Application of mulberry leaf powder in improving lipid metabolism, antioxidant capacity and relieving fatty liver syndrome of laying hens

Abstract

The application belongs to the technical field of poultry breeding and feed additive, and particularly relates to application of mulberry leaf powder in improving lipid metabolism, antioxidant capacity of laying hens and relieving fatty liver syndrome. 5% mulberry leaf powder is added to basic daily ration of laying hens in peak egg production period or high-energy low-protein daily ration for inducing fatty liver hemorrhagic syndrome, the mulberry leaf powder is prepared from fresh leaf blades of feed mulberry by shade air drying for 24 hours, drying at 55 DEG C until constant weight, and crushing through a 40-mesh sieve, and the intervention period is not less than 8 weeks. The application can significantly improve laying rate and daily average egg weight, and optimize serum and liver lipid metabolism and antioxidant indexes by regulating the intestinal-liver axis of laying hens. Meanwhile, the application adjusts the microecology of cecum, improves the content of short-chain fatty acids, inhibits the expression of key genes for liver lipid synthesis, effectively relieves fatty liver syndrome, and restores egg production performance. Under the same active ingredient equivalent, the comprehensive effect of the mulberry leaf powder is significantly better than that of mulberry leaf extract.

Description

Technical Field

[0001] This invention belongs to the field of poultry farming and feed additive technology, specifically relating to the application of mulberry leaf powder in improving lipid metabolism, antioxidant capacity and alleviating fatty liver syndrome in laying hens. Background Technology

[0002] Egg-laying hen farming is a crucial pillar industry of animal husbandry, and the production performance of laying hens during peak egg production directly determines the economic benefits of the farming operation. During peak egg production, the liver of laying hens, as the core organ for lipid metabolism, needs to continuously synthesize large amounts of yolk lipids to meet the demands of egg production. This leads to a significant increase in the liver's lipid metabolic load, easily causing problems such as dyslipidemia and hepatic oxidative stress, which in turn results in a decrease in egg production rate and egg quality, thus hindering the improvement of farming efficiency.

[0003] Fatty liver hemorrhagic syndrome (FLHS) is one of the most common nutritional and metabolic diseases in laying hens during their peak laying period, primarily induced by prolonged feeding of high-energy, low-protein diets. Affected hens exhibit excessive lipid deposition in the liver, enlarged, pale, and fragile liver tissue, along with hemorrhage and necrosis. This is accompanied by abnormally elevated levels of serum liver damage markers and exacerbated oxidative stress. Simultaneously, there is a significant and irreversible decline in egg production, which can lead to acute death in severe cases, causing substantial economic losses to the poultry farming industry.

[0004] Currently, the prevention and control of lipid metabolism disorders and fatty liver syndrome in laying hens mainly rely on conventional methods such as adjusting the energy-to-egg ratio in the diet and optimizing feeding management. However, these methods have limited effectiveness and cannot fundamentally solve the problem of excessive metabolic burden on the liver. In some poultry farming scenarios, chemically synthesized additives are used for regulation; however, these additives pose potential drug residue risks, easily leading to food safety issues, which is inconsistent with my country's current policy direction of "limiting and reducing antibiotic use" in poultry farming and the development needs of green farming.

[0005] Mulberry leaves, as a natural plant material with abundant resources, are rich in various bioactive substances such as polysaccharides, flavonoids, polyphenols, and alkaloids. They have been proven to have multiple physiological functions such as anti-oxidation, anti-inflammation, and regulation of lipid metabolism. Preliminary application studies in the field of livestock and poultry breeding have shown that they can improve the growth performance of livestock and poultry to a certain extent. However, existing research still has significant shortcomings: First, there is a lack of clear and systematic industrial application plans for regulating lipid metabolism and antioxidant capacity in laying hens during peak egg production, and key technical parameters such as the optimal preparation process, addition ratio, and intervention cycle of mulberry leaf powder are not clearly defined. Second, existing studies mostly focus on the application of single active ingredients in mulberry leaf extract, neglecting the synergistic effect among all components of mulberry leaf powder, and have not conducted direct comparative verification of mulberry leaf powder and mulberry leaf extract at equivalent active ingredient levels, making it impossible to determine the optimal application form of mulberry leaves in laying hen farming. Third, the mechanism by which mulberry leaf powder alleviates fatty liver syndrome in laying hens through the "gut-liver axis" pathway has not been systematically elucidated, and there is a lack of research on the molecular correlation between intestinal microecological regulation and liver lipid metabolism and oxidative stress, resulting in a lack of solid theoretical support for the scientific application of mulberry leaf powder in laying hen farming.

[0006] Therefore, developing a natural, safe, efficient, stable, cost-controllable feed additive suitable for industrial application to precisely improve lipid metabolism in laying hens during peak egg production, enhance their antioxidant capacity, and effectively alleviate fatty liver syndrome has become an urgent need for the development of the green poultry farming industry. Summary of the Invention

[0007] The purpose of this invention is to provide an application of mulberry leaf powder in improving lipid metabolism, antioxidant capacity and alleviating fatty liver syndrome in laying hens. By regulating liver metabolism and intestinal microecology in laying hens, it can significantly improve the production performance of laying hens during peak egg production without changing the average daily feed intake of laying hens.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides the application of mulberry leaf powder in the preparation of feed additives for improving lipid metabolism and / or enhancing antioxidant capacity in laying hens.

[0009] The present invention also provides the application of mulberry leaf powder in the preparation of a feed additive for alleviating fatty liver hemorrhagic syndrome in laying hens.

[0010] Furthermore, the laying hens are Hailan Grey laying hens aged 33 weeks and above; the mulberry leaf powder is stored in a sealed container at 4°C and used within four weeks; the application involves uniformly mixing the mulberry leaf powder into the laying hen's basal diet using a horizontal mixer, allowing the laying hens to consume it freely, with an intervention period of no less than 8 weeks.

[0011] Furthermore, the improvement of lipid metabolism involves reducing serum triglyceride, low-density lipoprotein cholesterol, and liver triglyceride levels in laying hens, while increasing serum high-density lipoprotein cholesterol levels; the enhancement of antioxidant capacity involves increasing serum superoxide dismutase and glutathione peroxidase activities, liver total superoxide dismutase and glutathione peroxidase activities, and reduced glutathione content, while reducing serum and liver malondialdehyde levels.

[0012] Furthermore, the application involves adding mulberry leaf powder to a high-energy, low-protein diet that induces fatty liver hemorrhagic syndrome in laying hens; the high-energy, low-protein diet has a crude protein content of 12.02%~12.10% and a metabolizable energy of 12.98 MJ / kg.

[0013] Furthermore, the method for alleviating fatty liver hemorrhagic syndrome in laying hens involves reducing liver indices, improving liver pathological damage, and reducing lipid deposition in hepatocytes; lowering serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels; increasing the activity of serum and liver superoxide dismutase (SOD), catalase, and glutathione peroxidase (GLP-P), and reducing malondialdehyde (MDA) content; and restoring egg production rate in laying hens.

[0014] Furthermore, the mulberry leaf powder can increase the relative abundance of *Femtobacter* and *Bifidobacterium* in the cecum of laying hens, and decrease the relative abundance of *Desulfovibrio*; at the same time, it can increase the content of acetic acid, propionic acid, and butyric acid in the cecum; and the regulatory effect of mulberry leaf powder on the above indicators is significantly better than that of mulberry leaf extract with the same amount of active ingredients.

[0015] Furthermore, the mulberry leaf powder is obtained by drying fresh mulberry leaves for feed in a cool, dry place for 24 hours, drying at 55°C to constant weight, and then pulverizing them through a 40-mesh sieve.

[0016] The present invention also provides a diet for laying hens that improves lipid metabolism, enhances antioxidant capacity and alleviates fatty liver syndrome, wherein mulberry leaf powder is added to the diet.

[0017] Furthermore, the diet is a basic diet for laying hens or a high-energy, low-protein diet, wherein the proportion of mulberry leaf powder added is 5%; the mulberry leaf powder is obtained by drying fresh mulberry leaves in a cool, dry place for 24 hours, drying at 55°C to constant weight, and then crushing them through a 40-mesh sieve.

[0018] The beneficial effects of this invention are as follows: This invention applies mulberry leaf powder prepared using a specific process to the diet of laying hens. By regulating liver metabolism and intestinal microecology through the gut-liver axis, it significantly improves the production performance of laying hens during peak laying periods without altering their average daily feed intake. Egg production rate increases from 90.08% to 93.93%, and average daily egg weight increases from 56.76 g / d to 60.78 g / d, effectively improving the economic efficiency per unit of breeding space. Simultaneously, it precisely optimizes lipid metabolism homeostasis in laying hens, significantly reducing serum triglyceride, low-density lipoprotein cholesterol, and liver triglyceride levels while increasing serum high-density lipoprotein cholesterol levels during peak laying periods. For laying hens with fatty liver syndrome, it can completely reverse the abnormal elevations in serum and liver triglycerides, total cholesterol, and low-density lipoprotein cholesterol, restoring lipid metabolism balance.

[0019] This invention can significantly enhance the antioxidant defense capabilities of laying hens and effectively alleviate liver oxidative stress damage. For laying hens at peak laying age, it can increase serum superoxide dismutase (SOD) and glutathione peroxidase (GPSP) activities, as well as total SOD, GPSP, and reduced glutathione content in the liver, while simultaneously reducing serum and liver malondialdehyde (MDA) levels. For laying hens with fatty liver syndrome, it can comprehensively enhance the activities of antioxidant enzymes such as SOD, catalase, and GPSP in both serum and liver, significantly reduce MDA levels, and repair tissue damage caused by oxidative stress.

[0020] This invention can effectively alleviate fatty liver hemorrhagic syndrome in laying hens, significantly reduce liver index in affected hens, improve the pathological characteristics of pale, fragile, and hemorrhagic livers, reduce hepatocyte vacuolation and lipid droplet deposition, restore the egg production rate that has decreased due to fatty liver syndrome to normal levels, significantly reduce the levels of liver damage markers such as serum aspartate aminotransferase and alanine aminotransferase, alleviate liver inflammation and damage, and avoid economic losses caused by acute death of laying hens.

[0021] This invention can directionally regulate the cecal microecological structure of laying hens, significantly increasing the relative abundance of short-chain fatty acid-producing bacteria such as *Faecalibacterium* and *Bifidobacterium*, while decreasing the abundance of harmful bacteria such as *Desulfovibrio*. Simultaneously, it significantly increases the content of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid in the cecum. Through the gut-liver axis signaling pathway, these beneficial bacteria and their metabolites can significantly inhibit the expression of key genes in liver lipid synthesis, such as FASN, ACACA, and SCD, reducing the nascent synthesis of fatty acids in the liver and fundamentally reducing the lipid deposition load in the liver, thus achieving systemic regulation of the metabolic health of laying hens.

[0022] Under the same active ingredient equivalent, mulberry leaf powder, due to the synergistic effect of all components, significantly outperforms mulberry leaf extract in multiple aspects, including lipid metabolism regulation, antioxidant capacity enhancement, intestinal microecology improvement, short-chain fatty acid generation, and fatty liver syndrome repair. This solves the problem of limited efficacy of single active ingredients in existing technologies. The mulberry leaf powder used in this invention is a natural plant-derived additive with no drug residue risk. Its preparation process is simple, raw material resources are abundant, and costs are controllable. It fully aligns with the current policy direction of "limiting and reducing antibiotics" in poultry farming in my country and the development needs of green farming, possessing broad prospects for industrial application. Attached Figure Description

[0023] 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.

[0024] Figure 1 The graphs show the differentially expressed genes in the livers of laying hens in the control group and the 5% mulberry leaf powder group, including: (A) KEGG bubble chart; (B) GO enrichment analysis graph; (C) KEGG enrichment analysis bar chart; (D) gene-pathway enrichment chord chart; and (E) differentially expressed gene-protein interaction network diagram. Figure 2 A heatmap showing the correlation between differentially expressed microbiota and short-chain fatty acids, lipid metabolism-related genes, and serum lipid / antioxidant indicators; Figure 3 The following are transcriptome analysis and enrichment analysis diagrams of differentially expressed genes in the liver of laying hens: (A) is a statistical diagram of the number of differentially expressed genes; (B) is a heatmap of differentially expressed genes; (C) is a Venn diagram of differentially expressed genes between different comparison groups; (D) is a KEGG enrichment analysis diagram of differentially expressed genes between the FLHS group and the control group (Con); and (E) is a KEGG enrichment analysis diagram of differentially expressed genes between the mulberry leaf powder treatment group (MLP) and the FLHS group.

[0025] Figure 4 The following diagram illustrates the effects of mulberry leaf powder (MLP) on the cecal microbial diversity and community composition of FLHS laying hens: (A) is the α-diversity analysis of gut microbiota; (B) is the principal coordinate analysis (PCoA) diagram based on Bray-Curtis distance; (C) is the stacked diagram of the relative abundance of major bacterial phyla in the three groups; (D) is the stacked diagram of the relative abundance of dominant bacterial genera in the cecal microbiota; and (E) is the bar chart of the top 10 genera with the highest differential abundance among the control group (Con), the FLHS group, and the MLP group.

[0026] Figure 5 The following diagram shows the effects of mulberry leaf powder (MLP) on cecal microbial markers, short-chain fatty acid (SCFA) content, and gut-liver axis correlation network in FLHS laying hens. Among them: (A) is a bar chart of LEfSe analysis; (B) is a bar chart of cecal SCFA composition and content in each group; and (C) is a correlation heatmap. Detailed Implementation

[0027] 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.

[0028] 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. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] 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.

[0030] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] 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.

[0032] Example 1: Application of mulberry leaf powder in laying hen farming during peak egg production period 1.1 Test Materials Mulberry leaf powder: Select fresh mulberry leaves for feed, wash them, and air dry them in a cool place for 24 hours. Then, transfer them to a 55℃ oven to dry to constant weight. After crushing, pass them through a 40-mesh sieve, seal them, and store them at 4℃. They should be used up within four weeks.

[0033] Mulberry leaf extract: prepared by hot water extraction, the main active ingredients are mulberry leaf polysaccharides (20%), mulberry leaf flavonoids (3%) and alkaloids (2%), and the total equivalent of its active ingredients is equivalent to the active ingredient content in 5% mulberry leaf powder.

[0034] 1.2 Experimental Design Three hundred Hy-Line Grey laying hens of similar weight and good health, aged 33 weeks, were selected and randomly divided into 5 groups, with 6 replicates per group and 10 hens per replicate. Control group: The chickens were fed a basal diet during their peak egg production period. The diet composition and nutritional levels were as follows: corn 58.00%, soybean meal 25.50%, soybean oil 2.00%, crude protein 16.20%, metabolizable energy 11.32 MJ / kg, calcium 3.50%, and total phosphorus 0.49%.

[0035] 3% Mulberry Leaf Powder Group: 3% mulberry leaf powder was added to the basal diet to replace an equal proportion of corn and soybean meal, maintaining the same nutritional level as the control group.

[0036] 5% Mulberry Leaf Powder Group: 5% mulberry leaf powder was added to the basal diet to replace an equal proportion of corn and soybean meal, maintaining the same nutritional level as the control group.

[0037] 8% Mulberry Leaf Powder Group: 8% mulberry leaf powder was added to the basal diet to replace an equal proportion of corn and soybean meal, maintaining the same nutritional level as the control group.

[0038] Mulberry leaf extract group: Mulberry leaf extract with the same active ingredient equivalent as 5% mulberry leaf powder was added to the basal diet to replace corn and soybean meal in the same proportion, maintaining the nutritional level of the diet consistent with the control group.

[0039] The experiment lasted for 8 weeks. During the experiment, all laying hens had free access to feed and water and were managed using conventional feeding methods. Daily egg production and weight were recorded in replicates, and weekly feed intake was recorded. Production performance indicators such as laying rate, average daily egg weight, and feed conversion ratio were calculated. At the end of the experiment, 12 hens were randomly selected from each group (2 hens per replicate). Blood was collected from the wing vein to separate serum. After slaughter, liver tissue and cecal contents were collected to determine lipid metabolism indicators, antioxidant indicators, gut microbiota structure, and short-chain fatty acid content. Simultaneously, liver tissue from the control group and the 5% mulberry leaf powder group was collected for transcriptome sequencing analysis.

[0040] 1.3 Results and Analysis 1.3.1 Effects on laying performance of hens The effects of adding different levels of mulberry leaf powder and mulberry leaf extract to the diet on the egg production performance of laying hens are shown in Table 1.

[0041] Table 1. Effects of adding mulberry leaf powder to the diet on egg production performance of laying hens.

[0042] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0043] The results showed that, compared with the control group, the egg production rate of the laying hens in the 5% mulberry leaf powder group significantly increased from 90.08% to 93.93%, and the average daily egg weight significantly increased from 56.76 g / d to 60.78 g / d (P<0.05); the egg production rate of the mulberry leaf extract group was 91.25%, and the average daily egg weight was 58.12 g / d, and its improvement effect was significantly lower than that of the 5% mulberry leaf powder group (P<0.05).

[0044] There were no significant differences in egg production rate and average daily egg weight between the 3% mulberry leaf powder group and the control group (P>0.05), and the 8% mulberry leaf powder group did not show any improvement in production performance. There were no significant differences in average daily feed intake and feed conversion ratio among the groups (P>0.05), indicating that 5% mulberry leaf powder can significantly improve the production performance of laying hens without increasing feed consumption.

[0045] 1.3.2 Effects on serum biochemical and antioxidant indicators of laying hens The effects of dietary supplementation with mulberry leaf powder on serum biochemical and antioxidant indicators of laying hens are shown in Table 2.

[0046] Table 2 Effects of dietary mulberry leaf powder supplementation on serum biochemical and antioxidant indicators in laying hens

[0047] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0048] The results showed that, compared with the control group, serum triglyceride (TG) in the 5% mulberry leaf powder group decreased from 1.79 mmol / L to 1.46 mmol / L, low-density lipoprotein cholesterol (LDL-C) decreased from 2.88 mmol / L to 2.28 mmol / L, and high-density lipoprotein cholesterol (HDL-C) increased from 1.61 mmol / L to 1.95 mmol / L, all with significant differences (P<0.05). Simultaneously, serum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities significantly increased, while malondialdehyde (MDA) content significantly decreased (P<0.05). Although the mulberry leaf extract group showed improvement in the above indicators, the degree of improvement was significantly weaker than that in the 5% mulberry leaf powder group (P<0.05).

[0049] It is noteworthy that the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and MDA levels in the 8% mulberry leaf powder group were significantly higher than those in the control group (P<0.05), while the activities of SOD, catalase (CAT), and GSH-Px were significantly reduced (P<0.05). This indicates that high-level mulberry leaf powder supplementation can lead to liver damage in laying hens and reduce the body's antioxidant capacity.

[0050] 1.3.3 Effects on lipid metabolism and antioxidant indicators in the liver of laying hens Further examination of liver lipid metabolism and antioxidant indicators in the control group, 5% mulberry leaf powder group and mulberry leaf extract group was conducted, and the results are shown in Table 3.

[0051] Table 3. Effects of dietary supplementation with mulberry leaf powder on liver lipid metabolism and antioxidant indicators in laying hens.

[0052] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0053] Compared with the control group, the liver TG content in the 5% mulberry leaf powder group decreased from 4.85 mmol / g prot to 4.27 mmol / g prot, and the MDA content decreased from 13.25 nmol / mg prot to 10.00 nmol / mg prot, with significant differences (P<0.05). Simultaneously, the activities of total superoxide dismutase (T-SOD), reduced glutathione (GSH), and GSH-Px in the liver significantly increased (P<0.05). The degree of improvement in all liver indicators in the mulberry leaf extract group was significantly lower than that in the 5% mulberry leaf powder group (P<0.05), further confirming that the effects of the whole component of mulberry leaf powder are superior to those of a single extract.

[0054] 1.3.4 Effects on cecal flora and short-chain fatty acid composition in laying hens The results of the detection of cecal flora and short-chain fatty acid content in the three groups of laying hens are shown in Table 4.

[0055] Table 4. Effects of dietary supplementation with mulberry leaf powder on cecal flora and short-chain fatty acid composition in laying hens.

[0056] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0057] Compared with the control group, the relative abundance of *Femtobacter* and *Bifidobacterium* in the cecum of the 5% mulberry leaf powder group significantly increased from 2.15% and 1.83% to 4.86% and 4.12%, respectively (P<0.05), while the relative abundance of *Desulfovibrio* significantly decreased from 1.56% to 0.62% (P<0.05). Simultaneously, the contents of acetic acid, propionic acid, and butyric acid in the cecum significantly increased from 2.12 mg / g, 1.05 mg / g, and 0.86 mg / g to 3.85 mg / g, 1.92 mg / g, and 1.65 mg / g, respectively (P<0.05). Although the abundance of beneficial bacteria and the content of short-chain fatty acids in the mulberry leaf extract group increased, they were significantly lower than those in the 5% mulberry leaf powder group (P<0.05), and the decrease in the abundance of *Desulfovibrio* was also smaller than that in the 5% mulberry leaf powder group.

[0058] 1.3.5 Differential Gene Expression Analysis in the Liver Transcriptome Transcriptome sequencing analysis was performed on the livers of laying hens in the control group and the 5% mulberry leaf powder group. The results are as follows: Figure 1 As shown in the figure. The analysis results showed that treatment with 5% mulberry leaf powder caused significant changes in the expression of genes related to lipid metabolism, especially energy metabolism, in the liver of laying hens. Among them, the expression of key lipid synthesis genes FASN, ACACA, and SCD was significantly downregulated (P<0.05).

[0059] 1.3.6 Intestine-Liver Axis Association Analysis Association analysis was performed on differentially expressed microbiota, short-chain fatty acids, and liver lipid metabolism, antioxidant markers, and differentially expressed genes. The results are as follows: Figure 2 The correlation heatmap shown indicates that acetic acid, propionic acid, butyric acid, and the expression of genes related to fatty acid regeneration such as Bifidobacterium, Faecalibacterium, and Prevotellaceae UCG-001 are significantly negatively correlated with liver TG and MDA levels, as well as the expression of genes related to fatty acid regeneration such as ACACA, FASN, SCD, and ELOVL6. They are also significantly positively correlated with liver GSH-Px, T-SOD, and T-AOC antioxidant indicators (P<0.05).

[0060] In summary, the addition of 5% mulberry leaf powder to the diet can regulate the intestinal microecology of laying hens, promote the production of short-chain fatty acids, and inhibit the expression of key genes for fatty acid synthesis in the liver via the gut-liver axis. This can optimize the regulation of lipid metabolism and redox homeostasis while ensuring egg production performance, and its overall effect is significantly better than that of mulberry leaf extract with the same amount of active ingredients.

[0061] Example 2: Application of mulberry leaf powder in alleviating fatty liver syndrome in laying hens 2.1 Test Materials Same as Example 1.

[0062] 2.2 Experimental Design Two hundred and forty Hy-Line Grey hens of similar weight and good health, aged 33 weeks, were selected and randomly divided into four groups, with six replicates per group and ten hens per replicate. Control group (Con): The chickens were fed the same basal diet during their peak egg production period, with the same diet composition and nutritional levels as in Example 1.

[0063] Fatty liver syndrome model group (FLHS): fed a high-energy, low-protein diet with the following composition and nutritional levels: corn 68.50%, soybean meal 12.80%, lard 4.00%, crude protein 12.02%, and metabolizable energy 12.98 MJ / kg.

[0064] Mulberry Leaf Powder Treatment Group (MLP): 5% mulberry leaf powder was added to the high-energy, low-protein diet of the FLHS group to replace an equal proportion of corn and soybean meal (63.50% corn, 11.20% soybean meal, and 5% mulberry leaf powder) to maintain the same nutritional level as the FLHS group.

[0065] Mulberry Leaf Extract Treatment Group (MLE): Mulberry leaf extract with the same active ingredient equivalent as 5% mulberry leaf powder was added to the high-energy, low-protein diet of the FLHS group to replace an equal proportion of corn and soybean meal, maintaining the same nutritional level of the diet as the FLHS group.

[0066] The experiment lasted for 8 weeks, with feeding management and testing indicators the same as in Example 1. At the end of the experiment, the successful construction of the fatty liver syndrome model was verified by observing liver pathological morphology and detecting serum liver injury indicators and lipid metabolism indicators; at the same time, the mechanism of action of mulberry leaf powder in alleviating fatty liver syndrome in laying hens was clarified by detecting liver transcriptome, cecal flora and short-chain fatty acids.

[0067] 2.3 Results and Analysis 2.3.1 Validation of the fatty liver syndrome model The experimental results showed that, compared with the Con group, the egg production rate of laying hens in the FLHS group was significantly reduced, and the serum levels of AST, ALT, TG, TC, and LDL-C were significantly increased (P<0.05). The activities of antioxidant enzymes such as SOD, CAT, and GSH-Px in serum and liver were significantly reduced (P<0.05). The liver showed obvious pathological characteristics such as enlargement, paleness, fragile texture, hemorrhage, and necrosis, indicating that the model of fatty liver hemorrhagic syndrome in laying hens was successfully established.

[0068] 2.3.2 Effects on egg production performance and liver damage indicators in FLHS laying hens The effects of mulberry leaf powder and mulberry leaf extract on egg production performance and liver damage indicators in FLHS laying hens are shown in Table 5.

[0069] Table 5. Effects of mulberry leaf powder and mulberry leaf extract on egg production performance and liver damage indicators in FLHS laying hens.

[0070] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0071] The results showed that, compared with the FLHS group, the egg production rate of hens in the MLP group significantly recovered from 78.32% to 91.46%, and the average daily egg weight significantly recovered from 54.26g to 60.12g, with no significant difference compared with the Con group (P>0.05). Simultaneously, the liver index significantly decreased from 4.85% to 3.26%, and serum AST and ALT levels significantly decreased from 68.52 IU / L and 71.26 IU / L to 45.11 IU / L and 46.23 IU / L, respectively (P<0.05). Although the egg production rate of the MLE group recovered to 85.71%, it was significantly lower than that of the MLP group (P<0.05), and the improvement in liver index, serum AST, and ALT levels was also significantly weaker in the MLE group (P<0.05).

[0072] 2.3.3 Effects on serum biochemical and antioxidant indicators of FLHS laying hens The effects of mulberry leaf powder and mulberry leaf extract on serum biochemical and antioxidant indicators of FLHS laying hens are shown in Table 6.

[0073] Table 6. Effects of mulberry leaf powder and mulberry leaf extract on serum biochemical parameters of FLHS laying hens

[0074] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0075] Compared with the FLHS group, the MLP group showed significantly lower serum TG, TC, LDL-C, and MDA levels, and significantly higher HDL-C levels and SOD, GSH-Px, and CAT activities (P<0.05). All indicators recovered to levels not significantly different from the Con group (P>0.05). Although the above indicators improved in the MLE group, the degree of improvement was significantly lower than that in the MLP group (P<0.05).

[0076] 2.3.4 Effects on liver-related indicators in FLHS laying hens Further testing was conducted on the liver lipid metabolism and antioxidant indicators of each group of laying hens, and the results are shown in Table 7.

[0077] Table 7. Effects of mulberry leaf powder and mulberry leaf extract on liver-related indicators in FLHS laying hens

[0078] Note: No letters or the same letters in the same row indicate no significant difference (P>0.05), while different letters indicate significant difference (P<0.05).

[0079] Compared with the FLHS group, the MLP group showed significantly lower levels of TG, TC, and MDA in the liver, and significantly higher activities of T-SOD, T-AOC, GSH, and GSH-Px in the liver (P<0.05), with no significant difference compared to the Con group (P>0.05). The improvement in all liver indicators in the MLE group was significantly lower than that in the MLP group (P<0.05), indicating that mulberry leaf powder has a significantly better effect on alleviating lipid deposition and oxidative stress in the liver of FLHS laying hens than mulberry leaf extract.

[0080] 2.3.5 Liver transcriptome analysis Transcriptome sequencing analysis was performed on the livers of three laying hens (Con, FLHS, and MLP groups), and the results are as follows: Figure 3 As shown in the figure. The results showed that MLP intervention induced reversal-like regulation of key genes involved in adipogenesis / lipid droplet dynamics and redox homeostasis in the liver of FLHS laying hens. These genes include ACACA, FASN, SCD, ELOVL6, SREBF1, ME1, PLIN2, APOA4, GLRX, and MSRB1.

[0081] 2.3.6 Effects on cecal microbial diversity and community composition in FLHS laying hens The microbial diversity and community structure of the cecum of laying hens in each group were analyzed, and the results are as follows: Figure 4 As shown in the figure. The results showed that, compared with the FLHS group, the relative abundance of *Femtobacter* and *Bifidobacterium* in the cecum of the MLP group was significantly increased, while the relative abundance of harmful bacteria such as *Desulfovibrio* was significantly decreased (P<0.05).

[0082] 2.3.7 Analysis of cecal microbial markers, short-chain fatty acids, and gut-hepatic axis network LEfSe analysis was used to identify biomarkers of differentially abundant gut microbiota among the three groups, and the content of short-chain fatty acids in the cecum was detected. Intestinal-liver axis correlation analysis was also performed. The results are as follows: Figure 5 As shown in the figure. The results showed that compared with the FLHS group, the contents of acetic acid, propionic acid, and butyric acid in the cecum of the MLP group were significantly increased (P<0.05). Correlation analysis showed that the changes in differentially expressed bacteria and SCFAs were negatively correlated with lipid indicators and the expression of adipogenic genes, and positively correlated with antioxidant parameters. The above results confirm that mulberry leaf powder can improve liver lipid metabolism and antioxidant capacity through the gut-liver axis pathway by increasing the abundance of short-chain fatty acid-producing bacteria in the cecum and increasing SCFA production, thereby alleviating FLHS-induced liver damage.

[0083] Based on the results of Examples 1 and 2, this invention, by adding 5% of mulberry leaf powder prepared by a specific process to the diet of laying hens, can effectively improve the lipid metabolism and antioxidant capacity of laying hens during their peak laying period, thereby enhancing their egg production performance. At the same time, it can significantly alleviate fatty liver hemorrhagic syndrome in laying hens by regulating the gut-liver axis pathway, thereby restoring their egg production performance. Moreover, its comprehensive application effect is significantly better than that of mulberry leaf extract with the same amount of active ingredients.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of mulberry leaf powder in the preparation of feed additives for improving lipid metabolism and / or enhancing antioxidant capacity in laying hens.

2. Application of mulberry leaf powder in the preparation of feed additives for alleviating hemorrhagic fatty liver syndrome in laying hens.

3. The application according to claim 1, characterized in that, The laying hens are Hailan Grey laying hens aged 33 weeks and above; the mulberry leaf powder is sealed and stored at 4°C and used within four weeks; the application involves uniformly mixing the mulberry leaf powder into the laying hen's basal diet using a horizontal mixer, allowing the laying hens to consume it freely, with an intervention period of no less than 8 weeks.

4. The application according to claim 1, characterized in that, The improvement of lipid metabolism is to reduce the levels of serum triglycerides, low-density lipoprotein cholesterol, and liver triglycerides in laying hens, and to increase the levels of serum high-density lipoprotein cholesterol. The enhancement of antioxidant capacity is to increase the activities of serum superoxide dismutase and glutathione peroxidase in laying hens, the activities of total superoxide dismutase and glutathione peroxidase in the liver, and the content of reduced glutathione, and to reduce the levels of malondialdehyde in serum and liver.

5. The application according to claim 2, characterized in that, The application involves adding mulberry leaf powder to a high-energy, low-protein diet that induces fatty liver hemorrhagic syndrome in laying hens; the high-energy, low-protein diet has a crude protein content of 12.02%~12.10% and a metabolizable energy of 12.98 MJ / kg.

6. The application according to claim 2, characterized in that, The method for alleviating hemorrhagic fatty liver syndrome in laying hens involves reducing liver indices, improving liver pathological damage, and reducing lipid deposition in hepatocytes; lowering serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides, total cholesterol, and low-density lipoprotein cholesterol levels; increasing the activity of serum and liver superoxide dismutase (SOD), catalase, and glutathione peroxidase (GLP-P), and reducing malondialdehyde (MDA) levels; and restoring egg production in laying hens.

7. The application according to claim 1 or 2, characterized in that, The mulberry leaf powder can increase the relative abundance of *Femtobacter* and *Bifidobacterium* in the cecum of laying hens, and decrease the relative abundance of *Desulfovibrio*; it also increases the content of acetic acid, propionic acid, and butyric acid in the cecum; and the regulatory effect of mulberry leaf powder on the above indicators is significantly better than that of mulberry leaf extract with the same amount of active ingredients.

8. The application according to claim 1 or 2, characterized in that, The mulberry leaf powder is obtained by drying fresh mulberry leaves for feed in a cool, dry place for 24 hours, drying at 55°C to constant weight, and then pulverizing them through a 40-mesh sieve.

9. A diet for laying hens that improves lipid metabolism, enhances antioxidant capacity, and alleviates fatty liver syndrome, characterized in that, The diet contains mulberry leaf powder.

10. The laying hen diet according to claim 9, characterized in that, The diet is a basic diet for laying hens or a high-energy, low-protein diet, wherein the proportion of mulberry leaf powder added is 5%; the mulberry leaf powder is obtained by drying fresh mulberry leaves for feed in a cool, dry place for 24 hours, drying at 55°C to constant weight, and then crushing them through a 40-mesh sieve.

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