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  • All the protocols of fish treatment were approved by the Fisheries and Oceans Canada Pacific Region Animal Care Committee (Nanaimo, BC, Canada). Fish, Rearing Conditions, Stress Performance, and Sampling: Coho salmon, Chehalis River (BC, Canada) broodstock, were raised in 3000L tanks supplied with well water in Center for Aquaculture and Environment Research (CAER)’s Aquarium Facility (West Vancouver, BC, Canada). The fish were fed by hand to apparent satiation twice a day with commercial feed (Skretting Canada, Canada). Healthy, mixed sex, juvenile fish were divided into 3 groups (n = 8). The fish in the first group were undisturbed (prestressed) fish used as a control, maintained under quiet and suitable conditions, and sampled at 9∶00. The fish in the second group were subjected to the stressor at 8∶30 and sampled at 1.5 h post-stress (at 10∶00). The fish in third group were also subjected to the stressor at 17∶30 and sampled at 16 h post-stress (at 9∶30). Accordingly, all tissues and blood for analysis were sampled at almost the same time in the morning, so that the effects of several factors, such as diurnal rhythm and photoperiod, on the expressions of growth-related factors should be minimized. Four fish were placed into each of 6 separate 180 L fiber glass tanks supplied with running 10–11°C well water and acclimated to laboratory conditions under natural photoperiod for 5 days prior to experiments. Food was withheld for 24 h prior to the experiment. Nutritional status, such as starving, has been reported to influence the GH-IGF-I axis in fish, but 2 to 4 weeks of starving was required to obtain a significant change in the expression of growth-related factors in fish [7], [8], [27]–[30]. Accordingly, the short-term starving performed in the present study should not affect the expressions of growth-related factors in coho salmon. Fish, with a body weight of 23.9±3.5 g (mean ± SD), were subjected to physical disturbance both from a 2 min of chasing by a hand-held dip net followed by a 0.5 min emersion from the water held within in the dip net. All stressed fish were returned to control conditions after this treatment. To minimize the effect of sampling protocol, five fish were sampled from each experimental tank at each sample time, thereby avoiding repeated sampling from the same tank. Total number of fish sampled at each time was 8 per group. Fish were anaesthetized with 100 mg/L tricane methane sulphonate (MS-222) buffered with 100 mg/L sodium bicarbonate and rapidly team-sampled for blood and tissues. Blood was collected from the caudal vein with using a heparinized syringe and plasma was separated by centrifugation at 1,000×g for 10 min, stored frozen at −80°C. All tissues were immediately immersed in RNA later (Ambion-Life Technologies, Austin, TX) and then stored at −80°C until later analyses. Measurements of Cortisol and Glucose Levels in Plasma: Plasma cortisol levels were measured using a commercially available enzyme-linked immunosorbent assay kit from Neogen Corp. (Lexington, KY) [31]. Plasma glucose was measured using an enzymatic-glucose oxidase assay method available in kit (Sigma-Aldrich, St. Louis, MO) [32]. Measurements of GH and IGF-1 Levels in Plasma: Circulating levels of both GH and IGF-1 were analyzed using a chloramine-T iodination (125I)-based radioimmunoassay (RIA) established by Swanson [33] and Moriyama et al. [34], respectively. In cases where plasma GH levels were below the detectable level, the assay’s lower detection limit of 0.8 ng/ml was used as values. Plasma IGF-1 was determined using components (IGF-1 and anti-IGF-1 antibody) from GroPep (Adelaide, Australia). Plasma samples for IGF-1 were extracted with acid ethanol to remove binding protein effects. RNA Extraction and cDNA Synthesis: Tissues were placed in TRIzol reagent (Invitrogen-Life Technologies, Carlsbad, CA) and immediately homogenized using a polypropylene pestle. The resulting RNA pellet was dissolved in RNase-free water (UltraPure, Gibco-Life Technologies, Grand Island, NY) and quantified by spectrophotometry (Spectronic 1001 PLUS, Milton Roy, Ivyland, PA) before being diluted to 500 ng/µl to be used in reverse transcription reactions. RNA samples were stored at -80°C. Complementary DNA (cDNA) was synthesized using the Multiscribe Reverse Transcriptase Kit (Applied Biosystems-Life Technologies, Foster City, CA) with random hexamer primer or with a gene specific primer for salmon GH (gh-reverse primer, Table 1), and 250 ng RNA [20]. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0071421.t001 Primers and probes used for the quantitative real-time PCR (qPCR) analysis in this study. Determination of gh, ghr, igf, and β-actin mRNA Levels: The levels of gh, ghr, and igf1 mRNA expressions in the tissues were determined by real-time quantitative PCR (qPCR) with an equipment of ABI Prism 7000 Sequence Detection System (Applied Biosystems-Life Technologies, Foster City, CA) using β-actin as an internal standard according to Raven et al. [20]. Primers and TaqMan probes for β-actin, gh, ghr, and igf1 genes were designed from alignments of teleost cDNA sequences from GenBank to allow amplification using conserved regions (Table 1) [20], [35]. Values for gh, ghr, and igf1 were normalized with those of β-actin. Levels of β-actin did not change with respect to treatment. Accordingly, each sample amplification value for each gene was expressed as a relative gene expression ratio (relative mRNA value). All samples were run in duplicate and results are reported as mean ± SEM. All data were subjected to one-way analysis of variance (ANOVA). Means were compared with Fisher’s least-square difference (LSD) multiple comparison test.
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