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), <i>Ampelopsis grossedentata</i> 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 (<i>p</i> < 0.05), whereas AGF reduced urea concentrations (<i>p</i> < 0.05). Only GAA decreased ruminal pH (<i>p</i> < 0.05). All additives enhanced microbial richness (<i>p</i> < 0.05), with 5,6-DMB + Co inducing the most pronounced community restructuring. Taxonomically, AGF and 5,6-DMB + Co decreased <i>Firmicutes</i> and enriched <i>Verrucomicrobiota</i>, while AGF uniquely increased <i>Cyanobacteria</i>. GAA selectively reduced <i>Quinella</i>, AGF decreased <i>Christensenellaceae_R-7_group</i> and <i>NK4A214_group</i>, and 5,6-DMB + Co markedly enriched <i>Rikenellaceae_RC9_gut_group</i> (<i>p</i> < 0.05). Microbial shifts were closely associated with biochemical indices, including positive associations of <i>Prevotella</i> with AST and <i>Rikenellaceae_RC9_gut_group</i> with total protein (<i>p</i> < 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.