Effects of Supplementing Rumen-Protected Lysine and Methionine on Apparent Digestibility, Rumen Fermentation Parameters, and Microbial Profiles in Lactating Dairy Cows Under Different Environmental Conditions

This study investigated the effects of supplementing rumen-protected lysine (RPL; 60 g/d) and methionine (RPM; 30 g/d) on nutrient digestibility, rumen fermentation, and microbial composition in 30 early-lactation Holstein dairy cows under both heat stress (HS) and non-heat stress (NHS) conditions. Cows were paired based on parity, milk yield, and body weight, and then assigned to one of two dietary treatments: the control group (CON), receiving the basal diet only, or the RPLM group, which received the basal diet supplemented with 60 g/d of RPL and 30 g/d of RPM (<i>n</i> = 15 per group). All animals underwent a transition from HS to NHS conditions during the experimental period, allowing for within-animal comparison across thermal environments. Results demonstrated that RPLM supplementation significantly improved the apparent digestibility of dry matter (DM), crude protein (CP), and neutral detergent fiber (NDF) (<i>p</i> < 0.05), with a significant treatment × environment interaction observed for DM digestibility (<i>p</i> < 0.01). Under HS conditions, RPLM reduced ruminal NH₃-N concentration (<i>p</i> < 0.05) and increased total volatile fatty acids (TVFA), acetate, and butyrate levels (<i>p</i> < 0.01), suggesting enhanced nitrogen utilization and energy metabolism. No significant effects were observed on ruminal pH or the acetate-to-propionate ratio. 16S rRNA sequencing revealed that RPLM supplementation increased the relative abundance of fiber-degrading and butyrate-producing taxa, including <i>NK4A214_group</i>, <i>Christensenellaceae_R-7_group</i>, <i>norank_f__Muribaculaceae</i>, and <i>norank_f__F082</i>. These microbial taxa exhibited strong positive correlations with DM and CP digestibility (<i>p</i> < 0.001). LEfSe analysis further confirmed their status as key microbial biomarkers in the RPLM-NHS group. Collectively, concurrent supplementation of RPL and RPM modulates the ruminal microbiota, alleviates HS-induced reductions in digestive efficiency, and enhances overall nutrient utilization in dairy cows.