IntroductionAs a core nutrient regulating ruminant metabolic networks, immune homeostasis, and rumen microbial community structure, the source and supply of protein influence the adaptive capacity of breeding cows to metabolic stress during physiological transitions in the peripartum period.MethodsSixty healthy, periparturient cows with similar body weights (573.14 ± 76.67 kg) and gestation lengths (228.77 ± 37.86 d) were selected and randomly divided into four groups (n = 15), each group received an isocaloric, isonitrogenous diet supplemented with soybean meal (Control Group, CG), rapeseed meal (Test Group 1, TG1), linseed cake (Test Group 2, TG2), or sunflower cake (Test Group 3, TG3) as the primary protein source. Rumen fermentation parameters, microbial composition and community structure, serum indicators, apparent nutrient digestibility, nitrogen utilization, and differential metabolites in feces and urine were measured to investigate the effects of different protein sources on rumen fermentation-microbial characteristics, nutrient utilization, and fecal/urinary metabolites in periparturient breeding cows.Results(1) pH levels in the TG2 increased significantly at 30 days postpartum (PP30 d), peptidyltransferase (PLT) counts in the TG2 and TG3 were significantly higher than those in the CG and TG1 at calving day (0 d); Proteolytic enzymes (Pro), urease (URE), and PLT levels in the TG3 increased significantly at PP30 d; beneficial bacteria such as Fibrobacter and Akkermansia were significantly enriched in the TG2 and TG3 (p < 0.05). (2) In the TG3, Urea and triglycerides (TG) levels were significantly elevated at PP30 d, while blood ammonia levels were reduced; in the TG2, energy metabolism was active at 0 d and stabilized at PP30 d, exhibiting the strongest humoral immune response and significantly elevated Gonadotropin-releasing hormone (GnRH) levels. The TG3 played a prominent role in the phasic regulation of insulin (INS) and growth hormone (GH). (3) Compared with the CG, 498, 285, and 225 differentially expressed metabolites were identified in the feces of the experimental groups, respectively; the TG1 was enriched in steroids, riboflavin, and the NF-κB pathway; the TG2 was enriched in riboflavin, steroids, retinol, and alkaloid pathways; and the TG3 showed a regulatory trend toward bile secretion and steroid synthesis; A total of 259, 456, and 234 differentially expressed metabolites were identified in urine, respectively. The TG1 primarily affected protein and carbohydrate metabolism; the TG2 focused on amino acid metabolism and biosynthesis; and the TG3 showed significant enrichment in the biosynthesis of unsaturated fatty acids, biotin metabolism, and folate biosynthesis pathways.DiscussionThe rapeseed meal group caused excessive protein fermentation, leading to the production of endogenous mycotoxins and triggering oxidative stress and metabolic burden. The sunflower meal group exhibited elevated levels of intestinal toxins and permeability markers, which impaired reproductive performance. The linseed cake group maintains ruminal fermentation and intestinal metabolism in cows, making it a high-quality protein source for feeding periparturient breeding cows as a substitute for soybean meal.