Gas emission measurement system and method, methane generation inhibitor and its evaluation method, and bacterial quantification method

Abstract

This invention enables accurate measurement of greenhouse gas emissions from animals. It also allows for easy evaluation of the reduction effect of greenhouse gas emissions from animals. Furthermore, it provides a methane emission inhibitor that suppresses methane production from Methanobrevibacter bacteria. [Solution] A bacterial quantification method for quantifying Methanobrevibacter species contained in the rectal feces of animals includes the steps of: collecting rectal feces from an animal; holding the rectal fecal sample in an anaerobic porter under anaerobic conditions; replacing the gas in the anaerobic porter with nitrogen gas, diluting the sample with a culture medium, and culturing the bacteria in a test tube; recovering the cultured bacteria; extracting DNA from the recovered bacteria; and quantifying the Methanobrevibacter species from the extracted DNA by quantitative PCR or a gene analysis method using next-generation sequencing analysis.

Description

Technical Field

[0001] The present disclosure relates to a gas emission measurement system and a gas emission measurement method, a methane generation inhibitor and an evaluation method thereof, and a bacterial quantification method. For example, it relates to an apparatus and a method capable of measuring the emission amount of greenhouse gases emitted by livestock, and a methane generation inhibitor that can be added to livestock feed and contributes to the reduction of greenhouse gas emissions.

Background Art

[0002] Along with the stable supply of food and the sustainable development of livestock farming, the compatibility with the global environment is strongly demanded. In particular, the reduction of greenhouse gases derived from fossil fuels (Green House Gas such as carbon dioxide and methane: hereinafter referred to as "GHG") is being carried out worldwide. As is well known, the Kyoto Protocol, which defines the GHG reduction obligations of each country, has been concluded for the prevention of global warming, and immediate action is required for the signatory countries including Japan. It is said that the proportion affecting the greenhouse effect is about 64% for carbon dioxide, 19% for methane gas, and 10% for fluorocarbon gas, and the greenhouse effect of methane is said to be about 25 times that of carbon dioxide. Also, it is known that 15% of the emitted methane gas is contained in the eructation (eructation: so-called belching) of living organisms, especially ruminants such as cows and goats. Therefore, in order to prevent global warming, there is a high demand for reducing methane gas derived from ruminants, and research is being advanced in various countries.

[0003] On the other hand, for pigs, since they are not ruminants, eructation is not a problem, but GHG is contained in the exhaust gas (so-called flatulence) and the gas derived from feces. Pork is consumed approximately twice as much as beef worldwide, and considering the number of individuals being raised, it is known that the total amount of GHG emitted in the production process is higher for pigs than for cows. Therefore, it can be said that the countermeasure against GHG derived from pigs is an urgent issue, but the current situation is that not much research has been conducted compared to cows and the like. Especially in pig houses, there is a problem that GHG emitted not only from living organisms but also from manure and compost is mixed, and it is difficult to specify and evaluate the GHG directly emitted from living organisms.

[0004] To control animal-derived GHGs, it is necessary to accurately monitor the emissions of GHGs from animals, such as methane and carbon dioxide. However, measuring methane and other gases emitted from exhaust fumes and feces in livestock farms is not easy, and accurate measurement methods have not been established. For ruminant animals, methods such as the chamber method, in which cattle and goats are kept in a completely enclosed chamber for several days, the mask method using green feed, in which GHGs are measured while animals are grazing and feeding in feed boxes, and the sniffer method, in which GHGs are measured in a semi-open room during milking and feeding, are known. However, none of these methods have been able to accurately and easily measure GHG emissions. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent No. 7402580 [Overview of the project] [Problems that the invention aims to solve]

[0006] The primary objective of this disclosure is to provide a gas emission measurement system and a gas emission measurement method capable of accurately measuring GHGs (particularly methane gas) directly emitted from living animals.

[0007] The second objective of this disclosure is to provide a method for evaluating the GHG reduction effect of substances that reduce GHGs (particularly methane gas) emitted from living animals.

[0008] Furthermore, by using the above GHG reduction effect evaluation methods as a screening test, it becomes possible to easily obtain evaluation results of the methane emission suppression effect of various feed additives without feeding animals.

[0009] Since directly measuring the amount of GHGs actually emitted from animals is time-consuming and costly, a method that allows for a simpler evaluation of GHG reduction effects across a wide range of substances is useful.

[0010] The third objective of this disclosure is to provide a methane-producing inhibitor that can be added to animal feed and reduces the amount of Methanobrevibacter species, which are methanogenic bacteria present in the intestines of animals, thereby suppressing methane production.

[0011] Furthermore, the description of these objectives and issues in this disclosure does not preclude the existence of other objectives and issues. Also, one aspect of this disclosure is not required to solve all of these issues. Moreover, it is possible to extract other issues from the description, drawings, and claims of this disclosure. [Means for solving the problem]

[0012] A bacterial quantification method according to Embodiment 1 of the present disclosure is a bacterial quantification method for quantifying Methanobrevibacter species contained in the rectal feces of an animal, comprising the steps of: collecting rectal feces from an animal; holding the rectal fecal sample in an anaerobic porter under anaerobic conditions; replacing the gas in the anaerobic porter with nitrogen gas, diluting the sample with a culture medium, and culturing the bacteria in a test tube; recovering the cultured bacteria; extracting DNA from the recovered bacteria; and quantifying the Methanobrevibacter species from the extracted DNA by quantitative PCR or next-generation sequencing analysis.

[0013] Furthermore, the bacterial quantification method according to Embodiment 2 of the present disclosure further includes, in the above embodiment, a step of feeding a first animal a first feed containing the test substance before the step of collecting rectal feces from the animal, and a step of feeding a second animal of the same species as the first animal a second feed not containing the test substance, wherein the step of quantifying the Metanobrevibacter species includes a step of quantifying the Metanobrevibacter species in bacteria collected from the rectal feces of the first animal to determine a first amount, a step of quantifying the Metanobrevibacter species in bacteria collected from the rectal feces of the second animal to determine a second amount, and a step of evaluating the increase or decrease of the Metanobrevibacter species due to the addition of the methane generation inhibitor based on the change in the first amount relative to the second amount. Here, the first animal and the second animal may be the same or different. Also, when the first animal and the second animal are the same, the period during which the first feed is fed and the period during which the second feed is fed do not overlap. That is, they are performed in different periods.

[0014] Furthermore, in any of the above embodiments, the bacterial quantification method according to Embodiment 3 of the present disclosure comprises, as an active ingredient, at least one of an organic acid, an organic acid salt, or an oligosaccharide.

[0015] Furthermore, the method for quantifying bacteria according to Embodiment 4 of the present disclosure, in any of the above embodiments, comprises an organic acid containing at least one of citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, and phosphoric acid.

[0016] Furthermore, the method for determining bacterial quantity according to Embodiment 5 of the present disclosure, in any of the above embodiments, comprises at least one of the following: citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, phosphate, and potassium, calcium, zinc, and magnesium salts thereof.

[0017] Furthermore, in any of the above embodiments, the method for determining bacterial quantity according to Embodiment 6 of the present disclosure comprises at least one of potassium diformate or sodium formate.

[0018] Furthermore, in any of the above embodiments, the bacterial quantification method according to Embodiment 7 of the present disclosure comprises, in any of the above embodiments, at least one of galactooligosaccharides or fructooligosaccharides.

[0019] Furthermore, the bacterial quantification method according to Embodiment 8 of the present disclosure is, in any of the above embodiments, an additive added to the feed of livestock (industrial animals), companion animals and exhibition animals, which are monogastric animals, such as pigs, dogs, cats, mammals, or birds.

[0020] Furthermore, a gas emission measurement method according to Embodiment 9 of the present disclosure is a gas emission measurement method for measuring the emission of greenhouse gases generated from animals, or from animal excrement or metabolites, and includes the steps of: placing an animal in a cage-like cage section that defines a breeding space for housing an animal inside; surrounding the cage section with a translucent cover section; connecting an air supply section through an air supply hole opened in a part of the surface constituting the cover section to continuously supply air into the cover section, and discharging a portion of the gas inside the cover section to the outside through a plurality of exhaust holes opened in another part of the surface constituting the cover section; connecting a detection section through a detection hole opened in yet another part of the surface constituting the cover section to begin pre-detection of at least greenhouse gases among the gases contained in the cover section; and ending the pre-detection when the amount of greenhouse gases pre-detected by the detection section has stabilized over time, and starting the main detection by the detection section. This makes it possible to accurately measure greenhouse gases emitted by animals such as livestock, which was previously difficult. In particular, by waiting for the gas inside the cover to reach equilibrium before starting detection, the accuracy of greenhouse gas measurement can be improved.

[0021] Furthermore, in any of the above embodiments, the gas emission measurement method according to Embodiment 10 of the present disclosure is performed 30 minutes or more after the detection unit has started detecting an animal in the cage, covering it with the cover unit, and starting to supply air with the air supply unit.

[0022] Furthermore, the method for measuring gas emissions according to Embodiment 11 of the present disclosure is, in any of the above embodiments, a method in which the animal is any one of livestock (industrial animals), companion animals, or exhibition animals, and is a non-ruminant monogastric animal. The monogastric animal is a mammal or a bird, and the mammals are, for example, pigs, dogs, and cats.

[0023] Furthermore, the method for evaluating a methane generation inhibitor according to Embodiment 12 of the present disclosure is a method for evaluating the effect of a test substance added to feed fed to an animal on the amount of greenhouse gas emissions emitted by the animal, the method comprising: a step of collecting rectal feces of an animal fed a basal diet not containing the test substance; a step of holding a sample of the rectal feces in an anaerobic port in an anaerobic state; a step of replacing the gas in the anaerobic port with nitrogen gas, diluting the sample of the rectal feces with a medium, and culturing bacterial flora in test tubes, with and without the addition of the test substance; a step of recovering the cultured bacterial flora; a step of extracting DNA from the recovered bacterial flora; and a step of evaluating an increase or decrease in Methanobrevibacter bacteria by a gene analysis method using quantitative PCR or next-generation sequence analysis from the extracted DNA. Thereby, a screening test capable of easily evaluating a methane generation inhibitor in a test tube becomes possible.

[0024] Furthermore, the greenhouse gas emission measurement system according to Embodiment 13 of the present disclosure is a greenhouse gas emission measurement system capable of measuring the greenhouse gas emissions generated from animals, or animal excrement or metabolites, and includes a cage-shaped cage part that defines a breeding space for accommodating animals inside, a cover part having translucency for surrounding the periphery of the cage part, an air supply part connected to the cover part surrounding the cage part for continuously supplying air into the cover part, a flow meter capable of measuring the amount of air supplied by the air supply part to the cover part, and a detection part capable of detecting at least greenhouse gases among the gases contained in the cover part. The cover part is provided with an air supply hole for connecting to the air supply part, a detection hole for connecting to the detection part, and a plurality of exhaust holes for discharging a part of the gas in the cover part to the outside on a part of the surface constituting the cover part. With the above configuration, it becomes possible to accurately measure the greenhouse gases emitted by animals such as livestock, which has been difficult in the past.

[0025] Furthermore, the greenhouse gas emission measurement system according to another embodiment of the present disclosure is, in any of the above embodiments, configured such that the inside of the cover part is maintained at a pressure higher than the outside. With the above configuration, by continuously supplying air from the air supply part into the cover part, the inside of the cover part can be maintained at a pressurized state higher than the outside, and by continuously discharging gas from the plurality of exhaust holes, it is possible to avoid the intrusion of outside air while providing an opening in the cover part, and to realize an environment for accurately measuring the greenhouse gases emitted by the animals in the breeding space. In particular, if the breeding space is sealed, the respiration of the animals will be hindered and growth will be difficult, while if it is opened, outside air will enter and it will be impossible to accurately measure the greenhouse gases emitted by the animals. On the other hand, with the above configuration, an environment that inhibits the intrusion of outside air can be realized with a simple configuration without adding cumbersome members such as valves through which gas flows only in one direction, while not sealing the cover part.

[0026] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 14 of the present disclosure has the plurality of exhaust holes opening on opposing sides of the cover portion. With this configuration, when gas is released from the cover portion to the outside in proportion to the amount of air supplied into the cover portion from the air supply portion, the gas is discharged from both sides of the cover portion, which has the advantage of enabling smooth gas discharge.

[0027] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 15 of the present disclosure is configured such that the plurality of exhaust holes, the intake holes, and the detection holes are arranged on the upper part of the opposing sides of the cover portion, at positions where they are spaced apart on each side.

[0028] Furthermore, in any of the above embodiments, the gas emission measurement system according to embodiment 16 of the present disclosure is configured such that the opening area of ​​the plurality of exhaust holes or detection holes is smaller than that of the air intake holes.

[0029] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 17 of the present disclosure has a diameter of 2 to 22 mm for the plurality of exhaust holes or detection holes.

[0030] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 18 of the present disclosure is configured such that the detection unit starts detection only when at least greenhouse gases among the gases contained in the cover are in a stable state. With this configuration, the accuracy of greenhouse gas measurement can be improved by waiting for the gas in the cover to reach equilibrium before starting detection.

[0031] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 19 of the present disclosure is configured such that the detection unit is an optical acoustic system.

[0032] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 20 of the present disclosure is such that the animal housed in the cage is a micro-miniature pig or a pig raised for food (piglet stage).

[0033] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 21 of the present disclosure comprises a cage section having a slatted floor section and a receiving section provided on the lower surface of the floor section. With this configuration, by making the floor section slatted as a double floor, animal feces and other waste can be collected in the receiving section below through the floor section, making it possible to keep the breeding space above the floor section clean.

[0034] Furthermore, in the gas emission measurement system according to embodiment 22 of the present disclosure, in any of the above embodiments, the cover portion is formed in the shape of a box with an open bottom, and a packing is provided at the open end of the bottom. With the above configuration, the cage portion is placed on the surface on which the gas emission measurement system is mounted, and the cover portion is placed over it so as to insert the cage portion from the open end, thereby easily surrounding the cage portion with the cover portion.

[0035] Furthermore, in any of the above embodiments, the gas emission measurement system according to Embodiment 23 of the present disclosure has a handle provided on a part of the opposing side of the cover. With this configuration, when covering the cage with the cover or removing the cover, workers can be positioned on the sides of the cover, and each person can grasp the handle provided on the side of the cover and lift the cover, making it easier to surround or remove the cage with the cover.

[0036] Furthermore, the methane emission inhibitor according to Embodiment 24 of the present disclosure is a methane emission inhibitor for addition to livestock feed, and contains at least one of an organic acid or organic acid salt, or an oligosaccharide as an active ingredient.

[0037] Furthermore, the methane generation inhibitor according to Embodiment 25 of this disclosure has a bacterial load suppression rate of 80% or more for Methanobrevibacter species in any of the above embodiments.

[0038] Furthermore, the methane generation inhibitor according to Embodiment 26 of this disclosure has a pH change of less than 1 before and after addition to the feed in any of the above embodiments.

[0039] Furthermore, in any of the above embodiments, the methane generation inhibitor according to Embodiment 27 of the present disclosure comprises at least one of the following organic acids: citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, phosphoric acid, and potassium, calcium, zinc, and magnesium salts thereof.

[0040] Furthermore, in any of the above embodiments, the methane generation inhibitor according to Embodiment 28 of the present disclosure comprises at least one of the following: citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, phosphoric acid, and potassium, calcium, zinc, and magnesium salts thereof.

[0041] Furthermore, in any of the above embodiments, the methane generation inhibitor according to Embodiment 29 of the present disclosure contains at least one of potassium diformate and sodium formate as the organic acid salt.

[0042] Furthermore, in any of the above embodiments, the methane generation inhibitor according to Embodiment 30 of the present disclosure comprises at least one of galactooligosaccharides or fructooligosaccharides as the oligosaccharide.

[0043] Furthermore, the methane emission inhibitor according to Embodiment 31 of the present disclosure is an additive added to the feed of livestock (industrial animals), companion animals, or exhibition animals, which are monogastric animals, such as pigs, dogs, cats, mammals, or birds, in any of the above embodiments. [Brief explanation of the drawing]

[0044] [Figure 1] This is a system configuration diagram of the gas emission measurement system according to Example 1. [Figure 2] Figure 1 is a perspective view showing the gas emission measurement device. [Figure 3] Figure 2 is a front view of the gas emission measurement device. [Figure 4] Figure 2 is an exploded perspective view showing the gas emission measurement device with the cover removed. [Figure 5] Figure 4 is a perspective view showing the cage section with the door open. [Figure 6] Figure 4 is a perspective view of the cover section, taken from a diagonal downward angle. [Figure 7] Figure 6 is a horizontal cross-sectional view along line VII-VII. [Figure 8] Figure 4 is a side view of the cover section. [Figure 9] This flowchart shows a method for quantifying methanogenic bacteria. [Figure 10] This graph shows the time course of the total bacterial count in each test substance. [Figure 11] This graph shows the time course of the amount of Methanobrevibacter bacteria in each test substance. [Figure 12] This graph shows the change in carbon dioxide concentration in Example 2. [Figure 13] This graph shows the change in methane concentration in Example 2. [Figure 14] This graph shows the relationship between body weight and carbon dioxide emissions in Example 2. [Figure 15] This graph shows the relationship between body weight and methane production in Example 2. [Figure 16] This graph shows the relationship between the average methane production and the percentage of Metanobrevibacter bacteria in Example 2. [Modes for carrying out the invention]

[0045] The embodiments of this disclosure will be described below with reference to the drawings. However, the embodiments shown below are examples for concretizing the technical concept of this disclosure, and this disclosure is not limited to the following. Furthermore, this specification does not limit the members shown in the claims to the members of the embodiments. In particular, the dimensions, materials, shapes, relative arrangements, etc. of the components described in the embodiments are not intended to limit the scope of this disclosure to those, unless otherwise specifically stated, but are merely illustrative examples. Note that the size and positional relationships of the members shown in each drawing may be exaggerated for clarity of explanation. Furthermore, in the following description, the same name and reference numeral indicate the same or similar members, and detailed explanations are omitted as appropriate. Furthermore, each element constituting this disclosure may be configured such that multiple elements are made of the same member, with one member serving multiple elements, or conversely, the function of one member may be shared among multiple members.

[0046] To accelerate efforts to reduce methane emissions from animal exhaust, aerosols, and feces, systems for monitoring methane gas under various conditions and measures to reduce methane emissions are needed. Currently, systems used for large animals such as cattle and other ruminants are not airtight, and there are no reported examples of greenhouse gas emission monitoring systems suitable for monogastric animals such as pigs. Furthermore, livestock feed currently fails to suppress methane gas emissions from livestock.

[0047] The inventors have developed a gas emission measurement system 1000 with the aim of clarifying the amount of GHGs emitted from animals. The overall configuration is shown in Figure 1, the external appearance of the main part, the gas emission measurement device 100, is shown in Figure 2, the front view is shown in Figure 3, and the exploded perspective view is shown in Figure 4. As shown in Figure 1, the gas emission measurement system 1000 comprises a gas emission measurement device 100, an air supply unit 30, a flow meter 40, and a detection unit 50. The gas emission measurement device 100 comprises a cage unit 10 and a cover unit 20. This gas emission measurement system 1000 is capable of measuring greenhouse gas emissions from animals while taking into consideration the animal welfare of the test animals, such as ensuring painless breathing.

[0048] The air supply unit 30 is connected to the cover unit 20 surrounding the cage unit 10 via a piping tube 70 or the like, and is a component that continuously supplies air into the cover unit 20 so that the animal AN inside the cage unit 10 can maintain respiration. A compressor or the like can be used for such an air supply unit 30. Also, as shown in Figure 1, a buffer unit 80 such as a bubbling device or trap may be interposed as needed. For example, ammonia can be removed by the adsorption removal effect of bubbling in water.

[0049] The flow meter 40 is a component capable of measuring the amount of air supplied by the air supply unit 30 to the cover unit 20. A mass flow meter or the like can be used for such a flow meter 40. A recorder 60 is also connected to the flow meter 40 as needed. The recorder 60 stores the flow rate data of the air supplied to the cover unit 20. A data logger can be suitably used for such a recorder 60.

[0050] The detection unit 50 is a component capable of detecting at least greenhouse gases among the gases contained within the cover unit 20. The detection unit 50 is connected to the detection hole 25 (details described later) of the cover unit 20 and detects the concentration of components contained in the air discharged from the cover unit 20 in real time. Such a detection unit 50 can utilize known sensors such as real-time gas monitors, photoacoustic systems, electrochemical sensors, semiconductor sensors, infrared absorption sensors, thermal conduction sensors, and mass spectrometers. Here, the concentration of GHGs such as methane and carbon dioxide is measured using a photoacoustic system. Preferably, it is also possible to measure nitrous oxide, ammonia, etc. (Cage section 10)

[0051] The cage section 10 is constructed in a cage-like shape, defining a breeding space inside for housing the animal AN. In examples such as Figure 2, it is formed in a rectangular box shape that extends in one direction. This cage section 10 functions as a cage (hereinafter referred to as the measurement cage) for sampling and analyzing the exhaled breath and feces / urine from the animal. Such a cage section 10 is made of a material that has sufficient strength to withstand the weight of the animal AN, such as stainless steel or other metal.

[0052] The size of the cage section 10 is designed according to the size of the animal AN to be housed in the rearing space. For example, when housing micro mini pigs and fattening piglets weighing 5 kg to 25 kg, the dimensions should be 800 mm to 1000 mm in length, 500 mm to 800 mm in width, and 500 mm to 900 mm in height. (Floor section 11)

[0053] As shown in Figures 3 and 4, the cage section 10 comprises a slatted floor section 11 and a receiving section 12 provided on the underside of the floor section 11. By making the floor section 11 a slatted structure as a double floor, animal feces and other waste accumulate in the receiving section 12 below through the floor section 11, thus creating a structure that separates feces and urine, and making it possible to keep the breeding space above the floor section 11 clean. The height of the floor section 11 should be high enough to cleanly separate feces and urine, for example, about 100 mm to 200 mm. The floor section 11 is made of a strong material such as a perforated plate material like perforated metal, metal mesh, or a resin slatted structure. (Door section 13)

[0054] The cage section 10 is provided with a door section 13 on its side that can be opened and closed. In the example shown in Figure 5, a pivot-type door section 13 is provided on one of the longitudinal end faces of the cage section 10. The door section 13 is opened to allow the animal AN to enter the enclosure, and the door section 13 is closed to lock it. The door section 13 may be provided with an opening / closing handle 14 or an opening / closing knob as needed. (Tray section 16)

[0055] Furthermore, the cage section 10 may be equipped with a tray section 16 for holding water and food. In the example shown in Figure 5, a tray holder section 15 for holding the tray section 16 is provided on the back side of the door section 13. The tray holder section 15 is formed in the shape of a circular cutout hole, and the bowl-shaped tray section 16 is inserted into the tray holder section 15 and secured so that it does not come off. Water is placed in each of the two tray sections 16. (Base part 17)

[0056] The cage section 10 may also be configured to be placed on a base section 17. In the examples shown in Figures 2 and 3, the cage section 10 is fixed to the upper surface of a table-shaped base section 17, which has legs 18 at its four corners. This allows necessary components to be placed at a higher level, improving work efficiency. The base section 17 is formed to be slightly larger than the bottom surface of the cage section 10, providing space around the cage section 10 for the open end of the cover section 20 to rest. (Cover section 20)

[0057] The cover portion 20 is a component that surrounds the cage portion 10. The cover portion 20 is formed in a box shape with an open bottom, and its interior is formed to be slightly larger than the cage portion 10, following the outer shape of the cage portion 10, so that the cage portion 10 can be housed inside. The cover portion 20 is also translucent. This makes it easier to see the animal AN being kept from the outside. This cover portion 20 is made of polycarbonate resin or the like.

[0058] As shown in Figure 6, the cover portion 20 is provided with a cushioning material 21 such as a packing at the open end of its lower surface. This makes it easy to surround the cage portion 10 with the cover portion 20 by placing the cage portion 10 on the surface on which the gas emission measurement device 100 is mounted and then placing the cover portion 20 over it, inserting the cage portion 10 from the open end.

[0059] Furthermore, the cover portion 20 is provided with handles 22 on parts of its opposing sides. In the example shown in Figure 4, U-shaped handles 22 are fixed to two locations on the left and right ends of the longitudinal end face. With this configuration, when covering the cage portion 10 with the cover portion 20 or removing the cover portion 20, workers can be positioned on each side of the cover portion 20, and each person can grasp the handles 22 provided on the side of the cover portion 20 and lift the cover portion 20, making it easier to surround or remove the cage portion 10 with the cover portion 20. (Fixed structure 19)

[0060] The cage portion 10, which is fixed to the upper surface of the base portion 17, is placed so as to be surrounded by the cover portion 20, and the cover portion 20 is secured with a fixing structure 19 to prevent it from coming off. In the example in Figure 2, the fixing structure 19 is a strip-shaped strap that fastens around the base portion 17 and the cover portion 20. The fixing structure is not limited to such a strip-shaped strap, and known connecting structures can be used as appropriate, such as a structure in which a flange portion is provided at the open end of the cover portion and the flange portion is clamped to the periphery of the base portion, or a structure in which a groove is provided in the base portion into which the open end of the cover portion is inserted and fixed by inserting a bolt or pin. (hole 23)

[0061] Furthermore, the cover portion 20 has openings 23 on a part of the surface that makes up the cover portion 20, which include an air intake hole 24, a detection hole 25, and multiple exhaust holes 26. The air intake hole 24 is used to supply air to the cover portion 20, and the detection hole 25 and exhaust holes 26 are used to exhaust air from the cover portion 20. The air intake hole 24 is a hole for connecting to the air supply unit 30. The detection hole 25 is a hole for connecting to the detection unit 50. These air intake hole 24 and detection hole 25 are provided with fittings 27 such as bulkhead unions and nipples for connecting to the piping tube 70.

[0062] It is preferable that the gas inside the cover section 20 be in equilibrium before the detection unit 50 begins detecting the gas contained within the cover section 20. That is, instead of placing the cage section 10 surrounded by the cover section 20 and immediately starting detection by the detection unit 50 after starting the air supply, the system waits until the gas concentration inside the cover section 20 stabilizes over time. Then, the detection unit 50 starts the actual detection once at least the carbon dioxide concentration has stabilized. By doing so, it is possible to avoid unstable measurement results due to measurements during transient periods when the air supply and circulation are unstable, and to expect high accuracy in measuring greenhouse gases. For example, after starting the air supply by the air supply unit 30, the system waits for about 30 minutes to 1 hour before starting measurement by the detection unit 50 and starting recording by a data logger or other recorder 60.

[0063] Furthermore, the GHG concentration of the incoming gas is measured and used as a baseline value. Specifically, to confirm the baseline value of the measurement gas before starting the measurement, the detection unit performs a pre-detection of the GHG concentration. Then, the generated GHG is calculated by subtracting the GHG concentration of the incoming gas from the concentration after a predetermined time (in this case, more than 1 hour) has elapsed since the animal was placed in the cage unit 10. In practice, the concentration of the supply gas is measured and used as the baseline value of the supply gas concentration, and the concentration of gas generated by the animal is obtained by subtracting the baseline value from the value measured after the animal is placed inside. The data used for analysis is the data after the predetermined time has elapsed.

[0064] Furthermore, while the measurement time by the detection unit 50 can be performed continuously for a long period, such as 24 hours, it is preferable to limit it to within 8 hours. By dividing the time for GHG measurement, it is possible to avoid the influence of methane gas generated by fecal fermentation and ammonia from urine, and to focus on the gases emitted by the animal AN. It is also expected that the effects of stress from being confined in the narrow cage section 10 for a long period of time can be reduced. For example, it is desirable to perform the measurement during a time when the animal AN's behavior is relatively stable, such as between 9 a.m. and 3 p.m., after feeding the animal AN during the day, taking into consideration the pig's circadian rhythm, and avoiding the sleep period.

[0065] The flow meter 40 is a component capable of measuring the amount of air supplied by the air supply unit 30 to the cover unit 20. A mass flow meter or the like can be used for such a flow meter 40. A recorder 60 is also connected to the flow meter 40 as needed. The recorder 60 stores the flow rate data of the air supplied to the cover unit 20. A data logger can be suitably used for such a recorder 60. (Exhaust port 26)

[0066] The exhaust holes 26 are holes for discharging a portion of the gas inside the cover 20 to the outside. Preferably, the exhaust holes 26 are opened on opposite sides of the cover 20. This has the advantage of allowing for smooth gas discharge when gas is released from the cover 20 to the outside in proportion to the amount of air supplied into the cover 20 from the air supply unit 30, by discharging from both sides of the cover 20. In the examples shown in Figures 7 and 8, a total of 12 holes are opened on each longitudinal side of the cover 20: 3 at the top and 3 at the bottom. The holes at the top and bottom are at the same height and are opened at equal intervals in the middle and on both sides. Of the 12 holes, 2 are designated as air supply holes 24 and detection holes 25, and the remaining 10 are designated as exhaust holes 26. By discharging from opposite sides, especially the longitudinal sides, the airflow inside the cover 20 can be made uniform.

[0067] In the example shown in Figure 8, of the two upper and lower holes 23 opened on the side of the cover portion 20, the upper hole 23 is designated as the air supply hole 24 or detection hole 25. The gas mainly generated is methane (0.66), which has a specific gravity lighter than air. By providing the air supply hole 24 on the upper side, the air is allowed to flow and settle inside the cover portion 20, creating an airflow and stirring the gas inside the cover portion 20. Also, since the gas mainly detected by the detection hole 25 is methane, which is lighter than air, providing the detection hole 25 on the upper side makes it easier to reliably detect methane.

[0068] Furthermore, as shown in the horizontal cross-sectional view of Figure 7, of the six openings (three at the top and three at the bottom of the figure), it is preferable to designate one of the corner openings (top left in Figure 7) as an air intake hole 24 and the remaining ones as exhaust holes 26. By using one of the multiple openings, one at the corner as an air intake hole 24 to pressurize air in, and the others as exhaust holes 26 to allow natural release, it is possible to mix and diffuse the methane generated by the animal AN without providing a forced stirring member such as a fan inside the cover 20. In addition, one of the multiple exhaust holes 26 (bottom center in Figure 7) can also be used as a detection hole 25, allowing for the detection of the components of the air stirred inside the cover 20 when it is exhausted. Note that the positions of the air intake holes 24, detection holes 25, and exhaust holes 26, as well as the number of exhaust holes 26, are not limited to the above example and should be appropriately designed according to the size of the cover 20, the type and weight of the animal AN to be housed, etc.

[0069] It is preferable to maintain a higher pressure inside the cover section 20 than outside. By continuously supplying air from the air supply section 30 to the cover section 20, the inside of the cover section 20 is pressurized higher than outside, and gas is continuously discharged from multiple exhaust holes. This allows for the opening of exhaust holes 26 in the cover section 20 while preventing outside air from entering, thus creating an environment in which greenhouse gases emitted by animals AN in the rearing space can be accurately measured. In particular, if the rearing space is sealed, the animals' respiration is hindered, making rearing difficult, while if it is left open, outside air enters, making it impossible to accurately measure greenhouse gases emitted by animals AN. In contrast, in this embodiment, the cover section 20 is a semi-closed space that is not sealed, and while multiple holes 23 are opened, an environment that prevents the intrusion of outside air can be easily achieved with a simple configuration without adding troublesome components such as check valves that allow gas to flow in only one direction.

[0070] The diameter of the hole 23 is preferably 2 mm to 22 mm. The air supply hole 24 is preferably 8 to 12 mm, more preferably 10 mm. On the other hand, the opening area of ​​the exhaust hole 26 and the detection hole 25 is preferably the same as or smaller than that of the air supply hole 24. By ensuring a sufficient amount of air supply by increasing the cross-sectional area, while keeping the exhaust hole 26 and the detection hole 25 small, it is possible to prevent the intrusion of outside air. Preferably, the diameter of each exhaust hole 26 and detection hole 25 is 2 mm to 10 mm, more preferably 2 mm to 6 mm, and even more preferably 4 mm. Furthermore, as shown in the enlarged cross-sectional view of Figure 7, the detection hole 25 is connected to the tube by connecting a fitting 27 to one of the exhaust holes 26. In addition, the air supply flow rate supplied from the air supply hole 24 is 20 to 30 L / min, preferably 23 to 28 L / min. (Method for measuring gas emissions)

[0071] Next, the procedure for measuring the greenhouse gas emissions emitted by an animal AN using the gas emission measurement system 1000 will be explained. First, the animal AN to be measured is placed in the rearing space of the cage section 10. Next, the cage section 10 is surrounded by a translucent cover section 20. In this state, the cover section 20 is fixed so that it does not come off the cage section 10.

[0072] On the other hand, the air supply unit 30 is connected to the air intake hole 24 opened in the cover unit 20, and the detection unit 50 is connected to the detection hole 25 via a piping tube 70 or the like. In this state, air is continuously supplied into the cover unit 20, and a portion of the gas inside the cover unit 20 is discharged to the outside from the cover unit 20 through a plurality of exhaust holes 26 opened in the cover unit 20.

[0073] Furthermore, the detection unit 50 pre-detects at least GHG from the gas contained within the cover unit 20. This state is maintained until the gas contained within the cover unit 20, particularly the carbon dioxide concentration, reaches a stable state. In other words, the detection unit 50 waits to record detections until the gas within the cover unit 20 reaches equilibrium. Here, the data from the first hour is not used in the analysis. By waiting for the gas within the cover unit 20 to reach equilibrium before starting the detection recording, transient fluctuations can be avoided, and the accuracy of greenhouse gas measurement can be improved. The waiting time is set until the time change in GHG concentration falls below a predetermined value. The waiting time is generally set according to the size of the cover unit 20 and the weight of the animal AN. For example, the waiting time can be set to 1 hour.

[0074] Then, after the carbon dioxide concentration value previously detected by the detection unit 50 has stabilized over time, the detection unit 50 begins the main detection process.

[0075] In this way, it becomes possible to accurately verify greenhouse gases emitted by livestock and other animals at the individual level, which was previously difficult. In particular, the accuracy of greenhouse gas measurement can be improved by waiting for the gas inside the cover 20 to reach equilibrium before starting detection. Furthermore, by using a simple configuration that does not completely seal the rearing space and leaves holes open without installing check valves, it becomes possible to analyze the gas inside and accurately measure gases emitted from animal breath, exhaust gases, skin gases, and feces.

[0076] Thus, the gas emission measurement method described herein is not limited to animal exhaust gases (so-called flatulence), but can also measure GHG emissions from excrement such as feces and urine, sweat and breath, and metabolites such as sebum. Some animals lack sweat glands. For example, cows sweat, but pigs do not. Regardless of the type of animal, the GHG emitted from the animal can be accurately measured using the gas emission measurement method and equipment. [Example 1]

[0077] The inventors hypothesized that reducing the amount of methanogenic bacteria in the intestinal flora of animals could reduce the amount of methane excreted by the animals, and therefore investigated a method to evaluate the effect of a substance that reduces the amount of methanogenic bacteria present in the rectal feces of animals.

[0078] Specifically, the objective is to quantify the amount of methanogenic bacteria in animal rectal feces by adding a test substance to the rectal feces in a test tube that replicates the environment of the digestive tract, and to observe the changes in the bacterial count. (Method for quantifying methanogenic bacteria)

[0079] The procedure for this quantitative method will be explained based on the flowchart in Figure 9.

[0080] First, in step S1, a rectal stool sample is collected from the target animal.

[0081] Next, in step S2, the rectal stool sample is held in an anaerobic porter. Here, the collected rectal stool is placed in an anaerobic porter and transported to the laboratory.

[0082] Furthermore, in step S3, the sample is diluted with culture medium and the bacteria are cultured in a test tube. Here, the fecal sample is diluted 20 to 50 times (40 times in this case) with diluent in an anaerobic chamber, and then added to a pressurized culture test tube containing culture medium to finally create a culture medium diluted 200 to 500 times (400 times in this case). Brainheart Infusion was used as the culture medium. The culture medium was prepared in a pressurized culture test tube, purged with nitrogen using a gas exchange device, and then autoclaved before use. Culture in the test tubes was carried out at 37°C.

[0083] Next, in step S4, the cultured bacteria are harvested. Here, harvesting is performed at 48 hours and 72 hours after the start of cultivation.

[0084] Then, in step S5, DNA is extracted from the recovered bacteria.

[0085] Finally, in step S6, the gut microbiota is analyzed using quantitative PCR or next-generation sequencing, and the amount of methanogenic bacteria is quantified from the extracted DNA. (Identification of methanogenic bacteria in the gut microbiota of animals)

[0086] Next, since the methane contained in the exhaust gas (so-called flatulence) of microminiature pigs is produced by methanogenic bacteria in the lower digestive tract of microminiature pigs, we focused on methanogenic bacteria and performed an analysis of the gut microbiota following the procedure described above. The relative abundance of methanogenic bacteria found in the gut microbiota is shown in Table 1 below.

[0087] [Table 1]

[0088] Analysis of the gut microbiota revealed that at most five genera of methanogenic bacteria were detected. Among these, the genus Methanobrevibacter was the most abundant, accounting for 1.233 ± 0.421%.

[0089] Therefore, it was found that quantifying the amount of Methanobrevibacter bacteria is appropriate in order to confirm the effect of the test substance on reducing the amount of methanogenic bacteria. (Screening of candidate substances)

[0090] Next, using the quantitative method described above, a screening test was conducted to select substances (candidate substances) that, when added to the feed given to animals, have the potential to reduce the amount of methane contained in the exhaust gas (so-called flatulence) from those animals.

[0091] Specifically, by feeding different animals a test diet with the test substance added and a basic diet without the test substance, and comparing the amount of metanobrevibacter bacteria in the rectal feces of the first animal fed the test diet with the amount of metanobrevibacter bacteria in the rectal feces of the second animal fed the basic diet, the effect of the test substance in suppressing metanobrevibacter bacteria can be evaluated. Alternatively, the same animal can be fed the basic diet for a first period and the test diet for a second period that does not overlap with the first period, and the effect of the test substance in suppressing metanobrevibacter bacteria can be evaluated by comparing the amount of metanobrevibacter bacteria in the rectal feces during each period. (Selection of candidate substances)

[0092] Culture tests were conducted on Methanobrevibacter bacteria present in the rectal feces of microminiature pigs. Using rectal feces from microminiature pigs fed a basic diet, the amount of Methanobrevibacter bacteria in the culture medium after culturing in a culture medium without the addition of each test substance was set as the baseline (100%). The change in the amount of Methanobrevibacter bacteria in the culture medium after culturing in a culture medium with each test substance added is shown, and the decrease from 100% is defined as the inhibition rate. For example, when the bacterial amount becomes 20%, the inhibition rate is 80%.

[0093] As test substances, we used organic acids, organic acid salts, oligosaccharides, polysaccharides, and oligosaccharides (all at a concentration of 0.5%) that are approved as feed additives. Specifically, we used seven types of organic acids: formic acid, acetic acid, propionic acid, butyric acid, lactic acid, citric acid, and sorbic acid; six types of organic acid salts: sodium formate, sodium acetate, sodium propionate, sodium butyrate, potassium diformate, and potassium formate; two types of oligosaccharides: galactooligosaccharide (product name: Oligomate) and fructooligosaccharide (product name: Meioligo); and one type of polysaccharide: indigestible dextrin (product name: Fibersol). The inhibition rate for each test substance and the pH change of the culture are shown in Table 2.

[0094] [Table 2]

[0095] The results above suggest that organic acids, their organic acid salts, or oligosaccharides are effective as methane production inhibitors to be added to animal feed. Specifically, citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, and lactic acid were shown as organic acids; sodium formate and potassium diformate were shown as organic acid salts; and lactoligosaccharides and fructooligosaccharides were shown as oligosaccharides, all demonstrating high inhibitory rates of over 80% against Metanobrevibacter species.

[0096] On the other hand, it was confirmed that dextrin does not have an inhibitory effect on methanobrevibacter bacteria.

[0097] Furthermore, citric acid, sorbic acid, galactooligosaccharides, and fructooligosaccharides lowered the pH of the culture medium. On the other hand, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, citric acid, sodium formate, and potassium diformate did not lower the pH. (Verification of factors affecting changes in the genus Metanobrevibacter)

[0098] Figures 10 and 11 show the changes in the bacterial count of Methanobrevibacter species in the rectal feces of microminiature pigs cultured in either an additive-free culture medium or a culture medium to which one of the test substances, galactooligosaccharide, fructooligosaccharide, or dextrin, was added. At the start of the culture test, the pH of the culture was 6.0 or higher.

[0099] Figure 10 shows the time course of the total bacterial count in each test substance, with the total bacterial count at time 0 set to 1.

[0100] Figure 11 shows the time course of the amount of Methanobrevibacter bacteria, with the bacterial load at 0 hours set as the baseline (100%). The decrease from 100% is defined as the inhibition rate. For example, when the bacterial load falls to 20%, the inhibition rate is 80%.

[0101] In all test substances used for cultivation, the overall bacterial count increased up to 72 hours, while the number of Methanobrevibacter bacteria decreased. This confirms that the candidate methane-producing inhibitors specifically affect the number of Methanobrevibacter bacteria. [Example 2] (Accurate measurement of GHG emissions directly from living animals)

[0102] Using the gas emission measurement system 1000, four littermate micro-miniature pigs weighing approximately 10-15 kg were used to conduct GHG emission measurement tests for each animal, clarifying the amount of GHG emitted from the pigs. In this study, the cage section 10 was set to a size of 84 cm x 59 cm x 64 cm, large enough to accommodate one micro-miniature pig weighing up to approximately 15 kg. (1) Real-time measurement of GHG using a measurement chamber

[0103] First, as preparation, the changes in the concentration of each gas inside the measurement chamber were investigated. First, one micro mini pig was placed in each cage section 10 and enclosed in the case section, and each was kept for approximately 6 hours. Ventilation volume [m³ 3 The flow rate [mg / m³ / h] was measured using a flow meter 40, specifically a mass flow meter. The ventilation rate and the components of the supply and exhaust gases, specifically the concentrations of methane and carbon dioxide [mg / m³ / h], were measured. 3 The gas was analyzed every two minutes. Based on the ventilation rate and each concentration, the amount of carbon dioxide and methane generated every two minutes was calculated. Furthermore, the total amount for one hour was taken as the amount of carbon dioxide and methane generated for each hour. Here, (gas generation per unit time) = (gas concentration in exhaust air - gas concentration in inlet air) × ventilation rate. The supply air flow rate from the air intake port 24 was set to approximately 25 L / min. (Waiting time)

[0104] Examples of gas concentration changes in the above test are shown in the graphs in Figures 12 and 13. Figure 12 shows the change in carbon dioxide concentration [mg / m³]. 3 Figure 13 shows the change in methane concentration [mg / m³], confirming that the carbon dioxide concentration stabilized approximately one hour after the start.3 The results showed that the methane concentration fluctuated, increasing and decreasing repeatedly. Based on these results, it is thought that stable data can be obtained by setting the waiting time to approximately 1 hour. (Administration of test substance and measurement items)

[0105] First, before administering the basic feed, each microminiature pig will be fed the basic feed and raised for at least two weeks. A first GHG measurement will be performed and a first rectal stool sample collected before administration begins. The weight of the microminiature pigs will also be measured. The same procedures will be followed for all measurements after administration begins.

[0106] Next, administration of the test feed will begin. Two weeks after the start of administration, a second measurement will be taken, and a second rectal stool sample will be collected.

[0107] Finally, the basic feed is resumed. After the end of administration, a third measurement and rectal stool collection are performed as post-measurements. (Relationship between bio-derived carbon dioxide and methane emissions and body weight)

[0108] First, Figures 14 and 15 show graphs plotting body weight on the horizontal axis and GHG levels per hour measured in the test on the vertical axis. In these figures, Figure 14 shows carbon dioxide emissions, and Figure 13 shows methane emissions. From Figure 14, the correlation coefficient between body weight and carbon dioxide emissions is 0.71, indicating a statistically strong correlation. On the other hand, from Figure 15, the correlation coefficient between body weight and methane emissions is -0.24, indicating no correlation. From these results, a strong positive correlation was confirmed between carbon dioxide emissions and the body weight of micro-miniature pigs, indicating that the amount of carbon dioxide emissions from living organisms depends on body weight. On the other hand, there is almost no correlation between methane emissions and body weight, indicating that methane emissions do not depend on body weight. (Correlation between methane production and the rate of suppression of metanobrevibacter bacteria)

[0109] Figure 16 shows the results of plotting the amount of methane produced per hour, measured in an animal GHG emission measurement test, on the x-axis and the percentage of Methanobrevibacter bacteria in the intestines on the y-axis. The correlation coefficient was 0.688, indicating a statistically significant correlation. This clearly shows a positive correlation between the amount of methane produced per hour and the percentage of Methanobrevibacter bacteria in the intestines.

[0110] In other words, the amount of methane produced by living organisms depends on the proportion of Methanobrevibacter bacteria in the digestive tract. Therefore, it has been found that suppressing Methanobrevibacter bacteria in the digestive tract may reduce methane production from living organisms.

[0111] By using this method for evaluating methane production suppression effects to screen for substances that may reduce the amount of Methanobrevibacter bacteria, and then using those substances as test materials to perform a correlation evaluation between methane production and Methanobrevibacter bacteria occupancy in microminiature pigs, methane production suppressants can be efficiently identified. On the other hand, directly measuring GHG levels by feeding animals candidate methane-producing inhibitors to verify the effect of reducing Methanobrevibacter bacteria in animal rectal feces is time-consuming and labor-intensive. Therefore, a simpler screening method has been established to evaluate the GHG-inhibiting effect of methane-producing inhibitors by analyzing the fecal microbiota measured by in vitro culture. [Industrial applicability]

[0112] The gas emission measurement system and method, methane emission inhibitor and its evaluation method, and bacterial quantification method described herein can accurately measure the emissions of greenhouse gases such as methane and carbon dioxide produced by animals and accurately evaluate the effectiveness of methane emission inhibitors. Furthermore, the methane emission inhibitor can be used as a feed additive with a high methane emission inhibitory effect. As a result, it can contribute to the reduction of emissions of carbon dioxide and methane, which has a higher greenhouse effect, and thus contribute to avoiding global warming.

[0113] As a result of reducing the environmental impact in this way, it contributes to the following of the 17 goals and 169 targets set forth in the SDGs (Sustainable Development Goals), which were adopted at the UN Summit in September 2015. Regarding "13. Take concrete action to address climate change" and "13.2 Incorporate climate change measures into national policies, strategies and plans," these measures will serve as practical means to achieve the goal of mitigating climate change and its impacts around the world caused by global warming due to greenhouse gas emissions. In particular, it will be possible to reduce emissions of methane, which has a greenhouse effect 25 times higher than carbon dioxide, thereby curbing global warming. Furthermore, reducing GHG emissions from pigs will enhance the mitigation effect on global warming. Regarding "11. Make cities and human settlements inclusive, safe, resilient and sustainable," "11.6 By 2030, reduce the adverse per capita environmental impacts of cities, including by paying special attention to air quality and the management of general and other wastes," and "11.7 By 2030, provide universal access to green spaces and public areas that are safe, inclusive and accessible to all, including women, children, older persons and persons with disabilities," these measures can contribute to reducing greenhouse gas emissions and mitigating global warming and the urban heat island effect. • "8. Decent Work and Economic Growth," "8.4 By 2030, progressively improve resource efficiency in global consumption and production and, led by developed countries, decouple economic growth from environmental degradation in accordance with the 10-Year Framework of Programmes on Sustainable Consumption and Production." • "9. Build resilient infrastructure, promote inclusive and sustainable industrialization, and foster industry and innovation." "9.4 By 2030, improve sustainability by upgrading infrastructure and industries through increased resource efficiency and the adoption of clean and environmentally friendly technologies and industrial processes. All countries will take action according to their respective capabilities."

[0114] As described above, this disclosure is a technology that contributes to achieving these goals and targets. [Explanation of Symbols]

[0115] 1000... Gas emission measurement system 100...Gas emission measuring device 10... Cage section 11…Floor part 12... Receiving part 13... Door section 14…Opening / closing grip 15...Tray holding section 16...Tray section 17…Base 18...legs 19…Fixed structure 20...Cover part 21...Cushioning material 22...Handle 23...hole 24…Air intake vent 25…Detection hole 26… Exhaust port 27... Fittings 30...Air supply unit 40...Flowmeter 50...Detection unit 60... Recorder 70... Piping tubes 80... Buffer section AN…animal

Claims

1. A method for quantifying Methanobrevibacter species contained in the rectal feces of a monogastric animal which is a mammal or bird selected from pigs, dogs, or cats, The process of collecting rectal feces from monogastric animals, The process involves holding the rectal stool sample in an anaerobic porter under anaerobic conditions, The process involves replacing the gas in the anaerobic porter with nitrogen gas, diluting the sample with a culture medium, and culturing the bacterial flora in a test tube. The process of recovering the cultured bacterial flora, The process involves extracting DNA from the recovered bacterial flora, A step of quantifying the Methanobrevibacter species from the extracted DNA by quantitative PCR or next-generation sequencing analysis, This includes, and furthermore, before the step of collecting the rectal feces of the monogastric animal, The process involves feeding the first animal with a first feed containing the test substance, A step of feeding a second animal of the same species as the first animal with a second feed that does not contain the test substance, Includes, The step of quantifying the Methanobrevibacter species is, A step of determining the first quantity by quantifying the Methanobrevibacter species in bacteria collected and cultured from the rectal feces of the first animal, The process involves determining the second quantity by quantifying the Methanobrevibacter species in bacteria collected and cultured from the rectal feces of the second animal, A step of evaluating the increase or decrease of the Methanobrevibacter species due to the addition of a methane generation inhibitor based on the variation of the first amount relative to the second amount, Includes, A method for determining bacterial count, wherein the test substance is an additive added to the feed of a mammal or bird selected from pigs, dogs, or cats.

2. A method for determining bacterial quantity according to claim 1, A method for quantifying bacteria, wherein the test substance contains at least one of an organic acid, an organic acid salt, or an oligosaccharide as an active ingredient.

3. A method for determining bacterial quantity according to claim 2, A method for determining the number of bacteria, wherein the organic acid comprises at least one of citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, and lactic acid.

4. A method for determining bacterial quantity according to claim 2, A method for determining bacterial quantity, wherein the organic acid salt comprises at least one of the salts of potassium, calcium, zinc, or magnesium of citric acid, sorbic acid, formic acid, acetic acid, propionic acid, butyric acid, or lactic acid.

5. A method for determining bacterial quantity according to claim 2, A method for determining bacterial quantity, wherein the organic acid salt comprises at least one of potassium diformate or sodium formate.

6. A method for determining bacterial quantity according to claim 2, A method for quantifying bacteria, wherein the oligosaccharide comprises at least one of galactooligosaccharides or fructooligosaccharides.

7. A method for evaluating the methane emission suppression ability of a test substance added to feed given to a monogastric animal, which is a mammal or bird selected from pigs, dogs, or cats, to evaluate the effect of the greenhouse gas emissions emitted by the monogastric animal, A step of collecting rectal feces from monogastric animals fed a basic diet that does not contain the aforementioned test substance, The process involves holding the rectal stool sample in an anaerobic porter under anaerobic conditions, The process involves replacing the gas in the anaerobic porter with nitrogen gas, diluting the rectal stool sample with a culture medium, and culturing the bacterial flora in test tubes with and without the test substance added. The process of recovering the cultured bacterial flora, The process involves extracting DNA from the recovered bacterial flora, A step of evaluating the increase or decrease of Methanobrevibacter species from the extracted DNA by quantitative PCR or next-generation sequencing analysis, A method for evaluating methane emission suppression ability, including the ability to suppress methane generation.

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