Aquaculture Nutrition
2013 19; 1–24
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1 2
5 2
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-
5
gies of the Sea, Tunis, Tunisia; 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
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ª 2013 John Wiley & Sons Ltd
doi: 10.1111/anu.12091
2 3 4
3
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
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%
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Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd
 60 (a) 1.0 (b) y = 0.68x – 0.25
R² = 0.71

Ovary protein (g/kg0.8/d)

Protein, lipid (g/kg initial body mass)
0.8
50

0.6
40

0.4
30
0.2
20
0.0
0.0 0.5 1.0 1.5 2.0
10
Protein use (g/kg0.8/d)

0
0 10 20 30 40 50 (c)
60 y = 0.37x – 1.04

Ovary energy (kJ/kg0.8/d)

GSI (%) R² = 0.43
50

40

30

20

10

0
0 50 100 150
Energy use (kJ/kg0.8/d)

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).

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
types are recognized: eggs without and eggs with
droplet(s), the latter being the most common, in
in temperate and warmwater species (Ahlstrom
..............................................................................................
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
eggs, two the spectrum (Table 1). If and how this position, which is
visible oil extended in the fatty acid and FAA profiles (sections Lip-
particular ids and Protein and amino acids), is related to the large
& Moser perivitelline space is not clear (Unuma et al. 2005).

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd
Table 1 Size and composition of marine pelagic fish eggs Lipids In most instances, reproduction of wild fish, or of
Other marine pelagic fish fed with natural food, is more successful than of
farmed fish. This has also been shown for A. anguilla
Anguilla No oil
(Tomkiewicz 2012), but less so for A. japonica, at least
spp.1 Oil globule2 globule3
when feminized eels are used (Yamada et al. 2006). One of
Egg diameter (mm) 1.1–1.8 0.8–1.5 (5) 0.8–2 (6) the differences between wild and farmed eels (and between
Oil glob diameter 0.25–0.35 0.15–0.4 –
Egg dm ug egg 1 60–70 35–120 150
males and females) are the body lipid levels (Fig. 2). In
Moisture (g kg 1) 880–920 880–930 900–930 A. anguilla, males start to silver, that is, initiating maturity,
Total lipids 350–440 130–450 70–150 at 80–150 g, while for females this is at sizes from 300 to
(g kg 1 dm)
800 g (Tesch 2003). Silvering eels, farmed and wild eels of
NL (%TL) 80–83 40–84 25–40
TG (%NL) 30–40 30–84 25–45 both sexes, attain lipid levels of 25–35% [a.o. (Bo€etius &
CH (%NL) 6–20 20–50 Bo€etius 1985; Larsson et al. 1990; Kamstra & Van He-
WSE (%NL) 6–65 6–15
eswijk 1996; Garcia-Gallego & Akharbach 1998; Kn€ osche
PL (%TL) 17–20 16–60 60–75
PC (%PL) 60–86 65–88 2009; Clevestam et al. 2011)]. Both farmed and wild males
PE (%PL) 12–20 7–25 reach these levels early, at masses of 70–100 g. Wild female
PI (%PL) 2–6 1–2 eels seem to follow another trajectory: they first invest in
Total N (g kg 1 dm) 95 90–110 100–120
Protein (g kg 1 dm) 300–460 250–500 350–550
body mass growth and reach these higher lipid levels at
FAA (g kg 1 dm) 30–50 100–170 150–220 higher body masses (Fig. 2). Although most farmed
Carbohydrates 3–24 6–20 females also have lower body lipid levels than males of
(g kg 1 dm)
comparable mass (Kamstra & Van Heeswijk 1996), their
Ash (g kg 1 dm) 83 60–100 60–150
trajectory is clearly advanced compared with wild females.
1
Data on A. anguilla (Kokhnenko et al. 1977; Boe €tius & Boe€tius
Anguilla anguilla silver eels generally have much
1980; Bezdenezhnykh & Prokhorchik 1984; Prokhorchik 1987; Pe-
dersen 2004; Palstra et al. 2005; Corraze et al. 2011), A. rostrata higher body lipid levels than other temperate eels
(Edel 1975; Oliveira & Hable 2010), A. australis (Lokman & Young (150–230 g kg 1, Han et al. 2000, 2001; Tremblay 2009;
2000) and A. japonica (Seoka et al. 2003, 2004; Unuma et al. De Silva et al. 2002; Hopkirk et al. 1975). In vertebrates,
2005; Furuita et al. 2006, 2007; Tanaka et al. 2006; Ohkubo et al.
2008; Ozaki et al. 2008; Kagawa et al. 2009).
the body lipid mass, through adiposity signals leptin and
2
Data on Dicentrarchus labrax (Devauchelle & Coves 1988; insulin, is thought to influence reproduction in a number of
Cerda et al. 1994a; Bell et al. 1997; Navas et al. 1997, 2001; ways (Caprio et al. 2001). On the one hand, a minimum
Rønnestad et al. 1998b), Sparus auratus (Mourente & Odriozola

ndez-Palacios et al. 1995,
lipid mass seems to be required to initiate puberty. Such an
1990; Rønnestad et al. 1994; Ferna
1997; Rodr
ıguez et al. 1998; Almansa et al. 1999, 2001), Pagrus effect was also observed in Oncorhynchus mykiss (Weil
major (Watanabe et al. 1984c, 1985b; Seoka et al. 1997), Den- et al. 2008). Furthermore, Peyon et al. (2001) showed in
tex dentex (Tulli & Tibaldi 1997; Mourente et al. 1999;
Dicentrarchus labrax that (recombinant mouse) leptin stim-
Gime
nez et al. 2008; Samaee et al. 2009a,b, 2010), Diplodus sar-

rez et al. 2007), Scophthalmus maximus
gus (Cejas et al. 2003; Pe ulated in vitro pituitary LH release in the prepubertal
(McEvoy et al. 1993; Rainuzzo et al. 1994; Silversand et al. 1996), stage, but much less in later stages. For A. anguilla, Lars-
Scophthalmus rhombus (Cruzado et al. 2011), Paralichthys olivac- son et al. (1990) even hypothesized that body lipid content
eus (Furuita et al. 2000, 2002, 2003c), Seriola quinqueradiata
(Verakunpiriya et al. 1996), Seriola lalandi (Moran et al. 2007;
might be the trigger for silvering, but as silver A. anguilla
Hilton et al. 2008), Sciaenops ocellata (Vetter et al. 1983), Latris are prepubertal and also some silver eels have (very) low
lineata (Morehead et al. 2001; Brown et al. 2005), Pseudocaranx body lipid levels (Sved€ ang & Wickstr€ om 1997), this seems
dentex (Vassallo-Agius et al. 1998, 2001a), Lates calcarifer
not so. On the other hand, excessive body lipid stores nega-
(Southgate et al. 1994; Sivaloganathan et al. 1998; Dayal et al.
2003) and Rachycentron canadum (Faulk & Holt 2003, 2008; tively affect reproduction, through impairment of gonadal
Nguyen et al. 2010, 2012). steroidgenesis (Caprio et al. 2001). Evidence for a negative
3
Data on Gadidae, Gadus morhua (Craik & Harvey 1984; Fraser
effect of increased adiposity on reproduction in fish is
et al. 1988; Finn et al. 1995a,b; Salze et al. 2005; Penney et al.
2006) Melanogrammus aeglefinus (Craik & Harvey 1984; Reith mostly anecdotal, in Indian (Chaudhuri, 1960) and Chinese
et al. 2001) Theragra chalcogramma (Ohkubo et al. 2006) and carps (Chen et al. 1969, cited by Rath et al. 1999). How-
Pleuronectidae, Hippoglossus hippoglossus (Falk-Petersen et al. ever, the observed negative effects of a low protein brood-
1986, 1989; Rainuzzo et al. 1992; Bruce et al. 1993; Evans et al.
1996; Mazorra et al. 2003) Pleuronectes platessa (Craik & Harvey
stock diet in Dicentrarchus labrax (Cerd a et al. 1994b)
1984; Rainuzzo et al. 1992; Thorsen & Fyhn 1996), Microstomus could well also have originated from a lower DP/DE ratio,
kitt (Thorsen & Fyhn 1996) and Verasper moseri (Ohkubo & as the gonads (but not the eggs) of the deficient fish
Matsubara 2002).
showed higher lipid levels during peak spawning. Finally,
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Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd
 (a)
400

Body/Muscle lipid (g/kg)

350

300

250

200

150

100

50

0
0 200 400 600 800 1000 1200 1400 1600
Body mass (g)

(b)

Figure 2 Whole body or muscle lipid 400

percentage of wild (a) and farmed (b)
Body/Muscle lipid (g/kg)

350
A. anguilla in relation to body mass,
and sex (females = circles; males = tri- 300
angles). The light grey symbols in (a)
are for yellow eels, and the dark grey 250
symbols are for silver eels. Data on
200
wild eels are from the study by Bo€etius
& Bo€etius (1985, 1989) and Larsson 150
et al. (1990) and Heinsbroek, unpub-
lished, IMARES, unpublished. Data 100
on farmed eels are from the study by 50
Kamstra & Van Heeswijk (1996), Sch-
mitz (1982), Corraze et al. (2011), 0
Støttrup et al. (2013) and Heinsbroek, 0 200 400 600 800 1000 1200 1400 1600
unpublished. Body mass (g)

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

Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd
the muscle of migrating Oncorhynchus keta. As further spe- Table 2 Fatty acid profile (% of total FA) of egg total and polar
cific FA (n-6 and n-9) are implicated in swimming capacity lipids of wild (or fed with raw fish and/or squid) A. japonica and
of other marine pelagic spawners
(McKenzie et al. 1998; Chatelier et al. 2006), it might be
that FA are selectively allocated to migration and repro- Other marine pelagic
duction and that if some FA are not used for migration, Oil
they might negatively influence the egg composition. Liu A. japonica1 globule2 No oil globule3
et al. (2009) did find in A. japonica that fasting and swim-
Total lipids
ming led to a selective retention of ARA in the muscle. 16:0 18.9 (18.1–21.6) 18.9 (13–21.5) 20 (17.3–23.5)
This was not the case in eels that were induced to matu- 18:1 33.5 (31.4–35) 19.2 (9.7–25.7) 14.8 (11.2–17.6)
rate, indicating a selective incorporation in the ovary. 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)
Total lipid levels in eggs of A. japonica are normally ARA 2.1 (0.9–3.8) 2 (0.5–3.7) 1.9 (1–3)
reported to be 300–450 g kg 1 dm (Furuita et al. 2003a, EPA 2.9 (2.1–3.7) 6.4 (2.4–11) 13.4 (8.7–15.5)
2006; Tanaka et al. 2006) although Unuma et al. (2005) 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)
also mention TL levels as low as 200 g kg 1 dm. Furuita
EPA/ARA 2.0 (0.7–3.3) 3.7 (0.6–8.6) 7.8 (4.4–14)
et al. (2006) did find a negative correlation between egg TL DHA/EPA 3.4 (2–4.1) 4.1 (2–6.8) 2.2 (1.8–2.9)
and fertilization, hatching and survival. Surprisingly this Polar lipids
16:0 21.2 (18.9–23.1) 21.0 (18.5–24.1) 21.4 (20.9–22)
effect was mainly caused by higher levels of PL. A similar
18:1 22.3 (15.4–24.2) 11.7 (10.7–13) 13.2 (11.1–14.3)
effect was also described in Hippoglossus hippoglossus by 18:2 n-6 0.7 (0.1–1.9) 0.6 (0.03–0.9)
Evans et al. (1996), but for the relative amount of PL. 18:3 n-3 0.3 (0.1–0.5) 0.2 (0.01–0.4)
These authors therefore suggested that this was more an 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)
indication of a lack of NL. Total lipid levels remain stable 22:5n-3 2.4 (1.2–4.6) 1.0 (0.5–1.4)
350–400 g kg 1 dm in A. japonica eggs until hatching and DHA 17.2 (13.6–21.4) 32.5 (27–37.3) 32.2 (29.3–34.8)
decrease during yolk sac and oil droplet resorption to c. 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)
160 g kg 1 dm (Tanaka et al. 2006). Ohkubo et al. (2008)
1
showed that during this period, TG decreased stronger Furuita et al. (2003a) and Ozaki et al. (2008).
2
Data on Dicentrarchus labrax (Bruce et al. 1999; Navas et al.
(80%) than PL (40%). A shortage of neutral lipids has
2001), Sparus aurata (Mourente & Odriozola 1990), Scophthalmus
been implied as a cause for larval mortality in Seriola la- maximus (Peleteiro et al. 1995; Silversand et al. 1996; Lavens
landi (Hilton et al. 2008) and Latris lineata (Morehead et al. 1999), Rachycentron canadum (Faulk & Holt 2003, 2008;
et al. 2001). This might well also have contributed to mor- Nguyen et al. 2010, 2012), Plectorhynchus cinctus (Li et al. 2005)
Centropomus undecimalis (Yanes-Roca et al. 2009), Pseudocaranx
talities before first feeding, or even mouth formation, in dentex (Vassallo-Agius et al. 1998, 2001a), Lutjanus campechanus
larvae of A. australis (Lokman & Young 2000) and A. ro- (Papanikos et al. 2008), Coryphaena hippurus (Divakaran & Os-
strata (Oliveira & Hable 2010). trowski 1989; Ostrowski & Divakaran 1989), Solea senegalensis
(Mourente & Va
zquez 1996), Solea solea (Lund et al. 2008), Para-
Another striking difference between wild and farmed fish
lichthys adspersus (Wilson 2009) and Centropristis striata (Bentley
lies in the fatty acid profile of the eggs. Clear relations et al. 2009).
3
between FA profile and egg quality have however not Data on Hippoglossus hippoglossus (Falk-Petersen et al. 1989;
Bruce et al. 1993; Mazorra et al. 2003) and Gadus morhua (Fraser
always been apparent (Fern
andez-Palacios et al. 2011). The
et al. 1988; Pickova et al. 1997; Salze et al. 2005; Penney et al.
egg fatty acid profile of wild A. japonica is compared with 2006; Lanes et al. 2012).
other marine pelagic spawners, for both type I and II, in
Table 2. The differences can partly be explained by differ- et al. 1980). It is a very specialized high-density serum
ences in lipid class composition (Table 1), but again eels phospho-lipo-glyco-protein consisting 830–860 g kg 1 pro-
are at the far end, or even outside, the spectrum of type II tein and 130–170 g kg 1 lipid. PL and TG account for
eggs. A. japonica eggs do have lower levels of EPA and 650–720 and 170–270 g kg 1, respectively, of the lipids
especially of DHA, and much higher levels of 18 : 1, also (Ando & Matsuzaki 1996; Komatsu et al. 1996). The fatty
in the PL. acid profile of eel VTG is not known, but Silversand &
The lipid class profile of eel eggs (Table 1) suggests that Haux (1995) showed for a number of fish species that the
the majority of the PL in the eggs originate from vitelloge- fatty acid profile of VTG and the egg PL were highly cor-
nin, as also shown in other fish (Silversand & Haux 1995; related. They did find species-specific differences in VTG
Johnson 2009)]. Vitellogenin (VTG) of A. japonica was FA profiles. In general, egg PL FA seem to be less affected
actually one of the first teleost VTGs characterized (Hara by the broodstock diet (Mourente & Odriozola 1990;
..............................................................................................

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

0 7–11
Week2 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: Pe
rez 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
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
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
 (a) (b)

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).

(a) (b) 100

35 Sunflower oil-fed group
30 Bonito oil-fed group 80
% total fatty acids

Percentage (%)
25
60
20

15 40
10
20
5

0 0
d

ng
an
te

tio

hi
la

oy

za

tc
vu

Bu

ili

Ha
O

rt
Fe

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).

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).
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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; Cerda 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.

(a) (b)
200 30 100 30
OLv (nmol AA individual–1)
PAA (nmol individual–1)

FAA (nmol individual–1)

FAA (nmol individual–1)

80
150
20 20
60
100
40
10 10
50
20

0 00 0 0
OC I OC II OV 0 1 2 3 4
Maturation stage dpf

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.
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Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd
 (a) 18

16

Amino Acid content (mol%)

14

12

10

(b) 35
Free Amino Acid content (mol%)

30

25

20

15

10

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).

Vitamins The essentiality of vitamins for metabolism although also specific transport proteins have been
makes them required for the process of gonad development described (Brooks et al. 1997; Lubzens et al. 2010). Little is
and also for embryonic development, after incorporation in known about the transport and incorporation of water-sol-
the eggs (Brooks et al. 1997; Lubzens et al. 2010). Trans- uble vitamins. In Scophthalmus maximus, Salmo salar
port and incorporation of lipid soluble vitamins is thought (Cowey et al. 1962; Albrektsen et al. 1994; Sandnes et al.
to be analogue to lipid transport (section Lipids), that is, 1998) and Gadus morhua (Mangor-Jensen et al. 1994) vita-
bound to VTG and other lipoproteins (Lie et al. 1994), min B6, pantothenic acid, nicotinic acid and vitamin C
..............................................................................................

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
..............................................................................................
Aquaculture Nutrition 19; 1–24 ª 2013 John Wiley & Sons Ltd
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
(Fontagn
e-Dicharry et al. 2010) to 80 (Furuita et al. 2001, Table 4 Mineral composition of ovaries and eggs (g or
2003b) mg kg 1. Furuita et al. (2009b) found in A. japon- mg kg 1 dm)
ica no correlations of egg VA concentrations with Ovaries Eggs
fertilization, hatching and survival. The authors claimed a
Anguilla Unfertilized/
tendency for lower survival at higher VA (>40 mg spp1 Other2 washed3 Unwashed4
kg 1 dm) in the eggs, but again also at lower levels, vari-
Ash (g kg 1) 70–120 32–60 50–80 150–200
ability was very high. These eels were further fed with
Ca (g kg 1) 0.2 0.3–0.5 0.3–0.8 1.5–2
frozen krill before maturation, but egg levels of carotenoids P (g kg 1) 11 8–15 6–13 7–10
were not measured. It has been shown that hypervitamin- Na (g kg 1) 2–3 3–5 23–35
K (g kg 1) 5–8 17–25 15–20
osis of VA in later stages of larval development can cause
Mg (g kg 1) 0.4–1 0.7–1.1 4–9
malformations, particularly on the head (jaws and opercu- Fe (mg kg 1) 15–60 11–23 17–60
lae) structures (Furuita et al. 2001; Hamre et al. 2010; Zn (mg kg 1) 20–400 102 80–140
Haga et al. 2011), but this is probably less of concern in Mn (mg kg 1) 2–3 0.7–1.5
Cu (mg kg 1) 1–6 8–11 3–5
eels as ossification in leptocephali is largely delayed till
1
metamorphosis (Miller 2009). Data on ash content from the study by Boe €tius & Boe€tius
(1980), Lau (1987) and Amin (1991), data on Ca and P content
Vitamin D and possibly vitamin K, also involved in
from (Yamada et al. 2001).
calcium transport, have been implied the demineralization 2
Gadus morhua (Rome
o 1987; Hellou et al. 1992; Saxholt et al.
of the bones during ovary development and maturation 2008), Dicentrarchus labrax (Devauchelle & Coves 1988), Mugil
cephalus (Rome
o 1987; Olgunog  lu & Olgunog
 lu 2011).
(Lopez et al. 1980; Hamre et al. 2010; Lock et al. 2010; 3
Pagrus major (Watanabe et al. 1984c, 1985a), Gadus morhua
Krossøy et al. 2011). (Craik 1986; Lanes et al. 2012), Pleuronectes platessa (Craik & Har-
For the water-soluble vitamins (other than VC), Mæland vey 1984; Craik 1986).
4
et al. (2003) reported that folate levels in Hippoglossus hip- Dicentrarchus labrax (Devauchelle & Coves 1988), Pagrus major
(Watanabe et al. 1985b, 1991b), Scophthalmus maximus (Finn
poglossus eggs were highly variable, and in some instances et al. 1995a).
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 observed in A. anguilla (Lopez et al. 1980; Sbaihi et al.
number of fish species, thiamine deficiency has been 2009). Somewhat unexpectedly, in view of the often
reported as cause of decreased larval survival (Fisher et al. reported association of vitellogenin with Ca (Lopez et al.
1996; Czesny et al. 2009; Rinchard et al. 2010), but paren- 1980; Lau 1987; Sbaihi et al. 2009; Palstra et al. 2010), the
tal and/or egg thiamine depletion only occurs with specific primary goal of this bone demineralization does not seem
broodstock diets, that is, raw seafood products containing to be the provision of Ca to the ovary. Ovary Ca content
thiaminase (Kreutzmann & Lehmitz 1976). was only 0.02% dm (Yamada et al. 2001), giving a Ca/P
ratio of 0.02, which is however in accordance with other
Minerals Reported ash contents of late stage eel ovaries fish species (Table 4). Furthermore, in the marine environ-
vary from 70 g kg 1 dm (A. anguilla) (Bo€etius & Bo€etius ment, requirements for Ca (and Mg, K) can be met by
1980; Amin 1991) to 120 g kg 1 dm (A. japonica) (Lau absorption through the gills and the intestine (from drink-
1987). These estimates are somewhat higher than for other ing water), even in starving eels (NRC 2011).
marine pelagic spawners (Table 4). In accordance with Total ash content of A. japonica eggs was reported by
these other fish, the most abundant mineral in the ovary is Tanaka et al. (2006) as 83 g kg 1 dm. Information on
P, with 11 g kg 1 dm (Yamada et al. 2001). Watanabe whether these were unfertilized or washed/unwashed fertil-
et al. (1984a) showed for Pagrus major that the (dietary) ized eggs was not given. Data on the mineral composition
provision of P was critical for good-quality eggs, despite of eel eggs are lacking. Craik & Harvey (1984) showed that
the fact that the egg P content was not much influenced in Pleuronectes platessa, the hydration of the oocytes dur-
(Watanabe et al. 1984c). Lanes et al. (2012) did find a posi- ing maturation was accompanied by a large influx of K. In
tive relationship between P levels in the eggs and the fertil- unfertilized eggs of Pleuronectes platessa and Gadus morhua
ization rate for wild broodstock of Gadus morhua. For (Craik & Harvey 1984) and washed fertilized eggs of Pag-
A. japonica, Yamada et al. (2001) showed that c. 40% of rus major (Watanabe et al. 1984c, 1985a), K is therefore
the total amount of P in the ovary was provided through the most abundant mineral, still followed by P. The cho-
extensive demineralization of the bones, a process also rion remains permeable to small osmolytes, and the mineral
..............................................................................................

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