Impact of lysozyme on ruminal fermentation and microbial population in Rusitec fermenters

The objective of this study was to determine the effects of geraniol and camphene at three dosages (300, 600, and 900 mg l-1) on rumen microbial fermentation and methane emission in in vitro batch culture of rumen fluid supplied with a 60 : 40 forage : concentrate substrate (16.2% crude protein, 33.1% neutral detergent fibre). The ionophore antibiotic monensin (8 mg/l) was used as positive control. Compared to control, geraniol significantly (P < 0.05) reduced methane production with increasing doses, with reductions by 10.2, 66.9, and 97.9%. However, total volatile fatty acids (VFA) production and in vitro dry matter digestibility were also reduced (P < 0.05) by all doses of geraniol. Camphene demonstrated weak and unpromising effects on rumen fermentation. Camphene did not decrease (P > 0.05) methane production and slightly decreased (P < 0.05) VFA production. Due to the strong antimethanogenic effect of geraniol a careful selection of dose and combination with other antimethanogenic compounds may be effective in mitigating methane emission from ruminants. However, if a reduction in total VFA production and dry matter digestibility persisted in vivo, geraniol would have a negative effect on animal productivity.

Interactions between methane mitigation additives and basal substrates on in vitro methane and VFA production

BackgroundThe objectives of this study were to determine the effect of commercial slow-release urea (SRU) on in vitro fermentation characteristics, nutrient digestibility, gas production, microbial protein synthesis and bacterial community using a rumen simulation technique (RUSITEC). The experiment was a completely randomized design with four treatments and four replications of each treatment. Treatments were: control diet (no SRU addition), control diet plus 0.28% SRU (U28), or plus 0.56% SRU (U56), and control diet that was modified substituting a part of soybean meal equivalent to 0.35% SRU (MU35; dry matter [DM] basis). The experiment consisted of 8 d of adaptation and 7 d of data and sample collection. Rumen inoculum was obtained from three ruminally fistulated Angus cows fed the same diet to the substrate incubated.ResultsDigestibility of DM, organic matter (OM), crude protein (CP), fibre and starch was not affected, but daily production of gas (P < 0.07) and methane (P < 0.05) was quadratically increased with increasing SRU supplementation. The increase of SRU addition did not affect fermentation pH and total volatile fatty acid (VFA) production, whereas linearly (P < 0.01) decreased proportion of propionate, and linearly (P < 0.01) increased acetate to propionate ratio and ammonia nitrogen (N) concentration. The microbial N efficiency was also linearly (P < 0.03) improved with increasing supplementation of SRU. In comparison with control diet, the dietary substitution of SRU for part of soybean meal increased (P < 0.05) the digestibility of DM, OM and CP and decreased (P < 0.02) the total gas production. The total VFA production and acetate to propionate ratio did not differ between control and MU35, whereas the proportion of butyrate was lower (P < 0.05) and that of branched-chain VFA was greater (P < 0.05) with MU35 than control diet. Total and liquid-associated microbial N production as well as ammonia N concentration were greater (P < 0.03) with MU35 than control diet. Observed operational taxonomic units (OTUs), Shannon diversity index, and beta diversity of the microbial community did not differ among treatments. Taxonomic analysis revealed no effect of adding SRU on the relative abundance of bacteria at the phylum level, while at the genus level, the beneficial impact of SRU on relative abundance of Rikenellaceae and Prevotellaceae in feed particle-associated bacteria, and the abundance of Roseburia in liquid associate bacteria was greater (P < 0.05) with MU35.ConclusionsSupplementation of a dairy cow diet with SRU showed potential of increase in ammonia N concentration and microbial protein production, and change fermentation pattern to more acetate production. Adding SRU in dairy cow diet also showed beneficial effect on improving digestibility of OM and fibre. The results suggest that SRU can partially substitute soybean meal in dairy cow diet to increase microbial protein production without impairing rumen fermentation.

This study aimed to evaluate the effects of fennel essential oils at two particle sizes (normal and nano) and three levels (10, 20, and 30 µL/g of substrate) on total and individual volatile fatty acid (VFA) production in an in vitro batch culture system. The experiment followed a 2 × 3 + 1 factorial design, with fennel essential oils incorporated into a substrate mixture of corn silage (64.35%), alfalfa (3.48%), and concentrate (32.17%). The rumen liquor was collected from four mature cannulated Holstein Friesian cows and incubated for 48 h, with VFAs analyzed at 48 h using gas chromatography-mass spectrometry (GC/MS). Data analysis was done using SAS Studio, while Tukey’s mean separation at 5% significance level indicated no significant trends in VFA production associated with particle size or oil inclusion level. The results revealed that neither particle size nor inclusion level significantly (P &amp;gt;0.05) affected VFA concentrations, including acetate, propionate, butyrate, iso-butyric acid, iso-valerate, and valerate at 48 h of incubation. Specifically, for VFA, no significant linear or quadratic trends were observed for either particle size condition. Acetate concentrations also showed no significant differences across levels or particle sizes. Similarly, Propionate did not exhibit any significant trend for particle size or level. No significant effects were observed for iso-butyric acid, butyrate, iso-valerate, valerate, or acetate-propionate ratio. These findings suggest that neither particle size nor level significantly influences VFA production at 48 h under the conditions tested. It is concluded that the inclusion of fennel essential oils at varying particle sizes and levels, under the tested conditions, had minimal effects on ruminal fermentation and VFA profiles in Holstein Friesian cows. Further studies may be needed to explore different dosages.

Values of stoichiometric coefficients were estimated to describe the production of volatile fatty acids (VFA) from fermented substrate in the rumen of lactating cows. Coefficient estimates were derived by regression of in vivo data of rumen digestion to a stoichiometric model with non-linear regression techniques. The model explained only part of the simulated range of VFA molar proportions. The theoretical potential of the statistical procedure was investigated by simulation studies. These studies revealed that, although the model explained only a small part of the simulated range of VFA molar proportions, coefficient estimates were fitted accurately when the ideal model is used. Simulation results closely corresponded to those obtained with regression of the in vivo data. An evaluation of the in vivo coefficient estimates with independent in vivo data showed again similar results and seemed to confirm the predictive potential of the coefficient estimates. Additionally, a model was developed which describes VFA metabolism by stomach epithelia. Inputs and outputs of the model are amounts of VFA produced in the rumen and VFA released to portal blood. The current model describes the absorption and the activation of VFA by CoAsynthetases as the first step in VFA metabolism. Activation of VFA was described by competitive inhibition between individual VFA. Parameters were derived from in vitro studies of CoA-synthetase activities in tissue samples of ovine or bovine rumen epithelium. The model is a first attempt to describe the interaction between production, absorption and metabolism of VFA, and it assists in relating VFA molar proportions or production rates in the rumen to appearance rates or concentrations of VFA in portal blood.

The effects of sulfide, sulfite and sulfate on the production of volatile fatty acid (VFA) in an upflow anaerobic sludge blanket process were investigated by using serum bottle assays. The seed sludges were acclimated with glucose by continuous feeding. It is known that the activity of acidogenic granular sludge is affected by the sulfur compounds. Inhibitions at high concentrations while low concentrations stimulated VFA production were observed in the present study. The effects were observed to be VFA dependent and the relative toxicity of sulfur compounds to total VFA production was found to vary as SO3 2‐‐S>SO4 2‐‐S>S2‐ for bed as well as blanket zone sludges. Different degrees of toxicity were, however, observed for individual VFA and sludges. At small concentrations these compounds stimulated the production of individual VFA. This stimulation was observed to depend on the types of sulfur compound, zones of sludge as well as types of VFA.

Rumen fermentation, bacterial and total volatile fatty acid (TVFA) production rates in cattle fed on urea-molasses-mineral block licks supplement

SUMMARYTwoin vitroexperiments were conducted to analyse the effects of replacing dietary barley grain with wastes of tomato and cucumber fruits and a 1 : 1 tomato : cucumber mixture on rumen fermentation characteristics and microbial abundance. The control (CON) substrate contained 250 g/kg of barley grain on a dry matter (DM) basis, and another 15 substrates were formulated by replacing 50, 100, 150, 200 or 250 g of barley grain/kg with the same amount (DM basis) of tomato or cucumber fruits or 1 : 1 tomato : cucumber mixture. In Expt 1, all substrates were incubated in batch cultures with rumen micro-organisms from goats for 24 h. Increasing amounts of tomato, cucumber and the mixture of both fruits in the substrate increased final pH and gas production, without changes in final ammonia-nitrogen (NH3-N) concentrations, substrate degradability and total volatile fatty acid (VFA) production, indicating that there were no detrimental effects of any waste fruits on rumen fermentation. Therefore, in Expt 2 the substrates including 250 g of waste fruits (T250, C250 and M250 for tomato, cucumber and the mixture of both fruits, respectively) and the CON substrate were incubated in single-flow continuous-culture fermenters for 8 days. Total VFA production did not differ among substrates, but there were differences in VFA profile. Molar proportions of propionate, isobutyrate and isovalerate were lower and acetate : propionate ratio was greater for T250 compared with CON substrate. Fermentation of substrates containing cucumber (C250 and M250) resulted in lower proportions of acetate, isobutyrate and isovalerate and acetate : propionate ratio, but greater butyrate proportions than the CON substrate. Carbohydrate degradability and microbial N synthesis tended to be lower for substrates containing cucumber than for the CON substrate, but there were no differences between CON and T250 substrates. Abundance of total bacteria,Fibrobacter succinogenesandRuminococcus flavefaciens, fungi, methanogenic archaea and protozoa were similar in fermenters fed T250 and CON substrates, but fermenters fed C250 and M250 substrates had lower abundances ofR. flavefaciens, fungi and protozoa than those fed the CON substrate. Results indicated that tomato fruits could replace dietary barley grain up to 250 g/kg of substrate DM without noticeable effects on rumen fermentation and microbial populations, but the inclusion of cucumber fruits at 250 g/kg of substrate DM negatively affected some microbial populations as it tended to reduce microbial N synthesis and changed the VFA profile. More studies are needed to identify the dietary inclusion level of cucumber which produces no detrimental effects on rumen fermentation and microbial growth.

This study was taken up to assess the impact of supplementing herbal feed additives [HFAs; fruit of Myristica fragrans (Jayphall), seeds of Anethum sowa (Suva), fruit of Apium graveolens (Ajmo), fruit of Cuminum cyminum (Jeera), bark of Cinnamonum zeylanicum (Dalchini), or whole plant of Eclipta alba (Bhangro)] containing essential oils as active component on the nutrient utilization and methane production using wheat straw-based total mixed ration (TMR) as a substrate by in vitro gas production technique. The essential oil content was the highest (P < 0.01) in M. fragrans followed by E. alba and A. sowa. In addition to essential oils, these HFAs also contained saponins, tannins, and antioxidants. The HFAs were supplemented at 1-3% of substrate dry matter (DM). The data were analyzed by 6 × 4 factorial design. Irrespective of level of HFA, the net gas production (NGP) and metabolizable energy (ME) availability was the highest (P < 0.01) in TMR supplemented with C. zeylanicum comparable with E. alba, but higher than TMR supplemented with other HFAs. Supplementation of TMR with different HFAs did not affect the digestibility of neutral detergent fiber (NDF) and true organic matter (TOM) and partitioning factor (PF). The total volatile fatty acids (VFAs), acetate, propionate (P < 0.01), and butyrate (P < 0.05) production was the highest in TMR supplemented with A. sowa, and the lowest was observed in TMR supplemented with C. cyminum. The isobutyrate and valerate production was also the highest (P < 0.01) in diet supplemented with A. sowa, but isovalerate production was the highest (P < 0.01) in diet supplemented with C. zeylanicum. The A:P ratio was the best in TMR supplemented with A. sowa. The efficiency of rumen fermentation was the highest, and efficiency of conversion of hexose to methane was the lowest in diet supplemented with A. sowa as compared to all other supplements. The in vitro methane production expressed as either percent of NGP, ml/100mg DM of substrate/24h, or as ml/100mg of digestible OM/24h was the lowest in TMR supplemented with A. sowa. The ammonia nitrogen production from TMR supplemented with M. fragrans and A. sowa was comparable, but significantly (P < 0.01) lower than TMR supplemented with other HFAs. Irrespective of the nature of HFA, the NGP and ME availability were significantly (P < 0.01) higher in TMR supplemented with HFAs at all levels as compared to un-supplemented TMR. As compared to control, the digestibility of NDF and that of TOM was depressed slightly in all the HFA-supplemented TMRs. The supplementation of HFAs at 2% of substrate DM improved (P < 0.01) the production of total VFAs, acetate, and propionate, and that of isovalerate in comparison to the un-supplemented TMR. The acetate to propionate ratio increased (P < 0.01) with the increase in the level of supplementation of HFAs containing essential oils. The methane and ammonia productions were depressed significantly when TMR was supplemented at 2% level of HFAs as compared to control TMR. It was concluded that supplementation of TMR with A. sowa at 2% of substrate was fermented better as indicated by the production of total and individual VFA, methane, and ammonia as compared to TMR supplemented with other HFA or un-supplemented TMR.

An in vitro study was conducted to determine effects of reducing pH of porcine in vitro fermentation medium on compositions of glucosinolate degradation products and porcine in vitro fermentation characteristics for solvent-extracted canola meal (SECM) and cold-pressed canola cake (CPCC). Two canola co-products were subjected to porcine in vitro fermentation for 72 h. Accumulated gas production during microbial fermentation was recorded and modeled to estimate kinetics of gas production. Glucosinolate degradation products after microbial fermentation were determined and fermentation medium pH after incubation was recorded. Total and individual volatile fatty acids (VFA) concentrations per unit weight of dry matter (DM) of feedstuffs were determined. On DM basis, SECM and CPCC contained 6.15 and 11.1 µmol/g of glucosinolates, respectively. Goitrin concentration for CPCC was lower (P < 0.05) than that for SECM. Isothiocyanate and indole-3-acetonitrile concentrations for CPCC were lower (P < 0.05) than those for SECM, whereas thiocyanate concentration for CPCC was greater (P < 0.05) than that for SECM. A reduction in fermentation medium pH from 6.2 to 5.2 increased (P < 0.05) goitrin and indole-3-acetonitrile concentrations and decreased (P < 0.05) isothiocyanates concentration for SECM, but did not affect concentration of these 2 glucosinolate degradation products for CPCC. Fermentation medium pH after in vitro fermentation for SECM tended to be greater (P = 0.081) than that for CPCC. Canola co-product type and fermentation medium pH did not interact on gas production parameters. However, a reduction in fermentation medium pH from 6.2 to 5.2 resulted in increased (P < 0.05) lag time and reduced (P < 0.05) fractional rate of degradation and total gas production. Canola co-product type and fermentation medium pH did not interact on total or individual VFA production per gram of DM of feedstuff fermented. However, reducing fermentation medium pH from 6.2 to 5.2 increased (P < 0.05) total VFA and acetate productions, and tended to reduce (P = 0.083) branched-chain VFA production SECM and CPCC. In conclusion, a reduction in fermentation medium pH from 6.2 to 5.2 resulted in increased production of goitrin and indole-3-acetonitriles from SECM-derived glucosinolates, but did not affect production of thiocyanate from SECM-derived glucosinolates. Glucosinolates in CPCC were less affected by the fermentation medium pH used in the current study. It appears that there are other factors other than pH that affect the degradation of canola-derived glucosinolates by microorganisms from hindgut of pigs.

In vivo production and molar percentages of volatile fatty acids in the rumen: a quantitative review by an empirical approach

A hybrid dry-fermentation and membrane contactor system: Enhanced volatile fatty acid (VFA) production and recovery from organic solid wastes

Control of protein degradation in the rumen for improving protein efficiency and reducing polluting emissions

BackgroundThe awareness of environmental and socio-economic impacts caused by greenhouse gas emissions from the livestock sector leverages the adoption of strategies to counteract it. Feed supplements can play an important role in the reduction of the main greenhouse gas produced by ruminants—methane (CH4). In this context, this study aims to assess the effect of two biochar sources and inclusion levels on rumen fermentation parameters in vitro.MethodsTwo sources of biochar (agro-forestry residues, AFB, and potato peel, PPB) were added at two levels (5 and 10%, dry matter (DM) basis) to two basal substrates (haylage and corn silage) and incubated 24-h with rumen inocula to assess the effects on CH4 production and main rumen fermentation parameters in vitro.ResultsAFB and PPB were obtained at different carbonization conditions resulting in different apparent surface areas, ash content, pH at the point of zero charge (pHpzc), and elemental analysis. Relative to control (0% biochar), biochar supplementation kept unaffected total gas production and yield (mL and mL/g DM, p = 0.140 and p = 0.240, respectively) and fermentation pH (p = 0.666), increased CH4production and yield (mL and mL/g DM, respectively, p = 0.001) and ammonia-N (NH3-N, p = 0.040), and decreased total volatile fatty acids (VFA) production (p < 0.001) and H2 generated and consumed (p ≤ 0.001). Biochar sources and inclusion levels had no negative effect on most of the fermentation parameters and efficiency. Acetic:propionic acid ratio (p = 0.048) and H2 consumed (p = 0.019) were lower with AFB inclusion when compared to PPB. Biochar inclusion at 10% reduced H2 consumed (p < 0.001) and tended to reduce total gas production (p = 0.055). Total VFA production (p = 0.019), acetic acid proportion (p = 0.011) and H2 generated (p = 0.048) were the lowest with AFB supplemented at 10%, no differences being observed among the other treatments. The basal substrate affected most fermentation parameters independently of biochar source and level used.DiscussionBiochar supplementation increased NH3-N content, iso-butyric, iso-valeric and valeric acid proportions, and decreased VFA production suggesting a reduced energy supply for microbial growth, higher proteolysis and deamination of substrate N, and a decrease of NH3-N incorporation into microbial protein. No interaction was found between substrate and biochar source or level on any of the parameters measured. Although AFB and PPB had different textural and compositional characteristics, their effects on the rumen fermentation parameters were similar, the only observed effects being due to AFB included at 10%. Biochar supplementation promoted CH4 production regardless of the source and inclusion level, suggesting that there may be other effects beyond biomass and temperature of production of biochar, highlighting the need to consider other characteristics to better identify the mechanism by which biochar may influence CH4 production.

Household food waste (HFW), which is rich in organic matter, is a good candidate for producing added-value bio-based chemicals, such as volatile fatty acids (VFAs), by acidogenic fermentation processes. However, the lack of design tools, such as appropriate kinetic models, hinders the implementation of this technology because the results of these processes are affected by operational factors. In this work, VFA production by the acidogenic fermentation of HFW under uncontrolled pH levels (4–5) was studied at thermophilic (55 °C) and mesophilic (35 °C) temperature conditions. Batch reactors were used to digest HFW, and VFA production and the individual acid distributions were measured at different fermentation times from 0 to 624 h. The results showed higher individual and total VFA production at 35 °C and 120 h of fermentation time as a consequence of the competition between the VFA production and decomposition reactions. Acetic and valeric acids were VFAs mainly produced as a result of a high content of proteins in the initial substrate, and a small amount of propionic and butyric acids were present. A simplified kinetic model was successfully developed to represent the complex process of VFA formation from the acidogenic fermentation of HFW. A simple mechanism for the production–decomposition of VFAs, corresponding to a zero-order reaction for the first 48 h and a single consecutive reaction from that time on, was proposed. For both mesophilic and thermophilic conditions, the suggested kinetic model was able to predict the individual and total concentrations of VFAs along the fermentation time.