Early post-weaning probiotic supplementation alters gut microbial community structure and predicted metabolic function in female juvenile mice

To characterize the effects of early post-weaning multi-strain probiotic supplementation on gut microbiota composition, predicted metabolic function, and short-chain fatty acid (SCFA) profiles in female juvenile mice at the pubertal window.

Female C57BL/6 mice were randomly assigned to probiotic (Pro) or control (Con) groups; after gavage-related losses, final sample sizes were Pro: n = 9 and Con: n = 6. The probiotic suspension contained Bifidobacterium longum, Lactobacillus bulgaricus, and Streptococcus thermophilus and was administered from postnatal day 21 to 27. Fecal samples were collected during the three days preceding vaginal opening for 16S rRNA gene sequencing (V3-V4 region, DADA2/ASV pipeline, SILVA 138.1 database), PICRUSt2 functional prediction, and targeted SCFA quantification. PERMANOVA with multivariate dispersion testing was used for beta-diversity analysis, and Benjamini-Hochberg correction was applied for multiple comparisons.

A total of 2,946 ASVs were identified from 15 fecal samples. Probiotic supplementation significantly decreased alpha diversity (Observed species, Chao1, ACE, and Shannon; all P < 0.05) and altered overall community structure (PERMANOVA: Bray-Curtis R² = 0.193, P = 0.013; Jaccard R² = 0.158, P = 0.008), with no difference in multivariate dispersion. No genus remained significant after FDR correction. At a nominally significant level, Prevotellaceae UCG-001 and an unclassified Bacteroidales genus were depleted, while Dubosiella, Parabacteroides, and an unclassified Lachnospiraceae genus were enriched. Akkermansia showed a trend toward enrichment (9.39% vs. 45.85%; ANCOM-BC2: log2FC = +2.15, P = 0.179, FDR = 0.395). PICRUSt2 identified 63 MetaCyc pathways at nominal significance, none surviving FDR correction (minimum FDR = 0.106). SCFA analysis revealed nominally significant reductions in caproic acid (P = 0.039) and elevations in acetic acid (P = 0.039), neither surviving FDR correction (FDR = 0.121). Two correlations remained significant at the exploratory FDR < 0.1 threshold: Prevotellaceae UCG-001-acetic acid (rho = -0.693) and Akkermansia-valeric acid (rho = -0.670).

A brief post-weaning probiotic intervention significantly altered overall gut microbial community structure in female juvenile mice, with exploratory shifts in predicted functional capacity and a trend toward Akkermansia enrichment, without disrupting normal pubertal development. These findings should be considered exploratory given the small sample size.