Use of gossypol and methane inhibitors to reduce methane formation resulting from ruminant digestive activity

Abstract

The present invention relates to the field of reducing methane emissions in ruminant animals, and in particular to the administration of gossypol and methane inhibitors to ruminant animals to reduce the production of methane resulting from the digestive activity of said ruminant animals.

Description

Detailed Description of the Invention

[0001] The present invention relates to the field of reducing methane emissions in ruminants, and in particular to the administration of gossypol and at least one methane inhibitor to ruminants to reduce the production of methane resulting from the digestive activity of said ruminants.

[0002] The present invention further relates to animal feed compositions and feed additives comprising a methane inhibitor and gossypol, and the use of said feed compositions or feed additives for reducing methane production resulting from the digestive activity of ruminant animals.

[0003] The temperature of the air surrounding the Earth is increasing, a process called 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 man-made; however, the two most important sources are agriculture and the fossil fuel industry. Within agriculture, ruminant animals, especially cattle, are the major contributors to biogenic methane formation. It has been estimated that preventing methane formation from ruminant animals would nearly stabilize atmospheric methane concentrations.

[0004] Methane emissions from the ruminant livestock sector (a by-product from the enteric fermentation of plant biomass in the ruminant digestive system) are caused by methanogenic archaea. Various attempts to mitigate methane production from ruminants have been made in recent decades. Approaches vary, but the most widespread methods so far are feed additives that act in the ruminal fluid by reducing and inhibiting methane production by methanogenic archaea.

[0005] The term methane inhibitor as used herein relates to all compounds suitable for reducing methane emission in ruminants (i.e. ruminal methane inhibitors). Such compounds are well known to those skilled in the art.

[0006] Gossypol is, for example, an isoform of the formula (I) found in the Indian tulip plant (genus Thespesia) [ka] It is a natural phenol.

[0007] Surprisingly, it has now been found that the combination of gossypol and a methane inhibitor synergistically reduces methane formation.

[0008] Therefore, the combination of methane inhibitors and gossypol has great potential to mitigate climate change by significantly lowering methane emissions generated during ruminant digestive activity.

[0009] Thus, in a first embodiment, the present invention provides the use of gossypol and methane inhibitors to reduce the formation of methane resulting from the digestive activity of ruminants.

[0010] In a second embodiment, the present invention further provides a method for reducing methane production resulting from digestive activity in a ruminant animal, comprising orally administering to the animal an effective amount of gossypol and a methane inhibitor.

[0011] In a third embodiment, the present invention relates to a (ruminant) feed composition or feed additive comprising gossypol and a methane inhibitor.

[0012] Suitable methane inhibitors according to the present invention include, but are not limited to, garlic extract, allicin, diallyl disulfide, propanediol mononitrate, bromoform, chloroform, bromoethanesulfonate sodium salt, nitrate, nitroethane, lauric acid, lauricidin, monensin, Coriandrum sativum seed oil, eugenol, geranyl acetate, vanillin, limonene, myristic acid, palmitic acid, linoleic acid, tannin, tannic acid, thymol, and geraniol, and marine algae such as the Hawaiian micro-algae Chaetoceros.

[0013] While it is appreciated that the gossypol and the methane inhibitor may be administered to the ruminant either mixed together (i.e., premixed) or separately, it is appreciated that in the latter case, administration of the methane inhibitor and gossypol occurs within a particular time window, i.e., within no more than 6 hours, preferably within 3 hours, more preferably within 1 hour, e.g., within 0.5 hours. Most preferably, if administered separately, both components are administered concomitantly to the ruminant, e.g., by their concomitant addition to the animal's feed (diet) or to the feed rack.

[0014] Preferably, in all embodiments of the present invention, the gossypol and the methane inhibitor are administered together in the form of a feed composition or feed additive comprising the gossypol and the methane inhibitor.

[0015] In all embodiments of the present invention, it is well understood that the gossypol and methane inhibitors are administered to the ruminant in an effective amount, i.e., an amount that preferably results in at least a 10% methane reduction when compared to a control, i.e., a ruminant not supplemented with the gossypol and methane inhibitors.

[0016] Therefore, the present invention also relates to the (combined) use of gossypol and a methane inhibitor, whereby methane production in ruminants is reduced by at least 10% compared to the control (ie, in the absence of gossypol and the respective methane inhibitor).

[0017] Propanediol mononitrate [CAS No: 100502-66-7] is a known compound that can be prepared, for example, as outlined in WO2004043898 or WO2012084629 and is available under the trademark Bovaer® from DSM Nutritional Products Ltd.

[0018] Eugenol (CAS No: 97-53-0) is a known compound that can be used in all embodiments of the present invention as such (i.e. as a distinct compound). However, preferably, in all embodiments of the present invention, eugenol is used in the form of an essential oil mixture containing eugenol, which is commercially available, for example, as Agolin Ruminant and Crina Ruminant (a mixture of essential oils containing up to 7% eugenol).

[0019] Allicin (CAS No: 539-86-6) is a known compound that can be used in all embodiments of the present invention as such (i.e. as a different compound). However, preferably, in all embodiments of the present invention, allicin is used in the form of garlic extract, e.g. commercially available as Mootral.

[0020] Bromoform (CAS No: 75-25-2) is a known compound that can be used in all embodiments of the present invention by itself (i.e. as a different compound). However, preferably, in all embodiments of the present invention, bromoform is used in the form of seaweed-containing bromoform. Such seaweeds are well known to those skilled in the art and include, for example, Hawaiian microalgae Chaetoceros, Asparagopsis species, such as the red algae A. taxiformis or A. armata, and brown algae such as Ascophyllum nodosum. The reported concentrations of bromoform in the Asparagopsis genus are in the range of 1-7 mg / g.

[0021] Gossypol (CAS No: 303-45-7) exhibits axial chirality resulting in two enantiomers. Hence, gossypol can be used in the form of a single enantiomer or as a mixture thereof. Furthermore, the term gossypol in all embodiments of the present invention can also be used as gossypol acetic acid solvate (CAS 12542-36-8). Preferably, in all embodiments of the present invention, gossypol is used in the form of an enantiomeric mixture, i.e., as (±) gossypol. Said gossypol is commercially available, for example, from Sigma-Aldrich as (±) gossypol from cottonseed ≧95% (HPLC).

[0022] In a preferred embodiment of the invention, gossypol is used as such (i.e. as a single compound) or in the form of a (±) gossypol acetic acid solvate according to the invention, both compounds being available from Merck. Particularly preferred in all embodiments according to the invention is the use of a gossypol acetic acid solvate obtained as (±) gossypol acetic acid.

[0023] In another preferred embodiment, gossypol is used / administered in all embodiments of the present invention in the form of a plant product containing gossypol. The plant product is well known to those skilled in the art and is commonly used, for example, as a cost-effective feed of choice for lactating cows and other ruminants. The gossypol content of such a plant product can reach up to about 6%. However, generally, the gossypol content of the plant product is in the range of about 50-10,000 ppm.

[0024] The term "effective amount" as used herein refers to the amount necessary to obtain a reduction in methane emissions resulting from the digestive activity of ruminants. It is well understood that said reduction can be achieved by one single (daily) dose or by repeated (daily) doses. Furthermore, it is well understood by those skilled in the art that the effective amount of gossypol and methane inhibitors in the uses, methods and compositions according to the present invention can vary according to known factors, such as the characteristics of a particular composition and its mode and route of administration, the methane inhibitor content of each plant product or essential oil mixture, the age, health and weight of the ruminant, the frequency of treatment, all of which can be determined by experts in the field through standard clinical trials or usual considerations regarding inoculation regimes and / or formulations.

[0025] Preferably, in all embodiments of the present invention, the effective amount of methane inhibitor to be administered to a ruminant is selected in the range of 0.01-100 g methane inhibitor / animal / day, more preferably in the range of 0.01-50 g methane inhibitor / animal / day, most preferably in the range of 0.01-25 g methane inhibitor / animal / day, for example in the range of 0.01-10 g methane inhibitor / animal / day, 0.01-5 g methane inhibitor / animal / day, or 0.01-2.5 g methane / animal / day.

[0026] Preferably, in all embodiments of the present invention, the effective amount of propanediol mononitrate to be administered to a ruminant is selected within the range of 0.05-5 g PDMN / animal / day, more preferably within the range of 0.1-4 g PDMN / animal / day, most preferably within the range of 0.25-3 g PDMN / animal / day. Further suitable effective amounts are selected within the range of 0.5-3 g PDMN / animal / day, 1-3 g PDMN / animal / day, or 1-2 g PDMN / animal / day.

[0027] With respect to the feed, preferably in all embodiments of the present invention, an effective amount of propanediol mononitrate in the feed is selected within the range of 10 mg to 300 mg PDMN / kg DM / day, more preferably within the range of 50 to 150 g PDMN / kg DM / day, and most preferably within the range of 60 to 100 g PDMN / kg DM / day.

[0028] Preferably, in all embodiments of the present invention, an effective amount of eugenol to be administered to a ruminant is selected within the range of 0.01 to 5 g eugenol / animal / day, more preferably within the range of 0.02 to 4 g eugenol / animal / day, most preferably within the range of 0.025 to 3 g eugenol / animal / day. Further suitable effective amounts are selected within the range of 0.01 to 1 g eugenol / animal / day, or 0.02 to 0.5 g eugenol / animal / day.

[0029] Preferably, in all embodiments of the present invention, the effective amount of gossypol to be administered to a ruminant is selected within the range of 0.05 to 100 g gossypol / animal / day, more preferably within the range of 0.1 to 50 g gossypol / animal / day, and most preferably within the range of 0.2 to 25 g gossypol / animal / day, for example, within the range of 0.2 to 10 g gossypol / animal / day, 0.2 to 5 g gossypol / animal / day, or 0.2 to 2.5 g gossypol / animal / day.

[0030] For cattle, more particularly suitable ranges are within the range of 0.2-100 g gossypol / animal / day, more preferably within the range of 0.5-90 g gossypol / animal / day, and most preferably within the range of 1-90 g gossypol / animal / day, for example, 1-50 g gossypol / animal / day, 1-25 g gossypol / animal / day, or 2-25 g gossypol / animal / day.

[0031] In all embodiments of the present invention, it is further advantageous if the molar ratio of gossypol to methane inhibitor (i.e. moles of gossypol / methane inhibitor) is comprised within the range of 100 to 0.1 (e.g. 100 μM gossypol / 1 μM methane inhibitor to 1 mM gossypol / 10 mM methane inhibitor), preferably 75 to 0.25, most preferably 60 to 0.35, for example 50 to 0.5. Further suitable ranges include 50:1 to 1:5, 20:1 to 1:5, 20:1 to 3:1, 15:1 to 1:2.5, and 10:1 to 1:2.

[0032] Preferably, in all embodiments of the present invention, the methane inhibitor is propanediol mononitrate (PDMN).

[0033] In all embodiments of the present invention, the propanediol mononitrate is preferably administered in the form of its powderous formulation.

[0034] Preferably, the powder formulation is a powder formulation comprising PDMN and a carrier material. Suitable carriers include, but are not limited to, any carrier well known in the food and feed industry, such as silicon dioxide (silica).

[0035] Powdered formulations comprising PDMN and a carrier material are typically prepared by spraying or mixing the PDMN onto or with the carrier material by standard methods in the art, for example by diluting the PDMN in an organic solvent suitable for the preparation of food or feed products, such as dichloromethane, spraying / mixing the solution onto the carrier, and then evaporating the organic solvent.

[0036] Alternatively, the PDMN may be diluted in a suitable edible oil before being sprayed or mixed with the carrier material. In the latter case, the respective edible oil is usually not removed. The powder formulation may additionally contain the usual additives used in the preparation of powder formulations for feed applications.

[0037] The amount of PDMN in the powder formulation according to the present invention is preferably selected in the range of 1 to 20% by weight, preferably in the range of 2 to 15% by weight, most preferably in the range of 4 to 12% by weight, based on the total weight of the composition.

[0038] A particularly suitable powdered formulation for use in all embodiments of the present invention consists essentially of PDMN, propylene glycol, and silica, as outlined, for example, in WO2018149756 and WO2018149755, and is commercially available as Bovaer® 10 from DSM Nutritional Products Ltd.

[0039] The gossypol and the methane inhibitor in all uses and methods according to the present invention are preferably administered concomitantly to the ruminant animal, either by prior mixing thereof or by separate addition to the animal's feed.

[0040] The gossypol and methane inhibitors are preferably administered to ruminants via a (ruminant) feed composition or feed additive, for example by mixing the individual components with the ruminant feed.

[0041] Thus, preferably, in all uses and methods according to the present invention, the methane inhibitors and gossypol, given all the definitions and preferences herein, are administered to ruminants by incorporation into a (ruminant) feed composition or feed additive.

[0042] The term feed composition or feed additive as used herein means any preparation, mixture, or composition suitable or intended for oral ingestion by an animal. Exemplary feeds for ruminant animals such as cattle include, but are not limited to, grass (grass, legumes, silage), hay, pasture, grain, and soybeans.

[0043] Said feed composition or feed additive may be prepared by methods known per se in the art of feed formulation and processing.

[0044] Said feed compositions and feed additives are still novel.Thus, further aspects of the present invention are (ruminant) feed compositions and feed additives comprising gossypol and a methane inhibitor, all of which are given herein with all the definitions and preferences.

[0045] In a preferred embodiment, the feed composition and feed additive is a mineral premix, a vitamin premix (containing vitamins and optionally minerals), or a bolus.

[0046] Gossypol and methane inhibitors may be used in animal feed compositions (feeds), such as in combination with forage (fresh grass, hay, silage), industry derived co-products (citrus pulp, soybean hulls, distillers, soluble dry or wet grains, traditional ingredients present including but not limited to minerals (calcium carbonate, electrolytes such as ammonium chloride, macro and trace minerals (all forms, inorganic and organic)), proteins such as soybean meal, sunflower meal, meat and bone meal, fish meal, amino acids etc., energy ingredients such as cereal grains, wheat, starch, barley, millet, sorghum, corn, animal and vegetable fats or oils, and vitamins).

[0047] Specific examples of feed compositions of the present invention include: - an animal feed additive comprising (a) a methane inhibitor and (b) gossypol, and (c) one or more of (c-1) fat-soluble vitamins, (c-2) water-soluble vitamins, (c-3) trace minerals, and (c-4) macrominerals; - an animal feed composition comprising (a) propanediol mononitrate and (b) a mixture of at least six phenolic substances, and (c) one or more of the following: (c-1) a crude protein content of 50-800 g / kg feed (50-80%), (c-2) a fat content of 5-100 g / kg feed (5-10%), (c-3) a NDF content of 150-700 g / kg feed (15-70%), (c-4) a TDN content of 300-800 (30-80%), and (c-5) a starch content of 150-700 g / kg feed (15-70%).

[0048] So-called premixes are examples of animal feed additives according to the invention. A premix preferably represents a homogeneous mixture of one or more micro-ingredients with a diluent and / or carrier. A premix is ​​used to facilitate homogeneous dispersion of the micro-ingredients in a larger mixture.

[0049] Apart from the active ingredients of the present invention (i.e., gossypol and methane inhibitor), 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 macromineral. In other words, the premix of the present invention comprises a methane inhibitor and gossypol together with at least one additional component selected from the group consisting of a fat-soluble vitamin, a water-soluble vitamin, a trace mineral, and a macromineral.

[0050] The macrominerals may be added separately to the feed. Thus, in certain embodiments, the premix comprises gossypol and a methane inhibitor with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, and trace minerals.

[0051] The following is a non-exhaustive list 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 pantothenates, such as Ca-D-pantothenate. - Examples of trace minerals include manganese, zinc, iron, copper, iodine, selenium, manganese, and cobalt. - Examples of macrominerals are calcium, phosphorus, potassium, magnesium, and sodium.

[0052] With regard to feed compositions for ruminant animals such as cattle, and their components, ruminant feed is usually composed of a readily degradable fraction (designated concentrate) and a fiber-rich, less readily degradable fraction (designated hay, forage, or roughage).

[0053] Hay is made of dried grasses, legumes, or whole grains. Grasses include temperate or tropical grasses, timothy, ryegrass, fescue, brachiaria, millet, tifton, among others. Legumes include clover, alfalfa or alfalfa, peas, beans, and vetch, among others. Whole grains include barley, maize (corn), oats, wheat, sorghum, among others. Other forage crops include sugarcane, sugarcane bagasse, citrus pulp, kale, rapeseed, and cabbage. Also, root crops such as turnip, swede, mangle, fodder beet, and sugar beet (including sugar beet pulp and beet molasses) are used to feed ruminants. Additional crops include tubers such as potato, cassava, and sweet potato. Silage is an ensiled version of fiber-rich fractions (e.g., from grass, legumes, or whole grains) and grains (e.g., high moisture corn silage) whereby the high water content material is treated (naturally fermented or additionally treated) through a controlled anaerobic fermentation process, but is not limited to this.

[0054] Concentrate feeds are composed mostly of grains (e.g., barley, corn, wheat, sorghum, including brewer's grains and distiller's grains), but often also contain protein-rich feed ingredients such as, but not limited to, soybean meal, rapeseed meal, palm kernel, and sunflower meal.

[0055] Ruminants (males, females (all stages and adults)) may also be fed a total mixed ration (TMR) diet, in which all dietary components, e.g., forage, silage, other feed ingredients, and concentrates, are mixed prior to feeding.

[0056] As mentioned above, a premix is ​​an example of a feed additive that may contain gossypol and a methane inhibitor. It is understood that the compound may be administered to the animal in other different forms. For example, the compound may be included in a bolus that is placed in the rumen and continuously releases a defined amount of the active compound over a specific period of time in a clearly defined dosage.

[0057] In certain advantageous embodiments, the feed composition according to the invention is a ruminant feed (often also referred to as a ruminant diet), including all dietary components, such as macro- and micro-ingredients, forage, silage, and concentrate feed, and additives including gossypol and methane inhibitors. Such ruminant feeds or diets are often also referred to as total mixed rations (TMR) or partial mixed rations (PMR) for herbivores, or as nutritional supplements.

[0058] Preferably, in all embodiments of the present invention, the amount of propanediol mononitrate in the ruminant feed is selected within the range of 1 mg to about 25 g per kg dry feed, preferably from about 1 mg to about 10 g per kg dry feed, more preferably from about 10 mg to about 1 g per kg dry feed, and most preferably from 20 mg to 500 mg per kg dry feed, such as from about 20 mg to 250 mg per kg dry feed, or even more preferably from 10 mg to 300 mg per kg dry feed, for example from 50 mg to 150 mg per kg dry feed, or from 60 mg to 100 mg per kg dry feed.

[0059] Preferably, in all embodiments of the present invention, the amount of gossypol in the ruminant feed is selected within the range of 25 mg to about 10 g gossypol per kg dry feed, preferably about 50 mg to about 10 g gossypol per kg dry feed, more preferably 50 mg to about 5 g gossypol per Kg dry feed, and most preferably 100 mg to 2.5 g gossypol per Kg dry feed. More particularly suitable ranges are 25 mg to 1 g gossypol per Kg dry feed, 50 mg to 1 g gossypol per Kg dry feed, or 100 mg to 1 g gossypol per Kg dry feed.

[0060] Daily dry matter intake for cattle is usually in the range of 1-3.5% dry matter per kg live weight. Dry matter intake (DMI) for dairy cattle is, for example, about 2-3% dry matter per kg live weight depending on the feeding system as land or grazing, and for beef cattle, dry matter intake is usually 1.0-3% of live weight.

[0061] It is also understood that in all uses and methods according to the present invention, gossypol and methane inhibitors must be supplemented together in a timely manner to excerpt the synergistic effect, but may be added separately to the animal's diet, for example, in the feeding rack taking into account.

[0062] Thus, the present invention relates to a method of supplementing ruminants with gossypol and methane inhibitors, comprising the step of adding gossypol and methane inhibitors (all definitions and preferences provided herein) concomitantly to the feed rack.

[0063] The amount of ruminant feed administered to a ruminant may vary depending on the species and age. Usually, the amount of dry matter fed to a beef or dairy cow is preferably selected within the range of 1.5% to 3.5% of the live weight, for example for an animal of 500 kg live weight, which means feeding 7.5 to 17.5 kg of dry matter per day.

[0064] In all embodiments of the present invention, this shall be understood to be by oral administration of a bolus, simple feeding, or manual administration, and it is appreciated that the methane inhibitors and gossypol may be premixed prior to administration or added separately to animal feed compositions and feed additives.

[0065] Methane emissions by ruminants can be easily measured in individual animals in metabolic chambers by methods known in the art (Grainger et al., 2007 J. Dairy Science; 90: 2755-2766). Furthermore, it can also be assessed at the burn level by laser beams (McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802) or emerging technologies using sulfur hexafluoride, or simply SF6 or GreenFeed systems. Besides this, methane produced by dairy ruminants can also be assessed by measuring the fatty acid profile in milk according to WO 2009 / 156453.

[0066] Ruminant 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.

[0067] For all embodiments of the present invention, domestic cattle, sheep and goats are the more preferred species. For the present purposes, the most preferred species is domestic cattle. The term includes all types of domestic cattle and all producing species of cattle, especially dairy and beef cattle. It is understood that the terms dairy and beef cattle encompass animals of all ages and physiological life stages and production systems, such as confined, semi-confined and herbivorous.

[0068] In a further embodiment, the present invention relates to the use of gossypol to synergistically enhance the methane lowering properties of a methane inhibitor in a ruminant, and it is fully understood that all definitions and properties defined herein apply to said use.

[0069] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0070] [Example] In vitro test for methane production: A modified version of the "Hohenheim Forage Value Test (HFT)" was used to test the effect of certain compounds on ruminal function mimicked by the in vitro system.

[0071] Principle: Feed is added into a syringe with an appropriate mixture of ruminal fluid composition and buffer. The solution is incubated at 39° C. After 8 hours, the amount (and composition) of the gas phase produced is measured and entered into a conversion equation.

[0072] [reagent:] [Mass element solution:] - 6.2g potassium dihydrogen phosphate (KH2PO4) - 0.6g Magnesium Sulfate Heptahydrate (MgSO4*7H2O) - 9 ml of concentrated phosphoric acid (1 mol / l) - Dissolve in distilled water to 1 liter (pH approx. 1.6)

[0073] [Buffer solution:] - 35.0 g sodium bicarbonate (NaHCO3) - 4.0 g ammonium bicarbonate ((NH4)HCO3) - Dissolve in distilled water up to 1 liter

[0074] [Trace element solution:] - 13.2g Calcium Chloride Dihydrate (CaCl2*2H2O) - 10.0 g Manganese(II) chloride tetrahydrate (MnCl2*4H2O) - 1.0g Cobalt(II) chloride hexahydrate (CoCl2*6H2O) - 8.0g of iron(III) chloride (FeCl3*6H2O) - Dissolve in distilled water up to 100ml

[0075] [Sodium salt solution:] - 100mg sodium salt - Dissolve in distilled water up to 100ml

[0076] [Reducing solution:] - First 3 ml of sodium hydroxide (c=1 mol / l) and then 427.5 mg of sodium sulfide hydrate (Na2S*H2O) are added to 71.25 ml of H2O. - The solution must be prepared shortly before adding to the vehicle solution.

[0077] [procedure:] Sample Weighing: The diet (i.e., TMR (44% concentrate, 6% hay, 37% corn silage, and 13% grass silage)) was sieved to 1 mm and accurately weighed into 64 syringes. Four of these syringes are substrate controls. These represent gas production without the effect of the compound being tested. The four other syringes are positive controls (3-NOP, 10 μM). If necessary, four syringes contain a carrier control (if the test compound requires a carrier). The remaining syringes contain the test substance in groups of four syringes in the amounts shown in Table 1.

[0078] [Preparation of vehicle solutions:] The components are mixed in a Woulff bottle in the following order: - 711ml of water - 0.18ml of trace element solution - 355.5ml of buffer solution - 355.5 ml of mass elemental solution

[0079] The completed solution is warmed to 39°C, followed by the addition of 1.83 ml of sodium salt solution and reducing solution at 36°C.

[0080] When the indicator becomes colourless, rumen fluid is added.

[0081] Ruminal fluid extraction: 750 ml of ruminal fluid is added to approximately 1,400 ml of the vehicle solution under continuous stirring and CO2 gassing.

[0082] Filling and incubating syringes to determine gas volume and VFA values: Diluted ruminal fluid (24 ml) is added to a glass syringe. The syringe is then incubated at 39° C. for 8 hours 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.

[0083] [result] The fermented diet was an artificial TMR (44% concentrate, 6% hay, 37% corn silage, and 13% grass silage). Gossypol was obtained from Sigma-Aldrich (G8761) and used at the concentrations outlined in Table 1. 3-Nitrooxy-propanol (3-NOP) was used at the concentrations outlined in Table 1.

[0084] The results are shown below in Table 1. When propanediol mononitrate was combined with gossypol, a clear synergistic effect was obtained on methane reduction, which translates into additional performance benefits for the animals.

[0085] [Table 1]

[0086] [Table 2]

Claims

1. 1. Use of gossypol and a methane inhibitor to reduce the formation of methane resulting from the digestive activity of a ruminant, wherein the methane inhibitor is administered to the ruminant in an amount selected within the range of 0.01 to 100 g of methane inhibitor / animal / day, and gossypol is administered to the ruminant in an amount selected within the range of 0.05 to 100 g of gossypol / animal / day.

2. 2. The use of claim 1, wherein the methane inhibitor is administered to the ruminant in an amount selected within the range of 0.01 to 50 g of methane inhibitor / animal / day.

3. The use described in claim 1, wherein the methane inhibitor is administered to the ruminant in an amount selected within the range of 0.01 to 25 g of methane inhibitor per animal per day.

4. The use according to claim 1, wherein gossypol is administered to the ruminant animal in an amount selected within the range of 0.1 to 50 g gossypol / animal / day.

5. The use described in claim 1, wherein gossypol is administered to the ruminant in an amount selected within the range of 0.2 to 25 g gossypol / animal / day.

6. The use according to any one of claims 1 to 5, wherein the molar ratio of gossypol to said methane inhibitor is comprised between 100 and 0.

1.

7. The use described in any one of claims 1 to 5, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 75 and 0.

25.

8. The use described in any one of claims 1 to 5, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 60 and 0.

35.

9. The use described in any one of claims 1 to 5, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 50 and 0.

5.

10. The use according to any one of claims 1 to 5, wherein the ruminant is selected from the group consisting of domestic cattle.

11. The use described in any one of claims 1 to 5, wherein the ruminant is selected from beef cattle or dairy cattle.

12. The use according to any one of claims 1 to 5, wherein the methane inhibitor is selected from the group consisting of eugenol, tannic acid, allicin, bromoform, chloroform, and propanediol mononitrate.

13. 1. A method for reducing methane production resulting from digestive activity in a ruminant animal, comprising orally administering to the ruminant effective amounts of gossypol and a methane inhibitor, wherein the effective amount of the methane inhibitor is selected within the range of 0.01 to 100 g of methane inhibitor / animal / day, and the effective amount of gossypol is selected within the range of 0.05 to 100 g of gossypol / animal / day.

14. The method of claim 13, wherein the effective amount of the methane inhibitor is selected in the range of 0.01 to 50 g of methane inhibitor per animal per day.

15. The method of claim 13, wherein the effective amount of the methane inhibitor is selected within the range of 0.01 to 25 g of methane inhibitor per animal per day.

16. The method according to any one of claims 13 to 15, wherein the effective amount of gossypol is selected within the range of 0.1 to 50 g gossypol / animal / day.

17. A method according to any one of claims 13 to 15, wherein the effective amount of gossypol is selected within the range of 0.02 to 25 g gossypol / animal / day.

18. A feed composition or feed additive comprising gossypol and a methane inhibitor, said feed composition being a ruminant feed comprising gossypol in an amount selected within the range of 25 mg to about 10 g per kg dry matter feed, and said methane inhibitor in an amount selected within the range of 1 mg to 5 g per kg dry matter feed.

19. A ruminant feed according to claim 18, wherein the molar ratio of gossypol to said methane inhibitor is comprised between 100 and 0.

1.

20. The ruminant feed of claim 18, wherein the molar ratio of gossypol to the methane inhibitor is between 75 and 0.

25.

21. A ruminant feed as described in claim 18, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 60 and 0.

35.

22. A ruminant feed as described in claim 18, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 50 and 0.

5.

23. A ruminant feed according to any one of claims 18 to 22, wherein the methane inhibitor is selected from the group consisting of eugenol, tannic acid, allicin, bromoform, chloroform and propanediol mononitrate.

24. The feed composition according to any one of claims 18 to 22, wherein the feed composition is a mineral premix, a vitamin premix or a bolus.

25. Use of gossypol to enhance the methane lowering properties of methane inhibitors in ruminants.

26. The use according to claim 25, wherein the molar ratio of gossypol to said methane inhibitor is comprised between 100 and 0.

1.

27. ​​The use described in claim 25, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 75 and 0.

25.

28. The use described in claim 25, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 60 and 0.

35.

29. The use described in claim 25, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 50 and 0.5.