Generational Succession of Rectal Microbiome and Metabolome in Holstein–Norwegian Red Crossbred Offspring and Their Regulatory Effects on Milk Quality

This study aimed to explore the generational succession rules of rectal microbes and metabolites in crossbred dairy cows, and clarify the regulatory mechanism underlying generational differentiation of milk quality traits, so as to provide a theoretical basis for improving milk quality and realizing precision breeding of dairy cows based on hindgut microecology. In this experiment, 10 healthy cows in mid-lactation were selected from each of three consecutive generations (F1, F2, and F3) of Holstein–Norwegian Red crossbred cattle, totaling 30 cows. High-throughput 16S rRNA sequencing combined with UPLC-MS/MS-based untargeted metabolomics was applied to systematically analyze the correlations between the rectal microbial community structure and metabolic profiles and milk quality indicators. The results indicated that milk quality traits exhibited obvious generational differentiation: daily milk yield increased gradually with generation and was significantly higher in F3 cows than in F1 cows (p < 0.05), while milk fat rate and milk protein rate showed an upward-then-downward trend and reached the highest level in the F2 generation. The rectal microbial community underwent directional generational succession, with the relative abundance of Firmicutes continuously increased and Actinobacteriota gradually decreased, and the microbial functions were mainly specialized in carbohydrate and amino acid metabolism. Significant generational differences were also observed in rectal metabolic profiles, and the differential metabolites screened between F1 and F3 generations were primarily enriched in steroid hormone biosynthesis, nucleotide sugar metabolism, bile acid metabolism, and amino acid metabolism pathways. Multi-omics correlation analysis further verified that the core rectal microbial genera could regulate the lactation performance of the mammary gland by affecting the abundance of nutritional metabolic substrates and signaling molecules, with metabolites acting as critical mediators in the regulatory relationship between rectal microbes and milk quality traits. This study systematically characterizes the generational succession characteristics of rectal microbes and metabolites in crossbred dairy cows and identifies potential correlative mediating links of rectal metabolites between microbes and milk-quality traits, providing a novel theoretical reference for exploring hindgut microecology to regulate dairy cow milk quality and assist elite breeding.