Content uploaded by Nigel Tomkins
Author content
All content in this area was uploaded by Nigel Tomkins on Oct 16, 2017
Content may be subject to copyright.
The red macroalgae Asparagopsis taxiformis is a potent natural
antimethanogenic that reduces methane production during
in vitro fermentation with rumen fluid
Robert D. Kinley
A,C
, Rocky de Nys
B
, Matthew J. Vucko
B
, Lorenna Machado
B
and Nigel W. Tomkins
A
A
CSIRO Agriculture, Australian Tropical Science and Innovation Precinct, James Cook University,
Townsville, Qld 4811, Australia.
B
MACRO-Centre for Macroalgal Resources and Biotechnology, College of Marine and Environmental Sciences,
James Cook University, Townsville, Qld 4811, Australia.
C
Corresponding author. Email: rob.kinley@csiro.au
Abstract. Livestock feed modification is a viable method for reducing methane emissions from ruminant livestock.
Ruminant enteric methane is responsible approximately to 10% of greenhouse gas emissions in Australia. Some species of
macroalgae have antimethanogenic activity on in vitro fermentation. This study used in vitro fermentation with rumen
inoculum to characterise increasing inclusion rates of the red macroalga Asparagopsis taxiformis on enteric methane
production and digestive efficiency throughout 72-h fermentations. At dose levels 1% of substrate organic matter there was
minimal effect on gas and methane production. However, inclusion 2% reduced gas and eliminated methane production in
the fermentations indicating a minimum inhibitory dose level. There was no negative impact on substrate digestibility for
macroalgae inclusion 5%, however, a significant reduction was observed with 10% inclusion. Total volatile fatty acids were
not significantly affected with 2% inclusion and the acetate levels were reduced in favour of increased propionate and, to a
lesser extent, butyrate which increased linearly with increasing dose levels. A barrier to commercialisation of Asparagopsis is
the mass production of this specific macroalgal biomass at a scale to provide supplementation to livestock. Another area
requiring characterisation is the most appropriate method for processing (dehydration) and feeding to livestock in systems
with variable feed quality and content. The in vitro assessment method used here clearly demonstrated that Asparagopsis can
inhibit methanogenesis at very low inclusion levels whereas the effect in vivo has yet to be confirmed.
Additional keyword: greenhouse gas, ruminant, seaweed.
Received 14 September 2015, accepted 23 November 2015, published online 9 February 2016
Introduction
Methane (CH
4
) in the atmosphere is a potent greenhouse gas
(GHG) with an IPCC Fifth Assessment Report (AR5) global
warming potential 28 times that of carbon dioxide (CO
2
, IPCC
2014). Between 2000 and 2009, agriculture and waste
management accounted for 62% of global anthropogenic CH
4
emissions (Kirschke et al.2013) with ruminant enteric
fermentation responsible for 58% of agricultural contributions
(Olivier et al.2005). In Australia, the contribution of CH
4
from
ruminant livestock is approaching 10% of total GHG emissions
(Henry et al.2012). These levels have resulted in a universal
effort to reduce enteric CH
4
emissions. Enteric CH
4
is a
consequence of anaerobic fermentation of feed organic matter
(OM) by a microbial consortium that produces substrate CO
2
and hydrogen in a reduction pathway used by methanogens
(Morgavi et al.2010). Feed additives have been used to
interfere with this pathway or otherwise reduce the numbers
of functional methanogens. Patra (2012) reviewed dietary
supplementation options for rumen enteric CH
4
management
that included ionophores, chemical compounds, legumes,
essential oils, fats, saponins, tannins, probiotics, and plant
secondary metabolites. Unfortunately, an antimethanogenic
effect may be concomitant with some detrimental impacts.
Most commonly there is a decrease in fermentation efficiency
leading to a decrease in feed intake and a measurable decline in
animal productivity.
Macroalgae also have potential for use as a supplement for
livestock feeds (Machado et al.2015a). The antimethanogenic
properties of macroalgae-based functional products in rumen
in vitro cultures has been demonstrated (Wang et al.2008;
Dubois et al.2013; Kinley and Fredeen 2015; Machado
et al.2014), however there is much variability in the
antimethanogenic potency between types and species of
macroalgae. Algae are generally classified by size (micro or
macro) whereas macroalgae are broadly classified based on
pigmentation (green, red or brown) and habitat (freshwater or
CSIRO PUBLISHING
Animal Production Science, 2016, 56, 282–289
http://dx.doi.org/10.1071/AN15576
Journal compilation CSIRO 2016 www.publish.csiro.au/journals/an
marine). Both freshwater and marine macroalgae are used in
human nutrition, cosmetics, and pharmaceutical products (Paul
and Tseng 2012). The opportunity to use macroalgae as a feed
additive for livestock is growing due to the increasing exploitation
of algae for other purposes such as bioremediation. Macroalgae
are unique in their rich and diverse lipid and tannin content and
secondary metabolites, which in some cases have demonstrated
antimethanogenic properties (Wang et al.2008; Kinley and
Fredeen 2015).
One novel antimethanogenic strategy seeks to harness the
effect of secondary metabolites found in some macroalgae.
These have been demonstrated to be variable in effect on
in vitro fermentation in a dose-dependent manner (Dubois et al.
2013; Machado et al.2015b). Machado et al.(2014) reported
the antimethanogenic effect of 20 different macroalgae species
in vitro when fermented with a low quality dry rangeland Rhodes
grass (Chloris qayana). This work demonstrated that at high
inclusion rates (17% OM basis) highly variable effects
on methanogenesis were possible. Asparagopsis is a marine
genus of red macroalgae characterised by secondary
metabolites with antibacterial properties (Paul et al.2006) that
demonstrates a potent antimethanogenic effect in vitro
(Machado et al.2015b).
It was hypothesised that the red macroalga Asparagopsis
taxiformis at low inclusion rates can dramatically reduce CH
4
emissions from in vitro fermentations with rumen fluid (RF)
without detrimental effects on fermentation while using a grass
feed substrate. The objective of this study was to demonstrate
the in vitro antimethanogenic potency of the red macroalga
Asparagopsis taxiformis at low inclusion rates over 72 h using
an irrigated Rhodes grass as the feed substrate. The effects on
parameters of rumen fermentation were examined using
standardised in vitro culture methods.
Materials and methods
Preparation of macroalgae and Rhodes grass substrate
The Asparagopsis taxiformis (hereafter Asparagopsis) was
harvested from Nelly Bay, Magnetic Island (19160S,
146850E) near Townsville, Qld, Australia. The macroalga
biomass was rinsed in seawater for 2 min then dipped in
freshwater to remove residual salt to maximise alga OM
content. The clean biomass was placed in 100-mm mesh and
centrifuged at 1000gfor 6 min at ambient temperature in a
commercial washing machine to remove excess water and then
stored at 10C. The biomass was then freeze-dried (SP
Industries VirTis K, Warminster, PA, USA) and ground to 1
mm and stored at 10C. The high quality Rhodes grass (HQR)
was grown under irrigation and harvested locally. Subsamples of
HQR were air-dried and ground to 1 mm. Table 1describes the
composition of the Asparagopsis and HQR biomass used as the
fermentation substrates. Dry matter was determined by
achievement of constant weight at 105C, and OM was
measured as loss on combustion at 550C for 8 h (Horwitz
2000). Neutral and acid detergent fibre were determined using
an Ankom (Macedon, NY, USA) model 200 fibre analyser.
Crude protein content was determined using a LECO (St
Joseph, MI, USA) model CHN628 series nitrogen analyser.
Donor animals and preparation of RF inoculum
Rumen fluid inoculum was collected from four fistulated
Brahman steers (Bos indicus; LW 490 45 kg) fitted with 10-
cm Bar Diamond (Parma, OH, USA) rumen cannulas. The steers
were maintained at the College of Public Health, Medical and
Veterinary Sciences at James Cook University (Townsville)
according to current guidelines (NHMRC 2013) and approved
by the local animal ethics committee (A5/2011). The steers were
maintained on Rhodes grass ad libitum for 6 months before the
collection of RF, which was extracted 2 h after morning feeding
by sampling from four quadrants of the rumen and hand-
squeezing to completely fill pre-warmed 1-L stainless steel
thermal flasks.
Inoculation and in vitro fermentation
The RF was pooled and immediately processed by filtration
through a 0.5-mm sieve and combined with Goering and van
Soest (1970) buffer (GVB) at a ratio of 1 : 4 (RF : GVB).
Maintenance of 39C and mixing of the RF buffer
fermentation media (RFB) was continuous to ensure
homogeneity (Major Science SWB 20 L-3; Saratoga, CA,
USA). The full system was N
2
purged and a Dose-It pump
(Integra Biosciences, Hudson, NH, USA) was used to aspirate
125 mL of RFB into incubation bottles containing the
Asparagopsis and HQR. The bottles were sealed with an
Ankom RF1 gas production module (Macedon, NY, USA) and
placed in an incubator (Ratek OM11; Boronia, Vic., Australia)
maintained at 39C and oscillating at 85 RPM.
Experimental design
To characterise the effect of Asparagopsis on in vitro rumen
fermentation a series of 20 incubation periods were conducted
using five Asparagopsis dose rates ranging from 0.5% to 10%
(OM basis) of the HQR substrate OM and compared with a
control (no macroalgae) and RFB blanks. Each dose level
was characterised in duplicated incubation periods containing
quadruplicate repetitions at each sampling time point (12, 24,
36, 48, and 72 h). Controls and blanks were included in all
periods and time points in duplicate. Each 72-h fermentation
series was split into two periods with the first monitoring at 12,
24 and 36 h [plus a 6-h sample for in vitro apparent digestibility
of substrate OM (IVD-OM only)] and the second monitoring
36, 48, and 72 h. The data was then combined to provide time
series curves covering the full 72 h. Each fermentation contained
1.0 g of the HQR (OM basis) and appropriate quantities of
Table 1. Nutritional composition of the Rhodes grass substrate and
Asparagopsis biomass (g/kg DM unless stated otherwise)
Composition Rhodes grass Asparagopsis
Dry matter (g/kg as used) 916 945
Organic matter 878 811
Crude protein 167 252
Neutral detergent fibre
A
645 –
Acid detergent fibre 315 –
A
Without aamylase.
Asparagopsis eliminates methane from fermentations Animal Production Science 283
Asparagopsis to achieve dose rates of 0.5%, 1%, 2%, 5% and
10% according to the biomass composition described in Table 1.
Fermentation monitoring and sample analyses
Total gas production
The methods used in this study were similar to those described
by Cone et al.(1996), Pellikaan et al.(2011) and Machado
et al.(2014). Total gas production (TGP) was measured
continuously for a maximum of 72 h. The Ankom parameter
settings were kept constant with maximum pressure in the
fermentation bottle of 3 psi, which when exceeded, would vent
for 250 ms and the pressure change accounted in the cumulative
pressure recording. Gas pressure was measured every 60 s and
cumulative pressure was recorded at 20-min intervals. The
cumulative TGP expressed in mL/g of substrate OM was
determined by application of the natural gas law to the
accumulation of the recorded gas pressure while accounting
for individual bottle volume.
Methane production
In vitro CH
4
production was determined and time series
production curves prepared by collection of samples at
multiple time points. Production in mL CH
4
/g of substrate OM
was estimated by application of CH
4
concentration in time series
samples using the relative TGP while assuming constant
homogeneity of bottle headspace. Concentration of CH
4
in
headspace collected in 10-mL Labco Exetainer vacuum vials
(Lampeter, Great Britain) were measured by gas chromatography
(GC) on a Shimadzu GC-2014 (Kyoto, Japan) equipped with a
Restek (Bellefonte, PA, USA) ShinCarbon ST 100/120 column
(2 m ·1mm·micropacked) with a flame ionisation detector
(FID). Column temperature was 150C, injector was 240C, and
380C in the FID. Ultra high purity N
2
was the carrier gas at
25 mL/min and injection volume was 250 mL.
In vitro apparent digestibility of substrate OM
The IVD-OM was quantified to coincide with CH
4
determinations (plus a 6-h sample for IVD-OM only). The
fermentation was chilled to terminate bacterial activity then
in vitro fluid (IVF) was vacuum filtered through a Duran No. 1
porosity glass fritted crucible with a 0.5-cm layer of sand filtration
aid. The crucible and fermentation residue was oven-dried to
constant weight at 105C for DM determination. Residue OM
was determined as loss on ignition in a muffle furnace at 550C
for 8 h (Carbolite AAF 11/18; Derbyshire, Great Britain).
Volatile fatty acid production
Volatile fatty acids (VFA) in the IVF were quantified after 72 h
of fermentation. The preparation of IVF for VFA analysis was
at a ratio of 4 mL of IVF to 1 mL of 20% metaphosphoric acid
spiked to 11 mM with 4-methylvaleric acid (Sigma-Aldrich;
Castle Hill, NSW, Australia) as internal standard and stored at
20C. A 1.5-mL subsample was centrifuged for 15 min at
13 500gand 4C (Labnet Prism R; Edison, NJ, USA). The
supernatant was filtered through 0.2-mm PTFE syringe tip
filters (Agilent; Santa Clara, CA, USA) and analysed using
a Shimadzu GC17A equipped with a Restek Stabilwax
(30 m ·0.25 mm ·0.25 mm) fused silica column and
FID. The column was ramped from 90C to 155Cat3
C/min
and held for 8.3 min. The temperature was 220C in the injector
and 250C in the FID. Ultra high purity N
2
was the carrier gas at
1.5 mL/min and the injection was 1.0 mL.
Statistical analyses
Two-factor repeated-measures permutational analysis of
variance (PERMANOVA) was used to test for significant
differences in the TGP, CH
4
production, and, IVD-OM over
time and a one-factor PERMANOVA was used to test for
significant differences in the production of VFA between the
treatments (fixed factor) using Primer 6 (version 6.1.13) statistical
software and PERMANOVA+ (version 1.0.3; Clarke and
Gorley 2006). Data were also fitted with generalised additive
models to predict the relationship and examine differences
between TGP over time between treatments and differences in
the changes in the rates of TGP. The generalised additive models
were produced using the mgcv package within the R language
(version 3.0.1; R Core Team 2013).
Results
Asparagopsis had consistent effects on in vitro fermentations
and these effects were dose dependent. This study applied
a HQR (Table 1) as substrate and the dose rate of 2%
Asparagopsis (OM basis) was near the optimum dose as
measured by decrease in TGP, CH
4
abatement, stability of
IVD-OM, and beneficial changes in VFA concentrations.
Using HQR, the 1% dose was no more effective than 0.5%
(Fig. 1). However, there was a significant reduction in TGP of
~30% with the inclusion at 2% of Asparagopsis (P<0.001).
There was good reproducibility of TGP lending to standard
error (s.e.) of <1.5 mL/g representing 0.7% of the TGP values
at 214 mL/g substrate OM.
The inclusion of Asparagopsis had the effect of reducing
CH
4
production in a dose and time-dependent manner (Fig. 2).
Methane production had a similar trend as TGP. There was
minimal CH
4
produced with 1% Asparagopsis inclusion in the
first 24 h, however after 24 h CH
4
production began to increase
rapidly. After 48 h there was no longer measurable difference in
CH
4
between the 0.5% and 1% dose rates. The prominent effect
occurred at dose levels 2% (P<0.001) and no detectable CH
4
was produced. Variability in CH
4
production between periods
was greater than that observed for other variables monitored in
this study.
Substrate degradability in vitro was not affected by the
inclusion of Asparagopsis 5.0% (OM basis). There was no
difference in IVD-OM over 72-h fermentations between the
control and dose rates up to 5% of substrate OM. However,
10% Asparagopsis induced significant reduction in IVD-OM
(P<0.001). The comparison of IVD-OM between dose rates
and the control was consistent and independent of time. The
IVD-OM variability within- and between periods was small as
reflected by the small s.e. (Fig. 3).
In Fig. 4it was shown that Asparagopsis at doses 2%
had little effect on total VFA (TVFA) after 72 h of
fermentation and little change was induced by dose rates <5%,
however with 5% the TVFA was decreased (P<0.05) and more
284 Animal Production Science R. D. Kinley et al.
so at 10% (P<0.001). Although TVFA was not acutely sensitive
to low level inclusion of Asparagopsis there was a trend towards
reduced TVFA with increased dose. For individual VFA a
significant change was demonstrated (P<0.05) and the effect
was magnified with increasing dose (P<0.001). As the dose rate
of Asparagopsis increased through the 0.5%, 1%, 2%, 5%, and
10% dose range the acetate concentrations decreased by 4%,
11%, 29%, 41%, and 61%, respectively, compared with the
control. For propionate the concentrations increased by 13%,
0%, 56%, 88%, and 106% for the same dose range, respectively.
For butyrate the increases were 67% and 116% for the 2% and
10% doses, respectively. These changes in acetate and propionate
concentrations reduced the acetate :propionate ratio. With little
effect on TVFA and a shift to lower acetate and greater
200
150
100
50
Total gas production (mL/g OM)
Time (h)
Control
0.5%
1%
2%
5%
10%
0
0 1224364872
Fig. 1. The time series effect of increasing dose rate of Asparagopsis on in vitro total gas production (mL/g organic matter).
Control was a high quality Rhodes grass hay. Error bars are not shown as they were smaller in size then the symbols used.
30
25
20
15
10
5
0
12 24 36 48 72
Time (h)
Control
0.5%
1%
2%
5%
10%
CH4 (mL/g OM)
Fig. 2. The time series effect of increasing dose rate of Asparagopsis on mean (s.e.m.) in vitro methane production
(mL/g organic matter). Control was a high quality Rhodes grass hay.
Asparagopsis eliminates methane from fermentations Animal Production Science 285
propionate, there was no negative impact on VFA production
due to low dose (<5%) Asparagopsis inclusion in vitro using
HQR substrate.
Discussion
The red seaweed Asparagopsis has a large CH
4
abatement
capacity compared with other natural products when included
at low dose in rumen fermentations in vitro. The effect of
Asparagopsis demonstrated in this study agreed with Machado
et al.(2015b), which described end-point results after 72 h of
fermentation that demonstrated dose sensitivity to Asparagopsis
using low quality Rhodes grass (LQR) substrate. Conspicuously
however, in their study there was an abrupt reduction in TGP
and CH
4
production occurring at 1% Asparagopsis rather than
the 2% in this study (Figs 1,2). Consequently, this effective dose
0.8
0.7
0.6
0.5
0.4
0.3
Coefficient of digestibility of OM
0.2
0.1
0
126024364872
Time (h)
Control
0.5%
1%
2%
5%
10%
Fig. 3. The time series effect of increasing dose rate of Asparagopsis on mean (s.e.m.) in vitro apparent organic matter
digestibility. Control was a high quality Rhodes grass hay.
45
40
35
30
25
20
VFA production (mM)
15
10
5
0
Total Acetic Propionic Butyric
Control
0.5%
1%
2%
5%
10%
Fig. 4. The effect of increasing dose rate of Asparagopsis on mean (s.e.m.) in vitro total volatile fatty acid, acetic, propionic
and butyric acid concentrations after 72 h of fermentation. Control was a high quality Rhodes grass.
286 Animal Production Science R. D. Kinley et al.
difference observed with variable grass quality may follow
a different pattern between grass types or when grain-based
substrates are used. Therefore, it is essential to evaluate the
effects using the various ruminant feeding systems. Using
HQR the 1% dose of Asparagopsis was no more effective than
0.5%, and thereafter TGP and CH
4
declined with increasing
dose levels. It is an important distinction that the fermentation
response to Asparagopsis may also be dependent upon the
quality of the substrate. Therefore, the requirements for
Asparagopsis biomass may be only half when feeding LQR
compared with HQR. Ruminant production systems utilising
low quality forage as the primary feed would require less
Asparagopsis to achieve equivalent CH
4
abatement.
The production of CH
4
was virtually undetectable at dose
rates 2% OM basis (Fig. 2). For this reason the CH
4
results
were not blank corrected, doing so would produce confusing
negative values because Asparagopsis-treated fermentations
produced less CH
4
than the blanks. However, occasionally
during other in-house rumen in vitro experiments using a 2%
dose (data not shown) a small rise in CH
4
was observed after
~36 h. This occasional rise in CH
4
still provided at minimum an
abatement of >85% compared with the control. Those
fermentations used the HQR substrate with the only difference
being the RFB. At the 1% Asparagopsis dose rate there was
a typical rise in CH
4
from undetectable to ~20 mL/g HQR
beginning between 24 and 36 h of fermentation. All
experimental periods demonstrated that at an Asparagopsis
dose rate 2% of substrate OM typically results in
undetectable in vitro methanogenesis. At 5% the CH
4
production was always undetectable. Feed energy is typically
lost as CH
4
at a rate of up to 12% of gross energy intake (Johnson
and Johnson 1995). Using Asparagopsis this energy may be
conserved in the rumen for productive use by the ruminant
animal at some undefined level which further reduces the cost
of abatement. This proportion of retained energy can be quantified
with in vivo feeding studies that closely monitor feed intake,
CH
4
production, and productivity.
It is known that the antibacterial defence mechanism of
Asparagopsis is predominantly a result of the secondary
metabolite bromoform (CHBr
3
) naturally present in the
macroalgal biomass (Paul et al.2006). Bromoform is similar
chemically and in antimethanogenic potency to that of
bromochloromethane (BCM; CH
2
BrCl). In previous in vivo
experiments investigating enteric CH
4
abatement, BCM
induced abatement in Brahman steers of 93% and 50% after
separate 28 and 90 days feeding regimes, respectively (Tomkins
et al.2009). However, BCM has been banned from manufacture
and use in Australia due to its contribution to ozone depletion.
The mode of action of BCM was described previously as
inhibition of the methanogenic pathway at the final step by
inhibition of the cobamide-dependent methyl transferase step
in release of CH
4
(Denman et al.2007). In that study inhibition of
methanogenesis occurred immediately however the methanogen
populations were only found to be reduced after several hours,
thus the observed lag in the population decline suggested that
the inhibition of methanogenesis directly affected growth of
methanogens. They also commented that BCM would be
removed from the rumen due to ruminal flow and unless it was
replaced CH
4
inhibition would decline, which could not be
observed during our in vitro batch culture. However, a decline
in inhibition was observed with the 1% inclusion (Fig. 2)
and was presumably associated with consumption of the
antimethanogenic capacity of Asparagopsis at very low dose
levels.
The naturally occurring secondary metabolites in
Asparagopsis armata a temperate species closely related to the
tropical Asparagopsis used in the present study include di-BCM
(CHBr
2
Cl) at low levels of <0.1% of algal DM, but also
bromoform, which at higher levels of ~1.7% (Paul et al.2006)
is considered to be the bioactive agent responsible for most
of the CH
4
abatement acting in the same way as BCM. The
naturally occurring secondary metabolites of Asparagopsis have
demonstrated activity in vitro at dose rates of 1% (Machado et al.
2015b) and 2% (substrate OM basis) in this study. Thus, the
inclusion rate for large abatement of CH
4
and degradation of the
bioactive metabolite may be managed. However, intensive study
of Asparagopsis CH
4
abatement efficacy in cattle and sheep is
required.
Reduction effect demonstrated in TGP and CH
4
production at low dose levels of Asparagopsis were not
reflected equivalently in the IVD-OM results, which remained
unchanged until the 10% dose was used. Inclusion of
Asparagopsis had little effect on IVD-OM at dose levels 5%
of substrate OM, compared with controls (Fig. 3). However, all
experiments and studies with Asparagopsis demonstrated a
significantly reduced IVD-OM (P<0.001) at doses of 10%
(Machado et al.2014,2015b). The IVD-OM represented a
demonstration of stability in the fermentation and suggests
fibre digesting microbes were not affected by Asparagopsis at
low dose.
The primary source of energy for ruminant animals is the
VFA produced by rumen microbes during digestion of
carbohydrates (Bergman 1990) thus negative effects against
their production during rumen fermentation is undesirable. The
production of TVFA, acetate, and propionate were affected by
increasing dose of Asparagopsis. At doses between 1% and
2% the decrease in TVFA was not significant with the HQR
substrate used in this study; however, this is not in agreement
with a study using LQR where there was a significant decrease
in TVFA (Machado et al.2015b). This may indicate that VFA
production may be more sensitive to Asparagopsis in LQR
possibly due to higher levels of indigestible fibre and lower
protein. However, IVD-OM was similar in both studies and
not affected at 1% versus 2% doses, thus the mechanism of the
effect is unclear. Generally, TVFA was reduced as the dose level
increased. The inclusion of Asparagopsis at low dose levels
induced a beneficial change in VFA in favour of propionate as
is common with antimethanogenic inclusions. This is believed
to be due to competition for the excess hydrogen and reductive
propionate production is more favourable than acetogenesis in
these conditions (Mitsumori et al.2012), which may be enhanced
by Asparagopsis. It was demonstrated for HQR that significant
changes (P<0.001) in individual VFA concentrations can be
achieved. In the present study acetate concentrations decreased
and propionate and butyrate increased with increasing doses of
Asparagopsis. Changes in acetate and propionate concentrations
also reduced the acetate : propionate ratio, which could be
partially responsible for corresponding decreases observed
Asparagopsis eliminates methane from fermentations Animal Production Science 287
in CH
4
production in vitro (Beauchemin et al.2009). The
reasoning is that propionate acts as a hydrogen sink; however,
production of acetate and butyrate liberates hydrogen thus
providing for greater ruminal reduction of CO
2
into CH
4
by
methanogens.
Recent studies reporting the antimethanogenic effect of
various macroalgae in rumen fermentations has demonstrated
variable responses (Dubois et al.2013; Kinley and Fredeen
2015; Machado et al.2014). Some macroalgae have
previously indicated potential for enteric CH
4
abatement;
however, Asparagopsis stands out as the most potent. Other
macroalgae, particularly the green species appear to be
most suitable as novel protein sources with little value as CH
4
abatement agents for ruminants. The production of Asparagopsis
at a scale large enough for feeding livestock requires development
before commercialisation as a functional feed ingredient. It is
unclear how various methods of drying Asparagopsis biomass
and subsequent storage will affect levels of secondary metabolites
and antimethanogenic potency thus characterisation of the most
appropriate methods is required.
Conclusions
A dose of Asparagopsis at 1% of substrate OM exhibited a
significant reduction of CH
4
in vitro, and at 2%
demonstrated virtual elimination of CH
4
with minimal effect
on fermentation efficiency of HQR. There was no impact on
IVD-OM at dose levels 5% and the effect on VFA was a
decrease in acetate with a concomitant increase in propionate
and to a lesser degree for butyrate. Other areas requiring
characterisation is the most appropriate method for processing
(dehydration) and feeding to livestock in systems with variable
feed quality and content. Nevertheless, using in vitro assessment
methods the use of Asparagopsis at low inclusion levels in
ruminant diets has demonstrated large CH
4
abatement as a
natural product.
References
Beauchemin KA, McAllister TA, McGinn SM (2009) Dietary mitigation of
enteric methane from cattle. CAB Reviews No. 035 Perspectives in
Agriculture, Veterinary Science, Nutrition and Natural Resources 4,
1–18. doi:10.1079/PAVSNNR20094035
Bergman EN (1990) Energy contributions of volatile fatty acids from the
gastrointestinal tract in various species. Physiological Reviews 70,
567–590.
Clarke KR, Gorley RN (2006) ‘Primer v6 user manual/tutorial.’(PRIMER-E
Ltd: Plymouth, UK)
Cone JW, van Gelder AH, Visscher GJW, Oudshoorn L (1996) Influence of
rumen fluid and substrate concentration on fermentation kinetics
measured with a fully automated time related gas production
apparatus. Animal Feed Science and Technology 61, 113–128. doi:org/
10.1016/0377-8401(96)00950-9
Denman SE, Tomkins NW, McSweeney CS (2007) Quantitation and
diversity analysis of ruminal methanogenic populations in response
to the antimethanogenic compound bromochloromethane. FEMS
Microbiology Ecology 62, 313–322. doi:10.1111/j.1574-6941.2007.
00394.x
Dubois B, Tomkins NW, Kinley RD, Bai M, Seymour S, Paul NA, de Nys R
(2013) Effect of tropical algae as additives on rumen in vitro gas
production and fermentation characteristics. American Journal of Plant
Sciences 4,34–43. doi:10.4236/ajps.2013.412A2005
Goering HK, van Soest PJ (1970) ‘Forage fiber analyses (apparatus, reagents,
procedures, and some applications) Agriculture Handbook no. 379.’
(ARS-USDA: Washington, DC)
Henry B, Charmley E, Eckard R, Caughan JB, Hegarty R (2012)
Livestock production in a changing climate: adaptation and mitigation
research in Australia. Crop and Pasture Science 63, 191–202. doi:10.
1071/CP11169
Horwitz W (Ed.) (2000) ‘Official methods of AOAC international.’17th edn.
(Association of Official Analytical Chemists International: Gaithersburg,
MD)
IPCC (2014) Climate change 2014: synthesis report. Contribution of
Working Groups I, II, and III to the Fifth Assessment Report of the
Intergovernmental Panel on Climate Change. (Eds RK Paachauri,
LA Meyer) (IPCC: Geneva, Switzerland)
Johnson KA, Johnson DE (1995) Methane emissions from cattle. Journal
of Animal Science 73, 2483–2492.
Kinley R, Fredeen A (2015) In vitro evaluation of feeding North Atlantic
stormtoss seaweeds on ruminal digestion. Journal of Applied Phycology
27, 2387–2393. doi:10.1007/s10811-014-0487-z
Kirschke S, Bousquet P, Ciais P, Saunois M, Canadell JG, Dlugokencky EJ,
Bergamaschi P, Bergmann D, Blake DR, Bruhwiler L, Cameron-Smith P
et al. (2013) Three decades of global methane sources and sinks. Nature
Geoscience 6, 813–823. doi:10.1038/ngeo1955
Machado L, Magnusson M, Paul NA, de Nys R, Tomkins NW (2014)
Effects of marine and freshwater macroalgae on in vitro total gas and
methane production. PLoS One 9, e85289. doi:10.1371/journal.pone.
0085289
Machado L, Kinley RD, Magnusson M, de Nys R, Tomkins NW (2015a)
The potential of macroalgae for beef production systems in
Northern Australia. Journal of Applied Phycology 27, 2001–2005.
doi:10.1007/s10811-014-0439-7
Machado L, Magnusson M, Paul NA, Kinley RD, de Nys R, Tomkins
NW (2015b) Dose-response effects of Asparagopsis taxiformis and
Oedogonium sp. on in vitro fermentation and methane production.
Journal of Applied Phycology. doi:10.1007/s10811-015-0639-9
Mitsumori M, Shinkai T, Takenaka A, Enishi O, Higuchi K, Kobayashi Y,
Nonaka I, Asanuma N, Denman SE, McSweeney CS (2012) Responses in
digestion, rumen fermentation and microbial populations to inhibition
of methane by a halogenated methane analogue. British Journal of
Nutrition 108, 482–491. doi:10.1017/S0007114511005794
Morgavi D, Forano E, Martin C, Newbold CJ (2010) Microbial ecosystem
and methanogenesis in ruminants. Animal 4, 1024–1036. doi:10.1017/
S1751731110000546
NHMRC (2013) ‘Australian code for the care and use of animals for scientific
purposes.’8th edn. (National Health and Medical Research Council:
Canberra)
Olivier JGJ, van Aardenne JA, Dentener FJ, Pagliari V, Ganzeveld LN, Peters
JAHW (2005) Recent trends in global greenhouse gas emissions: regional
trends 1970–2000 and spatial key sources in 2000. Environmental
Sciences 2,81–99. doi:10.1080/15693430500400345
Patra AK (2012) Enteric methane mitigation technologies for ruminant
livestock: a synthesis of current research and future directions.
Environmental Monitoring and Assessment 184, 1929–1952.
doi:10.1007/s10661-011-2090-y
Paul N, Tseng CK (2012) Seaweed. In ‘Aquaculture: farming aquatic
animals and plants’. 2nd edn. (Eds JS Lucas, PC Southgate)
pp. 268–284. (Blackwell Publishing Ltd: Oxford)
Paul N, de Nys R, Steinberg P (2006) Chemical defence against bacteria
in the red alga Asparagopsis armata: linking structure with function.
Marine Ecology Progress Series 306,87–101. doi:10.3354/meps30
6087
288 Animal Production Science R. D. Kinley et al.
Pellikaan WF, Hendriks WH, Uwimana G, Bongers LJGM, Becker PM, Cone
JW (2011) A novel method to determine simultaneously methane
production during in vitro gas production using fully automated
equipment. Animal Feed Science and Technology 68, 196–220. doi:
org/10.1016/j.anifeedsci.2011.04.096
R Core Team (2013) ‘R: A language and environment for statistical
computing.’(R Foundation for Statistical Computing: Vienna, Austria)
Available at http://www.R-project.org/ [Verified 11 November 2015]
Tomkins NW, Colegate S, Hunter R (2009) A bromochloromethane
formulation reduces enteric methanogenesis in cattle fed grain-based
diets. Animal Production Science 49, 1053–1058. doi:10.1071/EA08223
Wang Y, Xu Z, Bach S, McAllister T (2008) Effects of phlorotannins from
Ascophyllum nodosum (brown seaweed) on in vitro ruminal digestion of
mixed forage or barley grain. Animal Feed Science and Technology 145,
375–395. doi:10.1016/j.anifeedsci.2007.03.013
Asparagopsis eliminates methane from fermentations Animal Production Science 289
www.publish.csiro.au/journals/an
