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Animal Feed Science and Technology

123124 (2005) 197210

Estimation of direct and indirect gas production


in syringes: A tool to estimate short chain
fatty acid production that requires minimal
laboratory facilities
V. Fievez a, , O.J. Babayemi b , D. Demeyer a
a

Department of Animal Production, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium
b Department of Animal Science, University of Ibadan, Nigeria

Abstract
A technique is proposed that involves volumetric measurement of gas production in phosphate and
bicarbonate buffered in vitro incubations in syringes. Gas production is measured before and after
CO2 absorption by injection of NaOH (10 M) at the end of the incubation. After correction for the
amount of CO3 2 in the rumen inoculum, measurements allow calculation of direct and indirect CO2
production, reflecting metabolism and acidification, respectively, as well as methane (CH4 ) production. This allows estimation of approximate short chain fatty acid (SCFA) production, energy lost as
CH4 and it might provide information on the ratio of the glucogenic to lipogenic SCFA produced.
Results from in vitro gas production tests in syringes with straw (0.325 g), and eight different tropical
seeds (0.125 g) as protein supplements, were used to evaluate whether such calculations could indeed
predict measured amounts of SCFA and CH4 produced, as determined by gas chromatography. The
concordance correlation coefficient (CCC), representing both precision () and accuracy (Cb ) has
been used to assess effectiveness of this reproducibility. It was found that indirect CO2 production
equimolarly reflects total SCFA production (CCC = 0.79; = 0.86; Cb = 0.93), whereas absorption of
gaseous CO2 provided an accurate prediction of CH4 produced (CCC = 0.96; = 0.96; Cb = 1.00).
The ratio of CH4 /gasindirect allowed estimation of the ratio of lipogenic to glucogenic SCFA produced
(R2 = 0.73; R.S.D. = 0.20). However, the current approach did not allow accurate quantification of
proportions of individual SCFA produced (CCC = 0.19; = 0.83; Cb = 0.23 for propionate propor-

Abbreviations: 2Hr, hydrogen recovery; Ac, acetate; But, butyrate; CCC, concordance correlation coefficient;
Pr, propionate; SCFA, short chain fatty acids
Corresponding author. Tel.: +32 9 264 90 02; fax: +32 9 264 90 99.
E-mail address: Veerle.Fievez@Ugent.be (V. Fievez).
0377-8401/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2005.05.001

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V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

tions). The potential of a simple gas production technique to provide information on feed quality is
illustrated, with the only apparatus required being gas tight syringes and incubation facilities.
2005 Elsevier B.V. All rights reserved.
Keywords: In vitro gas production; Fermentation products; Methane; Concordance correlation

1. Introduction
Although gases produced during rumen fermentation are waste products and of no nutritive value to the ruminant, gas production tests are used routinely in feed research as gas
volumes are related to both the extent and rate of substrate degradation (e.g., Blummel et al.,
1997). Gas produced in vitro using a bicarbonate buffered system has a dual origin. Firstly,
CO2 and CH4 that are produced as a result of fermentation (i.e., direct gas production;
gasdirect ) and, secondly, CO2 is produced upon buffering of short chain fatty acids (SCFA)
generated (i.e., indirect gas production; gasindirect ; Blummel et al., 1997). Molar production of the latter equals molar SCFA production, provided that the incubation medium is
saturated with CO2 and the buffer is entirely based on bicarbonate (Blummel et al., 1997).
Moreover, stoichiometric relationships between SCFA, CO2 and CH4 (Wolin, 1960), indicated that direct gas production is the result of substrate fermentation to acetate (Ac) and
butyrate (But), but not to propionate (Pr). Accordingly, the following molar balances could
be derived (Blummel et al., 1997):
gasdirect = Ac + 2 But

(1)

gasindirect = total SCFA

(2)

Cone and van Gelder (1999) argued that the presence of protein in feeds might distort
the former relations, as Eq. (1) only considers carbohydrate fermentation and relation (2)
might be impaired by binding of H+ by NH3 that is released during protein degradation.
Nevertheless, Blummel et al. (1999 and 2003) confirmed these relationships for a wide range
of dairy compound feeds and forages, and suggested that protein contents below 400 g/kg
did not affect the established relations between SCFA production and gas volumes.
Clearly, based on fermentation stoichiometry, the distinction between direct and indirect
gas production could allow for immediate estimation of the molar productions of SCFA
from gasindirect . As the level of SCFA is an indicator of the energy value of diets, prediction of SCFA from in vitro gas measurements might be useful under circumstances where
laboratories lack gas chromatographic equipment.
An in vitro experiment was designed to serve the dual purpose of evaluating different
tropical seeds for their use as protein supplements to feeds of low nutritive value and,
to test the ability of gas production measurements in combination with stoichiometric
calculations to provide accurate information on the nutritive value of tropical ruminant
feeds. The former objective has been discussed elsewhere (Babayemi et al., 2004b), and
the latter is the focus here. Bicarbonate and phosphate buffered incubations were combined
in an attempt to estimate both direct and indirect gas production. With gas produced in
the phosphate buffered systems (gasphosphate ) assumed to be of fermentative origin only,

V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

199

we then evaluated whether gasindirect , calculated as gasbicarbonate gasphosphate , could


reproduce results of direct SCFA measurements by gas chromatography.
Moreover, when aiming at optimizing rumen fermentation, insight into the extent of energy losses as CH4 , is of extreme importance. Accordingly, a second aim of this experiment
was to evaluate whether CH4 production could be estimated accurately from gas volume
measurements after CO2 absorption at alkaline pH. For this purpose, gas volume readings
from incubations in syringes were compared to CH4 quantified by gas chromatography in
gas samples collected from incubations in gastight culture flasks.

2. Materials and methods


2.1. Seed collection
Mature seeds of Albizia saman, Albizia lebbeck, Albizia rhizonse, Leucaena leucocephala, Tephrosia candida, Tephrosia bracteolata, Lablab purpureus and Canavalia
ensiformis were harvested from different places, but within a radius of 15 km from Ibadan
(7 20N, 3 54E), Nigeria, with at least ten stands of seed containing trees sampled per
species. The legume trees, except A. saman, were sampled at the seed production site of
the Federal Ministry of Science and Technology (Moore Plantation); Tephrosia species
from the International Institute of Tropical Agriculture, while the other Albizia species,
L. leucocephala and L. purpureus and C. ensiformis were collected from the Teaching and
Research Farm of the University of Ibadan. Annual rainfall ranged from 1150 to 1500 mm
and temperatures during the periods of sampling in January 2004 were between 24 C
and 34 C.
2.2. Substrate
Wheat straw and seeds were milled in a Wiley mill (M100AN05828, T. Peppink &
Zn, Amsterdam, The Netherlands) to pass a 1 mm sieve and stored at room temperature.
Some chemical characteristics are shown in Table 1. In addition, qualitative evaluation
based on surfactant properties, and formation of blueviolet to dark green complexes with
ferric ions, revealed that in some of the seeds (i.e., A. lebbeck and A. rhizonse), there were
saponins as well as hydrolysable (i.e., L. leucocephala) or condensed (i.e., T. candida and
T. bracteolata) tannins (Babayemi et al., 2004a).
2.3. Chemical analysis
Chemical analysis was in duplicate. The moisture content was ascertained by drying
at 105 C for 24 h to constant weight. Crude protein was determined by a standard
Kjeldahl method (EC, 1993), using digestion equipment (Kjeldatherm System KT 40,
Gerhart Laboratory Instruments, Bonn, Germany) and associated titration apparatus (T
110-TR160-TA10-TM120, Schott-Gerate GmbH, Hofheim, Germany). Ether extract was
assessed, after hydrolysis (3 M HCl), by refluxing with petroleum ether in a soxhlet
system (Isopad Isomantle-HAT 1043, Borehamwood, Hertfordshire, UK) according to

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200

Table 1
Dry matter (g/100 g fresh material) and proximate chemical composition (g/100 g DM) of incubated tropical seeds
and straw
DMa

OMa

CPa

NDFa

EEa

Multipurpose trees
Albizia saman
Albizia lebbeck
Albizia rhizonse
Leucaena leucocephala

93.6
92.1
90.4
89.1

97.0
95.4
95.2
95.1

29.3
32.5
33.4
27.6

26.0
24.0
27.4
43.3

10.0
9.4
5.9
7.8

Shrubs
Tephrosia candida
Tephrosia bracteolate

92.6
93.5

94.8
94.6

38.2
38.1

27.0
22.1

15.3
17.0

Pulses
Lablab purpureus
Canavalia ensiformis

90.4
88.9

96.5
97.0

24.8
25.3

46.9
NDb

2.0
3.7

Straw

89.0

93.2

4.2

71.6

4.4

a
b

Dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fibre (NDF), ether extract (EE).
Not detectable due to clogging of the seed in the Fibertec apparatus.

International Standards Organisation (1973) method 1444. Organic matter content was
obtained as the difference from ash, determined according to EEC (1971). Neutral detergent
fibre (NDF) was expressed without residual ash (Van Soest et al., 1991).
2.4. Rumen contents
Rumen contents were obtained before the morning feeding from two fistulated wethers
(approximately 70 kg) fed at maintenance on a hay/pelleted grain based concentrate (7 MJ
NEL/kg DM) diet (650/350, w/w on DM basis) twice daily at 09:00 and 17:00 h. Only hay
was fed during the last 24 h before the collection of rumen liquor in order to exhaust rumen
NH3 concentrations in the inoculum.
2.5. Buffer
Per litre of distilled water, the phosphate buffer contained 28.8 g Na2 HPO4 12H2 O and
6.1 g NaH2 PO4 H2 O (Fievez et al., 2003). To avoid addition of extra (i.e., not desired)
N, ammonia bicarbonate was omitted from preparation of the bicarbonate buffer, which
consisted entirely of sodium bicarbonate (39.2 g/l of NaHCO3 ; Blummel et al., 2003). The
pH of both buffers was adjusted to 7.0 and buffers were gassed with CO2 for 1.5 h.
2.6. In vitro incubation
The experiment consisted of three types of in vitro incubations: in gastight culture
flasks using a phosphate buffer, or in syringes using either a phosphate or bicarbonate
buffer. Incubations in gastight culture flasks, and in syringes, were completed in triplicate
flasks/syringes on three different days, as summarised below, according to Van Nevel and

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201

Demeyer (1977) and Demeyer et al. (1988), respectively. The incubated substrate consisted
of 0.375 g of straw and 0.125 g of each of the eight seeds. These amounts are considerably
higher than used in the standard Hohenheim system (Menke et al., 1979), because of
the relatively low degradability of straw and the limited incubation time of 24 h. Under
continuous CO2 flushing, rumen contents were filtered through a 1 mm sieve, mixed 1:4
(v/v) with buffer and introduced (25 ml of the mixture) into 200 ml gastight culture flasks or
100 ml gastight plastic syringes (Egilabo, Kontich, Belgium). The headspace of the culture
flasks was flushed six times with CO2 (99.99% purity, Air Liquide, Aalter, Belgium),
whereas, all gas was expelled from the syringes, after which their lower end was closed.
Culture flasks were incubated in a shaking waterbath at 39 C for 24 h, while syringes were
placed in an incubator at 39 C for 24 h. Volumes of the gas produced were read after 2, 4,
6, 8, 10, 12 and 24 h of incubation, and syringes were turned carefully in order to ensure
complete mixing of the incubated contents. If plungers of the syringes with bicarbonate
buffer were above the 85 ml level in the evening, volumes were recorded and the plungers
pushed back. Syringes with phosphate buffer never reached these high volumes.
2.7. Determinations in non-incubated samples
For each incubation run, SCFA concentrations in rumen inoculum were determined
in non-incubated, immediately acidified (H2 SO4 , 10 M, final concentration 2%) samples,
after centrifugation (10 min at 22,000 g, MSE, Amsterdam, The Netherlands) and filtering
(Shimadzu GC-14A, s Hertogenbosch, The Netherlands; Van Nevel and Demeyer, 1977).
As release of CO2 from bicarbonate in the rumen inoculum might impair estimation of
gasdirect in the phosphate buffered incubations, its relevance was evaluated by gas volume
production (gasinoculum ) upon introduction of a 10-fold diluted (v/v) mixture of Ac/Pr/But
(625/230/145, v/v/v which corresponded to molar Ac/Pr/But ratios of 700/200/100) in nonincubated syringes with 5 ml of rumen inoculum and 20 ml of phosphate buffer. The amount
of acid introduced to the syringes corresponded to the volume causing a pH decline to 6.25,
as determined by a concomitant pH measurement (PHM Standard pH meter, Radiometer,
Copenhagen, Denmark) using a mixture of the same inoculum (5 ml) and phosphate buffer
(20 ml). The choice of the latter pH value was based on the average pH obtained after a 24 h
incubation during preliminary runs using the same substrates.
2.8. Determinations after incubation
Immediately after incubation, 1 ml of the gas phase was sampled from the culture flasks
with a gastight syringe and analysed for CH4 and H2 by gas chromatography (FM, Dual
Column GC 700, Avondale, Pennsylvania, USA) according to Van Nevel et al. (1970).
After gas sampling from the culture flasks and recording of the final gas volume of the syringes incubated with bicarbonate buffer, the pH of the incubation liquid was measured. The
sample was then acidified with H2 SO4 (10 M) to a final concentration of 2%, centrifuged
(10 min at 22000 g, MSE, Amsterdam, The Netherlands), filtered, and the filtrate used for
SCFA analysis by gas liquid chromatography (Shimadzu GC-14A, s Hertogenbosch, The
Netherlands; Van Nevel and Demeyer, 1977). To calculate the net amount of SCFA produced, the amount of SCFA in the non-incubated sample [mean S.D. (n = 3): 392 17.7,

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V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

665 24.2, 109 10.8 and 87.9 9.27 for total SCFA concentrations (mol/25 ml rumen
inoculum + buffer) and Ac, Pr and But proportions (mmol/mol total SCFA), respectively]
was subtracted. At the end of syringe incubations with phosphate buffer, and after recording
the final gas volume, the lower end of the syringe was connected to the lower end of another
syringe containing 4.0 ml of NaOH (10 M). NaOH (10 M), which was then introduced from
the latter into the incubated contents, thereby avoiding gas escape. Mixing of the contents
with NaOH allowed absorption of CO2 , with the gas volume remaining in the syringe considered to be CH4 (Demeyer et al., 1988). Five millilitres of H2 SO4 (10 M) was then added
and contents were centrifuged and pre-treated for SCFA determination and calculation,
as described above. Hydrogen recovery (2Hr) was calculated as (2 Pr + 2 But + 4 CH4 )/(2
Ac + Pr + 4 But) with Ac, Pr, But and CH4 expressed as net molar production rates (Marty
and Demeyer, 1973).
2.9. Statistical analysis
All statistical analyses used SPSS 11.0 (SPSS software for Windows, release 11.0., SPSS,
Inc., Chicago, IL, USA).
Effects on total net SCFA production and Ac, Pr and But proportions, induced by
differences in incubation environment (i.e., culture flask versus syringe and phosphate
versus bicarbonate buffer) and seed supplementation were evaluated using the model:
Yijk = + Fi + Pj + Ck + ijk , where Yijk is the individual observation, the overall mean, Fi
the effect of incubation recipient (i.e., culture flask versus syringe), Pj the effect of incubation buffer (i.e., phosphate versus bicarbonate buffer), Sk the effect of seed and ijk is the
residual error.
The concordance correlation coefficient (CCC) was introduced by Lin (1989) to assess
agreement in continuous data. This CCC often has been used to evaluate the extent of
agreement between two distinct methods of measuring the same response variable. In the
current experiment, this reproducibility index was used to determine whether calculations
based on gas volume measurements could reproduce SCFA and CH4 results obtained by
gas chromatography. This evaluation has been completed using individual observations
(n = 24) as well as averages per seed (n = 8). The CCC is calculated as: c = Cb with c
the concordance correlation coefficient, the Pearson correlation coefficient and Cb the
bias correction factor, which is calculated as: Cb = 2o p /(o2 + p2 + (o p )2 ) with
o , o , p and p the S.D. and mean of the values determined by gas chromatography
and calculated from gas volumes, respectively. The Pearson correlation coefficient reflects
precision (i.e., degree to which individual calculations from gas volumes cluster about
the regression line). The bias correction factor reflects accuracy (i.e., degree to which the
regression line adheres to the 45 line through the origin (i.e., the concordance line). The
CCC reflects the degree to which individual predictions adhere to the concordance line. The
bias correction factor consists of a scale shift (= o / p ) and a location shift relative to the
scale (=[o p ]/[ o p ]1/2 ) as per Lin (1989) and St-Pierre (2003). As there is, as yet, no
literature providing a descriptive scale for the degree of agreement based on the CCC, the
Landis and Koch (1977) scale has been used here to describe the degree of concordance,
with: 0.210.40 being Fair; 0.410.60 being Moderate; 0.610.80 being Substantial;
and 0.811.00 being Almost perfect.

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203

Linear regression analysis was used to determine regression coefficients of regression


lines, their standard error and significance. When regression coefficients were not statistically significant, they were removed from the equations. In none of the regressions did
the intercept significantly differ from zero, thereby forcing the regression line through the
origin. Accordingly, R2 measured the proportion of variability explained by the regression
of the dependent variable about the origin. As the statistical program used here does not
redefine the R2 to an uncentered one, R2 estimates cannot be compared to R2 values for
models including an intercept, and so are of poor statistical value. Hence, these values were
not reported.

3. Results and discussion


Study objectives required SCFA production and fermentation patterns to be independent
of the incubation environment (i.e., culture flask versus syringe and phosphate versus bicarbonate buffer), as results were combined (i.e., gas production in phosphate and bicarbonate
incubations) or compared (i.e., CH4 production in flasks and syringes). Accordingly, the
effect of the incubation environment has been evaluated and this comparison is in Table 2,
which shows similar total SCFA production and SCFA proportions when samples were
incubated in syringes or culture flasks and using a phosphate or bicarbonate buffer. Averages per seed (n = 3) of total and individual SCFA (Table 2) and gas and CH4 productions
(Table 3) are reported for the information of the reader but will not be discussed in detail,
as this was the emphasis of another paper (Babayemi et al., 2004b).
3.1. Net SCFA production
The validity of stoichiometric gas production calculations, and reproducibility of chromatographically measured SCFA productions by gas measures were assessed by the CCC
and its two components, reflecting precision () and accuracy (Cb ), the latter consisting of
a scale and location shift measure (Table 4).
Molar gas volumes in the phosphate buffered incubations (gasphosphate ) substantially reflected fermentative CO2 and CH4 (gasdirect ), as calculated from Ac and But productions
(Ac + 2 But). Nevertheless, the negative location shift (Table 4), indicates that measured
gas volumes (Y-axis) generally overestimated stoichiometrically calculated volumes (Xaxis, Ac + 2 But) (Fig. 1a). This might be related to the presence of bicarbonate in the
rumen inoculum, from which CO2 would be released upon buffering of SCFA produced.
Indeed, acidification of non-incubated samples (n = 3) to a pH of 6.25 caused release of
277 32 mol of gas (gasinoculum ), which is consistent with the location shift of the regression line versus the concordance line (Fig. 1a). Subtracting gasinoculum from gasphosphate
significantly improved the accuracy of the gasdirect (=Ac + 2 But, Eq. (1)) estimations, as
illustrated by the reduced location shift (Table 4, Fig. 1b). As gasphosphate combined with
gasinoculum measurements allowed a relatively accurate estimate of gasdirect , we further evaluated how well gasindirect , calculated as gasbicarbonate (gasphosphate gasinoculum ), predicted
SCFA production (as derived from Eqs. (1) and (2)). Bicarbonate and phosphate buffered
syringes with the same seed were combined by incubation day for these calculations. Ap-

204

Seed

Total SCFA (mmol/g OM)

Acetate (mmol/mol total SCFA)

Propionate (mmol/mol total SCFA)

Butyrate (mmol/mol total SCFA)

F/Sa

F
P

S
P

S
C

S.E.M.

F
P

S
P

S
C

S.E.M.

F
P

S
P

S
C

S.E.M.

F
P

S
P

S
C

S.E.M.

P/Cb
ALc
ARc
ASc
CEc
LLc
LPc
TBc
TCc

3.55
3.46
4.45
4.68
4.05
4.64
4.72
4.34

3.27
4.28
4.52
4.93
4.14
4.58
4.53
4.55

3.58
4.51
4.58
4.93
4.14
4.74
4.68
4.52

0.34
0.20
0.18
0.21
0.17
0.27
0.23
0.17

685
665
720
679
698
690
680
693

719
706
710
681
698
692
688
702

729
710
713
674
700
693
694
712

10.1
10.1
13.8
9.4
8.0
12.6
6.5
7.3

208
222
153
210
208
168
172
159

191
205
182
217
191
180
184
171

181
209
186
225
207
186
197
181

8.9
13.8
13.3
8.1
9.0
11.9
6.9
7.6

66.4
53.5
99.4
76.9
79.5
106
99.6
101

68.4
65.1
91.3
84.3
85.6
104
90.3
90.6

70.8
69.0
83.4
86.4
74.0
100
85.7
85.1

2.27
10.9
3.71
3.23
1.53
2.81
1.44
2.28

Mean
S.D.

4.24
0.50

4.35
0.50

4.46
0.42

0.22

689
22.8

699
12.8

703
22.1

186
30.0

192
18.0

197
21.0

86.1
20.0

84.2
12.8

82.1
10.4

Statd

F/S
ns

P/C
ns

Seed

F/S
ns

P/C
ns

Seed

F/S
ns

P/C
ns

Seed

***

**

***

F/S
ns

P/C
ns

Seed
***

Incubation performed in culture flask (F) or syringe (S).


Incubation using phosphate (P) vs. bicarbonate (C) buffer.
c Albizia lebbeck (AL), A. rhizonse (AR), A. saman (AS), Canavalia ensiformis (CE), Leucaena leucocephala (LL), Lablab purpureus (LP), Tephrosia candida (TC),
T. bracteolata (TB).
d *** P<0.001; ** P<0.01; ns: not significant; F/S, P/C, Seed: differences in fermentation characteristics provoked by incubation in flask vs. syringe, addition of phosphate
vs. bicarbonate buffer or supplementation of different seeds, respectively.
b

V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

Table 2
Total net production of SCFA and proportions of individual SCFA from straw (0.325 g) and seed supplements (0.125 g) during 24 h incubations in culture flasks or
syringes and buffered by a phosphate or bicarbonate buffer (n = 3 for averages and corresponding standard errors per seed and n = 8 for overall means and corresponding
standard deviations)

Seed

Total gas production (mmol/g OM)

Total CH4 production (mmol/g OM)

Hydrogen recovery (mol/mol)

F/Sa
P/Cb

S
P

S
C

F
P

S
P

F
P

S
P

ALc
ARc
ASc
CEc
LLc
LPc
TBc
TCc

2.93 (0.590)
3.87 (0.419)
4.04 (0.051)
4.45 (0.674)
4.14 (0.278)
4.40 (0.378)
4.25 (0.304)
4.30 (0.242)

5.94 (1.507)
7.88 (0.031)
8.16 (0.274)
9.66 (0.393)
7.55 (0.743)
8.71 (0.181)
9.05 (0.096)
8.09 (0.172)

0.77 (0.044)
0.80 (0.125)
1.18 (0.021)
1.13 (0.094)
1.10 (0.107)
1.25 (0.074)
1.20 (0.004)
1.24 (0.120)

0.83 (0.131)
0.80 (0.018)
1.13 (0.032)
1.08 (0.054)
1.07 (0.194)
1.37 (0.054)
1.21 (0.018)
1.26 (0.046)

0.90 (0.069)
0.93 (0.114)
0.93 (0.057)
0.96 (0.045)
1.00 (0.032)
0.95 (0.042)
0.95 (0.050)
0.99 (0.033)

0.94 (0.015)
0.79 (0.044)
0.91 (0.022)
0.90 (0.049)
0.96 (0.154)
1.01 (0.039)
0.98 (0.013)
0.97 (0.012)

Mean

4.05 (0.489)

8.13 (1.116)

1.08 (0.190)

1.09 (0.198)

0.95 (0.061)

0.94 (0.083)

Incubation in culture flask (F) or syringe (S).


b Incubation using phosphate (P) vs. bicarbonate (C) buffer.
c Albizia lebbeck (AL), A. rhizonse (AR), A. saman (AS), Canavalia ensiformis (CE), Leucaena leucocephala (LL), Lablab purpureus (LP), Tephrosia candida (TC),
T. bracteolata (TB).

V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

Table 3
Total gas and CH4 production and hydrogen recovery in 24 h incubations with straw (0.325 g) and seed supplements (0.125 g) in culture flasks or syringes and buffered
by a phosphate or bicarbonate buffer [mean (S.D.)] (n = 3 for averages and corresponding standard deviations per seed and n = 8 for overall means and corresponding
standard deviation)

205

206
V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210
Fig. 1. Plot of molar SCFA and CH4 productions obtained using gas chromatography (X-axis) vs. productions calculated from molar gas volume measurements (Y-axis)
(regression line: y = intercept + slope(standard error) x).

V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

207

Table 4
Accuracy, bias and correlation measures for the assessment of the reproducibility of molar SCFA and CH4 results
using gas chromatography (reference) by molar gas volume measurements (gas production derivates), with the
evaluation performed on individual observations (n = 24) as well as averages per seed (n = 8)
Gas production derivates

Reference

CCC

Evaluation based on n = 24
gasphosphate
gasphosphate gasinoculum
gasbicarbonate gasphosphate + gasinoculum [A]
gasphosphate at alkaline pH [B]
(0.67[A]1.33[B])/[A]

Ac + 2 But
Ac + 2 But
SCFA
CH4
Pr/SCFA

0.733
0.831
0.657
0.800
0.202

0.838
0.838
0.730
0.804
0.718

Evaluation based on n = 8
gasphosphate
gasphosphate gasinoculum
gasbicarbonate gasphosphate + gasinoculum [A]
gasphosphate at alkaline pH [B]
(0.67[A]1.33[B])/[A]

Ac + 2 But
Ac + 2 But
SCFA
CH4
Pr/SCFA

0.825
0.936
0.793
0.955
0.194

0.937
0.937
0.859
0.956
0.832

Cb

Shift
Scale

Location

0.876
0.992
0.899
0.997
0.282

0.879
0.879
0.629
0.917
0.180

0.518
0.023
0.065
0.039
1.17

0.881
0.998
0.934
0.999
0.234

0.983
0.983
0.699
0.963
0.211

0.520
0.051
0.104
0.031
1.90

plication of the CCC evaluation approach (St-Pierre, 2003) to the dataset with individual
measurements (n = 24) showed gasindirect and SCFA measured by gas chromatography are
concordant, but lack some precision (Table 4). Most probably, differences between the three
repetitions of the same seed, observed for the SCFA measured by gas chromatography, were
too small to be precisely reflected in gas volumes, which are read with a precision of 0.5 ml
from the graduated syringes. Indeed, the lack of precision could be largely overcome if
estimations were based on average gas and SCFA productions (Table 4, Fig. 1c). It is clear
that this approach allows a relatively accurate estimation of SCFA production, which, on
average, differed by 6.4 5.1% from SCFA determined by gas chromatography.
3.2. Methane production and SCFA proportions
As our gas syringes did not allow accurate removal of gas samples for GC analysis,
gas was sampled from concomitant incubations in culture flasks and analysed for CH4 ,
H2 , CO2 and N2 + O2 . Gas volumes, after absorption of CO2 , almost perfectly reproduced
CH4 measurements by gas chromatography (Table 4, Fig. 1d). When based on individual
measurements (n = 24), relative differences between the two methods were 8.1 6.8%.
Based on the means of the three repetitions per seed (n = 8), the gas volume and gas chromatography approach only differed by 3.5 2.7%. It should be stressed, however, that this
approach requires complete gas collection, without the necessity to push back the plunger,
as a preliminary experiment showed CH4 to total gas ratios were not constant throughout the
incubation period (data not shown). Besides being an estimate of the relative energy loss during fermentation, CH4 relative to gasindirect might provide insight into the ratio of lipogenic
(i.e., Ac + But) to glucogenic (i.e., Pr) SCFA produced, as suggested from their good
relationship [(Ac + But)/Pr = 2.32(0.477) + 5.88(1.51) CH4 /gasindirect ; R2 = 0.73; R.S.D. =
0.20]. The ratio of lipogenic to glucogenic SCFA is of high importance for optimised diet

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formulation, as all nutrients are readily exchangeable for animal labour and maintenance, but
not for lactation or growth (Baldwin and Donavan, 2000). Hence, more detailed insight into
the fermentation pattern (i.e., proportions of individual SCFA, is highly relevant in ruminant
nutrition. Theoretically, the currently proposed approach in which gasinoculum , gasphosphate ,
gasbicarbonate and CH4 are concomitantly quantified should allow direct quantification of
individual SCFA, as based on rumen stoichiometry, which now could be summarised as:
gasphosphate gasinoculum = gasdirect = Ac + 2 But

(3)

gasbicarbonate gasphosphate + gasinoculum = gasindirect


= total SCFA Ac + Pr + But

(4)

CH4 = 0.5 Ac 0.25 Pr + 0.5 But

(5)

Eq. (5) was derived directly from the 2Hr [(2 Pr + 2 But + 4 CH4 )/(2 Ac + Pr + 4 But);
Marty and Demeyer, 1973], assuming a completely balanced (2Hr = 1 mol/mol) metabolic
H2 transfer within the rumen fermentation reactions, causing production of Ac, Pr, But
and CH4 . With total SCFA assumed to be mainly the sum of Ac, Pr and But productions (Eq. (4)), molar Pr proportions (mmol/mol) should equal (0.67 gasindirect 1.33
CH4 )1000/gasindirect as derived from the stoichiometric relations (3)(5). Although there
is some correlation between Pr proportions, as determined by the latter equation and by
the GC methodology (rpearson = 0.80), the accuracy is very poor (Table 4). Similarly, Eqs.
(3)(5) allowed calculation of Ac and But proportions. As for Pr proportions, the latter were
correlated to the chromatographically measured proportions, but did not allow an accurate
estimate of these measurements (data not shown). Accumulation of residual errors of Eqs.
(3)(5) leads to erroneous estimation of individual SCFA productions, and these might be
partly due to the assumption of perfect stoichiometric balances (2Hr = 1 mol/mol) which,
in practice, is not the case, as suggested by the 2Hr as calculated from the CH4 and SCFA
measurements (Table 3). Indeed, the Cb increased from 0.23 (Table 4) to 0.50 when using
these actual 2Hr values (Table 3), instead of a 2Hr of 1. The latter consideration is theoretical, and of no practical importance when chromatographic equipment is not available, as
calculation of the actual 2Hr requires chromatographic determination of individual SCFA.
Finally, it should be noted that the approach proposed here only gives quantitative insight into factors related to apparent rumen digestibility. Further complementation of gas
measurements by true degradability measurements, based on separation of microbes and
undegraded feed by a neutral detergent solution treatment (Van Soest, 1994), should allow
more complete assessment of feed quality (Blummel et al., 1997; Blummel et al., 2003).
4. Conclusion
A relatively simple gas production technique has been illustrated, in which phosphate
and bicarbonate buffered incubations are combined to provide accurate information on total
SCFA and CH4 production, as well as some insight into the ratio of lipogenic to glucogenic
SCFA. However, this approach does not allow accurate estimation of the proportions of
individual SCFA. Nevertheless, as no apparatus other than gas tight syringes and incubation

V. Fievez et al. / Animal Feed Science and Technology 123124 (2005) 197210

209

facilities are required, this approach may be relevant under circumstances where laboratories
lack gas chromatographic equipment.

Acknowledgments
O.J. Babayemi is grateful to the John D. and Catherine T. MacArthur Foundation Grant
through the University of Ibadan (Nigeria) as support for Staff Development and Training.
We acknowledge Andy Deprez for technical assistance.

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