Effects on Rumen Microbial Population and Serum Biochemical Responses to Guanidinoacetic Acid, Ampelopsis grossedentata Flavonoids, and 5,6-Dimethylbenzimidazole Plus Cobalt in Lanping Black-Boned Sheep

To mitigate antimicrobial resistance and drug residues, the use of growth-promoting antibiotics in livestock has been prohibited, prompting interest in safe natural alternatives. Lanping black-boned sheep, an indigenous high-altitude breed in Yunnan, exhibit unique physiological traits but relatively low feed efficiency. This study evaluated the effects of three natural additives—guanidinoacetic acid (GAA, 1 g/sheep/day), Ampelopsis grossedentata flavonoids (AGF, 1 g/sheep/day), and 5, 6-dimethylbenzimidazole plus cobalt (5, 6-DMB + Co; 100 mg 5, 6-DMB + 0. 5 mg Co/sheep/day) —on serum biochemistry and rumen microbiota in twenty-four Lanping black-boned sheep fed under grazing conditions for 90 days following a 10-day adaptation period. GAA and 5, 6-DMB + Co increased serum globulin (p < 0. 05), whereas AGF reduced urea concentrations (p < 0. 05). Only GAA decreased ruminal pH (p < 0. 05). All additives enhanced microbial richness (p < 0. 05), with 5, 6-DMB + Co inducing the most pronounced community restructuring. Taxonomically, AGF and 5, 6-DMB + Co decreased Firmicutes and enriched Verrucomicrobiota, while AGF uniquely increased Cyanobacteria. GAA selectively reduced Quinella, AGF decreased ChristensenellaceaeR-7group and NK4A214group, and 5, 6-DMB + Co markedly enriched RikenellaceaeRC9gutgroup (p < 0. 05). Microbial shifts were closely associated with biochemical indices, including positive associations of Prevotella with AST and RikenellaceaeRC9gutgroup with total protein (p < 0. 05). Overall, GAA mainly affected protein-related indices, AGF lowered serum urea, and 5, 6-DMB + Co increased within-sample diversity with accompanying community reweighting, providing a theoretical basis for developing sustainable feeding strategies to enhance both productivity and health in Lanping black-boned sheep.