149
Miyagi, Japan. After acclimatization for 2 weeks
at the aquarium facility of Tohoku University,
fi sh (approx. body weight, 144 g) were reared in
60-L fl ow-through glass tanks at 8 °C (light/
dark = 12 h/12 h). The fi sh were fed by hand to
apparent satiation twice a day with commercial
feed (Nosan Co., Japan). Food was withheld for
over 48 h before each sampling period. Fish
were exposed to heat shock (+11 °C for 2 h) and
sampled at 2.5, 17.5, and 48 h post-stress. Blood
was collected from the caudal vessels under
MS222 (m-aminobenzoic acid ethyl ester methanesulfonate) anesthesia. The plasma was separated by centrifugation and frozen at
−80 °C. Fish were gutted; the tissues were
quickly removed and frozen at −80 °C in RNA
later (Ambion, Life Technologies, Austin, TX)
until analysis.
Our experiments were conducted in accordance with the principles and procedures
approved by the Animal Care Committee at
Tohoku University (Sendai, Japan).
2.2
Plasma Cortisol and Glucose
Levels
Plasma cortisol levels were measured using an
enzyme-linked immunosorbent assay kit
(Oxford Biomedical Research, UK) (Basu et al.
2001 ). Plasma glucose was measured using an
enzymatic assay method with a Glucose CIITest Wako kit (Wako Pure Chemical Industries,
Ltd., Japan).
2.3
Plasma Lipid Peroxides,
Glutathione, and Superoxide
Dismutase Levels
Lipid peroxides (LPO) were determined as thiobarbituric acid-reactive substances (TBARS) by a
HPLC-fl uorescence method (Wong et al. 1987 ;
Morliere et al. 1991 ). Glutathione (GSH) levels in
plasma were determined by a glutathione reductase-recycling method with a Total Glutathione
Quantifi cation Kit (Dojindo Laboratories, Japan).
This kit can measure the total amount of reduced
GSH and oxidized form of GSH. The superoxide
dismutase (SOD) activity was assayed by the
formazan-WST method (Total SOD Assay Kit,
Dojindo Laboratories, Japan).
2.4
RNA Extraction and cDNA
Synthesis
Tissues were suspended in TRIzol Reagent
(Invitrogen, Life Technologies, CA) and immediately homogenized using a polypropylene pestle.
The resulting RNA pellet was dissolved in
RNase-free water (UltraPure, Gibco, Life
Technologies, NY), quantifi ed by spectrophotometry (V-630-Bio, JASCO, Japan), and then
diluted to 500 ng/µL for use in reverse transcription reactions. RNA samples were stored at
−80 °C. Complementary DNA (cDNA) was synthesized using a ReverTra Ace qPCR RT Kit
(Toyobo, Japan) with a mixture of random hexamers and oligo-dT primers or with a genespecifi c primer for salmon GH (gh-reverse
primer) and 250 ng of RNA (Nakano et al. 2013 ).
2.5
Real-Time qPCR for gh, ghr,
igf1, and arp mRNA Levels
The mRNA expression levels of gh, ghr, and
igf1 in tissues were determined by a real-time
quantitative PCR (qPCR) with an ABI Prism
7300 Sequence Detection System (Applied
Biosystems, Life Technologies, Foster City, CA)
using acidic ribosomal phosphoprotein P0 gene
(arp) as an internal standard (Pierce et al. 2004 ;
Nakano et al. 2013 ). mRNA values for gh, ghr,
and igf1 were normalized to those for arp.
Accordingly, each sample amplifi cation value for
each gene was expressed as a relative gene
expression ratio (relative mRNA level).
2.6
Statistical Analysis
All samples were run in duplicate and results
were expressed as means ± SEM. All data were
subjected to one-way analysis of variance
Effects of Thermal Stressors on Growth-Related Gene Expressions in Cultured Fish
Miyagi, Japan. After acclimatization for 2 weeks
at the aquarium facility of Tohoku University,
fi sh (approx. body weight, 144 g) were reared in
60-L fl ow-through glass tanks at 8 °C (light/
dark = 12 h/12 h). The fi sh were fed by hand to
apparent satiation twice a day with commercial
feed (Nosan Co., Japan). Food was withheld for
over 48 h before each sampling period. Fish
were exposed to heat shock (+11 °C for 2 h) and
sampled at 2.5, 17.5, and 48 h post-stress. Blood
was collected from the caudal vessels under
MS222 (m-aminobenzoic acid ethyl ester methanesulfonate) anesthesia. The plasma was separated by centrifugation and frozen at
−80 °C. Fish were gutted; the tissues were
quickly removed and frozen at −80 °C in RNA
later (Ambion, Life Technologies, Austin, TX)
until analysis.
Our experiments were conducted in accordance with the principles and procedures
approved by the Animal Care Committee at
Tohoku University (Sendai, Japan).
2.2
Plasma Cortisol and Glucose
Levels
Plasma cortisol levels were measured using an
enzyme-linked immunosorbent assay kit
(Oxford Biomedical Research, UK) (Basu et al.
2001 ). Plasma glucose was measured using an
enzymatic assay method with a Glucose CIITest Wako kit (Wako Pure Chemical Industries,
Ltd., Japan).
2.3
Plasma Lipid Peroxides,
Glutathione, and Superoxide
Dismutase Levels
Lipid peroxides (LPO) were determined as thiobarbituric acid-reactive substances (TBARS) by a
HPLC-fl uorescence method (Wong et al. 1987 ;
Morliere et al. 1991 ). Glutathione (GSH) levels in
plasma were determined by a glutathione reductase-recycling method with a Total Glutathione
Quantifi cation Kit (Dojindo Laboratories, Japan).
This kit can measure the total amount of reduced
GSH and oxidized form of GSH. The superoxide
dismutase (SOD) activity was assayed by the
formazan-WST method (Total SOD Assay Kit,
Dojindo Laboratories, Japan).
2.4
RNA Extraction and cDNA
Synthesis
Tissues were suspended in TRIzol Reagent
(Invitrogen, Life Technologies, CA) and immediately homogenized using a polypropylene pestle.
The resulting RNA pellet was dissolved in
RNase-free water (UltraPure, Gibco, Life
Technologies, NY), quantifi ed by spectrophotometry (V-630-Bio, JASCO, Japan), and then
diluted to 500 ng/µL for use in reverse transcription reactions. RNA samples were stored at
−80 °C. Complementary DNA (cDNA) was synthesized using a ReverTra Ace qPCR RT Kit
(Toyobo, Japan) with a mixture of random hexamers and oligo-dT primers or with a genespecifi c primer for salmon GH (gh-reverse
primer) and 250 ng of RNA (Nakano et al. 2013 ).
2.5
Real-Time qPCR for gh, ghr,
igf1, and arp mRNA Levels
The mRNA expression levels of gh, ghr, and
igf1 in tissues were determined by a real-time
quantitative PCR (qPCR) with an ABI Prism
7300 Sequence Detection System (Applied
Biosystems, Life Technologies, Foster City, CA)
using acidic ribosomal phosphoprotein P0 gene
(arp) as an internal standard (Pierce et al. 2004 ;
Nakano et al. 2013 ). mRNA values for gh, ghr,
and igf1 were normalized to those for arp.
Accordingly, each sample amplifi cation value for
each gene was expressed as a relative gene
expression ratio (relative mRNA level).
2.6
Statistical Analysis
All samples were run in duplicate and results
were expressed as means ± SEM. All data were
subjected to one-way analysis of variance
Effects of Thermal Stressors on Growth-Related Gene Expressions in Cultured Fish