Simple SummaryAlfalfa (Medicago sativaL.) is considered the queen of forages, and hay is an important source of protein and fiber for livestock, while guanidinoacetic acid (GAA) is a feed additive that can improve growth performance and energy metabolism in animals and reduce the population of methanogenic microorganisms. However, the percentage of alfalfa hay (AH) in the diet can cause variations in greenhouse gas (GHG) production, the rumen fermentation profile and methane (CH4) conversion efficiency, which, in turn, influences the effectiveness of GAA. In this regard, this study demonstrates that the percentage of AH in the diet affects the effectiveness of GAA and that the addition of GAA in diets with 25 and 100% AH presents low effectiveness, a diet with 10% AH can improve the mitigation of GHG and the rumen fermentation profile without compromising the CH4conversion efficiency using a dose of 0.0015 or 0.0020 g GAA g−1DM in the diet.AbstractThe objective of this study was to evaluate the effect of different percentages of alfalfa (Medicago sativaL.) hay (AH) and doses of guanidinoacetic acid (GAA) in the diet on the mitigation of greenhouse gas production, the in vitro rumen fermentation profile and methane (CH4) conversion efficiency. AH percentages were defined for the diets of beef and dairy cattle, as well as under grazing conditions (10 (AH10), 25 (AH25) and 100% (AH100)), while the GAA doses were 0 (control), 0.0005, 0.0010, 0.0015, 0.0020, 0.0025 and 0.0030 g g−1DM diet. With an increased dose of GAA, the total gas production (GP) and methane (CH4) increased (p= 0.0439) in the AH10 diet, while in AH25 diet, no effect was observed (p= 0.1311), and in AH100, GP and CH4levels decreased (p= 0.0113). In addition, the increase in GAA decreased (p= 0.0042) the proportion of CH4in the AH25 diet, with no influence (p= 0.1050) on CH4in the AH10 and AH100 diet groups. Carbon monoxide production decreased (p= 0.0227) in the AH100 diet with most GAA doses, and the other diets did not show an effect (p= 0.0617) on carbon monoxide, while the production of hydrogen sulfide decreased (p= 0.0441) in the AH10 and AH100 diets with the addition of GAA, with no effect observed in association with the AH25 diet (p= 0.3162). The pH level increased (p< 0.0001) and dry matter degradation (DMD) decreased (p< 0.0001) when AH was increased from 10 to 25%, while 25 to 100% AH contents had the opposite effect. In addition, with an increased GAA dose, only the pH in the AH100 diet increased (p= 0.0142 andp= 0.0023) the DMD in the AH10 diet group. Similarly, GAA influenced (p= 0.0002) SCFA, ME and CH4conversion efficiency but only in the AH10 diet group. In this diet group, it was observed that with an increased dose of GAA, SCFA and ME increased (p= 0.0002), while CH4per unit of OM decreased (p= 0.0002) only with doses of 0.0010, 0.0015 and 0.0020 g, with no effect on CH4per unit of SCFA and ME (p= 0.1790 andp= 0.1343). In conclusion, the positive effects of GAA depend on the percentage of AH, and diets with 25 and 100% AH showed very little improvement with the addition of GAA, while the diet with 10% AH presented the best results.