Gemma González-Ortiz

The impact of dietary inert digestibility markers on gut microbiota and intestinal fermentation remains poorly understood. This study investigated the effects of dietary titanium dioxide (TiO₂) supplementation at 4 kg/t feed, representing a typical dose used in animal nutrition studies, on fermentation dynamics and microbial composition in broiler chickens using combined ex vivo and in vivo approaches. Ex vivo fermentations were conducted using ileal and caecal microbiota and substrates collected from 32-day-old broiler chickens. Titanium dioxide (TiO₂) was supplemented directly to the fermentations, and gas production and short-chain fatty acid (SCFA) profiles were used as the main outcome measures. In parallel, 392 broiler chickens were fed diets with or without TiO₂ for 32 days, and ileal and caecal digesta were analysed for fermentation end-products and microbial composition using shotgun metagenomic sequencing. A second ex vivo experiment was performed using microbiota adapted to dietary TiO₂. In the first ex vivo model, TiO₂ reduced gas production and acetic acid concentration in the ileum (p < 0.05), whereas in the caecum it increased gas production, total eubacterial counts, and branched-chain fatty acids (BCFAs) (p < 0.05). In vivo, TiO₂ did not affect growth performance or organ development but significantly increased isobutyric acid and total BCFA concentrations in the caecum (p < 0.05). Metagenomic analysis revealed increased caecal alpha diversity (Shannon index) and enrichment of taxa associated with amino acid metabolism, including Massilicoli timonensis, Blautia merdavium, Rubneribacter badeniensis, and Mediterraneibacter caccavium. The second ex vivo experiment showed similar trends, with increased gas and BCFA production. Collectively, these findings indicate that TiO₂ can modulate intestinal fermentation and microbial composition in a segment-specific manner, suggesting that dietary markers may not be biologically inert.

Numerous studies have reported the beneficial effects of supplemental xylo-oligosaccharides (XOS) on broiler performance and nutrient utilization. These effects are attributed to their prebiotic action, directly fueling beneficial bacterial species, and their ability to stimulate fibre-degrading bacteria in the gastrointestinal tract. Commercial XOS products typically contain mixtures with varying degrees of polymerisation (DP), ranging from xylobiose (X2) to xylohexaose (X6). This study aimed to determine the optimum DP range of XOS for broiler chickens. Two trials were conducted using 0 d-old Cobb 500 broilers (300 per trial, 60 birds per treatment, initial body weight 45.80 ± 0.15 g) fed wheat-based diets over 35-d period as three phases (starter d 0-12, grower d 12-23, and finisher d 23-35). In both trials, birds were fed one of five XOS treatments (150 g/tonne); no XOS (control), low-DP XOS, medium-DP XOS, high-DP XOS, or a standard commercial XOS mixture. In Trial 1, the XOS treatments were fed without xylanase, and in Trial 2 the XOS treatments were fed with xylanase (16,000 birchwood xylanase units [BXU]/kg). In Trial 1, birds fed the high-DP XOS had significantly lower feed conversion ratio corrected for mortality (cFCR) at d 0 to 35 (P < 0.001) compared to birds fed the control and medium-DP XOS treatments. Additionally, birds fed the control diet presented higher caecal concentrations of branched-chain fatty acids (iso-butyric and iso-valeric acids) compared to those fed the high-DP XOS (P = 0.025 and P = 0.035, respectively). In Trial 2, birds receiving the high-DP XOS showed improved cFCR compared to those fed the low-DP XOS (P = 0.037). Low-DP XOS fed birds also presented increased caecal Enterobacteriaceae abundance compared to those fed the control diet, medium-DP XOS or commercial XOS treatments (P = 0.008). In conclusion, supplementation with high-DP XOS was more effective in improving feed efficiency and modulating caecal fermentation than feeding no XOS or low-DP XOS.

The impact of dietary inert digestibility markers on gut microbiota and intestinal fermentation remains poorly understood. This study investigated the effects of titanium dioxide (TiO₂) supplementation at 4 kg/ton feed, representing a typical dose used in animal nutrition studies, on fermentation dynamics and microbial composition in broiler chickens using combined ex vivo and in vivo approaches. Ex vivo fermentations were conducted using ileal and caecal microbiota and substrates collected from 32-day-old broiler chickens, with direct TiO₂ supplementation, with gas production and volatile fatty acid (VFA) profiles as main measurements. In parallel, 392 broiler chickens were fed diets with or without TiO₂ for 32 days, and ileal and caecal digesta were analysed for fermentation end-products and microbial composition using shotgun metagenomic sequencing. A second ex vivo experiment was performed using microbiota adapted to dietary TiO₂. In the first ex vivo model, TiO₂ reduced gas production and acetic acid concentration in the ileum (p < 0.05), whereas in the caecum it increased gas production, total eubacterial counts, and branched-chain fatty acids (BCFA) (p < 0.05). In vivo, TiO₂ did not affect growth performance or organ development but significantly increased isobutyric acid and total BCFA concentrations in the caecum (p < 0.05). Metagenomic analysis revealed increased caecal alpha diversity (Shannon index) and enrichment of taxa associated with amino acid metabolism, including Massilicoli timonensis, Blautia merdavium, Rubneribacter badeniensis, and Mediterraneibacter caccavium. The second ex vivo experiment showed similar trends, with increased gas and BCFA production. Collectively, these findings indicate that TiO₂ can modulate intestinal fermentation and microbial composition in a segment-specific manner, suggesting that dietary markers may not be biologically inert.

Including additional dietary fibre fractions in broiler feed has gained renewed interest due to its potential for stimulating (gut) health and performance. The beneficial effects of fibre are mainly due to its fermentation in the caecal lobes, but the influx of digesta into these lobes seems restricted to small particles and solubles. To boost caecal fermentation via fibre inclusions, more knowledge on how fibre particle size in feed can affect the fibre particle size distribution along the gastrointestinal tract (GIT), and hence the caecal influx and the fermentation of fibre, is required. To this end, 78 one-day-old male broilers (Ross 308, average initial weight of 43.96 ± 2.97 g) were divided into 3 treatment groups (n = 26 birds per treatment group, 1 replicate pen per treatment), receiving a wheat-based diet with a 10% replacement of the wheat fraction by wheat bran with an average particle size of 1026 (coarse bran), 506 (medium bran) or 106 μm (fine bran), respectively, over a period of d 1 to 24. Digesta of the gizzard, duodenum-jejunum and ileum was collected at d 7 and 24 to determine the fibre particle size distribution in it. The overall ileal and caecal digesta particle size distribution and short-chain fatty acid (SCFA) content were also determined. An increase in the relative content of arabinoxylan along the GIT in the size fractions corresponding to the included bran particle size in the feed was observed for each dietary treatment (P < 0.05), indicating that the particle size differences induced by the bran incorporations were conserved along the GIT. Bran particle size did not affect the gizzard's development or grinding action. The coarse bran treatment resulted in the smallest caecal digesta average particle size (50th percentile, D50) (P = 0.088) and the highest caecal SCFA contents at d 7 (P < 0.001). At d 24, no differences in caecal digesta D50 were observed (P = 0.298), and the fine bran treatment resulted in the highest caecal SCFA contents (P = 0.026). The different effects of bran size on caecal fermentation as a function of broiler age can be ascribed to the limitations of caecal influx that also change with age. The fibre particle size distribution along the GIT can be steered by including the desired fibre particle size in the feed, and the effect of broiler age on caecal influx should be considered when aiming to improve caecal fibre fermentation through fibre inclusion.

A promising strategy to support broiler health and performance in a sustainable way is the enhancement of microbial fibre fermentation in broilers. This fermentation mainly occurs in the caeca, but the actual particle size range that allows caecal influx has not yet been described. This study aimed to understand the physical limitations of caecal influx as a function of broiler age by using both solid and soluble markers. In the first trial, the caecal filter mechanism was studied by microscopically visualising the caecal entrance and measuring caecal lobe development and digesta particle size as a function of age (d 8–36) for 44 broilers (Ross 308) receiving a conventional wheat-based diet. In two consecutive trials, microcrystalline cellulose beads (100–700 μm) and a combination of fluorescent polystyrene beads (5–30 μm) and chromium-ethylenediamine tetraacetic acid (Cr-EDTA) were administered to 176 and 189 broilers, respectively, at different ages (d 8–36). Results showed that the actual caecal entrance diameter is significantly reduced due to a dense villi network acting as a filter for digesta inflow. This explains the size gap between the average digesta particle size (D50) of the ileum (451–322 μm) and caeca (5–19 μm), and the outer diameter of the caecal entrance (2000–4000 μm) on d 8 to 36. In contrast to the caecal D50, cellulose beads of 700 μm already entered the caeca at 8 d of age, even though the general caecal influx of digesta particles larger than 100 μm seemed very limited. The caecal influx of the markers further exhibited large individual variation among birds. A maximum of 13.2% (d 9) and 4.3% (d 29) of the total administered soluble marker (Cr-EDTA) was detected in the caeca, 5 h after bolus administration. Both solid and soluble markers showed a larger concentration in the caeca at a young age compared to older ages (P < 0.01), possibly related to the limited caecal functioning early in life. These findings highlight the importance of carefully selecting the physical properties of fibres to be added as a function of age to further improve caecal fibre fermentation in broilers.

A 42-day study was conducted with 720-day-old Cobb male broiler chicks allocated to treatments in a 3 × 2 factorial, with the factors as wheat bran (WB) inclusion (no WB, 50 g/kg coarse WB, or 50 g/kg fine WB) and stimbiotic (STB) supplementation in corn-based diets. The inclusion of WB (p < 0.05) or STB supplementation (p < 0.05) increased the FCR and feed intake in the day 0–10 phase. During the day 0–28 phase, coarse-WB inclusion increased (p < 0.05) the FCR, compared to fine WB or diets without WB. In the day 0–42 phase, WB marginally decreased weight gain in diets without STB supplementation, but the STB-supplemented diet, weight gain was greater (p < 0.05) the diet with fine WB compared with diets with coarse WB. Fine-WB inclusion increased the ileal nitrogen and energy digestibility determined at day 18 compared to coarse WB or diets without WB. Supplementation with STB (p < 0.05) or fine WB (p < 0.05) inclusion increased the villi height compared to diets without STB supplementation or coarse WB, or the diet without WB. Coarse or fine WB decreased (p < 0.05) cecal branched-chain fatty acids compared to diets without WB. In conclusion, stimbiotic supplementation to fine WB improved the performance and nutrient digestibility of broilers compared to coarse WB with no effects on the caeca total SCFA concentration.

Xylo-oligosaccharides (XOS) stimulate proliferation of beneficial bacteria in the gastrointestinal tract of broiler chickens. This results in enhanced utilisation of dietary non-starch polysaccharides and increased production of valuable short-chain fatty acids. However, these positive effects do not always translate into improved bird productive performance, with inconsistent performance responses observed between bird trials.A holo-analysis was conducted to determine the effects of supplementing XOS into broiler diets on bird feed intake, body weight gain, feed conversion and mortality. This was done by comparing the XOS supplemented treatment to the control treatment. A total of 53 studies which met the criteria for inclusion were used in the analysis.The results showed that XOS had a notable positive impact on bird mortality; XOS reduced mortality by 0.69% for every 1% increment in the control group. XOS supplementation induced a positive effect on the feed conversion ratio (FCR). However, the efficacy of XOS at improving FCR was dependent on the efficiency of the control group (performance of the flock), and the concentration of total arabinoxylan, protein and phytase in the diet. There were insufficient data points to predict the effect of XOS on body weight and feed intake.In conclusion, the holo-analysis revealed that supplementing XOS to broiler chicken diets reduces bird mortality. XOS can also improve FCR, but the scale of response is dependent on the diet composition and control flock performance. Additional studies are required to confirm the effects of XOS on body weight and feed intake.