Necrotic enteritis challenge regulates peroxisome proliferator-1 activated receptors signaling and β-oxidation pathways in broiler chickens

Necrotic enteritis (NE) is an important enteric disease in poultry and has become a major concern in poultry production in the post-antibiotic era. The infection with NE can damage the intestinal mucosa of the birds leading to impaired health and, thus, productivity. To gain a better understanding of how NE impacts the gut function of infected broilers, global mRNA sequencing (RNA-seq) was performed in the jejunum tissue of NE challenged and non-challenged broilers to identify the pathways and genes affected by this disease. Briefly, to induce NE, birds in the challenge group were inoculated with 1 mL of Eimeria species on day 9 followed by 1 mL of approximately 10⁸ CFU/mL of a NetB producing Clostridium perfringens on days 14 and 15. On day 16, 2 birds in each treatment were randomly selected and euthanized and the whole intestinal tract was evaluated for lesion scores. Duodenum tissue samples from one of the euthanized birds of each replicate (n = 4) was used for histology, and the jejunum tissue for RNA extraction. RNA-seq analysis was performed with an Illumina RNA HiSeq 2000 sequencer. The differentially expressed genes (DEG) were identified and functional analysis was performed in DAVID to find protein–protein interactions (PPI). At a false discovery rate threshold <0.05, a total of 377 DEG (207 upregulated and 170 downregulated) DEG were identified. Pathway enrichment analysis revealed that DEG were considerably enriched in peroxisome proliferator-activated receptors (PPAR) signaling (P < 0.01) and β-oxidation pathways (P < 0.05). The DEG were mostly related to fatty acid metabolism and degradation (cluster of differentiation 36 [CD36], acyl-CoA synthetase bubblegum family member-1 [ACSBG1], fatty acid-binding protein-1 and -2 [FABP1] and [FABP2]; and acyl-coenzyme A synthetase-1 [ACSL1]), bile acid production and transportation (acyl-CoA oxidase-2 [ACOX2], apical sodium–bile acid transporter [ASBT]) and essential genes in the immune system (interferon-, [IFN-γ], LCK proto-oncogene, Src family tyrosine kinase [LCK], zeta chain of T cell receptor associated protein kinase 70 kDa [ZAP70], and aconitate decarboxylase 1 [ACOD1]). Our data revealed that pathways related to fatty acid digestion were significantly compromised which thereby could have affected metabolic and immune responses in NE infected birds.     

Dietary hydrolysed yeast cell wall extract is comparable to antibiotics in the control of subclinical necrotic enteritis in broiler chickens

ABSTRACT

1. The aim of this study was to examine the effect of yeast cell wall (YCW) on performance and physiological responses of broiler chickens under subclinical necrotic enteritis challenge.

2. Six treatments in a 2 × 3 factorial arrangement (non-challenged or challenged plus no supplement, YCW or antibiotics (AB)) was used. Each treatment was replicated eight times with 12 birds per replicate. The treatments included: (1) Positive control (PC; no additive, not challenged); (2) Negative control (NC; no additive, with challenge); (3) YCWN = yeast cell wall (2.0 g/kg diet, not challenged; (4) YCWC = yeast cell wall (2.0 g/kg diet, challenged); (5) ABN = zinc bacitracin 50 ppm + Salinomycin 60 ppm, not challenged); (6) ABC = zinc bacitracin 50 ppm + Salinomycin 60 ppm, challenged).

3. Eimeria challenge at 9 d of age did not affect feed intake (FI), body weight gain (BWG), FCR or liveability at 10 d. The BWG and FCR at 10 d were greater (P < 0.05) in birds fed YCW or AB (AB) diets relative to the PC or NC groups. On 24 and 35 d, FI, BWG, FCR and flock uniformity (28 d) were greater (P < 0.05) in the challenged groups fed YCW or AB diets compared to NC group.

4. Supplementation with YCW ameliorated the negative effects of NE on liver, spleen and bursa weight of birds.

5. Necrotic enteritis challenge decreased (P < 0.05) caecal Lactobacillus and Bifidobacterium spp. counts, and increased ileum lesion score and caecal Clostridium perfirngens counts. This was reversed by the addition of either YCW or AB.

6. Supplementation with YCW and AB resulted to a greater (P < 0.05) dressing percentage and meat yield (35 d).

7. The results indicated that YCW plays a vital role in improving the physiological response and performance of broiler chickens under subclinical necrotic enteritis challenge.     

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.     

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 x-phosphate, 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-to-villi ratio (P < 0.05). The over-processed MBM reduced the villi-to-crypt ratio (P < 0.05). A 3-way challenge × phytase × 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.     

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.     

Over-processed meat and bone meal and phytase effects on broilers challenged with subclinical necrotic enteritis: Part 3. Bone mineralization and litter quality

This study was conducted to determine the effect of necrotic enteritis (NE), phytase level and meat and bone meal (MBM) processing on bone mineralization of broilers and litter quality. Ross 308 male broiler chicks (n = 768) were allotted to 48 pens with 16 birds each. There were 8 dietary treatments in a 2 × 2 × 2 factorial arrangement. Factors were NE challenge (no or yes), phytase level (500 or 5,000 FTU/kg), and MBM (as-received or over-processed). Half of the birds were challenged with field strains of Eimeria spp. at d 9 and 10⁸ CFU per mL of Clostridium perfringens strain EHE-NE18 on d 14 and 15. The middle toe, tibia and femur of 2 birds per pen were excised at d 16 and 29 for determination of ash, breaking strength (BS) and bone mineralization. At d 42, all were assessed for hock burns and litter was scored and assessed for dry matter (DM). At d 16, challenged birds had lower toe ash (P < 0.01), femur ash (P < 0.001), tibia ash (P < 0.001) and tibial BS (P < 0.001) than unchallenged birds. At d 16, challenged birds fed high phytase and over-processed MBM had higher toe Mn than those fed low phytase and as-received MBM. At d 29 unchallenged birds fed high phytase and as-received MBM had a higher toe Mn than those fed over-processed MBM. At d 16, a phytase × MBM interaction was detected for femur Zn concentration (P < 0.05), where a higher level of Zn was observed in the high phytase group fed over-processed MBM. At d 16, tibial Ca (P < 0.05) and P (P < 0.05) were lower in the challenged whereas the femur K (P < 0.001), Mn (P < 0.01) and Na (P < 0.001) were higher in the challenged at d 16. At d 42, challenged birds had higher litter DM (P = 0.058) and fewer hock burns than those unchallenged (P < 0.05). In conclusion, NE impaired bone traits but high phytase and over-processed MBM increased bone mineral contents. Cases of hock burns may be lower under NE incidences due to lower livability of birds reducing litter wetness.     

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, unchallenged 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 digestibility (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.     

Multi-carbohydrase enzymes improve feed energy in broiler diets containing standard or low crude protein

This study evaluated the effect of multi-carbohydrase (MC) on energy and nitrogen (N) balance and gene expression in broilers fed diets with different crude protein (CP) contents. The study employed a 2 × 2 factorial arrangement of treatments. The factors were presence or absence of MC, and standard (SCP) or low (LCP) dietary CP concentration. A 3-phase feeding program was used, including starter (0 to 7 d), grower (8 to 17 d) and finisher (18 to 28 d) phases. The study was undertaken in closed calorimetry chambers. Each of the 4 dietary treatments was replicated 8 times in total across 2 runs, with 2 birds per replicate (n = 64). Data for energy partitioning and N balance were collected from d 25 to 28. On d 28, birds were euthanized to collect muscle and intestinal tissue samples for gene expression. The results showed that the MC increased apparent metabolizable energy (AME, P < 0.01) and net energy (NE, P < 0.05), and reduced the feed conversion ratio (FCR, P < 0.01) in all diets. The proportion of energy retained as fat per total energy retention (REf/RE) was positively correlated with feed AME and NE (r = 0.541, P < 0.01 and r = 0.665, P < 0.001, respectively), suggesting that feed energy augmented with increased fat gain. Muscle ATP synthase subunit alpha (ATP5A1W) gene expression had a positive correlation with REf/RE and feed NE (r = 0.587, P < 0.001 and r = 0.430, P < 0.05, respectively). Similarly, muscle peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1A) expression was negatively correlated with weight gain and positively correlated with FCR (r = −0.451, P < 0.05 and r = 0.359, P < 0.05, respectively). These correlations show that over-expressions of muscle genes related to energy production reduce bird performance. This study demonstrated that MC increase dietary energy utilization, regardless of dietary CP concentration. However, the energy released by the enzymes increases feed energy-to-CP ratio, meaning there is excess energy that is then deposited as body fat. This suggests that supplemental MC in broiler feeds is beneficial if diets are formulated to contain marginal energy levels.