In vitro aflatoxins recovery after changing buffer or protozoa concentrations in the rumen fermentation fluid.

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.

A new equipment for continuous measurement of methane production in a batch in vitro rumen system.

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.

Dynamics of in vitro rumen methane production after nitrate addition.

The present study aimed to assess the dynamics of rumen methane (CH4) production following the addition of NaNO3. This was done using an in vitro rumen fermentation system that ensures continuous gas and methane assessments. Four different levels of NaNO3 were used to get the final nitrate concentrations of 0.5, 1.0, 1.5, and 2.0 mg/ml of rumen fluid. For each dose, corresponding controls contained sodium chloride and urea were realised to ensure comparable levels of sodium and nitrogen. The addition of nitrates had slight effect on the intensity of fermentation because the total gas produced minus CH4 (total methane-free gas) only went down at the highest dose (2.0 mg/ml), and the final concentrations of SCFA were the same at all doses. The most evident effect was a modification of the SCFA profile (low concentrations of propionate and valerate, progressive increments of acetate, and decreases of butyrate) and a reduction in overall CH4 production. The CH4 yield for the 0.5 mg/ml dose was not different from control in the entire fermentation. Yield of the 1.0 mg/ml dose was significantly lower than the control group (p < 0.05) only within the initial 24-h period, and higher dosages (1.5 and 2.0 mg/ml) were lower during the entire fermentation (p < 0.01). Methane yields were well fitted with the Gompertz model, but only the highest level of nitrate inclusion had a significant impact on the majority of model parameters (p < 0.01). The linear regressions between CH4 yields (y) and the amounts of nitrates (x) at progressive fermentation durations (e.g. 6, 12, 24, and 48 h) produced equations with increasing absolute slopes (from -0.069 to -0.517 ml/mg of nitrate). Therefore, nitrate reduced rumen CH4 yield in a dose-dependent manner: the impact of low doses was primarily observed at the initial stages of fermentation, whereas high doses exhibited effectiveness throughout the entire fermentation process. In conclusion, in batch fermentation systems, the dose effect of nitrates on methane yield was time dependent.

Evaluation of dietary addition of 2 essential oils from Achillea moschata, or their components (bornyl acetate, camphor, and eucalyptol) on in vitro ruminal fermentation and microbial community composition.

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.

Supplementation of diets with tannins from Chestnut wood or an extract from Stevia rebaudiana Bertoni and effects on in vitro rumen fermentation, protozoa count and methane production.

The aim of the trial was to evaluate the effect of dietary additions of Stevia rebaudiana Bertoni extract (SB) and Chestnut wood tannin (CWT) on the in vitro rumen fermentability, protozoal population and methane yield. Both plant products were tested at 3 different levels of inclusion (0.75, 1.50 and 3.00% of incubated dry matter, DM) in a total mixed ration (TMR) for ruminants by using rumen batch culture systems and a rumen inoculum collected from sheep. Total volatile fatty acid concentration, their proportions and gas production were not modified by the plant extracts inclusion, except a significant linear increment of gas production at 24 hr for SB (p = .049). Ammonia concentration decreased (p < .05) of about 17% when 1.50 or 3.00% of CWT were included into TMR. Rumen protozoa population was depressed by the SB inclusion (p = .002) with a maximum reduction of 40% at the highest SB dosage, whereas CWT negatively affected total protozoa counts (-19%) only at the dose of 3.00%. In vitro DM and NDF degradability were not affected by the supplementation of SB and CWT, as well as the methane yield. Thus, the addition of SB and CWT decreases the in vitro protozoa population of the rumen with different intensity and without effects on fermentation parameters, apart from a reduction of nitrogen degradability caused by CWT. Despite the effect on protozoa, no decreasing effect on methane production was detected.

In vitro rumen fermentation of feed substrates added with chestnut tannins or an extract from Stevia rebaudiana Bertoni.

Rumen fermentation parameters and microbiota were evaluated in 3 in vitro rumen fermentation experiments after addition of chestnut tannins (CWT) or an extract from Stevia rebaudiana Bertoni (SB) to substrates. A control (CTR) substrate was fermented alone or added with 1.5% of CWT or SB extracts in a batch culture system (Exp. 1, fermentation in 500 mL for 24 h) and in a subsequent continuous culture system (Exp. 2, fermentation in 2 L bottles for 9 d). Experiment 3 used the fermentation system of Exp. 1 and tested 7 doses of each extract added to CTR (additions of 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2% and 1.4% for 48 h). The addition of CWT lowered (P < 0.01) the in vitro rumen ammonia concentration in all experiments and reduced the protozoa counts in Exp. 1 (P < 0.05). In contrast, the SB extract did not modify the ammonia concentrations, but significantly lowered the protozoa counts in all 3 experiments (reduction of 47% and 20% in Exp. 1 and 2, P < 0.05; and a quadratic reduction in Exp. 3, R 2 = 0.63, P < 0.01). Neither extract affected the fermentation in terms of gas production (Exp. 1 and 3) nor volatile fatty acids (VFA) yield (Exp. 1 and 2), if we exclude a reduction at the highest CWT concentration in Exp. 3. Changes in VFA profile were induced by CWT and were limited to reductions in the iso-valerate (P < 0.01, in Exp. 2) and iso-butyrate levels (P < 0.01, Exp. 2). The CWT increased the abundance of Prevotella ruminicola and Selenomonas ruminantium and decreased that of Ruminobacter amylophilus (P < 0.01, P < 0.05 and P < 0.05, respectively). The SB extract increased the relative abundance of Treponema saccarophylum (P < 0.05). Both of the studied substances had an impact on rumen metabolism, with SB reducing protozoa counts and CWT lowering the rumen ammonia concentration. The effects of both extracts on the rumen were appreciable at low dietary doses, and the negative impacts on fermentation were limited to the reduction in protein degradation with the addition of CWT.

Rumen Inoculum Collected from Cows at Slaughter or from a Continuous Fermenter and Preserved in Warm, Refrigerated, Chilled or Freeze-Dried Environments for In Vitro Tests.

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.

A nutritional and rumen ecological evaluation of the biorefinery by-product alfalfa silage cake supplemented with Scrophularia striata extract using the rumen simulation technique.

BACKGROUND: By-products of the food production chain are gaining importance as feedstuffs for ruminants. Alfalfa silage cake (AC) is an unexploited biorefinery by-product rich in fiber. The aim of this study was to test AC, using an in vitro rumen simulation technique (Rusitec), for its suitability as a fiber source for cattle. Three diets with similar crude protein (CP) content were formulated; they contained the biorefinery by-product AC, the original alfalfa silage (OA), or a fiber-rich hay. As fibrous feedstuffs are known to promote ruminal methanogenesis, we additionally tested a plant extract of Scrophularia striata (60 mg g-1 dry matter) for its methane mitigation and antimicrobial properties. RESULTS: Diets containing AC displayed lower nutrient degradability, with the largest difference in CP degradation (P < 0.001). Sequencing of microbial DNA revealed several effects of the diet and of the addition of S. striata extract, but no inhibitory effect on methanogens. Likewise, methane production, which, in general, is lower with AC and OA diets, was not inhibited by S. striata extract, while the short chain fatty acid (SCFA) profiles were unaffected. CONCLUSION: Although CP degradation of the AC diet was lower, degradation of the fiber fractions was similar among diets. According to the present results, AC can be used as fibrous feedstuff for ruminants. Supplementation with S. striata extract did not inhibit methane formation. © 2019 Society of Chemical Industry.

Social context modulates digestive efficiency in greylag geese (Anser anser).

In group-living animals, social context is known to modulate physiology, behaviour and reproductive output as well as foraging and nutritional strategies. Here we investigate the digestive efficiency of 38 individuals belonging to different social categories of a semi-feral and individually marked flock of greylag geese (Anser anser). During 9 consecutive days in winter 2017, when the ground was fully covered with snow (i.e. no grass or other natural forage available) and the accessible food was standardized, 184 individual droppings were collected and analysed to estimate the apparent digestibility of organic matter (ADOM). Lignin was used as an indigestible internal marker in the food and droppings. The digestive efficiency was higher in pairs with offspring as compared to pairs without offspring or unpaired birds. Furthermore, individuals with high ADOM were more likely to breed successfully in the following season than those with low ADOM. Our findings demonstrate that social status modulates digestive efficiency, probably via a chain of physiological mechanisms including a dampened stress response in individuals enjoying stable social relationships with and social support by their family members (i.e. their own pair-partner and offspring). Our findings underline the importance of the social network in modulating physiology, such as digestive efficiency, and ultimately reproductive success.