Integrated Gene–Metabolite Network Analysis Identifies Pathways Associated with Immune Regulation and Metabolic Remodeling in Subfertile Beef Heifers

Background/Objectives: Reproductive inefficiency remains a major contributor to heifer culling and reduced herd longevity in beef systems. This study examined the molecular basis of fertility by analyzing granulosa cells and follicular fluid from Angus–Simmental crossbred heifers classified as fertile or subfertile. Methods: Granulosa cells and follicular fluid were collected for RNA sequencing and metabolomic analysis. Differential expression, network, and gene–metabolite integration analyses identified genes, metabolites, and pathways associated with fertility differences between groups. Results: We identified 90 differentially expressed genes from the granulosa cells, including CXCL12 and HOXD family members, CALCRL, DNER, GADD45G, immune-related genes, and nine metabolites differentially abundant in follicular fluid, including parabanic acid, pimelic acid, α-tocopherol, glycine, and arachidonic acid. Network analysis revealed extensive rewiring in the network from the subfertile heifers for both genes and metabolites as compared to the fertile heifers. The gene–metabolite integration revealed a high degree of connectivity between arachidonic acid and α-tocopherol, correlating with immune and signaling genes, and between CXCL12 and carbohydrate intermediates. Pathway over-representation analysis highlighted carbohydrate and purine metabolism in fertile heifers, and aminoacyl-tRNA biosynthesis, alanine/aspartate/glutamate metabolism, and Hippo signaling in the subfertile heifers. ABC transporters, ferroptosis, mineral absorption, and glyoxylate/dicarboxylate metabolism were common pathways identified in both groups. Integrative granulosa-follicular fluid analysis revealed coordinated gene–metabolite rewiring in subfertility, including functional candidates (CXCL12, CALCRL, DNER; α-tocopherol, arachidonic acid, fucose, parabanic acid) and fertility-associated pathways. Conclusions: These signatures provide novel targets and pathways underlying beef heifer fertility.