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The objective of this study was to evaluate the effect of 2 different doses of a partially buffered formic acid product (Amasil NA; 61% formic acid, 20.5% sodium formate), and a monoglyceride blend of short- and medium-chain fatty acids (BalanGut LS P) on necrotic enteritis ( NE ) infected broilers in terms of performance, intestinal microbial population and short-chain fatty acids concentrations in the gastrointestinal tract. A total of 528-day-old as hatched Ross 308 broilers were allocated to 48 pens with 11 birds in each pen. Six dietary treatments applied in the study were: T1) nonsupplemented diet (Control); T2) antibiotic supplemented diets; T3) and T4) high (Starter: 0.5%; Grower and Finisher: 0.5%) and low (Starter: 0.3%; Grower and Finisher: 0.2%) dose of Amasil NA; and groups T5) and T6) high (Starter: 0.3%; Grower and Finisher: 0.2%) and low dose (Starter: 0.3%; Grower: 0.15%; Finisher: 0.075%) of (BalanGut LS P). All birds in this study were fed starter (d 0-10), grower (d 11–24) and finisher (d 25–35) diets and challenged with NE. To induce subclinical NE, oral administrations of Eimeria oocysts (d 9) followed by inoculation of Clostridium perfringens strains (d 14 and 15) were applied. Results showed that birds fed the high dose of Amasil NA, had a higher feed conversion ratio ( FCR, P < 0.05) compared to the nonsupplemented group during the starter period. Antibiotic supplementation reduced FCR during the grower ( P < 0.001), finisher ( P < 0.05) and overall ( P < 0.001) periods of the experiment. Both levels of BalanGut LS P and low levels of Amasil NA enhanced overall FCR ( P < 0.05) compared to the birds in the nonsupplemented group. Compared to the nonsupplemented group, high levels of Amasil NA and low levels of BalanGut LS P improved FCR in the finisher stage ( P < 0.05). On d 16, cecum digesta of birds fed with antibiotic supplemented diets showed a significantly lower number of C. perfringens ( P < 0.001) compared to the nonsupplemented and high level of BalanGut LS P group. Bacillus ( P < 0.01) and Ruminococcus numbers were significantly lower in the birds fed with high level of Amasil NA ( P < 0.05) compared to the antibiotic supplemented diets. High doses of Amasil NA, showed the highest propionate concentration in the cecum ( P < 0.001). The study suggests that supplementation of BalanGut LS P and Amasil NA at different feeding phases may achieve optimal performance improvement in broilers under NE challenge.

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.

Two studies were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 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 × 10 6 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 × 10 6 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 supplemented 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.

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The primary cause of necrotic enteritis ( NE ) disease in chickens is the NetB-positive Clostridium perfringens bacterium. Many factors are known to affect the severity of NE in the challenge models of broiler chickens, and one of these factors is the virulence of C. perfringens strain. This study was conducted to evaluate the effect of 2 pathogenic C. perfringens strains in a NE challenge model on gut health and mRNA expression of genes encoding apoptosis, tight junction, immunity, and nutrient transporters in broilers. Day-old Ross-308 male broilers ( n = 468) were allocated in a 2 × 3 factorial arrangement of treatments with in-feed antibiotics (no or yes) and challenge (Non, C. perfringens strain NE18, and C. perfringens strain NE36) as the factors. The birds in the challenged groups were inoculated with Eimeria species on day 9 and with a fresh suspension of C. perfringens NE18 or NE36 on day 14 and 15. Sample collection was performed on 2 birds of each pen on day 16. Necrotic enteritis challenge, impaired feed conversion ratio during day 0 to 16 compared with the control group where the effect of the NE36 challenge was more severe than that with NE18 ( P < 0.001). The mRNA expression of mucin-2, immunoglobulin-G, occludin ( P < 0.001), and tight junction protein-1 ( P < 0.05) genes were downregulated in both challenged groups compared with the nonchallenged counterparts. Antibiotic supplementation, on the other hand, increased weight gain, and feed intake in all challenged birds ( P < 0.01), but upregulated mucin-5ac and alanine, serine, cysteine, and threonine transporter-1 ( P < 0.05) only in the NE18 challenged birds. The challenge with NE36 significantly upregulated caspase-8 and claudin-1 ( P < 0.001), but downregulated glucose transporter-2 ( P < 0.001) compared with the NE18 challenge. These results suggest that NE challenge is detrimental to the performance of broilers through compromised intestinal health, and different C. perfringens strains can affect the severity of the disease through modulating the expression of intestinal genes encoding proteins responsible for apoptosis, gut integrity, immunity, mucus production, and nutrient transporters.

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 10 8 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.

Identification of sex in broiler chickens allows researchers to reduce the level of variation in an experiment caused by the sex effect. Broiler breeds commonly used in research are no longer feather sexable because of the change in their genetics. Other alternate sexing methods are costly and difficult to apply on a large scale. Therefore, a sexing method is required that is both cost effective and highly sensitive as well as having the ability to offer high throughput genotyping. In this study, high-resolution melting ( HRM ) analysis was used to detect DNA variations present in the gene chromodomain helicase DNA binding 1 protein ( CHD1 ) on the Z and W chromosomes ( CHD1Z and CHD1W , respectively) of chickens. In addition, a simplified DNA extraction protocol, which made use of the basal part of chicken feathers, was developed to speed up the sexing procedure. Three pairs of primers, that is, CHD1UNEHRM1F/R, CHD1UNEHRM2F/R, and CHD1UNEHRM3F/R, flanking the polymorphic regions between CHD1Z and CHD1W were used to differentiate male and female chickens via distinct melting curves, typical of homozygous or heterozygous genotypes. The assay was validated by the HRM-sexing of 1,318 broiler chicks and verified by examining the sex of the birds after dissection. This method allows for the sexing of birds within a couple of days, which makes it applicable for use on a large scale such as in nutritional experiments.