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Aim: The aim of this study was to evaluate the effects of various combinations of tropical grass-legume species in rations on the biohydrogenation (BH) activity of unsaturated fatty acids (FAs), C18:0 composition, and fermentation profile in an in vitro rumen system. Materials and Methods: Samples of the following five fodder plants were used: One species of grass (Pennisetum purpureum) and four species of tree legumes (Leucaena leucocephala, Gliricidia sepium, Calliandra calothyrsus, and Indigofera zollingeriana). The following eight experimental diets were evaluated: 50% P. purpureum + 50% L. leucocephala (LL I); 50% P. purpureum + 50% G. sepium (GS I); 50% P. purpureum + 50% C. calothyrsus (CC I); 50% P. purpureum + 50% I. zollingeriana (IZ I); 75% P. purpureum + 25% L. leucocephala (LL II); 75% P. purpureum + 25% G. sepium (GS II); 75% P. purpureum + 25% C. calothyrsus (CC II); and 75% P. purpureum + 25% I. zollingeriana (IZ II). Each ration was replicated 3 times. In vitro rumen incubation was performed for 48 h, according to the Tilley and Terry method. Determination of the FA profiles of the forage materials and rumen fluid samples was performed using gas chromatography. Results: The percentage of polyunsaturated FA (PUFA) in the forage materials ranged from 34.18% (P. purpureum) to 74.51% (C. calothyrsus). The percentage of monounsaturated FA (MUFA) ranged from 5.06% (P. purpureum) to 8.71% (L. leucocephala). The percentage of saturated FA (SFA) was the lowest at 19.12% (C. calothyrsus) and highest at 60.76% (P. purpureum). In vitro BH of C18:3 n-3, C18:2 n-6, C18:1 n-9, and C18 PUFA in the experimental diets ranged from 72% to 100%. The BH of C18:1 n-9 in GS I (80%) and IZ I (72%) was significantly different (p<0.05). The percentage of C18:0 was 10-50% and significantly different (p<0.05) among treatments, with the highest (of 50%) in GS II. No significant differences (p>0.05) were observed in the fermentation parameters (pH, total volatile FAs, in vitro dry matter digestibility, and in vitro organic matter digestibility) among the treatments, except in NH3 concentration (p<0.05). Conclusion: The various combinations of tropical legumes do not have significant inhibitory effects on the BH of C18:2 n-6, C18:3 n-3, and C18 PUFA after in vitro incubation for 48 h. Furthermore, an increase in the tropical legume ratio in the ration tends to suppress C18:0 formation after the fermentation simulation process. IZ I has the potential to reduce C18:1 n-9 (MUFA) disappearance and yield an ideal rumen fermentation profile. [ABSTRACT FROM AUTHOR]