The effects of three emulsifying methods on ruminal fatty acid biohydrogenation (BH) in vitro were compared. Using a static in-vitro gas test system, four replicates of each treatment were incubated in buffered ruminal fluid. Hemicellulose (300mg dry matter) was supplemented either with or without linoleic acid (9c12c-18:2, 5% in diet dry matter) and incubated for 4 and 24h. Three methods of emulsifying 9c12c-18:2 were tested: (1) ethanol, (2) Tween® 80, and (3) sonication. The products were then compared to non-emulsified 9c12c-18:2. Out of the three emulsifying methods tested, ethanol and sonication resulted in stable 9c12c-18:2 emulsions, indicating good 9c12c-18:2 distribution, while the Tween® 80 emulsion was less stable. BH was strongly inhibited by treating 9c12c-18:2 with ethanol and sonication at different steps of the BH-pathway, resulting in changed concentrations of certain BH intermediates. The fatty acid profile generated from the major BH-pathways of 9c12c-18:2 with Tween® 80 was comparable to that without emulsification after 24h of incubation. We conclude that it is not recommended to emulsify lipids before incubating them in vitro when investigating fatty acid BH. If emulsification of 9c12c-18:2 is necessary, Tween® 80 seems to be the method that interferes least with B
