| Cold stress can affect the health and welfare of animals by changing serum biochemical indices and glycolipid metabolism levels.The previous results of our research group confirmed that broilers can establish cold adaptation and improve immune function after a period of cold training.Substance metabolism and immune response are highly unified,so it is more practical to evaluate cold adaptation mechanism from the aspects of immunity and substance metabolism.In order to explore the molecular mechanism of substance metabolism in the establishment of cold adaptation model of broilers,288 broilers were randomly divided into 3 groups(CC,C3 and C9)with 6 replicates per group(n=6).1-14 days,they were raised at conventional temperature.From15-36 days,the CC group continued to receive conventional feeding,while the C3 and C9 groups,as cold stimulation groups,were treated at 3℃ and 9℃ below the temperature of the CC group for5 h at one day intervals,respectively.37-43 days,the temperature of all groups was maintained at20℃.44 d,broilers in all groups were treated with acute cold stimulation at 10℃ for 12h(Y12)and 24h(Y24),respectively.Blood,hypothalamus and heart tissue samples of broilers were collected at 22 d,29d,36 d,43d,Y12 and Y24.Transcriptome analysis was performed on hypothalamic tissue samples.The contents of serum biochemical indices(E,NE,INS,ADPN,LEP,TG,and FFA),the m RNA and protein levels of m TOR pathway(PI3K,Akt,m TOR,SREBP-1c,FASN,ACC,and SCD),FOXO1 pathway(FOXO1,PEPCK,G6 P,GLUT1,HK1,PFK,and LDHB),PPARα pathway(PPARα,CPT1,ACO,ACSL1,and PGC-1α)and heat shock proteins(HSP27,HSP40,HSP60,HSP70,and HSP90)in the heart were detected.The experimental results were as follows:1.Transcriptome results: 100 DEGs were obtained by transcriptome analysis of hypothalamus in CC and C3 groups,including 52 up-regulated genes and 48 down-regulated genes.GO items with high enriched DEGs include: response to external stimulus and cellular response to organonitrogen compensation.KEGG pathways with high enriched DEGs include: Adrenergic signaling pathways in cardiomyocytes,PPARα signaling pathway,FOXO signaling pathway.The results of RT-q PCR and transcriptome of DEGs were consistent.Among them,cardiomyocyte adrenergic signaling pathway activates the hypothalamic-sympathetic adrenal medulla system by participating in cold stimulation,which secretes a large amount of E and NE and then acts on adrenergic receptors of cardiomyocytes,further affecting the substance metabolism of cardiomyocytes.2.Blood biochemical indices: At 22 days of age,compared with CC group,the contents of ADPN,E,and NE in C3 group were significantly increased,while TG content was significantly decreased(P<0.05).The contents of FFA,NE,and ADPN in C9 group were significantly increased,while the contents of TG,INS,LEP,and E were significantly decreased compared to CC group(P<0.05).At 36 day,the contents of INS and ADPN in C3 group and TG,INS and LEP in C9 group were significantly lower than those in CC group,while the contents of ADPN,E and NE in C9 group were significantly increased compared with CC group(P<0.05).At 43 day,the contents of TG and E in C3 group and TG,FFA,INS,E,and NE in C9 group were increased significantly(P<0.05).After 12 h of acute cold stress,compared with CC group,the contents of TG,FFA,INS,NE,LEP in C3 were significantly increased(P<0.05).3.m TOR signaling pathway: At 22 day,compared with CC group,the m RNA levels of Akt and SREBP-1c in C3 group and PI3 K,Akt,m TOR,SREBP-1c,FASN,ACC and SCD in C9 group were significantly decreased,while the SCD m RNA level in C3 group was significantly increased(P<0.05).At 36 days of age,compared with CC group,the m RNA expressions of PI3 K,SCD,Akt,m TOR,SREBP-1c,ACC and FASN in C9 group and m TOR in C3 group were significantly decreased(P<0.05).At 43 days of age,the levels of PI3 K,Akt,m TOR,ACC,SCD in C3 group and m TOR,SREBP-1c,Akt,FASN,ACC,PI3 K and SCD in C9 group were significantly higher than those in CC group(P<0.05).The m RNA levels of PI3 K,Akt,FASN,m TOR,SREBP-1c,and ACC in CC group were significantly increased after Y24(P<0.05).After Y12,the levels of SREBP-1c and ACC in C3 group and PI3 K,ACC,Akt,FASN,m TOR,SREBP-1c,and SCD in C9 group were significantly higher than those before acute cold stress(P<0.05).4.PPARα signaling pathway: At 22 days of age,compared with CC group,the m RNA levels of PPARα,CPT1,ACO and ACSL1 and protein levels of PPARα and PGC-1α in C3 and C9 groups were significantly increased,while the PGC-1α m RNA level in C9 group was significantly decreased(P<0.05).At 36 days of age,compared with CC group,the m RNA expressions of PGC-1α in C3 and PPARα and PGC-1α in C9 group were significantly increased,while the m RNA expressions of ACO and ACSL1 in C9 group were significantly decreased(P<0.05).At 43 days of age,the m RNA and protein levels of PPARα,PGC-1α protein level in C3 group and m RNA and protein levels of PPARα,PGC-1α in C9 group were significantly higher than those in CC group(P<0.05).Compared with before acute cold stimulation,the m RNA levels of PPARα,PGC-1α,CPT1 and ACO in C9 group,CPT1 in C3 group and ACSL1 in CC group were significantly increased after Y12(P<0.05).5.FOXO1 signaling pathway: At the 22 days of age,compared with CC group,the m RNA levels of FOXO1,PEPCK,G6 P,PFK and LDHB in C3 and C9 groups were significantly increased,while GLUT1 expression level in C9 group was significantly decreased(P<0.05).At 36 days of age,compared with CC group,the levels of PEPCK and HK1 in C9 group and HK1 in C3 group were significantly decreased,while the m RNA levels of FOXO1,PFK and LDHB in C9 group were significantly increased(P<0.05).At 43 days of age,the m RNA expressions of FOXO1,G6 P and PFK in C3 group and FOXO1,PEPCK and G6 P in C9 group were significantly higher than those in CC group(P<0.05).Compared with before acute cold stress,m RNA levels of GLUT1 in CC group,PFK in C3 group and GLUT1,HK1,PFK and LDHB in C9 group were significantly increased after Y12,while m RNA levels of FOXO1,PEPCK and G6 P in CC group,G6 P in C3 group and PEPCK,G6 P in C9 group were significantly decreased(P<0.05).6.The expression levels of HSPs: At 43 days of age,the m RNA and protein levels of HSP27 and HSP60 in cold stimulation group were significantly increased compared to CC group.Compared with C9 group,the m RNA and protein expressions of HSP60 and HSP90 in C3 group were significantly increased(P<0.05).HSP40 protein and m RNA levels in C9 group were significantly higher than those in CC group(P<0.05).After Y12,compared with CC and C9 group,the levels of HSP40 and HSP90 m RNA in C3 group were significantly increased(P<0.05).After Y24,the m RNA and proteins levels of HSP70 and HSP90 in C3 and C9 groups were significantly increased compared with CC group(P<0.05).HSP27 and HSP60 m RNA and protein levels in C9 group were significantly higher than those in CC group(P<0.05).7.Pathological section observation of myocardial tissue: At 36 d,a few inflammatory cells were produced in C3 group compared to CC group.In C9 group,the size of nucleus was uneven,inflammatory cells infiltrated severely,the myocardial fibers were disordered.At 43 d,myocardial cells and the morphology structure of fibers in C3 group were basically restored to normal,and the cells were orderly arranged.After acute cold stimulation,compared with CC and C9 groups,the structure of C3 group was slightly damaged,and the myocardial fibers were in order.However,inflammatory cell infiltration was severe and myocardial fiber was broken significantly in CC and C9 groups.Conclusions:(1)Cold training of 3℃ lower than normal temperature can adjust the contents of lipid metabolism related indices by hypothalamus in serum of broilers to adapt to low temperature environment.(2)Cold training of 3℃ lower than normal temperature can improve the levels of glucose and lipid metabolism by activating FOXO1 and PPARα signaling pathways,and reduce lipid synthesis by inhibiting m TOR signaling pathway in cold-stress broilers,so as to maintain the energy balance in vivo to adapt to cold environment.(3)The cold training method of an interval of 1 day at 3℃ lower than normal temperature in early stage can improve the levels of HSPs to alleviate the injury of myocardial tissue caused by late acute cold stress at later period,and improve cold stress resistance of broilers. |
