Ž .Animal Feed Science and Technology 72 1998 261–281

In vitro gas measuring techniques for assessment ofnutritional quality of feeds: a review

G. Getachew, M. Blummel, H.P.S. Makkar, K. Becker )¨( )Institute for Animal Production in the Tropics and Subtropics, UniÕersity of Hohenheim 480 , D-75593

Stuttgart, Germany

Received 25 March 1997; accepted 26 November 1997

Abstract

The close association between rumen fermentation and gas production has been recognised forover a century, but it is only since the 1940s that quantification techniques for measuring gasproduction have been evolved. The gas measuring technique has been widely used for evaluationof nutritive value of feeds. More recently, the upsurge of interest in the efficient utilisation ofroughage diets has led to an increase in the use of this technique due to the advantage in studyingfermentation kinetics. Gas measurement provides a useful data on digestion kinetics of bothsoluble and insoluble fractions of feedstuffs. This review describes the available in vitro gasmeasuring techniques used for feed evaluation with emphasis on assessing their relative advan-tages and disadvantages. Origin of gas, stoichiometry of gas production, and various areas forapplication of gas measurement in feed evaluation are discussed. Some important results obtainedusing gas measuring techniques have been highlighted, and the potential of gas techniques fortackling some interesting areas of research are presented. The need to consider substrateincorporation into microbial cells in gas measuring technique is pointed out. q 1998 ElsevierScience B.V. All rights reserved.

Keywords: Gas production; Feed evaluation; Rumen fermentation; Microbial biomass

1. Introduction

Both milk yield and growth of ruminants are largely limited by forage quality whichŽ .is mainly reflected in low voluntary intake and digestibility Minson, 1990 . The

importance of these parameters in animal nutrition has long been recognised. The

) Corresponding author. Fax: q49-711-459-3702; e-mail: kbecker@uni-hohenheim.de

0377-8401r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0377-8401 97 00189-2

( )G. Getachew et al.rAnimal Feed Science and Technology 72 1998 261–281262

determination of intake and digestibility of feedstuffs in vivo is time-consuming,laborious, expensive, requires large quantities of feed and is unsuitable for large-scale

Ž .feed evaluation Coelho et al., 1988; Carro et al., 1994 . Therefore, many attempts havebeen made to predict intake and digestibility using laboratory techniques. Much efforthas been directed towards the development of regression equations to predict digestibil-ity from forage chemical composition, but a regression equation which satisfactorily

Žpredicts a wide range of forages has not yet been derived McLeod and Minson, 1971;.Van Soest, 1994 .

The digestibility of feeds can be estimated by biological methods which simulate theŽ .digestion process. Three major digestion techniques biological methods currently

Ž .available to determine the nutritive value of ruminant feeds are: 1 digestion withŽ . Žrumen microorganisms as in the work of Tilley and Terry 1963 or gas method Menke

. Ž . Ž .et al., 1979 , 2 cell-free fungal cellulase, and 3 in situ incubations of samples innylon bags in the rumen.

Biological methods are more meaningful since microorganisms and enzymes aremore sensitive to factors influencing the rate and extent of digestion than are chemical

Ž .methods Van Soest, 1994 . However, some important points need to be addressed in thedevelopment of a viable in vitro technique. An efficient laboratory method should bereproducible and should correlate well with actually measured in vivo parameters. Invitro methods have the advantage not only of being less expensive and less time-con-suming, but they allow one to maintain experimental conditions more precisely than doin vivo trials.

Ž .The technique of Tilley and Terry 1963 became an important tool for the evaluationof ruminant feeds and is used widely because of its convenience, particularly whenlarge-scale testing of feedstuffs is required. This method is employed in many forageevaluation laboratories and involves two stages in which forages are subjected to 48 hfermentation in a buffer solution containing rumen fluid, followed by 48 h of digestionwith pepsin in an acid solution. The method was modified by Goering and Van SoestŽ .1970 , in that the residue after 48 h incubation was treated with neutral detergentsolution to estimate true dry matter digestibility. Although the method of Tilley and

Ž . Ž .Terry 1963 has been extensively validated with in vivo values Van Soest, 1994 , themethod appears to have several disadvantages. The method is an end-point measurementŽ .gives only one observation thus, unless lengthy and labour-intensive time-coursestudies are made, the technique does not provide information on the kinetics of foragedigestion; the residue determination destroys the sample and therefore a large number ofreplicates are needed. The method is therefore difficult to apply to materials such astissue culture samples or cell-wall fractions.

ŽEnzymatic digestibility assays Jones and Hayward, 1975; Dowman and Collins,.1982; De Boever et al., 1986 which use enzymes instead of microorganisms have

appeared largely as a result of the increased availability of commercially producedenzymes. Enzymatic methods of evaluation are routinely used as end-point digestibility

Ž .procedures and suffer from similar disadvantages as the Tilley and Terry 1963technique. The enzymatic method may be insensitive to factors such as associativeeffects and toxins which can affect microbial degradation. The main advantage of theenzymatic method over the rumen fluid methods is that it does not require a fistulated

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animal as inoculum donor. However, recent studies have indicated that faecal inoculumhas a potential to replace the rumen fluid and therefore reduce the dependence of in vitro

Žtechnique on fistulated animal as inoculum donor Jones and Barnes, 1996; Macheboeuf. Ž .and Jestin, 1997; Mauricio et al., 1997 . Unlike the method of Tilley and Terry 1963 ,

results from the enzymatic method have not been extensively validated with in vivovalues.

The nylon bag technique has been used for many years to provide estimates of bothŽ .the rate and extent of disappearance of feed constituents Mehrez and Ørskov, 1977 .

This technique provides a useful means to estimate rates of disappearance and potentialdegradability of feedstuffs and feed constituents. The disadvantage of the method is thatonly a small number of forage samples can be assessed at any one time, and it alsorequires at least three fistulated animals to account for variations due to animal. It istherefore of limited value in laboratories undertaking routine screening of a largenumbers of samples. It is also laborious, and requires large amounts of samples.Substantial error could result in values obtained at early stages of digestion due to a lowweight loss, and for poor quality roughages adherence of microbes at early stages can

Ž .even lead to higher weights and thus distortion of results. Dewhurst et al. 1995Ž .compared the nylon bag technique with in vitro Tilley and Terry 1963 and found that

the nylon bag method overestimated the fermentation. The extent of overestimation wasstrongly related to the carbohydrate composition of feeds, particularly at short incubationtimes suggesting that it was caused mainly by a rapidly fermentable fraction which was

Ž .lost from bags before it was fermented. On the other hand, Ørskov and Ryle 1990indicated the possible underestimation of dry matter loss from the nylon bag at early

Žperiods of incubation due to adherence of microbes. Both the in vitro method Tilley and. Ž .Terry, 1963 and the nylon bag technique Mehrez and Ørskov, 1977 which are based

on residue determinations may result in overestimation dry matter digestibilities fortannin-rich feeds. In such systems, tannins are solubilised but might be indigestibleŽ .Makkar et al., 1993 .

The close association between rumen fermentation and gas production has long beenŽ .recognised Tappeiner, 1884 as cited by Marston, 1948 , but the history of the rumen

Ž .fermentative gas measuring technique started in the early 1940s Quin, 1943 . The gasmeasuring technique was considered to be a routine method of feed evaluation after the

Ž .work of Menke et al. 1979 , where a high correlation between gas production in vitroand in vivo apparent digestibility was reported.

2. Origin of gas

When a feedstuff is incubated with buffered rumen fluid in vitro, the carbohydratesŽ . Ž .are fermented to short chain fatty acids SCFA , gases mainly CO and CH and2 4

microbial cells. Gas production is basically the result of fermentation of carbohydrates toŽacetate, propionate and butyrate Wolin, 1960; Beuvink and Spoelstra, 1992; Blummel¨

.and Ørskov, 1993 . Gas production from protein fermentation is relatively small asŽ .compared to carbohydrate fermentation Wolin, 1960 . The contribution of fat to gas

production is negligible. When 200 mg of coconut oil, palm kernel oil andror soybean

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oil were incubated, only 2.0 to 2.8 ml of gas were produced while a similar amount ofŽcasein and cellulose produced about 23.4 ml and 80 ml gas Menke and Steingass, 1988;

.Getachew et al., 1997 .The gas produced in the gas technique is the direct gas produced as a result of

Ž .fermentation CO and CH and the indirect gas produced from the buffering of SCFA2 4Ž .CO released from the bicarbonate buffer . For roughages, when bicarbonate buffer is2

used, about 50% of the total gas is generated from buffering of the SCFAs and the rest isŽ .evolved directly from fermentation Blummel and Ørskov, 1993 . At very high molar¨

Ž .proportion of propionate concentrate diets the amount of CO generated from buffer-2Ž .ing of SCFA is about 60% of total gas production Table 1 . Each mmol of SCFA

produced from fermentation releases 0.8–1.0 mmol of CO from the buffered rumen2Žfluid solution, depending on the amount of phosphate buffer present Beuvink and

.Spoelstra, 1992; Blummel and Ørskov, 1993 . Highly significant correlation has been¨Žobserved between SCFA and gas production Beuvink and Spoelstra, 1992; Blummel¨

.and Ørskov, 1993; Makkar et al., 1995a .Gas is produced mainly when substrate is fermented to acetate and butyrate. Substrate

fermentation to propionate yields gas only from buffering of the acid and, therefore,Žrelatively lower gas production is associated with propionate production Wolin, 1960;

.Hungate, 1966; Van Soest, 1994 . The gas which is released with the generation ofpropionate is only the indirect gas produced from buffering. The molar proportions of

Ž .different SCFA acetate, propionate and butyrate produced is dependent on the type ofŽ .substrate Beuvink and Spoelstra, 1992; Blummel and Ørskov, 1993 . Therefore, the¨

molar ratio of acetate to propionate was used to evaluate substrate related differences.Rapidly fermentable carbohydrates yield relatively higher propionate as compared toacetate, and the reverse takes place when slowly fermentable carbohydrates are incu-bated. Many workers reported more propionate and thus a lower acetate to propionate

Ž .ratio in the ruminal fluid of cows fed a high grain diet see Ørskov and Ryle, 1990 . Iffermentation of feeds leads to a higher proportion of acetate, there will be a concomitantincrease in gas production compared with a feed with a higher proportion of propionate.In other words, a shift in the proportion of SCFA will be reflected by changes in gasproduction.

There are a number of factors which affect fermentation of feeds by rumen microor-ganisms and hence gas production. It is not the aim of this review to deal with all factorsaffecting rumen fermentation. However, an attempt is made to discuss some importantfactors which have considerable influence on in vitro gas production. Anaerobiosis,proper temperature, suitable pH and adequate buffering are important in affecting in

Ž .vitro fermentation. Using 3 h incubation, Trei et al. 1970 found a significant decreasein gas production per gram dry matter as sample size increased from 1 to 3 g and thiswas reflected by a corresponding decrease in dry matter disappearance, probably due toexhaustion of buffer and low proportion of microbes to substrate. No mention was madeconcerning any increase in the volume of buffered rumen fluid-medium as sample size

Ž .increased. Raab et al. 1983 reported a highly significant linear correlation between theamount of substrate incubated and the amount of gas produced at 24 h. However, thelinearity was lost when the gas volume exceeded 90 ml probably due to the exhaustion

Ž .of buffer of the medium. Beuvink and Spoelstra 1992 suggested that the amount of

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Table 1Influence of molar proportion of SCFA on gas production, substrate requirement, ATP yield, and partitioning factor

B.A. ProportionallyProportionally high

Components high acetate propionate

1 mmol of 1 mmol ofSCFA SCFAcomposed of composed of0.748, 0.459, 0.4620.194, and and 0.0790.058 mmol mmol ofof acetate, acetate,propionate propionateand andbutyrate, butyrate,respectively respectively

Fermentative CO 0.5095 mmol yielding 13.04 ml CO 0.4635 mmol yielding 11.87 ml CO2 2 2

Fermentative CH 0.3545 mmol yielding 9.08 ml CH 0.1535 mmol yielding 3.93 ml CH4 4 4

Buffering CO 1.0 mmol yielding 25.6 ml CO 1.0 mmol yielding 25.6 ml CO2 2 2

Total gas 47.7 ml 41.4 mlaSubstrate for production of SCFA, gases and water 105.3 mg 97.2 mg

bmmol of ATP produced 2.61 2.69Substrate for microbial biomassYATPs10 26.1 26.9YATPs20 52.2 53.8Total substrate requirementYATPs10 131.4 124.1YATPs20 157.5 151.0Partitioning factorYATPs10 2.8 3.0YATPs20 3.3 3.6

a Ž .Calculated from molar proportions of the fermentation products SCFA, CO , CH and H O .2 4 2b Ž . Ž .Calculated from the ATP yield; assuming each mmol of acetate, propionate, butyrate and methane would yield 2 Baldwin, 1970 , 3 De vries et al., 1973 , 3Ž . Ž . Ž Ž ..Baldwin et al., 1970 , and 1 Stadman, 1967 mmol of ATP, respectively modified from Blummel et al. 1997c .¨

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ŽSCFA produced must not exceed 4.5 mmol 1 mmol of bicarbonate buffer releases 1.mmol gas which is equivalent to 25.63 ml at 398C gasr60 ml buffered rumen fluid to

avoid exhaustion of the buffer which leads to lowering of pH and hence decrease inmicrobial activity. Using similar incubation medium as described by Beuvink and

Ž . Ž .Spoelstra 1992 , Cone et al. 1996 also reported exhaustion of buffer when more than0.5 g corn cob mix was incubated. Therefore, the quantity of feed incubated in in vitrosystems must be set in relation to the volume of buffered rumen fluid-medium. AnimalŽ . Ž .Trei et al., 1970 and batch Beuvink and Spoelstra, 1992 of inoculum also have a

Ž .considerable influence on in vitro gas production. Beuvink and Spoelstra 1992incubated glucose, rice starch and cellulose and found significant differences in gasproduction due to the different activity of rumen fluid taken on different days. Thiscould be corrected by introducing standards with known gas production.

3. Stoichiometry of gas production

An important aspect of the anaerobic system is that stoichiometric laws of fermenta-tion balance can be applied since fermentation products must be derived from thesubstrate incubated. High correlation between stoichiometrically calculated gas and

Ž .actually recorded values have been reported by Beuvink and Spoelstra 1992 , Blummel¨Ž . Ž .and Ørskov 1993 and Opatpatanakit et al. 1994 . The stoichiometric balance allows

Ž .the theoretical calculation of equilibrated amounts of the products SCFA and gasesŽ .Van Soest, 1994 . If the molar proportion and amount of SCFA are known, thetheoretical amounts of CH and CO expected from the rumen fermentation can be4 2

Ž .calculated Table 1 .

4. In vitro gas measuring techniques

ŽMuch of the earlier work on gas measurement McBee, 1953; El-Shazly and Hungate,.1965; Czerkawski and Breckenridge, 1969, 1970 centred on investigations of rumen

Ž .microbial activities using manometric measurements. McBee 1953 developed a mano-metric method of gas measurement for the evaluation of rumen microbial activity withrespect to cellulose and hemi-cellulose fermentation and concluded that the rate offermentation of various substrates in the rumen is not constant but subject to widefluctuations following changes in the diet of the animal. Using this method, microbial

Ž .preference for different components of feedstuff was examined. McBee 1953 foundthat organisms which are capable of fermenting cellulose are also able to fermenthemi-cellulose but not all of the hemi-cellulose fermenters ferment cellulose.

Ž .El-Shazly and Hungate 1965 measured rumen fermentation rates using the constantvolume manometric method where different amounts of hay were incubated with rumen

Ž .contents and mineral salt solutions. Czerkawski and Breckenridge 1969 developed agas measuring manometric apparatus to investigate the effect of fatty acids on thefermentation of sugar-beet pulp and sucrose by mixed rumen microorganisms. By using

Ž .this method, the authors reported that during short-term incubation 6–8 h the fermenta-tion pattern of sugar-beet pulp was similar to in vivo while the addition of linseed oilacids temporarily inhibited gas production. In this method, the apparatus consisted of

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five fermentation units and with these, it was not easy to make more than two multipleincubations during any one working week and analyse all the samples. To overcome this

Ž .problem, Czerkawski and Breckenridge 1970 developed a syringe system with thecapacity of ten units for studying rumen fermentation. However, even these ten unitswere not sufficient to be accepted as a routine method for feed evaluation, so thismethod has not been widely used.

Although the manometric method permits a quantitative determination of acids andgases evolved during fermentation, and allows incubation of a large amount of sample

Ž .by increasing the volume of the manometric vessels Hungate et al., 1955 , a largenumber of samples cannot be handled easily. In the manometric system, no provisionwas made for the mechanical stirring of the sample during incubation. Therefore,manometric methods of gas measurement do not seem to have wide applicability inroutine feed evaluation.

Ž .Wilkins 1974 developed an automated pressure transducer method for measuringgas production by microorganisms. The system consisted of a pressure transducer and apressure equaliser valve attached to the metal cap of a test tube containing inoculum andculture medium, and gas pressure was recorded on a strip-chart recorder. In this study,cultures shaken at 200 oscillationsrmin showed a marked increase in rate of gas releaseover stationary cultures. The importance of shaking of samples was also demonstrated

Ž .by Pell and Schofield 1993 where the coefficient of variation among the plateau gasreadings in stirred sample was reduced by half as compared to those of unstirred

Ž .samples. Although this method Wilkins, 1974 was developed only for the detection ofmicroorganisms in clinical samples and sterility testing of foods, it created a basis forthe development of the pressure transducer method for feed evaluation.

ŽThe advantages of the gas measuring techniques over other in vitro techniques Tilley. Ž .and Terry, 1963 for feed evaluation have been outlined by Blummel and Ørskov 1993¨

Ž .and Makkar et al. 1995b . Other in vitro methods are based on gravimetric measure-Žments which follow disappearance of the substrate the components which may or may

.not necessarily contribute to fermentation , whereas gas measurement focuses on theŽappearances of fermentation products soluble but not fermentable products do not

.contribute to gas production . In the gas method, kinetics of fermentation can be studiedon a single sample and therefore a relatively small amount of sample is required or alarger number of samples can be evaluated at a time.

Ž .There are basically two approaches for measuring gas volumes: 1 measuring gasŽ .collected at atmospheric pressure and its volume determined directly or 2 measuring

gas accumulated in a fixed volume container, and the volume is calculated from pressurechanges. Within these broad groups, there are different gas measuring techniques

Ž .developed for the evaluation of feed quality Table 2 . These techniques have been usedas a method for feed evaluation, for understanding mechanisms of microbial fermenta-tion, and for studying the mode of action of various anti-nutrients and feed supplements.

Ž .The available gas measuring techniques are: a Hohenheim gas method or Menke’sŽ . Ž . Ž .method Menke et al., 1979 ; b Liquid displacement system Beuvink et al., 1992 ;

Ž . Ž . Ž .c Manometric method Waghorn and Stafford, 1993 ; d Pressure transducer systems:Ž . Ž .manual Theodorou et al., 1994 ; computerised Pell and Schofield, 1993 , combination

Ž .of pressure transducer and gas release system Devies et al., 1995; Cone et al., 1996 .

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Table 2Comparison of incubation systems in commonly used gas measuring methods

Ž . Ž . Ž . Ž .Menke et al. 1979 Beuvink et al. 1992 Pell and Schofield 1993 Waghorn and Stafford 1993

Microbial inoculum liquid phase liquid phase liquid phase liquid q solid phaseRumen fluid collection time before feeding before feeding 2 h after feeding 2 h after feeding

Ž .Rumen fluid ml rincubation 10 20 2 20Ž .Sample mg 200–300 400 100 1250

Ž .Sample container size ml 100 100 50 250Incubation conducted in syringes kept in rotor bottle kept in shaking bottle and stirrer flask connected to a manometer,

Ž . Ž . Ž .50 cm diameter, 1 rotationrmin water bath 50 rpm 48 rpm , kept in incubator shaking water bathŽ .Incubation volume ml 30 60 9 80

Number of fermentation vessels 60 24 15 not availableŽ .HCO mmol rincubation 2.3 4.6 0.76 7.023Ž .Sample mg :rumen liquor 100:5:1.15 100:5:1.15 100:2:0.76 100:1.6:0.56

Ž . Ž .ml :HCO mmol3

a Na CO .2 3

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Ž .Table 2 continuedComparison of incubation systems in commonly used gas measuring methods

Ž . Ž . Ž .Blummel 1994 Theodorou et al. 1994 Cone et al. 1996¨

Microbial inocolum liquidqsolid phase liquidqsolid phase liquid phaseRumen fluid collection time before feeding before feeding 2 h after feedingRumen fluid collection time 10 10 20

Ž .Sample mg 500 500–1000 400–500Sample container size 100 125 250

Ž .Incubation conducted in syringes kept in waterbath bottle in incubator bottle in shaking water bath 50 rpmŽ .Incubation volume ml 40 100 60

Number of fermentation vessels 60–120 as required 12aŽ .HCO mmol rincubation 4.6 3.76 4.63

Ž . Ž . Ž .Sample mg :rumen ml :HCO mmol 100:2:0.92 100:5:1.88 100:4:1.153

a Na CO .2 3

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( )4.1. Hohenheim gas test Menke’s method

Ž .Menke et al. 1979 developed a feed evaluation system using an in vitro gasŽ .measuring technique. Fermentations are conducted in large 100 ml calibrated glass

syringes containing the feedstuff and a buffered rumen fluid. In this system, gasproduction in 24 h observed on incubation of 200 mg feed dry matter correlated wellwith digestibility of organic matter determined in vivo with sheep. The volume of gas in24 h from 200 mg feed DM was used together with the concentration of other chemical

Ž .constituents to predict metabolisable energy Menke and Steingass, 1988 . When a 200mg sample is used, the method is relatively simple to handle and a relatively largenumber of samples can be analysed at one time. However, the syringes are narrow and it

Ž .is difficult to place a large amount )500 mg of sample, particularly those which areŽ .bulky in nature e.g., straws and stovers . The syringes are of 100 ml capacity and can

accommodate gas produced from 200 mg sample without any push-back of thesyringe-piston to remove gas from the syringes. When larger amount of samples areincubated, more gas is produced and this necessitates frequent push-back of the plungerwhich is inconvenient and is also a potential source of error.

Ž . Ž .The method of Menke et al. 1979 was modified by Blummel and Ørskov 1993 in¨that feeds were incubated in a thermostatically controlled water bath instead of a rotor in

Ž . Ž .an incubator. Blummel et al. 1993 and Makkar et al. 1995b modified the method¨further by increasing the amount of sample from 200 to 500 mg and increasing theamount of buffer two-fold. The main advantages of the modified method over the

Ž . Ž .original method of Menke et al. 1979 are: i there is only a minimum drop intemperature of the medium during the period of recording gas readings on incubation ofsyringes in a waterbath. This is particularly useful for studying the kinetics of fermenta-

Ž .tion where gas volumes must be recorded at various time intervals, and ii an increasein amount of sample from 200 to 500 mg reduces the inherent error associated withgravimetric determination needed to determine concomitant in vitro apparent and true

Ž .digestibility Blummel et al., 1997a; Makkar et al., 1995a .¨

4.2. Liquid displacement system

Ž .Beuvink et al. 1992 used a closed system where gas production was measured byliquid displacement. Feed is incubated with 60 ml buffered rumen fluid in fermentation

Ž .bottles for 24 h. A bottle is placed in a water bath 398C and connected to the waterdisplacement system. The amount of liquid displaced by the gas is collected andweighed and the information is transmitted to a computer. The system needs to beequilibrated before the actual measurement starts. The time required to set the system toequilibrium does not allow the use of the method for measuring fermentation rates at theinstant of inoculating substrate. Even though the gas production was registered automati-cally, the preparation before starting measurements are reported to be laborious and

Ž .complicated Beuvink, 1993 .

4.3. Manometric method

Ž .Waghorn and Stafford 1993 measured gas production by incubating 1.25 g sampleŽ .with 20 ml rumen liquor and 60 ml of artificial saliva McDougall, 1948 . Incubation

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was carried out in 250 ml flask at 398C in a shaking water bath for 9.5 h with rates ofgas production recorded at 30 min intervals. Each flask was connected to a manometricmeasuring device which enabled gas volumes to be measured at atmospheric pressure.Similar to other manometric methods, this method seems to be of limited applicability inroutine feed evaluation as only a small number of samples can be handled at a time.

4.4. Pressure transducer systems

Ž .Pell and Schofield 1993 used a computerised system to study kinetics of foragedigestion by measuring gas production. Individual pressure sensors which remain inplace throughout incubation are used to transmit data to a computer. The bottlescontaining sample and incubation medium are stoppered with butyl rubber stoppers and

Ž .crimp sealed. After the medium is equilibrated for five min in an incubator 398C ,rumen fluid is added by injection, and a pressure sensor is inserted in each bottle. Thesensors are then plugged to the computer leads, and readings are initiated. In this systemthe temperature of the incubator is controlled by light bulbs which may affect thefermentation of feeds rich in phenolic substances and, this method may not be suitablefor studying the fate, effects and mechanism of action of phenolic compounds as these

Ž .can be sensitive to light Tunner et al., 1996 . Gas readings are carried out at hourlyinterval during a 48 h incubation. Using this method, the authors examined the effect ofsample, inoculum, and vessel size on gas production. When a small amount of sampleŽ .100 mg was used, a significant sampling error in gas production was observed

Ž .compared to a larger sample 200 mg .Ž .Although Pell and Schofield 1993 reported no difference in total gas production

when smaller or larger amounts of inoculum were used, the smaller inoculum appearedŽ .to have a slightly longer lag time than a larger inoculum. Hidayat et al. 1993 showed

that increasing bacterial density resulted in an increase in the rate of gas production inthe first 24 h, although the total gas production was not affected. This computerised

Ž .system of Pell and Schofield 1993 allows frequent recording of gas production.However, the pressure sensor used in this system has the capacity of 45 ml gas and,

Ž .therefore, it does not seem to allow incubation of a larger amount )100 mg ofsample. Incubation of larger amount of sample appears to be necessary as a small

Žsample resulted in significant sampling error in gas production Pell and Schofield,.1993 .

Ž .The method of Theodorou et al. 1994 also uses a pressure transducer to measure gasreleased upon fermentation of feeds. A hypodermic needle connected to a pressuretransducer with digital readout meter is inserted manually through a butyl rubber stopperto measure head-space gas pressure. Gas volume is determined by recording the volumeof gas displaced into the syringe barrel and on withdrawal of the syringe plunger untilthe head-space gas pressure returned to ambient pressure. A modified method of

Ž . Ž .Theodorou et al. 1994 was used by Williams et al. 1996 in which 0.8 g of feed wasincubated in a 100 ml serum bottle containing 75 ml of medium and 5 ml rumen liquor.

Ž .The method of Theodorou et al. 1994 requires only one pressure transducer while inŽ .the technique described by Pell and Schofield 1993 each incubation bottle has its own

Ž .pressure sensor. The advantage of the method of Theodorou et al. 1994 is that a larger

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number of samples can be handled at a time at a lower cost. However, mediumpreparation is complicated by the large number of ingredients used and the method alsoinvolves aseptic technique which makes initial preparation cumbersome.

Ž . Ž .In the method of Theodorou et al. 1994 and Pell and Schofield 1993 , theaccumulated pressure is not released and this may negatively affect microbial fermenta-tion. In addition, increase in pressure may change solubility of gases in the medium,which may cause error in gas measurement. Using the pressure transducer methodŽ . Ž .Theodorou et al., 1994 , Lowman et al. 1997 reported a significant influence ofreading interval on gas production.

Ž .Cone et al. 1996 used a pressure transducer in combination with an electric gasrelease valve. Gas measurement is carried out with an electronic control unit connectedto a personal computer. In this method fermentation bottles are closed with screw capson which electronic pressure meters are mounted and connected to an electronic valve.When gas pressure reaches the pre-set upper value, the electronic valve opens, allowing

Ž .the pressure to fall back to the pre-set lower value atmospheric pressure resulting inŽclosing of the valve. Every valve-opening represents a known amount of gas set at

.approx. 0.7 ml and the number of valve openings are recorded in a data logger. Themethod allows frequent recording of gas production, which offers an advantage overmanual recording of gas volume.

A close agreement between the gas experimentally observed and stoichiometricallyŽcalculated has been established by different workers Beuvink and Spoelstra, 1992;

.Blummel and Ørskov, 1993; Makkar et al., 1995b for the method of Menke et al.¨Ž .1979 where the samples are incubated in syringes and the gas produced on fermenta-tion is at the same pressure as that of the atmosphere. It appears there is a need toestablish these relationships for pressure transducer methods.

5. Applicability and significant achievements using gas methods

5.1. In ÕiÕo organic matter digestibility, prediction of metabolisable energy and rumenprotein degradability

Regression equations have been used to predict digestibility from chemical composi-Ž .tion McLeod and Minson, 1971; Van Soest, 1994 . Using the in vitro gas measurement

Ž . Žand chemical composition in multiple regression equation, Menke et al. 1979 data. Ž .from 89 experiments , found a high precision Rs0.98; S.D.s0.25 in prediction of in

vivo organic matter digestibility. This group further extended this work by examiningdata from about 400 in vitro experiments and used a correlative approach to predict themetabolizable energy content of feed by in vitro gas volume measurements and chemicalconstituents. Based on these extensive studies, the authors concluded that the predictionof metabolizable energy is more accurate when based on gas and chemical constituentsmeasurements as compared to calculations based on chemical constituents only. These

Ž .studies have been reviewed by Steingass and Menke 1986 and Menke and SteingassŽ . Ž1988 . Other workers Chenost et al., 1997; Fernandez-Rivera, 1997; Macheboeuf et al.,

.1997; Romney et al., 1997 have also reported significant correlation between in vitro

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gas measurement and in vivo digestibility. Inclusion of crude protein content to gasŽvolume improved precision of prediction of in vivo organic matter digestibility Chenost

.et al., 1997; Macheboeuf and Jestin, 1997; Macheboeuf et al., 1997 .Gas measurement was also employed for evaluation of the interaction between basal

and supplementary diets by incubating both basal diet and supplementary diet separatelyas well as in combination and monitoring gas production at different hours of incubation

Ž .using the pressure transducer system Sampath et al., 1995 . This will indicate theavailability of readily fermentable material as a ready energy source which willstimulate the activity of the rumen microorganisms which in turn would accelerate thedigestion of roughages. These workers, by incubating the basal diet and the supplement,observed a positive interaction in gas production in the early hours of incubation, whichaccording to the authors can be an approach to study the synergetic effects ofsupplementation. However, it must be pointed out that measurement of gas only, could

Ž .lead to misleading results see Section 6 .Ž . ŽAiple et al. 1996 compared three laboratory methods enzymatic, crude nutrient and

. Žgas measuring technique as predictors of net energy as estimated by equations based.on in vivo digestibility content feeds and found that for predicting net energy content of

individual feeds, the gas method was superior to the other two methods.Ž .Raab et al. 1983 developed a method to determine the rumen degradability of

Ž .proteins. This method is based on the in vitro gas method of Menke et al. 1979 andmeasurement of ammonia. The feed being studied is incubated in the absence andpresence of a carbohydrate source. A negative linear relationship between gas produc-tion and in vitro ammonia concentration in the medium is obtained which allows themathematical calculation of ammonia–nitrogen released when no fermentable carbo-hydrate was available. The net ammonia–nitrogen released from the feed is calculated

Žby subtracting the ammonia–nitrogen of the blank rumen liquor without the substrate.incubated from the mathematically derived value and this net ammonia–nitrogen

released is divided by the feed nitrogen in the system for determination of in vitrodegradability of feed protein. This method was used for various agro-industrial by

Ž . Ž .products Krishna and Gunther, 1987 and oil seed cakes Krishnamoorthy et al., 1990 ,¨and the values obtained in this method were in close agreement with those reported

Ž . Ž .using other methods Raab et al., 1983 . Recently, Getachew et al. 1997 modified thismethod for quantification of in vitro degradability of protein in low quality roughages.

5.2. Kinetics of fermentation and mathematical description of gas production profiles

Since the utilisation of roughages is largely dependent upon microbial degradationwithin the rumen, description of roughages in terms of their degradation characteristics

Ž .would provide a useful basis for their evaluation Hovell et al., 1986 . Kinetics offermentation feedstuffs can be determined from fermentative gas and the gas releasedfrom buffering of the SCFAs. Kinetics of gas production is dependent on the relativeproportions of soluble, insoluble but degradable, and undegradable particles of the feed.Mathematical descriptions of gas production profiles allows analysis of data, evaluationof substrate- and media-related differences, and fermentability of soluble and slowlyfermentable components of feeds. Various models have been used to describe gas

( )G. Getachew et al.rAnimal Feed Science and Technology 72 1998 261–281274

Ž .production profiles. The exponential model Ørskov and McDonald, 1979 is widelyused in ruminant feed evaluation to describe degradation kinetics as measured with thenylon bag technique, but the model has also been used to describe kinetics of gas

Ž .production data Blummel et al., 1990; Siaw et al., 1993; Khazaal et al., 1993a . This¨model is based on first-order kinetics, assuming a constant fractional rate fermentationŽ .Groot et al., 1996 . Since some feed particles ferment at different rates, the assumption

Ž .in exponential model is not universally valid. Beuvink and Kogut 1993 evaluatedvarious curve fitting models and reported that the exponential model resulted in larger

Ž .residual mean squares as compared to sigmoidal models. Groot et al. 1996 introducedthree-phasic model which differentiates soluble, insoluble but fermentable, and micro-bial turnover. Conceptually, this model should provide useful data, however, it requiressophisticated equipment to record gas production at different time of incubation.Furthermore, the model performed poorly when recently used in the prediction of

Ž .voluntary feed intake of 24 roughages from Ethiopia Blummel et al., 1998 .¨

5.3. Prediction of Õoluntary intake

The main constraint to the utilisation of roughages by ruminants is voluntary feedŽ .intake Hovell et al., 1986; Minson, 1990 so prediction of feed intake, particularly of

Žfibrous roughage, is one of the important aspects of ruminant nutrition Ørskov and.Ryle, 1990 . In vitro gas production has been used to predict dry matter intake. Various

Žworkers Blummel and Becker, 1997; Chenost et al., 1997; Fernandez-Rivera, 1997;¨.Romney et al., 1997 have reported significant correlation between in vitro gas produc-

tion and dry matter intake. Forage cell walls have considerable influence on voluntaryŽ .feed intake through rumen fill mechanism Van Soest, 1994 . Gas production from

extracted neutral detergent fibre was shown to be better correlated to voluntary feedintake than the values obtained from the incubation of whole roughage. The use ofvarious models for intake prediction was investigated and it currently appears that

Ž .combination of gas volume measurements 4–8 h with concomitant determination ofŽ .the amount of substrate degraded ) 24 h is superior to the models based on kinetics

Ž .of gas production only Blummel et al., 1997a,b; Blummel and Becker, 1997 . The in¨ ¨Ž .vitro gas production from NDF explained more 82% vs. 75% of the variation in dry

Ž .matter intake than gas production from whole roughage Blummel and Becker, 1997 .¨

5.4. EÕaluation of anti-nutritiÕe factors

In vitro gas methods have several advantages over the in sacco or other in vitromethods which are based on gravimetric determination of residues to study the action ofanti-nutritive factors. The latter techniques based on gravimetric determination ofresidues, lead to the solubilization of anti-nutritional factors, thus, making no contribu-tion to energy production in the system but being measured as dry matter digestibility.This could lead to misleading conclusions, whereas in the in vitro gas method the effectsof anti-nutritional factors on rumen fermentation are reflected in the gas production.Furthermore, the in vitro gas method is also expected to be better than chemical methods

( )G. Getachew et al.rAnimal Feed Science and Technology 72 1998 261–281 275

for quantification of anti-nutritional factors. Generally, chemical methods measureanti-nutritional factors related to one or another standard. The nature of the standard and,hence, their biological effects could be different from the anti-nutritional factors presentin feeds. This is particularly true for heterogeneous classes of anti-nutritional compoundssuch as tannins, saponins, alkaloids, etc. In addition, chemical assays do not indicate thepossible interaction of different anti-nutritional factors that take place during fermenta-tion.

The gas method has been used to assess the actions of anti-nutritive factors on rumenŽ .fermentation of Mediterranean browses Khazaal et al., 1994 and African browses

Ž .Siaw et al., 1993; Nsahlai et al., 1994; Bonsi et al., 1995 . Recently, we used the gasmethod to study interactions of tannins and saponins and to determine their effects on

Ž 15efficiency of microbial protein synthesis expressed as the ratio of N incorporation per. Ž .unit of SCFA production Makkar et al., 1995a , and found that the effects of

simultaneous presence of tannins and saponins on rumen fermentation were additive anddid not counteract effects of either tannins or saponins. Concentration of phenolics wererelated negatively but more significantly to gas production than to dry matter degrada-

Ž . Ž .tion in nylon bag Khazaal et al., 1993b . Longland et al. 1995 reported a significantinverse relationship between gas accumulation at different time of incubation and tannincontents of feed samples. In a study which compared the gas production and nylon bagtechniques for assessing the effect of phenolic related anti-nutritive factors on degrad-

Žability of feed, gas measurement was found to be more efficient than nylon bag Khazaal.et al., 1994 .

Various commercially available chemicals which have an affinity to tannins wereŽevaluated for their binding capacity of tannins using the gas method Makkar et al.,

.1995b; Khazaal et al., 1996 , so as to study the potential adverse effects of tannins onŽrumen fermentation and improve the digestibility of tannin-rich feeds Khazaal et al.,

.1994; Khazaal and Ørskov, 1994; Makkar et al., 1995b . Recently, Makkar and BeckerŽ .1996 developed a bioassay for tannins which is based on incubation of a feed in the

Ž .absence and presence of polyethylene glycol 6000 PEG 6000 , the most effectiveŽ .tannin-complexing agent Makkar et al., 1995b in in vitro gas method. The PEG 6000

binds to tannins forming inert PEG-tannin complexes which results in increase in gasproduction. The higher the biological activity of tannins on rumen microbes, the higherthe increase in gas production in presence of PEG. Using these gas volume and the

Ž . Ž .equations of Menke and Steingass 1988 , the metabolizable energy ME and organicŽ .matter digestibility OMD were quantified, which allowed expression of tannin activity

in terms of their potential to decrease ME and OMD in tannin-rich feeds.

6. Suggested modification in the use of in vitro gas method

Ž .It has been pointed out Sections 2 and 3 that gas production is a reflection ofgeneration of SCFA. The other nutritionally important fermentation product is microbialbiomass. Even though both these products are linked by ATP production, it is wellknown that varying amounts of microbial biomass can be produced per unit of ATPŽY . Ž .ATP Hespell and Bryant, 1979; Harrison and McAllan, 1980; Demeyer, 1981 . This

( )G. Getachew et al.rAnimal Feed Science and Technology 72 1998 261–281276

Žcan impose an inverse relationship upon SCFA and microbial biomass yield Leng,.1993 . It was recently shown that this relationship applies also for gas production in

vitro and microbial biomass yield when both variables were related to a unit of substrateŽ . Ž .fermented see, for review, Blummel et al., 1997c . Blummel and Bullerdick 1997¨ ¨

suggested to complement the in vitro gas production with residue determination inevaluation of the nutritive value of feeds. In this approach, the residue determinationreveals how much substrate is used in the fermentation and the gas measurement reflectshow much of this fermented substrate is converted into the SCFA and gases. The ratio ofsubstrate truly degraded to gas volume produced, defined as ‘partitioning factors’ wasfound to be valuable in predicting voluntary feed intake. Partitioning factor can vary

Ž . Ydepending on molar proportions SCFA acetate to propionate ratio and ATP. Highpropionate production would lead to higher partitioning factor as compared to acetateproduction. The production of microbial biomass per unit of ATP may vary from 10 to

Ž . Y32 mg Van Soest, 1994 . At similar ATP, proportionally higher propionate leads toŽhigher partitioning factor as compared to higher acetate production 3.0 vs. 2.8 and 3.6

Y . Ž .vs. 3.3 at ATP of 10 and 20, respectively Table 1 .The stoichiometrical relationship between variation in YATP, gas production and

Ž .partitioning factor can be illustrated Fig. 1 from incubation of 200 mg substrate usingŽ .the gas method of Menke et al. 1979 , assuming true degradability of 65%, i.e., 130 mg

would be fermented to SCFA, gases and microbial cells. Using the data for proportion-ally high propionate production in Table 1 and assuming YATP of 10, about 22% of thefermented substrate would be incorporated in to microbial cells which would yield about

Ž . Y28.6 mg microbes 101.4 mg substrate would be used for acids and gases . When ATPof 20 was considered, about 35.6% of the fermented substrate used for microbial cellswhich would yield about 46.3 mg microbes leaving 83.7 mg for the acids and the gases.From the substrate and fermentation product relationship outlined by Blummel et al.¨Ž . Y1997c , about 43.3 and 35.5 ml of gas would be produced at ATP of 10 and 20,respectively. In accordance with the accepted concept of ruminant nutrition to select

Ž .substrates for high microbial biomass production Leng, 1993; Van Soest, 1994 ,

Y Ž .Fig. 1. Stoichiometrical relationships between ATP yields, gas production and partitioning factors PF .ŽCalculations are for 200 mg of substrate incubated having 65% true degradability 130 mg substrate truly

.degraded . Short chain fatty acids proportions are as stated in Table 1.

( )G. Getachew et al.rAnimal Feed Science and Technology 72 1998 261–281 277

Ž .Blummel et al. 1997c suggested selection of roughages for high substrate degradabil-¨ity, but proportionally low gas production.

Ž .For conventional feeds roughages , the ratio of substrate truly degraded to gasy1 Ž .volume ranges from 2.74–4.65 mg ml Blummel et al., 1997c . Unfortunately,¨

partitioning factors can not be determined by the approach of residue determinationŽ .using neutral detergent solution Blummel et al., 1997b for tannin-rich forages due to¨

Ž .various artifacts Makkar et al., 1997a . The partitioning factor as high as 9.93 has beenŽ .recorded for a tannin rich leaf sample Dichostachys cinerea which is well beyond the

Ž . Ž .theoretical 2.75–4.41 or previously observed 2.74–4.65 ranges of partitioning fac-Ž .tors. This high partitioning factor of 9.93 could be due to: i leaching of tannins from

the feed during fermentation, contributing to the dry matter loss but without contributingŽ . Ž . Ž .to the gas, and ii inhibition of cell solubles by tannins, or combination of i and ii . In

tannin-containing feedstuffs, gas measurements should be combined with microbial massŽ .determination using either internal e.g., purines, 2,6-diaminopimelic acid or external

Ž 15 32 . Ž .markers e.g., N or P incorporation Makkar et al., 1997b .

7. Conclusion

Several systems are available for measuring gas evolved as a result of fermentation.Although the computerised systems have an advantage over the manual recording in thatgas can be measured without any interruption, it requires higher inputs and thereforeseems to be difficult to adopt as a routine method of feed evaluation. The pressure

Ž .transducer method such as Theodorou et al., 1994 is cheaper compared to thecomputerised system and seems to be promising as it offers flexibility regarding sample

Ž .number and sample size. The method of Menke et al. 1979 is relatively simple tohandle, and does not require sophisticated equipment. We have outlined that gasmeasurement alone is not satisfactory and needs to be complemented by residuedetermination. This should be kept in mind in selecting a gas method. Currently,non-automated methods like those based on pressure transducers and gas syringesappear to be better suited as these are cheaper and more robust, besides being capable ofaccommodating residue determination.

Acknowledgements

Ž .G. Getachew is grateful to the DAAD Deutscher Akademischer Austauschdienst forthe financial assistance during the course of this work.

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