Aquaculture research, tập 42, số 3, 2011

Aquaculture Research, 2011, 42, 313^321 doi:10.1111/j.1365-2109.2010.02622.x Growth, feed utilization and liver histology of juvenile common sole (Solea solea L.) fed isoenergetic diets with increasing protein levels Pier Paolo Gatta1, Luca Parma1, Ilaria Guarniero2, Luciana Mandrioli2, Rubina Sirri2, Ramon Fontanillas3 & Alessio Bonaldo1 Dipartimento di MorfoÂsiologiaVeterinaria e Produzioni Animali, Bologna, Italy Dipartimento di Sanitaỉ PubblicaVeterinaria e Patologia Animale, Bologna, Italy Skretting Aquaculture Research Centre, Stavanger, Norway Correspondence: A Bonaldo, Dipartimento di MorfoÂsiologia Veterinaria e Produzioni Animali,Via Tolara di Sopra 50, 40064 Bologna E-mail: alessio.bonaldo@unibo.it Abstract Introduction This study was undertaken to determine the inÊuence of dietary protein levels on growth, feed utilization and liver histology in common sole Four isoenergetic diets were formulated to contain four diĂerent crude protein levels: 39 (P39), 45 (P45), 51 (P51) and 57 (P57) % dry weight Fifty animals per tank (initial weight 10.2 ặ 0.4 g) were randomly distributed into twelve 500 litre square tanks (bottom surface: 5600 cm2) connected to a closed recirculation system The diets were tested in triplicate for 84 days At the end of the experiment, the Ânal weight ranged from 19.6 (P39) to 25.4 g (P57) The speciÂc growth rate showed statistical diĂerences between groups, with the best results in the group fed diet P57 (1.07% day 1) SigniÂcant diĂerences 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 eciency was also signiÂcantly aĂected by the dietary treatment, with the highest value (42.07%) found in P57 Ammonia excretion, expressed as g100 g protein intake, was signiÂcantly 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 Salmonids, gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) play a lead role in European Âsh production; however, other farmed Âsh species are required to diĂerentiate and widen the market supply From emerging candidate species, Senegalese sole (Solea senegalensis, Kaup 1858) and common sole (Solea solea L.) show promise, as stated formerly by Howell (1997) and more recently by Imsland, Foss, Conceicaỡo, Dinis Delbare Schram Kamstra Rema and White (2003) Hatchery production of Solea spp has been accomplished quite easily, and this has been a common bottleneck in the commercial production of other potential marine Âsh species However, it is during the juvenile stages that several factors combine to reduce the growth performance and thereby reduce the potential for commercial farming activities These factors include feeding behaviour, susceptibility to disease and stocking density (Day, Howell & Jones 1997; Imsland et al 2003; Schram, Van der Heul, Kamstra & Verdegem 2006; Piccolo, Marono, Bovera, Tudisco Caricato & Nizza 2008; Sanchez, Ambrosio & Flos 2010) Focusing on the nutrition and feeding of common sole juveniles and ongrowing, which seem to be particularly critical points, some signiÂcant steps forward have recently been achieved for S senegalensis (Rema, Conceicaỡo, Evers, Castro-Cunha Dinis & Dias 2008; Borges, Oliveira, Casal, Dias Conceicaỡo & Valente 2009; Rubio, Boluda Navarro, Madrid & Sanchez-Vazquez 2009), establishing optimal protein and lipid Keywords: common sole, growth, protein utilization, lipid eciency, liver histology r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd 313 Dietary protein levels and Solea solea performance P P Gatta et al levels for growth Other recent Ândings on juveniles of S senegalensis (Silva, Espe, Conceicaỡo, Dias & Valente 2009) and Solea aegyptiaca (Chabanaud 1927) (Bonaldo, Roem, Pecchini, Grilli & Gatta 2006) demonstrated the ability of Solea spp to grow equally well when fed diets containing vegetable proteins in the partial substitution of Âshmeal Less information is currently available for common sole (S solea) nutrition (Piccolo et al 2008) and it is inappropriate to rely on data from Senegalese sole, given diĂerences 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 recognized as key factors inÊuencing both adequate Âsh nutrition and feeding costs (Watanabe 2002), and considering the lack of speciÂc knowledge on S solea, the Ârst aim of this research was to assess growth response and feed utilization on feeding common sole juveniles isoenergetic diets with diĂerent protein levels Furthermore, considering the importance of the liver as an indicator of the nutritional and physiological status of Âsh (Bell, Tocher, MacDonald & Sargent 1995; Robaina, Moyano, Izquierdo, Socorro, Vergara & Montero 1997; Caballero, LoÔpez-Calero, Socorro, Roo, Izquierdo & FeÔrnandez 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 dry matter (DM)] were formulated to contain diĂerent protein concentrations: 39 (P39), 45 (P45), 51 (P51) and 57 (P57) % DM The energy level was chosen based on those used in previous trials on juvenile Senegalese sole (Dias, Rueda-Jasso, Panserat, Conceicaỡo, Gomes & Dinis 2004; Rema et al 2008) Protein sources of the diets were represented by Âshmeal 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 amino acid ratio constant among the diets In the absence of speciÂc data on the vitamin, mineral and trace mineral requirements of common sole, requirement data for other species were applied (National Research Council 1993) The ingredients and the proximate composition are given in Table Pellets of mm were produced using an experimental extru- 314 Aquaculture Research, 2011, 42, 313^321 Table Ingredients and proximate composition of the experimental diets Diet P39 P45 P51 P57 Dietary ingredients (%) Fishmeal LT Soybean protein concentrate Wheat Fish oil Wheat gluten meal Vitamin and mineral premix Proximate analysis Dry matter (DM) (%) Crude protein (% DM) Crude fat (% DM) Ash (% DM) Gross energy (MJ kg 1) Crude protein/gross energy (kg MJ 1) 26.7 10.7 31.1 19.0 10.7 1.8 32.0 12.8 24.5 16.2 12.8 1.8 34.7 13.9 21.0 14.7 13.9 1.8 40.0 16.0 14.4 11.9 15.9 1.8 91.8 39.0 23.3 6.1 23.5 16.6 93.2 45.2 22.8 6.4 23.5 19.2 92.7 51.0 20.4 7.0 23.8 21.4 91.4 56.6 18.1 7.6 23.0 24.6 Skretting standard vitamin and mineral premix der by the Skretting Aquaculture Research Center, Stavanger, Norway Pellets were crumbled and sieved to obtain suitable particle sizes Fish, experimental set-up and sampling The experiment was carried out at the Laboratory of Aquaculture, Faculty of Veterinary Medicine, Cesenatico, Italy The common sole (S solea) juveniles with an initial average weight 10.2 ặ 0.4 g were obtained from the hatchery Solea B.V, IJmuiden, the Netherlands Before the experiment, the Âsh were acclimatized for weeks to the experimental facilities and fed commercial Âshmeal-based diets (Skretting,Verona, Italy; crude protein 55%, crude fat 18%) At the start of the trial, 50 Âsh were randomly distributed into each of twelve 500 litre square tanks (bottom surface: 5600 cm2) Each diet was fed to triplicate tanks for 84 days Tanks were provided with natural seawater and connected to a unique closed recirculation system consisting of a mechanical sand Âlter, an ultraviolet light and a bioÂlter The water exchange rate per tank was 100% every h The overall water renewal of the system was 5% daily Temperature was maintained constant at 20 ặ 1C throughout the experiment; the photoperiod was held constant at a 12-h day length through artiÂcial light (200 lx at the water surface ^ Delta Ohm luxmeter HD-9221, Delta-Ohm, Padua, Italy).Water temperature and dissolved 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- r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 Aquaculture Research, 2011, 42, 313^321 Dietary protein levels and Solea solea performance P P Gatta et al trate (NO3 50 mg L 1) were determined spectrophotometrically 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 determined Fish were hand-fed twice daily (at 9.00 a.m and 5.00 p.m.) at a Âxed rate of 1.3% body weight day 1, days week According to the appetite of Âsh registered in previous trials conducted in our facilities (unpubl data), this ratio was considered to be close to satiation The feeding levels were recalculated daily for each tank according to the feed conversion ratio (FCR) and speciÂc growth rate (SGR) obtained at each intermediate weighing Feed losses were minimal throughout the trial but, when necessary, 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 Âsh 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 former case, Âve pooled samples of Âve Âsh each were sampled to determine the initial proximate composition, while in the latter case, one pooled sample of Âve Âsh from each tank was collected to determine the Ânal proximate composition Furthermore, at the end of the trial, wet weight, viscera and liver weight were individually recorded from Âve Âsh per tank for the determination of visceral and hepatosomatic indices At this time, two Âsh 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 measured in all tanks during a 24-h cycle, integrating repeated collected values according to the following formula given by Kaushik (1980): Et ẳ V DC ỵ Ct DW where V0 is the volume of water in the tank, DC the diĂerence in total nitrogen ammonia (Ci Ci t), Ct the mean of the total nitrogen ammonia concentration between two consecutive intervals (Ci1Ci t/2), DW the Êow rate/unit of time t, t the unit of increment in time in which concentration variation is considered to be minimal and Et the total nitrogen ammonia excreted by Âsh per unit of time retained The water inÊow was held constant at 250 L h 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 Ârst meal A tank without Âsh was used as a blank Samples were immediately stored at 32 1C until analysis The ammonia concentration in the samples was measured using the indophenol method (KoroleĂ 1983), and the overall data were expressed as g of total nitrogen ammonia per 100 g protein intake All experimental procedures were evaluated and approved by the Ethical-scientiÂc Committee for Animal Experimentation of the University of Bologna, in accordance with the European Community Council directive (86/609/ECC) Analyses of diets and body composition The experimental diets and carcasses were analysed for DM (drying to a constant weight in a stove at 105 1C), crude protein (N 6.25, determined using the Kjeldahl method), fat (Folch, Lees & Sloane Stanley 1957) and ash content (incineration to a constant weight in a muƠe oven at 450 1C) Calculations The formulae used were calculated as follows: SGR (% day 1) 100 (ln FBW ln IBW)/days, where FBWand IBW represent the Ânal and the initial weights (tank means) respectively FCR feed given/ weight gain Condition factor (CF) 100 (body weight/total length3) Viscerosomatic index (VSI) (%) 100 (viscera weight/body weight) Hepatosomatic index (HSI) (%) 100 (liver weight/body weight) Protein eciency ratio (PER) body weight gain/protein intake Gross protein eciency (GPE) (%) 100 [(% Ânal body protein Ânal body weight) (% initial body protein initial body weight)]/total protein intake Âsh Gross lipid eciency (GLE) (%) 100 [(% Ânal body lipid Ânal body weight) (% initial body lipid initial body weight)]/total lipid intake Âsh Liver histology At the end of the trial, two Âsh per tank were sampled for liver histology Samples were Âxed in 10% buĂered formalin, dehydrated in a graded ethanol series and embedded in paran Section series of mm were stained with haematoxylin and eosin (H&E) From each Âsh, a sample of liver was snap-frozen in liquid nitrogen Quenching was accomplished by placing a small amount of embedding medium for frozen tissue specimens (OCT) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 315 Dietary protein levels and Solea solea performance P P Gatta et al (Tissue-Tek, Sakura Ânetek, 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 mm 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 Âelds were selected; according to the grade of severity and the distribution of the fatty inÂltration within the cells (hepatic steatosis), and through the histological section (from multifocal to diĂuse distribution), cases were classiÂed as mildly aĂected (cytoplasmic Âlling of a clear, optically empty content forming microvesicular spaces), moderately aĂected (cytoplasmic Âlling of a clear, optically empty content pulling the nucleus at the cell periphery and forming macrovesicular spaces) or severely aĂected (cytoplasmic Âlling of a clear, optically empty content, conferring an appearance of signet-ring cells) If these changes were not present, the livers were considered to be normal Statistical analyses The performance response and the proximate composition of Âsh fed the experimental diets were analysed using nested ANOVA and the Newman^Keuls post hoc test The presence of morphological changes Aquaculture Research, 2011, 42, 313^321 in the distal intestinal structure of diĂerent groups of Âsh at the end of the trials was compared using the Kendall t rank correlation coecient All statistical analyses were performed using SAS computer software (SAS 2004) Tank was used as the experimental unit for analyzing the growth performance and ammonia excretion; a pool of Âve sampled Âsh was considered to be the experimental unit for analysing carcass composition, whereas individual Âsh was used for analysing CF,VSI and HSI and morphological changes SigniÂcant diĂerences were assumed when P 0.05 Results All Âsh readily accepted the experimental diets and feed intake was determined directly by the feeding regime The overall mean mortality in terms of the number of Âsh was o2% and no signiÂcant diĂerences were recorded between groups (P 0.9707) Growth and feed utilization are shown in Table The Ânal weight, weight gain and SGR in Âsh fed diet P57 were signiÂcantly higher than those found in the other groups Similarly, Âsh fed diet P57 had signiÂcantly lower FCR in comparison with the other Âsh groups Fish fed diets P39 and P45 showed similar results, while the group fed diet P51had intermediate values, with statistical diĂerences for weight gain, SGR and FCR Data on nutrient retention eciency and ammonia excretion are presented in Table Protein eciency Table Growth, feed utilization and ammonia excretion of sole fed with experimental diets Diet Parameters P39 IBW FBW WG SGR FCR PER GPE GLE Total ammonia nitrogen excretion 10.1 19.6 9.6 0.80 1.31 1.93 35.06 26.23 2.46 P45 ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.3 0.2a 0.4a 0.04a 0.02a 0.10 0.42 2.50a 0.26a 10.1 20.0 9.9 0.81 1.28 1.73 29.68 27.29 3.51 P51 ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.7 1.6a 0.9a 0.02a 0.01a 0.03 5.25 4.60a 0.21ab 10.2 21.6 11.4 0.89 1.12 1.73 30.33 32.19 4.70 P57 ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.3 1.2a 0.9b 0.04b 0.07b 0.09 1.80 3.37ab 0.89b 10.3 25.4 15.1 1.07 0.94 1.86 33.72 42.07 4.75 ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.3 0.3b 0.3c 0.03c 0.02c 0.03 7.82 5.25b 0.23b Data (mean ặ SD, n 3) in the same row with diĂerent superscript letters are signiÂcantly diĂerent (P 0.05) IBW, initial body weight (g); FBW, Ânal body weight (g); WG, weight gain (g) FBW IBW; SGR, speciÂc growth rate (% day 1) 100 (ln FBW ln IBW) days 1; FCR, feed conversion rate feed given/WG; PER, protein eciency ratio body weight gain/protein intake; GPE, gross protein eciency (%) 100 [(% Ânal body protein Ânal body weight) (% initial body protein initial body weight)]/total protein intake Âsh; GLE, gross lipid eciency (%) 100 [(% Ânal body lipid Ânal body weight) (% initial body lipid initial body weight)/total lipid intake Âsh)]; total ammonia nitrogen excretion (g100 g protein intake) 100 [(total ammonia nitrogen (g)/protein intake (100 g)] 316 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 Aquaculture Research, 2011, 42, 313^321 Dietary protein levels and Solea solea performance P P Gatta et al Table Whole body proximate composition and biometric parameters of sole fed with the experimental diets P39 Proximate composition Moisture Protein Lipid Ash Biometric parameters CF VSI HSI 75.35 16.56 6.93 2.83 P45 ặ ặ ặ ặ 0.38 0.35 0.38 0.07 1.04 ặ 0.12 4.88 ặ 0.48 1.07 ặ 0.22 75.20 16.05 6.85 2.48 P51 ặ ặ ặ ặ 0.58 1.58 0.61 0.20 1.05 ặ 0.13 5.04 ặ 0.59 1.06 ặ 0.18 75.56 16.39 6.67 2.17 P57 ặ ặ ặ ặ 0.19 0.78 0.35 0.07 1.04 ặ 0.13 5.31 ặ 0.95 1.11 ặ 0.26 75.23 16.79 6.61 2.39 ặ ặ ặ ặ 0.88 2.40 0.49 0.45 1.07 ặ 0.12 5.42 ặ 0.77 1.19 ặ 0.17 Data are shown as mean ặ SD Moisture, protein, lipid and ash (% wet weight), n 51 pool of Âve Âsh per tank; CF, condition factor 5100 (body weight/total length3), n 50 per tank; VSI, viscerosomatic index (%) 100 (viscera weight/body weight), n 510 per tank; HSI, hepatosomatic index (%) 100 (liver weight/body weight), n 510 per tank ratio and GPE were not statistically diĂerent among groups Gross lipid eciency was higher in Âsh fed diet P57 and signiÂcantly diĂerent from Âsh fed diets P39 and P45 Ammonia excretion was not statistically diĂerent between groups fed diets P45, P51 and P57 and signiÂcant diĂerences were only recorded between group P39 and both P51 and P57 Carcass proximate composition and biometric parameters were very similar between groups, with no signiÂcant diĂerences (Table 3) According to the histology observation, the livers were pink or white, and the rich vascular network, which constitutes a physiological feature of these Âsh, was evident Twelve cases, which did not show intracytoplasmic lipidic droplets in the hepatocytes, both on H&E and Oil red O staining, were classiÂed as normal (Fig.1a^d) In six cases, there was a cytoplasmic Âlling of a clear, optically empty content forming microvesicular spaces (mild hepatic fatty inÂltration); in these cases, Oil red O stain demonstrated small, intracellular orange droplets (Fig.1e and f) In the other six cases, this material, markedly pulling the nucleus at the cell periphery, showed a macrovesicular appearance to the hepatocytes, and showed a multifocal to diĂuse distribution through the histological section (moderate, multifocal to diĂuse hepatic fatty inÂltration) (Fig 1g); Oil red O stain showed larger orange droplets Occasionally, very large extracellular orange droplets were found, due to the rupture of cytoplasmic membranes during freezing and condensation of the lipidic content (Fig 1h) In all cases, a mild, diĂuse cytoplasmic swelling was detected (hepatic hydropic degeneration) All these Ândings were statistically more frequent in Âsh fed diets P51 and P57 than in those fed diets P39 and P45 Discussion While the growth of common sole has been studied for many years (Howell 1997) and many attempts have been made to overcome low growth rates, poor results have been achieved so far Sole have two unusual features that may limit growth potential They have a peculiar gut morphology, which presents a relatively small stomach without pyloric caecae, and a long intestine; furthermore, feeding behaviour in the natural environment occurs almost entirely by means of chemoreception and with a frequent ingestion of small prey items (de Groot1971) In juveniles of Senegalese sole fed isoenergetic diets with Âve diĂerent protein levels (CP 43; 48; 53; 57; and 60), Rema et al (2008) found SGR ranging from 0.93 to 1.22% day with statistical diĂerences related to dietary protein levels In particular, at least 53% CP was necessary to obtain the maximum growth rate In the present trial, SGR ranged from 0.80 to 1.07% day SpeciÂc growth rate was signiÂcantly aĂected by the dietary protein level, with the highest values recorded for Âsh fed diet D57 These Ândings, although the experiment was shorter, are comparable to the growth rate described by Howell (1997) in common sole, indicating that diet P57 is able to sustain good performance of S solea juveniles Concerning feed utilization, the present study demonstrated the inÊuence of dietary protein levels on FCR and GLE but not on PER and GPE A comparison with other data on common sole is quite dicult because of the paucity of sources Piccolo et al (2008) evaluated the performance of 30 g common sole fed diets containing 50% (diet A) and 54% (diet B) crude protein for 300 days The FCR were 2.65 and 2.49 for r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 317 Dietary protein levels and Solea solea performance P P Gatta et al Figure (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 inÂltration Hepatocytes contain a clear, optically empty content forming microvesicular intracytoplasmic spaces (e) Oil red O stain demonstrated small, single, intracellular orange droplets (f), 40 lens (g and h) Hepatic parenchyma from sole fed diet P57 was considered to have moderate hepatic fatty inÂltration Hepatocytes are Âlled with clear, optically empty content that pulls the nucleus at the cell periphery and gives a macrovesicular appearance (g) Oil red O stain detected large orange droplets (h), 40 lens diets A and B, respectively, with a statistical diĂerence between treatments The authors reported the presence of uneaten feed throughout the experimental period and hence those values could not represent the real FCR Rema et al (2008) carried out a trial on Senegalese sole juveniles to deÂne the optimal protein levels with experimental diets containing a crude protein percentage of 43, 48, 53, 57 and 60 They found feed eciency very close to one for the last three groups In the present trial, FCR decreased 318 Aquaculture Research, 2011, 42, 313^321 progressively from 1.31 in Âsh fed the lowest protein level to 0.94 in Âsh fed the highest protein level In the current trial, the highest protein level (diet P57) resulted in the lowest FCR This was lower than that reported previously for Senegalese sole fed optimal dietary protein levels (Rema et al 2008) No data are available on protein utilization for common sole and hence a comparison can only be made with other Âsh species The present study showed no inÊuence of dietary protein level on PER and GPE and this is in accordance with previous Ândings obtained by Rema et al (2008) for juvenile of Senegalese sole fed diets containing diĂerent protein levels ranging from 43% to 60% Cadena-Roa (1983) studied the protein requirements of common sole using semi-puriÂed diets based on casein and gelatin, with protein levels ranging from 24% to 77%, and the best performance was obtained at 57^58% dietary protein This Ânding supports the results of the present trial for Âsh fed diet P57, where SGR and FCR were not only the best among the other groups but also very similar to what was found for Senegalese sole The data shown in the present study cannot clarify whether a higher dietary protein level could improve common sole growth performance; however, it seems reasonable to consider 57% crude protein to be a reference point quite close to the requirements for juvenile common sole In addition to growth results, GLE increased signiÂcantly with increasing dietary protein levels (Table 2) Because higher dietary protein levels were balanced by a proportional lipid and carbohydrate reduction (Table 1) and assuming that higher protein levels led to good nitrogen utilization in this experiment, it is probable that lipid retention is mainly related to the dietary lipid and carbohydrate levels, with a negligible inÊuence of dietary protein levels This hypothesis is in accordance with the results found by Rema et al (2008), where a lower dietary lipid content (between 10% and 13%) resulted in higher lipid retention These data may suggest that a lower dietary lipid content than those used in the present trial is capable of fulÂlling common sole lipid requirements, irrespective of the dietary protein levels, as already stated for Senegalese sole (Rema et al 2008; Borges et al 2009) Whole-body composition (Table 3) was in accordance with the values found for Senegalese sole (Dias et al 2004; Rema et al 2008) Surprisingly, the proximate carcass composition was not aĂected by the dietary treatments in contrast to Rema et al (2008), who found that increased dietary protein levels tended to decrease r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 Aquaculture Research, 2011, 42, 313^321 Dietary protein levels and Solea solea performance P P Gatta et al whole-body fat deposition The carcass proximate composition in the present study is similar to previously reported data for other ÊatÂsh species, such as Atlantic halibut (Aksnes, Hjertnes & Opstvedt 1996) Hepatosomatic index observed in this study, ranging from1.06 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 intracytoplasmic lipidic droplets (steatosis) in Âsh 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,Conceicaỡo, Dias, De Coen, Gomes, Rees, Soares, Dinis & Sorgeloos 2004; Myers & Mc Gavin 2007), or to a deÂciency in lipotropic factors that are able to prevent or remove an excessive accumulation of fat in the liver (Mato, Mart|Ô nez-Chantar & Lu 2008) In this case, the inverse correlation between dietary lipid levels 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 Âsh (Lin, Romsos,Tack & Leveille 1977; Henderson & Sargent, 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 hepatic lipogenesis This is supported by the elevated activity of lipogenic enzymes, i.e FAS, G6PDH, ME and 12 ACC, which have been described previously for a variety of Âsh species fed similar diets, including channel catÂsh (Likimani & Wilson 1982), carp (Shimeno, 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, Lefevre & Medale 2008) Conclusion In conclusion, the dietary protein levels used in this trial demonstrated a considerable inÊuence 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 and 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 Table Number of animals per dietary treatment that displayed of morphological changes in liver (n 6) Diet Normal P39 P45 P51 P57 t P 0.421 0.019 Mild lipidic morphological changes Moderate lipidic morphological changes 0 3 protein/energy ratio requirements of common sole juveniles and the protein level inÊuences on GLE Regarding liver histology, the presence of lipid droplets in hepatocytes suggested a lipogenesis enhancement when the dietary protein levels increase and/or the dietary lipids decrease Finally, the overall performances of common sole juvenile, especially those related to the growth and the FCR of group P57, allow us to conÂrm the good potential of this Âsh species for intensive aquaculture Acknowledgments This research was supported by grants from the Italian Region Emilia-Romagna.We thank Lorenzo Mariani, Marina Silvi and Sara Giuliani for technical assistance, Elettra Pignotti for statistical analyses and Leo Nankervis for English editing The diets were kindly provided by Skretting ARC, Stavanger, Norway References Aksnes A., Hjertnes T & Opstvedt J (1996) EĂect of dietary protein level on growth and carcass composition in Atlantic halibut (Hippoglossus hippoglossus L) Aquaculture 145, 225^233 Arnesen P., Krogdahl & Kristiansen I.ề (1993) Lipogenic enzyme activities in liver of Atlantic salmon (Salmo salar, L) Comparative Biochemistry and Physiology B 105, 541^546 Bell J.G., Tocher D.R., MacDonald F.M & Sargent J.R (1995) EĂects of dietary borage oil [enriched in g-linolenic acid,18: 3(n-6)] or marine Âsh oil [enriched in eicosapentaenoic acid,20: 5(n-3)] on growth, mortalities, liver histopathology and lipid composition of juvenile turbot (Scophthalmus maximus) Fish Physiology and Biochemistry 14, 373^383 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 319 Dietary protein levels and Solea solea performance P P Gatta et al Bonaldo A., Roem A.J., Pecchini A., Grilli E & Gatta P.P (2006) InÊuence of dietary soybean meal levels on growth, feed utilization and gut histology on Egyptian sole (Solea aegyptiaca) juveniles 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A.G (1995) Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis Physiological Review 75, 47^76 Howell B.R (1997) A re-appraisal of the potential of the sole, Solea solea (L.), for commercial cultivation Aquaculture 155, 355^365 Imsland A.K., Foss A., Conceicaỡo L.E.C., Dinis M.T., Delbare D., Schram E., Kamstra A., Rema P & White P (2003) A review of the culture potential of Solea solea and S senegalensis Reviews in Fish Biology and Fisheries 13, 379^408 320 Aquaculture Research, 2011, 42, 313^321 Kaushik S.J (1980) InÊuence of nutritional status on the daily patterns of nitrogen excretion in the carp (Cyprinus Carpio L.) and the rainbow trout (Salmo gairdneri R.) Reproduction Nutrition Development 20, 1751^1765 Kolditz C., Borthaire M., Richard N., Corraze G., Panserat S., Vachot C., Lefevre F & Medale F (2008) Liver and muscle metabolic changes induced by dietary energy content and genetic selection in rainbow trout (Oncorhynchus mykiss) American Journal of Physiology ^ Regulatory, Integrative and Comparative Physiology 294, 1154^1164 KoroleĂ F (1983) Determination of ammonia In: Methods of Seawater Analysis, 2nd edn (eds by R GrasshoĂ, M Ehrhardt & R Kromling), pp 150^162 Verlag Chimic, Weinheim, Germany Likimani T.A & Wilson R.P (1982) EĂects of diet on lipogenic enzyme activities in channel catÂsh hepatic and adipose tissue adipose tissue Journal of Nutrition 112, 112^117 Lin H., Romsos D.R.,Tack P.I & Leveille G.A (1977) InÊuence of diet on in vitro and in vivo rates of fatty acid synthesis in Coho salmon [Oncorhynchus Kisutch (Walbaum)] Journal of Nutrition 107,1677^1682 Mato J.M., Mart|Ô nez-Chantar M.L & Lu S.C (2008) Methionine metabolism and liver disease Annual Review of Nutrition 28, 273^293 Myers R.K & Mc Gavin M.D (2007) Cellular and tissue responses to injury In: Pathologic Basis ofVeterinary Disease, 4th edn (eds by M.D Gavin & J.F Zachary), pp.3^62 MosbyYear Book, St Louis, MO, USA National Research Council (NRC) (1993) Nutrient Requirements of Fish National Academy Press, Washington, DC, USA Palazzi R., Richard J., Bozzato G & Zanella L (2006) Larval and juvenile rearing of common sole (Solea solea L.) in the Northern Adriatic (Italy) Aquaculture 255, 495^506 Piccolo G., Marono S., Bovera F., Tudisco R., Caricato G & Nizza A (2008) EĂect of stocking density and protein/fat ratio of the diet on the growth of Dover sole (Solea solea) Aquaculture Research 39, 1697^1704 Rema P., Conceicaỡo L.E.C., Evers F., Castro-Cunha M., Dinis M.T & Dias J (2008) Optimal dietary protein levels in juvenile Senegalese sole (Solea senegalensis) Aquaculture Nutrition 14, 263^269 Robaina L., Moyano F.J., Izquierdo M.S., Socorro J., Vergara J.M & Montero D (1997) Corn gluten and meat and bone meals as protein sources in diets for gilthead seabream (Sparus aurata): Nutritional and histological implications Aquaculture 157, 347^359 Rubio V.C., Boluda Navarro D., Madrid J.A & Sanchez-Vazquez F.J (2009) Macronutrient self-selection in Solea senegalensis fed macronutrient diets and challenged with dietary protein dilutions Aquaculture 291, 95^100 Rueda-Jasso R., Conceicaỡo L.E.C., Dias J., De CoenW., Gomes E., Rees J.F., Soares F., Dinis M.T & Sorgeloos P (2004) Effect of dietary non-protein energy levels on condition and r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 Aquaculture Research, 2011, 42, 313^321 Dietary protein levels and Solea solea performance P P Gatta et al oxidative status of Senegalese sole (Solea senegalensis) juveniles Aquaculture 231, 417^433 SAS (2004) SAS Institute Inc, 2004 SAS Version 9.0 SAS Institute, Cary, NC, USA Sanchez P., Ambrosio P.P & Flos R (2010) Stocking density and sex inÊuence individual growth of Senegalese sole (Solea senegalensis) Aquaculture 300, 93101 Schram E.,Van der Heul J.W., Kamstra A & Verdegem M.C.J (2006) Stocking density-dependent growth of Dover sole (Solea solea) Aquaculture 252, 339^347 Shimeno S., Kheyyali D & Shikata T (1995) Metabolic response to dietary lipid to protein ratios in common carp Fisheries Science 61, 977^980 Silva J.M.G., Espe M., Conceicaỡo L.E.C., Dias J & Valente L.M.P (2009) Senegalese sole juveniles (Solea senegalensis Kaup, 1858) grow equally well on diets devoid of Âshmeal provided the dietary amino acids are balanced Aquaculture 296, 309^317 Watanabe T (2002) Strategies for further development of aquatic feeds Fisheries Science 68, 242^252 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 313^321 321 Shelter and lighting in the intensive rearing of juvenile crayÂsh R Gonzalez et al Figure Survival rates at various periods of juvenile crayÂsh breeding under three lighting conditions during 120 days Error bars represent the standard errors of the means Figure Average carapace length (CL) at various periods of juvenile crayÂsh breeding under three lighting conditions during120 days Error bars represent the standard errors of the means 1.7% and 13.3%, with no signiÂcant diĂerences among groups Discussion It is generally accepted that providing shelters is necessary in the intensive rearing of juvenile crayÂsh, and it has been suggested that aggressiveness and cannibalism can be reduced considerably with increasing availability of shelters, allowing an increase in the survival and growth rates (Nystrm 1994; Saez-Royuela et al 1995, 2001; Streissl & Hdl 2002; 454 Aquaculture Research, 2011, 42, 450456 Figure Average weight at various periods of juvenile crayÂsh breeding under three lighting conditions during 120 days Error bars represent the standard errors of the means Savolainen et al 2003) It should be taken into account that refuges not only have to provide defence against aggressive behaviour but should also be hygienic, allowing easy access for cleaning and management without damaging water quality In this sense, PVC pipes are commonly used in crayÂsh farms and oĂer an individualized refuge (Saez-Royuela et al 1995, 2001) From the results of the present study with PVC pipes, each juvenile should have the possibility to Ând at least one shelter, which may be enough as there were no signiÂcant diĂerences between one, two and four pipes per crayÂsh Providing adequate shelter conditions is especially important in P leniusculus because this species shows clear shelter-related territoriality (Peeke, Sippel & Figler 1995; Edsman & Jonsson 1996) and tends not to share the same shelter (Ranta & Lindstrm 1992) Regarding lighting conditions, it has been reported that continuous or extended lighting had a positive eĂect on crayÂsh juvenile survival and decreased chelae lacking (Mason 1979; Taugbệl & Skurdal 1992; Nystrm 1994; Saez-Royuela et al 1996; Ulikowski & Krzywosz 2004) However, our present results are in disagreement with those reports While no signiÂcant diĂerences were found in survival or lack of chelae among the lighting conditions tested, the crayÂsh reared under continuous darkness grew signiÂcantly faster than those reared under continuous lighting This could be because crayÂsh are nocturnal animals and the duration of their activity is related to the length of the dark period (Gherardi 2002), in such a way that food Ânding takes place mainly during the night Thus, continuous darkness could have made r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 450^456 Aquaculture Research, 2011, 42, 450^456 Shelter and lighting in the intensive rearing of juvenile crayÂsh R Gonzalez et al animals keep eating a better-quality diet for longer than those used in the mentioned studies From the present results, it can be concluded that under the improved feeding conditions of this study, a minimum initial number of shelters can be provided, in such a way that each juvenile should have the opportunity to Ând at least one shelter, making management and hygienic practices easier Also, faster growth can be achieved in continuous darkness Acknowledgements Funding of this study was provided by the Plan Nacional de I1D1i, Ministerio de EducacioÔn y Ciencia, Spain, Research Project AGL2005-01127 We thank the Ânancing of a grant for Programa Nacional de FormacioÔn de Profesorado Universitario, Ministerio de EducacioÔn y Ciencia, Spain, reference AP20054860 We also thank the QuinỡoÔn S.A crayÂsh farm for their collaboration References Ackefors H & Lindqvist O.V (1994) Cultivation of freshwater crayÂshes in Europe In: Freshwater CrayÂsh Aquaculture (ed by J.V Huner), pp 157^216 The Haworth Press, Binghamton, NY, USA Blake M., Nystrm P & Hart P (1994) The eĂect of weed cover on juvenile signal crayÂsh (Pacifastacus leniusculus Dana) exposed to adult crayÂsh and non-predatory Âsh Annales Zoologici Fennici 31, 297^306 Celada J.D., Carral J.M., Gaudioso V.R., Gonzalez J., LopezBaissoÔn C & Fernandez R (1993) Survival and growth of juvenile freshwater crayÂsh Pacifastacus leniusculus Dana fed two raw diets and two commercial formulated feeds Journal of theWorld Aquaculture Society 24,108^111 Edsman L & Jonsson A (1996) The eĂect of size, antennal injury, ownership, and ownership duration on Âghting success in male signal crayÂsh, Pacifastacus leniusculus (Dana) Nordic Journal of Freshwater Research 72, 80^87 Gherardi F (2002) Behaviour In: Biology of Freshwater CrayÂsh (ed by D.M Holdich), pp 258^290 School of Life and Environmental Sciences, University of Nottingham, Nottingham, UK Gonzalez R., Celada J.D., Carral J.M., Gonzalez A., Saez-Royuela M & Garc|Ô a V (2009) Decapsulated Artemia cysts as dietary supplement for juvenile crayÂsh (Pacifastacus leniusculus, Astacidae) at diĂerent food supply frequencies from the onset of exogenous feeding under controlled conditions Aquaculture 295, 200^204 Gonzalez R., Celada J.D., Gonzalez A., Garc|Ô aV., Carral J.M & Saez-Royuela M (2010) Stocking density for the intensive rearing of juvenile crayÂsh, Pacifastacus leniusculus (Astacidae), using Artemia nauplii to supplement a dry diet from the onset of exogenous feeding Aquaculture International 18, 371^378 Mason J.C (1979) EĂects of temperature, photoperiod, substrate and shelter on survival, growth and biomass accumulation of juvenile Pacifastacus leniusculus in culture Freshwater CrayÂsh 4,73^82 Melendre P.M., Celada J.D., Carral J.M., Saez-Royuela M & Aguilera A (2006) EĂectiveness of antifungal treatments during artiÂcial incubation of the signal crayÂsh eggs (Pacifastacus leniusculus Dana Astacidae) Aquaculture 257, 257^265 Nystrm P (1994) Survival of juvenile signal crayÂsh (Pacifastacus leniusculus) in relation to light intensity and density Nordic Journal of Freshwater Research 69, 162^166 Nystrm P (2002) Ecology In: Biology of Freshwater CrayÂsh (ed by D.M Holdich), pp 192^235 School of Life and Environmental Sciences, University of Nottingham, Nottingham, UK Peeke H.V.S., Sippel J & Figler M.H (1995) Prior residence effects in shelter defense in adult signal crayÂsh (Pacifastacus leniusculus (Dana)): results in same- and mixed-sex dyads Crustaceana 68, 873^881 Ranta E & Lindstrm K (1992) Power to hold sheltering burrows by juveniles of the signal crayÂsh, Pacifastacus leniusculus Ethology 92, 217^226 Saez-Royuela M., Carral J.M., Celada J.D & Munỡoz C (1995) EĂects of management on survival and growth of stage juvenile freshwater crayÂsh (Pacifastacus leniusculus Dana) under laboratory conditions Aquaculture 133, 123^133 Saez-Royuela M., Carral J.M., Celada J.D., Munỡoz C & PeÔrez J.R (1996) ModiÂed photoperiod and light intensity inÊuence on survival and growth of stage juvenile signal crayÂsh Pacifastacus leniusculus Journal of Applied Aquaculture 6, 33^37 Saez-Royuela M., Carral J.M., Celada J.D & PeÔrez J.R (2001) EĂects of shelter type and food supply frequency on survival and growth of stage juvenile white-clawed crayÂsh (Austropotamobius pallipes Lereboullet) under laboratory conditions Aquaculture International 9, 489^497 Savolainen R., Ruohonen K & Tulonen J (2003) EĂects of bottom substrate and presence of shelter in experimental tanks on growth and survival of signal crayÂsh, Pacifastacus leniusculus (Dana) juveniles Aquaculture Research 34, 289^297 Savolainen R., Ruohonen K & Railo E (2004) EĂect of stocking density on growth, survival and cheliped injuries of stage juvenile signal crayÂsh Pacifastacus leniusculus Dana Aquaculture 231, 237^248 Streissl F & Hdl W (2002) Habitat and shelter requirements of the stone crayÂsh, Austropotamobius torrentium Schrank Hydrobiologia 477, 195^199 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 450^456 455 Shelter and lighting in the intensive rearing of juvenile crayÂsh R Gonzalez et al Taugbệl T & Skurdal J (1992) Growth, mortality and molting rate of noble crayÂsh Astacus astacus L juveniles in aquaculture experiments Aquaculture and Fisheries Management 23, 411^420 Ulikowski D & Krzywosz T (2004) The impact of photoperiod and stocking density on the growth and survival of 456 Aquaculture Research, 2011, 42, 450456 narrow-clawed crayÂsh (Astacus leptodactylus Esch.) larvae Archives of Polish Fisheries 12, 81^86 Van Stappen G (1996) Use of cysts In: Manual on the Production and Use of Live Food for Aquaculture FAO Fisheries Technical Paper 361 (ed by P Lavens & P Sorgeloos), pp.107^136 FAO, Rome, Italy r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 450^456 Aquaculture Research, 2011, 42, 457^468 doi:10.1111/j.1365-2109.2010.02643.x Effects of dietary phospholipids and highly unsaturated fatty acids on the precocity, survival, growth and hepatic lipid composition of juvenile Chinese mitten crab, Eriocheir sinensis (H Milne-Edwards) Xugan Wu1, Zongkai Wang1,Yong Xu Cheng1, Chaoshu Zeng1,2, Xiaozhen Yang1 & Jianfeng Lu3 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Shanghai, China Tropical Crustacean Aquaculture Research Group, School of Marine and Tropical Biology, James Cook University, Townsville, Qld, Australia School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, China Correspondence: Y X Cheng, Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, 999# Huchenghuan Road, Shanghai 201306, China E-mail: chengyongxucrablab@hotmail.com Abstract Precocious puberty is one of the major constraints to the further development of Chinese mitten crab (Eriocheir sinensis) farming industry Although dietary phospholipids (PL) and highly unsaturated fatty acids (HUFA) supplementation have been shown to enhance the growth of larval E sinensis in other studies, it is still unknown whether this also leads to a higher precocity rate for juvenile E sinensis This study was conducted to investigate the eĂects of dietary PL and HUFA on precocity, survival, growth and hepatic lipid composition of juvenile E sinensis Two diets were formulated with PL [3.95% dry weight (DW)] and HUFA (0.98% DW) supplementation (diet A) and without PL and HUFA supplementation (diet B) and fed to juvenile E sinensis Although dietary PL and HUFA levels did not signiÂcantly aĂect the survival and growth performance of juvenile E sinensis, compared with crabs fed diet A, a higher precocity rate was found among juvenile E sinensis fed diet B (P 0.051) A higher total lipid content, but signiÂcantly lower levels of HUFA and PL (Po0.05) were found in the hepatopancreas of crabs fed diet B than in those fed diet A Meanwhile, the precocious females had signiÂcantly lower hepatosomatic index , arachidonic acid (20:4n-6), eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) contents in their hepatopancreas when compared with that of the normal immature juveniles (Po0.05) The r 2010 Blackwell Publishing Ltd results suggest that the occurrence of precocious puberty among farmed juvenile E sinensis could be reduced by the inclusion of appropriate level of dietary PL and HUFA Keywords: Eriocheir sinensis, phospholipid, highly unsaturated fatty acid, precocious puberty, growth, lipid composition Introduction The Chinese mitten crab, Eriocheir sinensis, is an important aquaculture species in China (Zhang & Li 2002a) Because of high market demand and price, this freshwater crab species has been widely farmed in China (Chen, Zhang & Shrestha 2007; Wu, Cheng, Sui,Yang, Nan & Wang 2007), with aquaculture production having reached 474 948 tonnes and valued more than US$ billion in 2006 (China Fisheries Yearbook 2007) In China, the production cycle of E sinensis normally takes years: during the Ârst year, hatcheryproduced megalopae are stocked at high densities in freshwater nursery ponds to grow them onto coinsizedcrabs of 3^10 g individual Then, depending on the farm and location, either later in the year before winter sets in or early the following year, the coin-sized crabs are transferred to large grow-out 457 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Aquaculture Research, 2011, 42, 457^468 ponds, reservoirs or lakes to further grow-out to a market size of 80^200 g individual (Cheng, Wu, Yang & Anson 2008) However, it is commonly observed that at the end of the Ârst year of nursery culture, about 15^30% of crabs reach precocious puberty and the terminal molt These crabs, weighing between 15 and 35 g, have little or no market value (Wang, Li, Li & Zhao 2001) Because of the cessation of growth after reaching precocious puberty, precocious crabs are normally discarded and not used for stocking grow-out ponds in the following year (Jin & Li 2001; Jin, Li & Xie 2002) Obviously, this has serious impacts on the proÂtability of the E sinensis farming industry (Du, Zhang, Zhao & Zheng 2000; Li, Dong, Lei & Li 2007) Precocious puberty in Chinese mitten crabs has been associated with various factors, including temperature (Zhang & Li 2002b), salinity (Wei, Wu & Wei 2007), genetic degeneration (Du et al 2000;Wang et al 2001) and imbalanced nutrition (Liu 1998; He, Yin & Zhu 1999; Zhu, Wang & Zhang 1999; Zhu, Zhang, Wang & Chen 1999; Chen 2002) It has further been shown that manipulating dietary nutrition, particularly lipid composition, could reduce the occurrence of precocious crabs (Zhu, Wang et al 1999; Zhu, Zhang et al 1999; Du et al 2000) Phospholipids (PL) and highly unsaturated fatty acids (HUFA) are known as essential nutrients for many crustaceans (Kanazawa, Teshima & Sakamoto 1985; Cavalli, Lavens & Sorgeloos 1999; Sheen & Wu 2003; Sui, Mattieu, Cheng & Sorgeloos 2007; Wu, Cheng, Sui, Zeng, Southgate & Yang 2007); therefore, the dietary supplementation of PL and HUFA is crucial for many cultured crustaceans (Kontara, Coutteau & Sorgeloos 1997; Cheng, Yan, Wang, Shi & Tan 1998; Gong, Lawrence, Jiang, Castille & Gatlin III 2000; Gonzalez-Felix, Gatlin III, Lawrence & PerezVelazquez 2002; Gonzalez-Felix, Lawrence, Gatlin III & Perez-Velazquez 2002; Suprayudi, Takeuchi & Hamasaki 2004; Wang, Li, Hu & Jiang 2004) Previous studies have shown that supplementation of dietary PL and HUFA not only enhances survival and growth in larvae and early juveniles but also improves ovarian development and reproductive performance of adult E sinensis (Cheng et al 1998; Wang, Hu, Li & Jiang 2003; Wang et al 2004; Sui et al 2007; Wu, Cheng, Sui, Zeng, et al 2007) However, it is still unclear whether dietary PL and HUFA supplementation may also lead to a higher incidence of precocious puberty due to their well-known eĂects of enhancing ovarian maturation in crustaceans Based on growth performance, the optimal levels of dietary PL and 458 HUFA have been identiÂed for early juvenile E sinensis in previous studies Unfortunately, due to a relatively short experimental duration (25^60 days) of those studies, no information is available on the potential eĂects of dietary PL and HUFA on the occurrence of precocious E sinensis (Wang et al 2003, 2004) Meanwhile, the potential gender eĂects on the growth performance and incidence of precocity were not considered in those studies as male and female crabs were not identiÂed and/or separated (Mu, Shim & Guo 1998; Wang et al 2003, 2004) The current experiment was set up to investigate the potential eĂects of dietary PL and HUFA supplementation on the precocity rate, survival and growth performance of juvenile E sinensis Throughout the experiment, male and female crabs were cultured separately to evaluate gender diĂerences Further comparisons on the growth parameters and hepatic lipid composition between precocious and normal immature juvenile crabs, as well as between male and female crabs, were also carried out Materials and methods Experimental diets Two semi-puriÂed diets were formulated to contain two levels of PL and HUFA but remained almost isolipidtic and isonitrogenous (Table 1) The basal composition of the experimental diet was based on previous research on juvenile E sinensis (Cheng et al 1998; Wang et al 2004) The source of PL was defatted soy lecithin, a light yellow powder (Haerbin Luyuan, Xiangfang, Haerbin, China) containing 18% phosphatidylethanolamine (PE), 23% phosphatidylcholine (PC), 15% phosphatidylinositol (PI) and with 490% acetone insolubility, while the HUFA source was Âsh oil Diet A was supplemented with the optimized level of PL [3.95% dry weight (DW)] and HUFA (0.98% DW) for juvenile E sinensis, while diet B was lower in PL (0.35% DW) and HUFA (0.15% DW) (i.e without supplemental Âsh oil and soy lecithin) The levels of PL and HUFA in diet Awere based on results of previous research (Wang et al 2003, 2004) The ingredients of both experimental diets are shown in Table To prepare the diets, the dry ingredients of each diet were Ârst thoroughly mixed, and the lipid ingredients were then added drop by drop to the dry mixture while blending The diets were subsequently extruded through a 1.5 mm die disc and air-dried at room temperature (22^24 1C) for 48 h The diets were then stored at 20 1C until use r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 Aquaculture Research, 2011, 42, 457^468 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Table Diet formulation and analysed crude protein, total lipid, crude Âber, ash, cholesterol and phospholipid contents (% dry weight) Ingredients Casein Dextrin Yeast extract Cholesterol Vitamin mix Mineral mixw Inositol Choline chloride (purity 450%) Betaine Glycin Sodium alginate Butylated hydroxytoluene Cellulose Fish oil Soy lecithin Pork lard Analysed composition Moisture Crude protein Total lipid Crude fibre Ash Phospholipids Cholesterol Highly unsaturated fatty acids Source Diet A Diet B A A A A B B B B 40.00 25.64 3.00 0.50 2.60 3.00 0.60 1.00 40.00 25.64 3.00 0.50 2.60 3.00 0.60 1.00 B C C A 0.15 0.50 3.00 0.01 0.15 0.50 3.00 0.01 A B D B 10.00 60.0 40.0 10.00 0 100.0 8.36 36.23 9.51 8.52 3.74 3.95 0.33 0.98 8.89 37.25 10.51 8.43 4.05 0.35 0.36 0.15 Source of ingredient: (A) Sigma-Aldrich, St Louis, MO, USA (B) Jiangsu Jiawei Feed, Wuxi, Jiangsu, China; (C) Shanghai Chemical Reagent, Huangpu, Shanghai, China; (D) Haerbin Luyuan, Xiangfang, Haerbin, China Vitamin mix: 1kg of diet contained vitamin A 10 000 IU; vitamin D 2500 IU; vitamin K 64 mg; thiamin 60 mg; riboÊavin 250 mg; pyridoxine 60 mg; a-tocopherol acetate 500 mg; L -ascorbic acid 4.5 g; calcium panthothenic 240 mg; niacin 60 mg; folic acid 12 mg; biotin 50 mg; cyanocobalamine mg wMineral mix: 1kg of diet contained Ca(H2PO4)2 10 g; MgSO4 7H2O 2.4 g; KCl 4.5 g; NaCl 2.1g; FeSO4 H2O 155 mg; CuSO4 5H2O 40 mg; ZnSO4 H2O 80 mg; MnSO4 H2O 30 mg; KI 11.7 mg; CoCl2 6H2O 4.8 mg; Na2SeO3 2.4 mg Bold highlights the level diĂerence between two diets Source of crabs and experimental setup The experiment was conducted at the aquarium facility of Shanghai Ocean University, Shanghai, China In early August 2005, approximately 400 pond-reared normal immature juvenile E sinensis were obtained from a local crab farm Among them, half were males and half were females The mean wet body weights for the female and male crabs were 3.27 ặ 0.25 and 3.33 ặ 0.22 g (around eight crab stage, C8) respectively Active and intact crabs were randomly selected and stocked into 16 tanks (length width depth 560 cm 50 cm 45 cm, water volume 120 L) with a recirculating water supply The Âltering medium was composed of polyester wool, activated charcoal and gravel in six connected reservoir tanks (length width depth 90 cm 50 cm 40 cm, water volume 140 L) The Âltered water was recirculated at a speed of 1L to each rearing tank via a water pump (12 times per day) Eight tanks were used for stocking males, while the other eight were used for stocking females Each tank was initially stocked with 20 crabs Before the commencement of the experiment, the crabs were acclimated for weeks to the experimental conditions and fed diet B [low PL (0.35% DW) and HUFA (0.15% DW) contents] As diets A and B were fed to both male and female crabs, four treatments were formed, i.e diet A fed to female (AF) and male crabs, and diet B fed to female and male crabs Each treatment consisted of four replicate tanks and all experimental tanks were arranged in a randomized complete block design The experimental crabs were fed the designated diets twice daily at 8:00 and 19:00 hours with a ratio of approximately 5^8% of the total biomass The feeding trail lasted for 120 days and all throughout, the amount of diet given was adjusted based on both the body weight changes and observations of residual feed Before feeding, faeces and uneaten feed were removed by siphoning while the dead crab was removed During the experiment, a water depth of 40 cm was maintained and light was provided by overhead Êuorescent ceiling lights on a 12 h:12 h (light:dark) cycle All tanks were gently aerated and 10 cm PVC tubes (diameter cm) were provided as shelters Mortalities were checked and recorded twice per day The water temperature was controlled at 25 ặ 1.0 1C The pH, dissolved oxygen (DO), ammonia and nitrite concentration were monitored regularly: pH: 7.0^9.0; DO: 45 mg L 1; ammonia: o0.5 mg L and nitrite: o0.10 mg L respectively These water quality parameters were all within the suitable range for juvenile E sinensis (Zhang & Li 2002b) Crab sampling and data collection At the end of the experiment, the surviving crabs in each tank were counted to determine the Ânal survival The precocious individuals were identiÂed based onWang et al (2001) BrieÊy, precocious female crabs are characterized by a semi-circular shape of the abdomen Êap covered by short hair, which is in contrast r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 459 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Aquaculture Research, 2011, 42, 457^468 to the normal immature females whose abdomen appears to be more triangular In male crabs, the claws of precocious crabs are completely covered by thick and long hairs, while in the normal immature males, the claws are only partially covered with shorter hair To measure wet weight, an individual crab was Ârst blotted dry with a paper towel before being weighed on a digital balance (HA1001, Shanghai Eletronic Balance, Shanghai, China) The precocity rate, weight gain and SGR were calculated based on the following equations: Precocity rate %ị ẳ 100 Survival %ị ẳ 100 Total number of precocious crabs at the end of experiment Total number of surviving crabs at the end of the experiment Total number of survived crabs at the end of experiment 20 Weight gain %ị ẳ 100 Wt Wo ị Wo Specific growth rate SGR; % day1 ị ẳ 100 ln Wt ln Wo ị D where Wt is the Ânal body wet weight of the experimental crabs,Wo is the initial body wet weight of the crabs and D is the duration (120 days) of the experiment After weighing, hepatopancreas of all crabs were excised and weighed to determine the hepatosomatic index (HSI 5100 Hepatopancreas wet weight/ Body wet weight) The hepatopancreas of normal juveniles and precocious crabs from each tank were then pooled and stored separately at 70 1C for later biochemical analysis Biochemical analysis Before biochemical analysis, hepatopancreas pooled from each replicate were freeze-dried and homogenized separately The moisture content of each sample was obtained by drying the tissue in an oven set at 70 1C for 48 h and then the pre-dried sample weight was subtracted by the post-dried sample weight Total lipid (TL) was extracted with chloroform^methanol (2:1, v/v) based on the method described by Folch, Lees and Stanley (1957) The neutral lipids (NL) and polar lipids (PL) were separated by one developing solvent system (hexane/diethyl ether/ formic acid 42/28/0.3, v/v/v) as described in detail according to Wu, Cheng, Tang, Zhou, Hu, Yu and Yang (2007) BrieÊy, the TL was Ârstly dissolved in chloroform at 10^20 mg mL 1, then mL lipid 460 solution was added as a spot to the lower part of the chromatography rod (SIII, 10 cm 0.2 cm) using a micro-syringe After the development of the lipid sample, the rod was dried at 60 1C for to evaporate the solvents The individual lipid classes were quantiÂed by thin layer chromatography (Iatroscan MK-6 s, Iatron Laboratories, Tokyo, Japan) using a hydrogen Êame ionization detector IdentiÂcation of lipid classes was carried out by comparison with authentic standard reference mixtures (Nu- Chek-Prep., Elysian, MN, USA) Because the PL was not further separated into diĂerent PL classes, lipid classes of the experimental diets and crab tissues were only quantiÂed for total PL (including PC, PE, PI and others PL) and NL [including triacylglycerol (TG), free fatty acids and cholesterol] The level of total PL and NL were expressed as the percentage of TL (% TL) For fatty acid analysis, fatty acid methyl esters (FAME) were prepared by transesteriÂcation with14% borontriÊuoride/methanol (w/w) following the method of Morrison and Smith (1964) Fatty acid methyl esters were separated by Êame ionization detection after injecting a sample into an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) Âtted with an Omegawax 320 fused silica capillary column (30 m 0.32 mm; Supelco, Billefonte, PA, USA) The injector and detector temperature was maintained at 260 1C The column temperature was initially set at 60 1C, and then increased at a rate of 50 1C to 170 1C, followed by an increase at a rate of 1C to 180 1C and then held for It was further increased at the same rate of 1C to 230 1C and held for 1min before another increase at a rate of1 1C to the Ânal temperature of 240 1C until all FAME had been eluted The total time was 46.2 The carrier gas was helium with a Êow velocity of 25 cm s Peaks were identiÂed by comparing retention times with known standards (SigmaAldrich) Fatty acid contents are expressed as the percentage of each fatty acid to the total fatty acids (%) The moisture (930.15), crude protein (Kjeldahl method, using a 6.25 N to protein conversion factor 976.05), crude Âbre (978.10) and ash content (using a muƠe furnace at 550 1C until constant weight was reached r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 Aquaculture Research, 2011, 42, 457^468 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al 942.05) of the diets were analysed according to AOAC (1995) The formulated diets were analysed in duplicate for proximate, lipid class and fatty acid composition For crab hepatopancreas, four replicates (from each replicate tanks) were analysed for each treatment Statistical analysis Homogeneity of variance of data was tested using Levenes test When necessary, arc-sine, square root or logarithmic transformation was performed before analysis Two-way analysis of variance (ANOVA) was used to determine the diĂerences between diets, genders and their interactions If any signiÂcant diĂerence was detected, Duncans multiple range test was used as the means separation procedure When a normal distribution and/or homogeneity of the variances were not achieved, data were subjected to the Kruskal^Wallis H nonparametric test followed by the Games^Howell nonparametric multiple comparison test Independentsamples t-test was used to examine the diĂerences between normal immature juveniles and precocious crabs, diet A and diet B, as well as precocious male and female crabs Po0.05 was regarded as the statistically signiÂcant level (Sokal & Rohlf 1995) All statistics were performed using SPSS package (version12.0) Results Diet chemical composition Table shows the analysed proximate composition, PL, cholesterol and HUFA of diets A and B The crude protein, TL, ash content and the cholesterol level of the two diets were all within a small range However, as a consequence of PL and HUFA supplementation, diet A contained a substantially higher level of PL (3.95% DW) and HUFA (0.98% DW) than diet B (PL: 0.35% DW; HUFA: 0.15% DW) The most dominant polyunsaturated fatty acid (PUFA) in diet B was found to be the 18:2n-6 fatty acid (10.25% total fatty acid), with levels of all other PUFA being lower than 1% (Table 2) Meanwhile, although diet A contained higher PUFA and HUFA, it had substantially lower levels of saturated fatty acids (SFA) and monosaturated fatty acids (MUFA) as compared with diet B (Table 2) Survival and precocity rate of the crabs At the end of the 120-day experiment, the survival of juvenile crabs from the four treatments ranged from Table Fatty acid compositions (% total fatty acids, mean of duplicated samples) of the two experimental diets containing two levels of phospholipids and highly unsaturated fatty acids Fatty acids Diet A Diet B 14:0 15:0 16:0 17:0 18:0 23:0 SSFA 14:1n-7 16:1n-5 16:1n-7 17:1n 18:1n 20:1n SMUFA 18:2n-6 18:4n-3 18:3n-3 20:2n-6 20:5n-3 20:4n-6 22:5n-3 22:6n-3 SPUFA (! 18:2n) n-3PUFA n-6PUFA n-3/n-6 SHUFA (! 20:3n) Unidentified 5.74 0.69 21.50 0.59 4.34 0.27 33.12 3.13 0.60 6.03 0.35 14.54 2.03 26.10 15.60 1.71 2.61 0.18 6.76 0.63 0.94 5.93 33.81 18.33 16.41 1.12 14.64 5.44 3.47 0.48 24.35 0.51 16.48 45.30 1.91 0.28 2.57 0.38 33.15 0.81 38.82 10.25 0.20 0.79 0.47 0.58 0.38 0.14 0.61 13.41 2.30 11.18 0.21 1.78 2.05 SFA, saturated fatty acids; MUFA, monosaturated fatty acids; PUFA, polyunsaturated fatty acid; HUFA, highly unsaturated fatty acids Bold highlights the level diĂerence between two diets 57.5% to 72.5% (Fig 1) Two-way ANOVA showed that crab survival was not signiÂcantly aĂected by diet (P 0.366), gender (P 0.213) or by their interactions (P 0.366) However, for both male and female crabs, a higher precocity rate was recorded among those fed diet B when compared with those fed diet A (Fig 2) Statistical analysis showed that the precocity rate of the experimental crabs was not signiÂcantly aĂected by either gender (P 0.432) or an interaction between diet and gender (P 0.466) Therefore, the data of both male and female crabs were pooled for an independent-samples T-test; the result showed that the diĂerence of precocity rates between the two diet treatments were marginally signiÂcant (P 0.051), and this indicates that the precocity of juvenile E sinensis was aĂected by the dietary PL and HUFA level (Fig 2) r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 461 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Aquaculture Research, 2011, 42, 457^468 were not signiÂcantly diĂerent (P40.05) among the four treatments (Table 3) However, a two-way ANOVA showed that the percentage weight gain and SGR of precocious crabs were signiÂcantly aĂected by diet, gender and their interactions (Po0.05, Table 3) The highest growth performance was recorded for the AF treatment, which was signiÂcantly higher than that of the other three treatments (Po0.05, Table 3) When Growth performance of the crabs Precocious crabs were found in all experimental tanks Because the mean Ânal body wet weight of precocious crabs was signiÂcantly higher than that of normal immature crabs from both diet treatments (Po0.01,Table 3), the growth parameters were calculated separately for precocious and normal crabs The results showed that the Ânal weight, the percentage weight gain and SGR of the normal immature crabs Diet A Diet B AF BF AM Treatment The precocious rate (%) Survival (%) 24 80 70 60 50 40 30 20 10 BM 20 16 12 Figure Survival of Eriocheir sinensis juveniles at the end of the 120-day experiment AF, BF and AM, BM represent treatment of diets A and B fed to female (AF, BF) and male crabs (AM, BM) respectively Data are represented as mean ặ SE No signiÂcant diĂerence can be observed among the four treatments (P40.05) Female Pooled crabs Male Figure The precocity rate of juvenile Eriocheir sinensis at the end of the120-day experiment Data are represented as mean ặ SE The diĂerence in precocity rates between the two diet treatments were marginally signiÂcant (P 0.051) Table Initial weight, Ânal weight, weight gain and speciÂc growth rate (SGR) of normal immature and precocious Eriocheir sinensis from AF, BF, AM and BM treatments Treatments Normal immature crabs AF (n 41) BF (n 40) AM (n 43) BM (n 47) ANOVA (P-value) Diet type Gender Diet type Gender Precocious crabs AF (n 5) BF (n 6) AM (n 5) BM (n 11) ANOVA (P-value) Diet type Gender Diet type Gender Initial weight (g) 3.10 3.34 3.32 3.31 ặ ặ ặ ặ 0.22 0.17 0.17 0.19 0.642 0.543 0.316 3.10 3.34 3.32 3.31 ặ ặ ặ ặ 0.642 0.543 0.316 Final weight (g) 8.60 8.34 8.57 8.19 ặ ặ ặ ặ 2.26 2.06 2.19 1.94 0.395 0.892 0.764 0.22 0.17 0.17 0.19 18.88 14.42 12.59 14.21 0.066 0.000 0.000 Weight gain (%) 174.80 149.61 159.00 146.78 ặ ặ ặ ặ 71.58 61.80 66.03 58.44 0.066 0.358 0.522 ặ ặ ặ ặ 2.48b 1.55a 1.24a 1.81a 508.90 331.64 279.28 329.44 0.010 0.000 0.000 SGR (% day 1) 0.82 0.73 0.76 0.73 ặ ặ ặ ặ 0.21 0.23 0.21 0.20 0.075 0.425 0.501 ặ ặ ặ ặ 80.27b 46.44a 37.23a 54.72a 1.50 1.21 1.11 1.21 ặ ặ ặ ặ 0.11b 0.09a 0.08a 0.11a 0.035 0.000 0.000 Values are presented as means ặ SD Values in a same column that not share same superscripts are signiÂcantly diĂerent (Po0.05) Two-way analysis of variance was used to determine the diĂerences among the diets (diet A and diet B), gender and their interactions n, the number of samples; AF and BF, the treatments of diet A and B fed to females; AM and BM, the treatments of diet A and B fed to males 462 r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 Aquaculture Research, 2011, 42, 457^468 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al HSI and hepatic lipid composition The HSI of normal immature crabs of the four treatments ranged from 9.45% to 10.29% and no signiÂcant diĂerence was found (Fig 3) However, the HSI of precocious crabs was signiÂcantly aĂected by gender (Po0.05) as the precocious female crabs had substantially lower HSI than the male crabs (Fig 3) When compared between the precocious and the normal crabs, the female precocious crabs also had signiÂcantly lower HSI than immature females within the same dietary treatment (Po0.05) The moisture, TL, NL and total PL contents in the hepatopancreas of the normal immature and the precocious E sinensis are presented in Table For normal immature crabs, diet type had signiÂcant eĂects on the total hepatic lipid, NL and total PL for both males and females Regardless of gender, crabs fed diet B (low PL and HUFA contents) had signiÂcantly higher levels of TL and NL, but lower total PL content than crabs fed PL- and HUFA-supplemented diet A (Po0.05) No interaction between diet and gender was observed (P40.05) Similar results were found for the precocious crabs, except that the TL content was not signiÂcantly diĂerent (Table 4) The hepatic fatty acid composition of the crabs are shown in Tables and The hepatic fatty acid composition of both normal immature and precocious crabs were dramatically inÊuenced by their dietary fatty acids content (Po0.05) For both female and Hepatosomatic index (%) compared between the precocious and normal immature crabs within the same treatment, the precocious crabs had signiÂcantly higher percentage weight gain and SGR (Po0.05) than the immature crabs 12 10 Normal immature crabs * Precocious crabs b b BF AM Treatment BM * a a AF Figure Hepatosomatic index of normal immature and precocious Eriocheir sinensis at the end of the 120-day experiment AF and BF, treatments of diets A and B fed to females, respectively; AM and BM, treatments of diets A and B fed to males respectively Data are represented as mean SE For the female and male precocious crabs, the columns that not share same superscripts are signiÂcantly different (Po0.05) For normal immature and precocious crabs fed the same diet, the column of normal immature crabs with indicates signiÂcant diĂerence between normal immature and precocious crabs (Po0.05) Table Moisture (% wet weight), total lipid (% dry weight), neutral lipids and total phospholipids (% total lipids) in the hepatopancreas of normal immature and precocious E sinensis at the end of the experiment from AF, BF, AM and BM treatments Treatments Normal immature crabs AF (n 4) BF (n 4) AM (n 4) BM (n 4) ANOVA (P-value) Diet type Gender Diet type Gender Precocious crabs AF (n 4) BF (n 4) AM (n 4) BM (n 4) ANOVA (P-value) Diet type Gender Diet type Gender Moisture (%) 40.49 43.16 47.09 42.01 ặ ặ ặ ặ 2.05a 2.09ab 3.24b 2.77ab 0.548 0.126 0.036 50.68 42.37 46.23 44.74 0.296 0.741 0.147 Total lipid (%) 67.77 83.81 74.09 81.63 ặ ặ ặ ặ 4.36a 6.40b 3.87a 4.98b 0.000 0.370 0.082 ặ ặ ặ ặ 7.25 4.91 3.69 4.61 77.20 80.47 64.25 70.96 Neutral lipids (%) 91.21 94.89 91.20 95.32 ặ ặ ặ ặ 1.17a 2.81b 1.97a 0.66b 0.001 0.825 0.812 ặ ặ ặ ặ 5.36 8.11 2.08 13.95 0.975 0.226 0.215 89.06 97.01 82.60 93.62 0.000 0.010 0.313 Total phospholipids (%) 8.79 5.11 8.80 4.68 ặ ặ ặ ặ 1.17b 2.81a 1.97b 0.66a ặ ặ ặ ặ 3.75b 1.01a 4.99c 1.52ab 0.001 0.825 0.812 ặ ặ ặ ặ 3.75b 1.01c 4.99a 1.52bc 10.94 2.99 17.40 6.38 0.000 0.010 0.313 For treatment abbreviations, see Table Values are presented as means ặ SD Values in a same column that not share same superscripts are signiÂcantly diĂerent (Po0.05) n, number of replicates r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 463 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Aquaculture Research, 2011, 42, 457^468 Table Fatty acid composition (% total fatty acids) of hepatopancreas of the normal immature Eriocheir sinensis at the end of the experiment from AF, BF, AM and BM treatments AF Fatty acids 14:0 15:0 16:0 17:0 18:0 SFA 14:1n7 16:1n7 16:1n5 17:1n 18:1n9 18:1n7 20:1n9 20:1n7 MUFA 18:2n6 18:3n4 18:3n3 18:4n3 20:2n6 20:4n6 20:5n3 22:5n3 22:6n3 PUFA n-3PUFA n-6PUFA n-3/n-6 HUFA Unidentified 3.04 0.60 19.62 0.30 3.36 26.93 1.84 14.78 0.52 0.73 31.71 3.53 1.86 0.29 55.27 7.24 0.28 1.01 0.61 0.41 0.58 2.20 0.27 2.33 15.25 6.50 8.23 0.79 5.38 2.78 BF ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.08b 0.00b 0.25a 0.07b 0.06a 0.39 0.12b 0.12d 0.04b 0.02b 0.36a 0.21b 0.10b 0.03b 0.72b 0.07a 0.00b 0.01c 0.26 0.02a 0.05b 0.04b 0.02b 0.19d 0.29b 0.48c 0.15a 0.07c 0.08b 0.08b AM 2.21 0.38 20.37 0.22 4.21 27.38 1.70 12.50 0.39 0.60 38.42 2.70 1.16 0.20 57.67 8.71 0.17 0.71 0.54 0.70 0.40 0.65 0.19 0.60 12.90 2.68 9.82 0.28 1.84 2.27 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.18a 0.05a 0.47b 0.02ab 0.20bc 0.45 0.49ab 0.49c 0.05a 0.07a 2.02c 0.37a 0.16a 0.03a 0.89c 0.66b 0.03a 0.04b 0.08 0.08b 0.04a 0.28a 0.02a 0.21b 0.12a 0.64a 0.70b 0.09a 0.56a 0.43a 3.21 0.95 19.73 0.53 3.83 28.23 1.64 9.27 0.76 1.05 32.79 3.68 2.52 0.61 52.33 7.67 0.75 0.75 0.41 1.08 1.07 2.06 0.46 1.61 16.40 5.36 9.82 0.55 5.20 3.48 BM ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.20b 0.08c 1.21ab 0.05c 0.42ab 1.71 0.32ab 0.83a 0.09c 0.17c 0.55b 0.21b 0.09c 0.04c 1.49a 0.34a 0.36c 0.08b 0.10 0.03c 0.08c 0.14b 0.31c 0.33c 0.30c 0.10b 0.31b 0.01b 0.22b 0.38c 2.12 0.34 21.33 0.21 5.28 29.27 0.97 11.28 0.33 0.52 39.78 2.36 0.95 0.19 56.38 8.44 0.14 0.60 0.54 0.78 0.37 0.41 0.16 0.26 11.93 1.97 9.62 0.21 1.21 2.62 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.06a 0.01a 1.23b 0.00a 1.20c 2.45 0.44a 0.50b 0.05a 0.03a 0.41c 0.36a 0.03a 0.05a 1.03c 0.33b 0.01a 0.03a 0.02 0.08b 0.04a 0.11a 0.05a 0.07a 0.22a 0.12a 0.35b 0.02a 0.17a 0.56abc For treatment abbreviations, see Table Values are presented as means ặ SD (n 4) Values in the same line that not share same superscripts are signiÂcantly diĂerent (Po0.05) SFA, saturated fatty acids; MUFA, monosaturated fatty acids; PUFA, polyunsaturated fatty acid; HUFA, highly unsaturated fatty acids male crabs, the levels of14:0,16:0,18:0,18:1n-9, 20:5n3 eicosapentaenoic acid (EPA) and 22:6n3 docosahexaenoic acid in the crab hepatopancreas tissue appeared to generally reÊect their relative content in their diets However, the 18:2n-6 fatty acid is an exception Although diet B contained substantially lower 18:2n-6 than diet A (Table 2), crabs fed diet B had an opposite eĂect of slightly higher 18:2n-6 contents in their hepatopancreas than crabs fed diet A (Tables and 6), indicating possible important biological roles of 18:2n-6 fatty acids when crabs were fed low levels of PL and HUFA (diet B) It is also noteworthy that the female precocious crabs had signiÂcantly lower 20:4n6 arachidonic acid (ARA), EPA and HUFA content in hepatopancreas than the normal immature females when fed both diets A and B 464 (Po0.05); however, no signiÂcant diĂerences were found among male crabs (P40.05) Discussion Although PL and HUFA are essential nutrients for many crustaceans (Kanazawa et al 1985; Teshima, Kanazawa & Kakuta 1986; Kontara et al 1997; Gong et al 2000; Gonzalez-Felix, Gatlin III et al 2002; Gonzalez-Felix, Lawrence et al 2002; Holme, Southgate & Zeng 2007), our results indicate that when juvenile E sinensis were fed a diet (diet B) containing low PL and HUFA levels, this did not signiÂcantly aĂect the survival and growth of the juvenile crabs (3^ 10 g individual 1, from the initial weight to the Ânal r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 Aquaculture Research, 2011, 42, 457^468 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Table Fatty acid composition (% total fatty acids) of hepatopancreas of precocious E sinensis at the end of the experiment from AF, BF, AM and BM treatments AF Fatty acids 14:0 15:0 16:0 17:0 18:0 SFA 14:1n7 16:1n7 16:1n5 17:1n 18:1n9 18:1n7 20:1n9 20:1n7 MUFA 18:2n6 18:3n4 18:3n3 18:4n3 20:2n6 20:4n6 20:5n3 22:5n3 22:6n3 PUFA n-3PUFA n-6PUFA n-3/n-6 HUFA Unidentified 3.10 0.56 19.33 0.34 3.87 27.20 1.32 10.74 0.44 0.70 34.67 3.16 2.57 0.54 54.16 7.79 0.33 0.90 0.66 0.62 0.36 1.50 0.28 2.35 15.13 5.79 8.77 0.66 4.40 3.75 BF ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.80bc 0.10b 1.70ab 0.07b 0.77a 3.36 0.04b 0.96 0.02b 0.06c 4.00ab 0.64b 0.15a 0.21b 5.67ab 1.24ab 0.01b 0.31b 0.03b 0.21b 0.12ab 0.09b 0.01b 0.49b 1.76b 0.89b 0.91ab 0.04b 0.28b 0.31c 1.94 0.32 19.81 0.19 5.84 28.11 1.29 10.97 0.40 0.62 40.99 2.18 1.34 0.34 58.13 7.96 0.18 0.53 0.49 1.01 0.32 0.37 0.12 0.34 11.56 1.86 9.32 0.20 1.16 2.56 AM ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.08a 0.02a 0.44a 0.01a 0.69b 1.19 0.12b 1.62 0.07ab 0.01b 2.35c 0.30a 0.27b 0.20b 1.55b 0.75ab 0.03a 0.12a 0.03a 0.18c 0.04a 0.05a 0.02a 0.07a 0.80a 0.16a 0.67ab 0.02a 0.18a 0.13ab 3.23 0.66 20.06 0.38 3.79 29.18 0.72 11.78 0.46 0.70 31.48 2.92 2.26 0.36 50.69 7.67 0.48 1.36 0.90 0.49 0.59 2.32 0.32 2.77 17.29 7.73 8.76 0.88 6.00 3.12 BM ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.42c 0.07b 0.81ab 0.09b 0.40a 1.57 0.46a 2.13 0.01b 0.03c 2.18a 0.08b 0.10a 0.02b 4.53a 0.17a 0.29c 0.56b 0.21c 0.01a 0.08b 0.25c 0.05b 0.27b 1.62b 1.29c 0.14a 0.14c 0.58c 0.58bc 2.29 0.36 21.03 0.22 5.88 29.77 1.28 12.18 0.36 0.52 38.17 2.11 0.98 0.13 55.71 8.30 0.16 0.66 0.53 0.67 0.36 0.49 0.11 0.45 12.36 2.22 9.76 0.23 1.40 2.35 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.04b 0.03a 0.36b 0.02a 1.58b 1.29 0.12b 0.36 0.05a 0.03a 1.71bc 0.23a 0.23b 0.05a 2.45ab 0.23b 0.03a 0.04a 0.04a 0.02b 0.09a 0.30a 0.02a 0.28a 1.09a 0.69a 0.36b 0.06a 0.68a 0.18a For treatment abbreviations, see Table Values are presented as means ặ SD (n 4).Values in the same line that not share same superscripts are signiÂcantly diĂerent (Po0.05) SFA, saturated fatty acids; MUFA, monosaturated fatty acids; PUFA, polyunsaturated fatty acid; HUFA, highly unsaturated fatty acids weight) during the 120-day culture period However, for the larvae of E sinensis, a lack of dietary PL and HUFA could lead to high incidences of molting death syndrome (MDS) (Sui et al 2007; Wu, Yu, Cheng, He, Yang, Lu & Sheng 2007), a phenomenon for crab larvae or early juvenile mortality due to their inability to completely shed the old carapace (Holme et al 2007) In our experiment, no mortalities were observed due to MDS This suggests that a low dietary level of PL (3.5 g kg 1) and HUFA (1.5 g kg 1) may in fact be sufÂcient for the survival and growth of juvenile E sinensis with a body weight between and 10 g In contrast, our previous research has shown that dietary PL and HUFA levels could signiÂcantly aĂect survival, weight gain, carapace size as well as the intermoult period of larval E sinensis (Cheng et al 1998; Sui et al 2007; Wu, Yu, Cheng et al 2007) It was suggested that for normal growth and development of E sinensis larvae, a dietary PL level of 20^40 g kg 1and HUFA level of approximately 15 g kg are required (Cheng et al 1998; Sui et al 2007) These diĂerences of dietary PL and HUFA requirements at diĂerent life stages may be explained by the more rapid growth and shorter intermoult period of larval E sinensis compared with juveniles (Sui et al 2007) However, it may also reÊect the habitat transition from brackish water to freshwater occurring during the development from larvae to juveniles in E sinensis For example, previous research on juvenile freshwater crustaceans, such as the giant freshwater prawn Macrobrachium rosenbergii and red claw crayÂsh Cherax quadricarinatus, generally reported a lower PL and HUFA requirement than that of marine species (Briggs, Jauncey & Brown 1988; Reigh & Stickney1989; DAbramo & Sheen1993; Thompson, Muzinic, Christian, Webster, Manomatis r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 465 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al Aquaculture Research, 2011, 42, 457^468 & Rouse 2003) From the juvenile stage onward, E sinensis inhabit freshwater systems; therefore, it is not totally unexpected to Ând that E sinensis juveniles require lower levels of HUFA and PL Although urvival and growth were not signiÂcantly aĂected by dietary PL and HUFA levels, surprisingly, juvenile E sinensis fed low levels of PL and HUFA (diet B) led to a higher precocity rate than those fed a HUFAand PL-supplemented diet and the diĂerence was at the margin of statistical signiÂcance (P 0.051, Fig 2) It suggests that feeding E sinensis with diets formulated with appropriate levels of PL and HUFA could effectively reduce the precocity rate, leading to improved productivity in E sinensis culture Such a result may reÊect the relationship of lipid nutrition and endocrine regulation of sex maturation of the crab (Chen 2002) The occurrence of precocity in E sinensis was reported to be regulated by hormones, especially methyl farnesoate (MF), which is secreted by the mandibular organ (MO) (Zhao & Lu 2003) It was suggested that deÂciencies of dietary PL and HUFA may induce the advanced development of MO as higher MF biosynthetic rates were detected in the crabs fed diet deÂcient of PL and HUFA (J.F Lu, G.L Chang & X.G.Wu, unpubl data) Interestingly, the crabs fed diet A with optimal levels of dietary PL and HUFA still experienced precocity, albeit at a lower percentage (ca 10%) Therefore, this clearly indicates that such a phenomenon in E sinensis is aĂected not only by lipid nutrition but also by other factors, such as social interactions and/or population genotype of the crabs (Wang et al 2001) It is worth noting that the expensive and puriÂed soy lecithin (food grade, purity 490%) was used in this study to formulate the experimental semipuriÂed diet for research purposes, while for the practical diet used in commercial mitten crab culture, cheap and crude soy lecithin oil (feed grade, purity around 60%) could be used to obtain appropriate PL levels and reduce the precocity rate Although the initial body weight was similar, it is interesting to note that at the end of the experiment, the Ânal body weight of the precocious crabs were signiÂcantly higher than that of the normal juveniles for both male and female crabs within the same treatment (Table 3) This paradox may be explained by the shorter molting interval of the precocious crabs (20^ 30 days molting interval 1) than that of the normal immature crabs (25^38 days molting interval 1) (Zhang & Li 2002b) At the end of the experiment, normal immature crabs fed diet B were detected to have a higher hepatic lipid content than normal crabs fed diet A A likely 466 explanation for this is that the low level of dietary HUFA leads to relatively higher levels of SFA and/or MUFA in the diet This could have accelerated the biosynthesis of TG in the hepatopancreas, resulting in an increase in the HSI and in the TL content (Jump & Clarke 1999; Kumaraguru, Ramesh & Balasubramanian 2005) Alternatively, because past studies have demonstrated that the addition of dietary PL increases the transport rates of lipids out of the hepatopancreas (Kanazawa et al.1985;Teshima et al.1986), it may also reÊect the result of insucient dietary PL impacting the emulsiÂcation and transportation process of lipids from hepatopancreas to other tissues The precocious females also had lower ARA, EPA and HUFA contents in hepatopancreas than the normal juvenile females regardless of being fed diet A or B (Table and 6) This result may reÊect selective absorption and preferential accumulation of HUFA during ovarian development of precocious E sinensis, leading to a high proportion of hepatic HUFA being transferred to the ovaries (Chen, Cheng & Wang 2003) Conclusion The present study is the Ârst to investigate the eĂect of lipid nutrition on the occurrence of precocity in E sinensis while also taking potential gender diĂerences into consideration The results show that juvenile crabs fed low levels of PL and HUFA (diet B) had a higher percentage of precocity than crabs fed the PLand HUFA-supplemented diet A This indicates that diets formulated with appropriate dietary PL and HUFA levels could eĂectively reduce the precocity rate, and therefore, lead to improved productivity in juvenile E sinensis culture Acknowledgments We would like to thank Mr ZhiyongYu from Shanghai Ocean University for his assistance with chemical analysis This study was supported by two general projects from the Natural Science Foundation of China (No 30471349 and No 30871927) Infrastructure costs were also partially supported by the innovation research group (nutrition, feed and environment of animal aquaculture) developing project in the universities of Shanghai from Shanghai Municipal Education Commission and two extension projects (N0 2008-4-3 and No 2009-2-1) from Shanghai Agriculture Committee r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 457^468 Aquaculture Research, 2011, 42, 457^468 Dietary PL and HUFA on the precocity of juvenile Eriocheir sinensis X Wu et al References AOAC (1995) In: Ocial Methods of Analysis of theAssociation of 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The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 322^330 Aquaculture Research, 2011, 42, 331^340 doi:10.1111/j.1365-2109.2010.02627.x Influence 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... The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 331^340 Aquaculture Research, 2011, 42, 341^350 doi:10.1111/j.1365-2109.2010.02628.x The use of biochemical, sensorial and chromaticity attributes as indicators of postmortem changes in commercial-size, cultured red porgy Pagrus pagrus, stored on ice George Vardanis1,2, Liliana S¢chi-Duke1, Lluis Tort3, Pascal... 67.928, P 5 0.0001; Fig 2b] Total protein assessment showed signi¢cantly higher values in the stressed HC group than in the r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 322^330 325 Aquaculture Research, 2011, 42, 322–330 Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al (a) 100 b 95 90 85 80 a Cortisol (ng mL–1) 75 a 70 65... lactate) were higher than those in HC This indicates that a co-e¡ect of cormorant presence and harvesting was not apparent r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 322^330 Aquaculture Research, 2011, 42, 322^330 Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al Conclusion Blood plasma stress indicators (glucose, cortisol... randomly in the littoral zone by a r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 322^330 323 Stress responses of carp pond ¢sh stock upon great cormorant J Kortan et al lift net (1 Â 1m, mesh size 10 Â 10 mm), with 30 replicates on both ponds Fulton’s condition coe⁄cient (FCC) Aquaculture Research, 2011, 42, 322–330 biochemical analyser (Roche Diagnostics,... per group and per sampling level (i.e 24 larvae in total) were routinely dehydrated in a graded ethanol series, cleared in r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 331^340 Aquaculture Research, 2011, 42, 331^340 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al xylol and, ¢nally, embedded in para⁄n The entire larva body was transversely... at 14 1C (Po0.05) (Tables 2 and 3) However, in the larvae reared at 18 1C, these di¡erences seem to be inexistent (Table 3) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 331^340 Aquaculture Research, 2011, 42, 331^340 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Table 2 Total cross-sectional muscle area [A (muscle)], total number of... larger at the post-opercular level of the larvae from the 18 1C group (Po0.05) (Tables 2 and 3) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 331^340 335 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340 Figure 2 Total cross-sectional muscle area [A (muscle)], total number of ¢bres [N (¢bres)] and cross-sectional... Cole, Abercromby & Vieira 1998; Ayala, Lo¤pez-Albors, Gil, Garc|¤ aAlcaŁzar, AbellaŁn, Alarco¤n, Alvarez, Ramirez-Zarzosa & r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 331^340 Aquaculture Research, 2011, 42, 331^340 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Table 3 Signi¢cance of di¡erences between Pagellus bogaraveo muscle location... to juvenile, where a pause in muscle growth of both ¢bre types was observed at mouth opening r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 42, 331^340 337 Temperature e¡ect on Pagellus bogaraveo larvae P Silva et al Aquaculture Research, 2011, 42, 331^340 lower temperatures (14 1C) The presence of more red ¢bres in the post-anal part of the newly hatched