CN113866304A - Method for measuring content of volatile fatty acid in rumen fluid of ruminant - Google Patents
Method for measuring content of volatile fatty acid in rumen fluid of ruminant Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N30/68—Flame ionisation detectors
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Abstract
The invention discloses a method for measuring the content of volatile fatty acid in rumen fluid of a ruminant, which comprises the following steps: preparing a standard solution, carrying out headspace sample injection, carrying out gas chromatography, processing a sample and the like. The invention adopts headspace gas chromatography to measure the VFA content in ruminant rumen fluid, and the method does not need sample pretreatment and directly uses headspace bottle for sample injection analysis, thereby furthest reserving original components and leading the detection result to be closer to the real components of the sample. Compared with the common sample injection analysis, the method effectively reduces the pollution of sample impurities to the chromatographic column, reduces the maintenance cost and saves the experimental consumables used for sample pretreatment.
Description
Technical Field
The invention relates to the technical field of livestock breeding, in particular to a method for measuring the content of volatile fatty acid in rumen fluid of ruminants.
Background
Volatile fatty acids, also known as short chain fatty acids, are the major products of carbohydrate fermentation in the rumen of ruminants by a variety of microorganisms. VFA in the ruminant body is mainly acetic acid, propionic acid and butyric acid, and accounts for 90-95% of the total amount of the VFA. The energy provided by the ruminant animal is about 2/3 which is the total energy absorbed and utilized by the ruminant animal. Therefore, the VFA measurement is of great significance for researching rumen fermentation of ruminants and improving nutrient absorption and energy conversion rate of feed.
At present, detection methods of VFA mainly include gas chromatography, high performance liquid chromatography, atomic absorption spectrophotometry, capillary electrophoresis, and the like, and among them, gas chromatography is most commonly used. Gas chromatography has the advantage of high reliability, but it requires complex sample pretreatment processes such as centrifugation and membrane filtration. In addition, the complex ruminant rumen fluid components easily cause pollution to the sample inlet and the chromatographic column of the chromatograph, reduce the accuracy of analysis, shorten the service life of the chromatographic column and increase the maintenance cost.
The headspace sampling method is a method for analyzing short-chain substances in a complex matrix sample by using a headspace technology (gas extraction). The method can be used for qualitative and quantitative analysis of the sample without complex sample pretreatment process, has unique advantages in measuring the ruminant rumen fluid VFA, avoids interference of non-volatile components on analysis, reduces instrument pollution, and makes up for the defects of gas chromatography detection method. However, no method for measuring the content of volatile fatty acid in rumen fluid of ruminants by adopting the technology exists at present.
Disclosure of Invention
Aiming at the problems, the invention provides a method for measuring the content of volatile fatty acid in rumen fluid of a ruminant, which does not need sample pretreatment, has simple and quick detection method, good reproducibility and high accuracy and can be widely applied.
According to the purpose of the invention, the invention provides the following technical scheme:
a method for measuring the content of volatile fatty acid in rumen fluid of ruminants comprises the following steps:
s1, preparing standard solution
Respectively and accurately weighing acetic acid, propionic acid and butyric acid standard substances, fixing the volume by pure water, and preparing mixed standard stock solution of 320mmol/L acetic acid, 32mmol/L propionic acid and 32mmol/L butyric acid for later use; respectively transferring standard stock solutions with different volumes to prepare mixed standard substance solutions with the concentrations of 80%, 100% and 120% of the measured concentration of the sample;
s2, headspace sample injection conditions
Headspace injector conditions: the balance temperature is 85 ℃, the temperature of a sample flow path is 180 ℃, the temperature of a transmission line is 200 ℃, the balance time is 30min, and the sample introduction time is 1 min;
s3, gas chromatography conditions
The chromatographic column is an SH-Stabilwax quartz capillary chromatographic column; the temperature of a sample inlet is 200 ℃, the carrier gas is nitrogen, the pressure of the carrier gas is 100kPa, and the split ratio is 50: 1;
temperature rising procedure: keeping the temperature at 80 ℃ for 1min, heating to 170 ℃ at 8 ℃/min, heating to 220 ℃ at 20 ℃/min, and keeping the temperature for 4 min; the temperature of the FID detector is 220 ℃;
s4, sample treatment
The detection sample comprises natural rumen fluid and artificial rumen fermentation liquor of dairy cows and Hu sheep, 1mL of sample is accurately measured in a 20mL headspace bottle, the sample is sealed by a silica gel spacer, sealed by an aluminum cover, uniformly mixed and placed in an automatic headspace sample injector, and balanced gas is taken for determination after balanced preheating is carried out for a period of time at a certain temperature.
Further, to determine the optimal equilibrium temperature, the 6 temperatures of 60, 65, 70, 75, 80 and 85 ℃ were selected for HS-GC analysis in this experiment, with the peak reaching a maximum at 85 ℃.
Further, comparing the response signal intensities at the equilibrium times of 20, 25, 30, 35, 40 and 45min, respectively, the peak area reached the maximum at 30 min.
Furthermore, in the same day, the same batch of samples was measured every 2h, and the precision in the day was calculated by measuring 6 times per day.
Further, the same sample was stored at 4 ℃ and measured continuously for 3d, and the daytime precision of the sample was examined.
Further, a standard solution of known concentration was stored at 4 ℃ and sampled continuously for 3 days for HS-GC analysis, and the stability of the standard solution within 3 days of storage was examined.
Further, after taking a fasting sample of the hu-sheep rumen fluid before morning feeding, and measuring the concentration of the volatile fatty acid therein, adding the volatile fatty acid into the sample at concentrations of 80%, 100% and 120% of the measured concentration of the sample respectively, and calculating the standard recovery rate according to the following formula:
the recovery rate in normalized form is (normalized sample measurement value-initial amount)/normalized amount × 100%.
Further, taking 3 parts of the rumen fluid of the dairy cow, the rumen fluid of the Hu sheep and a product obtained after artificial fermentation of the rumen fluid, and determining the content of the volatile fatty acid.
The invention has the beneficial effects that:
the invention adopts headspace gas chromatography to measure the VFA content in ruminant rumen fluid, and the method does not need sample pretreatment and directly uses headspace bottle for sample injection analysis, thereby furthest reserving original components and leading the detection result to be closer to the real components of the sample. Compared with the common sample injection analysis, the method effectively reduces the pollution of sample impurities to the chromatographic column, reduces the maintenance cost and saves the experimental consumables used for sample pretreatment.
Drawings
FIG. 1 is a graph of equilibrium temperature versus response signal according to the present invention;
FIG. 2 is a graph of equilibrium time versus response signal strength in accordance with the present invention;
FIG. 3 is a chromatogram of volatile fatty acids and other organic solvents according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for measuring the content of volatile fatty acid in rumen fluid of ruminants comprises the following steps:
s1, preparing standard solution
Respectively and accurately weighing acetic acid, propionic acid and butyric acid standard substances, fixing the volume by pure water, and preparing mixed standard stock solution of 320mmol/L acetic acid, 32mmol/L propionic acid and 32mmol/L butyric acid for later use; respectively transferring standard stock solutions with different volumes to prepare mixed standard substance solutions with the concentrations of 80%, 100% and 120% of the measured concentration of the sample;
s2, headspace sample injection conditions
Headspace injector conditions: the balance temperature is 85 ℃, the temperature of a sample flow path is 180 ℃, the temperature of a transmission line is 200 ℃, the balance time is 30min, and the sample introduction time is 1 min;
s3, gas chromatography conditions
The chromatographic column is an SH-Stabilwax quartz capillary chromatographic column; the temperature of a sample inlet is 200 ℃, the carrier gas is nitrogen, the pressure of the carrier gas is 100kPa, and the split ratio is 50: 1;
temperature rising procedure: keeping the temperature at 80 ℃ for 1min, heating to 170 ℃ at 8 ℃/min, heating to 220 ℃ at 20 ℃/min, and keeping the temperature for 4 min; the temperature of the FID detector is 220 ℃;
s4, sample treatment
The detection sample comprises natural rumen fluid and artificial rumen fermentation liquor of dairy cows and Hu sheep, 1mL of sample is accurately measured in a 20mL headspace bottle, the sample is sealed by a silica gel spacer, sealed by an aluminum cover, uniformly mixed and placed in an automatic headspace sample injector, and balanced gas is taken for determination after balanced preheating is carried out for a period of time at a certain temperature.
Further, to determine the optimal equilibrium temperature, the 6 temperatures of 60, 65, 70, 75, 80 and 85 ℃ were selected for HS-GC analysis in this experiment, with the peak reaching a maximum at 85 ℃.
Further, comparing the response signal intensities at the equilibrium times of 20, 25, 30, 35, 40 and 45min, respectively, the peak area reached the maximum at 30 min.
Furthermore, in the same day, the same batch of samples was measured every 2h, and the precision in the day was calculated by measuring 6 times per day.
Further, the same sample was stored at 4 ℃ and measured continuously for 3d, and the daytime precision of the sample was examined.
Further, a standard solution of known concentration was stored at 4 ℃ and sampled continuously for 3 days for HS-GC analysis, and the stability of the standard solution within 3 days of storage was examined.
Further, after taking a fasting sample of the hu-sheep rumen fluid before morning feeding, and measuring the concentration of the volatile fatty acid therein, adding the volatile fatty acid into the sample at concentrations of 80%, 100% and 120% of the measured concentration of the sample respectively, and calculating the standard recovery rate according to the following formula:
the recovery rate in normalized form is (normalized sample measurement value-initial amount)/normalized amount × 100%.
Further, taking 3 parts of the rumen fluid of the dairy cow, the rumen fluid of the Hu sheep and a product obtained after artificial fermentation of the rumen fluid, and determining the content of the volatile fatty acid.
Example 2
The embodiment provides a specific test method as follows:
a method for measuring the content of volatile fatty acid in rumen fluid of ruminants comprises the following steps:
1 materials and methods
1.1 instruments and reagents
HSS 86.50 headspace sampler (DANI corporation, italy); GC-2010Plus gas chromatograph (Shimadzu, Japan) equipped with a hydrogen Flame Ionization Detector (FID); an Eppendorf series pipettor; the headspace bottle capper was purchased from feihong packaging materials factory (south china, Jiangsu province); glacial acetic acid, propionic acid, butyric acid were chromatographically pure and purchased from alatin (Shanghai); ethanol, propanol, and butanol were chromatographically pure and purchased from carbofuran corporation (beijing); air, nitrogen and hydrogen are all purchased from Hangzhou modern chemical and material Co.Ltd; the ultrapure water was produced from a NANO pure UV/UF ultrapure water system.
1.2 Standard solution preparation
Respectively and accurately weighing acetic acid, propionic acid and butyric acid standard substances, fixing the volume by pure water, and preparing mixed standard stock solution of 320mmol/L acetic acid, 32mmol/L propionic acid and 32mmol/L butyric acid for later use. And respectively transferring the standard stock solutions with different volumes to prepare mixed standard solution with different concentrations.
1.3 headspace sample introduction conditions
Headspace injector conditions: the balance temperature is 85 ℃, the sample flow path temperature is 180 ℃, the transmission line temperature is 200 ℃, the balance time is 30min, and the sample injection time is 1 min.
1.4 gas chromatography conditions
The chromatographic column was an SH-Stabilwax quartz capillary chromatographic column (30 mx 0.25mm,
) (ii) a The injection port temperature is 200 ℃, the carrier gas is nitrogen (99.999%), the pressure of the carrier gas is 100kPa, and the split ratio is 50: 1. Temperature rising procedure: keeping the temperature at 80 ℃ for 1min, heating to 170 ℃ at 8 ℃/min, heating to 220 ℃ at 20 ℃/min, and keeping the temperature for 4 min; FID detector temperature 220 ℃.
1.5 sample treatment
The detection sample comprises natural rumen fluid and artificial rumen fermentation liquor of dairy cows and Hu sheep, 1mL of sample is accurately measured in a 20mL headspace bottle, the sample is sealed by a silica gel spacer, sealed by an aluminum cover, uniformly mixed and placed in an automatic headspace sample injector, and balanced gas is taken for determination after balanced preheating is carried out for a period of time at a certain temperature.
2 results and analysis
2.1 equilibrium temperature
In order to determine the optimal equilibrium temperature, the 6 temperatures of 60, 65, 70, 75, 80 and 85 ℃ are selected for HS-GC analysis in the experiment, and the peak area response values of the VFA components are increased along with the increase of the temperature, and the peak value of the sample reaches the maximum value at 85 ℃; as the water vapor content is increased along with the further rise of the temperature, the vapor pressure of the headspace bottle is increased, so that the pressure bearing of the headspace bottle is increased, the potential safety hazard problem exists, meanwhile, the air tightness of the headspace bottle is deteriorated due to the increase of the vapor pressure, and the repeatability of data is influenced (figure 1). The headspace equilibrium temperature ultimately selected for the present invention is therefore 85 ℃.
2.2 equilibration time
Comparing the response signal intensities at the equilibrium times of 20, 25, 30, 35, 40 and 45min, respectively, it was found that the peak area response value became larger as the equilibrium time was longer, and the peak area reached the maximum value at 30 min. With further extension of the equilibration time, the peak area of the sample decreased while the reproducibility of the data became worse (RSD > 5%). Based on the stability of data and considering both time efficiency and peak area, the invention finally selects 30min as the optimal sample injection time (figure 2).
2.3 Linear Range and detection Limit (LOD)
And (3) performing HS-GC (high-speed gas chromatography) determination on the mixed standard solution subjected to gradient dilution to obtain a linear regression equation of acetic acid, propionic acid and butyric acid, and analyzing the detection limits of 3 volatile acids according to the signal-to-noise ratio S/N (signal-to-noise ratio) of not less than 3, wherein the results are shown in table 1.
TABLE 1 Retention time, Linear Range, Linear regression equation and detection limits for volatile fatty acids
As can be seen from Table 1, the linear correlation coefficients of acetic acid, propionic acid and butyric acid are all greater than 0.9997 in the corresponding concentration ranges, which shows that the linear relation of the standard curve of the volatile fatty acid is good in the concentration range of the method; the detection limits calculated by a signal-to-noise ratio method are respectively 0.329, 0.087 and 0.036mmol/L, and the detection limit is lower, which indicates that the method can be used for measuring the VFA content of rumen fluid of ruminants.
2.4 precision investigation
Precision within 2.4.1 days
And measuring the same batch of samples once every 2h in the same day, continuously measuring 6 times every day, and calculating the precision in the day. The results show that the Relative Standard Deviation (RSD) of the peak areas of the three volatile fatty acids is not higher than 3.3% (Table 2), indicating that the precision of the method is good.
TABLE 2 precision in day (n ═ 6)
2.4.2 daytime precision
The same sample was stored at 4 ℃ for 3 days, and the daytime precision of the sample was examined, and it was found that the daytime precision of the peak area of volatile fatty acids in 3 days was good, and the RSD range was 0.333% to 2.303% (table 3).
TABLE 3 precision between days (n ═ 3)
n＝3.
2.4.3 stability
Standard solutions of known concentration were stored at 4 ℃ and sampled continuously for 3 days for HS-GC analysis to investigate the stability of the standard solutions within 3 days of storage. The results are shown in Table 4.
TABLE 4 stability of the standards
n＝3.
As can be seen from Table 4, the RSD of the peak areas of the mixed standards is below 8.318%, which indicates that the volatile fatty acid has good stability within 3d under the condition, and the test sample can be used for detection within 3 d.
2.4.4 recovery rate with standard addition
Taking a fasting Hu sheep rumen fluid sample before morning feeding, measuring the concentration of volatile fatty acid in the sample, adding low, medium and high 3 mixed standard solutions with different concentrations into the sample, wherein the concentrations are respectively 80%, 100% and 120% of the measured concentration of the sample, and calculating the standard recovery rate according to the following formula.
The recovery rate in standard addition is (measured value of standard addition sample-initial amount)/standard addition x 100%
The recovery of the sample with the standard is determined, and the result is shown in Table 5.
TABLE 5 recovery of spiked samples
As can be seen from Table 5, the normalized recovery rates of acetic acid and butyric acid ranged between 94.09% -108.13% and 93.54% -100.35%, respectively. The method is suitable for quantitative determination of acetic acid and butyric acid in ruminant rumen fluid.
2.6 specificity test
The rumen fluid of ruminant may contain other organic compounds, and for the special purpose of the investigation method, ethanol, propanol, butanol which may exist are mixed with acetic acid, propionic acid and butyric acid in certain amount to prepare a mixed solution, and the result is measured under the selected chromatographic condition, and is shown in figure 3. As can be seen from fig. 3, the 3 volatile fatty acids were well separated from ethanol, propanol, butanol, etc. The presence of these organic compounds does not interfere with the detection of acetic acid, propionic acid and butyric acid.
2.7 sample determination
Taking 3 parts of cow rumen fluid, Hu sheep rumen fluid and products obtained after artificial fermentation of the rumen fluid respectively, and measuring the content of volatile fatty acid, and finding that the content of volatile fatty acid in the cow rumen fluid, the Hu sheep rumen fluid and the artificial rumen fermentation fluid are different due to different species and different proportion of fed fine and coarse fodder. The Relative Standard Deviation (RSD) ranges of the measured concentrations of acetic acid, propionic acid and butyric acid are 0.127-3.333% (Table 6).
TABLE 63 content of volatile fatty acids in rumen fluid of ruminant
VFA is a main nutrient substance generated after the feed is fermented by microorganisms in the rumen of the ruminant, and the determination of VFA in rumen fluid of the ruminant is carried out, so that the important significance is realized on the research on the fermentation, nutrient absorption and energy conversion rate of intestinal microorganisms and feed. In the detection process, the volatile characteristic of VFA often causes inaccurate sample quantification and poor repeatability, and the complex components in rumen fluid also influence the stability of the peak shape and baseline of the VFA chromatogram.
In the existing VFA measuring method, most experimental samples need to be subjected to complex pretreatment operation and then can be subjected to sample injection analysis. Liu stands to stand etc[11]The goat rumen fluid is treated by a filtration and centrifugation mode, and the content of VFA is measured by a gas chromatography external standard method, and the results show that the linear correlation coefficients are not more than 0.999, presumably due to the loss of VFA in the filtration and centrifugation process of the sample. Daihei Wei and the like[12]The sample is pretreated by using an acidification filtration method, a capillary column gas chromatography analysis method is established to measure the VFA in the synthesis gas anaerobic fermentation liquid, and experiments show that the base line drift of a sample chromatogram occurs after 7.5min, which is presumed to be probably because impurities in the sample cause the pollution of a chromatographic column or a detector.
The invention adopts a headspace sampling mode, thereby not only simplifying the pretreatment step of the sample, but also avoiding the volatility loss of VFA and improving the accuracy of the result. The test result shows that the standard curve linear relation of the VFA is good, and the correlation coefficients of the regression equation are all larger than 0.9997. In addition, the headspace sampler eliminates pollution and interference of non-volatile impurities in the sample to the gas chromatograph, and is beneficial to the stability of chromatographic peak shape and base line. Zhang Jun Yu et al[13]The VFA in rumen fluid was pre-treated by 6 different methods and experimental errors were reduced by adding crotonic acid as an internal standard. Except different pretreatment and sample introduction modes, the experimental apparatus and the detection method used by the invention are the same or close to the same, and the detection limit, the linear correlation coefficient, the intra-day precision, the inter-day precision, the stability and other results of the 3 VFAs are all equivalent to the results, which shows that the headspace is equivalent to the resultsThe gas chromatography external standard method is a VFA detection method of rumen fluid, which is simple and convenient to operate and effective.
The invention adopts headspace gas chromatography to measure the VFA content in ruminant rumen fluid. The method does not need sample pretreatment, and directly uses a headspace bottle for sample injection analysis, thereby furthest retaining the original components and leading the detection result to be closer to the real components of the sample. Compared with the common sample injection analysis, the headspace sample injection mode effectively reduces the pollution of sample impurities to the chromatographic column, reduces the maintenance cost, saves the experimental consumables used for sample pretreatment, and is an analysis means meeting the requirement of green analytical chemistry.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (8)
1. A method for measuring the content of volatile fatty acid in rumen fluid of ruminants is characterized by comprising the following steps:
s1, preparing standard solution
Respectively and accurately weighing acetic acid, propionic acid and butyric acid standard substances, fixing the volume by pure water, and preparing mixed standard stock solution of 320mmol/L acetic acid, 32mmol/L propionic acid and 32mmol/L butyric acid for later use; respectively transferring standard stock solutions with different volumes to prepare mixed standard substance solutions with the concentrations of 80%, 100% and 120% of the measured concentration of the sample;
s2, headspace sample injection conditions
Headspace injector conditions: the balance temperature is 85 ℃, the temperature of a sample flow path is 180 ℃, the temperature of a transmission line is 200 ℃, the balance time is 30min, and the sample introduction time is 1 min;
s3, gas chromatography conditions
The chromatographic column is an SH-Stabilwax quartz capillary chromatographic column; the temperature of a sample inlet is 200 ℃, the carrier gas is nitrogen, the pressure of the carrier gas is 100kPa, and the split ratio is 50: 1;
temperature rising procedure: keeping the temperature at 80 ℃ for 1min, heating to 170 ℃ at 8 ℃/min, heating to 220 ℃ at 20 ℃/min, and keeping the temperature for 4 min; the temperature of the FID detector is 220 ℃;
s4, sample treatment
The detection sample comprises natural rumen fluid and artificial rumen fermentation liquor of dairy cows and Hu sheep, 1mL of sample is accurately measured in a 20mL headspace bottle, the sample is sealed by a silica gel spacer, sealed by an aluminum cover, uniformly mixed and placed in an automatic headspace sample injector, and balanced gas is taken for determination after balanced preheating is carried out for a period of time at a certain temperature.
2. The method for determining the content of volatile fatty acids in rumen fluid of ruminants according to claim 1, wherein 6 temperatures of 60, 65, 70, 75, 80 and 85 ℃ are selected for HS-GC analysis in order to determine the optimal equilibrium temperature, and the peak value of the sample reaches the maximum value at 85 ℃.
3. The method of claim 1, wherein the peak area reaches a maximum at 30min by comparing the intensity of the response signal at equilibrium times of 20, 25, 30, 35, 40 and 45 min.
4. The method for measuring the content of volatile fatty acid in rumen fluid of ruminants according to claim 1, wherein the same batch of samples is measured every 2 hours on the same day, and the measurement is continuously carried out 6 times a day to calculate the precision in the day.
5. The method for measuring the content of the volatile fatty acid in the rumen fluid of the ruminant as claimed in claim 1, wherein the same sample is stored at 4 ℃, and the daytime precision of the sample is examined after 3 days of continuous measurement.
6. The method of claim 1, wherein the standard solution with known concentration is stored at 4 ℃, and 3d samples are continuously taken for HS-GC analysis, and the stability of the standard solution in 3d storage is examined.
7. The method of claim 1, wherein the method comprises the steps of taking a sample of the rumen fluid of a fasting hu sheep before morning feeding, measuring the concentration of the volatile fatty acid in the sample, adding the volatile fatty acid into the sample at a concentration of 80%, 100% and 120% of the measured concentration of the sample, and calculating the normalized recovery rate according to the following formula:
the recovery rate in normalized form is (normalized sample measurement value-initial amount)/normalized amount × 100%.
8. The method for determining the content of the volatile fatty acid in the rumen fluid of the ruminant as claimed in claim 1, wherein the content of the volatile fatty acid is determined by taking 3 parts of the rumen fluid of the cow, the rumen fluid of the hu sheep and the product of the artificial fermentation of the rumen fluid respectively.
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