A method for predicting the optimal ratio of crude protein, crude fat and starch in piglet feed and application thereof

Abstract

This invention belongs to the fields of information technology and animal nutrition and feed science and technology, specifically relating to a method for predicting the optimal ratio of crude protein, crude fat, and starch in piglet diets and its application. This invention uses crude protein, crude fat, and starch in piglet diets as three factors. Under conditions of consistent proportions of digestible amino acids in the standard ileum and an ideal amino acid profile, extreme vertex mixing designs are used to establish experimental combinations. Average daily weight gain, average daily feed intake, and feed conversion ratio are collected through a nursery piglet feeding trial. A response surface regression model is constructed and optimized to ultimately determine the optimal ratio of crude protein, crude fat, and starch in piglet diets. Furthermore, different crude fat levels are set at the optimal protein level, and the starch ratio is adjusted simultaneously. Growth performance, serum indicators, intestinal morphology, tight junction proteins, mucins, immune factors, expression of antioxidant-related genes, and colonic microbial structure are detected to comprehensively verify the prediction results.

Description

Technical Field

[0001] This invention belongs to the fields of information technology and animal nutrition and feed science and technology, and specifically relates to a method for predicting the optimal ratio of crude protein, crude fat and starch in piglet feed and its application. Background Technology

[0002] Protein, fat, and starch are key nutrients in piglet diets, collectively determining energy and nitrogen supply, digestibility and absorption efficiency, metabolic homeostasis, intestinal barrier function, and hindgut microecological structure. Current nutritional research often employs a single-nutrient approach, examining the effects of increases or decreases in crude protein, energy, or a single feedstock on piglet production performance. However, significant interactions exist among multiple nutrients, and examining only single nutrient levels often fails to accurately reflect the true nutritional needs of piglets.

[0003] Studies have shown that while higher crude protein levels can meet the needs of tissue deposition, excessively high protein levels can increase hindgut protein fermentation, ammonia and biogenic amine production, thus increasing the rate of diarrhea in piglets. Appropriately reducing crude protein and supplementing with crystalline amino acids can help reduce nitrogen emissions and improve piglet gut health. On the other hand, fat not only provides a high-energy-density substrate but also affects gastric emptying, small intestinal digestion and absorption, inflammatory responses, and gut microbiota structure; starch digestion kinetics also directly affect glucose supply, hindgut microbial fermentation, and protein deposition efficiency. Animal growth performance is not determined by a single nutrient factor but is shaped by the synergistic, antagonistic, and balanced effects of multiple nutrients in terms of quantity and proportion. Currently, this theory has limited direct application in the optimization of nursery piglet diets, especially lacking technical solutions based on the synergistic design of crude protein, crude fat, and starch, while simultaneously combining growth performance, intestinal barrier, and microbial structure for comprehensive verification. Summary of the Invention

[0004] The purpose of this invention is to predict the optimal ratio of crude protein, crude fat and starch in piglet diets under the comprehensive nutritional regulation target that takes into account both the intestinal barrier and intestinal microbial homeostasis, so as to be used for precise nutritional regulation of nursery piglets, feed formulation optimization and feed product development.

[0005] This invention provides a method for predicting the optimal ratio of crude protein, crude fat, and starch in piglet diets, comprising the following steps: Step 1: Set the range of proportions of crude protein, crude fat and starch in the feed, and control the total amount of the three factors (crude protein, crude fat and starch) to be constant; Step 2: Establish experimental combinations using extreme vertex mixing design; Step 3: Formulate experimental feed for nursery piglets according to the experimental combination; Step 4: Conduct feeding trials using weaned piglets as the experimental subjects; Step 5: Using crude protein level, crude fat level, and starch ratio as independent variables, and average daily weight gain, average daily feed intake, and feed conversion ratio as response values, establish a multinomial regression model and plot the response surface. Step 6: With the optimization objective of maximizing average daily weight gain and minimizing feed conversion ratio, predict the optimal combination of crude protein, crude fat and starch in piglet diet.

[0006] Preferably, step 1 is designed according to the condition of consistent standard ileal digestible amino acid ratio, or according to ideal amino acid pattern.

[0007] Preferably, under the condition that the proportion of standard ileal digestible amino acids is consistent, the experimental diet for weaned piglets described in step 3, by weight percentage, includes 14.0%~18.0% crude protein, 2.0%~4.0% crude fat, 45.0%~51.0% starch and 1.23% standard ileal digestible lysine; Under ideal amino acid profile conditions, the experimental diet for weaned piglets described in step 3, by weight percentage, comprises 14.5%–18.5% crude protein, 1.0%–5.0% crude fat, and 44.5%–52.5% starch.

[0008] Preferably, the experimental diet for weaned piglets further includes corn, corn starch, soybean meal, soybean hulls, soybean oil, corn gluten meal, wheat bran, beet meal, dicalcium phosphate, limestone powder, and sodium chloride. L -Lysine hydrochloride, DL -Methionine, L -Threonine, L -Tryptophan, L -valine, L -Leucine, L -Isoleucine, L -histidine, L - Phenylalanine and premix.

[0009] Preferably, the test combination in step 2 consists of 9 treatment groups, including 1 center point, 4 vertices and 4 axis points; In step 4, the feeding experiment for nursery piglets lasts for 21 days. On the start and end days of the experiment, the piglets are weighed on an empty stomach, and the amount of feed given, the amount of leftover feed, and the occurrence of diarrhea are recorded by pen. In step 5, Design-Expert 13 software is used for model fitting and response surface plotting, and the model determination coefficient is used. R 2 and corrected coefficient of determination Radj 2 Evaluate the goodness of fit of the model; Step 6 uses Minitab 21 software for response optimization to obtain the predicted optimal combination of crude protein, crude fat and starch in piglet diet.

[0010] Preferably, the following steps are also included: Step 7: Based on the optimal crude protein level obtained in Step 6, fix the crude protein level and the standard ileal digestible lysine level, set different crude fat levels and adjust the starch ratio simultaneously to formulate a trial diet for weaned piglets with consistent net energy levels. Step 8: Detect piglet growth performance, serum biochemical indicators, serum inflammation and antioxidant indicators, intestinal permeability, intestinal morphology, intestinal tight junction protein gene expression, mucin gene expression, immune factor expression, antioxidant-related gene expression, and colonic microbial structure to determine the optimal combination of proportions that balance growth performance, intestinal barrier function, and intestinal microbial homeostasis.

[0011] This invention provides the application of the prediction method described in the above technical solution in the preparation of piglet feed.

[0012] Preferably, the piglet feed has one or more of the following functions; (1) Nutritional regulation; (2) Improve feed conversion efficiency; (3) Maintain or improve intestinal barrier function; (4) Regulate colonic microbial structure.

[0013] Preferably, maintaining or improving intestinal barrier function includes increasing the ratio of villus height to crypt depth in the jejunum and ileum, and upregulating... ZO-1 , Occludin , Claudin-1 and MUC2 Gene expression, increasing goblet cell number, enhancing anti-inflammatory factor expression, or increasing the expression of one or more genes related to intestinal antioxidant activity; The regulation of colonic microbial structure includes increasing the number of Lactobacillus spp. ( Lactobacillus Relative abundance, enriched in Lactobacillus (Lactobacillus order) Lactobacillales Lactobacillus family ( Lactobacillaceae Bacilli, Lactobacillus Lactobacillus ) and Lactobacillus johnsonii ( Lactobacillus_johnsonii ) The present invention provides a piglet diet, which, by weight percentage, comprises 17.5% crude protein, 3.0% crude fat and 1.23% standard ileal digestible lysine; the net energy of the piglet diet is 2449 kcal / kg.

[0014] Beneficial effects: This invention uses crude protein, crude fat, and starch in piglet diets as three factors. Extreme vertex mixing designs were used to establish experimental combinations under both standard ileal digestible amino acid ratios and ideal amino acid profiles. Average daily weight gain, average daily feed intake, and feed conversion ratio were collected through a piglet rearing trial. A response surface regression model was constructed and optimized. The optimal ratio was determined to be 17.6% crude protein, 2.0% crude fat, and 47.4% starch under standard ileal digestible amino acid ratios, and 17.7% crude protein, 5.0% crude fat, and 45.3% starch under ideal amino acid profiles. The predictive method provided by this invention can be used for precise nutritional regulation of piglets, diet formulation optimization, and feed product development.

[0015] Furthermore, this invention sets different crude fat levels at the optimal protein level and simultaneously adjusts the starch ratio, detecting growth performance, serum indicators, intestinal morphology, tight junction proteins, mucins, immune factors, and antioxidant-related gene expression, as well as colonic microbial structure, to comprehensively verify the predicted results. Under the comprehensive nutritional regulation goal of balancing intestinal barrier function and intestinal microbial homeostasis, the preferred combination is 17.5% crude protein, 3.0% crude fat, and 1.23% standard ileal digestible lysine, while simultaneously controlling the starch ratio to maintain a net energy of 2449 kcal / kg. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 This is a layout diagram of the nine treatment groups in Example 1; Figure 2 Mixed contour plot (A) and mixed surface plot (B) of average daily weight gain (ADG) in Example 1; Figure 3 Mixed contour plot (A) and mixed surface plot (B) of average daily feed intake (ADFI) in Example 1; Figure 4 The mixed contour plot (A) and mixed surface plot (B) of feed conversion ratio (FCR) in Example 1 are shown. Figure 5 This is a layout diagram of the nine treatment groups in Example 2; Figure 6 Mixed contour plot (A) and mixed surface plot (B) of average daily weight gain (ADG) in Example 2; Figure 7 Mixed contour plot (A) and mixed surface plot (B) of average daily feed intake (ADFI) in Example 2; Figure 8The mixed contour plot (A) and mixed surface plot (B) of feed conversion ratio (FCR) in Example 2 are shown. Figure 9 Example 3: Effects of different crude fat levels on intestinal permeability in piglets under optimal crude protein levels; Figure 10 HE staining image of the effect of different crude fat levels on the morphology of piglet intestinal tissue under the optimal crude protein level in Example 3; Figure 11 This is an AB-PAS staining image showing the effect of different crude fat levels on the number of goblet cells in the piglet intestine at the optimal crude protein level in Example 3. Figure 12 Example 3: Effects of different crude fat levels on the expression of tight junction protein genes in piglet intestines at the optimal crude protein level; Figure 13 Example 3: Effects of different crude fat levels on the expression of intestinal mucin genes in piglets under optimal crude protein levels; Figure 14 Example 3: Effects of different crude fat levels on the expression of intestinal immune factors in piglets at the optimal crude protein level; Figure 15 Example 3: Effects of different crude fat levels on the expression of antioxidant-related genes in the piglet intestine at the optimal crude protein level; Figure 16 Example 3: Effects of different crude fat levels on alpha diversity of colonic microorganisms in piglets at the optimal crude protein level; Figure 17 Example 3: Analysis of the effects of different crude fat levels on beta diversity of colonic microorganisms in piglets under optimal crude protein levels; Figure 18 This is a stacked bar chart showing the effect of different crude fat levels on the composition of the colonic microbiota of piglets at the genus level under the optimal crude protein level in Example 3; where A is a stacked bar chart of each independent sample and B is a stacked bar chart of the mean. Figure 19 Example 3: LDA score histograms of differential colonic microbiota analysis (LEfSe) in the Lfat and Mfat groups for piglets at the optimal crude protein level. Figure 20 This is a phylogenetic tree diagram of the differential colonic microbiota analysis of piglets in the Lfat and Mfat groups under the optimal crude protein level in Example 3. Figure 21 Example 3: LDA score histograms of differential microbial community analysis (LEfSe) in the Lfat and Hfat groups for piglet colon at the optimal crude protein level. Figure 22This is a phylogenetic tree diagram of the differential microbial community (LEfSe) in the colon of piglets in the Lfat and Hfat groups at the optimal crude protein level in Example 3. Detailed Implementation

[0018] This invention provides a method for predicting the optimal ratio of crude protein, crude fat, and starch in piglet diets, comprising the following steps: Step 1: Set the range of proportions of crude protein, crude fat and starch in the feed, and control the total amount of the three factors (crude protein, crude fat and starch) to be constant; Step 2: Establish experimental combinations using extreme vertex mixing design; Step 3: Formulate experimental feed for nursery piglets according to the experimental combination; Step 4: Conduct feeding trials using weaned piglets as the experimental subjects; Step 5: Using crude protein level, crude fat level, and starch ratio as independent variables, and average daily weight gain, average daily feed intake, and feed conversion ratio as response values, establish a multinomial regression model and plot the response surface. Step 6: With the optimization objective of maximizing average daily weight gain and minimizing feed conversion ratio, predict the optimal combination of crude protein, crude fat and starch in piglet diet.

[0019] As one implementation method, the present invention is designed according to the condition of consistent standard ileal digestible amino acid ratio, or according to ideal amino acid pattern, setting the ratio range of crude protein, crude fat and starch in the diet, and controlling the total sum of the three factors of crude protein, crude fat and starch to be constant.

[0020] In one implementation method, under the condition of a consistent standard ileal digestible amino acid ratio, the crude protein level varies from 14.0% to 18.0%, the crude fat level varies from 2.0% to 4.0%, the starch ratio varies from 45.0% to 51.0%, and the sum of the three factors remains constant at 67.0%. The standard ileal digestible amino acids described in this invention include standard ileal digestible lysine, standard ileal digestible sulfur-containing amino acids (Met+Cys), standard ileal digestible threonine, standard ileal digestible tryptophan, standard ileal digestible valine, standard ileal digestible leucine, and standard ileal digestible histidine.

[0021] As one implementation method, under ideal amino acid pattern conditions, the crude protein level varies from 14.5% to 18.5%, the crude fat level varies from 1.0% to 5.0%, the starch ratio varies from 44.5% to 52.5%, and the sum of the three factors is constant at 68.0%.

[0022] This invention uses an extreme vertex mixing design to establish experimental combinations, and formulates experimental diets for nursery piglets based on these experimental combinations.

[0023] In one implementation, the present invention uses Design-Expert 13 software for extreme vertex mixing design. In another implementation, the experimental combination of the present invention consists of 9 processing groups, including 1 center point, 4 vertices, and 4 axis points.

[0024] As one implementation method, under the condition of a consistent standard ileal digestible amino acid ratio, the experimental diet for weaned piglets, by weight percentage, comprises 14.0%–18.0% crude protein, 2.0%–4.0% crude fat, 45.0%–51.0% starch, and 1.23% standard ileal digestible lysine. As another implementation method, the experimental diet for weaned piglets comprises 0.66% standard ileal digestible sulfur-containing amino acids (Met+Cys), 0.76% standard ileal digestible threonine, 0.28% standard ileal digestible tryptophan, 0.79% standard ileal digestible valine, 1.23% standard ileal digestible leucine, and 0.42% standard ileal digestible histidine.

[0025] As one implementation method, under ideal amino acid pattern conditions, the experimental diet for weaned piglets includes, by weight percentage, 14.5% to 18.5% crude protein, 1.0% to 5.0% crude fat and 44.5% to 52.5% starch.

[0026] As one implementation method, the experimental feed for nursery piglets according to the present invention further includes corn, corn starch, soybean meal, soybean hulls, soybean oil, corn gluten meal, wheat bran, beet meal, dicalcium phosphate, limestone powder, and sodium chloride. L -Lysine hydrochloride, DL -Methionine, L -Threonine, L -Tryptophan, L -valine, L -Leucine, L -Isoleucine, L -histidine, L - Phenylalanine and premix.

[0027] After formulating experimental feed for nursery piglets, this invention uses nursery piglets as the experimental subjects to conduct feeding experiments.

[0028] In one implementation method, the piglet feeding experiment of the present invention has a cycle of 21 days. On the start and end days of the experiment, the piglets are weighed on an empty stomach, and the amount of feed given, the amount of leftover feed, and the occurrence of diarrhea are recorded by pen.

[0029] During the feeding trials, this invention used crude protein level, crude fat level, and starch ratio as independent variables, and average daily weight gain, average daily feed intake, and feed conversion ratio as response values ​​to establish a multinomial regression model and plot the response surface.

[0030] As one implementation method, this invention uses Design-Expert 13 software for model fitting and response surface plotting, and employs the model determination coefficient. R 2 and corrected coefficient of determination Radj 2 Evaluate the goodness of fit of the model.

[0031] After plotting the response surface methodology, this invention aims to maximize average daily weight gain and minimize feed conversion ratio to predict the optimal combination of crude protein, crude fat, and starch in piglet diets.

[0032] As one implementation method, the present invention uses Minitab 21 software for response optimization to obtain the predicted optimal combination of crude protein, crude fat and starch in piglet diet.

[0033] In this invention, under the condition of consistent digestible amino acids in the standard ileum, the predicted optimal ratio combination after response optimization is 17.6% crude protein, 2.0% crude fat, and 47.4% starch. Under the ideal amino acid mode, the predicted optimal ratio combination after response optimization is 17.7% crude protein, 5.0% crude fat, and 45.3% starch.

[0034] As one implementation method, after predicting the optimal combination of crude protein, crude fat and starch in piglet diet, the present invention, based on the obtained optimal crude protein level, fixes the crude protein level and the standard ileal digestible lysine level, sets different crude fat levels and simultaneously adjusts the starch ratio to formulate a nursery piglet experimental diet with consistent net energy level.

[0035] In one embodiment, the present invention fixes the crude protein level at 17.5%, the standard ileal digestible lysine level at 1.23%, and the crude fat levels at 1.0%, 3.0%, and 5.0%, respectively. The net energy level is maintained consistently by adjusting the proportions of corn, corn starch, soybean oil, and wheat bran. In one embodiment, the net energy is 2449 kcal / kg.

[0036] In one implementation method, the present invention detects the growth performance of piglets, serum biochemical indicators, serum inflammation and antioxidant indicators, intestinal permeability, intestinal morphology, expression of intestinal tight junction protein genes, expression of mucin genes, expression of immune factors, expression of antioxidant-related genes, and colonic microbial structure, and determines a comprehensive and optimal combination of proportions that take into account growth performance, intestinal barrier function, and intestinal microbial homeostasis.

[0037] In this invention, with a net energy of 2449 kcal / kg, a fixed crude protein level of 17.5%, and a standard ileal digestible lysine level of 1.23%, the crude fat level was 3.0%. Under the optimal crude protein level, feeding with 1.0%, 3.0%, and 5.0% crude fat levels had no significant effect on ADG and diarrhea rate in weaned piglets; however, ADFI and FCR decreased linearly and significantly with increasing crude fat levels. The 5.0% EE group showed significantly increased serum total cholesterol and DAO activity, suggesting that high crude fat levels may increase intestinal adaptive stress. The 3.0% crude fat level, compared to the other two groups, optimized the intestinal morphology of weaned piglets, upregulated the expression of tight junction proteins and mucin-related genes, and improved intestinal immunity and antioxidant capacity. Colonic microbiota results showed that the 3.0% crude fat level significantly increased Lactobacillus spp. Lactobacillus The relative abundance and enrichment of differentially expressed flora, primarily Lactobacilli, corresponded to a superior intestinal barrier phenotype. While a 5.0% crude fat level reduced the free flow rate (FCR), it was accompanied by... Megasphaera rise, Streptococcus equinus Enrichment, elevated DAO, and decreased jejunal villus-cryptosome ratio suggest that high crude fat levels may lead to decreased gut microbiota homeostasis and increased intestinal adaptive pressure. Therefore, under conditions of 17.5% crude protein, 1.23% SIDLys, and consistent net energy, a crude fat level of around 3.0% is the optimal level that balances growth performance, intestinal barrier function, and colonic microbiota structure. This invention provides the application of the prediction method described in the above technical solution in the preparation of piglet feed.

[0038] As one embodiment, the piglet diet of the present invention has one or more of the following functions: (1) nutritional regulation; (2) improving feed conversion efficiency; (3) maintaining or improving intestinal barrier function; and (4) regulating colonic microbial structure. As one embodiment, maintaining or improving intestinal barrier function according to the present invention includes increasing the ratio of villus height to crypt depth in the jejunum and ileum, and upregulating... ZO-1 , Occludin , Claudin-1 and MUC2 Gene expression, increasing goblet cell number, enhancing anti-inflammatory factor expression, or increasing the expression of gut antioxidant-related genes are all considered. As one embodiment, the regulation of colonic microbiota structure described in this invention includes increasing the expression of Lactobacillus spp. (…). Lactobacillus Relative abundance, enriched in Lactobacillus (Lactobacillus order) Lactobacillales ).

[0039] The present invention provides a piglet diet, which, by weight percentage, comprises 17.5% crude protein, 3.0% crude fat and 1.23% standard ileal digestible lysine; the net energy of the piglet diet is 2449 kcal / kg.

[0040] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for predicting the optimal ratio of crude protein, crude fat, and starch in piglet feed and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1 1. Selection and grouping of experimental animals One hundred and eighty-nine castrated nursery piglets of the Landrace × Large White breed, weighing 11.04 ± 0.11 kg, were selected and randomly divided into nine treatment groups (T1 to T9) according to a completely randomized block design. Each treatment group had seven replicates (pens), with three piglets per pen. The crude protein (CP) content was set at 14.0%–18.0%, crude fat (EE) at 2.0%–4.0%, and starch (ST) at 45.0%–51.0%, with the total content of these three components kept constant at 67.0%. The standard ileum digestible amino acid levels were kept consistent across all treatment groups (Table 2). The experimental period was 21 days. The experimental design is shown in Table 1, and the distribution of the nine treatment groups is shown in Table 1. Figure 1 The experimental diet for weaned piglets was formulated according to the NRC (2012) swine nutrition standards. The composition and nutrient levels of the diet are shown in Table 2.

[0042] Table 1 Experimental Design

[0043] Table 2. Composition and nutrient levels of the basal diet

[0044] Note: 1) The premix provided the following per kg of diets: VA 7.80 KIU, VD3 3.20 KIU, VE 24.60 mg, VK3 3.07 mg, VB1 1.88 mg, VB2 7.33 mg, VB6 2.60 mg, VB12 0.026 mg, nicotinamide 31.30 mg, pantothenic acid 17.72 mg, folic acid 1.81 mg, biotin 0.346 mg, choline chloride 600 mg, Cu (CuSO4) 10 mg, Fe (FeSO4) 60 mg, Zn (ZnSO4) 40 mg, Mn (MnSO4) 15 mg, I (KI) 0.20 mg, Se (Na2SeO3) 0.66 mg. 2) Nutrient levels are calculated values. 3) Starch content was determined according to the national standard GB 5009.9-2023, using the amylase hydrolysis method. The starch data in the table are calculated values. The same applies below.

[0045] 2. Determination of growth performance and diarrhea rate During the experiment, piglets' feces were observed and diarrhea was recorded half an hour after feeding each day. The feces scoring criteria were: 1 point for severe constipation, 2 points for mild constipation, 3 points for normal, 4 points for mild diarrhea, and 5 points for diarrhea. A score of 4-5 for two consecutive days was considered diarrhea. On day 1 and day 22 at 08:00, all experimental piglets were weighed on an empty stomach, and their initial and final weights were recorded. The daily feed intake was recorded, and the remaining feed was recorded before the end of the experiment. The following indicators were calculated, and the specific results are shown in Table 3.

[0046] Average daily weight gain (ADG, g / d) = (final weight - initial weight) (g) / number of days of the trial (d); Average daily feed intake (ADFI, g / d) = Total feed intake during the experimental period (g) / Number of experimental days (d); Feed conversion ratio (FCR) = Total feed consumed (g) / Total weight gain (g); Diarrhea rate (%) = Total number of diarrhea episodes in each group / (Total number of piglets in each group × Number of days in the experiment) × 100.

[0047] Table 3. Effects of crude protein, crude fat, and starch levels on growth performance of piglets.

[0048] Note: In the same row of data, if the superscript does not have a letter or has the same letter, it indicates that the difference between groups is not significant. P >0.05); if different lowercase letters are used, it indicates that the difference between groups has reached a significant level ( P <0.05). Same as the table below.

[0049] As shown in Table 3, the ADG of group T6 was the highest (557 g / d), which was significantly higher than that of treatment groups T1, T2, T3, and T4, which had low crude protein content (CP). P <0.01). ADG generally increased with increasing crude protein levels. In the T1 and T2 treatment groups with 14.0% CP, crude fat (EE) levels increased from 2.0% to 4.0%, significantly increasing ADG and decreasing FCR ( P <0.05), while in the T6 and T7 treatment groups with a CP of 17.0% or the T8 and T9 treatment groups with a CP of 18.0%, increasing the EE level had no significant effect on the ADG and FCR of piglets. P >0.05). The FCR was highest in group T1 (2.07) and lowest in group T8 (1.52). There was no significant difference in diarrhea rates among the groups during the trial. P >0.05) 3. Response Surface Model Establishment and Analysis Extreme vertex feed design was performed using Design-Expert 13 software. With CP level (X), EE level (Y), and ST ratio (Z) as independent variables, and average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) as response values, a multinomial regression model was established, and regression analysis and analysis of variance were performed to evaluate the main effects and interaction effects of each factor. The coefficient of determination of the model was used. R 2 ) and correction determination coefficient ( Radj 2 The goodness of fit of the model was evaluated. Based on the established regression model, response surface plots were generated using Design-Expert 13 software. The optimal nutrient combination ratio was selected using the Minitab 21 response optimizer, with the optimization objectives of maximizing ADG and minimizing FCR. The results are shown in Tables 4-6.

[0050] Table 4. Regression and ANOVA of Average Daily Weight Gain (ADG)

[0051] Table 5. Regression and ANOVA of Average Daily Feed Intake (ADFI)

[0052] Table 6. Regression and ANOVA of Feed Conversion Rate (FCR)

[0053] As shown in Tables 4-6, both the ADG and FCR regression models are significant. P <0.05), while the ADFI regression model was not significant ( P >0.05). The polynomial regression equations for ADG and FCR are as follows: ADG = -345.431 X+120.773 Y-44.652 Z+10.198 XZ; FCR = 0.965 X-0.315 Y+0.179 Z-0.028 XZ; The regression model of ADG was significant (F=9.11). P <0.05), model determination coefficient R 2 =84.53%, Corrected coefficient of determination Radj 2 =75.26%. There is a significant interaction between CP and ST ( ). F =6.66, P <0.05. The regression model of ADFI is not significant ( F =1.08, P >0.05, indicating poor model fit ( R 2 =26.39%, Radj 2 =1.85%). The regression model of FCR is highly significant ( F =51.72, P <0.01, indicating excellent fitting results ( R 2 =96.88%, Radj 2 =95.00%) and the interaction between CP and ST is extremely significant ( F =18.17, P <0.01).

[0054] 4. Visualization analysis of nutrient ratio response surface methodology Figures 2-4The combined contour plot and combined surface plot illustrate the influence of three factors—CP, EE, and ST—on the response values. In the ADG contour plot, the maximum value appears in the upper right region (higher CP, lower EE, moderate ST), and the minimum value appears in the lower right region (…). Figure 2 (A); The peak of the ADG surface appears in the region corresponding to the contour map ( Figure 2 (B) The contour lines of ADFI are nearly parallel ( Figure 4 (A) and no curved surface is formed ( Figure 4 (Middle B). For example Figure 3 As shown in Figures A and B, the low-value areas of FCR are mainly distributed in regions with higher CP, lower EE, and moderate ST. The range of response values ​​varies little with changes in the ratios of the three nutrients, indicating that the levels of CP, EE, and ST have little effect on ADFI under these conditions.

[0055] 5. Predict the optimal ratio of crude protein, crude fat, and starch in piglet diets. With the optimization objectives of maximizing ADG (lower limit 418, expected target 800) and minimizing FCR (upper limit 2.07, expected target 1.40), the optimal ratio combination was predicted to be 17.6% crude protein, 2.0% crude fat, and 47.4% starch. Under the response optimization conditions, the predicted values ​​of ADG, ADFI, and FCR were 553 g / d, 852 g / d, and 1.56, respectively, with a composite desirability of 0.63.

[0056] This embodiment uses SAS 9.4 software for data analysis. Growth performance data were analyzed using one-way ANOVA, with Tukey's post-hoc multiple comparison test performed when differences were significant. Diarrhea rate was statistically analyzed using a generalized linear mixed model (GLMM), with the treatment group as a fixed effect and replication as a random effect. A binomial distribution was used to fit the data, and pairwise comparisons between groups were performed using Tukey's post-hoc multiple comparison test. The significance level was set at: P <0.01 indicates a highly significant difference. P <0.05 indicates a significant difference, 0.05≤ P A value <0.10 indicates a divergent trend.

[0057] Example 2 Predicting the optimal ratio combination under ideal amino acid mode 1. Selection and grouping of experimental animals Two hundred and fifty-two castrated nursery piglets of the Landrace × Large White breed, weighing 10.23 ± 1.46 kg, were selected and randomly divided into nine treatment groups according to a completely randomized block design. Each treatment group had seven replicates (pens), with four piglets per pen. The crude protein content was set at 14.5%–18.5%, crude fat at 1.0%–5.0%, and starch at 44.5%–52.5%, with the total content of these three components kept constant at 68.0%. All groups were kept within the ideal amino acid profile. The experimental period was 21 days. The experimental design is shown in Table 7, and the distribution of the nine treatment groups is shown in [Table 7]. Figure 5 The experimental diet for weaned piglets was formulated according to the NRC (2012) swine nutrition standards. The composition and nutrient levels of the diet are shown in Table 8.

[0058] Table 7 Experimental Design

[0059] Table 8. Composition and Nutritional Levels of Basal Diets

[0060] 2. Results (1) The effect on piglet growth performance was tested according to the method of Example 1, and the results are shown in Table 9.

[0061] Table 9. Effects of crude protein, crude fat, and starch levels on growth performance of piglets under the ideal amino acid pattern.

[0062] As shown in Table 9, the T9 group (18.5% CP, 5.0% EE, 44.5% ST) had the highest ADG (660 g / d), which was significantly higher than that of the low CP treatment groups T1 and T4. P <0.01). In the T1 and T2 treatment groups with a CP of 14.5%, an increase in EE level from 1.0% to 5.0% significantly increased ADG in piglets. P <0.01), while in the T6 and T7 groups with CP of 17.5% or the T8 and T9 groups with CP of 18.5%, increasing EE level had no significant effect on piglet ADG ( P >0.05). Pairwise comparisons of treatment groups at the same CP level revealed that increasing EE levels could reduce ADFI to some extent. FCR was highest in group T1 (1.81) and lowest in group T9 (1.34). In the low CP T1 and T2 groups (14.5% CP), increasing EE from 1.0% to 5.0% significantly reduced FCR in piglets ( P <0.01); In the high-CP T8 and T9 groups with 18.5% CP, increasing EE levels also significantly reduced the FCR of piglets ( P<0.01. During the trial, there was no significant difference in diarrhea rates among the groups ( P >0.05).

[0063] (2) The response surface model was established in accordance with the method of Example 1, and the data analysis was performed. The results are shown in Tables 10-12.

[0064] Table 10. Regression and ANOVA of Average Daily Weight Gain (ADG)

[0065] Table 11 Regression and ANOVA of Average Daily Feed Intake (ADFI)

[0066] Table 12 Regression and ANOVA of Feed Conversion Rate (FCR)

[0067] As shown in Tables 10-12, the ADG, ADFI, and FCR regression models all reached a significant level. P <0.05). The polynomial regression equations for ADG, ADFI, and FCR are as follows: ADG = -100.260 X+55.541 Y-12.972 Z+3.406 XZ; ADFI = 156.785 X-46.083 Y+30.542 Z-3.835 XZ; FCR = 0.823 X-0.207 Y+0.139 Z-0.005 XY-0.023 XZ; ADG's regression model is significant ( F =9.50, P <0.05), R 2 =85.08%, Radj 2 =76.13%. The ADFI regression model is also significant ( F =6.91, P <0.05), R 2 =80.56%, Radj 2 =68.90%. The regression model of FCR is significant ( F =10.27, P <0.05, indicating a good fitting effect ( R 2 =91.12%, Radj 2 =82.25%).

[0068] (3) Visualization analysis of nutrient ratio response surface methodology Figures 6-8 To create a mixed contour map and a mixed surface map, such as Figure 6 As shown, the contour plot of ADG indicates that ADG is relatively high in the upper left region of the feasible region and gradually decreases towards the lower right corner. Figure 6 (A) The overall curvature of the surface is relatively small, indicating a smoother mixing effect and weaker component interactions. Figure 6 (B) The contour lines of ADFI show a distinct low-value "valley" (dark blue area, located near the lower left of the feasible region), and gradually increase to the right ( Figure 7 (A) The mixed surface plot shows that the nonlinear interaction effect of ADFI is stronger than that of ADG. Figure 7 (B) The lowest value of FCR (blue area) is mainly distributed on the left side of the feasible region, gradually increasing towards the lower right corner. Figure 8 (A and B in the middle).

[0069] (4) Predict the optimal combination of crude protein, crude fat and starch in piglet diet. With the optimization objectives of maximizing ADG (lower limit 500, desired target 800) and minimizing FCR (upper limit 1.81, desired target 1.30), the optimal ratio combination was predicted to be 17.7% crude protein, 5.0% crude fat, and 45.3% starch. Under the response optimization conditions, the predicted values ​​of ADG, ADFI, and FCR were 646 g / d, 854 g / d, and 1.31, respectively, with a composite desirability of 0.81.

[0070] This embodiment uses SAS 9.4 software for data analysis. Growth performance data were analyzed using one-way ANOVA, with Tukey's post-hoc multiple comparison test performed when differences were significant. Diarrhea rate was statistically analyzed using a generalized linear mixed model (GLMM), with the treatment group as a fixed effect and replication as a random effect. A binomial distribution was used to fit the data, and pairwise comparisons between groups were performed using Tukey's post-hoc multiple comparison test. The significance level was set at: P <0.01 indicates a highly significant difference. P <0.05 indicates a significant difference, 0.05≤ P A value <0.10 indicates a divergent trend.

[0071] Example 3 1. Eighty-four weaned piglets weighing 10.70 ± 0.92 kg were selected and randomly assigned to three treatment groups (7 replicates per pen) using a completely randomized block design. Each pen housed four piglets. The experimental diets were configured with a crude protein level of 17.5%, a SID Lys level of 1.23%, and crude fat levels of 1.0%, 3.0%, and 5.0%, respectively. The experiment lasted 21 days, and the experimental design is shown in Table 13. The weaned piglet diets were formulated according to the NRC (2012) swine nutrition standards, and the diet composition and nutrient levels are shown in Table 14.

[0072] Table 13 Experimental Design

[0073] Table 14 Composition and Nutritional Levels of Basal Diets

[0074] 2. Sample Collection (1) Blood sample collection and serum sample preparation On day 22 of the experiment, blood was collected from six healthy piglets of near-average weight in different pens in each treatment group. The piglets were fasted for 12 hours prior to blood collection, but had free access to water. After being restrained, blood samples were collected using the anterior vena cava method. 10 mL of blood was collected, and after standing for 1 hour, the sample was centrifuged at 3000 r / min for 10 min to separate the supernatant serum. The serum was transferred to 1.5 mL centrifuge tubes and stored at -80℃.

[0075] (2) Collection of tissue and organ samples On day 22 of the experiment, six healthy piglets of near-average weight from different pens in each treatment group were slaughtered and sampled. After euthanasia, the piglets underwent rapid aseptic dissection. Samples were collected from the jejunum, ileum, and colon. The jejunum was taken from the mid-small intestine, the ileum from approximately 10 cm from the ileocecal junction, and the colon from the proximal mid-colon, with each segment approximately 2-5 cm long. The intestinal segments were longitudinally cut along the opposite side of the mesentery, and the intestinal lumen was gently rinsed with pre-cooled physiological saline to remove residual contents. Intestinal segments used for histomorphological analysis were immediately fixed in 4% paraformaldehyde. For mucosal sample collection, the rinsed intestinal segments were laid flat on an ice plate, and the intestinal mucosa was gently scraped off with a sterile glass slide, collected in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for later use. For colonic contents sample collection, the target intestinal segment was ligated at both ends, cut open, and the intestinal contents were aseptically collected, placed in sterile cryovials, flash-frozen in liquid nitrogen, and stored at -80°C. The entire sampling process avoids cross-contamination between samples from different intestinal segments.

[0076] 3. Measurement Indicators (1) Growth performance The effects on piglet growth performance were tested according to the method in Example 1, and the results are shown in Tables 15-16.

[0077] Table 15 Effects of crude fat level on growth performance of piglets

[0078] Table 16. Effects of crude fat level on diarrhea rate in weaned piglets

[0079] As can be seen from Tables 15-16, the differences in initial weight, final weight, and average daily weight gain among the piglets in each group were not statistically significant. P >0.05). ADG was highest in the Mfat group with 3.0% crude fat (EE) and lowest in the Hfat group with 5.0% EE. The ADFI in the Hfat group was significantly lower than that in the Mfat and Lfat groups; ADFI decreased linearly and significantly with increasing crude fat level. P <0.01. The FCR of the Mfat and Hfat groups was significantly lower than that of the Lfat group; with increasing crude fat levels, the FCR of piglets also decreased linearly and significantly. P <0.01). As shown in Table 20, the diarrhea rate did not differ significantly among the groups during the trial period ( P >0.05), with the lowest diarrhea rate at a crude fat level of 3.0%.

[0080] (2) Serum biochemical indicators After thawing, serum samples were thoroughly mixed by vortexing and centrifuged at 3000 rpm for 3 min at 4°C. The supernatant was then collected. Various biochemical indicators were measured using a BK-400 fully automated biochemical analyzer, including glucose (GLU), triglycerides (TG), total cholesterol (TC), total protein (TP), albumin (ALB), high-density lipoprotein (HDL), low-density lipoprotein (LDL), urea (UREA), and non-esterified fatty acids (NEFA). The results are shown in Table 17.

[0081] Table 17 Effects of crude fat levels on serum biochemical parameters of weaned piglets

[0082] As shown in Table 17, the total cholesterol (TC) content in the Hfat group was significantly higher than that in the Mfat and Lfat groups. P <0.05), and with increasing crude fat levels, TC content increased linearly and significantly ( P <0.01); serum glucose (GLU) levels were highest in the Hfat group and lowest in the Mfat group. Other serum biochemical indicators showed no significant differences among the groups. P >0.05).

[0083] (3) Serum inflammatory factors and antioxidant indicators Serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and transforming growth factor-β1 (TGF-β1) were detected using an enzyme-linked immunosorbent assay (ELISA) kit manufactured by Jiangsu Jingmei Biotechnology Co., Ltd. Serum levels of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), catalase (CAT), and malondialdehyde (MDA) were detected using a commercial kit manufactured by Nanjing Jiancheng Bioengineering Institute. The assays were performed strictly according to the instructions. The results are shown in Table 18.

[0084] Table 18 Effects of crude fat levels on serum antioxidant capacity and immune factor content in weaned piglets

[0085] As shown in Table 18, the serum total antioxidant capacity (T-AOC) level of piglets in the Hfat group was significantly higher than that in the Lfat group. P <0.05), with the increase of dietary crude fat level, T-AOC level increased linearly and significantly ( P <0.01. The catalase (CAT) content in the Mfat and Hfat groups was significantly higher than that in the Lfat group ( P <0.05%. There were no significant differences in the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) among the groups. P >0.05), MDA had the lowest content in the 3.0% EE group. Among the immune markers, tumor necrosis factor-α (TNF-α) showed no significant difference among the three groups. P >0.05), interleukin-6 (IL-6) was significantly higher in the Hfat group than in the Lfat group ( P<0.05), with increasing crude fat levels, IL-6 levels increased linearly and significantly. P <0.01). Compared to the other two groups, a crude fat level of 3.0% significantly increased the content of transforming growth factor β1 (TGF-β1). P <0.01), the content of TGF-β1 in the Hfat group was significantly higher than that in the Lfat group ( P <0.05), with increasing crude fat levels, the concentration of TGF-β1 showed a quadratic curve change of first increasing and then decreasing. P <0.01).

[0086] (4) Serum intestinal permeability markers The levels of D-lactic acid and diamine oxidase (DAO), indicators of intestinal permeability, in serum were determined using a commercially available kit manufactured by Nanjing Jiancheng Bioengineering Institute. The measurements were performed strictly according to the manufacturer's instructions. Results are as follows: Figure 9 As shown, the serum diamine oxidase (DAO) activity in the Hfat group with a crude fat level of 5.0% was significantly higher than that in the Lfat group with a crude fat level of 1.0%. P <0.05, different crude fat levels had no significant effect on the D-lactic acid content in piglet serum ( P >0.05).

[0087] (5) HE staining to observe intestinal morphology Jejunum, ileum, and colon tissue samples were fixed in 4% paraformaldehyde for 24–48 h, and slide preparation and staining were performed by Xi'an Yike Biotechnology Co., Ltd. Intestinal tissue morphology was observed and photographed under an optical microscope. Ten structurally intact and clearly oriented villi and crypts were selected from each slide for measurement. Villus height (VH) and crypt depth (CD) were measured and recorded using ImageJ software, and the villus height to crypt depth ratio (VH / CD) was calculated to evaluate intestinal morphological development. The results are shown in Table 19. Figure 10 As shown.

[0088] Table 19 Effects of crude fat levels on intestinal tissue morphology in weaned piglets

[0089] According to Table 19 and Figure 10 It can be seen that different crude fat levels have no significant effect on jejunal villus height. P >0.05); the jejunal crypt depth in the Lfat and Mfat groups was significantly lower than that in the Hfat group ( P<0.01), with the Mfat group having the lowest jejunal crypt depth. The jejunal VH / CD ratio was significantly higher in the Lfat and Mfat groups than in the Hfat group ( P <0.01), with increasing crude fat levels, the jejunal VH / CD exhibited a quadratic curve change, first increasing and then decreasing. P <0.01), with the 3.0% EE group exhibiting the highest jejunal villus-to-vaginal ratio. In the ileum tissue, villus height increased linearly and significantly with increasing crude fat levels. P <0.01, there were no significant differences in ileal recess depth and villous-to-crypt ratio among the groups ( P >0.05).

[0090] (6) AB-PAS staining to detect the number of goblet cells AB-PAS staining of jejunal, ileal, and colonic tissue sections was performed by Xi'an Yike Biotechnology Co., Ltd. The sections were observed and photographed under an optical microscope. Ten intact, undamaged small intestinal villi or large intestinal glands were randomly selected from each section, and cell counting was performed using ImageJ software. The cell counts for the jejunum, ileum, and colon were statistically analyzed separately, and the results are shown in Table 20. Figure 11 As shown.

[0091] Table 20 Effects of crude fat levels on the number of goblet cells in the intestine of weaned piglets

[0092] Figure 20 The cells stained blue or blue-purple are goblet cells, according to Table 20 and... Figure 11 It can be seen that the Mfat group significantly increased the number of goblet cells in the jejunum compared to the other two groups. P <0.01), with increasing crude fat levels, the number of jejunal goblet cells showed a quadratic curve change, first increasing and then decreasing. P <0.01. In the ileum and colon, the overall treatment effect of crude fat level on goblet cell number was not significant ( P >0.05), but as the crude fat level increases, the number of goblet cells shows a quadratic curve trend of first increasing and then decreasing, and reaches its maximum value at a crude fat level of 3.0%.

[0093] (7) RT-qPCR detection of transcriptional levels of gut-related genes After removing jejunal, ileal, and colonic mucosal samples from experimental piglets stored at -80℃, total RNA was extracted from the intestinal mucosa, RNA purity was determined, reverse transcription was performed, and real-time quantitative polymerase chain reaction (RT-qPCR) was conducted sequentially. Two [samples were used]. -△△CT The relative expression level of the target gene was calculated using a method, and the primers used were synthesized by a biotechnology company.

[0094] Intestinal tight junction protein gene expression results as follows Figure 12 As shown, the Mfat group significantly upregulated the jejunal mucosa of piglets compared to the other two groups. ZO-1 and Claudin-1 mRNA expression, in the Hfat group jejunum Claudin-1 The mRNA expression level of the gene was significantly downregulated compared to the other two groups. In ileal tissue, the Hfat group showed a significant upregulation compared to the Lfat group. ZO-1 Gene transcription level, crude fat level on ileal tissue Occludin and Claudin-1 Gene transcription levels were not significantly affected. Crude fat levels significantly affected the expression of genes related to the colonic physical barrier, particularly in the Mfat group. ZO-1 , Occludin and Claudin-1 Gene transcription levels were significantly higher in the Mfat group than in the Hfat group, and the colon of both groups was significantly different. ZO-1 , Occludin and Claudin-1 There was no significant difference in gene expression levels.

[0095] Intestinal mucin gene expression results as follows Figure 13 As shown, the transcriptional levels of protein genes exhibit different trends in different intestinal segments. In the jejunal mucosa, the Hfat group... MUC1 The mRNA expression level was significantly upregulated compared to the Mfat group. P <0.01); compared to the other two groups, it can significantly improve at a crude fat level of 3.0%. MUC2 Gene mRNA expression level ( P <0.01), while Lfat group MUC2 Gene expression was significantly upregulated compared to the Hfat group. P <0.01. Different crude fat levels had no significant effect on the expression of ileal mucin-related genes. P >0.05). In colonic tissue, a crude fat level of 3.0% can be significantly upregulated. MUC2 Gene expression ( P <0.01).

[0096] Results of intestinal immune factor expression, as follows Figure 14 As shown, the Mfat group significantly downregulated the jejunum TNF-α , IL-6 , IL-1β and its upstream regulatory genes TLR4 The mRNA expression level was significantly downregulated in the Hfat group compared to the Lfat group. TNF-α , IL-6 and TLR4 Gene expression ( Figure 14 (A) Jejunal anti-inflammatory factor TGF-β and IL-4 Compared to the other two groups, anti-inflammatory factors were significantly upregulated in the Mfat group. IL-10 Gene expression was significantly higher in the Lfat and Mfat groups than in the Hfat group. Figure 14 (Middle B). Ileum TNF-α , IL-6 , IL-1β and TLR4 Gene expression levels were significantly lower in both the Mfat and Hfat groups than in the Lfat group. Figure 14 The 3.0% crude fat level significantly improved ileum compared to the other two groups (C). TGF-β , IL-4 and IL-10 mRNA expression level ( Figure 14 (D). The transcriptional levels of pro-inflammatory factors and Toll-like receptors in the piglet colon showed the same trend as their expression in the ileum. Figure 14 (E), colonic anti-inflammatory factor TGF-β The 3.0% EE group showed a significant upregulation compared to the other two groups, while IL-4 The expression level was highest in the 5.0%EE group, significantly higher than that in the 1.0%EE group. Figure 14 (Middle F). TLR2 The transcriptional level showed no significant difference across the three crude fat levels. P >0.05) The results of antioxidant capacity gene expression are as follows Figure 15 As shown, compared to the other two groups, the Mfat group was able to significantly upregulate the jejunum. CAT , SOD1 and SOD2 The transcriptional level of genes. In ileal tissue, CAT Gene expression tended to be higher in the Mfat group than in the Lfat group. P = 0.05), the Mfat and Hfat groups were significantly upregulated compared to the Lfat group. SOD1 and SOD2 The expression of [something]. In colon tissue. CAT The expression of [a substance] was significantly higher in the Mfat and Hfat groups than in the Lfat group. SOD1 and SOD2 The transcriptional levels of genes were highest in the 5.0% EE group, significantly higher than the other two groups. Gut antioxidant-related enzymes GPX The mRNA levels in the three treatment groups were not significantly different. P >0.05).

[0097] (8) Detection of colonic microbial diversity 16S rDNA amplicon sequencing was performed by Wuhan Maiwei Metabolic Biotechnology Co., Ltd. The experimental and sequencing analysis workflow mainly included five parts: DNA extraction from colon contents, PCR amplification, PCR product purification, library construction and quality control, and sequencing. After sequencing, the obtained raw data was filtered and quality-controlled to obtain clean data, which was then used for bioinformatics analysis. The results are as follows: Figures 16-17 As shown.

[0098] according to Figure 16 It can be seen that, compared with the other two groups, the Mfat group showed an increase in both ACE and chao1 indices, although the statistical significance was not significant. P >0.05, but still indicates an increasing trend in the number of gut microbiota species in the 3.0% fat group of weaned piglets. Neither the Shannon nor the Simpson index showed significant differences among the groups. P The value >0.05 indicates that the species diversity levels of the gut microbiota in the three groups of piglets are relatively similar. Beta diversity mainly reflects the degree of difference in microbial community structure among different treatment groups. Figure 17 PCoA results showed significant grouping effects in the Lfat, Mfat, and Hfat groups. P The value <0.05 indicates that feeding different levels of crude fat can significantly alter the composition of the gut microbiota in piglets. The differences in microbiota composition between the Lfat and Hfat groups were more pronounced, while the Mfat group showed intermediate transitional characteristics.

[0099] At the genus level, a total of 87 genera were detected across the three groups. Based on the species annotation results, each group was selected from the genera ( genus The top 10 genera in terms of relative abundance at the [missing information] level were identified, and a relative abundance stacking plot was constructed. The results are shown in Table 21 and [missing information]. Figure 18 As shown, compared with the Lfat group, the Mfat group had a higher proportion of Lactobacillus spp. ( Lactobacillus Abundance increased significantly ( P <0.01), *Macrococcus* genus in group Hfat ( Megasphaera The relative abundance of ) was significantly higher than that of the Mfat group and the Lfat group ( P <0.05).

[0100] Differential bacterial comparison analysis was performed with an LDA score threshold of 4. The Mfat group was enriched with Lactobacillus orders (Lactobacillus) compared to the Lfat group. Lactobacillales A hierarchical group centered on ), including the Lactobacillus family ( Lactobacillaceae Bacilli, Lactobacillus Lactobacillus ) and Lactobacillus johnsonii ( Lactobacillus_johnsonii () Figures 19-20 The Hfat group was significantly enriched in Lactobacillus species compared to the Lfat group. Lactobacillales ) and Streptococcus equi ( Streptococcus equinus )member( Figures 21-22 The phylogenetic clade diagram further reveals the distribution characteristics of differentially expressed microorganisms at each taxonomic level, indicating that altering crude fat levels can reshape the functional structure of the piglet colonic microbiota.

[0101] This embodiment uses Microsoft Excel 2021 to organize and summarize experimental data, and employs SAS 9.4 (Version 9.4, SAS Institute Inc, Cary, NC, USA) statistical software for data analysis. Growth performance data were analyzed using ANOVA one-way ANOVA, with Duncan's method used for multiple comparisons when differences were significant. Orthogonal polynomial tests were used to examine the linear and quadratic effects of increased dietary crude fat levels; when both linear and quadratic effects were significant, only the quadratic effect was discussed. Diarrhea rates were statistically analyzed using a generalized linear mixed model (GLMM), and Tukey's method was used for multiple comparisons between groups. Results are presented as follows: P <0.01 indicates a highly significant difference. P <0.05 indicates a significant difference, 0.05 < P <0.1 indicates a trend of difference. Colonic microbiota sequencing was performed using the Maiwei Metabolic Cloud platform. Alpha and Beta diversity analyses were conducted using the phyloseq and vegan packages in R software. LEfSe (Linear Discriminant Analysis Effect Size) analysis was used to screen key differentially expressed microbiota in different groups and to assess the contribution of abundance of each taxonomic unit to the inter-group difference effect. Parametric and non-parametric tests were used for inter-group difference analysis. T-tests and Wilcoxon tests were used for comparisons between two groups, and Tukey and Kruskal-Wallis tests were used for comparisons between three groups. Microbiota abundance differences were analyzed using ANOVA in SAS 9.4 statistical software, combined with Duncan's method for multiple comparisons.

[0102] As can be seen from the above, under the conditions of 17.5% crude protein, 1.23% SID Lys, and consistent net energy, a crude fat level of around 3.0% is the optimal level that balances growth performance, intestinal barrier function, and colonic microbiome structure. This invention provides a replicable and scalable technical pathway for improving the growth performance of weaned piglets, enhancing feed conversion efficiency, maintaining the intestinal barrier, regulating colonic microecology, and refining feed formulation.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for predicting the optimal ratio of crude protein, crude fat, and starch in piglet feed, characterized in that, Includes the following steps: Step 1: Set the range of proportions of crude protein, crude fat and starch in the feed, and control the total amount of the three factors (crude protein, crude fat and starch) to be constant; Step 2: Establish experimental combinations using extreme vertex mixing design; Step 3: Formulate experimental feed for nursery piglets according to the experimental combination; Step 4: Conduct feeding trials using weaned piglets as the experimental subjects; Step 5: Using crude protein level, crude fat level, and starch ratio as independent variables, and average daily weight gain, average daily feed intake, and feed conversion ratio as response values, establish a multinomial regression model and plot the response surface. Step 6: With the optimization objective of maximizing average daily weight gain and minimizing feed conversion ratio, predict the optimal combination of crude protein, crude fat and starch in piglet diet.

2. The prediction method according to claim 1, characterized in that, Step 1 is designed according to the condition of consistent standard ileal digestible amino acid ratio, or according to ideal amino acid pattern.

3. The prediction method according to claim 2, characterized in that, Under the condition of consistent standard ileal digestible amino acid ratio, the experimental diet for weaned piglets described in step 3, by weight percentage, includes 14.0%~18.0% crude protein, 2.0%~4.0% crude fat, 45.0%~51.0% starch, and 1.23% standard ileal digestible lysine; Under ideal amino acid profile conditions, the experimental diet for weaned piglets described in step 3, by weight percentage, comprises 14.5%–18.5% crude protein, 1.0%–5.0% crude fat, and 44.5%–52.5% starch.

4. The prediction method according to claim 3, characterized in that, The experimental diet for piglets also included corn, corn starch, soybean meal, soybean hulls, soybean oil, corn gluten meal, wheat bran, beet meal, dicalcium phosphate, limestone powder, and sodium chloride. L -Lysine hydrochloride, DL -Methionine, L -Threonine, L -Tryptophan, L -valine, L -Leucine, L -Isoleucine, L -histidine, L - Phenylalanine and premix.

5. The prediction method according to claim 4, characterized in that, The test combination in step 2 consists of 9 treatment groups, including 1 center point, 4 vertices and 4 axis points; In step 4, the feeding experiment for nursery piglets lasts for 21 days. On the start and end days of the experiment, the piglets are weighed on an empty stomach, and the amount of feed given, the amount of leftover feed, and the occurrence of diarrhea are recorded by pen. In step 5, Design-Expert 13 software is used for model fitting and response surface plotting, and the model determination coefficient is used. R 2 and corrected coefficient of determination Radj 2 Evaluate the goodness of fit of the model; Step 6 uses Minitab 21 software for response optimization to obtain the predicted optimal combination of crude protein, crude fat and starch in piglet diet.

6. The prediction method according to any one of claims 1 to 5, characterized in that, It also includes the following steps: Step 7: Based on the optimal crude protein level obtained in Step 6, fix the crude protein level and the standard ileal digestible lysine level, set different crude fat levels and adjust the starch ratio simultaneously to formulate a trial diet for weaned piglets with consistent net energy levels. Step 8: Detect piglet growth performance, serum biochemical indicators, serum inflammation and antioxidant indicators, intestinal permeability, intestinal morphology, intestinal tight junction protein gene expression, mucin gene expression, immune factor expression, antioxidant-related gene expression, and colonic microbial structure to determine the optimal combination of proportions that balance growth performance, intestinal barrier function, and intestinal microbial homeostasis.

7. The application of the prediction method according to any one of claims 1 to 6 in the preparation of piglet feed.

8. The application according to claim 7, characterized in that, The piglet feed has one or more of the following functions; (1) Nutritional regulation; (2) Improve feed conversion efficiency; (3) Maintain or improve intestinal barrier function; (4) Regulate colonic microbial structure.

9. The application according to claim 8, characterized in that, Maintaining or improving intestinal barrier function includes increasing the ratio of jejunal and ileal villus height to crypt depth, and upregulating... ZO-1 , Occludin , Claudin-1 and MUC2 Gene expression, increasing goblet cell number, enhancing anti-inflammatory factor expression, or increasing the expression of one or more genes related to intestinal antioxidant activity; The regulation of colonic microbial structure includes increasing the number of Lactobacillus spp. ( Lactobacillus Relative abundance, enriched in Lactobacillus (Lactobacillus order) Lactobacillales Lactobacillus family ( Lactobacillaceae Bacilli, Lactobacillus Lactobacillus ) and Lactobacillus johnsonii ( Lactobacillus_johnsonii ).

10. A piglet feed, characterized in that, The piglet diet comprises, by weight percentage, 17.5% crude protein, 3.0% crude fat and 1.23% standard ileal digestible lysine; the net energy of the piglet diet is 2449 kcal / kg.

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