NZ562783A - Method of reducing greenhouse gas production - Google Patents

Abstract

Disclosed is a method of suppressing methanogen archaea activity in a non-human animal by the step of introducing an extract derived from the plant species Impatiens balsamina into an environment in which methanogen archaea are present in the non-human animal.

Description

562783 *10056641880* PATENTS FORM NO. 5 Fee No. 4. $250.00 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional No: 562783 Dated: 24 October 2007 METHOD OF REDUCING GREENHOUSE GAS PRODUCTION We, Methane Reduction Technologies Limited, 27b Lodge Place, Christchurch, New Zealand, a New Zealand Company, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: 2* OU 2008 562783 METHOD OF REDUCING GREENHOUSE GAS PRODUCTION TECHNICAL FIELD The invention relates to a method of reducing greenhouse gas production. More 5 specifically, the invention relates to the use of a plant extract administered to an animal which reduces the amount of greenhouse gases including methane and/or carbon dioxide produced by the animal. The method also reduces or prevents bloating in animals.

BACKGROUND ART Greenhouse gases and ways to reduce such emissions are a topical issue at present especially in view of increasing environmental awareness. Methane gas is used below to illustrate the issue although it should be appreciated that other gases such as carbon dioxide also contribute to greenhouse gas emissions.

Methane is a chemical compound with the molecular formula CH4. It is the simplest alkane, and the principal component of natural gas. Methane is a relatively potent greenhouse gas with a high global warming potential (i.e., warming effect compared to carbon dioxide). When averaged over 100 years, each kg of CH4 warms the Earth 25 times as much as the same mass of C02. Put 20 another way, methane in the Earth's atmosphere is an important greenhouse gas with a global warming potential to the point where 1 tonne of methane emission will have 25 times the impact on temperature of a 1 tonne carbon dioxide emission during the following 100 years. The methane concentration has increased by about 150% since 1750 and it accounts for 20% of the total radiative forcing from 25 all of the long-lived and globally mixed greenhouse gases.

Large amounts of methane are produced by methanogenesis as part of metabolism in animals and particularly for ruminant animals. Methanogensis is the formation of methane by microbes known as methanogens. The production of methane is a widespread form of microbial metabolism and occurs as part of 30 decomposition of biomass. Methanogens perform anaerobic respiration and their carbon sources (food) include C02, methanol and various simple carbon polymers. 562783 The biochemistry of methanogenesis is relatively complex, involving a number of coenzymes and cofactors including coenzyme B, coenzyme M, methanofuran and methanopterin.

Methanogenesis occurs in the gut of humans and other animals especially ruminants. In the rumen, anaerobic organisms digest cellulose into forms usable by the animal and by methanogens. The methane produced is released from the animal mainly by belching (eructation). Another potential release method is via the lungs whereby methane diffuses into the lungs and is exhaled as well as belched. The average cow emits around 600 litres of methane per day.

As illustrated in the table below, methane emissions due to ruminant animals account for approximately 19% per annum of total methane emissions. Given that methane emissions from human activity accounts for just over half of the methane emissions, this figure of 19% is highly significant.

Table 1 - Global Average Methane Emissions Origin CH4 Emission Mass (Tg/a) Type (%/a) Total (%/a) Natural Emissions Wetlands (incl. Rice agriculture) 225 83 37 Termites 7 3 Ocean 6 3 Hydrates 4 2 Natural Total 270 100 45 Anthropogenic Emissions Energy 110 33 18 Landfills 40 12 7 Ruminants (Livestock) 115 19 Waste treatment 8 4 Biomass burning 40 12 7 Anthropogenic Total 330 100 55 Sinks Soils -30 -5 -5 Tropospheric OH -510 -88 -85 Stratospheric loss -40 -7 -7 Sink Total -580 -100 -97 Emissions + Sinks Imbalance (trend) +20 -2.78 Tg/ppb +7.19 ppb/a 3 562783 The number of ruminant animals is increasing with estimates of 1.3 billion cattle at present and 1.1 billion sheep and goats. Based on one reference, about 6% of the food a cow eats (a single dairy cow consumes about 20 kilograms of high quality feed daily) is lost as methane.

It should therefore be appreciated from the above discussion that methane may be a significant contributor to global warming. It should further be appreciated that animals including ruminant animals, their propensity to generate methane, and their importance in the food chain, means that methods of modifying the amount of methane produced by these animals will be of benefit to the environment.

New Zealand needs to significantly reduce emissions from livestock to meet its targets under the Kyoto Treaty, which requires developed countries to reduce greenhouse gases by about 5% from 1990 levels by 2012. Recent figures suggest that 49% of New Zealand's green house gas emissions are attributable to agriculture, mainly as methane. Whilst a New Zealand example is provided, 15 worldwide, similar problems also exist, particularly in other agricultural based regions.

A further benefit is that, as making methane uses significant amounts of energy, if methane production is reduced, that energy may then be redirected to animal production. Methanogen microbes responsible for methane production represent a 20 parasitic mode of life. For example, if methane production was reduced, there may be potential gains in the amount and quality of meat and milk that a cow may produce and/or a reduction in feed requirements.

Many solutions have been proposed to adjust the amount of methane production by variations in what the animal eats. For example, one reference teaches that the 25 addition of nitrogen-rich urea to a cow's diet may lower methane exhalations by more than 25% as the nitrogen rich feed is easier to digest and produces this methane. Feeding a cow this way is only possible though where the cows eat a high-grain diet and are not pasture fed. In addition the feed itself may be expensive and therefore this method may not be feasible on a commercial scale.

Many other examples exist including by way of example US 3,615,649 which describes feeding halohemiacetal derivatives of polysaccharides. US 3,934,037 describes feeding a composition containing 2,3-dibromopropanol. US 3,949,090 describes feeding a composition containing dithiooxamide. 4 562783 Another document describes feeding the animal chlorinated hydrocarbons, commonly found in solvents and gasoline, and altering the chemistry of the rumen, (the fore-stomach where initial digestion occurs), to more closely resemble that of a kangaroo.

Scientists in Australia are studying eastern grey kangaroos, which eat a diet similar to that of cows and have similar digestive tracts, but produce no methane. Work being done in Australia has been centred on identifying the bacterium in the kangaroo's gut responsible for this difference and adding it to cow rumen to get non-polluting bovines via a vaccine. As noted though by the scientists, microbial 10 activity in the rumen is highly diverse and extremely complex and in a single millilitre of rumen fluid you expect to see a thousand million bacteria, a hundred million methanogens, and a hundred thousand protozoa cells per one millilitre. Therefore, the proposed method may be difficult to complete on a large scale in existing herds which are in uncontrolled environments.

In Scotland, scientists have identified a bacterium in soil that breaks methane down into hydrogen and carbon dioxide. If the bacterium could be transferred to cows, predictions are that British methane emissions could be reduced by 6%.

Canadian researchers are experimenting with vegetable-oil feed supplements that cut livestock methane by 15%.

US 5,985,907 describes use of synthetic HMG-CoA reductase inhibitors to inhibit growth of methanogens. Example inhibitors are described as being atorvastatin, fluvastatin, lovastatin, mevastatin, pravastatin, simvastatin and metabolites thereof. Natural extracts are not described or contemplated.

As should be appreciated from the above, the area of reducing methane emissions 25 from animals is developing and driven by the realisation of the significant volumes of methane produced by animals.

Herbal extracts have been known for centuries to impart healing or medicinal properties on animals including humans.

One plant genus Impatiens and in particular the species Impatiens balsamina are 30 native to southern Asia. Impatiens balsamina are annual plants growing to 20-75 cm tall, with a thick, but soft stem. The leaves are spirally-arranged, 2.5-9 cm long and 1-2.5 cm broad, with a deeply toothed margin. The flowers are red, pink, 562783 purple, or white, and 2.5-5 cm diameter; they are pollinated by bees and other insects, and also by nectar-feeding birds.

According to one reference, different parts of the plant are used to treat disease and skin afflictions; the leaves, seeds, and stems are also edible if cooked. Juice 5 from the leaves treat warts and also snakebites while the flower can be applied to burns to cool the skin.

Another reference describes that the plant is cathartic, diuretic and emetic [Indian uses]. It is used in the treatment of pains in the joints [Nepal uses]. The leaf juice is used as a treatment against warts [China uses]. The flowers are cooling, 10 mucilaginous and tonic [China, Nepal]. They are useful when applied to burns and scalds [China]. The juice of the flowers is used to treat snakebites [Nepal]. The flowers, and their alcoholic extract, possess marked antibiotic activity against some pathogenic fungi and bacteria [China]. The seed is expectorant and has been used in the treatment of cancer [India]. The powdered seeds are given to women 15 during labour in order to provide strength [Nepal].

In general though, the medicinal properties of this plant are considered weak and the plant is primarily cultivated as an ornamental plant.

Further uses described in the art include JP 09255583 which teaches that Impatiens balsamina may be used as an anti-pruritic agent and JP 10114668 20 which teaches that Impatiens balsamina may be used as a pharmacodynamic agent.

A further use of an extract from Impatiens balsamina termed 'Impress' has also been used in Japan to remove odours with the extract essentially acting as a chemical suppressant.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their 30 authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common 6 562783 general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an 5 inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent 10 from the ensuing description which is given by way of example only.

DISCLOSURE OF THE INVENTION The inventor has developed a method of suppressing certain anaerobic metabolic pathways in the lumen of the digestive tract of an animal resulting in the slowing or 15 stopping of methanogenesis in the gut. It is understood that as well as slowing or stopping methanogenesis, the method may also allow an increase in useful metabolites to pass across the animal's gut wall and be metabolised internally. The above effects have the ability to reduce methane and other greenhouse gas production from animals.

According to one aspect of the present invention there is provided a method of suppressing methanogen archaea activity in an animal by the step of introducing an extract derived from the plant species Impatiens balsamina in the environment in which methanogen archaea are present.

According to a further aspect of the present invention there is provided a method of 25 reducing greenhouse gas production from an animal by the step of administering an extract derived from the plant species Impatiens balsamina.

According to a further aspect of the present invention there is provided a method of altering the metabolism of an animal to inhibit greenhouse gas production by the step of administering an extract derived from the plant species Impatiens 30 balsamina.

According to a further aspect of the present invention there is provided a method of inhibiting methanogenesis resulting from the digestion of lignocellulosics by mixing 7 562783 the lignocellulosics with an extract derived from the plant species Impatiens balsamina.

According to a further aspect of the present invention there is provided a method of increasing the meat production of an animal by the step of administering an extract 5 derived from the plant species Impatiens balsamina to the animal.

According to a further aspect of the present invention there is provided a method of increasing the milk production of a lactating animal by the step of administering an extract derived from the plant species Impatiens balsamina to the animal.

According to a further aspect of the present invention there is provided a method of 10 preventing or reducing the severity of bloat in an animal by the step of administering an extract derived from the plant species Impatiens balsamina to the animal.

According to a further aspect of the present invention there is provided a method of reducing the maintenance feed requirement of an animal by the step of 15 administering an extract derived from the plant species Impatiens balsamina to the animal.

According to a further aspect of the present invention there is provided a composition for use in suppressing methanogen archaea activity in an animal which includes an extract derived from the plant species Impatiens balsamina.

According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition to suppress methanogen archaea activity According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture 25 of a composition formulated for administration to an animal in order to reduce greenhouse gas production from the animal.

According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition for administration to an animal to alter the metabolism of an 30 animal and inhibit greenhouse gas production. 8 562783 According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition to inhibit methanogenesis during digestion.

According to a further aspect of the present invention there is provided the use of 5 an extract derived from the plant species Impatiens balsamina in the manufacture of a composition to increase the meat production of an animal.

According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition to increase the milk production of a lactating animal.

According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition to prevent or reduce the severity of bloat in an animal.

According to a further aspect of the present invention there is provided the use of an extract derived from the plant species Impatiens balsamina in the manufacture 15 of a composition to reduce the maintenance feed requirement of an animal.

Preferably, the greenhouse gases may be selected from: methane, carbon dioxide, and combinations thereof. In one embodiment, the greenhouse gas may be methane.

Preferably, administration of the extract reduces the amount of greenhouse gas 20 released by between 10 and 100% w/v. In initial trials completed by the inventor, the reduction in greenhouse gas production is at least 20% w/v.

As noted above, the extract is derived from Impatiens balsamina species (also called rose balsam or jewelweed). One extract known by the inventors is termed 'Impress' which is derived from Impatiens balsamina species.

It is understood that the extract termed Impress contains compounds associated with aerobic microbe inhibition effects, plant growth promotants, antioxidants/coenzymes, anti-aerobic bacterial effects, odour eliminating compounds, and combinations thereof.

More specifically, these compounds include phenolic compounds.

In preferred embodiments the extract is formulated for oral administration and is administered orally to the animal. More preferably, the extract may be administered in methods selected from: a liquid, a paste, a tablet, a capsule, and g 562783 combinations thereof. In one embodiment, the animal may be a ruminant and the extract is administered as a bolus tablet. Examples of liquid or paste administration include via a drench, via a drinking trough, or via a feed. One option for administration found useful by the inventors in milking operations is to include 5 the extract of the present invention in the drinking water for animals immediately post-milking. Animals tend to drink more post-milking and are in a controlled environment hence administration via drinking trough at this point in time is an ideal opportunity.

Preferably, the dose administered ranges from at least 1 part extract per 1000 10 parts water or other liquid or solid diluents. In one embodiment, dosage administered may be approximately 1 part extract to 150 parts diluent. In a further embodiment, the ratio may be 1 part extract to 50 parts diluent. It should be appreciated that dosage may vary depending on species, animal metabolism, frequency of drinking (administration) and other factors such as any compromise 15 between cost versus response.

In one embodiment the administration occurs on a regular basis such as a daily dose of the extract. In an alternative embodiment, the extract is formulated so that administration may occur over greater intervals. For example, the extract may be formulated into a slow release formulation such as a slow release capsule or bolus. 20 The extract may then be released over an extended period of time.

In one embodiment, administration occurs daily and the full greenhouse gas reducing effect is noted once a time period of 3 days passes after commencing administration.

Preferably the extract is produced from the aerial portion of the plant although this 25 should not be seen as limiting as portions of the plant such as the leaves or flowers may also be used without departing from the scope of the invention.

In one embodiment known to the inventor, the extract is manufactured via a cold alcohol extraction steep to form a tincture. Other methods of extraction are also envisaged and this method is given by way of illustration only.

Preferably, the animal is a mammal. In one embodiment, the animal is a non- human animal. Preferred animals include farmed livestock including but not limited to cattle, sheep, goats, pigs, chickens, llamas, ostrich and deer. Fish species may also be treated in a similar manner to livestock for example by addition to feed 562783 used in carp or salmon farms. From the above it should be seen that ruminant animals are one key commercial application but this should not be seen as limiting as non-ruminant animals such as pigs also produce methane and the same methods may be used for non-ruminants without departing from the scope of the 5 invention.

As noted above, it is understood that the extract of the present invention manipulates methanogen archaea activity. It is understood by the inventor that the change in metabolism occurs in the animal's digestive metabolism microflora. A useful result from suppressing methanogen activity is that other organisms 10 (including the animal) associated with metabolism get a chance to process the cellulose material via other biochemical pathways e.g. the Krebs cycle. Given that the Krebs cycle is more efficient, there is therefore more energy available for other biological processes such as meat and milk production. It should be appreciated that maintenance feed is referred to. Animals may be fed less or more in order to 15 fatten or reduce weight. For the purposes of this specification, maintenance feed is that required to keep the animal at a healthy weight.

A further advantage of the present invention is that the extract is only active in the rumen and that therefore there is no withholding required before use of the milk or meat from the animal.

It should be appreciated from the above description that there is described various uses for an extract from the species Impatiens balsamina. The advantages which should be apparent to those skilled in the art include reduced greenhouse gas production, additional energy for other biological process such as meat and milk production and the prevention of bloat. A further advantage is that there is no with-25 holding requirement for ruminant animals as the extract is active in the rumen. A yet further advantage is that the present invention provides a 'natural' remedy to greenhouse gas production and does not require use of specific synthetically derived chemicals that may have a negative image or impact on the organic status of the farm. 11 562783 BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the ensuing description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 15 Figure 6 BEST MODES FOR CARRYING OUT THE INVENTION The invention is now described with reference to two trials completed by the inventor to determine the effects of the extract on methanogenesis.

EXAMPLE 1 A first trial was completed using cow pats or excrement as the substrate. Cow pat 25 material was selected because it releases large amounts of methane due to the presence of large numbers of methanogens and the presence of a large amount of methanogen metabolite. Cow pats therefore are typical of the contents of the animal's gut. Fresh cow pats were collected from a farm and stored in sealed black plastic bags until use. shows a graph of methane produced in a first trial completed by the inventor; shows a graph of methane produced in a second trial completed by the inventor; shows a graph of methane produced in a third trial completed by the inventor; shows a graph of methane produced in a fourth trial completed by the inventor; shows a graph illustrating the effects of dose rate on methane gas production expressed in terms of volume; and, shows a graph illustrating the effects of dose rate on methane gas production in treated cows (average shown by bar A) expressed as a percentage of an untreated control group of cows. 12 562783 Multiple samples were prepared with each sample including 150ml of cow pat mixed with 150ml of deoxygenated water and 5ml of Impatiens balsamina extract solution in a conical flask. Each flask was wrapped in aluminium foil to keep the contents dark and the flask opening was sealed and gas produced collected in a 5 gasometer adjusted to standard temperature and pressure (STP). The flasks were then incubated at 38°C while the gas was collected.

The flask was designed to emulate stomach conditions especially with regard to stomach temperature as methane production is highly sensitive to temperature.

As shown in Figure 1, the volume of methane gas produced steadily increased 10 over the first 3 hours and then levelled off at a maximum of 16ml produced.

EXAMPLE 2 A second cow pat trial was completed using the same conditions as above. A control solution was included using the same mixture of cow pat and water but with 15 no inhibitor added.

Results are shown in Figure 2 where a similar steady increase was again observed in all cases with the control sample increasing as expected much faster than the inhibitor added solutions (Marked 'Experimental' and 'EXP1). Total inhibition effect was noted as ranging from 21 to 30% with an average reduction in production of 20 approximately 25% EXAMPLE 3 A third trial was completed to further investigate the effect of C02 on gas emission from cow pat cultures and to establish a dose-response curve.

Fresh cow pat was homogenised and mixed with an equal volume of water. This medium was used in subsequent experiments below.

Conical Flask Experiments Four 250ml conical flasks were set up with medium as shown in Table 1 below: 13 562783 Table 1 - Flask Set Up Flask Label Medium Used Amount of Impatiens balsamina Extract Used C1 150m! None C2 150ml None E1 150ml 1:300 on a raw pat volume basis of extract E2 150ml 1:60 on a raw pat volume basis of extract The flasks were left open initially and incubated until the mixture temperature reached approximately 38°C. Once they reached 38°C, the flasks were sealed and attached to a gasometer to collect and measure the gas released. The headspace 5 and tubing in each flask was flushed with C02 prior to sealing.

The results showed that initially, all flasks lost volume in their headspace during the first day. This is attributed to the provision of nutrient in the form of C02. Once the C02 nutrient was utilised (Days 2 to 4) the amount of methane subsequently produced was significantly more for the control samples than the samples which 10 included extract as shown in Figure 3. Also, as shown in Figure 3, the extract is clearly active even at very low dose rates (1:300).

U-Tube Experiments The same medium as above was used (and similar methodology) but instead, U-tubes were used instead of flasks as U-tubes do not contain any headspace like 15 the flasks. Anaerobic fermentation in the sealed closed arm of the U-tube releases gases which indicate the rate of fermentation. The composition of these gases can be determined by gas chromatography and micro manometry.

In this experiment, one U-tube was kept as a control with no extract added and a second U-tube had extract added at a ratio of 1:150 parts Impatiens balsamina 20 extract.

Like with the flasks, the U-tubes were incubated at 38°C although C02 flushing was not required. 14 562783 The U-tube experiments had no initial inhibition phase like that observed with the flasks. The control sample produced significantly more methane gas than the sample which included the extract as shown in Figure 4.

Interestingly with both experiments above, the extract inhibited all gas production, 5 so it affected all anaerobic gas production. This is thought to have occurred by forcing anaerobiosis into non-gas forming metabolic pathways. This could be a very favourable result since the products of these pathways remain as solutes such as lactic acid, proprionic acid, formate and pyruvate etc, which are easily absorbed across the cow's intestinal membranes to be used by the cow through aerobic 10 means such as the Krebs Cycle. If gut metabolism goes toward methane production as it does in the absence of the invention extract, then these metabolic end products would be lost. Coupled with the increase in the cow's metabolic efficiency through the use of the extract is the possibility that the cow's own C02 production would be reduced through the more efficient capture of carbon into 15 metabolic compounds other than C02. In addition, lower feeding rates would be required because anaerobic respiration is about 1/11th as efficient as aerobic respiration and the products of anaerobic respiration in the cow's gut are not available to the cow in the absence of the invention extract. In the presence of the invention extract, a considerable increase in digestible food would be available to 20 an animal. Diverting more food to aerobic processes is envisaged to result in greater food absorption and digestion and greater metabolic efficiency.

Given the results above it should be appreciated that the extract used has a suppression/inhibition effect on methanogenesis as observed by a decrease in methane production. Given the reduced methane production, other metabolic 25 processes are likely to become more prominent such as the Krebs cycle which is more energy efficient and therefore may free up energy for other processes including meat and milk production. In addition, as methane production is associated with bloat, a reduction in methane and total rumen gases produced may also reduce the occurrence or severity of bloat.

EXAMPLE 4 In this trial, rumen fluid taken from live cows is tested to determine the methane reducing effects of the extract of the present invention. 562783 Rumen was collected from fistulated cows that had been treated with less than 5ml Impatiens balsamina extract per litre introduced daily into their drinking water.

They were allowed access to the treated water ad libitum. An equal sized group of cows were given untreated water.

The experimental design was a cross-over with four cows in each group. Thus each cow acted as its own control as well as there being a control group not administered Impress-treated water.

Rumen fluid was obtained at around 11am from the cows by hand collection through a fistula. Both control and treated cows were sampled at each collection 10 time.

The rumen collected was wrung out and the liquid collected in pre-warmed (38°C) Dewar flasks. The solids were discarded.

Within two hours of collection the fermentation trial commenced. Three fermentation tubes from each of the two groups, treated and control, were filled 15 with rumen fluid and 1g each of dry, sterile powdered grass added to each to provide a food source to mimic the actual rumen.

The results found are shown in Table 2 below.

Table 2 - Fermentation results of rumen fluid taken from fistulated cows either treated with less than 5ml/i Impress or untreated. Results are expressed as 20 percentages of the values given by the mean of the control (untreated) cows.

Treatment Total Gas Carbon dioxide Methane Untreated cow rumen fluid (no Impress added) 100 100 100 Treated cow rumen fluid (Impress added to the cows' drinking water at a rate of 5ml/l) 43 56 39 As can be seen from Table 2, the volume of gas produced from treated rumen fluids was significantly lower than from the control (untreated) animals. These results reinforce the results observed in earlier Examples for a more realistic in vivo 16 562783 environment.

EXAMPLE 5 In this example, rumen fluid was obtained from freshly slaughtered cows and a similar 5 trial to that completed in Example 4 was completed.

The rumen of freshly slaughtered cows was slit and the rumen contents collected and sampled. The activity of the fluid was tested in fermentation tubes as above, but with invention extract added to an experimental set of tubes and no extract to an equal number of tubes. One gram of dry, sterilised and powdered grass was added to each 10 tube as a food source.

Results, expressed as a percentage of the mean of the control group, are given in Table 3 below.

Table 3 - Slaughtered Cow Rumen Fluid Trials. Results are expressed as percentages of the values given by the mean of the control (untreated) cows.

Treatment Total Gas Carbon dioxide Methane Control (no extract added) 100% 100% 100% Extract added 72% 39% 64% As can be seen in Table 3, use of the extract of the invention caused a reduction in the total gas produced as compared to the control. This result further illustrates that use of the invention extract causes an increase in rumen efficiency. In this experiment the reduction was mainly caused by a reduction in C02 production, but also a very useful reduction of 36% in methane production.

EXAMPLE 6 In this example, experiments are shown describing the dose response using the extract of the present invention.

Dose response experiments were completed using varying concentrations of the 17 562783 invention extract and then measuring the resulting decrease in methane. A typical result is shown in Figure 5 where the gas volume produced decreases significantly initially for doses up to 0.5ml/100ml and then reaches an inhibition limit as dose increases.

The data above can also be expressed as the percentage of inhibition with dose which is shown in Figure 6 which illustrates the rapid rise in inhibition which reaches a peak as dose increases. Results for Example 4 are plotted on Figure 6 shown by arrow A. A bar is used to illustrate the fact that the dose the cows received varied as each cow may have consumed varying amounts of the drinking 10 water and used other water sources in the paddock/field.

EXAMPLE 7 In this example, experiments are shown to prove that the extract works to reduce greenhouse gas emissions in a variety of animals.

Rumen fluid or faecal material from cows, sheep and pigs were collected and then tested using protocols described in earlier examples. Cows were included to provide a positive control while sheep were included to show use in another ruminant animal and pigs were included to show effects in a non-ruminant animal.

The results found are shown below in Table 4.

Table 4 - In Vitro Trials on Cow, Sheep and Pig Faeces Species Rumen Faeces Cow Inhibition ranged from 100% to 60% (average 77%).

Inhibition ranged from 96% to 86% (average 90.2%). Cow pats were the most active of pats tested for faecal methane production.

Sheep Inhibition averaged 50%.

Inhibited (75% though little gas was produced) 18 562783 Pig (not applicable) Inhibition was complete (100%), although the rate of gas production was slower than in cow pats and the total amount of gas produced was less The above results illustrate that the invention extract has the ability to reduce methane production in all animals including ruminants and non-ruminants.

EXAMPLE 8 In this example, formulations are described to illustrate alternative methods of delivering the extract.

Formulation 1: A tablet formulation containing Impatiens balsamina extract with tableting carriers and aids.

Formulation 2: A bolus formulation containing Impatiens balsamina extract with 10 bolus constituents. The bolus may be designed so as to deliver a continuous dose of extract or may be configured so as to deliver a pulsed dose of extract.

Formulation 3: An oral drench formulation containing Impatiens balsamina extract optionally with viscosity increasing constituents so that the drench may be either a liquid or more viscous paste.

The above formulations may be further adapted to be administered with other agents known in the art. By way of example, the drench described above may also include anthelmintic agents. 19 562783 The above examples illustrate the greenhouse gas reducing effects of Impatiens balsamina species. Uses of this invention are wide and at the very least may assist in agriculture meeting environmental controls around greenhouse gas emissions.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Received at IPONZ 30 MarclfffffiS

Claims (38)

WHAT WE CLAIM IS:

1. A method of suppressing methanogen archaea activity in a non-human animal by the step of introducing an extract derived from the plant species Impatiens balsamina into an environment in which methanogen archaea are present in the a non-human animal.

2. A method of reducing greenhouse gas production from a non-human animal by the step of administering an extract derived from the plant species Impatiens balsamina.

3. The method as claimed in claim 2 wherein the greenhouse gas is selected from: methane, carbon dioxide, and combinations thereof.

4. A method as claimed in any one of claims 1 to 3 wherein the extract ranges in concentration from at least 1 part extract to 1,000 parts diluent.

5. The method as claimed in any one of the above claims wherein the meat production of the animal is increased.

6. The method as claimed in any one of claims 1 to 4 wherein the animal is a lactating animal and the amount of milk produced by the animal is increased.

7. The method as claimed in any one of claims 1 to 4 wherein the method prevents or reduces the severity of bloat in the animal.

8. The method as claimed in any one of claims 1 to 4 wherein the method results in a decrease in the amount of animal maintenance feed required.

9. The method as claimed in any one of the above claims wherein the extract is administered orally to the animal. 21 Received at IPONZ 30 Marctfftfffi

10. The method as claimed in any one of the above claims wherein the extract is formulated as: a liquid, a paste, a tablet, a capsule, and combinations thereof.

11. The method as claimed in any one of the above claims wherein the extract is formulated as a bolus tablet.

12. The method as claimed in any one of the above claims wherein the animal is a mammal.

13. The method as claimed in any one of the above claims wherein the animal is selected from the group comprising: cattle, sheep, deer, goats, pigs, llamas, poultry and farmed fish species.

14. The method as claimed in any one of the above claims wherein the extract reduces the amount of greenhouse gas produced from the animal by between 10 and 100% w/v.

15. The method as claimed in any one of the above claims wherein the extract includes compounds associated with: aerobic microbe inhibition effects, plant growth promotants, antioxidants/coenzymes, anti-aerobic bacterial effects and odour eliminating compounds, and combinations thereof.

16. The method as claimed in any one of the above claims wherein the extract includes phenolic compounds.

17. Use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition for suppressing methanogen archaea activity in an animal.

18. A composition for use in suppressing methanogen archaea activity in an animal which includes an extract derived from the plant species Impatiens 22 Received at IPONZ 30 MarclfffffiS balsamina wherein the composition includes the extract at a concentration from at least 1 part extract to 1,000 parts diluent.

19. The composition as claimed in claim 18 wherein the composition is formulated as: a liquid, a paste, a tablet, a capsule, and combinations thereof.

20. The composition as claimed in any one of claim 18 or 19 wherein the composition is formulated as a bolus tablet.

21. Use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition formulated for administration to an animal to suppress methanogen archaea activity in the animal.

22. Use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition formulated for administration to an animal to reduce greenhouse gas production from the animal.

23. The use as claimed in claim 22 wherein the greenhouse gas is selected from: methane, carbon dioxide, and combinations thereof.

24. The use as claimed in any one of claims 21 to 23 wherein the composition contains a dose of extract from at least 1 part extract to 1,000 parts diluent

25. The use as claimed in any one of claims 21 to 24 wherein the meat production of the animal is increased.

26. The use as claimed in any one of claims 21 to 24 wherein the amount of milk produced by the animal is increased wherein the animal is a lactating animal.

27. The use as claimed in any one of claims 21 to 24 wherein the use prevents or reduces the severity of bloat in the animal. 23 Received at IPONZ 30 Marclff^ffiS

28. The use as claimed in any one of claims 21 to 24 wherein the use reduces the animal's maintenance feed requirement.

29. The use as claimed in any one of claims 21 to 28 wherein the extract is formulated for oral administration to the animal.

30. The use as claimed in any one of claims 21 to 29 wherein the extract is formulated for administration as: a liquid, a paste, a tablet, a capsule, and combinations thereof.

31. The use as claimed in any one of claims 21 to 29 wherein the extract is formulated for administration as a bolus tablet.

32. The use as claimed in any one of claims 21 to 31 wherein the animal is a mammal.

33. The use as claimed in any one of claims 21 to 32 wherein the animal is selected from the group comprising: cattle, sheep, deer, goats, pigs, llamas, poultry and farmed fish species.

34. The use as claimed in any one of claims 21 to 33 wherein administration of the extract reduces the amount of greenhouse gas produced by the animal by between 10 and 100% w/v.

35. The use as claimed in any one of claims 21 to 34 wherein the extract includes compounds associated with: aerobic microbe inhibition effects, plant growth promotants, antioxidants/coenzymes, antibacterial effects, odour eliminating compounds, and combinations thereof.

36. The use as claimed in any one of claims 21 to 35 wherein the extract includes phenolic compounds. Received at IPONZ 30 MarchFftfffi

37. A method of reducing greenhouse gas production from a non-human animal by administration of an extract from the plant species Impatiens balsamina substantially as hereinbefore described with reference to the Examples and Figures.

38. Use of an extract derived from the plant species Impatiens balsamina in the manufacture of a composition to reduce greenhouse gas production from an animal substantially as hereinbefore described with reference to the Examples and Figures. METHANE REDUCTION TECHNOLOGIES LIMITED 25