4.1 Ethical statement
Animal handling and subsequent procedures complied with European laws (2010/63/EU) and Portuguese legislation for the use of laboratory animals (DL nº113/2013, 7 August). This study was approved by the ORBEA Animal Welfare Committee of CCMAR. This experiment was performed by trained scientists and followed the European Directive 2010/63/EU of European Parliament and of the Council of European Union on the protection of animals used for scientific purposes under authorization reference number 0421/000/000/2016. CCMAR facilities and their staff are certified to house and conduct experiments with live animals (‘group-1’ license by the ‘Direção Geral de Veterinaria’, Ministry of Agriculture, Rural Development and Fisheries of Portugal).
4.2 Experimental diets
Four diets were tested in this study, including a commercial diet (WINFlat, SPAROS Lda., Portugal) that was used as the control (CTRL diet). This diet contains ingredients such as krill meal, squid meal, wheat gluten, fish meal, shrimp meal, fish hydrolysate, pea protein concentrate, fish gelatine, fish oil, lecithin and a micronutrient premix comprising vitamins, minerals, and other additives. Moreover, three experimental diets were prepared by supplementing the CTRL diet with an extract of either curcumin (CC diet) at 46 g/kg of the micronutrient premix, green tea (GT diet) at 12g/kg of the micronutrient premix or grape seed (GS diet) at 12 g/kg of the micronutrient premix. These selected doses of each antioxidant extract are under a patent pending application (PCT/IB2020/056001) and were chosen based on preliminary trials conducted in the Centre of Marine Science of Algarve (CCMAR, unpublished data). All diets were prepared by SPAROS Lda. (Olhão, Portugal). Feed samples were freeze-dried, ground and analysed for dry matter (105 °C for 24 h), crude protein by automatic flash combustion (Leco FP-528, Leco, St. Joseph, USA; N× 6.25), lipid content by petroleum ether extraction using a Soxtherm Multistat/SX PC (Gerhardt, Königswinter, Germany; 150 °C), and gross energy in an adiabatic bomb calorimeter (Werke C2000; IKA, Staufen, Germany)21. Diets proximal composition was identical for all 4 diets (Table 1), and they only changed in the supplementation with extracts, and this supplementation did not exceed 1% of the diets.
4.3 Senegalese sole husbandry and experimental set-up
Senegalese sole postlarvae were reared for 25 days, starting at 45 days after hatching (DAH), in a recirculation aquaculture system at CCMAR (Portugal), under optimized environmental and zootechnical conditions. Sole postlarvae were kept in flat-bottom tanks (21 L), each tank stocking 630 individuals (corresponding to a 3,000 ind m-2). The dietary treatments (CTRL, CC, GT and GS) were randomly assigned to replicate tanks (n=3 tanks per treatment). Abiotic parameters were measured, and mortality was recorded daily. Dead fish were removed, and the rearing units were carefully cleaned with minimal disturbance. Dissolved oxygen in water was maintained at 96.6 ± 7.2% of saturation, temperature at 19.6 ± 0.5 °C and salinity at 35.4 ± 0.7 g.L-1. A 10:14 h light:dark photoperiod was maintained, and the light intensity was 400 lx at the water surface. Inert diet was delivered semi-continuously with automatic feeders. The amount of feed distributed to each tank was based on predicted maximum growth and daily adjustments were done based on visual inspection to avoid a large excess of uneaten food50.
4.4. Thermal stress test
A thermal stress test was conducted at the end of the experimental period (70 DAH) to assess how the dietary treatments modify the animal physiological responses. Two challenging periods were analysed in fish response: an acute stress (at 72 DAH) and a chronic stress (at 78 DAH). The seawater temperature of the rearing system was raised from 19.6 ºC until 24.0 ± 0.5 ºC (~5 ºC over the experimental temperature) during a 24h period. For the acute stress challenge fish were sampled after remaining at this temperature during 24h (72 DAH), whist for the chronic stress test fish were maintained at this temperature for one week (78 DAH). At the end of each thermal stress fish from the different dietary treatments were sample for biomarkers response assessment.
4.5 Key performance indicators
At the beginning (45 DAH, n = 60) and at the end of the experiment (70 DAH, n = 120) fish were killed by using an overdose of anaesthetic 2- phenoxyethanol (1000 ppm; Prolabo) then individually collected for dry weight (DW, mg) and body standard length (SL, mm) determination. These juveniles were frozen at -80ºC, photographed for measuring SL using Axio Vision L.E. 4.8.2.0 (Carl Zeiss Micro Imaging GmbH) and freeze-dried for DW determination (Denver Instrument, 0.001 mg precision). Survival rate (%) was calculated as the percentage of fish counted at the end of the trial relative to their initial number in each replicate. Growth, expressed as relative growth rate (RGR, %/day), was calculated, at the end of the experiment, using the formula: (eg−1)×100, with g=[(ln final weight−ln initial weight)/time]51. The condition factor (K) was calculated as (fish wet weight/total length3) x 100.
4.6 Preparation of diet extracts for antioxidant capacity assessment
Methanol extracts were prepared from the four diets CTRL, CC, GT and GS. For that purpose, the diets were freeze-dried, mixed with methanol (1:40, w/v) and maintained in an ultrasonic bath for 30 min. Then, samples were extracted overnight, with stirring, at room temperature (RT, approximately 20 ºC)52. The extracts were then filtered (Whatman no 4) to remove solid debris, and methanol was removed by using a rotary evaporator (60 ºC; 337 mbar). The obtained dried extracts were weighed, dissolved in methanol at 50 mg/mL and stored at -20 °C.
4.6.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azino-bis-(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)
Methanol extracts from diet samples were tested for radical scavenging activity against the DPPH and ABTS radicals at concentrations of 50 mg/mL, as described previously52. Ascorbic acid was used as a positive control at the same concentrations of the samples. Results were expressed as a percentage of inhibition, relative to a control containing methanol in place of the sample.
4.6.2. Total phenolic (TPC) and Flavonoids (TFC) content
The TPC and TFC were determined in the methanol extracts at the concentration of 50 mg/mL and absorbance was measured in a microplate reader (Biotek Synergy 4). The TPC was assessed by the Folin-Ciocalteu assay and TFC was estimated by the aluminium chloride colorimetric method adapted to 96-well microplates. Results were expressed respectively as gallic acid equivalents (GAE) and quercetin equivalents (QE) in milligrams per gram of diet (dry weight, DW). All methods were performed as previous described52.
4.7 Preparation of fish sample for biomarkers response assessment
For biomarkers analysis, 3 pools (n=3 juveniles/pool) per replicate (n=9 per dietary treatment) were sampled at the end of growth trial (70 DAH) and thermal stress (72 and 78 DAH). The region from the operculum cavity until the end of the visceral cavity was selected in each fish. Samples were homogenised through sonication (Brason Sonifier 250) on ice using 1500µl of ultra-pure water. From each sample, 2 aliquots of the supernatant were taken. One aliquot of 200µl containing 4 µl of 4 % butylated hydroxytoluene (BHT) in methanol was used for the determination of endogenous lipid peroxidation (LPO). The other aliquot of 500 µl was diluted (1:1) 0.2 M K-phosphate buffer, pH 7.4, and centrifuged for 10 min at 10,000 g (4 ºC). The post-mitochondrial supernatant (PMS) was divided into microtubes and kept in –80 ºC until further analyses. All biomarkers were determined spectrophotometrically, in micro-assays set up in 96 well flat bottom plates, with the Microplate reader MultiSkan Spectrum (Thermo Fisher Scientific).
4.7.1 Antioxidant biomarkers assessment
Protein concentration of PMS was determined according to the Bradford method53, using bovine γ-globulin as a standard. Catalase (CAT) activity was determined in PMS by measuring the decomposition of the substrate H2O2 at 240 nm54. Glutathione-S-transferase (GST) activity was determined following the conjugation of GSH with 1-chloro-2,4- dinitrobenzene (CDNB) at 340 nm55. Total glutathione (GSH) content was determined at 412 nm using a recycling reaction of reduced glutathione (GSH) with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence of glutathione reductase (GR) excess56,57. GSH content was calculated as the rate of TNB2- formation with an extinction coefficient of DTNB chromophore formed, ε = 14.1 x 103M-1cm-157,58. LPO was determined by measuring thiobarbituric acid-reactive substances (TBARS) at 535 nm59. Protein carbonylation was measured by the quantification of carbonyl groups based on the reaction of 2,4-dinitrophenylhydrazine (DNPH) with carbonyl groups, according to the DNPH alkaline method60. The amount of carbonyl groups was quantified spectrophotometrically at 450 nm (22,308 mM−1cm−1extinction coefficient).
4.7.2 Total Antioxidant Capacity (TAC)
The 2,2V-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS*+) is decolorised by antioxidants according to their concentrations and antioxidant capacities. This change in colour was measured as a change in absorbance at 660 nm and the assay was calibrated with Trolox61.
4.7.3 Heat shock proteins (HSP70) response
HSP70/HSC70 content was assessed by ELISA, adapted from Rosa62. Samples were added to coated 96 well microplates and allowed to incubate overnight at 4 °C. Microplates were then washed (3×) with 0.05% PBS-Tween-20, the blocking solution (1% BSA, Sigma-Aldrich) was added and left to incubate at RT for 2 h. Microplates were washed and 5 μg ml−1 primary antibody (1º Anti-HSP70 mouse mAB (C92F3A-5) Millipore), detecting 72 and 73 kDa proteins corresponding to the molecular mass of inducible hsp and hsc70, was added to each well and then incubated overnight at 4 °C. The non-linked antibodies were removed by washing the microplates again, which were then incubate for 2 h at RT with 1 μg ml−1 of the secondary antibody, anti-mouse IgC (2º Anti-mouse IgC (fab specific) Sigma). After another wash, the substrate p-nitrophenyl phosphate was added and incubated for 30 min at RT. Then, the stop solution (3 mol l−1 NaOH) was added to each well and the absorbance was read at 405 nm, using as a standard the purified HSP70 active protein (HSP70 protein Millipore).
4.8 Reverse transcription–quantitative real-time PCR (qPCR)
Gene expression analysis was performed in 70, 72 and 78 DAH soles from each dietary treatment. Fish were kept at -80ºC until analysis. A region from the operculum cavity until the end of the visceral cavity was select in each larva (n=4 per dietary treatment). This process was realised in each larva without thawing. Selected genes for oxidative stress defences and cellular stress proteins are described in detail in Table 2.
Samples were homogenised using a Fast-prep FG120 instrument (Bio101) and Lysing Matrix D (Q- Bio- Gene) with 1 ml Tri Reagent (Sigma-Aldrich) for 60s at speed setting 6. Chloroform (0.2 ml) was added to each sample before centrifuging at 14.000 rpm for 15 min. The supernatant content was transferred to columns of the Isolate II RNA Mini Kit (Bioline) and total RNA was treated twice for 30 min with DNase I following the manufacturer’s protocols. Total RNA quality was checked by agarose gel electrophoresis and a Nanodrop ND-8000 (Thermo Scientific) was used to determine its concentration. One μg of total RNA was reverse-transcribed using the iScriptTM cDNA Synthesis kit (Bio-Rad) according to the manufacturer's protocol.
The qPCR assays were performed in duplicate in a 10 μL volume containing cDNA generated from 10 ng of the original RNA template, 300 nM of each specific forward and reverse primers, and 10 µl of iQ™ SYBR® Green Supermix (Bio-Rad). The genes analysed were involved in the oxidative stress defences: catalase (cat), glutathione peroxidase 1 (gpx1), glutathione peroxidase 3 (gpx3), superoxide dismutase [Cu-Zn] (sod3)) and cellular stress proteins (heat shock protein 70 (hsp70), heat shock protein 90 alpha (hsp90aa), heat shock protein 90 beta (hsp90ab). Primers for Senegalese sole hsp70, hsp90aa and hsp90ab were previously published63,64 and new species-specific primers for qPCR were designed for remaining genes (Table 2). The qPCR amplification protocol was as follows: 7 min for denaturation and enzyme activation at 95 ºC followed by 40 cycles of 30 s at 95 ºC and 1 min at 60 ºC. Expression data were normalised using the geometric mean of two reference genes, ubiquitin (ubi) and glyceraldehyde-3-phosphate dehydrogenase 2 (gadph2)65 and the relative mRNA expression calculated using the comparative Ct method66.
4.9 Data analyses
All data were tested for normality using a Kolmogorov-Smirnov (whenever n > 30) or Shapiro-Wilk (whenever n < 30) test and for homogeneity of variance using a Levene's test using STATISTICA v13 (StatSoft). Data were log transformed when required and percentages were arcsin transformed prior analysis. Comparisons between groups fed different diets were made using one-way ANOVA followed by a Tukey post-hoc test for growth performance and oxidative stress biomarkers at the end of the growth trail. To assess the response of oxidative stress biomarkers to thermal stress exposure by each treatment group a two-way ANOVA was made and the analysis of the delta variation between pre- and post-stress indicators was performed by a one-way ANOVA, both followed by a Tukey post-hoc test. Significance levels were set at P < 0.05.