α-Ketoglutarate enhances milk protein yield in dairy cows accompanied by increased rumen microbial protein synthesis and improved dietary crude protein digestibility.

Alpha-ketoglutarate (AKG), a key intermediate in the tricarboxylic acid cycle, has been demonstrated to exert multiple physiological benefits, including promoting growth, enhancing nitrogen utilization, improving immunity, and optimizing intestinal health in monogastric livestock and aquatic animals. However, studies investigating its effects in ruminants remain limited. This study systematically evaluated the effects of AKG on rumen fermentation characteristics, production performance, nutrient digestibility, and health parameters in mid-lactation dairy cows through a combination of in vitro fermentation and in vivo feeding trials. The in vitro experiment was conducted using 4 AKG concentrations (0, 5, 15, and 45 mg/dL), with each treatment performed in quadruplicate and repeated across 3 batches. Rumen fermentation parameters and microbial crude protein (MCP) were measured to determine the optimal AKG dose. Results showed that AKG supplementation linearly increased total gas production, MCP, and total VFA concentrations, while linearly decreasing pH and NH3-N levels. All AKG supplementation levels improved in vitro rumen fermentation characteristics, with stronger responses observed at higher doses. Based on these in vitro findings, an in vivo experiment was performed using 24 multiparous Holstein cows, stratified by milk yield into 2 blocks. Within each block, cows were randomly assigned to one of 2 treatment groups (n = 12 per group): a control group fed the basal diet, and an AKG group fed the basal diet supplemented with 25 g of AKG per cow per day. The trial lasted 10 weeks, including a 2-week adaptation period and an 8-week experimental period. AKG supplementation significantly increased milk protein yield, while numerical increases were observed for DMI, milk yield, ECM, and lactose yield that did not reach statistical significance. Additionally, dietary AKG markedly enhanced ruminal MCP, butyrate, valerate, isobutyrate, isovalerate, and total branched-chain volatile fatty acid concentrations, with a tendency to increase total VFA. Compared with the control group, cows in the AKG group exhibited significantly higher apparent digestibility of DM, OM, and CP. Plasma biochemical and immune analyses revealed that AKG supplementation significantly decreased aspartate aminotransferase and γ-glutamyl transferase activities, while markedly increasing IgG levels, suggesting a more favorable hepatic metabolic profile and enhanced humoral immune response. AKG supplementation did not affect the population of bacteria, protozoa, methanogens, or fungi, but reduced the α-diversity of rumen bacteria. At the phylum level, AKG supplementation did not affect rumen bacterial abundance, but at the genus level, it tended to increase the abundance of Prevotella while decreasing that of Barnesiella, Coprobacter, and Desulfovibrio. In summary, the in vitro experiments showed that 15-45 mg/dL of AKG was identified as an appropriate supplementation level. The in vivo feeding trial further demonstrated that dietary supplementation with 25 g/d AKG enhanced rumen fermentation, improved nutrient digestibility and plasma immune-related biomarkers, and increased milk protein production in dairy cows. These changes were associated with decreased bacterial α-diversity and a trend toward increased Prevotella abundance.