Impact of Dietary Fiber on Ruminal Microbiome in Free-Range Ruminants

Thesis Abstract

This study investigates how dietary fiber shapes the ruminal microbiome in free-range ruminants, addressing the rising concern that heterogeneous foraging patterns and fiber diversity in pastoral systems influence microbial ecology, fermentation efficiency, and animal productivity. The aim is to quantify relationships between dietary fiber fractions and rumen microbial composition and function, and to link these to feed efficiency, methane emission potential, and nutrient utilization under real-world grazing conditions. Specific objectives are (i) to characterize the dietary fiber profile (neutral detergent fiber, acid detergent fiber, lignin, and soluble non-starch polysaccharides) of grazing ruminants across seasonal foraging cycles; (ii) to determine the ruminal microbial taxonomic and functional profiles using 16S rRNA gene amplicon sequencing and shotgun metagenomics; (iii) to quantify rumen fermentation parameters (pH, volatile fatty acids, ammonia-N) and methane emissions using open-path lasers and gas chromatography; (iv) to assess associations between dietary fiber variables and microbial community structure, functional gene abundance (carbohydrate-active enzymes), and fermentation outcomes; (v) to evaluate the influence of microbial profiles on feed intake quality, growth performance or body condition, and nitrogen utilization. The study adopts a longitudinal field design conducted on three large-scale pastoral herds in a temperate grassland region, involving 120 adult ewes and 60 castrated males over a 12-month cycle to capture seasonal variability. Data collection integrates nutrition analysis of forage samples, in situ rumen sampling via rumen-cannulated subsamples (n=60 animals at each sampling period), and non-invasive sampling for microbial and metabolite assessment. For microbial analyses, high-throughput 16S rRNA sequencing (Illumina MiSeq) will profile bacterial and archaeal communities, complemented by shotgun metagenomics (Illumina NovaSeq) on a subsample (n=24) to reveal functional genes, especially glycoside hydrolases and polysaccharide lyases. Fermentation and gas data will be measured monthly, with VFA concentrations determined by gas chromatography, and methane quantified using open-path Fourier-transform infrared spectroscopy calibrated against portable gas analyzers. Statistical analyses will employ linear mixed models to test associations between dietary fiber fractions and microbial alpha and beta diversity, PERMANOVA for community dissimilarities, and generalized linear models to relate microbial features to fermentation metrics and animal performance. Multivariate approaches, including redundancy analysis and canonical correspondence analysis, will elucid how fiber composition explains variation in microbial function. Structural equation modeling will be used to explore causal pathways linking forage fiber, microbiome, fermentation, and production outcomes. The theoretical framework integrates the Community Assembly Theory and the Host-Microbiome Interaction model to interpret how dietary fiber shapes microbial ecology and host physiology in free-range systems, while the study tests propositions derived from the Functional Redundancy and Niche Theory hypotheses regarding microbiome resilience to forage variability. Expected findings include a clear relationship between higher soluble fiber fractions and increased relative abundance of fibrolytic bacteria and cellulolytic enzymes, accompanied by shifts in VFA profiles toward acetate and butyrate with potential reductions in methane yield per unit of feed intake. It is anticipated that distinct fiber spectra will correlate with improved crude protein utilization and body condition scores in spring and autumn, moderated by microbial community stability across seasons. The study contributes to knowledge by providing empirical evidence on foraging-driven microbiome dynamics in free-range ruminants, informing diet–microbiome–production models, and guiding pasture management aimed at optimizing fiber quality to enhance fermentation efficiency and reduce greenhouse gas emissions. The main conclusion is that tailored management of forage fiber composition within pastoral systems can steer rumen microbial ecology toward more efficient fermentation and better animal performance, while recommendations emphasize adaptive grazing strategies, periodic forage quality assessments, and integration of microbial monitoring into herd nutrition programs to sustain productivity and environmental sustainability in free-range ruminants.