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 (CT) or an extract from Stevia rebaudiana Bertoni (SB) to substrates. A control (CTR) substrate was fermented alone or added with 1.5% of CT 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 CT 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, R2 = 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 CT concentration in Exp. 3. Changes in VFA profile were induced by CT 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 CT 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 CT 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 CT

Impacts of rumen fluid, refrigerated or reconstituted from a refrigerated pellet, on gas production measured at 24h of fermentation

3noRumen fluid is used as fresh inoculum for gas production fermentations to predict the nutritional value of feeds and rations for ruminants. However, collection of rumen fluid from animal donors is invasive, expensive, time consuming and results in fluids of variable quality. The general aim was to identify a procedure to manipulate rumen inoculum in order to facilitate its storage and transfer between laboratories. This strategy would also limit fluid collections from animals. Two experiments were completed based on gas production from graduated 100 mL glass syringe with five feeds as substrates. In experiment 1, the gas production and some fermentation parameters of fresh rumen fluids were compared with those preserved at 4 °C for 24, 48, 72 and 96 h. Refrigeration did not modify concentration of volatile fatty acids and pH, but ammonia in liquids refrigerated for 48–96 h was higher (P < 0.05) compared to fresh. In contrast, rumen fluid refrigeration for 24, 48 or 72 h did not depress gas production at 24 h, but it was lower at 96 h. In experiment 2, the rumen fluid was centrifugated at 13,000 x g and sedimented material (i.e., pellet) was refrigerated for 48 h at 4 °C. The asymptote of gas production kinetics from rumen fluid regenerated from the pellet was 8 % lower (P < 0.05) than that from fresh. However for 24 h gas production, the correlation between fresh liquid and pellet inoculum, calculated for five ingredients, was high (R2 = 0.94). Results support the use of rumen fluid preserved by refrigeration for up to 72 h, and rumen fluid reconstituted from refrigerated pellet, as an alternative to fresh. This would reduce the need for laboratories to maintain animal donors and/or frequently collect rumen fluid.openopenFabro C.; Sarnataro C.; Spanghero M.Fabro, C.; Sarnataro, C.; Spanghero, M

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 ammonia release of urea-treated high moisture barley and maize grain

Rumen nitrogen (N) release from ammoniated wet barley and maize kernels by urea treatment (UT) at harvesting was studied. Untreated samples (CTR) were compared to UT and to samples combined with urea just before the experiment (UA). In Experiment 1, ground CTR, UT and UA samples were fermented in a ruminal in vitro system, and ammonia of fermentation fluid was analysed at 0, 2, 4, 6 and 8 h. The effect of incubation time was observed as ammonia peaked at 4 h of fermentation (10.24 vs 9.01 and 7.20 mg \ub7 dl 121, respectively at 0 and 8 h, P < 0.01). Also, the effect of treatment was stated when UT released less ammonia than UA treatment (9.76 vs 10.52 mg \ub7 dl 121, P < 0.05), while the CTR samples showed the least ammonia N concentrations (P < 0.01). In Experiment 2, the water N solubility of CTR and UT of both cereal samples prepared in three physical forms (whole grain, coarsely ground and milled) was examined. Samples were incubated in flasks with distilled water for 1, 2, 4, 6 and 8 h and N was measured in filtered residues to calculate N solubility. The UT samples, regardless cereal type, solubilised more N in the milled than in the whole form with the coarse form in the middle (43.7 vs 15.3%, 32.4 vs 14.0% and 20.3 vs 9.2% for milled, coarse and whole form, respectively; treatment 7 physical form interaction: P < 0.01). The N added to wet cereal kernels by the urea treatment was released in the rumen fermentation liquid more slowly than that simply added as urea before incubation. Based on solubility data, the treated whole or cracked kernels exhibited a slower N release than milled ones

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

The Effect of the Stirring Speed on the In Vitro Dry Matter Degradability of Feeds

In vitro methods have been standardized and tested to correctly simulate the rumen environment and fermentation process. A few studies have verified that the feed degradability achieved as a result of stirring the samples is higher when the samples are incubated under continuous stirring than when they are only stirred twice daily. The objective of this study has been to verify the effect of the speed of stirring on feed degradability during In vitro incubation. For this purpose, the apparent and true dry matter degradability (ADMD and TDMD) of grass hay, pelleted alfalfa, corn silage, barley meal, straw, and a total mixed ration (TMR) were measured after 48 h of incubation in jars under different rotation speeds. The same types of feed were placed in the four jars of each instrument, and the rotation system of the machine was modified to ensure the simultaneous rotation of a pair of original jars (which sometimes stopped and/or rotated slowly and irregularly) together with a pair of modified jars under regular and continuous rotation. A rev counter data logger was mounted onto the jars, and the rotation speeds of the original and modified jars were measured and compared under different conditions (empty jars, jars with liquid, jars with rumen fluid, and sample bags). The modifications to the instruments stabilized the rotation of the jars, thereby making the stirring more regular during incubation. The degradability was partly influenced by the regular stirring, albeit with just one instrument, and for grass hay, barley meal, corn silage, and TMR. In short, it has been found that the regular stirring of sample bags is not essential to obtain reliable degradability measurement during incubation, although it is better to maintain a constant rotation to ensure a regular and standardized In vitro incubation process and therefore to allow reproducibility and comparisons of the results on feed degradability

Celiac disease in pediatric patients according to HLA genetic risk classes: a retrospective observational study

Background: Celiac disease (CD) is an autoimmune enteropathy in which HLA-DQ haplotypes define susceptibility. Our aim was to evaluate if belonging to a certain HLA-DQ class risk could be associated to the clinical, serological and histological presentation of CD. Methods: We performed a retrospective observational monocentric study including all 300 patients diagnosed with CD, who underwent HLA typing. Clinical, serological and histological data was collected from clinical records and their association with HLA-DQ class risk was verified through statistical tests. Results: In our sample mean age at onset was 6.7 ± 4.2 years, with a prevalence of females (n = 183; 61%), typical symptoms (n = 242; 80.6%) and anti-tTG IgA ≥ 100 U/mL (n = 194; 64.7%). Family history was present only in 19% (n = 57) of patients, and it was not significantly associated with any of the clinical and demographical data analyzed or the belonging to a certain HLA-DQ class risk. We found in the male population more frequently a coexistence of CD and atopic syndrome (males: n = 47; 40.2%; females: n = 50; 27.3%; p = 0.020). Early age of onset, instead, was associated with typical symptoms (m = 6.4 ± 4; p = 0.045) and elevated liver enzymes (m = 5 ± 3.8; p < 0.001), while later age of onset was associated with presence of other autoimmune diseases (m = 8.2 ± 4; p = 0.01). We observed statistically significant influences of HLA class risk on antibodies and liver enzymes levels: G1, G4 and G2 classes showed more frequently anti-tTG IgA ≥ 100 U/mL (n = 44; 80%, n = 16; 69.6%, n = 48; 67.6% respectively; p-value = 0.037), and in patients from G2 class we found enhanced liver enzymes (n = 28; 39.4%; p-value = 0.005). HLA class risk was still significantly associated with anti-tTG ≥ 100 (p = 0.044) and with hypertransaminasemia (p = 0.010) after a multiple logistic regression adjusted for the effect of gender, age at onset and family history. Conclusions: We failed to prove an association between HLA-DQ genotypes and the clinical features in our CD pediatric patients. Although, our results suggest an effect of the DQB1–02 allele not only on the level of antibodies to tTG, but possibly also on liver involvement

Environmental quality of the operating theatres in Campania: long lasting monitoring results

In this study the microbiological, physical and chemical results of an investigation concerning the environmental conditions of operating theatres in 38 public hospitals of the Campania Government are presented. The analysis of the results has been made by considering specific standards suggested by national and international regulations. The results showed that 84% of the operating theatres presented normal microbiological values, in relation to the total bacterial load, while 16% did not. By considering the microclimatic monitoring 55% of the operating theatres showed normal values while 45% at least a microclimatic index did not. In relation to the concentrations of anaesthetics gases the survey pointed out that the nitrous oxides was within non prescribed environmental limits (50 ppm for N2O); while 15% of the halogenated was not in normal values

PLoS One

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication