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Title:
AZIDO ALKANOIC ACIDS AND DERIVATIVES THEREOF IN FEED FOR REDUCING METHANE FORMATION EMANATING FROM THE DIGESTIVE ACTIVITIES OF RUMINANTS
Document Type and Number:
WIPO Patent Application WO/2011/045418
Kind Code:
A1
Abstract:
The present invention relates to a method for reducing the production of methane emanating from the digestive activities of a ruminant by using as active compound at least one azido alkanoic acid and/or derivative thereof, which is administrated to the animal together with the feed. The invention also relates to the use of these ingredients in feed and feed additives such as premix, concentrates and total mixedration (TMR) or in the form of a bolus.

Inventors:
DUVAL, Stephane (2 rue due Tilleul, Bartenheim, Bartenheim, F-68870, FR)
KINDERMANN, Maik (Arlesheimerstrasse 7, Basel, CH-4053, CH)
Application Number:
EP2010/065533
Publication Date:
April 21, 2011
Filing Date:
October 15, 2010
Export Citation:
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Assignee:
DSM IP ASSETS B.V. (Het Overloon 11, TE Heerlen, NL-6411, NL)
DUVAL, Stephane (2 rue due Tilleul, Bartenheim, Bartenheim, F-68870, FR)
KINDERMANN, Maik (Arlesheimerstrasse 7, Basel, CH-4053, CH)
International Classes:
A23K1/18; A23K1/16
Attorney, Agent or Firm:
DSM NUTRITIONAL PRODUCTS LTD. (Wurmisweg 576, Kaiseraugst, CH-4303, CH)
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Claims:
Claims

1 . Use of at least one azido alkanoic acid or derivative thereof as defined by the formula (I) as an active compound in animal feeding for reducing the formation of methane emanating from the digestive activities of a ruminant animal,

wherein

n signifies 0,1 , 2, 3, 4, or 5,

X is independently oxygen (O) or amine (NH), wherein if R1 ≠ H, X-R1- represents an ester or a secondary amide group of the compound,

R1 is independently, hydrogen or a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 10 carbon atoms, and

R2 is independently, hydrogen or a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 8 carbon atoms.

2. Use according to claim 1 , wherein

n is comprised between 0 and 3,

R1 is independently hydrogen, or a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 8 carbon atoms, and R2 is independently, hydrogen or a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 3 carbon atoms.

3. Use according to claim 1 or 2, wherein X is oxygen (O), wherein if R1≠ H, X- R1 - represents an ester of the compound.

4. Use according to any of claims 1 to 3, wherein R2 is hydrogen (H).

5. Use according to claim 4, wherein the azido alkanoic acid or derivative thereof is selected from the group consisting of 3-azido propionic acid, 4-azido butanoic acid, 5- azido pentanoic acid, ethyl-3-azido propionate, ethyl-4-azido bu- tanoate, ethyl-5-azido pentanoate, 3-azido-N-ethyl-propionic amide, 4-azido-N-ethyl- butanoic amide and 5-azido-N-ethyl-pentanoic amide.

6. Use according to any of claims 1 to 3, wherein R2 is a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 3 carbon atoms. 7. Use according to claim 6, wherein the azido alkanoic acid or derivative thereof is selected from the group consisting of 2-azido propionic acid, 2-azido butanoic acid, 3-azido butanoic acid, 2- azido pentanoic acid, 3- azido pentanoic acid, 4- azido pentanoic acid, 2-azido-2-isopropyl ethanoic acid, ethyl-2-azido propionate, ethyl-2-azido butanoate, ethyl-3-azido butanoate, ethyl-2-azido pentanoate, ethyl-5- azido pentanoate, 3-azido-N-ethyl-propionic amide and 4-azido-N-ethyl-butanoic amide.

8. Use according to any of claims 1 to 7, wherein the azido alkanoic acid or derivative thereof is combined with at least one additional active substance selected from the group consisting of diallyl disulfide, garlic acid, allyl isothiocyanate, deoxy- cholic acid, chenodeoxycholic acid and derivatives thereof.

9. Use according to any of claims 1 to 8, wherein the ruminant animal is selected from the group consisting of: cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

10. Use according to claim 9 wherein the ruminant animal is a domestic cattle. 1 1 . Use according to any of claims 1 to 10, wherein the amount of the at least one azido alkanoic acid or derivative thereof as defined in formula (I) administered to the ruminant animal is from about 50 mg to about 1000 mg per kg feed.

12. A feed composition or feed additive comprising at least one azido alkanoic acid and derivative thereof according to any of claims 1 to 8.

13. The composition of claim 12 which is a mineral premix, a vitamin premix, or a premix including vitamins as well as minerals or a bolus.

14. A method for reducing the production of methane emanating from the digestive activities of a ruminant animal comprising orally administering a sufficient amount of at least one active compound as defined in formula (I)

wherein

n signifies 0,1 , 2, 3, 4, or 5,

X is independently oxygen (O) or NH, wherein if R1≠ H, X-R1 - represents an ester or a secondary amide group of the compound,

R1 is independently, hydrogen or a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 10 carbon atoms, and R2 is independently, hydrogen or a saturated straight or branched chain of an alkyl or alkenyl group containing 1 to 8 carbon atoms. 15. A method according to claim 14, wherein the active compound is administered to the animal in combination with at least one additional active substance selected from the group consisting of diallyl disulfide, garlic acid, allyl isothiocyanate, deoxycholic acid, chenodeoxycholic acid and derivatives thereof. 16. A method according to claim 14 wherein the ruminant animal is selected from the group consisting of: cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

17. A method according to Claim 16 wherein the ruminant animal is a domestic cattle.

18. A method according to Claim 14 wherein the amount of the at least one ac- tive compound as defined in formula (I) administered to the ruminant animal is from about 50 mg to about 1000 mg per kg feed.

Description:
Azido Alkanoic Acids and Derivatives thereof in Feed for Reducing Methane Formation emanating from the Digestive Activities of Ruminants

The present invention relates to the use of at least one azido alkanoic acid and/or derivative thereof for reducing the production of methane emanating from the digestive activities of ruminants.

The present invention also relates to animal feed or animal feed composition and feed additives comprising these active compounds. The term feed or feed composi- tion means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.

In the present context, a ruminant is a mammal of the order Artiodactyla that digests plant-based food by initially softening it within the animal's first stomach, known as the rumen, then regurgitating the semi-digested mass, now known as cud, and chewing it again. The process of again chewing the cud to further break down plant matter and stimulate digestion is called "ruminating". Ruminating mammals include cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

For the present purposes, domestic cattle are the most preferred species. For the present purposes the term includes all races of domestic cattle, and all production kinds of cattle, in particular dairy cows and beef cattle.

Rumen fermentation brings some disadvantages. Methane is produced as a natural consequence of the anaerobic fermentation, which represents an energy loss to the host animal. Carbohydrate makes up 70 to 80 % of the dry matter in a typical dairy cattle ration and in spite of this the absorption of carbohydrates from the gastrointestinal tract is normally very limited. The reason for this is the extensive fermentation of carbohydrates in the rumen resulting in production of acetate, propionate and butyrate as the main products. These products are part of the so called volatile fatty acids, VFA.

Besides the energy loss, methane is also a greenhouse gas, which is many times more potent than CO2. Its concentration in the atmosphere has doubled over the last century and continues to increase alarmingly. Ruminants are the major contributors to the biogenic methane formation, and it has been estimated that the prevention of methane formation from ruminants would almost stabilize atmospheric methane concentrations. Furthermore, the assessment of the Kyoto protocol places increased priority in decreasing methane emissions as part of a multi-gas strategy. The most effective additives currently used for reducing the formation of methane contain antibiotics which diminish the proliferation of microorganisms providing hydrogen (H 2 ) to the methanogenes. However, the effect of antibiotics on the formation of methane has some disadvantages because of rapid adaptation of the microflora and/or resistance development leading to a complete loss of the intended effect.

In recent years there has been an intense debate about the use of antibiotics in animal feed and in many countries a ban on this type of additions to feed additives is being considered or already in place.

Under these circumstances there is a need to develop new substances which reduce the formation of methane and which are in line with reliable and generally accepted practice and not of a medicinal nature.

The present inventors surprisingly found that the compounds specified herein after have a great potential for use in animal feed in order to essentially reduce the for- mation of methane without affecting microbial fermentation in a way that would be detrimental to the host animal.

In particular, it has been observed that the addition of at least one azido alkanoic acid and/or derivative thereof according to the invention are highly effective in decreasing methane formation and methanogenic bacteria in in vitro experiments. The mechanism how the compounds according to the present invention reduce the formation of methane is not completely understood, but the inventors believe that these compounds are effective as inhibitors of methyl -coenzyme-M reductase, an enzyme catalyzing the last step of the methane formation in methanogenic microorganisms (Archaea).

Therefore, the present invention provides the use of at least one azido alkanoic acid and/or derivative thereof for suppressing methane formation in ruminants.

The invention further provides a method for reducing the production of methane emanating from the digestive activities of ruminants comprising orally administering a sufficient amount of at least one azido alkanoic acid and/or derivative thereof to the animal.

Azido alkanoic acids and/or derivatives thereof according to the invention are defined by the following formula (I)

wherein

n signifies 0,1 , 2, 3, 4, or 5, preferably 0, 1 , 2 or 3

X is independently O or NH, wherein if R1 ≠ H, X-R1 - represents an ester or a secondary amide group of the compound,

R1 is independently, hydrogen or a saturated straight or branched chain of an al- kyl or alkenyl group containing 1 to 10, preferably 1 to 8 carbon atoms, and R2 is independently, hydrogen or a saturated straight or branched chain of an alkyi or alkenyl group containing 1 to 8, preferably 1 to 3 carbon atoms.

In the above definition of derivatives of the formula (I) a preferred alkyl group is methyl, ethyl, propyl, isopropyl, butyl, sec. butyl, isobutyl, pentyl, neopentyl, hexyl, 2- ethyl-hexyl and octyl. Furthermore any alkyl or alkenyl group containing three or more carbon atoms can be straight chain or branched. In addition for the straight chain or branched C2-C10-alkenylene group, this is understood to encompass al- kenylene groups with one or (from C4) more double bonds; examples of such alkenylene groups are those of the formulae -CH=CH-, -CH=CH-CH2-, -CH=CH- (CH2)3- and -(CH=CH)2-.

In a particular embodiment, preferred esters according to the present invention are methyl, ethyl, propyl, isopropyl, butyl, sec. butyl, isobutyl, pentyl, neopentyl, hexyl, 2- ethyl-hexyl and octyl. Moreover, preferred amide derivatives are ethyl-amides, pro- pyl-amides, butyl-amides and pentyl-amides.

More preferred compounds of formula (I) in accordance with the present invention are compounds in which the R2 group is a hydrogen. These compounds comprise the following azido alkanoic acids, and esters thereof: 2-azido ethanonic acid, 3- azido propionic acid, 4-azido butanoic acid, 5-azido pentanoic acid, ethyl-2-azido ethanonate, ethyl-3-azido propionate, ethyl-4-azido butanoate, and ethyl-5-azido pentanoate. In the above definition of derivatives of the formula I preferred amide derivatives according to the present invention are 2-azido-N-ethyl ethanoic-amide, 3-azido-N- ethyl-propionic amide, 4-azido-N-ethyl-butanoic amide and 5-azido-N-ethyl- pentanoic amide. The compounds 4-azido butanoic acid, 5- azido pentanoic acid, ethyl-3-azido propionate, ethyl-4-azido butanoate, and ethyl-5-azido pentanoate which are disclosed in table 1 , are especially preferred compounds in view of their properties in decreasing methane formation.

Table 1 :

In yet another embodiment, more preferred compounds of formula (I) in accordance with the present invention are compounds in which the R2 group is a methyl, ethyl, propyl, or isopropyl. These compounds comprise the following azido alkanoic acids, and esters thereof: 2-azido propionic acid, 2-azido butanoic acid, 2-azido pentanoic acid, 2-azido-2-isopropyl ethanoic acid, 3-azido butanoic acid, 3-azido pentanoic acid, 3-azido n-hexanoic acid, 3-azido-3-isopropyl propanoic acid, 4-azido pentanoic acid, ethyl-2-azido propionate, ethyl-2-azido butanoate, ethyl-2-azido pentanoate, ethyl-2-azido-2-isopropyl ethanoate acid, ethyl-3-azido butanoate, ethyl-3-azido pentanoate, ethyl-3-azido n-hexanoate, ethyl-3-azido-3-isopropyl propanoate, ethyl- 4-azido pentanoate. In the above definition of derivatives of the formula I, preferred amides according to the present invention are 2-azido-N-ethyl propionic-amide, 2-azido-N-ethyl-butanoic amide, 3-azido-N-ethyl-butanoic amide and 4-azido-N-ethyl-pentanoic amide. The term "a derivative thereof as herein also comprises salts of the azido alkanoic acids. Preferred cations for salt preparation may be selected from the group consisting of sodium (Na+), potassium (K+), lithium (Li+), magnesium (Mg2+), calcium (Ca2+), barium (Ba2+), strontium (Sr2+), and ammonium (NH4+). Salts may also be prepared from an alkali metal or an alkaline earth metal.

The compounds of the present invention can be manufactured in principle according to synthetic methods known per se for azido alkanoic acids, and derivatives thereof. In the case of the synthesis of esters of an azido alkanoic acid compound, the reaction is generally conducted as described in examples 1 to 3.

In the case of the synthesis of an azido alkanoic acid compound, the reaction is generally conducted as described in examples 4 to 6.

In the case of amidation of an azido alkanoic acid compound, the reaction is gener- ally conducted as described in examples 7 and 8.

In all these cases the product, i.e. the compound of formula I, can be isolated and purified by methods known per se, e.g. by adding a solvent such as diethyl-ether or ethyl acetate to induce the separation of the crude product from the mixture after re- action, and drying over Na 2 SO 4 of the collected crude product.

As indicated above, the compounds of the present invention are useful as compounds for feed additives and animal feed compositions for ruminants, and accordingly are useful as the active ingredients in such feed to reduce methane formation in the digestive tract of the animal. For the realisation of their use as such ingredients for the feed of ruminants the compounds may be incorporated in the feed by methods known per se in the art of feed formulation and processing. Further aspects of the present invention are therefore formulations, i.e. feed additives and animal feed compositions containing compounds as herein above defined.

The normal daily dosage of a compound according to the invention provided to an animal by feed intake depends upon the kind of animal and its condition. Normally this dosage should be in the range of from about 50 to about 1000 mg, preferably from about 100 to about 500 mg compound per kg of feed.

For the use in animal feed, however, azido alkanoic acids and derivatives thereof need not be that pure; it may e.g. include other compounds and derivatives.

The at least one azido alkanoic acid and/or derivative thereof may be used in combination with conventional ingredients present in an animal feed composition (diet) such as calcium carbonates, electrolytes such as ammonium chloride, proteins such as soya bean meal, wheat, starch, sunflower meal, corn, meat and bone meal, amino acids, animal fat, vitamins and trace minerals.

Particular examples of compositions of the invention are the following:

- An animal feed additive comprising (a) at least one compound selected from table 1 (b) at least one fat-soluble vitamin, (c) at least one water-soluble vitamin, (d) at least one trace mineral, and/or (e) at least one macro mineral;

- An animal feed composition comprising at least one compound selected from table 1 and a crude protein content of 50 to 800 g/kg feed.

The so-called premixes are examples of animal feed additives of the invention. A premix designates a preferably uniform mixture of one or more micro-ingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix. Apart from the active ingredients of the invention, the premix of the invention contains at least one fat-soluble vitamin, and/or at least one water soluble vitamin, and/or at least one trace mineral, and/or at least one macro mineral. In other words, the premix of the invention comprises the at least one compound according to the invention together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals. Macro minerals may be separately added to the feed. Therefore, in a particular embodiment, the premix comprises the active ingredients of the invention together with at least one additional component selected from the group consisting of fat- soluble vitamins, water-soluble vitamins, and trace-minerals. The following are non-exclusive lists of examples of these components:

- Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.

- Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D- panthothenate.

- Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.

- Examples of macro minerals are calcium, phosphorus and sodium. As regards feed compositions for ruminants such as cows, as well as ingredients thereof, the ruminant diet is usually composed of an easily degradable fraction (named concentrate) and a fibre-rich less readily degradable fraction (named hay, forage, or roughage). Hay is made of dried grass, legume or whole cereals. Grasses include among others timothy, ryegrasses, fescues. Legumes include among others clover, lucerne or alfalfa, peas, beans and vetches. Whole cereals include among others barley, maize (corn), oat, sorghum. Other forage crops include sugarcane, kales, rapes, and cabbages. Also root crops such as turnips, swedes, mangels, fodder beet, and sugar beet (including sugar beet pulp and beet molasses) are used to feed ruminants. Still further crops are tubers such as potatoes, cassava and sweet potato. Silage is an ensiled version of the fibre-rich fraction (e.g. from grasses, legumes or whole cereals) whereby material with a high water content is treated with a controlled anaerobic fermentation process (naturally-fermented or additive treated). Concentrate is largely made up of cereals (such as barley including brewers grain and distillers grain, maize, wheat, sorghum), but also often contain protein-rich feed ingredients such as soybean, rapeseed, palm kernel, cotton seed and sunflower.

Cows may also be fed total mixed rations (TMR), where all the dietary components, e.g. forage, silage and concentrate, are mixed before serving.

As mentioned above a premix is an example of a feed additive which may comprise the active compounds according to the invention. It is understood that the compounds may be administered to the animal in different other forms. For example the compounds can also be included in a bolus that would be placed in the rumen and that would release a defined amount of the active compounds continuously in well defined dosages over a specific period of time.

The present invention also relates to the use of azido alkanoic acids and derivatives thereof in combination with at least one additional active substance which shows similar effects with regard to methane formation in the rumen and which is selected from the group consisting of diallyl disulfide, garlic oil, allyl isothiocyanate, deoxy- cholic acid, chenodeoxycholic acid and derivatives thereof.

Further components that could be given together with the compound according to the invention are for example yeasts and ionophores like Monensin, Rumensin. It is at present contemplated that diallyl disulfide, garlic oil, allyl isothiocyanate deoxycholic acid, chenodeoxycholic acid and derivatives thereof are independently administered in dosage ranges of for example 0.01 -500 mg active substance per kg feed (ppm). These compounds are either commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art.

The present invention is further described by the following example which should not be construed as limiting the scope of the invention.

Examples

Example 1 : Synthesis of ethyl-3-azido propionate

0.5M in

181.03 H 2 0/DMF 143.15

C 5 H 9 Br0 2 C 5 H 9 N 3 0 2

35.8 mmol Natriumazide was dissolved in 36 ml of water and 36 ml DMF was added. To this solution, 32.5 mmol Ethyl-3-Bromopropionate was added and the bi- phasic mixture was stirred at 55 °C for 18 hours. The solution turned into yellow. After cooling to room temperature the crude reaction mixture was extracted with di- ethylether (500 ml) and the combined organic phase was washed with water and dried over Na 2 SO 4 . The solvent was removed in vacuo leaving 3.24 g of ethyl-3- azido propionate (22.6 mmol, yield = 82.0 %). Example 2: Synthesis of ethyl-4-azido butanoate

0.5M in

195.06 H 2 0/DMF 157.17

CeH^BrC^ C 6 H 11 N 3 0 2 35.8 mmol Natriumazide was dissolved in 36 ml of water and 36 ml DMF was added. To this solution, 32.5 mmol Ethyl-4-Bromobutyrate was added and the bi- phasic mixture was stirred at 55 °C for 18 hours and at 67 °C for 6 hours. After cooling to room temperature the crude reaction mixture was extracted with diethylether (500 ml) and the combined organic phase was washed with water and dried over Na 2 SO 4 . The solvent was removed in vacuo leaving 4.6 g of ethyl-4-azido butanoate (29.6 mmol, yield = 91 .0 %).

Example 3: Synthesis of ethyl-5-azido pentanoate

35.8 mmol Natriumazide was dissolved in 36 ml of water and 36 ml DMF was added. To this solution, 32.5 mmol Ethyl-5-Bromovalerate was added and the bi- phasic mixture was stirred at 55 °C for 18 hours and at 67 °C for 6 hours. After cooling to room temperature the crude reaction mixture was extracted with diethyl-ether (500 ml) and the combined organic phase was washed with water and dried over Na 2 SO 4 . The solvent was removed in vacuo leaving 5.1 g of ethyl-5-azido pentanoate (29.9 mmol, yield = 92.0 %). Example 4: Synthesis of 3-azido propionic acid

143.15 MeOH, THF 1 15-0 9

C 5 H 9 N 3 0 2 C 3 H 5 N 3 0 2 29.1 mmol Lithium hydroxide monohydrate (1 M in Water), 90 ml THF and 90 ml methanol was added to 15.4 mmol ethyl-3-azido propionate and stirred at room temperature for 18 hours. The solvent was removed in vacuo and a small amount of water and HCI 37 % was added until pH 1 . After that the reaction mixture was extracted with ethyl acetate (250 ml) and the combined organic phase was dried over Na 2 SO 4 . The solvent was removed in vacuo leaving 1 .22 g of 3-azido propionic acid (10.6 mmol, yield = 69 %).

Example 5: Synthesis of 4-azido butanoic acid

157.17 MeOH, THF

129.12

C 6 H 11 N 3 0 2 C 4 H 7 N 3 0 2

29.1 mmol Lithium hydroxide monohydrate as a 1 M solution in Water, 90 ml THF and 90 ml Methanol was added to 15.4 mmol Ethyl-4-Azido butanoate and stirred at room temperature for 18 hours. The solvent was removed in vacuo and a small amount of water and HCI 37 % was added until pH 1 . After that the reaction mixture was extracted with ethyl acetate (250 ml) and the combined organic phase was dried over Na 2 SO 4 . The solvent was removed in vacuo leaving 1 .87 g of 4-azido butanoic acid (14.5 mmol, yield = 94 %). Example 6: Synthesis of 5-azido pentanoic acid

MeOH, THF

171.20 143.15

C 7 H 13 N 3 0 2 C 5 H 9 N 3 0 2 29.1 mmol Lithium hydroxide monohydrate as a 1 M solution in Water, 90 ml THF and 90 ml methanol was added to 15.4 mmol Ethyl-5-Azido pentanoate in a flask and stirred at room temperature for 18 hours. The solvent was removed in vacuo and a small amount of water and HCI 37 % was added till pH 1 . After that the reaction mixture was extracted with Ethyl acetate (250 ml) and the combined organic phase was dried over Na 2 SO 4 . The solvent was removed in vacuo leaving 2.18 g of 5-azido pentanoic acid (15.2 mmol, yield = 99 %).

Example 7: Synthesis of 3-azido-N-ethyl-propionic amide

C 3 H 5 N 3 0 2 142.16

C 5 H 10 N 4 O

13.1 mmol Thionyl chloride was added to 8.7 mmol 3-Azidoproponic acid and stirred for 2 hours at 50 °C. After cooling to room temperature, to this mixture was added 2ml THFabs-

This solution was added drop wise to 26.1 mmol Triethylamine and 26.1 mmol Ethylamine in THF (2M) over 10 min at 10 °C. After stirring for 20min at 10 °C and 1 h at room temperature the solvent was removed in vacuo. The crude product was dissolved in 70ml Dichloromethane and extracted with Water (100 ml) until the water was pH neutral. The water phase was rewashed with Dichloromethane (70 ml). The combined organic phase was washed with brine, dried over Na2SO 4 and the solvent was removed in vacuo leaving 0.53 g of 3-azido-N- ethyl-proponic amide (3.73 mmol, yield = 43 %).

Example 8: Synthesis of 4-azido-N-ethyl-butanoic amide

129 12 ^ 19

C4H7N302 C6H12N40

16.3 mmol 4-Azido butanoic acid was dissolved in 21 ml Dichloromethaneabs- 28.8 mmol Oxalylchloride was added over 5 min, followed by 2 drops of DMF abS and stirred over 45 min at room temperature. The solvent and remaining Oxalylchloride was removed in vacuo.

The residue was dissolved in 20 ml Dichloromethaneabs- To this solution 16.3 mmol Triethylamine and 19.6 mmol Ethylamine in THF (2M) was added over 10min and stirred 1 h at room temperature.

The crude reaction mixture was extracted with 1 M HCI (100 ml), washed neutral with water (150 ml) and rewashed with brine. The combined organic phase was dried over Na2SO 4 and the solvent was removed in vacuo leaving 0.93 g of 4-azdio- N-ethyl-butanoic amide (5.95 mmol, yield = 36.6 %) Example 9: In vitro test for methane production

A modified version of the "Hohenheim forage value test/HFT" is used for testing the effect of specific compounds on the rumen functions mimicked by this in vitro sys- tern.

Principle:

Feed is given into a syringe with a composition of rumen liquor and an appropriate mixture of buffers. The solution is incubated at 39 °C. After 24 hours the quantity (and composition) of methane produced is measured and put into a formula for conversion.

Reagents: Mass element solution:

- 6.2 g potassium dihydrogen phosphate (KH 2 PO 4 )

- 0.6 g magnesium sulfate heptahydrate (MgSO 4 * 7H 2 O)

- 9 ml concentrated phosphoric acid (1 mol/l)

- dissolved in distilled water to 1 I (pH about 1 .6)

Buffer solution:

- 35.0 g sodium hydrogen carbonate (NaHCOs)

- 4.0 g ammonium hydrogen carbonate ((NH 4 )HCOs)

- dissolved in distilled water to 1 I

Trace element solution:

- 13.2 g calciumchloride dihydrate (CaCI 2 * 2H 2 O)

- 10.0 g manganese(ll) chloride tetrahydrate (MnCI 2 * 4H 2 O)

- 1 .0 g cobalt(ll) chloride hexahydrate (CoCI 2 * 6H 2 O)

- 8.0 g iron(lll) chloride (FeCI 3 * 6H 2 O)

- dissolved in distilled water to 100 ml Sodium salt solution:

- 100 mg sodium salt

- dissolved in distilled water to 100 ml Reduction solution:

- first 3 ml sodium hydroxide (c = 1 mol/l), then 427.5 mg sodium sulfide hydrate (Na 2 S * H 2 O) are added to 71 .25 ml H 2 O

- solution must be prepared shortly before it is added to the medium solution Procedure:

Sample weighing:

The feed stuff is sieved to 1 mm - usually TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage) - and weighed exactly into 64 syringes. 4 of these syringes are the substrate controls, which display the gas production without the effect of the tested compounds. 4 other syringes are positive control, in which bromoethane sulfonate has been added to 0.1 mM. When needed, 4 syringes contain a carrier control (if the test compounds need a carrier). The remaining syringes contain the test substances, by groups of 4 syringes.

Preparation of the medium solution:

The components are mixed in a Woulff bottle in following order:

- 71 1 ml water

- 0.18 ml trace element solution

- 355.5 ml buffer solution

- 355.5 ml mass element solution

The completed solution is warmed up to 39 °C followed by the addition of 1 .83 ml sodium salt solution and the addition of reduction solution at 36 °C. The rumen liquor is added, when the indicator turns colourless. Extraction of the rumen liquor:

750 ml of rumen liquor are added to approximately 1 ,400 ml of medium solution under continued agitation and CO2-gassing. Filling the syringes, incubation and determining gas volumes and VFA values:

The diluted rumen fluid (24 ml) is added to the glass syringe. The syringes are then incubated for 8 hours at 39 °C under gentle agitation. After 8 hours, the volume of gas produced is measured, and the percentage of methane in the gas phase is determined by gas chromatography.

Results

The azido alkanoic acids and esters disclosed in Table 1 were tested using the methodology described above.

The food fermented was artificial TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage).

The compounds produced as described in examples 1 to 5 were added to the fer- mentation syringes to a concentration of 2 and 0.5 % of dry matter (DM).

The results are presented in the following Table 2.

Table 2: Methane reduction effect resulting from the average of two experiments with some compounds disclosed in the present invention (a minus sign in the column CH change (%) means a reduction in methane produced when compared to control).

Example 1 1 : Effect of ethyl-3-azido propionate in an in vitro continuous rumen simulation system: "Rusitec". In vitro system and experimental diets:

The in vitro experiment was conducted using a Rusitec continuous rumen simulation system as described in detail by Soliva & Hess (2007) In Measuring Methane Production from Ruminants, pp. 15-135. With this in vitro system digestion of basal diets was tested at 15 g dry matter / day (DM/d) both with and without ethyl-3-azido propionate supplemented at 1 % (on a DM basis) in a completely randomised design in four replicates per treatment. The basal diets composition is described in Table 3. Table 3. Composition of the dietary substrates

Basal diet Control

Supply to the fermenter (g DM/d)

Hay 7.47

Barley 5.23

Soybean meal 2.24

Vitamin -Mineral mixture * 0.08

Total dry matter supply 15.00

Analyses nutrient composition (g/kg DM)

Organic matter 826

Crude protein 182

Neutral detergent fibre (NDF) 343

contained (per kg) 140 g Ca; 70 g P; 80 g Na; 30 g Mg; 15 mg Se; 500Ό00 IU vitamin A; 120Ό00 IU vitamin D 3 ; 2'500 IU vitamin E.

Experimental procedures and sampling:

In four experimental runs, each lasting for 10 days, the daily portions of experimental feeds were put into nylon bags (70 x 140 mm) with a pore size of Ι ΟΟμιτι. Before that, hay and straw were ground to pass a 5mm sieve whereas the grains were ground to a size of 3 mm. Ruminal fluid was obtained from a lactating rumen- fistulated Brown Swiss cow which was fed hay ad libitum and concentrate (1 kg/d administered in two portions). The cow was kept according to the Swiss guidelines for animal welfare. Before inoculation, ruminal fluid was strained through four layers of medicinal gauze with a pore size of about 1 mm. At the beginning of each ex- perimental run the fermenters were filled with 100 ml pre-warmed buffer and 900 ml strained ruminal fluid. Thereafter, two nylon bags were administered whereby the first one was filled with solid ruminal content (about 40 g fresh matter) and the second one with the respective experimental diet. On the second experimental day the bag containing the solid ruminal content was exchanged with another bag contain- ing the experimental diet. Each feed bag was incubated for 48 h. To maintain an- aerobic conditions the system was flushed with gaseous nitrogen for 3 min after exchanging the feed bags. The incubation temperature was kept constant at 39.5 °C. Buffer flow to the fermenters was continuous and averaged 397 ml/d, resulting in a dilution rate of about 40 % per day. The resulting incubation fluid outflow was col- lected in bottles chilled at -20 °C. Incubation fluid samples, collected directly from the fermenters via a three-way valve using a syringe equipped with a plastic tube 3 hours before exchanging the feed bags, were analysed daily for redox potential and pH using the respective electrodes connected to a pH meter (model 634; Methrom AG, Herisau, Switzerland). Part of the incubation fluid samples taken were centri- fuged for 5 min at 4000 rpm (Varifugew K; Heraeus, Osterode, Germany) and the supernatant fraction was stored at -20 °C before being analysed for SCFA concentrations. After 48 h of incubation, dietary residues were washed with cold water in a washing machine and frozen at -20 °C until nutrient analyses were performed. Later the lyophilised and ground residues were analysed for DM and organic matter, via total ash (automatically by TGA-500; Leco Corporation, St Joseph, Ml, USA), N (C/N analyser, Leco-Analysator Typ FP-2000; Leco Instrumente GmBH, Kircheim, Germany; crude protein ¼ 6-25 £ N) and neutral-detergent fibre. Analyses of neutral-detergent fibre were carried out with the Fibretec System M (Tecator, 1020 Hot Extraction, Hoganas, Sweden) with the addition of D-amylase but without sodium sulfite as suggested by Van Soest et al. (1991 ) J Dairy Sci. 74, 3583-3597. The fermentation gases produced during 24 h were collected in gas-tight aluminium bags (TECOBAG 8 litres, PETP/AL/ PE - 12/12/75 quality; Tesserau Container GmbH, Burstadt, Germany). Gas was analysed daily for concentrations of CH , CO2 and H 2 with a GC (model 5890 Series II; Hewlett Packard, Avondale, PA, USA) equipped with a flame ionisation detector (to determine CH ), a thermal conductivity detector (to determine CO2 and H 2 ) and a 2-34m x 2-3mm column, 80/100 mesh (Porapak Q; Fluka Chemie AG, Buchs, Switzerland). The total amount of gas produced was quantified by water displacement. This was accomplished by pressing the fermentation gas out of the gas-tight aluminium bags using plates of 2 kg weight. Fermentation gas was flushed into an Erlenmeyer flask and the water displaced from this flask then was collected in a second, graduated, flask.

Results: All results demonstrating the methane reduction effect of ethyl-3-azido propionate are summarized in Table 4.

Table 4. Effects of ethyl-3-azido propionate on fermenter fluid traits, rumen micro- bial counts, degradation parameters and gas production (averages of days 6-10). SEM, standard error of means.

Control Ethyl-3-azido propionate SEM

Fermenter fluid traits

Redox potential (mV) -192 -194 5.8

PH 6.52 6.40 0.025

Ammonia (mmol/l) 1 1 .5 8.8 0.34

Short chain fatty acids

mmol/d 1 16 1 17 2.9 molar porportions (%)

Acetate 52.0 52.3 0.53

Propionate 19.9 16.8 1 .00 n-Butyrate 21 .3 25.0 0.92

/ ' so-Butyrate 0.628 0.567 0.0781 n-Valerate 5.82 4.97 0.2445

/ ' so-Valerate 0.355 0.364 0.0802

Acetate:propionate 2.66 3.18 0.209

Ruminai microbes

Entodiniomorphs (x10 3 /ml) 2.37 7.75 0.603

Holotrichs (x10 3 /ml) 1 .18 1 .77 0.275

Bacteria (x10 8 /ml) 5.59 6.33 0.274

Control Ethyl-3-azido propionate SEM

Nutrient degradation (%)

Organic matter 71 .5 71 .7 0.95

Crude protein 70.4 68.4 1 .39 Neutral detergent fibre 42.3 42.7 1 .58

Gaseous emissions

(mmol/d)

Methane 7.96 0.19 0.310 Carbon dioxide 73.3 72.7 2.38 Hydrogen 0.20 1 .51 0.245

CH 4 (mmol/g of degraded

nutrient)

Organic matter 0.789 0.018 0.0343

Neutral detergent fibre 3.70 0.09 0.234