Corticosterone-Simulated Stress Induces Liver Lipid Metabolism Disorders And Intestinal Flora Imbalance In Broiler Chickens And The Alleviating Effect Of Curcumin

In the process of broiler intensive farming,chronic stress seriously affects broiler health and performance,resulting in a series of stress reactions such as reduce growth rate,lower feed remuneration,weak immune function,fatty liver and structural changes of intestinal flora,which seriously restrict the healthy development of broiler farming.The disruption of the intestinal epithelial tight junction(TJ)barrier and the imbalance of intestinal flora caused by chronic stress are the main reasons for the reduce performance of broiler chickens.Curcumin(CUR)is the main active ingredient of the ginger plant turmeric,which has various pharmacological functions such as hypoglycemia and lipid lowering,antioxidant,inflammation inhibition,immune system enhancement and intestinal health improvement.It has been shown that CUR can significantly improve livestock performance both product quality and improve intestinal microbial structure.However,the mitigating effects of CUR on chronic stress broiler growth,liver lipid metabolism disorders,and intestinal flora imbalance need further study.In this experiment,we investigated the effects of CORT on growth,liver lipid metabolism,liver oxidative damage,small intestine mucosal barrier and cecum flora composition of growing broiler chickens by treating them with Corticosterone(CORT)to simulate chronic stress,and examined the effects of CORT on growth,liver lipid metabolism,broiler intestinal morphology,oxidative stress and cecum flora composition of growing broiler chickens.The effects of CORT on growth,liver lipid metabolism,broiler intestinal morphology,oxidative stress and cecum flora composition were investigated,and the alleviating effects of CUR were revealed,in order to provide a theoretical basis for the application of CUR as a novel feed additive in broiler breeding to alleviate the damage of stress-induced intestinal barrier structure and immune disorders.The main research contents are as follows:Experiment 1:Effect of corticosterone-simulated stress on growth and plasma biochemical parameters of broiler chickens and the regulatory effect of curcuminIn this experiment,120 1-day-old yellow-feathered broilers were used,and after 2 days of acclimatization(3 days old,day 0 of the experiment),they were randomly divided into 4 treatment groups,with 3 replicates per treatment and 10 chickens per replicate.The groups were:control group(CON group):fed the basal full-price diet and subcutaneously injected with 20%saline on days 43-49;CUR group:fed the basal full-price diet with 200 mg/kg CUR and subcutaneously injected with 20%saline on days 43-49;CORT group:fed the basal fullprice diet and subcutaneously injected with 4 mg/kg CORT on days 43-49;CORT+CUR group:fed the basal full-price diet with 200 mg/kg CUR and subcutaneously injected with 4 mg/kg CORT on days 43-49,for a total of 7 weeks,including 1 week of CORT injection(2 times/day).All broilers were weighed and organ weights were measured on days 0,43 and 50 of the experiment,and blood was collected on day 50 of the experiment,and plasma was prepared for biochemical analysis.The results showed that there was no significant difference in the body weight of broilers between the four groups at day 0(initial body weight)and day 43(initial body weight by injection)of the test.Compared with the CON group,at day 50 of the trial(end of injection),the broilers in the CORT-injected group had a highly significant decrease in body weight(P<0.001),but the liver index(P<0.001)and plasma total cholesterol(TC)(P<0.001),high-density lipoprotein cholesterol(HDLc)(P<0.001),lowdensity lipoprotein cholesterol(LDLc)(P<0.001),and triglyceride(TG)(P<0.001)levels were significantly increased;However,the plasma levels of TC(P<0.05),HDLc(P<0.05),and LDLc(P<0.001)were significantly lower in the CORT+CUR group compared to the CORT group.The above results suggest that dietary supplementation of CUR can improve the disorders of glycolipid metabolism in broilers caused by stressful conditions through regulating glycolipid metabolism.Experiment 2:Effect of corticosterone-simulated stress on liver lipid deposition and oxidative damage in broiler chickens and the modulating effect of curcuminBlood and liver tissues were collected at the end of the experiment,on the 50th day of the test,from 1-day-old SPF-grade.To measure malondialdehyde(MDA)levels and superoxide dismutase(SOD)activity in plasma and liver,as well as the expression of antioxidant genes in the liver.Observed the number of vacuolar degenerations,lipid droplet deposition and glycogen content,and determination of gene expression related to lipid metabolism in liver tissue,so as to comprehensively assess the improvement of liver lipid deposition and oxidative damage in broiler chickens by dietary supplementation of CUR.The results showed that CORT treatment significantly increased plasma MDA(P<0.001)and decreased liver tissue SOD activity(P<0.001)compared with the CON group;compared with the CORT group,the CORT+CUR group had significantly lower plasma MDA and liver tissue MDA contents(P<0.05),while liver tissue SOD contents were significantly higher(P<0.05).Meanwhile,the results of antioxidant genes assay showed,compared with the CON group,the mRNA expression of GXP1 in liver tissues was significantly lower in the CORT group,(P<0.05),while the mRNA expression of SOD1 and GSTa3 did not change significantly(P=0.1727,P=0.8694),compared with the CORT group,the mRNA expression of SOD1 was significantly higher(P<0.05),and the mRNA expression of GSTa3 and GXP1 was not significantly changed in the CORT+CUR group(P=0.4957,P=0.9794).Pathological observations showed that CORT treatment significantly increased the number of vacuolar degeneration and oil-red O-stained lipid droplets and decreased glycogen content in liver tissue compared with the CON group,and significantly decreased the number of vacuolar degeneration and oil-red O-stained lipid droplets,and significantly increased glycogen content in the liver of the CORT+CUR group compared with the CORT group.The results of lipid metabolism-related genes showed that the expression of lipid synthesis-limiting enzymes FAS,ACC and SCD in liver tissue was highly significantly increased in the CORT group compared with the CON group(P<0.001,P<0.001,P<0.01),while the expression of lipid hydrolases ATGL,Acox1 and CPT were significantly decreased(P<0.001,P<0.01,P<0.01).The expression of Acoxl and CPT was significantly higher in the CORT+CUR group compared to the CORT group(P<0.01,P<0.01),and the expression of CGL-58 showed an increase tendency(P=0.0811).It showed that dietary addition of CUR significantly improved oxidative damage caused by CORT stress and alleviated stress-induced disorders of liver lipid metabolism in broiler chickens by regulating the expression of key genes of lipid metabolism.Experiment 3:Effect of corticosterone-simulated stress on intestinal development and intestinal flora structure of broiler chickens and the ameliorating effect of curcuminIn this study,the intestinal tissues and intestinal contents collected on the 50th day of the experiment were used to determine the structural of intestinal mucosal barrier in duodenum,jejunum,ileum,cecum and cecum flora,and to analyze the structural changes of cecum microflora.The results showed that compared with the CON group,the duodenal villus height was highly significantly lower(P<0.001),the ileal villus height and villus crypt ratio were highly significantly higher(P<0.001),and the crypt depth was significantly lower(P<0.05)in the CORT group of broilers.Compared with the CORT group,the duodenal villus height and villus crypt ratio were highly significant(P<0.001)and significantly(P<0.05)higher,the ileal villus height(P<0.001)and villus crypt ratio(P<0.01)were highly significant lower,and the crypt depth was highly significant higher(P<0.001)in the CORT+CUR group of broilers.Analysis of intestinal flora diversity showed that Bray-Curtis distance analysis in cecum contents flora beta diversity was significantly different among the four groups compared to the CON group,the CORT group was farthest from the CON group,and the CORT+CUR group was positioned between the CON and CORT groups.The abundance of cecum contents flora showed that at the phylum level,at the phylum level the Proteobacteria had a significantly higher percentage in the CORT group,and the Firmicutes had a lower percentage.At the genus level,the dominant genera in the CON group were Faecalibacterium and Alistipes,compared to the CON group,unidentified_Clostridia_vadinBB60 group was increase in the percentage of the CUR group.Faecalibacterium was significantly lower in the CORT group,Escherichia-Shigella,UGG-005,Bacillus,[Ruminococcus]_torques_group and Eisenbergiella was increase in the percentage of the CORT group.Compared to the CORT group.The percentage of Faecalibacterium,Escherichia-Shigella was increase,and the percentage of Alistipes and Eisenbergiella was decreased in the CORT+CUR.Functional prediction of cecum contents flora showed that,compared with the CON group,the genes related to Amino acid metabolism,Infectious_diseases,Lipid_metabolism,Aging,Energy_metabolism and Metabolish_of_terpenoids_and_polyketides of CUR group had significantly higher than CON group,but the genes related to Immune_system,Endocrine_system,and Endocrin_and_metabolic_diseases were lower than CON in those group.Compared to the CON group,the CORT group had higher level of genes related to Poorly_characterized,Carbohydrate_Metabolism,Cellular_processes_and_signaling,Translation,Cancers,Xenobiotics_biodegradation_and_matebolism,Cellular_community_prokaryotes,but genes related to translation,cancers,xenobiotics biodegradation and matebolism,and cellular community_prokaryotes were reduced.Compared to CORT,the genes related to Endocrine_system,Translation and Signal_transduction in CORT+CUR group were lower than CORT group,but Metabolism_of_cofactors_and_vitamins vitamins,Carbohydrate_Metabolism and Drug_resistance related genes were higher than CON group.Finally,Spearman correlation analysis was performed on the intestinal flora,and the phylum level Spearman correlation analysis showed that Acidobacteriota,the dominant bacterium in the intestine,was significantly and positively correlated with BW,and Desulfobacterota was significantly and highly significantly negatively correlated with LDLc and ACC.Spearman correlation analysis at the genus level showed that Christensenellaceae_R.7_group was significant positive correlation with BW and significant negative correlation with LDLc,while both Merdibacter and Fournierella was significant negative correlation with BW,X.ruminococcus._torques_group was significant positive correlation with LW,LDLc and ACC.Faecalibacterium showed significant negative correlation with TC,Escherichla-Shlgella was significant positive correlation with TC,HDLc and LDLc,both Erysipelatoclostridium and Subdoligraanulum showed significant positive correlation with LDLc,Eisenbergiella was significantly negatively correlated with Acoxl and CPT,Lachnooclostridium was highly significantly positively correlated with ACC.In conclusion,chronic stress simulated by CORT can lead to weight loss,liver glycolipid metabolism disorders,oxidative stress and intestinal flora imbalance in broilers,and the addition of CUR to the diet can significantly reduce the weight loss,liver glycolipid metabolism disorders and reshape the intestinal flora in broilers caused by CORT simulated stress.It shows that CUR can be a potentially safe and effective feed additive to improve livestock intestinal health,alleviate stress-induced intestinal barrier damage and growth disorders in broilers,the results provide a theoretical reference for CUR to prevent or treat injury caused by stress in broilers.