Feed for reducing methane in the rumen of ruminants

Abstract

To provide a naturally occurring plant-derived component that has the effect of reducing methane production in the rumen of ruminants and can be used as feed for ruminants. [Solution] A rumen methane reduction feed for ruminants containing the stems and leaves of plants belonging to the genus Vicia, their silage fermented products, or extracts thereof.

Description

Technical Field

[0001] The present invention relates to a feed for reducing methane in the rumen of ruminants and a method for reducing methane generation.

Background Art

[0002] The impact of methane gas on global warming accounts for 23% of all greenhouse gases and is more than 20 times that of carbon dioxide. Also, 5% of the world's methane gas emissions are derived from the digestive organs of ruminants, and reducing these emissions has become an urgent issue.

[0003] As components for reducing methane in the rumen of ruminants such as cows, monensin, saponin, cashew nut shell liquid, 3-nitrooxypropanol (3-NOP), Gracilaria lemaneiformis, etc. have been reported (Non-Patent Documents 1 to 5).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0005] However, monensin is an antibiotic and should not be used in large quantities due to concerns about the emergence of resistant bacteria. Cashew nut shell extract presents problems because cashews can only be cultivated in limited areas, and cashew hulling is mainly done in India, resulting in high transportation costs for the raw materials used in formulation. 3-NOP is a synthetic substance, and its effects on the rumen bacterial flora are unknown. In addition, administering large amounts of saponins carries risks such as foamy bloat. Therefore, the object of the present invention is to provide a plant-derived component that exists naturally, can be cultivated in a wide range of weather and soil conditions including Japan, has the effect of reducing methane production in the rumen of ruminants, and can be used as feed for ruminants. [Means for solving the problem]

[0006] Therefore, the inventors investigated various plants that could be used as animal feed and discovered that by including the stems and leaves of plants belonging to the genus Vicia, their silage fermented products, or extracts thereof in the feed, a feed with excellent methane reduction effects in the rumen of ruminants can be obtained, thus completing the present invention.

[0007] In other words, the present invention provides the following [1] to

[10] . [1] A rumen methane reduction feed for ruminants containing the stems and leaves of plants belonging to the genus Vicia, their silage fermentation products, or extracts thereof. [2] The rumen methane reduction feed for ruminants according to [1], wherein the extract is an extract of water, an aqueous organic solvent, or a water-aqueous organic solvent mixture. [3] The rumen methane reduction feed for ruminants according to [2], wherein the extract of water, aqueous organic solvent, or water-aqueous organic solvent mixture is the component obtained by washing the extract with n-butanol and water by solvent extraction and distilling off n-butanol from the resulting aqueous phase. [4] The extract of water, aqueous organic solvent, or water-aqueous organic solvent mixture is a component obtained by adsorbing the extract onto a porous adsorption resin and distilling off the alcohol from the fraction eluted with 99% alcohol, as described in [2], for reducing methane in the rumen of ruminants. [5] The extract of water, aqueous organic solvent, or water-aqueous organic solvent mixture is the fraction of the extract that does not adsorb to the porous adsorption resin, as described in [2], for reducing methane in the rumen of ruminants. [6] The rumen methane reduction feed for ruminants according to [2], wherein the extract obtained by further high-pressure steam treatment of any of the components or fractions described in [3] to [5] is water, aqueous organic solvent, or water-aqueous organic solvent mixed solvent. [7] A rumen methane reduction feed for ruminants described in any of [1] to [6], wherein the plant belonging to the genus Vicia is Vicia villosa or Vicia sativa L. [8] A rumen methane reduction feed for ruminants according to any one of [1] to [7], wherein the content of stems and leaves of plants belonging to the genus Vicia, silage fermented products thereof, or extracts thereof is 0.01 to 50% by mass on a dry solid basis. [9] The silage fermented product is further fermented by adding lactic acid bacteria. A rumen methane reduction feed for ruminants as described in any of [1] to [8].

[10] A method for reducing methane generation in the rumen of ruminants, characterized by feeding ruminants a feed containing the stems and leaves of plants belonging to the genus Vicia, silage fermented products thereof, or extracts thereof. [Effects of the Invention]

[0008] Feeding the feed of the present invention to ruminants such as cattle can reduce methane production in the rumen of ruminants. The feed of the present invention is derived from plants belonging to the genus Vicia, which are widely distributed throughout the world and used as pasture grass and green manure, and is therefore easily supplied. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows the effect of various types of pasture grasses on the amount of methane produced in the rumen fluid. [Figure 2] This figure shows the effect of various solvent extracts of hairy vetch (Mamesuke) on the amount of methane generated in the rumen fluid. [Figure 3] This figure shows the effect of hairy vetch (Mamesuke) water-ethanol extract on the amount of methane produced in the rumen fluid. [Figure 4]It is a diagram showing the effect on the methane generation amount in the rumen fluid of the aqueous extract of hairy vetch (bean assistant). [Figure 5] It is a diagram showing the effect on the methane generation amount in the rumen fluid of various solvent extracts of hairy vetch (bean assistant) silage. [Figure 6] It is a diagram showing the effect on the methane generation amount in the rumen fluid of various solvent extracts of common vetch. [Figure 7] It is a diagram showing the effect on the methane generation amount in the rumen fluid of the purified product of the common vetch extract. [Figure 8] It is a diagram showing the effect on the methane generation amount in the rumen fluid of the purified product of the hairy vetch extract. [Figure 9] It is a diagram showing the effect on the methane generation amount in the rumen fluid of the purified product of the hairy vetch extract. [Figure 10] It is a diagram showing the effect on the methane generation amount in the rumen fluid of the hairy vetch extract.

Mode for Carrying Out the Invention

[0010] The terms used in this specification are used in the meanings commonly used in the art, unless otherwise specified.

[0011] One aspect of the present invention is a feed for reducing methane in the rumen of ruminants, which contains the stems and leaves of plants belonging to the genus Vicia, their silage fermented products or their extracts. In the present invention, ruminants are animals that return the food they have swallowed once back into their mouths and re-chew it, and usually have four stomachs. Examples of ruminants include cows, sheep, and goats, and cows have the largest number of breeding heads. The first stomach among these four stomachs is the rumen. A very large number of microorganisms inhabit the rumen, decompose cellulose and hemicellulose, and generate methane. Therefore, if the generation of methane in the rumen can be suppressed, the amount of methane released from ruminants into the atmosphere can be suppressed.

[0012] The active ingredients of the present invention's feed are the stems and leaves of plants belonging to the genus Vicia, their silage fermented products, or extracts thereof. Plants belonging to the genus Vicia are also known as vetch in English. While some plants in the genus Vicia, like broad beans, are edible, most are used as pasture or green manure. Check out the latest version of Vicia The product is based on the Vicia americana, Vicia amoena, Vicia amurensis Oett.(ノハラクサフジ), Vicia andicola Kunth, Vicia articulata Hornem., Vicia bakeri Ali, Vicia basaltica Plitmann, Vicia benghalensis L., Vicia biennis L., Vicia bithynica(L.) L. Vicia bungei Ohwi(Vicia) Vicia canescens Labill.Vicia cappadocica Boiss. & Balance、Vicia caroliniana Walter、Vicia cassubica L.、Vicia cracca(クサフジ)、Vicia cuspidata Boiss.、Vicia cusnae、Vicia cypria Unger & Kotschy、Vicia disperma DC.、Vicia dumetorum L.Vicia ervilia、Vicia esdraelonensis Warb. & Eig Scop.(Placeae) Vicia hassei S.Watson L.. Vicia japonica A.Gray(Ch.) Vicia lathyroides(Ch.) Vicia lens. Vicia lilacina Ledeb. Vicia linearifolia Hook. & Arn. 、Vicia loiseleurii (M.Bieb.) Litv., Vicia lutea, Vicia menziesii Spreng., Vicia minutiflora FG Dietr., Vicia monantha Retz., Vicia nana Vogel, Vicia narbonensis L., Vicia nigricans, Vicia nigricans ssp. Gigantea, Vicia onobrychioides L., Vicia oroboides Wulfen, Vicia orobus DC., Vicia palaestina Boiss., Vicia pannonica, Vicia parviflora Cav., Vicia peregrina L., Vicia pisiformis L., Vicia pseudo-orobus Fisch. & CA Mey., Vicia pubescens (DC.) Link, Vicia pyrenaica, Vicia sativa, Vicia Examples include *Sepium* (Japanese pea), *Vicia sericocarpa* Fenzl, *Vicia serratifolia* Jacq., *Vicia sylvatica* L., *Vicia tenuifolia* Roth., *Vicia tenuifolia* ssp. dalmatica (A.Kern.) Greuter, *Vicia tetrasperma* (Japanese pea), *Vicia tsydenii* Malyschev, *Vicia unijuga* A.Br. (Japanese bush clover), and *Vicia villosa* (hairy vetch), among which *Vicia villosa* (hairy vetch) and *Vicia sativa* L. (common vetch) are preferred.

[0013] In this invention, the stems and leaves of plants belonging to the genus Vicia, their silage fermentation products, or extracts thereof are used. The stems and leaves can be any part of the plant other than the roots, and any part that is normally harvested as pasture grass is acceptable. Furthermore, the stems and leaves may be used fresh or dried. Silage fermented products of stems and leaves are obtained by fermenting stems and leaves in a silo. For example, they can be obtained by cutting stems and leaves, filling them into a silo, and allowing them to ferment with lactic acid bacteria present on the stems and leaves. At this time, additional lactic acid bacteria may be added to promote fermentation.

[0014] The extract of the aforementioned stems and leaves or their silage fermentation product is preferably extracted using water, an aqueous organic solvent, or a water-aqueous organic solvent mixture. Here, the water can be cold or hot, but extraction at room temperature (5-30°C) is preferable. Examples of aqueous organic solvents include alcohols, polyhydric alcohols, ketones, esters, ethers, aliphatic hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons, but alcohols, polyhydric alcohols, ketones, and esters are preferred. In particular, alcohols such as ethanol, polyhydric alcohols such as ethylene glycol and glycerin, and ketones such as acetone are preferred. In addition, a water-hydrous organic solvent mixture can also be used. Furthermore, extraction with these solvents may be combined several times.

[0015] Specific examples of extracts include (1) aqueous extracts, (2) aqueous extracts that did not adsorb onto the porous adsorption resin, (3) extracts obtained by adsorbing the aqueous extract onto a porous adsorption resin, washing with water or aqueous ethanol, and then eluting with high-concentration ethanol, and (4) extracts obtained by extracting with an aqueous organic solvent, removing the organic solvent by distillation, and then recovering the remaining aqueous phase after solvent extraction with an organic solvent. Here, examples of porous adsorbent resins used in extract (3) include polystyrene-based synthetic adsorbents, styrene-divinylbenzene-based synthetic adsorbents, or methacrylic-based synthetic adsorbents. As for aqueous ethanol, 20-40% ethanol is preferred, and 25-35% ethanol is more preferred. As for high-concentration ethanol, 90% or higher is preferred, and 95% or higher is more preferred. As the aqueous organic solvent used in the extract (4), ketones such as acetone are preferred. Furthermore, it was confirmed that the activity of these active ingredients is not lost even when the extracts are subjected to autoclaving, which is equivalent to heat sterilization or high-pressure steam treatment. This means that the extraction efficiency can be increased by boiling during the extraction process.

[0016] The present invention's feed for reducing methane in the rumen of ruminants preferably contains 0.01 to 50% by mass, more preferably 0.1 to 50% by mass, even more preferably 1 to 30% by mass, and still more preferably 3.5 to 17% by mass, of the stems and leaves of plants belonging to the genus Vicia, their silage fermented products, or extracts thereof, on a dry solid basis.

[0017] In addition to the active ingredients mentioned above, the methane-reducing feed may also include ordinary ruminant feed, such as roughage like fresh grass, hay, or silage of grasses, fresh grass, hay, or silage of legumes, and corn silage, as well as concentrated feed ingredients. Cereals include corn, barley, wheat, flour, soybean flour, oats, milo, feed rice, brown rice, heated soybeans, and extruded soybeans. Other types of feed include bran, rice bran, wheat bran, corn gluten feed, hominy feed, brewer's waste, corn distiller's grain soluble, and soybean hulls.Vegetable oilseeds include soybean oilseed meal, rapeseed oilseed meal, flaxseed oilseed meal, sweetened and heat-treated soybean meal, palm kernel oilseed meal, corn gluten meal, and corn jam meal. Other ingredients that can be added include alfalfa hay cubes, alfalfa meal, beet pulp, cottonseed, molasses, vitamins, and minerals. In addition to being used individually, concentrated feed can also be used as a pre-mixed product (compound feed) prepared by mixing multiple concentrated feeds at a feed factory.

[0018] Feeding the aforementioned feed to ruminants can reduce methane production in the rumen of ruminants. In other words, another aspect of the present invention is a method for reducing methane production in the rumen of ruminants, characterized by feeding ruminants a feed containing the stems and leaves of plants belonging to the genus Vicia, their silage fermented products, or extracts thereof. This feed is the same as the feed described above. Preferably, the means of feeding this feed is the same as the means of feeding ordinary feed. [Examples]

[0019] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0020] Manufacturing Example 1 (Manufacturing of dried plant products) At Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, 5 kg of hairy vetch (product name: Mamesuke, variety: Namoi (Snow Brand Seed Co., Ltd.) and product name: Fujiemon, variety: Massa (Snow Brand Seed Co., Ltd.)) seeds were sown per 10 ares in May 2021. 5 kg of nitrogen, 5 kg of phosphorus, and 5 kg of potassium were applied, and the plants were cultivated for two months before sampling. As a control, the first cutting of grass in 2021 was used from a grassland at Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, where white clover (varieties: Abala Sting, Ababaarl (Snow Brand Seed Co., Ltd.)) and red clover (variety: Ares (Snow Brand Seed Co., Ltd.)) were sown at a rate of 3 kg per 10 ares on April 24, 2019. For fertilization of the grassland, 6 kg of nitrogen, 6 kg of phosphorus, and 4 kg of potassium per 10 ares were applied in early spring, and 3 kg of nitrogen, 3 kg of phosphorus, and 2 kg of potassium per 10 ares were applied after each harvest. Sampling was taken from the first cutting in June 2021. The sample was dried at 75°C for two days and then ground.

[0021] Manufacturing Example 2 (Preparation of Silage) Hairy vetch (variety: Mamesuke), harvested at Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, was shredded to a theoretical cutting length of 2 cm using a mounted cutter (IHI Agritech SCR1900 cylinder cutter) and then silaged. Specifically, 780g of chopped raw material was mixed with the manufacturer's recommended amount of lactic acid bacteria (Snow Brand Seed Co., Ltd., Cymaster LP, Cymaster AC), packed into a 1-liter plastic bottle (Sanplatec Co., Ltd., PE wide-mouth bottle) using a wooden stick, and stored at 25 degrees Celsius for two months. 750g of silage-processed hairy vetch was immersed in approximately four times its volume of water, crushed in a mixer, filtered through double-layered gauze, and the filtrate was centrifuged (6,500 rpm, 30 minutes, room temperature) to obtain the supernatant. This supernatant was concentrated five times.

[0022] Manufacturing Example 3 (Production of Vicia plant extracts) (1) Preparation of hairy vetch solvent extract fraction At Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, 5 kg of nitrogen, 5 kg of phosphorus, and 5 kg of potassium were applied per 10 ares, and 5 kg of seeds were sown per 10 ares. 1 kg of hairy vetch (product name: Mamesuke, variety: Namoi (Snow Brand Seed Co., Ltd.)) was cultivated for two months and then shredded into 5 cm widths to create a frozen sample. This was then immersed overnight in 3 L of 80% acetone and pulverized in a mixer. This was then filtered through a double layer of gauze and filter paper (Advantec Toyo Co., Ltd. Qualitative Filter Paper No. 1 and No. 5). The mixture was filtered using B) to obtain the filtrate. The residue was extracted twice with acetone to obtain the filtrate, and all the filtrates (9 L) were combined and concentrated, and the acetone was removed by distillation to obtain 1 L. This was adjusted to pH 8.0 using an aqueous sodium hydroxide solution, and the n-hexane phase, obtained by extraction three times with 1 L of n-hexane, was designated as the NH phase. The aqueous phase was adjusted to pH 2.5 with hydrochloric acid, and extracted three times with 1 L of n-hexane, and the resulting n-hexane phase was designated as the AH phase. The aqueous phase was treated with sodium hydroxide The pH of the aqueous phase was adjusted to 8.0 using an aqueous sodium hydroxide solution, and the resulting ethyl acetate phase was extracted three times with 1 L of ethyl acetate. The resulting ethyl acetate phase was designated as the NE phase. The pH of the aqueous phase was adjusted to 2.5 using hydrochloric acid, and the resulting ethyl acetate phase was extracted three times with 1 L of ethyl acetate. The resulting ethyl acetate phase was designated as the AE phase. The pH of the aqueous phase was adjusted to 8.0 using an aqueous sodium hydroxide solution, and the resulting n-butanol phase was extracted three times with 1 L of saturated n-butanol. The resulting n-butanol phase was designated as the NB phase. The pH of the aqueous phase was adjusted to 2.5 using hydrochloric acid, and the resulting n-butanol phase was extracted three times with 1 L of saturated n-butanol. The resulting n-butanol phase was designated as the AB phase. The remaining aqueous phase was designated as the AQ phase. The NH, AH, NE, and AE phases were dehydrated by adding anhydrous magnesium sulfate, filtered through filter paper (Advantec Toyo Co., Ltd. Qualitative Filter Paper No. 5B), and the filtrate was concentrated to dryness and dissolved in 50 mL of methanol to prepare the sample. The NB and AB phases were neutralized, concentrated in an evaporator, and then dissolved in 50 mL of aqueous methanol to prepare the sample.For the AQ phase, after neutralization with an aqueous sodium hydroxide solution, the mixture was concentrated using an evaporator to remove n-butanol by distillation. The mixture was then adsorbed onto Diaion HP-20 (Mitsubishi Chemical Corporation) packed into a glass chromatography tube (inner diameter φ40 mm × length 500 mm), washed and desalted with distilled water, eluted with 500 mL each of 50% isopropyl alcohol and 99% isopropyl alcohol, combined and concentrated using an evaporator, and then dissolved in 50 mL of aqueous methanol to obtain the sample.

[0023] (2) Purified water extracted hairy vetch A. At Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, 100g of hairy vetch (product name: Mamesuke, variety: Namoi (Snow Brand Seed Co., Ltd.)), which was sown after applying 5kg of nitrogen, 5kg of phosphorus, and 5kg of potassium and cultivated for 50 days, was cut into 5cm wide strips and used as a frozen sample. This was immersed in 700mL of water and crushed in a mixer, and the filtrate filtered through double-layered gauze was centrifuged (6,500rpm, 30 minutes, room temperature) to obtain the supernatant. This supernatant was 660mL and had a pH of 7.0. This was adsorbed onto a glass chromatography tube (inner diameter φ15mm × length 300mm) filled with Diaion HP-20 (Mitsubishi Chemical Corporation), washed with 30% ethanol, and then eluted with 500ml each of 99% ethanol. The eluted material was concentrated and dissolved in 5mL of aqueous ethanol to obtain the sample.

[0024] I. 5x concentrated unpurified water extract hairy vetch At Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, 100g of hairy vetch (product name: Mamesuke, variety: Namoi (Snow Brand Seed Co., Ltd.)), which was sown after applying 5kg of nitrogen, 5kg of phosphorus, and 5kg of potassium and cultivated for 50 days, was cut into 5cm wide strips and used as a frozen sample. This was immersed in 700mL of water and crushed in a mixer, and the filtrate, filtered through double-layered gauze, was centrifuged (6,500 rpm, 30 minutes, room temperature) to obtain the supernatant. This supernatant was 660mL and had a pH of 7.0. This was concentrated to 20mL (5-fold concentrated sample). This sample was centrifuged (14,000×g, 5 minutes, 4℃) to collect the supernatant, and then subjected to high-pressure steam treatment in an autoclave at 121℃ for 20 minutes (centrifugation supernatant autoclave).

[0025] Solvent-extracted fraction of common vetch At Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, 1 kg of common vetch (variety: AISIAI), which was sown after applying 5 kg of nitrogen, 5 kg of phosphorus, and 5 kg of potassium and cultivated for 60 days, was shredded into 5 cm widths and used as a frozen sample. This was immersed overnight in 3 L of 80% acetone and then pulverized in a mixer. This was filtered through double-layered gauze and filter paper (Advantec Toyo Co., Ltd. qualitative filter paper No. 1 and No. 5B) to obtain the filtrate. The residue was extracted twice with acetone to obtain the filtrate, and all the filtrates (9 L) were combined and concentrated, and the acetone was removed by distillation to obtain 1 L. This was adjusted to pH 8.0 using an aqueous sodium hydroxide solution, and the n-hexane phase, obtained by extraction three times with 1 L of n-hexane, was designated as the NH phase. The aqueous phase was adjusted to pH 2.5 with hydrochloric acid, and extracted three times with 1 L of n-hexane, and the resulting n-hexane phase was designated as the AH phase. The aqueous phase was adjusted to pH 8.0 using an aqueous sodium hydroxide solution and extracted three times with 1 L of ethyl acetate. The resulting ethyl acetate phase was designated as the NE phase. The aqueous phase was adjusted to pH 2.5 using hydrochloric acid and extracted three times with 1 L of ethyl acetate. The resulting ethyl acetate phase was designated as the AE phase. The aqueous phase was adjusted to pH 8.0 using an aqueous sodium hydroxide solution and extracted three times with 1 L of saturated water-normal butanol. The resulting normal butanol phase was designated as the NB phase. The aqueous phase was adjusted to pH 2.5 using hydrochloric acid and extracted three times with 1 L of saturated water-normal butanol. The resulting normal butanol phase was designated as the AB phase. The remaining aqueous phase was designated as the AQ phase. The NH, AH, NE, and AE phases were dehydrated by adding anhydrous magnesium sulfate, filtered through filter paper (Advantec Toyo Co., Ltd. Qualitative Filter Paper No. 5B), and the resulting filtrate was concentrated to dryness and dissolved in 50 mL of methanol to prepare the sample. For the NB and AB phases, neutralization and concentration in an evaporator were performed, and then the mixture was dissolved in 50 mL of aqueous methanol to obtain the sample. For the AQ phase, neutralization with aqueous sodium hydroxide solution was performed, and then the mixture was concentrated in an evaporator to remove n-butanol. The mixture was adsorbed onto Diaion HP-20 (Mitsubishi Chemical Corporation) packed in a glass chromatography tube (inner diameter φ40 mm × length 500 mm), washed and desalted with distilled water, and then eluted with 500 mL each of 50% isopropyl alcohol and 99% isopropyl alcohol. The combined solution was concentrated in an evaporator, and then dissolved in 50 mL of aqueous methanol to obtain the sample.

[0026] E. Fractionation of purified water-extracted hairy vetch using porous adsorbent resin. At Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm in Naganuma-cho, Yubari-gun, 100g of hairy vetch (product name: Mamesuke, variety: Namoi (Snow Brand Seed Co., Ltd.)), which was sown after applying 5kg of nitrogen, 5kg of phosphorus, and 5kg of potassium and cultivated for 50 days, was shredded into 5cm widths and used as a frozen sample. This was immersed in 700mL of water and crushed in a mixer, and the filtrate filtered through double-layered gauze was centrifuged (6,500rpm, 30 minutes, room temperature) to obtain the supernatant. This supernatant was 660mL and had a pH of 7.0. This was adsorbed onto a glass chromatography tube (inner diameter φ15mm x length 300mm) filled with Diaion HP-20 (Mitsubishi Chemical Corporation), washed with water, and then eluted with 500ml of 10% ethanol, 30% ethanol, or 99% ethanol. The eluent was concentrated and dissolved in 5mL of aqueous methanol to obtain the sample. Furthermore, the fraction of the supernatant that did not adsorb onto the Diaion HP-20 was also used as a sample (non-adsorbed phase).

[0027] Example 1 (Suppression of methane generation in rumen liquid) 127 mg of dried and pulverized concentrated feed (Farm King 18 Neo, Hokuren Kumiai / Snow Brand Feed Co., Ltd.), 63 mg of roughage (timothy first cut silage, harvested at Snow Brand Seed Co., Ltd. Hokkaido Research Farm (Naganuma-cho, Yubari-gun)), and various additives were weighed into 15 mL screw-cap test tubes (ST-16.5M, Nichiden Rika Glass Co., Ltd.) as shown in Tables 1 and 2.

[0028] [Table 1]

[0029] [Table 2]

[0030] Positive control: In the monensin-supplemented group, 12 μL of a solution of monensin sodium 34.4 mg / mL MeOH diluted 100-fold with 70% MeOH was added (equivalent to the addition of 3.6 μg of monensin). In the 3-nitrooxypropanol (3-NOP)-supplemented group, 19 μL of 3-NOP 1 mg / mL MeOH was added. Cashew nut shell liquid mixed feed (Luminap, Kyoritsu Pharmaceutical Co., Ltd.) was added at a dose of 1 mg per sample.

[0031] Rumen fluid was collected from fistulous cows being raised at the Hokkaido Research Farm. The fistula was surgically implanted by a veterinarian one month before the first calving. A 500 mL bottle was inserted into the lumen's liquid layer through the fistula to collect lumen fluid. The lumen fluid was mixed with an equal amount of McDougall artificial saliva (McDougall, 1948). Screw-cap test tubes containing various samples were held in a 39°C water bath, and 10 mL of the lumen fluid sample was dispensed into each tube. Under aeration with N2 gas, the tubes were capped with butyl packing (84-1882 NEG Butyl Packing C-2, for KC-2, Sansho Co., Ltd.) and fitted with caps (CH-2 perforated white cap, Nichiden Rika Glass Co., Ltd.). The test tubes were inverted and mixed once every hour and incubated for 24 hours. After incubation, a gas pressure gauge (HNT general-purpose pressure gauge AT1 / 4-60: 0.3 MPa, Daiichi Keiki) was inserted into the butyl packing to measure the headspace gas pressure. With the gas pressure gauge still inserted, insert a gas-tight syringe (1010RN, Hamilton Co., Ltd.) and aspirate the gas from the headspace into a 5 ml vacuum vial. *3 A 10 mL sample was filled into the sample. 1 mL of gas was drawn from this sample using a gas-tight syringe (1005RN, Hamilton Corporation) and subjected to a gas chromatograph (GC-2014GC-8A, Shimadzu Corporation) equipped with a thermal conductivity detector. The headspace gas volume was calculated using the following formula. The volume of methane gas produced (mL) was calculated from the proportion of methane gas output by the gas chromatograph.

[0032]

number

[0033] *3: A vial degasser (EN-1004, Endo Rika Co., Ltd.) was used to prepare the vacuum vials. The vacuum pump was connected to the degasser body, the exhaust cock was closed, and the vacuum hose cock was opened. The rubber stopper fixing plate was placed on the stopper. Gas chromatography vials (SVG-5, Nichiden Rika Glass Co., Ltd.) were lined up on the bottom plate, and butyl rubber stoppers (freeze-drying butyl rubber stopper A (medium), Nichiden Rika Glass Co., Ltd.) were lightly placed on top, leaving a gap. The degasser body was placed on the bottom plate, and the vacuum pump was started. When the vacuum gauge reading was approximately -0.1 MPa, the rubber stopper fixing plate was removed from the stopper, and the lightly placed rubber stoppers were pressed down to seal the vials. After closing the vacuum hose cock, the exhaust cock was opened. The degassing device was removed from the base plate, and the sealed vial was tightly secured with a cap (perforated cap for SVG-5, manufactured by Nichiden Rika Glass Co., Ltd.).

[0034] The amount of methane produced (mL) in the obtained lumen liquid is shown in Figures 1-9. The error bars represent the standard deviation. As is clear from Figures 1-10, extracts from *Vicia* species *Fujiemon* and *Mamesuke*, both belonging to the genus *Vicia*, and extracts from *Vicia* species *Common Vetch* showed strong methane production inhibitory activity in rumen fluid, comparable to monensin and 3-NOP. In particular, high specific activity was observed after purification to remove hydrophobic components and obtain a water-soluble fraction. The activity did not decrease even after silage fermentation with lactic acid bacteria or high-pressure steam treatment. That is, raw materials with improved preservation through silage may be used, and extraction efficiency may be increased by performing high-temperature steam treatment in the extraction process.

Claims

1. A rumen methane-reducing feed for ruminants containing the stems and leaves of plants belonging to the genus Vicia, their silage fermentation products, or extracts thereof.

2. The feed for reducing methane in the rumen of ruminants according to claim 1, wherein the extract is an extract obtained from water, an aqueous organic solvent, or a water-aqueous organic solvent mixture.

3. The rumen methane reduction feed for ruminants according to claim 2, wherein the extract obtained by washing the extract with water, an aqueous organic solvent, or a water-aqueous organic solvent mixture is a component obtained by washing the extract with n-butanol and water by solvent extraction and distilling off n-butanol from the resulting aqueous phase.

4. The feed for reducing methane in the rumen of ruminants according to claim 2, wherein the extract with water, an aqueous organic solvent, or a water-aqueous organic solvent mixture is a component obtained by adsorbing the extract onto a porous adsorption resin and distilling off the alcohol from the fraction eluted with 99% alcohol.

5. The rumen methane reduction feed for ruminants according to claim 2, wherein the extract obtained from the water, aqueous organic solvent, or water-aqueous organic solvent mixture is a fraction of the extract that does not adsorb to the porous adsorption resin.

6. The rumen methane reduction feed for ruminants according to claim 2, wherein the extract obtained by further high-pressure steam treatment of the component or fraction according to any one of claims 3 to 5 is the component obtained according to claim 2.

7. A rumen methane-reducing feed for ruminants according to any one of claims 1 to 6, wherein the plant belonging to the genus Vicia is Vicia villosa or Vicia sativa L.

8. A rumen methane-reducing feed for ruminants according to any one of claims 1 to 5, 7, wherein the content of stems and leaves of plants belonging to the genus Vicia, silage fermented products thereof, or extracts thereof is 0.01 to 50% by mass on a dry solid basis.

9. The rumen methane reduction feed for ruminants according to any one of claims 1 to 5, 7 to 8, wherein the silage fermented product is further fermented by adding lactic acid bacteria.

10. A method for reducing methane production in the rumen of ruminants, characterized by feeding ruminants a feed containing the stems and leaves of plants belonging to the genus Vicia, their silage fermented products, or extracts thereof.

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