| The most important stressor for animals living in northern regions is cold stress.Many studies have found that cold stress affected the health and welfare of animals,thus restricting the development of livestock industry.Therefore,in this experiment,different intermittent cold stimulation schemes were used for cold adaptation training of broilers,with glucose metabolism,inflammatory response and cold resistance as the main contents,aiming at finding the best cold training methods to improve the resistance to low temperature environment of broilers,so as to reduce the adverse effects of cold stress on livestock and poultry.In this study,288 similar weight and healthy AA male broilers selected from 300 1-day-old broilers were randomly divided into 1control group(CC group)and 2 cold stimulation groups(C3 group and C9 group)at 15 days of age,with 6 replicates in each group and 16 broilers in each replicate.The CC group was raised at a normal thermal temperature,and the C3 and C9 groups were raised at 3℃ and 9℃,respectively,below the temperature of the CC group for 5 h every other day from 15 to 35 days of age.At 44 days of age,all broilers were simultaneously treated with acute cold stress(ACS)at 10℃ for 12 h and 24 h,respectively.The performance during cold training(22,29,36 and 43 days of age)and the liver morphology,key enzymes of glucose metabolism,the m RNA and protein expression levels of AMPK and PI3K/AKT/m TOR pathways related genes,inflammatory factors and heat shock proteins,as well as the content of blood glucose indicators of broilers were detected before and after ACS.The experimental results are as follows:1.Performance: At the last week of cold adaptation training(36 d),the body weight of broilers in C3 group was significantly higher than that in CC and C9 groups(P < 0.05).The body weight of broilers in C9 group was significantly lower than that in CC and C3 groups(P < 0.05)one week after cold adaptation training(43 d).There was no significant difference in feed intake among the three groups during cold training(P > 0.05).Meanwhile,the body weight and feed intake of all broilers were significantly increased with the increase of day age(P < 0.05).2.Liver morphology: During cold adaptation training(22-43 d),the liver morphology and structure of broilers in CC group were complete.The cell arrangement was gradually loose and the inflammatory infiltration was more serious with the increase of age in C3 and C9 groups,but they were relieved one week after cold adaptation training(43 d).After ACS,the liver morphology of broilers in all groups was more severely damaged than before,but the C3 group was relatively intact and the inflammatory infiltration phenomenon was less.3.Inflammatory factors: At the last week of cold adaptation training(36 d),compared with CC group,the m RNA expressions of IL-1β,IL-4,IL-10 and i NOS and TNF-α concentration in the liver of broilers in C3 and C9 groups were significantly increased(P < 0.05).Compared with pre-ACS,the m RNA expressions of IL-1β,IL-4,IL-6,IL-10 and i NOS in C3 and C9 groups were significantly increased after 12 h of ACS(P < 0.05).After 24 h of ACS,the m RNA expressions of IL-1β,IL-6,IL-10 and IFN-γ in C3 group were markedly lower than in CC group(P < 0.05).4.Indicators related to glucose metabolism: During cold adaptation training(22-43 d),the GC concentration in serum of broilers in C3 and C9 groups was markedly higher than in CC group(P <0.05),and the m RNA expressions of HK1 and PDH in the liver of broilers in C3 group were significantly increased with the increase of age(P < 0.05),and GLUT1 m RNA expression was not significantly changed among the three groups(P > 0.05).No specific differences in INS concentration were found in CC and C3 group after ACS(P > 0.05).Compared with pre-ACS,INS concentrations in CC and C9 groups were significantly decreased after ACS(P < 0.05).The m RNA expressions of PFK,PDH and GLUT1,and the contents of LDH,CS and Glu in C3 group were not significantly different during pre-ACS or after ACS(P > 0.05).5.AMPK and PI3K/AKT/m TOR pathways related genes: After one week of cold adaptation training(22 d),the AMPK m RNA expression in the liver of broilers in C3 and C9 groups was markedly increased compared with CC group(P < 0.05),and m TOR m RNA expression in C9 group was significantly lower than in CC and C3 groups(P < 0.05).The m RNA expressions of LKB1,AMPK and AKT and the protein expressions of AMPK and AKT in C3 group were upregulated compared with CC group(P < 0.05),and the m RNA expression levels of SIRT1 and SIRT6 in C3 group were significantly increased with the extended time of cold adaptation training(P < 0.05),but there was no significant difference in HIF-1α m RNA expression in C3 group(P > 0.05)(36-43d).After 12 h of ACS,the m RNA expressions of VEGFR3,LKB1,AMPK and Caspase-1 and pAMPK/AMPK protein level in C3 and C9 groups were significantly higher than in CC group(P <0.05).The AKT m RNA expression level and p-AKT/AKT level in C9 group were significantly higher than in C3 group(P < 0.05).After 24 h of ACS,the m RNA expressions of LKB1,AMPK,PI3 K,AKT and m TOR in C3 group were significantly lower than after 12 h of ACS(P < 0.05),and there was no significant difference in p-AMPK /AMPK in C3 group(P < 0.05).6.Heat shock protein: Compared with 43 d,m RNA expressions of HSP40,HSP60,HSP70 and HSP90 in the liver of broilers in C3 and C9 groups and protein expressions of HSP60,HSP70 and HSP90 in C3 group were found to be significantly increased after 12 h of ACS(P < 0.05),and the m RNA expressions of HSPs in C3 and C9 groups were significantly higher than in CC group(P <0.05).The m RNA expression levels of HSP40,HSP60,HSP70 and HSP90 in C3 and C9 groups and the protein expression level of HSP90 in C3 group in the liver of broilers were significantly decreased with the extended time of ACS(P < 0.05),while HSP60 and HSP70 protein expressions were significantly increased(P < 0.05).In conclusion,the present study draws the following conclusions:(1)Intermittent cold stimulation of 3℃ lower than normal temperature can improve the glucose metabolism in the liver of broilers to a certain extent.A temperature of 9℃ lower than normal temperature can break the balance of glucose metabolism and damage the body’s energy metabolism.(2)When broilers were raised at a temperature of 3℃ lower than the normal thermal temperature,the inflammatory response in vivo was alleviated.On the contrary,a temperature as low as 9℃ induced an increased inflammatory response.(3)The expressions of heat shock proteins in the liver of broilers exposed to a temperature of3℃ lower than the normal thermal temperature were increased to a certain extent,which could enhance the short-term ACS resistance.A temperature of 9℃ lower than the normal thermal temperature can damage the ability of body to resist ACS,so that the broilers were lack of a good environmental adaptability.Therefore,intermittent mild cold stimulation can enhance the ability of broilers to resist cold environment by improving the glucose metabolism and reducing inflammatory response to a certain extent,so as to alleviate the adverse effects of ACS on the liver of broilers. |
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