Citation: Terry, S.A.; Krüger, A.M.; Lima, P.M.T.; Gruninger, R.J.; Abbott, D.W.; Beauchemin, K.A. Evaluation of Rumen Fermentation and Microbial Adaptation to Three Red Seaweeds Using the Rumen Simulation Technique. Animals 2023, 13, 1643. https://doi.org/10.3390/ ani13101643 Academic Editor: Alexandros Mavrommatis Received: 8 February 2023 Revised: 19 April 2023 Accepted: 20 April 2023 Published: 15 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article Evaluation of Rumen Fermentation and Microbial Adaptation to Three Red Seaweeds Using the Rumen Simulation Technique Stephanie A. Terry 1, * , Ana M. Krüger 2 , Paulo M. T. Lima 2,† , Robert J. Gruninger 1 , D. Wade Abbott 1 and Karen A. Beauchemin 1 1 Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 3B6, Canada 2 Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, São Paulo 13400-970, Brazil * Correspondence: stephanie.terry@agr.gc.ca † Current Address: Department of Animal Science, University of Wyoming, Laramie, WY 82071, USA. Simple Summary: There 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 only Asparagopsis taxiformis was effective at decreasing methane production, this study provides new information on the requirements for adapting animals to seaweed feeding. Abstract: Several 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, and Palmaria 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 by A. taxiformis during the adaptation phase, but returned to control levels in the stable phase. A. taxiformis supplementation 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. taxiformis was the only seaweed to suppress CH4 production (p < 0.001), with the suppressive effect increasing (p < 0.001) across phases. Similarly, A. taxiformis increased (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. japonica and P. mollis did not impact rumen fermentation or inhibit CH4 production within the RUSITEC. In contrast, we conclude that A. taxiformis is an effective CH4 inhibitor and its introduction to the ruminal environment requires a period of adaptation; however, the large magnitude of CH4 suppression by A. taxiformis inhibits VFA synthesis, which may restrict the production performance in vivo. Keywords: rumen simulation technique; methane production; seaweed; rumen fermentation 1. Introduction Increased greenhouse gas (GHG) concentrations in the atmosphere have resulted in alterations to the ozone layer, consequently raising the global surface temperature [1]. The agricultural sector contributes to 26.0% of anthropogenic global GHG, mainly CO2, N2O, Animals 2023, 13, 1643. https://doi.org/10.3390/ani13101643 https://www.mdpi.com/journal/animals