Linoleic acid enhances ruminal carbohydrate metabolism and biohydrogenation to promote rumen epithelial development in calves.

Unlike mature ruminant, pre-weaned calves have limited ruminal biohydrogenation during early microbial establishment. Since linoleic acid (LA) is known to promote calf growth, elucidating whether dietary LA supplementation during this plastic phase can actively shape rumen biohydrogenation function, restructure the microbiota, and drive epithelial development represents a pivotal unanswered question. This study determined the optimal dietary supplementation dose of LA and elucidated its regulatory effects on the rumen microbiota, metabolites and gene expression in preweaning calves. LA supplementation reshaped the rumen microbial community of calves and enhanced microbial carbohydrate metabolism by increasing the abundance of carbohydrate-active enzymes, thereby promoting acetate synthesis. Hydrogen generated during carbohydrate fermentation further facilitated LA biohydrogenation. The metabolites generated from this process activate the microbial phosphatidylcholine synthesis pathway in the rumen, increasing PC concentrations in both rumen fluid and tissue. CYP3A24 was identified as a core rumen gene involved in the PC-CYP3A24 regulatory axis. PC promoted bovine rumen epithelial cells (BRECs) proliferation, barrier function, antioxidant capacity and inflammation suppression, while CYP3A24 silencing diminishes these effects. In conclusion, optimal LA supplementation regulates rumen metabolism, enhances PC synthesis via functional microbiota, and activates the PC-CYP3A24 axis, thereby improving rumen development and calf growth performance.