Gilthead seabream fry (average body size 9.5 g) were obtained from a commercial hatchery (Piscimar SL, Andromeda Group, Spain) and transported by road to IRTA-Sant Carles de la Rapita research facilities (Sant Carles de la Ràpita, Spain), where they were acclimated in two 2000-L tanks for two weeks. After their acclimation, all fish were anesthetized (tricaine methanesulfonate [MS-222], 150 mg/L) and individually weighed for initial body weight (BWi)

and measured for standard length (SLi) to the nearest 0.1 g and 1 mm, respectively, and then distributed into eight 500-L cyclindroconical tanks at a density of 50 fish per tank (4 tanks/replicates per diet).

Fish (BWi = 10.6 ± 0.1 g, n = 400, mean ± standard deviation, SD) were fed for 95 days with both experimental diets by means of automatic feeders (ARVO-TEC T Drum 2000; Arvotec, Huutokosk, Finland) at the rate of 2.5% of the stocked biomass, which approached apparent satiation. Feed ration was evenly distributed in seven meals per day from 08:00 to 18:00 h. Fish were regularly sampled at a monthly basis in order to evaluate their growth in BW and adjust the feeding ratio to stocked biomass. During the trial, water temperature and pH (pH meter 507; Crison Instruments, Barcelona, Spain), salinity (MASTER-20T; ATAGO Co., Ltd., Tokyo, Japan), and dissolved oxygen (OXI330; Crison Instruments) were 22.1 ± 0.4°C, 7.0 ± 0.01, 36 mg/L, and 7.2 ± 0.3 mg/L (mean ± SD), respectively. Water flow rate in experimental tanks was maintained at approximately 9.0–10.1 liter/min via a recirculation system (IRTAmar®; Spain) that maintained adequate water quality (total ammonia and nitrite were ≤0.15 and 0.6 mg/L, respectively) through UV, biological, and mechanical filtration. Photoperiod followed natural changes according to the season of the year (November to February; 40°37′41″ N).

At the end of the trial, fish were anaesthetized as previously described; mucus was gently scrapped off from the skin surface (n = 15 per diet) using a sterile glass slide avoiding blood, urine and feces during collection and transferred into 2-ml Eppendorf tubes and stored at -80 °C as is described in Fernández-Alacid et al. (38). All fish in experimental tanks were measured for final BW and SL as indicated. Then, 10 fish per tank were sacrificed with an overdose of MS-222 (200 mg/L) for tissue sampling purposes. Skin samples (1 cm2 of anterior dorsal body region) were dissected and immediately transferred into RNAlater (Ambion®), fixed overnight and then frozen at -80°C until further RNA extraction. Fish growth and feed utilization from experimental groups was evaluated by means of the following indices: Fulton’s condition factor (K) = (BW/SL3) × 100; specific growth rate in BW (SGR, %) = ((ln BWf −ln BWi) × 100)/time (d) and feed conversion ratio (FCR, g/g) = FI/(Bf −Bi), where FI was the total feed intake during the experimental period (g) and, Bi and Bf were the initial and final biomass (g), respectively.

All animal experimental procedures were conducted in compliance with the experimental research protocol approved by the Committee of Ethics and Animal Experimentation of the Institut de Recerca i Tecnologia Agroalimentàries and in accordance with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals.

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