A new approach for in vitro bioassay to measure tannin biological effects based on a gas production technique

The aim of this paper was to study a method based on gas production technique to measure the biological effects of tannins on rumen fermentation. Six feeds were used as fermentation substrates in a semi-automated gas method: feed A - aroeira (Astronium urundeuva); feed B - jurema preta (Mimosa hostilis), feed C - sorghum grains (Sorghum bicolor); feed D - Tifton-85 (Cynodon sp.); and two others prepared mixing 450 g sorghum leaves, 450 g concentrate (maize and soybean meal) and 100 g either of acacia (Acacia mearnsii) tannin extract (feed E) or quebracho (Schinopsis lorentzii) tannin extract (feed F) per kg (w:w). Three assays were carried out to standardize the bioassay for tannins. The first assay compared two binding agents (polyethylene glycol - PEG - and polyvinyl polypirrolidone - PVPP) to attenuate the tannin effects. The complex formed by PEG and tannins showed to be more stable than PVPP and tannins. Then, in the second assay, PEG was used as binding agent, and this assay was done to evaluate levels of PEG (0, 500, 750, 1000 and 1250 mg/g DM) to minimize the tannin effect. All the tested levels of PEG produced a response to evaluate tannin effects but the best response was for dose of 1000 mg/g DM. Using this dose of PEG, the final assay was carried out to test three compounds (tannic acid, quebracho extract and acacia extract) to establish a curve of biological equivalent effect of tannins. For this, five levels of each compound were added to I g of a standard feed (Lucerne hay). The equivalent effect showed not to be directly related to the chemical analysis for tannins. It was shown that different sources of tannins had different activities or reactivities. The curves of biological equivalence can provide information about tannin reactivity and its use seems to be important as an additional factor for chemical analysis. (C) 2007 Elsevier B.V. All rights reserved.

Preliminary study of synthesis gas production from water electrolysis, using the ELECTROFUEL (R) concept

This paper describes preliminary work on the generation of synthesis gas from water electrolysis using graphite electrodes without the separation of the generated gases. This is an innovative process, that has no similar work been done earlier. Preliminary tests allowed to establish correlations between the applied current to the electrolyser and flow rate and composition of the generated syngas, as well as a characterisation of generated carbon nanoparticles. The obtained syngas can further be used to produce synthetic liquid fuels, for example, methane, methanol or DME (dimethyl ether) in a catalytic reactor, in further stages of a present ongoing project, using the ELECTROFUEL® concept. The main competitive advantage of this project lies in the built-in of an innovative technology product, from RE (renewable energy) power in remote locations, for example, islands, villages in mountains as an alternative for energy storage for mobility constraints.

Mortality of Migratory Birds from Marine Commercial Fisheries and Offshore Oil and Gas Production in Canada

There is an imminent need for conservation and best-practice management efforts in marine ecosystems where global-scale declines in the biodiversity and biomass of large vertebrate predators are increasing and marine communities are being altered. We examine two marine-based industries that incidentally take migratory birds in Canada: (1) commercial fisheries, through bycatch, and (2) offshore oil and gas exploration, development, and production. We summarize information from the scientific literature and technical reports and also present new information from recently analyzed data to assess the magnitude and scope of mortality. Fisheries bycatch was responsible for the highest levels of incidental take of migratory bird species; estimated combined take in the longline, gillnet, and bottom otter trawl fisheries within the Atlantic, including the Gulf of St. Lawrence, and Pacific regions was 2679 to 45,586 birds per year. For the offshore oil and gas sector, mortality estimates ranged from 188 to 4494 deaths per year due to the discharge of produced waters resulting in oil sheens and collisions with platforms and vessels; however these estimates for the oil and gas sector are based on many untested assumptions. In spite of the uncertainties, we feel levels of mortality from these two industries are unlikely to affect the marine bird community in Canada, but some effects on local populations from bycatch are likely. Further research and monitoring will be required to: (1) better estimate fisheries-related mortality for vulnerable species and populations that may be impacted by local fisheries, (2) determine the effects of oil sheens from produced waters, and attraction to platforms and associated mortality from collisions, sheens, and flaring, so that better estimates of mortality from the offshore oil and gas sector can be obtained, and (3) determine impacts associated with accidental spills, which are not included in our current assessment. With a better understanding of the direct mortality of marine birds from industry, appropriate mitigation and management actions can be implemented. Cooperation from industry for data collection, research to fill knowledge gaps, and implementation of mitigation approaches will all be needed to conserve marine birds in Canada.

Technical note: A proposed method to determine the extent of degradation of a feed in the rumen from the degradation profile obtained with the in vitro gas production technique using feces as the inoculum

A method is proposed to determine the extent of degradation in the rumen involving a two-stage mathematical modeling process. In the first stage, a statistical model shifts (or maps) the gas accumulation profile obtained using a fecal inoculum to a ruminal gas profile. Then, a kinetic model determines the extent of degradation in the rumen from the shifted profile. The kinetic model is presented as a generalized mathematical function, allowing any one of a number of alternative equation forms to be selected. This method might allow the gas production technique to become an approach for determining extent of degradation in the rumen, decreasing the need for surgically modified animals while still maintaining the link with the animal. Further research is needed before the proposed methodology can be used as a standard method across a range of feeds.

In vitro cumulative gas production techniques: History, methodological considerations and challenges

Methodology used to measure in vitro gas production is reviewed to determine impacts of sources of variation on resultant gas production profiles (GPP). Current methods include measurement of gas production at constant pressure (e.g., use of gas tight syringes), a system that is inexpensive, but may be less sensitive than others thereby affecting its suitability in some situations. Automated systems that measure gas production at constant volume allow pressure to accumulate in the bottle, which is recorded at different times to produce a GPP, and may result in sufficiently high pressure that solubility of evolved gases in the medium is affected, thereby resulting in a recorded volume of gas that is lower than that predicted from stoichiometric calculations. Several other methods measure gas production at constant pressure and volume with either pressure transducers or sensors, and these may be manual, semi-automated or fully automated in operation. In these systems, gas is released as pressure increases, and vented gas is recorded. Agitating the medium does not consistently produce more gas with automated systems, and little or no effect of agitation was observed with manual systems. The apparatus affects GPP, but mathematical manipulation may enable effects of apparatus to be removed. The amount of substrate affects the volume of gas produced, but not rate of gas production, provided there is sufficient buffering capacity in the medium. Systems that use a very small amount of substrate are prone to experimental error in sample weighing. Effect of sample preparation on GPP has been found to be important, but further research is required to determine the optimum preparation that mimics animal chewing. Inoculum is the single largest source of variation in measuring GPP, as rumen fluid is variable and sampling schedules, diets fed to donor animals and ratios of rumen fluid/medium must be selected such that microbial activity is sufficiently high that it does not affect rate and extent of fermentation. Species of donor animal may also cause differences in GPP. End point measures can be mathematically manipulated to account for species differences, but rates of fermentation are not related. Other sources of inocula that have been used include caecal fluid (primarily for investigating hindgut fermentation in monogastrics), effluent from simulated rumen fermentation (e.g., 'Rusitec', which was as variable as rumen fluid), faeces, and frozen or freeze-dried rumen fluid (which were both less active than fresh rumen fluid). Use of mixtures of cell-free enzymes, or pure cultures of bacteria, may be a way of increasing GPP reproducibility, while reducing reliance on surgically modified animals. However, more research is required to develop these inocula. A number of media have been developed which buffer the incubation and provide relevant micro-nutrients to the microorganisms. To date, little research has been completed on relationships between the composition of the medium and measured GPP. However, comparing GPP from media either rich in N or N-free, allows assessment of contributions of N containing compounds in the sample. (c) 2005 Published by Elsevier B.V.

Inter-laboratory variation of in vitro cumulative gas production profiles of feeds using manual and automated methods

A study was conducted to estimate variation among laboratories and between manual and automated techniques of measuring pressure on the resulting gas production profiles (GPP). Eight feeds (molassed sugarbeet feed, grass silage, maize silage, soyabean hulls, maize gluten feed, whole crop wheat silage, wheat, glucose) were milled to pass a I mm screen and sent to three laboratories (ADAS Nutritional Sciences Research Unit, UK; Institute of Grassland and Environmental Research (IGER), UK; Wageningen University, The Netherlands). Each laboratory measured GPP over 144 h using standardised procedures with manual pressure transducers (MPT) and automated pressure systems (APS). The APS at ADAS used a pressure transducer and bottles in a shaking water bath, while the APS at Wageningen and IGER used a pressure sensor and bottles held in a stationary rack. Apparent dry matter degradability (ADDM) was estimated at the end of the incubation. GPP were fitted to a modified Michaelis-Menten model assuming a single phase of gas production, and GPP were described in terms of the asymptotic volume of gas produced (A), the time to half A (B), the time of maximum gas production rate (t(RM) (gas)) and maximum gas production rate (R-M (gas)). There were effects (P<0.001) of substrate on all parameters. However, MPT produced more (P<0.001) gas, but with longer (P<0.001) B and t(RM gas) (P<0.05) and lower (P<0.001) R-M gas compared to APS. There was no difference between apparatus in ADDM estimates. Interactions occurred between substrate and apparatus, substrate and laboratory, and laboratory and apparatus. However, when mean values for MPT were regressed from the individual laboratories, relationships were good (i.e., adjusted R-2 = 0.827 or higher). Good relationships were also observed with APS, although they were weaker than for MPT (i.e., adjusted R-2 = 0.723 or higher). The relationships between mean MPT and mean APS data were also good (i.e., adjusted R 2 = 0. 844 or higher). Data suggest that, although laboratory and method of measuring pressure are sources of variation in GPP estimation, it should be possible using appropriate mathematical models to standardise data among laboratories so that data from one laboratory could be extrapolated to others. This would allow development of a database of GPP data from many diverse feeds. (c) 2005 Published by Elsevier B.V.

The in vitro gas production technique: limitations and opportunities

This preface outlines the reasons for undertaking the special issue, comments on the review process that was used, provides a brief summary of the papers included and discusses some of the implications of these papers on in vitro gas technique methodology and its applications.

In vitro gas production and nitrogen degradability of tannin-containing tree fruits in response to incremental levels of polyethylene glycol

This study was designed to determine the response of in vitro fermentation parameters to incremental levels of polyethylene glycol (PEG) when tanniniferous tree fruits (Dichrostachys cinerea, Acacia erioloba, A. erubiscens, A. nilotica and Piliostigma thonningii) were fermented using the Reading Pressure Technique. The trivalent ytterbium precipitable phenolics content of fruit substrates ranged from 175 g/kg DM in A. erubiscens to 607 g/kg DM in A. nilotica, while the soluble condensed tannin content ranged from 0.09 AU550nm/40mg in A. erioloba to 0.52 AU550nm/40 mg in D. cinerea. The ADF was highest in P. thonningii fruits (402 g/kg DM) and lowest in A. nilotica fruits (165 g/kg DM). Increasing the level of PEG caused an exponential rise to a maximum (asymptotic) for cumulative gas production, rate of gas production and nitrogen degradability in all substrates except P. thonningii fruits. Dry matter degradability for fruits containing higher levels of soluble condensed tannins (D. cinerea and P. thonningii), showed little response to incremental levels of PEG after incubation for 24 h. The minimum levels of PEG required to maximize in vitro fermentation of tree fruits was found to be 200 mg PEG/g DM of sample for all tree species except A. erubiscens fruits, which required 100 mg PEG/g DM sample. The study provides evidence that PEG levels lower than 1 g/g DM sample can be used for in vitro tannin bioassays to reduce the cost of evaluating non-conventional tanniniferous feedstuffs used in developing countries in the tropics and subtopics. The use of in vitro nitrogen degradability in place of the favoured dry matter degradability improved the accuracy of PEG as a diagnostic tool for tannins in in vitro fermentation systems.

Evaluation of in vitro gas production of roughages and their mixtures using the curves subtraction method

The in vitro gas production of four single roughages and their paired combinations (1:1 on dry matter basis) were evaluated. Two roughage samples (100 mg) per treatment were fermented with ruminal fluid during a 48 h incubation period. Total 48 h gas volumes of fermentation dry matter (DM), neutral detergent fiber (NDF) and soluble compounds in neutral detergent (NDS) were for sugarcane = 16.8, 11.2, 6.9 mL; sugarcane + corn silage = 20.1, 12.6, 9.1 mL; sugarcane + 60-day elephantgrass = 16.5, 17.6 mL; sugarcane + 180-day elephantgrass = 13.8, 8.2, 5.9 mL; corn silage = 18.8, 16.8, 4.7 mL; corn silage + 60-day elephantgrass = 16.3, 15.4, 2.4 mL; corn silage + 180-day elephantgrass = 16.1, 11.8, 4.2 mL; 60-day elephantgrass = 16.9, 19.0 mL and 180-day elephantgrass = fermented 10.7, 12.2 mL, respectively. The NDS gas production was not possible to estimate for sugarcane + 60-day elephantgrass, 60-day elephantgrass and 180-day elephantgrass. The present data shows that the curves subtraction method can be an option to evaluate the contribution of the soluble fractions in roughages to digestion kinetics. However, this method underestimates the NDS gas contribution when roughages are low in crude protein and soluble carbohydrates. It is advisable to directly apply the two-compartmental mathematical model to the digestion curves for roughage DM, when determining the NDS gas volume and the digestion rate. This method is more straightforward and accurate when compared to the curve subtraction method. Non-structural carbohydrates combined with fiber and protein promoted a positive associative effect in sugarcane + corn silage (50:50) mixture. Therefore, it can be concluded that the soluble fraction of roughages greatly contributes to gas production. (C) 2004 Elsevier B.V. All rights reserved.

Fermentation characteristics of several carbohydrate sources for dog diets using the in vitro gas production technique

Fermentable carbohydrates are an important part of the canine diet. They can improve gastrointestinal health by modifying gut microbial population and metabolic activity. The present study compared the fermentation characteristics and kinetic patterns of 10 carbohydrate sources using the in vitro gas production technique (IVGPT) with dog faecal inoculum. The substrates tested were: pure cellulose (PC), carboxymethylcellulose (CMC), sugar-cane fibre (SCF), beet pulp (BP), wheat bran (WB), fructooligosaccharides (FOS), inulin, yeast cell wall (YCW), ground psyllium seed (PS), pea hulls (PH). All substrates were incubated at 39°C under anaerobic conditions with faeces collected from dogs as microbial inoculum. Gas production of fermenting cultures was recorded and after 48 h, pH, shortchain fatty acids (SCFA) and organic matter disappearance (OMD) were determined. The results confirm high fermentation by dog faecal bacteria of FOS and inulin that produced high amounts of propionate and that underwent very rapid fermentation. Three substrates (SCF, CMC and PC) were not able to support bacterial growth, with low gas and SCFA production, and high BCFA formation. The PH and BP showed moderate OMD and SCFA production. Wheat bran B underwent rapid fermentation and generated a high proportion of butyrate. Psyllium seeds underwent slow fermentation with delayed gas production, supporting a high formation of SCFA, with an adequate amount of butyrate for bacterial growth while YCW, which showed a delayed fermentation, gave moderate SCFA production. The fermentation characteristics of PS and YCW suggest their potential use in promoting a more distal fermentation on intestinal tract. © Copyright S. Calabrò et al., 2013 Licensee PAGEPress, Italy.

Chemical composition, in vitro digestibility and gas production of Brachiaria managed under different forage allowances

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)