A composition and its use in the preparation of a feed additive for laying hens

Abstract

The application discloses a kind of composition and its application in preparation of laying hen feed additive, it is related to the field of biotechnology.The composition includes the following weight parts of components: Ganoderma lucidum polysaccharide 50-70 parts, jerusalem artichoke polysaccharide 25-35 parts, lentinan 25-35 parts, polygonatum officinale powder 15-25 parts and cypress leaf 15-35 parts.The application research finds that, adding 200~600 mg / kg composition in diet, it does not affect egg laying rate, egg weight and eggshell quality, can effectively improve the feed palatability of laying hen, can effectively improve production performance.The composition can promote intestinal tissue development, increase intestinal villus height, improve villus crypt ratio, strengthen intestinal physical barrier function;While up-regulating intestinal tight junction protein and short-chain fatty acid receptor gene expression, regulating intestinal flora structure, enriching beneficial bacteria, inhibiting harmful bacteria.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composition and its application in the preparation of feed additives for laying hens. Background Technology

[0002] Poultry farming is an important component of the livestock industry. Eggs produced by laying hens are rich in high-quality protein, minerals, and various vitamins, making them a crucial ingredient in the dietary nutrition system. The stable development of the poultry farming industry also has a significant impact on the overall operation of the livestock economy. Currently, the poultry farming industry is continuously developing towards large-scale and intensive operations, with increasing stocking densities and standardized and continuous feeding and management models. This places higher demands on the health of laying hens, production stability, and the quality of the final egg products.

[0003] In conventional poultry farming, various functional additives are commonly used to optimize breeding results, with some antibacterial agents being particularly prevalent. These substances can effectively reduce the incidence of diseases in laying hens and minimize losses from common intestinal and respiratory illnesses, playing a role in improving breeding efficiency and reducing the difficulty of feeding and management. However, long-term and continuous use of these agents can gradually lead to numerous negative effects. The components of these agents can easily leave residues in the laying hens and eggs, which can be transmitted along the food chain, posing a health risk to consumers and contradicting increasingly stringent food safety standards.

[0004] Meanwhile, these substances can disrupt the gut microbiota of laying hens. The gut is the core site for nutrient digestion, absorption, and transport in laying hens, and also an important line of immune defense. Maintaining a dynamic balance of gut microbiota is fundamental to ensuring the normal functioning of the gut. The introduction of exogenous preparations can disrupt the original structure of the microbiota, causing a decrease in the activity of beneficial microorganisms and an imbalance in the microbiota ratio. This directly affects the efficiency of nutrient decomposition and absorption, disrupts the body's normal metabolic rhythm, and consequently leads to unstable egg production. Furthermore, long-term use can also induce adaptive changes in harmful microorganisms, gradually increasing the difficulty of disease control. Components that are not absorbed and utilized by the body can also enter the natural environment through excrement, impacting the surrounding water and soil environment and hindering the coordinated development of the livestock industry and the ecological environment.

[0005] Developing functional feed additives based on natural substances has become an important direction for optimizing farming models in the industry. Currently, most existing feed additives suffer from problems such as limited functionality, fluctuating effects, and restricted application scenarios. Some products also have high production costs, failing to meet the comprehensive needs of large-scale farming. Based on the current state of industry development, in-depth exploration of natural active substances to develop compound feed additives that integrate intestinal regulation, immune modulation, performance optimization, and egg quality improvement, addressing the functional shortcomings of current products and meeting the comprehensive needs of intensive layer hen farming, has become a key research direction in the poultry feed and animal nutrition field. Summary of the Invention

[0006] The purpose of this invention is to provide a composition and its application in the preparation of laying hen feed additives, thereby solving the problems existing in the prior art. This invention develops a composition that can improve laying hen production performance and egg quality, promote intestinal development, enhance intestinal physical barrier defense function, improve immune function, and optimize intestinal flora.

[0007] To achieve the above objectives, the present invention provides the following solution: The present invention provides a composition for laying hen farming, comprising the following components in parts by weight: 50-70 parts of Ganoderma lucidum polysaccharide, 25-35 parts of Jerusalem artichoke polysaccharide, 25-35 parts of shiitake mushroom polysaccharide, 15-25 parts of Polygonatum odoratum powder, and 15-35 parts of Platycladus orientalis leaves.

[0008] Preferably, the composition comprises the following components in parts by weight: 60 parts of Ganoderma lucidum polysaccharide, 30 parts of Jerusalem artichoke polysaccharide, 30 parts of shiitake mushroom polysaccharide, 20 parts of Solomon's seal powder, and 20 parts of Platycladus orientalis leaf.

[0009] The present invention also provides the use of the above-described composition in the preparation of a layer hen feed additive, wherein the layer hen feed additive has at least one of the following functions: (1) Improve the production performance of laying hens; (2) Improve egg quality; (3) Promotes intestinal development in laying hens; (4) Enhance the intestinal physical barrier defense function; (5) Improve immune function; (6) Optimize gut microbiota.

[0010] Furthermore, the egg production performance of the hens includes the egg production rate.

[0011] Furthermore, the egg quality includes egg weight, eggshell thickness, and / or eggshell color.

[0012] The present invention also provides a laying hen feed additive, wherein the active ingredients include the above-described composition.

[0013] Furthermore, the laying hen feed additive has at least one of the following functions: (1) Improve the production performance of laying hens; (2) Improve egg quality; (3) Promotes intestinal development in laying hens; (4) Enhance the intestinal physical barrier defense function; (5) Improve immune function; (6) Optimize gut microbiota.

[0014] The present invention also provides the application of the above-mentioned layer hen feed additive in the preparation of layer hen feed.

[0015] The present invention also provides a layer hen feed, comprising the above-mentioned layer hen feed additives.

[0016] Furthermore, the composition content in the laying hen feed is 200-600 mg / kg.

[0017] Preferably, the composition content in the laying hen feed is 600 mg / kg.

[0018] The present invention discloses the following technical effects: This invention provides a composition that, as a feed additive for laying hens, exhibits multiple positive effects with a clear dose-dependent effect. Adding 200-600 mg / kg of the composition to the diet does not affect feed intake or palatability in laying hens, but effectively improves egg production rate, egg weight, and eggshell quality, optimizing production performance. This composition promotes intestinal tissue development, increases intestinal villus height and villus-cryptotropic ratio, and strengthens the intestinal physical barrier function; simultaneously, it upregulates the expression of intestinal tight junction proteins and short-chain fatty acid receptor genes, regulates the intestinal flora structure, enriches beneficial bacteria, and inhibits harmful bacteria. Furthermore, this composition improves the state of immune organs, enhances serum and intestinal mucosal immune indicators, inhibits the activation of inflammatory pathways, and comprehensively enhances the body's immunity. The optimal addition dose is 600 mg / kg, which provides the most outstanding comprehensive regulatory effect, enabling simultaneous improvement in laying hen health, production capacity, and egg quality.

[0019] Production performance and egg quality are core economic indicators for evaluating the application value of feed additives, and their changes directly reflect the body's nutritional metabolic efficiency and overall health status. This invention shows that there was no significant difference in the average daily feed intake among the laying hens in each group throughout the experiment, indicating that different dosages of the composition did not affect feed palatability and feeding behavior, eliminating the interference of feed intake fluctuations on the experimental results. The performance differences among the groups all stemmed from the intrinsic regulation of intestinal health and metabolic function by the composition. Regarding egg production performance, the composition exhibited typical low-promoting, medium-superior, and high-inhibiting dose effects. Appropriate dosages of the composition can improve nutrient utilization by improving intestinal health, thereby enhancing egg production performance, while high doses have no positive effect.

[0020] Regarding egg quality, the composition of this invention has no significant effect on egg shape index, albumen height, yolk color, and Haugh units, indicating that its regulation is targeted and does not change the basic physicochemical properties of eggs; however, its improvement effect on eggshell thickness, egg weight, and eggshell color shows a significant dose-dependent effect. Considering both production performance and egg quality results, the 600 mg / kg composition balances high yield and high quality, making it the optimal dosage for industrial application; 200 mg / kg can be used as a mild additive for maintaining steady egg production, and higher dosages have no promotional value.

[0021] The gut is the core organ for nutrient digestion and absorption in poultry. Intestinal weight index, tissue morphology and structure, tight junction barrier function, and short-chain fatty acids (SCFAs) metabolites from the gut microbiota collectively constitute the evaluation system for gut health and are also the core targets for the probiotic effects of the composition. This invention integrates and analyzes intestinal development, morphology and structure, barrier genes, and SCFA metabolism to systematically reveal the dose-regulation mechanism of the composition's effect on gut health. Regarding intestinal development, the 600 mg / kg composition significantly increased the weight index of the duodenum and cecum, while the 200 mg / kg composition significantly increased the cecum weight index, although the high-dose group showed no significant promoting effect.

[0022] The morphology and structure of the jejunum are the core manifestation of the intestinal physical barrier. Villus height and villus-crypt ratio directly determine nutrient absorption capacity, while crypt depth reflects the epithelial cell renewal rate. The experimental results of this invention show no significant difference in crypt depth among the groups, excluding pathological intestinal damage. The 200 and 600 mg / kg groups showed significantly increased jejunal villus height and villus-crypt ratio, and significantly increased intestinal absorptive area, while the 1200 and 1800 mg / kg groups showed no difference compared to the control group. These results indicate that low-to-medium dose compositions can physiologically optimize intestinal morphology and structure without cell damage, providing a structural basis for nutrient absorption and barrier defense. At the intestinal barrier function level, tight junction proteins ZO-1, Occludin, and Claudin-1 are core molecules maintaining intestinal epithelial integrity; their upregulation can reduce intestinal permeability and block endotoxin and pathogen invasion. All tight junction genes were significantly upregulated in the 600 mg / kg group, showing a significant improvement trend at 200 mg / kg, while high doses had no effect.

[0023] Short-chain fatty acids (SCFAs) are core metabolites of carbohydrate fermentation by gut microbiota. They regulate gut health by activating GPR41 and GPR43 receptors, and possess energy, anti-inflammatory, and barrier protection functions. The experimental results of this invention show that GPR41 / 43 genes were significantly upregulated in the low-to-medium dose group. Combined with the gut microbiota function prediction results, this suggests that the 600 mg / kg group had enhanced carbohydrate metabolism capacity and increased SCFA synthesis, forming a positive regulatory axis of "microbiota fermentation acid production - GPR receptor activation - enhanced intestinal barrier." The high-dose group showed no significant upregulation of receptor genes, stemming from a reduction in beneficial fermenting bacteria and insufficient SCFA synthesis, resulting in ineffective barrier function activation. In summary, the composition of this invention synergistically improves gut health by promoting intestinal development, optimizing morphological structure, strengthening tight junctions, and activating the SCFA signaling pathway. This effect is strictly dependent on the appropriate dose, with 600 mg / kg showing the most comprehensive regulatory effect.

[0024] Maintaining immune homeostasis is crucial for laying hens to resist stress and maintain high egg production. This invention systematically analyzes the immune regulatory mechanism of the composition from four dimensions: immune organ development, peripheral blood humoral immunity, intestinal mucosal immunity, and the TLRs / NF-κB inflammatory pathway, achieving a synergistic evaluation of systemic and local immunity. Immune organs are the sites of immune cell proliferation and differentiation; the spleen index reflects peripheral immune reserve capacity; and the liver participates in the body's immune defense and toxin clearance. The experimental results of this invention show that the spleen index significantly increased in the 600 mg / kg group, and showed an improving trend at 200 mg / kg, confirming that appropriate doses of the composition can physiologically promote the maturation of immune organs and enhance the body's innate immunity.

[0025] At the level of peripheral blood immune markers, immunoglobulin IgG and cytokines are core markers of humoral immunity, IL-4 is an anti-inflammatory factor, and IFN-γ is a pro-inflammatory factor; their balance determines the body's inflammatory homeostasis. In the experiments of this invention, 200 mg / kg significantly increased serum IL-4 and IgG levels, while 600 mg / kg simultaneously upregulated IgG, IL-2, and IL-4, achieving dual optimization of immune enhancement and anti-inflammatory homeostasis; the high-dose group showed no significant immune activation effect. These results indicate that the low-to-medium dose composition can systematically activate humoral immunity throughout the body without inducing excessive inflammation, consistent with the physiological immune characteristics of healthy animals.

[0026] Intestinal mucosal immunity is the body's largest immune barrier. IgA and pIgR constitute the core antibacterial defense line of the mucosa, while the TLRs / NF-κB pathway is the core hub for intestinal inflammation regulation. The intestinal gene expression results of this invention showed that 200 mg / kg significantly upregulated the anti-inflammatory factor IL-4 and downregulated the pro-inflammatory genes TLR4 and IFN-γ; 600 mg / kg significantly upregulated IgA, pIgR, and all anti-inflammatory genes, and significantly inhibited the activation of the TLR4 and NF-κB pro-inflammatory pathways; high doses showed only a weak regulatory trend. The slight upregulation of TLR2 was due to enhanced immune surveillance mediated by the composition, not inflammation activation. Comparative analysis revealed that the magnitude of intestinal immune regulation was significantly higher than that in peripheral blood, suggesting that the intestine is the core target organ for the immune regulation of the composition of this invention, and its effect follows the transmission law of "local intestinal activation—systemic immune enhancement." The inability to effectively inhibit inflammatory pathways in the high-dose group stemmed from the increased endotoxin load caused by dysbiosis, which continuously stimulated inflammatory pathways and counteracted the anti-inflammatory effect. In summary, appropriate dosage of the composition can promote the development of immune organs, activate humoral immunity throughout the body, strengthen mucosal defense, and inhibit inflammatory pathways, thereby building immune homeostasis. The optimal immunomodulatory effect is achieved at 600 mg / kg.

[0027] The gut microbiota is the core medium for host-composition interaction, and its structure and function directly determine the strength of the composition's probiotic effect. This invention combines five methods for cross-validation: α-diversity, β-diversity, genus-level TOP10 microbiota, independent samples t-test, and LEfSe differential marker analysis. This precisely analyzes the differential regulation of the gut microbiota by compositions at 200, 600, 1200, and 1800 mg / kg. The results clearly support the core thesis of this invention: 200 mg / kg maintains natural homeostasis, 600 mg / kg provides physiological optimization, and 1200 and 1800 mg / kg cause pathological dysregulation.

[0028] At the α-diversity level, the Chao1 index reflects the richness of the gut microbiota, while the Shannon index reflects the evenness of the community. In the experiments of this invention, the gut microbiota diversity in the 200 mg / kg group was not significantly different from that in the control group, maintaining the natural homeostasis of the gut microbiota without excessive intervention, representing an ideal mild dose for long-term culture. The diversity in the 600 mg / kg group was moderately increased, indicating physiological enrichment of beneficial bacteria without any risk of dysbiosis. The diversity in the 1800 mg / kg group was abnormally increased, indicating pathological diversity caused by excessive proliferation of pathogenic and mixed bacteria, and not a sign of gut health. The PCoA and ANOSIM tests for β-diversity showed that the 200 mg / kg group and the control group samples were highly clustered, with the highest similarity in microbiota structure. The 600 mg / kg group showed a moderate shift in the microbiota, indicating directional beneficial remodeling. The 1200 and 1800 mg / kg samples showed the greatest dispersion, indicating severe dysbiosis of the microbiota structure, confirming that high-dose intervention excessively disrupted the microecological balance.

[0029] Differential microbiota analysis is the core of this invention, with five methods cross-identifying the regulatory characteristics of beneficial and harmful bacteria at each dosage. At 200 mg / kg, only beneficial bacteria producing short-chain fatty acids, such as *Romebutzimia*, were significantly enriched, with no fluctuations in the abundance of harmful bacteria, resulting in the most stable microbiota structure, perfectly matching the "homeostasis maintenance" positioning. At 600 mg / kg, precise bidirectional regulation was achieved, significantly enriching anti-inflammatory and acid-producing beneficial bacteria such as *Romebutzimia* and *Weisseria*, while inhibiting potential opportunistic pathogens. Although there were minor microbiota fluctuations, the overall result was a physiological remodeling dominated by beneficial bacteria, effectively activating microbiota metabolism and immune regulation functions, making it the golden dose for microbiota regulation. At 1200 and 1800 mg / kg, negative regulation was observed, significantly reducing the abundance of core probiotics such as *Lactobacillus*, while heavily enriching opportunistic pathogens and harmful bacteria such as *Helicobacter* and *Desulfovibrio*, completely disrupting the microbiota balance and resulting in the loss of probiotic effects, confirming the harm of high doses.

[0030] The Tax4Fun gut microbiota function prediction results and species composition form a complete closed loop. The core functions of each group are enriched in basic pathways such as carbohydrate metabolism and membrane transport, suggesting that the composition does not alter the core metabolic types of the gut microbiota. The 600 mg / kg group showed stable and balanced abundance of functional pathways and healthy gut metabolic activity, achieving synergistic optimization of structure and function. The 200 mg / kg group showed high functional consistency with the control group, maintaining basal metabolic homeostasis. The 1200 mg / kg group showed abnormally hyperactive functional pathways, representing compensatory metabolism after gut microbiota dysbiosis. The 1800 mg / kg group showed a comprehensive decrease in metabolic activity, indicating impaired gut microbiota function. In summary, the composition of this invention exhibits a significant dose-dependent regulation of the structure and metabolic function of the intestinal microbiota in laying hens, and the orderly remodeling of the microbiota composition is the core mechanism mediating intestinal health and metabolic regulation. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 Principal coordinate analysis (PCoA, based on OTU level) plot of β diversity of cecal microbiota in laying hens under different doses of the composition; Figure 2 Bar chart showing the relative abundance of cecal microbial species at the genus level in laying hens treated with different dosage combinations; Figure 3 Wilcoxon rank-sum test analysis of significantly different levels of bacteria in the cecum of laying hens between the control group and the 200 mg / kg combination group; Figure 4Wilcoxon rank-sum test analysis of significantly different levels of bacteria in the cecum of laying hens between the control group and the 600 mg / kg combination group; Figure 5 Wilcoxon rank-sum test analysis of significantly different levels of bacteria in the cecum of laying hens between the control group and the 1200 mg / kg composition group; Figure 6 Wilcoxon rank-sum test analysis of significantly different levels of bacteria in the cecum of laying hens between the control group and the 1800 mg / kg combination group; Figure 7 LEfSe analysis diagram of differential species in the cecal microbiota of laying hens under different doses of the composition; Figure 8 Heatmap of functional abundance of KEGG Level 2 pathway in the cecal microbiota of laying hens under different doses of the composition. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Terminology Explanation: Yu Zhu, a Chinese medicinal herb, is the rhizome of Polygonatum odoratum (Mill.) Druce, a plant belonging to the genus Polygonatum in the family Liliaceae.

[0039] Platycladus orientalis (L.) franco, a Chinese medicinal herb, refers to the branches and leaves of Platycladus orientalis (L.) franco, a plant belonging to the genus Platycladus in the family Cupressaceae.

[0040] The Ganoderma lucidum polysaccharide used in the following examples was purchased from Shaanxi Senfu Natural Products Co., Ltd.; Jerusalem artichoke polysaccharide was purchased from Shandong Yidelai Biotechnology Co., Ltd.; and Lentinan was purchased from Shandong Xinde Technology Co., Ltd.

[0041] Example 1 A method for preparing a composition for egg-laying hen farming: (1) After crushing the Solomon's Seal and the leaves of Platycladus orientalis into powder, pass them through a 60-mesh sieve to obtain Solomon's Seal powder and Platycladus orientalis leaf powder.

[0042] (2) Mix 60g of Ganoderma lucidum polysaccharide, 30g of Jerusalem artichoke polysaccharide, 30g of shiitake mushroom polysaccharide, 20g of Solomon's seal powder and 20g of Platycladus orientalis leaf powder evenly to obtain the composition.

[0043] Example 2 A method for preparing a composition for egg-laying hen farming: (1) After crushing the Solomon's Seal and the leaves of Platycladus orientalis into powder, pass them through a 60-mesh sieve to obtain Solomon's Seal powder and Platycladus orientalis leaf powder.

[0044] (2) Mix 50g of Ganoderma lucidum polysaccharide, 35g of Jerusalem artichoke polysaccharide, 25g of shiitake mushroom polysaccharide, 15g of Solomon's seal powder and 35g of Platycladus orientalis leaf powder evenly to obtain the composition.

[0045] Example 3 A method for preparing a composition for egg-laying hen farming: (1) After crushing the Solomon's Seal and the leaves of Platycladus orientalis into powder, pass them through a 60-mesh sieve to obtain Solomon's Seal powder and Platycladus orientalis leaf powder.

[0046] (2) Mix 70g of Ganoderma lucidum polysaccharide, 25g of Jerusalem artichoke polysaccharide, 35g of shiitake mushroom polysaccharide, 25g of Solomon's seal powder and 15g of Platycladus orientalis leaf powder evenly to obtain the composition.

[0047] Example 1 of effect verification 1. Materials and Methods 1.1 Test Materials The composition prepared in Example 1; the serum immune marker ELISA kit, total RNA extraction and qPCR detection reagents were all commercially available analytical grade products; 16S rRNA high-throughput sequencing was performed by a professional biotechnology company; the remaining reagents were all domestically produced analytical grade.

[0048] 1.2 Experimental Animals and Experimental Design The experiment employed a single-factor, completely randomized design, selecting 240 healthy 70-week-old Hy-Line Brown laying hens, who were randomly divided into 5 treatment groups, with 6 replicates per group and 8 hens per replicate. All groups received the same basal diet, with the only difference being the addition of different dosages of the combined ingredients to the diet. The experiment lasted for 9 weeks, including a 1-week pre-trial period and an 8-week formal trial period. The experimental groups are as follows: CON group: Basic diet, without added ingredients; G200 group: basal diet + 200mg / kg combination; G600 group: basal diet + 600mg / kg combination; G1200 group: basal diet + 1200mg / kg combination; G1800 group: basal diet + 1800mg / kg combination.

[0049] 1.3 Basic Diet and Feeding Management The basal diet was formulated according to the "NY / T33-2021 Chicken Feeding Standard" as a corn-soybean meal complete diet. The diet was in powder form, and the formula and nutrient levels are shown in the appendix to the main text. The experimental chickens were housed in three-tiered cages, one chicken per cage, with free access to feed and water, and standardized feeding and management were implemented throughout the process. The temperature in the chicken house was controlled at 22-26℃, the relative humidity at 55-65%, and the lighting regime was 16 hours of light: 8 hours of darkness, with a light intensity of 15-20 lx. Manure was removed and ventilation was carried out regularly every day to keep the house clean and dry. No antibiotics, probiotics, or other feed additives were used during the experiment to prevent external factors from interfering with the experimental results. The formula and nutrient levels of the laying hen diet are shown in Table 1.

[0050] Table 1. Dietary formulation and nutrient levels (%, air-dried basis) Note: ª Premix provides vitamins, trace elements, and carriers per kilogram of ration (formulated according to laying hen feeding standards); ᵇ All nutrient levels are calculated values.

[0051] 1.4 Sample Collection and Processing On the day the experiment concluded, one laying hen of similar weight was randomly selected from each replicate, and slaughtered by exjugation via the jugular vein. Under aseptic conditions, the mid-jejunum tissue, cecal contents, and immune organs such as the spleen and thymus were rapidly separated. The intestinal tissue was rinsed thoroughly with sterile saline, and surface moisture was blotted dry with filter paper. A portion was fixed in 4% paraformaldehyde solution for histological morphology analysis; the other portion was aliquoted into enzyme-free cryovials, flash-frozen in liquid nitrogen, and then stored at -80°C for gene expression and microbial sequencing analysis. Cecal contents were directly and aseptically collected and stored at -80°C. Organ indices were calculated after weighing the immune organs.

[0052] 1.5 Measurement Indicators and Methods 1.5.1 Production performance and egg quality During the experiment, the number of eggs laid, egg weight, number of dead eggs, and feed intake were recorded daily for each group. The laying rate, average egg weight, average daily feed intake, and feed conversion ratio were calculated and statistically analyzed weekly. Fresh egg samples were collected from each group every 4 weeks, and conventional egg quality indicators such as eggshell thickness, eggshell strength, albumen height, Haugh unit, and yolk color were measured using an egg quality analyzer.

[0053] 1.5.2 Organ Index After weighing the organs, calculate the organ index (%) = organ weight (g) / live body weight (g) × 100%.

[0054] 1.5.3 Serum immune markers The serum immunoglobulin G (IgG), interleukin-2 (IL-2), IL-4, and interferon-gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., and the assay was performed according to the instructions.

[0055] 1.5.4 Intestinal Morphology Examination Jejunal tissue was dehydrated stepwise with ethanol, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and mounted. Observation was performed under an optical microscope (DM2000 LED, Leica, Germany) at 100x magnification, and photographs and analysis were conducted using ZEN software (Zeiss, Germany). Villus height was defined as the vertical distance from the villus tip to the villus-crypt junction, and crypt depth was defined as the indentation depth between adjacent villi. For each tissue section, 10 intact, straight-oriented villi were selected under the microscope, and their villus height and crypt depth were measured. The average value was used as the measurement for that sample, and the villus height to crypt depth ratio (V / C) was calculated.

[0056] 1.5.5 Relative expression levels of gene mRNA Total RNA was extracted from the samples using Trizol reagent (Invitrogen Biotechnology Inc., USA). The purity and concentration of total RNA were determined using a nanophotometer NP80 (Germany). RNA was reverse transcribed into cDNA using a PrimeScript RT kit and a gDNA Eraser (Takara Biotechnology Inc., Japan). Real-time quantitative PCR was performed on the sample genes using a TB Green® Premix Ex Taq™ (Takara Biotechnology Inc., Japan) kit on a CFX96 Real-Time PCR instrument (Bio-rad, USA). A 25 μL reaction mixture was prepared, consisting of 12.5 μL of TB Green Premix Ex Taq (Tli RnaseH Plus) (2×), 0.5 μL each of forward and reverse primers (10 μmol / L), 2 μL of cDNA, and 9.5 μL of sterile, nuclease-free water. PCR conditions were 95°C for 30 s, followed by 95°C for 5 s and 60°C for 30 s, for a total of 40 cycles. The specificity of PCR products was assessed using melting curves. Two [methods were used]. -ΔΔCt The method calculates the relative mRNA expression level of the gene.

[0057] 1.5.6 Analysis of the cecal microbiota Total microbial DNA was extracted from the cecal contents, and the V3-V4 region of the 16S rRNA gene was amplified. After library construction, high-throughput sequencing was performed using the Illumina platform. After sequence quality control, OTU clustering analysis was performed, and Alpha diversity indices (Chao1, Shannon) were calculated. Beta diversity analysis, species annotation, and LEfSe differential microbial community analysis were conducted. Tax4Fun was used for microbial function prediction analysis.

[0058] 1.6 Data Statistical Analysis One-way ANOVA was performed using SPSS 26.0 statistical software, and Duncan's method was used for multiple comparisons between groups. Data are expressed as mean ± standard error (SEM). P <0.05 indicates a significant difference. P <0.001 indicates a highly significant difference, and 0.05 < P <0.10 indicates a divergent trend.

[0059] 2 Results 2.1 Production performance The effects of different dosages of dietary additives on the production performance of laying hens from week 1 to week 8 are shown in Table 2. As shown in Table 2, there was no significant difference in the average daily feed intake among the groups during the experiment. P >0.05%, indicating that the composition of the present invention has no adverse effect on the feeding behavior of laying hens. The composition exhibits a regulatory trend on the egg production rate of laying hens (<0.05). P <0.10), showing an overall dose-dependent characteristic, with the 600 mg / kg (G600) group showing the best effect. Compared with the control group (CON), after 4 weeks of experimentation, the egg production rate of laying hens in the G600 group was significantly increased. P <0.05), the egg production rate of the 200 mg / kg (G200) group showed an improving trend (0.05 < P <0.10), the egg production rate of the medium- and high-dose groups (1200 mg / kg (G1200) and 1800 mg / kg (G1800) decreased to varying degrees, returning to the control group level. There was no significant difference in average egg production among the different groups each week. P >0.05), but the values ​​for the G600 group were generally higher than those for the other groups. Regarding the feed conversion ratio (FCR), the composition showed a significant regulatory effect in week 5 of the experiment ( P <0.05, the feed conversion ratio of the G600 group was significantly lower than that of the G1800 group ( P <0.05); in the remaining weeks, the feed conversion ratio of the G600 group was the lowest among all groups, demonstrating the best feed conversion efficiency.

[0060] Table 2 Effects of different dosages of dietary additives on laying hen production performance (weeks 1-8) Note: The P-value column in the table represents the overall significance test value between groups in a one-way ANOVA; different lowercase letters in the subscripts of the data in the same column indicate significant differences. P <0.05), the presence of both different and identical letters on the shoulder insignia indicates a trend of discrepancy (0.05 < P <0.10, with the same letter or no letter on the shoulder label indicating no significant difference ( P >0.05). Same as the table below.

[0061] 2.2 Egg quality The effects of different dosages of the dietary supplementation composition on egg quality are shown in Table 3. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly increased eggshell thickness at week 4, egg weight at week 8, and eggshell color A value (redness). P <0.05), egg weight in week 4 and eggshell thickness, eggshell color (L and B values) in week 8 showed an increasing trend (<0.05). P <0.10). Dietary supplementation with the 600 mg / kg composition significantly increased egg weight and shell thickness at weeks 4 and 8, as well as the L-value (brightness) of eggshell color at week 8.P <0.05), in week 8, the eggshell color A (redness) and B (yellowness) showed an increasing trend (<0.05). P <0.10). Dietary supplementation with the 1200 mg / kg composition significantly increased eggshell thickness in week 4, and egg weight, eggshell strength, egg weight in week 4, eggshell thickness, and eggshell color (L, A, and B values) showed an increasing trend in week 8 (<0.05). P <0.10. Dietary supplementation with a 1800 mg / kg composition significantly increased the B-value of eggshell color at week 8. P <0.05), showing a trend of increasing egg weight and shell thickness in week 4 and egg weight, shell color (L and A values) in week 8 (0.05 < P <0.10%. The addition of any dosage of the composition had no significant effect on egg shape index, albumen height, Haugh units, or yolk color. P >0.05).

[0062] Table 3 Effects of different dosages of dietary additives on egg quality 2.3 Organ Index The effects of different doses of the dietary supplementation composition on organ indices in laying hens at week 8 are shown in Table 4. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly increased the cecal weight index (CPI). P <0.05), indicating a tendency for an increase in the spleen index (0.05 < P <0.10. Dietary supplementation with 600 mg / kg of the composition significantly increased spleen index, duodenal weight index, and cecal weight index ( ). P <0.05). Dietary supplementation with a 1200 mg / kg composition tended to increase the spleen index (<0.05). P <0.10. Dietary supplementation with 1800 mg / kg of the composition had no significant effect on the weight indices of any organs or the intestine. P >0.05).

[0063] Table 4. Effects of different dosages of dietary additives on organ index in laying hens 2.4 Serum immune markers The effects of different doses of the dietary supplementation with the composition on serum immune indicators in laying hens are shown in Table 5. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly increased IL-4 levels at week 4 and IgG levels at week 8. P <0.05), showing an increasing trend in IL-4 and IL-2 levels at week 8 (0.05 < P<0.10. Dietary supplementation with a 600 mg / kg composition significantly increased IgG and IL-4 levels at week 4 and IgG, IL-4, IL-2, and IFN-γ levels at week 8. P <0.05. Dietary supplementation with the 1200 mg / kg composition significantly increased IL-2 levels at week 8 (P<0.05), and showed an increasing trend in IgG levels at week 4 and IFN-γ levels at week 8 (P<0.05). P <0.10). Dietary supplementation with a 1800 mg / kg composition showed a trend of increasing IgG levels in week 4 and IL-2 and IFN-γ levels in week 8 (<0.05). P <0.10).

[0064] Table 5 Effects of different dosages of dietary additives on serum immune indicators in laying hens 2.5 Jejunum Morphology and Structure The effects of different doses of the dietary supplementation with the composition on the morphology and structure of the jejunum in laying hens are shown in Table 6. Compared with the control group, dietary supplementation with 200 and 600 mg / kg of the composition significantly increased the villus height and villus-to-crypto ratio in the jejunum. P <0.05%. Adding 1200 and 1800 mg / kg of the composition had no significant effect on jejunal villus height or villus-to-crypto ratio. P >0.05). There was no significant difference in jejunal crypt depth among the groups. P >0.05).

[0065] Table 6. Effects of different dosages of dietary additives on the morphology and structure of jejunal tissue in laying hens. 2.6 Jejunal barrier function and expression of genes related to short-chain fatty acid receptors The effects of dietary supplementation with different doses of the composition on jejunal barrier function and short-chain fatty acid receptor-related gene expression in laying hens are shown in Table 7. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly improved jejunal barrier function. ZO-1, GPR41 and GPR43 Gene expression ( P <0.05), for Occludin, Claudin-1 Gene expression showed an increasing trend (0.05 < P <0.10). Adding 600 mg / kg of the composition significantly improved jejunal function. ZO-1, Occludin, Claudin-1, GPR41 and GPR43 Gene expression ( P <0.05). Adding 1200 mg / kg of the composition to ZO-1 Claudin-1 and GPR43 gene expression There is an upward trend (0.05 < P<0.10). Adding 1800 mg / kg of the composition did not significantly affect the expression of the above-mentioned genes in the jejunum ( P >0.05).

[0066] Table 7. Effects of different dietary additives on jejunal barrier function and short-chain fatty acid receptor-related gene expression in laying hens. 2.7 Expression of genes related to jejunal immunity and TLRs / NF-κB signaling pathway The effects of dietary supplementation with different doses of the composition on jejunal immunity and the expression of genes related to the TLRs / NF-κB signaling pathway in laying hens are shown in Table 8. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly increased jejunal immunity. IL-4 Gene expression ( P <0.05), significantly reducing jejunal effusion. TLR4, IFN-γ Gene expression ( P <0.05), for pIgR , IL-2 , IL-10 Gene expression showed an increasing trend (0.05 < P <0.10). Adding 600 mg / kg of the composition significantly improved jejunal function. IgA, pIgR, IL-2, IL-4, IL-10 Gene expression ( P <0.05), significantly reducing jejunal effusion. TLR4, NF-κB, IFN-γ Gene expression ( P <0.05). The addition of 1200 mg / kg of the composition tended to decrease the expression of jejunal TLR4 and IFN-γ genes (<0.05). P <0.10), showing a trend of increasing the expression of IgA, pIgR, IL-2, and IL-10 genes (0.05 <). P <0.10). The addition of 1800 mg / kg of the composition tended to decrease the expression of jejunal TLR4 and IFN-γ genes (<0.05). P <0.10), showing a trend of increasing IL-2 gene expression (0.05< P <0.10).

[0067] Table 8 Effects of different dietary supplements on jejunal immunity and expression of genes related to the TLRs / NF-κB signaling pathway in laying hens 2.8 Cecal Microbiota 2.8.1 α-diversity As shown in Table 9, compared with the control group, dietary supplementation with the 1800 mg / kg composition significantly increased the Chao1 index of the cecal microbiota (P<0.05). Supplementation with the 200, 600, and 1200 mg / kg composition showed an increasing trend in the Chao1 index (P<0.05). P <0.10).

[0068] Table 9. Effects of different dietary additives on cecal microbial α-diversity in laying hens 2.8.2 β-diversity To investigate the effect of the composition of this invention on the overall structure of the gut microbiota in laying hens, principal coordinate analysis (PCoA) was performed based on the OTU level. The results are as follows: Figure 1 As shown in the figure, the first principal component (PC1) explained 28.59% and the second principal component (PC2) explained 16.16%, with the two axes cumulatively explaining 44.75% of the differences in gut microbiota structure. The ANOSIM intergroup difference test results showed R=0.1252, P=0.018<0.05, indicating a statistically significant difference in the overall gut microbiota structure among the different dosage groups. From the sample distribution, the control group and the low-dose G200 group showed good clustering, mainly distributed on the left side of the coordinate axis, indicating high similarity in microbiota structure. The G600, G1200, and G1800 groups gradually shifted to the right with increasing dosage, showing increased differentiation from the control group's microbiota structure, and the high-dose group samples exhibited higher dispersion. These results indicate that the composition of this invention can significantly alter the overall structure of the gut microbiota in laying hens, and the regulatory effect shows a clear dose-dependent relationship, with medium- and high-dose additions having a more prominent effect on microbiota remodeling.

[0069] 2.8.3 Top 10 microorganisms at the genus level The effects of different doses of dietary additives on the top 10 microorganisms in the cecum of laying hens are shown in [see below]. Figure 2 The effects on the relative abundance of key microorganisms are shown in Table 10. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly increased the abundance of *Romebutzim* spp. (…). Romboutsia Relative abundance ( P <0.05%. Adding 600 mg / kg of the composition significantly increased the levels of *Romusbacterium* and *Weissella* spp. (…). Weissella Relative abundance ( P <0.05%, for Lactobacillus spp. ( Lactobacillus The relative abundance showed a decreasing trend (0.05 < P <0.10). Adding 1200 mg / kg of the composition significantly reduced the relative abundance of *Lactobacillus* and *Weisseria* species. P<0.05%. Adding 1800 mg / kg of the composition significantly increased the relative abundance of *Rhombus* spp. ( P <0.05, significantly reducing the relative abundance of the genus *Weissella* ( P <0.05), indicating a decreasing trend in the relative abundance of Lactobacillus (0.05 < P <0.10).

[0070] Table 10 Effects of different dietary additives on the relative abundance (%) of key bacteria in the top 10 microorganisms of the cecum in laying hens 2.8.4 T-test for differentially expressed microorganisms Wilcoxon rank-sum test was used to analyze the differential bacterial genus in the cecum of laying hens in each group. The results are as follows: Figure 3 As shown. Compared with the control group, dietary supplementation with 200 mg / kg of the composition significantly reduced Escherichia coli spp. (as shown). Aeriscardovia The relative abundance was significantly increased. UCG-005 genus Pseudomonas ( ) Pseudomonas ), NK4A136 group of Trichophyceae ( Lachnospiraceae_ NK4A136_group ) and Parabacterium genus ( Parabacteroides Relative abundance ( P <0.05).

[0071] Depend on Figure 4 It can be seen that, compared with the control group, the addition of 600 mg / kg of the composition significantly improved... UCG-005 genus *Bacillus*, genus *Koala* ( Phascolarctobacterium ) and Rosomonas genus ( Roseomonas The relative abundance of *Cyclocarya* was significantly reduced. Nannocystis Relative abundance ( P <0.05).

[0072] Depend on Figure 5 It can be seen that, compared with the control group, the addition of 1200 mg / kg of the composition significantly reduced Lactobacillus spp. ( Lactobacillus ), Pediococcus ( Pediococcus ), Ehrlich. genus ( Aeriscardovia ) and Leuconostoc genus ( Leuconostoc The relative abundance of *Romebuts* spp. was significantly increased. Romboutsia ), Shigella spp. Escherichia-Shigella ), Campylobacter spp. Campylobacter ), genus *Faecalibacterium* Faecalibacterium ), Koala spp. ( Phascolarctobacterium )and UCG-005 Relative abundance of fungal genera ( P <0.05).

[0073] Depend on Figure 6 It can be seen that, compared with the control group, the addition of 1800 mg / kg of the composition significantly reduced Corynebacterium spp. ( Corynebacterium ) and Escherichia coli spp. Aeriscardovia Relative abundance ( P <0.05%, significantly increasing the levels of *Rhombus* spp. ( Romboutsia Bacteroides ( Bacteroides Clostridium UCG-014 Genus, Fusobacterium ( Fusobacterium ), Koala spp. ( Phascolarctobacterium ), relative abundance of UCG-005 genus and Parabacterium genus ( P <0.05).

[0074] 2.8.5 Analysis of LEfSe differential markers LEfSe analysis was used to screen for marker differential species (LDA > 2) in the cecal gut microbiota of laying hens in different dosage groups. The results are as follows: Figure 7 As shown, each group exhibited specifically enriched differential bacterial communities, and the differential species covered multiple taxonomic levels, including phylum, class, order, family, and genus, indicating that the composition of this invention can significantly shape the characteristic bacterial community structure of the intestinal flora of laying hens.

[0075] The main differentially identified bacteria in the control group (CON) were from the order Corynebacteriales, family Corynebacteriaceae, and genus Corynebacterium. Corynebacterium The bacteria included species from the families Bifidobacteriaceae and Bifidobacteriales, and the abundance of these species was significantly higher in the control group than in the groups with added compositions; the 600 mg / kg composition group was dominated by Actinobacteria. UCG-005 Genus *Phytococcus* Pediococcus The core marker bacteria were Fusobacterium spp., which were specific enriched species at this dosage; the 1200 mg / kg combination group had the richest characteristic differential bacteria, mainly enriched in Fusobacterium spp. (…). Fusobacterium ), Fusobacteriales, Fusobacteriota, Bacteroidetes, Shigella genus ( Escherichia-Shigella Bacteroides ( Bacteroides) and other bacterial groups; the characteristic species of the 1800 mg / kg combination group are Erysipelotrichales, Veillonellales-Selenomonadales, Selenomonasaceae, and Megamonas genus ( Megamonas Different dosages of the combination can induce the formation of specific marker bacteria in the gut of laying hens, with medium to high doses showing a more significant effect on the remodeling of gut characteristic bacteria.

[0076] 2.8.6 Results of Microbial Community Function Prediction A heatmap of the relative abundance of the KEGG Level 2 pathway in the cecal microbiota was obtained using Tax4Fun functional prediction. The results are as follows: Figure 8 As shown in the figure, the functions of the gut microbiota in each group were mainly enriched in high-abundance basic pathways such as global metabolism, carbohydrate metabolism, and membrane transport. The composition of this invention did not change the core functional types of the gut microbiota. From the perspective of dosage changes, the G1200 group had the highest abundance in pathways such as signal transduction, energy metabolism, and nucleotide metabolism, showing the strongest microbial metabolic and signaling activity; the G600 group had moderate abundance of each functional pathway with the least fluctuation, and the most stable and balanced microbial functional state; the G200 group was closest to the control group, maintaining the homeostasis of basic intestinal function; the G1800 group showed a significant decrease in the abundance of various metabolic and regulatory pathways, and the microbial functional activity was significantly inhibited. Although the G1200 group had the highest abundance of functional pathways, combined with the results of microbial structure, it can be seen that at this dose, the beneficial bacteria decreased and the harmful bacteria increased, which is a compensatory hyperactivity after microbial dysbiosis, not a healthy state; while the microbial structure of the G600 group did not show dysbiosis, and the functional pathways remained stable and active, achieving a synergistic balance between microbial structure and functional activity. Therefore, 600 mg / kg is a comprehensive and appropriate dose that balances the stability of the gut microbiota structure and normal function. Excessive doses (1200~1800 mg / kg) will disrupt the homeostasis of the gut microbiota.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composition for egg-laying hen farming, characterized in that, It includes the following components by weight: 50-70 parts of Ganoderma lucidum polysaccharide, 25-35 parts of Jerusalem artichoke polysaccharide, 25-35 parts of shiitake mushroom polysaccharide, 15-25 parts of Solomon's seal powder, and 15-35 parts of Platycladus orientalis leaf.

2. The composition according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 60 parts of Ganoderma lucidum polysaccharide, 30 parts of Jerusalem artichoke polysaccharide, 30 parts of shiitake mushroom polysaccharide, 20 parts of Polygonatum odoratum powder, and 20 parts of Platycladus orientalis leaf.

3. The use of the composition as described in claim 1 or 2 in the preparation of a laying hen feed additive, characterized in that, The egg-laying hen feed additive has at least one of the following functions: (1) Improve the production performance of laying hens; (2) Improve egg quality; (3) Promotes intestinal development in laying hens; (4) Enhance the intestinal physical barrier defense function; (5) Improve immune function; (6) Optimize gut microbiota.

4. The application as described in claim 1, characterized in that, The egg production performance of the hens includes the egg production rate; The egg quality includes egg weight, eggshell thickness, and / or eggshell color.

5. A feed additive for laying hens, characterized in that, The active ingredient includes the composition according to claim 1 or 2.

6. The layer hen feed additive as described in claim 5, characterized in that, The egg-laying hen feed additive has at least one of the following functions: (1) Improve the production performance of laying hens; (2) Improve egg quality; (3) Promotes intestinal development in laying hens; (4) Enhance the intestinal physical barrier defense function; (5) Improve immune function; (6) Optimize gut microbiota.

7. The use of a layer hen feed additive as described in claim 5 or 6 in the preparation of layer hen feed.

8. A type of laying hen feed, characterized in that, Includes the layer hen feed additives as described in claim 5 or 6.

9. The laying hen feed as described in claim 8, characterized in that, The content of the composition in the laying hen feed is 200-600 mg / kg.

10. The laying hen feed as described in claim 9, characterized in that, The content of the composition in the laying hen feed is 600 mg / kg.

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