阅读说明：本技术 生命早期营养 (Early life nutrition ) 是由 斯蒂芬·杜瓦尔 迈克·金德曼 莎拉·贾登·梅亚莱 迪亚高·巴勃罗·马尔加维 于 2020-04-14 设计创作，主要内容包括：本发明涉及一种减少反刍动物中甲烷排放的方法,所述方法包括用3-硝基氧基丙醇处理预反刍动物/幼龄反刍动物。(The present invention relates to a method for reducing methane emission in ruminants, said method comprising treating a pre-ruminant/young ruminant with 3-nitrooxypropanol.)

1. A method of reducing methane emissions in adult ruminants, the method comprising administering to the ruminant an effective amount of 3-nitro ester propanol from birth up to 6 months of age, preferably up to 5 months of age, most preferably up to 4 months of age.

2. The method of claim 1, wherein the 3-nitro ester propanol is administered orally.

3. The method of any one of the preceding claims, wherein the 3-nitro ester propanol is administered at least once daily.

4. Method according to any of the preceding claims, wherein the amount of 3-nitrooxypropanol (3-NOP) administered to the ruminant is selected in the range of 0.1mg to 15mg 3-NOP per kg Body Weight (BW), preferably in the range of 0.5mg to 10mg 3-NOP per kg BW, most preferably in the range of 1mg to 5mg 3-NOP per kg BW.

5. A method according to any preceding claim, wherein the 3-nitrooxypropanol is administered mixed into a pre-ruminant/young-ruminant feed.

6. The method of any preceding claim, wherein the pre-ruminant feed is milk or a milk substitute preparation.

7. The method according to any of the preceding claims, wherein the methane emission is reduced by at least 5%, preferably by at least 10% after 1 year of birth compared to an untreated control group.

8. The method according to any of the preceding claims, wherein the ruminant is selected from a domesticated cow, a domesticated sheep and a domesticated goat, preferably from a domesticated cow.

9. A milk or milk substitute formulation for use in pre-ruminants, wherein the milk or milk substitute formulation comprises 3-nitrooxypropanol.

10. A feeding method for reducing methane emissions from adult ruminants, comprising:

(1) administering a diet comprising 3-nitrooxypropanol to a ruminant at a pre-ruminant and optionally young ruminant stage, and then,

(2) adult ruminants were given a diet without 3-nitrooxypropanol.

11. A kit for reducing methane emissions from a ruminant, the kit comprising:

a.) at least one dosage form of 3-nitrooxypropanol; and

b.) instructions to demonstrate to a potential user the benefit of administering 3-nitrooxypropanol to a ruminant in a pre-ruminant and optionally a young ruminant stage to reduce methane emissions in an adult ruminant.

12. A low methane emission ruminant, wherein the ruminant is obtained by supplementing the ruminant with 3-nitrooxypropanol at a pre-ruminant stage.

Use of 3-nitrooxypropanol in pre-ruminants to mitigate climate change by reducing the lifetime total methane emissions of said ruminants.

14. A method of affecting the microbial colonization pattern of the rumen of a ruminant, the method comprising the step of administering to the ruminant an effective amount of 3-nitrooxypropanol at a pre-ruminant stage.

15. The method according to claim 14, wherein the methane emission is reduced by at least 5%, preferably by at least 10%, after 1 year of birth compared to an untreated control group.

Technical Field

The present invention relates to a method for reducing methane emission in ruminants, said method comprising treating a pre-ruminant/young ruminant with 3-nitrooxypropanol.

Background

It is estimated that livestock production accounts for approximately 14% of the man-made methane production released into the atmosphere annually, most of which is formed in ruminants as a result of rumen methanogen (archaea) activity in the rumen of the host animal during intestinal fermentation, resulting in the formation of methane excreted from the animal. Methane is not only a potent Greenhouse Gas (GHG) and environmental problem, it also represents an energy loss from the host, estimated to be in the range of 2% to 12% of total energy dietary intake.

Currently, the agricultural sector is striving to find ways to reduce methane emissions from ruminants. One method involves administering to adult ruminants an anti-methanogenic feed additive such as chloroform, 3-nitrooxypropanol (3-NOP), linseed oil, inorganic nitrates, garlic extract/oil and/or certain seaweeds. Another approach is to select cows that are genetically inclined to emit less methane than others, with the hope that their offspring will also exhibit reduced methane emissions, or to alter the microbiome early in life, i.e. at the so-called pre-ruminant stage, with the hope that methane emissions will continue to be reduced for a long period of time in such early life treated ruminants as compared to untreated ruminants.

Anti-methanogenic feed additives, while having been shown to have consistent and significant effects on reducing methane emissions in ruminants, typically require supplementation daily and in significant amounts to maintain such effects. Therefore, some ruminants, such as grazing cattle, are difficult to target and are currently not amenable to such treatment. Furthermore, the additives may adversely affect the milk and/or meat: it is known that for example garlic derived products can produce an unpleasant taste to milk.

Previous experiments have demonstrated that, in terms of bacterial community structure, alterations of microbiome are possible in the pre-ruminant stage, the effects of which remain even after challenge or cessation of stress (Yanez-Ruis et al in FEMS Microbiol Ecol 72(2010)272-278and in Front. Microbiol.6:1133 (2015)). However, for methanogen communities belonging to the field of archaea, several attempts to use anti-methanogenic compounds, even if methane emission reduction is achieved during the replenishment treatment, failed to establish a lasting effect after removal of the selection pressure, as for example described by Saro et al (front. microbiol.9:1273 (2018)). The authors concluded that although there was a degree of plasticity within the rumen bacterial community that allowed changes in the development of the bacterial community, the resistance of these organisms to changes relative to the methanogenic community, and the absence of interference with methanogenesis by the persistence of early life, indicates the elasticity of this function. The authors even demonstrated that early-life lambs treated with the anti-methanogenic feed supplement had a long-term increase in methane emissions compared to untreated lambs.

Thus, there is a continuing need for solutions to reduce methane emissions in ruminants.

Drawings

FIG. 1: the x axis is as follows: the week age of the animal; and a y axis: in g CH₄The methane emission per unit of/d; stage I: 3-NOP supplementation; stage II: 3-NOP was not supplemented; dotted line: a treatment group; solid line: and (4) a control group. More detailed information is described in the embodiments.

Examples

Experimental setup

18 female calves born right before, respectively Holstein (Holstein) calves (n-12), montbelyand (Montb leiarde) calves (n-4) and Holstein x Montb leiarde (Holstein x Montb liaarde) calves (n-2), were randomized to the treatment group (3mg 3-NOP (activity)/kg BW) or to the control group (placebo supplement, i.e. without 3-NOP but containing silica and propylene glycol). The 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) in order to balance breed distribution and birth weight between groups. The treatment of administration (mixed in a small amount of water and delivered by oral gavage) was performed daily from birth to three weeks post weaning (week 14). Calves were weighed weekly. Milk and concentrate intake prior to weaning was recorded using an automatic feeder. Animals were housed in groups in individual pens post weaning. Rumen fluid samples were collected at weeks 1, 4, 11, 14, 23 and 60 of life, and DNA was extracted from these rumen fluid content samples and Illumina MiSeq sequencing was performed. The methane emission from calves was measured from 11 to 23 weeks weaning and then from 56 to 60 weeks using the GreenFeed system (C-Lock, Rapid City, SD USA).

Results

Although treatment was discontinued three weeks after weaning, methane emissions (g CH) of treated calves throughout the post-weaning period₄The reduction in/d) continued (11.6% in total, P ═ 0.03) (table 1). When methane emissions were measured almost one year later (weeks 56 to 60 of life), the overall methane emissions of the treatment groupsThe release is still reduced by about 19 percent (P)<0.05), indicating the persistence of the effect (see fig. 1).

Throughout the experiment, calf physiological parameters (body weight (BW) and Average Daily Gain (ADG)) were similar in each group, indicating no adverse effect on early life performance (see tables 2 and 3). Furthermore, no significant changes in the relative abundance of methanogens were observed with 3-NOP treatment.

Table 1: the effect of 3-nitrooxypropanol supplementation on the methane emission (g/day) of calves at the late stage of weaning to 23 weeks and 56-60 weeks at the age of 11 weeks.

Table 2: calf Body Weight (BW) (kg)

No significant difference in body weight was observed.

Table 3: average Daily Gain (ADG) (kg/d) from week 0 to week 23

	ADG(kg/d)
		Control (n ═ 8)	0.84
Treatment (n as 10)	0.79

No significant difference in average daily weight gain was observed.

Detailed Description

It has been found that according to the invention, a continuous reduction of methane emission in supplemented animals can be achieved by supplementing calves with 3-nitrooxypropanol immediately after birth until three weeks after weaning (14 weeks old), even long after the supplementation of 3-nitrooxypropanol has ceased. Treated animals showed lower methane emissions even after 60 weeks of age, indicating that the effect persists until later life. Interestingly, the total number of methanogens was not reduced. Furthermore, no adverse effects on the performance of the host animals were observed.

Thus, in a first embodiment, the present invention relates to a method of reducing methane emissions in adult ruminants, the method comprising administering to the ruminant an effective amount of 3-nitrooxypropanol at the pre-ruminant stage, and optionally evaluating the methane-reducing effect, preferably the long-term methane-reducing effect. Optionally, administration is extended to a stage of young ruminants.

Even more preferably, the 3-nitrooxypropanol is administered to the ruminant from birth up to 6 months of age, preferably up to 5 months of age, most preferably up to 4 years of age.

The present invention also relates to a method of reducing methane emissions in adult ruminants, the method comprising administering an effective amount of 3-nitrooxypropanol to a healthy newborn ruminant in which reduction of methane emissions is desired as an adult ruminant.

Another aspect of the invention is the reduction of methane from digestive activities of healthy ruminants by the use of orally administered 3-nitrooxypropanol in the pre-ruminant stage, optionally extended to the young ruminant stage, of said ruminants.

Another aspect of the invention is the (non-therapeutic) use of 3-nitrooxypropanol in pre-ruminants to mitigate climate change (i.e. to help reduce global atmospheric warming) by reducing the lifetime total methane emissions (as compared to untreated ruminants) of said ruminants.

The invention also relates to a feeding method for reducing methane emissions in adult ruminants and/or for reducing the lifetime total methane emissions of ruminants (compared to corresponding untreated ruminants), comprising:

(1) administering a diet comprising 3-nitrooxypropanol to a ruminant at a pre-ruminant and optionally young ruminant stage, and then,

(2) adult ruminants were given a diet without 3-nitrooxypropanol.

The present invention also includes a kit for reducing methane emissions in a ruminant animal, the kit comprising:

a.) at least one dosage form of 3-nitrooxypropanol, preferably an oral dosage form; and

b.) instructions to demonstrate to a potential user the benefit of administering 3-nitrooxypropanol to a ruminant in a pre-ruminant stage and optionally a young ruminant stage to reduce methane emissions in an adult ruminant.

In another embodiment, the invention relates to a low methane emission ruminant, characterized in that said ruminant is obtained by supplementing said ruminant with 3-nitrooxypropanol at the pre-ruminant stage. Said ruminants are characterized in particular by the fact that: the ruminant has a lower lifetime total methane emission compared to a corresponding untreated ruminant.

In another embodiment, the present invention relates to a method of affecting the microbial colonization pattern of the rumen of a ruminant, said method comprising the step of administering to said ruminant an effective amount of 3-nitrooxypropanol at the pre-ruminant stage.

Definition of

The so-called "pre-ruminant phase" is the time during which digestive functions in ruminant life are more similar to those of monogastric animals. The pre-ruminant stage typically occurs during the first few weeks after birth of the calf, when the calf ingests primarily milk or milk replacers. The pre-ruminant stage may be shorter or longer depending on the type of food provided and the intended use of the animal. During the second phase, the so-called young ruminant phase or the "post-weaning phase", the rumen is developing, but still does not fully reach the level of efficiency of an adult animal. The final stage of adult ruminant development occurs when the Rumen is considered fully functional, typically at 12 weeks of age (see, e.g., Teagosc 2017: Rumen depth, Section 4; Chapter 12, page 60(https:// www.teagasc.ie/media/web/publications/2017/Section 4-Rumen-depth. pdf) or reading Young storage media farm in Asia (John, Moran), channel 3: digest of feeds in the mill feed: page 27).

Within the meaning of the present invention, the term "pre-ruminant stage" refers to an animal that is still (at least partially) not yet weaned, whereas the term "young ruminant" refers to a stage where the rumen is still not fully developed, but the calf has been weaned (i.e. no longer receiving any milk).

Ruminants, as used herein, are animals of the order Artiodactyla of the mammalia class, which digest vegetable-based feed, first soften the vegetable-based feed in the first stomach of the animal, called the rumen, and then ruminate the semi-digested material, now called ruminant (cud), and re-chew. The process of chewing the cud again to further break down plant matter and stimulate digestion is called "rumination". Ruminants include cows, goats, sheep, giraffes, deer, gazelle, and antelope. Preferred ruminants for all embodiments of the present invention are domestic cattle, domestic sheep and domestic goats. In all embodiments according to the present invention, the most preferred ruminants are cattle and calves thereof. The term "cattle" includes all breeds of domesticated cattle and all production types of cattle (e.g., cows, grazing cattle and beef cattle). Grazing cattle are particularly preferred because they are difficult to obtain supplementation later in life (i.e., the year).

As used herein, a "methanogen" is a microorganism that produces methane as a metabolic byproduct. They are prokaryotes and belong to the field of archaea. Generally, they are also known as methanogenic archaea. They are present in the rumen along with various bacteria, protozoa and fungi.

An "anti-methanogenic compound" is a compound that can be used to inhibit the activity of such methanogenic archaea to reduce methane emissions, such as methane derived from the digestive activities of ruminants.

3-Nitrooxypropanol (CAS-No: 100502-66-7) is a known compound which can be prepared, for example, as described in WO-2004043898 or WO-2012084629. Suitable formulations thereof are disclosed, for example, in WO-2018149755, WO-2018149756, WO-2018091643 or WO-2019007740.

As used herein, the term "feed" refers to any compound, formulation, mixture, or composition suitable for or intended for ingestion by a corresponding animal and is not limited solely to nutritional materials.

As used herein, the term "edible oil or solvent" 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, canola oil, sunflower oil, Medium Chain Triglycerides (MCT) and glycerol and mixtures thereof.

Methane emission and methane reduction as referred to herein can be measured in individual animals in a metabolic chamber (metabolic chamber) by methods known in the art (Grainger et al, 2007j. dairy Science; 90: 2755-. Furthermore, evaluation can be performed at the stall level by emerging techniques using laser beams (McGinn et al, 2009, Journal of environmental-technical Quality; 38: 1796-. Alternatively, the GreenFeed system (C-Lock, Rapid City, SD USA) can be used to assess methane production by ruminants, which is preferred.

The term "microbial colonization pattern" refers to a specific composition of rumen microorganisms (also known as the rumen microflora) that is formed within the first hours, days and weeks of a ruminant animal and then persists throughout the animal's life cycle. The rumen microbiota is mainly composed of bacteria, archaea (e.g. in particular methanogenic archaea), protozoa and fungi.

As used herein, the term "from birth" refers to a time span of up to 3 days after birth, preferably up to 2 days after birth, most preferably up to 1 day after birth. Most preferably, the supplementation of 3-nitrooxypropanol is started from the first supplementation of the feed, not by lactation.

Dosage and administration

The 3-nitrooxypropanol is administered at least at the pre-ruminant stage. More preferably, however, the administration of 3-nitrooxypropanol is extended at least partially to the post-weaning (i.e. young ruminant) phase, e.g. further administration for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks.

Thus, advantageously, 3-nitrooxypropanol is administered to ruminants from birth up to 8 weeks after weaning.

Alternatively, in all embodiments of the invention, 3-nitrooxypropanol is administered to the ruminant, preferably from birth to up to 6 months of age, preferably to up to 5 months of age, most preferably to up to 4 months of age, e.g. to up to 3 months of age.

In all embodiments of the invention, it is preferred to stop the supplementation of 3-nitrooxypropanol completely after said determined administration period (i.e. not extending to adulthood).

The 3-nitrooxypropanol may be administered daily or every two days or weekly. Preferably, in all embodiments of the invention, the 3-nitrooxypropanol is administered daily, e.g. at least once daily.

Furthermore, preferably, 3-nitrooxypropanol is orally administered to pre-ruminant/young-ruminant animals.

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

In another and more preferred embodiment, the amount of 3-nitrooxypropanol administered to the pre-ruminant/young ruminant is selected in the range of 0.1mg to 15mg 3-NOP per kg Body Weight (BW), more preferably in the range of 0.5 to 10mg 3-NOP per kg BW, most preferably in the range of 1 to 5mg 3-NOP per kg BW, e.g. in the range of 2mg to 4mg 3-NOP per kg BW.

It will be appreciated by those skilled in the art that the total (daily) amount of 3-nitrooxypropanol fed may vary depending on the animal species, animal size and type of feed to which the 3-nitrooxypropanol or formulation or composition thereof is added.

The total (daily) amount of 3-nitrooxypropanol may be administered in a single dose, or in multiple doses, such as one dose in the morning and one dose in the evening.

In all embodiments of the invention, 3-nitrooxypropanol may be used as such. Preferably, however, the 3-nitrooxypropanol is used in the form of its formulation, i.e. in the form of a composition comprising the 3-nitrooxypropanol and a suitable carrier material (also referred to herein as "3-NOP formulation"). The 3-nitrooxypropanol or formulation thereof may be administered to the pre-ruminant/young ruminant as such or may be mixed into the pre-ruminant/young ruminant feed.

Suitable carriers for the 3-NOP formulation include any well known in the food and feed industry such as water, edible oils or solvents such as, preferably, propylene glycol, silica (silica) silica (silica dioxide), diatomaceous earth and sepiolite, but are not limited thereto. The most preferred carrier materials of the present invention are silica, propylene glycol or water and mixtures thereof.

The 3-NOP formulation may be formulated in any form suitable for direct administration to a pre-ruminant/young ruminant or for incorporation into a pre-ruminant/young ruminant feed. Suitable solid forms for direct administration or mixing are, for example, powders, granules or pills. Suitable liquid forms for direct administration or mixing are, for example, solutions or suspensions. The 3-NOP formulation may also be in the form of a controlled (delayed) release formulation.

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

The 3-NOP formulation of the invention is preferably administered in admixture with or together with feed for pre-ruminants or young ruminants.

Solid 3-NOP formulations

In an advantageous embodiment, the 3-NOP formulation is a solid formulation, such as a dry, free-flowing powder (also referred to herein as a 3-NOP powder formulation) or pellets comprising 3-nitrooxypropanol, preferably comprising 3-NOP and a solid carrier material. Such solid formulations are suitable for direct feeding to pre-ruminant/young-ruminant animals, or for inclusion in commercially available pre-ruminant/young-ruminant animal feeds or as a supplement to a total mixed ration or diet. When supplemented by pre-ruminant/young-ruminant feed, the solid formulation is typically mixed with solid or liquid feed or with (drinking) water.

Powdered formulations comprising 3-nitroxylpropanol and a solid carrier material are typically prepared by mixing 3-NOP with a solid carrier material by methods standard in the art. Alternatively, the 3-NOP may be sprayed onto a solid support material. For example, the 3-NOP may be sprayed onto the solid carrier material using a solvent suitable for preparing food or feed products, such as dichloromethane, and the organic solvent evaporated.

Particularly suitable solid carriers for the preparation of 3-NOP powdered formulations include, but are not limited to, silica (silica), diatomaceous earth and sepiolite. The most preferred solid support material for the present invention is silica.

This silica is a well-known carrier material in the feed and food industry and refers to white microspherical amorphous silica (also known as silica). Particularly suitable silicas of the present invention are amorphous precipitated silicas such as Ibersil D-250 from IQE group, Sipernat 2200 from Evonik or Tixosil 68 from Solvay.

It is well known that 3-NOP may be diluted in a suitable edible oil or solvent (liquid carrier) before being sprayed onto or mixed with a solid carrier material.

Furthermore, the 3-NOP pulverous formulation of the invention may additionally comprise usual additives used in the preparation of pulverous formulations for feeds, such as in particular thickeners, advantageously selected from gums or cellulose derivatives such as xanthan gum, karaya gum and/or ethylcellulose and (traces) of water.

The amount of 3-nitrooxypropanol in the 3-NOP pulverous formulation of the invention is preferably selected in the range of 1 to 20 wt. -%, preferably in the range of 2 to 15 wt. -%, most preferably in the range of 4 to 12 wt. -%, based on the total weight of the formulation.

Preferably, at least one edible oil or solvent is present in the 3-NOP pulverous formulation of the present invention. The amount of edible oil and/or solvent in such powdered 3-NOP formulations is preferably selected in the range of 0.1 to 50 wt. -%, preferably in the range of 1 to 45 wt. -%, most preferably in the range of 10 to 40 wt. -%, based on the total weight of the formulation.

Particularly suitable 3-NOP powder formulations of the invention consist essentially of 3-nitrooxypropanol, propylene glycol and silica (silicon dioxide), for example consisting essentially of: (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 disclosed, for example, in WO-2018149756 or WO-2018149755.

Alternatively, the 3-nitrooxypropanol may be supplemented in the form of a compressed tablet, for example as disclosed in WO-2019007740.

Liquid 3-NOP formulations

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

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

It is well known that the 3-NOP liquid formulation of the present invention may further comprise common additives used in the preparation of liquid formulations for feed or drinking water additives for ruminants, such as vitamins, sugars, salts and flavorings.

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

Particularly suitable 3-NOP liquid formulations consist essentially of a 10 to 30% by weight solution of 3-nitrooxypropanol in water and/or propylene glycol.

Feed for pre-ruminant/young ruminant

To achieve the use according to the invention, 3-nitrooxypropanol or its formulations, such as preferably its powder or liquid formulation, can be incorporated into pre-ruminant/young-ruminant feed by methods known per se in the field of feed formulation and processing.

Thus, other aspects of the invention relate to pre-ruminant and/or young ruminant feed containing 3-nitrooxypropanol or a formulation thereof.

Suitable feeds for pre-ruminants are milk or milk substitute preparations (milk substitutes). Such milk and milk substitute formulations comprising 3-nitrooxypropanol or formulations thereof remain new.

The invention therefore also relates to a milk or milk substitute for pre-ruminants, characterized in that it comprises 3-nitrooxypropanol or a preparation thereof.

The term milk or milk substitute refers to whole milk, reduced fat milk and skim milk, marketing milk and reconstituted milk substitutes. "marketing milk" is defined as milk that cannot be sold for human consumption by law. The milk substitute is a formulated feed intended to replace the milk of a cow during the critical, early lactation or breastfeeding stage of life for a calf.

The milk replacer typically comprises 18 to 30 wt.% protein and 10 to 28 wt.% fat, and optionally further additives and/or medicaments, wherein 18 wt.% to 22 wt.% is the most preferred fat content. The milk replacer was reconstituted with water, mixed and fed according to the manufacturer's instructions.

Exemplary protein sources for use in the milk replacer are milk proteins such as dried whey protein concentrate, dried whey product, skimmed milk, dried skimmed milk powder, casein and sodium or calcium caseinate. Sources of alternative proteins include soy protein isolate, protein modified soy flour, soy protein concentrate, soy flour, animal plasma, and wheat gluten or isolate, pulse protein, and pulse protein isolate. Suitable fats include vegetable and animal fats and fractions thereof. Exemplary additives are in particular carbohydrates, mainly from lactose, amino acids, (essential) vitamins and trace minerals and other materials considered acceptable by local authorities for use in animal feed.

The amount of 3-nitrooxypropanol in the milk or reconstituted milk is preferably selected in the range of from about 1mg to about 1000mg, preferably from about 1mg to about 500mg, most preferably from about 1mg to 250mg of 3-NOP per litre of milk or reconstituted milk.

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

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

After weaning, the 3-nitrooxypropanol or the composition or formulation comprising 3-nitrooxypropanol of the invention is preferably mixed into a corresponding feed for a young ruminant, for example preferably a concentrated feed for a young ruminant. Such a concentrate should preferably be rough in texture, high in energy and protein content, and low (> 15%) in roughage. Cereals (oat, barley, wheat, corn, triticale) may also be used in admixture with molasses, dehydrated alfalfa or grass pellets, bran or other by-products from the food, beverage and feed industries.

The young ruminant feed of the present invention may also contain conventional young ruminant feed additives and adjuvants, excipients or diluents, including, but not limited to, water, molasses, calcium carbonate, electrolytes such as ammonium chloride, proteins such as soybean meal, rapeseed meal, lentils, lupins, peas, legume wheat, starch, sunflower seed meal, corn, amino acids, fats, vitamins and trace minerals and other materials recognized by local authorities as useful in animal feed.

Typically, a feed for a young ruminant will be formulated from, but not limited to, the following ingredients: barley, wheat, triticale, oats, peas, lupins, lentils, beans, soybeans, rape, sunflowers and products derived from these ingredients. Vegetable oils, molasses, limestone, dicalcium phosphate, salts, bentonite, magnesium oxide, acidifying agents, lysine, methionine, antioxidants and anticoccidial agents and other drugs may also be included. Certain by-products may also be included, such as Palm Kernel Extract (PKE), corn flakes, and distillers grains.

The amount of 3-nitrooxypropanol in the feed for young ruminants is preferably selected in the range of from about 1mg to about 1000mg, preferably from about 1mg to about 500mg, most preferably from about 1mg to 250mg of 3-NOP per litre of milk or reconstituted milk.

It is well known that the amount of 3-NOP administered may be further adjusted to the specific needs of the individual pre-ruminant/young ruminant to be treated, e.g., calf, lamb or goat.

In a particularly advantageous embodiment of the invention, the 3-nitrooxypropanol is supplemented in the form of a liquid preparation, for example incorporated into milk or a liquid milk substitute preparation (i.e. reconstituted milk).

Thus, the present invention also relates to a method of supplying a liquid feed for pre-ruminants, most preferably milk or a liquid milk substitute formulation comprising 3-nitrooxypropanol or a formulation thereof, to a pre-ruminant calf, the 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.

In a further 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 (non-therapeutic) long-term, long-lasting reduction of methane emission by ruminants during their adult life's digestive activities and/or for reduction of ruminant lifetime total methane emission, preferably wherein additional administration of 3-nitrooxypropanol to adult ruminants is not required or desired.

The present invention further provides a method for the long-term, sustained reduction of methane produced by the digestive activity of an adult ruminant, the method comprising orally administering to a pre-ruminant and/or a young ruminant a sufficient amount of a pre-ruminant and/or a young ruminant feed comprising 3-nitropropanol or a composition or formulation thereof.

The present invention solves the problems unsolved so far: with the aid of the present invention, the gastrointestinal tract of pre-ruminant/young ruminants is adjusted in such a way that the lifetime total methane emission of said ruminants is lower than that of the respective untreated ruminants. This means, inter alia, that even after the supplementation of 3-nitrooxypropanol or a composition or formulation thereof has ceased, the methane reduction (compared to a corresponding untreated ruminant) persists into the adult life of the ruminant and is programmed during the adult life of the ruminant.

Thus, 3-nitrooxypropanol may be used as an active ingredient in feed additives and animal feed compositions for pre-ruminants and young ruminants and thus may be used as an active ingredient to adapt long term to the gastrointestinal tract of pre-ruminants/young ruminants, continuously reducing methane emissions (compared to untreated animals), in particular by all methods and uses disclosed herein.

In all embodiments of the invention, the long-lasting methane reduction is preferably characterized by a methane reduction of at least 5%, more preferably at least 10%,most preferably at least 15% (in g CH)₄// day). It is understood that the control group is the same breed raised under the same management conditions (same farm, same herd, similar genetic and vaccination origin and similar age).