Note: Descriptions are shown in the official language in which they were submitted.
tVC. 3026955078 10!08 ' 01 16:40 N0.973 04/1 US0027822
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POLYCXCLIC QU1NONE AND IONOPHORE COMPOSITION FOR THE
SYN~~tCISTIC REDUCTION Ok' MET'IiANE FORIf~A'TItIN IN RUNDNANT
A~I~TI~fI,cA~LS
B,Al,CKGItOITND 4~~ INVENTIUN
Control of methane production by metbanogenic bacteria in ruminant anirn,als
bas
important agronomic impact. Usc of inhibitors to control the methane produced
by
ruminants bas been recognized as a part of the mechanism ~or feed effxcicncy
that results
when mined with cattle feed fox both dairy and meat production. ,An effective
additive to
boost ruminant feed efficiency is a wcll-established part of the agronomic
practice for
commercial ruminant fatmittg.
Mathaaogenic bacteria form methane by an a~,aerobi.c process. The group
comprises the
genera Metlianvcoccus, Methanobactarium, Mathanosarcina, Meihanobrcylbactcr,
Methanotlrpxmus, Methanothri~ Metlutnvxpirillum, M.ethano~rsicrab#urn,
MethanococcoldeAs. Methanogenlum and Methanoplanuc.
inhibitors of methanogenesis in rumen perfozm two important functions. Cows
and
sheep loco 5-10°!0 of thoir caivric make to the fam~,ation of nnethane
and the resulting
loss of a carbon molecule that could have been incorpozatod iti short chain
Fatty acid
production. It~hibitian of metbaoe will., tberefG~xe, have a direct effect on
the formation of
short chain fatty acids in the rumen. ~Itber investigator have reported the
positive sect
of ivbibiting methane in rumern fer~ts~i..an (C. J. Van ~Tevel,13. L Demeyer,
Manipulatio~a of rutncn ferai~tativn, !h: ~ T'hc JLtumen h~F.crobial
F.,cosystecn., P. N.
~obsott, and (Ed) Elsevier=L'ublishing Co. ~198g);.
l~Hthrarre iar,~°X~itars f~uve previously beset deve'.crped fctr
feed°~lock additives to increase
' ~ feed effirier~cy. The iuhilritvrs fall gP~~,wa~lly i~.to tam clas:~e,~:
To.e lust class induces
' those ti~,aat affect me'tls~e ,formation indixectly'by intcxfering with the
electron tlvw
' upstream of the metbanogen in the microbial food cba.'xn,. Examples of this
group would
be ~trates arid rtit~ites. The second class anciudes~thc~se tb~ait affect
metbanogens directly.
' ' ' ' ~x$~iples oif such couapc~unds are ionophu~ca, arreit~ioGice;; tad
polycyclic quinanes.
Iot~opho~es irt~lude, fof Gxart~lc, Itl3mensin~'~;~ lasalc~cid .A;
salinonaycin, avoparccin,
~Tew !Page
Empfanssc~AMENDCI? SHEET
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WO 01/26482 PCT/US00/27822
actaplanin, and penicillin. A more complete list is cited in: C.J. Van Nevel,
D.I.
Demeyer, Manipulation of rumen fermentation, In: The Rumen Microbial
Ecosystem,
P.N.I-lobson, and (Ed) Elsevier Publishing Co. (1988). Polycyclic quinone
activity in this
regard are referenced in U.S. Patent 5,648,258 (Odour).
The inhibition of methane in rumen by polycyclic quinones (PCQ) operates by a
different
mechanism than ionophores. PCQ's are redox catalysts that block reduction of
electron
receptors at the cytochrome c-3 site in the cell wall of anaerobic bacteria,
such as
methanogens and sulfate reducers. Weimer reveals the action of 9,10-
anthraquinone in
U.S. Patent No. 5,385,844 as it applies to reducing sulfate by sulfate
reducing bacteria.
Ionophores act as antibiotics with the result that target bacteria
concentrations in the
rumen are reduced. Since 9,10-anthraquinone does not reduce target bacteria
concentration in the rumen, the two mechanisms are clearly distinct.
Garcia-Lopez et al. has demonstrated the use of PCQ's and ionophores each
separately
can reduce biogenic methane. (P.M. Garcia-Lopez, L. Kung, Jr., J.M. Odour. "In
Vitro
Inhibition of Microbial Methane Production by 9,10-anthraquinone".
Journal of Animal Science 1996, 74:2276 - 2284).
SUMMARY OF THE INVENTION
In its primary aspect, the invention is directed to a synergistic method for
reducing
methane formation in the rumen of ruminants comprising administering to the
ruminant
at least one ionophore compound, and at least one polycyclic quinone compound.
DEFINITIONS
As used herein, the term "rumen" refers to the gastrointestinal section found
in ruminants
(i.e. cattle, deer, moose, camels, sheep, goats, oxen, water buffalo, and musk
oxen)
where food is partially digested through bacterial fermentation.
As used herein, the term "Animal feed" refers to a prepared solid or liquid
given to a
ruminant animal for sustenance, health maintenance or supplementary food.
2
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DETAILED DESCRIPTION OF THE INVENTION
A. In General
It is recognized that the administration of an ionophore compound or the
administration
of a polycyclic quinine (PCQ) to a ruminant will reduce methane and boost feed
efficiency in the ruminant. However, applicant has discovered that when the
two classes
of compounds (ionophores and PCQ's) are administered simultaneously to a
ruminant, a
synergistic reduction of methane occurs. The advantage of employing this
technique is
to provide additional feed efficiency for agronomic benefits in ruminant
raising. In
addition, the levels of antibiotics in feed can be reduced which helps lower
the adaptive
challenge by non-target bacteria in the rumen and, thereby, lessens the
likelihood of
adaptation and resistance by rumen bacteria to the antibiotic.
B. Polycyclic Quinones (PCQ's)
A wide variety of polycyclic quinones can be used in the invention. As used
herein, the
term "polycyclic quinone" or "PCQ" refers to bicyclic, tricyclic and
tetracyclic
condensed ring quinones and hydroquinones, as well as precursors thereof. On
the
whole, the non-ionic polycyclic quinones and polycyclic hydroquinones (herein
referred
to collectively as PCQ's) have very low solubility in water at ambient
temperatures. For
use in the invention, it is preferred that such PCQs have water solubility no
higher than
about 1000 ppm by weight.
In addition, as noted above, certain precursors of such PCQ's can also be used
in the
invention either combined with the relatively insoluble PCQ's or by
themselves. Such
precursors are anionic salts of PCQ's, which are water soluble under alkaline
anaerobic
conditions. However, these materials are not stable and are easily converted
to the
insoluble quinone form upon exposure to oxygen.
Among the water-insoluble PCQ's, which can be used in the invention, are
anthraquinone compounds. As used herein, the term "anthraquinone" or "AQ"
refers to
3
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WO 01/26482 PCT/US00/27822
9,10-anthraquinone, naphthoquinone, anthrone (9,10-dihydro-9-oxo-anthracene),
10-
methylene-anthrone, phenanthrenequinone and the alkyl, alkoxy and amino
Derivatives of such quinones, 6,11-dioxo-1H-anthra[1,2-c]pyrazine, 1,2-
benzanthraquinone, 2,7-dimethylanthraquinone, 2-methylanthraquinone, 3-
methylanthraquinone, 2-aminoanthraquinone and 1-methoxyanthraquinone. Of the
foregoing cyclic ketones, 9,10-anthraquinone and methylanthraquinone are
preferred
because they appear to be more effective. Naturally occurring anthraquinones
can be
used as well as synthetic anthraquinones.
"Anthraquinone" or "AQ" compounds can further include insoluble anthraquinone
compounds, such as 1, 8-dihydroxy-anthraquinone, 1-amino-anthraquinone, 1-
chloro-
anthraquinone, 2-chloro-3-carboxyl-anthraquinone, 1-hydroxy-anthraquinone and
unsubstituted anthraquinone. Various ionic derivatives of these materials can
be
prepared by catalytic reduction in aqueous alkali.
In addition, a wide variety of anthrahydroquinone compounds can be used in the
method
of the invention. As used herein, the term "anthrahydroquinone compound"
refers to
compounds comprising the basic tricyclic structure, such as 9,10-
dihydroanthrahydroquinone, 1,4-dihydroanthrahydroquinone, and 1,4,4a,9a-
tetrahydroanthrahydroquinone. Anthrahydroquinone itself is 9,10-
dihydroxyanthracene.
More particularly, both water-insoluble and water-soluble forms can be used.
The non-
ionic compounds are largely insoluble in aqueous systems, while ionic
derivatives, such
as di-alkali metal salts, are largely soluble in water. The water-soluble
forms are stable
only in high pH anaerobic fluids. Low pH fluids (pH less than about 9-10) will
result in
the formation of the insoluble molecular anthrahydroquinone. Aerobic solutions
will
incur oxidation of the anthrahydroquinones to anthraquinone. Thus,
anthrahydroquinones will not exist for long periods of time in an aerated
environment.
For these reasons, anthrahydroquinone treatments are usually implemented with
the
soluble ionic form in a caustic solution. Sodium hydroxide solutions are
preferred over
the hydroxides of other alkali metals for economic reasons. Rumen physiology
may limit
the pH of such a preparation, but use of sodium hydroxide in ruminant feed is
an
established practice.
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CA 02382732 2002-04-02
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The extraordinary effectiveness of various forms of anthraquinone lies in
their non-
reactivity. These products are transported into the biofilm, diffuse through
the biofilm
voids, and then diffuse or are randomly transported by Brownian motion into
the
bacterial microcolonies without reduction in concentration as a consequence of
a
exopolysaccharide matrix present in the biofilm.
Even though solid particles of polycyclic quinone (PCQ) are required to
inhibit the
methane-producing bacteria, the PCQ can be introduced into the microbial
environment
in several physical forms. The PCQ can be introduced as a dispersion of these
solid
particles throughout the feed at the appropriate dose. The ionic (sodium salt)
form of the
PCQ will allow it to be solubilized in an anaerobic caustic solution as long
as the pH is
greater than 12 and preferably greater than 13. The salt stays soluble if the
pH of the
solution remains above about 12, with precipitation of solid PCQ taking place
as the pH
is reduced below this value. In the soluble form or with a slight amount of
precipitated
PCQ (typically in colloidal form), anthraquinone is in molecular form or
consists as
extremely small (submicron-sizes) particles. When the PCQ added to the water
is in the
form of a suspension of finely divided particles, it is preferred that their
largest
dimension be no greater than 50 micrometers, and preferably no greater than 5-
10
micrometers so that they can more easily pass through biofilm.
Whether the soluble or insoluble anthraquinone is used, it has been observed
that the
functional attachment of the anthraquinone particles to the bacteria is
limited in time by
metabolism of the particles by the sulfate-reducing bacteria. Thus,
application of the
treating medium must be repeated periodically in order to maintain inhibition
effectiveness.
Unlike antibiotics, which are lethal to rumen based bacteria, especially
methanogens,
PCQ's are non-lethal in their mechanism. Studies by Cooling et al. have
revealed the
mechanism of action of anthraquinones in sulfate-reducing bacteria (F.B.
Cooling III,
C.L. Maloney, En. Nagel, J. Tabinowski and J. M. Odom. "Inhibition of Sulfate
Respiration by 1, 8-Dyhydroxy-Anthraquinone and other Anthraquinone
Derivatives".
Applied And Environmental Microbiology, August 1996, p. 2999 - 3004). PCQ's
block
the production of adenosine triphosphate by the bacteria and thereby inhibit
respiration
using sulfate as an electron acceptor. The sulfate-reducing bacteria respire
by alternate
US002782~
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mechanisms under these conditions and are not killed. SltBs and methaaogens
are
closely licked in their ecological niche in the roman, and other anaerobic
eavironnaents.
?he PCQ effect on mcthanogans is either a direct e$ect sinniiar to flue SRB
mode of
action or indirect since methaaogens are dependent on SItB for micro-
nutrients. In both
conditions, nnethaaogcms thrive in the presence of PCQs without forming the
normal
levels of. methane.
C. Iononbores
Compounds known as ionophores are generally defined as substanced that
facilitate
TO tran~nission of an iotr, (such as sodium), across a lipid barrier each as a
cell membrane.
Two ionophore compounds particularly suited to this invention are the
Ruanoosin'~''~
product ~&om Eli Lilly which, is a sodium salt of a complex molecule of the
general
formula C3GHGl0I 1NA (foxmula weight 692.9) and tasaloeid A fiom Ho~msa
LaRoebe. Other ianophore compounds axe discussed in the Background Section oP
this
application, ax<d include salino~myein,, avoparcin, aridcin, actaplania
andpenicillin among
othexs. /u the rumen, ionbphores act as effective aatibactezial agents.
Killiuag me'khane
producing bacteria in the rumen of cattle decreases the toss bf carbon fxom
the rumen
fluid as rnethanc which is a si»~ilar action, to AQ.
Inhibition of methane by ionophones follows a mode of action where methanogens
and
other bacteztia that pmduce port hydxngen arid carbon: dioxide are reduced in
concentration. The antibacterial acao~a of iocwphores is the di,~t cause ofthe
reduction
in thethaaogenesis (/'.M. Ga~rcia-Lapez et a1.,1996 Ia Vittb xnbibition of
lVlirrobial
Methane l~duetion by 9,1,0 antbxa~quinone: Delaware Agricultmral >rxpczimental
Station;
paper no. 15G7). Reduction un bactezia conccntratian, iu tlae iumo~o. cap.~a~
a~:eot ~othec
W nicrolifd that is genec~y halpful in rumen digestiau attd '~hs ~hn ~of short
Chain.
fatty acids. The short cbaiu fatty acids are the souxce of emcrg~r r~uixc~d by
nuni.~~nts,
Increases ixi conc~rations of propxxonate and soPn~:~times bu~r2ate are
accoxnpani;ed by
reductions ira acetate 3n. xucn~, affected by ionopT~r~. Ionoplnoi~cs tend. to
louver. ,
. 30 concentrations of'bactexia that produce hydro~on, which is contrary to
the results i~een
with JPCQ's. . Hydxogen values tend to increase with PCQ's, which. should lead
to'
stimulation of bacteria levels that process lu~;~ci~nogan into butyxate.
($.~brssolv~s).
Acetate forming b~c~~cia are also reduced with ioo.~~phor~e~,; ~rhatv PCQ's
would tend. to
stimulate tl,~ formation of rnorz ac~~tat~ a~ acetoger~ic b~ctex~ s~,wcla as
(Acetitomaculwon
. hTew Page . G ~ . °
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CY-OlI4 PCT
runainis)Z (Gnxrting and Leedle,19$9 Enrichment anti Isolation of
A.cetitomacutum
Ruzninis gen.nov.sp. Nov; Acetogenic Bacteria from the Bovine Rumen. Arch.
' Micrabial. 151:399) are present. The advantage of increased bacterial
formation of short
chain fatty acids i s a boost in the food value of the feel ruminants.
D. lV~e~thods of Operation
The function of the PCQ i s to act as an inhibitor specific for methanogens
and sulfate
reducers found naturally in rumen fluid. Anthraduinone (AQ) is the preferred
PCQ to be
IO used in the invention. The inhibition of methane by AQ is a scpuate and
distinct
mechanism from the antibiotic effect of an ionopbore compound, such as
Rumensin'~'"1.
Bacteria counts of methanogens ere not affected by 9,10-aathxaquinone while
ionophores
reduce the viability o~'metb~aaogens. Therefore, the actions of the two
classes of
cornpoun~d.s are distinct and an additive effect would be expected. Contrary
to
expectations, the results show synergistic effects.
The custon~aty method of addi~ag a ~eed additive is to premix the compound
with a binder
anal a carrier so that the pre~anin carries a diiutEd coaeen~ration of alive
ingredient. The
premix is blended with the rations fior the animal in a subsequent process so
that there is a
certified. final concentration of active ingredient in the feed. A further
method of adding
PCQ to animal rations would be a direct admixture of active ingredient with
the rations
by means of a liquid formulation sprayed onto the feed or by a dry fwrmulation
admixed
by blending. The use of a sodium salt of anthraquixione in a high pH medium
could also
be 'used as a way to tnhaace the distribution of AQ ins animal feed. Certain
feeds would .
2S have putritive improvement due to the delignifi.cation of the fibs caused
by the well
known action df a high phi mcsddium and the catalytic action of AQ.ozt the
lignin bands
that m;akc fiber less di$~estibl~. .
. The preferred oonncentgalion, of.ionophores such as ~umensin'1'~; 2,2-
dxchlozacetamide is
preferably in, the nauge ~~ 0.5 ppm - 35 pp~n and more preferably in the range
of 5 - IO
ppm is the nlmen ~uadTof the rtt~minant. AQ is preferably in the range of 10 -
500 ppm
. . , a~.d. more p~ref~rably in .tbe range. of 10 - I 04. ppm i~. the rumen
Iluid o~ the ruminant.
. .. , .
I~ewv Page , 7
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CA 02382732 2002-04-02
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The advantageous properties of this invention can be further observed by
reference to the
following examples, which illustrate the invention.
EXAMPLES
Example 1
Culture Conditions
Effects of the potential methane inhibiting compounds were studied in in vitro
batch
culture ruminal fermentations. In all experiments, the treatment designated as
"control",
was a complete early market lamb feed (Agway, Inc., Tully, NY) that was ground
to pass
through a 1-mm screen of a Wiley Mill (Arthur H. Thomas, Co., Philadelphia,
PA) and
contained 0.29% sulfur (dry matter basis). In treatments with "high sulfur"
levels, the
control was supplemented with Na2S04 to yield a final concentration of 1.09%
(dry
matter basis). Sufficient control and high sulfur feed was prepared at the
start of the
study and was used in all experiments. A representative sample of each diet
was
analyzed for nutrient content by a commercial laboratory (Cumberland Valley
Analytical
Services, Maugansville, MD). The composition of the diets is shown in Table 1.
Batch
culture fermentations were replicated in triplicate 60 ml serum bottles for
each treatment
and (or) sampling point and contained 0.375 ~ 0.005 g of appropriate diet (air
dry basis).
Rumen fluid was obtained from a 300 kg fistulated steer with a rumen fistula.
The steer
had limited access to a commercial calf starter (18% CP) via a computer feeder
and had
ad libitum access to a medium quality alfalfa hay. Care and handling of the
steer
followed the standards outlines in the Agricultural Animal Care and Use
Handbook
(Consortium, 1989). Ruminal fluid was collected approximately 4 h after the
morning
allocation of hay and contents were placed in a sealed thermos while being
transported to
the lab for processing. Within 15 min of collection, ruminal fluid was
filtered through
four layers of cheese cloth and placed into a re-pipette dispenser that had
been purged
with anaerobic grade C02 (<1 ppm 02). An equal volume of warm (39°C)
mineral-
buffer solution (Goering and Van Soest, 1970) was added to the rumen fluid. A
reducing
solution was added where noted. In all experiments, 29.5 ml of the rumen fluid
- buffer
solution and 0.5 ml of appropriate treatment solution (when called for) was
added to each
serum bottle for each treatment. The serum bottles were then purged with
anaerobic
8
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CV-0114 PC°r CA 02382732 2002-04-02
grade C42 for 10 sec and sealCd with a butyl-rubber stopper $ad seal crimp.
Serum
bottles were incubated in a shaking water bath (Nevv Hruaswick Scientific,
model C376,
set at speed 2.5) for 24 h at 40°C.
S Treatment _ ' '
In order to establish an optimum sampling time nn subsequent experiments, the
high
sulfur diet was placed in nine serum bottles, incubated as described, and
three bottles
sampled at 6, 12, and~24 hoots (Experiment 1).
Treatments were: l) C; 2) HS; 3) HS plus 10 ppm AQ; 4) HS plus 5 ppm
Rumensin~''"~
(lrlaaco, Greeafxeld, ~ and 5) HS plus 10 ppm AQ. Fermentations 6upplemented
with
Rumettsin'rM were pzepared by fast dissolving the Rumeasin'~'M in 96% ethanol
and then
adding the samte volume of deioni2ed water, yielding a concentration of
znonensin (in
48% ethanol) that would result is 5 ppm in the rumen fluid buffer mix when
0.25~m1' of
the solution was added to 29.5 ml of the rumen fluid-buffer solution; finally,
0.25 mi,~of
deionized water was added to yield a final volume of 30 ml. Each of the other
treatment
conditions (including the control fermentations) were ritodi~,ed by adding the
same ' .
amount of ethanol to each as was present in the Rwmensia'~'"s fermentation.
This was .
dons by doubling the concentration of the stock solutions, adding 0.25 ml of
the stock
solution (or deionized water for tha controls) and 0.25 ml of 46% ethanol.
Analpses
~.eubatioa was stopped after 24 hr by immersing the serum bottles in ice. The
total
vdlume of gas produced was measured. by'noting the volume o~water displaced
i.e. as
inverted burette anal adding the amount of volume. Gas volume mea~urc~ments
were
conip~etcld within 20 min 'of cacb~ other. ~'o this value, was added the
volume of the head
space of the serum bottle. ~baec »ailliliters of the gas were then -
transferred to a
vaeutainer f~ibe for imethane mid hydxog~:o. aa~alysi.s. Tlte presence of
methane and
hydrogen were detcrrsni~ed by gas cluromatography. 'Two hundred microliters of
the gas
,,_
sample was infjected. onto a 1-Ie'~vlett Packard (Avondale, P~) 5880A gas
chrnmatograph
fitted with a Pors~pak. Q vc~umn using argon as abe carrier gas with a flow
rata of 11.1
~llmix~,; and a thE;r~nal conductivity detrcter. baitial oven, settings were
at 90°C for 1 _
' m3:nutc followed by a rats increas~e~ ef 30°Clmixa. unt3J. a final
temperature of 190°C was
Near page
' Emafangste~MENDED SHE=ET
CA 02382732 2002-04-02
WO 01/26482 PCT/US00/27822
reached. This temperature was maintained for 6 min. Analyses of gas and liquid
sulfide
were completed within two hours of the sample collection.
The pH of the final fermentation fluid was determined by pH probe. The
fermentation
fluid was then acidified with 1.0 ml of 25% meta-phosphoric acid (containing
10 ppm
isocaproic acid as an internal standard) to 5.0 ml of the fermentation fluid.
The acidified
fermentation fluid was analyzed for ammonia via a phenol-hypochlorite method
as
described by Okuda et al. (1965). The VFA were determined on a Hewlett Packard
5890A gas chromatograph using a 530 ~m macro bore Carbowax M column (Supelco,
Bellfonte, PA). The chromatograph oven was programmed as follows 70° C,
for 1 min,
5° C increase/min to 100° C, 45° C increase/min to
170° C, and final holding time of 5
min. Total VFA (TVFA) concentration was calculated as the sum of all VFA. The
molar proportions of VFA was calculated by dividing the individual VFA by the
sum of
the TVFA.
Statistical Analysis
The experiments were replicated on two separate days. Data were analyzed using
the
general linear model procedure of SAS (1985). The main effect of treatments
were tested
using the type III mean squares as the error term. When a significant F test
was detected,
means were compared by Turkey's test. Significance was declared at P<0.05
unless
otherwise noted.
Dose Effect of 9,10 Anthraguinone on In Yitro Ruminal Fermentation
Experiment 1
The effects of 9,10 AQ on in vitro fermentation are shown in Table 1: Total
VFA was
numerically but not statistically decreased by AQ relative to the control and
untreated
high sulfur diet. In general, the low level of 9,10 AQ (1 ppm) had no effect
on
fermentation end-products when compared to the untreated diets. However, both
the
intermediate and high levels of 9,10 AQ decreased (P<.OS) the molar proportion
of C2
and total gas produced but increased the percentages of C3, C4 and C5. These
amounts
of 9,10 AQ also increased (P<.OS) pH and the highest level decreased (P<.OS)
ammonia-
N. When compared to the control diet, addition of 10 and 25 ppm 9,10 AQ
decreased
methane production by 21 and 41 %, respectively, but hydrogen concentration
was
unaffected by treatment.
A-1 f'1 ~C. .
3026955078 'I 0/08 ' 0'1 16: 42 N0. 973 pg/1 US0027822
-0x~4 ~~ CA 02382732 2002-04-02
'.y~'ed of'Yutrlous Cnn~pounds oa Ire Y'uro Fermenladton
~,~''1'be eat of ionophore alone and in combination with 9,10 AQ compared to
9,I0 .AQ
alone and Mo04 alone in diets with high sulfvx are shown in Tablc 1. The
matbane
production shows the most dramatic synergy. Methacle inhibition is the best
measurement of how compounds will impxove the dige,~tive bcn~-'t of methane
inhibitors
~~ such as AQ and ionophores.
eble 1
Et~ect of Com~~nnd.~ on in vuro fermentation (Experiment 1)
~A -- ~~
- ~
Treatment TYF. C2 C3 Q4 G C95 CS pH mgldlTotw!Methtne~lyd~o8ca
A
Gne'
Contitnl 12?.8'55.324.1 1.1 15.2'1.6 2. 6.02 38.1 94.1'403 3.77
High 129.556.1"23.7 1. 15. 1.6 2.6 6.00'3G.8 97.1 465 4.15'
5ulftn'
~~~
T~igh 10 118.048.428,1 1.2 17.3t.7'33 G.13 37.2'89.2 265 4.2
+
PPm
High 25 123.955.224.2'1.2~15.11.6b2.T 6.00'37.9'94.1'451' 4.13
S +
Mo04 ppm ' .
High 5 11?.552.4'28.0 1.3 i3.6.1..73.0 G30'"'36.0'85.2 299 4:17
S + .
M9 1~ .
'
Aigb 5 107.344.2'33.1 1.4 13.42.0"4. 6.36 38.7"74.2 155 7.33'
S + +
~+AQ 10
SE . 0.8 I I 1 , <O.l~l I ~ I 13 ~O.ZS
- 0.1 0.1 <0.10.1 X0.10.03 1.G 1.6 .
_ ~ ~
°' b' °' °' a Means within a colunm with di.~~arcat
superscript letters dif~r (Pv.05) .
. ~ t ~'otal volatile fatty acids, »M
Z volatile: fatty acids; molesll00 moi of C2 ~ acetdtey C3 = propionate, Ci4 =
isobuiyrate,
C4 ~ butyrate, Ci,S = isoval~erate, C$ ~ va3.erate ,
3 mLlZ4, h, fe~enta>:~osx . .
1~ 4 ~.Molesl~4 h fermentrdtion .
S~.Molesr''24 b, ferme~atati.on .
~ Contained 0.~9% Sulfux on a d~MB .
~Ccutained 1.09% Sulfur on a IAMB r
a
R 9,10.A;nthrac~uizione
1
g Tturn~~in,T'~ (Ionophore)
New f agP~ 11
EmpfangsZe A~ENQEU SHEEP
CA 02382732 2002-04-02
WO 01/26482 PCT/US00/27822
i o n=3
Results
Methane Concentration
Umole/24hr. fermentationPercent of Control
Control 403 --
AQ 10 ppm 265 66%
Ionophores 5 ppm 299 74%
AQ plus Ionophore 10+5 155 38%
ppm
AQ plus Ionophore is synergistic by the following calculation:
Methane Concentration
AQ alone: 66% of control
Ionophore alone: 74% of control
Expected result if
Additive in effect: >50% of control (66% times 74% = 49%)
Actual result of
Combined effect: 38%
38% is statistically significant and lower than expected
12
