CA2382732A1 - Polycyclic quinone and ionophore composition for the synergistic reduction of methane formation in ruminant animals - Google Patents
Polycyclic quinone and ionophore composition for the synergistic reduction of methane formation in ruminant animals Download PDFInfo
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- CA2382732A1 CA2382732A1 CA002382732A CA2382732A CA2382732A1 CA 2382732 A1 CA2382732 A1 CA 2382732A1 CA 002382732 A CA002382732 A CA 002382732A CA 2382732 A CA2382732 A CA 2382732A CA 2382732 A1 CA2382732 A1 CA 2382732A1
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- ruminant
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/01—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing oxygen
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/111—Aromatic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C50/00—Quinones
- C07C50/16—Quinones the quinoid structure being part of a condensed ring system containing three rings
- C07C50/18—Anthraquinones, i.e. C14H8O2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/22—Methane [CH4], e.g. from rice paddies
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Abstract
A composition, preferably used to reduce methane formation in a ruminant, wherein the composition is a combination of a polycyclic quinone compound and an ionophore compound.
Description
tVC. 3026955078 10!08 ' 01 16:40 N0.973 04/1 US0027822 CV 0114 PC'f CA 02382732 2002-04-02 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
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
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.
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 9,10-anthraquinone, naphthoquinone, anthrone (9,10-dihydro-9-oxo-anthracene), 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.
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.
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 9,10-anthraquinone, naphthoquinone, anthrone (9,10-dihydro-9-oxo-anthracene), 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.
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-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~
08-10-2001 ENO. 3026955078 'i01O8~ '01 18:41 N0.9?3 05/10 CY-01141PG"P -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 ~ . °
Emvfansste AMENDED SF~EET
08-10-2001 tNO. _. 302695507$ 10/08 ' 01 16:41 N0.973 06/1t) 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 EmpfanasnAMENDED SHEET
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 OS-10-2001 ~C' .
30Z6g~5078 10/08 '01 16:42 N0.973 07/7 US0027822 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
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 g Tturn~~in,T'~ (Ionophore) New f agP~ 11 EmpfangsZe A~ENQEU SHEEP
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
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-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~
08-10-2001 ENO. 3026955078 'i01O8~ '01 18:41 N0.9?3 05/10 CY-01141PG"P -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 ~ . °
Emvfansste AMENDED SF~EET
08-10-2001 tNO. _. 302695507$ 10/08 ' 01 16:41 N0.973 06/1t) 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 EmpfanasnAMENDED SHEET
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 OS-10-2001 ~C' .
30Z6g~5078 10/08 '01 16:42 N0.973 07/7 US0027822 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
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 g Tturn~~in,T'~ (Ionophore) New f agP~ 11 EmpfangsZe A~ENQEU SHEEP
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
Claims (15)
POLYCYCLIC QUINONE AND IONOPHORE COMPOSITION FOR THE
SYNERGISTIC REDUCTION OF METHANE FORMATION IN RUMINANT
ANIMALS
CLAIMS:
1, A composition comprising at least one polycyclic quinone and at least one ionophore compound.
2. The composition of claim 1 wherein, the polycyclic quinone further comprises an anthraquinone or anthrahydroquinone compound.
3. The composition of claim 1 wherein the ionophore compound is selected from the group consisting of Rumensin.TM., lasalocid A, salinomycin, avoparcin, aridcin, actaplanin and penicillin.
4. The composition of claim 1 wherein the ionophore compound is Rumensin.TM..
5. A method of reducing methane formation in the rumen of a ruminant, the method comprising administering to the ruminant at least one polycyclic quinone and at least one ionophore compound.
6. The method of claim 5 wherein the polycyclic quinone and the ionophore are administered to the ruminant approximately simultaneously.
7. The method of claim 5 wherein the polycyclic quinone further comprises an anthraquinone or anthrahydroquinone compound.
8. The method of claim 5 wherein the ionophore compound is selected from the group consisting of Rumensin.TM., lasalocid A, salinomycin, avoparcin, aridcin, actaplanin and penicillin.
9. The method of claim 5 wherein the quantity of the polycyclic quinone is approximately within the range of 10 - 500 ppm in rumen fluid of the ruminant.
10. The method of claim 5 wherein the quantity of the ionophore compound is approximately within the range of 0.5 - 35 ppm in rumen fluid of the ruminant.
11. A method for reducing methane formation in the rumen of a ruminant, the method comprising administering to the ruminant a composition comprising at least one polycyclic quinone and at least one ionophore compound.
12. The method of claim 11 wherein the polycyclic quinone further comprises an anthraquinone or anthrahydroquinone compound.
13. The method of claim 11 wherein the ionophore compound is selected from the group consisting of Rumensin.TM., lavealocid A, salinomycin, avoparcin, aridcin, actaplanin and paricillin.
14. The method of claim 11 wherein the quantity of the polycyclic quinone is approximately within the range of 10 - 500 ppm in rumen fluid of the ruminant.
15. The method of claim 11 wherein the quantity of the ionophore compound is approximately within the range of 0.5 - 35 ppm in rumen fluid of the ruminant.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US15947899P | 1999-10-14 | 1999-10-14 | |
| US60/159,478 | 1999-10-14 | ||
| PCT/US2000/027822 WO2001026482A1 (en) | 1999-10-14 | 2000-10-10 | Polycyclic quinone and ionophore composition for the synergistic reduction of methane formation in ruminant animals |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2382732A1 true CA2382732A1 (en) | 2001-04-19 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002382732A Abandoned CA2382732A1 (en) | 1999-10-14 | 2000-10-10 | Polycyclic quinone and ionophore composition for the synergistic reduction of methane formation in ruminant animals |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1231844A1 (en) |
| JP (1) | JP2003529333A (en) |
| KR (1) | KR20020042866A (en) |
| AU (1) | AU7874000A (en) |
| CA (1) | CA2382732A1 (en) |
| NZ (1) | NZ518372A (en) |
| WO (1) | WO2001026482A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7966971B2 (en) * | 2008-05-23 | 2011-06-28 | C-Lock Inc. | Method and system for monitoring and reducing ruminant methane production |
| WO2011130538A2 (en) | 2010-04-16 | 2011-10-20 | C-Lock Inc. | Method and system for monitoring and reducing ruminant methane production |
| AU2011347802B2 (en) | 2010-12-20 | 2015-05-07 | Dsm Ip Assets B.V. | Use of nitrooxy organic molecules in feed for reducing methane emission in ruminants, and/or to improve ruminant performance |
| EP2713766B1 (en) * | 2011-05-26 | 2015-06-24 | DSM IP Assets B.V. | Use of a feed composition for reducing methane emission in ruminants, and/or to improve ruminant performance |
| US10085419B2 (en) | 2015-07-13 | 2018-10-02 | C-Lock Inc. | Modular livestock feed system for measuring animal intake and monitoring animal health |
| US11793768B2 (en) | 2018-01-24 | 2023-10-24 | Dsm Ip Assets B.V. | Methods for the non-therapeutic alleviation or prophylaxis of symptoms of oxidative stress, reduced immune system or cartilage damage of ruminants |
| AU2019212738A1 (en) * | 2018-01-24 | 2020-07-23 | Dsm Ip Assets B.V. | Novel use |
| WO2021038832A1 (en) * | 2019-08-30 | 2021-03-04 | 株式会社メニコン | Livestock feed efficiency improving agent, livestock weight gain promoting feed, livestock breeding method, and methane generation limiting method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5175319A (en) * | 1983-04-14 | 1992-12-29 | Burroughs Wellcome Co. | Naphthoquinone derivatives |
| WO1994008738A1 (en) * | 1992-10-22 | 1994-04-28 | Bio-Technical Resources Lp | Anthraquinone inhibition of methane production in methanogenic bacteria |
| AUPM922594A0 (en) * | 1994-11-04 | 1994-11-24 | Commonwealth Scientific And Industrial Research Organisation | Reduction of methane production in animals |
-
2000
- 2000-10-10 KR KR1020027004752A patent/KR20020042866A/en not_active Application Discontinuation
- 2000-10-10 EP EP00968885A patent/EP1231844A1/en not_active Withdrawn
- 2000-10-10 CA CA002382732A patent/CA2382732A1/en not_active Abandoned
- 2000-10-10 WO PCT/US2000/027822 patent/WO2001026482A1/en not_active Application Discontinuation
- 2000-10-10 JP JP2001529281A patent/JP2003529333A/en active Pending
- 2000-10-10 NZ NZ518372A patent/NZ518372A/en unknown
- 2000-10-10 AU AU78740/00A patent/AU7874000A/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| KR20020042866A (en) | 2002-06-07 |
| WO2001026482A1 (en) | 2001-04-19 |
| EP1231844A1 (en) | 2002-08-21 |
| AU7874000A (en) | 2001-04-23 |
| NZ518372A (en) | 2003-10-31 |
| JP2003529333A (en) | 2003-10-07 |
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