Lyophilized rumen fluid from goats fed different roughage-based diets: fermentation characteristics, microbial communities, and post-reconstitution in vitro fermentation performance

Introduction Fresh rumen fluid is a promising donor material for rumen fluid transplantation (RFT), but poor storage stability and batch variability limit its use. Methods In this study, lyophilized rumen fluid samples from goats fed alfalfa hay or corn stover diets were designated AHP and CSP, respectively. For the in vitro fermentation assay, portions of lyophilized rumen fluid from each batch within each dietary group were pooled and reconstituted; the substrate-supplemented groups were designated AH and CS, whereas AH-CON and CS-CON served as the corresponding substrate-free controls. Results Lyophilization yields were 6.29 and 6.38%, and culturable carbohydrate-utilizing bacterial counts were 3.79 × 10⁷ and 3.93 × 10⁷ CFU/g, respectively, with no significant differences between AHP and CSP. AHP contained higher propionate and valerate concentrations ( p < 0.05), whereas CSP contained higher acetate and isovalerate concentrations and had a higher acetate-to-propionate ratio (A/P; p < 0.05). Bacterial, fungal, and methanogen community structures differed significantly between groups ( p < 0.01), and CSP showed higher bacterial and fungal Shannon indices ( p < 0.01). AHP was significantly enriched in the bacterial genera Xylanibacter , Segatella , Prevotellaceae_UCG-001 , and Succinivibrio ; the fungal genera Neocallimastix , Piromyces , Feramyces , and Aspergillus ; and the methanogen genera Methanoculleus and Methanosphaera . CSP was significantly enriched in the bacterial genera norank_f__F082 , Rikenellaceae_RC9_gut_group , norank_f__Bacteroidales_BS11_gut_group , norank_f__[Eubacterium]_coprostanoligenes_group , Ruminococcus , Monoglobus , norank_f__UCG-010 , and Candidatus Saccharimonas ; the fungal genera Orpinomyces , unclassified_f__Neocallimastigaceae , Cyllamyces , Caecomyces , Pecoramyces , Anaeromyces , Joblinomyces , and Alternaria ; and the methanogen genera Methanobrevibacter and Methanolacinia . Cross-domain correlation analysis showed predominantly positive associations among genera enriched within the same roughage-source group and negative associations between AHP- and CSP-enriched genera. Compared with their substrate-free controls, AH and CS showed increased gas production, bacterial crude protein (BCP), total volatile fatty acids (TVFA), acetate, propionate, and butyrate, and decreased pH ( p < 0.05). CS showed higher pH, acetate, isovalerate, TVFA, and A/P, whereas AH showed higher gas production, butyrate, and valerate ( p < 0.01). Discussion Overall, the roughage source in the donor diets influenced the fermentation characteristics and microbial composition of lyophilized rumen fluid. After reconstitution, both inocula fermented exogenous substrate and exhibited different fermentation profiles, supporting their potential as storable inocula for standardization in RFT.