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.

This study investigated the effects of 2 Achillea moschata essential oils extracted from plants collected in 2 different valleys of the Italian Alps and 3 pure compounds of oils — bornyl acetate (BOR), camphor (CAM), and eucalyptol (EUCA) — on in vitro ruminal fermentation and microbiota. An in vitro batch fermentation experiment (Exp. 1) tested the addition of all of the substances (2 essential oils and 3 compounds) in fermentation bottles (120 mL) at 48 h of incubation, whereas a subsequent in vitro continuous culture experiment (Exp. 2) evaluated the pure compounds added to the fermenters (2 L) for a longer incubation period (9 d). In both experiments, total mixed rations were incubated with the additives, and samples without additives were included as the control (CTR). Each treatment was tested in duplicate and was repeated in 3 and 2 fermentation runs in Exp. 1 and 2, respectively. Gas production (GP) in Exp. 1 was similar for all of the treatments, and short chain volatile fatty acid (SCFA) production was similar in both experiments except for a decrease of SCFA produced (P = 0.029) due to EUCA addition in Exp. 2. Compared to CTR, BOR and CAM reduced the valerate proportion (P = 0.04) in Exp. 1, and increased (P < 0.01) the acetate proportion in Exp. 2. All treatments increased (P < 0.01) total protozoa counts (+36.7% and +48.4% compared to CTR on average for Exp. 1 and 2, respectively). In Exp. 1, all of the treatments lowered the Bacteroidetes and Firmicutes and increased the Proteobacteria relative abundances (P < 0.05), whereas in Exp. 2, the EUCA addition increased (P = 0.012) the Ruminococcus. In Exp. 1, methane (CH4) as a proportion of the GP was lowered (P = 0.004) by the addition of CAM and EUCA compared to CTR, whereas in Exp. 2, EUCA reduced the amount of stoichiometrically calculated CH4 compared to CTR. Overall, essential oils extracted from A. moschata and the pure compounds did not depress in vitro rumen fermentation, except for EUCA in Exp. 2. In both experiments, an increase of the protozoal population occurred for all the additives.

A new rumen batch fermentation system that allows continuous measures of total gas (GP) and methane production (MP) was tested. The fermentation system is composed of glass bottles connected to gas counters (Ritter Apparatebau GmbH & Co. KG) and an infrared gas analyser that measures the methane concentration. The system allows direct and continuous measurement of GP and MP for accurate kinetic studies. The aim of the work was to test the rumen fermentation system and compare the GP and MP kinetics obtained. Barley meal (BM), alfalfa hay (AH), corn silage (CS), and soya bean hulls (SH) were used as substrates in four consecutive fermentation runs. Cumulative volumes of GP and MP and the percentage of methane on total GP were recorded continuously until 48 h and average values at 1 h intervals were fitted with an exponential model with a lag phase reaching a good fit (R² > 0.992). GP and MP reached the highest plateau levels for SH (1836 and 370 ml, respectively; p < 0.01) and the lowest for AH (1000 and 233 ml, respectively). The remaining substrates showed intermediate values. MP kinetics showed a discrete lag phase (from 0.09 to 1.12 h), whereas it was equal to zero for the total GP (except for SH). The methane concentration in gas flowing increased rapidly at the beginning of fermentation (from 0.35 to 0.95 h⁻¹) and reached a plateau after approximately 8–12 h. In conclusion, the rumen fermentation system evaluated generates methane data comparable to those reported in the literature and allows simple continuous measurement of methane release throughout fermentation.

This study simulates in vitro the effects of (i) rumen acidity and (ii) change in rumen protozoa numbers on the recovery of aflatoxins (AFs). Two 24‐h fermentation experiments were carried out using the same batch in vitro fermentation systems and substrate (dried corn meal) containing 11.42, 2.42, 7.65 and 1.70 µg/kg of AFB1, AFB2, AFG1 and AFG2 respectively. In Experiment 1, two buffer concentrations (normal salts dosage or lowered to 25%) were tested. Buffer reduction decreased gas production (730 vs. 1101 mL, p < 0.05), volatile fatty acids (VFA) and NH₃ concentrations in the fermentation liquid (39.8 vs. 46.3 mmol/L, and 31.7 vs. 46.5 mg/dL respectively, p < 0.01). Recovery of all four AFs types was higher (p < 0.01) in the reduced buffer fermentation fluid, both as a percentage of total AF incubated (73.6% vs. 62.5%, 45.9% vs. 38.1%, 33.6% vs. 17.9% and 18.9% vs. 6.24% for AFB1, AFB2, AFG1 and AFG2 respectively) and as amounts relative to VFA production (163.4 vs. 123.5, 22.1 vs. 15.7, 48.8 vs. 22.5 and 6.16 vs. 1.86 ng/100 mmol of VFA, for AFB1, AFB2, AFG1 and AFG2 respectively). In Experiment 2, Stevia rebaudiana Bertoni extracts (S) or a Camphor essential oil (Cam) were added to fermenters and compared to the control (no additives, C). S and Cam addition resulted in a 25% reduction (p < 0.05) and a 15% increase (p < 0.05) in protozoa counts respectively, when compared to C. Both plant additives slightly reduced (p < 0.05) AFB1 recovery as a percentage of total AFB1 incubated (68.5% and 67.7% vs. 74.9% for S, Cam and C respectively). Recoveries of all other AFs were unaffected by the additives. In conclusion, the rumen in vitro AFB1 recovery (63%–75%) was higher than other AFs (3%–46%) and the acidic fermentation environment increased it. In our conditions, changes in protozoa numbers did not affect AFs recovery.