Feed compositions and uses thereof

Abstract

The present invention relates to a feed additive for reducing methane production in the rumen of a ruminant animal comprising an isoflavone. In particular, the isoflavone comprises one or more of formononetin, biochanin A and / or precursors thereof. The present invention also relates to a method of reducing methane production in the rumen of a ruminant animal comprising administration of an isoflavone.

Description

[0001] FEED COMPOSITIONS AND USES THEREOF Field of the Invention The present invention relates to feed additives and compositions for modulating methane production in ruminant animals and in industrial anaerobic digestion. The feed additives and compositions comprise isoflavones, in particular biochanin A and formononetin, and their conjugates. The invention also relates to methods of reducing methane production in ruminant animals. The invention further relates to methods of decreasing and increasing methanogenesis in industrial processes by varying the isoflavone concentration. Background to the Invention Methanogenesis in the rumen of a ruminant animal has profound effects upon nutrient cycling by livestock, and affects both local animal production metrics, and anthropogenic climate change on a global scale. The ecosystems and biochemical pathways involved in ruminal digestion and methanogenesis are complex. The main process involves the conversion of lignocellulosic materials to the volatile fatty acids (VFAs) acetate, propionate and butyrate. In ruminant livestock, these VFAs are available for absorption across the alimentary epithelium and provide for the bulk of the animals’ energy requirement. This fermentation involves the oxidation of redox coenzymes (NADH, NADPH, FADH), thereby liberating free hydrogen and carbon dioxide into the rumen. Members of the domain archaea utilise the endogenous CO2 and H2 as metabolic substrates, and release methane as a by-product according to the equation: CO2 + 8H++ 8e−→ CH4 + 2H2O The removal of methane from the ruminal ecosystem is a necessary prerequisite for continued oxidative respiration, and therefore fermentation (1) and is usually achieved via eructation to the atmosphere. However, methane is one of the most potent greenhouse gases contributing to global warming. Therefore, reducing methane production is an important area of global agricultural research, and crucial for sustainable livestock production and mitigating climate change. Recently, the United Kingdom Government has mandated the inclusion of anti-methanogenic compounds in ruminant livestock rations from 2025 (2). It is the object of the present invention to address one or more issues outlined above. In particular, it is an object of the present invention to provide a feed additive for reducing methane production in ruminant animals. Summary of the Invention In accordance with a first aspect of the present invention, there is provided a feed additive for reducing methane production in the rumen of a ruminant animal comprising an isoflavone. Advantageously the inventors have found that the addition of isoflavones to an anaerobic digestion media, such as rumen fluid, reduced methanogenesis and delayed the peak methane producing period compared to a control. The addition of isoflavones also altered the microbial populations present including changing their population size and the ratio of the different microbial populations present. Isoflavones are a class of phytoestrogenic compounds commonly found in members of the Fabaceae family. In some embodiments, the isoflavone comprises an O-methylated isoflavone. Preferably, the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA. The precursors may comprise daidzein and / or genistein. Advantageously, the inventors of the present invention have found that FMN and BCA display high anti-methanogenic activity in anaerobic microbial fermentative systems. Anaerobic microbial fermentative systems include any in vivo, in vitro or ex vivo environment with an absence of free oxygen and the presence of fermentative microbes. The fermentative microbes may comprise bacteria and / or fungi. Examples of an anaerobic microbial fermentative system include the rumen of a ruminant animal and an industrial anaerobic digestion plant. Other anaerobic microbial fermentative systems include those inoculated with anaerobic digestate or rumen liquor. Trifolium pratense (red clover) contains high levels of polyphenolic components including the phytoestrogenic compounds isoflavones. Red clover mainly contains the isoflavones formononetin (FMN) and biochanin A (BCA), with smaller quantities of their respective precursors, daidzein and genistein, which are more predominant in soy. In leguminous plants, isoflavones may be found in either an aglycone or conjugated form. Hexosides and malonyl-hexosides are the predominant conjugates encountered in leguminous herbage, and may be more abundant than the aglycone form (3). Although subject to enzymatic hydrolysis (4), conjugated isoflavones are more water soluble than their aglycone counterparts, and may demonstrate superior anti-methanogenic properties. The inventors demonstrated that in an inoculation medium an extract from Avisto red clover comprising 85% hexoside and malonyl hexoside conjugates and 15% aglycones reduced cumulative methane yield compared to that produced by a control containing no isoflavones. The reduction in cumulative methane yield was greater in an inoculation medium with an extract comprising the isoflavone conjugates compared to aglycone forms of the isoflavones alone. The isoflavone may comprise an aglycone and / or a conjugated form of the isoflavone. The isoflavone may comprise a hexoside conjugate and / or a malonyl-hexoside conjugate of the isoflavone. For instance the isoflavone may comprise an aglycone form of FMN, BCA and / or precursors of FMN and / or BCA. In other embodiments the isoflavone may comprise a conjugated form of FMN, BCA and / or precursors of FMN and / or BCA. The conjugated isoflavone may comprise a hexoside conjugate form of FMN, BCA and / or precursors of FMN and / or BCA. The conjugated isoflavone may comprise a malonyl-hexoside form of FMN, BCA and / or precursors of FMN and / or BCA. Banik et al, 2017 teaches that certain plants with isoflavone contents have been found to mitigate ruminal methane emissions (5). Liu et al., 2020 describes the effect of biochanin A (BCA) on gas production, urease activity, amino acid decomposition and rumen microbial populations (6). Harlowe et al., 2020 teaches the effect of transruminally-administered BCA on the rumen microbial community of Holstein steers in the treatment of subacute rumen acidosis (7). Moorby et al., 2004 discloses the effect of orally administered red clover formononetin on body weight and growth rates in lambs (8). However, none of these studies investigated the changes in methanogenesis nor the effect this might have upon animal performance, as the energy lost from production systems due to methanogenesis leads to a reduction in resource efficiency and productivity. Red clover has been shown historically to be non-toxic, and to have no negative implications upon either product quality or animal performance. The natural credentials of an extract from red clover, an existing forage crop which is commonly grown and fed to cattle, may be attractive to the consumer. In addition, isoflavones are easily harvested and extracted from red clover. Preferably, formononetin and / or the biochanin A, and in smaller amounts their precursors Daidzein and Genistein, are derived from Trifolium pratense (red clover). However, formononetin and biochanin A may be derived from other herbaceous, leguminous or plant- based sources. For example, biochanin A may be derived from soy, alfalfa sprouts or other legumes, such as peanuts and chickpeas. In alternative embodiments, formononetin and / or the biochanin A are synthetically produced. The isoflavones may be purified and added to the animal feed at levels which would exceed the trace levels found in any natural food sources. They are commonly found in red clover at rates of 1-2% of dry matter. The feed additive may be added to an animal feed and therefore should be formulated into a form that is suitable for oral consumption by the animal along with the animal feed. In some embodiments, the feed additive and / or animal feed may be in the form of an intraruminal bolus, a liquid, flakes, pellets or a powder. In some embodiments, the feed additive is homogenously mixed with the animal feed. In other embodiments, the feed additive is admixed to the animal feed prior to the feeding of the animal or as a supplemental feed to the general animal feed ration. The feed additive will preferably be added to the feed of a ruminant animal. Ruminant animals may comprise any of one cattle, goat, sheep, deer, bison, camel, alpaca and llama. The incorporation of the feed additive into a livestock ration enables regular feeding, to allow for consistency of effects upon ruminal microbes. This ration may include forage or concentrated feedstuffs, and the exact method of administration of the additive will depend upon the livestock species, age and its level of production, all of which dictate its feed regime. The thorough incorporation of additive into feed will allow intake concurrent with the other dietary components in the feed, and therefore encourage the desired effect within the ruminal environment. Incorporation of the additive into concentrated feed may be the most convenient way to achieve this. The rumen is the largest stomach compartment of a ruminant. The rumen is a site of microbial fermentation of ingested feed. The uppermost area of the rumen, the headspace, is filled with gases (such as methane, carbon dioxide, and, to a much lower degree, hydrogen) released from fermentation and anaerobic respiration of food. These gases are regularly expelled from the reticulorumen through the mouth, in a process called eructation. Archea present in the rumen produce methane through anaerobic respiration. Hydrogen produced by bacteria, protozoa and fungi is used by the archea to reduce carbon dioxide to methane. The dosage of the feed additive administered to the animal will vary depending upon many different factors including species, size, age and gender. The dosage of the feed additive administered to the animal preferably comprises about 0.025 to 1g isoflavone per kg bodyweight per day. For instance the dosage of the feed additive administered to the animal is 0.25g isoflavone per kg bodyweight per day. In embodiments the dosage of the feed additive administered to the animal comprises 0.025 to 0.1g isoflavone per kg bodyweight per day. The feed additive reduces methane production in comparison to a control. For instance methane production is reduced in the rumen of a ruminant animal compared to the methane production in the rumen of a ruminant animal which has not consumed the feed additive. In a second aspect of the present invention, there is provided a feed composition comprising the feed additive of the first aspect. In some embodiments, the feed additive comprises between about 0.1% and about 4% of the feed composition. In preferred embodiments, the isoflavone comprises between about 0.2% and about 1.5% of the feed composition. In alternative embodiments, the isoflavone comprises at least about 0.1%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, about 1.75%, about 1.8%, about 1.85%, about 1.9%, about 1.95% or about 2% of the feed volatile dry matter composition. The isoflavone may comprise about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9% or about 4% of the feed volatile dry matter composition. The concentration of isoflavone present in the food composition may be from 1g to 40g isoflavone per kg feed dry matter. The skilled person would understand that the concentration of isoflavones present in the food composition depends on the system in which the animal is being fed. The percentage of isoflavones in a concentrated feed will depend on the percentage of the concentrated feed within the food ration. The feed composition may include one or more further ingredients, which are commonly found in animal feed. Such ingredients will be well-known to those skilled in the art. Examples of these ingredients include ryegrass, hay, grass, grain, maize, wheat, starch, barley, oats, rapeseed meal, soya, citrus pulp, sunflower meal, groundnut meal, cottonseed meal, palm kernel, wheatfeed, oatfeed, distillers grains, maize gluten, alfalfa, sorghum, beet pulp, malt residual pellets, molasses, pearl millet and / or micronized peas. In another aspect of the present invention, there is provided the use of a feed additive or a feed composition of the first or second aspect for reducing methanogenesis in the rumen of a ruminant animal. The ruminant animal may comprise any one of cattle, goat, sheep, deer, bison, camel, alpaca and llama. In a further aspect of the invention, there is provided the use of an isoflavone in reducing methane production in the rumen of a ruminant animal. Advantageously the inventors have demonstrated a reduction in methane emissions of 17% when fed isoflavones. The isoflavones may be fed daily at a rate of 1% isoflavones. In this aspect, the isoflavone may be in accordance with an earlier aspect and / or formulated in accordance with an earlier aspect. In another aspect of the present invention, there is provided a method of reducing methane production in the rumen of a ruminant animal comprising the steps of combining an effective amount of isoflavone with one or more ingredients to form a feed and providing the feed to the ruminant animal. In some embodiments, the isoflavone comprises an O-methylated isoflavone. Preferably, the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA. The precursors may comprise daidzein and / or genistein. The ruminant animals may comprise any one of cattle, goat, sheep, deer, bison, camel, alpaca, and llama. The one or more ingredients to form a feed will be well-known to those skilled in the art. Examples of these ingredients include ryegrass, hay, grass, grain, maize, wheat, starch, barley, oats, rapeseed meal, soya, citrus pulp, sunflower meal, groundnut meal, cottonseed meal, palm kernel, wheatfeed, oatfeed, distillers grains, maize gluten, alfalfa, sorghum, beet pulp, malt residual pellets, molasses, pearl millet and / or micronized peas. In a further aspect of the present invention, there is provided a method of modulating microbiota through administering an isoflavone. Preferably, the isoflavone comprises one or more of biochanin A (BCA), formononetin (FMN) and / or one or more precursors of BCA and / or FMN. In some embodiments, modulating the microbiota comprises modulating the population of any of Firmicutes, Bacteroidetes and / or Spirochaetes. In specific embodiments, modulating the microbiota comprises reducing the population of Firmicutes and / or increasing the population of Bacteroidetes as a proportion of the total microbial population. Such modulations are dose-dependent. Microbial analysis using 16S rRNA revealed shifts in microbial communities with isoflavone inclusion rate. Advantageously, the inventors of the present invention found that the bacterial phylum Firmicutes decreased and the phylum Bacteroidetes increased following administration of each isoflavone. The effect of isoflavone supplementation upon methanogenic archaea appeared to be less pronounced than that upon the ruminal bacteria. Surprisingly, the inventors also found that increasing BCA concentration led to a fivefold increase in Spirochaetes. In yet a further aspect of the present invention, there is provided a method of modulating methane production in anaerobic digestion, comprising the step of adding an isoflavone to an anaerobic digestion system. Anaerobic digestion is the decomposition of waste material by microorganisms in the absence of oxygen resulting in the production of methane and carbon dioxide. Methane is a greenhouse gas and contributes to global warming and so its reduction from such anaerobic digestion systems may be desired. Additionally, methane production may result in a loss of resources from an industrial process, such as the industrial production of volatile fatty acids. In other industrial anaerobic digestion systems, such as biogas production for energy generation, an increase in methane production, methanogenesis, may be desired. The anaerobic digestion system includes the rumen of a ruminant animal. The anaerobic digestion system also includes industrial digestion systems which involve the decomposition of sewage or other waste material by anaerobic mircoorganisms. Anaerobic digestion systems can be used for industrial or domestic purposes to manage waste or produce fuels. The isoflavone may be added to any anaerobic digestion system in vitro, in vivo or ex vivo for which a reduction in methane production is desired. The anaerobic digestion system may include biotechnological applications where methane is not a desired end product of the fermentation process. A modulation in methane production, which may comprise a reduction or increase in methane production, is assessed in comparison to a control anaerobic digestion system to which no isoflavone has been added. The isoflavone may comprise an O-methylated isoflavone. Preferably, the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA. The precursors may comprise daidzein and / or genistein. In embodiments the method of modulating methane production in anaerobic digestion comprises reducing methane production, the method comprising a step of adding an isoflavone to an anaerobic digestion system wherein the isoflavone is present at a concentration of greater than 0.1%. The isoflavone may be present at a concentration of greater than 0.25%. The isoflavone may comprise an O-methylated isoflavone. Preferably, the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA. The precursors may comprise daidzein and / or genistein. Preferably the isoflavone may comprise one or more of FMN and / or BCA. The isoflavone may be added to an anaerobic digestion system at a concentration of 0.25% to 1.25% in a method of reducing methane production in anaerobic digestion. In embodiments the method of modulating methane production in anaerobic digestion comprises increasing methane production, the method comprising a step of adding an isoflavone to an anaerobic digestion system wherein the isoflavone is present at a concentration of 0.1% or less. The isoflavone may be added to an anaerobic digestion system wherein the isoflavone is present at a concentration of 0.25% or less. The isoflavone may comprise an O-methylated isoflavone. Preferably, the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA. The precursors may comprise daidzein and / or genistein. Preferably the isoflavone may comprise one or more of FMN and / or BCA. The isoflavone may be added to an anaerobic digestion system at a concentration of 0.01% to 1% in a method of increasing methane production in anaerobic digestion. The isoflavone may be added to an anaerobic digestion system at a concentration of 0.01% to 0.25% in a method of increasing methane production in anaerobic digestion. The isoflavone may be added to an anaerobic digestion system at a concentration of 0.01% to 0.1% in a method of increasing methane production in anaerobic digestion. It will be appreciated that numerous modifications to the above-described embodiments may be made without departing from the scope of the invention as defined in the appended claims. Detailed Description of the Invention Embodiments of the invention are described below, by way of example only, with reference to the accompanying figures in which: Figure 1 is a graph showing the net daily methane yield for 5 levels of biochanin A treatment and a control over 17 days following inoculation, measured by automatic methane potential system (AMPTS). The digestion medium comprises 390mL digestate and 10g dry matter (DM) ryegrass. The concentrations of biochanin A are 0.25%, 0.5%, 0.75%, 1% and 1.25%. Figure 2 is a graph showing the cumulative net methane yield for 5 levels of biochanin A treatment and a control over 17 days following inoculation, measuring by AMPTS. The digestion medium comprises 390mL digestate and 10g (DM) ryegrass. The concentrations of biochanin A are 0.25%, 0.5%, 0.75%, 1% and 1.25%. Figure 3 is a graph showing the net daily methane yield for 5 levels of biochanin A treatment and a control over 8 days following inoculation, measuring by AMPTS. The digestion medium comprises 80mL digestate and 2g (DM) ryegrass. The concentrations of biochanin A are 0.01%, 0.02%, 0.05%, 0.1% and 1%. Figure 4 is a graph showing the net daily methane yield for 5 levels of formononetin treatment and a control over 17 days following inoculation, measuring by AMPTS. The digestion medium comprised 390mL digestate and 10g (DM) ryegrass. The concentrations of formononetin are 0.25%, 0.5%, 0.75%, 1% and 1.25%. Figure 5 is a graph showing the cumulative net methane yield for 5 levels of formononetin treatment and a control over 17 days following inoculation, measuring by AMPTS. The digestion medium comprises 390mL digestate and 10g (DM) ryegrass. The concentrations of formononetin are 0.25%, 0.5%, 0.75%, 1% and 1.25%. Figure 6 are LB agar plates following 24h incubation at 39oC of 1% BCA (A), no treatment (B) and 1% FMN (C). The cultures were not speciated. Figure 7 are graphs showing the genomic assemblage by phylum after 8 days of incubation of a) 5 levels of BCA and control and b) 5 levels of FMN and control. Figure 8 is a graph showing the cumulative net methane yield for 5 levels of formononetin treatment and a control over 8 days following inoculation, measuring by AMPTS. The concentrations of formononetin are 0.01%, 0.02%, 0.05%, 0.1% and 1%. Figure 9 is a graph showing the mean methane yield for 4 levels of BCA treatment and a control over 15 days following inoculation. The concentrations of BCA are 25%, 0.5%, 0.75% and 1%. Figure 10 is a graph showing the cumulative methane yield for 2 levels of BCA and 2 levels of FMN and a control over 9 days following inoculation. The BCA and FMN are in an aglycone form. The concentrations of BCA and FMN are 0.5% and 1%. Figure 11 is a graph showing the cumulative methane yield for 4 levels (0.25%, 0.5%, 0.75% and 1%) of 70% methanol extract from Avisto red clover hay, and a control over 9 days following inoculation. Figure 12 is a bar chart showing the effect of BCA at 5 levels (0.25%, 0.5%, 0.75%, 1%, 1.25%) upon microbial diversity in anaerobic culture at 38°C over 17 days. Figure 13 is a bar chart showing the effect of BCA and FMN upon residual total volatile solid content in a digestion medium at 38°C over 9 days. Figure 14 is a bar chart showing the effect of BCA and FMN upon the volatile fatty acid composition of digesta at 38°C over 9 days. Figure 15 is a graph showing the metabolic products arising from 0.75% BCA addition to anaerobic culture over 7 days. Examples An experiment to quantify the anti-methanogenic potential of a single dose of the aglycones biochanin A and formononetin was conducted as detailed below. Ryegrass was used as a substrate for digestion due to the absence of isoflavones. This was harvested at the Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, and oven-dried before chopping to approximately 25mm length for use. Formononetin (98% purity) and Biochanin A (98% purity) were purchased from Sigma- Aldrich (UK) Ltd. Liquid anaerobic digestate was used as a microbial inoculum and was obtained from Leen Farm, Pembridge, Herefordshire. Feedstocks for the digestate were 50% cow slurry, 30% maize silage, 20% poultry litter, and digestion conditions were 38oC for an average duration of 30 days. Solids were removed through a cheesecloth and the liquid retained as an inoculant. The automatic methane potential test system (AMPTS) was used as the platform for this experiment. Liquid digestate from a biogas plant was used as a proxy for rumen fluid, to allow for the quantities of a consistent inoculum required in a short timeframe. Volatile solids were set at a ratio of 3:2 from digestate and ryegrass respectively. This corresponded to bottle loading of 390mL of digestate, and 10g dried ryegrass for each treatment.15 x 1 litre bottles were used for the experiment. Individual isoflavones were dissolved in dimethyl sulfoxide (DMSO), and included at rates of 0, 0.25%. 0.5%, 0.75%, 1% and 1.25% of ryegrass dry matter (see Table 1 below). DMSO inclusion was set at 0.5% to avoid potential toxic effects upon microflora (Galvao et al., 2014). Before commencement of measurement, 15 x 1 litre bottles were flushed with nitrogen gas. One replicate of each treatment was performed. Table 1: Bottle contents for AMPTS. Details describe biochanin A (BCA), formononetin (FMN) and dimethyl sulfoxide (DMSO) inclusion rates. The bottles were stirred continually, and digestion temperature was set at 37°C. A CO2fixer comprising NaOH with phenolphthalein was incorporated in the gas flow line, and this was checked every day and the solution replaced when the indicator showed acidic conditions. Methane yield was measured for 17 days, at which point peak yield had been achieved for all treatments. In order to maintain integrity of the anaerobic digestion, a parallel trial was conducted to provide for post-hoc analyses, which was sampled after 8 days of digestion to allow for analysis of the active digesta. A range of concentrations of both isoflavones was used, and methane production was measured at these range of lower concentrations over the 8 day trial. Samples from this trial were plated on LB agar plates and were also taken pre and post digestion for 16s RNA sequencing. Illumina bridge amplification technology was used to identify and quantify the metagenomic assemblage present. Example 1 – Inhibitory effects of Biochanin A on methane production in anaerobic fermentative systems Five inclusion levels of the aglycone forms of biochanin A (BCA), and five of formononetin (FMN) and controls (DMSO) were added to bottles containing standardised quantities of liquid digestate and ryegrass. Methane production was then measured using AMPTS following inoculation of ryegrass with each isoflavone. Daily methane production over a period of 17 days and cumulative yield were recorded and samples were taken for post-hoc analysis of metagenomic assemblage. DMSO was found not to inhibit methane production at the inclusion rate of 0.5%. A peak in methane yield in the controls was observed on day 1 of around 10ml g-1total volatile solids (TVS) as shown in Figure 1. A subsequent peak in methane production was observed on day 5 and 6 in the control of 13.45 ml g-1TVS. Following BCA treatment, a peak in methane yield was observed on day 1. As illustrated in Figure 1, daily methane production was inhibited by BCA inclusion in a dose-dependent manner. The inclusion rates of BCA also displayed a dose-dependent delay in peak methane yield, and significant reductions in daily methane production. The 0.25% BCA treatment displayed a delay in the peak methane yield by one day, with only a slight inhibition of total methane production. 0.5% and 0.75% BCA inclusions displayed a progressive delay in peak methane yield, although the daily quantity produced at peak was similar (9.6 ml g-1TVS).1% and 1.25% BCA inclusion peaked at day 12, and it appeared that an upper threshold of BCA inclusion upon methane yield was being approached at this level. By day 17, methane production from control and 0.25% BCA had virtually ceased. However, the higher concentration treatments were still producing methane. The results between BCA treatments were significant (ANOVA; p=0.006). The effects of reducing methane emission and delaying peak methane emission upon total methane production are clearly displayed in the cumulative yield as shown in Figure 2. The effect of BCA addition upon methane production from a digestion medium containing 3 parts TVS from anaerobic digestate:2 parts TVS from dried ryegrass are shown. Five rates of BCA were added, and digestion occurred at 38°c over 17 days, until methane emission had ceased. The automated methane potential test system (AMPTS) was used to measure methane production. Mean values from triplicated samples. Concentrations quoted are as % of ryegrass DM. 0.25% BCA yielded marginally less cumulative CH4than the control throughout the trial. 0.5%, 0.75% and 1% BCA treatments all yielded significantly lower cumulative CH4until day 16 in a dose-dependent manner.1.25% BCA yielded marginally less CH4than 1% BCA, and it appeared that a threshold was being approached at this level. After 7 days of digestion, the 1.25% BCA treatment had yielded 25% of the total CH4compared to the control, and after 17 days, it had yielded 75% of the total CH4 compared to the control. This highlights the delay in reaching peak methane yield with increasing BCA inclusion. Example 2 – Enhancement of methanogenesis at low concentrations of BCA Although doses of BCA at 0.25% and above were shown to inhibit methanogenesis, lower inclusions of BCA were demonstrated to enhance methanogenesis. As shown in Figure 3, inclusions at or below 0.1% of the digestion substrate were shown to enhance methanogenesis (p=.013). Example 3 - Inhibitory effects of Formononetin on methane production in anaerobic fermentative systems Similarly, to BCA, FMN treatments showed a peak in methane yield on day 1. All formononetin treatments inhibited methane production compared to the control for the first 7 days as shown in Figure 4. However, the reduction was not as great as that observed in the BCA treatments. The treatment did not delay peak methane yield, as it did with BCA, with all treatments peaking at day 6. The effect of FMN upon methane yield was found not to be dose dependent and there was no clear ranking of methane yield according to FMN inclusion rate. There was no statistical significance attributed to this effect (p=0.19). When compared to BCA treatments, the cumulative total CH4yield displayed smaller treatment effect between control and FMN treatments. The differences between the treatments were not ranked by FMN concentration, and the anti-methanogenic effect of FMN was lower than that for BCA as shown in Figure 5. Example 4 – The effect of BCA and FMN treatment on microfloral populations The digestion media was incubated with either BCA, FMN or a blank treatment, with no added isoflavones, and then cultured on LB agar plates. There were clear differences observed in constituent microflora populations between the treatments. The blank treatment displayed significant population growth of species which presented as opaque circular cultures, with a plate coverage of approximately 30% as shown in Figure 6B. The 1% FMN plate (figure 6C) displayed approximately 1% coverage of species that presented much smaller cultures than those on the blank plate whereas the 1% BCA plate (figure 6A) displayed virtually no cultures under these conditions. 16S rRNA genomic data did not show any reduction in methanogenic archaea over the course of the experiment at any inclusion rate of BCA. Following 8 days of incubation, digesta including 1% BCA displayed a 345% increase in archaea as a proportion of total reads, compared to an isoflavone free control. Methane production was however greatly reduced by the BCA treatment over this period. Digesta including 1% FMN displayed a 66% decrease in archaea compared to control. The main effect of increasing isoflavone inclusion after 8 days digestion was observed at the phylum level. As BCA inclusion increased from 0% to 1%, bacteria of the phyla Firmicutes and Bacteroidetes displayed an inverse relationship with each other. Firmicutes varied between 55% of total reads at 0% BCA, to 70% at 0.05% BCA before falling to 34% at 1% BCA. Bacteroidetes varied from 26% to 13% before rising to 41% at the same concentrations as shown in Figure 7a. A diminished effect was also observed in FMN treatments with increasing concentrations. Firmicutes fell from 74% to 51% over the range of concentrations, whilst Bacteroidetes rose from 13% to 38% as shown in Figure 7b. The majority of the fall in the Firmicutes with isoflavone inclusion was due to bacteria of the order Clostridiales, whilst much of the rise in the Bacteroidetes was due to bacteria of the order Bacteroidales. Example 5- Enhancement of methanogenesis at low concentrations of FMN As shown in figure 8 a similar effect to that observed for low concentrations of BCA was observed for FMN with concentrations in the range of 0.01%-0.1% FMN displaying enhanced methanogenesis whilst methanogenesis was inhibited at the 1% FMN level (p=.008). Example 6- Reduction in mean methane yield following daily feeding of BCA A digestion medium comprising 40ml digestate, 8.6ml macronutrient solution, 8.6ml buffer solution, 0.04ml micronutrient solution, 23 ml water, 1g ryegrass (DM), plus 0.2ml DMSO. A control containing no BCA was prepared and BCA was added to the other samples at a concentration of 0.25%, 0.5%, 0.75% and 1%. Daily gas readings were taken. 20ml of digestion medium was removed under anaerobic conditions. This was replaced with 4.3ml macronutrient solution, 4.3ml buffer solution, 0.02ml micronutrient solution, 11.5 ml water, 0.25g ryegrass (DM), 0.05ml DMSO plus a relevant amount of BCA. Mean methane yields were measured as a % of the control. The mean methane yield results are shown in figure 9. As shown in figure 9 a daily feeding of BCA at 1% BCA resulted in a 17% reduction in methane emissions. Example 7- Methane yield from aglycone forms of isoflavones and conjugated isoflavones The cumulative methane yield from a control containing no isoflavones, 0.5% BCA (aglycone), 1% BCA (aglycone, 0.5% FMN (aglycone) and 1% FMN (aglycone) over 9 days following inoculation was measured. The results are shown in figure 10. The digestion medium comprised 2g ryegrass (DM), 80ml digestate, 0.4ml DMSO and 12.8ml total water. The concentrations are expressed as the total isoflavones as a percentage of ryegrass volatile solids (RGVS). As shown in figure 10 as concentrations of the aglycone forms of BCA and FMN reduced methane yield compared to a control. The cumulative methane yield from a control containing no isoflavones and four concentrations (0.25%, 0.5%, 0.75% and 1%) of 70% methanol extract from Avisto red clover hay over 9 days following inoculation. The results are shown in figure 11. The digestion medium comprised 2g ryegrass (DM), 80ml digestate, 0.4ml DMSO and 12.8ml total water. The concentrations are expressed as the total isoflavones as a percentage of ryegrass volatile solids (RGVS). Isoflavones were present as 85% hexoside and malonyl hexoside conjugates and 15% aglycones. Isoflavones were present as 60% BCA and 40% FMN. Example 8- Microbial diversity The effect of BCA addition upon microbial diversity in a digestion medium containing 3 parts total volatile solids (TVS) from anaerobic digestate to 2 parts TVS from dried ryegrass. Five rates of BCA were added (0.25%-1.25%) and digestion occurred at 38°C over 17 days, until methane emission had ceased. Illumina NGS technology was used to evaluate the relative abundance of microbiota at all levels of taxonomy, with phylum level data presented in figure 12. Of the variation in Firmicutes, the principal effect was upon Clostridia III and Clostridia XIV. Concentrations quoted are as % of ryegrass DM. Example 9- Residual TVS following digestion The effect of BCA and formononetin (FMN) upon the residual TVS following digestion is shown in figure 13. The digestion medium comprised 3 parts TVS from anaerobic digestate to 2 parts TVS from dried ryegrass. Two different rates of BCA and FMN were added and sampled after 9 days digestion at 38°C. The figures do not reflect digestibility per se as residual TVS includes the VFA content present following digestion in a sealed system. In essence, these figures reflect the %TVS not converted to gaseous products. Mean values are from triplicated samples and concentrations are as % of ryegrass DM. Example 10- Volatile fatty acids (VFA) The effect of BCA and FMN treatment upon the volatile fatty acid content following digestion, using a medium comprising 3 parts TVS from anaerobic digestate to 2 parts TVS from dried ryegrass is shown in figure 14. Two different rate of BCA and FMN were added and sampled after 9 days digestion at 38°C. VFA analysis was conducted using JASCO HPLC. Mean values are from triplicated samples and concentrations are as % of ryegrass DM. Example 11- Metabolic fate of BCA during digestion The metabolic fate of BCA during a multi-day feeding trial of a digestion medium comprising 3 parts TVS from anaerobic digestate to 2 parts TVS from dried ryegrass and 0.75% BCA at 38°C is shown in figure 15. Each day 25% of digesta was removed and replaced pro rata with buffer solution, nutrient solutions, ryegrass and BCA, before bottles were flushed with CO2and resealed. Analysis was carried out on a Waters C18 HPLC column. The biota rapidly adapted to metabolise BCA to genistein, then to methoxy-phenylacetic acid. However, mitigation of methane production persisted during this period. Mean values are from triplicated samples. Conclusions The peak in methane yield on day 1 was observed in both BCA and FMN samples and at all rates of inclusion. This reflects the rapid digestion of simple compounds such as sugars and peptides which do not require hydrolytic cleavage (9). The early peak in methane yield observed at day 1 was ranked by the inclusion rate in the BCA trial. There was no evidence of a similar ranking effect on day 1 in the FMN trial, however, the control was the highest yielding treatment. Divergence occurred between treatment rates on day 2 for both isoflavones. During the experiments, FMN was visibly less soluble than BCA in both digestate and DMSO. FMN did appear to have a small anti-methanogenic effect, and the differences in methane abatement between the two isoflavones may, in part, be due to the differences in their solubility, an effect previously proposed by Braden et al., (10). The reduction in Firmicutes and increase of Bacteroidetes as a percentage of total genomic reads is a potentially significant finding and may aid in the explanation of the anti- methanogenic effect of both BCA and FMN. It is noteworthy that the relative abundance of Firmicutes and Bacteroidetes mirrors each other with isoflavone inclusion rate for both BCA and FMN. Increasing Firmicutes lead to reduced Bacteroidetes and vice-versa. Inverse correlations between Firmicutes and Bacteroidetes populations have often been described in vitro, with Bacteroidetes being the superior phylum in respect of volatile fatty acid (VFA) production, whilst high Firmicutes populations produce more methane (11). In biogas plants, increases in relative abundance of both Firmicutes and Bacteroidetes by 1% each have been associated with enhanced methane production of 4.5 mL / g [VS] and 7.0 mL / g [VS] respectively. However, due to the interdependent niche roles played by individual species within complex ecosystems, a balance of species may be required to optimise methanogenesis (12). Both Firmicutes and Bacteroidetes are classified as generalist microbes and are well adapted to the degradation of a wide range of polysaccharides. However, Firmicutes are primarily associated with the degradation of lignocellulosic carbohydrates to simpler sugars and volatile fatty acids. This leads to an increased efficiency in overall digestion, which is often associated with higher levels of Firmicutes. In order to achieve this, Firmicutes employ enzyme complexes known as the cellulosome, which are associated with the cell wall as well as free enzymes (13). Bacilli are a member of the Firmicutes, and are responsible for the production of fermentative hydrogen, an important substrate for methanogenesis by archaea (14). Bacteroidetes, on the other hand, are associated with the breakdown of proteins and peptides, as well as long chain carbohydrates, thereby producing ammonia and branched chain fatty acids. Their cellulolytic enzymes are clustered together into polysaccharide utilisation loci (13). Bacteroidetes are not thought to be significant hydrogen producers and members of the phylum, such as Bacteroides ruminicola and Selenomonas ruminantium , have been shown to use ruminal hydrogen as a reducing agent in the conversion of fumarate to succinate (15). This clearing of free hydrogen from the rumen may be essential in the prevention of hydrogen toxicity by providing an alternative hydrogen sink to methane production (16). The experiments conducted herein illustrate that isoflavone addition to the digestion media clearly influences the Firmicute:Bacterioidete ratio, which may mediate hydrogen emission, and subsequent methane emission by the archaea. The mechanisms underlying the microbial effects of these isoflavones are unclear. It may be mitigated through the phytoestrogenic properties of the isoflavones. The estrobolome has been described as the aggregate of microbial genes involved in mammalian estrogen metabolism in human gut microflora (17) however there is no mention of the potential effects of phytoestrogens such as isoflavones on the estrobolome in a ruminant animal. Estrogen and progesterone can both enhance bacterial growth (18), and host derived estrogen enhances faecal microbiome diversity in humans (19). On the other hand, BCA and FMN may act through antimicrobial properties. Bacteroidetes are gram-negative bacteria, possessing a complex cell wall with a peptidoglycan layer surrounded by an outer membrane consisting of lipopolysaccharides. BCA has been found to be capable of penetrating and disrupting of hydrophobic components of the cell membranes in the gram-negative bacteria E. coli, thereby leading to potential outflow of cell contents and cell death (20). However, Firmicutes have a gram-positive cell wall including a glycolipid layer, which is vulnerable to attack, notably by ionophore antibiotics. BCA may work synergistically with other undetermined compounds to achieve antibiotic effects. Both FMN and BCA are reported to possess antimicrobial properties, possibly due to their inhibition of efflux pump mechanisms, thereby enhancing the activity of other antibiotic compounds (21-23). Ionophore antibiotics are themselves microbial in origin, and synergies with such compounds may be a potential mode of action of BCA and FMN upon Firmicutes (24). Surprisingly, a fivefold increase in the population Spirochaetes was observed between 0.1 and 1% BCA inclusion. Spirochaetes are a more specialist phylum of bacteria than Bacteroidetes and Firmicutes . They possess enzyme complexes facilitating the breakdown of the cell wall components xyloglucans, peptidoglycans and pectins (13), producing hydrogen in the process. However, they are classified as neither gram positive nor negative as they possess a unique cell wall. The antibiotic mechanisms of isoflavones upon Firmicutes may therefore not be applicable to Spirochaetes, allowing the phylum to move into the niche vacated by a declining Firmicutes population Alternatively, the delicate balance between communities of Firmicutes and Bacteroidetes may be due to competition for resources such as the complex carbohydrates upon which they feed. The formation of microbial communities called biofilms can lead to the reduction of availability of such substrates to competing organisms, and lead to shifts in populations. From the results presented in these experiments, it appears that treatment differences between 1% and 1.25% BCA are less than those between lower inclusions, and that possibly some upper threshold of BCA inclusion upon methanogenesis is being approached. However, when considering the economics of the inclusion of anti- methanogenic additives in livestock rations, an inclusion of 0.5% BCA elicits a reduction in methanogenesis of 72% that of a 1.25% inclusion on day 2. The experiments conducted herein provide evidence that formononetin (FMN) and biochanin A (BCA), two isoflavones found to high levels in red clover, display anti- methanogenic activity in vitro when included in anaerobic microbial fermentative systems. Methanogenesis, the production of methane by archaea in microbial ecosystems during digestion processes, has significant implications for livestock production, both in terms of commercial metrics, and its contribution to global anthropogenic climate change. The results showed that the inclusion of BCA and FMN to the digestion medium led to a reduction in daily methane production, with BCA demonstrating a more pronounced effect compared to FMN. Increasing the concentration of BCA led to a dose-dependent delay in peak methane yield, leading to significant reductions in total cumulative methane production. FMN treatments however inhibited methane production during the early days of digestion but did not delay peak methane yield. BCA demonstrated significant inhibitory effects upon methane production (p=0.006) FMN had an anti-methanogenic effect however the results were not significant (p=0.19). The experiments also revealed shifts in microbial populations due to the inclusion of BCA and FMN. The relative abundance of Firmicutes and Bacteroidetes, two major phyla of ruminal bacteria, showed an inverse relationship with each other based on the inclusion rate of BCA and FMN. BCA led to a proportional reduction in Firmicutes and an increase in Bacteroidetes, while FMN treatments showed a similar trend, although the effect was less pronounced. Significantly, there was no evidence here that populations of ruminal archaea were inhibited by inclusion of isoflavones. It appears that the mitigation of methanogenesis is a consequence of the isoflavones mediation of hydrogen-producing bacterial populations. The reduction in Firmicutes likely leads to reduced hydrogen available for archaeal synthesis into methane. The potential of BCA and FMN as an anti-methanogenic agents holds promise for mitigating methane emissions as a feed additive in ruminant production systems. The findings of the experiments show that methanogenesis in anaerobic microbial fermentative systems, such as the rumen, can be mitigated. Although a few preferred embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications might be made without departing from the scope of the invention, as defined in the appended claims. Throughout this specification, the term “comprising” or “comprises” means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of” or “consists essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. Typically, when referring to compositions, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight of non-specified components. The term “consisting of” or “consists of” means including the components specified but excluding addition of other components. Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to encompass or include the meaning “consists essentially of” or “consisting essentially of”, and may also be taken to include the meaning “consists of” or “consisting of”. The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments. Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. All of the features disclosed in this specification (including any accompanying claims, and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0002] References 1. McAllister, T. A., & Newbold, C. J. (2008). Redirecting rumen fermentation to reduce methanogenesis. Australian Journal of Experimental Agriculture, 48(2), 7-13. 2. Department for Energy Security and Net Zero (2023) (online). Accessed via: Powering Up Britain: Net Zero Growth Plan - GOV.UK (www.gov.uk). Accessed on 26thMay 2023. 3. Krenn, L., Unterrieder, I., & Ruprechter, R. (2002). Quantification of isoflavones in red clover by high-performance liquid chromatography. Journal of Chromatography B, 777(1-2), 123-128 4. Křížová, L., Dadáková, K., Kašparovská, J., & Kašparovský, T. (2019). Isoflavones. Molecules, 24(6), 1076. 5. Banik, B. K. (2017). Understanding the in vitro bioactivity of the forage legume Biserrula pelecinus L.-variability and mechanisms (Doctoral dissertation, The University of Western Australia). 6. Liu, S., Zhang, Z., Hailemariam, S., Zheng, N., Wang, M., Zhao, S., & Wang, J. (2020). Biochanin A inhibits ruminal nitrogen-metabolizing bacteria and alleviates the decomposition of amino acids and urea in vitro. Animals, 10(3), 368. 7. Harlow, B. E., Flythe, M. D., Klotz, J. L., Harmon, D. L., & Aiken, G. E. (2021). Effect of biochanin A on the rumen microbial community of Holstein steers consuming a high fiber diet and subjected to a subacute acidosis challenge. Plos one, 16(7), e0253754. 8. Moorby, J. M., Fraser, M. D., Theobald, V. J., Wood, J. D., & Haresign, W. (2004). The effect of red clover formononetin content on live-weight gain, carcass characteristics and muscle equol content of finishing lambs. Animal Science, 79(2), 303-313. 9. Kryvoruchko, V., Machmüller, A., Bodiroza, V., Amon, B., & Amon, T. (2009). Anaerobic digestion of by-products of sugar beet and starch potato processing. Biomass and bioenergy, 33(4), 620-627. 10. Braden, A. W. H., Hart, N. K., & Lamberton, J. A. (1967). Oestrogenic activity and metabolism of certain isoflavones in sheep. Australian Journal of Agricultural Research, 18(2), 355-348. 11. Chen, S., Cheng, H., Wyckoff, K. N., & He, Q. (2016). Linkages of Firmicutes and Bacteroidetes populations to methanogenic process performance. Journal of Industrial Microbiology and Biotechnology, 43(6), 771-781. 12. Ma, G., Chen, Y., & Ndegwa, P. (2021). Association between methane yield and microbiota abundance in the anaerobic digestion process: A meta-regression. Renewable and Sustainable Energy Reviews, 135, 110212. 13. Gharechahi, J., Vahidi, M. F., Sharifi, G., Ariaeenejad, S., Ding, X. Z., Han, J. L., & Salekdeh, G. H. (2023). Lignocellulose degradation by rumen bacterial communities: new insights from metagenome analyses. Environmental Research, 115925. 14. Zhang, L., Tian, H., Shi, H., Pan, S., Chang, J., Dangal, S. R., ... & Jackson, R. B. (2022). A 130-year global inventory of methane emissions from livestock: Trends, patterns, and drivers. Global Change Biology, 28(17), 5142-5158. 15. Henderson, C. (1980). The influence of extracellular hydrogen on the metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium. Microbiology, 119(2), 485-491. 16. McAllister, T. A., & Newbold, C. J. (2008). Redirecting rumen fermentation to reduce methanogenesis. Australian Journal of Experimental Agriculture, 48(2), 7-13. 17. Kwa, M., Plottel, C. S., Blaser, M. J., & Adams, S. (2016). The intestinal microbiome and estrogen receptor–positive female breast cancer. Journal of the National Cancer Institute, 108(8), djw029. 18. Kornman, K. S., & Loesche, W. J. (1982). Effects of estradiol and progesterone on Bacteroides melaninogenicus and Bacteroides gingivalis. Infection and immunity, 35(1), 256- 263. 19. Adlercreutz, H., Pulkkinen, M. O., Hämäläinen, E. K., & Korpela, J. T. (1984). Studies on the role of intestinal bacteria in metabolism of synthetic and natural steroid hormones. Journal of steroid biochemistry, 20(1), 217-229. 20. He, M., Wu, T., Pan, S., & Xu, X. (2014). Antimicrobial mechanism of flavonoids against Escherichia coli ATCC 25922 by model membrane study. Applied Surface Science, 305, 515- 521. 21. Flythe, M. D., Davis, B. E., & Kagan, I. A. (2023). Reduction in Rumen Tetracycline- Insensitive Bacteria during a Grain Challenge Using the Isoflavone Biochanin A. Veterinary Sciences, 10(4), 273. 22. Hanski, L., Genina, N., Uvell, H., Malinovskaja, K., Gylfe, Å., Laaksonen, T., ... & Vuorela, P. M. (2014). Inhibitory activity of the isoflavone biochanin A on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation. PLoS One, 9(12), e115115. 23. Lo, Y. L., & Wang, W. (2013). Formononetin potentiates epirubicin-induced apoptosis via ROS production in HeLa cells in vitro. Chemico-biological interactions, 205(3), 188-197. 24. Flythe, M., & Kagan, I. (2010). Antimicrobial effect of red clover (Trifolium pratense) phenolic extract on the ruminal hyper ammonia-producing bacterium, Clostridium sticklandii. Current Microbiology, 61, 125-131.

Claims

Claims 1. A feed additive for reducing methane production in the rumen of a ruminant animal comprising an isoflavone.

2. The feed additive according to claim 1, wherein the isoflavone comprises an O- methylated isoflavone.

3. The feed additive according to claim 2, wherein the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA.

4. The feed additive according to any previous claim, wherein the isoflavone comprises an aglycone and / or a conjugated form of the isoflavone.

5. The feed additive according to claim 4 wherein the isoflavone comprises a hexoside or a malonyl-hexoside conjugate of the isoflavone.

6. The feed additive according to any preceding claim, wherein the feed additive is in the form of an intraruminal bolus, a liquid, flakes, pellets, or a powder.

7. The feed additive according of any preceding claim, wherein the ruminant animal comprises an animal selected from cattle, goat, sheep, deer, bison, camel, alpaca and llama.

8. The feed additive according to claim 7, wherein the feed additive is administered at a dosage in the range of about 0.025g to about 1g isoflavone per kg bodyweight per day.

9. A feed composition comprising the feed additive of claims 1 to 6.

10. The feed composition according to claims 7 to 9, wherein the feed additive comprises between about 0.1% to about 4% of the feed composition.

11. The feed composition according to claims 9 or 10, wherein the feed additive comprises between about 0.2% to about 1.5% of the feed composition.

12. The feed composition according to claims 9 to 11, wherein the feed further comprises one or more of ryegrass, hay, grass, grain, maize, wheat, starch, barley, oats, rapeseed meal, soya, citrus pulp, sunflower meal, groundnut meal, cottonseed meal, palm kernel, wheatfeed, oatfeed, distillers grains, maize gluten, alfalfa, sorghum, beet pulp, malt residual pellets, molasses, pearl millet and / or micronized peas.

13. The feed composition according to any of claims 9 to 12 wherein the concentration of isoflavone present in the food composition is from 1g to 40g isoflavone per kg feed dry matter.

14. Use of the feed additive of claims 1 to 6 or the feed composition of claims 9 to 13 in reducing methane production in the rumen of a ruminant animal.

15. The use according to claim 14, wherein the ruminant animal comprises an animal selected from cattle, goats, sheep, deer, bison, camels, alpacas and llamas.

16. Use of an isoflavone in reducing methane production in the rumen of a ruminant animal.

17. Use of the isoflavone of claim 16, wherein the isoflavone comprises an O-methylated isoflavone.

18. Use of the isoflavone of claim 17, wherein the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors thereof.

19. Use of the isoflavone according to claims 16 to 18, wherein the isoflavone is added to a ruminant animal feed.

20. Use of the isoflavone according to claim 19, wherein the ruminant animal feed comprises an animal feed for cattle, goat, sheep, deer, bison, camel, alpaca and llama.

21. A method of reducing methane production in the rumen of a ruminant mammal, the method comprising the steps of combining an effective amount of isoflavone with one or more ingredients to form a feed and providing the feed to the ruminant animal.

22. The method according to claim 21, wherein the isoflavone comprises one or more of formononetin (FMN), biochanin A (BCA) and / or precursors of FMN and / or BCA.

23. The method according to claims 21 or 22, wherein the ruminant animal comprises an animal selected from cattle, goat, sheep, deer, bison, camel, alpaca and llama.

24. The method according to claims 21 to 23, wherein the one or more ingredients comprise ryegrass, hay, grass, grain, maize, wheat, starch, barley, oats, rapeseed meal, soya, citrus pulp, sunflower meal, groundnut meal, cottonseed meal, palm kernel, wheatfeed, oatfeed, distillers grains, maize gluten, alfalfa, sorghum, beet pulp, malt residual pellets, molasses, pearl millet and / or micronized peas.

25. A method of modulating methane production in anaerobic digestion, the method comprising a step of adding an isoflavone to an anaerobic digestion system.

26. The method according to claim 25 wherein the method comprises reducing methane production.

27. The method according to claim 26 wherein the isoflavone is present at a concentration of greater than 0.1%.

28. The method according to claim 25 wherein the method comprises increasing methane production.

29. The method according to claim 28 wherein the isoflavone is present at a concentration of 0.1% or less.

30. The method according to any one of claims 25 to 29 wherein the anaerobic digestion system comprises an industrial anaerobic digestion system.

Images