animal
Animal (2012), 6:3, pp 518–525 & The Animal Consortium 2011
doi:10.1017/S175173111100156X
Effect of release rate of the SF6 tracer on methane emission
estimates based on ruminal and breath gas samples
C. Martin1a, J. Koolaard2, Y. Rochette1, H. Clark2, J. P. Jouany1 and C. S. Pinares-Patiño21
UR1213 Herbivores, INRA Clermont-Ferrand/Theix, 63122 Saint-Genès-Champanelle, France; 2AgResearch Limited, Grasslands Research Centre, Tennent Drive,
Private Bag 11008, Palmerston North, New Zealand
(Received 11 April 2011; Accepted 19 July 2011; First published online 19 September 2011)
The release rate (RR) of sulphur hexafluoride (SF6) gas from permeation tube in the rumen appears to be positively related with
methane (CH4) emissions calculated using the SF6 tracer technique. Gas samples of breath and ruminal headspace were collected
simultaneously in order to evaluate the hypothesis that transactions of SF6 in the rumen are the source for this relationship.
Six non-lactating dairy cows fitted with rumen cannulae were subdivided into two groups and randomly assigned to a two-period
crossover design to permeation tubes with low RR (LRR 5 1.577 mg/day) or two-times higher RR (HRR 5 3.147 mg/day) RR. The
cows were fed limited amounts of maize silage (80% ad libitum) split into two meals (40% at 0800 h and 60% at 1600 h). Each
period consisted of 3-day gas sampling. Immediately before the morning feed and then each hour over 8 h, ruminal gas samples
(50 ml) were withdrawn through the cannula fitted with stoppers to prevent opening. Simultaneously, 8-h integrated breath gas
samples were collected over the same period. Ratios of concentration of CH4/SF6, CO2/SF6 and CO2/CH4 and emission estimates of
CH4 and CO2 were calculated for each sample source using the SF6 tracer technique principles. The LRR treatment yielded higher
( P , 0.001) ruminal CH4/SF6 (by 1.79 times) and CO2/SF6 (by 1.90 times) ratios than the HRR treatment; however, these
differences were lower than the 2.0 times difference expected from the RR between the LRR and HRR. Consequently, the LRR
treatment was associated with lower ( P , 0.01) ruminal emissions of CH4 over the 8-h collection period than with the HRR
treatment (111%), a difference also confirmed by the breath samples (111%). RR treatments did not differ ( P 5 0.53) in ruminal
or breath CO2 emissions; however, our results confirm that the SF6 tracer seems inappropriate for CO2 emissions estimation in
ruminants. Irrespective of the RR treatment, breath samples yielded 8% to 9% higher CH4 emission estimates than the ruminal
samples ( P 5 0.01). The relationship between rumen and breath sources for CH4 emissions was better for LRR than for HRR
treatment, suggesting that tracer performance decreases with the highest RR of SF6 tested in our study (3.1 mg/day). A hypothesis
is discussed with regard to the mechanism responsible for the relationship between RR and CH4 emission estimates. The use of
permeation tubes with small range in RR is recommended in animal experiments to decrease variability in CH4 emission estimates
using the SF6 tracer technique.
Keywords: SF6 tracer, permeation rate, methane, breath, ruminal gas
Implications
The sulphur hexafluoride (SF6) tracer technique is a method
of choice to estimate individual methane (CH4) emissions
from large groups of animals under production conditions
such as grazing. However, the CH4 emissions estimates
appear related to the SF6 release rate (RR) in the rumen. This
suggests the need to use permeation tubes with a small
range in RR in animal experiments, and that the RR is
balanced across treatments. The performance of the SF6 tracer
a
Present address: AgResearch Limited, Grasslands Research Centre, Tennent
Drive, Private Bag 11008, Palmerston North, New Zealand.
E-mail: Cesar.Pinares-Patiñ[email protected]
technique to estimate CH4 emissions from ruminants
diminishes with the increase of SF6 RR in the rumen.
Introduction
The production of greenhouse gases (GHG) from livestock
and their impact on climate changes are a major concern
worldwide (Gill et al., 2010). In their review, Martin et al.
(2010) reported that enteric methane (CH4) is the most
important GHG emitted (50% to 60%) at the farm scale in
ruminant production systems. The sulphur hexafluoride (SF6)
tracer technique (Johnson et al., 1994) is the method of
choice in all situations where simultaneous estimations of
518
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Release rate of SF6 tracer and methane emission estimates
individual CH4 emissions on large number of free ranging
animals are required (Clark et al., 2005). Most comparative
studies show that mean estimates of CH4 production using
the SF6 technique are not statistically different from those
obtained using calorimetric methods (e.g. Boadi et al., 2002;
McGinn et al., 2006; Grainger et al., 2007; Johnson et al.,
2007) or using the micrometeorological technique (Leuning
et al., 1999). The tracer technique relies on the release of a
known quantity of SF6 tracer gas from a pre-calibrated permeation tube inserted into the reticulo-rumen, and the
CH4/SF6 ratio of concentrations (above the background air)
on the representative breath samples collected from the
tested animal. Recent studies reported a positive relationship between the pre-calibrated release rate (RR) of SF6 and
the estimated CH4 emissions on cattle (Vlaming et al., 2007;
Pinares-Patiño et al., 2008b). This could suggest that the
stability of permeation tubes at high rates is not as robust as
lower rates, and/or that the molar proportion of SF6 in the
breath sample, relative to that of CH4, decreases as the RR of
SF6 in the rumen increases. As gas-mixing processes within
the rumen headspace, as well as during eructation, are
highly turbulent, it would not be expected that such mixing
could discriminate between CH4 and the nine-fold heavier
SF6 molecules (Johnson et al., 2007). Alternatively, it can be
hypothesised that the SF6 gas released in the reticulum or
the ventral sac of the rumen, where the permeation tube sits,
does not reach the ruminal headspace gas pool available for
eructation. A loss of the tracer to the lower digestive tract
with the liquid and/or the solid fraction of the ruminal content, by absorption through the ruminal wall, and/or by
‘trapping’ within the rumen, might be implicated in a systematic lower abundance of SF6 (relative to that of CH4) in
the breath sample. This study explored the above hypothesis
by comparing ratios of the concentration of fermentation
gases (CH4 and CO2) and SF6, and estimated volumes of CH4
and CO2 in the samples collected from the rumen gas
headspace and from the breath of cows deployed with permeation tubes with low RR (LRR) and high RR (HRR) of SF6.
Material and methods
The experiment was conducted at the animal experimental
facilities of the INRA’s Herbivores Research Unit (SaintGenès-Champanelle, France). Procedures on animals were in
accordance with the guidelines for animal research of the
French Ministry of Agriculture and all other applicable
national and European guidelines and regulations for
experimentation with animals (see http://www2.vet-lyon.fr/
ens/expa/acc_regl.html for details).
Animals, experimental design and diets
Six non-lactating Holstein cows fitted with a ruminal cannula
(made of polyvinyl chloride (PVC) and polyamide, 120 mm
internal diameter, Synthesia, Nogent-sur-Marne, France)
were used in this experiment. The surgery had been performed aseptically at least 1 year before the experiment,
under general anaesthesia using halothane (ICI Pharma,
Paris, France).
Animals were used in a crossover design consisting of two
treatments (rates of release of the SF6 tracer, RR) and two
periods during which gas measurements were carried out.
Ruminal cannulae were equipped with stoppers, allowing
collection of rumen gas headspace samples (hereafter
referred as ‘ruminal samples’) without having to open the
cannula (Jouany and Senaud, 1979). Ruminal liquid and gas
leakages throughout the fistula were minimised by fitting
an inner rubber tube around it. No visual leakages have
been postponed during the experiment. The study involved
21 days adaptation, which was followed by an 18-day
experimental period (days 1 to 18). Gas measurements were
carried over days 1 to 3 and 16 to 18.
The cows were randomly allocated into two groups (three
animals each) balanced for live weight (average 816 6 101
and 832 6 56 kg for groups A and B, respectively). Each
group was allocated to permeation tubes with either LRR
(1.577 6 0.283 mg/day) or HRR (3.147 6 0.561 mg/day) of
SF6 in the first period, and the permeation tubes were
swapped between groups in the second period. Permeation
tubes were introduced in the rumen 5 days before each gas
measurement period. Brass permeation tubes (12.5 mm 3
40 mm) were charged with 733 6 6 mg and calibrated by
regular weighing (two times a week) for a 10-week period
on average while they were kept in an Erlenmeyer glass in
398C water bath. Coefficients of variation of the SF6 RR
estimated during the period of calibration were similar
between the LRR and HRR tubes (Table 1). Before each gas
collection period, the calibrated permeation tubes were
dosed via fistula into the reticulum of the cows 7 days earlier
Table 1 Characteristics of individual SF6 permeation tubes used in this experiment
Permeation tube
code number
LRR-W
LRR-19
LRR-X
HRR-Y
HRR-U
HRR-O
SF6 release rate (mg/day)
Duration of
calibration (days)
Mean 6 s.d.
Coefficient of variation (%)
79
40
82
82
72
79
1.3824 6 0.0867
1.4482 6 0.0580
1.9027 6 0.1322
2.7440 6 0.1522
2.9103 6 0.0825
3.7886 6 0.2319
6.27
4.00
6.95
5.54
2.83
6.12
LRR 5 low release rate; HRR 5 high release rate.
519
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Martin, Koolaard, Rochette, Clark, Jouany and Pinares-Patiño
and retrieved at the end of each measurement period. To find
permeation tubes easily in the rumen, they were tied at
the end of a twine measuring 1 m, and the other end of the
twine being fixed to the ruminal cannula. Between the two
periods of gas measurement, the permeation tubes were
kept in the laboratory in the same conditions as during the
calibration period. The concentration of the residual SF6
in the rumen before switching the tubes for the second
period of gas measurement was determined. It averaged
19.4 6 9.4 ppt and represented less than 0.1% of the SF6
concentration detected in the rumen loaded with permeation
tubes during the first period of gas measurement.
Animals were individually fed a 100% maize silage
diet obtained from a single batch and were supplemented
(180 g/day) with a commercial mineral–vitamin premix
(Galaphos Midi Duo GR, CCPA, Aurillac, France) and with
bicarbonate (140 g/day). Feed allowance was ad libitum
during the adaptation period, and then was restricted to
80% of ad libitum intake during the experimental period to
ensure no refusals. Daily feed allowance was split into two
meals delivered at 0800 and 1600 h. The amount of feed
delivered during the morning and afternoon feedings were,
respectively, 40% and 60% of the daily feed allowance.
Drinking water was available ad libitum. Animals were kept
in individual stalls in a barn (30 000 m3 of volume). Ventilation of the barn was a natural ventilation system with side(shutters) and ridge tile-opening.
Feed intake
Dry matter intake (DMI) was measured twice daily at 0800
and 1600 h throughout the experiment. Maize silage dry
matter (DM) content was measured at 1038C for 24 h, and its
chemical composition (crude protein, NDF, starch and gross
energy) was determined on pooled samples taken daily on
the gas measurement periods after drying (608C for 48 h)
and grinding (0.8-mm screen). Analytical methods used for
determination of maize silage chemical composition were as
described by Martin et al. (2008). The maize silage contained
285 g DM/kg, 233 g starch, 74 g crude protein, 442 g NDF
and 18.5 MJ gross energy/kg DM.
Gas collection and analyses
At each experimental period, breath and ruminal gases were
simultaneously collected during 3 consecutive days. Both
breath and ruminal gas samples were collected over an 8-h
period beginning just before the morning feeding and ending
just before the afternoon feeding.
Breath samples. Breath samples from each animal were
continuously collected into pre-evacuated (20.9 atm) yokeshaped PVC collection devices (,2.5 l). Sample flow was
set by means of capillary and Teflon tubing calibrated for a
gas collection period of 8 h, and fitted to a halter as described by Martin et al. (2008). Samples of background air inside
the barn were also collected over the same period. Collections of both breath and background samples were stopped
at 1600 h just before the afternoon feeding and collection
devices were immediately transported to the laboratory
where they were over-pressured with N2 gas (,1.4 atm) and
analysed for concentrations of SF6, CH4 and CO2.
Ruminal gas samples. On each animal, ruminal gas samples
were collected before the morning feeding and at 1-h intervals following the feeding (i.e. nine samples per cow). At
each sampling time, 50 ml of gas was collected from the
rumen headspace gas (dorsal sac) using a plastic syringe
(one per sample) on standing animals to avoid contamination with ruminal juice as described by Jouany and Senaud
(1979). Gas samples were immediately sub-sampled into
two vacutainer tubes of 10 ml without additives (red cap;
VT-100SU; Terumo Europe N.V. 3001 Leuven, Belgium) and
transported to the laboratory. Ruminal gas samples were
diluted with N2 gas (1/200 to 1/300 vol/vol) to obtain gas
concentrations within the range of concentration of the
standards. Because of varying dilution rates of samples,
comparisons between treatments (LRR v. HRR) and sampling
sites (breath v. ruminal) were based on ratios of gas concentrations rather than on their specific concentrations.
Gas analyses. Concentrations of SF6, CH4 and CO2 in the
breath, ruminal and background air samples were determined using gas chromatography as described by Martin
et al. (2008). A gas chromatograph (Varian-Chrompack, CP9003, Les Ulis, France) fitted with electron capture detector
was used to determine concentrations of SF6, whereas
another gas chromatograph (Perkin Elmer instruments;
Autosystem XL, Courtaboeuf, France) fitted with a flame
ionisation and a thermal conductivity detector, was used to
determine the concentrations of CH4 and CO2, respectively.
For determination of SF6, the samples were run on the
chromatograph using a Molecular Sieve 0.5 nm column
(3 m 3 3.2 mm internal diameter) at 508C, whereas a Porapack N 80–100 mesh column (3 m 3 3.2 mm internal diameter) maintained at 408C was used for determination of
CH4 and CO2. The carrier gas used for determination of SF6
was N2 (30 ml/min), whereas He (40 ml/min) was used as
carrier for CH4 and CO2. Chromatographic analyses were
performed after calibration with standard gases (Air Liquide,
Mitry-Mory, France) for SF6 (55 and 195 ppt), CH4 (100 ppm)
and CO2 (1490 ppm).
Data calculation and statistical analysis
Data calculation. Ruminal gas samples were uniformly
spaced in time separated by a period of 1 h. Ruminal concentrations of SF6, CH4 and CO2 of each 1-h period were
estimated by averaging their concentrations from gas samples taken at two consecutive time points, and then were
used to calculate the ratios of concentrations (CH4/SF6 and
CO2/SF6). Subsequently, these ratios in conjunction with
the known RR of SF6 were used to estimate the hourly
emissions of CH4 and CO2 arising into the rumen gas headspace. The total ruminal emissions of CH4 and CO2 over the
8-h sampling period were obtained by adding their estimates
of hourly emissions.
520
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Release rate of SF6 tracer and methane emission estimates
Table 2 Concentration of tracer gas (SF6), methane (CH4) and carbon dioxide (CO2) in ambient air of the barn across days and periods of
measurement
SF6
Periods (P) and days (d) of measurement
P1 – d1
P1 – d2
P1 – d3
Mean 6 s.d.
P2 – d1
P2 – d2
P2 – d3
Mean 6 s.d.
CH4
CO2
ppt
% breath1
ppm
% breath1
ppm
% breath1
21.1
29.6
52.4
34.4 6 16.2
29.7
31.8
35.8
32.4 6 3.1
3.2
3.3
5.1
3.9 6 1.1
5.4
6.2
5.7
5.8 6 0.4
82.0
65.3
75.3
74.2 6 8.4
66.5
44.9
26.6
46.0 6 20.0
9.1
8.1
8.8
8.7 6 0.6
9.7
8.7
5.8
8.1 6 2.0
1555.3
1372.9
1520.0
1482.7 6 96.7
1276.7
1024.2
761.2
1020.7 6 257.8
89.1
89.1
87.8
88.7 6 0.8
88.8
88.2
89.4
88.8 6 0.6
1
Expressed as % of gas concentration detected in the breath gas samples of the cows (n 5 6).
ruminal CH4 ðl=hÞ ¼ RR of SF6 ðl=hÞ hourly ½CH4 =½SF6 measurements of ANOVA, with the same block structure as
above, to compare the two RRs of SF6.
Means for each combination of RR and emission source
were presented together with least significant difference.
Most of the gas and ratios data had variances that tended to
increase with the mean; this meant that a log transformation
had to be applied to the data before analysis in order to
stabilise the variance. In such cases, log-transformed and
back-transformed means were presented.
ruminal CO2 ðl=hÞ ¼ RR of SF6 ðl=hÞ hourly ½CO2 =½SF6 Results
The 8-h period breath emissions of CH4 and CO2 were
calculated using the net concentrations of SF6, CH4 and CO2
in the breath samples (i.e. subtracting background air concentrations reported in Table 2). Both the hourly emissions of
CH4 and CO2 in the rumen, and the emissions of CH4 and
CO2 in the breath over the 8-h period, were calculated
according to Johnson et al. (1994):
breath CH4 ðl=8-h periodÞ ¼ RR of SF6 ðl=8-h periodÞ
8-h period ½CH4 =½SF6 breath CO2 ðl=8-h periodÞ ¼ RR of SF6 ðl=8-h periodÞ
8-h period ½CO2 =½SF6 Statistical analysis. The statistical software GenStat 10th
edition (Payne et al., 2007) was used to analyse the gas and
ratio data with analysis of variance (ANOVA). The model
comprised emission source (S: breath or rumen), RR (high or
low), S 3 RR as fixed effects, and cow, period, cow 3 period,
period 3 day, cow 3 period 3 day as random effects. Data
were analysed as a 2-period crossover design, where RR was
the crossover treatment and was tested against the periodwithin-cow variability, whereas S and SS 3 R were tested
against day-within-period-within-cow variability. DMI during
the 8-h gas measurement period was used as a covariate
only for those measurements for which it explained a significant portion of the variability as assessed from the
ANOVA. The nature of the relationship between CO2/CH4
ratios and DMI differed for breath and rumen sources, and
thus it was not possible to incorporate DMI as a covariate in
a combined ANOVA; therefore, the CO2/CH4 ratios data from
the two sources were analysed separately.
The gas and ratio time-trend data for the rumen emissions
measured over the 8-h period were analysed using repeated
Feed intake
The mean feed intake during the 8-h gas measurement period was 2.94 6 0.49 kg DM per cow. Because feeding level
was fixed and restricted, feed intake did not differ (P . 0.05)
between LRR and HRR treatments (data not shown).
Post-feeding kinetics of ruminal gas concentration
ratios and emissions
Ruminal post-feeding kinetics of the different gas concentration
ratios (CH4/SF6, CO2/SF6 CO2/CH4) and of the hourly gas production (CH4 and CO2) for the two RRs of SF6 tracer are presented in Figure 1. No interaction effect of sampling time 3 RR
treatment (P . 0.05) was observed for these parameters (data
not shown). Irrespective of the post-feeding hour, both CH4/SF6
and CO2/SF6 ratios of gas concentrations were higher (P , 0.01)
for the LRR treatment than for the HRR, whereas the CO2/CH4
ratio did not differ (P . 0.05) between LRR and HRR. Through
the entire period of sample collection, the estimated hourly
ruminal emissions of CH4 were numerically higher (P . 0.05) for
the HRR than for the LRR treatment (Figure 1). The estimated
hourly emissions of ruminal CO2 showed a similar post-feeding
pattern to that of CH4 without difference between the two RR
treatments, except towards the start of the 8-h sampling period
where values were numerically higher for HRR than for LRR.
Ruminal v. breath gas sampling
The mean ratios of gas concentrations and estimated emissions
of CH4 and CO2 over the 8-h sampling period for ruminal and
breath sampling sources are presented in Table 3.
521
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1
2
3
4
5
6
7
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
3.2
3
CO2/CH4
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
CO2/SF6 (x106)
CH4/SF6 (x106)
Martin, Koolaard, Rochette, Clark, Jouany and Pinares-Patiño
3
4
5
6
7
8
1
Hours post feeding
20
18
CO2 (l/h)
16
CH4 (l/h)
2.4
2
2
Hours post feeding
14
12
10
8
6
1
2.6
2.2
1
8
2.8
2 3 4 5 6 7
Hours post feeding
3
4
5
6
7
8
Hours post feeding
60
55
50
45
40
35
30
25
20
15
10
1
8
2
2
3 4 5 6 7
Hours post feeding
8
Figure 1 Post feeding kinetics of gas concentration ratios and estimated hourly productions of CH4 and CO2 based on gases sampled from the rumen
headspace with low (LRR, o) and high (HRR, ’) release rates of SF6. Dashes bars represent approximate LSD (5%).
Table 3 Effects of RR of SF6 tracer (LRR v. HRR) and source of gas samples (ruminal v. breath) on mean ratios of concentrations of gases and
estimated emissions of CH4 and CO2 over a 8-h sampling period
Ruminal
Ratios of concentration (vol/vol)
CH4/SF6 (3106)
CO2/SF6 (3106)
Log value
CO2/CH4
Log value
Emission estimates (l/8-h period)
CH4
CO2
Log value
P-value
Breath
LRR
HRR
LRR
HRR
LSD1
Source
RR
Source 3 RR
1.283
3.353
1.210
2.563
0.941
0.716
1.752
0.561
2.509
0.920
1.389
21.85
3.084
15.67
2.752
0.783
11.51
2.443
15.47
2.739
0.124
NA2
0.180
NA
0.089
0.004
NA
,0.001
NA
,0.001
,0.001
NA
,0.001
NA
0.619
0.479
NA
0.911
NA
0.865
100.9
266.9
5.587
113.5
278.4
5.629
109.6
1738.9
7.461
122.6
1828.0
7.510
7.48
NA
0.184
0.013
NA
,0.001
0.003
NA
0.535
0.968
NA
0.911
RR 5 release rate; LRR 5 low release rate; HRR 5 high release rate; LSD 5 least significant difference.
1
Least significant difference for comparing ruminal v. breath (d.f. between 6 and 9).
2
NA 5 not applicable because the statistical analysis was conducted on natural log data.
Overall, the effect of RR of SF6 tracer, both on ratios of gas
concentrations and estimated emissions of CH4 and CO2,
was similar for ruminal and breath gas samples (source 3 RR
treatment: P . 0.05).
Treatment differed significantly (P , 0.001) in CH4/SF6
and CO2/SF6 ratios, but not in CO2/CH4 ratios (P 5 0.619). As
expected, the rumen and breath samples had significantly
higher means CH4/SF6 and CO2/SF6 concentrations ratios
with LRR compared with HRR (31.78 and 1.90, respectively;
P , 0.001). However, the differences were lower than twice
the difference in the tracer RR between LRR and HRR. The
ruminal and breath estimated emissions of CH4 were lower
with LRR than with HRR (105 v. 118 l/8-h period on average,
respectively). RR treatment did not differ in ruminal or breath
CO2 emissions (P 5 0.535).
Irrespective of the RR treatment, a significant effect of
source of sampling (ruminal v. breath) on CH4/SF6 ratios was
observed (P , 0.01), with breath sampling yielding higher
ratios and consequently higher estimated emissions of CH4
(18.3% on average). Because of respiratory CO2 output, the
source of sampling affected both CO2/SF6 and CO2/CH4
ratios (P , 0.001), and breath sampling yielded higher CO2
emission estimates than the ruminal source (P , 0.001).
Discussion
Kinetics of ruminal gas emission estimates
The estimated hourly ruminal CH4 and CO2 production profiles determined in this study are in agreement with previous
findings that ruminal fermentation gases evolve rapidly with
feeding and that eructation frequency increases with
increasing rates of ruminal gas production (Waghorn and
Reid, 1983; Rémond et al., 1993; Moate et al., 1997). In this
study, the mean CO2/CH4 ratio in the rumen increased from
522
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Release rate of SF6 tracer and methane emission estimates
2.2 at the pre-feeding stage to 3.6 at the first hour following
feeding, with an increase of the rate of production of CO2 by
3.3 times (0.37 v. 1.20 l/min on average) and that of CH4 by
2.4 times (0.16 v. 0.39 l/min on average). These results
confirm the rates of increase of gases previously observed
in sheep fed twice daily on hay (Rémond et al., 1993), and in
cattle fed on perennial ryegrass/white clover pastures
(Moate et al., 1997). The latter authors reported that, from
the pre-feeding to the first hour post-feeding, the rate
of entry into the rumen gas headspace of CO2 increased by
3.5 times (0.43 v. 1.45 l/min) and that of CH4 by 2.4 times
(0.18 v. 0.41 l/min). The consistency of our results with those
of the literature supports the joint utilisation of the SF6 tracer
technique and of the ruminal headspace gas sampling to
study the kinetics of gas production in the rumen.
SF6 tracer technique and CH4 emission estimates
The 8-h production estimates of CH4 based on ruminal and
breath samples were both significantly higher for the HRR than
for the LRR treatment. This is in agreement with previous
studies on rumen-fistulated steers pen-fed on lucerne silagebased diets (Vlaming et al., 2007; Pinares-Patiño et al., 2008b),
mentioning that there is a positive association between the RR
of SF6 and the CH4 emission estimates (both on absolute and
per unit of intake basis) based on breath samples. Vlaming
et al. (2007) reported an increase in the estimated CH4 yield of
4.3% for each 1 mg/day increase in RR of SF6, irrespective of
the level of intake of animals assuming the RR from permeation tubes was linear over the range from 2.88 to 7.34 mg/day.
Pinares-Patiño et al. (2008b) confirmed the relationship
with permeation tubes releasing four different rates of SF6
(1.91, 3.62, 5.34 and 11.34 mg/day). Both the absolute
CH4 emission (g/day) and CH4 yield (g/kg DMI) increased linearly with increasing RR of SF6, each 1 mg/day increase in RR
of SF6 accounting for an increase of 6.6% in CH4 emission
estimates from breath samples. The same difference in CH4
emission estimates between the two tested RR of SF6 (1.58 v.
3.15 mg/day) was reported in our experiment for the two
sources of gas samples (breath v. rumen) and averaged 7.2%
for each 1 mg/day increase in RR of SF6.
The calibrated tracer source, the collection of representative breath sample and the subsequent analyses of CH4
and SF6 gases are the three major factors of variation of the
SF6 tracer technique (Lassey et al., 1997). Because all these
components are tightly controlled, in any study, their
potential role on the observed association between RR of SF6
and CH4 emission estimates can be ruled out. The calculation
of daily CH4 emission estimates is based on the CH4/SF6 ratio
of concentrations in breath samples (corrected for background concentrations), and the specific RR of SF6 from a
particular permeation tube deployed in the animal. In our
study, the differences in CH4/SF6 ratios of concentration in
ruminal and breath samples between the RR treatments
were much less (1.8) than a two-fold, as expected from the
difference in RR between HRR and LRR, whereas the CO2/CH4
ratio of concentration (in ruminal and breath samples) did
not differ between HRR and LRR treatments. Thus, the fact
that the HRR treatment was associated with higher CH4
production estimates than the LRR treatment suggests that,
as the RR of SF6 increased, a greater proportion of the SF6
supposed to be released from its source (i.e. the permeation
tube) was not accounted for in the rumen gas headspace and
therefore in the breath.
The effect of SF6 RR observed in the CH4/SF6 ratios was
similar from the collected breath and ruminal samples. This
indicates that SF6 and CH4 are well mixed, with similar
behaviour and collection efficiency in the two sampling
sources, even though most of the eructated gases (77% to
99%) are inhaled and absorbed into the blood and then
excreted with exhalation gases (Dougherty and Cook, 1962;
Hoernicke et al., 1965). Thus, it is unlikely that the 93 difference between the molecular weights of SF6 and CH4
would compromise proper mixing and dispersion, and
representative sampling. As suggested by Johnson et al.
(2007), because of the forceful eructation process and the
turbulent mixing during excretion, the molecular diffusion of
gases becomes unimportant, and consequently the SF6
emission would exactly simulate the CH4 emission.
An assumption for the lower SF6 recovery at HRR is that
the tracer gas may be released from the rumen via another
route than the mouth and nose. The unaccounted fraction of
SF6 may be trapped in the rumen, absorbed from the rumen
to the blood or bypassed to the lower digestive tract and
then subsequently absorbed or excreted with undigested
material or flatus. It is known that at body temperatures SF6
has lower solubility than CH4 (Meyer et al., 1980); however,
solubility of a gas increases as its partial pressure increases.
Therefore, despite the minute amounts of SF6 in the rumen, it
can be postulated that as RR of SF6 increases a larger
proportion of SF6 solubilises in the ruminal liquid phase.
Then, this dissolved fraction flows to the lower digestive
tract where it is absorbed into the blood or passed out
with the faecal material. Studies have reported diffusion
of SF6 into the lungs from venous blood (Schimmel et al.,
2004), suggesting that SF6 absorption takes places from
the digestive tract. As long as the SF6 is excreted via the
breath and not stored in the body, this route of excretion
per se could not be responsible for the association between
RR of SF6 and the CH4 emission estimates. Detectable
concentrations of SF6 in either gas trapped in faeces and in
urine of animals dosed with SF6 release sources have been
reported, but in minute quantities (Vlaming et al., 2007).
The possible excretion of SF6 via flatus or in solution or
adhering to faecal material cannot be excluded. The relative
importance of these alternative pathways of excretion of
SF6 remains to be determined.
By confirming the relationship between RR of SF6 and CH4
estimates, this study suggests that in order to obtain precision in CH4 measurement, permeation tubes used should
have a narrow range in the RR and be balanced between
treatments of the same animal experimentation. In the literature, the RR of SF6 from permeation tubes mostly reported vary between 1 and 5 mg/day with an average RR of
3 mg/day, which was represented by the HRR in this study.
523
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Martin, Koolaard, Rochette, Clark, Jouany and Pinares-Patiño
1.0
0.5
25
30
25
20
15
10
1.0
Ruminal CH4/SF6 (x106)
2
3
3000
170
140
110
80
50
50
80
4
1.5
6
5
2.0
Ruminal CO2/SF6 (x106)
Breath CO2 (l/8-hours)
Breath CH4 (l/8-hours)
1
All: r = 0.47; P<0.01
LRR: r = 0.66; P<0.01
HRR: r = 0.35; P=0.167
200
15
10
0
2.0
1.5
20
5
0
0.5
All: r = 0.61; P<0.001
LRR: r = 0.64; P<0.01
HRR: r = 0.63; P<0.01
35
Breath CO2 /CH4
1.5
0.0
0.0
All: r = 0.89; P<0.001
LRR: r = 0.84; P<0.001
HRR: r = 0.62; P<0.01
40
Breath CO2 /SF6 ( x106)
Breath CH4 /SF6 ( x106)
2.0
All: r = 0.89; P<0.001
LRR: r = 0.74; P<0.001
HRR: r = 0.34; P=0.183
110
140
170
200
Ruminal CH4 (l/8-hours)
2.5
3.0
3.5
Ruminal CO2/CH4
All: r = 0.55; P<0.001
LRR: r = 0.71; P<0.001
HRR: r = 0.48; P=0.05
2500
2000
1500
1000
100
200
300
400
500
Ruminal CO2 (l/8-hours)
Figure 2 Relationships between breath and ruminal ratios of gas concentrations and emission estimates of CH4 and CO2 for cows deployed with permeation
tubes with low (LRR, o) and high (HRR, ’) release rates of SF6 tracer. Pearson’s correlation coefficients are presented for all data (n 5 34) and each level of
release rate of SF6 tracer (n 5 17 each). Total number of points is 34 instead of 36 due to 2 breath gas samples lost.
Given that most of the ruminal CH4 and CO2 are excreted
(eructed and exhaled) to the mouth and nostrils where the
breath samples were taken, a close correlation between
ruminal and breath sources for ratios of concentration of
gases and emission estimates could be expected. However,
results of this study (Figure 2) indicate that contrary to the
good relationships for the LRR treatment, the relationships
for the HRR treatment were either not significant or were of
lower magnitude. These findings suggest that, in our
experimental conditions, the SF6 tracer has better performance at LRR (1.5 mg/day) than at HRR (3.0 mg/day) of
tracer. Parallel measurements of CH4 emissions using both
respiration chamber and tracer technique and involving a
range of RR treatments may yield a final answer on the most
optimal RR of SF6.
In this study, the breath samples yielded systematic 8% to
9% higher CH4 emission estimates than the ruminal samples, suggesting that the breath sources accounted for the
hindgut production of CH4, which is mostly exhaled. Studies
have reported that CH4 produced in the large intestine
accounted for 8% to 13% of total daily CH4 production in
sheep fed different diets based on lucerne or grain (Murray
et al., 1976; Torrent and Johnson, 1994), 89% being absorbed into the blood, then released into the lungs and exhaled
in the breath (Murray et al., 1976). However, it can be suggested that the non-significant differences between tracer
and chamber CH4 emission estimates observed in validation
studies (e.g. Grainger et al., 2007; Johnson et al., 2007;
Pinares-Patiño et al., 2008a) may be because of the efficacy
of gas mixing in rumen and lungs during the eructation and
exhalation processes rather than the strict compliance of the
SF6 with the tracer principles.
SF6 tracer technique and CO2 emission estimates
In this study, the effect of RR of SF6 tracer on both ruminal and
breath CO2 emission estimates was not significant, contrary to
what was observed with CH4. However, the latter observation
does not imply that SF6 is a better tracer for CO2 than for CH4.
In fact, the tracer principles compliance to trace CO2 seems
worst than for CH4. Ruminal CO2 is composed of fermentation
CO2, salivary CO2 and urea CO2 (Hoernicke et al., 1965). CO2
and CH4 are absorbed at different rates in the rumen, the
absorption of ruminal CO2 being absolutely and relatively
higher than the absorption of CH4. In addition, whereas
the ruminal flux of CH4 is one way, there is a large exchange
of CO2 between rumen and blood (Veenhuizen et al., 1988).
It has also been reported that increased concentrations of
ruminal CO2 following feeding increases the flux of blood
urea across the ruminal epithelium (Rémond et al., 1993).
Breath CO2 corresponds to both exhaled and eructed
CO2 (metabolic 1 fermentation). In this study, the CO2/CH4
ratio in breath gases of 15.5 was much higher than those
(CO2/CH4 5 9 to 12) reported previously for dairy cows and
measured using the open-circuit indirect respiration technique (Kinsman et al., 1995; Sauer et al., 1998), which suggests an obvious overestimation. Higher estimates of total
CO2 emissions in ruminants with the tracer technique
(,120%) than those obtained using open-circuit calorimetry have already been reported (Boadi et al., 2002). The
overestimation of CO2 productions with the tracer technique
may be explained by a difference in the rate of emission
between enteric and metabolic CO2, and therefore between
the SF6 tracer gas and the metabolic CO2. Our results confirm
that SF6 tracer seems inappropriate for CO2 emissions estimation in ruminants.
524
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Release rate of SF6 tracer and methane emission estimates
Conclusion
This study found a positive relationship between RR of SF6
and CH4 production estimates based on rumen headspace
gas samples. It also confirmed the positive relationship
between RR of SF6 and CH4 emission estimates from breath
samples. In our experimental conditions, the performance of
the SF6 tracer to estimate CH4 emissions diminished with the
highest RR of SF6. Nevertheless, the mechanism by which the
relationship occurs is not known, but it seems that this has
ruminal origins. We hypothesise that at HRR an increasing
proportion of SF6 is washed out to the lower digestive tract
and then not accounted for in the breath because of losses in
faecal material. Experimental work, involving a bigger range
of RR treatments, on transactions of SF6 in the digestive
tract, blood and other tissues is required to comprehend the
limitations of the SF6 tracer technique for accurate and precise estimation of enteric CH4 emissions.
References
Boadi DA, Wittenberg KM and Kennedy AD 2002. Validation of the sulphur
hexafluoride (SF6) tracer gas technique for measurement of methane and carbon
dioxide production by cattle. Canadian Journal of Animal Science 82, 125–131.
Clark H, Pinares-Patiño CS and deKlein C 2005. Methane and nitrous oxide
emissions from grazed grasslands. In Grassland: a global resource (ed. DA
McGilloway), pp. 279–293. Wageningen Academic Publishers, Wageningen,
The Netherlands.
Dougherty RW and Cook HM 1962. Routes of eructed gas expulsion in cattle – a
quantitative study. American Journal of Veterinary Research 23, 997–1000.
Lassey KR, Ulyatt MJ, Martin RJ, Walker CF and Shelton D 1997. Methane
emissions measured directly from grazing livestock in New Zealand. Atmospheric Environment 31, 2905–2914.
Leuning R, Baker SK, Jamie IM, Hsu CH, Klein L, Denmead OT and Griffith DWT
1999. Methane emission from free-ranging sheep: a comparison of two
measurements methods. Atmospheric Environment 33, 1357–1365.
Martin C, Rouel J, Jouany JP, Doreau M and Chilliard Y 2008. Methane
output and diet digestibility in response to feeding dairy cows with crude
linseed, extruded linseed or linseed oil. Journal of Animal Science 86,
2642–2650.
Martin C, Doreau M and Morgavi DP 2010. Methane mitigation in ruminants:
from microbe to the farm scale. Animal 4, 351–365.
McGinn SM, Beauchemin KA, Iwassa AD and McAllister TA 2006. Assessment of
the sulfur hexafluoride (SF6) tracer technique for measuring enteric methane
emissions from cattle. Journal of Environmental Quality 35, 1686–1691.
Meyer M, Tebbe U and Piiper J 1980. Solubility of inert gases in dog blood and
skeletal muscle. European Journal of Physiology 384, 131–134.
Moate PJ, Clarke T, Davis LH and Laby RH 1997. Rumen gases and bloat in
grazing dairy cows. Journal of Agricultural Science 129, 459–469.
Murray RM, Bryant AM and Leng RA 1976. Rates of production of methane in
the rumen and large intestine of sheep. British Journal of Nutrition 36, 1–14.
Payne RW, Murray DA, Harding SA, Baird DB and Soutar DM 2007.
GenStat for Windows introduction, 10th edition. VSN International, Hemel
Hempstead, UK.
Pinares-Patiño CS, Holmes CW, Lassey KR and Ulyatt MJ 2008a. Measurement
of methane emission from sheep by the sulphur hexafluoride tracer
technique and by the calorimetric chamber: failure and success. Animal 2,
141–148.
Pinares-Patiño CS, Machmüller A, Molano G, Smith A, Vlaming JB and Clark H
2008b. The SF6 tracer technique for measurement of methane emissions from
cattle – effect of tracer permeation rate. Canadian Journal of Animal Science 88,
309–320.
Gill M, Smith P and Wilkinson JM 2010. Mitigating climate change: the role of
domestic livestock. Animal 4, 323–333.
Rémond D, Chaise JP, Delval E and Poncet C 1993. Net flux of metabolites across
the ruminal wall of sheep fed twice a day with orchardgrass hay. Journal of
Animal Science 71, 2529–2538.
Grainger C, Clarke T, McGinn SM, Auldist MJ, Beauchemin KA, Hannah MC
Waghorn GC, Clark H and Eckard RJ 2007. Methane emissions from dairy cows
measured using the sulfur hexafluoride (SF6) tracer and chamber techniques.
Journal of Dairy Science 90, 2755–2766.
Sauer FD, Fellner V, Kinsman R, Kramer JK, Jackson HA, Lee AJ and Chen S
1998. Methane output and lactation response in Holstein cattle with
monensin or unsaturated fat added to the diet. Journal of Animal Science 76,
906–914.
Hoernicke H, Williams WF, Waldo DR and Flatt WP 1965. Composition and
absorption of rumen gases and their importance for the accuracy of respiration
trials with tracheostomized ruminants. In Energy metabolism (ed. KL Blaxter),
pp. 165–178. Academic Press, London.
Schimmel C, Bernard SL, Anderson JC, Polissar NL, Lakshminarayan S and
Hlastala MP 2004. Soluble gas exchange in the pulmonary airways of sheep.
Journal of Applied Physiology 97, 1702–1708.
Torrent J and Johnson DE 1994. Methane production in the large intestine of
sheep. In Energy metabolism of farm animals (EAAP Publication No. 76) (ed. JF
Aguilera), pp. 391–394. Servicio de Publicaciones, Consejo Superior de
Investigaciones Cientificas, Granada, Spain.
Johnson KA, Huyler M, Westberg H, Lamb B and Zimmerman P 1994.
Measurement of methane emissions from ruminant livestock using a SF6 tracer
technique. Environmental Science and Technology 28, 359–362.
Johnson KA, Westberg H, Michal JJ and Cossalman MW 2007. The SF6 tracer
technique: methane measurement from ruminants. In Measuring methane
production from ruminants (ed. HPS Makkar and PE Vercoe), pp. 33–67.
Springer, The Netherlands.
Veenhuizen JJ, Russell RW and Young JW 1988. Kinetics of metabolism of
glucose, propionate and CO2 in steers as affected by injecting phlorizin and
feeding propionate. Journal of Nutrition 118, 1366–1375.
Jouany JP and Senaud J 1979. Description d’une technique permettant
d’effectuer des prélèvements répétés de gaz dans le rumen. Annales Biologie
Animale Biochimie Biophysique 19, 1007–1010.
Vlaming JB, Brookes IM, Hoskin SO, Pinares-Patiño CS and Clark H 2007. The
possible influence of intra-ruminal sulphur hexafluoride release rates on
calculated methane emissions from cattle. Canadian Journal of Animal Science
87, 269–275.
Kinsman R, Sauer FD, Jackson HA and Wolynetz MS 1995. Methane and carbon
dioxide emissions from dairy cows in full lactation monitored over a six month
period. Journal of Dairy Science 78, 2760–2766.
Waghorn GC and Reid CSW 1983. Rumen motility in sheep and cattle
giving different diets. New Zealand Journal of Agricultural Research 26,
289–295.
525
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 15 Jun 2017 at 15:36:02, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S175173111100156X