Feeding regime to reduce methane emissions in adult ruminants

Abstract

Nutrition for early life. The present invention relates to a method for reducing methane emissions in ruminants, said method encompassing the treatment of young pre- / ruminants with 3-nitrooxypropanol.

Description

1 / 23 Feeding regimen for reducing methane emissions in adult ruminants. Brief description of the invention

[001] The present invention relates to a method for reducing methane emissions in ruminants, said method encompassing the treatment of pre- / young ruminants with 3-nitrooxypropanol. Background of the invention

[002] It is estimated that livestock production is responsible for approximately 14% of anthropogenic methane released annually into the atmosphere, with the majority formed in ruminants as a result of the activity of ruminal methanogens (archaea) in the rumen of the host animal during enteric fermentation, leading to the formation of methane that is eructated by the animal. Not only is methane a potent greenhouse gas (GHG) and a problem for the environment, but it also represents a loss of energy for the host, with estimated losses ranging from 2 to 12% of the gross energy intake of the diet.

[003] Currently, there are significant efforts in the agricultural sector to find ways to reduce methane emissions from ruminants. One method includes administering anti-methanogenic feed additives such as chloroform, 3-nitrooxypropanol (3-NOP), linseed oil, inorganic nitrate, garlic extracts / oils, and / or certain marine algae to adult ruminants. Another approach is to select cows that are genetically predisposed to emit less methane than others, hoping that their offspring will also exhibit reduced methane emissions, or to alter the microbiome early in life, i.e., in the so-called pre-methane phase. Petition 870250074624, dated 08 / 22 / 2025, page 14 / 67 2 / 23 ruminant, in the hope of persistently lower long-term methane emissions in ruminants treated early in life compared to untreated ruminants.

[004] Anti-methanogenic feed additives, while demonstrating a consistent and significant effect in reducing methane emissions in ruminants, generally need to be supplemented daily and in significant quantities to maintain this effect. Certain ruminants, for example, pasture-raised cattle, are therefore difficult to target and currently beyond the reach of such treatment. In addition, the additive may have an undesirable impact on milk and / or meat: garlic-derived products, for example, are known to impart an unpleasant taste to milk.

[005] Previous experiments have demonstrated that an alteration of the microbiome in the pre-ruminant stage is possible in terms of the structure of the bacterial community, with effects persisting even after the challenge or pressure has ceased (Yanez-Ruis et al. in FEMS Microbiol Ecol 72 (2010) 272-278 and in Front. Microbiol. 6: 1133 (2015)). However, with regard to the methanogenic community, which belongs to the domain of archaea, several attempts, also using antimethanogenic compounds, have failed to establish a persistent effect once the selection pressure has been removed, although during treatment supplementation a reduction in methane emission was achieved, as illustrated for example by Saro et al. (Front. Microbiol. 9: 1273 (2018)). The authors concluded that although there was a degree of plasticity in the ruminal bacterial community that allowed for an alteration in the development of the bacterial community, with respect to Petition 870250074624, dated 08 / 22 / 2025, page 15 / 67 3 / 23 methanogenic community, these organisms resisted the change, and there was no persistence of the early-life perturbation in methanogenesis, indicating resilience for this function. The authors even demonstrated that lambs treated early in life with an anti-methanogenic feed additive exhibited a long-term increase in methane emissions compared to untreated lambs.

[006] Therefore, there is an ongoing need for solutions to persistently reduce methane emissions from ruminants. Detailed description of the invention

[007] It has been discovered, according to this invention, that by supplementing calves with 3-nitrooxypropanol immediately after birth up to three weeks after weaning (14 weeks of age), a persistent reduction in methane emissions from supplemented animals could be achieved, even long after 3-nitrooxypropanol supplementation had ceased. Treated animals exhibited lower methane emissions even after 60 weeks of age, indicating that the effect persisted later in life. Interestingly, the total number of methanogens was not reduced. Furthermore, no detrimental effects on the performance of the host animal were observed.

[008] Therefore, in a first embodiment, the present invention relates to a method for reducing methane emissions in adult ruminants, said method encompassing the administration to said ruminant in the pre-ruminant phase of an effective amount of 3-nitrooxypropanol, and optionally appreciating the methane-reducing effect, preferably in the long term. Optionally, the Petition 870250074624, dated 08 / 22 / 2025, p. 16 / 67 4 / 23 administration is extended to the young ruminant phase.

[009] Even more preferably, 3-nitrooxypropanol is administered to ruminants from birth to a maximum age of 6 months, preferably to a maximum age of 5 months, more preferably to a maximum age of 4.

[0010] The invention further relates to a method for reducing methane emissions in adult ruminants, comprising administering to a healthy newborn ruminant desired to have reduced methane emissions as an adult ruminant an effective amount of 3-nitrooxypropanol.

[0011] Another aspect of this invention is the reduction of methane from the digestive activities of a healthy ruminant through the use of 3-nitrooxypropanol administered orally in the pre-ruminant phase of said ruminant, optionally extended to the young ruminant phase.

[0012] Another aspect of this invention is the (non-therapeutic) use of 3-nitrooxypropanol in pre-ruminants to mitigate climate change (i.e., to help reduce the warming of the planet's atmosphere) by reducing the total methane emission over the lifetime of said ruminants (compared to a respective untreated ruminant).

[0013] The invention also relates to a feeding regimen for reducing methane emissions in adult ruminants and / or for reducing the total lifetime methane emission of a ruminant (compared to a respective untreated ruminant), comprising (1) administering a diet comprising 3 Petition 870250074624, dated 08 / 22 / 2025, p. 17 / 67 5 / 23 nitroxypropanol to the said ruminant in its preruminant phase and optionally young ruminant phase followed by (2) administration of a diet that does not contain 3 nitroxypropanol to adult ruminants.

[0014] The invention also includes a kit for reducing methane emissions in ruminants comprising a.) at least one dosage form of 3-nitrooxypropanol, preferably oral; and b.) Information that informs a potential user about the benefits of administering 3-nitrooxypropanol to ruminants in the pre-ruminant phase and optionally in the young ruminant phase, in order to reduce methane emissions in the adult ruminant.

[0015] In another embodiment, the present invention relates to a ruminant with low methane emissions, characterized in that said ruminant is obtained by supplementing said ruminant in the pre-ruminant phase with 3-nitrooxypropanol. Said ruminant is particularly characterized by the fact that the total methane emission over the lifetime of said ruminant is lower compared with a respective untreated ruminant.

[0016] In a further embodiment, the present invention relates to a method for influencing the microbial colonization pattern of the rumen of a ruminant, said method comprising the step of administering an effective amount of 3-nitrooxypropanol to said ruminant in the pre-ruminant phase. Definitions

[0017] The so-called pre-ruminant phase is the period in a ruminant's life when digestive functions are Petition 870250074624, dated 08 / 22 / 2025, p. 18 / 67 6 / 23 more similar to those of monogastric animals. The pre-ruminant phase generally occurs during the first weeks of life, when the calf mainly ingests milk or milk replacer. Depending on the type of feed offered and the intended use of the animals, the pre-ruminant phase may be shorter or longer. During the second stage, the so-called 'young ruminant phase' or 'post-weaning phase', the rumen is developing but is not yet capable of full performance at the efficiency level of an adult animal. The final stage in the development of the adult ruminant occurs when the rumen is considered fully functional, which generally occurs at 12 weeks of age. (see, for example, Teagasc 2017: Rumen development, Section 4; Chapter 12, page 60 (https: / / www.teagasc.ie / media / website / publications / 2017 / Section4-Rumen-development.pdf) or Rearing Young Stock on Tropical Dairy Farms in Asia (John, Moran), chapter 3: Digestion of feeds in the milk fed calf: page 27).

[0018] Within the meaning of the present invention, the term 'pre-ruminant' refers to an animal that has not yet (at least partially) been weaned, while the term 'young ruminant' refers to the stage in which the rumen is not yet fully developed, but the calf has been weaned (i.e., no longer receives milk).

[0019] In the present context, a ruminant is a mammal of the order Artiodactyla that digests plant-based foods by initially softening them in the animal's first stomach, known as the rumen, then regurgitating the semi-digested mass, now known as the ruminal bolus, and chewing it again. The process of ruminating again to break down Petition 870250074624, dated 08 / 22 / 2025, p. 19 / 67 7 / 23 further consuming plant matter and stimulating digestion is called rumination. Ruminant mammals include cattle, oxen, goats, sheep, giraffes, deer, gazelles, and antelopes. Preferred ruminants for all embodiments of the present invention are domestic cattle, sheep, and goats. The most preferred ruminants in all embodiments according to the present invention are domestic cattle and, consequently, their calves. The term cattle includes all breeds of domestic cattle and all types of production livestock (such as, for example, dairy cows, pasture cattle, and beef cattle). Particularly preferred are pasture cattle, since they are difficult to reach with supplementation later in life (i.e., in adulthood).

[0020] In the present context, 'methanogens' are microorganisms that produce methane as a metabolic byproduct. They are prokaryotic and belong to the domain Archaea. They are also frequently called methanogenic archaea. Along with various bacteria, protozoa, and fungi, they are present in the rumen.

[0021] Antimethanogenic compounds are compounds that can be used to inhibit the activity of such methanogenic archaea to reduce methane emissions, for example, emanating from the digestive activities of ruminants.

[0022] 3-Nitrooxypropanol (CAS-No: 100502-66-7) is a known compound that can, for example, be manufactured as described in WO-2004043898 or WO-2012084629. Suitable formulations thereof are, for example, disclosed in WO2018149755, WO-2018149756, WO-2018091643 or WO-2019007740.

[0023] The term ration, as used herein, refers to any compound, preparation, mixture or composition. Petition 870250074624, dated 08 / 22 / 2025, p. 20 / 67 8 / 23 suitable for, or intended for ingestion by the respective animal, and is not limited to nutritional materials.

[0024] The term 'edible oil(s) or solvent(s)', as used herein, refers to oils or solvents commonly used in feed applications. Preferred edible oils or solvents in all embodiments of the present invention are propylene glycol, corn oil, rapeseed oil, sunflower oil, medium-chain triglyceride (MCT), and glycerol, as well as mixtures thereof.

[0025] Methane emissions and methane reductions as referred to herein can be measured in individual animals in metabolic chambers by methods known in the art (Grainger et al., 2007 J. Dairy Science; 90: 2755-2766). Additionally, it can also be assessed at the barn level by an emerging technology using laser beams (McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802). Alternatively, methane produced by a ruminant can also be assessed with the GreenFeed system (C-Lock, Rapid City, SD USA), which is the preferred method.

[0026] The standard term microbial colonization refers to a specific ruminal microbial composition (also called ruminal microflora) that develops during the first hours, days, and weeks of a ruminant and then persists throughout the animal's life. This ruminal microflora consists mainly of bacteria, archaea (such as methanogenic archaea in particular), protozoa, and fungi.

[0027] The term "from birth" as used here refers to the time interval up to a maximum of 3 days after birth, preferably up to a maximum of 2 Petition 870250074624, dated 08 / 22 / 2025, page 21 / 67 9-23 days after birth, more preferably up to 1 day after birth at the latest. Ideally, 3-nitrooxypropanol supplementation should begin with the first feed supplementation other than breastfeeding. Dosage and administration

[0028] 3-Nitrooxypropanol is administered at least during the pre-ruminant phase. More preferably, however, the administration of 3-nitrooxypropanol is at least partially extended into the post-weaning phase (i.e., young ruminant), such as for a further 1, 2, 3, 4, 5, 6, 7 or 8 weeks. Thus, advantageously, 3-nitrooxypropanol is administered to the ruminant from birth until 8 weeks after weaning.

[0029] Alternatively, in all embodiments of the present invention, 3-nitrooxypropanol is preferably administered to the ruminant from birth to a maximum age of 6 months, preferably to a maximum age of 5 months, more preferably to a maximum age of 4 months, or to a maximum age of 3 months.

[0030] In all embodiments according to the present invention, it is preferred that supplementation with 3-nitrooxypropanol be completely discontinued after the aforementioned defined period of administration (i.e., not extend into adulthood).

[0031] 3-Nitrooxypropanol can be administered daily, every other day, or weekly. Preferably, in all embodiments according to the present invention, the 3-Nitrooxypropanol of the invention is administered daily at least once a day.

[0032] It is still preferred that 3-nitrooxypropanol be Petition 870250074624, dated 08 / 22 / 2025, p. 22 / 67 10 / 23 administered orally to pre-ruminant / young ruminant.

[0033] The amount of 3-nitrooxypropanol to be administered to the pre-ruminant / young ruminant is preferably selected in the range of 10 mg to 10 g of 3-NOP per animal per day, preferably 50 mg to 5 g of 3-NOP per animal per day, more preferably 75 mg to 2.5 g of 3-NOP per animal per day. Other suitable ranges include 100 mg to 2 g of 3-NOP per animal per day or 100 to 500 mg of 3-NOP per animal per day.

[0034] In another and more preferred embodiment, the amount of 3-nitrooxypropanol to be administered to the preruminant / young ruminant is selected in the range of 0.1 mg to 15 mg of 3-NOP / kg of body weight (BW), more preferably in the range of 0.5 to 10 mg of 3-NOP / kg of BW, more preferably in the range of 1 to 5 mg of 3-NOP / kg of BW, as well as in the range of 2 mg to 4 mg of 3-NOP / kg of BW.

[0035] A person skilled in the art may appreciate that the total (daily) amount of 3-nitrooxypropanol fed may vary depending on the animal species, animal size, and type of feed to which 3-nitrooxypropanol or a formulation or composition thereof is added.

[0036] The total (daily) amount of 3-nitrooxypropanol can be administered in a single dose or distributed over several doses, for example one dose in the morning and one dose at night.

[0037] In all embodiments of the present invention, 3-nitrooxypropanol can be used as such. Preferably, however, 3-nitrooxypropanol is used in the form of a formulation thereof, that is, in the form of a composition comprising 3-nitrooxypropanol and a carrier material. Petition 870250074624, dated 08 / 22 / 2025, page 23 / 67 11 / 23 suitable (also referred to here as '3-NOP formulation'). 3-Nitrooxypropanol or a formulation thereof may be administered to the pre-ruminant / young ruminant as such or may be mixed into the pre-ruminant / young ruminant feed.

[0038] Suitable carriers for 3-NOP formulations include any carrier well known in the food and feed industry, such as water, edible oil(s) or solvent(s), preferably propylene glycol, silica (silicon dioxide), diatomaceous earth, and sepiolite, but not limited to these. The most preferred carrier materials according to the present invention are silica, propylene glycol, or water, as well as mixtures thereof.

[0039] 3-NOP formulations can be formulated in any form suitable for direct administration to preruminants / young ruminants or for mixing into preruminant / young ruminant feed. Suitable solid forms for direct administration or mixing are, for example, a powder, a granule, or a pellet. Suitable liquid forms for direct administration or mixing are, for example, a solution or a suspension. 3-NOP formulations can also be in the form of a controlled-release (delayed-release) formulation.

[0040] The amount of 3-nitrooxypropanol in 3-NOP formulations according to the present invention is preferably selected in the range of 0.1 to 20% by weight, more preferably in the range of 0.5 to 15% by weight, most preferably in the range of 1 to 12% by weight, based on the total weight of the formulation.

[0041] The 3-NOP formulations according to the present invention are preferably administered mixed in Petition 870250074624, dated 08 / 22 / 2025, page 24 / 67 12 / 23 or with pre-ruminant or young ruminant feed. Solid 3-NOP formulations

[0042] In an advantageous embodiment, the 3-NOP formulation is a solid formulation, preferably a dry free-flowing powder comprising 3-NOP and a solid carrier material (also referred to herein as a 3-NOP powder formulation) or a pellet comprising 3-nitrooxypropanol. These solid formulations are suitable for direct feeding to preruminant / young ruminant or for inclusion in a commercially available preruminant / young ruminant ration or as a supplement to a total mixed diet or ration. When supplemented with the preruminant / young ruminant ration, the solid formulation is generally mixed with the solid or liquid ration or with (potable) water.

[0043] Powder formulations comprising 3-nitrooxypropanol and a solid carrier material are generally prepared by mixing 3-NOP with a solid carrier material by standard methods in the art. Alternatively, 3-NOP can be sprayed onto the solid carrier material. For example, 3-NOP can be sprayed onto a solid carrier material using a solvent suitable for the preparation of feed or food products, such as, for example, dichloromethane followed by evaporation of the organic solvent.

[0044] Solid carriers particularly suitable for the preparation of 3-NOP powder formulations include silica (silicon dioxide), diatomaceous earth, as well as sepiolite, without being limited to them. The most preferred solid carrier material according to the present invention is silica.

[0045] This silica is a well-known carrier material in Petition 870250074624, dated 08 / 22 / 2025, page 25 / 67 13 / 23 feed and food industry and refers to white amorphous silica microspheres (also referred to as silicon dioxide). The silica particularly suitable according to the present invention is precipitated amorphous silica, for example available as Ibersil D-250 at IQE Group, Sipernat 2200 at Evonik or Tixosil 68 at Solvay.

[0046] It is well understood that 3-NOP can be diluted in a suitable edible oil or solvent (liquid carrier) before being sprayed or mixed with the solid carrier material.

[0047] Furthermore, the 3-NOP powder formulation according to the present invention may additionally contain usual additives used in the preparation of powder formulations for feed application, such as, in particular, thickeners which are advantageously selected from gums or cellulose derivatives such as xanthan gum, karaya gum and / or ethylcellulose as well as (traces) of water.

[0048] The amount of 3-nitrooxypropanol in the 3-NOP powder formulation according to the present invention is preferably selected in the range of 1 to 20% by weight, preferably in the range of 2 to 15% by weight, more preferably in the range of 4 to 12% by weight, based on the total weight of the formulation.

[0049] Preferably, at least one edible oil or solvent is present in the 3-NOP powder formulations according to the present invention. The amount of edible oil(s) and / or solvent(s) in such 3-NOP powder formulations is preferably selected in the range of 0.1 to 50% by weight, preferably in the range of 1 to 45% by weight, more preferably in the range of 10 to 40% by weight, with Petition 870250074624, dated 08 / 22 / 2025, page 26 / 67 14 / 23 based on the total weight of the formulation.

[0050] A suitable 3-NOP powder formulation according to the present invention essentially consists of 3-nitrooxypropanol, propylene glycol and silica (silicon dioxide), such as (i) 2 to 12% by weight of 3-nitrooxypropanol, (ii) 20 to 40% by weight of propylene glycol, (iii) 0 to 7% by weight of water and (iv) silica, wherein the total amount of ingredients (i) to (iv) amounts to 100% by weight. Such formulations are for example disclosed in WO2018149756 or WO-2018149755.

[0051] Alternatively, 3-nitrooxypropanol can be supplemented in tablet form, as disclosed in NO WO-2019007740. Liquid formulations of 3-NOP

[0052] In another embodiment, the 3-NOP formulation may be a liquid formulation suitable for direct administration to pre-ruminant / young ruminant or suitable for inclusion in a commercially available pre-ruminant / young ruminant ration or as a supplement to a total mixed diet or ration.

[0053] Suitable liquid formulations of 3-NOP include, for example, solutions of 3-NOP in water or in an edible oil or solvent (or mixtures thereof), preferably in propylene glycol.

[0054] It is well understood that the liquid formulation of 3-NOP according to the present invention may also contain usual additives used in the preparation of liquid formulations for application in feed or drinking water additives for ruminants, such as, for example, vitamins, sugars, salts and flavoring agents. Petition 870250074624, dated 08 / 22 / 2025, page 27 / 67 15 / 23

[0055] The amount of 3-nitrooxypropanol in the liquid formulation of 3-NOP is preferably selected in the range of 1 to 30% by weight, preferably in the range of 5 to 28% by weight, more preferably in the range of 10 to 25% by weight, based on the total weight of the formulation.

[0056] A suitable liquid 3-NOP formulation essentially consists of a 10 to 30% by weight solution of 3-nitrooxypropanol in water and / or propylene glycol. Feed for pre-ruminants / young ruminants

[0057] For the purpose of carrying out its use in accordance with the present invention, 3-nitrooxypropanol or a formulation thereof, preferably a powder or liquid formulation thereof, can be incorporated into preruminant / young ruminant feed by methods known per se in the art of feed formulation and processing.

[0058] Other aspects of the present invention therefore relate to pre-ruminant and / or young ruminant feeds containing 3-nitrooxypropanol or a formulation thereof.

[0059] Suitable feeds for preruminants are milk or milk substitute preparations (milk replacements). These milk and milk replacement preparations comprising 3-nitrooxypropanol or a formulation thereof are still new.

[0060] Thus, the present invention also relates to milk or milk substitutes for pre-ruminants, characterized in that said milk or milk substitutes comprise 3-nitrooxypropanol or a formulation thereof.

[0061] The term milk or milk substitutes refers to whole milk, reduced-fat milk and nonfat milk, non-saleable milk and milk substitutes. Petition 870250074624, dated 08 / 22 / 2025, p. 28 / 67 16 / 23 reconstituted. “Unsaleable milk” is defined as milk that cannot, by law, be sold for human consumption. Milk substitutes are formulated feeds intended to replace mother's milk for calves during the critical early stages of suckling or milk feeding.

[0062] Milk substitutes generally contain 18 to 30% by weight of protein and 10 to 28% by weight of fat, with 18% to 22% by weight being the most preferred fat level, and optionally other additives and / or medications. Milk substitutes are reconstituted with water, mixed, and supplied according to the manufacturer's instructions.

[0063] Exemplary protein sources to be used in milk substitutes are milk proteins, such as dehydrated whey protein concentrate, dehydrated whey, dehydrated whey product, skimmed milk, dehydrated skimmed milk powder, casein, as well as sodium or calcium caseinate. Alternative protein sources include soy protein isolate, soy protein-modified flour, soy protein concentrate, soy flour, animal plasma, as well as wheat gluten or isolate, legume proteins, and isolated legume proteins. Suitable fats include vegetable and animal fats and their fractions. Exemplary additives are in particular carbohydrates, mainly lactose, amino acids, (essential) vitamins and minerals, and other materials considered acceptable for use in animal feed by local authorities.

[0064] The amount of 3-nitrooxypropanol in milk or Petition 870250074624, dated 08 / 22 / 2025, page 29 / 67 17 / 23 The reconstituted milk substitute is preferably selected in the range of about 1 mg to about 1000 mg, preferably from about 1 mg to about 500 mg, more preferably from about 1 mg to 250 mg 3-NOP per liter of milk or reconstituted milk substitute.

[0065] For calves, the amount of 3-nitrooxypropanol in milk or reconstituted milk replacer is most preferably selected in the range of 10 to 100 mg / L.

[0066] For small ruminants, such as lambs and kids, the amount of 3-nitrooxypropanol in milk or reconstituted milk replacer is most preferably selected in the range of 1 mg to 100 mg / L.

[0067] Post-weaning, 3-nitrooxypropanol or a composition or formulation comprising 3-nitrooxypropanol according to the present invention is preferably mixed into the respective feed for young ruminants, such as preferably concentrated feed for young ruminants. Such concentrated feed should preferably be of coarse texture, high in energy and protein and low in forage (> 15%). Cereal grains (oats, barley, wheat, corn, triticale) may also be used, mixed with molasses, dehydrated alfalfa or grass pellets, bran or other co-products derived from the food, beverage and feed industries.

[0068] The young ruminant feed according to the present invention may also contain conventional young ruminant feed additives and adjuvants, excipients or diluents, including, but not limited to, water, molasses, calcium carbonates, electrolytes such as ammonium chloride, proteins such as soybean meal, canola meal, lentils, lupins, peas, wheat, beans, starch, flour of Petition 870250074624, dated 08 / 22 / 2025, page 30 / 67 18 / 23 sunflower, corn, amino acids, fats, vitamins and trace minerals and other materials considered acceptable for use in animal feed by local authorities.

[0069] Typically, young ruminant feed would be formulated from, but not limited to, a selection of the following ingredients: barley, wheat, triticale, oats, peas, lupins, lentils, beans, soybeans, canola, sunflower, and products derived from these ingredients. Vegetable oil, molasses, limestone, dicalcium phosphate, salt, bentonite, magnesium oxide, acidifier, lysine, methionine, antioxidants and coccidiostats, and other medications may also be included. Certain co-products, such as palm kernel extract (PKE), ground grits, and distillers grains may also be included.

[0070] The amount of 3-nitrooxypropanol in the ration of young ruminants is preferably selected in the range of about 1 mg to about 1000 mg, preferably from about 1 mg to about 500 mg, more preferably from about 1 mg to 250 mg of 3-NOP per liter of milk or reconstituted milk replacer.

[0071] It is well understood that the amount of 3-NOP administered may still be adjusted to the specific needs of the respective pre-ruminant / young ruminant being treated, such as calves, lambs or kids.

[0072] In a particularly advantageous embodiment according to the present invention, 3-nitrooxypropanol is supplemented in the form of a liquid preparation, such as mixed with milk or a liquid milk substitute preparation (i.e., a reconstituted milk substitute).

[0073] Thus, the present invention also relates to a Petition 870250074624, dated 08 / 22 / 2025, p. 31 / 67 19 / 23 method of providing a liquid pre-ruminant feed, more preferably milk or a liquid milk replacement preparation comprising 3-nitrooxypropanol or a formulation thereof to a pre-ruminant calf, said method comprising dissolving 3-nitrooxypropanol or a formulation thereof in the liquid feed and feeding the liquid feed containing 3-nitrooxypropanol or a formulation thereof to the pre-ruminant calf.

[0074] In another embodiment, the present invention also provides the use of a pre-ruminant and / or young ruminant feed comprising 3-nitrooxypropanol or a formulation thereof for a persistent, long-term (non-therapeutic) reduction of methane formation emanating from the digestive activities of ruminants in their adult life and / or to reduce the total methane emission throughout the life of a ruminant, preferably where it is not necessary or desired to additionally administer 3-nitrooxypropanol to the adult ruminant.

[0075] The invention further provides a method for the persistent, long-term reduction of methane production from the digestive activities of adult ruminants, said method comprising the oral administration of a sufficient quantity of a pre-ruminant and / or young ruminant feed comprising 3-nitrooxyropanol or a composition or formulation thereof for a pre-ruminant and / or young ruminant.

[0076] The invention solves a problem that has not been solved until now: With the help of the present invention, the gastrointestinal tract of a pre-ruminant / young ruminant is adapted in such a way that the total emission of methane over Petition 870250074624, dated 08 / 22 / 2025, page 32 / 67 20 / 23 of the lifespan of the aforementioned ruminant is shorter compared to a respective untreated ruminant. This especially means that the reduction in methane (compared to a respective untreated ruminant) lasts until and throughout the adult life of a ruminant even after supplementation with 3-nitrooxypropanol or a composition or formulation thereof is discontinued.

[0077] Thus, 3-nitrooxypropanol is useful as an active ingredient for feed additives and animal feed compositions for pre-ruminants and young ruminants and consequently is useful as an active ingredient for adapting the gastrointestinal tract of a pre-ruminant / young ruminant in the long term, persistently reduced methane emissions (compared to untreated animals), in particular by all methods and uses as disclosed herein.

[0078] The persistent long-term reduction of methane in all embodiments of the present invention is preferably characterized by at least 5% less, more preferably at least 10% less, most preferably at least 15% less methane (calculated in g CH4 / day) after 1 year of age, when compared to an untreated control group (measured using the GreenFeed system (C-Lock, Rapid City, SD USA)). It is understood that the control group is of the same breed raised under the same management conditions (same farm, same herd, similar genetics and inoculation source, and similar age). Figure

[0079] Figure 1: x-axis: animal age in weeks; y-axis: methane emission in g CH4 / d; Period I: 3-NOP supplementation; Period II: No 3-NOP supplementation Petition 870250074624, dated 08 / 22 / 2025, page 33 / 67 21 / 23 NOP; dashed line: treatment group; solid line: control group. For more details, see the Examples. Examples Experimental configuration

[0080] At birth, 18 female calves of the Holstein breed (n = 12), Montbéliarde (n = 4) and Holstein x Montbéliarde (n = 2) Calves were randomly assigned to a treatment group (3 mg of 3-NOP (active) / kg of BW) or a control group (placebo supplement, i.e., without 3-NOP but with silica and propylene glycol). 3-NOP was supplemented in the form of a 3-NOP powder formulation (10% by weight of 3-NOP mixed with silica and propylene glycol) so that the distribution of breed and birth weight was balanced between the groups. Treatment was administered daily from birth until three weeks post-weaning (week 14) (mixed in a small volume of water and administered by oral gavage). Calves were weighed weekly. Milk and concentrate intake before weaning was recorded in automated feeders. Animals were housed in groups in a single pen after weaning. Rumen fluid samples were collected at weeks 1, 4, 11, 14, 23, and 60 of age, and DNA from these rumen fluid samples was extracted and subjected to Illumina MiSeq sequencing.Methane emissions from calves were measured using the GreenFeed system (C-Lock, Rapid City, SD USA) from weaning at week 11 to week 23 and subsequently from week 56 to 60 of life. Results

[0081] The treated calves showed a persistent reduction (overall 11.6%, P = 0.03) in methane emissions. Petition 870250074624, dated 08 / 22 / 2025, page 34 / 67 22 / 23 (g of CH4 / d) throughout the post-weaning period, despite treatment ceasing three weeks after weaning (Table 1). When methane emissions were measured almost a year later (weeks 56 to 60 of life), overall methane emissions in the treatment group were still significantly reduced by about 19% (P < 0.05), indicating persistence of the effect (see Figure 1).

[0082] The calf's physiological parameters (body weight (BW) and average daily gain (ADG)) were similar between groups throughout the trial, indicating no adverse effect on performance from early-life treatment (see Tables 2 and 3). Furthermore, no significant change in the relative abundance of methanogens as a result of 3-NOP treatment was observed. Table 1: Effect of 3-nitrooxypropanol supplementation on methane emissions (g / day) of calves measured after weaning at 11 to 23 weeks of age and at advanced age from 1 to 60 weeks of age. Animal age [in weeks] 11 14 17 20 23 56 to 60 Daily supplementation No supplementation Control (n=8) 48.4 72.4 81.4 101.9 111.2 238.7 Treatment (n=10) 45.0 63.4 68.2 88.9 97.13 193.0 Δ CH4 emissions [g CH4 / d] -3.4 -9.00 -13.2 -13 -14 -45.7 Δ CH4 emissions [%] -7.0 -12.4 -16.2 -12.7 -12.7 -19.2 Table 2: Body weight (BW) of calves (in kg) Animal age [in weeks] 0 11 14 17 20 23 56 to 60 Control (n=8) 40.8 104.7 123.1 140.2 158.1 184.2 357 Treatment (n=10) 41.6 102.2 119.5 134.9 151.8 189.5 339

[0083] No significant difference in body weight was observed. Petition 870250074624, dated 08 / 22 / 2025, page 35 / 67 23 / 23 Table 3: Average Daily Gain (ADG) (kg / d) from week 0 to 23 weeks of age GMD (kg / d) Control (n=8) 0.84 Treatment (n=10) 0.79

[0084] No significant difference in average daily gain was observed. Petition 870250074624, dated 08 / 22 / 2025, pp. 36 / 67

Claims

1 / 3 CLAIMS 1. Feeding regimen for reducing methane emissions in adult ruminants, characterized in that it comprises (1) administering a diet comprising 3-nitrooxypropanol to said ruminant in its pre-ruminant phase ingesting mainly milk or milk replacer and optionally young ruminant phase, followed by (2) administering a diet that does not contain 3-nitrooxypropanol to adult ruminants.

2. Feeding regimen according to claim 1, characterized in that 3-nitrooxypropanol is administered orally.

3. Feeding regimen, according to claim 1 or 2, characterized in that the amount of 3-nitrooxypropanol (3-NOP) administered to the ruminant is selected in the range of 0.1 mg to 15 mg of 3-NOP / kg of body weight (BW), preferably in the range of 0.5 to 10 mg of 3-NOP / kg of BW, more preferably in the range of 1 to 5 mg of 3-NOP / kg of BW.

4. Feeding regimen, according to any one of claims 1 to 3, characterized in that 3-nitrooxypropanol is administered mixed with the feed of preruminant / young ruminant.

5. Feeding regimen, according to any one of claims 1 to 4, characterized in that the pre-ruminant feed is milk or a milk replacement preparation.

6. Feeding regimen, according to any one of claims 1 to 5, characterized in that methane emissions are reduced by at least 5%, preferably by at least 10% after 1 year of age, compared with an untreated control group.

7. Feeding regime, according to any one of claims 1 to 6, characterized in that the ruminant is selected from the group of domestic cattle, sheep and goats, preferably from domestic cattle.

8. Feeding regimen, according to any one of claims 1 to 7, characterized in that feeding is extended to the young ruminant stage for an additional 1, 2, 3, 4, 5, 6, 7 or 8 weeks.

9. Feeding regimen, according to any one of claims 1 to 8, characterized in that 3-nitrooxypropanol is administered to the ruminant from birth until 8 weeks after weaning.

10. Feeding regimen, according to any one of claims 1 to 9, characterized in that 3-nitrooxypropanol is administered to the ruminant from birth to 6 months of age, preferably to 5 months of age, more preferably to 4 months of age, for example, to 3 months of age. Petition 870250074624, dated 22 / 08 / 2025, pp. 61 / 67

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