| Summary: | This study explores whether guanidinoacetic acid (GAA) addition can regulate nutrient degradability, rumen fermentation characteristics, and gas composition in two sheep-fattening diets. A 2 × 8 factorial in vitro culture was examined to determine the effects of GAA addition at the following levels of 0%, 0.03%, 0.05%, 0.07%, 0.09%, 0.11%, 0.13%, and 0.15% of two total mixed rations (T1 diet: early fattening stage diet; T2 diet: late fattening stage diet). After 72 h in vitro incubation of two diets with mixed rumen liquid obtained from six rumen-cannulated lambs, the T2 diet exhibited higher dry matter (DM) digestibility, higher cumulative gas production at 72 h (GP<sub>72</sub>), higher asymptotic gas production(A), and longer the time at which half of A is reached (C). However, it exhibited a lower acetic acid and a lower ratio of acetate to propionate than the diet of T1. A quadratic increase occurred in neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility, with a maximum point occurring at the 0.09% GAA group. The gas production kinetic result indicated that increasing the level of GAA addition resulted mainly in an increase of GP<sub>72</sub> and A, with the maximum point occurring at 0.09% for the T1 diet and 0.07–0.09% for the T2 diet. Moreover, the levels of GAA addition did not affect pH, the proportion of any of the volatile acid, or gas composition, but when the levels of GAA addition were increased, the microbial crude protein (MCP), ammonia nitrogen (NH<sub>3</sub>-N), and total volatile fatty acid (TVFA) content exhibited a quadratic relationship. The highest MCP contents were seen in the 0.07%, 0.09%, and 0.11% groups, while NH<sub>3</sub>-N and TVFA were in the 0.07% group. In summary, the appropriate level of GAA addition in early and late fattening stage diets ranged from 0.07% to 0.11%.
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