New Regulations

Abstract

The field of the invention relates to methods for producing asparagopsis oil compositions, comprising extracting at least one bioactive from asparagopsis biomass into oil to form compositions suitable for reducing total gas production and / or methane production in ruminant or pseudo-ruminant animals.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a composition suitable for reducing total gas production and / or methane production in ruminants. [Background technology]

[0002] Methane (CH4) is a greenhouse gas (GHG) produced primarily in natural ecosystems (e.g., wetlands, oceans, and lakes) and by methanogenic microorganisms in the gastrointestinal tract of animals, such as termites and ruminants. Approximately 429–507 Tg of CH4 are removed from the atmosphere each year; approximately 40 Tg by reaction with hydroxyl (OH) radicals in the stratosphere and approximately 30 Tg by CH4-oxidizing bacteria in the soil.

[0003] Nevertheless, anthropogenic GHG emissions are increasing rapidly, with current atmospheric CH4 concentrations more than double what they were in the early 1800s. Methane absorbs infrared radiation from the sun very efficiently and has a global warming potential 25 times greater than that of CO2. Therefore, the accumulation of methane in the atmosphere contributes significantly to climate change. One of the main sources of anthropogenic CH4 can be attributed to agricultural activities, including ruminant livestock.

[0004] According to a recent UN report, raising cattle generates more global-warming greenhouse gases, measured in CO2 equivalents, than transportation. In Australia, ruminants are estimated to contribute approximately 10% of all GHG emissions. Ruminants produce CH4 as a by-product of anaerobic microbial fermentation of their feed in the rumen and, to a lesser extent, the large intestine. Ruminant microorganisms are diverse and comprise bacteria, protozoa, fungi, and bacteriophages, which act collectively to ferment ingested organic matter (OM) to produce CO2, H2, volatile fatty acids (VFAs), and formic acid. Methanogenic archaea present in the rumen use these end products to produce CH4. CH4 production reduces the partial pressure of H2, which can inhibit ruminal fermentation, but it also reduces the amount of energy and carbon available for the formation of VFAs essential for ruminant nutrition. The majority of CH4 produced in ruminants is released as breath or burps by the animals, representing a loss of up to 12% of total energy intake.

[0005] Strategies to reduce enteric CH4 formation are important, and methods to reduce total gas production and / or methane production in ruminants are a major challenge. Summary of the Invention

[0006] In one aspect, the present invention provides a method for producing an Asparagopsis oil composition, the method comprising the steps of providing an Asparagopsis biomass; providing at least one oil; and contacting the biomass with the at least one oil under conditions to extract at least one bioactive agent from the biomass into the at least one oil to form the Asparagopsis oil composition.

[0007] In one aspect of this method, the step of contacting the biomass with at least one oil can include homogenizing the biomass in the at least one oil. In another aspect of this method, the step of contacting the biomass with the at least one oil can be followed by separating the biomass from the at least one oil.

[0008] In another aspect of this method, the biomass (in grams):at least one oil (in mL) may be greater than 0.3:1. In one embodiment, the biomass (in grams):at least one oil (in mL) is greater than 0.6:1. In another embodiment, the biomass (in grams):at least one oil (in mL) is greater than 0.9:1. In another embodiment, the biomass (in grams):at least one oil (in mL) is greater than 1.2:1.

[0009] In another aspect of this method, the conditions for extracting at least one bioactive agent from the biomass into at least one oil may be carried out for at least 1 day. In one embodiment, the extraction is carried out for 2 days. In another embodiment, the extraction is carried out for 3 days. In another embodiment, the extraction is carried out for 4 days. In another embodiment, the extraction is carried out for 5 days. In another embodiment, the extraction is carried out for 6 days. In another embodiment, the extraction is carried out for 7 days. In another embodiment, the extraction is carried out for 8 days. In another embodiment, the extraction is carried out for 9 days. In another embodiment, the extraction is carried out for 10 days. In yet another embodiment, the extraction is carried out for 11 days.

[0010] In another aspect of this method, the conditions for extracting at least one bioactive agent from the biomass into at least one oil may occur at about 4°C.

[0011] In one embodiment of this method, the biomass in contact with the at least one oil may be heated prior to the step of separating the biomass from the at least one oil. In this embodiment, the heating is performed so that the gel, which may include the at least one bioactive agent, releases the at least one bioactive agent into the at least one oil. In one embodiment, the heating may be performed to 60°C. In the above embodiment, the heating may be performed for 1 hour.

[0012] In one aspect of the invention, the asparagopsis biomass may be Asparagopsis taxiformis. In another aspect, the asparagopsis biomass may be Asparagopsis armata. In another aspect, the asparagopsis biomass may be Asparagopsis taxiformis and Asparagopsis armata.

[0013] In one aspect of this method, the step of preparing the asparagopsis biomass does not include air-drying the biomass. In another aspect, the step of preparing the asparagopsis biomass includes collecting the biomass into at least one oil.

[0014] In another aspect, the at least one oil comprises an edible oil. In one embodiment, the edible oil may be 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, 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 combinations thereof.

[0015] In another aspect, the at least one bioactive agent may be an anti-methanogenic agent. In one embodiment, the at least one bioactive agent may be selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM). In another embodiment, the anti-methanogenic agent is BF. In another embodiment, the anti-methanogenic agent is DBCM. In another embodiment, the anti-methanogenic agents are BF and DBCM.

[0016] In another aspect, the amount of at least one bioactive agent extracted from the biomass into the at least one oil is increased compared to the amount of the at least one bioactive agent extracted from an equivalent amount of biomass into water. In one embodiment, the at least one bioactive agent is an antimethanogenic agent, and the amount of the antimethanogenic agent extracted from the biomass into the at least one oil does not decrease by more than 20% after 65 weeks of storage at 25°C, and the at least one antimethanogenic agent is BF and / or DBCM.

[0017] In one embodiment, the amount of at least one bioactive agent extracted from the biomass into the at least one oil is not significantly reduced after 12 weeks of storage at 25° C. In another embodiment, the amount of at least one bioactive agent extracted from the biomass into the at least one oil is not significantly reduced after 65 weeks of storage at 4° C.

[0018] In another aspect, the asparagopsis oil composition formed by this method comprises at least 0.1 mg of bromoform per mL of extract. In one embodiment, the asparagopsis oil composition formed by this method comprises at least 1 mg of bromoform per mL of extract. In another embodiment, the asparagopsis oil composition formed by this method comprises at least 2 mg of bromoform per mL of extract. In another embodiment, the asparagopsis oil composition formed by this method comprises at least 3 mg of bromoform per mL of extract. In another embodiment, the asparagopsis oil composition formed by this method comprises at least 4 mg of bromoform per mL of extract.

[0019] In another aspect, the present invention provides an asparagopsis oil composition comprising at least one oil and at least one antimethanogenic agent. In one embodiment, the asparagopsis can be Asparagopsis armata. In another embodiment, the asparagopsis can be Asparagopsis armata and Asparagopsis armata.

[0020] In the above embodiment, the at least one oil may comprise an edible oil, which may be 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, 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 a combination thereof.

[0021] In another aspect of the asparagopsis oil composition, the at least one antimethanogenic agent may be selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

[0022] In one embodiment of the asparagopsis oil composition, the amount of the at least one antimethanogenic agent does not decrease significantly after 12 weeks of storage at 25° C. In another embodiment, the amount of the at least one antimethanogenic agent does not decrease significantly after 65 weeks of storage at 4° C. In another embodiment, the amount of the at least one antimethanogenic agent does not decrease by more than 20% after 65 weeks of storage at 25° C. In the above embodiment, the at least one antimethanogenic agent may be BF and / or DBCM. In another embodiment, the amount of the at least one antimethanogenic agent does not decrease by more than 50% after 65 weeks of storage at 4° C. or 25° C. In this embodiment, the at least one antimethanogenic agent may be DBA.

[0023] In one embodiment, the asparagopsis oil composition may have a concentration of at least one antimethanogenic agent per mL of extract of at least 0.1 mg. In another embodiment, the asparagopsis oil composition may have a concentration of at least one antimethanogenic agent per mL of extract of at least 1 mg. In another embodiment, the asparagopsis oil composition may have a concentration of at least one antimethanogenic agent per mL of extract of at least 2 mg. In another embodiment, the asparagopsis oil composition may have a concentration of at least one antimethanogenic agent per mL of extract of at least 3 mg. In another embodiment, the asparagopsis oil composition may have a concentration of at least one antimethanogenic agent per mL of extract of at least 4 mg.

[0024] In another aspect, the present invention provides a feed supplement for reducing total gas production and / or methane production in a ruminant, the supplement comprising an effective amount of an asparagopsis oil composition described herein.

[0025] In another aspect, the present invention provides a diet for a ruminant animal, the diet being supplemented with a dietary supplement as described herein.

[0026] In another aspect, the present invention provides a method for reducing total gas production and / or methane production in a ruminant, comprising administering to the ruminant an effective amount of an asparagopsis oil composition described herein. In one embodiment, the method may comprise maintaining an effective amount of a desired volatile fatty acid. In this embodiment, the desired volatile fatty acid may comprise acetate and propionate, and maintaining comprises reducing the ratio of acetate to propionate. In another embodiment, the method may comprise maintaining the amount of organic matter and / or dry matter decomposed.

[0027] In another aspect of this method, the asparagopsis oil composition may be administered in an amount equivalent to at least 3% of the organic matter administered to the ruminant. In one embodiment, the asparagopsis oil composition may be administered in an amount equivalent to at least 2% of the organic matter administered to the ruminant. In another embodiment, the asparagopsis oil composition may be administered in an amount equivalent to at least 1% of the organic matter administered to the ruminant. In another embodiment, the asparagopsis oil composition may be administered in an amount equivalent to at least 0.5% of the organic matter administered to the ruminant. In another embodiment, the asparagopsis oil composition may be administered in an amount equivalent to at least 0.25% of the organic matter administered to the ruminant. In another embodiment, the asparagopsis oil composition may be administered in an amount equivalent to at least 0.125% of the organic matter administered to the ruminant. In another embodiment, the asparagopsis oil composition may be administered in an amount equivalent to at least 0.067% of the organic matter administered to the ruminant.

[0028] In another aspect of the above method, the ruminant may be selected from members of the suborders Ruminantia and Nucleopoda. In one embodiment of this aspect, the ruminant may be a cow or a sheep. In another embodiment, the ruminant is a cow. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 shows the amount of bioactive bromoform (an antimethanogenic agent) in compositions prepared by extracting Asparagopsis biomass in oil or water for 1, 3, 5, 7, and 10 days, including compositions prepared by homogenizing Uncaria nigra biomass in oil or water ("Fractured") or without homogenizing the biomass in oil or water ("Untreated"). × indicates the standard method of extraction, in which freeze-dried biomass was soaked in methanol for 72 hours. Data are shown as mean ± SE, n=3. [Figure 2]Figure 2 shows the loss (%) of the bioactive compound bromoform (an antimethanogenic agent) from the composition after 6 months of storage under various conditions: ambient conditions (25°C ± 10°C), an air-conditioned room, a 4°C refrigerator, and a -20°C freezer. Data are presented as mean ± SE, n = 3. [Figure 3] Figure 3 shows the retention of the bioactive bromoform (an antimethanogenic agent) in compositions prepared by extracting Asparagopsis biomass into oil or water, including compositions prepared by homogenizing Uncaria bicolor biomass in oil or water ("Homogenized") or compositions prepared without homogenizing the biomass in oil or water ("Untreated"), after storage for 0, 4, 8, 12, and 65 weeks (at 4°C or 25°C). Data are shown as mean ± SE, n=3. [Figure 4] Figure 4 shows the retention of the bioactive DBCM (an antimethanogenic agent) in a composition made by extracting intact asparagopsis biomass with oil after storage for 0, 4, 8, 12, and 65 weeks (at 4°C; "refrigerator" or 25°C; "RT"). The ratio of DBCM:naphthalene (IS) in the oil is shown. There are no significant differences between bars with the same superscript. Data are presented as mean ± SE, n=3. [Figure 5] Figure 5 shows the retention of the bioactive DBCM (an antimethanogenic agent) in a composition made by homogenizing Uncaria nigra biomass in oil after storage for 0, 4, 8, 12, and 65 weeks (at 4°C; "refrigerator" or 25°C; "RT"). The DBCM:naphthalene (IS) ratio in the oil is shown. Data are presented as mean ± SE, n=3. The data do not show statistical differences in the DBCM:IS ratio. [Figure 6] Figure 6 shows the amount of bioactive DBA in a composition produced by extracting intact asparagopsis biomass with oil for 10 days, followed by immediate storage (0 weeks) at 25°C (RT) or 4°C (refrigerator). The DBA:naphthalene (IS) ratio in the oil is shown. Data are presented as mean ± SE, n=3. [Figure 7]Figure 7 shows the amount of bioactive DBA in a composition prepared by extracting homogenized asparagopsis biomass with oil for 10 days, followed by immediate storage (0 weeks) at 25°C (RT) or 4°C (refrigerator). The DBA:naphthalene (IS) ratio in the oil is shown. Data are presented as mean ± SE, n=3. [Figure 8] Figure 8 shows the amount of bioactive bromoform (an antimethanogenic agent) extracted into oil by homogenizing various amounts of biomass in the same volume of oil. Heating the gel sample assisted in the transfer of the bioactive from the gel to the oil. Data are shown as mean ± SE, n = 3. [Figure 9] Figure 9 shows the amount of the bioactive bromoform (an antimethanogenic agent) (mg / g dw biomass) in compositions prepared by homogenizing A. nigricans biomass in oil, with varying amounts of biomass homogenized in the same volume of oil. Heating the gel samples aided in the transfer of the bioactive from the gel to the oil. Data are shown as mean ± SE, n = 3. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to methods for producing bioactive asparagopsis oil compositions, including asparagopsis oil compositions suitable for reducing total gas production (TGP) and / or methane (CH4) production by ruminants. In particular, the inventors have shown that asparagopsis oil compositions can be produced that contain halogenated secondary metabolites with bioactive properties, and importantly, the inventors have also shown that the amount of halogenated secondary metabolites in the asparagopsis oil compositions is stable over an extended period of time.

[0031] Figure 1 shows that asparagopsis oil compositions containing bioactive molecules can be produced by contacting asparagopsis biomass with oil, and the amount of bioactives is increased by homogenizing the biomass in the oil. Figure 3 shows that asparagopsis oil compositions produced by contacting asparagopsis biomass with oil, with or without homogenization, retain high levels of bioactives for extended periods of time when stored at room temperature (25°C) or 4°C.

[0032] Therefore, the present invention provides a method for producing an asparagopsis oil composition, the method comprising: (a) preparing asparagopsis biomass; (b) providing at least one oil; and (c) contacting the biomass with at least one oil under conditions to extract at least one antimethanogenic agent from the biomass into the at least one oil. The present invention relates to a method comprising:

[0033] Asparagopsis has a heterotypic life cycle, with two free-living life stages: a gametophyte (a large, leaf-like form) and a sporophyte (or tetrasporophyte, a small, filamentous form). Historically, the tetrasporophyte was recognized as a separate genus (Falkenbergia). Therefore, the term "Asparagopsis" refers to the genus Asparagopsis and other taxonomic divisions currently known to belong to the genus.

[0034] There are at least two recognized species of Asparagopsis, one tropical / subtropical (Asparagopsis armata) and one temperate (Asparagopsis armata), which occur worldwide.

[0035] In one aspect, the Asparagopsis species is selected from Asparagopsis nigra or Asparagopsis armata.

[0036] In another aspect, biomass of at least one species of red marine macroalgae selected from species belonging to other genera of red algae in the Bonnemaisoniaceae family, to which Asparagopsis belongs (e.g., Bonnemaisonia, Delisea, Ptilonia, Leptophyllis, and Pleuroblepharidella), is used in place of Asparagopsis biomass in the methods and compositions of the invention. Without wishing to be bound by theory, six genera of red algae in the Bonnemaisoniaceae family (e.g., Asparagopsis, Delisea, Delisea, Ptilonia, Leptophyllis, and Pleuroblepharidella) produce and store bioactive halogenated secondary metabolites with bioactive properties, including the antimethanogenic compounds described herein.

[0037] The term "preparing a biomass" includes the preparation or use of asparagopsis biomass removed from water immediately prior to contacting the biomass with at least one oil. In one embodiment, the asparagopsis biomass is harvested from its natural environment and directly placed in at least one oil. In another embodiment, the asparagopsis biomass is contacted with at least one oil within 5, 4, 3, 2, or 1 hour of removing the biomass from its natural environment.

[0038] In one embodiment, water is removed from the biomass by blotting the biomass dry prior to contacting the biomass with at least one oil.

[0039] In one aspect, the asparagopsis biomass is not a freeze-dried asparagopsis biomass.

[0040] In another embodiment, the asparagopsis biomass is not air-dried asparagopsis biomass.

[0041] The term "at least one oil" includes a single type of oil, a composition comprising a single type of oil, a mixture of two or more oils, or a composition comprising a mixture of two or more oils. The at least one oil includes oils suitable for application, administration, or feeding to an animal.

[0042] In one embodiment, the at least one oil comprises an edible oil.

[0043] In one aspect, 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, 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 combinations thereof.

[0044] The term "oil" generally refers to a non-polar, hydrophobic substance that is liquid at ambient temperature and pressure. Oils may be of animal, vegetable, or petrochemical origin and generally have a high carbon and hydrogen content. The oil is preferably an edible oil and is preferably easily digestible by ruminants. Oils of vegetable origin are typically extracted from plant seeds or fruits and consist primarily of triglycerides. The term "vegetable oil" is a generic term indicating that the oil is primarily or exclusively of vegetable origin and may include a mixture of one or more oils of vegetable or different origins.

[0045] The inventors have demonstrated that bioactive agents are rapidly extracted from asparagopsis biomass into oil. For example, Figure 1 shows that the amount of bromoform extracted from a homogenized biomass / oil mixture in 24 hours is greater than the amount of bromoform extracted from freeze-dried biomass with methanol. Figure 1 also shows that the amount of bromoform extracted from an unhomogenized biomass / oil mixture in 72 hours is the same as the amount of bromoform extracted from freeze-dried biomass. Figure 1 also shows that the amount of bromoform extracted from an unhomogenized biomass / oil mixture in 120 hours is greater than the amount of bromoform extracted from freeze-dried biomass with methanol.

[0046] "Contacting" herein includes mixing a biomass with at least one oil to form a biomass / oil mixture and maintaining the biomass / oil mixture for a period of time and at a temperature suitable to extract at least one bioactive agent from the biomass into the oil of the biomass / oil mixture.

[0047] The term contacting includes maintaining the biomass oil mixture at various suitable temperatures.

[0048] The temperature at which the biomass can be contacted with at least one oil is not limited, as long as the antimethanogenic agent does not significantly evaporate / sublime from the oil and / or significantly decompose at the temperature at which the contacting is carried out. The temperature may be selected from the group consisting of -78°C to -50°C, -50°C to -20°C, -20°C to -5°C, -5°C to 0°C, -5°C to 4°C, 0°C to 4°C, 4°C to 10°C, 10°C to 20°C, 20°C to 25°C, 25°C to 30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, and 90°C to 100°C. In some embodiments, the temperature may be selected from the group consisting of -78°C, -20°C, -5°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C. In one embodiment, the temperature is preferably about 4°C. In another embodiment, the temperature is preferably about 25°C. Suitable temperatures can be achieved using freezers, refrigerators, air conditioners, and the like.

[0049] The term "room temperature" is used to refer to normal ambient temperature. As one skilled in the art will appreciate, ambient conditions vary depending on geographic location and season, and room temperature may range from below 0°C to above 40°C. Room temperature is typically considered to be between about 20°C and about 25°C, and in experiments conducted in Townsville, Queensland, room temperature is understood to mean about 25°C.

[0050] The inventors have demonstrated that when a large amount of asparagopsis biomass is contacted with a relatively small volume of oil, gel formation occurs, preventing the extraction of one or more bioactive agents into the oil. To increase the yield of extracted bioactive (e.g., antimethanogenic) agents, the gel, biomass, and / or oil can be heated to release more of the at least one bioactive (e.g., antimethanogenic) agent into the at least one oil. For example, Figures 8 and 9 show that heating a sample of biomass in contact with at least one oil at 60°C for 1 hour extracted up to 20% more bioactive (e.g., antimethanogenic) agent into the oil compared to a sample that was not heated (see "120g + Heat" and "120g" in Figures 8 and 9).

[0051] The temperature to which biomass in contact with at least one oil can be heated to release at least one bioactive (e.g., antimethanogenic) agent into the at least one oil is not limited, so long as the at least one bioactive (antimethanogenic) agent does not significantly evaporate / sublimate from the oil and / or significantly decompose at the temperature at which heating is performed. The temperature may be selected from the group consisting of 25°C to 30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, and 90°C to 100°C. In some embodiments, the temperature may be selected from the group consisting of 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C. In one embodiment, the temperature is preferably about 60°C.

[0052] The time period for which biomass in contact with at least one oil can be heated to release at least one bioactive (e.g., anti-methanogenic) agent into the at least one oil is not limited, so long as the at least one bioactive (anti-methanogenic) agent does not significantly evaporate / sublimate from the oil and / or significantly decompose during heating. The time period may be selected from the group consisting of 1 second to 1 minute, 1 minute to 2 minutes, 2 minutes to 5 minutes, 5 minutes to 10 minutes, 10 minutes to 20 minutes, 20 minutes to 30 minutes, 30 minutes to 40 minutes, 40 minutes to 50 minutes, 50 minutes to 60 minutes, 60 minutes to 70 minutes, 70 minutes to 80 minutes, 80 minutes to 90 minutes, 1.5 hours to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, and 4 hours to 5 hours. In some embodiments, the time may be selected from the group consisting of 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, and 3 hours. In one embodiment, the time for which the biomass in contact with the at least one oil is heated is preferably about 1 hour.

[0053] If the biomass / oil mixture is not heated to release the at least one bioactive (e.g., anti-methanogenic) agent into the at least one oil, the amount of time the biomass can be contacted with the at least one oil under conditions to extract the at least one bioactive (e.g., anti-methanogenic) agent from the biomass into the at least one oil is not particularly limited. As one of ordinary skill in the art will understand, it is desirable for the concentration of the at least one bioactive (e.g., anti-methanogenic) agent in the oil to reach or nearly reach an equilibrium level, maximizing the amount of the at least one bioactive (e.g., anti-methanogenic) agent in the at least one oil. Thus, contacting the biomass with at least one oil under conditions to extract at least one bioactive (e.g., anti-methanogenic) agent from the biomass into the at least one oil can be carried out for a time period selected from the group consisting of 1 minute to 1 hour, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 2 days, 2 days to 3 days, 3 days to 4 days, 4 days to 5 days, 5 days to 6 days, 6 days to 7 days, 7 days to 8 days, 8 days to 9 days, 9 days to 10 days, 10 days to 14 days, 2 weeks to 3 weeks, and 3 weeks to 1 month. In some embodiments, the contacting can be performed for a period of time selected from the group consisting of 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, and 2 weeks. In some embodiments, the contacting can be performed for a period of at least 1, 2, 3, 4, 5, 4, 6, 7, 8, 9, or 10 days.

[0054] The step of contacting the biomass with at least one oil may also include homogenizing the biomass in the at least one oil. The term "homogenizing" refers to breaking up the biomass to facilitate the release of at least one antimethanogenic agent from the biomass, which may be extracted into the at least one oil. Homogenization may be accomplished by means known in the art, such as crushing, grinding, milling, mixing, cutting, chopping, or dicing.

[0055] In one aspect, homogenization may be carried out to obtain a uniform mixture to facilitate uniform distribution of the biomass, bioactive agent, and / or oil in the composition into a food source for the animal.

[0056] In one aspect, the bioactive agent is an agent that has a biological effect on animals, preferably ruminants. The biological effect may be an effect on the behavior or physiology of the animal, or may further affect the microorganisms within the animal. For example, the biological effect may be the inhibition of methanogenesis. The bioactive agent responsible for inhibiting methanogenesis is not particularly limited and is intended to include all agents that inhibit methanogenesis, including, but not limited to, secondary metabolites. Asparagopsis produces secondary metabolites, including halogenated low-molecular-weight compounds, particularly brominated and chlorinated haloforms. Many of these compounds have strong antibacterial properties and inhibit the activity of a wide range of microorganisms, including Gram-positive and Gram-negative bacteria, as well as mycobacteria and fungi, and therefore may be involved in contributing to the effects described herein. Secondary metabolites from Asparagopsis also inhibit protozoa.

[0057] In one aspect, the bioactive agent is an agent that reduces total gas production (TGP) and / or an agent that reduces methane produced by ruminants.

[0058] 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 gases and / or methane produced by a ruminant fed an asparagopsis oil composition described herein is relative to an animal not fed the asparagopsis oil composition. Reductions can be measured in vitro using an artificial rumen system that simulates anaerobic fermentation, or in vivo using animals confined to a respiration chamber. Estimating intestinal methanogenesis by ruminants is within the knowledge and skill of one of ordinary skill in the art.

[0059] The term "reducing total gas production" refers to reducing the total amount of gas produced, e.g., the total amount of gas produced in the gastrointestinal tract. This term, for example, as used herein, includes the collective volume of all gases generated as a result of anaerobic fermentation. Fermentation in the rumen and digestive tract of ruminants results in 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. Estimating total gas production by ruminants is within the knowledge and skill of one of ordinary skill in the art.

[0060] The term "reducing methane production" refers to a reduction in methane produced in the gastrointestinal tract. This term, for example, as used herein, includes the specific volume of methane produced as a result of anaerobic fermentation. Fermentation in the rumen and digestive tract of ruminants leads to the production of methane. The present invention aims to reduce this process, for example, to reduce the total amount of methane produced in the gastrointestinal tract. Estimating methane production by ruminants is within the knowledge and skill of one of ordinary skill in the art.

[0061] The term "anaerobic fermentation" is intended to include anaerobic fermentation in vivo, for example in ruminants.

[0062] The term "antimethanogenic agent" refers to a bioactive compound that inhibits methanogenesis in ruminants. Such compounds are typically halogenated secondary metabolites, including at least bromochloroacetic acid, bromochloromethane, 2-bromoethanesulfonic acid, chloral hydrate, chloroform, iodopropane, bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

[0063] For example, the inventors have shown in Figures 4, 5, 6, and 7 that asparagopsis oil compositions containing dibromochloromethane (DBCM) and / or dibromoacetic acid (DBA) can be produced by homogenizing asparagopsis biomass and contacting it with oil. Many bioactives are extracted into the oil, and these amounts, as shown with DBCM, are maintained over time when stored at either room temperature (RT) or 4°C (refrigerated).

[0064] In one embodiment, the anti-methanogenic agent is selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

[0065] In one embodiment, the biomass is contacted with at least one oil under conditions to extract the at least one antimethanogenic agent from the biomass into the at least one oil, and then the biomass is separated from the at least one oil. Separation of the biomass from the at least one oil may be accomplished by means known in the art, including, but not limited to, centrifugation, decantation, filtration, distillation, or the use of a separatory funnel, or other equivalent means.

[0066] The amount of biomass and at least one oil contacted can be varied to vary the amount of at least one bioactive entity extracted into the at least one oil.

[0067] The inventors also demonstrated that the amount of at least one bioactive substance may be controlled by varying the ratio of asparagopsis biomass to at least one oil. For example, Figures 8 and 9 show that the amount of bromoform can be increased by increasing the ratio of asparagopsis biomass to at least one oil.

[0068] Thus, in some embodiments, the biomass:at least one oil may be selected from the group consisting of 0.01g:1mL, 0.05g:1mL, 0.1g:1mL, 0.2g:1mL, 0.3g:1mL, 0.4g:1mL, 0.5g:1mL, 0.6g:1mL, 0.7g:1mL, 0.8g:1mL, 0.9g:1mL, 1g:1mL, 1.1g:1mL, 1.2g:1mL, 1.3g:1mL, 1.4g:1mL, and 1.5g:1mL. Preferably, the biomass:at least one oil is selected from the group consisting of more than 0.3g:1mL, more than 0.6g:1mL, more than 0.9g:1mL, and more than 1.2g:1mL.

[0069] In one aspect of the present invention, there is provided an asparagopsis oil composition produced by the methods described herein.

[0070] The inventors have previously shown that freeze-dried milled Asparagopsis species can effectively reduce in vivo total gas and methane production in cattle compared to a dehulled cottonseed (DSC) positive control. Importantly, dehulled cottonseed is used as a dietary supplement for cattle because it significantly reduces CH4 production compared to other high-energy grains. The reduction in total gas production was similar across species, indicating that macroalgae such as Asparagopsis reduce TGP and CH4 production in ruminants compared to high-energy grains, and that some macroalgae reduce TGP and CH4 production in ruminants compared to the DCS positive control. Furthermore, the inventors showed that CH4 production generally followed the same pattern as TGP.

[0071] Thus, in one aspect, the asparagopsis oil compositions produced by the methods described herein can be used to reduce total gas and / or methane produced by ruminant animals.

[0072] Thus, in one aspect, the present invention relates to a method for reducing total gas production and / or methane production in a ruminant, comprising administering to the ruminant an effective amount of an asparagopsis oil composition of the present invention.

[0073] In preferred embodiments of the present invention, the amount of total gas produced is reduced by at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to a baseline. In one embodiment, the baseline is the amount of total gas produced when the animal is not administered an effective amount of an asparagopsis oil composition of the present invention. In another embodiment, the baseline is the amount of total gas produced when the animal is administered dehulled cottonseed. In another embodiment, the baseline is the amount of total gas produced when the dehulled cottonseed is subjected to in vitro anaerobic fermentation.

[0074] In preferred embodiments of the present invention, the amount of methane produced is reduced by at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or 10% compared to a baseline. In one embodiment, the baseline is the amount of methane produced when the animal is not administered an effective amount of an asparagopsis oil composition. In another embodiment, the baseline is the amount of methane produced when the animal is administered dehulled cottonseed. In another aspect, the benchmark is the amount of methane produced when animals are fed a pelleted commercial shipper diet based on lupin, oats, barley, and wheat, with cereal straw as a roughage ingredient [chemical composition: ash 72 (g / kg DM), crude protein (CP) 112; neutral detergent fiber (aNDFom) 519; acid detergent fiber (ADFom) 338, no cobalt, selenium, or rumen improvers], with an additional amount of crushed lupin, referred to herein as the "lupin diet". In another aspect, the benchmark is the amount of methane produced when the lupin diet is subjected to in vitro anaerobic fermentation.

[0075] In one aspect, the amount of methane produced in vitro by ruminal fermentation is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the amount of methane produced when dehulled cottonseed is subjected to ruminal fermentation in vitro.

[0076] In one aspect, the amount of methane produced is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the amount of methane produced when the ruminant is fed dehulled cottonseed.

[0077] The inventors have also previously demonstrated that air-dried milled asparagopsis can effectively reduce methane production compared to a positive control of a lupine diet in sheep.

[0078] In one aspect, the amount of methane produced is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the amount of methane produced when the ruminant is fed the lupin diet.

[0079] "Effective amount" refers to an amount of the asparagopsis oil composition of the present invention sufficient to improve, for example, reduce the amount of methane production compared to a reference or control, reduce the amount of total gas produced compared to a reference or control, maintain an effective amount of one or more desired volatile fatty acids compared to a reference or control, reduce the acetate to propionate ratio compared to a reference or control, maintain live weight, dry matter intake, and / or organic matter intake compared to a reference or control. Within the meaning of the present invention, methane reduction effects can be measured in the rumen by an artificial rumen system, such as that described in T. Hano, J. Gen. Appl. Microbiol., 1993, 39, 35-45, or in vivo by oral administration to ruminants.

[0080] Thus, in one aspect, the asparagopsis oil compositions of the present invention are preferably administered at a dose equivalent to at least 16.67, 10, 5, 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant.

[0081] To calculate the volume of oil required to administer to a ruminant animal to achieve a desired organic matter equivalent dose, the % organic matter content of the asparagopsis oil composition of the present invention is calculated from the fresh weight (fw) of asparagopsis biomass in contact with a specified volume of at least one oil. The fresh weight to dry weight (dw) ratio of imbibed asparagopsis is 10 (i.e., 30 g fw = 3 g dw). Based on previous data, assuming an organic matter (OM) content of 80% dw, the corresponding OM content of asparagopsis origin in the biomass / oil can be calculated. For example, an asparagopsis oil composition containing 30 g fresh weight (fw) asparagopsis biomass in 100 mL of at least one oil corresponds to 0.024 asparagopsis organic matter / mL. If the desired amount in the diet is 0.1% Asparagopsis OM in 100 g of diet, then 4.17 mL of the 30 g fw / 100 mL Asparagopsis oil composition would be required per 100 g of diet.

[0082] The corresponding OM content of Asparagopsis origin in the biomass / oil can be calculated from the % OM of different contents of dw, for example, using the OM content of 50%, 55%, 60%, 70%, and 75% of the dw based on previous data, the corresponding OM content of Asparagopsis origin in the biomass / oil can be calculated.

[0083] As indicated above, in preferred embodiments, the asparagopsis oil composition of the present invention is preferably administered in an amount corresponding to at least 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to ruminants. Thus, when using 30 g fw / 100 mL of the asparagopsis oil composition of the present invention, at least 125 mL, 83.3 mL, 41.7 mL, 20.8 mL, 10.4 mL, 5.2 mL, or 2.8 mL per 100 g of feed is required. When using 60 g fw / 100 mL of the asparagopsis oil composition of the present invention, at least 62.5 mL, 41.7 mL, 20.8 mL, 10.4 mL, 5.2 mL, 2.6 mL, or 1.4 mL per 100 g of feed is required. When using 120 g fw / 100 mL of the asparagopsis oil composition of the present invention, at least 31.25 mL, 20.8 mL, 10.4 mL, 5.2 mL, 2.6 mL, 1.3 mL or 0.69 mL per 100 g of feed is required.

[0084] For example, if a 400 kg ruminant (e.g., a steer) consumes 2.5% to 3% of its body weight in feed per day, the asparagopsis oil composition of the present invention is administered at a dose proportional to the amount of organic matter administered to the ruminant. For a 400 kg ruminant and 80% of the feed is organic matter, if the animal consumes approximately 2.5% of its body weight per day (10 kg, containing 8 kg of organic matter), 3.3 L of 30 g fw / 100 mL of the asparagopsis oil composition of the present invention would be required to provide the equivalent of 1% of the organic matter administered to the ruminant. 1.6 L of 60 g fw / 100 mL of the asparagopsis oil composition of the present invention would be required to provide the equivalent of 1% of the organic matter administered to the ruminant. To achieve the equivalent of 1% of the organic matter administered to ruminants, 1.1 L of a 90 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 1% of the organic matter administered to ruminants, 833 mL of a 120 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 1% of the organic matter administered to ruminants, 666 mL of a 150 g fw / 100 mL asparagopsis oil composition of the present invention is required.

[0085] To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 330 mL of a 30 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 167 mL of a 60 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 111 mL of a 90 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 83 mL of a 120 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 67 mL of a 150 g fw / 100 mL asparagopsis oil composition of the present invention is required.

[0086] To calculate the composition containing the biomass required to achieve a desired organic matter equivalent dose to be administered to ruminants, the organic matter percentage of the asparagopsis oil composition of the present invention is calculated from the fresh weight (fw) of asparagopsis biomass in contact with a specified volume of at least one oil. The fresh weight to dry weight (dw) ratio of imbibed asparagopsis is 10 (i.e., 30 g fw = 3 g dw). Based on previous data, assuming an organic matter (OM) content of 80% of dw, the corresponding OM content of asparagopsis origin in the biomass / oil can be calculated. For example, an asparagopsis composition containing 30 g fresh weight (fw) asparagopsis biomass in 100 mL of at least one oil corresponds to 0.024 asparagopsis organic matter / mL, and the amount of organic matter in the final overall volume is calculated and included in the calculation.

[0087] Effective amounts of the asparagopsis oil compositions of the present invention may be determined by the methods described herein, including the in vitro and in vivo dose-response experiments described herein. For example, the inventors have demonstrated that in vitro ruminal fermentation can be used to investigate the effect of the amount of freeze-dried, ground asparagopsis on the amount of volatile fatty acids, including acetate and propionate, methane production, and total gas production. Thus, in vitro ruminal fermentation can be used to characterize a dose of the asparagopsis oil compositions of the present invention that may be effective enough to improve, for example, reduce the amount of methane production compared to a baseline or control, reduce the amount of total gas produced compared to a baseline or control, maintain an effective amount of one or more desired volatile fatty acids compared to a baseline or control, or reduce the acetate-to-propionate ratio compared to a baseline or control.

[0088] Ruminants are even-toed cloven-hoofed mammals that digest plant-based foods by first softening and partially fermenting them in the animal's rumen chamber, and then regurgitating the semi-digested mass (now known as regurgitation) and re-chewing it.

[0089] The process of re-chewing the cud to further break down plant material and stimulate digestion is called "rumination." Ruminants have four digestive tracts: the rumen, abomasum, omasum, and abomasum. In the first two chambers, the rumen and abomasum, food is mixed with saliva and separated into layers of solid and liquid material. The solids clump together to form the cud, or bolus. The cud is then regurgitated and slowly chewed to thoroughly mix with saliva and further break down the fiber. Fiber, particularly cellulose, is broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa, and fungi. The broken down fiber, the liquid portion of the contents, passes through the rumen to the next stomach chamber, the abomasum. Food in the abomasum is digested similarly to that in the stomach of monogastric animals. The digested digestive tract contents ultimately pass to the small intestine, where nutrient absorption occurs. Nearly all of the glucose produced by cellulose breakdown is used by symbiotic bacteria, and ruminants obtain their energy from the volatile short-chain fatty acids (VFAs) produced by the bacteria: acetate, propionate, butyrate, valerate, and isovalerate.

[0090] Importantly, the inventors have shown that freeze-dried milled asparagopsis has the ability to reduce total gas production and / or methane production in ruminants without impairing ruminal fermentation.

[0091] For example, the inventors have shown that freeze-dried milled asparagopsis has the ability to reduce total gas and / or methane production in ruminants without compromising ruminal fermentation, e.g., while maintaining an effective amount of one or more desired volatile fatty acids. The inventors have also shown that freeze-dried milled asparagopsis has the ability to reduce total gas and / or methane production in ruminants without compromising ruminal fermentation, e.g., without significantly affecting daily feed intake and / or animal live weight.

[0092] The term "maintaining an effective amount" means that the amount of a substance in an animal after treatment (e.g., administration of an asparagopsis oil composition of the present invention) is not significantly different from a control or baseline, including the amount of the substance in an animal not administered an asparagopsis oil composition of the present invention.

[0093] For example, an "effective amount of a desired volatile fatty acid" is intended to mean that the amount of the desired volatile fatty acid is sufficient to improve a condition, such as reducing the amount of methane production compared to a baseline or control; reducing the amount of total gas produced compared to a baseline or control; reducing the acetate to propionate ratio compared to a baseline or control; or maintaining live weight, dry matter intake, and / or organic matter intake compared to a baseline or control.

[0094] Carbohydrate metabolism provides energy for the growth of ruminal microorganisms, primarily through the fermentation of cellulose and starch. Insoluble polymers are converted by extracellular enzymes from ruminal microorganisms into oligosaccharides and soluble sugars. The resulting sugars are then fermented to one of various forms of volatile fatty acids, carbon dioxide, and hydrogen. Herein, volatile fatty acids—acetic acid, propionic acid, and butyric acid—are also referred to as acetate, propionate, and butyrate, respectively.

[0095] Volatile fatty acids are utilized as a major source of carbon and energy by animals with varying efficiencies. High levels of propionic acid are desirable because it is the primary metabolic precursor for gluconeogenesis in animals. Fermentation of hexoses to acetate is relatively inefficient because in this process, carbon and hydrogen are lost via burping in the form of carbon dioxide or, importantly, methane. On the other hand, propionic acid production utilizes hydrogen and does not result in the loss of carbon or methane.

[0096] Thus, by increasing the molar ratio of propionic acid to acetic acid, or in another aspect, by increasing the total volatile fatty acid concentration in the rumen (i.e., the sum of acetic acid, propionic acid, and butyric acid), it is possible to improve the efficiency of feed utilization and / or the rate of growth of ruminants.

[0097] The inventors have demonstrated that the presence of freeze-dried milled asparagopsis reduces total gas and / or methane produced during in vitro and in vivo anaerobic fermentation without adversely affecting total VFA production in cattle. Importantly, the inventors have shown that asparagopsis does not reduce VFA production in cattle at doses of asparagopsis that do not reduce organic or dry matter uptake / degradation; at doses that reduce the acetate to propionate ratio; at doses that reduce acetate; at doses that increase propionate; and / or at doses that inhibit total gas and methane production in vitro and in vivo.

[0098] Importantly, the inventors demonstrated that freeze-dried milled asparagopsis did not reduce the amount of VFAs at doses of asparagopsis that inhibited total gas and methane production in cattle. The inventors also demonstrated that freeze-dried milled asparagopsis did not reduce organic matter or dry matter intake / decomposition in sheep at doses that reduced the acetate-to-propionate ratio; at doses that reduced acetate; at doses that increased propionate; and / or at doses that inhibited methane production in vitro and in vivo. For example, the inventors demonstrated that freeze-dried milled asparagopsis did not reduce organic matter or dry matter intake / decomposition in sheep fed 1.2 times the maintenance energy.

[0099] Thus, in one aspect, the present invention relates to a method for reducing total gas production and / or methane production in a ruminant, comprising administering to the ruminant an effective amount of an asparagopsis oil composition of the present invention, wherein an effective amount of desired volatile fatty acids is maintained.

[0100] In one embodiment, the desired volatile fatty acids are acetate and propionate.

[0101] The term "volatile fatty acids" ("VFAs") includes the end products of anaerobic microbial fermentation of feed in the rumen. Common VFAs include acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate. VFAs are absorbed by the rumen and used by the animal for energy and lipid synthesis.

[0102] In a preferred embodiment of the present invention, the total VFAs produced in ruminal fermentation in the presence of an effective amount of the asparagopsis oil composition of the present invention is at least 80 mmol / L.

[0103] In another embodiment of the present invention, the total VFAs produced in ruminal fermentation in the presence of an effective amount of the asparagopsis oil composition of the present invention is at least 65 mmol / L.

[0104] The inventors have also demonstrated that freeze-dried milled asparagopsis does not reduce the amount of VFAs in cattle at doses of freeze-dried milled asparagopsis that do not reduce the amount of organic matter or dry matter degraded from ruminal fermentation or dry matter intake. The inventors have also demonstrated that freeze-dried milled asparagopsis does not reduce the dry matter intake or live weight of sheep. For example, the inventors have demonstrated that freeze-dried milled asparagopsis does not reduce the dry matter intake or live weight of sheep fed 1.2 times maintenance energy. This indicates that red marine macroalgae such as asparagopsis reduce total gas production and / or methane production in ruminants without impairing ruminal fermentation.

[0105] Thus, in one aspect, the present invention relates to a method for reducing total gas production and / or methane production in a ruminant, comprising administering to the ruminant an effective amount of an asparagopsis oil composition of the present invention. In one aspect, an effective amount of a desired volatile fatty acid is maintained. In another aspect, the acetate to propionate ratio is reduced. In another aspect, the amount of organic matter and / or dry matter decomposed is maintained. In another aspect, the present invention relates to a method for reducing total gas production and / or methane production in a ruminant, comprising administering to the ruminant an effective amount of an asparagopsis oil composition of the present invention, wherein the amount of dry matter intake is maintained.

[0106] The terms "organic matter" and "dry matter" refer to the amount of feed (on an organic matter or moisture-free basis, respectively) consumed by an animal in a given period of time, typically 24 hours. Methods for calculating intake and / or decomposition of organic matter and dry matter are known in the art. For example, dry matter and organic matter may be 90% and 80% of the amount of feed, respectively.

[0107] In one embodiment, the asparagopsis oil compositions of the present invention are preferably administered at a dose equivalent to at least 16.67, 10, 5, 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant.

[0108] In preferred embodiments, the asparagopsis oil compositions of the present invention are administered in an amount equivalent to at least 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant, preferably to maintain the amount of organic matter and / or dry matter decomposed.

[0109] In another embodiment, the amount of organic matter or dry matter decomposed is maintained together with an effective amount of the desired volatile fatty acids.

[0110] In preferred embodiments, the asparagopsis oil compositions of the present invention are preferably administered in an amount equivalent to at least 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant to maintain effective amounts of the desired volatile fatty acids.

[0111] Importantly, the inventors demonstrated that freeze-dried milled asparagopsis with increasing amounts of propionate at the dose of asparagopsis inhibited total gas and methane production, and freeze-dried milled asparagopsis with increasing amounts of propionate at the dose of freeze-dried milled asparagopsis did not reduce the amount of organic matter or dry matter decomposed.

[0112] Thus, in another aspect, the present invention relates to a method for reducing total gas production and / or methane production in a ruminant animal, comprising administering to the ruminant an effective amount of an asparagopsis oil composition of the present invention, wherein the amount of organic matter or dry matter decomposed is maintained and / or the acetate to propionate ratio is reduced.

[0113] In preferred embodiments, the asparagopsis oil compositions of the present invention are administered in an amount equivalent to at least 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant to reduce the acetate to propionate ratio.

[0114] In one embodiment, the acetate to propionate ratio (C2 / C3 ratio) is not adversely affected after administration of the asparagopsis oil composition of the present invention, hi another embodiment, the acetate to propionate ratio (C2 / C3 ratio) is reduced after administration of the asparagopsis oil composition of the present invention.

[0115] In another embodiment, the propionate molarity is not adversely affected.

[0116] Ruminal fermentation of low-quality fiber diets is the major source of methane production in ruminants.

[0117] Examples of ruminants are listed below. However, preferably, the asparagopsis oil composition of the present invention is used as an additive for feed for domestic livestock such as cattle, goats, sheep and llamas. The present invention is particularly useful for cattle and sheep. Thus, in one embodiment, the ruminant is selected from the suborder Ruminantia and Nucellopoda. In another embodiment, the ruminant is a cattle or a sheep. In a further embodiment, the ruminant is a cattle.

[0118] "Administering" and "administered" refer to the act of introducing the asparagopsis oil composition of the present invention into the gastrointestinal tract of an animal. More specifically, this administration is by the oral route. This administration can be carried out, in particular, by supplementing the asparagopsis oil composition of the present invention into feed intended for the animal, which feed is then ingested by the animal. Administration can also be carried out by stomach tube or other means that allow the asparagopsis oil composition of the present invention to be introduced directly into the gastrointestinal tract of the animal.

[0119] As noted above, in preferred embodiments of the present invention, the effective amount of the asparagopsis oil composition of the present invention is at least 16.67, 10, 5, 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant.

[0120] For example, the asparagopsis oil compositions of the present invention are preferably administered at a dose of at least 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter available in the diet of a ruminant.

[0121] For example, if a ruminant consumes approximately 2.5-3% of its live weight per day, a 400 kg ruminant may consume 10-12 kg of feed per day. For example, if a 400 kg ruminant (e.g., a steer) consumes 2.5-3% of its body weight per day, the asparagopsis oil composition of the present invention is administered at a dose proportional to the amount of organic matter administered to the ruminant. For a 400 kg ruminant and 80% of the feed is organic, if the animal consumes approximately 2.5% of its body weight per day (10 kg, containing 8 kg of organic matter), 3.3 L of a 30 g fw / 100 mL asparagopsis oil composition of the present invention would be required to provide the equivalent of 1% of the organic matter administered to the ruminant. To achieve 1% of the organic matter administered to ruminants, 1.6 L of a 60 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve 1% of the organic matter administered to ruminants, 1.1 L of a 90 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve 1% of the organic matter administered to ruminants, 833 mL of a 120 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve 1% of the organic matter administered to ruminants, 666 mL of a 150 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve 0.1% of the organic matter administered to ruminants, 330 mL of a 30 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 167 mL of a 60 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 111 mL of a 90 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 83 mL of a 120 g fw / 100 mL asparagopsis oil composition of the present invention is required. To achieve the equivalent of 0.1% of the organic matter administered to ruminants, 67 mL of a 120 g fw / 100 mL asparagopsis oil composition of the present invention is required.

[0122] An effective amount can be administered to the ruminant in one or more doses.

[0123] An effective amount can also be administered to a ruminant in one or more doses per day.

[0124] The dosage defined herein as the amount per kg body weight per day refers to the average amount of the asparagopsis oil composition of the present invention administered during a given period of treatment, for example, during a week or month of treatment. Thus, the asparagopsis oil composition of the present invention may be administered daily, every other day, etc., without departing from the scope of the present invention. Preferably, however, the method involves daily administration of the asparagopsis oil composition of the present invention at a specified dosage. Even more preferably, the asparagopsis oil composition of the present invention is administered each time the animal is fed in an amount that achieves the above-mentioned daily dosage during the animal's feeding.

[0125] The methods of the present invention may comprise administration of the asparagopsis oil compositions of the present invention for at least 5, 10, 25, 50, 100, 250, or 350 days according to the dosage regimen described above. An aspect of the present invention resides in the fact that the methods of the present invention provide very sustained effectiveness in reducing intestinal methanogenesis, e.g., do not decrease in effectiveness over long periods of treatment, e.g., increase the resistance of ruminal or digestive tract microorganisms, thereby making long-term treatment of ruminants particularly viable.

[0126] Thus, given the significant benefits of freeze-dried milled asparagopsis, including reduced total gas production and CH4 production, in one aspect, the asparagopsis oil compositions of the present invention are preferably administered in a form that provides the benefits described herein (e.g., reduced CH4 production) without affecting nutritionally important fermentation parameters.

[0127] Among the diverse secondary metabolites produced by Asparagopsis, bromoform (CHBr3) is one of the most abundant and biologically active. However, as a purified chemical, bromoform and chemically related compounds (e.g., bromochloromethane) are unsafe and not approved for human or animal applications, including the inhibition of methanogenesis in ruminants.

[0128] Intact, freeze-dried, ground asparagopsis biomass can be used as a feed supplement to inhibit methanogenesis in ruminants, but careful processing is required before feeding to minimize loss of bioactive compounds, such as bromoform, and maintain their activity. The currently most effective method for producing asparagopsis biomass for use as a feed supplement is rapid freezing followed by freeze-drying. However, we have previously found that if the intact biomass is not rapidly frozen and / or air-dried, significant losses of bromoform occur. Furthermore, freeze-dried biomass stored at ambient temperature (66% loss) or refrigerated conditions (32% loss) suffer significant losses after 6 months, making freezing the freeze-dried material (20% loss) the only practical option for long-term storage (>6 months) (Figure 2). These processing and storage conditions are described in Vucko et al. (J. Appl. Phycol. 2017, 29, 1577-1586).

[0129] Surprisingly, the inventors have found that asparagopsis oil compositions produced by contacting asparagopsis biomass with oil retain high levels of bioactives for extended periods of time, even when stored at either room temperature or 4° C. For example, as discussed above, Figure 3 shows that asparagopsis oil compositions, with or without homogenization, produced by contacting asparagopsis biomass with oil retain high levels of bioactives for extended periods of time when stored at room temperature or 4° C. Thus, the methods of the present invention can produce asparagopsis oil compositions in a form suitable for long-term storage (greater than 6 months) at room temperature.

[0130] The present invention results in a significant reduction in the loss of bioactive components that would otherwise occur during transportation, drying, and / or storage of wild-harvested Asparagopsis biomass. The present invention also reduces the number of steps required to obtain a blended product of preserved intact biomass or biomass extract, and therefore represents a substantial improvement over current processing techniques.

[0131] In another aspect, the asparagopsis oil compositions of the present invention are preferably administered in a form in which the secondary metabolites remain effective (eg, therapeutically effective).

[0132] The asparagopsis oil composition of the present invention may be administered to ruminants in one of many ways. The asparagopsis oil composition of the present invention can be administered in solid form as a veterinary formulation, dispersed in an excipient and fed directly to the animal, physically mixed with feed material in dry form, or the asparagopsis oil composition of the present invention may be provided as a solution and then sprayed onto the feed material. Methods for administering the asparagopsis oil composition of the present invention to animals are considered to be within the skill of those skilled in the art.

[0133] When used in combination with a feed material, the feed material is preferably grain / hay / silage / grass-based. Included among such feed materials are improved and / or tropical grass or legume-based forages, feed ingredients, and by-products of the food or feed industry and by-products of the biofuel industry, as well as corn meal, and mixtures thereof or feedlot and dairy feeds, such as those with a high grain content, either fed directly or produced as preserved feed hay.

[0134] The time of administration is not critical as long as the methane production reducing effect is demonstrated. Administration can be at any time as long as the feed is retained in the rumen. However, since the asparagopsis oil composition of the present invention is preferably present in the rumen at approximately the time when methane is produced, the asparagopsis oil composition of the present invention is preferably administered with or immediately before feeding.

[0135] In a particular embodiment of the present invention, an effective amount of the asparagopsis oil composition of the present invention is administered to a ruminant by supplementing the feed intended for the animal with an effective amount of the asparagopsis oil composition of the present invention. "Supplementing" in the sense of the present invention refers to the act of directly incorporating an effective amount of the asparagopsis oil composition of the present invention into the feed intended for the animal. Thus, when fed, the animal ingests the asparagopsis oil composition of the present invention, which can then act, for example, to increase the digestibility of the fiber and / or grain contained in the animal's feed.

[0136] Another subject of the present invention therefore relates to a feed supplement for ruminants comprising the asparagopsis oil composition of the present invention.

[0137] In another aspect, the present invention also provides a feed supplement for reducing total gas production and / or methane production in a ruminant, the supplement comprising an effective amount of an asparagopsis oil composition of the present invention.

[0138] In one embodiment, an effective amount of the asparagopsis oil composition of the present invention is administered to a ruminant by supplementing food intended for the animal with an effective amount of the asparagopsis oil composition of the present invention.

[0139] In one aspect, the present invention maintains the amount of VFAs in ruminants, thus allowing ruminants to maintain energy from, for example, a fiber and grain based diet while reducing total gas and CH production, resulting in maintained metabolically available energy even when starting from the same caloric intake.

[0140] This is advantageous to the farmer as it allows for the optimization of feed cost per unit of available metabolizable energy, which also represents a substantial economic benefit.

[0141] The inventors have previously demonstrated that administration of an effective amount of freeze-dried, ground asparagopsis to ruminants does not adversely affect voluntary feed intake, and therefore the present invention includes methods in which the amount of organic matter and / or dry matter decomposed is maintained.

[0142] The term "animal feed supplement" refers to a concentrated additive premix containing an active ingredient; the premix or supplement may be added to an animal feed or feed to form a supplemented 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 supplement of the present invention is in the form of a powder or a compressed or granulated solid. In practice, livestock may be fed supplemented animal feed by adding the supplement directly to the feed, usually as a so-called top dressing, although the supplement may also be used in the manufacture or production of products such as formulated animal feed or lick blocks, which are described in more detail below. The present invention is not particularly limited in this respect. The supplement according to the present invention is typically fed to animals at a rate of 16 to 2500 g / animal / day.

[0143] In one embodiment, a supplement according to the present invention is administered in an amount based on the actual individual animal intake (eg, g / kg DM intake).

[0144] The animal feed supplements of the present invention comprise the asparagopsis oil composition of the present invention and are formulated so that, when added to the feed, the asparagopsis oil composition is present in an amount equivalent to at least 0.067, 0.125, 0.25, 0.5, 1, 2, 3, 5, 10, or 16.67% of the organic matter of the feed.

[0145] For example, if a ruminant consumes approximately 5 kg of organic matter per day, a feed supplement containing the asparagopsis oil composition of the present invention is formulated to be added to the feed so that 208 mL of the asparagopsis oil composition of the present invention is administered per day. To achieve 0.1% of the organic matter administered to the ruminant, 208 mL of 30 g fw / 100 mL of the asparagopsis oil composition of the present invention is required. To achieve 0.1% of the organic matter administered to the ruminant, 104 mL of 60 g fw / 100 mL of the asparagopsis oil composition of the present invention is required. To achieve 0.1% of the organic matter administered to the ruminant, 52 mL of 120 g fw / 100 mL of the asparagopsis oil composition of the present invention is required. To achieve the equivalent of 1% of the organic matter administered to ruminants, 2082 mL of a 30 g fw / 100 mL asparagopsis oil composition of the present invention would be required, 1041 mL of a 60 g fw / 100 mL asparagopsis oil composition of the present invention would be required, and 520 mL of a 120 g fw / 100 mL asparagopsis oil composition of the present invention would be required to achieve the equivalent of 1% of the organic matter administered to ruminants.

[0146] In a preferred embodiment of the invention, the supplement comprises 10-100% by weight of the asparagopsis oil composition of the invention, and preferably this amount is greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97 or 99% by weight.

[0147] It is within the skill of one of ordinary skill in the art to accurately determine the ideal amounts of ingredients to include in the supplement and the amounts of supplement to be used in the manufacture of feed or compound animal feed, taking into account the specific animal type and circumstances under which production will occur. Preferred dosage amounts for each of the ingredients are given herein.

[0148] The animal feed supplement of the present invention may contain additional ingredients without departing from the scope of the present invention. This may include well-known excipients that are generally necessary to produce the desired product form, and may also include additional additives aimed at improving the quality of the feed and / or the performance of the animal consuming the supplement. Suitable examples of such excipients include carriers or fillers such as lactose, sucrose, mannitol, starch, crystalline cellulose, sodium bicarbonate, and sodium chloride, as well as binders such as gum arabic, gum tragacanth, 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 enhancers, and gastrointestinal flora stabilizers.

[0149] The inventors have demonstrated that freeze-dried and ground Dictyota, Oedogonium, and Cladophora patentiramea reduce total gas production and CH4 production from ruminal fermentation. Thus, in another aspect, the method further comprises administering to the ruminant an effective amount of at least one species of macroalgae selected from the group consisting of Asparagopsis armata, Asparagus nigricans, Dictyota (e.g., Dictyota bartayresii), Dictyota, Ulva, and Cladophora patentiramea.

[0150] Thus, in one aspect, the present invention relates to a method for reducing total gas production and / or methane production in a ruminant animal, comprising administering to the ruminant an effective amount of an Asparagopsis oil composition of the present invention, wherein the composition comprises marine macroalgae or a marine macroalgae oil extract.

[0151] In one aspect, the marine macroalgae of the composition is selected from the group consisting of Asparagopsis armata, Asparagus nigricans, Dictyostelium (e.g., Dictyostelium macrocarpa), Asparagus, Ulva, and Cladophora patentyramea.

[0152] A further aspect of the present invention relates to products such as formulated animal feeds and lick blocks that contain the supplements described hereinbefore.

[0153] The term "formulated animal feed composition" refers to a composition suitable for use as an animal feed and containing a mixture of various natural or non-natural bases or ingredients and / or additives. Therefore, the term "formulated" is used specifically herein to distinguish the animal feed compositions of the present invention from natural ingredients. These mixtures or compound feeds are formulated according to the specific requirements of the target animal. The main ingredients used in commercially produced compound feeds typically include bran, rice bran, corn meal, cereal grains such as barley, wheat, rye, oats, soybean meal, alfalfa meal, cottonseed meal, wheat flour, etc. Commercially available compound feeds will typically contain 15% or more crude protein and 70% or more digestible total nutrients, although the present invention is not particularly limited in this regard. Liquid, solid, and semi-solid compound 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 produced as meal types, pellets, or crumbles. In practice, livestock may be fed a combination of a compound feed such as that of the present invention with silage, hay, or the like. Typically, compound animal feed is fed at 0.3 to 10 kg / animal / day. It is within the skill of one skilled in the art to determine the appropriate amounts of these ingredients to include in a compound animal feed, taking into account the type of animal and the circumstances under which feeding occurs.

[0154] The formulated animal feed composition of the present invention may contain additional feed additives commonly used in the art. As known to those skilled in the art, the term "feed additive" in this context refers to products used in animal nutrition to improve the quality of feed and food of animal origin or to improve animal performance, for example, by enhancing the digestibility of feed ingredients. Non-limiting examples include technical additives such as preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators, and silage additives; sensory additives, especially flavorings and colorants; (additional) nutritional additives, such as vitamins, amino acids, and trace elements; and (additional) animal husbandry additives such as digestibility enhancers and gut flora stabilizers.

[0155] As will be apparent to one skilled in the art, the formulated animal feed compositions of the present invention can include additional ingredients or additives without departing from the scope of the present invention.

[0156] In a further aspect, the present invention provides lickstones or lickblocks comprising the asparagopsis oil composition of the present invention. As known to those skilled in the art, such lickstones or blocks are particularly advantageous for providing mineral supplements (as well as protein and carbohydrates) to ruminants grazing on either or both natural and cultivated grasses. Such lickblocks or lickstones according to the present invention typically contain, in addition to the asparagopsis oil composition of the present invention, various types of binders, such as cement, gypsum, lime, calcium phosphate, carbonate, and / or gelatin; and additional additives such as vitamins, trace elements, mineral salts, sensory additives, etc.

[0157] A further aspect of the present invention relates to a method of reducing gastrointestinal methane production in ruminants, the method comprising administering a composition comprising the asparagopsis oil composition of the present invention.

[0158] The terms "reducing gastrointestinal methanogenesis" and "reducing gastrointestinal methane production" refer to the reduction of methane gas production in the gastrointestinal tract. As mentioned above, fermentation in the rumen and digestive tract of ruminants leads to the production of methane gas by so-called methanogens. The present invention aims to reduce this process, for example, by directly reducing methane emissions from the gastrointestinal tract. Estimating methane emissions by animals is within the knowledge and skill of a person skilled in the art. As mentioned above, methane production in the rumen and digestive tract is a normal process in healthy animals, and reducing methanogenesis does not change the general health of the ruminant.

[0159] The method of treatment of the present invention is therefore a non-therapeutic method of treatment, i.e., it does not improve the health of animals suffering from a particular condition, does not prevent a particular disease or condition, and does not affect the health of ruminants in any other way to any extent, i.e., compared to ruminants not subjected to the method of treatment of the present invention. The advantages of the method of the present invention are, as explained previously, limited to environmental and / or economic aspects.

[0160] As will be apparent from the foregoing, the methods of the present invention involve oral administration of the asparagopsis oil compositions of the present invention. Preferably, the treatment involves oral administration of the formulated animal feed composition and / or animal feed supplement product described hereinabove, although one of skill in the art will appreciate that other liquid, solid, or semi-solid orally ingestible compositions may be used without departing from the scope of the present invention.

[0161] A still further aspect of the present invention relates to the use of a composition comprising the asparagopsis oil composition of the present invention for the non-therapeutic reduction of gastrointestinal methane production in ruminants.

[0162] In another aspect, the present invention also provides a feed for a ruminant animal, the feed being supplemented with the feed supplement of the present invention.

[0163] In another aspect, the present invention provides a method for reducing methane production by a ruminant animal, the method comprising administering to the animal a feed supplement of the present invention or a feed of the present invention.

[0164] The term "shelf life" is used to indicate that the amount of bioactive (e.g., antimethanogenic) agent in the at least one oil does not decrease significantly over that period of time at the temperature at which the composition is stored. In other words, the time that elapses before there is a significant reduction in the amount of bioactive (e.g., antimethanogenic) agent is understood to be indicative of shelf life. The term "stability" is also used as an indicator of shelf life. The "stability" of at least one bioactive (e.g., antimethanogenic) agent can be estimated by measuring the amount of antimethanogenic agent over time.

[0165] The amount of bioactive (e.g., antimethanogenic) agent extracted into the oil typically decreases over time, although this will depend on storage conditions. The decrease in amount can be due to a number of reasons, such as evaporation or sublimation of the bioactive (e.g., antimethanogenic) agent from the oil. This can also be due to reaction of the bioactive (e.g., antimethanogenic) agent with itself, with water remaining in the oil extract, with oxygen or other gases in the air, with other components in the oil, or with the oil itself, which may be mediated by light and / or heat, as is typical for many halogenated compounds. The amount of bioactive (e.g., antimethanogenic) agent reduced that is still acceptable for consumption will vary depending on several factors, such as the end use of the product and any degradation by-products that may be produced. For example, after storage at either 4° C. or 25° C., the amount of at least one bioactive (e.g., anti-methanogenic) agent may be reduced by no more than 5%, 10%, 20%, 30%, 40%, or 50%, and still be acceptable for use. In one embodiment, the amount of at least one bioactive (anti-methanogenic) agent may be reduced by no more than 20% after 65 weeks of storage at 25° C. In another embodiment, the amount of at least one anti-methanogenic agent may be reduced by no more than 50% after 65 weeks of storage at either 4° C. or 25° C.

[0166] Abbreviations Throughout this patent specification, several abbreviations are used. For the avoidance of doubt, these abbreviations are defined below. ANOVA analysis of variance BCA Bromochloroacetic acid BF Bromoform d day DBA Dibromoacetic acid DBCM Dibromochloromethane DCM dichloromethane dw dry weight eq. equivalent quantity fw fresh weight GC Gas Chromatography HPLC High-Performance / High-Pressure Liquid Chromatography IS internal standard MS mass spectrometry MTBE Methyl tert-butyl ether n number of samples OM organic matter RT room temperature VFA Volatile Fatty Acids wk week

[0167] The present invention is further illustrated by the examples, which serve to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. [Example]

[0168] Example 1 Preparation of an Asparagopsis Oil Composition Asparagopsis taxiformis (gametophyte stage) was collected from Magnetic Island (QLD, Australia). Asparagopsis armata was collected from Cloudy Bay (Bruny Island, Tasmania, 43°.43'94"S; 147°.21'.52"E). Fresh biomass was blotted dry and samples (6 x 30.0 g fresh weight (fw)) were placed in 250 mL glass bottles (Schott) pre-filled with 100 mL of vegetable oil (Homebrand blend vegetable oil; 95% canola oil, 5% sunflower oil). Control samples (6 x 30.0 g fresh weight (fw)) were also placed in bottles filled with Milli-Q water instead of vegetable oil. All bottles were tightly capped, kept on ice, and promptly transported to the laboratory for further processing.

[0169] Biomass samples (6 x 30.0 g fw) were also collected in Ziploc bags for determination of dry weight (dw) and bromoform content in biomass (3 x 30.0 g fw).

[0170] In the laboratory (<4 h after collection), biomass in each solvent (oil or water) was homogenized by crushing for 60 seconds with an IKA Ultra-Turrax T-25 (n = 3 for each solvent). The remaining three biomass samples for each solvent were not homogenized. All bottles (n = 12) were then stored in a refrigerator (4 °C). At 1, 3, 5, 7, and 10 days after collection (day 0), the content of bromoform, a typical antimethanogenic agent present in algae, was analyzed.

[0171] For bromoform analysis, 1.5 mL of extraction solvent (oil or water) was removed from each bottle and centrifuged (12,000 g, 1 min) to remove solids. Bromoform (BF) was extracted from the resulting 1.0 mL clarified solution using either methyl tert-butyl ether (MTBE) or methanol, as described below.

[0172] For samples using water as the solvent, bromoform was extracted from 1.0 mL of sample using 1.0 mL of naphthalene-ether solution (10 μg / mL naphthalene in methyl tert-butyl ether (MTBE)). Naphthalene served as the internal standard, and MTBE was the extraction solvent. The MTBE phase was collected and analyzed by GC / MS, as described below.

[0173] For samples using oil as the solvent, bromoform was extracted from 1.0 mL of sample using 1.0 mL of naphthalene-methanol solution (10 μg / mL naphthalene in HPLC-grade methanol). Samples were partitioned for 2 h at 4 °C before analysis of the methanol phase. Partition conditions were selected based on previous experiments with various solvents (hexane, methanol, MTBE, DCM) and times (30 min to 48 h). The methanol phase was collected and analyzed by GC / MS as described below.

[0174] The experimental design for oil or water extraction of halogenated metabolites in unhomogenized (untreated) or homogenized Uncaria spp. biomass is shown below.

[0175] [ka]

[0176] For determination of dry weight and bromoform content, the biomass was quickly frozen and then freeze-dried (Virtiz benchtop 2K, -55°C, 120 μbar, 48 h). The dried biomass was weighed to determine the dry weight, then ground to 1 mm particles and stored in sealed jars at -20°C.

[0177] Bromoform was quantified by GC / MS (Agilent 7890c equipped with a Zebron ZB-wax capillary column (30 m × 0.25 mm × 0.25 μm) Phenomenex, Australia) according to the method of Paul et al. (Mar. Ecol. Prog. Ser. 306 (2006) 87-101), modified by Machado et al. (J Appl Phycol. 28 (2016) 3117-3126). Briefly, analytical conditions were pulse injection (1 μL, 35 psi) in splitless mode, with temperature parameters as described by Paul et al.: injection port: 250 °C, GC / MS interface: 300 °C, and oven (40 °C for 1 min, ramp to 250 °C at 16 °C / min, 2 min at 250 °C). Helium was used as the carrier gas at 2 mL / min. Separate standard curves were completed for each method (water, oil, biomass), and the concentration of the target compound in each sample was calculated from the ratio of the target compound's peak area relative to the internal standard. Bromoform was identified by comparison with a commercial standard (Sigma-Aldrich, Australia) and based on a characteristic ion fragment (molecular ion cluster at m / z 250, 252, 254, 256 [1:2:2:1]). The concentration of bromoform in solution was normalized to the amount of dry biomass extracted and reported as mg bromoform / g dw biomass.

[0178] The dry weight of a 30 g sample of absorbed and dried asparagopsis was determined to be approximately 3 g. Figure 1 shows the results of various extraction methods (solvent - water, oil; biomass - untreated, homogenized / crushed) in which the biomass was contacted with a solvent (water or oil) for up to 10 days. Results are plotted as the amount of BF extracted (mg) per gram of dry weight of algae. For comparison, BF was also extracted using a previously described method. In this method, the biomass sample was freeze-dried and then contacted with methanol for 72 hours, resulting in the extraction of BF into methanol.

[0179] Figure 1 shows that extraction of asparagopsis biomass with water yields less BF than extraction with oil, and that the amount of BF extracted from asparagopsis biomass into oil is increased compared to the amount of BF extracted from the same amount of biomass into water. This data demonstrates that the bioactive agent BF was extracted from asparagopsis biomass into oil, forming an asparagopsis oil composition.

[0180] Similar results were obtained with untreated (unhomogenized) and homogenized biomass when water was used as the extraction solvent: the amount of BF extracted from these samples was approximately 50% less after 24 hours and approximately 30–40% less over the soaking period (up to 10 days) compared to when oil was used as the solvent.

[0181] Figure 1 also shows that homogenizing the biomass in oil maximized bromoform extraction in the shortest period (1 day), and that the BF content in oil from untreated (unhomogenized) biomass reached a similar level to that of homogenized biomass after approximately 7 days of soaking / extraction. This data demonstrates that the extraction rate of the bioactive agent BF from asparagopsis biomass to oil is increased by homogenizing the biomass in at least one oil.

[0182] Figure 1 also shows that the extraction efficiency using oil depends on whether the biomass was intact or homogenized. Surprisingly, extraction of homogenized biomass with oil for 24 hours yielded greater BF than extraction of freeze-dried biomass with methanol for 72 hours. As can be seen from Figure 1, the freeze-dried sample extracted by soaking in methanol for 72 hours yielded only approximately 15 mg BF / g algae (dw), whereas the homogenized algae sample soaked in oil for only 24 hours yielded approximately 17.8 mg BF / g algae (dw), a significant improvement over extraction of freeze-dried biomass with methanol. This data demonstrates that the amount of bioactive BF extracted from Asparagopsis biomass into oil is increased by homogenizing the biomass in at least one oil.

[0183] Example 2 Shelf Life of Asparagopsis Oil Compositions The shelf life (e.g., stability) of the asparagopsis oil compositions of the present invention was investigated. The percentage of bioactive compounds, such as bromoform (BF) and dibromochloromethane (DBCM), was examined as a function of time and compared to an internal standard (IS: naphthalene). A decrease in percentage indicates loss of compound and a decrease in the stability of the bioactive oil composition, leading to a shortened shelf life.

[0184] Asparagopsis biomass was extracted in oil or water for 10 days, after which the biomass was removed. The asparagopsis oil composition (including the biomass) was filtered through a 100 μm nylon mesh, followed by centrifugation (3200 g, 15 min). The clarified solution extracted with oil or water was divided into two 30 mL glass bottles (Schott), one of which was stored at 4°C and the other at 25°C (n=3 per temperature, total of 24 bottles). After 0, 4, 8, 12, and 65 weeks of storage, samples (1 mL) were collected and analyzed as described above. The day of the start of storage was designated as week 0, and the experimental design is shown below.

[0185] [ka]

[0186] The bromoform content of the freeze-dried biomass was also analyzed on the same schedule, with week 0 being the day the biomass was removed from the freeze-dryer.

[0187] When freeze-dried biomass stored at -20°C according to standard storage methods was extracted using standard methanol extraction methods, no loss was observed over time (data not shown).

[0188] Figure 3 shows the stability of the asparagopsis oil composition of the present invention over time. Water extracts showed significant loss of the bioactive compound bromoform after 12 weeks of storage at both 4°C and 25°C. Therefore, the water samples were not further analyzed.

[0189] In contrast, the amount of the oil-extracted bioactive bromoform did not decrease significantly over 12 weeks, whether homogenized or unprocessed, regardless of storage temperature (4°C or 25°C) (Figure 3). Surprisingly, there was no significant decrease in bromoform after 65 weeks in asparagopsis oil compositions stored at 4°C, either homogenized or unprocessed. Asparagopsis oil compositions stored at 25°C for 65 weeks lost 15-20% of the extracted bromoform. The apparent "increase" in BF over time may be due to analytical variance. This data demonstrates that asparagopsis oil compositions formed by extracting asparagopsis with oil are stable over long periods of time. In particular, this data demonstrates that the amount of the bioactive BF is stable over long periods of time in asparagopsis oil compositions formed by extracting asparagopsis with oil.

[0190] The DBCM:IS ratio over time is shown in Figure 4 (untreated biomass) and Figure 5 (homogenized biomass). DBCM was detected throughout the 65-week storage period. Importantly, there was no significant difference in the DBCM:IS ratio between treatments (untreated vs. homogenized, 4°C vs. 25°C), indicating excellent shelf life for all samples when used as a marker for antimethanogenic agents. The apparent "increase" in the DBCM:IS ratio over time may be due to analytical variance. This data indicates that the amount of DBCM is stable over time in asparagopsis oil compositions formed by extracting asparagopsis with oil.

[0191] Figures 6 and 7 demonstrate that the bioactive agent DBA was extracted from asparagopsis biomass into oil to form an asparagopsis oil composition.

[0192] These results demonstrate that the clarified oil composition (i.e., from which the asparagopsis biomass has been removed) has a stable shelf life of at least one year (65 weeks) at 4°C and at least 12 weeks at room temperature (25°C). In contrast, the water extract was stable for 8 weeks but deteriorated thereafter, regardless of storage temperature. This result is surprising, as storage at lower temperatures would be expected to extend shelf life, regardless of the storage medium (water or oil).

[0193] Example 3 Maximizing Biomass Extraction to Oil The maximum amount of extractable Asparagopsis biomass was determined using the extraction method of Example 1 (e.g., homogenization and extraction into oil) which provided the highest concentration of bioactive (bromoform) detected in the oil in the shortest amount of time.

[0194] Asparagopsis taxiformis (gametophyte stage) was collected on Magnetic Island (QLD, Australia). Fresh biomass was blotted dry, and the following samples (30.0 g, 60.0 g, 90.0 g, and 120.0 g fw) were placed in 250 mL glass bottles pre-filled with 100 mL of vegetable oil (n = 3 each). All bottles were tightly capped, stored on ice, and promptly transported to the laboratory. Samples (n = 3) of the blotted dry biomass (30.0 g fw) were also collected for dry weight determination, as described in the experimental design below.

[0195] [ka]

[0196] Biomass was homogenized in oil (IKA ultra-turrax T-25) in the laboratory as described above, and samples were stored at 4°C for 24 h before bromoform quantification.

[0197] Figures 8 and 9 show the amount of bioactive (bromoform) extracted from various amounts of asparagopsis biomass (in the same volume of oil). Figure 8 shows that the amount of bioactive extracted into the oil increased with increasing asparagopsis biomass to oil ratio. The data show that increasing the biomass to oil ratio increases the amount of the bioactive agent, bromoform, transferred from the asparagopsis biomass to the oil.

[0198] At higher biomass-to-oil ratios, a solid, opaque gel formed, making it difficult to fully homogenize the biomass in the oil. For example, when 90 g or 120 g of homogenized biomass was homogenized in 100 mL of oil, a solid, opaque gel formed. Because of this gelation, separating even 1 mL of clarified oil was extremely laborious and required partitioning with methanol for GC / MS analysis.

[0199] A 120 g sample containing the gel was heated (1 hour, 60°C, water bath) prior to centrifugation. This resulted in a 20% increase in the yield of bioactives in the oil compared to the unheated case (see Figures 8 and 9). Asparagopsis typically contains water-soluble sulfated cell wall polysaccharides. Without wishing to be bound by theory, it is hypothesized that a gel / emulsion formed between the extracted polysaccharides, the oil, and the external and internal water remaining on and within the biomass. At higher biomass-to-oil ratios, more polysaccharides were extracted, leading to the formation of a gel. The gel apparently contained bioactive agents (e.g., bromoform), and heating caused the gel to release the bromoform, which then migrated to the oil in the asparagopsis oil composition.

[0200] Example 4: Investigation of Asparagopsis oil compositions for reducing methane production in vivo In vivo feeding was performed on fistulated steers (Bos indicus). All animals were fistulated and trained in a breathing chamber prior to the start of the experiment. Initially, steers were housed on dry Flinders grass in a group pen for four days. Steers were then assigned to two groups: a control group (dry Flinders grass only) and a group supplemented with the asparagopsis oil composition. The dosage of the asparagopsis oil composition was selected based on the results of previous in vitro experiments examining its potential for methane reduction.

[0201] The blotted-dried asparagopsis had a fresh-to-dry weight ratio of 10 (i.e., 30 g fw = 3 g dw). Assuming an organic matter (OM) content of 80% of dw (Machado et al., 2014), the OM content of asparagopsis-derived OM in the tested biomass / oil mixtures ranged from 0.024 to 0.096 g OM / mL (Table 1).

[0202] [Table 1]

[0203] ** This volume is based on the oil separated from the biomass. For example, adding 100 mL of oil to 120 g of seaweed (fw) increases the final volume of the mixture. Assuming that the mixture is 90% water (fw:dw ratio of 10) and that the density of asparagopsis is equal to that of water for ease of calculation, the volume of the biomass and water mixture increases according to the amount of biomass added (i.e., 100 mL of oil + 150 g of asparagopsis (fw) = 250 mL of mixture, whereas 15 g of asparagopsis (dw) = 12 g of asparagopsis (OM) / 250 mL = 0.048 g of asparagopsis (OM) / mL of homogeneous mixture).

[0204] Based on the data presented above, a biomass:oil ratio greater than 150 g fw:100 mL cannot form a homogeneous mixture. A dose of 1% OM Asparagopsis is practical for biomass:oil ratios of 120 g or more per 100 mL. A dose of 0.2% OM Asparagopsis has been demonstrated to be effective in inhibiting in vivo methanogenesis in animals; therefore, for a 120 g fw biomass / 100 mL oil mixture, only 2.08 mL of oil (or 4.6 mL of biomass / oil mixture) needs to be added to 100 g of feed.

[0205] To calculate the volume of oil required to administer to a ruminant animal to achieve a desired organic matter equivalent, the organic matter percentage of the asparagopsis oil composition of the present invention is calculated from the fresh weight (fw) of asparagopsis biomass in contact with a specified volume of at least one oil. The fresh weight to dry weight (dw) ratio of imbibed asparagopsis is 10 (i.e., 30 g fw = 3 g dw). Based on previous data, assuming an organic matter (OM) content of 80% of dw, the corresponding OM content of asparagopsis origin in the biomass / oil can be calculated. For example, an asparagopsis oil composition containing 30 g fresh weight (fw) asparagopsis biomass in 100 mL of at least one oil corresponds to 0.024 asparagopsis organic matter / mL. If the desired amount in the diet is 0.1% asparagopsis OM in 100 g of diet, then 4.17 mL of the 30 g fw / 100 mL asparagopsis oil composition would be required per 100 g of diet.

[0206] Steers were assigned to individual pens in the experimental building with ad libitum access to flindersgrass hay and water. Animals receiving the asparagopsis oil composition were fed directly to the rumen before the morning feeding, or the asparagopsis oil composition was added to the diet as described above, to ensure complete intake and consistency of treatment intake between animals. A 14-day acclimation period to the different diets was allowed before placing the animals in an open-type respiration chamber for 48-hour methane production measurements. Animal methane production was measured 7, 14, 21, and / or 29 days after treatment to assess the efficacy of the asparagopsis oil composition in reducing animal methane production over time. After 31 days, the asparagopsis oil composition was discontinued, and the animals were reassigned to paddocks. To assess changes in VFA production and acetate to propionate ratios, rumen samples are collected 4 hours after ruminal insertion of the asparagopsis oil composition or after feeding (e.g., on days 1, 15, 22, and 30 of asparagopsis oil composition treatment). Live weights and offered and refused feed are measured daily, and total dry matter (DM) intake and total organic matter (OM) intake are calculated to determine average individual DM and OM intake. Average methane production is calculated for all time points tested.

[0207] Example 5: Investigation of Asparagopsis oil compositions for in vivo reduction of methane production in sheep method Merino-cross steers were assigned to one of five groups based on the daily dose of Asparagopsis oil composition (organic, OM basis): 0 (control), 0.1, 0.5, 1.0, 2.0, 3.0%. The dose (% OM intake) was calculated using the calculations shown in Table 1 and Example 4.

[0208] The sheep were maintained under the same conditions as in the animal housing and fed a pelleted commercial sipper diet based on lupin, oats, barley, and wheat, with cereal straw as the roughage component [chemical composition: ash 72 (g / kg DM), crude protein (CP) 112, neutral detergent fiber (aNDFom) 519, acid detergent fiber (ADFom) 338, no cobalt, selenium, or rumen improvers] at a maintenance rate of 1.2 times throughout the experiment. All sheep were fed Co-Brett before the start of the experiment.

[0209] Over the first two weeks, sheep are gradually adapted to the asparagopsis oil composition by mixing the ground material with 200 g of crushed lupin (lupin diet). The asparagopsis oil composition / lupin mix is then added to the pelleted feed, mixed, and fed for a further 75 days.

[0210] Food intake is recorded daily and live weight (LW) is measured at 14 day intervals for the duration of the experiment.

[0211] Methane production (g / kg DM intake) was measured for each individual animal three times, once 30 days after inclusion of the asparagopsis oil composition and then at 21-day intervals throughout the experiment. Diet (pellets / lupin) was proportionally reduced to 1.0x maintenance dose to ensure consistent intake during 24-hour methane measurements using an open respiration chamber, as described in Li (2013) [PhD thesis; Eremophila glabra reduced methane production in sheep, University of Western Australia].

[0212] Following methane measurements, up to 50 mL of ruminal fluid is collected using a stomach tube for measurement of volatile fatty acid (VFA) concentrations.

[0213] statistical analysis Statistical analyses were performed by fitting linear mixed models to each response variable. These models could account for the experimental design (assignment of animals to specific groups and chambers), the structure of the data (repeated measures), and any missing values that may arise. The "fixed effects" in the mixed models consisted of the effect of treatment (various doses of Asparagopsis taxiformis), the effect of time (date of sampling), the interaction between treatment and time, and covariates. When analyzing live weights, initial live weight was included as a covariate. It was also tested as a potential covariate for the other response variables.

[0214] The mean is calculated for all combinations of treatment and time, adjusting for all other terms in the model. P values are calculated to test the global effects of time, treatment, and their interactions. The least significant difference (P=0.05) is calculated for pairwise comparisons of means.

[0215] Measure the following: Intake of food containing asparagopsis oil composition Dry matter degraded in vivo Animal live weight Total VFA concentration Molar percentage of individual VFA I (including acetate and propionate), excluding isobutyrate Methane emissions (g / kg DM intake) The following claims as originally filed in this application are appended as embodiments. [1] A method for producing an Asparagopsis oil composition, the method comprising: (a) providing Asparagopsis biomass; (b) providing at least one oil; and (c) contacting the biomass with the at least one oil under conditions to extract at least one bioactive agent from the biomass into the at least one oil to form the Asparagopsis oil composition. [2] The method according to [1], wherein step (c) comprises homogenizing the biomass in the at least one oil. [3] The method according to [1], further comprising, after step (c), separating the biomass from the at least one oil. [4] The method according to [1], wherein the ratio of the biomass to the at least one oil is more than 0.3 g:1 mL, more than 0.6 g:1 mL, more than 0.9 g:1 mL, or more than 1.2 g:1 mL. [5] The method according to [1], wherein step (c) is carried out for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. [6] The method according to [1], wherein step (c) is carried out at about 4°C. [7] The method according to any one of [3] to [6], wherein, prior to the step of separating the biomass from the at least one oil, the biomass in contact with the at least one oil is heated so that a gel containing the at least one bioactive agent releases the at least one bioactive agent into the at least one oil. [8] The method of [7], wherein the biomass in contact with the at least one oil is heated to 60°C. [9] The method of [7] or [8], wherein the biomass in contact with the at least one oil is heated for 1 hour.

[10] The method according to [1], wherein the Asparagopsis is Asparagopsis taxiformis and / or Asparagopsis armata.

[11] The method according to [1], wherein the step of preparing Asparagopsis biomass does not include air-drying the biomass.

[12] The method of [1], wherein the step of preparing Asparagopsis biomass includes collecting the biomass into the at least one oil.

[13] The method of [1], wherein the at least one oil comprises an edible oil.

[14] The method of

[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, 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 a combination thereof.

[15] The method according to [1], wherein the at least one bioactive agent is selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

[16] The method of [1], wherein the amount of the at least one bioactive agent extracted from the biomass into the at least one oil is increased relative to the amount of the at least one bioactive agent extracted into water from an equivalent amount of biomass.

[17] The method of [1], wherein the amount of the at least one bioactive agent extracted from the biomass into the at least one oil does not significantly decrease after storage at 25°C for 12 weeks.

[18] The method of [1], wherein the amount of the at least one bioactive agent extracted from the biomass into the at least one oil does not significantly decrease after 65 weeks of storage at 4°C.

[19] The method according to any one of [1] to

[18] , wherein the at least one physiologically active agent is an anti-methanogenic agent.

[20] The method according to

[19] , wherein the antimethanogenic agent is BF and / or DBCM.

[21] The method of

[19] , wherein the amount of the antimethanogenic agent extracted from the biomass into the at least one oil does not decrease by more than 20% after 65 weeks of storage at 25°C, and the at least one antimethanogenic agent is BF and / or DBCM.

[22] The method of [1], wherein the Asparagopsis oil composition contains at least 0.1 mg of bromoform per mL of extract.

[23] The method of [1], wherein the Asparagopsis oil composition contains at least 1 mg of bromoform per mL of extract.

[24] The method of [1], wherein the Asparagopsis oil composition contains at least 2 mg of bromoform per mL of extract.

[25] The method of [1], wherein the Asparagopsis oil composition contains at least 3 mg of bromoform per mL of extract.

[26] The method of [1], wherein the Asparagopsis oil composition contains at least 4 mg of bromoform per mL of extract.

[27] An Asparagopsis oil composition produced by the method according to any one of [1] to

[26] .

[28] An Asparagopsis oil composition comprising at least one oil and at least one antimethanogenic agent.

[29] The Asparagopsis oil composition according to

[28] , wherein the Asparagopsis is Asparagopsis taxiformis and / or Asparagopsis armata.

[30] The Asparagopsis oil composition of

[28] , wherein the at least one oil comprises an edible oil.

[31] The Asparagopsis oil composition of

[30] , 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, 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 combinations thereof.

[32] The Asparagopsis oil composition of

[28] , wherein the at least one antimethanogenic agent is selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

[33] The Asparagopsis oil composition according to

[28] , wherein the amount of the at least one antimethanogenic agent does not significantly decrease after storage at 25°C for 12 weeks.

[34] The Asparagopsis oil composition according to

[28] , wherein the amount of the at least one antimethanogenic agent does not significantly decrease after storage at 4°C for 65 weeks.

[35] The Asparagopsis oil composition according to

[28] , wherein the at least one antimethanogenic agent is BF and / or DBCM.

[36] The Asparagopsis oil composition according to

[28] , wherein the amount of the at least one antimethanogenic agent does not decrease by more than 20% after 65 weeks of storage at 25°C, and the at least one antimethanogenic agent is BF and / or DBCM.

[37] The Asparagopsis oil composition of

[28] , wherein the amount of the at least one antimethanogenic agent does not decrease by more than 50% after 65 weeks of storage at 4°C or 25°C, and the at least one antimethanogenic agent is DBA.

[38] The Asparagopsis oil composition of

[28] , wherein the concentration of the at least one antimethanogenic agent is at least 0.1 mg per mL of extract.

[39] The Asparagopsis oil composition of

[28] , wherein the concentration of the at least one antimethanogenic agent is at least 1 mg per mL of extract.

[40] The Asparagopsis oil composition of

[28] , wherein the concentration of the at least one antimethanogenic agent is at least 2 mg per mL of extract.

[41] The Asparagopsis oil composition of

[28] , wherein the concentration of the at least one antimethanogenic agent is at least 3 mg per mL of extract.

[42] The Asparagopsis oil composition of

[28] , wherein the concentration of the at least one antimethanogenic agent is at least 4 mg per mL of extract.

[43] A feed supplement for reducing total gas production and / or methane production in ruminants, the feed supplement comprising an effective amount of the Asparagopsis oil composition of

[27] or

[28] .

[44] A feed for ruminants, the feed being supplemented with the feed supplement according to

[43] .

[45] A method for reducing total gas production and / or methane production in a ruminant, comprising administering to the ruminant an effective amount of the Asparagopsis oil composition of

[27] or

[28] .

[46] The method of

[45] , wherein the method comprises maintaining an effective amount of a desired volatile fatty acid.

[47] The method of

[46] , wherein the desired volatile fatty acids include acetate and propionate, and the maintaining includes reducing the ratio of acetate to propionate.

[48] The method according to any one of

[45] to

[47] , wherein the method comprises maintaining the amount of organic matter and / or dry matter decomposed.

[49] The method according to any one of

[45] to

[48] , wherein the Asparagopsis oil composition is administered in an amount equivalent to at least 3, 2, 1, 0.5, 0.25, 0.125, or 0.067% of the organic matter administered to the ruminant.

[50] The method according to any one of

[45] to

[49] , wherein the ruminant is selected from the suborder Ruminantia and the suborder Nucellopoda.

[51] The method according to any one of

[45] to

[49] , wherein the ruminant is a cow or a sheep.

[52] The method according to any one of

[45] to

[49] , wherein the ruminant is a cow.

Claims

1. 1. A method for producing an Asparagopsis oil composition, the method comprising: (a) providing Asparagopsis biomass; (b) providing at least one oil; and (c) contacting the biomass with the at least one oil under conditions to extract at least one bioactive agent from the biomass into the at least one oil to form the Asparagopsis oil composition; The method, wherein the at least one bioactive agent comprises bromoform.

2. 10. The method of claim 1, wherein step (c) comprises homogenizing the biomass in the at least one oil.

3. 10. The method of claim 1, further comprising, after step (c), separating the biomass from the at least one oil.

4. 2. The method of claim 1, wherein the biomass:at least one oil is greater than 0.3 g:1 mL, greater than 0.6 g:1 mL, greater than 0.9 g:1 mL, or greater than 1.2 g:1 mL.

5. 10. The method of claim 1, wherein step (c) is carried out for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

6. 10. The method of claim 1, wherein step (c) is carried out at about 4°C.

7. 4. The method of claim 3, wherein prior to the step of separating the biomass from the at least one oil, the biomass in contact with the at least one oil is heated such that a gel comprising the at least one bioactive agent releases the at least one bioactive agent into the at least one oil.

8. 8. The method of claim 7, wherein the biomass in contact with the at least one oil is heated to 60°C.

9. 9. The method of claim 7 or claim 8, wherein the biomass in contact with the at least one oil is heated for 1 hour.

10. 2. The method according to claim 1, wherein the Asparagopsis is Asparagopsis taxiformis and / or Asparagopsis armata.

11. 10. The method of claim 1, wherein the step of providing Asparagopsis biomass does not include air-drying the biomass.

12. 10. The method of claim 1, wherein said step of providing Asparagopsis biomass comprises harvesting said biomass into said at least one oil.

13. The method of claim 1 , wherein the at least one oil comprises an edible oil.

14. 14. The method 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, 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 combinations thereof.

15. 10. The method of claim 1, wherein the at least one bioactive agent further comprises bromochloroacetic acid (BCA), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

16. 10. The method of claim 1, wherein the amount of the at least one bioactive agent extracted from the biomass into the at least one oil is increased relative to the amount of the at least one bioactive agent extracted into water from an equivalent amount of biomass.

17. 10. The method of claim 1, wherein the Asparagopsis oil composition comprises at least 0.1 mg of bromoform per mL of extract, at least 1 mg of bromoform per mL of extract, at least 2 mg of bromoform per mL of extract, at least 3 mg of bromoform per mL of extract, or at least 4 mg of bromoform per mL of extract.

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