Associations between volatile fatty acid profiles, methane emissions, and rumen microbiota in sheep fed Ethiopian forage

This study was part of an <i>in vivo</i> investigation of methane (CH₄) abatement feed on local Menz breed sheep in Ethiopia, conducted over 90 days period using a randomized complete block design. Sheep were subjected to four dietary treatments: Control, Acacia (<i>Acacia nilotica</i>), BSG (Brewer's Spent Grain), and Ziziphus (<i>Ziziphus spina-christi</i>). The aim of the study was to investigate the rumen microbial community composition, diversity, and their relationships with CH₄ intensity. Rumen fluid was collected on days 0 (SD_0), 45 (SD_45), and 90 (SD_90), using an esophageal tube. The dynamics of the bacterial and archaeal domains were assessed by 16S rRNA gene sequencing. The sequencing results showed that 92.9% of ASVs were Bacteria, and 0.05% Archaea. At the genus level, <i>Rikenellaceae RC9 gut</i> group (18%), <i>Prevotella</i> (17%), and <i>Candidatus</i> Saccharimonas (8.9%) were the most abundant Bacteria, while <i>Methanobrevibacter</i> (88%) dominated the Archaeal genera across all treatment groups. Treatment feed significantly altered microbial profiles, notably reducing <i>Methanobrevibacter</i> abundance in CH₄ abatement diets and increasing the presence of <i>Methanosphaera</i>. Shannon diversity increased in the abatement diet and decreased when the sheep were fed BSG. CH₄ intensity was most strongly associated with the archaeal genus <i>Methanomicrobium</i>, but did not associate strongly with any other Bacteria or Archaea, although <i>Methanobrevibacter</i> and <i>Methanosphaera</i> were correlated negatively (<i>r</i> = -0.97). CH₄ intensity also did not covary with volatile fatty acids (VFAs), of which Acacia yielded the highest acetate (772 mmol/mol) and BSG the highest propionate (172 mmol/mol) concentration. The volatile fatty acids (VFAs) showed a strong correlation: a positive correlation between acetate and butyrate (<i>r</i> = 0.80) and a strong negative correlation between acetate and propionate (<i>r</i> = -0.92). These findings highlight the complex relationship between diet, rumen microbiota, and fermentation products, with implications for CH₄ mitigation strategies in sheep.