The technique of sheep faeces preservation affects the microbiome activity and associated gas production kinetics <i>in vitro</i>

Abstract In vitro systems involving microbial fermentation typically require freshly obtained inocula, such as rumen fluid or faeces. The objective of this study was to test whether preserved faeces can be used instead of fresh faeces in the Hohenheim gas test (HGT). Fresh faeces from sheep (control, C) was compared with seven differently preserved faeces by using nine different feeds and studying in vitro gas production (GP) ( n = 6–9 per treatment). Preservation involved freezing at −20°C (FR), shock-freezing with liquid nitrogen (N) and additional freezing at −20°C (FRN), FRN followed by defrosting (FRNdef), shock-freezing with liquid N and freeze-drying (FDN), freeze-drying (FD) and freeze-drying with storage for 3 weeks (FD3W) or 6 months (FD6M). Metaproteomics was used to analyse microbiome composition and function in treatments C, FR, FRN, FD, and FDN (n = 3 per treatment). On average across all feeds, the potential GP with FR and FRN (61 mL/200 mg DM) was comparable to that of C (62 mL/200 mg DM), whereas values for FRNdef, FDN, FD, FD3W, and FD6M were 85, 78, 76, 78 and 71% of C, respectively. All estimated GP kinetic parameters were affected by feed and preservation interactions ( P &lt;0.001). Microbiomes from C, FR, and FRN differed from those of FD and FDN based on the relative abundance of the core proteins ( P&lt; 0.001). FD and FDN showed a significant decline of Bacteroidota, functional redundancy values, and specific proteins such as carbohydrate esterases (CE) ( P &lt;0.05) and glycoside hydrolases (GH) ( P &lt;0.01). Overall, frozen faeces closely resembled fresh faeces and can serve as a viable alternative inoculum source in the HGT. This may reduce animal numbers used for scientific purposes, but preservation and storage must be strictly standardised to maintain an active microbiome for GP-based in vitro tests.