3-nitrooxypropanol with metronidazole for reducing methane formation resulting from digestive activity in ruminants

Abstract

The present invention relates to the field of reducing methane emissions from ruminants. In particular, the present invention relates to administering to a ruminant a combination of 3-nitrooxypropanol and / or a derivative thereof and metronidazole for reducing methane production resulting from digestive activities of said ruminant.

Description

Technical Field

[0001] This invention relates to the field of reducing methane emissions from ruminants. Specifically, this invention relates to the administration of 3-nitrooxypropanol and / or its derivatives in combination with metronidazole to ruminants to reduce methane production resulting from the digestive activity of said ruminants.

[0002] The present invention also relates to an animal premix comprising essentially (a) 3-nitrooxypropanol and / or its derivatives with metronidazole, and optionally one or more of (b-1) fat-soluble vitamins, (b-2) water-soluble vitamins, (b-3) trace minerals and (b-4) macro minerals, and (c) a carrier. The present invention also relates to the use of said premix for reducing methane generation from the digestive activities of ruminants. Background Technology

[0003] The temperature of the air surrounding the Earth is rising, a process known as global warming. One of the main focuses to mitigate this warming effect is reducing the amount of greenhouse gases emitted into the atmosphere. Greenhouse gases are emitted from a variety of sources, both natural and anthropogenic; however, the two most concerning sources are agriculture and the fossil fuel industry. In agriculture, ruminants, particularly cattle, are major contributors to biogenic methane formation, and it is estimated that preventing methane formation by ruminants could almost stabilize atmospheric methane concentrations.

[0004] Methane emissions from ruminant livestock farming are byproducts of intestinal fermentation of plant biomass in the ruminant digestive system, produced by methanogenic archaea. Over the past decade, various attempts have been made to reduce methane production in ruminants. Despite the varying approaches, the most popular method to date has been feed additives, which work by either reducing or inhibiting methane production by methanogenic archaea in the rumen fluid.

[0005] Metronidazole (CAS 443-48-1) is an antibiotic and antiprotozoal drug. It can be used alone or in combination with other antibiotics to treat pelvic inflammatory disease, endocarditis, and bacterial vaginosis. Metronidazole is commercially available under the brand name Flagyl. Summary of the Invention

[0006] It has now been surprisingly found that the use of 3-nitrooxypropanol and / or its derivatives in combination with metronidazole can substantially reduce methane formation caused by the digestive activity of the ruminants, wherein the combination of 3-nitrooxypropanol and / or its derivatives with metronidazole exhibits a synergistic effect in reducing methane formation.

[0007] Therefore, the combination of 3-nitrooxypropanol and / or its derivatives with metronidazole has great potential in mitigating climate change by significantly reducing methane emissions generated during the digestive activities of ruminants.

[0008] Therefore, in a first embodiment, the present invention provides the use of a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole for reducing methane formation resulting from the digestive activity of ruminants.

[0009] In a second embodiment, the present invention also provides a method for reducing methane production caused by the digestive activity of ruminants, the method comprising orally administering to the animal a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, the method comprising orally administering to the animal at a dose of at least 0.5 g metronidazole per animal per day and at least 0.025 g 3-nitrooxypropanol and / or its derivatives per animal per day.

[0010] In a third embodiment, the present invention relates to a (ruminant) premix comprising a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, and optionally one or more of (b-1) fat-soluble vitamins, (b-2) water-soluble vitamins, (b-3) trace minerals and (b-4) macro minerals and (c) a carrier.

[0011] It should be fully understood that, in all embodiments of the invention, the methane reduction achieved by administering a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole is preferably at least 10%, more preferably at least 20%, and most preferably at least 30% compared to a control (i.e., ruminants not supplemented with a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole).

[0012] Therefore, the present invention also relates to the use of 3-nitrooxypropanol and / or its derivatives in combination with metronidazole, wherein methane production in ruminants is reduced by at least 10% compared to a control. Detailed Implementation

[0013] Preferably, 3-nitrooxypropanol and / or its derivatives are 3-nitrooxypropanol (CAS No.: 100502-66-7). 3-nitrooxypropanol and / or its derivatives may be incorporated into the solid dosage form according to the invention in the form of a powder formulation (also referred to herein as a powder formulation or PDMN) containing 3-nitrooxypropanol and / or its derivatives and a carrier before being added to the premix or animal feed.

[0014] Preferably, in all embodiments of the invention, the effective amount of metronidazole applied to ruminants is selected from the range of 0.5 to 200 g metronidazole / animal / day, more preferably from 1 to 150 g metronidazole / animal / day, and most preferably from 5 to 100 g metronidazole / animal / day, for example from 10 to 100 g metronidazole / animal / day, 15 to 100 g metronidazole / animal / day, or 20 to 100 g metronidazole / animal / day.

[0015] 3-Nitrooxypropanol and / or its derivatives are preferably administered to ruminants at a rate of 0.025 to 5 g / animal / day, more preferably 0.05 to 4 g / animal / day, and most preferably 0.125 to 3 g / animal / day. Other suitable effective amounts are selected from the range of 0.25 to 3 g / animal / day or 0.5 to 3 g / animal / day.

[0016] The combination of 3-nitrooxypropanol and / or its derivatives with metronidazole is preferably administered to ruminants as a combination of pure (separate) chemical compounds (i.e., each with a purity ≥95% (HPLC)) or via animal premix, and the two are subsequently dispersed throughout the animal feed (diet) using any of a variety of common mixing techniques.

[0017] Such premixes are well known to those skilled in the art. Such premixes mainly consist of an active ingredient, namely 3-nitrooxypropanol and / or its derivatives, in combination with metronidazole, a carrier, and other nutrients optionally selected from, for example, vitamins, trace minerals, and macrominerals.

[0018] A premix is ​​a preferred homogeneous mixture of one or more trace components with a diluent and / or a carrier. Premixes are used to promote the uniform dispersion of trace components in larger mixtures.

[0019] The particularly advantageous premix according to the invention consists essentially of (a) a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, and optionally one or more of (b-1) fat-soluble vitamins, (b-2) water-soluble vitamins, (b-3) trace minerals and (b-4) macro minerals and (c) a carrier.

[0020] Commonly used carriers in this field include soybean grits, clay, (corn) sugar, silica, starch, and similar products.

[0021] The premix can be prepared by methods known in the art itself.

[0022] The premix is ​​still novel. Therefore, other aspects of the invention are (ruminant) premixes comprising a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, and their use in all uses and methods according to the invention.

[0023] Preferred animal premixes include: a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, a carrier, and optionally one or more ingredients selected from vitamins, trace minerals, and macro minerals.

[0024] In an advantageous embodiment, the premix of the present invention is substantially composed of (a) a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, a carrier, and one or more of (b-1) fat-soluble vitamins, (b-2) water-soluble vitamins, (b-3) trace minerals and (b-4) macro minerals.

[0025] In a preferred embodiment, the premix is ​​a mineral premix, a vitamin premix containing vitamins and optionally minerals, or a pellet.

[0026] In addition to the combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, the premix of the present 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 present invention comprises a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole and at least one other component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals.

[0027] Major minerals can be added to the feed separately. Therefore, in one specific embodiment, the premix comprises a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, and at least one other component selected from fat-soluble vitamins, water-soluble vitamins, and trace minerals.

[0028] Here is a list of examples of the non-exclusivity of these components: Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

[0029] Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenates, such as Ca-D-pantothenate.

[0030] Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, manganese, and cobalt.

[0031] Examples of essential minerals are calcium, phosphorus, potassium, magnesium, and sodium.

[0032] As described above, premix is ​​one example of a feed additive. It should be understood that the combination of 3-nitrooxypropanol and / or its derivatives with metronidazole can be administered to animals in other different forms. For example, the combination of 3-nitrooxypropanol and / or its derivatives with metronidazole can also be contained in pellets, which will be placed in the rumen and continuously release a defined amount of the combination of 3-nitrooxypropanol and / or its derivatives with metronidazole at a defined dose over a defined time period.

[0033] In all embodiments of the present invention, it should be understood that the large pills are administered orally, by simple feeding, or by manual application.

[0034] Methane emissions from ruminants can be readily measured in the metabolic chamber of individual animals using methods known in the art (Grainger et al., 2007 J. Dairy Science; 90: 2755-2766). Alternatively, assessment can be performed at the barn level using emerging techniques with laser beams (McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802), sulfur hexafluoride, SF6 alone, or the GreenFeed system. Alternatively, methane production by milk-producing ruminants can also be assessed by measuring the fatty acid profile in milk according to WO 2009 / 156453.

[0035] The present invention also relates to the use of feed additives according to the invention, the feed additive comprising one or more other active substances that exhibit similar effects in rumen methane formation, the active substances being selected from diallyl disulfide, garlic oil, allyl isothiocyanate, deoxycholic acid, chenodeoxycholic acid and its derivatives.

[0036] Other components that can be given in combination with metronidazole along with 3-nitrooxypropanol and / or its derivatives are, for example, yeast, oregano extract, tannins and tannic acid, and essential oils such as thymol, 3-methylphenol, limonene, vanillin, guaiacol, and eugenol.

[0037] It is currently envisioned that diallyl disulfide, garlic oil, allyl isothiocyanate, deoxycholic acid, chenodeoxycholic acid, and their derivatives be administered independently at dosages, for example, ranging from 0.01 to 500 mg of active substance (ppm) per kilogram of feed. These compounds are either commercially available or readily prepared by those skilled in the art using processes and methods well known in the prior art.

[0038] The ruminant mammals according to the present invention include cattle, goats, sheep, giraffes, bison, European bison, yaks, buffalo, deer, camels, alpacas, llamas, wildebeest, antelopes, pronghorn, and blue takin.

[0039] For all embodiments of the invention, domestic cattle, sheep, and goats are preferred species. For the purposes of the invention, the most preferred species is domestic cattle. This term includes domestic cattle of all breeds, as well as cattle of all production types, particularly dairy and beef cattle. It should be fully understood that the terms dairy and beef cattle encompass animals of all ages and physiological stages of life, as well as those produced using systems such as penning, semi-penning, and grazing.

[0040] The present invention is further described through the following embodiments, which should not be construed as limiting the scope of the invention.

[0041] Example In vitro testing of methane production A modified version of the Hohenheim Forage value Test (HFT) can be used to test the effect of a specific compound on rumen function simulated by the in vitro system.

[0042] principle: The feed mixture, along with rumen fluid and a suitable buffer solution, was loaded into a syringe. The solution was incubated at 39°C. After 8 hours, the amount (and composition) of the generated gas phase was measured and converted into the formula.

[0043] Reagents: Macro-element solutions: 6.2 g potassium dihydrogen phosphate (KH2PO4) 0.6 g magnesium sulfate heptahydrate (MgSO4*7H2O) 9 ml concentrated phosphoric acid (1 mol / L) Dissolve in distilled water to a volume of 1 L (pH approximately 1.6). Buffer solution: 35.0 g sodium bicarbonate (NaHCO3) 4.0 g ammonium bicarbonate ((NH4)HCO3) Dissolve in distilled water to a final volume of 1 L. Trace element solution: 13.2 g calcium chloride dihydrate (CaCl2*2H2O) 10.0 g manganese(II) chloride tetrahydrate (MnCl2*4H2O) 1.0 g cobalt(II) chloride hexahydrate (CoCl2*6H2O) 8.0 g ferric chloride (III) (FeCl3 * 6H2O) Dissolve in distilled water to a final volume of 100 ml. Sodium salt solution: 100 mg sodium salt Dissolve in distilled water to a final volume of 100 ml. Reducing solution: First, add 3 ml of sodium hydroxide (c = 1 mol / L), then add 427.5 mg of sodium sulfide hydrate (Na2S*H2O) to 71.25 ml of H2O. This solution must be prepared shortly before it is added to the culture medium solution. process: Sample weighing: The feed ingredients (i.e., TMR (44% concentrate, 6% hay, 37% corn silage, and 13% forage silage)) were sieved to 1 mm and accurately weighed into 64 syringes. Four syringes served as substrate controls to demonstrate gas generation in the absence of the test compound. The remaining syringes contained the test substance, with four syringes in each group.

[0044] Preparation of culture medium solution: Mix the components in the Woulff bottle in the following order: 711 ml water 0.18 ml trace element solution 355.5 ml buffer solution 355.5 ml macro-element solution The prepared solution was heated to 39°C, then 1.83 ml of sodium salt solution was added, and the reducing solution was added at 36°C.

[0045] When the indicator turns colorless, add rumen fluid.

[0046] Extraction of rumen fluid: With continuous stirring and CO2 gas introduction, 750 ml of rumen fluid was added to approximately 1,400 ml of culture medium solution.

[0047] Syringe filling, incubation, and determination of gas volume and VFA value: Diluted rumen fluid (24 ml) was added to a glass syringe. The syringe was then incubated at 39°C with gentle stirring for 8 hours. After 8 hours, the volume of the generated gas was measured, and the percentage of methane in the gas phase was determined by gas chromatography.

[0048] result The fermented food was artificial TMR (44% concentrate, 6% hay, 37% corn silage, and 13% forage silage). Metronidazole was used at two different concentrations, 10 µM and 20 µM. 3-Nitrooxypropanol (3-NOP) was used at two different concentrations, 1.5 µM and 1 µM.

[0049] The results are presented in Table 1 below. A clear synergistic effect was obtained.

[0050] Table 1: Methane-reducing effects of metronidazole, 3-nitrooxypropanol, or a combination of both.

Claims

1,3-Nitrooxypropanol and / or its derivatives in combination with metronidazole are used to reduce methane formation resulting from the digestive activity of ruminants.

2. The use according to claim 1, wherein metronidazole is applied to the ruminant in an amount selected from the range of 0.5 to 200 g metronidazole / animal / day, preferably from the range of 1 to 150 g metronidazole / animal / day, and most preferably from the range of 10 to 100 g metronidazole / animal / day.

3. The use according to claim 1 or 2, wherein the 3-nitrooxypropanol and / or its derivatives are applied to the ruminant in an amount selected from the range of 0.025 to 5 g of 3-nitrooxypropanol and / or its derivatives per animal per day, more preferably from the range of 0.05 to 4 g of 3-nitrooxypropanol and / or its derivatives per animal per day, most preferably from the range of 0.125 to 3 g of 3-nitrooxypropanol and / or its derivatives per animal per day, and other suitable effective amounts selected from the range of 0.25 to 3 g of 3-nitrooxypropanol and / or its derivatives per animal per day or 0.5 to 3 g of 3-nitrooxypropanol and / or its derivatives per animal per day.

4. The use according to any one of the preceding claims, wherein the ruminant is selected from cattle, preferably from domestic cattle, and most preferably from beef cattle or dairy cattle.

5. The use according to any one of the preceding claims, wherein the 3-nitrooxypropanol and / or its derivatives are supplemented with metronidazole, each in the form of a pure (separate) 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-nitrooxypropanol and / or its derivatives with metronidazole, a carrier, and optionally one or more components selected from vitamins, trace minerals and macrominerals.

7. The use according to claim 6, wherein the vitamin is selected from fat-soluble vitamins and water-soluble vitamins.

8. The use according to claim 6, wherein the premix is ​​substantially composed of (a) a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, a carrier, and one or more of (b-1) fat-soluble vitamins, (b-2) water-soluble vitamins, (b-3) trace minerals and (b-4) macro minerals.

9. A method for reducing methane production from the digestive activity of a ruminant, the method comprising orally administering to the animal at a dose of at least 0.5 g metronidazole per animal per day and at least 0.025 g 3-nitrooxypropanol and / or its derivatives per animal per day.

10. The method of claim 9, wherein the amount of metronidazole is selected from the range of 0.5 to 200 g metronidazole / animal / day, preferably from the range of 1 to 150 g metronidazole / animal / day, and most preferably from the range of 10 to 100 g metronidazole / animal / day.

11. The method according to claim 9 or 10, wherein the 3-nitrooxypropanol and / or its derivatives are applied to the ruminant in an amount selected from 0.025 to 5 g of 3-nitrooxypropanol and / or its derivatives per animal per day, more preferably from 0.05 to 4 g of 3-nitrooxypropanol and / or its derivatives per animal per day, most preferably from 0.125 to 3 g of 3-nitrooxypropanol and / or its derivatives per animal per day, and other suitable effective amounts selected from 0.25 to 3 g of 3-nitrooxypropanol and / or its derivatives per animal per day or 0.5 to 3 g of 3-nitrooxypropanol and / or its derivatives per animal per day.

12. An animal premix comprising (a) a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, a carrier, and optionally one or more ingredients selected from vitamins, trace minerals and macrominerals.

13. The animal premix according to claim 12, wherein the vitamin is selected from fat-soluble vitamins and water-soluble vitamins.

14. The animal premix according to claim 12, wherein the premix is ​​substantially composed of a combination of 3-nitrooxypropanol and / or its derivatives with metronidazole, a carrier, and one or more of (b-1) fat-soluble vitamins, (b-2) water-soluble vitamins, (b-3) trace minerals and (b-4) macro minerals.

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