CN117665209A - Method for estimating feed efficiency of beef cattle before fattening through artificial rumen in-vitro gas production - Google Patents

Abstract

The invention provides a method for estimating the efficiency of beef cattle feed before fattening through artificial rumen in-vitro gas production, which comprises the steps of collecting rumen fluid of ruminants to be tested, preparing an in-vitro rumen fermentation system, placing a stable daily ration air-dried sample in a gas production tube, and then carrying out in-vitro artificial rumen gas production fermentation; after fermentation for 24 hours and 48 hours, the ammonia nitrogen increasing value and the pH decreasing value of the fermentation system are measured to judge the feed efficiency of the ruminant by calculating the gas yield and the methane yield of the rumen liquid fermentation system, and the higher the daily ration fermentation gas yield, the higher the digestibility, the higher the ammonia nitrogen increasing amount and the lower the pH decreasing value of the fermentation system, the higher the methane yield and the feed efficiency of the ruminant are. The invention establishes an operable and repeatable ruminant feed efficiency judging method, which is beneficial to selectively breeding high-feed efficiency ruminants so as to improve the economic benefit of breeding enterprises and reduce the feed resource consumption and the emission of greenhouse gases.

Description

Method for estimating feed efficiency of beef cattle before fattening through artificial rumen in-vitro gas production

Technical Field

The invention relates to the technical field of ruminant feed efficiency judgment, in particular to a method for estimating the feed efficiency of beef cattle before fattening through artificial rumen in-vitro gas production.

Background

Meat and dairy products produced by ruminants (e.g., cattle, sheep, and goats) are a very important source of high quality protein for human ingestion. Ruminants possess a unique rumen with a large population of microorganisms within them that are able to break down cellulose and convert it into readily digestible material. The rumen digestion not only improves the utilization efficiency of plant cellulose by ruminants, but also can digest more plant feed. However, ruminants consume large amounts of feed and water resources during growth, and thus the sustainability and production efficiency of the animal industry depend on the feed efficiency of ruminants. One of the main objectives of modern animal husbandry is to improve production efficiency and economic efficiency.

Residual Feed Intake (RFI), which is the difference between the actual feed intake of an animal and its predicted feed intake based on maintenance energy demand and growth rate, is a negative-going selective trait and has higher genetic power, with lower RFI, higher animal feed efficiency. Although feed to weight ratio (F/G) and weight gain feed ratio (G/F) are also common measures of ruminant feed efficiency, it is not possible to measure the feed efficiency differences between individuals of equi-ratio or weight negative growing animals, whereas RFI is not affected by the stage of animal growth and daily gain. The ruminant with Low Residual Feed Intake (LRFI) can improve the feed efficiency of the ruminant for a long time, and the ruminant with high feed efficiency can enable the animal husbandry to produce more meat and dairy products, and meanwhile, the waste of feed and water resources is reduced. This is very important to reduce the negative environmental impact of animal husbandry and to improve the economic benefits of animal husbandry, and can bring long-term economic benefits.

However, until now, the work of collecting and analyzing the individual feed intake of large ruminants is still cumbersome and expensive. Therefore, how to rapidly and effectively screen ruminants with high feed efficiency is a technical problem to be solved. The in vitro gas production method (In Vitro Gas Production Method) is developed from animal nutrition at the university of Huo Enhai M in GermanyEstablished by K.H. Menke et al (1979), the foreign literature mostly refers to the HFT (Hohenheimer Futterwert Test or Hohenheimcastest) technique. The in vitro gas yield can be used to accurately estimate the rumen organic matter digestibility of the feed, and the gas (CO) generated by in vitro fermentation of rumen microorganism of the feed sample ₂ 、CH ₄ And H ₂ ) Is a metabolite of microorganisms, and thus, under certain metabolic pathways, the amount or rate of gas production can be utilized to reflect the extent and rate of nutrient fermentation by rumen microorganisms. However, a method for estimating the feed efficiency of beef cattle before fattening by using the gas production rate is not yet known.

Disclosure of Invention

The invention aims to provide a method for estimating the feed efficiency of beef cattle before fattening through artificial rumen fermentation gas production, which can rapidly measure the feed efficiency of ruminants, and select and feed high feed efficiency (LRFI) ruminants to improve the economic benefit of breeding enterprises and reduce resource consumption.

In order to achieve the above purpose, the invention provides a method for estimating the feed efficiency of beef cattle before fattening through artificial rumen in-vitro gas production, which comprises the following steps:

s1, collecting rumen fluid of ruminants to be tested, which is suitable for a feeding environment, and mixing the rumen fluid with artificial saliva to prepare in-vitro artificial rumen fermentation liquor; the volume ratio of the rumen fluid to the artificial saliva is 1:2;

s2, drying the stable daily ration with the granularity of 0.5-1mm to obtain a stable daily ration air-dried sample;

s3, placing the stable daily ration air-dried sample into the in-vitro artificial rumen fermentation liquid, culturing for 48 hours in a water bath shaking table at 39 ℃, taking out half of gas production pipes after fermentation for 24 hours, and ending fermentation in an ice water bath; after fermentation, calculating one or more parameters of net gas yield, methane gas yield, net pH reduction value, net increase of volatile acid and net increase of ammoniacal nitrogen; the feed efficiency of the ruminant to be tested is in direct proportion to the net gas yield, methane gas yield, net increase in volatile acid and net increase in ammoniacal nitrogen, and in inverse proportion to the net decrease in pH. The principle is that the higher the gas yield is, the higher the digestibility of gastric juice of the animal tumor to be detected on the feed is, and the better the production performance is.

The invention cultures a certain amount of feed samples by inoculating a mixture of rumen fluid and buffer fluid (artificial saliva) in an in vitro gas production tube (injector), and the culture conditions simulate the rumen temperature, pH value, buffer capacity, micronutrients, nitrogen source and anaerobic environment thereof. The dry feed material measured by the method has high positive correlation between 24-hour gas production rate and the digestibility of organic substances in ruminant living bodies, so that the feed efficiency of beef cattle before fattening can be estimated.

Further, in step S1, the method for collecting rumen fluid includes: collecting rumen fluid of the ruminant to be tested before morning feeding through an oral gastric tube, discarding the rumen fluid at the front section, squeezing and filtering the content by using medical gauze, then placing the rumen fluid in a threaded freezing storage tube, storing the rumen fluid in a heat-preserving barrel containing warm water at 39 ℃, and placing the rumen fluid in an in-vitro rumen fermentation system for fermentation for 48 hours.

Further, in step S1, the preparation method of the artificial saliva includes: taking a culture solution split charging bottle, putting into a stirring magnetic rod, and putting into a circulating water bath; then distilled water, trace element solution, buffer solution, macroelement solution and resazurin solution are added in sequence and uniformly mixed, and the solution is blue at the moment; heating to 39deg.C, adding reducing solution, and introducing CO ₂ Saturated, the solution gradually turns from blue to pink, and then turns to light red or colorless, and the artificial saliva is obtained;

the volume fraction of the trace element solution is 0.025 percent based on the distilled water content; the volume fraction of the buffer solution is 50%; the volume fraction of the macroelement solution is 50%; the volume fraction of the resazurin solution is 0.25%.

Further, the trace element solution comprises calcium chloride, manganese chloride, cobalt chloride and ferric chloride.

Further, the buffer solution comprises ammonium bicarbonate and sodium bicarbonate.

Further, the macroelement solution comprises anhydrous disodium hydrogen phosphate, anhydrous monopotassium phosphate and magnesium sulfate.

Further, in step S1, the ruminant to be tested is a beef cattle of 6-12 months of age;

and/or the ruminant to be tested adapting to the feeding environment is to feed the ruminant to be tested in the feeding environment for 7-14 days by adopting the stable ration.

Further, in step S2, the method for drying the stable ration includes: and (5) drying the stabilized grains in an oven for 48+/-5 hours at 65+/-5 degrees.

Further, in the step S3, the mass-volume ratio of the stable daily ration air-dried sample to the in-vitro artificial rumen fermentation broth is 0.22 g/30 mL.

Further, the net gas yield=the gas yield of the ruminant to be tested at a certain time point-the average gas yield of the blank tube at the time point is in mL;

methane yield, mL = total gas yield x percentage of methane;

net methane yield = loading ration gas production tube methane yield-corresponding animal blank gas production tube methane yield;

net pH decrease = loading ration gas production tube pH-loading ration gas production tube pH corresponding to animal blank gas production tube;

Ammonia nitrogen increment = loading ration gas production tube ammonia nitrogen concentration-corresponding to animal blank gas production tube loading ration gas production tube ammonia nitrogen concentration.

Further, the dynamic fermentation parameter calculation model is as follows:

Y＝B(1–e ^-ct )

wherein: y is the accumulated gas yield of the t time point, and the unit is mL; b is theoretical maximum gas yield per mL; c is the gas production rate, unit h ^-1 T is the culture time, unit h; the gas production tube was taken out at each time point of 0, 2, 4, 6, 8, 12, 16, 20, 24, 32, 40, 48h, and the scale value was recorded, and when the scale value exceeded 70mL, the gas was exhausted.

The gas in the gas generating tube is extracted by a needle tube of the injector to measure the gas component. The fermentation broth in the gas production tube was discharged to a 50mL plastic centrifuge tube, and a part of the fermentation broth was taken out and immediately measured for pH value by a pH meter. Using Lei Ci PHS-3C pH meter (Shanghai Reed magnetic Instrument factory, display accuracy 0.01)And (5) determining the pH value of the fermentation liquor. Volatile fatty acid content was determined using a gas chromatograph (SP-3420, beijing analytical instrument) and an internal standard 2-ethylbutyrate method. Another part of the fermentation broth was centrifuged at low temperature (4 ℃,800g,10 min) and the supernatant was frozen for further fermentation parameters: volatile acid (VFA) and ammoniacal Nitrogen (NH) ₃ -N). The rumen fermentation was terminated and the pH of the fermentation broth was measured using a pH meter.

Further, in step S3, rumen fluid and artificial saliva are prepared into an artificial rumen fermentation broth in a volume ratio of 1:2, and CO is introduced ₂ Maintaining an anaerobic environment; specifically, 220mg of stable grain air-dried sample is weighed, sent to the front end of a 100mL gas production pipe by a long-handle medicine spoon and placed in an incubator at 39 ℃ for preheating; injecting 20mL of artificial rumen fermentation liquid from the front end of the gas production pipe by using a liquid separating device, and injecting 10mL of rumen liquid of ruminant to be tested by using an injector; the gas production tube of the blank control group is not added with a substrate, 20mL of artificial rumen fermentation liquid is injected from the front end of the gas production tube by a liquid separating device, and 10mL of rumen liquid of ruminant to be detected is injected by an injector;

exhausting bubbles in the gas production tube, recording initial scale values of the gas production tube, then placing the gas production tube in a 39 ℃ constant-temperature water bath box for culture, wherein 2n blank tubes of each ruminant to be tested, 2n test tubes and at least 2 repetition are carried out, and n is an integer; weighing 0.22g of samples respectively; the scale values of the gas production pipes are read and recorded at 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 and 48 hours, one half of the gas production pipes in each group of experiments are cultivated to 24 hours, the rest gas production pipes are used for stopping fermentation in an ice bath at 48 hours to measure volatile acid, ammonia nitrogen and pH, and the net gas production measurement is carried out through accumulated gas production.

The beneficial effects of the invention are as follows:

the method for estimating the feed efficiency of the beef cattle before fattening through the artificial rumen in-vitro fermentation gas production provided by the invention is characterized in that a perfect and reasonable in-vitro digestion system is constructed, the rumen liquid fermentation system gas production amount and methane yield are calculated, and the ammonia Nitrogen (NH) of the fermentation system is determined ₃ -N) increasing value, pH decreasing value, dry Matter (DM), crude Protein (CP) and neutral washing insoluble fiber (NDF) digestibility, and further judging the feed efficiency of ruminant, wherein the higher the ration yield of 24h and 48h ration fermentation, the DM and CThe higher the digestibility of P and NDF, the higher the methane yield and NH ₃ The higher the N increase, the smaller the pH decrease, and the higher the feed efficiency of the ruminant. The invention establishes an operable and repeatable ruminant feed efficiency judging method, which is beneficial to selectively breeding high-feed efficiency ruminants so as to improve the economic benefit of breeding enterprises and reduce the feed resource consumption and the emission of greenhouse gases. Therefore, the method can rapidly measure the feed efficiency of ruminants, and select ruminants with high feed efficiency to improve the economic benefit of breeding enterprises and reduce the resource consumption.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method for estimating the feed efficiency of beef cattle before fattening through artificial rumen in-vitro fermentation gas production;

FIG. 2 is a schematic diagram of a ruminant rumen fluid sampling and preservation method;

FIG. 3 is a schematic structural diagram of an artificial saliva preparation system;

FIG. 4 is a flow chart of a gas production fermentation test;

figure 5 is a graph showing the gas production of different RFI animals as a function of fermentation time.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Feed intake is a gold standard for measuring feed efficiency of ruminants, and ruminants fed with Low Residual Feed Intake (LRFI) can improve the feed efficiency of ruminants for a long time, reduce feeding cost and bring long-term economic benefit. However, the measurement of residual food intake in the prior art needs to rely on a complex prediction model, which is inconvenient for practical application of farms. The method for estimating the feed efficiency of the beef cattle before fattening through the artificial rumen in-vitro digestion is obtained through the construction and the test of an in-vitro digestion system. The method comprises the following steps:

A method for estimating the feed efficiency of beef cattle before fattening through artificial rumen fermentation gas production, which comprises the following steps:

s1, in order to ensure the feasibility of measurement, in a normal feeding environment, the ruminant to be tested needs to be fed for 7-14 days in an adaptive manner, and the diet is the stable fattening diet of the beef farm. In addition, fattening beef cattle in a fattening factory are generally in the stage of 6-12 months of age (240-400 kg) and have little difference in age.

S2, uniformly taking 3-5kg of stable fattening daily ration, airing at 65 ℃ for 48 hours, sieving with a 1mm sieve, and weighing 0.22g of air-dried sample in a gas production tube by using a special ladle for later use.

S3, preparing an in-vitro artificial rumen fermentation system.

Instrument and equipment

1. Gas production pipe: glass gas production tube: the culture of 1 batch requires 57, the length of the special glass syringe is 27cm, the inner diameter is 3cm, the scale display is provided in the range of 100mL, and the minimum scale is 1mL. A silicone rubber hose is sleeved outside a 3.5cm extension tube at the front end of the injector, and the silicone rubber hose is clamped by a special PVC water stop clamp.

2. Oscillating the water bath shaking table: an oscillating water bath shaking table (manufactured by Jiangsu Taicang instrument factory) customized by the beef cattle research center of China agricultural university is selected, the oscillating speed is adjustable, and each incubator can be loaded with 63 gas production pipes.

3. Carbon dioxide gas tank and gas communication device: the purity of the canned carbon dioxide gas is more than 99.99 percent, and the canned carbon dioxide gas is connected with a liquid separating device through a salt-tolerant pipeline.

4. Liquid separating device: a PYTIFIX type Universal liquid separation device (Universal) manufactured by Germany was usedDispenser) is used for subpackaging the culture solution, the subpackaging range is 10-50 mL, the minimum scale is 1mL. The liquid separating device is provided with an air inlet and an air outlet, and CO can be introduced into the liquid separating device ₂ The anaerobic environment of the culture solution is ensured.

5. Constant temperature magnetic stirring device: a p/1 type thermostat device manufactured by JulaboLabortechnik GmbH D-7633Seelbach, germany, and a TypRCONr type magnetic stirrer manufactured by Janke & Kunkel Gmbh & Co.KGIKA-Werk.D7813stautent T.Breisgau were used for the constant temperature and uniform mixing of the culture solution. The constant temperature device adopts a circulating water system to ensure that the temperature of the culture solution is kept at 39 ℃, and the frequency of the circulating water and the frequency of the magnetic stirrer are both 50Hz.

6. Oral cavity stomach tube: tumor gastric juice is collected orally.

7. Medical gauze: a standard medical gauze bag is adopted for filtering rumen juice.

8. Thermal insulation barrel: store and maintain rumen fluid activity.

9. And (3) sampling tube: a100 mL sampling tube stores rumen fluid collected orally.

Reagents and solutions

TABLE 1 microelement solution (A liquid)

Calcium chloride	CaCl 2 ·2H 2 O	13.2g
			Manganese chloride	MnCl 2 ·4H 2 O	10.0g
Cobalt chloride	CoCl 2 ·6H 2 O	1.0g
			Ferric chloride	FeCl 3 ·6H 2 O	8.0g
Distilled water is fixed to volume		100mL

TABLE 2 buffer solution (solution B)

Distilled water	1000mL
			Ammonium bicarbonate	NH 4 HCO 3	4.0g
Sodium bicarbonate	NaHCO 3	35.0g

TABLE 3 macroelement solution (solution C)

Distilled water		1000mL
			Anhydrous disodium hydrogen phosphate	Na 2 HPO 4	5.7g
Anhydrous potassium dihydrogen phosphate	KH 2 PO 4	6.2g
			Magnesium sulfate	MgSO 4 ·7H 2 O	0.6g

TABLE 4 Reductant solution

1N sodium hydroxide	1N-NaOH	4mL
			Sodium sulfide	Na 2 S·9H 2 O	625mg
Distilled water is fixed to volume		100mL

Resazurin solution: 0.1% (w/v).

1. Sample weighing

Making air-dried sample of TMR (total mixed ration) fed during ruminant animal feeding, sieving with 20 mesh or 1mm sieve, weighing feed sample dry matter 200mg (or air-dried sample 220 mg) with small medicine spoon, feeding into 100mL glass syringe, uniformly smearing proper amount of medical vaseline at 1/2 position in front of syringe piston, and preheating at 39deg.C. To ensure that the test samples are representative, 3 replicates per ruminant to be tested are recommended. At least 4 (two blank pipes, two loading ration pipes, 24h for taking out one blank pipe and gas producing pipe, 48h for taking out the rest) N (parallel measurement number) gas producing pipes are needed for each ruminant to be tested, and the ruminant to be tested is placed in a constant temperature incubator at 39 ℃.

2. Preparation of artificial saliva

A clean 5500mL jar was placed in a stirring bar 1 particle in a circulating water bath. The artificial rumen nutrient solution is prepared according to the following proportion and sequence: 2000mL of distilled water, 0.5mL of solution A, 1000mL of solution B, 1000mL of solution C, 5mL of solution Resazurin, and 200mL of solution of reducing agent. Fresh prepare before use, reuse CO ₂ Saturated and preheated to 39 ℃. Distilled water, A liquid, B liquid, C liquid and resazurin solution are sequentially added into a glass bottle according to the proportion. Adding resazurin solution, adding 200mL of reducing solution, and introducing anaerobic CO ₂ And preheated to 39 ℃ for about 30 minutes, the solution was observed for a reduction process, and the color was changed from red (oxidized state) to light or colorless (reduced state). And before the rumen fluid of the ruminant to be tested is added, the rumen fluid is split into gas production pipes, 20mL of the rumen fluid of the ruminant to be tested is added into each gas production pipe, 10mL of rumen fluid of the ruminant to be tested is added into a syringe, and the rumen fluid is placed into 39 ℃ water bath for fermentation.

3. Gastric juice collection and preservation of animal tumor to be tested

After the ruminant to be tested is suitable for fattening or daily feed, rumen fluid is collected before morning feeding through an oral gastric tube, forepart rumen fluid is discarded, about 100mL is taken, 4 layers of medical gauze are used for squeezing and filtering the content and placing the content into a 100mL threaded freezing tube, a tube cover is screwed to keep an anaerobic environment after the freezing tube is in an overflow state, and the tube cover is placed into a heat-preserving barrel for containing warm water at 39 ℃ and is sent to a laboratory as soon as possible.

4. Configuration, fermentation and judgment standard of in vitro digestive system

Under the conditions of heat preservation at 39 ℃ and avoiding contact with oxygen as much as possible, a sterile medical injector is used for placing rumen fluid of ruminants to be tested into a gas production tube added with artificial saliva, 30mL of artificial rumen culture fluid (20 mL of artificial saliva and 10mL of oral rumen fluid) is added, the rumen fluid is cultured in a 39 ℃ water bath shaking table, and after the liquid adding is finished, the rumen fluid is transferred into an artificial rumen incubator for culture in batches.

5. Reading gas production

When the culture is carried out for each time point of 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42, 48 hours and the like, the gas production tube is taken out, and the scale value (mL) of the piston is quickly read and recorded. If the reading exceeds 80mL at a certain time point, in order to prevent the gas from exceeding the scale and failing to read, the gas should be timely exhausted after the reading and the scale value after the exhausting should be recorded.

6. Terminating fermentation and sampling

After 48h of in vitro culture, the gas generating tubes (syringes) were taken out separately and placed in an ice-water bath to stop fermentation. The fermentation broth in the gas production tube was discharged to a 50mL plastic centrifuge tube, and the pH value of the fermentation broth was measured immediately with a pH meter. The fermentation broth was centrifuged at low temperature (4 ℃,8,000Xg, 15 min) and the supernatant was frozen for further fermentation parameters (VFA, ammoniacal nitrogen etc.) measurement.

3 result calculation

3.1 calculation formula of cumulative net gas production (0.200 gDM) of gas production tube in a certain time period

Net gas production (mL) =gas production (mL) at a time point of ruminant to be tested-average gas production (mL) for the ruminant blank tube to be tested at that time point;

methane yield (mL) =total gas yield x percent methane;

net methane yield = loading ration gas production tube methane yield-corresponding animal blank gas production tube methane yield;

Ammonia nitrogen increment = loading ration gas production tube ammonia nitrogen concentration-corresponding to animal blank gas production tube loading ration gas production tube ammonia nitrogen concentration;

pH reduction = loaded ration gas production tube pH-loaded ration gas production tube pH corresponding to animal blank gas production tube.

3.2 dynamic fermentation parameter calculation

The in vitro gas production method provides a good data source for calculating in vitro dynamic fermentation parameters. In calculating the dynamic fermentation parameters, a suitable mathematical model needs to be selected. In general, for feeds without fermentation delay (lag.ltoreq.0), suitable models are (1), (2) and (3); for feeds with fermentation delay (lag > 0), suitable models are selected from (4) and (5):

Y＝B(1–e ^-ct ) (1)

Y＝B/(1+e ^(2–4ct ) (2)

Y＝B1(1–e ^-c1t1 )+B2(1–e ^-c2t2 ) (3)

Y＝B(1–e ^-c(t-lag) ) (4)

Y＝B/(1+e ^{(2–4c(t-lag)} ) (5)

wherein: y is the cumulative gas production (mL) of 0.2000g DM samples at time t; b is the theoretical maximum gas production (mL) of 0.2000g sample; c is the gas production rate (h) of 0.2000g sample ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the B1 is 0.2000g of theoretical maximum gas yield (mL) of the sample rapid fermentation component; c1 is the gas production rate (h) of 0.2000g sample rapid fermentation component ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the B2 is 0.2000g of theoretical maximum gas yield (mL) of the sample slow fermentation component; c2 is the gas production rate (h) of the sample slow-fermentation component of 0.2000g ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the t is the incubation time (h).

Formulas (1) and (4) are exponential curve models, and are most widely used at present. Formulas (2) and (5) are empirical models developed by the university of Cornell Schofield, usa, and formula (3) is a two-component model, i.e., the fermented feed contains a fast-digesting component B1 (e.g., starch or sugar) and a slow-digesting component B2 (e.g., fiber).

After such a set of data is obtained, dynamic fermentation parameters can be calculated using the NON-LINEAR method in SAS (1996) statistical software.

4. Notice that:

(1) During sample weighing, the sample should be sent to the scale of 0-30mL of the syringe as much as possible, so that the sample is prevented from being sprinkled on the wall of the syringe.

(2) The device used for collecting rumen fluid is preheated and as anaerobic as possible.

(3) The reducing agent must be prepared on site, and the reducing agent should be added to the mixed nutrient solution before the rumen fluid is added, and carbon dioxide is introduced until the solution becomes colorless, and then the rumen fluid can be added.

(4) In the whole sample adding process, the magnetic stirrer must work normally, so that on one hand, stirring can uniformly mix substances in the inoculum, and on the other hand, CO can be avoided ₂ The gas production read during the test, which resulted from the formation of supersaturation in the liquid solution, was not representative of the actual gas production and was subject to error.

The feed efficiency of ruminants is positively correlated with the rumen digestibility, and the in-vitro culture in rumen fermentation and gas production can reflect the digestibility of ruminants, which indirectly indicates that the higher the feed efficiency, the higher the ration produced by 24h and 48h ration fermentation, the higher the digestibility of DM, CP and NDF, the higher the methane yield, the higher the digestibility of DM and CP, and the NH ₃ The higher the N increase, the smaller the pH decrease, the higher the ruminant feed efficiency, the better the ruminant feed efficiency.

Example 1

Adaptive feeding, artificial saliva preparation, rumen fluid collection, in-vitro rumen fermentation gas production system preparation and feed efficiency judging method for ruminants

In vitro digestion experiments are carried out on beef cattle with Low Residual Feed Intake (LRFI) and High Residual Feed Intake (HRFI) respectively, and feasibility of judging results of the invention is verified, as shown in figures 1-4.

1.1 adaptive feeding and feed Material treatment for ruminants

The ruminant (beef cattle) to be tested is driven into a feeding environment, the ruminant is adaptively fed for 7-14D, daily ration is fed for 2-3D, stable daily ration is obtained, the stable daily ration is obtained through air-drying samples, the stable daily ration is sieved by a 20-mesh (1 mm) sieve, 200mg (or 220mg of air-dried samples) of feed sample dry matter is weighed by a small medicine spoon, the feed sample dry matter is fed into a 100mL glass injector, and a proper amount of medical vaseline is uniformly smeared at the 1/2 part in front of a piston of the injector, and the feed sample is preheated at 39 ℃. To ensure that the test samples are representative, 3 replicates per ruminant to be tested are recommended. At least 4 (two blank pipes, two loading ration pipes, 24h for taking out one blank pipe and gas producing pipe, 48h for taking out the rest) N (parallel measurement number) gas producing pipes are needed for each ruminant to be tested, and the ruminant to be tested is placed in a constant temperature incubator at 39 ℃.

1.2 preparation of Artificial rumen fermentation System

A clean 5500mL jar was placed in a stirring bar 1 particle in a circulating water bath. The artificial rumen nutrient solution is prepared according to the following proportion and sequence: 2000mL of distilled water, 0.5mL of solution A, 1000mL of solution B, 1000mL of solution C, 5mL of solution Resazurin, and 200mL of solution of reducing agent. Fresh prepare before use, reuse CO ₂ Saturated and preheated to 39 ℃. Distilled water, A liquid, B liquid, C liquid and resazurin solution are sequentially added into a glass bottle according to the proportion. Adding resazurin solution, adding 200mL of reducing solution, and introducing anaerobic CO ₂ And preheated to 39 ℃ for about 30 minutes, the solution was observed for a reduction process, and the color was changed from red (oxidized state) to light or colorless (reduced state). And before the rumen fluid of the ruminant to be tested is added, the rumen fluid is split into gas production pipes, 20mL of the rumen fluid of the ruminant to be tested is added into each gas production pipe, 10mL of rumen fluid of the ruminant to be tested is added into a syringe, and the rumen fluid is placed into 39 ℃ water bath for fermentation.

1.3, collecting and preserving gastric juice of animal tumor to be tested

After the ruminant to be tested is suitable for fattening or daily feed, rumen fluid is collected before morning feeding through an oral gastric tube, forepart rumen fluid is discarded, about 100mL is taken, 4 layers of medical gauze are used for squeezing and filtering the content and placing the content into a 100mL threaded freezing tube, a tube cover is screwed to keep an anaerobic environment after the freezing tube is in an overflow state, and the tube cover is placed into a heat-preserving barrel for containing warm water at 39 ℃ and is sent to a laboratory as soon as possible.

1.4, configuration, fermentation and judgment criteria of in vitro digestive System

Under the conditions of heat preservation at 39 ℃ and avoiding contact with oxygen as much as possible, a sterile medical injector is used for placing rumen fluid of ruminant to be tested into a gas producing pipe added with artificial saliva, 30mL of artificial rumen culture fluid (20 mL of artificial saliva and 10mL of oral rumen fluid) is added, the rumen culture fluid is cultured in a 39 ℃ water bath shaking table, and the rumen culture fluid is transferred into an artificial rumen incubator for culture in batches after the liquid addition is completed.

1.5 reading gas production

When the culture is carried out for each time point of 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42, 48 hours and the like, the gas production tube is taken out, and the scale value (mL) of the piston is quickly read and recorded. If the reading exceeds 80mL at a certain time point, in order to prevent the gas from exceeding the scale and failing to read, the gas should be timely exhausted after the reading and the scale value after the exhausting should be recorded.

1.6 termination of fermentation and sampling

After 48h of in vitro culture, the gas generating tubes (syringes) were taken out separately and placed in an ice-water bath to stop fermentation. The fermentation broth in the gas generating tube was discharged to a 50mL plastic centrifuge tube, and the pH value of the fermentation broth was measured immediately with a pH meter. The fermentation broth was centrifuged at low temperature (4 ℃,8,000Xg, 15 min) and the supernatant was frozen for further fermentation parameters (VFA, ammoniacal nitrogen etc.) measurement.

2. Result calculation

2.1 calculation formula of cumulative net gas production (0.200 gDM) of gas production tube in a certain time period

Net gas production (mL) =gas production (mL) at a time point of ruminant to be tested-average gas production (mL) for the ruminant blank tube to be tested at that time point;

methane yield (mL) =total gas yield x percent methane;

net methane yield = loading ration gas production tube methane yield-corresponding animal blank gas production tube methane yield;

ammonia nitrogen increment = loading ration gas production tube ammonia nitrogen concentration-corresponding to animal blank gas production tube loading ration gas production tube ammonia nitrogen concentration;

pH reduction = loaded ration gas production tube pH-loaded ration gas production tube pH corresponding to animal blank gas production tube.

Generally, the higher the 24h and 48h ration gas yield, the higher the digestibility of DM, CP and NDF, NH ₃ The higher the N increase, the smaller the pH decrease, the higher the methane yield and the higher the feed efficiency of the ruminant.

Table 1: different RFI animal in vitro fermentation parameters

As can be seen from FIG. 5 and the table above, the method for determining the feed efficiency of ruminants in vitro established by the invention can be used for identifying the RFI of different ruminants, the feed passing through a 1mm sieve and the fermentation time of 24 hours and 48 hours, and the net gas yield, the methane yield, the nutrient increment and the ethylene-propylene ratio are positively correlated with the feed efficiency.

Appendix NH ₃ N, VFA and CH ₄ Measurement method

1. Colorimetry for measuring NH by phenol-sodium hypochlorite ₃ N content (Broderickan dKang, 1980)

1 instrument

An ultraviolet visible spectrophotometer (UV-VIS 8500, shanghai Tianmei science instruments ltd); constant temperature water bath

2 reagent

Phenol reagent: 0.05g of sodium nitrosoferricyanide was dissolved in 500mL of distilled water, 9.9g of crystalline phenol was added thereto, and the solution was stored in a brown glass reagent bottle after being fixed to a volume of 1L.

Sodium hypochlorite reagent: 5g NaOH was dissolved in 200mL distilled water, and 50.6g Na was added ₂ HPO ₄ ·12H ₂ O(37.9g Na ₂ HPO ₄ ·7H ₂ O, or 20.1g anhydrous Na ₂ HPO ₄ ) The mixture is heated by medium fire and stirred continuously until the mixture is completely dissolved. After cooling, 26.3mL of 10% sodium hypochlorite is added, and the mixture is uniformly mixed, the volume is fixed to 1L, and finally the filtrate filtered by the filter paper is stored in a brown reagent bottle for standby.

Standard ammonium solution: 0.6607g (NH) ₄ ) ₂ SO ₄ (dried at 100 ℃ C. For 24 h) dissolved in 0.1mol/L hydrochloric acid and fixed to 100mL to obtain 100mmol/L standard ammonium stock solution. The stock solution is diluted and prepared into five standard solutions with different concentration gradients of 0.5, 1.0, 1.5, 2.0 and 2.5 mmol/L.

3 operation

(1) 100 mu L of 10-fold diluted sample liquid or standard liquid is added into each test tube, and the blank is 100 mu L of distilled water;

(2) Adding 5mL of phenol reagent into each test tube, and shaking uniformly;

(3) Adding 4mL of sodium hypochlorite reagent into each test tube, and uniformly mixing;

(4) Heating the mixed solution in a water bath at 95 ℃ for color development reaction for 5 minutes;

(5) After the solution cooled (cold water bath), it was colorized at 630nm wavelength with an ultraviolet-visible spectrophotometer.

4 results calculation

Fermentation broth sample NH ₃ -N (mg/100 mL) =c×1.4×sample dilution;

and C in the formula is a standard curve check or a spectrophotometer workstation to calculate the ammonia nitrogen concentration (mmol/L) of the sample.

2. Gas chromatography analysis of volatile fatty acid content in rumen fluid and silage

1. Purpose(s)

The experiment aims to rapidly and accurately analyze the volatile fatty acid content in rumen fluid by adopting a gas chromatography analysis method with direct sample injection and an internal calibration amount calculation method.

2. Principle of

The assay protocol is based on the method of direct gas chromatography of volatile fatty acids established by Erwin et al (1961) and is optimized for the choice of chromatographic column and chromatographic operating conditions.

The gas chromatography method uses gas as a mobile phase (carrier gas) to carry a sample entering from a sample inlet into a separation column (packed column or capillary column). Because of the difference in partition or adsorption coefficients between the stationary phase (liquid or solid) and the mobile phase (gas) of the chromatographic column, the time to reach the detector is also ordered by repeating the partition of the different component samples between the two phases. The gas chromatograph is widely applied to quantitative analysis work of gas, volatile liquid substances and solid samples, and the volatile organic substances are usually directly sampled and analyzed, and derivatization pretreatment is needed for the substances with low volatility and easy decomposition, so that the volatility and the stability of the analysis samples are improved.

2.1. Preparation of chromatographic samples

The gas chromatographic analysis method of the volatile short-chain fatty acid can adopt two methods of direct loading and methyl ester derivatization. The preparation method of the methyl ester derivatization can improve the volatility and the stability of the tested sample, reduce the tailing phenomenon which is easy to generate due to the strong polarity of the organic acid, and simultaneously enable the tested sample to be transferred from the water phase to the organic phase. However, this preparation method increases sample preparation time and cost, and may cause inaccuracy of the measurement result due to problems such as sample loss and incomplete derivatization. Considering that volatile acids in rumen fluid or silage fermentation broth are mainly acetic acid (118 ℃), propionic acid (141 ℃), isobutyric acid (154.5 ℃), butyric acid (163.5 ℃), isovaleric acid (176.5 ℃) and valeric acid (187 ℃), the present experiment used a direct loading chromatographic method.

Pretreatment of microbial fermentation liquid of rumen fluid and silage mainly comprises removal of solid particles, proteins and other interference components. The experiment was carried out by removing feed residues from the sample by first centrifugation, denaturing the protein by acidification and freezing, changing its sedimentation coefficient, and removing protein precipitate by re-centrifugation.

2.2. Selection of chromatographic columns

The experimental sample is a low molecular organic acid present in the aqueous phase and is therefore separated using an HP-INNOWax capillary chromatographic column. The HP-INNOWax column is a strong polar polyethylene glycol (PEG) cross-linked capillary column that can withstand sample separation with aqueous solvents.

2.3. Working principle of hydrogen flame ionization detector

The experiment utilizes a hydrogen flame ionization detector of gas chromatography to detect various volatile fatty acids.

A hydrogen flame ionization detector is one of the ionization type detectors that has the common feature of using some form of energy to ionize the detected components in the gas phase in the detector, the overall detector ionization efficiency being dependent on the ionization efficiency and ion collection efficiency of the ionization source.

The hydrogen flame ionization detector (FlameIonizationDetector, FID) is a hydrogen flame detector for short, and is the most widely used detector for gas chromatography. FID detector utilization H ₂ The sample is ionized at the flame to generate carbon positive ions by the heat energy and chemical energy of hydrogen flame generated by combustion in air, and weak ion current which moves directionally is formed under the action of an external electric field of a polarized electrode and an insulating gasket of a collector, and then amplified by a microcurrent amplifier and output to a chromatographic data workstation. The magnitude of the current is proportional to the amount of sample ionization.

2.4. Chromatographic quantification method

2.4.1 multistage correction of Standard samples

The chromatographic quantification method is to determine the content of the compound in a sample by using the peak height or peak area of a certain chromatographic peak. "correction" is the process of determining the corresponding component content or concentration using a certain peak height or peak area and establishing a regression curve. Correction is necessary when the detector has different detection sensitivities to different components in the mixed sample, or when the detector changes in response to different amounts of the same component.

Single stage correction: that is, single point correction, only one concentration level standard sample, no matter how many times the sample is sampled and averaged, is equivalent to only one correction as long as the concentration is the same, and the response curve must pass through the origin. The single-stage correction is suitable for detection experiments which do not need to accurately quantify an unknown sample and only concern whether the unknown sample meets the standard or not, and can also be applied to experiments in which the concentration of the unknown sample is smaller than and close to that of a standard sample.

Multistage correction: by analyzing standard compound samples at multiple concentration levels, nonlinear response of the detector to different content sample components can be compensated, a multi-stage calibration curve can be obtained, and the response curve can be over-origin. Each calibration level of the multi-level calibration corresponds to the concentration of each compound of one calibration standard. The concentration range of the unknown sample should be considered when determining each correction level. Firstly, preparing a concentrated standard sample (standard stock solution) with the concentration higher than that of an unknown sample, then establishing other correction samples of various grades by a progressive dilution method, and finally, enabling the concentration of the test sample to be contained in the grade of the correction standard sample. The least squares method is the most common fitting method in multi-stage correction.

2.4.2 internal standard correction method

In the experiment, 2-ethylbutyric acid (2 EB) is used as an internal standard substance, and the content of a certain component in an unknown mixed sample is calculated by adopting an internal standard correction and quantification method (ISTD).

The correction method can be simply classified into a normalization method, an external standard method and an internal standard method. When analyzing the content of a certain component in an unknown sample, an internal standard substance is added into the standard and the sample to eliminate the influence on the analysis result caused by fluctuation of the operation condition and improve the accuracy of the analysis result. The internal standard method working curve consists of the content ratio and the response value ratio of each level of standard of a certain compound in a standard sample, and the abscissa of the curve is the content ratio (AmourtRatio) and the area ratio (ArearRatio) respectively. The content ratio refers to the content of the grade compound divided by the content of the internal standard, and the response value ratio refers to the area of the grade compound divided by the area or height of the internal standard. And calculating a slope formula of a certain point according to the curve fitting type. The response value of the component of the unknown sample is divided by the response value of the internal standard of the unknown sample to obtain the response value ratio of the unknown sample, and the content of the component to be measured in the unknown sample is obtained by a curve fitting equation and the actual value of ISTD in the sample.

RF = content/area, RF value is an absolute correction factor, independent of other components of the sample and the content of that component. The response factor takes into account the fact that the same content does not necessarily yield the same detector response value.

The internal standard method has the advantages that: (1) Because the ratio of the internal standard substance to the content of the measured component is constant, the sample injection amount is not required to be accurate; (2) The ratio of the area of the detected component to the area of the internal standard substance is adopted for calculation, so that the response of the detector can be corrected, and therefore, the result is not affected by slight change of the operation condition, and the quantitative result is relatively accurate; (3) only the component of interest may be analyzed.

The key to the internal standard method is the choice of the appropriate internal standard, which must be met: (1) The internal standard substance is a pure substance which does not exist in the sample originally, has the property similar to that of the measured substance, can be completely dissolved in the sample, and can not react with the sample chemically. (2) The peak position of the internal standard should be as close as possible to the peak of the component to be measured or be located in the middle of the peaks of several objects to be measured and completely separated from these chromatographic peaks. (3) The mass of the internal standard should be close to that of the detected substance, and the size of the chromatographic peak can be kept close.

3. Instrument and materials

3.1. Instrument for measuring and controlling the intensity of light

Agilent6890N gas chromatograph, hydrogen flame ionization detector, HP-CHEM chromatographic workstation

TGL-16G type desk type high-speed centrifuge and GL-20B high-speed refrigerated centrifuge (Shanghai Anting scientific instrument factory)

Single-pass pipette: 200-1000 mu L of one, 40-200 mu L of one, 5-40 mu L of one

3.2. Chromatographic standard

4. Operating procedure

4.1. Preparation of standard solution

4.1.1 preparation of deproteinized solution containing internal Standard 2-ethylbutyric acid (2 EB)

25g of metaphosphoric acid and 0.217mL of 2-ethylbutyric acid were accurately weighed and fixed to a 100mL volumetric flask to prepare a 25% (w/v) metaphosphoric acid deproteinized solution containing 2g/L of internal standard 2 EB.

4.1.2 Preparation of 100mL Standard stock solution

Name of the name	Acetic acid	Propionic acid	Isobutyric acid	Butyric acid	Isopentanoic acid	Valeric acid
							Additive amount (mu L)	330	400	30	160	40	50
Final concentration (g/L)	3.46	3.97	0.29	1.53	0.38	0.47
							Molar concentration (mmol/L)	57.65	53.63	3.29	17.45	3.67	4.61

Preparation of 4.1.3VFA gradient dilution standard solution

0.2mL of the deproteinized metaphosphoric acid solution containing 2EB was added to 5 1.5mL centrifuge tubes, and 1mL, 0.8mL, 0.6mL, 0.4mL, and 0.2m of the mixed standard stock solution, and 0mL, 0.2mL, 0.4mL, 0.6mL, and 0.8mL of distilled water were added thereto, respectively, to prepare 5-stage VFA gradient standard solutions. The concentration of each component in the gradient standard solution is shown in the following table:

4.2. sample pretreatment

Rumen fluid or silage extract was centrifuged at 5400rpm for 10 min (radius r=14.5 cm, relative centrifugal force rcf= 1.119 ×10) ^-5 ×14.5×(5400) ² =4731g+). 1mL of the centrifugal supernatant and 0.2mL of 25% (w/v) metaphosphoric acid solution containing the internal standard 2EB are accurately added into a 1.5mL centrifuge tube, evenly mixed and placed in an ice water bath for more than 30 minutes. Centrifugation at 10000rpm for 10 min (r=5, rcf= 1.119 ×10) ^-5 ×5×(10000) ² ＝5595g)。

4.3. Gas chromatograph procedure

4.3.1 starting-up of the gas chromatograph

Checking the states of the gas pipeline connector and the gas bottle pressure reducing valve. The carrier gas (N) of the gas chromatograph was turned on ₂ ) And the fuel gas and combustion-supporting gas (H) required by the FID detector ₂ And air), and a leak check is performed. And opening the computer, and entering a Windows picture. The 6890NGC power switch is turned on. After the instrument self-checking is finished, the GCOnline icon is double-clicked, and the chemical workstation automatically communicates with 6890N.

4.3.2 editing of data acquisition methods

Selecting an item of 'edition EntitreMethod' from a menu of 'Method', selecting three items except 'Dataanalysis', clicking OK, and entering a next screen. In the "methods", information of the method (e.g., use of the method, etc.) is entered, and a click Ok is entered into the instrument operation parameter setting screen. The specific operating parameters are as follows:

sample inlet parameters:

carrier gas N ₂ Split ratio 40:1, sample injection amount 0.6 μl, temperature: 220 DEG C

Chromatographic column parameters:

model: HP-INNOWax capillary chromatography column (30.0mX320.mu.m X0.5. Mu.m, catalogNo: 19091J-213)

Constant flow mode, flow rate 2.0mL/min, average linear velocity 38cm/sec, column pressure 11.3psi column oven programming temperature:

120℃(3min)——10℃/min——180℃(1min)

detector parameters:

FID, temperature 250 ℃, H ₂ Flow rate 40mL/min, air flow rate 450mL/min, column flow rate + tail blow flow rate 45mL/min

Signal parameters:

signal 1 recorded post detector, signal 2 recorded post detector-column compensation curve 1, saved all data.

After editing the instrument operating conditions, "SaveMethodAs …" is selected in the "Method" menu, and the Method name is entered.

4.3.3 collecting Standard spectrogram and sample spectrogram

"Onlinesignal" is selected from the menu "View", windows1 is selected, then the Change button is clicked, the desired post-detector signal is moved to the right box, and OK is clicked. The "SampleInfo …" option is selected from the "RunControl" menu, the operator name is entered, and "Prefix" is selected from the "Datafile". A Prefix is entered in the Prefix box and a start bit of a Counter is entered in the Counter box (e.g., prefix is the date 40822 of the day of acquisition, and the Counter starts at 000). Clicking OK waits for instrument Ready, and the baseline is stable.

The experiment adopts the operation condition of temperature programming of a column temperature box, so that a column compensation curve is firstly adopted after a base line is stable so as to correct the base line drift. Clicking the COLComp1 button on the instrument panel of the chromatograph determines the detector compensated by the column compensation curve 1 as a post detector, starts to collect the column compensation curve by pressing the ENTER key, and the curve is invisible.

After the column compensation curve is collected, selecting 'RunMETHOD' from a Method menu, sampling the sample one by using standard samples with 5-level gradient, and collecting a chromatogram of the gradient standard.

And collecting chromatograms of unknown samples to be detected under the same chromatograph condition.

4.3.3 editing of data analysis methods

After the standard spectrogram is collected, clicking the data analysis from the View menu to enter a data analysis picture.

Selecting a 'LoadSignal' option from a 'File' menu, selecting a data File name of you, clicking OK, and performing spectrogram optimization firstly: selecting a SignalOptions option from a Graphics menu, selecting Autoscale and appropriate display time from Ranges, clicking OK or selecting UseRange adjustment. The process is repeated until the ratio of the figures is appropriate.

And (3) integral optimization: the standard sample chromatogram of the low concentration level that has been collected is called, and "Autointegration" is selected from "integration". If the integral result is not ideal, parameters such as slope sensitivity, chromatographic peak width, area rejection value, integrator opening and the like are optimized by means of an integral event table (integral) and a manual integral, and the integral attitude of the spectrogram is determined. When the satisfactory integral parameters are reached, clicking the left-hand 'V' icon to store the integral parameters into the method.

Establishing a multi-stage correction table (quantitative analysis): and respectively calling chromatograms of standard samples at all levels, inputting names and contents of the corresponding standard samples, identifying internal standard peaks, and automatically establishing a correction curve step by the instrument. And selecting the calization setting in the calization menu, setting correction parameters, inputting AmountUnits as mmol/L, and setting the curve Type as Linear.

Setting a report format: the specifiyReport is selected in the Report menu, and the calculation method in the quantitive result is selected as the ISTD internal standard method.

And saving a method file subjected to spectrogram optimization and curve correction.

Determination of unknown samples: and calling or collecting a spectrogram of the unknown sample, and obtaining the content of each component of the unknown sample by an internal standard calculation method according to the established integral parameters and the correction curve.

The prior art has shown that VFA has a good linear response at FID detectors, so the present laboratory also often uses simplified single point calibration for quantitative calculations. The following table gives the concentration (mmol/l), integrated area of the various VFAs in the Standard (STD) and integrated area of the various VFAs in the Sample (SAM), taking the determination of the acetic Acid (ACE) content in the sample as an example, the procedure for single point correction is shown in steps:

The method comprises the steps of establishing a single-stage correction curve by using a standard sample, namely establishing a single-point correction curve of ACE by using 2EB correction concentration of ACE in the standard and 2EB correction area of ACE in the standard.

Calculating ACE concentration in sample by using single-point calibration curve slope of ACE

To a third point, simplify the formula

Since the standard and the concentration of the internal standard in the sample are equal, the formula is finally simplified into

Similarly, the content of other components in the sample is determined by a single-point correction method. The sum of the VFA contents is the total volatile acid (TVFA) content of the sample, and the percentage of each VFA to TVFA is the percentage of the component in the sample (all the above steps are completed in the exell software).

4.3.4 instrument operation end shutdown

After the experiment is completed, a shutdown method which is programmed in advance is called up, and the method comprises the steps of switching off the FID detector, cooling each heat source (Olettemp, dettemp), and switching off the FID gas (H) ₂ Air). And after the temperature of each place is lower than 50 ℃, the chemical workstation is exited, the computer is closed, the GC power supply is turned off, and finally the carrier gas is turned off.

5. Notice matters

5.1. Sample pretreatment: the cut-off sample must be pre-treated to prevent clogging of the column by protein entering the column, high temperature coking.

5.2. Aging of the chromatographic column: when newly purchased columns, columns that have not been used for a long time, and analysis conditions change, aging of the columns is required. The aging process needs to separate the tail end of the chromatographic column from the detector, and the detector end is plugged to prevent the detector from being polluted; the aging of the chromatographic column should be carried out by introducing carrier gas at room temperature for 10min and then raising the temperature to prevent damage to the chromatographic column.

5.3. And (3) gas circuit inspection: FID detector requires H ₂ And the support is that the gas path leakage detection is required to be carried out before the instrument is operated so as to prevent the hydrogen leakage from being dangerous.

3. Determination of gas Components

1. Instrument and equipment

1. Gas chromatograph, beijing North Fentianpu Instrument technology Co.Ltd SP-2060T.

2.5A stainless steel column (Φ3mmx3m, 60-80 mesh support chromasorb) and Tbx-01 stainless steel column (Φ3mmx1m, 60-80 mesh support chromasorb).

2. Standard gas composition

Component name	Content V/V
		Methane	25.0％
Carbon dioxide	65.0％
		Hydrogen gas	2.03％
Oxygen gas	2.00％
		Nitrogen gas	Balance air

3. Measurement procedure

1. Firstly, a gas cylinder for protecting gas and argon is opened, the pressure is 0.5Mpa, two barometers above carrier gas on an instrument panel are checked, whether the two barometers are consistent or not is displayed, if the two barometers are different in pressure, the corresponding black valve below is regulated, and the values of the two barometers are consistent.

2. And (3) turning on a power supply of the instrument 2060T, turning on a small red power button on a panel when the column temperature and the detector on the instrument reach set values, turning on a computer, turning on an online workstation of N2000 software, and then enabling the machine to be stably preheated for 1h.

3. Taking out a standard gas cylinder, connecting a hose of the gas cylinder with one end of the left side sample of the instrument, opening a standard gas valve, screwing a silver valve beside the left side sample end of the instrument clockwise, rapidly screwing back anticlockwise, clicking a data acquisition button in an N2000 online workstation, performing sample measurement, and clicking and storing after the sample is tested;

4. And opening an offline workstation of the N2000 software, and processing the tested data in the offline workstation. The standard gas needs to be tested for several times until the two newly tested peak shapes of the standard gas can be completely overlapped, and the areas of the peaks are pasted through manual integration. And then testing the sample, wherein the steps are the same as the above, and finally calculating the content of each gas by using an external standard method.

4. Result calculation

And (3) after the standard gas is collected, the standard gas enters an N-2000 chromatographic workstation for data collection, and the peak areas of all components of the standard gas are obtained. Collecting a spectrogram of an unknown sample, and obtaining the content of each component of the unknown sample according to the standard gas peak area and the following calculation formula.

Wherein: c, sample, the concentration of a certain component in the measured sample; c standard sample, concentration of certain component in standard gas; a sample, the peak area of the tested component; a standard, the corresponding peak area in the standard gas.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A method for estimating the feed efficiency of pre-fattening beef cattle through artificial rumen fermentation gas production, which is characterized by comprising the following steps:

s1, collecting rumen fluid of ruminants to be tested, which is suitable for a feeding environment, and mixing the rumen fluid with artificial saliva to prepare in-vitro artificial rumen fermentation liquor; the volume ratio of the rumen fluid to the artificial saliva is 1:2;

s2, drying the stable daily ration with the granularity of 0.5-1mm to obtain a stable daily ration air-dried sample;

s3, placing a stable daily ration air-dried sample into the in-vitro artificial rumen fermentation liquid, culturing for 48 hours in a water bath shaking table at 39 ℃, taking out half of gas production pipes when fermenting for 24 hours, and ending the fermentation in an ice water bath; after fermentation, calculating one or more parameters of net gas yield, methane gas yield, net pH reduction value, net increase of volatile acid and net increase of ammoniacal nitrogen; the feed efficiency of the ruminant to be tested is in direct proportion to the net gas yield, methane gas yield, net increase in volatile acid and net increase in ammoniacal nitrogen, and in inverse proportion to the net decrease in pH.

2. The method for estimating the feed efficiency of pre-fattening beef cattle according to claim 1, wherein in step S1, the rumen fluid collection method comprises: collecting rumen fluid of the ruminant to be tested before morning feeding through an oral gastric tube, discarding the rumen fluid at the front section, squeezing and filtering the content by using medical gauze, then placing the rumen fluid in a threaded freezing storage tube, storing the rumen fluid in a heat-preserving barrel containing warm water at 39 ℃, and placing the rumen fluid in an in-vitro rumen fermentation system for fermentation for 48 hours.

3. The method for estimating the efficiency of pre-fattening beef cattle feed according to claim 1, wherein in step S1, the method for preparing artificial saliva comprises: taking a culture solution split charging bottle, putting into a stirring magnetic rod, and putting into a circulating water bath; then distilled water, trace element solution, buffer solution, macroelement solution and resazurin solution are added in sequence and uniformly mixed, and the solution is blue at the moment; heating to 39deg.C, adding reducing solution, and introducing CO ₂ Saturated, the solution gradually turns from blue to pink, and then turns to light red or colorless, and the artificial saliva is obtained;

the volume fraction of the trace element solution is 0.025 percent based on the distilled water content; the volume fraction of the buffer solution is 50%; the volume fraction of the macroelement solution is 50%; the volume fraction of the resazurin solution is 0.25%.

4. The method for estimating the efficiency of pre-fattening beef cattle feed according to claim 3, wherein the trace element solution comprises calcium chloride, manganese chloride, cobalt chloride and ferric chloride;

and/or, the buffer solution comprises ammonium bicarbonate and sodium bicarbonate;

And/or the macroelement solution comprises anhydrous disodium hydrogen phosphate, anhydrous monopotassium phosphate and magnesium sulfate.

5. The method for estimating a feed efficiency of a pre-fattening beef cattle according to any one of claims 1 to 4, wherein in step S1, the ruminant to be tested is a beef cattle of generally 6 to 12 months of age;

and/or the ruminant to be tested adapting to the feeding environment is to feed the ruminant to be tested in the feeding environment for 7-14 days by adopting the stable ration.

6. The method for estimating the feed efficiency of pre-fattening beef cattle according to claim 1, wherein in step S2, the method for drying the stable ration comprises: and (5) drying the stabilized grains in an oven for 48+/-5 hours at 65+/-5 degrees.

7. The method for estimating the efficiency of pre-fattening beef cattle feed according to claim 1, wherein in step S3, the mass-volume ratio of the stable daily ration air-dried sample to the in-vitro artificial rumen fermentation broth is 0.22 g/30 ml.

8. The method for estimating the efficiency of pre-fattening beef cattle feed according to claim 1, wherein the net gas production = gas production of ruminants to be tested at a certain time point-the average gas production of a blank tube at the time point is in mL;

Methane yield, mL = total gas yield x percentage of methane;

net methane yield = loading ration gas production tube methane yield-corresponding animal blank gas production tube methane yield;

net pH decrease = loading ration gas production tube pH-loading ration gas production tube pH corresponding to animal blank gas production tube;

ammonia nitrogen increment = loading ration gas production tube ammonia nitrogen concentration-corresponding to animal blank gas production tube loading ration gas production tube ammonia nitrogen concentration.

9. The method for estimating a feed efficiency of a pre-fattening beef cattle according to claim 8, wherein the dynamic fermentation parameter calculation model is as follows:

Y＝B(1–e ^-ct )

wherein: y is the accumulated gas yield of the t time point, and the unit is mL; b is theoretical maximum gas yield per mL; c is the gas production rate, unit h ^-1 T is the culture time, unit h;

the gas production tube is taken out at each time point of 0, 2, 4, 6, 8, 12, 16, 20, 24, 32, 40 and 48 hours respectively, the scale value is recorded, when the scale value exceeds 70mL, the gas is exhausted, and the gas in the gas production tube is extracted by a syringe needle tube to measure the gas component.

10. The method for estimating the efficiency of beef cattle feed before fattening by artificial rumen fermentation gas production according to any one of claims 1 to 9, wherein in step S3, 220mg of stable grain air is weighed and dried, and sent to the front end of a 100mL gas production tube by a long handle medicine spoon, and preheated in an incubator at 39 ℃; injecting 20mL of artificial rumen fermentation liquid from the front end of the gas production pipe by using a liquid separating device, and injecting 10mL of rumen liquid of ruminant to be tested by using an injector; the gas production tube of the blank control group is not added with a substrate, 20mL of artificial saliva is injected from the front end of the gas production tube by a liquid separating device, and 10mL of rumen liquid of ruminant to be tested is injected by a syringe;

Exhausting bubbles in the gas production tube, recording initial scale values of the gas production tube, then placing the gas production tube in a 39 ℃ constant-temperature water bath box for culture, wherein 2n blank tubes of each ruminant to be tested, 2n test tubes and at least 2 repetition are carried out, and n is an integer; weighing 0.22g of samples respectively; the scale values of the gas production pipes are read and recorded at 2, 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36 and 48 hours, one half of the gas production pipes in each group of experiments are cultivated to 24 hours, the rest gas production pipes are used for stopping fermentation in an ice bath at 48 hours to measure volatile acid, ammonia nitrogen and pH, and the net gas production measurement is carried out through accumulated gas production.