Application of paeonol in preparation of product for improving liver function and ovarian function of laying hens in later laying period

Abstract

The invention discloses application of paeonol in preparation of a product for improving liver functions and ovarian functions of laying hens in the later egg laying period, belongs to the technical field of feed additives, and particularly relates to application of paeonol in preparation of a product for improving liver functions and ovarian functions of laying hens in the later egg laying period. It is found that paeonol can significantly reduce the activity of glutamic-pyruvic transaminase in the liver, significantly reduce the content of cholesterol and the content of triglyceride in serum, significantly reduce hepatic cell fatty degeneration and lipid droplet aggregation in a hepatic tissue pathological section, relieve liver injury and lipid metabolism disorder, and significantly improve the liver function. The paeonol can effectively improve the fatty liver hemorrhagic syndrome of the laying hens in the later egg laying period, and the paeonol can recover the storage capacity of ovary by improving the survival rate of follicles, improve the egg laying performance of the laying hens and improve the egg laying rate.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, and in particular relates to the application of paeonol in the preparation of products that improve liver and ovarian function in laying hens in the late laying period. Background Technology

[0002] In the later stages of egg production, the ovarian function of laying hens gradually declines, and the egg production rate gradually decreases from its peak (usually above 85%). However, their feed intake often remains at a high level. If the proportion of energy substances (such as corn and wheat bran) in the feed is too high, while the supply of functional nutrients that promote fat metabolism (such as choline, vitamin E, and B vitamins) is insufficient, the excess energy will be converted into triglycerides in the liver. Under normal circumstances, triglycerides combine with apolipoproteins to form very low-density lipoproteins, which are transported to the ovary via the blood to synthesize egg yolks. However, when apolipoprotein synthesis is blocked, triglycerides cannot be effectively transported, leading to lipid accumulation in the liver, increasing the metabolic burden on the liver, and seriously affecting the laying hen's production performance and health. This results in decreased egg production rate, reduced egg quality, and increased mortality, causing huge economic losses to the poultry industry. Currently used drug treatments have limited effectiveness and may pose a risk of drug residues.

[0003] Paeonol is a phenolic ketone compound extracted and isolated from the root bark of Paeonia suffruticosa (Ranunculaceae family) or the whole plant of Cynanchum paniculatum (Asclepiadaceae family). Paeonol is generally a white or slightly yellow, lustrous needle-like crystal, soluble in organic solvents such as methanol and ethanol, soluble in hot water, but insoluble in cold water. In recent years, the pharmacological effects of paeonol on various diseases have been extensively studied, revealing its anti-inflammatory, antitumor, neuroprotective, immunomodulatory, and cardiovascular disease-treating activities. However, there are currently no reports on the use of paeonol in poultry, and its effects on poultry remain unclear. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes the application of paeonol in the preparation of products that improve liver and ovarian function in laying hens during the late laying period. This addresses the technical challenge of existing technologies in meeting the dual requirements of controlling the production performance and liver health of laying hens during the late laying period. By incorporating paeonol into high-energy, low-protein diets for laying hens during the late laying period, this invention aims to prevent disease, improve the liver and ovarian condition of laying hens, increase egg production rate, and improve egg quality.

[0005] To achieve the above objectives, the present invention provides the application of paeonol in the preparation of products that improve liver and ovarian function in laying hens during the late laying period.

[0006] Preferably, paeonol is added to the basal diet at a dosage of 200-280 mg / kg and fed to laying hens in the late laying period for 7-9 weeks.

[0007] Preferably, paeonol can alleviate and treat fatty liver hemorrhage syndrome in laying hens in the late laying period, promote the recovery of ovarian reserve, and improve the breakage resistance of eggs produced by laying hens in the late laying period.

[0008] The present invention also provides a product for improving liver and ovarian function in laying hens during the late laying period, the product comprising paeonol and acceptable excipients.

[0009] This invention also provides the application of paeonol in the preparation of products for alleviating and treating fatty liver hemorrhage syndrome in laying hens in the late laying period.

[0010] Preferably, paeonol reduces abdominal fat percentage, liver L value, and liver b value in laying hens during the late laying period, improves lipid accumulation in the abdomen, improves liver appearance and color, reduces serum aspartate aminotransferase activity and cholesterol content, reduces fat vacuoles and lipid droplets in liver tissue, and alleviates liver tissue damage.

[0011] This invention also provides the application of paeonol in the preparation of products that promote the recovery of ovarian reserve in laying hens during the later stages of egg production.

[0012] Preferably, paeonol can restore ovarian reserve by improving follicle survival rate.

[0013] The present invention also provides a product for promoting the recovery of ovarian reserve capacity in laying hens during the late laying period, the product comprising paeonol and acceptable excipients.

[0014] The present invention also provides the application of paeonol in the preparation of products that improve the breakage resistance of eggs produced by laying hens in the late laying period, specifically: paeonol is added to the basal diet at an addition rate of 200~280mg / kg and fed to laying hens in the late laying period for 7~9 weeks.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides the application of paeonol in the preparation of products that improve liver and ovarian function in late-laying laying hens. The invention discovers that paeonol can significantly reduce the liver color L value and abdominal fat percentage in late-laying laying hens, improve the degree of liver jaundice and the body's lipid metabolism status, thereby significantly improving the liver health level of late-laying laying hens and maintaining a more stable physiological state, providing a fundamental guarantee for the stable performance of subsequent egg production in late-laying laying hens. This invention also finds that paeonol can significantly increase eggshell thickness, eggshell strength, and Haugh units, enhancing eggshell resistance to breakage and improving egg freshness. Furthermore, paeonol can significantly reduce the activity of alanine aminotransferase in the liver, significantly reduce serum cholesterol and triglyceride levels, and significantly alleviate hepatocyte steatosis and lipid droplet aggregation in liver tissue pathological sections, alleviating liver damage and lipid metabolism disorders, thus effectively improving fatty liver hemorrhage syndrome in late-laying laying hens. Attached Figure Description

[0016] 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.

[0017] Figure 1 The statistical results of average egg weight, average daily egg production rate, average daily feed intake, and feed conversion ratio of laying hens in the late laying period of each group are shown. In the figure, A is the average egg weight, B is the egg production rate, C is the average daily feed intake, and D is the feed conversion ratio. NC represents the control group, MC represents the model group, and Pae represents the paeonol group. Different lowercase letters a and b indicate significant differences between groups, P<0.05. Figure 2 The results of egg shape index, shell thickness, shell strength, albumen height, Haugh unit, and yolk color of eggs produced by hens in the late laying period of each group were statistically analyzed. In the figure, A is egg shape index, B is shell thickness, C is shell strength, D is albumen height, E is Haugh unit, and F is yolk color. NC represents the control group, MC represents the model group, and Pae represents the paeonol group. Different lowercase letters a and b indicate significant differences between groups, P<0.05. Figure 3 The statistical results of follicle morphology and follicles at different stages in the late laying period of each group of laying hens are shown. Among them, A is the follicle morphology of the control group, B is the follicle morphology of the model group, C is the follicle morphology of the paeonol group, D is the number of small white follicles, E is the number of large white follicles, F is the number of small yellow follicles, G is the number of large yellow follicles, H is F2~F5, I is F1, J is the total number of graded follicles, K is the total number of pre-graded follicles. In the figure, NC represents the control group, MC represents the model group, and Pae represents the paeonol group. Different lowercase letters a and b indicate significant differences between groups, P<0.05. Figure 4 The results of liver index, abdominal fat percentage, liver L value and liver b value of laying hens in the late laying period of each group were statistically analyzed. In the figure, A is liver index, B is abdominal fat percentage, C is liver L value and D is liver b value. NC represents control group, MC represents model group and Pae represents paeonol group. Different lowercase letters a, b and c indicate significant differences between groups, P<0.05. Figure 5The appearance and pathological morphology of liver tissue in laying hens during the late laying period of each group were determined by H&E staining (40×) and Oil Red O staining (40×). Among them, A represents the appearance of liver tissue in the control group, B represents the appearance of liver tissue in the model group, C represents the appearance of liver tissue in the paeonol group, D represents the pathological morphology of liver tissue in the control group, E represents the pathological morphology of liver tissue in the model group, F represents the pathological morphology of liver tissue in the paeonol group, G represents the pathological morphology of liver tissue in the control group, H represents the pathological morphology of liver tissue in the model group, and I represents the pathological morphology of liver tissue in the paeonol group. Figure 6 The statistical results of serum alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, cholesterol content, and triglyceride content of laying hens in the late laying period of each group are presented. In the figure, A represents ALT activity, B represents AST activity, C represents cholesterol content, and D represents triglyceride content. NC represents the control group, MC represents the model group, and Pae represents the paeonol group. Different lowercase letters a, b, and c indicate significant differences between groups, P < 0.05. Detailed Implementation

[0018] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. 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. The specification and embodiments of this invention are merely exemplary. The instruments, equipment, reagents, and materials used in the embodiments are all commercially available, and the experimental methods in the embodiments, unless otherwise specified, are conventional methods in the art.

[0019] Example 1 I. Materials and Methods: 1. Experimental animals and experimental design: This experiment was conducted at an animal testing facility, using 432 55-week-old GL laying hens. All the chickens were healthy individuals of similar weight, sourced from the same hatchery, to ensure the reproducibility and reliability of the experiment. The animals were fed a basal diet for a one-week acclimatization period before the start of the experiment to ensure the flock was in good physiological condition at the start of the experiment.

[0020] A fatty liver model of hemorrhagic fatty liver syndrome (FLHS) in laying hens was established using a high-energy, low-protein diet (12.55 MJ / kg metabolizable energy, 13% crude protein) for 8 weeks. The chickens were randomly divided into three groups, with six replicates per group and twelve chickens per replicate. The experimental groups were: a control group fed a corn-soybean meal basal diet with 11.13 MJ / kg metabolizable energy and 15.59% crude protein; a model group fed a high-energy, low-protein diet with 12.55 MJ / kg metabolizable energy and 13% protein; and a paeonol group fed the above high-energy, low-protein diet supplemented with paeonol (240 mg / kg). This feed formulation, by increasing fat content and decreasing protein levels, simulated the pathogenesis of fatty liver in laying hens, thus inducing fatty liver disease. The corn-soybean meal basal diet fed to the control group of laying hens was formulated according to the nutritional requirements of laying hens in the Chinese Chicken Feeding Standard NY / T33-2004. The diets fed to the model group and the paeonol group of laying hens were formulated according to NY / T33-2004, except that the energy and protein nutritional standards were not formulated according to the nutritional requirements of NY / T33-2004. The composition and nutritional levels of the two different diets are shown in Table 1 and Table 2.

[0021] During the experiment, the light duration was guaranteed to be 16 hours per day, the temperature was maintained at 18~25℃, the humidity was maintained at 50%~70%, and the animals were fed twice a day (8:00 am and 4:00 pm), with free access to water.

[0022] Table 1 Composition of Dietary Feed

[0023] Note: 1: Each kilogram of premixed feed provides: Vitamin A 9500 IU, Vitamin D3 4000 IU, Vitamin E 25 IU, Vitamin K3 2.5 mg, Vitamin B1 2.5 mg, Vitamin B2 5.5 mg, Vitamin B6 4 mg, Vitamin B12 0.02 mg, Biotin 0.2 mg, Pantothenic Acid 8 mg, Folic Acid 1 mg, Choline 450 mg, Iron 60 mg, Copper 10 mg, Manganese 110 mg, Zinc 100 mg, Iodine 1.0 mg, Selenium 0.3 mg.

[0024] Table 2 Nutritional levels of diet feed

[0025] 2. Main reagents and instruments: Total cholesterol (T-CHO) test kit (catalog number A111-1-1), triglyceride (TG) test kit (catalog number A110-1-1), alanine aminotransferase (ALT / GPT) test kit (catalog number C009-2-1), and aspartate aminotransferase (AST / GOT) test kit (catalog number C010-2-1) were all purchased from Nanjing Jiancheng Technology Co., Ltd.; hematoxylin-eosin staining solution (catalog number B1002), universal tissue fixative (catalog number G1101-15ML) were purchased from Wuhan Saiweier Biotechnology Co., Ltd.; and oil red staining solution (catalog number G1015) was purchased from Servicebio. Eggshell strength tester and multifunctional egg quality tester were also used.

[0026] 3. Tissue sample collection: At the end of the 8th week of the experiment, 12 chickens were randomly selected from each group for slaughter and sampling. Before sampling, the chickens were fasted for 12 hours. Then, about 3 mL of blood was drawn from the wing vein using a disposable sterile syringe. The blood sample was immediately placed in a test tube containing anticoagulant, gently inverted to mix, and stored in a 4°C refrigerator for subsequent testing. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC) were measured using a fully automated biochemical analyzer. After blood collection, the chickens were euthanized, and their abdominal cavity was dissected. The liver and abdominal fat were completely removed, photographed, and the appearance of the liver was observed, including its color, texture, size, and the presence of hemorrhages. The surface moisture of the liver was blotted dry with filter paper, and its weight was weighed and recorded. From each laying hen, a liver tissue of the same size was surgically removed from the edge of the liver lobules. This portion of liver tissue was fixed in a 4% paraformaldehyde solution for the preparation of pathological sections. The remaining liver tissue was minced, placed in cryovials in liquid nitrogen, and then transferred to a -80°C freezer for subsequent determination of lipid content in the liver and routine pathological histological sections and routine oil red staining of the sections.

[0027] 4. Measurement indicators and methods: 1) Measurement of production performance: On day 0 of the formal trial, the number of eggs laid was recorded at 16:00 every day. The laying hens were weighed weekly, and the remaining feed was tallied once a week. Using replicates as the unit, the number of eggs laid and the weight of eggs were recorded daily, and the weight of the laying hens and the amount of feed consumed were tallied weekly. Production performance indicators such as average egg weight (AEW), average daily feed intake (ADFI), laying rate, and feed conversion ratio (F / E) were calculated.

[0028] Average egg weight (g / egg) = Average daily total egg weight / Total number of eggs; Average daily feed intake (g / bird) = Total daily feed consumption / Number of chickens; Egg production rate (%) = (Average daily total number of eggs / Number of chickens) × 100%; Feed conversion ratio = Feed intake / Egg weight.

[0029] 2) Determination of egg quality: The day before the end of the experiment, all eggs from each treatment were collected for egg quality testing. The major and minor diameters of the eggs were recorded using vernier calipers, and the eggshell thickness and strength were measured. Egg weight, yolk color, albumen height, and Haugh units were measured using a multi-functional egg quality analyzer.

[0030] 3) Determination of the number of reproductive follicles: After slaughter, the ovary and follicles were completely removed using sterile surgical forceps and scissors to avoid follicle rupture. Referring to the classic grading standard for follicle development in laying hens, the follicle diameter was measured using digital calipers (accuracy 0.01 mm), and counted as follows: small white follicles (SWFs, 2-4 mm); large white follicles (LWFs, 4-6 mm); small yellow follicles (SYFs, 6-8 mm); large yellow follicles (LYFs, 8-12 mm); F2-F5 (12-25 mm); F1 (25-28 mm). In this invention, the graded follicle count refers to the total number of follicles in the F1 and F2-F5 stages. During the counting process, follicles with abnormal morphology were individually marked and recorded. After counting, follicles of different sizes were arranged in a specific order and photographed.

[0031] 4) Measurement of liver index, liver color, and abdominal fat percentage: Before slaughter, the weight of each chicken was recorded. During sampling, the abdominal cavity was opened, and the complete liver was removed and placed on a petri dish. After absorbing the surface moisture with filter paper, the liver weight was recorded. Similarly, as much abdominal fat as possible was removed and placed on a petri dish for weighing. After weighing the liver tissue, the complete liver specimen was laid flat on a specimen photography and measurement board. The shooting angle and lighting conditions were adjusted to ensure that the liver outline, color, and surface lesion characteristics were clearly visible. After taking the pictures, the images were classified and stored according to the test number. Colorimetric analysis of the liver tissue was performed using a colorimeter to measure the L value (brightness), a value (red-green hue), and b value (yellow-blue hue). To reduce systematic errors, three non-lesion areas were randomly selected for each sample for repeated measurements, and the average value of the three measurements was recorded.

[0032] Liver index = (liver weight / chicken live weight) × 100%; Abdominal fat percentage = (abdominal fat weight / chicken live weight) × 100%.

[0033] 5) Histopathological morphological observation of liver tissue: (1) Paraffin sections and HE staining of liver tissue: Liver tissue paraffin embedding and sectioning: Fresh liver tissue was fixed in 4% paraformaldehyde for more than 24 hours, dehydrated with graded ethanol, routinely embedded in paraffin, and sectioned at 4μm. Dewaxing of paraffin sections: The sections were sequentially immersed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and washed with distilled water.

[0034] Hematoxylin staining of cell nuclei: Slices were stained with Harris hematoxylin for 5 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia solution, and rinsed with running water. Eosin staining of cytoplasm: Slices were stained with eosin solution for 2 min. Dehydration and mounting: Slices were sequentially immersed in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min, until dehydrated and cleared. The slices were removed from the xylene solution, allowed to dry slightly, and mounted with neutral resin. Observation, statistics, and analysis were performed under an optical microscope.

[0035] (2) Paraffin sections of liver tissue and Oil Red O staining: Embedding: Cut the frozen liver tissue into small pieces as needed and place them on an ice tray. Completely cover the tissue with OTC embedding medium and freeze it in a frozen section. Sectioning: Fix the frozen tissue and ice tray on a cryostat and adjust the thickness as needed to make sections, generally 4~8μm.

[0036] Fixation: Place the cut tissue sections onto a glass slide and fix them in formaldehyde for 30 seconds to 1 minute. Freeze after fixation for later use.

[0037] Frozen tissue sections embedded in OCT were removed and rewarmed. Oil Red O stock solution and distilled water were mixed in a 3:2 ratio and allowed to stand for 10 minutes. The mixture was then filtered to obtain the working solution. After washing the sections with distilled water, the working solution was added to the tissue for staining for 10 minutes. The sections were then immersed in 60% isopropanol for differentiation and rinsed with 60% isopropanol. The staining time was controlled under a microscope, followed by rinsing with distilled water. The sections were then stained with Harris hematoxylin for 5 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, and then blued with 0.6% ammonia solution. The sections were rinsed with running water. The water on the sections was shaken off, and the sections were mounted with glycerol. The sections were examined and photographed as soon as possible after mounting to observe the lipid deposition in the liver.

[0038] 6) Measurement of blood lipids and liver function indicators: The collected blood was centrifuged to obtain serum, and the levels of TG and TC in the serum were measured, as well as the activities of ALT and AST.

[0039] 5. Statistical Analysis: All data were initially organized and recorded using Excel 2010, and then analyzed using SPSS 26.0 software. Data on production performance, egg quality, liver index, and abdominal fat percentage were analyzed using one-way ANOVA and Duncan's method for multiple comparisons. Results are expressed as mean ± standard error. Different lowercase letters a, b, and c in the text indicate significant differences between groups (P < 0.05), while the same letter indicates no significant difference (P > 0.05). 0.05 ≤ P < 0.10 indicates a trend of change. GraphPad Prism 9.50 software was used for graphing.

[0040] II. Results and Analysis: 1. Effects of the plant extract paeonol on the laying performance of hens in the late laying period: The effects of paeonol on the laying performance of hens in the late laying period, such as Figure 1 China A Figure 1 B, Figure 1 C and Figure 1 As shown in Figure D, compared with the control group (NC), the model group (MC) showed a significant decrease in average daily egg production rate and average daily feed intake (P<0.05). Compared with the model group, the paeonol group (Pae) ​​showed an increase in both average egg production rate and average daily feed intake, but the difference was not statistically significant. There were no significant differences in average egg weight and feed conversion ratio among the three groups, but the feed conversion ratio of the model group was slightly higher than that of the control group. A high-energy, low-protein diet pattern easily leads to an imbalance in the energy intake of laying hens. Excess energy is converted into lipid deposition and also significantly inhibits the feed intake of laying hens, ultimately resulting in a decline in egg production performance.

[0041] 2. The effect of the plant extract paeonol on the quality of eggs in the later stages of egg production: One day before the end of the experiment, all eggs from each group were collected and their quality was measured. The results showed that paeonol had the following effect on egg quality in a high-energy, low-protein diet-induced hepatic steatosis model in laying hens: Figure 2 China A~ Figure 2 As shown in Tables F and 3, compared with the control group (NC), the eggshell thickness and Haugh units of the paeonol group (Pae) ​​were significantly increased (P<0.05); compared with the model group (MC), the eggshell thickness, eggshell strength, and Haugh units of the paeonol group were significantly increased (P<0.05). Compared with the control group, the yolk color of the eggs fed a high-energy, low-protein diet was darker, and the yolk color of the eggs in the model group was significantly higher than that in the control group (P<0.05). There were no significant differences in egg weight, egg shape index, and albumen height among the groups.

[0042] Table 3. Determination of Egg Quality

[0043] 3. Effects of the plant extract paeonol on follicles in laying hens during the late laying period: The effect of paeonol on follicles in laying hens during the late laying period is shown in [reference needed]. Figure 3 China A~ Figure 3 As shown in Figure K, compared with the control group (NC) and the paeonol group (Pae), the model group (MC) had a significantly reduced number of large white follicles (P<0.05), fewer small white follicles, and fewer small yellow follicles than the control and paeonol groups, but these differences were not statistically significant. Furthermore, the total number of graded follicles in the model group was significantly less than that in the control and paeonol groups (P<0.05). The lack of significant differences in the number of small white follicles among the groups indicates that the basal reproductive potential of the laying hens in the model and paeonol groups was the same, excluding differences caused by congenital ovarian hypoplasia. Compared with the model group, paeonol treatment significantly increased the number of large white follicles. Large white follicles are the stage where follicles are most sensitive to oxidative stress and nutritional deficiencies, and are also the high-risk period for atresia. Paeonol may restore ovarian reserve by improving follicle survival rate. The total number of graded follicles in the paeonol group was significantly higher than that in the model group (P<0.05). In the physiological studies of laying hens, F1 to F5 are the gold standard for measuring egg production performance. Therefore, paeonol can improve the egg production performance of laying hens and increase the egg production rate.

[0044] 4. Effects of the plant extract paeonol on liver morphology and indicators in late-laying hens: The effects of paeonol on liver index, abdominal fat percentage, and liver color in late-laying hens are shown in the table below. Figure 4 China A Figure 4 B, Figure 4 C and Figure 4 In the study, compared with the control group (NC), the model group (MC) showed significantly higher liver index, abdominal fat percentage, liver color L value, and liver color b value (P<0.05). Compared with the model group, the paeonol group (Pae) ​​significantly reduced liver index, abdominal fat percentage, liver color b value (P<0.05), and liver color L value (P<0.01). The color values ​​are L, a, and b values ​​measured by a colorimeter, belonging to the parameters of the CIE Lab color space. This space is a standard system developed by the International Commission on Illumination (CIE) that accurately describes all colors visible to the human eye. The three values ​​represent different color dimensions: L value: represents the brightness of the color; a value: represents the degree of color shift on the red-green axis; b value: yellow-blue hue, representing the degree of color shift on the yellow-blue axis. The livers of laying hens fed a high-energy, low-protein diet in the model group appeared pale yellow or yellowish-brown due to abnormal lipid accumulation. In terms of color parameters, the L value, which represents brightness, increased, while the b value of the yellow-blue dimension decreased relatively. The livers of the paeonol group were significantly darker than those in the model group, which may have the effect of reducing liver fat accumulation.

[0045] 5. Effects of the plant extract paeonol on the microstructure of liver tissue in laying hens during the late laying period: Changes in the microstructure of liver tissue in laying hens: The effect of paeonol on liver tissue structure in a high-energy, low-protein diet-induced model of steatosis in laying hens. Figure 5 China A~ Figure 5 As shown in Figure I, the livers of the control group were firm and without obvious hemorrhages, while those of the model group were dark yellow, oily, and fragile, with varying degrees of hemorrhage on the surface. HE staining results showed that the livers of the model group had a large number of fatty vacuoles, extensive hepatocyte degeneration, and blurred cell boundaries. Compared with the model group, the liver tissue damage in the paeonol group was milder, the structure was relatively intact, and the overall condition was slightly improved. Oil Red O staining results showed that the model group had a large number of lipid droplet aggregations, while the control group and the paeonol group only had a small number of lipid droplets stained red. These results indicate that paeonol can alleviate liver damage induced by a high-energy, low-protein diet in chickens and reduce lipid accumulation.

[0046] 6. Effects of the plant extract paeonol on blood lipids and liver function in laying hens during the late laying period: The effects of paeonol on blood lipids and liver function in laying hens during the late laying period are as follows: Figure 6 China A~ Figure 6 As shown in Figure D, compared with the control group (NC), the model group (MC) showed significantly higher levels of aspartate aminotransferase (AST) activity, alanine aminotransferase (ALT) activity, triglyceride (TG) content, and total cholesterol (TC) content (P<0.05). This is because long-term feeding of high-energy, low-protein diets may lead to excessive accumulation of energy substances in the liver, exceeding the normal metabolic load of hepatocytes. This prompts the liver to convert excess energy into lipids and store it, thus inducing hepatic steatosis. Abnormal lipid deposition in the liver damages the integrity and normal physiological function of hepatocytes, leading to the release of large amounts of intracellular AST and ALT marker enzymes into the blood, resulting in elevated serum transaminase activity. Disorders of hepatic lipid metabolism further affect the synthesis and transport efficiency of lipoproteins, causing abnormally high levels of triglycerides, cholesterol, and other lipid components in the blood, ultimately forming a pathological state centered on hepatocyte damage and accompanied by dyslipidemia. Compared with the model group, the paeonol group (Pae) ​​showed significantly lower AST activity and cholesterol levels (P<0.05), while ALT activity and triglyceride levels showed a decreasing trend but no significant difference. Paeonol can improve liver damage and reduce blood lipids. Paeonol can improve the pathological state of the liver by inhibiting hepatocyte damage and reducing intracellular AST release; at the same time, it can effectively regulate the body's lipid metabolism process and reduce the accumulation of cholesterol in the blood. Although the regulatory effect of paeonol on ALT activity and triglyceride levels has not yet reached a statistically significant level, it still shows a clear decreasing trend.

[0047] 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. Application of paeonol in the preparation of products that improve liver and ovarian function in laying hens during the late laying period.

2. The application according to claim 1, characterized in that, Specifically, paeonol is added to the basal diet at a dosage of 200-280 mg / kg and fed to laying hens in the late laying period for 7-9 weeks.

3. The application according to claim 1, characterized in that, Paeonol can alleviate and treat fatty liver hemorrhage syndrome in laying hens in the late laying period, promote the recovery of ovarian reserve, and improve the breakage resistance of eggs produced by laying hens in the late laying period.

4. A product for improving liver and ovarian function in laying hens during the late laying period, characterized in that, The product includes paeonol and acceptable excipients.

5. Application of paeonol in the preparation of products for alleviating and treating fatty liver hemorrhage syndrome in laying hens in the late laying period.

6. The application according to claim 5, characterized in that, Paeonol reduces abdominal fat percentage, liver L value, and liver b value in laying hens during the late laying period, improves lipid accumulation in the abdomen, improves liver appearance and color, reduces serum aspartate aminotransferase activity and cholesterol content, reduces fat vacuoles and lipid droplets in liver tissue, and alleviates liver tissue damage.

7. Application of paeonol in the preparation of products that promote the recovery of ovarian reserve in laying hens during the later stages of egg production.

8. The application according to claim 7, characterized in that, Paeonol can restore ovarian reserve by improving follicle survival rate.

9. A product that promotes the recovery of ovarian reserve in laying hens during the later stages of egg production, characterized in that, The product includes paeonol and acceptable excipients.

10. The application of paeonol in the preparation of products that improve the breakage resistance of eggs laid by hens in the later stages of laying, characterized in that, Specifically, paeonol is added to the basal diet at a dosage of 200-280 mg / kg and fed to laying hens in the late laying period for 7-9 weeks.

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