Method for evaluating feed digestibility through rumen in-vitro digestion model

Abstract

The invention provides a method for evaluating feed digestibility through a rumen in-vitro digestion model, and belongs to the technical field of feed digestion evaluation. The method comprises the following steps: pretreating a fiber bag, loading a feed sample, sealing to obtain a sample bag, and placing the sample bag in a fermentation bottle; respectively preparing two buffer solutions, mixing the two buffer solutions in proportion to form a mixed buffer solution simulating a rumen environment, and placing the mixed buffer solution in a fermentation bottle with a sample bag; rumen fluid is prepared and treated, then the rumen fluid is inoculated into a fermentation bottle with a sample bag, and anaerobic culture is carried out; treating the sample bag after culture is finished, measuring the weight of the neutral detergent fiber, and calculating the real in-vitro digestibility of the feed through a correction formula. The method provided by the invention is simple to operate, the reagent formula is simplified, the labor cost and the consumable consumption are remarkably reduced, the measurement precision is improved by simulating the rumen fermentation environment, and a low-cost and standardized technical scheme is provided for rapid evaluation of the digestibility of the ruminant feed.

Description

Technical Field

[0001] The present invention relates to the technical field of feed digestion evaluation, and particularly to a method for evaluating the digestibility of feed by using an in vitro rumen digestion model. Background Art

[0002] In the field of evaluating ruminant feed digestion technology, the digestibility of feed is an important indicator for measuring the quality and nutritional value of feed. Accurately evaluating the digestibility of feed is of crucial significance for rationally formulating feed, improving breeding efficiency, reducing breeding costs, and ensuring the healthy growth of animals.

[0003] Currently, there are mainly three methods for evaluating the digestibility of ruminant feed, namely in vivo method, nylon bag method, and in vitro method. And from the commonly used digestion and metabolism tests, nylon bag tests, etc., artificial rumen tests, gas production method, moving nylon bag method have been derived. In vitro tests are favored for their simple operation, no need for animals, short determination time, low cost, easy standardization and other characteristics. The traditional method for evaluating feed digestibility is mainly through in vivo digestion tests, that is, letting animals actually eat feed, and then collecting and analyzing excreta such as feces to calculate the digestibility.

[0004] However, the existing methods for evaluating feed digestibility have the following problems: (1) In vivo digestion tests require a long time. From the adaptation period to the formal test period of animals, the whole process may take several weeks or even months. This not only increases the test cost, but also makes the efficiency of feed evaluation low and unable to provide a basis for adjusting the feed formula in a timely manner. (2) In vivo digestion tests need to use a large number of animals as test subjects, involving costs in many aspects such as animal purchase, feeding, and management. In addition, professional test sites and equipment are required, further increasing the test cost. (3) There are physiological and metabolic differences among different animal individuals, and these differences will affect the measurement results of feed digestibility, resulting in a decrease in the accuracy and reliability of test results. (4) Although the rumen environment in animals is relatively stable, in actual breeding, the rumen environment of animals will be affected by various factors, such as feed types, feeding and management methods. It is difficult to control these factors in traditional in vivo tests, so it is difficult to accurately simulate the complex and changeable rumen environment.

[0005] Therefore, there is an urgent need for a method for evaluating feed digestibility that is fast, accurate and low-cost, which has important practical significance. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for evaluating the digestibility of feed using an in vitro rumen digestion model. This method is simple to operate, simplifies the reagent formulation, significantly reduces labor costs and consumable consumption, and improves the measurement accuracy by simulating the rumen fermentation environment, providing a low-cost and standardized technical solution for the rapid evaluation of ruminant feed digestibility.

[0007] To achieve the above object, the present invention provides the following solutions: A method for evaluating the digestibility of feed using an in vitro rumen digestion model, comprising the following steps: S1. Pretreat the fiber bag, load the feed sample, seal to obtain a sample bag, and place it in a fermentation bottle; S2. Prepare two buffer solutions and mix them in proportion to form a mixed buffer solution simulating the rumen environment, and place it in the fermentation bottle containing the sample bag; S3. Prepare rumen fluid and process it, then inoculate it into the fermentation bottle containing the sample bag for anaerobic culture; S4. After the culture is completed, process the sample bag, measure the weight of neutral detergent fiber, and calculate the true in vitro digestibility of the feed through a correction formula.

[0008] Preferably, in S1, the pretreatment process of the fiber bag is as follows: pre-rinse the fiber bag with acetone for 3 - 5 min, remove the surfactant, air-dry and weigh it, denoted as W 1; then weigh 0.5 - 1.0 g of the feed sample and load it into the treated fiber bag, and denote the sample weight as W 2; finally, heat-seal the mouth of the fiber bag containing the feed sample, and simultaneously prepare a blank correction fiber bag.

[0009] Preferably, in S2, the two buffer solutions include: Buffer A, with components and concentrations: KH2PO4 10.0 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, CaCl2·2H2O 0.1 g / L, urea 0.5 g / L; Buffer B, with components and concentrations: Na2CO3 15.0 g / L, Na2S·9H2O 1.0 g / L.

[0010] Preferably, in S2, the mixing process of the two buffer solutions is as follows: pre-heat Buffer A and Buffer B to 39 °C respectively, mix them according to the volume ratio of Buffer B:Buffer A of 1:5, and adjust the pH to 6.8 at 39 °C to obtain a mixed buffer solution; then add the mixed buffer solution to the fermentation bottle containing the sample bag and place it in a constant temperature air bath shaker to balance the temperature for 20 - 30 min to obtain a mixed buffer solution simulating the rumen environment.

[0011] Preferably, in S3, the treatment process of the rumen fluid is as follows: collect the rumen fluid and place it in a thermos flask at 39°C, pour it into a stirrer, purge the container with CO2, and quickly stir for 30 s to disperse the microorganisms; then filter through four layers of gauze to obtain the rumen fluid as the inoculum.

[0012] Preferably, in S3, the process of anaerobic culture is as follows: add the rumen fluid to a fermentation flask with a sample bag, purge the inside of the fermentation flask with CO2 for 30 s to form an anaerobic gas covering layer, and seal the fermentation flask; then place the fermentation flask in a constant temperature air bath shaker at a temperature of 39 - 40°C and culture for 36 - 48 h for anaerobic culture.

[0013] Preferably, the constant temperature air bath shaker continuously stirs the fermentation flask during the culture process to simulate the rumen peristalsis environment and ensure sufficient contact between the microorganisms and the sample in the sample bag.

[0014] Preferably, in S4, the process of treating the sample bag after the culture is completed is as follows: after the culture is completed, drain the fermentation broth in the fermentation flask and rinse the sample bag in the fermentation flask; then mechanically stir to avoid loss of the sample in the sample bag; finally, put the rinsed sample bag into a fiber analyzer, and according to the standard NDF determination procedure, determine the neutral detergent fiber weight and record the final weight as .

[0015] Preferably, when rinsing the sample bag, cold tap water is used and rinsed until the effluent is clear to remove the residual fermentation broth and soluble components on the sample bag.

[0016] Preferably, in S4, the correction formula is: ; where IVTD is the in vitro true digestibility of the feed, W 1 is the tare weight of the fiber bag, W 2 is the total weight of the sample bag, that is, the sum of the weights of the fiber bag and the feed sample, is the weight of the sample bag after measurement, C 1 is the correction coefficient of the blank correction fiber bag, and the correction coefficient of the blank correction fiber bag is obtained by calculating a blank correction fiber bag processed in the same steps as the sample bag.

[0017] According to the specific embodiments provided by the present invention, the following technical effects of the present invention are disclosed: (1) Compared with the solution of publication number CN114350475B, the present invention simplifies the experimental process significantly by using a simplified formulation of only two buffers, combining fiber bag pretreatment with direct determination of NDF by a fiber analyzer. The reagent preparation steps are reduced by 60%, the consumable cost is reduced by 30%, and there is no need for complex pH adjustment or live animals. Compared with the traditional nylon bag method, such as publication number CN116908038A, the present application eliminates the carrier error by blank-correcting the fiber bag, improving the measurement accuracy by about 3.3 times, and there is no need for repeated experiments, significantly reducing the labor cost.

[0018] (2) The present invention restores the rumen physico-chemical conditions by using a thermostatic air bath shaker (39 - 40 °C), purging the anaerobic environment with CO2, and buffer ratio; and eliminates the carrier error through the correction coefficient of the blank fiber bag, directly correlating NDFv the degradation amount with the sample weight, improving the measurement accuracy. At the same time, the results are more intuitive and reliable, avoiding indirect calculation errors.

[0019] (3) The culture period of the present invention is shortened to 36 - 48 hours. By combining a standardized fermentation bottle with a general-purpose thermostatic air bath shaker, ≥20 samples can be processed simultaneously in a single batch. For example, the commonly used THZ-300C shaker in the laboratory can accommodate 24 fermentation bottles, supporting rapid screening of feed formulations; completely eliminating the need for live animals, only a small amount of rumen fluid is required, solving the problems of long cycle and large individual differences in the traditional in vivo method. Brief Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a flowchart of a method for evaluating feed digestibility by a rumen in vitro digestion model of the present invention. Detailed Embodiments

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0023] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0024] As Figure 1 shown, the present invention provides a method for evaluating the feed digestibility by using an in vitro rumen digestion model, comprising the following steps: S1. Pretreat the fiber bag, load the feed sample, seal to obtain a sample bag, and place it in a fermentation bottle; S2. Prepare two kinds of buffer solutions respectively and mix them in proportion to form a mixed buffer solution simulating the rumen environment, and place it in the fermentation bottle with the sample bag; S3. Prepare rumen fluid, process it, and then inoculate it into the fermentation bottle with the sample bag for anaerobic culture; S4. After the culture is completed, process the sample bag, measure the weight of neutral detergent fiber, and calculate the true in vitro digestibility of the feed through a correction formula.

[0025] The specific process of the above steps is as follows: (1) First, take the fiber bag, mark the osmotic number, pre-rinse it with acetone for 3 - 5 minutes to remove the surfactant, and then place it in a ventilated place to dry completely; then use a balance to weigh the weight of the dried fiber bag, denoted as W 1, with the unit of g; (2) Secondly, weigh 0.5 - 1.0 g of the feed sample, load it into the pretreated fiber bag, and weigh and record the sample weight as W 2, with the unit of g; then use a heat sealer to seal the mouth of the fiber bag to ensure that no sample leaks out. At the same time, prepare at least 1 blank correction fiber bag according to the same steps, without loading the sample, only seal the empty bag; finally, put the sealed sample bag and the blank correction fiber bag into the fermentation bottle respectively for standby; (3) Prepare buffer solution A and buffer solution B respectively. Among them, the components and concentrations of buffer solution A are: KH2PO4 10.0 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, CaCl2·2H2O 0.1 g / L, urea 0.5 g / L; The components and concentrations of buffer solution B are: Na2CO3 15.0 g / L, Na2S·9H2O 1.0 g / L; Dissolve each component in deionized water, stir until completely dissolved, make up to the required volume, and preheat the two buffer solutions to 39°C in advance.

[0026] (4) Mix according to the volume ratio of buffer solution B: buffer solution A of 1:5, adjust the pH of the mixed solution to 6.8 at 39°C to obtain a mixed buffer solution, then add 1600 mL of the mixed buffer solution to the fermentation bottle containing the sample bag, put it into a constant temperature air bath shaker, and balance the temperature for 20 - 30 minutes; (5)Collect and prepare the rumen fluid, place it in a thermos flask at 39 °C, pour it into a blender, purge the container with CO2, and stir at high speed for 30 seconds to disperse the microorganisms. Then filter through four layers of gauze, measure 400 mL of the filtrate as the inoculum, and add it to the fermentation flask. Purge the inside of the flask with CO2 for 30 seconds to form an anaerobic gas overlay, and seal the fermentation flask. Place the fermentation flask in a constant temperature air bath shaker and incubate at 39 - 40 °C for 36 - 48 hours. Continuously stir the fermentation flask during the incubation to simulate rumen peristalsis.

[0027] It should be noted that the above-mentioned rumen fluid is taken from healthy adult cows (or sheep). The specific collection method is as follows: Through conventional animal experiment operations, rumen intubation or aseptic collection of rumen fluid is carried out on healthy adult cows (or sheep). After collection, immediately filter through four layers of gauze to remove impurities such as food residues. The filtrate is used as the inoculum for standby. The role of the inoculum is that the rumen fluid contains the microbial community in the rumen of ruminants (such as bacteria, protozoa, etc.). As the inoculum, it can simulate the digestive function of rumen microorganisms in an in vitro fermentation system, decompose components such as fiber in the feed sample, and thus realize the evaluation of feed digestibility.

[0028] (6)After the incubation, drain the fermentation broth, rinse the sample bag with cold tap water until the water is clear, and minimize mechanical stirring to avoid sample loss. During the rinsing process, minimize mechanical stirring, that is, control the stirring speed through experimental equipment (such as low-speed stirring can be used) to avoid sample loss caused by fiber bag breakage or sample detachment due to violent stirring. Put the rinsed sample bag into a fiber analyzer, operate according to the standard NDF determination procedure, and record the final weight as , in grams; (7)Process the blank correction fiber bag according to the same steps, calculate the correction coefficient C1, that is, the ratio of the dried weight of the blank bag after in vitro fermentation and NDF analysis to the original blank bag weight; Calculate the in vitro true digestibility according to the formula ( IVTD , in %): ; Among them, IVTD is the in vitro true digestibility of the feed, W 1is the tare weight of the fiber bag, W 2is the total weight of the sample bag, that is, the sum of the weights of the fiber bag and the feed sample, is the weight of the sample bag after measurement, C 1is the correction coefficient of the blank correction fiber bag, that is: ; The correction coefficient of the blank correction fiber bag is obtained by calculating the blank correction fiber bag processed in the same steps as the sample bag.

[0029] Therefore, the method for evaluating the feed digestibility using the above-mentioned in vitro rumen digestion model is simple to operate, simplifies the reagent formula, significantly reduces the labor cost and consumable consumption, and improves the measurement accuracy by simulating the rumen fermentation environment, providing a low-cost and standardized technical solution for the rapid evaluation of the feed digestibility of ruminants.

[0030] In this article, specific examples are used to elaborate on the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A method for evaluating feed digestibility using an in vitro rumen digestion model, characterized in that: The following steps are involved: S1, pre-treating the fiber bag, filling it with the feed sample, sealing the sample bag and placing it in a fermentation bottle; S2. Prepare two buffer solutions respectively and mix them in proportion to form a mixed buffer solution simulating the rumen environment, and place it in a fermentation bottle with a sample bag; the two buffer solutions include: Buffer A, composition and concentration: KH2PO4 10.0 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, CaCl2·2H2O 0.1 g / L, urea 0.5 g / L; Buffer B, composition and concentration: Na2CO3 15.0 g / L, Na2S·9H2O 1.0 g / L; S3, preparing rumen fluid and treating it, and then inoculating it into a fermentation bottle with a sample bag for anaerobic culture; S4. After the culture is completed, the sample bag is processed, the weight of the neutral detergent fiber is measured, and the in vitro true digestibility of the feed is calculated using a correction formula.

2. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 1, characterized in that: In S1, the pretreatment process of the fiber bag is: pre-rinsing the fiber bag with acetone for 3-5 minutes, removing the surfactant, air-drying and weighing, which is recorded as W 1; Then weigh 0.5~1.0g of feed sample and put it into the treated fiber bag, and the sample weight is recorded as W 2. Finally, heat seal the mouth of the fiber bag containing the feed sample and prepare a blank calibration fiber bag at the same time.

3. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 1, characterized in that: In S2, the mixing process of the two buffer solutions is as follows: the buffer solution A and the buffer solution B are preheated to 39°C respectively, and mixed at a volume ratio of buffer solution B:buffer solution A of 1:5, and the pH is adjusted to 6.8 at the mixing temperature of 39°C to obtain a mixed buffer solution; then, the mixed buffer solution is added to the fermentation bottle containing the sample bag, and the mixed buffer solution is placed in a constant temperature air bath shaker to balance the temperature for 20 to 30 minutes to obtain a mixed buffer solution that simulates the rumen environment.

4. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 1, characterized in that: In S3, the rumen fluid processing process is as follows: collect the rumen fluid and place it in a 39°C thermos bottle, pour it into a blender and purge the container with CO2, and quickly stir for 30 seconds to disperse the microorganisms; then filter through four layers of gauze to obtain the rumen fluid as an inoculum.

5. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 1, characterized in that: In S3, the anaerobic culture process is as follows: adding the rumen fluid to the fermentation bottle containing the sample bag, purging the fermentation bottle with CO2 for 30 seconds to form an anaerobic gas covering layer, and sealing the fermentation bottle; then placing the fermentation bottle in a constant temperature air bath shaker at a temperature of 39 to 40°C for 36 to 48 hours for anaerobic culture.

6. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 5, characterized in that: The constant temperature air bath shaker continuously stirs the fermentation bottle during the culture process to simulate the rumen peristalsis environment, so as to ensure that the microorganisms are fully in contact with the sample in the sample bag.

7. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 1, characterized in that: In S4, the process of processing the sample bag after the cultivation is completed is as follows: after the cultivation is completed, the fermentation liquid in the fermentation bottle is discharged, and the sample bag in the fermentation bottle is rinsed; Then mechanical stirring is used to avoid sample loss in the sample bag; finally, the rinsed sample bag is placed in a fiber analyzer, and the neutral detergent fiber weight is measured according to the standard NDF determination procedure, and the final weight is recorded as .

8. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 7, characterized in that: The sample bag is rinsed with cold tap water until the water is clear, so as to remove the fermentation liquid and soluble components remaining on the sample bag.

9. The method for evaluating feed digestibility using an in vitro rumen digestion model according to claim 7, characterized in that: In S4, the correction formula is: ； in, IVTD is the actual in vitro digestibility of feed, W 1 is the tare weight of the fiber bag, W 2 is the total weight of the sample bag, i.e. the sum of the weight of the fiber bag and the feed sample. is the weight of the sample bag after measurement, C 1 is the correction coefficient of the blank correction fiber bag, which is obtained by calculating the blank correction fiber bag processed in the same step as the sample bag.

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