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. 2014 Jul 29;9(7):e102204.
doi: 10.1371/journal.pone.0102204. eCollection 2014.

Differential expression of heat shock transcription factors and heat shock proteins after acute and chronic heat stress in laying chickens (Gallus gallus)

Jingjing Xie  1 , Li Tang  1 , Lin Lu  1 , Liyang Zhang  1 , Lin Xi  2 , Hsiao-Ching Liu  2 , Jack Odle  2 , Xugang Luo  1
Affiliations

Differential expression of heat shock transcription factors and heat shock proteins after acute and chronic heat stress in laying chickens (Gallus gallus)

Jingjing Xie et al. PLoS One. .

Abstract

Heat stress due to high environmental temperature negatively influences animal performances. To better understand the biological impact of heat stress, laying broiler breeder chickens were subjected either to acute (step-wisely increasing temperature from 21 to 35°C within 24 hours) or chronic (32°C for 8 weeks) high temperature exposure. High temperature challenges significantly elevated body temperature of experimental birds (P<0.05). However, oxidation status of lipid and protein and expression of heat shock transcription factors (HSFs) and heat shock proteins (HSPs) 70 and 90 were differently affected by acute and chronic treatment. Tissue-specific responses to thermal challenge were also found among heart, liver and muscle. In the heart, acute heat challenge affected lipid oxidation (P = 0.05) and gene expression of all 4 HSF gene expression was upregulated (P<0.05). During chronic heat treatment, the HSP 70 mRNA level was increased (P<0.05) and HSP 90 mRNA (P<0.05) was decreased. In the liver, oxidation of protein was alleviated during acute heat challenge (P<0.05), however, gene expression HSF2, 3 and 4 and HSP 70 were highly induced (P<0.05). HSP90 expression was increased by chronic thermal treatment (P<0.05). In the muscle, both types of heat stress increased protein oxidation, but HSFs and HSPs gene expression remained unaltered. Only tendencies to increase were observed in HSP 70 (P = 0.052) and 90 (P = 0.054) gene expression after acute heat stress. The differential expressions of HSF and HSP genes in different tissues of laying broiler breeder chickens suggested that anti-heat stress mechanisms might be provoked more profoundly in the heart, by which the muscle was least protected during heat stress. In addition to HSP, HSFs gene expression could be used as a marker during acute heat stress.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Illustration of thermal application in the acute heat stress treatment.
During thermal application to the acute heat stress group (AS, full line), the control group (AC, dotted line) were maintained at 21°C. The increment in the AS chamber temperature started at 7 PM. The environmental temperature was increased by 4°C in 1 hour and held for another 4 hours. After holding at 33°C for 4 hours, the temperature was gradually decreased to 21°C in 3 hours on the first day for adaptation. All 24 birds rested at 21°C for 24 hours and then the procedure was repeated. On the final day (Day 3) of the experiment, instead of returning to 21°C, the AS chamber temperature was continuously increased from 33°C to 35°C and held for 4 hours before sampling.
Figure 2
Figure 2. Rectal temperatures of laying broiler breeder chickens in response to the chronic heat challenge at 32°C over 8 weeks.
In the first week, the rectal temperature of laying broiler breeder chickens in the chronic heat-stressed group (CS) was not elevated by the cyclic environmental temperature (32-35-32°C) and only a tendency of increase in rectal temperature was observed in the second week. After exposed to the consistent 32°C thermal treatment from the third week, CS birds had a significantly higher average body temperature than those in the chronic control group (CC). Values are expressed as means ± SE of data from 10–12 individual birds. * indicates significant differences (P<0.05) between CS (line with squares) and CC (line with diamonds) at the same time of the trial.
Figure 3
Figure 3. Changes in MDA, PCC and SOD activity after acute and chronic heat challenges.
Malondialdehyde (MDA) was used as a biomarker of lipid peroxidation and was determined by the thibabituric acid (TBA) method. Protein carbonyl content (PCC) was employed as a protein oxidation biomarker and was determined using a spectrophotometric 2, 4-dinitrophenylhydrazine (DNPH) assay. Activities of total superoxide dismutase (TSOD) and MnSOD were measured with the nitrite method. Values are expressed means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P<0.05) between controls and heat-stressed groups. AC, acute control group; AS, acute heat-stressed group; CC, chronic control group; CS, chronic heat-stressed group.
Figure 4
Figure 4. Normalized gene expression of HSF after acute and chronic heat challenges.
Expression of heat shock factor (HSF) genes was determined by real time PCR using SYBR green dye and the 2−ΔΔCt method was used to calculate mRNA level of each gene, where the average mean of the unstressed group was used as the calibrator. Each PCR reaction was conducted in triplicate and the geometric mean of internal references, β-actin and GAPDH, was used to normalize the expression of targets genes. Values are expressed as means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P<0.05) between controls and heat -stressed groups. AC, acute control group; AS, acute heat-stressed group; CC, chronic control group; CS, chronic heat-stressed group.
Figure 5
Figure 5. Normalized gene expression of HSP 70 and 90 after acute and chronic heat challenges.
Expression of HSP 70 and 90 genes was determined by real time PCR using SYBR green dye and the 2−ΔΔCt method was used to calculate mRNA level of each gene, where the average mean of the unstressed group was used as the calibrator. Each PCR reaction was conducted in triplicate and the geometric mean of internal references, β-actin and GAPDH, was used to normalize the expression of targets genes. Values are expressed as means ± SE of data from 5–6 individual tissue samples. * indicates significant differences (P<0.05) between controls and heat -stressed groups. AC, acute heat control group; AS, acute heat-stressed group; CC, chronic control group; CS, chronic heat-stressed group.
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Grants and funding

This study was supported by the Key International Cooperation Program of the National Natural Science Foundation of China (project No. 31110103916; Beijing, P. R. China), The Agricultural Science and Technology Innovation Program (ASTIP-IAS08; Beijing, P. R. China), and China Agriculture Research System (project No. CARS-42; Beijing, P. R. China). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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