Inclusion of a Bacillus-based probiotic in non-starch polysaccharides-rich broiler diets.

This study examined the effects of a 3-strain Bacillus-based probiotic (BP; Bacillus amyloliquefaciens and two Bacillus subtilis) in broiler diets with different rye levels on performance, mucus, viscosity, and nutrient digestibility. We distributed 720 one-d-old female broilers into 72 pens and designed nine diets using a 3 × 3 factorial approach, varying BP levels (0, 1.2 × 106, and 1.2 × 107 CFU/g) and rye concentrations (0, 200, 400 g/kg). On d 35, diets with 200 or 400 g/kg rye reduced broiler weight gain (BWG). Diets with 400 g/kg rye had the highest FCR, while rye-free diets had the lowest (p ≤0.05). Adding BP increased feed intake and BWG in weeks two and three (p ≤0.05). It should be noted that the overall performance fell below the goals of the breed. Including rye in diets reduced the coefficient of apparent ileal digestibility (CAID) for protein, ether extract (EE), calcium, phosphorus, and all amino acids (p ≤0.05). Rye-free diets exhibited the highest CAID for all nutrients, except for methionine, EE, and calcium, while diets with 400 g/kg of rye demonstrated the lowest CAID (p ≤0.05). BP in diets decreased phosphorus CAID (p ≤0.05). Diets containing 1.2 × 107 CFU/g (10X) of BP exhibited higher CAID of methionine than the other two diets (p ≤0.05). Diets containing 10X of BP showed higher CAID of cysteine than diets with no BP (p ≤0.05). Ileal viscosity increased as the inclusion level of rye in the diets increased (p ≤0.05). The ileal concentration of glucosamine in chickens fed diets with 400 g/kg of rye was higher than in those fed diets with no rye (p ≤0.05). Furthermore, ileal galactosamine concentrations were elevated in diets with 200 and 400 g/kg of rye when compared to rye-free diets (p ≤0.05). However, BP in diets had no impact on ileal viscosity, galactosamine, or glucosamine (p > 0.05). In conclusion, the applied Bacillus strains appeared to have a limited capacity to produce arabinoxylan-degrading enzymes and were only partially effective in mitigating the negative impacts of rye arabinoxylans on broilers.

Alterations in bacterial metabolites, cytokines, and mucosal integrity in the caecum of broilers caused by feed additives and host-related factors.

A total of 2,880 one-day-old male and female broiler chicks from two breeds, Ross308 and Cobb500 were randomly assigned to 72 pens. Broilers were offered three diets: a wheat-soybean diet without (CO), or with either a probiotic (probiotic; 2.4 x 109 CFU/kg diet of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (phytobiotic; grape extract with 165 ppm procyanidin and 585 ppm polyphenol) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed and sex in a completely randomized design and consisted of 6 replicate-pens per treatment (40 birds per pen). At day 7, 21, and 35, one chicken per pen was slaughtered for caecal sampling to quantify bacterial metabolites (digesta) as well as evaluate mRNA abundance and histomorphology (tissue). Data were subjected to ANOVA using GLM procedure to evaluate age, diet, breed and sex and their interactions. Spearman's correlation (r) was analyzed between metabolite concentration and mRNA abundance. Overall, the concentration of short chain fatty acids increased with age, while lactate decreased from day 7 to 21 (p < 0.05). The mRNA abundance of IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17α, IL-18, IFN-γ and TGF-ß2 increased with age but IL-1ß and TNF-α increased in abundance from day 7 to 21 and then decreased (p < 0.05). Abundance of MUC2 and CLDN5 increased after day 21 (p < 0.05). Caecal crypt depth increased with age (p < 0.05). Acidic goblet cell (GC) number peaked at day 21 (p < 0.05), while mixed GC number was not affected by age. A few impacts of breed, diet and interactions on the investigated variables showed no meaningful biological pattern. Propionate positively correlated with all cytokines investigated (r = 0.150-0.548), except TNF-α. Lactate negatively correlated with pro-inflammatory cytokines like IL-1ß (r = -0.324). Aging affected caecal histomorphology, bacterial activity and genes responsible for barrier integrity and inflammatory response. This effect could be attributed to the interaction between gut microbiota and immune system as well as the direct effect of metabolites on gut histomorphology and cytokine mRNA abundance.

Novel strategies to improve chicken performance and welfare by unveiling host-microbiota interactions through hologenomics.

Fast optimisation of farming practices is essential to meet environmental sustainability challenges. Hologenomics, the joint study of the genomic features of animals and the microbial communities associated with them, opens new avenues to obtain in-depth knowledge on how host-microbiota interactions affect animal performance and welfare, and in doing so, improve the quality and sustainability of animal production. Here, we introduce the animal trials conducted with broiler chickens in the H2020 project HoloFood, and our strategy to implement hologenomic analyses in light of the initial results, which despite yielding negligible effects of tested feed additives, provide relevant information to understand how host genomic features, microbiota development dynamics and host-microbiota interactions shape animal welfare and performance. We report the most relevant results, propose hypotheses to explain the observed patterns, and outline how these questions will be addressed through the generation and analysis of animal-microbiota multi-omic data during the HoloFood project.

Development and Functional Properties of Intestinal Mucus Layer in Poultry.

Intestinal mucus plays important roles in protecting the epithelial surfaces against pathogens, supporting the colonization with commensal bacteria, maintaining an appropriate environment for digestion, as well as facilitating nutrient transport from the lumen to the underlying epithelium. The mucus layer in the poultry gut is produced and preserved by mucin-secreting goblet cells that rapidly develop and mature after hatch as a response to external stimuli including environmental factors, intestinal microbiota as well as dietary factors. The ontogenetic development of goblet cells affects the mucin composition and secretion, causing an alteration in the physicochemical properties of the mucus layer. The intestinal mucus prevents the invasion of pathogens to the epithelium by its antibacterial properties (e.g. ß-defensin, lysozyme, avidin and IgA) and creates a physical barrier with the ability to protect the epithelium from pathogens. Mucosal barrier is the first line of innate defense in the gastrointestinal tract. This barrier has a selective permeability that allows small particles and nutrients passing through. The structural components and functional properties of mucins have been reviewed extensively in humans and rodents, but it seems to be neglected in poultry. This review discusses the impact of age on development of goblet cells and their mucus production with relevance for the functional characteristics of mucus layer and its protective mechanism in the chicken's intestine. Dietary factors directly and indirectly (through modification of the gut bacteria and their metabolic activities) affect goblet cell proliferation and differentiation and can be used to manipulate mucosal integrity and dynamic. However, the mode of action and mechanisms behind these effects need to be studied further. As mucins resist to digestion processes, the sloughed mucins can be utilized by bacteria in the lower part of the gut and are considered as endogenous loss of protein and energy to animal. Hydrothermal processing of poultry feed may reduce this loss by reduction in mucus shedding into the lumen. Given the significance of this loss and the lack of precise data, this matter needs to be carefully investigated in the future and the nutritional strategies reducing this loss have to be defined better.

Screening of Host Specific Lactic Acid Bacteria Active Against Escherichia coli From Massive Sample Pools With a Combination of in vitro and ex vivo Methods.

A novel three-step combination of in vitro and ex vivo screening was established to massively screen host derived lactic acid bacteria (LAB) from the broiler chicken intestine with inhibitory activity against Escherichia coli. In a first step, a massive sample pool consisting of 7102 broiler-derived colonies from intestinal contents were established and sub-cultured. Supernatants thereof were incubated with an E. coli model strain to screen suitable isolates with inhibitory activity. A total of 76 isolates of interest were subsequently further studied based on either pH dependent or -independent activity in the second step of the assay. Here, in-depth growth inhibition of the E. coli model strain and the potential of isolates for lactic acid production as inhibitory substance were indexed for all isolates. Resulting scatter plots of both parameters revealed five isolates with exceptional inhibitory activity that were further studied under ex vivo condition in the third step of the assay. These isolates were taxonomically classified as strains of the species Lactobacillus agilis, Lactobacillus salivarius, and Pediococcus acidilactici. Samples from the broiler chicken intestine were inoculated with the Lactobacillus isolates and the E. coli model strain. After 8 and 24 h incubation, respectively, growth of the E. coli model strain was monitored by cultivation of the E. coli strain in antibiotic supplemented medium. By their superior inhibitory activity against the E. coli model strain, one L. agilis and one L. salivarius strain were selected and characterized for further application as probiotics in broiler chicken. Additionally, their antibiotic resistance patterns and resilience under gastric stress of isolates were also characterized. The results of this study demonstrate that the novel isolation procedure was able to efficiently and rapidly isolate and identify bacterial strains from a massive sample pool with inhibitory potential against specific types of bacteria (here E. coli). The introduction of the final ex vivo selection step additionally confirmed the inhibitory activity of the strains under conditions simulating the intestinal tract of the host. Furthermore, this method revealed a general potential for the isolation of antagonistic strains that active against other pathogenic bacteria with specific biomarker.

Bacillus subtilis in broiler diets with different levels of energy and protein.

The present study evaluated the impacts of Bacillus subtilis (BAS) inclusion in broiler diets with standard nutrient content or nutrient deficiency (ND) on growth performance (GP) and nutrient digestibility. The 42 d experiment consisted of 6 experimental diets, a diet with standard nutrient content, and 2 diets with different levels of energy and protein deficiency, without or with BAS. At the end of experiment, apparent ileal digestibility coefficients (AIDC) of starch, crude protein (CP), and gross energy (GE) were determined. Furthermore, impacts of BAS supplementation in standard diets on gut histomorphology, bacterial metabolic activity, and composition were evaluated. Performance and AIDC data were subjected to ANOVA using GLM procedure with a 3 (nutrient levels) × 2 (BAS presence/absences) factorial arrangement of treatments. Gut histomorphology and microbiology data, obtained from broilers fed standard diets without (S) and with BAS (SB), were assessed by an independent Student's t-test. The ND in diets was effective enough to cause nutritional stress and negatively affect performance. Inclusion of BAS in both types of diet improved GP, which was due to the fact that adding BAS in these diets led to improvements in AIDC of CP, starch, and GE (P ≤ 0.05). Comparing only 2 experimental groups, S and SB, revealed no impact on bacterial composition and metabolism in the ileum and cecum, except a reduction in ileal lactobacilli number for SB group. Adding BAS to standard diet reduced crypt depth (CD) and increased villus length to CD ratio in the duodenum, whereas it had no impact on other histomorphological variables in the duodenum, jejunum, and ileum. In conclusion, supplementation of broiler diets with probiotic BAS can positively affect growth performance and nutrient digestibility and this positive impact might even be more pronounced in nutrient-deficient diets. However, the extent of the alleviating ability of BAS in nutrient-deficient diets as well as the biological mechanisms for such a phenomenon needs to be studied further.

The impacts of Macleaya cordata extract and naringin inclusion in post-weaning piglet diets on performance, nutrient digestibility and intestinal histomorphology.

The current study investigated the impacts of supplementation of post-weaning piglet diets with Macleaya cordata extract (MCE) and naringin (NAR) on performance, nutrient digestibility and intestinal histomorphology. Post-weaning crossbred piglets (28 males and 28 females; age at weaning 25 d) were randomly allotted to 28 pens. The experiment consisted of a control and three treatment groups (MCE60, MCE120 and NAR). For diets MCE60 and MCE120, the control diet was supplemented with 60 and 120 mg Sangrovit® Extra (a standardised premixture of MCE) per kg diet, respectively. Group NAR received 50 mg pure NAR per kg diet. The experiment lasted 42 d (d 25 - 66 of age). At d 66, apparent pre-caecal digestibility (APD) of nutrients was determined and histomorphological changes in mid-jejunum were evaluated. Feeding diets MCE120 and NAR improved body weight gain and feed conversion ratio of piglets. After feeding diets MCE120 and NAR, the APD of phosphorus and different single and total amino acids were greater than after feeding the control diet. The present data demonstrated that supplementation of post-weaning piglet diets with 120 mg MCE or 50 mg NAR per kg diet could improve growth performance and nutrient digestibility and had no impact on histomorphological variables in the jejunum. These findings indicate the potential of these products to be used as growth promoters in pig nutrition.

The effects of particle size, milling method, and thermal treatment of feed on performance, apparent ileal digestibility, and pH of the digesta in laying hens.

Various milling methods result in different particle size distributions and, in combination with mash and thermal treatment (expandate) of the feed, may have an impact on nutrient digestibility, pH of the digesta and subsequently the performance of an animal. Since this aspect has not been widely considered in laying hens, the objective of the present study was to investigate the effects of milling method, expansion, and particle size of feed on performance, apparent ileal nutrient digestibility, and pH of digesta in laying hens. Twelve variants of the same diet were produced. Four different milling techniques (hammer mill, roller mill, disc mill, and wedge-shaped disc mill) were used to grind the feed cereals. Coarse feed was obtained from all four mills. Additionally, fine feed was obtained from the hammer mill and the roller mill. Each of the six feed variants was offered as mash or expandate, resulting in a total of 12 treatments. The duration of the experimental period was 21 days. A total of 576 layers, each 19 weeks of age, were used in eight replicates. The statistical analysis for the four milling methods and two thermal treatments was performed using a 4×2 factorial arrangement. The effect of particle size was investigated using a 2×2×2 factorial arrangement including the coarse and fine particle sizes that were produced with the hammer mill and the roller mill as well as the mash and expandate. The animal performance and the pH of the digesta were not affected by the treatments. Ileal digestibility of starch was significantly improved by feeding mash compared to expandate (P=0.013) and by feeding coarse compared to fine feed (P=0.028). Based on this study, the tested milling methods can be used for the production of feed for laying hens without affecting performance and digestibility of nutrients.