Novel composition for reducing methane production in ruminants

Abstract

The present invention relates to a method for preparing a composition suitable for reducing total gas production and / or methane production and / or improving growth performance in ruminants, said composition comprising artificial bromides and bromides stabilizing excipients.

Description

Field of the Invention

[0001] The field of the invention relates to methods for preparing compositions suitable for reducing total gas production and / or methane production and / or growth performance in ruminants. Background of the Invention

[0003] Methane (CH4) is a greenhouse gas (GHG) produced primarily by methanogenic microorganisms present in natural ecosystems (e.g., swamps, oceans, and lakes) and in the gastrointestinal tracts of invertebrates and vertebrates (such as termites and ruminants). ˜429-507 Tg of CH4 are removed from the atmosphere and ˜40 Tg from the stratosphere each year by reactions with hydroxyl (OH) radicals; and ˜30 Tg by CH4-oxidizing bacteria in the soil.

[0004] However, GHG emissions from human activities have been increasing rapidly, with atmospheric CH4 concentrations now more than twice as high as in the early 19th century. Methane is very efficient at absorbing solar infrared radiation and has a global warming potential 28 times higher than CO2. Therefore, its accumulation in the atmosphere contributes significantly to climate change. A major source of anthropogenic CH4 can be attributed to agricultural activities, including ruminant animal husbandry.

[0005] According to a recent UN report, cattle farming produces more global warming greenhouse gases than transportation, as measured in CO2 equivalents. In Australia, it is estimated that ruminants contribute ~10% of total GHG emissions. Ruminants produce CH4 as a byproduct of anaerobic microbial fermentation of feed in the rumen, and, to a lesser extent, as a byproduct of anaerobic microbial fermentation of feed in the large intestine. The rumen microbial community is highly diverse and consists of bacteria, protozoa, fungi and bacteriophages, which work together to ferment ingested organic matter (OM) to produce CO2, H2, volatile fatty acids (VFA) and formate. Methanogenic archaea present in the rumen utilize these end products and produce CH4. Although the production of CH4 reduces the partial pressure of H2, which may otherwise inhibit rumen fermentation, it also reduces the amount of energy and carbon available for the formation of VFA essential for ruminant nutrition. Most of the CH4 produced in ruminants is exhaled and burped by the animal, and represents a loss of up to 12% of total energy intake.

[0006] Previous work has focused on using Asparagopsis species as feed supplements to reduce total gas production and / or methane production in ruminants. This work has led to the use of Asparagopsis biomass and products derived from Asparagopsis to reduce methane, but further mitigation strategies to reduce enteric CH4 formation are still needed. Summary of the invention

[0007] In one aspect, the present invention provides a method for reducing total gas production and / or methane production in ruminants, comprising the step of providing to the ruminants an effective amount of a composition comprising artificial bromoform (artificial bromoform or manufactured bromoform) and a bromoform stabilizing excipient (bromoform stabilizing excipient).

[0008] In one embodiment, the step of providing an effective amount of a composition to the ruminant comprises administering to the ruminant an effective amount of a composition comprising synthetic bromoform in a bromoform stabilizing excipient.

[0009] In another embodiment, the step of providing an effective amount of a composition to the ruminant comprises preparing a composition comprising artificial bromoform in a bromoform stabilizing excipient that is usable in a feed system comprising the ruminant.

[0010] In another embodiment, the composition is administered at a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

[0011] In another embodiment, the composition is prepared to be available in an amount that provides a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

[0012] In another embodiment, the ruminant is selected from members of the suborders Ruminantia and Tylopoda.

[0013] In another embodiment, the ruminant is cattle or sheep.

[0014] In another embodiment, the bromoform stabilizing excipient is an edible non-polar substance.

[0015] In another embodiment, the edible non-polar substance is edible oil.

[0016] In another embodiment, the edible oil is selected from the group consisting of almond oil, apricot oil, argan oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, colza oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil. oil, pecan oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0017] In another embodiment, the bromoform stabilizing excipient is an edible carbohydrate or water.

[0018] In another embodiment, the carbohydrate is cyclodextrin or molasses.

[0019] In another embodiment, the composition is in solid, semisolid or liquid form.

[0020] In another aspect, the present invention provides a composition for use in reducing total gas production and / or methane production in a ruminant, wherein the composition comprises synthetic bromoform and a bromoform stabilizing excipient.

[0021] In one embodiment, the invention provides a composition for use as described herein, wherein the bromoform stabilizing excipient is an edible non-polar substance.

[0022] In one embodiment, the invention provides a composition for use as described herein, wherein the edible non-polar substance is an edible oil.

[0023] In one embodiment, the invention provides a composition for use as described herein, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olein oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0024] In one embodiment, the invention provides a composition for use as described herein, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

[0025] In one embodiment, the invention provides a composition for use as described herein, wherein the carbohydrate is cyclodextrin or molasses.

[0026] In one embodiment, the invention provides a composition for use as described herein, wherein the composition is formulated to be provided to a ruminant at a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

[0027] In one embodiment, the invention provides a composition for use as described herein, wherein the composition is in solid, semi-solid or liquid form.

[0028] In one aspect, the present invention provides a feed supplement for use in reducing total gas production and / or methane production in ruminants, the supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0029] In one embodiment, the invention provides a feed supplement for use as described herein, wherein the feed supplement is formulated to provide a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

[0030] In another embodiment, the present invention provides a feed supplement for use as described herein, wherein the supplement further comprises one or more edible excipients.

[0031] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the ruminant is selected from members of the suborder Rumina and the suborder Callosopoda.

[0032] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the ruminant is a cattle or sheep.

[0033] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the bromoform stabilizing excipient is an edible non-polar substance.

[0034] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the edible non-polar substance is an edible oil.

[0035] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olein oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0036] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

[0037] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the carbohydrate is cyclodextrin or molasses.

[0038] In another embodiment, the invention provides a feed supplement for use as described herein, wherein the composition is in solid, semi-solid or liquid form.

[0039] In one aspect, the present invention provides a method of producing a methane reducing ruminant animal feed comprising mixing a ruminant feed with a feed supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0040] In one embodiment, the invention provides a method as described herein, wherein the animal feed comprises a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter of the ruminant feed.

[0041] In one aspect, the present invention provides an animal feed for reducing total gas production and / or methane production in a ruminant when used, the animal feed comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient and a ruminant feed.

[0042] In one embodiment, the invention provides an animal feed for use as described herein, wherein the composition is formulated to provide a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter of the ruminant feed.

[0043] In one embodiment, the invention provides an animal feed when used as described herein, wherein the ruminant is selected from members of the suborder Rumina and the suborder Callosopoda.

[0044] In another embodiment, the invention provides an animal feed for use as described herein, wherein the ruminant is a cattle or sheep.

[0045] In another embodiment, the invention provides an animal feed for use as described herein, wherein the bromoform stabilizing excipient is an edible non-polar substance.

[0046] In another embodiment, the invention provides an animal feed for use as described herein, wherein the edible non-polar substance is an edible oil.

[0047] In another embodiment, the invention provides an animal feed for use as described herein, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olein oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0048] In another embodiment, the present invention provides an animal feed for use wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

[0049] In another embodiment, the present invention provides an animal feed for use wherein the carbohydrate is cyclodextrin or molasses.

[0050] In another embodiment, the invention provides an animal feed for use as described herein, wherein the composition is in solid, semi-solid or liquid form.

[0051] In one embodiment, the present invention provides a stabilized bromoform composition comprising synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of an edible solid, an edible semisolid or an edible liquid.

[0052] In one embodiment, the invention provides a stable bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates and water.

[0053] In one embodiment, the invention provides a stable bromoform combination as described herein, wherein the composition does not comprise one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid and dibromoacetic acid.

[0054] In one embodiment, the present invention provides a method of preparing a stable bromoform composition, the method comprising contacting synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of an edible solid, an edible semisolid, or an edible liquid.

[0055] In one embodiment, the present invention provides a method of preparing a stable bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates and water.

[0056] In one embodiment, the present invention provides a method for preparing a stable bromoform composition, wherein the composition does not contain one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1Data relating to the effects of addition (inclusion) of an Asparagopsis-derived composition comprising Asparagopsis biomass extracted into oil (Asp-Oil) and a composition comprising synthetic bromoform in oil (Syn-Oil) on methane, hydrogen, carbon dioxide, and total gas production (mL / g fed) in vitro are presented. Addition rates were based on bromoform concentrations of 0.08-0.26 mg. Gas results are expressed in mL / gram of Rhodes grass substrate fed, as digestibility was not measured in Experiment 1. Standard errors are not shown (experiment was performed once).

[0058] Figure 2 The effects of low (0.04 mg), medium (0.07 mg) and high (0.11 mg) addition of a composition derived from Asparagaceae comprising Asparagaceae biomass extracted into oil (Asp-Oil) and a composition comprising artificial bromoform in oil (Syn-Oil) are shown on methane, hydrogen, total gas production and in vitro dry matter digestibility (IVDMD). Gas results are expressed as mL / gram dry matter digestibility (mL / g IVDDM). Standard errors are not presented (the experiment was performed once).

[0059] Figure 3 The methane (CH4) produced by the control (no bromoform; CHBr3), canola oil containing artificial synthetic CHBr3 ["Canola Oil"], natural source CHBr3 from Asparagus ["Asp-Oil"], and freeze-dried Asparagus ["FD-Asp"] (supplemented with different levels of CHBr3 [0.03, 0.05, and 0.08 mg CHBr3 / g feed matrix]) during 24, 48, and 72 hours of in vitro rumen fermentation is shown. Values ​​are expressed as mL / g digested dry matter (IVDDM). Lowercase letters indicate statistical significance at P < 0.05 within each time period.

[0060] Figure 4 The hydrogen (H2) produced by the control (no bromoform; CHBr3), canola oil containing artificial synthetic CHBr3 [Canola Oil], natural source CHBr3 from Asparagaceae [Asp-Oil], and freeze-dried Asparagaceae [FD-Asp] (supplemented with different levels of CHBr3 [0.03, 0.05, and 0.08 mg CHBr3 / g feed matrix]) during 24, 48, and 72 hours of in vitro rumen fermentation is shown. The values ​​are expressed as mL / g digested dry matter (IVDDM). Lowercase letters indicate statistical significance at P<0.05 in each time period, while ns indicates not significant.

[0061] Figure 5 In vitro digested dry matter (IVDMD) [A], total volatile fatty acid production (tVFA) in millimoles (mM) [B], acetate (Acetate), propionate (Propionate), butyrate (Butyrate) in percentage of tVFA ​​[C], and acetate to propionate ratio (A:P) [D] produced by control (no bromoform; CHBr3), canola oil containing artificial synthetic CHBr3 [Canola Oil], natural source CHBr3 from Asparagus [Asp-Oil], and freeze-dried Asparagus [FD-Asp] (supplemented with different levels of CHBr3 [0.03, 0.05, and 0.08 mg CHBr3 / g feed matrix] during 72 hours of in vitro rumen fermentation are shown. Lowercase letters indicate statistical significance at P < 0.05 in each time period, while ns indicates not significant.

[0062] Figure 6 Methane (CH4) produced by a control (no bromoform; CHBr3) and four CHBr3-stabilizing excipients [corn oil, cyclodextrin, molasses, and water] (added at different levels of CHBr3 [0.01, 0.04, and 0.07 mg CHBr3 / g feed matrix]) during 24, 48, and 72 hours of in vitro rumen fermentation is shown. Values ​​are expressed as mL / g digested dry matter (IVDDM). Lowercase letters indicate statistical significance at P < 0.05 for each time period.

[0063] Figure 7 Shown is the production of hydrogen (H2) during 24, 48 and 72 hours of in vitro rumen fermentation for a control (no bromoform; CHBr3) and four CHBr3 stabilizing excipients [corn oil, cyclodextrin and molasses] (added at different levels of CHBr3 [0.01, 0.04 and 0.07 mg CHBr3 / g feed matrix]). Values ​​are expressed as mL / g digested dry matter (IVDDM). Lowercase letters indicate statistical significance at P < 0.05 for each time period.

[0064] Figure 8In vitro digestible dry matter (IVDMD) [A], total volatile fatty acid yield (tVFA) in millimoles (mM) [B], acetate, propionate, butyrate in percentage of tVFA ​​[C], and acetate to propionate ratio (A:P) [D] produced by a control (no bromoform; CHBr3) and four CHBr3-stabilizing excipients [corn oil, cyclodextrin, and molasses] (added at different levels of CHBr3 [0.01, 0.04, and 0.07 mg CHBr3 / g feed matrix]) during 72 hours of in vitro rumen fermentation are shown. Lowercase letters indicate statistical significance at P < 0.05 for each time period.

[0065] Fig. 9 Shown are the percent reductions in methane (CH4) during 24 (#1), 48 (#2), and 72 (#3) hours of in vitro rumen fermentation using artificial synthetic bromoform (CHBr3) [grey bars; corn oil, cyclodextrin, molasses, canola oil, and water] and two Asparagus products [black bars] containing natural CHBr3 (lyophilized [FD-Asp] or in canola oil [Asp-Oil]) as stabilizing excipients (supplemented with different levels of CHBr3 [0.01-0.08 mg CHBr3 / g feed matrix]).

[0066] Fig.10 Shown are the reductions (expressed as a percentage) in methane (CH4) production (grams CH4 / day) and CH4 yield (grams CH4 / kg DMI) of artificial synthetic bromoform (CHBr3) stabilized in canola oil [Canola Oil] and natural source CHBr3 stabilized in canola oil [Asp-Oil] (at 4 supplementation levels; 6, 12, 18 and 20 mg CHBr3 / kg dry matter intake) when fed to lactating dairy cows in a pulse feeding system.

[0067] Fig.11 The bromoform (CHBr3) content (mg / g) is shown after storage for 1, 2, 4 weeks at 40°C, 25°C, 4°C and -20°C under airtight conditions and after storage for 1, 2, 4 weeks at 40°C and 25°C under open air conditions in A) corn oil, B) molasses and C) cyclodextrin. Values ​​are mean ± SE (n = 3). DETAILED DESCRIPTION

[0068] As will be discussed below, the present invention is based in part on the characterization of compositions that are capable of inhibiting methane production by rumen fermentation using synthetic bromoform. These compositions have anti-methanogenic activity at doses of synthetic bromoform that have previously been demonstrated (in different compositions) to not significantly inhibit methane production. Developing compositional technologies for delivering Asparagaceae products to livestock diets on pastures allows for maximum distribution and / or environmental benefits, particularly in countries where the red meat, dairy and wool industries (by animal head count) are the primary livestock industries.

[0069] The present invention provides stable compositions comprising synthetic bromoform. The ability to prepare stable compositions comprising synthetic bromoform allows the use of low doses of synthetic bromoform to inhibit methane production from ruminants, including at doses of bromoform that have previously been shown not to inhibit methane production.

[0070] Since low doses of synthetic bromoform can be used, a bioavailable formulation can be delivered to animals in livestock systems such as pasture systems.

[0071] The ability to prepare stable compositions comprising synthetic bromoform also allows for the preparation of compositions with known sustained release characteristics, including at different temperatures.

[0072] The compositions described herein allow high levels of undesirable trace elements and minerals to be avoided.

[0073] In vitro studies using rumen fluid have demonstrated that CH4 production is significantly affected by synthetic methyl halides when these synthetic compounds are added to rumen fluid. For example, bromoform and dibromochloromethane in dimethyl sulfoxide (DMSO) significantly inhibited CH4 production when added to rumen fluid compared to controls when added to rumen fluid (at concentrations of ≥5 μM) (Machado, L., Magnusson, M., Paul, NA et al. J Appl Phycol (2016) 28: 3117).

[0074] However, previous work has demonstrated that the effects of Asparagaceae biomass containing secondary metabolites - or products derived from Asparagaceae - both in rumen fluid and in vivo are variable and contradictory due to differences in the type and quality of effect of the extracts / compositions containing such compounds, the dose of the compounds, and the basal diet.

[0075] Bromoform, one of the secondary metabolites contained in Asparagaceae biomass, is volatile, and its physical properties, including its volatility, are thought to make it impractical for use in vivo or in vitro. Preliminary work demonstrated that synthetic bromoform in DMSO was unable to significantly inhibit methane production at low doses (e.g., doses below 5 uM) (Machado et al. (2016) J Appl Phycol 28:3117–3126). Subsequent work by Machado et al. (2018) (Microbial Ecology 75 (D1)) demonstrated that biomass addition at 2% OM was more effective in inhibiting methane production than comparable doses of synthetic bromoform in DMSO, suggesting that other components of Asparagaceae contribute to its anti-methanogenic activity.

[0076] As noted above, bromoform is volatile, and thus studies using volatile bromoform (eg, unstabilized) in closed systems have not demonstrated that synthetic bromoform can be delivered so as to be bioavailable in the rumen.

[0077] Importantly, more recent work has demonstrated that synthetic bromoform does not inhibit methane production (Stefenoni et al. (2019) J. Dairy Sci. Vol. 102, Suppl. 1, p. 378). Without wishing to be bound by theory, the inventors propose that bromoform must be made in a form that is bioavailable in the rumen for utilization.

[0078] Exposure to high levels of concentrated bromoform is considered harmful to animals, and synthetic bromoform and chemically related compounds (e.g., bromochloromethane) are unsafe as manufactured / synthesized / purified chemicals and are not approved for use in human and animal applications, including inhibition of methane production in ruminants.

[0079] Recently, Muizelaar et al., Foods (2021) 10:584 published results presenting data that claim that after feeding Asparagaceae to cattle, bromoform was detected in the milk of some cows on the first day of feeding only in the low and medium treatment groups (9.1 and 11 μg / L, respectively), and bromoform (35 μg / L) was detected in the milk of a single sample only in the high treatment group on the 9th day, and two animals were killed and their rumen walls showed abnormalities. After histological examination, signs of inflammation became visible. The authors of the study inferred that within the scope of their experiments, CHBr3 did not accumulate in animal tissues, but could be excreted in urine and milk. This article expresses many concerns about human consumption after Asparagaceae use, and further work is needed to examine the effects of ruminants consuming materials containing bromoform. Although this article teaches away from the use of materials containing bromoform, as part of the work described herein, the inventors propose that the composition of the present invention allows the use of significantly lower amounts of bromoform.

[0080] The present inventors also consider that the work described by Muizelaar et al. does not directly link inflammation to Asparagaceae.

[0081] The present inventors have examined the effects of compositions comprising synthetic bromoform and a bromoform stabilizing excipient. In particular, the present inventors have Figure 1 , 2 , 3, 6 and 9, it was demonstrated that compositions comprising artificial bromoform and bromoform-stabilizing excipients can reduce methane production in in vitro fermentation and have been used in Fig.10 It was demonstrated in the literature that the compositions described herein can inhibit methane production in vivo. In some embodiments, the compositions comprising synthetic bromoform and a bromoform stabilizing excipient can eliminate methane production from in vitro and / or in vivo fermentation.

[0082] Importantly, the inventors have demonstrated in Example 8 that the in vivo effects of the compositions described herein include improvements in growth performance. In particular, the in vivo effects observed include increased average daily gain and increased feed utilization efficiency.

[0083] In stark contrast to the data presented herein, previous work demonstrated that the addition of bromoform at doses of 0.25%, 1%, and 1.5% of feed dry matter had no effect on methane production (Stefenoni et al. (2019) J. Dairy Sci. Vol. 102, Suppl. 1, p. 378, Dose-response effect of the macroalga Asparagopsis taxiformis on enteric methane emission in lactating dairy cows), whereas in the same experiment, Asparagopsis taxiformis (which contains a large number of antimethanogenic compounds, including bromoform) linearly reduced methane (CH4) yields at doses of 0.25%, 0.5%, 0.75%, 1.0%, and 1.5% of feed dry matter when added in in vitro fermentation. This indicates that bromoform alone had no effect on methane production, whereas Asparagaceae added at corresponding doses inhibited methane production.

[0084] Thus, in one aspect, the present invention provides a method for reducing total gas production and / or methane production in a ruminant, comprising the step of providing to the ruminant an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0085] Machado et al. (2016) J Appl Phycol 28:3117–3126 demonstrated that Asparagopsis taxiformis contains a number of antimethanogenic compounds, including bromoform, dibromochloromethane, and bromochloromethane. This work demonstrated that artificial bromoform formulated in DMSO significantly inhibited methane production at doses of 5 μM, 10 μM, and 25 μM, but at 1 μM, artificial bromoform did not significantly inhibit methane production in in vitro fermentation. Machado et al. (2016) showed that bromoform at 1.3 μM was equivalent to the amount of bromoform added to Asparagopsis taxiformis at 2% OM (24.7 mg) in vitro. Since 1 μM artificial bromoform did not significantly inhibit methane production, Machado et al. showed that more than 1 μM of bromoform was required for significant inhibition of methane production. The compositions described herein allow for the provision of compositions effective in reducing methane production at doses of bromoform significantly lower than those previously demonstrated in Machado et al. (2016) J Appl Phycol 28:3117-3126 to provide no significant effect.

[0086] The present invention demonstrates that the use of 0.01 mg of artificial bromoform and bromoform stabilizing excipients per gram of feed matrix reduces total gas production and / or methane production, and that total gas production and / or methane production can be eliminated at higher doses of compositions comprising artificial bromoform and bromoform stabilizing excipients (e.g., more than 0.04 mg of artificial bromoform and bromoform stabilizing excipients). This data contrasts with the data of Machado et al., which demonstrate that significantly more bromoform is required (e.g., more than 1 μM of artificial bromoform is required).

[0087] There are many problems in using Asparagaceae biomass and products derived from Asparagaceae to suppress methane production. One problem is that the amount of secondary metabolites (bromoform, dibromochloromethane and bromochloromethane) known to suppress methane production is different between Asparagaceae batches, and therefore the composition of bromoform, dibromochloromethane and bromochloromethane with a desired amount formed by Asparagaceae needs to pay great efforts. In contrast to Asparagaceae biomass and products derived from Asparagaceae, the composition described herein allows providing a composition comprising a controlled amount of artificial bromoform.

[0088] In one embodiment, the present invention provides a stable bromoform composition comprising artificial bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates and water.

[0089] In one embodiment, the stable bromoform compositions described herein do not comprise dibromochloromethane, bromochloroacetic acid, and / or dibromoacetic acid.

[0090] Machado et al. (2016) showed that 1.3 μM bromoform is equivalent to the amount of bromoform added to Asparagaceae in vitro with 2% OM (24.7 mg). Since more than 1 μM bromoform is required to significantly inhibit methane production (based on bromoform content and data from Machado et al.), it will be necessary to add more than 2% OM (24.7 mg) of Asparagaceae to significantly inhibit methane production. However, it is known that adding Asparagaceae to feeds with high % OM levels can lead to animals that avoid consuming feed (Muizelaar et al. Foods (2021) 10: 584). The compositions described herein allow for the provision of compositions that avoid the use of Asparagaceae biomass or products derived from Asparagaceae that are unpalatable to ruminants.

[0091] In addition, and without wishing to be bound by theory, the inventors propose that other secondary metabolites of Asparagaceae, including dibromochloromethane and bromochloromethane, may contribute to the anti-methanogenic effect of Asparagaceae biomass or products derived from Asparagaceae when provided in combination. For example, Machado et al. (2016) J Appl Phycol 28:3117–3126 Figure 2 It was demonstrated that total gas production was significantly reduced by Asparagaceae biomass, but the extract at a dose equivalent to the same amount of biomass did not reduce total gas production, and Asparagaceae biomass had the greatest inhibition of methane production compared to the extract at the same amount of biomass, indicating that other secondary metabolites contribute to the antimethanogenic effect.

[0092] In line with this, Figure 1 Preliminary data in show that Asparagaceae biomass in oil inhibits methane production to a greater extent than compositions comprising synthetic bromoform at the same concentration (0.08 mg / g OM), and that Asparagaceae biomass in oil and synthetic bromoform in oil inhibit methane production to the same extent and in the same manner at higher doses. Importantly, however, the inventors have now demonstrated that synthetic bromoform in a stabilizing excipient inhibits methane production to the same extent as Asparagaceae biomass in oil.

[0093] It is not clear whether metabolites (when provided alone) can inhibit methane production. For example, as discussed above, previous work demonstrated that synthetic bromoform does not inhibit methane production. The compositions described herein allow for the provision of compositions that are effective in reducing methane production in the absence of dibromochloromethane and bromochloromethane and other secondary metabolites of Asparagus. Without wishing to be bound by theory, the inventors propose that the compositions described herein comprising synthetic bromoform and a bromoform stabilizing excipient provide bioavailable bromoform that is capable of inhibiting methane production at low doses. Fig. 9As summarized in , the present invention provides for the use of many types of bromoform stabilizing excipients which, when combined with bromoform, provide bioavailable bromoform capable of inhibiting methane production at low levels.

[0094] The inventors have demonstrated in Example 6 that a composition comprising bromoform and a bromoform stabilizing excipient, wherein the bromoform is a bromoform derived from Asparagaceae, can inhibit methane production in animals. Example 8 demonstrates that the in vivo effects of the compositions described herein include improvements in growth performance metrics. In particular, the observed in vivo effects include increased average daily weight gain and increased feed utilization efficiency. In another embodiment provided herein, the invention provides a composition comprising bromoform and a bromoform stabilizing excipient, wherein the bromoform is a bromoform derived from Asparagaceae.

[0095] In one embodiment, the invention provides a composition comprising bromoform and a bromoform stabilizing excipient, wherein the bromoform is bromoform derived from Asparagaceae, and wherein the composition further comprises synthetic bromoform.

[0096] Figure 3 Preliminary work is presented showing that an Asparagaceae-derived composition comprising Asparagaceae biomass extracted into a bromoform stabilizing vehicle (Asp-Oil) inhibits methane production to the same extent as synthetic bromoform in canola oil, even though the canola oil composition does not contain other antimethanogenic compounds present in Asp-Oil. The ability to deliver bioavailable synthetic bromoform in controlled amounts in the absence of other contaminants of Asparagaceae allows for new uses of the compositions described herein.

[0097] Another problem is that increasing the level of addition of Asparagaceae biomass or products derived from Asparagaceae also requires attention to trace elements and minerals that may accumulate in the seaweed. Asparagaceae, like many other seaweeds, can contain iodine at concentrations that can exceed the maximum tolerable level in the feeding system. The compositions described herein that include artificial bromoform and bromoform stabilizing excipients allow for the elimination of trace elements and minerals (such as iodine) from Asparagaceae biomass or products derived from Asparagaceae.

[0098] In addition, like many other seaweeds, Asparagaceae can contain bromine in concentrations that can exceed the maximum tolerable level in the feeding system. The compositions described herein comprising artificial bromoform and bromoform stabilizing excipients allow for the elimination of trace elements and minerals (such as bromine) from Asparagaceae biomass or products derived from Asparagaceae.

[0099] As used herein, the term "reduction" includes a reduction in the amount of a substance compared to a reference. For example, relative to an animal or multiple animals that do not use a composition comprising an effective amount of a composition comprising an artificial bromoform and a bromoform-stabilizing excipient as described herein, a ruminant or multiple ruminants that use an effective amount of a composition comprising an artificial bromoform and a bromoform-stabilizing excipient according to the present invention produces a total gas and / or methane reduction. Reduction can be measured in vitro with an artificial rumen system that simulates anaerobic fermentation, or measured in an animal that is confined to a respiratory chamber. Evaluating the intestinal methane production of ruminants is within the knowledge and skills of trained personnel in this area.

[0100] As used herein, the term "anaerobic fermentation" is intended to include in vitro anaerobic fermentation (eg, in an experimental system) and in vivo anaerobic fermentation (eg, in ruminants).

[0101] As used herein, the term 'reducing total gas production' refers to reducing the total amount of gas produced, such as reducing the amount of total gas produced in the gastrointestinal tract. The term includes the overall volume of all gases generated, such as due to anaerobic fermentation in the systems described herein. Fermentation in the rumen and intestine of ruminants causes gas (including methane) production. The present invention is intended to reduce this process, such as reducing the total amount of gas produced in the gastrointestinal tract. Estimating the total gas production of ruminants is within the knowledge and skills of trained personnel in the field.

[0102] As used herein, the term 'reducing methane production' refers to reducing methane produced in the gastrointestinal tract. The term includes, for example, the specific volume of methane produced due to anaerobic fermentation in the systems described herein. Fermentation in the rumen and intestine of ruminants causes methane production. The present invention is intended to reduce this process, such as reducing the total amount of methane produced in the gastrointestinal tract. Estimating methane production in ruminants is within the knowledge and skills of a person trained in the art.

[0103] In a preferred embodiment of the invention, the amount of total gas produced is reduced by at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% compared to a reference. In one embodiment, the reference is the amount of total gas produced when an effective amount of a composition comprising artificial bromoform and bromoform stabilizing excipients is not administered to an animal. In another embodiment, the reference is the amount of total gas produced when a control feed is administered to an animal. In another embodiment, the reference is the amount of total gas produced when a control feed is subjected to in vitro anaerobic fermentation.

[0104] In a preferred embodiment of the invention, the amount of methane produced is reduced by at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% compared to a reference. In one embodiment, the reference is the amount of methane produced when an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient is not administered to an animal. In another embodiment, the reference is the amount of methane produced when a control feed is administered to an animal. In another embodiment, the reference is the amount of methane produced when a control feed is subjected to in vitro anaerobic fermentation.

[0105] "Effective amount" means an amount of a composition comprising artificial bromoform and bromoform stabilizing excipients as described herein that is sufficient to allow improvements, such as: reducing the amount of methane produced compared to a reference or control, reducing the amount of total gas produced compared to a reference or control, maintaining effective levels of required volatile fatty acids compared to a reference or control, reducing the acetate to propionate ratio compared to a reference or control, maintaining live weight, dry matter intake and / or organic matter intake compared to a reference or control, or improving growth performance compared to a reference or control. Within the meaning of the present invention, the methane reduction effect can be measured in the rumen using an artificial rumen system (such as described in T. Hano., J. Gen. Appl. Microbiol., 39, 35-45, 1993), or in vivo by oral administration to ruminants.

[0106] The effective amount of the composition comprising artificial bromoform and bromoform stabilizing excipients can be determined by the methods described herein, including in vitro and in vivo studies and in vivo dose-response studies described in Kinley et al. (2020) Journal of Cleaner Production 259: 120836 and WO2015109362, which are incorporated herein by reference. For example, the inventors have confirmed that in vitro rumen fermentation can be used to examine the effect of the amount of the composition comprising artificial bromoform and bromoform stabilizing excipients on total gas, hydrogen and methane production, and can be used to examine the level of volatile fatty acids (including acetate and propionate). The in vivo studies described herein can be used to measure the measurement of growth performance. Therefore, in vitro rumen fermentation can be used to characterize the dosage of the composition comprising artificial bromoform and bromoform stabilizing excipients, and the dosage can be an effective amount sufficient to allow improvement, such as, compared with a reference or control, to reduce the amount of methane produced, compared with a reference or control, to reduce the amount of total gas produced, compared with a reference or control, to maintain the effective level of the desired volatile fatty acids compared with a reference or control, or to reduce the acetate to propionate ratio compared with a reference or control.

[0107] For example, Figure 4 and 7It was shown that at high doses, compositions comprising synthetic bromoform and bromoform stabilizing excipients can increase hydrogen levels, consistent with inhibition of methane production. Figure 5 It was confirmed that no negative impact on fermentation was observed in terms of IVDDM and tVFA ​​production.

[0108] As used herein, the term "providing" includes providing a composition comprising artificial bromoform and a bromoform stabilizing excipient as a feed additive in a feed provided to an animal or animal system (e.g., an animal in a feedlot or an animal in a livestock system, etc.). Providing also includes administering a composition as described herein.

[0109] In one embodiment, the livestock system is a pastoral, a feedlot, a regular supplementation system, or a combination thereof.

[0110] "Administering" and "administered" mean the action of introducing a composition as described herein into the gastrointestinal tract of an animal. More particularly, this administration is administration by an oral route. This administration can be particularly carried out in the following manner: supplementing the feed intended for the animal with a composition comprising artificial bromoform and a bromoform-stabilizing excipient, and then the feed so supplemented is ingested by the animal. This administration can also include providing a composition comprising artificial bromoform and a bromoform-stabilizing excipient in a form consumed by the animal (e.g., a lick block, a supplement, etc.). Administration can also be carried out using a gastric tube or any other means that makes it possible to introduce the composition (e.g., a dosage form of the composition (e.g., a bolus)) directly into the gastrointestinal tract of the animal.

[0111] Thus, the present invention provides a method as described herein, wherein the step of providing to the ruminant an effective amount of a composition comprises administering to the ruminant an effective amount of a composition comprising artificial bromoform in a bromoform stabilizing excipient.

[0112] Compositions comprising artificial bromoform and bromoform-stabilizing excipients can be provided to ruminants in one of many ways. Compositions comprising artificial bromoform and bromoform-stabilizing excipients can be provided in solid form as veterinary drugs, can be distributed in excipients, and directly fed to animals, can be physically mixed with feed materials in any suitable form (e.g., dry form, solution, or suspension, etc.), or compositions comprising artificial bromoform and bromoform-stabilizing excipients can be formed into solutions and then sprayed onto feed materials. For example, dry mixtures prepared by adsorbing or depositing a solution on / in a dry excipient can be used.

[0113] Methods of administering to animals compositions comprising synthetic bromoform and bromoform stabilizing excipients are deemed to be within the skill of the artisan.

[0114] When used in combination with feed materials, the feed materials are preferably cereal / hay / silage / grass based. Included in such feed materials are improved and / or grass or legume based forages (either directly grazed or made into stored forage hay), any feed ingredients and food or feed industry by-products and biofuel industry by-products and corn meal and mixtures thereof, or feedlot and dairy rations such as those high in cereal content.

[0115] The composition comprising artificial bromoform and bromoform stabilizing excipients can be provided as an inclusion in an animal feed supplement for consumption in an animal system (e.g., a feedlot, natural grassland / pasture livestock system) in any suitable form, including mineral loose licks, wet licks, pellets, aqueous suspensions or lick blocks. As known to those skilled in the art, such loose licks, wet licks, pellets, aqueous suspensions or lick blocks are particularly convenient for feeding mineral supplements (as well as proteins and carbohydrates) to ruminants grazing in pastures. In addition to the composition comprising artificial bromoform and bromoform stabilizing excipients of the present invention, such loose licks, wet licks, pellets, aqueous suspensions or lick blocks, etc., can also contain various types of binders, such as cement, gypsum, lime, calcium phosphates, carbonates and / or gelatin; and optional additional additives, such as vitamins, trace elements, mineral salts, sensory additives, etc.

[0116] The time of administration is not critical, as long as a reduction in methane production and / or growth performance is shown. Administration can be performed at any time as long as the feed is retained in the rumen. However, since the composition comprising artificial bromoform and a bromoform stabilizing excipient is preferably present in the rumen at about the time when methane is produced, the composition comprising artificial bromoform and a bromoform stabilizing excipient is preferably administered with the feed or immediately prior to feeding.

[0117] In a particular embodiment of the present invention, the composition comprising artificial bromoform and bromoform stabilizing excipients is supplemented with a composition intended for ruminants, and the composition comprising artificial bromoform and bromoform stabilizing excipients is administered to the animal in an effective amount. Within the meaning of the present invention, "supplementation" means the action of directly incorporating an effective amount of a composition comprising artificial bromoform and bromoform stabilizing excipients according to the present invention into a feed intended for an animal. Therefore, when feeding, the animal ingests a composition comprising artificial bromoform and bromoform stabilizing excipients according to the present invention, which can then act to maintain the digestibility of the fiber and / or cereals contained in the animal feed. Alternatively, supplements such as loose licks, wet licks, pellets, aqueous suspensions or lick blocks and other feed supplements can be provided in the animal system without being directly incorporated into the animal feed.

[0118] Therefore, another subject of the present invention relates to a feed supplement for ruminants comprising a composition containing artificial bromoform and a bromoform stabilizing excipient.

[0119] In another aspect, the present invention also provides a feed supplement for reducing total gas production and / or methane production in ruminants, the supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0120] In one embodiment, an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient is administered to the ruminant by supplementing a food intended for the animal with the composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0121] As discussed above, the inventors have demonstrated in Example 6 that a composition comprising bromoform and a bromoform stabilizing excipient, wherein the bromoform is bromoform derived from Asparagus, is capable of inhibiting methane production in vivo. Example 8 demonstrates that the in vivo effects of the compositions described herein include improved growth performance metrics. In particular, the in vivo effects observed include increased average daily gain and increased feed utilization efficiency.

[0122] Thus, in one embodiment, the present invention provides a method for improving the growth performance of a ruminant, comprising the step of providing to the ruminant an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0123] In a preferred embodiment, the growth performance is increased average daily gain and / or increased feed utilization efficiency.

[0124] In one embodiment, the present invention provides a method of increasing the average daily gain of a ruminant, comprising the step of providing to the ruminant an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0125] In one embodiment, the present invention provides a method of increasing feed utilization efficiency of a ruminant, comprising the step of providing to the ruminant an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0126] In one embodiment, the present invention provides a method as described herein, wherein an effective amount of a composition comprising synthetic bromoform and a bromoform stabilizing excipient is provided in a livestock system, thereby reducing the total gas production and / or methane production and / or improving the growth performance of livestock animals in the livestock system.

[0127] In one embodiment, the invention provides a method as described herein, wherein an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient is provided to a stocker operation so that livestock animals in the stocker operation can consume the composition comprising artificial bromoform and a bromoform stabilizing excipient, thereby reducing the total gas production and / or methane production and / or improving the growth performance of the livestock animals in the stocker operation.

[0128] In one embodiment, the invention provides a method as described herein, wherein an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient is provided to a feedlot system so that livestock animals in the feedlot system can consume the composition comprising artificial bromoform and a bromoform stabilizing excipient, thereby reducing the total gas production and / or methane production and / or improving the growth performance of livestock animals in the feedlot system.

[0129] In one embodiment, the present invention provides a method as described herein, wherein an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient is provided to a pasture system so that livestock animals in the pasture system can consume the composition comprising artificial bromoform and a bromoform stabilizing excipient, thereby reducing the total gas production and / or methane production and / or improving the growth performance of the livestock animals in the pasture system.

[0130] Ruminants are mammals of the order Artiodactyla that digest plant-based foods by initial softening and partial fermentation in the animal's first stomach chamber, followed by regurgitation of the semi-digested material (now called the cud) and chewing it again.

[0131] The process of chewing the cud to further break down plant matter and stimulate digestion is called "rumination". The digestive tract of ruminants has four chambers, namely the rumen, reticulum, omasum and abomasum. In the first two chambers, namely in the rumen and reticulum, food is mixed with saliva and separated into layers of solid and liquid materials. The solids are massed together to form the rumen or food bolus. The rumen is then regurgitated and chewed slowly to mix it thoroughly with saliva, which further breaks down the fiber. In these chambers, fiber (especially cellulose) is broken down into glucose by symbiotic anaerobic bacteria, protozoa and fungi in the unique process of digestion and fermentation by rumen microorganisms. Then, the decomposed fiber, now in the liquid part of the contents, passes through the rumen into the next stomach chamber, the omasum, where further fermentation occurs and water and dissolved nutrients are absorbed. The next stage is the abomasum, where undigested and bypassed rumen feed is digested, just like in a single stomach. Finally, the digestive tract contents are transferred to the small intestine, where nutrient absorption occurs. Almost all of the glucose produced by cellulose fermentation is utilized by the symbiotic microbial consortium. Ruminants obtain their energy from volatile short-chain fatty acids (VFAs) produced by bacteria, namely acetate, propionate, butyrate, valerate and isovalerate. Most of the protein utilized by ruminants is microbial protein. The byproducts of bacterial fermentation of feed are CO2 and H2, which are used by anaerobic archaea for reduction processes, which in turn have a waste byproduct, CH4. The production of CH4 is an energy inefficiency of digestive fermentation in ruminants.

[0132] Importantly, the inventors have demonstrated that the compositions described herein have the property of reducing total gas production and / or methane production in ruminants without compromising rumen fermentation (as measured by dry matter digestibility).

[0133] Examples of ruminants are listed below. However, preferably, the compositions described herein are used as feed food additives for livestock (such as cattle, goats, sheep and llamas). In particular, the present invention can be used for cattle and sheep. Therefore, in one embodiment, the ruminant is selected from members of the suborder Ruminans and the suborder Callosopoda. In another embodiment, the ruminant is a cattle or sheep. In another embodiment, the ruminant is a cattle.

[0134] Bromoform (CHBr3) is also referred to by many synonyms, including bromoform, see PubChem CID: 5558.

[0135] As used herein, the term "artificial bromoform" refers to bromoform that is not derived from Asparagaceae biomass, and the term includes artificially synthesized bromoform. Artificial bromoform can be artificially synthesized by chemical means by known methods, including haloform reaction using acetone and sodium hypobromite, electrolysis of potassium bromide in ethanol, or by treating chloroform with aluminum bromide, or reacting chloroform with sodium hydroxide to generate bromoform and sodium chloride.

[0136] Artificial bromoform can also be artificially synthesized by biological means, such as using enzymes or genetically modified organisms (including in a manufacturing system), and the produced bromoform is collected from the headspace gas or captured using chemical or mechanical means. For example, WO2020243792 (incorporated herein by reference) describes a recombinant yeast capable of producing bromoform.

[0137] In a preferred embodiment, the synthetic bromoform is free bromoform provided in a purified form. For example, in one embodiment, the synthetic bromoform is 90% pure and may contain a low level (e.g., 1-10%) of stabilizers such as ethanol or pentene, etc. In another embodiment, the synthetic bromoform is at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, or at least 99% pure.

[0138] In a preferred embodiment, the compositions described herein do not contain iodine.

[0139] In a preferred embodiment, the compositions described herein do not contain bromine.

[0140] In one embodiment, the present invention provides a stable bromoform composition comprising synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of an edible solid, an edible semisolid, or an edible liquid.

[0141] In one embodiment, the invention provides a stable bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates and water.

[0142] In one embodiment, the invention provides a stable bromoform combination as described herein, wherein the composition does not comprise one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid and dibromoacetic acid.

[0143] In one embodiment, the present invention provides a method of preparing a stable bromoform composition comprising contacting synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of an edible solid, an edible semisolid, or an edible liquid.

[0144] In one embodiment, the present invention provides a method of preparing a stable bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates and water.

[0145] In one embodiment, the present invention provides a method for preparing a stable bromoform composition, wherein the composition does not contain one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

[0146] The inventors herein have demonstrated that stabilizing excipients can be used to stabilize volatile bromoform so that it is bioavailable for rumen fermentation to inhibit methane production. Importantly, the inventors have demonstrated that carbohydrates (including cyclodextrins and molasses), water, and edible non-polar substances (such as oils) can be used to stabilize bromoform and allow for the formation of a composition that makes bromoform bioavailable for rumen fermentation.

[0147] Stabilizing excipients are discussed below and include edible solids, liquids, and semisolids that are capable of stabilizing bromoform.

[0148] Importantly, the ability to use different stabilizing excipients allows for the formation of compositions that are solid, semisolid or liquid and that contain bromoform that is bioavailable for rumen fermentation.

[0149] Furthermore, the ability to use different stabilizing excipients allows for the formation of compositions with defined stability.

[0150] For example, Fig.10 The stability of the compositions described herein over time under different storage conditions is demonstrated.

[0151] Fig.10 It has also been shown that the sustained release properties of the compositions can be determined (including under different conditions). Thus, these sustained release properties can be used to provide methods for improving growth performance or reducing methane production that take into account different feed intake times and / or different temperatures (e.g. climate).

[0152] The present inventors have surprisingly found that a stabilizing excipient in combination with synthetic bromoform delays the release of the volatile synthetic bromoform, thereby providing an extended or sustained release of the synthetic bromoform.

[0153] As used herein, the term "volatile" refers to the tendency of an agent to evaporate and typically refers to a fluid substance.

[0154] The term "so as to provide sustained release of the agent" means that the synthetic bromoform and the stabilizing excipient are able to dissociate, whereby the synthetic bromoform is released at a reduced rate compared to synthetic bromoform administered alone.

[0155] Thus, in one aspect, the present invention provides an antimethanogenic composition for use in animals comprising synthetic bromoform together with a stabilizing excipient such that a sustained release of the synthetic bromoform is provided.

[0156] In another aspect, the present invention provides an animal feed comprising the above-mentioned composition of the present invention together with a nutrient source.

[0157] In another aspect, the present invention provides a medicament for administering artificial bromoform to an animal over an extended period of time, comprising a composition comprising artificial bromoform together with a stabilizing excipient, so as to provide a sustained release of the artificial bromoform in a manner such that the composition is retained by the animal over the period of time.

[0158] The term "over an extended period of time" refers to a period of time that is longer than the time it takes for the synthetic bromoform to evaporate when the synthetic bromoform is not present in the composition.

[0159] The term "in such a way that the composition is retained by the animal over the said period of time" means that the composition is administered in a suitable manner allowing sustained release. For example, in ruminants, administration may be provided in the form of a controlled release device or may be provided in the feed.

[0160] As demonstrated herein, where the animal is a ruminant, the method results in increased weight gain (e.g., increased average daily gain). The method also has benefits for anaerobic fermentation of ruminant and non-ruminant feces because it reduces greenhouse gas emissions.

[0161] In a particularly preferred embodiment, the present invention is directed to a method of reducing methane production in an animal over an extended period of time comprising administering a methane reducing effective amount of an antimethanogenic composition comprising synthetic bromoform together with a stabilizing excipient such that sustained release of the agent is provided.

[0162] As used herein, the term "stabilizing excipient" refers to an excipient that prevents the loss of bromoform from the composition and that allows the synthetic bromoform to be bioavailable in ruminants. Stabilizing excipients include those described herein, which include edible solids, liquids, and semisolids that are capable of stabilizing bromoform.

[0163] Suitable stabilizing excipients include alcohols (e.g., ethanol), benzene, chloroform, ether, petroleum ether, DCM, diethyl ether, hexane, pyridine, toluene, xylene, acetone, and oils. Preferably, the bromoform stabilizing excipient is safe for consumption by animals at the final concentration in the dose of the composition to be provided to the animal.

[0164] In a preferred embodiment, when the composition comprises a low dose of synthetic bromoform, the bromoform stabilizing excipient allows the synthetic bromoform to be bioavailable in ruminants.

[0165] In a preferred embodiment, when the composition comprises synthetic bromoform at a dose of less than 5 μM bromoform, the bromoform stabilizing excipient allows the synthetic bromoform to be bioavailable in a ruminant.

[0166] In a preferred embodiment, when the composition comprises synthetic bromoform at a dose of less than 1 μM bromoform, the bromoform stabilizing excipient allows the synthetic bromoform to be bioavailable in ruminants.

[0167] In a preferred embodiment, the bromoform stabilizing excipient is not DMSO.

[0168] In one aspect, the present invention provides a method for preparing a stable bromoform composition comprising contacting synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils and edible carbohydrates and water.

[0169] In one aspect, the present invention provides a method for preparing a composition comprising artificial bromoform and a bromoform stabilizing excipient, the method comprising the steps of:

[0170] (a) providing artificial bromoform;

[0171] (b) providing a bromoform stabilizing excipient; and

[0172] (c) contacting the synthetic bromoform with a bromoform stabilizing excipient under conditions to form a composition.

[0173] In one embodiment, the bromoform stabilizing excipient is selected from the group consisting of edible waxes, greases, oils, cyclodextrins, molasses, and saturated fats.

[0174] In one embodiment, the bromoform stabilizing excipient is an edible protein.

[0175] The inventors herein have demonstrated that edible carbohydrates, including cyclodextrins and molasses, can be used as stabilizing excipients.

[0176] Thus, in one embodiment, the stabilizing excipient is a carbohydrate.

[0177] Carbohydrates include molasses, cyclodextrin, lactose, dextrose, sucrose, glucose, fructose, galactose, xylose, arabinose, β-glucan, galactan, pectin, etc.

[0178] Figure 6 and 9 It was demonstrated that a composition comprising synthetic bromoform and cyclodextrin (as a bromoform stabilizing excipient) can be formed and can inhibit methane production from rumen fermentation.

[0179] In another embodiment, the stabilizing excipient is a cyclodextrin.

[0180] Cyclodextrins (sometimes referred to as cycloamylose) are cyclic oligosaccharides containing glucose units (i.e., (α-1,4) linked aD-pyranose glucose units) bonded together in a ring. Cyclodextrins are typically produced from starch by enzymatic conversion. Typical cyclodextrins contain many glucose monomers (in the range of six to eight units) in the ring, forming a cone or hollow plug shape; for example, α-cyclodextrin contains 6-membered sugar ring molecules, β-cyclodextrin contains 7-membered sugar ring molecules; and γ-cyclodextrin contains 8-membered sugar ring molecules. If desired, cyclodextrins suitable for use in the compositions of the present invention can be modified by adding substituents. As used herein, "cyclodextrin" includes modified and unmodified cyclodextrins. The substituents typically replace hydrogen atoms on the entire hydroxyl group of the cyclodextrin ring or one or more hydroxyl groups of the cyclodextrin ring.

[0181] The cyclodextrin may be selected from α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or derivatives thereof, which may be naturally occurring and / or synthetically produced.

[0182] In one embodiment, the invention provides a composition as described herein, wherein the composition is prepared by a method comprising the steps of:

[0183] (a) contacting synthetic bromoform and an aqueous solution of cyclodextrin; and

[0184] (b) preparing a solid by step a).

[0185] In another embodiment, the present invention provides a composition as described herein, prepared by a method comprising the steps of:

[0186] (a) contacting artificial bromoform and a cyclodextrin aqueous solution;

[0187] (b) preparing a solid by step a); and

[0188] (c) preparing a powder from the solid from step b).

[0189] In another embodiment, the stabilizing excipient is molasses.

[0190] Figure 6 , 9 and 10 demonstrated that a composition comprising artificial bromoform and molasses (as a bromoform stabilizing excipient) could be formed and could inhibit methane production from rumen fermentation in vitro and in vivo.

[0191] As used herein, "molasses" includes syrups produced as a byproduct of processing sugar cane or other plant products. Some examples include sugar cane waste (a byproduct of sugar cane sugar production); high-quality (sugar cane) molasses (the main product extracted from sugar cane); cane molasses (a byproduct of the process of refining unrefined brown sugar into white sugar); sugar beet molasses (a byproduct of sugar production from beets); citrus molasses (the juice extracted from the production of dried citrus pulp), etc. In one embodiment, the bromoform stabilizing excipient is an edible non-polar substance.

[0192] Figure 1 , 2 , 6, 9 and 10 demonstrated that a composition comprising artificial bromoform and edible oil (as a bromoform stabilizing excipient) can be formed and can inhibit methane production from rumen fermentation.

[0193] Figure 5 It was demonstrated that a composition comprising artificial bromoform and edible oil (as a bromoform stabilizing excipient) can be formed without negatively affecting rumen fermentation, such as measured by IVDDM and tVFA ​​production. For example, in one embodiment, the edible non-polar substance is selected from the group consisting of oil or molasses.

[0194] In another embodiment, the edible non-polar substance is an edible oil.

[0195] As used herein, the term "edible oil" includes a single type of oil, or a composition comprising a single type of oil, or a mixture of two or more oils, or a composition comprising a mixture of two or more oils. The oil includes oils suitable for providing to, applying to animals.

[0196] In another embodiment, the edible non-polar substance is an edible fat that is solid at ambient temperature.

[0197] Figure 6 and 9 It was demonstrated that a composition comprising synthetic bromoform and water (as a bromoform stabilizing excipient) can be formed and can inhibit methane production from rumen fermentation.

[0198] This result is surprising because bromoform is considered only slightly soluble in water and is thought to be soluble at less than 0.1 g bromoform / 100 g water at 20 degrees (Mackison, FW, RSS Tricoff, and LJ Partridge, Jr. (eds.). NIOSH / OSHA-Occupational Health Guidelines for Chemical Hazards. DHHS (NIOSH) Publication No. 81-123 (3 VOLS). Washington, DC: US ​​Government Printing Office, Jan. 1981., p. 2).

[0199] In another embodiment, the bromoform stabilizing excipient is water.The inventors have prepared bromoform in water at a concentration of 3.0 ± 0.10 mg bromoform per gram of deionized water.

[0200] In one embodiment, the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olive oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0201] As used herein, the term "oil" means any nonpolar, hydrophobic substance, which is typically liquid at ambient temperature and pressure. Oil can be derived from animal, plant or petrochemical products, and typically has high carbon and hydrogen content. Oil is preferably edible oil, and is preferably digestible by ruminants. Typically, the oil of plant origin is extracted from the seed or fruit of the plant, and is typically mainly composed of triglycerides. The term "vegetable oil" is a general term to represent that the oil is mainly or completely plant-derived, and can comprise a mixture of plant-derived or one or more oils from different sources.

[0202] As used herein, the term "contacting" includes mixing synthetic bromoform with a bromoform stabilizing excipient to form a synthetic bromoform and bromoform stabilizing excipient mixture, and maintaining the synthetic bromoform, and maintaining the synthetic bromoform and bromoform stabilizing excipient composition at a temperature suitable for forming the composition for a certain period of time.

[0203] The term contacting includes maintaining the synthetic bromoform and the bromoform stabilizing excipient at different suitable temperatures.

[0204] There is no limit to the temperature to which the synthetic bromoform in contact with the bromoform stabilizing excipient may be heated, provided that the synthetic bromoform does not significantly evaporate / sublime from the bromoform stabilizing excipient and / or does not significantly degrade at the temperature at which the heating is performed.

[0205] There is no limit to the time that the synthetic bromoform is in contact with the contacted bromoform-stabilizing excipient, so long as the synthetic bromoform does not significantly evaporate / sublime from the bromoform-stabilizing excipient and / or does not significantly degrade during the contact time.

[0206] The inventors herein have demonstrated that the compositions described herein maintain levels of volatile synthetic bromoform over extended periods of time.

[0207] For example, Example 7 demonstrates that a composition comprising synthetic bromoform and corn oil (as bromoform stabilizing excipients) maintains bromoform for four weeks in storage at -20°C, 4°C, 25°C, and 40°C.

[0208] Example 7 also demonstrates that a composition comprising synthetic bromoform and corn oil (as a bromoform stabilizing excipient) maintained over 46% of the initial bromoform concentration over four weeks at 25°C and 40°C in open air conditions.

[0209] Example 7 demonstrates that a composition comprising synthetic bromoform and molasses (as a bromoform stabilizing excipient) maintains bromoform for four weeks in storage at -20°C, 4°C, 25°C, and 40°C.

[0210] Example 7 demonstrates that a composition comprising synthetic bromoform and molasses (as a bromoform stabilizing excipient) maintains bromoform for four weeks in storage at -20°C and 4°C.

[0211] Example 7 also demonstrates that a composition comprising artificial bromoform and molasses (as a bromoform stabilizing excipient) maintains more than 38% of the initial bromoform concentration over four weeks of storage at 25°C.

[0212] Example 7 also demonstrates that a composition comprising artificial bromoform and molasses (as a bromoform stabilizing excipient) maintains more than 24% of the initial bromoform concentration over four weeks of storage at 40°C.

[0213] Surprisingly, Example 7 also demonstrates that a composition comprising artificial bromoform and molasses (as a bromoform stabilizing excipient) maintains more than 58% of the initial bromoform concentration over four weeks at 25°C in open air conditions.

[0214] Furthermore, surprisingly, Example 7 also demonstrates that a composition comprising artificial bromoform and molasses (as a bromoform stabilizing excipient) maintains more than 62% of the initial bromoform concentration over four weeks at 40°C in open air conditions.

[0215] Example 7 also demonstrates that a composition comprising synthetic bromoform and cyclodextrin (as a bromoform stabilizing excipient) maintains the starting bromoform concentration over four weeks of storage at -20°C, 4°C, 25°C, and 40°C.

[0216] Example 7 also demonstrates that a composition comprising synthetic bromoform and cyclodextrin (as a bromoform stabilizing excipient) maintains the starting bromoform concentration over four weeks at 25°C and 40°C in open air conditions.

[0217] In one embodiment, the present invention provides a stable bromoform composition comprising synthetic bromoform and a bromoform stabilizing excipient.

[0218] In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after one week of storage at -20°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after two weeks of storage at -20°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after three weeks of storage at -20°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after four weeks of storage at -20°C.

[0219] In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after one week of storage at 4°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after two weeks of storage at 4°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after three weeks of storage at 4°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after four weeks of storage at 4°C.

[0220] In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after one week of storage at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after two weeks of storage at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after three weeks of storage at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after four weeks of storage at 25°C.

[0221] In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after one week of storage at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after two weeks of storage at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after three weeks of storage at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after four weeks of storage at 40°C.

[0222] In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after one week in open air at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after two weeks of storage in open air at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after three weeks of storage in open air at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after four weeks of storage in open air at 25°C.

[0223] In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after one week in open air at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after two weeks of storage in open air at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after three weeks of storage in open air at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of artificial bromoform in the composition is maintained after four weeks of storage in open air at 40°C.

[0224] In one aspect, the present invention provides a composition as described herein, wherein after one week of storage at -20°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after two weeks of storage at -20°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after three weeks of storage at -20°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after four weeks of storage at -20°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform.

[0225] In one aspect, the present invention provides a composition as described herein, wherein after one week of storage at 4°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after two weeks of storage at 4°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after three weeks of storage at 4°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after four weeks of storage at 4°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform.

[0226] In one aspect, the present invention provides a composition as described herein, wherein after one week of storage at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after two weeks of storage at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after three weeks of storage at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after four weeks of storage at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform.

[0227] In one aspect, the present invention provides a composition as described herein, wherein after one week of storage at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after two weeks of storage at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after three weeks of storage at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after four weeks of storage at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform.

[0228] In one aspect, the present invention provides a composition as described herein, wherein after one week in open air at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after two weeks of storage in open air at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after three weeks of storage in open air at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after four weeks of storage in open air at 25°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform.

[0229] In one aspect, the present invention provides a composition as described herein, wherein after one week in open air at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after two weeks of storage in open air at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after three weeks of storage in open air at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform. In one aspect, the present invention provides a composition as described herein, wherein after four weeks of storage in open air at 40°C, the level of artificial bromoform in the composition is reduced by no more than 10%, 20%, 30%, 40%, 50% or 60% relative to the initial concentration of bromoform.

[0230] The present invention provides compositions that release bromoform over time. As shown in Example 7, in one embodiment, the present invention provides a sustained release composition comprising synthetic bromoform and a bromoform stabilizing excipient.

[0231] Previous work has demonstrated that synthetic bromoform formulated in DMSO significantly inhibits methane production at doses of 5 μM, 10 μM, and 25 μM, but at 1 μM, synthetic bromoform does not significantly inhibit methane production in in vitro fermentation. In contrast, the present inventors have surprisingly demonstrated that synthetic bromoform at a dose of 0.04 mg / gram of feed OM inhibits methane production from rumen fermentation.

[0232] Importantly, Fig. 9 It was demonstrated that even lower doses of synthetic bromoform can be used in the methods and compositions described herein and inhibit rumen fermentation. For example, 0.01 mg of synthetic bromoform per gram of feed matrix can be used in the compositions and methods of the invention to inhibit methane production from rumen fermentation.

[0233] In one embodiment, a composition according to the invention is administered in an amount based on actual individual animal intake (e.g. g / kg OM or g / kg DM intake). Compared to Asparagaceae, which is a natural product with variable bromoform content, the present invention allows for compositions with defined artificial bromoform content.

[0234] Therefore, in one embodiment, the present invention provides a method as described herein, wherein the composition is administered at a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of artificial bromoform / gram of feed provided to ruminants. In a preferred embodiment, the present invention provides a method as described herein, wherein the composition is administered at a dosage of at least 0.03, 0.04 or 0.05 mg of artificial bromoform / gram of feed provided to ruminants.

[0235] In a preferred embodiment, the invention provides a method as described herein, wherein the composition is administered at a dosage of at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of artificial bromoform / gram of organic matter provided to the ruminant. In another embodiment in which the composition according to the invention is administered in an amount based on dry matter intake, the composition is administered at a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of artificial bromoform / about 1.19 grams of dry matter provided to the ruminant.

[0236] In another embodiment, the invention provides a method as described herein, wherein the composition is available in an amount that provides a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg bromoform / gram of organic matter provided to the ruminant. In another embodiment in which the composition according to the invention is available in an amount based on dry matter intake, the composition is administered in a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg synthetic bromoform / about 1.19 grams of dry matter provided to the ruminant.

[0237] For example, if a ruminant consumes approximately 2.5-3% of its live weight in feed per day, a 400 kg ruminant may consume 10-12 kg of feed per day.

[0238] As discussed above, in preferred embodiments of the invention, the effective amount of synthetic bromoform is, for example, at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of synthetic bromoform per gram of organic matter provided to the ruminant per day, or, for example, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of synthetic bromoform per about 1.19 grams of dry matter provided to the ruminant per day.

[0239] Therefore, if a 400 kg ruminant consumes about 10 kg of organic matter per day, the artificial bromoform in the composition of the effective amount is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 g of the composition as described herein per day. These doses are equivalent to 0.0005, 0.00075, 0.001, 0.00125, 0.0015, 0.00175 or 0.002 g of artificial bromoform / kg body weight / day. Suitable doses can be calculated as described herein.

[0240] An effective amount may be administered to the ruminant in one or more doses.

[0241] The effective amount may also be administered to the ruminant in one or more doses on a daily basis.

[0242] In another preferred embodiment, there is provided a method as defined above, wherein the dose of synthetic bromoform is in the range of 0.00025-0.0065 g / kg body weight / day, more preferably in the range of 0.0005-0.0065 g / kg body weight / day, most preferably in the range of 0.001-0.00275 g / kg body weight / day.

[0243] The dosages defined herein in terms of amount per kg body weight per day relate to the average amount of artificial bromoform during a given treatment period (e.g., during a treatment period of one week or one month). Thus, the compositions described herein may be provided or administered every day, every other day, every two days, etc., without departing from the scope of the present invention. However, preferably, the method comprises administering the compositions defined herein every day at a specified dosage. Even more preferably, the composition is provided or administered during the animal feeding period at each feeding of the animal in an amount that produces the above-mentioned daily dosage.

[0244] The methods of the invention may comprise providing or administering the composition according to the above dosage regimen for a period of at least 5, 10, 25, 50, 100, 250 or 350 days.

[0245] In animal systems (including pasture-based systems), providing systems with supplements that allow effective doses to be applied to animals may be a problem with using Asparagaceae biomass or products derived from Asparagaceae. For example, if only a small portion of the feed intake of pasture-based animals is feed supplements, the dose of bromoform in the supplement is required to be high to provide an effective dose. Because the high addition rate of Asparagaceae biomass or products derived from Asparagaceae in the feed supplement may make the supplement unpalatable to the animal, resulting in ineffective doses being consumed. The present invention allows the preparation of feed supplements containing artificial bromoform at levels that allow effective doses to be delivered in animal systems (such as pasture-based systems / grazing systems).

[0246] The ability to prepare feed supplements containing synthetic bromoform at higher levels to allow delivery of effective doses to animals in smaller amounts (e.g., feed volumes) also allows delivery of effective amounts of the compositions described herein while the animals are at a feeding machine or during milking, etc.

[0247] The ability to prepare feed supplements containing higher levels of synthetic bromoform relative to Asparagaceae-based or Asparagaceae-derived oil products to allow for delivery of an effective dose to an animal in a smaller amount allows for avoidance of high levels of oil in animal feed. Use of high levels of oil in animal feed can cause the oil-containing feed to partially liquefy, be unpalatable, have a foul odor, and / or cause problems with feeding machinery.

[0248] In another aspect, the present invention provides a composition for reducing total gas production and / or methane production and / or improving growth performance of ruminants, wherein the composition comprises artificial bromoform and a bromoform stabilizing excipient.

[0249] In another aspect, the present invention provides a composition for use in reducing total gas production and / or methane production and / or improving growth performance of ruminants, wherein the composition comprises artificial bromoform and a bromoform stabilizing excipient.

[0250] In one embodiment of the composition for use, the bromoform stabilizing excipient is an edible non-polar substance.

[0251] In one embodiment of the composition for use, the edible non-polar substance is an edible oil.

[0252] In one embodiment of the composition for use, the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olive oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0253] In one embodiment of the composition for use, the composition is formulated to be provided to the ruminant at a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

[0254] In one embodiment of the composition for use, the composition is in solid, semi-solid or liquid form.

[0255] In another aspect, the present invention provides a feed supplement for use in reducing total gas production and / or methane production in ruminants, the supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0256] As used herein, the term "animal feed supplement" refers to a concentrated additive premix containing active ingredients, which premix or supplement can be added to animal feed or rations to form a supplemented feed according to the present invention. It is generally believed that the terms "animal feed premix", "animal feed supplement" and "animal feed additive" have similar or identical meanings and are generally considered to be interchangeable. Typically, the animal feed supplement of the present invention is in the form of a powder or a compacted or granulated solid. In another embodiment, the animal feed supplement of the present invention is in the form of a mineral loose lick, a wet lick, a dry lick, a pellet, an aqueous suspension, a liquid supplement (e.g., a molasses blend) or a lick block. In practice, the animal feed supplement can typically be fed to livestock by adding the animal feed supplement directly to the ration (e.g., as a so-called surface fertilizer), or it can be used to prepare or manufacture products, such as compound animal feed or lick blocks, which will be described in more detail below. The present invention is not particularly limited in this regard. The supplement according to the present invention is typically fed to animals in an amount within the range of 16-3000g or more / animal / day.

[0257] Without departing from the scope of the present invention, the animal feed supplement of the present invention may include any additional ingredients. It may typically include the known excipients required for preparing the desired product form, and it may include additional additives intended to improve feed quality and / or improve the performance of the animal consuming the supplement. Suitable examples of such excipients include carriers or fillers, such as lactose, sucrose, mannitol, starch crystalline cellulose, sodium bicarbonate, sodium chloride, etc., and adhesives, such as gum arabic, tragacanth, sodium alginate, starch, PVP and cellulose derivatives, etc. Examples of feed additives known to those skilled in the art include vitamins, amino acids and trace elements, digestion enhancers and intestinal flora stabilizers, etc.

[0258] As used herein, the term 'compound animal feed composition' means a composition suitable for use as an animal feed and admixed with a variety of natural or non-natural matrices or raw materials and / or additives. Thus, in particular, the term 'compound' is used herein to distinguish the animal feed composition of the present invention from any naturally occurring raw material. These admixtures or compound feeds are formulated according to the specific needs of the target animal. The main ingredients of commercially prepared compound feeds typically include wheat bran, rice bran, corn meal, cereal grains (such as barley, wheat, rye and oats), soybean meal, alfalfa meal, cottonseed meal, wheat flour, etc. Commercial compound feeds will typically contain not less than 15% crude protein and not less than 70% digestible total nutrients, but the present invention is not particularly limited in this regard.

[0259] The present invention includes liquid, solid and semi-solid compound animal feed compositions, solid and semi-solid forms being particularly preferred. These compositions are typically manufactured as meal types, pellets or crushed materials. In practice, livestock can typically be fed with a combination of compound feeds (such as the compound feeds of the present invention) and silage or hay, etc. Typically, compound animal feeds are fed in an amount within the range of 0.3-10 kg / animal / day. Taking into account the type of animal and the environment in which it is raised, it is within the skill of a trained professional to determine the appropriate amount of these components contained in the compound animal feed.

[0260] The compound animal feed composition of the present invention may contain any additional feed additives typically used in the art. As known to those skilled in the art, in this context, the term 'feed additive' refers to a product used for animal nutrition for the purpose of improving the quality of feed and the quality of food from animal sources, or for improving animal performance, for example providing enhanced digestibility of feed materials. Non-limiting examples include technical additives such as preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators, non-protein nitrogen (NPN) and silage additives; sensory additives, especially flavorings and colorants; (additional) nutritional additives such as vitamins, amino acids and trace elements; and (additional) zootechnical additives such as digestion enhancers and intestinal flora stabilizers. As will be clear to those skilled in the art, the compound animal feed composition of the present invention may contain any additional ingredients or additives without departing from the scope of the present invention.

[0261] In another aspect, the present invention provides a lick stone or lick block comprising the supplement of the present invention. As known to those skilled in the art, such lick stones or lick blocks are particularly convenient for feeding mineral supplements (as well as proteins and carbohydrates) to ruminants grazing on one or both of natural pastures and cultivated pastures. In addition to the composition of the present invention, such lick blocks or lick stones according to the present invention typically also contain various types of binders, such as cement, gypsum, lime, calcium phosphate, carbonates and / or gelatin; and optional additional additives, such as vitamins, trace elements, mineral salts, sensory additives, etc.

[0262] In one embodiment of the feed supplement for use, the feed supplement is formulated to provide a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of synthetic bromoform per gram of organic matter provided to the ruminant.

[0263] In another embodiment of the feed supplement for use, the feed supplement is formulated to provide a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of synthetic bromoform per gram of feed matrix provided to the ruminant.

[0264] In one embodiment of the feed supplement used, the feed supplement further comprises a feed additive.

[0265] In one embodiment of the feed supplement for use, the ruminant is selected from members of the suborder Ruminans and the suborder Callospoda.

[0266] In one embodiment of the feed supplement for use, the ruminant is cattle or sheep.

[0267] In one embodiment of the feed supplement for use, the bromoform stabilizing excipient is an edible non-polar substance.

[0268] In one embodiment of the feed supplement for use, the edible non-polar substance is an edible oil.

[0269] In one embodiment of the feed supplement for use, the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olive oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0270] In one embodiment of the feed supplement for use, the composition is in solid, semi-solid or liquid form.

[0271] Methods of preparing methane-reduced ruminant feeds are described herein.

[0272] Thus, in one aspect, the present invention provides a method of producing a methane-reduced ruminant feed comprising mixing a ruminant feed with a feed supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

[0273] In one embodiment of the method of producing a methane-reduced ruminant feed, the animal feed comprises a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter of the ruminant feed.

[0274] In another aspect, the present invention provides an animal feed for reducing total gas production and / or methane production in ruminants when used, the supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient and a ruminant feed.

[0275] In one embodiment of an animal feed for use in reducing total gas production and / or methane production in ruminants, the feed supplement is formulated to provide a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter of the ruminant feed.

[0276] In one embodiment of the animal feed for reducing total gas production and / or methane production in a ruminant when used, the ruminant is selected from members of the suborder Ruminans and the suborder Callospoda.

[0277] In one embodiment of the animal feed for reducing total gas production and / or methane production in a ruminant when used, the ruminant is a cattle or sheep.

[0278] In one embodiment of an animal feed for use in reducing total gas production and / or methane production in ruminants, the bromoform stabilizing excipient is an edible non-polar substance.

[0279] In one embodiment of the animal feed for use in reducing total gas production and / or methane production in ruminants, the edible non-polar substance is an edible oil.

[0280] In one embodiment of the animal feed for reducing total gas production and / or methane production in ruminants when used, the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olive oil, avocado oil, Brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

[0281] In one embodiment of an animal feed for use in reducing total gas production and / or methane production in ruminants, the composition is in solid, semi-solid or liquid form.

[0282] Now, the present invention will be further described by way of examples, which are intended to help those of ordinary skill in the art to practice the present invention and are not intended to limit the scope of the present invention in any way.

[0283] Example

[0284] Example 1: Materials and Methods

[0285] Preparation of feed matrix and oil products

[0286] Tiger grass hay was used as the main feed substrate and was first dried and ground to 1 mm and then supplied to each container at 1.25 g DM (1.0 g OM).

[0287] Asparagaceae seaweed was prepared by FutureFeed Pty Ltd of Townsville, Queensland, Australia, and was sourced from the middle reef of Magnetic Island, Queensland, Australia. Approximately 20 kg of freshly harvested Asparagaceae was first rinsed in seawater for two minutes, spun dry three times [1-2 minutes each], and then added to 20 L of refined canola oil for 20 days. Another batch of fresh Asparagaceae was collected from the same area, and the process was repeated as above using the same 20 L of refined canola oil to achieve a final CHBr concentration of 3.60 mg / g in a composition of Asparagaceae biomass ("Asp-Oil") in oil (Table 1).

[0288] Compositions containing artificial bromoform in oil (synthetic CHBr3 oil; "Syn-Oil") were produced by FutureFeed Pty Ltd using the same refined canola oil used to prepare Asp-Oil. Synthetic CHBr3 stabilized in pentene (Sigma-Aldrich, Product No.: 36972) was dissolved and homogenized into canola oil by repeated inversion and left to stabilize overnight before analysis. The oil was then diluted in volume to achieve comparable CHBr3 concentrations between Asp-Oil and Syn-Oil (Table 1).

[0289] A subsample of freeze-dried Asparagus spp., which has been previously shown to reduce enteric CH4 emissions in vitro and in vivo (Roque et al., 2019; Kinley et al., 2020; Roque et al., 2021), was added as a positive control during Experiment 2.

[0290] The bromoform concentration of Asp-Oil was 3.60 ± 0.05 mg / g DW, that of Syn-Oil was 3.69 ± 0.05 mg / g DW, and that of freeze-dried Asparagine was 7.7 mg / g DW. The bromoform concentration was confirmed using a modified analytical protocol as previously described by Paul et al. (2006).

[0291] Table 1. Dry matter, organic matter and bromoform content of the Chloris unmansii matrix, freeze-dried Asparagaceae and Asparagaceae oils and synthetic bromoform oil.

[0292]

[0293] Donor animals and in vitro preparation

[0294] Donor animals were maintained at the Lansdown Research Station of the Commonwealth Scientific and Industrial Research Organization (CSIRO) near Townsville, Queensland, Australia (coordinates; 19°39'27.000"S, 146°50'04.60"E) in accordance with current guidelines (NHMRC 2013) and approved by the CSIRO Animal Ethics Committee (Ethics Permit 2018-37). Rumen fluid (RF) was collected from four fistulated Brahman steers equipped with 10-cm Bar Diamond (Parma, OH, USA) rumen cannulas. All four steers were allowed to graze on pasture and supplemented with unawned tricholoma hay ad libitum, and RF was then collected, which was extracted two hours after the morning feeding, then placed in a 1-L pre-warmed insulated thermos, and transported back to the laboratory. All RFs were collected, filtered through a 0.5 mm mesh, and then mixed with artificial saliva buffer (Goering & van Soest 1970) at a ratio of 1 part RF to 4 parts buffer. Approximately 125 mL of the RF buffer mixture and 1.25 g of DM were dispensed into a 250 mL Ankom incubation bottle, the headspace was purged with N2, and then sealed with an AnkomRF1 gas generation module (Macedon, New York, United States). The incubation bottle was placed in a Ratek OM11 dry incubator (Boronia, Victoria, Australia) and maintained at a constant temperature of 39°C and a shaking speed of 85 RPM.

[0295] Sampling and analysis

[0296] Total gas, CH4 and hydrogen production

[0297] Ankom RF gas production technology (Macedon, NY, USA) is used to determine total gas production (TGP), CH4, hydrogen and carbon dioxide production. In brief, the Ankom RF module is set to a maximum pressure of 3psi, and 250 milliseconds will be exhausted when the pressure is exceeded. The real-time interval (LI) is set at 60 seconds (monitoring gas production), and each measurement is corrected for ambient pressure changes via the environmental Ankom RF monitor. The recording interval (RI) is set to 20min, so the LI cumulative pressure change is recorded at each RI as a 20min contribution to the cumulative pressure change in the duration (24, 48 or 72h) of the fermentation. The total cumulative pressure change is converted to TGP using the natural gas law, and the absolute volume of a single fermentation bottle is corrected. The external CH4 production is determined by analyzing the headspace gas relative to TGP while assuming that the bottle headspace has a constant uniformity. At the end of each incubation time period, the headspace gas samples from the individual fermentation bottles were collected into 10-mL Labco Exetainer vacuum vials (Lampeter, Great Britain) by the Ankom RF module exhaust pipe. The gas samples were analyzed by gas chromatography (GC) on a Shimadzu GC-2014 (Kyoto, Japan) equipped with Restek (Bellefonte, PA, USA) ShinCarbon ST 100 / 120 columns (2m 1mm microfilling) using a flame ionization detector (FID). The column temperature was set to 150°C, the injector was set to 240°C, and the FID was set to 380°C. Ultra-high purity N2 was the carrier gas (at 25mL / min) and the total injection volume was 250 μL.

[0298] Apparent digestibility

[0299] After the fermentation bottles were sacrificed [at 24, 48 or 72 h], they were frozen in a -20°C freezer to stop bacterial activity, and the rumen fluid was then vacuum filtered through a 50 mL Gooch sintered disk glass filter crucible (porosity 1) (GLASSCO; Haryana, India) with a 0.5-cm sand filter aid layer. The crucible containing the digested rumen fluid residue was then dried at 105°C until a constant weight was reached and then subtracted from the total DM added to the bottle to determine the in vitro apparent digestibility of the substrate dry matter (IVD-DM). The resulting dry residue was then ashed at 530°C to determine the apparent digestibility of the substrate organic matter (IVD-OM).

[0300] Example 2: Compositions comprising bromoform and a bromoform stabilizing excipient reduce methane production in rumen fermentation.

[0301] In vitro setup and experimental design

[0302] Asparagus oil ("Asp-oil") was tested at four addition levels [0.08 mg, 0.11 mg, 0.15 mg and 0.18 mg], and synthetic CHBr3 oil (Syn-oil) was tested at six addition levels [0.08 mg, 0.11 mg, 0.15 mg, 0.18 mg, 0.22 mg and 0.26 mg / gram of organic matter (OM of feed)], along with four control treatments (which had varying canola oil additions to match levels 1, 2, 4 and 6 of the Asp-Oil and Syn-Oil products). Unsaturated oils themselves can serve as CH4-reducing feed ingredients (Grainger & Beauchemin, 2011), so it was also necessary to test a range of additions for the oils. All experimental treatments [14 in total] were tested in duplicate at two fermentation time points (24 and 48 hours, which were performed during the in vitro incubation time period). The data collected for each treatment, fermentation time point, and incubation were then combined and analyzed. Note that CH4 production based on digested feed cannot be reported here because IVDDM was not measured in Example 2, which was designed only to demonstrate CH4 production. However, all fermentations received the same tricholoma and canola oil feed substrates at comparable levels. Reporting based on digested feed is not Figure 1 The shape of the feeding base has little effect.

[0303] result

[0304] Figure 1 Asp-Oil and a composition comprising bromoform and a bromoform stabilizing excipient (Syn-Oil) were both demonstrated to be highly antimethanogenic in vitro at comparable levels of CHBr3 delivery.

[0305] Hydrogen (H2) emissions (also in Figure 1 ) is generally absent, as indicated by the absence of hydrogen concentration in the control, because H2 is utilized by methanogens to produce CH4 (which contains four hydrogens (H + ) atoms and act as a sink for hydrogen. Consistent with CH4 mitigation, H2 concentration increased at all CHBr3 addition levels for both Asp-Oil and Syn-Oil products, except for Syn-Oil 0.08 mg at the 48 hour fermentation time point, as CH4 mitigation efficacy decreased at 48 hours.

[0306] Figure 1Total gas production (TGP) was also included to confirm that microbial activity was maintained in all treatments, including those with CHBr3 content. A slight decrease in TGP could be expected due to the proportion of CH4 that constituted TGP in the control treatment.

[0307] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions comprising bromoform and bromoform stabilizing excipients are able to reduce methane production from rumen fermentation in the absence of other secondary metabolites of Asparagaceae (and other components).

[0308] Example 3: Dose-dependent response of oil products on gas production and digestibility during a 72 hour time period.

[0309] In vitro setup and experimental design

[0310] Two oil products [matrix + Asp-Oil or Syn-Oil] were used along with a negative control (C1-) [matrix + 0.02 g canola oil], a positive control (C2+) [matrix + 0.01 g freeze-dried Asparagine + 0.02 g canola oil] and a blank [no matrix or treatment, rumen fluid only].

[0311] Three levels of Asp-Oil or Syn-Oil addition were tested based on the CHBr3 content and weight of the material and are referred to as low [0.04 mg], medium [0.07 mg] and high [0.11 mg] addition levels. All experimental treatments [9 in total] were tested in duplicate at three fermentation time points (24, 48 and 72 hours, which were performed during an in vitro incubation time period). The data collected for each treatment, fermentation time point and incubation were then combined and the time series results were analyzed.

[0312] Results and discussion

[0313] The design of Example 3 was based on Asp-Oil and Syn-Oil addition levels, as learned from Example 2, to provide the lowest effective addition level to consistently reduce CH4 over a 72 hour period. Based on the results of Example 2, the inventors hypothesized that a CHBr3 addition level of 0.04 mg for both Asp-Oil and Syn-Oil would suppress methane production, but to a lesser extent than higher doses of bromoform.

[0314] Figure 2 The CH4 results are presented in A, demonstrating that the 0.07 mg / g addition level exhibited greater CH4 mitigation capabilities than expected based on the data from Example 2. The 0.11 mg addition for both Asp-Oil and Syn-Oil completely inhibited CH4 production throughout the duration of the study.

[0315] This data demonstrates that compositions comprising bromoform and a bromoform stabilizing excipient are able to inhibit methane production in the absence of other secondary metabolites (and other components) of Asparagaceae.

[0316] At 48 hours, an Asparagaceae-derived composition comprising Asparagaceae biomass extracted into a bromoform stabilizing excipient (Asp-Oil), which included other antimethanogenic secondary metabolites of Asparagaceae (and other components), was able to reduce methane production to a greater extent than Syn-Oil, which did not include other antimethanogenic secondary metabolites of Asparagaceae (and other components).

[0317] In addition to the oil treatments, a positive control of freeze-dried (FD) Asparagus was included in this study with a CHBr3 addition of 0.07 mg and functioned similarly to the medium (0.07 mg) and high (0.11 mg) oil addition levels.

[0318] Figure 2 It was also confirmed that hydrogen increased significantly with the downregulation of CH4 production, and then decreased in unison with when CH4 production increased again. Therefore, with the reduction of the CH4 component of TGP, there was a corresponding general reduction in total gas production (TGP), and thus TGP was confirmed to be lower than the control without FD-Asp, Asp-Oil or Syn-Oil. In addition to this, in vitro dry matter digestibility (IVDMD) was measured to ensure that there was no negative impact on dietary digestibility due to the addition of antimethanogenic products, and it appeared to be unaffected in these treatment groups (bottom right, Figure 2 The maintenance of TGP and IVDMD strongly suggests that rumen microbial activity is stable with the addition of CH4-mitigated oil products.

[0319] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions comprising bromoform and bromoform stabilizing excipients are able to reduce methane production from rumen fermentation in the absence of other secondary metabolites of Asparagaceae (and other components).

[0320] Example 4: Dose-dependent response of oil products on in vitro gas production and digestibility over a 72 hour time period.

[0321] To compare enteric methane mitigation and the effects on enteric fermentation (eg, in vitro digestible dry matter (IVDDM) and rumen volatile fatty acid production (VFA) as fermentation markers), the present inventors examined the response in in vitro fermentation.

[0322] method

[0323] Two oil products [substrate + Canola Oil or Asp-Oil] were used together with a negative control (CON) [substrate + 0.02g canola oil], a positive control (FD-Asp) [substrate + freeze-dried Asparagine fed with 0.05mg CHBr3 / g substrate] and a blank [no substrate or treatment, rumen fluid only]. Three levels of Canola Oil and Asp-Oil addition were tested based on the CHBr3 content and weight of the material and were referred to as 0.03 [mg / g substrate], 0.05 [mg / g substrate] and 0.08 [mg / g substrate] addition levels. All experimental treatments [a total of 9] were tested in duplicate at 3 fermentation time periods (24, 48 and 72 hours, which were repeated during three in vitro incubation time periods). The data collected for each treatment, fermentation time period and incubation were then merged and the time series results were analyzed.

[0324] Asparagaceae seaweed was prepared by FutureFeed Pty Ltd in Townsville, Queensland, Australia, and was sourced from the coast of Magnetic Island, Queensland, Australia. Approximately 20 kg of freshly harvested Asparagaceae was first rinsed in seawater for two minutes, dried three times [1-2 minutes each], and then added to 20 L of refined canola oil for 20 days. After soaking for 3 weeks, the Asparagaceae biomass was removed, and then another 20 kg of fresh Asparagaceae (collected from the same area) was added to the canola oil, and the above process was repeated to reach a final CHBr3 concentration of [3.03 mg / g ± 0.03].

[0325] Synthetic CHBr3 (Sigma-Aldrich, Product No. 36972) with 98% purity and stabilized with amylene was dissolved and homogenized in canola oil by repeated inversion and left to stabilize overnight before analysis. The oil was then diluted in volume to achieve a CHBr3 concentration comparable to that of Asparagine oil.

[0326] As previously described, the total gas production (TGP), CH4, hydrogen (H2) and carbon dioxide production (CO2) were determined using the Ankom RF gas generation technology (Macedon, NY, USA). Briefly, the total cumulative pressure change was converted to TGP using the natural gas law and corrected for the absolute volume of the individual fermentation bottles. At the end of each incubation period, headspace gas samples from individual fermentation bottles were collected through the Ankom RF module exhaust tube into 10-mL Labco Exetainer vacuum vials (Lampeter, Great Britain), and in vitro CH4, CO2 and H2 production were determined while assuming constant homogeneity of the bottle headspace.

[0327] In addition, IVDDM and VFA were determined. Briefly, after the fermentation bottles were removed [at 24, 48, or 72 h], they were frozen in a -20 °C freezer to stop bacterial activity, and the rumen fluid was then vacuum filtered through a sintered glass disk filter crucible with a 0.5-cm sand filter aid layer. The crucible containing the digested rumen fluid residue was then dried at 105 °C until a constant weight was reached and then subtracted from the total DM added to the bottle to determine IVDDM. 1.5 mL rumen fluid samples were collected from 72 h fermentation bottles to determine total VFA (tVFA) as well as acetate, propionate, and butyrate production.

[0328] result

[0329] The design of Example 4 was based on Asp-Oil and Canola Oil addition levels, as learned from in vitro experiments 1 and 2, to provide a range of effective addition levels (ie, dose response curve) that consistently reduced CH4 over a 72 h fermentation period.

[0330] CH4 results in Figure 3 , which demonstrates that Asparagaceae biomass extracted into oil ("Asp-Oil") and synthetic bromoform in oil as a stabilizing excipient ("Canola Oil") behaved in a similar manner.

[0331] The 0.05 addition level showed even stronger CH4 mitigation ability than expected for Asp-Oil, Canola Oil and freeze-dried Asparagine biomass ("FD-Asp") during the 24 hour fermentation time period, however, as expected, there appeared to be a small general decrease in effectiveness within the 48h and 72h fermentations. In addition, during the 48 and 72 hour time periods, FD-Asp appeared to be more effective in mitigating CH4 than Asp-Oil and Canola Oil at the same addition level of 0.05 [mg / g substrate]. As expected based on Examples 1 to 3, 0.08 additions of both Asp-Oil and Canola Oil completely inhibited CH4 production during the entire study period. This shows the stability of mitigation effectiveness when CHBr3 is provided within the optimal range.

[0332] As demonstrated in in vitro and in vivo studies, Figure 4 showed that H2 increased as CH4 production was downregulated, and then decreased consistently when CH4 production was restored ( Figure 4 No negative effects on the production of IVDDM and tVFA ​​were observed due to the addition of Asp-Oil or Canola Oil. Figure 5A and B), and appeared to be unaffected in these addition groups. In addition, at the corresponding addition rates, the reduction in acetate and the increase in propionate and butyrate production occurred in the same proportion between Asp-Oil and Canola Oil, providing evidence that Asp-Oil and Canola Oil act similarly in an in vitro fermentation environment.

[0333] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions comprising bromoform and bromoform stabilizing excipients are able to reduce methane production from rumen fermentation in the absence of other secondary metabolites of Asparagaceae (and other components).

[0334] Example 5: A composition comprising artificially synthesized bromoform stabilized in corn oil, molasses, cyclodextrin and water inhibits methane production in enteric fermentation.

[0335] To examine the ability of synthetic bromoform to be stabilized in different excipients and to inhibit methane production in enteric fermentation, synthetic CHBr3 was stabilized in edible oil (corn oil), solid carbohydrate (cyclodextrin), liquid form (water), or semi-liquid carbohydrate (molasses).

[0336] method

[0337] Four CHBr3 stabilizing excipients [matrix + corn oil, molasses, cyclodextrin, or water] were used, along with a negative control ("CON") [matrix only] and a blank [no matrix or treatment, rumen fluid only]. Three addition levels of each excipient were tested based on the CHBr3 content and weight of the material, and are referred to as the 0.01 [mg / g matrix], 0.04 [mg / g matrix], and 0.07 [mg / g matrix] addition levels. The experiment consisted of three in vitro incubation replicates (n=3), which included each of the CHBr3 product experimental treatments [a total of 14], which were tested in duplicate within each replicate over three fermentation time periods (24, 48, and 72 hours). The data collected for each treatment, fermentation time period, and incubation were then combined, and the time series results were analyzed.

[0338] Man-made CHBr3 (Sigma-Aldrich, product number: 241032) with 98% purity and stabilized with 2-methyl-2-butene was used to prepare four excipients. Corn oil, molasses and water were prepared using a similar scheme to that of Canola Oil in Example 4. In brief, CHBr3 was dissolved and homogenized in retail corn oil, molasses or deionized water, and homogenized by repeated inversions, and left to stabilize overnight, then reaching a concentration of 3.0 ± 0.10 mg / g. As previously described, cyclodextrins (patent WO 96 / 14062, PCT AU95 / 00733) were prepared. In brief, CHBr3 was added to an α-cyclodextrin (Sigma-Aldrich, product number: 779008) aqueous solution and mixed overnight. The solid was then separated, washed, and then dried. The CHBr3 concentration of the dry powder was determined and then diluted with additional α-cyclodextrin to adjust the CHBr3 concentration to 3.0±0.10 mg / g. The protocol described in Example 4 was used to determine TGP, CH4, H2 and CO2 production, IVDDM and VFA.

[0339] result

[0340] The design of Example 5 used addition levels as learned from preliminary exploratory experiments (data not shown, similar experimental design as Example 1) to provide a range of effective addition levels (i.e., dose response curves) for each CHBr3 stabilizing excipient used over a 72 h fermentation time period.

[0341] CH4 results in Figure 6 It is presented in , and demonstrates that synthetic bromoform can be stabilized in edible oils other than canola oil (corn oil), solid excipients (cyclodextrins), liquid excipients (water), or semi-liquid excipients (molasses).

[0342] The potency levels from molasses and water excipients at each addition level and over the 24, 48, and 72 h fermentation time periods were unexpected, showing similar CH4 reductions to corn oil and cyclodextrin. This is particularly interesting because CHBr3 is not well retained in water-based compounds (see Example 6), so it was expected that potency would decrease over the course of the 4 incubation time periods.

[0343] At 24 h of fermentation, there appeared to be an increase in H2 production on all excipients at the 0.04 and 0.07 addition rates, however due to the large variability in H2 measurements, the results were not found to be statistically significant ( Figure 7). During the 48h time period, cyclodextrin and water at 0.04 and 0.07 addition rates resulted in a significant increase in H2, while molasses and corn oil only showed a significant increase at 0.07 addition rate. During the 72h time period, cyclodextrin, water, and molasses at 0.07 addition rate resulted in a significant increase in H2.

[0344] Corn oil showed a potential reduction in IVDDM at 0.07 addition compared to the control during the 24 and 48 h fermentation periods, however there was no difference over the 72 h period ( Figure 8 A). Other excipients did not show any effect on IVDDM at any addition rate or time period. tVFA ​​production at 72 h of fermentation ( Figure 8 B) appears to be affected by the addition of the four excipients, with the highest addition rate of 0.07 [mg / g substrate] showing the greatest reduction in tVFA. Without wishing to be bound by theory, the inventors propose that this trend may be a reflection of decreased acetate production, which is typically the most abundant VFA in the rumen, but when CH4 is suppressed, acetate is downregulated. The reduction in acetate from all excipients and the increase in propionate and butyrate production ( Figure 8 C) showed significant individual effects on individual VFA production at specific addition rates, with cyclodextrin having the largest effect. As a result, the same trend was observed for the A:P ratio ( Figure 8 D) This may be due to the high methane reduction efficacy of cyclodextrin at each addition rate compared to corn oil, molasses, and water.

[0345] Fig. 9 A summary of the data from Examples 4 and 5 relating to different CHBr3 stabilizing excipients (grey bars) and a comparative Asparagaceae product (black bars) at various addition rates over 24, 48 and 72 h fermentations is presented. This summary of data is of particular interest as it illustrates that CHBr3 addition at 0.03-0.05 mg CHBr3 / gram feed matrix [30-50 mg / kg] resulted in complete inhibition of enteric CH4 in the intestine.

[0346] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions comprising bromoform with solid, liquid and semisolid bromoform stabilizing excipients are able to reduce methane production from rumen fermentation in the absence of other secondary metabolites of Asparagaceae (and other components).

[0347] These results also demonstrate that the compositions and methods of the present invention can be used to inhibit methane production at significantly lower doses of bromoform than has been previously demonstrated.

[0348] Example 6: Asparagaceae biomass extracted into oil ("Asp-Oil") and synthetic bromoform stabilized in oil ("Canola Oil") inhibit methane production in vivo.

[0349] To examine the ability of the stabilized compositions to inhibit methane production in vivo, the inventors provided animals with feed supplements containing either Asparagaceae biomass extracted into oil ("Asp-Oil") or synthetic bromoform stabilized in canola oil ("CanolaOil").

[0350] method

[0351] A total of 60 early lactation cows were randomly assigned to one of 10 treatment groups: Asp-Oil [addition level 0, 1, 2, 3 or 4] or Canola Oil [addition level 0, 1, 2, 3 or 4]. Based on previous experimental studies using Asp-Oil in lactating cows, the addition level was initially set to achieve 6, 12, 18 and 20 mg CHBr3 / kg DMI (on a daily basis). Asp-Oil and Canola Oil were mixed into the cows' grain supplement, which was provided twice a day at milking as two pulse doses separated from the main daily feed intake measured after milking. The initial introduction of Asp-Oil and Canola Oil was gradual over a 15-day adaptation period, consisting of a 2-day increment of the corresponding oil into its feed supplement. This standard precaution maximizes the possibility of rumen discomfort and refusal to consume supplements. After this time period, the cows were fed their corresponding diets and treatments for another 21 days, and then CH4 measurements were performed using sulfur hexafluoride (SF6) measurement technology. Briefly, an inert permeation tube is inserted into the rumen, which constantly and consistently emits a standardized tracer of SF6. The tracer provides accurate separation of gas components by dilution of background gas, rumen gas, and respiratory gas. Representative respiratory and belching gases are collected in a 24h sampling set for 5 consecutive days. Gas is extracted through the sampling tube from a sampling point above the cow's nostrils using a vacuum canister, and each cow is sampled continuously during the collection period. Background gas is collected and calculated by extracting gas from the cow's flank using a second canister.

[0352] result

[0353] Fig.10The CH4 reduction results before statistical analysis are shown, and the reduction in CH4 production [g CH4 / day] compared to the control (no CHBr3) was demonstrated to be up to 32% (for Canola Oil) and 38% (for Asp-Oil) at high CHBr3 addition levels [20 mg CHBr3 / kg DMI]. When CH4 was normalized (CH4 yield) on a per kg dry matter intake (DMI) basis, Canola Oil resulted in a reduction of up to 28%, and Asp-Oil resulted in a reduction of 36%.

[0354] The range of CH4 reduction was consistent with the CH4 reduction demonstrated in vitro after 24 h fermentation and the corresponding addition rates, and Fig.11 Shown in.

[0355] These results demonstrate that compositions comprising synthetic bromoform and stabilizing excipients that inhibit methane production from in vitro fermentation are able to inhibit methane production in vivo. This is in contrast to the work of Stefenoni et al., which showed that synthetic bromoform was unable to inhibit methane production.

[0356] These results also demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions comprising bromoform and bromoform stabilizing excipients similarly reduce in vivo methane production in the absence of other antimethanogenic metabolites of Asparagaceae (and other components).

[0357] Example 7: Shelf life / stability of compositions comprising synthetic bromoform and bromoform stabilizing excipients.

[0358] To examine the retention of synthetic bromoform (CHBr3) in a stable composition, bromoform levels in the following excipients were monitored over a 4-week period under different storage temperatures and air exposure conditions: corn oil, molasses, and cyclodextrin.

[0359] method

[0360] The ability of the Asparagine products (FD-Asp and Asp-Oil) to maintain CHBr3 content over a range of different temperatures, exposure to UV irradiation, and exposure to open air was used as a framework to test the shelf life stability of artificially synthesized CHBr3 in the current study. The preparation of three excipients [corn oil, molasses, and cyclodextrin] is described in Example 5. The initial concentration of CHBr3 in each excipient was between 2.5-3.0 mg / g (mg CHBr3 / g excipient).

[0361] Individual excipient products were stored at four gradient temperatures [-20°C, 4°C, 25°C, or 40°C] using amber test vials, either hermetically sealed and at 25°C or 40°C, or unsealed and exposed to open air conditions [6 treatment groups per excipient]. Triplicate samples were taken from each excipient, temperature, and air exposure at the beginning of the project (week 0) and then used to determine CHBr3 content at weeks 1, 2, and 4, analyzed using GC-MS as described in Tan et al. (2022). The weight of the open air vials was measured at each measurement time period to determine moisture loss. Molasses samples exposed to open air conditions lost a significant amount of moisture over 4 weeks, and the CHBr3 results were subsequently adjusted to account for weight loss within the sample. Means and standard errors were calculated for each storage temperature / condition after each storage period, and the effect of each treatment over time was analyzed by one-way ANOVA using Tukey's honestly significant difference (HSD). Results were declared significant at P < 0.5.

[0362] result

[0363] The CHBr3 content in corn oil was stable over 4 weeks when stored at -20°C, 4°C, and 25°C. Fig.11 , Table 2). However, when stored at 40°C, the concentration of CHBr3 decreased by 6.3% after 2 weeks. Extending the duration of exposure will help to better understand the effect of extended storage on the retention of CHBr3 in corn oil when stored in airtight vials at 40°C. When exposed to open air conditions, CHBr3 decreased significantly each week at 25°C and 40°C, with total losses of 41.1% and 53.6% over a 4-week period, respectively.

[0364] In an airtight container, the CHBr3 content of molasses appears to be stable at both -20°C and 4°C. However, at higher temperatures, the CHBr3 content in molasses appears to be less stable than that of canola oil or corn oil. The CHBr3 in molasses decreases significantly at both 25°C and 40°C under both airtight and open air conditions. In airtight vials, the CHBr3 content decreased by 41.4% and 75.4% at 25°C and 40°C, respectively, after 4 weeks of storage. Surprisingly, when exposed to open air conditions during storage, the CHBr3 content in molasses more successfully retains CHBr3 compared to the airtight counterpart, with only 14.8% and 37.9% CHBr3 lost at 25°C and 40°C storage temperatures, respectively. Without wishing to be bound by theory, the molasses product forms a dehydrated shell when exposed to open air conditions, which can effectively prevent CHBr3 from escaping by creating a natural seal. This shell does not form in the gas-tight sample, so CHBr3 can gradually escape into the headspace of the vial and is subsequently released when the vial is opened for sampling.

[0365] The CHBr3 content in cyclodextrins was the most stable of the excipients tested, with no significant changes over the 4-week period at any temperature and exposure condition. By week 4, cyclodextrin-bound CHBr3 did not appear to be negatively affected by higher temperatures (25°C and 40°C) or open air conditions as observed with corn oil or molasses products. Small changes in CHBr3 over time were observed under all storage conditions ( Fig.11 ) can be attributed to analytical changes.

[0366] These results demonstrate that synthetic CHBr3 can be stabilized and stored in liquid (corn oil), semi-liquid (molasses), and solid (cyclodextrin) bromoform stabilizing excipients, thereby providing stable bromoform compositions.

[0367] These results also demonstrate that the stable bromoform compositions described herein allow for appropriate concentrations of synthetic bromoform for effective methane (CH4) mitigation in vitro and in vivo and are suitable for ruminant diet formulation.

[0368] These results also demonstrate that the compositions described herein allow for the sustained release of synthetic bromoform.

[0369] Table 2: Mean bromoform (CHBr3) content in each excipient over time (weeks) at different storage temperatures (degrees Celsius) in airtight containers and exposed to open air conditions (25°C and 40°C only). Superscript letters indicate significantly different mean CHBr3 content within the excipient (P<0.05). In treatments where there was a significant difference in CHBr3 over time, the % change between week 0 and week 4 has been indicated. The molasses open air samples were adjusted to account for the percent weight loss of each vial at each time point.

[0370]

[0371] Example 8: Asp-Oil improves growth performance of feedlot cattle

[0372] Based on the results of Example 6, which demonstrated that Asp-Oil and compositions comprising synthetic bromoform and a stabilizing excipient (canola oil) performed similarly in vivo, the inventors sought to determine whether the in vivo effects of the compositions described herein included changes in growth performance.

[0373] method

[0374] The experimental design was a randomized block design, including 30 pens, 10 animals per pen: a total of 300 cattle. All cattle were from the same farm, similar in breed (British cross) and sex (bull), with a target entry weight between 330-380 kg. The experimental design followed the Australian feedlot retail meat standard, which was raised for 80 days to achieve an ideal exit weight of 480-540 kg. A total of 3 incremental diets were used to transition bulls from a high forage diet before entry to a high grain concentrate finishing ration, which reflects current feedlot practices. During these transitions, the corresponding total oil content of each ration was increased in a stepwise procedure. The pre-start ration was used only in the first few days after arriving at the feedlot. The starter feed, transition 1 (T1) feed, and transition 2 (T2) feed were fed in 7-day segments (total 21 days), respectively.

[0375] Individual whole body live weight (LW) measurements were made upon arrival, at trial induction (Day -5), starting weight (Day 0), intermediate weight (Day 40), and ending weight (Day 80). ADWG was calculated for the entire 80 days of feeding (ending weight - starting weight / # days) and the last 40 days of feeding (intermediate weight - ending weight / 40 to represent the effect on the finishing diet). Bulls were fed with zero / low rejection rates based on the previous day's intake. FCE was calculated for the entire 80 days of feeding (DMI / ADWG) and the last 40 days of feeding (DMI Day 40-Day 80 / ADWG Day 40-Day 80).

[0376] result

[0377] During the course of the experiment, the feedlot was hit with a severe weather event which resulted in pens flooding midway through the trial. Twelve pens (of 30) were relocated to other areas of the feedlot which may have caused issues with feed intake and weight gain, therefore we report results in two formats [with and without flooded pens]. Table 2 reports data for all 30 pens, while Table 3 reports data for pens that were not flooded. This study demonstrated that ADWG representing the entire study duration (Days 1 to 81) increased by 2.2% (all pens) to 5.6% (removal of flooded pens) and FCE increased by 4.8% (all pens) to 5.2% (removal of flooded pens) compared to control bulls (no Asp-Oil). Feed intake on Days 1 to 81 had a differential effect between all pens and the removal of flooded pens, with all Asp-Oil pens decreasing by 2.2% compared to controls, while feed intake increased by 0.46% in Asp-Oil pens that were not flooded compared to controls. While this is an unusual effect, Asp-Oil fences still saw improvements in ADWG and FCE.

[0378] During the time period between the last LW measurements, Asp-Oil was at its full feed inclusion level (Days 40 to 81; 34 mg CHBr3 / kg DMI), and ADWG improved by 2.6% (all pens) to 6.6% (removing flooded pens), DMI decreased by 4.5% (all pens) to 1.24% (removing flooded pens), and FCE improved by 7.3% (all pens) to 8.0% (removing flooded pens). These results indicate that there is an improvement in ADWG and FCE of beef bulls in animals fed Asparagaceae biomass extracted into a bromoform stabilizing excipient (Asp-Oil). Example 6 demonstrates that compositions comprising bromoform and the same bromoform stabilizing excipient similarly reduce in vivo methane production; the inventors propose that compositions comprising bromoform and a bromoform stabilizing excipient improve growth performance on the same basis.

[0379] Table 3. Intake and growth parameters of feedlot bulls in control and Asparagaceae oil treated groups. This data table includes animals from all 30 pens.

[0380] Day 1-81 unit Comparison Asp Oil difference Ingestion kg fed / day 14.67 14.33 -2.3％ Average daily weight gain kg / day 2.38 2.43 2.2％ Feed conversion efficiency ADG / Intake 0.160 0.170 4.8％ Days 40-81 unit Comparison Asp Oil difference Ingestion kg fed / day 15.65 14.95 -4.5％ Average daily weight gain Total live weight gain / #day 2.11 2.16 2.6％ Feed conversion efficiency ADG / Intake 0.130 0.140 7.3％

[0381] Table 4. Intake and growth parameters of feedlot steers in control and Asparagaceae oil treated groups. This data table does not include the 12 pens (of 30) that were flooded midway through the experiment.

[0382]

Claims

1. A method for reducing total gas production and / or methane production in a ruminant, the method comprising the step of providing to the ruminant an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

2. The method of claim 1, wherein the step of providing an effective amount of a composition to the ruminant comprises administering to the ruminant an effective amount of a composition comprising artificial bromoform in a bromoform stabilizing excipient.

3. The method of claim 1, wherein the step of providing an effective amount of a composition to the ruminant comprises preparing a composition comprising artificial bromoform in a bromoform stabilizing excipient that is usable in a feed system including the ruminant.

4. The method of claim 1 or claim 2, wherein the composition is administered at a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

5. The method of claim 3, wherein the composition is prepared to be available in an amount that provides a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

6. The method according to any one of claims 1 to 5, wherein the ruminant is selected from members of the suborder Rumina and the suborder Callosopoda.

7. The method according to any one of claims 1 to 6, wherein the ruminant is a cattle or a sheep.

8. The method according to any one of claims 1 to 7, wherein the bromoform stabilizing excipient is an edible non-polar substance.

9. The method of claim 8, wherein the edible non-polar substance is an edible oil.

10. The method of claim 9, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, caprine oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil, and vegetable oil, or any combination thereof.

11. The method according to any one of claims 1 to 7, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

12. The method of claim 11, wherein the carbohydrate is cyclodextrin or molasses.

13. The method according to any one of claims 1 to 13, wherein the composition is in solid, semi-solid or liquid form.

14. A composition for use in reducing total gas production and / or methane production in a ruminant, wherein the composition comprises synthetic bromoform and a bromoform stabilizing excipient.

15. The composition for use according to claim 14, wherein the bromoform stabilizing excipient is an edible non-polar substance.

16. The composition for use according to claim 15, wherein the edible non-polar substance is an edible oil.

17. The composition for use according to claim 16, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olein oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

18. A composition for use according to claim 14 or claim 15, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

19. A composition for use according to claim 18, wherein the carbohydrate is cyclodextrin or molasses.

20. A composition for use according to any one of claims 14 to 19, wherein the composition is formulated to be provided to the ruminant at a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

21. A composition for use according to any one of claims 14 to 20, wherein the composition is in solid, semi-solid or liquid form.

22. A feed supplement for use in reducing total gas production and / or methane production in a ruminant, the supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

23. A feed supplement for use according to claim 22, wherein the feed supplement is formulated to provide a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter provided to the ruminant.

24. A feed supplement for use according to claim 22 or claim 23, further comprising one or more edible excipients.

25. A feed supplement for use according to any one of claims 22 to 24, wherein the ruminant is selected from members of the suborder Ruminans and the suborder Callospoda.

26. A feed supplement for use according to any one of claims 22 to 25, wherein the ruminant is a cattle or sheep.

27. A feed supplement for use according to any one of claims 22 to 26, wherein the bromoform stabilizing excipient is an edible non-polar substance.

28. A feed supplement for use according to claim 27, wherein the edible non-polar substance is an edible oil.

29. A feed supplement for use according to claim 28, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, cape olein oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

30. A feed supplement for use according to any one of claims 22 to 26, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

31. A feed supplement for use according to claim 18, wherein the carbohydrate is cyclodextrin or molasses.

32. An animal feed supplement for use according to any one of claims 22 to 31 , wherein the composition is in solid, semi-solid or liquid form.

33. A method of producing a methane-reduced ruminant feed, the method comprising mixing a ruminant feed with a feed supplement comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient.

34. The method of claim 27, wherein the animal feed comprises a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter of the ruminant feed.

35. An animal feed for use in reducing total gas production and / or methane production in a ruminant, the animal feed comprising an effective amount of a composition comprising artificial bromoform and a bromoform stabilizing excipient and a ruminant feed.

36. An animal feed for use according to claim 35, wherein the composition is formulated to provide a dosage of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg bromoform per gram of organic matter of the ruminant feed.

37. An animal feed for use according to claim 35 or claim 36, wherein the ruminant is selected from members of the suborder Rumina and the suborder Callosopoda.

38. An animal feed for use according to any one of claims 35 to 37, wherein the ruminant is a cattle or sheep.

39. An animal feed for use according to any one of claims 35 to 38, wherein the bromoform stabilizing excipient is an edible non-polar substance.

40. An animal feed for use according to claim 39, wherein the edible non-polar substance is an edible oil.

41. The animal feed for use according to claim 40, wherein the edible oil is selected from the group consisting of almond oil, apricot kernel oil, caprine oil, avocado oil, brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, coconut shell oil, cottonseed oil, diglyceride oil, linseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, walnut oil, pine nut oil, pistachio oil, pumpkin seed oil, rapeseed oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

42. An animal feed for use according to any one of claims 35 to 38, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

43. A composition for use according to claim 42, wherein the carbohydrate is cyclodextrin or molasses.

44. An animal feed for use according to any one of claims 35 to 43, wherein the composition is in solid, semi-solid or liquid form.

45. A stable bromoform composition comprising synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of an edible solid, an edible semisolid, or an edible liquid.

46. ​​The stable bromoform composition of claim 45, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates, and water.

47. The stable bromoform composition of claim 45 or claim 46, wherein the composition does not comprise one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

48. A method of preparing a stable bromoform composition, the method comprising contacting synthetic bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of an edible solid, an edible semisolid, or an edible liquid.

49. The method for preparing a stable bromoform composition according to claim 48, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates and water.

50. The method of claim 48 or claim 49, wherein the composition does not comprise one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

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