This study investigated the potential of feed additives for very long-chain fatty acids (VLCFA) and α-linolenic acid (ALA), which were identified in sea staghorn (Codium fragile) as natural feed additives to reduce enteric methane emissions in ruminants. An in vitro rumen fermentation model was used to evaluate the synergistic effects of VLCFAs and ALA, both individually and in combination, on methane emissions, dry matter digestibility, and the microbial community in the rumen. The results demonstrated that treatments with VLCFAs or ALA alone, at concentrations of up to 1.5% of the feed, did not significantly affect methane production, and significantly reduced the digestibility in the VLCFAs treatment. However, a mixture of 1.0% VLCFAs and ALA, particularly at a 2:1 ratio, significantly reduced methane emissions by 21% without compromising dry matter digestibility synergistically. Methanobrevibacter was over 96% of the total archaeal population, and Methanosphaera accounted for 2–3% of the total archaea. Furthermore, Prevotella and Succinivibrio were identified as predominant bacteria in the bacterial community. The treatment of VLCFAs or ALA alone slightly decreased the total gene counts, while the mixture of VLCFA-ALA treatment increased the total gene counts. In addition, the VLCFA-ALA mixture treatment increased the overall archaeal population and the archaea-to-bacteria ratio, while preserving the total bacterial population and digestibility; these were both negatively affected by VLCFAs alone.
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