Reduction of methane emissions

Abstract

The present invention provides a method of reducing methane emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen.

Description

[0001] Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Reduction of Methane Emissions Field of the Invention The present invention concerns methods, systems and apparatus to reduce the methane emissions of ruminant animals. More particularly, the present invention concerns methods, systems and apparatus for the delivery of drinking water to ruminant animals, wherein the drinking water reduces the methane emissions of the ruminant animals. The present invention also concerns methods, systems and apparatus to reduce the emissions of other greenhouse gases of ruminant animals (e.g. carbon dioxide) and to improve the health of ruminant animals. the Invention Climate change is a well-documented phenomenon involving complex changes to local and global environmental systems as a result of increasing average global temperatures. These changes are largely detrimental and include among other things largescale disruption to weather systems, increases in desertification, rising sea levels which threaten the safety of coastal settlements and disruption of food and crop growth. Average global temperatures have been increasing at a significant rate since the late 19th Century, with a primary contributor to this increase being anthropogenic greenhouse gas emissions. Methane is a very potent greenhouse gas, and although the concentration of methane in the Earth’s atmosphere is much lower than other greenhouse gases such as carbon dioxide, methane has a disproportionately high contribution to the warming effect of greenhouse gases and is estimated to be responsible for 30% of the increase in average global temperatures since the industrial revolution. One of the largest sources of methane emissions is the methane produced by methanogenic microorganisms that reside in the digestive tracts of ruminants (approximately 10% of anthropogenic warming to date). These microorganisms produce methane as a byproduct of their metabolism in the in the digestive tracts of ruminants. Since ruminant animals (e.g. in particular cows and sheep) are important livestock animals on a global scale (it is estimated that the global populations of cows and sheep are 1 and 1.2 billion respectively), reducing methane emissions from Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO ruminants (even by a small percentage) is a key goal in reducing overall methane emissions and thus combatting global warming. The present invention seeks to address the problem of reducing methane emissions from ruminants. The present invention also seeks to address certain other problems associated with raising ruminants in a domesticated setting, as explained in further detail below. Summary of the Invention The present invention provides, according a first aspect, a method of reducing methane emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen. According to a second aspect, the present invention also provides a system suitable for providing to a ruminant drinking water as defined according to the first aspect of the invention (i.e. drinking water containing gas nanobubbles comprising oxygen), comprising a water source operably connected to an apparatus which is configured to produce the drinking water as defined according to the first aspect of the invention. According to a third aspect, the present invention also provides a system which provides drinking water containing gas nanobubbles comprising oxygen as defined herein, comprising a water source operably connected to an apparatus which is configured to produce said drinking water. It will of course be appreciated that features described in relation to one aspect of the present invention may be incorporated into other aspects of the present invention. For example, the system of the invention may incorporate any of the features described with reference to the method of the invention and vice versa. Description of the Drawings Figure 1 shows a schematic diagram of a system according to an embodiment of the present invention. Figure 2 shows a graphical depiction of oxygen inhibition of methanogenesis in the digestive tract of a ruminant according to an embodiment of the present invention. Figure 3 is a graph showing the effect of nanobubble treated water on methane production from rumen fluid in vitro. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Detailed Description The present inventors have surprisingly found that providing a ruminant with drinking water comprising nanobubbles comprising oxygen can reduce the methane emissions of that ruminant. Methane production by ruminants is due to the presence of methane-producing microorganisms (referred to herein as methanogens) in the digestive tract, e.g. the stomach, of the ruminant. A key enzyme catalyst in the production of methane by methanogens is methyl-coenzyme M reductase. However, that enzyme is highly sensitive to oxygen (the methanogens’ normal environment in the digestive tract of the ruminant is essentially anaerobic, i.e. devoid of oxygen). The amount of methane that is produced by these microorganisms can be reduced by exposing them to oxygen. Cattle drink between 50 and 150 litres of water per day depending on production cycle (e.g., in, milk, dry etc.) and multiple times (7-10 times) per day. If drinking water is oxygenated as described herein, regular drinking could help deploy oxidative stress to the rumen throughout the day and more consistently instead of in a single dose. The average fluid content of the rumen is 80 litres and on average a cow takes up 12% of the rumen volume of water per drinking event. Nanobubbles are nanoscopic gaseous cavities in liquids, e.g. water, that have unusual properties compared to bubbles of a larger size. In particular, nanobubbles can stay within the liquid for much longer than normal bubbles, on the scale of hours, days or weeks whereas normal bubbles usually do not last more than a few minutes. The present invention makes use of nanobubbles as a vector to introduce oxygen into the digestive tract of ruminants via drinking water, in order to increase the concentration of oxygen in the environment of the methanogens and thus disrupt their methanogenesis. This will reduce the amount of methane produced by these microorganisms and, when practiced on a large scale, help to reduce the effect of climate change overall. Without being bound by theory, the present inventor considers that nanobubbles are a particularly effective way of delivering oxygen to the digestive tract of ruminants because, as noted above, their small size allows them to persist in the drinking water. Furthermore, as well as boosting the amount of dissolved oxygen in the drinking water, the nanobubbles can also act as a reservoir of oxygen that can replenish the dissolved oxygen in the drinking water as the dissolved oxygen is consumed or Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO otherwise leaves the drinking water, thereby working to maintain an elevated concentration of oxygen in the drinking water over a longer period of time. A further advantage of the present invention is that it can improve the cleanliness of containers, e.g. drinking troughs, and pipework used to deliver the drinking water of the invention to ruminants, by reducing the amount of microorganism contamination in the containers and pipework, thereby reducing the microbial load of ruminants drinking the water of the invention. This leads to a reduced chance of infection by an infectious disease and a corresponding decrease in the disease burden in these ruminants. Where these ruminants are given antibiotics, for example, if they are farmed, this also allows for lower amounts of antibiotics to be administered to the ruminants, thereby reducing the amount of antibiotics present in milk and meat products from said ruminants and reducing the likelihood of the development of antibiotic resistance. Accordingly, in a further aspect, the present invention provides drinking water containing gas nanobubbles comprising oxygen as defined herein, for use in reducing infectious disease risk in a ruminant or for use in preventing infectious disease in a ruminant. Infectious disease is for example infectious disease caused by a microorganism, such as a bacterium, virus, parasite or fungus, especially a bacterium. The improved cleanliness of the containers and pipework supplying water to the containers as a result of the invention will also lead to reduced cleaning of the containers and pipework, reducing associated costs. Thus, in a further aspect, the present invention also provides a method of reducing microorganism contamination in a drinking container for a ruminant and, optionally, associated pipework, comprising providing drinking water to said drinking container, wherein the drinking water contains gas nanobubbles comprising oxygen as described herein. Cleaner drinking water also leads to increased uptake of food and water by the ruminants, which increases the total amount of growth and the growth rate of the ruminants. A further advantage of the present invention is that it can promote the activity of aerobic, methanotrophic bacteria, which consume methane, in ruminants, thus resulting in further reductions of methane emissions from said ruminants supplied with drinking water of the invention. Thus, in a further aspect, the present invention also provides a method of promoting the activity of methanotrophic bacteria comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen as described herein. Similarly, the present invention can promote the colonisation and activity of direct-fed microbes and probiotics in the Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO ruminants, which improves the growth and health of the ruminants. Thus, in a further aspect, the present invention also provides a method of promoting the colonisation and activity of direct-fed microbes and probiotics comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen as described herein. A further advantage of the present invention is that it can also be combined with other strategies for reducing methane emissions from a ruminant including, for example, methane-reducing substances such as 3-nitrooxypropanol (3-NOP), bromoform, peroxide treatments or anti-methanogen lytic enzymes. The nanobubbles present in the drinking water of the invention can disrupt microbial communities in the biofilm, which makes them more susceptible to intervention by additional methane- reducing substances. The combination of at least one additional methane-reducing substance with the drinking water of the invention is at least as good at reducing methane emissions as each strategy in isolation. Furthermore, in certain embodiments, when used in combination, the drinking water of the present invention can prolong or enhance the effect of the additional methane-reducing substances, which, therefore, results in fewer administrations of the additional methane-reducing substances. In certain embodiments, the combination of the drinking water of the present invention and one or more of the additional methane-reducing strategies as described herein may have a synergistic effect on the reduction of methane emissions from the ruminant. A further advantage of the present invention is that it can also improve the profile of volatile fatty acids in the rumen, for example, by increasing the total production of volatile fatty acids, which are a source of energy for the ruminant, and by increasing the ratio of propionate to acetate, which is associated with lower methane emissions. The present invention can also increase the production of butyrate in the rumen, which is a component of milk fat. An advantage of the present invention is that it can also increase milk production in the ruminant. Thus, in a further aspect, the present invention provides a method of increasing milk production in a ruminant comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen as described herein. Butyrate also reacts with molecular hydrogen, therefore, increasing the production of butyrate also leads to a reduction in molecular hydrogen in the rumen, removing one of the cofactors required for methanogenesis by methanogenic microorganisms, leading to further reductions in methane emissions. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO The present invention can also lead to improvements in the production and utilisation of ammonia and nitrogen. The present invention can also lead to improvements in mineral solubility, and neutral detergent fibre digestibility. The present invention can also lead to an increase in the pH of the rumen. This is important in animals who are fed highly digestible diets as more complex forage diets generally result in greater methane production by the ruminant. Additionally, high consumption of feed, as is the goal in farmed animals, can result in the production of acid in the rumen, which can lead to rumen acidosis, thus disrupting the rumen environment and negatively affecting the health and growth of the ruminant. Therefore, the present invention can help to resist rumen acidosis by increasing the pH of the rumen and thus reduce or eliminate any potential negative effects associated with decreased pH due to high consumption of feed. Accordingly, in an embodiment of the methods of the invention, the methods also result in an increase in the pH of the rumen. Further, in an embodiment of the methods of the invention, the provision of the drinking water of the invention to the ruminant resists, reduces or eliminates rumen acidosis. The present invention also provides drinking water containing gas nanobubbles comprising oxygen as described herein for use in reducing rumen acidosis in a ruminant. In a first aspect, the invention provides a method of reducing methane emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen (i.e. the nanobubbles comprise gas of which a certain percentage is oxygen, e.g. at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% oxygen). References to oxygen herein refer to molecular oxygen, i.e. O2. In a further embodiment of the invention, the method also reduces emissions of CO2derived from the ruminant. In another aspect, the present invention also provides a method of reducing CO2 emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen as described herein. In another aspect, the present invention also provides a method of reducing greenhouse gas emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen, as described herein. In an embodiment of this aspect, the greenhouse gases are selected from the group consisting of methane, carbon dioxide Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO and nitrous oxide. In an embodiment of this aspect, the greenhouse gases are selected from the group consisting of methane and carbon dioxide. In an embodiment of the invention, the drinking water provided to the ruminant has a concentration of oxygen greater than 7 ppm. In another embodiment of the invention, the drinking water provided to the ruminant has a concentration of oxygen of from 7 ppm to about 31 ppm. In further embodiments of the invention, the drinking water provided to the ruminant has a concentration of oxygen of from 7 ppm to about 30 ppm, or from 7 ppm to about 29 ppm, or from 7 ppm to about 28 ppm, or from 7 ppm to about 27 ppm, or from 7 ppm to about 26 ppm, or from 7 ppm to about 25 ppm, or from 7 ppm to about 24 ppm, or from 7 ppm to about 23 ppm, or from 7 ppm to about 22 ppm, or from 7 ppm to about 21 ppm, or from 7 ppm to about 21 ppm, or from 7 ppm to about 20 ppm, or from 7 ppm to about 19 ppm, or from 7 ppm to about 18 ppm, or from 7 ppm to about 17 ppm, or from 7 ppm to about 16 ppm, or from 7 ppm to about 15 ppm, or from 7 ppm to about 14 ppm, or from 7 ppm to about 13 ppm, or from 7 ppm to about 12 ppm, or from 7 ppm to about 11 ppm, or from 7 ppm to about 10 ppm, or from 7 ppm to about 10 ppm, or from 7 ppm to about 9 ppm, or from 7 ppm to about 8 ppm. In further embodiments of the invention, the drinking water provided to the ruminant has a concentration of oxygen of from about 8 ppm to about 31 ppm, or from about 9 ppm to about 31 ppm, or from about 10 ppm to about 31 ppm, or from about 11 ppm to about 31 ppm, or from about 12 ppm to about 31 ppm, or from about 13 ppm to about 31 ppm, or from about 14 ppm to about 31 ppm, or from about 15 ppm to about 31 ppm, or from about 16 ppm to about 31 ppm, or from about 17 ppm to about 31 ppm, or from about 18 ppm to about 31 ppm, or from about 19 ppm to about 31 ppm, or from about 20 ppm to about 31 ppm, or from about 21 ppm to about 31 ppm, or from about 22 ppm to about 31 ppm, or from about 23 ppm to about 31 ppm, or from about 24 ppm to about 31 ppm, or from about 25 ppm to about 31 ppm, or from about 26 ppm to about 31 ppm, or from about 27 ppm to about 31 ppm, or from about 28 ppm to about 31 ppm, or from about 29 ppm to about 31 ppm, or from about 30 ppm to about 31 ppm, or about 31 ppm. In an embodiment of the invention, the drinking water comprises both dissolved oxygen and nanobubble cavities comprising oxygen. In further embodiments of the invention, the drinking water provided to the ruminant has a concentration of oxygen of from about 15 ppm to about 31 ppm, or from about 15 ppm to about 30 ppm, or from about 15 ppm to about 29 ppm, or from about Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO 15 ppm to about 28 ppm, or from about 15 ppm to about 27 ppm, or from about 15 ppm to about 26 ppm, or from about 15 ppm to about 25 ppm, or from about 15 ppm to about 24 ppm, or from about 15 ppm to about 23 ppm, or from about 15 ppm to about 22 ppm, or from about 15 ppm to about 21 ppm, or from about 15 ppm to about 20 ppm, or from about 15 ppm to about 19 ppm, or from about 15 ppm to about 18 ppm, or from about 15 ppm to about 17 ppm, or from about 15 ppm to about 16 ppm. In an embodiment of the invention, the concentration of oxygen in the drinking water refers to the amount of dissolved oxygen in the drinking water. In another embodiment of the invention, the concentration of oxygen in the drinking water refers to the total amount of oxygen in the drinking water, including both dissolved oxygen and oxygen comprised in the gas nanobubble cavities comprising oxygen. For example, the total concentration of oxygen in the drinking water may be from about 7 ppm to about 100 ppm, or from about 15 ppm to about 100 ppm, or from about 30 ppm to about 100 pm, or from about 35 ppm to about 100 ppm, for example from about 35 ppm to about 75 ppm, or from about 35 ppm to about 65 ppm, for example from about 35 ppm to about 60 ppm, for example from about 45 ppm to about 60 ppm. For example, the total concentration of oxygen in the drinking water may be from about 7 ppm to about 80 ppm, or from about 7 ppm to about 60 ppm, or from about 15 ppm to about 80 ppm, or from about 15 ppm to about 60 ppm, or from about 30 ppm to about 80 ppm, or from about 35 ppm to about 80 ppm, for example from about 35 ppm to about 75 ppm, or from about 35 ppm to about 65 ppm, for example from about 35 ppm to about 60 ppm, for example from about 45 ppm to about 60 ppm. The concentration of oxygen in the water may be described herein in parts per million (ppm) or mg / L. Both of those units are equivalent. In an embodiment of the invention, the drinking water provided to the ruminant has a concentration of oxygen that is the maximum that can be incorporated in the drinking water via the introduction of nanobubbles, given the prevailing conditions (temperature, altitude etc.) under which the drinking water of the invention is generated. It is well known that the solubility of oxygen in water can vary depending on certain prevailing environmental conditions, therefore, the maximum amount of oxygen that can be dissolved in the drinking water may be different under different prevailing conditions. For example, conditions such as water temperature, salinity, altitude, atmospheric pressure, and contamination with impurities or microbes can impact the amount of oxygen that can be dissolved in water. In further embodiments of the invention, the drinking water of the invention (containing gas nanobubbles comprising Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO oxygen) which is provided to the ruminant has a concentration of oxygen that is ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥8 times, ≥10 times, ≥12 times, ≥15 times, ≥18 times, or ≥20 times, for example from 2 to 20 times, or from 2 to 10 times, or from 2 to 15 times, or from 5 to 10 times, or from 10 to 20 times, or from 15 to 20 times the concentration of oxygen that would be found in untreated water under the same prevailing environmental conditions (e.g. temperature, altitude, salinity etc.). By untreated water, it is meant water that has not been exposed to oxygen nanobubbles as described herein (such as may be available from a municipal water source or a natural water source) and thus which does not comprise gas nanobubbles comprising oxygen. In an embodiment of the invention, the concentration of oxygen in the drinking water is measured when the surrounding air is at ambient temperature, for example, the ambient temperature of a cattle feedlot, for example at a temperature of from about 5ºC to about 30ºC, for example at about 5ºC, or about 10ºC, or about 15ºC, or about 20ºC, or about 25ºC, or about 30ºC. In an embodiment of the invention, the concentration of oxygen in the drinking water is measured at a water temperature of from about 1ºC to about 30ºC, for example at about 1ºC, or about 5ºC, or about 10ºC, or about 15ºC, or about 20ºC, or about 25ºC, or about 30ºC. In an embodiment of the invention, the concentration of oxygen in the drinking water is measured at an altitude of from 0 to 1500 metres above sea level, e.g. at an altitude of from 0 to 200 metres above sea level e.g. at sea level, or at 10, 50, 100, 150 or 200 metres above sea level. In an embodiment of the invention, the concentration of oxygen in the drinking water is measured when the drinking water is clean, i.e. not contaminated with significant levels of impurities. The term nanobubbles can be used to describe any gaseous cavity in a liquid with a diameter of less than one micron (1000 nm). However, conventionally, a nanobubble is used to describe any gaseous cavity in a liquid with a diameter of less than about 200 nm, e.g. about 10 nm to about 200 nm. In an embodiment of the invention, the nanobubbles in the drinking water have a diameter of less than 1000 nm, less than 800 nm, less than 500 nm, less than 300 nm, less than 200 nm, or less than 100 nm e.g. from about 10 nm to about 1000 nm, from about 10 nm to about 800 nm, from about 10 nm to about 500 nm, from about 10 nm to about 300 nm, from about 10 nm to about 200 nm, or from about 10 nm to about 100 nm. In an embodiment of the invention, the nanobubbles have a diameter of from about 35 nm to about 250 nm. Nanobubbles are characterized by their extremely small size and high surface area relative to their volume. Nanobubbles with smaller diameters are more stable and last Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO for a longer period of time in the liquid than nanobubbles with larger diameters. In an embodiment of the invention, the nanobubble diameter distribution in the drinking water has a D90 of about 1000 nm, about 800 nm, about 500 nm, about 300 nm, about 200 nm, or about 100 nm. The D90 value specifies that 90% of the distribution of nanobubbles by number has a diameter that is less than the indicated value, i.e. if the distribution contains 100 nanobubbles, then 90 of those nanobubbles will have a diameter of less than the indicated D90 value. In an embodiment, the nanobubble diameter distribution has a mean in the range 100-140 nm, a mode in the range 80-100 nm, SD in the range 30-50, D10 in the range 80-100, D50 in the range 100-120 and D90 in the range 160-200. In an embodiment of the invention, the methods described herein comprise a step of introducing the gas nanobubbles comprising oxygen into the drinking water. In further embodiments of the methods described herein, the gas nanobubbles comprising oxygen are introduced into the drinking water using an apparatus 102 configured for that purpose, for example, a nanobubble generator, for example, a nanobubble generator from Moleaer Inc. (Hawthorne, CA, USA) or from Trident Bubble Technologies LLC (Sheridan, WY, USA), e.g. the Trident Nanobubble System 1. In an embodiment of the methods of the invention, the drinking water is provided to the ruminant in a container, for example, a drinking trough. In an embodiment of the invention, the concentration of oxygen in the drinking water is measured in the container, for example, a drinking trough, which provides the drinking water to the ruminant (i.e. the container, such as a drinking trough, from which the ruminant drinks). In a further embodiment of the invention, the concentration of oxygen in the drinking water is measured at the output of the apparatus 102 which generates the nanobubbles, for example, the nanobubble generator. In an embodiment of the invention, the method further comprises a step of recirculating the drinking water from the container to the apparatus 102. Such a recirculation step can be used to boost or at least maintain the level of oxygen in the drinking water. In an embodiment of the invention, the drinking water containing gas nanobubbles comprising oxygen is not magnetised, i.e. it has not been exposed to a magnetic field (beyond the natural magnetic field of the earth), such as a magnetic field produced by a permanent magnet or an electromagnet. In an embodiment of the invention, the drinking water containing gas nanobubbles does not contain significant amounts of oxygen radicals. In an embodiment of the invention, the drinking water Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO containing gas nanobubbles does not contain oxygen radicals when generated. In an embodiment of the invention, the drinking water containing gas nanobubbles is not magnetised and does not contain significant amounts of oxygen radicals. In an embodiment of the methods of the invention, said methods do not include a step of exposing the drinking water containing gas nanobubbles comprising oxygen to an artificial magnetic field (i.e. a magnetic field other than the natural magnetic field of the earth). In an embodiment of the invention, the ruminant drinks the drinking water at least once a day for at least one day. In other embodiments of the invention, the ruminant drinks the drinking water at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or 20 times a day, and the ruminant drinks the drinking water for at least one, two, three, four, five, six or seven days, or for at least one, two, three or four weeks, or for at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or 12 months, or for at least one, two, three, four or five years. In an embodiment of the invention, the ruminant drinks the drinking water between about 5 and about 15 times per day, for example between about 6 and about 12 times per day or between about 7 and about 10 times per day. In an embodiment of the invention, the ruminant drinks between about 3 and about 200 litres per day, for example between about 10 and about 200 litres per day, between about 25 and about 200 litres of the drinking water per day, for example between about 35 and about 175 litres of the drinking water per day, for example between about 50 and about 150 litres of the drinking water per day, for example between about 80 and about 120 litres of the drinking water per day, for example about 100 litres per day. In an embodiment of the invention, the amount of oxygen delivered to the ruminant, for example in the drinking water of the invention, is from about 0.015 moles to about 3.5 moles of oxygen per day, for example from about 0.015 moles to about 3 moles of oxygen per day, from about 0.015 moles to about 2.5 moles of oxygen per day, from about 0.015 moles to about 2 moles of oxygen per day, from about 0.015 moles to about 1.5 moles of oxygen per day, from about 0.015 moles to about 1 mole of oxygen per day, from about 0.015 moles to about 0.5 moles of oxygen per day, from about 0.015 moles to about 0.25 moles of oxygen per day, from about 0.015 moles to about 0.1 moles of oxygen per day, from about 0.015 moles to about 0.05 moles of oxygen per day, from about 0.015 moles to about 0.03 moles of oxygen per day, from Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO about 0.03 moles to about 3.5 moles of oxygen per day, from about 0.05 moles to about 3.5 moles of oxygen per day, from about 0.06 moles to about 3.5 moles of oxygen per day, from about 0.09 moles to about 3.5 moles of oxygen per day, from about 0.1 moles to about 3.5 moles of oxygen per day. As will be apparent, the amount of oxygen delivered to the ruminant can be calculated by determining the concentration of oxygen in the drinking water of the invention which is provided to the ruminant and then multiplying that figure by the amount of water drunk by the ruminant. The amount of water drunk by the ruminant can be based on an average figure (see above), or can be calculated more precisely by measuring, at the start and end of the day, the amount of water in the container (e.g. a drinking trough) which provides the drinking water of the invention to the ruminant. To the amount of oxygen delivered in the drinking water of the invention must be added any oxygen delivered via an alternative strategy, e.g. via a feed additive such as a peroxide, e.g. calcium peroxide. The amount of oxygen delivered to the ruminant is an important criterion, since delivery of too little oxygen will have an insufficient inhibitory effect on methane production or will fail to realise the other advantages of the invention as described herein. On the other hand, delivery of too much oxygen will upset the delicate redox balance in the rumen and may impact digestibility negatively, particularly anaerobic digestion. Depending on prevailing conditions, achieving delivery of higher levels of oxygen (e.g >0.1 moles of oxygen per day) may require additional administration of oxygen, e.g. via the administration of a peroxide such as calcium peroxide to the ruminant, as described elsewhere herein. In an embodiment of the invention, the method of reducing methane emissions derived from a ruminant comprises reducing the activity of methyl-coenzyme M reductase (MCR). The activity of MCR relies on a nickel-based coenzyme F430. The nickel in the coenzyme must be in the +1 oxidation state in order for MCR to initiate catalysis. In an embodiment of the invention, MCR is present in microorganisms, e.g. methanogens, in the stomach, rumen or hindgut of the ruminant. Ruminants are herbivorous animals that derive their nutrition from a plant-based diet via a specialised, multi-chambered stomach. The plant material the ruminant eats passes through the chambers of the stomach and is regurgitated so that the ruminant can chew the regurgitated plant material and re-digest it. Ruminants belong to the suborder Ruminantia, and in one embodiment include animals such as cattle, goats, sheep, buffalo, deer, antelope and reindeer. In an embodiment of the invention, the ruminant Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO is selected from the group consisting of cattle and sheep. In an embodiment of the invention, the ruminant is cattle, i.e. an individual of the species Bos taurus. Mature female cattle are referred to as cows and mature male cattle are referred to as bulls. Young female cattle are called heifers, young male cattle are bullocks, and castrated male cattle are known as steers. An ox (plural oxen) is a bovine trained as a draft or riding animal. Oxen are usually castrated mature bulls. All of those terms are incorporated within the term cattle. In an embodiment of the invention, the ruminant is a steer. In an embodiment of the invention, MCR is present in microorganisms that are present in the digestive tract of the ruminant. These microorganisms can be found, for example, in the rumen or hindgut of the ruminant. In an embodiment of the invention, these microorganisms are methanogens. Methanogens are anaerobic archaea that produce methane as a byproduct of their energy metabolism. All known methanogens belong exclusively to the domain Archaea, i.e., in an embodiment of the invention the MCR is present in methanogens of the domain Archaea. Archaea is a domain of organisms distinct from both the Bacteria and Eukaryota domains, and archaeal cells have unique properties which make them distinct from Bacteria and Eukaryota. In an embodiment of the invention, the methods as described herein, e.g. a method of reducing methane emissions derived from a ruminant, further comprise an additional strategy for reducing methane emissions from the ruminant. For example, in an embodiment of the invention, said methods further comprise providing an additional methane reducing substance to said ruminant. Examples of methane reducing substances include, for example, 3-nitrooxypropanol (3-NOP), bromoform (tribromomethane or CHBr3), a peroxide, such as calcium peroxide or hydrogen peroxide, or anti-methanogen lytic enzymes. Additional methane-reducing substances can be administered to the ruminant in a number of different ways, for example, as a feed additive, or via injection, or as an orally ingested tablet or pill, or combined with the drinking water of the ruminant, e.g. the drinking water of the present invention. Methane-reducing substances such as a 3-NOP, bromoform and a peroxide are conveniently administered to the ruminant as a feed additive. One particularly convenient way to provide bromoform to a ruminant is to provide feed containing the seaweed Asparagopsis taxiformis, which naturally contains high concentrations of bromoform. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO In a second aspect, the present invention provides a system suitable for providing to a ruminant drinking water as defined in the first aspect of the invention (i.e. drinking water containing gas nanobubbles comprising oxygen), comprising a water source 101 operably connected to an apparatus 102 which is configured to produce the drinking water as defined in the first aspect of the invention. In a third aspect, the present invention is directed to a system which provides drinking water containing gas nanobubbles comprising oxygen as defined herein to a ruminant, comprising a water source 101 operably connected to an apparatus 102 which is configured to produce said drinking water. In an embodiment of the invention, the apparatus 102 comprises a first inlet for receiving water. In an embodiment of the invention, the apparatus 102 comprises a second inlet for receiving gas. In an embodiment of the invention, the gas is atmospheric air or oxygen, e.g. atmospheric air as found at or near sea level, for example in the range between sea level and 2000 m above sea level, or a gas comprising a proportion of oxygen which is greater than that in atmospheric air. As is well-known to a skilled person, atmospheric air comprises approximately 21% oxygen. In an embodiment, the gas comprises at least 20% oxygen, at least 21% oxygen, at least 30% oxygen, at least 40% oxygen, at least 50% oxygen, at least 55% oxygen, at least 60% oxygen, at least 65%, at least 70% oxygen, at least 75% oxygen, at least 80% oxygen, at least 85% oxygen, at least 90% oxygen, at least 95% oxygen or 100% oxygen. In an embodiment of the invention, the apparatus 102 further comprises an outlet from which said drinking water containing gas nanobubbles comprising oxygen can exit the apparatus 102. In an embodiment of the invention, the apparatus 102 comprises baffles. In an embodiment of the invention, the apparatus 102 comprises a membrane. In an embodiment of the invention, the apparatus 102 is a manifold. In an embodiment of the invention, the apparatus 102 introduces nanobubbles into the drinking water. In an embodiment of the invention, the system of the second or third aspect comprises a pump 103 for pumping water from the source of water 101 into the apparatus 102. In an embodiment of the invention, the pumping of the drinking water through the apparatus 102 causes the introduction of nanobubbles into the drinking water, e.g. the physical act of pumping the water through the membrane in the apparatus 102 causes the nanobubbles to be introduced into the drinking water. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO In an embodiment of the invention, the pump 103 provides water to the apparatus 102 at a pressure not exceeding 7 bar, for example not exceeding 5 bar, for example not exceeding 4 bar or not exceeding 3 bar. In an embodiment of the invention, the system of the second or third aspect comprises a container into which water can flow from the apparatus 102. In an embodiment of the invention, the apparatus 102 further comprises an outlet operably connected to the container, e.g. a drinking trough for a ruminant. In an embodiment of the invention, the drinking water can flow from the apparatus 102 to the container via the outlet. In an embodiment of the invention, the container can hold at least 10 L of drinking water, for example, at least 20 L of drinking water, at least 30 L of drinking water, at least 40 L of drinking water, or at least 50 L of drinking water. In an embodiment of the invention, the system of the second or third aspect comprises a recirculation stream from the apparatus 102 to the source of water 101. In an embodiment of the invention, the system of the second or third aspect comprises a recirculation stream or flow which recirculates water produced by the apparatus 102 back into the apparatus, for example so that the oxygen concentration in the water can be further increased. In an embodiment of the invention, the system of the second or third aspect comprises a recirculation stream from the outlet of the apparatus 102 to the first inlet of the apparatus 102 or to the water pump 103. In an embodiment of the invention, the system of the second or third aspect comprises an outlet from the container, which is operably connected to the apparatus 102, such that the drinking water in the container can be recirculated through the apparatus 102, thus allowing for further increases in the amount of nanobubbles and, therefore, concentration of oxygen in the drinking water. In further embodiments, the system comprises a pump configured to achieve recirculation of drinking water from the container to the apparatus 102. In further embodiments, the system comprises an oxygen sensor configured to detect the concentration of oxygen in the drinking water in the container. This oxygen sensor may be the oxygen sensor 105 which is configured to detect the concentration of oxygen in the drinking water produced by the apparatus 102, or it may be an additional oxygen sensor. In further embodiments, the output from the oxygen sensor configured to detect the concentration of oxygen in the drinking water in the container controls a mechanism which regulates the recirculation from the container to the apparatus 102. Accordingly, the system can therefore maintain a Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO particular concentration of oxygen in the drinking water present in the container within certain defined parameters. In an embodiment of the invention, the system of the second or third aspect further comprises an oxygen sensor 105 configured to detect the amount of oxygen in the drinking water produced by the apparatus 102. In an embodiment of the invention, an output from the oxygen sensor 105 controls a mechanism 106, e.g. a solenoid or a valve, which regulates the flow of gas into the apparatus 102. The amount or concentration of oxygen in the drinking water produced by the apparatus 102 can therefore be detected and varied by regulating the gas flow entering the apparatus 102, thus allowing the system to maintain a concentration or amount of oxygen in the drinking water produced by the apparatus 102 within certain defined preset parameters. In some embodiments, the desired concentration of oxygen in the drinking water is not reached with a single pass of water and gas through the apparatus 102. Therefore, in one embodiment, the drinking water is recirculated through the apparatus 102 so that the oxygen concentration in the drinking water can be further increased. In an embodiment of the invention, the system of the second or third aspect comprises a source of oxygen gas 107 operably connected to the second inlet of the apparatus 102, i.e. the inlet for receiving gas. In an embodiment of the invention, the composition of the source of oxygen gas 107 is at least 20% oxygen, at least 21% oxygen, at least 30% oxygen, at least 40% oxygen, at least 50% oxygen, at least 55% oxygen, at least 60% oxygen, at least 65%, at least 70% oxygen, at least 75% oxygen, at least 80% oxygen, at least 85% oxygen, at least 90% oxygen, at least 95% oxygen or 100% oxygen. In an embodiment of the invention, the system of the second or third aspect comprises the drinking water as described in any of the embodiments herein. In an embodiment of the invention, the system of the second or third aspect comprises the ruminant. In an embodiment of the invention, the ruminant is selected from the group consisting of cattle, goats, sheep, buffalo, deer, antelope and reindeer. In an embodiment of the invention, the ruminant is selected from the group consisting of cattle and sheep. In an embodiment of the invention, the ruminant is cattle, i.e. an individual of the species Bos taurus. Mature female cattle are referred to as cows and mature male cattle are referred to as bulls. Young female cattle are called heifers, young male cattle are bullocks, and castrated male cattle are known as steers. An ox (plural oxen) is a bovine trained as a draft or riding animal. Oxen are usually castrated mature Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO bulls. All of those terms are incorporated within the term cattle. In an embodiment of the invention, the ruminant is a steer. In an embodiment of the invention, the system of the second or third aspect comprises reduced microbial contamination compared to a similar system which provides non-nanobubble aerated water to a ruminant. In a particular embodiment of the invention, it is the container and the operable connection between the apparatus 102 and the container of the system which comprise reduced microbial contamination. Reduced microbial contamination may take the form of an overall reduced level of microbial contamination and / or it may take the form of a reduction in certain microbial species. Non-nanobubble aerated water is normally aerated water, for example, water supplied from a municipal water supply, or a natural water supply such as a stream or river. Non-nanobubble aerated water typically has an oxygen content of less than or equal to 7 ppm. In an embodiment of the invention, the system of the second or third aspect is suitable for use in the method of the invention. Experimental Examples Example 1 An experiment is performed to determine the difference in methane emissions derived from ruminants that are provided with drinking water of the invention from ruminants provided with normal drinking water, e.g. water as available from municipal water supply facilities. The amount of methane produced by ruminants is measured using a closed system respiration chamber. Ruminants occupy the respiration chamber for a period of time, for example five days, during which time all methane emissions from the animals are measured and their dietary and water intake is closely controlled. The amount of methane emitted by each ruminant is measured over the period of time and is divided by the bodyweight of the ruminant in kilograms and the number of days the ruminant spends in the respiration chamber. An average amount of methane produced per kilogram of feed per day is calculated for the ruminants provided with drinking water of the invention and for the ruminants provided with municipal drinking water. These two values are compared using an appropriate statistical comparator, for example 95% confidence intervals, to determine the difference in methane emissions derived from the two groups of ruminants. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Example 2 A system for providing drinking water of the invention to ruminants is constructed from constituent parts. A first part is a source of water 101, for example a water tank containing water, which may be, for example, rainwater collected for the purpose, or water pumped from an underground aquifer. Water is pumped from the source of water 101 using a pump 103 into the apparatus 102 for producing nanobubbles in the water. The apparatus 102 draws in air from the surrounding air and forces the air and water through a membrane. As the water is forced through the membrane along with the air, the water becomes infused with nanobubbles with the same gaseous composition of the surrounding air. The water can then leave the apparatus 102 and enters a container in which the water containing nanobubbles can be stored, or from which can be provided to a ruminant for drinking. The system is configured so that the water can, instead of passing from the apparatus 102 to the container, be recirculated back into the source of water 101. This allows for the amount of nanobubbles in the water to be increased, and thus increasing the concentration of oxygen contained within the water. Example 3 In this example, an in vitro experiment was carried out to investigate the effect of nanobubble-aerated water on gas production in bovine rumen fluid. As is demonstrated below, the administration of nanobubble-aerated water to rumen fluid in a volume that mimics a single drinking event reduced enteric methane production by up to 15%, and had no effect on digestibility, total gas production, or pH. Materials & Methods Dry matter (DM) of the forage substrate (grass hay) was weighed into acetone- washed, pre-weighed filter bags (F57 ANKOM bag; Ankom Technology Corp., Macedon, NY, USA) in an amount of 0.7 g per bag. After sealing the bags, they were placed into empty 120 mL serum vials (1 bag / vial). Rumen fluid from 3 cannulated cows was collected 2 h after morning feeding and filtered through four layers of cheesecloth. Cows were fed on a total mixed ration (TMR) basis - 50% forage:50% concentrate dry matter (DM). Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Inoculum was prepared by mixing rumen fluid at a ratio of 1:3 with mineral buffer (Goering and Van Soest, 1970, Agric. Handbook Number 379. ARS-USDA, Washington, DC).50 mL of inoculum was transferred to each 120 mL serum vial under a stream of O2-free CO2. The vials were sealed with a rubber stopper and incubated at 39°C on a shaker at 120 rpm for 12 h. Water (maintained at 39°C) was delivered to each vial at 6 h using a syringe injection, thus mimicking a drinking event. Distilled water was delivered to the blank and regular control vials, and fully oxygenated water or mixtures of fully oxygenated water and distilled water was delivered to the treatment group vials. Fully oxygenated water was generated using a 4 gallon per minute (GPM) Trident Nanobubble System Model TNS-1 (nanobubbler). A 30-liter reservoir of water was re-circulated though the nanobubbler using a diaphragm pump for 60 minutes and the gas source was lab-grade oxygen supplied at 8 PSI, resulting in a maximum nanobubble oxygenation of 31 ppm oxygen. Nanobubble output diameter statistics were – Mean: 121.0 + / - 0.7 nm; Mode: 92.4 + / - 3.1 nm; SD: 43.2 + / - 1.4 nm; D10: 79.5 + / - 0.7 nm; D50: 109.5 + / - 1.5 nm and D90: 182.6 + / - 3.0 nm. The error values refer to the standard error. The D50, the median, is the diameter where half of the distribution of nanobubbles by number has a diameter below this value. Similarly, 90 percent of the distribution by number has a diameter below the D90 value, and 10 percent of the distribution by number has a diameter below the D10 value. The semi-automated in vitro gas production technique as described by Mauricio et al. (1999, Anim Feed Sci Technol 79:321–330) was used to measure the gas produced in each vial. Briefly, gas pressure (GP) in each vial was measured after 6 h and 12 h of incubation by inserting a 23 gauge (0.6 mm) needle attached to a pressure transducer (Fisherbrand Traceable Manometer model 06-664-21; Fisher Scientific, Pittsburgh, PA, USA). After measurement of GP, a 20 mL sample of headspace gas was removed using a gas-tight syringe and transferred into a 12 mL evacuated exetainer (Labco Ltd, High Wycombe, UK) for later analysis of CH4 content. After 12 h of incubation and after gas measurements and sampling, the vials were placed in ice to stop the fermentation process, and then the filter bags were removed from each vial and the pH measured. Bags were thoroughly rinsed with cold water until the water ran clear, dried at 55ºC for 48 h, and weighed for DM disappearance calculation. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO The concentration of CH4in headspace gas samples was analyzed by gas chromatography as described by Sarich et al. (2022) using a Scion 456-Gas Chromatograph (Goes, the Netherlands, EU) with hydrogen (6 mL / min) as the carrier gas and a packed column of 3.4 m filled with Hayesep N. The gas volume (GV) at each time point was calculated from the measured gas pressure (GP in psi) using the Boyle’s law equation adapted to our laboratory conditions (Lopez et al. 2007, Anim. Feed Sci. Technol. 135:139-156. doi:10.1016 / j.anifeedsci.2006.06.005). The CH4 production was calculated based on total GV and CH4 concentration and is expressed as mL g–1substrate DM incubated and DM disappeared. CH4remaining in the headspace from previous gas measurements was corrected for the previous CH4 concentration using the equation described by Lopez et al. (2007, Anim. Feed Sci. Technol.135:139-156. doi:10.1016 / j.anifeedsci.2006.06.005). Treatment and Control Groups Treatment group 1: inoculum, substrate and 6 mL of fully oxygenated water. Treatment group 2: inoculum, substrate, 4 mL of fully oxygenated water and 2 mL of distilled water. Treatment group 3: inoculum, substrate, 2 mL of fully oxygenated water and 4 mL of distilled water. Regular control group: inoculum, substrate and 6 mL of distilled water. Blank control group: inoculum and 6 mL of distilled water. Each group of samples had 3 vials / incubation time / run. Table 1: Record of the experimental parameter results for the control rumen fluid and nanobubble treated rumen fluid at 2 mL, 4 mL, and 6 mL volumes.

[0002] Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO

[0003] Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Results As shown in Figure 3 and Table 1, in vitro analyses of rumen fluid treated with nanobubble-aerated water support the hypothesis that such water is able to reduce enteric methane production. Data indicate that the response tends to be dose dependent, and enteric methane production can be decreased with increasing doses of nanobubble treated water. The treatment, which simulated a single drinking event, resulted in a 12% to 15% reduction of methane production, had no effect on dry matter digestibility, no effect on cumulative total gas production, and no effect on pH under the tested conditions. These data support the use of nanobubble treated drinking water as a promising method to reduce enteric methane from ruminants. Example 4 In this example, an in vivo experiment was conducted to determine the effect of enriched water with oxygen nanobubbles on gas flux, rumen fermentation dynamics, and ruminal microbial communities in cannulated beef steers. Approach and Research Procedures 8 ruminally cannulated steers and 8 intact cows were used to test the effect of water enriched with oxygen nanoparticles over 35 days at the Climate Smart Research Facility at Colorado State University, CO. The altitude of the facility was 1524 m above sea level. The steers and cows were located in two pens containing five SmartFeed units and one GreenFeed unit (C-Lock, Rapid City, SD). One SmartFeed in each pen was used to provide water to the group of steers and cows. Water from one pen was enriched with oxygen nanobubbles using a custom nanobubble generator set-up, which included a recirculation stream to enable greater oxygen enrichment of the water, while water from the other pen did not receive oxygen enrichment. Nanobubble output diameter statistics were – Mean: 121.0 + / - 0.7 nm; Mode: 92.4 + / - 3.1 nm; SD: 43.2 + / - 1.4 nm; D10: 79.5 + / - 0.7 nm; D50: 109.5 + / - 1.5 nm and D90: 182.6 + / - 3.0 nm. The error values refer to the standard error. The D50, the median, is the diameter where half of the distribution of nanobubbles by number has a diameter below this value. Similarly, 90 percent of the distribution by number has a diameter below the D90 value, and 10 percent of the distribution by number has a diameter below the D10 value. Steers and cows received a radio frequency electronic ID (RFID, Allflex, USA Inc.), and were adapted to the units for two weeks. Steers and Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO cows were blocked by body weight and randomly distributed in one of two treatments (Table 2). Treatments were 1) normal water (CTL), and 2) oxygen-enriched water (OEW). Table 2. Description of the initial body weight (kg) of steers and cows used in the experiment. Animals were observed daily to assess their health and well-being. Dissolved oxygen was evaluated three times per day. The average oxygen concentration for the control water was 2.1 ± 0.51 mg / L, while it was 10.6 ± 2.59 mg / L in the oxygenated water. This represents an approximately five-fold higher concentration of oxygen in the oxygen-enriched water compared to the control water, which was an encouraging result under normal farm conditions. On days 1 and 26, a 24-h ruminal fluid collection was conducted every 4 hours (at 00.00, 04.00, 08.00, 12.00, 16.00, and 20.00), to measure the concentrations of volatile fatty acids (VFA), ammonia nitrogen (NH3-N), mineral solubility, and microbial communities. In addition, blood samples were collected at 800, 1200, and 1600, to determine mineral solubility and NH3-N concentration. On days 21 to 25, apparent total tract digestibility was evaluated. Samples of feed were collected daily during days 21 to 24, while fecal samples were collected twice daily by rectal grab during days 22 to 25. Feed and fecal samples were dried for further analysis. Indigestible neutral detergent fiber (iNDF) will be used as an internal digestibility marker. Steers were fed a high-forage diet of corn silage, wheat straw, dry distillers' grains, and a salt and vitamin supplement (Table 3). Feed samples were collected, dried and composited, for analysis. Daily water and feed intake and gas flux were individually recorded during the experiment via SmartFeed and GreenFeed technology. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Table 3. Ingredient inclusion in the experimental diet (on DM basis) Ruminal sample of the 24-h collections In the cannulated steers, the redox potential was determined by inserting a manual probe into the ventral sac of the rumen (HI98195, Hanna Instruments, Inc, Woonsocket, RI). Then, a representative sample of ruminal digesta was collected at each time and strained through four layers of cheesecloth. Dissolved oxygen and pH were measured using manual sensors (HI98198 and HI98195, Hanna Instruments, Inc, Woonsocket, RI). One 40-mL sample was acidified with 0.4 mL of 20% (vol / vol) H2SO4 to halt fermentation and frozen at -20°C until further analysis of VFA and NH3- N concentration. Additionally, one 40 mL sample was frozen at -20°C until further mineral solubility analysis. Finally, a 15 mL sample was collected and frozen at -20°C until further microbial analysis. Gas flux determination Animals were allowed to visit the GreenFeed units every 4 hours (up to 6 visits per day) and consume up to 6 drops of alfalfa pellet (approximately 35 g / drop) with 30- second spacing between drops. This strategy encourages animals to visit the units regularly throughout the day and ensures animals stay at the GreenFeed for an appropriate gas flux collection. The emission rate of gases (Qc) is calculated using the following equation (Huhtanen et al., 2015, J Dairy Sci 98(5):3394-3409; doi: 10.3168 / jds.2014-9118): ^c= [^p × (^onc − ^conc) × ^air] ÷ 106Where Cp is the fractional capture rate of air, Conc is the concentration of captured gas, BConc is the background concentration of gas, and Qair is the volumetric airflow. Thus, the gas flux (Qm) is calculated using the following equation: Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO ^m = ^c × 273.1 ÷ (273.15 + ^air) × ^D Where Tair is the air temperature, and GD is the density of gas at 1 atm and 273.5 K To ensure the whole system's performance, CO2recovery tests were performed monthly throughout the experiment and at the beginning and end of each experiment. Additionally, zero and span calibrations of the CH4, CO2, and O2gas analyzers were performed every three days via an onboard autocalibration system. Raw collection data was validated by C-Lock Inc., which includes checking head proximity, visit length, and airflow and wind corrections. Additionally, data was excluded when the length of the visit is less than 2 min, and the airflow is lower than 26L / s (Vargas et al., 2024a). Statistical analysis Intake and gas flux data were analyzed for cannulated steers and intact cows as a complete randomized design using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). The animal was considered the experimental unit, and the model included the fixed effects of treatment and the initial values of intake and gas flux as covariate. For ruminal fluid data, the model included the fixed effects of treatment, collection time, the interaction of treatment and collection time, and the initial values rumen fermentation as covariate. Significance was determined at P ≤ 0.05, and tendencies were considered when 0.10 > P > 0.05. Results Ruminal fermentation dynamics There were no treatment × time interactions (P > 0.10) for any of the rumen fluid variables analyzed (Table 4). Cannulated steers consuming oxygenated water had greater (P = 0.04) rumen fluid pH than those consuming control water. In addition, time was a significant (P < 0.0001) source of variation of oxidation-reduction potential and pH. Dissolved oxygen and oxidation-reduction potential in the rumen fluid were not different (P > 0.10) between treatments. Finally, dissolved oxygen was not different (P > 0.10) among collection time points. Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Table 4. Fermentation parameters in the rumen fluid of cannulated steers receiving control and oxygenated water. Dry matter and water intake Total intake and total mixed ration (TMR) intake were not different (P > 0.10) in cannulated steers or intact cows consuming oxygenated or control water (Table 5). Only intact cows exposed to oxygenated water consumed more pellets (P > 0.036) than those exposed to control water. Water consumption was similar among steers and cows consuming control and oxygenated water. Table 5. Dry matter and water intake of cannulated steers and intact cows receiving control and oxygenated water. Gas flux determination The production of CH4 tended (P < 0.10) to be lower (16.2% and 10.7%) in cannulated steers and intact cows consuming oxygenated water than those consuming control water, respectively (Table 6). Methane yield was lower (P = 0.032) in Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO cannulated steers consuming oxygenated water, but similar (P = 0.665) in intact cows between treatments. The production of CO2 was lower (P <0.05) in cannulated steers and intact cows consuming oxygenated water than in those consuming control water. The consumption of O2was lower (P = 0.011) in cannulated steers and tended to be (P = 0.082) lower in intact cows consuming oxygenated water than in those consuming control water. The production of hydrogen was not different in cannulated steers and intact cows consuming either oxygenated or control water. Table 6. Gas flux evaluation of cannulated steers and intact cows receiving control and oxygenated water.

Claims

Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO Claims 1. A method of reducing methane emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen.

2. The method of claim 1, wherein the method also reduces CO2 emissions derived from said ruminant.

3. The method of claim 1 or 2, wherein the drinking water has a concentration of oxygen of greater than 7 ppm.

4. The method of any of claims 1 to 3, wherein the drinking water has a concentration of oxygen of from 7 ppm to about 31 ppm.

5. The method of any of claims 1 to 4, wherein the drinking water has a concentration of oxygen of from 7 ppm to about 25 ppm, or from 7 ppm to about 20 ppm, or from 7 to about 15 ppm, or from 7 ppm to about 10 ppm.

6. The method of any of claims 1 to 4, wherein the drinking water has a concentration of oxygen of from about 15 ppm to about 31 ppm, or from about 20 ppm to about 31 ppm, or from about 25 ppm to about 31 ppm.

7. The method of any of claims 1 to 4, wherein the drinking water has a concentration of oxygen of from about 10 ppm to about 31 ppm, or from about 10 ppm to about 25 ppm, or from about 15 ppm to about 25 ppm, or from about 20 ppm to about 25 ppm.

8. The method of any of claims 1 to 7, wherein the nanobubbles have a diameter of less 1000 nm, optionally the nanobubbles have a diameter of less than 500 nm, or less than 300 nm, or less than 200 nm, or less than 100 nm.

9. The method of any of claims 1 to 8, wherein the nanobubbles have a diameter of from about 35 nm to about 250 nm.

10. The method of any of claims 1 to 9, wherein the drinking water has a concentration of oxygen that is ≥2 times, for example ≥5 times, the concentration of oxygen that would be found in otherwise equivalent water that has not been exposed to oxygen nanobubbles.

11. The method of any of claims 1 to 10, wherein the amount of oxygen delivered to the ruminant is from about 0.015 moles to about 3.5 moles of oxygen per day.

12. The method of any of claims 1 to 11, wherein the ruminant drinks the drinking water at least once a day, for at least one day.Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO 13. The method of any of claims 1 to 12, wherein the method comprises reducing the activity of methyl-coenzyme M reductase present in a ruminant.

14. The method of claim 13, wherein the methyl-coenzyme M reductase is contained within microorganisms present in the ruminant.

15. The method of claim 14, wherein the microorganisms are present in the digestive tract of the ruminant, e.g. the rumen and / or the hindgut.

16. The method of claim 14 or 15, wherein the microorganisms are methanogens.

17. The method of any of claims 1 to 16, wherein the ruminant is selected from the group consisting of cattle, goats, sheep, buffalo, deer, antelope and reindeer.

18. The method of any of claims 1 to 17, wherein the method further comprises an additional strategy for reducing methane emissions from said ruminant, for example, administering an additional methane-reducing substance to said ruminant.

19. The method of any of claims 1 to 18, wherein the method also results in an increase in the pH in the rumen of said ruminant.

20. The method of any of claims 1 to 19, wherein the method also results in a reduction of acidosis in the rumen of said ruminant.

21. A system suitable for providing to a ruminant drinking water containing gas nanobubbles comprising oxygen as defined in any of claims 1 to 11, comprising a water source operably connected to an apparatus which is configured to produce said drinking water.

22. A system which provides drinking water containing gas nanobubbles comprising oxygen as defined in any of claims 1 to 11 to a ruminant, comprising a water source operably connected to an apparatus which is configured to produce said drinking water.

23. The system of claim 21 or 22, wherein the apparatus comprises a first inlet for receiving water.

24. The system of any of claims 21 to 23, wherein the apparatus further comprises a second inlet for receiving gas.

25. The system of claim 24, wherein the gas is atmospheric air or a gas comprising a proportion of oxygen which is greater than that in atmospheric air.

26. The system of any of claims 21 to 25, wherein the apparatus further comprises an outlet from which said drinking water containing gas nanobubbles comprising oxygen can exit the apparatus.Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO 27. The system of any of claims 21 to 26, wherein the apparatus comprises baffles.

28. The system of any of claims 21 to 27, wherein the apparatus comprises a membrane.

29. The system of any of claims 21 to 28, wherein the apparatus introduces nanobubbles into the drinking water.

30. The system of any of claims 21 to 29, wherein the apparatus is a manifold.

31. The system of any of claims 21 to 30, wherein the system comprises a pump for pumping water from the source of water into the apparatus.

32. The system of any of claims 26 to 31, wherein the outlet of the apparatus is operably connected to a container, e.g. a drinking trough for a ruminant, and wherein the drinking water can flow from the apparatus to the container via the outlet.

33. The system of claim 32, wherein the container can hold at least 10 L of drinking water, at least 20 L of drinking water, or at least 50 L of drinking water.

34. The system of any of claims 21 to 33, wherein the system comprises a recirculation stream from the apparatus to the source of water.

35. The system of any of claims 21 to 34, wherein the system further comprises an oxygen sensor configured to detect the amount of oxygen in the drinking water produced by the apparatus.

36. The system of claim 35, wherein an output from the oxygen sensor controls a mechanism which regulates the flow of gas into the apparatus.

37. The system of any of claims 21 to 36, wherein the system comprises a source of oxygen gas operably connected to the second inlet of the apparatus.

38. The system of claim 37, wherein the source of oxygen gas comprises at least 50% oxygen, optionally at least 70%, at least 80%, at least 90% or 100% oxygen.

39. The system of any of claims 21 to 38, comprising drinking water as defined in any of claims 1 to 11.

40. The system of any of claims 21 to 39, which comprises said ruminant.

41. The system of any of claims 21 to 40, wherein the ruminant is selected from the group consisting of cattle, goats, sheep, buffalo, deer, antelope and reindeer.

42. The system of any of claims 21 to 41, which comprises reduced microbial contamination compared to a similar system which provides non-nanobubble aerated water to a ruminant.Attorney Docket Reference: 41165 / 2 ABEL-002 / 01WO 43. The system of any of claims 21 to 42 that is suitable for use in the method of any of claims 1 to 20.

44. Water containing gas nanobubbles comprising oxygen for use in reducing rumen acidosis in a ruminant.

45. Water for use as claimed in claim 44, wherein said water is drinking water as described in any of claims 2 to 11.

46. A method of reducing carbon dioxide emissions derived from a ruminant, comprising providing drinking water to said ruminant, wherein the drinking water contains gas nanobubbles comprising oxygen.

47. A method as claimed in claim 46, wherein said drinking water is as described in any of claims 2 to 11.

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