Novel composition

Abstract

The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to a combination of 3-nitrooxypropanol and / or derivatives thereof in the drinking water and to its administration to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

Description

[0001] DSM IP Assets B.V., 34816-EP-EPA121

[0002] NOVEL COMPOSITION

[0003] The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to a combination of 3-nitrooxypropanol and / or derivatives thereof with improved storage-stability and to its administration to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

[0004] The present invention further relates to a liquid composition of 3-nitrooxypropanol and / or derivatives thereof, wherein the liquid composition preferably comprises 3-ni- trooxypropanol and / or derivatives thereof in water, as well as the use of said composition in the drinking water of ruminants for reducing the production of methane emanating from the digestive activities of ruminants.

[0005] The temperature of the air surrounding the earth is increasing, a process referred to as global warming. One of the main focuses to reduce this warming effect is to reduce the amount of greenhouse gases emitted into the atmosphere. Greenhouse gases are emitted from several different sources, both natural and artificial; however, the two sources with the most emphasis are the agricultural and fossil fuel industries. Within agriculture, ruminants and in particular cattle are the major contributors to the biogenic methane formation, and it has been estimated that the prevention of methane formation from ruminants would almost stabilize atmospheric methane concentrations.

[0006] Methane emission from the ruminant livestock sector — a by-product from enteric fermentation of plant biomass in the ruminant digestive system — is produced by methanogenic archaea. Various attempts have been made in the last decade to mitigate methane production from ruminant animals. Although the approaches vary, the most popular method so far are feed additives which act in the rumen fluid by reducing respectively inhibiting the methane production by methanogenic archaea.

[0007] It has been found, that the use of 3-nitrooxypropanol and / or derivatives thereof leads to a substantial reduction of the methane formation emanating from the digestive 34816-EP-EPA activities of said ruminant. However, 3-nitrooxypropanol and / or derivatives thereof have been found not to be effectively retained under conventional storage conditions. The lack of retention of the active in conventional product forms for the feed industry is, however, highly unwanted as accordingly an appropriate dosage is not possible without undue burden, i.e. sophisticated packaging, analysis of the active content before use or overdosing. Moreover, all these methods add significant additional costs- in-use which are not readily accepted by the end user.

[0008] Thus, there is an ongoing need for product forms and methods, which overcome the above-mentioned problems by enabling the storage over a period of time without significant losses of the active, i.e. of 3-nitrooxypropanol respectively derivatives thereof.

[0009] It has surprisingly been found that that drinking water supplementation with 3-nitroox- ypropanol to ruminants results in a substantial reduction in CH4 production, wherein a lower concentration of 3-nitrooxypropanol showed a higher methane reduction throughout the day compared to higher concentration of 3-nitrooxypropanol. The reduction of treatments provided in drinking water are related to sustained and long inhibition even after feeding times

[0010] Thus, the invention relates in a first aspect to a liquid composition of 3-nitrooxypropa- nol and / or derivatives thereof, preferably a composition comprising 3-nitrooxypropa- nol and / or derivatives thereof in water, for reducing the formation of methane emanating from the digestive activities of ruminants.

[0011] Thus, the use of a combination of 3-nitrooxypropanol and / or derivatives thereof has a great potential in the mitigation of climate change by significantly reducing the methane emissions emanating during the digestive activities of ruminants.

[0012] Therefore, in a first embodiment, the present invention provides a composition comprising 3-nitrooxypropanol and / or derivatives thereof. Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof is a liquid. Preferably, the 34816-EP-EPA liquid composition comprises 3-nitrooxypropanol and / or derivatives thereof in water. The liquid composition may be diluted further before administration to the animal.

[0013] In a second embodiment, the invention further provides the use of a combination of 3-nitrooxypropanol and / or derivatives thereof for reducing the formation of methane emanating from the digestive activities of ruminants.

[0014] In a third embodiment, the invention further provides a method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the animal a combination of 3-nitrooxypropanol and / or derivatives thereof, said method comprising orally administering to the animal in the drinking water 3-nitrooxypropanol and / or derivatives thereof in an amount of at least 0.1 g 3-nitrooxypropanol and / or derivatives thereof / animal / day.

[0015] It is well understood, that in all embodiments of the present invention the methane reduction by the administration of a combination of 3-nitrooxypropanol and / or derivatives thereof is preferably at least 10 %, more preferably at least 20 %, most preferably at least 30 % when compared to control, i.e. to ruminants not supplemented with a combination of 3-nitrooxypropanol and / or derivatives thereof.

[0016] Thus, the present invention also relates to the use of a combination of 3- nitrooxypropanol and / or derivatives thereof, wherein the methane production in ruminants is reduced by at least 10 % compared to control.

[0017] Preferably, 3-nitrooxypropanol and / or derivatives thereof is 3-nitrooxypropanol (CAS- No: 100502-66-7). 3-nitrooxypropanol and / or derivatives thereof may be applied to the animal in the drinking water of the animal.

[0018] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof, is a liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water. 34816-EP-EPA

[0019] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof, is a liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water, wherein the concentration of 3-nitrooxypropanol and / or derivatives thereof in water is at least 0.00001 wt%, preferably at least 0.00002 wt%, preferably at least 0.00003 wt%, preferably at least 0.00003 wt%, preferably at least 0.00004 wt%, preferably at least 0.00005 wt%, preferably at least 0.00006 wt%, preferably at least 0.00007 wt%, preferably at least 0.00008 wt%, preferably at least 0.00009 wt%, preferably at least 0.0001 wt%, preferably at least 0.0002 wt%, preferably at least 0.0003 wt%, preferably at least 0.0004 wt%, preferably at least 0.0005 wt%, preferably at least 0.0006 wt%, preferably at least 0.0007 wt%, preferably at least 0.0008 wt%, preferably at least 0.0009 wt%, preferably at least 0.001 wt%.

[0020] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof, is a liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water, wherein the concentration of 3-nitrooxypropanol and / or derivatives thereof in water is below 0.05 wt%, preferably below 0.04 wt%, preferably below 0.03 wt%, preferably below 0.02 wt%, preferably below 0.01 wt%, preferably below 0.009 wt%, preferably below 0.008 wt%, preferably below 0.007 wt%, preferably below 0.006 wt%, preferably below 0.005 wt%, preferably below 0.007 wt%, preferably below 0.006 wt%, preferably below 0.005 wt%, preferably below 0.004 wt%, preferably below 0.003 wt%, preferably below 0.002 wt%, preferably below 0.001 wt%.

[0021] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof, is a liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water, wherein the concentration of 3-nitrooxypropanol and / or derivatives thereof in water is 0.00001 to 0.05 wt%, preferably 0.00002 to 0.05 wt%, preferably 0.00003 to 0.05 wt%, preferably 0.00003 to 0.05 wt%, preferably 0.00003 to 0.04 wt%, preferably 0.00003 to 0.02 wt%, preferably 0.00004 to 0.02 wt%, preferably 0.00005 to 0.02 wt%, preferably 0.00006 to 0.02 wt%, preferably 0.00007 to 0.02 wt%, preferably 0.00008 to 0.01 wt%, preferably 0.00009 to 0.01 wt%, preferably 0.0001 to 0.01 wt%, preferably 0.0001 to 0.01 wt%. 34816-EP-EPA

[0022] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof in water, comprises 3-nitrooxypropanol and / or derivatives thereof in a different concentration depending on if it is intended for administration to Dairy cattle, dairy, ewes or goats or to Beef cattle or ruminating lambs.

[0023] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof, is a liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water for administration to Dairy cattle, dairy and / or ewes, wherein the concentration of 3-nitrooxypropanol and / or derivatives thereof in water is 0.0001 to 0.01 wt%, preferably 0.0002 to 0.01 wt%, preferably 0.0003 to 0.01 wt%, preferably 0.0003 to 0.009 wt%, preferably 0.0003 to 0.008 wt%, preferably 0.0003 to 0.007 wt%.

[0024] Preferably, the composition comprising 3-nitrooxypropanol and / or derivatives thereof, is a liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water for administration to Beef cattle and / or ruminating lambs, wherein the concentration of 3-nitrooxypropanol and / or derivatives thereof in water is 0.0001 to 0.05 wt%, preferably 0.0002 to 0.05 wt%, preferably 0.0003 to 0.05 wt%, preferably 0.0004 to 0.05 wt%, preferably 0.0004 to 0.04 wt%, preferably 0.0004 to 0.03 wt%, preferably 0.0004 to 0.02 wt%.

[0025] The composition comprising 3-nitrooxypropanol and / or derivatives thereof preferably is administered to the ruminants, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered in an amount selected in the range from 0.01 to 3 g 3-ni- trooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.02 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.03 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 3 g 3-nitrooxypropa- nol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.9 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.8 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.7 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.6 g 3- 34816-EP-EPA nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.05 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.06 to 2.5 g 3-nitroox- ypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.07 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.08 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.09 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.4 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.2 g 3-nitrooxypropa- nol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.1 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.2 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.3 to 2 g 3-nitrooxypropa- nol and / or derivatives thereof / animal / day, more preferably in the range from 0.4 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.5 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.6 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.7 to 2 g 3-nitrooxypropa- nol and / or derivatives thereof / animal / day, more preferably in the range from 0.8 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.9 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 1 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day.

[0026] The composition comprising 3-nitrooxypropanol and / or derivatives thereof preferably is administered to Dairy cattle, dairy and / or ewes, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered in an amount selected in the range from 34816-EP-EPA

[0027] 0.1 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.1 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.1 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.2 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.3 to 2 g 3-ni- trooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.4 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.5 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.6 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, preferably in the range from 0.7 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.8 to 2 g 3-nitrooxypro- panol and / or derivatives thereof / animal / day.

[0028] The composition comprising 3-nitrooxypropanol and / or derivatives thereof preferably is administered to Beef cattle and / or ruminating lambs, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered in an amount selected in the range from 0.1 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.1 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.1 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.2 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.3 to 2 g 3-ni- trooxypropanol and / or derivatives thereof / animal / day, preferably in the range from 0.4 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day.

[0029] The combination of 3-nitrooxypropanol and / or derivatives thereof is preferably administered through the drinking water of the animal.

[0030] When ruminants are stressed, such as in heat stress or during lactation, they consume larger amounts of drinking water. Such special cases lead to the liquid composition comprising 3-nitrooxypropanol and / or derivatives thereof in water to be dosed down to a lower concentration of 3-nitrooxypropanol and / or derivatives thereof to keep the desired amount of 3-nitrooxypropanol and / or derivatives thereof / animal / day. 34816-EP-EPA

[0031] The term ‘liquid composition’ as used herein refers to a formulation which is liquid at ambient temperature (i.e. about 22°C) (i.e. a solution, wherein the 3-nitrooxypropanol and / or derivatives thereof is completely solubilized), preferably dissolved in water.

[0032] The drinking water may optionally comprise further nutritional ingredients selected from the group consisting of such as vitamins, trace minerals and macro minerals, preferably water-soluble vitamins, macro minerals, and / or trace minerals.

[0033] The following are non-exclusive lists of examples of these components:

[0034] Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D- panthothenate.

[0035] Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, manganese, and cobalt.

[0036] Examples of macro minerals are calcium, phosphorus, potassium, magnesium and sodium.

[0037] Methane emission by ruminants can easily be measured in individual animals in metabolic chambers by methods known in the art (Grainger et al., 2007 J. Dairy Science; 90: 2755-2766). Moreover, it can also be assessed at barn level by an emerging technology using laser beam (McGinn et al., 2009, Journal of Environmental Quality; 38: 1796-1802) or Sulfur hexafluoride or just SF6 or GreenFeed system. Alternatively, methane produced by a dairy ruminant can also be assessed by measurement of fatty acid profiles in milk according to WO 2009 / 156453.

[0038] Ruminating mammals according to the present invention include cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and Nilgai.

[0039] For all embodiments of the present invention, domestic cattle, sheep and goat are the more preferred species. For the present purposes most preferred species are 34816-EP-EPA domestic cattle. The term includes all races of domestic cattle, and all production kinds of cattle, in particular dairy cows and beef cattle. It is well understood, that the term dairy cows and beef cattle encompasses animals in all ages and physiological stage of life and production systems such as confined, semi-confined and grazing.

[0040] Figures

[0041] Figure 1 : CH4 profile related to feed and water intakes comparing all treatments in animal 1 over 24 hrs. The dotted line indicates feeding times (9:00 and 13:30 hours). Figure 2: CH4 profile related to feed and water intakes comparing all treatments in animal 2 over 24 hrs. The dotted line indicates feeding times (9:00 and 13:30 hours). Figure 3: CH4 profile related to feed and water intakes comparing all treatments in animal 3 over 24 hrs. The dotted line indicates feeding times (9:00 and 13:30 hours). Figure 4A: Diurnal dynamics of CH4 emissions (symbols above curves indicated significant differences between treatments (p<0.05).

[0042] Figure 4B: Diurnal dynamics of H2 emissions (symbols above curves indicated significant differences between treatments (p<0.05).

[0043] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0044] Examples

[0045] Example 1

[0046] Efficacy of 3-NOP applied in Drinking Water on Enteric Methane Reduction in Sheep The objective of the study was to evaluate the effect of 2 different doses of 3-nitroox- ypropanol (3-NOP), when applied in drinking water, on the enteric methane (CH4) and hydrogen (H2) emissions in sheep.

[0047] Methodology

[0048] Treatments

[0049] Two formulations were used: 34816-EP-EPA

[0050] A liquid preparation of 3-NOP (minimum 23% w / w) on propylene glycol was prepared. 2 different doses of the liquid preparation (DW80 & DW150) were mixed with the daily water offered.

[0051] Animals were trained to the application of delivery method via drinking water two weeks prior to trial start.

[0052] The following treatments and dosages were tested.

[0053] 1. Control: No supplementation but application of syringe with molasses.

[0054] 2. DW80: supplementation referred to 521.74 mg / day of liquid preparation of 3-NOP, calculated per dose of 80 mg / kg DM intake, based on an estimated DMI of 1 .5 kg / d. The total amount of daily water was estimated based on the average water intake of the 2 previous days.

[0055] 3. DW150: supplementation referred to 978.26 mg / day of liquid preparation of 3-NOP, calculated per dose of 150 mg / kg DM intake, based on an estimated DMI of 1 .5 kg / d. The total amount of daily water was estimated based on the average water intake of the 2 previous days.

[0056] Study design

[0057] 3 non-lactating female healthy adult sheep were tested. The experimental periods consisted of 7 days of adaptation to the treatments and 3 days (periods 1 and 2) or 4 days (periods 3 and 4) of gas production measurements (CH4, H2, and CO2) in the respirometry chambers, followed by a wash-out period of 4 days. The additional measurements day for periods 3 and 4 is due to some drinking water time points data recovery issues and to ensure proper data in a 24-hour span. Animals were housed individually throughout the experiment to monitor daily dry matter and water intakes.

[0058] Feeding management

[0059] Animals were offered a diet consisting of 70 % grass hay and 30 % concentrate, they were fed twice daily at 9:00 and 13:30 hours, (e.g.,500 g of hay and 250 g of concentrate per feeding). 34816-EP-EPA

[0060] Water management

[0061] Animals were provided with new water daily at 9:00 hours. The total amount of daily water supplied was estimated based on the average water intake of the 2 previous days. Animals in the Control treatments were offered only water. Animals in DW80 and DW150 were offered water plus a concentration of liquid preparation of 3-NOP, which was calculated according to the treatments and dosages laid out above. The total water intake was recorded daily, and the number and duration and times of drinking events were monitored thanks to the presence of an electromagnetic caudalimeter (Endress+Hauser, Spain) located in the pipeline of the respirometry chambers. Voltage changes were transformed and stored in a data logger DaqLab (v. 1.40.01 , Omega, USA) for further analysis. Changes superior to 0.5 in voltage were considered as a drinking event. Daily volt data was used to calculate total water intake and the amount of intake per drinking.

[0062] Sampling and measurements

[0063] Enteric emissions (CH4 and H2) were measured in respirometry chambers over a period of 72h using 4 open circuit respiration chambers constructed of metal frame and polycarbonate (Figures 3 and 4) (Abecia et al., 2012; Martinez-Fernandez et al., 2014). Each chamber measured 1 .8 m wide x 1.8 m deep x 1.5 m tall. Chamber air temperature was 21.7 ± 0.66 °C throughout the experiment. Interruptions occurred daily at 0900 h, when the chamber floor was cleaned and the sheep received the morning feed, and at 1330 h, when animals received the afternoon feed. These interruptions had little effect on the daily emissions as they lasted for around 4 min for each chamber and occurred when the corresponding exhaust duct was not being sampled. Fluxes were calculated twice a day and then summed to derive the 24-h emission value. Airflow (L / min) and concentration of CH4 and H2 were measured for the intake and exhaust ducts of each chamber. Airstream in each duct was subsampled, and methane concentration was measured continuously using a gas analyzer (Ultramat 23, Siemens AG Karlsruhe, Germany). The measurements comprised cycles of 15 minutes, in which the air from each chamber was sampled and analysed 34816-EP-EPA for 3 minutes and then 3 more minutes to analyse the concentrations in the room, which was considered the basal level of CH4 and H2. The production of CH4 and H2 for each chamber was calculated for 2 days period of measurement from the fresh-air intake and chamber exhaust concentrations and mean airflow.

[0064] The daily CH4 and H2 emission values using 2 days per animal were then averaged for a given period and treatment to provide a single value per animal.

[0065] Statistical analyses

[0066] Data were analyzed in Python 3.10.12 (main, Nov 20 2023, 15:14:05) [GCC 11.4.0] via the Google Colab platform, including the libraries numpy (v. 1.25.2), pandas (v. 2.0.3), and scipy (v. 1.11 .4). The T test was used to assess the nulled hypothesis that treatments and controls samples have identical expected values. The function ttest_ind in the stats module of the scipy library was used to estimate the T test. Data were considered statistically different at a P value lower than 0.05 (P < 0.05).

[0067] Results

[0068] Sheep remained in good health throughout the experiment and did not show any sign of discomfort during the adaptation and measurement periods. There were no significant differences (P > 0.05) in the amount of feed or water intakes across treatments (Table 1 ). Methane production was significantly lower in DW80 and DW150 than in Control.

[0069] The analysis of the daily 24-h pattern of emissions helps to better understand the impact of each treatment. The CH4 profile (CH4 average concentrations over 24 h) for individual animals related to feed and water intakes comparing all treatments for different times intervals during the day covering the time after the morning feeding (t1 - 8:30-11 :00 and t2-11 :01 -13:30), afternoon feeding (t3-13:31 -16:00 and t4-16:01 - 18:30) and then the second half of the day (t5-18:31 -22:00, t6-22:01 -1 :30, t7-1 :31 - 5:00 and t8-5:01 -8:30) are shown in Figures 1 -3. The impact of treatments in the drinking water (DW80 and DW 150) lasted longer after intake, covering both the first and second interval after feeding times (t1 , t2, t3 and t4). 34816-EP-EPA

[0070] Conclusions

[0071] The drinking water supplementation with DW80 and DW150 to sheep fed a 70:30 forage: concentrate diet results in a substantial reduction in CH4 production (41 .4 and 35.7 % for DW80 and DW150, respectively). The reduction of treatments provided in drinking water are related to sustained and long inhibition after feeding times. The treatments DW80 and DW150 displayed a sharp reduction after feeding times and flat evolution for the rest of the day. In drinking water treatments the reduction of CH4 concentrations lasted long after the feeding times. Surprisingly, the lower concentration of 3-nitrooxypropanol (DW80) showed a higher methane reduction throughout the day compared to the higher concentration of 3-nitrooxypropanol (DW150) as shown in figures 1 -3. In none of the treatments dry matter or water intakes were impaired.

[0072] Example 2

[0073] In vitro test for methane production: A modified version of the “Hohenheim Forage value Test (HFT)” may be used for testing the effect of specific compounds on the rumen functions mimicked by this in-vitro system.

[0074] Principle: Feed is gadded into a syringe with a composition of rumen liquor and an appropriate mixture of buffers. The solution is incubated at 39°C. After 8 and 24 hours the quantity (and composition) of gas phase produced is measured and put into a formula for conversion.

[0075] Reagents:

[0076] Mass element solution:

[0077] 6.2 g potassium dihydrogen phosphate (KH2PO4)

[0078] 0.6 g magnesium sulfate heptahydrate (MgSCk * 7H2O)

[0079] 9 ml concentrated phosphoric acid (1 mol / l) dissolved in distilled water to 1 I (pH about 1 .6)

[0080] Buffer solution:

[0081] 35.0 g sodium hydrogen carbonate (NaHCOs)

[0082] 4.0 g ammonium hydrogen carbonate ((NH4)HCO3) dissolved in distilled water to 1 I 34816-EP-EPA

[0083] Trace element solution:

[0084] 13.2 g calcium chloride dihydrate (CaCl2 * 2H2O)

[0085] 10.0 g manganese(ll) chloride tetrahydrate (MnCl2 * 4H2O) 1 .0 g cobalt(ll) chloride hexahydrate (C0CI2 * 6H2O) 8.0 g iron(lll) chloride (FeCh * 6H2O) dissolved in distilled water to 100 ml

[0086] Sodium salt solution:

[0087] 100 mg sodium salt dissolved in distilled water to 100 ml

[0088] Reduction solution: first 3 ml sodium hydroxide (c = 1 mol / l), then 427.5 mg sodium sulfide hydrate (Na2S * H2O) are added to 71 .25 ml H2O solution must be prepared shortly before it is added to the medium solution

[0089] Procedure:

[0090] Sample weighing: The feed stuff (i.e. TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage) is sieved to 1 mm and weighed exactly into syringes. 4 of these syringes are the substrate controls, which display the gas production without the effect of the tested compounds. The remaining syringes contain the test substances, by groups of 4 syringes.

[0091] Preparation of the medium solution:

[0092] The components are mixed in a Woulff bottle in following order:

[0093] 711 ml water

[0094] 0.18 ml trace element solution

[0095] 355.5 ml buffer solution

[0096] 355.5 ml mass element solution

[0097] The completed solution is warmed up to 39 °C followed by the addition of 1 .83 ml sodium salt solution and the addition of reduction solution at 36 °C. The rumen liquor is added, when the indicator turns colourless.

[0098] Extraction of the rumen liquor: 750 ml of rumen liquor are added to approximately 1 ,400 ml of medium solution under continued agitation and CO2-gassing. 34816-EP-EPA

[0099] Filling the syringes, incubation and determining gas volumes and VFA values: The diluted rumen fluid (24 ml) is added to the glass syringe. The syringes are then incubated for 24 hours at 39 °C under gentle agitation. After 8 hours and 24 hours, the volume of gas produced at each timepoint is measured, and the percentage of methane in the gas phase at each timepoint is determined by gas chromatography.

[0100] Results. Data collected from this study is presented in Table 1 below.

[0101] Table 1 .

[0102] Example 3

[0103] Objectives. The study aimed to (1 ) investigate the effect of 3-NOP applied via water for drinking in dairy cows, on methane and hydrogen emission (proof of concept), and to (2) examine the diurnal pattern of methane and hydrogen emission when 3-NOP is administered through water for drinking. The synchronization effects between feed supply, 3-NOP delivery via water, and observed methane emissions were compared to a continuous intake of 3-NOP when provided through TMR.

[0104] Experimental Design. The experiment followed a double Latin-square design with 8 animals and 4 treatments.

[0105] Animals. In total, 8 lactating Holstein Friesian dairy cows were enrolled. These dairy cows originated from the dairy herd of the research facility ‘Dairy Campus’ (Leeuwar- den, the Netherlands). The dairy cows were in mid lactation (between 120 and 170 34816-EP-EPA days in milk at the start of the trial) and were clinically healthy. No heifers were enrolled.

[0106] Dietary Treatments. There were 4 dietary treatments: a) Placebo (negative control) (“CON”) b) BovaerW in TMR (positive control) with a target close of 60 mg 3-NOP / kg DM (“TMR”) c) 3-NOP water application at a target dose equivalent to 60 mg 3-NOP / kg DM as provided via feed (“Water LD”) d) 3-NOP water application at a target dose equivalent to 90 mg 3-NOP / kg DM as provided via feed (“Water HD”)

[0107] The basal TMR was identical for all treatments and consisted of grass silage, com silage, and concentrate (in meal form). For the positive control treatment (BovaerW in TMR), a fraction of the concentrate was replaced with a premix containing BovaerW. This premix had a similar ingredient composition as that of the concentrate (except for BovaerW) and was produced by Research Diet Services (Wijk bij Duurstede, the Netherlands). For the 2 treatments in which 3- NOP was applied in the water for drinking, 3-NOP was manually dosed into the water for drinking.

[0108] This trial had a double Latin-square design, with 4 cows each and 8 cows in total. Table 2 shows the order of the treatments given to each cow per experimental period.

[0109] Table 2. Order of the treatments given to the 8 dairy cows, divided over 2 Latin Square designs with 4 cows each 34816-EP-EPA

[0110] Feeding. The dairy cows were fed twice daily throughout the experiment, simultaneously with milking around 05:00h and 16:00h. Cows had ad libitum access to the TMR, targeting a 10% feed residual.

[0111] Drinking. The dairy cows had free access to clean water for drinking throughout the experiment. For the 2 control treatments (positive and negative), the water was provided directly from the drinking water infrastructure. For the 2 treatments in which 3- NOP was applied in the water for drinking, the water was stored in a large tank (~100L) to which 3-NOP was dosed. For these 2 treatments, the water and the 3-NOP were replenished daily.

[0112] Timeline. The experiment ran for 13 weeks and consisted of 8 experimental periods. Each experimental period lasted 19 days, consisting of 14 days of adaptation to their assigned dietary treatments in the tie stall of the dairy bam followed by 5 days of measurement in the climate respiration chambers. Group 1 and 2 (each consisting of 4 cows; each representing a Latin Square), and therefore the treatment periods, followed each other in a staggered approach, as shown in Table 3, due to the limited capacity of the climate respiration chambers (housing only 4 animals simultaneously). No covariate period was included because a negative control (placebo) was given to all cows.

[0113] Table 3. The experimental timeline of the double Latin-square design, with each Latin- square consisting of 4 dairy cows. “TS” represents the adaptation in the tie-stalls and “CRC” represents the measurement in the climate respiration chambers (CRC). 34816-EP-EPA

[0114] Measurements. The following measurements were taken (per individual animal):

[0115] - Clinical observations (incl. general health, morbidity, and mortality) daily.

[0116] - Water intake daily. During the adaptation period in the tie stall, this was measured on a daily basis (intake in L / d), whereas in the CRC, water intake was measured in a 10-second interval allowing to determine daily intake (L / d), amount of drinking events (number / d), time of drinking event, duration of drinking event (seconds), and intake per drinking event (L / event).

[0117] - Dry matter intake daily (in kg / d), both during the adaptation period and the measurement period.

[0118] - Milk yield daily, both during the adaptation period and the measurement period.

[0119] - Milk composition (i.e., milk fat, protein, lactose, and urea content, and somatic cell count) once weekly during the adaptation period in the tie stall, and daily during the measurement period in the CRC.

[0120] - Kinetics of methane and hydrogen emissions as well as daily emissions during the measurement period in the CRC.

[0121] - Chemical composition of the dietary components per measurement period.

[0122] - Samples of the TMR and water for 3-NOP recovery, per measurement period.

[0123] Analysis. Each cow was considered the experimental unit for all variables. All variables were averaged per cow per measurement period. Data was analyzed using the MIXED procedure using SAS (version 9.4, SAS Institute Inc.), containing treatment as fixed effect, and cow and period as random effects. Kinetic variables (e.g., emission and drinking water) were subjected to repeated measures ANOVA to account for repeated sampling within the same cow per treatment period into account. This model 34816-EP-EPA included the treatment and time as fixed effects, and cow and period as random effects. For both models, differences were considered significant at P < 0.050 and tendencies at 0.050 < P < 0.100. Multiple comparisons between treatment means were made using the Tukey-Kramer method when an effect of treatment (or time) is detected at P < 0.050.

[0124] Ftesu / ts. Postprandial peaks in methane production were absent for all 3-NOP treatments. Postprandial peaks in hydrogen were present for all 3-NOP treatments. 3-NOP treatments reduced methane emissions. Water intake was unaffected by 3-NOP. The data is presented below in Table 4. Additionally, Figures 4A and 4B show diurnal dynamics of methane and hydrogen emissions, respectively.

[0125] Table 4.

[0126] Example 4

[0127] Groups of fifteen animals were randomly assigned to one of four groups resulting in groups with an average live weight of 278 ± 15 kg (mean ± SD) with no difference 34816-EP-EPA between groups or treatments (Table 2). Animals grazed in four paddocks containing typical tropical and sub-tropical grasses and legumes. Each paddock included an individual GreenFeed Emission Monitoring (GEM) unit and animals were provided access to a single water point connected to a Smartdosing+ D25+ unit used to deliver 3-NOP or control treatment through drinking water.

[0128] The Smartdosing+ equipment recorded the amount of water consumed by the herd and the quantity of 3-NOP dose delivered throughout the day. Daily individual water intake estimates were calculated by dividing the total daily water consumed within a paddock by 15 animals.

[0129] Water consumption patterns by animals were broadly separated into a major drinking event in the morning between 0:07:00 and 11 :00:00 in which close to 50% of the daily water intake was consumed, followed by smaller intakes across the rest of the day with another ~ 25% consumed in the afternoon.

[0130] For block 1 the Smartdosing+ units consistently supplied the prescribed dose level of 3-NOP to the treatment groups with an average dose level of ~ 2.5 mg I kg LW per animal per day (Table 3, Figure 5). Pump errors with the Smartdosing+ units became frequent in block 2 and a portion of block 3 leading to dosing levels below the prescribed value. While errors were less prevalent in block 3, paddock 1 was compromised with a lower water intake from the trough due to the animals consuming the addition of mangoes in their diet (Table 2). Block 4 dosing levels returned close to the prescribed dose level but were confounded by a requirement to increase the 3-NOP stock solution to overcome the decrease in water intake due to a series of rain events. The stock solution dose was increased for the last week of block 4 only and would likely not be accurately reflected in any emission measurement due to the lower number of GreenFeed visits for this week period.

[0131] Table 5. Estimated daily 3-NOP intake for animals at each block and paddock 34816-EP-EPA

[0132] Paddock

[0133] Daily 3-NOP intake I7273747p-value2

[0134] Block 1 2.5 (0.01) 0.0 (0.00) 2.4 (0.01) 0.0 (0.00) <0.001

[0135] Block 2 0.0 (0.00) 1.9 (0.01) 0.0 (0.00) 1.7 (0.01) <0.001

[0136] Block 3 1.1 (0.01) 0.0 (0.00) 2.3 (0.01) 0.0 (0.00) <0.001

[0137] Block 4 0.0 (0.00) 3.0 (0.04) 0.0 (0.00) 2.3 (0.04) <0.001

[0138] 1Estimated daily 3 -NOP intake (mg / kg LW) calculated as total treated water consumed in paddock divided by 15 head, mean (std. error)

[0139] 2Kruskal-Wallis rank sum test

[0140] Animals consistently visited the GreenFeed units across the spread of a day and provided an average 2.66 ± 0.056 (mean ± se) valid emission measurements per day throughout the trial. There was some variance in the average daily visits for the block and paddocks, with paddock 1 consistently providing the least number of visits per day (Table 4).

[0141] Table 6. Average daily visits to a GreenFeed unit for animals at each block and paddock

[0142] Paddock

[0143] Daily visits I7273747p-value2

[0144] Block 1 1.7 (0.06) 2.6 (0.15) 3.1 (0.15) 2.1 (0.14) <0.001

[0145] Block 2 1.9 (0.09) 3.0 (0.10) 2.6 (0.11) 2.5 (0.09) <0.001

[0146] Block s 1.9 (0.05) 3.7 (0.12) 3.5 (0.09) 2.5 (0.09) <0.001

[0147] Block 4 2.2 (0.09) 2.4 (0.10) 2.0 (0.09) 2.4 (0.05) 0.007

[0148] 1Number of daily visits per animal per day, mean (std. error)

[0149] 2Kruskal -Wallis rank sum test 34816-EP-EPA

[0150] The majority of the emission measurements were collected between 06:00 and 19:00 hours, with extra measurements between 22:00 - 23:00 and 01 :00 - 02:00. This distribution of measurements across 24 hrs provides adequate coverage across the day to produce accurate estimates for daily emissions from grazing animals to account for grazing and ruminating.

[0151] There was a total of 9,143 valid GreenFeed measurements across the trial. However, the days in which animals were rotated between paddocks at the end of each block were removed resulting in 8,836 measurements across 75 days. Methane emissions from treated animals were observed to be directly linked with the dose rate of 3-NOP supplied through the Smartdosing + units.

[0152] A linear mixed-effects model was fitted to assess the effect of treatment on methane emissions (CH4) across the trial while accounting for the Animal, block and paddock design. Animals in the 3-NOP treatment groups showed an 11 % decrease in emissions, producing 131.06 g / day of CH4 which was significantly decreased by 16.72 g I day compared to the control groups 147.78 g I day.

[0153] Methane production and estimated methane yields for each block showed the greatest reduction was achieved in block 1 when treated animals had a consistent daily dose of 3-NOP at the target level of 2.5 mg / kg LW I day (Table 5). Treated animals were significantly inhibited by ~26.5 % producing less methane at 114 g / day compared to the control animals at 155 g / day. A similar result was observed for the methane yield estimate with a ~26% decrease in methane yield for treated animals. Hydrogen was observed to be significantly increased with levels of 5.6 g / day compared to the controls with 0.5 g / day. These results are consistent with previous trials and our understanding of the effect of methane inhibition on increased levels of hydrogen loss within the rumen.

[0154] Block 2 experienced some pump errors to the Smartdosing+ equipment resulting in the lowest dose rates of 3-NOP to the treated groups (Table 5). Methane production was still numerically lower in the treated group with a 5% decrease in methane 34816-EP-EPA production of 143 g / day compared to the control at 151 g / day. Likewise, there was only a minor decrease in estimated methane yield. The hydrogen production in block 2 was significantly higher at 1 .53 g / day compared to control animals at 0.74 g I day, however the treated animals were 3.6-fold less than block 1 hydrogen levels.

[0155] Block 3 also experienced some pump errors for the Smartdosing+ equipment, which again resulted in sub-optimal delivery of 3-NOP to the animals. Treated animals showed a ~13.5% decrease in both methane emissions and estimated methane yield. However, paddock 1 had a reduced level of water intake and at this time contained animals from a 3-NOP group for this block 3. Hydrogen was also significantly increased at 2.3 g / day, however was still half the level of block 1 .

[0156] Methane emissions in block 4 were not different between the treated and control animals and reflects the ~ 50% lower water consumption from the troughs in this period due to a series of rain events that increased moisture content of feed and provided small sources of surface water. To reach the target level of 2.5 mg I kg LW the concentration of the 3-NOP stock solution was increased in the last week of block 4 to counteract the decrease in water intake. Methane production from the 3-NOP treated animals for this week were lower, 136 g / day compared to the control at 153 g / day (p = 0.2) and hydrogen was observed to increase to 2.3 g I day compared to 1 .36 g / day for control animals (p < 0.001 ). The lower number of observations in this period (mean 2 visits per day I animal for 7 days) may not possess enough power to detect a true effect but numerically indicated an 11 % reduction in daily emission from 3-NOP animals. In addition, with only ~ 14 visits per animal across this week it is below the recommended 30 visits advised to be necessary based on the manufacturer’s guidelines to accurately estimate emissions from an animal.

[0157] Table 7. Emission data for animals across the trial

[0158] _ Treatment groups _

[0159] 3-NOP1Control1p-value2

[0160] Block 1

[0161] Live Weight (kg) 302 (3) 301 (3) 0.8

[0162] Estimated DMI (kg / day) 7.18 (0.09) 7.18 (0.10) 0.7 34816-EP-EPA

[0163] Analysis using data from the treated groups that was collected on days in which animals received at least 80% of the prescribed 3-NOP dose (>= 2 mg / kg LW) was also fitted to a mixed-effect model across the trial while accounting for the animal, block and paddock design. Animals in the 3-NOP treatment groups showed a 15% decrease in emissions, producing 125.65 g / day of CH4 which was significantly decreased by 22.12 g / day compared to the control groups 147.78 g I day (p < 0.001 ). Blocks 1 - 3 all produced significant reductions in methane emissions with an ~ 28%, 10% and 15% respectively (Table 8). Block 4 also produced a 4.6% reduction in methane production (p =0.6), but as mentioned above, this was confounded by the decrease in water intake across this block which resulted in a lower consumption of 3-NOP. 34816-EP-EPA

[0164] Conversely, H2 expelled by treated animals showed a significant increase 3.32 g H2 / day / head compared with control animals 0.69 g / day / head (p <0.001 ).

[0165] Table 8. Emission data filtered for 3-NOP animals receiving >= 2 mg / kg Lw of 3- NOP per day _

[0166] Treatment groups

[0167] Block 1

[0168] Live Weight (kg) 301 (3) 301 (3) >0.9

[0169] Estimated DMI (kg / day) 7.18 (0.09) 7.18 (0.10) 0.7

[0170] CH4(g / day) 112 (4) 155 (4) <0.001

[0171] CH4(g / day kg DMI) est. 16.0 (0.6) 21.6 (0.5) <0.001

[0172] H2(g / day) 5.8(0.8) 0.5 (0.0) <0.001

[0173] Block 2

[0174] Live Weight (kg) 317 (3) 317 (3) >0.9

[0175] Estimated DMI (kg / day) 6.87 (0.06) 6.89 (0.08) 0.7

[0176] CH4(g / day) 136 (5) 151 (5) 0.034

[0177] CH4(g / day kg DMI) est. 19.8 (0.7) 22.1 (0.7) 0.034

[0178] H2(g / day) 1.85 (0.17) 0.74 (0.10) <0.001

[0179] Block 4

[0180] Live Weight (kg) 334 (3) 334 (4) 0.9

[0181] Estimated DMI (kg / day) 5.78 (0.08) 5.91 (0.08) 0.2

[0182] CH4(g / day) 125 (5) 131 (6) 0.6

[0183] CH4(g / day kg DMI) est. 21.8 (0.8) 22.0 (0.9) 0.8

[0184] H2(g / day) 1.94 (0.17) 1.13 (0.19) 0.002

[0185] 1Mean (std. error)

[0186] 2Wilcoxon rank sum exact test

[0187] No significant (P>0.05) treatment effects were observed on live weight, average daily weight gain or faecal dry matter or organic matter digestibility (Table 9). This indicated 34816-EP-EPA a non-detrimental effect on animal performance with the 3-NOP levels supplemented through drinking water to the grazing animals in this study.

[0188] Table 9. Treatment effect on animal performance (live weight (LW), average daily weight gain (ADWG)) and faecal digestibility (NIRS) in steers grazing a grass-based production system in northern Australia. _

[0189] Control 3-NOP SEM P-value

[0190] Initial LW (Kg) to paddock 278.4 276.9 2.03 0.707

[0191] LW (Kg) prior rain event1336.3 335.7 1.45 0.794

[0192] LW (Kg) whole trial2332.0 331.9 1.47 0.661

[0193] ADG (Kg / day) prior rain event10.851 0.865 0.02 0.794

[0194] ADG (Kg / day) whole trial20.595 0.611 0.02 0.661

[0195] Faecal DMD % (NIRS)360.4 61.3 0.35 0.237

[0196] Faecal OMD % (NIRS)362.2 62.8 0.33 0.291

[0197] 1Animals rotated three times through paddocks (Block 1-3)

[0198] 2Animals rotated four times through paddocks (Block 1-4), last rotation coincided with significant rain events which reduced the animal water intake from water point.

[0199] Collected at the end of the trial in Block 4.

[0200] Rumen fermentation profile of steers that consumed the 3-NOP in water showed a shift in fermentation from acetate to propionate (Table 10). As a consequence, the ratio of acetate : propionate (A:P) was significantly reduced.

[0201] Table 10 Treatment effect on rumen fermentation parameters in steers grazing a grass-based production system in northern Australia.

[0202] Control13-NOP1SEM P-Value

[0203] Rumen pH 6.78 6.92 0.05 0.217

[0204] Total VFA mM 88.6 85.8 2.71 0.601

[0205] Individual VFA (%)

[0206] Acetate 72.9 70.1 0.32 0.001

[0207] Propionate 14.2 16.5 0.25 0.001 iso-Butyrate 1.33 1.31 0.03 0.709 n-Butyrate 9.37 9.78 0.14 0.152 iso-Valerate 1.37 1.43 0.03 0.218 n- Valerate 0.73 0.75 0.01 0.411 n-Caproate 0.17 0.16 0.01 0.793

[0208] Ratio A:P 5.16 4.36 0.07 0.001

[0209] Collected at the end of the trial (Block 4) after the morning drinking event. 34816-EP-EPA

[0210] Accelerometer data collected from the eGrazor collars was used to classify individual cattle behaviour at each five-second interval as grazing, ruminating, resting, walking, drinking or other. The typical behaviour profile for cattle included two intensive periods of grazing from 05:00 - 08:00 and then 15:00 - 19:00. The period outside of the graz- ing peaks is largely characterised by ruminating and resting behaviours.

[0211] The mean time spent grazing per animal was not different between the treated and untreated animals for all blocks (Table 11 ). Table 11 . Mean daily grazing time (hours) per treatment and block.

[0212] Treatment

[0213] Daily Grazing 3-NOP1Control1p-value2

[0214] Block 1

[0215] 7.4 (0.18) 7.3 (0.21) 0.9

[0216] Block 2

[0217] 7.9 (0.12) 7.8 (0.22) 0.8

[0218] Block 3

[0219] 7.8 (0.29) 8.0 (0.19) >0.9

[0220] Block 4

[0221] 8.1 (0.19) 8.2 (0.17) 0.9

[0222] 1Hours spent grazing / day, mean(std. error)

[0223] 2Wilcoxon rank sum exact test

[0224] Conclusions. This study demonstrates that 3-nitrooxypropanol (3-NOP), commercially known as Bovaer®, can be effectively administered to grazing cattle via drinking water under extensive pastoral conditions. The results confirm that when cattle consistently receive the targeted dose of 3-NOP (>2 mg / kg LW / day), significant reductions in enteric methane emissions up to 28% can be achieved without compromising animal performance, feed intake, or digestibility. 34816-EP-EPA

[0225] Emission data segmented by time of day revealed that the most substantial reductions occurred shortly after peak drinking periods. Between 07:00 and 11 :00 (when the largest single intake of water occurred) methane emissions dropped by approximately 20%. Additional reductions of ~16% were observed during subsequent drinking windows between 11 :00-15:00 and 15:00-18:00. Outside of these periods, particularly after 18:00 when water intake was minimal, no significant differences in methane emissions were detected between treated and control groups.

[0226] Methane mitigation efficacy was closely linked to the consistency of 3-NOP delivery, which was influenced by environmental factors such as rainfall, pasture moisture content, and equipment reliability. Periods of reduced water intake or dosing errors corresponded with diminished methane reduction.

[0227] The trial found no adverse effects on live weight gain, average daily weight gain, or faecal digestibility, suggesting that 3-NOP supplementation via water is a safe and non-disruptive intervention. Behavioural data further supported that grazing patterns remained unaffected, and methane reductions were most pronounced during peak drinking periods following grazing events.

Claims

34816-EP-EPAClaims1 . A liquid composition of 3-nitrooxypropanol and / or derivatives thereof for reducing the formation of methane emanating from the digestive activities of ruminants.

2. The composition of any of the preceding claims, wherein the composition comprises 3-nitrooxypropanol and / or derivatives thereof in water.

3. The composition of any of the preceding claims, wherein the concentration of 3- nitrooxypropanol and / or derivatives thereof is at least 0.00001 wt%, preferably at least 0.00002 wt%, preferably at least 0.00003 wt%, preferably at least 0.00003 wt%, preferably at least 0.00004 wt%, preferably at least 0.00005 wt%, preferably at least 0.00006 wt%, preferably at least 0.00007 wt%, preferably at least 0.00008 wt%, preferably at least 0.00009 wt%, preferably at least 0.0001 wt%, preferably at least 0.0002 wt%, preferably at least 0.0003 wt%, pref-erably at least 0.0004 wt%, preferably at least 0.0005 wt%, preferably at least 0.0006 wt%, preferably at least 0.0007 wt%, preferably at least 0.0008 wt%, pref-erably at least 0.0009 wt%, preferably at least 0.001 wt%.

4. The composition of any of claims 1 to 3, wherein the concentration of 3-nitrooxy- propanol and / or derivatives thereof is below 0.05 wt%, preferably below 0.04 wt%, preferably below 0.03 wt%, preferably below 0.02 wt%, preferably below 0.01 wt%, preferably below 0.009 wt%, preferably below 0.008 wt%, preferably below 0.007 wt%, preferably below 0.006 wt%, prefera-bly below 0.005 wt%, preferably below 0.007 wt%, preferably below 0.006 wt%, pref-erably below 0.005 wt%, preferably below 0.004 wt%, preferably below 0.003 wt%, preferably below 0.002 wt%, preferably below 0.001 wt%.

5. The composition of any claims 1 to 3, wherein the concentration of 3-nitrooxypro- panol and / or derivatives thereof is 0.00001 to 0.05 wt%, preferably 0.00002 to 0.05 wt%, preferably 0.00003 to 0.05 wt%, preferably 0.00003 to 0.05 wt%, preferably 0.00003 to 0.04 wt%, preferably 0.00003 to 0.02 wt%, preferably 0.00004 to 0.02 wt%, preferably 0.00005 to 0.02 wt%, preferably 0.00006 to 0.02 wt%, preferably 0.00007 to 0.02 wt%, preferably 0.00008 to 0.01 wt%, preferably34816-EP-EPA0.00009 to 0.01 wt%, preferably 0.0001 to 0.01 wt%, preferably 0.0001 to 0.01 wt%.

6. The composition according to anyone of the preceding claims, wherein the ruminant animal is selected from the group consisting of cattle, preferably from the group consisting of domestic cattle, most preferably from beef cattle or dairy cows7. Use of a composition of 3-nitrooxypropanol and / or derivatives thereof in the drinking water of ruminants for reducing the formation of methane emanating from the digestive activities of ruminants.

8. The use according to claim 7, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered to the ruminant in an amount selected in the range from 0.00001 to 0.05 wt%, preferably 0.00002 to 0.05 wt%, preferably 0.00003 to 0.05 wt%, preferably 0.00003 to 0.05 wt%, preferably 0.00003 to 0.04 wt%, preferably 0.00003 to 0.02 wt%, preferably 0.00004 to 0.02 wt%, preferably 0.00005 to 0.02 wt%, preferably 0.00006 to 0.02 wt%, preferably 0.00007 to 0.02 wt%, preferably 0.00008 to 0.01 wt%, preferably 0.00009 to 0.01 wt%, preferably 0.0001 to 0.01 wt%, preferably 0.0001 to 0.01 wt%.

9. The use according to claim 7 or 8, wherein the 3-nitrooxypropanol and / or derivatives thereof is administered to the ruminant in an amount selected in the range from 0.01 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.02 to 3 g 3-nitrooxypropanol and / or derivatives thereof I ani-mal / day, more preferably in the range from 0.03 to 3 g 3-nitrooxy- propanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.9 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.8 g 3-nitrooxy- propanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 2.7 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 2.6 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.5 g 3-34816-EP-EPA nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.05 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.06 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.07 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.08 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.09 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.5 g 3- nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2.4 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2.3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.2 g 3- nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2.1 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.2 to 2 g 3-nitroox- ypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.3 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.4 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.5 to 2 g 3-nitrooxypro- panol and / or derivatives thereof I animal / day, more preferably in the range from 0.6 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.7 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.8 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.9 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 1 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day.

10. The use according to anyone of the preceding claims, wherein the ruminant animal is selected from the group consisting of cattle, preferably from the group consisting of domestic cattle, most preferably from beef cattle or dairy cows.34816-EP-EPA11. A method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the animal 3-nitrooxypropanol and / or derivatives thereof in the drinking water of the animal.

12. The method according to claim 11 , wherein the 3-nitrooxypropanol and / or derivatives thereof is administered to the ruminant in an amount selected in the range from 0.01 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.02 to 3 g 3-nitrooxypropanol and / or derivatives thereof I ani-mal / day, more preferably in the range from 0.03 to 3 g 3-nitrooxy- propanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.9 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.04 to 2.8 g 3-nitrooxy- propanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 2.7 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 2.6 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.04 to 2.5 g 3- nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.05 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.06 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.07 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.08 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.09 to 2.5 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.5 g 3- nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2.4 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2.3 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.1 to 2.2 g 3- nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2.1 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.1 to 2 g 3-nitrooxypropanol and / or34816-EP-EPA derivatives thereof I animal / day, more preferably in the range from 0.2 to 2 g 3- nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.3 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.4 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.5 to 2 g 3-nitrooxypro- panol and / or derivatives thereof I animal / day, more preferably in the range from 0.6 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.7 to 2 g 3-nitrooxypropanol and / or derivatives thereof / animal / day, more preferably in the range from 0.8 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 0.9 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day, more preferably in the range from 1 to 2 g 3-nitrooxypropanol and / or derivatives thereof I animal / day.* * *

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