Simple SummaryThere is a global focus on the search for identifying species of macroalgae that can be fed to livestock as a feedstock, or as a methane mitigant. This study evaluated the effect of three red seaweeds to assess their potential for decreasing ruminant enteric methane production. While onlyAsparagopsis taxiformiswas effective at decreasing methane production, this study provides new information on the requirements for adapting animals to seaweed feeding.AbstractSeveral red seaweeds have been shown to inhibit enteric CH4 production; however, the adaptation of fermentation parameters to their presence is not well understood. The objective of this study was to examine the effect of three red seaweeds (Asparargopsis taxiformis,Mazzaella japonica, andPalmaria mollis) on in vitro fermentation, CH4 production, and adaptation using the rumen simulation technique (RUSITEC). The experiment was conducted as a completely randomized design with four treatments, duplicated in two identical RUSITEC apparatus equipped with eight fermenter vessels each. The four treatments included the control and the three red seaweeds added to the control diet at 2% diet DM. The experimental period was divided into four phases including a baseline phase (d 0–7; no seaweed included), an adaptation phase (d 8–11; seaweed included in treatment vessels), an intermediate phase (d 12–16), and a stable phase (d 17–21). The degradability of organic matter (p= 0.04) and neutral detergent fibre (p= 0.05) was decreased byA. taxiformisduring the adaptation phase, but returned to control levels in the stable phase.A. taxiformissupplementation resulted in a decrease (p< 0.001) in the molar proportions of acetate, propionate, and total volatile fatty acid (VFA) production, with an increase in the molar proportions of butyrate, caproate, and valerate; the other seaweeds had no effect (p> 0.05) on the molar proportions or production of individual VFA.A. taxiformiswas the only seaweed to suppress CH4 production (p< 0.001), with the suppressive effect increasing (p< 0.001) across phases. Similarly,A. taxiformisincreased (p< 0.001) the production of hydrogen (H2, %, mL/d) across the adaptation, intermediate, and stable phases, with the intermediate and stable phases having greater H2 production than the adaptation phase. In conclusion,M. japonicaandP. mollisdid not impact rumen fermentation or inhibit CH4 production within the RUSITEC. In contrast, we conclude thatA. taxiformisis an effective CH4 inhibitor and its introduction to the ruminal environment requires a period of adaptation; however, the large magnitude of CH4 suppression byA. taxiformisinhibits VFA synthesis, which may restrict the production performance in vivo.