RESULTS

Productive performance

No mortality was observed during the experimental period. The average body weight of hens was 1992.95 ± 79.95 g and did not differ significantly over time or among dietary treatments (p > 0.05). Egg production was not significantly affected by FC supplementation. During the 12-week experimental period, hens fed the D4-RS diet showed the highest mean egg production; however, differences among treatments were not significant (Table 2). Egg mass did not differ among dietary treatments in any period or in the overall analysis. However, FCR was significantly affected by diet during period 2 and during the overall experimental period (p < 0.001 and p = 0.028, respectively). Hens fed the D4-RS diet consistently exhibited the lowest FCR values across all evaluated periods (Table 2).

Serum oxidative status

Dietary supplementation significantly affected serum oxidative status at 48 weeks of age (p < 0.05) (Table 3). Serum ORAC was highest in hens fed the D4-RS diet, followed by those fed D3-FOS and D2-MOS, whereas the lowest ORAC value was observed in the D1-Control group (p < 0.05). Serum lipid peroxidation, assessed using TBARS, was significantly lower in the D3-FOS and D4-RS groups than in the D1-Control group (p < 0.05). The D2-MOS group showed intermediate TBARS values (Table 3).

Intestinal permeability

Serum FITC-d concentration was significantly affected by dietary treatment (p < 0.001). Hens fed the D1-Control diet showed the highest serum FITC-d concentration (380.99 ± 46.28 ng/mL), whereas those fed the D4-RS diet showed the lowest concentration (101.89 ± 25.15 ng/mL; p < 0.05). Intermediate FITC-d concentrations were observed in the D2-MOS (296.24 ± 34.87 ng/mL) and D3-FOS (154.87 ± 25.05 ng/mL) groups, both of which differed significantly from the D1-Control group (Figure 1).

Intestinal morphometry

Dietary treatments differentially affected intestinal morphology after 12 weeks of supplementation (Table 4). In the duodenum, significant differences were observed in CD (p = 0.029) and V:C ratio (p = 0.001). Hens fed the D2-MOS and D3-FOS diets showed higher V:C ratios than those fed the D1-Control and D4-RS diets.

Diet | Period 1 (37–42 weeks) | Period 2 (43–48 weeks) | Total (37–48 weeks)
Egg production (%) |  |  | 
D1-Control | 93.70 | 92.17 | 92.90
D2-MOS | 93.77 | 88.52 | 90.91
D3-FOS | 91.14 | 90.11 | 90.58
D4-RS | 94.74 | 92.70 | 93.63
SEM | 1.740 | 1.608 | 1.216
p-value | 0.520 | 0.262 | 0.228
Egg mass (g/hen/day) |  |  | 
D1-Control | 55.67 | 55.15 | 55.36
D2-MOS | 55.44 | 53.39 | 54.21
D3-FOS | 53.72 | 52.98 | 53.28
D4-RS | 54.37 | 55.78 | 55.21
SEM | 1.045 | 1.330 | 0.884
p-value | 0.532 | 0.400 | 0.321
FCR |  |  | 
D1-Control | 1.58 | 1.60ᵇ | 1.59ᵃ
D2-MOS | 1.55 | 1.61ᵇ | 1.58ᵃ
D3-FOS | 1.62 | 1.63ᵃ | 1.63ᵃ
D4-RS | 1.55 | 1.57ᶜ | 1.56ᵇ
SEM | 0.030 | 0.003 | 0.015
p-value | 0.337 | <0.001 | 0.028

Diet | ORAC (µmol TE/mL) | TBARS (µmol MDA/mL)
D1-Control | 1686.54ᵇ | 0.39ᵃ
D2-MOS | 1720.47ᵃᵇ | 0.31ᵇ
D3-FOS | 1722.25ᵃᵇ | 0.27ᶜ
D4-RS | 1731.16ᵃ | 0.25ᶜ
SEM | 4.23 | 0.01
p-value | 0.019 | <0.001

Figure 1: Serum fluorescein isothiocyanate-dextran (FITC-d) concentration in 48-week-old laying hens after 12 weeks of dietary supplementation with different prebiotics. Error bars represent the standard error of the mean. Bars that do not share a letter differ significantly according to Tukey’s test (p < 0.05). n = 5 experimental units per treatment. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).

In the jejunum, the V:C ratio differed significantly among treatments (p = 0.003), with the highest value in the D4-RS group and the lowest in the D1-Control group. No significant differences were detected in VH, VW, or CD in this segment. In the ileum, dietary treatments did not significantly affect any morphometric variable.

Intestinal segment | Diet | VH (µm) | VW (µm) | CD (µm) | V:C ratio
Duodenum | D1-Control | 2431.85 | 264.07 | 221.32ᵃ | 10.99ᵇ
 | D2-MOS | 2692.53 | 368.15 | 197.79ᵃᵇ | 13.61ᵃ
 | D3-FOS | 2566.44 | 327.57 | 187.45ᵇ | 13.69ᵃ
 | D4-RS | 2483.98 | 299.90 | 256.27ᵃ | 9.69ᵇ
 | SEM | 68.21 | 14.89 | 8.17 | 1.46
 | p value | 0.681 | 0.235 | 0.029 | 0.001
Jejunum | D1-Control | 1890.54 | 302.98 | 223.72 | 8.45ᵇ
 | D2-MOS | 1475.84 | 241.09 | 162.75 | 9.07ᵃᵇ
 | D3-FOS | 1569.01 | 310.51 | 175.67 | 8.93ᵃᵇ
 | D4-RS | 1472.72 | 250.55 | 143.27 | 10.28ᵃ
 | SEM | 72.89 | 9.77 | 14.99 | 0.89
 | p value | 0.067 | 0.409 | 0.104 | 0.003
Ileum | D1-Control | 933.63 | 285.48 | 127.77 | 7.31
 | D2-MOS | 1218.40 | 287.84 | 135.45 | 9.00
 | D3-FOS | 1129.53 | 264.65 | 195.02 | 5.79
 | D4-RS | 978.32 | 280.52 | 157.57 | 6.21
 | SEM | 66.63 | 8.38 | 15.99 | 2.92
 | p value | 0.264 | 0.251 | 0.211 | 0.091

Goblet cell counts

The relative proportion of goblet cells was not affected by dietary treatments in the duodenum, jejunum, or ileum. However, in the cecum, hens fed the D3-FOS (14.9 ± 5.6%) and D4-RS (13.8 ± 2.7%) diets showed significantly lower goblet cell percentages than those fed the D1-Control (26.9 ± 1.6%) and D2-MOS (25.1 ± 3.1%) diets (p < 0.05) (Figure 2).

Figure 2: Percentage of goblet cells relative to enterocytes in the duodenum (A), jejunum (B), ileum (C), and cecum (D) of 48-week-old laying hens after 12 weeks of dietary supplementation with different prebiotics.Error bars represent the standard error of the mean. Bars that do not share a letter differ significantly according to Tukey’s test (p < 0.05). n = 5 hens per treatment. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligo-saccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).

Cecal microbial communities

Core microbiota: Core microbiome analysis identified 25 bacterial genera that met the established prevalence and detection thresholds. Genera such as Ligilactobacillus, _Clostridium _sensu stricto, Cloacibacillus, Gemmiger, and Lactiplantibacillus showed high prevalence across all dietary treatments (Figure 3A). Venn diagram analysis revealed a shared core of 139 genera among all diets, while each treatment also showed unique genera (Figure 3B). The D3-FOS diet had the highest number of unique genera, followed by D2-MOS, D4-RS, and D1-Control.

Figure 3: Determination of the cecal microbiome core in laying hens under different dietary treatments. (A) Heatmap showing the prevalence (≥50%) and relative abundance (≥0.0001) of amplicon sequence variants (ASVs) detected in cecal samples. (B) Venn diagram illustrating unique and shared bacterial genera, with relative abundance ≥0.0001, among cecal microbial communities of commercial laying hens supplemented with different prebiotics. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).

Alpha and beta diversity: No significant differences were detected among diets or sampling weeks for the Chao1 richness index, although a trend associated with diet was observed (p = 0.058). The Shannon diversity index differed significantly across evaluation weeks (p = 0.019), with higher diversity observed at week 48. At this time point, hens fed the D3-FOS diet showed higher microbial diversity than those fed the D1-Control diet (p < 0.05) (Figure 4).

Figure 4: Alpha diversity of cecal microbial communities in commercial laying hens. Box-and-whisker plots show the Chao1 richness index and Shannon diversity index for hens at 43 weeks of age, W43, and 48 weeks of age, W48, supplemented with different prebiotics. Significant differences among diets within each time point, as determined by Tukey’s test (α ≤ 0.05), are indicated by different letters. n = 5 hens per treatment. W43: 43 weeks of age; W48: 48 weeks of age. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS). Principal coordinates analysis (PCoA) based on Bray–Curtis distances showed increased dissimilarity among microbial communities at week 48 (Figure 5). PERMANOVA confirmed significant effects of evaluation week (p = 0.006) and diet (p = 0.047) on microbial community composition.

Figure 5: Beta diversity of cecal microbial communities in commercial laying hens. Principal coordinates analysis (PCoA) based on Bray–Curtis distances showing the microbial composition of hens at 43 weeks of age, W43, and 48 weeks of age, W48, supplemented with different prebiotics. Points represent individual samples, and 95% confidence ellipses are shown for each dietary group. n = 5 hens per treatment. W43: 43 weeks of age; W48: 48 weeks of age. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).

Taxonomic composition: At the phylum level, Bacillota was dominant, with a mean relative abundance of 72.4% across all treatments and sampling times (Figure 6). Together with Pseudomonadota (9.6%) and Synergistota (7.2%), these three phyla represented nearly 90% of the cecal microbiota. A notable temporal shift was observed for Fusobacteriota, which decreased from 4.5% at week 43 to 0.5% at week 48. At week 43, Fusobacteriota was particularly enriched in the D3-FOS group. The relative abundance of Verrucomicrobiota was higher in the D2-MOS (2.7%) and D4-RS (3.7%) groups at week 43 and in the D2-MOS group (3.1%) at week 48. At week 48, Actinomycetotawas most abundant in the D3-FOS group (11.1%).

Figure 6: Phylum-level taxonomic composition of cecal microbial communities in commercial laying hens. Stacked bar charts show the relative abundance (%) of the main bacterial phyla in hens fed four different diets. Samples were collected at 43 weeks of age, W43, and 48 weeks of age, W48, corresponding to 7 and 12 weeks of experimental supplementation, respectively. n = 5 hens per treatment. W43: 43 weeks of age; W48: 48 weeks of age. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).At the genus level, Clostridiumsensu stricto (10.7%) and Ligilactobacillus (10.6%) were the most abundant genera at week 43, whereas Eubacteriales (12.7%) and Ligilactobacillus (12.6%) dominated at week 48 (Figure 7). Dietary effects were evident at week 43, when the highest relative abundances of Clostridiumsensu stricto were observed in the D1-Control (14.6%) and D2-MOS (12.5%) groups. By week 48, the D1-Control group showed the highest abundance of Ligilactobacillus (14.1%), whereas the D3-FOS group was enriched in Eubacteriales (16.7%), and the D4-RS group showed predominant abundance of Ligilactobacillus (17.7%). Regarding the Escherichia/Shigella genus, the highest relative abundance at week 43 was observed in the D1-Control group (5.3%), followed by the D4-RS group (4.6%). The D2-MOS and D3-FOS groups showed lower abundances of 2.5% and 3.3%, respectively, at week 43, and 4.0% and 3.5%, respectively, at week 48.

Figure 7: Relative abundance of the top 10 most abundant genera in cecal microbial communities of commercial laying hens. The chart shows genus-level composition for hens at 43 weeks of age, W43, and 48 weeks of age, W48, supplemented with different prebiotics. n = 5 hens per treatment. W43: 43 weeks of age; W48: 48 weeks of age. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).

Diet-associated microbial biomarkers: linear discriminant analysis effect size (LEfSe) identified specific bacterial taxa that were significantly enriched in each dietary group using an LDA score >3.0 and p < 0.05 (Figure 8). At week 43, the cecal microbiota of hens fed the D4-RS diet showed significant enrichment of Ligilactobacillus aviarius and Lactiplantibacillus_ plantarum_, whereas the D3-FOS diet was particularly enriched with Aminipila butyrica. By week 48, hens fed D4-RS showed enrichment of Lachnospiraceae and Blautia, whereas those fed D3-FOS showed enrichment of Cloacibacillus. The D1-Control group showed significant and consistent enrichment of Eggerthellaceae at both evaluation weeks.

Figure 8: Identification of diet-specific microbial biomarkers using linear discriminant analysis effect size (LEfSe) in commercial laying hens. The histogram shows linear discriminant analysis (LDA) scores for taxa whose abundance differed significantly among diets at 43 weeks of age, panel A, and 48 weeks of age, panel B. Biomarkers were identified using the Kruskal–Wallis rank-sum test (p < 0.05) with an LDA score threshold >3.0. n = 5 hens per treatment. D1-Control: basal diet without additives; D2-MOS: basal diet supplemented with 2,000 mg/kg mannanoligosaccharides (MOS); D3-FOS: basal diet supplemented with 700 mg/kg fructooligosaccharides (FOS); D4-RS: basal diet supplemented with 700 mg/kg potato resistant starch (RS).