Effects of different nitrate sources on in vitro rumen fermentation, methane production, and the microbiome

Nitrate (NO₃⁻) serves as a microbial non-protein nitrogen source and as a methanogen and methanogenesis inhibitor. Nitrate salts exhibit varying solubilities depending on their constituent cations, which could influence their metabolic utilisation by rumen microorganisms. These cations also potentially influence rumen environments and microbial functions. This study aimed to investigate the effects of seven different nitrates [NaNO₃, KNO₃, Ca(NO₃)₂, Cr(NO₃)₃, Mn(NO₃)₂, Fe(NO₃)₃, and Zn(NO₃)₂] on <i>in vitro</i> rumen fermentation, methane production, and the microbiome in south Korea. Supplementation of a basal diet with NO₃⁻ at 10 g/kg of dry matter significantly reduced methane production. Zn(NO₃)₂ uniquely decreased fibre digestibility, total volatile fatty acids, acetate proportion, and acetate-to-propionate ratio while most other nitrate treatments only reduced gas production and butyrate proportion compared to the control. Microbial analysis revealed that Zn(NO₃)₂ treatment led to significant decreases in fungal abundance and tended to decrease the abundance of methanogenic archaea (<i>p</i> = 0.0581), while simultaneously resulting in substantial increases in <i>Fibrobacter</i> abundance and <i>Streptococcus</i> enrichment. Functional analysis showed upregulation of sulphur metabolism pathways, including dissimilatory sulphate reduction (M00596) and sulphate–sulphur assimilation (M00616), as well as cysteine biosynthesis pathways (M00021 and M00338). These findings suggest that nitrate cations, particularly zinc, trigger complex microbial and metabolic changes in the rumen environment, enhancing methane mitigation while potentially activating biogenic sulphide production pathways as a detoxification mechanism against zinc-induced stress. NO₃⁻ supplementation at 10 g/kg dry matter resulted in significant methane reduction across all treatments.Zn(NO₃)₂ treatment had the most pronounced methane-reducing effect among the nitrate salts.Zn(NO₃)₂ treatment altered the rumen microbial community and metabolic pathways in response to zinc-induced stress. NO₃⁻ supplementation at 10 g/kg dry matter resulted in significant methane reduction across all treatments. Zn(NO₃)₂ treatment had the most pronounced methane-reducing effect among the nitrate salts. Zn(NO₃)₂ treatment altered the rumen microbial community and metabolic pathways in response to zinc-induced stress.