IN VITRO EVALUATION OF ESSENTIAL OILS AND ORGANIC ACIDS: TOXICITY AND RUMINAL FERMENTATION

Ruminant livestock play an essential role in global food security and economic sustainability, providing approximately one-third of humanity's protein consumption and supporting the livelihoods of 1.3 billion people. However, ruminant livestock are also a major source of greenhouse gas (GHG) emissions, primarily methane (CH₄). On an individual level, beef cattle emit approximately 60 to 66 kilograms of methane per year, while dairy cows produce between 84 and 123 kilograms annually. This difference may be partly due to the higher proportion of concentrate in beef cattle diets, which is known to reduce methane production compared to forage-based diets. Methane is primarily produced during rumen fermentation, where methanogenic archaea convert available hydrogen into methane. High-concentrate diets help mitigate these emissions by shifting fermentation toward propionate production, which competes with methanogenesis for hydrogen utilization. Beyond environmental concerns, reducing methane emissions is also crucial for improving feed efficiency, as methane losses can account for 2% to 12% of a ruminant’s dietary energy, representing a significant inefficiency in nutrient utilization. This thesis evaluates the effects of plant-based compounds, including essential oils (EOs) and organic acids (OAs), on the animal’s immune system, reducing Greenhouse gas emissions while maintaining or improving ruminant productivity and rumen microbiome modulation. The research conducted a series of in vitro experiments to evaluate the effects of these compounds. The EO and OA were selected following a cooperation with the SILA company (https://www.silaonline.com/en/), in the frame of the PON program (DM 1061 del 10 agosto 202IV.4 – Dottorati e contratti di ricerca su tematiche dell’innovazione -Azione IV.5 – Dottorati su tematiche Green). In the first set of experiments, the potential toxicity of the compounds was tested on White Blood Cells. The viability and apoptosis assays was measured on bovine peripheral mononuclear cells (PBMCs), and the results demonstrated that OAs (AO1, AO2, AO3) maintained cell viability across all concentrations, whereas AO4 showed cytotoxicity at concentrations above 50 μM. Meanwhile, essential oils (EO1 to EO5) exhibited minimal toxicity up to 400 μM but became toxic at higher doses. Mixtures of EOs and OAs retained high cell viability, confirming their safety. In a second set of experiments, the effects of EO and OA were tested using an in vitro model of ruminal fermentation. After concluding the cell viability and toxicity experiments, the company developed 10 different mixtures of the previously tested essential oils and organic acids for fermentation experiments. Using batch bottles, the fermentation experiments evaluated the impacts of these encapsulated mixtures on methane emissions and rumen fermentation with two distinct diets: a high-forage diet representative of dairy production systems (Diet 1) and a high-concentrate diet typical of growing and fattening systems (Diet 2). This distinction provides insight into how these additives function in different ruminant production contexts. The study found that Diet 1 did not demonstrate favorable outcomes, whereas Diet 2 showed promising methane mitigation and productivity metrics results. Overall, most additives demonstrated potential in reducing methane emissions, which can be related to the impact of the specific compound on the fermentation, given that some also reduced VFA production, especially with Diet 1. Concentrated formulations (Additives 8 to 10) were tested at reduced doses, with Additive 10 demonstrating the best results by achieving significant methane reduction and maintaining fermentation efficiency. High-concentrate diets further enhanced the efficacy of these additives in mitigating methane emissions, suggesting their particularly relevant application in growing systems. This effect was evident when compared to the control group, where diets without additives exhibited higher methane emissions, confirming the contribution of supplementation beyond the inherent effects of diet composition. Meanwhile, their impact on high-forage diets requires further investigation to optimize methane reduction without compromising fermentation efficiency.