WO2024133548A1

WO2024133548A1 - Method for reduction of methane production in and/or for improving performance of a ruminant

Info

Publication number: WO2024133548A1
Application number: PCT/EP2023/087079
Authority: WO
Inventors: Mette Olaf Nielsen; Mirka Mølgaard THORSTEINSSON; Marie RØNN; Natalja NØRSKOV
Original assignee: Dansk Landbrugs Grovvareselskab AMBA
Current assignee: Dansk Landbrugs Grovvareselskab AMBA
Priority date: 2022-12-21
Filing date: 2023-12-20
Publication date: 2024-06-27
Anticipated expiration: 2025-06-21
Also published as: CL2025001812A1; AU2023409568A1; MX2025007226A; EP4637383A1; CN120712022A

Abstract

The present invention relates to a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering to the ruminant one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane in an effective amount in the range of 2-50 mg of dihalomethane/kg DM. The present invention also relates to a feed additive for a ruminant and to one or more dihalomethanes for use in a method of reducing methane production in and/or for improving performance of a ruminant.

Description

METHOD FOR REDUCTION OF METHANE PRODUCTION IN AND/OR FOR IMPROVING PERFORMANCE

OF A RUMINANT

FIELD OF THE INVENTION

The present invention relates to a method of reducing methane production in and/or for improving performance of a ruminant and to a feed additive for a ruminant. The present invention also relates to one or more dihalomethanes for use in a method of reducing methane production in and/or for improving performance of a ruminant.

BACKGROUND OF THE INVENTION

Ruminants account for as much as 30% of global anthropogenic methane emissions or 44% of total greenhouse gas (GHG) emissions from livestock (Gerber et al. (2013); Tackling Climate Change Through Livestock - A global assessment of emissions and mitigation opportunities. Rome: Food and Agriculture Organization of the United Nations (FAO), 2013). Methane is a GHG 25 times more powerful than CO2. There is a substantial pressure to reduce climate footprint from milk and beef production. Methane (CH4) is synthesised as follows: CO2+4H2-> CH4+2H2O. Rumen archaea are the microorganisms responsible for this synthesis (Patra et al. (2017); Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances, J Anim Sci Biotechnol 8: 13, DOI 10.1186/s40104-017— 145-9). Archaea represents a distinct domain of living organisms, differing from both bacteria and eukaryotes.

Broucek, J. Options to Methane Production Abatement in Ruminants: A Review, The Journal of Animal & Plant Sciences, 28(2) : 2018, p. 348-364, analyses published data on practices that mitigate enteric methane emissions from ruminants. The study of Broucek is divided into nine sections (defaunation and inhibition of archaea, bacteriocins, methane inhibitors and analogues, probiotics, saponins, tannins, ionophores, organic acids, and lipids).

Lanigan, G. W., Metabolism of pyrrolizidine alkaloids in the ovine rumen, Aust. J. Agric. Res., (1972), 23, 1085-91 report results showing that five halogenated methane analogues (bromoform, chloroform, iodoform, carbon tetrabromide, and carbon tetrachloride) inhibit methane formation in the sheep's rumen as well as in rumen fluid in vitro.

Lanigan, G. W. et aL, Antimethanogenic Drugs and Heliotropium europaeum Poisoning in Penned Sheep, Aust. J. Agric. Res., (1978), 29, 1281-92 report results showing an anti-methanogenic effect of oral dosing with iodoform pellets to sheep. Tomkins, N. W. et al, A bromochloromethane formulation reduces enteric methanogenesis in cattle fed grain-based diets, Animal Production Science, 2009, 49, 1053-1058 reports experiments showing an antimethanogenic effect of bromochloromethane (BCM). The authors note that uncomplexed BCM has an ozone-depleting effect and were prohibited by the Australian Government in 2004.

Song et aL, Control of Methane Emission in Ruminants and Industrial Application of Biogas from Livestock Manure in Korea, Asian-Aust. J. Anim. Sci. Vol. 24, no.l : 130136, January 2011 discusses the effects of 2-bromoethansulfonic acid on methane production.

WO 2012/084629A1 relates to a method for reducing the production of methane emanating from the digestive activities of a ruminant and/or for improving ruminant animal performance by using, as an active compound, at least one organic molecule substituted at any position with at least one nitrooxy group, or a salt thereof, which is administrated to the animal together with the feed.

WO 2021/205420 Al discloses compositions comprising algae and methods of using same for increasing animal product production.

WO 2022/136857 Al discloses compositions for reducing methane emission, and methods for improving the metabolic efficiency of ruminant animals, wherein the compositions comprise an organohalogen compound and an organosulfur compound, preferably bromoform and allicin. Experiments showed that bromoform had practically no effect on inhibition of methane, when administered alone.

EP 0 782 442 Bl relates to a delivery system for antimethanogenic agents comprising cyclodextrin and antimethanogenic agents. As antimethanogenic agent is disclosed bromochloromethane.

There is still a need for a method of reducing methane formation in and/or for improving performance of a ruminant, which provides an efficient reduction in methane formation without deleterious side-effects.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide a method for reducing methane production in and/or for improving performance of a ruminant. It is a further object of embodiments of the invention to provide a feed additive for a ruminant for use for reducing methanogenesis in and/or for improving performance of said ruminant.

It is a further object of embodiments of the invention to provide one or more dihalomethanes for use in a method of reducing methanogenesis in and/or for improving performance of a ruminant while avoiding compromising milk production of the ruminant to any significant extent.

SUMMARY OF THE INVENTION

It has been found by the present inventors that by providing one or more dihalomethanes to a ruminant in an effective amount in the range of 2-50 mg/kg dry matter (DM) a reduction in methane production of at least 10% may be obtained without significantly compromising milk production of the ruminant.

So, in a first aspect the present invention relates to a method of reducing methane production in and/or for improving performance of a ruminant, comprising the step of administering to said ruminant one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane in an effective amount in the range of 2-50 mg/kg feed dry matter (DM).

In a second aspect the present invention relates to a feed additive for a ruminant, comprising one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane in an effective amount in the range of 5-100 mg/kg dry matter (DM).

In a third aspect the present invention relates to one or more dihalomethanes selected from the group consisting of dibromomethane ^iiodomethane, bromoiodomethane, and chloroiodomethane for use in a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering to the ruminant the dihalomethane in an effective amount in the range of 2-50 mg/kg feed (DM).

LEGENDS TO THE FIGURE

Fig. 1 shows the methane production (CH4) per gram of dry matter (DM) maize silage at 24 hours for negative control, iodoform, and dihalomethane treatments. Fig. 2 shows the hydrogen (H2) production per gram of dry matter (DM) maize silage at 24 hours for iodoform and dihalomethane treatments.

Fig. 3 shows the concentration of iodoform (IF) (Black dot) and diiodomethane (DIM) (grey dot) over time in rumen fluid added iodoform+ethanol solution from in vitro fermentation.

Fig. 4 shows the concentration of diiodomethane (DIM) (grey dot) over time in rumen fluid added diiodomethane+ethanol solution from in vitro fermentation.

Fig. 5 shows the daily methane emission for cows fed with and without diiodomethane at two different dose levels (10 and 20 mg/kg DM).

Fig. 6 shows the daily hydrogen emission for cows fed with and without diiodomethane at two different dose levels (10 and 20 mg/kg DM).

Fig. 7 shows the methane emission per kg dry matter intake for cows fed with and without diiodomethane at two different dose levels (10 and 20 mg/kg DM).

Fig. 8 shows the hydrogen emission per kg dry matter intake for cows fed with and without diiodomethane at two different dose levels (10 and 20 mg/kg DM).

Fig. 9 shows the daily feed intake (kg dry matter) for cows fed with and without diiodomethane at two different dose levels (10 and 20 mg/kg DM).

Fig. 10 shows methane production (CH4) per gram of dry matter (DM) maize silage at 24 hours for negative control and dihalomethane treatments in Example 5.

Fig. 11 - Hydrogen (H2) production per gram of dry matter (DM) maize silage at 24 hours for control and dihalomethane treatments in Example 5.

Fig. 12 - Dry matter intake (DMI) and Energy corrected milk yield (ECM) for treatments, Control (CON), Bromoiodomethane (BIM), Chloroiodomethane (CIM), Diiodomethane (DIM) in Example 6.

Fig. 13 - Methane gas yield (g CH4/day) for treatments, Control (CON), Bromoiodomethane (BIM), Chloroiodomethane (CIM), Diiodomethane (DIM) in Example 6. Control diet relative to test diets. Fig. 14 - Methane gas exchange per kg dry matter intake (g CH4/kg DMI) for treatments, Control (CON), Bromoiodomethane (BIM), Chloroiodomethane (CIM), Diiodomethane (DIM) in Example 6. Control diet relative to test diets.

Fig. 15 - Methane gas intensity per kg energy corrected milk (g CH4/kg ECM) for treatments, Control (CON), Bromoiodomethane (BIM), Chloroiodomethane (CIM), Diiodomethane (DIM) in Example 6. Control diet relative to test diets.

Fig. 16 - Total gas production per gram DM maize silage for Control and at different dose levels for the two treatments (Dibromomethane (DBM) and Diiodomethane (DIM) in example 7.

Fig. 17 - Methane production per gram DM maize silage for Control (MS) and at different dose levels for the two treatments (Dibromomethane (DBM) and Diiodomethane (DIM) in example 7.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

The term "methanogenesis", "methane production" and "methane emission" is used interchangeably in the present context to describe the production of methane emanating from the digestive activities of ruminants.

The term "improving performance" is used in the present context to refer to improving overall performance of a ruminant in terms of e.g. feed conversion ratio and feed efficiency, such as improvement of milk yield as a function of feed intake.

The term "ruminant" refers to any of a subfamily of bovinae including cattle and cows, sheep, goats, buffaloes, bison, and other antelopes.

The term "feed dry matter" or "feed DM" is used in the present context to refer to the material remaining after all of the water is evaporated out of a feed.

Specific embodiments of the invention

In an embodiment of aspects of the invention one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane is for use in a method of inhibiting methanogenesis of a ruminant, said method comprising administering one or more dihalomethanes to said ruminant.

In an embodiment of aspects of the invention the one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane is for use in a method of improving metabolic efficiency in a ruminant, said method comprising administering one or more dihalomethanes to said ruminant.

In an embodiment of the invention the one or more dihalomethanes is for use in a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane to said ruminant in an amount of 5-50 mg/kg feed DM, such as 10-50 mg/kg feed DM, such as 10-40 mg/kg feed DM, such as 10-30 mg/kg feed DM, such as 10-25 mg/kg feed DM, such as 10-20 mg/kg feed DM, such as 10-15 mg/kg feed DM, such as 12-15 mg/kg feed DM, such as an amount in the range of 8-15 mg/ kg feed DM.

In an aspect of the invention the one or more dihalomethanes is for use in a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering dibromomethane to said ruminant in an amount of 1-32 mg/kg feed DM, e.g. 3-32 mg/kg feed DM, such as 6-32 mg/kg feed DM, such as 6-26 mg/kg feed DM, such as 6-19 mg/kg feed DM, such as 6-16 mg/kg feed DM, such as 6-13 mg/kg feed DM, such as 6-10 mg/kg feed DM, such as 8-10 mg/kg feed DM, such as an amount in the range of 5-10 mg/ kg feed DM.

In an aspect of the invention the one or more dihalomethanes is for use in a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering diiodomethane to said ruminant in an amount of 2-50 mg/kg feed DM, e.g. 5-50 mg/kg feed DM, such as 10-50 mg/kg feed DM, such as 10-40 mg/kg feed DM, such as 10-30 mg/kg feed DM, such as 10-25 mg/kg feed DM, such as 10-20 mg/kg feed DM, such as 10- 15 mg/kg feed DM, such as 12-15 mg/kg feed DM, such as an amount in the range of 8-15 mg/ kg feed DM.

In an aspect of the invention the one or more dihalomethanes is for use in a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering bromoiodomethane to said ruminant in an amount of 2-41 mg/kg feed DM, e.g. 4-41 mg/kg feed DM, such as 8-41 mg/kg feed DM, such as 8-33 mg/kg feed DM, such as 8-25 mg/kg feed DM, such as 8-21 mg/kg feed DM, such as 8-16 mg/kg feed DM, such as 8-12 mg/kg feed DM, such as 10-12 mg/kg feed DM, such as an amount in the range of 7-12 mg/ kg feed DM.

In an aspect of the invention the one or more dihalomethanes is for use in a method of reducing methane production in and/or for improving performance of a ruminant, said method comprising the step of administering chloroiodomethane to said ruminant in an amount of 1-33 mg/kg feed DM, e.g. 3-33 mg/kg feed DM, such as 7-33 mg/kg feed DM, such as 7-26 mg/kg feed DM, such as 7-20 mg/kg feed DM, such as 7-16 mg/kg feed DM, such as 7-13 mg/kg feed DM, such as 7-10 mg/kg feed DM, such as 8-10 mg/kg feed DM, such as an amount in the range of 5-10 mg/ kg feed DM.

Of the dihalomethanes, dibromomethane, diiodomethane and bromoiodomethane are preferred, dibromomethane and diiodomethane are more preferably, wherein diiodomethane is most preferred.

It has surprisingly been found that by administering one or more dihalomethanes to a ruminant at such amounts it is possible to achieve a significant reduction in methane production in said ruminant without significantly influencing milk production and/or feed intake. While a significant reduction in methanogenesis is achieved through the administration of one or more dihalomethanes accompanied by some or a substantial reduction in feed intake a much less pronounced reduction in milk yield is observed, and hence an improved feed conversion rate - measured as amount of milk produced per unit of ingested feed - is obtained and thereby an improved feed efficiency.

In an embodiment of the method according to the invention the one or more dihalomethanes is administered to the ruminant by supplementing feed intended for said ruminant with an amount in the range of 2-50 mg of dihalomethane/kg DM.

In an embodiment of the method according to the invention a single dihalomethane selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane is administered to said ruminant. Thus, it has been found that dihalomethanes are efficient methanogenesis inhibitors, reducing the amount of methane produced by ruminants. The use of a dihalomethane has been shown to result in a significant reduction of methane production of a ruminant without substantially influencing milk production of said ruminant.

In an embodiment of the invention one or more dihalomethanes is for use in a method of reducing methane production in a ruminant selected from the group consisting of cattle, sheep and goats, such as lactating cows. In an embodiment of the invention one or more dihalomethanes is for use in a method of reducing methane production in and/or for improving performance of a ruminant, wherein the methane production, calculated in liters per kilogram of dry matter intake, is reduced by at least 10% when measured in metabolic chambers, such as wherein the methane production is reduced by at least 15%, such as at least 20%, such as at least 25%, such as at least 30%.

In an embodiment of the invention one or more dihalomethanes is comprised in a feed additive intended for a ruminant. This allows a convenient method of administration of a dihalomethane to the ruminant.

In an embodiment of the feed additive according to the invention, said feed additive comprises one or more dihalomethanes in an amount in the range of 5-50 mg/kg feed DM, such as 10-50 mg/kg feed DM, such as 10-40 mg/kg feed DM, such as 10-30 mg/kg feed DM, such as 10-25 mg/kg feed DM, such as 10-20 mg/kg feed DM, such as 10-15 mg/kg feed DM, such as 12-15 mg/kg feed DM, such as an amount in the range of 8-15 mg/ kg feed DM.

One or more dihalomethanes may be administered to the ruminant in different forms. For example, a dihalomethane may be included in a bolus that would be placed in the rumen and that would release a defined amount of the active compound continuously in well-defined dosages over a specific period of time as known to a person skilled in the art.

In an embodiment of the feed additive according to the invention one or more dihalomethanes is in the form of a bolus dose corresponding to an amount in the range of 2-50 mg of dihalomethane/kg DM. Thereby an efficient uptake by the ruminant is secured.

In an embodiment of the feed additive according to the invention a single dihalomethane selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane is comprised in said feed additive.

Methane emission by ruminants can easily be measured in individual animals in metabolic chambers by methods known in the art, see e.g. Ding et al 2006: Chapter 10: Emissions from Livestock and Manure Management. 2006 IPCC Guidelines for National Greenhouse Gas Inventories. Volume 4: Agriculture, Forestry and Other Land Use. https://www.ipcc-nggip.iges.or.jP/public/2006gl/pdf/4.

In an embodiment of the feed additive according to the invention one or more dihalomethanes is supplied in pure form. In an embodiment of the method according to the invention the methane production in ruminants, calculated in liters per kilogram of dry matter intake, is reduced by at least 10% when measured in metabolic chambers, such as wherein the methane production is reduced by at least 15%, such as at least 20%, such as at least 25%, such as at least 30%.

As indicated above, one or more dihalomethanes is useful as a compound for feed additives and animal feed compositions for ruminants for reducing methane formation in and/or for improving performance of said ruminants. The feed additive or feed composition according to the invention is preferably a ruminant base mix, such as a mineral premix or a vitamin premix comprising vitamins and minerals. Vitamin and mineral premixes are designed to provide ruminants with all their nutritional needs. Non-limiting commercially available examples thereof include fat-soluble vitamins as A, D and E, trace minerals such as manganese, zinc, cobalt, iron, iodine and selenium, macrominerals as calcium, phosphorous and sodium.

In an embodiment of the feed additive according to the invention one or more dihalomethanes is admixed to a total mixed ration (TMR) feed. In a TMR all dietary components, e.g. forage, silage and concentrate, are mixed before serving. Forage is a plant material, silage is grass or other green fodder made from green foliage crops which have been preserved such as by acidification, achieved through fermentation, and concentrate refers to a product mainly consisting of cereals, such as, but not limited to, barley, maize, wheat, but may also include protein-rich feed ingredients such as soybean, rapeseed, and sunflower.

One or more dihalomethanes may also be provided in encapsulated form. Encapsulation is a technique by which one material or a mixture of materials is coated with or entrapped within another material or system. Materials for encapsulation include proteins, carbohydrates, lipids, gums and cellulose. Non-limiting examples include polysaccharide, maltodextrin, corn syrup solid, modified starch, gum arabic, modified cellulose, gelatine, cyclodextrin, lecithin, whey protein, hydrogenated starch.

Non-limiting methods for encapsulation include pelleting, coacervation, spray drying, fluid bed drying, spray cooling, molecular inclusion as known to a person skilled in the art.

One or more dihalomethanes may also be dissolved into a liquid solution. This liquid solution can be added to the TMR ration or through water for drinking.

In an embodiment of the feed additive for a ruminant according to the invention the ruminant is selected from the group consisting of cattle, sheep and goats, such as lactating cows. In an embodiment of the invention, the one or more dihalomethanes for use in a method of reducing methane production in and/or improving performance of a ruminant are administered in an amount in the range of 5-50 mg/kg feed DM, such as 10-50 mg/kg feed DM, such as 10-40 mg/kg feed DM, such as 10-30 mg/kg feed DM, such as 10-25 mg/kg feed DM, such as 10-20 mg/kg feed DM, such as 10-15 mg/kg feed DM, such as 12-15 mg/kg feed DM, such as an amount in the range of 8-15 mg/ kg feed DM.

In an embodiment of the invention, the one or more dihalomethanes is to be administered to said ruminant by supplementing feed intended for said ruminant with an amount in the range of 2-50 mg of dihalomethane/kg feed DM or as a bolus dose corresponding to an amount in the range of 2- 50 mg of dihalomethane/kg feed DM, preferably wherein the dihalomethane is a single dihalomethane, wherein said dihalomethane is selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane, and preferably wherein improving performance of a ruminant comprises improving feed efficiency in the form of milk yield per unit of ingested feed.

In one aspect of the method of the invention, said one or more dihalomethanes is not administered in a composition together with an organosulfur compound. In one aspect of the feed additive of the invention, said feed additive does not comprise an organosulfur compound. Such organosulfur compounds may include allicin (C6H10S2O), diallyl sulfide (CeHioS), diallyl sulfide (CeHioS?) and allyl mercaptan (CsHeS).

EXAMPLES

EXAMPLE 1 - In vitro test of efficiency of methane reduction with use of diiodomethane and iodoform.

Rumen fluid was collected from three rumen cannulated maintenance cows before morning feeding and filtered through two layers of cheese cloth. A buffer solution was prepared according to Menke, K. H., and H. Steingass. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28 (7-55) and was flushed with N2 for lh before the reduction agent and rumen fluid was added in a ratio of 1 :3 rumen fluid : buffer solution. Ninety mL of inoculum was dispensed into 100 mL bottles, whereafter 100 pL of iodoform or diiodomethane in an ethanol solution (17.5 pg per 100 pL, 35 pg per 100 pL or 52.5 pg per 100 pL) was added. Thus, a total of 20 bottles were used all containing 0.5 g of maize silage; 2 of these bottles were used as control including 100 pg EtOH. 18 bottles included either diiodomethane and iodoform at 3 different concentrations (17.5 pg, 35.0 pg and 52.5 pg). Cumulative gas production was measured after 24 hours. The samples were analyzed on GC- MS/MS.

The control treatment had a methane production on 56.45 ml CH g maize silage after 24-hour incubation. All other treatments had significantly lower methane production. Inclusion of diiodomethane and iodoform reduced the methane concentration from 80 % to 99 %. This demonstrates a high efficiency of both the diiodomethane and the iodoform to reduce methane emission.

EXAMPLE 2 - In vitro testing of dihalomethanes

Three different dihalomethanes (diiodomethane, bromoiodomethane, and chloroiodomethane) were tested via in vitro gas production for their potential to reduce methane production. For comparison both a negative and positive control treatment were included. Another halomethane, iodoform, was used as the positive control. The four halomethanes were each tested at three different dose levels. Rumen fluid was collected from three rumen cannulated maintenance cows before morning feeding and filtered through two layers of cheese cloth. A buffer solution was prepared according to Menke, K. H., and H. Steingass. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28 (7-55) and was flushed with N2 for lh before reduction agent and rumen fluid was added in a ratio of 1 :3 rumen fluid : buffer solution. Ninety mL of inoculum was dispensed into 100 mL bottles, whereafter 100 pL of test substance in an ethanol solution with three different halomethane concentrations (17.5 pg-100pL ^-1, 35 pg-100pL ¹ or 52.5 pg-100pL ^-1) was added. A total of 38 bottles were used all containing 0.5 g of maize silage; 2 of these bottles were used as negative control added 100 pg EtOH and each treatment (halomethane X dose level) was made in triplets yielding 36 bottles. Cumulative gas production was tracked, and gas sample were collected in gas bags during fermentation. Cumulative gas production was measured by the ANKOM pressure sensor module. Gas samples were analyzed on GC-MS/MS. Mean values for each treatment (halomethane X dose level) are presented in the results.

Dihalomethanes showed a lower amount of methane produced per gram of maize silage (dry matter level) compared to negative control (12.9 ml-g DM maize silage ^-1) and iodoform (Figure 1). The positive control (iodoform) showed a linear decrease in methane production with increasing dose level. Similarly, dihalomethanes generally depressed methane production to the same level at all three dose levels with exception of diiodomethane at 17.5 pg, where methane production was at 3.5 ml-g DM maize silage ^-1.

Hydrogen is the main substrate for production of methane and is expected to increase when methane production is suppressed. For dihalomethanes, hydrogen produced per gram of maize silage (dry matter level) was increased compared to corresponding dose level for positive control (iodoform) (Figure 2). The development in hydrogen production in relation to dose level were different for the three dihalomethanes.

In conclusion, dihalomethanes showed a similar potential to reduce methane production from fermentation as iodoform at similar dose levels followed by an increase in hydrogen production.

EXAMPLE 3 - In vitro clearance rate of iodoform and diiodomethane from rumen liquid

To investigate whether it is the iodoform or diiodomethane which is responsible for the reduction of the methane the kinetic for the two components were investigated.

A timeseries in vitro study tested the disappearance of iodoform and diiodomethane from rumen liquid. Rumen fluid was sampled from three cows and filtered for the in vitro testing. Rumen fluid was collected from three rumen cannulated maintenance cows before morning feeding and filtered through two layers of cheese cloth. A buffer solution was prepared according to Menke, K. H., and H. Steingass. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28 (7-55) and was flushed with N2 for lh before reduction agent and rumen fluid was added in a ratio of 1 :3 rumen fluid : buffer solution. The liquid mixture was portioned into 90 mL and added 100 pL of iodoform or diiodomethane in an ethanol solution (35 pg/mL). Bottles with controlled gas release caps were incubated at 39 °C in times of 0, 1, 2, 5, 10, 15, 20, 25, 30, 45, 60, 120, 180, 240, 360 and 1440 minutes in a controlled incubator shaker. Fermentation was stopped by placing the bottles on ice, whereafter two samples of 1 mL were collected from each bottle and immediately treated with 1 mL of methanol to make sure the fermentation was stopped completely. The samples were analyzed on GC-MS/MS. From the in vitro fermentation results show when iodoform is added to the rumen fluid it rapidly disappears from the liquid and diiodomethane is formed (Figure 3). Whereas, when diiodomethane is directly added to the rumen fluid the rate of disappearance is slower compared to iodoform. Even after 1500 minutes (24h) diiodomethane is still present in the rumen fluid (Figure 4). The reduction of methane on cows supplemented with iodoform has earlier shown to be decreased through around 8 hours (results not shown) after. Thus, this example suggests that the active component is the diiodomethane and not the iodoform as iodoform only exists for a very short time in the rumen.

EXAMPLE 4 - In vivo testing of diiodomethane supplemented to dairy cows

In an experiment 2 cows were administered diiodomethane for two days through mixing into the total feed ration at two different levels (10 and 20 mg/kg feed (dry matter basis)). Results of feed intake and gas emission were compared to the levels measured the day prior to testing. Measurements of gas emission in respiration chambers showed that diiodomethane mixed into the feed ration can reduce the methane emission from dairy cows in a dose-response dependent manner based on the current results by 127 L/day (516 vs. 389 L/day) up to 24.8% per kg dry matter intake for the highest dose (20 mg/kg dry matter feed) level (Figure 5 and 7). The decrease in methane emission resulted in an increase in total hydrogen emission of 92 L/day (8.8 vs. 100.8 L/day) or about 1040% on dry matter intake level for the highest dose (20 mg/kg DM) (Figure 6 and 8). The reduction in methane emission did not result in decreasing effects on feed intake. Levels of feed intake on dry matter level are presented in Figure 9, showing a minor increase in feed intake when administered diiodomethane at both levels of diiodomethane.

EXAMPLE 5 - In vitro testing of diiodomethane, bromoiodomethane, and chloroiodomethane

Three different dihalomethanes (diiodomethane, bromoiodomethane, and chloroiodomethane) were tested via in vitro gas production for their potential to reduce methane production. For comparison a negative control treatment was included. The three halomethanes were each tested at three different dose levels. Rumen fluid was collected from three rumen cannulated maintenance cows before morning feeding and filtered through two layers of cheese cloth. A buffer solution was prepared according to Goering and Van Soest (1970) and was flushed with N2 for lh before reduction agent and rumen fluid was added in a ratio of 1 :3 rumen fluid :buffer solution. Ninety mL of inoculum was dispensed into 100 mL bottles, whereafter 100 pL of test substance in an ethanol solution with two different halomethane concentrations (0.75 mM-lOOpL ¹ or 1.0 mM-100pL ^-1) was added. The dihalomethane concentrations calculated as mg/kg DM feed is presented in Table 2. A total of 38 bottles were used all containing 0.5 g of maize silage; 2 of these bottles were used as negative control added 100 pg EtOH and each treatment (halomethane X dose level) was made in triplets yielding 36 bottles. Cumulative gas production was tracked, and gas sample were collected in gas bags during fermentation. Cumulative gas production was measured by the ANKOM pressure sensor module. Gas samples were analyzed on GC-MS/MS. Mean values for each treatment (halomethane X dose level) are presented in the results.

Dose levels at dihalomethanes 0.75 and 1.0 mM-lOOpL ¹ generally depressed methane production with diiodo- and bromoiodomethane being more efficient than chloroiodomethane (Figure 10).

Hydrogen, is the main substrate for production of methane and is expected to increase with decreasing methane production. For dihalomethanes, hydrogen produced per gram of maize silage (dry matter level) was increased compared to negative control (Figure 11). The development in hydrogen production in relation to dose level were different for the three dihalomethanes.

Table 2 - Dose level as mg/kg DM feed for dihalomethanes at molar concentration

Millimolar concentration

Dihalomethane 0.75 mM 1.0 mM

Diiodomethane 40.2 53.6

Bromoiodomethane 33.1 44.2

Chloroiodomethane 26.5 35.3

In conclusion, the dihalomethanes tested showed a greater potential to reduce methane production from fermentation at similar dose levels followed by an increase in hydrogen production.

EXAMPLE 6 - In vivo test of test of bromoiodo-, chloroiodo- and diiodomethane

Description

The three tested compounds shown anti-methanogenic potential in vitro, but they have never been tested in dairy cows. The compounds are added to a standard total mixed ration, resulting in four different diets including the control diet, and fed to four dairy cows in four periods.

The aim of this experiment was to quantify effects of dihalomethanes on methane emission from dairy cows. The effects on milk yield and feed intake were also investigated. The experiment was conducted as a 4x4 complete latin square design. The experiment consists of 4 periods of 2 weeks duration with 4 cows and 4 dietary treatments Control (CON), Bromoiodomethane (BIM), Chloroiodomethane (CIM), Diiodomethane (DIM). Dose level for dihalomethanes were 0.0508 mmol/kg DM corresponding to 11.22, 8.96, and 13.61 mg/kg DM feed for BIM, CIM and DIM, respectively.

A TMR ration was made from feedstuffs available for Danish farmers and nutritional values were based on recommendations to Danish dairy cows. The control feed ration aimed to give a milk yield of around 10900 kg ECM per year. In the three test diets, the dihalomethanes were mixed into the control ration after being dissolved in 99% ethanol. A similar amount of ethanol was mixed into the CON diets as well. The feed additives were stored at 5°C until mixing.

The rations were offered ad libitum as a Total Mixed Ration (TMR). Forty percent of the daily allocated feed was fed in the morning and 60% was fed in the afternoon. Water intake was automatically recorded in the chamber period.

A recovery-test was done before the experiment started. The cows were placed in respiration chambers for 4 days. The cows were shifted between chambers after 2 days. Milk yield and feed intake was recorded every day in the chamber period, and samples of new and residual feed are taken for dry matter determination. Milk samples for nutrient analysis were sampled at every milking when cows were in chambers.

Gas exchange was measured on day 11-14 using four individual transparent polycarbonate respiration chambers based on open-circuit indirect calorimetry. Airflow was measured using a mass flow meter (HFM-200 with laminar flow element, Teledyne Hastings Instruments, Hampton, Virginia, USA). The concentrations of gases (CH4, CO2, and 02; Columbus Instruments, Columbus, Ohio, USA) in outlet air, and temperature, humidity, and pressure (Veng Systems, Roslev, Denmark) in the chambers were also measured. Recovery tests were performed before and during the experiment by infusing a known amount of pure CO2 or CH4 into the chambers and comparing it with the amount of gas measured by the system. Throughout the experiment, the cows were assigned to the same specific respiration chamber for the first 48 h of gas measurements. For the latter 48 h of gas measurement, the cows were changed to the opposite chamber to counteract eventual differences in background air composition.

Results

Results for the experiment are shown in Table 3. Adding dihalomethanes to the diet reduced dry matter intake (DMI) by 5-7% compared to the control treatment. Energy corrected milk (ECM) yield also decreased for BIM and DIM treatments by 3%, whereas CIM treatment increased with 1% compared to control (31.8 kg/d) (Figure 12). Diiodomethane had the highest reduction of all three dihalomethanes on methane exchange, yield and intensity compared to control. Bromoiodomethane was intermediate and Chloroiodomethane had the lowest effect on methane compared to negative control. Daily methane (CFU) exchange from cows when fed BIM or DIM treatment was reduced by 10 and 12%, respectively, compared to control diet with a methane exchange of 370 g/d (Figure 13). Methane yield was 17 g/kg DMI for cows on control diet, where BIM and DIM reduced the methane yield by 10 and 12% according to control (Figure 14). Methane intensity was 11.6 g/kg ECM for cows on control diet, where BIM and DIM reduced the methane intensity by 6 and 9% (Figure 15).

In conclusion, bromoiodo- and diiodomethane had the most efficient methane emission reduction based on dry matter intake and milk yield.

Table 3: Dry matter intake (DMI), energy corrected milk yield (ECM), and gas exchange of dairy cows fed a control diet (CON) or a diet supplemented with one of three dihalomethanes.

Treatments

Example 7 - In vitro testing of dibromomethane and diiodomethane

Two different dihalomethanes (dibromomethane and diiodomethane) were tested via in vitro gas production for their potential to reduce methane production. The two halomethanes were each tested at two different dose levels. Rumen fluid was collected from three rumen cannulated maintenance cows before morning feeding and filtered through two layers of cheese cloth. A buffer solution was prepared according to Goering and Van Soest (1970) and was flushed with N2 for lh before reduction agent and rumen fluid was added in a ratio of 1 :3 rumen fluid : buffer solution. Ninety mL of inoculum was dispensed into 100 mL bottles, whereafter 100 pL of test substance in an ethanol solution with two different halomethane concentrations (1.6 mM-100pL ¹ or 2.4 mM-lOOpL ¹) was added. The dihalomethane concentration calculated as mg/kg DM feed is presented in Table 4. A total of 20 bottles were used all containing 0.5 g of maize silage; 2 of these bottles were used as control added 100 pg EtOH and each treatment (halomethane X dose level) was made in triplets yielding 18 bottles. Cumulative gas production was tracked, and gas sample were collected in gas bags during fermentation. Cumulative gas production was measured by the ANKOM pressure sensor module. Gas samples were analyzed on GC-MS/MS. Mean values for each treatment (halomethane X dose level) are presented in the results. Dibromomethane and diiodomethane showed a lower level of both total gas and methane produced per gram of maize silage (dry matter level) compared to negative control (11.31 ml-g DM maize silage ^-1), when dose was at 1.6 and 2.4 (Figure 16 and 17).

In conclusion dibromo- and diiodomethane were able to reduce the amount of both total gas and methane produced at dose level of 1.6 and 2.4.

Table 4 - Dose level as mg/kg DM feed for dihalomethanes at molar concentration

Millimolar concentration

Dihalomethane 1-6 mM 2.4 mM

Dibromomethane 55.6 83.4

Diiodomethane 85.7 129

List of references

Lanigan, G. W., Metabolism of pyrrolizidine alkaloids in the ovine rumen, Aust. J. Agric. Res., (1972), 23, 1085-91

Lanigan, G. W. et aL, Antimethanogenic Drugs and Heliotropium europaeum Poisoning in Penned Sheep, Aust. J. Agric. Res., (1978), 29, 1281-92

Gerber et al. (2013); Tackling Climate Change Through Livestock - A global assessment of emissions and mitigation opportunities. Rome: Food and Agriculture Organization of the United Nations (FAO), 2013

Kamra D N et aL : "Manipulation of Rumen Microbial Ecosystem for Reducing Enteric Methane Emission in Livestock", Climate Change Impact on Livestock: Adaptation and Mitigation, 1 January 2015, p. 255-272, XP009531560, DOI: 10.1007/978-81-322-2265-l_16 ISBN :978- 81-2265-1

Patra et al. (2017); Rumen methanogens and mitigation of methane emission by antimethanogenic compounds and substances, J Anim Sci Biotechnol 8: 13, DOI 10.1186/s40104- 017-145-9

Broucek, J. Options to Methane Production Abatement in Ruminants: A Review, The Journal of Animal & Plant Sciences, 28(2) : 2018, p. 348-364

Song et aL, Control of Methane Emission in Ruminants and Industrial Application of Biogas from Livestock Manure in Korea, Asian-Aust. J. Anim. Sci. Vol. 24, no.l : 130136, January 2011

AU 2018 229 465 Al, WO 2012/084629A1, WO 2021/205420 Al, WO 2022/136857 Al, EP 0 782 442 Bl

Claims

1. A method of reducing methane production in and/or for improving performance of a ruminant, comprising the step of administering to said ruminant one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane in an effective amount in the range of 2-50 mg of dihalomethane/kg feed dry matter (DM).

2. The method according to claim 1, said method comprising administering to the ruminant one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane in an effective amount in the range of 5-50 mg/kg feed DM, such as 10-50 mg/kg feed DM, such as 10-40 mg/kg feed DM, such as 10-30 mg/kg feed DM, such as 10-25 mg/kg feed DM, such as 10- 20 mg/kg feed DM, such as 10-15 mg/kg feed DM, such as 12-15 mg/kg feed DM, such as an amount in the range of 8-15 mg/ kg feed DM.

3. The method according to any one of claims 1-2, wherein said one or more dihalomethanes is administered to said ruminant by supplementing feed intended for said ruminant with an amount in the range of 2-50 mg of dihalomethane/kg feed DM or as a bolus dose in an amount corresponding to the range of 2-50 mg of dihalomethane/kg feed DM.

4. The method according to any one of the preceding claims, wherein a single dihalomethane is administered to said ruminant, wherein said dihalomethane is selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane and chloroiodomethane.

5. The method according to any one of the preceding claims, wherein the ruminant is selected from the group consisting of cattle, sheep, and goats, such as lactating cows.

6. The method according to any one of the preceding claims, wherein the methane production in the ruminant animal, calculated in liters per kilogram of feed dry matter, is reduced by at least 10% when measured in metabolic chambers, such as wherein the methane production is reduced by at least 15%, such as at least 20%, such as at least 25%, such as at least 30%.

7. A feed additive for a ruminant comprising one or more dihalomethanes selected from the group consisting of dibromomethane, diiodomethane, bromoiodomethane, and chloroiodomethane in an effective amount in the range of 2-50 mg of dihalomethane/kg feed DM.

8. The feed additive according to claim 7, comprising one or more dihalomethanes in an amount in the range of 5-50 mg/kg feed DM, such as 10-50 mg/kg feed DM, such as 10-40 mg/kg feed DM, such as 10-30 mg/kg feed DM, such as 10-25 mg/kg feed DM, such as 10- 20 mg/kg feed DM, such as 10-15 mg/kg feed DM, such as 12-15 mg/kg feed DM, such as an amount in the range of 8-15 mg/ kg feed DM.

9. The feed additive according to any one of claims 7-8, wherein the feed additive is an additive for a total mixed ration (TMR.) feed.

10. The feed additive according to any one of claims 7-8, wherein the one or more dihalomethanes is in the form of a bolus dose in an effective amount corresponding to a range of 2-50 mg of dihalomethane/kg feed DM.

11. The feed additive according to any one of claims 7-10, comprising a single dihalomethane, wherein said dihalomethane is selected from the group consisting of dibromomethane, diiodomethane and bromoiodomethane, preferably dibromomethane and diiodomethane and more preferably diiodomethane.

12. The feed additive according to any one of claims 7-11, wherein the ruminant is selected from the group consisting of cattle, sheep, and goats, such as lactating cows.

13. The method according to any one of claims 1-6, or the feed additive according to any one of claims 7-12, wherein said one or more dihalomethanes are provided in encapsulated form, suitably, wherein the material for encapsulation is selected from proteins, carbohydrates, lipids, gums and cellulose.

14. The method according to any one of claims 1-6, wherein said one or more dihalomethanes is not administered in a composition together with an organosulfur compound, such as e.g. allicin, diallyl sulfide, diallyl disulfide or allyl mercaptan.

15. The feed additive according to any one of claims 7-13, wherein said feed additive does not comprise an organosulfur compound, such as e.g. allicin, diallyl sulfide, diallyl disulfide or allyl mercaptan.