Plant-derived post-egg laying period layer feed additive and use thereof

Abstract

This invention belongs to the field of egg-laying hen breeding technology, specifically relating to a plant-based feed additive for late-laying egg-laying hens and its uses. This invention provides a plant-based feed additive for late-laying egg-laying hens, comprising sappanwood extract and gallnut extract. This feed additive can replace antibiotics in the egg-laying hen breeding process, significantly improving and / or enhancing the production and reproductive performance of late-laying egg-laying hens, which is of great significance for promoting the development of the egg-laying hen industry.

Description

Technical Field

[0001] This invention belongs to the field of egg-laying hen breeding technology, specifically relating to a plant-based late-laying hen feed additive and its uses. Background Technology

[0002] Antibiotics are widely used as feed additives in the global livestock industry, making significant contributions to the large-scale development of animal husbandry. However, antibiotics are not completely metabolized in animals, with most being excreted unchanged or as metabolites in feces and urine. Once in the environment, these excretions can induce the development and spread of drug-resistant bacteria.

[0003] The long-term addition of antibiotics to livestock and poultry feed has led to problems such as bacterial resistance, drug residues, and environmental pollution, which have become major challenges in the global public health field. In recent years, my country has also been vigorously promoting the reduction and replacement of antibiotics in livestock farming. In the later stages of egg production, due to the decline in physiological function, reduced antioxidant capacity, and imbalance in immune inflammation, laying hens experience a significant decrease in production performance and egg quality. At the same time, the risk of infection by opportunistic pathogens such as E. coli increases, further exacerbating the loss of farming efficiency.

[0004] Therefore, finding new feed additives that can effectively improve the health of laying hens in the late laying period, enhance their antioxidant and immune functions, and have antibacterial activity as an alternative to antibiotics is an urgent problem to be solved. Summary of the Invention

[0005] In view of this, the present invention provides a plant-based late-laying hen feed additive and its uses.

[0006] In one aspect, the present invention provides the use of sappanwood extract and gallnut extract in the preparation of feed additives.

[0007] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:1 to 4.

[0008] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:4.

[0009] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:1.5.

[0010] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:1.

[0011] In some embodiments, the feed additive also includes a carrier.

[0012] In some embodiments, the carrier includes at least one of corn starch, maltodextrin, rice husk powder, wheat bran, peanut shell powder, and corn cob powder.

[0013] In some preferred embodiments, the carrier is corn starch and maltodextrin.

[0014] In another aspect, the present invention provides a feed additive comprising sappanwood extract and gallnut extract, and a carrier.

[0015] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:1 to 4.

[0016] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:4.

[0017] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:1.5.

[0018] In some embodiments, the ratio of the sappanwood extract to the gallnut extract is 1:1.

[0019] In some embodiments, the carrier includes one or more of corn starch, maltodextrin, rice husk powder, wheat bran, peanut shell powder, and corn cob powder.

[0020] In some preferred embodiments, the carrier is corn starch and maltodextrin.

[0021] In another aspect, the present invention provides the use of the aforementioned feed additives in the preparation of laying hen feed.

[0022] In some embodiments, the feed additive is used to improve and / or enhance the production and / or reproductive performance of laying hens in the later stages of egg production.

[0023] In some embodiments, the feed additive has an inhibitory effect on Escherichia coli.

[0024] In another aspect, the present invention provides a layer hen feed, the layer hen feed comprising the aforementioned feed additives and basal feed.

[0025] In some embodiments, the basic feed includes one or more of the following: corn, soybean meal, soybean oil, straw powder, wheat bran, limestone powder, dicalcium phosphate, sodium chloride, methionine, and premix.

[0026] In some preferred embodiments, the basic feed includes corn, soybean meal, soybean oil, wheat bran, limestone powder, dicalcium phosphate, sodium chloride, methionine, and premix.

[0027] In some preferred embodiments, the basal feed comprises: 61.5 wt% corn, 25 wt% soybean meal, 0.5 wt% soybean oil, 1.46 wt% wheat bran, 8.98 wt% limestone powder, 1.1 wt% dicalcium phosphate, 0.37 wt% sodium chloride, 0.09 wt% methionine, and 1.0 wt% premix.

[0028] Plant-based feed additives are considered one of the most promising antibiotic alternatives due to their advantages such as natural sources, multi-target action, low incidence of drug resistance, and low residues. This invention is the first to identify gallnut extract and sappanwood extract as the main components of a feed additive. Experimental results show that their antibacterial, antioxidant, and anti-inflammatory effects are close to or better than antibiotics. They can replace antibiotics in improving and / or enhancing the production and reproductive performance of laying hens in the later stages of egg production, which is of great significance for promoting the development of the laying hen industry.

[0029] In vitro antibacterial experiments confirmed that the combination of gallnut and sappanwood has a synergistic effect against Escherichia coli (FICI ≤ 0.5). The tannins in gallnut and the flavonoids such as brassinolide in sappanwood can directly disrupt bacterial cell walls and inhibit biofilm formation. The combination of the two significantly enhances the inhibitory effect on pathogens through multi-target attack. This synergistic antibacterial effect translates into beneficial regulation of the intestinal microecology in animals: inhibiting potentially harmful bacteria in the cecum (such as *Dethiobacillus thermophilus*) while promoting the proliferation of beneficial bacteria (such as the RC9 intestinal flora of the Rikenidae family), thus increasing gut microbiota diversity. In addition, the combination's promoting effect on the intestinal barrier (significantly increasing jejunal villus height and crypt depth) creates a low-oxidative-stress, low-inflammatory-interference internal environment for the reproductive organs, thereby promoting the secretion of key reproductive hormones in laying hens.

[0030] Compared with the prior art, the present invention has at least the following beneficial effects: (1) In terms of production performance, the addition of 0.1% gallnut extract to the basal diet significantly reduced ADFI and FCR in laying hens at 1-2 weeks and 2-4 weeks of late laying, and the rate of deformed eggs in each plant extract group decreased significantly. In terms of egg quality, the eggshell thickness of the compound group increased significantly on day 14, while the yolk weight of the antibiotic group and the sappanwood group increased significantly on day 14. In terms of nutrient metabolism rate, both the gallnut group and the compound group significantly increased the metabolism rate; the dry matter metabolism rate of each experimental group was significantly increased.

[0031] (2) The results of serum biochemical index studies showed that the antibiotic group and the gallnut group significantly reduced TG content, while the compound group significantly reduced TC content. In terms of antioxidant capacity, the gallnut group significantly increased serum CAT activity, and the serum SOD activity of all experimental groups was significantly increased. The liver SOD activity of each plant extract group was significantly increased, and the GSH-ST activity was significantly decreased. The liver TAOC level of the sappanwood group was significantly increased, and the liver CAT activity of the antibiotic group and the compound group was significantly increased. Therefore, the addition of gallnut extract and sappanwood extract can effectively regulate lipid metabolism in laying hens in the late laying period and enhance the body's antioxidant capacity.

[0032] (3) Regarding reproductive hormones, the levels of FSH, LH, PROG, and E2 in each plant extract group were significantly increased. This indicates that gallnut extract and sappanwood extract can effectively activate the reproductive endocrine axis of laying hens in the late laying period and promote follicle development and ovulation.

[0033] (4) In terms of histological morphology, the number of mature follicles in the ovaries of laying hens in the plant extract group and the antibiotic group was significantly increased; the length of villi in the isthmus of the oviduct and the length of secondary villi were significantly increased in all plant extract groups; the liver tissue of all plant extract groups showed intact hepatocyte structure, with no obvious pathological phenomena except for a small amount of hemorrhage; the height of jejunal villi, crypt depth and villi-to-crypt ratio were significantly increased in the sappanwood group, and the height of jejunal villi and crypt depth were significantly increased in the compound group. This indicates that gallnut extract and sappanwood extract can improve the histological structure of the reproductive and digestive organs of laying hens in the late laying period to a certain extent.

[0034] (5) Regarding gut microbiota, the Simpson index was significantly decreased and the Shannon index was significantly increased in the gallnut group, indicating increased microbial diversity and more even distribution. At the phylum level, each experimental group showed... Bacteroidota Relative abundance increased, Bacillota The relative abundance trend was observed in the Galla chinensis group, the Hematoxylin and argyi group, and the composite group. Thermodesulfobacteriota Relative abundance decreased significantly. At the genus level, the experimental groups showed... Rikenellaceae_RC9_ gut_group The relative abundance of both was significantly increased. This indicates that the addition of gallnut and sappanwood extracts can improve the abundance of beneficial bacteria. Thermodesulfobacteriota Abundance, reducing harmful bacteria Thermodesulfobacteriota Abundance promotes the improvement of gut microbiota structure in laying hens. Correlation analysis shows that... Bacteroides It was positively correlated with serum reproductive hormones LH, FSH, E2, and PROG. Rikenellaceae_RC9_gut_group The positive correlation between gallnut extract and sappanwood extract and FSH, E2, and PROG suggests that gallnut extract and sappanwood extract may promote the growth of beneficial bacteria. Bacteroides and Rikenellaceae_RC9_gut_group The proliferation of these hormones regulates their metabolic products, affecting the function of the hypothalamus-pituitary-gonadal axis and promoting the secretion of reproductive hormones. Attached Figure Description

[0035] Figure 1 The effects of different treatments on the ovarian morphology of laying hens in the late laying period are shown (magnification 3×, scale bar 125 μm).

[0036] Figure 2 The effects of different treatments on the oviduct morphology of laying hens in the late laying period are shown (scale bar 125 μm).

[0037] Figure 3 The effects of different treatments on the liver morphology of laying hens in the late laying period are shown (magnification 20×, scale bar 20 μm).

[0038] Figure 4 The effects of different treatments on the jejunal morphology of laying hens in the late laying period are shown (magnification 4×, scale bar 20 μm).

[0039] Figure 5 The dilution curves of the cecal contents microbiota of late-laying laying hens under different treatments are shown.

[0040] Figure 6 The results show the alpha diversity analysis of the cecal microbiota of late-laying laying hens under different treatments. A represents the ace diversity analysis results; B represents the chao diversity analysis results; C represents the shannon diversity analysis results; and D represents the simpson diversity analysis results.

[0041] Figure 7 PCoA analysis of the cecal contents of laying hens under different treatments is shown.

[0042] Figure 8 The study showed the relative abundance of bacterial phyla in the cecal contents of late-laying laying hens under different treatments.

[0043] Figure 9 The study showed the relative abundance of bacterial genera in the cecal contents of laying hens during the late laying period under different treatments.

[0044] Figure 10 This study demonstrates the Lefse multilevel species difference analysis of the cecal contents microbiota of late-laying laying hens under different treatments.

[0045] Figure 11 The study demonstrates the correlation between different treatments and genus-level microbiota in the cecal contents of late-laying hens and environmental factors. Detailed Implementation

[0046] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0047] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0048] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0049] Experimental Materials and Methods 1. Experimental Materials The *Escherichia coli* (ATCC25922), tryptone soybean broth (TSB), tryptone soybean agar (TSA), and MH broth were all purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The medicinal materials *Coptis chinensis*, *Scutellaria baicalensis*, *Phellodendron chinense*, *Zanthoxylum bungeanum*, *Caesalpinia sappan*, *Rheum palmatum*, and *Rhus chinensis* were purchased from Henan Yixingyong Pharmaceutical Co., Ltd. (Henan, China). Chlortetracycline hydrochloride (effective content >90%) was provided by Pucheng Zhengda Biochemical Co., Ltd. (Fujian, China).

[0050] The gallnut and sappanwood used in animal experiments were purchased from Henan Yixingyong Pharmaceutical Co., Ltd. (Henan, China). The extraction method was as follows: Sappanwood or gallnut was crushed and sieved (80 mesh) using a pulverizer. 5 kg of the pulverized material was added to 10 times its volume of distilled water and soaked for 30 min. The decoction was heated at 100℃ for 2 h. The filtrate was collected, and the residue was extracted again. The residue was filtered, and the two filtrates were combined and concentrated to 5 kg, so that the ratio of raw material to liquid was 1:1. The raw material was mixed with the liquid using corn starch and maltodextrin as carriers. The mixture was then granulated into Chinese medicine granules using a shaking granulator. After drying, the granules were sieved (20 mesh) and stored for later use. The proportions of the gallnut extract granules were as follows: gallnut extract: corn starch: maltodextrin = 1:2:0.45; the proportions of the sappanwood extract granules were as follows: sappanwood extract: corn starch: maltodextrin = 1:2:0.3; and the proportions of the compound extract granules were as follows: compound extract: corn starch: maltodextrin = 1:2:0.5.

[0051] 2. Experimental apparatus The main instruments and equipment involved in this invention are shown in Table 1.

[0052] Table 1 Main Instruments and Equipment Example 1: Effects of Gallnut Extract and Sappanwood Extract on Laying Hen Performance in Late Laying Period 1.1 Experimental Design The experiment selected 330 healthy 61-week-old Nongjin No. 1 laying hens and divided them into 5 treatment groups, with 6 replicates in each group and 11 hens in each replicate. The specific grouping is as follows:

[0053] Control group (CON): fed with basal diet; Antibiotic group (ANT): fed basal diet + chlortetracycline hydrochloride 75 mg / kg; Gallnut Group (GAE): Feed basal diet + 0.1wt% gallnut extract Chinese medicine granules; Caesal group (CAE): fed with basal diet + 0.1wt% Caesal extract Chinese medicine granules; Compound group (CPE): Feeding basal diet + 0.1wt% compound plant extract Chinese medicine granules (of which, the ratio of sappanwood extract to gallnut extract in the compound extract is 1:1).

[0054] 1.2 Experimental Diets The feed was formulated according to the production and formulation of the farm, and its composition and nutritional level are shown in Tables 2 and 3.

[0055] Table 2 Composition of the basal diet (air-dried basal diet) Table 3. Nutritional levels of the basal diet (air-dried basal diet) (1) The premix provides the following per kilogram of complete feed: VA 60,000 IU, VD3 20,000 IU, VE 200 IU, VK3 20 mg, VB6 60 mg, VB2 65 mg, VB12 20 mg, nicotinamide 30 mg, calcium pantothenate 100 mg, manganese 80 mg, zinc 60 mg, ferric sulfate 80 mg, and copper 20 mg.

[0056] (2) Metabolizable energy, lysine and methionine are calculated values, and the rest are measured values.

[0057] 1.3 Feeding and Management The feeding experiment was conducted at a poultry farm in Tongxu County, Kaifeng City, Henan Province, and lasted for 4 weeks. The experimental laying hens were housed in three-tiered stacked cages at a temperature of 20℃~25℃ with a light exposure of 16 h / d. They were fed twice a day (7:00 and 17:00) and had free access to feed and water. The hens' mental state, daily feed intake, and mortality rate were observed and recorded daily.

[0058] 1.4 Production performance measurement During the experimental period, the number of eggs laid, egg weight, number of broken eggs, number of dirty eggs, and number of deformed eggs (including bloody eggs, small eggs, large eggs, shell-less eggs, and rough-shelled eggs) were accurately recorded daily. The remaining feed weight was weighed weekly. The average daily feed intake (ADFI), average laying rate, average egg weight, feed conversion ratio (FCR), and deformed egg rate were calculated on a replicate basis. The calculation formulas are as follows:

[0059] Average daily feed intake (g) = total feed consumption / (number of days in the experiment × number of chickens); Egg production rate (%) = 100% × total number of eggs / (number of days in the experiment × number of chickens); Average egg weight (g) = Total egg weight / Total number of eggs laid; Feed conversion ratio = Total feed consumption / Total egg weight; Deformed egg rate = 100% × number of deformed eggs / total number of eggs; Broken egg rate = 100% × number of broken eggs / total number of eggs; Dirty egg rate = 100% × number of dirty eggs / total number of eggs.

[0060] The effects of adding gallnut extract granules and sappanwood extract granules to the diet on the laying performance of laying hens in the late laying period are shown in Table 4. Compared with the control group, the feed conversion ratio of gallnut group broilers was significantly lower in weeks 1-2 and weeks 2-4. P <0.05), with no significant difference in average egg production rate and average egg weight; the average egg weight was significantly reduced in the antibiotic group ( P <0.05); the rates of dirty eggs and deformed eggs in different plant extract groups decreased significantly.

[0061] Eggshells are the first line of defense against microbial invasion, and eggshell quality is crucial for biosecurity in poultry production. As laying hens age, the morphology of their endometrium changes, leading to microstructural defects in the eggshell and affecting its quality. Simultaneously, laying hens require more calcium in the later stages of laying, but their absorption capacity decreases, easily causing calcium deficiency, which in turn thins the eggshell, increasing the rate of broken and deformed eggs (Qu Yuan, Yang Haiming, Shao Dan, et al. China Poultry, 2023, 45(12): 95-100.; Zhi Shiquan, Zhang Hengtong, Wei Wuqi, et al. Feed Expo, 2023, (02):66-68+77.). As shown in Table 4, compared with the control group, the eggshell thickness of the compound group increased significantly on day 14, the yolk weight of the sappanwood group and the antibiotic group increased significantly on day 14, and the rates of dirty and deformed eggs in each plant extract group decreased significantly. The rate of unqualified eggs in each plant extract group was significantly lower than that in the antibiotic group.

[0062] Table 4. Effects of Gallnut Extract and Sappanwood Extract on Laying Hen Performance in Late Laying Period Example 2: Effects of Gallnut Extract and Sappanwood Extract on Egg Quality in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0063] Egg quality assessment: On days 14 and 28, three eggs were randomly selected from each replicate, totaling 18 eggs per group, for a total of 90 eggs, to determine egg quality. Egg quality indicators included: egg weight, egg shape index, eggshell strength, eggshell thickness, albumen height, yolk color, and Haugh units. Egg weight: Weighed using a balance. Egg shape index: The longitudinal and transverse diameters of the egg were measured using calipers, and the egg shape index was calculated using the formula "longitudinal diameter / transverse diameter". Eggshell strength: Measured using an eggshell strength tester. Eggshell thickness: 0.25 cm was taken from the blunt end, acute end, and equatorial portion of the eggshell. 2 For eggs of different sizes, the thickness is measured using a micrometer, and the average value is the eggshell thickness. Egg white height, yolk color, and Haugh unit are determined using a multi-functional egg product analyzer. Yolk specific gravity: After removing the shell, gently separate the yolk and egg white, removing the egg white and chalaza. Weigh the yolk using a 0.01g balance and calculate the yolk weight / egg weight. Eggshell specific gravity: After careful washing and removing any remaining egg white, weigh the eggshell using a balance and calculate the shell weight / egg weight.

[0064] The effects of dietary supplementation with gallnut extract and sappanwood extract on egg quality in the later laying period are shown in Table 5. Compared with the control group, the eggshell thickness of the compound group was significantly increased on day 14. P <0.05), the proportion of egg yolk in the antibiotic group and the hematoxylin and eosin group increased significantly on day 14 ( P <0.05), with no significant differences in other indicators ( P >0.05).

[0065] Table 5. Effects of Gallnut Extract and Sappanwood Extract on Egg Quality in Late Laying Period Example 3: Effects of Gallnut Extract and Sappanwood Extract on Nutrient Metabolism Rate in Laying Hens in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0066] Nutrient metabolism rate determination: The dry matter metabolism rate and organic matter metabolism rate were determined using the total fecal collection method. Excrement was collected from day 26 to day 28 of the experiment. Feathers, feed, and other debris were removed, and the mixture was thoroughly mixed. 10% sulfuric acid nitrogen fixation solution was added, and the mixture was dried at 60℃-65℃. After fully rehydrating under natural conditions for 24 hours, the mixture was pulverized and passed through a 40-mesh sieve for later use. Crude protein (Cp) in the fecal samples was determined according to national standard GB / T 6432-2018; crude fat (Cf) was determined according to national standard GB / T 6433-2006; calcium (C) was determined according to national standard GB / T 6436-2018; and phosphorus (P) was determined according to national standard GB / T 6437-2018.

[0067] The effects of dietary supplementation with gallnut extract and sappanwood extract on the nutrient metabolic rate of laying hens in the late laying period are shown in Table 6. Compared with the control group, the dry matter metabolic rate of each experimental group was significantly increased. P <0.05); the CP metabolic rate was significantly increased in the antibiotic group ( P <0.05), while the difference was not significant in the plant extract group ( P >0.05); the metabolic rate of P was significantly increased in the antibiotic group, gallnut group, and compound group. P <0.05).

[0068] Table 6. Effects of Gallnut Extract and Sappanwood Extract on Nutrient Metabolism Rate in Laying Hens During Late Laying Period (%) Example 4: Effects of Gallnut Extract and Sappanwood Extract on Serum Biochemistry of Laying Hens in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0069] Serum biochemical index detection: Total protein (TP), albumin (ALB), globulin (GLB), glucose (GLU), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDLC), and low-density lipoprotein (LDLC) in serum were measured using a fully automated biochemical analyzer.

[0070] Serum biochemical indicators are important parameters reflecting the metabolic state and health level of the body. The levels of total cholesterol (TC) and triglycerides (TG) in serum are important indicators for measuring blood lipids. The TG level reflects the body's absorption, utilization, and metabolism of lipids, while the TC level reflects the fat deposition status in the animal's body (Cheng Wentao. Yangzhou University, 2025.). In the late laying period, laying hens often experience elevated blood lipids and increased lipid deposition due to the gradual decline in liver function and lipid metabolism. This not only affects production performance but may also induce metabolic diseases such as fatty liver syndrome (Shini A, Shini S, L Bryden W L. Avianpathology: journal of the WVPA, 2018, 48(1): 1-32.). Glucose is the body's main energy source, providing immediate energy for various life activities and is crucial for maintaining brain function, muscle activity, and egg formation. Indicators such as total protein and albumin are related to protein synthesis and immune function.

[0071] The effects of gallnut extract and sappanwood extract on serum biochemistry of laying hens in the late laying period are shown in Table 7. Compared with the control group, the TG content in the antibiotic group, gallnut group and compound group was significantly reduced. P <0.05), the TC content in the compound group was significantly reduced ( P <0.05); GLU levels were significantly increased in the antibiotic group, gallnut group, and sappanwood group ( P <0.05).

[0072] Table 7. Effects of Gallnut Extract and Sappanwood Extract on Serum Biochemical Indicators in Laying Hens in Late Laying Period Example 5: Effects of Gallnut Extract and Sappanwood Extract on Antioxidant Function of Laying Hens in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0073] Serum antioxidant assay: Serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), catalase (CAT), and total antioxidant capacity (TAOC) were measured using a kit (Nanjing Jiancheng Bioengineering Institute).

[0074] Liver antioxidant assay: Liver tissue was prepared into a 10% homogenate with PBS. The protein concentration of the homogenate was determined using a total protein quantification kit (Nanjing Jiancheng Bioengineering Institute). The levels of superoxide dismutase (SOD), glutathione S-transferase (GSH-ST), malondialdehyde (MDA), catalase (CAT), and total antioxidant capacity (TAOC) in the liver were determined using a kit (Nanjing Jiancheng Bioengineering Institute).

[0075] Oxidative stress is one of the main physiological challenges faced by laying hens in the later stages of egg production. The decline in reproductive system function is closely related to oxidative damage to the ovarian and oviduct tissues. As laying hens age, the balance between the generation and scavenging of free radicals is disrupted. Excessive reactive oxygen species attack unsaturated fatty acids in biological membranes, triggering lipid peroxidation, leading to damage to cell structure and function, and consequently affecting egg production performance and egg quality. Antioxidant enzymes in the body are the first line of defense against oxidation. Among them, CAT is a key enzyme for scavenging hydrogen peroxide, SOD's main function is to scavenge superoxide free radicals in the body, converting them into more stable oxygen and hydrogen peroxide to reduce oxidative damage, and T-AOC is a comprehensive indicator for measuring the functional status of the antioxidant system (He Junjin. The effects of tea polyphenols on laying performance, egg quality and health of laying hens [D]. Sichuan Agricultural University, 2017.).

[0076] The effects of gallnut extract and sappanwood extract on the antioxidant function of laying hens in the late laying period are shown in Table 8. Compared with the control group, the serum CAT content in the antibiotic group and gallnut group was significantly increased, and the SOD content in all experimental groups was significantly increased. P <0.05); the liver antioxidant capacity of each plant extract group showed a significant increase in SOD content ( P <0.05), GSH-ST content decreased significantly ( P The changes were <0.05); the TAOC content in the sappanwood group was significantly increased, and the CAT content in both the antibiotic group and the compound group was significantly increased ( P <0.05).

[0077] Table 8. Effects of Gallnut Extract and Sappanwood Extract on Antioxidant Function in Laying Hens in Late Laying Period Example 6: Effects of Gallnut Extract and Sappanwood Extract on Serum Reproductive Hormones in Laying Hens in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0078] Reproductive hormone assay: The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (PROG), estradiol (E2), and gonadotropin-releasing hormone (GnRh) in serum were measured using an ELISA kit (Jiangsu Enzyme Immunosorbent Assay Co., Ltd.).

[0079] Ovarian development and egg production performance in poultry are closely related to the regulation of the hypothalamic-pituitary-ovarian axis (HPO), among which GnRH, FSH, LH, E2, and PROG are key factors in the HPO regulation process (Peng Jianling. Jiangxi Agricultural University, 2024.). As laying hens age, the sensitivity of the hypothalamus to E2 negative feedback changes, leading to a decrease in GnRH secretion, which in turn results in a decrease in FSH and LH levels, a reduction in E2 and PROG secretion in the ovary, and follicular development is inhibited (Brady K, Long JA, Liu HC, et al. Poultry Science, 2020, 99(11): 6221-6232.).

[0080] The effects of gallnut extract and sappanwood extract on reproductive hormones in laying hens during the late laying period are shown in Table 9. Compared with the control group, the levels of reproductive hormones in each experimental group were significantly increased. P <0.05); while the GnRH level in the gallnut group was significantly increased ( P <0.05), while the levels in the antibiotic group, sappanwood group, and compound group were significantly reduced ( P <0.05).

[0081] Table 9. Effects of Gallnut Extract and Sappanwood Extract on Serum Reproductive Hormones in Laying Hens in Late Laying Period Example 7: Effects of Gallnut Extract and Sappanwood Extract on Ovarian and Oviduct Development in Laying Hens in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0082] Ovarian and oviduct development assessment: After slaughter, the ovaries and oviducts of laying hens were removed, weighed separately, and the ovarian index and oviduct index were calculated: Ovarian index (%) = (ovarian weight / live body weight) × 100%, Oviduct index (%) = (oviduct weight / live body weight) × 100%. The number of follicles in the ovaries was statistically analyzed according to the following criteria: Grade follicles (diameter > 10 mm); Large yellow follicles (diameter 8 mm–10 mm); Small yellow follicles (diameter 5 mm–7 mm); Large white follicles (diameter 2 mm–5 mm); Small white follicles (diameter < 2 mm).

[0083] The ovary and oviduct are core components of the laying hen's reproductive system, and their developmental status directly determines egg production performance and reproductive efficiency. In the later stages of egg production, the reproductive organs of laying hens gradually degenerate, the ovary loses weight, follicle atresia increases, and the oviduct atrophies and its function declines. This is a significant reason for the decrease in egg production.

[0084] The effects of gallnut extract and sappanwood extract on ovarian development in late-laying hens are shown in Table 10. Compared with the control group, the oviduct length in each experimental group was significantly increased. P <0.05), the fallopian tube index in the sappanwood group increased significantly ( P <0.05); there was no significant change in the number of follicles in any group ( P >0.05).

[0085] Table 10. Effects of Gallnut Extract and Sappanwood Extract on Ovarian and Oviduct Development in Laying Hens During Late Laying Period Example 8: Effects of Gallnut Extract and Sappanwood Extract on the Morphology of Ovary, Oviduct, Liver and Jejunum in Laying Hens in the Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0086] Histological observation: 1 cm samples were cut from the same location. 2 Fallopian tube, liver, ovary, and jejunum tissues were fixed in 4% paraformaldehyde solution and sent to Zhengzhou Baiping Biotechnology Co., Ltd. for section preparation. Hematoxylin-eosin (H&E) staining was used to stain the fixed tissues. Microscopic observation was performed, and several fields of view with intact morphology and clear orientation were selected. K-Viewer software was used to measure intestinal villus height (VH) and crypt depth (CD), and the villus-crypt ratio (VH / CD) was calculated. Morphological analysis of fallopian tube (dilatation, isthmus, and uterus) tissue samples was performed, including the lengths of primary and secondary villi in the dilatation and the villi in the isthmus, as well as the intervillous space within the uterus. At least six different fields of view were selected for each section.

[0087] Animal histomorphometry is an important method for evaluating organ and tissue health. The oviduct is an important site for egg formation and can be divided into the infundibulum, medulla oblongata, isthmus, uterus, and vagina according to function. The medulla oblongata is the largest part of the oviduct and produces egg white protein that surrounds the yolk; the isthmus is the main site for the formation of the eggshell membrane, and its well-developed villi help the normal formation of the shell membrane; the uterus is responsible for completing the eggshell mineralization and pigment deposition process (Arias JL, Fernandez MS, Dennis JE, et al. Matrix, 1991, 11(5): 313-320.; Gautron J, Stapane L, Le R, et al. BMC Molecular and Cell Biology, 2021, 22(1): 11.). The liver is the most metabolically active organ in laying hens, undertaking multiple physiological functions such as lipid synthesis and transport, protein metabolism, detoxification, and bile secretion (Tian WJ, Gonzales GB, Wang H, et al. Journal of Animal Science and Biotechnology, 2025, 16(1): 49.). The intestine is the main site of nutrient digestion and absorption, and also the largest immune organ in the body. The height of the intestinal villi determines the size of the absorptive surface area, and the crypt depth reflects the rate of epithelial cell renewal.

[0088] Effects of gallnut extract and sappanwood extract on ovarian morphology in late-laying hens, such as Figure 1 As shown, by Figure 1 It can be seen that, compared with the control group, the number of mature follicles in the ovaries of laying hens in the plant extract group and the antibiotic group was significantly increased.

[0089] The effects of gallnut extract and sappanwood extract on oviduct morphology in late-laying hens, such as Figure 2 As shown in Table 12, compared with the control group, the length of the isthmus villi and the length of the secondary villi in each plant extract group were significantly increased. P <0.05).

[0090] Table 11 Effects of Gallnut Extract and Sappanwood Extract on Oviduct Villi in Laying Hens in Late Laying Period Effects of gallnut extract and sappanwood extract on liver morphology in late-laying hens, such as Figure 3 As shown, the liver cells of laying hens in the late laying period of each experimental group had intact structures, and no obvious pathological phenomena were observed except for a small amount of bleeding.

[0091] The effects of gallnut extract and sappanwood extract on the morphology of the jejunum in laying hens during the late laying period are shown in the figure. Figure 4 As shown in Table 12, compared with the control group, the down height, crypt depth, and down-to-crypt ratio of laying hens in the Sumu group were significantly increased. P <0.05), the down height and crypt depth of the composite group of laying hens were significantly increased ( P <0.05), while there was no significant difference between the gallnut group and the antibiotic group ( P >0.05).

[0092] Table 12 Effects of Gallnut Extract and Sappanwood Extract on Jejunal Villus Height and crypt Depth in Laying Hens During Late Laying Period Example 11 Effects of Gallnut Extract and Sappanwood Extract on the Intestinal Microbiota of Laying Hens in Late Laying Period The experimental design, experimental diet, and feeding management were the same as in Example 1.

[0093] Cecal contents microbiota determination: Cecal contents samples from laying hens were sent to Shanghai Meiji Biotechnology Co., Ltd. for 16S rRNA sequencing analysis. Total genomic DNA of the microbial community was extracted using the EZNA® soil DNA kit (Omega Bio-tek, Norcross, Georgia, USA). The quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis. The DNA concentration and purity were determined using a NanoDrop2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, USA). The extracted DNA was used as a template for PCR amplification of the V3-V4 variable region of the 16S rRNA gene. Library construction was performed on the purified PCR products using the NEXTFLEX Rapid DNA-Seq Kit: (1) adapter ligation; (2) removal of adapter self-ligated fragments using magnetic beads; (3) enrichment of library templates using PCR amplification; (4) recovery of PCR products using magnetic beads to obtain the final library. Sequencing was performed using the Illumina Nextseq2000 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.), and all analyses were completed through the Meiji Cloud platform.

[0094] This experiment analyzed 16S rDNA gene sequencing of cecal contents samples collected from five treatment groups. The RDP classifier Bayesian algorithm was used to perform taxonomic analysis on representative OTU (Operational Taxonomic Units) sequences at a 97% similarity level. The community species composition of each sample was statistically analyzed at each taxonomic level: Domain, Kingdom, Phylum, Class, Order, Family, Genus, and Species. Species annotation, richness, and diversity analyses were also performed. Dilution curves for cecal contents samples from each treatment group are shown below. Figure 5 As shown, the dilution curve tends to plateau and saturate at the end, while the number of OTUs remains basically unchanged as the sequencing depth increases, indicating that the sequencing data of the experimental samples is reasonable and the sequencing depth is sufficient, basically covering all species in the tested samples.

[0095] The gut microbiota plays a central role in maintaining animal health by participating in host nutrient metabolism, immune regulation, and intestinal barrier maintenance. The cecum, as the most active segment of the laying hen's intestine, directly influences the fermentation and utilization of nutrients and the generation of harmful metabolites through its microbial structure and function. The Alpha diversity index is a comprehensive indicator of species richness and evenness; a higher Shannon index and a lower Simpson index indicate a richer and more evenly distributed microbial community (Liu Chun, Li Zhijian, Guan Xiuqiong. Fine Chemicals, 2021, 38(06): 1162-1170.).

[0096] Alpha diversity analysis results of gallnut extract and sappanwood extract on the cecal microbiota of late-laying laying hens are as follows: Figure 6 As shown, compared with the control group, the Simpson index of the antibiotic group and the gallnut group was significantly reduced ( P <0.05), while the Shannon index of the gallnut group was significantly increased ( P <0.05); the ace index and chao index of each experimental group showed an upward trend, but the differences were not significant. P >0.05).

[0097] The results of principal coordinate analysis (PCoA) of gallnut extract and sappanwood extract on the cecal microbiota of laying hens in the late laying period are as follows: Figure 7As shown, the Bray-Curtis distance algorithm was used to analyze each experimental group at the OTU level, and the nonparametric ANOSIM test was used to assess the significance of differences between groups. Different colored points represent samples from different groups, and the proximity of points between samples reflects the magnitude of differences in species composition. Principal component 1 (PC1) explained 17.3% of the variation, and principal component 2 (PC2) explained 13.44% of the variation.

[0098] Studies have shown that the gut microbiota of laying hens exhibits distinct patterns of change at different growth stages. During the brooding period, the gut microbiota is mainly composed of Proteobacteria (…). Proteobacteria )and Bacillota Composition, as age increases, Bacillota and Bacteroidota The relative abundance of increased, while Proteobacteria The relative abundance decreased; until the peak egg production period, Bacillota and Bacteroidota Beyond Proteobacteria They became the two most abundant dominant bacterial groups in the cecal flora of laying hens; and in the later stages of egg production, Bacteroidota Relative abundance exceeds Bacillota Becoming the dominant bacteria, at the same time Proteobacteria The abundance decreased again (Dai D, Wu SG, Zhang HJ, et al. Journal of Animal Science Biotechnol, 2020, 11: 19; Joat N, Van TTH, Stanley D, et al. Applied Microbiology and Biotechnology, 2021, 105(11): 4719–4730.). Bacillota It mainly produces metabolic products such as lactic acid and butyric acid through the fermentation of carbohydrates, while Bacteroidota They are responsible for degrading complex plant polysaccharides to produce short-chain fatty acids such as propionic acid. The synergistic metabolism between the two phyla maintains the energy supply and pH homeostasis of the intestine (Theresa S, Isabel K, R.Segura MR, et al. Gut Microbes, 2020, 13(1): 1-21; Lin Miao, Feng Limei, Wang Kuopeng, et al. Bulletin of Microbiology, 2021, 48(02): 555-564.).

[0099] The results of the differential analysis of the phylum level of the cecal contents of laying hens by extracts of gallnut and sappanwood are as follows: Figure 8 As shown in Table 13. The results show that, at the phylum level, the cecal microbiota of laying hens is mainly composed of... Bacteroidota (Bacteroidetes) Bacillota (Firmwallis) Thermodesulfobacteriota (Thermal desulfurobacteria) Spirochaetota (Spirome) and Actinobacteriota Composed of (Actinomycetes), etc., among which Firmicutes and Bacteroidota These two phyla account for over 90% of the total bacterial phyla, making them the dominant bacterial groups. Compared to the control group, all experimental groups showed... Bacteroidota The relative abundance increased, while Bacillota The relative abundance showed a decreasing trend, but the results were not significantly different. P >0.05); and at the same time, each plant extract group Thermodesulfobacteriota The relative abundance decreased significantly ( P <0.05).

[0100] Table 13. Relative abundance of bacterial flora at the phylum level in the cecal contents of laying hens. The results of the differential analysis of the bacterial genus level of the cecal contents of laying hens in the late laying period by gallnut extract and sappanwood extract are as follows: Figure 9 As shown in Table 14. The results indicate that, at the genus level, the cecal microbiota of laying hens is mainly composed of... Bacteroides (Bacteroides) Rikenellaceae_RC9_gut_group (RC9 intestinal flora of Riken Bacteria Family) unclassified_o__ Bacteroidales (Unclassified Bacteroidetes) Mediterraneibacter (Mediterranean bacteria) and norank_o__ Clostridia_UCG-014 Composed of (unranked Clostridium orders, UCG-014 group), etc. Among them, Bacteroides and Rikenellaceae_RC9_gut_group It is the dominant genus. Compared with the control group, the experimental groups... Rikenellaceae_RC9_ gut_group The relative abundance was significantly increased ( P <0.05), Bacteroides The relative abundance also showed an increasing trend, but the difference was not significant. P >0.05).

[0101] Table 14. Relative abundance of bacterial flora at the genus level in the cecal contents of laying hens. The results of the Lefse multilevel species difference analysis of the cecal flora of laying hens in the late laying period were as follows: (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) Figure 10 As shown, compared with the control group, the main enriched bacterial flora in the antibiotic group was... Bacteroidescaecigallinarum (Bacteroides cecum) Parabacteroides (Parabacteroides) Marseille-P3108 (Bacteroides vitiformis); the main enriched bacterial groups in the Galla section are Lactobacillus salivarius (Lactobacillus salivarius) NK4A214 group The main bacterial communities enriched in the Hematoxylin section are: Spirochaetales (Spirometra); the main enriched bacterial groups in the complex group are unclassified Rikenellaceae , Colidextribacter .

[0102] Results regarding the relationship between the genus-level microbial community of the cecal contents of laying hens and environmental factors, as follows: Figure 11 As shown. This invention performs correlation analysis on the top 15 abundant genus-level bacterial communities and serum reproductive hormone indicators (GnRH, FSH, LH, PROG, E2). The results show that... Bacteroides It was positively correlated with serum reproductive hormones LH, FSH, E2, and PROG; Rikenellaceae_ RC9_gut_group It is positively correlated with FSH, E2, and PROG; Extibacter (Exobramyl) was negatively correlated with FSH, E2, and PROG.

[0103] Comparative Example 1: Effect of different plant extracts on the diameter of the inhibition zone of Escherichia coli Preparation of plant water extract and bacterial culture: The plant extract raw material was pulverized and sieved (80 mesh) using a pulverizer; 30 g was weighed and placed in a food-grade filter bag, and soaked in 10 times its volume of distilled water in a beaker for 30 min. After boiling on an electric stove, it was simmered for 30 min. The filtrate was collected, and the residue in the bag was extracted again in the same way. The two filtrates were combined, concentrated over a low flame, and brought to a final volume of 30 mL to obtain an extract concentration of 1 g / mL. The extract was then stored at 4℃ for later use. The frozen bacterial culture was inoculated onto a solid culture medium and incubated statically at 37℃ for 24 h. A single intact colony was picked up with an inoculation loop and transferred to a liquid culture medium, and then incubated again at 37℃ and 180 r / min on a shaker for 12 h. The viable count of E. coli after the third generation of liquid culture was determined for subsequent use.

[0104] The effects of different plant extracts on the inhibition zones of *Escherichia coli* were determined using the perforation method. Approximately 20 mL of solid culture medium cooled to about 50°C was poured into autoclaved plates. After horizontal solidification, a sterile cotton swab was immersed in a solution diluted to 1×10⁻⁶. 8After removing excess bacterial solution by squeezing the test tube wall, the solution was evenly spread onto a plate using a CFU / mL E. coli culture. Four holes were punched in each plate using a sterile pipette tip, ensuring a center-to-center distance of at least 24 mm and a distance from the edge of the plate of at least 15 mm. 200 μL of plant aqueous extract (1 g / mL) was added to each plate. Sterile physiological saline was used as a blank control, and chlortetracycline hydrochloride and florfenicol were used as antibiotic controls. Each treatment was repeated three times. The plates were incubated at 37°C for 16–20 h, and the diameter of the inhibition zone was measured using calipers. The criteria for determining the sensitivity of the inhibition zone diameter were: <10 mm for low sensitivity, 10 mm ≤ <15 mm for moderate sensitivity, 15 mm ≤ <20 mm for high sensitivity, and ≥20 mm for extremely sensitive.

[0105] Table 15 shows that the effect of different plant extracts on the inhibition zone of Escherichia coli was determined by the perforation method. Compared with physiological saline, except for Phellodendron bark, Sichuan pepper and rhubarb, the other four plant extracts all had a significant inhibitory effect on Escherichia coli. The diameter of the inhibition zone from largest to smallest was Scutellaria baicalensis > Gallnut > Sappanwood > Coptis chinensis > Phellodendron bark.

[0106] Table 15. Diameter of inhibition zones of different plant extracts against Escherichia coli Comparative Example 2: Minimum inhibitory concentrations of different plant extracts against Escherichia coli The effect of different plant extracts on the minimum inhibitory concentration (MIC) of Escherichia coli was determined using the broth microdilution method. Following the definition and method of MIC in CLSI M07 (Wayne, PA: Clinical and Laboratory Standards Institute, 2015), the concentration corresponding to the lowest dilution of the antimicrobial drug with visible turbidity pores was used as the MIC value, and the broth microdilution method was employed. The experimental setup and specific procedures were based on the methods of Gao et al. (Su Mengru, Ma Peipei, Li Xinxin, et al. Advances in Animal Medicine, 2020, 41(03): 52-56; Gao Yu. Study on the antibacterial activity of 35 medicinal plant extracts [D]. Kashgar University, 2021). The plant extracts were diluted in 96-well plates, and the extract test group, negative control, positive growth control, and blank culture medium control were set up. The plates were incubated at 37℃ for 16-20 h. Because most plant water extracts are dark in color and difficult to observe, to ensure the accuracy of the MIC (minimum inhibitory concentration) determined by visual inspection, the OD 600 nm value of each well was measured using a microplate reader to determine the bacterial inhibition rate. A value was considered reliable when the inhibition rate was greater than 90%, and the corresponding concentration was defined as the minimum inhibitory concentration (MIC) of that plant water extract. kThe calculation formula is as follows: where O k The OD value in the k-th hole; O p The average OD value of the positive control wells; O n The OD value of the negative control well; O b This refers to the average OD value of the blank control wells.

[0107] Inhibition rate I k (k=1, 2...5, 6)(%)=1-(O k -O n ) / (O p -O b )×100%.

[0108] The preparation of plant water extracts and bacterial solutions was the same as in Comparative Example 1.

[0109] All blank and negative control wells were clear, indicating that the experiment was conducted under normal conditions. Table 16 shows that the minimum inhibitory concentrations (MICs) of different plant extracts against *E. coli* were determined using the micro-broth dilution method. Low concentrations of *Galla chinensis*, *Caesalpinia sappan*, and *Scutellaria baicalensis* extracts showed inhibitory effects, with MICs of 7.8125 mg / mL, 15.625 mg / mL, and 15.625 mg / mL, respectively. Based on the above inhibition zone test results, *Galla chinensis*, *Caesalpinia sappan*, and *Scutellaria baicalensis* extracts, which exhibited the strongest inhibitory effects against *E. coli*, were subsequently combined.

[0110] Table 16 Minimum inhibitory concentrations of different plant extracts against Escherichia coli Note: "+" indicates significant growth of indicator bacteria, while "-" indicates insignificant growth of indicator bacteria. Comparative Example 3: Effect of different combinations of plant extracts on the combined antibacterial concentration index of Escherichia coli Following the method described by Li Yan et al. (Clemente I, Aznar M, Silva F, et al. Innovative FoodScience and Emerging Technologies, 2016, 36: 26-33; Li Yan. Study on the antibacterial activity of compound plant essential oils and their protective effect against ETEC K99-infected mice [D]. Beijing Agricultural College, 2023.), the plant water extracts were diluted to 2MIC, MIC, 1 / 2MIC, 1 / 4MIC, 1 / 16MIC, and 1 / 32MIC using MH broth. In a sterile operating table, 50 μL of different concentrations of plant extract 1 were added horizontally from top to bottom in a 96-well plate, and 50 μL of different concentrations of plant extract 2 were added vertically from left to right. The treatment group received 100 μL of the final concentration of 5 × 10⁻⁶. 5CFU / mL bacterial suspension was used to set up a negative control (drug solution + MH broth) and a blank control (MH broth). The plates were sealed and incubated at 37℃ for 16-20 h. OD600 was measured using a microplate reader. The combined inhibitory concentration index (FICI) was used to determine the combined effect of different plant extracts. FICI ≤ 0.5 indicated a synergistic effect; 0.5 < FICI ≤ 1 indicated an additive effect; 1 < FICI ≤ 2 indicated an indifferent effect; and FICI > 2 indicated an antagonistic effect. The FICI calculation formula is as follows: MIC1 refers to the concentration of plant extract 1 in the minimum inhibitory concentration obtained when plant extract 1 and plant extract 2 are used together; MIC... A MIC1 refers to the minimum inhibitory concentration (MIC) when plant extract 1 is used alone; MIC2 refers to the concentration of plant extract 2 in the minimum inhibitory concentration obtained when plant extract 1 and plant extract 2 are used in combination; MICB refers to the minimum inhibitory concentration (MIC) when plant extract 2 is used alone.

[0111] FICI = FIC A +FIC B =MIC1 / MIC A +MIC2 / MIC B .

[0112] The preparation of plant water extracts and bacterial solutions was the same as in Comparative Example 1.

[0113] The results are shown in Table 17. The antibacterial effects of each combination of gallnut, sappanwood, and scutellaria against Escherichia coli were determined by the checkerboard dilution method. According to the FICI calculation results, the combination of gallnut and sappanwood, as well as the combination of gallnut and scutellaria, showed a synergistic effect on the inhibition of Escherichia coli, while the combination of sappanwood and scutellaria showed an antagonistic effect.

[0114] Table 17 FICI of compound plant extracts against Escherichia coli Comparative Example 4: Effect of different proportions of plant extract combinations on the antibacterial effect of Escherichia coli Based on the FICI results, two to three plant extracts with synergistic and additive effects were screened out, and different combinations and levels were set for formulation. The specific groupings are shown in Table 18. The diameter of the inhibition zone and the minimum inhibitory concentration of the compound plant extracts with different ratios were measured, and the combination with the best antibacterial effect was screened out.

[0115] The preparation of plant water extracts and bacterial solutions was the same as in Comparative Example 1.

[0116] Using sappanwood extract, gallnut extract, and scutellaria baicalensis extract as three experimental factors, each with five levels, and based on the FICI values ​​of the compound plant extracts against Escherichia coli, sappanwood extract × gallnut extract and gallnut extract × scutellaria baicalensis extract were selected as experimental combination schemes. The FICI value of sappanwood extract × scutellaria baicalensis extract was greater than 2, indicating antagonistic effect, and no further combination was performed. Table 19 shows that compared with single plant extracts, the ratio of the two compound plant extracts increased the diameter of the inhibition zone against Escherichia coli, decreased the MIC value, and significantly enhanced the antibacterial effect. Furthermore, the overall antibacterial effect of the sappanwood extract × gallnut extract combination was stronger than that of gallnut extract × scutellaria baicalensis extract. Among these, combination A (sappanwood extract to gallnut extract ratio of 1:4), B (sappanwood extract to gallnut extract ratio of 2:3), and C (sappanwood extract to gallnut extract ratio of 2.5:2.5) were considered the optimal combinations.

[0117] Table 18 Compound Plant Extract Formulation Scheme Note: The numbers 0, 1, ... 4 in the column corresponding to plant extracts indicate the proportion of that Chinese herbal medicine in the compound plant extract.

[0118] Table 19. Antibacterial effects of different ratios of compound plant extracts on Escherichia coli Escherichia coli is one of the most common opportunistic pathogens in poultry farming. It readily induces diseases such as salpingitis, peritonitis, and septicemia, leading to a significant decline in the laying performance of hens (Heidemann OR, Bisgaard M, Christensen JP, et al. Avian Dis, 2016, 60(1): 1-7.). E. coli infection in laying hens significantly reduces ADFI, laying rate, and the percentage of qualified eggs (Wang Puhui, Guo Fangshen, Hu Zeqiong, et al. Chinese Journal of Animal Husbandry, 2023, 59(06): 222-230.). Escherichia coli infection activates the body's immune system and inflammatory response, releasing large amounts of pro-inflammatory cytokines (such as IL-1β and TNF-α), which inhibit the activity of the feeding center through central action, reducing feed intake. Nutrients are used extensively for the immune defense system, leading to a decline in egg production performance. At the same time, it causes oviduct inflammation and tissue damage, directly affecting eggshell formation and albumen secretion, resulting in an increased rate of deformed eggs (Wu Y, Wang W, Kim IH, et al. Poultry Science, 2022, 101(2): 101615.; Fang H, Quan H, Zhang Y, et al. Pathogens, 2021, 10(6):755.).

[0119] The results of comparative examples 1–4 showed that *Gnaphalium affine*, *Caesalpinia sappan*, and *Scutellaria baicalensis* all exhibited significant antibacterial activity against *Escherichia coli*, with MICs of 7.8125 mg / mL, 15.625 mg / mL, and 15.625 mg / mL, respectively. The combination of *Gnaphalium affine* × *Caesalpinia sappan* and *Gnaphalium affine* × *Scutellaria baicalensis* showed a synergistic effect against *Escherichia coli*. The combination of *Caesalpinia sappan* and *Gnaphalium affine* showed a better overall antibacterial effect than that of *Gnaphalium affine* and *Scutellaria baicalensis* alone, indicating that the combined antibacterial effect was superior to that of any single herb. Combination A showed the best antibacterial effect, followed by combinations B and C.

[0120] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0121] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A feed additive, characterized in that, The feed additive contains sappanwood extract and gallnut extract, as well as a carrier; The ratio of the sappanwood extract to the gallnut extract is 1:1 to 4.

2. The feed additive according to claim 1, characterized in that, The ratio of the sappanwood extract to the gallnut extract is 1:

4.

3. The feed additive according to claim 1, characterized in that, The ratio of the sappanwood extract to the gallnut extract is 1:1.

5.

4. The feed additive according to claim 1, characterized in that, The ratio of the sappanwood extract to the gallnut extract is 1:

1.

5. The feed additive according to any one of claims 1 to 4, characterized in that, The carrier includes one or more of the following: corn starch, maltodextrin, rice husk powder, wheat bran, peanut shell powder, and corn cob powder.

6. The feed additive according to claim 5, characterized in that, The carrier is corn starch and maltodextrin.

7. Use of the feed additive according to any one of claims 1 to 6 in the preparation of laying hen feed.

8. The use according to claim 7, characterized in that, The feed additive is used to improve and / or enhance the production and / or reproductive performance of laying hens in the later stages of egg production.

9. A type of laying hen feed, characterized in that, Includes the feed additives described in any one of claims 1 to 6 and the basic feed; The mass ratio of the feed additive to the basic feed is 1:1000.

10. The laying hen feed according to claim 9, characterized in that, The basic feed includes one or more of the following: corn, soybean meal, soybean oil, wheat bran, limestone powder, dicalcium phosphate, sodium chloride, methionine, and premix.

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