Functional guild analysis characterizes gut microbiome responses to heat stress in thermotolerant and thermosensitive lactating sows across three omics layers

Heat stress (HS) reduces milk production, feed intake, and piglet growth in lactating sows, causing substantial economic losses. Genomic selection for thermotolerance is viable, yet how the gut microbiome responds to HS in genetically divergent sows is unclear. Multi-omics profiling could resolve this, but in small mechanistic studies molecular features vastly outnumber animals, defeating per-feature analysis. We compared thermotolerant (TOL, n = 13) and thermosensitive (SEN, n = 12) lactating sows from the top and bottom 1% of genomic estimated breeding values (GEBVs) for thermotolerance in a reference population of 1,381 commercial Landrace × Yorkshire sows. Sows underwent controlled cyclic HS from days 4–18 of lactation, and fecal samples were profiled by metagenomics (MG; days 4, 8, 14), metatranscriptomics (MT; days 0, 18), and metabolomics (MB; days 0, 8, 18). Features across all layers were aggregated into KEGG-Orthology-defined functional guilds and analyzed with linear mixed-effects models under false-discovery-rate (FDR) control; the framework was validated against random feature groupings and standard KEGG-module profiling. Guild aggregation captured differentiated temporal structure beyond random groupings (permutation p = 0.012) and reproduced module-level profiling. Gut functional responses to HS were dominated by temporal reorganization, robust to FDR correction across the metatranscriptome (13 of 22 guilds, q < 0.05) and metabolome (14 of 31 guilds; sphingolipids q = 2.3 × 10 −7 ). The functional guild response was shared across genetic lines, with line differences non-significant at baseline and at every timepoint (PERMANOVA p ≥ 0.21) and genetic line explaining at most 1.2% of guild-level variance versus 16–22% for timepoint — consistent with function tracking physiology and the shared temporal response rather than genotype. Rendered analyzable by a validated functional-guild framework, the lactating sows’ gut microbiome responds to HS through coordinated temporal reorganization that is shared across thermotolerant genetic lines rather than differing constitutively between them. Host genotype shapes gut function only indirectly, through physiology; between-line divergence is expressed taxonomically, not functionally.