The utilization of animal donors of rumen fluid for laboratory experiments can raise ethical concerns, and alternatives to the collection of rumen fluids from live animals are urgently requested. The aim of this study was to compare the fresh rumen fluid (collected at slaughter, W) with that obtained from a continuous fermenter (RCF) and three methods of rumen fluid preservation (refrigeration, R, chilling, C, and freeze-drying, FD). The fermentability of different inoculum was evaluated by three in vitro tests (neutral detergent fiber (NDF) and crude protein (CP) degradability and gas production, NDFd, RDP and GP, respectively) using six feeds as substrates. Despite the two types of inoculum differed in terms of metabolites and microbiota concentration, the differences in vitro fermentability between the two liquids were less pronounced than expected (−15 and 20% for NDFd and GP when the liquid of fermenter was used and no differences for RDP). Within each in vitro test, the data obtained from rumen and from fermenter liquids were highly correlated for the six feeds, as well as between W and R (r: 0.837–0.985; p < 0.01). The low fermentative capacity was found for C and, particularly, FD for liquids. RCF could be used to generate inoculum for in vitro purposes and short-term refrigeration is a valuable practice to manage inoculum.

Cow faeces have been investigated as alternative inoculum to replace rumen fluid to determine neutral detergent fibre (NDF) digestibility (NDFD). Aims of this study were to estimate: (1) the NDFD (48 h) of feed ingredients using a rumen inoculum in comparison with faecal inocula from cows fed diets with different forage basis; (2) the undigestible NDF (uNDF) at 240 and 360 h with ruminal fluid and faecal inocula from lactating cows fed two different diets. At 48 h incubation, the NDFD was affected both by feed and type of inoculum (p < 0.01) and by their interaction (p = 0.03). Overall, the mean NDFD was higher for rumen inoculum than for faecal inocula (585 vs. 389 g/kg NDF, p < 0.05), and faecal inoculum obtained from cows fed hay-based diets gave lower NDFD than those from cows fed maize silage (367 vs. 440 g/kg, p < 0.05). At long incubation times, the average uNDF was affected by substrate, inoculum and incubation time (p < 0.01), but not by their interactions. For each inoculum, significantly lower values were obtained at 360 than at 240 h. Regressions between uNDF with rumen and with the tested faecal inocula resulted in r² ≥ 0.98. Despite the differences at 48 h, the uNDF showed that faecal inoculum could replace rumen fluid at longer incubation times.