The aim of this study was to determine the effect of milk thistle (Silybum marianum L., SM) oil supplementation on proinflammatory cytokines, acute phase proteins, rumen metagenomic profile, rumen fluid variables and performance during the milk feeding period of Holstein calves. In the present study, 24 calves that consumed quality and sufficient amount of colostrum (>= 50 mg/ml IgG) after birth were divided into three groups, with 8 animals in each group (4 males + 4 females). Individually fed calves were given added SM oil as 0 mu L/day/calf (Control group, SM0), 100 mu L/day/calf (SM100) and 200 mu L/day/calf (SM200). The ration containing concentrated feed mix (90%) + wheat straw (10%) was offered to the calves. SM oil did not change the ammonia nitrogen and pH values of the rumen fluid of calf (P>0.05). The molarities of PA, IBA, IVA and BSCFA in the rumen fluid increased linearly with SM oil dose (P<0.05). The relative abundance of Firmicutes linearly increased and the relative abundance of Actinobacteriota decreased with the addition of SM oil (P<0.05). Relative abundances of Erysipelotrichaceae_UCG_002, Eubacterium_coprostanoligenes_group, Clostridia_UCG_014, Lachnospiraceae_Unknown_1, Lachnospiraceae_NK3A20_group, Shuttleworthia, Selenomonadaceae_Uncultured_1, Rikenellaceae_RC9_gut_group and Succinivibrionaceae_UCG_001 linearly increased with SM oil (P<0.05). Relative abundances of Methanobrevibacter, Acetitomaculum, Olsenella and Megasphaera in calf rumen fluid linearly decreased with SM oil (P<0.05). Concentrations of TNF-alpha, IFN-gamma, and SAA of calf' serum at weaning stage linearly decreased with SM oil doses (P<0.05). Serum IgA concentration increased with 100 <mu>L SM oil /day (P<0.05). As a result, the addition of SM oil to calves has the potential to reduce the immune suppression of calves during the milk feeding period and at weaning time, has a positive effect on the microbiome involved in starch and protein catabolism in the rumen fluid, and increases the fermentation end products (PA, IVA and BA). Milk thistle oil has an inhibitory effect on methanogenic archaea and can be used as an anti-methanogenic feed additive and will contribute to the effective use of feed energy.
