Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

The effect of sex and dietary crude protein level on nutrient transporter gene expression and cecal microbiota populations in broiler chickens.

It is well known that male and female broilers differ in their growth performance and that many physiological factors contribute to this difference. The aim of this experiment is to investigate if there are differences between male and female broilers in cecal microbiota and nutrient transporter gene expression and if these differences play a role in the growth performance of broilers. The possible effect of protein level and its interaction with sex on microbiota and expression of the nutrient transporters were also investigated. Samples were collected from male and female birds fed either standard crude protein (SCP) or reduced crude protein diets (RCP) at the age of d 35. The experiment was designed as a 2 × 2 factorial arrangement of treatments consisting of 448 Cobb 500 broilers assigned to 32-floor pens with 4 treatments, 8 replicates, and 14 birds per pen for performance measurements. The factors were sex (male or female) and dietary crude protein (CP) level (standard or reduced). Body weight gain (BWG), feed intake and feed conversion ratio were recorded for each pen. Sex had a significant effect on BWG and FCR (P < 0.001) where males had a significantly higher BWG and better FCR compared to females. There was a significant interaction between sex and protein level on feed intake (FI) (P < 0.05), where male birds had a higher FI compared to female birds only when the birds were fed SCP but not RCP diets. There was a significant interaction between CP level and sex on the expression of CAT2 (P = 0.02) and PEPT2 (P = 0.026) where the genes were significantly upregulated in females but only when the RCP diet was fed. The RCP diet upregulated the expression of BoAT (P = 0.03) as a main effect. Female birds had significantly higher expression of the PepT-2 gene compared to the males. The alpha diversity of the cecal microbiota showed differences among the treatments. The Shannon diversity index was statistically higher (P = 0.036) for males fed the SCP diet and the Chao1 index for evenness was statistically higher (P = 0.027) in females fed the SCP diet. There was also a difference in the relative abundance of the 15 most common genera found in the cecal content of the broilers in this experiment and lastly, the differential composition of microbiota between the different treatments was also significantly different. This study suggests that chickens are able to compensate for a reduction in AA substrates when fed a low CP diet through the upregulation of certain AA transporters, females may adapt to low CP diets better by such upregulation compared to males, and lastly, sex has an effect on the cecal microbial population and these differences contribute towards the performance differences between male and female broilers.

Sex proportion as a covariate increases the statistical test power in growth performance based experiments using as-hatched broilers.

The availability of sexed day-old broiler chicks is becoming an issue as feather sexing is no longer possible. This has great implications for broiler researchers as the use of randomly distributed mixed-sex birds may result in a greater between-pen variation and thus less statistical power than the use of single-sex birds. The objective of this study was to evaluate the effect of including sex proportion as a covariate in an analysis of covariance (ANCOVA) on the statistical power compared to analysis of variance (ANOVA) where sex was not considered. The statistical parameters examined include mean square error (MSE), the F-statistic, model fit, model significance and observed power. A total of 4 separate experiments that used mixed-sex broilers with unequal numbers of male and female birds per pen were conducted during which performance of the birds was measured. The male % in each pen was recorded during each experiment and corrected for mortality. The performance results were analysed by ANOVA and the statistical parameters were then compared to ANCOVA where sex proportion was included as a covariate. The results showed that a set of assumptions first needed to be met to run ANCOVA. In addition, if the ANOVA results show a high level of model significance and power, then ANCOVA may not be necessary. In other circumstances where the assumptions are met and model significance and observed power are low, the inclusion of sex proportion as a covariate in the analysis will help to reduce MSE, increase the F-statistic value and improve the model significance, model fit and observed power. Therefore, it is suggested that sex proportion should be considered as a covariate in ANCOVA to improve statistical power in nutritional experiments when male and female broilers are unequally and randomly distributed in pens.

Influence of sex and rearing method on performance and flock uniformity in broilers-implications for research settings.

Male and female broiler chickens differ in their growth performance, carcass part weights and nutrient requirements. The potential reasons for these differences have been explored by looking at differences in nutrient digestibility, nutrient transporter gene expression as well as gut microbiota populations between male and female birds. Studies have shown that male broilers have higher crude protein requirements compared to female broilers. The expression of monosaccharide and amino acid transporters show conflicting results as expression depends on the interactions between sex and bird age and breed as well as which tissue is sampled. Differences in microbiota populations between the genders were reported which may contribute towards performance differences, however research in this area is limited. The differences observed between the sexes contribute to increased variation in nutrition trials, and the potential to rear birds as equally mixed-sex becomes an option to reduce the variation introduced by the sex effect. Difference in rearing options obviously would only be feasible provided a quick, practical and cost-effective method of sexing birds is available, a topic that is also discussed in this review.

Rearing broilers as mixed or single-sex: relevance to performance, coefficient of variation, and flock uniformity.

With known variation in performance between male and female broilers and the fact that sourcing single-sex birds for use in research is becoming increasingly difficult, it becomes important to determine the effect of rearing method with male and female broilers on between-pen variation and body weight (BW) uniformity. We evaluated the performance response of broilers reared as single or mixed-sex to standard and reduced crude protein (CP) diets. The study was designed as a 2 × 3 factorial arrangement of treatments consisting of 672 Cobb-500 broilers assigned to 48 floor pens with 6 treatments, 8 replicates, and 14 birds per pen. The factors were rearing method (male single-sex, female single-sex, or equally mixed-sex) and dietary CP level (standard or reduced). For the overall period of the trial (d 0-35) there was a significant effect (P < 0.001) of rearing method and CP level on feed intake (FI) and feed conversion ratio (FCR). There was also a significant interaction between rearing method and CP level for BWG during d 0 to 35 (P < 0.01). There was a significant interaction between CP level and sex on d 34 BW (P < 0.01) where the reduced CP diet decreased the BW of both males and females, but to a greater extent the BW of the female birds. Dietary CP level had a significant effect on relative breast and drumstick weights with birds fed the reduced CP diet having significantly lower breast weights (P < 0.001) and higher drumstick weights (P < 0.01).This study suggests that male and female broilers have different CP requirements, and rearing birds as equally mixed-sex results in the lowest CV% for performance parameters and best BW uniformity compared to single-sex birds. Furthermore, when low CP diets are fed to broilers, they will prioritize the growth of more important body parts such as the legs.

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 2 × 2 × 4 factorial arrangement of treatments was used. Factors were the following: NE challenge, no or yes; diet type, wheat- or maize-based; and supplemental enzymes, control (no enzyme), family 10 xylanase (XYN10), family 11 xylanase (XYN11) or ß-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 wheat-based diet, resulting in a 2-way diet type × enzyme 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.

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 ß-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 wheat-based 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 maize-based 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 maize-based diet. Supplementing wheat-based diets with fibre-degrading enzymes ameliorates production losses induced by NE.

Buffered formic acid and a monoglyceride blend coordinately alleviate subclinical necrotic enteritis impact in broiler chickens.

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.

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 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 × 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 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.

Necrotic enteritis challenge regulates peroxisome proliferator-1 activated receptors signaling and ß-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 ß-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.

Differential expression of intestinal genes in necrotic enteritis challenged broiler chickens with 2 different Clostridium perfringens strains.

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.

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.