Influence of substrate freshness on metabolite dynamics and methane emissions from pig slurry during laboratory incubation.

Freeze/thaw pretreatment is commonly applied to animal slurry samples in laboratory studies, but its impact on organic matter transformation and methane (CH4) emissions remains uncertain. In this study, slurry incubations were performed at 20°C with three treatments differing in freshness of feces, urine, and inoculum. CH4 production and metabolite dynamics were monitored over 30 days. Freeze/thaw of feces and urine combined with fresh inoculum yielded the highest and most stable CH4 production (20 g kg-1 volatile solids), supported by the gradual transformation of protein-derived metabolites and no inhibition. In contrast, fully fresh material led to rapid hydrolysis and volatile fatty acids accumulation and slight inhibition of methanogenesis, whereas freezing the inoculum caused a pronounced delay likely due to impairment of methanogens. Metabolomics identified protein-related compounds (e.g., L-phenylalanine, 5-aminovaleric acid, and L-norleucine) as early substrates correlated to CH4 formation. In contrast, lignin/carboxyl-rich aliphatic molecules and lipid compounds persisted as residual fractions. These results demonstrate that inoculum freshness is the decisive factor controlling freeze/thaw effects on CH4 production and should be prioritized when preparing substrates for laboratory incubations. When fresh materials are not practical, freeze/thaw feces and urine can substitute it when combined with fresh inoculum.