Lipid characterization of both wild and cultured eggs of cuttlefish ( Sepia officinalis L.)...

1,2 3 2 1

1 C.C.MAR, Universidade do Algarve, Faculdade de Ciencias do Mar e do Ambiente, Faro, Portugal; 2 Departamento de

Biologıa Animal, Facultad de Biologıa, Universidad de La Laguna, La Laguna, Tenerife, Canary Islands, Spain; 3 Centro

Oceanografico de Canarias, Instituto Espanol de Oceanografia, Tenerife, Spain

The present work reports a characterization of mean wet

weight and moisture, the lipid class and fatty acid (FA)

composition from the total lipids (TL), of both culture and

wild eggs of the cuttlefish, Sepia officinalis, throughout the

embryonic development. Additionally, reproductive data,

such as the number of spawnings, number and mean weight

of eggs and duration of spawning period of cultured cuttle-

fish is provided. Both types of eggs were similar in mean wet

weight, moisture content, TL content and lipid composition

throughout embryonic development. Females from the cul-

tured group spawned 13 times and laid 8654 eggs in 64 days,

with a mean weight of 0.607 ± 0.179 g. A sex ratio of 1.57

(11$ for 7#) promoted an individual fecundity of 787 eggs/$

(the biggest until now on our culture facilities), which might

be related to increased bottom areas. The TL increased with

day/stage of embryonic development (P < 0.05) only in the

cultured egg group. However, no differences were found on

TL between culture and wild eggs at the same day/stage

(P > 0.05). Eggs displayed predominant levels of phospha-

tidylcholine, phosphatidylethanolamine (PE), cholesterol and

triacylglycerol at the end of embryonic development. Polar

and neutral lipids of both eggs groups remained consistently

proportional (�50% for each lipid fraction) and a significant

increase (P < 0.05) was observed in phosphatidylserine, PE

and free FA throughout the embryonic development. In

either egg type and day, 16:0, 18:0, 20:5n-3 and 22:6n-3

accounted for approximately 70 g Kg)1 of all FA and satu-

rated and n-3 totals seemed to have the same proportion in

the cuttlefish eggs. The present results suggest that lipids are

not used as energetic substrate but as structural components

in cuttlefish egg.

KEY WORDSKEY WORDS: cuttlefish, eggs, embryonic development, life cy-

cles, lipid composition, n-3 highly unsaturated fatty

acids

Received 20 September 2007, accepted 4 January 2008

Correspondence: Antnio V. Sykes, C.C.MAR, Universidade do Algarve,

Faculdade de Ciencias do Mar e do Ambiente, Campus de Gambelas, 8000-

810 Faro, Portugal. E-mail: [email protected]

Cephalopods have been defined as aquaculture candidates for

the past 20 years (Vaz-Pires et al. 2004; Sykes et al. 2006a),

and this was first acknowledged by the inclusion of a chapter

dedicated to cuttlefish in the �Bases Biologiques et ecologiques

de l�aquaculture� de Barnabe (1996). The constraints in devel-

oping cephalopod culture technology are many and have been

described by Sykes et al. (2006a) for the European cuttlefish,

Sepia officinalis. The development of culture technology for

cephalopod species faces similar problems to those found for

fish in the early years. Like in fish, one of the most important

challenges in aquaculture is larval or first stage nutrition,

whereas in most species a limited knowledge of their nutri-

tional requirements creates bottlenecks. Theoretically, wild

eggs have all the nutrients needed for both correct development

and homeostasis of the egg and larvae until their first exoge-

nous feeding. Therefore, an estimation of the use of endoge-

nous nutrients provided by the yolk reserves during embryonic

development should be used as an approach to nutritional

requirement of the embryonic and post-hatching-phase (Sar-

gent 1995; Mourente & Vazquez 1996; Rainuzzo et al. 1997;

Cejas et al. 2004). This nutrient composition and its use during

embryonic development, however, seem to be species-specific,

2009 15; 38–53. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

doi: 10.1111/j.1365-2095.2008.00566.x

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� 2008 Blackwell Publishing Ltd

No claim to original US government works

Aquaculture Nutrition

leading to differences in both quantity and quality of larvae

used during that stage (Mourente & Vazquez 1996). Energy

metabolism in fish during embryonic development is based on

protein, carbohydrates, free amino acids and lipids (Finn 1994;

Finn et al. 1995, 1996; Rønnestad et al. 1998). The choice for

the substrate varies according to species and between different

stages of embryonic development and depends on ambient/

culture conditions, physiological events and energy demands

(Rainuzzo 1993; Sargent 1995; Mourente & Vazquez 1996;

Rainuzzo et al. 1997; Almansa et al. 1999). For instance, the

lipid fraction of some fish eggs has been described as substrate

for energy metabolism and as structural components in

membrane biogenesis (Tocher & Sargent 1984; Falk-Petersen

et al. 1986; Cejas et al. 2004).

According to Boletzky (1987a), in cephalopods, �the

embryonic phase tends to be a black box, succinctly called

the egg stage�. Although there is information regarding the

distinct phases of the embryonic development in cuttlefish

(Naef 1928; Lemaire 1970), data regarding the composition,

physiology and metabolism during this phase are scarce or

inexistent. Besides, it has been assumed that cuttlefish

metabolism is mainly protein and amino acid driven (Lee

1994) and the lipid fraction represent <2% of their body

weight (Boucaud-Camou 1990); thus neglecting the study of

its lipid composition. Nevertheless, some studies have already

been conducted regarding lipid nutrition in cephalopods.

To determine the ideal lipid profile during the early life of

cephalopods, Navarro & Villanueva (2000) studied the lipid

composition of the mature ovary and late eggs and wild

hatchlings and juveniles. Additionally, the same authors also

studied the lipid composition of natural preys used in suc-

cessful cultures. From these data they stated that cephalo-

pods in their early stages of growth show a high requirement

for polyunsaturated fatty acids (PUFA), with docosahexoe-

noic acid (DHA, 22:6n-3) representing 20–30% of the total

FA of the total lipids (TL) of cuttlefish, squid and octopus

hatchlings. The importance of PUFA in larval fish nutrition

has been thoroughly researched in the last 20–30 years

(Watanabe 1993; Watanabe & Kiron 1994; Sargent et al.

1999). According to Sargent et al. (1999), teleost eggs are

generally rich in highly unsaturated FA of the n-3 series (n-3

HUFA), mainly eicosapentaenoic acid (EPA, 20:5n-3) and

DHA. Furthermore, there is strong evidence that, in fish, n-3

HUFA are crucial to female fecundity, to embryo and to

early larval development, growth and survival (Watanabe &

Kiron 1994; Sargent 1995; Silversand et al. 1996). Like in

fish, in cuttlefish n-3 HUFA are also involved in the organ-

ogenesis of structural components such as brain (Dumont

et al. 1992, 1994), and probably will have a similar degree of

importance in the eye development (Boyle et al. 2001), and

will be precursors of physiological active molecules such as

eicosanoids (Sargent 1995).

In fish, yolk reserves are obtain from vitelogenin, a phos-

pholipoprotein which are synthesized in the liver and trans-

ported to the gonads to be included in the eggs. An increase

in the liver lipid content during gonadal maturity has been

observed in fish that is related to the previous (Almansa et al.

1999). Cephalopods share some similar facts, such as, the

digestive gland (which has a similar role to the liver in fish)

usually yields a large volume of lipids during the sexual

maturity in similar way to their gonads, and these are rich in

n-3 HUFA, especially EPA and DHA (Rosa et al. 2005).

According to Blanchier & Boucaud-Camou (1984) and

Blanchier et al. (1986), there is an increase of cholesterol

(CHO) and phospholipids content of the digestive gland in

cuttlefish females at sexual maturity. However, these were

proved to be not significant. The same authors also noted a

slight increase and a marked increase in CHO and phos-

pholipids respectively, in the ovary at this time that might be

related to yolk and steroid synthesis. They suggested that the

increase in CHO must be related to membrane synthesis

during gametogenesis (Blanchier & Boucaud-Camou 1984),

while the increase in phospholipids is, according to Fujii

(1960), related to the synthesis of the vitellus. These data

suggest the importance of lipids in the egg reserves, probably

to be used along the embryonic development as energy sub-

strate and/or structural components.

In cuttlefish, the influence of temperature in the TL and LC

yolk content during the embryonic development was studied

byBouchaud&Galois (1990). The results obtained suggest the

importance of lipid content in the embryogenesis. According

to these authors, TL represents about 14% of dry weight in

eggs, despite of egg size and incubation temperature range.

However, these eggs were from the French-English Channel

population cuttlefish, displaying higher mean wet weight and

longer embryonic development times than those commonly

found in southern Portugal (for a review on this problematic,

see Perez-Losada et al. 1999, 2002, 2007; Sykes et al. 2006a).

These differences in the reproduction biology could imply

differences in the biochemical composition of the eggs. It is

therefore of interest to investigate the lipid composition of

ovulated eggs from different cuttlefish populations.Moreover,

the study of the FA composition of wild and cultured eggs is of

extreme importance as egg composition may be altered by the

breeder�s diet (Morehead et al. 2001). Therefore, a comparison

between wild and cultured cuttlefish could be useful to obtain

further insight into the nutritional requirements of embryos

and yolk sac hatchlings of the species.

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� 2008 Blackwell Publishing Ltd Aquaculture Nutrition 15; 38–53


In the present study, egg mean wet weight and moisture,

TL, lipid classes and FA of wild and cultured eggs were

examined throughout the embryonic phase until hatching.

Also, reproduction data regarding the number of spawnings,

number and mean weight of eggs and duration of spawning

period of cultured cuttlefish was studied.

The eggs used in the present study were laid in the laboratory

by both culture- and wild-captured adult individuals. Culture

spawners (n = 18; mean weight of 147.05 ± 55.09 g) were

selected from a captive F2 generation, already cultured in the

laboratory (using exclusively grass shrimp as prey), while

already reproducing wild spawners (n = 18; mean weight of

156.56 ± 51.40 g) were captured in the Ria Formosa La-

goon (south Portugal), using a small bottom trawl (6 m wide;

0.5 m high; 10-mm mesh size at the cod end) for 10 min haul

in shallow water (�4 m). Both groups were placed in white

400-L rectangular plastic tanks (with a bottom area of

1.2 · 1.0 cm2) of a culture system similar to that previously

described by Domingues et al. (2001). These tanks were

placed in a low-disturbance room of CCMAR�s Ramalhete

Field Station (Faro, south Portugal). Water flow was of

12 L h)1 for both tanks. Temperature and salinity were

measured daily for the culture-spawners tank. Oxygen was

kept at optimal values that ranged approximately 100%

saturation. Mean water temperature was 19.5 ± 1.09 �C,while mean salinity was 38.8 ± 0.42 g L)1. The conditions

of the used spawning system ensured an excellent water

quality from a physical, chemical and bacteriological point of

view. Cultured cuttlefish were fed live adult grass shrimp

(0.090 ± 0.022 g; n = 200) throughout their life cycle. Food

was captured from ponds surrounding the research facility

and supplied ad libitum.

Although the objective of the current work was to study

differences between wild and cultured eggs, only cultured

cuttlefish were conditioned before spawning. Wild specimens

were captured during the spawning season, so it was proba-

ble that the wild broodstock had started spawning before

their capture. Therefore, cultured spawners were the only

group where growth and reproduction data were acquired.

Data were collected at the beginning of the experiment, the

start of spawning and at the end of the experiment (when the

last animal died) and were used to calculate: (i) mean wet

weight (MWW); (ii) mean instantaneous growth rate (IGR)

(%body weight day)1) = (LnW2)LnW1)/t*100, where W2

and W1 are the final and initial weight respectively, Ln the

natural logarithm and t the number of days of the

time-period; (iii) total biomass (TB) =P

weight (g) of the

animals at time of death; (iv) duration of the spawning period

(DSP, days); (v) TB Increase During Spawning Period

(TBIDSP) = (TB-BBSP)/DSP, where TB is defined above,

BBSP is biomass at the beginning of the spawning period; (vi)

sex ratio = number of $/#; and (vii) individual fecundity

(F) = eggs/$. The DSP as well as the amount of times that

egg laying occurred (batches) were also recorded.

After being conditioned in the present conditions, adult-

cultured sepia took about 18 days to mature and start

spawning. Captured individuals started spawning on the first

day after being collected and placed under laboratory con-

ditions. This provided the excellent opportunity to obtain

wild eggs in the laboratory without culture interference, such

as feeding. When spawning started, eggs were collected from

both wild and culture tanks. Collection was made using a

green-plastic-rectangular-supportive net (1 cm2 holes), sus-

pended inside both wild and cultured spawners tanks. These

nets were checked daily and if eggs were present (day spawn),

they were carefully removed (as freshly laid eggs are very soft

and gelatinous) and individualized. After this, all of them

were accounted for and weighed (100%, if n < 100 or 50% if

n > 100). Also, eggs were separated based on shape and

colour. Black oval eggs were considered to be viable, while

eggs of any other shape and/or colour were considered non-

viable and discarded. Total egg numbers from both viable

and non-viable were accounted for and the percentage of

non-viable egg presence in each batch of spawned eggs

determined. The viability of both black oval eggs from both

wild and culture spawners was then investigated using 500

eggs of each group of breeders throughout the embryonic

development in the incubation tanks and at the conditions

described below.

Embryonic development of both wild and cultured eggs

(n = 1000 eggs each) was carried out in 250 L bowl-shaped

incubation tanks with clean natural seawater in a similar

seawater system described by Domingues et al. (2001) and in

the egg stage conditions described by Sykes et al. (2006b).

Water flow was of 12 L h)1. Both group of eggs were incu-

bated at a mean temperature of 21.7 ± 0.93 �C and mean

salinity of 39.2 ± 0.21 g L)1. Light intensity was 200 lux, at

the top of the water column of the incubation tanks, and

photoperiod resembled natural geographical conditions in

spring (14 hD : 10 hN).

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While going through the embryonic development and at

every 5 days, 50 eggs from both groups were weighed and used

formoisture determination.Also, at every 10 days, samples for

biochemical analysis from both wild and cultured eggs were

taken. From these samplings, on bothwild and culture groups,

15 eggs were used to determine moisture and another 70 eggs

for later lipid determinations.

Moisture content was determined from individual egg samples

using the method of Horwitz (1980). TL was extracted with

chloroform : methanol (2 : 1 v/v) containing 0.01% of butyl-

ated hydroxytoluene (BHT) as antioxidant (Christie 1982).

The organic solventwas evaporated under a streamof nitrogen

and the lipid content determined gravimetrically. Lipid classes

were separated by one dimensional double development high-

performance thin layer chromatography (HPTLC), using

methyl acetate/isopropanol/chloroform/methanol/0.25% (w/

v) KCl (25 : 25 : 25 : 10 : 9 by vol.) as the polar solvent sys-

tem, and hexane/diethyl ether/glacial acetic acid (80 : 20 : 2 by

vol.) as the neutral solvent system. Lipid classes were quanti-

fied by charring with a copper acetate reagent followed by

calibrated scanning densitometry using a Shimadzu CS-

9001PC dual wavelength flying spot scanner (Olsen & Hen-

derson 1989). TL extracts were subjected to acid-catalysed

transmethylation for 16 h at 50 �C, using 1 mL of toluene and

2 mLof 1%sulphuric acid (v/v) inmethanol. The resultantFA

methyl esters (FAME) were purified by TLC, and visualized

under UV light with 2¢,7¢-dichlorofluorescein in 98% (v/v)

methanol, containing 0.01% BHT (Christie 1982). Prior to

transmethylation 19:0 was added to TL as internal standard.

FAME were separated and quantified by using a Shimadzu

GC-14A gas chromatograph equipped with a flame ionization

detector (250 �C) and a fused silica capillary column Supe-

lcowaxTM 10 (30 m · 0.32 mm I.D.) (Supelco, Bellefonte,

USA). Helium was used as carrier gas and samples were

applied by on-column injection at an initial temperature of

50 �C. During each analysis, the oven was programmed to rise

from 60 to 150 �C at a rate of 39 �C min)1, and then to a final

temperature of 215 �C at 2.5 �C min)1. Individual FAME

were identified by reference to authentic standards and to a

well-characterized fish oil (PUFA-3, Biosigma, Barcelona,

Spain).

Lipid samples, for each sampled day triplicate, were com-

posed of amaximum of three eggs andweight dependable. TL,

LC and FAME are expressed as lg/egg. Individual means

were calculated on a dry weight basis. BHT, potassium

chloride, potassium bicarbonate, 2¢,7¢-dichlorofluorescein

were supplied by Sigma Chemical Co. (St Louis, MO, USA).

TLC (20 · 20 cm · 0.25 mm) and HPTLC (10 · 10 cm ·0.15 mm) plates, precoated with silica gel (without fluorescent

indicator) were purchased from Macheren-Nagel (Duren,

Germany). All organic solvents for GC used were of reagent

grade and were purchased from Panreac (Barcelona, Spain).

Results are presented as means ± standard deviations (SD)

for triplicate experiments. The data were checked for normal

distribution with the one-sample Kolmogorov–Smirnoff test

(Zar 1999) as well as for homogeneity of variances with the

Levene�s test (Zar 1999) and, when necessary, arcsin trans-

formation was performed. When a normal distribution and/

or homogeneity of the variances were not achieved, non-

parametric tests were used.

A Kruskal–Wallis test (Zar 1999) was used to compare

differences in MWW between the different egg batches ob-

tained from cultured spawners, followed by a post hoc

Dunn�s test (Zar 1999) to determine which were statistically

different. A Mann–Whitney test (Zar 1999) was used to

compare differences among culture and wild eggs in terms of

MWW and moisture throughout the embryonic develop-

ment. A one-way ANOVAANOVA (Zar 1999), comparing the eggs at

different stages but within the same type of egg, was per-

formed for TL, LC and FA profiles, followed up by a pos-

teriori post hoc multi-comparison Scheffe test (Zar 1999) to

determine differences within samples. When lipid data failed

normal distribution and/or homogeneity, first arcsin trans-

formation (Fowler et al., 2002) was applied and the same

parametric test was used. After this and if data still failed to

comply to either normality and/or homogeneity, then a

Kruskal–Wallis test (Zar 1999) was used, followed up by a

posteriori post hoc multi-comparison Games–Howell test

(Zar 1999) to determine differences. A student t-test (Zar

1999) was applied to determine differences of TL, LC and

FAME between egg types at the same egg stage. If, after

arcsin transformation, data still did not comply with nor-

mality and/homogeneity, then a Mann–Whitney test (Zar

1999) was used. In all statistical tests used, P < 0.05 was

considered statistically different.

After 18 days of being set apart in the breeding tank, cul-

tured spawners MWW was 183.84 ± 72.47 g, from which #

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displayed a MWW of 172.46 ± 45.54 g and $ of

191.08 ± 86.81 g. From these, the maximum weight re-

corded was of 325.6 g from a $, while the minimum was of

82.6 g from a #.

The cultured group of 18 cuttlefish intermittent-spawned

13 times and laid 8654 eggs in 64 days, with a mean weight

of 0.607 ± 0.179 g (Table 1). From these, 418 eggs were

considered non-viable, which corresponded to 4.83% of to-

tal egg numbers. All egg batches were found to be statisti-

cally different between them (P < 0.05). However, Dunn�s

test revealed that not every batch was different from the

other. Table 1 presents a resume of the cultured egg data

obtained, and those that were not statistically different be-

tween them (P > 0.05). We also noticed that the amount of

eggs laid over the spawning period had a normal distribu-

tion. Both wild and cultured egg batches used for the study

of weight evolution during embryogenesis displayed MWW

(g) of 0.627 ± 0.131 and 0.766 ± 0.310 respectively, at

spawning.

After the 64 DSP, culture cuttlefish MWW was

293.63 ± 131.08 g, from which # displayed a MWW of

280.69 ± 128.40 g and $ of 301.87 ± 138.29 g. From these,

the maximum and minimum weights recorded were 513.20 g

and 102.30 g, both from #s. Mean IGR during the spawning

period was 0.79% BW day)1 for # and 0.74% BW day)1 for

$. At the end of reproduction a TB of 5285.33 g was

achieved, which corresponded to a TBIDSP of 24.70% dur-

ing that time interval. A sex ratio of 1.57 (11$ for 7 #), in this

type of culture conditions, promoted an individual fecundity

of 787 eggs/$.

Growth and moisture Embryonic development duration (until

the last egg hatched) of both wild and cultured eggs was

30 days. The evolution of egg mean wet weight for both wild

and cultured eggs throughout the embryonic development is

shown in Fig. 1. Both wild and cultured egg batches used for

the study of weight evolution during embryogenesis displayed

MWW (g) of 0.627 ± 0.131 and 0.766 ± 0.310 respectively,

at spawning. Statistical differences (P < 0.05) were only

found between eggs (wild versus culture) at the day/stage 1, 5

and 25 of embryonic development. Figure 2 shows the evo-

lution of egg moisture (%) of both wild and cultured eggs

throughout the embryonic development. Here, no statistical

differences (P > 0.05) were found between both type of eggs

at the same day/stage of embryonic development.

Both MWW and moisture trends () showed a similar

lowering tendency during the first 10 days, after which this

was inverted until the end of the embryonic development.

However, the evolution of moisture curves displayed a sig-

moidal trend during embryonic development. Non-viability

of eggs was 18.6% and 8.8% for culture and wild eggs

respectively.

Lipids The TL increased with day/stage of embryonic

development (P < 0.05) only in the cultured egg group

(Table 3), but in the wild egg group this increase was not

significant (P > 0.05; Table 2). No differences were found on

TL between wild and cultured eggs at the same day/stage

(P > 0.05).

Table 1 Cultured egg batches, numbers of viable and non-viable, sampled, mean wet weights (MWW) and Dunn�s post hoc results

Egg Batch n Sampled N MWW ± SD SE Min WW Max WW Non-viable Non-viable % Dunn�s

1 219 110 0.541 ± 0.0690 0.0066 0.430 0.920 0 0.00 20, 50, 61

2 241 120 0.705 ± 0.1248 0.0114 0.480 1.030 0 0.00 14, 18, 54, 56

14 1042 500 0.742 ± 0.1480 0.0066 0.420 1.790 0 0.00 2, 18, 26, 54, 56

18 524 260 0.724 ± 0.1663 0.0103 0.420 1.190 0 0.00 2, 14, 26, 54, 56

20 69 69 0.519 ± 0.0436 0.0053 0.400 0.590 14 16.87 1, 50, 61

26 1047 524 0.716 ± 0.1450 0.0063 0.390 1.430 160 13.26 2, 14, 18, 54, 56

36 1761 900 0.445 ± 0.0977 0.0033 0.240 0.880 100 5.37 –

42 1259 660 0.622 ± 0.1159 0.0045 0.360 1.070 60 4.55 –

50 1267 635 0.556 ± 0.1909 0.0076 0.140 1.260 76 5.66 1, 20, 61

54 494 250 0.722 ± 0.1499 0.0095 0.470 1.170 0 0.00 2, 14, 18, 26, 56

56 222 115 0.770 ± 0.1435 0.0134 0.520 1.350 8 0.03 2, 14, 18, 26, 54

61 381 185 0.531 ± 0.1182 0.0087 0.390 0.870 0 0.00 1, 20, 50

64 128 64 0.345 ± 0.0723 0.0090 0.180 0.520 0 0.00 –

Total 8654 4392 0.607 ± 0.1793 0.0027 0.140 1.790 418 4.83

Egg batch corresponds to egg batch days starting from the first day of egg laying. n, total egg number; sampled N, eggs sampled; MWW,

mean wet weight (g); SD, standard deviation; SE, standard error; Min WW, minimum wet weight (g); Max WW, maximum wet weight (g).

Comparisons between MWW of different egg batches were conducted using a Kruskal–Wallis test. Numbers in the Dunn�s column identify

those who were not different in MWW against the egg batch in that row for P < 0.05.

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Table 2 resumes the LC (lg/egg) for wild eggs throughout

the embryonic development, while Table 3 resumes it for

cultured eggs. Polar and neutral lipids of both wild and

cultured eggs remained consistently proportional (�50% for

each lipid fraction) despite their increase in content

throughout the embryonic development. Higher levels of

phosphatidylcholine (PC), PE, CHO and triacylglycerol

(TAG) were found in both types of eggs along the embryonic

development. Within the same group of eggs and for wild

eggs, a significant increase (P < 0.05) was only found be-

tween stages in terms of phosphatidylserine (PS), PE and

FFA content. In cultured eggs, an increase along the

embryogenesis (P < 0.05) was found in polar and neutral

totals and in the PS, phospatidylinositol (PI), PE, CHO and

FFA contents. Other LC, such as SM, PC and TAG

increased between 1 and 20 days but not between 1 and

30 days. In terms of the percentage profile (data not shown),

all LC presented constant values along the embryonic

development (1–30 days), in both type of eggs, except for a

reduction in sterol esters (SE) in wild eggs (10.1–4.6%,

P < 0.05) and PC in cultured eggs (30.7–22.2%, P < 0.05).

When comparing wild and cultured eggs, at a given day of

embryogenesis (Table 3), the more relevant differences

(P < 0.05) were found in the higher contents of SM in cul-

tured eggs after 10 days old and the higher contents of PE in

wild eggs at 30 days old.

Tables 4 and 5 resume the overall FA composition of TL

of wild and cultured eggs (respectively) throughout the

embryonic development. In both type of eggs, although with

different amounts, a similar FA profile of TL was obtained,

1·5

2·5

2

1

0·5

01 5 10 15 20 25 30

Mea

n w

et w

eigh

t (g

)

CultureWild

Days

Figure 1 Egg mean wet weight (MWW)

trends over embryonic development.

Vertical bars represent standard devia-

tion. Stars represent differences for

P < 0.05.

96

94

92

90

88

86

84

8250 1510 2520 30

Days

Moi

stur

e (%

)

Culture eggsWild eggs

Figure 2 Egg moisture trends over

embryonic development. Vertical bars

represent standard deviation. Stars rep-

resent differences for P < 0.05.

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



where the main FA present were 16:0, 18:0, 20:5n-3 and

22:6n-3. These FA accounted for approximately 70% of all

FA and saturated and n-3 totals seemed to have the same

proportion in the cuttlefish eggs.

The most relevant FA changes (P < 0.05) in wild eggs,

during embryonic development, were present in 18:0, 21:5n-

3, 20:1n-9 and in n-9 totals, which increased between 1 and

30 days. Both 20:5n-3 and n-3 HUFA totals showed signifi-

cant differences (P < 0.05) according to ANOVAANOVA but a poste-

riori test did not revealed differences between day groups.

Although less variable, cultured egg FA also presented a

significant increase (P < 0.05) along embryonic development

for 16:1, 17:0, 16:3n-3, 20:1n-9, 20:3n-3 and n-9 totals. Other

FA, such as saturated totals (including 18:0 and 20:0) and

monoenes totals (among them 18:1n-9), showed an increase

(P < 0.05) until the 20th day. However, at the final stage

(30 days) a higher egg data variability was noted and, con-

sequently, significant differences, with respect to day 1, dis-

appeared. 16:0 showed significant differences (P < 0.05), but

the a posteriori test applied was not able to distinguish the

variables in question. The n-3/n-6 ratio showed an increased

tendency, between 1 and 20 days old, in cultured eggs but not

in wild eggs, which produced differences (P < 0.05) between

both kinds of eggs at different days. The EPA/DHA ratio

presented quite steady and similar values of 0.6–0.7

throughout the embryonic development, whereas AA/EPA

Table 2 Total lipids and lipid classes in

lg/egg for wild eggs through the

embryonic developmentLipid classes

W1 W10 W20 W30

Mean ± SD Mean ± SD Mean ± SD Mean ± SD

LPC 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00

SM 26.6 ± 4.28 31.6 ± 7.23 27.6 ± 10.69 30.4 ± 5.29

PC 391.6 ± 100.13 427.9 ± 52.12 578.9 ± 60.30 535.0 ± 80.84

PS 20.3 ± 3.34a 30.7 ± 10.44ab 74.1 ± 5.99b 150.8 ± 28.68c

PI 62.1 ± 27.10 62.4 ± 9.36 85.7 ± 13.64 100.4 ± 25.20

PE 313.9 ± 98.80a 341.5 ± 36.27a 456.3 ± 44.08ab 527.2 ± 57.76b

CHO 503.5 ± 132.69 564.3 ± 62.01 655.5 ± 135.14 666.7 ± 103.60

FFA 0.0 ± 0.00a 0.0 ± 0.00a 31.7 ± 5.20b 19.9 ± 6.49b

TAG 210.8 ± 43.47 245.4 ± 42.39 295.4 ± 18.78 269.7 ± 56.64

SE 171.2 ± 48.51 129.6 ± 19.47 198.0 ± 101.49 111.3 ± 30.73

Polar lipids 814.5 ± 229.97a 894.0 ± 111.31ab 1222.7 ± 115.31ab 1343.7 ± 197.15b

Neutral lipids 885.5 ± 215.39 939.3 ± 114.90 1180.6 ± 53.58 1067.6 ± 188.71

Total lipid 1700.0 ± 444.41 1833.3 ± 225.46 2403.3 ± 152.59 2411.3 ± 383.03

Results represent means SD (n = 3). Superscript letter represent differences within the same row

(lipid class) for P < 0.05.

LPC, lisophophatidylcholine; SM, sphingomyelin; PC, phosphatidylcholine; PS, phosphatidylser-

ine; PI, phospatidylinositol; PE, phosphatidylethanolamine; CHO, cholesterol; FFA, free fatty

acids; TAG, triacylglicerides; SE, sterol esters.

Table 3 Total lipids and lipid classes in

lg/egg for cultured eggs through the

embryonic developmentLipid classes

C1 C10 C20 C30


LPC 4.1 ± 3.91 9.4 ± 0.91* 4.5 ± 3.93 2.0 ± 3.46

SM 30.9 ± 12.39a 61.0 ± 11.34b* 64.6 ± 5.82b* 48.8 ± 3.33ab*

PC 335.3 ± 110.6a 572.6 ± 87.29ab 688.0 ± 22.08b* 521.8 ± 95.91ab

PS 17.9 ± 6.62a 30.6 ± 3.58a 104.2 ± 27.97b 138.5 ± 17.31b

PI 42.1 ± 15.28a 57.3 ± 9.92ab 113.7 ± 28.36c 102.2 ± 19.13bc

PE 146.0 ± 54.07a 255.4 ± 44.13ab 372.5 ± 43.82b 383.5 ± 51.78b*

CHO 250.4 ± 95.72a 446.3 ± 75.98ab 688.1 ± 54.71c 656.9 ± 75.57bc

FFA 8.5 ± 8.29a 5.7 ± 9.84a 19.8 ± 1.74ab* 31.9 ± 3.50b*

TAG 137.6 ± 53.19a 265.4 ± 47.04ab 333.1 ± 34.06b 263.1 ± 53.19ab

SE 127.2 ± 54.08 190.7 ± 44.97 211.3 ± 96.44 201.2 ± 169.81

Polar lipids 572.2 ± 197.91a 976.9 ± 155.33ab 1343.1 ± 110.86b 1194.8 ± 183.87b

Neutral lipids 523.7 ± 195.94a 908.2 ± 159.52ab 1252.4 ± 106.99b 1153.2 ± 161.91b

Total lipid 1095.9 ± 393.27a 1885.0 ± 312.86ab 2595.5 ± 3.94b 2348.0 ± 253.00b

Footnotes as in Table 2. Asterisk represent differences between wild and cultured eggs at the

same period for P < 0.05.

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



presented similar values of 0.2–0.3 (P > 0.05) consistently in

both wild and cultured eggs and at different days. Table 5

also presents a FA comparison (marked as *) between both

types of eggs at a given day of embryonic development. No

differences were found on the most expressive FA (P > 0.05),

but only found in some minor components (P < 0.05). Sev-

eral FA, such as 16:1n-5, 17:0 and 22:5n-3, only showed

differences (P < 0.05) at the first days/stages, because of the

initial lower content found in cultured eggs that increased at

the end of embryogenesis.

While obtaining eggs to the present experiment, intermittent

spawning [as reported by Boletzky (1987b)] was observed

and seemed to be directly related to the present culture

conditions. It seems that the use of a larger spawning tank

(with a larger bottom area), in the present experiment, also

enhanced several other biological aspects of the species, such

as the size of individual spawners (the biggest until now on

our culture facilities), the spawning period and both total

and individual fecundity (8654 eggs and 787 eggs/$) as well

as fertility (81.9%). Larger bottom areas probably stimu-

lated the animals to become bigger and to lay more eggs,

extending their spawning period (see Sykes et al. 2006b for

comparisons). Forsythe et al. (1994), by using larger tanks

with larger bottom areas, consistently obtained higher

fecundity values than those reported by Domingues et al.

(2001, 2002, 2003) and Sykes et al. (2006b). However, as no

replicas of spawning tanks were used in any of these studies,

Table 4 Fatty acid of total lipid (lg/egg)of wild eggs

Fatty acids

W1 W10 W20 W30


14:0 23.3 ± 11.7 32.5 ± 4.5 35.8 ± 2.1 39.3 ± 7.0

16:0 209.5 ± 81.3 230.9 ± 28.5 279.3 ± 37.9 341.0 ± 53.0

16:1n-7/n-9 3.4 ± 1.8 2.5 ± 0.2 3.1 ± 0.7 4.9 ± 0.7

16:1n-5 9.0 ± 4.4 7.8 ± 0.3 7.7 ± 1.3 11.1 ± 0.8

16:1 6.1 ± 4.5 3.5 ± 0.7 4.2 ± 0.7 7.6 ± 1.2

17:0 14.1 ± 4.4 13.2 ± 2.3 16.3 ± 2.6 18.3 ± 0.8

16:3n-3 3.7 ± 4.5 2.5 ± 0.3 5.0 ± 2.0 6.2 ± 0.8

18:0 75.3 ± 30.7a 79.3 ± 5.2a 100.9 ± 15.6ab 140.2 ± 24.1b

18:1n-9 25.3 ± 15.7 21.4 ± 0.9 30.6 ± 5.2 42.2 ± 7.3

18:1n-7 18.3 ± 9.2 17.0 ± 0.8 16.4 ± 1.5 24.6 ± 4.3

18:1n-5 4.2 ± 2.2a 3.4 ± 1.2ab 0.0 ± 0.0b 3.6 ± 0.5ab

18:2n-6 6.6 ± 5.4 2.2 ± 1.0 1.5 ± 1.4 4.7 ± 1.5

18:3n-3 5.0 ± 3.4 2.0 ± 0.3 2.3 ± 0.6 2.7 ± 2.4

20:0 7.0 ± 9.0 2.6 ± 0.3 3.9 ± 0.7 5.1 ± 0.4

20:1n-9/n-11 22.2 ± 8.6a 24.9 ± 3.6a 35.4 ± 7.1a 53.3 ± 6.7b

20:2n-6 1.2 ± 1.0ab 1.8 ± 0.3a 2.7 ± 1.1ab 4.0 ± 0.2b

20:4n-6 28.6 ± 11.5 26.6 ± 3.3 37.0 ± 10.4 42.3 ± 1.6

20:3n-3 1.9 ± 2.2ab 0.3 ± 0.6a 0.8 ± 1.4a 5.7 ± 1.0b

20:5n-3 122.9 ± 48.3 107.6 ± 22.8 142.5 ± 55.4 216.9 ± 29.7

22:1n-9/n-11 0.0 ± 0.0 0.9 ± 1.5 2.7 ± 0.9 3.4 ± 1.2

21:5n-3 0.6 ± 1.0a 1.5 ± 0.5ab 3.3 ± 0.5bc 3.8 ± 0.9c

22:4n-6 0.9 ± 0.8 0.9 ± 0.8 3.5 ± 1.3 4.4 ± 3.8

22:5n-6 5.1 ± 2.2 4.3 ± 1.0 7.5 ± 3.3 8.9 ± 2.6

22:5n-3 11.1 ± 2.8 10.4 ± 2.7 18.9 ± 8.9 20.7 ± 5.3

22:6n-3 221.0 ± 93.5 164.1 ± 40.2 198.7 ± 76.0 351.4 ± 68.1

UK 41.9 ± 41.1 28.3 ± 3.1 18.6 ± 0.9 37.1 ± 4.4

Totals

Saturated 339.6 ± 144.9 366.5 ± 41.5 447.5 ± 60.1 561.1 ± 81.4

Monoenes 102.9 ± 61.2 87.0 ± 4.3 101.4 ± 18.3 157.4 ± 19.5

n-3 374.0 ± 154.5ab 291.5 ± 64.1a 376.9 ± 143.8ab 618.3 ± 88.4b

n-6 43.2 ± 16.5 38.9 ± 2.2 53.5 ± 15.8 66.4 ± 6.1

n-9 47.5 ± 24.2a 47.2 ± 4.4a 69.3 ± 13.2ab 102.3 ± 16.1b

n-3 HUFA 358.1 ± 145.3 284.9 ± 64.0 365.5 ± 140.3 594.4 ± 91.1

n-3/n-6 8.6 ± 0.6 7.5 ± 1.5 6.9 ± 0.7 9.4 ± 2.1

EPA/DHA 0.6 ± 0.0a 0.7 ± 0.0ab 0.7 ± 0.0b 0.6 ± 0.0ab

AA/EPA 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.2 ± 0.0

Results represent means SD (n = 3). Totals include some minor components not shown. Super-

script letters represent differences within the same row (lipid class) for P < 0.05.

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



this subject still has to undergo detailed research before any

confirmation.

The duration of embryogenesis is temperature dependable

(Boletzky 1987a, 2003; Boletzky et al. 2006). The trend of

MWW of both wild and cultured eggs was similar to that

reported by Bouchaud & Daguzan (1990) for the species, but

showed lower embryonic development duration because of

the higher temperature of the present experiment. The dif-

ferences found in MWW of eggs for days 1 and 5 (increased

weight in wild eggs) may be explained by the bigger females

of the wild group (data not shown), which according to

Boletzky (1983), lay bigger eggs. The differences found in wet

weight at day 25 were probably because of the fact that the

cultured eggs started hatching at this day, while wild eggs

took five more days until the first cuttlefish hatched. In fact,

cultured eggs displayed a hatching window (from the first

born until the last one) of 5 days while wild eggs displayed a

smaller time of only 2 days. Nevertheless, both trends of

weight evolution over time were similar and characterized by

a drop during the first 10 days (while expelling the water

from the inside and consequent hardening of the egg). It was

then followed by a huge increase of the water content to

maintain osmolarity (Paulij et al. 1991; Boletzky 2003) until

hatching [which, according to Naef (1928) and Lemaire

(1970), is when most of the cuttlefish hatchling is already

basic formed, and only specialized structures like eyes,

digestive gland, chromatophores are in the final stages of

development]. This dehydration and re-hydration was

Table 5 Fatty acid of total lipid (lg/egg)of cultured eggs

Fatty acids

C1 C10 C20 C30


14:0 17.3 ± 6.7 27.5 ± 6.1 38.7 ± 8.2 34.8 ± 14.7

16:0 130.7 ± 40.1 219.7 ± 38.6 327.7 ± 53.0 286.7 ± 121.2

16:1n-7/n-9 1.7 ± 0.4 2.1 ± 0.4 3.7 ± 1.6 4.6 ± 2.2

16:1n-5 3.8 ± 1.0 6.0 ± 1.1* 7.5 ± 2.9 8.6 ± 3.7

16:1 2.6 ± 0.9a 2.3 ± 0.3a 10.4 ± 0.8b* 8.4 ± 2.7b

17:0 5.5 ± 1.2a* 8.9 ± 1.3ab* 13.1 ± 1.6b 13.2 ± 5.5b

16:3n-3 2.7 ± 0.4a 4.6 ± 1.2ab 4.4 ± 0.3ab 6.1 ± 1.5b

18:0 44.7 ± 13.0a 77.7 ± 14.8ab 122.9 ± 16.2b 123.4 ± 56.5ab

18:1n-9 17.4 ± 6.0a 25.0 ± 4.0ab 42.8 ± 8.0b 39.6 ± 17.1ab

18:1n-7 10.1 ± 2.9 17.4 ± 3.9 22.1 ± 7.2 24.5 ± 11.5

18:1n-5 2.2 ± 1.9 1.5 ± 0.3 4.7 ± 3.6 2.8 ± 1.2

18:2n-6 2.6 ± 1.0 2.6 ± 0.4 5.4 ± 2.9 5.6 ± 3.2

18:3n-3 1.3 ± 0.3 1.8 ± 0.3 2.5 ± 2.2 2.7 ± 0.9

20:0 1.9 ± 0.7a 3.2 ± 0.5ab 5.0 ± 0.6b 4.2 ± 1.6ab

20:1n-9/n-11 11.8 ± 4.4a 21.4 ± 5.3ab 37.7 ± 8.8ab 45.0 ± 21.8b

20:2n-6 1.7 ± 0.6 2.5 ± 0.4 3.5 ± 1.1 4.4 ± 2.7

20:4n-6 17.4 ± 7.8 24.0 ± 1.9 28.3 ± 13.5 25.5 ± 18.8

20:3n-3 0.2 ± 0.4a 1.2 ± 0.2ab 1.2 ± 1.1ab 7.2 ± 4.8b

20:5n-3 69.2 ± 24.0 128.0 ± 26.0 141.7 ± 64.1 131.4 ± 74.5

22:1n-9/n-11 1.4 ± 0.3* 1.8 ± 0.3 2.2 ± 0.8 1.5 ± 1.4

21:5n-3 1.8 ± 0.9ab 2.1 ± 0.4a 4.4 ± 1.9ab 3.6 ± 0.2b

22:4n-6 1.1 ± 1.0 0.8 ± 0.7 2.0 ± 1.8 2.6 ± 1.1

22:5n-6 3.2 ± 1.0 4.8 ± 0.5 4.4 ± 3.9 4.0 ± 2.3

22:5n-3 4.1 ± 1.2* 8.3 ± 1.3 10.4 ± 5.4 9.8 ± 6.2

22:6n-3 107.3 ± 37.3 196.5 ± 33.8 213.5 ± 112.4 187.3 ± 114.3

24:1n-9 1.3 ± 2.2 2.3 ± 1.1 * 0.0 ± 0.0 * 0.0 ± 0.0*

UK 9.2 ± 6.1 9.7 ± 1.5 29.8 ± 15.9 25.1 ± 11.5

Totals

Saturated 208.3 ± 59.7a 345.4 ± 62.0ab 522.2 ± 78.9b 475.5 ± 202.0ab

Monoenes 54.1 ± 13.3a 80.4 ± 16.9ab 134.2 ± 23.9b 139.2 ± 64.4ab

n-3 190.9 ± 62.8 346.0 ± 62.8 386.5 ± 181.8 354.7 ± 205.5

n-6 26.8 ± 9.2 34.8 ± 2.7 43.6 ± 22.3 42.1 ± 27.9

n-9 31.9 ± 12.7a 50.5 ± 10.3ab 82.7 ± 16.1b 86.2 ± 39.9b

n-3 HUFA 183.0 ± 62.5 334.9 ± 61.2 370.8 ± 178.6 332.7 ± 196.0

n-3/n-6 7.2 ± 0.6a* 9.9 ± 1.2ab 9.0 ± 0.6b* 8.7 ± 0.7ab

EPA/DHA 0.6 ± 0.0 0.6 ± 0.0 0.7 ± 0.1 0.7 ± 0.0

AA/EPA 0.2 ± 0.0 0.2 ± 0.0* 0.2 ± 0.0* 0.2 ± 0.0

Footnotes as in Table 4. Asterisk represent differences between wild and cultured eggs at the

same period *P < 0.05.

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



previously described by Bouchaud & Daguzan (1990), with

similar values of moisture content between 85% and 95%

(Fig. 2) but it is not temperature dependable. In fact, the

trend of the moisture curves obtained for both wild and

cultured eggs, in the present experiment, was similar to those

obtained by these authors at similar embryonic development

temperatures. While undergoing its development, the ceph-

alopod embryos use yolk as food, to obtain energy and

structural components. Its amount and quality is of para-

mount importance to achieve a successful embryonic and

post-embryonic development. The sepia yolk has been

described as consisting of soft globules, surrounded by a

membrane and composed by a water soluble glyco-lipo-

protein (Ito et al. 1962; Blanchier 1981). According to Ito &

Fujii (1962), the glyco-lipo-protein has 20% lipid (with 65%

phospholipid and minor or none CHO contents) and a high

amount of carbohydrate (12.6%). In cuttlefish, yolk is

available in the outer and inner yolk sacs. According to

Boletzky (1989, 2003), the outer yolk sac is an �organ� which

plays several functions such as respiratory and circulatory

(until the proper formation of both gills and hearts) and also

acts as a stirrer, that allow the continuous movement of the

perivitelline fluid around the embryo. The epithelium of the

inner yolk sac is functional from early stages, while the outer

yolk sac epithelium becomes progressively active with the

completion of the outer (ectodermic) cover (Boletzky 1987a).

Newly born hatchlings generally have yolk left in the inner

yolk sac, the amount of which varies greatly (Boucher-Ro-

doni et al. 1987), and its quantity may influence the matu-

ration of central nervous system (CNS; Dickel et al. 1997).

During embryonic development and until hatching, the yolk

platelets are �digested� and assimilated by the growing

embryo through its blood sinuses, which exist in the external

yolk sac (Boletzky 1975).

In the present study, after deposition by females, both wild

and cultured egg batches used for the study of weight evo-

lution during embryogenesis displayed MWW(g) of

0.626 ± 0.131 and 0.766 ± 0.310 respectively (Fig. 1); while

Bouchaud & Galois (1990) reported a MWW(g) of

1.31 ± 0.12. Several factors may be the cause for such a

difference in egg weight and we will address this issue by

discussing the influence of these factors in egg weight,

duration of embryonic development and lipid content.

It is known that bigger females lay bigger eggs (Boletzky

1983) and that English Channel cuttlefish females, such as

those used in the Bouchaud & Galois (1990) study, are bigger

in nature because of the increased life cycle [data from

Richard (1971) compared with that of Sykes et al. (2006b)].

It is also known that temperature plays a major role in

cuttlefish growth and life cycle (Domingues et al. 2001, 2002;

Sykes et al. 2006b) and that both geographical locations

(south Portugal and English Channel) have different tem-

peratures all year round. Higher temperature could explain

the lower egg weights that have been consecutively found in

the Faro populations [data from the current study and Sykes

et al. (2006b)]. For instance, eggs obtained by Sykes et al.

(2006b) during consecutive generations displayed bigger eggs

from females cultured at lower temperatures than those from

females cultured at higher temperatures, and in here the

premise �bigger females lay bigger eggs� is still confirmed.

However, the existence of contradictory data between both

English Channel and south Portugal populations also may be

as a result of the different genetic substructure found (Wol-

fram et al. 2006), which would imply different physiological

adaptations and eggs nutritionally different in content and

development. Sykes et al. (2006a) discussed, in its recent re-

view on the potential of cuttlefish culture, that there might

exist sub-speciation in the species, based on several reported

data on biological parameters such as egg and hatchling

weights, duration of the life cycle, etc.

Similarly, embryonic development is known to be tem-

perature dependent (Boletzky 1983; Bouchaud & Daguzan

1990). However, the geographical variations were not taken

into account until now and, although the period of embry-

onic development increases with lower temperatures, the

values diverge between Faro and Caen (see Bouchaud &

Galois 1990; Sykes et al. 2006b). A longer incubation period

demands for higher and/or different yolk content. In fact,

Bouchaud & Galois (1990) described higher yolk-TL con-

sumption at 12 �C (82% of initial yolk TL) than at 24 �C(only 26%). Thus, the embryo will have different metabolic

needs and rates at lower temperatures that also promote

higher available dissolved oxygen. However, at higher culture

temperatures there is an increased need for oxygen uptake by

the embryo (Wolf et al. 1985). Therefore, different physiol-

ogies may be applied in accordance. This is even more evi-

dent when, according to Bouchaud & Galois (1990),

cuttlefish embryos consume only 26% of their egg-yolk

during the embryonic development at 24 �C, while at 15 �Cegg-yolk consumption is close to 78%. Also, Bouchaud &

Daguzan (1990) reported data regarding the conversion of

the yolk in hatchling dry weight, where eggs cultured at 12 �Cwould convert as much as 91%, while those cultured at 21 �Cwould only convert 49%. So, at 15 �C, the embryo needs to

cope with a longer embryonic development, yolk must pro-

vide enough food (for growth and energetic demands) and

oxygen demands are more easily met due the high solubility,

thus not requiring so much outer yolk sac area/volume

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



available. According to the energetic budgets of Bouchaud

(1991), at 15 �C, 41% of egg-yolk are used for growth rate

and only 10% for catabolic processes (respiration and

excretion), while at 24 �C growth rates are smaller (15%) and

there is an increased catabolism (52%). In practice, English

Channel eggs cultured at 15 �C use about 7.8% of their yolk

as energy, while those cultured at 24 �C use as much as 13%.

All these data would be related with the smaller hatchlings

obtained at higher temperatures. It seems that lower tem-

peratures (12–15 �C), which promote extended incubation

because of lower growth rates but also higher use of yolk, will

also imply the requirement of the egg for higher and/or dif-

ferent nutritional content. Depending on the temperature,

life cycle and embryogenesis duration in cuttlefish are

adapted [lower temperatures promote extended periods; see

Sykes et al. (2006b) for data], and therefore geographical

locations probably stimulate physiological differences that

are reflected in the egg content and metabolism. Therefore,

studies that will use both Faro and Caen eggs cultured

through their embryogenesis at similar temperatures and

covering a wide range of temperatures needed to determine if

embryonic development follows the same time periods and if

hatchlings share similar weight at hatching. Also, the quan-

tity, quality and the use of the yolk should be investigated.

When we analysed the lipid content and composition in

both egg groups (from wild and cultured spawners),

throughout embryonic development, they showed similar li-

pid and moisture content. It was noteworthy an increase in

TL, and certain LC and FA, but different live feed (cultured

spawners were only fed live grass shrimp while wild spawners

usually feed on a wide variety of prey) did not promoted a

significantly different lipid profiles between eggs.

Sargent (1995) and Rainuzzo et al. (1997) stated that, in

fish, lipids are major sources of metabolic energy throughout

the embryonic development. However the TL amounts and

the LC used varies with the fish species. Normally, there is a

PC and TAG catabolism and a PS and PE synthesis. In the

present experiment, no lipid consumption, either in percent-

age or in absolute value (lg/egg), during embryonic devel-

opment was observed. The maintenance of lipid contents of

this study is in agreement with Bouchaud & Galois (1990),

who observed maintenance of TL between newly-laid eggs

and hatchlings. However, percentage values from the present

study (2–3%DW of TL, data not shown) were clearly lower

than that of the previous authors (14% DW). Also, our egg

absolute TL values were lower at spawning (�1.5 mg/egg;

mean from values of Tables 2 and 3) than those of the Bou-

chaud & Galois (1990) study (�8.5 mg/egg). Therefore the

differences found cannot be explained only by the elimination

of the outer gummy layers from the eggs, made by the latter

authors and not performed in the present study. From the

present data, we understand that the lipid value cannot be

independent from egg size and constant geographically, as

stated earlier by Bouchaud & Galois (1990) and, therefore,

this value is different between different populations of the

same species. The reason for the different amount of lipids in

cuttlefish eggs from Faro and Caen could be related with

differences in metabolism, temperature influence or genetic

sub-speciation, as mentioned before in this discussion.

According to Richard (1971), the egg size greatly influences

the amount of viteline reserves. Viteline assimilation provides

energy for breathing and, through catabolic processes, is used

in the formation of new tissues. Therefore, bigger eggs will

probably mean different content (in %) either in protein, lipid

and carbohydrate, and/or in their individual content [e.g. it

may refer to an increased lipid content and different LC of

eggs from the English Channel, that are used during

embryogenesis at similar temperatures (laboratory induced)

obtained naturally in the present study, which are not normal

in that geographical area], as seen when comparing data from

the latter authors and that of present study.

In the present study, SD obtained for both TL and LC

were quite high and seemed to reflect not only different

amounts of lipid content per egg (promoted by either dif-

ferent females and/or the individual content of each egg), but

also the fact that each female may lay eggs for several hours,

thus leading to different embryonic development times (to a

maximum of 24 h). The increase or maintenance of the egg�s

lipid fraction, in its total weight, points out to a possible

alternative use of the protein and/or carbohydrate fraction as

energy during this phase, which would be in accordance with

the findings of Hochachka (1994). This author stated that

cephalopod muscle mitochondria work under oxygen limi-

tations, because of the lack of several mechanism to upre-

gulate the capacities for oxygen flux from blood to

mitochondria, such as lack of intracellular myoglobin-ana-

logue and low-intracellular lipid concentrations (presented

that high-lipid concentrations supplied a higher oxygen sol-

ubility and diffusion). This situation would favour an oxy-

gen-efficient metabolic organization which maximizes the

ATP yield per mole of oxygen. In this sense, the oxidation of

carbohydrates and amino acids are more oxygen efficient

than lipids. This is also in accordance with O�Dor et al.

(1984), that states that cephalopods use protein as building

blocks for growth and carbohydrate as energy fuel, the later

being stored in small amounts as muscle glycogen. Similar

results were obtained by Castro et al. (1992), for starved

juvenile-adult cuttlefish. In the present study, the verified

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



lipid increase (although not statistical significant in wild eggs)

may perhaps be explained by de novo synthesis of some of its

components during embryogenesis, although further studies

are necessary to confirm this.

Both polar and neutral lipid fractions seemed to be equal

in the egg content (�50% each). A similar 1 : 1 ratio of PL

and NL was found by Silversand et al. (1996) in turbot eggs.

The absolute value (lg/egg) of several lipid classes such as

PS, PI, PE and CHO increased along the embryonic devel-

opment. Similar results were reported by Vazquez et al.

(1994), in Solea senegalensis eggs, for the PS and PI; this

could be explained by de novo synthesis of these LC, given

their importance as membrane constituents. Navarro & Vi-

llanueva (2000) reported similar findings for hatchlings and

Almansa et al. (2006) for juvenile and maturing cuttlefish.

Phospholipids and CHO are known to have a structural role

as components of cell membranes (Sargent et al. 1995a).

Phosphoglycerides (especially PC) are used not only for cell

division and organogenesis but also used as fuel by some fish

species (Sargent 1995), while neutral lipids (especially TAG

and SE) are generally considered the most important energy

reserves in marine fish eggs and larvae (Almansa et al. 1999).

Triacylglycerol content described in the present study

corresponded to >10% of TL. That is in agreement with

Almansa et al. (2006), who found low levels of TAG in the

mantle of maturing cuttlefish, despite the high level of this

LC content in fish used as diet. Bouchaud & Galois (1990)

reported a small decrease in TAG content between newly-laid

eggs and hatchlings of cuttlefish, which was not observed in

the current study in either group. It is known that TAG may

have a dual application: a metabolic, where it can be used as

a source for ATP production through oxidation, and/or

structural, where it can be a source of FA for polar lipid (PL)

biosynthesis (Sargent et al. 1995a). However, the statistical

differences found in TAG (only for cultured eggs) did not

allow us to determine a trend. Nonetheless, it was notewor-

thy the lower SD found in both groups at day 20. Finally, the

higher contents of PE present in wild eggs respect to cultured

eggs at 30 days old suggest a possible deficiency of this LC,

which has a relevant role in the nervous system and eye

formation.

The high levels of CHO obtained for both type of eggs in

the present study was similar to those found by Almansa

et al. (2006) and Domingues et al. (2004) in cuttlefish mantle.

CHO is an important LC as a component of cell membranes

with relevant properties (Crockett & Hazel 1997) and, in

cuttlefish, CHO (either endo or exogenous) is a precursor for

steroid biosynthesis (Blanchier 1981). In addition, it was re-

ported earlier by Goad (1981) that cuttlefish gonads possess

all seven enzymes required to produce the steroid interme-

diates that allow the biosynthesis of testosterone. Besides the

earlier Zandee�s (1967) findings regarding an absence of

endogenous synthesis of CHO for the species, we believe that

CHO should be included in the elaboration of a future diet

and must be present in the egg yolk for a correct develop-

ment. In fact, grass shrimp, which was used as optimal prey

during the first 30 DAH and the rest of the life cycle (Sykes

et al. 2006b), presents high levels of CHO. It is relevant that

Ito & Fujii (1962) reported the non-existence of CHO in

cuttlefish high-density lipoproteins (HDL) isolated from the

egg yolk. However, our results and previous data by Bou-

chaud & Galois (1990), found an 8–9% of CHO (%TL) in

cuttlefish eggs. Evidence for de novo sterol biosynthesis in the

species had been acknowledged by Kanazawa (2001). These

differences may perhaps be explained by the inclusion of

CHO in other egg-yolk components different from the HDL

studied by the later authors. Also, the presence of CHO, in

an esterified form, as SE (CHO esterified with a FA), would

explain the difficult of detection of CHO content in the HDL

by the experimental method used by Ito & Fujii (1962), al-

though these hypotheses should be confirmed with further

studies.

Lipid content of the eggs is supplied by the female, ob-

tained from diet and/or its lipid storage. Lee (1994) suggested

that if the lipid fraction of the mantle is so low, then the lipid

storage is made at the digestive gland. Blanchier & Boucaud-

Camou (1982) studied the lipid composition of the digestive

gland in wild-mature cuttlefish and found differences of TL

between males and females (9%WW and 13%WW respec-

tively). This is in accordance with the findings of Boucaud-

Camou (1971), who described the lipid fraction of the

digestive gland to be rich in TAG, sterols, sterol and wax

esters, plus phospholipids (PE and PC). Blanchier & Bou-

caud-Camou (1982) also found a similar LC content, for

both sexes, to be characterized by phospholipids, sterols,

FFA, TAG, wax and SE. Nevertheless, the authors stated

that the available lipid (in the digestive gland) does not

change in terms of percentage, but do not discard its use in

lipid and lipoprotein synthesis in the gonads. It must be

considered that cuttlefish presents an intermittent spawning,

which would allow the females to replace the lipids used

and taken from the digestive gland through food ingested

during the spawning period. As information regarding oocyte

lipid incorporation is inexistent, studies regarding the lipid

pathways involved in digestive gland storage and release

into gonad development and oocytes in females at matur-

ing and spawning age need to be conducted to clarify this

issue.

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



In terms of FA, no major differences were found between

wild and cultured eggs throughout the embryonic develop-

ment. Although 16:0, EPA and DHA are the most commonly

found FA in cephalopod flesh, whole body, digestive gland,

CNS and photoreceptors, cuttlefish eggs also displayed high

levels of 18:0 and intermediate levels of both 18:1n-9 and

20:1n-9. Dumont et al. (1992) studied the FA profiles in the

CNS of cuttlefish and found a similar predominance of

DHA, EPA, 16:0 and 18:0 in its CNS. The present results

revealed high levels of the n-3 HUFA (especially EPA and

DHA) in cuttlefish eggs, thus confirming the results already

obtained by Navarro & Villanueva (2000) that the dietary

requirements for n-3 HUFA are critical in early cephalopod

developmental stages because of the high demand for mem-

brane synthesis (Henderson & Sargent 1985; Sargent

et al.2002). DHA plays an important role in the synthesis,

maintenance and functional integrity of the structure of cell

membranes in fish, especially in brain and retina, where it can

account for up to 40% of the total FA (Sargent 1995). The

importance of DHA in general cephalopods and in S. offi-

cinalis specifically may be classified as extreme if one takes

into account the exclusivity of both their brain and retinas.

EPA is a precursor of active eicosanoids such as prosta-

glandins. Nonetheless, despite the growing importance of

EPA and DHA in cuttlefish nutrition, one should not fall in

the same mistake of not considering AA as an essential FA.

The importance of AA as the major eicosanoid precursor for

normal growth and development has been demonstrated in

fish (Bell et al. 1995, 2003; Sargent et al. 1995a,b; Bell &

Sargent 2003) and Almansa et al. (2006) suggested an n-6

HUFA metabolism in juvenile and maturing juvenile cuttle-

fish, where AA could be involved in a possible biosynthesis

pathway of 22:4n-6 and 22:5n-6.

When we analysed the variation of FA along the

embryonic development, it was relevant the increased of

several FA such as n-9 totals and particularly 20:1n-9,

found in both wild and cultured eggs. This tendency could

be as a result of de novo synthesis of these FA, which is a

usual pathway in marine species (Sargent 1995). In wild

eggs, despite being statistically non-significant, an increase

in 20:5n-3 was also noted. The synthesis of this FA is not as

usual as the one observed for monoenes and especially in a

carnivorous species as cuttlefish, which must obtain 20:5n-3

from the diet (Sargent 1995; Sargent et al. 2002). However,

no data regarding both elongation and desaturation (met-

abolic pathways) of FA are currently available for cepha-

lopods, and therefore additional studies are necessary to

provide further insight and confirmation of the current

data.

In conclusion, the present authors suggest that the dis-

crepancy in results obtained here (in terms of egg lipid con-

tent and possible lipid metabolism) might be related to the

existence of sub-species or at least two different populations

(see Sykes et al. 2006a). If this is proven to be true, then the

optimal embryonic development temperature of 15–18 �Csuggested by Bouchaud (1991) for the English Channel might

not be the best for the south Portugal population. One pos-

sible way to determine nutritional and physiological differ-

ences in eggs between populations, based on the above

assumptions, would be to determine both total amount of

energy per individual (J or cal/ind.) of south Portugal (as it

will indicate the energy available to a developing embryo),

and the caloric value (J or cal mg)1) of those eggs. Surely the

energy available in bigger eggs will be substantially higher

than in smaller eggs, as previously observed by Bouchaud

(1991), as the embryos will have higher energetic demands as

demonstrated above. Another way would be the study of

nutrient contents and metabolism in geographically different

eggs (embryo plus yolk) and hatchlings. This would allow a

better judgment of what really happens in terms of different

egg weights and if the egg energetic reserves are or not

completely exhausted before hatching.

The present results suggest that lipids are not used as ener-

getic substrate in cuttlefish eggs of south Portugal. The FA

extracted from eggs of wild cuttlefish were highly unsaturated,

thus indicating that embryos and hatchlings have nutritional

requirements for PUFA, especially EPA and DHA. An

increasing trendwas found during embryonic development for

lipid and FA content which was not statistically significant in

many components because of the high SDof lipids within eggs.

The present data suggest a possible anabolism of several LC

such as PS, PI, PE and CHO, which could be explained given

their importance for the cell membrane formation. Regarding

that, further studies, which should involve separation of cho-

rion, embryo and yolk, need to be conducted to provide ex-

tended knowledge of lipid metabolism at this stage and prove

the above. We believe, however, that lipid importance at

the reproduction, egg and hatchling stages must be under-

lined because of their structural function. The present

results provide new insight in what should be the lipid

requirements for a prepared diet for the hatchling stage, which

should have a balanced PL and NL fractions and have similar

FA profiles as the ones presented here.

The authors would like to thank the Fundacao para a Ci-

encia e a Tecnologia (FCT) from the Portuguese government,

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and the Agencia de Inovacao (project AQUASEPIA) for

the financial support for this research. This work was also

funded by a FCT PhD grant (SFRH/BD/12409/2003) to

Antonio Sykes.

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Lipid characterization of both wild and cultured eggs of cuttlefish ( Sepia officinalis L.)...

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