RU2821991C2 - New composition - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to agricultural industry. Method of producing an Asparagopsis oil composition, comprising the steps of: providing Asparagopsis biomass; providing at least one oil; and contacting the biomass with at least one oil under conditions of extracting at least one biologically active substance from the biomass into at least one oil to form an oil composition Asparagopsis, where at least one biologically active substance includes bromoform. Asparagopsis oil composition containing at least one exogenous oil and at least one antimethanogenic component, where at least one antimethanogenic component contains bromoform. Fodder additive for reducing the total gas and/or methane formation in ruminants, said additive contains an effective amount of the Asparagopsis oil composition. Fodder for ruminant animals, where said fodder is added with a fodder additive containing an effective amount of Asparagopsis oil composition.

EFFECT: method of reducing total gas and/or methane formation in a ruminant, involving administering to said ruminant an effective amount of Asparagopsis oil composition.

52 cl, 9 dwg, 1 tbl, 5 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The scope of the invention relates to methods for preparing compositions suitable for reducing overall gas production and/or methane formation in ruminant animals.

BACKGROUND OF THE ART

Methane ( _CH4 ) is a greenhouse gas (GHG) produced primarily by methanogenic microorganisms that occur in natural ecosystems (such as wetlands, oceans, and lakes) and the gastrointestinal tracts of invertebrates and vertebrates such as termites and ruminants . Every year ~429-507 Tg CH ₄ is removed from the atmosphere and ~40 Tg from the stratosphere due to reactions with hydroxyl (OH) radicals; and ~30 Tg by _CH4- oxidizing bacteria in the soil.

However, anthropogenic greenhouse gas emissions are increasing rapidly, with atmospheric _CH4 concentrations now more than twice as high as they were in the early 1800s. Methane is very efficient at absorbing solar infrared radiation and has a global warming potential 25 times greater than CO ₂ . Consequently, its accumulation in the atmosphere contributes significantly to climate change. One of the main sources of anthropogenic CH ₄ is agricultural activity, including the breeding of ruminant animals.

According to a recent UN report, livestock production produces more global warming greenhouse gas _CO2 equivalents than transportation. In Australia, ruminant animals are estimated to account for ~10% of total greenhouse gas emissions. Ruminants produce _CH4 as a by-product of anaerobic microbiological fermentation of feed in the rumen and, to a lesser extent, in the large intestine. The rumen microbial community is very diverse and consists of bacteria, protozoa, fungi and bacteriophages that act collectively to ferment consumed organic matter (OM), resulting in the formation of CO ₂ , H ₂ , volatile fatty acids (VFA) and formates. Methanogenic archaea present in the rumen utilize these end products and produce _CH4 . Although CH ₄ production reduces the partial pressure of H ₂ that might otherwise inhibit rumen fermentation, it also reduces the amount of energy and carbon available to produce VFAs needed for ruminant nutrition. Most of the CH ₄ produced by ruminants is exhaled and regurgitated by the animals and represents a loss of up to 12% of total energy intake.

Risk mitigation strategies that reduce intestinal CH ₄ production are important, and ways to reduce overall gas production and/or methane production in ruminants is a major concern.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method for producing an Asparagopsis oil composition, said method comprising the steps of: providing Asparagopsis biomass; providing at least one oil; and contacting the biomass with at least one oil under conditions of extracting at least one biologically active substance from the biomass into at least one oil to form an Asparagopsis oil composition.

In one embodiment of the method, the step of contacting the biomass with at least one oil may include homogenizing the biomass in the at least one oil. In another embodiment of the method, the biomass can be separated from the at least one oil after the step of contacting the biomass with the at least one oil.

In another aspect of the method, the ratio of biomass (in grams) to at least one oil (in milliliters) may be greater than 0.3:1. In one embodiment, the ratio of biomass (in grams) to at least one oil (in milliliters) may be greater than 0.6:1.

In another embodiment, the ratio of biomass (in grams) to at least one oil (in milliliters) may be greater than 0.9:1. In another embodiment, the ratio of biomass (in grams) to at least one oil (in milliliters) may be greater than 1.2:1.

In another aspect of the method, the conditions for extracting at least one biologically active substance from the biomass into at least one oil can be carried out for at least 1 day. In one embodiment, the extraction is carried out over 2 days. In another embodiment, the extraction is carried out over 3 days. In another embodiment, the extraction is carried out over 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.

In another aspect of the method, the conditions for extracting at least one biologically active substance from the biomass into at least one oil can be performed at a temperature of about 4°C.

In one embodiment of the method, prior to the step of separating the biomass from the at least one oil, the biomass in contact with the at least one oil may be heated. In this embodiment, heating is carried out in such a way that the gel, which may contain at least one biologically active substance, releases at least one biologically active substance into at least one oil. In one embodiment, heating can be carried out at a temperature of 60°C. In the above embodiments, heating can be carried out for one hour.

In one aspect of the present 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 .

In one embodiment of the method, the step of providing Asparagopsis biomass does not include air drying the biomass. In another embodiment, the step of providing Asparagopsis biomass includes introducing the biomass into at least one oil.

In another aspect, the at least one oil includes 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, rapeseed oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grapeseed oil, hazelnut oil, hemp oil, lemon oil, flaxseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, pecan oil , pine nut oil, pistachio oil, pumpkin seed oil, canola oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil or any combination thereof.

In another aspect, the at least one biologically active substance may be an antimethanogenic component. In one embodiment, the at least one biologically active substance can be selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA) and dibromochloromethane (DBCM). In another embodiment, the anti-methanogenic component is BF. In another embodiment, the anti-methanogenic component is DBCM. In another embodiment, the anti-methanogenic components are BF and DBCM.

In another aspect, the concentration of at least one biologically active substance extracted from the biomass into at least one oil is increased compared to the concentration of at least one biologically active substance extracted from an equivalent amount of biomass into water. In one embodiment, the at least one biologically active substance is an antimethanogenic component, wherein the concentration of the antimethanogenic component extracted from the biomass into the at least one oil does not decrease by more than 20% after storage for 65 weeks at 25 °C, wherein at least one anti-methanogenic component is BF and/or DBCM.

In one embodiment, the concentration of at least one biologically active substance extracted from the biomass into at least one oil does not decrease significantly after storage for 12 weeks at 25°C. In another embodiment, the concentration of at least one biologically active substance extracted from the biomass into at least one oil does not decrease significantly after storage for 65 weeks at 4°C.

In another aspect, the Asparagopsis oil composition obtained by this method contains at least 0.1 milligrams of bromoform per milliliter of extract. In one embodiment, the Asparagopsis oil composition obtained by this method contains at least 1 milligram of bromoform per milliliter of extract. In another embodiment, the Asparagopsis oil composition obtained by this method contains at least 2 milligrams of bromoform per milliliter of extract. In another embodiment, the Asparagopsis oil composition obtained by this method contains at least 3 milligrams of bromoform per milliliter of extract. In another embodiment, the Asparagopsis oil composition obtained by this method contains at least 4 milligrams of bromoform per milliliter of extract.

In another aspect, the present invention relates to an Asparagopsis oil composition containing at least one oil and at least one antimethanogenic component. In one embodiment, the Asparagopsis may be Asparagopsis taxiformis . In another embodiment, the Asparagopsis may be Asparagopsis armata . In another embodiment, the Asparagopsis may be Asparagopsis taxiformis and Asparagopsis armata .

In the above embodiments, the at least one oil may comprise an edible oil. In this 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, rapeseed oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flax seed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, nut oil pecan oil, pine nut oil, pistachio oil, pumpkin seed oil, canola oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof.

In another aspect of the Asparagopsis oil composition, the at least one anti-methanogenic component can be selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA) and dibromochloromethane (DBCM).

In one embodiment of the Asparagopsis oil composition, the concentration of at least one antimethanogenic component is not significantly reduced after storage for 12 weeks at 25°C. In another embodiment, the concentration of at least one antimethanogenic component does not decrease significantly after storage for 65 weeks at 4°C. In another embodiment, the concentration of at least one antimethanogenic component does not decrease by more than 20% after storage for 65 weeks at 25°C. In the above embodiments, the at least one anti-methanogenic component may be BF and/or DBCM. In another embodiment, the concentration of at least one antimethanogenic component does not decrease by more than 50% after storage for 65 weeks at 4°C or 25°C. In this embodiment, the at least one anti-methanogenic component may be a DBA.

In one embodiment, the Asparagopsis oil composition may have a concentration of at least one antimethanogenic component of at least 0.1 milligrams per milliliter of extract. In another embodiment, the Asparagopsis oil composition may have a concentration of at least one antimethanogenic component of at least 1 milligram per milliliter of extract. In another embodiment, the Asparagopsis oil composition may have a concentration of at least one antimethanogenic component of at least 2 milligrams per milliliter of extract. In another embodiment, the Asparagopsis oil composition may have a concentration of at least one antimethanogenic component of at least 3 milligrams per milliliter of extract. In another embodiment, the Asparagopsis oil composition may have a concentration of at least one antimethanogenic component of at least 4 milligrams per milliliter of extract.

In another aspect, the present invention relates to a feed additive for reducing overall gas production and/or methane production in ruminants, the additive containing an effective amount of an Asparagopsis oil composition as described herein.

In another aspect, the present invention relates to feed for ruminant animals, wherein said feed is fortified with a feed additive as described herein.

In another aspect, the present invention relates to a method of reducing overall gas production and/or methane production in ruminant animals, comprising administering to said ruminant animal an effective amount of an Asparagopsis oil composition as described herein. In one embodiment, the method may include maintaining an effective concentration of the desired volatile fatty acid. In this embodiment, the desired volatile fatty acid may include acetate and propionate, wherein maintenance involves reducing the acetate to propionate ratio. In another embodiment, the method may include maintaining a concentration of decomposed organic matter and/or dry matter.

In another aspect of the method, the Asparagopsis oil composition can be administered at a dose equivalent to at least 3% of the organic matter administered to the ruminant animal. In one embodiment, the Asparagopsis oil composition can be administered at a dose equivalent to at least 2% of the organic matter administered to the ruminant. In another embodiment, the Asparagopsis oil composition can be administered at a dose equivalent to at least 1% of the organic matter administered to the ruminant animal. In another embodiment, the Asparagopsis oil composition can be administered at a dose equivalent to at least 0.5% of the organic matter administered to the ruminant. In another embodiment, the Asparagopsis oil composition can be administered at a dose equivalent to at least 0.25% of the organic matter administered to the ruminant. In another embodiment, the Asparagopsis oil composition may be administered at a dose equivalent to at least 0.125% of the organic matter administered to the ruminant. In another embodiment, the Asparagopsis oil composition can be administered at a dose equivalent to at least 0.067% of the organic matter administered to the ruminant.

In another aspect of the above method, the ruminant can be selected from members of the suborders Ruminantia and Tylopoda. In one embodiment of this aspect, the ruminant animal may be cattle or sheep. In another embodiment, the ruminant animal is a cattle.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the amount of biologically active substance, bromoform (antimethanogenic component), in compositions prepared by extracting Asparagopsis biomass into oil or water, including compositions prepared by homogenizing A. taxiformis biomass in oil or water (“pulverized”), or non-homogenized biomass in oil or water (“intact”) for 1, 3, 5, 7 and 10 days. Coal represents a standard extraction method using freeze-dried biomass soaked in methanol for 72 hours. Data are presented as mean±standard error, n=3.

Figure 2 shows the loss (%) of the biologically active substance, bromoform (antimethanogenic component), in the composition when stored under various conditions for 6 months: at ambient conditions (25°C±10°C); in an air-conditioned room; in the refrigerator at 4°C; and in the freezer at -20°C. Data are presented as mean±standard error, n=3.

Figure 3 shows the retention of the biologically active substance, bromoform (antimethanogenic component), in compositions obtained by extracting Asparagopsis biomass into oil or water, including compositions obtained by homogenizing A. taxiformis biomass into oil or water (“pulverized”) or non-homogenized biomass into oil or water (“intact”) at t=0 and after 4, 8, 12 and 65 weeks of storage (at 4°C or 25°C). Data are presented as mean±standard error, n=3.

Figure 4 shows the retention of the biologically active substance, DBCM (antimethanogenic component), in compositions obtained by extracting intact Asparagopsis biomass into oil, at t=0 and after 4, 8, 12 and 65 weeks of storage (at or 4°C; "Refrigerator" or 25°C; "RT"). The ratio of DBCM:naphthalene (IS) in oil is given. Columns with the same superscript letters are not significantly different. Data are presented as mean±standard error, n=3.

Figure 5 shows the retention of the biologically active substance, DBCM (antimethanogenic component), in compositions prepared by extracting Asparagopsis biomass into oil, prepared by homogenizing A. taxiformis biomass in oil, at t=0 and after 4, 8, 12 and 65 weeks of storage (at 4°C; “Refrigerator” or 25°C; “RT”). The ratio of DBCM:naphthalene (IS) in oil is given. Data are presented as mean±standard error, n=3. These data show no statistical differences in DBCM:IS ratios.

Figure 6 shows the amount of biologically active substance, DBA, in compositions prepared by extracting intact Asparagopsis biomass into oil for 10 days followed by ongoing storage (week 0) at the indicated temperatures (25°C/"RT" or 4°C/ "Fridge"). The ratio of DBA:naphthalene (IS) in oil is given. Data are presented as mean±standard error, n=3.

Figure 7 shows the amount of biologically active substance, DBA, in compositions prepared by extracting homogenized Asparagopsis biomass into oil for 10 days followed by ongoing storage (week 0) at the indicated temperatures (25°C/"RT" or 4°C/ "Fridge"). The ratio of DBA:naphthalene (IS) in oil is given. Data are presented as mean±standard error, n=3.

Figure 8 shows the amount of biologically active substance, bromoform (antimethanogenic component), extracted into the oil using different amounts of biomass homogenized in the same volume of oil. Heating the gel-like samples promoted the release of the biologically active substance into an oil fraction, which could be separated from the gel. Data are presented as mean±standard error, n=3.

Figure 9 shows the concentrations (mg/g dry weight of biomass) of the biologically active substance, bromoform (antimethanogenic component), in compositions prepared by extracting Asparagopsis biomass into oil by homogenizing A. taxiformis biomass in oil using different amounts of biomass homogenized in the same volume oils Heating the gel-like samples promoted the release of the biologically active substance into an oil fraction, which could be separated from the gel. Data are presented as mean±standard error, n=3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for producing biologically active Asparagopsis oil compositions, including Asparagopsis oil compositions suitable for reducing total gas production (TGP) and/or methane production (CH ₄ ) in ruminant animals. In particular, the present inventors have shown that it is possible to produce Asparagopsis oil compositions containing halogenated secondary metabolites with bioactive properties, and importantly, the present inventors have also shown that the concentrations of halogenated secondary metabolites in Asparagopsis oil compositions are stable over long periods of time.

Figure 1 shows that Asparagopsis oil compositions containing a bioactive molecule can be prepared by contacting Asparagopsis biomass with oil, and that concentrations of the bioactive compound are increased by homogenizing the biomass in the oil. Figure 3 shows that Asparagopsis oil compositions prepared by contacting Asparagopsis biomass with oil, with or without homogenization, retain high concentrations of biologically active substance for long periods of time when stored either at room temperature (25°C), or at 4 °C.

Thus, the present invention relates to a method for producing an Asparagopsis oil composition, said method comprising the steps of:

(a) providing Asparagopsis biomass;

(b) providing at least one oil; and

(c) contacting the biomass with at least one oil under conditions of extracting at least one antimethanogenic component from the biomass into the at least one oil.

Asparagopsis has a heteromorphic developmental cycle with two phases of the free-living developmental cycle - gametophyte (macrothallic form) and sporophyte (or tetrasporophyte - smaller, filamentous form). Historically, the tetrasporophyte was recognized as a separate genus ( Falkenbergia ). Therefore, the term " Asparagopsis " as used herein refers to the genus Asparagopsis and other taxonomic classifications currently known to belong to the genus Asparagopsis.

There are at least two recognized species of Asparagopsis , one tropical/subtropical ( Asparagopsis taxiformis ) and one temperate ( Asparagopsis armata ), which are found throughout the world.

In one embodiment, the Asparagopsis species is selected from Asparagopsis taxiformis or Asparagopsis armata .

In another aspect, the biomass of at least one species of red sea macroalgae selected from species belonging to other genera of red sea algae of the family Bonnemaisoniaceae to which Asparagopsis belongs (for example, Bonnemaisonia , Delisea , Ptilonia , Leptophyllis and Pleuroblepharidella ) is used instead of Asparagopsis biomass in the methods and compositions presented here. Without wishing to be bound by theory, six genera of red seaweeds in the family Bonnemaisoniaceae (e.g., Asparagopsis , Bonnemaisonia , Delisea , Ptilonia , Leptophyllis , and Pleuroblepharidella ) produce and accumulate bioactive halogenated secondary metabolites with bioactive properties, including the antimethanogenic compounds described here.

As used herein, the term "providing biomass" includes providing or using Asparagopsis biomass removed from the water immediately prior to contacting the biomass with at least one oil. In one embodiment, Asparagopsis biomass is obtained from the environment and placed directly into at least one oil. In another embodiment, the Asparagopsis biomass is contacted with at least one oil for 5, 4, 3, 2, or 1 hour after the biomass is removed from the environment.

In one embodiment, water is removed from the biomass by blot drying the biomass until the biomass contacts at least one oil.

In one embodiment, the Asparagopsis biomass is not lyophilized Asparagopsis biomass.

In another embodiment, the Asparagopsis biomass is not air-dried Asparagopsis biomass.

As used herein, the term “at least one oil” includes one type of oil, or compositions containing one type of oil, or a mixture of two or more oils, or a composition containing a mixture of two or more oils. The at least one oil includes an oil suitable for use, administration or feeding to the animal.

In one embodiment, the at least one oil is an edible oil.

In one embodiment, the edible oil is selected from the group consisting of almond oil, apricot oil, argan oil, avocado oil, Brazil nut oil, canola oil, cashew oil, coconut oil, rapeseed oil, corn oil, copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flax seed 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, canola oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil or any combination thereof.

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

The present inventors have demonstrated that biologically active substances are rapidly extracted from Asparagopsis biomass into oil. For example, FIG. 1 shows that over a 24-hour period, concentrations of bromoform extracted from a homogenized biomass/oil mixture exceed concentrations of bromoform extracted from lyophilized biomass using methanol. Figure 1 also shows that over 72 hours the concentration of bromoform extracted from the non-homogenized biomass/oil mixture is the same as the concentration of bromoform extracted from the lyophilized biomass. Figure 1 also shows that over 120 hours, the concentrations of bromoform extracted from the non-homogenized biomass/oil mixture exceed the concentrations of bromoform extracted from the lyophilized biomass using methanol.

As used herein, the term "contacting" includes mixing the 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 for extracting at least one biologically active substance from biomass to oil biomass/oil mixtures.

The term contacting includes holding the biomass oil mixture at various suitable temperatures.

The temperature at which the biomass can contact the at least one oil is not limited as long as the anti-methanogenic component does not significantly evaporate/sublimate from the oil and/or does not significantly decompose at the temperature at which contact occurs. The temperature can be selected from a group consisting of intervals from -78°C to -50°C, from -50°C to -20°C, from -20°C to -5°C, from -5°C to 0° C, from -5°C to 4°C, from 0°C to 4°C, from 4°C to 10°C, from 10°C to 20°C, from 20°C to 25°C, from 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.

As used herein, the term “room temperature” is used to refer to the normal ambient temperature. As one skilled in the art will appreciate, environmental conditions will vary depending on geographic location and time of year such that room temperatures can vary from below zero Celsius to above 40°C. Room temperature is generally considered to be in the range of about 20°C to about 25°C, and for the purposes of the experiments conducted in Townsville, Queensland, room temperature was assumed to be about 25°C.

The present inventors have demonstrated that when a large amount of Asparagopsis biomass is contacted with a relatively small volume of oil, a gel is formed which prevents one or more bioactive substances from being extracted into the oil. To increase the yield of extracted bioactive (e.g., antimethanogenic) component, the gel and/or biomass and/or oil can be heated to release more of the at least one bioactive (e.g., antimethanogenic) component into at least one oil . For example, FIGS. 8 and 9 show that heating a biomass sample in contact with at least one oil at 60°C for 1 hour resulted in a 20% increase in the amount of biologically active (e.g., antimethanogenic) component , extracted into oil, compared to unheated samples (see data for “120 g+heat” versus “120 g” in Figures 8 and 9).

The temperature at which the biomass in contact with the at least one oil can be heated to release at least one biologically active (e.g., antimethanogenic) component into the at least one oil is not limited because at least At least one biologically active (eg, antimethanogenic) component does not significantly evaporate/sublimate from the oil and/or does not significantly decompose at the temperature at which heating occurs. The temperature can be selected from a 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.

The time during which the biomass in contact with at least one oil can be heated to release at least one biologically active (e.g., antimethanogenic) component into at least one oil is not limited, since at least one biologically active (eg, antimethanogenic) component does not significantly evaporate/sublimate from the oil and/or does not significantly decompose when heated. The time period can be selected from the group consisting of intervals from 1 second to 1 minute, from 1 minute to 2 minutes, from 2 minutes to 5 minutes, from 5 minutes to 10 minutes, from 10 minutes to 20 minutes, from 20 minutes to 30 minutes, from 30 minutes to 40 minutes, from 40 minutes to 50 minutes, from 50 minutes to 60 minutes, from 60 minutes to 70 minutes, from 70 minutes to 80 minutes, from 80 minutes to 90 minutes, from 1.5 hours to 2 hours, from 2 hours to 3 hours, from 3 hours to 4 hours and from 4 hours to 5 hours. In some embodiments, the time period 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 period of time during which the biomass in contact with the at least one oil is heated is preferably about 1 hour.

If the biomass/oil mixture is not heated to release at least one biologically active (e.g., antimethanogenic) component into at least one oil, then the time that the biomass can be in contact with at least one oil under conditions of extracting at least one biologically active (eg, antimethanogenic) component from the biomass into at least one oil is not particularly limited. As will be understood by one skilled in the art, preferably the concentration of at least one biologically active (e.g., antimethanogenic) component in the oil reaches or nearly reaches an equilibrium level, whereby the amount of at least one biologically active (e.g., antimethanogenic) component, at least in one oil will be maximum. Thus, contacting the biomass with at least one oil under conditions of extraction of at least one biologically active (for example, antimethanogenic) component from the biomass into at least one oil can be carried out for a period selected from the group, consisting of intervals from 1 minute to 1 hour, from 1 hour to 2 hours, from 2 hours to 3 hours, from 3 hours to 4 hours, from 4 hours to 5 hours, from 5 hours to 6 hours, from 6 hours to 12 hours, from 12 hours to 24 hours, from 1 day to 2 days, from 2 days to 3 days, from 3 days to 4 days, from 4 days to 5 days, from 5 days to 6 days, from 6 days to 7 days , from 7 days to 8 days, from 8 days to 9 days, from 9 days to 10 days, from 10 days to 14 days, from 2 weeks to 3 weeks and from 3 weeks to 1 month. In some embodiments, the contact may be conducted for a period 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, contacting may be conducted over a period of at least 1, 2, 3, 4, 5, 4, 6, 7, 8, 9, or 10 days.

The step of contacting the biomass with at least one oil may also include homogenizing the biomass in the at least one oil. As used herein, the term “homogenization” means breaking down the biomass to facilitate the release of at least one antimethanogenic component from the biomass, which can then be extracted into at least one oil. Homogenization can be accomplished by any methods known in the art, such as crushing, grinding, grinding, mixing, cutting, slicing or dicing.

In one embodiment, homogenization can be carried out to obtain a homogeneous mixture, thereby facilitating uniform distribution of the biomass, biologically active substance and/or oil in the composition into the animal feed source.

In one embodiment, the biologically active substance is a substance that has a biological effect on an animal, preferably a ruminant animal. A biological effect can be any effect on the behavior or physiology of an animal, or can even affect microorganisms within said animal. For example, the biological effect may be the inhibition of methanogenesis. The biologically active substance responsible for inhibiting methanogenesis is not particularly limited, and is intended to include all substances that inhibit methanogenesis, including, but not limited to, secondary metabolites. Asparagopsis produces secondary metabolites, including halogenated low molecular weight compounds, in particular brominated and chlorinated halo forms. Many of these compounds have potent antimicrobial properties and inhibit a wide range of microorganisms, including gram-positive and gram-negative bacteria, as well as mycobacteria and fungi, and may therefore contribute to the effects described here. Secondary metabolites of Asparagopsis also inhibit protozoa.

In one embodiment, the biologically active substance is a substance that reduces total gas production (TGP) and/or a substance that reduces methane produced by a ruminant animal.

As used herein, the term "reduction" includes a reduction in the amount of a substance compared to a reference. For example, a reduction in the total amount of gas and/or methane produced by a ruminant animal or animals that have been administered an Asparagopsis oil composition as described herein, compared to an animal or animals that have not been administered a composition containing an Asparagopsis oil composition as described herein. The reduction can be measured in vitro using an artificial rumen system simulating anaerobic fermentation, or in vivo using animals kept in breathing chambers. The assessment of methanogenesis in the intestines of ruminants is within the knowledge and competence of specialists in the field.

As used herein, the term “reducing total gas production” refers to reducing the total amount of gas produced, for example, the total amount of gas produced in the gastrointestinal tract. The term includes the total volume of all gases produced by anaerobic fermentation, such as in the systems described herein. Fermentation in the rumen and intestines of ruminants produces gas, including methane. The purpose of the present invention is to reduce this process, for example, to reduce the total amount of gas produced in the gastrointestinal tract. Estimation of total gas production in ruminant animals is within the knowledge and competence of those skilled in the art.

As used herein, the term “methane reduction” refers to the reduction of methane production in the gastrointestinal tract. The term includes the specific volume of methane produced by anaerobic fermentation, such as in the systems described herein. Fermentation in the rumen and intestines of ruminants produces methane. The purpose of the present invention is to reduce this process, for example, to reduce the total amount of methane produced in the gastrointestinal tract. The assessment of methane production by ruminant animals is within the knowledge and competence of those skilled in the field.

As used herein, the term "anaerobic fermentation" means anaerobic fermentation in vivo, for example, in a ruminant animal.

As used herein, the term "antimethanogenic component" means any biologically active compound that inhibits methanogenesis in a ruminant animal. Such compounds are typically halogenated secondary metabolites, including at least the following: bromochloroacetic acid, bromochloromethane, 2-bromoethanesulfonic acid, chloral hydrate, chloroform, iodopropane, bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

For example, the present inventors have demonstrated in FIGS. 4, 5, 6 and 7 that Asparagopsis oil compositions containing dibromochloromethane (DBCM) and/or dibromoacetic acid (DBA) can be prepared by contacting Asparagopsis biomass with oil through homogenization. High concentrations of the bioactive are extracted into the oil and, as shown for DBCM, these concentrations are maintained for extended periods of time when stored at either room temperature (RT) or 4°C (refrigerator).

In one embodiment, the antimethanogenic component is selected from the group consisting of bromochloroacetic acid (BCA), bromoform (BF), dibromoacetic acid (DBA), and dibromochloromethane (DBCM).

In one embodiment, after contacting the biomass with at least one oil under conditions of extracting at least one antimethanogenic component from the biomass into at least one oil, said biomass is separated from said at least one oil. Separation of the biomass from at least one oil can be accomplished by any method known in the art. Separation methods include, but are not limited to, centrifugation, decantation, filtration, distillation, or the use of a separatory funnel or other equivalent methods.

The amounts of biomass and at least one oil that contact may be varied so as to change the amount of at least one biologically active substance extracted into the at least one oil.

The present inventors have also demonstrated that the amounts of at least one biologically active substance can be adjusted by changing the ratio of Asparagopsis biomass to at least one oil. For example, FIGS. 8 and 9 show that bromoform concentrations can be increased by increasing the ratio of Asparagopsis biomass to at least one oil.

Accordingly, in some embodiments, the ratio of biomass to at least one oil can be selected from the group consisting of 0.01 g:1 ml, 0.05 g:1 ml, 0.1 g:1 ml, 0.2 g:1 ml, 0.3 g:1 ml, 0.4 g:1 ml, 0.5 g:1 ml, 0.6 g:1 ml, 0.7 g:1 ml, 0.8 g: 1 ml, 0.9 g:1 ml, 1 g:1 ml, 1.1 g:1 ml, 1.2 g:1 ml, 1.3 g:1 ml, 1.4 g:1 ml and 1 .5 g:1 ml. Preferably, the ratio of biomass to at least one oil is selected from the group consisting of more than 0.3 g:1 ml, more than 0.6 g:1 ml, more than 0.9 g:1 ml and more than 1 .2 g:1 ml.

In one embodiment, the invention provides an Asparagopsis oil composition prepared by the methods described herein.

The present inventors have previously shown that lyophilized ground Asparagopsis species can effectively reduce total gas production and in vivo methane production in cattle compared to the positive control hulled cotton seed (DCS). It is important to note that hulled cottonseed is used as a cattle feed additive because it significantly reduces _CH4 production compared to other high-energy grains. The reduction in total gas production compared to DCS was similar across species, indicating that macroalgae such as Asparagopsis reduce TGP and _CH4 production in ruminants compared to high-energy grains, and some macroalgae reduce TGP and _CH4 production in ruminants compared to the DCS positive control. In addition, the present inventors have shown that the formation of CH ₄ generally follows the same pattern as TGP.

Accordingly, in one embodiment, an Asparagopsis oil composition produced by the methods described herein can be used to reduce the total amount of gas and/or methane produced by a ruminant animal.

Thus, in one aspect, the invention relates to a method of reducing overall gas production and/or methane production in a ruminant animal, comprising the step of administering to said ruminant animal an effective amount of an Asparagopsis oil composition of the present invention.

In preferred embodiments of the invention, the total amount of gas produced is reduced by at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% compared to the control. In one embodiment, the control is the total amount of gas produced when animals are not administered an effective amount of an Asparagopsis oil composition as described herein. In another embodiment, the control is the amount of total gas produced when the animals are administered hulled cotton seeds. In another embodiment, the control is the total amount of gas produced when hulled cotton seeds are subjected to anaerobic fermentation in vitro.

In preferred embodiments of the invention, the amount of methane produced is reduced by at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or 10% compared to the control. In one embodiment, the control is the amount of methane produced when the animals are not administered an effective amount of the Asparagopsis oil composition. In another embodiment, the control is the amount of methane produced when the animals are administered hulled cotton seeds. In another embodiment, the control is the amount of methane produced when animals are fed a pelleted commercially available diet based on lupine, oats, barley, wheat with cereal straw as a crude component [chemical composition (g/kg DM) of ash, 72 ; crude protein (CP) 112; neutral detergent fiber (aNDFom) 519; acid detergent fiber (ADFom) 338 and without cobalt, selenium and rumen modifiers] with additional crushed lupine, referred to here as “lupine diet”. In another embodiment, the control is the amount of methane produced when the lupine diet is subjected to anaerobic fermentation in vitro.

In one embodiment, the amount of methane produced by in vitro rumen fermentation is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to with the amount of methane produced when hulled cotton seeds are fermented in the rumen in vitro.

In one embodiment, 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 administered to a ruminant animal of hulled cotton seeds.

The present inventors have also previously demonstrated that air-dried ground Asparagopsis can effectively reduce methane production compared to a positive control lupine diet in sheep.

In one embodiment, 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 administered to a ruminant animal lupine diet.

By "effective amount" is meant an amount of Asparagopsis oil composition as described herein sufficient to provide an improvement in, for example, a reduction in the amount of methane produced relative to a reference or control, a reduction in the total amount of gas produced relative to a reference or control, maintaining an effective concentration of one or more more required volatile fatty acids compared to the reference or control, reduction in the acetate to propionate ratio compared to the reference or control, maintenance of body weight, dry matter intake and/or organic matter intake compared to the reference or control. In accordance with the spirit of the present invention, reduced methane exposure can be measured in the rumen using an artificial rumen system, such as that described in T. Hano, J. Gen. Appl. Microbiol., 1993, 39, 35-45, or by oral administration in vivo to ruminants.

Therefore, in one embodiment, the Asparagopsis oil composition as described herein is administered at a dose equivalent to preferably at least 16.67, 10.5, 3, 2, 1, 0.5, 0.25, 0.125, or 0.067 % of organic matter administered to a ruminant animal.

To calculate the volume of oil required to achieve a dose equivalent to the required % organic matter concentration administered to ruminants, the % organic matter in the Asparagopsis oil compositions described herein is calculated based on the fresh weight (fw) of Asparagopsis biomass contacting the specified volume at least one oil. The wet weight to dry weight (dw) ratio of Asparagopsis blotted dry is 10 (ie 30 g wet weight=3 g dry weight). Assuming an organic matter (OM) content of 80% dry weight based on previous data, the corresponding Asparagopsis -derived OM content of the biomass/oil can be calculated. For example, for an Asparagopsis oil composition containing 30 g fresh weight (fw), the Asparagopsis biomass in 100 ml of at least one oil is equivalent to 0.024 Asparagopsis organic matter/ml. If the required inclusion concentration in the feed is 0.1% Asparagopsis OM in 100 g of feed, then 4.17 ml of Asparagopsis oil composition 30 g/100 ml is required per 100 g of feed.

The corresponding content of Asparagopsis -derived OM in the biomass/oil can be calculated from the different organic matter (OM) content in % dry weight. For example, organic matter (OM) content of 50%, 55%, 60%, 70%, 75% dry weight based on previously obtained data can be used to calculate the corresponding Asparagopsis -derived OM content of the biomass/oil.

As stated above, in a preferred embodiment, the Asparagopsis oil composition as described herein is administered at a dose equivalent to preferably at least 3, 2, 1, 0.5, 0.25, 0.125 or 0.067% organic matter administered to the ruminant animals. Accordingly, when using a 30 g wet weight/100 ml Asparagopsis oil composition as described herein, per 100 g of feed would require at least 125 ml, 83.3 ml, 41.7 ml, 20.8 ml, 10.4 ml, 5.2 ml or 2.8 ml. When using the Asparagopsis oil composition 60 g wet weight/100 ml as described here, per 100 g of feed, at least 62.5 ml, 41.7 ml, 20.8 ml, 10.4 ml, 5.2 are required. ml, 2.6 ml or 1.4 ml. When using an Asparagopsis oil composition of 120 g wet weight/100 ml as described herein, per 100 g of feed, at least 31.25 ml, 20.8 ml, 10.4 ml, 5.2 ml, 2.6 are required. ml, 1.3 ml or 0.69 ml.

For example, if a 400 kg ruminant (eg, a steer) consumes 2.5% to 3% of its live weight per day of feed, then the Asparagopsis oil composition as described herein is administered at a dose proportional to the amount of organic matter administered to the ruminant. In the case of a ruminant 400 kg, and 80% of the feed is organic matter, if the animal consumes about 2.5% of its live weight per day (10 kg containing 8 kg of organic matter), then 3.3 liters of Asparagopsis oil composition is required 30 g wet weight/100 ml as described to obtain a dose equivalent to 1% organic matter administered to the ruminant. 1.6 liters of Asparagopsis oil composition 60 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 1.1 liters of Asparagopsis oil composition 90 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 833 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 666 ml of Asparagopsis oil composition 150 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal.

330 ml of Asparagopsis oil composition 30 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 167 ml of Asparagopsis oil composition 60 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 111 ml of Asparagopsis oil composition 90 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 83 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 67 ml of Asparagopsis oil composition 150 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal.

To calculate the composition comprising the biomass required to achieve a dose equivalent to the required % organic matter concentration administered to the ruminant, the % organic matter in the Asparagopsis oil compositions described herein is calculated based on the fresh weight (fw) of the Asparagopsis biomass in contact with a certain volume of at least one oil. The wet weight to dry weight (dw) ratio of Asparagopsis blotted dry is 10 (ie 30 g wet weight=3 g dry weight). Assuming an organic matter (OM) content of 80% dry weight based on previous data, the corresponding Asparagopsis -derived OM content of the biomass/oil can be calculated. For example, for an Asparagopsis oil composition containing 30 g fresh weight (fw), the Asparagopsis biomass in 100 ml of at least one oil is equivalent to 0.024 Asparagopsis organic matter/ml, and the amount of organic matter in the total final volume is calculated and included in the calculation.

An effective amount of an Asparagopsis oil composition as described herein can be determined by the methods described herein, including the in vitro and in vivo dose-response studies described herein. For example, the present inventors have demonstrated that in vitro rumen fermentation can be used to study the effects of amounts of lyophilized ground Asparagopsis on concentrations of volatile fatty acids, including acetate and propionate, methane production, and total gas production. Therefore, in vitro rumen fermentation can be used to determine doses of the Asparagopsis oil composition as described herein that may be an effective amount sufficient to provide an improvement, for example, a reduction in the amount of methane produced compared to a reference or control, a reduction in the total amount of gas produced compared to a reference or control, maintaining an effective concentration of one or more desired volatile fatty acids compared to a reference or control, or reducing the acetate to propionate ratio compared to a reference or control.

A ruminant is a mammal of the order Artiodactyla , which digests plant food by first softening and partially fermenting it in the first chambers of the animal's stomach, then regurgitating the semi-digested mass, now known as the cud, and chewing it again.

The process of chewing gum to further break down plant matter and stimulate digestion is called “chewing.” In ruminants, the digestive tract consists of four chambers, namely the rumen, reticulum, book and abomasum. In the first two chambers, the rumen and the mesh, food is mixed with saliva and separated into layers of solid and liquid material. The solid particles clump together to form a gum or bolus. The gum is then regurgitated and chewed slowly to completely mix it with saliva, which further breaks down the fibers. Fiber, especially cellulose, is broken down into glucose in these chambers by symbiotic anaerobic bacteria, protozoa and fungi. The split fiber, which is now in the liquid portion of the contents, then passes through the rumen into the next chamber of the stomach, the book. Food in the abomasum is digested in the same way as in the monogastric stomach. The digested contents of the stomach are finally sent to the small intestine, where absorption of nutrients occurs. Almost all the glucose produced by the breakdown of cellulose is used by symbiotic bacteria. Ruminants get their energy from volatile short-chain fatty acids (VFAs) produced by bacteria, namely acetate, propionate, butyrate, valerate and isovalerate.

Importantly, the inventors have shown that lyophilized ground Asparagopsis has the property of reducing overall gas production and/or methane production in ruminants without interfering with ruminal fermentation.

For example, the inventors have shown that lyophilized ground Asparagopsis has the property of reducing overall gas production and/or methane production in ruminants without interfering with rumen fermentation, for example, while maintaining an effective concentration of one or more desired volatile fatty acids. The inventors have also shown that freeze-dried ground Asparagopsis has the property of reducing overall gas production and/or methane production in ruminants without interfering with rumen fermentation, for example, without significantly affecting daily feed intake and/or body weight of the animals.

As used herein, the term "maintenance of effective concentration" means the amount of substance in the animal or animals after treatment (for example, administration of the Asparagopsis oil composition as described herein) that is not significantly different from control or ethanol, including the amount of substance in the animal or animals that were not administered the Asparagopsis oil composition of the present invention.

For example, “effective concentration of desired volatile fatty acid” is intended to mean that the amount of required volatile fatty acid is sufficient to produce an improvement, such as a reduction in the amount of methane production, compared to a reference or control; reduction in the total amount of gas produced compared to the standard or control; decrease in the acetate to propionate ratio compared to the standard or control; or maintaining live weight, dry matter intake, and/or organic matter intake relative to a reference or control.

Carbohydrate metabolism provides energy for the growth of rumen microorganisms primarily through the fermentation of cellulose and starch. Insoluble polymers are converted into oligosaccharides and soluble sugars by extracellular enzymes of rumen microorganisms. The resulting sugars are then fermented into one of various forms of volatile fatty acids, carbon dioxide and hydrogen. The volatile fatty acids used here—acetic acid, propionic acid, and butyric acid—are also called acetate, propionate, and butyrate, respectively.

Volatile fatty acids are used by animals as major sources of carbon and energy with varying degrees of efficiency. High concentrations of propionic acid are required because propionic acid is the major metabolic precursor for gluconeogenesis in animals. The fermentation of 6-carbon sugars into acetic acid is relatively inefficient because in this process carbon and hydrogen are lost through regurgitation as carbon dioxide or, importantly, methane. On the other hand, the production of propionic acid uses hydrogen and does not result in the loss of carbon or methane.

It is thus possible to increase the feed efficiency and/or growth rate of ruminants by increasing the mole fraction of propionic acid relative to acetic acid or, in another embodiment, by increasing the total concentration of volatile fatty acids (i.e. the total amount of acetic, propionic and butyric acids). acids) in the rumen.

The present inventors have demonstrated a reduction in the total gas and/or methane produced by in vitro and in vivo anaerobic fermentation in the presence of lyophilized ground Asparagopsis without negatively affecting the total VFA production in cattle. It is important to note that the present inventors have shown that Asparagopsis does not reduce VFAs in cattle; at doses of Asparagopsis that do not reduce the amount of organic matter or dry matter consumed/broken down; in doses that reduce the ratio of acetate to propionate; in doses that reduce the amount of acetate; in doses that increase the amount of propionate; and/or at doses of Asparagopsis that inhibit general gas and methane production in vitro and in vivo.

It is important to note that the present inventors have demonstrated that lyophilized ground Asparagopsis does not reduce the amount of VFAs at doses of Asparagopsis that inhibit overall gas and methane production in cattle. The present inventors have also shown that lyophilized ground Asparagopsis does not reduce the amount of organic matter or dry matter intake/breakdown in sheep; in doses that reduce the ratio of acetate to propionate; in doses that reduce the amount of acetate; in doses that increase the amount of propionate; and/or at doses of Asparagopsis that inhibit methane formation in vitro and in vivo. For example, the present inventors have shown that freeze-dried crushed Asparagopsis does not reduce the amount of organic matter intake/breakdown or dry matter in sheep fed 1.2 times the maintenance energy.

Thus, in one aspect, the invention provides a method for reducing overall gas production and/or methane production in ruminant animals, comprising the step of administering to said ruminant animal an effective amount of an Asparagopsis oil composition of the present invention while maintaining effective concentrations of the desired volatile fatty acids.

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

As used herein, the term “volatile fatty acids” (“VFA”) includes the end product of anaerobic microbiological fermentation of feed components 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.

In preferred embodiments, the total amount of VFA produced by rumen fermentation in the presence of an effective amount of an Asparagopsis oil composition as described herein is at least 80 mmol/L.

In other embodiments, the total amount of VFA produced by rumen fermentation in the presence of an effective amount of an Asparagopsis oil composition as described herein is at least 65 mmol/L.

The present inventors have also demonstrated that lyophilized ground Asparagopsis does not reduce the amount of VFA in cattle at doses of lyophilized ground Asparagopsis that do not reduce the amount of organic matter or dry matter broken down by ruminal fermentation or dry matter intake. The present inventors have also demonstrated that freeze-dried ground Asparagopsis does not reduce dry matter intake or live weight of sheep. For example, the present inventors have demonstrated that freeze-dried crushed Asparagopsis does not reduce dry matter intake or body weight in sheep fed 1.2 times the maintenance energy. This indicates that red marine macroalgae, such as Asparagopsis , reduce overall gas production and/or methane production in ruminants without interfering with ruminal fermentation.

Thus, in one aspect, the invention relates to a method of reducing overall gas production and/or methane production in ruminant animals, comprising the step of administering to said ruminant animal an effective amount of an Asparagopsis oil composition as described herein. In one embodiment, an effective concentration of the desired volatile fatty acid is maintained. In another embodiment, the acetate to propionate ratio is reduced. In another embodiment, the concentration of degradable organic matter and/or dry matter is maintained. In another aspect, the invention relates to a method of reducing overall gas production and/or methane production in ruminants, comprising the step of administering to said ruminant animal an effective amount of an Asparagopsis oil composition as described herein while maintaining the amount of dry matter intake.

As used herein, the terms "organic matter" and "dry matter" refer to the amount of feed (organic based or dry matter, respectively) that an animal consumes over a specified period of time, typically 24 hours. It is known in the art to calculate the consumption and/or breakdown of organic matter and dry matter. For example, dry matter and organic matter may account for 90% and 80% of the feed amount, respectively.

In one embodiment, the Asparagopsis oil composition as described herein is administered at a dose equivalent to preferably at least 16.67%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0. 25%, 0.125% or 0.067% organic matter administered to the ruminant.

In a preferred embodiment, the Asparagopsis oil composition as described herein is administered at a dose equivalent to preferably at least 3%, 2%, 1%, 0.5%, 0.25%, 0.125% or 0.067% organic matter, administered to a ruminant animal to maintain the amount of broken down organic matter and/or dry matter.

In another embodiment, the amount of both degradable organic matter and dry matter is maintained, and effective concentrations of desired volatile fatty acids are maintained.

In a preferred embodiment, the Asparagopsis oil composition as described herein is administered at a dose equivalent to preferably at least 3%, 2%, 1%, 0.5%, 0.25%, 0.125% or 0.067% organic matter, administered to a ruminant animal to maintain effective concentrations of required volatile fatty acids.

It is important to note that the present inventors have demonstrated that lyophilized ground Asparagopsis increases the amount of propionate at doses of Asparagopsis that inhibit overall gas and methane production, and lyophilized ground Asparagopsis increases the amount of propionate at doses of lyophilized ground Asparagopsis that do not reduce the amount of degradable organic matter and dry matter .

Accordingly, in another aspect, the invention relates to a method of reducing overall gas production and/or methane production in a ruminant animal, comprising the step of administering to said ruminant animal an effective amount of an Asparagopsis oil composition as described herein, while maintaining the amount of degradable organic matter or dry matter and/ or the acetate to propionate ratio decreases.

In a preferred embodiment, the Asparagopsis oil composition as described herein is administered at a dose equivalent to preferably at least 3%, 2%, 1%, 0.5%, 0.25%, 0.125% or 0.067% organic matter, administered to a ruminant to reduce the acetate to propionate ratio.

In one embodiment, the acetate to propionate ratio (C2/C3 ratio) following administration of the Asparagopsis oil composition as described herein is not adversely affected. In another embodiment, the acetate to propionate ratio (C2/C3 ratio) is reduced following administration of the Asparagopsis oil composition as described herein.

In another embodiment, the molar concentration of propionate does not have a negative effect.

Rumen fermentation of low-quality fibrous feeds is a major source of methane production in ruminants.

Examples of ruminants are given below. However, preferably, the Asparagopsis oil composition as described herein is used as a feed additive for livestock such as cattle, goats, sheep and llamas. The present invention is particularly suitable for cattle and sheep. Therefore, in one embodiment, said ruminant is selected from members of the suborder Ruminantia and Tylopoda. In another embodiment, said ruminant animal is a cattle or sheep. In another embodiment, said ruminant animal is cattle.

By "administer" and "administration" is meant the act of introducing the Asparagopsis oil composition as described herein into the gastrointestinal tract of an animal. In particular, this administration is the oral route of administration. This administration can in particular be accomplished by adding an Asparagopsis oil composition as described herein to the animal's feed, the feed thus enriched being then consumed by the animal. The Asparagopsis oil composition as described herein is administered to the gastrointestinal tract of the animal.

As discussed above, in preferred embodiments of the invention, the effective amount of the Asparagopsis oil composition as described herein is at least 16.67%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.25%, 0.125% or 0.067% organic matter administered to a ruminant animal.

For example, an Asparagopsis oil composition as described herein is administered at a dose of preferably at least 3%, 2%, 1%, 0.5%, 0.25%, 0.125% or 0.067% organic matter available in the ruminant diet animal.

For example, if a ruminant consumes approximately 2.5-3% of its live weight in feed per day, then a 400 kg ruminant can consume 10-12 kg of feed per day. For example, if a 400 kg ruminant (e.g., a steer) consumes 2.5% to 3% of its body weight per day of feed, then the Asparagopsis oil composition as described herein is administered at a dose proportional to the amount of organic matter administered to the ruminant. animal. In the case of a ruminant weighing 400 kg and where 80% of the feed is organic matter, if the animal consumes about 2.5% of its body weight per day (10 kg containing 8 kg organic matter), 3.3 liters of Asparagopsis oil composition 30 g wet weight/100 ml as described is required to obtain a dose equivalent to 1% organic matter administered to a ruminant. 1.6 liters of Asparagopsis oil composition 60 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 1.1 liters of Asparagopsis oil composition 90 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 833 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 666 ml of Asparagopsis oil composition 150 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 330 ml of Asparagopsis oil composition 30 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 167 ml of Asparagopsis oil composition 60 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 111 ml of Asparagopsis oil composition 90 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 83 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 67 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal.

An effective amount can be administered to the ruminant animal in one or more doses.

An effective amount may also be administered to said ruminant animal in one or more doses daily.

Dosages defined herein as amounts per kg of body weight per day refer to the average amount of Asparagopsis oil composition as described herein administered during a given treatment period, such as a week or month of treatment. Thus, the Asparagopsis oil composition as described herein can be administered every day, every other day, and so on, without departing from the scope of the invention. However, preferably the method includes daily administration of the Asparagopsis oil composition as described herein, in the required doses. Even more preferably, the Asparagopsis oil composition as described herein is administered during feeding of the animal whenever the animal is fed amounts to provide the above daily dosages.

The present method may involve administering the Asparagopsis oil composition as described herein in accordance with the dosage regimens described above for a period of at least 5, 10, 25, 50, 100, 250 or 350 days. An aspect of the invention is that the present methods provide very consistent effectiveness in reducing intestinal methanogenesis, for example, the effect is not reduced over a long period of treatment, for example, as a result of increased resistance of rumen or intestinal microorganisms, which makes long-term treatment of ruminants particularly suitable.

Accordingly, given the significant effects of lyophilized ground Asparagopsis , including reduction in overall gas production and CH ₄ yield, in one embodiment, the Asparagopsis oil composition as described herein is preferably administered in a form that produces the effects described herein (e.g., to reduce CH yield ₄ ), without affecting nutritionally important fermentation parameters.

Of the variety of secondary metabolites produced by Asparagopsis , bromoform (CHBr ₃ ) is one of the most abundant and biologically active. However, bromoform and chemically related compounds (eg, bromochloromethane) as refined chemicals are unsafe and are prohibited for use in humans and animals, including inhibition of methanogenesis in ruminants.

While intact freeze-dried ground Asparagopsis biomass can be used as a feed additive to inhibit methanogenesis in ruminants, it requires careful processing before feeding to minimize the loss of bioactive compounds such as bromoform and maintain activity. The most effective method currently available for obtaining Asparagopsis biomass is immediate freezing and subsequent freezing of the dry biomass for use as a feed additive. However, the present inventors have previously discovered that there is significant loss of bromoform if the intact biomass is not immediately frozen and/or air dried. Additionally, after 6 months, significant losses occur if lyophilized biomass is stored at ambient temperature (66% loss) or even refrigerated conditions (32% loss), with freezing of lyophilized material (20% loss) being the only practical option for long-term storage (˃6 months) (Fig.2). These processing and storage conditions are described in Vucko et al. (J. Appl. Phycol. 2017, 29, 1577-1586).

Surprisingly, the inventors of the present invention have discovered that Asparagopsis oil compositions prepared by contacting Asparagopsis biomass with oil retain high concentrations of the bioactive compound for long periods of time, even when stored at either room temperature or 4°C. For example, as described above, FIG. 3 shows that Asparagopsis oil compositions prepared by contacting Asparagopsis biomass with oil, with or without homogenization, retain high concentrations of bioactive compound for long periods of time when stored either at room temperature or at 4°C. Thus, the methods of the present invention provide Asparagopsis oil compositions in a form suitable for long-term storage (˃6 months) at room temperature.

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 Asparagopsis biomass collected from the wild. The present invention also reduces the number of steps to obtain a mixed product from preserved intact biomass or biologically active extract and therefore represents a significant improvement to existing processing technologies.

In another embodiment, the Asparagopsis oil composition as described herein is preferably administered in a form in which the secondary metabolites remain effective (eg, therapeutically effective).

Asparagopsis oil compositions as described herein can be administered to ruminants in one of many ways. Asparagopsis oil compositions as described herein can be administered in solid form as a medicinal product for veterinary use, can be distributed in excipients and directly fed to the animal, can be physically mixed with feed material in dry form, or an Asparagopsis oil composition as described herein. can be provided as a solution and then sprayed onto the feed material. It is believed that the method of administering the Asparagopsis oil composition as described herein to an animal is within the skill of one skilled in the art.

When used in combination with a feed material, the feed material is preferably grain/hay/silage/grass. Such feed materials include improved feeds and/or feeds based on tropical grasses or legumes, or directly grown, prepared in the form of canned feed hay, any feed ingredients and by-products of the food or feed industry, as well as by-products of the biofuel industry and corn meal and mixtures thereof, or batches of feed and dairy cow rations, such as high grain.

The timing of administration is not critical as long as the methane reduction effect is demonstrated. If the feed remains in the rumen, administration is possible at any time. However, since the Asparagopsis oil composition as described herein is preferably present in the rumen around the time methane is produced, the Asparagopsis oil composition as described herein is preferably administered with the feed or immediately prior to feeding.

In a specific embodiment, said effective amount of an Asparagopsis oil composition as described herein is administered to a ruminant animal by adding said effective amount of an Asparagopsis oil composition as described herein to said animal's feed. By "addition" as used herein is meant the act of incorporating an effective amount of an Asparagopsis oil composition as described herein directly into a feed intended for an animal. Thus, the animal, when fed, consumes the Asparagopsis oil composition as described herein, which can then have the effect of increasing, for example, the digestibility of the fibers and/or grains contained in the animal's feed.

Thus, another aspect of the invention relates to a feed additive for ruminants containing an Asparagopsis oil composition as described herein.

In another aspect, the present invention also provides a feed additive for reducing overall gas production and/or methane production in ruminants, said additive containing an effective amount of an Asparagopsis oil composition as described herein.

In one embodiment, an effective amount of an Asparagopsis oil composition as described herein is administered to said ruminant animal by adding said effective amount of an Asparagopsis oil composition as described herein to said animal's feed.

As discussed above, in one embodiment, the present invention maintains VFA concentrations in ruminant animals. Thus, this method allows the ruminant to maintain energy from feed based on, for example, fiber and cereals, and as a result, starting with a similar caloric intake, maintain energy suitable for metabolism, while reducing overall gas and CH ₄ production.

This is beneficial for livestock producers, who can thus optimize the cost of feed per unit of corresponding metabolic energy. This also represents a significant economic benefit.

The present inventors have previously demonstrated that administration of an effective amount of lyophilized crushed Asparagopsis to a ruminant animal does not adversely affect voluntary feed intake. Therefore, the present invention includes methods in which the concentration of degradable organic matter and/or dry matter is maintained.

As used herein, the term "animal feed additive" refers to a concentrated supplemental premix containing active ingredients, which premix or supplement can be added to an animal's feed or diet to create a fortified feed in accordance with the present invention. The terms "animal feed premix", "animal supplementary feed" and "animal feed additive" are generally considered to have similar or identical meanings and are generally considered interchangeable. Typically, the animal feed additive of the present invention is in the form of a powder or a compressed or granular solid. In practice, livestock can usually be fed an animal feed additive by adding it directly to the diet, for example as a so-called top dressing, or it can be used in the preparation or production of products such as animal feed or lick blocks, which will be described in more detail. below. The invention is not particularly limited in this regard. The supplement of the invention is typically fed to an animal in an amount of 16-2500 g/animal/day.

In one embodiment, the supplement of the invention is administered in an amount based on the animal's actual individual intake (eg, g/kg DM intake).

This animal feed supplement contains the Asparagopsis oil composition as described herein and is formulated such 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% feed organic matter.

For example, if a ruminant animal consumes approximately 5 kg of organic matter per day, the animal feed supplement contains an Asparagopsis oil composition as described herein and is formulated such that, when added to the feed, the Asparagopsis oil composition as described herein is administered at a dose of 208 ml per day. 208 ml of Asparagopsis oil composition 30 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 104 ml of Asparagopsis oil composition 60 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 52 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 0.1% organic matter administered to a ruminant animal. 2082 ml of Asparagopsis oil composition 30 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 1041 ml of Asparagopsis oil composition 60 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal. 520 ml of Asparagopsis oil composition 120 g wet weight/100 ml, as described, is required to obtain a dose equivalent to 1% organic matter administered to a ruminant animal.

In preferred embodiments, the additive contains an Asparagopsis oil composition as described herein in an amount of 10-100% by weight, preferably the amount greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, or 99 wt.%.

It is within the competence of a qualified specialist to accurately determine the ideal amount of components that should be included in the additive, and the amount of additive that will be used in the preparation of diet or feed for animals, etc., taking into account the specific type of animal and the conditions under which it is kept. Here are the preferred dosages of each component.

The animal feed additives of the present invention may contain any additional component without departing from the scope of the invention. Typically, it may contain well-known excipients that are necessary to prepare the desired form of the product, and may contain additional components aimed at improving the quality of the feed and/or improving the performance of the animal consuming the supplement. Suitable examples of such excipients include carriers or excipients such as lactose, sucrose, mannitol, starchy crystalline cellulose, sodium bicarbonate, sodium chloride and the like, as well as binders such as gum acacia, gum tragacanth, sodium alginate, starch, derivatives PVP and cellulose and so on. Examples of feed additives known to those skilled in the art include vitamins, amino acids and trace elements, digestibility enhancers and intestinal microflora stabilizers, and the like.

The present inventors have demonstrated that freeze-dried crushed Dictyota , Oedogonium and Cladophora patentiramea reduce overall gas production and CH ₄ production due to rumen fermentation. Therefore, in another embodiment, the methods further comprise administering to said ruminant an effective amount of at least one species of macroalgae selected from the group consisting of Asparagopsis armata , Asparagopsis taxiformis , Dictyota spp (e.g., Dictyota bartayresii ), Oedogonium spp, Ulva spp and C. patentiramea .

Accordingly, in one aspect, the present invention relates to a method of reducing overall gas production and/or methane production in ruminant animals, comprising the step of administering to said ruminant animal an effective amount of an Asparagopsis oil composition as described herein, and a composition comprising marine macroalgae or marine macroalgae oil extract .

In one embodiment, the marine macroalgae included in the composition is selected from the group consisting of Asparagopsis armata , Asparagopsis taxiformis , Dictyota spp (eg, Dictyota bartayresii ), Oedogonium spp, Ulva spp and C.patntiramea .

Another aspect of the invention relates to products such as animal feed and lick blocks containing an additive as previously defined herein.

The term “animal feed composition” as used herein means a composition that is suitable for use as animal feed and which is mixed from various natural or non-natural starting or raw materials and/or additives. Therefore, in particular, the term "compound feed" is used here to distinguish real feed compositions from any natural raw materials. These mixtures or feeds are prepared according to the specific needs of the target animal. The main ingredients used in commercially prepared feed generally include wheat bran, rice bran, corn meal, grains such as barley, wheat, rye and oats, soybean meal, alfalfa meal, cottonseed meal, wheat starch and the like. Commercial feed typically contains at least 15% crude protein and at least 70% total digestible nutrients, although the invention is not particularly limited in this regard. Liquid, solid, as well as semi-solid animal feed compositions are within the scope of the present invention, solid and semi-solid forms being particularly preferred. These compositions are usually produced in the form of flour, granules or grits. In practice, livestock can typically be fed a combination of feed, such as the feed of the present invention and silage, hay or the like. Typically, animal feed is fed in quantities ranging from 0.3-10 kg/animal/day. It is within the competence of a qualified specialist to determine the required quantity of these components that must be included in animal feed, taking into account the type of animal and the conditions of its keeping.

The animal feed compositions of the invention may contain any additional feed additive commonly used in the art. As is known to those skilled in the art, the term "feed additive" in this context refers to products used in animal nutrition to improve the quality of feed and the quality of food products of animal origin or to improve animal performance, for example, providing increased digestibility of feed. Non-limiting examples include processing aids such as preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators and silage additives; sensory additives, in particular flavors and colours; (hereinafter) nutritional supplements such as vitamins, amino acids and microelements; and (further) zootechnical additives such as digestibility enhancers and intestinal flora stabilizers.

As will be appreciated by those skilled in the art, the present animal feed compositions may contain any additional component or additive without departing from the scope of the invention.

In a further aspect, the invention relates to a lick stone or lick block containing an Asparagopsis oil composition as described herein. As is known to those skilled in the art, such stones or lick blocks are particularly useful for feeding mineral supplements (as well as proteins and carbohydrates) to ruminant animals grazing either natural or cultivated pastures, or both. Such lick blocks or lick stones in accordance with the present invention typically contain, in addition to the Asparagopsis oil composition as described herein, various types of binders, for example cement, gypsum, lime, calcium phosphate, carbonate and/or gelatin; and optionally other additives such as vitamins, trace elements, mineral salts, sensory additives and the like.

Another aspect of the invention relates to a method of reducing methane production in the gastrointestinal tract of ruminants, the method comprising administering a composition containing an Asparagopsis oil composition as described herein.

As used herein, the terms “reducing gastrointestinal methanogenesis” and “reducing gastrointestinal methane production” refer to reducing the formation of methane gas in the gastrointestinal tract. As explained above, fermentation in the rumen and intestines of the ruminant leads to the formation of methane gas by so-called methanogens. The present invention is aimed at reducing this process, for example, reducing the release of methane directly from the gastrointestinal tract. Estimating methane emissions from animals is within the knowledge and competence of a person skilled in the field. As previously explained, methane production in the rumen and intestine is a process that normally occurs in healthy animals, and reducing methanogenesis does not improve or worsen the overall health or well-being of ruminants.

Thus, the present treatment methods are not therapeutic treatments, that is, the methods do not improve the health of an animal suffering from a specific disorder, do not prevent a specific disease or disorder, and do not in any way affect the health of a ruminant animal in any other way, i.e., compared to ruminants not subjected to these treatments. As explained previously, the advantages of the present methods are limited to environmental and/or economic aspects.

As will be clear from the foregoing, the present method involves the oral administration of an Asparagopsis oil composition as described herein. Preferably, the treatment involves oral administration of the animal feed composition and/or additional animal feed products as defined above, even though other liquid, solid or semi-solid oral compositions may be used without departing from the scope of the invention as will be understood by those skilled in the art. .

In accordance with the foregoing, another aspect of the invention relates to the use of a composition containing an Asparagopsis oil composition as described herein to non-therapeutically reduce methane production in the gastrointestinal tract of ruminants.

In another aspect, the present invention also relates to feed for ruminant animals, wherein said feed is enriched with a feed additive described herein.

In another aspect, the present invention relates to a method for reducing methane production in ruminant animals, said method comprising the step of administering to said animal a feed additive described herein or a feed described herein.

As used herein, the term "shelf life" is used to indicate that the amount or concentration of the biologically active (eg, antimethanogenic) component in at least one oil does not decrease significantly during the period of time and temperature at which the composition is stored. In other words, the amount of time that passes before a significant decrease in the concentration of a biologically active (eg, antimethanogenic) component occurs is considered an indicator of shelf life. The term "stability" is also used as an indicator of shelf life. The "stability" of at least one biologically active (eg, antimethanogenic) component can be assessed by measuring the concentration of the antimethanogenic component over time.

The concentration of the biologically active (eg antimethanogenic) component will generally decrease over time after it is extracted into the oil and will depend on storage conditions. A decrease in concentration can be caused by many reasons, such as evaporation or sublimation of a biologically active (eg antimethanogenic) component from the oil. It may also be due to the reaction of the biologically active (e.g., antimethanogenic) component with itself, residual water in the oil extract, oxygen or other gases in the air, other components in the oil, or reaction with the oil itself, and may be mediated by light and/or by heating, as is typical for many halogenated compounds. The concentration by which the biologically active (eg, antimethanogenic) component is reduced to remain acceptable for consumption will vary depending on a number of factors, such as the end use of the product and degradation by-products that may be generated. For example, the concentration by which at least one biologically active (e.g., antimethanogenic) component may be reduced may be no more than 5%, 10%, 20%, 30%, 40%, or 50% after storage at or 4°C or 25°C and this will still be acceptable for use. In one embodiment, the concentration of at least one biologically active (eg, antimethanogenic) component may decrease by no more than 20% after 65 weeks of storage at 25°C. In another embodiment, the concentration of at least one antimethanogenic component may be reduced by no more than 50% after 65 weeks of storage at 4°C or 25°C.

ABBREVIATIONS

A number of abbreviations are used in the description. For the avoidance of doubt, these abbreviations are defined below:

ANOVA analysis of variance B.C.A. bromochloroacetic acid B.F. bromoform d day(s) DBA dibromoacetic acid DBCM dibromoacetic acid DCM dichloromethane dw dry weight eq. equivalent(s) fw wet weight GC gas chromatography HPLC high performance liquid chromatography under pressure IS Internal standard MS mass spectrometry MTBE methyl tert-butyl ether n number of samples OB organic matter RT Room temperature LZHK Volatile fatty acids wk week(s)

The invention will now be further described by way of examples, which are intended to assist one skilled in the art in practicing the invention and are in no way intended to limit the scope of the invention.

EXAMPLES

EXAMPLE 1. OBTAINING ASPARAGOPSIS OIL COMPOSITION

Asparagopsis taxiformis (gametophyte stage) was collected from Magnetic Island (Queensland, Australia). Asparagopsis armata collected from Cloud Bay, Bruny Island, Tasmania, 43°43'94S, 147°21'52E. The raw biomass was blotted dry and samples (6 x 30.0 g fresh weight (fw)) were collected into individual 250 ml glass bottles (Schott), each pre-filled with 100 ml vegetable oil (Homebrand coupled vegetable oil, 95 canola oil %, sunflower oil 5%). Comparative samples (6 x 30.0 g fresh weight (fw)) were also collected in 100 ml Milli-Q water instead of vegetable oil. All bottles were tightly sealed and stored on ice for immediate transport to the laboratory for further processing.

Biomass samples (6 × 30.0 g fresh weight) were also collected in zip lock bags for dry weight (dw) determination and determination of bromoform content of the biomass (3 × 30.0 g fresh weight).

In the laboratory (less than 4 hours from collection), the biomass in each solvent (oil or water) was homogenized by grinding using an IKA ultra-turrax T-25 for 60 s (n=3 for oil and n=3 for water), while the biomass in the remaining three replicates for each solvent remained unchanged. All biomass bottles (n=12) were then stored in a refrigerator (4°C). Samples were analyzed for bromoform, an antimethanogenic component present in algae, on days 1, 3, 5, 7, and 10 after collection (day 0).

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

For samples in which water was used as a solvent, bromoform was extracted from 1.0 ml sub-samples from each bottle replicate using 1.0 ml 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.

For samples in which oil was used as the solvent, bromoform was extracted from 1.0 mL sub-samples from each bottle replicate using 1.0 mL naphthalene-methanol solution (10 μg/mL HPLC-grade naphthalene in methanol). Samples were allowed to separate for 2 hours at 4°C before analyzing the methanol layer. Separation conditions were chosen based on previous experiments using different solvents (hexane, methanol, MTBE, DCM) and time periods (from 30 minutes to 48 hours). The methanol phase was collected and analyzed by GC/MS as described below.

An experimental scheme for extracting halogenated metabolites from intact or homogenized A. taxiformis biomass into oil or water is shown below.

Experiment 1: Treatment

To determine dry weight and bromoform content, the biomass was immediately frozen and then lyophilized (Virtiz benchtop 2K, -55°C, 120 μbar, 48 h). The dried biomass was weighed to determine dry weight, then ground to 1 mm particles and stored at -20°C in sealed jars.

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 Paul et al. (Mar. Ecol. Prog. Ser. , 306 (2006) 87-101) with modifications described by Machado et al. (J Appl Phycol, 28 (2016) 3117-3126). Briefly, analytical conditions were pulse injections (1 μL, 35 psi) in splitless mode with the following temperature parameters: injection port (250°C), GC/MS interface (300°C), and oven (hold at 40°C for 1 min, rapidly increased by 16°C⋅min ^-1 to 250°C, then held at 250°C for 2 min) as described by Paul et al. Helium was used as carrier gas at 2 ml⋅min ^-1 . Separate standard curves were generated for each method (water, oil, biomass), and the concentration of target compounds in each sample was calculated from the ratio of the peak areas of the target compound to the internal standard. Bromoform was identified by comparison with a commercially available standard (Sigma Aldrich, Australia) and based on its characteristic fragment ions (molecular ion cluster at m/z 250, 252, 254, 256 [1:2:2:1]). The concentration of bromoform in solutions was standardized to the amount of dry biomass extracted and expressed as mg bromoform/g dry weight of biomass.

The dry weight of a 30 g sample of Asparagopsis blotted dry was determined to be about 3 g. Figure 1 shows the results of different extraction methods (using water as solvent versus oil as solvent; and using intact biomass versus homogenized/comminuted biomass), where the biomass is in contact with a solvent (water or oil) for a period of up to 10 days. The results are presented as the amount of extracted BF (mg) per gram of dry weight of algae. BF was also extracted using a previously described method for comparison. This method involved lyophilizing a biomass sample and extracting BF into methanol by contacting the biomass with methanol for 72 hours.

Figure 1 shows that water-extracted Asparagopsis biomass produced less BF than oil-extracted Asparagopsis biomass; the concentration of BF extracted from Asparagopsis biomass into oil increases compared to the concentration of BF extracted from an equivalent amount of biomass into water. These data demonstrate the extraction of BF, a bioactive compound, from Asparagopsis biomass into oil to form an Asparagopsis oil composition.

When used as an extraction solvent, intact or homogenized biomass gave similar results. The amount of BF extracted from these samples was approximately 50% lower than when using oil as solvent (after 24 h) and remained approximately 30-40% lower during the soaking time (up to 10 days).

Figure 1 also shows that homogenizing the biomass in oil resulted in the greatest amount of bromoform extracted in the shortest period of time (1 day), with the BF content of the intact biomass in oil reaching concentrations similar to the homogenized biomass only after about seven days of soaking/extraction. These data demonstrate that the rate of extraction of BF, a bioactive substance, from Asparagopsis biomass into oil is increased by homogenizing the biomass in at least one oil.

Figure 1 also shows that the extraction efficiency using oil depended on whether the biomass was intact or homogenized. Surprisingly, the oil-extracted homogenized biomass yielded much more BF even after 24 h compared to the methods of extracting lyophilized biomass with methanol for 72 h. As can be seen from Fig. 1, the lyophilized sample extracted by soaking in methanol for 72 h gave only about 15 mg BF/g algae (dw), while a homogenized algae sample soaked in oil for only 24 h gave about 17.8 mg BF/g algae (dw), which represents a significant improvement in compared to extracting lyophilized biomass with methanol. These data demonstrate that the extraction rate of BF, a bioactive substance, from Asparagopsis biomass into oil is increased by homogenizing the biomass in at least one oil.

EXAMPLE 2. STORAGE LIFE OF ASPARAGOPSIS OIL COMPOSITION

The shelf life (eg, stability) of the Asparagopsis oil compositions of the present invention was evaluated. The ratio of bioactive compounds such as bromoform (BF) and dibromochloromethane (DBCM) was examined and compared with the internal standard (IS; naphthalene) as a function of time. Any decrease in the ratio indicates a loss of compound and, therefore, a decrease in the stability of bioactive oil compositions, which leads to a reduction in shelf life.

After 10 days of extracting the Asparagopsis biomass into oil or water, the biomass was removed. Asparagopsis oil compositions (with biomass) were filtered through a 100 μm nylon mesh followed by centrifugation (3200 g, 15 min). The clarified solution from each replicate of each treatment (oil or water) was then divided into two 30 ml glass bottles (Schott), with one of these bottles stored at 4°C and the other at 25°C (n=3 for each temperature, 24 bottles in total). Sub-samples (1 ml) were collected after 0, 4, 8, 12 and 65 weeks of storage and analyzed as described above. Week 0 was counted from the start date of storage, as shown in the experimental design below:

Experiment 2: shelf life

The lyophilized biomass was also analyzed for bromoform content at the same time intervals, with week 0 counted from the day the biomass was removed from the freeze dryer.

Lyophilized biomass stored at -20°C according to the standard storage method showed no loss over time when using the standard methanol extraction method (data not shown).

Figure 3 shows the stability of the Asparagopsis oil composition of the present invention over time. As can be seen for the aqueous extracts, significant losses of the biologically active substance, bromoform, were observed by week 12 of storage at both 4°C and 25°C. Therefore, further analysis of water samples was not performed for these samples.

Conversely, the concentration of the bioactive compound, bromoform, extracted into the oil for any treatment (homogenized or intact) did not decrease significantly over 12 weeks regardless of storage at 4°C or 25°C (Figure 3). Surprisingly, after 65 weeks, there was no significant reduction in bromoform content in Asparagopsis oil formulations stored at 4°C for any treatment (homogenized or intact). Asparagopsis oil formulations stored at 25°C for 65 weeks lost 15% to 20% of the extracted parent bromoform. The apparent "increase" in BF over time can be attributed to analytical variance. These data demonstrate that Asparagopsis oil compositions formed by extracting Asparagopsis into oil are stable over long periods of time. In particular, these data demonstrate that the concentrations of the bioactive compound, BF, in Asparagopsis oil compositions formed by extracting Asparagopsis into oil are stable over long periods of time.

The results of the DBCM:IS ratio over time are shown in Figure 4 (intact biomass) and Figure 5 (homogenized biomass). DBCM was detected over a storage period of 65 weeks. It is important to note that there was no statistically significant difference in the DBCM:IS ratio in each treatment (intact biomass versus homogenized biomass or storage at 4°C versus 25°C), demonstrating superior shelf life for all samples using DBCM as a marker for anti-methanogenic components. The apparent "improvement" in the DBCM:IS ratio over time can be attributed to analytical variance. These data demonstrate that DBCM concentrations in Asparagopsis oil compositions formed by extracting Asparagopsis into oil are stable over long periods of time.

Figures 6 and 7 show the extraction of DBA, a biologically active substance, from Asparagopsis biomass into oil to form an Asparagopsis oil composition.

Considering the above results, it can be seen that the clarified oil compositions (i.e. with Asparagopsis biomass removed) had 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, aqueous extracts remained stable for 8 weeks and deteriorated thereafter, regardless of storage temperature. This result is unexpected since storage at lower temperatures, regardless of the carrier (water-based or oil-based), is expected to provide longer shelf life.

EXAMPLE 3. MAXIMUM EXTRACTION OF BIOMASS INTO OIL

To quantify the maximum amount of Asparagopsis biomass that can be extracted, the extraction method from Example 1 was used, which provided the highest concentration of bioactive compound (bromoform) found in the oil in the shortest period of time (eg, homogenization and extraction into oil).

Asparagopsis taxiformis (gametophyte stage) was collected from Magnetic Island (Queensland, Australia). The raw biomass was blotted dry and samples of the following weights were placed in separate 250 ml glass bottles pre-filled with 100 ml vegetable oil: 30.0 g, 60.0 g, 90.0 g and 120.0 g wet weight (n=3 for each value). All bottles were tightly sealed and stored on ice for immediate transport to the laboratory for further processing and analysis. Replicate samples (n=3) of dry-blotted biomass (30.0 g wet weight) were also collected to determine dry weight, as shown in the experimental design below:

Experiment 3: Maximum increase in biomass:oil ratio

In the laboratory, the biomass was homogenized (IKA ultra-turrax T-25) in oil and samples were stored at 4°C for 24 hours until bromoform was quantified as described above.

Figures 8 and 9 show the amount of biologically active substance (bromophore) extracted from different amounts of Asparagopsis biomass (in the same volume of oil). Figure 8 shows that increasing the Asparagopsis biomass:oil ratio resulted in a corresponding increase in the amount of bioactive substance extracted into the oil. These data demonstrate that the concentration of the bioactive compound, bromoform, from Asparagopsis biomass to oil is increased by increasing the biomass to oil ratio.

At higher biomass:oil ratios, a hard, opaque gel formed and it was not possible to completely homogenize the biomass into oil. For example, a solid, opaque gel was formed when 90 g or 120 g of homogenized biomass was homogenized in 100 ml of oil. This gelation also made it very difficult to recover the 1 mL of clarified oil required for methanol separation for GC/MS analysis.

Sub-samples from the 120 g treatment containing the gel were heated (1 h, 60°C, water bath) before centrifugation. This led to an increase in the yield of biologically active substances in oil by 20% compared to centrifugation of unheated samples (see Figs. 8 and 9). Asparagopsis contains sulfated cell wall polysaccharides that are generally water soluble. Without wishing to be bound by theory, it is believed that a gel/emulsion is formed between the extracted polysaccharides, oil and residual external and internal water on top and within the biomass. Extracting higher amounts of polysaccharides at higher biomass:oil ratios resulted in gel formation. It is understood that the gel contains biologically active substances (such as bromoform); when heated, the gel releases bromoform, which is then transferred into the oil from the Asparagopsis oil composition.

EXAMPLE 4. STUDY OF THE REDUCTION OF METHANE FORMATION OF ASPARAGOPSIS OIL COMPOSITION IN VIVO

Fistulated steers (Bos indicus) are used in in vivo feeding studies. Before the start of the experimental period, all animals undergo fistulation and training in breathing chambers. The steers are initially kept on Flinders grass hay in group pens (barnyards) for four days. The steers are then divided into two groups and allocated to treatment, control (Flinders grass hay only) and Asparagopsis oil supplementation groups. The dose selection of the Asparagopsis oil formulation was based on results obtained in a previous in vitro study examining methane reduction capabilities.

The wet weight to dry weight ratio of Asparagopsis blotted dry is 10 (ie 30 g wet weight=3 g dry weight). Assuming an organic matter (OM) content of 80% dry weight (Machado et al., 2014), the corresponding Asparagopsis -derived OM content of the tested biomass/oil mixtures ranges from 0.024-0.096 g OM/mL (Table 1):

Table 1.

Content of organic matter derived from Asparagopsis biomass in biomass/oil mixtures at increased ratios and volumes** required for a feed dose equivalent to 0.1-3% Asparagopsis OM

Quantity (g) Asparagopsis in oil Volume of oil (ml) required to obtain an equivalent dose of 0.1-3.0% Asparagopsis OM per 100 g of feed** fw/100 ml fw/ml dw/ml OM Asparagopsis/ml eq. 0.1 OB Asp eq. 1.0 OB Asp eq. 3.0 OB Asp thirty 0.3 0.03 0.024 4.17 41.7 125.0 60 0.6 0.06 0.048 2.08 20.8 62.5 90 0.9 0.09 0.072 1.39 13.9 41.7 120 1.2 0.12 0.096 1.04 10.4 31.3 150 1.5 0.15 0.12 0.83 8.3 25.0

**This volume depends on the oil separated from the biomass. For example, adding 120g wet weight of seaweed to 100ml oil increases the final volume of the mixture. Being 90% water (fw:dw ratio is 10), and for ease of calculation it is assumed that the density of Asparagopsis is equal to water, and therefore the volume of the biomass:oil mixture should increase according to the amount of biomass added (i.e. 100 ml oil + 150 g fresh weight of Asparagopsis = 250 ml of mixture, which corresponds to 15 g of fresh weight of Asparagopsis = 12 g of Asparagopsis OM / 250 ml = 0.048 g of Asparagopsis OM / ml of homogeneous mixture).

Based on the data discussed above, beyond the biomass:oil ratio of 150 g fresh weight: 100 ml, a homogeneous mixture may not be formed. A dose of 1% Asparagopsis OM is applicable at a biomass:oil ratio ≥120 g/100 ml. A dose of 0.2% Asparagopsis OM has been demonstrated to be effective in inhibiting methanogenesis in animals in vivo, so a volume of only 2.08 ml of oil (or 4.6 ml of biomass/oil mixture) needs to be added to 100 g of feed for a 120 g mixture biomass/100 ml oil.

To calculate the volume of oil required to produce a dose equivalent to the required % organic matter concentration administered to the ruminant, the % organic matter in the Asparagopsis oil compositions described herein is calculated from the fresh weight (fw) of Asparagopsis biomass contacting the specified volume, at least one oil. The wet weight to dry weight (dw) ratio of Asparagopsis blotted dry is 10 (ie 30 g fw=3 g dw). Assuming organic matter (OM) content to be 80% of dw based on previous data, the corresponding Asparagopsis -derived OM content of the biomass/oil can be calculated. For example, for an Asparagopsis oil composition containing 30 g fresh weight (fw) of Asparagopsis biomass in 100 ml, the at least one oil is equivalent to 0.024 Asparagopsis organic matter/ml. If the required inclusion concentration in the feed is 0.1% Asparagopsis OM per 100 g of feed, then 4.17 ml of Asparagopsis OM 30 g fw/100 ml are required per 100 g of feed.

Steers are allocated to individual pens at the research station with unlimited Flinders grass hay and water. Animals receiving an Asparagopsis oil composition supplement are dosed directly into the rumen prior to morning feeding to ensure complete consumption of the Asparagopsis oil composition and uniformity of dosage by the animals, or the Asparagopsis oil composition is added to the feed as described above. Before entering open-circuit breathing chambers to measure methane production over a 48-hour period, an adaptation period of 14 days to the different diets is carried out. Animal methane production was measured after 7, 14, 21 and/or 29 days of treatment to evaluate the effectiveness of the Asparagopsis oil composition in reducing methane production in animals over time. After 31 days, the Asparagopsis oil composition runs out and the animals are moved to pens. Rumen samples are collected 4 hours after intraruminal administration of the Asparagopsis oil composition or after feeding (eg, days 1, 15, 22, and 30 of Asparagopsis oil composition treatment) to assess changes in VFA production and acetate to propionate ratio. Live weight and feed offered and rejected 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. At all control points in time, the average methane production is calculated.

EXAMPLE 5. STUDY OF REDUCED METHANE FORMATION OF ASPARAGOPSIS OIL COMPOSITION IN VIVO IN SHEEP

Methodology

Merino crosses are divided into one of five groups based on the daily inclusion rate (organic matter in terms of OM) of the Asparagopsis oil composition [0 (control), 0.1, 0.5, 1.0, 2.0, 3.0% ]. Inclusion factors (% RH consumption) are calculated as shown using the calculations given in Table 1 and Example 4.

Sheep are kept in animal housing conditions and fed a pelleted commercially available diet based on lupine, oats, barley, wheat with cereal straw as a coarse component [chemical composition (g/kg DM) of ash, 72; crude protein (CP) 112; neutral detergent fiber (aNDFom) 519; acid detergent fiber (ADFom) 338 and without cobalt, selenium and rumen modifiers] at 1.2 times the content throughout the study. All sheep are dosed using a cobalt pill prior to the start of the experimental period.

Sheep are gradually adapted to the Asparagopsis oil formulation over the first two weeks by mixing the ground material with 200 g of ground lupine (lupine ration). A mixture of Asparagopsis oil composition and lupine is then added to the pelleted diet, mixed and fed for another 75 days.

Feed intake was recorded daily and live weight (LW) was measured at 14-day intervals throughout the trial.

Three measurements of individual methane production by animals (g/kg DM consumption) are carried out, first after 30 days of inclusion of the Asparagopsis oil composition, and then at 21 day intervals throughout the test period. When measuring methane over 24 hours using open circuit breathing chambers as described by Li (2013) [PhD thesis; Eremophila glabra reduced methane production in sheep, University of Western Australia] feed offered (pellets/lupine) is proportionally reduced to 1.0 times the content to ensure consistent intake.

After each methane measurement, up to 50 ml of rumen fluid is collected via gavage to determine the concentration of volatile fatty acids (VFA).

Statistical analysis

Statistical analyzes are performed by fitting linear mixed models to each response variable. These models can account for experimental design (assignment of animals to specific groups and chambers), data structure (repeated measurements), and any missing values that arise. “Fixed effects” in the mixed model included the treatment effect (different inclusion rates of Asparagopsis taxiformis ), the time effect (sampling dates), the treatment effect versus time, and any covariates. Initial live weight is included as a covariate in the analysis of live weight. It is also tested as a potential covariate for other response variables.

Averages are calculated for all treatment and time combinations, adjusting for all other terms in the model. P-values are calculated to test the overall effect of time, treatment, and their interaction. Least significant differences (P=0.05) are calculated to compare pairs of means.

The following indicators are measured:

consumption of feed containing Asparagopsis oil composition,

dry matter destroyed in vivo,

live weight of animals,

total VFA concentration,

molar ratios of individual VFAs (including acetate, propionate), excluding isobutyrate,

methane emissions (DM consumption g/kg).

Claims (55)

1. A method for producing an Asparagopsis oil composition, including the stages: (a) providing Asparagopsis biomass; (b) providing at least one oil; And (c) contacting the biomass with at least one oil under conditions of extracting at least one biologically active substance from the biomass into at least one oil to form an Asparagopsis oil composition, wherein the at least one biologically active substance includes bromoform. 2. The method according to claim 1, wherein step (c) includes homogenizing the biomass in at least one oil. 3. The method according to claim 1, further comprising, after step (c), the step of separating said biomass from said at least one Asparagopsis oil composition. 4. The method according to claim 1, wherein the ratio of biomass to 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 claim 1, wherein step (c) is carried out over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. 6. Method according to claim 1, wherein step (c) is carried out at a temperature of about 4°C. 7. The method according to any one of claims 3-6, in which, before the stage of separating said biomass from said at least one oil, the biomass in contact with at least one oil is heated in such a way that the gel containing at least one biologically active a substance that releases said at least one biologically active substance into at least one oil. 8. The method according to claim 7, in which the biomass in contact with at least one oil is heated to a temperature of 60°C. 9. Method according to claim 7 or 8, wherein the biomass in contact with at least one oil is heated for one hour. 10. The method according to claim 1, wherein said Asparagopsis is Asparagopsis taxiformis and/or Asparagopsis armata . 11. The method of claim 1, wherein the step of providing Asparagopsis biomass does not include air-drying the biomass. 12. The method of claim 1, wherein the step of providing Asparagopsis biomass includes introducing the biomass into at least one oil. 13. The method of claim 1, wherein the at least one oil comprises an edible oil. 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, rapeseed oil, corn oil, butter copra, cottonseed oil, diacylglycerol oil, linseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flax seed 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, canola oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil or any combination thereof. 15. The method according to claim 1, wherein the at least one biologically active substance further comprises bromochloroacetic acid (BCA), dibromoacetic acid (DBA) and/or dibromochloromethane (DBCM). 16. The method according to claim 1, wherein the concentration of at least one biologically active substance extracted from the biomass into at least one oil is increased relative to the concentration of at least one biologically active substance extracted from an equivalent amount of biomass into water. 17. The method according to claim 1, wherein the concentration of at least one biologically active substance extracted from the biomass into at least one oil does not decrease significantly after storage for 12 weeks at 25°C. 18. The method according to claim 1, wherein the concentration of at least one biologically active substance extracted from the biomass into at least one oil does not decrease significantly after storage for 65 weeks at 4°C. 19. Method according to any one of claims 1 to 18, in which at least one biologically active substance is an antimethanogenic component. 20. The method of claim 19, wherein the anti-methanogenic component further comprises DBCM. 21. The method of claim 19, wherein the concentration of the antimethanogenic component extracted from the biomass into the at least one oil does not decrease by more than 20% after storage for 65 weeks at 25°C, wherein the at least one antimethanogenic component represents BF and/or DBCM. 22. The method according to claim 1, in which the Asparagopsis oil composition contains at least 0.1 mg of bromoform per 1 ml of Asparagopsis oil composition. 23. The method according to claim 1, in which the Asparagopsis oil composition contains at least 1 mg of bromoform per 1 ml of Asparagopsis oil composition. 24. The method according to claim 1, in which the Asparagopsis oil composition contains at least 2 mg of bromoform per 1 ml of Asparagopsis oil composition. 25. The method according to claim 1, in which the Asparagopsis oil composition contains at least 3 mg of bromoform per 1 ml of Asparagopsis oil composition. 26. The method according to claim 1, in which the Asparagopsis oil composition contains at least 4 mg of bromoform per 1 ml of Asparagopsis oil composition. 27. Asparagopsis oil composition obtained by the method according to any one of claims 1 to 26. 28. An Asparagopsis oil composition containing at least one exogenous oil and at least one antimethanogenic component, wherein the at least one antimethanogenic component includes bromoform. 29. The Asparagopsis oil composition according to claim 28, wherein said Asparagopsis is Asparagopsis taxiformis and/or Asparagopsis armata . 30. The Asparagopsis oil composition of claim 28, wherein the at least one oil comprises an edible oil. 31. The Asparagopsis oil composition of claim 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, rapeseed oil, corn oil , copra oil, cottonseed oil, diacylglycerol oil, flaxseed oil, grapefruit seed oil, grape seed oil, hazelnut oil, hemp oil, lemon oil, flaxseed oil, macadamia oil, mustard oil, olive oil, orange oil, palm oil, palm kernel oil, peanut oil, pecan oil, pine nut oil, pistachio oil, pumpkin seed oil, canola oil, rice bran oil, safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil and vegetable oil, or any combination thereof. 32. The Asparagopsis oil composition of claim 28, wherein the at least one anti-methanogenic component 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 claim 28, wherein the concentration of at least one antimethanogenic component does not decrease significantly after storage for 12 weeks at 25°C. 34. The Asparagopsis oil composition of claim 28, wherein the concentration of the at least one antimethanogenic component does not decrease significantly after storage for 65 weeks at 4°C. 35. The Asparagopsis oil composition according to claim 28, wherein at least one anti-methanogenic component is BF and/or DBCM. 36. The Asparagopsis oil composition according to claim 28, wherein the concentration of at least one antimethanogenic component does not decrease by more than 20% after storage for 65 weeks at 25°C, wherein the at least one antimethanogenic component is BF and/ or DBCM. 37. The Asparagopsis oil composition of claim 28, wherein the concentration of the at least one antimethanogenic component does not decrease by more than 50% after storage for 65 weeks at 4°C or 25°C, wherein the at least one antimethanogenic component is DBA. 38. The Asparagopsis oil composition according to claim 28, wherein at least one antimethanogenic component has a concentration of at least 0.1 mg per 1 ml of extract. 39. The Asparagopsis oil composition according to claim 28, wherein at least one antimethanogenic component has a concentration of at least 1 mg per 1 ml of extract. 40. The Asparagopsis oil composition according to claim 28, wherein at least one antimethanogenic component has a concentration of at least 2 mg per 1 ml of extract. 41. The Asparagopsis oil composition according to claim 28, in which at least one antimethanogenic component has a concentration of at least 3 mg per 1 ml of extract. 42. The Asparagopsis oil composition according to claim 28, in which at least one antimethanogenic component has a concentration of at least 4 mg per 1 ml of extract. 43. A feed additive for reducing the overall gas formation and/or methane formation in ruminants, said additive contains an effective amount of the Asparagopsis oil composition according to claim 27 or 28. 44. Feed for ruminant animals, where the feed additive according to claim 43 is added to said feed. 45. A method of reducing overall gas production and/or methane production in a ruminant animal, comprising administering to said ruminant animal an effective amount of an Asparagopsis oil composition according to claim 27 or 28. 46. The method of claim 45, wherein the method includes maintaining an effective concentration of the desired volatile fatty acid. 47. The method of claim 46, wherein the desired volatile fatty acid comprises acetate and propionate and wherein maintenance includes reducing the ratio of acetate to propionate. 48. The method according to any one of claims 45-47, where the method includes maintaining the concentration of decomposed organic matter and/or dry matter. 49. The method according to any one of claims 45-48, wherein the Asparagopsis oil composition is administered at a dose equivalent to at least 3%, 2%, 1%, 0.5%, 0.25%, 0.125% or 0.067% organic substance administered to a ruminant animal. 50. The method according to any one of claims 45-49, in which the specified ruminant animal is selected from representatives of the suborder Ruminantia and callosopods (Tylopoda). 51. The method according to any one of claims 45 to 49, wherein said ruminant animal is cattle or sheep. 52. The method according to any one of claims 45 to 49, wherein said ruminant animal is cattle.

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