Rumen and saliva microbiome alterations in ketotic dairy cows

Ketosis is a common metabolic disorder in dairy cows which has been associated with altered rumen fermentation and potentially increased CH₄ production[1]. Metabolomic studies on serum of cows affected by ketosis have reported increased levels of methanogenesis-related substrates, suggesting a systemic metabolic shift potentially linked to rumen fermentation patterns and CH₄ production[2,3]. The aim of the study was to assess the alterations of rumen and saliva microbiome in dairy cows affected by ketosis. Procedures were approved by Ethical Committee for Animal Welfare of University of Padua (n.103549/2024). A total of 60 Holstein-Friesian multiparous dairy cows were enrolled from a single farm. Animals were divided into two groups according to BHB: CTR or control (BHB<1.0 mmol/L; n=43); KET or affected by ketosis (BHB≥1.0 mmol/L; n=17). Rumen fluid sampling via stomach tube and oral swabs were performed in KET cows at the time of diagnosis. The CTR, which remained clinically healthy during the trial, were sampled only once at 21 days in milk to ensure a physiologically comparable control group. The rumen and saliva microbiota were investigated using an amplicon-based metagenomic analysis amplifying the V3-V4 hypervariable regions of the 16S rRNA gene. Reads were clustered into zOTUs and taxonomically assigned to the latest version of the SILVA database (v.138). Alpha-diversity, beta-diversity (UniFrac), differential abundance, and rumen-saliva distance analyses were performed to evaluate microbiome structure and its association with disease status. A p≤0.05 was accepted. Biodiversity and composition analyses revealed significant differences between rumen and saliva, as well as between CTR and KET experimental groups. In rumen samples, phylogenetic diversity was higher in KET animals (Faith’s phylogenetic diversity, p=0.027). Differential abundance analysis showed enrichment of Prevotella, Treponema, Fibrobacter, and Ruminococcaceae-related taxa in rumen samples from KET, together with a reduction in Firmicutes and Lachnospiraceae groups. In saliva, ketosis was associated with a lower alpha-diversity (p<0.01 for all the considered metrics) and a different microbial composition (p=0.008 and p=0.021 for the unweighted and weighted UniFrac distances, respectively), with increased Actinobacteriota, Staphylococcus, Bibersteinia, and Kingella, and decreased Lachnospiraceae and Succinivibrionaceae. Distance analyses indicated greater rumensaliva microbial dissimilarity in KET cows (borderline significance, p=0.05), suggesting disrupted host-microbiome interactions. Overall, the observed shifts in bacterial Printed on 29/04/2026 12:01 – Page 2/2 communities’ support a disease-driven alteration of fermentation-related microbial pathways potentially linked to CH₄ production. These findings highlight the microbiome as a key component of the pathophysiology of ketosis and as a potential target for prevention strategies and mitigation of livestock-related environmental impacts. 1.Lisuzzo,A. et al. (2022)“Differences in the serum metabolome profile of dairy cows according to the BHB concentration revealed by proton nuclear magnetic resonance spectroscopy(1H-NMR),”Scientific Reports,12(1),pp.1–10 2.Mostert,P.F. et al. (2018)“The impact of subclinical ketosis in dairy cows on greenhouse gas emissions of milk production,”Journal of Cleaner Production,171,pp.773–782 3.Yanibada,B. et al. (2020)“Inhibition of enteric methanogenesis in dairy cows induces changes in plasma metabolome highlighting metabolic shifts and potential markers of emission,”Scientific Reports 2020 10:1,10(1),pp.15591