The Functional Roles And Mechanisms Of L-citrulline In Heat Stressed And Immune Challenged Chickens

L-Citrulline(L-Cit)is a non-essential amino acid that is involved in several physiological roles including nitric oxide(NO)synthesis,protein synthesis,gut function,and energy metabolism.Recent progress has been made to understand L-Cit's metabolism,however,its potential for utilization in animal production remains nascent.Through seven aspects of experimental research,we investigated the anti-heat stress effects and the functional roles of LCit in poultry production,especially its involvement in body temperature regulation and nitric oxide synthesis.These studies provide the theoretical background for the application of L-Cit in poultry production.1.Effects of L-Citrulline supplementation on nitric oxide production and redox balance in laying hens during summer seasonDuring summer heat(maximum temperature of 27.4°C to 34.8°C),Hy-Line Brown laying hens at 34 weeks old were fed basal diet(CON),or basal diet supplemented with 0.25%,0.50%,and 1.00% L-Cit for 8 weeks.The results showed that the rectal temperature of hens was not changed by L-Cit(P > 0.05).However,the plasma NO,nitric oxide synthase(NOS),antioxidant enzymes(SOD and CAT),and amino acids involved in arginine(Arg)metabolism(that is,Arg,Citrulline(Cit),and ornithine(Orn))were increased(P < 0.05)by L-Cit supplementation.Therefore,dietary L-Cit supplementation promoted arginine metabolism and redox balance of laying hens during the summer season.2.Effects of L-Citrulline supplementation to arginine deficient diets on the growth performance,amino acid profile,nitric oxide regeneration,and intestinal health of broiler chickensOne day-old Arbor acre broilers were randomly assigned to either an Arg-deficient basal diet(NC),NC diet + 0.50% L-Arg(PC),or NC diet + 0.50% L-Cit(NCL)until 21 days of age.Results showed that the production performance of broilers was unaffected(P > 0.05).NCL diet promoted plasma Arg,Cit,and Orn levels higher than the NC diet while the PC diet only promoted free Arg content(P < 0.05).The kidney and liver NO concentrations were increased(P< 0.05)by NCL diet.Also,the NCL diet upregulated(P < 0.05)the m RNA expression of intestinal nutrient transporters(PEPT1,EAAT3),tight junction proteins(Claudin 1,Occludin),and intestinal mucosal defenses(MUC2,p Ig R)higher than PC fed broilers.Thus,L-Cit may serve as a partial replacement for Arg in broiler's diet without detrimental effects on the growth performance and gut health.3.Effects of L-Citrulline supplementation on the thermotolerance,nitric oxide generation,and physiological responses of broiler chickens under acute and chronic heat stressOne day-old broilers were fed either CON or 1% L-Cit diets for 28 days,then they were exposed to thermoneutral(TNZ,24°C)or heat stress(HS,35°C)temperatures for 5 h(acute HS,AHS)or 14 days(8h HS per day,CHS).The ear temperature(ET),core body temperature(CBT),and rectal temperature(RT)of broilers were increased(P < 0.05)during AHS and CHS conditions,however,TNZ + L-Cit suppressed the CBT and mean CBT,initiating hypothermia in broilers.HS increased the plasma NO but decreased total NOS activity(P < 0.05).Contrastingly,L-Cit diminished plasma NO but stimulated NOS activity.During AHS,TNZ +L-Cit inhibited the i NOS-COX2-PGE2 signaling and during CHS,L-Cit partly influenced mitochondrial biogenesis in breast muscle tissues(P < 0.05).Altogether,this study revealed that L-Cit initiates hypothermia,promotes NOS activity,and moderately alleviates HS-induced dysfunctions in broilers.4.Dietary L-Citrulline influences the growth performance and protein metabolism in heat-stressed broilersOne day old arbor acre broilers were fed CON or L-Cit diet for 3 weeks and later subjected to TNZ(24°C)or HS(35°C,8 h per day)for another 3 weeks.The results showed that L-Cit alleviated HS depression of feed intake and body weight(P < 0.05).HS downregulated insulinlike growth factor-1(IGF-I)expression in the hypothalamus and breast muscle,whereas,L-Cit stimulated plasma growth hormone(GH)and the IGF-I concentration in the plasma and breast muscle of broilers.Also,L-Cit increased IGF-1R and total m TOR abundance in the breast muscle of HS broilers(P < 0.05).Thus,this study showed that L-Cit supplementation promoted the growth performance,circulating GH and IGF-1 levels,and selectively stimulated growth factors related to protein metabolism in HS broilers.5.Transcriptomics of hypothalamic responses in broilers fed dietary L-Citrulline supplementation during chronic heat stressOne day old Ross 308 broilers were randomized into four treatments as CON(TNZ + CON diet);A(TNZ + L-Cit diet);B(HS + CON diet);C(HS+ L-Cit diet)for 14 days and the hypothalamus(n = 6 per treatment)was collected for RNA sequencing.A total of 57 differentially expressed genes(DEGs)were identified between CON vs A group,whereas,a total of 38 DEGs were identified between B vs C groups.Potential candidate genes identified were associated with central nervous system development,energy metabolism,and feed intake in chickens.Therefore,this study unveiled that L-Cit influenced hypothalamic networks related to brain development under thermoneutrality whereas,hypothalamic networks controlling feeding behavior were affected during high-temperature conditions.6.Central and peripheral L-Citrulline injection influences the body temperature and nitric oxide generation in chickensBroiler chicks at 9 days old were given intracerebroventricular(ICV)injection of Saline(CON)or varying doses of L-Cit(1,2,or 4 ?g/ 10-?L)and monitored for 2 h.In another trial,broilers at 26 days old were given intraperitoneal(IP)injections of Saline(CON)or L-Cit(200mg/kg BW)and studied for 5h.Chicks responded to ICV L-Cit differentially with increased body temperature at the lowest dose,whereas,the body temperature was suppressed at higher doses(P< 0.05).Administration of IP L-Cit promoted the t NOS and i NOS enzymes(P < 0.05),and upregulated i NOS and n NOS m RNA expression in the hypothalamus(P< 0.05).Therefore,this study demonstrated that central and peripheral injection of L-Cit would influence the body temperature and nitric oxide synthesis in broilers.7.L-Citrulline influences body temperature and inflammation during nitric oxide synthase inhibition and endotoxin challengeIn experiment 1,one day old chicks were fed CON or L-Cit diets for 14 days,then IP injected with Saline or NW-nitro-L-arginine methyl ester(L-NAME,150 mg/kg BW).In experiment 2,21 days old broilers were fed CON or L-Cit diets until 42 days of age,then IP injected with Saline or Lipopolysaccharide(LPS,2mg/ kg BW).In experiment 3,one day old chicks were fed CON or L-Cit diets for 26 days,then IP injected with Saline,L-NAME,LPS,or L-NAME + LPS.Results showed that the RT,ET,and CBT were decreased by L-NAME injection,whereas,elevated by LPS injection.Similarly,L-NAME decreased plasma NO concentration,whereas LPS promoted NO levels and increased PGE2 concentration.L-Cit diet suppressed the mean CBT and interacted with L-NAME to lower the mean CBT of chickens in experiment 1.L-Cit also suppressed the RT,mean RT,NO and PGE2 concentration in experiment 3.Therefore,this study demonstrated that L-Cit-induced hypothermia was related to NOS inhibition,NO deficiency,and decreased PGE2 concentration in broiler chickens.In conclusion,these studies reveal that L-Citrulline directly regulates systemic arginine metabolism and nitric oxide synthesis in chickens.L-Citrulline supplementation induces arginine sparing effects by promoting intestinal morphology and amino acid transport,thus serving as a potential substrate for arginine replacement in poultry nutrition.L-Citrulline can exert anabolic effects on body growth via its stimulation of GH/IGF-1 in circulation,as well as anti-heat stress effects by promoting the redox balance and immune responses of chickens.Importantly,LCitrulline can lower the body temperature due to its inhibition of nitric oxide synthesis and prostaglandin E2 production via the i NOS-COX2-PGE2 signaling pathway.