ArticlePDF Available

Abstract and Figures

Asparagopsis taxiformis concentrates halogenated compounds which are known to inhibit cobamide-dependent methanogenesis in vitro and therefore has potential to mitigate enteric methane production. This study investigated the effect of Asparagopsis on methane (CH4) production from sheep offered a high fibre pelleted diet (offered at 1.2 x30 maintenance) at five levels of Asparagopsis for 72 d (0% (control), 0.5%, 1%, 2% and 3% organic matter -OM basis as offered ). Individual animal methane measurements were conducted at 21 d intervals using pen circuit respiration chambers. Asparagopsis inclusion resulted in a consistent and dose dependent reduction in enteric methane production over time, with up to 80% methane mitigation at the 3% offered rate compared with the group fed no Asparagopsis (P < 0.05). Sheep fed Asparagopsis had significantly lower concentration of total volatile fatty acids and acetate, but higher propionate concentration. No changes in live weight gain were identified. Supplementing Asparagopsis in a high fiber diet (< 2% OM) results in significant and persistent decreases in enteric methanogenesis over a 72 d period. Granulomatous and keratotic ruminal mucosa changes were identified in several sheep with Asparagopsis supplementation. While the outcomes of this study may be extrapolated to feedlot to achieve the antimethanogenic effect associated with Asparagopsis, further work is required to define the long term effects on productivity and animal health.
Content may be subject to copyright.
Asparagopsis taxiformis decreases enteric methane
production from sheep
Xixi Li
A,F
, Hayley C. Norman
A
, Robert D. Kinley
B
, Michael Laurence
C
, Matt Wilmot
A
,
Hannah Bender
C
, Rocky de Nys
D
and Nigel Tomkins
B,E
A
CSIRO Agriculture, Centre for Environment and Life Sciences, Floreat, WA 6014, Australia.
B
CSIRO Agriculture, Australian Tropical Sciences and Innovation Precinct James Cook University,
Townsville, Qld 4811, Australia.
C
College of Veterinary Medicine, Murdoch University, Murdoch, WA 6150, Australia.
D
MACRO, The Centre for Macroalgal Resources and Biotechnology, College of Marine and Environmental
Sciences, James Cook University, Townsville, Qld 4811, Australia.
E
Meat and Livestock Australia, 527 Gregory Terrace, Fortitude Valley, Qld 4006, Australia.
F
Corresponding author. Email: xixili.r@gmail.com
Abstract. Asparagopsis taxiformis concentrates halogenated compounds that are known to inhibit cobamide-dependent
methanogenesis in vitro and, therefore, has potential to mitigate enteric methane production. The present study investigated
the effect of Asparagopsis on methane (CH
4
) production from sheep offered a high-bre pelleted diet (offered at 1.2 ·
maintenance) at ve inclusion levels of Asparagopsis for 72 days (0% (control), 0.5%, 1%, 2% and 3% organic matter basis
as offered). Individual animal CH
4
measurements were conducted at 21-day intervals using open-circuit respiration
chambers. Asparagopsis inclusion resulted in a consistent and dose-dependent reduction in enteric CH
4
production over
time, with up to 80% CH
4
mitigation at the 3% offered rate compared with the group fed no Asparagopsis (P<0.05). Sheep
fed Asparagopsis had a signicantly lower concentration of total volatile fatty acids and acetate, but a higher propionate
concentration. No changes in liveweight gain were identied. Supplementing Asparagopsis in a high-bre diet (<2% organic
matter) resulted in signicant and persistent decreases in enteric methanogenesis over a 72-day period. Granulomatous
and keratotic ruminal mucosa changes were identied in several sheep with Asparagopsis supplementation. While the
outcomes of the present study may be extrapolated to feedlot to achieve the antimethanogenic effect associated with
Asparagopsis, further work is required to dene the long-term effects on productivity and animal health.
Additional keywords: halogenated compounds, macroalgae, methanogenesis, ruminal fermentation.
Received 20 December 2015, accepted 2 August 2016, published online 28 September 2016
Introduction
Enteric fermentation from ruminant livestock contributes
approximately 10% of total greenhouse-gas emissions across
Australia (Australians National Greenhouse Accounts 2014).
Use of an antimethanogenic compound in the diet of farmed
ruminants could reduce greenhouse-gas emissions from the
livestock sector. Diet manipulation is regarded as the most
direct and effective approach to lowering methane (CH
4
)
emissions from ruminant production systems (Beauchemin
et al.2008). Chemical additives, such as bromochloromethane
(BCM), have been shown to decrease CH
4
production from steers
(Tomkins et al.2009) and decreases methanogenic archaea by
34% (Denman et al.2007). BCM can react with reduced vitamin
B
12
; thus, BCM inhibits cobamide-dependent methyl group,
leading to methanogensis, and inhibits CH
4
production (Wood
et al.1968). However, BCM was prohibited by the Australian
Government in 2004 under the Ozone Protection and Synthetic
Greenhouse Gas Management Act 1989.
Macroalgae are used in the nutraceutical and health markets
and have demonstrated antibacterial, anti-viral, antioxidant or
anti-inammatory properties (OSullivan et al.2010). There has
been interest in their use to improve livestock health and
productivity. Antimethanogenic compounds are also known to
exist naturally in macroalgae in varying concentrations. The red
marine macroalgae Asparagopsis taxiformis produces haloforms
and dihalomethanes (Burreson et al.1976) in specialised glands,
as a natural defence against disease and marine herbivory
(Paul et al.2006b). The bioactives from Asparagopsis have
been identied and shown to reduce CH
4
production in vitro
(Machado et al.2016a). These include bromoform and
dibromochloromethane, which are likely to exhibit the same
mode of action as BCM, making Asparagopsis a potential
natural CH
4
inhibitor (Paul et al.2006a). In 72-h in vitro
fermentation studies, addition of Asparagopsis to Flinders
grass (Iseilema spp.) or Rhodes grass (Chloris gayana) hay
has been shown to decrease CH
4
production by over 90%
CSIRO PUBLISHING
Animal Production Science
http://dx.doi.org/10.1071/AN15883
Journal compilation CSIRO 2016 www.publish.csiro.au/journals/an
(Machado et al.2014,2016b). Machado et al.(2016b) observed
a 99% reduction in CH
4
and a decrease in volatile fatty acids
(VFAs) when Asparagopsis was included at 2% organic matter
(OM) in in vitro fermentations. It is hypothesised that inclusion
of Asparagopsis in the diet of livestock would reduce enteric
CH
4
emissions and that there would be an appropriate inclusion
level at which animals would maintain normal rumen function.
The aim of the present study was to characterise the effect of
inclusion rates of Asparagopsis on enteric CH
4
emissions from
Merino wethers fed a high-bre pelleted diet. The study also
considered the effect on rumen fermentation and general animal
health over a 72-day feeding period.
Materials and methods
The experiment was conducted at the CSIRO Centre for
Environment and Life Sciences, Floreat, Western Australia.
The experimental protocol was approved by the CSIRO
Centre for Environment and Life Sciences Animal Ethics
Committee (AEC 1404, 2014), and was conducted under the
Australian Code of Practice for Care and Use of Animals for
Scientic Purposes (NHMRC 2013).
Basal diet and Asparagopsis
The basal diet was a commercial pelleted ration (16% lupin seed,
8% oat grain, 8% barley grain, 8% wheat grain, 17% oat hulls,
40% cereal straw, 1.4% calcium hydroxide, 1% CSIRO pellet
pre-mix, 0.3% salt and 0.3% gypsum, Macco Feeds, Williams,
WA, Australia). Additional crushed lupin seed was fed to all
sheep as a carrier mixed with or without the Asparagopsis.
Biomass of wild Asparagopsis was harvested from a site near
Humpy Island, Keppel Bay (23130S, 15054.80E) on the
Capricorn Coast, central Queensland. The biomass was rinsed
in clean sea water to remove sand and fouling organisms then
dried in a forced-air solar kiln at 45C for 72 h. Prior to feeding,
it was ground in a mill with a 3-mm sieve.
Proximate analysis was determined on bulked samples
of Asparagopsis, pellets and lupins collected throughout the
feeding period (Table 1). Plant samples were dried at 65C for
48 h, ground through a 1-mm sieve using a Tecator grinder (Foss,
Hillerød, Denmark) and analysed for dry matter (DM) and ash
(Faichney and White 1983), neutral detergent bre and acid
detergent bre (AFIA 2009; Ankom 200/220 bre analyser;
Ankom Technology Co., Macedon, NY, USA). Nitrogen (N)
concentration was analysed according to AOAC method 990.03
using TruSpec Micro (AOAC 2005; LECO corporation, Sydney,
NSW, Australia). Crude protein was calculated as N ·6.25 for
lupin seed and the basal diet, and N ·4.59 for Asparagopsis
(Louren¸co et al.2002).
Animals and feeding
Merino-cross wethers (n= 29, 2-year old) were housed in
individual pens throughout the experimental period, in a
raised, enclosed animal house with slatted wooden oor.
Sheep had an initial mean (s.e.m.) liveweight (LW) of 66
1.03 kg and were dosed with a Co bullet (Coopers animal
Health, Sydney, NSW, Australia) before the experiment.
Sheep were fed once daily at 0830 hours and had free access
to water. Individual animal LW and body condition score (15)
were measured at 14-day intervals (Suiter 1994).
During the initial adaptation period, sheep were fed 1.2 kg
basal diet (~1.2 times their metabolisable-energy requirement
for maintenance) for 21 days and acclimatised to animal-house
conditions and periods of connement in respiration chambers.
During treatment period, total diet consisted of 1.2 kg basal
diet and 200 g of crushed lupin seed offered with the
following ve rates of Asparagopsis: 0 (control), 13, 26, 58
and 80 g/day (equivalent to 0%, 0.5%, 1.0%, 2.0%, 3.0% OM
basis respectively). Sheep were randomly allocated to one of
ve treatments in a randomised design, with ve sheep per
Asparagopsis inclusion (plus one spare) and four sheep on
control diet (plus one spare), and fed over a 72-day period.
Treatment diets were gradually introduced over 2 weeks to
ensure that sheep were each offered a full inclusion level of
Asparagopsis for the same period before measuring enteric
CH
4
production in open-circuit respiration chambers. On
the day of CH
4
measurement, feed on offer (pellets + lupins +
Asparagopsis) was proportionally reduced to ~1.0 ·maintenance
(0.5% Asparagopsis: 900 g pellet + 100 g lupins + 10 g
Asparagopsis;1%Asparagopsis: 900 g pellet + 100 g lupins +
19 g Asparagopsis;2%Asparagopsis: 900 g pellet + 100 g lupins
+39gAsparagopsis; and 3% Asparagopsis: 900 g pellet + 100 g
lupins + 58 g Asparagopsis) to encourage consistent intakes
for each measurement period and to reduce individual animal
variability in feed intake when conned in respiration chambers.
Feed intake was recorded daily as the difference between the
amount offered and the amount refused. Animals with greater
than 50% refusal over three consecutive days were removed
from the experiment according to the animal ethics protocol.
Some sheep would not consume all of the Asparagopsis,
particularly at the higher levels of inclusion. The range of
voluntary Asparagopsis intake for the two high-inclusion
rates of Asparagopsis (2.0% and 3.0% OM intake basis) was
determined on the refusals of Asparagopsis from 11 sheep
collected over 11 days.
At the completion of the experimental feeding period, 12
sheep were selected from the treatment groups (control: n=2;
0.5% Asparagopsis:n=2;1%Asparagopsis:n=3;2%
Asparagopsis:n= 2; and 3% Asparagopsis:n= 3), and
transported by road to the College of Veterinary Medicine,
Murdoch University. Animals were euthanised (pentobarbital
sodium, 160 mg/kg intravenously), necropsied and tissues
were collected for histopathological examination. Fat and
muscle from each sheep were collected, immediately frozen on
dry ice and stored in a freezer at 40C, before being sent to
Table 1. Composition of Asparagopsis taxiformis, lupin seeds and
pellets offered to Merino-cross wethers over 72 days (on a DM basis
unless otherwise noted)
Component Asparagopsis Lupins Pellet
DM (g/kg) 966 918 890
Organic matter 431 968 928
Crude protein 92 343 108
Neutral detergent bre 286 519
Acid detergent bre 251 338
BAnimal Production Science X. Li et al.
National Measurement Institute (Melbourne, Vic., Australia)
for analysis of bromoform and dibromochloromethane
concentrations by using gas chromatography and tandem mass
spectrometry.
Enteric CH
4
production using respiration chambers
Three measurement periods of CH
4
production (g/kg DM intake)
from individual animals were conducted at 21-day intervals
throughout the experimental period, using eight open-circuit
respiration chambers (Li 2014). CH
4
recovery was conducted
following the procedure of Klein and Wright (2006). Each
chamber was constructed of clear polycarbonate over an
aluminium frame, with an internal volume of 2.2 m
3
(interior:
length 1.6 m, width 0.86 m and height 1.6 m). Chambers were
tted with an automatic water supply and a feed bin. Slotted
ooring allowed urine and faeces to fall away from the animal and
underneath the chamber space. Mean airow through each
chamber was 0.35 L/min and chamber space was maintained
at 21C and 74% relative humidity. Data for ow rate,
temperature, chamber pressure and CH
4
concentrations in the
inlet and outlet air were managed in Excel to calculate methane
production expressed on a g/day basis. Air sampled from each
chamber and ambient air, at 5-min cycles, were analysed by two
gas chromatographs (GC2014; Shimadzu Corporation, Kyoto,
Japan). Sample injection, data acquisition and calculation of
CH
4
peak area were achieved using Shimadzu GC solutions
software (Ver2.3, Shimadzu Corporation, Kyoto, Japan). The
GCs were calibrated three times each day (0900 hours, 1200 hours
and 1530 hours) by using 100 mg/L CH
4
in a N Micromat-14 gas
standard mix and 10 mg/L CH
4
in a N standard mix (BOC Gas,
Perth, Australia).
Sheep were fed immediately after entry to the chambers at
0900 hours and removed after 23 h. Individual feed intake was
recorded for each chamber period and used for methane
calculation. Enteric CH
4
production (v/v) was converted to
g/kg DM intake for each animal.
Rumen uid and blood parameters
Following each chamber period and 3 h after feeding, samples
of rumen uid and jugular blood were collected from each
animal. Approximately 50 mL of rumen uid was collected by
stomach tubing and ltered through a 1-mm stainless steel
sieve. Two 3-mL subsamples were acidied using 200 mL
99% sulfuric acid, transferred to a 10-mL sealed tube on wet
ice and stored at 20C for the determination of VFAs and
rumen ammonia-N concentrations. The determination of VFA
concentrations in rumen uid was conducted using an Agilent
7890A GC (Agilennt Technologies, Santa Clara, Canada)
tted with a ame ionisation detector. Rumen ammonia-N
concentrations were determined by a direct enzymatic method
using an Olympus AU400 Auto analyser (Olympus Corporation,
Tokyo, Japan).
Jugular blood (5 mL) was collected by venepuncture into
10-mL lithiumheparin tubes (Vacuette; Greiner Bio-One,
Monroe, NC, USA) and 10-mL EDTA vacutainers (Vacutainers;
BD, Franklin Lakes, NJ, USA) from each sheep, mixed and
immediately placed on ice. Whole blood samples in heparin
tubes were centrifuged (2000gfor 10 min at 4C) and plasma
was stored at 20C for analyses of liver enzyme function, and
general wellbeing (kidney and muscle function) using an
Olympus AU400 auto analyzer (Beckman Coulter Inc.,
Krefeld, Germany). Whole blood samples were retained and
analysed for red and white blood cell counts (neutrophils,
lymphocytes, monocytes, basophils and eosinophils), haemoglobin
and packed cell volume by using the Cell-Dyn automated
hematology analyzer (Abbott Laboratories, Chicago, IL, USA).
Statistical analyses
Statistical analysis was conducted using GenStat 13.1 (VSN
International 2010, VSN International, Hemel Hempstead,
UK). The effect of including Asparagopsis in the diet was
examined using separate ANOVAs (mixed model with
restricted maximum likelihood, REML). The xed factors in
the mixed model consisted of the treatment effect (ve
inclusion levels of Asparagopsis), the time effect (three
sampling dates), the treatment by time interaction and
identied covariates. The response variables were DM intake
(DMI), DMI in the chamber, LW, CH
4
production,
concentrations of total and individual VFAs, concentration of
ammonia, parameters of blood chemistry and pathology. LW
recorded at the start of the experimental period was included as
an initial covariate, but was not signicant, and was therefore
removed from the nal model. If ANOVAs were signicant,
means were compared using least signicant difference (at
P= 0.05).
Results
Feed intake and LW
Sheep offered various inclusion levels of Asparagopsis had
similar LWs at the end of the experiment and average daily
DM intakes throughout the experimental period (pens and
chambers; P>0.05; Table 2). At the completion of the
experiment, the mean (s.e.m.) LW was 71.4 0.99 kg.
Neither the inclusion level of Asparagopsis nor the interaction
with time could be associated with LW (P>0.05). Three
animals offered the Asparagopsis (one each from sheep
offered 1%, 2% and 3% Asparagopsis) and one without the
Asparagopsis supplement were removed from the experiment
before CH
4
measurement according to the animal ethics protocol.
Sheep offered lower inclusion rates of Asparagopsis (1%)
consumed all the Asparagopsis on a daily basis when mixed with
the crushed lupins. Sheep offered 2% and 3% Asparagopsis
supplements did not always consume all the Asparagopsis,
which resulted in a range of Asparagopsis (Table 2). The
average value of Asparagopsis intake was calculated from
11 days of the Asparagopsis refusals for six (mean s.e.m.;
29.2 4.7 g/day) and ve sheep (32.0 3.2 g/day) selected
from the 2.0% and 3.0% groups respectively.
Enteric CH
4
production
There was no signicant (P>0.05) Asparagopsis offered ·time
interaction on CH
4
production. Offering Asparagopsis as
a dietary supplement signicantly (P<0.001) reduced the
overall CH
4
production compared with the animals that were
not offered Asparagopsis (Table 2, Fig. 1). There was a strong
relationship between average Asparagopsis intake (estimated
Methane mitigation and Asparagopsis Animal Production Science C
over 11 days for the 2% treatments) and enteric CH
4
emissions
(R
2
= 0.96, P<0.001; Fig. 2).
Rumen fermentation
Feeding animals with or without Asparagopsis in the daily
ration had no effect on the pH of rumen uid (6.8 0.25).
Total concentration and molar proportions of VFAs are shown
in Table 3. There was no signicant (P>0.05) Asparagopsis
offered ·time interaction on total VFAs and individual VFAs.
Increasing the inclusion levels of Asparagopsis had a signicant
(P<0.05) effect on total concentration and molar proportions
of individual VFAs, except iso-butyrate. Inclusions of
Asparagopsis in the daily ration resulted in a decrease in
acetate and total VFA concentration, and an increase in
propionate concentration compared with control animals.
The mean acetate : propionate ratio was signicantly lower
for sheep with access to Asparagopsis than that for sheep
without Asparagopsis. There was no signicant (P>0.05)
difference in the values of acetate : propionate ratio associated
with Asparagopsis inclusion rates. Rumen ammonia-N
concentrations (mg/L) decreased numerically with increasing
levels of Asparagopsis offered in the diet for the rst two
periods of the experiment and increased in the third period of
experiment, but this was not statistically signicant (P>0.05).
Pathology and necropsy
Values reported here for sheep offered increasing rates of
Asparagopsis in the diet were within haematological reference
ranges for sheep (as indicated by the laboratory reference, values
provided). Mean blood biochemistry and blood pathology
values as well as the reference values are summarised in
Table 4. There was no signicant treatment or time effect for
creatinine kinase activity. Liver function was considered normal
for sheep and was not inuenced by the treatments in the present
study.
There was no signicant interaction between Asparagopsis
offered and time for plasma creatinine, magnesium, phosphate
or b-hydroxybutyrate concentrations (P>0.05). Mean total
bilirubin and albumin concentrations in blood increased over
time (P<0.001) for all levels of Asparagopsis supplementation,
but remained within normal clinical limits. Mean blood urea
concentrations increased over time (P<0.001) by 26% with 1.0%,
Table 2. Mean Asparagopsis intake, DM intake (DMI), DMI during chamber measurement, methane production for sheep fed a pelleted diet, with
and without a supplement of Asparagopsis offered at different inclusion levels
Mean values shown are pooled means for three sampling events at 21-day intervals throughout the experimental period. P-values are for main effects only.
Means with different letters within a row differ signicantly (at P= 0.05)
Parameter Asparagopsis offered (% organic matter (OM) intake
per day)
s.e.m. P-value
0 0.5 1 2 3 Treatment Time
Asparagopsis offered (g/day DM) 0 13 26 58 80
Actual Asparagopsis intake range (% OM per day) 0 0.5 1 1.01.5 1.23.0
Actual Asparagopsis intake range (g/day DM)
A
013 26 2640 3080
DMI (kg/day) 1.04 1.06 1.04 1.05 1.02 0.014 n.s. 0.001
DMI in chamber (kg/day) 0.89 0.9 0.91 0.9 0.89 0.004 n.s. n.s.
Methane production (g/day) 13.4c 11.4c 6.4b 5.1ab 2.5a 0.65 <0.001 n.s.
Methane yield (g/kg DMI) 15.0c 12.7c 7.0b 5.6ab 2.9a 0.65 <0.001 n.s.
A
Actual Asparagopsis intake from 2% and 3% groups was measured from 11 animals (six animals from 2% group and ve animals from 3% group) over 11 days.
0
2
4
6
8
10
12
14
16
18
20
30 51 72
Methane (g/kg DM intake)
Time (days)
Control 0.5% Asp. 1% Asp. 2% Asp. 3% Asp.
Fig. 1. Mean (s.e.m.) methane emissions (g/kg DM intake) measured
at three intervals throughout the experimental period for sheep offered a
pelleted diet with 200 g of crushed lupins and increasing inclusion levels
of Asparagopsis (Asp.) (0% (control), 0.5%, 1.0%, 2.0% and 3.0% organic
matter basis), with and without Asparagopsis (Asp.) on a daily basis.
R
2
= 0.96
0
5
10
15
20
05
Methane (g/kg DM intake)
10 15 20 25 30
Asparagopsis (g/day)
y = 0.0023x
2
– 0.4189x + 14.982
Fig. 2. The relationship between mean (s.e.m.) methane production
(g/kg DM intake) and average Asparagopsis intake (g/day) for Merino-
cross wethers over 72 days. The Asparagopsis was fed with crushed lupins
in addition to a pelleted diet. Asparagopsis intake data for animals offered
2% and 3% organic matter intake basis was measured over 11 days only.
DAnimal Production Science X. Li et al.
2.0% or 3.0% Asparagopsis offered (OM basis). Blood
cholesterol concentrations also increased over time (P= 0.001)
by 39% for 0.5% Asparagopsis supplementation. Similar
increases were also recorded in the control group, and by 18%
for the 1.0% and 2.0% Asparagopsis inclusion rates. Plasma
calcium concentrations varied throughout the experimental
period (P= 0.007), but could not be associated with
Asparagopsis (P>0.05). For all groups, plasma total protein
Table 3. Mean ruminal fermentation parameters, including total volatile fatty acids (VFA), molar proportions of individual VFAs and rumen
ammonia-nitrogen (NH
3
-N), for sheep fed a pelleted diet with crushed lupins and increasing levels of Asparagopsis on offer
Mean values shown are pooled means for three sampling events at 21-day intervals throughout the experimental period. P-values are for main effects only. A : P,
acetate : propionate ratio. Means with different letters within a row differ signicantly (at P= 0.05)
Parameter Asparagopsis offered (% organic matter intake per day) s.e.m. P-value
Control 0.5 1.0 2.0 3.0 Treatment Time
Total VFA (mM) 92.0c 86.5bc 74.9ab 69.1a 65.4a 1.60 <0.05 n.s.
VFA proportions (% total VFA)
Acetate 65.0b 56.3a 54.4a 55.0a 54.5a 0.78 <0.001 0.035
Propionate 20.8a 27.7b 31.5c 30.8bc 32.0c 0.71 <0.001 0.026
Butyrate 11.6ab 13.0b 11.2a 11.1a 10.3a 0.25 0.017 n.s.
Iso-butyrate 0.41 0.36 0.32 0.42 0.47 0.003 n.s. n.s.
Valerate 1.00a 1.50b 1.66bc 1.87c 1.80bc 0.06 <0.001 n.s.
Iso-valerate 0.76b 0.46a 0.34a 0.55ab 0.53ab 0.04 0.022 n.s.
A : P 3.19b 2.10a 1.76a 1.86a 1.77a 0.08 <0.001 n.s.
NH
3
-N (mg/L) 220 203 192 191 165 9.97 n.s. n.s.
Table 4. Mean blood biochemistry and blood pathology for sheep fed a pelleted diet with and without a supplement of Asparagopsis, offered at
increasing iclusion levels
CK, creatin kinase; ALT, alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; GLDH, glutamate dehydrogenase; BHB, b-hydroxybutyrate; AG,
anion gap; PCV, packed cell volume; RBC, red blood cell count; WBS, white bloodcell count; neuts, neutrophils; lymphs, lymphocytes; mono, monocytes; eosis,
eosinophils; baso, basophils. Laboratory reference ranges are as follows: CK, <500 U/L; ALT, <30 U/L; GGT, 2367 U/L; GLDH, <20 U/L; total bilirubin, <15
mmol/L; urea, 3.38.0 mmol/L; creatinine, 50150 mmol/L; calcium, 2.23.0 mmol/L; magnesium, 0.821.44 mmol/L; phosphorus, 0.92.5 mmol/L; BHB, <0.7
mmol/L; total protein, 6075 g/L; albumin, 2834 g/L; iron, 3336 mmol/L; haemoglobin, 90150 g/L; haptoglobin, 0.032 mg/mL; PCV, 0.270.45 L/L; RBC,
9.015.0 ·10
12
/L; WBC, 4.012.0 ·10
9
/L. P-values are main effects only. Within each row, means with different letters differed signicantly (l.s.d., P= 0.05)
Parameter Asparagopsis offered (% organic matter intake per day) s.e.m. P-value
0 0.5 1.0 2.0 3.0 Treatment Time
CK (U/L) 63.8 77.8 70.5 70.7 79.8 5.71 n.s. n.s.
ALT (U/L) 8.34ab 8.50b 6.84a 6.85a 6.02a 0.26 0.005 n.s.
GGT (U/L) 60.2b 57.5a 69.2c 59.4ab 57.0a 1.66 0.018 n.s.
GLDH (U/L) 5.84 13.01 7.56 8.89 5.07 1.09 n.s. 0.005
Total bilirubin (mmol/L) 3.13 2.88 2.94 2.93 2.84 0.07 n.s. <0.001
Urea (mmol/L) 6.91 6.96 6.61 6.39 6.23 0.19 n.s. <0.001
Creatinine (mmol/L) 98.9 98.6 96.2 100.6 97.5 1.82 n.s. n.s.
Calcium (mmol/L) 2.67 2.64 2.57 2.59 2.67 0.02 n.s. 0.007
Magnesium (mmol/L) 0.93 0.94 0.97 0.93 0.93 0.01 n.s. n.s.
Phosphate (mmol/L) 1.94 1.96 1.85 1.77 1.91 0.08 n.s. n.s.
BHB (mmol/L) 0.41 0.42 0.36 0.39 0.36 0.03 n.s. n.s.
Cholesterol (mmol/L) 2.08 2.05 2.28 2.03 2.35 0.10 n.s. 0.001
Total protein (g/L) 65.7 66.1 64.9 65.9 66.7 0.44 n.s. 0.009
Albumin (g/L) 33.4 34.0 32.6 33.0 33.4 0.28 n.s. <0.001
Iron (mmol/L) 22.2ab 23.3b 25.8b 22.9ab 19.7a 1.670 0.047 n.s.
A:G ratio 1.02 1.08 1.00 1.02 1.01 0.04 n.s. <0.001
Haemoglobin (g/L) 112 118 100 112 108 2.25 n.s. <0.001
Haptoglobins (mg/mL) 0.37 0.39 0.32 0.43 0.52 0.11 n.s. <0.001
PCV (L/L) 0.34b 0.36b 0.29a 0.34b 0.33ab 0.007 0.026 0.002
RBC (·10
12
/L) 10.2 10.7 9.1 10.0 9.63 0.239 n.s. 0.002
WBS (·10
9
/L) 4.75 4.11 4.28 5.02 4.91 0.244 n.s. 0.011
Neuts (%) 28.5 26.1 23.0 33.4 29.5 1.91 n.s. n.s.
Lymphs (%) 60.3 63.6 70.1 54.3 58.0 2.07 n.s. 0.012
Mono (%) 2.20 3.07 2.00 3.87 3.40 0.330 n.s. n.s.
Eosis (%) 8.93 7.07 4.90 8.07 9.00 0.776 n.s. n.s.
Baso (%) 0.00 0.13 0.09 0.33 0.15 0.066 n.s. n.s.
Methane mitigation and Asparagopsis Animal Production Science E
decreased over time (P= 0.009). Although some of the gamma-
glutamyl transpeptidase (GGT) and glutamate dehydrogenase
(GLDH) values across all treatment groups were outside
laboratory reference ranges, there was no evidence of
hepatobiliary disease on postmortem examination. There were
signicant differences in packed cell volume with Asparagopsis
inclusion (P= 0.026) over the experiment period but all data were
within the clinically normal range. Mean red blood cell and white
blood cell counts and lymphocytes in blood increased with time
(P<0.05) for animals with and without Asparagopsis
supplementation.
On completion of the experiment, 12 sheep were presented
for necropsy. Tissue samples (muscle and adipose fat) were
collected for histopathological examination. The interval
between death and postmortem observations and sampling was
<1 h. Carcasses were in good body condition with minimal
autolysis. No detectible levels (<0.05 mg/kg) of bromform or
dibromochloromethane were found in any muscle or fat sample.
On gross postmortem examination, the mucosal lining of the
rumen oor in ve sheep that were offered Asparagopsis (of
10 sheep that had been offered Asparagopsis) was characterised
by a variably extensive area of nodular proliferation and
whitetan discolouration, with blunting of ruminal papillae.
No gross changes or histologic evidence of eosinophilic
inammation were noted in the two sheep that had not been
offered Asparagopsis.
In all sheep, the mucosa of the rumen, reticulum and omasum
was mildly hyperplastic, with hyperkeratosis and mild hydropic
degeneration of the surface epithelium. No additional signicant
lesions were noted. No evidence of nutritional neuropathy
or myopathy was present in any of the examined animals, and
additional individual changes (myocardial sarcocysts, rare
intestinal nematodes and rare cerebral protozoal cysts) were
unrelated to the feeding trial.
Discussion
Asparagopsis and CH
4
production
The present study has conrmed that inclusion of Asparagopsis
in the diet of sheep decreases enteric CH
4
production.
This decrease in CH
4
production persisted over the 72-day
experimental period. Including Asparagopsis, presumably as
a source of haloforms, to a high-bre pelleted diet can be an
effective CH
4
abatement strategy. There was evidence of
a doseresponse relationship for Asparagopsis consumed
(g/day) and CH
4
production, although the variation in
Asparagopsis intake for sheep offered 2% and 3% in their diet
(on an OM basis) constrained this conclusion. Animals that
consumed the most Asparagopsis had an 80% reduction in
CH
4
over 72 days, when compared with animals without
Asparagopsis. On the basis of voluntary intake levels, we
suggest that sheep will choose to consume ~30 g/day of
Asparagopsis and this will reduce CH
4
emissions by at least
50% when compared with sheep that have not eaten
Asparagopsis.
Machado et al.(2016a) reported that the concentration
of halogenated metabolites in the biomass of freeze-dried
Asparagopsis was ~1750 mg/kg DM, with the halogenated
metabolites including predominantly bromoform,
dibromochloromethane and bromochloroacetic acid. The
Asparagopsis biomass sample collected from the same site
was kept on racks in the dark, delivered to Townsville, and
frozen on arrival and then freeze-dried before analysis
(National Measurement Institute, Melbourne, Vic., Australia);
the sample was found to contain only 384 mg/kg DM
halogenated metabolites of similar composition. Ongoing work
(Kinley, unpubl. data) has conrmed that the processing of
collected biomass samples from wild harvest will affects
bioactivity. The Asparagopsis supplemented to the sheep was
kiln dried and can be expected to have a lower concentration
of halogenated metabolites than does freeze-dried material.
Freeze-drying will conserve volatile compounds and is likely
to provide a more consistent concentration in the biomass
sample, which could then be fed at lower inclusion rates if
based on the actual concentration of the bioactive component.
In comparison, Sawyer et al. (1974) reported that mature
wethers fed a maintenance diet with up to 4.5 mg BCM/kg
bodyweight per day had up to 85% decrease in CH
4
production compared with a control diet. Tomkins et al.
(2009) also showed signicant decreases in CH
4
production
for cattle given a BCM formulation, containing 1012% w/w
BCM, at rates of 3 mg/kg LW, twice daily. These studies
clearly demonstrated that the antimethanogenic potential of
halogenated, particularly brominated, analogues that naturally
accumulate in the gland cells of Asparagopsis (Paul et al.2006a)
is comparable to BCM.
Unlike BCM, which appears to decrease in efcacy over
90 days for cattle (Tomkins et al.2009), no rumen adaption
was observed during the present 72-day study in terms of CH
4
emissions. BCM can directly inhibit the terminal cobamide-
dependent methyl transferase step in the enzymatic pathway of
methanogenesis (Wood et al.1968), rather than act directly as a
toxin to methanogens (Johnson et al.1972). However, a delay in
rumen methanogen population decline, followed by a decrease
of 42% in methanogen populations, was reported for steers 8 h
after treatment with complexed BCM, suggesting that inhibition
affected the growth of methanogens (Denman et al.2007).
Additional ruminal microbial diversity and abundance analysis
will be required to fully understand the impact of Asparagopsis
supplementation on the rumen archaea population where
cobamide-dependent methanogenesis is inhibited.
The present study has demonstrated that the inclusion
of Asparagopsis in a high-bre diet decreases total VFA
concentration in the rumen, but increases the concentration of
propionate, seemingly at the expense of butyrate. This supports
the conclusions of a previous in vitro study using Asparagopsis
at 17% (OM basis) and Flinders grass as the forage substrate
(Machado et al.2014). In comparison, there were no signicant
changes in molar proportions of VFA; however, Sawyer et al.
(1974) reported changes in valerate concentrations when both
lambs and wethers were offered increasing inclusion levels
of BCM. The shift in theVFA prole suggests that inclusion of
Asparagopsis as a supplement leads to diversion of hydrogen to
propionate, creating less hydrogen available for methanogenesis.
Decreases in the concentration of total VFAs have been linked
to anti-nutritional factors that interfere with ruminal fermentation
(Getachew et al.1998). This may be associated with the reduced
feed intake reported for some sheep in the early adaption period.
FAnimal Production Science X. Li et al.
Any inhibitory effect on productivity from inclusion of an
antimethanogenic compound would be counter-productive and
reduce the incentive to reduce enteric emissions. Nevertheless,
Tomkins et al.(2009) demonstrated a slight increase in
productivity (100 g/day) over 85 days when BCM was
incorporated in the diet, with a signicant decrease in CH
4
production. While inhibitory effects on intake have been
reported with high concentrations of fats and oils to decrease
enteric CH
4
(Beauchemin et al.2008), this is unlikely to occur
with Asparagopsis due to the low levels of inclusion and
potential application in total mixed rations.
Effect on animal health
In the current study, sheep were fed a restricted diet (1.2 times
maintenance). This did not allow an assessment of the effect of
Asparagopsis inclusion on ad lib DMI. However, with restricted
intake and ability by individuals to sift out the Asparagopsis, there
were no treatment-related differences in the intake measured.
Previous studies with cattle (Tomkins et al. 2009) have reported
that the feeding of BCM is not associated with a decrease in daily
DMI of a grain-based ration. Similarly, Sawyer et al.(1974) did
not report a signicant effect on DMI with increasing levels of
BCM for lambs and wethers fed a commercial pelleted ration
ad lib. However, in our study, four animals decreased their
DMI over three consecutive days, to the point that they were
removed from the experiment. This included one animal without
Asparagopsis supplement and three offered the Asparagopsis
supplement. McCrabb et al.(1997) reported a reduction in
voluntary DMI for steers fed low- and medium-quality hay
diets over 1012 weeks with BCM supplementation, suggesting
a need to account for the impact on intake when developing an
abatement methodology based on halogenated analogues.
On gross examination, the mucosal lining of the rumen in 5 of
the 12 examined sheep was characterised by an extensive area of
nodular proliferation and whitetan discolouration with blunting
of ruminal papillae. All ve sheep were in Asparagopsis
treatments. Histologically, these changes were localised in the
ruminal submucosa, with a light to moderate inammatory
inltrate comprising predominantly eosinophils and macrophages.
The cause of these changes was not evident either on gross
examination or within sections of ruminal mucosa. The
predominance of eosinophils within the inammatory inltrate
is suggestive of hypersensitivity to luminal antigens or possibly
parasitism. No helminthes were detected on necropsy or within
histologic sections of the forestomachs and intestines. Likewise,
no infectious organisms were detected with additional
histochemical stains. Given the chronicity of the lesions, it is
possible the antigenic and allergic stimulus resolved before
the commencement of the study.
In all animals, there were changes in the rumen mucosa
consistent with mild ruminal acidosis secondary to carbohydrate
ingestion. These are likely associated with the pelleted diet
and unrelated to the eosinophilic inammation. The impact of
Asparagopsis ingestion on voluntary intake and the clinical
signicance of rumen-wall changes requires further investigation.
Bromochloromethane could be used as a model for the
antimethanogenic mode of action observed for Asparagopsis.
It is possible that dissociated bioactives derived from
Asparagopsis metabolites could similarly complex with reduced
vitamin B
12
in the rumen. It is also likely that dissociation and
availability of bioactives such as bromoform in the rumen results
in products that are either readily lost by eructation, or are
further metabolised in the gastro-intestinal tract. Bromoform and
dibromochloromethane could not be detected in the fat and
muscle tissue of the sheep fed Asparagopsis, suggesting that,
within the connes of this experiment, these metabolites were
not transferred to tissues.
Burreson et al. (1976) identied 42 volatile compounds in the
extracted oil of Asparagopsis taxiformis, of which 87% by weight
were haloforms of predominantly bromine and, to a lesser extent,
iodine and chlorine. Inorganic bromide has been shown to
accumulate in the blood of monogastrics when subjected to
inhalation of BCM over extended periods (Svirbely et al.
1947). The serum anion gap can be used in the detection of
metabolic acidosis and bromide intoxication (Kraut and Madias
2007). Although the normal value for serum anion gap can vary
widely, the absence of a signicant difference for this parameter
between the treatment groups and control suggests that sheep in
the present study were not subjected to either bromide toxicity
or metabolic acidosis induced by feeding Asparagopsis. The
absence of detectable concentrations of BCM in bovine tissues
reported by Tomkins et al. (2009) suggests that halogenated CH
4
analogues may not accumulate in ruminants fed the compound
over extended periods. The halogenated metabolites identied
in Asparagopsis are equally volatile, and attempting to
determine residue concentrations in tissue near the current
temporary minimum residue limit may be inconclusive.
Conclusions
Asparagopsis supplementation can reduce CH
4
emissions
by 5080% over a 72-day feeding period. This corresponds
to a voluntary inclusion rate of ~0.42 g/kg LW for sheep.
Nevertheless, inclusion rates will be dependent on the
concentration of key bioactives in the Asparagopsis biomass.
The effect of Asparagopsis supplementation on feed intake,
digestibility, animal productivity and animal health will need
further investigation. The exploitation of Asparagopsis as
a natural antimethanogenic agent has potential to benet
Australias ruminant livestock sector by providing a novel, yet
practical, strategy to achieve substantial abatement outcomes.
Acknowledgements
We thank Josh Hendry and Miranda Macintyre, for their assistance in
animal house experimentation and sample collection. Paul Young and
Elizabeth Hulm provided assistance with laboratory analyses. Dr Marie
Magnusson provided the chemical analysis of natural products from
Asparagopsis. The study was funded by Meat and Livestock Australia (MLA).
References
AFIA (2009) Australian Fodder Industry Association laboratory methods
manual.Publication No. 03/001 (AFIA: Melbourne)
AOAC (2005) Ofcial method 990.03. Protein (crude) in animal feed,
combustion method. In Ofcial methods of analysis of AOAC
International. pp. 3031. (AOAC International: Arlington, VA)
Australians National Greenhouse Accounts (2014) Quarterly update
of Australias National Greenhouse Gas Inventory: June 2014.
(Australian Government Department of the Environment and Energy)
Methane mitigation and Asparagopsis Animal Production Science G
Available at https://www.environment.gov.au/climate- change/greenhouse-
gas-measurement/publications/quarterly-update-australias-national-green
house-gas-inventory-june-2014 [Veried 10 September 2016]
Beauchemin KA, Kreuzer M, OMara F, McAllister TA (2008) Nutritional
management for enteric methane abatement: a review. Australian
Journal of Experimental Agriculture 48,2127. doi:10.1071/EA07199
Burreson BJ, Moore RE, Roller (1976) Volatile halogen compounds in the
alga Asparagopsis taxiformis (Rhodophyta). Journal of Agricultural
and Food Chemistry 24, 856861. doi:10.1021/jf60206a040
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, 313322. doi:10.1111/j.1574-6941.2007.
00394.x
Faichney GJ, White GA (1983) Methods for the analysis of feeds eaten by
ruminants.(CSIRO, Division of Animal Production, Ian Clunies Ross
Animal Research Laboratory: Melbourne)
Getachew G, Blümmel M, Makkar HPS, Becker K (1998) In vitro gas
measuring techniques for assessment of nutritional quality of feeds:
a review. Animal Feed Science and Technology 72, 261281.
doi:10.1016/S0377-8401(97)00189-2
Johnson ED, Wood AS, Stone JB, Moran ET Jr (1972) Some effects of
methane inhibition in ruminants (steers). Canadian Journal of Animal
Science 52, 703712. doi:10.4141/cjas72-083
Klein L, Wright AG (2006) Construction and operation of open-circuit
methane chambers for small ruminants. Australian Journal of
Experimental Agriculture 46, 12571262. doi:10.1071/EA05340
Kraut JA, Madias NE (2007) Serum anion gap: its uses and limitations
in clinical medicine. Clinical Journal of the American Society of
Nephrology 2, 162174. doi:10.2215/CJN.03020906
Li X (2014) Eremophila glabra reduces methane production in sheep. PhD
thesis. University of Western Australia, Perth.
Louren¸co SO, Barbarino E, De-Paula JC, Pereira LOdS, Marquez UML
(2002) Amino acid composition, protein content and calculation
of nitrogen-to-protein conversion factors for 19 tropical seaweeds.
Phycological Research 50, 233241. doi:10.1111/j.1440-1835.2002.
tb00156.x
Machado L, Magnusson M, Paul NA, de Nys R, Tomkins N (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, Magnusson M, Paul NA, Kinley R, de Nys R, Tomkins N
(2016a) Identication of bioactives from the red seaweed Asparagopsis
taxiformis that promote antimethanogenic activity in vitro.Journal of
Applied Phycology. doi:10.1007/s10811-016-0830-7
Machado L, Magnusson M, Paul N, Kinley R, de Nys R, Tomkins N (2016b)
Doseresponse effects of Asparagopsis taxiformis and Oedogonium
sp. on in vitro fermentation and methane production. Journal of
Applied Phycology 28, 14431452. doi:10.1007/s10811-015-0639-9
McCrabb GJ, Berger KT, Magner T, May C, Hunter RA (1997) Inhibiting
methane production in Brahman cattle by dietary supplementation with
a novel compound and the effects on growth. Australian Journal of
Agricultural Research 48, 323329. doi:10.1071/A96119
NHMRC (2013) Australian code for the care and use of animals for
scientic purposes.8th edn. (National Health and Medical Research
Council: Canberra)
OSullivan L, Murphy B, McLoughlin P, Duggan P, Lawlor PG, Hughes H,
Gardiner GE (2010) Prebiotics from marine macroalgae for human and
animal health applications. Marine Drugs 8, 20382064. doi:10.3390/
md8072038
Paul N, de Nys R, Steinberg P (2006a) Chemical defence against bacteria
in the red alga Asparagopsis armata: linking structure with function.
Marine Ecology Progress Series 306,87101. doi:10.3354/meps306087
Paul NA, Cole L, de Nys R, Steinberg PD (2006b) Ultrastracutre of the gland
of the red alga Asparagopsis armata (boonemaisoniaceae). Journal of
Phycology 42, 637645. doi:10.1111/j.1529-8817.2006.00226.x
Sawyer M, Hoover W, Sniffen C (1974) Effects of a ruminal methane inhibitor
on growth and energy metabolism in the ovine. Journal of Animal Science
38, 908914.
Suiter J (1994) Body condition scoring of sheep and goats. Farmnote 69.
Department of Agriculture and Food Western Australia.
Svirbely JL, Alford WC, Von Oettingen WF (1947) Toxicity and narcotic
action of mono-chloro-mono-bromo-methane with special reference to
inorganic and volatile bromide in blood, urine, and brain. Federation
Proceedings 6, 375.
Tomkins NW, Colegate SM, Hunter RA (2009) A bromochloromethane
formulation reduces enteric methanogenesis in cattle fed grain-based
diets. Animal Production Science 49, 10531058. doi:10.1071/EA08223
Wood JM, Kennedy FS, Wolfe RS (1968) Reaction of multihalogenated
hydrocarbons with free and bound reduced vitamin B12. Biochemistry 7,
17071713. doi:10.1021/bi00845a013
HAnimal Production Science X. Li et al.
www.publish.csiro.au/journals/an
... These include selective breeding, vaccines, methanogenesis inhibitors, and dietary measures. While the effective management of enteric methane emissions is likely to be integrated across strategies, methanogenesis inhibitors in the form of feed ingredients are the best performers to date with the highly bioactive seaweeds of the genus Asparagopsis having the highest activity [9][10][11][12][13]. ...
... Brown seaweed (kelp and fucoids) has traditionally been the main macroalgal group used to supplement animal feeds, however, select species from all seaweed phyla (Rhodophyta, Chlorophyta, and Ochrophyta) have been investigated for their capacity to reduce enteric methane emissions in ruminants [15][16][17][18][19]. Of these seaweeds, the species of the genus Asparagopsis (A. taxiformis and A. armata) stand out for their efficacy, inhibiting methane production (methanogenesis) through specific effects on rumen methanogenic archaea (methanogens) [9][10][11][12][13]. These species consistently and significantly reduce methane emissions from sheep [10] and cattle [9,[11][12][13] consuming Asparagopsis at inclusion levels of less than 1% of the feed organic matter (OM) intake. ...
... Of these seaweeds, the species of the genus Asparagopsis (A. taxiformis and A. armata) stand out for their efficacy, inhibiting methane production (methanogenesis) through specific effects on rumen methanogenic archaea (methanogens) [9][10][11][12][13]. These species consistently and significantly reduce methane emissions from sheep [10] and cattle [9,[11][12][13] consuming Asparagopsis at inclusion levels of less than 1% of the feed organic matter (OM) intake. Notably, the effective level of inclusion is dependent on the bioactive content of the seaweed and the formulation of the basal feed [9,12]. ...
Article
The agricultural production of ruminants is responsible for 24% of global methane emissions, contributing 39% of emissions of this greenhouse gas from the agricultural sector. Strategies to mitigate ruminant methanogenesis include the use of methanogen inhibitors. For example, the seaweeds Asparagopsis taxiformis and Asparagopsis armata included at low levels in the feed of cattle and sheep inhibit methanogenesis by up to 98%, with evidence of improvements in feed utilisation efficiency. This has resulted in an increasing interest in and demand for these seaweeds globally. In response, research is progressing rapidly to facilitate Asparagopsis cultivation at large scale, and to develop aquaculture production systems to enable a high quality and consistent supply chain. In addition to developing robust strategies for sustainable production, it is important to consider and evaluate the benefits and risks associated with its production and subsequent use as an antimethanogenic feed ingredient for ruminant livestock. This review focuses on the relevant ruminal biochemical pathways, degradation, and toxicological risks associated with bromoform (CHBr3), the major active ingredient for inhibition of methanogenesis in Asparagopsis, and the effects that production of Asparagopsis and its use as a ruminant feed ingredient might have on atmospheric chemistry.
... taxiformis and A. armata) are considered the most effective macroalgae due to high concentrations of bromoform (Machado et al., 2014;Kinley et al., 2016). Five in vivo studies are published using Asparagopsis in sheep (Li et al., 2016), dairy cattle (Roque et al., 2019a;Stefenoni et al., 2021), and beef steers (Kinley et al., 2020;Roque et al., 2021). Sheep fed A. taxiformis at 78.4 g/kg DM reduced CH 4 yield by 80% with no effects on ADG or DMI (Li et al., 2016). ...
... Five in vivo studies are published using Asparagopsis in sheep (Li et al., 2016), dairy cattle (Roque et al., 2019a;Stefenoni et al., 2021), and beef steers (Kinley et al., 2020;Roque et al., 2021). Sheep fed A. taxiformis at 78.4 g/kg DM reduced CH 4 yield by 80% with no effects on ADG or DMI (Li et al., 2016). Kinley et al. (2020) reported 98% less CH 4 yield and a 22% increase in ADG in beef steers fed 3.7 g/kg DM A. taxiformis. ...
... Rumen fermentation effects, such as total VFA production, are inconsistent. However, the reduction of acetate-to-propionate is consistently reported in vivo (Li et al., 2016;Kinley et al., 2020;Stefenoni et al., 2021). Additionally, only one study has showed long-term efficacy (21 weeks) (Roque et al., 2021), thus more long-term studies with greater animal numbers are needed. ...
Article
Full-text available
Mitigation of enteric methane (CH4) presents a feasible approach to curbing agriculture’s contribution to climate change. One intervention for reduction is dietary reformulation, which manipulates the composition of feedstuffs in ruminant diets to redirect fermentation processes toward low methane emissions. Examples include reducing the relative proportion of forages to concentrates, determining the rates of digestibility and passage rate from the rumen, and dietary lipid inclusion. Feed additives present another intervention for CH4 abatement and are classified based on their mode of action. Through inhibition of key enzymes, 3-nitroxypropanol (3-NOP) and halogenated compounds directly target the methanogenesis pathway. Rumen environment modifiers, including nitrates, essential oils, and tannins, act on the conditions that affect methanogens and remove the accessibility of fermentation products needed for CH4 formation. Low methane-emitting animals can also be directly or indirectly selected through breeding interventions, and genome-wide association studies are expected to provide efficient selection decisions. Overall, dietary reformulation and feed additive inclusion provide immediate and reversible effects, while selective breeding produces lasting, cumulative CH4 emission reductions.
... To abate enteric methanogenesis, different strategies such as feeding management and antimethanogenic feed ingredients have been proposed and assessed (e.g., Moate et al., 2016;Mayberry et al., 2019;Beauchemin et al., 2020). Some types of macroalgae have been demonstrated to mitigate production of CH 4 during in vitro and in vivo rumen fermentation significantly (Machado et al., 2014;Kinley and Fredeen 2015;Li et al., 2018;Kinley et al., 2020;Abbott et al., 2020). Among the different macroalgae species, Kinley et al. (2016a) concluded that the red algae Asparagopsis spp. ...
Article
Full-text available
To mitigate the rumen enteric methane (CH4) produced by ruminant livestock, Asparagopsis taxiformis is proposed as an additive to ruminant feed. During the cultivation of Asparagopsis taxiformis in the sea or in terrestrially based systems, this macroalgae, like most seaweeds and phytoplankton, produces a large amount of bromoform (CHBr3), which contributes to ozone depletion once released into the atmosphere. In this study, we focus on the impact of CHBr3 on the stratospheric ozone layer resulting from potential emissions from proposed Asparagopsis cultivation in Australia. The impact is assessed by weighting the emissions of CHBr3 with its ozone depletion potential (ODP), which is traditionally defined for long-lived halocarbons but has also been applied to very short-lived substances (VSLSs). An annual yield of ∼3.5 × 104 Mg dry weight is required to meet the needs of 50 % of the beef feedlot and dairy cattle in Australia. Our study shows that the intensity and impact of CHBr3 emissions vary, depending on location and cultivation scenarios. Of the proposed locations, tropical farms near the Darwin region are associated with the largest CHBr3 ODP values. However, farming of Asparagopsis using either ocean or terrestrial cultivation systems at any of the proposed locations does not have the potential to significantly impact the ozone layer. Even if all Asparagopsis farming were performed in Darwin, the CHBr3 emitted into the atmosphere would amount to less than 0.02 % of the global ODP-weighted emissions. The impact of remaining farming scenarios is also relatively small even if the intended annual yield in Darwin is scaled by a factor of 30 to meet the global requirements, which will increase the global ODP-weighted emissions up to ∼0.5 %.
... As described in the previous section, the fine grinding of forages and/or increasing the rapidly fermentable carbohydrates in the diet increases the rate of passage in the gut and decreases the rumen pH which contributes to the decreased rumen methanogenesis (reviewed in Hook et al. 2010). In a feeding trial on sheep, the supplementation of red alga (Asparagopsis taxiformis) in sheep diet up to the level of 3% linearly decreased the methane emission up to 80% and no recurrence of the original level of methane emission was observed which suggested that methanogens could not adapt to the alga in the diet (Li et al. 2016). In recent experiments with cattle, the supplementation of dried Asparagopsis at 0.2-1% level of organic matter reduced methane emission by 55-98% with no negative effects on the other growth parameters and product quality (Rouque et al. 2019;Kinley et al. 2020;Stefenoni et al. 2021). ...
Article
Full-text available
Much of the biomass in this world is rich in fibre which is utilised by the ruminants with the help of rumen microbes to produce a good quality protein for human consumption. However, this conversion of fibre to high-quality protein is paralleled by the production of methane which represents the wastage of feed energy and is a powerful greenhouse gas harmful to the global climate. The microbial community in the rumen has co-evolved with their host animal in a symbiotic relationship over millions of years and methanogenesis has emerged as a result of the refinement of the fermentation process in the rumen. The one-to-one relationship between the methanogen population and the methanogenesis has not been established yet, which indicates the role of associated rumen microbiota, substrate availability, and other functional parameters of the rumen. This review has focused on the total rumen microbial structure, methanogen structure, rumen fermentation process, methanogenesis, factors affecting methane production, and methane mitigation strategies. The balance between the H2 producers and H2 consumers in the rumen determines the level of methane production in the rumen. Therefore, decreasing the availability of H2 in the rumen by fostering alternative H2 sinks, such as propionate production, is very instrumental in reducing the rumen methane emissions. Any strategy of methane abatement should concurrently consider the enhancement of propionate production to prevent the inhibition of rumen functions. Although a great deal of information regarding the rumen microbial community structure, rumen physiology, and methane mitigation strategies is currently available, more research is still needed. The majority of the in vivo experiments pertaining to methane abatement strategies discussed in this review are the short term experiments in which long term unwanted effects could not be precisely predicted. Therefore, there is a need for long-term experiments to draw valid and logical conclusions on the methane abatement strategies.
... Seaweeds, also known as macroalgae, including brown (Phaeophyta), red (Rhodophyta), and green (Chlorophyta) seaweeds, have become preferable feed additives because of their anti-methanogenic properties [100,101]. Several in vitro studies of seaweed supplements showed a negative correlation with methane generation especially using Asparagopsis taxiformis [72,102,103] and its fellow Asparagopsis spp., which could cut back in vivo methane emission from 50% to over 80% in dairy cattle [104][105][106]. ...
Article
Full-text available
Human activities account for approximately two-thirds of global methane emissions, wherein the livestock sector is the single massive methane emitter. Methane is a potent greenhouse gas of over 21 times the warming effect of carbon dioxide. In the rumen, methanogens produce methane as a by-product of anaerobic fermentation. Methane released from ruminants is considered as a loss of feed energy that could otherwise be used for productivity. Economic progress and growing population will inflate meat and milk product demands, causing elevated methane emissions from this sector. In this review, diverse approaches from feed manipulation to the supplementation of organic and inorganic feed additives and direct-fed microbial in mitigating enteric methane emissions from ruminant livestock are summarized. These approaches directly or indirectly alter the rumen microbial structure thereby reducing rumen methanogenesis. Though many inorganic feed additives have remarkably reduced methane emissions from ruminants, their usage as feed additives remain unappealing because of health and safety concerns. Hence, feed additives sourced from biological materials such as direct-fed microbials have emerged as a promising technique in mitigating enteric methane emissions.
... The effectiveness of active plant compounds in the mission of reducing methane production is also influenced by animal species. Roque et al. (2019) stated that the utilization of the Asparagopsis genus plant which was included in 1% of the total feed of dairy cows succeeded in reducing 67% of energy CH 4 emissions, while Li et al. (2018) stated that the utilization of plants with the genus was tested on sheep with concentrations of 0.5%, 1%, 2 % and 3% succeeded in reducing enteric CH 4 to 80% compared to control cattle. Addition of tannin to the feed does not always have an impact on reducing methane production. ...
... However, it cannot be ruled out that other secondary metabolites, such as tannins and flavonoids, may exert this anti-methanogenic effect [173]. Concerning the use of Asparagopsis spp. in animal feed, some caution is recommended, since Li et al. [174] identified granulomatous and keratotic ruminal-mucosa changes in sheep supplemented with Asparagopsis, while Muizelaar et al. [175] detected bromoform in the milk of lactating cows that had Asparagopsis incorporated into their feed, and found abnormalities in their rumen wall, with visible signs of inflammation. It should be noted that Silva et al. [176] reported oxidative stress and neurotoxicity in the shrimp Palaemon elegans. ...
Article
Full-text available
Although the genus Asparagopsis includes only two taxonomically accepted species, the published literature is unanimous about the invasive nature of this genus in different regions of the globe, and about the availability of large amounts of biomass for which it is important to find a commercial application. This review shows that extracts from Asparagospsis species have already been evaluated for antioxidant, antibacterial, antifungal, antiviral, antifouling, cytotoxic, antimethanogenic and enzyme-inhibitory activity. However, the tables presented herein show, with few exceptions, that the activity level displayed is generally low when compared with positive controls. Studies involving pure compounds being identified in Asparagopsis species are rare. The chemical compositions of most of the evaluated extracts are unknown. At best, the families of the compounds present are suggested. This review also shows that the volatile halogenated compounds, fatty acids and sterols that are biosynthesized by the Asparagopsis species are relatively well known. Many other non-volatile metabolites (halogen compounds, flavonoids, other phenolic compounds) seem to be produced by these species, but their chemical structures and properties haven’been investigated. This shows how much remains to be investigated regarding the secondary-metabolite composition of these species, suggesting further studies following more targeted methodologies.
Chapter
Methane is the single largest source of anthropogenic greenhouse gases produced in ruminants. As global warming is a main concern, the interest in mitigation strategies for ruminant derived methane has strongly increased over the last years. Methane is a natural by-product of anaerobic microbial (bacteria, archaea, protozoa, and fungi) fermentation of carbohydrates and, to a lesser extent, amino acids in the rumen. This gaseous compound is the most prominent hydrogen sink product synthesized in the rumen. It is formed by the archaea, the so-called methanogens, which utilize excessive ruminal hydrogen. Different nutritional strategies to reduce methane production in ruminants have been investigated such as dietary manipulations, plant extracts, lipids and lipid by-products, plant secondary metabolites, flavonoids, phenolic acid, statins, prebiotics, probiotics, etc. With the range of technical options suggested above, it is possible to develop best nutritional strategies to reduce the ill effects of livestock on global warming. These nutritional strategies seem to be the most developed means in mitigating methane from enteric fermentation in ruminants and some are ready to be applied in the field at the moment.
Article
Full-text available
Asparagopsis taxiformis has potent antimethanogenic activity as a feed supplement at 2 % of organic matter in in vitro bioassays. This study identified the main bioactive natural products and their effects on fermentation using rumen fluid from Bos indicus steers. Polar through to non-polar extracts (water, methanol, dichloromethane and hexane) were tested. The dichloromethane extract was most active, reducing methane production by 79 %. Bromoform was the most abundant natural product in the biomass of Asparagopsis (1723 μg g−1 dry weight [DW] biomass), followed by dibromochloromethane (15.8 μg g−1 DW), bromochloroacetic acid (9.8 μg g−1 DW) and dibromoacetic acid (0.9 μg g−1 DW). Bromoform and dibromochloromethane had the highest activity with concentrations ≥1 μM inhibiting methane production. However, only bromoform was present in sufficient quantities in the biomass at 2 % organic matter to elicit this effect. Importantly, the degradability of organic matter and volatile fatty acids were not affected at effective concentrations.
Article
Full-text available
This study aimed to identify the optimal doses of the macroalgae Asparagopsis taxiformis and Oedogonium sp., individually and in combination, which would decrease the in vitro production of methane while minimizing adverse effects on fermentation, using rumen inoculant from Bos indicus steers. The dose-response experiment evaluated ten doses of Asparagopsis [ranging from 0 to 16.7 % of the organic matter (OM) incubated] and seven doses of Oedogonium (ranging from 0 to 100 % OM) using Rhodes grass hay as a basal substrate. Asparagopsis was highly effective in decreasing the production of methane with a reduction of 99 % at doses as low as 2 % OM basis. However, a dose of 2 % OM also decreased the production of volatile fatty acids (VFA). Oedogonium was less effective with doses ≥50 % OM significantly decreasing the production of methane. A combination of Asparagopsis (2 % OM) and Oedogonium (25 and 50 % OM) continued to suppress the production of methane, independent of the inclusion rate of Oedogonium. The effectiveness of Asparagopsis demonstrates its potential for the mitigation of methane emissions from ruminants at inclusion rates of ≤2 % OM. Oedogonium is a potential feed supplement due to its nutritional value, but supplements ≤25 % OM are recommended to avoid adverse effects on apparent in vitro fermentation.
Article
Full-text available
This study aimed to evaluate the effects of twenty species of tropical macroalgae on in vitro fermentation parameters, total gas production (TGP) and methane (CH4) production when incubated in rumen fluid from cattle fed a low quality roughage diet. Primary biochemical parameters of macroalgae were characterized and included proximate, elemental, and fatty acid (FAME) analysis. Macroalgae and the control, decorticated cottonseed meal (DCS), were incubated in vitro for 72 h, where gas production was continuously monitored. Post-fermentation parameters, including CH4 production, pH, ammonia, apparent organic matter degradability (OMd), and volatile fatty acid (VFA) concentrations were measured. All species of macroalgae had lower TGP and CH4 production than DCS. Dictyota and Asparagopsis had the strongest effects, inhibiting TGP by 53.2% and 61.8%, and CH4 production by 92.2% and 98.9% after 72 h, respectively. Both species also resulted in the lowest total VFA concentration, and the highest molar concentration of propionate among all species analysed, indicating that anaerobic fermentation was affected. Overall, there were no strong relationships between TGP or CH4 production and the >70 biochemical parameters analysed. However, zinc concentrations >0.10 g.kg(-1) may potentially interact with other biochemical components to influence TGP and CH4 production. The lack of relationship between the primary biochemistry of species and gas parameters suggests that significant decreases in TGP and CH4 production are associated with secondary metabolites produced by effective macroalgae. The most effective species, Asparagopsis, offers the most promising alternative for mitigation of enteric CH4 emissions.
Article
Full-text available
Enteric fermentation has been estimated to be responsible for 64.2 Mt CO2-e, or ∼16% of Australia's greenhouse gas emissions (Australian Greenhouse Office 2007). A bromochloromethane (BCM) formulation, previously shown to inhibit methanogenesis, was included in the diet of Brahman (Bos indicus) cross steers, twice daily in three separate experiments, to determine the effect on methane production, daily feed intake, liveweight (LW) gain and accumulation of residues of BCM in edible tissue. In the first experiment, the BCM formulation was fed at rates of 0, 0.15, 0.30, or 0.60 g/100 kg LW, twice daily, for 28 days. Methane production (mean s.e.), measured over 11 h after feed was first consumed on day 28, was 0.3 0.13 and 0.1 0.03 L/h for animals treated at a rate of 0.30 and 0.60 g/100 kg LW, respectively. This was significantly less (P 0.05) than for control animals (4.6 0.46 L/h) and animals treated at a rate of 0.15 g/100 kg LW (2.1 0.28 L/h). The dose rate of 0.30 g/100 kg LW was associated with a decrease in methanogenesis by ∼93% compared with the control group and was used in subsequent experiments. The second experiment evaluated the efficacy of the BCM formulation fed at rates of 0 or 0.30 g/100 kg LW, twice daily, for 90 days. Methane production was measured over 24-h periods, on days 30, 60 and 90. For days 30 and 90, methane production was reduced by 60% (P 0.05) to 4.2 1.82 L/h and by 50% (P 0.05) to 6.1 0.63 L/h, respectively, for treated animals compared with the control group. The final experiment determined the effect on LW gain and detectable residues in edible tissue, with animals given the BCM formulation at rates of 0 or 0.30 g/100 kg LW, twice daily, for 85 days. Liver, kidney, depot fat and muscle samples collected 1 and 10 days after the last day of treatment had concentrations of BCM that did not exceed 0.015 mg/kg and were less than the temporary maximum residue limit (0.02 mg/kg BCM), which applies to bovine meat, fat and edible offal. There were no significant differences in LW gain (1.4 0.10 v. 1.5 0.07 kg/day), feed conversion ratio (5.7 0.32 v. 5.4 0.09), hot carcass weight (235 5.0 v. 250 6.5 kg) or P8 fat depth (6.4 0.89 v. 8.1 1.15 mm) between control and treated animals. The experiments reported here were completed in 2004 before the Australian Government prohibited the manufacture and use of BCM. It is unlikely that the BCM formulation will be available for commercial use to mitigate livestock methane emissions in Australia. Nevertheless, the study has demonstrated that methane emissions were substantially reduced over a 90-day feedlot finishing period. This indicates that alternative antimethanogens with a similar mechanism of action may have practical commercial relevance.
Article
Full-text available
A variety of nutritional management strategies that reduce enteric methane (CH4) production are discussed. Strategies such as increasing the level of grain in the diet, inclusion of lipids and supplementation with ionophores (> 24 ppm) are most likely to be implemented by farmers because there is a high probability that they reduce CH4 emissions in addition to improving production efficiency. Improved pasture management, replacing grass silage with maize silage and using legumes hold some promise for CH4 mitigation but as yet their impact is not sufficiently documented. Several new strategies including dietary supplementation with saponins and tannins, selection of yeast cultures and use of fibre-digesting enzymes may mitigate CH4, but these still require extensive research. Most of the studies on reductions in CH4 from ruminants due to diet management are short-term and focussed only on changes in enteric emissions. Future research must examine long-term sustainability of reductions in CH4 production and impacts on the entire farm greenhouse gas budget.
Article
Full-text available
Although numerous algal products have antimicrobial activity, limited knowledge of metabolite localisation and presentation in algae has meant that ecological roles of algal natural products are not well understood. In this study, extracts of Asparagopsis armata had antibacterial activity against marine (Vibrio spp.) and biomedical (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus spp.) strains. The major natural products in both life-history stages of A. armata (as determined by gas chromatography-mass spectrometry analysis [GC-MS]) were bromoform (0.58 to 4.3 % of dry weight [DW]) and dibromoacetic acid [DBA] (0.02 to 2.6 % DW), and each compound was active against these same bacteria. To resolve whether this antibiotic activity was ecologically relevant, we examined the localisation of metabolites in the specialised cells of A. armata and observed a delivery mechanism for the release of metabolites to the surface. Bromoform and DBA were subsequently quantified in the surrounding medium of laboratory cultures, establishing their release from the alga. In a novel ecological test of algal natural products, halogenated metabolites in A. armata were manipulated by omitting bromine from an artificial seawater medium. Significantly higher densities of epiphytic bacteria occurred on algae that no longer produced halogenated metabolites. Both bromoform and DBA were more active against bacteria isolated from algae lacking brominated metabolites than algae producing normal amounts of these compounds. Taken together, these results indicate that halogenated metabolites of A. armata may be important in reducing epiphytic bacterial densities.
Article
Four steers were used in a 4 × 4 latin square experiment to assess comparative antimethanogenic effects of including bromochloromethane (BCM) (5.5 g once daily), unsaturated fat (corn oil), and saturated fat (tallow) at 5% in experimental diets. Basic dietary ingredients were citrus pulp and beet pulp plus hay. Average daily gain (ADG) and ruminal parameters including soluble carbohydrate and total volatile fatty acid (TVFA) concentrations, VFA’s percentages, and pH were measured. Only BCM proved significantly (P 0.05) by CH4 inhibition, although average daily gain showed the following trend: BCM > saturated fat > unsaturated fat > control. Inhibition caused lower (P < 0.05) ruminal acetate, and higher (P < 0.05) propionate over 24 hr post-feeding, higher (P < 0.05) butyrate from 3–15 hr post-fee...
Article
Three experiments were conducted to determine the effects of dietary supplementation with a novel antimethanogenic compound (AM) on methane production and growth in Brahman (Bos indicus) steers. The compound was a chemical complex of bromochloromethane (BCM) and alpha-cyclodextrin, which is chemically stable when added to feed, thus overcoming the highly volatile nature of BCM. In these experiments the AM compound was administered to steers as a mixture with different feed supplements. In Expt 1 the effect on in vivo methane production of feeding steers the AM compound was determined using a confinement-type respiration chamber. Methane production of AM-treated steers (0+/-2.4 mL/min) was lower (P < 0.001) than that of control steers (205+/-5.2 mL/min) over 28 days. In Expt 2 we determined the effect of AM treatment over 12 weeks on growth of steers fed on a low quality roughage diet. The most marked effect of AM treatment was reduced (P < 001) voluntary roughage dry matter intake (DMI), and reduced (P < 0.01) acetate:propionate molar ratio (A:P) in rumen fluid. Average daily liveweight gain (ADG) (0.22+/-0.01 kg/day) and feed:gain ratio (F:G) (20.7+/-1.46 kg DMI/kg liveweight) were not significantly affected by AM treatment. In Expt 3 we determined the effect of AM treatment over 10 weeks on growth of steers fed on a medium quality roughage diet, in steers that were either treated or not treated with a hormonal growth promotant (HGP; oestadiol 17 beta). AM treatment reduced (P < 0.05) DMI below that of steers not treated with AM, whereas DMI was not significantly affected by HGP treatment. Both AM (P < 0.01) and HGP (P < 0.05) treatments separately reduced A:P ratio in rumen fluid. AM treatment had no significant effect on ADG, whereas ADG of HGP-treated steers was higher (P < 0.05) than that of steers not treated with HGP (0.76+/-0.27 v. 0.60+/-0.027 kg/day). F:G was reduced (P < 001) by HGP treatment. F:G of both HGP-treated steers and those steers not treated with HGP was reduced (P < 0.05) by AM treatment. We conclude that feeding steers with this novel AM. compound enables the potent antimethanogenic properties of BCM to be realised under commercial conditions, and that prolonged use over 10-12 weeks is associated with an improved feed conversion efficiency in steers fed on better quality roughage diets.
Article
The close association between rumen fermentation and gas production has been recognised for over a century, but it is only since the 1940s that quantification techniques for measuring gas production have been evolved. The gas measuring technique has been widely used for evaluation of nutritive value of feeds. More recently, the upsurge of interest in the efficient utilisation of roughage diets has led to an increase in the use of this technique due to the advantage in studying fermentation kinetics. Gas measurement provides a useful data on digestion kinetics of both soluble and insoluble fractions of feedstuffs. This review describes the available in vitro gas measuring techniques used for feed evaluation with emphasis on assessing their relative advantages and disadvantages. Origin of gas, stoichiometry of gas production, and various areas for application of gas measurement in feed evaluation are discussed. Some important results obtained using gas measuring techniques have been highlighted, and the potential of gas techniques for tackling some interesting areas of research are presented. The need to consider substrate incorporation into microbial cells in gas measuring technique is pointed out.