Effects of methionyl-methionine dipeptide on fecal microbiota in transition dairy cows

Transition dairy cows undergo substantial metabolic stress, which increases the demand for methionine, a key amino acid involved in milk synthesis and metabolic regulation. Methionyl-methionine dipeptide is a methionine-containing dipeptide that has been shown to promote milk protein synthesis in vitro and improve milk protein production in vivo. Although the effects of methionine supplementation on ruminal metabolism in dairy cows have been widely studied, the effects of methionyl-methionine dipeptide on the fecal microbiota of transition dairy cows remain poorly characterized. At 28 d prepartum, 45 Holstein cows were blocked by parity and randomly assigned to control ( CON , basal diet), unprotected methionyl-methionine dipeptide ( MM , basal diet + 8 g/d MM), or rumen-protected methionyl-methionine dipeptide ( RPMM , basal diet + 22 g/d RPMM, providing 8 g/d intestinally available MM). At 21 ± 1 d postpartum, 6 representative cows per treatment were selected for fecal sampling and 16S rRNA gene sequencing. Fecal microbial diversity and community structure differed among treatments, whereas the dominant taxa at the phylum and genus levels remained largely consistent. Among the differentially abundant genera, Turicibacter was more abundant in the RPMM group than in the CON and MM groups and was positively associated with milk yield, plasma total protein, and plasma total antioxidant status. Howardella was less abundant in the RPMM group than in the CON group and was positively associated with plasma BHB and nonesterified fatty acids, but negatively associated with milk protein concentration. Compared with the RPMM group, the MM group had a higher relative abundance of [Eubacterium]_siraeum_grou p and Colidextribacter and a lower relative abundance of Frisingicoccus , with Colidextribacter positively associated with milk protein concentration and plasma GLU concentration. Overall, supplementation with MM or RPMM was associated with distinct shifts in the fecal microbiota, with RPMM-related taxa showing closer associations with lactation performance and plasma metabolic parameters. These findings advance our understanding of the links between methionyl-methionine supplementation, fecal microbiota, and metabolic adaptation in transition dairy cows.