Inorganic, organic, and encapsulated minerals in vegetable meal based diets for Sparus aurata (Linnaeus, 1758)

Submitted 5 December 2016 Accepted 28 July 2017 Published 27 October 2017 Corresponding author David Domínguez, [email protected] Academic editor Sadasivam Kaushik Additional Information and Declarations can be found on page 13 DOI 10.7717/peerj.3710 Copyright 2017 Domínguez et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Inorganic, organic, and encapsulated minerals in vegetable meal based diets for Sparus aurata (Linnaeus, 1758) David Domínguez1, Simona Rimoldi2, Lidia E. Robaina1, Silvia Torrecillas1, Genciana Terova2,3, María J. Zamorano1, Vasileios Karalazos4, Kristin Hamre5 and Marisol Izquierdo1 1Grupo de Investigación en Acuicultura (GIA), University Institute Ecoaqua, University of Las Palmas de Gran Canaria, Telde, Las Palmas, Canary Islands, Spain 2Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy 3Inter-University Centre for Research in Protein Biotechnologies, ‘‘The Protein Factory’’, Polytechnic University of Milan and University of Insubria, Varese, Italy 4BioMar Hellenic SA, Volos, Greece 5National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway ABSTRACT Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FMbased diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1),suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply. Subjects Aquaculture, Fisheries and Fish Science, Nutrition Keywords Inorganic, Organic, Encapsulated, Vegetable meals, Manganese, Selenium, Zinc, Sparus aurata, Vertebral morphology How to cite this article Domínguez et al. (2017), Inorganic, organic, and encapsulated minerals in vegetable meal based diets for Sparus aurata (Linnaeus, 1758). PeerJ 5:e3710; DOI 10.7717/peerj.3710 INTRODUCTION Gilthead sea bream (Sparus aurata) is one of the main marine finfish produced in the European aquaculture (APROMAR, 2015). Commercial feeds for gilthead sea bream have been traditionally based in fishmeal (FM) and fish oil (FO). These ingredients are limited resources with a tendency to decrease their production (Tacon & Metian, 2008;Tacon & Metian, 2009). Vegetable meals (VM) and oils (VO) can partially replace FM and FO in gilthead sea bream diets (Robaina et al., 1995;Montero et al., 2003;Gómez-Requeni et al., 2004;Izquierdo et al., 2005;Benedito-Palos et al., 2007). However, this replacement may decrease antioxidant status (Saera-Vila et al., 2009). These issues may arise even when diets are formulated to satisfy the recommended nutrient requirements for essential fatty acids or amino acids (NRC, 2011). This suggests possible nutritional imbalances, which can be partly related to differences in the content of zinc (Zn), manganese (Mn), and selenium (Se) between FM and VM (NRC, 2011;Hansen & Hemre, 2013), and can affect bone morphology and antioxidant status. Zn is involved in bone formation and mineralization by activating osteoblastic cells and inhibiting osteoclastic bone resorption (Yamaguchi, 1998). Zn also forms part of several metalloenzymes that are involved in antioxidant defence such as Cu/Zn-SOD. In Jian carp the activity of Cu/Zn-SOD was increased with increasing dietary Zn levels (Feng et al., 2011). Low dietary Zn may cause slower growth rates in several fish species including Nile tilapia (Do Carmo e Sá et al., 2004), hybrid striped bass (Buentello, Goff & Gatlin, 2009), Jian carp (Tan et al., 2011), grass carp (Liang et al., 2012), and Malabar grouper (Houng-Yung et al., 2014). In rainbow trout low Zn content in the feed may also cause cataracts, skin and fin erosion (Ogino & Yang, 1978), and dwarfism (Satoh et al., 1983). Studies focused on Zn requirement have been carried out on gilthead sea bream, using FM based diets (Serra et al., 1996). FMs contain high concentrations of Zn, whereas VMs are generally low in this mineral (NRC, 2011). For this reason, early studies in gilthead sea bream investigated the effects of increased Zn in diets in which FM was substituted by VM (Robaina et al., 1998). Mn is a cofactor for metal-enzyme complexes, essential for the antioxidant defence through Mn-SOD. Gibel carp and yellow catfish fed a Mn-deficient diet showed reduced growth (Pan et al., 2008;Tan et al., 2012), whereas cataracts and dwarfism were described in rainbow trout and common and Gibel carps (Ogino & Yang, 1980;Satoh et al., 1983; Yamamoto et al., 1983;Satoh et al., 1992;Pan et al., 2008). Similarly, Se plays an important role in reducing oxidative stress by being part of the selenoproteins such as glutathione peroxidase 1 (gpx-1), whose hepatic activity represent a robust and sensitive criterion to define Se deficiency (Pacitti et al., 2013;Fontagné-Dicharry et al., 2015). Se supplementation in diets for sea bass markedly reduced the occurrence of muscular dystrophy and oxidative risk, enhanced fish growth (Betancor et al., 2012) and adequate skeletal development (Saleh et al., 2014). Bone morphogenetic proteins (bmp) and osteocalcin (oc) are considered important indicators of bone development and mineralization and are positively related to Se inclusion (Saleh et al., 2014). Bmp are involved in a series of cascades that lead to osteoblast differentiation and osteogenesis Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 2/21 in fish (Smith et al., 2006). Oc, instead, is an osteoblast-specific gene encoding a secreted protein which represents the most abundant non-collagenous protein of bone matrix (Sommer et al., 1996). This gene is generally inactivated during osteoblast proliferation, while it is abundantly transcribed during osteoblast differentiation. Osteocalcin is released by osteoblasts during bone formation and binds to the mineralized bone matrix (Hauschka & Wians Jr, 1989). Inorganic minerals may be more effectively absorbed if they are present in their chelated organic forms (Apines et al., 2003;Apines et al., 2004). Some authors have described an increase in Se absorption in fish fed an organic source of Se (Paripatananont & Lovell, 1997). Similarly, several studies have shown that Zn and Mn are absorbed better when fish receive them in inorganic forms than organic or chelated to amino acids (Watanabe, Kiron & Satoh, 1997;Do Carmo e Sá et al., 2005). Other studies have reported higher bioavailability of these minerals when organic compounds are used instead of inorganic ones (Paripatananont & Lovell, 1995;Satoh et al., 2001;Apines et al., 2004;Fountoulaki et al., 2010). Minerals can also be delivered through encapsulation, thus reducing the interactions occurring with other minerals when they are supplied in excess. However, there is a lack of consistency in the repeatability of these results and important variations are found among different authors’ data (Antony Jesu Prabhu, Schrama & Kaushik, 2014). Production of gilthead sea bream is constrained by the high incidence of skeletal anomalies, commonly reaching up to 100% (as cited in Boglione et al., 2001). Radiological studies serve as a useful method for classifying vertebral anomalies (Boglione et al., 2001; Witten et al., 2009). However, the relationship between vertebral mineral content and bone morphology has not been extensively studied with respect to experimental diets (Roy & Lall, 2003;Poirier Stewart et al., 2014). Many radiological studies are based upon observations, and very few studies have actually used vertebral measurements to accurately describe vertebral morphology and anomalies (Fjelldal, Nordgarden & Hansen, 2007;Poirier Stewart et al., 2014). Accordingly, the objective of the present research was to further study the effects of Mn, Zn, and Se on bone development and oxidative stress markers by supplementing such minerals in different delivery forms in the gilthead sea bream diets for with low levels of FM and FO. MATERIAL AND METHODS The animal experiments described comply with the guidelines of the European Union Council (2010/63/EU) for the use of experimental animals and have been approved by the Bioethical Committee of the University of Las Palmas de Gran Canaria (REF: 007/2012 CEBA ULPGC). Diets In order to determine the effect of supplementing low FM diets with zinc (Zn) manganese (Mn) and selenium (Se) by using different mineral delivery forms, five diets (manufactured by BioMar Tech-Centre, Brande, Denmark) were formulated (Table 1). Specifically, a low-FM, plant based diet (15% FM) was formulated without any supplementation of Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 3/21 Table 1 Ingredients and analyzed proximate composition of the experimental diets supplemented with different sources of target minerals (Zn, Mn and Se). Ingredients (%) CDE DO DI FM Fish meala15 15 15 15 63 Corn gluten 22 22 22 22 Soya cakeb20 20 20 20 Soya protein concentrate 10 10 10 10 Wheat gluten 3.8 3.8 3.8 3.8 Wheat 11.73 11.43 11.33 11.33 20.52 Fish oilc7.5 7.5 7.5 7.5 8 Rapeseed oild7.5 7.5 7.5 7.5 8 Microingredientse2.02 2.02 2.02 2.02 0.3 Premix vitamins and mineralsf0.45 0.45 0.45 0.45 0.45 Inorganic target mineralsg0.4 Chelated (organic) target mineralsh0.4 Encapsulated (chitosan) target mineralsi0.3 Proximate composition (%) Moisture 8.1 8.2 8.1 8.1 7.0 Crude protein 44.2 43.7 43.5 42.5 45.0 Crude lipid 19.6 20.3 19.6 20.8 23.6 Ash 5.7 5.6 5.4 5.8 10.4 Mineral Composition Zn (mg kg−1) 39 120 150 140 53 Mn (mg kg−1) 22 62 52 52 13 Se (mg kg−1) 0.55 0.89 1.20 0.90 1.70 Ca (%) 0.95 0.98 0.86 1.00 2.20 P (%) 1.00 1.10 0.98 0.99 1.60 Ca:P 0.95 0.89 0.88 1.01 1.38 Notes. aSouth-American, Superprime. b48 Hi Pro Solvent Extr. cSTD 18. dEuropean, non-GM, double-low quality rapeseed oil. eContains monocalcium phosphate, lysine, methionine and yttrium. fContains vitamins and minerals to satisfy known nutritional requirements excluding the target minerals (Zn, Mn and Se) (DSM Nutritional Products, Basel, Switzerland). gZinc oxide, manganese oxide and sodium selenite (DSM Nutritional Products, Basel, Switzerland). hSe Methionine, Zn, and Mn chelated to amino acids (DSM Nutritional Products, Basel, Switzerland). iEncapsulated (chitosan) zinc oxide, manganese oxide and sodium selenite. Zn, Mn and Se (negative control, C-). This basal diet, was then supplemented with the aforementioned target minerals in inorganic (DI, zinc oxide as ZnO (Zn 72%), manganese oxide as MnO2(Mn 60%), and sodium selenite), organic (DO, Se-methionine, Mnand Zn-amino acids chelated) or inorganic encapsulated (DE). The encapsulated minerals were prepared according to Berthold, Cremer & Kreuter (1996) by SPAROS (Faro, Portugal) by precipitation-coacervation, in which chitosan was solved in a 2% (v/v) acetic acid solution and encapsulated particles were prepared by dropping the target mineral solution containing sodium selenite, manganese oxide and zinc oxide (Table 1). For comparison, Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 4/21 a FMand FO-based diet (FM) without supplementation of the target minerals was also included. For each of the supplemented experimental diets, a premix consisting of the target minerals in the different delivery forms (inorganic, organic and encapsulated) was prepared and added to the basal diet in order to ensure efficient mixing with the rest of the feed ingredients. Mineral composition of each diet was determined (Table 1). All diets were isoenergetic and isonitrogenous and were formulated to meet all known nutritional requirements for rainbow trout (NRC, 2011) including vitamins and minerals apart from the target ones (Table 1). Fish and experimental conditions For the study, 1,725 gilthead sea bream (Sparus aurata) juveniles, obtained by natural spawning from our own broodstock (University of Las Palmas de Gran Canaria Las Palmas, Spain), were randomly distributed into fifteen 500-L circular fiberglass tanks at a density of 115 fish/ tank. Initial mean body weight and total length (TL) were 22.5 ±1.5 g and 117 ±4 mm (mean ±SD), respectively. Tanks in a flow-through system were supplied with filtered seawater at 22.8–24.3 ◦C and kept under a natural photoperiod (July-October) of approximately 12 h of light. Water-dissolved oxygen ranged between 6.5 and 6.9 ppm. Each diet was fed to triplicate groups until apparent satiation three times per day for 12 weeks. To monitor growth, individual fish were anesthetized with clove oil (Guinama S.L.U., Valencia, Spain) and weighed after 47 and 84 days of feeding. At the end of the study, five fish per tank were collected for whole body chemical composition; 20 for radiographic study; eight fish per tank for vertebral axis weight, six fish per tank for vertebrae and liver gene expression studies, and three fish per tank for chemical and mineral composition of the vertebrae. Vertebrae gross chemical composition was determined from haemal vertebrae. Total vertebral weight was compared to total fish weight (vertebral weight/fish weight) to avoid the effect of weight differences. Biochemical analysis Chemical composition of diets, and vertebrae was determined by following standard procedures (Association of Official Analytical Chemists (AOAC, 2000). Crude lipid was extracted according to the method of Folch, Lees & Stanley (1957) and ash by combustion in a muffle furnace at 600 ◦C for 12 h and at 550 ◦C for vertebrae; protein content (N ×6.25) was determined by using the Kjeldahl method (AOAC, 2000) and dry matter content was determined after drying the sample in an oven at 105 ◦C until reaching constant weight. Chemical composition of fish was determined using near-infrared spectroscopy (FoodScan, Foss, Sweden). The evaluation of the mineral content was conducted by means of an inductively coupled plasma mass spectrometry (iCAPQ ICP-MS) at a private, certified laboratory (LDG, Barcelona, Spain), after submitting the sample to acid digestion. Vertebral morphometry Radiographs were taken using a fixed X-ray apparatus (Bennett B-OTC, Bennett X-ray Corp., Chicago, IL, USA) and a 35 ×43 cm digital film (Fujifilm FDR D-EVO (Fujifilm Corporation, Tokyo, Japan). Fish were radiographed in groups of ten. Radiographs were treated digitally (Onis 2.4, DigitalCore, Co.Ltd, Tokyo, Japan) and height, length Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 5/21 and intervertebral spaces of eight different vertebrae were measured (vertebrae 3–6 and 13–16) (Fjelldal et al., 2006). A series of measurements were used to describe vertebral morphometry, including vertebral height, vertebral length and vertebral surface area. Additionally, vertebral weight and length (mm) were studied in relation to total length (mm) of fish (vertebral weight/fish total length and vertebral length/fish total length) and the vertebral length and height relationship was calculated (vertebral length/vertebral height) to study vertebral shape. All these parameters were measured individually for each of the 20 fish per tank studied and served to define morphometric differences among fish fed the different diets. Gene expression RNA extraction Six fish were sampled from each tank and divided into two pools of three fish. Total RNA was extracted from 60 mg of liver and 150 mg of posterior vertebrae using TRI Reagent R Solution (Life Technologies, Carlsbad, CA, USA) and purified on RNeasy Mini Spin Columns (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Reverse transcription Reverse transcription of 1 µg total RNA from each experimental sample was performed with the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions with slight modifications. Briefly, 1 µg total RNA and nuclease-free water to a final volume of 15 µl were heated at 65 ◦C for 10 min and cooled in ice. Afterwards 1 µl of iScript reverse transcriptase and 4 µl of 5 ×iScript reaction mix were added, reaching a final reaction volume of 20 µl. The complete reaction mix was incubated for 5 min at 25 ◦C, 30 min at 42 ◦C, and then 5 min at 85 ◦C to inactivate reverse transcriptase. For gene quantification, the reverse transcription reactions were diluted 1:10. Quantitative PCR The nucleotide sequences of primers used in this study are reported in Table 2. A total of 2 µl of diluted cDNA was used in real-time PCR for gene expression quantification using IQTM SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA).Duplicate analyses were performed for each sample for both the housekeeping and the target gene in a final reaction volume of 20 µl. β-actin and ribosomal protein 27a (rpl-27a) were used as housekeeping genes to normalize the expression of oxidative stress genes (gpx-1,sod) in liver and of osteogenesis genes (oc,bmp2) in posterior vertebrae, respectively. Real-time quantitative PCR was performed using the iQ5 Multicolor Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The PCR conditions were as follows: 95 ◦C for 3 min and 30 sec, followed by 40 cycles of 95 ◦C for 15 sec, 58.1 ◦C for 30 sec, and 72 ◦C for 30 sec; 95 ◦C for 1 min, and a final denaturation step from 58 to 95 ◦C for 10 sec. The 2−11Ct method was applied to analyse the relative changes in gene expression. Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 6/21 Table 2 Sequences of primers used for gene expression analysis. Gene Symbol Nucleotide sequence F: 50-TCTGTCTGGATCGGAGGCTC-30 Alpha-actin α-act R: 50-AAGCATTTGCGGTGGACG-30 F: 50-ACAACTCACTGCCCCACCAT-30 Ribosomal protein 27a rpl-27a R: 50-CTTGCCTTTGCCCAGAACTT-30 F: 50-TTGGAGACCTGGGCAACGTGA-30 Cu/Zn superoxide dismutase sod R: 50-TCCTGCTTGCCTCCTTTTCCC-30 F: 50-GCTTTGAGCCAAAGATCCAG-30 Glutathione peroxidase 1 gpx-1 R:50-CTGACGGGACTCCAAATGAT-30 F: 50-GTGGCTTCCATCGTATCAACATTTT-30 Bone morphogenetic protein 2 bmp2 R: 50-GCTCCCCGCCATGAGT-30 F: 50-AGCCCAAAGCAGGTAAGCAAG-30 Osteocalcin oc R: 50-TTTCATCACGCTACTCTACGG-30 Table 3 Body weight (g) of gilthead sea bream fed diets with different mineral sources at 0, 47, and 84 days. Body weight (g) CDE DO DI FM 0 days 22.5 ±0.8 22.6 ±0.8 22.4 ±0.8 22.3 ±0.8 22.5 ±0.8 47 days 50.0 ±3.6a50.3 ±3.6a49.6 ±3.6a53.0 ±3.5b55.9 ±3.6c 87 days 81.4 ±6.1a79.1 ±6.1a80.8 ±6.0a84.2 ±6.1b90.0 ±6.1c Notes. *Different letters in a row denote significant differences between groups fed different diets for a given feeding period (mean ±SD, n=3, P<0.05). Statistics All data were statistically analysed using STATGRAPHICS Centurion XVI (Version 16.2.04), STATGRAPHICS plus 5.1 (Statpoint Technologies, Warrenton, VA, USA), or SPSS v21 (IBM Corp., Chicago, IL, USA) and means ±SD were calculated for every parameter measured. Data were tested for normality with the one-sample Kolmogorov– Smirnov test. For normally distributed data, one-way analysis of variance (ANOVA) was used to determine the effects of the different diets. Data were tested for homogeneity and post-hoc analysis was carried out using Tukey test if variances were the same or Games-Howell test whenever variances were different. Significant differences were considered for P<0.05. When data did not follow a normal distribution, logarithmic or arcsin transformation was carried out or non-parametric tests, such as Kruskal-Wallis, were used. RESULTS Growth Fish readily accepted experimental diets and no significant differences were found in feed intake between fish fed the different diets. From 47 days of feeding until the end of the trial, body weight was significantly lower in fish fed the VM-based diets, containing only 15% FM and 7.5% FO, than in fish fed the FM diet (Table 3). Whereas supplementation Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 7/21 Table 4 Vertebral weight/fish total length, vertebral length/fish total length, and vertebral length/vertebral height of gilthead sea bream fed diets with different mineral sources for 12 weeks*. Vertebral Morphometry CDE DO DI FM Vertebral weight/fish total length (mg mm−1) 8.51 ±2.10ab 8.48 ±2.29ab 9.15 ±1.76ab 9.55 ±2.03b8.24 ±1.56a Vertebral length/fish total length (mm mm−1)−22.47 ±0.11ab 2.51 ±0.07b2.49 ±0.07ab 2.47 ±0.07a2.44 ±0.09ab Vertebral length/vertebral height (mm mm−1) 1.33 ±0.07a1.39 ±0.06c1.38 ±0.07c1.36 ±0.07b1.33 ±0.08a Notes. *Different letters in a row indicate significant differences in vertebral weight (mean ±SD, n=3, P<0.05) between experimental groups for the same time period. Table 5 Initial and final whole body composition (% dry weight) of gilthead sea bream juveniles fed diets with different mineral sources for 12 weeks*. Initial CDE DO DI FM Lipid 29.0 ±0.3 33.3 ±2.1 34.1 ±1.9 33.1 ±0.3 34.6 ±0.6 33.8 ±1.2 Ash 11.4 ±0.7 10.2 ±0.6 9.5 ±0.1 9.7 ±0.1 10.2 ±0.4 8.9 ±0.9 Protein 36.6 ±0.6 36.6 ±2.0 34.3 ±2.0 35.1 ±1.5 33.5 ±0.4 35.4 ±1.6 Moisture 67.7 ±0.9 66.6 ±0.4 66.2 ±0.4 66.2 ±0.6 66.4 ±0.6 65.6 ±0.3 Notes. *mean ±SD, n=3. Table 6 Initial and final composition (% dry weight) of posterior vertebrae of gilthead sea bream juveniles fed diets with different mineral sources for 12 weeks*. Initial CDE DO DI FM Lipid 28.4 ±0.9 31.8 ±1.4 32.1 ±3.4 32.5 ±2.0 33.3 ±0.9 32.0 ±1.7 Ash 33.0 ±1.8 32.3 ±1.8a32.3 ±1.5a34.2 ±1.1b34.9 ±0.9b33.5 ±1.5ab Protein 33.5 ±1.8 28.4 ±0.7b26.9 ±1.4a29.6 ±0.9b29.8 ±1.3b26.5 ±0.9a Moisture 47.8 ±1.3 43.6 ±2.2 44.2 ±1.2 40.3 ±7.3 44.3 ±0.3 43.3 ±1.0 Notes. *Different letters in a row indicate significant differences (mean ±SD, n=3, P<0.05) between experimental groups for the same time period. with Zn, Mn and Se in organic (DO) or encapsulated forms (DE) did not affect sea bream growth, addition of the same minerals in inorganic form (DI) significantly improved final body weight (Table 3). Moreover, in fish fed DI diet, the ratio between vertebral weigh and total length ratio resulted significantly higher than in fish fed FM diet (Table 4). Biochemical analysis Fish belonging to different feeding groups did not differ in their whole body composition (Table 5). However, DI and DO diets significantly increased the ash content of posterior vertebrae in comparison to fish fed diet C- (Table 6). Feeding with diet DE did not increase ash content, but reduced protein content in the vertebrae compared to C-, DO and DI fed fish (Table 6). Zn content in vertebra was increased by dietary supplementation of minerals regardless of mineral delivery form (Table 7). Mn and Se content in the vertebrae did not reflect the amount of this mineral in the diet, which was lower in Cand FM diets, since no significant differences were found between the dietary fish groups (Table 7). Finally, among all fish groups, only sea bream fed the DI diet had a Ca and P vertebrae content significantly lower Domínguez et al. (2017), PeerJ, DOI 10.7717/peerj.3710 8/21 Table 7 Zn, Mn, Se, Ca, and P content in the posterior vertebrae of gilthead sea bream juveniles fed different mineral sources for 12 weeks*. Zn (mg kg−1) Mn (mg kg−1) Se (mg kg−1) Ca (%) P (%) Ca:P C31.4 ±1.0a10.9 ±0.2 0.15 ±0.01 6.6 ±0.5ab 3.2 ±0.2ab 2.07 DE 34.7 ±0.2b11.0 ±0.3 0.15 ±0.02 6.0 ±0.5ab 2.9 ±0.3ab 2.06 DO 34.9 ±0.5b11.1 ±0.5 0.17 ±0.03 6.3 ±0.6ab 3.0 ±0.3ab 2.07 DI 35.2 ±1.2b11.2 ±0.2 0.18 ±0.03 5.4 ±0.9a2.7 ±0.4a2.03 FM 33.4 ±1.4ab 10.7 ±0.7 0.17 ±0.03 7.0 ±0.2b3.4 ±0.2b2.04 Notes. *Different letters in a row indicate significant differences (mean ±SD, n=3, P<0.05) between experimental groups for the same time period. Table 8 Expression level of osteocalcin (oc) and bone morphogenetic protein 2 gene (bmp2) in vertebrae of gilthead sea bream juveniles fed diets with different mineral sources for 12 weeks*. Vertebral ossification related genes CDE DO DI FM oc 1.00 ±0.07a1.94 ±0.95ab 2.04 ±0.47ab 2.25 ±0.61ab 1.59 ±0.11b bmp2 1.10 ±0.58a3.31 ±1.93ab 7.75 ±1.00b2.56 ±0.49ab 4.07 ±1.38ab Notes. *Different letters in a row indicate significant differences in gene expression (mean ±SD, n=6, P<0.05) between experimental groups for the same time period. than fish receiving FM diet, although (Table 7). The dietary Ca and P levels did not differ significantly between the VM based diets (Table 1). Vertebral morphometry From our previous studies on vertebral morphology, the posterior vertebra V13 resulted to be more affected by the diet than anterior vertebrae (Supplemental File), and therefore it was used, in this study, to calculate vertebral morphometric parameters. Among the different parameters measured, the ratios between the vertebral length and fish total length and between the vertebral length and vertebral height were significantly affected by dietary treatments (Table 4). In general, inclusion of minerals to a VM-diet did not increase the vertebral length/fish total length ratio in comparison to fish fed diets Cand FM (Table 4). This value was significantly higher in fish fed DE diet compared to those receiving diet DI. Conversely, the shape of the vertebrae was significantly affected by mineral dietary inclusion. Specifically, compared to Cand FM groups, the vertebral length/vertebral height ratio was significantly increased by including encapsulated (DE) and organic (DO) target minerals, and to a lesser extent by inorganic minerals (DI) (Table 4). Only fish fed DI diet showed, instead, a vertebral weight/fish total length value higher than FM group. Gene expression Fish fed diet Cshowed the lowest oc gene expression in vertebrae, being significantly lower than in fish fed FM diet (Table 8). 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