The follow-ing parameters were estimated in transversal body sections at post-opercular and post-anal body levels: the total cross-sectional muscle area, the total num-ber of ¢bres and t
Trang 2Growth, feed utilization and liver histology of juvenile
with increasing protein levels
Pier Paolo Gatta1, Luca Parma1, Ilaria Guarniero2, Luciana Mandrioli2, Rubina Sirri2,
Ramon Fontanillas3& Alessio Bonaldo1
1 Dipartimento di Morfo¢siologiaVeterinaria e Produzioni Animali, Bologna, Italy
2
Dipartimento di SanitaØ PubblicaVeterinaria e Patologia Animale, Bologna, Italy
3 Skretting Aquaculture Research Centre, Stavanger, Norway
Correspondence: A Bonaldo, Dipartimento di Morfo¢siologia Veterinaria e Produzioni Animali,Via Tolara di Sopra 50, 40064 Bologna E-mail: alessio.bonaldo@unibo.it
Abstract
This study was undertaken to determine the
in£u-ence of dietary protein levels on growth, feed
utiliza-tion and liver histology in common sole Four
isoenergetic diets were formulated to contain four
di¡erent crude protein levels: 39 (P39), 45 (P45), 51
(P51) and 57 (P57) % dry weight Fifty animals
per tank (initial weight 10.2 0.4 g) were randomly
distributed into twelve 500 litre square tanks (bottom
surface: 5600 cm2) connected to a closed
recircula-tion system The diets were tested in triplicate for
84 days At the end of the experiment, the ¢nal
weight ranged from 19.6 (P39) to 25.4 g (P57) The
speci¢c growth rate showed statistical di¡erences
between groups, with the best results in the group
fed diet P57 (1.07% day 1) Signi¢cant di¡erences
between groups were also recorded for the feed
conversion ratio, with values of 1.31, 1.28, 1.12 and
0.94 in P39, P45, P51and P57 respectively Gross lipid
e⁄ciency was also signi¢cantly a¡ected by the
dietary treatment, with the highest value (42.07%)
found in P57 Ammonia excretion, expressed as
g100 g 1 protein intake, was signi¢cantly lower
for group P39 (2.46) than groups P51 (4.70) and
P57 (4.75) Increased incidence of lipid droplets
in hepatocytes was observed when the dietary
protein levels increased and/or dietary lipid
decreased
Keywords: common sole, growth, protein
utiliza-tion, lipid e⁄ciency, liver histology
IntroductionSalmonids, gilthead sea bream (Sparus aurata L.) andEuropean sea bass (Dicentrarchus labrax L.) play a leadrole in European ¢sh production; however, otherfarmed ¢sh species are required to di¡erentiate andwiden the market supply From emerging candidatespecies, Senegalese sole (Solea senegalensis, Kaup1858) and common sole (Solea solea L.) show promise,
as stated formerly by Howell (1997) and more cently by Imsland, Foss, Conceicao, Dinis DelbareSchram Kamstra Rema and White (2003) Hatcheryproduction of Solea spp has been accomplished quiteeasily, and this has been a common bottleneck in thecommercial production of other potential marine ¢shspecies However, it is during the juvenile stages thatseveral factors combine to reduce the growth perfor-mance and thereby reduce the potential for commer-cial farming activities These factors include feedingbehaviour, susceptibility to disease and stocking den-sity (Day, Howell & Jones 1997; Imsland et al 2003;Schram, Van der Heul, Kamstra & Verdegem 2006;Piccolo, Marono, Bovera, Tudisco Caricato & Nizza2008; SaŁnchez, Ambrosio & Flos 2010) Focusing onthe nutrition and feeding of common sole juvenilesand ongrowing, which seem to be particularly criti-cal points, some signi¢cant steps forward have re-cently been achieved for S senegalensis (Rema,Conceicao, Evers, Castro-Cunha Dinis & Dias 2008;Borges, Oliveira, Casal, Dias Conceicao & Valente2009; Rubio, Boluda Navarro, Madrid & SaŁnchez-VaŁz-quez 2009), establishing optimal protein and lipidAquaculture Research, 2011, 42, 313^321 doi:10.1111/j.1365-2109.2010.02622.x
re-r 2010 The Authors
Trang 3levels for growth Other recent ¢ndings on juveniles
of S senegalensis (Silva, Espe, Conceicao, Dias &
Va-lente 2009) and Solea aegyptiaca (Chabanaud 1927)
(Bonaldo, Roem, Pecchini, Grilli & Gatta 2006)
de-monstrated the ability of Solea spp to grow equally
well when fed diets containing vegetable proteins in
the partial substitution of ¢shmeal Less information
is currently available for common sole (S solea)
nutri-tion (Piccolo et al 2008) and it is inappropriate to rely
on data from Senegalese sole, given di¡erences in the
growth performance, optimal thermal regime,
broodstock behaviour and natural range (Imsland
et al 2003; Palazzi, Richard, Bozzato & Zanella
2006) Because dietary energy and protein are
recog-nized as key factors in£uencing both adequate ¢sh
nutrition and feeding costs (Watanabe 2002), and
considering the lack of speci¢c knowledge on S solea,
the ¢rst aim of this research was to assess growth
re-sponse and feed utilization on feeding common sole
juveniles isoenergetic diets with di¡erent protein
le-vels Furthermore, considering the importance of
the liver as an indicator of the nutritional and
physio-logical status of ¢sh (Bell, Tocher, MacDonald &
Sar-gent 1995; Robaina, Moyano, Izquierdo, Socorro,
Vergara & Montero 1997; Caballero, Lo¤pez-Calero,
So-corro, Roo, Izquierdo & Fe¤rnandez 1999), the second
purpose of this trial was to assess common sole liver
histology in response to the experimental diets
Materials and methods
Diets
Four isoenergetic diets (estimated gross energy:
23.5 MJ kg 1dry matter (DM)] were formulated to
contain di¡erent protein concentrations: 39 (P39),
45 (P45), 51 (P51) and 57 (P57) % DM The energy
le-vel was chosen based on those used in previous trials
on juvenile Senegalese sole (Dias, Rueda-Jasso,
Pan-serat, Conceicao, Gomes & Dinis 2004; Rema et al
2008) Protein sources of the diets were represented
by ¢shmeal and vegetable protein ingredients such as
soybean protein concentrate and wheat gluten meal
at an increasing ratio Their increase was in the same
proportion at each step in order to maintain the
ami-no acid ratio constant among the diets In the
ab-sence of speci¢c data on the vitamin, mineral and
trace mineral requirements of common sole,
require-ment data for other species were applied (National
Research Council 1993) The ingredients and the
proximate composition are given in Table 1 Pellets of
4 mm were produced using an experimental
extru-der by the Skretting Aquaculture Research Center,Stavanger, Norway Pellets were crumbled and sieved
to obtain suitable particle sizes
Fish, experimental set-up and samplingThe experiment was carried out at the Laboratory ofAquaculture, Faculty of Veterinary Medicine, Cesena-tico, Italy The common sole (S solea) juveniles with
an initial average weight 10.2 0.4 g were obtainedfrom the hatchery Solea B.V, IJmuiden, the Nether-lands Before the experiment, the ¢sh were acclima-tized for 4 weeks to the experimental facilities andfed commercial ¢shmeal-based diets (Skretting,Vero-
na, Italy; crude protein 55%, crude fat 18%) At thestart of the trial, 50 ¢sh were randomly distributedinto each of twelve 500 litre square tanks (bottomsurface: 5600 cm2) Each diet was fed to triplicatetanks for 84 days Tanks were provided with naturalseawater and connected to a unique closed recircula-tion system consisting of a mechanical sand ¢lter, anultraviolet light and a bio¢lter The water exchangerate per tank was 100% every 2 h The overall waterrenewal of the system was 5% daily Temperature wasmaintained constant at 20 1 1C throughout the ex-periment; the photoperiod was held constant at a12-h day length through arti¢cial light (200 lx atthe water surface ^ Delta Ohm luxmeter HD-9221,Delta-Ohm, Padua, Italy).Water temperature and dis-solved oxygen ( 6.5 mg L 1) were monitored daily
in each tank Ammonia (total ammonia nitrogen
0.1mg L 1), nitrite (NO2 0.2 mg L 1) and
ni-Table 1 Ingredients and proximate composition of the perimental diets
ex-Diet P39 P45 P51 P57
Dietary ingredients (%) Fishmeal LT 26.7 32.0 34.7 40.0 Soybean protein concentrate 10.7 12.8 13.9 16.0 Wheat 31.1 24.5 21.0 14.4 Fish oil 19.0 16.2 14.7 11.9 Wheat gluten meal 10.7 12.8 13.9 15.9 Vitamin and mineral premix 1.8 1.8 1.8 1.8 Proximate analysis
Dry matter (DM) (%) 91.8 93.2 92.7 91.4 Crude protein (% DM) 39.0 45.2 51.0 56.6 Crude fat (% DM) 23.3 22.8 20.4 18.1 Ash (% DM) 6.1 6.4 7.0 7.6 Gross energy (MJ kg 1 ) 23.5 23.5 23.8 23.0 Crude protein/gross energy (kg MJ 1 ) 16.6 19.2 21.4 24.6
Skretting standard vitamin and mineral premix.
Dietary protein levels and Solea solea performance P P Gatta et al Aquaculture Research, 2011, 42, 313^321
Trang 4trate (NO3 50 mg L 1) were determined
spectro-photometrically once weekly (Spectroquant Nova 60,
Merk, Lab business) at 12.00 p.m At the same time,
pH (7.8^8.2) and salinity (28^33 g L 1) were also
de-termined Fish were hand-fed twice daily (at 9.00 a.m
and 5.00 p.m.) at a ¢xed rate of 1.3% body
weight day 1, 7 days week 1 According to the
ap-petite of ¢sh registered in previous trials conducted
in our facilities (unpubl data), this ratio was
consid-ered to be close to satiation The feeding levels were
recalculated daily for each tank according to the feed
conversion ratio (FCR) and speci¢c growth rate (SGR)
obtained at each intermediate weighing Feed losses
were minimal throughout the trial but, when
neces-sary, the remaining feed was estimated and deducted
from the feed intake for the overall calculations At
the beginning and at the end of the experiment, all
the ¢sh of each tank were individually weighed and
the total length was recorded The total biomass was
also determined at day 28 and 56 by bulk weighing
Carcass proximate composition was determined at
the beginning and at the end of the trial In the
for-mer case, ¢ve pooled samples of ¢ve ¢sh each were
sampled to determine the initial proximate
composi-tion, while in the latter case, one pooled sample of ¢ve
¢sh from each tank was collected to determine the
¢-nal proximate composition Furthermore, at the end
of the trial, wet weight, viscera and liver weight were
individually recorded from ¢ve ¢sh per tank for the
determination of visceral and hepatosomatic indices
At this time, two ¢sh per tank were also randomly
sampled for liver histology
In order to evaluate nitrogen metabolism, after the
end of the growth trial, ammonia excretion was
mea-sured in all tanks during a 24-h cycle, integrating
re-peated collected values according to the following
formula given by Kaushik (1980):
Et¼ V0DCþ CtDW
where V0is the volume of water in the tank,DC the
di¡erence in total nitrogen ammonia (Ci Ci t), Ct
the mean of the total nitrogen ammonia
concentra-tion between two consecutive intervals (Ci1Ci t/2),
DW the £ow rate/unit of time ‘t’, t the unit of
incre-ment in time in which concentration variation is
con-sidered to be minimal and Et the total nitrogen
ammonia excreted by ¢sh per unit of time retained
The water in£ow was held constant at 250 L h 1
in each tank the day before and during ammonia
sampling Water of each tank was sampled from the
outlet at 0, 2, 4, 6, 8, 10, 12, 18 and 24 h after the ¢rst
meal A tank without ¢sh was used as a blank
Samples were immediately stored at 32 1C untilanalysis The ammonia concentration in the sampleswas measured using the indophenol method (Koro-le¡ 1983), and the overall data were expressed as g oftotal nitrogen ammonia per 100 g protein intake.All experimental procedures were evaluated andapproved by the Ethical-scienti¢c Committee for Ani-mal Experimentation of the University of Bologna, inaccordance with the European Community Councildirective (86/609/ECC)
Analyses of diets and body compositionThe experimental diets and carcasses were analysedfor DM (drying to a constant weight in a stove at
105 1C), crude protein (N 6.25, determined usingthe Kjeldahl method), fat (Folch, Lees & Sloane Stan-ley 1957) and ash content (incineration to a constantweight in a mu¥e oven at 450 1C)
CalculationsThe formulae used were calculated as follows:SGR (% day 1) 5 100 (ln FBW ln IBW)/days,where FBW and IBW represent the ¢nal and the initialweights (tank means) respectively FCR 5 feed given/weight gain Condition factor (CF) 5 100 (bodyweight/total length3) Viscerosomatic index (VSI)(%) 5 100 (viscera weight/body weight) Hepatoso-matic index (HSI) (%) 5 100 (liver weight/bodyweight) Protein e⁄ciency ratio (PER) 5 body weightgain/protein intake Gross protein e⁄ciency (GPE) (%)
5 100 [(% ¢nal body protein ¢nal body weight)
(% initial body protein initial body weight)]/totalprotein intake ¢sh Gross lipid e⁄ciency (GLE)(%) 5 100 [(% ¢nal body lipid ¢nal body weight)
(% initial body lipid initial body weight)]/totallipid intake ¢sh
Liver histology
At the end of the trial, two ¢sh per tank weresampled for liver histology Samples were ¢xed in10% bu¡ered formalin, dehydrated in a gradedethanol series and embedded in para⁄n Sectionseries of 4mm were stained with haematoxylinand eosin (H&E) From each ¢sh, a sample of liverwas snap-frozen in liquid nitrogen Quenching wasaccomplished by placing a small amount of embed-ding medium for frozen tissue specimens (OCT)Aquaculture Research, 2011, 42, 313^321 Dietary protein levels and Solea solea performance P P Gatta et al.
r 2010 The Authors
Trang 5(Tissue-Tek, Sakura ¢netek, Torrance, CA, USA) onto
a cork disc where the sample was positioned, and
then dropping it into a beaker containing isopentane
and liquid nitrogen for 1min and stored at 80 1C
Frozen sections of 3mm were cut in cryostate (Leica
Microsystems GmbH, Wetzlar, Germany) and stained
with Oil red O Histological samples were evaluated
objectively by two pathologists (R.S., L.M.) and
blindly with respect to the diet group; liver sections
were scanned at 40 lens with a light microscope
and 10 ¢elds were selected; according to the grade of
severity and the distribution of the fatty in¢ltration
within the cells (hepatic steatosis), and through the
histological section (from multifocal to di¡use
distri-bution), cases were classi¢ed as mildly a¡ected
(cyto-plasmic ¢lling of a clear, optically empty content
forming microvesicular spaces), moderately a¡ected
(cytoplasmic ¢lling of a clear, optically empty content
pulling the nucleus at the cell periphery and forming
macrovesicular spaces) or severely a¡ected
(cytoplas-mic ¢lling of a clear, optically empty content,
confer-ring an appearance of signet-confer-ring cells) If these
changes were not present, the livers were considered
to be normal
Statistical analyses
The performance response and the proximate
com-position of ¢sh fed the experimental diets were
ana-lysed using nested ANOVAand the Newman^Keuls
post hoc test The presence of morphological changes
in the distal intestinal structure of di¡erent groups of
¢sh at the end of the trials was compared using theKendallt rank correlation coe⁄cient All statisticalanalyses were performed usingSAS computer soft-ware (SAS 2004) Tank was used as the experimentalunit for analyzing the growth performance and am-monia excretion; a pool of ¢ve sampled ¢sh was con-sidered to be the experimental unit for analysingcarcass composition, whereas individual ¢sh wasused for analysing CF,VSI and HSI and morphologicalchanges Signi¢cant di¡erences were assumed when
P 0.05
ResultsAll ¢sh readily accepted the experimental diets andfeed intake was determined directly by the feeding re-gime The overall mean mortality in terms of thenumber of ¢sh waso2% and no signi¢cant di¡er-ences were recorded between groups (P 5 0.9707).Growth and feed utilization are shown in Table 2.The ¢nal weight, weight gain and SGR in ¢sh fed dietP57 were signi¢cantly higher than those found in theother groups Similarly, ¢sh fed diet P57 had signi¢-cantly lower FCR in comparison with the other ¢shgroups Fish fed diets P39 and P45 showed similarresults, while the group fed diet P51 had intermediatevalues, with statistical di¡erences for weight gain,SGR and FCR
Data on nutrient retention e⁄ciency and ammoniaexcretion are presented in Table 2 Protein e⁄ciency
Table 2 Growth, feed utilization and ammonia excretion of sole fed with experimental diets
Parameters
Diet
IBW 10.1 0.3 10.1 0.7 10.2 0.3 10.3 0.3 FBW 19.6 0.2 a 20.0 1.6 a 21.6 1.2 a 25.4 0.3 b
WG 9.6 0.4 a 9.9 0.9 a 11.4 0.9 b 15.1 0.3 c
SGR 0.80 0.04 a 0.81 0.02 a 0.89 0.04 b 1.07 0.03 c
FCR 1.31 0.02 a 1.28 0.01 a 1.12 0.07 b 0.94 0.02 c
PER 1.93 0.10 1.73 0.03 1.73 0.09 1.86 0.03 GPE 35.06 0.42 29.68 5.25 30.33 1.80 33.72 7.82 GLE 26.23 2.50 a 27.29 4.60 a 32.19 3.37 ab 42.07 5.25 b
Total ammonia nitrogen excretion 2.46 0.26 a
3.51 0.21 ab
4.70 0.89 b
4.75 0.23 b
Data (mean SD, n 5 3) in the same row with di¡erent superscript letters are signi¢cantly di¡erent (P 0.05).
IBW, initial body weight (g); FBW, ¢nal body weight (g); WG, weight gain (g) 5 FBW IBW; SGR, speci¢c growth rate (% day 1
)
5 100 (ln FBW ln IBW) days 1 ; FCR, feed conversion rate 5 feed given/WG; PER, protein e⁄ciency ratio 5 body weight tein intake; GPE, gross protein e⁄ciency (%) 5 100 [(% ¢nal body protein ¢nal body weight) (% initial body protein initial body weight)]/total protein intake ¢sh; GLE, gross lipid e⁄ciency (%) 5 100 [(% ¢nal body lipid ¢nal body weight) (% initial body lipid initial body weight)/total lipid intake ¢sh)]; total ammonia nitrogen excretion (g100 g 1 protein intake) 5 100 [(total ammonia nitrogen (g)/protein intake (100 g)].
gain/pro-Dietary protein levels and Solea solea performance P P Gatta et al Aquaculture Research, 2011, 42, 313^321
Trang 6ratio and GPE were not statistically di¡erent among
groups Gross lipid e⁄ciency was higher in ¢sh fed
diet P57 and signi¢cantly di¡erent from ¢sh fed diets
P39 and P45 Ammonia excretion was not
statisti-cally di¡erent between groups fed diets P45, P51 and
P57 and signi¢cant di¡erences were only recorded
between group P39 and both P51 and P57 Carcass
proximate composition and biometric parameters
were very similar between groups, with no
signi¢-cant di¡erences (Table 3) According to the histology
observation, the livers were pink or white, and the
rich vascular network, which constitutes a
physiolo-gical feature of these ¢sh, was evident Twelve cases,
which did not show intracytoplasmic lipidic droplets
in the hepatocytes, both on H&E and Oil red O
stain-ing, were classi¢ed as normal (Fig.1a^d) In six cases,
there was a cytoplasmic ¢lling of a clear, optically
empty content forming microvesicular spaces (mild
hepatic fatty in¢ltration); in these cases, Oil red O
stain demonstrated small, intracellular orange
dro-plets (Fig.1e and f) In the other six cases, this
materi-al, markedly pulling the nucleus at the cell periphery,
showed a macrovesicular appearance to the
hepato-cytes, and showed a multifocal to di¡use distribution
through the histological section (moderate,
multifo-cal to di¡use hepatic fatty in¢ltration) (Fig 1g); Oil
red O stain showed larger orange droplets
Occasion-ally, very large extracellular orange droplets were
found, due to the rupture of cytoplasmic membranes
during freezing and condensation of the lipidic
con-tent (Fig 1h) In all cases, a mild, di¡use cytoplasmic
swelling was detected (hepatic hydropic
degenera-tion) All these ¢ndings were statistically more
fre-quent in ¢sh fed diets P51 and P57 than in those fed
diets P39 and P45
DiscussionWhile the growth of common sole has been studiedfor many years (Howell 1997) and many attemptshave been made to overcome low growth rates, poorresults have been achieved so far Sole have two unu-sual features that may limit growth potential Theyhave a peculiar gut morphology, which presents a re-latively small stomach without pyloric caecae, and along intestine; furthermore, feeding behaviour in thenatural environment occurs almost entirely bymeans of chemoreception and with a frequent inges-tion of small prey items (de Groot1971) In juveniles ofSenegalese sole fed isoenergetic diets with ¢ve di¡er-ent protein levels (CP 43; 48; 53; 57; and 60), Rema
et al (2008) found SGR ranging from 0.93 to1.22% day 1with statistical di¡erences related todietary protein levels In particular, at least 53% CPwas necessary to obtain the maximum growth rate
In the present trial, SGR ranged from 0.80 to1.07% day 1 Speci¢c growth rate was signi¢cantlya¡ected by the dietary protein level, with the highestvalues recorded for ¢sh fed diet D57 These ¢ndings,although the experiment was shorter, are compar-able to the growth rate described by Howell (1997) incommon sole, indicating that diet P57 is able to sus-tain good performance of S solea juveniles
Concerning feed utilization, the present study monstrated the in£uence of dietary protein levels onFCR and GLE but not on PER and GPE A comparisonwith other data on common sole is quite di⁄cult be-cause of the paucity of sources Piccolo et al (2008)evaluated the performance of 30 g common sole feddiets containing 50% (diet A) and 54% (diet B) crudeprotein for 300 days The FCR were 2.65 and 2.49 for
de-Table 3 Whole body proximate composition and biometric parameters of sole fed with the experimental diets
Proximate composition
Moisture 75.35 0.38 75.20 0.58 75.56 0.19 75.23 0.88 Protein 16.56 0.35 16.05 1.58 16.39 0.78 16.79 2.40 Lipid 6.93 0.38 6.85 0.61 6.67 0.35 6.61 0.49 Ash 2.83 0.07 2.48 0.20 2.17 0.07 2.39 0.45 Biometric parameters
CF 1.04 0.12 1.05 0.13 1.04 0.13 1.07 0.12 VSI 4.88 0.48 5.04 0.59 5.31 0.95 5.42 0.77 HSI 1.07 0.22 1.06 0.18 1.11 0.26 1.19 0.17 Data are shown as mean SD.
Moisture, protein, lipid and ash (% wet weight), n 51 pool of ¢ve ¢sh per tank; CF, condition factor 5100 (body weight/total length 3
Trang 7diets A and B, respectively, with a statistical
di¡er-ence between treatments The authors reported the
presence of uneaten feed throughout the
experimen-tal period and hence those values could not represent
the real FCR Rema et al (2008) carried out a trial on
Senegalese sole juveniles to de¢ne the optimal
pro-tein levels with experimental diets containing a
crude protein percentage of 43, 48, 53, 57 and 60
They found feed e⁄ciency very close to one for the
last three groups In the present trial, FCR decreased
progressively from 1.31 in ¢sh fed the lowest proteinlevel to 0.94 in ¢sh fed the highest protein level Inthe current trial, the highest protein level (diet P57)resulted in the lowest FCR This was lower than thatreported previously for Senegalese sole fed optimaldietary protein levels (Rema et al 2008) No data areavailable on protein utilization for common sole andhence a comparison can only be made with other ¢shspecies The present study showed no in£uence ofdietary protein level on PER and GPE and this is inaccordance with previous ¢ndings obtained by Rema
et al (2008) for juvenile of Senegalese sole fed dietscontaining di¡erent protein levels ranging from 43%
to 60%
Cadena-Roa (1983) studied the protein ments of common sole using semi-puri¢ed dietsbased on casein and gelatin, with protein levels ran-ging from 24% to 77%, and the best performance wasobtained at 57^58% dietary protein This ¢ndingsupports the results of the present trial for ¢sh feddiet P57, where SGR and FCR were not only the bestamong the other groups but also very similar to whatwas found for Senegalese sole The data shown in thepresent study cannot clarify whether a higherdietary protein level could improve common solegrowth performance; however, it seems reasonable toconsider 57% crude protein to be a reference pointquite close to the requirements for juvenile commonsole In addition to growth results, GLE increased sig-ni¢cantly with increasing dietary protein levels(Table 2) Because higher dietary protein levels werebalanced by a proportional lipid and carbohydratereduction (Table 1) and assuming that higher proteinlevels led to good nitrogen utilization in this experi-ment, it is probable that lipid retention is mainlyrelated to the dietary lipid and carbohydrate levels,with a negligible in£uence of dietary protein levels.This hypothesis is in accordance with the resultsfound by Rema et al (2008), where a lower dietarylipid content (between 10% and 13%) resulted inhigher lipid retention These data may suggest that alower dietary lipid content than those used in thepresent trial is capable of ful¢lling common sole lipidrequirements, irrespective of the dietary proteinlevels, as already stated for Senegalese sole (Rema
require-et al 2008; Borges require-et al 2009) Whole-body tion (Table 3) was in accordance with the valuesfound for Senegalese sole (Dias et al 2004; Rema
composi-et al 2008) Surprisingly, the proximate carcass position was not a¡ected by the dietary treatments incontrast to Rema et al (2008), who found that in-creased dietary protein levels tended to decrease
com-Figure 1 (a^d) Hepatic parenchyma from sole fed diet
P39 (a and b) and diet P45 (c and d) was considered to be
‘Normal’ Absence of intracytoplasmic lipidic droplets in
the hepatocytes, both on H&E (a, c) and Oil red O stainings
(b, d), 40 lens (e and f) Hepatic parenchyma from sole
fed diet P51 was considered to have mild hepatic fatty
in-¢ltration Hepatocytes contain a clear, optically empty
content forming microvesicular intracytoplasmic spaces
(e) Oil red O stain demonstrated small, single,
intracellu-lar orange droplets (f), 40 lens (g and h) Hepatic
par-enchyma from sole fed diet P57 was considered to have
moderate hepatic fatty in¢ltration Hepatocytes are ¢lled
with clear, optically empty content that pulls the nucleus
at the cell periphery and gives a macrovesicular
appear-ance (g) Oil red O stain detected large orange droplets (h),
40 lens
Dietary protein levels and Solea solea performance P P Gatta et al Aquaculture Research, 2011, 42, 313^321
Trang 8whole-body fat deposition The carcass proximate
composition in the present study is similar to
pre-viously reported data for other £at¢sh species, such
as Atlantic halibut (Aksnes, Hjertnes & Opstvedt
1996) Hepatosomatic index observed in this study,
ranging from 1.06 to 1.19, are within the values found
in a trial conducted by Dias et al (2004) with juvenile
Senegalese sole, whereas VSI were slightly higher in
the present study
Liver histology showed a higher density of
intracy-toplasmic lipidic droplets (steatosis) in ¢sh fed diets
P51 and P57 (Table 4) Steatosis can be associated
with an excess of lipids in the diet, resulting in fat
accumulation in the liver (Rueda-Jasso,Conceicao,
Dias, De Coen, Gomes, Rees, Soares, Dinis &
Sorge-loos 2004; Myers & Mc Gavin 2007), or to a
de¢-ciency in lipotropic factors that are able to prevent
or remove an excessive accumulation of fat in the
li-ver (Mato, Mart|¤nez-Chantar & Lu 2008) In this
case, the inverse correlation between dietary lipid
le-vels and the presence of lipid droplets in hepatocytes
led us to lean towards a third mechanism The liver is
the major site of de novo fatty acid synthesis in ¢sh
(Lin, Romsos,Tack & Leveille 1977; Henderson &
Sar-gent, 1981) as in mammals (Hillgartner, Salati &
Goodridge 1995), and it is proposed that the elevated
incidence of intracytoplasmic lipidic droplets for the
high-protein/low-lipid diet is due to increased
hepa-tic lipogenesis This is supported by the elevated
ac-tivity of lipogenic enzymes, i.e FAS, G6PDH, ME and
12 ACC, which have been described previously for a
variety of ¢sh species fed similar diets, including
channel cat¢sh (Likimani & Wilson 1982), carp
(Shi-meno, Kheyyali & Shikata 1995), European sea bass
(Dias, Alvarez, Diez, Arzel, Corraze, Bautista &
Kaushik 1998), Atlantic salmon (Arnesen, Krogdahl
& Kristiansen 1993) or rainbow trout (Kolditz,
Borthaire, Richard, Corraze, Panserat, Vachot,
Le-fevre & Medale 2008)
Conclusion
In conclusion, the dietary protein levels used in this
trial demonstrated a considerable in£uence on
growth, feed utilization and nitrogen excretion in
common sole juveniles, with the highest SGR and
the lowest FCR achieved with a diet containing 57%
crude protein (1.07% day 1and 0.94 respectively)
Lipid retention increased when the dietary protein
increased and dietary lipids decreased Further
research is needed to clarify both the protein and
protein/energy ratio requirements of common solejuveniles and the protein level in£uences on GLE.Regarding liver histology, the presence of lipiddroplets in hepatocytes suggested a lipogenesis en-hancement when the dietary protein levels increaseand/or the dietary lipids decrease Finally, the overallperformances of common sole juvenile, especiallythose related to the growth and the FCR of groupP57, allow us to con¢rm the good potential of this ¢shspecies for intensive aquaculture
AcknowledgmentsThis research was supported by grants from the Ita-lian Region Emilia-Romagna.We thank Lorenzo Mar-iani, Marina Silvi and Sara Giuliani for technicalassistance, Elettra Pignotti for statistical analysesand Leo Nankervis for English editing The dietswere kindly provided by Skretting ARC, Stavanger,Norway
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r 2010 The Authors
Trang 11Stress responses of carp pond fish stock upon hunting activities of the great cormorant
Jiri Kortan1, Jana Blahova2, Kamila Kruzikova2& Zdenek Adamek1
1 Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceske Budejovice,Vodn›any, Czech Republic
2 Department of Veterinary Public Health and Toxicology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
Correspondence: J Kortan, Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protection of Waters, University
of South Bohemia in Ceske Budejovice, ZaŁtis›|¤ 738/II, 389 25 Vodn›any, Czech Republic E-mail: jiri.kortan@gmail.com
Abstract
In addition to direct predation, cormorants can a¡ect
carp pond ¢sh stock through disturbance and
wounding of the ¢sh Fish stress response was
evalu-ated by catch per unit e¡ort (CPUE) using lift-net
sam-pling A signi¢cantly higher (Po0.001) CPUE was
recorded in the littoral region of ponds a¡ected by
cor-morant visits (43.6 39.6) in comparison with a
con-trol pond (CP) without their occurrence (0.9 1.1)
Stress indices, spleen somatic index (SSI) and Fulton’s
condition coe⁄cient (FCC) were evaluated for ¢sh
subject to two stress factors, hunting cormorants
(HC) and pond harvesting (PH), and compared with
levels in ¢sh from the CP Both SSI and FCC decreased
signi¢cantly (Po0.05) in PH and HC ¢sh in
compari-son with the control group, while non-signi¢cant
dif-ferences were observed in both SSI and FCC indices
among the HC and PH groups Plasma biochemical
in-dices (cortisol, glucose and lactate concentrations)
were signi¢cantly higher (Po0.01) in the PH group
as compared with the HC and CP groups
Concentra-tions in the HC and CP groups did not di¡er
signi¢-cantly Total plasma protein levels were signi¢cantly
higher (Po0.01) in HC ¢sh compared with CP ¢sh,
but did not di¡er signi¢cantly from PH ¢sh
Keywords: cormorant, stress, cortisol, carp pond
harvesting
Introduction
The great cormorant (Phalacrocorax carbo L.) and
Eurasian otter (Lutra lutra L.) are the two predators
most commonly blamed for serious damages to ¢shstocks in ponds in Central Europe (Kranz 2000) Inaddition to direct consumption of ¢sh, cormorantsmay also cause indirect losses during their feedingactivities through wounding of ¢sh and stress (Kor-tan, AdaŁmek, Flajs›hans & Piac›kovaŁ 2008)
Fishing cormorants hunt in £ocks or as duals In the case of collective hunting near theshore, ¢sh are crowded along the shoreline where,due to high concentrations and panic behaviour, theybecome easy prey for cormorants and some otherpredatory birds [grey heron (Ardea cinerea L.) in parti-cular] This tactic is recorded frequently on eutrophicwater bodies such as ponds as it is more e¡ective un-der conditions of limited visibility (Veldkamp 1996).Systematic attacks on pond ¢sh stocks result in amore or less continual stress to ¢sh, which in turndecreases production (Berka 1989; AdaŁmek 1991).Stressed ¢sh, which hide in the littoral zone, may suf-fer for several weeks, with serious consequences forspring harvesting operations
indivi-Fish display a wide variation in their cal responses to stress Among these responses is achange in plasma corticosteroid levels (chie£y corti-sol in actinopterygian ¢sh) following a stressful event(Barton 2002).Various biochemical and haematologi-cal indices are used to indicate and assess the e¡ect ofstressors on ¢sh, e.g concentrations of glucose, corti-sol, lactate, ammonia and chlorides in blood plasma,the spleen somatic index (SSI), etc (Thomas 1990; Pa-l|¤kovaŁ & SvobodovaŁ 1995) Concentrations of cortisoland glucose are considered among the most impor-tant stress indicators in ¢sh Short-term intensiveAquaculture Research, 2011, 42, 322^330 doi:10.1111/j.1365-2109.2010.02624.x
Trang 12physiologi-stress (handling, netting, stocking) leads to
consider-ably elevated cortisol levels, followed by a gradual
de-cline to normal values that can take from hours to
weeks (Barton, Peter & Paulencu 1980; Pickering,
Pottinger & Christie 1982) Plasma glucose levels
be-come elevated in stressed ¢sh because of an increase
in blood catecholamine levels As plasma glucose
concentration is a function of several factors,
how-ever, such as diet, age or season, its value is more
equivocal than cortisol (Dobs›|¤kovaŁ, SvobodovaŁ,
Bla-hovaŁ, ModraŁ & Vel|¤s›ek 2006)
Changes in SSI have been used by a number of
authors to assess the intensity of stress in a variety
of ¢sh species (SvobodovaŁ, KalaŁb, Dus›ek, VykusovaŁ,
KolaŁr›ovaŁ & Janous›kovaŁ 1999; Mukhopadhyay 2003)
A decrease is recorded in SSI levels when ¢sh are
ex-posed to stressors such as pond harvesting (PH) due
to the washing out of blood elements into the
blood-stream (SvobodovaŁ, VykusovaŁ, ModraŁ, JarkovskyŁ &
SmutnaŁ 2006)
The aim of this study is to record the reactions of
¢sh stock associated with attacks by cormorants and
to evaluate the stress load in a¡ected ¢sh Any
mani-festation of stress in blood plasma and SSI of ¢sh
af-fected by cormorant presence will be compared with
samples of ¢sh subjected to the PH process Pond
har-vesting (as practised in Central Europe) consists of
slow pond draining, causing ¢sh to move slowly with
the descending water into the deepest part of the
pond known as the harvesting pit (as described, e.g.,
by HorvaŁth, TamaŁs & T˛lg 1984), from which ¢sh are
removed by netting (usually seining) This harvesting
procedure is stressful to ¢sh (SvobodovaŁ et al 2006)
For this reason, only ¢sh collected from the pond
with the minimum possible disturbance were
consid-ered as a control To date, no cogent data have been
published for stress caused by bird predation on ¢sh
stocks Such knowledge of the interactions between
protected bird predators and ¢sh is required in order
to understand and mitigate the cormorant/¢sheries
con£ict, which is becoming increasingly serious
throughout Europe
Material and methods
Sites
Two-year-old common carp (Cyprinus carpio L.),
raised in ¢shponds, were used to document
cormor-ant impact on behavioural and selected physiological
traits Monitoring of cormorant and ¢sh behaviour
was carried out in March 2008 on two ponds: Nade›je
(69 ha, mean depth 1.9 m) and LaŁska (17 ha, meandepth 2.1m) Both ponds are situated in the Tr›ebon›
¢shpond area (South Bohemia, Czech Republic) andare supplied from the same water in£ow
On the Nade›je pond, cormorant numbers were corded on a daily basis during both morning andafternoon Hunting cormorants (HC) were ¢rst re-corded on 28 February and the mean daily numbervisiting the pond during March was 50 birds or0.72 individuals ha 1 In the autumn and spring of2007/2008, the Nade›je pond was stocked with 2-year-old common carp with an average weight of
re-155 g (3704 individuals ha 1, 574 kg ha 1) and withsupplementary ¢sh species, including grass carp(Ctenopharyngodon idella Val.) and tench (Tinca tincaL.), at 788 individuals ha 1, 85 kg ha 1 Fishsamples were taken commencing on the third day (3March) of cormorant presence (HC group) and on the
¢rst day of harvesting at the same pond (PH group),which started 8 days later on 11 March Cormorantsleft the roost site 4 days after their arrival (on 4March) as ¢sheries sta¡ scared them o¡
The LaŁska pond, which served as the control pondsite (CP), was also stocked with 2-year-old carp, with
an average weight of 150 g (6478 individuals ha 1,
972 kg ha 1), and supplementary ¢sh species,such as grass carp, tench and zander (Sander lucioper-
ca L.) at 25 029 individuals ha 1 or around
40 kg ha 1 No cormorant occurrence was recorded
on this pond during March 2008
The HC and CP ¢sh samples were collected domly by a lift net from the shore zone at the pondfeeding-site, though no food was supplied Duringthe harvesting process, PH ¢sh samples were also ta-ken randomly using a dip net, directly from the har-vesting seine ¢shnet
ran-Basic water environmental parameters, such astemperature and dissolved oxygen, were regularlymeasured before sampling No £uctuations and/orunfavourable values were recorded During samplingevents, the mean values of temperature and dissolvedoxygen corresponded to 6.0, 6.0 and 5.8 1C, and 12.56,12.48 and 8.22 mg L 1on the CP, HC and PH sam-pling sites respectively
Catch per unit e¡ort (CPUE)The CPUE approach was used to assess the ¢sh den-sity resulting from ¢sh displacement close to theshore in response to cormorant feeding behaviour.Fish were caught randomly in the littoral zone by aAquaculture Research, 2011, 42, 322^330 Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al.
r 2010 The Authors
Trang 13lift net (1 1m, mesh size 10 10 mm), with 30
re-plicates on both ponds
Fulton’s condition coe⁄cient (FCC)
Fulton’s condition coe⁄cient was calculated for all
three groups (HC, PH and CP) as the ratio between
individual ¢sh weight (W in g) and total length (TL
in cm):
FCC¼ ðW TL3Þ 100
Blood sampling and SSI assessment
Twenty-two individuals were taken from all three
sample sites (HC, CP and PH) Blood samples were
im-mediately taken from all ¢sh caught by the lift net
and during PH An average of 0.7 mL of blood was
ta-ken from the caudal vein of each individual using a
heparinized syringe ¢tted with a 40 0.5 mm
sin-gle-use needle (SvobodovaŁ, Pravda & PalaŁc›kovaŁ
1986) Fish blood was centrifuged (10 min, 100 G)
im-mediately after sampling, and blood plasma was
transferred by a pipette (0.5 mL) into eppendorf test
tubes and stored at 80 1C for future analysis
Following blood sampling, ¢sh (length and weight
determinants in Table 1) were sacri¢ced by overdosing
with anaesthetic clove oil (0.2 mL L 1) The spleen
was removed and weighed to 0.001g precision Spleen
somatic index was calculated using the formula
[spleen mass (g)/¢sh mass (g)] 104
(Lai Jimmy, kuta, Mok Helen, Rummer & Randall 2006)
Ka-Plasma biochemical indices
Glucose and cortisol levels, assessed according to
SvobodovaŁ et al (1999), and total protein (TP) and
lac-tate levels were used to evaluate the stress load both
by cormorants and by harvesting Glucose, lactate
and TP were measured using the Cobas EMira
biochemical analyser (Roche Diagnostics, Basel,Switzerland) and commercial test kits The concen-tration of plasma cortisol was measured using theELISA method (Tizard 1996)
StatisticsAll values were examined for normal distribution(Kolmogorov^Smirnov test) and homoscedasticity(Levene test) Fulton’s condition coe⁄cient, glucoseand TP values were analysed using one-wayANOVA
with Tukey’s post hoc test, while SSI, cortisol and tate values were analysed using the non-parametricKruskal^Wallis test The Student t-test was used tocompare CPUE data All data were analysed using
lac-STATISTICAv 8.0 software for Windows (StatSoft 1999)
ResultsCormorant occurrenceAfter 3 days of bird presence and activity (huntingand resting on trees along the pond shore), ¢sh hadbeen driven to the pond margin opposite the cormor-ant roosting sites At the same time, ¢sh displayednormal behaviour on the LaŁska CP
CPUE
A signi¢cant di¡erence (Po0.001) was recordedbetween the number of ¢sh captured in one lift netsession in the littoral zone of the LaŁska pond(CP, mean standard deviation 5 0.9 1.1) andthat at the Nade›je pond (HC, 43.6 39.6)
SSI and FCCRelative spleen weight was signi¢cantly lower in PHand HC ¢sh (Po0.01) than in CP ¢sh (Po0.05), whilenon-signi¢cant di¡erences [ANOVA: H(2, n 5 66) 522.393, P 5 0.0001] were detected between the HCand PH groups (Fig 1a) Fulton’s condition coe⁄cientwas signi¢cantly higher (Po0.01) in the controlgroup in comparison with PH and HC, although
no signi¢cant di¡erences [ANOVA: F(2,63) 5 15.947,
P 5 0.0001] were observed between groups HC and
PH (Fig 1b)
Plasma biochemical indicesThe mean plasma cortisol level in PH ¢sh was signi¢-cantly higher (Po0.01) than that in the HC and CPgroups (Po0.01), while the di¡erence between
Table 1 Length and weight data in examined ¢sh samples
Pond Sampled group n TL (mm) W (g)
Nadeˇje HC 22 188.45 18.88 109.09 35.50
Nadeˇje PH 22 207.82 25.18 151.55 53.57
La´ska CP 22 198.45 19.75 149.20 54.51
Values are mean SD.
HC, hunting cormorants; PH, pond harvesting; CP, control group.
Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al Aquaculture Research, 2011, 42, 322–330
Trang 14groups HC and CP was not statistically signi¢cant
[ANOVA: H(2, n 5 66) 5 40.752, P 5 0.0001] (Fig 2a)
A similar situation was recorded with glucose
con-centration, where the values from harvested PH ¢sh
di¡ered signi¢cantly (Po0.01) from the HC and CP
(Po0.01) groups No signi¢cant di¡erence was foundbetween groups HC and CP [ANOVA: F(2,63) 5 67.928,
HC0.0
HC1.2
Trang 15CP group (Po0.01), while comparisons of values for the
PH and CP groups, and between the PH and HC groups,
did not di¡er signi¢cantly [ANOVA: F(2,63) 5 5.839,
P 5 0.0047] Lactate concentration di¡ered among
treatments in a pattern similar to those of blood
glu-cose and cortisol The value recorded for PH ¢sh
dif-fered signi¢cantly from the HC and CP groups [ANOVA:
F(2,63) 5 87.483, P 5 0.0001; Fig 3a and b]
DiscussionFish movements towards the pond shoreline due tocormorant feeding activities are well known onCzech ¢shponds, especially during the spring (Febru-ary^April) migration £ights of cormorants (¢sh farm-ers’ reports 2000^2008) However, there is a lack ofscienti¢c data to describe this behavioural phenom-
(a)
(b)
CPPH
HC20
253035404550556065707580859095100
HC0
12345678910
CP 2.58 0.76.Values that do not di¡er signi¢cantly (P40.05) share common letters
Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al Aquaculture Research, 2011, 42, 322–330
Trang 16enon In the present study, the CPUE results show
highly signi¢cant di¡erences between ¢sh numbers
in the littoral zone of the pond visited by cormorants
and the CP without cormorants, indicating an
im-mediate ¢sh evasion response into shallow littoral
areas
Studies of ¢sh wounded by cormorants while
hunt-ing have proved that the condition of wounded ¢sh is
signi¢cantly lower than that of ¢sh without ant beak marks (AdaŁmek, Kortan & Flajs›hans 2007;Kortan et al 2008) Fulton’s condition coe⁄cientshowed signi¢cantly higher values for ¢sh from the
cormor-CP group than for the HC and PH groups, probablydue to energy release without food intake, though thedi¡erence between harvested ¢sh and ¢sh stressed bycormorants was not statistically proved The environ-
(a)
(b)
CPPH
HC10
1214161820222426283032
HC0
123456789101112131415
Aquaculture Research, 2011, 42, 322^330 Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al.
r 2010 The Authors
Trang 17mental conditions in both ponds correspond to typical
conditions in Czech ¢shponds, and therefore, these are
unlikely to have a¡ected the coe⁄cient
The relative weight of spleen (SSI) is used
fre-quently as a stress indicator (Pal|¤kovaŁ & SvobodovaŁ
1995; SvobodovaŁ, KolaŁr›ovaŁ, MaŁchovaŁ, KalaŁb, Piac›ka,
VykusovaŁ, ModraŁ, Dus›ek & Raab 1998; Dobs›|¤kovaŁ
et al 2006; Lai Jimmy et al 2006) According to
Svobo-dovaŁ et al (1998), the SSI calculated for ¢sh
under-going long-distance transport did not show a
signi¢cant decrease, although a signi¢cant decrease
in SSI was observed during PH (SvobodovaŁ et al
2006) Our study indicates a signi¢cantly higher SSI
in the control CP group in comparison with the HC
and PH groups, i.e cormorant-disturbed and
har-vested ¢sh Therefore, PH appears to be a greater
stressor than cormorant attack, though the
di¡er-ences in SSI were not signi¢cant
Biochemical indices of ¢sh blood plasma are also
considered to be indicators of stress (Svoboda 2001)
Blood plasma glucose concentration is a characteristic
trait utilized for the evaluation of acute stress in ¢sh
(Pottinger 1998; Bau, Ferroni-Claverie & Parent 2001;
Ruane, Huisman & Komen 2001) SvobodovaŁ et al
(2006) proved experimentally that glucose levels
in-creased during PH, and that they returned to normal
levels1month after the ¢sh were transferred to storage
ponds In our study, the increase in glucose level was
signi¢cantly higher in HP ¢sh collected during
har-vesting, with no signi¢cant di¡erence recorded
be-tween the control CP sample and HC ¢sh impacted by
cormorant hunting Blood plasma samples were taken
from stressed ¢sh after the third day of cormorant
pre-sence This delay in sampling may explain why no
sta-tistically signi¢cant di¡erence was found between the
HC and CP groups, as glucose levels can both fall over a
3-day period and as the stress is unlikely to have been
less acute than that experienced during PH
Similar di¡erences were also found in plasma
cor-tisol The literature indicates that cortisol levels
in-crease in the initial stages of stress situations, but
return to baseline levels within a few hours due to
¢sh adaptation (Pickering & Pottinger 1989; Svoboda
2001) This has also been shown in the study by
SvobodovaŁ et al (2006), where high values of cortisol
during the harvesting period returned to normal
after 1 month of storage In the present study, high
values of cortisol were found in PH ¢sh Fish from
the CP showed lower levels of cortisol than HC
¢sh, though the di¡erence was not signi¢cant These
results are consistent with the ¢ndings for blood
glucose levels, i.e the presence of cormorants is not
as acute a stressor as PH, with cortisol levels ing slowly to normal values 3 days after exposure tothe stressor The increase in the plasma lactate level is
decreas-a result of decreas-andecreas-aerobic metdecreas-abolism decreas-and re£ects the position of severe exercise (Pottinger 1998) Lactate,
im-as a stress indicator, wim-as used in the study of khurst and Dedualj (1994), who evaluated the e¡ect
Pan-of capture and recovery in rainbow trout hynchus mykiss (Walbaum)] In their study, signi¢-cant elevation of lactate level was recorded within
[Oncor-15 min of capture, which decreased to normal levelswithin 24 h This ¢nding corresponds with our ex-periment, where the highest increase in lactate levelwas found in PH ¢sh, while HC ¢sh showed no statis-tically signi¢cant increase in lactate level comparedwith CP ¢sh This indicates a decline in blood lactatelevels due to acclimatisation to the stressor after 3days of exposure to cormorants The total plasma pro-tein in ¢sh ranges from 20 to 80 g L 1and appears to
be fairly constant within and among species nald & Milligan 1992) Stress can lead to an increase
(McDo-in the total plasma prote(McDo-in For example, (McDo-in ra(McDo-inbowtrout, an increase in TP in blood plasma was con-cluded to be a response to strenuous exercise(Milligan & Wood 1986) and exposure to low environ-mental pH (Milligan & Wood 1982) Total proteinconcentration in blood plasma decreases in the ¢rstshock phase of the stress reaction During the anti-shock reaction phase, TP concentrations increasedue to the e¡ect of cortisol, which is responsible forprotein catabolism (NovaŁk 2002) In our investiga-tion, the increase in TP level was signi¢cantly higher
in HC ¢sh than in CP ¢sh, while the TP value in PH
¢sh did not di¡er from HC ¢sh and/or the CP ¢sh.The results of TP assessment are consistent withthose reported in the literature, though it remains amatter of conjecture as to why they di¡ered fromother stress indicators (cortisol, glucose and lactate),which were higher in PH ¢sh (harvested) than in HC
¢sh (cormorant impacted) It is highly likely that this
is a matter of timing and of numbers of blood pling repetitions
sam-The in£uence of the time span between the ture of cormorants and the beginning of PH is also amatter for debate It is known that stress levels de-crease with time due to adaptation and/or stressordisappearance (Pickering & Pottinger 1989) In thecase of PH, which was initiated 8 days after the cor-morants left, stressor values (glucose, cortisol, lac-tate) were higher than those in HC This indicatesthat a co-e¡ect of cormorant presence and harvest-ing was not apparent
depar-Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al Aquaculture Research, 2011, 42, 322–330
Trang 18Blood plasma stress indicators (glucose, cortisol and
lactate concentrations) do not indicate a signi¢cant
impact of cormorant hunting activities on ¢sh
(com-mon carp) as their values did not di¡er signi¢cantly
from those found in control non-impacted ¢sh
Ac-cording to our results, the stress caused by PH is a
more important cause of physiological response in
¢sh than that resulting from a 3-day period of
cor-morant disturbance Avoidance behaviour of ¢sh,
with resulting high densities in the pond littoral
zone, was demonstrated by CPUE estimates These
phenomena indicate a distinct negative in£uence of
the presence of cormorant £ocks on ¢shpond stock
behaviour, with possible adverse e¡ects on PH,
starvation and subsequent ¢sh condition, along
with suppressed immunity when persisting for long
periods
Acknowledgments
This study was supported by the projects RIFCH USB
nos MSM6007665809 and SP/2d3/209/07 (Ministry
of Environment, Czech Republic), and COST OC090
42 Data on pond stocks and cormorant monitoring
was provided by the RybaŁr›stv|¤ Tr›ebon› (Tr›ebon›
Fish-eries) Hld Company The authors also thank
Vlasti-mil Stejskal (RIFCH USB) and Zuzana Kno£ic›kova
(USB) for their help with ¢eld monitoring and to
Da-vid Kortan (SEPA) for his critical comments
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Trang 20Influence of temperature on muscle fibre hyperplasia and hypertrophy in larvae of blackspot seabream,
Pagellus bogaraveo
Paula Silva1,2, Lu|¤sa Maria Pinheiro Valente1,2, Mercedes Olmedo3, Blanca AŁlvarez-BlaŁzquez3,Maria Helena Galante1, Roge¤rio Alves Ferreira Monteiro1,2& Eduardo Rocha1,2
1 ICBAS ^ Institute of Biomedical Sciences Abel Salazar, UPorto ^ University of Porto, Porto, Portugal
2 CIMAR/CIIMAR ^ Interdisciplinary Centre for Marine and Environmental Research, UPorto ^ University of Porto, Porto, Portugal
3
Instituto Espanol de Oceanogra¢a, Cabo Estay,Vigo, Spain
Correspondence: E Rocha, Laboratory of Histology and Embryology, ICBAS ^ Institute of Biomedical Sciences Abel Salazar, Lg Prof Abel Salazar 2, 4099-003 Porto, Portugal E-mail: erocha@icbas.up.pt
Abstract
The e¡ects of temperature on Pagellus bogaraveo
mus-cle cellularity were investigated, by morphometry,
throughout the endogenous feeding stage, testing
two rearing temperatures: 14 and 18 1C The
follow-ing parameters were estimated in transversal body
sections at post-opercular and post-anal body levels:
the total cross-sectional muscle area, the total
num-ber of ¢bres and the mean cross-sectional ¢bre area
At hatching, no signi¢cant in£uence of the
tempera-ture was observed on the morphometric parameters
measured in the white muscle At mouth opening, an
increase in the number of post-opercular white ¢bres
was promoted by the highest temperature During
embryonic development, the red muscle ¢bre number
in the post-anal part of the larvae increased with
higher temperature, but it appears that the di¡erence
was no longer present at mouth opening An increase
in the ¢bre area and in the total cross-sectional area
of red muscle at the post-anal level was promoted by a
4 1C increase in the temperature during the vitelline
phase In conclusion, the axial musculature of
black-spot seabream embryos/larvae reacted di¡erently to
temperature in£uence according to the body
loca-tion, strongly supporting the need to look at and
ac-count for di¡erent body locations when evaluating
muscle cellularity in ¢sh, namely in
growth/aquacul-ture-related studies
Keywords: temperature, Pagellus bogaraveo, ¢sh
larvae, muscle growth, blackspot seabream
IntroductionSeveral studies suggested that rearing temperaturecan in£uence muscle cellularity at hatching and/or
in the subsequent stages of ¢sh, but these e¡ects varywidely among and within ¢sh species Generally, stu-dies have taken eggs from individual or mixed fa-milies, incubated them at di¡erent temperatures andsampled the o¡spring at de¢ned life-history stages(e.g hatching or ¢rst feeding).Various patterns of re-sponse of muscle cellularity to temperature havebeen described at hatch and at the start of exogenousfeeding In recently hatched larvae of herring (Clupeaharengus, Linnaeus), higher temperatures increasedwhite muscle ¢bre hyperplasia (Vieira & Johnston1992; Johnston, Vieira & Abercromby 1995) Never-theless, in a di¡erent study with herring (Johnston1993) and in salmon (Salmo salar, Linnaeus) (Usher,Stickland & Thorpe 1994; Nathanailides, Lo¤pez-Al-bors & Stickland 1995), hypertrophy of the whitemuscle ¢bres was induced by an increase in the rear-ing temperature In sea bass (Dicentrarchus labrax,Linnaeus), the slight increase in the water tempera-ture during incubation had a positive e¡ect on bothhypertrophic and hyperplastic muscle growth, fromthe beginning of the exogenous feeding onwards(Ayala, Lo¤pez-Albors, Gil, Latorre, VaŁsquez, Garc|¤a-AlcaŁzar, AbellaŁn, Ram|¤rez & Moreno 2000) Anotherstudy, with the same species, showed that when thetemperature increase was maintained until mouthopening [5^6 days post-hatching (dph)], the larvaland early larval muscle growth was improved, andAquaculture Research, 2011, 42, 331^340 doi:10.1111/j.1365-2109.2010.02627.x
r 2010 The Authors
Trang 21that the histochemical maturity of white muscle was
advanced in prewarmed specimens (Lo¤pez-Albors,
Ayala, Gil, Garc|¤a-AlcaŁzar, AbellaŁn, Latorre,
Ram|¤r-ez-Zarzosa & VaŁzquez 2003) This inter- and
intraspe-ci¢c variability in the response to temperature may
re£ect genetic variation, abiotic factor e¡ects and/or
could be related to distinct body sampling sites, as
some of those studies were carried out in anterior
po-sitions of the larvae trunk (Vieira & Johnston 1992;
Johnston1993; Johnston et al.1995) and others in
pos-terior ones (Nathanailides et al 1995; Ayala et al
2000; Lo¤pez-Albors et al 2003) In gilthead sea bream
larvae (Sparus aurata, Linnaeus) of 20 days,
Rowler-son, Mascarello, Radaelli and Veggetti (1995) found
signi¢cant di¡erences between the muscle ¢bre size
distributions at the anal opening level and at a level
found 2 mm more caudally In Atlantic salmon,
di¡erent muscle ¢bre size distributions were also
observed among three longitudinal sampling points
(Johnston 2001) To the best of our knowledge,
previous works were not particularly focused on
comparing the e¡ects of temperature on muscle
cellularity between the distant zones of the trunk
musculature that display di¡erent gross morphology
The blackspot seabream (Pagellus bogaraveo,
Brun-nich) is common along the Southern European
Atlantic continental shelf and throughout the
Mediter-ranean, and its intensive production has increased in
the last decade In the natural environment, blackspot
seabream spawning occurs at the end of winter
be-tween January and April (Bauchot & Hureau 1990)
and the peak spawning activity is reached between
February and May (SaŁnchez1983; Krug1990) In
captiv-ity, spawning coincides more or less with these periods,
being at a controlled temperature of 14 1C from
Febru-ary through to May at north-west of the Iberian
Penin-sula (Olmedo, Peleteiro, Alvarez-BlaŁzquez & Go¤mez
1998) The present work was carried out to investigate
the in£uence of two di¡erent water temperatures (14
and 18 1C), used in incubating early embryos and
sus-taining the vitelline phase, on the blackspot seabream
axial muscle cellularity at two time points: at hatching
and at mouth opening Another question we wanted to
address concerned the hypothesis that the e¡ect
of temperature on muscle growth is di¡erent along a
‘rostro-caudal’gradient
Materials and methods
Fish
The experiment was carried out at the Instituto
Espa-nol de Oceanograf|¤a (IEO) (Centro OceanograŁ¢co de
Vigo, Spain), using eggs obtained from an adult stock
of blackspot seabream (68 reproducers) maintained
in a £ow-through seawater system at natural water temperature (14 1C) Eggs were obtained at theend of March of 2006, and were divided into twotanks (ca 6390 eggs for each tank): one tank wasmaintained at 14 0.1 1C and another was reared at18.0 0.1 1C from fertilization to mouth opening.Egg incubation and rearing were carried out in cylin-droconical tanks (150 L) and exposed to continuouslight (1500^2000 lx).Water quality variables (i.e tem-perature, oxygen level, pH, ammonia, nitrite and ni-trate) were checked daily The oxygen level was 99%saturation (8.1 0.1ppm in both tanks) The rate ofwater £ow, recirculation, pH (8.2 0.1 in the 14 1Ctank and 8.1 0.1 in the 18 1C tank, with no signi¢-cant di¡erences between tanks) and ad libitum supply
sea-of live plankton and commercial ¢sh food sea-of ate particle sizes were held constant (all according tothe standard procedures of the IEO ¢sh breeding la-boratory) Nitrite and nitrate were barely detectable
appropri-in all the tanks (i.e.o0.05 ppm)
The growth performance was described using thefollowing parameters:
Thermal-unit growth coefficientðTGCÞ
¼ 100 ½ðFBW1=3 IBW1=3Þ=SðT DaysÞSpecific growth rateðSGRÞ
¼ 100 ððLnFBW LnIBWÞ=DaysÞCondition factorðKÞ
¼ FBW=Length3 100where IBW is the mean initial body weight (mg) (athatching), FBW is the mean ¢nal body weight (mg) (atmouth opening) and T is the water temperature ( 1C)
Sample preparationLarvae sampled at hatching (Day 0 of the experimentwas de¢ned as the time when 50% of the eggs hadhatched) and at mouth opening (MO) were killed byproviding excess anaesthesia in a solution of MS-222(Sigma-Aldrich, St Louis, MO, USA), ¢xed in 4% paraf-ormaldehyde in phosphate bu¡er Larvae were laterpaper dried and bulk-weighed (a total of six groupseach with six larvae per treatment) using a digitalbalance (accuracy 0.001g) Thereafter, the totallength was individually measured under a micro-scope with a graduated eyepiece to estimate growth.For morphometric analysis, six larvae per group andper sampling level (i.e 24 larvae in total) were routi-nely dehydrated in a graded ethanol series, cleared inTemperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340
Trang 22xylol and, ¢nally, embedded in para⁄n The entire
larva body was transversely sectioned (7mm) and all
the sections were saved Every 10th section was then
sampled, stained with haematoxylin^eosin before
being coversliped and analysed under the
micro-scope to select the two body sections of individual
¢sh, at both post-opercular and post-anal locations
chosen to measure the morphometric parameters
As our primary goal was to perform comparisons
be-tween the two experimental groups, the same
labora-tory protocol was used for preparing tissue samples
(all animals being processed at the same time), to
minimize the shrinkage problem
Morphometry
Morphometric variables were measured in transversal
body sections of individual ¢sh, at both post-opercular
and post-anal locations, to estimate the total
cross-sectional muscle area [A (muscle)], the total number
of ¢bres [N (¢bres)] and the mean cross-sectional ¢bre
area [a (muscle ¢bre)], of the fast white muscle
(inner-most ¢bres) and for slow red muscle (outer(inner-most ¢bres)
The study was performed using an interactive
im-age analysis system (CAST-Grid; Olympus Denmark
A/S, Ballerup, Denmark; version 1.6), working with a
live-image captured using a CCD-video camera (Sony,
Tokyo, Japan) The light microscope (BX50; Olympus,
Tokyo, Japan) used was equipped with a fully
motor-ized stage (Prior Scienti¢c, Rockland, MA, USA), thus
allowing meander sampling with an (x^y axis)
accu-racy of 1mm The cross-sectional (half) white muscle
area [A (white muscle)] was automatically computed
using the software after the physical limits of interest
in the section were traced by the operator Precise
tra-cing was made on the monitor, at a magni¢cation
of 1608, using live-image primarily captured
under a 40 objective lens Because it had been
con-¢rmed that no signi¢cant di¡erences existed between
the right and the left side of the ¢sh, an estimate of
the total cross-sectional area [A (white muscle)] was
performed by doubling the computed value
Red muscle
The cross-sectional red muscle area [A (red muscle)]
was estimated as follows:
Aðred muscleÞ ¼ a ðred muscle fibreÞ N ðred fibresÞ
ð1Þwhere a (red muscle ¢bre) is the mean individual
red muscle ¢bre cross-sectional area and N (red
¢bres) is the total number of red muscle ¢bres percross-section
The mean individual red muscle ¢bre tional area [a(red muscle ¢bre)] of larvae was esti-mated using the 2D-nucleator technique The area
cross-sec-of any object irrespective cross-sec-of its size, shape and tation is rapidly and unbiasedly estimated using thattechnique by measuring the distance between a
orien-‘central point’ (a virtual one) within the object andthe intersections between the object boundary andradiating test lines (four in our case) (Larsen, Gun-dersen & Nielsen 1998; Savnik, Bliddal, Nyengaard &Thomsen 2002) The mathematical formula underly-ing this technique reads as follows:
aðred muscle fibreÞ ¼ pI2 ð2Þwhere I is the distance from the ‘central point’ to theobject boundary measured in a pre-determined num-ber of random directions For our case, the ‘centralpoint’ was chosen by the operator as the virtual mostapproximate centre of a cross-sectioned ¢bre All theprocedures and measurements were performedusing the Grid software (CAST-GRID) All ¢bres in abody cross-section were measured, also at a ¢nalmagni¢cation of 1608 ( 40 objective lens)
White muscleFor the sake of measuring e⁄ciency (high speed/ef-fort ratio), the mean individual white muscle ¢brecross-sectional area [a(white muscle ¢bre)] was indir-ectly estimated by unbiasilly combining A (whitemuscle) and N (white ¢bres), as follows:
aðwhite muscle fibreÞ ¼ A ðwhite muscleÞ
=Nðwhite fibresÞ
As with red muscle, the total number of white
¢bres [N (white ¢bres)] was obtained by counting allthe ¢bres that appeared in sections
Hypertrophy and hyperplasiaThe relative contribution of hypertrophy and hyper-plasia towards the increase in the cross-sectionalarea was estimated as applied previously (Valente,Rocha, Gomes, Silva, Oliveira, Monteiro & Faucon-neau 1999):
DAðmm2Þ ¼ NmDaðmm2Þ þ amðmm2ÞDNwhereD is calculated between two sampling times(t and t11) and Nm and amrefer to the mean totalnumber of ¢bres and ¢bre area at t
Aquaculture Research, 2011, 42, 331^340 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al.
r 2010 The Authors
Trang 23Data were analysed using the software STATISTICA
(version 6; StatSoft,Tulsa, OK, USA) For all statistical
tests, the signi¢cance level was set ata 5 0.05 The
analysis was carried out using the Mann^Whitney
U non-parametric test in order to determine the
in£u-ence of water temperatures on larvae growth
Be-cause ¢sh reared at di¡erent temperatures do not
necessarily hatch or start feeding at the same
devel-opmental stage, the e¡ects of the temperature on the
morphometric parameters were analysed for
covar-iance (ANCOVA), in which temperature was set as the
independent variable while the total length was set
as a covariate In the speci¢c cases, where theANCOVA
assumptions failed, data transformations (inverse
va-lue) were performed to meet the assumptions When
di¡erences were signi¢cant, individual means were
compared using the Tukey’s honest signi¢cant
di¡er-ence test Such pair comparisons were also tested
using the Mann^Whitney U-test, which showed the
same signi¢cant di¡erences; the P-values given are
from Tukey’s test For a particular age and
tempera-ture, comparisons between opercular and
post-anal morphometric values were performed using the
Student’s t-test for paired samples, after checking
normality and homogeneity of variance, using the
Kolmogorov^Smirno¡ and the Levene tests
respec-tively (Zar 1996)
Results
Larvae development and growth performance
Blackspot seabream embryos developed faster at
higher temperatures Embryonic life (from
fertiliza-tion to hatching) lasted 72 h at 18 1C and 96 h at
14 1C Pre-larval (hatching ^ mouth opening)
devel-opment was also accelerated by a higher cultivation
temperature (occurred at 6 dph at 18 1C versus 9 dph
at 14 1C) Data on growth are reported in Table 1 The
body weight of larvae reared at 18 1C was just higher
at mouth opening (Table 1) A di¡erent trend was
dis-played by the larvae length: larvae incubated at 18 1C
were signi¢cantly larger at hatching but according to
our experiment similar values at mouth opening
were obtained at both treatments (Table 1) The
growth of ¢sh expressed either as the speci¢c growth
rate (SGR) or as the thermal-unit growth coe⁄cient
(TGC) was signi¢cantly improved at 18 1C (Table 1)
The condition factor apparently was not signi¢cantly
a¡ected by di¡erent temperatures among ¢sh at both
hatching and mouth opening (Table 1)
Baseline growth dynamicsThe distinction between white and red muscle wasalways evident The larvae A (white muscle) increasedfrom hatching until mouth opening with both tem-peratures at the post-opercular level and with 14 1C
at the post-anal level (Po0.05) (Table 2) At the opercular level, the white muscle growth was mainlyachieved by hypertrophy (Fig 1; Table 2) The hyper-plastic process gained importance only at the post-anal level of the ¢sh, principally of the ones at a high-
post-er temppost-erature (Po0.05) (Fig 1; Table 2) Duringthe trial, the A (red muscle) increased at both tempera-tures, at the post-anal level (Po0.05), only (Table 2).This enlargement in the red muscle area was related
to the a(red muscle ¢bre) and N (red ¢bres) increase inlarvae reared at 18 1C (Po0.05) and to the totalnumber of ¢bres increase in larvae reared at 14 1C(Po0.05) (Table 2)
Temperature e¡ect on white muscle
No signi¢cant temperature-induced e¡ect wasobserved in A (white muscle) parameter during theexperiment (Fig 2a) Moreover, no signi¢cant di¡er-ences in A (white muscle) were found between thetwo muscle locations sampled (Table 3)
At mouth opening, the N (white ¢bres) at the opercular level (Fig 2c) was signi¢cantly (Po0.05)increased by the highest incubation temperature Atboth sampling time points, the N (white ¢bres) washigher in the post-anal zone of the larvae reared at
post-14 1C (Po0.05) (Tables 2 and 3) However, in the vae reared at 18 1C, these di¡erences seem to be inex-istent (Table 3)
lar-Table 1 Growth performance of Pagellus bogaraveo larvae reared at di¡erent temperatures [14 1C (T14) versus 18 1C (T18)], from hatching (H) to mouth opening (MO)
Initial body weight (H), mg 0.25 (0.02) 0.25 (0.02) Final body weight (MO), mg 0.27 (0.02) a 0.33 (0.03) b
Initial body length (H), mm 3.4 (0.02) a 3.9 (0.02) b
Final body length (MO), mm 4.9 (0.19) 4.9 (0.24) Initial condition factor (K) 0.22 (0.03) 0.22 (0.04) Final condition factor (K) 0.24 (0.03) 0.28 (0.04) Thermal-unit growth coefficient (TGC) 0.01 (0.02) a 0.05 (0.02) b
Specific growth rate (SGR) 0.97 (1.03) a 4.37 (1.53) b
Within a row, means without a common superscript letter di¡er signi¢cantly (P o0.05) The absence of a superscript indicates no signi¢cant di¡erence between treatments Values are means (standard deviation); n 5 6.
Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340
Trang 24Throughout the assay, no signi¢cant temperature
e¡ect was found in the a(white muscle ¢bre) (Fig 2e)
Moreover, at mouth opening, larvae reared at 14 1C
showed a greater a(white muscle ¢bre) at the
post-op-ercular level when compared with the post-anal one(Po0.05) (Tables 2 and 3)
Temperature e¡ect on red muscle
At mouth opening, the A (red muscle) (Fig 2b) was creased by the highest incubation temperature(Po0.05) Also at this stage, a higherA (red muscle) inthe post-anal level at 14 1C (Table 2) was the only sig-ni¢cant di¡erence observed between the muscle lo-cations (Po0.05) (Table 3)
in-At hatching, more red ¢bres were found at the anal level in the larvae reared at 18 1C (Fig 2d) com-pared with the larvae at a lower temperature(Po0.05), but this di¡erence was apparently inexis-tent at mouth opening On comparing muscle loca-tions, the N (red ¢bres) was superior at the post-anallevel at the two sampling time points and at both tem-peratures (Po0.05) (Tables 2 and 3)
post-During the vitelline phase, the 4 1C increase in thewater temperature induced a slight but signi¢cant in-crease in the red ¢bre cross-sectional area (Po0.05)(Fig 2f) On comparing both muscle locations, athatching, the a(red muscle ¢bre) was larger at thepost-opercular level of the larvae from the 18 1Cgroup (Po0.05) (Tables 2 and 3)
Table 2 Total cross-sectional muscle area [A (muscle)],
to-tal number of ¢bres [N (¢bres)] and cross-sectional ¢bre area
[ a (muscle ¢bre)] of white ¢bres and red ¢bres at hatching
(H) and mouth opening (MO) in Pagellus bogaraveo
Values are means (CV), n 5 6 The di¡erences in the
morpho-metric parameters between the two sampling time points
(hatching and mouth opening) for each body zone and each
temperature are shown; means without a common letter di¡er
signi¢cantly (P o0.05).
Figure 1 Relative contribution of hyperplasia and trophy to white (a) and red (b) to the total cross-sectionalmuscle area increase in Pagellus bogaraveo at two di¡erentbody locations (post-opercular and post-anal), fromhatching to mouth opening
hyper-Aquaculture Research, 2011, 42, 331^340 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al.
r 2010 The Authors
Trang 25One key factor in the commercial production of
mar-ine ¢sh species is the control of hatchery production
procedures (broodstock and egg managements,
lar-val rearing and feeding) Blackspot seabream studies
on larvae culture techniques began in the 1990s
(Peleteiro, Olmedo, Go¤mez & Alvarez-BlaŁzquez 1997;
Olmedo et al 1998; Micale, Maricchiolo & Genovese
2002), but this is the ¢rst study on the in£uence of
two water temperatures (14 and 18 1C) on the larvae
growth and on muscle cellularity from hatching to
mouth opening It was also the ¢rst study in ¢sh
aimed at establishing whether the temperature
in£u-ence is similar anteriorly and posteriorly in the body
The use of replicates for each treatment was not
pos-sible in this study, however, we decided to conduct
the experiment because the baseline e¡ects of
tem-perature on the muscle growth were anticipated byvery good background information and only a preli-minary estimate of this e¡ect was required More-over, all the husbandry conditions and waterparameters were accurately controlled so that thetemperature was the only parameter that could vary
in the two experimental groups All available datarevealed that this was the case Thus, our designallowed a reliable general overview of the e¡ect
of temperature on the blackspot seabream larvaemuscle growth
As hypothesized, the development rate of the bryos was improved by an increase in the rearing tem-perature within the tolerance range of the blackspotseabream, as reported for other species (Usher et al.1994; Nathanailides et al 1995; Johnston, Cole, Aber-cromby & Vieira 1998; Ayala, Lo¤pez-Albors, Gil, Garc|¤a-AlcaŁzar, AbellaŁn, Alarco¤n, Alvarez, Ramirez-Zarzosa &
em-Figure 2 Total cross-sectional muscle area [A (muscle)], total number of ¢bres [N (¢bres)] and cross-sectional ¢bre area [a(muscle ¢bre)] of white ¢bres (a, c, e) and red ¢bres (b, d, f) at hatching (H) and mouth opening (MO) in Pagellus bogaraveo.Values are means SE, n 5 6 The e¡ect of the incubating temperatures on the morphometric parameters estimated isshown; means without a common letter di¡er signi¢cantly (Po0.05) The absence of letters indicates no signi¢cant di¡er-ence between treatments
Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340
Trang 26Moreno 2001) Our data suggest that the yolk depletion
speed in blackspot seabream depends on the water
tem-perature This could mean that, at high temperatures,
this species may be better able to resist starvation and
may be reared with high survival and rapid growth
Comparison of blackspot seabream larvae at equivalent
development stages con¢rmed that growth di¡erences
were induced by temperature At hatching, larvae
in-cubated at a higher temperature were already longer
The same was observed in the sea bass, where the body
length was greater in larvae incubated at higher
tem-peratures (Lo¤pez-Albors et al 2003; Alami-Durante,
Rouel & Kentouri 2006) Larval length at hatch is an
important parameter in£uencing the initial
locomo-tion performance, such as swimming speed, escape
re-sponse and predator avoidance (Blaxter 1992) Further
temperature in£uences in the pre-larval period were
not related to body length but rather to weight, as
18 1C larvae reached the stage of mouth opening with
a higher body weight than 14 1C larvae Our
explana-tory hypothesis is that during the pre-larval phase, the
use of vitelline energy to satisfy the requirements
re-lated to organogenesis causing acceleration in vitelline
sac reabsorption was favoured by the higher
tempera-ture (Johnston 1993)
In general, the A (white muscle) increased until
mouth opening at both locations The A (red muscle),
however, increased with age only at the post-anal
re-gion These results di¡er from those included in a
pre-vious study with this ¢sh species (Silva, Valente,
Olmedo, Galante, Monteiro & Rocha 2009), covering
hatching to juvenile, where a pause in muscle growth
of both ¢bre types was observed at mouth opening
As to the increase observed at the post-opercular vel, the di¡erence in muscular growth observedcould be explained by the fact that the larvae dis-played di¡erent growth dynamics in the two studies.Accordingly, in the previous one (Silva et al 2009) lar-vae grew mainly in length (3.6^6.0 mm), reachingmouth opening faster, whereas in the current one,the larvae growth at mouth opening was mainly re-lated to a weight increase The present results showthat the white muscle hypertrophy was the maincontributor to the A (white muscle) growth at thepost-opercular body position, which relates well withthe above-mentioned weight increase As to the post-anal level, the increase observed in the present work
le-in both A (white muscle) and A (red muscle) was due toboth hyperplastic and hypertrophic mechanisms.Also, at the post-anal level, the current results weredi¡erent from those observed in the previous study(Silva et al 2009) This is probably related to the di¡er-ent position of the post-anal level resulting from thelarvae length, because as noted in this and in the pre-vious study (Silva et al 2009), and for the ¢rst time inthis species, di¡erences exist in the cellularity be-tween di¡erent body zones Previous researchshowed species di¡erences in muscle growth in the
¢rst days after hatching An uninterrupted myogenesis
in fast muscle after hatching, for example, was also parent in some teleost species including salmon (Stick-land, White, Mescall, Crook & Thorpe 1988), rainbowtrout (Oncorhynchus mykiss,Walbaum) (Stoiber & Sn-ger 1996; Xie, Mason,Wilkes, Goldspink, Fauconnueau
ap-& Stickland 2001), zebra¢sh (Danio rerio, Hamilton)(Barresi, D’Angelo, Hernandez & Devoto 2001) andpearl¢sh (Rutilus meidingeri, Heckel) (Steinbacher, Ha-slett, Six, Gollmann, Snger & Stoiber 2006) A pause
in the recruitment of new ¢bres in the ¢rst days afterhatching was, however, observed in herring (Johnston1993), turbot (Gibson & Johnston 1995; Johnston et al.1998), cod (Galloway, Korsvik & Kryvi 1999) and sole(Veggetti, Rowlerson, Radaelli, Arrighi & Domeneghini1999) Besides these inter-speci¢c di¡erences in ¢shmuscle growth in the ¢rst days after hatching, ourstudy showed that the in£uence of temperature inmuscle cellularity is somewhat variable even for thesame species This intra-speci¢c variability is probablyrelated to genetic variation and/or di¡erences in thespawning condition of the parents
The embryonic growth of blackspot seabreamaxial musculature was signi¢cantly a¡ected bytemperature Fish incubated at higher temperatures(18 1C) at hatching showed a higher number of red
¢bres at the post-anal level than those incubated at
Table 3 Signi¢cance of di¡erences between Pagellus
bogar-aveo muscle location (post-opercular versus post-anal) for
the estimated morphometric parameters at di¡erent
tem-peratures [14 1C (T14) versus 18 1C (T18)], evaluated using
the Student’ t-test for dependent samples
H, hatching; MO, mouth opening; A, muscle area; N, total
num-ber of ¢bres; a, cross-sectional ¢bre area; NS, non-signi¢cant
Aquaculture Research, 2011, 42, 331^340 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al.
r 2010 The Authors
Trang 27lower temperatures (14 1C) The presence of more red
¢bres in the post-anal part of the newly hatched
lar-vae suggested that, towards the end of embryonic life,
more red ¢bres were formed in the caudal myotomes
Also, Rowlerson et al (1995) suggested that during
larval life in sea bream, the hyperplastic growth of
slow muscle ¢bres occurred in a very restricted zone
of the post-anal muscle It is suggested by our data
that this process is not restricted to a species and is
modulated by temperature The majority of the works
studied the in£uence of incubation temperature in
white muscle only, and large inter-speci¢c di¡erences
in embryonic ¢sh muscle growth in response to
changes in water temperature are found The present
results in white muscle are in agreement with those
obtained in cyprinids, where higher pre-hatch
tem-peratures were found to have no signi¢cant e¡ect on
the ¢bre number and ¢bre size in common carp
(Cy-prinus carpio Linnaeus) at hatching (Alami-Durante,
Bergot, Rouel & Goldspink 2000) However, this
sce-nario is not universal in ¢sh and seems to depend on
the species Thus, in Atlantic salmon (Stickland et al
1988; Usher et al.1994; Nathanailides et al.1995),
com-mon white¢sh (Coregonus lavaretus, Linnaeus)
(Ha-nel, Karjalainen & Wiser 1996) and Arctic cod
(Gadus morhua, Linnaeus) (Galloway, Korsvik & Kryvi
1998), a higher phatch temperature was not
re-sponsible for modifying white muscle area but
de-creases in the ¢bre number and inde-creases in the ¢bre
size at hatching or 1day later were reported In turbot
(Scophthalmus maximus, Linnaeus) (Gibson &
John-ston 1995) and in sea bass (Alami-Durante et al
2006), it was observed that incubation at higher
tem-peratures increased white muscle size and white ¢bre
size at hatching, but the number of white ¢bres was
not altered In Atlantic halibut (Hippoglossus
hippo-glossus, Linnaeus), white muscle size, ¢bre number
and ¢bre size at hatching were reduced by higher
in-cubation temperatures (Galloway et al 1999) Our
re-sults further stress the complex nature of muscle
growth and development in ¢sh, and highlight the
species-speci¢c response of myogenesis to changes
in incubation temperature
At mouth opening, the total number of white ¢bres
was greater at the post-opercular level of larvae reared
at 18 1C The present results are in accordance with
those from herring, where ¢bre hyperplasia in the
anterior part of the larvae was augmented due to
war-mer temperatures (Vieira & Johnston 1992; Johnston
et al 1995) At the end of the endogenous feeding
peri-od, the total red muscle area at the post-anal level was
larger in larvae at 18 1C This signi¢cant outcome
re-sulted from the a(red muscle ¢bre), because a cally signi¢cant temperature e¡ect on this parameterwas observed when the ¢sh length was considered to
statisti-be a covariate Recent studies found that early perature produced early e¡ects on somatic bothgrowth and muscle ¢bre phenotype that persisted un-til adult life (Lo¤pez-Albors, Abdel, Periago, Ayala, AlcaŁ-zar, GraciaŁ, Nathanailides & VaŁzquez 2008; Macqueen,Robb, Olsen, Melstveit, Paxton & Johnston 2008) Webelieve that the temperature regime before ¢rst feed-ing a¡ected the number of undi¡erentiated myoblastsand hence the future growth potential If so, then itmight be possible to manipulate temperature duringembryonic development, to optimize the ¢nal muscle
tem-¢bre number and induce growth rate improvements.Comparing the results obtained in both body loca-tions in our experiment, the total number of white ¢-bres was always higher at the post-anal level in thelarvae from the 14 1C group Simultaneously, at ¢rstfeeding, the white ¢bres tended to have a smallermean cross-sectional area at the post-anal level Also,
at both sampling time points, and for both tures, the total number of red ¢bres was larger at thepost-anal level, supporting what was stated aboveabout the post-anal zone being most likely the mainlocation for the red ¢bre hyperplasia during larvalgrowth Our experiment emphasizes the importance
tempera-of looking at di¡erent body locations when studying
¢sh muscle growth If we had investigated only thepost-anal zone, an incorrect conclusion would havebeen made, i.e., that temperature had no e¡ect onthe white muscle ¢bre number
In conclusion, this study showed that the ment rate of blackspot seabream was accelerated by ahigher temperature (18 1C) Also, the larval musclegrowth dynamics was a¡ected by the temperaturevia the production of new white muscle ¢bres (hyper-plasia) at the post-opercular level and on the size ofthe red ¢bre and, consequently, on the size of the totalred muscle area at the post-anal level It was also de-monstrated that the axial musculature of larvae dis-played a non-similar response to the di¡erent earlytemperatures between the post-anal and the post-opercular levels Further investigation is required todetermine whether these di¡erent responses areparticular for that species or body size
develop-AcknowledgmentsThis work was partially supported by FCT PhD GrantSFRH-BD-14068-2003 awarded to P Silva and FCTTemperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340
Trang 28pluriannual funding awarded to CIIMAR-CIMAR
LA The authors are greatly indebted to the ‘Instituto
Espanol de Oceanograf|¤a’ (Centro OceanograŁ¢co de
Vigo, Espana), which provided the ¢sh Handling and
care of animals were conducted according to the EU
guiding principles for animal research (86/609/EU)
and the Portuguese law (Decreto Lei No 192/92 and
197/96, regulated by Portaria No.1005/92, No 466/95
and No 1131/97) The experiment was part of a
pro-ject approved by the Portuguese Foundation for
Science and Technology and also approved by the
Scienti¢c Council of the ICBAS ^ Univ Porto (which
is advised by an o⁄cial Ethics Committee)
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Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340
Trang 30The use of biochemical, sensorial and chromaticity attributes as indicators of postmortem changes in
stored on ice
George Vardanis1,2, Liliana S¢chi-Duke1, Lluis Tort3, Pascal Divanach2, Kiriakos Kotzabasis1&Michail Pavlidis1
1 Department of Biology, University of Crete, Heraklion, Crete, Greece
2 Hellenic Centre for Marine Research, Institute of Aquaculture, Heraklion, Crete, Greece
3 Department of Cell Biology and Physiology, Facultat de Sciencies, Universitat Autonoma de Barcelona, Cerdanyola, Spain
Correspondence: M Pavlidis, Department of Biology, University of Crete, PO Box 2208, GR-714 09 Heraklion, Crete, Greece E-mail: pavlidis@biology.uoc.gr
Abstract
The freshness of red porgy slaughtered in ice slurry
and stored in melting ice was evaluated
instrumen-tally, biochemically and sensorialy Additionally,
postmortem skin colour changes were monitored, in
an attempt to demonstrate the use of chromaticity
parameters as a reliable and convenient approach to
quality assessment Dielectric properties showed a
statistically signi¢cant decrease (Po0.05) from
12 0 on day 0 to 7.85 0.31 on day 7; there was
also a signi¢cant decrease in muscle polyamines
with more than two amino groups (spermidine and
spermine) and an increase in the di-amine
putres-cine The sensorial score was signi¢cantly decreased
from 30 0 to 4.33 0.21 on day 7 Minimal
light-ness (L) and hue (H1ab) at the dorsal skin area were
observed on days 1 and 3 following harvesting
Final-ly, there was a marked decrease in the entire colour
index (ECI ^ a combination of skin hue and chroma)
of both the dorsal and the ventral area in day 3
on-wards to day 7, as well as a statistically signi¢cant
correlation between all the estimated freshness
in-dices and ECI Based on all these, we inferred that
the total polyamines (or putrescine to spermidine
and spermine ratio) and ECI could be reliable
estima-tors of freshness, at least under the experimental
conditions applied
Keywords: colour, freshness, Pagrus pagrus,
polya-mines, skin, red porgy
IntroductionFollowing slaughter, physical and chemical processescaused by enzymes and microorganisms begin to oc-cur, resulting in spoilage and complete decay of the
¢sh (Bonnal, Raynaud, Astier, Lebart, Marcilhac,Coves, Corraze, Gelineau, Fleurence, Roustan & Be-nyamin 2001; Kjaersgard & Jessen 2003; Gramm &Huss 1996) In order to monitor spoilage, and assessend-product quality, which are of utmost importance,various methods have been established Traditionally,these include sensory analysis [e.g quality indexmethod (QIM)], biochemical (e.g total volatile basicamines, dimethylamine, trimethylamine, biogenicamines), microbiological (the number of speci¢c spoi-lage bacteria related to the remaining shelf-life) andphysical methods (changes in the electrical properties
of skin, in the pH of meat or in ¢sh muscle texture)(FAO 1995; Kyrana & Lougovois 2002; Parisi, Franci &Poli 2002; Chytiri, Chouliara, Savvaidis & Kontomi-nas 2004), all of which have certain advantages anddisadvantages Instrumental, non-destructive meth-ods to measure changes in physical properties of skinand tissues, when applicable, are rapid, convenientand more reliable, allowing repetition Therefore, thedevelopment of adequate instrumental methods is ofprimary interest for the aquaculture industry.One of the critical points in maintaining high end-product quality upon its introduction to the market isslaughtering Studies in farmed snapper Pagrus aura-tus (Lowe, Ryder, Carragher & Wells 1993), EuropeanAquaculture Research, 2011, 42, 341^350 doi:10.1111/j.1365-2109.2010.02628.x
r 2010 The Authors
Trang 31sea bass Dicentrarchus labrax (Parisi et al 2002), trout
Oncorhynchus mykiss, carp Cyprinus carpio, eel
Angu-illa anguAngu-illa (Marx, Brunner, Weinzierl, Ho¡man &
Stolle 1997) or Salmo salar (Sigholt, Ericsson, Rustad,
Johansen, Nordtvedt & Seland 1997; Skjervold, Fjaera,
Ostby, Isaksson, Einen & Taylor 2001) associate
har-vesting procedures and slaughter methods, which
trigger a stress response, and storage conditions with
end-product quality Thus, poor pre-slaughter
hand-ling or prolonged storage time may result in an
in-crease in both the microbiological metabolism
and the postmortem muscle degradation process,
causing changes in rigor mortis onset and resolution,
alteration in muscle pH and loss of water, protein
denaturation and lipid oxidation, ¢nally leading to
deterioration in £esh quality
The red porgy P pagrus is a sparid traditionally
consumed in the Mediterranean countries, due to its
distinct £avour traits The aquaculture industry has
shown interest in this species and the number of
scienti¢c references and basic knowledge of this
species has grown considerably (Kentouri, Pavlidis,
Papandroulakis & Divanach1995;
Mihelakakis,Yoshi-matsu & Tsolkas 2001; Mylonas, Papadaki, Pavlidis &
Divanach 2004; Pavlidis, Karkana, Fanouraki &
Pa-pandroulakis 2008), resulting in an increased
pro-duction within the last decade Additionally,
research has focused on the quality traits of the
end-product, by optimizing husbandry and feeding
proto-cols Nevertheless, it has been shown previously that
the red porgy may undergo relevant skin colour
changes when subjected to several stressors (Pavlidis,
Papandroulakis & Divanach 2006; Pavlidis et al
2008) This sensitivity of the species in colour
changes may also be observed postmortem and,
therefore, objective colour measurements may be
used as an indicator of product quality
The aim of the present work was to assess the
potential of monitoring postmortem changes by
measuring changes in skin colour and the muscle
polyamine content, in the red porgy, P pagrus, and
relating these changes to other instrumental
fresh-ness indicators (TorrymeterTMreadings) and sensory
analysis
Material and methods
Experimental set-up
Fifteen red porgies, with a mean body weight
( SEM) 907.2 g ( 63.6 g), were obtained from a
commercial Greek ¢sh farm (Inter¢sh S.A.) Fish were
reared in net cages, at a stocking density of13.8 kg m 3, and fed for 3 months on a commercialdiet (ProAqua, Dieta 1, Duenas, Spain) supplementedwith additional sources of astaxanthin in its esteri-
¢ed form (40 mg astaxanthin kg 1food) Red porgieswere starved before slaughter for 2 days, to ensurecomplete gut emptying and to reduce spoilage organ-ism charge Fish were harvested directly from a com-mercial sea cage according to the standard methodfor sea bass and bream, as described by Smart(2001) In brief, ¢sh were netted, lifted out of the cageand chilled in a few minutes from sea temperature(16 1C) to about 2^3 1C by immersion into an insu-lated plastic grey bin containing a mixture of ice andsea water Fish were then transferred (within 10 min)
to the laboratory installations of the ¢sh farm, wherethe skin colour, dielectric properties and cornealliquid turbidity were measured and a sensorialevaluation was performed Following the initialsampling (day 0), ¢shes were packed ‘belly-up’ into
an insulated polystyrene box provided with holes fordrainage and were covered by a nylon sheet Thebox was ¢lled with £aked ice and air-freighted withinthe same day to the Laboratory of Fish Physiology,University of Crete, and stored in a cold room(3^4 1C) throughout the experiment The ice to ¢shratio was maintained throughout the trial A thinplastic ¢lm covered and separated the ¢sh fromcoming in direct contact with the ice Measurementswere carried out on days 0, 1, 3, 5 and 7 from theharvest
Skin colour measurementsFour measurements per ¢sh were performed: two at thedorsal and two at the ventral left side skin area (Fig 1).Colour measurements were performed using a portablespectrocolorimeter (MiniScanTM XE, Hunter Assoc.Laboratory, Reston, VA, USA) Colour values mea-sured by the instrument are relative to the absolutevalue of a perfect re£ecting di¡user as measured un-der the same geometric conditions The model typeused is based on a large viewing area and 451/01 geo-metry Measurements were taken with reference tothe D65/101 illuminant/standard observer Illumi-nant D65 represents daylight with a correlated col-our temperature of approximately 6500 K The CIE
1976 Labsystem recommended by the tional Commission on Illumination (CIE) was chosen
Interna-as the colour scale Lmeasures lightness and variesfrom 100 for perfect white to 0 for black, whereas aFreshness indicators in red porgy G Vardanis et al Aquaculture Research, 2011, 42, 341–350
Trang 32values indicate redness when positive, grey when 0
and greenness when negative and bvalues show
yellowness when positive, grey when 0 and blueness
when negative; hue and chroma were calculated
ac-cording to the formulae (HunterLab MiniScanTMXE
Dielectric properties
A TorrymeterTMwas used to measure the dielectric
properties in every ¢sh This instrument allows
relat-ing the dielectric properties with tissue freshness, as
the reduction in the dielectric index is related to an
increase in body £uids originating from tissue
dete-rioration One measurement was performed per ¢sh
on the left side of the body, approximately at the level
of the lateral line in the middle of the anteroposterior
body axis The instrument converts the electrical
measurements of cellular activity into a centesimal
reading scale (Parisi et al 2002)
Quantitative and qualitative analyses of
polyamines
Muscle samples were dissected from the right side of
the body of the same six ¢sh in all consecutive
sam-plings Muscle samples were dried in a constant
tem-perature oven set at a temtem-perature of 90 1C for 12 h
Polyamines were extracted as described by S¢chi,
Ioannidis and Kotzabasis (2004) and analysed
fol-lowing the method of Kotzabasis,
Christakis-Hamp-sas and Roubelakis-Angelakis (1993) Brie£y, dried
¢sh samples were homogenized in 1 N NaOH A lume of 0.1mL from the homogenate was mixed with36% HCl at a ratio of 1:1 (v/v) and incubated at 110 1Cfor 18 h The hydrolysate was evaporated at 70^80 1C.The dried products were re-dissolved in 0.2 mL of 5%(v/v) perchloric acid To identify and estimate thepolyamines, the samples were derivatized by benzoy-lation For this purpose, 1mL of 2 N NaOH and 10mLbenzoylchloride were added to 0.2 mL of the hydroly-sate and the mixture was vortexed for 30 s After a20-min incubation at room temperature, 2 mL of sa-turated NaCl solution was added to stop the reaction.The benzoyl-polyamines were extracted three timesinto 2^3 mL diethylether; all ether phases were col-lected and evaporated to dryness The remainingbenzoyl-polyamines were re-dissolved in 0.2 mL of63% (v/v) methanol and 20mL aliquots of this solu-tion were injected into a high-performance liquidchromatography (HPLC) system for polyamine ana-lysis The analyses were performed using a ShimadzuLiquid Chromatography apparatus (LC-10AD, Kyoto,Japan) equipped with an SPD-M10A diode array de-tector (Shimadzu SPD-M10A) and a narrow-bore col-umn (C18, 2.1 200 mm,5 mm particle size Hypersil,Hewlett-Packard, Houston, TX, USA) To estimate theamount of each polyamine directly, the method ofKotzabasis et al (1993) was followed again
vo-Sensorial evaluationSensorial evaluation of freshness was performed fol-lowing a quality index approach, using the EU fresh-ness rating scheme for ¢n¢sh (EEC 1976) The end ofthe experiment was set at the time when ¢sh condi-tion reached the lower margin of consumption suit-
D1
D2
D: Dorsal area V: Ventral area
Figure 1 Sampling points for the measurement of the three-dimensional characteristics of colour appearance (L, a,
b ), using a portable spectrocolorimeter
Aquaculture Research, 2011, 42, 341^350 Freshness indicators in red porgy G Vardanis et al.
r 2010 The Authors
Trang 33ability as proposed by the scheme adopted Six ¢sh
were used randomly and analysis was performed by
a group of three judges The attributes examined
were (Table 1): the external appearance of ¢sh (skin,
eyes, gills, £esh from the abdomen, organs and skin
colour along the vertebral column); the condition of
£esh texture, the spinal cord and peritoneum; and
the odour of the gills, skin and abdominal cavity For
each parameter, a four-point scale, from 3 for freshly
slaughtered ¢sh to 0 for rancid ¢sh, was used
For each ¢sh, these points were summed to produce
a score, which was then used for the statisticalanalysis
Statistical analysisThe estimation of the mean value and standard de-viation for angular hue, being a circular variable,was performed using statistical methods for describ-ing and analysing data from circular distributions(Zar 1996) The Rayleigh test was applied to check
Table 1 Freshness ratings for sensory inspection of fresh, iced ¢sh (EEC 1976)
Skin Bright, iridescent pigmentation, no discoloration Aqueous, transparent, mucus 3
Pigmentation bright but not lustrous Slightly cloudy mucus 2 Pigmentation in the process of becoming discoloured and dull Milky mucus 1 Dull pigmentation Opaque mucus 0 Eyes Convex (bulging) Transparent cornea Black, bright pupil 3
Convex and slightly sunken Slightly opalescent cornea Black, dull pupil 2 Flat convex Opalescent cornea Opaque pupil 1 Concave in the centre Milky cornea Grey pupil 0
Less coloured Slight traces of clear mucus 2 Becoming discoloured Opaque mucus 1 Yellowish Milky mucus 0 Belly Bluish, translucent, smooth, shining No change in the original colour 3
Velvety, waxy, dull Colour slightly changed 2
Organs Kidneys and residues of the other organs should be bright red, as should the
blood inside the aorta.
3 Kidneys and residues of other organs should be dull red; blood becoming discoloured 2 Kidneys and residues of other organs and blood should be pale red 1 Kidneys and residues of other organs and should be brownish in colour 0 Flesh Firm and elastic Smooth surface 3
Peritoneum Sticks completely to flesh 3
Trang 34the uniformity of the circular distribution
Di¡er-ences in the hue variable between sampling points
(time) were tested using the Watson^Williams F-test
or the nonparametric Watson U2-test (when at least
one of the sampled populations was not unimodal)
Within groups of data with no signi¢cant
di¡er-ence in hue, the mean hue was estimated and a novel
variable, termed as the ‘entire colour index’ (ECI)
(Pavlidis et al 2006), was calculated as:
ECIi¼ CicosðHi HmeanÞ;
where Hmeanis the mean hue and (Ci, Hi) the chroma
and hue values of each measurement The variable
ECIiis therefore the projection of each ‘colour vector’
in the mean direction angle of the group and
de-scribes its contribution to the mean colour (Pavlidis
et al 2006) It is a combination of both hue and
chro-ma, and because it is a scalar variable, the classical
statistical approach can be further applied
Statistical di¡erences in skin lightness (L), ECI
va-lues, dielectric properties and muscle polyamine
con-tent were tested using one- or two-way repeated
measures analysis of variance (RM ANOVA) If
signi¢-cant (Po0.05), the Holm^Sidak test was applied to
identify groups that were signi¢cantly di¡erent
Sta-tistical di¡erences in the sensorial evaluation
be-tween the di¡erent sampling days were tested using
one-way analysis of variance (ANOVA) If signi¢cant
(Po0.05),Tukey’s signi¢cant means test was applied
to identify groups that were signi¢cantly di¡erent
Pearson product^moment correlation analysis was
applied to de¢ne the correlation coe⁄cient values of
the estimated parameters
Results
There were no statistically signi¢cant di¡erences in
the skin colour measurements between ¢sh used for
polyamine analysis and the rest of the ¢sh (data
not-shown) In all ¢sh, there was no signi¢cant di¡erence
in the mean values of the chromaticity attributes (L,
H1ab and ECI) between the two sampling points of
the dorsal and the two sampling points of the ventral
skin area, but a signi¢cant dorsoventral gradient
(Po0.05) Along the dorsal skin area of red porgies,
lightness (L) values on days 1 and 3 following
har-vesting were signi¢cantly lower (Po0.05), while
there were no signi¢cant changes in Lof the ventral
skin area for up to 7 days of storage (Fig 2) There was
a signi¢cant decrease (Table 2) in the hue (H1ab)
va-lue of the dorsal body area in days 1 and 3, followed
by an increase in days 5 and 7 to values similar to
those of the initial sampling (day 0), whereas alongthe ventral skin area, no signi¢cant e¡ect of time sto-rage was found on the hue values (Table 2) There was
a marked decrease (Po0.05) in ECI of both the dorsaland the ventral area on day 3 onwards to day 7 (Fig.3)
In the dorsal skin area, ECI decreased from an initialvalue of 11.00 1.03 to 6.85 0.61 on day 5 and to6.03 0.49 on day 7 In the ventral skin area, ECI de-creased from an initial value of 15.01 1.78 to10.65 0.95 on day 5 and to 7.56 0.83 on day 7.Statistically signi¢cant di¡erences (Po0.05) from the
Ventral Area
45.00 50.00 55.00 60.00 65.00
Time (DPH)
b b
b
Figure 2 Postmortem changes in skin lightness (L) ofthe dorsal and the ventral body area of red porgies slaugh-tered by hypothermia and stored on ice for a period of 7days post-harvest (DPH) Each point represents the meanvalue of 15 measurements and vertical bars represent thestandard error of means Means with di¡erent letters dif-fer signi¢cantly from one another (Po0.05) Note that y-axes are in a di¡erent scale
Table 2 Post-mortem changes in skin hue (H1ab) of the sal and ventral body area of red porgies slaughtered by hy- pothermia and stored on ice for a period of 7 days post- harvest (days 0^7)
dor-Days Dorsal H1ab Ventral H1ab
Aquaculture Research, 2011, 42, 341^350 Freshness indicators in red porgy G Vardanis et al.
r 2010 The Authors
Trang 35initial values were also observed from day 3 onwards
in all the freshness quality indicators used
Dielectric properties (TorrymeterTMreadings)
ran-ged from an initial value (day 0) of 12 0 to a
mini-mum of 7.85 0.31 on day 7 (Fig 4)
There was a clear postmortem pattern of changes
in the polyamines determined, with a tendency
to-wards a signi¢cant increase in the catabolism of
poly-amines with more than two amino groups (Fig 5) In
particular, a signi¢cant decrease in the tri-amine
spermidine (Spd) and tetra-amine spermine (Spm)
amount in muscle was observed from day 1 (Spd:
380.8 32.2 nmol mg 1dry weight; Spm: 339.834.6 nmol mg 1dry weight) to day 7 (Spd: 212.02.3 nmol mg 1dry weight; Spm: 107.2 37.6 nmol
mg 1dry weight) postmortem In contrast, the cle content of the di-amine putrescine (Put) showed asigni¢cant increase from day 1 (11.3 1.9 nmol
mus-mg 1dry weight) to day 7 (26.8 4.2 nmol mg 1dry weight) postmortem The increase in the Putcontent was more evident on days 5 and 7, wherethe ratio Put/(Spd1Spm) showed the highest value(Fig 5)
The sensorial analysis score showed a signi¢cantdecrease (Po0.05) in the score from an initialvalue of 30.0 0 on day 0 to 4.33 0.21 on day 7(Fig 6), thus reinforcing the assessment of the othermethods
There was no signi¢cant correlation between theestimated freshness indicators and skin lightness.However, there were statistically signi¢cant correla-tions between the dielectric properties and the sen-sorial analysis score and the ECI (Table 3) In boththe dorsal and the ventral skin area, ECI showed sig-ni¢cant positive correlations with the dielectric prop-erties and the sensorial analysis In addition, therewas a signi¢cant correlation between the dielectricproperties, sensorial analysis, polyamines and ECI ofthe ventral skin area (Table 3)
Discussion
A single, ¢rm and reliable indicator to evaluate mortem quality and freshness in ¢sh is not yet avail-able Sensorial evaluation is based on the analysis ofappearance, odour, £avour and texture using the hu-man senses Sensorial methods are based either ondiscriminative (is there a di¡erence?) or descriptivetests (what is the di¡erence or the absolute valueand how large is it?) (FAO 1995) Nowadays, the QIM,originally developed by Bremner, Olley and Vail(1987), is the most commonly used sensorial methodfor quality assessment In the present work, a sensor-ial evaluation was performed following the qualityindex approach Although subjective, this fast meth-
post-od could be used for preliminary quality tests chemical and chemical methods are able to setquantitative standards and may be used in resolvingissues regarding products of marginal quality Unfor-tunately, these methods are laborious, expensive, in-
Bio-£uenced by processing and not always correlatedwith sensory quality evaluations and the level of mi-crobial spoilage or autolysis (FAO 1995) Microbiologi-
Figure 3 Postmortem changes in the entire colour index
(ECI) of the dorsal and ventral skin area of red porgies
slaughtered by hypothermia and stored on ice for a period
of 7 days post-harvest (DPH) Each point represents the
mean value of 15 measurements, and vertical bars
repre-sent the standard error of means Means with di¡erent
let-ters di¡er signi¢cantly from one another (Po0.05) Note
that the y-axes are on a di¡erent scale
a a
Figure 4 Postmortem changes in the dielectric
proper-ties (DP) of red porgies slaughtered by hypothermia and
stored on ice for a period of 7 days post-harvest (DPH)
Each point represents the mean value of six
measure-ments, and vertical bars represent the standard error of
means Means with di¡erent letters di¡er signi¢cantly
from one another (Po0.05)
Freshness indicators in red porgy G Vardanis et al Aquaculture Research, 2011, 42, 341–350
Trang 36cal methods are also laborious and time-consuming,
but they can be used to detect the presence of
spoi-lage bacteria, which cause degradation, and
organ-isms that present risk to the public health However,
these methods are applied much more in assessing
hygiene, safety and conformance with standards
than in estimating the eating quality
Various studies on farmed sea bream (Sparus
aura-ta) (Huidobro, Pastor & Tejada 2000; Huidobro,
Pas-tor, Lopez-Caballero & Tejada 2001; Alasalvar,Taylor
& Shahidi 2002) and sea bass (D labrax) (Poli, Parisi,
Zampacavallo, Mecatti, Lupi, Gualtieri & Franci 2001;
Kyrana & Lougovois 2002; Parisi et al 2002) indicate
the subjective perception of skin colour as being an
important attribute for the sensory perception of
quality and freshness Our results suggested that theECI re£ected better the e¡ect of storage time on theskin colour of red porgies A comparison of the H1abvalues failed to con¢rm any quality changes Instead,ECI showed a statistically signi¢cant decrease(Po0.05) from day 3 onwards, in both the dorsaland the ventral areas It can be inferred that ECI is amore sensitive index for the estimation of postmor-tem skin colour changes related to storage time, be-cause it combines two chromaticity attributes,speci¢cally hue and chroma (saturation) Thesechanges in skin colour could be induced by the loss
of water content due to leakage or protein tion (Robb, Kestin & Warriss 2000) or rapid chroma-tosome movements (dispersion and aggregation) inchromatophores, which is a key factor in determin-ing skin colour in some ¢sh species Earlier works in
degrada-P major showed that arti¢cially induced aggregation
of melanosomes can prevent skin darkening mortem In this context, the L value in the dorsal areauntil day 3 might re£ect a reduction in melanosomeaggregation associated with skin darkening (Lin,Ushio, Ohshima, Yamanaka & Koizumi 1998) Also,changes in the refractive indices in post and pre rigormuscle might be responsible for the di¡erences in col-our, as it is demonstrated for salmon ¢llets (Skjervold
post-et al 2001) The strong correlation found bpost-etween ECIand the other indices used in this study suggestedthat ECI could be used to determine ¢sh freshness
At least in red porgy and for similar work conditions,this index could estimate the overall quality, usuallygiven by other single accepted indicators and also by
an array of them
0.00 10.00 20.00 30.00
0.00 0.05 0.10 0.15
Time (DPH)
c b a a
0.00 200.00 400.00 600.00 800.00
0.00 100.00 200.00 300.00 400.00 500.00
a
Figure 5 Postmortem changes in the polyamine pattern (Put, putrescine; Spd, spermidine; Spm, spermine) in muscle ofred porgies slaughtered by hypothermia and stored on ice for a period of 7 days post-harvest (DPH) Each point representsthe mean value of six measurements, and vertical bars represent the standard error of means Means with di¡erent lettersdi¡er signi¢cantly from one another (Po0.05) Note that the y-axes are on a di¡erent scale
Figure 6 Postmortem changes in the sensorial score of
red porgies slaughtered by hypothermia and stored on ice
for a period of 7 days post-harvest (DPH) Each point
repre-sents the mean value of six measurements, and vertical
bars represent the standard error of means Means with
di¡erent letters di¡er signi¢cantly from one another
(Po0.05)
Aquaculture Research, 2011, 42, 341^350 Freshness indicators in red porgy G Vardanis et al.
r 2010 The Authors
Trang 37Postmortem modi¢cations in the skin colour of
in-tensively reared red porgy stored on ice were
re-corded from day 3 onwards In parallel, there were
changes in other quality indicators such as dielectric
properties and sensorial analysis Also, these
altera-tions correlated to a redistribution of polyamines
to-wards an increase in the Put content and a decrease
in the Spd and Spm content In general, it is
consid-ered that putrescine (Put) is a precursor of spermine
(Spd) and spermidine (Spm), which would suggest the
level of Spd and Spm to follow the level of Put
How-ever, our data show an opposite pattern that could
have resulted from the reverse conversions of
sper-mine to spermidine and spermidine to putrescine in
reactions catalysed by diamine oxidase and
polya-mine oxidase (DAO and PAO), on the one hand, and
de novo putrescine production, on the other It is
known that ¢sh muscle has the ability to support
the bacterial formation of a wide variety of amine
compounds that result from the direct
decarboxyla-tion of amino acids (FAO 1995) Histamine, putrescine
and cadaverine are produced from the
decarboxyla-tion of histidine, ornithine and lysine respectively
Several studies showed that Put accumulation
dur-ing ice storage of ¢sh indicates microbial spoilage
(Koutsoumanis, Lampropoulou & Nychas 1999;
Pons-SaŁnchez-Cascado, Veciana-Nogue¤s, Bover-Cid,
Marine¤-Font & Vidal-Carou 2006) and suggested that
this polyamine, in association with other biogenic
amines, such as cadaverine and histamine, can be
used as an index of freshness Mietz and Karmas
(1977) proposed a chemical quality index (expressed
as the ratio of histamine1putrescine1cadaverine to
spermidine1spermine concentrations) based on
bio-genic amines that re£ected the quality loss in canned
tuna A high quality index ratio indicates that the
sensorial score of the canned product decreased
With respect to the above ¢ndings, our results dicated that the Put/Spd1Spm ratio could also pro-vide an estimate of red porgy muscle freshness.There was a signi¢cant negative correlation betweenthe ECI of the ventral skin area and the Put/Spd1Spmratio The same tendency was observed between ECI
in-of the dorsal skin area and the Put/Spd1Spm ratio,but with no statistically signi¢cant correlation Stu-dies on plants have shown that treatment with pu-trescine can delay colour changes in associationwith changes in the levels of abscisic acid (Valero,Mart|¤nez-Romero, Serrano & Riquelme 1998) How-ever, there are no data on any direct or indirect e¡ect
of polyamines on ¢sh skin colour The signi¢cant relation of the ECI with commonly used freshness in-dicators, on the one hand, and polyamine pattern, onthe other, showed that this index is sensitive enough
cor-to be used as an indicacor-tor for quality assessment TheECI could be more e⁄cient than other instrumentalmethods, because it is simple and reliable Neverthe-less, further research is still necessary in order tostandardize the use of this index for other reared ¢n-
¢sh species of commercial interest for the nean aquaculture, because postmortem changes arespecies dependent
Mediterra-Acknowledgments
We would like to thank the reviewers for their able comments The study has been carried out, inpart, with ¢nancial support from the Commission ofthe European Communities, DG Fisheries, QLRT pro-gramme, QLK53 2000-031629, ‘Environmental,nutritional and neuroendocrine regulation of skincoloration in the red porgy (Pagrus pagrus),towards the development of natural hue in cultured
valu-Table 3 Correlation coe⁄cient values, as de¢ned by Pearson’s product^moment correlation analysis (Po0.01), of freshness indexes and skin colour parameters in red porgy slaughtered by hypothermia and stored on ice for a period of 7 days post- harvesting
DP SSA TP Put/Spd1Spm L dorsal L ventral ECI dorsal ECI ventral
DP 0.908 0.770 0.771 NS NS 0.543 0.786 SSA 0.841 0.725 NS NS 0.628 0.813
Trang 38populations’ It does not necessarily re£ect its views
and in no way anticipates the Commission’s future
policy in this area Sincere thanks are due to Mr H
Bantavas (Inter¢sh SA) for providing ¢sh
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Trang 40Effects of partial and complete replacement of
soybean meal with cottonseed meal on growth, feed utilization and haematological indexes for mono-sex
Deyab M S D El-Saidy1& Amal S Saad2
1 Department of Poultry Production, Faculty of Agriculture, University of Minu¢ya, Shebin El-Kom, Egypt
2 National Institute of Oceanography and Fisheries, Fish Research Station, El-Qanater El-Khayria, Egypt
Correspondence: D M S D El-Saidy, Department of Poultry Production, Faculty of Agriculture, University of Minu¢ya, Shebin El-Kom, P.O Box 32516, Egypt E-mail: deyabelsaidy@yahoo.com
Abstract
The main objective of this study was to evaluate the
e¡ects of replacing soybean meal (SBM) with
cotton-seed meal (CSM) on the growth performance, feed
utilization and haematological parameters of
mono-sex male Nile tilapia ¢ngerlings Five isonitrogenous
diets (containing 31.82% crude protein) containing
graded levels of CSM to replace SBM protein were fed
to triplicate groups of ¢sh The diets were
supplemen-ted with lysine so that they were similar to the
con-trol diet After a 14-week feeding experiment, the
results revealed that up to 75% of SBM could be
re-placed by CSM without causing a signi¢cant
reduc-tion in growth Fish fed the diet highest in CSM had
a signi¢cantly lower protein e⁄ciency ratio and a
sig-ni¢cantly higher feed conversion ratio than ¢sh fed
the other diets High survival was observed in all the
dietary treatments, and no signi¢cant di¡erence was
observed among treatments The apparent
digestibil-ity coe⁄cients (ADC) of dry matter and phosphorus
decreased signi¢cantly with an increase in the
diet-ary CSM level, whereas the ADC of lipid was not
af-fected by the dietary treatment The hepatosomatic
index and the condition factor were signi¢cantly
af-fected by the replacement of SBM by CSM No
signi¢-cant di¡erences were detected in the moisture, lipid
and ash content in whole body and muscle samples,
but protein in whole-body samples was signi¢cantly
a¡ected by the CSM levels Signi¢cant di¡erences
were found in the haemoglobin, haematocrit, red
blood cell and white blood cell contents in ¢shfed diets with di¡erent CSM levels Therefore, these
¢ndings suggest that up to 41.25% CSM can be used
to replace 75% of SBM protein in diets for sex male Nile tilapia ¢ngerlings without any adversee¡ects on the growth performance, feed utilization,body composition and haematological indexes.Keywords: cottonseed meal, soybean meal, growth,haematology index, mono-sex male Nile tilapia
mono-IntroductionThe rapid expansion of the livestock industry inmany parts of the world is resulting in the rapid use
of almost all feedstu¡s produced, thus increasing thecost of certain feedstu¡s used in aquaculture feeds(FAO 2007) Soybean meal (SBM) is currently themost commonly used plant protein source in ¢shfeeds and comprises 50% of the diet of freshwateromnivorous ¢sh species (Yue & Zhou 2008) Soybeanmeal is one of the most nutritious of all plant proteinsources (Lovell 1988) Soybeans are the leading oil-seed crop produced globally, and its production for2004^2005 is expected to exceed 200 mmt (GatlinIII, Barrows, Brown, Dabrowski, Gaylord, Hardy, Her-man, Hu, Krogdahl, Nelson, Ovrturf, Rust, Sealey,Skonberg & Souza 2007) Because of its high proteincontent, high digestibility, relatively well-balancedAquaculture Research, 2011, 42, 351^359 doi:10.1111/j.1365-2109.2010.02629.x
r 2010 The Authors