New composition

Abstract

The present invention relates to a process for preparing a composition suitable for reducing total gas production and / or methane production and / or growth ability in ruminants.

Description

Detailed Description of the Invention

[0001] [Technical Field to Which the Invention Belongs] The field of the present invention relates to a process for preparing a composition suitable for reducing total gas production and / or methane production and / or growth ability in ruminants.

[0002] [Technical Field to Which the Invention Belongs] Methane (CH 4 ) is a greenhouse gas (GHG) produced mainly by methane-producing microorganisms present in natural ecosystems (e.g., wetlands, oceans, and lakes) and in the digestive tracts of invertebrate and vertebrate animals such as termites and ruminants. Every year, about 429 - 507 Tg of CH 4 is released from the atmosphere, about 40 Tg is removed from the stratosphere through reaction with hydroxyl (OH) radicals, and about 30 Tg is removed by methane-oxidizing bacteria in the soil.

[0003] Nevertheless, anthropogenic GHG emissions are increasing rapidly, and the atmospheric CH 4 concentration is now more than twice as high as in the early 1800s. Methane is very effective at absorbing solar infrared radiation and has a global warming potential 28 times greater than that of CO 2 . As a result, its accumulation in the atmosphere is a major contributor to climate change. One of the main sources of anthropogenic CH 4 can be attributed to agricultural activities, including ruminant livestock.

[0004] According to a recent United Nations report, cattle breeding generates more greenhouse gases contributing to global warming than transportation, measured in CO 2 equivalents. In Australia, ruminants are estimated to contribute about 10% of total GHG emissions. Ruminants produce CH 4 as a byproduct of anaerobic microbial fermentation of feed in the rumen and, to a lesser extent, in the large intestine. The rumen microbiota is very diverse and acts collectively to ferment the ingested organic matter (OM), producing CO 2 , H 2, and is composed of bacteria, protozoa, fungi, and bacteriophages that yield volatile fatty acids (VFA) and formate. Methanogenic archaea present in the rumen use these end products to produce CH 4 The production of CH 4 lowers the partial pressure of H 2 , which could otherwise inhibit rumen fermentation, but also reduces the amount of energy and carbon available for the formation of VFA, which are essential for ruminant nutrition. Most of the CH 4 produced in ruminants is exhaled and belched by the animal, corresponding to a loss of up to 12% of the total energy intake.

[0005] Previous studies have focused on the use of species as feed supplements to reduce total gas production and / or methane production in ruminants. This study has led to the use of Asparagopsis biomass and Asparagopsis-derived products to reduce methane, but there remains a need for additional mitigation strategies to reduce intestinal CH 4 formation.

[0006] [Summary of the Invention] In one aspect, the present invention is a method for reducing total gas production and / or methane production in a ruminant, the method comprising providing to the ruminant an effective amount of a composition comprising bromoform produced and a bromoform-stabilizing excipient.

[0007] In one embodiment, the step of providing an effective amount of the composition to the ruminant comprises administering to the ruminant an effective amount of the composition comprising bromoform produced in a bromoform-stabilizing excipient.

[0008] In another embodiment, the step of providing an effective amount of the composition to the ruminant comprises making available in a feed system comprising the ruminant a composition comprising bromoform produced in a bromoform-stabilizing excipient.

[0009] In another embodiment, 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 of bromoform per gram of organic matter provided to the ruminant animal.

[0010] In another embodiment, the composition is made available in an amount providing a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of bromoform per gram of organic matter provided to the ruminant animal.

[0011] In another embodiment, the ruminant animal is selected from members of the Ruminantia suborder and the Tylopoda suborder.

[0012] In another embodiment, the ruminant animal is a cow or a sheep.

[0013] In another embodiment, the bromoform stabilizing excipient is an edible nonpolar substance.

[0014] In another embodiment, the edible nonpolar substance is an edible oil.

[0015] 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, corza oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flaxseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0016] In another embodiment, the bromoform stabilizing excipient is an edible carbohydrate or water.

[0017] In another embodiment, the carbohydrate is cyclodextrin or molasses.

[0018] In another embodiment, the composition is in solid, semi-solid or liquid form.

[0019] In another aspect, the present invention provides a composition when used to reduce total gas production and / or methane production in ruminants, said composition comprising produced bromoform and a bromoform stabilizing excipient.

[0020] In one embodiment, the present invention provides a composition when used as described herein, wherein the bromoform stabilizing excipient is an edible non-polar substance.

[0021] In one embodiment, the present invention provides a composition when used as described herein, wherein the edible non-polar substance is edible oil.

[0022] In one embodiment, the present invention provides a composition when used as described herein, wherein 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, corza oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flaxseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0023] In one embodiment, the present invention provides a composition when used as described herein, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

[0024] In one embodiment, the present invention provides a composition in which the carbohydrate is cyclodextrin or molasses when used as described herein.

[0025] In one embodiment, the present invention provides a composition when used as described herein, the composition being 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 of bromoform per gram of organic matter provided to a ruminant.

[0026] In one embodiment, the present invention provides a composition in solid, semi-solid or liquid form when used as described herein.

[0027] In one aspect, the present invention provides a feed supplement when used to reduce total gas production and / or methane production in ruminants, said supplement comprising an effective amount of a composition comprising manufactured bromoform and a bromoform stabilizing excipient.

[0028] In one embodiment, the present invention provides a feed supplement when used as described herein, the feed supplement being 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 of bromoform per gram of organic matter provided to a ruminant.

[0029] In another embodiment, the present invention provides a feed supplement when used as described herein, the feed supplement further comprising one or more edible excipients.

[0030] In a further embodiment, the present invention provides a feed supplement when used as described herein, wherein said ruminant is selected from members of the Ruminantia suborder and the Tylopoda suborder.

[0031] In a further embodiment, the present invention provides a feed supplement for use as described herein, wherein the ruminant is a cow or a sheep.

[0032] In a further embodiment, the present invention provides a feed supplement for use as described herein, wherein the bromoform stabilizing excipient is an edible non-polar substance.

[0033] In a further embodiment, the present invention provides a feed supplement for use as described herein, wherein the edible non-polar substance is an edible oil.

[0034] In a further embodiment, the present invention provides a feed supplement for use as described herein, wherein 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, coriander oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0035] In a further embodiment, the present invention provides a feed supplement for use as described herein, wherein the bromoform stabilizing excipient is an edible carbohydrate or water.

[0036] In a further embodiment, the present invention provides a feed supplement for use as described herein, wherein the carbohydrate is cyclodextrin or molasses.

[0037] In a further embodiment, the present invention provides a feed supplement as used herein, wherein the composition is in solid, semi-solid or liquid form.

[0038] In one aspect, the present invention provides a method for producing a methane-reducing ruminant feed, comprising mixing a ruminant feed with a feed supplement comprising an effective amount of a composition comprising produced bromoform and a bromoform-stabilizing excipient.

[0039] In one embodiment, the present invention provides the method 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 of bromoform per gram of organic matter of the ruminant feed.

[0040] In one aspect, the present invention provides an animal feed for use in reducing total gas production and / or methane production in ruminants, said animal feed comprising an effective amount of a composition comprising produced bromoform and a bromoform-stabilizing excipient and a ruminant feed.

[0041] In one embodiment, the present invention provides an animal feed as used 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 of bromoform per gram of organic matter of the ruminant feed.

[0042] In one embodiment, the present invention provides an animal feed as used herein, wherein the ruminants are selected from members of the Ruminantia suborder and the Tylopoda suborder.

[0043] In another embodiment, the present invention provides an animal feed as used herein, wherein the ruminant is a cow or a sheep.

[0044] In a further embodiment, the present invention provides an animal feed when the bromoform stabilizing excipient is an edible non-polar substance, as used as described herein.

[0045] In a further embodiment, the present invention provides an animal feed when the edible non-polar substance is an edible oil, as used as described herein.

[0046] In a further embodiment, the present invention provides an animal feed when 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, coriander oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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 oils, or any combination thereof, as used as described herein.

[0047] In a further embodiment, the present invention provides an animal feed in use when the bromoform stabilizing excipient is an edible carbohydrate or water.

[0048] In a further embodiment, the present invention provides an animal feed in use when the carbohydrate is cyclodextrin or molasses.

[0049] In a further embodiment, the present invention provides an animal feed when the composition is in solid, semi-solid or liquid form, as used as described herein.

[0050] In one embodiment, the present invention provides a stabilized bromoform composition comprising produced bromoform and a bromoform stabilizing excipient, the bromoform stabilizing excipient being selected from the group consisting of an edible solid, an edible semi-solid or an edible liquid.

[0051] In one embodiment, the present invention provides a stabilized bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates, and water.

[0052] In one embodiment, the present invention provides a stabilized bromoform composition as described herein, which does not contain one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

[0053] In one embodiment, the present invention provides a method for preparing a stabilized bromoform composition, which includes contacting the produced bromoform with a bromoform stabilizing excipient, and the bromoform stabilizing excipient is selected from the group consisting of edible solids, edible semi-solids, or edible liquids.

[0054] In one embodiment, the present invention provides a method for manufacturing a stabilized bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates, and water.

[0055] In one embodiment, the present invention provides a method for preparing a stabilized bromoform composition that 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

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[0057] [Detailed Description] As discussed below, the present invention is partly based on the characterization of compositions that can inhibit methane production by rumen fermentation using manufactured bromoform. The compositions have been shown not to significantly inhibit methane production with anti - methane activity at doses of manufactured bromoform that have been demonstrated in previous - different compositions. The development of composition technologies for delivering Asparagopsis products into the diets of grazing livestock enables the greatest distribution and / or environmental benefits, particularly in countries where the red meat, dairy, and wool industries, by the number of animals, are mainly the forage industry.

[0058] The present invention provides a stabilized composition comprising manufactured bromoform. The ability to prepare a stabilized composition comprising manufactured bromoform enables the use of low doses of manufactured bromoform, including doses of bromoform that have previously been demonstrated not to inhibit methane production, to inhibit methane production from ruminants.

[0059] Since low dosages of manufactured bromoform can be used, biologically available formulations can be delivered to animals in agricultural systems such as pasture systems.

[0060] The ability to prepare stabilized compositions containing manufactured bromoform also allows for the preparation of compositions with known sustained release properties, including different temperatures.

[0061] The compositions described herein make it possible to avoid high levels of undesirable trace elements and minerals.

[0062] In vitro studies using rumen fluid have shown that the production of CH 4 is significantly affected by synthetic halomethanes when these synthetic compounds are added to rumen fluid. For example, bromoform and dibromochloromethane in dimethyl sulfoxide (DMSO), when added to ruminant gastric fluid, inhibit CH 4 production significantly compared to the control when added at concentrations of 5 μM or more (Machado, L., Magnusson, M., Paul, N. A. et al., J Appl Phycol (2016) 28:3117).

[0063] However, previous studies have shown that the effects of Asparagopsis biomass containing secondary metabolites - or products derived from Asparagopsis - vary and are contradictory in both rumen fluid and in vivo, due to differences in the type and quality of the extracts / compositions containing such compounds, the dosage of the compounds, and the basal diet.

[0064] One of the secondary metabolites contained in bromoform-Asparagopsis biomass is volatile and has physical properties (including its volatility) that render its use in vivo or in vitro impractical. Initial studies demonstrated that bromoform produced in DMSO could not significantly inhibit methane production at low doses (e.g., doses less than 5 μM) (Machado et al. (2016) J Appl Phycol 28:3117-3126). Subsequent studies by Machado et al. (2018) (Microbial Ecology 75(D1)) demonstrated that the inclusion of biomass at 2% OM inhibited methane production more effectively than an equivalent dose of bromoform produced in DMSO, indicating that other components of Asparagopsis contribute to its anti-methanogenic activity.

[0065] As noted above, bromoform is volatile, and studies using volatile bromoform (e.g., non-stabilized) in a closed system have not shown that the produced bromoform is supplied in a biologically available form within the rumen.

[0066] Importantly, more recent studies have demonstrated that produced bromoform does not inhibit methane production (Stefenoni et al. (2019) J. Dairy Sci. Vol.102, Suppl.1, page378). Without wishing to be bound by theory, the inventors propose that bromoform needs to be provided in a biologically available form within the rumen.

[0067] Exposure to high concentrations of concentrated bromoform is considered dangerous to animals, and synthetic bromoform and chemically related compounds (e.g., bromochloromethane) as manufactured / synthesized / purified chemicals are not safe and are not approved for human and animal uses, including methane production inhibition in ruminants.

[0068] Very recently, Muizelaar et al., Foods (2021) 10:584 published results presenting data claiming that bromoform was detected in the milk of some cows only on the first day of feeding in the low and medium treatment groups (9.1 and 11 μg / L, respectively), and only in the milk of a single sample in the high treatment group (35 μg / L) on day 9, and that two animals were slaughtered and their forestomachs showed abnormalities. Histological examination revealed signs of inflammation. The authors of that study concluded that, within the scope of their experiments, CHBr 3 does not accumulate in animal tissues but can be excreted in urine and milk. This paper represents numerous concerns about human consumption after the use of Asparagopsis, and the need for further research to investigate the effects of ruminants consuming bromoform-containing materials. Although this paper does not teach the use of bromoform-containing materials, as part of the research described herein, the inventors propose that the compositions of the present invention enable the use of significantly less bromoform.

[0069] The inventors believe that the study by Muizelaar et al. does not directly link inflammation to Asparagopsis.

[0070] The inventors tested the effects of compositions containing manufactured bromoform and bromoform-stabilizing excipients. In particular, the inventors demonstrated in FIGS. 1, 2, 3, 6, and 9 that the compositions containing manufactured bromoform and bromoform-stabilizing excipients can reduce methane produced in in vitro fermentation, and in FIG. 10, demonstrated that the compositions described herein can inhibit methane production in vivo. In some embodiments, the compositions containing manufactured bromoform and bromoform-stabilizing excipients can eliminate methane production from in vitro and / or in vivo fermentation.

[0071] Importantly, the inventors have demonstrated in Example 8 that the in vivo effects of the compositions described herein include an improvement in growth ability. In particular, the observed in vivo effects include an improvement in average daily weight gain and an improvement in feed utilization efficiency.

[0072] In contrast to the data presented herein, previous studies have shown that the inclusion of bromoform at doses of 0.25, 1, and 1.5% of the supplied dry matter had no effect on methane production (Stefenoni et al. (2019) J. Dairy Sci. Vol. 102, Suppl. 1, page 378, Dose-response effect of the macroalga Asparagopsis taxiformis on enteric methane emission in milking dairy cows). However, in the same experiment, Asparagopsis, which contains many anti-methanogenic compounds including bromoform, linearly decreased methane (CH 4 ) yields over doses of 0.25, 0.5, 0.75, 1.0, and 1.5% of the feed dry matter when included in in vitro fermentation. This indicates that bromoform alone does not affect methane production, but Asparagopsis included at the corresponding doses inhibits methane production.

[0073] Accordingly, in one aspect, the present invention provides a method for reducing total gas production and / or methane production in ruminants, the method comprising providing to the ruminant an effective amount of a composition comprising produced bromoform and a bromoform-stabilizing excipient.

[0074] Machado et al. (2016) J Appl Phycol 28:3117-3126 demonstrated that Asparagopsis taxiformis contains a number of anti-methanogenic compounds, including bromoform, dibromochloromethane, and bromochloromethane. This study demonstrated that manufactured bromoform formulated in DMSO significantly inhibits methane production at doses of 5 μM, 10 μM, and 25 μM, but at 1 μM, manufactured bromoform does not significantly inhibit methane production in in vitro fermentation. Machado et al. (2016) showed that bromoform at 1.3 μM is the amount of bromoform equivalent to the Asparagopsis content at 2% OM (24.7 mg) in vitro. Since manufactured bromoform at 1 μM did not significantly inhibit methane production, Machado et al. showed that bromoform requires more than 1 μM of bromoform to significantly inhibit methane production. The compositions described herein enable the provision of a composition effective to reduce methane production at a bromoform dose significantly lower than that previously demonstrated to provide no significant effect in Machado et al. (2016) J Appl Phycol 28:3117-3126.

[0075] The present invention shows that by using 0.01 mg of manufactured bromoform and a bromoform stabilizing excipient per gram of feed substrate, the total gas production and / or methane production is reduced, and at higher doses of the composition comprising manufactured bromoform and a bromoform stabilizing excipient (e.g., 0.04 mg or more of manufactured bromoform and a bromoform stabilizing excipient), the total gas production and / or methane production is eliminated. This data is in contrast to the study by Machado et al., which showed that much more bromoform (e.g., more than 1 μM of manufactured bromoform) is required.

[0076] There are many problems with the use of Asparagopsis biomass and Asparagopsis - derived products for inhibiting methane production. One problem is that the amounts of secondary metabolites (bromoform, dibromochloromethane, and bromochloromethane), which are known to inhibit methane production, vary between batches of Asparagopsis, and thus, forming a composition having a desired amount of bromoform, dibromochloromethane, and bromochloromethane from Asparagopsis requires a great deal of effort. In contrast to Asparagopsis biomass and Asparagopsis - derived products, the compositions described herein enable the provision of a composition containing a controlled amount of manufactured bromoform.

[0077] In one embodiment, the present invention provides a stabilized bromoform composition comprising manufactured 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.

[0078] In one embodiment, the stabilized bromoform composition described herein does not contain dibromochloromethane, bromochloroacetic acid, and / or dibromoacetic acid.

[0079] Machado et al. (2016) showed that bromoform at 1.3 μM is the amount of bromoform equivalent to the Asparagopsis content at 2% OM (24.7 mg) in vitro. Since bromoform above 1 μM is required to significantly inhibit methane production (based on bromoform content and the data of Machado et al.), a content of Asparagopsis exceeding 2% OM (24.7 mg) is required to significantly inhibit methane production. However, it is known that the inclusion of Asparagopsis at high % levels of OM in the feed can result in animals avoiding consuming the feed (Muizelaar et al. Foods (2021) 10:584). The compositions described herein enable the provision of compositions that can avoid the use of Asparagopsis biomass or Asparagopsis-derived products that are unpalatable to ruminants.

[0080] Furthermore, without wishing to be bound by theory, the inventors propose that other secondary metabolites of Asparagopsis (including dibromochloromethane and bromochloromethane), when provided in combination, may contribute to the anti-methanogenic effect of Asparagopsis biomass or Asparagopsis-derived products. For example, Machado et al. (2016) J Appl Phycol 28:3117-3126 showed in Figure 2 that the total gas production was significantly reduced by Asparagopsis biomass, but an extract at a dose equivalent to the same amount of biomass did not reduce the total gas production, indicating that Asparagopsis biomass has the highest inhibitory effect on methane production compared to an extract equivalent to the same amount of biomass, suggesting that other secondary metabolites contribute to the anti-methanogenic effect.

[0081] Consistent with this preliminary data in Figure 1, Asparagopsis biomass in oil inhibits methane production to a greater extent than compositions containing the same concentration (0.08 mg / g OM) of produced bromoform, and Asparagopsis biomass in oil and produced bromoform in oil were shown to inhibit methane production to the same extent in the same manner but at higher doses. However, importantly, the inventors have now demonstrated that produced bromoform in a stabilizing excipient inhibits methane production to a similar extent as Asparagopsis biomass in oil.

[0082] Whether the metabolites can inhibit methane production when provided separately - remains unclear. For example, as noted above, previous studies have demonstrated that produced bromoform does not inhibit methane production. The compositions described herein enable the provision of compositions effective for reducing methane production in the absence of dibromochloromethane, and bromochloromethane, as well as other secondary metabolites of Asparagopsis. Without wishing to be bound by theory, the inventors propose that the compositions described herein, which contain produced bromoform and a bromoform stabilizing excipient, provide bioavailable bromoform that can inhibit methane production at low levels. As summarized in Figure 9, the present invention provides the use of a number of classes of bromoform stabilizing excipients that provide bioavailable bromoform that can inhibit methane production at low levels when combined with bromoform.

[0083] In Example 6, the inventors demonstrated that a composition containing bromoform and a bromoform-stabilizing excipient, wherein the bromoform is bromoform derived from Asparagopsis, can inhibit methane production in animals. Example 8 demonstrates that the in vivo effects of the compositions described herein include improvement of growth performance metrics. In particular, the observed in vivo effects include an improvement in average daily weight gain and an improvement in feed utilization efficiency. In another embodiment provided herein, the present invention provides a composition comprising bromoform and a bromoform-stabilizing excipient, wherein the bromoform is bromoform derived from Asparagopsis.

[0084] In one embodiment, the present invention provides a composition comprising bromoform and a bromoform-stabilizing excipient, wherein the bromoform is bromoform derived from Asparagopsis, and the composition further comprises produced bromoform.

[0085] Figure 3 shows the first study demonstrating that an Asparagopsis-derived composition containing Asparagopsis biomass (Asp-Oil) extracted in a bromoform-stabilizing excipient inhibits methane production to the same extent as produced bromoform in canola oil, despite the canola oil composition not containing other anti-methane-producing compounds present in the Asp-Oil. The ability to deliver produced bromoform, which is biologically available, in a controlled amount in the absence of other contaminants of Asparagopsis enables new uses of the compositions described herein.

[0086] A further issue is to increase the level of inclusion of Asparagopsis biomass or products derived from Asparagopsis, and care must also be taken with trace elements and minerals that can accumulate in seaweed. Asparagopsis, like many other seaweeds, can contain iodine at concentrations that can exceed the maximum allowable levels in the feeding system. Compositions containing the bromoform and bromoform-stabilizing excipients produced as described herein make it possible to exclude trace elements and minerals of Asparagopsis biomass or products derived from Asparagopsis, such as iodine.

[0087] Furthermore, Asparagopsis, like many other seaweeds, can contain bromine at concentrations that can exceed the maximum allowable levels in the feeding system. Compositions containing the bromoform and bromoform-stabilizing excipients produced as described herein make it possible to exclude trace elements and minerals of Asparagopsis biomass or products derived from Asparagopsis, such as bromine.

[0088] As used herein, the term "reducing" includes a reduction in the amount of a substance compared to a reference. For example, the reduction in the amount of total gas and / or methane produced by a ruminant (s) administered an effective amount of a composition containing the bromoform and bromoform-stabilizing excipients produced according to the present invention is compared to an animal (s) not administered a composition containing an effective amount of a composition containing the bromoform and bromoform-stabilizing excipients described herein. This reduction can be measured in vitro using an artificial rumen system that simulates anaerobic fermentation or in vivo using animals confined in a respiration chamber. Evaluating enteric methane production by ruminants is within the knowledge and skill of those skilled in the art.

[0089] As used herein, the term "anaerobic fermentation" is intended to include anaerobic fermentation in vitro (e.g., experimental systems) and in vivo (e.g., ruminants).

[0090] As used herein, the term "reduction of total gas production" refers to a reduction in the total amount of gas produced, e.g., the amount of total gas produced in the gastrointestinal tract. This term includes, for example, the total volume of all gases produced as a result of anaerobic fermentation in the systems described herein. Fermentation in the rumen and intestines of ruminants causes the production of gases including methane. The present invention aims to reduce this process, e.g., to reduce the total amount of gas produced in the gastrointestinal tract. Evaluating total gas production by ruminants is within the knowledge and skill of those skilled in the art.

[0091] As used herein, the term "reduction of methane production" refers to a reduction in methane produced in the gastrointestinal tract. This term includes, for example, the specific volume of methane produced as a result of anaerobic fermentation in the systems described herein. Fermentation in the rumen and intestines of ruminants causes the production of methane. The present invention aims to reduce this process, e.g., to reduce the total amount of methane produced in the gastrointestinal tract. Evaluating methane production by ruminants is within the knowledge and skill of those skilled in the art.

[0092] In a preferred embodiment of the present 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 containing manufactured bromoform and a bromoform-stabilizing excipient is not administered to the animal. In another embodiment, the reference is the amount of total gas produced when a control feed is administered to the animal. In another embodiment, the reference is the amount of total gas produced when a control feed is subjected to in vitro anaerobic fermentation.

[0093] In a preferred embodiment of the present 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 bromoform and a bromoform-stabilizing excipient produced 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.

[0094] "Effective amount" means an amount of a composition comprising bromoform and a bromoform-stabilizing excipient produced as described herein sufficient to enable an improvement, e.g., a reduction in methane production compared to a reference or control, a reduction in total gas production compared to a reference or control, maintenance of an effective level of a desirable volatile fatty acid compared to a reference or control, a reduction in the acetate to propionate ratio compared to a reference or control, maintenance of live body weight, dry matter intake and / or organic matter intake compared to a reference or control, or an improvement in growth ability 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. Examples of methods include those described in Hano. Gen. Appl. Microbiol., 39, 35-45, 1993, or by in vivo oral administration to ruminants.

[0095] The effective amount of a composition comprising manufactured bromoform and a bromoform-stabilizing excipient can be determined by the methods described herein, including in vitro and in vivo studies and in vivo dose-response tests as described by Kinley et al. (2020) Journal of Cleaner Production 259:120836 and WO2015109362 (incorporated herein by reference). For example, the inventors were able to use in vitro rumen fermentation to examine the effect of the amount of a composition comprising manufactured bromoform and a bromoform-stabilizing excipient on total gas, hydrogen, and methane production and to examine the levels of volatile fatty acids including acetate and propionate. A measure of growth ability can be measured using the in vivo tests described herein. Thus, using in vitro rumen fermentation, an effective amount of a composition comprising manufactured bromoform and a bromoform-stabilizing excipient can be characterized that is sufficient to allow for an improvement, e.g., a reduction in methane production compared to a reference or control, a reduction in total gas production compared to a reference or control, maintenance of an effective level of a desired volatile fatty acid compared to a reference or control, or a reduction in the acetate to propionate ratio compared to a reference or control.

[0096] For example, Figures 4 and 7 show that at high doses, a composition comprising manufactured bromoform and a bromoform-stabilizing excipient can increase hydrogen levels in line with inhibition of methane production. Figure 5 demonstrates that no negative effect on fermentation was observed with respect to IVDDM and tVFA production.

[0097] As used herein, the term "providing" includes providing a composition comprising manufactured bromoform and a bromoform-stabilizing excipient as a feed additive in the feed provided to an animal or animal system (e.g., animals in a feedlot, animals in an agricultural system, etc.). Providing also includes administration of the compositions described herein.

[0098] In one embodiment, the agricultural system is a pasture, a feedlot, a system that is periodically replenished, or a combination thereof.

[0099] "Administer" and "administered" mean the act of introducing the compositions described herein into the gastrointestinal tract of an animal. More specifically, this administration is by the oral route. This administration can in particular be carried out by supplementing feed intended for animals with a composition comprising bromoform produced and a bromoform-stabilizing excipient, and the feed thus supplemented is then ingested by the animals. This administration can also include providing a composition comprising bromoform produced and an excipient that stabilizes bromoform in a form (lick block, supplement, etc.) that the animal consumes. Administration can also be carried out by directly introducing the composition (e.g., the dosage form of the composition (e.g., bolus)) into the digestive tract of the animal using a stomach tube or other means.

[0100] Accordingly, the present invention provides a method as described herein, wherein the step of providing an effective amount of a composition to the ruminant animal comprises administering to the ruminant animal an effective amount of a composition comprising bromoform produced in a bromoform-stabilizing excipient.

[0101] The composition comprising bromoform produced and a bromoform-stabilizing excipient can be provided to ruminant animals in one of many ways. The composition comprising bromoform produced and a bromoform-stabilizing excipient can be provided in solid form as a veterinary pharmaceutical, can be distributed in an excipient, and can be fed directly to the animal, or physically mixed with a feed material and provided in any suitable form (e.g., dry form, solution, or suspension, etc.). Alternatively, the composition comprising bromoform produced and a bromoform-stabilizing excipient can be formed into a solution and then sprayed onto a feed material. For example, a dry mixture prepared by adsorption or deposition of a solution onto / in a dry excipient can be used.

[0102] A method of administering to an animal a composition comprising manufactured bromoform and a bromoform-stabilizing excipient is believed to be within the skill of those in the art.

[0103] When used in combination with a feed material, the feed material is preferably of the grain / hay / silage / forage type. Among such feed materials are improved ones, and / or any pasture or legume-based feed, whether directly grazed or prepared as conserved feed hay, any feed ingredient and food or feed industry by-products, as well as biofuel industry by-products and corn meal and mixtures thereof, or feed lots and dairy feeds (e.g., those with a high grain content).

[0104] The composition comprising manufactured bromoform and a bromoform-stabilizing excipient can be provided for consumption in an animal system (e.g., feedlot, natural grassland / pasture agricultural system) and can be incorporated as an animal feed supplement in any suitable form such as a loose lick, wet lick, pellet, water suspension, or lick block of minerals. As is known to those skilled in the art, these loose licks, wet licks, pellets, water suspensions, or lick blocks are particularly convenient for feeding mineral supplements (and proteins and carbohydrates) to ruminants grazing on pasture. These loose licks, wet licks, pellets, water suspensions, or lick blocks, etc., in addition to the composition of the present invention comprising manufactured bromoform and a bromoform-stabilizing excipient, can contain various types of binders such as cement, gypsum, lime, calcium phosphate, carbonates, and / or gelatin. Further additives such as vitamins, trace elements, mineral salts, flavoring additives, etc. can optionally be included.

[0105] The time of administration is not critical as long as a reducing effect on methane production and / or growth performance is shown. Administration is possible at any time as long as the feed is retained in the rumen. However, compositions containing manufactured bromoform and bromoform stabilizing excipients are preferably present in the rumen when methane is produced, so compositions containing manufactured bromoform and bromoform stabilizing excipients are preferably administered with the feed or immediately prior to feeding.

[0106] In certain embodiments of the invention, the effective amount of the composition comprising manufactured bromoform and bromoform stabilizing excipients is administered to a ruminant animal by supplementing the feed for the animal with a composition comprising manufactured bromoform and bromoform stabilizing excipients. Within the meaning of the present invention, "supplementing" means the act of directly incorporating an effective amount of a composition comprising manufactured bromoform and bromoform stabilizing excipients according to the present invention into the feed for the animal. Thus, the animal ingests, at feeding time, a composition comprising manufactured bromoform and bromoform stabilizing excipients according to the present invention, which can then act to maintain the digestibility of the fiber and / or grain contained in the animal's feed. Alternatively, supplements such as loose licks, wet licks, pellets, aqueous suspensions, or lick blocks and other feed supplements can be provided to the animal system without directly incorporating them into the animal feed.

[0107] Accordingly, another subject of the present invention relates to a feed supplement for ruminant animals comprising a composition comprising manufactured bromoform and bromoform stabilizing excipients.

[0108] In another aspect, the present invention also provides a feed supplement for reducing total gas production and / or methane production in ruminant animals, said supplement comprising an effective amount of a composition comprising manufactured bromoform and bromoform stabilizing excipients.

[0109] In one embodiment, a composition comprising an effective amount of produced bromoform and a bromoform stabilizing excipient is administered to the ruminant by supplementing the food for the animal with the composition comprising the produced bromoform and the bromoform stabilizing excipient.

[0110] As discussed above, the inventors demonstrated in Example 6 that a composition comprising bromoform and a bromoform stabilizing excipient, wherein the bromoform is bromoform derived from Asparagopsis, can inhibit methane production in vivo. Example 8 demonstrates that the in vivo effects of the compositions described herein include improvement in growth performance metrics. In particular, the observed in vivo effects include an improvement in average daily weight gain and an improvement in feed utilization efficiency.

[0111] Accordingly, in one embodiment, the present invention provides a method for improving the growth ability of a ruminant, the method comprising providing to the ruminant an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient.

[0112] In a preferred embodiment, the growth performance is an improvement in average daily weight gain and / or an improvement in feed utilization efficiency.

[0113] In one embodiment, the present invention provides a method for increasing the average daily weight gain of a ruminant, the method comprising providing to the ruminant an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient.

[0114] In one embodiment, the present invention provides a method for increasing the feed utilization efficiency of a ruminant, the method comprising providing to the ruminant an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient.

[0115] In one embodiment, the present invention provides an agricultural system with an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient, thereby reducing total gas production and / or methane production in the agricultural system and / or improving the growth ability of livestock animals, and provides the method described herein.

[0116] In one embodiment, the present invention provides the method described herein, wherein an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient is provided to a stocker operation to enable consumption by livestock animals in the stocker operation of a composition comprising produced bromoform and a bromoform stabilizing excipient, thereby reducing total gas production and / or methane production and / or improving the growth ability of livestock animals in the stocker operation.

[0117] In one embodiment, the present invention is the method described herein, wherein an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient is provided to a feedlot system to enable consumption by livestock animals in the feedlot system of a composition comprising produced bromoform and a bromoform stabilizing excipient, thereby reducing total gas production and / or methane production and / or improving growth ability of livestock animals in the feedlot system, and provides a method.

[0118] In one embodiment, the present invention is the method described herein, wherein an effective amount of a composition comprising produced bromoform and a bromoform stabilizing excipient is provided in a forage system to enable consumption by livestock animals in the forage system of a composition comprising produced bromoform and a bromoform stabilizing excipient, thereby reducing total gas production and / or methane production in the forage system and / or improving the growth ability of livestock animals, and provides a method.

[0119] Ruminants are mammals of the order Artiodactyla that initially soften and partially ferment plant-based food in the first stomach chamber of the animal, then regurgitate the semi-digested mass, now known as the cud, and chew it again to digest the plant-based food.

[0120] The process of chewing the cud again to further break down plant matter and stimulate digestion is called "rumination". Ruminants have a digestive tract with four chambers, namely the rumen, reticulum, omasum, and abomasum. In the first two chambers, the rumen and reticulum, the food is mixed with saliva and separated into layers of solid and liquid substances. The solids aggregate with each other to form lumps or boluses. The cud is then regurgitated, chewed slowly and thoroughly mixed with saliva to further break down the fibers. Fibers, especially cellulose, are broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa, and fungi in the unique process of rumen microbial digestion fermentation. The now-decomposed fibers in the liquid part of the contents are then carried from the rumen to the next stomach chamber, the omasum, where further fermentation occurs and water and solubilized nutrients are absorbed. The next stage is the abomasum, where the undigested feed that bypassed the rumen is digested in much the same way as in the case of monogastric animals. The digested intestinal contents are finally sent to the small intestine, where nutrient absorption takes place. Almost all of the glucose produced by cellulose fermentation is used by the symbiotic microbial consortium. Ruminants obtain energy from volatile short-chain fatty acids (VFA) produced by bacteria, namely acetate, propionate, butyrate, valerate, and isovalerate. Most of the protein utilized by ruminants is microbial protein. The by-products of bacterial fermentation of feed are CO 2 and H 2 which are used in the reduction process by anaerobic archaea, and as a result, CH 4 is produced as a waste by-product. The production of CH 4 indicates the inefficiency of energy in ruminant digestive fermentation.

[0121] Importantly, the inventors have shown that the compositions described herein have the property of reducing total gas production and / or methane production in ruminants without impairing rumen fermentation, as measured by dry matter digestibility.

[0122] Examples of ruminants are listed below. However, preferably, the compositions described herein are used as additives for foods for domesticated livestock such as cows, goats, sheep, and llamas. The present invention is particularly useful in cows and sheep. Thus, in one embodiment, the ruminant is selected from members of the Ruminantia suborder and the Tylopoda suborder. In another embodiment, the ruminant is a cow or a sheep. In a further embodiment, the ruminant is a cow.

[0123] Bromoform (CHBr 3 ) is also known by several synonyms such as tribromomethane. See PubChem CID:5558 for details.

[0124] As used herein, the term "manufactured bromoform" refers to bromoform not derived from Asparagopsis biomass, and this term includes bromoform synthesized artificially. Manufactured bromoform can be synthesized artificially by chemical means by known methods including the haloform reaction using acetone and sodium hypobromite, electrolysis of potassium bromide in ethanol, or treating chloroform with aluminum bromide, or reacting chloroform with sodium hydroxide to obtain bromoform and sodium chloride.

[0125] The produced bromoform can also be artificially synthesized by biological means, for example, using enzymes or genetically modified organisms included in the production system, and the produced bromoform is collected from the headspace gas or captured using chemical or mechanical means. For example, International Publication No. 2020 / 243792 (incorporated herein by reference) describes recombinant yeast capable of producing bromoform.

[0126] In a preferred embodiment, the produced bromoform is free bromoform provided in a purified form. For example, in one embodiment, the produced bromoform is 90% pure and can contain low levels (e.g., 1 - 10%) of stabilizers such as ethanol and amylenes. In another embodiment, the produced 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.

[0127] In a preferred embodiment, the compositions described herein do not contain iodine.

[0128] In a preferred embodiment, the compositions described herein do not contain bromine.

[0129] In one embodiment, the present invention provides a stabilized bromoform composition comprising the produced bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of edible solids, edible semi - solids, or edible liquids.

[0130] In one embodiment, the present invention provides the stabilized bromoform composition described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates, and water.

[0131] In one embodiment, the present invention provides a stabilized bromoform composition as described herein that does not contain one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

[0132] In one embodiment, the present invention provides a method for producing a stabilized bromoform composition, the method comprising contacting the produced bromoform with a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of edible solids, edible semi-solids, or edible liquids.

[0133] In one embodiment, the present invention provides a method for producing a stabilized bromoform composition as described herein, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oils, edible carbohydrates, and water.

[0134] In one embodiment, the present invention provides a method for producing a stabilized bromoform composition that does not contain one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

[0135] The inventors herein demonstrate that a stabilizing excipient can be used to stabilize volatile bromoform, make it biologically available for rumen fermentation, and inhibit methane production. Importantly, the inventors demonstrate that carbohydrates (including cyclodextrin and molasses), water, and edible non-polar substances (such as oils) can be used to stabilize bromoform and enable the formation of compositions that make bromoform biologically available for rumen fermentation.

[0136] The stabilizing excipients are discussed below and include edible solids, liquids, and semi-solids that can stabilize bromoform.

[0137] Importantly, the ability to use different stabilizing excipients enables the formation of compositions that are solids, semi-solids, or liquids and contain bromoform that is biologically available for rumen fermentation.

[0138] Furthermore, the ability to use different stabilizing excipients enables the formation of compositions with defined stability.

[0139] For example, Figure 10 shows the stability of the compositions described herein over time under different storage conditions.

[0140] Figure 10 also shows that the sustained release characteristics of the composition, including under different conditions, can be determined. Thus, these sustained release characteristics can be used to provide methods for improving growth performance or reducing methane production in consideration of the feed intake time and / or different temperatures (e.g., climate).

[0141] Surprisingly, the inventors have found that a stabilizing excipient combined with the produced bromoform delays the release of the volatile produced bromoform and provides an extended or sustained release of the produced bromoform.

[0142] As used herein, the term "volatile" refers to the tendency of a drug to evaporate and generally refers to a fluid substance.

[0143] The term "such that a sustained release of the drug is provided" means that the produced bromoform and the stabilizing excipient can dissociate, and as a result, the produced bromoform is released at a lower rate compared to the produced bromoform administered by itself.

[0144] Thus, in one aspect, the present invention provides an anti-methane production composition for use in animals, the composition comprising a produced bromoform together with a stabilizing excipient such that a sustained release of the produced bromoform is provided.

[0145] In another aspect, the present invention provides an animal feed comprising the composition of the present invention as described above together with a nutrient source.

[0146] In another aspect, the present invention provides a medicament for administering produced bromoform to an animal over a long period of time, the medicament comprising a composition containing produced bromoform together with a stabilizing excipient in a manner such that the composition is retained by the animal over said period of time.

[0147] The term "over a long period of time" refers to a period longer than the time it takes for produced bromoform to evaporate when it is not present in the composition.

[0148] The term "in a manner such that the composition is retained by the animal over said period of time" means that the composition is applied in a suitable manner that allows for sustained release. In ruminants, for example, administration can be provided in the form of a controlled release device or in the feed.

[0149] When the animal is a ruminant, this method results in weight gain (e.g., improved average daily weight gain) as demonstrated herein. This method is also beneficial in the anaerobic fermentation of feces of ruminants and non-ruminants, reducing the emission of greenhouse gases.

[0150] In a particularly preferred embodiment, the present invention relates to a method for reducing methane production in an animal over a long period of time, the method comprising the step of administering an anti-methane production composition in an amount effective to reduce methane, the composition containing produced bromoform together with a stabilizing excipient such that sustained release of the agent is provided.

[0151] As used herein, the term "stabilizing excipient" refers to an excipient that prevents loss of bromoform from the composition and allows the produced bromoform to be biologically available in ruminants. Stabilizing excipients include those described herein, including edible solids, liquids, and semi-solids that can stabilize bromoform.

[0152] Suitable stabilizing excipients include alcohol (e.g., ethanol), benzene, chloroform, ether, petroleum ether, DCM, diethyl ether, hexane, pyridine, toluene, xylene, acetone, and oil. Preferably, the bromoform stabilizing excipient is safe for animals to ingest at the final concentration of the composition provided to the animals.

[0153] In a preferred embodiment, the bromoform stabilizing excipient enables the bromoform produced to be biologically available in ruminants when the composition contains bromoform produced at a low dose.

[0154] In a preferred embodiment, the bromoform stabilizing excipient enables the bromoform produced in ruminants to be biologically available when the composition contains bromoform produced at a dose of less than 5 μM.

[0155] In a preferred embodiment, the bromoform stabilizing excipient enables the bromoform produced in ruminants to be biologically available when the composition contains bromoform produced at a dose of less than 1 μM.

[0156] In a preferred embodiment, the bromoform stabilizing excipient is not DMSO.

[0157] In one aspect, the present invention provides a method for producing a stabilized bromoform composition, comprising contacting the produced bromoform with a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is selected from the group consisting of edible oil, and edible carbohydrates and water.

[0158] In one aspect, the present invention provides a process for preparing a composition comprising a produced bromoform and a bromoform stabilizing excipient composition, said process comprising a) providing the produced bromoform; and b) providing a bromoform stabilizing excipient. c) contacting the produced bromoform with the bromoform stabilizing excipient under conditions for forming the composition.

[0159] In one embodiment, the bromoform stabilizing excipient is selected from the group consisting of edible waxes, greases, oils, cyclodextrins, molasses, and saturated fats.

[0160] In one embodiment, the bromoform stabilizing excipient is an edible protein.

[0161] The inventors have demonstrated herein that edible carbohydrates including cyclodextrin and molasses can be used as stabilizing excipients.

[0162] Accordingly, in one embodiment, the stabilizing excipient is a carbohydrate.

[0163] Examples of carbohydrates include molasses, cyclodextrin, lactose, dextrose, sucrose, glucose, fructose, galactose, xylose, arabinose, beta-glucan, galactan, pectin, and the like.

[0164] Figures 6 and 9 demonstrate that a composition can be formed that includes the produced bromoform and cyclodextrin as a bromoform stabilizing excipient, and that methane production from foregut fermentation can be inhibited.

[0165] In another embodiment, the stabilizing excipient is cyclodextrin.

[0166] Cyclodextrin (sometimes called cycloamylose) is a cyclic oligosaccharide containing glucose units linked in a ring, i.e., (α-1,4)-linked α-D-glucopyranose units. Cyclodextrin is typically produced from starch by enzymatic conversion. Typical cyclodextrins contain a number of glucose monomers in the range of 6 to 8 units in the ring and form a conical or hollow plug shape. For example, α (alpha)-cyclodextrin contains a 6-membered sugar ring molecule, β (beta)-cyclodextrin contains a 7-membered sugar ring molecule, and γ (gamma)-cyclodextrin contains an 8-membered sugar ring molecule. The cyclodextrins suitable for the compositions of the present invention can be modified, if desired, by the addition of substituents. As used herein, "cyclodextrin" includes both modified and unmodified cyclodextrins. Substituents generally replace either the entire hydroxyl group or a hydrogen atom on one or more of the hydroxyl groups of the cyclodextrin ring.

[0167] Cyclodextrin can be selected from α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin or derivatives thereof that can be produced naturally and / or synthetically.

[0168] In one embodiment, the present invention provides a composition as described herein, the composition comprising a) contacting the produced bromoform with an aqueous cyclodextrin solution; and b) preparing a solid from step a), and is prepared by a method comprising.

[0169] In another embodiment, the present invention provides a composition as described herein, the composition comprising a) contacting the produced bromoform with an aqueous cyclodextrin solution b) preparing a solid from step a) c) preparing a powder from the solid from step b), and is prepared by a method comprising.

[0170] In another embodiment, the stabilizing excipient is molasses.

[0171] Figures 6, 9, and 10 demonstrate that compositions can be formed that include produced bromoform and molasses as a bromoform stabilizing excipient, and that can inhibit methane production from rumen fermentation, both in vitro and in vivo.

[0172] As used herein, "molasses" includes syrups produced as by-products of processing sugar cane or other vegetable products. Some examples include sugar cane waste (a by-product of sugar production from sugar cane), high-test (cane) molasses (a primary product squeezed from sugar cane), cane molasses (a by-product of the process of refining raw brown sugar to white sugar), sugar beet molasses (a by-product when sugar is manufactured from sugar beets), citrus molasses (juice squeezed in the manufacture of dried citrus pulp), and the like. In one embodiment, the bromoform stabilizing excipient is an edible nonpolar substance.

[0173] Figures 1, 2, 6, 9, and 10 demonstrate that compositions can be formed that include produced bromoform and edible oil as a bromoform stabilizing excipient, and that can inhibit methane production from rumen fermentation.

[0174] Figure 5 demonstrates that a composition including produced bromoform and edible oil as a bromoform stabilizing excipient can be formed without adversely affecting rumen fermentation, as measured, for example, by IVDDM and tVFA production. For example, in one embodiment, the edible nonpolar substance is selected from the group consisting of an oil or molasses.

[0175] In another embodiment, the edible nonpolar substance is an edible oil.

[0176] As used herein, the term "edible oil" includes a single type of oil, or a composition containing a single type of oil, or a mixture of two or more oils, or a composition containing a mixture of two or more oils. The oil includes an oil suitable for providing and administering to animals.

[0177] In another embodiment, the edible nonpolar substance is an edible fat that is solid at ambient temperature.

[0178] Figures 6 and 9 demonstrate that a composition containing bromoform and water as a bromoform stabilizing excipient can be formed and can inhibit methane production from rumen fermentation.

[0179] Bromoform is considered to be slightly soluble in water, being soluble in less than 0.1 g of bromoform per 100 g of water at 20 degrees, so this result is surprising (Mackison, F.W., S. Stricoff, and L.J. Partridge, Jr. (eds.). NIOSH / OSHA - Occupational Health Guidelines for Chemical Hazards. DHHS (NIOSH) Publication No. 81 - 123 (3 VOLS). Washington, DC: U.S. Government Printing Office, Jan. 1981., p. 2).

[0180] In another embodiment, the bromoform stabilizing excipient is water. The inventors prepared bromoform in water at a bromoform concentration of 3.0 ± 0.10 mg per gram of deionized water.

[0181] In one 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, coriander oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0182] As used herein, the term "oil" means any nonpolar hydrophobic substance that is typically liquid at ambient temperature and pressure. Oils can be derived from animals, vegetables, or petrochemicals and typically have a high carbon and hydrogen content. The oil is preferably an edible oil and is preferably digestible by ruminants. Typically, plant-derived oils are extracted from plant seeds or fruits and are typically composed primarily of triglycerides. The term "vegetable oil" is a general term indicating that the oil is primarily or exclusively of plant origin and can include a mixture of one or more oils of plant origin or different origins.

[0183] As used herein, the term "contacting" includes mixing the produced bromoform with a bromoform stabilizing excipient to form a mixture of the produced bromoform and the bromoform stabilizing excipient, as well as holding the produced bromoform, and holding the composition of the produced bromoform and the bromoform stabilizing excipient at a time and temperature suitable for forming the composition.

[0184] The term contacting includes holding the produced bromoform and the bromoform stabilizing excipient at different suitable temperatures.

[0185] The temperature at which the produced bromoform is contacted with the bromoform stabilizing excipient is not limited, as long as the produced bromoform does not significantly evaporate / sublime from the bromoform stabilizing excipient and / or does not significantly decompose at the temperature at which heating is carried out.

[0186] The time for which the produced bromoform is contacted with the bromoform stabilizing excipient is not limited, as long as the produced bromoform does not significantly evaporate / sublime from the bromoform stabilizing excipient and / or does not significantly decompose during the contact time.

[0187] The inventors have demonstrated herein that the compositions described herein maintain the level of volatile produced bromoform over a long period of time.

[0188] For example, Example 7 demonstrates that a composition containing produced bromoform and corn oil as a bromoform stabilizing excipient maintains bromoform for 4 weeks upon storage at -20°C, 4°C, 25°C and 40°C.

[0189] Example 7 also demonstrates that a composition containing produced bromoform and corn oil as a bromoform stabilizing excipient maintains more than 46% of the starting bromoform concentration over 4 weeks under ambient conditions of 25°C and 40°C.

[0190] Example 7 demonstrates that a composition containing produced bromoform and molasses as a bromoform stabilizing excipient maintains bromoform for 4 weeks upon storage at -20°C, 4°C, 25°C and 40°C.

[0191] Example 7 demonstrates that a composition containing produced bromoform and molasses as a bromoform stabilizing excipient maintains bromoform for 4 weeks upon storage at -20°C and 4°C.

[0192] Example 7 also demonstrates that a composition containing the produced bromoform and molasses as a bromoform stabilizing excipient maintains more than 38% of the starting bromoform concentration over 4 weeks of storage at 25°C.

[0193] Example 7 also demonstrates that a composition containing the produced bromoform and molasses as a bromoform stabilizing excipient maintains more than 24% of the starting bromoform concentration over 4 weeks of storage at 40°C.

[0194] Surprisingly, Example 7 also demonstrates that a composition containing the produced bromoform and molasses as a bromoform stabilizing excipient maintains more than 58% of the starting bromoform concentration over 4 weeks under ambient conditions of 25°C.

[0195] Also surprisingly, Example 7 also demonstrates that a composition containing the produced bromoform and molasses as a bromoform stabilizing excipient maintains more than 62% of the starting bromoform concentration over 4 weeks under ambient conditions of 40°C.

[0196] Example 7 also demonstrates that a composition containing the produced bromoform and cyclodextrin as a bromoform stabilizing excipient maintains the starting bromoform concentration over 4 weeks of storage at -20°C, 4°C, 25°C, and 40°C.

[0197] Example 7 also demonstrates that a composition containing the produced bromoform and cyclodextrin as a bromoform stabilizing excipient maintains the starting bromoform concentration over 4 weeks under ambient conditions of 25°C and 40°C.

[0198] In one embodiment, the present invention provides a stabilized bromoform composition comprising the produced bromoform and a bromoform stabilizing excipient.

[0199] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at -20°C for one week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at -20°C for two weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at -20°C for three weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at -20°C for four weeks.

[0200] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 4°C for one week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 4°C for two weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 4°C for three weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 4°C for four weeks.

[0201] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 25°C for one week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 25°C for two weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 25°C for three weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 25°C for four weeks.

[0202] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 40°C for one week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 40°C for two weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 40°C for three weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage at 40°C for four weeks.

[0203] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after one week under ambient conditions at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage for two weeks under ambient conditions at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage for three weeks under ambient conditions at 25°C. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage for four weeks under ambient conditions at 25°C.

[0204] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after one week under ambient conditions at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage for two weeks under ambient conditions at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage for three weeks under ambient conditions at 40°C. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition is maintained after storage for four weeks under ambient conditions at 40°C.

[0205] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at -20°C for 1 week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at -20°C for 2 weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at -20°C for 3 weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at -20°C for 4 weeks.

[0206] In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 4°C for 1 week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 4°C for 2 weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 4°C for 3 weeks. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 4°C for 4 weeks.

[0207] In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 25 °C for one week. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 25 °C for two weeks. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 25 °C for three weeks. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 25 °C for four weeks.

[0208] In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 40 °C for one week. In one aspect, the present invention provides a composition as described herein, wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 40 °C for two weeks. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 40 °C for three weeks. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after storage at 40 °C for four weeks.

[0209] In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 1 week under ambient conditions at 25°C. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 2 weeks of storage under ambient conditions at 25°C. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 3 weeks of storage under ambient conditions at 25°C. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 4 weeks of storage under ambient conditions at 25°C.

[0210] In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 1 week under ambient conditions at 40°C. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 2 weeks of storage under ambient conditions at 40°C. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 3 weeks of storage under ambient conditions at 40°C. In one aspect, the present invention provides a composition wherein the level of bromoform produced in the composition does not decrease by more than 10, 20, 30, 40, 50, or 60% relative to the starting concentration of bromoform after 4 weeks of storage under ambient conditions at 40°C.

[0211] As shown in Example 7, the present invention provides a composition that releases bromoform as a function of time. In one embodiment, the present invention provides a sustained-release composition comprising manufactured bromoform and a bromoform stabilizing excipient.

[0212] Previous studies have demonstrated that manufactured bromoform formulated in DMSO significantly inhibits methane production at doses of 5 μM, 10 μM, and 25 μM, but at 1 μM, manufactured bromoform does not significantly inhibit methane production in in vitro fermentation. In contrast, the inventors have surprisingly demonstrated that a dose of 0.04 mg of manufactured bromoform per gram of feed OM inhibits methane production from rumen fermentation.

[0213] Importantly, Figure 9 demonstrates that even lower doses of manufactured bromoform can be used in the methods and compositions described herein and can inhibit rumen fermentation. For example, 0.01 mg of manufactured bromoform per gram of feed substrate can be used in the compositions and methods of the present invention to inhibit methane production from rumen fermentation.

[0214] In one embodiment, the composition according to the present invention is administered in an amount based on the intake (e.g., body weight) of the actual individual animal. (g / kg OM or g / kg DM intake). In contrast to Asparagopsis, a natural product with a variable bromoform content, the present invention enables compositions with a defined manufactured bromoform content.

[0215] Thus, in one embodiment, the present invention provides a method as described herein, 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, 0.07 or 0.08 mg of produced bromoform per gram of feed provided to a ruminant. In a preferred embodiment, the present invention provides a method as described herein, wherein the composition is administered at a dose of at least 0.03, 0.04, or 0.05 mg of produced bromoform per gram of feed provided to a ruminant.

[0216] In a preferred embodiment, the present invention provides a method as described herein, wherein the composition is administered at a dose of at least 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of produced bromoform per gram of organic matter provided to a ruminant. In another embodiment, where the composition according to the invention is administered in an amount based on dry matter intake, the composition is administered at a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of produced bromoform per about 1.19 grams of dry matter provided to a ruminant.

[0217] In another embodiment, the present invention provides a method as described herein, wherein the composition is made 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, 0.07 or 0.08 mg of bromoform per gram of organic matter provided to a ruminant. In another embodiment, where the composition according to the invention is made available in an amount based on dry matter intake, the composition is administered at a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08 mg of produced bromoform per about 1.19 grams of dry matter provided to a ruminant.

[0218] For example, if a ruminant consumes about 2.5 - 3% of its body weight in feed per day, a 400 kg ruminant can consume 10 - 12 kg of feed per day.

[0219] As described above, in a preferred embodiment of the present invention, the effective amount of the produced 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 the produced 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 the produced bromoform per about 1.19 grams of dry matter provided to the ruminant per day.

[0220] Thus, if a 400 kg ruminant consumes about 10 kg of organic matter per day, the effective amount of the produced bromoform in the composition 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 described herein per day. These dosages are equivalent to 0.0005, 0.00075, 0.001, 0.00125, 0.0015, 0.00175 or 0.002 g of the produced bromoform / kg body weight / day. Appropriate dosages can be calculated as described herein.

[0221] The effective amount can be administered to the ruminant in one or more dosages.

[0222] The effective amount can also be administered to the ruminant in one or more dosages per day.

[0223] In another preferred embodiment, there is provided a method as previously defined herein, wherein the dosage of the produced bromoform is in the range of 0.00025 to 0.0065 g / kg body weight / day, more preferably in the range of 0.0005 to 0.0065 g / kg body weight / day, and most preferably in the range of 0.001 to 0.00275 g / kg body weight / day.

[0224] The dosage as defined herein as the amount per kilogram of body weight per day relates to the average amount of bromoform produced during a given treatment period (e.g., during a treatment of one week or one month). Thus, the compositions described herein may be provided or administered daily, every other day, every two days, etc., without departing from the scope of the invention. However, preferably, the method includes daily administration at the defined dosage of the composition as defined herein. Even more preferably, the composition is provided or administered during animal feeding, each time the animal is fed, in an amount that provides the above daily dosage amount.

[0225] The method of the invention may include providing or administering the composition according to the described dosing schedule for a period of at least 5, 10, 25, 50, 100, 250 or 350 days.

[0226] In animal systems that include forage-based systems, providing the system with supplements that enable the administration of an effective amount to the animal can be problematic when using Asparagopsis biomass or Asparagopsis-derived products. For example, if only a small percentage of the forage-based animal's feed intake is the feed supplement, the dosage of bromoform in the supplement needs to be high to provide an effective dosage. A high content of Asparagopsis biomass or Asparagopsis-derived products in the feed supplement may make the supplement unpalatable to the animal and render the dosage ingested ineffective. The present invention enables the preparation of feed supplements that contain the produced bromoform at a level that enables delivery of an effective dosage in animal systems such as forage-based / grazing systems.

[0227] The ability to prepare a feed adjuvant containing a high concentration of the produced bromoform enables the supply of an effective dosage in small amounts (e.g., feed amounts) to the animal, and also enables the supply of an effective amount of the composition described herein when the animal is at the feeder or during milking.

[0228] Compared to Asparagopsis-based or Asparagopsis-derived oil products, the ability to prepare a feed supplement that contains bromoform at a higher level and enables delivery of an effective dose to animals in a smaller amount makes it possible to avoid high levels of oil in animal feed. The use of high levels of oil in animal feed can partially liquefy the oil-containing feed, make it unpalatable, soiled, and / or cause problems in the feeder.

[0229] In another aspect, the present invention provides a composition for reducing total gas production and / or methane production and / or improving the growth performance of ruminants, the composition comprising produced bromoform and a bromoform stabilizing excipient.

[0230] In another aspect, the present invention provides a composition for use in reducing total gas production and / or methane production and / or improving the growth performance of ruminants, the composition comprising produced bromoform and a bromoform stabilizing excipient.

[0231] In one embodiment of the composition for use, the bromoform stabilizing excipient is an edible non-polar substance.

[0232] In one embodiment of the composition for use, the edible non-polar substance is an edible oil.

[0233] In one embodiment of the composition for use, 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, corozo oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0234] In one embodiment of the composition for use, the composition is formulated to provide to ruminants at a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of bromoform per gram of organic matter provided to the ruminant.

[0235] In one embodiment of the composition for use, the composition is in solid, semi-solid or liquid form.

[0236] In another aspect, the present invention provides a feed supplement when used to reduce total gas production and / or methane production in ruminants, said supplement comprising an effective amount of a composition comprising manufactured bromoform and a bromoform stabilizing excipient.

[0237] As used herein, the term "animal feed supplement" refers to a concentrated additive premix containing active ingredients, which premix or supplement may be added to an animal's feed or ration to form a supplementary feed according to the present invention. The terms "animal feed premix", "animal feed supplement", and "animal feed additive" are generally considered to have similar or identical meanings and are generally considered interchangeable. Typically, the animal feed supplements of the present invention are in the form of powders or compressed or granulated solids. In another embodiment, the animal feed supplements of the present invention are in the form of mineral loose licks, wet licks, dry licks, pellet agents, water suspensions, liquid supplements (e.g., molasses blends) or lick blocks. In practice, livestock may typically be fed the animal feed supplement by adding it directly to the feed, for example, as a so-called top-dress, or it may be used in the preparation or manufacture of products such as formulated animal feeds or lick blocks, as described in more detail below. The present invention is not particularly limited in this regard. The supplements according to the present invention are typically fed to animals in amounts in the range of 16 to 3000 g or more / animal / day.

[0238] The animal feed supplements of the present invention may contain any additional ingredients without departing from the scope of the present invention. It may typically contain well-known excipients necessary to prepare the desired product form, and may also contain additional additives for the purpose of improving the quality of the feed and / or the ability of the animals consuming the supplement. Suitable examples of such excipients include carriers or fillers such as lactose, sucrose, mannitol, starch, crystalline cellulose, sodium bicarbonate, sodium chloride, and binders such as gum arabic, tragacanth gum, sodium alginate, starch, PVP and cellulose derivatives. Examples of feed additives known to those skilled in the art include vitamins, amino acids and trace elements, digestibility improvers and intestinal flora stabilizers.

[0239] As used herein, the term "formulated animal feed composition" means a composition that is suitable for use as animal feed and is blended from various natural or non-natural bases or raw materials and / or additives. Thus, in particular, the term "formulated" is used herein to distinguish the animal feed compositions of the present invention from any naturally occurring raw materials. These blends or compound feeds are formulated according to the specific requirements of the target animal. The main components typically used in commercially prepared compound feeds include grains such as wheat bran, rice bran, cornmeal, barley, wheat, rye, and oats, soybean meal, alfalfa meal, cottonseed meal, wheat flour, and the like. Commercially available compound feeds typically contain at least 15% crude protein and at least 70% digestible total nutrients, but the present invention is not particularly limited in this regard.

[0240] Liquid, solid, and semi-solid formulated animal feed compositions are encompassed within the scope of the present invention, with solid and semi-solid forms being particularly preferred. These compositions are typically manufactured as meal type, pellet, or crumble. In practice, livestock can typically be fed a combination of a compound feed such as those of the present invention and silage or hay, etc. Typically, the formulated animal feed is fed in an amount within the range of 0.3 to 10 kg / animal / day. Determining the appropriate amounts of these components to be included in the formulated animal feed, taking into account the type of animal and the environment in which it is kept, is within the skill of a trained professional.

[0241] The formulated animal feed composition of the present invention may include any additional feed additives typically used in the art. As is known to those skilled in the art, the term "feed additive" in this context refers to products used in animal nutrition for the purpose of improving the quality of feed and food of animal origin, or for improving the capabilities of animals, for example, to enhance the 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, especially sensory additives such as flavors and colorants, (additional) nutritional additives such as vitamins, amino acids and trace elements, and (additional) animal production technical additives such as digestion promoters and intestinal flora stabilizers. As will be apparent to those skilled in the art, the formulated animal feed composition of the present invention can include any additional components or additives without departing from the scope of the present invention.

[0242] In a further aspect, the present invention provides a lickstone or lick block comprising a supplement of the present invention. As is known to those skilled in the art, such lickstones or blocks are particularly convenient for providing mineral supplements (as well as proteins and carbohydrates) to ruminants grazing on either or both natural and cultivated pastures. Such lick blocks or lickstones according to the present invention typically include, in addition to the composition of the present invention, various types of binders, such as cement, gypsum, lime, calcium phosphate, carbonates, and / or gelatin, and optionally further additives such as vitamins, trace elements, inorganic salts, sensory additives, and the like.

[0243] In one embodiment of the feed supplement for use, 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 of manufactured bromoform per gram of organic matter provided to ruminants.

[0244] In another embodiment of the feed supplement for use, 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 of manufactured bromoform per gram of feed substrate provided to ruminants.

[0245] In one embodiment of the feed supplement for use, the feed supplement comprises a feed additive.

[0246] In one embodiment of the feed supplement for use, the ruminant is selected from members of the Ruminantia suborder and the Tylopoda suborder.

[0247] In one embodiment of the feed supplement for use, the ruminant is a cow or a sheep.

[0248] In one embodiment of the feed supplement for use, the bromoform stabilizing excipient is an edible nonpolar substance.

[0249] In one embodiment of the feed supplement for use, the edible nonpolar substance is an edible oil.

[0250] In one embodiment of the feed supplement for use, 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, corza oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flaxseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0251] In one embodiment of the feed supplement for use, the composition is in solid, semi-solid or liquid form.

[0252] A method of preparing a methane-reducing ruminant feed is described herein.

[0253] Thus, in one aspect, the present invention provides a method of producing a methane-reducing ruminant feed, comprising mixing a ruminant feed with a feed supplement comprising an effective amount of a composition comprising produced bromoform and a bromoform-stabilizing excipient.

[0254] In one embodiment of the method of producing a methane-reducing ruminant feed, the animal feed comprises a dose of at least 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06 or 0.08 mg of bromoform per gram of organic matter of the ruminant feed.

[0255] In another aspect, the present invention provides an animal feed when used to reduce total gas production and / or methane production in ruminants, said supplement comprising an effective amount of a composition comprising produced bromoform and a bromoform-stabilizing excipient and a ruminant feed.

[0256] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, 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 of bromoform per gram of organic matter of the ruminant feed.

[0257] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, the ruminants are selected from members of the Ruminantia suborder and the Tylopoda suborder.

[0258] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, the ruminant is a cow or a sheep.

[0259] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, the bromoform stabilizing excipient is an edible non-polar substance.

[0260] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, the edible non-polar substance is an edible oil.

[0261] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, 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, corza oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flaxseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

[0262] In one embodiment of the animal feed when used to reduce total gas production and / or methane production in ruminants, the composition is in solid, semi-solid or liquid form.

[0263] Here, the present invention will be further described by examples, which are meant to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

[0264] [Examples] [Example 1: Materials and Methods] Preparation of Feed Substrates and Oil Products Rhodes grass dried hay was used as the primary feedstock, first dried and ground to 1 mm, and then supplied to each container at 1.25 g DM (1.0 g OM).

[0265] The seaweed Asparagopsis was prepared by FutureFeed Pty Ltd, Townsville, Queensland, Australia, supplied from Middle Reef at Magnetic Island, Queensland, Australia. Approximately 20 kg of freshly harvested Asparagopsis was first rinsed with seawater for 2 minutes and spin-dried 3 times [1 - 2 minutes each], then added to 20 L of refined canola oil for 20 days. Additional collections of fresh Asparagopsis were collected from the same area and the process repeated as above using the same 20 L of refined canola oil to achieve a final CHBr 3 concentration of 3.60 mg / g (Table 1).

[0266] The bromoform produced in oil (synthetic CHBr 3 oil; "Syn - Oil") was produced by FutureFeed Pty Ltd using the same refined canola oil used to make Asp - Oil. Synthetic CHBr 3 (Sigma - Aldrich, product number: 36972) was dissolved and homogenized into the canola oil by repeated inversion and stabilized overnight before analysis. The oil was then volumetrically diluted to achieve equivalent CHBr 3 concentrations between Asp - Oil and Syn - Oil (Table 1).

[0267] Both in vivo and in vitro intestinal CH 4Previously proven to reduce emissions, freeze-dried Asparagopsis subsamples (Roque et al., 2019; Kinley et al., 2020; Roque et al., 2021) were added as a positive control group during Experiment 2.

[0268] The bromoform concentration of Asp-Oil was 3.60 ± 0.05 mg / g DW, Syn-Oil was 3.69 ± 0.05 mg / g DW, and freeze-dried Asparagopsis was 7.7 mg / g DW. The bromoform concentration was confirmed using an improved analytical protocol as previously described by Paul et al. (2006). [Table 1]

[0269] Donor Animals and in vitro Preparations The donor animals were maintained at the Lansdown Research Station (coordinates; 19°39’27.000”S, 146°50’04.60”E), near Townsville, Queensland, Australia, maintained by the Commonwealth Scientific and Industrial Research Organization (CSIRO), and approved by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) animal ethics committee (Ethical Clearance Certificate 2018 - 37) in accordance with current guidelines (NHMRC 2013). Rumen fluid (RF) was collected from four fistulated Brahman bullocks fitted with 10 cm Bar Diamond (Parma, OH, USA) rumen cannulas. All four castrated bulls were allowed to graze, supplemented freely with Rhodes grass hay before RF collection, and it was withdrawn 2 hours after morning feeding and then placed in a 1 L pre-warmed insulated flask and returned to the laboratory. All RF was pooled, filtered through a 0.5 mm sieve, and then combined with artificial saliva buffer (Goering and van Soest 1970) in a ratio of 1 part RF and 4 parts buffer. Approximately 125 mL of the RF buffer mixture and 1.25 g DM of substrate were assigned to 250 mL Ankom incubation bottles, the headspace was purged with N 2 and then sealed with an Ankom RF1 gas production module (Macedon, New York, United States). The incubation bottles were 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.

[0270] Sampling and Analysis Total Gas, CH 4 , and Hydrogen Production Total gas production (TGP), CH 4, hydrogen and carbon dioxide production were determined using the Ankom RF gas production technology (Macedon, NY, USA). Briefly, the Ankom RF module was set to a maximum pressure of 3 psi and vented for 250 milliseconds if exceeded. The live interval (LI) was set to 60 seconds, gas production was monitored, and each measurement was corrected for ambient pressure changes via the surrounding Ankom RF monitor. The recording interval (RI) was set to 20 minutes, and thus, the LI cumulative pressure change was recorded at each RI as a 20-minute contribution to the cumulative pressure change over the fermentation period (24, 48, or 72 hours). The total cumulative pressure change was converted to TGP using the laws of natural gas and corrected for the absolute volume of each individual fermentation bottle. Assuming a constant homogeneity of the bottle headspace, in vitro CH 4 production was determined by analysis of the headspace gas for TGP. At the end of each incubation period, headspace gas samples from the individual fermentation bottles were collected into 10 mL Labco Exetainer vacuum vials (Lampeter, Great Britain) through the Ankom RF module vent tube. 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 column (2 m·1 mm· micropack) with a flame ionization detector (FID). The column temperature was set to 150 °C, the injector to 240 °C, and the FID to 380 °C. Ultra-high purity N 2 was the carrier gas at 25 mL / min and the total injection volume was 250 μL.

[0271] Apparent Digestibility After sacrificing the fermentation bottles [at either 24, 48, or 72 h], they were cooled in a -20 °C freezer to stop bacterial activity. Then, the rumen fluid was used with a 0.5 cm layer of sand filtration aid in a 50 mL Gooch sintered disk glass filtration crucible, porosity 1, (GLASSCO; Haryana, India). Next, the crucible containing the digested rumen fluid residue was oven dried at 105 °C until a constant weight was achieved 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).

[0272] [Example 2: Compositions containing bromoform and bromoform-stabilizing excipients reduce methane production in rumen fermentation.] in vitro Setup and Experimental Design Four control treatments with canola oil content varied to match levels 1, 2, 4, and 6 of Asp-oil and Syn-Oil products were used to test Asparagopsis oil (“Asp-oil”) at four inclusion levels [0.08 mg, 0.11 mg, 0.15 mg, and 0.18 mg] and synthetic CHBr 3 oil (Syn-oil) at six inclusion levels [0.08 mg, 0.11 mg, 0.15 mg, 0.18 mg, 0.22 mg, and 0.26 mg per gram of organic matter (OM) of the feed]. Unsaturated oils can serve as a mitigating feed component (Grainger and Mayer, 2011), and thus, it was necessary to test various inclusions for the oils. All experimental treatments [14 in total] were tested in duplicate over two fermentation time points: 24 h and 48 h during the in vitro incubation period:. Then, the data collected for each treatment, fermentation time point, and incubation were combined and analyzed. Since IVDDM was not measured in Example 2 which was designed to demonstrate only CH 4 production, CH 4 production was calculated on a feed digestion basis. 4Note that production could not be reported here. However, all fermentations received the same Rhodes grass and canola oil feed substrates at equivalent levels. Feed digestion-based reporting has little impact on the feeding-based profile of Figure 1.

[0273] Results Figure 1 demonstrates that both compositions containing Asp-Oil and bromoform and bromoform-stabilized excipients (Syn-Oil) are highly anti-methanogenic at equivalent levels in vitro in terms of CHBr 3 delivery.

[0274] As shown in Figure 1, the emission of hydrogen (H 2 ) is not normally present. This is because H2 is taken up by methanogens to produce CH 4 , and the chemical formula of CH 4 contains four hydrogen (H + ) atoms, indicating that it functions as a sink for hydrogen. Consistent with CH 4 mitigation, the H 2 concentration increases across all CHBr 4 content levels for both Asp-Oil and Syn-Oil products, except for Syn-Oil 0.08 mg at 48-hour fermentation timepoints due to the decrease in CH 3 mitigation effectiveness at 48 hours.

[0275] Total gas production (TGP) is also included in Figure 1 to demonstrate the maintenance of microbial activity within all treatments, including those with CHBr 3 content. A slight decrease in TGP may be expected due to the proportion of CH 4 that constitutes TGP in the control treatment.

[0276] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions containing bromoform and bromoform-stabilizing excipients can reduce methane production from rumen fermentation in the absence of other secondary metabolites (and other components) of Asparagopsis.

[0277] [Example 3: Dose-dependent response of oil products on gas production and digestibility over a 72-hour period.] in vitro Setup and Experimental Design Two oil products [substrate + Asp-Oil or Syn-Oil] were used along with one negative control (C1-) [substrate + 0.02 g of canola oil], one positive control (C2+) [substrate + 0.01 g of freeze-dried Asparagopsis + 0.02 g of canola oil], and one blank [no substrate or treatment, rumen fluid only].

[0278] Three levels of Asp-Oil or Syn-Oil inclusion were tested based on CHBr 3 content and weight of the material and designated as low [0.04 mg], medium [0.07 mg], and high [0.11 mg] inclusion levels. All experimental treatments [nine in total] were tested in duplicate over three fermentation time points: 24, 48, and 72 hours during one in vitro incubation period. The data collected for each treatment, fermentation time point, and incubation were then combined and analyzed for time-series results.

[0279] Results and Discussion The design of Example 3 was based on the Asp-Oil and Syn-Oil inclusion levels informed by Example 2 to provide the minimum effective inclusion level that consistently reduces CH 4 over 72 hours. Based on the results of Example 2, the inventors hypothesized that an inclusion level of 0.04 mg of CHBr 3 for both Asp-Oil and Syn-Oil inhibits methane production but to a lesser extent than higher doses of bromoform.

[0280] CH shown in Figure 2A 4 The results show that a content level of 0.07 mg / g exhibited a stronger CH 4 mitigation ability than predicted based on the data of Example 2. The 0.11 mg content in both Asp-Oil and Syn-Oil completely suppressed CH 4 production during the study period.

[0281] This data indicates that a composition containing bromoform and a bromoform-stabilizing excipient can reduce methane production in the absence of other secondary metabolites (and other components) of Asparagopsis.

[0282] After 48 hours, the Asparagopsis-derived composition (Asp-Oil) obtained by extracting Asparagopsis biomass with a bromoform-stabilizing excipient contains other anti-methanogenic secondary metabolites (and other components) of Asparagopsis and was able to significantly reduce methane production compared to Syn-Oil which does not contain other anti-methane-producing secondary metabolites (and other components) of Asparagopsis.

[0283] In addition to the oil treatment, a positive control of freeze-dried (FD) Asparagopsis was included in this study with a CHBr content of 0.07 mg and was made to act in the same way as the medium (0.07 mg) and high (0.11 mg) oil content levels. 3 and high (0.11 mg) oil content levels.

[0284] Figure 2 also demonstrates that hydrogen increases significantly along with the down-regulation of CH 4 production and then consistently decreases when CH 4 production increases again. Total gas production (TGP) is the CH of TGP 4Decreases generally in response to the reduction of ingredients, and thus, TGP is demonstrated to be equally 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 is no negative impact on diet digestibility due to the inclusion of anti-methane producing products and that it appears not to be affected across treatment groups (lower right, Figure 2). The maintenance of TGP and IVDMD is a strong indicator that the rumen microbial activity is CH 4 A strong indicator of being stable including the relaxation oil product.

[0285] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions containing bromoform and bromoform stabilizing excipients can reduce methane production from rumen fermentation in the absence of other secondary metabolites (and other ingredients) of Asparagopsis.

[0286] [Example 4: Dose-dependent response of oil products to in vitro gas production and digestibility over 72 hours.]

[0287] To compare the mitigation of enteric methane and its effect on enteric fermentation (e.g., in vitro digested dry matter (IVDDM) and rumen volatile fatty acid production (VFA) as fermentation markers), the inventors investigated the reaction in in vitro fermentation.

[0288] Methods Two oil products [substrate + canola oil or Asp-Oil] were used with one negative control (CON) [substrate + 0.02 g of canola oil], one positive control (FD-Asp) [substrate + 0.05 mg of CHBr 3 / g substrate freeze-dried Asparagopsis] and one blank [no substrate or treatment, only rumen fluid]. Three levels of canola oil and Asp-Oil inclusion were used with CHBr 3Tested based on the content and weight of the materials, and referred to as the content levels of 0.03 [mg / g substrate], 0.05 [mg / g substrate], and 0.08 [mg / g substrate]. All experimental treatments [total 9] were tested in duplicate over three fermentation periods: 24, 48, and 72 hours, and these were repeated during three in vitro incubation periods. Then, the data collected for each treatment, fermentation period, and incubation were combined and analyzed for time-series results.

[0289] The Asparagopsis seaweed was prepared by FutureFeed Pty Ltd, Townsville, Queensland, Australia (which was supplied from the coast of Magnetic Island, Queensland, Australia). Approximately 20 kg of freshly harvested Asparagopsis was first rinsed with seawater for 2 minutes, spin-dried 3 times [1 - 2 minutes each time], and then added to 20 L of refined canola oil for 20 days. After 3 weeks of immersion, the Asparagopsis biomass was removed, and then an additional 20 kg of fresh Asparagopsis collected from the same area was added to the canola oil, and this process was repeated as above to achieve a final CHBr 3 concentration [3.03 mg / g ± 0.03].

[0290] The synthesized CHBr stabilized in amylene with a purity of 98% 3 (Sigma - Aldrich, product number: 36972) was dissolved, homogenized in canola oil by repeated inversion, and stabilized overnight before analysis. Then, this oil was volumetrically diluted to achieve a CHBr 3 concentration equivalent to that of the Asparagopsis oil.

[0291] Total gas production (TGP), CH 4 , hydrogen (H 2 ), and carbon dioxide (CO 2)Production was determined using the Ankom RF gas production technology (Macedon, NY, USA) as described above. Briefly, the total cumulative pressure change was converted to TGP using the natural gas law and corrected for the absolute volume of each fermentation bottle. At the end of each incubation period, headspace gas samples from individual fermentation bottles were collected into 10 mL Labco Exetainer vacuum vials (Lampeter, Great Britain) through the Ankom RF module vent tube, and CH 4 and CO 2 production, and H 2 production were determined assuming a constant homogeneity of the bottle headspace.

[0292] Furthermore, IVDDM and VFA were determined. Briefly, after sacrificing the fermentation bottles [at either 24, 48, or 72 hours], they were cooled in a -20 °C freezer to stop bacterial activity, and then the rumen fluid was vacuum filtered through a sintered glass disk filtration crucible with a 0.5 cm layer of sand filter aid. Next, the crucible containing the digested rumen fluid residue was oven dried at 105 °C until a constant weight was achieved, and then subtracted from the total DM added to the bottle to determine IVDDM. To determine the production of total VFA (tVFA) as well as acetate, propionate, and butyrate, 1.5 mL samples of rumen fluid were collected from the 72-hour fermentation bottles.

[0293] Results The design of Example 4 was based on the inclusion levels of Asp-Oil and canola oil informed by in vitro Examples 1 and 2 to provide a range of effective inclusion levels (i.e., dose-response curve) that consistently decreased CH 4 over a 72-hour fermentation period.

[0294] CH 4The results are shown in Figure 3, which demonstrates that the Asparagopsis biomass extracted in oil ("Asp-Oil"), and bromoform produced in oil as a stabilizing excipient ("Canola Oil"), functioned in a similar manner.

[0295] The 0.05 inclusion level showed a stronger CH mitigation ability than expected for Asp-Oil, canola oil, and freeze-dried Asparagopsis biomass ("FD-Asp") over the 24-hour fermentation period. 4 However, as expected, a small universal decrease in effectiveness appeared over the 48- and 72-hour fermentations. Additionally, over the 48- and 72-hour periods, FD-Asp appeared to mitigate CH more effectively than Asp-Oil and canola oil at the same inclusion level of 0.05 [mg / g substrate]. 4 As predicted based on Examples 1-3, the 0.08 inclusion for both Asp-Oil and canola oil completely inhibited CH production throughout the study period. 4 This indicates the stability of the mitigation efficacy when CHBr is provided within the optimal range. 3 As demonstrated in previous in vitro and in vivo studies, Figure 4 shows that H increases with the downregulation of CH production and then consistently decreases when CH production resumes (Figure 4). No adverse effects on fermentation were observed for IVDDM and tVFA production (Figures 5A and B).

[0296] This is due to the inclusion of Asp-Oil or canola oil and appears to be unaffected across the inclusion groups. In addition, the decrease in acetate and the increase in the production of propionate and butyrate occur at equal rates between Asp-Oil and canola oil at their respective inclusion rates, providing evidence that Asp-Oil and canola oil act similarly in the in vitro fermentation setting. 2 4 4 4 4 4

[0297] These results demonstrate that Asp-Oil inhibits methane production from rumen fermentation and that compositions containing bromoform and bromoform-stabilizing excipients can reduce methane production from rumen fermentation in the absence of other secondary metabolites (and other components) of Asparagopsis.

[0298] [Example 5: Compositions containing manufactured synthetic bromoform stabilized in corn oil, molasses, cyclodextrin, and water inhibit methane production in enteric fermentation.]

[0299] To examine the ability of manufactured bromoform stabilized in different excipients to inhibit methane production in enteric fermentation, manufactured CHBr 3 was stabilized in edible oil (corn oil), solid carbohydrate (cyclodextrin), liquid form (water), or semi-liquid carbohydrate (molasses).

[0300] Methods Four CHBr 3 stabilizing excipients [substrate + corn oil, molasses, cyclodextrin, or water] were used with one negative control ("CON") [substrate only] and one blank [no substrate or treatment, rumen fluid only]. Three inclusion levels of each excipient were tested based on the CHBr 3 content and the weight of the material and designated as 0.01 [mg / g substrate], 0.04 [mg / g substrate], and 0.07 [mg / g substrate] inclusion levels. The experiment consisted of three in vitro incubation replicates (n = 3) for each of the CHBr 3 product experimental treatments [total 14], which were tested in duplicate within each replicate over three fermentation periods: 24, 48, and 72 hours. The data collected for each treatment, fermentation period, and incubation were then combined and analyzed for time series results.

[0301] Manufactured synthetic CHBr of 98% purity stabilized with 2-methyl-2-butene (Sigma-Aldrich, product number: 241032)3 was used for the preparation of four excipients. Corn oil, molasses and water were prepared following the same protocol as the canola oil in Example 4. Briefly, CHBr 3 was dissolved, homogenized in either retail corn oil, molasses, or deionized water, homogenized by repeating inversion, left to stabilize overnight, and then a concentration of 3.0 ± 0.10 mg / g was achieved. Cyclodextrin was prepared as previously described (Patent WO96 / 14062, PCT / AU95 / 00733). Briefly, CHBr 3 was added to an aqueous solution of α-cyclodextrin (Sigma-Aldrich, product number: 779008) and mixed overnight. The solid was then separated, washed, and then dried. The concentration of CHBr 3 in the dry powder was measured and diluted with additional α-cyclodextrin to adjust the CHBr 3 concentration to 3.0 ± 0.10 mg / g. TGP (Total Gas Production), CH 4 , H 2 and CO 2 production, IVDDM (in vitro digestible dry matter), and VFA (volatile fatty acids) were measured using the protocol described in Example 4.

[0302] Results The design of Example 5 used the content levels informed by an initial exploratory experiment (data not shown, experimental design similar to Example 1) to provide the range of effective content levels (i.e., dose-response curve) for each CHBr 3 stabilizing excipient over a 72-hour fermentation period.

[0303] The results of CH 4 shown in Figure 6 indicate that the bromoform produced can be stabilized in edible oils other than canola oil (corn oil), solid excipients (cyclodextrin), liquid excipients (water), or semi-liquid excipients (molasses).

[0304] The levels of efficacy from molasses and the aqueous excipients, at each concentration level and over fermentation periods of 24, 48, and 72 hours, were similar to those of corn oil and cyclodextrin for CH 4 showed a decrease, which was unexpected. This is because CHBr 3 does not have strong retention in water-based compounds (see Example 6), and thus it was particularly interesting as it was predicted that there would be a decrease in efficacy over the course of four incubation periods.

[0305] After 24 hours of fermentation, H production appeared to increase at concentrations of 0.04 and 0.07 for all excipients, but due to the large variability in H 2 measurements, no statistical significance was seen in the results (Figure 7). During the 48-hour period, cyclodextrin and water significantly increased H 2 at concentrations of 0.04 and 0.07, while molasses and corn oil showed a significant increase only at a concentration of 0.07. During the 72-hour period, cyclodextrin, water, and molasses significantly increased H 2 at a concentration of 0.07. 2 Corn oil at a concentration of 0.07 showed a potential decrease in IVDDM compared to the control during 24 and 48 hours of fermentation, but no difference was seen during the 72-hour period (Figure 8A). The other excipients did not show an effect on IVDDM at any concentration or period. The tVFA production at 72 hours of fermentation (Figure 8B) appears to be affected by the concentration of the four excipients, with the greatest decrease in tVFA shown at the highest concentration of 0.07 [mg / g substrate]. Without being bound by theory, the inventors propose that this trend may reflect a decrease in acetate production. Acetic acid is usually the most abundant VFA in the rumen, but CH

[0306] 4 ​When it is suppressed, it is down-regulated. The decrease in acetate and the increase in the production of propionate and butyrate (Figure 8C) indicate that all excipients individually have a significant impact on the production of individual VFAs at specific contents, and cyclodextrin has the most influence. As a result, the same trend applies to the A:P ratio (Figure 8D). This is likely due to the high methane reduction effect of cyclodextrin at each content compared to corn oil, molasses, and water.

[0307] Figure 9 shows different CHBr 3 Summary of the data from Examples 4 and 5 regarding various different contents of stabilized excipients (gray bars) and comparative Asparagopsis products (black bars), and over 24, 48, and 72 hours of fermentation. The aggregation of this data is particularly interesting, with a content of 0.03 - 0.05 mg of CHBr 3 [30 - 50 mg / kg] per gram of feed substrate shown to result in complete suppression of intestinal CH 4 .

[0308] These results demonstrate that Asp - Oil inhibits methane production from rumen fermentation, and that compositions containing bromoform and solid, liquid, and semi - solid bromoform - stabilized excipients can reduce methane production from rumen fermentation in the absence of other secondary metabolites (and other components) of Asparagopsis.

[0309] These results also demonstrate that the compositions and methods of the present invention can be used to inhibit methane production at bromoform dosages significantly lower than previously demonstrated.

[0310] [Example 6: Asparagopsis extracted in oil ("Asp - Oil") and manufactured bromoform stabilized in oil ("Canola Oil") suppress methane production in vivo.] To examine the ability of the stabilized compositions to suppress methane production in vivo, the inventors provided animals with a feed supplement containing Asparagopsis biomass extracted in oil ("Asp-Oil") or bromoform produced and stabilized in canola oil ("Canola Oil").

[0311] Methods A total of 60 early lactation dairy cows were randomly assigned to one of 10 treatment groups: Asp-Oil [at inclusion levels 0, 1, 2, 3, or 4] or Canola Oil [at inclusion levels 0, 1, 2, 3, or 4]. Inclusion levels were initially set to achieve 6, 12, 18, and 20 mg CHBr 3 / kg DMI (per day) based on previous experimental studies using Asp-Oil in lactating dairy cows. Asp-Oil and Canola Oil were mixed into the cows' grain supplement and provided as a pulse dose twice a day at milking, measured separately from the major daily feed intake which occurs after milking. The initial introduction of Asp-Oil and Canola Oil was done gradually over a 15-day adaptation period, increasing the respective oil in the feed supplement stepwise every two days. This standard precaution minimizes the potential for rumen upset and feed refusal. After the adaptation period, the cows received their respective diets and treatments for an additional 21 days, after which methane (CH 6 ) measurements were taken using sulfur hexafluoride (SF 4 ) measurement technology. Briefly, an inert permeation tube is inserted into the rumen and a standardized SF 6 tracer is released steadily and consistently. Dilution of the tracer by background and rumen and respiratory gases allows for accurate partitioning of the gas components. Representative respiratory and belch gases are collected in a 24-hour sampling set for 5 consecutive days. Continuous sampling of individual cows during collection is achieved using a vacuum canister that draws gas through a sampling tube from a sampling point on the cow's nostril. Background gas is collected using a second canister that draws gas from the cow's flank.

[0312] Results Figure 10 shows the CH reduction results before statistical analysis, 4 compared with the control (without CHBr). 3 The reduction in CH production [g CH / day] was demonstrated up to 32% for canola oil and up to 38% for Asp - Oil at high CHBr levels [20 mg CHBr / kg DMI]. When CH was standardized based on dry matter intake (DMI) per kg (CH yield), canola oil resulted in a maximum 28% decrease and Asp - Oil resulted in a 36% decrease. 4 [CH production] 4 [g / day] 3 [The high content level of CHBr [20 mg CHBr / kg DMI]] 3 [was demonstrated up to 32% for canola oil and up to 38% for Asp - Oil. When CH was standardized based on dry matter intake (DMI) per kg (CH yield), canola oil resulted in a maximum 28% decrease and Asp - Oil resulted in a 36% decrease.] 4 [When CH was standardized based on dry matter intake (DMI) per kg (CH yield)] 4 [yield], canola oil resulted in a maximum 28% decrease and Asp - Oil resulted in a 36% decrease.

[0313] [CH] 4 [The range of CH reduction was demonstrated in vitro 24 hours after fermentation and corresponded to the CH reduction and respective content rates, as shown in Figure 11.] 4 [The range of CH reduction was demonstrated in vitro 24 hours after fermentation and corresponded to the CH reduction and respective content rates, as shown in Figure 11.]

[0314] [These results demonstrate that a composition containing manufactured bromoform and a stabilizing excipient that inhibits methane production from fermentation in vitro can inhibit methane production in vivo. This is in contrast to the study by Stefenoni et al. which showed that manufactured bromoform cannot inhibit methane production.]

[0315] [These results also demonstrate that Asp - Oil inhibits methane production from rumen fermentation and that a composition containing bromoform and a bromoform - stabilizing excipient can similarly reduce methane production in vivo in the absence of other anti - methane - producing metabolites (and other components) of Asparagopsis.]

[0316] [Example 7: Shelf life / stability of a composition containing manufactured bromoform and a bromoform - stabilizing excipient.] [The manufactured synthetic bromoform (CHBr) in the stabilized composition] 3To examine the retention of [[ID=]], the bromoform levels in the following excipients: corn oil, molasses, and cyclodextrin were monitored over 4 weeks under different storage temperatures and air exposure conditions.

[0317] Methods The Asparagopsis products (FD-Asp and Asp-Oil) were investigated for their ability to retain CHBr 3 content over a range of temperatures, exposure to UV illumination, and exposure to open air, and this served as a framework for testing the shelf-life stability of synthetically produced CHBr 3 in the present study. The preparation of the three excipients [corn oil, molasses, and cyclodextrin] is described in Example 5. The starting CHBr 3 concentration of each excipient ranged from 2.5 - 3.0 mg / g (mg CHBr 3 / g excipient).

[0318] Individual excipient products were stored sealed at four gradient temperatures [-20 °C, 4 °C, 25 °C, or 40 °C], sealed at 25 °C or 40 °C, or exposed to open air without sealing [six treatment groups for each excipient] using amber test vials. Triplicate samples from each excipient, temperature, and air exposure were sacrificed at project start (week 0), then at weeks 1, 2, and 4, and the CHBr 3 content was measured. The CHBr 3 content was analyzed using GC-MS according to the method described by Tan et al. in 2022. The weight of the open-air vials was measured at each measurement period to measure moisture loss. The molasses samples exposed to open air lost a significant amount of moisture over 4 weeks, and then the CHBr 3 results were adjusted taking into account the weight loss within the samples. The mean and standard error for each storage temperature / condition were calculated after each storage period, and the effect of time progression for each treatment was analyzed by one-way analysis of variance (ANOVA) using Tukey's honestly significant difference (HSD). Results were considered significant at P < 0.5.

[0319] Results CHBr in Corn Oil 3 The content is stable when stored at -20°C, 4°C and 25°C for 4 weeks (Figure 11, Table 2). However, when stored at 40°C, CHBr 3 The concentration of CHBr in corn oil when stored at 40 °C in airtight vials decreased to 6.3% after 2 weeks. 3 This will contribute to a better understanding of the effect of long-term storage on the retention of CHBr when exposed to ambient conditions. 3 was significantly decreased every week at 25°C and 40°C, with an overall loss of 41.1% and 53.6%, respectively, over the 4-week period.

[0320] Molasses CHBr 3 The CHBr content appears to be stable at both -20°C and 4°C in sealed containers. However, at higher temperatures, the CHBr content in molasses is 3 The content appears to be less stable compared to canola and corn oils. 3 was significantly decreased at 25 and 40 °C under both closed and open air conditions. In closed vials, CHBr 3 The CHBr content of molasses decreased by 41.4% and 75.4%, respectively, when exposed to open air during storage. 3 The content was CHBr compared to the sealed control. 3 The results were successful in retaining CHBr by only losing 14.8% and 37.9% at storage temperatures of 25°C and 40°C, respectively. Without being bound by theory, when exposed to open air, the molasses product loses CHBr by creating a natural seal. 3 This crust did not form in the sealed samples, so the CHBr 3 can slowly escape into the headspace of the vial and then be released when the vial is opened for sampling.

[0321] CHBr in cyclodextrin 3The content was the most stable among the excipients tested, and no significant changes were observed under any temperature and exposure conditions during the four-week period. At the fourth week, CHBr bound to cyclodextrin 3 did not seem to be adversely affected by high temperatures (25 °C and 40 °C) or open-air conditions, as observed with corn oil and molasses products. The small fluctuations in CHBr 3 observed over time under all storage conditions (Figure 11) may be due to analytical variability.

[0322] These results indicate that the manufactured CHBr 3 can be stabilized and stored in liquid (corn oil), semi-liquid (molasses), and solid (cyclodextrin) bromoform stabilizing excipients, thereby providing a stabilized bromoform composition.

[0323] These results also demonstrate that the stabilized bromoform compositions described herein allow for an appropriate concentration of bromoform to be manufactured for effective methane (CH 4 ) reduction in vitro and in vivo and are suitable for use in ruminant dietary formulations.

[0324] These results also demonstrate that the compositions described herein allow for a sustained release of the manufactured bromoform.

Table 2

[0325] [Example 8: Asp-Oil increases the growth ability of feedlot cattle.] Based on the results of Example 6 demonstrating that compositions containing Asp-Oil as well as the manufactured bromoform and stabilizing excipient (canola oil) behave similarly in vivo, the inventors sought to determine whether the in vivo effects of the compositions described herein include changes in growth performance.

[0326] Methods The experimental design is a randomized block design including 30 pens with 10 animals each (total of 300 cattle). All cattle were sourced from the same farm, of similar breed (British Cross) and sex (castrated bulls), with a target entry weight of 330 - 380 kg. The experimental design followed the Australian feedlot standard for 80 days of feed intake to achieve a desired exit weight of 480 - 540 kg. A total of three step - by - step feeds were used, transitioning the castrated bulls from a high - fiber pre - entry feed to a high - grain finishing feed, reflecting current feedlot practices. During these transition periods, the total oil content of each feed increased step - by - step. The prestarter feed was used only for the first few days after arrival at the feedlot. The starter, transition 1 (T1), and transition 2 (T2) feeds were each fed in 7 - day blocks (total of 21 days).

[0327] Individual whole - body live weight (LW) measurements were taken at arrival, at introduction to the trial (day 5), start weight (day 0), mid - weight (day 40), and end weight (day 80). ADWG was calculated over the entire 80 days of feed intake (end weight - start weight / days) and for the last 40 days to represent the effect of the finishing feed (mid - weight - end weight / 40). Castrated bulls were fed with zero / low refusals based on the previous day's intake. FCE was calculated over the entire 80 days (DMI / ADWG) and for the last 40 days of feed intake (DMI 40 - 80 days / ADWG 40 - 80 days).

[0328] Results During the course of the experiment, the feedlot was affected by a major weather event that resulted in pens being flooded midway through the trial. Twelve pens (out of 30) were relocated to different areas of the feedlot, which may have caused issues regarding feed intake and weight gain. Therefore, the inventors are reporting the results in two forms [with and without flooded pens]. Table 2 reports data from all 30 pens, while Table 3 reports data from the non-flooded pens. This study showed that compared to control bulls (without Asp-Oil), ADWG was improved from 2.2% (all pens) to 5.6% (excluding flooded pens) over the entire study period (days 1 - 81), and FCE was improved from 4.8% (all pens) to 5.2% (excluding flooded pens). Feed intake between days 1 - 81 showed different effects among all pens. Excluding the flooded pens, there was a 2.2% decrease in the overall Asp-Oil pens compared to the control group, and a 0.46% increase in feed intake in the flooded Asp-Oil pens compared to the control group. Although such effects are anomalous, ADWG (average daily weight gain) and FCE (feed conversion efficiency) were still improved in the Asp-Oil pens.

[0329] During the period between the last LW measurement, Asp-Oil was at its full feed inclusion level (days 40 - 81; 34 mg CHBr 3 / kg DMI), ADWG was improved from 2.6% (all pens) to 6.6% (excluding flooded pens), DMI decreased from 4.5% (all pens) to 1.24% (excluding flooded pens), and FCE was improved from 7.3% (all pens) to 8.0% (excluding flooded pens). These results indicate that there is an improvement in ADWG and FCE in beef cattle when fed Asparagopsis biomass (Asp-Oil) extracted in a bromoform-stabilized excipient. Example 6 demonstrates that a composition containing bromoform and the same bromoform-stabilized excipient similarly reduces methane production in vivo. The inventors propose that a composition containing bromoform and a bromoform-stabilized excipient improves growth performance on the same basis.

Table 3

Table 4

Claims

1. 1. A method for reducing total gas and / or methane production in a ruminant, comprising providing to said ruminant an effective amount of a composition comprising manufactured bromoform and a bromoform-stabilizing excipient, wherein said bromoform-stabilizing excipient is an edible oil.

2. 10. 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 bromoform formulated in a bromoform-stabilizing excipient.

3. 10. The method of claim 1, wherein the step of providing an effective amount of a composition to the ruminant comprises making the composition comprising bromoform manufactured in a bromoform-stabilizing excipient available in a feed system comprising the ruminant.

4. 4. The method of any one of claims 1 to 3, wherein 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, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

5. The method of any one of claims 1 to 3, wherein the composition is in solid, semi-solid or liquid form.

6. 1. A composition for reducing total gas production and / or methane production in a ruminant, the composition comprising manufactured bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is an edible oil.

7. 7. The composition of claim 6, wherein 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, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

8. 1. A feed supplement for reducing total gas production and / or methane production in a ruminant, comprising an effective amount of a composition comprising manufactured bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is an edible oil.

9. 10. The feed supplement of claim 8, further comprising one or more edible excipients.

10. 9. The feed supplement of claim 8, wherein 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, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

11. The animal feed supplement of any one of claims 8 to 10, wherein the composition is in solid, semi-solid or liquid form.

12. 1. A method for producing a methane-reduced ruminant feed, comprising mixing a ruminant feed with a feed supplement comprising an effective amount of a composition comprising the produced bromoform and a bromoform-stabilizing excipient, wherein the bromoform-stabilizing excipient is an edible oil.

13. An animal feed for reducing total gas production and / or methane production in a ruminant, said animal feed comprising: an effective amount of a composition comprising manufactured bromoform and a bromoform-stabilizing excipient; and ruminant feed, wherein said bromoform-stabilizing excipient is an edible oil.

14. 14. The animal feed of claim 13, wherein 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, linseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, linseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut 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.

15. 15. The animal feed of claim 13 or 14, wherein the composition is in solid, semi-solid or liquid form.

16. A stabilized bromoform composition comprising the prepared bromoform and a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is an edible oil.

17. 17. The stabilized bromoform composition of claim 16, which is free of one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

18. A method for producing a stabilized bromoform composition, comprising contacting the produced bromoform with a bromoform stabilizing excipient, wherein the bromoform stabilizing excipient is an edible oil.

19. 20. The method of claim 18, wherein the composition does not include one or more compounds selected from the group consisting of iodine, bromine, dibromochloromethane, bromochloroacetic acid, and dibromoacetic acid.

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