ABSTRACT: It is well known that the methionine dipeptide (MD) could enhance the dairy cows milking performance. However, there is still a knowledge gap of the effects of MD on the rumen fermentation characteristics, microbiota composition, and digestibility. This experiment was conducted to determine the effect of different nitrogen sources with a total mixed ration on in vitro nutrient digestibility, fermentation characteristics, and bacterial composition. The treatments included 5 mg urea (UR), 25.08 mg methionine (Met), 23.57 mg MD, and no additive (CON) in fermentation culture medium composed of buffer solution, filtrated Holstein dairy cow rumen fluid, and substrate (1 g total mixed ration). Nutrient digestibility was measured after 24 h and 48 h fermentation, and fermentation parameters and microbial composition were measured after 48 h fermentation. Digestibility of dry matter, crude protein, neutral detergent fiber (NDF), and acid detergent fiber (ADF) in the MD group at 48 h were significantly higher than in the CON and UR groups. The total volatile fatty acid concentration was higher in the MD group than in the other groups. In addition, 16S rRNA microbial sequencing results showed MD significantly improved the relative abundances of Succinivibrio, Anaerotruncus, and Treponema_2, whereas there was no significant difference between Met and UR groups. Spearman's correlation analysis showed the relative abundance of Succinivibrio and Anaerotruncus were positively correlated with gas production, NDF digestibility, ADF digestibility, and acetate, propionate, butyrate, and total volatile fatty acid concentrations. Overall, our results suggested that the microbiota in the fermentation system could be affected by additional nitrogen supplementation and MD could effectively enhance the nutrient utilization in dairy cows.
