Paper-Mulberry-Silage-Enhanced Lipid Metabolism and Milk Polyunsaturated Fatty Acid Concentration in Dairy Cows: A Multi-Omics Study

Developing alternative forages that modify milk fatty acid composition is important for improving milk nutritional quality and diversifying feed resources in dairy production. Previous studies have demonstrated the potential of paper mulberry (PM, Broussonetia papyrifera) silage to partially replace conventional roughages and influence antioxidant status and milk fatty acid composition. However, its effects on milk polyunsaturated fatty acids (PUFAs) and the associated ruminal microbial and metabolic mechanisms remain insufficiently characterized. This study aimed to evaluate the effects of feeding PM silage on the concentration of polyunsaturated fatty acids in milk, the structure of the rumen microbial community, and lipid metabolic functions, as well as to analyze the associations between rumen microbial and metabolic profiles and the fatty acid composition of milk. Thirty lactating Holstein cows were randomly assigned to control and PM groups, with 15 cows per group. The PM group received diets containing 9% PM silage for the first 28 days and 18% for the subsequent 28 days on a dry matter basis. Rumen fluid, plasma, and milk were collected on day 56 for integrated analyses combining fatty acid profiling, ruminal fermentation measurements, 16S rRNA gene sequencing, metagenomics, and metabolomics. Milk PUFA concentration was significantly higher in the PM group than in controls, with no significant differences in dry matter intake or milk yield. PM feeding also altered ruminal microbial community composition and lipid-metabolism-related functional gene abundances, accompanied by increased glycerophosphocholine and glycerophosphoethanolamine levels. Integrated analyses linked these changes to fatty acid profiles in rumen fluid, plasma, and milk, supporting ruminal lipid remodeling as a candidate mechanism underlying the increase in milk PUFA concentrations. These findings provide evidence for the microbial and metabolic basis of the effects of PM silage on milk fatty acid composition and inform its development as a functional forage and the optimization of dairy cow feeding strategies.