Fermented broussonetia papyrifera-based laying hen feed additive and application thereof in improving production performance and intestinal health of aged laying hens

Abstract

The invention relates to the field of livestock and poultry breeding, and discloses a laying hen feed additive based on fermented broussonetia papyrifera and application of the laying hen feed additive. The additive is prepared by taking paper mulberry leaves as a main material, matching with rice husks, wheat bran and other auxiliary materials, inoculating a microecological preparation containing lactobacillus plantarum, candida utilis and bacillus subtilis, and performing anaerobic fermentation. 4% of the additive is used for daily ration of the aged laying hens, so that the laying rate can be remarkably increased, the dirty egg rate can be reduced, and the egg weight, the eggshell strength and the yolk color can be improved; the mechanism is to improve the form of the intestinal tract, enhance the expression of tight junction protein, regulate immune balance, optimize the structure of cecum flora and enrich beneficial microorganisms; the broussonetia papyrifera leaf feed additive realizes efficient utilization of broussonetia papyrifera leaf resources, provides a safe and effective functional additive for prolonging the laying period of laying hens and improving the breeding benefits, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of livestock and poultry breeding and feed technology, and in particular to a fermented mulberry-based feed additive for laying hens and its application in improving the production performance and intestinal health of older laying hens. Background Technology

[0002] The laying cycle of laying hens can generally be divided into three stages: the initial laying period (15-25 weeks of age), the peak laying period (25-50 weeks of age), and the late laying period (50-80 weeks of age). The late laying period (generally from 50 weeks of age to culling) is the longest, accounting for about half of the entire economic utilization cycle. During this stage, the physiological functions of laying hens undergo a systemic decline, particularly in gut health. Specifically, older laying hens often experience impaired intestinal mucosal barrier function, increased levels of chronic inflammation, and gut microbiota dysbiosis. This deterioration in gut function directly leads to reduced efficiency in nutrient digestion and absorption, resulting in decreased egg production and poorer eggshell quality (such as reduced eggshell strength, increased broken and dirty egg rates), causing significant economic losses to the poultry industry.

[0003] Antibiotics have long been widely used as feed additives to maintain animal health and production performance. Currently, common antibiotic alternatives include plant extracts, probiotics, and organic acids, but their effectiveness and cost are often unsatisfactory. Therefore, developing new and highly efficient functional feed resources is of great significance.

[0004] Paper mulberry (Broussonetia papyrifera) is a fast-growing, high-biomass woody plant. Its leaves are rich in protein, vitamins, and various bioactive substances, making it a potential unconventional feed resource. However, fresh paper mulberry contains anti-nutritional factors such as tannins and has a high cellulose content, resulting in poor palatability and low digestibility when fed directly, severely limiting its large-scale application. Fermentation is a mature bioprocessing technology. Through anaerobic fermentation by microorganisms such as lactic acid bacteria, the nutritional components of green fodder can be effectively preserved, some anti-nutritional factors and complex macromolecules can be degraded, and the palatability and nutritional value of the feed can be improved. Existing technologies have reported the application of paper mulberry in the feed of livestock and poultry such as pigs, cattle, and sheep. Preliminary studies have also explored its application effects in the diets of laying hens during peak egg production.

[0005] However, existing technologies have obvious limitations and gaps: First, most existing studies focus on the general effects of paper mulberry on animal growth performance or peak egg production performance, lacking in-depth research on the specific intestinal health decline problems faced by laying hens during the later stages of egg production.

[0006] Second, existing technologies have failed to elucidate the deep-seated mechanisms by which paper mulberry improves the production performance of laying hens, particularly lacking empirical data to support its systemic regulatory effects on the intestinal physical barriers (such as tight junction proteins), immune status, and microbial community structure in aging laying hens.

[0007] Third, there is a lack of systematic and clear technical solutions and experimental evidence regarding the appropriate proportion of paper mulberry added to the diet of aged laying hens, as well as its quantitative impact on key economic indicators such as eggshell quality and protein metabolism in the later stages.

[0008] Therefore, there is an urgent need in this field for a feed additive for laying hens based on fermented mulberry and its application in improving the production performance and intestinal health of older laying hens, so as to achieve a targeted approach with a clear mechanism of action that can effectively alleviate the decline in intestinal function in older laying hens, thereby stabilizing and prolonging their production performance in the later stages of egg production. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention aims to provide a feed additive and its application that can effectively improve the egg production performance, egg quality, and gut health of laying hens aged 50 weeks and older. This additive, with fermented mulberry tree as its core, achieves its goal of comprehensively improving the production performance of older laying hens by reshaping the gut microbiota and enhancing intestinal barrier function through specific formulations and addition ratios.

[0010] The solution of the present invention is: A feed additive for laying hens based on fermented paper mulberry, wherein the feed additive is prepared by fermentation of crushed paper mulberry leaves, and the addition amount in the complete feed for laying hens is 2% to 8% by mass.

[0011] As a preferred technical solution, the amount added to the complete feed for laying hens is 4% by weight.

[0012] As a preferred technical solution, the feed additive comprises the following raw materials in parts by weight: 50-70 parts of crushed paper mulberry leaves; 15-25 parts rice husks; 5-15 parts wheat bran; 2-8 parts corn flour; 0.5 to 2 parts glucose; Salt 0.5 to 2 parts; 1-5 parts zeolite powder; 1-3 parts of probiotic preparation.

[0013] As a preferred technical solution, the microecological preparation comprises *Lactobacillus plantarum*, *Candida utilis*, and *Bacillus subtilis*, wherein the *Lactobacillus plantarum* ≥ 1 × 10⁻⁶. 5 CFU / g, *Candida utilis* ≥ 4 × 10⁻⁴ 9cfu / g, Bacillus subtilis ≥2×10 9 cfu / g, wherein the Candida utilis accounts for 25% of the total mass of the microecological preparation.

[0014] This invention also discloses a method for preparing a feed additive for laying hens based on fermented paper mulberry, comprising the following steps: 1) Mix 50-70 parts by weight of crushed paper mulberry leaves, 15-25 parts by weight of rice husks, 5-15 parts by weight of wheat bran, 2-8 parts by weight of corn flour, 0.5-2 parts by weight of glucose, 0.5-2 parts by weight of salt, 1-5 parts by weight of zeolite powder and 1-3 parts by weight of microecological preparation evenly to obtain a mixture. 2) The mixture is placed into a fermentation container, compacted, sealed, and subjected to anaerobic fermentation. It can be used when the pH value is stable and a strong sour aroma is produced. The fermentation cycle is 14 to 37 days to obtain a layer hen feed additive based on fermented paper mulberry.

[0015] The present invention also discloses a compound feed for improving the egg production performance, egg quality and intestinal health of older laying hens, comprising a basic diet for laying hens and a feed additive for laying hens based on fermented mulberry.

[0016] As a preferred technical solution, the age of the laying hens is ≥50 weeks.

[0017] As a preferred technical solution, the age of the laying hens is ≥65 weeks.

[0018] This invention also discloses a method for improving the egg production performance, egg quality and intestinal health of older laying hens by adding a fermented mulberry-based feed additive to the feed of older laying hens.

[0019] As a preferred technical solution, improving egg production performance includes increasing the egg production rate and reducing the rate of dirty eggs; improving egg quality includes increasing egg weight, eggshell strength, eggshell weight and / or yolk color.

[0020] As a preferred technical solution, improving gut health includes one or more of the following effects: a) Improves intestinal morphology, manifested as an increase in the ratio of villus height to crypt depth in the jejunum; b) Enhances intestinal barrier function, manifested by upregulating the mRNA expression of tight junction proteins ZO-1 and / or ZO-2 in the jejunum; c) Regulate the gut microbiota, manifested by increasing the relative abundance of Verrucomicrobia and / or Butycoccidae in the cecal contents, and increasing the relative abundance of unclassified genus-level units under Butycoccidae.

[0021] The present invention also discloses the use of a fermented mulberry-based feed additive for laying hens in the preparation of compositions for extending the peak laying period of older laying hens, improving their laying performance, egg quality, or gut health.

[0022] Compared with the prior art, the advantages of the present invention are: 1. The target population and dosage are clearly defined, and the effects are direct and significant. This invention, for the first time, clarifies the optimal application dosage (4% of the total feed weight) of fermented mulberry feed additive for feeding older laying hens (65 weeks and older) in the late laying period, precisely targeting the core problems commonly encountered in this stage, such as declining intestinal function and reduced production performance. Rigorous animal trials have confirmed that after 8 weeks of continuous feeding, it can significantly increase egg production rate (an absolute increase of over 8 percentage points), effectively reduce the rate of dirty eggs, and simultaneously improve key commercial indicators such as eggshell strength, eggshell weight, and yolk color, comprehensively enhancing the production value and economic returns of older laying hens.

[0023] 2. The mechanism of action is clear, delving from the apparent phenomenon to the biological essence. This invention goes beyond improvements in apparent production performance; its underlying mechanism of action is elucidated through systematic biological evaluation. Examples demonstrate that the feed compositions of this invention can: Optimizes intestinal structure: Significantly improves the morphology of the jejunum and ileum, increases the ratio of villus height to crypt depth, thereby expanding the nutrient absorption area and enhancing digestive function.

[0024] Strengthening the intestinal barrier: Effectively upregulates the gene expression of tight junction proteins (such as ZO-1 and ZO-2) in the jejunum, enhances the physical barrier function of the intestinal epithelium, reduces the translocation of harmful substances, and maintains the stability of the internal environment.

[0025] Regulating the balance of gut microbiota: Specifically increases the relative abundance of beneficial microbial groups closely related to gut health, such as Verrucocephala and Butycoccus in cecal contents, thus promoting gut homeostasis and health from a microecological perspective.

[0026] The aforementioned multi-dimensional and multi-target synergistic effects together constitute a solid scientific basis for improving the health of laying hens and enhancing their production performance.

[0027] 3. The technical solution is green and safe, fully in line with the trend of healthy aquaculture industry. This invention uses widely distributed and renewable paper mulberry resources as the core raw material and employs a mature anaerobic fermentation (silage) process for biotransformation. This process effectively degrades anti-nutritional factors such as tannins, improves the bioavailability of nutrients, and produces a natural palatable flavor. The selected compound microecological preparation (containing Candida utilis, lactic acid bacteria, and Bacillus) are all safe microorganisms permitted for use in China. No chemical drugs or antibiotics are added throughout the entire production process, ensuring the final product's green, safe, and residue-free characteristics, perfectly aligning with modern healthy farming and the industry's development philosophy after the "antibiotic ban."

[0028] 4. The industrialization process is simple and yields significant economic benefits. The feed additive provided by this invention can be directly and uniformly mixed with the basic diet of laying hens in a predetermined ratio without requiring major changes to existing feed processing technology and feeding management procedures, making it easy to rapidly promote and apply in farms. By effectively extending the peak laying period of older laying hens, improving egg quality, and reducing the rate of defective products, it can significantly increase the total return throughout the laying cycle, with a high input-output ratio, broad prospects for industrial application, and significant economic and social benefits. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 This diagram illustrates the effect of fermented mulberry (SBP) feed additive from Example 5 of this invention on the intestinal morphology of laying hens in the late laying period. In the diagram, A represents villus height, B represents crypt depth, and C represents the ratio of villus height to crypt depth (VH:CD). Data are expressed as mean ± standard error (n=6). * indicates a significant difference in the t-test (*P<0.05). Villus height (VH) represents villus height; crypt depth (CD) represents crypt depth; Con represents the basal diet group; SBP represents the basal diet + SBP laying hen feed additive group; Jejunum represents the jejunum; Ileum represents the ileum; and duodenum represents the duodenum. Figure 2This diagram illustrates the effect of fermented mulberry bark (SBP) feed additive from Example 5 of the present invention on the expression of genes related to intestinal barrier function in laying hens during the late laying period. The diagram shows the mRNA expression of tight junction proteins in the jejunum (A), ileum (B), and duodenum (C). Data are expressed as mean ± standard error (n=6). * indicates significant difference in the T-test (*P<0.05, **P<0.01). Con: basal diet group; SBP: basal diet + SBP feed additive group. Figure 3 The effect of fermented mulberry (SBP) feed additive for laying hens in Example 5 of this invention on the gene expression of intestinal immune-related factors in late-laying laying hens; wherein, the mRNA expression of immune-related factors in the jejunum (A), ileum (B), and duodenum (C); data are expressed as mean ± standard error (n=6); * indicates significant difference in T-test (*P<0.05, **P<0.01); Con: basal diet group; SBP: basal diet + SBP feed additive group; Figure 4This diagram illustrates the effect of fermented mulberry bark (SBP) feed additive from Example 5 of the present invention on the bacterial microbial community of the cecal contents of laying hens in the late laying period. A represents the Venn diagram of the operational taxonomic units (OTUs) of the Con and SBP groups; B represents the Alpha diversity index analysis (Chao1, ACE, Shannon, Simpson); C represents the principal coordinate analysis (PCoA) diagram showing the differences in community structure between groups; D represents the relative abundance of the top ten bacterial taxa at the genus level; and E represents the species with significant differences in abundance at the phylum, family, and genus levels between the two groups, identified by a t-test. The species names involved in the diagram include: *Methanobrevibacter*, *Desulfovibrio*, *Phascolarctobacterium*, and *[Ruminococcus] torques* (family Trichophyceae). group), Lachnospiraceae, Faecalibacterium, Lactobacillus, Treponema, Rikenellaceae_RC9_gut_group, Bacteroides, Verrucomicrobiota, Prevotellaceae, Barnesiellaceae, Butyricicoccaceae, g__Butyricicoccaceae_unclassified (butyricicoccaceae unclassified), Flavoisolibacter; * indicates significant difference in T test (*P<0.05, **P<0.01); Con: basal feed group; SBP: basal feed + SBP layer hen feed additive group; Figure 5This is a diagram illustrating the effect of fermented mulberry tree (SBP) feed additive for laying hens (Example 5 of this invention) on the eukaryotic microbial community of the cecal contents of late-laying laying hens. A is a Venn diagram; B is a principal coordinate analysis (PCoA) diagram; C is a UPGMA clustering tree diagram, all used to show differences in community structure between groups, and relative abundance at the phylum level; D shows the relative abundance at the phylum level, phylum, and relative abundance. Abundance; E represents relative abundance at the genus level. Species names included in the figure include: Unidentified Eukaryota, Ascomycota, Chordata, Ciliophora, Cercozoa, Arthropoda, Protalveolata, Mucoromycota, Streptophyta, Basidiomycota, Diatoms, Others, Mollusca, Unidentified Streptophyta, Fusarium, Boeremia, Mortierella, Blastocystis, Unidentified chondrionid-vertebrate group. Choanozoa-Vertebrata, Actinomucor, Penicillium, Cyberlindnera-Candida clade; * indicates significant difference between the two treatments (*P<0.05, **P<0.01); Con: basal diet group; SBP: basal diet + SBP layer hen feed additive group; Figure 6The graph shows the correlation analysis results between the bacterial genera that caused significant changes in the cecal contents of laying hens after treatment with the feed additive (SBP) of the present invention and their egg production performance and eggshell quality indicators. The indicators involved include: laying rate, egg weight, egg-misshapen rate, dirtyeggs rate, feed conversion ratio, egg-broken rate, eggshell weight, eggshell strength, calcitonin, eggshell thickness, and parathyroid hormone. * indicates a significant difference when P < 0.05. The graph uses a dynamic correlation heatmap to show the strength and significance of the correlation between each bacterial genera and egg production performance and eggshell quality (*P < 0.05). Detailed Implementation

[0031] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0032] Example 1: After preparing the raw materials, proceed with the following steps: 1) Mix 50 parts by weight of crushed paper mulberry leaves, 15 parts by weight of rice husks, 5 parts by weight of wheat bran, 2 parts by weight of corn flour, 0.5 parts by weight of glucose, 0.5 parts by weight of salt, 1 part by weight of zeolite powder and 1 part by weight of microecological preparation evenly to obtain a mixture. 2) The mixture is placed into a fermentation container, compacted and sealed, and anaerobic fermentation is carried out. It can be used when the pH value is stable and a strong sour aroma is produced. The fermentation cycle is 14 days to obtain a layer chicken feed additive based on fermented paper mulberry.

[0033] The microecological preparation contains *Lactobacillus plantarum*, *Candida utilis*, and *Bacillus subtilis*, wherein *Lactobacillus plantarum* ≥ 1 × 10⁻⁶. 5 CFU / g, *Candida utilis* ≥ 4 × 10⁻⁴ 9 cfu / g, Bacillus subtilis ≥2×10 9 cfu / g.

[0034] Example 2: After preparing the raw materials, proceed with the following steps: 1) Mix 70 parts by weight of crushed paper mulberry leaves, 25 parts by weight of rice husks, 15 parts by weight of wheat bran, 8 parts by weight of corn flour, 2 parts by weight of glucose, 2 parts by weight of salt, 5 parts by weight of zeolite powder and 3 parts by weight of microecological preparation evenly to obtain a mixture. 2) The mixture is placed into a fermentation container, compacted, sealed, and subjected to anaerobic fermentation. It can be used when the pH value is stable and a strong sour aroma is produced. The fermentation cycle is 37 days to obtain a layer hen feed additive based on fermented paper mulberry.

[0035] The microecological preparation contains *Lactobacillus plantarum*, *Candida utilis*, and *Bacillus subtilis*, wherein *Lactobacillus plantarum* ≥ 1 × 10⁻⁶. 5 CFU / g, *Candida utilis* ≥ 4 × 10⁻⁴ 9 cfu / g, Bacillus subtilis ≥2×10 9 cfu / g.

[0036] Example 3: Preparation of feed additive samples Prepare the raw materials according to the following weight percentages: 58.82% crushed mulberry leaves, 19.62% rice husks, 9.80% wheat bran, 4.90% corn flour, 0.98% glucose, 0.98% salt, 2.94% zeolite powder, and 1.96% microecological preparation. The microecological preparation contains *Lactobacillus plantarum*, *Candida utilis*, and *Bacillus subtilis*, wherein the *Lactobacillus plantarum* content is ≥1×10⁻⁶. 5 CFU / g, *Candida utilis* ≥ 4 × 10⁻⁴ 9 cfu / g, Bacillus subtilis ≥2×10 9 cfu / g, wherein the *Candida utilis* species constitutes 25% of the total mass of the microecological preparation; All the above components are thoroughly mixed in a mixer to obtain a mixture.

[0037] The mixture was placed in a fermentation container, compacted, and sealed for anaerobic fermentation. It was ready for use once the pH stabilized and a strong sour aroma was produced. The fermentation cycle was 30 days, yielding a layer hen feed additive based on fermented mulberry leaves. The nutritional value of crushed mulberry leaves is shown in Table 1.

[0038] Table 1. Nutritional composition of hybrid paper mulberry (B. papyrifera) raw materials

[0039] Example 4: Experiment on the effects of fermented paper mulberry on the production performance and egg quality of aged laying hens

[0040] 4.1 Experimental Animals and Experimental Design Two hundred and forty healthy Hy-Line Brown laying hens aged 65 weeks with similar egg production rates and body weights were randomly divided into two treatment groups, with eight replicates per group and 15 hens per replicate. The control group (Con) was fed a basal diet, while the experimental group (SBP group) was fed an experimental diet supplemented with 4.0% (by weight) fermented mulberry (SBP) prepared in Example 3 (provided by Henan Zhongke Kanggou Technology Co., Ltd.). The composition and nutrient levels of the basal diet are shown in Table 2, and its formulation followed the guidelines of the U.S. National Research Council (1994), the Chinese Chicken Feed Standard (NY / T33-2004), and the Hy-Line Brown Laying Hen Feeding Manual. The experiment lasted for eight weeks, including a one-week pre-trial period and a seven-week formal trial period. The experimental area was the high-quality laying hen demonstration farm of Henan Guan'an Biotechnology Co., Ltd. in Shanxi Province. Feeding and management strictly followed the standard procedures of commercial farms. Chickens were housed in cages with a 12-degree floor inclination under specific environmental conditions (temperature 20–25°C, humidity 60–65%, light cycle 16 hours / day). Feeding was provided at 6:00 AM and 2:00 PM daily, with free access to water. Eggs were collected daily, and egg quality was assessed every 28 days. Husbandry and vaccination procedures followed standard procedures at the experimental site.

[0041] Table 2: Basal Diet Composition and Nutritional Levels (Air-Dried Basis) %

[0042] The mineral premix, added per kilogram of feed, consists of: dicalcium phosphate, limestone, feed-grade lysine, methionine, feed-grade vitamins (A, D, E, K, B1, B2, B6, B12, biotin, folic acid, niacin, pantothenic acid, choline chloride), feed-grade trace elements, bran powder, phytase, non-starch polysaccharide complex enzyme, and ethoxyquinoline. Vitamin A: 120,000-200,000 IU / kg; Vitamin D: 30,000-70,000 IU / kg; Vitamin B2: ≥90 mg / kg; Iron: 600-4400 mg / kg; Vitamin K3: ≥20 mg / kg; Copper: 80-550 mg / kg; Zinc: 900-2400 mg / kg; Manganese: 1000-3000 mg / kg.

[0043] 4.2 Measurement Indicators and Methods 4.2.1 Production performance: The number of eggs laid, egg weight, number of broken eggs, number of dirty eggs, and number of deformed eggs were recorded daily in replicates. The feed consumption for each replicate was calculated weekly. Egg production rate, average egg weight, average daily feed intake, and feed conversion ratio were calculated on day 28 (end of week 4) and day 56 (end of week 8) of the experiment.

[0044] 4.2.2 Egg quality: On days 28 and 56 of the experiment, 60 eggs were randomly collected from each group (approximately 5 eggs per replicate). Egg weight, albumen height, yolk color and Haugh units were measured using an egg analyzer (TM, Oka Technology).

[0045] The major and minor diameters of the egg were measured using an electronic vernier caliper (Deli, DL3944, Ningbo) to calculate the egg shape index (longitudinal diameter / transverse diameter), and the thickness of the eggshell at the tip, middle, and blunt ends was measured. The average eggshell thickness (mm) was calculated as (tip thickness + middle thickness + blunt end thickness) / 3.

[0046] Eggshell strength was measured using an eggshell strength tester (EFG-0503, Nanjing Mingao Instrument Equipment), and the weight of the eggshell and yolk was measured using an electronic balance (Leqi, DL3944, Ningbo). The yolk percentage (%) was calculated as: yolk weight / egg weight × 100%.

[0047] 4.2.3 Serum Biochemical Indicators: After the aforementioned measurements were completed, one chicken was randomly selected from each replicate group, fasted for 12 hours, weighed, and slaughtered. Sampling sites included the jejunum, ileum, duodenum, and cecum. Samples were frozen in liquid nitrogen and then transferred to the laboratory, placed on dry ice, and stored at -80°C. Blood samples were centrifuged for 15 minutes (2,000×g, 4°C) to obtain serum, which was stored at -80°C for subsequent analysis.

[0048] The levels of uric acid (UA), glucose (GLU), total cholesterol (CHOL), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C / LDL-C), calcium (Ca), phosphorus (P), total protein (TP), albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and diamine oxidase (DAO) in serum were measured using a fully automated biochemical analyzer.

[0049] 4.3 Test Results 4.3.1 Impact on production performance The experimental results are shown in Table 3. Compared with the control group, the SBP group fed a diet containing 4% fermented mulberry showed significantly higher egg production rates on days 28 and 56 of the experiment (P<0.05), and a highly significant decrease in the rate of dirty eggs on day 56 (P<0.01). The average egg weight of the SBP group showed an increasing trend, while the feed conversion ratio showed a decreasing trend.

[0050] Table 3: Effects of fermented paper mulberry on laying hen production performance

[0051] 4.3.2 Impact on Egg Quality The experimental results are shown in Table 4. On day 28, the egg weight and yolk color of the SBP group were significantly better than those of the control group (P<0.05). On day 56, the egg weight, shell strength, shell weight, and yolk color of the SBP group were all significantly or extremely significantly better than those of the control group (P<0.05 or P<0.01).

[0052] Table 4: Effects of fermented paper mulberry on egg quality

[0053] 4.3.3 Effects on serum biochemical indicators The experimental results are shown in Table 5. Compared with the control group, the serum triglyceride (TG) level in the SBP group was significantly lower (P<0.05), and the serum phosphorus (P) level was significantly higher (P<0.05). Other indicators such as uric acid (UA), glucose (GLU), total cholesterol (CHOL), high-density lipoprotein / low-density lipoprotein (HDL / LDL), calcium (Ca), total protein (TP), albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and diamine oxidase (DAO) levels showed no significant differences between the two groups (P>0.05).

[0054] Table 5: Effects of fermented paper mulberry on serum biochemical parameters of laying hens

[0055] Example 5: Mechanism study of the effects of fermented mulberry on intestinal health of aged laying hens 5.1 Sample Collection At the end of the experiment in Example 4, one chicken was randomly selected from each replicate group of Example 4 and euthanized by exsanguination via the carotid artery. Subsequently, the duodenum, jejunum, and ileum were removed. A 2-cm segment was taken from the middle of each segment, rinsed with sterile saline, and then stored in Bouin's solution in the dark for ≥48 hours to obtain samples.

[0056] Tissues from the jejunum, ileum, and duodenum, as well as cecal mucosa, were collected, aliquoted into cryovials, and stored at -20°C to obtain tissue samples for subsequent genomic RNA extraction, PCR amplification, purification, and sequencing.

[0057] 5.1.2 Measurement Indicators and Methods 5.1.2.1 Intestinal Morphological Analysis Samples were fixed, intestinal segments were trimmed with a scalpel, and then dehydrated, cleared, and embedded in paraffin. The sections were stained with hematoxylin and eosin (HE) and mounted with glue. Six sections were prepared from each intestinal segment, and five representative fields of view were randomly selected from each section. Villous height (VH) and crypt depth (CD) were measured using image analysis software (NIS-Elements, Nikon), and the ratio of villous height to crypt depth (V / C) was calculated.

[0058] 5.1.2.2 Intestinal barrier and expression of immune-related genes Approximately 30 mg of jejunal, ileal, and duodenal tissue samples were placed in centrifuge tubes, and total RNA was separated using MagZol reagent (0.05 g tissue / 0.6–0.8 mL MagZol; Magen). RNA purity and yield were assessed by measuring absorbance at 260 / 280 nm using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, New York, USA). RNA samples with an OD value of 1.8–2.0 at 260 / 280 nm were considered suitable for further analysis. Subsequently, cDNA was synthesized in a 20 µL reaction volume using 1 µg of RNA with the aid of a PrimeScript RT kit (Takara, Tokyo, Japan).

[0059] Primer design (Primer 5.0) was based on chicken sequences (Table 6). RT-qPCR was performed using an ABI Prism 7900 HT sequence detection system (Applied Biosystems, Foster City, California, USA), with a total reaction volume of 10 µL, including 5 µL LSYBR Premix Ex Taq (2x) mixture, 0.2 µL ROX (50x), 1 µL cDNA, 3 µL double-distilled water, and 0.4 µL forward and reverse primers (10 mmol / L). The thermal cycling program consisted of 40 cycles of 95 °C for 30 seconds, 95 °C for 5 seconds, and 60 °C for 20 seconds. β-actin was used as an internal reference gene for standardization at the target gene level, and the 2-ΔΔCt method was used to calculate the relative mRNA expression level of the target gene.

[0060] Table 6: Information on qPCR primers used

[0061] 5.1.2.3 Analysis of the cecal microbiota Total genomic DNA of microorganisms in cecal contents was extracted using the CTAB method.

[0062] 16S rRNA and 18S rDNA amplicon sequencing: To analyze bacterial community structure, the V4 region of the bacterial 16S rRNA gene was amplified using specific primers 515F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). To assess eukaryotic microbial community composition, the V4 region of eukaryotic 18S rDNA was amplified using specific primers 528F (5'-GCGGTAATTCCAGCTCCAA-3') and 706R (5'-AATCCRAGAATTTCACCTCT-3'), and sample-specific barcoding was employed. Amplified products were purified using a Qiagen gel extraction kit (Qiagen, Fernol, Netherlands). Sequencing libraries were constructed according to the manufacturer's instructions using the TruSeq DNA PCR-free Sample Preparation Kit (Illumina, USA), and index codes were added. Library quality was assessed using a Qubit 2.0 fluorometer (Thermo Fisher Scientific) and an Agilent Bioanalyzer 2100 system. Sequencing was then performed on the Illumina NovaSeq platform, yielding 250 bp paired-end reads.

[0063] Bioinformatics analysis: After quality control and noise reduction of the raw sequencing data, operational taxonomic units were clustered and species were annotated based on 97% similarity. QIIME2 and R software were used to perform α-diversity (Chao1, ACE, Shannon, Simpson indices), β-diversity (principal coordinate analysis based on Bray-Curtis distance, PCoA), and intergroup differential species analysis (t-test).

[0064] 5.1.3 Statistical Analysis All data are expressed as mean ± standard error (SEM). Differences between groups were analyzed using t-tests. *P < 0.05 was considered statistically significant, and **P < 0.01 was considered highly statistically significant. Spearman correlation analysis was used for correlation analysis.

[0065] 5.2 Test Results 5.2.1 Effects of fermented paper mulberry on intestinal morphology Compared with the control group, the villus height (VH) of the jejunum and ileum in the fermented paper mulberry group was significantly increased (P<0.05), and the crypt depth (CD) was significantly decreased (P<0.05), resulting in a significantly higher V / C ratio. Figure 1 This indicates that fermented mulberry can effectively improve the intestinal absorption structure of older laying hens.

[0066] 5.2.2 Effects of fermented mulberry on intestinal barrier function RT-qPCR results showed that fermented mulberry significantly upregulated the mRNA expression of tight junction proteins ZO-1 and ZO-2 in the jejunum (P<0.05), and the mRNA expression of Claudin-2 and ZO-2 in the duodenum (P<0.05). Figure 2 This indicates that fermented mulberry can enhance the tight junctions of intestinal epithelial cells and strengthen the intestinal physical barrier.

[0067] 5.2.3 Effects of fermented paper mulberry on intestinal immune function Fermented mulberry tree (Broussonetia papyrifera) modulated the local intestinal immune response. Compared with the control group, the fermented mulberry tree group significantly upregulated the mRNA expression of TLR4 in the jejunum and IL-6 in the ileum (P<0.05), while downregulating the expression of IL-6 in the jejunum (P<0.05). The expression of TLR4 and TNF-α in the duodenum was also significantly upregulated (P<0.05). Figure 3 This indicates that fermented mulberry has the potential to regulate intestinal immune homeostasis.

[0068] 5.2.4 Effects of fermented paper mulberry on the cecal microbial community Bacterial community analysis results: The bacterial community composition in the cecal contents was assessed by 16S rRNA gene sequencing. A total of 2,931 operational taxonomic units (OTUs) were identified, of which 1,947 were shared between the Con and SBP groups, and 424 and 560 were unique OTUs in the Con and SBP groups, respectively. Figure 4 A). There were no significant differences in the α-diversity indices (Shannon, ACE, Chao1, and Simpson) between the two groups. Figure 4 B, P>0.05). Principal coordinate analysis (PCoA) showed a trend of separation between the two bacterial community structures. Figure 4 C). No significant differences were found between groups in the ten most abundant taxa at the genus level. Figure 4 D). However, intergroup differential species analysis (t-test) showed that, compared with the control group, the SBP group had increased abundance of Verrucomicrobiota at the phylum level; significantly increased abundance of Prevotellaceae, Butyricicoccaceae, and Barnesiellaceae at the family level; and significantly increased abundance of g__Butyricicoccaceae_unclassified and Flavobacterium at the genus level. Figure 4 E).

[0069] Eukaryotic microbial community analysis results: A total of 1,595 eukaryotic OTUs were identified through 18S rDNA gene sequencing, of which 537 OTUs were shared between the two groups. The Con group and the SBP group contained 552 and 506 unique OTUs, respectively. Figure 5 A). PCoA ( Figure 5 B) and UPGMA cluster analysis ( Figure 5 C) Both groups showed a clear separation in their eukaryotic microbial community structures. Community abundance analysis showed that, compared with the Con group, the SBP group had a higher proportion of unidentified eukaryotes at the phylum level, while the proportions of Chordata, Ascomycota, Mucoromycota, and Streptophyta were decreased. Figure 5 D). At the family level, the SBP group had a higher proportion of Aspergillaceae and Dipodascaceae, while the proportion of Mucoraceae and Pezizaceae was lower. Figure 5 E).

[0070] 5.2.5 Correlation analysis of differentially expressed microorganisms and production performance To explore the association between microbial alterations and host phenotype, a correlation analysis was conducted on the differentially changed bacterial groups and egg production performance and eggshell quality indicators. Figure 6 The results showed that the abundance of related taxa in the genera *Flavobacterium*, *Prevotellaceae*, *Barnesiellaceae*, and *Verrucomicrobiota* was significantly positively correlated with egg production performance indicators. Simultaneously, the abundance of related taxa in the family *Butyricicoccaceae*, unclassified genus *Butyricicoccaceae*, *Flavobacterium*, *Prevotellaceae*, and *Verrucomicrobiota* was also significantly positively correlated with eggshell quality indicators such as eggshell strength and weight. Figure 6 ).

[0071] 5.3 Discussion This invention demonstrates that dietary supplementation with fermented mulberry can improve the gut health of aged laying hens through multiple mechanisms: Improving intestinal structure: Increasing the height of villi in the jejunum and ileum, decreasing crypt depth, and increasing the vitamin C / C ratio indicate that the intestinal absorption area and digestive efficiency are improved.

[0072] Enhanced physical barrier: Upregulation of the expression of key tight junction proteins (ZO-1, ZO-2, Claudin-2) strengthens the barrier function of the intestinal epithelium and helps prevent the translocation of pathogens and toxins.

[0073] Regulation of immune homeostasis: It differentially regulates the expression of immune-related factors such as TLR4 and IL-6, showing anti-inflammatory and immunomodulatory activities, which helps maintain intestinal immune balance.

[0074] Remodeling the gut microbiota: Significantly increased the abundance of beneficial bacteria (such as Butycocci and Prevotaceae). These bacteria are closely related to polysaccharide metabolism, short-chain fatty acid (especially butyrate) production, and immune regulation, and are key to improving host metabolism and health.

[0075] Correlation analysis confirmed that changes in specific beneficial microbiota induced by fermented mulberry were directly related to improved egg production performance and eggshell quality, elucidating the potential pathway through which they exert their effects via gut microbiota-host interactions.

[0076] Conclusion: Fermented mulberry effectively improves the gut health of older laying hens through a comprehensive mechanism involving improved intestinal morphology, enhanced barrier function, regulation of immune response, and remodeling of the cecal microbiota (enrichment of beneficial bacteria). This may be an important basis for improving the laying performance of hens in the later stages of egg production. This invention provides detailed mechanistic evidence for the application of fermented mulberry as a functional feed additive.

[0077] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A feed additive for laying hens based on fermented mulberry, characterized in that: The feed additive is prepared by fermentation of crushed paper mulberry leaves, and the amount added to the complete feed for laying hens is 2% to 8% by weight.

2. The feed additive as described in claim 1, characterized in that, The feed additive comprises the following raw materials in parts by weight: 50-70 parts of crushed paper mulberry leaves; 15-25 parts rice husks; 5-15 parts wheat bran; 2-8 parts corn flour; 0.5 to 2 parts glucose; Salt 0.5 to 2 parts; 1-5 parts zeolite powder; 1-3 parts of probiotic preparation.

3. The feed additive as described in claim 1, characterized in that: The microecological preparation contains *Lactobacillus plantarum*, *Candida utilis*, and *Bacillus subtilis*, wherein *Lactobacillus plantarum* ≥ 1 × 10⁻⁶. 5 CFU / g, *Candida utilis* ≥ 4 × 10⁻⁴ 9 cfu / g, Bacillus subtilis ≥2×10 9 cfu / g, wherein the Candida utilis accounts for 25% of the total mass of the microecological preparation.

4. A method for preparing the feed additive as described in any one of claims 1 to 3, characterized in that, Includes the following steps: 1) Mix 50-70 parts by weight of crushed paper mulberry leaves, 15-25 parts by weight of rice husks, 5-15 parts by weight of wheat bran, 2-8 parts by weight of corn flour, 0.5-2 parts by weight of glucose, 0.5-2 parts by weight of salt, 1-5 parts by weight of zeolite powder and 1-3 parts by weight of microecological preparation evenly to obtain a mixture. 2) The mixture is placed into a fermentation container, compacted, sealed, and subjected to anaerobic fermentation. It can be used when the pH value is stable and a strong sour aroma is produced. The fermentation cycle is 14 to 37 days to obtain a layer hen feed additive based on fermented paper mulberry.

5. A compound feed for improving the egg production performance, egg quality, and intestinal health of older laying hens, characterized in that: It includes a basic diet for laying hens and a fermented mulberry-based feed additive for laying hens as described in any one of claims 1 to 4.

6. The compound feed for improving egg production performance, egg quality, and intestinal health of aged laying hens as described in claim 5, characterized in that: Old laying hens must be at least 50 weeks old.

7. A method for improving the egg production performance, egg quality, and gut health of aged laying hens, characterized in that: Add the fermented mulberry-based feed additive for laying hens as described in any one of claims 1 to 4 to the feed for older laying hens.

8. The method as described in claim 7, characterized in that: Improving egg production performance includes increasing the egg production rate and reducing the rate of dirty eggs; improving egg quality includes increasing egg weight, eggshell strength, eggshell weight and / or yolk color.

9. The method as described in claim 7, characterized in that, Improving gut health includes one or more of the following effects: a) Improves intestinal morphology, manifested as an increase in the ratio of villus height to crypt depth in the jejunum; b) Enhances intestinal barrier function, manifested by upregulating the mRNA expression of tight junction proteins ZO-1 and / or ZO-2 in the jejunum; c) Regulate the gut microbiota, manifested by increasing the relative abundance of Verrucous and / or Butycoccidiale in the cecal contents.

10. The use of a fermented mulberry-based layer hen feed additive as described in any one of claims 1 to 4 in the preparation of a composition for prolonging the peak laying period of older layer hens, improving their laying performance, egg quality, or gut health.

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