Influence of induced heat stress on HSP70 in buffalo lymphocytes

Heat stress in farm animals, such as cattle and buffalo during summer and post-summer seasons is a problem for livestock producers. The effect of heat stress becomes pronounced when heat stress is accompanied with ambient humidity impairing the immune status, growth, production and reproductive performance of animals. Increase in HSP70 levels from cell cultures in presence of different stressors often does not reflect the physiological adaptability of animals governing thermal regulation. In this study we directly compared the effect of different heat stress conditions with the immune status and HSP70 expression patterns from buffalo lymphocytes both in vivo and in vitro. Murrah buffalo calves were exposed to induced heat stress with two experimental treatments: hot-dry (42 °C with existing relative humidity) or hot humid (35 °C with 70% relative humidity) condition in psychometric chamber, 4 h daily for 12 days and compared with control animals maintained in an experimental shed under natural conditions. There was >200-fold increase in serum-HSP70 levels in both heat stress conditions compared with control. Furthermore, the immune status of the calves failed to activate the level of HSP70 expression in serum lymphocytes. Lymphocytes cultured in vitro at higher temperature exert 2.5-fold increase in HSP70 concentration. This study is the first of its kind to demonstrate more complex expression pattern of buffalo serum-HSP70 level as a thermo adaptive response compared with in vitro treated cells. Results from this study indicate that serum-HSP70 levels could be used as a sensitive biomarker for heat stress management in large farm animals.     

夏季奶牛血液中热休克蛋白的表达量

选择40头体质健康的中国荷斯坦奶牛,根据胎次和泌乳天数,按照随机区组试验设计分为Ⅰ组（30kg/d）、Ⅱ组（30～35kg/d）、Ⅲ组（35～40kg/d）和Ⅳ组（40kg/d）。热应激前、热应激前期、热应激中期、热应激后期和热应激后分别于尾静脉采血,用ELISA试剂盒测定热休克蛋白（HSP）27,70,90的表达量。结果显示,Ⅳ组HSP27表达量最高,Ⅱ组表达量最低,Ⅳ组、Ⅲ组和Ⅰ组均显著高于Ⅱ组（P〈0.05）。HSP70表达量各组间没有明显差异,但随产奶量呈线性增加（P〈0.05）。HSP90的表达量,Ⅳ组和Ⅲ组明显高于Ⅱ组（P〈0.05）。HSP27的表达量热应激后差异较大;HSP70的表达量各组整个过程差异较大;HSP90的表达量在热应激前、热应激前期和热应激后差异较大。总之,在热应激过程,高产奶牛血清中热应激蛋白的表达量较高,HSP70表达量随产奶量呈线性增加,而不同热应激蛋白的变化规律差异较大。     

Synthesis of heat stress proteins in lymphocytes from livestock

Cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of a specific set of proteins known as heat stress proteins (HSP). Proteins with molecular weights of 70 and 90 kDa were synthesized in all species. Additional proteins were found in bovine, ovine and chicken lymphocytes. A time course of induction showed an increased synthesis of some of these proteins with only 30 min of heat stress and of several proteins with 60 min of heat stress. A specific monoclonal antibody was used to identify HSP70 as one of the stress proteins in bovine lymphocytes. Cells from these animals are capable of responding to heat stress by the production of heat stress proteins. These may be of physiological importance.     

热应激对肉鸡的影响及热休克蛋白70的研究进展

在现代化畜禽生产中,热应激已成为制约肉鸡生长、代谢、肉品质及生理功能的重要因素之一,给养殖业带来了严重的经济损失。热应激蛋白（heat shock protein,HSPs）是动物在不良因素作用下所产生的一组特异性蛋白质,而HSP70是其家族成员中最保守、最重要的蛋白之一;它具有分子伴侣、热耐性,抗过氧化损伤等特性,能够使机体迅速适应环境变化,在热应激保护中发挥了重要的作用。作者主要阐述了热应激对肉鸡的影响和HSP70的生物学特点及其抗热应激的作用机理,以期为从药物方面探讨HSP70的抗应激功能提供理论依据。     

运输应激对山羊血清和空肠中HSP27、HSP70和HSP90含量的影响

为探讨运输应激对山羊血清和空肠中HSP27、HSP70和HSP90 3种热休克蛋白含量的影响,试验选取12只赣西山羊,随机分为对照组、运输应激2 h组和运输应激6 h组,应用双抗体夹心ELISA法测定山羊血清和空肠中3种热休克蛋白浓度。结果表明:与对照组相比,运输2 h山羊血清中HSP70和HSP90极显著升高(P<0.01),空肠中HSP70和HSP90显著升高(P<0.05),血清和空肠中HSP27有升高趋势(P>0.05);与对照组相比,运输6 h后血清中HSP70和HSP90分别极显著和显著升高(P<0.05和P<0.01),血清中HSP27含量以及空肠中3种蛋白含量均无显著变化(P>0.05);与运输2 h相比,运输6 h后3种蛋白含量都极显著降低(P<0.01)。结果显示,山羊对运输应激在细胞水平的适应性调节与血清中HSP70和HSP90蛋白水平升高有密切联系,运输2 h山羊通过上调HSP70和HSP90对应激导致的空肠损伤发挥细胞保护作用。     

HSP90 gene expression induced by aspirin is associated with damage remission in a chicken myocardial cell culture exposed to heat stress

1. To understand the potential protection of heat shock protein 90 (HSP90) induced by aspirin against heat stress damage in chicken myocardial cells, enzyme activities related to stress damage, cytopathological changes, the expression and distribution of HSP90, and HSP90 mRNA levels in the myocardial cells exposed to heat stress (42°C) for different durations with or without aspirin administration (1 mg/ml, 2 h prior) in vitro were investigated.

2. Significant increase of enzyme levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterised by acute degeneration, karyopyknosis and karyorrhexis were observed, compared to non-treated cells. However, the lesions of cells treated with aspirin were milder, characterised by earlier recovery of enzyme levels to the control levels and no obvious heat stress-related cellular necrosis.

3. Stronger positive signals in the cytoplasm and longer retention of HSP90 signal in nuclei were observed in aspirin-treated myocardial cells than those of only heat-stressed cells. HSP90 level in the aspirin-treated myocardial cells was 11.1-fold higher than that in non-treated cells, and remained at a high level at the early stage of heat stress, whereas it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to a low level.

4. Overexpression of HSP90 mRNA in aspirin-treated cells was observed throughout the experiment, whereas HSP90 mRNA decreased significantly only in heat-stressed cells.

5. The early higher HSP90 expression induced by aspirin during heat stress was accompanied by decreased heat stress damage, suggesting that aspirin might play an important role in preventing myocardial cells from heat stress damage in vitro.

     

Comparative studies on temperature threshold for heat shock protein 70 induction in young and adult Murrah buffaloes

To know the temperature threshold for heat shock protein 70 (HSP70) induction in lymphocytes and to assess physiological changes, if any, in relation to HSP70 induction in young and adult Murrah buffaloes, this study was divided into two parts: I. In vivo study: where assay of HSP70 was performed in blood samples collected from acutely exposed young and adult Murrah buffaloes (n = 6) inside a climatic chamber at 40, 42 and 45 °C for 4 h and thermoneutral temperature (22 °C). Physiological parameters viz., rectal temperature, respiratory rate, pulse rate and skin temperature of different body parts were monitored to assess magnitude of stress in the animals owing to thermal exposure II. For in vitro study, equal numbers of lymphocyte cells were separated from blood collected from young and adult buffaloes and were subjected to four temperature treatments (38, 40, 42 and 45 °C) for 4 h. A significant increase (p < 0.05) in all the physiological parameters in both young and adult buffaloes was observed after exposure to 40, 42 and 45 °C for 4 h as compared to 38 °C. The average plasma HSP70 concentrations (ng/ml) were significantly higher (p < 0.05) at 40, 42 and 45 °C as compared to 38 °C in both young and adult and were higher in young than adult buffaloes at 38 and 45 °C. Heat shock protein 70 level in lymphocyte lysate showed highest concentration after 3-h exposure to all temperatures (40, 42 and 45 °C) in both young and adult buffaloes. The intensity of changes of all physiological parameters was more in young animals than in the adults indicating the greater susceptibility of younger animals to heat stress and was found to be changed at around 40 °C when animals were exposed to different temperatures, indicating the possibility that HSP70 production may be initiated at this temperature which is 2 or 3 °C higher than core body temperature.     

Research Progress of Effect of Oxidative Stress on Broiler and its Mitigation Technology

Oxidative stress is ubiquitous in livestock and poultry production.When the body is in the situation of harmful stimulation in vitro or in vivo,the oxidation system and antioxidant system are in a state of imbalance,leading to metabolic disorders,which depresses the growth and development of animals,decreases disease resistance and quality of livestock products seriously,and has a negative impact on the production and health of livestock and poultry.So it is important to find an effective measure to alleviate the health of livestock and poultry.Lipoic acid,tea polyphenol,VE and other antioxidants play significant roles in reducing oxidative stress.In this paper,the effects of oxidative stress on broiler and mitigation technology research were outlined.     

氧化应激对肉鸡的影响及其缓解技术研究进展

在畜禽生产中氧化应激无处不在,当机体受到体内外各种有害刺激时抗氧化系统失衡,从而导致机体代谢紊乱,使畜禽生长发育、抗病能力及畜产品品质受到严重影响,对畜禽的生产和健康有着负面的影响作用.因此,找到一种有效的缓解措施对畜禽的健康生产有着重要的感义.硫辛酸、茶多酚、VE等抗氧化剂在缓解氧化应激方面有显著的作用.作者就氧化应激对肉鸡产生的影响及其缓解技术对其改善作用的研究作一概述.     

Zinc alleviates the heat stress of primary cultured hepatocytes of broiler embryos via enhancing the antioxidant ability and attenuating the heat shock responses

Zinc (Zn) has been shown to attenuate the adverse effects of heat stress on broilers, but the mechanisms involving this process remain unclear. We aimed to investigate possible protective mechanisms of Zn on primary cultured hepatocytes of broiler embryos subjected to heat stress. Three experiments were conducted. In Exp. 1, hepatocytes were treated with 0, 50, 100, 200, or 400 μmol/L added Zn as inorganic Zn sulfate (iZn) for 12, 24 or 48 h. In Exp. 2, cells were exposed to 40 °C (a normal temperature [NT]) and 44 °C (a high temperature [HT]) for 1, 2, 4, 6, or 8 h. In Exp. 3, cells were preincubated with 0 or 50 μmol/L Zn as iZn or organic Zn lysine chelate (oZn) for 8 h under NT, and then incubated with the same Zn treatments under NT or HT for 4 or 6 h. The biomarkers of antioxidative status and heat stress in cells were measured. The results in Exp. 1 indicated that 50 μmol/L Zn and 12 h incubation were the optimal conditions for increasing antioxidant ability of hepatocytes. In Exp. 2, the 4 or 6 h incubation under HT was effective in inducing heat shock responses of hepatocytes. In Exp. 3, HT elevated (P < 0.01) malondialdehyde content and expressions of heat shock protein 70 (HSP70) mRNA and protein, as well as HSP90 mRNA. However, Zn supplementation increased (P < 0.05) copper zinc superoxide dismutase (CuZnSOD) activity and metallothionein mRNA expression, and effectively decreased (P < 0.05) the expressions of HSP70 mRNA and protein, as well as HSP90 mRNA. Furthermore, oZn was more effective (P < 0.05) than iZn in enhancing CuZnSOD activity of hepatocytes under HT. It was concluded that Zn (especially oZn) could alleviate heat stress of broiler hepatocytes via enhancing their antioxidant ability and attenuating heat shock responses.     

C-terminal heat shock protein 70 of Mycobacterium tuberculosis as a molecular adjuvant for DNA vaccination with the porcine circovirus type 2 ORF2 (capsid) gene in mice

Mycobacterium tuberculosis heat shock protein 70 (HSP70) and the peptide binding C-terminal portion of HSP70 (amino acids 359-610; HSP70c) exert an adjuvant effect when used in vaccines. To enhance the immunogenicity of a DNA vaccine against porcine circovirus type 2 (PCV2), recombinant plasmids encoding the PCV2 ORF2 (capsid) gene fused to full length hsp70 (pCA-TCH) or truncated C-terminal hsp70c (pCA-TCHc) were constructed. Immunisation of mice with pCA-TCHc induced higher serum immunoglobulin G antibody levels, stronger T helper 1 immune responses and lower PCV2 viral titres following challenge than immunisation with pCA-TCH or Cap plasmids only.     

哺乳动物HSP70及其与精子发生相关关系研究进展

HSP70为存在于所有生物细胞的、结构上高度保守的一组细胞应激蛋白,通常分为诱导型HSP70与非诱导型HSP70,其主要功能是参与细胞应激反应和作为细胞管家蛋白,二者均与哺乳动物的精子生成存在密切关系。深入研究HSP70与精子发生的相互关系,将有助于提高对哺乳动物精子发生机制的进一步了解。     

Purification, characterization and expression kinetics of heat shock protein 70 from Bubalus bubalis

The present work on Bubalus bubalis (buffalo) was designed to study heat shock protein 70 (HSP70) induction in lymphocytes, its purification and characterization. HSP70 induction and expression kinetics at different temperatures and time durations were also studied. HSP70 purification was carried out by immunoaffinity chromatography using adenosine di-phosphate (ADP-agarose column) and the characterization of the purified protein was done using western blotting by mouse monoclonal anti-HSP70. The molecular weight of HSP70 of buffalo lymphocytes was found to be approximately of 68 kDa and was less than that of bovine brain HSP70. The purified HSP70 was assessed using indirect inhibition enzyme-linked immunosorbent assay (ELISA). A good amount of HSP70 (1430 ηg HSP70/100 μl) was recovered after purification, out of the total 2040.40 ηg of HSP70/100 μl of cell supernatant. To assess the impact of temperature and time dependent variability in the induction and expression pattern of HSP70, buffalo lymphocytes were subjected to three different temperature treatments, viz.: (I) 38 °C for 48 h and further exposed the same cells at 45 °C for 3 h, (II) 42 °C for 3 h, and (III) 45 °C for 3 h, respectively. The respective cell viability was found to be 68%, 63%, and 51%. The HSP70 levels were 58.30 ± 4.37, 42.59 ± 9.04 and 21.95 ± 6.79 ng/million cells, respectively, at three temperature exposures. The results indicates that higher intensity and duration of temperature exposure cause higher HSP70 induction in buffalo lymphocytes to maintain cellular homeostasis with a threshold of thermal dose for maximum HSP70 expression. However immediate induction of HSP70 in the lymphocytes was dependent on magnitude of thermal exposure (stress level) and time of thermal exposure (stress duration). The present study on HSP70 and its induction will help likely to solve the problems related to the present scenario of thermo-adaptability in buffaloes.     

中药复方对慢性热应激蛋鸡生产性能和心脏组织HSP70表达量的影响

探讨中药复方对慢性热应激下蛋鸡生产性能和心脏组织HSP70表达量的影响。选取250只60日龄蛋鸡,随机分为空白对照组、维生素C组、复方Ⅰ、复方Ⅱ、复方Ⅲ组。每天记录各组的耗料量、蛋重、产蛋量、淘死数以及软、破蛋数量,采用ELISA法测定蛋鸡心脏组织HSP70表达量。结果显示,中药复方能够改善热应激状态下蛋鸡的生产性能,其中复方Ⅱ的效果最佳;中药复方组蛋鸡心脏组织HSP70表达量均低于空白对照组,其中方Ⅱ组下降的幅度最大,表明各中药复方均能提高热应激蛋鸡的生产性能,其作用机制可能与调控HSP70表达量有关。     

Physiological indicators of stress and meat and carcass characteristics in tail bitten slaughter pigs

Background

Tail biting is a common welfare problem in pig production and in addition to being a sign of underlying welfare problems, tail biting reduces welfare in itself. The aim of this study was to evaluate the effects of tail biting on different pre and post mortem indicators of stress in slaughter pigs and on carcass and meat characteristics. A total of 12 tail bitten (TB) and 13 control (C) pigs from a farm with a long-term tail biting problem were selected for salivary cortisol analyses before and after transport to the slaughterhouse. After stunning, samples were taken for the analysis of serum cortisol, blood lactate, intestinal heat shock protein 70 (HSP70), and meat quality characteristics. In addition, body temperature immediately after and muscle temperature 35 min after stunning were measured, as well as lean meat percentage and carcass weight.

Results

TB pigs showed a lower cortisol response to the transport-induced stress than C pigs and also had a lower serum cortisol concentration after stunning. HSP70 content in the small intestine was higher in the TB pigs than in C pigs. TB pigs had a considerably lower carcass weight therefore produced a lower total amount of lean meat per carcass than C pigs.

Conclusions

This study suggests that prolonged or repeated stress in the form of tail biting causes a blunted stress response, possibly a sign of hypocortisolism. In addition, it underlines the importance of reducing tail biting, both from an animal welfare and an economic point-of-view.     

热应激畜禽的营养调控

夏季高温环境下,畜禽极易发生热应激。畜禽发生热应激后其生产性能降低,严重影响养殖效益。营养调控措施是根据畜禽的发育规律,通过调配适宜的营养成分和含量,降低热应激对畜禽的生长过程的影响,使畜禽养殖朝着健康、绿色、持续、高效的方向发展。从调节电解质、矿物质、维生素等营养调控的角度综述了热应激防控的最新研究进展,以期为防控热应激和寻找新的措施、最大限度地降低畜牧业生产损失提供有益的思路。     

Prokaryotic expression of chicken interferon‐γ fusion protein and its effect on expression of poultry heat shock protein 70 under heat stress

Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full‐length chicken interferon‐γ (ChIFN‐γ ) gene was amplified by RT‐PCR. The full complementary DNA sequence of the ChIFN‐γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX?HT_b. The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN‐γ was induced by isopropyl β‐D‐1‐thiogalactopyranoside. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis and Western blot results showed the expressed fusion protein had a molecular weight of approximately 18 kDa and was recognized by an anti‐His mAb. Moreover, ChIFN‐γ was found to demonstrate anti‐viral activity in vitro . To test the in vivo function of ChIFN‐γ in broilers under heat stress, a total of 100 broilers were randomly assigned to either a control group or a treated group, in which they were hypodermically injected with recombinant ChIFN‐γ. Results demonstrated ChIFN‐γ affects the messenger RNA expression levels of heat shock protein 70 (HSP70) in the heart and lung tissues, and decreases the concentration of HSP70 in serum. Therefore, we conclude recombinant ChIFN‐γ can reduce heat stress to some extent in vivo .