The Study On Effects Of Dietary Capsaicin On Growth Performance And Antioxidant Function Of Broilers And Its Mechanism

Modern broilers have low immunity due to their characteristics of fast growth,high feed conversion,and short feeding period.Meanwhile,broilers are faced with many causes of oxidative stress:Environment stress,abnormal feed composition,animals’own state changes,and microbial infection,which will increase the morbidity and mortality of broilers,affecting the health of broilers and the economic benefits of breeding enterprises.Capsaicin(CAP),a kind of alkaloid extracted from pepper,has been proved to have anti-inflammatory,antibacterial,lipid metabolism regulation,antioxidant,and other biological functions.At present,the studies on the antioxidant mechanism of CAP in broilers are very rare.Therefore,this study investigated the effects and mechanisms of dietary CAP on growth performance and antioxidant function of broilers,and explored the antioxidant effect of capsaicin at the cellular level to provide scientific reference for the application of capsaicin in the broiler diet.Experiment 1:Effects of dietary capsaicin on growth performance,serum biochemical indices,and meat quality of broilersThis experiment was conducted to investigate the effects of dietary CAP on growth performance,serum biochemical and antioxidant indices,and meat quality of broilers.A total of 256 one-day-old Arbor Acre male broilers were randomly allocated into 4 treatments with 8 replicates of 8 birds,feeding a basal diet(control group),a basal diet supplemented with 2,4,and 6 mg/kg CAP for 42 d,respectively.The growth performance,and serum biochemical and antioxidant indices were measured at 21 and 42 d.The meat quality traits of breast muscles were determined at 42 d.The results showed dietary 4 mg/kg CAP supplementation decreased(P<0.05)the feed to gain ratio(F/G)in the grower phase(22-42 d)and overall(1-42 d)compared with the control group,and 2mg/kg CAP group also decreased(P<0.05)the F/G from 1-42 d.Compared with the control group,dietary 4 mg/kg CAP could significantly reduce(P<0.05)serum TG and LDL-C levels and increase the contents of HDL-C;All CAP groups could decrease(P<0.05)the TC content at 42 d;2 mg/kg CAP group significantly increased(P<0.05)the serum total bile acid(TBA)content of broilers at 42 d.The CAP linearly and quadratically increased serum GPX level(P<0.05)at 21 d,and linearly and quadratically decreased serum MDA level(P<0.05)at 42 d.Dietary 4 mg/kg CAP supplementation decreased(P<0.05)the drip loss at 48 h of breast muscles relative to the control group.The above results suggest that dietary supplementation of 2 and 4 mg/kg CAP could decrease the F/G and improve serum biochemical and antioxidant indices of broilers.Adding 4 mg/kg CAP in the diet could improve the meat quality of breast muscle.Experiment 2:Effects of dietary capsaicin on antioxidant function in liver of broilersThis experiment was conducted to investigate the effects of dietary CAP on antioxidant function in liver of broilers and its mechanism.The experimental design is the same as experiment 1.Bile acids not only promote the absorption of lipid metabolites,but also serve as a major substance in hepatoenteric circulation and a key signal molecule that can regulate a series of metabolism and play an antioxidant role.Therefore,in addition to detecting conventional antioxidant indexes,this study also investigated the effect of capsaicin on liver bile acid synthesis.The results showed that CAP could quadratically increase GPX and linearly and quadratically decrease MDA level in the liver at 21 and 42 d(P<0.05).Compared with the control group,adding 4 mg/kg CAP significantly increased(P<0.05)the gene expression levels of GPX at 21 d and Nrf2,HO-1,GPX1 and TRPV1 in liver at 42 d.2 mg/kg CAP group significantly upregulated(P<0.05)the genes expression of TRPV1 and avUCP in liver at 21 d,and TRPV1 in liver at 42 d.Compare with the control group,dietary supplementation of 2 and 4mg/kg CAP could up-regulate(P<0.05)the expression of CYP7A1 in liver at 21 d.The above results suggest that CAP could improve the antioxidant function by promoting Nrf2 and TRPV1 related pathway genes and GPX activity,and regulate the synthesis of bile acids in liver of broilers.Experiment 3:Effects of capsaicin on antioxidant function of chicken primary hepatocytesThe aim of this study was to explore the antioxidant effect of CAP on the chicken primary hepatocytes in vitro.Chicken primary hepatocytes were treated with 5 μmol/L CAP,and then collected at 12 h,24 h,and 48 h.Transcriptome sequencing(RNA-seq)was performed on chicken primary hepatocytes treated with CAP for 24 h.The contents of SOD,MDA and TBA in the culture medium were detected,and the expression of antioxidant and bile acid pathway related genes in all cells were detected.The results showed that RNA-seq results showed that capsaicin had significant effects on mitochondria,glutathione metabolism,peroxisome and synthesis of primary bile acids of chicken primary hepatocytes at 24 h.The CAP treatment for 12 h increased SOD content(P<0.05)and decreased MDA content(P<0.05)in the culture medium,upregulating Nrf2,SOD1,SOD2,TRPV1 and avUCP gene expression(P<0.05);The CAP incubation for 24 h upregulated Nrf2,HO-1,NQO1 and GPX1 gene expression in primary hepatocytes(P<0.05).The CAP incubation for 24 h increased(P<0.05)the content of TBA in the medium for 24 h,increase(P<0.05)the gene expression of CYP7A1 and CYP7B1,and decrease(P<0.05)the gene expression of CYP8B1,HMGCR and FXR.These results suggest that CAP could enhance the antioxidant function of chicken primary hepatocytes through Nrf2,TRPV1 and bile acid synthesis pathways.Experiment 4:Effects of dietary capsaicin on antioxidant function of the intestine of broilersThis experiment was conducted to investigate the effects of dietary CAP on antioxidant function of intestinal tissue of broilers.The experiment design is the same as experiment 1.The results showed that compared with the control group,dietary 2 mg/kg CAP supplementation increased(P<0.05)the jejunal villus height,villus width,and villous surface area at 21 d;The length of the jejunum segment at 42 d in the 4 mg/kg CAP group were higher(P<0.05)than the control group.The CAP could linearly and quadratically(P<0.05)increase the lipase activity at 21 d and quadratically(P<0.05)increase the trypsin activity at 21 d and 42 d in the jejunum contents.The CAP linearly and quadratically increased lipase activity(P<0.05)at 21 d and quadratically increased amylase and lipase activity(P<0.05)at 42 d in the ileum contents.The CAP could quadratically increase the level of SOD in jejunum(P<0.05)at 21 d and decrease the level of MDA in jejunum at 42 d.CAP could quadratically increase ileum GPX(P<0.05)at 21 d and quadratically decrease(P<0.05)its MDA content.The content of GPX in ileum at 42 d was increased linearly and quadratically(P<0.05),where the level of SOD was increased quadratically(P<0.05).The expression levels of GPX1 and TRPV1 genes in jejunum in 2 and 4 mg/kg CAP groups were significantly higher than those in the control group at 21 d(P<0.05).The gene expression of Keap1 in jejunum was significantly down-regulated(P<0.05)in all CAP groups at 42 d,and the gene expression of HO-1,TRPV 1,PKAcb and avUCP in the 4 mg/kg CAP group was significantly increased(P<0.05).2 mg/kg CAP group could upregulate(P<0.05)the gene expression of SOD1 in ileum at 21 d where 4 mg/kg CAP group could promote(P<0.05)the gene expression of HO-1 and NQO1,and all CAP groups could promote(P<0.05)the gene expression of TRPV1.The gene expression of Keap1 in ileum was decreased(P<0.05)in all CAP groups at 42 d where the gene expression of GPX1 and avUCP was also increased(P<0.05)in the 4 mg/kg CAP group.In addition,compared with the control group,the level of TB A in jejunal contents of broilers at 21 d in the 4 mg/kg CAP group was significantly increased(P<0.05).Dietary supplementation of 2 and 4mg/kg CAP could decrease(P<0.05)the gene expression of FXR in jejunum and FGF19 in ileum at 21 d.The level of FXR in 2 mg/kg CAP group was decreased(P<0.05)in ileum both at 21 and 42 d.These results suggest that CAP can up-regulate Nrf2 and TRPV1 pathway related genes,inhibit FXR/FGF19 pathway regulation of bile acid synthesis,and improve the antioxidant function of broiler intestinal tissue.Experiment 5:Effects of capsaicin on antioxidant function of chicken primary intestinal epithelial cellsThis experiment was conducted to investigate the effects of CAP on antioxidant function of chicken primary intestinal epithelial cells.Primary chicken intestinal epithelial cells were treated with 5 μmol/L CAP,and then collected at 12 h,24 h and 48 h to detect the content of SOD and MDA in the culture medium,and the expression of antioxidant,bile acid pathway,and mucosal barrier related genes in the cells.The results showed that:CAP increased SOD content at 24 h and decreased MDA content at 12 h in culture medium of chicken primary intestinal epithelial cells(P<0.05).CAP treatment for 24 h also up-regulated Nrf2,NQO1 and SOD2 gene expressions(P<0.05).The gene expression of HO-1 and TRPV1 was up-regulated by CAP treatment for 12 h and 24 h.CAP incubation for 12 and 24 h decreased the gene expression of FXR in primary intestinal epithelial cells(P<0.05),while CAP treatment for 24 h decreased the gene expression of FGF19(P<0.05).CAP upregulated the gene expression of Claudin1,Claudin2 and Occludin at 12 h and Occludin at 24 h in chicken primary intestinal epithelial cells treated with CAP(P<0.05).These results suggest that CAP could improve the antioxidant function of primary chicken intestinal epithelial cells,affect the expression of bile acid receptor related genes,and contribute to the maintenance of intestinal barrier.In conclusion,dietary supplementation of 2 or 4 mg/kg CAP can reduce the F/G,improve serum biochemical indices and meat quality of breast muscles of broilers.CAP can improve the antioxidant function of hepatoenteric system by activating Nrf2 pathway to upregulate antioxidant enzymes,activating TRPV1 pathway to upregulate the avUCP expression,and inhibiting intestinal FXR/FGF19 pathway to promote liver synthesis of bile acids.