Serine governs metabolic homeostasis and microbial crosstalk to promote the utilization of urea nitrogen in rumen fermentation in vitro

Abstract Background Urea is commonly used as a protein substitute in ruminant diets due to its nitrogen utilization efficiency and cost‐effectiveness. Serine‐derived downstream metabolism contributes to urea‐enriched metabolic pathways in rumen fluid. Results This study examined the effects of 7.50 g kg −1 dry matter serine supplementation on fermentation parameters and microbial communities in urea‐based substrates. Fifteen amino acids were detected in the fermentation fluid. Urea‐based substrates exhibited distinct amino acid profiles compared to the control basal substrate, with lower levels of most amino acids except cysteine (Cys), leucine (Leu), and phenylalanine (Phe). Serine supplementation increased propionate production, microbial protein synthesis, and dry matter digestibility, while decreasing isovalerate, isoleucine (Ile), and Leu levels. Additionally, serine supplementation altered microbial populations, increasing Ruminobacter and Bacteroidales_BS11_gut_group by 24.47% and 26.42%, respectively, while decreasing Prevotella and WCHB1 ‐41 by 9.30% and 24.15%, respectively. Glycine and folate, which are downstream metabolites of serine, exhibited partial similarity to serine regarding their influence on fermentation profiles. Notably, serine reduced the fermentation of Leu and Ile into isovalerate, irrespective of their supplementation, exhibiting a dosage‐dependent effect. Conclusions In short, serine supplementation in urea‐based substrates enhances fermentation, promoting the utilization of urea nitrogen and amino acids. Serine mediated crosstalk between the ‘serine‐glycine‐folate’ metabolic pathway and reshaped microbial communities to improve the utilization of urea nitrogen and amino acids. © 2025 Society of Chemical Industry.