Innovative Technologies and Laboratory Procedures for the Mitigation of Methane Emissions from Ruminants Using Plant Secondary Compounds

Abstract Enteric methane emissions from ruminant livestock are an important source of agricultural greenhouse gases and also represent a loss of dietary energy for the animal. Therefore, reducing methane emissions has both environmental and nutritional importance. However, progress in this area depends not only on effective mitigation strategies, but also on accurate methods to measure methane production. This PhD thesis focused on two main aspects: improving the reliability of in vitro methane measurement, and evaluating plant-based nutritional strategies that can reduce methane without negatively affecting protein utilisation. The first part of the thesis examined methodological factors affecting in vitro gas and methane measurements. Two automated systems, AnkomRF and Gas Endeavour (GES), were evaluated to study methane production over time. The results showed that methane production changes during fermentation and cannot be adequately described using a single end-point value. Factors such as CO₂ flushing time and headspace volume had a strong effect on measured gas and methane production. When these factors were not standardised, methane was either under- or over-estimated. After optimisation, the Gas Endeavour system allowed continuous and more consistent measurement of methane kinetics, making it suitable for in vitro methane mitigation studies. Using this optimised approach, the second part of the thesis evaluated plant secondary compounds as methane mitigation strategies. A sugarcane-derived polyphenol (Polygain™) was tested using a combined in vitro rumen fermentation and simulated intestinal digestion model. Polygain™ reduced methane production in a dose-dependent manner, especially at higher inclusion levels, without causing a clear reduction in total gas production. This suggests that Polygain™ affected specific fermentation pathways rather than broadly inhibiting microbial activity, which was supported by changes in volatile fatty acid profiles. An important finding of this work was that Polygain™ also influenced protein utilisation. Polygain reduced rumen degradable protein and increased rumen undegradable protein by altering protein degradation kinetics, while intestinal digestibility was maintained or improved. Increased in vitro intestinal digestibility of rumen undegradable protein supports the idea of reversible polyphenol–protein interactions. These effects were also reflected in amino acid profiles, with lower release of free amino acids during rumen fermentation and a tendency for higher availability of essential amino acids after simulated intestinal digestion. The results also showed that type of substrate (forage) characteristics influenced methane responses. Desmanthus meal had lower baseline methane production and showed a smaller response to supplementation, likely due to its natural polyphenol content. Therefore, Desmanthus has potential to be used as alternative forage. In contrast, the herein evaluated alpine plant extracts did not reduce methane production under the conditions tested, although some increased propionate concentration, suggesting a possible improvement in rumen energy efficiency rather than direct methane mitigation. Overall, this thesis highlights the importance of methodological optimisation before evaluating methane mitigation strategies. It also shows that methane reduction and protein utilisation should be considered together. Sugarcane-derived polyphenols, particularly Polygain™, appear to be promising candidates for reducing methane while improving nitrogen efficiency, although further validation under in vivo conditions is needed.