Dose–Response Effects of 3-Nitrooxypropanol Combined with Low- and High-Concentrate Feed Proportions in the Dairy Cow Ration on Fermentation Parameters in a Rumen Simulation Technique

Simple Summary Feeding strategies which aim at mitigating ruminal methane formation, a significant contributor to total greenhouse gas emissions, are being continuously developed, yet they need to be investigated in relation to their effectiveness and the mechanisms behind their effects in vitro before they undergo further assessment in vivo. In this context, the present study investigated the dose–response relationships of the methane inhibitor 3-nitrooxypropanol supplemented to varying concentrate feed proportions in a rumen simulation technique. Methane production was effectively reduced with an increasing dose of 3-nitrooxypropanol, which was, however, independent of concentrate feed proportion. Total gas production and fibre degradability were not affected by 3-nitrooxypropanol, indicating no negative side effects on fermentative capability. However, the hydrogen-liberating acetate production was reduced, whilst hydrogen gas was notably increased in a dose-dependent manner. The present in vitro study provides a deeper insight into a combined (3-nitrooxypropanol and high-concentrate feed proportions) methane abatement strategy under controlled conditions. The present combined approach reveals neither negative side effects nor additive effects between 3-nitrooxypropanol and varying concentrate feed proportions, which should be further investigated in future experiments in vivo. Abstract Methane (CH4) from ruminal feed degradation is a major pollutant from ruminant livestock, which calls for mitigation strategies. The purpose of the present 4 × 2 factorial arrangement was to investigate the dose–response relationships between four doses of the CH4 inhibitor 3-nitrooxypropanol (3-NOP) and potential synergistic effects with low (LC) or high (HC) concentrate feed proportions (CFP) on CH4 reduction as both mitigation approaches differ in their mode of action (direct 3-NOP vs. indirect CFP effects). Diet substrates and 3-NOP were incubated in a rumen simulation technique to measure the concentration and production of volatile fatty acids (VFA), fermentation gases as well as substrate disappearance. Negative side effects on fermentation regarding total VFA and gas production as well as nutrient degradability were observed for neither CFP nor 3-NOP. CH4 production decreased from 10% up to 97% in a dose-dependent manner with increasing 3-NOP inclusion rate (dose: p < 0.001) but irrespective of CFP (CFP × dose: p = 0.094). Hydrogen gas accumulated correspondingly with increased 3-NOP dose (dose: p < 0.001). In vitro pH (p = 0.019) and redox potential (p = 0.066) varied by CFP, whereas the latter fluctuated with 3-NOP dose (p = 0.01). Acetate and iso-butyrate (mol %) decreased with 3-NOP dose, whereas iso-valerate increased (dose: p < 0.001). Propionate and valerate varied inconsistently due to 3-NOP supplementation. The feed additive 3-NOP was proven to be a dose-dependent yet effective CH4 inhibitor under conditions in vitro. The observed lack of additivity of increased CFP on the CH4 inhibition potential of 3-NOP needs to be verified in future research testing further diet types both in vitro and in vivo.