Study On Effects Of β-sitosterol On Growth,and Antioxidant And Mitochondial Function Of Broilers Under Normal And Heat Stress Conditions

Heat stress induced by high ambient temperature is the most common environmental stress in broiler production.Mitochondron,the energy metabolic center of cell,is an organelle that can be attacked firstly by heat stress.Heat stress can induce mitochondrial dysfunction and therefore damage tissue function in broilers.Phytosterols are one of naturally plant-derived active substances,and have been approved as a functional feed additive by Chinese Ministry of Agriculture.Natural phytosterols are a kind of mixture,while its main component is β-sitosterol.Trials of cells and model animals have confirmed that β-sitosterol exerts antioxidant ability,and also regulates mitochondrial energy metabolism efficiency,which suggests that β-sitosterol supplemented into diet may improve antioxidant and mitochondrial function of tissues in broilers.Herein,this study investigated the effects of dietary β-sitosterol inclusion with different levels on antioxidant and mitochondrial function of tisues in broilers,and the optimum dosage of β-sitosterol was selected.On this basis,the heat stress model was constructed to explore the protective effect of β-sitosterol on the growth,and antioxidant and mitochondrial function of heat-stressed broilers and the related mechanism,in order to provide theoretical basis for rational application of β-sitosterol in broiler diet and technical reference for alleviating the injury of broilers induced by heat stress based on mitochondrial pathway.1 Effects of dietary β-sitosterol inclusion with different levels on growth performance and antioxidant function in broilersThis trial aimed to investigate the effects of β-sitosterol inclusion with different levels on growth performance,serum biochemical indexes,and antioxidant ability in broilers.One-day-old Arbor Acres(AA)male chicks with similar weight were allocated into 5 groups,and each group consisted of 6 replicates of 8 chicks each,and fed a basal diet supplemented with 0(Control group),40,60,80,and 100 mg/kg β-sitosterol for 42 d,respectively.The results showed that,β-sitosterol quadratically increased average daily gain(ADG)and average daily feed intake(ADFI)of broilers from 1 to 21 d(P<0.05),and linearly and quadratically reduced feed to gain ratio(F/G)during the whole period(P<0.05).Compared with the control group,60 and 80 mg/kg β-sitosterol decerased F/G from 1 to 42 d(P<0.05).β-Sitosterol linearly increased serum total protein(TP)and globulin(GLB)contents at 21 d(P<0.05),whereas linearly reduced serum total cholesterol(TC)level at 42 d(P<0.05).60,80,and 100 mg/kg β-sitosterol decreased serum TC content at 42 d(P<0.05);100 mg/kg β-sitosterol increased serum TP level at 21 d(P<0.05).β-Sitosterol linearly reduced malondialdehyde(MDA)content of serum at 21 d and liver and breast muscle at 42 d(P<0.05),linearly and quadratically decreased MDA content of jejunum and ileum at 42 d(P<0.05).β-Sitosterol linearly increased activities of superoxide dismutase(SOD)activity at 42-d serum and breast muscle,and 21-d jejunum,catalase(CAT)at serum and jejunum at 42 d,glutathione peroxidase(GPX)at 21-d liver,and glutathione(GSH)level of jejunum and liver at 42 d(P<0.05).80 mg/kg β-sitosterol increased SOD activity of breast muscle(P<0.05);100 mg/kg β-sitosterol reduced MDA content of 21-d serum and 42-d liver(P<0.05),whereas increased 42-d serum CAT and 21-d liver GPX(P<0.05);60 and 80 mg/kg β-sitosterol decreased MDA level of jejunum at 42 d(P<0.05);80 and 100 mg/kg β-sitosterol increased activities of SOD activity at serum and CAT at serum and jejunum,and GSH content of liver at 42 d(P<0.05);60,80 and 100 mg/kg β-sitosterol decreased MDA content of ileum and breast muscle at 42 d(P<0.05),but increased GSH level of ileum at 42 d(P<0.05).β-Sitosterol linearly upregulated mRNA abundances of nuclear factor E2 related factor 2(NRF2)at 42-d jejunum and liver,GPX1 at 42-d jejunum and 21-d liver,CAT at 42-d ileum,and SOD1 at breast muscle(P<0.05).80 mg/kg β-sitosterol upregulated GPX1 mRNA abundance of 42-d ileum(P<0.05);100 mg/kg β-sitosterol upregulated GPX1 mRNA abundance of 21-d liver and SOD1 mRNA abundance of breast muscle(P<0.05);80 and 100 mg/kg β-sitosterol increased NRF2 mRNA abundance of 42-d jejunum and liver(P<0.05).The results suggested that dietaryβ-sitosterol supplementation can improve feed efficiency and increase antioxidant enzymes activities,and GSH content,inhibit lipid peroxidation,and upregulate antioxidant related genes expression in the tissues of broilers.2 Effects of dietary β-sitosterol inclusion with different levels on mitochondrial function of tissues in broilersThis trial aimed to investigate the effects of β-sitosterol inclusion with different levels on enzymes activities involved in mitochondrial tricarboxylic acid cycle(TCA)and mitochondrial biogenesis in the tissues of broilers.The experimental design was the same to the trial 1.The results showed that,β-Sitosterol linearly increased malate dehydrogenase(MDH)activity of 21-d ileum and 42-d jejunum,mitochondrial isocitrate dehydrogenase(ICDHm)activity of 42-d liver,and succinate dehydrogenase(SDH)activity of breast muscle(P<0.05).80 and 100 mg/kg β-sitosterol increased ICDHm activity of 42-d liver(P<0.05);100 mg/kg β-sitosterol increased MDH activity of 21-d ileum and 42-d jejunum and SDH activity of breast muscle(P<0.05).β-Sitosterol linearly mitochondrial DNA(mtDNA)copy number of jejunum and liver at 42 d(P<0.05),and upregulated peroxisome proliferation activated receptor y coactivator-1α(PCG-1α)mRNA abundance of jejunum,liver,and breast muscle,nuclear factor E2 related factor 1(NRF1)mRNA abundance of jejunum,ileum,and liver at 42 d,and mitochondrial transcription factor A(TFAM)mRNA abundance of liver(21 d)and breast muscle(P<0.05).80 and 100 mg/kg β-sitosterol increased PCG-la mRNA abundance of liver and breast muscle,and TFAM mRNA abundance of breast muscle at 42 d(P<0.05);100 mg/kg β-sitosterol incresed PCG-la mRNA abundance of jejunum and NRF1 mRNA abundance of liver at 42 d(P<0.05).The results suggested that dietary β-sitosterol addition can enhance enzymes activities involved in TCA,and upregulate genes expression levels related to mitochondrial biosgenesis of tissues in broilers.3 Effects of dietary β-sitosterol on growth performance,serum biochemical indexes and hormone levels,and intestinal development and its mitochondrial function in heat-stressed broilersThis trial aimed to investigate the effects of dietary β-sitosterol on growth performance,serum biochemical indexes and hormone levels,and intestinal development and its mitochondrial function in broilers under heat stress.One-day-old AA male chicks with similar body weight were allocated into 3 treatment groups,and each groups consisted of 8 replicates of 8 chicks each.The feeding period lasted for 42 d.The 3 treatments were shown as follows:(1)Control group,broilers fed the basal diet were reared under thermal isothermal temperature;(2)Heat stress group,broilers fed the basal diet were raised under thermal isothermal temperature during the starter period and cyclic high ambient temperature during the finisher period;(3)β-sitosterol group,broilers were fed the basal diet supplemented with 80 mg/kg β-sitosterol,and the rearing temperature was the same to the heat stress group.The results showed that,compared with the control group,heat stress increased rectal temperature of broilers at 35 and 42 d(P<0.05),reduced final body weight,ADFI,and ADG of broilers(P<0.05),while F/G was increased(P<0.05);β-sitosterol increased final body weight and ADG(P<0.05),whereas reduced F/G of heat-stressed broilers(P<0.05).Heat stress reduced serum TP,GLB,triiodothyronine,thyroxine(T4)contents(P<0.05),whereas increased glucose,TC,low density lipoprotein cholesterol(LDL-C),and corticosterone levels of serum in broilers(P<0.05);β-sitosterol increased serum GLB and T4 levels(P<0.05),whereas decreased LDL-C and corticosterone contents of serum in heat-stressed broilers(P<0.05).Heat stress decreased Villus height(VH)of jejunum and ileum and villus height to crypt depth ratio(VH/CD)of jejunum in broilers(P<0.05);β-sitosterol increased VH of jejunum and ileum and VH/CD of jejunum in heat-stressed broilers(P<0.05).Heat stress increased H2O2 content of jejunum and ileum in broilers(P<0.05);β-sitosterol reduced jejunal H2O2 level of heat-stressed broilers(P<0.05).Heat stress increased MDA content but decreased GPX activity of jejunal and ileal mitochondria(P<0.05),and also reduced GSH content of jejunal mitochondria and MnSOD activity of ileal mitochondria of broilers(P<0.05);β-sitosterol reduced MDA level but increased GSH content of jejunal mitochondria(P<0.05),and also increased ileal mitochondrial MnSOD activity of heat-stressed broilers(P<0.05).Heat stress reduced mtDNA copy number of jejunum and ileum of broilers(P<0.05);β-sitosterol increased jejunal mtDNA copy number in heat-stressed broilers(P<0.05).Heat stress downregulated sirtuinl(SIRT1),PCG-1α,and TFAM mRNA abundances of jejunum,and PCG-1α,NFR1,and TFAM mRNA abundances of ileum(P<0.05),whereas increased jejunal heat shock protein 70(HSP70)and ileal HSP90 mRNA abundances of broilers(P<0.05);β-sitosterol upregulated mRNA abundance in PCG-1α of jejunum and ileum(P<0.05),whereas decreased HSP70 mRNA abundance of jejunum in heat-stressed broilers(P<0.05).The results suggested that dietary β-sitosterol can regulate serum lipid and hormone levels,and improve intestinal development and its mitochondrial antioxidant function and biogenesis,and therefore promote growth performance of heat-stressed broilers.4 Effects of dietary β-sitosterol on redox status and apoptosis in the liver of broilers under heat stress and related mechanismThis trial aimed to investigate the effects of dietary β-sitosterol on redox status and apoptosis in the liver of broilers under heat stress as well as related mechanism.The experimental design was the same to the Trial III.The results showed that,compared with the control group,heat stress decreased absolute and relative liver weight(P<0.05),but increased glutamic-pyruvic transaminase and glutamic oxaloacetic transaminase activities of serum in broilers(P<0.05);β-sitosterol increased absolute liver weight in heat-stressed broilers(P<0.05).Heat stress resulted in pathology changes of liver cells,such as central venous hemorrhage,and induced liver mitochondrial structural swelling and membrane rupture of in broilers,which were alleviated partly by β-sitosterol.Heat stress decreased O2·-and·OH scavenging ability of serum and O2·-scavenging ability of liver in broilers(P<0.05);β-sitosterol increased·OH scavenging ability of serum in heat-stressed broilers(P<0.05).Heat stress increased H2O2 and mitochondrial MDA contents(P<0.05),whereas reduced mitochondrial MnSOD and GPX activities and GSH level of liver in broilers(P<0.05);β-sitosterol decreased mitochondrial MDA content(P<0.05),whereas tended to increase GSH level of liver in heat-stressed broilers(P<0.1).Heat stress increased MDA level of serum and liver and protein carbonyl content of serum(P<0.05),whereas reduced activities of SOD at serum and liver,CAT at serum,and GPX at liver and GSH level at liver in broilers(P<0.05);β-sitosterol reduced liver MDA level(P<0.05),whereas tended to increase SOD activity of serum and liver,serum CAT activity,and liver GSH level in heat-stressed broilers(P<0.1).Heat stress downregulated mRNA abundances of NRF2,NAD(P)H dependency quinone oxidoreductase 1(NQO1),SOD1,SOD2,GPX4,y-glutathione cysteine ligase catalytic subunit(γ-GCLc),and y-GCL ligase modifier subunit(y-GCLm)(P<0.05),whereas mRNA abundances upregulated of HSP70 and HSP90 of liver in broilers(P<0.05);β-sitosterol upregulated mRNA abundances of NRF2,SOD2,and y-GCLc(P<0.05),whereas downregulated mRNA abundances of kelch like epichlorohydrin related protein 1(Keapl)and HSP70 of liver in heat-stressed broilers(P<0.05).Heat stress increased activities of Caspase-3 and Caspase-9(P<0.05),upregulated mRNA abundances of cytochrome C(Cytc),apoptotic protease activating factor 1(Aparf-1),B cell lymphoma 2 associated X protein(BAX),Caspase-3,and Caspase-9(P<0.05),whereas downregulated B cell lymphoma 2(Bcl-2)mRNA abundance of liver in broilers(P<0.05);β-sitosterol reduced Caspase-3 activity(P<0.05),downregulated Cytc,p53,and Caspase-3 mRNA abundances(P<0.05),whereas upregulated Bcl-2 mRNA abundance of liver in heat-stressed broilers(P<0.05).The results suggested that dietary β-sitosterol can improve redox status of liver,and therefore inhibit liver cell apoptosis via mitochondrial pathway in heat-stress broilers through activation of NRF2 signal pathway.5 Effects of dietary β-sitosterol on mitochondrial energy metabolism efficiency and biogenesis in the liver of broilers under heat stress and related mechanismThis trial aimed to investigate the effects of dietary β-sitosterol on mitochondrial energy metabolism efficiency and biogenesis in the liver of heat-stressed broilers,as well as related mechanism.The results showed that,compared with the control group,Heat stress reduced ATP level and mtDNA copy number(P<0.05),whereas increased TC and triglyceride(TG)contents of liver in broilers(P<0.05);dietary β-sitosterol increased ATP level and mtDNA copy number(P<0.05),whereas decreased TC level of liver in heat-stressed broilers(P<0.05).Heat stress decreased activities of ICDHm,SDH,and mitochondrial complex Ⅰ,Ⅲ,Ⅳ,and Ⅴ of liver in broilers(P<0.05);dietary β-sitosterol increased ICDHm and complex Ⅲ activities of liver in heat-stressed broilers(P<0.05).Heat stress downregulated SIRT1,PGC-lα,NRF1.TFAM,and carnitine palmitoyltransferase I(CPT-I)mRNA abundances(P<0.05),whereas upregulated sterol-regulatory element binding protein lc(SREBP-lc)and acetyl CoA carboxylase(ACC)mRNA abundances of liver in broilers(P<0.05);β-sitosterol upregulated AMP-activated protein kinaseα1(AMPKα1),peroxisome proliferator activated receptor a(PPARα),PGC-1α,CPT-I,and TFAM mRNA abundances of liver in heat-stressed broilers(P<0.05).The results suggested that dietary β-sitosterol can promote mitochondrial TCA,oxidative phosphorylation,and fatty acid β-oxidation efficiency as well as biogenesis,therefore contributing to improve energy homeostasis and inhibit fat deposition in the liver,which are related to the activation of AMPK/PGC-lα/PPARα signal pathway.On basis of abovementioned,it is concluded that:(1)Dietary β-sitosterol supplementation can improve feed efficiency,and enhance antioxidant and mitochondrial function in the tisues of broilers,and the recommended dosage β-sitosterol in broiler diet was 80 mg/kg.(2)Dietary β-sitosterol inclusion at a level of 80 mg/kg can improve growth performance of heat-stressed broilers through improving intestinal mitochondrial antioxidant capacity and biogenesis.(3)Dietary β-sitosterol addition at a level of 80 mg/kg can improve redox status of liver and therefore inhibit liver cell apoptosis via mitochondrial pathway through activation of NRF2 signal pathway;promote mitochondrial energy metabolism efficiency and biogenesis to improve energy homeostasis and inhibit fat deposition in the liver via activation of AMPK/PGC-1α/PPARα signal pathway of heat-stressed broilers.