Pasture diversity and regenerative management shape ruminant gut microbiome dynamics within a real multi-year farming system.

The ruminant gut microbiome is central to feed conversion efficiency, nutrient utilisation, and animal health, yet how pasture diversity and management shape its composition over time under real, working farm conditions remains poorly understood, as most previous studies have relied on short-term or more tightly controlled comparisons. A multi-year study (2022-2025) investigated gut microbial dynamics in cattle and sheep grazing on different pasture management systems; standard pastures under contemporary management (Std-Con), standard pastures under regenerative management (Std-Reg), diverse pastures under contemporary management (Div-Con), and diverse pastures under regenerative management (Div-Reg) at Massey University, Palmerston North, New Zealand, using 16S rRNA gene amplicon sequencing of faecal DNA. Microbial relative abundance remained broadly stable overall, with notable seasonal fluctuations. Faecalibacterium spp. were the most abundant taxon overall in both hosts, though its relative abundance varied seasonally and dropped below detection in cattle under the Std-Con treatment during winter (August 2023), where Bacteroides spp. became dominant instead. Alpha diversity was also stable across treatments for both hosts, however, a sharp decline was observed in Std-Con (1.6 ± 0.27) and Div-Con (1.6 ± 0.21) during winter (August 2023), while Div-Reg remained higher (2.3 ± 0.2) in cattle. Significant temporal signals were also detected in alpha diversity, with a peak across all treatments in cattle in spring (October 2022). In sheep, both regenerative treatments were higher in spring (November 2023) (Std-Reg: 2.59 ± 0.076, Div-Reg: 2.52 ± 0.077). Pairwise beta-diversity comparisons revealed significant divergence in microbial community composition between Std-Con and Div-Reg, most pronounced in cattle from December 2024 to April 2025 (p = 0.049-0.015) and in sheep from August to October 2023 (p = 0.003-0.001). These findings demonstrate that, under real, multi-year grazing conditions, seasonal effects played a greater role in shaping gut microbial dynamics than pasture treatment alone, with regenerative management buffering a marked winter decline in cattle. Future studies directly linking functional gene expression, forage-quality, and animal production data to microbiome composition within the same sampling framework are needed to determine whether these compositional shifts translate into measurable benefits for ruminant health and productivity.