Gas Production Profiles Research Articles

In vitro gas production profile and the formation of end products from non‐ washable, insoluble washable and soluble washable fractions in some concentrate ingredients

AbstractA procedure that mimics washing in the in situ incubation technique, combined with an in vitro gas and volatile fatty acids (VFAs) production technique, was used to verify the assumption that rumen degradation behaviour of material washed out of nylon bags is instantaneous and complete. In a 6 × 4 factorial arrangement of treatments with three replicates, fractions of maize, barley, milo, yellow peas, lupins (a mixture of white and spotted lupins) and round‐seeded brown faba beans were subjected to an in vitro incubation technique. Fractions were whole (WHO), non‐washable (NWF), insoluble washable (ISWF) and soluble washable (SWF). In a manually operated in vitro fermentation system, another 24 samples of the same substrates were fermented for VFA and ammonia analysis. Except in lupins, ISWF in the concentrate ingredients was very rich in starch. SWF was relatively rich in ash, crude protein, soluble sugars, and a residual unknown fraction but contained only a negligible quantity of starch. Thus, the fermentation characteristics of ISWF were more like WHO and NWF than SWF. Total gas production of SWF was considerably lower than the other fractions. A very rapidly degradable fraction was seen in the first phase of degradation of SWF. The pattern of fermentation end‐product formation for SWF differed from that of the other fractions. Copyright © 2007 Society of Chemical Industry

The effect of sample grinding procedures after processing on gas production profiles and end‐product formation in expander processed barley and peas

AbstractGrinding is a technological process widely applied in the feed manufacturing industry and is a prerequisite for obtaining representative samples for laboratory procedures (e.g. gas production analysis). When feeds are subjected to technological processes other than grinding (e.g. expander treatment), grinding afterwards may disturb the effect of processing, both in practice and when laboratory techniques are applied. Therefore, this study aimed to establish the possible effects of different grinding procedures and sample preparation on the degradative behaviour of expander processed barley and peas. Samples of expander processed barley and peas were subjected to six different sample preparation procedures (intact sample, dissolved sample, samples ground stepwise over 6 and 3 mm sieves, samples ground stepwise over 6, 3 and 1 mm sieves, samples ground over a 3 mm sieve and samples ground over a 1 mm sieve). The patterns of gas production in these samples were studied over a period of 72 h incubation using an automated in vitro gas production system. The particle size distribution determined by dry sieve analysis and the Coulter counter method changed due to the different grinding procedures. Grinding the samples of expander processed barley and peas changed the kinetics of gas production and led to a faster degradation, most pronounced after stepwise grinding. However, the formation of the fermentation end‐product was not affected by the method of sample preparation. In expander processed barley, the difference in the degradation pattern due to the different grinding procedures was small. Copyright © 2007 Society of Chemical Industry

Voluntary intake of silages in dairy cows depending on chemical composition and in vitro gas production characteristics

Abstract A study was conducted to investigate the possibilities to develop models for predicting the relative silage dry matter intake (SDMI) in dairy cows utilising information on chemical composition and in vitro gas production (GP) kinetics of silages. In five experiments, each with an average of 38 lactating dairy cows, SDMI was recorded for 15 grass silages made from primary growth and regrowth swards of timothy (Phleum pratense L.). The silages were characterised by chemical analysis and by utilising an automated in vitro GP recording technique with end point measurements of substrate residues. The silage samples were analysed both as dried and wet samples to evaluate the effects of sample preparation techniques on GP kinetics and their relations to SDMI. Relationships between feed variables and SDMI were investigated utilising simple linear and multiple regression. The wet silage samples had higher cumulative GP and different GP curves compared to the dried samples. The linear relationships between, GP variables, harvest number (first or second cut) of the grass, chemical characteristics of the silages and the relative SDMI show that the GP technique is a powerful tool to detect silage quality. By using the parameters from the dried samples the multiple regression analysis resulted in a relationship, relative SDMI (kg per 100 kg live weight (LW)) per day = 0.071 + 0.0029 × NDFD − 0.266 × C (R2 = 0.82, S.D. = 0.06). NDFD is the degradability of the neutral detergent fibre (NDF) (g/kg NDF) and C is the variable that regulates the switching characteristics of the GP profiles. By using the wet silage samples the multiple relationship did not include any GP variable; relative SDMI (kg per 100 kg LW per day) = 1.86 − 0.008 × acetic acid (g/kg DM) + 0.024 × ethanol (g/kg DM) (R2 = 0.62, S.D. = 0.08). The results from the regression analysis and the experience of the laborious sample preparation technique for wet samples, give the conclusion that dried silage samples are recommended for determining feed characteristics using the GP technique in intake studies.

Some methodological and analytical considerations regarding application of the gas production technique

The in vitro gas production technique is used widely in animal nutrition for feed evaluation and to study the kinetics of microbial fermentation processes in the digestive tract. This technique is based on the assumption that gas produced in batch cultures inoculated with mixed microorganisms from ruminal or hindgut contents or faeces is directly related to the amount of substrate fermented. The technique relies on mathematical modelling to estimate the rate and extent of feed digestion from cumulative gas production profiles. Section 1 of this article deals with the application of gas laws to calculate the volume of gas produced from pressure measurements when batch cultures are incubated in gas-tight bottles. Calculation of the amount of a specific gas (methane) produced at a given incubation time from total gas production and composition is presented in Section 2. Then, the definition of specific rate of substrate degradation and that of gas production are considered. Next, a piecewise linear model based on the assumption that microbial growth and fermentation follows zero-order kinetics is derived and evaluated by comparison with other non-linear models with regard to goodness-of-fit of experimental data and estimation of rate and extent of degradation in the rumen of some feedstuffs. Finally, the possibility of using procedures to account for the inevitable cyclic trends in gas production profiles by data smoothing are discussed. All these considerations provide an insight into some of the mathematical and methodological aspects of the in vitro gas production technique and should contribute to facilitating the description and interpretation of experimental data.

In vitro fermentation patterns and methane production as influenced by cultivar and season of harvest of Lolium perenne L.

AbstractIn some European countries, the majority of annual enteric methane (CH4) emissions by ruminants occur at pasture – a direct result of the predominance of grazing within ruminant production systems. However, there are only limited data available as to the effect of perennial ryegrass cultivar and season of harvest on CH4 production. Using the in vitro gas production technique, the effect of perennial ryegrass cultivar on fermentation characteristics and CH4 production was determined (Experiment 1) and the persistence of these traits throughout the growing season for two cultivars, identified from Experiment 1 as having either a high or low methanogenic potential, was examined (Experiment 2). In Experiment 1, organic matter (OM) digestibility and cumulative total gas production profiles were unaffected by cultivar but, with regard to the kinetics of CH4 production, the asymptote value (A), cumulative CH4 yield at 72 h, and the fractional rate (μ) of CH4 production at both time of 0·5A(T)(μCH4T) and at 48 h (μCH448h) were significantly (P < 0·05) different. The amount of digested OM, as a proportion of cumulative CH4 production (DigOM/CH4) at 24 and 72 h after commencement of inoculation, revealed that the amount of substrate required to produce 1 ml of CH4 also differed significantly between cultivars (P < 0·01). In Experiment 2, regrowth number significantly modified the majority of measured samples (P < 0·01); cultivar effects were limited to the lag phase of the cumulative CH4 production curve and DigOM/CH4 at 8 h only (P < 0·05). These results suggest that differences exist between cultivars in how OM is partitioned following microbial fermentation and that these differences demonstrate persistency throughout the growing season. In the course of time it may be possible to exploit these differences through cultivar selection and plant breeding programmes, and thereby reduce enteric CH4 emissions within pastoral production systems.

Combining in vitro data and modelling to predict in vivo animal response

SummaryExisting feed evaluation systems for ruminants assess the feed value in a rather empirical way, with a limited ability to integrate metabolism in a meaningful framework. For the quantitative description of the mechanisms, appropriate biological data can be obtained using in vitro methods. The aim of this paper is to examine the use of modelling and in vitro data to predict digestion processes in vivo. Suitable mathematical methods are required to describe and interpret substrate disappearance profiles or gas production profiles. The derivation of such models is important since this allows a clear definition of the underlying assumptions made. Such assumptions are related to the change in fractional rate of degradation (kd) during incubation that will determine the shape of the profile. Furthermore, the value of the fractional passage rate (kp) is of crucial importance in the prediction of extent of degradation in the rumen. The development and application of models, based on classic microbial growth equations, clearly shows that observed variation in microbial efficiency in batch cultures (including the gas production technique) is not necessarily related to that in vivo. Rather, kp is again a major factor contributing to explanation of variation in microbial efficiency. Similarly, the end products of fermentation (VFA) and the VFA molar proportions can be estimated in vitro, but its direct applicability to the in vivo situation is limited. It is concluded that some potential uses of in vitro techniques are ultimately misleading. Mechanistic models indicate that mechanisms governing microbial efficiency and VFA molar proportions in vitro are not necessarily valid for the in vivo situation. Therefore, the in vitro data cannot be used directly for a uniform system of feed evaluation to predict animal responses. Rather, the in vitro data obtained for substrate degradation may be used in whole rumen models as a basal input value to indicate the degradation potential.

Fermentation behaviour of the nylon bag washout and degradable fractions determined with the gas production technique

Most feed evaluation systems for ruminants are based on data obtained with the nylon bag technique. This technique divides the samples in a washout ( W) fraction, passing the pores of the nylon gauze of approximately 40 μm, and a degradable ( D) fraction, unable leaving the bags unfermented. Generally, it is assumed that the W-fraction is immediately and completely fermented in the rumen. Experiments were conducted to determine the percentage fast and moderate fermentable components in both the W- and the D-fraction, using the gas production technique. Ten starchy feed ingredients were separated in vitro by filtration over a nylon gauze of 40 μm. Gas production profiles were recorded of the obtained W- (mean 223 g/kg) and D-fractions (mean 777 g/kg). The results show that on average only 376 g/kg of the W-fraction, high in starch, can be regarded as fast fermentable and 624 g/kg as moderate fermentable. The moderate fermentable proportion showed a fermentation behaviour comparable with the D-fraction. On the contrary, also 143 g/kg of the D-fraction could be regarded as fast fermentable. The results obtained with the D-fractions obtained by an in vitro method were confirmed with D-fractions obtained according to the nylon bag protocol, using a washing machine with running tap water. It was concluded that the major part of the in situ W-fraction is not immediately fermentable and actually is a washout part of the D-fraction.

Nutritive value of selected grasses in North Sumatra, Indonesia

ABSTRACTThe nutritive values of seven native grass species collected in North Sumatra, Indonesia, during dry and rainy seasons were evaluated. The chemical composition, in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), in vitro crude protein digestibility (IVCPD), macro mineral concentrations of calcium, phosphorus and magnesium, in vitro gas production profile and metabolizable energy (ME) content of the grasses varied greatly among species and seasons. The crude protein content ranged from 6.6 (Andropogon gayanus) to 16.2% dry matter (Cynodon plectostachyus) in the rainy season, with a significant (P < 0.05) reduction in the dry season. Data on the fiber fraction showed that the grasses contained more neutral detergent fiber (NDF) and acid detergent fiber (ADF) in the dry season and it significantly (P < 0.05) decreased in the rainy season, except for Panicum maximum and Pennisetum purpureum for NDF content and C. plectostachyus, P. maximum and Brachiaria decumbens for ADF content. Data on the mineral concentration showed that C. plectostachyus in the dry and rainy seasons had a higher calcium content than those of other species. The overall means of the seven grasses for IVDMD, IVOMD and IVCPD were significantly higher (P < 0.05) in the rainy season than in the dry season. The in vitro gas production and ME concentration were numerically higher in the rainy season than in the dry season. In conclusion, the nutritive value of the observed grasses in North Sumatra was relatively higher during the rainy season compared with the dry season. Among the species of grass, P. purpureum and C. plectostachyus had a higher nutritive value in both the dry and rainy seasons.

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.

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 1 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.

Interference of indirect gas produced by grass silage fermentation acids in an in vitro gas production technique

Volatile fatty acids (VFA) resulting from rumen fermentation are measured in in vitro gas production techniques (IVGPT) by including bicarbonate in the incubation medium and measuring indirect gas produced. During ensiling, anaerobic fermentation of plant organic matter also produces VFA, along with lactic acid. The aim of this study was to establish whether silage fermentation acids (SFA) affected gas production profiles and subsequent carbohydrate parameter estimations of fresh and dried grass silage. Results indicate that a substantial volume of SFA gas is produced by fermentation of both fresh and dried grass silage. SFA gas production reaches an asymptote at 30 min post-inoculation and is 81% higher in fresh versus dried grass silage ( P<0.001). Cumulative gas productions were fitted to the model: gas = A(1 − exp(− c a t)) + B(1 − exp(− c b( t − l))) × exp(5( t − l))/(1 + exp(5( t − l))), where ‘ A’ is the asymptotic gas production (ml) from fermentation of quickly degraded carbohydrate (QCHO), ‘ c a’ the fractional rate of gas production of A (fixed at >0.2 h −1), ‘ B’ the asymptotic gas production (ml) from fermentation of slowly degraded carbohydrate (SCHO), ‘ c b’ the fractional rate of gas production of B (h −1), ‘ t’ time (h) of fermentation and ‘ l’ is the discrete time lag (h) before fermentation of SCHO begins. Results indicate that failure to correct for SFA gas resulted in overestimation of A, l and total rumen degraded carbohydrate ( P<0.001), as well as underestimation of B ( P<0.001) and c b ( P<0.05). Multiple linear regression equations were developed to predict SFA gas volume produced by fresh grass silage at 30 min of incubation ( R 2 = 0.91, SEE = 0.62) and to predict the proportion of gas produced in the first 30 min of IVGPT that was derived from SFA for fresh grass silage ( R 2 = 0.95, SEE = 0.046) and dried grass silage ( R 2 = 0.71, SEE = 0.125).

A general compartmental model for interpreting gas production profiles

Equations to describe gas production profiles, obtained using manual or automated systems for in vitro fermentation of ruminant feeds, are derived from first principles using a general compartmental model. The underlying pools represented are the potentially degradable and undegradable feed fractions, and accumulated gases. Equations derived and investigated mathematically include a Gompertz equation. They were obtained by allowing the fractional rate of degradation to vary with time. The equations permit the extent of ruminal degradation, and hence the supply of microbial protein to the duodenum, to be evaluated thereby linking the gas production technique to animal production.

Effects of particle size of a total mixed ration on in vivo ruminal fermentation patterns and inocula characteristics used for in vitro gas production

The objective was to investigate effects of particle size (PL) of a total mixed ration (TMR) on in vivo ruminal fermentation patterns and characteristics of the inocula used for in vitro gas production. A 3 × 3 randomized block design was used with three ruminally cannulated Holstein cows (640 kg BW, 63 DIM, 36 kg milk/d) fed ad libitum with TMR diets consisting of 450 g/kg grass silage (GS) with three PL levels (25, 11 and 5.5 mm), 500 g/kg cereal-based concentrate mixture and 50 g/kg of long hay. Replicates of samples of solid digesta from the dorsal (SRF-T) and ventral (SRF-B) rumen and free fluid (FRL) samples were collected 1 h before and 2, 8 and 12 h after the morning feeding. Samples were analyzed for pH and short chain fatty acids (SCFA), thiamine and protozoa concentrations. The same digesta collected 1 h before the morning feeding was used as inoculum for in vitro incubation in a gas test, and the gas production (GP) profile of all TMR were determined. The SCFA concentration of SRF-T increased with reduction of PL of TMR ( P < 0.05), but that of FLR decreased ( P < 0.05). There was no effect of PL of TMR on SCFA concentration in SRF-B. Reducing the PL of TMR to 5.5 mm decreased ruminal pH and the acetate: propionate ratio 1 h prior to the morning feeding ( P < 0.05), while 11 mm did not affect ruminal pH. Ruminal protozoa numbers increased by reducing the PL to 11 mm. These effects of PL on the ruminal milieu are also reflected in the digestive characteristics of inocula used for in vitro incubation. The inocula of SRF-T collected from cows fed TMR of 11 and 5.5 mm PL increased in vitro GP of blank and substrate (TMR) in comparison to the 25 mm PL treatment. GP positively correlated with concentrations of SCFA and thiamine, and negatively to pH of the ruminal digesta in vivo, 2 h after incubation and feeding ( P < 0.05) only (i.e. at the start of fermentation of feed). Studies are needed to explain effects of PL reduction on microbial activity in vivo and in vitro.

Comparative analysis of gas production profiles obtained with buffalo and sheep ruminal fluid as the source of inoculum

An in vitro study was conducted to investigate effects of inoculum source (i.e., sheep versus buffalo rumen fluid) on gas production profiles, and to evaluate the suitability of various mathematical equations to fit the profiles and provide accurate values of degradation attributes. Incubations were completed using ruminal fluid obtained from sheep or buffalos fed the same diet (600 g/kg grass hay and 400 g/kg concentrate). Kinetics of fermentation of five feeds commonly fed to ruminants, being maize silage, grass silage, wheat straw, barley grain and a mixed hay, were studied with a gas production technique using an automated pressure evaluation system (APES). Ruminal fermentation characteristics (i.e., substrate disappearance, pH and VFA production) were determined after 120 h of incubation. Five mathematical functions (i.e., exponential, France, Gompertz, logistic, Morgan) were fitted to the experimental data to estimate rate and extent of feed degradation. Model comparison was based on goodness-of-fit assessed from analysis of residual variance and Akaike's information criterion. The logistic and Morgan functions were best overall, although the goodness-of-fit attained with all models was considered acceptable. Except for the Morgan, there were small differences among models in values derived for extent of degradation. There were differences between sources of inoculum in gas production measured at intermediate times (i.e., gas volumes with buffalo rumen fluid of 133 and 164 ml/g organic matter (OM) at 24 h for grass hay and silage, respectively, were smaller than those with sheep rumen fluid of 182 and 208 ml/g OM), but not at earlier or later incubation times. As a result of this trend, shorter half times, faster fermentation rates and higher extents of degradation occurred when feeds were incubated in sheep (estimated OM degradability was 0.308 and 0.402 g/g OM for grass hay and silage, respectively) compared with buffalo rumen fluid (0.246 and 0.330 g/g OM). Differences were larger for more fibrous substrates (i.e., grass silage, straw and hay) and negligible for barley grain.

Prediction of forage intake using in vitro gas production methods: Comparison of multiphase fermentation kinetics measured in an automated gas test, and combined gas volume and substrate degradability measurements in a manual syringe system

This study investigated two approaches to in vitro analysis of gas production data, being a three phase model with long (≥72 h) incubation times, to obtain kinetics and asymptotic values of gas production, and combination of gas volume measurements with residue determinations after a relatively short incubation of 24 h. Dry matter intake (DMI) of 24 Ethiopian forages were predicted by these approaches. Combined gas volume and true degradability measurements in vitro were used to calculate a partitioning factor (PF), being the ratio of mg substrate degraded to ml of gas produced. In the automated test, kinetics of gas production were described by a three phase model: y = a 1 / ( 1 + ( b 1 / t ) c 1 ) + ⋯ + a 3 / ( 1 + ( b 3 / t ) c 3 ) , where y is the gas volume at time t and a 1– a 3 are the volumes of gas in the pools, b 1– b 3, the time when half of the gas volume of a given pool is produced and c 1– c 3 describe the switching characteristics of the curves by pool. The model fitted gas production profiles, but there was no relationship between curve parameters and DMI in multiple regressions ( R 2 = 0.24, P=0.06). In contrast, combined gas volume and substrate degradability measurements at 24 h of incubation accounted for 63, 62 and 56% of the variation ( P<0.0001) in DMI when PF was combined with gas volumes measured after 2, 4 and 6 h of incubation, respectively, with PF accounting for 31% of variation in DMI. Forages with high voluntary DMI have high gas volumes at early incubation times (i.e., 2–8 h) and high substrate true degradabilities at 24 h of incubation, but low gas production at later times of incubation.

Methodological considerations in using gas production techniques for estimating ruminal microbial efficiencies for silage-based diets

The relationship between in vivo and in vitro estimates of efficiency of microbial production (EMP) was tested using four silage-based diets. In vivo estimates were based on renal allantoin excretion in steers and in vitro estimates were based on a combination of gas volume and true substrate degradability (TSD OM) measures conducted at substrate-specific times. After an initial test to evaluate 96 h gas production profiles of diets incubated in both N-low and N-rich media, the time to half maximal gas production ( t 1/2) was calculated and a second incubation conducted in both media with fermentation stopped at t 1/2 for each substrate. True substrate degradability was measured from incubation residues and combined with gas volumes to estimate EMP. Additionally, microbial N (MN) production estimates were made using two equations from information on dietary N, N content of residues and NH 3–N content of media before and after incubation. In vitro estimates of EMP were related to in vivo EMP in both N-low ( R 2 = 0.94, P=0.03) and N-rich ( R 2 = 0.91, P=0.04) incubation media. No relationships occurred between in vitro efficiency of microbial N production and in vivo EMP when microbial N production was calculated from direct N determinations using N content of the apparently degraded residue after centrifugation and NDFN of the incubation residue. When in vitro microbial N production was estimated indirectly by accounting for dietary N, changes in NH 3–N concentrations in the incubation medium and NDFN in the incubation residue, efficiency of microbial N production relative to 100 mg TSD OM tended ( R 2 = 0.83, P=0.09) to be positively related to in vivo EMP. Silage fermentation acids appeared to interfere with relationships between gas production and in vitro substrate degradability measures because they were removed from the incubation residue without having contributed to fermentation. Therefore, silage acids should be accounted for when using combined in vitro gas production and true substrate degradability estimates to evaluate EMP of silage-based diets. N supplementation of incubation medium can influence substrate use for microbial growth, and attempts should be made to simulate in vivo N availability. It may be possible to rank silage-based diets for microbial efficiency using combined gas volume and true substrate degradability measures conducted at substrate-specific times ( t 1/2).

Ranking of in vitro fermentability of 20 feedstuffs with an automated gas production technique: Results of a ring test

A ring test was completed to determine within laboratory repeatability, and reproducibility among laboratories, of a gas production technique for measuring fermentation kinetics of feeds in rumen fluid. Eight laboratories in different European countries received 20 feed samples. All used the same gas production equipment and method. Gas production profiles were fitted to a multiphasic model and the most important parameters were statistically compared. Although the within laboratory repeatability of, and levels of, calculated parameters differed among laboratories, in most cases there was a high correlation between pairs of laboratories ( r = 0.93–1.00), although in some cases the correlation was only moderate ( r = 0.73–0.87). Within laboratory gas production profiles were influenced by variation in microbiological activity of the rumen fluid and air pressure changes at the end of the run. Among laboratory differences were observed in calibration factors, type of donor animal and diets fed. Variation sources can be minimized using within run standards.

In vitro biohydrogenation of n − 3 polyunsaturated fatty acids protected against ruminal microbial metabolism

The objective of this study was to investigate, in vitro, methods of protecting n − 3 polyunsaturated fatty acids from biohydrogenation in the rumen. Linseed oil (LO) was used as a source rich in α-linolenic acid (18:3 n − 3), and was compared with whole linseed (LS), formaldehyde-treated linseed (FL), formaldehyde-treated linseed that was pre-treated with sodium hydroxide (SFL), formic acid (FFL) or ammonium tetraformate (AFL), xylose treated linseed (XL) or linseed oil absorbed into a fibre source (ALO). Fish oil (FO) was used as an oil rich in eicosapentaenoic acid (20:5 n − 3) and docosahexaneoic acid (22:6 n − 3), and was compared with fish oil encapsulated with saturated fat (EFa and EFb), marine algae (AG) or fish oil absorbed into a fibre source (AFO). In addition, there was a control (C) with no added oil. The oil sources were incubated using a batch culture technique, and the fermentation terminated after 6, 12, 24 and 48 h. Gas production profiles were similar for the treatments high in 18:3 n − 3, whilst the potential extent of gas production was lower ( P < 0.01) in FO compared with C, EFa, EFb, AG or AFO. For treatments high in 18:3 n − 3, vessel fluid pH decreased ( P < 0.001) with time, and was highest ( P < 0.001) in C, but did not differ among treatments. Vessel pH also decreased ( P < 0.001) with time in treatments high in 20:5 n − 3and 22:6 n − 3, and was highest ( P < 0.001) in C and FO. Biohydrogenation of 18:3 n − 3 was extensive in treatments LO, LS, FL, AFL, XL and ALO, with mean values at 48 h of incubation of 966, 974, 919, 897, 874 and 904 g/kg respectively, and was lower ( P < 0.001) in SFL and FFL (values at 48 h of 557 and 693 g/kg, respectively). The major biohydrogenation intermediary product in treatments high in 18:3 n − 3 was trans 18:1, which was highest at 48 h in treatments LO and LS ( P < 0.001). Biohydrogenation of 20:5 n − 3, but not 22:6 n − 3, increased with time ( P < 0.01), and was lowest in AG ( P < 0.01). Pre-treatment of linseed using sodium hydroxide or formic acid, followed by formaldehyde treatment, offers the best protection against ruminal microbial biohydrogenation, whilst the degree of biohydrogenation of 22:6 n − 3 in marine algae was particularly low.

Estimation of protein fermentation in the large intestine of pigs using a gas production technique

Proteolytic fermentation in the colon of pigs and the caecum of poultry can have negative effects on their performance and health due to formation of harmful end products. To reduce this, rations can be formulated with expected carbohydrate fermentation being higher in level, and rate, than that of protein fermentation, as it is accepted that available energy is the limiting factor during fermentation in the hindgut. In this study, a modified version of a gas production technique was used to determine fermentation characteristics of protein in feedstuffs and concentrates after pre-digestion with pepsin and pancreatic enzymes in order to simulate digestion in the stomach and small intestine of pigs. Completing the incubations in an N-free environment, and with an excess of rapidly fermentable carbohydrates, made N the limiting factor to microbial growth, and so gas production profiles reflect availability of N. Results showed that fermentation of protein in the hindgut can be determined with this modified gas production technique, and that there were distinct differences in protein fermentation among the feed samples. Availability of protein for fermentation was highest in pea meal, solvent extracted soybean meal and feather meal, and lowest in palm kernel (expeller), wheat grain and meat meal. Between 5 and 15 h of incubation, gas production increased linearly. Urea was used as a positive control, as it is readily available to microbes. Gas production after 10 h of incubation, relative to gas production with urea, was assumed to be a measure of protein availability, allowing the content and proportion of fermentable crude protein (CP) to be estimated. Results show that this gas production technique ranked feed samples relative to their CP availability in the hindgut. Four concentrates were made with increasing proportions of expected fermentable CP, which was confirmed with the in vitro technique.

In vitro gas and volatile fatty acids production profiles of barley and maize and their soluble and washout fractions after feed processing

In vitro fermentation characteristics of soluble, washout, and non-washout fractions separated from barley and maize and their technologically processed (i.e., pressure toasting, pelleting and their combination) products were measured. The aim was to establish whether the soluble equaled the washout fraction, whether these fractions are rapidly and completely fermented, and whether their fermentation characteristics are the same. A simple fractionation method was developed to separate the whole grain into a soluble fraction, a washout fraction, an insoluble washout fraction, and a non-washout fraction. Proximate analysis of the different fractions revealed in the soluble fraction a large (>400 g kg −1 DM) residual organic matter component of unknown origin, while the insoluble washout fraction contained between 800 and 900 g starch/kg DM. Fermentation characteristics of all fractions as well as the whole grain were measured for 72 h using gas and volatile fatty acids (VFA) production in vitro. After fractionation, gas and VFA production profiles of the fractions with different chemical compositions were found to be different. Based on gas production profiles it was shown that the soluble fraction was easily fermentable and was fermented faster than the insoluble washout and the non-washout fraction. Differences were however much smaller than anticipated, which would suggest that the extent of fermentation of washout fractions in the rumen is incomplete. The maximum gas and VFA production from the soluble fraction was always lower than that of the insoluble washout, and the non-washout fraction. The combination of pressure toasting and pelleting reduced fermentability both in terms of gas and VFA production compared with the other treatments.