| Summary: | The objective of this study was to evaluate the effect of different percentages of alfalfa (<i>Medicago sativa</i> L.) 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 (CH<sub>4</sub>) 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<sup>−1</sup> DM diet. With an increased dose of GAA, the total gas production (GP) and methane (CH<sub>4</sub>) increased (<i>p</i> = 0.0439) in the AH10 diet, while in AH25 diet, no effect was observed (<i>p</i> = 0.1311), and in AH100, GP and CH<sub>4</sub> levels decreased (<i>p</i> = 0.0113). In addition, the increase in GAA decreased (<i>p</i> = 0.0042) the proportion of CH<sub>4</sub> in the AH25 diet, with no influence (<i>p</i> = 0.1050) on CH<sub>4</sub> in the AH10 and AH100 diet groups. Carbon monoxide production decreased (<i>p</i> = 0.0227) in the AH100 diet with most GAA doses, and the other diets did not show an effect (<i>p</i> = 0.0617) on carbon monoxide, while the production of hydrogen sulfide decreased (<i>p</i> = 0.0441) in the AH10 and AH100 diets with the addition of GAA, with no effect observed in association with the AH25 diet (<i>p</i> = 0.3162). The pH level increased (<i>p</i> < 0.0001) and dry matter degradation (DMD) decreased (<i>p</i> < 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 (<i>p</i> = 0.0142 and <i>p</i> = 0.0023) the DMD in the AH10 diet group. Similarly, GAA influenced (<i>p</i> = 0.0002) SCFA, ME and CH<sub>4</sub> conversion 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 (<i>p</i> = 0.0002), while CH<sub>4</sub> per unit of OM decreased (<i>p</i> = 0.0002) only with doses of 0.0010, 0.0015 and 0.0020 g, with no effect on CH<sub>4</sub> per unit of SCFA and ME (<i>p</i> = 0.1790 and <i>p</i> = 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.
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