| Stress is a nonspecific response to various kinds of stimulates in organism which can cause performance descends, induce various diseases, even result in death in livestock and poultries. The cold is one of the most important factor to restrict livestock farming development owing to the direct and indirect economic loss. Studying’the animal cold stress mechanism and investigating scientific stress monitor method have important meaning to elevate preventative level of stress and ensure livestock farming development. Cold stress has important effect on the HPA and HPT axis of nerve internal system, however, there was no correlated study in young goose. In this study,7d Wanxi White geese were used to study the effects on hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-thyroid (HPT) axis under cold stress (12℃±1℃), using the radioimmunity, fluorescence quantitative RT-PCR etc.Dynamic change rule of blood physiological and biochemical indicators, stress hormone, TRH, TSH, CRH, ACTH, HSP70mRNA by different times during cold exposure0,0.5,3,6,9,12,18,24hour and after terminate cold exposed0,0.5,3,6,9,12,24hour using radioimmunity, fluorescence quantitative PCR. Moreover, HSP70gene was cloned from Wanxi white goose by RT-PCR, the protein structure and function were further predicted by bioinformatics. The studies can provide experimental evidence for investigating reaction mechanism and earlier monitoring of animal cold stress. The main results were showed as following: 1. Analysis of blood routines7d Wanxi White geese were used to study the influence on some parameters of blood under cold stress (12℃±1℃). The results were as follows. The Red blood cell (RBC), Haematocrit (HCT), Hemoglobin (HGB), Red blood cell distribution width (RDW) were decreased collectively. RBC was significant lower at3h,6h and at0.5h,3h after terminating cold stress than pre-cold stress. HCT and HGB were obviously decreased at3h,6h during cold stress and at3h after terminating cold stress. RDW was lower at6h during cold stress and at12h after end cold stress than pre-cold stress significantly (P<0.01). WBC was increased at12h,24h during cold stress. The absolute value of Lymphoid cell (LYM) was lower at6h during cold stress than pre-cold stress significantly (P<0.05). LYM%was increased at3h,6h and12h after cold stress (P<0.01). however, at normal level at24h after cold stress. Collecting detected the hematological results from all test groups during cold stress, obtaining Wanxi white goose blood physiological and biochemical reference values, which can provide important proof for veterinary clinical diagnosis and therapy.2. Analysis of stress hormone in HPA and HPT anxisThe effect on HPA axis and HPT axis was analyzed under cold stress (12℃±1℃) in Wanxi White geese. The results showed as follows.The Free triiodothyronine (FT3) was significantly higher at6h and12h after acute cold stress than that of pre-cold stress. The Free thyroxine (FT4) was obviously higher at3h in12℃±1℃temperature, however, dropped gradually later, decreased at12h (P<0.05). The triiodothyronine (T3) was increased steadily during cold stress and significantly higher at6h after cold exposure and at0.5h,3h after end cold exposure. The Thyroxine (T4) got peak value at6h after cold exposure and6h after end cold exposure. The cortisone (Cor) retained in the higher level at more parts time of cold exposure and obviously higher at3h during cold exposure. The Adrenocorticotrophic hormone (ACTH) significant increased at6h after cold exposure and at3h after end cold exposure (P<0.01, P<0.05). The ACTH, Cor and FT3are sensitive to cold stimulus.They can probably be used as the supply indexes evaluating cold stress.3. Analysis dynamic expression of gene mRNA correlated with hormone in HPA and HPT axisTRH, TSH, CRH and ACTH gene in Wanxi white goose were coloned using RT-PCR. The sequences identity with that of chicken were94%,95%and95%, respectively. Dynamic expression of TRH, TSH, CRH and ACTH mRNA in HPA and HPT axis was analyzed by FQ-RT-PCR. Hypothalamic TRH mRNA transcription level went up significantly at0.5h,3h,6h,9h ad18h during cold stress; It increased again after cold exposure in1h,6h andl2h; It decreased to normal level in the other time. Pituitary TSH mRNA transcription level was increased significantly from1.5h to12h, moreover, it went up the highest level at12h; Hereafter it declined to normal level. It increased again after cold exposure from1h to6h and was decreased to normal level after6h. Hypothalamic CRH mRNA transcription level went up significantly at3h,9h,18h during cold stress and at1h,6h and12h after cold exposure; It was down-regulated to normal level at the other time. Adrenal gland ACTH gene mRNA transcription level went up significantly from1.5h to9h and at18h during cold stress; It increased again after cold exposure at0.5h and3h; It decreased to normal level in the other time.Hypothalamic TRH mRNA transcription level had fluctuation trend and it’s quanity was not over1.5, which indicated that Hypothalamic TRH was the down-regulation gene in cold stress reaction. The transcription quanity of TSH, CRH and ACTH was227-7.76、1.75-6.17、5.5-14.83and had ascend trend, which showed that they were the up-regulation gene in cold stress reaction. The results demonstrated that TRH, TSH, CRH and ACTH gene can be probably used as the molecular biomarker of animal cold stress.4. HSP70gene cloning and bioinformaticsHSP70gene cDNA was cloned, whose size was2181bp in Wanxi white goose for the first time. It’s1905bp of CDS and276bp of3’-UTR sequence, and encoded634amino acids. There were15nucleotides (in CDS) and only one amino acids difference, which were different between2populations. HSP70CDS sequence in goose had82-90%homology with mammals and fish, and up to99%with Chinese goose. Cluster analysis revealed that probably mammals, birds and fishes, each of these belonged to different categories separately.Bioinformatics analysis indicated that goose HSP70protein contained34phosphorylation sites. The diversity of CDS and the structure of amino acids in HSP70genes resulted in the differences of protein secondary structure. HSP70protein secondary structure had more Alpha and Coil region, without signal peptide and transmembrane helix; The hydrophobicity structure prediction of goose HSP70protein indicated that it had major hydrophobicity region. The protein had abundant B cell antigen location. There were more overlapping region in flexibility, surfaceprobability and antigencity, which made antigen and antibody combine easily, and it was probably antigen enrichment region. It was predicted that HSP70protein probably played an important role in cell signal transfer and control mechanisms.5. Analysis dynamic expression of HSP70mRNA in HPA and HPT axis during cold stressThe dynamic expression of HSP70mRNA in HPA and HPT axis of7d Wanxi White goose was got utilizing FQ-RT-PCR. Hypothalamic HSP70mRNA transcription level went up significantly atl.5h,6h and9h; It decreased to normal level in the other time; It increased again after cold exposed in6hours, moreover, it went up the highest level at6h;Hereafter it declined to normal level. Pituitary HSP70mRNA transcription level descended significantly at0.5h,1.5h,6h and12h. It went up again gradually to normal level in the next12hours. It decreased again significantly at0.5h,3h,6h after cold exposure, hereafter, gradually increased to normal level. Thyroid HSP70mRNA transcription level went up quickly at0.5h, hereafter decreased at3h,6h,12h significantly under cold stress. It went up significantly at0.5h and3h after cold exposure and it was down-regulated gradually to normal level in the other time. Adrenal gland HSP70mRNA transcription level was decreased at1.5h,6h,9h during cold stress and at3h,6h after end cold exposure. It was up-regulated gradually to normal level in the other time. The Adrenal gland HSP70mRNA transcription quanity was0.02-0.57, which was predicted that it was down-regulation gene in cold stress reaction.The expression rule of HSP70mRNA was different in Hypothalamic, Pituitary, Thyroid and Adrenal gland. Hypothalamic HSP70mRNA transcription level was the highest which showed going up trend as a whole. HSP70was the up-regulation gene of Hypothalamic in cold stress reaction. Pituitary, thyroid and adrenal gland HSP70mRNA transcription level was repressed, which showed that it was the down-regulation gene of pituitary, thyroid and adrenal gland in cold stress reaction. The results 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 lymphocyte HSP70can probably be used as the molecular biomarker of animal cold stress. |
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