| In alpine region,cold is one of the important factors restricting the development of livestock and poultry industry,which has adverse effects on animal growth performance,antioxidant system,immune system and neuroendocrine system.To alleviate the adverse effects of cold stress on broilers,we used moderate stimulation to train the broilers with the immune function as the screening index.The purpose of this study was to find the best cold stimulation method to improve the immune function and the resistant to cold stress of broilers.In this study,a total of 400 healthy one-day-old broilers were divided into one control group(CC)and four cold stimulation(CS)groups.The CC group was raised at a normal temperature,while the CS groups were raised at 3 °C below the temperature of the CC for 3,4,5,or 6 hour periods at 1 day intervals from 15 to 35 days of age(D35),denoted CS3,CS4,CS5,and CS6,respectively.Subsequently,they were raised at 20 °C from 36 to 49 days of age(D49).The growth performance,the immune organ growth index,immunoglobulins and cytokines content in serum and immune related genes(Toll-like receptors,?-defensins cytokines and immunoglobulins)m RNA expression in bursa,spleen and thymus of broilers were measured during cold stimulation.The heat shock proteins(HSPs)m RNA expression in bursa,spleen and thymus of broilers was measured before and after 6h and 12 h acute cold stress.The results are as follows:1.Growth performance: Intermittent cold stimulation had no significant effect on the average daily feed intake of broilers(P > 0.05),and there were no significant difference among all groups(P < 0.05).The average daliy weight gain of CS3 group was significantly higher than that of the control group(P < 0.05),CS4 and CS6 group(P < 0.05),and there was no significant difference between CS3 and CS5 group(P > 0.05).The average daliy weight gain of CS5 group was significantly higher than that of the control group(P < 0.05),and there was no significant difference between CS4 and CS6 group(P > 0.05).The feed conversion ratio of CS3 group was significantly lower than that of control group and other cold stimulation grou ps(P < 0.05).There was no significant difference in feed conversion ratio among CS4,CS5,CS6 groups and the control group(P > 0.05).2.The immune organ growth index: At the 22 nd,29th,36 th and 43 rd day,there was no significant difference in bursa growth index and spleen growth index in the cold stimulation groups and the control group(P > 0.05),and there was no significant difference among the cold stimulation groups(P > 0.05).The spleen growth index of CS5 group was significantly higher than that of CS3 group(P < 0.05).There was no significant difference among control group(P > 0.05),CS4,CS6 and CS3 group(P > 0.05).There was no significant difference among control group,CS4,CS6 and CS5 group(P > 0.05).3.The immunoglobulins and cytokines content in serum: After 21-day intermittent cold stimulation(i.e.,the 36 th day),the contents of Ig A and Ig G in serum of broilers were significantly higher than those in the control group(P < 0.05).The content of Ig G in serum of CS5 group was significantly higher than that of CS3 group(P < 0.05).The content of Ig G in CS4 group,CS5 group and CS6 group had no significant difference(P > 0.05).The content of Ig G in CS3 group,CS5 group and CS6 group had no significant difference(P > 0.05).There was no significant difference in serum Ig A in broilers among the cold stimulation groups(P > 0.05).The serum IL-2 content of cold stimulation groups was significantly higher than that of the control grou p(P < 0.05).There was no significant difference on serum IL-2 in cold stimulation group broilers(P > 0.05).There was no significant difference on serum IFN-? of broilers in the cold stimulation groups and the control group(P > 0.05).The serum IFN-? in CS4 group was significantly higher than that in CS6 group(P < 0.05).There was no significant difference in the other groups(P > 0.05).14 days after the end of cold stimulation(i.e.,49 th day),serum Ig A levels in the CS5 group were significantly higher than those in the control group and other cold stimulation groups(P > 0.05).There was no significant difference in Ig A content in other groups without CS5 group.The content of serum Ig G in CS4 group was significantly higher than that in contr ol group(P < 0.05).The content of serum Ig G in CS3,CS6,CS4 and CS5 group had no significant difference(P > 0.05).The content of serum Ig G in CS5 group was significantly higher than that in CS6 group(P < 0.05).There was no significant difference among CS5 group,CS3 group and control group(P > 0.05)and there was no significant difference among CS6 group,CS3 group and control group(P > 0.05).4.The m RNA expression levels of immune related genes:1)The m RNA expression levels of Toll-like receptors(TLRs): After 21 TLRday intermittent cold stimulation,the expression levels of TLRs in the bursa of broilers were significantly decreased in the cold stimulation groups(P < 0.05).The m RNA expression levels of TLR1,TLR5,TLR15 and TLR21 in the CS5 group were significantly lower than that in the CS3 group(P < 0.05),and the expression levels of TLR3,TLR4 and TLR7 in CS5 group were all lower than that in CS4 group(P < 0.05).Meanwhile,the expression levels of TLR1,TLR4 and TLR15 in bursa of CS6 group were all higher than that in CS5 group(P < 0.05).As for spleen,the expression levels of TLR4,TLR5,TLR15 and TLR21 in spleen of the cold stimulation broilers was significantly higher than that in control group(P < 0.05),and TLR4,TLR 5 and TLR 21 expression levels in CS5 group were significantly higher than that in other three cold stimulation groups(P < 0.05).14 days after the end of cold stimulation,the expression levels of TLRs in bursa in CS5 and CS6 groups were significantly lower than that in the control group(P < 0.05),except for TLR21,the other TLRs in CS5 group were significantly lower than those in CS3 group(P < 0.05);in addition,the expression levels of TLR1,TLR2,TLR3,TLR5 and TLR21 in CS5 were also significantly lower than that in CS4 group(P < 0.05).Meanwhile,the expression levels of TLR2,TLR5,TLR7 and TLR15 in CS5 were significantly lower than those in CS6.The expression levels of TLR3,TLR4,TLR7 and TLR15 in spleen of broilers in CS4,CS5 and CS6 groups were significantly lower than that in the control group(P < 0.05),and the expression levels of TLR7 and TLR15 in CS5 group were significantly lower than those in the other three cold stimulation groups(P < 0.05).As for thymus,the expression levels of TLR1,TLR2,TLR3,TLR5,TLR 7 and TLR15 in CS5 and CS6 group were significantly lower than that in the control group and CS4 group(P < 0.05),and the expression levels of TLR2,TLR3,TLR4 and TLR15 in CS6 group were higher than that of CS5 group(P < 0.05).2)The m RNA expression levels of avian ?-defensins(Av BD): After 21 Av BDday intermittent cold stimulation,the expression of Av BD8 in bursa of CS4 and CS6 groups decreased significantly(P < 0.05),but there was no significant difference on other Av BDs m RNA expression levels(P > 0.05).The expression levels of Av BD2 and Av BD8 in CS5 group were significantly lower than that in control group and CS6 group(P < 0.05).As for the spleen,the expression levels of Av BDs in CS3 group increased(P < 0.05),while that in CS4 and CS6 groups decreased(P < 0.05).The expression level of Av BD2 in CS5 group was significantly lower than that in the control group(P < 0.05).The expression levels of Av BDs in thymus of broilers in cold stimulation groups w ere generally lower than that in control group(P < 0.05),and the expression levels of Av BD4,Av BD5 and Av BD7 in CS3 and CS4 groups were decreased(P < 0.05),while the expression levels of Av BD5,Av BD7,Av BD8 and Av BD10 in CS5 and CS6 groups were significantly lower than that in control group(P < 0.05)and the expression level of Av BD8 in CS5 and CS6 groups was significantly lower than that in other cold stimulation groups(P < 0.05).14 days after the end of cold stimulation,the expression levels of ?Av BDdefensins in bursa of broilers were generally downregulated(P < 0.05),and the expression levels of Av BD2,Av BD4 and Av BD7 in bursa of Fabricius of CS5 group was significantly lower than that of other cold stimulation groups(P < 0.05).In terms of spleen,the expression levels of Av BD4 and Av BD7 in spleen of broilers in CS5 group and the expression level of Av BD5 in CS6 were significantly lower than that in control group(P < 0.05),and the expression levels of Av BD4 and Av BD7 in CS5 group were significantly lower than that in CS6 group(P < 0.05).The expression level of Av BD7 in thymus of broilers in cold stimulation groups were significantly decreased(P < 0.05).In addition,the expression of Av BD2 and Av BD10 in CS5 and CS6 groups were significantly lower than those in CS3 and CS4 groups(P < 0.05).3)The m RNA expression levels of cytokines: After 21-day intermittent cold stimulation,the expression level of interleukin(IL)-2 in spleen and thymus of broilers were significantly higher than that of the control group(P < 0.05),and the expression levels of IL-6 and interferon(IFN)-? were also significantly increased in spleen and thymus of CS5 group(P < 0.05).In addition,the expression levels of IL-2 and IL-8 in the spleen of CS5 group were significantly higher than those of CS3 and CS4 group(P < 0.05).The expression levels of IL-2,IL-8 and IL-17 in thymus were significantly higher than those of the other three cold stimulation groups(P < 0.05),and the expression levels of IL-17 in the thymus of the CS5 and CS6 groups were also increased(P < 0.05).14 days after the end of cold stimulation,the expression levels of IL-2 in spleen of broilers in the cold stimulation groups were significantly higher than that in the control group(P < 0.05),and IL-2 was significantly higher in the CS5 and CS6 groups than in the CS3 and CS4 groups(P < 0.05).As for thymus,the expression levels of IL-2,IL-17 and IFN-? in thymus of broilers in the CS4,CS5 and CS6 groups were significantly increased(P < 0.05)without CS3 group,and the expression levels of IL-6 in the CS5 and CS6 groups were significantly higher than the control group,CS3 and CS4 groups(P < 0.05).4)The m RNA expression levels of immunoglobulins: After 21-day intermittent cold stimulation,the expression levels of Ig A and Ig G in bursa of CS4 and CS5 groups were significantly higher than that of CS3 and CS6 groups(P < 0.05).The expression levels of Ig A in thymus of CS5 and CS6 groups were significantly higher than that of CS3 and CS4 groups(P < 0.05).There was no difference in the Ig A expression levels between CS5 and CS6 groups(P < 0.05),but the expression levels of Ig G in CS5 group were significantly higher than that in CS6 group.The expression levels of Ig G in spleen was significantly higher than that in control group(P < 0.05).There was no difference in Ig G expression levels between CS6 and CS5 groups(P > 0.05),but it was significantly higher than CS3 and CS4 group(P < 0.05).14 days after the end of cold stimulation,the expression levels of Ig G in bursa of broilers in CS5 and CS6 group were significantly higher than that in CS3 and CS4 group(P < 0.05).The expression levels of Ig A in spleen and thymus of CS5 group was significantly higher than that of control group and other cold stimulation groups(P < 0.05).5.The m RNA expression levels of heat shock proteins(HSPs)before and after 6h and 12 h cold stress:After cold stress for 6 hours,the expression levels of HSP40 and HSP70 in bursa of broilers were increased significantly in cold stimulation groups and control group(P < 0.05).The expression levels of HSP27,HSP40 and HSP60 in spleen of broilers in CS4,CS5 and CS6 groups were significantly increased(P < 0.05).However,the expression level of HSP27 in the spleen of broilers in the control group was significantly increased(P < 0.05),while there was no significant difference on HSP40 and HSP60(P > 0.05).The expression levels of HSP40,HSP60 and HSP70 in thymus of broilers in CS3 and CS4 groups and HSP60,HSP70 and HSP90 in CS5 and CS6 groups were significantly increased(P < 0.05).In the control group,the expression level of HSP60 was increased significantly(P < 0.05),while the expression levels of HSP70 and HSP90 were decreased significantly(P < 0.05).After cold stress for 12 hours,the expression levels of HSP40,HSP70 and HSP90 in bursa of broilers were increased in cold stimulation groups and control group(P < 0.05).The expression levels of HSP40,HSP60,HSP70 and HSP90 in spleen of broilers in cold stimulation group were increased(P < 0.05).In the control group,the expression levels of HSP40 and HSP60 were increased(P < 0.05),while HSP27 and HSP70 were decreased(P < 0.05).The expression levels of HSP60,HSP70 and HSP90 in thymus of broilers in CS4,CS5 and CS6 groups were increased(P < 0.05).In the control group,HSP70 was increased(P < 0.05),while HSP60 and HSP90 were decreased significantly(P < 0.05).Compared with 6 hours of cold stress,after 12 hours of cold stress,the expression levels of HSP40,HSP70 and HSP90 in bursa of Fabricius in cold stimulation group and control group were increased(P < 0.05).The expression levels of HSP40,HSP60,HSP70 and HSP90 in spleen of CS3,CS5 and CS6 groups were all increased(P < 0.05),and the expression levels of HSP40 and HSP90 in spleen of CS4 group and control group were increased significantly(P < 0.05).The expression levels of HSP40 and HSP70 in CS5 and CS6 groups and HSP27 in thymus of CS3 and CS4 groups were significantly increased(P < 0.05),but there was no significant difference in the expression of HSP27 and HSP40 in the control group(P < 0.05).The expression levels of HSP60 in cold stimulation groups and control group were decreased significantly(P < 0.05).To sum up all results above,the following conclusions are drawn from this study:(1)Intermittent cold stimulation had effect on growth performance of broilers.The average daliy weight gain of CS3 and CS5 groups increased,while the feed conversion ratio of CS3 group decreased.(2)Intermittent cold stimulation had no significant effect on immune organ growth index of broilers.The CS5 group showed a tendency to enhance bursa organ growth index and spleen organ growth index of broilers.(3)The CS5 group had certain enhancement effect on immune function of broilers.(4)Intermittent cold stimulation can alleviate the damage caused by cold stress to broilers to a certain extent,and the CS5 group had better cold resistance. |
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