| China is one of powerful poultry raising countries. The poultry industry often suffer great economic losses because of durative high temperature last 2-3 months during summer time in the most parts of China. Heat stress response is a systematical response, which has an important effect on nerve and endocrine system, digestion and absorption, blood circulation, urinary and reproductive system, immune system and substance metabolism. When living organisms are exposed to thermal and nonthermal stressors, a group of highly conserved proteins known as heat shock proteins (Hsps) are rapidly synthesized, which play important roles in the protection of cells and stabilization of internal environment. Therefore, it is very important to study the characteristics of the heat stress-induced injury and take reasonable measures to reduce injury in poultry industry. The objective of this study was to investigate the expressions and localizations of Hsps and their corresponding mRNA in the tissues of heat-stressed broilers, and to correlate these Hsps levels with tissue damages at various stressing times.One-day-old male AA broiler chicks (n=100) were housed in large coops (20 birds per coop) and the coops were placed in a controlled-climate chamber. The birds were given a 30-day period to acclimate to their new surroundings and to reduce the influence of environmental stressor. During this period, the broilers were raising under standard conditions by specified person to avoid environmental stress. At 30 days of age, the room temperature was suddenly increased from 22±1℃to 37±1℃within 30 min and the broilers were heat stressed for 0 (control),2,3,5, and 10 h, respectively. On terminating the heat treatment, the plasma samples were obtained from jugular vein for subsequent CK and GPT determination. Following exsanguinations, the birds were manually eviscerated, and the heart, liver, and kidneys were quickly dissected and fixed in freshly prepared 4% paraformaldehyde in phosphate-buffered saline, for histological analysis, immunohistochemistric analysis and in situ hybridization analysis. Further tissues samples were frozen in liquid nitrogen, and then stored at-80℃for ELISA detection and real time RT-PCR analysis. Enzyme results indicated that the concentrations of plasma CK and GPT increased obviously with heat stressing, implying damages to myocardial cells and hepatic cells were more serious as the time of heat stress prolonged. Histopathological results indicated that parenchymal cells of broiler heart, liver and kidney showed acute granular degeneration, fatty degeneration and even necrosis. The changes of parenchymal cells became more severe as the time of heat exposure prolonged. Enzyme results and histopathological results implied that broiler tissue damages were closely related to the detected plasma enzyme activities. The concentration of plasma enzyme can indirectly reflect stress-induced tissue injury and the degree of injury.Immunohistochemistry (IHC) results indicated that Hsps positive signals were located both in the heat stressed groups and the control group, however, the localization of different Hsps was varied in the three detected tissues. Hsp60 positive signals were observed in the cytoplasm of myocardial cells, hepatic cells and the renal tubular epithelial cells. Hsp60 expression was more prominent in the cytoplasm of intact tissue cells than in that of granular degenerated cells, vacuolar degenerated cells and collapsed cells. This maybe implied an association between tissue damage and the expression of Hsp60 in response to heat stress. Positive Hsp70 and Hsp90 protein signals were detected in the nucleus and cytoplasm of parenchymal cells in broiler heart, liver and kidney, however, the positive signals were stronger in the nucleus than the cytoplasm. This implied that Hsp70 and Hsp90 may effect as the important proteins for nucleus protection and message transmission. Hsp70 and Hsp90 positive signals were also detected in the blood vessel walls of heart, liver and kidney, which implied an close association between Hsp70 and Hsp90 expression and the function of vasoconstriction and vasodilatation. The expression of Hsps in vascular system may play an important role in the development of cardiovascular diseases, such as ischemia-perfusion injury during heat stress process. The localization of hsp70 mRNA in broiler tissues was studied by in situ hybridization method. Hybridization signals for hsp 70 mRNA were detected in the cytoplasm of myocardial cells, hepatic cells and the renal tubular epithelial cells, however, the nucleus of the three examined tissues in broilers exhibited no hsp70 mRNA positive signals. It gave us an implication that Hsp70 can shuttle from cytoplasm to nucleus after heat shock.The levels of Hsps and their mRNA were determined by the methods of ELISA and real time RT-PCR, respectively. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P＜0.01) at 2 h of heat stress but then decreased to normal level, while the level of Hsp60 protein in the heart increased (P＜0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicated that the expression of Hsp70,Hsp90 and Hsp60 showed different characteristics and fluctuations in the three tissues of heat stressed broilers. Especially, Hsp60 in heart, may be important markers reflecting tissue injury at the beginning of heat stress and act as protective proteins in adverse environments. The level of Hsp90 in liver of broilers increased significantly after 2 h heat exposure (P＜0.01), whereas the level of Hsp70 increased significantly after 5 h (P＜0.01) and 10 h (P＜0.05) heat exposure, which implied that Hsp90 acted an important role after short-term heat stress (2 h-3 h), whereas Hsp70 contributed more effect after long-term heat stress (5 h-10 h). However, the level of Hsp60 in the liver tissues decreased significantly during heat exposure. The levels of Hsp60 in the kidney fluctuated after 10 h of heat stressing, however, there was no significant difference between heat stressed groups and the control. After 3 h of heat stress, the levels of Hsp70 in kidney of broilers increased significantly (P＜0.01) compared with the control. Hsp90 levels increased significantly after 2 h (P＜0.01) and 10 h (P＜0.05) of heat stressing. It implied that Hsp70 and Hsp90 played important roles on kidney protection during 10 h of heat exposure.Real time RT-PCR results demonstrated that the transcription of hsps mRNA wasn't coincide with the translation of Hsps. It implied that the mechanism of hsps mRNA transcription and Hsps translation was so complicated. Different Hsps may have different mechanisms.The results indicated that pH30min and pH24h of pectoralis muscles significantly decreased (P<0.01) after heat stress for varying amounts of time compared with control. The values of pH3omin and pH24h reached their lowest point after 5 h of heat exposure. Broilers exposed to high temperature had a higher drip loss, expressible moisture and cooking loss, especially after 5 h of heat exposure. After heat stress, broilers had a higher shear value, L* value and lower a* value (P＜0.01) compared to control. Correlation analysis indicated that the decrease of pH value may have an important impact on water holding capability, tenderness and meat color of pectoralis muscles after heat exposure. High temperature would lead to PSE meat of broilers pectoralis muscles. |
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