Abstract
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 Christensenellaceae_R-7_group and NK4A214_group, and 5,6-DMB + Co markedly enriched Rikenellaceae_RC9_gut_group (p < 0.05). Microbial shifts were closely associated with biochemical indices, including positive associations of Prevotella with AST and Rikenellaceae_RC9_gut_group 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.