deposition of mass and energy

in the gonads, is not 100% efficient and in itself costs

energy From the initial body mass and composition and

the final mass (soma plus ovary) and composition, the

costs of deposition can be determined (Fig 1) Protein

deposition is quite efficient, 68%, while energy efficiency is

lower, 37%, although the latter could be determined with

less certainty, probably caused by uncertainty about the

& Bo€etius 1980) With this energy efficiency, the total

energy requirement for ovary development would become

body energy

types are recognized: eggs without and eggs with visible oil

droplet(s), the latter being the most common, in particular

in temperate and warmwater species (Ahlstrom & Moser

1980) Eels spawn marine pelagic eggs with an oil droplet,but also with a large perivitelline space, which is less com-mon (Ahlstrom & Moser 1980; Tsukamoto et al 2011).Rønnestad et al (1999) showed that eggs with oil droplets(by them classified as type II) differed in composition butalso in embryonic metabolism The type II eggs containmore lipids, and within these lipids, a (much) larger frac-tion is neutral lipids (Table 1) In the neutral lipids, theyfurther contain a larger (although variable) fraction ofwax- and sterol-esters (Wiegand 1996) All marine pelagiceggs contain similar amounts of total amino acids, butthese are more present as free amino acids (FAA) in thetype I eggs Apart from the role of FAA in early embry-onic energy metabolism (section Protein and amino acids),they also function as osmotic effectors in the acquisition of

Finn & Fyhn 2010)

Although eel eggs can be categorized as type II eggs,even within this group, eel eggs are at the extreme end ofthe spectrum (Table 1) If and how this position, which isextended in the fatty acid and FAA profiles (sections Lip-ids and Protein and amino acids), is related to the largeperivitelline space is not clear (Unuma et al 2005)

0 10 20 30 40 50 60

0 10 20 30 40 50 60

0.0 0.2 0.4 0.6 0.8 1.0

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

Lipids In most instances, reproduction of wild fish, or offish fed with natural food, is more successful than offarmed fish This has also been shown for A anguilla(Tomkiewicz 2012), but less so for A japonica, at leastwhen feminized eels are used (Yamada et al 2006) One ofthe differences between wild and farmed eels (and betweenmales and females) are the body lipid levels (Fig 2) In

800 g (Tesch 2003) Silvering eels, farmed and wild eels of

He-eswijk 1996; Garcia-Gallego & Akharbach 1998; Kn€osche2009; Clevestam et al 2011)] Both farmed and wild malesreach these levels early, at masses of 70–100 g Wild femaleeels seem to follow another trajectory: they first invest inbody mass growth and reach these higher lipid levels athigher body masses (Fig 2) Although most farmedfemales also have lower body lipid levels than males ofcomparable mass (Kamstra & Van Heeswijk 1996), theirtrajectory is clearly advanced compared with wild females

De Silva et al 2002; Hopkirk et al 1975) In vertebrates,the body lipid mass, through adiposity signals leptin andinsulin, is thought to influence reproduction in a number ofways (Caprio et al 2001) On the one hand, a minimumlipid mass seems to be required to initiate puberty Such aneffect was also observed in Oncorhynchus mykiss (Weil

stim-ulated in vitro pituitary LH release in the prepubertalstage, but much less in later stages For A anguilla, Lars-son et al (1990) even hypothesized that body lipid contentmight be the trigger for silvering, but as silver A anguillaare prepubertal and also some silver eels have (very) low

not so On the other hand, excessive body lipid stores tively affect reproduction, through impairment of gonadalsteroidgenesis (Caprio et al 2001) Evidence for a negativeeffect of increased adiposity on reproduction in fish ismostly anecdotal, in Indian (Chaudhuri, 1960) and Chinesecarps (Chen et al 1969, cited by Rath et al 1999) How-ever, the observed negative effects of a low protein brood-stock diet in Dicentrarchus labrax (Cerda et al 1994b)could well also have originated from a lower DP/DE ratio,

nega-as the gonads (but not the eggs) of the deficient fishshowed higher lipid levels during peak spawning Finally,

Table 1 Size and composition of marine pelagic fish eggs

Anguilla spp 1

Other marine pelagic

Oil globule 2

No oil globule 3 Egg diameter (mm) 1.1 –1.8 0.8 –1.5 (5) 0.8 –2 (6)

Oil glob diameter 0.25 –0.35 0.15 –0.4 –

1 Data on A anguilla (Kokhnenko et al 1977; Bo€etius & Bo€etius

1980; Bezdenezhnykh & Prokhorchik 1984; Prokhorchik 1987;

Pe-dersen 2004; Palstra et al 2005; Corraze et al 2011), A rostrata

(Edel 1975; Oliveira & Hable 2010), A australis (Lokman & Young

2000) and A japonica (Seoka et al 2003, 2004; Unuma et al.

2005; Furuita et al 2006, 2007; Tanaka et al 2006; Ohkubo et al.

2008; Ozaki et al 2008; Kagawa et al 2009).

2 Data on Dicentrarchus labrax (Devauchelle & Coves 1988;

Cerda et al 1994a; Bell et al 1997; Navas et al 1997, 2001;

Rønnestad et al 1998b), Sparus auratus (Mourente & Odriozola

1990; Rønnestad et al 1994; Fern
andez-Palacios et al 1995,

1997; Rodr
ıguez et al 1998; Almansa et al 1999, 2001), Pagrus

major (Watanabe et al 1984c, 1985b; Seoka et al 1997),

Den-tex dentex (Tulli & Tibaldi 1997; Mourente et al 1999;

Gim
enez et al 2008; Samaee et al 2009a,b, 2010), Diplodus

sar-gus (Cejas et al 2003; P
erez et al 2007), Scophthalmus maximus

(McEvoy et al 1993; Rainuzzo et al 1994; Silversand et al 1996),

Scophthalmus rhombus (Cruzado et al 2011), Paralichthys

olivac-eus (Furuita et al 2000, 2002, 2003c), Seriola quinqueradiata

(Verakunpiriya et al 1996), Seriola lalandi (Moran et al 2007;

Hilton et al 2008), Sciaenops ocellata (Vetter et al 1983), Latris

lineata (Morehead et al 2001; Brown et al 2005), Pseudocaranx

dentex (Vassallo-Agius et al 1998, 2001a), Lates calcarifer

(Southgate et al 1994; Sivaloganathan et al 1998; Dayal et al.

2003) and Rachycentron canadum (Faulk & Holt 2003, 2008;

Nguyen et al 2010, 2012).

3 Data on Gadidae, Gadus morhua (Craik & Harvey 1984; Fraser

et al 1988; Finn et al 1995a,b; Salze et al 2005; Penney et al.

2006) Melanogrammus aeglefinus (Craik & Harvey 1984; Reith

et al 2001) Theragra chalcogramma (Ohkubo et al 2006) and

Pleuronectidae, Hippoglossus hippoglossus (Falk-Petersen et al.

1986, 1989; Rainuzzo et al 1992; Bruce et al 1993; Evans et al.

1996; Mazorra et al 2003) Pleuronectes platessa (Craik & Harvey

1984; Rainuzzo et al 1992; Thorsen & Fyhn 1996), Microstomus

kitt (Thorsen & Fyhn 1996) and Verasper moseri (Ohkubo &

Matsubara 2002).

.

the link between adiposity signals and the dopaminergic

system (Baskin et al 1999) might explain (partly) the

dif-ference in responsiveness to maturation between farmed

and wild eels and the fact that for A anguilla, substantially

2006; Palstra & Thillart 2009), to complete maturation

2000; Kagawa et al 2005; Oliveira & Hable 2010)

Based on the above, one might have assumed that a

decrease in body lipids during starvation and swimming

Bo€etius (1985) and Van Ginneken et al (2005a) showed

that starvation had no effect on body composition,

indicat-ing that energy use from lipid and protein was in the same

proportion as in the body composition Van Ginneken

effect on the body composition of A anguilla This may be

due to the fact that they did used farmed eels with a very

lower body lipid levels, a decrease does occur, as indicated

by the results of Larsson & Lewander (1973) and Dave

reported to induce no (Palstra et al 2006) or only a slight(Mazzeo et al 2011) decrease in the muscle lipid content.Ozaki et al (2008) also reported no change in lipid content

in the muscle of A japonica during induced maturation;however, Lau (1987) and Liu et al (2009) did find a strongdecrease

In insects and birds, it has been shown that the demandsfor specific lipid classes and FA differ between migrationand reproduction (Zhao & Zera 2002) Sasaki et al (1989)also found a change in lipid class and FA composition in

0 50 100 150 200 250 300 350 400

Body mass (g)

0 50 100 150 200 250 300 350 400

Figure 2 Whole body or muscle lipid

percentage of wild (a) and farmed (b)

A anguilla in relation to body mass,

and sex (females = circles; males =

tri-angles) The light grey symbols in (a)

are for yellow eels, and the dark grey

symbols are for silver eels Data on

wild eels are from the study by Bo €etius

& Bo €etius (1985, 1989) and Larsson

et al (1990) and Heinsbroek,

unpub-lished, IMARES, unpublished Data

on farmed eels are from the study by

Kamstra & Van Heeswijk (1996),

Sch-mitz (1982), Corraze et al (2011),

Støttrup et al (2013) and Heinsbroek,

unpublished.

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

the muscle of migrating Oncorhynchus keta As further

spe-cific FA (n-6 and n-9) are implicated in swimming capacity

(McKenzie et al 1998; Chatelier et al 2006), it might be

that FA are selectively allocated to migration and

repro-duction and that if some FA are not used for migration,

they might negatively influence the egg composition Liu

swim-ming led to a selective retention of ARA in the muscle

This was not the case in eels that were induced to

matu-rate, indicating a selective incorporation in the ovary

Total lipid levels in eggs of A japonica are normally

2006; Tanaka et al 2006) although Unuma et al (2005)

and fertilization, hatching and survival Surprisingly this

effect was mainly caused by higher levels of PL A similar

effect was also described in Hippoglossus hippoglossus by

Evans et al (1996), but for the relative amount of PL

These authors therefore suggested that this was more an

indication of a lack of NL Total lipid levels remain stable

decrease during yolk sac and oil droplet resorption to c

showed that during this period, TG decreased stronger

(80%) than PL (40%) A shortage of neutral lipids has

been implied as a cause for larval mortality in Seriola

mor-talities before first feeding, or even mouth formation, in

larvae of A australis (Lokman & Young 2000) and A

Another striking difference between wild and farmed fish

lies in the fatty acid profile of the eggs Clear relations

between FA profile and egg quality have however not

always been apparent (Fern
andez-Palacios et al 2011) The

egg fatty acid profile of wild A japonica is compared with

other marine pelagic spawners, for both type I and II, in

Table 2 The differences can partly be explained by

differ-ences in lipid class composition (Table 1), but again eels

are at the far end, or even outside, the spectrum of type II

eggs A japonica eggs do have lower levels of EPA and

especially of DHA, and much higher levels of 18 : 1, also

in the PL

The lipid class profile of eel eggs (Table 1) suggests that

the majority of the PL in the eggs originate from

vitelloge-nin, as also shown in other fish (Silversand & Haux 1995;

Johnson 2009)] Vitellogenin (VTG) of A japonica was

actually one of the first teleost VTGs characterized (Hara

Haux (1995) showed for a number of fish species that thefatty acid profile of VTG and the egg PL were highly cor-related They did find species-specific differences in VTG

FA profiles In general, egg PL FA seem to be less affected

by the broodstock diet (Mourente & Odriozola 1990;

Table 2 Fatty acid profile (% of total FA) of egg total and polar lipids of wild (or fed with raw fish and/or squid) A japonica and

of other marine pelagic spawners

ARA 2.1 (0.9 –3.8) 2 (0.5 –3.7) 1.9 (1 –3) EPA 2.9 (2.1 –3.7) 6.4 (2.4 –11) 13.4 (8.7 –15.5)

DHA 8.9 (6.1 –12) 24.1 (13.7 –31.4) 28.8 (25.5 –31.1) EPA/ARA 2.0 (0.7 –3.3) 3.7 (0.6 –8.6) 7.8 (4.4 –14) DHA/EPA 3.4 (2 –4.1) 4.1 (2 –6.8) 2.2 (1.8 –2.9) Polar lipids

16:0 21.2 (18.9–23.1) 21.0 (18.5–24.1) 21.4 (20.9–22) 18:1 22.3 (15.4–24.2) 11.7 (10.7–13) 13.2 (11.1–14.3)

ARA 3.8 (2.5 –5) 3.6 (1.8 –4.9) 2.6 (1.5 –3.3) EPA 5.3 (3.7 –6.4) 9.1 (6.8 –10.1) 13.0 (10.9 –15)

DHA 17.2 (13.6 –21.4) 32.5 (27 –37.3) 32.2 (29.3 –34.8) EPA/ARA 1.4 (0.5 –2.2) 3.1 (1.4 –5.4) 5.4 (3.9 –8.8) DHA/EPA 3.3 (2.4 –3.9) 3.6 (3.2 –4) 2.5 (2.2 –2.9)

1 Furuita et al (2003a) and Ozaki et al (2008).

2 Data on Dicentrarchus labrax (Bruce et al 1999; Navas et al.

2001), Sparus aurata (Mourente & Odriozola 1990), Scophthalmus maximus (Peleteiro et al 1995; Silversand et al 1996; Lavens

et al 1999), Rachycentron canadum (Faulk & Holt 2003, 2008;

Nguyen et al 2010, 2012), Plectorhynchus cinctus (Li et al 2005) Centropomus undecimalis (Yanes-Roca et al 2009), Pseudocaranx dentex (Vassallo-Agius et al 1998, 2001a), Lutjanus campechanus (Papanikos et al 2008), Coryphaena hippurus (Divakaran & Os- trowski 1989; Ostrowski & Divakaran 1989), Solea senegalensis (Mourente & V
azquez 1996), Solea solea (Lund et al 2008), Para- lichthys adspersus (Wilson 2009) and Centropristis striata (Bentley

et al 2009).

3 Data on Hippoglossus hippoglossus (Falk-Petersen et al 1989;

Bruce et al 1993; Mazorra et al 2003) and Gadus morhua (Fraser

et al 1988; Pickova et al 1997; Salze et al 2005; Penney et al.

2006; Lanes et al 2012).

.

Wiegand 1996), although Silversand et al (1995) did find

higher levels of 18:2 and lower levels of EPA in both VTG

and eggs of farmed Gadus morhua Increased incorporation

of 18:2 was also noted in the egg PL (and NL) of farmed

and selectively incorporated in the PL (Table 2), suggesting

the importance of DHA for embryonic and larval

develop-ment (Sargent 1995; Wiegand 1996) Selective

incorpora-tion of DHA is also seen in A japonica, be it at a lower

level than for other fish (Furuita et al 2007; Ozaki et al

2008) Despite the selective incorporation of DHA into the

gonads and the eggs, both low levels of DHA and

imbal-anced LC-PUFA ratios in the broodstock diets can lead to

lower DHA levels in the egg lipids (Bell et al 1997;

Al-mansa et al 1999; Bruce et al 1999) However, no effect of

broodstock diet on the DHA content of the eggs was found

in A japonica (Furuita et al 2007; Ozaki et al 2008)) In

both studies, the EPA level in the eggs decreased with the

replacement of fish oil by corn oil in the broodstock diets,

similarly to the results of Yamada et al (2006) with

sun-flower oil (Fig 5), and those in Gadus morhua (Silversand

selec-tively incorporated, but even more in the gonad than in

eggs P
erez et al (2007) found high levels of ARA

accumu-lated in gonad PL of male and female Diplodus sargus and

selective retention of this fatty acid after gonad recession

There seem to be large species differences, however

(Table 2) In Lutjanus argentimaculatus, Emata et al (2003)

reported for eggs an already low EPA/ARA ratio of 0.9;

in the ovary this ratio was only 0.2, with an ARA level

of 10.4%FA Similarly high levels of ARA (4-20%FA)were observed in ovaries and testes of a number of tropicalreef species (Ogata et al 2004; Suloma & Ogata 2011) Ahigher level of ARA in the PL of the (immature) ovariesthan in the eggs of A japonica was also observed by Furui-

ta et al (2007) Remarkably, and contrary to the findings

in other marine fish, in A japonica, the egg ARA can beformed from conversion of dietary 18:2 (Yamada et al.2006; Furuita et al 2007; Ozaki et al 2008) The capacity

of anguillid eels to elongate and desaturate FA is well umented (Takeuchi et al 1980; Kissil et al 1987), soalthough of marine origin, they truly earn the name offreshwater eels (NRC 2011) This capacity is also reflected

doc-in the egg composition of A japonica Yamada et al.(2006) showed that A japonica fed sunflower oil, rich in18:2, produced eggs with twice as much ARA than eels fedwith a marine oil (Fig 5) A similar but less dramatic effect

of dietary 18:2 on egg ARA levels was observed by Furuita

(2008)

A selective incorporation of FA was also observed in thetestes and semen of farmed A anguilla (Mazzeo et al.2010) (Table 3) Whereas the FA profile of the immaturetestes is essentially the same as that of the muscle, in themature testes, the levels of EPA, DHA and especially ARAare increased Remarkably, in the semen, EPA and ARAare further increased, but not DHA Although P
erez et al.reported much lower levels of LC-PUFA in the semen offarmed A anguilla, they also found a low DHA/EPA ratiowhich seems to be unique among the few marine teleostsstudied, Dicentrarchus labrax (Bell et al 1996; Asturiano

Table 3 Fatty acid profile (% of total FA) of muscle, liver, testes and semen of farmed A anguilla before and after induction of tion1

1 0 and 7 –11 weeks: Mazzeo et al (2010) and 5–13 weeks: P
erez et al (2000).

2 In both studies, eels were weekly injected with HCG and started spermiating in week 4.

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

et al 2001) and Seriola quinqeradiata (Verakunpiriya et al.

1996) It is not known whether there are differences

between semen of wild and farmed A anguilla as shown in

2001), nor if and how the fatty acid profile of the semen

affects fertilization and embryonic development

In eels, most of the egg NL seem to be transported by

VLDL Endo et al (2011) showed that already during

pre-vitellogenesis, in the lipid droplet stage, both 11-KT and

VLDL were required to stimulate oocyte growth and lipid

incorporation Furthermore, Ando & Matsuzaki (1996)

found that the plasma lipoproteins of A japonica were

or more than 40% oftotal lipoproteins), even after induction of vitellogenesis

to deposition of transported lipids, a substantial amount of

NL in the ovary of A anguilla originates from de novo

lipid synthesis within the ovary Bo€etius et al (1991)

during a 24-h period after injection in male and female

that at early stages of maturation, gonad lipid synthesis

equalled that in the liver For females, this coincided with

the period of maximum lipid deposition in the ovaries, at

GSI of 5-13% (cf Fig 1) In this period, radioactivity was

mainly incorporated in TG, with 16:0 and 18:0 as major

FA In later stages of ovary development, also sterol esters

became important as well as monoenes and FA with more

than 18 carbon atoms Bo€etius et al (1991) did find only

minor synthesis of wax esters in the ovaries This might be

due to fatty alcohols being synthesized as FA (not

neces-sary de novo) in the liver and only transformed to alcohols

after transport to the ovary, as described by Bell et al

(1997) However, the low DHA level in the neutral lipids

of anguillid eggs is another indication that wax esters are

not abundant, because in other type II eggs, it was shown

that the fatty alcohols were mainly saturated (mainly 16:0),

or monoenes (mainly 18:1), but the FA consist for almost

half of n-3 LC-PUFA, of which 50-70% DHA (Joh et al

1995; Silversand et al 1996; Bell et al 1997)

The physiological and structural roles of the LC-PUFA

in the reproduction of fish are reasonably well documented

(Fern
andez-Palacios et al 2011) However, due to the

com-plex interactions and the fact that these roles vary with the

reproductive stage, that is, different in gonad development,

spawning and fertilization (fecundity), embryonic

develop-ment (egg quality, hatching) and larval developdevelop-ment (yolk

sac use/retention, survival), the picture is still far from

com-plete It has been recognized for some time that a (severe)

deficiency of n-3 LC-PUFA in the broodstock diet impairsreproduction in fish (Watanabe et al 1984a; Chou et al

More recently the importance of ARA was also recognized(Bell & Sargent 2003) Reproductive success is influenced

by not only the levels but also by the ratios between PUFAs in the broodstock diet, the gonad and gametes(Fern
andez-Palacios et al 2011) However, little is knownabout the physiological role of EPA and DHA duringgonad development, ovulation and fertilization For ARA,

LC-it is known that ARA-derived eicosanoids, in particular the

in the control of oocyte maturation and ovulation (Sorbera

embryogenesis (Bruce et al 1999) and larval development(Izquierdo & Koven 2011) and play a role in spermiation(Asturiano et al 2000) Kagawa et al (2003) showed in

DHP-induced ovulation Indomethacin, actinomycin D and

reversed the effects of these inhibitors Similar effects of theseries 2 PGs were observed by Sorbera et al (2001) with in

showed that addition of free ARA induced maturation ofthe oocytes Free ARA also enhanced GTH-induced matu-ration, while free EPA and DHA had the opposite effect

ARA is also known to stimulate testicular testosterone in

spermia-tion may be delayed causing reduced fertilizaspermia-tion rates due

to depressed steroidogenesis caused by broodstock EFAdeficiency or imbalance (Izquierdo et al 2001) In larvalfish during endogenous feeding, ARA is selectively retainedand has been shown to enhance survival and stress response(Tandler et al 1995) Fuiman & Ojanguren (2011) found in

lar-val survilar-val and growth, but did show a strong positiveeffect of the egg ARA level on the predator avoidance(escape) behaviour of the larvae

Based on the work on Pagrus major (Watanabe et al

1984b), Sparus aurata (Fern
andez-Palacios et al 1995;

Tandler et al 1995) and Paralichthys olivaceus (Furuita

diet and aminimum DHA/EPA ratio of 0.6 Lower levels of total n-3LC-PUFA reduced fecundity, fertilization, hatching and .

survival Both Fern
andez-Palacios et al (1995) and Furuita

studies, both EPA and DHA were increased, EPA even

(1995) In studies with Dicentrarchus labrax (Bruce et al

1999) and Hippoglossus hippoglossus (Mazorra et al 2003),

impair reproduction Based on this, it seems that the

will vary from 0.6 to 3, depending on the DHA level In

A japonica, although there was no notable effect of FA

composition of broodstock diet on the DHA content of the

eggs, low-quality eggs contained significantly less DHA in

the PL (Fig 3) (Furuita et al 2006; Ozaki et al 2008)

There are indications that the optimal EPA range is

fur-thermore also dependent on the dietary ARA level, and

par-ticular on ovulation (fecundity) and fertilization, have been

observed in Dicentrarchus labrax (Bell et al 1997; Bruce

2003) and Gadus morhua (Salze et al 2005; Norberg et al

2009; Sawanboonchun 2009) with an increase in dietary

diet, with a concomitant decrease

Watanabe et al (1984a,c) with Pagrus major, it could be

noted that their cuttlefish meal diet, and the eggs from

these fish, contained higher levels of ARA than their

fish-meal-based diet and eggs Improved fertilization, hatching

and larval survival were also observed in Paralichthys

the broodstock diet (Furuita et al 2003c) A further

opposite effect Similarly, negative effects of higher ARAlevels in both the PL and the NL of A japonica eggs havebeen shown (Furuita et al 2003a, 2006) (Fig 4), althoughthis is a bit puzzling in view of the fact that this ARA waslargely synthesized by the eels themselves These data indi-cate that while ARA is essential for larval development,excess ARA levels can be detrimental for embryonic andlarval development Broodstock origin and diet did nothave a large effect on egg ARA level of A japonica (Furui-

ta et al 2003a) (Fig 4a), although Ozaki et al (2008) didfind lower levels of ARA in eggs of farmed A japonica fedwith fish-oil-based diets

One notable exception to the above is formed by krill,which is often fed, either frozen or as meal incorporated

in the diet, to broodstock fish with excellent results(Watanabe et al 1985b; Watanabe & Kiron 1995; Mazorra

and EPA/ARA ratios of 0.5 and 25, respectively Also uita et al (2006) reported some positive effects of frozenkrill in A japonica broodstock, but egg FA compositionwas not affected

Fur-Apart from the levels and ratios of DHA, EPA andARA, ‘pollution’ by other FA in farmed fish has been sug-gested as a cause for impaired egg quality Almansa et al.(1999) reported for Sparus aurata that a high 18:1/n-3HUFA ratio in both the NL and PL of the eggs negativelyaffected the fertilization rate Such an effect does not seemlikely in eels, as even the eggs from wild eels show a high18:1/n-3HUFA ratio (Table 2) Also negative effects ofhigh levels of 18:2 in the eggs, as reported by Bell et al.(1997) and Palacios et al (2011), do not seem to occur ineels (Fig 5)

protein and amino acids, nucleic acids, and more, form alarge part of fish eggs (Table 1), but contrary to liposolu-ble compounds (and minerals, section Vitamins), there is

no specific storage for these nitrogen-containing nutrients

in the body There are a few studies on the effect ofN-containing nutrients in broodstock nutrition in fish spe-cies who keep feeding during (part of the) gonad develop-ment It has been shown that defatted squid or cuttlefishmeal has a positive effect on reproduction of Pagrus

Figure 3 Effect of DHA level in polar lipid (PL) on egg quality

(Furuita et al 2006) Lines are for hatching: y = 3.57 9 43.15

(P < 0.01), and for survival to 8 days posthatch:

y = 2.43 9 39.25 (P < 0.05) NB The arrows for survival and

hatching seem to have been exchanged in the original publication

(Reprinted with permission of John Wiley and Sons).

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

this was caused by the protein and/or the mineral fraction

is not known In Colisa lalia, Shim et al (1990) found

that deletion of certain amino acids from the broodstock

diet reduced spawning performance and hatchability Fish

fed the methionine deficient diet completely failed to

spawn Also Harel et al (1995) showed that Sparus aurata

broodstock fed a wheat gluten based diet (low in lysine)

had significantly lower VTG levels, resulting in a decrease

in larval survival by 50% In Plecoglossus altivelis,

addi-tional tryptophan in the broodstock diet advanced

spermi-ation in males and final maturspermi-ation in females, while a

serotonine depletor delayed gonad development (Akiyama

taurine to the diet during 5 months before spawning

improved the spawning performance, expressed as the

per-centage of females spawning, from nil to 86% Taurine

content of the ovaries was not different between

treat-ments In A japonica, Higuchi et al (2012) showed that

although taurine is essential in spermatogenesis, it is ofendogenous origin, through DHP-stimulated biosynthesisfrom cysteine in the testes Gonzalez-Vecino et al (2004)reported that broodstock diets enriched with nucleotidesimproved the first feeding success and survival of Melano-

incorporation in the eggs or through enhanced parentalphysiology was not reported

ovary and the eggs comes from VTG, the remainder largelyfrom the choriogenins, or zona pellucida (ZP) precursorproteins (Pati~no & Sullivan 2002; Lubzens et al 2010) Theamino acid composition of VTG (and therefore largely ofthe eggs) is highly conserved in teleost fish (Fig 7a),although the most abundant amino acid, alanine, seems to

be even more dominant in anguillid VTG (Hara et al

1980; Komatsu et al 1996) Compared with VTG, the ZPproteins of A japonica are relatively low in lysine and high

in proline and glycine (Sano et al 2010)

(b) (a)

Figure 4 Effect of ARA in PL on fertilization of A japonica eggs (a) (Furuita et al 2003a) and of ARA in NL on fertilization, hatching

and survival (b) (Furuita et al 2006) Lines in (b) are for fertilization: y = 18.38 9 +75 (P < 0.05), for hatching: y = 30.31 9 +54.16

(P < 0.01) and for survival to 8 days posthatch: y = 19.35 9 +35.63 (P < 0.05) NB in (b): AA = ARA (Reprinted with permission of

Springer Science & Business Media and John Wiley and Sons).

0

Ovulated Buoyant

20 40 60 80 100

The VTG is taken up by developing oocytes through

pinocytosis and is cleaved in the egg to generate the major

egg yolk proteins, lipovitellin and phosvitin (Matsubara

dur-ing final maturation is largely driven by an increase in

FAA, through hydrolysis of yolk proteins (section Marine

pelagic fish eggs) (Rønnestad et al 1999; Matsubara et al

2006; Cerda et al 2007; Finn & Fyhn 2010) Also anguillid

increase in volume, and an increase in moisture content

(Palstra et al 2005;

Kagawa et al 2009) However, the increase in FAA is only

limited, up till c 10% of the total AA, in A japonica

(Seoka et al 2004; Ohkubo et al 2008) (Fig 6), and

hydration, and buoyancy, seems to be related to other

osm-olytes (Seoka et al 2003; Unuma et al 2005) Another

striking difference between A japonica and other marine

pelagic spawners, even two other Anguilliformes, is the

FAA profile (Fig 7b) (Seoka et al 2004; Ohkubo et al

2008) Not alanine, but glutamine dominates the FAA

pool As a consequence, most amino acids are relatively

lower, but especially serine (from phosvitin) is much lower,

while the proportions of lysine, histidine, glycine and

pro-line are higher From these differences, and the SDS-PAGE

protein profile of eggs and embryos, it could be deduced

that in A japonica, neither lipovitellin nor phosvitin is

hy-drolysed during maturation (Matsubara et al 2003b, 2006;

Ohkubo et al 2008)

Apart from their role in hydration and buoyancy, (F)AA

are also important energy sources in developing embryos of

marine pelagic spawners Up till first feeding, amino acid

catabolism, initially from FAA and subsequently from yolk

proteins, supports c 75% of the energy expenditure of type

I eggs and c 50% for type II eggs (Finn & Fyhn 2010)

Although the FAA pool in A japonica is smaller than inother fish, its decline is linked to an even faster decline ofthe major yolk protein, lipovitellin (Fig 6) Further, someFAA, serine and phenylalanine, are actually increasingduring the first 4 days, indicating that also phosvitin ishydrolyzed (Ohkubo et al 2008) Due to this constantturnover of the FAA pool, correlations between levels andratios of specific AA and hatching rate and embryonicdevelopment as reported in Lates calcarifer (Nocillado

and Dentex dentex (Samaee et al 2010) are difficult tointerpret and might be spurious

fish eggs They are present in glycoproteins, both tural, in the zona pellucida, and functional, in the con-

Lubzens et al 2010) Free glucose, partly derived fromglycogen, has been shown to be the most importantenergy source in early embryonic development (first cleav-ages), both in type I (Finn et al 1995a) and in type II

2006) It was further suggested that the most dominantfree amino acid in most marine pelagic fish eggs, alanine,has an important role in embryonic gluconeogenesis (Finn

& Fyhn 2010) It is tempting to assume a similar role forthe large free glutamine pool in eel eggs (Fig 7), becausethis glutamine pool is almost completely used during thefirst day after fertilization (Ohkubo et al 2008) In Den-

(Lahnsteiner & Patarnello 2004), correlations were foundbetween hatchability and/or survival and levels of freeglucose and other carbohydrates, such as sialic acid andribose

0 10 20 30

0 20 40 60 80 100

10 20 30

0 50 100 150 200

Figure 6 Amounts of protein amino acids [as PAA or as OLv (ovarian lipovitellin)] and free amino acids (FAA) during maturation (a) and embryonic development (b) in A japonica Data on maturation are from the study by Seoka et al (2004), in two oocyte development stages (oocyte diameter OCI 700 –750 lm, OCII 800–850 lm) and ovulated eggs (OV 850–950 lm) Data after fertilization are from the study by Ohkubo et al (2008) Hatching occurred at 2 days postfertilization (dpf) See text for further explanation.

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

Vitamins The essentiality of vitamins for metabolism

makes them required for the process of gonad development

and also for embryonic development, after incorporation in

the eggs (Brooks et al 1997; Lubzens et al 2010)

Trans-port and incorporation of lipid soluble vitamins is thought

to be analogue to lipid transport (section Lipids), that is,

bound to VTG and other lipoproteins (Lie et al 1994),

described (Brooks et al 1997; Lubzens et al 2010) Little isknown about the transport and incorporation of water-sol-uble vitamins In Scophthalmus maximus, Salmo salar(Cowey et al 1962; Albrektsen et al 1994; Sandnes et al

1998) and Gadus morhua (Mangor-Jensen et al 1994) min B6, pantothenic acid, nicotinic acid and vitamin C

Figure 7 Amino acid profile of (a) vitellogenin and (b) free amino acids in eggs of A japonica (light grey) and other marine pelagic

spaw-ners (dark grey) Data are presented as mean  SE Data on vitellogenin are for A japonica (Hara et al 1980; Komatsu et al 1996) and

UniProtKB/TrEMBL accession numbers Q5WR04; Q5WR05; Q5U8V4) and for Conger myriaster (Q589G6), Melanogrammus aeglefinus

(Q98T86; Q98T87), Sparus aurata (Q3V7A1; Q3V7A2), Thunnus thynnus (D3U1X3; D4NUV4), Mugil cephalus (A6BLZ1; A6BLZ2),

Veras-per moseri (Q589T1; Q589T2) and Hippoglossus hipoglossus (A5JV30; A5JV31) Data on FAA are for A japonica (Seoka et al 2004;

Ohkubo et al 2008) and for Ariosoma anagoides, Ophichtus erabo (Seoka et al 2004), Lates calcarifer (Sivaloganathan et al 1998), Latris

lineata (Brown et al 2005), Seriola lalandi (Moran et al 2007), Dicentrarchus labrax (Rønnestad et al 1998b), Scophthalmus maximus

(Rønnestad et al 1992), Dentex dentex (Samaee et al 2010), Lutjanus campechanus (Hastey et al 2010), Pleuronectes platessa, Microstomus

kitt (Thorsen & Fyhn 1996), Hippoglossus hippoglossus (Evans et al 1996), Gadus morhua (Finn et al 1995a), Verasper moseri (Ohkubo &

Matsubara 2002) and Theragra chalcogramma (Ohkubo et al 2006).

.

(VC) in the ovaries did originate to a large extent from the

white muscle compartment Remarkably, both Yoshikawa

(1997, 1998) and Furuita et al (2009a) found that in

imma-ture A japonica, before the start of maturation, already

more than half of the body store of VC was located in the

ovary

Linked to the high levels of LC-PUFA in fish eggs,

which are vulnerable to lipid peroxidation, an efficient

anti-oxidant system is essential for gonad and embryonic

devel-opment (Mourente et al 1999; Palace & Werner 2006)

Most attention has been paid to vitamin E (VE) Vitamin

E protects unsaturated lipids against peroxidation by

donating a proton to the lipid peroxide radical, becoming a

VE radical in the process (Hamre et al 2010) In sparids, it

was shown that high levels of n-3 LC-PUFA actually

depressed hatchability and survival, but increased levels of

VE could obviate this to some extend (Watanabe et al

1985b, 1991a; Izquierdo et al 2001; Fern
andez-Palacios

found that the level of VE in the eggs, and in particular

the ratio VE/HUFA, was correlated with fertilization and

hatching rates This was corroborated in a later study

(Furuita et al 2009b), although at higher levels of VE and

higher VE/HUFA ratios

Other antioxidants implicated in embryonic development

and survival are vitamin C (VC), vitamin A (VA) and

car-otenoids (Blom & Dabrowski 1996; Palace & Werner 2006;

Waagbø 2010; Fern
andez-Palacios et al 2011) Vitamin C,

being water soluble, cannot be stored but maximized tissue,

liver and ovary, saturation proved beneficial in

Ciereszko 2001) Furuita et al (2009b) found in A japonica

positive correlations between egg VC and hatching and

sur-vival rates In another study, where the A japonica

brood-stock were injected with VC and VE combined (Furuita

high-quality eggs, and the percentage of normal larvae and

survival till 8dph was positively correlated with liver VC

levels The positive effect of krill (whole but also only the

NL) on reproduction of Pagrus major was ascribed by

Watanabe et al (1991a) to carotenoids Similar effects of

krill meal were observed in Seriola quinqueradiata

showed that this effect was most likely due to astaxanthin,

as addition of synthetic astaxanthin also improved

repro-duction in Seriola quinqueradiata Synthetic astaxanthin

also had positive effects on reproduction of Gadus morhua

(Salze et al 2005; Sawanboonchun et al 2008), and

carotenoids from paprika improved sperm and egg quality

of Sparus aurata (Scabini et al 2011) Although the

with frozen krill (Furuita et al 2003a, 2006, 2009b), oreven with added synthetic astaxanthin (Furuita et al 2007),carotenoid levels in the eggs were not measured Feedingthe A japonica broodstock frozen krill had a slight, butnot significant, positive effect on reproduction, but not onegg biochemical composition (Furuita et al 2006)

What all antioxidants have in common is that they takethe ‘burden’ of peroxidation of the unsaturated lipids,becoming radicals themselves The liposoluble antioxidantsalso contain highly unsaturated lipid structures themselves.Vitamin C interacts with VE, by regenerating VE radicals,forming a VC radical in the process, which in turn can beregenerated by glutathione (Palace & Werner 2006; Hamre

marginally adequate VE levels could be protected by VC injuvenile Salmo salar In A japonica, Furuita et al (2009a)found less effect of VE at high doses of (injected) VC Due

to these interactions between antioxidants and the fact thatcarotenoids are even more active radical scavengers than

VE (Palace & Werner 2006), too much antioxidants mightalso negatively affect reproduction Chou & Chien (2006)reported for Lateolabrax japonica that a combination of

VE and astaxanthin gave worse results than either VE orastaxanthin alone A similar effect might have caused thereported negative effects of an increase in krill meal to 20-30% of the broodstock diet for Seriola quinqueradiata(Verakunpiriya et al 1997b)

Apart from its role as antioxidant, VA also act as tor of the development of neural tissues, in retina forma-tion, organogenesis and differentiation of immune cells(Rønnestad et al 1998a; Palace & Werner 2006; Haga

(increased spawning period and fecundity, increased centage of normal larvae) when VA in broodstock diet for

not different Earlier, they showed that an increase from 33

concen-tration in ovaries and eggs was higher with the highestdietary level, but excess VA was mainly stored in the liver

broodstock diet an increased mortality in the offspring but

no malformations It is noteworthy that all tioned diets contained synthetic astaxanthin, from 50 .

above-men-Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

(Fontagn
e-Dicharry et al 2010) to 80 (Furuita et al 2001,

fertilization, hatching and survival The authors claimed a

vari-ability was very high These eels were further fed with

frozen krill before maturation, but egg levels of carotenoids

were not measured It has been shown that

hypervitamin-osis of VA in later stages of larval development can cause

malformations, particularly on the head (jaws and

opercu-lae) structures (Furuita et al 2001; Hamre et al 2010;

Haga et al 2011), but this is probably less of concern in

eels as ossification in leptocephali is largely delayed till

metamorphosis (Miller 2009)

Vitamin D and possibly vitamin K, also involved in

calcium transport, have been implied the demineralization

of the bones during ovary development and maturation

(Lopez et al 1980; Hamre et al 2010; Lock et al 2010;

Krossøy et al 2011)

For the water-soluble vitamins (other than VC), Mæland

critically low in view of the observed folate use during

embryonic and larval development Similar observations

have been described for vitamin B6 (Waagbø 2010) In a

number of fish species, thiamine deficiency has been

reported as cause of decreased larval survival (Fisher et al

1996; Czesny et al 2009; Rinchard et al 2010), but

paren-tal and/or egg thiamine depletion only occurs with specific

broodstock diets, that is, raw seafood products containing

thiaminase (Kreutzmann & Lehmitz 1976)

1987) These estimates are somewhat higher than for other

marine pelagic spawners (Table 4) In accordance with

these other fish, the most abundant mineral in the ovary is

provision of P was critical for good-quality eggs, despite

the fact that the egg P content was not much influenced

(Watanabe et al 1984c) Lanes et al (2012) did find a

posi-tive relationship between P levels in the eggs and the

fertil-ization rate for wild broodstock of Gadus morhua For

the total amount of P in the ovary was provided through

extensive demineralization of the bones, a process also

observed in A anguilla (Lopez et al 1980; Sbaihi et al

2009) Somewhat unexpectedly, in view of the oftenreported association of vitellogenin with Ca (Lopez et al

1980; Lau 1987; Sbaihi et al 2009; Palstra et al 2010), theprimary goal of this bone demineralization does not seem

to be the provision of Ca to the ovary Ovary Ca contentwas only 0.02% dm (Yamada et al 2001), giving a Ca/Pratio of 0.02, which is however in accordance with otherfish species (Table 4) Furthermore, in the marine environ-ment, requirements for Ca (and Mg, K) can be met byabsorption through the gills and the intestine (from drink-ing water), even in starving eels (NRC 2011)

Total ash content of A japonica eggs was reported by

whether these were unfertilized or washed/unwashed ized eggs was not given Data on the mineral composition

fertil-of eel eggs are lacking Craik & Harvey (1984) showed that

in Pleuronectes platessa, the hydration of the oocytes ing maturation was accompanied by a large influx of K Inunfertilized eggs of Pleuronectes platessa and Gadus morhua(Craik & Harvey 1984) and washed fertilized eggs of Pag-

the most abundant mineral, still followed by P The rion remains permeable to small osmolytes, and the mineral

cho-Table 4 Mineral composition of ovaries and eggs (g or

mg kg 1 dm)

Anguilla spp 1 Other 2

Unfertilized/

washed 3 Unwashed 4 Ash (g kg 1 ) 70 –120 32 –60 50 –80 150 –200

2 Gadus morhua (Rom
eo 1987; Hellou et al 1992; Saxholt et al.

2008), Dicentrarchus labrax (Devauchelle & Coves 1988), Mugil cephalus (Rom
eo 1987; Olgunoglu & Olgunoglu 2011).

3 Pagrus major (Watanabe et al 1984c, 1985a), Gadus morhua (Craik 1986; Lanes et al 2012), Pleuronectes platessa (Craik & Har- vey 1984; Craik 1986).

4 Dicentrarchus labrax (Devauchelle & Coves 1988), Pagrus major (Watanabe et al 1985b, 1991b), Scophthalmus maximus (Finn

et al 1995a).

.

profile of the perivitelline space is largely identical to

sea-water (Riis-Vestergaard 2002) In unwashed fertilized eggs

of Pagrus major, the contents of Na, Ca and Mg were

indeed increased (Watanabe et al 1985b, 1991b)

With regard to the trace minerals, Takeuchi et al (1981)

showed that Oncorhynchus mykiss fed on a white

fishmeal-based diet without a trace mineral mix produced less eggs,

of which hatchability was almost nil Within the eggs, only

diet A similar effect of Mn-deficient broodstock diets, high

observed for Salvelinus fontinalis by Lall and Hines [1985,

in (Luquet & Watanabe 1986)] Yamaguchi et al (2009)

showed in HGC-induced maturation of male A japonica

that Zn is essential for the maintenance of germ cells, the

progression of spermatogenesis and the regulation of sperm

motility

The negatively charged phosphates on the phosvitin part

of vitellogenin attract, apart from Ca and Mg, also other

metal cations such as Fe, Zn, Mn and Cu (Ghosh &

Thomas 1995) These minerals are required, but also act as

plasma lipoproteins by exerting an antioxidant action in

taking up Cu In Sander vitreus, Johnston et al (2007)

found that hatching success was negatively correlated with

the Cu level in the eggs Alsop et al (2007) showed that

high-dietary Cu levels resulted in a depletion of body

reti-noids (vitamers A) in Danio rerio This had no effect on

reproductive output, as dietary retinoids appeared sufficient

for normal reproduction Seemingly contrary to the above

findings, Le et al (2010) found however in immature

and GSI Furthermore, two dietary components with

reported positive effects on reproduction in fish, cuttlefish

meal and krill (raw or as meal), have high levels of Cu

(a.o Watanabe et al (1991b)

Two non-essential minerals, Cd and As, are worth

men-tioning, as they have been shown to accumulate also in

farmed fish Ure~na et al (2007) found higher Cd

concen-trations in liver and kidneys of farmed A anguilla than in

wild eels from La Albufera Lake in Spain These

concen-trations were actually higher than those reported by

Pierron et al (2008) to have a detrimental effect on

matu-ration of A anguilla Ghosh & Thomas (1995) showed for

could bind to vitellogenin and was effectively incorporated

in the ovaries For As, Boyle et al (2008) found a decrease

in egg production and hatchability of Danio rerio fed rally contaminated Nereis diversicolor Total As levels can

natu-be quite high in fishmeals and fish oils used in aquaculturefeeds (Sloth et al 2005) These authors claimed that this is

of little concern, because less than c 1% of the As is in thetoxic inorganic form However, Celino et al (2009) showed

inorganic As(V) inhibited matogenesis in A japonica, although it should be notedthat they worked in an in vitro testicular organ culture sys-tem with water exposure, which is often more critical thandietary element exposure The mode of action was throughinhibition of synthesis of 11-KT, which androgen also plays

sper-a centrsper-al role in silvering (previtellogenesis) sper-and in genesis in female eels (Rohr et al 2001; Matsubara et al.2003a; Lokman et al 2007; Divers et al 2010; Endo et al.2011) Further, Davey et al (2007) showed that low levels

vitello-of As disrupted the oestrogen receptor (ER), and therebyvitellogenesis, be it in a bird, Gallus gallus (chicken)

Freshwater eels spawn marine pelagic eggs with an oildroplet (type II) and with a large perivitelline space Thecomparison of the freshwater eels with other marine pelagicspawners revealed that even within this type II group, eeleggs are at the extreme end of the spectrum in terms of eggcomposition and therefore most likely also in embryonicmetabolism Eel eggs contain a large amount of total lipids

In A japonica, a negative correlation between egg totallipid and fertilization, hatching and survival is proposed to

be linked to shortage of neutral lipids The even higherdeposition of lipids in ovaries of A anguilla relative to

in ovary development and egg production in the Europeaneel Deficiency of long-chain PUFA has for some time beenrecognized as important for successful reproduction in fish,and more recently the importance of ARA has been recog-nized The high levels of ARA, indicative of selective incor-poration of ARA in the ovary during maturation of

A japonica, suggest an even more prominent role in lid eels Whether this is also the case for A anguillaremains to be revealed Requirements for essential fattyacids in marine broodstock have been documented butneed to be tested on A Anguilla, especially in the light ofthe reported elongation/desaturation capacity of the fresh-water eels Free amino acids are important for hydration,buoyancy and as important energy source The level andthe profile of these free amino acids in A japonica differdramatically from other marine pelagic spawners Their .

anguil-Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

levels, profile and role in A anguilla eggs still need to be

explored, especially whether they play an important role in

embryonic development Apart from the antioxidants,

vita-min E and C (but not carotenoids), the roles of vitavita-mins

and minerals in maturation, ovulation, fertilization, and

egg and larval quality have not been clearly mapped in eels

and most certainly would need to be examined for the

European eel

The comparison of farmed and wild eels revealed

differ-ences in egg composition, predominantly in the lipid

com-position and fatty acid profile Eggs from wild eels mostly

contain more ARA and less EPA than those of farmed

eels Nutritional intervention through the fatty acid profile

of the broodstock diet does seem feasible (Støttrup et al

2013), but due to the high variability in reproductive

suc-cess, these egg compositional differences could not clearly

be linked to egg and larval quality Another big difference

between farmed and wild A anguilla lies in the

responsive-ness to the maturation protocol of weekly injections for up

to 6 months Again this difference might have some links

to biochemical differences, such as the very high lipid level

of farmed eels, but is probably more related to the

physio-logical state (silvering stage) of the eels Durif et al (2006)

showed that farmed eels never advance beyond stage III

(premigrants), while the best results with maturation were

invariably achieved with wild eels in stage V (advanced

migrants) Next to the already routinely applied seawater

adaptation (Kagawa et al 1998), a lowered temperature

regime before (Sudo et al 2011b) and in the initial stage of

induced maturation (P
erez et al 2011) holds the most

promise to improve the silvering stage and the

responsive-ness to induced maturation of eels

The information reviewed here is important for

develop-ing optimal broodstock diets for the European eel to

improve the quality of broodstock under farming

condi-tions to enable the procurement of viable eggs and larvae,

once adequate protocols for induced maturation have been

developed

This work is part of the FP7 project PRO-EEL supported

by the EC (GA: 245257) www.pro-eel.eu

Ahlstrom, E.H & Moser, H.G (1980) Characters useful in

identification of pelagic marine fish eggs CalCOFI Rep 21,

121 –131.

Akiyama, T., Shiraishi, M., Yamamoto, T & Unuma, T (1996) Effect of dietary tryptophan on maturation of Ayu Plecoglossus altivelis Fish Sci., 62, 776–782.

Albrektsen, S., Waagbø, R., Lie, Ø & Sandnes, K (1994) tents and organ distribution of vitamin B6 in Atlantic salmon (Salmo solar) and turbot (Psetta maxima) during the reproduc- tive cycle Comp Biochem Physiol A Physiol., 109, 705–712.

Con-Almansa, E., Perez, M.J., Cejas, J.R., Badia, P., Villamandos, J.E.

& Lorenzo, A (1999) Influence of broodstock gilthead seabream (Sparus aurata L.) dietary fatty acids on egg quality and egg fatty acid composition throughout the spawning season Aqua- culture, 170, 323–336.

Almansa, E., Mart
ıan, M.V., Cejas, J.R., Bad
ı, P., Jerez, S &

Lorenzo, A (2001) Lipid and fatty acid composition of female gilthead seabream during their reproductive cycle: effects of a diet lacking n-3 HUFA J Fish Biol., 59, 267–286.

Alsop, D., Brown, S & Van Der Kraak, G (2007) The effects of copper and benzo[a]pyrene on retinoids and reproduction in ze- brafish Aquat Toxicol., 82, 281–295.

Amin, E.M (1991) Estimation of energy budget for gonadal opment, migration and spawning of eels (Anguilla anguilla L.) inhabiting the Egyptian lagoons Arab Gulf J Sci Res., 9, 129–

cop-Aoyama, J (2009) Life history and evolution of migration in Catadromous Eels (Genus Anguilla) Aqua-BioSci Monogr, 2, 1–

42.

Asturiano, J.F., Sorbera, L.A., Zanuy, S & Carrillo, M (2000) Effects of polyunsaturated fatty acids and gonadotropin on pros- taglandin series E production in a primary testis cell culture sys- tem for the European sea bass J Fish Biol., 57, 1563–1574.

Asturiano, J.F., Sorbera, L.A., Carrillo, M., Zanuy, S., Ramos, J., Navarro, J.C & Bromage, N (2001) Reproductive performance

in male European sea bass (Dicentrarchus labrax L.) fed two PUFA-enriched experimental diets: a comparison with males fed

a wet diet Aquaculture, 194, 173–190.

Baskin, D.G., Figlewicz Lattemann, D., Seeley, R.J., Woods, S.C., Porte, D Jr & Schwartz, M.W (1999) Insulin and leptin: dual adiposity signals to the brain for the regulation of food intake and body weight Brain Res., 848, 114–123.

Bell, J.G & Sargent, J.R (2003) Arachidonic acid in aquaculture feeds: current status and future opportunities Aquaculture, 218,

491 –499.

Bell, M.V., Dick, J.R., Thrush, M & Navarro, J.C (1996) Decreased 20:4n-6/20:5n-3 ratio in sperm from cultured sea bass, Dicentrarchus labrax, broodstock compared with wild fish Aqua- culture, 144, 189–199.

Bell, J.G., Farndale, B.M., Bruce, M.P., Navas, J.M & Carillo,

M (1997) Effects of broodstock dietary lipid on fatty acid positions of eggs from sea bass (Dicentrarchus labrax) Aquacul- ture, 149, 107–119.

com-Bentley, C.D., Watanabe, W.O., Rezek, T.C & Seaton, P.J (2009) Preliminary investigations on the effects of dietary lipid on the

.

spawning performance and egg quality of black sea bass

Centro-pristis striata L Aquacult Res., 40, 1873–1883.

Bezdenezhnykh, V.A & Prokhorchik, G.A (1984) Period of

oocyte maturation and assessment of egg quality of Eel, Anguilla

anguilla (Anguillidae), under Stimulation of Maturation with

Gonadotropic Hormones J Ichthyol., 24, 50–57.

Blom, J.H & Dabrowski, K (1995) Reproductive success of

female rainbow trout (Oncorhynchus mykiss) in response to

graded dietary ascorbyl monophosphate levels Biol Reprod., 52,

1073 –1080.

Blom, J.H & Dabrowski, K (1996) Ascorbic acid metabolism in

fish: is there a maternal effect on the progeny? Aquaculture, 147,

215 –224.

Bo €etius, I & Bo€etius, J (1967) Studies in the European eel

Angu-illa anguAngu-illa (L.) Experimental induction of the male sexual

cycle, its relation to temperature and other factors Meddr.

Danm Fiskeri- og Havunders., 4, 339–405.

Bo€etius, I & Bo€etius, J (1980) Experimental maturation of female

silver eels, Anguilla anguilla Estimates of fecundity and energy

reserves for migration and spawning Dana, 1, 1–28.

Bo€etius, I & Bo€etius, J (1985) Lipid and protein content in

Angu-illa anguAngu-illa during growth and starvation Dana, 4, 1–17.

Bo €etius, I & Bo€etius, J (1989) Ascending elvers, Anguilla anguilla,

from five European localities Analyses of pigmentation stages,

condition, chemical composition and energy reserves Dana, 7,

1 –12.

Bo €etius, I., Bo€etius, J & Hansen, H.J.M (1991) Studies on lipid

synthesis by incorporation of 14 C-acetate during experimental

maturation of silver eels, Anguilla anguilla Dana, 9, 1–14.

Boyle, D., Brix, K.V., Amlund, H., Lundebye, A.-K., Hogstrand,

C & Bury, N.R (2008) Natural arsenic contaminated diets

per-turb reproduction in fish Environ Sci Technol., 42, 5354–5360.

Brooks, S., Tyler, C.R & Sumpter, J.P (1997) Egg quality in fish:

what makes a good egg? Rev Fish Biol Fisheries, 7, 387–416.

Brown, M.R., Battaglene, S.C., Morehead, D.T & Brock, M.

(2005) Ontogenetic changes in amino acid and vitamins during

early larval stages of striped trumpeter (Latris lineata)

Aquacul-ture, 248, 263–274.

Bruce, M.P., Shields, R.J., Bell, M.V & Bromage, N.R (1993)

Lipid class and fatty acid composition of eggs of Atlantic

hali-but, Hippoglossus hippoglossus (L.), in relation to egg quality in

captive broodstock Aquacult Res., 24, 417–422.

Bruce, M., Oyen, F., Bell, G., Asturiano, J.F., Farndale, B.,

Carril-lo, M., Zanuy, S., Ramos, J & Bromage, N (1999)

Develop-ment of broodstock diets for the European Sea Bass

(Dicentrarchus labrax) with special emphasis on the importance

of n-3 and n-6 highly unsaturated fatty acid to reproductive

per-formance Aquaculture, 177, 85–97.

Burgerhout, E., Brittijn, S.A., Palstra, A.P & Van den Thillart,

G.E.E.J.M (2010) Swimming trials with male silver eels

indicat-ing a higher efficiency by swimmindicat-ing in groups In: Proceedindicat-ings of

the FitFish-Workshop on the Swimming Physiology of Fish.

(Palstra, A.P & Planas, J.V eds), pp 40 2 –3 July 2010

Barce-lona, Spain.

Caprio, M., Fabbrini, E., Isidori, A.M., Aversa, A & Fabbri, A.

(2001) Leptin in reproduction Trends Endocrinol Metab., 12,

65 –72.

Cejas, J.R., Almansa, E., Villamandos, J.E., Bad
ıa, P., Bola~nos, A.

& Lorenzo, A (2003) Lipid and fatty acid composition of

ova-ries from wild fish and ovaova-ries and eggs from captive fish of

white sea bream (Diplodus sargus) Aquaculture, 216, 299–313.

Celino, F.T., Yamaguchi, S., Miura, C & Miura, T (2009)

Arsenic inhibits in vitro spermatogenesis and induces germ cell

apoptosis in Japanese eel (Anguilla japonica) Reproduction, 138,

279 –287.

Cerda, J., Carrillo, M., Zanuy, S., Ramos, J & De La Higuera,

M (1994a) Influence of nutritional composition of diet on sea bass, Dicentrarchus labrax L., reproductive performance and egg and larval quality Aquaculture, 128, 345–361.

Cerd a, J., Carrillo, M., Zanuy, S., Ramos, J & de la Higuera, M (1994b) Influence of nutritional composition of diet on sea bass, Dicentrarchus labrax L., reproductive performance and egg and larval quality Aquaculture, 128, 345–361.

Cerd a, J., Fabra, M., Rald
ua, D (2007) Physiological and lar basis of fish oocyte hydration In: The Fish Oocyte From Basic Studies to Biotechnical Applications (Babin, P., Cerd a, J.

molecu-& Lubzens, E eds), pp 349 –396 Springer, The Netherlands Chatelier, A., McKenzie, D.J., Prinet, A., Galois, R., Robin, J., Zambonino, J & Claireaux, G (2006) Associations between tis- sue fatty acid composition and physiological traits of perfor- mance and metabolism in the seabass (Dicentrarchus labrax).

J Exp Biol., 209, 3429–3439.

Chaudhuri, H (1960) Experiments on induced spawning of Indian carps with pituitary extracts Ind J Fish., 7, 20–48.

Cheung, L.A (1983) Studies on endocrine and metabolic changes

in the Japanese eel (Anguilla japonica) following induced sexual maturation, pp 340 PhD thesis, University of Hong Kong Chou, Y & Chien, Y (2006) Effects of astaxanthin and vitamin E supplement in Japanese sea bass (Lateolabrax japonicus) brood- stock diet on their spawning performance and egg quality.

J Fish Soc Taiwan, 33, 157–169.

Chou, Y., Liou, C., Lin, S & Wu, C (1993) Effects of highly unsaturated fatty acids in broodstock diets on spawning and egg quality of black porgy (Acanthopagrus schlegeli) J Fish Soc Taiwan, 20, 167–176.

Chow, S., Kurogi, H., Katayama, S et al (2010) Japanese eel Anguilla japonica do not assimilate nutrition during the oceanic spawning migration: evidence from stable isotope analysis Mar Ecol Prog Ser., 402, 233–238.

Clevestam, P.D., Ogonowski, M., Sj€oberg, N.B & Wickstr€om, H (2011) Too short to spawn? Implications of small body size and swimming distance on successful migration and maturation of the European eel Anguilla anguilla J Fish Biol., 78, 1073–1089 Corraze, G., Støttrup, J., Holst, L.K., Larroquet, L., Jacobsen, C., Tybjerg, L., Kaushik, S & Tomkiewicz, J (2011) Modulation of stored lipid reserves through broodstock nutrition and reproduc- tive success in European eel (Anguilla anguilla) In: Aquaculture Europe 11 18 –21 October, 2011 Rhodes, Greece.

Cottrill, R.A., McKinley, R.S., Van Der Kaak, G., Dutil, J.D., Reid, K.B & McGrath, K.J (2001) Plasma non-esterified fatty acid profiles and 17 b-oestradiol levels of juvenile immature and maturing adult American eels in the St Lawrence River J Fish Biol., 59, 364–379.

Cowey, C.B., Daisley, K.W & Parry, G (1962) Study of amino acids, free or as components of protein, and of some B vitamins

in the tissues of the atlantic salmon, Salmo salar, during ing migration Comp Biochem Physiol., 7, 29–38.

spawn-Craik, J.C.A (1986) Levels of calcium and iron in the ovaries and eggs of cod Gadus morhua L and plaice Pleuronectes platessa L Comp Biochem Physiol A Physiol., 83, 515–517.

Craik, J.C.A & Harvey, S.M (1984) Biochemical changes ring during final maturation of eggs of some marine and fresh- water teleosts J Fish Biol., 24, 599–610.

occur-Cruzado, I.H., Herrera, M., Quintana, D., Rodiles, A., Navas, J.I., Lorenzo, A & Almansa, E (2011) Total lipid and fatty acid composition of brill eggs Scophthalmus rhombus L relationship

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

between lipid composition and egg quality Aquacult Res., 42,

1011 –1025.

Czesny, S., Dettmers, J.M., Rinchard, J & Dabrowski, K (2009)

Linking egg thiamine and fatty acid concentrations of lake

Michigan lake trout with early life stage mortality J Aquat.

Anim Health, 21, 262–271.

Dabrowski, K & Ciereszko, A (2001) Ascorbic acid and

repro-duction in fish: endocrine regulation and gamete quality

Aqua-cult Res., 32, 623–638.

Dave, G., Johansson-Sj €obeck, M.-L., Larsson,  A., Lewander, K &

Lidman, U (1975) Metabolic and hematological effects of

star-vation in the European eel, Anguilla anguilla L –I Carbohydrate,

lipid, protein and inorganic ion metabolism Comp Biochem.

Physiol A Physiol., 52, 423–430.

Davey, J.C., Bodwell, J.E., Gosse, J.A & Hamilton, J.W (2007)

Arsenic as an endocrine disruptor: effects of arsenic on estrogen

receptor –mediated gene expression in vivo and in cell culture.

Toxicol Sci., 98, 75–86.

Dayal, J., Ahamad Ali, S., Thirunavukkarasu, A.R., Kailasam, M.

& Subburaj, R (2003) Nutrient and amino acid profiles of egg

and larvae of Asian seabass, Lates calcarifer (Bloch) Fish

Phys-iol Biochem., 29, 141–147.

De Silva, S.S., Gunasekera, R.M & Collins, R.O (2002) Some

morphometric and biochemical features of ready-to-migrate

sil-ver and pre-migratory yellow stages of the shortfin eel of

south-eastern Australian waters J Fish Biol., 61, 915–928.

Devauchelle, N & Coves, D (1988) The characteristics of sea bass

(Dicentrarchus labrax) eggs: description, biochemical

composi-tion and hatching performances Aquat Living Resour., 1,

223 –230.

Divakaran, S & Ostrowski, A.C (1989) Fatty acid analysis of fish

eggs without solvent extraction Aquaculture, 80, 371–375.

Divers, S.L., McQuillan, H.J., Matsubara, H., Todo, T &

Lok-man, P.M (2010) Effects of reproductive stage and

11-ketotes-tosterone on LPL mRNA levels in the ovary of the shortfinned

eel J Lipid Res., 51, 3250–3258.

Durif, C.M.F & Elie, P (2008) Predicting downstream migration

of silver eels in a large river catchment based on commercial

fishery data Fish Manage Ecol., 15, 127–137.

Durif, C.M.F., Dufour, S & Elie, P (2006) Impact of silvering

stage, age, body size and condition on reproductive potential of

the European eel Marine Ecology-Progress Series, 327, 171–181.

Edel, R.K (1975) The induction of maturation of female American

eels through hormone injections Helgol Mar Res., 27, 131–138.

Emata, A.C., Ogata, H.Y., Garibay, E.S & Furuita, H (2003)

Advanced broodstock diets for the mangrove red snapper and a

potential importance of arachidonic acid in eggs and fry Fish

Physiol Biochem., 28, 489–491.

Endo, T., Todo, T., Lokman, P.M., Kudo, H., Ijiri, S., Adachi, S.

& Yamauchi, K (2011) Androgens and very low density

lipopro-tein are essential for the growth of previtellogenic oocytes from

Japanese eel, Anguilla japonica, in vitro Biol Reprod., 84, 816–

825.

Evans, R.P., Parrish, C.C., Brown, J.A & Davis, P.J (1996)

Bio-chemical composition of eggs from repeat and first-time

spawn-ing captive Atlantic halibut (Hippoglossus hippoglossus).

Aquaculture, 139, 139–149.

Falk-Petersen, S., Falk-Petersen, I.-B., Sargent, J.R & Haug, T.

(1986) Lipid class and fatty acid composition of eggs from the

Atlantic halibut (Hippoglossus hippoglossus) Aquaculture, 52,

207 –211.

Falk-Petersen, S., Sargent, J.R., Fox, C., Falk-Petersen, I.B.,

Haug, T & Kjørsvik, E (1989) Lipids in atlantic halibut

(Hip-poglossus hip(Hip-poglossus) eggs from planktonic samples in ern Norway Mar Biol., 101, 553–556.

North-Faulk, C.K & Holt, G.J (2003) Lipid nutrition and feeding of cobia Rachycentron canadum Larvae J World Aquacult Soc., 34,

Fern
andez-Palacios, H., Izquierdo, M., Robaina, L., Valencia, A., Salhi, M & Montero, D (1997) The effect of dietary protein and lipid from squid and fish meals on egg quality of broodstock for gilthead seabream (Sparus aurata) Aquaculture, 148, 233–

246.

Fern
andez-Palacios, H., Norberg, B., Izquierdo, M & Hamre, K.

(2011) Effects of broodstock diet on eggs and larvae In: Larval Fish Nutrition (Holt, G.J ed), pp 151 –181 Wiley-Blackwell, Oxford.

Finn, R.N & Fyhn, H.J (2010) Requirement for amino acids in ontogeny of fish Aquacult Res., 41, 684–716.

Finn, R.N., Fyhn, H.J & Evjen, M.S (1995a) Physiological getics of developing embryos and yolk-sac larvae of Atlantic cod (Gadus morhua) I Respiration and nitrogen metabolism Mar.

ener-Biol., 124, 355–369.

Finn, R.N., Henderson, J.R & Fyhn, H.J (1995b) Physiological energetics of developing embryos and yolk-sac larvae of Atlantic cod (Gadus morhua) II Lipid metabolism and enthalpy balance.

Mar Biol., 124, 371–379.

Finn, R.N., Widdows, J & Fyhn, H.J (1995c) Calorespirometry

of developing embryos and yolk-sac larvae of turbot mus maximus) Mar Biol., 122, 157–163.

(Scophthal-Fisher, J.P., Fitzsimons, J.D., Combs, G.F & Spitsbergen, J.M.

(1996) Naturally occurring thiamine deficiency causing tive failure in Finger Lakes Atlantic Salmon and Great Lakes Lake Trout Trans Am Fish Soc., 125, 167–178.

reproduc-Fontagn
e-Dicharry, S., Lataillade, E., Surget, A., Breque, J., bonino-Infante, J.-L & Kaushik, S.J (2010) Effects of dietary vitamin A on broodstock performance, egg quality, early growth and retinoid nuclear receptor expression in rainbow trout (On- corhynchus mykiss) Aquaculture, 303, 40–49.

Zam-Fraser, A.J., Gamble, J.C & Sargent, J.R (1988) Changes in lipid content, lipid class composition and fatty acid composition of developing eggs and unfed larvae of cod (Gadus morhua) Mar.

Takeu-398.

Furuita, H., Tanaka, H., Yamamoto, T., Shiraishi, M & chi, T (2001) Effects of high dose of vitamin A on reproduction and egg quality of Japanese flounder Paralichthys olivaceus Fish.

Takeu-Sci., 67, 606–613.

Furuita, H., Tanaka, H., Yamamoto, T., Suzuki, N & Takeuchi,

T (2002) Effects of high levels of n-3 HUFA in broodstock diet

on egg quality and egg fatty acid composition of Japanese der, Paralichthys olivaceus Aquaculture, 210, 323–333.

floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun- floun-.

Furuita, H., Ohta, H., Unuma, T., Tanaka, H., Kagawa, H.,

Su-zuki, N & Yamamoto, T (2003a) Biochemical composition of

eggs in relation to egg quality in the Japanese eel, Anguilla

japonica Fish Physiol Biochem., 29, 37–46.

Furuita, H., Tanaka, H., Yamamoto, T., Suzuki, N & Takeuchi,

T (2003b) Supplemental effect of vitamin A in diet on the

repro-ductive performance and egg quality of the Japanese flounder

Paralichthys olivaceus (T & S) Aquacult Res., 34, 461–468.

Furuita, H., Yamamoto, T., Shima, T., Suzuki, N & Takeuchi, T.

(2003c) Effect of arachidonic acid levels in broodstock diet on

larval and egg quality of Japanese flounder Paralichthys

olivac-eus Aquaculture, 220, 725–735.

Furuita, H., Unuma, T., Nomura, K., Tanaka, H., Okuzawa, K.,

Sugita, T & Yamamoto, T (2006) Lipid and fatty acid

composi-tion of eggs producing larvae with high survival rate in the

Japa-nese eel J Fish Biol., 69, 1178–1189.

Furuita, H., Hori, K., Suzuki, T., Sugita, T & Yamamoto, T.

(2007) Effect of n-3 and n-6 fatty acids in broodstock diet on

reproduction and fatty acid composition of broodstock and eggs

in the Japanese eel Anguilla japonica Aquaculture, 267, 55–61.

Furuita, H., Ishida, T., Suzuki, T., Unuma, T., Kurokawa, T.,

Su-gita, T & Yamamoto, T (2009a) Vitamin content and quality

of eggs produced by broodstock injected with vitamins C and E

during artificial maturation in Japanese eel Anguilla japonica.

Aquaculture, 289, 334–339.

Furuita, H., Unuma, T., Nomura, K., Tanaka, H., Sugita, T &

Yamamoto, T (2009b) Vitamin contents of eggs that produce

larvae showing a high survival rate in the Japanese eel Anguilla

japonica Aquacult Res., 40, 1270–1278.

Garcia-Gallego, M & Akharbach, H (1998) Evolution of body

composition of European eels during their growth phase in a fish

farm, with special emphasis on the lipid component Aquacult.

Int., 6, 345–356.

Ghosh, P & Thomas, P (1995) Binding of metals to red drum

vitellogenin and incorporation into oocytes Marine

Environmen-tal Research, 39, 165–168.

Gim
enez, G., Est
evez, A., Lahnsteiner, F., Zecevic, B., Bell, J.G.,

Henderson, R.J., Pi~nera, J.A & Sanchez-Prado, J.A (2006) Egg

quality criteria in common dentex (Dentex dentex) Aquaculture,

260, 232–243.

Gim
enez, G., Est
evez, A., Henderson, R.J & Bell, J.G (2008)

Changes in lipid content, fatty acid composition and lipid class

composition of eggs and developing larvae (0 –40 days old) of

cultured common dentex (Dentex dentex Linnaeus 1758).

Aquacult Nutr., 14, 300–308.

van Ginneken, V.J.T & Maes, G.E (2005) The european eel

(Angu-illa angu(Angu-illa, Linnaeus), its lifecycle, evolution and reproduction: a

literature review Rev Fish Biol Fisheries, 15, 367–398.

Gonzalez-Vecino, J.L., Cutts, C.J., Batty, R.S., Greenhaff, P.L &

Wadsworth, S (2004) The effects of nucleotide enriched

brood-stock diet on first feeding success and survival of haddock

(Mel-anogrammus aeglefinus L.) larvae In: XI International

Symposium on Nutrition and Feeding in Fish, pp 268 May 2 –

7, 2004 Phuket Island, Thailand.

Haga, Y., Du, S.-J., Satoh, S., Kotani, T., Fushimi, H &

Takeu-chi, T (2011) Analysis of the mechanism of skeletal deformity in

fish larvae using a vitamin A-induced bone deformity model.

Aquaculture, 315, 26–33.

Hamre, K (2011) Metabolism, interactions, requirements and

functions of vitamin E in fish Aquacult Nutr., 17, 98–115.

Hamre, K., Waagbø, R., Berge, R.K & Lie, Ø (1997) Vitamins C

and E interact in juvenile Atlantic salmon (Salmo salar, L.) Free

Radical Biol Med., 22, 137–149.

Hamre, K., Krossøy, C., Lock, E.J & Moren, M (2010) Roles of lipid-soluble vitamins during ontogeny of marine fish larvae Aquac Res., 41, 745–750.

Han, Y.S., Tzeng, W.N., Huang, Y.S & Liao, I.C (2000) The vering of the Japanese eel Anguilla japonica: season, age, size and fat J Taiwan Fish Res., 8, 37–45.

sil-Han, Y.S., Huang, Y.S., Liao, I.C & Tzeng, W.N (2001) Age and energy store of the silvering Japanese eel in the Kaoping River

of Southwestern Taiwan In: Asian Fisheries Society, 6th Asian Fisheries Forum, Book of Abstracts, pp 89 Nov 25–30, Kaoh- siung, Taiwan.

Hara, A., Yamauchi, K & Hirai, H (1980) Studies on cific serum protein (vitellogenin) and egg yolk protein in Japa- nese eel (Anguilla japonica) Studies on female-specific serum p Comp Biochem Physiol B, 65, 315–320.

female-spe-Harel, M., Tandler, A., Kissil, G.W & Applebaum, S.W (1995) The role of broodstock dietary protein in vitellogenin synthesis and oocyte development, and its effects on reproductive perfor- mance and egg quality in gilthead seabream Sparus aurata In: Proceedings of the Fifth International Symposium on the Repro- ductive Physiology of Fish (Goetz, F & Thomas, P eds), pp.

105 –107 FishSymp 95, The University of Texas at Austin Hastey, R., Phelps, R., Davis, D & Cummins, K (2010) Changes

in free amino acid profile of red snapper Lutjanus campechanus, eggs, and developing larvae Fish Physiol Biochem., 36, 473–481 Hellou, J., Warren, W.G., Payne, J.F., Belkhode, S & Lobel, P (1992) Heavy metals and other elements in three tissues of cod, Gadus morhua from the Northwest Atlantic Mar Pollut Bull.,

24, 452–458.

Higuchi, M., Celino, F., Tamai, A., Miura, C & Miura, T (2012) The synthesis and role of taurine in the Japanese eel testis Amino Acids, 43, 773–781.

Hilton, Z., Poortenaar, C & Sewell, M (2008) Lipid and protein utilisation during early development of yellowtail kingfish (Seri- ola lalandi) Mar Biol., 154, 855–865.

Hopkirk, G., Wills, R & Townsend, P (1975) Seasonal variations

in lipid content of eels (Anguilla australis) Mar Freshw Res.,

26, 271–273.

Izquierdo, M & Koven, W (2011) Lipids In: Larval Fish tion (Holt, G.J ed), pp 47 –82 Wiley-Blackwell, Oxford Izquierdo, M.S., Fern
andez-Palacios, H & Tacon, A.G.J (2001) Effect of broodstock nutrition on reproductive performance of fish Aquaculture, 197, 25–42.

Nutri-Joh, Y.-G., Brechany, E & Christie, W (1995) Characterization of wax esters in the roe oil of amber fish, Seriola aureovittata.

J Am Oil Chem Soc., 72, 707–713.

Johnson, R.B (2009) Lipid deposition in oocytes of teleost fish during secondary oocyte growth Rev Fish Sci., 17, 78–89 Johnston, T.A., Wiegand, M.D., Leggett, W.C., Pronyk, R.J., Dyal, S.D., Watchorn, K.E., Kollar, S & Casselman, J.M (2007) Hatching success of walleye embryos in relation to mater- nal and ova characteristics Ecol Freshw Fish, 16, 295–306 Kagawa, H., Iinuma, N., Tanaka, H., Ohta, H & Okuzawa, K (1998) Effects of rearing period in seawater on induced matura- tion in female Japanese eel Anguilla japonica Fish Sci., 64,

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

Kagawa, H., Horiuchi, Y., Kasuga, Y & Kishi, T (2009) Oocyte

hydration in the Japanese eel (Anguilla japonica) during meiosis

resumption and ovulation J Exp Zool A Ecol Genet Physiol.,

311A, 752–762.

Kamstra, A & Van Heeswijk, J.C.F (1996) The sex ratio and fat

content in a cohort of eels reared under semi-commercial

condi-tions In: EIFAC Working Party on Eel, pp 8 23 –27 September

1996 IJmuiden, The Netherlands.

Kissil, G.W., Youngson, A & Cowey, C.B (1987) Capacity of the

European eel (Anguilla anguilla) to elongate and desaturate

die-tary linoleic acid J Nutr., 117, 1379–1384.

Kn €osche, R (2009) Aalr€uckgang – Schl€usselfaktor Fettgehalt?

Fisch Teichwirt, 8, 284–287.

Kokhnenko, S.V., Bezdenezhnykh, V.A & Gorovaya, S.L (1977)

Maturation of the European eel (Anguilla anguilla) when

artifi-cially reared J Ichthyol., 17, 878–882.

Komatsu, M., Matsumoto, W & Hayashi, S (1996) Protease

activity appeared after trypsin treatment of the purified

vitelloge-nin from eel Anguilla japonica Comp Biochem Physiol B:

Bio-chem Mol Biol., 113, 561–571.

Kreutzmann, H.-L & Lehmitz, R (1976) Influence of vitamin B1

deficiency on some physiological and morphological blood

char-acteristics of Anguilla anguilla Archiv f €ur Tierern€ahrung, 26,

293 –303.

Krossøy, C., Waagbø, R & Ørnsrud, R (2011) Vitamin K in fish

nutrition Aquacult Nutr., 17, 585–594.

Lahnsteiner, F & Patarnello, P (2004) Egg quality determination

in the gilthead seabream, Sparus aurata, with biochemical

parameters Aquaculture, 237, 443–459.

Lanes, C.F.C., Bizuayehu, T.T., Bolla, S., Martins, C., de Oliveira

Fernandes, J.M., Bianchini, A., Kiron, V & Babiak, I (2012)

Biochemical composition and performance of Atlantic cod

(Ga-dus morhua L.) eggs and larvae obtained from farmed and wild

broodstocks Aquaculture, 324–325, 267–275.

Larsson,  A & Lewander, K (1973) Metabolic effects of starvation

in the eel, Anguilla anguilla L Comp Biochem Physiol A

Phys-iol., 44, 367–374.

Larsson, P., Hamrin, S & Okla, L (1990) Fat content as a factor

inducing migratory behavior in the eel (Anguilla anguilla L.) to

the Sargasso Sea Naturwissenschaften, 77, 488–490.

Lau, E.Y.L (1987) Changes in intermediary metabolism of the eel,

Anguilla japonica (Temminck & Schlegel) during artificial

induc-tion of sexual maturainduc-tion, pp 393 PhD thesis, University of

Hong Kong.

Lavens, P., Lebegue, E., Jaunet, H., Brunel, A., Dhert, P &

Sorge-loos, P (1999) Effect of dietary essential fatty acids and vitamins

on egg quality in turbot broodstocks Aquacult Int., 7, 225–240.

Le, D.Q., Chino, N., Shirai, K & Arai, T (2010) Trace metals in

Japanese eel Anguilla japonica in relation to ecological migratory

types and growth stages Estuar Coast Shelf Sci., 87, 405–410.

Li, Y.-Y., Chen, W.-Z., Sun, Z.-W., Chen, J.-H & Wu, K.-G.

(2005) Effects of n-3 HUFA content in broodstock diet on

spawning performance and fatty acid composition of eggs and

larvae in Plectorhynchus cinctus Aquaculture, 245, 263–272.

Lie, Ø., Sandvin, A & Waagbø, R (1994) Transport of

alpha-tocopherol in Atlantic salmon (Salmo salar) during

vitellogene-sis Fish Physiol Biochem., 13, 241–247.

Liu, L., Li, R., Zhang, J.-M., Guo, F & Zhang, T (2009)

Compo-sition and metabolizing of lipids and fatty acids in muscle of

female Anguilla japonica during the period of artificial inducing

ovarian maturation Acta Hydrobiol Sin., 33, 1011–1019.

Lock, E.J., Waagbø, R., Bonga, S.W & Flik, G (2010) The

signifi-cance of vitamin D for fish: a review Aquacult Nutr., 16, 100–116.

Lokman, P.M & Young, G (2000) Induced spawning and early ontogeny of New Zealand freshwater eels (Anguilla dief- fenbachii and A australis) NZ J Mar Freshwat Res., 34,

135 –145.

Lokman, P.M., Vermeulen, G.J., Lambert, J.G.D & Young, G.

(1998) Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachii and A australis) before and at the onset of the natural spawning migration I.

Females * Fish Physiol Biochem., 19, 325–338.

Lokman, P.M., George, K.A.N., Divers, S.L., Algie, M & Young,

G (2007) 11-Ketotestosterone and IGF-1 increase the size of previtellogenic oocytes from shortfinned eel, Anguilla australis, in vitro Reproduction, 133, 955–967.

Lopez, E., Mac Intyre, I., Martelly, E., Lallier, F & Vidal, B.

(1980) Paradoxical effect of 1.25 dihydroxycholecalciferol on osteoblastic and osteoclastic activity in the skeleton of the eel Anguilla anguilla L Calcif Tissue Int., 32, 83–87.

Lubzens, E., Young, G., Bobe, J & Cerda, J (2010) Oogenesis in teleosts: How fish eggs are formed Gen Comp Endocrinol., 165,

367 –389.

Lund, I., Steenfeldt, S.J., Suhr, K.I & Hansen, B.W (2008) A comparison of fatty acid composition and quality aspects of eggs and larvae from cultured and wild broodstock of common sole (Solea solea L.) Aquacult Nutr., 14, 544–555.

Luquet, P & Watanabe, T (1986) Interaction tion” in fish Fish Physiol Biochem., 2, 121–129.

“nutrition-reproduc-Mæland, A., Rønnestad, I & Waagbø, R (2003) Folate in eggs and developing larvae of Atlantic halibut, Hippoglossus hippog- lossus L Aquacult Nutr., 9, 185–188.

Mangor-Jensen, A., Holm, J.C., Rosenlund, G., Lie, Ø & nes, K (1994) Effects of dietary vitamin C on maturation and egg quality of Cod Gadus morhua L J World Aquaculture Soc.,

Sand-25, 30–40.

Matsubara, M., Lokman, P.M., Senaha, A et al (2003a) Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.) Fish Physiol Biochem., 28, 353–354.

Matsubara, T., Nagae, M., Ohkubo, N., Andoh, T., Sawaguchi, S., Hiramatsu, N., Sullivan, C.V & Hara, A (2003b) Multiple vitellogenins and their unique roles in marine teleosts Fish Phys- iol Biochem., 28, 295–299.

Matsubara, T., Andoh, T., Ohkubo, N & Sawaguchi, S (2006) Biochemical characteristics of yolk proteins in Japanese eel – involvement of selective yolk proteolysis in modulation of egg buoyancy Bull Fish Res Agency, (Suppl 5), 45 –50.

Matsunari, H., Hamada, K., Mushiake, K & Takeuchi, T (2006) Effects of taurine levels in broodstock diet on reproductive per- formance of yellowtail Seriola quinqueradiata Fish Sci., 72,

955 –960.

Mazorra, C., Bruce, M., Bell, J.G., Davie, A., Alorend, E., Jordan, N., Rees, J., Papanikos, N., Porter, M & Bromage, N (2003) Dietary lipid enhancement of broodstock reproductive perfor- mance and egg and larval quality in Atlantic halibut (Hippoglos- sus hippoglossus) Aquaculture, 227, 21–33.

Mazzeo, I., Gallego, V., P
erez, L., Pe~naranda, D.S., Jover, M &

Asturiano, J.F (2010) Variations in fatty acids composition in different tissues of the European eel (Anguilla anguilla L.) males during induced sexual maturation J Appl Ichthyol., 26,

763 –774.

Mazzeo, I., Baeza, R., Gallego, V., V
ılchez, M.C., P
erez, L & turiano, J.F (2011) Study of thermal regime effect on fatty acid mobilization in European eel (Anguilla anguilla, L.) females dur- ing induced sexual maturation In: Aquaculture Europe 11 18 –

As-21 October 2011 Rhodes, Greece.

.

McEvoy, L., Holland, D & McEvoy, J (1993) Effect of spawning

fast on lipid and fatty acid composition of eggs of captive turbot

(Scophthalmus maximus L.) Aquaculture, 114, 131–139.

McKenzie, D.J., Higgs, D.A., Dosanjh, B.S., Deacon, G &

Ran-dall, D.J (1998) Dietary fatty acid composition influences

swim-ming performance in Atlantic salmon (Salmo salar) in seawater.

Fish Physiol Biochem., 19, 111–122.

Miller, M.J (2009) Ecology of anguilliform leptocephali:

remark-able transparent fish larvae of the ocean surface layer

Aqua-BioSci Monogr 2, 1–94.

Moran, D., Gara, B & Wells, R.M.G (2007) Energetics and

metabolism of yellowtail kingfish (Seriola lalandi Valenciennes

1833) during embryogenesis Aquaculture, 265, 359–369.

Morehead, D.T., Hart, P.R., Dunstan, G.A., Brown, M &

Pank-hurst, N.W (2001) Differences in egg quality between wild

striped trumpeter (Latris lineata) and captive striped trumpeter

that were fed different diets Aquaculture, 192, 39–53.

Mourente, G & Odriozola, J (1990) Effect of broodstock diets on

lipid classes and their fatty acid composition in eggs of gilthead

sea bream (Sparus aurata L.) Fish Physiol Biochem., 8, 93–101.

Mourente, G & V
azquez, R (1996) Changes in the content of

total lipid, lipid classes and their fatty acids of developing eggs

and unfed larvae of the Senegal sole, Solea senegalensis Kaup.

Fish Physiol Biochem., 15, 221–235.

Mourente, G., Tocher, D.R., Diaz, E., Grau, A & Pastor, E.

(1999) Relationships between antioxidants, antioxidant enzyme

activities and lipid peroxidation products during early

develop-ment in Dentex dentex eggs and larvae Aquaculture, 179, 309–

324.

Navas, J.M., Bruce, M., Thrush, M., Farndale, B.M., Bromage,

N., Zanuy, S., Carrillo, M., Bell, J.G & Ramos, J (1997) The

impact of seasonal alteration in the lipid composition of

brood-stock diets on egg quality in the European sea bass J Fish Biol.,

51, 760–773.

Navas, J.M., Trush, M., Zanuy, S., Ramos, J., Bromage, N &

Ca-rillo, M (2001) Total lipid in the broodstock diet did not affect

fatty acid composition and quality of eggs of sea bass

(Dicen-trarchus labrax L.) Scientia Marina, 65, 11–19.

Nguyen, H.Q., Tran, T.M., Reinertsen, H & Kjørsvik, E (2010)

Effects of dietary essential fatty acid levels on broodstock

spawning performance and egg fatty acid composition of cobia,

Rachycentron canadum J World Aquaculture Soc., 41, 687–699.

Nguyen, H.Q., Reinertsen, H., Rustad, T., Tran, T.M & Kjørsvik,

E (2012) Evaluation of egg quality in broodstock cobia

Rachy-centron canadum L Aquac Res., 43, 371–385.

Nocillado, J.N., Pe ~naflorida, V.D & Borlongan, I.G (2000)

Mea-sures of egg quality in induced spawns of the Asian sea bass,

Lates calcarifer Bloch Fish Physiol Biochem., 22, 1–9.

Norberg, B., Hamre, K., Araujo, P & Rosenlund, G (2009) Effect

of arachidonic acid on prostanoid synthesis and reproductive

physiology in Atlantic cod In: Larvi 2009: 5th Fish & Shellfish

Larviculture Symposium 7 –11 September 2009 Ghent, Belgium.

NRC (2011) Nutrient Requirements of Fish and Shrimp National

Academies Press, Washington.

Ogata, H.Y., Emata, A.C., Garibay, E.S & Furuita, H (2004)

Fatty acid composition of five candidate aquaculture species in

Central Philippines Aquaculture, 236, 361–375.

Ohkubo, N.O & Matsubara, T.M (2002) Sequential utilization of

free amino acids, yolk proteins and lipids in developing eggs and

yolk-sac larvae of barfin flounder Verasper moseri Mar Biol.,

140, 187–196.

Ohkubo, N., Sawaguchi, S., Hamatsu, T & Matsubara, T (2006)

Utilization of free amino acids, yolk proteins and lipids in

devel-oping eggs and yolk-sac larvae of walleye pollock Theragra cogramma Fish Sci., 72, 620–630.

chal-Ohkubo, N., Sawaguchi, S., Nomura, K., Tanaka, H &

Matsubar-a, T (2008) Utilization of free amino acids, yolk protein and ids in developing eggs and yolk-sac larvae of Japanese eel Anguilla japonica Aquaculture, 282, 130–137.

lip-Olguno glu, I.A & Olgunoglu, M.P (2011) Concentrations of metal contaminants, vitamin and mineral in waxed caviar from Mugil cephalus (L.,1758) Afr J Agric Res., 6, 1041–1046 Oliveira, K & Hable, W.E (2010) Artificial maturation, fertiliza- tion, and early development of the American eel (Anguilla ro- strata) Can J Zool., 88, 1121–1128.

Ostrowski, A.C & Divakaran, S (1989) The amino acid and fatty acid compositions of selected tissues of the dolphin fish (Cory- phaena hippurus) and their nutritional implications Aquaculture,

80, 285–299.

Ozaki, Y., Koga, H., Takahashi, T., Adachi, S & Yamauchi, K (2008) Lipid content and fatty acid composition of muscle, liver, ovary and eggs of captive-reared and wild silver Japanese eel Angu- illa japonica during artificial maturation Fish Sci., 74, 362–371 Palace, V.P & Werner, J (2006) Vitamins A and E in the maternal diet influence egg quality and early life stage development in fish:

a review Scientia Marina, 70, 41–57.

Palacios, E., Santamar
ıa, A., Trevi, L., Morales, C., Dumas, S., Rojas, R & Ibarra-Castro, L (2011) Fatty acid variation in eggs from wild and captive snappers (Lutjanus peru, L guttatus,

L argentiventris) In: Aquaculture America 2011 New Orleans, Louisiana.

Palstra, A., Thillart, G (2009) Artificial maturation and tion of the European eel In: Spawning Migration of the Euro- pean Eel (Thillart, G., Dufour, S & Rankin, J.C eds), Vol 30,

reproduc-pp 309 –331 Springer, The Netherlands.

Palstra, A & van den Thillart, G (2010) Swimming physiology of European silver eels (Anguilla anguilla L.): energetic costs and effects on sexual maturation and reproduction Fish Physiol Bio- chem., 36, 297–322.

Palstra, A.P., Cohen, E.G.H., Niemantsverdriet, P.R.W., van Ginneken, V.J.T & van den Thillart, G.E.E.J.M (2005) Artifi- cial maturation and reproduction of European silver eel: devel- opment of oocytes during final maturation Aquaculture, 249,

533 –547.

Palstra, A., van Ginneken, V., Murk, A & van den Thillart, G (2006) Are dioxin-like contaminants responsible for the eel (Anguilla anguilla) drama? Naturwissenschaften, 93, 145–148 Palstra, A., van Ginneken, V & van den Thillart, G (2008) Cost

of transport and optimal swimming speed in farmed and wild European silver eels (Anguilla anguilla) Comp Biochem Physiol., Part A Mol Integr Physiol., 151, 37–44.

Palstra, A.P., Schnabel, D., Nieveen, M.C., Spaink, H.P & van den Thillart, G.E.E.J.M (2010) Temporal expression of hepatic estrogen receptor 1, vitellogenin1 and vitellogenin2 in European silver eels Gen Comp Endocrinol., 166, 1–11.

Palstra, A., Guerrero, M., de Laak, G., Klein Breteler, J & van den Thillart, G (2011) Temporal progression in migratory status and sexual maturation in European silver eels during down- stream migration Fish Physiol Biochem., 37, 285–296.

Papanikos, N., Phelps, R.P., Davis, D.A., Ferry, A & Maus, D (2008) Spontaneous spawning of captive red snapper, Lutjanus campechanus, and dietary lipid effect on reproductive perfor- mance J World Aquaculture Soc., 39, 324–338.

Pati ~no, R & Sullivan, C (2002) Ovarian follicle growth, tion, and ovulation in teleost fish Fish Physiol Biochem., 26,

matura-57 –70.

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

Pedersen, B.H (2004) Fertilisation of eggs, rate of embryonic

development and hatching following induced maturation of the

European eel Anguilla anguilla Aquaculture, 237, 461–473.

Peleteiro, J.B., Lavens, P., Rodr
ıguez-Ojeja, G & Iglesias, J.

(1995) Relationship between egg quality and fatty acid content

of various turbot broodstocks (Scophthalmus maximus L.) ICES

Mar Sci Symp., 201, 51–56.

Penney, R.W., Lush, P.L., Wade, J., Brown, J.A., Parrish, C.C &

Burton, M.P.M (2006) Comparative utility of egg blastomere

morphology and lipid biochemistry for prediction of hatching

success in Atlantic cod, Gadus morhua L Aquac Res., 37, 272–

283.

P
erez, L., Asturiano, J.F., Tom
as, A., Zegrari, S., Barrera, R.,

Espin
os, F.J., Navarro, J.C & Jover, M (2000) Induction of

maturation and spermiation in the male European eel:

assess-ment of sperm quality throughout treatassess-ment J Fish Biol., 57,

1488 –1504.

P
erez, M.J., Rodr
ıguez, C., Cejas, J.R., Mart
ın, M.V., Jerez, S &

Lorenzo, A (2007) Lipid and fatty acid content in wild white

seabream (Diplodus sargus) broodstock at different stages of the

reproductive cycle Comp Biochem Physiol B: Biochem Mol.

Biol., 146, 187–196.

P
erez, L., Pe~naranda, D.S., Dufour, S., Baloche, S., Palstra, A.P.,

Van Den Thillart, G.E.E.J.M & Asturiano, J.F (2011) Influence

of temperature regime on endocrine parameters and

vitellogene-sis during experimental maturation of European eel (Anguilla

anguilla) females Gen Comp Endocrinol., 174, 51–59.

Peyon, P., Zanuy, S & Carrillo, M (2001) Action of leptin on in

vitro luteinizing hormone release in the European Sea Bass

(Dicentrarchus labrax) Biol Reprod., 65, 1573–1578.

Pickova, J., Dutta, P.C., Larsson, P.O & Kiessling, A (1997)

Early embryonic cleavage pattern, hatching success, and

egg-lipid fatty acid composition: comparison between two cod

(Ga-dus morhua) stocks Can J Fish Aquat Sci., 54, 2410–2416.

Pierron, F., Baudrimont, M., Dufour, S., Elie, P., Bossy, A.l.,

Bal-oche, S., Mesmer-Dudons, N., Gonzalez, P., Bourdineaud, J.-P.

& Massabuau, J.-C (2008) How cadmium could compromise

the completion of the European eel’s reproductive migration.

Environ Sci Technol., 42, 4607–4612.

Prokhorchik, G.A (1987) Postembryonic development of

Euro-pean eel, Anguilla anguilla, under experimental conditions J.

Ichthyol., 26, 121–127.

Rainuzzo, J.R., Reitan, K.I & Jørgensen, L (1992) Comparative

study on the fatty acid and lipid composition of four marine fish

larvae Comp Biochem Physiol B, 103, 21–26.

Rainuzzo, J.R., Reitan, K.I., Jørgensen, L & Olsen, Y (1994)

Lipid composition in turbot larvae fed live feed cultured by

emulsions of different lipid classes Comp Biochem Physiol A

Physiol., 107, 699–710.

Rath, S.C., Gupta, S.D & Dasgupta, S (1999) Non-stripping

induced spawning and double spawning of grass carp in a

hatch-ery system with foliage-free brood diet Vet Arh., 69, 7–15.

Reith, M., Munholland, J., Kelly, J., Finn, R.N & Fyhn, H.J.

(2001) Lipovitellins derived from two forms of vitellogenin are

differentially processed during oocyte maturation in haddock

(Melanogrammus aeglefinus) J Exp Zool., 291, 58–67.

Riis-Vestergaard, J (2002) Energy density of marine pelagic fish

eggs J Fish Biol., 60, 1511–1528.

Rinchard, J., Ware, K., Dabrowski, K., Van Tassell, J., Marschall,

E & Stein, R (2010) Egg thiamine concentration affects embryo

survival in Lake Erie walleye Environ Biol Fishes, 90, 53–60.

Rodr
ıguez, C., Cejas, J.R., Mart
ın, M.V., Bad
ıa, P., Samper, M &

Lorenzo, A (1998) Influence of n-3 highly unsaturated fatty acid

deficiency on the lipid composition of broodstock gilthead ream (Sparus aurata L.) and on egg quality Fish Physiol Bio- chem., 18, 177–187.

seab-Rohr, D.H., Lokman, P.M., Davie, P.S & Young, G (2001) Ketotestosterone induces silvering-related changes in immature female short-finned eels, Anguilla australis Comp Biochem.

11-Physiol., Part A Mol Integr 11-Physiol., 130, 701–714.

Rom
eo, M (1987) Trace metals in fish roe from the Mauritania coast Mar Pollut Bull., 18, 507–508.

Rønnestad, I., Fyhn, H.J & Gravningen, K (1992) The tance of free amino acids to the energy metabolism of eggs and larvae of turbot (Scophthalmus maximus) Mar Biol., 114,

impor-517 –525.

Rønnestad, I., Koven, W.M., Tandler, A., Harel, M & Fyhn, H.J.

(1994) Energy metabolism during development of eggs and larvae of gilthead sea bream (Sparus aurata) Mar Biol., 120,

187 –196.

Rønnestad, I., Hemre, G.I., Finn, R.N & Lie, O (1998a) nate sources and dynamics of vitamin A and its incorporation into the eyes during the early endotrophic and exotrophic larval stages of Atlantic halibut (Hippoglossus hippoglossus L.) Comp.

Alter-Biochem Physiol., Part A Mol Integr Physiol., 119, 787–793.

Rønnestad, I., Koven, W., Tandler, A., Harel, M & Fyhn, H.J.

(1998b) Utilisation of yolk fuels in developing eggs and larvae of European sea bass (Dicentrarchus labrax) Aquaculture, 162,

Res., 36, 1488–1499.

Samaee, S.-M., Est
evez, A., Gim
enez, G & Lahnsteiner, F.

(2009a) Evaluation of quantitative importance of egg lipids and fatty acids during embryos and larvae development in marine pelagophil teleosts: with an emphasis on Dentex dentex J Exp.

Zool A Ecol Genet Physiol., 311A, 735–751.

Samaee, S.-M., Lahnsteiner, F., Gim
enez, G., Est
evez, A., Sarg, B.

& Lindner, H (2009b) Quantitative composition of derived yolk proteins and their effects on viability of embryos and larvae of common dentex (Dentex dentex), a marine pelago- phil teleost J Exp Zool A Ecol Genet Physiol., 311A,

vitellogenin-504 –520.

Samaee, S.M., Mente, E., Est
evez, A., Gim
enez, G & Lahnsteiner,

F (2010) Embryo and larva development in common dentex (Dentex dentex), a pelagophil teleost: the quantitative composi- tion of egg-free amino acids and their interrelations Theriogenol- ogy, 73, 909–919.

Sandnes, K., Rosenlund, G., Mangor-Jensen, A & Lie, Ø (1998) Contents and organ distribution of pantothenic acid in maturing turbot (Psetta maxima) Aquac Nutr., 4, 285–286.

Sano, K., Kawaguchi, M., Yoshikawa, M., Iuchi, I & Yasumasu,

S (2010) Evolution of the teleostean zona pellucida gene inferred from the egg envelope protein genes of the Japanese eel, Anguilla japonica FEBS J., 277, 4674–4684.

Sargent, J.R (1995) Origins and functions of egg lipids: nutritional implications In: Broodstock Management and Egg and Larval Quality (Bromage, N.R & Roberts, R.J eds), pp 353 –372.

Blackwell Science Ltd, Oxford.

Sasaki, S., Ota, T & Takagi, T (1989) Compositions of fatty acids

in the lipids of chum salmon during spawning migration Nippon Suisan Gakk., 55, 2191–2197.

.

Sawanboonchun, J (2009) Atlantic Cod (Gadus morhua L.)

brood-stock nutrition: the role of arachidonic acid and astaxanthin as

determinants of egg quality pp 212 PhD thesis, University of

Stirling.

Sawanboonchun, J., Roy, W.J., Robertson, D.A & Bell, J.G.

(2008) The impact of dietary supplementation with astaxanthin

on egg quality in Atlantic cod broodstock (Gadus morhua, L.).

Aquaculture, 283, 97–101.

Saxholt, E., Christensen, A.T., Møller, A., Hartkopp, H.B., Hess

Ygil, K & Hels, O.H (2008) Danish food composition

data-bank, revision 7 Department of Nutrition, National Food

Insti-tute, Technical University of Denmark.

Sbaihi, M., Rousseau, K., Baloche, S., Meunier, F.,

Fouchereau-Peron, M & Dufour, S (2009) Cortisol mobilizes mineral stores

from vertebral skeleton in the European eel: an ancestral origin

for glucocorticoid-induced osteoporosis? J Endocrinol., 201,

241 –252.

Scabini, V., Fern
andez-Palacios, H., Robaina, L., Kalinowski, T.

& Izquierdo, M.S (2011) Reproductive performance of gilthead

seabream (Sparus aurata L., 1758) fed two combined levels of

carotenoids from paprika oleoresin and essential fatty acids.

Aquac Nutr., 17, 304–312.

Schmitz, O (1982) Beitr €age zur Entwicklung einer Bilanzmethodik

f €ur Aale (Anguilla anguilla, L.) pp 85 PhD thesis, Rheinischen

Friedrich-Wilhelms-Universit €at.

Seoka, M., Takii, K., Takaoka, O., Nakamura, M & Kumai, H.

(1997) Biochemical phases in embryonic red sea bream

develop-ment Fish Sci., 63, 122–127.

Seoka, M., Yamada, S., Iwata, Y., Yanagisawa, T., Nakagawa, T.

& Kumai, H (2003) Differences in the biochemical content of

buoyant and non-buoyant eggs of the Japanese eel, Anguilla

japonica Aquaculture, 216, 355–362.

Seoka, M., Yamada, S & Kumai, H (2004) Free amino acids in

Japanese eel eggs obtained by hormonal inducement J Fish

Biol., 65, 592–596.

Shim, K.F., Lam, T.J & Landesman, L (1990) Effect of deletion

of amino acid from supplement on the growth and reproductive

performance of the dwarf gourami, Colisa lalia (Hamilton) J.

Aqua Tropics, 5, 149–154.

Silversand, C & Haux, C (1995) Fatty acid composition of

vitel-logenin from four teleost species J Comp Physiol B, Biochem.

Syst Environ Physiol., 164, 593–599.

Silversand, C., Norberg, B., Holm, J.C., Lie, Ø & Haux, C (1995)

Dietary influence on the fatty acid composition of vitellogenin

and the subsequent effect on the egg composition in Cod (Gadus

morhua) In: Proceedings of the Fifth International Symposium

on the Reproductive Physiology of Fish (Goetz, F & Thomas,

P eds.), pp 375 FishSymp 95, The University of Texas at

Aus-tin.

Silversand, C., Norberg, B & Haux, C (1996) Fatty-acid

composi-tion of ovulated eggs from wild and cultured turbot

(Scophthal-mus maxi(Scophthal-mus) in relation to yolk and oil globule lipids Mar.

Biol., 125, 269–278.

Sivaloganathan, B., Walford, J., Ip, Y.K & Lam, T.J (1998) Free

amino acids and energy metabolism in eggs and larvae of

sea-bass, Lates calcarifer Mar Biol., 131, 695–702.

Sloth, J.J., Julshamn, K & Lundebye, A.K (2005) Total arsenic

and inorganic arsenic content in Norwegian fish feed products.

Aquac Nutr., 11, 61–66.

Sorbera, L.A., Asturiano, J.F., Carrillo, M & Zanuy, S (2001)

Effects of polyunsaturated fatty acids and prostaglandins on

oocyte maturation in a marine teleost, the European Sea Bass

(Dicentrarchus labrax) Biol Reprod., 64, 382–389.

Southgate, P.C., Lee, P.S & Rimmer, M.A (1994) Growth and biochemical composition of cultured Sea Bass (Lates calcarifer) Larvae Asian Fish Sci., 7, 241–247.

Støttrup, J.G., Jacobsen, C., Tomkiewicz, J & Jarlbæk, H (2013) Modification of essential fatty acid composition in broodstock

of cultured European eel Anguilla anguilla L Aquacult Nutr.,

19, 172–185.

Sudo, R., Suetake, H., Suzuki, Y., Utoh, T., Tanaka, S., Aoyama,

J & Tsukamoto, K (2011a) Dynamics of reproductive mones during downstream migration in females of the Japanese eel, Anguilla japonica Zoolog Sci., 28, 180–188.

hor-Sudo, R., Tosaka, R., Ijiri, S., Adachi, S., Suetake, H., Suzuki, Y., Horie, N., Tanaka, S., Aoyama, J & Tsukamoto, K (2011b) Effect of temperature decrease on oocyte development, sex ste- roids, and gonadotropin b-subunit mRNA expression levels in female Japanese eel Anguilla japonica Fish Sci., 77, 575–582 Suloma, A & Ogata, H.Y (2011) Arachidonic acid is a major component in gonadal fatty acids of tropical coral reef fish in the Philippines and Japan J Aquac Res Development, 2, 111 Sved€ang, H & Wickstr€om, H (1997) Low fat contents in female silver eels: indications of insufficient energetic stores for migra- tion and gonadal development J Fish Biol., 50, 475–486 Takeuchi, T., Arai, S., Watanabe, T & Shimma, Y (1980) Requirement of eel Anguilla japonica for essential fatty acids Nippon Suisan Gakk., 46, 345–353.

Takeuchi, T., Watanabe, T., Ogino, C., Saito, M., Nishimura, K.

& Nose, T (1981) Effects of low protein-high calory diets and deletion of trace elements from a fish meal diet on reproduction

of rainbow trout Nippon Suisan Gakk., 47, 645–654.

Tanaka, H., Nomura, K., Yamamoto, T & Oku, H (2006) opment of artificial diets and rearing systems for eel larvae – the first success of production of glass eel in captivity Bull Fish Res Agency, (Suppl 5), 63 –69.

Devel-Tandler, A., Harel, M., Koven, W.M & Kolkovski, S (1995) Broodstock and larvae nutrition in gilthead seabream Sparus au- rata – new findings on its mode of involvement in improving growth, survival and swimbladder inflation Israeli J Aquac., 47,

95 –111.

Taranger, G.L., Carrillo, M., Schulz, R.W et al (2010) Control of puberty in farmed fish Gen Comp Endocrinol., 165, 483–515 Tesch, F.W (2003) The Eel Blackwell Science, Oxford [etc.] Thorsen, A & Fyhn, H.J (1996) Final oocyte maturation in vivo and in vitro in marine fishes with pelagic eggs; yolk protein hydrolysis and free amino acid content J Fish Biol., 48, 1195– 1209.

Todd, P.R (1981) Morphometric changes, gonad histology, and fecundity estimates in migrating New Zealand freshwater eels (Anguilla spp.) NZ J Mar Freshwat Res., 15, 155–170 Tomkiewicz, J ed (2012) Reproduction of European Eel in Aqua- culture (REEL): consolidation and new production methods DTU Aqua Report No 249-2012 pp 47 National Institute of Aquatic Resources, Technical University of Denmark.

Tremblay, V (2009) Reproductive strategy of female American eels among five subpopulations in the St Lawrence River Watershed In: Eels at the Edge: Science, Status, and Conservation Concerns (Casselman, J.M & Cairns, D.K eds), Vol 58, pp 85–102 American Fisheries Society Symposium 58, Bethesda, Maryland Tsukamoto, K., Chow, S., Otake, T et al (2011) Oceanic spawn- ing ecology of freshwater eels in the western North Pacific Nat Commun., 2, 1–9.

Tulli, F & Tibaldi, E (1997) Changes in amino acids and essential fatty acids during early larval rearing of dentex Aquacult Int.,

5, 229–236.

.

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd

Unuma, T., Kondo, S., Tanaka, H., Kagawa, H., Nomura, K &

Ohta, H (2005) Relationship between egg specific gravity and

egg quality in the Japanese eel, Anguilla japonica Aquaculture,

246, 493–500.

Ure ~na, R., Peri, S., del Ramo, J & Torreblanca, A (2007) Metal

and metallothionein content in tissues from wild and farmed

Anguilla anguilla at commercial size Environ Int., 33, 532–539.

Van Ginneken, V., Antonissen, E., Muller, U.K., Booms, R.,

Eding, E., Verreth, J & van den Thillart, G (2005a) Eel

migra-tion to the Sargasso: remarkably high swimming efficiency and

low energy costs J Exp Biol., 208, 1329–1335.

Van Ginneken, V., Vianen, G., Muusze, B et al (2005b) Gonad

development and spawning behaviour of artificially-matured

European eel (Anguilla anguilla L.) Anim Biol., 55, 203–218.

Vassallo-Agius, R., Watanabe, T., Mushiake, K., Kawano, K &

Satoh, S (1998) Chemical components of eggs and yolksac

lar-vae obtained from striped jack broodstock fed on a raw fish mix

or dry pellets Fish Sci., 64, 759–765.

Vassallo-Agius, R., Imaizumi, H., Watanabe, T., Yamazaki, T.,

Satoh, S & Kiron, V (2001a) Effect of squid meal in dry pellets

on the spawning performance of striped jack Pseudocaranx

den-tex Fish Sci., 67, 271–280.

Vassallo-Agius, R., Imaizumi, H., Watanabe, T., Yamazaki, T.,

Satoh, S & Kiron, V (2001b) The influence of astaxanthin

sup-plemented dry pellets on spawning of striped jack Fish Sci., 67,

260 –270.

Vassallo-Agius, R., Watanabe, T., Imaizumi, H., Yamazaki, T.,

Satoh, S & Kiron, V (2001c) Effects of dry pellets containing

astaxanthin and squid meal on the spawning performance of

striped jack Pseudocaranx dentex Fish Sci., 67, 667–674.

Verakunpiriya, V., Watanabe, T., Mushiake, K., Kiron, V., Satoh,

S & Takeuchi, T (1996) Effect of broodstock diets on the

chem-ical components of milt and eggs produced by yellowtail Fish.

Sci., 62, 610–619.

Verakunpiriya, V., Mushiake, K., Kawano, K & Watanabe, T.

(1997a) Supplemental effect of astaxanthin in broodstock diets

on the quality of yellowtail eggs Fish Sci., 63, 816–823.

Verakunpiriya, V., Watanabe, K., Mushiake, K., Kawano, K.,

Ko-bayashi, I., Hasegawa, I., Kiron, V., Satoh, S & Watanabe, T.

(1997b) Effect of krill meal supplementation in soft-dry pellets

on spawning and quality of egg of yellowtail Fish Sci., 63,

433 –439.

Vetter, R.D., Hodson, R.E & Arnold, C (1983) Energy

metabo-lism in a rapidly developing marine fish egg, the red drum

(Sci-aenops ocellata) Can J Fish Aquat Sci., 40, 627–634.

Waagbø, R (2010) Water-soluble vitamins in fish ontogeny.

Aquac Res., 41, 733–744.

Watanabe, T & Kiron, V (1995) Red sea bream (Pagrus major).

In: Broodstock Management and Egg and Larval Quality

(Bro-mage, N.R & Roberts, R.J eds), pp 398 –413 Blackwell Science

Ltd, Oxford.

Watanabe, T., Arakawa, T., Kitajima, C & Fujita, S (1984a)

Effect of nutritional quality of broodstock diets on reproduction

of red sea bream Nippon Suisan Gakk., 50, 495–501.

Watanabe, T., Itoh, A., Murakami, A., Tsukashima, Y., Kitajima,

C & Fujita, S (1984b) Effect of nutritional quality of diets giets

given to broodstock on the verge of spawning on reproduction

of red sea bream Nippon Suisan Gakk., 50, 1023–1028.

Watanabe, T., Ohhashi, S., Itoh, A., Kitajima, C & Fujita, S.

(1984c) Effect of nutritional composition of diets on chemical

components of red sea bream broodstock and eggs produced.

Nippon Suisan Gakk., 50, 503–515.

Watanabe, T., Itoh, A., Satoh, S., Kitajima, C & Fujita, S.

(1985a) Effect of dietary protein levels and feeding period before spawning on chemical components of eggs produced by red sea bream broodstock Nippon Suisan Gakk., 51, 1501–1509.

Watanabe, T., Koizumi, T., Suzuki, H., Satoh, S., Takeuchi, T., Yoshida, N., Kitada, T & Tsukashima, Y (1985b) Improvement

of quality of red sea bream eggs by feeding broodstock on a diet containing cuttlefish meal or on raw krill shortly before spawn- ing Nippon Suisan Gakk., 51, 1511–1521.

Watanabe, T., Fujimura, T., Lee, M.-J., Fukusho, K., Satoh, S &

Takeuchi, T (1991a) Effect of polar and nonpolar lipids from krill on quality of eggs of red seabream Pagrus major Fish Sci.,

57, 695–698.

Watanabe, T., Lee, M.-J., Mizutani, J., Yamada, T., Satoh, S., Takeuchi, T., Yoshida, N., Kitada, T & Arakawa, T (1991b) Effective components in cuttlefish meal and raw krill for improvement of quality of red seabream Pagrus major eggs Fish.

Sci., 57, 681–694.

Weil, C., Goupil, A.-S., Quillet, E., Labbe, L & Le Gac, F (2008) Two-way selection for muscle lipid content modifies puberty and gametogenesis in rainbow trout Cybium, 32(Suppl 2), 198.

Wiegand, M.D (1996) Composition, accumulation and utilization

of yolk lipids in teleost fish Rev Fish Biol Fisheries, 6, 259–286.

Wilson, R (2009) Dietary effects of n-3 highly unsaturated fatty acid levels on egg and larval quality, and the fatty acid composi- tion of the eggs of Chilean flounder Paralichthys adspersus broodstock Aquac Res., 40, 1400–1409.

Yamada, Y., Okamura, A., Tanaka, S., Utoh, T., Horie, N., awa, N & Oka, H.P (2001) The roles of bone and muscle as phosphorus reservoirs during the sexual maturation of female Japanese eels, Anguilla japonica Temminck and Schlegel (An- guilliformes) Fish Physiol Biochem., 24, 327–334.

Mik-Yamada, S., Hori, K & Seoka, M (2006) Toward the ments of broodfish management and egg quality of the Japanese eel, Anguilla japonica, for larviculture – egg collection from young broodfish and effects of broodfish diet on egg fatty acid contents Bull Fish Res Agency, (Suppl 5), 33 –37.

improve-Yamaguchi, S., Miura, C., Kikuchi, K., Celino, F.T., Agusa, T., Tanabe, S & Miura, T (2009) Zinc is an essential trace element for spermatogenesis Proc Natl Acad Sci U.S.A., 106, 10859–

45, 519–526.

Yoshikawa, M (1998) Distribution and accumulation of bic acid and a-tocopherol in Japanese eel, Anguilla japonica dur- ing the induced final maturation with extract of pituitary glands.

L-ascor-Aquac Sci (Suisan Zoshoku), 46, 261–267.

Zhao, Z & Zera, A.J (2002) Differential lipid biosynthesis lies a tradeoff between reproduction and flight capability in a wing-polymorphic cricket Proc Natl Acad Sci U.S.A., 99,

under-16829 –16834.

.

1,2,3 1,2 3,4 3,4 1,2 1

2

Repeated handling was employed in this study as a

chronic stressor to verify whether its attributed

immuno-suppressive effects could be minimized by dietary arginine

supplementation Therefore, turbot (Scophthalmus

maxi-mus) were air-exposed for 3 min (mild hypoxia) every

other day during 15 or 60 days (handled) or left

undis-turbed (control) In addition, control and handled

speci-mens were fed one of three diets with graded levels of

11, respectively) Repeated handling increased plasma

cor-tisol levels in handled fish fed the Arg 6 diet when

com-pared with control groups, while those levels decreased in

handled fish fed Arg 7 and Arg 11 diets after both 15 and

60 days Moreover, repeated handling stress decreased

nitric oxide production, lysozyme activity and peripheral

lymphocyte numbers after 15 days, while more profound

effects were observed after 60 days Furthermore,

circulat-ing monocytes, nitric oxide production and humoral

parameters increased in both control and handled fish fed

arginine supplements In conclusion, dietary arginine

sup-plementation may present valuable effects in chronically

stressed turbot by decreasing plasma cortisol levels and

enhancing most aspects of the innate immune mechanisms

assessed after 60 days

peripheral neutrophils, respiratory burst activity

Received 8 August 2012; accepted 1 April 2013

Correspondence: B Costas, Centro Interdisciplinar de Investigac ß~ao

Mar-inha e Ambiental (CIIMAR), Rua dos Bragas 289, 4050-123 Porto,

Portugal E-mail: bcostas@ciimar.up.pt

Turbot (Scophthalmus maximus) production has increasedsignificantly over the last decade in Europe During grow-out, turbot culture relies solely on intensive productionmethods as this species can tolerate rearing at high stock-ing densities (up to four layers of large fish stacked ontop of each other) This is a typical stressor in fish farm-ing which can be intensified during periods of handling(Person-Le Ruyet 2010) The effect of stress on the

although acute stress can have beneficial effects, chronicstress was found to inhibit an optimal immune response

in teleost fish (Fast et al 2008), leading to increased ceptibility to pathogens and parasites (Wendelaar Bonga1997; Tort 2011) In fact, cortisol can influence multipleaspects of the innate immune defence mechanisms in fish(Verburg-van Kemenade et al 2009) For instance, phago-cytosis of peripheral blood leucocytes from common carp(Cyprinus carpio) and hybrid tilapia (Oreochromis niloti-

cortisol administration (Law et al 2001) Chronic dling stress induced lymphocytopenia in gilthead seabream(Sparus aurata), while high stocking density decreasedmRNA levels of g-type lysozyme and hepcidin antimicro-bial peptide-1 genes in Senegalese sole (Solea senegalensis)liver and kidney (Sunyer et al 1995; Salas-Leiton et al.2010) Furthermore, high plasma cortisol levels resulted inreduction in leucocyte proliferation, numbers of antibodyproducing cells and levels of virus neutralizing antibodies

han-in fish (Weyts et al 1999) Stress and cortisol treatmenthave also been shown to increase susceptibility to para-sitic infections and nodavirus (Saeij et al 2003; Varsamos

Stress conditions that induced high plasma cortisol levels

also modified amino acid metabolism in several fish species

2012a) Moreover, it has been suggested that fish under

stressful conditions present additional amino acid

require-ments, due to either increased energetic demands or for the

synthesis of stress-related proteins and other compounds

related to the stress response (Arag~ao et al 2008; Costas

metabolites in key metabolic pathways that are necessary for

growth, immunity or resistance to environmental stressors

and pathogens has been recently reviewed in fish (Li et al

serve as constituents of proteins and energy sources, but also

can be converted into important biochemically active

sub-stances in vivo In particular, arginine is the precursor for the

synthesis of nitric oxide (NO) and polyamines in terrestrial

animals (Wu & Morris 1998) In fish, macrophage NO

pro-duction plays an important role in the cellular defence

mecha-nisms, and the release of NO by stimulated macrophages has

been demonstrated in several fish species (Neumann et al

1995; Tafalla & Novoa 2000; Costas et al 2011) A positive

effect of an arginine-enriched diet on the resistance of channel

catfish (Ictalurus punctatus) to infection with Edwardsiella

2001) Similarly, dietary arginine increased some innate

immune mechanisms and disease resistance of Senegalese sole

following challenge with Photobacterium damselae subsp

in the availability of dietary indispensable amino acids

trans-lated in higher plasma lysozyme activity in Senegalese sole

fed a high-protein diet for 14 days (Costas et al 2012a)

The interactive effects of dietary amino acids and stressful

husbandry conditions on the fish immune system are a

com-plex and important issue for the aquaculture industry that has

received little attention Therefore, the central objective of this

study was to assess whether increased availability of dietary

arginine can minimize the immunosuppressive effects

attrib-uted to chronic stress in fish Similarly, it is also intended to

verify to what extent the dietary arginine supplementation can

influence some aspects of the innate immune system in turbot

diets were formulated to meet the indispensable amino acidrequirements estimated for turbot (Kaushik 1998) One dietjust met the estimated arginine requirement (Arg 6), whiletwo other experimental diets (Arg 7 and Arg 11) were pre-

dietary protein, respectively) Amino acid nitrogen wasequilibrated among diets by adding to the Arg 6 and Arg 7

acids are ubiquitous odorants for fish, lysine was chosen toavoid differences in feed intake due to its moderate olfac-tory sensitivity in marine fish (Hubbard et al 2011) After

until use Formulation and proximate composition of tary treatments are presented in Table 1

die-Diets were analysed for total amino acids content Diet

nitrogen-flushed glass vials Afterwards, samples were column derivatized with phenylisothiocyanate (PITC; Pierce,Rockford, IL, USA), using the Pico-Tag method (Waters,Milford, MA, USA) described by Cohen et al (1989) Exter-nal standards were prepared along with the samples, usingphysiological amino acid standard solutions (acid/neutraland basics from Sigma, Steinheim, Germany) and a gluta-mine solution Norleucine was used as an internal standard

pre-Samples and standards were analysed by high-performanceliquid chromatography (HPLC) in a Waters Reversed-PhaseAmino Acid Analysis System equipped with a Pico-Tag col-

Cohen et al (1989) Resulting peaks were analysed with theBreeze software (Waters) During analytical procedures,asparagine was converted to aspartate and glutamine to glu-tamate during acid hydrolysis, so the reported values forthese amino acids (Asx and Glx) represent the sum of therespective amine and acid Tryptophan was not determined

as it is destroyed by acid hydrolysis The amino acid profile

of experimental diets is presented in Table 2

Specimens originated from a commercial fish farm RIA, Aveiro, Portugal) Prior to the experiment, fish weretransported to the CIIMAR facilities (Porto, Portugal) andmaintained in two separate recirculated seawater systems

; and a 12/

12 fluorescent light/dark cycle with the light period from 8:00

to 20:00 h was adopted) comprised of flat-bottomed, round .

tanks (volume: 300 L; bottom surface= 0.64 m2) for

14 days During this acclimation period, fish were fed daily

to apparent satiety (based on the assessment of feed

remain-ing in the tanks) with the Arg 6 diet

in two separate recirculated seawater systems as indicated

above In one of the systems, fish were reared in triplicate

tank cleaning procedures, and served as controls In theother system, handled fish were chased with a net everyother day to capture all fish inside each tank (triplicates;

3 min Immediately after air exposure, the fish werereturned to the experimental tanks Both control and han-dled fish were fed with the Arg 6, Arg 7 and Arg 11 diets.Tanks were cleaned, and temperature, salinity, dissolvedoxygen and ammonia and nitrite levels in the water weremeasured daily Fish were fed by hand four times a day at

a daily feeding rate of 1.5% biomass

Two samplings were performed after 15 and 60 days ofexperiment As experimental sampling procedures wereidentical during both periods, they will be described once.Fish were fasted for 24 h prior to sampling to avoid anyinfluence of feeding on cortisol and glucose levels (Arends

at a time and anaesthetized with 2-phenoxyethanol (0.5 mL

each fish using heparinized syringes The blood collection

manipula-tion during sampling Fresh blood was transferred to aheparinized Eppendorf tube for haematocrit determination

Table 2 Amino acid composition of experimental diets (g 16 g N)

Table 1 Ingredients and proximate composition of experimental

LT, low temperature; CPSP G, fish soluble protein concentrate

(hydrolysed fish meal); DCP, dibasic calcium phosphate; DM, dry

matter; NFE, nitrogen-free extracts = 1000 – (CP + CL + CA).

1 Minerals (g kg 1 diet): Mn (manganese oxide), 0.02; I

(potas-sium iodide), 0.0015; Cu (copper sulphate), 0.005; Co (cobalt

sul-phate), 0.0001; Mg (magnesium sulsul-phate), 0.5; Zn (zinc oxide),

0.03; Se (sodium selenite), 0.0003; Fe (iron sulphate), 0.06; Ca

(cal-cium carbonate), 2.15; dibasic cal(cal-cium phosphate, 5; KCl

(potas-sium chloride), 1; NaCl (sodium chloride), 0.4.

2 Vitamins (g or IU kg 1 diet): vitamin A (retinyl acetate),

8000 IU; vitamin D3 (DL-cholecalciferol), 1700 IU; vitamin K3

(menadione sodium bisulphite), 0.01 g; vitamin B12

(cyanocobala-min), 0.00002 g; vitamin B1 (thiamine hydrochloride), 0.008 g;

vitamin B2 (riboflavin), 0.02 g; vitamin B6 (pyridoxine

hydrochlo-ride), 0.01 g; folic acid, 0.006 g; biotin, 0.0007 g; inositol, 0.3 g;

nicotinic acid, 0.07 g; pantothenic acid, 0.03 g; vitamin E (Lutavit

E50), 0.3 g; vitamin C (Lutavit C35), 0.5 g; betaine (Betafin S1),

0.5 g.

.

Aquaculture Nutrition 19; 25–38 ª 2013 John Wiley & Sons Ltd

temperature; Hawksley & Sons, Lancing, UK), total blood

cell counts and blood smear preparations The remaining

blood was used for plasma collection Plasma was obtained

blood collection, fish were individually weighed and the

head-kidney subsequently dissected over an ice bed Liver

form each fish was also dissected and weighed for

hepato-somatic index calculation Experimental procedures were

directed by trained scientists (following FELASA category

C recommendations) and were conducted according to the

guidelines on the protection of animals used for scientific

purposes from the European directive 2010/63/UE

Total cell counts were performed with a haemocytometer

from fresh blood previously diluted in phenol-red-free

Hank’s balanced salt solution (HBSS; Gibco, Paisley, UK)

Blood smears were prepared from one drop of

homoge-nized fresh blood, air-dried and stained with Wright’s stain

(Hemacolor, Merck, Darmstadt, Germany) after fixation

for 1 min with formol-ethanol (10% of 37% formaldehyde

in absolute ethanol) Detection of peroxidase activity to

label neutrophils was performed according to Afonso et al

(1998) The slides were examined under oil immersion

clas-sified as thrombocytes, lymphocytes, monocytes or

neu-trophils The relative percentage of each leucocyte type was

also calculated

Leucocytes from head-kidney were isolated and

main-tained essentially as described by Secombes (1990) Briefly,

the head-kidney was removed under aseptic conditions,

Leibovitz L-15 medium (L15; Gibco) supplemented with

2% foetal bovine serum (FBS; Gibco), penicillin (100 IU

then loaded onto a 34 : 51% Percoll (Sigma) density

band of cells lying at the interface of the Percoll gradient

for 5 min in L15, 2% FBS, P/S and heparin The viable

cell concentration was determined by the Trypan blue

exclusion test Cells were counted in a haemocytometer and

non-adherent cells were washed off and the monolayers weremaintained with L15 supplemented with 5% FBS, until therespiratory burst assays were conducted after 24 h of incu-

Respiratory burst activity of head-kidney leucocytes wasbased on the reduction in ferricytochrome C method for

monolayers were washed twice with phenol-red-free HBSS,

phenol-red-free HBSS) was added to each well Ferricytochrome C

phorbol myristate acetate(PMA; Sigma) was added as a soluble stimulant of therespiratory burst Ferricytochrome C with PMA and

used to confirm the specificity of the reaction For each

three or more wells of leucocytes per fish were assayed

Plates were read immediately after the addition of reagents

to the leucocytes, and optical densities (OD) were then

micro-plate spectrophotometer (BioTek, Winooski, VT, USA) at

were determined after respiratory burst assays by removing

acid, 1% Tween 20 and 0.05% crystal violet; Sigma) After

2 min, the nuclei were counted in a haemocytometer Themean of four wells was used as the total cell count per well

technical constrains, respiratory burst was only performed

in fish from the final sampling (60 days)

Plasma cortisol levels were measured with a commercially

(SIEMENS Medical Solutions Diagnostics, Los Angeles,

CA, USA) as described by Irwin et al (1999) Briefly,

bath The contents of all tubes were decanted and allowed

to drain for 5 min before being read on a gamma counter

; Inc., Zaventem, gium) for 1 min A calibration curve was constructed on .

Bel-logit-log graph paper and used to convert results from

Coat-A-Count cortisol antiserum cross-reacts 100% with

cortisol, 11.4% with 11-deoxycortisol, 0.98% with

corti-sone, 0.94% with corticosterone and 0.02% with

progester-one All analyses were conducted in duplicates

Plasma glucose, lactate and triglycerides were assessed

using commercially available Spinreact kits (Glucose HK

Ref 1001200; Lactate Ref 1001330; Triglycerides Ref

1001311; Sant Esteve d’en Bas, Girona, Spain), adapted for

96-well microplates Plasma total proteins were determined

in 1 : 50 (v/v) diluted plasma samples using the

bicinchoni-nic acid (BCA) Protein Assay Kit (Pierce #23225, Rockford,

IL, USA) for microplates Bovine serum albumin served as a

standard All analyses were conducted in triplicates

Total nitrite plus nitrate in serum was analysed using a

GmbH, Mannheim, Germany) Briefly, nitrate was reduced

to nitrite with nitrate reductase, and nitrite was determined

N-naphthyl-ethylenediamine Nitrite concentration was

cal-culated by comparison with a sodium nitrite standard curve

As nitrite and nitrate are endogenously produced as

oxida-tive metabolites of the messenger molecule NO, these

com-pounds are considered as indicative of NO production (Saeij

Lysozyme activity was measured using a turbidimetric

assay as described by Costas et al (2011) Briefly, a

sodium phosphate buffer; pH 6.2; Sigma) was prepared In

suspension were added The reaction was carried out at

0.5 and 4.5 min Lyophilized hen egg white lysozyme

(Sigma) was serially diluted in sodium phosphate buffer

(0.05 M; pH 6.2) and used to develop a standard curve

The amount of lysozyme in the sample was calculated

using the formula of the standard curve All analyses were

conducted in triplicates

Alternative complement pathway (ACP) activity was

esti-mated as described by Sunyer & Tort (1995) The following

buffers were used: GVB (Isotonic veronal-buffered saline),

pH 7.3, containing 0.1% gelatin; ethylenediaminetetraacetic

acid (EDTA)-GVB, as previous one but containing 20 mM

EDTA; and Mg-ethylene glycol-bis (B-aminoethyl ether)-N,

N, N’-tetra acetic acid (EGTA)-GVB, which is GVB with

(RaRBC; Probiologica Lda, Belas, Portugal) were used for

ACP activity determination RaRBC were washed four

times in GVB and resuspended in GVB to a concentration

Mg-EGTA-GVB buffer Samples were incubated at roomtemperature for 100 min with occasional shaking The reac-

Samples were then centrifuged, and the extent of sis was estimated by measuring the optical density of thesupernatant at 414 nm The reciprocal of the plasma dilu-tion causing 50% lysis of RaRBC was designated theACH50 titre, and the results are presented as ACH50 units

Total peroxidase activity in plasma was measured ing the procedure described by Quade & Roth (1997)

acid, and the OD was read at 450 nm The wells withoutplasma were used as blanks The peroxidase activity (units

peroxidase produces an absorbance change of 1 OD

Data on proximate composition of diets and initial weight,final weight, total length and liver weight were used to cal-culate nitrogen-free extracts (NFE), relative growth rate(RGR), weight gain, condition factor (K), voluntary feedintake (VFI) and hepatosomatic index (HSI) as follows:

Crude ash)

final wet weights, respectively

VFI = Crude feed intake/average body weight ((initialbody weight + final body weight)/2)/days

(SD) Data were analysed for normality (Kolmogorov–Smirnov test) and homoscedasticity of variance (Levene’s .

Aquaculture Nutrition 19; 25–38 ª 2013 John Wiley & Sons Ltd

test), and, when necessary, they were log-transformed

before being treated statistically Data from experimental

chronic stress and dietary treatment as dependent variables

Tukey’s post hoc tests were carried out to identify

signifi-cantly different groups fed the experimental diets In

comparisons to analyse the effect of chronic stress All

sta-tistical analyses were performed using the computer

pack-age SPSS 15.0 for WINDOWS The level of significance

Feed ingredients and the analysed feed composition are

given in Table 1 Protein, lipids, DM, ash and energy levels

N) presented significant differences among dietary

treat-ments As expected, arginine and lysine showed an increase

directly proportional to the level of supplementation

(Table 2) Tryptophan could not be analysed by the

method used and thus is not part of the evaluation of the

amino acid profile Experimental diets were well accepted,

and no significant differences were found in growth and

feed intake among the experimental groups (Table 3)

Plasma cortisol levels increased significantly in handled fishfed the Arg 6 diet compared with either control or handledspecimens fed different dietary treatments after 15 days(Fig 1a), while those levels increased significantly only inhandled fish fed the Arg 6 diet compared with control spec-imens fed the same diet after 60 days (Fig 1b) Plasma tri-glycerides followed the opposite pattern than that observedfor cortisol with significantly lower values in handled fishfed the Arg 6 diet after both 15 and 60 days Furthermore,plasma glucose, lactate and total proteins were not signifi-

(Table 4)

Haematocrit and total peripheral blood cells were notaltered by different treatments after 15 days However, hae-matocrit and total red blood cell numbers increased signifi-cantly in handled groups regardless of dietary treatment,while total white blood cells showed the opposite patternafter 60 days (Table 5)

Regarding differential leucocytes counting, more drasticchanges were observed after 60 days of treatment Whilethe percentage of thrombocytes increased significantly inhandled fish regardless of dietary treatment at 15 days,absolute numbers decreased in stressed specimens at theend of the experimental period, with no effect of dietary

Table 3 Relative growth rate (RGR), weight gain, condition factor (K), voluntary feed intake (VFI) and hepatosomatic index (HSI) in

Scophthalmus maximus after 14 or 60 days held at different treatments

Parameters

Dietary treatment

treatment (Table 6) Moreover, absolute numbers and

pro-portion of lymphocytes decreased significantly in handled

fish after 15 and 60 days with respect to control specimens

Interestingly, the percentage of circulating lymphocytes

increased significantly in handled fish fed the Arg 7 and

Arg 11 diets compared with fish fed the Arg 6 diet after

15 days of treatment (Table 6) While the percentage of

cir-culating monocytes were not affected by chronic stress,

absolute numbers followed the same pattern observed for

both thrombocytes and lymphocytes and decreased in

han-dled specimens after 60 days The percentage of circulating

monocytes increased significantly in both control and

han-dled fish fed the Arg 11 diet after 60 days (Table 6)

Although circulating neutrophils were not altered aftereither 15 or 60 days, the percentage increased significantly

in handled fish regardless of dietary treatment with respect

to control specimens after 60 days (Table 6)

Respiratory burst activity and NO production were cantly affected by both chronic stress and dietary treat-ments Respiratory burst activity decreased significantly inhead-kidney leucocytes from handled specimens fed theArg 6 diet compared with fish from all other treatments(Fig 2) While NO production increased significantly infish fed the Arg 7 and Arg 11 diets after 15 and 60 days,values decreased after 15 days in handled specimens com-pared with unstressed fish regardless of dietary treatment

signifi-At the end of the experimental period, handled fish fed theArg 7 and Arg 11 diets presented significantly higher valuesthan handled specimens fed the Arg 6 diet (Figs 3a,b).Plasma lysozyme activity followed a similar pattern tothat observed in the respiratory burst, with decreased val-ues in handled specimens fed the Arg 6 diet compared withfish from all other treatments after both 15 and 60 days

increased significantly in handled fish regardless of dietarytreatment after 60 days, with no significant changes after

15 days (Table 7) ACP values increased significantly inboth control and handled fish fed the Arg 11 diet after

15 days, while levels increased in control fish fed the Arg 7and Arg 11 diets after 60 days In addition, handled groups

unstressed fish fed the Arg 6 diet after 60 days (Table 7)

Lysine and arginine are known to interact antagonistically

in higher vertebrates (Anderson et al 1984) However, theeffects of feeding disproportionate levels of arginine andlysine have only been evaluated in a few fish species, andthe interaction between these two indispensable aminoacids is still not conclusive Some fish species have beenreported to be insensitive to an excess of dietary arginineand lysine (Tibaldi et al 1994; Small & Soares 2000; Alam

2002; Zhou et al 2011) In the present study, growth formance and feed intake were not affected when fish werefed diets with excessive lysine levels along with an adequatedietary arginine level, suggesting an absence of antagonismbetween lysine and arginine in turbot Still, differences in

per-A

B

Figure 1 Plasma cortisol levels in Scophthalmus maximus after 15

(a) and 60 (b) days held at different treatments Control ( ■) or

handled () fish Values are means  SD (n = 6) Different letters

stand for significant differences within chronically stressed fish

(two-way ANOVA ; P < 0.05), and * stands for significant differences

attributed to chronic stress (t-Test; P < 0.05).

.

Aquaculture Nutrition 19; 25–38 ª 2013 John Wiley & Sons Ltd

arginine and lysine metabolism due to inhibition from

lysine or excess lysine cannot be ruled out as free amino

acids in plasma and tissues and/or arginase activity have

not been measured

An interesting result from the present study was the

decrease in plasma cortisol levels in handled fish fed Arg 7

and Arg 11 diets In contrast, cortisol levels increased

sig-nificantly in handled fish fed the Arg 6 diet after both 15

and 60 days of treatment, clearly indicative of a stressful

status in these specimens Plasma cortisol levels are

consid-ered to be a good indicator of the stress levels in fish after

(Wendelaar Bonga 1997; Barton 2002) Costas et al

(2012a) observed that an increase in all dietary

indispens-able amino acids appears to minimize some negative effects

attributed to cortisol release in chronically stressed

Senegal-ese sole Moreover, dietary arginine supplementation also

decreased the level of cortisol in serum of finishing pigs

and weaned piglets (Ma et al 2010; Yao et al 2011)

Therefore, dietary arginine supplementation beyond

mini-mum requirement for optimal growth appears to attenuate

the stress response in turbot, at least under these particular

experimental conditions It is possible that arginine

metab-olites or arginine itself may have minimized the release of

the adrenocorticotropic hormone (ACTH) from pituitary

and/or interfered in the brain monoaminergic system In

fish, ACTH cells appear to be under negative dopaminergic

control in vivo (Flik et al 2006) In addition, Suenaga et al

(2008) observed that an intracerebroventricular injection of

arginine induced sedative or hypnotic effects in chicks

exposed to a social isolation stress Still, the underlying

mechanisms are unknown, and the function of endogenousarginine under stressful husbandry conditions in fishdeserves further attention

Increased plasma cortisol levels are frequently associatedwith the subsequent elevation of plasma glucose and lactatevalues (Barton 2002) However, plasma glucose and lactateconcentrations were not significantly affected by the han-dling stress imposed on the fish after either 15 or 60 days

of treatment This may be due to a possible habituation tothe stressful conditions When the acute stressor is repeatedover a period of time, some habituation may occur, whichmay result in reductions in poststress cortisol and glucoselevels (Tort et al 1996; Jentoft et al 2005) However, thislack of increase in plasma glucose and lactate levels is morelikely due to a strategy representative of genetic adapta-tions that turbot may have developed in its natural habitat

The distribution of fishes to diversified habitats makesthem unique with regard to their physiological andbehavioural responses to stress (Peter 2011) In fact, anincrease in plasma-free fatty acids instead of glucose andlactate has been observed in stressed turbot submitted toair exposure (Waring et al 1996) In the present study, thedecrease in plasma triglyceride levels may reflect greater tri-glyceride uptake from the blood by tissues in handled fishfed the Arg 6 diet, suggesting that these specimens pre-sented higher metabolic needs than handled fish fed theArg 7 and Arg 11 diets Moreover, this hypothesis is con-gruent with the higher plasma cortisol levels observed inhandled fish fed the Arg 6 diet Similarly, an increase in alldietary indispensable amino acids influenced the secondarystress response in handled Senegalese sole by minimizing

Table 4 Plasma glucose, lactate, triglycerides and protein levels in Scophthalmus maximus after 15 and 60 days held at different treatments

Plasma parameters

Dietary treatment

Values are means  SD (n = 6).

* Significant differences attributed to chronic stress (t-test; P < 0.05).

.

the subsequent mobilization of energy substrates after

14 days of feeding (Costas et al 2012a)

In the present study, chronic stress decreased total

circu-lating leucocytes in handled fish regardless of dietary

treat-ment after 60 days Chronic stress normally involves the

switch of energetic metabolism to cope with the demands

of the stressor Therefore, mechanisms that imply

continu-ous energy availability by the immune system such as

pro-duction and differentiation of different types of leucocytes

will be subjected to a lack of resources, thus decreasing its

efficiency and therefore leading to immune depression or

suppression (Tort 2011) For instance, handling and

trans-port of channel catfish (I punctatus) induced a decrease in

circulating B-lymphocytes, while Atlantic salmon (Salmo

salar) macrophages isolated from chronically handled

speci-mens showed decreased survival when exposed to

Verburg-van Kemenade et al (2009) suggested that these

mechanisms are probably modulated by cortisol, mediated

by one or several of the glucocorticoid receptors present in

immune cells In the present study, decreases in plasma

lysozyme after both 15 and 60 days and head-kidney

respi-ratory burst activity after 60 days are clearly attributed to

cortisol action However, the observed leucopenia and

lower NO levels in handled fish fed the Arg 7 and Arg 11

diets suggest that other hormones and proteins related to

the stress response appear to modulate the turbot immune

machinery Indeed, the effects of stress on the immune tem are difficult to interpret in vivo, as a number of differ-ent hormones are involved via the HPI axis In fact, this

actions of various hormones and cytokines, as well as ontheir interactions Leucocytes express adrenergic and cho-linergic receptors, which make them ‘sensitive’ to catechol-amines (Verburg-van Kemenade et al 2011) Moreover,Chadzinska et al (2012) observed that adrenaline down-

A

B

Figure 3 Nitric oxide levels in Scophthalmus maximus after 15 (a) and 60 (b) days held at different treatments Control ( ■) or han- dled ( ) fish Data are presented as nitrite concentration in plasma, expressed as means  SD (n = 6) Different small letters stand for significant differences due to dietary treatment within control fish, while different capital letters stand for significant dif- ferences due to dietary treatment within handled fish (two-way ANOVA ; P < 0.05) * stands for significant differences attributed to chronic stress (t-test; P < 0.05).

Figure 2 Respiratory burst activity of head-kidney leucocytes from

Scophthalmus maximus after 60 days held at different treatments.

Control ( ■) or handled () fish Data are presented as V max rates of

ferricytochrome C reduction, expressed as means  SD (n = 3).

Different letters stand for significant differences within chronically

stressed fish (two-way ANOVA ; P < 0.05), and * stands for

signifi-cant differences attributed to chronic stress (t-Test; P < 0.05).

.

synthase) expression and decreased respiratory burst

activ-ity and NO production in carp head-kidney leucocytes

expres-sion levels in gilthead seabream head-kidney cells (Castillo

In the present study, most changes in turbot immune

parameters were observed after 60 days In particular,

decreased, while the relative percentage of neutrophils was

augmented regardless of dietary treatment Interestingly,

neutrophils seem to be protected from stress in fish For

instance, a change in ambient temperature reduced the

number of circulating lymphocytes, whereas circulating

neutrophils increased in carp and rainbow trout (Engelsma

Go-mes (2009) observed that social subordination decreased

the percentage of thrombocytes and lymphocytes, while

neutrophil values were augmented in matrinx~a (Brycon

amazonicus) Plasma ACP levels also increased in handled

specimens regardless of dietary treatment In fact, increased

plasma ACP activity has been associated with augmented

neutrophil numbers in the Senegalese sole peritoneal cavity

increased in Senegalese sole submitted to repeated handling

after 15 days (Costas et al 2011) Therefore, these immune

mechanisms appear to be modulated by stress but not

nec-essarily by cortisol, as already mentioned above Weyts

sensitivity to cortisol-induced apoptosis is subpopulation

specific as B cells are very sensitive, whereas the T-cell

frac-tion was hardly affected, and thrombocytes were unaffected

by addition of cortisol

Dietary arginine supplementation may have increasedsome aspects of the turbot immune system in both handledand unstressed specimens For instance, an increase in therelative percentage of circulating lymphocytes was observed

in handled specimens fed the Arg 7 and Arg 11 diets after

15 days, while the relative percentage of peripheral cytes augmented in both control and handled fish fed theArg 11 diet after 60 days This increase in lymphocyte andmonocyte numbers could be related to an increased avail-ability of polyamines for leucocyte growth and differentia-tion Arginine is a precursor substrate for polyaminebiosynthesis, which is required for animal cell growth anddifferentiation and is also involved in various steps ofDNA, RNA and protein synthesis (Satriano et al 1999;Soda et al 2005) Therefore, considering the role of lym-phocytes and monocytes in acquired and innate immunity,together with the increased cellular (respiratory burst activ-ity and NO production) and humoral immune parametersobserved in control and handled fish fed arginine supple-ments, the present study suggests that dietary arginineappears to be of great importance for the turbot immunemechanisms

mono-During stressful periods, plasma arginine levels decrease

in both mammals and fish (Komarov & Reddy 1998; tas et al 2008) Consequently, high doses (i.e levels abovethose found in standard diets) of arginine are added to ent-eral formulas or to experimental animal diets to improveimmune outcomes during critical illness (Butler et al 2005;Costas et al 2011) In fact, Senegalese sole fed a diet witharginine supplementation increased resistance to a bacterialchallenge when compared with specimens fed a control diet(Costas et al 2011) Therefore, results from this study are

Cos-Table 7 Plasma lysozyme, superoxide dismutase and alternative complement pathway (presented as ACH50 values) activities of mus maximus after 15 and 60 days held at different treatments

Scophthal-Humoral parameters

Dietary treatments

* Significant differences attributed to chronic stress (t-test; P < 0.05).

Values are means  SD (n = 6) Different small letters stand for significant differences due to dietary treatment within control fish, while different capital letters stand for significant differences due to dietary treatment within handled fish (two-way ANOVA ; P < 0.05).

.

Aquaculture Nutrition 19; 25–38 ª 2013 John Wiley & Sons Ltd

particularly important for the aquaculture industry as

peri-ods of chronically stressful conditions are sometimes

unavoidable, and dietary arginine supplements would thus

be a valuable nutraceutical tool to attenuate immune

sup-pression in chronically stressed fish

In summary, the present study corroborates the

suppres-sive effects attributed to chronic stress on the fish immune

system Although those effects are generally attributed to

increased glucocorticoid levels, increasing evidence suggests

that other stress-related hormones may also suppress

immune function In the current study, repeated handling

stress decreased NO production, lysozyme activity and

peripheral lymphocyte numbers already after 15 days, while

more profound effects were observed after 60 days

How-ever, not all effects were detrimental as ACP activity and

circulating neutrophils increased in handled specimens after

60 days Significantly, dietary arginine supplementation

decreased plasma cortisol levels after both 15 and 60 days

mechanisms assessed after 60 days However, as arginine

and lysine may have interacted antagonistically, not all

effects are necessarily due solely to increased arginine alone

The exact mechanisms by which arginine decreased cortisol

levels in chronically stressed turbot remain to be elucidated,

and future studies should address mechanisms of action and

regulation of the monoaminergic system and

immune-related genes in chronically stressed fish It is suggested that

diets containing 4% of arginine (DM basis) may be used

during stressful aquaculture practices to minimize negative

effects of chronic stress on turbot immune system and

there-fore attenuate eventual infections due to opportunistic

pathogens

The authors are deeply thankful to Jonathan Wilson

(CIIMAR, Porto, Portugal) for a careful linguistic review

of the manuscript SPAROS participation in this study had

the support of project ANYFISH, cofunded by PO Algarve

21, QREN and FEDER (European Union) This study was

partially supported by the European Regional

Develop-ment Fund (ERDF) through the COMPETE - Operational

Competitiveness Programme and national funds through

FCT – Foundation for Science and Technology, under

the project “PEst-C/MAR/LA0015/2011“C/MAR/LA0015/

2011 Benjamın Costas and Paulo Cesar N.P R^ego

Tecnolo-gia, Portugal (SFRH/BPD/77210/2011 and SFRH/BD/

30907/2006, respectively)

Afonso, A., Lousada, S., Silva, J., Ellis, A.E & Silva, M.T (1998) Neutrophil and macrophage responses to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss A light and electron microscopic cytochemical study Dis Aquat Org.,

34, 27–37.

Ainsworth, A.J., Dexiang, C & Waterstrat, P.R (1991) Changes

in peripheral blood leucocyte percentages and function of trophils in stressed channel catfish J Aquat Anim Health, 3,

neu-41 –47.

Alam, M.D.S., Teshima, S.I., Ishikawa, M & Koshio, S (2002) Effects of dietary arginine and lysine levels on growth perfor- mance and biochemical parameters of juvenile Japanese flounder Paralichthys olivaceus Fish Sci., 68, 509–516.

Anderson, L.C., Lewis, A.J., Peo, E.R.J & Crenshaw, J.D (1984) Effect of various dietary arginine: lysine ratios on performance, carcass composition and plasma amino acid concentrations of growing-finishing swine J Anim Sci., 58, 362–368.

Arag ~ao, C., Corte-Real, J., Costas, B., Dinis, M.T & Conceicß~ao, L.E.C (2008) Stress response and changes in amino acid require- ments in Senegalese sole Solea senegalensis Kaup 1758 Amino Acids, 34, 143–148.

Arends, R.J., Mancera, J.M., Mu ~noz, J.L., Wendelaar Bonga, S.E.

& Flik, G (1999) The stress response of the gilthead sea bream (Sparus aurata L.) to air exposure and confinement J Endocr.,

163, 149–157.

Barton, B.A (2002) Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids.

Integ Comp Biol., 42, 517–525.

Berge, G.E., Sveier, H & Lied, E (2002) Effects of feeding tic salmon (Salmo salar L.) imbalanced levels of lysine and argi- nine Aquac Nutr., 8, 239–248.

Atlan-Buentello, V.E.R & Gatlin, D.M (2001) Effects of elevated tary arginine on resistance of channel catfish to exposure to Ed- wardsiella ictaluri J Aquat Anim Health, 13, 194–201.

die-Butler, M.F.S., Langkamp-Henken, B., Herrlinger-Garcia, K.A., Klash, A.E., Szczepanik, M.E., Nieves, C., Cottey, R.J &

Bender, B.S (2005) Arginine supplementation enhances induced splenocyte proliferation but does not affect in vivo indicators of antigen-specific immunity in mice J Nutr., 135,

mitogen-1146 –1150.

Castillo, J., Teles, M., Mackenzie, S & Tort, L (2009) related hormones modulate cytokine expression in the head kid- ney of gilthead seabream (Sparus aurata) Fish Shellfish Immu- nol., 27, 493–499.

Stress-Chadzinska, M., Tertil, E., Kepka, M., Hermsen, T., Scheer, M &

Verburg-van Kemenade, L.B.M (2012) Adrenergic regulation of the innate immune response in common carp (Cyprinus carpio L.) Dev Comp Immunol., 36, 306–316.

Cohen, S.A., Meys, M & Tarvin, T.L (1989) The Pico-Tag Method – A Manual of Advanced Techniques for Amino Acids Analysis Waters, Division of Millipore, Bedford, USA.

Conceic ß~ao, L.E.C., Arag~ao, C., Dias, J., Costas, B., Terova, G., Martins, C & Tort, L (2012) Dietary nitrogen and fish welfare.

Fish Physiol Biochem., 38, 119–141.

Costas, B., Arag ~ao, C., Mancera, J.M., Dinis, M.T & Conceicß~ao, L.E.C (2008) High stocking density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegal- ensis (Kaup 1858) juveniles Aquac Res., 39, 1–9.

Costas, B., Conceic ß~ao, L.E.C., Dias, J., Novoa, B., Figueras, A &

Afonso, A (2011) Dietary arginine and repeated handling increase disease resistance and modulate innate immune mechanisms of

.

Senegalese sole (Solea senegalensis Kaup, 1858) Fish Shellfish

Immunol., 31, 838–847.

Costas, B., Arag~ao, C., Soengas, J.L., Mıguez, J.M., Rema, P.,

Dias, J., Afonso, A & Conceic ß~ao, L.E.C (2012a) Effects of

die-tary amino acids and repeated handling on stress response and

brain monoaminergic neurotransmitters in Senegalese sole (Solea

senegalensis) juveniles Comp Biochem Physiol., 161A, 18–26.

Costas, B., R ^ego, P.C.N.P., Sim~oes, I., Marques, J.F.,

Castro-Cunha, M & Afonso, A (2012b) Cellular and humoral immune

responses of Senegalese sole (Solea senegalensis Kaup, 1858)

fol-lowing challenge with two Photobacterium damselae subsp

pisci-cida strains from different geographical origins J Fish Dis , in

press doi: 10.1111/jfd12033.

Engelsma, M.Y., Hougee, S., Nap, D., Hofenk, M., Rombout,

J.H., van Muiswinkel, W.B & Verburg-van Kemenade, B.M.L.

(2003) Multiple acute temperature stress affects leucocyte

popu-lations and antibody responses in common carp Cyprinus carpio

L Fish Shellfish Immunol., 15, 397–410.

Fast, M.D., Hosoya, S., Johnson, S.C & Afonso, L.O.B (2008)

Cortisol response and immune-related effects of Atlantic salmon

(Salmo salar Linnaeus) subjected to short- and long-term stress.

Fish Shellfish Immunol., 24, 194–204.

Ferraz, F.B & Gomes, L.C (2009) Social relationship as inducer

of immunological and stress responses in matrinx ~a (Brycon

amazonicus) Comp Biochem Physiol., 153A, 293–296.

Flik, G., Klaren, P.H.M., Van den Burg, E.H., Metz, J.R &

Huis-ing, M.O (2006) CRF and stress in fish Gen Comp Endocrinol.,

146, 36–44.

Hubbard, P.C., Barata, E.N., Oz orio, R.O.A., Valente, L.M.P &

Can ario, A.V.M (2011) Olfactory sensitivity to amino acids in

the blackspot sea bream (Pagellus bogaraveo): a comparison

between olfactory receptor recording techniques in seawater.

J Comp Physiol A., 197, 839–849.

Irwin, S., Kenny, A.P., O’Halloran, J., FitzGerald, R.D &

Dug-gan, P.F (1999) Adaptation and validation of a

radioimmunoas-say kit for measuring plasma cortisol in turbot Comp Biochem.

Physiol., 124C, 27–31.

Jentoft, S., Aastveit, A.H., Torjesen, P.A & Andersen, Ø (2005)

Effects of stress on growth, cortisol and glucose levels in

non-domesticated Eurasian perch (Perca fluviatilis) and non-domesticated

rainbow trout (Oncorhynchus mykiss) Comp Biochem Physiol.,

141A, 353–358.

Kaushik, S.J (1998) Whole body amino acid composition of

Euro-pean seabass (Dicentrarchus labrax), gilthead seabream (Sparus

aurata) and turbot (Psetta maxima) with an estimation of their

IAA requirement profiles Aquat Living Resour., 11, 355–358.

Komarov, A.M & Reddy, M.N (1998) Effect of septic shock on

nitrate, free amino acids, and urea in murine plasma and urine.

Clin Biochem., 31, 107–111.

Law, W.Y., Chen, W.H., Song, Y.L., Dufour, S & Chang, C.F.

(2001) Differential in vitro suppressive effects of steroids on

leu-kocyte phagocytosis in two teleosts, tilapia and common carp.

Gen Comp Endocrinol., 121, 163–172.

Li, P., Mai, K., Trushenski, J & Wu, G (2009) New developments

in fish amino acid nutrition: towards functional and

environmen-tally oriented aquafeeds Amino Acids, 37, 43–53.

Ma, X., Lin, Y., Jiang, Z., Zheng, C., Zhou, G., Yu, D., Cao, T.,

Wang, J & Chen, F (2010) Dietary arginine supplementation

enhances antioxidative capacity and improves meat quality of

finishing pigs Amino Acids, 38, 95–102.

Neumann, N.F., Fagan, D & Belosevic, M (1995) Macrophage

activating factor(s) secreted by mitogen stimulated goldfish

kid-ney leucocytes synergize with bacterial lipopolysaccharide to

induce nitric oxide production in teleost macrophages Dev Comp Immunol., 19, 473–482.

Person-Le Ruyet, J (2010) Turbot culture In: Practical Flatfish Culture and Stock Enhancement (Daniels, H.V & Watanabe, W.O eds), pp 125 –139 Wiley-Blackwell Ltd., Ames, IA Peter, M.C.S (2011) The role of thyroid hormones in stress response in fish Gen Comp Endocrinol., 172, 198–210.

Quade, M.J & Roth, J.A (1997) A rapid, direct assay to measure degranulation of bovine neutrophil primary granules Vet Immu- nol Immunopathol., 58, 239–248.

Saeij, J.P.J., Verburg-van Kemenade, L.B.M., van Muiswinkel, W.B & Wiegertjes, G.F (2003) Daily hangling stress reduces resistance of carp to Trypanoplasma borreli: in vitro modulatory effects of cortisol on leukocyte function and apoptosis Dev Comp Immunol., 27, 233–245.

Salas-Leiton, E., Anguis, V., Mart ın-Antonio, B., Crespo, D., nas, J.V., Infante, C., Ca~navate, J.P & Manchado, M (2010) Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole (Solea senegalensis): potential effects on the immune response Fish Shellfish Immunol., 28,

Pla-296 –302.

Satriano, J., Ishizuka, S., Archer, D.C., Blantz, R.C & Kelly, C.J (1999) Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-alpha and IFN-gamma.

Am J Phys., 276C, 892–899.

Secombes, C.J (1990) Isolation of salmonid macrophages and analysis of their killing activity In: Techniques in Fish Immu- nology (Stolen, J.S., Fletcher, T.C., Anderson, D.P., Roberson, B.S & van Muiswinkel, W.B eds), pp 137 –154 SOS Publica- tions, Fair Haven, NJ.

Small, B.C & Soares, J.R (2000) Quantitative dietary lysine requirement of juvenile striped bass Morone saxatilis Aquac Nutr., 6, 207–212.

Soda, K., Kano, Y., Nakamura, T., Kasono, K., Kawakami, M.

& Konishi, F (2005) Spermine, a natural polyamine, suppresses LFA-1 expression on human lymphocyte J Immunol., 175, 237– 245.

Suenaga, R., Tomonaga, S., Yamane, H., Kurauchi, I., shi, Y., Sato, H., Denbow, D.M & Furuse, M (2008) Intracere- broventricular injection of L-arginine induces sedative and hypnotic effects under an acute stress in neonatal chicks Amino Acids, 35, 139–146.

Tsuneyo-Sunyer, J.O & Tort, L (1995) Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are affected by the alternative complement pathway Vet Immunol Immunopathol.,

45, 333–345.

Sunyer, J.O., Gomez, E., Navarro, V., Quesada, J & Tort, L (1995) Physiological responses and depression of humoral com- ponents of the immune system in gilthead sea bream (Sparus au- rata) following daily acute stress Can J Fish Aquat Sci., 52,

2339 –2346.

Tafalla, C & Novoa, B (2000) Requirements for nitric oxide duction by turbot (Scophthalmus maximus) head kidney macro- phages Dev Comp Immunol., 24, 623–631.

pro-Tibaldi, E., Tulli, F & Lanari, D (1994) Arginine requirement and effect of different dietary arginine and lysine levels for fingerling sea bass (Dicentrarchus labrax) Aquaculture, 127, 207–218 Tort, L (2011) Stress and immune modulation in fish Dev Comp Immunol., 35, 1366–1375.

Tort, L., Sunyer, J.O., G omez, E & Molinero, A (1996) Crowding stress induced changes in serum haemolytic and agglutinating activity in the gilthead sea bream Sparus aurata Vet Immunol Immunopathol., 51, 179–188.

.

Aquaculture Nutrition 19; 25–38 ª 2013 John Wiley & Sons Ltd

Valenzuela, A.E., Silva, V.M & Klempau, A.E (2008) Effects of

different artificial photoperiods and temperatures on

haemato-logical parameters of rainbow trout (Oncorhynchus mykiss) Fish

Physiol Biochem., 34, 159–167.

Varsamos, S., Flik, G., Pepin, J.F., Wendelaar Bonga, S.E &

Bre-uil, G (2006) Husbandry stress during early life stages affects

the stress response and health status of juvenile sea bass

Dicen-trarchus labrax Fish Shellfish Immunol., 20, 83–96.

Verburg-van Kemenade, L.B.M., Stolte, E.H., Metz, J.R &

Chad-zinska, M (2009) Neuroendocrine-immune interactions in teleost

fish In: Fish Physiology Vol 28 (Bernier, N.J., Van Der Kraak,

G., Farrell, A.P & Brauner, C.J eds), pp 313 –364 Academic

Press Inc., San Diego, CA.

Verburg-van Kemenade, L.B.M., Ribeiro, C.M.S & Chadzinska,

M (2011) Neuroendocrine –immune interaction in fish:

differen-tial regulation of phagocyte activity by neuroendocrine factors.

Gen Comp Endocrinol., 172, 31–38.

Vijayan, M.M., Pereira, C., Gordon Grau, E & Iwama, G.K.

(1997) Metabolic response associated with confinement stress in

tilapia: the role of cortisol Comp Biochem Physiol., 116C, 89–95.

Waring, C.P., Stagg, R.M & Poxton, M.G (1996) Physiological

responses to handling in the turbot J Fish Biol., 48, 161–173.

Wendelaar Bonga, S.E (1997) The stress response in fish Physiol.

Rev., 7, 591–625.

Weyts, F.A., Flik, G., Rombout, J.H.W.M & Verburg-van nade, B.M (1998) Cortisol induces apoptosis in activated B cells,

Keme-no in other lymphoid cells of the common carp, Cyprinus carpio

L Dev Comp Immunol., 22, 551–562.

Weyts, F.A.A., Cohen, N., Flik, G & Verburg-van Kemenade, B.M.L (1999) Interactions between the immune system and the hypothalamo-pituitary-interrenal axis in fish Fish Shellfish Immunol., 9, 1–20.

Wu, G & Morris, S.M (1998) Arginine metabolism: nitric oxide and beyond Biochem J., 336, 1–17.

Yao, K., Guan, S., Li, T., Huang, R., Wu, G., Ruan, Z & Yin,

Y (2011) Dietary L-arginine supplementation enhances intestinal development and expression of vascular endothelial growth fac- tor in weanling piglets Br J Nutr., 105, 703–709.

Zhou, F., Shao, Q., Xiao, J., Peng, P., Ngandzali, B.-O., Sun, Z.

& Ng, W.-K (2011) Effects of dietary arginine and lysine levels

on growth performance, nutrient utilization and tissue cal profile of black sea bream, Acanthopagrus schlegelii, finger- lings Aquaculture, 319, 72–80.

biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi- biochemi-.

The Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao, China

An eight-week feeding experiment was conducted to

inves-tigate the effects of dietary crystalline methionine (CMet)

or oligo-methionine (OMet) on growth performance and

feed utilization of white shrimp, Litopenaeus vannamei A

practical diet was used as control diet The other four

diets replacing 30% and 60% fish meal by plant meal

were formulated To balance the methionine content, 1 g

CMet (SPP60-CMet) or OMet (SPP60-OMet) was added

in 60% fish meal replacing diets Results showed that

methionine source significantly affected growth, body

com-positions and hepatosomatic indices (HSI) of white shrimp

sig-nificantly lower weight gain, body crude protein content

However, no significant difference in these indices was

observed between the control and OMet supplemented

showed significantly higher feed efficiency ratio and

pro-tein efficiency ratio than those in SPP30-CMet treatment

CMet, dietary OMet resulted in better growth and feed

efficiency of L vannamei fed with plant protein-enriched

diets

pro-tein, shrimp

Received 12 August 2012; accepted 15 April 2013

Correspondence: W.B Zhang, The Key Laboratory of Mariculture

(Ministry of Education), Ocean University of China, 5 Yushan Road,

Qingdao, Shandong, 266003, China E-mail: wzhang@ouc.edu.cn

At present, despite the growth of aquaculture, the globalproduction of fish meal has remained fairly static Con-sumption of fish meal is increasingly being concentrated inAsia with China continuing as by far the single largestmarket (IFFO, 2011) Commercial shrimp feed containsabout 25% fish meal (Tacon & Barg 1998) The global use

of fish meal for commercial shrimp feed was more than 800thousand tons in recent years Due to the limited produc-tion and increasing price of fish meal, the amount of fishmeal can be used in shrimp feed has to be decreased(Tacon & Metian 2008) Alternative dietary protein sources

to replace fish meal in shrimp feed are important for thefuture of the shrimp farming industry Plant proteinsources hold promise because of their secure supply andlower price However, they are less suitable for aquatic ani-mal feed than fish meal because of the presence of anti-nutrients and suboptimal essential amino acid profile(Tacon 1994)

Addition of crystalline amino acids (CAAs) to lated aquatic feed is used to balance the amino acid com-position and is likely to become an increasingly commonpractice in aquatic industry (Yuan et al 2011) Severalstudies have shown that CAAs were well utilized in meetingamino acid requirements of fish (Espe & Lied 1994;Rodehutscord et al.1995; Williams et al 2001; Rollin et al.2003; Espe et al 2006) However, there are still someobstacles to be overcome when using CAAs in shrimp feed.CAAs represent high potential for leaching from feed pel-lets and may have different adsorption rate in the hostcompared with intact protein Consequently, it couldreduce the efficiency of protein synthesis (Yuan et al.2011) Several studies in different fish and shrimp specieshave indicated that CAAs appear to be utilized with alower efficiency than amino acid supplied by intact protein(Espe & Njaa 1991; Schuhmacher & Gropp 1997; Zarate & .

formu-ª 2013 John Wiley & Sons Ltd

.

Aquaculture Nutrition

Lovell 1997; Refstie et al 2001; Liu et al 2002; Dabrowski

2007) Therefore, it is necessary to find substitute with low

leaching loss and high utilization efficiency to replace

CAAs

has been studied by many researchers for a wide array of

proteases and amino acid ester over the years (Schwab

oligo-methionine from methionine ethyl ester using papain

as a catalyser High availability of this synthesized

oligo-methionine (OMet) has been reported in rats (Chiji et al

1990; Hara & Kiriyama 1991; Kasai et al 1996) The

absorbability and bioavailability of oligo-methionine have

been also studied in fish (Masumoto et al 1999) However,

no data on using this enzymatic synthesized methionine in

shrimp feed have been published This synthesized OMet

has the potential to be used as substitute for crystalline

methionine (CMet) in shrimp feed because of its form as

the combined amino acid, and the water-insoluble

charac-ter, which may reduce leaching loss in water Thus, the

objective of this study was to compare the effects of two

different dietary methionine sources (CMet and OMet) on

growth performance and feed utilization of white shrimp

(Litopenaeus vannamei) fed with plant protein-enriched

diets

The oligo-methionine was prepared by ourselves in

labora-tory scale from methionine ethyl ester (Sigma-Aldrich, St

Louis, MO, USA) using papain (A3824, Applichem) as a

catalyser according to the method of Jost et al (1980) The

product was a mixture of peptides from dipeptide to

octa-peptide confirmed by mass spectrometry analysis The

aver-age peptide chain length of the oligo-methionine is 5.02

calculated based on the method of Schwab et al (2012)

fish meal, which used as control diet (Table 1) Based on

the control diet, the other four isonitrogenous and isolipid

diets were formulated Thirty per cent or sixty per cent fish

meal in the control diet was replaced by a mixture of

soy-bean meal protein and peanut meal protein (SPP) In 30%

SPP30-OMet) was added In 60% fish meal replacing diets,

added The five experimental diets had the same tion of methionine Dietary cysteine concentration was alsoadjusted to the similar level with that of control diet Die-tary lysine was adjusted to meet the requirement of whiteshrimp Even in the SPP60-CMet and SPP60-OMet diets,

Ingredients were ground into fine powder through

mixed with the fish oil, and water was added to producestiff dough The dough was then pelleted with an experi-mental feed mill (F-26; South China University of Technol-ogy, Guangdong, China) and dried for about 12 h in a

Leaching loss of methionine from diets of SPP30-CMet,SPP30-OMet, SPP60-CMet and SPP60-OMet was mea-

30 min according to Smith et al (2007) with some cations Briefly, about 1 g diet was put into the 500-mLbeaker containing 400 mL of sea water and mechanically

and rinsed gently and briefly in distilled water, then dried

White shrimps L vannamei were obtained from a cial hatchery and acclimated to the system for 2 weeksbefore trials During this period, the shrimps were fed thecontrol diet

ran-domly distributed into 5 treatments, each of which has 3 licates Each replicate has 30 shrimps in a 300-l tank (filledwith 200 L seawater) The shrimps were fed with experimen-tal diets to apparent satiation four times daily (06 : 00,

rep-12 : 00, 18 : 00 and 24 : 00) for 8 weeks Seawater in eachtank was changed for 2/3 every day Photoperiod was pro-vided using a 12-h light: 12-h dark regime The uneaten feedwas collected 60 min after each meal, dried to constant

consump-tion During the experimental period, water temperature was

. .