Method for estimating methane gas generation amount from ruminant stomach of ruminant

Abstract

To provide a methane gas generation amount estimation method in which a gastric juice of ruminant is cultured to properly estimate a methane generation amount from a stomach of a target individual.SOLUTION: A methane gas generation amount estimation method includes: culturing a ruminant gastric juice collected from a first stomach of a ruminant individual in the presence of a culture substrate in a culture bottle for a predetermined time; measuring total gas quantity (mL / gDM) per culture substrate (g), generated from a culture fluid of the ruminant gastric juice until the elapse of the predetermined time and a methane gas molar ratio HSCH4 in a gas collected from an air layer in the culture bottle at the elapse of the predetermined time; and estimating a methane gas generation amount MP (mL / gDM) per culture substrate (g) from the total gas quantity (mL / gDM) and the methane gas molar ratio HSCH4, in which the predetermined time is selected in a range of 4 hours or more and 8 hours or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for estimating the amount of methane gas produced from the rumen of a ruminant. [Background technology]

[0002] There are two main methods for measuring methane generated from the rumen: one is to directly measure the methane contained in the breath of a target individual such as a dairy cow, and the other is to collect rumen fluid from the target individual, culture it under certain conditions, and then measure the methane contained in the gas generated (Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Animal Feed Science and Technology 174,2012,p190-200 [Non-patent document 2] J.Dairy Sci.100,2017,p8881-8894 Summary of the Invention [Problem to be solved by the invention]

[0004] According to the method of culturing ruminal fluid collected from a subject, as described in Non-Patent Documents 1 and 2, it is simple and convenient because it does not require equipment for collecting breath from the subject. However, methods for appropriately evaluating differences in methane gas production due to individual differences, differences in rearing conditions, etc. have not yet been fully explored. The present invention has been made in consideration of the above circumstances, and provides a method for estimating the amount of methane gas produced by culturing ruminal fluid and appropriately estimating the amount of methane produced from the stomach of a subject individual. [Means for solving the problem]

[0005] In order to achieve the above object, the present invention employs the following configuration. [1] Ruminal fluid collected from the first stomach of a ruminant animal is cultured in a culture bottle in the presence of a culture substrate for a predetermined period of time, and the total amount of gas (mL / gDM) generated from the ruminal fluid culture solution per g of culture substrate until the predetermined period of time has elapsed and the molar ratio of methane gas (HSCH4) in the gas collected from the gas layer in the culture bottle at the predetermined period of time are measured; A method for estimating the amount of methane gas generated, which estimates the amount of methane gas generated per culture substrate (g) MP (mL / gDM) from the obtained total gas amount GP (mL / gDM) and the methane gas molar ratio HSCH4, A method for estimating the amount of methane gas generated, characterized in that the predetermined time is selected within a range of 4 hours to 8 hours. [2] The methane gas molar ratio HSCH4 is determined by analyzing gas collected from the gas layer in the culture bottle after the predetermined time has elapsed by gas chromatography; The method for estimating methane gas generation amount described in [1], wherein the value is calculated based on the following formula (1). HSCH4=S CH4 / (S CH4 +S CO2 ) ···(1) However, in equation (1), S CO2 is the mole fraction calculated from the peak area of ​​carbon dioxide in the obtained gas chromatogram, S CH4 is the mole fraction calculated from the peak area of ​​methane in the obtained gas chromatogram. [Effects of the Invention]

[0006] According to the method of estimating the amount of methane gas produced from the ruminant stomach of a ruminant of the present invention, the amount of methane produced from the stomach of a target individual can be appropriately estimated by culturing rumen fluid. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram illustrating an example of a collection device. [Figure 2] FIG. 10 is a schematic diagram showing another example of a collection device. [Figure 3] 3 is a schematic diagram showing the collection device of FIG. 2 with the switching valve switched. FIG. [Figure 4] 1 is a graph showing the cumulative total gas volume GP (mL / gDM) generated per culture substrate (g) over time in each culture bottle containing diluted rumen fluid. [Figure 5] 1 is a graph showing the cumulative amount of methane gas generated over time (MP (mL / gDM) per g of culture substrate) in each culture bottle containing diluted rumen fluid. [Figure 6] 1 is a graph showing the relationship between the estimated concentration of methane gas based on saturated fatty acids in ruminal fluid and the amount of methane gas generated MP estimated based on the culture medium of ruminal fluid. DETAILED DESCRIPTION OF THE INVENTION

[0008] The method for estimating the amount of methane gas produced from the ruminant rumen of this embodiment is a method for estimating the amount of methane gas produced from the ruminant rumen, MP (mL / gDM), by culturing rumen fluid collected from the first stomach of an individual ruminant in a culture bottle in the presence of a culture substrate for a predetermined period of time and analyzing the cultured rumen fluid.

[0009] <Method for collecting ruminal fluid> To collect rumen fluid, a fasting period is required prior to collection to eliminate the influence of the feed intake immediately before collection. The fasting period depends on the type and amount of feed consumed last, but it is generally recommended to fast for 30 minutes, preferably 1 hour, before collection.

[0010] Ruminal fluid can usually be collected using a collection device shown in Fig. 1. The device in Fig. 1 includes a sonde 10, a collection bottle 20, and a pump 30. It is also roughly composed of a liquid flow path 41 whose upstream end is connected to the sonde 1 and whose downstream end is inserted up to near the bottom of the collection bottle 20, and a liquid flow path 42 whose upstream end is inserted up to the top of the collection bottle 20 and whose downstream end is connected to the pump 30.

[0011] The sonde 10 is a gastric juice collector that is inserted into the stomach of a ruminant. For animals weighing approximately 400 kg or more or aged 12 months or more, a commercially available gastric juice collector (for example, Luminar manufactured by Fujidaira Kogyo Co., Ltd.) can be used as is. For nursing and growing cattle that do not meet these requirements, use an appropriate type of rubber tube, and use a plastic pipe with a thick tube inserted through it to prevent the cow from biting through. In addition, a weight may be attached near the tip of the sonde 10 so that the sonde 10 can easily fall into the liquid layer after reaching the solid phase portion in the upper part of the first stomach.

[0012] The collection bottle 20 is a container for collecting gastric juice for culturing. A typical glass reagent bottle (100 ml to 500 ml capacity) is suitable for the collection bottle 20, but a thermos-type bottle with a vacuum layer is preferable. Filling the collection bottle 20 with warm water at about 45°C in advance and discarding the warm water immediately before collecting gastric juice makes it easier to keep the collected stomach warm.

[0013] The pump 30 is designed to suck gas from the air layer in the collection bottle 20. Any pump that can suck gas can be used as the pump 30, but it is preferable that the pump is capable of discharging gas in addition to suction. By being capable of discharging gas, it is possible to remove feed particles adhering to the sonde 10 during suction. For example, a bicycle pump that can also suck gas can be used.

[0014] To collect rumen fluid using the collection device shown in Figure 1, first insert the probe 10 into the rumen of a ruminant and wait about 5 seconds after the probe 10 reaches the rumen. The reason why gastric fluid is not collected immediately after the probe 10 reaches the rumen is to wait for the probe 10 to pass through the solid phase in the upper part of the rumen and fall into the liquid phase. Thereafter, a suction operation is performed by the pump 30. This causes gastric juice to be sucked up from the sonde 10 and collected in the collection bottle 20. Note that a short discharge operation may be performed as appropriate between suction operations to prevent the sonde from being clogged with feed particles.

[0015] Because gastric juice contains a large amount of saliva at the start of collection, the suction operation by the pump 30 is temporarily stopped, the collection bottle 20 is removed, and the gastric juice is discarded. The timing to stop the suction operation is determined by the color and viscosity of the collected liquid. Generally, the longer it takes to insert the sonde 10, the more likely it is that saliva will be mixed in. Therefore, to improve workability, it is preferable to insert the sonde 10 smoothly.

[0016] After discarding the gastric juice containing a large amount of saliva from the collection bottle 20, the collection bottle 20 is reattached and a suction operation is performed using the pump 30. This allows the gastric juice to be collected into the collection bottle 3 for culture. Note that even at this time, a short discharge operation may be performed between suction operations as appropriate to prevent the feed particles from clogging the probe. Alternatively, a different collection bottle 20 may be prepared in advance to replace the first collection bottle 20 from which gastric juice containing a large amount of saliva was collected, and this may be replaced when the collection bottle 20 is detached.

[0017] Ruminal fluid can also be collected by a collection device shown in Figures 2 and 3. The device in Figures 2 and 3 includes a sonde 1, a switching valve 2, a collection bottle 3, a second bottle 4, and a pump 5. It is also roughly composed of a liquid flow path 11 connecting the sonde 1 and the switching valve 2, a liquid flow path 12 connecting the second bottle 4 and the switching valve 2, a liquid flow path 13 connected to the switching valve 2 and inserted up to near the bottom of the collection bottle 3, a liquid flow path 14 connected to the switching valve 2 and inserted up to the top of the collection bottle 3, and a gas flow path 15 connected to the pump 5 and inserted up to the top of the second bottle 4.

[0018] The sonde 1 is a gastric juice collector that is inserted into the stomach of a ruminant, and its desired specifications are the same as those of the sonde 10 in FIG. The switching valve 2 has four ports, each oriented at a 90-degree angle, and is configured to switch between a state in which liquid flow path 11 is connected to liquid flow path 12 and liquid flow path 13 is connected to liquid flow path 14, as shown in Figure 2, and a state in which liquid flow path 11 is connected to liquid flow path 13 and liquid flow path 14 is connected to liquid flow path 12, as shown in Figure 3. The collection bottle 3 is a container for collecting gastric juice for culturing, and the desired specifications are the same as those of the collection bottle 20 in FIG.

[0019] The second bottle 4 is a container for collecting gastric juice containing a large amount of saliva at the start of collection (gastric juice that is unsuitable for culture) and gastric juice that overflows from the collection bottle 3. The second bottle 4 also serves to prevent gastric juice from entering the pump 5. The pump 5 is configured to suck gas from the gas space in the second bottle 4. The specifications desired for the pump 5 are the same as those for the pump 30 in FIG.

[0020] To collect rumen fluid using the collection device shown in Figures 2 and 3, first insert the probe 1 into the rumen of a ruminant and wait about 5 seconds after the probe 1 reaches the rumen. The reason why gastric fluid is not collected immediately after the probe 1 reaches the rumen is to wait for the probe 1 to pass through the solid phase in the upper part of the rumen and fall into the liquid layer.

[0021] Thereafter, a suction operation is performed by the pump 5 in the state shown in Figure 2. As a result, negative pressure is created inside the second bottle 4, and gastric juice is sucked up from the sonde 1 connected to the second bottle 4 via the switching valve 2 and collected in the second bottle 4. In other words, gastric juice containing a large amount of saliva at the start of collection can be collected in the second bottle 4. Note that a short discharge operation may be performed as appropriate between suction operations to prevent the feed pieces from clogging the sonde.

[0022] After that, the switching valve 2 is switched to the state shown in Figure 3, and the pump 5 performs the suction operation. The timing of the switch is determined by the color and viscosity of the collected liquid. Generally, the longer it takes to insert the sonde 1, the more likely it is that saliva will be mixed in. Therefore, to improve workability, it is preferable to insert the sonde 1 smoothly.

[0023] When negative pressure is created inside the second bottle 4 by performing a suction operation with the pump 5 in the state shown in Figure 3, negative pressure is also created inside the collection bottle 3 connected to the second bottle 4 via the switching valve 2, and gastric juice is sucked up from the sonde 1 connected to the collection bottle 3 via the switching valve 2 and collected in the collection bottle 3. In other words, gastric juice to be used for culture can be collected in the collection bottle 3. Note that, even in this case, a short discharge operation may be performed as appropriate between suction operations to prevent the feed pieces from clogging the sonde.

[0024] When the gastric juice in the collection bottle 3 reaches the bottom end of the liquid flow path 14, the gastric juice collected thereafter overflows from the liquid flow path 14, passes through the switching valve 2 and the liquid flow path 12, and is introduced into the second bottle 4, without damaging the pump 5. Once it is confirmed that overflow has occurred, the pump 5 is stopped, and the collection of gastric juice is completed.

[0025] It is preferable to collect gastric juice at 80% or more, and preferably 90% or more, of the internal volume of the collection bottle 3. This prevents the collected gastric juice from coming into contact with the air layer. The upper limit of the amount of gastric juice that can be collected is determined by the lower end position of the liquid flow path 14, so the liquid flow path 14 should be positioned so that the volume of the collection bottle 3 above its lower end is 20% or less, and preferably 10% or less, of the total volume of the collection bottle 3.

[0026] In the devices shown in Figures 2 and 3, the gastric juice introduction destination can be switched from the second bottle 4 to the collection bottle 3 simply by operating the switching valve 2. In other words, there is no need to interrupt the collection process, which occurs when gastric juice is collected using a normal device such as that shown in Figure 1, so gastric juice collection can be carried out quickly and it is easier to collect gastric juice suitable for culture.

[0027] Once the collection of gastric juice is complete, an inert gas is immediately blown into the gas space of the collected collection bottle 20 or 3 to prevent the gastric juice from coming into contact with oxygen. Carbon dioxide (purity 99% or higher) is preferred as the inert gas. Carbon dioxide is readily available in small containers that can be brought to the collection site and is easy to handle. Furthermore, it is preferable that the collected collection bottle 20 or collection bottle 3 is kept warm using a cooler box or a polystyrene foam box, and kept sufficiently warm until it is used for culturing.

[0028] <Ruminal fluid culture method> The gastric juice collected in the collection bottle 20 or 3 contains minute feed particles, which are removed by filtration using gauze prior to culture. The gauze may be made of cotton or nonwoven fabric. To ensure that feed particles in the collected gastric juice are captured, it is preferable to use four layers of cotton and two or three layers of nonwoven fabric.

[0029] The filtered gastric juice can be used for culture as is, or it can be mixed with an equal volume of artificial gastric juice made from 5mM-10mM phosphate buffer with added sodium bicarbonate. The substrate is the same as the one given to the ruminant at the time. It is dried, crushed, and sieved beforehand, and a fixed amount (0.5-1.0% by weight) is added to the culture bottle. Nitrogen gas is then sprayed to remove oxygen from the air space, and the bottle is then sealed. If it is desired to examine the average tendency of multiple ruminant animals, equal amounts of gastric juice collected from multiple animals may be mixed.

[0030] During the culture, the culture bottle is kept warm while being shaken. A shaking constant temperature water bath or a shaking incubator with a constant temperature air bath may be used. The culture temperature is preferably 39 to 41°C, more preferably 39 to 40°C. The incubation time should be between 4 and 8 hours. The inventors have confirmed that incubation for 4 hours or more produces a sufficient amount of methane gas. Furthermore, if incubation is continued for more than 8 hours, the differences between specimens become smaller, so it was found that 4 to 8 hours is appropriate. During the culture, the pressure inside the culture bottle is kept constant by degassing. The amount of gas generated during the culture can be determined by measuring the amount of gas released.

[0031] <Estimation based on analysis of gases generated from the culture medium> The gas generated from the culture solution is sampled from the gas layer in the culture bottle after a predetermined time selected from the range of 4 to 8 hours has elapsed, and the molar ratio of methane gas HSCH4 in the sampled gas is measured. Additionally, the total amount of gas GP (mL / gDM) generated from the culture solution per culture substrate (g) until the predetermined time has elapsed is determined. Sampling can be performed by inserting a needle into the gas layer from the branched collection port of the culture bottle to collect the gas.

[0032] The methane gas molar ratio HSCH4 can be calculated based on the following formula (1) by analyzing gas collected from the gas layer in the culture bottle by gas chromatography. HSCH4=S CH4 / (S CH4 +S CO2 ) ···(1) However, in equation (1), S CO2 is the mole fraction calculated from the peak area of ​​carbon dioxide in the obtained gas chromatogram, S CH4 is the mole fraction calculated from the peak area of ​​methane in the obtained gas chromatogram.

[0033] The total amount of gas generated from the culture solution per culture substrate (g) during the time from the start of culture to sampling after a predetermined time has elapsed (culture time) can be calculated as follows: That is, gas pressure inside the culture flask increases due to gas generation during the culture period, and the amount of gas generated is recorded as this gas pressure increase at regular intervals (every 5 to 30 minutes). To prevent the gas pressure from increasing too much, a valve automatically opens and the gas is released when the gas pressure reaches a certain value. The amount of pressure increase due to gas generated after the gas is released is integrated into the recorded value of gas pressure increase. At any given time, the amount of gas generated can be calculated from this integrated value of gas pressure increase according to Boyle's law.

[0034] The amount of methane gas generated per g of culture substrate (g) MP (mL / gDM) is estimated from the total gas amount GP (mL / gDM) and the methane gas molar ratio HSCH4. Various known regression equations can be used as appropriate for this estimation. Furthermore, depending on the purpose, a regression equation may be adopted that includes other factors, such as temperature, liquid volume, VFA concentration, and hydrogen molecule concentration, in addition to the total gas volume GP (mL / gDM) and the methane gas molar ratio HSCH4. [Example]

[0035] <Collection conditions> Twenty-four Holstein lactating dairy cows raised at Northern Farm Co., Ltd. were fasted for one hour before sampling, and then gastric juice was orally collected from each cow using a collection device consisting of a Luminar gastric juice collector (Fujidaira Kogyo Co., Ltd.), a gastric juice collection bottle (250 mL reagent bottle), and a commercially available bicycle pump connected by a tube. Sampling was started 5 seconds after the sampling part was submerged in the gastric juice. During sampling, the pushing and pulling of a commercially available bicycle pump was adjusted appropriately to prevent feed particles from adhering to the sampling part of the sampler.

[0036] The first liquid collected was discarded as it contained a lot of saliva, and gastric juice was then collected into collection bottles until the bottles were at least 80% full. Immediately after collection, carbon dioxide gas was blown into the collected gastric juice and then the bottles were sealed. The collection bottles were warmed in advance in a commercially available cooler box filled with 45°C water before use. The warm water in the cooler box was then replaced with 40°C water, and the collection bottles containing the collected gastric juice from each individual were placed in this to prevent the temperature from dropping. The collected gastric juice was transported to the laboratory within 20 minutes and filtered using triple-layered nonwoven gauze. 80 mL of this filtered gastric juice was mixed with an equal volume of McDougal's artificial saliva (pH 7.0) and used as diluted rumen fluid.

[0037] <Culture process> A branched culture bottle (320 mL volume) was used for the culture. 1.6 g (dry matter equivalent) of the mixed feed being fed to the lactating dairy cow used for gastric juice collection was used as the culture substrate. 160 mL of the diluted rumen fluid was added, and an ANKOM RF Gas Production System Measurement Module (ANCOM Technology) was attached while nitrogen gas was blown into the headspace. An anaerobic culture was performed in a 40°C water bath.

[0038] <Measurement of total gas volume GP> The degassing and measurement of the total amount of gas generated were performed automatically by the gas production system measurement module. Gas pressure inside the culture flask increased due to gas generation during the incubation period, and the amount of gas generated was recorded as this gas pressure increase at regular intervals (every 30 minutes). To prevent the gas pressure from increasing too much, the valve was set to automatically open and release the gas when it reached a certain value (10 pounds per square inch, psi).

[0039] The pressure increase due to the gas generated after degassing is integrated with the gas pressure at the time of degassing. At any given time, the total amount of gas generated is calculated from the integrated value of this gas pressure increase according to Boyle's law using the following formula (2).

[0040] PV=nRT (2) In equation (2), P is the increase in gas pressure (kPa), V is the volume of the gas layer in the culture flask (L), n is the total amount of gas generated (mol), T is the temperature during culture (K), and R is the gas constant (8.314 L·kPa / (K·mol)).

[0041] In this experiment, V = 0.16 (L) and T = 313 (K), so the total amount of gas generated (mol) was calculated using the following formula (3). Next, the total amount of gas generated (mL) was calculated using the following formula (4).

[0042]

number

[0043] Figure 4 shows the total gas volume (GP) (mL / gDM) generated per g of culture substrate in each culture bottle containing diluted rumen fluid from 6 of the 24 animals after 20 hours. As shown in Figure 4, the total amount of gas GP (integrated value) generated from the diluted rumen fluid continued to increase for all individuals up to 20 hours after the start of the experiment. This confirmed that all diluted rumen fluids maintained their activity at least after 20 hours.

[0044] <Analysis of atmospheric gas> After 4, 6, 8, and 20 hours, gas samples were taken from the air space of culture bottles containing diluted rumen fluid from 24 animals and analyzed by TCD gas chromatography (LC Sciences, Inc. INORGA: PolarPAQ 3 μm column). The methane gas molar ratio (HSCH4) was calculated using the following formula (1).

[0045] HSCH4=S CH4 / (S CH4 +S CO2 ) ···(1) However, in equation (1), S CO2 is the mole fraction calculated from the peak area of ​​carbon dioxide in the obtained gas chromatogram, S CH4 is the mole fraction calculated from the peak area of ​​methane in the obtained gas chromatogram.

[0046] <Calculation of methane gas generation amount MP> The methane gas generation rate MP (mL / gDM) was estimated using the following equation (5): Equation (5) is a regression equation proposed in a non-patent document (J Sci Food Agric, 2019, 99, pp. 109-116).

[0047]

number

[0048] In equation (5), HSV is the volume of the gas layer in the culture bottle per g of culture substrate (mL / gDM), GP is the total amount of gas generated (mL / gDM), and HSCH4 is the methane gas molar ratio HSCH4.

[0049] Figure 5 shows the amount of methane gas produced per g of culture substrate (MP) (mL / gDM) in each culture bottle containing diluted rumen fluid from six of the 24 individuals (the same six individuals as in Figure 4). As shown in Figure 5, the amount of methane gas produced (integrated value) MP reached its peak after 4 hours of cultivation. Furthermore, no individual differences were observed after 20 hours of cultivation.

[0050] <Estimated value based on saturated fatty acids in ruminal fluid> Before culturing, a portion of the filtered gastric fluid from 24 Holstein dairy cows used in the culture was separated and the concentrations of volatile fatty acids (VFA), including acetic acid (carbon number 2), propionic acid (carbon number 3), and butyric acid (carbon number 4), were measured using high-performance liquid chromatography (JASCO Corporation LC-2000 system). The measurement conditions were as follows, and the estimated methane gas concentration was calculated from the measured VFA concentration according to the following equation (6).

[0051] Column: Inertsil ODS-3 250 mm × 4.6 mm (GL Sciences Inc.). Column oven: 40°C. Mobile phase: 10% acetonitrile, 0.02% perchloric acid. Flow rate: 1mL / min. Detector: 210 nm (ultraviolet light).

[0052]

number

[0053] <Checking correlation> Figure 6 shows the relationship between the estimated methane gas concentration based on saturated fatty acids in rumen fluid and the estimated methane gas production MP based on the culture medium of rumen fluid for 24 individuals. The horizontal axis of Figure 6 represents the estimated methane gas concentration (mmol / L) calculated by the method described above in <Estimated Value Based on Saturated Fatty Acids in Ruminal Fluid>, and the vertical axis represents the methane gas generation amount MP (integrated value) after 4 hours (4H), 6 hours (6H), and 8 hours (8H) of culture, calculated based on formula (5) as described above in <Calculation of Methane Gas Generation Amount MP>.

[0054] The regression equations and correlation coefficients after 4 hours (4H), 6 hours (6H), and 8 hours (8H) of culture were as follows: After 4 hours of incubation: Regression equation y = 0.6192x + 9.9125 Correlation coefficient r = 0.818 After 6 hours of incubation: Regression equation y = 0.5175x + 15.504 Correlation coefficient r = 0.828 After 8 hours of incubation: Regression equation y = 0.4462x + 19.108 Correlation coefficient r = 0.807

[0055] Thus, it was found that there is a very high correlation between the amount of methane gas produced MP estimated based on the culture medium of rumen fluid and the estimated concentration of methane gas based on saturated fatty acids in rumen fluid. It is known that the estimated concentration of methane gas based on saturated fatty acids in ruminal fluid is highly correlated with direct measurement of methane emitted from cattle. Therefore, it was confirmed that the estimation method of the present invention is a reliable estimation method that can replace direct measurement.

[0056] <Confirmation of reproducibility> Gastric juice was collected from 22 Holstein lactating dairy cows raised at Northern Farm Co., Ltd. on two consecutive days under the same conditions as described above in <Collection Conditions>, and cultured using the same culture process as described above in <Cultivation Process>. The total gas volume GP generated was determined under the same conditions as described above in <Measurement of Total Gas Volume GP>, and the methane gas molar ratio HSCH4 was determined under the same conditions as described above in <Analysis of Air Layer Gas>. The methane gas generation volume MP (mL / gDM) was estimated using equation (5) in the same manner as described above in <Calculation of Methane Gas Generation Volume MP>. Ta.

[0057] Analysis of variance was performed on data on methane gas production (mL / gDM) for 22 lactating dairy cows over two days to compare the error for each factor. Table 1 shows the results after 4 hours, Table 2 shows the results after 6 hours, and Table 3 shows the results after 8 hours.

[0058] [Table 1]

[0059] [Table 2]

[0060] [Table 3]

[0061] As shown in Tables 1 to 3, while differences between individuals were clearly evident, the repetition error was very small, confirming high reproducibility. [Explanation of symbols]

[0062] 1, 10... Sonde, 2... Switching valve, 3, 20... Collection bottle, 4... Second bottle, 5, 30... Pump

Claims

1. Ruminant fluid collected from the first stomach of a ruminant animal is cultured in a culture bottle for a predetermined time in the presence of a culture substrate, and the total amount of gas (mL / gDM) generated from the culture solution of the ruminal fluid per g of culture substrate until the predetermined time has elapsed and the molar ratio (HSCH) of methane gas in the gas collected from the gas layer in the culture bottle after the predetermined time has elapsed are measured. ４ Measure The obtained total gas amount GP (mL / gDM) and methane gas molar ratio HSCH ４ A method for estimating methane gas generation amount MP (mL / gDM) per culture substrate (g) from A method for estimating the amount of methane gas generated, characterized in that the predetermined time is selected within a range of 4 hours to 8 hours.

2. The methane gas molar ratio HSCH ４ a gas sample collected from the gas layer in the culture bottle after the predetermined time has elapsed is analyzed by gas chromatography; The method for estimating the amount of methane gas generated according to claim 1, wherein the value is calculated based on the following formula (1): HSCH ４ =S ＣＨ４ / (S ＣＨ４ +S ＣＯ２ ) ・・・(1) However, in formula (1), S ＣＯ２ is the mole fraction calculated from the peak area of ​​carbon dioxide in the obtained gas chromatogram, S ＣＨ４ is the mole fraction calculated from the peak area of ​​methane in the obtained gas chromatogram.

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