Method for measuring metabolic energy of shelduck feed raw materials by using in-vitro digestion method

Abstract

The invention discloses a method for measuring metabolic energy of shelduck feed raw materials by using an in-vitro digestion method. The method comprises the steps of preparing a buffer solution and a digestive juice, simulating stomach digestion, simulating duodenum digestion, simulating empty ileum digestion, calculating metabolic energy and the like. The method is simple and convenient, low in price, wide in application scene and capable of meeting the requirement for rapid detection of metabolic energy of a large number of samples; the digestion integrating degree with the real in-vivo digestion of ducks is higher, and the metabolic energy measurement is more stable and accurate.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition technology, specifically relating to a method for determining the metabolizable energy of duck feed ingredients using in vitro digestion. Background Technology

[0002] Energy is the most important nutrient in feed, a key element for promoting animal growth, production, and maintaining life activities. The energy level of duck feed is assessed using a metabolizable energy system. Rapid and accurate evaluation of the metabolizable energy of feed ingredients is of great significance for the development of unconventional feed resources and the application of low-protein feeds, and is a technical means to optimize feed formulation, reduce feed costs, and improve farming efficiency.

[0003] Currently, the following methods are used to determine the metabolizable energy of duck feed ingredients: ① Calculation based on physicochemical composition and prediction formula. This method is based on measuring the physicochemical composition of raw materials, such as grain density, grain weight, endosperm hardness, moisture, crude ash, crude fat, crude protein, and crude fiber, and then using prediction formulas to calculate the metabolizable energy of the raw materials. Organizations such as the Chinese Academy of Agricultural Sciences, the National Research Council (NRC) of the United States, the French National Institute for Agricultural Research (INRA), and the Dutch Federation of Animal Feed Chains (FND) have established databases of feed raw material physicochemical composition and provided formulas and values ​​for estimating metabolizable energy values. The drawback of this method is that the physicochemical measurement results only reflect the nutritional components of the raw materials and do not consider the digestibility and utilization rate of nutrients in the duck's body. Furthermore, different researchers have different measurements of the physicochemical composition of raw materials, and the physicochemical composition of raw materials from different origins varies. Therefore, the metabolizable energy value estimated using this method has a significant error.

[0004] ② Near-infrared reflectance spectroscopy. This method estimates the metabolizable energy by measuring the infrared absorption of chemical bonds in organic compounds. Its disadvantages include the high cost of the equipment and the complex setup and calibration required. Furthermore, the accuracy of this method's estimation of metabolizable energy depends heavily on the volume and quality of the local reference data, making it unsuitable for applications with low detection frequency. Additionally, sample particle size and density affect the infrared radiation detection depth, thus this method has specific requirements regarding the physical properties of the sample.

[0005] ③ Forced feeding method. This is a classic method for determining the metabolizable energy of duck feed. It involves forcibly feeding a known amount of feed, collecting the animals' excrement, and analyzing the energy differences to calculate the metabolizable energy value. The disadvantages of this method are that the experimental animals must be strictly fasted, force-fed, and their excrement collected at regular intervals, making the experiment complex and requiring high operational skills. Furthermore, fasting and forced feeding may cause stress in the ducks, affecting their digestive function and causing the measured metabolizable energy value to deviate from the true situation. Secondly, individual differences in ducks' tolerance to forced feeding and their digestive and absorptive abilities may increase data variability and reduce experimental repeatability. Finally, this method is not suitable for high-fiber or low-palatability feed ingredients, as it may cause digestive abnormalities in the animals, leading to deviations in the measured values.

[0006] ④ Bionic Digestion Method. This method simulates the digestive tract environment of ducks (such as pH, enzymes, and temperature) and uses computer programming to control the entire digestion process, measuring the metabolizable energy of feed ingredients. The disadvantages of this method are: the bionic digestive system and its equipment are expensive, and its applicability is limited. Secondly, this method only simulates the stomach and intestines, ignoring the differences in digestive and absorptive capacity among different intestinal segments of ducks; therefore, the measured values ​​differ significantly from the actual values. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of the prior art and provide a method for determining the metabolizable energy of duck feed ingredients using in vitro digestion.

[0008] To achieve the above objectives, the technical solution provided by this invention is as follows: The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion includes the following steps: (1) Preparation of buffer solutions and digestive juices: Prepare gastric digestive buffer solution, duodenal digestive buffer solution, jejunal and ileal digestive buffer solution, as well as gastric digestive juice, duodenal digestive juice, and jejunal and ileal digestive juice respectively; (2) Simulated gastric digestion: Insert one end of the dialysis bag into the first silicone stopper and secure it with a rubber band. Place the feed raw material sample to be tested and the gastric digestion fluid into the dialysis bag. Then insert the other end of the dialysis bag into the second silicone stopper with a glass tube and secure it with a rubber band. Place the dialysis bag into an empty screw-cap test tube. Fill the screw-cap test tube with gastric digestion buffer and tighten the cap. The screw cap has a small hole. The glass tube of the second silicone stopper extends out from the small hole in the screw cap to form a gastric digestion chamber. Place the gastric digestion chamber in a constant temperature shaking water bath at 37°C for gastric digestion reaction. After the reaction is completed, remove the gastric digestion fluid from the dialysis bag. (3) Simulated duodenal digestion: Add duodenal digestion fluid to the dialysis bag after the gastric digestion reaction in step (2), place the dialysis bag in an empty screw-cap test tube, fill the screw-cap test tube with duodenal digestion buffer and tighten the cap. The screw cap has a small hole, and the glass tube of the second silicone stopper extends out of the small hole in the screw cap to form a duodenal digestion chamber. Place the duodenal digestion chamber in a constant temperature shaking water bath at 37°C to carry out the duodenal digestion reaction. After the reaction is completed, remove the duodenal digestion fluid from the dialysis bag. (4) Simulated ileal digestion: Add ileal digestion solution to the dialysis bag after the duodenal reaction in step (3), place the dialysis bag in an empty screw-cap test tube, fill the screw-cap test tube with ileal digestion buffer solution and tighten the screw cap. The screw cap has a small hole, and the glass tube of the second silicone stopper extends out from the small hole of the screw cap to form an ileal digestion chamber. Place the ileal digestion chamber in a constant temperature shaking water bath at 37°C to carry out the ileal digestion reaction. After the reaction is completed, remove the ileal digestion solution from the dialysis bag. (5) Metabolizable energy calculation: Collect the undigested residue remaining after the digestion reaction in step (4), dry it to constant weight, preferably at 105℃, measure the dry matter weight and energy of the undigested residue, and then calculate the metabolizable energy of the feed raw material sample to be tested by combining the dry matter weight and total energy of the feed raw material sample to be tested.

[0009] Preferably, the pH value of the gastric digestive buffer and gastric digestive fluid is 4.3, and the pH value of the duodenal digestive buffer, duodenal digestive fluid, jejunal and ileal digestive buffer, and jejunal and ileal digestive fluid is 6.0.

[0010] Preferably, each 5 mL of the gastric digestive fluid contains 1593 U of trypsin, 8825.5 U of amylase, 1507 U of lipase, and 3076 U of chymotrypsin; each 5 mL of the duodenal digestive fluid contains 1236.5 U of trypsin, 10701.5 U of amylase, 6946 U of lipase, and 1003.5 U of chymotrypsin; and each 5 mL of the jejunal and ileal digestive fluid contains 2077 U of trypsin, 11261.5 U of amylase, 3035.5 U of lipase, and 541.5 U of chymotrypsin.

[0011] Preferably, the gastric digestion reaction time is 3 hours, the duodenal digestion reaction time is 3 hours, and the jejunal and ileal digestion reaction time is 12 hours.

[0012] Preferably, the shaking condition in the constant temperature shaking water bath is 90 rpm, and the stomach digestion chamber, duodenal digestion chamber, and jejunum digestion chamber are all placed at an angle of 45° in the constant temperature shaking water bath.

[0013] Preferably, the formula for calculating metabolizable energy is: Total energy of the feed ingredient sample to be tested: GE1 = E1 × M1 GE1 is the total energy of the feed ingredient sample to be tested, in J; E1 is the dry matter energy value of the feed ingredient sample to be tested, in J / g; M1 is the dry matter mass of the ingredient sample, in g. Total energy of undigested residue: GE2 = E2 × M2 GE2 is the total energy of the undigested residue sample, in J; E2 is the dry matter energy value of the undigested residue sample, in J / g; M2 is the dry matter mass of the undigested residue sample, in g. Metabolizable energy of duck feed ingredients: In vitro ME = (GE1 - GE2) ÷ M1 ÷ 1000 InvitroME represents the in vitro digestible metabolizable energy of the tested duck feed ingredients on a dry matter basis, expressed in MJ / kg.

[0014] Preferably, in step (2), the mass-to-volume ratio of the feed raw material sample to be tested and the gastric digestive fluid in the dialysis bag is 2±0.05g:5±0.5mL, more preferably 2g:5mL; in step (3), the amount of duodenal digestive fluid added to the dialysis bag is equal to the amount of gastric digestive fluid in step (2); in step (4), the amount of jejunal and ileal digestive fluid added to the dialysis bag is equal to the amount of duodenal digestive fluid in step (3).

[0015] The present invention also provides an in vitro digestion device for the above-described method. The in vitro digestion device includes a screw-capped test tube, a dialysis bag, a rubber band, a first silicone stopper, and a second silicone stopper. One end of the dialysis bag is fitted onto the first silicone stopper and secured with a rubber band, and the other end is fitted onto the second silicone stopper and secured with a rubber band. A glass tube is inserted into the second silicone stopper. The dialysis bag contains a sample of the feed ingredient to be tested and digestive fluid. The sealed dialysis bag is placed inside the screw-capped test tube, which is filled with digestive buffer solution. The screw cap of the test tube has a pre-drilled hole, and the glass tube extends from the hole in the screw cap.

[0016] The present invention will be further described below: This invention addresses the problem of expensive instruments and equipment in existing technologies by using commonly used laboratory instruments and equipment, such as test tubes, beakers, and water bath constant temperature shaking pots, to simulate the duck digestive tract and measure the metabolizable energy of feed ingredients in vitro.

[0017] Existing biomimetic simulations of the duck digestive tract are mainly divided into one-stage and two-stage methods. One-stage in vitro digestion integrates the duck's stomach, duodenum, jejunum, and ileum into a single digestive cavity to complete the in vitro digestion process. Two-stage in vitro digestion designs the duck's stomach digestion as one digestive cavity, and integrates the duodenum, jejunum, and ileum into a second digestive cavity. Both technologies consider the digestive differences between the duck's duodenum, jejunum, and ileum. This invention collects digestive fluids from the duck's stomach, duodenum, jejunum, and ileum at different time points, accurately measuring the digestive enzyme activity, pH, temperature, and digestion time in different digestive segments. Data analysis revealed a significant difference in duodenal digestive enzyme activity compared to jejunal and ileal digestive enzyme activity, but no significant difference in pH, temperature, and digestion time. Therefore, this invention adopts a three-stage method, simulating gastric digestion, duodenal digestion, and jejunoileal digestion respectively, improving the completeness of in vitro digestion and the accuracy of metabolizable energy measurement. The gastric, duodenal, and jejunal digestive chambers all consist of screw-cap test tubes, dialysis bags, glass tubes, silicone stoppers, and rubber bands. The difference between the gastric, duodenal, and jejunal digestive chambers lies in the different digestive fluids and buffer solutions they contain.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is simple, convenient, inexpensive, and widely applicable, and can meet the needs of rapid detection of metabolic energy in a large number of samples.

[0019] 2. Based on the different digestive tract characteristics of ducks, such as pH, temperature, digestive enzyme activity, and digestion time, a three-stage in vitro digestion method is adopted to simulate the food digestion process of the stomach, duodenum, jejunum, and ileum respectively. This method has a higher degree of consistency with the actual digestion of ducks in vivo, and the measurement of metabolizable energy is more stable and accurate. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the operation of the method of the present invention; Figure 2 This is a photograph of the oscillating water bath used in the method of the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and beneficial effects of the embodiments of the present invention clearer, further descriptions will be provided below in conjunction with specific implementations of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Other implementation methods obtained by those skilled in the art based on the embodiments disclosed in the present invention without creative effort should all fall within the scope of protection of the present invention.

[0022] Example 1 See Figure 1 The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion includes the following steps: (1) Preparation of buffer solution and digestion solution: Preparation of gastric digestion buffer: 800 mL of deionized water was used to adjust the pH of the solution to 4.3 with 6 mol / L hydrochloric acid at 37 °C, and then the volume was adjusted to 1000 mL to prepare gastric digestion buffer. Preparation of duodenal digestion buffer: Adjust the pH of 800 mL of deionized water to 6.0 with 6 mol / L phosphoric acid or 6 mol / L sodium hydroxide at 37 °C, and then make up to 1000 mL to prepare duodenal digestion buffer. Preparation of jejunal and ileal digestion buffer: Adjust the pH of 800 mL of deionized water to 6.0 with 6 mol / L phosphoric acid or 6 mol / L sodium hydroxide at 37 °C, and then make up to 1000 mL to prepare jejunal and ileal digestion buffer. Preparation of gastric digestive juice: 1593U trypsin, 8825.5U amylase, 1507U lipase and 3076U chymotrypsin were dissolved in 5mL of 37℃ gastric digestive buffer to prepare gastric digestive juice; Preparation of duodenal digestive fluid: 1236.5U trypsin, 10701.5U amylase, 6946U lipase and 1003.5U chymotrypsin were dissolved in 5mL of 37℃ duodenal digestion buffer to prepare duodenal digestive fluid; Preparation of jejunal and ileal digestive fluid: 2077U trypsin, 11261.5U amylase, 3035.5U lipase and 541.5U chymotrypsin were dissolved in 5mL of jejunal and ileal digestive buffer at 37℃ to prepare jejunal and ileal digestive fluid; (2) Sample loading Accurately weigh 2g of the pulverized feed sample, which has passed through a 60-mesh standard sieve. Secure one end of the dialysis bag with a silicone stopper and a rubber band, then place 2g of the feed sample and 5mL of gastric digestion fluid inside. Secure the other end with a silicone stopper fitted with a glass tube and a rubber band. Place the dialysis bag into a screw-top test tube, fill it with gastric digestion buffer, and tighten the cap. The glass tube should extend from the pre-drilled hole in the cap. Each test tube represents one gastric digestion chamber. Perform five replicates for each sample, using one gastric digestion chamber per replicate.

[0023] (3) Simulating gastric digestion The gastric digestive chamber was tilted at 45° and placed in a 37°C constant temperature shaking water bath (see...). Figure 2In this process, a 90 rpm vortex is used to provide a hybrid mixture of gastric digestive fluid and sample, initiating a simulated gastric digestion process that lasts for 3 hours. After digestion, the dialysis bag is removed, the silicone stopper with the glass tube is taken out, and the gastric digestive fluid in the dialysis bag is aspirated using a pipette. Then, 5 mL of duodenal digestive fluid is injected into the dialysis bag, the silicone stopper with the glass tube is replaced, and the bag is placed into an empty screw-capped test tube. The test tube is filled with duodenal buffer and the cap is tightened, with the glass tube extending from the pre-drilled hole in the cap. Each test tube represents one duodenal digestion chamber.

[0024] (4) Simulating duodenal digestion The duodenal digestion chamber was tilted at 45° and placed in a 37°C constant-temperature shaking water bath. A 90 rpm vortex was used to provide a mixed solution of duodenal digestion fluid and sample, initiating a simulated duodenal digestion process for 3 hours. After digestion, the dialysis bag was removed, and the silicone stopper with the glass tube was taken out. The duodenal digestion fluid in the dialysis bag was aspirated using a pipette. 5 mL of ileal digestion fluid was then injected into the dialysis bag, and the silicone stopper with the glass tube was replaced. The bag was then placed in an empty screw-capped test tube. The test tube was filled with ileal buffer solution and the cap was tightened, with the glass tube extending from the pre-drilled hole in the cap. Each test tube represents one ileal digestion chamber.

[0025] (5) Simulated ileum digestion The jejunal and ileal digestion chamber was tilted at 45° and placed in a 37°C constant-temperature shaking water bath. A hybrid mixing process of jejunal and ileal digestion fluid and sample was provided by 90 rpm vortex shaking, initiating the simulated jejunal and ileal digestion process for 12 hours. After digestion, the dialysis bag was removed, the silicone stopper with the glass tube end was taken out, and the duodenal digestion fluid in the dialysis bag was aspirated using a pipette.

[0026] (6) Treatment of undigested residue and calculation of metabolizable energy The undigested residue was rinsed onto filter paper with deionized water, filtered, and then transferred to a glass sand pot with a known oven-dry weight. It was dried at 105°C to constant weight, cooled, and weighed. 1.0 g of oven-dry undigested residue sample was accurately weighed and its energy was determined using an oxygen bomb calorimeter. The metabolizable energy was then calculated based on the weight of the undigested residue and the total energy of the feed ingredient sample.

[0027] Total energy of the feed ingredient sample to be tested: GE1 = E1 × M1 GE1 represents the total energy of the feed ingredient sample to be tested, in joules (J); E1 represents the dry matter energy value of the feed ingredient sample to be tested, in joules per gram (J / g); M1 represents the dry matter mass of the feed ingredient sample to be tested, in grams (g).

[0028] Total energy from undigested residue: GE2 = E2 × M2 GE2 is the total energy of the undigested residue sample, in joules (J); E2 is the dry matter energy value of the undigested residue sample, in joules per gram (J / g); M2 is the dry matter mass of the undigested residue sample, in grams (g).

[0029] Metabolizable energy of raw materials from Muscovy ducks: In vitro ME (dry matter basis) = (GE1 - GE2) ÷ M1 ÷ 1000 In vitro ME represents the metabolizable energy of the tested raw material after in vitro digestion, expressed in megajoules per kilogram (MJ / kg).

[0030] Metabolizable energy of maize from 15 different producing areas was determined using the method described in Example 1 and the existing forced emptying feeding method. A one-way completely randomized design was used, with six replicates for each maize variety. The differences between the two methods were compared. One-way ANOVA was performed on the data using SPSS 25. The results are shown in Table 1. Table 1

[0031] 1 Deviation = (Value measured by in vitro digestion method - Value measured by emptying force-feeding method) ÷ Value measured by emptying force-feeding method × 100% Results analysis: As shown in Table 1, there was no significant difference between the metabolizable energy values ​​determined by the in vitro digestion method and the results determined by the emptying forced feeding method for the 15 maize varieties. P The absolute values ​​of the relative deviations between the two methods were both between 0.02% and 2.75%, with an average ratio of 1.00, meaning that the value measured by the in vitro digestion method is close to 100% of that measured by the emptying forced feeding method. This indicates that the in vitro digestion method can simulate the digestion and metabolism of corn by ducks, and the metabolizable energy measured by this method is very close to that measured by the traditional emptying forced feeding method, enabling accurate and sensitive measurement of the metabolizable energy value of corn by ducks.

[0032] Metabolizable energy of maize from 15 different origins was determined using the method described in Example 1 and an existing biomimetic digestion method. A one-way completely randomized design was used, with six replicates for each maize variety. The differences between the two methods were compared. One-way ANOVA was performed on the data using SPSS 25. The results are shown in Table 2. Table 2

[0033] 1 Deviation = (Value measured by in vitro digestion method - Value measured by biomimetic digestion method) ÷ Value measured by biomimetic digestion method × 100% Results Analysis: As shown in Table 2, there were significant differences between the metabolizable energy values ​​determined by the in vitro digestion method and the results determined by the biomimetic digestion method for 11 of the 15 corn varieties. P< 0.05). The absolute values ​​of the relative deviations between the two methods were both between 0.06% and 20.69%, with an average ratio of 2.81, meaning that the value measured by the in vitro digestion method was 281% of that measured by the biomimetic digestion method. Meanwhile, the biomimetic digestion method showed a larger dispersion in its measurement results (11.004~14.552 MJ / kg), reflecting significant differences in the results. In contrast, the method described in Example 1 showed very concentrated and stable measurement results (13.092~13.845 MJ / kg), indicating that the method of the present invention is more stable, accurate, and has better repeatability than the biomimetic digestion method.

Claims

1. A method for determining the metabolizable energy of duck feed ingredients using in vitro digestion, characterized in that, The method includes the following steps: (1) Preparation of buffer solutions and digestive juices: Prepare gastric digestive buffer solution, duodenal digestive buffer solution, jejunal and ileal digestive buffer solution, as well as gastric digestive juice, duodenal digestive juice, and jejunal and ileal digestive juice respectively; (2) Simulated gastric digestion: Insert one end of the dialysis bag into the first silicone stopper and secure it with a rubber band. Place the feed raw material sample to be tested and the gastric digestion fluid into the dialysis bag. Then insert the other end of the dialysis bag into the second silicone stopper with a glass tube and secure it with a rubber band. Place the dialysis bag into an empty screw-cap test tube. Fill the screw-cap test tube with gastric digestion buffer and tighten the cap. The screw cap has a small hole. The glass tube of the second silicone stopper extends out from the small hole in the screw cap to form a gastric digestion chamber. Place the gastric digestion chamber in a constant temperature shaking water bath at 37°C for gastric digestion reaction. After the reaction is completed, remove the gastric digestion fluid from the dialysis bag. (3) Simulated duodenal digestion: Add duodenal digestion fluid to the dialysis bag after the gastric digestion reaction in step (2), place the dialysis bag in an empty screw-cap test tube, fill the screw-cap test tube with duodenal digestion buffer and tighten the cap. The screw cap has a small hole, and the glass tube of the second silicone stopper extends out of the small hole in the screw cap to form a duodenal digestion chamber. Place the duodenal digestion chamber in a constant temperature shaking water bath at 37°C to carry out the duodenal digestion reaction. After the reaction is completed, remove the duodenal digestion fluid from the dialysis bag. (4) Simulated ileal digestion: Add ileal digestion solution to the dialysis bag after the duodenal reaction in step (3), place the dialysis bag in an empty screw-cap test tube, fill the screw-cap test tube with ileal digestion buffer solution and tighten the screw cap. The screw cap has a small hole, and the glass tube of the second silicone stopper extends out from the small hole of the screw cap to form an ileal digestion chamber. Place the ileal digestion chamber in a constant temperature shaking water bath at 37°C to carry out the ileal digestion reaction. After the reaction is completed, remove the ileal digestion solution from the dialysis bag. (5) Metabolizable energy calculation: Collect the undigested residue remaining after the digestion reaction in step (4), dry it to constant weight, measure the dry matter weight and energy of the undigested residue, and then combine it with the dry matter weight and total energy of the feed raw material sample to be tested to calculate the metabolizable energy of the feed raw material sample to be tested.

2. The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion as described in claim 1, characterized in that, The pH value of the gastric digestive buffer and gastric digestive fluid is 4.3, and the pH value of the duodenal digestive buffer, duodenal digestive fluid, jejunal and ileal digestive buffer, and jejunal and ileal digestive fluid is 6.

0.

3. The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion as described in claim 1, characterized in that, Each 5 mL of the gastric digestive fluid contains 1593 U of trypsin, 8825.5 U of amylase, 1507 U of lipase, and 3076 U of chymotrypsin; each 5 mL of the duodenal digestive fluid contains 1236.5 U of trypsin, 10701.5 U of amylase, 6946 U of lipase, and 1003.5 U of chymotrypsin; each 5 mL of the jejunal and ileal digestive fluid contains 2077 U of trypsin, 11261.5 U of amylase, 3035.5 U of lipase, and 541.5 U of chymotrypsin.

4. The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion as described in claim 1, characterized in that, The time for the gastric digestion reaction is 3 hours, the time for the duodenal digestion reaction is 3 hours, and the time for the jejunal and ileal digestion reaction is 12 hours.

5. The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion as described in claim 1, characterized in that, The shaking condition in the constant temperature shaking water bath is 90 rpm, and the gastric digestion chamber, duodenal digestion chamber, and jejunal digestion chamber are all placed at an angle of 45° in the constant temperature shaking water bath.

6. The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion as described in claim 1, characterized in that, The formula for calculating metabolizable energy is: Total energy of the feed ingredient sample to be tested: GE1 = E1 × M1 GE1 is the total energy of the feed ingredient sample to be tested, in J; E1 is the dry matter energy value of the feed ingredient sample to be tested, in J / g; M1 is the dry matter mass of the ingredient sample, in g. Total energy of undigested residue: GE2 = E2 × M2 GE2 is the total energy of the undigested residue sample, in J; E2 is the dry matter energy value of the undigested residue sample, in J / g; M2 is the dry matter mass of the undigested residue sample, in g. Metabolizable energy of duck feed ingredients: In vitro ME = (GE1 - GE2) ÷ M1 ÷ 1000 In vitro ME represents the in vitro digestible metabolizable energy of the tested duck feed ingredients on a dry matter basis, expressed in MJ / kg.

7. The method for determining the metabolizable energy of duck feed ingredients using in vitro digestion as described in any one of claims 1 to 6, characterized in that, In step (2), the mass-to-volume ratio of the feed raw material sample to be tested and the gastric digestive fluid in the dialysis bag is 2±0.05g:5±0.5mL; in step (3), the amount of duodenal digestive fluid added to the dialysis bag is equal to the amount of gastric digestive fluid in step (2); in step (4), the amount of jejunal and ileal digestive fluid added to the dialysis bag is equal to the amount of duodenal digestive fluid in step (3).

8. An in vitro digestion apparatus for the method of determining the metabolizable energy of duck feed ingredients by in vitro digestion as described in claim 7, characterized in that, The in vitro digestion device includes a screw-capped test tube, a dialysis bag, a rubber band, a first silicone stopper, and a second silicone stopper. One end of the dialysis bag is fitted onto the first silicone stopper and secured with a rubber band, and the other end is fitted onto the second silicone stopper and secured with a rubber band. A glass tube is inserted into the second silicone stopper. The dialysis bag contains the feed sample to be tested and digestive fluid. The sealed dialysis bag is placed inside the screw-capped test tube, which is filled with digestive buffer solution. The screw cap of the test tube has a pre-drilled hole, and the glass tube extends out from the hole in the screw cap.

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