Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis

Background Necrotic enteritis(NE)is a major enteric disease in poultry,yet effective mitigation strategies remain elusive.Deoxycholic acid(DCA)and butyrate,two major metabolites derived from the intestinal microbiota,have independently been shown to induce host defense peptide(HDP)synthesis.However,the potential synergy between these two compounds remains unexplored.Methods To investigate the possible synergistic effect between DCA and butyrate in regulating HDP synthesis and barrier function,we treated chicken HD11 macrophage cells and jejunal explants with DCA and sodium butyrate(NaB),either individually or in combination,for 24 h.Subsequently,we performed RNA isolation and reverse transcrip-tion-quantitative PCR to analyze HDP genes as well as the major genes associated with barrier function.To further determine the synergy between DCA and NaB in enhancing NE resistance,we conducted two independent trials with Cobb broiler chicks.In each trial,the diet was supplemented with DCA or NaB on the day-of-hatch,followed by NE induction through sequential challenges with Eimeria maxima and Clostridium perfringens on d 10 and 14,respectively.We recorded animal mortality after infection and assessed intestinal lesions on d 17.The impact of DCA and NaB on the microbiota in the ileum and cecum was evaluated through bacterial 16S rRNA gene sequencing.Results We found that the combination of DCA and NaB synergistically induced multiple HDP genes in both chicken HD11 cells and jejunal explants.Additionally,the gene for claudin-1,a major tight junction protein,also exhibited synergistic induction in response to DCA and NaB.Furthermore,dietary supplementation with a combination of 0.75 g/kg DCA and 1 g/kg NaB led to a significant improvement in animal survival and a reduction in intestinal lesions compared to either compound alone in a chicken model of NE.Notably,the cecal microbiota of NE-infected chickens showed a marked decrease in SCFA-producing bacteria such as Bacteroides,Faecalibacterium,and Cuneatibacter,with lactobacilli becoming the most dominant species.However,supplementation with DCA and NaB largely restored the intestinal microbiota to healthy levels.Conclusions DCA synergizes with NaB to induce HDP and claudin-1 expression and enhance NE resistance,with potential for further development as cost-effective antibiotic alternatives.

Dietary Macleaya cordata extract supplementation improves the growth performance and gut health of broiler chickens with necrotic enteritis

Background The poultry industry needs effective antibiotic alternatives to control outbreaks of necrotic enteritis(NE)caused by Clostridium perfringens.Methods The aim of this study was to investigate the effects of dietary supplementation with Macleaya cordata extract(MCE)on the immune function and gut microbiota of broilers with NE.A total of 2881-day-old broiler chicks were randomly assigned to a 2×2 factorial arrangement with two concentrations of dietary MCE supplementation(0 or 350 mg/kg of diet)and two disease challenge statuses(control or NE).Results The results revealed that NE significantly increased the feed conversion rate(FCR),mortality,intestinal lesion score,the levels of IL-1β,IL-17 and IFN-γ/IL-4 in serum and IL-17/IL-10 in the jejunal mucosa,m RNA levels of TLR2,IFN-γand p Ig R in the jejunum,and Clostridium perfringens concentrations in the cecum.NE significantly decreased the body weight(BW),body weight gain(BWG),jejunal villus height,V/C,m RNA level of AMPK-α1 in jejunum,IL-4 level in the jejunal mucosa and lactic acid bacteria abundance in the cecum.MCE significantly increased BW,BWG,jejunal villus height,V/C,m RNA levels of occludin,ZO-1 and AMPK-α1 in the jejunum,the levels of Ig A and Ig G in serum and IL-10 in the jejunal mucosa and m RNA levels of NF-κB,IL-10 and MHC-II in the jejunum.Additionally,MCE significantly decreased the FCR,mortality,intestinal lesion score,jejunal crypt depth,the levels of IFN-γand IL-17 in serum and IL-17/IL-10 in the jejunal mucosa,Clostridium perfringens concentrations in the cecum,and m RNA levels of IL-17/IL-10 in the jejunum.Moreover,NE significantly increased the abundance of bacteria that are associated with inflammation,obesity and depression(Alistipes,Barnesiella,Intestinimonas,RF39 and UCG-005)and significantly decreased the abundance of short-chain fatty acid(SCFA)-producing bacteria(Anaerotruncus,Butyricicoccus and Bacteroides)in the cecum.MCE significantly increased the abundance of SCFA-producing bacteria(Streptococcus,Ruminococcus_torques_group and Lachnospiraceae_NK4A136_group)and significantly reduced the abundance of bacteria that are associated with inflammation and obesity(Alistipes,Barnesiella and UCG-010)in the cecum.In the cecum of broilers with NE,the relative abundance of Barnesiella and Alistipes was higher and that of Lachnoclostridium and Shuttleworthia was lower.Interestingly,these trends were reversed by the addition of MCE to the diet.Spearman correlation analysis showed that Barnesiella and Alistipes were associated with enhanced intestinal inflammation and inhibited growth performance,whereas Lachnoclostridium and Shuttleworthia were associated with anti-inflammatory effects.Conclusions MCE ameliorated the loss of growth performance in broiler chickens with NE,probably by regulating the intestinal barrier,immune function,and gut microbiota.

Effects of tannic acid on the immunity and intestinal health of broiler chickens with necrotic enteritis infection

Background In broiler chickens,necrotic enteritis(NE)infection can reduce production performance.Tannic acid has shown great potential as a treatment of NE in broilers.However,the appropriate dosage of tannic acid in NE of broilers and the improvement effect on intestinal health are not very clear.In this study,we aimed to investigate the effects of different doses of tannic acid on the production performance,immunity,and intestinal health of broilers by constructing an NE model with C.perfringens infection and determining the appropriate dosage of tannic acid with regard to NE.Results Challenged birds showed significant reduction in body weight,villus height,and the ratio of villus height to crypt depth(P<0.05)and increase in the feed consumption gain ratio,intestinal lesion score,and crypt depth(P<0.05).The infection significantly reduced the relative Bacteroidota and Ligilactobacillus abundance(P<0.05)and increased the ratio of Firmicutes/Bacteroidota and cecal content of C.perfringens(P<0.05).Challenged birds fed diets supplemented with tannic acid showed significantly increased mRNA expression of nutrient transport carriers and intestinal barrier genes and growth performance and reduced serum zonulin and endotoxin levels(P<0.05).Addi-tion of tannic acid to the diet inhibited the inflammatory response by reducing the number of coccidia oocysts in feces and the content of C.perfringens in the cecum.Specifically,tannic acid reduced the serum levels of C reactive protein,myeloperoxidase,and specific IgY and ileal mucosal secretory immunoglobulin A levels in the ileal mucosa compared with those in the NE-infected birds.NE-infected birds fed diets supplemented with tannic acid also showed significantly increased relative Anaerocolumna,Thermoanaerobacterium,and Thermosinus abundance(P<0.05);their microbial composition and functional predictions were similar to those of the NC group.Conclusions Tannic acid in the diet alleviated NE by enhancing the intestinal barrier and absorption function.The recommended dietary tannic acid additive level is 500–750 mg/kg.Our study findings would be useful in reducing related economic losses in the broiler industry.

Stimbiotic supplementation modulated intestinal inflammatory response and improved broilers performance in an experimentally-induced necrotic enteritis infection model

Background:Two experiments were conducted to establish an optimal NE challenge model and evaluate the efficacy of stimbiotic(STB)supplementation in necrotic enteritis(NE)challenged broilers.In Exp.1,a total of 120 Arbor Acres(AA)broilers(45.0±0.21 g)were randomly assigned to 6 treatments in a 3×2 factorial arrangement.Vaccine treatments included non-challenge(0),×10 the recommended dose(×10)or×20 the recommended dose(×20)by the manufacturer.Clostridium perfringens(CP)treatments were non-challenge(No)or 3 mL of 2.2×10^(7)CFU CP challenge(Yes).In Exp.2,a total of 72 AA broilers(40.17±0.27 g)were randomly assigned to 6 treatments in a 3×2 factorial arrangement.Dietary treatments included non-additive(CON),100 mg/kg STB(STB)and 100 mg/kg STB on top of a typical commercial blend including an essential oil,probiotics,and enzyme(CB).Challenge treatments included non-NE challenge(No)and NE challenge(Yes)as established in Exp.1.Results:In Exp.1,CP and vaccine challenge decreased(P<0.05)body weight(BW),body weight gain(BWG)and feed intake(FI),and increased(P<0.05)the number of broilers with diarrhea and intestinal lesions.The oral administration of×20 recommended dose of vaccines coupled with 3 mL of 2.2×10^(7)CFU CP resulted in(P<0.01)a significantly increased incidence of wet litter and intestinal lesions.Thus,this treatment was chosen as the challenge model for the successful inducement of NE in Exp.2.In Exp.2,the NE challenge negatively affected(P<0.01)growth performance,ileal morphology,immunoglobulin contents in blood,caecal microbiota in the caecum,footpad dermatitis,intestinal lesion scores,tumour necrosis factor(TNF-α)and endotoxin in the serum compared with the non-NE challenged birds.The supplementation of STB and CB in diets enhanced(P<0.05)growth performance,intestinal microbiota,and blood profiles by stimulating ileal morphology(VH and VH:CD)and propionate production in the cecum,and there were no differences in measured variables between STB and CB supplemented birds.Conclusion:Overall,these results indicate that STB supplementation was able to reduce the inflammatory response and improve the performance of NE challenged birds,and the supplementation of STB alone was as effective as a typical commercial blend containing a number of other additives.

Essential Oils as an Alternative to Antibiotics to Reduce the Incidence and Severity of Necrotic Enteritis in Broiler Chickens: A Short Review

Due to the removal of antibiotic growth promoters (AGPs) and consumer pressure for antibiotic-free (ABF) or no antibiotics ever (NAE) poultry production, there is a need for sustainable alternatives to prevent disease in commercial poultry operations. Without AGPs, there has been a rise in diseases that were traditionally controlled by subtherapeutic levels of antibiotics in the diet. This has impacted the health of commercial poultry and has been a significant cost to poultry producers. To mitigate this, the industry has started to investigate alternatives to antibiotics to treat these forthcoming health issues, such as necrotic enteritis (NE). NE is an enteric disease caused by an over proliferation of toxigenic Clostridium perfringens (CP) in the gastrointestinal tract. Although CP is a commensal in the avian intestinal tract, dysbiosis caused by inflammation and impaired intestinal integrity facilitates uncontrolled replication of CP. Infectious agents, such as Eimeria maxima, appear to be a predominant predisposing factor that promotes NE. However, non-infectious stressors, including dietary changes, have also been associated with NE to some degree. As a result of increased pressure to restrict the use of antibiotics, there is a need for research evaluating the efficacy of alternatives, such as plant-derived essential oils, as potential tools to mitigate NE in commercial poultry flocks. The aim of this study is to review the effects of essential oils as an alternative to antibiotics to reduce the incidence and severity of necrotic enteritis in broiler chickens.

Modulations of genes related to gut integrity,apoptosis,and immunity underlie the beneficial effects of Bacillus amyloliquefaciens CECT 5940 in broilers fed diets with different protein levels in a necrotic enteritis challenge model

Background:The ban of in-feed antimicrobial additives has negatively affected the poultry industry by causing necrotic enteritis(NE)to emerge in the flocks.Alternatives such as Bacillus probiotics have shown to be effective on eliminating the negative effects of this disease.Two experiments were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940(BA)in broiler chickens under NE challenge and/or fed diets with different protein levels.Methods:In both experiments,480 day-old mix-sexed Ross-308 broilers were arranged in a 2×2 factorial arrangement of treatments.In experiment 1,the factors were NE challenge(yes or no)and probiotic(yes or no).In experiment 2,the factors were dietary crude protein levels(standard or reduced)and probiotic(yes or no)and were used under NE challenge condition.Oral administration of Eimeria oocysts(day 9)followed by inoculation with Clostridium perfringens(day 14 and 15)was used to induce NE challenge.On day 16,two birds from each treatment were gavaged with fluorescein isothiocyanate-dextran(FITC-d)and blood samples were collected for gut integrity evaluation,and jejunal samples were collected for gene expression assay.Results:In experiment 1,BA supplementation decreased caspase-3(CASP3)(P<0.001)and caspase-8(CASP8)(P<0.05)and increased occludin(OCLD)(P<0.05)expression regardless of the challenge.Additionally,BA supplementation downregulated interfron-γ(IFN-γ)expression(P<0.01)and upregulated immunoglobulin-G(IgG)(P<0.01)and immunoglobulin-M(IgM)(P<0.05)only in challenged birds.In experiment 2,the expression of genes encoding mucin-2(MUC2)(P<0.001),tight junction protein-1(TJP1)(P<0.05)and OCLD(P<0.05)were upregulated by the addition of BA in the diet,regardless of the crude protein level.Further,BA supplementation downregulated INF-γ(P<0.01)and upregulated immunoglobulin-A(IgA)(P<0.05),IgM(P<0.05)and IgG(P<0.01)regardless of the crude protein level.Conclusion:These findings suggest that supplementation of BA in broiler diets can improve gut health by modulation of genes related to the mucosal barrier,tight junction,and immunity in broilers challenged by unfavourable conditions such as NE challenge.

Butyrate in combination with forskolin alleviates necrotic enteritis,increases feed efficiency,and improves carcass composition of broilers

Background:The emergence of antimicrobial resistance has necessitated the development of effective alternatives to antibiotics for livestock and poultry production.This study investigated a possible synergy between butyrate and forskolin(a natural labdane diterpene)in enhancing innate host defense,barrier function,disease resistance,growth performance,and meat quality of broilers.Methods:The expressions of representative genes involved in host defense(AvBD9 and AvBD10),barrier function(MUC2,CLDN1,and TJP1),and inflammation(IL-1β)were measured in chicken HD11 macrophages in response to butyrate and forskolin in the presence or absence of bacterial lipopolysaccharides(LPS).Intestinal lesions and the Clostridium perfringens titers were also assessed in C.perfringens-challenged chickens fed butyrate and forskolincontaining Coleus forskohlii(CF)extract individually or in combination.Furthermore,growth performance and carcass characteristics were evaluated in broilers supplemented with butyrate and the CF extract for 42 d.Results:Butyrate and forskolin synergistically induced the expressions of AvBD9,AvBD10,and MUC2 in chicken HD11 cells(P<0.05)and the synergy was maintained in the presence of LPS.Butyrate and forskolin also suppressed LPS-induced IL-1βgene expression in HD11 cells in a synergistic manner(P<0.05).The two compounds significantly reduced the intestinal lesions of C.perfringens-challenged chickens when combined(P<0.05),but not individually.Furthermore,butyrate in combination with forskolin-containing CF extract had no influence on weight gain,but significantly reduced feed intake(P<0.05)with a strong tendency to improve feed efficiency(P=0.07)in a 42-d feeding trial.Desirably,the butyrate/forskolin combination significantly decreased abdominal fat deposition(P=0.01)with no impact on the carcass yield,breast meat color,drip loss,or pH of d-42 broilers.Conclusions:Butyrate and forskolin has potential to be developed as novel antibiotic alternatives to improve disease resistance,feed efficiency,and carcass composition of broilers.

Dietary ellagic acid ameliorated Clostridium perfringens-induced subclinical necrotic enteritis in broilers via regulating inflammation and cecal microbiota

Background: Subclinical necrotic enteritis(SNE), a common intestinal disease of broiler caused by Clostridium perfringens, could reduce production performance of broilers by chronic intestinal damage and poor absorption of nutrients. Ellagic acid(EA) has been reported to present antioxidant and anti-inflammatory properties on human and animals in many aspects. This study was conducted to evaluate the effect and mechanism of EA in relieving SNE in broilers induced by C. perfringens.Results: C. perfringens challenge decreased body weight(BW), average daily gain(ADG), jejunal villi height/crypt depth(V/C) ratio, the activity of catalase(CAT) and the mRNA expression of zonula occludens 1(ZO-1) in jejunal mucosa of broilers. While feed conversion ratios(FCR), jejunal crypt depth(CD), the activities of myeloperoxidase(MPO) and diamine oxidase(DAO), as well as the concentrations of interleukin 6(IL-6), C-reactive protein(CRP) and procalcitonin(PCT) in serum, the activities of inducible nitric oxide synthase(iNOS) and lysozyme(LZM), the concentration of malondialdehyde(MDA), and the mRNA expressions of claudin-2, TNF-α, IL-1β, TLR-4, TLR-2, NF-κB,JAK3, STAT6 and iNOS in jejunal mucosa of broilers were increased by C. perfringens challenge. Dietary EA supplement relieved these adverse effects, and heightened jejunal villi height(VH), the concentration of D-xylose in plasma, activity of superoxide dismutase(SOD), and the mRNA expression of occludin in jejunal mucosa of broilers.The alpha diversity of cecal microbiota indicated that dietary EA supplement increased observed species and Shannon index. C. perfringens challenge increased the relative abundance of Firmicutes and decreased the relative abundance of Desulfobacterota in cecal microbiota. EA increased the relative abundance of Firmicutes in cecal microbiota. LEfSe analysis showed that C. perfringens challenge triggered the imbalance of cecal microbiota in broilers, dietary EA supplementation led to a small beneficial effect on microbiota, while the simultaneous effect of them seemed to stimulate the immune function of broilers by improving the microbiota balance.Conclusions: Dietary EA ameliorated C. perfringens-induced SNE in broilers via regulating jejunal inflammation signaling pathways TLR/NF-κB and JAK3/STAT6, relieving jejunal oxidative stress and balancing cecal microbiota to inhibit intestinal barrier damage, prevent systemic inflammatory response and improve nutrient absorption capacity,finally protect and enhance growth performance of broilers.

Perturbations of the ileal mycobiota by necrotic enteritis in broiler chickens

Background:Intestinal microbiota is critical for maintaining animal health and homeostasis.However,involvement of the fungal community,also known as the mycobiota,in animal health and disease is poorly understood.This study was aimed to examine the association between the intestinal mycobiota and the severity of necrotic enteritis(NE),an economically significant poultry disease.Methods:A total of 90 day-of-hatch Cobb broilers were infected with Eimeria maxima on d 10,followed by an oral challenge with C.perfringens on d 14 to induce NE,while another 10 broilers were served as mock-infected controls.On d 17,the lesions in the jejunum were scored,and the ileal digesta were subjected to DNA isolation and real-time PCR quantification of total bacterial and fungi populations.Internal transcribed spacer 2(ITS2)amplicon sequencing was also performed to profile the ileal mycobiota composition.Changes in the ileal mycobiota in response to NE were investigated.Spearman correlation analysis was further conducted to identify the correlations between relative abundances of individual ileal fungi and the severity of NE.Results:While the total bacterial population in the ileum was increased by 2-to 3-fold in NE chickens,the total fungal population was progressively declined in more exacerbated NE,with the most severely infected chickens showing a nearly 50-fold reduction relative to mock-infected controls.Richness of the ileal mycobiota also tended to reduce in chickens with NE(P=0.06).Compositionally,among 30 most abundant fungal amplicon sequence variants(ASVs),11 were diminished and 7 were enriched(P<0.05),while 12 remained largely unchanged in NEafflicted chickens(P>0.05).Multiple Wallemia and Aspergillus species were markedly diminished in NE(P<0.05)and also showed a significant negative correlation with NE severity(P<0.05).Conclusions:Dysbiosis of the ileal mycobiota is induced evidently by NE and the extent of the dysbiosis is positively correlated with disease severity.These findings suggest a possible role of the intestinal mycobiota in NE pathogenesis and highlight the mycobiota as a new potential target for NE mitigation in poultry.

Necrotic enteritis and antibiotic-free production of broiler chickens:Challenges in testing and using alternative products

The global trend towards raising broiler chickens without the use of in-feed antibiotics(IFAs)means that there is an ongoing need to develop alternative treatments capable of delivering the benefits that IFAs previously provided.IFAs supported the productivity performance of chickens and played a key role in maintaining their health.Necrotic enteritis(NE)is an important disease of broilers that affects health,productivity,and welfare,and was previously well controlled by IFAs.However,with the reduction in IFA use,NE is resurgent in some countries.Vaccines and various feed additives,including pre-,pro-,and postbiotics,phytobiotics,fatty acids,and phage therapies have been introduced as alternative methods of NE control.While some of these feed additives have specific activity against the NE pathogen,Clostridium perfringens,most have the more general goal of reinforcing gut health.Extensive reviews of the effects of many of these feed additives on gut health have been published recently.Hence,rather than cover previously well reviewed areas of research this review focuses on the challenges and pitfalls in undertaking experimental assessment of alternative NE treatments and translating laboratory research to real world commercial production settings.The review is based on the author's particular experience,reading,thoughts,and analysis of the available information and inevitably presents a particular understanding that is likely to be at odds with others thinking on these issues.It is put forward to stimulate thinking and discussion on the issues covered.

Effect of oat hulls as a free choice feeding on broiler performance,short chain fatty acids and microflora under a mild necrotic enteritis challenge

Structure of fibre has been reported to enhance performance,intestinal function and modify the composition and quantity of the microbial population in the chicken gastrointestinal tract.It is hypothesised that insoluble fibre in oat hulls may improve gut health and reduce intestinal Clostridium perfringens number.This research assessed the effect of free choice oat hulls(OH) on performance and gut microbiota in broilers during a mild(subclinical) necrotic enteritis(NE) challenge.A total of 240 dayold male Ross 308 broiler chickens were assigned to 24 cages in a 2×2 factorial arrangement of treatments.Factors were challenge - or +; and OH - or +.On d 16,challenged broilers had lower weight gain and feed intake(P < 0.05) compared with unchallenged broilers.Ond 16,broilers given OH had lower feed intake(P < 0.05) and tended to have lower(P=0.062) feed conversion ratio(FCR) compared with those without access to OH.Broiler performance,however,was not affected by OH nor by challenge on d 24 and 35.The broilers given OH had heavier gizzards(P < 0.05) compared with those without OH at d 35 but not at d 13 or 16.Increased numbers of C.perfringens(P < 0.001) and reduced numbers(P < 0.05)of Lactobacillus and Salmonellae were observed in the caecal contents of challenged broilers on d 16.Challenged broilers had a lower concentration of caecal acetic acid(P < 0.01) compared with unchallenged broilers at d 16.The broilers given OH had lower concentrations of caecal acetic acid(P < 0.05),propionic acid(P < 0.05),and valeric acid(P < 0.01) compared with those without access to OH.An OH by challenge interaction on succinic acid concentration was observed on d 16(P < 0.05).Oat hulls elevated the caecal succinic acid concentration only in the unchallenged broilers.This study indicated a positive role of OH through improved gizzard function and increased succinic acid in the gut but its role in controlling NE was not conclusive.

Effects of yeast cell wall on growth performance, immune responses and intestinal short chain fatty acid concentrations of broilers in an experimental necrotic enteritis model

Subclinical necrotic enteritis(NE) causes devastating economic losses in the broiler chicken industry,especially in birds raised free of in-feed antibiotics. Prebiotics are potential alternatives to in-feed antibiotics. Yeast cell wall extract(YCW) derived from Saccharomyces cerevisiae is a prebiotic with known immune modulating effects. This study examined the effects of YCW and antibiotics(AB) during subclinical NE on broiler growth performance, intestinal lesions, humoral immune response and gut microflora metabolites. The study employed a 2 × 3 factorial arrangement of treatments. Factors were:NE challenge(yes or no) and feed additive(control, AB, or YCW). Each treatment was replicated in 8 floor pens with 15 birds per pen. Challenged birds had higher feed conversion ratio(FCR) than unchallenged birds on d 35(P < 0.05). Dietary inclusion of AB decreased FCR regardless of challenge(P < 0.05) on d 24 and 35. Inclusion of YCW reduced serum interleukin-1(IL-1) concentration in NE challenged birds(P < 0.01) and increased immunoglobulin(Ig) G(P < 0.05) and Ig M(P < 0.05) levels compared to other dietary treatments regardless of challenge. Yeast cell wall extract increased formic acid concentration in cecal contents during challenge and increased butyric acid concentration in unchallenged birds on d 16.This study indicates YCW suppressed inflammatory response, promoted generation of immunoglobulin and increased short chain fatty acid production suggesting potential benefits to bird health.

Potential of blended organic acids to improve performance and health of broilers infected with necrotic enteritis

Organic acids(OA)and their blends have been shown to positively affect performance and health of broilers.However,the data in the literature are not consistent.This study examined the potential of blended short-chain fatty acids(SCFA)with medium-chain fatty acids(MCFA)as alternatives to antibiotic growth promoters(AGP)on performance,health and welfare of broilers infected with necrotic enteritis(NE).The additives used were:A)a blend of SCFA,MCFA,and a phenolic compound(SMP);B)a blend of free and buffered SCFA with MCFA(SMF);C)a blend of free and buffered SCFA with a high concentration of MCFA(SHM).A total of 1,404 Ross 308 one-day-old male parental chicks were randomly distributed into 78-floor pens with 13 replicates of 18 birds each.Six treatments were the following:T1,unchal-lenged control(UCC);T2,challenged control(CHC);T3,challenged group plus zinc bacitracin(BAC);T4,challenged group plus additive SMP;T5,challenged group plus additive SMF;T6,challenged group plus additive SHM.Challenged birds were gavaged with Eimeria spp.on d 9 and Clostridium perfringens EHE-NE18 on d 14.Post NE challenge and cumulatively,BWG,FCR,and nutrient digestibility of birds were compromised(P<0.05)by NE challenge indicating a successful induction of sub-clinical NE.Additive SHM had higher BWG compared to CHC and BAC groups(P=0.001;d 10 to 24)but not different from SMP and SMF groups(P>0.05).All the 3 additive groups had lower FCR compared to CHC(P=0.001;d 0 to 35),and exhibited similar jejunal lesions(d 16)compared to BAC and apparent ileal protein di-gestibility(d 21)compared to UCC and BAC groups(P>0.05).Birds in additive SHM group had a higher concentration of serum IgA compared to all groups(P=0.001)except additive SMF(P>0.05;d 21).All the additive groups had lower footpad dermatitis and hock burns compared to CHC(P<0.05).The findings suggest the potential of blended OA as alternatives to BAC to protect broilers from NE indicated by improved FCR,immunity,digestibility,and bird welfare.

Influence of feeding crimped kernel maize silage on the course of subclinical necrotic enteritis in a broiler disease model

This experiment was carried out with 375 male broilers(Ross 308) from days 1 to 28 to evaluate the influence of crimped kernel maize silage(CKMS) on the manifestation of subclinical necrotic enteritis,microbiota counts, organic acid production and relative weights of gastrointestinal segments. A necrotic enteritis disease model was applied. Birds were allocated into 3 different dietary treatments: a maizebased feed(MBF, control diet), and 2 diets supplemented with 15%(CKMS15) or 30%(CKMS30) of crimped ensiled kernel maize. The disease model involved a 10-time overdose of an attenuated live vaccine against coccidiosis given orally on day 17, followed by oral inoculation of Clostridium perfringens Type A(S48,10~8 to 10~9 bacteria/bird) twice daily on days 18,19, 20 and 21. Scoring of intestinal lesions was performed on days 22, 23, 25 and 28. Ileal and caecal digesta samples were collected for the quantification of selected bacterial groups and organic acids. The results showed that there was no effect of dietary treatments on small intestinal lesion scores(P> 0.05). Lesions scores peaked on days 23 and 25 and decreased again on day 28(P = 0.001). No effect of age on microbiota counts was observed, but feeding of CKMS30 reduced the number of coliforms in ileal contents(P = 0.01). Dietary treatments did not affect organic acid concentrations in ileum and caeca, but there was an effect of age; butyric acid was higher on days 22, 23 and 25 than on day 28(P = 0.04). Acetic acid and propionic acid concentrations in caeca were the highest on days 22 and 28 but the lowest on days 23 and 25. Relative gizzard and caeca weights were increased, and relative ileum weights were decreased when birds were fed CKMS30(P < 0.05). In conclusion, the inclusion of CKMS in broiler diets had no effects on the course of necrotic enteritis but had potential benefits in terms of inhibition of potentially harmful microorganisms.

Bacillus amyloliquefaciens CECT 5940 improves performance and gut function in broilers fed different levels of protein and/or under necrotic enteritis challenge

Two studies were conducted to investigate the effect of Bacillus amyloliquefaciens CECr 5940(BA)as a probiotic on growth performance,amino acid digestibility and bacteria population in broiler chickens under a subclinical necrotic enteritis(NE)challenge and/or fed diets with different levels of crude protein(CP).Both studies consisted of a 2×2 factorial arrangement of treatments with 480 Ross 308 mix-sexed broiler chickens.In study 1,treatments included 1)NE challenge(+/),and 2)BA(1.0×106 CFU/g of feed)supplementation(+/-).In study 2,all birds were under NE challenge,and treatments were 1)CP level(Standard/Reduced[2%less than standard])and 2)BA(1.0×106 CFU/g of feed)supplementation(+/-).After inducing NE infection,blood samples were taken on d 16 for uric acid evaluation,and cecal samples were collected for bacterial enumeration.In both studies,ileal digesta was collected on d 35 for nutrient digestibility evaluation.In study 1,the NE challenge reduced body weight gain(BWG),supressed feed conversion ratio(FCR)and serum uric acid levels(P<0.001).Supplementation of BA increased BWG(P<0.001)and reduced FCR(P=0.043)across dietary treatments,regardless of challenge.Bacillus(P=0.030)and Ruminococcus(P=0.029)genomic DNA copy numbers and concentration of butyrate(P=0.017)were higher in birds fed the diets supplemented with BA.In study 2,reduced protein(RCP)diets decreased BWG(P=0.010)and uric acid levels in serum(P<0.001).Supplementation of BA improved BWG(P=0.001)and FCR(P=0.005)and increased Ruminococcus numbers(P=0.018)and butyrate concentration(P=0.033)in the ceca,regardless of dietary CP level.Further,addition of BA reduced Clostridium perfringens numbers only in birds fed with RCP diets(P=0.039).At d 35,BA sup-plemented diets showed higher apparent ileal digestibility of cystine(P=0.013),valine(P=0.020),and lysine(P=0.014).In conclusion,this study suggests positive effects of BA supplementation in broiler diets via modulating gut microflora and improving nutrient uptake.

Organic acid blends improve intestinal integrity,modulate short-chain fatty acids profiles and alter microbiota of broilers under necrotic enteritis challenge

Controlling enteric diseases of broilers is crucial.Among many additives,organic acids(OA)and their blends are gaining attention to combat diseases in the post-antibiotic era.The current study evaluated the potentials of short-chain fatty acids(SCFA)and medium-chain fatty acids(MCFA)blends and/or phenolic compounds on intestinal integrity,intestinal pH,caecal microbiota,and caecal SCFA profiles of broilers under necrotic enteritis(NE)challenge.The additives used were:(A)a blend of SCFA,MCFA,and a phenolic compound(SMP),(B)a blend of free and buffered SCFA with MCFA(SMF),and(C)a blend of free and buffered SCFA with a high concentration of MCFA(SHM).A total of 1,404 male parental chicks of Ross 308 broilers were randomly allocated to 78 floor pens on hatching day with 6 treatments replicated 13 times with 18 birds per pen.The treatments were:UCC,unchallenged control;CHC,challenged control;BAC,challenged group plus zinc bacitracin;SMP,challenged group plus additive SMP;SMF,challenged group plus additive SMF;SHM,challenged group plus additive SHM.Birds were challenged with field-strain Eimeria spp.on d 9 and Clostridium perfringens on d 14.Birds challenged with NE increased fluorescein isothiocyanate dextran(FITC-d)concentration in serum,reduced acetate and butyrate concentrations,and increased Bacteroides and C.perfringens load in the caeca(P<0.05).Birds fed additives decreased FITC-d from gut to serum,reduced Bacteroides(d 16,P<0.05)and numerically reduced C.perfringens load compared to CHC group.Birds fed additive SHM had higher concentrations of acetate and butyrate(d 21,P<0.05)than CHC group but were not different from SMP and SMF groups.All the additives exhibited similar intestinal protection against NE compared to the BAC group indicated by FITC-d concentration in serum,acetate,propionate and butyrate concentrations in the caeca,and caecal bacterial loads except for the C.perfringens(P>0.05).The SMP group had a higher load compared to BAC(P<0.05).These findings suggest the promising effects of OA blends as alternatives to BAC to ameliorate the impact of NE challenge of broilers as indicated by improved intestinal health.

Non-starch polysaccharide-degrading enzymes may improve performance when included in wheat- but not maize-based diets fed to broiler chickens under subclinical necrotic enteritis challenge

The present study investigated whether supplementing fibre-degrading enzymes can ameliorate the severity of subclinical necrotic enteritis(NE)in broiler chickens offered wheat-or maize-based diets.A total of 1,544 mixed-sex broiler chickens were assigned to 16 experimental treatments as a 2×2×4 factorial arrangement of treatments.The factors were the following:NE challenge,yes or no;diet type,wheat-or maize-based;and enzyme supplementation,control(no enzyme),family 10 xylanase(XYN10),family 11 xylanase(XYN11)or b-mannanase(MAN).Each treatment was replicated 6 times,with 16 birds per replicate pen.A three-way challenge×diet type×enzyme interaction occurred for body weight at 21 d of age(P=0.025)and overall feed conversion ratio(P=0.001).In the non-challenged birds fed the wheat-based diet,supplementing MAN increased d 21 body weight compared to the control.In challenged birds fed the maize-based diet,supplemental XYN11 impeded body weight and overall FCR compared to the control.Birds offered the maize-based diet presented heavier relative gizzard weights at both 16 and 21 d of age(P<0.001)and reduced liveability(P=0.046)compared to those fed the wheatbased diet.Enzyme supplementation reduced ileal and jejunal digesta viscosity at 16 d of age only in birds fed the wheat-based diet(P<0.001).XYN11 increased ileal digesta viscosity in birds fed the maizebased diet,and MAN reduced it in birds fed the wheat-based diet at 21 d of age(P=0.030).Supplementing XYN11 improved ileal soluble non-starch polysaccharides(NSP)digestibility in birds fed the wheat-based diet compared to non-supplemented birds(P<0.001).Birds fed the wheat-based diet displayed a higher abundance of Bifidobacterium,Lactobacillus and Enterobacteriaceae and butyric acid in the caeca at 16 d of age compared to birds fed the maize-based diet(P<0.05).In conclusion,supplemental XYN11 exacerbated the negative impact of NE on growth performance in birds fed the maizebased diet.Supplementing wheat-based diets with fibre-degrading enzymes ameliorates production losses induced by NE.

Potential of a mixture of eugenol and garlic tincture to improve performance and intestinal health in broilers under necrotic enteritis challenge

Plant extracts(PE)are gaining increased attention as potential alternatives to in-feed antimicrobials(AM)due to their known antimicrobial activities.This study was conducted to examine the potential of PE,a microencapsulated product composed of eugenol and garlic tincture as an alternative to AM-agent on performance and intestinal health in broilers under necrotic enteritis(NE)challenge.A total of 960 dayold mixed-sex Cobb 500 chicks were randomly distributed to 48-floor pens with 6 treatments replicated 8 times with 20 birds each.The 6 treatments were as follows:UC,unchallenged control;CC,challenged control;PE,challenged group plus PE;AM,challenged group plus AM;FAP,challenged group plus a full dose of AM with PE;HAP,challenged group plus a half dose of AM with PE in starter,grower and finisher phases.Birds in the challenged groups were inoculated with Eimeria spp.on d 9 and Clostridium perfringens on d 14.The body weight gain(BWG),feed intake(FI),feed conversion ratio(FCR),and livability of birds were compromised,and intestinal lesions and mortality were increased(P<0.05)by NE challenge,illustrating a successful clinical NE challenge.Birds fed AM had higher BWG and FI,and lower FCR,mortality,and intestinal lesions compared to the CC group(P<0.05).Birds fed PE had improved FCR(P<0.05)and livability(5.8%)in an overall period compared to the CC group.On d 16,PE supplementation reduced ileal lesion scores in only male birds(P<0.05).Birds fed PE had decreased Eimeria maxima and Eimeria acervulina oocyst counts in caecal content(P<0.05).Birds fed PE had decreased Eimeria brunetti and total oocyst counts in caecal content,and E.acervulina oocyst counts in ileal content in only female birds(P<0.05).On d 35,PE supplementation reduced variation of BW in both male and female birds and increased yellowness(b*value,14.4%)in the thigh.These findings suggest the potential of PE supplementation in diets to improve the performance and intestinal health of birds under clinical NE as indicated by improved FCR,livability,uniformity,reduced ileal lesions,oocyst counts and increased skin yellowness.However,the protective effect of PE may not be apparent in the presence of AM in the feed.

Ileal profile of non-starch polysaccharides and oligosaccharides in response to exogenous enzymes in broiler chickens offered wheat-or maize-based diets under subclinical necrotic enteritis challenge

The present study evaluated the impacts of fibre-degrading enzymes on the profiles of non-starch polysaccharides(NSP)and oligosaccharides(OS)in the ileum of broiler chickens offered wheat-or maize-based diets under subclinical necrotic enteritis(NE)challenge.A 224 factorial arrangement of treatments was used.Factors were the following:NE challenge,no or yes;diet type,wheat-or maizebased;and supplemental enzymes,control(no enzyme),family 10 xylanase(XYN10),family 11 xylanase(XYN11)or b-mannanase(MAN).Birds in the challenged group were inoculated with Eimeria on d 9 and Clostridium perfringens on d 14 and 15.A 3-way interaction(P=0.047)occurred on overall(d 0 to 16)weight gain.When NE was present,all the supplemental enzymes increased weight gain in birds fed the wheat-based diet;whereas in those fed the maize-based diet supplemental XYN10 and XYN11 decreased weight gain.When NE was absent,birds fed the wheat-based diet supplemented with XYN10 or MAN presented increased weight gain compared to non-supplemented birds,but no improvements with enzyme addition were observed in birds fed the maize-based diet.A 3-way interaction(P=0.002)was observed on insoluble NSP level in the ileum.When NE was absent,all the supplemental enzymes reduced the ileal level of insoluble NSP,regardless of diet type.In the challenged birds,supplementing XYN10 and MAN reduced insoluble NSP level in the ileum,but only in birds fed the wheat-based diet.Ileal soluble NSP level was reduced by supplemental XYN11 and MAN,but only in birds fed the wheatbased diet,resulting in a 2-way diet typeenzyme interaction(P<0.001).Ileal OS arabinose(P=0.030)level was highest in birds offered the wheat-based diet supplemented with XYN11.Collectively,supplementation of NSP-degrading enzymes to the wheat-based diet enhanced bird performance regardless of NE challenge,with XYN11 significantly increasing oligosaccharide release.However,enzyme addition did not improve growth performance in birds fed maize-based diet,with supplemental XYN10 and XYN11 impeding weight gain when NE was present.

Over-processed meat and bone meal and phytase effects on broilers challenged with subclinical necrotic enteritis: Part 2. Inositol phosphate esters hydrolysis, intestinal permeability, hematology,jejunal gene expression and intestinal morphology

This study investigated the hypothesis that feeding broilers over-processed meat and bone meal(MBM)would impair gut health in the absence of phytase and in turn,affect inositol phosphate(inositol xphosphate,IPx:IP3,IP4,IP5 and IP6)ester hydrolysis,intestinal permeability,hematology,jejunal gene expression and intestinal morphology during necrotic enteritis(NE).Ross 308 male broilers(n=768)were assigned to one of 8 dietary treatments in a 2×2×2 factorial arrangement,with 6 replicate pens per diet and 16 birds per pen in a completely randomized design.Factors were:NE challenge(no or yes),phytase level(500 or 5,000 FTU/kg)and MBM processing(as-received or over-processed).For the NE challenge,half of the birds were challenged with field strains of Eimeria spp.on d 9 and 108 CFU/mL of Clostridium perfringens strain EHE-NE18 on d 14 and 15.A 3-way challenge,phytase and MBM processing interaction was detected for IP5(P<0.05)and IP6(P<0.05)levels in the ileum.Birds fed low phytase had increased IP5 and IP6 in unchallenged birds only when diets contained over-processed MBM.Challenge with NE increased intestinal permeability as measured by serum fluorescein isothiocyanate dextran(FITC-d;P<0.001),increased white blood cells(WBC;P<0.001),decreased mean corpuscular volume(MCV;P<0.001)and mean corpuscular hemoglobin(MCH;P<0.05),and decreased crypt-tovilli ratio(P<0.05).The over-processed MBM reduced the villi-to-crypt ratio(P<0.05).A 3-way challenge x phytase x MBM processing interaction was detected for mucin 2(MUC-2)expression(P<0.05)where only in unchallenged birds fed over-processed MBM did high phytase reduce MUC-2 expression.A lower expression of aminopeptidase N(APN;P<0.001)and vitamin D receptor(VDR;P<0.001)were recorded in NE challenged birds.In conclusion,NE has a negative impact on the gut and hematology of broilers,but its effect on phytate hydrolysis is minimal.