Aquaculture research, tập 41, số 12, 2010

Aquaculture Research, 2010, 41, 1727^1740 doi:10.1111/j.1365-2109.2009.02450.x Natural zooplankton as larval feed in intensive rearing systems for juvenile production of Atlantic cod (Gadus morhua L.) Kjersti Eline Tệnnessen Busch1, Inger-Britt Falk-Petersen1, Stefano Peruzzi1, Nora Arctander Rist2 & Kristin Hamre3 Department of Arctic and Marine Biology, University of Tromsệ,Tromsệ, Norway LoÂlab AS, Steine, Stamsund, Norway National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway Correspondence: K E T Busch, Department of Aquatic BioSciences, Norwegian College of Fishery Science, University of Tromsệ, N-9037 Tromsệ, Norway E-mail: Kjersti.Busch@uit.no Abstract Introduction The growth potential of cod larvae is not fully achieved when rotifers (Brachionus spp.) are used as live feed In this experiment, we studied the effect of natural zooplankton (mainly copepods) on the growth of cod (Gadus morhua L.) larvae reared in intensive systems Using a growth model developed for cod larvae, the growth rates observed could be evaluated and compared with growth rates reported previously The cod larvae showed optimal growth rates until age 19 days post hatch (DPH) when they reached 9.77 ặ 0.25 mm standard length (SL) Early weaning (20^25 DPH) resulted in signiÂcantly longer larvae at age 30 DPH compared with late weaning (25^32 DPH); however, in this period, the zooplankton concentrations were low The experimental larvae showed considerably higher growth rates compared with rotifer (Brachionus spp.)-reared cod larvae in previous experiments The nutritional composition of cod larvae was analysed and compared with published results on rotifer-reared larvae The levels of iodine, manganese, selenium and n-3 PUFA were considerably higher in larvae fed copepods compared with larvae fed rotifers The diĂerences in nutritional status may well explain the diĂerences in growth observed between copepod and rotiferreared larvae Aquaculture of cod (Gadus morhua L.) is a growing industry in Canada, Faroe Islands, Iceland, Norway, Scotland, United Kingdom and United States (Brown, MinkoĂ & Puvanendran 2003; Watson, Sales, Cumming, Fitzsimmons, Walden, Arthur, Saravanan & McEvoy 2006; Bjrnsson, Steinarsson & Arnason 2007; Rosenlund & Halldorsson 2007) Norway is by far the largest producer of farmed cod, and in 2006 the domestic production was 11087 tonnes whole Âsh and10.5 million juveniles (Directorate of Âsheries 2007) A reliable production of high-quality cod juveniles is essential to ensure an economically sustainable production There are in principle two diĂerent approaches to juvenile production of cod; the extensive and the intensive method Extensive Âsh farming relies on an enclosed ecosystem where the food is produced within the system, while an intensive rearing system requires a constant food supply due to its small size and high larval densities (van der Meeren & Naas 1997) There is a long tradition for extensive production of cod fry in Norway, the Ârst attempt being carried out at the Flệdevigen research station in 1885 (Solemdal, Dahl, Danielssen & Moksness 1984) After 1975, a number of experiments have been carried out in ponds and lagoons with the aim of studying Âsh larval feeding, growth and survival (van der Meeren & Naas 1997; Svễsand, Otterễ & Taranger 2004) In the 1980s, relatively high numbers of cod juveniles were produced in ponds by commercial companies and Keywords: Gadus morhua, copepod, rotifer, growthrate, nutrition, fatty acid r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd 1727 Farmed cod larvae fed natural zooplankton K E T Busch et al government-Ânanced institutions for sea ranching and aquaculture purposes After some years of scarce production due to low prices, production increased again after 2000 (Engelsen, Asche, Skjennum & AdoĂ 2004) At present, there is production of cod juveniles in ponds with or without submerged bags at three to four sites in Norway For a description of the bag-in-pond system, see van der Meeren and Naas (1997) Among the main challenges that cod farmers face relying on the extensive method are the seasonality of plankton production and the low stocking densities of cod larvae resulting in a limited production of juveniles To avoid the seasonality and to increase production, development in cod culture has moved towards an intensive production in indoor systems with a year-round production of larvae fed rotifers (Brachionus spp.) (Rosenlund & Halldorsson 2007) At present, there are 10 intensive cod juvenile producers in Norway Production methods have, to a large extent, been adopted from intensive production of other marine species like European seabass (Diecentrarchus labrax L.) and gilthead seabream (Sparus aurata L.) with a reliance on rotifers and Artemia salina L as live feed during the Ârst weeks of culture (Svễsand et al 2004) A growing body of scientiÂc studies has contributed to the intensive rearing methods of cod larvae (Puvanendran & Brown 1999, 2002; Baskerville-Bridges & Kling 2000a, b, c; Brown et al 2003; Puvanendran, Burt & Brown 2006; Fletcher, Roy, Davie, Taylor, Robertson & Migaud 2007); however, there are still challenges to be met in optimizing production and juvenile quality Although not well documented in the scientiÂc literature, there might be large diĂerences in the quality, in particular the growth potential, between intensively and extensively reared cod juveniles Higher rates of normal pigmentation and a higher stress resistance in marine larvae reared on natural zooplankton compared with those reared on rotifers or Artemia have been reported (reviewed in Stệttrup 2000) To our knowledge, only one study has compared long-term diĂerences in growth rates and deformities of extensively vs intensively reared cod Imsland, Foss, Koedijk, Folkvord, Stefansson and Jonassen (2006) concluded that extensively reared cod grew faster and had a lower rate of deformities compared with intensively reared cod, but the two experimental groups were initially raised at diĂerent production sites, rendering a direct comparison dicult (Imsland et al 2006) Based on the assumption that the fast growth rates of extensively reared cod 1728 Aquaculture Research, 2010, 41, 1727^1740 juveniles are due to the feed provided during the Ârst weeks of life, we hypothesized to obtain similar high growth rates in an intensive system if natural zooplankton replaced rotifers as the start feed For this purpose, we combined the two technologies by stocking cod larvae at high densities in tanks where natural zooplankton were provided as feed during the Ârst weeks of life and where weaning occurred at an early age The nutritional composition of cod larvae reared on natural zooplankton was analysed to serve as a reference for rotifer-fed larvae A size- and temperature-dependent growth model (STDG model) was developed for cod larvae by Folkvord (2005) and the use of this model allows comparison of growth rates of cod larvae reared at diĂerent temperatures.We applied the STDG model to evaluate the growth rates of cod larvae in this experiment Further, to test if the high growth rates achieved by extensively reared cod larvae are likely due to the feed (i.e natural zooplankton) or to the rearing protocols (low density of larvae, very large volumes), the growth rates of cod larvae in a commercial semi-extensive production was compared with the intensively reared experimental larvae by the use of the STDG model Finally, the STDG model (Folkvord 2005) was applied to evaluate published growth rates of rotifer-reared larvae Materials and methods Cod larvae and natural zooplankton This experiment was conducted at the cod hatchery LoÂlab AS in Lofoten, Northern Norway Eggs were collected from common spawning tanks of the Ârstgeneration selected broodstock at the National Breeding Programme, Tromsệ in subsequent days and incubated at 5.5 1C At the age of 110^115 day degrees post fertilization and days post hatch (DPH), the larvae were transported by airplane to the experimental site Here, the larvae were stocked at 50 larvae L in three outdoor tanks of 3000 L, at a total of 150 000 larvae per tank Natural zooplankton, primarily copepods, was supplied from a 300.000 m3 enclosed seawater pond regularly fertilized to enhance primary production The zooplankton was harvested from the pond by three plankton concentrators where the pond water was Âltered through a 120 mm Unik Âlter (Unik Filtering Systems, Oslo, Norway) The concentrated plankton was then separated into the following size fractions: small (80^150 mm), medium (150^290 mm) r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 Aquaculture Research, 2010, 41, 1727^1740 and large (290^1000 mm) using a Unik Âlter The small size fraction contained mostly copepod nauplii, the medium fraction was dominated by copepodites, while the large fraction contained mainly adult copepods The small fraction of zooplankton was supplied to the larval tanks from day to DPH, followed by a mixture of the small and medium fraction from day to 12 and a mixture of all three plankton fractions from day 13 to the end of live feeding Daily samples of the zooplankton mixture fed to the experimental tanks were collected and later analysed under a dissection microscope to determine the proportions of nauplii, copepodites and copepods Natural zooplankton was fed to the tanks at 08:00, 14:00 and 20:00 hours The objective was to supply the tanks with 1500 prey items L at each feeding Unfortunately, at age 14 DPH the zooplankton concentration in the pond dropped dramatically and the experimental tanks were thereafter supplied with the maximum amount of zooplankton available Farmed cod larvae fed natural zooplankton K E T Busch et al an eye-piece micrometer At the day of sampling, 10 larvae from each tank were measured for SL (live SL) before being freeze-stored at 20 1C The other 10 larvae were measured after freezing (frozen SL) The shrinkage caused by freezing was estimated by measuring 10 larvae from each sampling before and after freezing and the following relationship was found: live SL (frozen SL 0.6491)/0.8652, R 50.98 All lengths were converted to live SL (hereafter SL) To obtain the dry mass (DM), all larvae were dried at 60 1C for 24 h and subsequently weighed using a precision balance (MX5 microbalance; Mettler-Toledo AS, Oslo, Norway) Because of the large size of the tanks, daily mortality could not be easily established At the day of termination, the biomass of larvae from each tank was measured, and the number of juveniles was calculated by dividing the biomass by the mean wet weight of the juveniles The total mortality was corrected for sampling mortality, where each sampled larvae was counted as 0.5 survivors Weaning experiment At age 19 DPH, larvae in each of the three original tanks were split in two and placed into six new tanks of 3000 L volume In three tanks, the larvae were weaned on formulated feed (Gemma micro 150 and Gemma micro 300, Skretting, Norway) from day 20 to 25 DPH (Early weaning, EW) while in the other three tanks larvae were weaned from age 27 to 32 DPH (late weaning, LW) The experiment was terminated at age 41DPH Seawater was supplied from the surface outside the pond and Âltered through a 60 mm Unik Âlter The water exchange was 16% h from the beginning of the experiment until age 32 DPH, and was then gradually increased to 36% h at age 41DPH The salinity was 35 g L throughout the experiment Oxygen was measured daily and saturation never dropped below 90% in any tank The temperature was measured daily at 08:00, 14:00 and 20:00 hours Sampling Upon arrival at the experimental site,10 larvae were sampled for length and mass measurement N 20 larvae from each tank were sampled for length and mass measurements at age 6, 9, 16, 19, 23, 26, 30, 33, 37 and 41 DPH All larvae were over-anaesthetized in metacaine (MS-222) before the measurements were undertaken Standard length (SL) was measured under a dissecting microscope equipped with Semi-extensive production For the commercial production at LoÂlab AS, 35 m impermeable plastic bags were submerged in the pond and Âlled with Âltered pond water Newly hatched larvae were stocked at a density of larvae L Filtered natural zooplankton of chosen size-fractions was constantly supplied to the bags In addition, rotifers were supplied to the bags three times per day to ensure a sucient supply of food The larvae were weaned on formulated feed from age 27 to 36 DPH A growth series of the commercial semi-extensive production at LoÂlab was obtained by weekly samples of 10 larvae from two rearing bags from age to 44 DPH Standard length and DM were obtained as described above Growth rates of cod larvae reared on rotifers In order to compare growth rates of cod larvae reared on copepods vs larvae reared on rotifers, the growth rates of eight published experiments where rotifers were used as the start feed were evaluated by the use of an STDG model (described in Growth model) (Baskerville-Bridges & Kling 2000b; Puvanendran & Brown 2002; Callan, Jordaan & Kling 2003; Monk, Puvanendran & Brown 2006; Park, Puvanendran, Kellett, Parrish & Brown 2006; Fletcher et al 2007; Garcia, Parrish & Brown 2008) The protocols in the r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 1729 Farmed cod larvae fed natural zooplankton K E T Busch et al diĂerent experiments varied regarding tank volume, stocking densities, feeding densities, rotifer feeding period and temperature (Table 1) However, using the STDG model a comparison of growth rates across different rearing temperatures is facilitated Aquaculture Research, 2010, 41, 1727^1740 the treatment with the highest growth rates from each of the published results Modelled DM was converted to SL by the following relationships given by Finn, Rệnnestad, van der Meeren and Fyhn (2002): Size range : 0:031 0:175 mg ln SL ẳ ln DM ỵ 7:799ị=3:109 Growth model The growth rates of the cod larvae in this experiment, in the semi-extensive production and in published reports were evaluated by the use of the STDG model (Folkvord 2005) The model was developed based on growth rates obtained by cod larvae reared on natural zooplankton provided ad libitum and seems to represent the growth capacity of cod larvae (Folkvord 2005) The input of the model is the DM at start and the temperatures observed throughout the experiment For all comparisons, the modelled growth for northeast Arctic cod was used The modelled daily instantaneous growth rate [speciÂc growth rate (SGR)] is given as: SGR 51.0811.79T 0.074T ln DM 0.0965aT(ln DM)210.0112T(ln DM)3 (Folkvord 2005) As the growth in DM is exponential, published graphs presenting DM might be dicult to read We therefore chose to present the comparisons of modelled and observed growth as SL An exception was the results of Garcia et al (2008), where the DM sampling series was longer than the SL series; hence, here we used DM data converted to SL (see description of conversion below) We chose to use the average SL of Size range : 0:175 1:829 mg ln SL ẳ ln DM ỵ 9:657ị=4:129 Size range : 1:829 409 mg ln SL ẳ ln DM ỵ 7:932ị=3:406 Analysis of nutritional composition At age 27 DPH, larvae from the LW treatment were sampled for analyses of nutritional composition As a limited number of larvae were available from each tank, pooled samples from the three tanks were used The larvae were Âltered and rinsed in tap water, dried with a paper towel under the sieve and frozen in aliquots on dry ice for the diĂerent analyses The larvae were transported on dry ice to the lab and stored at 80 1C until analyses Dry weight was determined gravimetrically after drying the samples at 105 1C overnight Protein was calculated as N 6.25 Nitrogen was measured using a nitrogen analyzer (Leco FP-528, St Joseph, MI, USA).Vitamin A and E were determined using normal-phase HPLC after saponiÂcation and extraction of the sample using hexane (Lie, Table Summary of rearing protocols in studies reporting growth rates of cod larvae Experiment Puvanendran and Brown (2002) Garcia et al (2008) Duration (days) Temperature ( 1C) Tank Stocking volume density (L) (L 1) Rotifer period (days) N per feeding (L 1) Number of feedings Rotifer (day 1) enrichment 42 30 40 342 4000 37 1113 3000 50 037 4000 OBrien-MacDonald et al (2006) Park et al (2006) 65 3000 50 065 4000 43 11 30 50 043 4000 Callan et al (2003) 63 10 22 75 021 9000 Monk et al (2006) 58 10.5 4500 50 045 4000 34 Baskerville-Bridges & Kling 2000ac Fletcher et al (2007) 71 22 55 035 11 000 80 75 054 800024 000 70 1011 6.511 DHA protein selco (INVE) Pavlova and Algamac s 2000 High Lipid Rotifer Enrichment Spray-dried Crypthecodinium sp DHA-selco (INVE) s Algamac 2000 Isochrysis and Algamac s 2000 DHA-selco (INVE) Culture Selco 3000 (INVE) Only the protocol of the treatment that resulted in the highest growth rates is hereby reported 1730 r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 Statistical analyses The eĂect of early and late weaning on SL and DM was tested using an analysis of variance (ANOVA), where early/late weaning was the Âxed eĂect while larvae nested in the tank and the original tank were the random eĂects Separate analyses were run for SL and DM, and each sampling day was tested separately Values are represented as means ặ SE of means Level of signiÂcance was set to Po0.05 The analyses were performed with the statistical software R (2.1.0) (R development core team 2008) Results Temperature and feeding The average temperature from hatching to the age of 25 DPH was 10.2 1C; it then rose to 15.8 1C at day 30 DPH, and to 18.8 1C by the end of the experiment (Fig.1a) On average, zooplankton was supplied at a density of 1400 prey items L between age and 14 DPH In this period, the zooplankton consisted of a majority of copepod nauplii, some copepodites and some adult copepods The copepod species observed were Pseduocalanus spp., Eurytemora longicornis, Centropages hamatus and some harpacticoids (Fig 1b) At age 15 DPH, a major drop in the zooplankton concentration in the pond occurred, and an average feeding between age 15 and age 32 DPH was 200 prey items L 1; in this period, copepodites dominated the zooplankton (Fig.1b) Survival The average survival from the start to the end (2^ 41DPH) of the experiment was 7.2 ặ 3.98% in the EW tanks and 7.2 ặ 0.42% in the LW tanks An elevated mortality was observed on the last day of the experiment Growth The growth rates in all tanks were high from the beginning of the experiment and until age 19 DPH At this time, the average SL was 9.77 ặ 0.25 mm and the DM was 0.54 ặ 0.07 mg (Fig 2) From age 19 to 26 DPH, the growth rates declined in all tanks before increasing again from age 26 to 41DPH In all tanks, (a) 20 Temperature (C ) Sandvin & Waagbệ 1994; Nll 1996; Moren, Naess & Hamre 2004) Ascorbic acid was analysed using HPLC with amperiometric electrochemical detector, as described in Mìland and Waagbệ (1998) Thiamine analysis was carried out using HPLC according to Comite' Europe'en de Normalisation (2003) For trace element analyses, freeze-dried samples of diet ingredients and rotifers were wet digested in nitric acid with 30% hydrogen peroxide using a microwave technique (Julshamn, Thorlasius & Lea 2000) The samples were then analysed for Fe, Mn, Cu, Zn and Se according to Julshamn, Lundebye, Heggstad, Berntssen and Bệe (2004), and for P according to Liaseth, Julshamn and Espe (2003) Iodine was analysed according to Julshamn, Dahl and EckhoĂ (2001) Fatty acid composition of total lipids was analysed according to Lie and Lambertsen (1991), using 19:0 as the internal standard BrieÊy, the lipid was extracted with chlorform:methanol 2:1, with the internal standard added and methylated in methanol/NaOH with BF3 The extract was separated by GLC and detected by Êame ionization (FID) The columns used were a head column (Silica 0.53 mm ID) and a CP-sil-88 column of 50 m WCOT, with an inner diameter of 0.32 mm The fatty acids were quantiÂed by an integration of the peak areas The temperature programme of the GC was 50 1C for 1min, an increase in 25 1C to 155 1C, hold for 20 min, increase of 1C to 220 1C and hold for 10 Farmed cod larvae fed natural zooplankton K E T Busch et al 15 10 (b) 2000 Prey items (L1) Aquaculture Research, 2010, 41, 1727^1740 1600 1200 800 Others Adult copepods Copepodites Nauplii 400 0 10 15 20 25 30 35 Age (days post hatch) 40 45 Figure Average daily temperatures (a) and average densities of natural zooplankton (b) supplied at each feeding to Gadus morhua L larvae in a weaning experiment In the tanks where larvae received early weaning, zooplankton feeding was stopped after day 25 post hatch r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 1731 Farmed cod larvae fed natural zooplankton K E T Busch et al the growth rate was similar to the modelled growth rate until age 19 DPH (Fig 2a), and declined compared with the modelled growth from age 19 to 26 DPH When the observed SL was $ 10 mm, the observed and modelled SL were similar, while at SL $ 20 mm the average observed SL was 30% lower than the modelled growth (Table 2) Aquaculture Research, 2010, 41, 1727^1740 vord 2005) until age 25 DPH After this time, SL and DM increased at rates close to the model in bag and somewhat slower in bag (Fig 3) The observed and modelled SL were similar at $ 10 mm, while the average observed SL was 14% lower compared with the modelled SL at $ 20 mm (Table 2) Growth of rotifer-reared larvae Weaning experiment The SL of larvae in the EW treatment was signiÂcantly higher than in the LW treatment at age 30 DPH (ANOVA, P 0.0015) No signiÂcant diĂerences between treatments were found on other sampling days (Fig 2a) The DM was signiÂcantly higher in the EW treatment at age 30 DPH (ANOVA, P 0.0069), whereas no signiÂcant diĂerences between treatments were found on the other sampling days (Fig 2b) The eĂect of tank of origin was not signiÂcant on either DM or SL on any sampling day Nutritional composition Semi-extensive production 30 Standard length (mm) In the two bags from the commercial production, SL and DM increased as predicted by the model (Folk(a) 25 20 15 10 Dry mass (mg) (b) The increase in SL of cod larvae reared on rotifers was lower than the modelled growth in the published results (Fig 4) The diĂerence between the observed growth and modelled growth appears evident from the start of the experiments (Fig 4) When the observed SL was $ 10 mm, it was on average 44% shorter than the modelled SL In three out of the eight published experiments, the larvae reached SL longer than 20 mm At SL of $ 20 mm, these larvae were on average 45% shorter compared with the modelled SL (Table 2) 10 Protein constituted 51% and nitrogen 8.1% of the larval DM (Table 3) Of the minerals found in copepodfed larvae, iodine, manganese and selenium were considerably higher, copper and zinc were similar and phosphorus was lower than in intensively reared cod larvae fed rotifers.Vitamin A also appeared lower in the larvae fed copepods than in those fed rotifers, although the latter showed large variation The levels of thiamine, vitamin C and vitamin E are given for the copepod-fed larvae, but were not analysed in the larvae fed rotifers (Table 3) The fatty acid composition in the larvae fed copepods was characterized by lower levels of monounsaturates and n-6 polyunsaturatted fattyacid (PUFA) and higher levels of n-3 PUFA than in larvae fed rotifers This trend was also seen in the levels of ARA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and in the EPA/ARA ratio (Table 4) 0.1 Discussion 0.01 14 21 28 35 Age (days post hatch) 42 Figure Growth of Gadus morhua L larvae measured as standard length (mm) (a) and dry mass (mg) (b) in common rearing tanks (ALL) and in early weaning (EW) and late weaning (LW) treatments The dashed line in the upper graph shows the modelled growth at the temperatures observed Data represent mean values ặ SE of means 1732 Combining the use of intensive techniques and natural zooplankton The use of natural zooplankton as live feed for cod larvae has traditionally been associated with extensive and semi-extensive rearing methods (van der Meeren & Naas 1997) Some experiments on cod larvae have been conducted using indoor tanks and natural zooplankton (Otterlei, Nyhammer, Folkvord & r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 Aquaculture Research, 2010, 41, 1727^1740 Farmed cod larvae fed natural zooplankton K E T Busch et al Table A comparison between observed and estimated SL for Gadus morhua L larvae of SL $ 10 and $ 20 mm Standard length $ 10 mm Standard length $ 20 mm Experiment Age (DPH) Obs SL (mm) Mod SL (mm) Diff (%) Puvanendran & Brown (2002) Garcia et al (2008) OBrien-MacDonald et al (2006) Park et al (2006) Callan et al (2003) Monk et al (2006) Baskerville-Bridges & Kling (2000b) Fletcher et al (2007) Weaning experiment (this study) Commercial production (this study) 35 30 49 29 36 36 29 33 19 25 10.6 9.8 9.8 9.5 11 10.5 9.5 9.8 10.5 14.5 20.7 23 16.25 19.6 20.8 15.13 16.53 9.29 11 26.9 52.9 57.4 41.5 43.9 49.5 37.2 45.6 5.5 4.5 Age (DPH) Obs SL (mm) Mod SL (mm) Diff (%) 64 23.5 40.5 42.0 64 62 41 38 20.9 22.5 20.3 19 42.1 40 28.1 22 50.4 43.8 27.8 13.6 (a) 30 Temperature and survival Standard length (mm) SL was estimated by a growth model for cod larvae (Folkvord 2005) The cod larvae in the eight published experiments were fed Brachionus (spp.) while the larvae in this study were fed natural zooplankton DPH, days post hatch; SL, standard length; Obs.,observed; Mod.,modelled; DiĂ., diĂerence The temperature rose to high levels at the end of the experiment, but only at age 41DPH when the average temperature reached 18.8 1C, high mortalities were observed Yin and Blaxter (1987) found that the temperature tolerance (where 50% of the larvae survived 24 h) was 18 1C for newly hatched cod larvae and 15.5 1C at yolk exhaustion for unfed cod larvae Our results indicate that the temperature tolerance of fed cod larvae is higher than 17.8 1C and close to 18.8 1C The observed survival was in the lower range of reported survivorships in intensive and extensive systems In intensive systems, survival of cod larvae of 3^40% have been reported for experiments terminated between age 43 and 72 DPH (BaskervilleBridges & Kling 2000b; Callan et al 2003; Monk et al 2006; OBrien-MacDonald, Brown & Parrish 2006; Park et al 2006; Fletcher et al 2007) Survival of cod larvae from hatching until metamorphosis in marine ponds have been reported to be on average 23% (range 3^42%) (reviewed in Blom 1995) In the present experiment, the relatively high mortality may have been caused by the low feeding densities from age 14 DPH and by the high temperatures at the end of the experiment 25 20 15 10 Dry mass (mg) (b) 100 10 0.1 0.01 14 21 28 35 Age (days post hatch) 42 49 Figure Growth of Gadus morhua L larvae measured as standard length (mm) (a) and dry weight (mg) (b) in two diĂerent rearing bags (1and 2) of the commercial semi-extensive production The dashed line in the upper graph shows the modelled growth at the observed temperatures Data represent mean values ặ SE of means Stefansson 1999) However, the larval densities have been low ($ larvae L 1) compared with intensive rearing systems To our knowledge, the present experiment is the Ârst attempt to combine the use of large-scale intensive rearing methods with natural zooplankton as live feed for cod Weaning Until age 19 DPH (SL 9.77 mm), the growth rates were optimal compared with the growth model (Folkvord 2005) Such high growth rates would probably allow a very early weaning Baskerville-Bridges and Kling (2000b) managed to complete weaning on a micro- r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 1733 Standard length (mm) Farmed cod larvae fed natural zooplankton K E T Busch et al 50 30 20 20 10 10 0 Standard length (mm) 10 20 30 40 50 60 70 50 10 20 30 40 50 60 70 30 40 50 60 70 40 50 60 70 20 30 40 50 60 Age (days post hatch) 70 50 OBrienMacDonald et al.2006 (C) 40 Park et al 2006 11 (C) 40 30 30 20 20 10 10 0 Standard length (mm) Garcia et al 2008 1113 (C) 40 30 50 10 20 30 40 50 60 70 50 Callan et al 2003 10 (C) 40 30 20 20 10 10 10 20 Monk et al 2006 10.5 (C) 40 30 0 Standard lenght (mm) 50 Puvanendran & Brown 2002 (C) 40 Aquaculture Research, 2010, 41, 1727^1740 10 20 30 40 50 60 70 50 10 20 30 50 BaskervilleBridges & Kling 2000b 1011 (C) 40 Fletcher et al 2007 6.511 (C) 40 30 30 20 20 10 10 0 10 20 30 40 50 60 Age (days post hatch) 70 10 Figure Growth in standard length (SL) (mm) from eight published results where Gadus morhua L larvae were fed Brachionus spp The points represent the average SL for the treatment with the highest growth rates from the reported experiments In the graph from the experiment reported by (Garcia et al 2008), dry mass has been converted to SL The dashed lines are the modelled growth at the temperatures observed particulate diet at a larval SL of 8.5 mm In Norwegian commercial hatcheries, cod larvae are now commonly weaned directly from rotifers onto formulated feed Weaning may start when the larvae are SL of $ 7.5 mm and 25 days old and Ânish when the larvae reach SL of $ 8.0 mm (T A Hangstad, pers comm.) With the high growth rates observed in our experiment, weaning could have been Ânished well before the age of 16 DPH, something which could substantially shorten the time depending on live feed using current rearing protocols Poor growth was observed between age 19 and 26 DPH in both the EW and LW treatment These low growth rates may have been caused by stress from the transfer of larvae to the ex- 1734 perimental tanks at age 19 DPH However, the low feed density in this period is a probable cause of the poor growth rates The prey concentrations needed to support optimal growth rates are likely to vary with larval densities, larval sizes and prey type Puvanendran and Brown (1999) reported higher growth rates for cod larvae that received 4000 rotifers L than for larvae that received 2000 rotifers L Further, prey concentrations of 1000 rotifers L did not support survival beyond age 32 DPH (Puvanendran & Brown 1999) Rajkumar and Kumaraguru vasagam (2006) fed Acartia clausi (Giesbrecht) at densites of $ 1700 L with no signs of limited growth rates in Lates calcarifer (Bloch) r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 Aquaculture Research, 2010, 41, 1727^1740 Farmed cod larvae fed natural zooplankton K E T Busch et al Table Protein, nitrogen, selected minerals and vitamins on dry weight from Gadus morhua L larvae aged 27 DPH reared on natural zooplankton Dry matter (% of wet wt) Protein (% of dry wt) Nitrogen (% of dry wt) mg kg dry wt Iodine Manganese Copper Zinc Selenium Phosphorus mg kg dry wt Vitamin A (sum retinol) Thiamine (B1) Vitamin C Vitamin E (a-tokopherol) Experimental larvae Larvae fed rotifers 15.3 51 8.1 12.5 ặ 0.2 na na 29 13 142 5.1 16 497 1.76 ặ 0.51 4.7 ặ 0.4 6.1 ặ 1.2 146 ặ 1.1 ặ 0.3 19 470 ặ 520 5.2 9.9 594 76 12.6 ặ 4.7 na na na Each nutrient was analysed from a pooled sample of larvae from three rearing tanks Data from larvae reared on industrially produced Brachionus plicatilis (Mller) (26 DPH) are given for comparison (mean ặ SD, n 4; Hamre et al 2008b) na, not analysed; wt, weight Table Fatty acid composition (% of total fatty acids) from Gadus morhua L larvae aged 27 DPH reared on natural zooplankton 16:0 16:1 (n-7) 18:0 18:1 (n-9) 18:1 (n-7) 18:2 (n-6) 20:4 (n-6) ARA 20:4 (n-3) 20:5 (n-3) EPA 22:5 (n-3) 22:6 (n-3) DHA Total saturates Total monounsaturated Total (n-3) PUFA Total (n-6) PUFA DHA/EPA EPA/ARA Experimental larvae Larvae fed rotifers 16.5 1.4 6.2 4.9 2.5 0.8 3.3 0.8 11.3 1.6 37.6 24.8 10.3 53.6 5.1 3.33 3.42 14.4 3.0 8.6 9.6 2.7 3.4 2.8 1.3 7.2 2.7 25.4 25.1 19.9 38.2 7.1 3.5 2.5 ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ ặ 0.1 0.2 0.1 0.1 0.0 0.1 0.0 0.0 0.1 0.0 0.7 0.2 0.4 0.6 0.1 0.1 0.1 Fatty acid composition was analysed from a pooled sample of larvae from three rearing tanks Data from larvae reared on industrially produced Brachionus plicatilis (Mller) (26 DPH) are given for comparison (mean ặ SD, n 4; Hamre et al 2008b) The total sums include fatty acids not reported in this table PUFA, polyunsaturatted fattyacid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid In a pond experiment, prey densities in the range of 1.5^32.3 prey L supported optimal growth of cod larvae with an initial stocking density of $ 30 larvae m (Busch, Folkvord, Otterễ, Hutchinson & Svễsand 2009) The present results indicate that prey densities below 200 L not support optimal growth of cod larvae when natural zooplankton is used as feed in intensive rearing systems After a period of slow growth, the larvae again started to grow fast from age 26 and 30 DPH for the EW and LW treatment respectively (Fig 2) Larvae in the EW tanks were signiÂcantly longer and heavier at age 30 DPH, indicating that it was an advantage to receive formulated feed at an early age in this experiment The limited larval growth in the LW treatment may have been caused by the low densities of prey organisms The weaning period was short compared with what has been reported previously (Fletcher et al 2007; Baskerville-Bridges & Kling 2000b); but with the high growth rates observed at the end of the weaning periods, we consider weaning to have been successful To our knowledge, this is the earliest weaning reported for cod larvae reared on natural zooplankton Such an early weaning reduces the quantity of natural zooplankton needed and would make it possible to increase the number of larvae produced Additionally, an early weaning reduces the risks associated with the use of live feed, e.g sudden drops in zooplankton availability Natural zooplankton vs rotifers The growth rates of the intensively reared experimental larvae and the commercial semi-extensive production were both optimal until the larvae reached $ 10 mm (Figs and 3) As the larvae in these two systems were produced by two entirely diĂerent protocols, but with the same food, we suggest that the high growth rates observed in ponds and semi-extensive systems might be caused rather by the natural zooplankton than by low densities of larvae or large volumes We evaluated reported growth rates of cod larvae reared on rotifers (Fig and Table 2) and found that despite a wide range of protocols regarding light regimes, feed densities, rotifer enrichment and temperature (Table 1), all of the reported growth rates were sub-optimal and surprisingly similar across the studies The modelled maximum SGR occurs at $ 10 mm (Folkvord 2005), being 19% at 10 1C At this length, rotifer- r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 1735 Oocyte growth stimulation by methyl farnesoate B C Paran et al Aquaculture Research, 2010, 41, 1887^1897 vels had similar eĂects on the GSI results The concentration of Vg declined signiÂcantly in the shortterm treatment with 1.0 mg MF (Fig 2); however, no signiÂcant trend in the Vg level was observed in any other treatment (Fig.3) In contrast, the haemolymph protein concentration decreased signiÂcantly in all the treatment groups, except in the long-term treatment with 0.1 mg MF (Figs 4, 5) Statistical analysis Treatment comparison of data was performed using two-way repeated measures ANOVA, and subsequent diĂerences were determined using Tukeys test at Po0.05 The GSI was analysed using a standard t-test analysis Results Response to MF administration Response to eyestalk ablation in adult females The mean values of GSI were higher in MF-treated shrimp than the control shrimps in all the treatment groups after 21 days (short term and long term; 0.1 and 1.0 mg MF injections) (Fig 1) Both MF dosage le- On day 0, ovaries were in the pre-vitellogenic stage (GSI 51.3 ặ 0.2; Figs and 8b) in both intact and eyestalk ablated groups In response to unilateral eyestalk ablation, GSI started to increase (Po0.05) and reached a maximum of 13.5 ặ 0.6 on day 21 In contrast, control shrimp remained in the pre-vitellogenic stage as shown in Figs and 8b, without signiÂcant changes in GSI (P40.05, Fig 6) The levels of Vg in eyestalk ablated and intact females on day were similar (4.12 ặ 1.0 and 3.90 ặ 0.22 mg mL 1, respectively, Fig 6) The levels started increasing and reached a maximum of 7.5 ặ 0.3 mg mL at day14, when the GSI increased to 8.6%, and remained at the same level until the end of the experimental period (Fig.6) Although eyestalk ablation did not alter the Vg levels during the experimental period, the Vg levels in eyestalk ablated females were elevated compared with intact females (Po0.05) Unilateral eyestalk ablation caused an increase in the total haemolymph protein concentration (Po0.05) (Fig 6) 18 * 16 14 GSI(%) 12 * * * 10 S 0.1 àg L 0.1 àg S 1.0 àg L 1.0 àg Treatments Vitellogenin levels (mg /ml) Figure Gonadosomatic index (GSI) of Sicyonia ingentis in response to short- and long-term treatment of 0.1 and 1.0 mg methyl farnesoate (MF) injection Methyl farnesoate increased GSI after a 25-day treatment when compared with controls DiĂerences between means (t-test, Po0.05) 10 Control (0.1 àg) Control (1 àg) c cc c c c d d MF (0.1 àg) MF (1 àg) d d e d d e e aa a b e a b b b b b Days bb bb e b b 14 e b b 21 Figure Haemolymph vitellogenin levels (mg mL 1) of Sicyonia ingentis in response to a short-term treatment with 0.1 and 1.0 mg methyl farnesoate (MF) over 25 days Vitellogenin levels decreased between and 21 days in both the MFtreated and the control shrimp Vitellogenin levels decreased signiÂcantly in 1.0 mg MF-treated shrimp compared with control Means and SEM followed by the same letter are similar (two-way ANOVA,Tukeys test; Po0.05) 1890 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 Aquaculture Research, 2010, 41, 1887^1897 Oocyte growth stimulation by methyl farnesoate B C Paran et al 10 Vitellogenin levels (mg / ml) Control (0.1 àg) MF (0.1 àg) Control (1 àg) MF (1 àg) a a a a a aa a a a a a b a b b b b b b b b b b b b b b b b b b b bb b b b b c b bb b b b b b 18 21 Days 12 15 Figure Haemolymph vitellogenin levels (mg mL 1) of Sicyonia ingentis in response to a long-term treatment with 0.1 and 1.0 mg methyl farnesoate (MF) over 25 days Vitellogenin levels decreased between and 21 days in both the MFtreated and the control shrimp No diĂerence was found in the Vg levels between MF and control shrimp Means and SEM followed by the same letter are similar (two-way ANOVA,Tukeys test; Po0.05) Total hemolymph protein (mg/ml) 60 a a a a 50 a b b a a a Control (0.1 àg) Control (1 àg) c b b c 40 d d c b c e MF (0.1 àg) MF(1 àg) b b d d b e c 30 d b e c e c e c e 20 10 2 Days 14 21 Figure Haemolymph protein levels (mg mL 1) of Sicyonia ingentis in response to a short-term treatment with 0.1 and 1.0 mg methyl farnesoate (MF) over 25 days Protein levels decreased between and 21 days in both the MF-treated and the control shrimp Haemolymph protein levels decreased in both 0.1 and 1.0 mg MF-treated shrimp compared with control Means and SEM followed by the same letter are similar (two-way ANOVA,Tukeys test; Po0.05) Response of eyestalk ablation in juveniles Intact and unilaterally ablated juveniles remained at the oogonial stage (Fig 8a) throughout the experimental period In contrast, bilaterally ablated females developed ovaries and showed signiÂcantly elevated GSI on days 7, 14 and 21 days (GSI: 0.12 ặ 0.04, 1.46 ặ 0.17 and 2.76 ặ 0.3, respectively, Po0.05; Figs and 8d) On day after bilateral ablation, ovaries were at the endogenous vitellogenic stage, with follicle cells enveloping oocytes, and at the mature stage on day 21 with yolk granules and cortical rods (Fig 8d) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 1891 Oocyte growth stimulation by methyl farnesoate B C Paran et al Total hemolymph protein (mg/ ml) 60 a cc 50 a a cc a b cc b b dd b d b b Aquaculture Research, 2010, 41, 1887^1897 Control (0.1 àg) MF (0.1 àg) Control (1 àg) MF(1 àg) d e d b b e d d d b b e b b b 40 e d b d b b b b b b e 30 e e e 20 10 2 Days 12 15 18 21 Figure Haemolymph protein levels (mg mL 1) of Sicyonia ingentis in response to a long-term treatment with 0.1 and 1.0 mg methyl farnesoate (MF) over 25 days Protein levels decreased between and 21 days in both the MF-treated and the control shrimp Shrimp haemolymph protein levels decreased with 1.0 mg MF treatment compared with the control Means and SEM followed by the same letter are similar (two-way ANOVA,Tukeys test; Po0.05) Discussion We demonstrated for the Ârst time that administration of MF increased GSI in S ingentis when compared with the control After 21 days, MF-treated shrimp ovaries increased in mass such that they were similar to naturally maturing S ingentis (Anderson, Clark & Chang 1985) The role of MF in reproduction was originally inferred by correlating oocyte size and MF levels in the haemolymph (Borst, Laufer, Landau, Chang, Hertz, Baker & Schooley1987; Laufer et al 1987) Elevated titres of haemolymph MF were correlated with higher ovarian weight in the freshwater prawn M rosenbergii (Wilder et al 1994) and in the spider crab, L emarginata (Laufer & Biggers 2001) The increase in GSI by MF administration supports previous studies in decapod crustaceans (Table 1) Collectively, these data suggest that MF acts to increase oocyte size and consequently ovarian weight An increase in ovary weight suggests that MF may play a role in crustacean reproduction similar to that of JH-III in insects, where uptake of protein into oocytes is increased by widening follicle cell spacing (Chapman 1998) The data presented here constitute another piece of evidence suggesting that MF is a key regulatory factor involved in reproductive development in crustaceans Most studies on the eĂect of MF in female crustacean reproduction used an increase in GSI or oviposition as endpoints; however, it is generally believed that these endpoints might obscure immediate eĂects 1892 from injected hormone (Tsukimura 2001) Haemolymph Vg level is one of the easily determined endpoints to evaluate the vitellogenic phase of the animal (Tuberty et al 2002) In this study, therefore, we evaluated haemolymph Vg as an additional endpoint to determine the reproductive status of the animal The present data on the eĂect of MF administration show that MF did not increase the Vg level in any of the treatments (short term and long term/0.1 and 1.0 mg MF) Conversely, the eyestalk ablation data show that haemolymphVg levels increased compared with both the initial control and the concurrent controls The heterogeneity of the initial reproductive stage of the animal (GSI of ES experiments:1.4 ặ 0.6, for adults and undetectable for juveniles; MF treatment experiments: 4^6%) used in this experiments possibly caused a diĂerence in the dynamics of Vg in both experiments In Fenneropenaeus indicus, the haemolymph Vg levels decreased at a GSI 3.4^7.1% after an initial upsurge during the early maturation phase (Vazquez-Boucard, Levy, Ceccaldi & Brogren 2002) Similarly, the haemolymph Vg levels declined in advanced ovarian stages of F merguiensis (Auttarat, Phiriyangkul & Utarabhand 2006) and Marsupenaeus japonicus (Tahara, Suitoh & Hattori 2005) These studies, in line with our present Ândings, indicate that haemolymph Vg levels are poor biomarkers for estimating reproductive activity in shrimps with advanced ovarian stages Declining Vg levels observed in response to MF treatment may be due to the massive sequestration r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 Aquaculture Research, 2010, 41, 1887^1897 Figure Changes in the gonadosomatic index (GSI) (a), haemolymph vitellogenin levels (b) and haemolymph protein levels (c) at diĂerent intervals after unilateral eyestalk ablation in adult Sicyonia ingentis females Means and SEM followed by the same letter are similar (one-way ANO VA, Duncans multiple range test) SigniÂcant diĂerence from the concurrent control (t-test, Po0.05) of yolk protein into oocytes, a decrease in synthesis or both Further, this study indicates that MF decreased the total haemolymph protein in all treatments, except in the long-term treatment with 0.1 mg MF, when compared with controls, suggesting that the total haemolymph protein was incorporated into oocytes, and resembling the JH model by active uptake of protein by Vg receptors found throughout the oocyte Despite the considerable evidence that supports the gonadotropic eĂect of MF, several studies report Oocyte growth stimulation by methyl farnesoate B C Paran et al Figure Changes in the gonadosomatic index (GSI) (a), haemolymph vitellogenin levels (b) and haemolymph protein levels (c) at diĂerent intervals after bilateral eyestalk ablation in juvenile Sicyonia ingentis females Means and SEM followed by the same letter are similar (one-way ANO VA, Duncans multiple range test) SigniÂcant diĂerence from the concurrent control (t-test, Po0.05) conÊicting results, suggesting either no eĂect or inhibition of reproduction (Table 1; Mak, Choi, Tiu, Hui, He, Tobe & Chan 2005; Tiu, Hui, He, Tobe & Chan 2006; Tsukimura, Nelson & Linder 2006; Marsden, Hewitt, Boglio, Mather & Richardson 2008) These results may be inÊuenced by species-speciÂc diĂerences, the mode of action of MF, pre-capture physiological and environmental conditions and differences in the formulation of the MF integrated diet (Marsden et al 2008) Furthermore, the half-life of MF in the haemolymph is less than an hour (Tsukimura 2001), suggesting that the concentration may have Êuctuated with the periodicity of feeding with the MF integrated diet (Marsden et al 2008) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 1893 Oocyte growth stimulation by methyl farnesoate B C Paran et al Aquaculture Research, 2010, 41, 1887^1897 Figure Histology of ovaries in eyestalk ablated adult and juvenile Sicyonia ingentis (a) Control female on day 21; (b) unilaterally ablated female on day 7; (c) unilaterally ablated female on day 14; (d) bilaterally ablated juvenile on day 21 Oocyte development was classiÂed into oogonial cells (OO) pre-vitellogenic oocytes (PR), yolky oocytes (YO) and cortical oocytes (CO); arrows indicate cortical rods Table EĂect of methyl farnesoate on crustacean reproduction Species Mode of study Nature of response Triops longicudatus In In In In In In In In Litopenaeus vannamei Sicyonia ingentis Penaeus monodon Macrobrachium rosenbergii Homarus americanus Procambarus clarkii Cherax quadricarinatus vitro vivo vitro vivo vivo vivo vivo vivo In vivo In vivo In vivo No effects in adults Riley and Tsukimura (1998) Inhibition in juveniles Tsukimura et al (2006) Reproductive stimulation Tsukimura and Kamemoto (1991) Improvement of reproductive performance Laufer (1992) Reproductive stimulation Present study Improvement of reproductive performance Hall, Mastro and Prestwich (1999) Reproductive inhibition Marsden et al (2008) No effect Wilder et al (1994) No effect Tsukimura, Kamemoto and Borst (1993) Reproductive stimulation Laufer et al (1998) Reproductive stimulation Rodriguez et al (2002) No effect Abdu, Barki, Karplus, Barel, Takac, Yehezkel, Laufer and Sagi (2001) The main Ânding of our eyestalk ablation experiment is that bilateral eyestalk ablation induces vitellogenesis in juvenile S ingentis (19.1^25 mm CL and 3.4^8.3 g BW) The earlier studies in penaeoidean shrimps report that bilateral ablation resulted in high mortality, such that this procedure is no longer practiced in commercial penaeid hatcheries, as unilateral ablation is sucient to attain acceptable reproduction in a commercial production system (Muthu & Laxminarayana 1982) The most accepted consensus 1894 References regarding the endocrine control of reproduction in crustaceans is that GIH, synthesized in the X-organ sinus gland complex of eyestalk, suppresses vitellogenesis Once the animal is released from this inhibitory action (by eyestalk ablation), animals become reproductive (Adiyodi & Adiyodi 1970) The present study strongly suggests that in juvenile shrimp, the remaining X-organ/sinus gland complex in unilaterally ablated females shrimp is able to secrete sucient amount of GIH to maintain the r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 Aquaculture Research, 2010, 41, 1887^1897 suppression of Vg synthesis, and that only bilateral eyestalk ablation eliminates endogenous GIH The induction of vitellogenesis in juveniles by bilateral eyestalk has not been reported so far in penaeoidean shrimp, although vitellogenesis in juvenile caridean shrimp after bilateral eyestalk ablation has been reported (Jayasankar, Jasmani,Tsutsui, Aida & Wilder 2006) Studying the eĂect of uni- and bilateral eyestalk ablation in F indicus (as Penaeus indicus), Mohamed and Diwan (1991) suggested that while unilateral eyestalk ablation leads to vitellogenesis, bilateral ablation leads to moulting In the present study, however, bilateral eyestalk ablation in juvenile shrimps resulted in vitellogenesis similar to that seen in adults, although the per cent of females completing vitellogenesis was low (23%) Our study also demonstrated that juvenile S ingentis can form a useful in vivo model system to evaluate the endocrine control of reproduction This study clearly indicates that MF accelerates ovarian growth in adult females, and it oĂers a potential crustacean gonadotropin that could be used in commercial penaeid hatchery However, further studies on spawning performance (e.g fecundity and hatching rate), as compared with eye stalk ablation, are essential to develop a commercial reproductive technology using MF An alternative delivery system for MF administration has to be developed as mortality due to the injection of hormones is found to be high In eyestalk ablation studies, atretic oocytes became apparent These results indicate that the induction of ovarian development by eyestalk ablation does not induce the condition of natural gametogenesis, suggesting that in addition to GIH, other hormones probably play a role in regulating Vg synthesis and oocyte development (Okumura 2007) Recent experimental evidences on the involvement of neurotransmitters (5-hydroxytryptamine, HT) in vitellogenesis and oocyte maturation of closed and open thelycum shrimps (Alfaro, ZuÔnỡiga & Komen 2004; Wongprasert, Asuvapongpatana, Poltana, Tiensuwan & Withyachumnarnkul 2006; Santhoshi, Sugumar & Munuswamy 2009) further elaborate the multi-hormonal hypothesis regulating the female gametogenesis Further studies on ovarian cytology and Vg mRNA expression proÂle in response to MF treatment could help to understand the endocrinological control of vitellogenesis, and it would yield a comprehensive hormonal treatment protocol to improve the captive maturation and spawning in penaeid commercial hatchery facilities Oocyte growth stimulation by methyl farnesoate B C Paran et al Acknowledgments This study was partially supported by the Department of Biology, CSU, Fresno A scholarship grant for B C P was provided by the Department of Biotechnology, Government of India We thank Dr Ryan L Earley for his assistance in histological preparation We are grateful to Dr A.G Ponniah, Director, CIBA, Chennai, for his constant encouragement We are grateful to Nagaraju Kotagiri for his assistance in shrimp husbandry.We are grateful to anonymous reviewers for their critical comments on the initial version of the manuscript References Abdu U., Barki A., Karplus I., Barel S.,Takac P., Yehezkel G., Laufer H & Sagi A (2001) Physiological eĂects of methyl farnesoate and pyriproxfen on wintering female crayÂsh Cherax quadricarinatus Aquaculture 202,163^175 Adiyodi K.G & Adiyodi R.G (1970) Endocrine control in decapod crustacea Biological Review 45,121^165 Alfaro J., ZuÔnỡiga G & Komen J (2004) Induction of ovarian maturation and spawning by 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350^360 Tahara D., Suitoh K & Hattori H (2005) Hemolymph vitellogenin levels during maturation and post-spawning in the female kuruma prawn, Marsupenaeus japonicus Aquaculture 245, 311^319 Tiu S.H., Chan S & Tobe S.S (2009) The eĂect of farnesoic acid and 20-hydroxyecdysone on vitellogenin gene expression in the lobster, Homarus americanus, and possible roles in the reproductive process General and Comparative Endocrinology 166, 337345 Tiu S.H.K., Hui J.H.L., He J.-G.,Tobe S.S & Chan S.-M (2006) Characterization of vitellogenin in the shrimp Metapenaeus ensis: expression studies and hormonal regulation of MeVg1 transcription in vitro Molecular Reproduction and Development 73, 424^436 Tsukimura B (2001) Crustacean vitellogenesis: its role in oocyte development American Zoologists 41, 465^476 Tsukimura B & Kamemoto F.I (1991) In vitro stimulation of oocytes by presumptive mandibular organ secretions in the shrimp, Penaeus vannamei Aquaculture 92, 59^66 Tsukimura B., Kamemoto F.I & Borst D.W (1993) Cyclic nucleotide regulation of methyl farnesoate synthesis by r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 Aquaculture Research, 2010, 41, 1887^1897 mandibular organ of the lobster, Homarus americanus The Journal of Experimental Zoology 265, 427^431 Tsukimura B., Bender J.S & Linder C.J (2000) Development of an anti vitellin ELISA for the assessment of reproduction in the ridge back shrimp, Sicyonia ingentis Comparative Biochemistry and Physiology Part A 127, 215^224 Tsukimura B., NelsonW.K & Linder C.J (2006) Inhibition of ovarian development by methyl farnesoate in the tadpole shrimp, Triops longicaudatus Comparative Biochemistry and Physiology Part A 144,135^144 Tsutsui N., KimY.K., Jasmani S., Ohira T.,Wilder M.N & Aida K (2005) The dynamics of vitellogenin gene expression diĂers between intact and eyestalk ablated kuruma prawn Penaeus (Marsupenaeus) japonicus Fisheries Science 71, 249^256 Tuberty S.R., Nates S.F & McKenney C.L Jr (2002) Polyclonal antisera against estuarine crustacean vitellins: a molecular approach to reproductive endocrinology and toxicology In: Modern Approaches to the Study of Crustacean (ed by E Escobar-Brionen & F Abwarez), pp 29 37 Kluwer Academic/Plenum Publishers, New York, NY, USA Van Herp F & Soyez D (1997) Arthropoda ^ crustacea In: Progress in Reproductive Endocrinology (ed by K.G Adiyodi & R.G Adiyodi), pp 247^275 Oxford and IBH, New Delhi, India Vazquez-Boucard C.G., Levy P., Ceccaldi H.J & Brogren C.H (2002) Developmental changes in concentrations of vitellin, vitellogenin, and lipids in hemolymph, hepatopancreas, and ovaries from diĂerent ovarian stages of Indian Oocyte growth stimulation by methyl farnesoate B C Paran et al white prawn Fenneropenaeus indicus Journal of Experimental Marine Biology and Ecology 281, 63^75 Wainwright G., Prescott M.C., Rees H.H & Webster S.G (1996) Mass spectrometric determination of methyl farnesoate proÂles and correlation with ovarian development in the edible crab, Cancer pagurus Journal of Mass Spectrometry 31, 1338^1344 Wilder M.N., Okumura T., Suzuki Y., Fusetani N & Aida K (1994) Vitellogenin production induced by eyestalk ablation in juvenile giant freshwater prawn Macrobrachium rosenbergii and trial methyl farnesoate administration Zoological Science 11, 45^53 Wonglapsuwan M., Phongdara A & Chotigeat W (2009) Dynamic changes in gene expression during vitellogenic stages of white shrimp: Fenneropenaeus merguiensis de Man Aquaculture Research 40, 633^643 Wongprasert K., Asuvapongpatana A., Poltana P., Tiensuwan M & Withyachumnarnkul B (2006) Serotonin stimulates ovarian maturation and spawning in the black tiger shrimp, Penaeus monodon Aquaculture 261, 1447^ 1454 Wyban J (2009) World shrimp farming revolution: industry impact of domestication breeding and widespread use of speciÂc pathogen free Penaeus vannamei In: Rising tide Proceedings of Special Session of Sustainable Shrimp Farming (ed by C.L Browdy & D.E Jory), pp 12^ 21 The world Aquaculture Society, Baton Rouge, LA, USA Yano I (1987) Oocyte development in the Kuruma prawn Penaeus japonicus Marine Biology 99, 547^553 r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1887^1897 1897 Aquaculture Research, 2010, 41, 1898^1904 doi:10.1111/j.1365-2109.2010.02556.x SHORT COMMUNICATION Effect of density on Scortum barcoo (McCulloch & Waite) juvenile performance in circular tanks Liu Gang,Tan Hongxin, Luo Guozhi & Sun Da Chuan Ministry of Education Key Laboratory of Exploration and Utilization of Aquatic Resources, Shanghai Ocean University, Shanghai, China Correspondence: L Guozhi, Ministry of Education Key Laboratory of Exploration and Utilization of Aquatic Resources, Shanghai Ocean University, Shanghai 201306, China E-mail: gzhluo@shou.edu.cn In recent years, Âsh welfare has been a much-debated topic Stocking density used in commercial aquaculture has been highlighted as an area of speciÂc concern Fish farmers are keen to rear Âsh at higher stocking densities in order to reduce the production costs and enhance production eciency Stocking Âsh at a high density (HD) may have both positive and negative eĂects on farmed Âsh (Abbott, Dunbrack & Orr 1985) High stocking densities have been shown to have a positive eĂect on the behaviour of African catÂsh; increasing densities reduced the occurrence of agonistic behaviour in African catÂsh larvae and juveniles (Hecht & Appelbaum 1988; Kaiser, Weyl & Hecht 1995a, b; Hecht & Uys 1997; Almazan 2004) The negative eĂects of HD include competition for space and food; changes in growth, energy metabolism, behaviour, physiology and immunologic function; decrease in the viability, population growth rate and survival rate of the population; and increased possibility of sickness (Andrews, Knight, Page, Matsuda & Brown 1971; Allen 1974; Fagerlund, McBride & Stone 1981; Suresh & Lin 1992) Although stocking density has been demonstrated to aĂect various aspects of the welfare of farmed Âsh, diĂerences among species are distinct (Nieuwegiessen, van de Olwo, Khong,Verreth & Schrama 2009) High densities may impair the welfare of some Âsh species, such as trout (Ellis, James, Scott & Stewart 2004), salmon (Ewing & Ewing 1995) and seabream (Montero, Izquierdo, Tort, Robaina & Vergara 1999) On the other hand, high densities have positive eĂects on other species, such as Arctic charr (Jệrgensen, Christiansen & Jobing 1993) and African catÂsh (Almazan 2004)) Moreover, the results of high 1898 stocking density can diĂer even within the same species For example, published data on the eĂect of stocking density on the growth performance of African catÂsh are contradictory Several studies reported decreasing growth performance with increasing density (Hecht & Appelbaum 1988; Haylor 1991, 1993; Hengsawat, Ward & Jaruratjamorn 1998), whereas others showed increasing growth performance with increasing density (Hecht & Uys 1997; Almazan 2004) or no eĂect of density (Hengsawat et al.1998) The barcoo grunter or jade perch (Scortum barcoo) is a freshwater ÂnÂsh native to Lake Eyre and the BullooBancannia catchments It is a new species now being cultured in the developing freshwater ÂnÂsh aquaculture industry Over the last few years, this species has been farmed by several individuals in recirculating systems (Liu, Luo, Sun,Tan & Zhu 2003) and by a handful of pond farmers Because an artiÂcial environment is used when culturing these Âsh, knowledge of the appropriate rearing conditions is necessary to design the most eĂective and productive operation Currently, no research has been carried out on the eĂect of stocking density on jade perch Therefore, the purpose of this study was to assess the inÊuence of stocking density on the welfare indicators of juvenile jade perch and to provide a theoretical basis for the production of jade perch Material and methods The adaptation period This study was conducted at the Fishery Machinery and Instrument Research Institute, Chinese Academy r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd Aquaculture Research, 2010, 41, 1898^1904 The eĂect of density on Scortum barcoo L Gang et al pipe and a submerged bioÂlter (Fig 1) A moderate Êow-through rate allowed renewal of water to the system 12 times per day During both the adaptation and the experimental period, a certain amount of water was changed and unconsumed food and faeces were removed to maintain water quality Water was sampled daily at 10:00 and 16:00 hours to assess water quality and to decide whether the water needed to be changed; the volume of the changed water was recorded The water quality in the experimental aquaria was maintained at pH 7.4^ 7.5; total ammonia nitrogen o5 mg L (ammonia molecules NH3 o0.08 mg L 1); NO2-N o1.5 mg L 1, DO 44.5 mg L 1; and temperature 23.5 ặ 0.1 1C Fish were fed a commercial pellet diet (moisture, 3%; crude protein, 45.0%; crude lipid, 8.0%; Ca, 1.8%; P, 1.5%; lysine, !2.9%; methionine, 1.4%), Suzhou Tong Wei Special Feed (Wujiang, China) three times each day at 9:00, 15:00 and 19:00 hours by hand The aquaria were kept under ambient lighting, and the photoperiod was not controlled of Fisheries Science Juvenile jade perch were obtained from a commercial Âsh farm on July 2008 The average weight (ặ SD) was 3.35 ặ 0.33 g Before the experiment, Âsh were maintained for months in the experimental recirculating aquaculture system to allow them to adapt to their new environment During this 3-month adaptation period, the aquaria were Âlled with water with the following characteristics: tempera1 ture 23 ặ 1C; pH 7.0^7.5; NH1 (NH3-N o4.5 mg L o0.06 mg L ); nitrite nitrogen (NO2-N) o1mg L 1; salinity o5; and dissolved oxygen (DO) 4^6 mg L Experimental design Fish (mean body weight 50 ặ 0.03 g SD) were starved for 24 h, anaesthetized with tricaine methanesulphonate (MS-222) and then sorted for allocation to experimental tanks randomly The Âsh were held in separate tanks at three diĂerent densities: low density (LD) at 100 Âsh m 3, kg m 3; moderate density (MD) at 260 Âsh m 3, 13 kg m 3; and HD at 360 Âsh m 3, 18 kg m Three tanks per density were used The experiment was started after the handling and the experimental Âsh started consuming food normally The experiment was carried out from 16 October to December 2008 The experiment was divided into three periods of 15 days each On day 15 (named P1, 31 October), day 30 (named P2,15 November) and day 45 (named P3,1 December) eight Âsh were sampled from each tank for the measurement of the welfare parameters After sampling, the water volume of each tank was adjusted to maintain the proper Âsh density Measured parameters Fish selection and blood sampling For each sampling time point, Âsh were starved for 24 h, and then eight Âsh were randomly and quickly collected with nets from every tank They were then anaesthetized with 0.25 g L tricaine methanesulphonate (MS-222) for min, weighed (ặ 0.01g) and measured (fork length ặ 1mm), and a blood sample was taken Blood was taken from the caudal blood vessels using a1mL hypodermic syringe containing mg of Na2 EDTA The procedure of catching, anaesthetizing and collecting blood was completed for all sampled Âsh within to minimize handling stress Serum was separated by centrifugation (1505 g, min, 1C) and stored at 20 1C until analysis Water quality control and experimental condition The experiment was conducted in 0.5 m3 Âbre-reinforced plastic aquaria, each with a drain, an inÊow Figure Experimental aquaria with bioÂlters.1, tank; 2, bioÂlter; 3, drain pipe r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1898^1904 1899 The eĂect of density on Scortum barcoo L Gang et al Aquaculture Research, 2010, 41, 1898^1904 Water quality parameters Data analysis pH, water temperature and DO were measured using a YSI556 (YSI,Yellow Springs, OH, USA) Total ammonia nitrogen and NO2-N were analysed spectrophotometrically according to standard methods (American Public Health Association1998) In this study, aquaria were used as the experimental units for analysing growth performance data For physiological data, Âsh were used as the experimental unit Growth performance and physiological data were analysed using a one-way analysis of variance (ANOVA), followed by the Tukey HSD post hoc test The error terms of these ANOVA analyses were tested for homogeneity of variances and normality Results were considered to be statistically signiÂcant at Po0.05 Fish performance parameters The recorded body weight, body length and food intake were used to calculate the following growth indexes: Daily weight gain DWGị : DWG ẳ W2 W1 ị=t2 t1 ị Specific growth rate SGRị : SGR ẳ ẵln W2 ln W1 ị=t2 t1 ị 100 Feed conversion ratio FCRị : FCR ẳ feed supply kgị=fish biomass increase kgị Growth efficiency GEị : GE ẳ ẵW2 W1 ị=F 100 Survival %ị ẳ 100Nf Ni ị=Ni In these equations, W1 and W2 are the weights at t1 and t2, n is the number of Âsh, F is the weight of the total feed (g), Ni initial number of Âsh and Nf Ânal number of Âsh Blood serum parameters Serum cortisol (ng mL ) levels were measured using enzyme-linked immunosorbent assays (Ellis et al.2004) (ELISA analyzer, BIO-TEK ELX-800, BIO-TEK Instruments,Winooski,VT, USA) using a kit developed for the quantitative measurement of cortisol in Âsh serum and serum (QRCT-3013331321131 EIA\UTL, from SiRuike Biotechnology, Shanghai, China) Serum C3 (mg mL 1) levels were measured using ELISAU g L using a kit (QRCT-301330010021 EIA\ UTL) developed for the quantitative measurement of C3 Âsh serum and plasma from SiRuike Biotechnology Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured using the UV rate method (U L 1) (Hitachi 7600-010 Automatic Biochemical Analyzer, Hitachi HighTechnologies, Tokyo, Japan); the kits were the AST Kit and the ALT Kit (Wako Pure Chemical Industries, Osaka, Japan) Spleen weight/body index (SWBI) For each Âsh sampled, the spleen was isolated, weighed and expressed as per cent body weight 1900 Results Blood parameters Cortisol level The serum cortisol level of the experimental Âsh was aĂected by stocking density at the Ârst and the second sampling time; the diĂerence was signiÂcant between LD and HD groups but not between LD and MD groups or MD and HD groups At the third sampling time, the signiÂcant diĂerence had disappeared (Table 1) Table SigniÂcant diĂerences in the physiology parameters P1 ALT LD MD 0.012 MD HD o0.01 LD HD 0.069 AST LD MD 0.768 MD HD 0.003 LD HD 0.006 C3 LD MD 0.949 MD HD 0.137 LD HD 0.128 Spleen weight/body index LD MD 0.162 MD HD 0.516 LD HD 0.447 Cortisol LD MD 0.055 MD HD 0.798 LD HD 0.026 P2 P3 0.029 o0.01 0.043 0.635 0.235 0.103 0.705 0.006 0.016 0.629 0.166 0.061 o0.01 0.035 o0.01 o0.01 0.091 o0.01 0.096 0.426 0.014 0.001 0.113 o0.01 0.062 0.753 0.040 0.571 0.855 0.743 LD MD represents the comparison between LD and MD, and so on SigniÂcant diĂerence Po0.05 LD, MD and HD show the low-density group (100 Âsh m 3), the moderate-density group (260 Âsh/m3) and the high-density group (360 Âsh m 3); P1, P2 and P3 show day 15, day 30 and day 45 ALT, alanine aminotransferase; AST, serum aspartate aminotransferase r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1898^1904 The eĂect of density on Scortum barcoo L Gang et al Serum ALT (u/L) Serum cortisol (ng / ml) Aquaculture Research, 2010, 41, 1898^1904 Sampling time Sampling time Figure Serum alanine aminotransferase (ALT) levels of juvenile jade perch stocked at three rearing densities at times P1, P2 and P3 LD, MD and HD indicate the low-density group (100 Âsh m 3), the moderate-density group (260 Âsh m 3) and the high-density group (360 Âsh m 3); P1, P2 and P3 show day 15, day 30 and day 45 Serum C3 (ng/L) Serum AST (u/L) Figure Serum cortisol levels of juvenile jade perch stocked at three rearing densities at times P1, P2 and P3 LD, MD and HD represent the low-density group (100 Âsh m 3), the moderate-density group (260 Âsh m 3) and the high-density group (360 Âsh m 3); P1, P2 and P3 indicate day 15, day 30 and day 45 Sampling time Sampling time Figure Serum C3 levels of juvenile jade perch stocked at three rearing densities at times P1, P2 and P3 LD, MD and HD show the low-density group (100 Âsh m 3), the moderate-density group (260 Âsh m 3) and the high-density group (360 Âsh m 3); P1, P2 and P3 show day 15, day 30 and day 45 Figure Serum aspartate aminotransferase (AST) levels of juvenile jade perch stocked at three rearing densities at times P1, P2 and P3 LD, MD and HD indicate the lowdensity group (100 Âsh m 3), the moderate-density group (260 Âsh m 3) and the high-density group (360 Âsh m 3); P1, P2 and P3 show day 15, day 30 and day 45 During the experiment, the serum cortisol level of every density decreased with time Low density, MD and HD decreased from 78.5 to 24.13, 79.21 to 25.24 and 83.37 to 24.17 ng mL 1, respectively, from P1 to P3 (Fig 2) groups, and the LD and HD groups at P2 and P3 but not at P1 Serum C3 level During the experiment period, the serum C3 level of the experimental Âsh ranged from 2.58 to 7.72 mg mL The C3 level of all densities increased early and decreased later (Fig 3) The C3 level of HD Âsh was lower than that of LD and MD throughout the experiment A signiÂcant diĂerence in the C3 level was detected between the LD and the MD ALT and AST levels During the experiment, the ALT of the Âsh serum ranged from 6.8 to 19.8 U L and the AST ranged from 49.9 to 108.6 U L The ALT and the AST levels of the LD and MD groups increased early and decreased later (Fig 4); in the HD group, the levels decreased continuously from P1 to P3 When comparing the diĂerent density groups, a signiÂcance diĂerence was detected between the LD and the HD groups and the MD and HD groups, but not between the LD and MD groups, at P1 and r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1898^1904 1901 Spleen weight / Body weight The eĂect of density on Scortum barcoo L Gang et al Table Growth parameters of jade perch in the stocking density experiment Stocking density (fish m 3) Sampling time Figure The spleen weight/body weight of juvenile jade perch stocked at three rearing densities at times P1, P2 and P3 LD, MD and HD indicate the low-density group (100 Âsh m 3), the moderate-density group (260 Âsh m 3) and the high-density group (360 Âsh m 3); P1, P2 and P3 show day 15, day 30 and day 45 P2; however, at P3, no signiÂcant diĂerence was detected among the three densities Thus, stocking density inÊuenced the serum ALT and AST of the experimental Âsh (Fig 5) SWBI The SWBI of the experimental Âsh ranged from 0.05% to 0.15% The SWBI of the HD group was signiÂcantly lower than that of the LD and MD groups (Fig 6) There was no signiÂcant diĂerence among the three densities at P1 A signiÂcant diĂerence appeared between the LD and the HD groups at P2 and P3, but not between MD and HD or MD and LD at P2 and not between LD and MD at P3 Thus, stocking densities in this experiment inÊuenced the SWBI of the experimental Âsh Growth performance Table shows the growth performance responses of jade perch to stocking density The highest and lowest Ânal body weight were observed at the stocking density of LD (99.62 g) and HD (69.18 g), respectively, and GE, Ânal body weight, SGR and FCR diĂered signiÂcantly between treatments The survival rate of all treatments was 100% Discussion Stocking density can aĂect Âsh in the following ways (1) Stress from worsened water quality The water quality declines with increasing density (e.g decreased DO, increased CO2 and ammonia) (2) Stress from the HD itself, including the increased chance of 1902 Aquaculture Research, 2010, 41, 1898^1904 Item LD (100) MD (260) HD (360) Initial body weight (g) Final body weight GE (%) SGR (%) FCR DWG (g day 1) Survival (%) 65.62 ặ 1.97a 68.82 ặ 2.94a 64.63 ặ 3.49a 96.62 ặ 2.99a 88.62 ặ 6.81ab 75.54 ặ 6.36b 89.17 0.864 1.122 0.692 100a 35.30 0.561 2.833 0.439 100a 25.67 0.346 3.896 0.242 100a DiĂerent letters on the parameters in a given row indicate significant diĂerence (Po0.05); otherwise, whereas the same letter indicate no signiÂcant diĂerence LD, MD and HD show the low-density group (100 Âsh m 3), the moderate-density group (260 Âsh m 3) and the high-density group (360 Âsh m 3); P1, P2 and P3 show day 15, day 30 and day 45 encountering aggression, the intensiÂed eĂects of community stress and changes in physiological conditions due to chronic stress Because the water quality in this experiment was controlled and maintained at a suitable level, the physiological stress of the Âsh could be attributed directly to the crowding stress, which in turn inÊuenced feeding, growth, upgrowth and energy metabolism Jade perch can tolerate high nitrate and ammonia concentrations in the water, and throughout the experiment, the values of the key water quality parameters were maintained close to the recommended values for jade perch aquaculture in all density treatments (Liu et al 2003) Thus, the diĂerences observed among the density treatments could be attributed to the density itself In response to stressors such as handling or crowding, a Âsh undergoes a series of biochemical and physiological changes in an attempt to compensate for the challenge and cope with the stress (Iwama, Afonso & Vijayan 2004) The overall eĂect of stress may be considered to be the change in the biological condition beyond the normal resting state that challenges homoeostasis and, thus, presents a threat to the Âshs health These stress-induced changes are categorized as primary and secondary (including metabolic, haematological, hydromineral and structural changes) and tertiary (or whole animal) responses Many of these responses can be used as quantitative indicators of stress, although investigators need to be aware of the various non-stress factors that can also inÊuence these conditions (Pickering 1981) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1898^1904 Aquaculture Research, 2010, 41, 1898^1904 Because the body weight of the experimental Âsh in this study was o100 g and the blood volume sampled was no more than 1mL, a range of welfare indicators (i.e physiological and performance factors) were used to assess the eĂects of stocking density on Âsh welfare Growth reduction is generally considered to be a good indicator of chronic stress, and in some species, density acts as a chronic stressor Some studies have shown decreasing growth with increasing density, whereas others have shown increasing growth performance with increasing density (Hecht & Uys 1997; Almazan 2004) or no eĂect of density (Hengsawat et al 1998) The diĂerences in outcome are due to the varying abilities of diĂerent species to counteract environmental stress Even within the same species, diĂerent individuals can exhibit a diĂerent response Thus, the eĂect of density stress on a Âsh population may not be manifested solely as an increase or a decrease in growth, and this issue requires further study The high production rate generated by HD aquaculture is based not only on the high average growth rate but also on the number of Âsh present Most farmers cannot create a completely stress-free environment and thus cannot attain the ideal growth rate The proÂts of aquaculture depend on the production and the costs associated with it, such as water consumption and illness For this reason, it is important to determine the Âsh density that is most economical for the farmer Researchers often measure the serum cortisol level to assess stress in Âsh because of its responsiveness to acute stressors, its relative ease of measurement and its functional signiÂcance in the physiological processes that aĂect Âsh health (Barton & Iwama 1991) The serum cortisol level of the experimental Âsh in this study ranged from 24.13 to 82.37 ng mL 1, which was similar to that reported for some Âsh species (e.g 9^60 ng mL in Cyprinus carpio (Pottinger 1998; Ruane, Huisman & Komen 2001), higher than that reported for other Âsh species (e.g 1^13 ng mL 1in Salmo trutta (Sumpter, Pickering & Pottinger1985; Pickering & Pottinger1987) and lower than that of the African catÂsh (64.3^138.9 ng mL (Almazan 2004; Martins, Schrama, & Verreth 2006; Nieuwegiessen, van de Zhao, Verreth & Schrama 2009) Generally, resting or unstressed levels of circulating corticosteroids in Âsh are o30^40 ng mL 1, but ideally, they should be o5 ng mL The experimental results of this study showed that the serum cortisol levels of the experimental Âsh pulled through at the end of the experiment The eĂect of density on Scortum barcoo L Gang et al Variations among species and strains and between wild and hatchery-reared Âsh are evident in their responses to stressors Such diĂerences appear, at least in part, to have a genetic basis In addition, the magnitude of stress responses may be inÊuenced by the Âshs developmental or maturational state In this study, the serum C3 level of the experimental Âsh declined with increasing density, which is similar to the results reported by Yin, Lam and Sin (1995), Rotllant, Pavlidis, Kentouri, Abad and Tort (1997) and Montero et al (1999) The spleen is an important immune organ of Âsh, and the SWBI of the experimental Âsh increased with increasing density at P2 and P3; this Ânding is diĂerent from that reported by Wang, Li,Wang, Cai and Wu (2004) All Âsh responded over time with a decline in serum cortisol and an early increase, followed by a decline in serum C3, ALT and AST levels It was shown that the Âsh was motion-adaptive to the experimental condition However, this process involved consumption of energy, and so growth diĂerences appeared Conclusion Under experimental conditions and with controlled water quality, the densities examined in this study (5, 13 and 18 kg m 3) signiÂcantly inÊuenced the serum cortisol, serum C3 and serum ALTand AST levels The growth, Ânal body weight and SGR also showed signiÂcant diĂerences depending on the stocking density Although the juvenile jade perch in this study were able to withstand the negative eĂects of a high stocking density, it still inÊuenced the Âshs growth Acknowledgments This study was funded by the Shanghai Key Discipline Construction Project number S30701 and Start-up funding for Dr of Shanghai Ocean University Project number B-8201-08-0216 The authors would sincerely like to thankWang Zhewei, LuoYuwen and Li Chun for their help during Âsh rearing and sampling References Abbott J.C., Dunbrack R.L & Orr C.D (1985) The interaction of size and experience in dominance relationships of juvenile steelhead trout (Salmo gairdneri) Behaviour 92, 241^253 Allen K.O (1974) EĂects of stocking density and water exchange rate on growth and 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F-test, Po0.001) (Fig 2) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1741^1747 1743 Aquaculture Research, 2010, 41, 1741–1747 Temperature e¡ects on tawny pu¡er eggs and larvae Y-H Shi et al Table 1 Hatch rate, incubation period, viability of 24... from 23 to 26 1C This optimal temperature range was similar to the water temperature observed from May to June in Bohai Bay, China r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1741^1747 Aquaculture Research, 2010, 41, 1741^1747 Temperature e¡ects on tawny pu¡er eggs and larvae Y-H Shi et al Table 2 Survival rate and growth rate of tawny pu¡er larvae... survival and r 2009 The Authors Aquaculture Research r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 1727^1740 Aquaculture Research, 2010, 41, 1727^1740 normal development of cod larvae Aquaculture Nutrition 14, 51^60 Hamre K., Mollan T.A., S×le Ò & Erstad B (2008b) Rotifers enriched with iodine and selenium increase survival in Atlantic cod (Gadus morhua) larvae Aquaculture 284, 190^195 Imsland... survival of larvae from 3 to 19 dah The temperatures of the experimental treatments were 20, 23, 26 and 29 1C About 2400 larvae (3 dah; r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1741^1747 Aquaculture Research, 2010, 41, 1741^1747 Temperature e¡ects on tawny pu¡er eggs and larvae Y-H Shi et al 3.26 Æ 0.02 mm total length, mean Æ SEM, n 5 20) were haphazardly... respective portions of the polypary and the stalk Their dry weight, inorganic weight and percentage of organic weight were determined r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S glaucum I Sella & Y Benayahu Comparison between cuttings reared in the reef, £ow-through... cuttings reared under the di¡erent salinities (one-way ANOVA, P40.05) The survival of cuttings at the end of this experiment ranged r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S glaucum I Sella & Y Benayahu Dry weight (g) 0.03 (a) 0.025 0.02 0.015 0.01 0.005 0... percentage of organic weight under di¡erent feeding regimes on day 60 of the experiment (Æ SE, number of cuttings indicated in Table 1) r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1748^1758 1753 Aquaculture Research, 2010, 41, 1748–1758 0.08 30 0.07 25 0.06 Dry weight (g) 35 20 15 10 5 0 10-15 cm Disc diameter 20-23 cm Figure 6 Samples removed from reef-collected... to develop a protocol for rearing miniature cuttings removed from colonies of S glaucum in a closed seawater system We compared the r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 1748^1758 Aquaculture Research, 2010, 41, 1748^1758 Rearing cuttings of the soft coral S glaucum I Sella & Y Benayahu derived colonies with those reared in a £ow-through seawater