Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site Archimer Aquaculture July 2007,Volume 267, Issues 1-4, Pages 199-212 http://dx.doi.org/10.1016/j.aquaculture.2007.01.011 © 2007 Elsevier B.V. All rights reserved. Archive Institutionnelle de l’Ifremer http://www.ifremer.fr/docelec/ Combined replacement of fish meal and oil in practical diets for fast growing juveniles of gilthead sea bream (Sparus aurata L.): Networking of systemic and local components of GH/IGF axis Laura Benedito-Palosa, Alfonso Saera-Vilaa, Josep-Alvar Calduch-Ginera, Sadasivam Kaushikb and Jaume Pérez-Sáncheza, * a Instituto de Acuicultura de Torre de la Sal (CSIC), Department of Biology, Culture and Pathology of Marine Species, 12595 Ribera de Cabanes, Castellón, Spain b UMR Nutrition, Aquaculture and Genomics, INRA, Unité-Mixte INRA-IFREMER-Univ. Bordeaux I, 64310 SaintPée-sur-Nivelle, France *: Corresponding author : Jaume Pérez-Sánchez, email address : jperez@iats.csic.es Abstract: Growth performance and growth regulatory pathways were examined in juvenile gilthead sea bream fed diets containing largely plant-based ingredients. Four isonitrogenous and isolipidic extruded diets with a low level (20%) of fish meal inclusion were formulated with graded levels of a vegetable oil mixture (17:58:25 of rapeseed: linseed: palm oils) replacing fish oil at 33, 66 and 100% (33VO, 66VO and VO diets). All diets were supplemented with lysine (0.55%) and contained soy lecithin (1%). Daily growth coefficients and feed efficiency over the course of an 11-week trial were almost identical in fish fed the FO, 33VO and 66VO diets. The VO diet reduced feed intake and growth without significant effects in proximate whole body composition, nitrogen or energy retentions. The highest concentration of plasma levels of insulin-like growth factor-I (IGF-I) was found in fish fed the 33VO diet. The lowest concentration was attained in fish fed the VO diet, whereas intermediate values were found in fish fed FO and 66VO diets. An opposite trend was found for circulating levels of growth hormone (GH), probably as a result of a reduced negative feedback inhibition from circulating IGF-I. Hepatic expression of IGF-I and GH receptor type I (GHR-I) was regulated in concert and mRNA levels paralleled plasma levels of IGF-I. Hepatic IGF-II and GHR-II were expressed in a more constitutive manner and no changes at the mRNA level were detected. In the skeletal muscle, IGF-I and GHR-I mRNAs did not vary significantly among groups. By contrast, IGF-II mRNA was up-regulated in fish fed the control diet, whereas the highest amount of GHR-II mRNA was attained in fish fed the 66VO diet. All together, these results suggest different growth compensatory mechanisms mediated by IGFII and GHR-II at the local tissue level. These new insights prompted us to propose that practical diets low in marine ingredients can be used over the productive cycle of gilthead sea bream when essential fatty acids are supplied above the requirement levels. Keywords: Sparidae; Fish oil; Vegetable oil; Plant proteins; Growth hormone; Growth hormone receptors; Insulin-like growth factors; Endocrine disrupters; Contaminants 1 4 50 12 ! 51 52 Currently, aquaculture is the major consumer of fish meal, a protein4dense feedstuff 53 that approximates the ideal amino acid profile of most cultured livestock. However, fish 54 meal is a limited resource whose availability has remained stable from the late 1980s at 55 approximately 6 million metric tonnes per annum, which limits the continuous growth of 56 aquaculture production (FAO, 2004). Furthermore, inherent variability in fish meal 57 composition due to species, season, geographic origin and processing leads to variation in 58 quality (Opstvedt et al., 2003; Bragadóttir et al., 2004), and most of the future changes in 59 developing novel aquafeeds should be focused on alternative protein sources. 60 The n43 long4chain highly unsaturated fatty acids (n43 HUFA) are naturally 61 abundant in the marine environment, and fish oil is the major source of eicosapentaenoic 62 acid (EPA, 20:5n43) and docosahexaenoic acid (DHA, 20:6n43) for aquafeeds. Besides the 63 scarcity of fish oil, which is of great concern for marine fish, these animals have a limited 64 capacity to biosynthesize n43 HUFA from the shorter chain linolenic acid (18:3n43), and 65 both EPA and DHA become critical dietary constituents to ensure successful survival, 66 growth, and development of these fish (Sargent et al., 1999, 2002). At this standpoint, it 67 must be noted that fish meal also contains certain amounts of oil rich in n43 HUFA, and the 68 fish oil added to energized diets can be totally replaced by vegetable oils when fish meal is 69 included at a high level in diets for Atlantic salmon (Bell et al., 2003; Bransden et al., 2003; 70 Torstensen et al., 2004), rainbow trout (Richard et al., 2006a), and the freshwater African 71 catfish (Ng et al., 2004). Similar results have been achieved in a typically marine fish such 72 as turbot (Regost et al., 2003). A high fish oil replacement is also feasible in the Murray 73 cod using casein4based diets (Francis et al., 2006). Likewise, up to 60% of fish oil added to 5 74 diets has been replaced successfully in juvenile European sea bass (Montero et al., 2005; 75 Mourente et al., 2005) and gilthead sea bream (Izquierdo et al., 2005), but the diets used in 76 these studies also contained 35 to 40% fish meal. 77 Marine derived feedstuffs are also possible vectors of contaminants, such as PCBs, 78 dioxins and other harmful chemicals affecting the safety of farm4raised fish (Jacobs et al., 79 2002). It is clear that reduction in fish oil levels can lead to a decrease in the contaminant 80 levels of feed and consequently on fish filets (Berntssen et al., 2005; Bethune et al., 2006). 81 Thus, the general consensus is that alternative protein and oil sources are needed to 82 supplement or replace fish meal and fish oil in aquafeeds, contributing to long4term 83 sustainability of the aquaculture industry (Hardy, 2004). In the present study, our objective 84 was hence to maximize the combined replacement of fish meal and fish oil in practical diets 85 for fast growing juveniles of gilthead sea bream. In earlier studies, we had shown that a 86 good proportion of fish meal can be replaced by a mixture of plant protein sources in 87 gilthead sea bream diets (Gómez4Requeni et al., 2003, 2004; Sitjà4Bobadilla et al., 2005). 88 Based on these results, we attempted here to replace fish oil by a blend of vegetable oils, 89 which have been already shown to be very effective in other fish species (Torstensen et al., 90 2005; Mourente and Bell, 2006; Richard et al., 2006a,b). To address this issue, growth and 91 nutrient retention were analyzed in a conventional manner. Circulating levels of growth 92 hormone (GH) and insulin4like growth factor4I (IGF4I) were used as markers of growth and 93 nutrient status (see Pérez4Sánchez and Le Bail, 1999; Dyer et al., 2004). Also, transcripts of 94 IGFs and GH receptors (GHR) were measured in liver and skeletal muscle by means of 95 real4time PCR assays. 6 96 32 4 97 98 99 100 As shown in Table 1, three diets (33VO, 66VO and VO) with relatively low fish meal 101 inclusion (20%) levels were formulated with practical plant protein ingredients for the graded 102 replacement (33, 66 and 100%) of the added fish oil by a blend of vegetable oils (rapeseed oil: 103 linseed oil: palm oil). A fish oil4based diet (FO diet) equal in lipid content (220 g kg41) was 104 used as the reference diet. Diets were supplemented with lysine (0.55%) and contained soy 105 lecithin (1%). EPA plus DHA content varied on a dry matter basis between 2.3% (FO diet) and 106 0.3% (VO diet), and the DHA/EPA ratio (1.141.2) remained constant. All diets were 107 manufactured using a twin4screw extruder (Clextral, BC 45) in the INRA experimental research 108 station of Donzacq (Landes, France), dried under hot air, sealed and kept in air4tight bags until 109 use. 110 Diet samples were hydrolysed (6N HCl, 110 °C) and amino acid analysis was performed 111 using high4performance liquid chromatography. Tryptophan was determined by the 112 colorimetric method of Basha and Roberts (1977) after alkaline hydrolysis of each sample (see 113 Table 2). Fatty acid methyl esters (FAME) were prepared from aliquots of total lipid by acid4 114 catalysed transmethylation for 16 h at 50 °C (Christie, 1982) after the addition of 115 nonadecaenoic fatty acid (19:0) as an internal standard. FAMEs were extracted and separated in 116 a Fisons Instruments GC 8000 Series (Thermo Electron Co., Rodano, Italy) gas chromatograph, 117 equipped with a fused silica 30 m x 0.25 mm open tubular column (Tracer TR4WAX, film 118 thickness: 0.25 Nm4Teknockroma, Spain) and a cold column injection system, using helium as 119 carrier and 50 to 220 °C thermal gradient. Peaks were recorded with Chrom4Card for Windows 7 120 software (Fisons CE Instruments, Milan, Italy) and identified by comparison with known 121 standards (see Table 3). 122 123 124 125 Gilthead sea bream ( L.) fingerlings of Atlantic origin (Ferme Marine 126 de Douhet, Ile d’Oléron, France) were acclimated to laboratory conditions for 20 days 127 before the start of the growth study. Fish of 16 g initial mean body weight were distributed 128 into 12 fibreglass tanks (5004l capacity) in groups of 60 fish each. Water (37.5 ‰ salinity) 129 flow was 20 l/min, and oxygen content of outlet water remained higher than 85% 130 saturation. Day length increased over the course of the trial (May4August) following natural 131 changes at our latitude (40º 5’ N; 0º 10’ E). Water temperature also varied naturally 132 increasing from 17 to 25 ºC. 133 The growth study was undertaken over 11 weeks (74 days) and each diet was 134 randomly allocated to triplicate groups of fish. Feed was offered by hand to apparent visual 135 satiety in two meals per day (0900 and 1400 h), and feed consumption was recorded daily. 136 Every 3 weeks, fish were counted and group4weighed under moderate anaesthesia (34 137 aminobenzoic acid ethyl ester, MS 222; 100 µg/ml). Blood and tissue sampling was done at 138 the end of the growth trial from randomly selected fish killed by a blow to the head. Five h 139 after the morning meal (12 animals per diet; 4 animals per tank), blood samples were taken 140 from caudal vessels with heparinised syringes. Following overnight fasting (20 h after the 141 second daily meal), 12 additional fish per dietary treatment were taken for sampling of 142 blood, liver and white skeletal muscle. Plasma was drawn after centrifugation at 3000 x 143 for 20 min at 4 °C, and stored at 430 °C until further hormone analyses. Liver and white 8 144 muscle were rapidly excised, frozen in liquid nitrogen and stored at 480 °C for RNA 145 extraction. 146 147 148 149 Proximate analysis of diets was made by the following procedures: dry matter by 150 drying at 105 °C for 24 h, ash by combustion at 550 °C for 12 h, protein (N x 6.25) by the 151 Kjeldahl method, fat after dichloromethane extraction by the Soxhlet method and gross 152 energy in an adiabatic bomb calorimeter (IKA). Specimens for whole body analyses (a 153 pooled sample of 10 fish at the beginning and pools of 5 fish per tank at the end of trial) 154 were ground, and small aliquots were dried to estimate moisture content. The remaining 155 samples were freeze4dried and chemical analyses were performed as indicated for 156 experimental diets. 157 158 ! 159 160 Plasma GH levels were assayed by a homologous gilthead sea bream 161 radioimmunoassay (RIA), using recombinant GH as tracer and standard (Martínez4 162 Barberá et al., 1995). Sensitivity and midrange of the assay were 0.1 ng/ml and 2.1 to 2.3 163 ng/ml, respectively. 164 After acid4ethanol precipitation, circulating levels of IGF4I were measured with a 165 generic fish IGF4I RIA (Vega4Rubín de Celis et al., 2004). The assay was based on the use 166 of recombinant red sea bream IGF4I (GroPep, Adelaide, Australia) as tracer and standard, 167 and anti4barramundi (Asian sea bass) IGF4I serum (GroPep, Adelaide, Australia) (1:8000) 9 168 as first antibody. A goat anti4rabbit IgG (1:20) (Biogenesis, Poole, UK) was used as 169 precipitating antibody. The sensitivity and midrange of the assay were 0.05 and 0.7 to 0.8 170 ng/ml, respectively. 171 172 " #$% & #' 173 174 Total RNA extraction was performed with the ABI PRISM™ 6100 Nucleic Acid 175 PrepStation (Applied Biosystems, CA, USA). Briefly, liver and white skeletal muscle were 176 homogenized at a ratio of 25 mg/ml with a guanidine4detergent lysis reagent. The reaction 177 mixture was treated with protease K, and RNA purification was achieved by passing the 178 tissue lysate (0.5 ml) through a purification tray containing an application4specific 179 membrane. Wash solutions containing DNase were applied, and total RNA was eluted into 180 a 964well PCR plate. The RNA yield was 40450 Ng with absorbance measures (A260/280) of 181 1.9 to 2.1. 182 Reverse transcription (RT) with random decamers was performed with the High4 183 Capacity cDNA Archive Kit (Applied Biosystems). For this purpose, 500 ng total RNA 184 were reverse transcribed in a final volume of 100 Nl. RT reactions were incubated 10 min at 185 25 ºC and 2 h at 37 ºC. Control reactions were run without reverse transcriptase and were 186 used as negative real4time PCR controls. 187 188 ( # ! ) # 189 190 Real4time PCR was performed using an iCycler IQ Real4time Detection System 191 (Bio4Rad, Hercules, CA, USA) as previously described (Calduch4Giner et al., 2003). 10 192 Diluted RT reactions were used for PCR reactions in 25 Vl volume. Each PCR4well 193 contained SYBR Green Master Mix (Bio4Rad) with specific primers for target and 194 reference genes at a final concentration of 0.9 VM (see Table 4). 195 The efficiency of PCR reactions for target and reference genes varied between 87 196 and 97%. The dynamic range of standard curves (serial dilutions of RT4PCR reactions) 197 spanned five orders of magnitude, and the amount of product in a particular sample was 198 determined by interpolation of the cycle threshold (Ct) value. The specificity of reaction 199 was verified by analysis of melting curves and by electrophoresis and sequencing of PCR 200 amplified products. Reactions were performed in triplicate and fluorescence data acquired 201 during the extension phase were normalized to β4actin, using the delta4delta method (Livak 202 and Schmittgen, 2001). 203 204 * 205 206 Tank average values of growth, feed intake and nutrient retention were used as 207 experimental units in one way analysis of variance followed by Student4Newman4Keuls 208 test at a significance level of P<0.05. Plasma levels of GH and IGF4I were analysed by one 209 and two4way analysis of variance, followed by Student4Newman4Keuls test. Correlation 210 analyses between hepatic transcripts and plasma hormone levels were made by Pearson 211 Product Moment correlations (P<0.05). 212 11 213 52 6 214 215 Diets 33VO and 66VO were well accepted by fish, and animals grew rapidly from 216 16 to 91492 g over the course of the 114week growth study (Table 5). No differences in feed 217 intake (69 to 67.5 g/fish), daily growth indices (2.66 to 2.68%), and feed (1.09 to 1.11) or 218 protein (2.21 to 2.25) efficiencies were found among control fish (FO) and fish fed 33VO 219 and 66VO diets. Total replacement of fish oil by the vegetable oil blend (diet VO) reduced 220 feed intake (61 g/fish) and daily growth indices (2.43%) without any significant effect on 221 whole body composition. Nitrogen (35 to 37%) and energy (50 to 52%) retentions were not 222 altered by dietary treatments, remaining high in all experimental groups. Lipid deposition 223 in mesenteric and liver depots was not affected significantly by dietary treatments, although 224 there was a trend for liver fat to increase with fish oil replacement. 225 At the end of the growth study, plasma levels of IGF4I were decreased over the 226 course of the post4pandrial period (P<0.05) (Fig. 1). The highest IGF4I concentration was 227 found in fish fed the 33VO diet and the lowest in fish fed the VO diet irrespective of 228 sampling time (5 to 20 h postfeeding). Intermediate values were found in control fish and 229 fish fed the 66VO diet. 230 There was no significant effect of dietary treatment on plasma GH levels (Fig. 2). 231 However, the trend was opposite to that of plasma IGF4I levels. First, the overall plasma 232 GH concentration increased over the course of post4pandrial period (P<0.05). Secondly, the 233 lowest GH concentration was found in fish fed the 33VO diet whereas increased values 234 were observed in fish fed the VO diet. 235 Hepatic IGF4I mRNA and plasma levels of IGF4I (20 h postfeeding) were positively 236 correlated (P< 0.05). The highest amount of IGF4I mRNA was found in fish fed the 33VO 12 237 diet with a progressive and significant decrease with additional fish oil replacement, 238 whereas control fish remained at intermediate values (Fig. 3A). IGF4II was expressed at a 239 reduced level and no significant changes were found with dietary treatments, although the 240 trend for IGF4II mRNA was similar to that reported for IGF4I mRNA (Fig. 3B). 241 Hepatic levels of GHR4I mRNA correlated positively with hepatic transcripts of 242 IGF4I and plasma levels of IGF4I (Fig. 4A). Thus, GHR4I mRNA decreased progressively 243 and significantly with the graded replacement of fish oil in fish fed 33VO, 66VO and VO 244 diets. Intermediate values were found in fish fed diet FO. The overall expression of GHR4II 245 was of the same order of magnitude, but no significant changes in GHR transcripts were 246 detected with dietary treatments (Fig. 4B). 247 Muscle expression of IGF4I was lower in comparison to that of IGF4II, and no 248 significant effect of dietary treatments on IGF4I mRNA levels were detected (Fig. 5A). By 249 contrast, IGF4II mRNA was down4regulated in fish fed vegetable oils irrespective of the 250 degree of replacement (Fig. 5B). 251 The overall muscle expression of GHR4I and II was of the same order of magnitude. 252 There was no consistent change on GHR4I mRNAs with dietary treatment (Fig. 6A). By 253 contrast, transcripts of GHR4II were progressively up4regulated in fish fed 33VO and 66VO 254 diets, decreasing thereafter with the 100% of replacement of fish oil (VO diet) (Fig. 6B). 255 13 256 72 8 257 258 The overall growth indices attained in the current work by juvenile gilthead sea 259 bream are higher than those reported for fish of the same age under similar light and 260 temperature conditions (Gómez4Requeni et al., 2003, 2004). This excellent growth 261 performance in all experimental groups could be attributed to improved diet formulation, 262 fish management and culture conditions. However, fish fed the VO diet showed a reduced 263 feed intake and increased liver fat deposition, which is characteristic of a wide range of 264 dietary and hormonal imbalances (see McClain et al., 2004; Avramoglu et al., 2006). 265 Indeed, in juvenile gilthead sea bream fed diets with amino acid imbalances, peripheral 266 lipolysis and tissue expression of lipoprotein lipase are regulated in concert to increase the 267 flux of dietary fatty acids through the liver (Albalat et al., 2005; Saera4Vila et al., 2005a). 268 This can be of special relevance during fasting and over4wintering, and extensive work is 269 now underway for this risk assessment. 270 Quantitative requirements of essential fatty acids (EFA) appear to vary depending 271 on fish species and growth stage (Sargent et al., 2002). Thus, the biological demand for n43 272 HUFA was at least 1.3% for flatfish larvae (Le Milinaire et al., 1983), whereas 273 requirements for juvenile and grower fish were reduced to 0.8% (Gatesoupe et al., 1977; 274 Lee et al., 2003; Kim and Lee, 2004) and 0.6% (Lèger et al., 1979), respectively. Similar 275 requirements have been reported for juveniles of European sea bass (Skalli and Robin, 276 2004) and gilthead sea bream (Kalogeropoulos et al., 1992) fed defatted fish meal and 277 casein4based diets, respectively. Likewise, no detrimental growth effects were found in the 278 present study in fish fed the 66VO diet (0.9% EPA + DHA, see Table 3), which indicates 279 that fish oil replacement by alternative vegetable oils is feasible at a high level when EFA 14 280 requirements are covered. Partial fish oil replacement has been conducted successfully in a 281 wide variety of fish species, but this is the first report that maximizes the simultaneous 282 replacement of fish meal and fish oil in practical aquafeeds for fast growing juvenile marine 283 fish. 284 Fish growth rates vary with season, age and nutritional status and most of these 285 regulatory events are mediated by the GH/IGF axis (Company et al., 2001; Pérez4Sánchez 286 et al., 2002). The wide tissue distribution of GHRs supports the pleiotropic action of GH, 287 although the liver is the most important target tissue of GH and the primary source of 288 systemic IGF4I (endocrine form). In this scenario, changes on the plasma binding capacity 289 of the 33447 kDa IGF4binding protein represents in rainbow trout an effective mechanism 290 to limit biologically active IGFs (free IGF fraction), keeping growth and GH secretion 291 under control (Gómez4Requeni et al., 2005). Likewise, circulating levels of IGF4I are 292 positively correlated with growth rates and dietary protein levels in Atlantic salmon and 293 Asian sea bass (Dyer et al., 2004). Plasma IGF4I levels are also a good indicator of growth 294 in channel catfish (Silverstein et al., 2000; Li et al., 2006). Similarly, in gilthead sea bream, 295 circulating GH and IGF4I are good markers of nutritional disorders arising from changes in 296 ration size (Pérez4Sánchez et al., 1995, 2002), dietary energy/ratio (Martí4Palanca et al., 297 1996; Company et al., 1999) and dietary protein source (Gómez4Requeni et al., 2003, 298 2004). In the current work, the decreased growth of fish fed the VO diet were accordingly 299 paralleled by decreased plasma levels of IGF4I. Since IGF4I mRNA and GHR4I mRNA 300 were also reduced, the reduction in growth could be attributed to a transcriptional defect in 301 the signal transduction of GHR in spite of increased plasma levels of GH. This metabolic 302 feature leads to liver GH resistance as is now widely accepted in several fish species 15 303 (Pérez4Sánchez et al., 1995; Beckman et al., 2004; Pierce et al., 2005; Wilkinson et al., 304 2006). 305 Growth in fish fed the VO diet (0.3% EPA + DHA; see Table 3) was only 90% of 306 the maximum observed, and there was no mortality in this group over the course of the 307 study. Similar results were reported for juvenile European sea bass fed defatted fish meal 308 diets (Skalli and Robin, 2004), which suggests that marine fish are relatively tolerant to 309 dietary fish oil restriction despite of the recognized essentiality of n43 HUFA. As stated 310 very early by Watanabe (1982), the triacyglycerol and polar lipid fractions of lipids, both 311 containing adequate amounts of EPA and DHA, have the same EFA value. Takeuchi and 312 Watanabe (1979) have shown that a level of EFA exceeding four times the requirement of 313 rainbow trout leads to poor growth and feed utilisation. Detrimental growth effects have 314 also been reported in juvenile flounder when dietary n43 HUFA becomes excessive (Kim 315 and Lee, 2004), and 25% replacement of fish oil by palm oil fatty acid distillate improved 316 weight gain of African catfish (Ng et al., 2004). In the present study, growth performance 317 of fish fed FO, 33VO and 66VO diets was almost identical, but the balance between 318 endocrine and locally produced IGFs differed depending on dietary treatment. Thus, in fish 319 fed the FO diet, the reduced gene expression and protein production of hepatic IGF4I was 320 apparently compensated by the increased expression of IGF4II at the local tissue level. 321 In mammals, IGF4II mRNA is detected in many fetal tissues but decreases quickly 322 during postnatal development (Daughaday and Rotwein, 1989). Accordingly, IGF4II null 323 mice are small at birth but continue to growth postnatally at a rate similar to wild4type. By 324 contrast, IGF4I null mice born were small and most died in the early neonatal stages. All 325 this strongly supports the key role of IGF4I during prenatal and postnatal growth. However, 326 hepatic IGF4I is not crucial for postnatal growth in mammals, and liver4specific IGF4I 16 327 knockout mice show normal growth due to the compensatory action of autocrine/paracrine 328 IGF4I (see Le Roith et al., 2001a,b). As postulated above in the present study, 329 compensatory increases of systemic IGF4I also occur in fish but, in this case, most of these 330 effects are dependent on local IGF4II. Indeed, substantial amounts of IGF4II are expressed 331 later in life in a wide range of fish species, including common carp (Vong et al., 2003), 332 rainbow trout (Chauvigné et al., 2003), Nile tilapia (Caelers et al., 2004), channel catfish 333 (Peterson et al., 2004), and gilthead sea bream as already evidenced in previous studies 334 (Duguay et al., 1996; Radaelli et al., 2003) and confirmed here. Furthermore, as found for 335 IGF4I, the main site for fish IGF4II expression is the liver, but in contrast to IGF4I, other 336 organs such as skeletal muscle also express quite high levels of IGF4II mRNA. Thus, fast 337 growing families of channel catfish express hepatic and muscle IGF4II at a high rate 338 (Peterson et al., 2004), and the growth spurt of juvenile rainbow trout during refeeding 339 could be mediated by muscle IGF4II (Chauvigné et al., 2003). Accordingly, it is reasonable 340 to assume that IGF4II acts in fish as an important growth4promoting factor through all the 341 life cycle, although most of these regulatory capabilities might have been lost during the 342 evolution of higher vertebrates. 343 To our knowledge, the precise mechanism(s) regulating the relative contribution of 344 systemic and local IGFs on fish growth remains unexplored. However, we suspect that 345 some results of the current study could be mediated by factors other than dietary fatty acids. 346 One of these factors might be the reduction in unwanted feed4borne lipid soluble 347 contaminants with the reduction in fish oil level. This assumption is based on our 348 complementary data (unpublished results) showing that dioxin4like PCBs in the FO diet 349 were markedly reduced with the graded fish oil replacement, as shown in previous studies 350 (Berntssen et al., 2005; Bethune et al., 2006). Experimental evidence also indicates that the 17 351 wasting syndrome caused in mice by 34methylcholanthrene is mediated by aromatic 352 hydrocarbon receptors (AHRs) that interact with xenobiotic responsive elements (XREs) in 353 the GHR promoter, disrupting the liver GH signalling pathway (IGF production) (Nukaya 354 et al., 2004). Likewise, several XREs have been identified in the 5´4flanking region of 355 gilthead sea bream GHR4I (unpublished results), although further studies are needed to 356 determine whether these cis4regulatory elements are functional in fish. Besides, it has been 357 proven that expression levels of GH and prolactin (PRL) are regulated in rainbow trout by 358 persistent xenoestrogens and antiestrogenic pollutants (Elango et al., 2006), and both 359 estradiol and 44nonylphenol suppress growth and plasma levels of IGF4I in juvenile 360 Atlantic salmon (Arsenault et al., 2004). It is not surprising, therefore, that gonadal steroids 361 modulate hepatic production of IGF4I and IGFBPs in tilapia (Riley et al., 2004) and coho 362 salmon (Larsen et al., 2004). All this provides regulatory mechanisms for dimorphic growth 363 patterns in fish, but at the same time makes the GH/IGF axis more vulnerable to potential 364 anthropogenic feed4borne contaminants. 365 Transgenic models in mice also indicate that major effects of GH on growth are 366 dependent on IGF4I expression, which requires intact insulin and IGF4I receptor signalling 367 in skeletal muscle (Kim et al., 2005). However, GH regulates other mitogenic factors, and 368 there is now experimental evidence supporting the up4regulated expression of GHRs during 369 muscle repair and maintenance (Casse et al., 2003). In fish, it is believed that genetic 370 duplication and divergence of two GHR subtypes (GHR type I and II) would take place on 371 an early ancestor of fish lineage (Saera4Vila et al., 2005b; Jiao et al., 2006). GHR4I was 372 first described in non4salmonid fish (Calduch4Giner et al., 2001), conserving most of the 373 structural features of mammalian GHRs. By contrast, GHR4II (also named somatolactin 374 receptor by Fukada et al., 2005) is unique to teleosts and encompasses most of the 18 375 published GHR sequences of salmonid fish. These two GHR subtypes are conserved in a 376 wide range of fish species, although apparent silencing and/or genomic loss of GHR4II was 377 reported in the flatfish lineage with the occurrence of truncated variants of GHR4I (Pérez4 378 Sánchez et al., 2002; Saera4Vila et al., 2005b). In this scenario, the current study confirms 379 and extends the notion that major GH effects on growth and hepatic IGF expression are 380 mediated by GHR4I in gilthead sea bream. By contrast, GHR4II emerges as a more 381 constitutive gene that does not necessarily require intact IGF4pathways to exert a protective 382 and/or growth promoting action. This is consistent with the up4regulated expression of 383 GHR4II in skeletal muscle of fish fed the 66VO diet. In this way, we previously reported 384 that GHR4II is up4regulated in the skeletal muscle of fasted juvenile gilthead sea bream 385 (Calduch4Giner et al., 2003). Similarly, Fukada et al. (2004) indicated that transcript levels 386 of GHR4II are not related in masu salmon to decreased expression of hepatic IGF4I during 387 fasting. 388 In summary, our data strongly support that combined replacement at a high level of 389 fish meal and oil is possible in diets of gilthead sea bream, contributing to the development 390 of sustainable aquafeeds. Data also bring new insights on the compensatory regulation of 391 systemic and local components of the GH/IGF axis (see Fig. 7 for a comprehensive survey) 392 in growing fish. Additional studies are underway to further explore the potential of 393 practical diets low in marine ingredients over the full cycle of gilthead sea bream farming, 394 addressing also issues related to potential feed4borne endocrine disruption of GH/IGF axis. 19 395 ( 396 397 This research was funded by EU (FOOD4CT42006416249; Sustainable Aquafeeds to 398 Maximise the Health Benefits of Farmed Fish for Consumers, AQUAMAX), and Spanish 399 (AGL20044063194CO2) projects. A.S4V was recipient of a PhD fellowship from the 400 Diputación Provincial de Castellón. The authors are grateful to M.A. Gónzalez for excellent 401 technical assistance in real4time PCR assays. 20 402 6 403 404 Albalat, A., Gómez4Requeni, P., Rojas, P., Médale, F., Kaushik, S., Vianen, J., Van den 405 Thillart, G., Gutiérrez, J., Pérez4Sánchez, J., Navarro, I., 2005. 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Aquaculture 171, 2794292. 457 Company, R., Astola, A., Pendón, C., Valdivia, M.M., Pérez4Sánchez, J., 2001. 458 Somatotropic regulation of fish growth and adiposity: growth hormone (GH) and 459 somatolactin (SL) relationship. Comp. Biochem. Physiol. 130C, 4354445. 460 Daughaday, W.H., Rotwein, P., 1989. Insulin4like growth factors I and II. Peptide, 461 messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. 462 Rev. 10, 68491. ): risks and benefits of high 23 463 Duguay, S.J., Lai4Zhang, J., Steiner, D.F., Funkenstein, B., Chan, S.J., 1996. 464 Developmental and tissue4regulated expression of IGF4I and IGF4II mRNAs in 465 . J. Mol. Endocrinol. 16, 1234132. 466 Dyer, A.R., Barlow, C.G., Bransden, M.P., Carter, C.G., Glencross, B.D., Richardson, N., 467 Thomas, P.M., Williams, K.C., Carragher, J.F., 2004. Correlation of plasma IGF4I 468 concentrations and growth rate in aquacultured finfish: a tool for assessing the potential of 469 new diets. Aquaculture 236, 5834592. 470 Elango, A., Shepherd, B., Chen, T.T., 2006. Effects of endocrine disrupters on the 471 expression of growth hormone and prolactin mRNA in the rainbow trout pituitary. Gen. 472 Comp. Endocrinol. 145, 1164127. 473 FAO, Food and Agriculture Organization, 2004. The state of world fisheries and 474 aquaculture. Fisheries department, Rome, 168pp. 475 Francis, D.S., Turchini, G.M., Jones, P.L., De Silva, S.S., 2006. Effects of dietary oil 476 source on growth and fillet fatty acid composition of Murray cod, + 477 . Aquaculture 253, 5474556. 478 Fukada, H., Ozaki, Y., Pierce, A.L., Adachi, S., Yamauchi, K., Hara, A., Swanson, P., 479 Dickhoff, W.W., 2004. Salmon growth hormone receptor: molecular cloning, ligand 480 specificity, and response to fasting. Gen. Comp. Endocrinol. 139, 61471. 481 Fukada, H., Ozaki, Y., Pierce, A.L., Adachi, S., Yamauchi, K., Hara, A., Swanson, P., 482 Dickhoff, W.W., 2005. Identification of the salmon somatolactin receptor, a new member 483 of the cytokine receptor family. Endocrinology 146, 235442361. 24 484 Gatesoupe, F.J., Lèger, C., Boudon, M., Métailler, R., Luquet, P., 1977. Lipid feeding of 485 turbot ( 486 esters of linolenic acid and fatty acids of ω−9 series. Ann. Hydrobiol. 8, 2474254. 487 Gómez4Requeni, P., Mingarro, M., Kirchner, S., Calduch4Giner, J.A., Médale, F., Corraze, 488 G., Panserat, S., Martin, S.A.M., Houlihan, D.F., Kaushik, S.J., Pérez4Sánchez, J., 2003. 489 Effects of dietary amino acid profile on growth performance, key metabolic enzymes and 490 somatotropic axis responsiveness of gilthead sea bream ( 491 7494767. 492 Gómez4Requeni, P., Mingarro, M., Calduch4Giner, J.A., Médale, F., Martin, S.A.M., 493 Houlihan, D.F., Kaushik, S., Pérez4Sánchez, J., 2004. Protein growth performance, amino 494 acid utilisation and somatotropic axis responsiveness to fish meal replacement by plant 495 protein sources in gilthead sea bream ( 496 Gómez4Requeni, P., Calduch4Giner, J., Vega4Rubín de Celis, S., Médale, F., Kaushik, S.J., 497 Pérez4Sánchez, J., 2005. Regulation of the somatotropic axis by dietary factors in rainbow 498 trout (- 499 Hardy, M.L., 2004. A comparison of the fish bioconcentration factors for brominated flame 500 retardants with their nonbrominated analogues. Environ. Toxicol. Chem. 23, 6564661. 501 Izquierdo, M.S., Montero, D., Robaina, L., Caballero, M.J., Rosenlund, G., Ginés, R., 2005. 502 Alterations in fillet fatty acid profile and flesh quality in gilthead seabream ( 503 fed vegetable oils for a long term period. Recovery of fatty acid profiles by fish oil feeding. 504 Aquaculture 250, 4314444. & . L). 2. Influence on growth of supplementation with methyl ). Aquaculture 220, ). Aquaculture 232, 4934510. ). Br. J. Nutr. 94, 3534361. ) 25 505 Jacobs, M.N., Covaci, A., Schepens, P., 2002. Investigation of persistent organic pollutants 506 in farmed Atlantic salmon ( 507 of the feed. Environ. Sci. Technol. 36, 279742805. 508 Jiao, B.W., Huang, X.G., Chan, C.B., Zhang, L., Wang, D.S., Cheng, C.H.K., 2006. The 509 co4existence of two growth hormone receptors in teleost fish and their differential signal 510 transduction, tissue distribution and hormonal regulation of expression in seabream. J. Mol. 511 Endocrinol. 36, 23440. 512 Kalogeropoulos, N., Alexis, M.N., Henderson, R.J., 1992. Effects of dietary soybean and 513 cod4liver oil levels on growth and body composition of gilthead bream ( 514 Aquaculture 104, 2934308. 515 Kaushik, S.J., 1998. Whole body amino acid composition of European seabass 516 ( 517 with an estimation of their IAA requirement profiles. Aquat. Living Resour. 11, 3554358. 518 Kim, K.D., Lee, S.M., 2004. Requirement of dietary n43 highly unsaturated fatty acids for 519 juvenile flounder () 520 Kim, H., Barton, E., Muja, N., Yakar, S., Pennisi, P., LeRoith, D., 2005. Intact insulin and 521 insulin4like growth factor4I receptor signaling is required for growth hormone effects on 522 skeletal muscle growth and function in vivo. Endocrinology 146, 177241779. 523 Larsen, D.A., Shimizu, M., Cooper, K.A., Swanson, P., Dickhoff, W.W., 2004. Androgen 524 effects on plasma GH, IGF4I, and 414kDa IGFBP in coho salmon (- 525 Gen. Comp. Endocrinol. 139, 29437. ), salmon aquaculture feed, and fish oil components / &), gilthead seabream ( , ). ) and turbot () & ) ). Aquaculture 229, 3154323. . ). 26 526 Le Milinaire, C., Gatesoupe, F.J., Stephan, G., 1983. Quantitative needs for n43 series poly4 527 unsaturated long chain fatty acids requirement in turbot ( 528 C.R. Acad. Sci. Paris 296, 9174920. 529 Le Roith, D., Bondy, C., Yakar, S., Liu, J.L., Butler, A., 2001a. The somatomedin 530 hypothesis: 2001. Endocr. Rev. 22, 53474. 531 Le Roith, D., Scavo, L., Butler, A., 2001b. What is the role of circulating IGF4I? Trends 532 Endocrinol. Metab. 12, 48452. 533 Lee, S.M., Lee, J.H., Kim, K.D., 2003. Effect of dietary essential fatty acids on growth, 534 body composition and blood chemistry of juvenile starry flounder () 535 Aquaculture 225, 2694281. 536 Lèger, C., Gatesoupe, F.J., Métailler, R., Luquet, P., Fremont, L., 1979. Effect of dietary 537 fatty acids differing by chain lengths and ω series on the growth and lipid composition of 538 turbot 539 Li, M.H., Peterson, B.C., Janes, C.L., Robinson, E.H., 2006. Comparison of diets 540 containing various fish meal levels on growth performance, body composition, and insulin4 541 like growth factor4I of juvenile channel catfish 542 Aquaculture 253, 6284635. 543 Livak K.J., Schmittgen T.D., 2001. Analysis of relative gene expression data using real4 544 time quantitative PCR and the 24∆∆ Ct method. Methods 25, 4024408. & & ) larvae. ). L. Comp. Biochem. Physiol. 64B, 3454350. of different strains. 27 545 Martí4Palanca, H., Martínez4Barberá, J.P., Pendón, C., Valdivia, M.M., Pérez4Sanchez, J., 546 Kaushik, S., 1996. Growth hormone as a function of age and dietary protein: energy ratio in 547 a marine teleost, the gilthead sea bream ( 548 Martínez4Barberá, J.P., Pendón, C., Martí4Palanca, H., Calduch4Giner, J.A., Rodríguez, 549 R.B., Valdivia, M.M., Pérez4Sánchez, J., 1995. The use of recombinant gilthead sea bream 550 ( 551 radioimmunoassay. Comp. Biochem. Physiol. 110A, 3354340. 552 McClain, C.J., Mokshagundam, S.P., Barve, S.S., Song, Z., Hill, D.B., Chen, T., Deaciuc, 553 I., 2004. Mechanisms of non4alcoholic steatohepatitis. Alcohol 34, 67479. 554 Montero, D., Robaina, L., Caballero, M.J., Ginés, R., Izquierdo, M.S., 2005. Growth, feed 555 utilization and flesh quality of European sea bass ( 556 containing vegetable oils: A time4course study on the effect of a re4feeding period with a 557 100% fish oil diet. Aquaculture 248, 1214134. 558 Mourente, G., Dick, J.R., Bell, J.G., Tocher, D.R., 2005. Effect of partial substitution of 559 dietary fish oil by vegetable oils on desaturation and β4oxidation of [1414C] 18:3n43 (LNA) 560 and [1414C] 20:5n43 (EPA) in hepatocytes and enterocytes of European sea bass 561 ( 562 Mourente, G., Bell, J.G., 2006. Partial replacement of dietary fish oil with blends of 563 vegetable oils (rapeseed, linseed and palm oils) in diets for European sea bass 564 ( 565 acid composition and effectiveness of a fish oil finishing diet. Comp. Biochem. Physiol. 566 145B, 3894399. ). Growth Regul. 6, 2534259. ) growth hormone for radioiodination and standard preparation in / &) fed diets / & L.). Aquaculture 248, 1734186. / & L.) over the whole production cycle: effects on flesh and liver fatty 28 567 Ng, W.K., Wang, Y., Ketchimenin, P., Yuen, K.H., 2004. Replacement of dietary fish oil 568 with palm fatty acid distillate elevates tocopherol and tocotrienol concentrations and 569 increases oxidative stability in the muscle of African catfish, 570 Aquaculture 233, 4234437. 571 Nukaya, M., Takahashi, Y., González, F.J., Kamataki, T., 2004. Aryl hydrocarbon receptor4 572 mediated suppression of GH receptor and Janus kinase 2 expression in mice. FEBS Lett. 573 558, 964100. 574 Opstvedt, J., Nygård, E., Samuelsen, T.A., Venturini, G., Luzzana, U., Mundheim, H., 575 2003. Effect on protein digestibility of different processing conditions in the production of 576 fish meal and fish feed. J. Sci. Food Agric. 83, 7754782. 577 Pérez4Sánchez, J., Le Bail, P.Y., 1999. Growth hormone axis as marker of nutritional status 578 and growth performance in fish. Aquaculture 177, 1174128. 579 Pérez4Sánchez, J., Martí4Palanca, H., Kaushik, S.J., 1995. Ration size and protein intake 580 affect circulating growth hormone concentration, hepatic growth hormone binding and 581 plasma insulin4like growth factor4I immunoreactivity in a marine teleost, the gilthead 582 bream ( 583 Pérez4Sánchez, J., Calduch4Giner, J.A., Mingarro, M., Vega4Rubín de Celis, S., Gómez4 584 Requeni, P., Saera4Vila, A., Astola, A., Valdivia, M.M., 2002. Overview of fish growth 585 hormone family. New insights in genomic organization and heterogeneity of growth 586 hormone receptors. Fish Physiol. Biochem. 27, 2434258. Special Issue, E.M. Plisetskaya 587 (Ed.), Fish Growth and Metabolism. Experimental, Nutritional and Hormonal Regulation, 588 published in 2004. . ). J. Nutr. 125, 5464552. 29 589 Peterson, B.C., Waldbieser, G.C., Bilodeau, L., 2004. IGF4I and IGF4II mRNA expression 590 in slow and fast growing families of USDA103 channel catfish ( 591 Comp. Biochem. Physiol. 139A, 3174323. 592 Pierce, A.L., Shimizu, M., Beckman, B.R., Baker, D.M., Dickhoff, W.W., 2005. Time 593 course of the GH/IGF axis response to fasting and increased ration in chinook salmon 594 (- 595 Radaelli, G., Patruno, M., Maccatrozzo, L., Funkenstein, B., 2003. Expression and cellular 596 localization of insulin4like growth factor4II protein and mRNA in 597 development. J. Endocrinol. 178, 2854299. 598 Regost, C., Arzel, J., Robin, J., Rosenlund, G., Kaushik, S.J., 2003. Total replacement of 599 fish oil by soybean or linseed oil with a return to fish oil in turbot () 600 Growth performance, flesh fatty acid profile, and lipid metabolism. Aquaculture 217, 4654 601 482. 602 Richard, N., Kaushik, S., Larroquet, L., Panserat, S., Corraze, G., 2006a. Replacing dietary 603 fish oil by vegetable oils has little effects on lipogenesis, lipid transport and tissue lipid 604 uptake in rainbow trout (- 605 Richard, N., Mourente, G., Kaushik, S., Corraze, G., 2006b. Replacement of a large portion 606 of fish oil by vegetable oils does not affect lipogenesis, lipid transport and tissue lipid 607 uptake in European sea bass ( 608 Riley, L.G., Hirano, T., Grau, E.G., 2004. Estradiol417β and dihydrotestosterone 609 differentially regulate vitellogenin and insulin4like growth factor4I production in primary ). ). Gen. Comp. Endocrinol. 140, 1924202. . during & ): 1. ). Br. J. Nutr. 96, 2994309. / & L.). Aquaculture 261, 107741087. 30 610 hepatocytes of the tilapia - 611 186. 612 Saera4Vila, A., Calduch4Giner, J.A., Gómez4Requeni, P., Médale, F., Kaushik, S., Pérez4 613 Sánchez, J., 2005a. Molecular characterization of gilthead sea bream ( 614 lipoprotein lipase. Transcriptional regulation by season and nutritional condition in skeletal 615 muscle and fat storage tissues. Comp. Biochem. Physiol. 142B, 2244232. 616 Saera4Vila, A., Calduch4Giner, J.A., Pérez4Sánchez, J., 2005b. Duplication of growth 617 hormone receptor (GHR) in fish genome: gene organization and transcriptional regulation 618 of GHR type I and II in gilthead sea bream ( 619 1934203. 620 Sargent, J., McEvoy, L., Estevez, A., Bell, G., Bell, M., Henderson, J., Tocher, D., 1999. 621 Lipid nutrition of marine fish during early development: current status and future 622 directions. Aquaculture 179, 2174229. 623 Sargent, J., Tocher, D.R., Bell, J.G., 2002. The lipids. In: Halver, J.E., Hardy, R.W. (Eds.), 624 Fish Nutrition. Academic Press, San Diego, pp. 1814257. 625 Silverstein, J.T., Wolters, W.R., Shimizu, M., Dickhoff, W.W., 2000. Bovine growth 626 hormone treatment of channel catfish: strain and temperature effects on growth, plasma 627 IGF4I levels, feed intake and efficiency and body composition. Aquaculture 190, 77488. 628 Sitjà4Bobadilla, A., Peña4Llopis, S., Gómez4Requeni, P., Médale, F., Kaushik, S., Pérez4 629 Sánchez, J., 2005. Effect of fish meal replacement by plant protein sources on non4specific / . Comp. Biochem. Physiol. 138C, 1774 ) ). Gen. Comp. Endocrinol. 142, 31 630 defence mechanisms and oxidative stress in gilthead sea bream ( ). 631 Aquaculture 249, 3874400. 632 Skalli, A., Robin, J.H., 2004. Requirement of n43 long chain polyunsaturated fatty acids for 633 European sea bass ( 634 Aquaculture 240, 3994415. 635 Takeuchi, T., Watanabe, T., 1979. Effect of excess amounts of essential fatty acids on 636 growth of rainbow trout. Bull. Jpn. Soc. Sci. Fish. 45, 151741519. 637 Torstensen, B.E., Froyland, L., Ørnsrud, R., Lie, Ø., 2004. Tailoring of a cardioprotective 638 muscle fatty acid composition of Atlantic salmon ( 639 Chem. 87, 5674580. 640 Torstensen, B.E., Bell, J.G., Rosenlund, G., Henderson, R.J., Graff, I.E., Tocher, D.R., Lie, 641 Ø., Sargent, J.R., 2005. Tailoring of Atlantic salmon ( 642 composition and sensory quality by replacing fish oil with vegetable oil blend. J. Agric. 643 Food Chem. 53, 10166410178. 644 Vega4Rubín de Celis, S., Gómez4Requeni, P., Pérez4Sánchez, J., 2004. Production and 645 characterization of recombinantly derived peptides and antibodies for accurate 646 determinations of somatolactin, growth hormone and insulin4like growth factor4I in 647 European sea bass ( 648 Vong, Q.P., Chan, K.M., Leung, K., Cheng, C.H.K., 2003. Common carp insulin4like 649 growth factor4I gene: complete nucleotide sequence and functional characterization of the 650 5'4flanking region. Gene 322, 1454156. / &) juveniles: growth and fatty acid composition. ) fed vegetable oils. Food L.) flesh lipid / &). Gen. Comp. Endocrinol. 139, 2664277. 32 651 Watanabe, T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol. 73B, 3415. 652 Wilkinson, R.J., Porter, M., Woolcott, H., Longland, R., Carragher, J.F., 2006. Effects of 653 aquaculture related stressors and nutritional restriction on circulating growth factors (GH, 654 IGF4I and IGF4II) in Atlantic salmon and rainbow trout. Comp. Biochem. Physiol. 145A, 655 2144224. 33 656 9 1. Ingredients and chemical composition of experimental diets. Ingredient (g/kg) Fish meal (CP 70%) 1 CPSP 90 2 Corn gluten meal Soybean meal Extruded wheat Fish oil 3 Rapeseed oil Linseed oil Palm oil Soya lecithin Binder (sodium alginate) Mineral premix 4 Vitamin premix 5 CaHPO4.2H2O (18%P) L4Lysine FO 15 5 40 14.3 4 15.1 0 0 0 1 1 1 1 2 0.55 33VO 15 5 40 14.3 4 10.1 0.85 2.9 1.25 1 1 1 1 2 0.55 66VO 15 5 40 14.3 4 5.1 1.7 5.8 2.5 1 1 1 1 2 0.55 VO 15 5 40 14.3 4 0 2.58 8.8 3.8 1 1 1 1 2 0.55 93.4 48.9 22.2 6.5 2.3 24.7 94.2 48.7 22.3 6.6 1.6 24.7 94.8 49.0 22.1 6.6 0.9 24.6 95.4 48.6 22.3 6.4 0.3 24.5 ) & Dry matter (DM, %) Crude protein (% DM) Crude fat (% DM) Ash (% DM) EPA + DHA (% DM) Gross energy (kJ/g DM) 657 658 659 660 661 662 663 664 665 666 667 668 669 1 Fish meal (Scandinavian LT) Fish soluble protein concentrate (Sopropêche, France) 3 Fish oil (Sopropêche, France) 4 Supplied the following (mg / kg diet, except as noted): calcium carbonate (40% Ca) 2.15 g, magnesium hydroxide (60% Mg) 1.24 g, potassium chloride 0.9 g, ferric citrate 0.2 g, potassium iodine 4 mg, sodium chloride 0.4 g, calcium hydrogen phosphate 50 g, copper sulphate 0.3, zinc sulphate 40, cobalt sulphate 2, manganese sulphate 30, sodium selenite 0.3 5 Supplied the following (mg / kg diet): retinyl acetate 2.58, DL4cholecalciferol 0.037, DL4α tocopheryl acetate 30, menadione sodium bisulphite 2.5, thiamin 7.5, riboflavin 15, pyridoxine 7.5, nicotinic acid 87.5, folic acid 2.5, calcium pantothenate 2.5, vitamin B12 0.025, ascorbic acid 250, inositol 500, biotin 1.25 and choline chloride 500 2 34 670 671 672 713 714 715 716 9 3. Amino acid, mineral and trace element composition of the diets along with data on amino acid needs (Kaushik 1998). 673 Needs 674 Amino acid (%) FO/VO diets 1.86 675 Arg 2.18 676 0.86 677 His 0.93 1.11 678 Ile 1.92 1.73 679 Leu 5.58 2.23 680 Lys 2.42 681 Met 1.05 682 Cys 0.61 683 1.24 684 Cys + Met 1.66 685 Phe 2.47 686 Tyr 2 687 2.23 688 Phe + Tyr 4.47 0.99 689 Thr 1.69 0.25 690 Trp 0.39 1.49 691 Val 2.15 692 Ser 2.23 693 Ala 3.36 694 695 Asp 3.69 696 Glu 8.37 697 Gly 2.01 698 Pro 3.02 699 700 701 + (%, Ng/g) 702 Phosphorous (%) 1.08 703 Magnesium (%) 0.18 704 705 Potassium (%) 0.74 706 Iron (Ng/g) 216 707 Copper (Ng/g) 15 708 Manganese (Ng/g) 19 709 Zinc (Ng/g) 50 710 711 Selenium (Ng/g) 0.9 712 35 717 9 5. Fatty acid composition of experimental diets (% total FAME). Values 718 are means of two determinations; tr = trace value < 0.05 Fatty acid FO 33VO 66VO VO 14:0 5.02 3.70 1.89 0.59 15:0 0.35 0.22 0.13 0.12 16:0 16.7 16.9 16.9 16.7 16:1n47 4.63 2.97 1.96 0.76 16:1n49 0.22 0.15 tr tr 16:3 0.49 0.35 0.26 0.14 16:3n43 0.19 0.13 0.08 tr 16:4 0.40 0.29 0.17 tr 17:0 0.41 0.29 0.23 0.10 18:0 2.55 2.92 3.43 3.73 18:1n49 12.5 17.5 21.9 25.9 18:1n47 1.92 1.69 1.49 1.21 18:2n46 12.1 15.7 19.2 21.3 18:3n43 1.58 8.94 16.3 23.2 18:4n43 2.16 1.47 0.82 0.20 20:0 0.30 0.30 0.31 0.29 20:1n49 7.24 5.12 3.05 1.06 20:1n47 0.21 0.16 0.09 tr 20:2n46 0.17 0.12 0.11 tr 20:3n43 0.08 0.07 tr tr 20:4n46 0.31 0.22 0.13 tr 20:4n43 0.43 0.28 0.15 tr 20:5n43 (EPA) 6.86 4.68 2.75 0.94 22:0 tr 0.16 0.16 0.17 22:1n411 10.19 6.74 3.68 0.74 22:1n49 0.56 0.43 0.29 0.16 22:2n46 0.24 0.17 tr tr 22:5n43 0.64 0.40 0.18 tr 22:6n43 (DHA) 8.34 5.68 3.38 1.06 Total Saturates Monoenes n46 HUFA1 n43 HUFA1 719 1 96.9 25.3 37.6 0.31 16.3 97.7 24.5 34.8 0.22 11.9 98.9 22.9 32.4 0.12 6.5 Fatty acids with more than 20 carbon atoms and more than 3 double bonds. 98.4 21.7 29.8 0.7 2 36 720 7. Primers for real4time PCR. Forward primer, f; reverse primer, r. 9 Gene 721 722 Accession Primer sequence Position β4actin X89920 f r 5’4 5’4 TCC TGC GGA ATC CAT GAG A GAC GTC GCA CTT CAT GAT GCT 8114829 8614841 GHR4I AF438176 f r 5’4 5’4 ACC TGT CAG CCA CCA CAT GA TCG TGC AGA TCT GGG TCG TA 127541294 137341354 GHR4II AY573601 f r 5’4 5’4 GAG TGA ACC CGG CCT GAC AG GCG GTG GTA TCT GAT TCA TGG T 169041709 176441743 IGF4I AY996779 f r 5’4 5’4 TGT CTA GCG CTC TTT CCT TTC A AGA GGG TGT GGC TAC AGG AGA TAC 1124133 1954172 IGF4II AY996778 f r 5’4 5’4 TGG GAT CGT AGA GGA GTG TTG T CTG TAG AGA GGT GGC CGA CA 4064427 5144495 37 723 724 725 726 9 :2 Data on growth performance, whole body composition, and nutrient gain and retention of gilthead sea bream fed the four experimental diets for 11 weeks. Each value is the mean ± SEM of data from triplicate groups. Data on viscera, liver and mesenteric fat indices were calculated from 16 fish. FO 33VO 66VO VO ) Initial body weight (g) 16.1 ± 0.09 16.3 ± 0.01 16.3 ± 0.03 16.1 ± 0.09 Final body weight (g) 91.7 ± 0.45 b 91.3 ± 0.90 91.1 ± 1.20 a 10 00 Viscera (g) 8.28 ± 0.45 8.50 ± 0.33 8.37 ± 0.31 8.35 ± 0.46 0 ** Mesenteric fat (g) 1.72 ± 0.24 1.66 ± 0.11 1.79 ± 0.15 1.52 ± 0.14 0 Liver (g) b b 80.9± 0.28 0 1.78 ± 0.12 1.82 ± 0.09 1.92 ± 0.08 1.72 ± 0.13 0 (2 VSI (%) 2 9.36 ± 0.30 9.12 ± 0.23 9.10 ± 0.24 9.89 ± 0.55 0 2 MFI (%) 3 1.78 ± 0.23 1.73 ± 0.39 1.95 ± 0.17 1.78 ± 0.15 0 " HSI (%) 4 1.85 ± 0.07 15.9 ± 0.83 1.93 ± 0.11 17.7 ± 0.94 2.09 ± 0.09 18.7 ± 1.05 2.02 ± 0.16 0 2 19.3 ± 0.51 0 0( a 10 00 Liver fat (%) DM intake (g/fish) 68.8 ± 0.60 b Weight gain (%) DGI (%) b 467.6 ± 6.2 2.68 ± 0.03 5 68.9 ± 0.66 460.3 ± 5.2 b b 2.66 ± 0.03 67.6 ± 0.25 460.1 ± 7.1 b b b b 2.66 ± 0.05 61.3 ± 0.77 401.9 ± 3.1 b a 2.43 ± 0.02 10 00 a 10 00 1.10 ± 0.01 1.09 ± 0.01 1.11 ± 0.02 1.06 ± 0.01 0 0* 2.21 ± 0.01 2.23 ± 0.01 2.25 ± 0.04 2.14 ± 0.02 0 0( 64.1 ± 0.48 16.0 ± 0.52 64.1± 0.28 16.7 ± 0.43 63.9 ± 0.32 0 *0 Crude protein 64.3 ± 0.29 15.9 ± 0.46 16.9 ± 0.12 0 0 Crude fat 14.1 ± 0.67 14.3 ± 0.34 14.4 ± 0.42 14.4 ± 0.10 0 7" Ash 2.88 ± 0.11 3.44 ± 0.27 3.38 ± 0.14 3.61 ± 0.16 0 07 Nitrogen 35.4 ± 1.27 35.1 ± 1.12 36.9 ± 1.92 37.3 ± 0.63 0 "( Energy 50.1 ± 2.01 50.3 ± 1.06 52.5 ± 0.55 51.9 ± 0.76 0 "2 FE PER 7 3 / 45 Moisture # 6 45 . 6 727 728 Initial body composition: water, 70.9%; protein, 15.1%; lipid, 9.3%; ash, 3.4% 729 1P values result from analysis of variance. Different superscript letters in each row indicate significant 730 differences among dietary treatments (Student Newman4Keuls test, P<0.05). 731 2Viscerosomatix index = (100 × viscera wt) / fish wt 732 3Mesenteric fat index = (100 × mesenteric fat wt) / fish wt 733 4Hepatosomatic index = (100 × liver wt) / fish wt 734 5Daily growth index = [100 × (final fish wt1/3 − initial fish wt1/3)] / days 735 6Feed efficiency = wet weight gain / dry feed intake 736 7Protein efficiency ratio = wet weight gain / protein intake 38 737 738 739 Figure 1. Plasma levels of insulin4like growth factor4I (IGF4I) in fish fed experimental diets 740 5 h after the meal (A) and following overnight fasting (B). Each value is the mean ± SEM 741 of 10 to 12 animals. Values with different letters are significantly different (P<0.05). 742 743 Figure 2. Plasma growth hormone (GH) levels in fish fed experimental diets 5 h after the 744 meal (A) and following overnight fasting (B). Each value is the mean ± SEM of 10 to 12 745 animals. 746 747 Figure 3. Normalized mRNA levels of IGF4I (A) and IGF4II (B) in the liver of fish fed 748 experimental diets (20 h postfeeding). Each value is the mean ± SEM of 6 to 8 animals. 749 Values with different letters are significantly different (P<0.05). 750 751 Figure 4. Normalized mRNA levels of GHR4I (A) and GHR4II (B) in the liver of fish fed 752 experimental diets (20 h postfeeding). Each value is the mean ± SEM of 6 to 8 animals. 753 Values with different letters are significantly different (P<0.05). 754 755 Figure 5. Normalized mRNA levels of IGF4I (A) and IGF4II (B) in the skeletal muscle of 756 fish fed experimental diets (20 h postfeeding). Each value is the mean ± SEM of 6 to 8 757 animals. Values with different letters are significantly different (P<0.05). 758 39 759 Figure 6. Normalized mRNA levels of GHR4I (A) and GHR4II (B) in the skeletal muscle of 760 fish fed experimental diets (20 h postfeeding). Each value is the mean ± SEM of 6 to 8 761 animals. Values with different letters are significantly different (P<0.05). 762 763 Figure 7. Proposed model for the balanced regulation of systemic and local components of 764 GH/IGF axis. Growth dysfunction occurs when the reduced production of systemic IGF4I is 765 not compensated at the local tissue level (fish fed VO diet). Compensatory IGF4II 766 production occurs at the local tissue level in fish fed FO diet. Alternatively, other 767 compensatory mechanisms of GH/IGF axis could be mediated at the local tissue level by 768 GHR4II via unknown factors, X, (66VO diet). 40 769 Figure 1 ! "%! : 11; 1;; ?; >; =; <; :; 7; & a A ab ab b ! "%! 3; 11; 1;; ?; >; =; <; :; 7; B a & % a ab b "@ 770 % 55*@ <<*@ *@ AB&A6!4AC9( 8!A9 41 771 Figure 2 1< : A & % 17 13 1; > < 7 3; 1< & % B 17 13 1; > < 7 "@ 772 55*@ <<*@ *@ AB&A6!4AC9( 8!A9 42 Figure 3 123 A 12; a b ;2< ;27 ;23 6C( 6D ;2; 123 ;27 B 12; ;2> ;2< ;23 ;2; "@ 774 a a ;2> ! "%!! ! "%! 6C( 6D 773 55*@ <<*@ *@ AB&A6!4AC9( 8!A9 43 6C( 6D Figure 4 123 6%! 775 ;27 A a 12; ab ;2> ab b ;2< ;23 6C( 6D 123 6%!! ;2; ;27 B 12; ;2> ;2< ;23 ;2; "@ 776 55*@ <<*@ *@ AB&A6!4AC9( 8!A9 44 Figure 5 123 A 12; ;2> ;2< ;27 ;23 6C( 6D ;2; 123 ! "%!! ! "%! 6C( 6D 777 ;27 B a 12; b ;2> b ;2< ;23 ;2; "@ 778 b 55*@ <<*@ *@ AB&A6!4AC9( 8!A9 45 6C( 6D Figure 6 123 6%! 779 ;27 A 12; ;2> ;2< ;23 6C( 6D 123 6%!! ;2; ;27 B a 12; ;2> ab ab b ;2< ;23 ;2; "@ 780 55*@ <<*@ *@ AB&A6!4AC9( 8!A9 46 781 Figure 7 COMPENSATORY NETWORKING OF GH/IGF AXIS 782 DISRUPTION OF GH/IGF AXIS 783 GH GH GH GH GH GH GH GH GH GHR-I GHR-I IGF-I GHR-I IGF-I - - IGF-I IGF-I IGF-I IGF-I IGF-I IGF-I IGF-I IGF-II IGF-II GHR-II IGF-II IGF-II IGF-II 33VO Dietd GHR-II FO 66VO GHR-II VO 47 784