Aquaculture Nutrition
doi: 10.1111/j.1365-2095.2009.00661.x
1 2
2
1
Universidade do Algarve, Faro, Portugal; 2 IFAPA CIFPA-Agua del Pino, Huelva, Spain; 3 Centro Oceanograco de
Canarias, IEO, Santa Cruz de Tenerife, Spain; 4 Departamento de Biologia Animal, Facultad de Biologa, Universidad de La
Laguna, La Laguna, Spain
(Correction added on 1 June 2009, after rst online publication: The header LETTER TO THE EDITOR was removed from
the rst page of this article.)
The eects of feeding three natural frozen diets, grass
shrimp (Palaemonetes sp.), craysh (Procambarus clarkii)
and sh (Sardina pilchardus) and two semi-humid articial
diets (based on sh or shrimp powder) to the cuttlesh,
Sepia ofcinalis, were analysed. Growth rate and feeding
rate [FR; % body weight (BW) day)1] and food conver-
sions (FC, %) were determined. Cuttlesh fed shrimp grew
larger (3.8% BW day)1) and had the highest FC, followed
by those fed craysh, and sardine. The highest FR was
obtained for cuttlesh fed craysh (10.5% BW day)1).
Although both articial diets were accepted, none pro-
duced growth. Digestive gland-to-body weight ratio (DG/
BW ratio) was calculated for animals fed each diet. A
positive correlation (r = 0.94) between cuttlesh ingestion
FR and DG weight was obtained. Mortality occurred
mainly during the last week, and some cannibalism
occurred among cuttlesh fed articial diets. Finally, lipid
composition of diets, DG and mantle of each group were
analysed. Sardine diet was characterized by high levels of
triacylglycerol (TG), whereas the main dierence between
shrimp and craysh was the higher n-3/n-6 ratio found in
shrimp. Changes in the lipid composition of DG were
related to diet, but did not correlate with growth data. A
strong loss of TG in the DG of articial diets groups was
notable. No dierences in mantle lipid composition among
the natural diets were found, but articial diet groups
showed higher contents of neutral lipids in their mantle
respect to natural diets. According to results obtained,
craysh (P. clarkii) could be used as an alternative prey for
rearing S. ofcinalis compared with shrimp. Articial diets
2010 16; 262275
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2,3 1 4
showed the worst eects in growth and mortality as well as
the stronger inuence on DG and mantle lipid composition
of cuttlesh.
KEY WORDS: alternative diets, culture, cuttlesh, digestive
gland, growth, lipid composition, mantle
Received 19 August 2008, accepted 22 January 2009
Correspondence: Eduardo Almansa, Centro Oceanograco de Canarias,
IEO, Avd. 3 de Mayo n73, 38005, Santa Cruz de Tenerife, Canary Islands,
Spain. E-mail: eduardo.almansa@ca.ieo.es
Sepia ofcinalis is one of the most easily cultured cephalo-
pods. This species has been maintained and/or cultured in
aquaria world over since the late 1960s (Richard 1971;
Pascual 1978; Boletzky & Hanlon 1983; Forsythe et al. 1994;
Lee et al. 1998; Domingues et al. 2001a,b, 2002, 2003a); it is
highly adaptable to life in captivity and suitable for aqua-
culture because it has (1) large eggs, (2) high hatchling sur-
vival, (3) sedentary behaviour, (4) tolerance to crowding,
handling and shipping, (5) acceptance of non-living foods
and (6) most importantly, it is easy to reproduce in captivity
(Forsythe et al. 1994).
Appropriate and inexpensive diets are basic requirements
for the success in commercial aquaculture (Chen & Long
1991). Cephalopods can be maintained or grown with several
natural live or dead preys (Boletzky & Hanlon 1983; Toll &
Strain 1988; DeRusha et al. 1989; DiMarco et al. 1993;
Domingues et al. 2002, 2005). Saltwater crustaceans, mainly
shrimp, have been the diets that promote better growth
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Journal compilation  2009 Blackwell Publishing Ltd
 (Domingues et al. 2003a,b, 2004), but the existence of very
few alternative diets that can be successfully used to culture
cephalopods is an important limitation that has prevented
large-scale culture until the present days (DeRusha et al.
1989; DiMarco et al. 1993; Lee 1994; Domingues et al. 2005).
Craysh (Procambarus clarkii) and sardine (Sardina pilchar-
dus) are two possible alternative preys to shrimp, when
considering the large-scale culture of some cephalopod spe-
cies, because of their considerable lower price, compared with
shrimp, if acceptable growth and survival could be obtained
when feeding these alternative preys to cephalopods.
Over 50% of the labour required in cephalopod culture or
maintenance involves collecting, holding and feeding live
prey (Lee 1994). Collecting and immediately freezing the prey
reduces labour costs by one-third. On the contrary, labour
required to prepare articial diets can be signicantly lower.
When prepared diets replace natural prey, additional cost
reduction higher than 20% can be estimated. Greater cost
reductions might be obtained by using commercial produc-
tion methods to obtain large quantities of articial diet
(Domingues et al. 2005).
Articial diets for cephalopods are being tested over the
past 15 years in order to lower cephalopod culture costs (Lee
et al. 1991; Domingues 1999; Domingues et al. 2005). To the
present day, growth and survival of cephalopods are com-
parable to sh larvae being transitioned from natural to
prepared diets (Dabrowski et al. 1978; Lindberg & Doroshov
1986). The inexistence of an inexpensive and storable arti-
cial diet for cephalopod culture has inhibited cephalopod
mariculture on a commercial basis (Lee et al. 1991; Domin-
gues et al. 2005, 2008). Therefore, the formulation of such a
diet is one of the primary objectives for the successful large-
scale culture of cephalopods (Lee 1994). In the last 15 years,
moist or dry pellets (Lee et al. 1991; Castro et al. 1993) or
surimi (sh myobrillar protein concentrate; Castro et al.
1993; Castro & Lee 1994; Domingues 1999; Domingues et al.
2005) have been experimented. Cuttlesh readily accepted
these prepared diets. These characteristics make this species
as a good research cephalopod to be used in the development
of articial diets.
Research on lipid and fatty acid (FA) requirements for
cephalopods has been neglected because of their main protein
metabolism, where lipids are not used for energy production
(Hochachka et al. 1975). Nevertheless, cephalopod paralar-
vae and juveniles most likely require prey rich in polyun-
saturated fatty acids (PUFA), phospholipids and cholesterol
(CHO; Navarro & Villanueva 2000; Koueta et al. 2002;
Domingues et al. 2003b, 2004). Therefore, in the present
study, the lipid composition of cuttlesh fed each diet and the
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Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
dierent prey and articial diets used were analysed in order
to get a better evaluation of dietary lipid sources, and their
possible relation with the obtained growth rates (GR).
In cephalopods, lipids usually account for less than 2% in
mantle wet weight composition (Almansa et al. 2006), but
digestive gland (DG) present a four-to-ve times higher lipid
content and high lipase activity (Moltschaniwskyj &
Johnston 2006). These data suggest that DG could be implied
in the lipid digestion and metabolism, but it is remarkable
that no evidence of a long-term lipid storage has been found
in this tissue. The data have been conrmed by Fluckiger
et al. (2008) who has observed a rapid turnover in the FA
prole of DG reecting changes in the diet. So, DG is a good
candidate to study the eects of diet changes in cuttlesh,
especially on lipid composition. Besides, we have included
mantle analysis because of the importance of this issue for
human consumption.
The present research had three main objectives. The rst
was to compare two alternative preys (craysh and sardine)
with the normally used prey (shrimp), which produces opti-
mal growth and survival, in order to determine if the
obtained GR and survival rate could be acceptable enough in
order to successfully replace shrimp with any of the two
alternative diets. The second objective was to determine the
acceptance of two articial diets prepared in the laboratory,
and their eect on growth and survival juvenile of cuttlesh.
The third objective was to determine the eect of each of the
ve diets used in lipid composition of the DG and mantle of
juvenile cuttlesh.
Sepia ofcinalis used in this experiment were born from eggs
collected in El Portil beach (Huelva, Spain South Atlantic
coast). Cuttlesh hatchlings were cultured in the IFAPA
Centro Agua del Pino in Cartaya, Spain. Animals were fed
live mysid shrimps (Mesopodopsis slabberi), during the rst
3 weeks of their life cycle in a ow through system, with
water temperatures of 21 1 C, salinity of 37 1 g L)1
and dissolved oxygen of 97 1%. Mysid shrimp were
captured with small hand nets in the salt marshes of the Rio
Piedras, Cartaya, close to the research facility (about 10 km),
and placed directly in the rearing tanks. Afterwards, juveniles
were fed frozen shrimps (Palaemonetes sp.) and maintained
in the same conditions, until the start of the experiment.
A total of 150 juveniles were used (75 days old;
13.8 2.3 g). Ten cuttlesh were randomly placed in each
of the 15 tanks used; there were no dierences (P > 0.05) in
the average body weight (BW) of the cuttlesh in each tank
at the start of the experiment. All tanks had a bottom area of
0.34 m2 (84 cm 40 cm), with water depth of 8 cm. Culture
density was of 30 cuttlesh m)2 (411 g m)2).
Each tank had an independent water inlet, outlet and
aeration, and was covered with a plastic mesh in order to
prevent cuttlesh from jumping out. Low light intensity,
which promotes better growth (Koueta & Boucaud-Camou
2003) was used to maintain low stress levels. Lights were
turned on only during feeding and cleaning periods. The
photoperiod cycle was 16 L : 8 D.
Three natural diets and two articial diets were tested. The
natural diets used were: (1) frozen shrimp (Palaemonetes sp.),
used as the control diet; (2) frozen sh (S. pilchardus) and (3)
frozen craysh (P. clarkii). All natural diets were purchased
from a frozen-product company in Sevilla, Spain (Seafood
Sevilla). The composition of the two articial semi-humid
diets is shown in Table 1. The sh commercial powder was
obtained from Harinas de Andaluca, S. A. (Cadiz, Spain).
The shrimp powder was made in the laboratory by drying
frozen grass shrimp at 60 C during 24 h. The protein hy-
drolysed (CPSP 90%) came from Cooperative de Traitement
de produits de la pesce (CTPP) (Le Portel, France); minerals
and vitamins came from Roche (Madrid, Spain). Both
articial diets were prepared on a weekly basis. Both frozen
prey and articial diets were stored at )20 C, and the por-
tions to be used were defrosted just before each feeding period.
Feeding rate (FR) was set at 15% BW day)1, which is
considered to be a good ration for this species, at this tem-
perature (DeRusha et al. 1989; Koueta & Boucaud-Camou
1999; Domingues et al. 2002, 2003a, 2004). Food was pro-
vided two times per day at 09:00 and 17:00. Domingues et al.
(2001a) report that several feeding periods throughout the
day promote higher feeding rates. Each tank was provided
with at least one piece of food per animal, to assure all ani-
mals had food available. The food was presented for 2 h and
Table 1 Composition of semi-moisture articial diets tested on
experiment 1 (expressed as wet weight)
Diet 1 Diet 2
Components (g kg)1) (g kg)1)
Fish commercial powder 550
Shrimp powder 550
Protein hydrolysed (CPSP)1 150 150
Wheat powder 100 100
Vitaminic premix 25 25
Mineral premix 10 10
Vitamin C 15 15
Fresh squid2 750 750
1
860 g kg)1 of fish protein and 94 g kg)1 total lipid content.
2
The amount of fresh squid added represents 15% dry weight
(DW).
Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
then uneaten remains were removed and weighed; the weights
were multiplied by a factor correcting for articial water
intake. This factor was determined by weighing pieces of
each of the ve diets, then soaking them in water for 2 h and
then weighing again, to determine bleaching and soaking.
Cuttlesh were weighed individually every 7 days and data
were used to calculate: (1) mean instantaneous growth rate
(MIGR), (2) FR (% body weight day)1) = [FI/average
W(t)] 100, where FI is the food ingested and average W(t)
is the average wet weight of the cuttlesh during the time
period (t) and (3) food conversion (FC) = (W2 ) W1)/FI,
where W2 ) W1 is the weight gained by the cuttlesh during
the time period. The cumulative mortality was accounted in
all diets tested.
The experiment lasted for 4 weeks. At the end of that
period, nine animals from each of the natural diets, 10 ani-
mals fed the rst articial diet and 11 animals fed the second
articial diet were sacriced. Animals were placed over trit-
urated ice until cessation of motility. Afterwards, they were
decapitated. Samples of the mantle tissues and DG were
collected for posterior analysis. For every animal sacriced,
BW and DG weight were recorded in order to establish the
DG weight/BW relation.
Moisture, total lipids (TL), lipid classes and FA of TL from
diets, DG and mantle of cuttlesh were analysed. Moisture
content was determined from 500 mg samples using the
method of Horwitz (1980). TL was extracted with chloro-
form : methanol (2 : 1 v/v) containing 0.1 g kg)1 of butylated
hydroxytoluene (BHT) as antioxidant (Christie 1982). The
organic solvent was evaporated under a stream of nitrogen
and the lipid content determined gravimetrically. Lipid classes
were separated by one-dimensional double development high
performance thin layer chromatography (HPTLC) using me-
thyl acetate/isopropanol/chloroform/methanol/0.25% (v/v)
KCl (25 : 25 : 25 : 10 : 9 by vol.), as the polar solvent system
and hexane/diethyl ether/glacial acetic acid (80 : 20 : 2 by
vol.), as the neutral solvent system. Lipid classes were quan-
tied by charring with a copper acetate reagent followed by
calibrated scanning densitometry using a Shimadzu CS-
9001PC (Shimadzu Europe, Duisburg, Germany) dual wave-
length ying spot scanner (Olsen & Henderson 1989). TL
extracts were subjected to acid-catalysed transmethylation for
16 h at 50 C, using 1 ml of toluene and 2 ml of 1% sulphuric
acid (v/v) in methanol. The resultant fatty acid methyl esters
(FAMEs) were puried by thin layer chromatography (TLC),
and visualized under UV light with 2,7-dichlorouorescein in
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 2009 The Authors
 98% (v/v) methanol, containing 0.01% BHT (Christie 1982).
FAMEs were separated and quantied by using a Shimadzu
GC-14A gas chromatograph equipped with a ame ionization
detector (250 C) and a fused silica capillary column Supe-
lcowax 10 (30 m 0.32 mm I.D.) (Supelco BelLefonte,
PA, 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 nal
temperature of 215 C at 2.5 C min)1. Individual FAMEs
were identied by reference to authentic standards and to well
characterize sh oil (PUFA-3, Biosigma, s/c de Tenerife,
Spain).
Butylated hydroxytoluene, potassium chloride, potassium
bicarbonate, 27-dichlorouorescein were supplied by Sigma
Chemical Co (St Louis, MO, USA). TLC (20 cm 20 cm
0.25 mm) and HPTLC (10 cm 10 cm 0.15 mm) plates,
precoated with silica gel (without uorescent indicator) were
purchased from Macherey-Nagel (Duren, Germany). All
organic solvents for GC used were of reagent grade and were
sh fed the two articial diets and ANOVA test for the three
natural diets. The same statistical tests were performed to
compare GR and FR and food conversions between the three
natural diets (ANOVA), as well as the groups fed the two
articial diets (t-test).
When comparing natural and articial diets, dierences
found in lipid compositional variables are usually much more
moderate than those generally reported for growth variables.
For all these reasons, we have included natural and articial
diets into a single statistical comparison for this section of the
study. Lipid composition results are presented as mean
SD for triplicate experiments. The data were checked for
normal distribution with the one-sample Kolmogorov
Smirnov test as well as for homogeneity of the variances with
Levenes test and, when necessary, arcsin transformation
(Fowler et al. 2002) was applied to data on lipid proportional
composition; compositional data expressed as g kg)1 was
divided by 1000 before applying the arcsin transformation,
because it needs gures in the interval [0, 1]. A one-way
ANOVA (Zar 1999) and Duncans multiple range post hoc test,

purchased from Panreac (Barcelona, Spain).
At the present state of research, cephalopod articial diets
show much lower palatability or growth variables (ingestion,
conversion or GR) than comparable natural diets. In that
respect, we chose a separate statistical analysis for each kind
of diet, provided that, as expected, their performances were
clearly dierent.
After every weighing period, statistical analysis was per-
formed to determine dierences in weight between groups.
ANOVAs (Zar 1999) were performed between the three repli-
cates of each group, and if no signicant dierences were
found between the three replicates, all cuttlesh in those
groups fed the same diet were gathered, and a t-test was
performed to compare dierences in weight between cuttle-
comparing the eects of the ve dierent diets was per-
formed. When a normal distribution and/or homogeneity of
the variances were not achieved, data were subjected to a
non-parametric KruskallWallis test. In all statistical tests
used, P < 0.05 was considered statistically dierent. The
statistical analysis was performed using the SPSS package
from SPSS Inc (Chicago, IL, USA).
Average cuttlesh wet weight is showed in Table 2. When
analysing the three natural diets, signicant dierences
(P < 0.05) were found between all of them, in every weighing
period, even at the end of the rst week of the experiment.
The control diet (grass shrimp) promoted the highest growth,

Table 2 Average cuttlesh wet weight

Time
(g)
Diet Day 1 Day 8 Day 15 Day 22 Day 29

Average wet weight (g SD)

Shrimp 14.2 0.4 18.6 0.8 24.4 0.6 31.5 0.6 40.8 1.5
Sardine 13.7 1.0 14.8 1.1 15.8 1.0 15.6 1.0 17.8 1.5
Crayfish 14.2 0.7 15.8 0.7 18.3 1.0 22.6 2.5 27.7 2.9
Diet 1 13.9 0.1 14.1 0.4 12.5 0.5 8.4 1.2 13.3 3.1
Diet 2 13.2 0.4 13.1 0.1 12.1 1.1 10.3 1.6 13.6 0.4

The animals were weighed individually on a weekly basis. Triplicates from each diet group were
considered as one, if no significant differences were found initially among the correspondent
wet weights.
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Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
followed by the craysh, with sardine promoting poor Palaemonetes sp.
S. pilchardus
growth. On the contrary, no signicant dierences 14.0 P. clarkii
Diet 1
(P > 0.05) were found in growth of cuttlesh fed the two 12.0
Diet 2
articial diets, which lost weight at the end of the experiment.
10.0
8.0
6.0
4.0
Growth rates of cuttlesh fed the ve experimental diets are
2.0
shown in Fig. 1. When comparing the natural diets, highest
0.0
overall GR was obtained with the shrimp used as control diet 8 15 22 29
(3.8 0.1% BW day)1), followed by the craysh Time (Day)

(2.3 0.6% BW day)1), with sardine promoting very low
GR (0.8 0.7% BW day)1). Signicant dierences
(P < 0.05) were found between the three treatments even
from the end of the rst week.
No signicant dierences (P > 0.05) were found in GR of
cuttlesh fed the two articial diets. Both of them promoted
negative growth ()1.8 2.8% BW day)1 for diet 1 and
)0.3 2.1% BW day)1 for diet 2). GR obtained with the
articial diets in the fourth week were not considered,
because of high cannibalism during this period.
The cumulative mortality, mainly because of cannibalism,
was measure each week by diet group. Data of rst week: 0%
in all groups; second week: 3% in all groups; third week: 3%
shrimp, 17% sardine, 7% craysh, 10% articial diet 1 and
7% articial diet 2. Finally, a relevant increase in mortality
was observed in both articial diet groups (47%) during the
fourth week, whereas natural diets maintained lower levels
(3% shrimp, 20% sardine and 13% craysh.
Feeding rates of cuttlesh fed the ve experimental diets are
shown in Fig. 2. When comparing the natural diets, highest
Figure 2 Mean feeding rates (% body weight day)1) of juveniles of
S. ofcinalis cultured for a four week period, under ve dierent
diets. Growth rates were determined every 7 days.
overall FR was obtained with the craysh (10.5 1.6% BW
day)1), followed by the control diet, the grass shrimp
(8.8 0.7% BW day)1), with sardine promoting lower FR
(6.6 2.1% BW day)1). Signicant dierences (P < 0.05)
were only found between cuttlesh being fed sardines and the
craysh.
No signicant dierences (P > 0.05) were found in FR of
cuttlesh fed the two articial diets, which were of
4.5 2.6% BW day)1 for diet 1 and 2.4 0.9% BW day)1
for diet 2.
Food conversions of cuttlesh fed the ve experimental diets
are shown in Fig. 3. When comparing the natural diets, the
highest overall food conversion was obtained with the con-
trol diet (42.6 2.4%), followed by the craysh
(20.9 3.5%), with sardine promoting very low food con-
version (13.6 12.6%). Grass shrimp promoted higher food
80

4.0 50

2.0 20

0.0 10

2.0 40 Palaemonetes sp.

Palaemonetes sp. S. pilchardus
S. pilchardus P. clarkii
4.0 70
P. clarkii
Diet 1
Diet 1
6.0 Diet 2
Diet 2 100

8.0 130
8 15 22 29
8 15 22 29
Time (Day) Time (Day)

Figure 1 Mean growth rates (% body weight day)1) of juveniles of S. Figure 3 Mean food conversions (%) of juveniles of S. ofcinalis
ofcinalis cultured for a four week period, under ve dierent diets. cultured for a four week period, under ve dierent diets. Growth
Growth rates were determined every 7 days. rates were determined every 7 days.
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Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
 conversion (P < 0.05) compared with the two alternative sardine, 5.1 1.1 for cuttlesh fed craysh, and signicantly
natural diets, whereas these two were not dierent lower (P < 0.05) for those fed the rst (2.5 0.4) and sec-
(P > 0.05). ond (3.6 0.8) articial diets. There was a positive corre-
No signicant dierences (P > 0.05) were found in food lation (r = 0.96) between DG and BW (Fig. 4).
conversion of cuttlesh fed the two articial diets, which were
negative. Food conversion obtained with the articial diets in
the fourth week was not considered, because of high canni-
balism during this period. The main compositional features of the ve diets are shown in
Table 3. Moisture (H) was similar among natural preys (722
852 g kg)1), but clearly lower in articial diets (400 g kg)1).
The TL content was about six times higher in sardine with
The ratio (%) between DG and BW was of 5.2 0.6 for respect to the rest of the diets. This strong dierence was due to
cuttlesh fed the control diet, 5.8 1.0 for cuttlesh fed the high level of triacylglycerol (TG; 290 g kg)1), which
implies lower percentages of polar lipids (PL), CHO and free
4.5
Correlation: r = 0.95743 fatty acids (FFA) in this diet. In addition, it was observed a
4.0
Digestive gland wet weight (g)

high content of FFA both in natural and articial diets.

3.5
In general terms, FA composition of articial diets
3.0 resemble the prole of the powder used as main component,
2.5 so diet 2 based on grass shrimp powder showed a similar
2.0 composition to shrimp diet, with lower levels of saturated
1.5 and monoenes and higher levels of polyunsaturated (PUFA),
1.0
especially n-3 series, respect to diet 1 (based on commercial
sh powder) and sardine diet. Craysh diet is characterized
0.5
by high variability and, similar to shrimp diet, showed high
0.0
10 20 30 40 50 60 70 80 percentages of PUFA but reduced the FA levels from n-3
Cuttlefish wet body weight (g)
series, especially n-3 highly unsaturated FA (n-3 HUFA) and
higher levels of FA from n-6 series (Table 4).
Figure 4 Correlation between the digestive gland weight (g) and
Among the individual FA, it is remarkable the higher
cuttlesh body weight (g). Results were obtained using 9 animals
from each of the natural diets, 10 animals fed the rst articial diet,
content of 20:1n-9 and 22:1n-9 in sardine diet and both
and 11 animals fed the second articial diet. articial diets despite of powder origin. The predominance of

Table 3 Composition of moisture (g kg)1), total lipids (g kg)1 DW) and lipid classes (g kg)1 DW) of natural and articial diets

Diet composition Shrimp Crayfish Sardine Diet 1 Diet 2

a a a b
Moisture 756.6 0.7 852.9 7.0 722.3 0.6 392.9 216.2 412.0 4.4b
Total lipid 53.8 12.0b 56.4 8.8b 410.3 63.3a 85.2 30.5b 83.4 4.5b
Sphingomyelin 0.1 0.0 0.6 0.1 4.5 4.2 1.4 1.1 0.8 0.1
Phosphatidylcholine 8.5 1.1b 7.3 2.1b 21.3 3.7a 10.2 4.1b 9.2 2.8b
Phosphatidylserine 0.6 0.1 0.9 0.2 3.7 1.6 0.7 0.4 1.0 0.5
Phosphatidylinositol 0.3 0.2* 1.3 0.3 2.2 2.3 1.6 0.3 0.2 0.1
Phosphatidylglycerol1 0.8 0.2b 0.3 0.1b 5.5 3.7a 0.6 0.4b 0.9 0.4b
Phosphatidylethanolamine 6.7 1.0 6.5 1.8 8.8 3.9 5.2 1.7 6.9 0.8
Cholesterol 14.0 2.0b 18.3 2.9ab 27.4 3.4a 22.8 10.2ab 23.8 2.2ab
Free fatty acids 16.3 2.6b 14.2 0.9b 43.5 16.0a 17.9 8.2b 29.4 2.2ab
Triacylglycerol 5.6 4.6b 4.4 3.3b 291.7 72.8a 20.1 6.5b 8.3 2.0b
Esterol ester 0.8 0.7 2.6 4.3 1.7 2.9 4.7 1.0 2.9 0.4
Polar lipids 17.1 2.2b 16.9 4.8b 46.0 18.5a 19.7 7.3b 19.0 2.8b
Neutral lipids 36.7 9.9b 39.4 5.4b 364.2 77.5a 65.6 23.9b 64.4 2.6b

Results represent mean SD (n = 3).

Values in the same row bearing different superscript letters are significantly different (P < 0.05).
1
Contain phosphatidylglycerol, phosphatidic acid and cardiolipin.
* KruskalWallis showed differences, but Duncans test could not locate them.
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Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
 Table 4 Fatty acid of total lipid (%
Diet
weight) of natural and articial diets
composition Shrimp Crayfish Sardine Diet 1 Diet 2
cd d a b
14:0 1.3 0.1 0.5 0.3 6.5 0.8 3.9 0.5 2.0 0.2c
16:0 15.4 1.0b 14.3 1.2b 22.3 1.6a 24.0 1.4a 16.5 0.4b
16:11 6.1 1.9 6.2 5.5 7.2 1.5 5.3 0.2 6.9 1.2
16:3n-3 2.1 0.9a 0.5 0.1b 0.3 0.1b 0.6 0.0b 0.7 0.4b
16:4 1.4 0.3a 1.4 0.1a 0.8 0.2b 0.3 0.0c 0.9 0.1b
18:0 5.6 0.4bc 6.2 0.7ab 3.7 0.5d 6.7 0.4a 4.8 0.4c
18:1n-9 8.2 2.1c 19.2 6.9a 10.0 1.2c 16.4 0.7ab 10.7 0.7bc
18:11 9.0 0.5a 4.1 0.3c 2.9 0.2d 4.3 0.2c 7.5 0.1b
18:2n-6 4.2 0.8b 8.3 1.8a 1.1 0.3c 2.4 0.2c 4.5 0.2b
18:3n-3 0.8 0.0b 3.0 1.7a 0.7 0.2b 0.5 0.0b 0.9 0.3b
18:4n-3 0.3 0.2bc 0.1 0.2c 1.7 0.4a 0.6 0.1b 0.4 0.0bc
20:1n-9 0.5 0.0d 0.8 0.0d 6.2 0.6a 4.9 0.3b 3.1 0.3c
20:2n-6 0.5 0.1b 1.1 0.4a 0.2 0.0b 0.4 0.0b 0.5 0.0b
20:4n-6 2.6 0.1b 6.3 1.9a 0.6 0.1c 1.2 0.2bc 2.0 0.0bc
20:4n-3 0.0 0.0c 0.1 0.2c 0.7 0.0a 0.3 0.0b 0.2 0.1bc
20:5n-3 22.0 2.7a 15.1 5.8b 9.7 1.8c 5.2 0.6c 16.1 0.6b
22:1n-9 0.0 0.0d 0.0 0.0d 7.3 0.6a 4.2 0.2b 2.2 0.4c
22:5n-6 0.5 0.1ab 0.8 0.4a 0.3 0.1b 0.6 0.2ab 0.5 0.1ab
22:5n-3 0.8 0.1bc 0.5 0.2c 1.3 0.1a 1.0 0.2b 0.7 0.1bc
22:6n-3 10.9 2.1a 4.9 2.7b 10.9 2.0a 11.2 1.9a 13.8 0.6a
UK 2.9 0.5 2.3 0.2 1.5 0.5 1.4 0.3 1.5 0.8
Totals
Saturates 26.6 1.3b 23.7 0.8b 34.1 1.9a 36.9 2.1a 25.6 1.2b
Monoenes 23.9 4.4b 30.7 12.3ab 34.7 1.0ab 36.6 1.6a 31.2 1.5ab
PUFA 46.7 3.9a 43.5 12.1a 29.8 2.4b 25.3 3.6b 41.8 1.0a
n-3 36.9 3.9a 25.0 10.7bc 25.6 2.1bc 20.0 2.9c 33.0 1.2ab
n-6 7.9 0.5b 16.5 1.9a 2.7 0.3d 4.6 0.6c 7.6 0.2b
n-9 8.6 2.1c 20.1 6.9ab 23.5 2.4a 25.4 1.1a 16.0 0.7b
n-3 HUFA 33.6 4.7a 20.6 8.7c 22.5 1.7bc 17.6 2.7c 30.8 1.2ab
n-3/n-6 4.7 0.7b 1.5 0.6c 9.6 0.4a 4.4 0.2b 4.4 0.2b
EPA/DHA 2.1 0.2b 3.4 0.9a 0.9 0.3c 0.5 0.0c 1.2 0.0c
AA/EPA 0.1 0.0c 0.4 0.1a 0.1 0.0d 0.2 0.0b 0.1 0.0c

Results represent mean SD (n = 3).

Totals include some minor components not shown.
Values in the same row bearing different superscript letters are significantly different (P < 0.05).
1
Contains n-7 and n-5 isomers.

n-6 FA in craysh diet was mainly because of 18:2n-6 and
20:4n-6 (8.3 and 6.3%, respectively). On the contrary, pre-
dominance of n-3 FA in shrimp diet was due to 20:5n-3
(Eicosapentaenoic acid EPA) levels, whereas 22:6n-3 (Doc-
osahexaenoic acid DHA) showed similar levels in all diets
except in craysh diet (Table 4).
The TL of DG was similar between shrimp and sardine diet
group, but showed lower levels in craysh and in both arti-
cial diets group. The lower TL was related to lower neutral
lipid (NL) contents, especially in articial diets group. In
general terms, total PL showed lower level in craysh diet
group, which was reected in several lipid classes as phos-
phatidylcholine (PC), phosphatidylserine and phosphatidyl-
ethanolamine (PE). It was noteworthy the higher contents of
..............................................................................................
CHO in articial diets groups (4050 g kg)1 in articial diets
versus 1530 g kg)1 in natural preys; Table 5).
The total content of FA in the DG of cuttlesh fed with
articial diets was lower respect to cuttlesh fed with natural
preys as a result of the lower contents of TL and higher
proportion of CHO in the DG of articial diets groups. This
tendency was clearly observed in monoenes contents. Satu-
rated and PUFA contents also resembled this tendency but
showed lower contents in craysh diet group respect to ini-
tial, shrimp and sardine diet groups. The inuence of diet FA
composition could be observed in the higher contents of
20:1n-9 in sardine diet group and higher contents of 18:2n-6
and 20:2n-6 in craysh group, resembling the higher per-
centages of these FA in both diets. On the contrary, n-3
HUFA, especially EPA and DHA showed higher contents in
shrimp and sardine diet groups with respect to craysh and
articial diet groups. Finally, it was noteworthy the higher

 2009 The Authors

Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
 Table 5 Composition of moisture (g kg)1), total lipids and lipid classes (g kg)1 DW) of cuttlesh digestive gland

Digestive gland
composition Initials Shrimp Crayfish Sardine Diet 1 Diet 2
bc d cd b a
Moisture 650.6 74.6 542.7 5.9 597.8 25.4 662.3 16.4 737.0 15.5 757.6 11.1a
Total lipid 232.1 22.5a 167.6 42.4b 116.1 21.6cd 152.8 2.16bc 105.1 11.3d 107.6 8.5d
Sphingomyelin 0.5 0.1 0.7 0.1 1.0 0.7 0.4 0.1 0.4 0.1 0.8 0.2
Phosphatidylcholine 19.5 6.1a 14.0 1.9ab 10.2 1.4b 16.8 2.9a 15.2 2.2ab 18.2 2.5a
Phosphatidylserine 2.0 0.0abc 1.5 0.2bc 1.3 0.2c 3.0 1.7ab 3.3 1.1a 2.0 0.2abc
Phosphatidylinositol 4.1 0.6a 3.3 0.7ab 2.5 0.3b 3.6 1.2ab 3.3 1.0ab 2.7 0.1ab
Phosphatidylglycerol1 1.7 0.3 1.0 0.3 1.5 0.0 1.6 1.1 1.1 0.6 1.7 0.5
Phosphatidylethanolamine 13.1 4.0a 9.6 1.3ab 7.4 1.2b 12.9 0.3a 12.9 3.5a 10.7 0.2ab
Cholesterol 44.6 8.9ab 30.1 4.6cd 22.3 4.5de 15.7 1.9e 47.6 1.6a 38.4 4.0bc
Free fatty acids 3.9 0.7a 1.7 0.4bc 1.8 0.6b 1.0 0.1c 0.0 0.0d 0.0 0.0d
Triacylglycerol 98.6 1.4a 69.4 26.3b 33.1 10.2c 71.6 18.9b 2.5 1.3d 4.3 1.5d
Esterol ester 44.1 3.5a 36.2 8.4ab 35.1 3.8ab 24.9 1.4cd 18.8 3.0d 28.9 7.3bc
Polar lipids 40.9 10.4a 30.2 3.6ab 23.8 2.9b 38.4 5.6a 36.2 6.4a 36.0 2.7a
Neutral lipids 191.2 12.1a 137.4 38.9b 92.2 18.9cd 113.1 18.9bc 68.9 5.1d 71.6 9.7d

Results represent mean SD (n = 3).

Values in the same row bearing different superscript letters are significantly different (P < 0.05).
1
Contain phosphatidylglycerol, phosphatidic acid and cardiolipin.

Table 6 Fatty acid of total lipid

DG
(lg mg)1 tissue DW) of cuttlesh
composition Initials Shrimp Crayfish Sardine Diet 1 Diet 2
digestive gland
b b c a c
14:0 1.9 0.1 1.4 0.5 0.1 0.2 2.8 0.8 0.3 0.1 0.3 0.1c
16:0 16.0 1.9a 10.6 3.4b 5.6 3.2c 13.3 3.5ab 3.5 0.5c 3.4 0.6c
16:11 12.9 0.7a 9.1 3.7b 3.1 2.1cd 4.1 1.1c 0.3 0.1d 0.5 0.1d
a
16:3n-3 1.4 0.2 18 0.6a 0.4 0.2 b
0.4 0.2b 0.0 0.0b 0.0 0.0b
16:4 1.3 0.4a 1.0 0.2ab 0.6 0.1b 0.8 0.1b 0.8 0.2b 0.7 0.3b
18:0 9.3 0.4a 5.4 1.6b 4.1 0.8 bc
5.5 0.8b 2.6 0.1cd 2.4 0.7d
18:1n-9 13.1 2.5a 8.5 3.4ab 10.2 5.1ab 5.6 2.3bc 0.7 0.4c 0.8 0.1c
18:11 12.0 1.9 a
8.2 3.3b 2.9 1.0cd 4.1 1.1c 0.4 0.1d 0.8 0.1d
18:2n-6 6.2 0.3a 3.4 1.3bc 5.5 3.0ab 1.6 0.8cd 0.1 0.1d 0.2 0.0d
a
18:3n-3 1.9 0.4 1.3 0.5a 1.8 0.4a 0.7 0.1b 0.0 0.0c 0.0 0.0c
20:0 0.9 0.1b 1.2 0.2a 0.3 0.1cd 0.4 0.1c 0.1 0.0d 0.1 0.0d
b
20:1n-9 0.8 0.2 0.6 0.1b 0.9 0.2 b
3.0 0.8a 1.1 0.4b 1.1 0.2b
20:2n-6 1.4 0.1a 0.9 0.4bc 1.3 0.2ab 0.7 0.2c 0.2 0.0d 0.2 0.0d
a
20:4n-6 5.1 0.3 3.5 1.0b 2.7 0.3b 1.8 0.1c 0.5 0.1d 0.5 0.1d
20:4n-3 0.3 0.2ab 0.3 0.2ab 0.1 0.1b 0.5 0.2a 0.0 0.0b 0.0 0.0b
a
20:5n-3 32.6 1.5 21.9 8.9b 10.8 2.3 cd
17.0 2.8bc 4.6 0.4d 5.0 1.3d
22:1n-9 0.0 0.0b 0.0 0.0b 0.0 0.0b 0.0 0.0b 0.1 0.1a 0.1 0.0a
a
22:5n-6 1.0 0.1 0.6 0.2b 0.3 0.1 c
0.5 0.1b 0.1 0.0c 0.1 0.0c
22:5n-3 1.4 0.0a 1.1 0.3a 0.5 0.1b 1.4 0.5a 0.4 0.0b 0.4 0.1b
a
22:6n-3 17.6 0.9 11.3 4.0b 2.3 1.0 c
20.6 5.8a 4.8 0.3c 4.7 1.2c
UK 2.7 0.4 2.6 0.9 1.5 0.4 1.3 0.5 0.5 0.2 0.4 0.1
Totals
Saturates 30.8 2.7a 20.8 6.0b 10.9 4.2c 22.9 5.1b 7.0 0.4c 6.7 1.6c
Monoenes 39.3 4.2a 26.5 10.6b 17.3 7.5b 17.2 5.3b 2.8 1.2c 3.4 0.4c
PUFA 71.2 2.9a 48.0 17.8b 26.8 6.1c 46.7 11.0b 11.7 0.8c 12.2 3.2c
n-3 55.9 2.1a 38.2 14.8b 16.3 3.9c 41.2 9.9b 10.0 0.5c 10.4 2.9c
n-6 13.8 0.8a 8.5 2.9b 9.9 3.5b 4.5 1.0c 0.9 0.2d 1.1 0.1d
n-9 14.0 2.7a 9.2 3.5a 11.1 5.3a 8.5 3.2a 1.9 0.8b 1.9 0.3b
n-3 HUFA 51.9 2.2a 34.6 13.2b 13.7 3.4c 39.5 9.3b 9.7 0.4c 10.1 2.5c
n-3/n-6 4.1 0.1b 4.5 0.4b 1.7 0.5b 9.1 0.8a 11.8 2.7a 9.8 2.7a
EPA/DHA 1.9 0.1b 1.9 0.3b 5.0 1.1a 0.9 0.1c 1.0 0.1c 1.1 0.1bc
AA/EPA 0.2 0.0bc 0.2 0.0b 0.3 0.0a 0.1 0.0d 0.1 0.0abc 0.1 0.0cd

Results represent mean SD (n = 3).

Totals include some minor components not shown.
Values in the same row bearing different superscript letters are significantly different (P < 0.05).
1
Contains n-7 and n-5 isomers.
..............................................................................................

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Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
Table 7 Composition of moisture (g kg)1), total lipids and lipid classes (g kg)1 DW) of cuttlesh mantle

Mantle composition Initials Shrimp Crayfish Sardine Diet 1 Diet 2

b d d c a
Moisture 799.5 11.0 749.8 3.0 760.1 3.2 780.0 4.4 839.3 11.6 830.9 15.1a
Total lipid 49.5 3.5abc 44.5 3.4bc 40.3 6.1c 37.1 14.4c 63.2 1.5a 55.8 9.4ab
Sphingomyelin 0.5 0.1b 0.5 0.0b 0.7 0.2b 0.4 0.2b 1.5 0.1a 1.3 0.3a
Phosphatidylcholine 12.3 0.9 11.5 0.4 10.5 1.9 8.6 3.6 10.1 1.2 9.1 1.9
Phosphatidylserine 2.8 0.5 2.3 0.2 2.5 0.5 2.6 1.3 3.2 0.1 3.1 0.4
Phosphatidylinositol 1.5 0.3b 2.3 0.7ab 1.8 0.8b 1.6 0.6b 3.0 0.5a 2.2 0.1ab
Phosphatidylglycerol1 1.5 0.2a 0.5 0.9ab 1.4 0.5a 0.3 0.5b 0.7 0.4ab 1.4 0.1a
Phosphatidylethanolamine 9.4 0.8 8.9 1.2 7.7 1.2 7.5 3.1 8.2 0.3 6.9 1.2
Cholesterol 17.5 1.3ab 16.1 2.4ab 13.6 1.8b 13.3 5.1b 21.7 2.3a 18.2 3.9ab
Free fatty acids 1.1 0.2 0.7 0.4 0.9 0.3 1.1 0.4 0.8 0.3 0.6 0.2
Triacylglycerol 1.3 0.4c 0.7 0.1c 0.3 0.0c 0.1 0.1c 8.0 2.6a 5.9 0.8b
Esterol ester 1.5 0.5b 1.0 0.1b 0.7 0.1b 1.2 0.5b 6.1 2.0a 7.1 0.9a
Polar lipids 28.0 2.5 26.1 1.4 24.6 4.6 20.9 8.3 26.6 0.8 24.0 3.8
Neutral lipids 21.5 1.0b 18.4 2.2b 15.7 1.6b 16.2 6.1b 36.5 2.3a 31.8 5.6a

Results represent mean SD (n = 3).

Values in the same row bearing different superscript letters are significantly different (P < 0.05).
1
Contain phosphatidylglycerol, phosphatidic acid and cardiolipin.

Table 8 Fatty acid of total lipid

Mantle
(lg mg)1 tissue DW) of cuttlesh mantle
composition Initials Shrimp Crayfish Sardine Diet 1 Diet 2
a a a ab bc
14:0 0.3 0.0 0.2 0.0 0.2 0.1 0.2 0.1 0.2 0.0 0.1 0.0c
16:0 3.4 0.3a 3.2 0.6a 2.3 0.6b 2.1 0.6b 2.4 0.4b 2.2 0.3b
16:11 0.2 0.0a 0.2 0.0ab 0.2 0.1bc 0.1 0.0d 0.1 0.0cd 0.1 0.0bcd
16:4 0.4 0.1ab 0.5 0.2a 0.3 0.1b 0.3 0.1b 0.5 0.1ab 0.4 0.1ab
18:0 1.1 0.2ab 1.2 0.3ab 0.8 0.2b 0.9 0.3b 1.4 0.1a 1.3 0.2a
18:1n-9 0.6 0.1a 0.5 0.0abc 0.5 0.1ab 0.4 0.1c 0.5 0.1bc 0.5 0.1abc
18:11 0.5 0.1a 0.6 0.1a 0.3 0.1b 0.3 0.1b 0.3 0.0b 0.3 0.1b
18:2n-6 0.2 0.0ab 0.1 0.0ab 0.2 0.0a 0.1 0.0b 0.1 0.0ab 0.1 0.0ab
20:0 0.1 0.0a 0.1 0.0a 0.1 0.0b 0.1 0.0b 0.1 0.0a 0.1 0.0a
20:1n-9 0.6 0.1b 0.5 0.0b 0.5 0.1b 0.5 0.1b 0.8 0.0a 0.8 0.1a
20:2n-6 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0
20:4n-6 0.3 0.0ab 0.5 0.2a 0.4 0.2a 0.2 0.0b 0.3 0.1ab 0.4 0.1ab
20:5n-3 3.6 0.4 3.3 0.5 3.0 0.9 2.5 0.8 3.0 0.2 3.1 0.5
22:1n-9 0.0 0.0c 0.0 0.0c 0.0 0.0c 0.0 0.0c 0.0 0.0a 0.0 0.0b
22:5n-6 0.1 0.0ab 0.1 0.0a 0.1 0.0a 0.0 0.0b 0.1 0.0ab 0.1 0.0b
22:5n-3 0.4 0.1 0.3 0.0 0.3 0.1 0.3 0.1 0.3 0.0 0.3 0.1
22:6n-3 4.6 0.2a 4.3 0.7a 2.6 0.6b 3.4 1.1ab 4.0 0.4a 4.1 0.6a
UK 0.3 0.0 0.3 0.1 0.2 0.0 0.2 0.0 0.4 0.1 0.2 0.1
Totals
Saturates 5.3 0.4a 5.1 1.0ab 3.7 1.0bc 3.4 1.0c 4.5 0.3abc 4.1 0.7abc
Monoenes 1.9 0.2a 1.8 0.1a 1.5 0.4ab 1.3 0.3b 1.8 0.1a 1.8 0.2a
PUFA 9.9 0.8 9.4 1.7 7.2 1.9 7.0 2.2 8.7 0.5 8.8 1.3
n-3 8.8 0.7a 8.1 1.3ab 6.1 1.6b 6.3 2.0ab 7.6 0.6ab 7.7 1.1ab
n-6 0.7 0.1ab 0.8 0.3a 0.8 0.3a 0.4 0.1b 0.6 0.1ab 0.6 0.1ab
n-9 1.2 0.1a 1.0 0.0ab 1.0 0.3ab 0.9 0.2c 1.3 0.0a 1.3 0.2a
n-3 HUFA 8.6 0.7a 7.9 1.2ab 6.0 1.5b 6.1 2.0ab 7.4 0.6ab 7.5 1.1ab
n-3/n-6 13.2 0.8ab 10.1 2.0bc 7.7 1.1c 15.2 1.9a 13.4 3.9ab 12.0 0.3ab
EPA/DHA 0.8 0.1b 0.8 0.1b 1.2 0.1a 0.8 0.1b 0.8 0.0b 0.8 0.0b
AA/EPA 0.1 0.0bc 0.2 0.0a 0.1 0.0ab 0.1 0.0c 0.1 0.0abc 0.1 0.0ab

Results represent mean SD (n = 3).

Totals include some minor components not shown.
Values in the same row bearing different superscript letters are significantly different (P < 0.05).
1
Contains n-7 and n-5 isomers.

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

 2009 The Authors

Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
 EPA/DHA ratio showed by the DG in craysh diets group,
as well as the higher monoenes/saturates and monoenes/n-3
HUFA ratios in the same diet (data not shown; Table 6).
The mantle of cuttlesh fed with articial diets (1 and 2)
showed, in general terms, a higher moisture and TL content
respect to cuttlesh fed with natural preys (Table 7). These
dierences were mainly because of TG and sterol esters (EE)
accumulation, whereas PL as PC and PE did not show any
dierences among the dietary treatments.
Fatty acid composition of cuttlesh mantle expressed in
lg mg)1 tissue (dry weight, DW) is showed in Table 8. In
general terms, mantle showed a conservative FA
composition, regardless of the dierent diets. The dierences
observed in total saturated and monoenes contents were not
important and more related to the levels of mantle TL than
to diet FA composition. PUFA total contents did not show
dierences among treatments. Mantle FA of the n-6 series
showed higher contents in crustacean groups with respect to
sardine ones; this reects the higher contents of this FA in
crustaceans used as diet. Within saturates, 16 : 0 showed the
higher content, mainly in initial and shrimp diet groups.
Contents of 20:1n-9 and 22:1n-9 were slightly higher in arti-
cial diets group with respect to natural diets. It is remark-
able that n-3 HUFA and especially EPA were maintained at
similar contents in spite of the dierences in the dietary
treatments.
One of the bottlenecks of large-scale cephalopod culture in
general, and cuttlesh in particular, is the dependence on
natural diets, especially high priced crustaceans such as several
species of shrimp. Lower production costs could be obtained
using less expensive natural diets, or articial diets, even with
lower growth or survival. The craysh (P. clarkii), an infesting
freshwater crustacean and one of the most adaptable species in
the world (Elmossalami & Emara 1999), and sardine (S. pil-
chardus), are two low market value species; they could there-
fore be good candidates for large-scale cuttlesh culture, if
they would promote acceptable growth and survival, com-
pared with more expensive usual diets such as shrimp.
Results obtained from the present research indicate that
sardine is not a good alternative prey for large-scale cuttlesh
culture. When feeding sardine, GR and food conversion were
considerably lower, compared with previous studies
(Domingues et al. 2002, 2003a,b, 2004). They were even
..............................................................................................
 2009 The Authors
Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
lower when compared with results obtained when feeding
with other sh species (Domingues et al. 2003a). A possible
explanation could be the high TG content (300 g kg)1)
showed by sardine lipid classes prole. First studies carried
out by Vonk (1962) and ODor et al. (1984) suggested that,
the lack of biliary salts in the digestive tract of cephalopods
would imply diculties to assimilate high contents of NL in
the diets. Garca & Gimenez (2002) proposed that dierences
in growth of Octopus vulgaris could be due to dierent NL
content of the diet.
A higher content of NL in sh, respect to shrimp, has been
reported in previous studies (Almansa et al. 2006), as well as
a similar content of TG (587 g kg)1) has been observed in
sardine lipids analysed by our group (D. Quintana et al.,
unpublished results). Sardine lipid content varies according
to a seasonal cycle, with an increase in NLs during hot
months (De Leonardis & Macciola 2004): this suggests that
sardine used in this experiment were captured with a fat-rich
reserve previous to winter months. So, the almost exclusive,
protein metabolism of cuttlesh with high requirement in
proteins and amino acids (Lee 1994; Lee et al. 1998), with
lipids having mainly a structural function, could explain, at
least partially, the lower food conversions and poor growth
obtained in sardine group.
Another aspect to be taken into account in the lipid class
composition of diets is the high levels of FFA. These levels
used to be less than 10% of TL. On the contrary, they are
ranging from 43.5 g kg)1 in sardine diet (10% of TL) to
29.4 g kg)1 in articial diet 2 (35% of TL; see Table 3). All
our diets were based on commercial frozen products (from
dierent rms), so our data could indicate that diets were not
correctly conserved and oxidation processes were activated
before the experiment. Another explanation could be related
with problems in analytical methodology or samples con-
servation; this second explanation is improbable, provided
that both processes would be identical in mantle and DG
analysis, but FFA levels were in the normal physiological
range. Therefore, oxidative problems detected in the used
diets should come from previous processes.
It is possible that these FFA levels could make the
experimental diets worse, reducing their nutritional value.
However, our growth and survival data with shrimp diet are
similar to previous studies using shrimp (Domingues et al.
2004). Besides, the levels of FFA in the diets were not cor-
related with harmful eects to cuttlesh (e.g. shrimp diet
showed one of the highest levels of FFA together with the
best GR and food conversion). From our point of view, the
eects of high levels of FFA in the diets are not relevant to
the results reported in this study, although it should be
considered in the selection of commercial diets in future
studies.
Other dierences are related to the FA prole of the sar-
dine diet, which showed a clearly dierent prole with respect
to the shrimp diet, with higher percentages of saturates and
monoenes (especially from n-9 series) and lower percentage
of PUFA, especially EPA in the sardine diet respect to the
shrimp diet. Similar data were reported in previous studies
for other cephalopod species, at dierent life stages (Sina-
noglou & Miniadis-Meimaroglou 1998; Navarro & Villanu-
eva 2000, 2003; Domingues et al. 2003b, 2004; Almansa et al.
2006).
Monoenes are a common energy substrate in the marine
species (Sargent et al. 1995), but the amino acid-based
metabolism of cephalopods (ODor 1984; Lee 1994) and the
low percentage of this FA group in mantle lipid composition,
ranging from 1.3 to 1.9 g kg)1 DW (around 10% of total
FAs: see Table 8), points out that monoenes were not rele-
vant as diet component in cuttlesh physiology.
There are other nutritional components as proteins, oli-
goelements or carbohydrates that have not been considered
in this study. The inuence of these components cannot be
discarded to explain the dierences among the experimental
diets. Copper content could be a good candidate in this sense,
as a haemocyanin component (a respiratory pigment in
cephalopods). Copper content is low in shes when com-
pared with crustaceans and potentially inuences cuttlesh
growth (Villanueva & Bustamante 2006).
Craysh seems to be an acceptable candidate to be used as
an alternative diet for the culture of S. ofcinalis. GR
obtained, although signicant lower than the ones for
cuttlesh fed, the control diet could be suitable enough
to replace grass shrimp. Although food conversions with
craysh were half compared when feeding grass shrimp, the
considerably lower market value of craysh compared with
commercial shrimp reduces production costs. Dierences
observed in GR of both diet groups were not clearly corre-
lated with lipid composition of the diets. Lipid class prole
did not show signicant dierences between craysh and
shrimp. In addition, both diets showed similar contents of
saturate, monoenes and PUFA. However, the prole of FAs
series displayed a clear dierence with higher levels of 18:2n-6
and 20:4n-6 and lower levels of n-3 FA series (especially EPA
and DHA) in craysh diet respect to shrimp diet, which is
clearly reected in n-3/n-6 ratio. It has to be taken into
account that 20:4n-6 (AA) is an important eicosanoid pre-
cursor in sh (Sargent et al. 1995) and competes with EPA
for the enzymes (cyclooxygenase and lipoxygenase), both
implied in eicosanoid production. Thus, variations in the
Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
concentrations of AA and EPA could have profound eects
for both spectrum and quantity of eicosanoid production
with subsequent alterations in several physiological func-
tions, such as growth and reproduction, among other
(Mustafa & Srivastava 1989). The ratio AA/EPA is clearly
higher in craysh respect to shrimp (0.4 versus 0.1).
Both articial diets tested in the present experiment pre-
sented lower FR, negative food conversion and consequently
negative GR, besides taking into account the reserve accu-
mulation role of DG (Semmens 1998) a reduction in the ratio
between DG and BW suggested lower reserves content in the
specimens fed with both articial diets.
The proximate compositions of articial diets were char-
acterized by lower moisture content (400 g kg)1) compared
with natural diet (720850 g kg)1). This could be one of the
factors related with the bad acceptation of articial diets,
despite the inclusion of fresh calamar (150 g kg)1 of DW) in
the diet formulation to improve diet acceptability. The lipid
composition of these diets showed similar contents of PE
compared with natural diets. The presence of PE in the diet
could be a positive factor given the importance of this lipid
class in the mantle percentage composition (200 g kg)1),
besides, this lipid class used to content high proportions of
n-3 HUFA, especially DHA, which are one of the main
components in cuttlesh mantle composition (Almansa et al.
2006).
When we compared lipid and FA composition between
both articial diets, sh powder-based diet showed higher
levels of TG, saturates and monoenes and lower contents of
PUFA respect to shrimp powder-based diet. These dier-
ences are in accordance with the lipid prole for shrimp or
sh described in this and previous studies (Domingues et al.
2003a). However, these dierences had no signicant inu-
ence in the food conversion and GR, despite sh powder-
based diet display a theoretical more inadequate prole than
shrimp powder-based diet. Therefore, our data suggested
that dierences in lipid composition in articial diets were
not relevant for the bad results showed by these diets, so
other parameters such as organoleptic characteristics, which
could inuence diet acceptability, should be studied.
The DG of S. ofcinalis has been shown to play an out-
standing role in digestive processes, being the main organ for
enzyme synthesis, digestive absorption and fat depot (Bidder
1966; Boucaud-Camou et al. 1985), which imply an active
metabolism. This organ reects the nutritional condition of
cuttlesh better than other tissues as mantle. Therefore,
Domingues (1999) described a higher level of amino acid
content in DG from cuttlesh fed with shrimp respect to
cuttlesh fed with articial diets or after a fast period. In
..............................................................................................
 2009 The Authors
 similar ways, ODor et al. (1984) described a drop in the lipid
content of DG from 0.3 to 0.1% of BW after a 6-day fasting.
Furthermore studies related to lipid composition of DG
should be carried out.
TL content of DG decreased in cuttlesh analysed at the
end of the experiment respect to initial cuttlesh. Moreno
et al. (1998) found a decrease in TL during maturation
process in squid (Illex argentinus). Among the experimental
cuttlesh, articial diets groups showed a lower TL com-
pared with natural diets groups, except for craysh group, in
which TL was similar to articial diets groups. It is note-
worthy that the decrease in the craysh group was related to
lower content in both PL and NL, whereas in the articial
diet groups were more related to NL, especially TG. These
data suggest that cuttlesh fed with articial diets used TG
depots during experimental periods but maintained PL and
CHO, showing a lower DG/BW ratio, FR and food con-
versions. These nding are in agreement with lipid depletion
in DG and GR described by ODor et al. (1984) after a
fasting period. The maintenance of PL and CHO could be
related with the structural role of these lipids and their
importance in the mantle lipid composition. Besides, Zandee
(1967) suggested the absence of CHO synthesis in cuttlesh,
which points out to a necessity to maintain this lipid class.
Decreases in TL depots in the craysh groups are more dif-
cult to explain. A general reduction in all lipid classes was
observed, but was not reected on growth and DG/BW ratio,
which was similar to those ones found in the shrimp groups.
Digestive gland of articial diet groups showed the lower
contents of saturates, monoenes and PUFA respect to nat-
ural diets groups. But, in the craysh group, the TL reduc-
tion was more related to saturates and n-3 HUFA than to
monoenes. Diet FA composition appeared to be related to
DG FA composition, at least in natural diets groups, with
higher contents of n-3 HUFA in shrimp and sardine groups
respect to craysh group. The percentage of n-6 FA (%TL)
was higher in craysh group compared with shrimp and
sardine groups (15.8 versus 8.0 and 4.8, respectively; data not
shown). This is in agreement with Fluckiger et al. (2008) who
found a clear inuence of diet FA composition in the DG
only 14 days after a diet change.
Therefore, DG lipid composition showed similar prole
between shrimp and sardine groups but did not in craysh
group. Taking into account, that GR were higher in craysh
group respect to sardine group, our data suggest that lipid
composition of DG was not related with GR observed in the
present study. In fact, n-3 HUFA contents of the DG do not
seem related with the correct development of juvenile cut-
tlesh.
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 2009 The Authors
Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
Mantle is the main edible part of cuttlesh, so changes in
the nutritional composition could be relevant considering its
human nutrition values. Therefore, changes in the diet could
inuence the biochemical composition of mantle. Regarding
lipid composition, in general terms, cephalopods mantle is
characterized by small TL content without signicant varia-
tion, despite the lipid composition of the diet, food ratios or
maturation cycle (Moreno et al. 1998; Sinanoglou & Miniadis-
Meimaroglou 1998; Moltschaniwskyj & Jackson 2000;
Almansa et al. 2006).
Data obtained with natural diets in the present experiment
resembled results cited by the authors mentioned above, in
spite of the dierences observed in GR among the diet groups.
When we fed articial diets, an increase in mantle TL content
was observed. This increase was due to a strong rise in TG and
EE levels of around 14 times for TG and seven times for EE
respect to natural diet groups. The reason of this dierence is
not clear but could be related with the negative GR, poor food
conversion and high mortality observed in both articial diet
groups. Accumulation of these NL could indicate a dysfunc-
tion in mantle metabolism caused by poor feeding.
The lack of data on cephalopods mantle lipid composition,
under dierent diets, made it dicult to compare these data.
A TG level of 10% of TL was described for wild specimens of
S. ofcinalis captured in Greece (Sinanoglou & Miniadis-
Meimaroglou 1998). This value was similar to TG (% TL,
not shown) described for articial diets groups, but in this
case EE levels were lower (0.4% of TL) and more similar to
natural diet groups. However, those studies did not compare
dierent diets and specimens were not maintained in cap-
tivity during a long period of time. Therefore, direct com-
parisons with the present study are not possible.
Fatty acid composition of cuttlesh mantle did not show
relevant dierences under dierent diets, according to pre-
vious studies carried out by our group (Almansa et al. 2006)
and other data already published on octopus (Navarro &
Villanueva 2000), cuttlesh (Sinanoglou & Miniadis-
Meimaroglou 1998) and squid (Moreno et al. 1998), dis-
playing higher PUFA contents, mainly n-3 HUFA. It is
notorious, that the dierences in NL prole in natural and
articial diets groups were not reected in FA prole. Fur-
ther studies would be necessary to explain the reason of these
dierences.
An overall view of lipid analysis suggests a stronger diet
inuence on DG with respect to mantle; this inuence was
more notable in FA composition than in lipid classes.
Finally, shrimp was the diet whose FA composition was
more similar to those described in GD and mantle. These
data reinforce the suitability of this diet for cuttlesh feeding.
 In conclusion, according to results obtained, craysh
(P. clarkii) could be used as an alternative prey for rearing
S. ofcinalis because of their low market value, in spite of the
lower GR and FC compared with shrimp. High lipid content
of sardine could be a relevant factor to explain the bad result
showed by this diet and an unbalance between n-3/n-6 FAs
could be related with the lower growth eects of craysh diet.
However, the inuence of other nutritional components
(proteins, oligoelements, carbohydrates, etc.) that have not
been considered in this study cannot be discarded. Finally,
articial diets showed the worst eects in growth and mor-
tality as well as the stronger inuence on DG and mantle
lipid composition of cuttlesh.
Ana Ferreira wishes to thank the IFAPA (CIFPA Agua del
Pino), from the Junta de Andaluca, Cartaya, Spain, who
provided the animals, diets and facilities for the present
research.
Almansa, E., Domingues, P., Sykes, A., Tejera, N., Lorenzo, A. &
Andrade, J.P. (2006) The eects of feeding with shrimp or sh fry
on growth and mantle lipid composition of juvenile and adult
cuttlesh (Sepia ofcinalis). Aquaculture, 256, 403413.
Bidder, A. (1966) Feeding and digestion in cephalopods. In: Physi-
ology of Mollusca (Wilbur, K.M. & Young, C.M. eds), Vol. 2, pp.
97124. Academic Press, New York/London.
Boletzky, S. & Hanlon, R.T. (1983) A review of the laboratory
maintenance, rearing and culture of cephalopod mollusks. Mem.
Nat. Mus. Vic., 44, 147187.
Boucaud-Camou, E., Yim, M. & Tresgot, A. (1985) Feeding and
digestion of young Sepia ofcinalis L. (Mollusca: Cephalopoda)
during post-hatching development. Vie Milieu, 35, 263266.
Castro, B. & Lee, P. (1994) The eects of semi-puried diets on
growth and condition of Sepia ofcinalis L. (Mollusca: Cephalo-
poda). Comp. Biochem. Physiol., 109A, 10071016.
Castro, B., DiMarco, P., DeRusha, R. & Lee, P. (1993) The eects of
surimi and pelleted diets on the laboratory survival, growth and
feeding rate of the cuttlesh Sepia ofcinalis L. J. Exp. Mar. Biol.
Ecol., 170, 241252.
Chen, X.Q. & Long, L.J. (1991) Research and production of live
feeds in China. In: Rotifer and Microalgae Culture Systems. Pro-
ceedings of a U.S. Asia Workshop, Honolulu, HI (Fulks, W. &
Main, K.L. eds), pp. 187202. Argent Laboratories, Redmond.
Christie, W.W. (1982) Lipids Analysis, 2nd edn. Pergamon Press,
Oxford.
Dabrowski, K., Dabrowska, H. & Grundniewski, C. (1978) A study
of the feeding of the common carp larvae with articial food.
Aquaculture, 13, 257264.
De Leonardis, A. & Macciola, V. (2004) A study on the lipid fraction of
Adriatic sardine lets (Sardina pilchardus). Nahrung, 48, 209212.
DeRusha, R., Forsythe, J., DiMarco, F. & Hanlon, R. (1989)
Alternative diets for maintaining and rearing cephalopods in
Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
captivity, American Association for Laboratory Animal Science.
Lab. Anim. Sci., 39, 306312.
DiMarco, F.P., Turk, P.E., Scimeca, J.M., Browning, W.J. & Lee,
P.G. (1993) Laboratory survival, growth and digestive gland his-
tology of squids reared on living and non-living sh diets. J. Lab.
Anim. Sci., 43, 226231.
Domingues, P. (1999) Development of Alternative Diets for the Mass
Culture of the European Cuttlesh Sepia ocinalis, p. 95. PhD
Thesis, University of the Algarve, Algarve, 195.
Domingues, P., Kingston, T., Sykes, A. & Andrade, J. (2001a)
Growth of young cuttlesh, Sepia ofcinalis (Linnaeus, 1758) at
the upper end of the biological distribution temperature range.
Aquac. Res., 32, 923930.
Domingues, P., Sykes, A. & Andrade, J. (2001b) The use of Artemia
or mysids as food for hatchlings of the cuttlesh Sepia ofcinalis
Linnaeus, 1758; eects on growth and survival throughout the life
cycle. Aquac. Int., 9, 319331.
Domingues, P., Sykes, A. & Andrade, J. (2002) The eects of tem-
perature in the life cycle of two consecutive generations of the
cuttlesh Sepia ofcinalis (Linnaeus, 1758), cultured in the Algarve
(South Portugal). Aquac. Int., 10, 207220.
Domingues, P., Poirier, R., Dickel, L., Almansa, E., Sykes, A. &
Andrade, P. (2003a) Eects of culture density and live prey on
growth and survival of juvenile cuttlesh, Sepia ofcinalis. Aquac.
Int., 11, 225242.
Domingues, P., Sykes, A., Sommereld, A. & Andrade, P. (2003b)
The eects of feeding live or frozen prey on growth, survival and
the life cycle of the cuttlesh, Sepia ofcinalis (Linnaeus, 1758).
Aquac. Int., 11, 397410.
Domingues, P., Sykes, A., Sommereld, A., Almansa, E., Lorenzo,
A. & Andrade, J. (2004) Growth and survival of cuttlesh (Sepia
ofcinalis) of dierent ages fed crustaceans and sh. Eects of
frozen and live prey. Aquaculture, 229, 239254.
Domingues, P., DiMarco, F., Andrade, J. & Lee, P. (2005) The
eects of diets with amino acid supplementation on the survival,
growth and body composition of the cuttlesh Sepia ofcinalis.
Aquac. Int., 13, 423440.
Domingues, P.M., Marquez, L., Lopez, N. & Rosas, C. (2008)
Eects of food thermal treatment on growth, absorption, and
assimilation eciency of juvenile cuttlesh, Sepia ofcinalis.
Aquac. Int., DOI:10.1007/s10499-008-9200-z.
Elmossalami, M.K. & Emara, M.T. (1999) Safety and quality of
fresh water craysh Procambarus clarkii in the river Nile. Nahrung,
43, 126128.
Fluckiger, M., Jackson, G.D., Nichols, P., Virtue, P., Daw, A. &
Wotherspoon, S. (2008) An experimental study of the eect of diet
on the fatty acid proles of the European cuttlesh (Sepia ofci-
nalis). Mar. Biol., 154, 363372.
Forsythe, J., DeRusha, R. & Hanlon, R. (1994) Growth, reproduc-
tion and life span of Sepia ofcinalis (Cephalopoda: Mollusca)
cultured through seven consecutive generations. J. Zool. Lond.,
233, 175192.
Fowler, J., Cohen, L. & Jarvis, P. (2002) Practical Statistics for Field
Biology, 2nd edn, p. 259. John Wiley & Sons, Chichester, West
Sussex.
Garca, B.G. & Gimenez, F.A. (2002) Inuence of diet on ongrowing
and nutrient utilization in the common octopus (Octopus vulgaris).
Aquaculture, 211, 171182.
Hochachka, P.W., Moon, T.W., Mustafa, T. & Storey, K.B. (1975)
Metabolic sources of power for mantle muscle of a fast swimming
squid. Comp. Biochem. Physiol., 52B, 151158.
Horwitz, W. (1980) Methods of Analysis, 13th edn. Association of
Ocial Analytical Chemists, Washington.
..............................................................................................
 2009 The Authors
 Koueta, N. & Boucaud-Camou, E. (1999) Food intake and growth in
reared early juvenile cuttlesh Sepia ofcinalis L. (Mollusca:
Cephalopoda). J. Exp. Mar. Biol. Ecol., 240, 93109.
Koueta, N. & Boucaud-Camou, E. (2003) Combined eects of
photoperiod and feeding frequency on survival and growth of
juvenile cuttlesh Sepia ofcinalis L. in experimental rearing.
J. Exp. Mar. Biol. Ecol., 296, 215226.
Koueta, N., Boucaud-Camou, E. & Noel, B. (2002) Eects of
enriched natural diet on survival and growth of juvenile cuttlesh
Sepia ofcinalis L. Aquaculture, 203, 293310.
Lee, P. (1994) Nutrition of cephalopods: fuelling the system. Mar.
Freshw. Behav. Phy., 25, 3551.
Lee, P., Forsythe, J., DiMarco, F., DeRusha, R. & Hanlon, R.
(1991) Initial palatability and growth trials on pelleted diets for
cephalopods. Bull. Mar. Sci., 49, 362372.
Lee, P., Turk, P., Forsythe, J. & DiMarco, F. (1998) Cephalopod
culture: physiological, behavioural and environmental require-
ments. Suisanzoshoku, 46, 417422.
Lindberg, J.C. & Doroshov, S.I. (1986) Eects of diets switch
between natural and prepared foods on growth and survival of
white sturgeon juveniles. Trans. Am. Fish. Soc., 115, 166171.
Moltschaniwskyj, N.A. & Jackson, G.D. (2000) Growth and tissue
composition as a function of feeding history in juvenile cephalo-
pods. J. Exp. Mar. Biol. Ecol., 253, 229241.
Moltschaniwskyj, N.A. & Johnston, D. (2006) Evidence the lipid can
be digested by the dumpling squid Euprymna tasmanica, but is not
stored in the digestive gland. Mar. Biol., 149, 565572.
Moreno, J.E.A., Moreno, V.J., Ricci, L., Roldan, M. & Gerpe, M.
(1998) Variations in the biochemical composition of the squid Illex
argentinus from the South Atlantic Ocean. Comp. Biochem.
Physiol., 119B, 631637.
Mustafa, T. & Srivastava, K.C. (1989) Prostaglandins (eicosanoids)
and their role in ectothermic organisms. Adv. Comp. Environ.
Physiol., 5, 157207.
Navarro, J.C. & Villanueva, R. (2000) Lipid and fatty acid compo-
sition of early stages of cephalopods: an approach to their lipid
requirements. Aquaculture, 183, 161177.
Navarro, J.C. & Villanueva, R. (2003) The fatty acid composition
of Octopus vulgaris paralarvae reared with live and inert food:
..............................................................................................
 2009 The Authors
Journal compilation  2009 Blackwell Publishing Ltd Aquaculture Nutrition 16; 262275
deviation from their natural fatty acid prole. Aquaculture, 219,
613631.
ODor, R.K., Mangold, K., Boucher-Rodoni, R., Wells, M.J. &
Wells, J. (1984) Nutrient absorption, storage and remobilizaion in
Octopus vulgaris. Mar. Behav. Physiol., 11, 239258.
Olsen, R.E. & Henderson, R.J. (1989) The rapid analysis of
neutral and polar marine lipids using double development
HPTLC and scanning densitometry. J. Exp. Mar. Biol. Ecol.,
129, 189197.
Pascual, E. (1978) Crecimiento y alimentacion de tres generaciones
de Sepia ofcinalis en cultivo. Inv. Pesq., 42, 421442.
Richard, A. (1971) Contribuition aletude experimentale de la crois-
sance et de la maturation sexuelle de Sepia ocinalis L. (Mollus-
que, Cephalopode), p. 264. These de 3eme cycle, Universite de
Lille, Lille, 1264.
Sargent, J.R., Bell, M.V., Bell, J.G., Henderson, R.J. & Tocher, D.R.
(1995) Origins and functions of n-3 polyunsaturated fatty acids in
marine organisms. In: Phospholipids: Characterization, Metabolism
and Novel Biochemical Applications (Ceve, G. & Paltauf, F. eds),
pp. 248259. American Oil Chemical Society Press, Champaign.
Semmens, JM. (1998) An examination of the role of the digestive
gland of two loliginid squids, with respect to lipid: storage or
excretion? Proc. R. Soc. Lond. B Biol. Sci., 265, 16851690.
Sinanoglou, V.J. & Miniadis-Meimaroglou, S. (1998) Fatty acid of
neutral and polar lipids of (edible) Mediterranean cephalopods.
Food Res. Int., 31, 467473.
Toll, R.B. & Strain, C.H. (1988) Freshwater and terrestrial food
organisms as an alternative diet for laboratory culture of cepha-
lopods. Malacologia, 29, 195200.
Villanueva, R. & Bustamante, P. (2006) Composition in essential and
non-essential elements of early stages of cephalopods and dietary
eects on the elemental proles of Octopus vulgaris paralarvae.
Aquaculture, 261, 225240.
Vonk, H.J. (1962) Emulgators in the digestive uids of invertebrates.
Arch. Int. Physiol. Biochim., 70, 6785.
Zandee, D.J. (1967) Absence of cholesterol synthesis in Sepia ofci-
nalis L. Arch. Internat. Arch. Physiol. Biochem., 75, 487491.
Zar, J. (1999) Biostatistical Analysis, 4th edn (Ryu, T., ed.), chapter
11, p. 162184, Prentice-Hall Inc., Upper Saddle River.