DISCUSSION

Productive performance response to FC supplementation

Supplementation with potato RS in laying hens during the production phase (37–48 weeks of age) improved productive efficiency, as evidenced by a significantly lower FCR, while maintaining laying percentage at levels comparable to the D1-Control group. Although evidence regarding the effects of potato RS on productive parameters in laying hens remains limited, studies modifying starch structure, particularly amylose-to-amylopectin ratios, have shown beneficial effects on cecal microbiota modulation and nitrogen utilization, with consequent improvements in performance [35]. Similarly, in broilers, dietary inclusion of MOS at 0.5% and retrograded potato RS at 1% resulted in lower FCR values than those observed in control birds (1.69 and 1.64 vs. 1.79, respectively); however, yeast inclusion at 0.5% did not affect productive parameters in the same study [36]. Therefore, the improved FCR observed in the D4-RS group may be associated with enhanced nutrient utilization linked to improved intestinal integrity.

The modest effect of MOS inclusion on productive parameters, such as laying percentage, has also been reported in hens of different ages. In 34-week-old hens, dietary inclusion of probiotics and prebiotics, including β-glucans and MOS at 0.2%, resulted in lower egg production percentage than that observed in controls [37]. Similarly, studies comparing MOS at 1% with essential oils in 36-week-old birds [12] and evaluating MOS at 0.5% in 73-week-old birds reported no significant effects on productive performance [38]. These findings suggest that the efficacy of MOS may be more evident under specific conditions, such as sanitary challenges on commercial farms, or may depend on dose, bird age, and physiological stage.

Similarly, birds receiving FOS did not show improvements in laying performance or FCR. This finding agrees with previous observations in 30-week-old Hy-Line Brown hens supplemented with 0.3%–0.5% FOS, in which positive effects on intestinal morphology did not translate into marked improvements in productivity [39]. Such physiological benefits may not necessarily be reflected in productive efficiency, possibly because the effects are localized mainly to the cecum or because the birds in the present study were maintained under optimal health conditions with a low challenge level.

Intestinal permeability and barrier function

Serum FITC-d concentrations were significantly higher in the D1-Control group than in the prebiotic-supplemented groups, suggesting that FC supplementation reduced intestinal permeability. This effect may be attributed to enhanced cecal fermentation and increased SCFA production. SCFAs activate adenosine monophosphate-activated protein kinase and strengthen tight junctions, thereby reducing paracellular passage, as demonstrated in vitro for FOS [40]. Although serum FITC-d values >200 ng/mL have been suggested as a “leaky gut” threshold in broilers [41], adult laying hens have different metabolic and physiological requirements, which may result in higher baseline serum FITC-d concentrations without necessarily indicating intestinal dysfunction [41, 42]. Thus, this study is among the first to assess this biomarker in laying hens, and further research is required to establish reference values for this species and production phase.

In line with this interpretation, Baxter et al. [43] reported higher serum FITC-d concentrations under feed restriction challenges (~468.1 ng/mL) than in unchallenged birds. Wiersema et al. [42] reported serum FITC-d ranges between 101.31 and 114.23 ng/mL in laying hens reared under different housing systems. They observed high individual variability, possibly related to the wide age range of the birds (26–70 weeks), and attributed the generally low serum detection to good sanitary status and the absence of major nutritional or environmental challenges [42].

Oxidative status and its relationship with intestinal integrity

In the present study, the redox profile of hens showed an inverse pattern with intestinal permeability. Higher serum FITC-d concentrations coincided with lower ORAC values and higher MDA concentrations across treatment groups. Because ORAC estimates the overall serum capacity to neutralize peroxyl radicals involved in lipid peroxidation [44], the combination of reduced ORAC and elevated MDA in the D1-Control group may indicate compromised antioxidant defense and increased oxidative damage. Inflammation may represent a common mechanistic link, as it promotes pro-oxidant processes and disrupts tight junction proteins, thereby contributing to increased intestinal permeability and oxidative stress [45, 46]. However, these findings remain associative, and longitudinal studies including complementary markers are required to confirm causality and exclude confounding factors.

In 30-week-old Hy-Line layers, FOS inclusion at 0.3%–0.6% reduced MDA concentrations compared with controls [10, 39]. In broilers, RS supplementation also reduced MDA and increased ORAC in prebiotic-supplemented groups. These effects have been attributed to enhanced microbial fermentation and SCFA production, which activate the Nrf2–Keap1 pathway and stimulate antioxidant gene expression [47].

In the present study, potato RS increased serum ORAC from 1686.54 to 1731.16 µmol TE/mL and reduced MDA from 0.39 to 0.25 µmol MDA/mL compared with the D1-Control group, demonstrating an inverse relationship between antioxidant capacity and lipid peroxidation. A similar pattern was reported by Zhou et al. [48] in broilers, where different MOS levels increased total antioxidant activity and reduced MDA concentrations. These findings suggest that the reduced intestinal permeability and improved antioxidant capacity observed, particularly in D4-RS-fed birds, may reflect a more intact intestinal barrier and lower oxidative stress. This improved physiological state may partly explain the enhanced productive efficiency observed in this group.

Intestinal morphology and epithelial development

Favorable intestinal morphology is characterized by longer, structurally intact villi and shallower crypts, which provide greater absorptive surface area and reduce the metabolic cost of epithelial turnover. In the present study, birds supplemented with MOS and FOS showed shallower crypts and higher V:C ratios in the duodenum than those in the D1-Control group, whereas RS supplementation was the most effective treatment for increasing the V:C ratio in the jejunum. VW remained unchanged, indicating that FCs primarily affected epithelial length and proliferative dynamics rather than villus thickness. These findings are consistent with previous studies reporting that FOS supplementation in layers increases VH and V:C ratio and that MOS inclusion in broilers typically increases VH and reduces CD [39, 49].

These effects are consistent with prebiotic mechanisms involving competitive exclusion of pathogenic bacteria and increased fermentation by beneficial bacteria, which enhance SCFA production. SCFAs directly nourish enterocytes, activate cell signaling pathways, and increase tight junction protein expression, thereby improving intestinal structure [39, 50]. Segment-specific effects are expected because distal small intestinal segments exhibit lower bacterial activity and SCFA production. In addition, FOS and MOS may be metabolized before reaching these segments, whereas residual fractions may reach the cecum, the primary site of fermentation [51]. In this study, FOS and MOS reduced duodenal CD compared with the D1-Control group, whereas RS exerted its greatest effect in the jejunum.

Goblet cell responses and mucosal protection

Goblet cell counts relative to enterocytes did not differ significantly among diets in the duodenum, jejunum, or ileum. This finding is consistent with a study in 50-week-old layers supplemented with xylooligosaccharides [52], although a non-significant trend toward higher counts was observed in the prebiotic groups, similar to the present study. In the cecum, however, FOS and RS treatments reduced the goblet cell-to-enterocyte ratio compared with the D1-Control group.

In the intestinal epithelium, a common progenitor cell from the crypt stem cell niche differentiates into either a secretory or an absorptive lineage. The Notch pathway acts as a key molecular switch in this fate decision. Inhibition of Notch signaling as precursor cells exit the crypt promotes differentiation into goblet cells, whereas Notch activation promotes differentiation into absorptive enterocytes [53]. These differentiation pathways are modulated by the intestinal microenvironment. Factors such as microbiota composition, diet, SCFAs, and cytokines can alter the balance between goblet cells and enterocytes. In addition, a subset of sentinel goblet cells has been identified that can rapidly adjust mucus secretion in response to enteric challenges, thereby reinforcing the mucosal barrier [54].

Previous studies have demonstrated that enteric challenges can modulate goblet cell numbers. Kinstler et al. [55] reported that a subclinical necrotic enteritis challenge induced by Clostridium perfringens increased goblet cell density and MUC2 expression in broilers, suggesting compensatory thickening of the mucus layer. Conversely, challenge with Eimeria alone or co-infection with C. perfringens reduced goblet cell numbers and mucus production. Thus, the level and type of enteric challenge can differentially alter the mucus layer response. Severe challenges, particularly co-infections, can cause profound epithelial damage, goblet cell depletion, and mucosal barrier impairment, as evidenced by overall gut dysfunction and barrier damage observed in challenged laying hens [56].

Cecal microbial diversity and community structure

The inclusion of different FCs modulated cecal microbial ecology. Notably, the D3-FOS diet significantly increased the Shannon diversity index relative to unsupplemented birds at the end of the experimental period (week 48). This pattern aligns with previous reports. Supplementation with 0.04% xylooligosaccharides in adult Hy-Line layers significantly increased microbial richness, as indicated by the ACE index, and showed a trend toward higher Chao1 values, without altering the Shannon or Simpson indices, suggesting expansion of subpopulations without major redistribution of abundance [57]. Similarly, in broilers, corn RS supplementation tended to increase alpha diversity based on Chao1 values without affecting the Simpson index [58].

Regarding beta diversity, the effect of FCs was more pronounced at week 48, indicating a time-dependent effect. This suggests that several weeks of supplementation are required to modulate the microbiome and shift community structure, as also reported in layers supplemented with MOS [59]. Zhou et al. [57] similarly found significant dietary effects using ANOSIM and PCoA with xylooligosaccharides, alongside improved intestinal morphology and barrier integrity, underscoring that bacterial remodeling through dietary intervention is a gradual process.

Taxonomic shifts induced by FC supplementation

Marked shifts in microbial composition were observed during the study. Bacillota increased, whereas Pseudomonadota and Fusobacteriota decreased, accompanied by expansion of Verrucomicrobiota, particularly in MOS- and RS-supplemented groups. In layers supplemented with xylooligosaccharides, increased Akkermansia (Verrucomicrobiota) and reduced Erysipelatoclostridium were correlated with improved intestinal structure, increased goblet cell numbers, enhanced expression of tight junction proteins (ZO-2, CLDN1, and CLDN5), reduced endotoxins, and increased fermentative families such as Ruminococcaceae and Lachnospiraceae [57]. In broilers, corn RS increased Bacillota abundance while slightly reducing Bacteroidota and Pseudomonadota. This was associated with improved metabolic responses linked to higher SCFA production, which favors lower cecal pH and reduced inflammation [60], a dynamic consistent with the intestinal permeability findings of the present study.

In healthy hens, the most frequently detected cecal genera include Bacteroides, Bifidobacterium, Clostridia UCG-014, Alistipes, Prevotellaceae, Faecalibacterium, Escherichia, Lactobacillus, and Ruminococcus. At the phylum level, Bacillota and Actinomycetota predominate, followed by Bacteroidota and Pseudomonadota. The cecum harbors the greatest microbial richness and diversity in the avian gastrointestinal tract [61]. The predominance of Bacillota followed by Bacteroidota is typical in adult layers, where these two phyla can constitute 85%–93% of the cecal community [62].

Prebiotic inclusion has been linked to shifts in lactic acid metabolism and competitive reduction of potential pathogens. In broilers supplemented with 0.25% FOS and 0.05% MOS, ileocecal C. perfringens and Escherichia coli were reduced, whereas Lactobacillus increased [56]. In layers, mannan-rich fractions reduced _Campylobacter _jejuni levels and had concurrent effects on production parameters [59, 63].

RS-associated butyrogenic taxa and microbial biomarkers

RS reaches the cecum intact and is expected to stimulate butyrate production through enrichment of butyrogenic taxa. In broilers, corn RS modulated the cecal microbiota by reducing Pseudomonadota abundance and increasing markers of SCFA production [58]. In the present study, RS consumption at week 48 was associated with improved FCR. LDA further supported the role of RS as a substrate for butyrogenic families, including Lachnospiraceae, and for genera such as Blautia and Subdoligranulum [64].

Among the taxa enriched by RS, Blautia showed a marked increase. This genus is associated with SCFA production, primarily acetate and butyrate, which are crucial for colonic mucosal integrity and function [65]. Furthermore, RS supplementation favored the enrichment of Ligilactobacillus aviarius and Lactiplantibacillus_ plantarum_. Both species are associated with lactic acid metabolism, which acidifies the intestinal environment, supports competitive exclusion of pathogens, and has been linked to improved production parameters in poultry [35, 66]. Notably, birds receiving RS showed better intestinal barrier integrity, improved redox status, and more efficient FCR. Collectively, these pronounced shifts in microbial communities and their potential metabolic effects may underlie the observed improvements in intestinal health and productivity, although the directionality of this relationship requires further elucidation.

Finally, it is noteworthy that the D1-Control group showed consistent enrichment of the family Eggerthellaceae (phylum Actinomycetota) at both evaluation ages in the LEfSe analysis. In chickens, the presence of this taxon has been associated with increased intramuscular fat and proposed as a potential pathobiont capable of eliciting pro-inflammatory stimuli [67]. This enrichment is consistent with the phenotype observed in the D1-Control group, which showed higher intestinal permeability, elevated MDA concentrations, and lower ORAC values, suggesting oxidative stress and low-grade inflammation.

Strengths, limitations, and future perspectives

This study demonstrated clear effects of FCs on intestinal health and cecal microbiota in Babcock Brown laying hens under controlled conditions. However, several considerations should be acknowledged when interpreting the findings. The experiment was conducted under optimal health conditions without deliberate sanitary or environmental challenges; therefore, outcomes may differ under commercial stressors. Microbial analysis focused on cecal communities using 16S rRNA gene sequencing, which provided a robust taxonomic profile, but direct measurements of microbial metabolites and specific host immune markers were not included. In particular, SCFAs and host immune markers were not quantified, which should be addressed in future studies to better link observed microbial shifts, such as the enrichment of butyrogenic taxa, to their functional effects in the host. Intestinal barrier integrity was evaluated using the validated FITC-d biomarker, although complementary measures could provide a more comprehensive understanding of intestinal barrier function. Finally, the reported responses correspond to the specific types and doses of FCs evaluated in this study, and effects may vary with alternative prebiotics, doses, supplementation durations, or production conditions. These considerations clarify the scope of the current results and indicate promising directions for future research.