Effect Of Alpha-lipoic Acid On Relieving Ammonia Stress And Hepatic Proteomic Analyses Of Broilers

Ammonia is the most harmful gas in poultry house.Ammonia concentration is always higher than the standard during the late stage of broiler breeding,which will cause disease in broilers and seriously affect broiler production.Previous studies have focused on the harmful effects of ammonia on the broiler performance,serum biochemical and so on.It is necessary to carry out research to reduce ammonia toxicity.Alpha-lipoic acid(LA)is an effective antioxidant that protects cells against oxidative injury during various toxic and pathological processes.However,the protection effect of LA against ammonia-induced adverse effects in broilers remains unclear.In this experiment,environmental control cabin that can precise control of ammonia concentration was used to pose ammonia stress for broilers,and the mitigating effect of LA on ammonia stress was detected.Additionally,iTRAQ quantitive technology was used to investigate the liver proteome changes of broilers under ammonia stress with or without LA supplementation.The comparative proteomics technology was selected to investigate the protein responses associated with ammonia stress and the sensitive proteins related to its relieve effect.The main results were summarized as follows:(1)The mitigating effects of LA supplementation on ammonia stress of broilers:18022-day-old male AA broilers were allocated to 3 groups with 6 replicates of 10 birds each:1)a control group without ammonia stress(CTRL);2)exposure to 70±3 mg/kg ammonia(AM);3)exposure to 70±3 mg/kg ammonia and administration of 300 mg/kg LA(AM+LA).After 3 wk,the growth performance,hormone levels,antioxidant indexes,plasma biochemistry were determined.The results showed that exposure of broilers to ammonia for three weeks significantly reduced average daily gain(ADG)and feed conversion rate(F/G)(P<0.05);increased plasmalevels of corticosterone(CORT),insulin(INS),pancreatic glucagon(GC)and malondialdehyde(MDA)(P<0.05);decreased activities of total superoxidc dismutase(T-SOD),glutathione peroxidase(GSH-PX)in the Plasma(P<O.05),and glutathione(GSH),GSH-PX in the liver(P<0.05);for the plasma biochemistryparameters,ammonia exposure significantly increased alkaline phosphatase(AKP),lactate dehydrogenase(LDH),creatine kinase(CK),aspertate aminotransferase(AST),alanine aminotransferase(ALT)and nitric oxide synthase(iNOS)activities(P<0.05),and increased blood ammonia and glucose levels(P<0.05).Compared with ammonia group,addition of 300 mg/kg LA to diet of broilers in 22-42 d improved the production performance of broilers under ammonia stress,significantly increased ADGand decreased F/G(P<0.05);reduced contents of blood CORT,INfS and free thyroid hormone fT3(P<0.05);and improved the antioxidant levels and reduced oxidative stress,the plasma T-SOD and GSH-PX activity were significantly increased(P<0.05),MDA content in plasma were significantly reduced(P<0.05),and GSH content in liver tissue were significantly increase(P<0.05);Cell toxicity and liver injury caused by ammonia stress was reduced to a certain extent,which was proved by the significant lower contents of plasma ALT,LDH,CK and TNOS(P<0.05).(2)Hepatic proteomic analyses of broilers:At day 42,six broilers(one broiler from each replicate)were randomly selected from each group,liver samples from same position were obtained for proteomic analysis.Using iTRAQ proteomic research techniques,2301 reliable proteins were successfully identificatd.Based on a threshold of?1.8-fold or ?0.56-fold change,a total of 105 proteins were differentially expressed between AM and CTRL groups,including 41 up-regulated proteins and 64 down-regulated proteins.A total of 216 proteins were differentially expressed between the AM+LA and AM groups,including 28 up-regulated proteins and 188 down-regulated proteins.Among these differentially expressed proteins,30 proteins were shared between AM/CTRL and(AM+LA)/AM comparisons.Among the 30 differentially expressed proteins,14 proteins that were up-regulated in the AM group were down-regulated by LA in the AM+LA group;of the 16 proteins were down-regulated in the AM group,13 were up-regulated in the AM+LA group.The abundance of 24 proteins was similar in the AM+LA and CTRL groups(fold change<1.8 or>0.56).The function of these proteins involved in transcriptional regulation,stress response,detoxification,protein translation,protein degradation,immune response,etc.Among thenl,the glutathione S-transferase(GSTA3 and GSTM2)was associated with the oxidative stress,ethanol dehydrogenase 6(ADH6)and carboxylic ester hydrolase 1(CES1)was associated with detoxification,high mobility group protein(HMGB1 and HMGB3)was involved in the inflammatory response,collagen protein(COL6A1 and COL6A3)was involved in liver injury.Protein abundances of these proteins were regulated to control group levels,indicating that oxidative stress,cell damage and inflammation caused by ammonia stress were reduced after LA supplementation.The mitigating effects of LA on ammonia toxicity may be through the regulation of these proteins.From the above,long-term ammonia stress can reduce broiler production performance,induce protein abundance changes in liver;addition of a certain amount of LA in the diet of broilers could improve broiler production performance under ammonia stress,reduce oxidative stress,and alleviate the liver injury to a certain extent.LA could restore the expression of certain proteins to control levels,such as GST,ADH6,CES1,HMGB1,HMGB3,COL6A1 and COL6A3.