Dynamic Analysis Of Hormones, HSP70 And HSP70 MRNA During Piglets Cold Stress

Stress is a nonspecific response to various kinds of stimulates in organism,which can cause performance descends, induce various diseases, even result in to diein livestock and poultries. The cold is one of the most universal stressgen in northernanimal, which is an important factor to restrict northern livestock farmingdevelopment owing to the direct and indirect economic loss. Studying the animalcold stress mechanism and investigating scientific stress monitor method, whichfocus on the body biochemical indicator in cold stress reaction, have importantmeaning to elevate preventative level of stress and ensure livestock farmingdevelopment. The former research concentrates primarily on the energy materialmetabolism and stress hormones. Molecular biomarkers can provide theenvironmental biological effects and information. They have the unique advantagefor accurate, sensitive evaluate the early reactions of animal stress. Combining thebiomarkers and its mechanism may get breakthrough to research animal cold stress.Heat shock protein 70 (HSP70) is the most representative member in HSP familywhich can increase express in cell stress, and maintain cell construction and functionby molecular chaperones. HSP70 has the center regulation effect on stress reaction,high conservatism in evolution and the characteristics of feel directly cell inside andexternal environment charge, so it may be used as the biomarker in evaluating thecold stress reaction. Dynamic studies on HSP70 by continuous variables have moremeaning to approach cold stress reaction mechanism and reveal the intensity(time)-effect relation. This research choose breed "JunMu-1" piglet (normal feedingtemperature at 15℃~18℃), using the radioimmunity, western-blotting, fluorescencequantitative PCR etc, dynamic inspected change rule of blood physiological andbiochemical indicators, stress hormone, HSP70 and HSP70 mRNA by different coldstress intensity (-10℃~-4℃, -3℃~3℃, 4℃~10℃) and different times (cold expose0, 0.5, 3, 6, 12, 24 hour and after terminate cold expose 0, 0.5, 3, 6, 12, 24 hour),moreover, detected and analyzed HSP70 gene sequence of "JunMu-1" breed swine.The studies can provide experimental evidence for investigating reaction mechanismand earlier monitoring of animal cold stress.First, we detected eighteen blood routines and ten blood biochemical indicators.The results showed that the red blood cell, haematocrit and hemoglobin reducedgradually after cold stress, and just went up at experiment ending. At -10℃~-4℃cold stress, white blood cell and lymphocyte increased at 12 hour and 24 hour aftercold exposure. That may have something to do with body immunity and guardingmechanism enhancement. The blood glucose obviously increased at 3 hour and 6hour and creatinine obviously reduced after cold exposure. The results that indicatedbody increased the energy material requirement in cold environment. Collectingdetected the hematological results from all test groups before cold stress, obtaining"Jun Mu-1"weaned piglet's blood physiological and biochemical reference values.They can provide important proof for veterinary clinical diagnosis and therapy.The cold stress has the important influence on neuroendocrine function such ashypothalamic –pituitary -adrenal cortex axis (HPA) and hypothalamic –pituitary-thyroid (HPT) axis. The radioimmunity assay showed that the ACTH got peakvalue at 6 hour after -10℃~-4℃ cold exposure and 24 hour after end cold exposure.The cortisone (Cor) retained in the higher level at more parts time of cold exposure,and obviously high at 12 hour and 24 hour after end cold exposure. The FT3 washigher at 6 hour and 12 hour, and FT4, T3 and T4 did not charged obviously after coldexpose. But FT4, T3, and T4 had the trend of reduced gradually after cold exposureending. This study discovered that animal Cor concentration increase did not dependon HPA axial regulation, but not indicated that the glucocorticoid (GC) effect wasenhanced. That may be concerned with GC receptors decreased, angiotonin andcatecholamine increased, and HSP70 expression enhanced. That signify alsohistiocyte function was damaged at different degree. At -3℃~3℃ cold stress, TheACTH significant increased at 6, 12 hour after cold expose and at 0.5, 6 hour afterend cold exposure. The Cor significant increased at 12 hour after cold exposure andat 0.5 hour, 6 to 24 hour after cold expose ending. FT3, FT4, T3, T4 were effected notnotable. At this time, HPA axis regulates GC secretion and the ACTH and Cor levelsheightened have the time order. From cold environment in to ordinary temperature,equal to exert a new stress stimulus, so ACTH and Cor will go up quickly in a shorttime. Weak cold environment of 4℃~10℃ has not obviously influences to six kindsof hormones. This indicated that "JunMu-1"has certain degree of resistant ability tothe cold. The lower temperature was, the bigger hormone levels changed. TheACTH, Cor and FT3 are sensitive to cold stimulus. They can as the supply indexesevaluating cold stress.This article detected HSP70 express amounts in cold stress piglets histiocyteusing western-blotting method. At -10℃~-4℃ cold environment, the lymphocyteHSP70 express levels increased quickly and retained high level. It went upobviously again at 6 to 24 hour after cold exposure ending. The liver HSP70 levelschanged in a bigger extent and emerged highest value after cold exposure 6 hour, butlater near to the normal level. The HSP70 went up quickly at 0.5 hour after endingcold expose, afterward tend gradually to normal levels through wave up and down.The spleen HSP70 was basic normal in period of cold exposure, but appeared twopeak values at 0.5 and 6 hour after cold expose ending. The change of muscleHSP70 not very large. At -3℃~3℃ cold expose, the lymphocyte HSP70 levelsincreased obviously at 6 hour, and went up again at 12 hour after ending coldexposure. The liver HSP70 levels were higher at 6 and 12 hour, and increased at 0.5hour after cold expose. The changes of HSP70 in spleen and muscle did notsignificant at total. At 4℃~10℃, the histiocyte HSP70 did not obviously changed.The research discovered, the lower temperature, the faster lymphocyte HSP70expression, and the longer HSP70 keep in high levels. The liver was sensitive tocold stress reaction, the lower temperature, the higher HSP70 expressing amounts.The muscle HSP70 did not obviously change in three temperature ranges. TheHSP70 increased again after piglets into the normal temperature. That havingsomething to do with body facilitates subsequence stress. The high expressed ofHSP70 in different tissue cells has the physiological foundation respectively. Theresults demonstrated that it play a part in protective cell function, on the other hand,denoted that histiocyte function was placed in the urgent condition. The lymphocyteHSP70 can be used as the molecular biomarker of animal cold stress.According to the other animal HSP70 gene sequence, primers and probe weredesigned, and quantitative research piglets histiocyte HSP70 mRNA after cold stressusing real-time fluorescence quantitative PCR. The result showed, at -10℃~-4℃cold exposure, that lymphocyte HSP70 mRNA transcription amounts went upquickly at 0.5 hour and kept high levels in 6 hours. It went up again gradually aftercold expose end and achieved the high peak at 12 hour. The liver HSP70 mRNAwent up quickly and significantly in 0.5 hour, and returned to the high levels again at6 and 12 hour through transient decline, hereafter gradually declined to normal level.The HSP70 mRNA in spleen and muscles retained in higher level at the initial stageof cold expose and ending cold expose, and the changed extent was fairly small. At-3℃~3℃ cold expose, lymphocyte HSP70 mRNA increased notable until 12 and24 hour, and the high level can keep on to 3 hour after ending cold exposure. Theliver HSP mRNA also went up quickly at 0.5 hour and retained to 3 hour, and had apeak values at 12 hour. It went up significantly at 3 hour and recovered to normallevel at 6 hour after ending cold exposure. The spleen HSP70 mRNA did not change.The muscle HSP70 mRNA went up significantly at 6 hour after cold expose andincreased at 3 hour after ending cold exposure. At 4℃~10℃ cold expose, theHSP70 mRNA in four kinds of histiocytes did not change at total. The researchdiscovered that lymphocyte HSP70 mRNA was influenced notable by cold stress,and reacted quickly and changed regular during cold expose time, so it can be usedas index of evaluating cold stress degree. The liver HSP70 mRNA respondedquickly to cold stress, its changed range was bigger. The general trend of the HSP70mRNA transcription amounts is similar to that of HSP70 expression amounts duringcold stress.Total mRNA was extracted from lymphocyte of "JunMu-1" piglet and HSP70cDNA was obtained by RT-PCR. By the product was cloned and sequenced, thecDNA complete reading frame of coding HSP70 was obtained. The coding geneincluded 1926 nucleotides. The nucleotides identity was 81%~99%, and amino acidsequence identity was 81%~98% by comparing "JunMu-1" with other animals. Theidentity of "JunMu-1" and Sus scrofa was the highest. The result confirmed that theHSP70 has highly conservatism in different organism. This studies providedmaterials for research the association of HSP70 gene polymorphism and biomomicsof "JunMu-1", and established foundation for further research and application ofHSP70.