In vitro gas production (GP) techniques, such as the extended Hohenheim gas test (eHGT), are being used to estimate the protein values of ruminant feeds and commonly apply rumen fluid obtained from rumen-cannulated animals as inoculum. This study aimed to compare rumen fluid inoculum (RI) with faecal inoculum (FI) concerning in vitro ammonia-nitrogen (NH3-N) release and microbially bound nitrogen (mN). Rumen fluid was obtained from lactating dairy cows and faeces from adult wether sheep. Six feeds for ruminants, including rapeseed meal, soybean meal, sunflower meal, pea grain, maize grain, and rye grain, were incubated for 8, 24, and 48 h with and without an additional carbohydrate (CHO) source. NH3-N increased with the incubation time for all feeds and both inocula. For NH3-N values after 8, 24 and 48 h of incubation, the two-way interaction between feed and inoculum source was significant (p < 0.001). Across the six feeds, the NH3-N values did not differ between RI and FI after 8 h, whereas RI-NH3-N was 17% and 23% higher than FI-NH3-N after 24 and 48 h, respectively. For most feeds, the mN values decreased with RI as incubation time increased. In contrast, mN in FI remained constant or increased between 8 and 24 h before decreasing at 48 h. Overall, the NH3-N and mN values were lower with FI than RI. After 24 and 48 h, the two inocula did not significantly differ in the response to the CHO supply, studied as the ratio of mg NH3-N decrease per 1 mL of GP. Linear regression analysis showed that FI-NH3-N can be used to predict RI-NH3-N using the six feeds or the CHO-supplemented feeds at all incubation times. The highest estimation accuracy was found at 24 h for the feeds alone (slope = 1.39, R2 = 0.98, CV = 3.9%) and the feeds plus CHO source (slope = 1.55, R2 = 0.99, CV = 4.1%). In conclusion, FI has the potential to be applied as an inoculum to assess the protein values of ruminant feeds in vitro.
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