Flaxseed is the richest source of secoisolariciresinol diglucoside, which is converted by ruminal microorganisms primarily to the mammalian lignan enterolactone. Our objective was to investigate the effect of diets containing soybean meal or flaxseed meal (FM) supplemented with sucrose, flaxseed oil, or both, on milk enterolactone concentration yield, diversity, and relative abundance of ruminal bacterial taxa, ruminal fermentation profile, production performance, milk fatty acids (FA) yield, and nutrient utilization in dairy cows. Sixteen Holstein cows (8 multiparous [4 ruminally cannulated] and 8 primiparous) averaging (mean ± SD) 134 ± 54.1 DIM and 679 ± 78.9 kg of BW in the beginning of the study were assigned to treatment sequences in a replicated 4 × 4 Latin square design. Each experimental period lasted 25 d, with 18 d for diet adaptation and 7 d for data and sample collection. Diets were formulated to contain a 60:40 forage:concentrate ratio and included the following (DM basis): (1) 8% soybean meal and 23% ground corn (SBM); (2) 15% FM, 10.7% ground corn, and 5% sucrose (FLX+S); (3) 15% FM, 15.4% ground corn, and 3% flaxseed oil (FLX+O); and (4) 15% FM, 10.2% ground corn, 5% sucrose, and 3% flaxseed oil (FLX+SO). Compared with SBM, the concentration and yield of milk enterolactone increased in cows fed the FM diets but did not differ among FLX+S, FLX+O, and FLX+SO. The relative abundances of the phyla Firmicutes, Verrucomicrobiota, and Actinobacteriota, and those of the bacterial genera Lachnospiraceae NK3A20 group, Eubacterium coprostanoligenes group, Anaeromusa-Anaeroarcus, WCHB1-41, and p-251-o5 decreased, whereas Prevotella and NK4A214 group increased when comparing SBM against at least 1 diet containing FM. Furthermore, the relative abundances of Firmicutes and Actinobacteriota and those of Prevotella, Lachnospiraceae NK3A20 group, Eubacterium coprostanoligenes group, Acetitomaculum, Lachnospiraceae unclassified, NK4A214 group, and Anaeromusa-Anaeroarcus changed (increased or decreased) across the FLX+S, FLX+O, and FLX+SO diets. However, all these changes in the relative abundance of the ruminal bacterial taxa were not conclusively associated with the effect of diets on milk enterolactone. Diets did not affect ruminal pH and concentrations of NH3-N and total VFA. Dry matter intake and yields of milk, milk fat, and milk true protein all decreased in cows fed FLX+O or FLX+SO. Yields of milk total odd-chain FA, branched-chain FA, total <16C FA, and total 16C FA decreased with feeding FLX+O and FLX+SO. The apparent total-tract digestibilities of DM and OM were lowest in the FLX+S and FLX+O diets, with CP and ADF digestibilities lowest in cows receiving FLX+S or FLX+O, respectively. Urinary excretion of total N was lowest with feeding SBM. Contrarily, diets did not affect the urinary excretion of total purine derivatives. In brief, despite the effect of diets on the relative abundance of several ruminal microbiota phyla and genera, we were unable to conclusively associate these changes with increased milk enterolactone in FM-containing diets versus SBM.
