Feed for ruminants obtained by combining methane emission inhibitor and lumen-protected amino acid

Abstract

The objective of the present invention is to provide, for example, a feed enabling effective suppression of methane emission by ruminants.　The present invention relates to, for example, a feed for ruminants, which is obtained by combining a methane emission inhibitor and a lumen-protected amino acid.

Description

Feed for ruminants comprising a combination of a methane emission inhibitor and rumen-protected amino acids

[0001] The present invention relates to a feed for ruminants. The present invention also relates to a method for raising ruminants and a method for suppressing methane emissions from ruminants, which include feeding the feed to ruminants.

[0002] Methane (CH ４ Methane is a greenhouse gas that has the second largest impact on global warming after carbon dioxide. Methane is produced from a variety of sources, but it is known to be produced in large quantities by ruminants such as cattle. Methane production in ruminants is carried out by multiple methanogens in the rumen, and the methane produced is mainly excreted as burps.

[0003] At the 26th Conference of the Parties to the United Nations Framework Convention on Climate Change (COP26), held in the UK in November 2021, more than 100 countries, including Japan, the US, and the EU, agreed to reduce methane emissions by at least 30% by 2030 compared to 2020 levels. As a result, there is a global demand for technologies that can effectively reduce methane emissions from ruminants.

[0004] To date, in order to suppress methane emissions from ruminants, it has been proposed to feed ruminants mannosylerythritol lipids and / or rhamnolipids, or to feed bromoform and allicin (Patent Documents 1 and 2). It has also been proposed to feed ruminants an amino acid balanced feed that satisfies the amino acid requirements of ruminants (Patent Document 3). However, there has been a need for a technology that can more effectively suppress methane emissions from ruminants.

[0005] JP 2009-5676 A International Publication No. 2022 / 136857 International Publication No. 2022 / 039942

[0006] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a feed that can effectively suppress methane emission by ruminants. Another object of the present invention is, in one aspect, to provide a new method for raising ruminants, which enables ruminants to be raised while suppressing methane emission by ruminants. Another object of the present invention is, in one aspect, to provide a new method for suppressing methane emission by ruminants. Another object of the present invention is, in one aspect, to provide a feed that can increase the propionic acid concentration in the rumen of a ruminant. Another object of the present invention is, in one aspect, to provide a feed that can increase the proportion of bacteria of the family Selenomonadaceae among the bacteria that constitute the ruminal bacterial flora of a ruminant. Another object of the present invention is, in one aspect, to provide a feed that can increase the butyric acid concentration in the rumen of a ruminant. Another object of the present invention is to provide a feed that can decrease the acetic acid concentration in the rumen of a ruminant. An object of one aspect of the present invention is to provide a feed capable of increasing the proportion of bacteria of the family Lachnospiraceae in bacteria constituting the rumen bacterial flora of ruminants.Another object of the present invention is to provide a feed capable of increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) in bacteria constituting the rumen bacterial flora of ruminants.

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that feeding ruminants a feed comprising a combination of a methane emission inhibitor and a rumen-protected amino acid (a feed containing a combination of a methane emission inhibitor and a rumen-protected amino acid) can effectively suppress methane emission from ruminants, and through further research have completed the present invention.

[0008] [1] A feed for ruminants comprising a combination of a methane emission inhibitor and a rumen-protected amino acid. [2] The feed according to [1], which has a higher effect of increasing the propionic acid concentration in the rumen of ruminants than the methane emission inhibitor. [3] The feed according to [1] or [2], which has a higher effect of increasing the butyric acid concentration in the rumen of ruminants than the methane emission inhibitor. [4] The feed according to any one of [1] to [3], which has a higher effect of decreasing the acetic acid concentration in the rumen of ruminants than the methane emission inhibitor. [5] The feed according to any one of [1] to [4], which has a higher effect of increasing the proportion of bacteria of the family Selenomonadaceae in the bacteria constituting the rumen bacterial flora of ruminants than the methane emission inhibitor. [6] The feed according to any one of [1] to [5], which has a higher effect of increasing the proportion of bacteria of the family Lachnospiraceae in the bacteria constituting the rumen bacterial flora of ruminants than the methane emission inhibitor. [7] The feed according to any one of [1] to [6], which has a stronger effect of increasing the proportion of at least one selected from the group consisting of Bacteroidota, Proteobacteria, Firmicutes, and Fibrobacterota bacteria in the ruminal bacterial flora of ruminants than the methane emission inhibitor. [8] The feed according to any one of [1] to [7], wherein the amino acid comprises an essential amino acid. [9] The feed according to any one of [1] to [8], wherein the amino acid comprises at least one selected from the group consisting of lysine, methionine, arginine, and histidine.

[10] The feed according to any one of [1] to [9], wherein the methane emission inhibitor comprises at least one selected from the group consisting of an organic compound having a nitrooxy group, cashew nut shell liquid, polyphenols, nitrates, essential oils, plant extracts, halogenated methane analogs, fatty acid calcium, saponins, and organic acids.

[11] The feed according to any one of [1] to

[10] , wherein the methane emission inhibitor comprises at least one selected from the group consisting of 3-nitrooxypropanol, cashew nut shell liquid, tannic acid, essential oils, plant extracts, and bromoform.

[12] The feed according to any one of [1] to

[11] , wherein the methane emission inhibitor comprises at least one selected from the group consisting of 3-nitrooxypropanol, cashew nut shell liquid, tannic acid, essential oils, and plant extracts.

[13] The feed according to any one of [1] to

[12] , wherein the crude protein content of the feed is 0.1 to 10% DM lower than the crude protein content of feed used until the start of feeding of the feed.

[14] The feed according to any one of [1] to

[13] , wherein the crude protein content is 10 to 20% DM.

[15] A method for raising ruminants, comprising feeding the feed according to any one of [1] to

[14] to ruminants.

[16] A method for suppressing methane emissions by ruminants, comprising feeding the feed according to any one of [1] to

[14] to ruminants.

[17] A method for improving the effectiveness of a methane emission inhibitor, comprising adding a rumen-protected amino acid to feed for ruminants fed with the methane emission inhibitor.

[18] The method according to

[17] , comprising reducing the crude protein content of the feed by 0.1 to 10% DM.

[19] The method according to

[17] or

[18] , wherein the amino acid comprises an essential amino acid.

[20] The method according to any one of

[17] to

[19] , wherein the amino acid comprises at least one selected from the group consisting of lysine, methionine, arginine, and histidine.

[21] The method according to any one of

[17] to

[20] , wherein the methane emission inhibitor comprises at least one selected from the group consisting of an organic compound having a nitrooxy group, cashew nut shell liquid, polyphenols, nitrates, essential oils, plant extracts, halogenated methane analogs, fatty acid calcium, saponins, and organic acids.

[22] The method according to any one of

[17] to

[21] , wherein the methane emission inhibitor comprises at least one selected from the group consisting of 3-nitrooxypropanol, cashew nut shell liquid, tannic acid, essential oils, plant extracts, and bromoform.

[23] The method according to any one of

[17] to

[22] , wherein the methane emission inhibitor comprises at least one selected from the group consisting of 3-nitrooxypropanol, cashew nut shell liquid, tannic acid, essential oils, and plant extracts.

[24] The method according to any one of

[17] to

[23] , comprising adjusting the crude protein content of the feed to 10 to 20% DM.

[25] The method according to any one of

[17] to

[24] , wherein the improved effect of the methane emission inhibitor is an improvement in the effect of increasing the propionic acid concentration in the rumen of a ruminant.

[26] The method according to any one of

[17] to

[25] , wherein the improved effect of the methane emission inhibitor is an improvement in the effect of increasing the butyric acid concentration in the rumen of a ruminant.

[27] The method according to any one of

[17] to

[26] , wherein the improved effect of the methane emission inhibitor is an improvement in the effect of decreasing the acetic acid concentration in the rumen of a ruminant.

[28] The method according to any one of

[17] to

[27] , wherein the improved effect of the methane emission inhibitor is an improvement in the effect of increasing the proportion of bacteria of the family Selenomonadaceae in the bacteria constituting the rumen bacterial flora of a ruminant.

[29] The method according to any one of

[17] to

[28] , wherein the improved effect of the methane emission inhibitor is an improvement in the effect of increasing the proportion of bacteria of the family Lachnospiraceae in the bacteria constituting the rumen bacterial flora of a ruminant.

[30] The method according to any one of

[17] to

[29] , wherein the improved effect of the methane emission inhibitor is an improvement in the activity of increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota, bacteria of the phylum Proteobacteria, bacteria of the phylum Firmicutes, and bacteria of the phylum Fibrobacterota in the bacteria constituting the rumen bacterial flora of a ruminant.

[0009] According to the present invention, a feed capable of effectively suppressing methane emission by ruminants can be provided. Furthermore, according to the present invention, a method for raising ruminants can be provided, which enables ruminants to be raised while suppressing methane emission by ruminants. Furthermore, according to the present invention, a method for suppressing methane emission by ruminants can be provided. Furthermore, according to the present invention, a feed capable of increasing the propionic acid concentration in the rumen of a ruminant can be provided. The present invention can provide a feed capable of increasing the proportion of bacteria of the family Selenomonadaceae among the bacteria constituting the rumen bacterial flora of a ruminant. Furthermore, according to the present invention, a feed capable of increasing the butyric acid concentration in the rumen of a ruminant can be provided. The present invention can provide a feed capable of decreasing the acetic acid concentration in the rumen of a ruminant can be provided. The present invention can provide a feed capable of increasing the proportion of bacteria of the family Lachnospiraceae among the bacteria constituting the rumen bacterial flora of a ruminant. Furthermore, according to the present invention, it is possible to provide a feed that can increase the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) in the bacteria that constitute the rumen bacterial flora of ruminants.

[0010] The ruminant feed of the present invention (sometimes referred to as "the feed of the present invention" in this specification) comprises a combination of a methane emission inhibitor and a rumen-protected amino acid, or in other words, contains a combination of a methane emission inhibitor and a rumen-protected amino acid. In the present invention, "ruminant" is a general term for animals belonging to the class Mammalia, order Artiodactyla, suborder Ruminata, and more specifically refers to herbivorous mammals having a rumen divided into three or four chambers and having the habit of ruminating their food. Specific examples of ruminant animals include, but are not limited to, cows, goats, and sheep.

[0011] In the present invention, the term "methane emission inhibitor" is a general term for a substance that has the effect of inhibiting methane emission by ruminants (sometimes referred to herein as "methane emission inhibitory activity"). By feeding a methane emission inhibitor to a ruminant (e.g., cattle, etc.), the amount of methane contained in the ruminant's belching can be reduced. The methane emission inhibitor that can be used in the present invention is not particularly limited as long as it has a methane emission inhibitory activity (i.e., an activity of inhibiting methane emission by ruminants), and examples thereof include organic compounds having a nitrooxy group, cashew nut shell liquid, polyphenols, nitrates, essential oils, plant extracts, halogenated methane analogs, calcium fatty acids, saponins, organic acids, etc. These methane emission inhibitors may be used alone or in combination of two or more.

[0012] The nitrooxy group (—O—NO) that can be used in the present invention ２The organic compound having the formula (I) is not particularly limited as long as it has a methane emission suppressing effect, and examples thereof include 3-nitrooxypropanol, racemic-4-phenylbutane-1,2-diyldinitrate, 2-(hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol, N-ethyl-3-nitrooxypropionic acid sulfonylamide, 5-nitrooxypentanenitrile, 5-nitrooxypentane, 3-nitrooxypropyl propionate, 1,3-bis-nitrooxypropane, 1,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, 3-nitrooxypropyl benzoate, 3-nitrooxypropyl hexanoate, 3-nitrooxypropyl 5-nitrooxyhexanoate, benzyl nitrate, isosorbide dinitrate, N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide, 3-nitrooxypropyl Examples of the nitrooxy-N-methyl-pentanoic acid include methyl 3-nitrooxypropionate, ethyl 3-nitrooxypropionate, ethyl 4-nitrooxybutanoate, ethyl 3-nitrooxybutanoate, 5-nitrooxypentanoic acid, ethyl 5-nitrooxypentanoate, 6-nitrooxyhexanoic acid, ethyl 6-nitrooxyhexanoate, ethyl 4-nitrooxycyclohexylcarboxylate, 8-nitrooxyoctanoic acid, ethyl 8-nitrooxyoctanoate, 11-nitrooxyundecanoic acid, ethyl 11-nitrooxyundecanoate, 5-nitrooxypentanoic acid amide, and 5-nitrooxy-N-methyl-pentanoic acid amide, of which 3-nitrooxypropanol, ethyl 3-nitrooxypropionate, methyl 3-nitrooxypropionate, and 3-nitrooxypropionic acid are preferred, and 3-nitrooxypropanol is particularly preferred. These compounds may be used alone or in combination of two or more.

[0013] 3-nitrooxypropanol (3-NOP) is a nitric acid ester of 1,3-propanediol represented by the following formula (CAS registration number: 100502-66-7).

[0014]

[0015] The organic compound having a nitrooxy group that can be used in the present invention may be in the form of a salt, and the term "organic compound having a nitrooxy group" in the present invention includes such salts. The salt is not particularly limited as long as it can be fed to ruminants, and examples thereof include salts with inorganic bases, salts with inorganic acids, and salts with organic acids.

[0016] Cashew nut shell liquid (CNSL) is an oily liquid obtained by squeezing the shells of cashew nuts, and is also commonly called cashew nut shell oil or cashew nut oil.

[0017] Polyphenols are antioxidants produced by plants during photosynthesis and are components that may contribute to the bitterness, astringency, and color of plants. The polyphenols that can be used in the present invention are not particularly limited as long as they have the effect of suppressing methane emissions, and examples include tannins, chlorogenic acid, catechin, isoflavones, and flavonoids. Tannin is a type of natural polyphenol compound derived from plants and is a general term for compounds that have the property of forming sparingly soluble salts by binding with proteins, metal ions, etc. Tannins can be classified into hydrolyzable tannins in which an aromatic compound (e.g., gallic acid, ellagic acid, etc.) forms an ester bond with a sugar (e.g., glucose, etc.) and condensed tannins in which a compound having a flavanol skeleton is polymerized. The tannins that can be used in the present invention are preferably hydrolyzable tannins, with tannic acid being particularly preferred. The tannins that can be used in the present invention are not particularly limited in terms of the plant they are derived from, as long as they have a methane emission-inhibiting effect, and examples include Chinese gallnut, gall nut, tea, chestnut, persimmon, mimosa, eucalyptus, quebracho, etc. The form of the tannins that can be used in the present invention is not particularly limited, and for example, a liquid containing tannin extracted from a plant (extract) may be used as is, or an extract containing plant-derived tannins may be dried or otherwise used in the form of a powder.

[0018] The nitrate is not particularly limited as long as it can be fed to ruminants, and examples thereof include potassium nitrate, sodium nitrate, calcium nitrate, ammonium nitrate, etc. The nitrate may be used in combination with cysteine ​​and / or cystine.

[0019] Essential oils are volatile oils obtained from plants and have a fragrance specific to the plant. The essential oils used in the present invention are not particularly limited in terms of the plant from which they are derived, as long as they have the effect of inhibiting methane emission. Examples of essential oils include coriander, eucalyptus, basil, rosemary, etc.

[0020] The term "plant extract" refers to an extract derived from a plant, and more specifically, refers to a substance or a processed product thereof obtained by subjecting a plant to an extraction process. The plant extract usable in the present invention is not particularly limited in terms of its plant origin, as long as it has a methane emission inhibitory effect. Examples include Allium plants such as garlic, onion, and leek; Cinnamon plants such as cinnamon, Cinnamon cinnamon, and Cinnamon cinnamon; and Citrus plants such as oranges, lemons, and citrus fruits. The plant extract usable in the present invention is not particularly limited in terms of its components, as long as it has a methane emission inhibitory effect. Examples of the components of plant extracts usable in the present invention include aromatic compounds (e.g., aromatic aldehydes such as cinnamaldehyde), sulfur-containing compounds (e.g., allyl sulfide such as allicin, sulfur-containing amino acids such as alliin), and the like. The form of the plant extract usable in the present invention is not particularly limited. For example, the liquid extracted from the plant (extract) may be used as is, or the plant-derived extract may be dried or otherwise processed into a powder.

[0021] Halogenated methane analogues (HMA) are halogenated compounds found in the red algae Asparagopsis (Christopher R.K. Glasson et al., Algal Research 64 (2022) 102673). Halogenated methane analogues that can be used in the present invention are not particularly limited as long as they have the effect of inhibiting methane emissions, and examples include bromoform and dibromochloromethane. The form of the halogenated methane analogues that can be used in the present invention is not particularly limited; for example, red algae can be freeze-dried and used as a powder.

[0022] Fatty acid calcium is a processed oil or fat product obtained by subjecting oils and fats (e.g., palm oil, soybean oil, linseed oil, etc.) to processing (such as the addition of calcium hydroxide) to protect them from ruminal microorganisms, with the aim of increasing the availability of oil and fat nutrients in ruminants. The fatty acid calcium usable in the present invention is not particularly limited in terms of its components, as long as it has a methane emission inhibitory effect. For example, the fatty acids contained in the fatty acid calcium may be either saturated or unsaturated. Examples of saturated fatty acids include lauric acid, myristic acid, palmitic acid, and stearic acid, while examples of unsaturated fatty acids include palmitoleic acid, oleic acid, linoleic acid, and linolenic acid. However, from the viewpoint of methane emission inhibitory effect, unsaturated fatty acids (e.g., oleic acid, linoleic acid, linolenic acid, etc.) are preferred.

[0023] Saponin is a type of glycoside found in plants and has surfactant properties. The plant from which the saponin that can be used in the present invention is derived is not particularly limited as long as it has a methane emission inhibitory effect, and examples thereof include soybean, Astragalus membranaceus, ginseng, olive, and grape skin.

[0024] The organic acid is an organic compound that exhibits acidity. The organic acid that can be used in the present invention is not particularly limited as long as it has the effect of suppressing methane emission, and examples thereof include acetic acid, lactic acid, malic acid, succinic acid, and fumaric acid.

[0025] The methane emission inhibitor that can be used in the present invention is preferably an organic compound having a nitrooxy group (e.g., 3-nitrooxypropanol, etc.), cashew nut shell liquid, polyphenols (e.g., tannic acid, etc.), essential oils, plant extracts, or halogenated methane analogs (e.g., bromoform, etc.), and more preferably 3-nitrooxypropanol, cashew nut shell liquid, tannic acid, essential oils, plant extracts, or bromoform.

[0026] The method for producing the methane emission inhibitor that can be used in the present invention is not particularly limited, and the methane emission inhibitor may be produced by a method known per se or a method equivalent thereto. In addition, the methane emission inhibitor may be a commercially available product (commercial product).

[0027] The amount of the methane emission inhibitor contained in the feed of the present invention (i.e., the amount of the methane emission inhibitor combined with the rumen-protected amino acid in the feed of the present invention) may be an amount effective for suppressing methane emission by ruminants. In other words, the feed of the present invention may contain the methane emission inhibitor in an amount effective for suppressing methane emission by ruminants. Here, "an amount effective for suppressing methane emission by ruminants" means an amount that can bring about a methane emission suppression effect in ruminants fed the feed of the present invention (i.e., an amount that can reduce the methane contained in the belching of ruminants). The effective amount for suppressing methane emission by ruminants will vary depending on the type of methane emission inhibitor, the type and body weight of the ruminant fed the feed of the present invention, etc., and can be appropriately determined taking these factors into consideration.

[0028] In the present invention, a "rumen-protected amino acid" (RPAA) used in combination with a methane emission inhibitor refers to an amino acid that has been treated or processed to reduce degradation in the rumen (first stomach of a ruminant), and is a concept that encompasses rumen-bypassed amino acids, rumen-protected amino acids, rumen-protected amino acids, and the like. In this specification, a "rumen-protected amino acid" may be referred to as "RPAA." Furthermore, the "treatment or processing to reduce degradation in the rumen" that has been applied to an amino acid contained in RPAA may be referred to as "rumen-protection treatment."

[0029] The types of amino acids contained in RPAA (i.e., amino acids that have been subjected to rumen protection treatment) are not particularly limited, but preferably contain essential amino acids appropriate for the type of ruminant to which the feed of the present invention is fed. Taking cows, a type of ruminant, as an example, there are 10 essential amino acids for cows: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. When the feed of the present invention is fed to cows, it is preferable that the RPAA contain at least one of these 10 amino acids. In particular, it is preferable that the RPAA contain an essential amino acid (limiting amino acid) that has a relatively low sufficiency relative to the amino acid requirement of ruminants among the essential amino acids contained in the components other than RPAA of the feed of the present invention. Specifically, it is preferable that the RPAA contain lysine, methionine, arginine, and histidine, more preferably lysine and methionine, and particularly preferably lysine. The amino acids contained in RPAA may be in the L-, D-, or DL- configuration, preferably the L- or DL- configuration, and more preferably the L- configuration. The form of the amino acids contained in RPAA is not particularly limited, and may be, for example, in the free form or salt form, or in a form that can generate free amino acids through enzymatic reaction, hydrolysis, or the like in the living body of a ruminant (e.g., constituent amino acids of peptides, constituent amino acids of proteins, etc.).

[0030] The RPAA used in the present invention can be obtained by subjecting amino acids (e.g., free amino acids, salt amino acids, peptide amino acids, protein amino acids, etc.) to rumen protection treatment (i.e., treatment or processing to reduce degradation in the rumen). The rumen protection treatment is not particularly limited as long as it can reduce degradation of amino acids in the rumen, and may be performed by a method known per se or a method equivalent thereto. For example, the method for preparing RPAA used in the present invention may include coating amino acid particles with a protective agent mainly composed of hydrogenated oil, wax, wax, etc., or granulating a mixture of the protective agent and amino acids. In one embodiment, the protective agent used to prepare RPAA may further contain, in addition to the main component (hydrogenated oil, wax, wax, etc.), for example, oils and fats other than hydrogenated oil, surfactants (e.g., lecithin, etc.), etc.

[0031] In one embodiment, the RPAA used in the present invention can be any of the RPAAs described in WO 2008 / 041371, U.S. Patent Application Publication No. 2009 / 232933, U.S. Patent Application Publication No. 2013 / 095206, U.S. Patent No. 9,173,420, U.S. Patent Application Publication No. 2014 / 308418, U.S. Patent No. 9,241,503, WO 2009 / 122750, U.S. Patent Application Publication No. 2011 / 081444, U.S. Patent No. 9,204,660, U.S. Patent Application Publication No. 2014 / 308412, U.S. Patent No. 9,265,273, WO 2018 / 030476, U.S. Patent Application Publication No. 2019 / 166879, and WO 2018 / 07974. No. 8, U.S. Patent Application Publication No. 2019 / 246666, U.S. Patent No. 11076618, U.S. Patent Application Publication No. 2021 / 321642, U.S. Patent No. 11805793, WO 2018 / 079747, U.S. Patent Application Publication No. 2019 / 246665, U.S. Patent No. 11083209, WO 2019 / 189605, U.S. Patent Application Publication No. 2021 / 007371, U.S. Patent No. 11582988, U.S. Patent Application Publication No. 2023 / 138420, WO 2021 / 060388, U.S. Patent Application Publication No. 2022 / 211076 It may be prepared by a method described therein or a method equivalent thereto. In one embodiment, the RPAA used in the present invention may be a feed additive composition (preferably a dispersion-type feed additive composition) described in WO 2008 / 041371, which contains (A) at least one selected from hydrogenated vegetable oils and hydrogenated animal oils having a melting point higher than 50°C and lower than 90°C, (B) lecithin (preferably 0.05 to 6% by weight of lecithin), (C) 40% by weight or more and less than 65% by weight of a basic amino acid (preferably lysine hydrochloride), and (D) water (preferably 0.01 to 6% by weight of water).

[0032] In one embodiment, the RPAA used in the present invention may be a commercially available product (commercially available product). Examples of commercially available RPAA products include, but are not limited to, "AjiPro (registered trademark)-L" from Ajinomoto Co., Inc., "Smartamine M" and "Smartamine ML" from Adisseo, Inc., and "Mepron" from Evonik.

[0033] The form of RPAA used in the present invention is not particularly limited, and may be, for example, granules, powder, capsules, tablets, gels, solids, etc.

[0034] The amount of RPAA contained in the feed of the present invention (i.e., the amount of RPAA combined with the methane emission inhibitor in the feed of the present invention) can be set appropriately taking into consideration the type of amino acid contained in the RPAA, the type, purpose, breed, weight, growth stage, and age at parturition of the ruminant to be fed the feed of the present invention, etc., but in one embodiment, when the amino acids contained in the RPAA include essential amino acids for the ruminant to be fed the feed of the present invention, the amount of RPAA contained in the feed of the present invention may be set taking into consideration the essential amino acid requirement of the ruminant to be fed the feed of the present invention.

[0035] The amount of RPAA contained in the feed of the present invention will be described below using as an example a case where the ruminant animal fed the feed of the present invention is a cattle and the amino acids contained in the RPAA include essential amino acids for cattle. In this case, the amount of RPAA contained in the feed of the present invention can be set so that the feed of the present invention satisfies 80% or more (preferably 90-500%, more preferably 100-150%) of the essential amino acid requirement of cattle (the requirement of essential amino acids contained in RPAA). In one embodiment, when the RPAA contained in the feed of the present invention contains lysine, the amount of RPAA contained in the feed of the present invention can be set so that the feed of the present invention satisfies 80% or more (preferably 90-500%, more preferably 100-150%) of the lysine requirement of cattle.

[0036] In the present invention, the nutrient requirements of ruminants (e.g., essential amino acid requirements, etc.) may be calculated based on, for example, feeding standards established and used in any country. In one embodiment, the essential amino acid requirements of cattle may be calculated based on the Japanese Feeding Standards, academic literature on cattle nutrition, or a feed design program. That is, in the above-mentioned example (where the ruminant fed the feed of the present invention is a cattle and the amino acids contained in RPAA include essential amino acids for cattle), the amount of RPAA contained in the feed of the present invention may be set so that the sufficiency rate of the feed of the present invention relative to the essential amino acid requirements of cattle based on the Japanese Feeding Standards, academic literature on cattle nutrition, or a feed design program is the above-mentioned specific rate (usually 80% or more, preferably 90 to 500%, more preferably 100 to 150%). Generally, nutrient requirements (e.g., requirements for metabolic energy, metabolic protein, metabolic amino acids, etc.) are calculated based on each country's feeding standards and feed design programs, and feed is designed so that the calculated nutrient requirements are met at approximately 100%. In feed design, various feed ingredients are used to meet the calculated nutrient requirements, and for example, RPAA may be used to meet the calculated metabolic amino acid requirements. In this specification, "Japanese Feeding Standard" refers to "Japanese Feeding Standard for Beef Cattle (2022 Edition)" and "Japanese Feeding Standard for Dairy Cattle (2017 Edition)" (both compiled by the National Agriculture and Food Research Organization and published by the Japan Livestock Industry Association). Examples of academic literature on bovine nutrition include Nutrient Requirements of Dairy Cattle: Eighth Revised Edition (2021) (sometimes referred to as "NASEM" in this specification), and examples of feed design programs include AMTS (Agriculture Modeling and Training System, manufactured by AMTS), NDS (Nutritional Dynamic System, manufactured by RUM&N), and CPM-Dairy (Cornell-Penn-Miner-Dairy).To calculate the lysine and methionine requirements of beef cattle, the "Japanese Feeding Standards for Beef Cattle (2022 Edition) Feed Design Diagnostic Program (Amino Acid Balance Compatible)" (https: / / www.naro.go.jp / laboratory / nilgs / contents / shiryo_hyojyun / nikyuyou2022 / download / index.html) may be used.

[0037] The feed of the present invention may further contain ingredients other than the methane emission inhibitor and RPAA. The components other than the methane emission inhibitor and RPAA are not particularly limited as long as they do not impair the object of the present invention, and examples include feed materials such as corn, corn silage, grass silage, alfalfa, alfalfa hay, timothy, sudan grass, orchard grass, Italian ryegrass, Italian ryegrass silage, perennial ryegrass, sorghum, oat hay, barley, rye, rice bran, wheat bran, corn gluten feed, beet pulp, soybean meal, linseed meal, whole milk, and salt; feed additives such as antioxidants, fungicides, binders, emulsifiers, conditioners, amino acids that have not been treated with rumen protection, vitamins, minerals, color enhancers, synthetic antibacterial agents, antibiotics, flavorings, flavoring agents, enzymes, probiotics, and organic acids; milking robot compound feed, and PMR (partially mixed feed). Any one of these components may be used in combination with the methane emission inhibitor and RPAA, or any two or more of these components may be used in combination with the methane emission inhibitor and RPAA.

[0038] The crude protein content of the feed of the present invention may be appropriately set taking into consideration the type, purpose, breed, body weight, growth stage, calving age, etc. of the ruminant to be fed the feed of the present invention, but is preferably set taking into consideration the crude protein requirement of the ruminant to be fed the feed of the present invention. The crude protein requirement of a ruminant can be calculated based on feeding standards established and used in any country, academic literature on ruminant nutrition, etc. For example, the crude protein requirement of a cow may be calculated based on the Japanese Feeding Standards, NASEM, etc. The crude protein content of the feed of the present invention may be an amount in accordance with feeding standards established and used in any country (e.g., Japanese Feeding Standards, etc.) or academic literature on ruminant nutrition (e.g., NASEM, etc.), but in one embodiment, it may be an amount less than the amount in accordance with the feeding standards or academic literature (e.g., 1% DM or more less). Furthermore, when the crude protein content of the feed of the present invention is set to a lower amount (for example, 1% or more lower) than the amount in accordance with the feeding standards established and used in any country or academic literature on ruminant nutrition, the resulting reduction in the sufficiency rate of the feed of the present invention for the essential amino acid requirements of ruminants can be compensated for by adjusting the amount of RPAA contained in the feed of the present invention, etc.

[0039] The crude protein content of the feed of the present invention will be specifically explained below using as an example a case where the ruminant animal to be fed the feed of the present invention is a cattle (beef cattle, dairy cattle). For example, when the feed of the present invention is fed to lactating dairy cows, the crude protein content is preferably 14 to 20% DM, more preferably 15 to 18% DM, and even more preferably 15 to 16% DM, based on the feed (dry matter) of the present invention. As used herein, "% DM" means the percentage by weight of dry matter (DM). When the feed of the present invention is fed to dry dairy cows, the crude protein content is preferably 10 to 16% DM, more preferably 12 to 14% DM, based on the feed (dry matter) of the present invention. When the feed of the present invention is fed to beef cattle, the crude protein content is preferably 10 to 17% DM, based on the feed (dry matter) of the present invention. For example, when the feed of the present invention is fed to beef cattle weighing less than 400 kg, the crude protein content thereof is preferably 12 to 17% DM of the feed of the present invention (dry matter), and when the feed of the present invention is fed to beef cattle weighing 400 kg or more, the crude protein content thereof is preferably 11 to 13% DM of the feed of the present invention (dry matter). Alternatively, the crude protein content of the feed of the present invention may be 0.1 to 10% DM lower than the crude protein content of the feed used by the cattle fed the feed of the present invention until the start of feeding the feed of the present invention (for example, until the day before the start of feeding the feed of the present invention) (hereinafter, sometimes referred to as "pre-feed"). The feeding period of the pre-feed is not particularly limited and may be, for example, one year or more, one year, six months, three months, one month, one week, or the like. Furthermore, the crude protein content of the feed of the present invention may be 0.2 to 7.5% DM lower than the crude protein content of the pre-feed, 0.5 to 5% DM lower than the crude protein content of the pre-feed, or 1 to 3% DM lower than the crude protein content of the pre-feed.Alternatively, the agricultural methodology of J-Credit (https: / / japancredit.go.jp), "Feeding of feed with improved amino acid balance to cattle, pigs and broilers" (methodology number: AG-001), stipulates as application conditions that conventional feed must be fed before the project is implemented, and that after the project is implemented, feed with improved amino acid balance, whose CP content is DM reduced by 1 to 3% from the CP content (%) of the conventional feed, and the crude protein content of the feed of the present invention may be set in accordance with this.

[0040] In the present invention, the crude protein content of the feed is measured and calculated by measuring the components by near-infrared spectroscopy (NIR) or wet chemical analysis.

[0041] The rumen degradable protein (RDP) content of the feed of the present invention may be set appropriately taking into consideration the type, purpose, breed, body weight, growth stage, calving age, etc. of the ruminant to be fed the feed of the present invention, but in one embodiment, it may be an amount in accordance with feeding standards established and used in any country (e.g., Japanese Feeding Standards, etc.) or academic literature on ruminant nutrition (e.g., NASEM, etc.). Here, "rumen degradable protein" means proteins in the feed that are degraded and utilized by microorganisms in the rumen. In this specification, the rumen degradable protein content may be referred to as "RDP content."

[0042] The RDP content of the feed of the present invention will be specifically explained below using as an example a case where the ruminant animal to be fed the feed of the present invention is a cattle (beef cattle, dairy cattle). For example, when the feed of the present invention is fed to dairy cattle, the RDP content of the feed of the present invention (dry matter) is preferably 8% DM or more, more preferably 8.8% DM or more, and even more preferably 9% DM or more. Furthermore, when the feed of the present invention is fed to beef cattle, the RDP content of the feed of the present invention (dry matter) is preferably 6% DM or more, more preferably 7% DM or more, and even more preferably 8% DM or more.

[0043] In the present invention, the RDP content of a feed (i.e., the rumen-degradable protein content) can be determined by calculating component values ​​based on a feed design program (e.g., AMTS, NDS, CPM Dairy, etc.) or a feed composition table published in NASEM, etc.

[0044] In one embodiment, the content of metabolic protein from bacterial protein (MP from Bacteria) in the feed of the present invention may be an amount that conforms to feeding standards established and used in any country (e.g., Japanese Feeding Standards, etc.) or academic literature on ruminant nutrition (e.g., NASEM, etc.). It is desirable that the content of metabolic protein from bacterial protein in the feed of the present invention is not lower than that of the feed used before the start of feeding of the feed of the present invention. In this specification, the content of metabolic protein from bacterial protein may be referred to as the "MP content from BP."

[0045] In the present invention, the MP content from BP of the feed (i.e., the content of metabolic protein from bacterial protein) can be determined by calculating component values ​​based on a feed design program (e.g., AMTS, NDS, CPM Dairy, etc.) or a feed composition table published in NASEM, etc.

[0046] The method for producing the feed of the present invention is not particularly limited, and the feed of the present invention can be produced by a method known per se or a method similar thereto.

[0047] The form of the feed of the present invention is not particularly limited, and the methane emission inhibitor and RPAA may be combined when fed to ruminants. In one embodiment, the feed of the present invention may be a single feed containing the methane emission inhibitor and RPAA. The feed of the present invention may also be, for example, a combination of a feed containing RPAA and a methane emission inhibitor formulated separately therefrom. In this case, the RPAA-containing feed and the methane emission inhibitor may be fed to ruminants simultaneously, or the RPAA-containing feed and the methane emission inhibitor may be fed to ruminants with a time lag (for example, feeding the RPAA-containing feed and the methane emission inhibitor in that order, or vice versa).

[0048] The method for feeding the feed of the present invention to ruminants is not particularly limited, and the feed may be fed by a method known per se or a method similar thereto. The type of feed animal to which the feed of the present invention can be fed is not particularly limited, but is preferably cattle. The use of the feed animal to which the feed of the present invention can be fed is not particularly limited. When the feed of the present invention is fed to cattle in one embodiment (cattle feed), the cattle to which the feed of the present invention can be fed may be either beef cattle or dairy cattle. The breed of feed animal to which the feed of the present invention can be fed is not particularly limited. When the feed of the present invention is fed to dairy cows in one embodiment (dairy cow feed), examples of breeds of dairy cows to which the feed of the present invention can be fed include Holstein, Jersey, Brown Swiss, etc., and the feed of the present invention can be fed to any of these breeds. In this case, the calving age of the dairy cow is also not particularly limited, and the dairy cow to which the feed of the present invention can be fed may be in the early lactation, late lactation, dry period, etc.

[0049] The feeding period of the feed of the present invention is not particularly limited, but is usually one week or more, preferably four weeks or more. The number of times the feed is fed per day is also not particularly limited, and may be, for example, one or more times, or the feed of the present invention may be fed ad libitum.

[0050] The feed of the present invention can suppress methane emission by ruminants. The feed of the present invention can suppress methane emission by ruminants more effectively than the methane emission inhibitor contained in the feed of the present invention. The method for evaluating the inhibitory effect of the feed of the present invention on methane emission by ruminants is not particularly limited, and may be evaluated by a method known per se or a method equivalent thereto. For example, as in the Examples described below, evaluation can be performed by performing a culture test using rumen fluid (gastric fluid) collected from ruminants fed the feed of the present invention as a sample, and measuring the amount of gas (e.g., methane) generated from the rumen fluid. The amount of gas generated can be measured by a method known per se (e.g., gas chromatography, high-performance liquid chromatography, etc.) or a method equivalent thereto.

[0051] In one embodiment, the feed of the present invention may have a stronger effect of increasing the propionic acid concentration in the rumen of a ruminant than the methane emission inhibitor contained in the feed of the present invention. Methane production in the rumen of a ruminant can be inhibited by preventing the use of hydrogen generated in the rumen for methane synthesis. Because hydrogen is used when propionic acid is produced in the rumen, promoting propionic acid production and increasing the propionic acid concentration in the rumen reduces the amount of hydrogen available for methane synthesis, thereby inhibiting methane production in the rumen and suppressing methane emission by ruminants. The method for evaluating the effect of the feed and methane emission inhibitor of the present invention on increasing the propionic acid concentration in the rumen of a ruminant is not particularly limited, and may be performed using a known method or a method equivalent thereto. For example, as in the Examples described below, the effect can be evaluated by performing a culture test using ruminal fluid (gastric fluid) collected from a ruminant fed the feed of the present invention as a sample, and measuring the amount of gas (propionic acid, etc.) generated from the rumen fluid. The amount of gas generated can be measured by a method known per se (eg, gas chromatography, high performance liquid chromatography, etc.) or a method similar thereto.

[0052] In one embodiment, the feed of the present invention may have a stronger effect of increasing the butyric acid concentration in the rumen of a ruminant or an effect of decreasing the acetic acid concentration in the rumen of a ruminant than the methane emission inhibitor contained in the feed of the present invention. As described above, methane production in the rumen of a ruminant can be suppressed by preventing hydrogen generated in the rumen from being used for methane synthesis. Because hydrogen is used when butyric acid is produced in the rumen, promoting butyric acid production and increasing the butyric acid concentration in the rumen reduces the hydrogen available for methane synthesis, thereby suppressing methane production in the rumen and methane emission by ruminants. Furthermore, because hydrogen is produced when acetic acid is produced in the rumen, suppressing acetic acid production and decreasing the acetic acid concentration in the rumen reduces the hydrogen available for methane synthesis, thereby suppressing methane production in the rumen and methane emission by ruminants. The method for evaluating the effect of the feed and methane emission inhibitor of the present invention to increase the butyric acid concentration and decrease the acetic acid concentration in the rumen of a ruminant is not particularly limited, and may be performed by a method known per se or a method equivalent thereto, but for example, as in the Examples described below, the effect can be evaluated by performing a culture test using ruminal fluid (gastric fluid) collected from a ruminant that has been fed the feed, etc. of the present invention as a sample, and measuring the amount of gas (butyric acid, acetic acid, etc.) generated from the rumen fluid. The amount of gas generated can be measured by a method known per se (e.g., gas chromatography, high-performance liquid chromatography, etc.) or a method equivalent thereto.

[0053] In one embodiment, the feed of the present invention may have a stronger effect of increasing the proportion of bacteria of the family Selenomonadaceae among the bacteria constituting the rumen bacterial flora of ruminants than the methane emission inhibitor contained in the feed of the present invention. Bacteria of the family Selenomonadaceae are involved in propionic acid production in the rumen of ruminants, and increasing the proportion of bacteria of the family Selenomonadaceae among the bacteria constituting the rumen bacterial flora can change the bacterial flora to promote propionic acid production. Such a change in the rumen bacterial flora reduces hydrogen available for methane synthesis, suppressing methane production in the rumen and thereby suppressing methane emission by ruminants. The method for evaluating the effect of the feed and methane emission inhibitor of the present invention on increasing the proportion of bacteria of the family Selenomonadaceae among the bacteria that constitute the rumen bacterial flora of ruminants is not particularly limited, and may be performed by a method known per se or a method equivalent thereto, but for example, as in the Examples described below, evaluation can be performed by performing a culture test using rumen fluid (gastric fluid) collected from a ruminant that has been fed the feed, etc. of the present invention as an inoculum, and analyzing the bacterial flora of the rumen fluid, etc. Analysis of the bacterial flora (measurement of bacterial count, etc.) can be performed by a method known per se (e.g., quantitative PCR, amplicon sequencing, etc.) or a method equivalent thereto.

[0054] Bacteria that can exert an effect similar to that of the above-mentioned Selenomonadaceae bacteria include bacteria of the Prevotellaceae family and Bacteroidales RF-16 group family belonging to the Bacteroidota phylum, bacteria of the Succinivibrionaceae family belonging to the Proteobacteria phylum, bacteria of the Ruminococceae family belonging to the Firmicutes phylum, and bacteria of the Fibrobacteraceae family belonging to the Fibrobacterota phylum. By increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) among the bacteria constituting the rumen bacterial flora, the bacterial flora can be altered to promote propionic acid production. Such an alteration of the rumen bacterial flora reduces hydrogen available for methane synthesis, and as described above, methane production in the rumen is suppressed, and methane emission by ruminants can be suppressed.

[0055] In one embodiment, the feed of the present invention may have a stronger effect of increasing the proportion of bacteria of the family Lachnospiraceae of the phylum Firmicutes among the bacteria constituting the rumen bacterial flora of ruminants than the methane emission inhibitor contained in the feed of the present invention. Bacteria of the family Lachnospiraceae are involved in butyric acid production in the rumen of ruminants, and increasing the proportion of bacteria of the family Lachnospiraceae among the bacteria constituting the rumen bacterial flora can change the bacterial flora to promote butyric acid production. Such a change in the rumen bacterial flora reduces hydrogen available for methane synthesis, suppressing methane production in the rumen and thereby suppressing methane emission by ruminants. The method for evaluating the effect of the feed and methane emission inhibitor of the present invention on increasing the proportion of bacteria of the family Lachnospiraceae in the bacteria that constitute the rumen bacterial flora of ruminants is not particularly limited, and may be performed by a method known per se or a method equivalent thereto. For example, as in the Examples described below, evaluation can be performed by performing a culture test using rumen fluid (gastric fluid) collected from a ruminant that has been fed the feed, etc. of the present invention as an inoculum, and analyzing the bacterial flora of the rumen fluid. Analysis of the bacterial flora (measurement of bacterial count, etc.) can be performed by a method known per se (e.g., quantitative PCR, amplicon sequencing, etc.) or a method equivalent thereto.

[0056] Examples of bacteria that can exert an effect similar to that of the above-mentioned Lachnospiraceae family bacteria include bacteria of the Prevotellaceae family and Bacteroidales RF-16 group family belonging to the Bacteroidota phylum, bacteria of the Succinivibrionaceae family belonging to the Proteobacteria phylum, bacteria of the Ruminococcaeaceae family belonging to the Firmicutes phylum, and bacteria of the Fibrobacteraceae family belonging to the Fibrobacterota phylum. By increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) in the bacteria constituting the rumen bacterial flora, the bacterial flora can be altered to promote butyric acid production. Such an alteration of the rumen bacterial flora reduces hydrogen available for methane synthesis, and as described above, methane production in the rumen is suppressed, and methane emission by ruminants can be suppressed.

[0057] In one embodiment, the feed of the present invention may have a stronger effect of increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) in the bacteria constituting the rumen bacterial flora of ruminants than the methane emission inhibitor contained in the feed of the present invention. The feed and methane emission inhibitor of the present invention are effective against Bacteroidota bacteria (e.g., Prevotellaceae bacteria, Bacteroidales RF-16) which are bacteria constituting the rumen flora of ruminants. The method for evaluating the effect of increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae) is not particularly limited, and may be evaluated by a method known per se or a method equivalent thereto. For example, as in the Examples described below, evaluation can be performed by performing a culture test using rumen fluid (gastric fluid) collected from a ruminant fed the feed of the present invention as an inoculum, and analyzing the bacterial flora of the rumen fluid. Analysis of the bacterial flora (measurement of bacterial count, etc.) can be performed by a method known per se (e.g., quantitative PCR, amplicon sequencing, etc.) or a method equivalent thereto.

[0058] The present invention also provides a method for raising ruminants (sometimes referred to herein as the "raising method of the present invention"). The raising method of the present invention is characterized by feeding the feed of the present invention to ruminants. The raising method of the present invention is not particularly limited other than that it includes feeding the feed of the present invention to ruminants, and may further include other steps (for example, normal ruminant raising steps, etc.) as long as the object of the present invention is not impaired. According to the raising method of the present invention, ruminants can be raised while suppressing methane emission by the ruminants.

[0059] The present invention also provides a method for suppressing methane emission by ruminants (sometimes referred to herein as the "suppression method of the present invention"). The suppression method of the present invention is characterized by feeding a feed of the present invention to a ruminant. The suppression method of the present invention is not particularly limited other than that it includes feeding a feed of the present invention to a ruminant, and may further include other steps as long as the object of the present invention is not impaired. For example, the suppression method of the present invention may be performed in combination with another method for suppressing methane emission that is different from the suppression method of the present invention.

[0060] The present invention also provides a method for improving the effectiveness of a methane emission inhibitor (sometimes referred to herein as the "improvement method of the present invention"). The improvement method of the present invention may comprise adding RPAA to feed for a ruminant that is fed the methane emission inhibitor (i.e., feed fed to a ruminant).

[0061] The RPAA (i.e., rumen-protected amino acids) that can be used in the enhancement method of the present invention may be the same as those contained in the feed of the present invention described above, and the preferred embodiments are also the same. Furthermore, the methane emission inhibitor to which the enhancement method of the present invention can be applied (i.e., the methane emission inhibitor whose effect can be improved by the enhancement method of the present invention) may be the same as those contained in the feed of the present invention described above, and the preferred embodiments are also the same.

[0062] The amount of RPAA that can be used in the improvement method of the present invention (i.e., the amount of RPAA added to feed) can be set appropriately taking into consideration the type of amino acids contained in RPAA, the type, use, breed, weight, growth stage, and age at parturition of the ruminant to which the methane emission inhibitor is fed, etc., but in one embodiment, when the amino acids contained in RPAA include essential amino acids for the ruminant to which the methane emission inhibitor is fed, the amount of RPAA that can be used in the improvement method of the present invention may be set taking into consideration the essential amino acid requirement of the ruminant to which the methane emission inhibitor is fed.

[0063] The amount of RPAA that can be used in the improvement method of the present invention will be described below using an example in which the ruminant fed the methane emission inhibitor is a cattle and the amino acids contained in the RPAA include essential amino acids for cattle. In this case, the amount of RPAA that can be used in the improvement method of the present invention can be set so that the feed (the feed to which RPAA is added) satisfies 80% or more (preferably 90-500%, more preferably 100-150%) of the cattle's essential amino acid requirement (the required amount of essential amino acids contained in RPAA). In one embodiment, when the RPAA that can be used in the improvement method of the present invention contains lysine, the amount of RPAA that can be used in the improvement method of the present invention can be set so that the feed satisfies 80% or more (preferably 90-500%, more preferably 100-150%) of the cattle's lysine requirement. Furthermore, as described below, when the improvement method of the present invention involves adjusting the crude protein content of feed for ruminants fed with a methane emission inhibitor to reduce it, the amount of RPAA that can be used in the improvement method of the present invention can be set to compensate for the resulting reduction in the feed's sufficiency for the ruminant's essential amino acid requirement. In other words, when the improvement method of the present invention involves adjusting the crude protein content of feed for ruminants fed with a methane emission inhibitor to reduce it, the amount of RPAA that can be used in the improvement method of the present invention can be an amount that maintains the sufficiency for the essential amino acid requirement (e.g., lysine requirement) before adjusting the crude protein content.

[0064] The method for improving protein content of the present invention may include adjusting the crude protein content of feed for ruminants to which the methane emission inhibitor is administered so as to reduce it. The crude protein content may be reduced by 0.1 to 10% DM, by 0.2 to 7.5%, by 0.5 to 5%, or by 1 to 3% DM. The method for improving protein content of the present invention may include adjusting the crude protein content of feed for ruminants to which the methane emission inhibitor is administered in the same manner as the crude protein content of the feed of the present invention described above.

[0065] The method for improving methane emission of the present invention may involve adjusting the RDP content (i.e., the rumen-degradable protein content) and the MP content from BP (i.e., the content of metabolic proteins from bacterial proteins) of feed for ruminants fed with a methane emission inhibitor in the same manner as the RDP content and the MP content from BP of the feed of the present invention described above, respectively.

[0066] The improvement method of the present invention can improve the effect of a methane emission inhibitor. For example, the improvement method of the present invention can improve the inhibitory effect on methane emission by ruminants (e.g., cattle, etc.). Furthermore, the improvement method of the present invention can improve the effect of increasing the propionic acid concentration in the rumen of ruminants (e.g., cattle, etc.) and the effect of increasing the proportion of bacteria of the family Selenomonadaceae among the bacteria that constitute the ruminal bacterial flora of ruminants (e.g., cattle, etc.). The method for evaluating these effects is not particularly limited, and may be evaluated by a method known per se or a method equivalent thereto. For example, the effect may be evaluated by the same method as that for the feed of the present invention described above.

[0067] The improvement method of the present invention can improve the effect of increasing the butyric acid concentration in the rumen of a ruminant (e.g., cows, etc.), the effect of decreasing the acetic acid concentration in the rumen of a ruminant (e.g., cows, etc.), and the effect of increasing the proportion of Lachnospiraceae bacteria in the bacteria that make up the ruminal bacterial flora of a ruminant (e.g., cows, etc.). Furthermore, the improvement method of the present invention can improve the effect of increasing the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) in the bacteria constituting the rumen bacterial flora of ruminants (e.g., cattle, etc.). The method for evaluating these effects is not particularly limited, and evaluation may be performed by a method known per se or a method equivalent thereto, and for example, evaluation may be performed by the same method as for the feed of the present invention described above.

[0068] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples. In this specification, "%" means "% by weight" unless otherwise specified.

[0069] The 3-nitrooxypropanol reagent used in the following test examples was manufactured by MedChemExpress USA, and the tannic acid, sodium nitrate and bromoform reagents were all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.

[0070] <Test Example 1> Feeding test Four Holstein cows (dry cows, average weight 815±36 kg) were divided into two groups of two cows each, and each group was fed a low-protein feed (Feed A) containing a bovine preparation containing rumen-protected lysine ("AjiPro (registered trademark)-L", manufactured by Ajinomoto Co., Inc.) or a control feed (Feed C) every day for three weeks. After three weeks of feeding, gastric juice was orally collected from the four cows. After gastric juice collection, the feeds fed to the two groups were interchanged, and the two groups were again fed every day for three weeks. After three weeks of feeding, gastric juice was again orally collected from the four cows. The collected gastric juice was used in the culture test described below.

[0071] The feed compositions of Feeds A and C are shown in Table 1. The component values ​​of Feeds A and C are also shown in Table 2. The component values ​​shown in Table 2 were obtained by multiplying the component values ​​obtained by measuring the components of each feed ingredient shown in Table 1 using near-infrared spectroscopy (NIR) or wet chemical analysis, or the component values ​​calculated based on the feed component table included in the AMTS feed design software, according to the composition shown in Table 1. The energy content of Feeds A and C was adjusted to approximately 1.3 times the value recommended by the Japanese Feeding Standards (calculated based on a cow's body weight of 800 kg). The crude protein content of Feed A ("CP" in Table 2) was approximately 4% DM less than that of Feed C. A bovine preparation containing rumen-protected lysine (AjiPro®-L) was added to Feed A so that the lysine requirement fulfillment rate was similar to that of Feed C (both Feeds A and C fulfilled more than 100% of the cow's lysine requirement).

[0072]

[0073]

[0074] The component values ​​listed in Table 2 have the following meanings: CP: Crude Protein RDP: Rumen Degradable Protein ME: Metabolizable Energy MP: Metabolizable Protein MP from Bacterium: Metabolizable Protein from Bacterial Protein MP from RUP: Metabolizable Protein from Rumen Undegradable Protein aNDFom: Thermostable α-amylase-treated neutral detergent fiber (amylase neutral detergent fiber organic matter) Met: Methionine Lys: Lysine Here, ME, MP, MP from Bact, MP from RUP, Met, and Lys are each amounts per 9.6 kg of feed (approximate daily intake (dry matter) of a cow).

[0075] Culture Test: Equal amounts of 25 mL of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 3 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 3 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. Six and 24 hours after the end of the culture, the gas generated in the vials was collected using a syringe, and the combined samples were used to measure the total gas generation and the amount of propionic acid generation. The total amount of gas generated was measured using a gas chromatograph, and the amount of propionic acid generated was measured using a high performance liquid chromatograph (HPLC). The measurement results are shown in Table 4. In Table 4, the amount of propionic acid generated is shown as a relative amount of propionic acid generated, with the amount of propionic acid generated in a sample of the same feed to which no methane emission inhibitor had been added being set at 100%.

[0076]

[0077]

[0078] As shown in Table 4, the amount of propionic acid generated was greater in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol, plant extract) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0079] <Test Example 2> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0080] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 5 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 5 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial mass of the Selenomonadaceae family bacteria (the proportion of Selenomonadaceae bacteria in the bacteria constituting the bacterial flora) was calculated from the obtained analysis results. The results are shown in Table 6. In Table 6, the bacterial mass of the Selenomonadaceae family bacteria is shown as a relative bacterial mass of the Selenomonadaceae family, with the bacterial mass of the Selenomonadaceae family bacteria in a sample of the same feed to which no methane emission inhibitor had been added being set at 100%.

[0081]

[0082]

[0083] As shown in Table 6, the relative bacterial count of Selenomonadaceae bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (cashew nut shell liquid, essential oil, 3-nitrooxypropanol, plant extract, sodium nitrate) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0084] <Test Example 3> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0085] Culture Test: Equal amounts of 25 mL of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 7 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 7 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these ingredients, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. Six and 24 hours after the start of culture, the amount of gas generated in the vials was measured and collected using a syringe, and the amount of methane generated was measured using a gas chromatograph. The measurement results are shown in Table 8. The amount of methane generated shown in Table 8 is the sum of the amount of methane generated during 6 hours of culture and the amount of methane generated during the subsequent 18 hours of culture. In Table 8, the amount of methane generated is shown as a relative amount of methane generated, with the amount of methane generated from a sample to which no methane emission inhibitor was added being set at 100%.

[0086]

[0087]

[0088] As shown in Table 8, the amount of methane generated was lower in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C to which methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol, plant extract) were added. Methane generation was synergistically suppressed in the sample of gastric juice collected from cattle fed Feed A to which the methane emission inhibitors were added.

[0089] Test Example 4 Feeding Test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0090] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 9 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 9 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these ingredients, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. Six and 24 hours after the end of the culture, the gas generated in the vials was collected using a syringe, and the combined samples were used to measure the total gas and butyric acid production. The total gas production was measured using a gas chromatograph, and the butyric acid production was measured using a high-performance liquid chromatograph (HPLC). The measurement results are shown in Table 10. In Table 10, the amount of butyric acid generated is shown as a relative amount of butyric acid generated, with the amount of butyric acid generated in a sample to which no methane emission inhibitor was added being taken as 100%.

[0091]

[0092]

[0093] As shown in Table 10, the amount of butyric acid produced was greater in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol, sodium nitrate, plant extract) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0094] <Test Example 5> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0095] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 11 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 11 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. Six and 24 hours after the end of culture, the gas generated in the vials was collected using a syringe, and the collected samples were combined to measure the total gas and acetic acid production. The total gas production was measured using a gas chromatograph, and the acetic acid production was measured using a high-performance liquid chromatograph (HPLC). The measurement results are shown in Table 12. In Table 12, the amount of acetic acid generated is shown as a relative amount of acetic acid generated, with the amount of acetic acid generated from a sample to which no methane emission inhibitor was added being set at 100%.

[0096]

[0097]

[0098] As shown in Table 12, the amount of acetic acid produced was lower in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (cashew nut shell liquid, essential oil) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0099] <Test Example 6> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0100] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 13 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 13 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial load of Bacteroidota bacteria (the proportion of Bacteroidota bacteria in the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 14. Table 14 shows the relative bacterial load of Bacteroidota bacteria, with the bacterial load of Bacteroidota bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0101]

[0102]

[0103] As shown in Table 14, the relative abundance of Bacteroidota bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (essential oil, 3-nitrooxypropanol) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0104] <Test Example 7> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0105] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 15 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 15 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial mass of Proteobacteria (the proportion of Proteobacteria bacteria among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 16. Table 16 shows the relative bacterial mass of Proteobacteria, with the bacterial mass of Proteobacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0106]

[0107]

[0108] As shown in Table 16, the relative abundance of Proteobacteria bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C to which methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol, plant extract, sodium nitrate) were added.

[0109] <Test Example 8> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0110] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 17 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 17 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial mass of Firmicutes bacteria calculated from the measured bacterial flora analysis (the proportion of Firmicutes bacteria in the bacteria constituting the bacterial flora) is shown in Table 18. Table 18 shows the relative bacterial mass of Firmicutes bacteria, with the bacterial mass of Firmicutes bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0111]

[0112]

[0113] As shown in Table 18, the relative bacterial count of Firmicutes bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C to which methane emission inhibitors (cashew nut shell liquid, essential oil, 3-nitrooxypropanol, plant extract, sodium nitrate) were added.

[0114] <Test Example 9> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0115] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 19 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 19 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial load of Fibrobacterota bacteria (the proportion of Fibrobacterota bacteria among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 20. Table 20 shows the relative bacterial load of Fibrobacterota bacteria, with the bacterial load of Fibrobacterota bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0116]

[0117]

[0118] As shown in Table 20, the relative bacterial count of Fibrobacterota bacteria was higher in samples of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (tannic acid, sodium nitrate) were added, compared to samples of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0119] <Test Example 10> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0120] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) made from crushed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 21 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 21 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Prevotellaceae family bacteria calculated from the measured bacterial flora analysis (the proportion of bacteria of the Prevotellaceae family among the bacteria constituting the bacterial flora) is shown in Table 22. Table 22 shows the relative bacterial count of the Prevotellaceae family bacteria, with the bacterial count of the Prevotellaceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0121]

[0122]

[0123] As shown in Table 22, the relative bacterial count of Prevotellaceae family bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (essential oil, 3-nitrooxypropanol) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0124] <Test Example 11> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0125] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 23 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 23 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Bacteroidales RF-16 group family bacteria calculated from the measured bacterial flora analysis (the proportion of bacteria of the Bacteroidales RF-16 group family among the bacteria constituting the bacterial flora) is shown in Table 24. Table 24 shows the relative bacterial count of the Bacteroidales RF-16 group family bacteria, with the bacterial count of the Bacteroidales RF-16 group family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0126]

[0127]

[0128] As shown in Table 24, the relative bacterial count of Bacteroidales RF-16 group bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C to which methane emission inhibitors (cashew nut shell liquid, essential oil) were added.

[0129] <Test Example 12> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0130] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 25 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 25 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Succinivibrionaceae family bacteria calculated from the measured bacterial flora analysis (the proportion of Succinivibrionaceae family bacteria in the bacteria constituting the bacterial flora) is shown in Table 26. Table 26 shows the relative bacterial count of the Succinivibrionaceae family bacteria, with the bacterial count of the Succinivibrionaceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0131]

[0132]

[0133] As shown in Table 26, compared to samples of gastric juice collected from cattle fed Feed C to which methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol, plant extract, sodium nitrate) had been added, samples of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors had been added had a higher relative bacterial count of bacteria in the family Succinivibrionaceae.

[0134] <Test Example 13> Feeding test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0135] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 27 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 27 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Lachnospiraceae family bacteria calculated from the measured bacterial flora analysis (the proportion of Lachnospiraceae bacteria in the bacteria constituting the bacterial flora) is shown in Table 28. Table 28 shows the relative bacterial count of the Lachnospiraceae family bacteria, with the bacterial count of the Lachnospiraceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0136]

[0137]

[0138] As shown in Table 28, the relative bacterial count of Lachnospiraceae family bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C to which methane emission inhibitors (cashew nut shell liquid, essential oil, 3-nitrooxypropanol, sodium nitrate) were added.

[0139] Test Example 14 Feeding Test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0140] Culture Test: Equal amounts of 25 mL of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 29 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 29 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the family Ruminococcaceae (the proportion of bacteria of the family Ruminococcaceae among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 30. Table 30 also shows the relative bacterial count of the family Ruminococcaceae, with the bacterial count of the family Ruminococcaceae in the sample to which no methane emission inhibitor was added being set at 100%.

[0141]

[0142]

[0143] As shown in Table 30, the relative abundance of bacteria in the family Ruminococcaceae was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (cashew nut shell liquid, essential oil, plant extract, sodium nitrate) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0144] Test Example 15 Feeding Test A feeding test was carried out in the same manner as in Test Example 1. The collected gastric juice was used in the culture test described below.

[0145] Culture Test: Equal amounts of 25 mL of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the feed (Feed A or Feed C) and each methane emission inhibitor listed in Table 31 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 31 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Fibrobacteraceae family (the proportion of bacteria of the Fibrobacteraceae family among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 32. Table 32 shows the relative bacterial count of the Fibrobacteraceae family, with the bacterial count of the Fibrobacteraceae family in the sample to which no methane emission inhibitor was added being set at 100%.

[0146]

[0147]

[0148] As shown in Table 32, the relative bacterial count of Fibrobacteraceae family bacteria was higher in the sample of gastric juice collected from cattle fed Feed A to which the same methane emission inhibitors (tannic acid, sodium nitrate) were added, compared to the sample of gastric juice collected from cattle fed Feed C to which the same methane emission inhibitors were added.

[0149] Test Example 16 Feeding Test Six Holstein cows (lactating dairy cows) were fed a low-protein feed (Feed A1) containing a bovine preparation containing rumen-protected lysine ("AjiPro (registered trademark)-L", manufactured by Ajinomoto Co., Inc.) every day for three consecutive weeks. After three weeks of feeding, gastric juice was orally collected from the six cows. After gastric juice collection, the cows were fed a control feed (Feed C1) every day for three consecutive weeks. After three weeks of feeding, gastric juice was again orally collected from the six cows. The collected gastric juice was used in the culture test described below.

[0150] The feed compositions of Feed A1 and Feed C1 are shown in Table 33. The component values ​​of Feed A1 and Feed C1 are also shown in Table 34. The component values ​​shown in Table 34 were obtained by integrating the component values ​​obtained by measuring the components of each feed ingredient shown in Table 33 using near-infrared spectroscopy (NIR) or wet chemical analysis, or the component values ​​calculated based on the feed composition table included in the AMTS feed design software, according to the composition shown in Table 33. The crude protein content of Feed A1 ("CP" in Table 34) was set to about 1.5% DM less than that of Feed C1. A bovine preparation containing rumen-protected lysine (AjiPro (registered trademark)-L) was added to Feed A1 so that the lysine requirement fulfillment rate was similar to that of Feed C1.

[0151]

[0152]

[0153] The component values ​​listed in Table 34 have the following meanings: CP: Crude Protein RDP: Rumen Degradable Protein ME: Metabolizable Energy MP: Metabolizable Protein MP from Bacterium: Metabolizable Protein from Bacterial Protein MP from RUP: Metabolizable Protein from Rumen Undegradable Protein aNDFom: Thermostable α-amylase-treated neutral detergent fiber (amylase neutral detergent fiber organic matter) Met: Methionine Lys: Lysine Here, ME, MP, MP from Bact, MP from RUP, Met, and Lys are each amounts per 27 kg of feed (approximate daily intake (dry matter) of a cow).

[0154] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) made from crushed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 35 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 35 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as vials without these, were sealed and placed in an incubator, where they were anaerobic cultured at 39°C for 24 hours with shaking. Six and 24 hours after the end of the culture, the gas generated in the vials was collected using a syringe, and the combined samples were used to measure the total gas generation and the amount of propionic acid generation. The total amount of gas generated was measured using a gas chromatograph, and the amount of propionic acid generated was measured using a high performance liquid chromatograph (HPLC). The measurement results are shown in Table 36. In Table 36, the amount of propionic acid generated is shown as a relative amount of propionic acid generated, with the amount of propionic acid generated from a sample to which no methane emission inhibitor was added being set at 100%.

[0155]

[0156]

[0157] As shown in Table 36, the amount of propionic acid generated was greater in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol) were added, compared to the sample of gastric juice collected from cattle fed Feed C1 to which the same methane emission inhibitors were added.

[0158] Test Example 17 Feeding Test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0159] Culture Test: Equal amounts of 25 mL of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in 100 mL vials, and a substrate (0.5 g per sample) prepared by pulverizing the feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 37 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 37 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. Six and 24 hours after the end of the culture, the gas generated in the vials was collected using a syringe, and the combined samples were used to measure the total gas generation and the amount of butyric acid generation. The total amount of gas generated was measured using a gas chromatograph, and the amount of butyric acid generated was measured using a high performance liquid chromatograph (HPLC). The measurement results are shown in Table 38. In Table 38, the amount of butyric acid generated is shown as a relative amount of butyric acid generated, with the amount of butyric acid generated in a sample to which no methane emission inhibitor was added being taken as 100%.

[0160]

[0161]

[0162] As shown in Table 38, the amount of butyric acid produced was greater in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (cashew nut shell liquid, essential oil) were added, compared to the sample of gastric juice collected from cattle fed Feed C1 to which the same methane emission inhibitors were added.

[0163] <Test Example 18> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0164] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) prepared by pulverizing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 39 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 39 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. Six and 24 hours after the end of culture, the gas generated in the vials was collected using a syringe, and the collected samples were combined to measure the total gas and acetic acid production. The total gas production was measured using a gas chromatograph, and the acetic acid production was measured using a high-performance liquid chromatograph (HPLC). The measurement results are shown in Table 40. In Table 40, the amount of acetic acid generated is shown as a relative amount of acetic acid generated, with the amount of acetic acid generated in a sample to which no methane emission inhibitor was added being taken as 100%.

[0165]

[0166]

[0167] As shown in Table 40, the amount of acetic acid produced was lower in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (essential oil, tannic acid) were added, compared to the sample of gastric juice collected from cattle fed Feed C1 to which the same methane emission inhibitors were added.

[0168] <Test Example 19> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0169] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) made from crushed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 41 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 41 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Selenomonadaceae family bacteria (the proportion of Selenomonadaceae bacteria in the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 42. Table 42 shows the relative bacterial count of the Selenomonadaceae family bacteria, with the bacterial count of the Selenomonadaceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0170]

[0171]

[0172] As shown in Table 42, the relative bacterial count of Selenomonadaceae bacteria was higher in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C1 to which methane emission inhibitors (cashew nut shell liquid, 3-nitrooxypropanol, tannic acid) were added.

[0173] <Test Example 20> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used for the culture test described below.

[0174] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 43 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 43 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial load of Bacteroidota bacteria (the proportion of Bacteroidota bacteria among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 44. Table 44 shows the relative bacterial load of Bacteroidota bacteria, with the bacterial load of Bacteroidota bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0175]

[0176]

[0177] As shown in Table 44, the relative bacterial count of Bacteroidota bacteria was higher in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (cashew nut shell liquid, essential oil, 3-nitrooxypropanol) were added compared to the sample of gastric juice collected from cattle fed Feed C1.

[0178] <Test Example 21> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0179] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 45 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 45 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial mass of Proteobacteria (the proportion of Proteobacteria bacteria among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 46. Table 46 also shows the relative bacterial mass of Proteobacteria, with the bacterial mass of Proteobacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0180]

[0181]

[0182] As shown in Table 46, the relative abundance of Proteobacteria bacteria was higher in samples of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (tannic acid, 3-nitrooxypropanol, bromoform) were added compared to samples of gastric juice collected from cattle fed Feed C1.

[0183] <Test Example 22> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0184] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 47 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 47 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial load of Fibrobacterota bacteria (the proportion of Fibrobacterota bacteria among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 48. Table 48 shows the relative bacterial load of Fibrobacterota bacteria, with the bacterial load of Fibrobacterota bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0185]

[0186]

[0187] As shown in Table 48, the relative bacterial count of Fibrobacterota bacteria was higher in samples of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (cashew nut shell liquid, tannic acid, bromoform) were added compared to samples of gastric juice collected from cattle fed Feed C1.

[0188] <Test Example 23> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0189] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 49 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 49 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Prevotellaceae family bacteria calculated from the measured bacterial flora analysis (the proportion of bacteria of the Prevotellaceae family in the bacteria constituting the bacterial flora) is shown in Table 50. Table 50 also shows the relative bacterial count of the Prevotellaceae family, with the bacterial count of the Prevotellaceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0190]

[0191]

[0192] As shown in Table 50, the relative bacterial count of Prevotellaceae family bacteria was higher in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors were added, compared to the sample of gastric juice collected from cattle fed Feed C1 to which methane emission inhibitors (cashew nut shell liquid, essential oil, 3-nitrooxypropanol) were added.

[0193] Test Example 24 Feeding Test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0194] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 51 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 51 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Bacteroidales RF-16 group family bacteria calculated from the measured bacterial flora analysis (the proportion of bacteria of the Bacteroidales RF-16 group family among the bacteria constituting the bacterial flora) is shown in Table 52. Table 52 shows the relative bacterial count of the Bacteroidales RF-16 group family bacteria, with the bacterial count of the Bacteroidales RF-16 group family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0195]

[0196]

[0197] As shown in Table 52, compared to samples of gastric juice collected from cattle fed Feed C1 to which methane emission inhibitors (cashew nut shell liquid, essential oil, tannic acid, 3-nitrooxypropanol, bromoform) had been added, samples of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors had been added had a higher relative abundance of bacteria of the Bacteroidales RF-16 group.

[0198] Test Example 25 Feeding Test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0199] Culture Test: Equal amounts of 25 mL of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in 100 mL vials. Substrates (0.5 g per sample) prepared by pulverizing the feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 53 were added. The methane emission inhibitors were added in amounts that achieved the concentrations shown in Table 53 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting cultures were then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Succinivibrionaceae family bacteria calculated from the measured bacterial flora analysis (the proportion of Succinivibrionaceae family bacteria in the bacteria constituting the bacterial flora) is shown in Table 54. Table 54 shows the relative bacterial count of the Succinivibrionaceae family bacteria, with the bacterial count of the Succinivibrionaceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0200]

[0201]

[0202] As shown in Table 54, the relative abundance of Succinivibrionaceae bacteria was higher in samples of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (tannic acid, 3-nitrooxypropanol, bromoform) were added compared to samples of gastric juice collected from cattle fed Feed C1.

[0203] <Test Example 26> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used for the culture test described below.

[0204] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 55 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 55 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator. Anaerobically cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Lachnospiraceae family bacteria calculated from the measured bacterial flora analysis (the proportion of Lachnospiraceae bacteria in the bacteria constituting the bacterial flora) is shown in Table 56. Table 56 shows the relative bacterial count of the Lachnospiraceae family bacteria, with the bacterial count of the Lachnospiraceae family bacteria in the sample to which no methane emission inhibitor was added being set at 100%.

[0205]

[0206]

[0207] As shown in Table 56, the relative bacterial count of Lachnospiraceae bacteria was higher in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitor (bromoform) was added, compared to the sample of gastric juice collected from cattle fed Feed C1 to which the same methane emission inhibitor was added.

[0208] Test Example 27 Feeding Test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0209] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 57 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 57 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The amount of bacteria of the family Ruminococcaceae (the proportion of bacteria of the family Ruminococcaceae among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 58. Table 58 shows the amount of bacteria of the family Ruminococcaceae relative to the amount of bacteria of the family Ruminococcaceae in the sample to which no methane emission inhibitor was added, which is set to 100%.

[0210]

[0211]

[0212] As shown in Table 58, the relative abundance of Ruminococcaceae bacteria was higher in samples of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (essential oil, 3-nitrooxypropanol) had been added, compared to samples of gastric juice collected from cattle fed Feed C1.

[0213] <Test Example 28> Feeding test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0214] Culture Test: Gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed in equal amounts (25 mL per sample) in 100 mL vials, and a substrate (0.5 g per sample) prepared by crushing the fed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 59 were added. The methane emission inhibitor was added in an amount that achieved the concentration shown in Table 59 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as the vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. The resulting culture solution was then used for bacterial flora analysis. The bacterial flora analysis was performed using amplicon sequencing targeting the bacterial 16S ribosomal RNA gene. The bacterial count of the Fibrobacteraceae family (the proportion of bacteria of the Fibrobacteraceae family among the bacteria constituting the bacterial flora) calculated from the measured bacterial flora analysis is shown in Table 60. Table 60 shows the relative bacterial count of the Fibrobacteraceae family, with the bacterial count of the Fibrobacteraceae family in the sample to which no methane emission inhibitor was added being set at 100%.

[0215]

[0216]

[0217] As shown in Table 60, the relative bacterial count of Fibrobacteraceae family bacteria was higher in the sample of gastric juice collected from cattle fed Feed A1 to which the same methane emission inhibitors (cashew nut shell liquid, tannic acid, bromoform) were added compared to the sample of gastric juice collected from cattle fed Feed C1 to which the same methane emission inhibitors were added.

[0218] Test Example 29 Feeding Test A feeding test was carried out in the same manner as in Test Example 16. The collected gastric juice was used in the culture test described below.

[0219] Culture Test: Using 100 mL vials, equal amounts of gastric juice collected in the feeding test (described above) and McDougall's buffer (McDougall, 1948) were mixed at 25 mL per sample, and a substrate (0.5 g per sample) made from crushed feed (Feed A1 or Feed C1) and each methane emission inhibitor listed in Table 61 were added. The methane emission inhibitor was added in an amount that would result in the concentration shown in Table 61 relative to the substrate. The vials containing the substrate and each methane emission inhibitor, as well as vials without these, were sealed and placed in an incubator and anaerobic cultured at 39°C for 24 hours with shaking. Six and 24 hours after the start of culture, the amount of gas generated in the vials was measured and collected using a syringe, and the amount of methane generated was measured using a gas chromatograph. The measurement results are shown in Table 62. The amount of methane generated shown in Table 62 is the sum of the amount of methane generated during 6 hours of culture and the amount of methane generated during the subsequent 18 hours of culture. In Table 62, the amount of methane generated is shown as a relative amount of methane generated, with the amount of methane generated from a sample to which no methane emission inhibitor was added being set at 100%.

[0220]

[0221]

[0222] As shown in Table 62, the amount of methane generated was lower in samples of gastric juice collected from cattle fed Feed A1 to which the same methane output inhibitors (tannic acid, 3-nitrooxypropanol, bromoform) were added, compared to samples of gastric juice collected from cattle fed Feed C1 to which the same methane output inhibitors were added. Methane generation was synergistically suppressed in samples of gastric juice collected from cattle fed Feed A1 to which the methane output inhibitors were added.

[0223] The results of Test Examples 1 to 29 suggest that methane emission from ruminants can be effectively suppressed by feeding the ruminants feed containing a combination of a methane emission inhibitor and RPAA.

[0224] According to the present invention, a feed capable of effectively suppressing methane emission by ruminants can be provided. Furthermore, according to the present invention, a method for raising ruminants can be provided, which enables ruminants to be raised while suppressing methane emission by ruminants. Furthermore, according to the present invention, a method for suppressing methane emission by ruminants can be provided. Furthermore, according to the present invention, a feed capable of increasing the propionic acid concentration in the rumen of a ruminant can be provided. The present invention can provide a feed capable of increasing the proportion of bacteria of the family Selenomonadaceae among the bacteria constituting the rumen bacterial flora of a ruminant. Furthermore, according to the present invention, a feed capable of increasing the butyric acid concentration in the rumen of a ruminant can be provided. The present invention can provide a feed capable of decreasing the acetic acid concentration in the rumen of a ruminant can be provided. The present invention can provide a feed capable of increasing the proportion of bacteria of the family Lachnospiraceae among the bacteria constituting the rumen bacterial flora of a ruminant. Furthermore, according to the present invention, it is possible to provide a feed that can increase the proportion of at least one selected from the group consisting of bacteria of the phylum Bacteroidota (e.g., bacteria of the family Prevotellaceae, bacteria of the family Bacteroidales RF-16 group, etc.), bacteria of the phylum Proteobacteria (e.g., bacteria of the family Succinivibrionaceae, etc.), bacteria of the phylum Firmicutes (e.g., bacteria of the family Ruminococceae, etc.), and bacteria of the phylum Fibrobacterota (e.g., bacteria of the family Fibrobacteraceae, etc.) in the bacteria that constitute the rumen bacterial flora of ruminants.

[0225] This application is based on patent application No. 2023-219983 filed in Japan (filing date: December 26, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A feed for ruminants, comprising a methane emission inhibitor and a rumen-protected amino acid.

2. The feed according to claim 1, which has a high effect of increasing the concentration of propionic acid in the rumen of ruminants compared to the methane emission inhibitor.

3. The feed according to claim 1, which has a high effect of increasing the proportion of bacteria of the family Selenomonadaceae in the bacteria constituting the rumen microflora of ruminants compared to the methane emission inhibitor.

4. The feed according to claim 1, wherein the amino acid contains essential amino acids.

5. The feed according to claim 1, wherein the amino acid contains at least one selected from the group consisting of lysine, methionine, arginine, and histidine.

6. The feed according to claim 1, wherein the methane emission inhibitor contains at least one selected from the group consisting of an organic compound having a nitrooxy group, cashew nut shell liquid, polyphenol, nitrate, essential oil, plant extract, methane halide analog, calcium fatty acid, saponin, and organic acid.

7. The feed according to claim 1, wherein the methane emission inhibitor contains at least one selected from the group consisting of 3-nitrooxypropanol, cashew nut shell liquid, tannic acid, essential oil, plant extract, and bromoform.

8. The feed according to claim 1, wherein the crude protein content of the feed is 0.1 to 10% DM lower than the crude protein content of the feed used until the start of feeding of the feed.

9. The feed according to claim 1, wherein the crude protein content is 10 to 20% DM.

10. A method for raising ruminants, comprising feeding the ruminants with the feed according to any one of claims 1 to 9.

11. A method for suppressing methane emission by ruminants, comprising feeding the ruminants with the feed according to any one of claims 1 to 9.