Method for determining digestibility of mineral element nutrients in pig feed

Abstract

The invention discloses a method for determining the digestibility of mineral element nutrients in pig feed. The method comprises the following steps: selecting a test pig of which the ileum tail end and the cecum tail end are provided with T-shaped fistula, and preparing a feed containing an indicator; test periods including a pre-test period, an excrement collection period, a cecum chyme collection period and an ileum chyme collection period are set, and the test pigs are fed with the prepared feed; in the collection period, respectively collecting a feed sample, an excrement sample, a cecum chyme sample and an ileum chyme sample; measuring the content of mineral elements and the content of indicators in the feed sample, the excrement sample, the cecum chyme sample and the ileum chyme sample; and calculating the apparent digestibility of the mineral elements at different parts of the pig digestive tract according to a formula.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition assessment technology, specifically relating to a method for determining the digestibility of mineral elements in pig feed. Background Technology

[0002] In the field of precision nutrition and green farming for pigs, accurately assessing the in vivo digestibility and metabolic rate of mineral elements (such as iron, zinc, copper, and manganese) in feed is a core prerequisite for optimizing feed formulation, improving element utilization efficiency, reducing environmental pollution, and ensuring animal health. Traditionally, the effectiveness of mineral elements in pig diets is often assessed using the slope ratio method to obtain the relative biological value of mineral elements. However, the differences in standard mineral element sources greatly limit the accuracy of this evaluation system and cannot truly reflect the pig's needs for mineral elements. The digestive and absorptive status of intestinal nutrients can be characterized by measuring the apparent digestibility and digestibility (ATTD) of nutrients throughout the entire digestive tract by collecting feces. The results can reflect the end-point digestibility value of the entire digestive tract, but this method faces significant challenges in applying it to the digestibility and metabolic rate of mineral elements. Due to the complex microbial fermentation activities in the hindgut (cecum and colon) and the unavoidable secretion and excretion of endogenous minerals, the ATTD value is severely interfered with. Because of the low mineral element content, endogenous excretion makes it impossible to truly reflect the actual digestive and absorptive efficiency of mineral elements in the small intestine, the main site of digestion and absorption.

[0003] To overcome hindgut interference, a T-shaped fistula can be installed at the terminal ileum to directly collect ileal chyme and measure apparent ileal digestibility (AID), thus more accurately assessing the digestion and absorption of minerals in the small intestine. However, this method only provides data on foregut digestibility and still has the following key limitations: First, it ignores the digestion and absorption of minerals by hindgut microorganisms, failing to answer questions about changes in minerals in the hindgut. Second, when exploring novel nutritional strategies such as low-protein and high-fiber diets, dietary composition significantly affects the composition and fermentation activity of hindgut microorganisms, potentially influencing the solubility, chemical form, and excretion of minerals. Therefore, ileal digestibility data alone cannot reveal the complete metabolic profile of this foregut-hindgut interaction.

[0004] There is a methodological gap in existing technologies: either generalized overall digestibility (ATTD, heavily influenced by the hindgut) or isolated foregut absorption (AID, lacking hindgut metabolic information). This results in a lack of in vivo research methods that can simultaneously and continuously analyze the flow patterns of mineral elements in key parts of the digestive tract when accurately assessing mineral element nutrition and studying nutrient interactions. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a method for determining the digestibility of mineral elements in pig feed.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows: The method for determining the digestibility of mineral elements in pig feed includes the following steps: (1) Select experimental pigs with T-shaped fistulas at the end of the ileum and cecum, and prepare feed containing indicators (the feed for experimental growing pigs was prepared according to the standard of "Nutritional Requirements of Pigs" (2012). (2) Set up an experimental period including a pre-trial period, a fecal collection period, a cecal digest collection period and an ileal digest collection period, and feed the experimental pigs described in step (1) with the feed prepared in step (1); (3) During the collection period described in step (2), feed samples, fecal samples, cecal digest samples and ileal digest samples are collected respectively; (4) Determine the mineral element content and indicator content in feed samples, fecal samples, cecal digest samples and ileal digest samples; (5) Calculate the apparent digestibility of mineral elements in different parts of the pig's digestive tract according to the following formula:

[0007] Preferably, the indicator includes, but is not limited to, one of TiO2, Cr2O3, Fe2O3, Ti2O3, or BaSO4.

[0008] More preferably, the indicator is TiO2.

[0009] Preferably, the indicator has a mass percentage content of 0.2-0.5% in the feed.

[0010] More preferably, the indicator has a mass percentage content of 0.3% in the feed.

[0011] Preferably, the pre-test period is no less than 5 days, the fecal collection period is no less than 3 days, the cecal chyme collection period is no less than 2 days, and the ileal chyme collection period is no less than 2 days.

[0012] Preferably, the mineral element includes one of calcium, phosphorus, iron, zinc, copper, and manganese.

[0013] More preferably, based on the AID, ACD, and ATTD calculated using the indicator method, the highest value is selected as the standard for the digestibility or nutritional requirement parameter of the mineral element.

[0014] More preferably, the apparent digestibility of calcium and phosphorus in feed is referenced by ACD or ATTD, the apparent digestibility of iron in feed is referenced by ATTD, and the apparent digestibility of zinc, copper and manganese in feed is referenced by ACD.

[0015] Preferably, the ATTD values ​​measured by the indicator method and the total manure collection method are cross-checked, and the relative error of the ATTD values ​​obtained by the two methods should be ≤10%.

[0016] The present invention will be further described below: This invention employs a revolutionary solution using an ileocecal double-fistula model. The core advantage of this model lies in its ability to obtain chyme from the terminal ileum and the beginning of the cecum in chronological order within the same animal and experimental period. By combining this with an indicator method (such as TiO2), the following can be calculated: ① Apparent digestibility of the terminal ileum (AID): accurately reflecting the digestive and absorptive efficiency of the small intestine (the main absorption site). ② Apparent digestibility of the cecum (ACD): characterizing the digestion or changes of chyme from the terminal ileum to the beginning of the cecum (i.e., the first half of the hindgut). ③ Apparent total intestinal digestibility (ATTD): a traditional overall value calculated from feces. This method, through dual-site sampling, multi-phase segmented collection, and dual-method verification, achieves spatial analysis and temporal tracking of the mineral element digestion process, significantly improving the accuracy and physiological relevance of the measurement results. This provides reliable key technical support for establishing precise mineral element nutritional requirements, developing efficient and environmentally friendly diets, and conducting in-depth research on intestinal mineral metabolism mechanisms.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: by comparing the data differences of the terminal ileum (AID), the beginning of the cecum (ACD), and feces (ATTD) in the same animal, the high value is selected as the standard for its digestibility or nutritional requirement parameter. This not only accurately reflects the digestibility of mineral elements, but also clarifies the contribution of endogenous metabolism and microbial metabolism by comparing the element content in the ileum, cecum, and feces, and minimizes the interference of endogenous excretion and microbial metabolism on the determination of mineral element digestibility and metabolism.

[0018] The core of this invention lies in using growing pigs equipped with double T-shaped fistulas at the terminal ileum and cecum as an experimental model. Indicators such as TiO2 are added to the diet. An experimental procedure is designed to include a pre-feeding period, a complete fecal collection period, and a phased collection period of digesta from the ileum and cecum. Feed, digesta, and fecal samples are systematically collected. After determining the content of mineral elements and indicators in each sample, the apparent digestibility of mineral elements in the ileum, cecum, and entire intestine is calculated using both the indicator method and the complete fecal collection method.

[0019] In summary, this invention achieves precise collection of chyme from different parts of the digestive tract and segmented digestibility calculation through a double-fistula design. It can effectively distinguish the influence of foregut absorption and hindgut metabolism, providing a reliable method for accurately evaluating the true biological value of dietary mineral elements and studying their digestive and metabolic patterns under different nutritional conditions. Attached Figure Description

[0020] Figure 1 This study investigated the digestive and metabolic status of iron, zinc, copper, manganese, calcium, and phosphorus in different intestinal segments of three-way crossbred pigs under varying protein and mineral levels. Detailed Implementation

[0021] Example 1: Determination of mineral element digestibility in three-way crossbred pig diets I. Experimental Methods Six Duroc × Landrace × Large White crossbred pigs with a starting body weight (BW) of 56.12 ± 3.07 kg, which underwent double fistula surgery at the terminal ileum and cecum, were divided into two groups. A 4×4 Latin square design (4 diets, 4 cycles) was used, including a two-factor (protein, mineral) experimental protocol. Each group was fed diets with protein levels of 12% (LP), 15% (NP), 12% CP + mineral elements (LPM), and 15% CP + mineral elements (NPM), respectively. 0.3% titanium dioxide (TiO2) was added to each diet as an indicator. Each experimental cycle lasted 12 days (5 days pre-trial period, 3 days fecal and urine collection, 2 days cecal digestion collection, and 2 days ileal digestion collection). Each group was rotated between the four diets within each cycle, for a total of 48 days across the four experimental cycles (n=6). According to the Chinese guidelines on nutritional requirements for pigs (2020), growing pigs should be provided with a mixed premix of minerals and vitamins to meet their recommended nutritional needs (Table 1).

[0022] Table 1. Feed Formulation Composition in Implementation Cases

[0023] The premix provides the following per kilogram of feed: 3250 IU Vitamin A; 80000 IU Vitamin E; 10g Vitamin B1; 8g Vitamin B6; 5g Folic Acid; 100g Nicotinamide; 10 million IU Vitamin D3; 10g Vitamin K3; 25g Vitamin B2; 75mg Vitamin B12; 600mg Biotin; 50g D-Pantothenic Acid (moisture ≤6%); 3g TiO2.

[0024] II. Feeding Management and Sample Collection The experiment was conducted at the Institute of Subtropical Ecological Agriculture, Chinese Academy of Sciences. Experimental pigs were housed individually in metabolic cages (1.4m × 0.7m × 0.5m), with the temperature and humidity controlled at 22-24℃ and 55-65%, respectively. Before each cycle, the pigs were weighed, and the daily feed amount was determined based on 4% of the pig's average body weight, divided into two equal portions, fed at 8:00 AM and 5:00 PM respectively. The pigs had free access to water throughout the cycle. During the experiment, the pigs were inspected at irregular intervals daily to observe their feeding, drinking, defecation, and fistula care. During the main trial period, the pigs' feed intake (including the amount fed, spilled feed, and leftover feed) was accurately recorded. Any remaining wet feed in the troughs was dried and converted to dry matter (DM) weight. Cleaning was carried out after the pre-feeding period and sample collection for each cycle.

[0025] After each batch of feed was prepared, 500 g of feed samples from each group were stored at -20℃. Finally, all samples were mixed according to the group using the quartering method, and the samples were stored at -20℃ for testing.

[0026] On days 6-8 of each experimental cycle, the daily feed intake and fecal output of each pig were collected and accurately recorded. Sampling time was from 8:00 a.m. on the day of sampling to 8:00 a.m. the next day. The collected feces were bagged and numbered. For fecal samples from days 3, 10 mL of 10% sulfuric acid nitrogen fixation solution was added to 100 g of each sample and mixed well. 400 g of fecal sample was placed in a 65°C oven and dried for 72 h. After rehydration for 24 h, the sample was weighed and crushed through a 40-mesh sieve to prepare fecal samples.

[0027] On days 9-12 of each experimental cycle, food residue was collected from the terminal ileum and cecum of pigs. Sampling began at 8:00 AM and continued for 8 hours. The sample collection bag was secured to the fistula with a rubber band, and the bag was changed every 30-40 minutes to prevent fermentation. Each collected food residue sample was placed in a plastic bag corresponding to the pig's number and immediately placed at -20°C. After the experiment, the food residue samples from each pig for each cycle were thawed and mixed at room temperature. 500 mL of the mixture was then freeze-dried in a vacuum freeze-drying process. The freeze-dried sample was promptly pulverized, passed through a 60-mesh sieve, bagged, and placed at -20°C for analysis.

[0028] III. Detection Methods The feed, feces and digest samples were pretreated according to the method of GB / T18246-2019. The contents of Ti, Fe, Zn, Cu, Mn, Ca and P in the feed, digest and feces were determined by inductively coupled plasma atomic emission spectrometry.

[0029] IV. Calculation Formula

[0030] V. Test Results Table 2 shows the apparent digestibility of each mineral element in the ileum (AID), cecum (ACD), and whole intestine (ATTD) of three-way crossbred pigs under different protein and mineral element levels. Figure 1 This study demonstrates the digestive and metabolic processes of iron, zinc, copper, manganese, calcium, and phosphorus in different intestinal segments of three-way crossbred pigs under varying protein and mineral levels.

[0031] Table 2. Apparent digestibility of mineral elements in the ileum (AID), cecum (ACD), and whole intestine (ATTD) of three-way crossbred pigs, %

[0032] Table 1 shows the apparent digestibility of different intestinal segments of pigs under different protein and trace element levels in the diet. The ATTD data calculated by the indicator method and the total manure collection method have an error of ≤10%, indicating that the AID and ACD data calculated by the indicator method are accurate and effective.

[0033] Figure 1 This study demonstrates the digestibility of iron, zinc, copper, manganese, calcium, and phosphorus in different intestinal segments of three-way crossbred pigs under different protein and mineral element levels. Based on this, it can be considered that ATTD for iron, ACD for zinc, copper, and manganese, and ACD or ATTD for calcium and phosphorus can be used as reference standards for the apparent digestibility of feed mineral elements.

Claims

1. A method for determining the digestibility of mineral elements in pig feed, characterized in that, The method includes the following steps: (1) Select experimental pigs with T-shaped fistulas at the end of the ileum and cecum, and prepare feed containing indicators; (2) Set up an experimental period including a pre-trial period, a fecal collection period, a cecal digest collection period and an ileal digest collection period, and feed the experimental pigs described in step (1) with the feed prepared in step (1); (3) During the collection period described in step (2), feed samples, fecal samples, cecal digest samples and ileal digest samples are collected respectively; (4) Determine the mineral element content and indicator content in feed samples, fecal samples, cecal digest samples and ileal digest samples; (5) Calculate the apparent digestibility of mineral elements in different parts of the pig's digestive tract according to the following formula:

2. The method for determining the digestibility of mineral elements in pig feed as described in claim 1, characterized in that, The indicator includes one of TiO2, Cr2O3, Fe2O3, Ti2O3, or BaSO4.

3. The method for determining the digestibility of mineral elements in pig feed as described in claim 2, characterized in that, The indicator is TiO2.

4. The method for determining the digestibility of mineral elements in pig feed as described in claim 1, characterized in that, The indicator has a mass percentage content of 0.2-0.5% in the feed.

5. The method for determining the digestibility of mineral elements in pig feed as described in claim 4, characterized in that, The indicator has a mass percentage content of 0.3% in the feed.

6. The method for determining the digestibility of mineral elements in pig feed as described in claim 1, characterized in that, The experimental period shall include a pre-test period of no less than 5 days, a fecal collection period of no less than 3 days, a cecal chyme collection period of no less than 2 days, and an ileal chyme collection period of no less than 2 days.

7. The method for determining the digestibility of mineral elements in pig feed as described in claim 1, characterized in that, The mineral elements include one of calcium, phosphorus, iron, zinc, copper, and manganese.

8. The method for determining the digestibility of mineral elements in pig feed as described in claim 7, characterized in that, Based on the AID, ACD, and ATTD values ​​calculated using the indicator method, the highest value is selected as the standard for the digestibility or nutritional requirement parameter of the mineral element.

9. The method for determining the digestibility of mineral elements in pig feed as described in claim 7, characterized in that, The apparent digestibility of calcium and phosphorus in feed is referenced by ACD or ATTD, the apparent digestibility of iron in feed is referenced by ATTD, and the apparent digestibility of zinc, copper and manganese in feed is referenced by ACD.

10. The method for determining the digestibility of mineral elements in pig feed as described in claim 1, characterized in that, The ATTD values ​​obtained by the indicator method and the total manure collection method should be cross-checked, and the relative error of the ATTD values ​​obtained by the two methods should be ≤10%.

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