2.1. Experimental diets
Raw materials for preparation of the experimental fish diets were purchased from 21 Beyza Feed Mill, Shiraz, Iran. Three iso-caloric, iso-lipid and iso-nitrogenous diets were prepared using the same raw materials (Table 1) based on the NRC nutrient requirement for rainbow trout fingerling, including a control diet which contained only the base diet without any supplementation. The other two diets were supplemented with either selenium yeast or the chestnut polyphenol as below. Selenium yeast (SelPlex®, Alltech, KY) at 5 mg/kg and chestnut polyphenol extract (Silvateam s.p.a., Italy) at 2 g/kg were each added to the other ingredients for their respective experimental diet and then mixed and prepared separately. The exploratory testing of these two dietary supplements was based on previous research (Hoseinifar et al., 2020; Pacitti et al., 2015; Pérez-Valenzuela et al., 2021). For all three diets, pellets with a 2 - 2.5 mm diameter and 3 - 5 mm length were prepared, dried at 40°C for 24 hours and manually sieved to remove dust and stored at 4°C until use. Fish were fed three times a day (i.e., 08:00, 13:30 and 16:00 hours) at rate of 3% of the total fish biomass in each tank.
2.2. Fish rearing condition and sampling
A total of 756 rainbow trout fingerlings with a mean initial weight of 9.1 ± 0.4 g were provided by a local fish farm (Yasuj, Iran, natal temperature 13-16 ºC) and transported to the aquaculture facility at Shiraz University. The fish handlings were conducted with minimal suffering of experimental animals based on the regulations of the Institutional Animal Care and Ethics Committee of Shiraz University.
Fish were randomly allocated into 18 fiberglass tanks (ca. 900 l volume) supplied with thoroughly aerated well-water (14-16ºC). Fish were acclimated for 20 days to laboratory conditions and during this period, they were fed with the basal diet (21 Beyza Feed Mill, Shiraz, Iran).
This study was conducted in two phases, including an acclimation phase and a heat shocks phase:
A) Acclimation phase. At the end of the initial 20 days of acclimation to laboratory conditions, the water supplied into nine tanks (as Cold water category) set to 14°C and for the rest nine tanks (as Warm water category) was set to 20°C (in 48 hours, 2-3°C increase per day).
The tanks were monitored every two hours for temperature (14 ± 0.5°C and 20 ± 0.5°C) as well as daily for pH and dissolved oxygen.
Treatments in the Cold water category were as: (1) fish fed on basal diet with no supplement (Cold-B), (2) fish fed on a diet supplemented with selenium (Cold-Se) and (3) fish fed on a diet supplemented with polyphenol (Cold-P). Similarly, treatments in the Warm water category were as: (4) fish fed on basal diet with no supplement (Warm-B), (5) fish fed on a diet supplemented with selenium (Warm-Se) and (6) fish fed on a diet supplemented with polyphenol (Warm-P). Each treatment consisted of three replications each of 42 individuals.
B) Heat shocks phase. In this phase, after 60-day acclimation, fish in all treatments (in both cold and warm categories) were subjected to heat shocks up to 30°C. To this end, an aquarium heater (RS 408-E - 200 W, Risheng - China) was used to gradually increase the water temperature of all tanks up to 30°C at a rate of 2°C h-1.
The tanks were well aerated and the behaviour of the fish was closely monitored during this period and the timing of any mortalities recorded. Fish were not fed during heat shocks phase.
The survival rate (SUR) after heat shocks was calculated as below:
Survival Rate (%) = (No. of fish after each heat shock / No. of fish at the end of 60 days) × 100
At the end of 60-day acclimation phase and during the heat shock phase when the temperature of tanks reached 24, 28 and 30°C, three fish per tank were sampled (nine fish at each sampling point from each treatment). At each sampling point, live fish were immediately anesthetized with clove powder (150 mg/l) and blood samples were collected with non-heparinized 2.5 ml plastic syringes (23-gauge needles) via the caudal vein. Serum was obtained by centrifuging blood samples at 6000 g for 15 minutes (K241R, Centurion, UK) and kept at -80°C for further analysis.
At the same time, whole liver tissue samples were taken and snap-frozen in liquid nitrogen. Samples were stored at -80°C until RNA extraction.
In addition, specific growth rate (SGR) and feed conversion ratio (FCR) were calculated at the end of 60-day acclimation phase for the fish in each treatment as described in Islam et al. (2020a) and Vazirzadeh et al. (2020).
2.3. Biochemical parameters
Serum total protein and albumin were measured by using commercial kits (Pars Azmun, Iran, Cod 96001-95003) according to manufacturers’ manuals.
2.4. Serum cortisol
The serum cortisol level was measured using ELISA commercial kit (DiaMetra, Italy, DKO 001) containing antigenic cortisol conjugated with horseradish peroxidase. During the assay, a blue colour complex was created that its intensity was proportional to the amount of cortisol in the sample (Arakawa et al., 1979). Optical absorption of the samples was read using a 490 and 630 nm wavelength in a plate reader (BioTek, UK).
2. 5. Immunological parameters
Respiratory burst activity was measured using nitro blue tetrazolium (NBT)- a substrate internalized by macrophages from fresh blood. This assay was carried out as described in Vazirzadeh et al. (2020). Briefly, 50 µl of blood was mixed with equal volume of 0.2% NBT (Merck, Germany) solution and incubated for 30 minutes at 25°C, then 50 µl of NBT and blood mixture was mixed with 1 ml dimethyl formamide and centrifuged (K241R, Centurion, UK) at 6000 g for 15 minutes. The absorbance of the supernatant was read at 540 nm using a spectrophotometer (PG Instruments Ltd, UK).
Lysozyme activity was also measured as described in Vazirzadeh et al. (2020). Briefly, 10 µl of serum were added to 90 µl suspension of Micrococcus luteus cell wall (0.2 mg ml-1 in sodium phosphate buffer with pH 7.4) and the absorbance was measured at 450 nm after 0 and 10 min using a plate reader (BioTek, UK). The level of lysozyme activity (unit/min) was defined as the amount of enzyme that caused a decrease in absorbance of 0.001 per min.
2.6. Haematological parameters
The RBC of fresh blood was assayed by Natt and Herrick's staining method as detailed in Svobodová et al. (2012). Briefly, 10 µl of fresh blood was mixed with 1990 µl of Natt and Herrick solution (Bioanalytic GmBH, Germany) and then 10 µl of this mixture was used for cell counts using haemocytometer (Marien Feld, Germany) under a light microscope (Leica, Germany) with 40× magnification.
2.7. RNA extraction and RT-qPCR
The total RNA was extracted from the liver using a whole RNA extraction column kit (DenaZist Asia, Iran, S-1020). RNA degradation and contamination with DNA were assayed on 1% agarose gels electrophoresis (Thermo Fisher Scientific, MA). The RNA concentration and purity were also checked using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, MA). A total of 1 µg from each RNA sample was used to synthesize cDNA using a first strand cDNA synthesis kit (Sinnaclon, Iran, RT5201) according to the manufacturer's protocol and stored at -80°C until further use.
The gene expressions were quantified in StepOneTM Real-Time PCR (Thermo Fisher Scientific, MA) using 2x SYBR Green (RealQ Plus Master Mix Green, Amplicon A/S, Denmark). The β-actin gene (181 bp) was used as the housekeeping gene to normalize the level of expression for HSP70α, HSP70β, HSP90β, and IL-1β in different samples amplified using the specific primers (Table 2). Reaction mixtures contained 5 µl of 2x SYBR Green, 0.5 µM of each primer (forward and reverse), 1 µl of template cDNA, and 3 µl of distilled water in a final volume of 10 µl. For all PCR primer pairs, optimal annealing temperatures and reaction conditions were determined by examining the annealing temperature in gradients PCR using StepOne thermocycler (Thermo Fisher Scientific, MA). Cycling condition was 95°C for 15 min, 95°C for 15 s, 64°C for 30 s, and 72°C for 15 s. A melting curve analysis (95°C for 5 s and 64°C for 30 s) was performed after each qPCR run to verify the amplification specificity. The relative levels of HSPs and IL-1β mRNA were expressed using the 2 (- ∆∆CT ) method (Livak and Schmittgen, 2001).
2.8. Statistical analyses
The normality and homogeneity of data were tested using Shapiro-Wilk’s and Levene’s tests, respectively. Data were log (gene expression) or arcsine (survival rate) transformed to meet analyses of variance (ANOVA) assumptions of normality and homogeneity of variance if necessary. Two-way ANOVA was used to detect differences among treatments using the R version 3.6.3 software (R Core Team 2019). When ANOVA detected any differences among treatments, means of treatments were compared using Tukey’s post hoc tests. Data are presented as least-squares means ± SEM (pooled standard error mean of the experiment). The significance level was considered at P≤0.05. When the interaction of Temperature×Supplement was significant, the main effects of treatments and supplements were not presented.
Ethics statement
The fish handlings were conducted with minimal suffering of experimental animals based on the regulations of the Institutional Animal Care and Ethics Committee of Shiraz University.
The experimental protocols (Clinical examination, dissection, sampling, sample processing, microscopical examination, physiological and immunological analyses) were carried out in accordance with relevant guidelines and regulations supported by relevant references throughout the manuscript materials and methods section.
The current study was carried out in compliance with the ARRIVE guidelines when relevant methods were applied.