Cecal Microbiota of Free-Range Hens Varied With Different Rearing Enrichments and Ranging Patterns

Abstract

Free-range pullets are reared indoors but the adult hens can go outside which is a mismatch that may reduce adaptation in the laying environment. Rearing enrichments might enhance pullet development and adaptations to subsequent free-range housing with impact on behavior and health measures including gut microbiota. Adult free-range hens vary in range use which may also be associated with microbiota composition. A total of 1,700 Hy-Line Brown^® chicks were reared indoors across 16 weeks with three enrichment treatment groups: "control" with standard litter housing, "novelty" with weekly changed novel objects, and "structural" with custom-designed perching structures in the pens. At 15 weeks, 45 pullet cecal contents were sampled before moving 1,386 pullets to the free-range housing system. At 25 weeks, range access commenced, and movements were tracked via radio-frequency identification technology. At 65 weeks, 91 hens were selected based on range use patterns ("indoor": no ranging; "high outdoor": daily ranging) across all rearing enrichment groups and cecal contents were collected for microbiota analysis via 16S rRNA amplicon sequencing at V3-V4 regions. The most common bacteria in pullets were unclassified Barnesiellaceae, Prevotella, Blautia and Clostridium and in hens Unclassified, Ruminococcus, unclassified Lachnospiraceae, unclassified Bacteroidales, unclassified Paraprevotellaceae YRC22, and Blautia. The microbial alpha diversity was not significant within the enrichment/ranging groups (pullets: P ≥ 0.17, hen rearing enrichment groups: P ≥ 0.06, hen ranging groups: P ≥ 0.54), but beta diversity significantly varied between these groups (pullets: P ≤ 0.002, hen rearing enrichment groups: P ≤ 0.001, hen ranging groups: P ≤ 0.008). Among the short-chain fatty acids (SCFAs), the propionic acid content was higher (P = 0.03) in the novelty group of pullets than the control group. There were no other significant differences in the SCFA contents between the rearing enrichment groups (all P ≥ 0.10), and the ranging groups (all P ≥ 0.17). Most of the genera identified were more abundant in the indoor than high outdoor hens. Overall, rearing enrichments affected the cecal microbiota diversity of both pullets and adult hens and was able to distinguish hens that remained inside compared with hens that ranging daily for several hours.

Keywords: 16S rRNA; RFID; bacteria; gut; indoor; novel objects; outdoor; perching structures.

FIGURE 1

Microbial community composition of pullets at 15 weeks of age from different rearing enrichments (control, novelty, structural). The top 20 abundant microbial genera are shown (clustered bar chart) in different enrichment groups of pullets.

FIGURE 2

(A) Relative abundance of cecal microbiota of pullets at 15 weeks of age from different rearing enrichments (control, novelty, structural) showing linear discriminant analysis (LDA) effect size method (LEfSe). (B) Differences in the relative abundance of cecal microbiota of pullets at 15 weeks of age from different rearing enrichments (control, novelty, structural) at genus level. One-way ANOVA followed by post hoc comparisons between rearing enrichment groups showed differences *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIGURE 3

Microbial diversity of cecal microbiota of pullets at 15 weeks of age from different rearing enrichments (control, novelty, structural) at ASV level. Alpha diversity: (A) Shannon index, P = 0.17; (B) Richness, P = 0.51; (C) Chao1, P = 0.54; (D) Simpson’s index, P = 0.34; and Beta diversity: (E) Redundancy analysis (RDA), P = 0.002; (F) ANOSIM (Bray-Curtis), P = 0.004.

FIGURE 4

Microbial community composition of free-range hens at 65 weeks of age from different rearing enrichments (control, novelty, structural). Clustered bar chart showing 20 most abundant genera.

FIGURE 5

Microbial community composition of free-range hens at 65 weeks of age from different ranging patterns (high outdoor, indoor). Clustered bar chart showing 20 most abundant genera.

FIGURE 6

(A) Relative abundance of cecal microbiota of free-range hens at 65 weeks of age from different rearing enrichments (control, novelty, structural) showing linear discriminant analysis (LDA) effect size method (LEfSe). (B) Relative abundance of cecal microbiota of free-range hens at 65 weeks of age from different rearing enrichments (control, novelty, structural) at genus level. One-way ANOVA followed by post hoc comparisons between rearing enrichment groups showed differences *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIGURE 7

(A) Relative abundance of cecal microbiota of free-range hens at 65 weeks of age from different ranging patterns (high outdoor, indoor) showing linear discriminant analysis (LDA) effect size method (LEfSe). The most abundant genera of microbiota from different ranging groups are shown. (B) Relative abundance of cecal microbiota of free-range hens at 65 weeks of age from different ranging patterns (high outdoor, indoor) at genus level. One-way ANOVA followed by post hoc comparisons between ranging patterns showed differences *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIGURE 8

Microbial diversity (alpha-diversity) of cecal microbiota of free-range hens at 65 weeks of age from different rearing enrichments (control, novelty, structural) at ASV level. (A) Shannon index, P = 0.07; (B) Richness, P = 0.74; (C) Chao1, P = 0.72; (D) Simpson’s index, P = 0.07.

FIGURE 9

Microbial diversity (alpha-diversity) of cecal microbiota of free-range hens at 65 weeks of age from different ranging patterns (high outdoor, indoor) at ASV level. (A) Shannon index, P = 0.74; (B) Richness, P = 0.66; (C) Chao1, P = 0.66; (D) Simpson’s index, P = 0.54.

FIGURE 10

Microbial diversity (Beta-diversity) of cecal microbiota of free-range hens at 65 weeks of age from different rearing enrichments (control, novelty, structural) and ranging patterns (high outdoor, indoor) at ASV level. (A) Redundancy analysis (RDA), P = 0.001; (B) ANOSIM (Bray-Curtis), P = 0.001. Microbial diversity of cecal microbiota of free-range hens at 65 weeks of age from different ranging patterns (high outdoor, indoor); (C) Redundancy analysis (RDA), P = 0.006; (D) ANOSIM (Bray–Curtis), P = 0.008.