Novel use

Abstract

The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to the administration of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

Description

[0001] NOVEL USE

[0002] The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to the administration of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

[0003] The present invention further relates to animal premix consisting essentially of (a) 3-nitroox- ypropanol and / or derivatives thereof and Linolic acid and optionally one or more of (b-1) fat soluble vitamin(s), (b-2) water soluble vitamin(s), (b-3) trace mineral(s) and (b-4) macro minerals) and (c) a carrier as well as the use of said pre-mix for reducing the production of methane emanating from the digestive activities of ruminants.

[0004] The temperature of the air surrounding the earth is increasing, a process referred to as global warming. One of the main focuses to reduce this warming effect is to reduce the amount of greenhouse gases emitted into the atmosphere. Greenhouse gases are emitted from several different sources, both natural and artificial; however, the two sources with the most emphasis are the agricultural and fossil fuel industries. Within agriculture, ruminants and in particular cattle 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.

[0005] Methane emission from the ruminant livestock sector — a by-product from enteric fermentation of plant biomass in the ruminant digestive system — is produced by methanogenic archaea. Various attempts have been made in the last decade to mitigate methane production from ruminant animals. Although the approaches vary, the most popular method so far are feed additives which act in the rumen fluid by reducing respectively inhibiting the methane production by methanogenic archaea.

[0006] Linoleic acid (CAS No: 60-33-73) is a polyunsaturated omega-6 fatty acid. It is a fatty acid sometimes denoted 18:2 (n-6) or 18:2 cis-9,12. A linoleate is a salt or ester of this acid. It is a colorless liquid that is virtually insoluble in water but soluble in many organic solvents. It typically occurs in nature as a triglyceride (ester of glycerin) rather than as a free fatty acid.

[0007] Surprisingly, it has now been found, that the use of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid leads to a substantial reduction of the methane formation emanating from the digestive activities of said ruminant, wherein the combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid exhibits a synergistic effect with regard to the reduction of the methane formation.

[0008] Thus, the use of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid has a great potential in the mitigation of climate change by significantly reducing the methane emissions emanating during the digestive activities of ruminants.

[0009] Therefore, in a first embodiment, the present invention provides the use of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid for reducing the formation of methane emanating from the digestive activities of ruminants.

[0010] In a second embodiment, the invention further provides a method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the animal a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, said method comprising orally administering to the animal Linolic acid in an amount of at least 0.5 g Linolic acid / animal / day and 3-nitrooxypropanol and / or derivatives thereof in an amount of at least 0.025 g 3-nitrooxypropanol and / or derivatives thereof / animal / day.

[0011] In a third embodiment, the present invention relates to a (ruminant) pre-mix comprising a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid and optionally one or more of (b-1) fat soluble vitamin(s), (b-2) water soluble vit-amin(s), (b-3) trace minerals) and (b-4) macro mineral(s) and (c) a carrier.

[0012] It is well understood, that in all embodiments of the present invention the methane reduction by the administration of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid is preferably at least 10 %, more preferably at least 20 %, most preferably at least 30 % when compared to control, i.e. to ruminants not supplemented with a combination of 3- nitrooxypropanol and / or derivatives thereof and Linolic acid.

[0013] Thus, the present invention also relates to the use of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, wherein the methane production in ruminants is reduced by at least 10 % compared to control.

[0014] Preferably, 3-nitrooxypropanol and / or derivatives thereof is 3-nitrooxypropanol (CAS-No: 100502-66-7). 3-nitrooxypropanol and / or derivatives thereof may be incorporated into the solid dosage forms according to the present invention in the form of a powderous formulation comprising the 3-nitrooxypropanol and / or derivatives thereof and a carrier (also referred to herein as powderous formulation or PDMN) before being added to a premix or animal feed.

[0015] Preferably, in all embodiments of the present invention, the effective amount of Linolic acid to be administered to the ruminants is selected in the range from 0.5 to 200 g Linolic acid / animal / day, more preferably in the range from 1 to 150 g Linolic acid / animal / day, most preferably in the range from 5 to 100 g Linolic acid / animal / day, such as in the range from 10 to 100 g Linolic acid / animal / day, 15 to 100 g Linolic acid / animal / day or 20 to 100 g Linolic acid / animal / day.

[0016] 3-nitrooxypropanol and / or derivatives thereof preferably is administered to the ruminants in the range from 0.025 to 5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.05 to 4 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, most preferably in the range from 0.125 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day. Further suitable effective amounts are selected in the range from 0.25 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day or from 0.5 to 3 g 3-nitroox- ypropanol and / or derivatives thereof I animal / day.

[0017] The combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid is preferably administered either as a combination of pure (distinct) chemical compounds (i.e. each having a purity of >95% (HPLC)) or via an animal premix to the ruminant, both of which are then dispersed throughout the animal feed (diet) by any of a number of ordinary mixing techniques.

[0018] Such premixes are well known to a person skilled in the art. In the main, such premixes comprise the active ingredient, i.e. the combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, a carrier, and optionally further nutritional ingredients selected from the group consisting of such as vitamins, trace minerals and macro minerals.

[0019] A premix designates a preferably uniform mixture of one or more micro-ingredients with diluents and / or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix.

[0020] Particular advantageous premixes according to the present invention consist essentially of (a) a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid and optionally of one or more of (b-1) fat soluble vitamin(s), (b-2) water soluble vitamin(s), (b-3) trace mineral(s) and (b-4) macro mineral(s) and (c) a carrier Usual carriers employed in the art are soy grits, clays, (corn) sugars, silica, starch and similar products.

[0021] Said pre-mix may be prepared by methods known per se in the art.

[0022] Said premix are still novel. Thus, further aspects of the present invention are (ruminant) premix comprising a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid as well as the use thereof in all uses and methods according to the present invention. Preferred animal premixes are animal premixes comprising a combination of 3-nitrooxypro- panol and / or derivatives thereof and Linolic acid, a carrier, and optionally one or more ingredients selected from the group consisting of vitamins, trace minerals and macro minerals.

[0023] In one advantageous embodiment, the premixes of the present invention consist essentially of (a) a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, a carrier and one or more of (b-1) fat soluble vitamin(s), (b-2) water soluble vitamin(s), (b-3) trace mineral(s) and (b-4) macro mineral(s).

[0024] In one preferred embodiment, the premix is a mineral premix, a vitamin premix including vitamins and optionally minerals or a bolus.

[0025] Apart from a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid the premix of the invention preferably 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 combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals.

[0026] Macro minerals may be separately added to the feed. Therefore, in a particular embodiment, the premix comprises a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid with at least one additional component selected from the group consisting of fatsoluble vitamins, water-soluble vitamins, and trace-minerals.

[0027] The following are non-exclusive lists of examples of these components:

[0028] 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, manganese, and cobalt.

[0029] Examples of macro minerals are calcium, phosphorus, potassium, magnesium and sodium.

[0030] As mentioned above a premix is an example of a feed additive. It is understood that the combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid may be administered to the animal in different other forms. For example the combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid can also be included in a bolus that would be placed in the rumen and that would release a defined amount ofthe combination of 3-nitrooxypropa- nol and / or derivatives thereof and Linolic acid continuously in the defined dosages over a specific period of time.

[0031] In all embodiments of the present invention, it is to be understood by oral administration, a simple feeding, or manual administration of a bolus.

[0032] Methane emission by ruminants can easily be measured in individual animals in metabolic chambers by methods known in the art (Grainger et al., 2007 J. Dairy Science; 90: 2755- 2766). Moreover, it can also be assessed at barn level by an emerging technology using laser beam (McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802) or Sulfur hexafluoride or just SF6 or GreenFeed system. Alternatively, methane produced by a dairy ruminant can also be assessed by measurement of fatty acid profiles in milk according to WO 2009 / 156453.

[0033] The present invention also relates to the use of a feed additive according to the present invention which feed additive comprises one or more 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, deoxycholic acid, che- nodeoxycholic acid and derivatives thereof.

[0034] Further components that could be given together with the combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid are for example yeasts, oregano extracts, tannins and tannic acids and essential oils e.g. thymol, 3-methylphenol, limonene, vanillin, guajacol and eugenol. 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.

[0035] Ruminating mammals according to the present invention include cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and Nilgai.

[0036] For all embodiments of the present invention, domestic cattle, sheep and goat are the more preferred species. For the present purposes most preferred species are domestic cattle. The term includes all races of domestic cattle, and all production kinds of cattle, in particular dairy cows and beef cattle. It is well understood, that the term dairy cows and beef cattle encompasses animals in all ages and physiological stage of life and production systems such as confined, semi-confined and grazing.

[0037] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0038] Examples

[0039] In vitro test formethane production: A modified version of the “Hohenheim Forage value Test

[0040] (HFT)” may be used for testing the effect of specific compounds on the rumen functions mimicked by this in-vitro system. is gadded into a syringe with a composition of rumen liquor and an appropriate mixture of buffers. The solution is incubated at 39°C. After 8 hours the quantity (and composition) of gas phase produced is measured and put into a formula for conversion.

[0041] Mass element solution:

[0042] 6.2 g potassium dihydrogen phosphate (KH2PO4)

[0043] 0.6 g magnesium sulfate heptahydrate (MgSC>4 * 7H2O)

[0044] 9 ml concentrated phosphoric acid (1 mol / l) dissolved in distilled water to 1 I (pH about 1 .6)

[0045] Buffer solution:

[0046] 35.0 g sodium hydrogen carbonate (NaHCCh)

[0047] 4.0 g ammonium hydrogen carbonate ((NH4)HCC>3) dissolved in distilled water to 1 I

[0048] Trace element solution:

[0049] 13.2 g calcium chloride dihydrate (CaCh * 2H2O)

[0050] 10.0 g manganese(ll) chloride tetrahydrate (MnCh * 4H2O)

[0051] 1.0 g cobalt(ll) chloride hexahydrate (C0CI2 * 6H2O)

[0052] 8.0 g iron(lll) chloride (FeCI3* 6H2O) dissolved in distilled water to 100 ml

[0053] Sodium salt solution:

[0054] 100 mg sodium salt dissolved in distilled water to 100 ml

[0055] Reduction solution: first 3 ml sodium hydroxide (c = 1 mol / l), then 427.5 mg sodium sulfide hydrate (Na2S

[0056] * H2O) are added to 71.25 ml H2O solution must be prepared shortly before it is added to the medium solution Procedure:

[0057] Sample weighing: The feed stuff (i.e. TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage) is sieved to 1 mm 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. The remaining syringes contain the test substances, by groups of 4 syringes.

[0058] Preparation of the medium solution:

[0059] The components are mixed in a Woulff bottle in following order:

[0060] 711 ml water

[0061] 0.18 ml trace element solution

[0062] 355.5 ml buffer solution

[0063] 355.5 ml mass element solution

[0064] 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.

[0065] Extraction of the rumen liquor: 750 ml of rumen liquor are added to approximately 1 ,400 ml of medium solution under continued agitation and CCh-gassing.

[0066] 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.

[0067] Results

[0068] The food fermented was artificial TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage). Linolic acid was used at a concentration of 100 pM. 3-nitrooxy propanol (3-NOP) was used at two different concentrations 3.5 pM and 5 pM.

[0069] The results are presented in the following Table 1. Clear synergistic effects were obtained. Table 1 : Effect on Methane reduction resulting from linolic acid, 3-nitrooxy propanol, or combination of both.

Claims

Claims1 . Use of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid for reducing the formation of methane emanating from the digestive activities of ruminants.

2. The use according to claim 1 , wherein Linolic acid is administered to the ruminant in an amount selected in the range from 0.5 to 200 g Linolic acid I animal / day, preferably in the range from 1 to 150 g Linolic acid I animal / day, most preferably in the range from 10 to 100 g Linolic acid I animal / day.

3. The use according to claim 1 or 2, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered to the ruminant in an amount selected in the range from 0.025 to 5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.05 to 4 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, most preferably in the range from 0.125 to 3 g 3-nitrooxypropanol and / or derivatives thereof I animal / day. Further suitable effective amounts are selected in the range from 0.25 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day or from 0.5 to 3 g 3-nitroox- ypropanol and / or derivatives thereof I animal / day.

4. The use according to anyone of the preceding claims, wherein the ruminant animal is selected from the group consisting of cattle, preferably from the group consisting of domestic cattle, most preferably from beef cattle or dairy cows.

5. The use according to anyone of the preceding claims, wherein the 3-nitrooxypropanol and / or derivatives thereof and Linolic acid are each supplemented in the form of the pure (distinct) chemical compound or in the form of an animal premix.

6. The use according to claim 5, wherein the premix comprises a combination of 3-nitroox- ypropanol and / or derivatives thereof and Linolic acid, a carrier, and optionally one or more ingredients selected from the group consisting of vitamins, trace minerals and macro minerals.

7. The use according to claim 6, wherein the vitamins are selected from the group consisting of fat soluble vitamins and water soluble vitamins.

8. The use according to claim 6, wherein the premix consist essentially of (a) a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, a carrier and one ormore of (b-1) fat soluble vitamin(s), (b-2) water soluble vitamin(s), (b-3) trace mineral(s) and (b-4) macro mineral(s).

9. A method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the animal Linolic acid in an amount of at least 0.5 g Linolic acid / animal / day and 3-nitrooxypropanol and / or derivatives thereof in an amount of at least 0.025 g 3-nitrooxypropanol and / or derivatives thereof / animal / day.

10. The method according to claim 9, wherein the amount of Linolic acid is selected in the range from 0.5 to 200 g Linolic acid I animal / day, preferably in the range from 1 to 150 g Linolic acid I animal / day, most preferably in the range from 10 to 100 g Linolic acid I animal / day.11 . The method according to claim 9 or 10, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered to the ruminant in an amount selected in the range from 0.025 to 5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.05 to 4 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, most preferably in the range from 0.125 to 3 g 3-nitrooxypropanol and / or derivatives thereof I animal / day. Further suitable effective amounts are selected in the range from 0.25 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day or from 0.5 to 3 g 3-nitroox- ypropanol and / or derivatives thereof I animal / day.

12. An animal premix comprising (a) a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, a carrier, and optionally one or more ingredients selected from the group consisting of vitamins, trace minerals and macro minerals.

13. The animal premix according to claim 12, wherein the vitamins are selected from the group consisting of fat soluble vitamins, and / s water soluble vitamins14. The animal premix according to claim 12, wherein the premix consists essentially of a combination of 3-nitrooxypropanol and / or derivatives thereof and Linolic acid, a carrier and one or more of (b-1) fat soluble vitamin(s), (b-2) water soluble vitamin(s), (b-3) trace mineral(s) and (b-4) macro mineral(s).

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