L-carvone supplementation reduces methane production and modulates rumen fermentation, digestibility and microbial...

Natural feed additives are increasingly explored to reduce ruminal methane emissions. This study evaluated L-carvone (LC), a monoterpene compound present in essential oils, for its effects at different inclusion levels on rumen fermentation, in vitro dry matter digestibility (IVDMD), methane production, and microbiota composition.

Three treatments were tested: a basal diet (total mixed ration; TMR + 0 μL/L LC), LC250 (TMR + 250 μL/L LC), and LC500 (TMR + 500 μL/L LC), using a 59:41 concentrate-to-forage substrate. Fermentation parameters were analysed using the Gas Endeavour system and 16 S rRNA gene sequencing was applied to investigate the ruminal microbiota profile. Data were analysed using linear mixed models, with treatment included as a fixed effect and experimental runs as a random effect. Pairwise comparisons were conducted using Bonferroni correction, with p value below 0.05 considered significant.

Total gas production decreased progressively with increasing LC inclusion (p < 0.001), whereas methane production was significantly reduced only in LC500 (p < 0.001). LC500 reduced methane production by 28% compared with TMR (p < 0.001) and decreased IVDMD by approximately 15%, while LC250 showed a numerical but non-significant reduction in methane production without affecting IVDMD. Total volatile fatty acid (TVFA) concentration was lower in LC500compared with TMR (p < 0.01), whereas LC250 showed intermediate values. This reduction was mainly due to lower propionate, iso-butyrate, and iso-valerate concentrations. The proportion of butyrate increased in LC500 (p < 0.01). In addition, ammonia nitrogen concentration was lower in LC500 than in LC250 (p = 0.040), while TMR remained intermediate. At the phylum level, increasing LC supplementation reduced the abundance of Bacteroidota and increased Firmicutes. At the genus level, Prevotella abundance decreased with LC inclusion, whereas butyrate-producing genera including Butyrivibrio, Pseudobutyrivibrio, and Ruminococcus increased. Alpha diversity analysis showed that richness indices (observed OTUs and Faith's phylogenetic diversity) were highest in LC250, whereas Shannon diversity and Pielou's evenness were similarly higher in LC250 and LC500 compared with TMR.

The LC reduced methane production during a 24-h in vitro rumen fermentation while altering rumen fermentation characteristics, as evidenced by lower total gas production, IVDMD, and VFA concentrations at the highest inclusion level. These changes were accompanied by shifts in the rumen microbiota, including a reduced abundance of Euryarchaeota, a phylum associated with methanogenic archaea. Therefore, optimizing the inclusion level of LC is essential to achieve methane mitigation while minimizing adverse effects on feed digestion.