Apple pomace and hempseed cake can reduce methane intensity (CH₄/DMI) and alter the rumen microbiome in dairy cows: a shotgun metagenomic approach

Abstract Background With growing attention to environmental impacts, the dairy sector is increasingly focused on implementing strategies that lower methane emissions and enhance sustainability while maintaining productivity and economic viability. Utilizing agro-industrial by-products as alternative feed ingredients supports circular economy goals, lowers feed costs, and may benefit rumen fermentation and environmental performance in dairy cows. Methods Forty-five mid-lactation Holstein cows were assigned to three diets, Control, Apple Pomace (AP), or Hempseed Cake (HC) for 24 d. Feed intake, milk yield, rumen fermentation, methane emissions, and nutrient use were measured. Rumen samples underwent shotgun metagenome sequencing and bioinformatics analysis to assess microbial and functional changes. Results Values are reported as mean ± SEM. Shotgun metagenomic sequencing revealed that both supplements significantly increased the relative abundance of Bacteroidota (AP: 56.7% ± 2.8%, P = 0.032; HC: 54.5% ± 3.4%, P = 0.048) compared to the Control (48.2% ± 3.1%). Concurrently, Bacillota (formerly Firmicutes) abundance decreased, significantly reducing the Bacillota/Bacteroidota ratio (formerly the Firmicutes/Bacteroidetes ratio) from 0.81 ± 0.06 (Control) to 0.58 ± 0.05 for AP ( P = 0.012) and 0.64 ± 0.05 for HC ( P = 0.034). Functional analysis showed that AP increased the abundance of Segatella bryantii (2.1-fold, P < 0.01), associated with a 1.52-fold enrichment in propionate metabolism pathways ( P = 0.019). Phenotypically, AP significantly reduced the acetate-to-propionate ratio (AP: 2.41 vs. Control: 4.50; P = 0.0075) and methane emissions per unit of dry matter intake (CH 4 /DMI) (AP: 20.33 vs. Control: 24.27 g/kg; P = 0.016). HC supplementation upregulated fiber-degrading taxa such as Xylanibacter ruminicola (1.6-fold) and enriched xylanase families (GH10: 1.58-fold, P = 0.035), alongside a significant reduction in methane intensity (CH 4 /DMI). Total methane output, feed intake, and milk yield were not significantly changed by treatments ( P > 0.05). Conclusions In this short-term (24-d) controlled feeding study in mid-lactation Holstein cows, AP and HC were associated with distinct microbial and functional shifts alongside lower methane intensity, with AP linked to propanoate-related signals and HC to fiber-degrading functions; however, ruminal H 2 concentration and methanogenesis/hydrogen-metabolism markers were not quantified, so the proposed mechanisms should be interpreted as plausible inferences rather than direct physiological evidence.