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.

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.

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.

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.

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.

Effect of dietary nitrogen level and source on mRNA expression of urea transporters in the rumen epithelium of fattening bulls.

This paper aims to study the effect of the dietary treatments on mRNA expression of urea transporter B (UT-B) and some aquaporins (AQP) in rumen epithelium of Italian Simmental young bulls. Eighty animals allocated to 16 pens were fed from about 500 to 650 kg body weight with four experimental diets, which resulted from the combination of two crude protein levels (125 and 110 g/kg dry matter, diets M and L, respectively) and two nitrogen sources (soybean meal (SBM) or SBM partly replaced by an isonitrogenous mixture of corn and urea; diets -U and +U, respectively). At slaughtering samples of blood and rumen epithelium were collected from six bulls for each diet. Blood samples were analysed for haematological parameters and quantitative PCR was carried out on the mRNA extracted from the rumen epithelium samples. The bulls fed diets M had lower plasma concentrations of aspartate aminotransferase than those receiving diets L (78.9 vs. 88.3 U/l, p = 0.04). Plasma urea was higher (p = 0.03) for diets M and lower for diets +U (2.0 vs. 2.5 and 1.73 vs. 2.00 mmol/l, respectively, in M and L diets, p = 0.04). The effect of dietary treatments on rumen UT expression were limited to AQP3, which was down regulated (p = 0.01) in diets +U. Finally, a high positive correlation (R2 = 0.871) between the expressions of AQP7 and AQP10 was found. In conclusion, the AQP3 appears very responsive to dietary treatments and therefore it is a candidate to be further studied in rumen metabolism experiments. The close relationship between mRNA expression of AQP7 and AQP10 indicates a similar function of these two proteins.

In vitro effects of different levels of quebracho and chestnut tannins on rumen methane production, fermentation parameters, and microbiota.

Both condensed and hydrolysable tannins (CTs and HTs, respectively) have the ability to reduce enteric CH4 production in ruminants. However, the precise mechanism of action is not fully understood. Among the proposed hypotheses are the reduction of ruminal digestibility, direct control action on protozoa, reduction of archaea, and a hydrogen sink mechanism. In this in vitro study, which simulated rumen fermentation, two additives, one containing CTs (70% based on DM) from quebracho and one with HTs (75% based on DM) from chestnut, at four levels of inclusion (2, 4, 6, 8% on an as-fed basis) were added to the fermentation substrate and tested against a negative control. Both types of tannins significantly reduced total gas (GP) and CH4 (ml/g DM) production during the 48 h of incubation. The lower GP and CH4 production levels were linked to the reduction in dry matter digestibility caused by CTs and HTs. Conversely, no significant differences were observed for the protozoan and archaeal populations, suggesting a low direct effect of tannins on these rumen microorganisms in vitro. However, both types of tannins had negative correlations for the families Bacteroidales_BS11 and F082 and positive correlations for the genera Prevotella and Succinivibrio. Regarding the fermentation parameters, no differences were observed for pH and total volatile fatty acid production, while both CTs and HTs linearly reduced the NH3 content. CTs from quebracho were more effective in reducing CH4 production than HTs from chestnut. However, for both types of tannins, the reduction in CH4 production was always associated with a lower digestibility without any changes in archaea or protozoa. Due to the high variability of tannins, further studies investigating the chemical structure of the compounds and their mechanisms of action are needed to understand the different results reported in the literature.

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.

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.

Short note: Infliximab recovery in a simulated intestinal fluid of the upper intestine tract.

BACKGROUND: The oral administration of Infliximab (IFX) antibody would ensure a direct action on inflamed intestinal tissues without side effects. Thus, investigations about its resilience within the intestinal environment are required. OBJECTIVE: Quantify the IFX recovery in a simulated upper intestinal environment. METHODS: IFX was incubated for different times until 120 min in simulated intestinal fluid (SIF) which differed (i) for pH (7.2 vs 6.8, Exp 1), (ii) for addition or not with pancreatin (Exp 2) and (iii) for addition or not with bovine serum albumin in presence of pancreatin (BSA, Exp 3). RESULTS: In Exp 1 the IFX incubated without pancreatin was degraded by about 15% by SIF pH change from 7.2 to 6.8 and after 120 min it was reduced by about 20%. In Exp 2 the presence of pancreatin determined an intense and rapid IFX degradation (recovery < 33%, within 30 min), but when BSA was added to simulate the presence of food protein (Exp 3) the IFX half-life ranged between 59 and 70 min. CONCLUSIONS: A discrete in vitro stability of IFX in the upper intestine environment was demonstrated, if food protein is available and competes with pancreatin proteases.

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.

Topographic distribution of gastritis in heavy pigs investigated by a geographic information system approach.

The aim of this paper was to determine the topographic distribution of gastritis lesions in pigs through an open source geographic information system (GIS) software analysis. The stomachs of 146 Italian heavy pigs were collected at slaughter and subjected to macroscopic pathological examination of the internal mucosa. A total of 623 lesions were either classified as hyperplastic or follicular (97%) with the remaining minority of lesions categorised as atrophic and simple. The hyperplastic gastritis lesions had an average surface of 77.8 cm2 and were mainly located in an oval shaped area of the fundus region of the stomach near the Curvatura ventriculi major. The follicular gastritis lesions had generally a smaller surface (40.3 cm2) and were concentrated in two distinct small areas of the pyloric region. The GIS analysis provided the opportunity to produce useful maps showing the distribution and characteristics of gastritis in pigs.

Effect of dietary inclusion of whole ear corn silage on stomach development and gastric mucosa integrity of heavy pigs at slaughter.

The effect of dietary inclusion of whole ear corn silage on stomach development and on the incidence of gastric lesions was studied in heavy pigs. Three groups of 14 castrated male pigs were fed a control cereal-based diet and two diets containing whole ear corn silage (15% or 30% DM) from 90 kg bodyweight to slaughter at 170 kg. The diets with whole ear corn silage increased the amount of neutral detergent fibre in the stomach contents, the weight of the organs and the area of the pyloric region. Follicular gastritis was significantly lower and gastritis less severe in pigs fed the whole ear corn silage diets than pigs fed the control diet. The inclusion of whole ear corn silage in the diet influenced the development of the stomach and reduced the incidence of gastritis in heavy pigs.