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

Abstract

This study was part of an in vivo investigation of methane (CH 4 ) 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 ( Acacia nilotica ), BSG (Brewer's Spent Grain), and Ziziphus ( Ziziphus spina-christi ). The aim of the study was to investigate the rumen microbial community composition, diversity, and their relationships with CH 4 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, Rikenellaceae RC9 gut group (18%), Prevotella (17%), and Candidatus Saccharimonas (8.9%) were the most abundant Bacteria, while Methanobrevibacter (88%) dominated the Archaeal genera across all treatment groups. Treatment feed significantly altered microbial profiles, notably reducing Methanobrevibacter abundance in CH 4 abatement diets and increasing the presence of Methanosphaera . Shannon diversity increased in the abatement diet and decreased when the sheep were fed BSG. CH 4 intensity was most strongly associated with the archaeal genus Methanomicrobium , but did not associate strongly with any other Bacteria or Archaea, although Methanobrevibacter and Methanosphaera were correlated negatively ( r = –0.97). CH 4 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 ( r = 0.80) and a strong negative correlation between acetate and propionate ( r = –0.92). These findings highlight the complex relationship between diet, rumen microbiota, and fermentation products, with implications for CH 4 mitigation strategies in sheep.