A review on broodstock nutrition of marine pelagic spawners: the curious case of the freshwater eels...

1 2 2 3 4

5 2 3

1 Aquaculture and Fisheries Group, Wageningen University, Wageningen, The Netherlands; 2 Section for Coastal Ecology,

DTU Aqua, National Institute of Aquatic Resources, Danish Technical University, Charlottenlund, Denmark; 3 INRA,

UR1067 Nutrition Metabolism Aquaculture, Saint-P�ee-sur-Nivelle, France; 4 National Institute of Sciences and Technolo-

gies of the Sea, Tunis, Tunisia; 5 Biomar A/S, Brande, Denmark

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 eels 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.

KEY WORDS: amino acids, Anguilla spp., broodstock 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 (Anguilla

anguilla, 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 as

causes for the current decline of the eel population (van

Ginneken & Maes 2005). The major part of eel production

now 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, because

broodstock eels stop feeding when silvering in nature.

Although silvering is reversible and feeding can be resumed

when migration is not initiated (Sved€ang & Wickstr€om

1997; Durif & Elie 2008), it has been shown for A. japonica

that eels caught in the spawning area had not been eating in

the marine phase of the migration (Chow et al. 2010). Also

in captivity, feeding is terminated after transfer to saltwater

prior to induction of maturation. Thus, for eels, all the

qualitative and quantitative requirements for reproduction

have to be met from their body reserves highlighting the

2013 19; 1–24 doi: 10.1111/anu.12091. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ª 2013 John Wiley & Sons Ltd

Aquaculture Nutrition

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

et al. 2010), is not yet started (Durif et al. 2006; Palstra

et al. 2011). Other temperate eels, A. japonica (Sudo et al.

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

transport (COT, kJ kg�1 km�1) are influenced by both the

distance to the spawning area and the swimming speed (Pal-

stra & van den Thillart 2010). At optimal swimming speeds

of 0.4–0.6 m s�1, both females and males eels have a COT

of 0.4–0.7 kJ kg�1 km�1, when measured by oxygen

consumption, or a COT of 0.6–1 kJ kg�1 km�1, when 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,

A. anguilla uses between 15% and 40% of its initial energy

reserves for migration. Of this energy, about 70–80% is 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 to

40% have been reported (Lau 1987; Tsukamoto et al.

2011), but it is not clear whether this is a true difference or

the result of a further advanced emaciation of these eel’s

soma. Maturing female eels reach GSI of 40–60% or, due

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

matter (dm dm�1) basis (Bo€etius & Bo€etius 1980; Lau

1987; Yamada et al. 2001).

Little is known about the composition of the testes and

the semen of eels. Cheung (1983) and Lau (1987) reported

for A. japonica testes lipid levels of 8–11 g kg�1

(30–60 g kg�1 dm), for both immature and ripe testes. Tes-

tes lipid levels were a factor 10 lower than ovary lipid lev-

els. The few data available for A. anguilla suggest that

testes lipid levels are higher in this species, 90–160 g kg�1

(430–540 g kg�1 dm), with little variation between imma-

ture and ripe testes (Kokhnenko et al. 1977; Amin 1991).

Indirect evidence for a high lipid level of the immature tes-

tes of A. anguilla can also be deduced from the testes fatty

acid profile (Mazzeo et al. 2010); Table 3). Due to the high

lipid level, testes protein levels are a little bit lower in

A. anguilla, but total energy deposited in the testes seems

similar between species, 0.8–1 MJ kg�1 initial body mass

or 5–10% of the initial body energy (Lau 1987; Amin

1991).

Lipid levels in the immature and early stage ovaries of

A. anguilla are higher (150–300 g kg�1 or 500–

650 g kg�1 dm, Palstra et al. 2006; Bo€etius & Bo€etius

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

2011) than of A. japonica (50–150 g kg�1 or 300–

500 g kg�1 dm (Lau 1987; Cheung 1983; Ozaki et al.

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

between species, 50–60 g kg�1 or 400-500 g kg�1 dm

(Bo€etius & Bo€etius 1980; Ozaki et al. 2008). Still, also egg

lipid levels in A. anguilla seem to be somewhat higher,

60–65 g kg�1 (Corraze et al. 2011) versus 42–55 g kg�1 for

A. japonica (Furuita et al. 2006; Tanaka et al. 2006; Ozaki

et al. 2008). There are no indications that the protein levels

in the ovaries and the eggs differ between species. Deposi-

tion of total lipid and (crude) protein in the ovaries of

A. anguilla (Bo€etius & Bo€etius 1980) is shown in Fig. 1.

Initially more lipid is deposited, but in mature ovaries,

both lipid and protein converge at c. 50 g kg�1 initial body

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

57 � 22 g kg�1. Based on these lipid and protein deposi-

tions, energy deposition in the ovary of A. anguilla is esti-

mated to be 3–3.5 MJ kg�1 initial body mass, or 17–23%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

individual initial lipid contents of these wild eels (Bo€etius

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

energy requirement for ovary development would become

8–9.5 MJ kg�1 initial body mass, or 46–62% of the initial

body energy.

Marine pelagic fish eggs In marine pelagic fish eggs, two

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 but

also in embryonic metabolism. The type II eggs contain

more lipids, and within these lipids, a (much) larger frac-

tion is neutral lipids (Table 1). In the neutral lipids, they

further contain a larger (although variable) fraction of

wax- and sterol-esters (Wiegand 1996). All marine pelagic

eggs contain similar amounts of total amino acids, but

these are more present as free amino acids (FAA) in the

type 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

egg buoyancy (Rønnestad et al. 1999; Cerd�a et al. 2007;

Finn & Fyhn 2010).

Although eel eggs can be categorized as type II eggs,

even within this group, eel eggs are at the extreme end of

the spectrum (Table 1). If and how this position, which is

extended in the fatty acid and FAA profiles (sections Lip-

ids and Protein and amino acids), is related to the large

perivitelline space is not clear (Unuma et al. 2005).

0

10

20

30

40

50

60

Prote

in,

lipid

(g/k

g initia

l body

mas

s)

GSI (%)y = 0.37x – 1.04

R² = 0.43

0

10

20

30

40

50

60

Ova

ry e

ner

gy

(kJ/

kg0.8

/d)

Energy use (kJ/kg0.8/d)

y = 0.68x – 0.25R² = 0.71

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50

0 50 100 150

0.0 0.5 1.0 1.5 2.0

Ova

ry p

rote

in (

g/k

g0.8

/d)

Protein use (g/kg0.8/d)

(a) (b)

(c)

Figure 1 Lipid and (crude) protein deposition in the ovary of artificially matured wild silver A. anguilla Calculated from Bo€etius & Bo€etius

(1980), who matured a series of 27 female eels, and afterwards determined the mass and the composition of the ovaries and the remaining

soma. Data on initial ovary composition from Palstra et al. (2006, 2011).

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

Lipids In most instances, reproduction of wild fish, or of

fish fed with natural food, is more successful than of

farmed fish. This has also been shown for A. anguilla

(Tomkiewicz 2012), but less so for A. japonica, at least

when feminized eels are used (Yamada et al. 2006). One of

the differences between wild and farmed eels (and between

males and females) are the body lipid levels (Fig. 2). In

A. anguilla, males start to silver, that is, initiating maturity,

at 80–150 g, while for females this is at sizes from 300 to

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

both sexes, attain lipid levels of 25–35% [a.o. (Bo€etius &

Bo€etius 1985; Larsson et al. 1990; Kamstra & Van He-

eswijk 1996; Garcia-Gallego & Akharbach 1998; Kn€osche

2009; Clevestam et al. 2011)]. Both farmed and wild males

reach these levels early, at masses of 70–100 g. Wild female

eels seem to follow another trajectory: they first invest in

body mass growth and reach these higher lipid levels at

higher body masses (Fig. 2). Although most farmed

females also have lower body lipid levels than males of

comparable mass (Kamstra & Van Heeswijk 1996), their

trajectory is clearly advanced compared with wild females.

Anguilla anguilla silver eels generally have much

higher body lipid levels than other temperate eels

(150–230 g kg�1, Han et al. 2000, 2001; Tremblay 2009;

De Silva et al. 2002; Hopkirk et al. 1975). In vertebrates,

the body lipid mass, through adiposity signals leptin and

insulin, is thought to influence reproduction in a number of

ways (Caprio et al. 2001). On the one hand, a minimum

lipid mass seems to be required to initiate puberty. Such an

effect was also observed in Oncorhynchus mykiss (Weil

et al. 2008). Furthermore, Peyon et al. (2001) showed in

Dicentrarchus labrax that (recombinant mouse) leptin stim-

ulated in vitro pituitary LH release in the prepubertal

stage, but much less in later stages. For A. anguilla, Lars-

son et al. (1990) even hypothesized that body lipid content

might be the trigger for silvering, but as silver A. anguilla

are prepubertal and also some silver eels have (very) low

body lipid levels (Sved€ang & Wickstr€om 1997), this seems

not so. On the other hand, excessive body lipid stores nega-

tively affect reproduction, through impairment of gonadal

steroidgenesis (Caprio et al. 2001). Evidence for a negative

effect of increased adiposity on reproduction in fish is

mostly anecdotal, in Indian (Chaudhuri, 1960) and Chinese

carps (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 (Cerd�a et al. 1994b)

could well also have originated from a lower DP/DE ratio,

as the gonads (but not the eggs) of the deficient fish

showed higher lipid levels during peak spawning. Finally,

Table 1 Size and composition of marine pelagic fish eggs

Anguilla

spp.1

Other marine pelagic

Oil globule2No oil

globule3

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 –

Egg dm ug egg�1 60–70 35–120 150

Moisture (g kg�1) 880–920 880–930 900–930

Total lipids

(g kg�1 dm)

350–440 130–450 70–150

NL (%TL) 80–83 40–84 25–40

TG (%NL) 30–40 30–84 25–45

CH (%NL) 6–20 20–50

WSE (%NL) 6–65 6–15

PL (%TL) 17–20 16–60 60–75

PC (%PL) 60–86 65–88

PE (%PL) 12–20 7–25

PI (%PL) 2–6 1–2

Total N (g kg�1 dm) 95 90–110 100–120

Protein (g kg�1 dm) 300–460 250–500 350–550

FAA (g kg�1 dm) 30–50 100–170 150–220

Carbohydrates

(g kg�1 dm)

3–24 6–20

Ash (g kg�1 dm) 83 60–100 60–150

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;

Cerd�a 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).

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

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

more weekly injections are needed, 12–25 (Durif et al.

2006; Palstra & Thillart 2009), to complete maturation

than for other eels, 8–14 injections (Lokman & Young

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

could be stimulating for reproduction. However, Bo€etius &

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

et al. (2005a) found that even prolonged swimming had no

effect on the body composition of A. anguilla. This may be

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

high body lipid content (340 g kg�1). It might be that at

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

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

et al. (1975) who reported a decline in muscle lipids from

90 to 30 g kg�1 in yellow A. anguilla fasted for more than

3 months at 2–10 °C. Induced maturation of A. anguilla is

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 strong

decrease.

In insects and birds, it has been shown that the demands

for specific lipid classes and FA differ between migration

and 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/

Musc

le lip

id (

g/k

g)

Body mass (g)

0

50

100

150

200

250

300

350

400

0 200 400 600 800 1000 1200 1400 1600

0 200 400 600 800 1000 1200 1400 1600

Body/

Musc

le lip

id (

g/k

g)

Body mass (g)

(a)

(b)

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.

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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

et al. (2009) did find in A. japonica that fasting and 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

reported to be 300–450 g kg�1 dm (Furuita et al. 2003a,

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

also mention TL levels as low as 200 g kg�1 dm. Furuita

et al. (2006) did find a negative correlation between egg TL

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

350–400 g kg�1 dm in A. japonica eggs until hatching and

decrease during yolk sac and oil droplet resorption to c.

160 g kg�1 dm (Tanaka et al. 2006). Ohkubo et al. (2008)

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 la-

landi (Hilton et al. 2008) and Latris lineata (Morehead

et al. 2001). This might well also have contributed to mor-

talities before first feeding, or even mouth formation, in

larvae of A. australis (Lokman & Young 2000) and A. ro-

strata (Oliveira & Hable 2010).

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

et al. 1980). It is a very specialized high-density serum

phospho-lipo-glyco-protein consisting 830–860 g kg�1 pro-

tein and 130–170 g kg�1 lipid. PL and TG account for

650–720 and 170–270 g kg�1, respectively, of the lipids

(Ando & Matsuzaki 1996; Komatsu et al. 1996). The fatty

acid profile of eel VTG is not known, but Silversand &

Haux (1995) showed for a number of fish species that the

fatty 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

A. japonica1

Other marine pelagic

Oil

globule2 No oil globule3

Total lipids

16:0 18.9 (18.1–21.6) 18.9 (13–21.5) 20 (17.3–23.5)

18:1 33.5 (31.4–35) 19.2 (9.7–25.7) 14.8 (11.2–17.6)

18:2 n-6 2 1.8 (0.3–7.5) 1 (0.3–2.7)

18:3 n-3 0.9 0.6 (0.1–1.1) 0.3 (0.2–0.5)

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)

22:5n-3 2.4 3 (1–4.6) 1.5 (1.2–1.8)

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)

18:2 n-6 0.7 (0.1–1.9) 0.6 (0.03–0.9)

18:3 n-3 0.3 (0.1–0.5) 0.2 (0.01–0.4)

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)

22:5n-3 2.4 (1.2–4.6) 1.0 (0.5–1.4)

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).

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

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

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

Scophthalmus maximus (Silversand et al. 1996) and Dicen-

trarchus labrax (Bell et al. 1997). DHA is highly conserved

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

et al. 1995). In most marine fish species, ARA is also 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 tropical

reef species (Ogata et al. 2004; Suloma & Ogata 2011). A

higher level of ARA in the PL of the (immature) ovaries

than 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 be

formed 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 doc-

umented (Takeuchi et al. 1980; Kissil et al. 1987), so

although of marine origin, they truly earn the name of

freshwater eels (NRC 2011). This capacity is also reflected

in the egg composition of A. japonica. Yamada et al.

(2006) showed that A. japonica fed sunflower oil, rich in

18:2, produced eggs with twice as much ARA than eels fed

with a marine oil (Fig. 5). A similar but less dramatic effect

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

et al. (2007) and, although less pronounced, by Ozaki et al.

(2008).

A selective incorporation of FA was also observed in the

testes and semen of farmed A. anguilla (Mazzeo et al.

2010) (Table 3). Whereas the FA profile of the immature

testes is essentially the same as that of the muscle, in the

mature testes, the levels of EPA, DHA and especially ARA

are increased. Remarkably, in the semen, EPA and ARA

are further increased, but not DHA. Although P�erez et al.

reported much lower levels of LC-PUFA in the semen of

farmed A. anguilla, they also found a low DHA/EPA ratio

which seems to be unique among the few marine teleosts

studied, 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 matura-

tion1

Week20 7–11

5–13

Tissue Muscle Liver Testis Muscle Liver Testis Semen Semen

Fatty acid

16:0 18 21.7 18.1 18.3 24.5 19.8 20 28.2

18:1 28.9 25.3 29.2 31 24.7 21.5 14 13.4

18:2n-6 7.2 5.3 6.7 7.1 6.5 4.2 3.3 2.5

18:3n-3 1.6 0.7 1.1 0.9 0.9 0.3 0.2

20:1 8.9 5.3 9.1 8.5 4.2 5 3.6 2.2

ARA 0.6 1.5 0.7 0.7 1.1 4.5 7 5.2

EPA 3.7 4.4 3.6 3.2 4.5 8.5 16 8.5

22:5n-3 2 2.4 2.1 2.1 2.2 2.2 1.6 0.8

DHA 7.8 14 7.7 7.6 15 19.1 22 7.7

EPA/ARA 6.2 2.9 5.1 4.6 4.1 1.9 2.3 1.6

DHA/EPA 2.1 3.2 2.1 2.4 3.3 2.2 1.4 0.9

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

Dicentrarchus labrax (Bell et al. 1996; Asturiano et al.

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

dominated by VLDL (25–30 g L�1 or more than 40% of

total lipoproteins), even after induction of vitellogenesis

with E2 injections, which brought VTG to 20 g L�1. Next

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) moni-

tored the incorporation of radioactivity from 14C-acetate

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

A. anguilla in different stages of maturation. They found

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 development (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 impairs

reproduction in fish (Watanabe et al. 1984a; Chou et al.

1993; Fern�andez-Palacios et al. 1995; Almansa et al. 1999).

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 LC-

PUFAs in the broodstock diet, the gonad and gametes

(Fern�andez-Palacios et al. 2011). However, little is known

about the physiological role of EPA and DHA during

gonad development, ovulation and fertilization. For ARA,

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

series 2 prostaglandins (PGE2 and PGF2a), are important

in the control of oocyte maturation and ovulation (Sorbera

et al. 2001; Kagawa et al. 2003), are probably involved in

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

A. japonica oocytes in vitro that PGF2a enhanced DHP-

induced ovulation. Indomethacin, actinomycin D and

cycloheximide blocked DHP-induced ovulation and PGF2a

reversed the effects of these inhibitors. Similar effects of the

series 2 PGs were observed by Sorbera et al. (2001) with in

vitro Dicentrarchus labrax oocytes. These authors further

showed that addition of free ARA induced maturation of

the 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

Carassius auratus testis in vitro through its conversion to

PGE2. Again both EPA and DHA blocked the steroido-

genic action of ARA and PGE2 (Wade et al., 1994, cited

by Fern�andez-Palacios et al. 2011). The timing of spermia-

tion may be delayed causing reduced fertilization rates due

to depressed steroidogenesis caused by broodstock EFA

deficiency or imbalance (Izquierdo et al. 2001). In larval

fish during endogenous feeding, ARA is selectively retained

and has been shown to enhance survival and stress response

(Tandler et al. 1995). Fuiman & Ojanguren (2011) found in

Sciaenops ocellatus no relation with egg FA profile and lar-

val survival and growth, but did show a strong positive

effect 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

et al. 2000), the minimal amount of n-3 LC-PUFA in the

broodstock diet seems to be 15-20 g kg�1 diet, with a mini-

mal level of DHA no more than 6–7 g kg�1 diet and a

minimum DHA/EPA ratio of 0.6. Lower levels of total n-3

LC-PUFA reduced fecundity, fertilization, hatching and

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

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

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

et al. (2002) found that also higher levels of n-3 LC-PUFA

(>20 g kg�1) impaired reproduction. However, in these

studies, both EPA and DHA were increased, EPA even

more than DHA in the study by Fern�andez-Palacios et al.

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

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

it was shown that DHA levels up to 40 g kg�1 diet did not

impair reproduction. Based on this, it seems that the opti-

mal range for EPA is much narrower, from 7–10 to c.

15 g kg�1 diet. Consequently, the optimal DHA/EPA ratio

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

vice versa (Fern�andez-Palacios et al. 2011). Most fish-oil-

based broodstock diets have ARA levels of 0.5–1.2 g kg�1

diet, with EPA/ARA ratios of 8–15. Positive effects, in par-

ticular on ovulation (fecundity) and fertilization, have been

observed in Dicentrarchus labrax (Bell et al. 1997; Bruce

et al. 1999), Hippoglossus hippoglossus (Mazorra et al.

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

2009; Sawanboonchun 2009) with an increase in dietary

ARA (up to 2–3 g kg�1 diet, with a concomitant decrease

in EPA/ARA ratio till 1.5–6). Already in the early work of

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 oli-

vaceus with increasing ARA levels from 1 to 6 g kg�1 in

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

increase in ARA dietary levels to 12 g kg�1 diet gave the

opposite effect. Similarly, negative effects of higher ARA

levels in both the PL and the NL of A. japonica eggs have

been shown (Furuita et al. 2003a, 2006) (Fig. 4), although

this is a bit puzzling in view of the fact that this ARA was

largely 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 and

larval development. Broodstock origin and diet did not

have a large effect on egg ARA level of A. japonica (Furui-

ta et al. 2003a) (Fig. 4a), although Ozaki et al. (2008) did

find lower levels of ARA in eggs of farmed A. japonica fed

with 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

et al. 2003). Not only has krill a low level of ARA (c.

1 g kg�1 or 0.8% of FA), but also a very high level of

EPA (c. 25 g kg�1 or 20% of FA), resulting in DHA/EPA

and EPA/ARA ratios of 0.5 and 25, respectively. Also Fur-

uita et al. (2006) reported some positive effects of frozen

krill in A. japonica broodstock, but egg FA composition

was not affected.

Apart from the levels and ratios of DHA, EPA and

ARA, ‘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-3

HUFA ratio in both the NL and PL of the eggs negatively

affected the fertilization rate. Such an effect does not seem

likely in eels, as even the eggs from wild eels show a high

18:1/n-3HUFA ratio (Table 2). Also negative effects of

high levels of 18:2 in the eggs, as reported by Bell et al.

(1997) and Palacios et al. (2011), do not seem to occur in

eels (Fig. 5).

Protein and amino acids Nitrogen-containing nutrients,

protein and amino acids, nucleic acids, and more, form a

large 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 of

N-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 cuttlefish

meal has a positive effect on reproduction of Pagrus

major (Watanabe et al. 1991b) and Sparus aurata (Tandler

et al. 1995; Fern�andez-Palacios et al. 1997), but whether

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 maturation in females, while a

serotonine depletor delayed gonad development (Akiyama

et al. 1996). Matsunari et al. (2006) showed for Seriola

quinqueradiata broodstock that addition of 10 g kg�1 of

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 of

endogenous origin, through DHP-stimulated biosynthesis

from cysteine in the testes. Gonzalez-Vecino et al. (2004)

reported that broodstock diets enriched with nucleotides

improved the first feeding success and survival of Melano-

grammus aeglefinus larvae. Whether this was through

incorporation in the eggs or through enhanced parental

physiology was not reported.

The bulk (85–90%) of the (total) amino acids in the

ovary and the eggs comes from VTG, the remainder largely

from the choriogenins, or zona pellucida (ZP) precursor

proteins (Pati~no & Sullivan 2002; Lubzens et al. 2010). The

amino acid composition of VTG (and therefore largely of

the 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 ZP

proteins 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).

0Ovu

late

dBu

oyan

tFe

rtiliza

tion

Hatc

hing

20

40

60

80

100

Perc

enta

ge

(%)

(b)

0

5

10

15

20

25

30

35

% t

ota

l fa

tty

acid

s

(a)Sunflower oil-fed group

Bonito oil-fed group

Figure 5 Fatty acid profile in the total lipids of eggs (a) and reproductive performance (b) of A. japonica fed with different oils. Data from

Yamada et al. (2006).

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

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

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

et al. 2006). In marine pelagic spawners, the hydration 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; Cerd�a et al. 2007; Finn & Fyhn 2010). Also anguillid

oocytes are hydrated during maturation, with a 3–4 fold

increase in volume, and an increase in moisture content

from 500–600 to 880–920 g kg�1 (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 in

other fish, its decline is linked to an even faster decline of

the major yolk protein, lipovitellin (Fig. 6). Further, some

FAA, serine and phenylalanine, are actually increasing

during the first 4 days, indicating that also phosvitin is

hydrolyzed (Ohkubo et al. 2008). Due to this constant

turnover of the FAA pool, correlations between levels and

ratios of specific AA and hatching rate and embryonic

development as reported in Lates calcarifer (Nocillado

et al. 2000), Rachycentron canadum (Nguyen et al. 2012)

and Dentex dentex (Samaee et al. 2010) are difficult to

interpret and might be spurious.

Carbohydrates Carbohydrates form a minor fraction in

fish eggs. They are present in glycoproteins, both struc-

tural, in the zona pellucida, and functional, in the con-

tents of the cortical alveoli (Pati~no & Sullivan 2002;

Lubzens et al. 2010). Free glucose, partly derived from

glycogen, has been shown to be the most important

energy source in early embryonic development (first cleav-

ages), both in type I (Finn et al. 1995a) and in type II

eggs (Finn et al. 1995c; Seoka et al. 1997; Gim�enez et al.

2006). It was further suggested that the most dominant

free 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 for

the large free glutamine pool in eel eggs (Fig. 7), because

this glutamine pool is almost completely used during the

first day after fertilization (Ohkubo et al. 2008). In Den-

tex dentex (Gim�enez et al. 2006) and Sparus aurata

(Lahnsteiner & Patarnello 2004), correlations were found

between hatchability and/or survival and levels of free

glucose and other carbohydrates, such as sialic acid and

ribose.

0

10

20

30

0

20

40

60

80

100

0 1 2 3 4

FAA (

nm

ol in

div

idual

–1)

OLv

(nm

ol AA indiv

idual

–1)

dpf

00

10

20

30

0

50

100

150

200

OC I OC II OV

FAA (

nm

ol in

div

idual

–1)

PAA (

nm

ol in

div

idual

–1)

Maturation stage

(b)(a)

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),

although also specific transport proteins have been

described (Brooks et al. 1997; Lubzens et al. 2010). Little is

known 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) vita-

min B6, pantothenic acid, nicotinic acid and vitamin C

0

2

4

6

8

10

12

14

16

18

Am

ino A

cid c

onte

nt

(mol%

)

0

5

10

15

20

25

30

35

Free

Am

ino A

cid c

onte

nt

(mol%

)

(a)

(b)

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).

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

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

(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

et al. 2011). Also in A. japonica, Furuita et al. (2003a)

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 Oncorhyn-

chus mykiss (Blom & Dabrowski 1995; Dabrowski &

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

et al. 2009a), egg VC levels tended to be higher in 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 (Vera-

kunpiriya et al. 1997b). Verakunpiriya et al. (1997a)

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

Pseudocaranx dentex (Vassallo-Agius et al. 2001b,c), while

carotenoids from paprika improved sperm and egg quality

of Sparus aurata (Scabini et al. 2011). Although the

A. japonica in most of the studies of Furuita et al. were fed

with frozen krill (Furuita et al. 2003a, 2006, 2009b), or

even with added synthetic astaxanthin (Furuita et al. 2007),

carotenoid levels in the eggs were not measured. Feeding

the A. japonica broodstock frozen krill had a slight, but

not significant, positive effect on reproduction, but not on

egg biochemical composition (Furuita et al. 2006).

What all antioxidants have in common is that they take

the ‘burden’ of peroxidation of the unsaturated lipids,

becoming radicals themselves. The liposoluble antioxidants

also 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 be

regenerated by glutathione (Palace & Werner 2006; Hamre

et al. 2010; Hamre 2011). Hamre et al. (1997) found that

marginally adequate VE levels could be protected by VC in

juvenile 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 that

carotenoids are even more active radical scavengers than

VE (Palace & Werner 2006), too much antioxidants might

also negatively affect reproduction. Chou & Chien (2006)

reported for Lateolabrax japonica that a combination of

VE and astaxanthin gave worse results than either VE or

astaxanthin alone. A similar effect might have caused the

reported 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 regula-

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

et al. 2011). Furuita et al. (2003b) found a positive effect

(increased spawning period and fecundity, increased per-

centage of normal larvae) when VA in broodstock diet for

Paralichthys olivaceus was increased from 0.75 to

16.9 mg kg�1. Vitamin A concentrations in the eggs were

not different. Earlier, they showed that an increase from 33

to 1000 mg kg�1 had no effect on reproduction of Para-

lichthys olivaceus (Furuita et al. 2001). Vitamin A concen-

tration in ovaries and eggs was higher with the highest

dietary level, but excess VA was mainly stored in the liver.

In Oncorhynchus mykiss, Fontagn�e-Dicharry et al. (2010)

found at a high (170 mg kg�1) VA concentration in the

broodstock diet an increased mortality in the offspring but

no malformations. It is noteworthy that all above-men-

tioned diets contained synthetic astaxanthin, from 50

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

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

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

2003b) mg kg�1. Furuita et al. (2009b) found in A. japon-

ica no correlations of egg VA concentrations with

fertilization, hatching and survival. The authors claimed a

tendency for lower survival at higher VA (>40 mg

kg�1 dm) in the eggs, but again also at lower levels, 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

et al. (2003) reported that folate levels in Hippoglossus hip-

poglossus eggs were highly variable, and in some instances

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).

Minerals Reported ash contents of late stage eel ovaries

vary from 70 g kg�1 dm (A. anguilla) (Bo€etius & Bo€etius

1980; Amin 1991) to 120 g kg�1 dm (A. japonica) (Lau

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

P, with 11 g kg�1 dm (Yamada et al. 2001). Watanabe

et al. (1984a) showed for Pagrus major that the (dietary)

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

A. japonica, Yamada et al. (2001) showed that c. 40% of

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 often

reported association of vitellogenin with Ca (Lopez et al.

1980; Lau 1987; Sbaihi et al. 2009; Palstra et al. 2010), the

primary goal of this bone demineralization does not seem

to be the provision of Ca to the ovary. Ovary Ca content

was only 0.02% dm (Yamada et al. 2001), giving a Ca/P

ratio of 0.02, which is however in accordance with other

fish species (Table 4). Furthermore, in the marine environ-

ment, requirements for Ca (and Mg, K) can be met by

absorption 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

Tanaka et al. (2006) as 83 g kg�1 dm. Information on

whether these were unfertilized or washed/unwashed fertil-

ized eggs was not given. Data on the mineral composition

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

in Pleuronectes platessa, the hydration of the oocytes dur-

ing maturation was accompanied by a large influx of K. In

unfertilized eggs of Pleuronectes platessa and Gadus morhua

(Craik & Harvey 1984) and washed fertilized eggs of Pag-

rus major (Watanabe et al. 1984c, 1985a), K is therefore

the most abundant mineral, still followed by P. The cho-

rion remains permeable to small osmolytes, and the mineral

Table 4 Mineral composition of ovaries and eggs (g or

mg kg�1 dm)

Ovaries Eggs

Anguilla

spp1 Other2Unfertilized/

washed3 Unwashed4

Ash (g kg�1) 70–120 32–60 50–80 150–200

Ca (g kg�1) 0.2 0.3–0.5 0.3–0.8 1.5–2

P (g kg�1) 11 8–15 6–13 7–10

Na (g kg�1) 2–3 3–5 23–35

K (g kg�1) 5–8 17–25 15–20

Mg (g kg�1) 0.4–1 0.7–1.1 4–9

Fe (mg kg�1) 15–60 11–23 17–60

Zn (mg kg�1) 20–400 102 80–140

Mn (mg kg�1) 2–3 0.7–1.5

Cu (mg kg�1) 1–6 8–11 3–5

1 Data on ash content from the study by Bo€etius & Bo€etius

(1980), Lau (1987) and Amin (1991), data on Ca and P content

from (Yamada et al. 2001).2 Gadus morhua (Rom�eo 1987; Hellou et al. 1992; Saxholt et al.

2008), Dicentrarchus labrax (Devauchelle & Coves 1988), Mugil

cephalus (Rom�eo 1987; Olguno�glu & Olguno�glu 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).

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

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

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

the level of Mn was strongly reduced, 1.6 mg kg�1 com-

pared with 4.1 mg kg�1 for eggs of trout fed a commercial

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

embryo mortalities and reduced hatching rates, was

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

pro-oxidants. Ando & Yanagida (1999) showed in

A. japonica that vitellogenin actually protected other

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

A. japonica a positive correlation between liver Cu level

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

Sciaenops ocellata and Micropogonias undulatus that Cd

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 natu-

rally contaminated Nereis diversicolor. Total As levels can

be quite high in fishmeals and fish oils used in aquaculture

feeds (Sloth et al. 2005). These authors claimed that this is

of little concern, because less than c. 1% of the As is in the

toxic inorganic form. However, Celino et al. (2009) showed

that as little as 7.5 lg L�1 inorganic As(V) inhibited sper-

matogenesis in A. japonica, although it should be noted

that they worked in an in vitro testicular organ culture sys-

tem with water exposure, which is often more critical than

dietary element exposure. The mode of action was through

inhibition of synthesis of 11-KT, which androgen also plays

a central role in silvering (previtellogenesis) and in vitello-

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

of As disrupted the oestrogen receptor (ER), and thereby

vitellogenesis, be it in a bird, Gallus gallus (chicken).

Freshwater eels spawn marine pelagic eggs with an oil

droplet (type II) and with a large perivitelline space. The

comparison of the freshwater eels with other marine pelagic

spawners revealed that even within this type II group, eel

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

composition and therefore most likely also in embryonic

metabolism. Eel eggs contain a large amount of total lipids.

In A. japonica, a negative correlation between egg total

lipid and fertilization, hatching and survival is proposed to

be linked to shortage of neutral lipids. The even higher

deposition of lipids in ovaries of A. anguilla relative to

A. japonica suggests lipids to be an important component

in ovary development and egg production in the European

eel. Deficiency of long-chain PUFA has for some time been

recognized 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 anguil-

lid eels. Whether this is also the case for A. anguilla

remains to be revealed. Requirements for essential fatty

acids in marine broodstock have been documented but

need to be tested on A. Anguilla, especially in the light of

the reported elongation/desaturation capacity of the fresh-

water eels. Free amino acids are important for hydration,

buoyancy and as important energy source. The level and

the profile of these free amino acids in A. japonica differ

dramatically from other marine pelagic spawners. Their

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

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 vitamins

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.

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