The role of rumen Fluid adaptation: an in vitro comparison of diet-adapted and non–diet-adapted rumen fluid for continuous CO2 and CH4 monitoring with Gas Endeavour

In vitro gas production techniques, such as the Gas Endeavour system, are widely used to assess rumen fermentation kinetics, providing an effective tool to evaluate rumen function and possible methane mitigation strategies. Donor animal diet and forage characteristics shape rumen microbiota, fermentation, pH, microbial activity, and gas production in vitro. The study aimed to evaluate the effect of ruminal fluid (RF), adapted or non-adapted to the four experimental diets used in the in vivo trial, on in vitro fermentation parameters. All diets, formulated to have the same NDF and net energy, included 2000 g/head/d of hay, while varying concentrate and prickly pear peel silage: the control diet (CTR) contained 450 g/head/d of concentrate; diet A included 300 g/head/d of concentrate + 500 g/head/d of prickly pear peel silage; diet B included 150 g/head/d of concentrate + 1000 g/head/d of silage; diet C included 1500 g/head/d of silage. Diets (3 replicates) were incubated for 24 hours using the Gas Endeavour with two rumen inocula collected via an oro-esophageal probe: adapted rumen fluid (ARF) from 2 sheep fed each experimental diet, and common rumen fluid (CRF) from 2 sheep fed hay and concentrate, managed under identical conditions. A 2-way ANOVA (diet and RF as fixed factors) was used. Methane production in 24 h did not differ among diets, whereas total gas production was affected by both diet and RF: CTR diet produced the highest total gas, while ARF resulted in higher total gas production than CRF (276 vs. 267 mL/3g incubated feed). RF type influenced early gas production kinetics (first 7 h), after which curves converged. During this early phase, total gas and methane production were higher with CRF, whereas ARF reduced both parameters, particularly in diets B and C with highest silage inclusion. These findings show that dietary adaptation of the rumen microbiome significantly affects early fermentation, reducing total gas and methane production in initial stages, and highlight the necessity of using diet-adapted rumen fluid in in vitro studies for accurate and biologically meaningful results.