Aquaculture nutrition, tập 17, số 3, 2011

Aquaculture Nutrition 2011 17; 235–243 doi: 10.1111/j.1365-2095.2009.00734.x DE School of Marine and Tropical Biology, James Cook University, Townsville, Qld, Australia Cultured barramundi, Lates calcarifer, suffer from abnormalities affecting the jaw, opercula and spine The aim of this study was to quantify for the first time the effects of supplemented dietary vitamin C, vitamin D3 and ultraviolet (UV) light on the development of jaw, opercula and spinal deformities Four diets were formulated to contain (i) no vitamin C or vitamin D3, (ii) only vitamin D3, (iii) only vitamin C and (iv) both vitamin C and vitamin D3 In addition, two commercial diets (diets and 6) were also tested These diets were replicated in the presence, and in the absence, of ultraviolet (UV) light as this may also affect skeletal development Diets formulated with 170 ± mg kg)1 and 195 ± 0.5 mg kg)1 of vitamin C (diets and 4, respectively) and the commercial diets (diets and 6) had significantly lower incidences of spinal deformities (0–2.5%; P < 0.01) and opercula deformities (nil detected) Spinal deformities were Ôbroken backÕ syndrome, which was found only in the precaudal vertebrae, and lordosis which was only in the caudal vertebrae UV light and vitamin D3 did not affect spinal or opercula deformities There was no change in the occurrence of jaw deformities in vitamin C, vitamin D3 or UV light treatments KEY WORDS: deformities, Lates calcarifer, ultraviolet light, vitamin C, vitamin D3 Received March 2009, accepted 30 September 2009 Correspondence: Matthew Fraser, School of Marine and Tropical Biology, James Cook University, Townsville, QLD, Australia, 4811 E-mail: matthew.fraser@jcu.edu.au Morphological deformities pose a problem for many propagated fish species as they affect the survival and commercial value of the product (Divanach et al 1997) As such, Ó 2010 Blackwell Publishing Ltd investigating the development of skeletal deformities in cultured finfish has been the focus of recent studies (Fjelldal et al 2007; Sullivan et al 2007; Kamler et al 2008) Species with a documented incidence of abnormal development include the Red sea bream, Pagrus major (Temminck and Schlegel) (Kihara et al 2002; Hattori et al 2003), European sea bass, Dicentrarchus labrax (Linnaeus) (Paperna 1978; Barahona-Fernandes 1982; Daoulas et al 1991; Koumoundouros et al 2001), Rainbow trout, Oncorhynchus mykiss (Walbaum) (Dabrowski et al 1990), Milkfish, Chanos chanos (Forsskal) (Gapasin et al 1998), Tilapia, Oreochromis mossambicus (Peters) (Soliman et al 1986) and Barramundi, Lates calcarifer (Block) (Fraser et al 2004; Fraser & de Nys 2005) The three major regions of deformation are the jaw (Roberts et al 2001), opercula (Galeotti et al 2000) and spine (Koumoundouros et al 2001) with environmental, nutrition and genetic factors proposed to be causal mechanisms (Andrades et al 1996) Because deformities affect both bone and cartilage tissue, investigating the requirements for their correct development has the potential to determine and mitigate the underlying causes of morphological deformities Vitamin C is required in the formation of collagen, a principle component of bone and cartilage (Horton et al 1993) Vitamin C is an essential dietary nutrient (Soliman et al 1986; Dabrowski et al 1990; Alexis et al 1997; Li & Robinson 2001), because teleosts are unable to produce, L-gulonolactone, the enzyme required for vitamin C biosynthesis (Dabrowski et al 1990) Many studies have demonstrated the requirement of dietary vitamin C for correct skeletal development in teleosts including Coho salmon, Oncorhynchus kisutch (Walbaum), Rainbow trout, O mykiss (Halver et al 1969), Channel catfish, Ictalurus punctatus (Rafinesque), (Wilson & Poe 1973), Spotted snakehead, Channa punctatus (Bloch) (Mahajan & Agrawal 1979) and Tilapia, O mossambicus (Soliman et al 1986) Vitamin D3 is also suggested to play a role in teleost skeletal deformation (Galeotti et al 2000) with fish reportedly having a higher vitamin requirement than terrestrial animals (NRC 1993) Vitamin D3 regulates calcium absorption and bone mineralization in vertebrates and requires ultraviolet (UV) light exposure for its formation in most terrestrial vertebrates (Webb 1993) However, the function of vitamin D3 in teleost skeletal metabolism and the role of UV radiation in regulating the synthesis of vitamin D3 from its precursors are not clearly defined (Graff et al 2002; Lall & Lewis-McCrea 2007) Studies on vitamin D3 requirements in cultured fish have investigated its effects of on growth and mortality in salmonids (Hilton & Ferguson 1982), but none have reported a relationship between vitamin D3 and the development of deformities The barramundi, L calcarifer is a developing species for finfish aquaculture production in Australia and southeast Asia (Tucker et al 2002) and suffers from spinal, jaw and opercula deformities during the larval and postlarval phases (Fraser et al 2004; Fraser & de Nys 2005) Furthermore, juvenile Lates calcarifer fed diets devoid of vitamin C develop skeletal deformities including lordosis, scoliosis and Ôbroken backÕ syndrome (Phromkunthong et al 1997) The minimum requirement of vitamin C in juvenile L calcarifer to prevent pathological signs of deficiency is 30 mg kg)1 (as ascorbal phosphate) (Phromkunthong et al 1997); however, the quantification and specific histological identification of spinal deformities that result from a dietary vitamin C deficiency has not been determined Similarly, the roles of vitamin D3 and UV light in the development of morphological deformities in barramundi have not been addressed By documenting the nature and frequency of deformities this study quantifies, for the first time, the quantitative effects of vitamin C, vitamin D3 and UV light on the development of morphological deformities in juvenile L calcarifer Four dietary treatments varying in vitamin C (ascorbic acid) and vitamin D3 (cholecalciferol) levels (diets 1, 2, and 4), and two commercial diets (diet and 6), were fed in the presence of, and in the absence of, ultraviolet (UV) light (Table 1) Each diet was replicated with five tanks per light treatment Diets to were prepared from individual base ingredients (Table 2) Each vitamin and mineral was individually weighed and combined to form a premix used in diet preparation (Tables & 4) with vitamin C and vitamin D3 individually added as required (Table 3) Vitamin C in the form Table Experimental design used for the manipulation of diet and ultraviolet light exposure for juvenile Lates calcarifer Treatment Diet UV light No UV light 51 62 No Vit C or D3 Vit D3 Vit C Vit C and D3 Commercial diet Commercial diet No Vit C or D3 Vit D3 Vit C Vit C and D3 Commercial diet Commercial diet 2 Skretting barramundi pellet (3 mm) Ridley Aqua-feed barramundi pellet (3 mm) Table The base ingredients used in formulating diets to Ingredient Quantity in (g kg)1) Fish meal Cellulose Casein Fish oil Gelatine Starch1 469.8 217.9 175.6 52.2 34.1 16 Gelatinized by autoclaving at 110 °C for 20 Ingredients obtained from: Fish meal, Fish oil; Ridleys Aqua-Feed Company, Brisbane, Qld, Australia Cellulose; Hahnflock Hahn and Co., Germany Casein; Dairy Farmers Association, Malanda, Qld, Australia Gelatine; Trumps Pty Ltd., Brisbane, Qld, Australia Starch; Poppy cornflour, Australia of free ascorbic acid was used in this study because it has the highest level of bioavailability (Li & Robinson 2001) Because free ascorbic acid is readily oxidized during feed manufacture, a supplement of 2000 mg kg)1 was added to allow for losses (as quantified below) Previous work has shown that vitamin C supplements of less than 2000 mg kg)1 are not toxic in L calcarifer (Boonyaratpalin et al 1989) The minimum requirement of vitamin D3 for normal growth in L calcarifer has not been determined Therefore, supplemental vitamin D3 levels were formulated according to previous work with Atlantic salmon, Salmo salar The level of 100 000 IU of vitamin D3 was taken from Graff et al (2002) who found this level of vitamin D3 to be non-toxic in S salar All ingredients were combined in a Hobart mixer (Model A120, Hobart Corporation, Troy, OH, USA) and mixed for a minimum of 30 with distilled water being added to the mixture until the appropriate consistency was achieved for pelletization Each diet was extruded (Model A120) to form a 3-mm pellet for the first weeks of the trial and a 4-mm pellet for the remaining weeks of culture All diets were Aquaculture Nutrition 17; 235–243 Ó 2010 Blackwell Publishing Ltd Table The vitamins combined to form a premix for diets to Vitamin Quantity (g kg)1 of diet) Retinylacetate Menadione Alpha-tocopherol Choline chloride Myo-inositol PABA Thiamin Riboflavin Pyridoxine HLC Pantothenate acid Nicotine acid Biotin Cyanocobalamin Folic acid Ethoxyquin Citric acid Cellulose 0.012 0.033 0.375 1.2 0.255 0.105 0.018 0.021 195 · 10)4 555 · 10)4 0.075 · 10)4 · 10)5 45 · 10)4 1275 · 10)4 18.174 Vitamin C levels present after processing were 30 ± mg kg)1 for diet 1; 40 ± mg kg)1 for diet 2; 170 ± mg kg)1 for diet 3; 195 ± 0.5 mg kg)1 for diet 4; 50 ± mg kg)1 for diet 5; 45 ± 0.5 mg kg)1 for diet Vitamin D3 levels present after processing were 11 200 ± 800 IU kg)1 for diet 1; 164 000 ± 32 000 IU kg)1 for diet 2; 8600 ± 200 IU kg)1 for diet 3; 82 000 ± 6000 IU kg)1 for diet 4; 8200 ± 200 IU kg)1 for diet 5; 14 200 ± 600 IU kg)1 for diet Table The minerals combined to form a premix for diets to Mineral Quantity g kg)1 of diet Aluminium chloride Cobalt chloride Copper sulphate Magnesium sulphate Manganese sulphate Sodium selenate Zinc sulphate Cellulose 25 · 10)4 0.001 15 · 10)4 1.5 375 · 10)4 · 10)4 0.185 31625 · 10)4 oven dried (50 °C) overnight and broken into designated sized pellets and stored ()12 °C) between feeding Two samples of all diets were analysed for vitamin C (free ascorbate) and vitamin D3 (cholecalciferol) content using HPLC (2-6-dichloroindopheno; titrimetric method) analysis (performed by Dairy Technical Services Ltd., Melbourne, Vic., Australia) Commercial diets (diets & 6) also contain supplemented vitamin C as a stabilized monophosphate salt (ascorbal-2-monophosphate 30–40 mg kg)1) which is not quantifiable using this method (Dairy Technical Services), but is nutritionally available (Lin & Shiau 2004) Aquaculture Nutrition 17; 235–243 Ó 2010 Blackwell Publishing Ltd Juvenile L calcarifer (25–30 mm total length) was supplied from a commercial hatchery in Mourylian, Queensland and housed at the Marine and Aquaculture Facilities Unit at James Cook University The fish were kept in a freshwater recirculating system consisting of 60 tanks (70 L in capacity) Eight fish were weighed (0.01 g) and allocated to a tank randomly assigned a dietary treatment (n=5) Each tank received gentle and constant aeration with 100% water exchange every hour supplied through a biological and sand filter Temperature was maintained between 24–30 °C and photoperiod at 13L:11D The maximum levels for ammonia, nitrite and nitrate attained during the trial were · 10)4, 0.001 and 0.08 g L)1, respectively Water from the recirculating system was exchanged by 25–70% daily as required to maintain water quality Fish were acclimated for 50 days before salt bathing and anaesthetizing (0.08 g L)1 benzocaine solution; Sigma E 1501) The weight of fish per tank was recorded (0.01 g), and fish were visually assessed for jaw, opercula and spinal deformities Only fish without visually identifiable deformities were used All fish were fed twice daily to satiety, and each tank was cleaned daily by siphoning Mortalities were removed, weighed and stored at )12 °C The visible lighting in both the UV light and non-UV light treatments was supplied using three cool white fluorescent tubes (Philips Fluorotone, 36 W, ÔTLÕD 36W/33, wavelength 400–700 nm) Ultraviolet light was supplied in the UV light treatment using six UV fluorescent tubes (NEC black light, 40 W, wavelength 300–425 nm) The non-UV light treatment was separated from the UV light treatment using a nontransparent black screen The experiment ran for weeks after which time all fish were euthanized in ice slurry Each fish was visually assessed for spinal, jaw and opercula deformities and the total mass of fish for each tank recorded (0.01 g) Growth was measured as % mass gain and was determined by dividing the increase in mass by the initial mass multiplied by 100 (for each tank) Growth was measured only to indicate fish were fed appropriately and that normal growth was achieved The fish were immediately transported on ice to Townsville General Hospital for X-raying (Philips Optimus Digital Radiography Unit, set at 50 kV and 4.00 mAs, processed on Agfa laser film) All spinal deformities were recorded for each treatment including the location of the vertebrae and number of vertebrae affected Vertebral numbers are referenced as one being cranial to 24 being caudal Mean incidence of deformities (%) The effects of UV light and supplimented vitamin C and vitamin D3 on spinal deformity development were analysed 30 25 20 15 10 5 Diet Mean incidence of deformities (%) Figure The incidence of spinal deformities (Ôbroken backÕ syndrome and lordosis) (mean ± SE pooled from UV+ and UV) treatments) in juvenile Lates calcarifer fed formulated diets varying in vitamin C and vitamin D content (diets to 4) and two commercial diets (diets and 6) over weeks by a three-factor ANOVA Diets and were not incorporated in the analysis as they contained vitamin C as stabilized monophosphate salt (see Analysis of vitamin C and D3 in Methods section) and therefore could not be compared with diets to However, they provide a useful control for comparison with standard commercial diets Log (ln + 1) transformations of spinal deformity data minimized the heterogeneity of variance but not completely As a result, a was set at P = 0.01 (Quinn & Keough 2002) Opercula deformity data were analysed only for the effects of UV light and dietary vitamin D with two-factor ANOVA because of the occurrence of zeros in most diet treatments Scatter plots and histograms of residuals were used to examine the ANOVA assumptions of homogeneity of variances and normality, respectively (Quinn & Keough 2002) The effects of UV light, vitamin C and vitamin D on growth were analysed by a threefactor ANOVA Scatter plots and histograms of residuals were used to examine the ANOVA assumptions of homogeneity of variances and normality, respectively (Quinn & Keough 2002) Frequency distributions of Ôbroken backÕ syndrome and lordosis along the vertebral column were compared using a Kolmogorov-Smirnov test All analyses were performed using SPSS (Version11, SPSS Inc., Chicago, IL, USA) 50 40 30 20 10 Diet Figure The incidence of opercula deformities (mean ± SE pooled from UV+ and UV) treatments) in juvenile Lates calcarifer fed formulated diets varying in vitamin C and vitamin D content (diets to 4) and two commercial diets (diets and 6) over weeks (n = 5) Only non-supplemented vitamin C diets affected the incidence of skeletal deformities in barramundi Supplemented vitamin C had a significant affect on the development of spinal (threefactor ANOVA, df = 1, P < 0.01) (Fig 1) and opercula deformities (Fig 2) (Table 5) Diets supplemented with vitamin C (diets and 4) had no or very low occurrence of spinal deformity (diet 3, 0%; diet 4, 1.25 ± 1.25%) (Fig 1; Table 5) and there were no deformities of the opercula (Fig 2; Table 5) Similarly, both commercial diets (diets and 6) had Table Three-factor Analysis of Variance for the effects of vitamin C, vitamin D and UV light on the development of spinal deformities and one-factor Analysis of Variance for the effects of UV light on the development of opercula deformities in juvenile Lates calcarifer (P = 0.01) F -value P -value Degrees of freedom Mean square Factor Spinal Opercula Spinal Opercula Spinal Opercula Spinal Opercula UV light Vitamin C Vit D3 UV light · Vit C UV light · Vit D3 Vit C · Vit D3 UV light · Vit C · Vit D3 1 1 1 1 – – – – – – 1.939 3259.117 7.629 27.324 4.127 43.518 3.252 0.270 – – – – – – 0.010 17.603 0.041 0.147 0.022 0.235 0.017 1.522 – – – – – – 0.919 0.05) (Table 5), and levels of vitamin C and D3 did not affect the formation of jaw deformities Similarly, UV light did not affect the incidence of spinal (three-factor ANOVA, df = 1, P > 0.05) or opercula deformities (two-factor ANOVA, df = 1, P > 0.05) (Table 5) There was no interaction between UV light, Vitamin C and Vitamin D3 in the development of spinal deformities (three-factor ANOVA, df = 1, P > 0.01) (Table 5) Table Three-factor Analysis of Variance for the effects of vitamin C, vitamin D and UV light on growth in juvenile Lates calcarifer (P = 0.05) Factor UV light Vitamin C Vit D3 UV light · Vit C UV light · Vit D3 Vit C · Vit D3 UV light · Vit C · Vit D3 Degrees of freedom Mean square F-value P-value 1 1 1 23 925 200 984 33 22 228 34 696 19 530 1359 2.10 17.64 0.002 1.95 3.04 1.71 0.1 0.157 30 mm in length, 60 DAH) did not affect the incidence of spinal deformities, suggesting that they develop predominantly during the larval phase of growth in fish reared under standard industry protocols In contrast to the developing understanding of the minimum requirements of vitamin C, the requirement and role of vitamin D3 in fish nutrition remains unclear (Lall & LewisMcCrea 2007) and the outcomes of this study not advance the current understanding A lack of supplemented dietary vitamin D3 does not influence the rate of deformities in cultured juvenile L calcarifer One reason may be the naturally high levels of vitamin D3 found in the fish meal and oil components of artificial diets (see Results section) The results of this study allow for the development of hypotheses for the physical mechanisms underlying the development of skeletal deformities of the spine Lordotic deformities occur primarily in the caudal vertebrae, while Ôbroken backÕ syndrome occurs in the precaudal vertebrae The underlying cause of this pattern is unknown; however, analysis of vertebral column function and the mechanics of swimming may provide some explanation In scombrids, swimming motion is achieved through muscular forces exerted on the caudal vertebrae causing a lever action between the caudal and precaudal vertebrae (Westneat & Wainwright 2001) The forces exerted on the caudal vertebrae are distributed across several of the caudal centra at multiple attachment points causing it to bend while there is minimal flexing between the precaudal centra Forces exerted on the caudal vertebrae, which are developing without the correct collagen content, may undergo buckling failure of the caudal centra resulting in a U-shaped curvature This is anecdotally supported by changes in the detailed architecture of lordotic vertebrae of D labrax which appeared to have undergone locally increased bending movements (Kranenbarg et al 2005) Furthermore, Divanach et al (1997) demonstrated that P major reared in high water currents to induce excessive swimming activity induced lordotic deformities in the caudal vertebrae Additionally, in many teleost fishes, the neural and haemal spines are bound by collagen fibres This forms a vertical septum used for the transmission of energy when swimming and for the prevention of vertebrae rotating out of the sagittal plane (Westneat & Wainwright 2001) Without sufficient levels of vitamin C, it may be expected that structural integrity of the collagen fibres could be compromized Therefore, forces exerted by the surrounding musculature on precaudal vertebrae without correct structural rigidity supplied by collagen fibres and correct bone formation may induce Ôbroken backÕ syndrome In conclusion, L calcarifer fed a diet not supplemented with vitamin C develop high levels of spinal deformities with lordosis occurring predominantly in caudal vertebrae and Ôbroken backÕ syndrome in the precaudal vertebrae The precise nature of spinal deformities also suggests that biomechanical forces play a key role in their development Supplementing diets with vitamin C is suggested as a safeguard mechanism to prevent the development of deformities in juvenile barramundi, and may also be critical in larval feeding This study was financially supported by Cell Aquaculture Ltd We would like to thank Skretting, Ridley Aqua-feed and Bluewater Barramundi for supporting this project Alexis, M.N., Karanikolas, K.K & Richards, R.H (1997) Pathological findings owing to the lack of ascorbic acid in cultured gilthead sea bream (Sparus aurata L.) Aquaculture, 151, 209–218 Andrades, J.A., Becerra, J & Fernandez-Llebrez, P (1996) Skeletal deformities in larval, juvenile and adult stages of cultured sea bream (Sparus aurata L.) Aquaculture, 141, 1–11 Barahona-Fernandes, M.H (1982) Body deformation in hatchery reared European sea bass Dicentrarchus labrax (L) Types, prevalence and effect on fish survival J Fish Biol., 21, 239–249 Boonyaratpalin, M (1997) Nutrient requirements of marine food fish cultured in Southeast Asia Aquaculture, 151, 283–313 Boonyaratpalin, M & Phromkunthong, W (2001) Bioavailability of ascorbyl phosphate calcium in hybrid catfish, Clarias macrocephalus (Gunther) · Clarias gariepinus (Burchell) feed Aquacult Res., 32(Suppl 1) 126–134 Boonyaratpalin, M., Unprasert, N & Buranapanidgit, J (1989) Optimal supplementary vitamin C level in seabass fingerling diet In: The Current Status of Fish Nutrition in Aquaculture (Takeda, M & Wantanabe, T eds), pp 149–157 Tokyo University of Fisheries, Tokyo, Japan Chavez De Martinez, M.C (1990) Vitamin C requirement of the Mexican Native Cichlid Cichlasoma urophthalmus (Gunther) Aquaculture, 86, 409–416 Dabrowski, K., El-Fiky, N., Kock, G., Frigg, M & Wieser, W (1990) Requirement and utilisation of ascorbic acid and ascorbic sulphate in juvenile rainbow trout Aquaculture, 91, 317–337 Dabrowski, K., Moreau, R., El-Saidy, D & Ebeling, J (1996) Ontogenetic sensitivity of channel catfish to ascorbic acid deficiency J Aquat Anim Health, 8, 22–27 Daoulas, C., Economou, A.N & Bantavas, I (1991) Osteological abnormalities in laboratory reared seabass (Dicentrarchus labrax) fingerlings Aquaculture, 97, 169–180 Divanach, P., Papandroulakis, N., Anastasiadis, P., Koumoundouros, G & Kentouri, M (1997) Effects of skeletal deformities in sea bass (Dicentrarchus labrax L.) with functional swimbladder during postlarval and nursery phase Aquaculture, 156, 145–155 Fjelldal, P.G., Hansen, T.J & Berg, A.E (2007) A radiological study on the development of vertebral deformities in cultured Atlantic Salmon (Salmo salar L.) Aquaculture, 273, 721–728 Fraser, M.R & de Nys, R (2005) The morphology an occurrence of jaw and operculum deformities in cultured barramundi (Lates calcarifer) larvae Aquaculture, 250, 496–503 Fraser, M.R., Anderson, T & de Nys, R (2004) Ontogenic development of the spine and spinal deformities in larval barramundi (Lates calcarifer) culture Aquaculture, 242, 697–711 Galeotti, M., Beraldo, P., de Dominis, S., DÕAngelo, L., Ballestrazzi, R., Musetti, R., Pizzolito, S & Pinosa, M (2000) A preliminary histological and ultrastructural study of opercular anomalies in gilthead sea bream larvae (Sparus aurata) Fish Physiol Biochem., 22, 151–157 Gapasin, R.S.J., Bombeo, R., Lavens, P., Sorgeloos, P & Nelis, H (1998) Enrichment of live food with essential fatty acids and vitamin C: effects on milkfish (Chanos chanos) larval performance Aquaculture, 162, 269–286 Graff, I.E., Hoie, S., Totland, G.K & Lie, O (2002) Three different levels of dietary vitamin D3 fed to first-feeding fry of Atlantic salmon (Salmo salar L.): effect on growth, mortality, calcium content and bone formation Aquacult Nutr., 8, 103–111 Halver, J.E., Ashley, L.M & Smith, R.R (1969) Ascorbic acid requirements of Coho Salmon and Rainbow Trout Trans Am Fish Soc., 98, 762–771 Hattori, M., Sawada, Y., Takagi, Y., Suzuki, R., Okada, T & Kumai, H (2003) Vertebral deformities in cultured red sea bream, Pagrus major, Temminck and Schlegel Aquacult Res., 34, 1129– 1137 Hilton, J.W & Ferguson, H.W (1982) Effect of excess vitamin D3 on calcium metabolism in rainbow trout Salmo gairdneri (Richardson) J Fish Biol., 21, 373–379 Horton, H., Moran, L.A., Ochs, R.S., Rawn, D.J & Scrimgeour, G.K (1993) Principles of Biochemistry N Patterson PublisherÕs, Eaglewood Cliffs, New Jersey, pp 412–415 Kamler, E., Wolnicki, J & Kaminski, R (2008) Fatty acid composition, growth and morphological deformities in juvenile cyprinid, Scardinius erythropthalmus fed formulated diet supplemented with natural food Aquaculture, 278, 69–76 Kihara, M., Ogata, S., Kawano, N., Kubota, I & Yamaguchi, R (2002) Lordosis induction in juvenile red sea bream, Pagarus major, by high swimming activity Aquaculture, 212, 149–158 Koumoundouros, G., Maingot, E., Divanach, P & Kentouri, M (2001) Kyphosis in reared sea bass (Dicentrarchus labrax L.): ontogeny and effects on mortality Aquaculture, 209, 49–58 Kranenbarg, S., Waarsing, J.H., Muller, M., Weinans, H & van Leeuwen, J.L (2005) Lordotic vertebrae in sea bass (Dicentrarchus labrax L.) are adapted to increased loads J Biomech., 38, 1239– 1246 Lall, S.P & Lewis-McCrea, L.M (2007) Role of nutrients in skeletal metabolism and pathology in fish – An overview Aquaculture, 267, 3–19 Li, M.H & Robinson, E.H (2001) Dietary ascorbic acid requirement for growth and health in fish In: Nutrition and Fish Health (Lim, C & Webster, C.D eds), The Haworth Press, Inc., Binghamto, New York, pp 163–187 Lin, M.F & Shiau, S.Y (2004) Requirements of vitamin C (L-ascorbyl-2-monophosphate-Mg and L-ascorbyl-2-monophosphate-Na) Aquaculture Nutrition 17; 235–243 Ó 2010 Blackwell Publishing Ltd and its effects on immune responses of grouper, Epinephelus malabaricus Aquacult Nutr., 10, 327–333 Lovell, R.T (1973) Essentiality of vitamin – C in feeds for intensively fed caged channel catfish J Nutr., 103, 134–138 Mahajan, C.L & Agrawal, N.K (1979) Vitamin C deficiency in Channa punctatus Bloch J Fish Biol., 15, 613–622 Mahajan, C.L & Agrawal, N.K (1980) Nutritional requirement of ascorbic acid by Indian major carp, Cirrhina mrigala, during early growth Aquaculture, 19, 37–48 NRC (National Research Council) (1993) Nutrient Requirements of Fish National Academy Press, Washington, DC, USA 114 p Paperna, I (1978) Swim bladder and skeletal deformations in hatchery bred Sparus aurata J Fish Biol., 12, 109–114 Phromkunthong, W., Boonyaratpalin, M & Storch, V (1997) Different concentrations of ascorbyl-2-monophosphate-magnesium as dietary sources of vitamin C for seabass, Lates calcarifer Aquaculture, 151, 225–243 Quinn, G.P & Keough, M.J (2002) Experimental Design and Data Analysis for Biologists Cambridge University Press, Cambridge Roberts, R.J., Hardy, R.W & Sugiura, S.H (2001) Screamer diseases in Atlantic salmon, Salmo salar L., in Chile J Fish Dis., 24, 543–549 Soliman, A.K., Jauncey, K & Roberts, R.J (1986) The effect of dietary ascorbic-acid supplementation on hatchability, survival Aquaculture Nutrition 17; 235–243 Ó 2010 Blackwell Publishing Ltd rate and fry performance in Oreochromis-mossambicus (Peters) Aquaculture, 59, 197–208 Sullivan, M., Reid, S.W.J., Ternent, H., Manchester, N.J., Roberts, R.J., Stone, D.A.J & Hardy, R.W (2007) The aetiology of spinal deformity in Atlantic Salmon, Salmo salar L.: influence of different commercial diets on the incidence and severity of the preclinical condition in salmon parr under two contrasting husbandry regimes J Fish Dis., 30, 759–767 Tucker, J.W., Russell, J.D & Rimmer, M.A (2002) Barramundi culture: a success story for aquaculture in Asia and Australia World Aquac., 33, 53–59 Wang, X., Kim, K.-W., Bai, S.C., Huh, M.-D & Cho, B.-Y (2003) Effects of the different levels of dietary vitamin C on growth and tissue ascorbic acid changes in parrot fish (Oplegnathus fasciatus) Aquaculture, 215, 203–211 Webb, A.R (1993) Vitamin D synthesis under changing UV spectra In: Environmental UV Photobiology (Young, A.R., Bjorn, L.O., Moan, J & Nultsch, W eds), Plenum Press, New York, pp 185– 202 Westneat, M.W & Wainwright, S.A (2001) Mechanical design for swimming: muscle, tendon and bone In: Tuna: Physiology, Ecology and Evolution (Block, B.A & Stevens, E.D eds), Academic Press, San Diego, California, pp 272–308 Wilson, R.P & Poe, W.E (1973) Impaired collagen formation in the scorbutic channel catfish J Nutr., 103, 1359–1364 Aquaculture Nutrition doi: 10.1111/j.1365-2095.2009.00736.x 2011 17; 244–247 Dependencia de Educacio´n Superior Ciencias Naturales y Exactas, Universidad Auto´noma del Carmen, Ciudad del Carmen, Campeche, Me´xico Because of the filter-feeding behavior of shrimp larvae, it is important to define precisely the size of the particle ingested in the different stages until postlarval stage where raptorial habits are more evident than the filter-feeding lifestyle Selectivity assays were conducted by using Polystyrene DVB particles with diameter between and 50 lm as food A group of organisms from each stage were put into the particle suspension for 15 to let the polystyrene particles be ingested The particle distribution in the media and the content of the gut of the larvae were characterized with digital image processing analysis The results were compared using Ivlev selectivity formula, which compares the frequency distribution of each size of the particle in the media and in the gut of larvae The results of selectivity were adjusted with a third-order polynomial regression to determine the optimum and preferred size of the food particles for each larval stage between Zoea I and Postlarva I It is concluded that the different larval stages of Litopenaeus vannamei may be considered as a single group of larvae who ingest foods with size between 5.71 and 20.33 lm The optimal size of the food ingested was 14.42 lm wide KEY WORDS: feeding, larvae, particle size, selectivity, shrimp Received 23 June 2009, accepted 28 September 2009 Correspondence: R Gelabert, Dependencia de Educacio´n Superior Ciencias Naturales y Exactas, Universidad Auto´noma del Carmen, Calle 56 No Esq Ave Concordia Col Benito Jua´rez, C.P 24180, Ciudad del Carmen, Campeche, Me´xico E-mail: rgelabert@pampano.unacar.mx Formulated feeds play an important role in semi-intensive shrimp production, constituting nearly 55% of the total operating cost (Mohanty 2001), so it is necessary for food producers to carefully handle not only the ingredients to support the appropriate nutritional value and stability in the water, but also the portion size The food not ingested sinks to the bottom of the culture tank and affects the water quality Furthermore, the cost of larval production is increased when the food is not utilized by larvae, raising the total cost of the hatchery The nutritional effectiveness of a food organism is in the first place determined by its ingestibility and as a consequence by its size and configuration (Leger et al 1986) Food ration and food selectivity are the basis for intensive rearing technology for zooplankton-fed fish (Szlaminska et al 1999) Partial or total replacement of microalgae by artificial food in the assays conducted by Robinson et al (2005) indicated that further investigations are necessary to determine whether inert feeds offer a less nutritionally balanced diet, a less digestible diet, or inadequate particle sizes that are rejected by shrimp larvae Feeding shrimp larviculture has been studied to evaluate replacement of live food by artificial diets (Brito et al 2001; Pedroza- Islas et al 2004; DÕAbramo et al 2006; Pin˜a et al 2006), but artificial diets for the total replacement of live feeds for rearing marine larvae have not been developed despite many years of research The types of microparticles used to deliver nutrients to larvae need to be carefully evaluated and improved (Langdon 2003) Considering the filter-feeding behavior of shrimp larvae as well as the scarce information about the size of the food ingested by shrimp larvae and the importance for the larviculture, the present paper evaluated the particle size ingested by Litopenaeus vannamei larvae from Zoea I to the first postlarval stage The larvae, obtained from a commercial hatchery at the Nauplius stage, were reared to the postlarva (PL-1) stage Ó 2009 Blackwell Publishing Ltd No claim to original US government works Aquaculture Nutrition doi: 10.1111/j.1365-2095.2010.00848.x 2011 17; e781–e788 1 Institute of Marine Research (IMR), Matre Aquaculture Research Station, Matredal, Norway; University of Stirling, Stirling, Scotland, UK; NOFIMA, Fyllingsdalen, Norway 2 Institute of Aquaculture, environmental temperature on tissue lipids were more pronounced in fish fed the CO diets than FO diets Copepod oil (CO) from the marine zooplankton, Calanus finmarchicus, is a potential alternative to fish oils (FOs) for inclusion in aquafeeds The oil is composed mainly of wax esters (WE) containing high levels of saturated fatty acids (SFAs) and monounsaturated fatty alcohols that are poorly digested by fish at low temperatures Consequently, tissue lipid compositions may be adversely affected in salmon-fed CO at low temperatures This study examined the lipid and FA compositions of muscle and liver of Atlantic salmon reared at two temperatures (3 and 12 °C) and fed diets containing either FO or CO, supplying 50% of dietary lipid as WE, at two fat levels (330 g kg)1, high; 180 g kg)1, low) Fish were acclimatized to rearing temperature for month and then fed one of four diets: high-fat fish oil (HFFO), high-fat Calanus oil (HFCO), low-fat fish oil (LFFO) and low-fat Calanus oil (LFCO) The fish were grown to produce an approximate doubling of initial weight at harvest (220 days at °C and 67 days at 12 °C), and lipid content, lipid class composition and FA composition of liver and muscle were determined The differences in tissue lipid composition between dietary groups were relatively small The majority of FA in triacylglycerols (TAG) in both tissues were monounsaturated, and their levels were generally higher at °C than 12 °C Polyunsaturated fatty acids (PUFA), particularly (n-3) PUFA, predominated in the polar lipids, and their level was not significantly affected by temperature The PUFA content of TAG was highest (26%) in the muscle of fish fed the HFCO diet at both temperatures Tissue levels of SFAs were lower in fish-fed diets containing HFCO than those fed HFFO, LFFO or LFCO, particularly at °C The results are consistent with Atlantic salmon being able to incorporate both the FA and fatty alcohol components of WE into tissue lipids but, overall, the effects of Ó 2011 Blackwell Publishing Ltd KEY WORDS: copepod, fatty acids, lipid classes, liver, muscle, temperature Received 10 September 2010, accepted 30 November 2010 Correspondence: A.S Bogevik, Institute of Marine Research, Austevoll Aquaculture Research Station, N-5392 Storebø, Norway E-mail: andre.bogevik@imr.no The oil produced by the naturally abundant zooplankton, Calanus finmarchicus, is considered a possible alternative to marine FOs as a lipid source for use in aquafeeds However, the lipids in this copepod exist mainly as wax esters (WE), rather than the triacylglycerols (TAG) that predominate in fish (Sargent et al 1976) Chemically, WE consist of a longchain fatty acid (FA) esterified to a long-chain fatty alcohol (FAlc), and are intrinsically more hydrophobic than TAG As a consequence, WE are more difficult to digest than TAG by fish (Bauermeister & Sargent 1979) Nevertheless, feeds formulated with oil extracted from C finmarchicus (CO) have been previously shown to produce growth rates in Atlantic salmon (Salmo salar) similar to those obtained for fish given feeds containing fish oil (FO), provided the level of WE is below 40% of the total dietary lipid (Olsen et al 2004; Bogevik et al 2009) Lipid digestion was relatively unaffected at these levels of WE, and dietary CO had only minor effects on FA compositions of salmon muscle and liver (Olsen et al 2004) The high level of monounsaturated FAlc appeared to be readily absorbed and converted to the corresponding monounsaturated fatty acid (MUFA) with subsequent incorporation into tissue lipids Although, the rate of uptake of FAlc into Atlantic salmon enterocytes is lower than the rate of uptake of FA (Bogevik et al 2008), the increased feed conversion rate, bile volume and enzyme activity upon feeding WE at levels below 40% of total dietary lipid appear sufficient for similar amounts of energy for growth and storage to be generated as in fish fed a diet containing FO (Bogevik et al 2009) High levels of WE in dietary lipid could be particularly challenging for salmonids farmed at high latitudes where they are exposed to low sea temperatures during winter At low environmental temperature, feed intake is reduced and the digestive processes are slowed down, which will inevitably lower nutrient utilization However, the gastrointestinal holding time is also increased at low temperature by the way of compensation to enable good nutrient availability at any temperature (Olsen & Ringø 1998) In addition, lipid digestibility is inversely related to the type of lipid and its melting point (Olsen & Ringø 1997) For FA, data clearly show that digestibility of saturated fatty acid (SFA) in dietary TAG is reduced at lower temperature, while the digestibility of MUFA and polyunsaturated fatty acids (PUFA) is less affected (Olsen & Ringø 1998; Ng et al 2003, 2004) In addition to the temperature effect on FA digestion, salmon-fed CO at an inclusion level of 500 g kg)1 showed a massive reduction in digestibility of monounsaturated FAlc in WE at °C compared to 12 °C, whereas digestibility of saturated and polyunsaturated FAlc was less affected (Bogevik et al 2010) Their lower digestibility at °C resulted in a higher proportion of dietary WE remaining in faecal lipid It can be expected that these observed alterations in utilization of WE at low temperature will lead to alterations in tissue and cell membrane FA compositions The above has demonstrated that low water temperatures that occur over the winter months during the normal production cycle of Atlantic salmon demand that feeds are readily absorbed to provide energy for growth and optimal tissue composition The slower digestive processes, absorption of SFA and conversion of FAlc to FA at lower temperature could thus be challenging issues with feeds containing WE In this study, we tested the hypothesis that temperature would significantly affect tissue lipid and FA compositions of Atlantic salmon-fed diets containing high levels of WE in their dietary lipid and that dietary fat content would be an important influence on this affect Therefore, we determined the lipid and FA compositions of muscle and liver of Atlantic salmon reared at two temperatures (3 and 12 °C) and fed Calanus oil comprising 50% WE at two dietary fat levels (330 g kg)1 high; 180 g kg)1 low) Three hundred and sixty Atlantic salmon (S salar L., Mowi strain; Norwegian breeding programme, 13-month-old postsmolts) originally held at °C and averaging 447 g were anaesthetized in 0.1% (w/v) tricaine methane sulphonate (MS-222; Norwegian Medical Depot, Bergen, Norway) and measured for weight and length The fish were then distributed equally between 24 fibreglass tanks (1.5 · 1.5 · 1.0 m) supplied with aerated seawater and acclimatized to the experimental temperatures by gradually adjusting from °C to either or 12 °C over a period of month, with twelve tanks in each temperature group The fish grew through the acclimation period to an average of 485 g in the coldwater group (3.1 ± 0.4 °C) and 599 g in the warm water group (12.3 ± 0.4 °C) Four diets were prepared at NOFIMA (Bergen, Norway) as outlined in detail previously (Olsen et al 2004), with the only exception being that lipid-extracted fish meal (23 g kg)1 lipid) was used (TripleNine Fish protein amba, Esbjerg, Denmark) The diets were formulated to be low (180 g kg)1) and high (330 g kg)1) in lipid (fat) with the added oils being either exclusively FO or oil extracted from the marine copepod C finmarchicus (CO) Thus, the four diets were low-fat FO (termed LFFO) low-fat CO (LFCO), high-fat fish oil (HFFO) or high-fat CO (HFCO) Further details of feed composition are given in Table and in Bogevik et al (2010) Triplicate tanks of fish were fed the four diets at both temperatures All fish were fed to satiation twice a day using Arvo Tec TD2000 feeders (Arvotec, Huutokoski, Finland) To compensate for the different growth rates at the two temperatures, fish were fed to an approximate doubling of initial weight Fish at °C grew from 485 to 899 g (SGR, 0.28) over 220 days, while fish at 12 °C grew from 599 to 985 g (SGR, 0.74) over 67 days Fish-fed CO (HFCO and LFCO feeds) had an overall lower SGR than those fed FO (HFFO and LFFO) as the primary lipid source (Bogevik et al 2010) After the experimental period had elapsed, fish were starved for 72 h, anaesthetized in 0.1% MS-222 and measured for weight and length Three fish from each tank (nine per treatment) were killed by a sharp blow to the head and muscle (Norwegian Quality Cut) and liver sampled and stored at )80 °C prior to biochemical analysis Total lipid of diets, muscle and liver was extracted with chloroform/methanol (2 : 1, v/v) according to the method of Aquaculture Nutrition 17; e781–e788 Ó 2011 Blackwell Publishing Ltd Table Formulation (g kg)1 diet) of the experimental diets Triple Nine fish meal1 Fish oil2 Calanus oil Soya lecithin Soya protein3 Wheat 230/054 Wheat gluten 156/055 Vitamin mixture6 Mineral mixture7 Carophyll Pink (10%) Yttrium oxide (Y2O3) HFFO HFCO LFFO LFCO 417 289 60 140 80 10 0.3 0.1 417 289 60 140 80 10 0.3 0.1 575 131 60 140 80 10 0.3 0.1 575 131 60 140 80 10 0.3 0.1 HFFO, high-fat fish oil; HFCO, high-fat Calanus oil; LFCO, low-fat Calanus oil Triple Nine, Denmark (891 g kg)1 dry matter, 765 g kg)1 crude protein, 23 g kg)1 fat (soxhlet) and 131 g kg)1 ash) NorSalmOil, Norsildmel, Bergen, Norway Soya protein concentrate (SPC 70), Sopropeche, Boulogne, France Norgesmøllene, Bergen, Norway Received from Ewos Innovation, Dirdal, Norway Supplied per kg diet: vitamin D3, 3000 I.E.; vitamin E (Rovimix, 50%), 160 mg; thiamine, 20 mg; riboflavin, 30 mg; pyridoxine–HCl, 25 mg; vitamin C (Riboflavin Stay C 35%), 200 mg; calcium pantothenate, 60 mg; biotin, mg; folic acid, 10 mg; niacin, 200 mg; vitamin B12, 0.05 mg; menadione bisulphite, 20 mg Supplied per kg diet: magnesium, 500 mg; potassium, 400 mg; zinc, 80 mg; iron, 50 mg; manganese, 10 mg; copper, mg Folch et al (1957) The chloroform phase was evaporated to dryness in vacuo at room temperature and the lipid extract resuspended at 10 mg mL)1 in chloroform/methanol (2:1, v/v) containing 0.05% (w/v) butylated hydroxytoluene (BHT) as antioxidant and stored under nitrogen at )80 °C prior to further analysis The lipid class composition of total lipid was determined by double-development high-performance thin-layer chromatography (HPTLC) coupled with scanning densitometry, as described by Olsen & Henderson (1989) HPTLC plates were initially developed to halfway in methyl acetate/ isopropanol/chloroform/methanol/0.25% aqueous KCl (25 : 25 : 25 : 10 : 9, v/v) before developing fully with isohexane/ diethyl ether/acetic acid (85 : 15 : 1, v/v) Lipid classes were visualized by spraying the plate with 3% copper acetate (w/v) in 8% phosphoric acid (v/v) and charring at 160 °C for 15 (Olsen & Henderson 1989) Lipid classes were quantified using a CAMAG TLC Scanner and WinCATS software (CAMAG, Muttenz, Switzerland) Identities of individual lipid classes were confirmed by running authentic standards alongside samples on HPTLC plates which also compensated for interplate variation when quantifying each lipid class within a linear area utilizing established standard equations Aquaculture Nutrition 17; e781–e788 Ó 2011 Blackwell Publishing Ltd To determine FA and FAlc compositions of diets and fish tissues, total lipid was subjected to acid-catalysed transesterification using 1% (v/v) H2SO4 in methanol with 17 : FA and FAlc added as internal standards Resultant fatty acid methyl esters (FAME) were extracted and purified by TLC on 20 · 20 cm plates as described previously (Tocher & Harvie 1988) Long-chain FAlc present in total lipid extracts from the CO diets of fish fed these diets were identified on the TLC plates as a single component and recovered from silica by elution with chloroform/methanol (2 : 1, v/v) before conversion to acetate derivatives by reaction with acetic anhydride/pyridine (1 : 2, v/v) (Farquhar 1962) Prior to GC analysis, FAlc acetates were purified on TLC plates as described for FAME Both FAME and FAlc acetates were separated and quantified by gas liquid chromatography using a 60 m · 0.32 mm i.d fused silica capillary column coated with ZB-Wax (Phenomenex, Macclesfield, UK) and a Thermo Finnigan Trace gas chromatograph Hydrogen was used as carrier gas, and temperature programming was from 50 to 150 °C at a rate of 40 °C min)1, from 150 to 170 °C at a rate of °C min)1, from 170 to 199 °C at a rate of 0.5 °C min)1, and then to a final temperature of 220 °C at 40 °C min)1 Individual components were identified by comparison with known standards The absolute amounts of individual FA and long-chain FAlc present were calculated by reference to the internal standard (Olsen et al 2004) Tissue lipid compositions are given as means of nine individuals per dietary treatment with sum standard error mean P ( SE) for comparison between fish given the same diet at two temperatures All statistical analyses were performed using STATISTICA software for Windows (Tulsa, OK USA) Data were checked for homogeneity of variances by the Levene test and, where necessary, transformed via arcsine (percentage data) or Ln functions Differences in tissue lipid compositions were analysed by one-way ANOVA for mean effect of temperature for fish given the same diet The major difference in lipid class composition between the diets was observed in neutral lipids with TAG predominating in the feeds formulated with FO (Tables & 3) and WE in the feeds formulated with CO (Tables & 5) The FO feeds were rich in MUFA with lower levels of SFA and PUFA, whereas the CO feeds contained high levels of n-3 PUFA Table Gross composition (g kg)1) and total fatty acid composition (% of lipid) of the HFFO diet and of TAG and PL in muscle and liver from Atlantic salmon-fed HFFO and kept at and 12 °C Muscle lipids Total lipid PL1 TAG1 Liver lipids Diet 12 P SE 333 64 623 29 293 628 35 254 653 14 18 TAG P 12 P SE P – – – 39 467 262 41 481 285 19 23 – – – PL Fatty acids FA 12 14:0 16:0 16:1n-7 18:1n-9 20:1n-9 22:1n-11 18:2n-6 20:4n-6 18:4n-3 20:5n-3 22:6n-3 SFA MUFA n-6 PUFA n-3 PUFA PUFA 6.4 13.4 5.1 9.0 10.4 15.2 4.0 0.3 4.2 7.1 11.4 22.8 44.7 5.1 26.7 32.5 5.1 14.5 5.4 14.4 8.9 10.3 4.3 0.4 2.1 4.3 8.9 23.4 45.2 5.6 20.3 25.9 5.5 14.6 5.1 14.0 8.3 10.3 3.9 0.4 2.0 4.6 8.4 25.0 43.8 5.3 20.0 25.3 P SE 0.1 1.0 0.1 0.4 0.2 0.4 4.3 0.0 0.2 0.1 0.4 1.1 0.7 0.2 0.6 0.8 TAG P 12 P SE ** – – – – – – – – – – – – – – – 1.7 23.9 1.1 4.2 1.2 0.4 1.1 1.1 0.3 8.2 39.2 31.8 9.9 2.9 51.2 54.2 1.6 20.8 1.1 4.7 1.2 0.5 1.1 1.2 0.5 8.9 39.5 28.6 10.8 3.1 53.2 56.3 0.1 1.3 0.1 0.2 0.1 0.0 0.0 0.0 0.0 0.3 1.4 1.5 0.5 0.1 1.7 1.7 PL P 12 – – – – – – – * * – – – – – – – 4.6 15.8 6.0 27.3 12.1 7.1 3.5 0.2 0.5 1.7 3.0 24.9 62.4 4.5 8.2 12.7 3.6 21.3 4.5 23.1 8.8 6.4 2.9 0.2 0.7 3.1 5.2 31.3 50.3 5.2 13.2 18.4 P SE 0.3 2.3 0.2 1.1 0.7 0.5 0.3 0.0 0.1 0.4 0.7 2.8 2.2 0.4 1.7 2.0 P 12 P SE P * – *** * ** – – – – * * – ** – * – 2.8 20.5 2.3 9.5 3.9 0.8 1.6 2.4 0.2 9.0 30.7 30.5 21.8 5.0 42.6 47.6 2.0 20.4 1.6 9.9 3.9 1.0 1.7 2.5 0.2 9.0 29.9 30.8 20.4 5.6 43.0 48.6 0.1 0.8 0.1 0.5 0.2 0.1 0.0 0.1 0.0 0.3 1.5 1.2 0.7 0.1 1.7 1.8 *** – *** – – – – – – – – – – *** – – MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acid; TAG, triacylglycerol; PL, polar lipids; HFFO, high-fat fish oil *[...]... size of the particles ingested and the size of the larvae in neither the zoea nor the mysis Larval size (lm) N particles analyzed in the gut R2 F value P value 19.47 F3,29 61.46 F3,27 20.33 F3,46 10.14 F3,31 18.85 F3,28 37.23 F3,33 28.75 F3,26 0.000 Stage N larvae ZI 31 945.3 33.4 1 031 0.668 ZII 60 1 344.4 103.2 1 948 0.872 ZIII 32 1 838.4 147.4 2 012 0.542 MI 35 2 730 180.3 1 601 0.495 MII 11 3 154.4... either stage PZ2 or M1 to PL1 Aquaculture, 261, 13561362 Kolkovski, S (2001) Digestive enzymes in sh larvae and juvenilesimplications and applications to formulated diets Aquaculture, 200, 181201 Langdon, C (2003) Microparticle types for delivering nutrients to marine sh larvae Aquaculture, 227, 259275 Lee, C.S (2003) Biotechnological advances in nsh hatchery production: a review Aquaculture, 227, 439458... (Hippoglossus hippoglossus L.) Aquaculture, 285, 174178 Olafsen, J.A (2001) Interactions between sh larvae and bacteria in marine aquaculture Aquaculture, 200, 223247 Olsen, A.I., Attramadal, Y., Jensen, A & Olsen, Y (1999) Inuence of size and nutritional value of Artemia franciscana on growth and quality of halibut larvae (Hippoglossus hippoglossus) during the live feed period Aquaculture, 179, 475487 Pittman,... sablesh (Anoplopoma mbria Pallas) Aquaculture, 119, 4761 Yufera, M., Parra, G., Santiago, R & Carrascosa, M (1999) Growth, carbon, nitrogen and caloric content of Solea senegalensis (Pisces: Soleidae) from egg fertilization to metamorphosis Mar Biol., 134, 4349 Aquaculture Nutrition 2011 17; 258266 doi: 10.1111/j.1365-2095.2009.00747.x 1 1 2 2 Department of Aquaculture, National Kaohsiung... 6380 Soft Science, Tokyo Aquaculture Nutrition 17; 258266 ể 2010 Blackwell Publishing Ltd Aquaculture Nutrition doi: 10.1111/j.1365-2095.2009.00749.x 2011 17; 267277 1 2 2 1 1 2 1 1 2 3 Science of Marine Resources, The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan; 2 Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima... system of Solea senegalensis (Kaup, 1858) larvae Aquaculture, 171, 293308 Ribeiro, L., Zambonino-Infante, J.L., Cahu, C & Dinis, M.T (1999b) Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858 Aquaculture, 179, 465473 Ribeiro, A.R.A., Moren, M., Ribeiro, L., Dinis, M.T & Hamre, K (2007) Iodine Enrichment of Rotifers Brachionus plicatilis Aquaculture Europe 07, October 2007, Istanbul,... Stappen, G (1996) Artemia In: Manual on the Production and Use of Live Food for Aquaculture (Lavens, P & Sorgeloos, P eds), 1295 FAO, Rome VernerJereys, D.W., Joiner, C.L., Bagwell, N.J., Reese, R.A., Husby, A & Dixon, P.F (2008) Development of bactericidal and virucidal testing standards for aquaculture disinfectants Aquaculture, 286, 190197 Watanabe, T (1993) Importance of docosahexaenoic acid in... a histochemical and immunohistochemical approach Aquaculture, 260, 346356 Dinis, M.T., Ribeiro, L., Soares, F & Sarasquete, C (1999) A review on the cultivation potential of Solea senegalensis in Spain and in Portugal Aquaculture, 176, 2738 Eales, J.G & Brown, S.B (1993) Measurement and regulation of thyroidal status in teleost sh Rev Fish Biol Fish., 3, 299347 Einarsdottir, I., Silva, N., Power, D.,... August 2009, accepted 23 October 2009 Correspondence: Y.-H Chien, Department of Aquaculture, National Taiwan Ocean University, Keelung 202, Taiwan E-mail: yhchien@mail ntou.edu.tw 2 Department of Aquaculture, Various synthetic and natural carotenoids (CD) have been used as dietary supplement to improve pigmentation of aquaculture animals (Storebakken & No 1992; Gouveia et al 2003; Chien & Shiau 2005;... Litopenaeus vannamei protozoea larvae, fed with monoalgal and mixed diets Aquaculture, 2 53, 523530 Robinson, C.B., Samocha, T.M., Fox, J.M., Gandy, R.L & McKee, D.A (2005) The use of inert articial commercial food sources as replacements of traditional live food items in the culture of larval shrimp, Farfantepenaeus aztecus Aquaculture, 245, 135 147 Salazar, O & Gonzalez, M.A (1986) Intensidad de la