A review and simplification of the in vitro incubation medium

Animal Feed Science and Technology 123–124 (2005) 155–172

A review and simplification of the in vitro incubation medium F.L. Mould ∗ , R. Morgan, K.E. Kliem, E. Krystallidou Department of Agriculture, The University of Reading, Earley Gate, P.O. Box 237, Reading RG6 6AR, UK

Abstract The requirement to rapidly and efficiently evaluate ruminant feedstuffs places increased emphasis on in vitro systems. However, despite the developmental work undertaken and widespread application of such techniques, little attention has been paid to the incubation medium. Considerable research using in vitro systems is conducted in resource-poor developing countries that often have difficulties associated with technical expertise, sourcing chemicals and/or funding to cover analytical and equipment costs. Such limitations have, to date, restricted vital feed evaluation programmes in these regions. This paper examines the function and relevance of the buffer, nutrient, and reducing solution components within current in vitro media, with the aim of identifying where simplification can be achieved. The review, supported by experimental work, identified no requirement to change the carbonate or phosphate salts, which comprise the main buffer components. The inclusion of microminerals provided few additional nutrients over that already supplied by the rumen fluid and substrate, and so may be omitted. Nitrogen associated with the inoculum was insufficient to support degradation and a level of 25 mg N/g substrate is recommended. A sulphur inclusion level of 4–5 mg S/g substrate is proposed, with S levels lowered through omission of sodium sulphide and replacement of magnesium sulphate with magnesium chloride. It was confirmed that a highly reduced medium was not required, provided that anaerobic conditions were rapidly established. This allows sodium sulphide, part of the reducing solution, to be omitted. Further, as gassing with CO2 directly influences the quantity of gas released, it is recommended that minimum CO2 levels be used and that gas flow and duration, together with the volume of medium treated, are detailed in experimental procedures. It is considered that these simplifications will improve safety

∗

Abbreviations: HCl, hydrochloride; iOMD, in vitro organic matter degradability Corresponding author. Tel.: +44 118 987 5123; fax: +44 118 935 2421. E-mail address: [email protected] (F.L. Mould).

0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.05.002

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and reduce costs and problems associated with sourcing components, while maintaining analytical precision. © 2005 Elsevier B.V. All rights reserved. Keywords: Buffer; Carbon dioxide; Reducing solution; Microminerals; Gas production

1. Introduction Development of in vitro gas production techniques has resulted in each laboratory favouring a particular technique modified to suit their specific requirements. These systems are broadly similar with the accumulation of headspace gas, from the microbial fermentation of a feedstuff in a buffered medium, measured either as volume or pressure. This is accomplished using manual or automatic methodologies that vary in both complexity and capacity. It is to be hoped that “ring tests” of these systems will rank feedstuffs in a similar order with respect to characteristics such as rate or extent of gas release, however the outcomes of such comparisons are unreliable (e.g., Madsen and Hvelplund, 1994; Rymer et al., 1988). It is not the purpose of this paper to compare these systems; however given the extent of developmental work undertaken, together with the widespread application of in vitro gas techniques, it is remarkable how little attention has been paid to the incubation medium itself. That is, how does the chemical composition of the buffer, macro- and microminerals and reducing solution components, plus the degree of initial anaerobiosis achieved, influence microbial activity, as measured by degradation or fermentation gas release? An improved understanding of these mechanisms should lead to refinement and simplification of the in vitro medium, thereby permitting alternative uses and assays. The requirement to rapidly and efficiently evaluate potential ruminant feedstuffs, especially in developing countries with respect to the “livestock revolution” (Delgado, 1999) places increasing emphasis on in vitro evaluation systems. Much of this research is conducted in resource-poor regions that have difficulties associated with technical expertise, sourcing chemicals or funding to cover analytical and equipment costs. Such limitations have, to date, restricted large-scale feed evaluation programmes in these areas. This paper will, therefore, review the development of in vitro media, the role or action of the various components, together with an examination of their relevance and adequacy. Finally, a suggestion for a simplified in vitro medium in terms of buffer, supplementary nutrients and reducing solution, will be offered. To avoid confusion, the following designations have been used. The in vitro incubation medium is considered to comprise three major components: a mixed inorganic solution, which contains buffer salts, macro- and micro-nutrients and the reducing solution, the microbial inoculum and the test substrate (generally a feedstuff together with any experimental treatments).

2. In vitro media The function of an in vitro incubation medium is to create and maintain an environment suitable for the fermentative process. This is achieved through supply of supplementary

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nutrients and a buffering system to maintain medium pH, such that degradation is not compromised. The major components are combined in sufficient volume to ensure that the concentration of intermediary or end-products released (e.g., cellobiose or glucose) do not inhibit degradation, but not so dilute that the nutrients themselves are limiting (Hungate, 1966). With the exception of studies to determine the basic degradation or fermentation properties of a substrate, it is common for these factors to be manipulated to produce a specific fermentative environment. For example, a medium with a low N content may be used to evaluate ammonia treatment of cereal crop residues, or variable levels of polyethylene glycol may be added to mitigate the suppressive effects of tannins (Mlambo et al., 2002).

3. Medium development Initial in vitro media were similar to those used in the early rumen microbial studies, which identified and evaluated individual species in pure culture. As these were selective media, designed to reflect niche environments thought to favour the bacterial species to be isolated, they tended to be complex and highly specific. In contrast, habitat-simulating media (Hungate, 1962) are used in feed evaluation to stimulate growth of all rumen microorganisms without favouring a particular group or species. Hungate (1966) indicated that early studies to culture cellulolytic rumen microorganisms failed due to deficiencies in the medium, including insufficient carbon dioxide (CO2 ), incorrect type and level of cellulose, and an absence of what Bryant and Doetsch (1955) had described earlier as “growth factors” (e.g., n-valeric and the branched chain iso-butyric and iso-valeric fatty acids). In other studies, these factors were supplied by including sterilised rumen contents (Bryant and Burkey, 1953; Bryant and Robinson, 1961). Media consisting of rumen fluid plus an energy source (i.e., carbohydrate) generally failed because of inhibition by microbial waste (especially acidic fermentative end-products), which would normally have passed out of the rumen or been absorbed across the rumen epithelium. To compensate, in vitro media tend to be relatively dilute, with dry matter contents of about 10 g/l, less than a tenth of that of in the rumen. 3.1. Composition 3.1.1. Buffer The inorganic salt component maintains incubation medium pH, ensures an osmotic pressure similar to that of rumen fluid and provides nutrients, such as trace elements (e.g., cobalt, magnesium and manganese). Both Hungate (1947) and Gall et al. (1947), and later Bryant and Burkey (1953), used phosphate-only buffered media. However, Tilley and Terry (1963) incorporated the “synthetic saliva” suggested by McDougall (1948) that included bicarbonate (Table 1). Hungate (1966) later argued that, as long as the total mixture was isosmotic with rumen fluid, the exact composition of the salts used was unimportant, provided that “sufficient bicarbonate” was included. The inorganic salt composition of the incubation media used in many in vitro gas assays is essentially that described by Goering and Van Soest (1970). It is relatively complex in that both cysteine hydrochloride (HCl) and sodium sulphide are included to lower the redox

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Table 1 In vitro incubation media (quantities/l final solution) Component

PO4 3− (g) HCO3 2− (g) N (g) S (mg) Mg (mg) Ca (mg) Mn (mg) Co (mg) Fe (mg) PO4 :HCO3 N:S

Mediuma Theodorou et al. (1994)

Goering and Van Soest (1970)

Menke et al. (1979)

Beuvink and Spoelstra (1992)

Tilley and Terry (1963)

1.307 5.451 0.542 89 12 3.2 2.5 0.2 1.5 0.240 6.1

1.549 5.422 0.539 90 11 2.9 2.2 0.2 1.3 0.286 6.0

1.291 4.492 – 38 9 2.4 1.9 0.2 1.1 0.287 –

1.360 4.753 0.473 154 9 3.1 2.8 0.3 1.7 0.286 3.1

1.973 5.693 – – 12 3.2 – – – 0.347 –

potential (Bryant and Robinson, 1961) with N and microminerals added to ensure their supply is not adversely affecting microbial activity. To contrast the more common media, these are detailed in Table 1 in terms of their chemical components and, as final media preparation varies, composition has also been expressed relative to the quantity of substrate incubated (Table 2). All five media described use a phosphate:carbonate buffer at inclusion rates of between 7.5 and 9.5 g/l and ratios of 0.24–0.35:1. Differences in the buffer composition (i.e., phosphate:bicarbonate ratio) and the type, rate and quantity of acidic fermentation end-products generated, all affect gas production. It follows, and in contrast to the fact that small deviations in media composition are unlikely to affect end-point degradation values, that the sensitivity of in vitro gas systems is such that slight variations in gas production will significantly impact their ability to accurately estimate fermentation kinetics. Table 2 In vitro incubation media (mg/g substrate incubated, unless stated) Component

PO4 3− HCO3 2− N S Mg Ca Mn Co Fe Buffer (ml g−1 ) Inoculum (ml g−1 )

Mediuma Theodorou et al. (1994)

Goering and Van Soest (1970)

Menke et al. (1979)

Beuvink and Spoelstra (1992)

Tilley and Terry (1963)

130.7 545.1 54.2 8.95 1.05 0.32 0.23 0.02 0.14 90 10

154.9 542.2 53.9 8.98 1.05 0.29 0.18 0.02 0.10 80 20

193.6 673.5 – 5.74 1.31 0.36 0.19 0.02 0.11 100 50

203.8 712.6 70.8 23.15 1.38 0.46 0.28 0.03 0.17 100 50

256.5 740.1 – – 1.59 0.46 – – – 104 26

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Table 3 Direct and indirect gas production resulting from the fermentation of 1 mol hexose, as influenced by acidic end product Acidic end-products (mol)

Acetic (2) Butyric (1) Propionic (2) Lactic (2)

Carbon dioxide production (mol) Direct

Indirect

Total

2 2 0 0

2 1 2 2

4 3 2 2

Source: Beuvink and Spoelstra (1992).

Beuvink and Spoelstra (1992) used the stoichiometric assumptions of Wolin (1960) and Hungate (1966) to calculate the quantities of direct and indirect gas released per mol of hexose fermented, in a bicarbonate buffered medium, to either acetic, butyric, propionic or lactic acids (Table 3). While CO2 may be reduced to methane (CH4 ), this does not influence the total quantity of gas evolved. As can be seen a shift in the fermentation pattern from, for example, one predominantly acetic to one predominantly propionic, as found with a change from a high fibre to a high starch diet (McDonald et al., 2002), will alter the total quantity of gas released. However, as buffers contain both bicarbonate and phosphate, the quantity of indirect gas released will be related to the extent that volatile fatty acids (VFA) are neutralised by bicarbonate or phosphate, as only interaction with the former will liberate CO2 . With their buffer, Beuvink and Spoelstra (1992) estimated indirect gas to be 0.87 of theoretical, suggesting that 0.13 of the acid load was neutralised by phosphate. The lack of direct gas production, associated with the formation of lactic or propionic acids, provides a partial explanation as to why readily degradable substrates such as glucose or molasses often exhibit gas kinetic profiles, more commonly associated with poorly degraded substrates, e.g., low peak rate of gas release (Fig. 1). The three substrates in Fig. 1 (glucose, starch and cellulose) had cumulative gas yields of 267, 304 and 249 ml after 48 h, respectively. Further, as identified by Russell and Wallace (1997), some rumen bacterial species, such as Streptococcus bovis and Selenomonas ruminantium, utilise different

Fig. 1. In vitro gas release kinetics (ml/h/g organic matter incubated) [glucose - - - - -, starch – – – – – and cellulose —]. Source: Mould et al. (2000).

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metabolic pathways depending on nutrient availability. Where energy supply is non-limiting, as occurs in vitro immediately post-inoculation, growth rate is maximised and lactic acid is produced, in addition to VFA and gas. As substrate becomes limiting, fermentation reverts to VFA and gas alone (Beuvink and Spoelstra, 1992). Thus, with readily fermentable carbohydrates, although bacteria will be growing rapidly in the initial incubation phase, gas release kinetics is low. The finding of Beuvink and Spoelstra (1992) that gas release became non-linear as fermentation medium pH fell below 6.2 emphasised that buffering capacity is limited, and can become exhausted if excess fermentation end-products are generated. Studies in this laboratory (Kliem et al., 2005) have indicted that the incubation of 1.0 g ground wheat in 100 ml buffered rumen fluid (9:1 buffer:inoculum), where the buffer (Theodorou et al., 1994) concentration has been reduced to 50%, results in pH of the medium decreasing from 6.7 to 5.6 after 24 h, compared with a reduction to only 6.0 with 100% buffer concentration. 3.1.2. Microminerals Magnesium content of the incubation media varies from 1.05 to 1.59 mg/g substrate and calcium from 0.32 to 0.46 mg/g (Table 2), with the Tilley and Terry (1963) medium providing the highest levels. Manganese, cobalt and iron contents were similar between media (1–3) and slightly higher than the Beuvink and Spoelstra (1992) medium. Tilley and Terry (1963) omitted these three elements, and also supplemental N and S, arguing that “adequate levels of accessory factors and trace elements” would be provided by the rumen fluid inoculum or substrate. Grant and Mertens (1992) examining NDF degradability found a variable response to microminerals and concluded that they should be included to ensure their adequacy with respect to fibre digestion of poor quality substrates. Although levels such as 0.2 mg cobalt/g substrate (Table 2) appear adequate, it has to be remembered that these are offered in a dilute medium with the result that actual concentrations are nearer 0.25 mg/l. While not “limiting” (Hungate, 1966), such levels are unlikely to make a substantial contribution to the nutrient status of the medium, relative to that supplied by the incubated substrate and inoculum, and can, therefore, be omitted. Further the media used in microbial studies were designed to provide all required nutrients. However, with in vitro fermentations, nutrients are also supplied via the inoculum and substrate. Only under extreme conditions where, for example, hay obtained from cobalt deficient pastures was to be examined with rumen fluid from donor animals offered a similar substrate, is it likely that a response to micromineral supplementation would be identified. 3.2. Nitrogen supplementation In terms of N supplementation, both Theodorou et al. (1994) and Goering and Van Soest (1970) included trypticase (2.2 and 2.4 g/l, respectively), in addition to that added as ammonium carbonate. Neither Menke et al. (1979) nor Tilley and Terry (1963) added N; the rationale being that strained rumen liquor and incubated substrate would supply sufficient N for bacterial growth. Later, Menke and Steingass (1988) included ammonium carbonate at the same rate as Goering and Van Soest (1970). Using the assumption that, approximately, 25 mg N is required to degrade 1.0 g carbohydrate (based on the ARC (1980) estimate that 1.25 g degradable N is required to degrade 1 MJ metabolizable energy), and considering

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Fig. 2. In vitro gas release kinetics (ml/h/g organic matter incubated) of maize starch supplemented with 0–16.8 mg N as urea).

only the N supplied in the inorganic component of the buffer, the media of Theodorou et al. (1994), Goering and Van Soest (1970) and Beuvink and Spoelstra (1992) provide over twice the calculated N requirement (Table 2). While this in itself can be considered inefficient, it generates a further confounding factor for, as identified by Pichard et al. (1998), protein supply is a source of error in gas pressure measurements. Their results showed a consistent inverse relationship between gas production and level of protein supplementation, with gas release decreasing proportionally as ammonium sulphate was substituted with organic N. Cone and van Gelder (1999) estimated that 0.21 mmol NH3 was released for every 10 mg protein degraded, and that this inhibited the release of 4.4 ml indirect gas. Mould et al. (2004) examined the influence of supplementary N on fermentation by incubating maize starch in a reduced, N-free basal medium. Increasing concentrations of urea were used, with the highest equivalent to the quantity of N supplied by the Goering and Van Soest (1970) medium. The rumen fluid inoculum, obtained from two dry cows offered a poor quality grass hay-based diet, contained 1 mg N/ml. Gas release was measured over 48 h (in triplicate) using the methodology of Mauricio et al. (1999). Without supplemental N fermentation gas production plateaued after 8 h at 6 ml/h and remained constant (Fig. 2). This represents the level of microbial activity that can be supported by N in the rumen fluid inoculum. In contrast, N supplemented gas production rate profiles increased to 10 h post-inoculation, and then declined. Gas production rate increased with each increment of urea (Fig. 2), and significant differences in cumulative gas volume were identified (Table 4). A highly significant regression (P<0.001, r2 = 0.929) between supplemental N (y, mg) and rate of gas release (x, ml/h) at 10 h post-inoculation was observed as: y = 1.0376x + 0.0008x2 − 5.062 indicating that gas production rate could be used to assay N availability. A second study examined degradability of wheat straw, grass silage and maize silage using the same N-free basal medium and rumen fluid source, as detailed in Mould et al. (2004).

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Table 4 Cumulative and rate of gas release (at 10 h post-inoculation) from the fermentation of maize starch as influenced by the addition of urea Urea (N g/flask) 0 1.7 3.4 5.0 6.7 8.4 10.1 11.8 13.4 15.1 16.8 S.E. mean

Rate of gas release (ml/h/g OM) 5.5f

Cumulative gas (ml/g OM)

5.7f 8.7e 8.69e 10.5d 13.0d 13.1d 14.1cd 15.7bc 16.1b 18.4a

39.3ef 35.7f 52.0d 50.5de 59.7dc 76.6b 79.7ab 68.9bc 80.8ab 80.6ab 90.7a

0.19

1.21

Values within columns with common superscripts do not differ (P<0.05). Source: Mould et al. (2004).

Four concentrations of urea were included to provide 0, 10, 20 and 30 mg N/g substrate, with the highest concentration equivalent to 1.5 times the basal concentration of N in the Goering and Van Soest (1970) medium (without trypticase). The methodology of Mauricio et al. (1998) was used to estimate gas release and degradation kinetics simultaneously, with 1 g milled substrate (2 mm), added to each flask. All procedures were conducted in triplicate. In vitro organic matter degradability (iOMD) was assessed by recovering fermentation residues after 6, 12, 18, 24, 36 and 48 h of incubation. In contrast to the previous study, these substrates varied both in their inherent degradability and N content. All substrates showed an increase (P<0.05) in the volume of gas produced with the lowest level of additional N (10 mg), in comparison to 0 mg, which represented the level of fermentation supported by substrate N (Table 5). Higher levels of urea-N supplementation resulted in no further improvement. No effect of supplementation on iOMD occurred when grass silage was supplemented with urea N (Table 6). While an increase in wheat straw degradation occurred at incubation periods longer than 12 h with 10 mg N, higher urea inclusion rates had no further effect. In contrast, maize silage iOMD increased at both 10 and 20 mg N, although 30 mg N had no additional effect. These results agree with Dryhurst and Wood (1998) who reported supplementation response to be dependent on both N content of the feed and its degradability. Thus maize silage, a highly degradable substrate with a low N content, showed the greatest response to supplementation. In contrast, while the N content of wheat straw was low, the inherent degradability of this material, rather than the low N content per se, limited response. The grass silage examined was highly degradable and contained sufficient N such that supplementation levels above 10 mg N would have been in excess. The conclusions from these studies are that the level of supplemental N required is dependent on both the inherent degradability of the substrate and its N content. Rumen fluid alone supplies insufficient N, although an incubation medium N content of 25 mg N/g substrate can be considered adequate.

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Table 5 Influence of nitrogen supplementation and substrate type on in vitro cumulative gas production (ml/g organic matter) Substrate

Substrate (N mg/g)

Hours post-inoculation 6

24

36

48

0 10 20 30

24.7c

73.6c

139.7b

191.1b

31.3b 29.8b 33.8a

85.9ab 84.6b 90.7a

155.9a 155.0a 159.7a

203.1a 200.8ab 205.8a

210.7a 220.0a 219.4a 220.4a

Maize silage

0 10 20 30

18.3b 24.2a 23.0a 23.7a

65.5b 107.1a 109.1a 111.7a

142.2b 210.0a 207.8a 210.7a

237.2b 267.1a 269.7a 278.5a

272.1c 281.4bc 291.3ab 303.3a

Wheat straw

0 10 20 30

4.6b 6.7a 5.7a 6.2a

18.1a 26.9a 25.6a 26.3a

55.8b 87.8a 84.0a 86.7a

107.7b 147.2a 142.3a 145.8a

143.3b 168.2a 166.4a 168.1a

–

0.21

0.71

1.72

1.28

1.44

Grass silage

S.E. mean

12

Values in columns within substrate with similar superscripts do not differ (P<0.05).

3.3. Sulphur Sulphur contents of in vitro media are variable and in excess requirements. Using a ratio of 10:1 N:S (ARC, 1980) a level of 4 mg S/g substrate should be adequate based on N requirement concentrations of 25 mg N/g substrate. Data in Table 1 shows inclusion levels of 6–23 mg S/g substrate, up to six times that required and N:S ratios of between 6.1:1 Table 6 Influence of nitrogen supplementation and substrate type on in vitro organic matter degradation (g/kg) Substrate

Substrate (N mg/g)

Hours post-inoculation 6

12

0 10 20 30

232 273 260 262

389b 402ab 417a 413a

626 607 619 622

679 691 656 684

707ab 727a 726a 692b

Maize silage

0 10 20 30

67 96 89 87

241c 316b 350a 343ab

414c 577b 615a 611a

629b 707a 713a 721a

700b 729a 744a 746a

Wheat straw

0 10 20 30

12 28 22 35

94 101 124 117

260b 335a 338a 339a

360b 465a 472a 468a

453b 525a 528a 498a

Grass silage

S.E. mean

–

3.2

24

3.5

36

7.4

Values in columns within substrate with similar superscripts do not differ (P<0.05).

48

4.5

3.4

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(Theodorou et al., 1994) and 3.1:1 Beuvink and Spoelstra (1992). While an allowance has to be made with respect to availability, this S is present in a soluble inorganic form. It is, therefore, recommended that S inclusion levels be reduced. This could be achieved by supplying Mg, for example, as a chloride salt rather than as magnesium sulfate (e.g., Tilley and Terry, 1963,) or by omitting sodium sulphide, as will be discussed later.

4. Anaerobiosis—reducing solutions and carbon dioxide With the exception of the Tilley and Terry (1963) medium, others (Table 1) include a reducing solution to lower the redox potential and generate an anaerobic environment at the point of inoculation. Hungate (1966) recommended that, in pure culture work, cysteine HCl, sodium sulphide or other reducing agents should be used. Menke et al. (1979) and Beuvink and Spoelstra (1992) used sodium sulphide, while Goering and Van Soest (1970), and later Theodorou et al. (1994) based on Bryant and Robinson (1961), combined equal portions of cysteine HCl and sodium sulphide. Fukushima et al. (2003) reported that, because of its lower toxicity to many anaerobic bacteria, cysteine HCl was preferred to sodium sulphide, although achieving a low redox potential could take longer. Tilley and Terry (1963) emphasised that it was essential to maintain good anaerobic conditions throughout the fermentation phase and saturated their solutions with CO2 prior to use. However, their medium contained no reducing agents, relying instead on “gas production during the digestion to maintain anaerobic conditions”. Leedle and Hespell (1983) identified losses of both cellulolytic and amyloytic bacterial species and a decrease in their activity when anaerobiosis was not maintained, while when Minson and McLeod (1972) omitted CO2 they observed degradation to be slightly, although significantly, depressed. Consequently both Goering and Van Soest (1970), and later Grant and Mertens (1992), recommended continuous gassing with CO2 , when determining substrate degradability. However, where gas production is used as a proxy for ruminal degradation, it is clearly inappropriate to continually flush with CO2 . Equally, as Pell et al. (1988) indicated, gas systems are more sensitive to buffer compositional differences than in vitro degradation techniques, and thus any factor which influences gas release kinetics will impact on the estimation of this parameter. Menke et al. (1979), Beuvink and Spoelstra (1992) and Cone et al. (1996) all prepared media under CO2 ; Pell and Schofield (1993) heated their medium to near boiling under CO2 , while Theodorou et al. (1994) and Mauricio et al. (1999) passed CO2 through the medium (as suggested by Minson and McLeod, 1972) following addition of reducing solution. However, Hungate (1966) stated that, for the cultivation of some rumen bacteria, extreme CO2 gassing precautions were not necessary and that fastidious anaerobes may grow in mixed culture “where no great pains are made to exclude oxygen”, simply because the medium is reduced by the accompanying microorganisms which are less sensitive to oxygen. This suggests that a highly reduced medium need not be produced, in a sealed in vitro fermentation system, as utilisation of oxygen by facultative anaerobes will rapidly create an environment with a low redox potential. In practice, this means that preparation of the media can be greatly simplified, thereby, reducing costs. Potentially toxic ingredients, such as sodium sulphide, which releases hydrogen sulphide in an acidic environment (e.g., carbonic acid following over-gassing with CO2 ), could be omitted. However, saturation

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of the medium with CO2 to displace oxygen will result in elevated gas release during the initial fermentation period. Further, not only is it likely that dissolved CO2 concentrations will differ between experiments over time, but the possible generation of carbonic acid may lower incubation medium pH and depress microbial activity, negatively impacting repeatability. Grant and Mertens (1992) examined substrate degradation characteristics using the Goering and Van Soest (1970) technique, omitting (either singly or both) continuous CO2 gassing or the reducing solution. Purging with gas, but not continuous gassing, resulted in the rate of neutral detergent fibre degradation being decreased with the initial lag phase extended, a finding similar to that reported by Johnson (1966). Grant and Mertens (1992) also found that use of a reduced medium decreased lag phase but had no effect on extent of degradation. Rymer et al. (1998) identified that purging with CO2 decreased initial and final pH values and increased gas release, although this was considered to be gas leaving solution rather than to increased losses following neutralisation of propionic acid. Morgan et al. (2004) examined effects of omitting the reducing solution on fermentation characteristics of maize starch, grass hay, wheat grain and wheat straw. The Goering and Van Soest (1970) medium was used either in a reduced (R) or non-reduced (NR) state. In the latter, cysteine HCl, sodium hydroxide and sodium sulphide were omitted, with urea added (0.075 g/l) to maintain N content. No other adjustments were made. The solutions were prepared in 10 l flasks and gassed with CO2 for 30 min. The inoculum was prepared from rumen fluid collected pre-feeding at 07:00 h from four cows offered a grass hay/grass silage ration ad libitum. The fluid, which was strained through a double layer of muslin, had a dry matter content of 25 mg/g and contained 1 mg N/ml. Head-space gas pressure readings were collected over a 96 h incubation period with accumulated gas released after each measurement. Organic matter degradation was determined by recovering fermentation residues at 6, 12, 24, 48 and 96 h post-inoculation. While no overall effect of medium reduction on the extent of OM degradation was identified (Table 7), feeds incubated in the non-reduced medium produced more gas at extended incubation periods (i.e., >12 h, Table 8). No effect of treatment on either degradation or cumulative gas volume was identified in the first 6 h post-inoculation, with the exception of maize silage, indicating that the large difference in the redox potential generated had comparatively little effect on initial microbial activity. In a second study, Morgan et al. (2004) examined four media, based on Goering and Van Soest (1970), generated through omission of either or both the reducing solution and gassing with CO2 . Grass silage, wheat straw and wheat grain were used with rumen fluid preparation, gas release and degradation determined over a 48 h period, as described in the first study. Differences were found between media in terms of both iOMD and gas production (Table 9), although no treatment×substrate interactions occurred. Inclusion of the reducing solution decreased iOMD and gas production, while gassing the media with CO2 resulted in no effect on iOMD, but increased initial gas release (i.e., 12 h post-inoculation). This difference (approximately, 19 ml) was maintained over the entire incubation period, and thus can be attributed to CO2 leaving the supersaturated incubation medium. The negative effect of reducing solution inclusion on microbial activity (i.e., fermentation/degradation) occurred with all substrates throughout the incubation period. This is in agreement with Fukushima et al. (2003), who reported poor activity of anaerobic bacteria in media reduced

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Table 7 Effect of omitting reducing solution on organic matter degradation (g/kg) Substrate

Reduced mediuma

Hours post-inoculation 6

12

24

48

96

Grass hay

+ −

231 224

296 273

455 450

635 636

712a 701b

Maize silage

+ −

339 226

513a 438b

669a 639b

770 774

812 813

Maize starch

+ −

442 562

856 801

952 942

986 984

999 998

Wheat grain

+ −

536 579

751 725

871 882

935 940

956a 949b

Wheat straw

+ −

93 93

155 162

330 348

523 534

618a 588b

Overall

+ −

328 337

514a 480b

655 652

770 774

820a 810b

S.E. mean

49.1

9.2

4.4

1.9

1.0

Means in columns within substrate with similar superscript do not differ (P<0.05). Source: Morgan et al. (2004). a (+) Reduced; (−) non-reduced.

Table 8 Cumulative gas release (ml/gorganic matter incubated) Substrate

Reduced mediuma

Hours post-inoculation 6

12

24

48

96

Grass hay

+ −

16.3 16.0

44.1 42.8

109.8 116.8

177.0 188.a

206.3 216.9

Maize silage

+ −

43.1a 25.4b

122.6a 100.5b

186.0 169.4

234.8b 248.8a

253.0b 276.1a

Maize starch

+ −

26.7b 31.8a

136.8b 154.5a

272.2 291.3

334.1b 358.7a

358.6b 381.9a

Wheat grain

+ −

40.4 38.7

152.2 149.3

247.9 255.8

296.4 299.8

314.1 311.7

Wheat straw

+ −

6.1 8.0

24.8 28.5

81.3 96.5

149.7 160.1

183.2 189.9

Overall

+ −

26.5a 24.0b

96.1 95.1

179.4 186.0

238.4b 251.2a

263.0b 275.3a

6.05

8.76

S.E. mean

0.74

3.03

5.54

Means in columns within substrate with similar superscripts do not differ (P<0.05). Source: Morgan et al. (2004). a (+) Reduced; (−) non-reduced.

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167

Table 9 Organic matter degradation and fermentation gas release (hours post-inoculation) as influenced by inclusion of a reducing solution or gassing of the medium with carbon dioxide (CO2 ) Reducing solution

CO2

+ − + −

+ + − −

S.E. mean

1.4

Significance§

***

+ – S.E. mean

3.3

Significance§

*** + –

OM degradation (g/kg)

Cumulative gas (ml/g OM)

12 h

24 h

48 h

24 h

48 h

361b

601b

711b

84.6b

167.5b

396a 344b 383a

610b 576c 633a

723ab 721b 734a

93.7a 66.6d 72.9c

181.1a 143.5d 159.5c

218.5b 241.8a 206.4c 214.2bc

0.60

1.02

1.60

1.8

2.2

12 h

***

*

352a

588b

716b

390b

621a

729a

75.7b 83.2a 1.03

1.53

2.52

717 727

** 69.8b 89.0a

*** 152.5b 174.0a

** 210.3b 230.2a

0.71

1.33

2.48

3.2 *** 378 363

***

2.6 * 605 604

S.E. mean

4.6

4.3

2.4

Significance§

n.s.

n.s.

n.s.

***

***

***

155.9b 170.4a

212.5b 228.0a

***

***

Main effects derived from incubations using three substrates (grass silage, wheat grain and wheat straw). Values in columns within treatment group with similar superscripts do not differ (P>0.05), § *, **, *** and n.s. represent P>0.05, 0.01 and 0.001 and not significant, respectively.

with agents such as sodium sulphide, possibly due to toxic intermediaries or precipitation of metal ions. In contrast to the basal medium, when both reducing solution and gassing with CO2 were omitted, not only was iOMD increased and gas losses reduced but, fermentation efficiency (expressed as iOMD (mg)/cumulative gas (ml)) was enhanced from 3.25 to 3.43, for media where both components were included or omitted, respectively. The argument that higher gas release following CO2 addition resulted from a change in microbial fermentation efficiency can be refuted by considering the alternative, that the omission of CO2 increased efficiency (i.e., reduced fermentation gas losses), under conditions less favourable to microbial fermentation. Equally, the lack of change in degradation suggests that the level of CO2 added was insufficient to adversely affect medium pH. While there does not appear to be a requirement for a highly reduced incubation medium, in agreement with Tilley and Terry (1963) and Hungate (1966), a degree of initial anaerobiosis may be beneficial. While this should not be achieved solely through saturation with CO2 , due to potential problems associated with additional gas release, the depressed microbial activity when the reducing solution was included suggests that its use should be modified (e.g., the quantity included lowered, or a component, such as sodium sulphide, omitted). This would have additional benefits through decreasing analytical costs, improving safety and easing problems associated with sourcing medium ingredients.

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5. Neutralisation of fermentation acids Where an in vitro system has insufficient capacity for degradation kinetics to be estimated simultaneously, attempts have been made to generate these data from gas release profiles using mathematical models (e.g., Groot et al., 1998; France et al., 2000). An assumption with this approach is that a constant relationship exists between the two parameters. This is despite the fact that fermentation of individual feedstuff components over time results in an uneven release of acidic end products, which affect direct and indirect gas production. A further assumption is that gas release from neutralisation of VFA is independent of their composition or rate of production, with 1 mol acid liberating 1 mol CO2 from a bicarbonate buffer according to: H+ + HCO3 − ↔ H2 CO3 ↔ CO2 + H2 O. ·

·

However as Beuvink and Spoelstra (1992) suggested, this should be modified to consider those acids neutralised by phosphate and through interaction with components such as ammonia. Rymer et al. (1998) found variation (P<0.001) in medium pH among four incubation media, following addition of 12 mmol propionic acid. Volumes of gas produced also differed, and appeared to be related to the bicarbonate: phosphate composition of the medium. Rymer et al. (1998) concluded that for a given quantity of fermentation acids, a constant amount of gas would be liberated from a specific medium. However, this will vary with the pKa of the VFA mixture produced. Mauricio et al. (1998) added 45, 60 or 75 mmol acetic acid/l to the medium of Theodorou et al. (1994) and concluded that gas released from neutralisation varies with the quantity and rate of addition, and that it was important to accurately partition gas between fermentation and neutralisation. Krystallidou et al. (2002) compared gas release kinetics following the addition of three VFA mixtures (0.68:0.23:0.09; 0.53:0.35:0.12 and 0.42:0.42:0.16 acetic:propionic:butyric, designated as 1, 2 and 3, respectively), to two media. These were based on Beuvink and Spoelstra (1992) and Mauricio et al. (1999), identified as A and B, respectively, but comprised only the buffer and macromineral components. They differed in terms of phosphate and carbonate content (i.e., 3.26 and 8.11 and 2.98 and 9.75 g/l, for buffers A and B, respectively). Three final acid concentrations (45, 60 and 75 mmol/l) were generated by adding 0.1 ml aliquots of VFA solutions of different concentrations at 30 min intervals over a 9-h period. Each treatment was replicated five times. A difference in gas release was observed with the higher phosphate buffer (A) generating less gas than buffer B. (Table 10). Similarly, cumulative gas release increased with VFA concentration from 33.1 to 52.2 ml (45 and 75 mmol, respectively). In contrast, while equal quantities of gas were released from neutralisation of VFA mixtures 1 and 2, mixture 3 generated more (P<0.05). Although the pH of buffer A was initially higher (8.12) than B (7.74) it declined to a greater (P<0.05) extent (6.56 and 6.78, respectively), indicating that the ability of this buffer to maintain pH values was poorer. It was concluded that gas production varied with the buffer composition, and both the proportion of VFA and rate of addition (i.e., fermentation), and that such factors must be considered when attempts are made to model degradation from gas production.

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169

Table 10 Influence of buffer type, volatile fatty acid mixture and concentration added on gas release and medium pH Factor

Level

Gas release (ml) 6h

9h

3h

6h

9h

Buffera

A B

9.3b 9.9a

24.2b 25.2a

41.1a 42.2a

7.16b 7.26a

6.81b 6.96a

6.56b 6.78a

VFA mixa

1 2 3

9.9a 9.7ab 9.2b

24.6b 24.0b 25.6a

41.1b 40.7b 43.1a

7.30a 7.16b 7.18b

6.93a 6.88b 6.84b

6.66b 6.73a 6.63b

20.7c 23.0b 30.65a

33.1c 39.6b 52.2a

7.40a 7.15b 7.08c

7.11a 6.85b 6.69c

6.94a 6.66b 6.41c

0.053

0.042

0.044

3h

Concentration (␮mol l−1 )

S.D.

45 60 75 0.69

8.8b 8.6b 11.3a 1.33

Medium Ph

1.91

Means in columns within factor without similar superscripts differ (P<0.05). a See text for details.

6. Conclusions The purpose of this review was to assess the ability of in vitro media to provide a suitable environment for microbial fermentation of feedstuffs and to identify where changes to the buffer component could be made. There appears to be no requirement to change the carbonate and phosphate salts, which comprise the main buffer components. However, it is important to ensure that sufficient buffering capacity exists to maintain pH of the incubation medium within a range such that degradation is not compromised. It is vital that, where degradation profiles are generated from gas production data, the relationship between these factors is known for each buffer type and that the impact of ammonia release from protein degradation is considered. Calculations suggest that inclusion of microminerals provide few additional nutrients over that already supplied by rumen fluid and test substrate and thus, in agreement with Tilley and Terry (1963), can be omitted. The lack of an effect found by Grant and Mertens (1992) is considered insufficient grounds to merit their incorporation. A major difference between current incubation media is their N content. The studies reviewed identified that N contribution of inoculum was insufficient to fully support degradation. Thus for N supplied directly by the media (i.e., ammonium carbonate and cysteine HCl), it is recommended that inclusion levels of 25 mg N/g substrate are achieved, although it is recognised that, with the exception of highly fermentable low protein substrates, such as cereal grains and maize silage, this level could be reduced. Use of additional N is unnecessary plus excess ammonia released could interfere with indirect gas production. By limiting N inclusion, and using recommended N:S ratios, a modified S inclusion level (i.e., 4–5 mg S/g substrate) is proposed. Sulphur levels can be reduced through omission of sodium sulphide and replacement of magnesium sulphate with magnesium chloride. The data reviewed not only indicated that highly reduced media are not required for feedstuff degradation, provided that anaerobic conditions are rapidly established, but that a component of the reducing solution apparently suppressed microbial activity. On the

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170

Table 11 A simplified in vitro incubation medium Component

Chemical

Composition Final solution (g/l)a

Substrate (mg/g)b

Buffer 1

Na2 HPO4 ·12H2 0 KH2 PO4 MgCl2 ·6H2 O

1.985 1.302 0.105

– – –

Buffer 2

NH4 HCO3 NaHCO3

1.407 5.418

– –

Reducing solution

Cysteine HCl NaOH

0.390 0.100

– –

Composition

PO4 3− HCO3 2− N S Mg PO4 3− :HCO3 2− N:S

1.435 5.020 0.280 0.071 0.013 0.286 3.95

129 452 25 6 1

a b

0.01 mg resazurin included per litre medium. Assuming an inclusion rate of 90 ml medium and 10 ml rumen fluid per 1.0 g substrate.

grounds of both safety and that sodium sulphide may impair microbial growth (Fukushima et al., 2003), it is recommended that this component be omitted. As gassing with CO2 to displace dissolved O2 has been shown to directly influence the quantity of gas released, it is recommended that, where CO2 is used, minimal amounts are added and that gas flow and duration of gassing, together with the volume of medium treated are described. 6.1. A simplified in vitro medium To summarise these conclusions, an alternative medium based on Goering and Van Soest (1970) is suggested (Table 11). The microminerals have been omitted, as has sodium sulphide from the reducing solution. The level of cysteine HCl remains unchanged, while magnesium chloride is used in place of magnesium sulphate to aid the reduction of sulphur levels. While the N:S ratio is not ideal (3.95), S levels cannot be lowered further without a reduction in cysteine HCl. The N levels have been decreased to 25 mg/g substrate, by omitting supplementary N sources such as trypticase. The phosphate:carbonate ratio has been maintained (0.29), as has the total buffer (phosphate + carbonate) content. Resazurin is included to provide a visual indication of the redox status of the medium. It is suggested that this simplification will improve safety and reduce costs and problems associated with sourcing components, without negatively impacting analytical precision.

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