| Appropriate low temperature treatment can enhance the immune function and disease resistance of the animals.The purpose of this study was to explore the mechanism of cold acclimation promoted by appropriate cold stimulation and cold tolerance range in broilers,in order to enhance the immunity and resistance to acute cold stress in broilers.A total of 600 1-d old healthy Arbor Acres female broiler chicks were randomly divided into 5 equal groups,and were housed in 5 climatic chambers,respectively.Each group had 4 replicates of 30 birds.The birds in the control group were managed under normal room temperature scheme from 1 to 42 d,namely 34? for 1 to 3 d,33? for 4 to 7 d,then reduced gradually by 1? every 2 d till 20? at 32 d and maintained at 20? until 42 d(07:00 h).As the cold stimulation groups,air temperature in the other four groups was the same as in the control group for the first 7 d of age(1 to 7 d),and cold stimulation started at 07:00 h at 8 d.The cold stimulation condition in the other four groups was,3?,6?,9? and 12? lower than that of the control,respectively.Therefore,the temperature in the other four groups was reduced to 17? at 07:00 h at 32 d,26 d,20 d and 14 d,respectively,and maintained at 17? until 07:00 h at 42 d.Commencing at 07:00 h on 42 d,chicks in all the groups were subjected to acute cold stress(ACS)of 7? for 24 h.Part 1: Screening of cold stimulation modelsThe production performance indexes(average daily feed intake,average daily weight gain,feed conversion ratio,body weight of 42 d and the total livability rate),behavior indexes(state behaviors such as feeding,lying,walking and standing;event behaviors such as drinking,stretching and comfort behaviors),and welfare indexes(fluctuating asymmetry of leg and wing)of broilers in each group were compared.The following results drawn from part 1:(1)Compared with the control group,the cold stimulation environment of 9? and 12? lower than the normal temperature significantly reduced the average daily weight gain,body weight of 42 d and total livability rate of broilers(P<0.05),and significantly increased the average daily feed intake and feed conversion ratio(P<0.05).While the cold stimulation environment of 3? lower than the normal temperature had no significant effects on average daily feed intake,average daily weight gain,feed conversion ratio and body weight of 42 d(P>0.05),and significantly increased the livability rate of broilers(P<0.05).(2)During the entire behavioral observation cycle(2-6 weeks),compared with the control group,cold stimulation environment of lower 9? and lower 12? significantly increased the feeding,drinking,standing and walking behaviors(P<0.05),and significantly reduced lying,stretching and comfort behaviors(P<0.05).Cold stimulation environment of lower 6? significantly increased feeding,standing and drinking behaviors(P<0.05),and significantly reduced lying,stretching and comfort behaviors(P<0.05).Cold stimulation environment of lower 3? had little effects on feeding,lying,walking,drinking and comfort behaviors of broilers(P>0.05),and significantly increased standing behavior(P<0.05),and significantly reduced stretching behavior(P<0.05).(3)After a 34-day cold stimulation,compared with the control group,cold stimulation environment of lower 12?,lower 9? and lower 6? increased the fluctuating asymmetry of leg(P>0.05).However,cold stimulation environment of lower 3? significantly reduced the fluctuating asymmetry of leg(P<0.05).Cold stimulation temperature had no significant effect on fluctuating asymmetry of wing in broilers(P>0.05).The conclusion was as follows: With the decreasing of cold stimulation temperatures,the degree of cold stress response was gradually increased,while cold stimulation condition of lower 3? could promote the establishment of cold acclimation in broiler chickens.Through the analysis of production performance,behavior and welfare indexes in broilers of the five groups,we found that cold stimulation of 12? lower than the normal temperature led to cold stress of broilers,accompanied by markedly reduced production performance and livability rate,as well as significantly changed behavior pattern.However,cold stimulation of 3? lower than the normal temperature not only had no adverse effects on production performance and main behaviors of the broilers,but also significantly improved the livability rate and welfare.Therefore,we next mainly studied the group managed under normal room temperature(the control group),the group managed in 3? lower than the normal temperature(the selected suitable cold stimulation group,hereinafter referred to as lower 3? group)and the group managed in 12? lower than the normal temperature(cold stress group,the negative control group,hereinafter referred to as lower 12? group).We investigated the changes in physiological indexes,antioxidant function,immune function,and the resistance to cold stress in broilers of the three groups under long-term cold stimulation conditions.Part 2: Effects of long-term cold stimulation on physiological indexes,antioxidant function,immune function and the resistance to cold stress in broiler chickens.Serum of the birds were collected at 14 d,21 d,28 d,35 d,42 d and 43 d to detect the levels of immunoglobulins(Ig A,Ig M,Ig G),cytokines(IL-2,IL-4,IFN-?,TNF-?)and acute phase proteins(AGP,CP,OVT).Trachea tissues,ileum tissues and thymus tissues of each group were collected at 14 d,21 d,28 d,35 d,42 d and 43 d to detect the levels of oxidation(MDA,i NOS,NO,H2O2)and antioxidant(SOD,CAT,GSH-Px)indexes,and the m RNA expression levels of immune-related genes(Ig A,Ig M,Ig G,Av BD1,Av BD6,Av BD7,Av BD9,Av BD10,Av BD12),cytokines(IL-4,IFN-?,TNF-?),heat shock proteins(HSP27,HSP40,HSP60,HSP70,HSP90)and pro-inflammatory factors(NF-?B,i NOS,COX-2,PTGEs).Trachea tissues,ileum tissues and thymus tissues of each group were collected at 42 d and 43 d to detect the protein expression levels of heat shock proteins(HSP60,HSP70,HSP90),microstructure and ultrastructure.The results were as follows:(1)After a 34-day cold stimulation,compared with the control group,the levels of immunoglobulins(Ig A,Ig M,Ig G)reduced(P<0.05),and the levels of acute phase proteins(AGP,CP)and inflammatory cytokines(IL-4,TNF-?)increased(P<0.05)in serum of lower 12? group.However,the levels of immunoglobulins(Ig A,Ig M,Ig G)increased(P<0.05),and the levels of acute phase proteins(AGP,CP,OVT)and inflammatory cytokines(IL-4,TNF-?)in serum of lower 3? group had no significant difference(P>0.05).(2)After a 34-day cold stimulation,compared with the control group,the activities of antioxidant enzymes(SOD and GSH-Px in trachea and ileum;SOD,CAT and GSH-Px in thymus)decreased(P<0.05),and the levels of free radicals(i NOS,MDA and H2O2 in trachea;i NOS,MDA,NO and H2O2 in ileum;MDA,NO and H2O2 in thymus)increased in broilers of lower 12? group(P<0.05).However,the activities of antioxidant enzymes(SOD,CAT and GSH-Px in trachea;GSHPx in ileum;SOD and GSH-Px in thymus)increased(P<0.05),and the levels of free radicals did not increase in broilers of lower 3? group(P>0.05).(3)After a 34-day cold stimulation,compared with the control group,the m RNA expression levels of immunoglobulins(Ig A and Ig G in trachea;Ig A,Ig M and Ig G in ileum and thymus)and antimicrobial peptides(Av BD1,Av BD6,Av BD7,Av BD9 and Av BD10 in trachea and ileum;Av BD1,Av BD6,Av BD7,Av BD10 and Av BD12 in thymus)decreased in broilers of lower 12? group(P<0.05).However,the m RNA expression levels of immunoglobulins(Ig A,Ig M and Ig G in trachea,ileum and thymus)and antimicrobial peptides(Av BD1,Av BD6,Av BD7,Av BD9,Av BD10 and Av BD12 in trachea and ileum;Av BD6,Av BD7,Av BD9,Av BD10 and Av BD12 in thymus)increased in broilers of lower 3? group(P<0.05).(4)After a 34-day cold stimulation,compared with the control group,the m RNA expression levels of cytokines(IL-4 and TNF-? in trachea,ileum and thymus)and pro-inflammatory factors(NF-?B,i NOS,COX-2 and PTGEs in trachea,ileum and thymus)increased in broilers of lower 12? group(P<0.05).However,the m RNA expression levels of inflammatory cytokines(IL-4 and TNF-? in trachea,ileum and thymus)and pro-inflammatory factors(NF-?B,i NOS,COX-2 and PTGEs in trachea,ileum and thymus)did not increase in broilers of lower 3? group(P>0.05).(5)After a 34-day cold stimulation,compared with the control group,the m RNA expression levels of heat shock proteins(HSP27,HSP40,HSP60,HSP70 and HSP90 in trachea,ileum and thymus)and protein expression levels of heat shock proteins(HSP60,HSP70 and HSP90 in trachea,ileum and thymus)increased in broilers of lower 12? group(P<0.05).However,the m RNA expression levels of heat shock proteins(HSP27,HSP40,HSP60,HSP70 and HSP90 in trachea,ileum and thymus)and protein expression levels of heat shock proteins(HSP60,HSP70 and HSP90 in trachea;HSP60 in ileum;HSP70 and HSP90 in thymus)did not increase in broilers of lower 3? group(P>0.05).(6)After a 34-day cold stimulation,trachea,ileum and thymus tissues of the broilers in lower 12? group were severely damaged,with destroyed cell structure,and even apoptosis.However,trachea,ileum and thymus tissues and cell structure of the broilers in lower 3? group were normal,and no tissue injuries were observed.(7)After the acute cold stress,the levels of immunoglobulins(Ig A,Ig M,Ig G)and immunerelated cytokines(IL-2,IFN-?)in serum of lower 3? group were higher than those of control group and lower 12? group(P<0.05).While the levels of cytokines(IL-4,TNF-?)and CP in serum of lower 3? group were lower than those of control group and lower 12? group(P<0.05).(8)After the acute cold stress,the activities of antioxidant enzymes(SOD in trachea and thymus,CAT and GSH-Px in all the tissues)in lower 3? group were higher than those of control group and lower 12? group(P<0.05).While the levels of free radicals(MDA in trachea and thymus,NO in ileum and thymus,H2O2 in ileum)in lower 3? group were lower than those of control group and lower 12? group(P<0.05).(9)After the acute cold stress,the m RNA expression levels of immunoglobulins(Ig A,Ig M,Ig G),antimicrobial peptides(Av BD1,Av BD6,Av BD7,Av BD9,Av BD10,Av BD12)and IFN-? in trachea and thymus of lower 3? group were higher than those of control group and lower 12? group(P<0.05).(10)After the acute cold stress,the m RNA expression levels of cytokines(IL-4 in ileum and thymus,TNF-? in all the tissues),and pro-inflammatory factors(NF-?B in ileum,i NOS in trachea and ileum,COX-2 and PTGEs in ileum and thymus)in lower 3? group were lower than those of control group and lower 12? group(P<0.05).(11)After the acute cold stress,the m RNA expression levels of heat shock proteins(HSP27,HSP40,HSP90)in all the tissues,HSP60 in trachea and thymus,and HSP70 in trachea and ileum in lower 3? group were lower than those of control group and lower 12? group(P<0.05).The protein expression levels of heat shock proteins(HSP60,HSP70 and HSP90 in trachea,HSP60 and HSP90 in ileum and thymus)in lower 3? group were lower than those of control group and lower 12? group(P<0.05).(12)After the acute cold stress,trachea,ileum and thymus tissues of the broilers in control group and lower 12? group were severely damaged,with destroyed cell structure,and even apoptosis.However,the tissue structure and nucleus morphology of trachea,ileum and thymus of broilers in lower 3? group were relatively intact and normal,and mitochondrial cristae was slightly reduced in trachea and thymus.The following conclusions were obtained:(1)The cold stimulation environment of 3? lower than normal temperature improved immune function,antioxidant capacity of the trachea,ileum and thymus tissues as well as the resistance to acute cold stress of the birds to a certain extent.However,cold stimulation environment of 12? lower than the normal temperature led to cold stress of the broilers,accompanied by oxidative stress and inflammatory response in trachea,ileum and thymus tissues,which reduced immune function of broilers.(2)Cold acclimation alleviated oxidative stress and the high levels of heat shock proteins as well as pro-inflammatory factors caused by cold stress to a certain extent,which may be a protective mechanism of cold acclimation.In conclusion,cold stimulation of 3? lower than normal temperature for 34 days had no adverse effects on production performance and behaviors of the broilers from day 8 to day 42,and improved the livability rate.Cold stimulation of 3? lower than the normal temperature enhanced the antioxidant capacity and immune function as well as the resistance to acute cold stress of the birds to a certain extent.It provided theoretical and technical support for the suitable temperature of broiler rearing in the cold region of northeast in China. |
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