Method for improving intestinal health of broiler chickens by using earthworm powder and application thereof

Abstract

This invention provides a method and application for improving the intestinal health of broilers using earthworm powder, belonging to the field of animal nutrition and feed technology. This invention solves the problems of poor growth performance, impaired intestinal health, and soybean meal substitution caused by nutritional deficiencies in broiler mixed meal diets. It includes the following steps: (1) raising earthworms in a substrate formed by mixing fermented chicken manure and Chinese medicine residue at a mass ratio of 2:3, and adding 2000 ppm sodium citrate to the substrate, controlling the substrate humidity at 50% to 55% and the temperature at 23 to 25°C; (2) preparing earthworm powder by freeze-drying the earthworms obtained in step (1) after disinfection; (3) replacing part of the soybean meal with 5.0% to 5.5% of the protein such as earthworm powder in the mixed meal broiler diet, and feeding the broilers. This invention significantly improves the growth performance, feed conversion ratio, immune function, intestinal digestive enzyme activity, intestinal morphology and barrier function of broilers, and optimizes the cecal microbiota, ultimately achieving efficient reduction and substitution of soybean meal and healthy and efficient broiler breeding.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition and feed technology, and to a method and application of using earthworm powder to improve the intestinal health of broilers. Specifically, it relates to feed formulation technology and its application of replacing conventional protein raw materials in mixed meal diets with earthworm powder to improve the production performance, intestinal morphology, digestive function and cecal microbiota of broilers. Background Technology

[0002] In my country's livestock production, soybean meal is a core source of high-quality plant protein, with a balanced amino acid composition and wide application in livestock and poultry production. Replacing soybean meal with other widely available and lower-cost meals such as cottonseed meal is an important direction for addressing this issue. However, these other meals themselves have problems such as amino acid imbalances and the presence of anti-nutritional factors, which can inhibit the activity of digestive enzymes in animals, reduce nutrient utilization, and damage intestinal health, thus limiting their large-scale application in diets.

[0003] Against this backdrop, developing new environmentally friendly and nutritionally balanced protein alternatives has become a research hotspot. Insect protein, due to its high protein content, relatively balanced amino acid composition, and low-carbon and environmentally friendly breeding process, is considered an ideal alternative to traditional protein raw materials. Among them, earthworms and cockroaches (such as the American cockroach) are not only rich in protein but also contain various bioactive components such as antimicrobial peptides. Furthermore, they can be produced by converting organic waste such as livestock and poultry manure and kitchen waste, thus possessing both environmental and economic value. However, existing technologies mostly focus on exploring the application effects of single insect proteins in conventional corn-soybean meal diets, lacking a complete integrated technology from efficient insect breeding and standardized processing to systematic application in complex diets (such as mixed meal diets).

[0004] More specifically, current technology has not yet resolved the unclear issues regarding the differentiated effects of various insect proteins on broiler gut health, digestive enzyme systems, and cecal microbiota, as well as their regulatory mechanisms. Furthermore, in the specific context of mixed meal diets, how to utilize insect proteins to compensate for the nutritional deficiencies of mixed meals through precise diet formulation technology, achieving feeding results comparable to or even better than conventional soybean meal diets, still requires in-depth research. Therefore, developing a complete technical solution encompassing efficient earthworm farming, high-value processing, and the scientific formulation of mixed meal diets is of great significance for improving broiler growth performance, enhancing gut health, and promoting the reduction and substitution of soybean meal. Summary of the Invention

[0005] This invention aims to solve problems such as the tight supply of traditional protein raw materials, low utilization rate of miscellaneous meal diets, and poor intestinal health of broilers. It provides an integrated technology for efficient earthworm farming, high-value processing of earthworm powder, and protein replacement of miscellaneous meal diets, so as to achieve the goals of rapid growth, low feed conversion ratio, strong immunity, good intestinal health, and high nutrient utilization rate in broilers.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution: This invention provides a method for improving the intestinal health of broiler chickens using earthworm powder, comprising the following steps: (1) Earthworms were raised in a substrate formed by mixing fermented chicken manure and Chinese medicine residue at a mass ratio of 2:3, and 2000 ppm sodium citrate was added to the substrate. The substrate humidity was controlled at 50% to 55% and the temperature at 23 to 25℃. (2) After disinfection, the earthworms obtained in step (1) are prepared into earthworm powder by freeze drying. (3) In a mixed meal type broiler diet, 5.5% of the protein such as earthworm powder mentioned above is used to replace part of the soybean meal, and the mixture is prepared and fed to broilers.

[0007] Preferably, in step (1), the Chinese herbal residue includes: extract residues of medicinal and edible Chinese herbal medicines such as Astragalus membranaceus, Codonopsis pilosula, Lycium barbarum, and Taraxacum mongolicum, prepared in a mass ratio of 1:1:1:1.

[0008] Preferably, in step (2), the crude protein content of the earthworm powder prepared by the freeze-drying method is not less than 60.34%, and the content of lysine, methionine and tryptophan is significantly higher than that of earthworm powder prepared by sun-drying and oven-drying methods.

[0009] Preferably, in step (3), the mixed meal broiler diet includes corn, soybean meal, cottonseed meal, whole wheat flour and compound premix, wherein the crude protein, metabolizable energy, calcium, available phosphorus and amino acid nutritional levels of each group of diets are consistent.

[0010] The second aspect of this invention provides a feed for improving broiler production performance, intestinal health, and meat quality. The feed contains freeze-dried earthworm powder prepared by the method described in claim 1 or 2, and the earthworm powder is added at 5.0%-5.5% to a mixed-meal broiler diet, used to replace a portion of soybean meal with equal protein. The feed significantly increases the body weight and average daily weight gain of broilers at 21 and 42 days of age, reduces the feed conversion ratio, increases the bursal index, enhances the activity of amylase, protease, and lipase in the broiler intestine, improves intestinal villus morphology, and increases the villus-cryptomorphic ratio. The feed optimizes the cecal microbiota of broilers, specifically by increasing the abundance of Lactobacillus, Bifidobacterium, and Ruminococcus family UCG-014, and decreasing the abundance of Escherichia coli and Shigella. Feeding this feed enables broilers to achieve growth performance, slaughter performance, and meat quality comparable to broilers fed a conventional corn-soybean meal diet.

[0011] The third aspect of this invention provides an application of the above method in reducing and replacing soybean meal in broiler mixed meal diets, improving broiler gut health, enhancing nutrient utilization, and improving broiler slaughter performance and meat quality.

[0012] The fourth aspect of this invention provides the application of the above-mentioned feed in the 42-day full-process breeding of Ross 308 broiler chickens.

[0013] The fifth aspect of the present invention provides an additive composition for earthworm farming, comprising a substrate of fermented chicken manure and Chinese herbal medicine residue mixed in a mass ratio of 2:3, and sodium citrate at a concentration of 2000 ppm, for promoting the growth and reproduction of earthworms under conditions of 50% to 55% humidity and 23 to 25°C.

[0014] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a technology for raising earthworms using fermented chicken manure, and the earthworm powder, after appropriate processing, is applied to broiler chicken meal diets to replace part of the protein raw materials such as soybean meal, which has the following significant beneficial effects: (1) Adding sodium citrate can significantly promote the growth and reproduction of earthworms, realize the harmless and resource-based utilization of chicken manure, and reduce breeding pollution; (2) Freeze-drying retains crude protein and limiting amino acids such as lysine, methionine, and tryptophan to the maximum extent, and its nutritional quality is significantly better than sun-drying and oven-drying. (3) The earthworm powder group significantly increased the body weight and average daily weight gain of broilers at 21 days and 42 days, and reduced the feed conversion ratio throughout the period, with better results than fish meal and cockroach powder; (4) Significantly increases the bursa of Fabricius index, has no negative impact on immunoglobulins, and enhances the body's disease resistance; (5) Significantly increases the activity of intestinal amylase, protease and lipase, improves the morphology of villi in the jejunum and ileum, increases the villi-to-crypt ratio, and enhances absorption function; (6) Improves cecal microbial diversity, enriches beneficial bacteria such as lactobacilli, bifidobacteria, and rumenococci UCG-014, and reduces Escherichia coli. Abundance of harmful bacteria such as Shigella, Merdibacter, and Ruminococcus_torques_group; (7) It can significantly improve the slaughter rate, eviscerated rate, breast muscle rate, and leg muscle rate of broilers, and reduce the abdominal fat rate; at the same time, it reduces the drip loss, cooking loss, and shear force of breast muscle, and improves the water retention and tenderness of muscle. The meat quality is comparable to that of conventional corn. The soybean meal group is comparable.

[0015] (8) Earthworm powder has been proven to have no toxic side effects through safety tests and has no adverse effects on hematological and blood biochemical indicators. It can stably replace soybean meal in mixed meal diets, reduce feed costs, and achieve soybean meal reduction and replacement, which is in line with the national policy of saving grain and reducing consumption and is suitable for large-scale broiler farming. Attached Figure Description

[0016] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0017] Figure 1 The effect of earthworm meal replacing part of the protein on digestive enzyme activity is shown in the figure. AC: intestinal amylase, protease, and lipase activities of 21-day-old broilers; DF: intestinal amylase, protease, and lipase activities of 42-day-old broilers. Con: control group; FM: fish meal group; EM: earthworm meal group; CM: cockroach meal group.

[0018] Figure 2 The effect of replacing part of the protein with earthworm powder on the microbial diversity of the cecum in 21-day-old broiler chickens.

[0019] Figure 3 The effect of replacing part of the protein with earthworm powder on the cecal microbial diversity of 42-day-old broiler chickens.

[0020] Figure 4 The effect of replacing part of the protein with earthworm powder on the phylum and genus level colony composition of the cecal microbiota in 21-day-old broilers.

[0021] Figure 5 The effect of replacing part of the protein with earthworm powder on the phylum and genus level colony composition of the cecal microbiota in 42-day-old broiler chickens.

[0022] Figure 6 LEfSe analysis was used to identify differentially abundant microbiota in the cecum of 21-day-old broilers.

[0023] Figure 7 LEfSe analysis was used to identify differentially abundant microbiota in the cecum of 42-day-old broilers. Detailed Implementation

[0024] The following are specific embodiments of the present invention, described in conjunction with the accompanying drawings, to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments. Specific details, such as particular configurations, are provided in the following description merely to aid in a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0026] Example 1: Fermentation of chicken manure for earthworm farming Approximately 40%–70% of the nutrients in chicken feed cannot be absorbed and are excreted, making chicken manure the most nutrient-rich of all poultry and livestock manure. Currently, chicken manure is mostly used for microbial fermentation to produce organic fertilizer or biogas, which is the main way to harmlessly treat and utilize chicken manure. However, both methods share common drawbacks: secondary environmental pollution and high input costs. Utilizing the wide diet, high reproductive capacity, and strong adaptability of higher organisms like insects to treat chicken manure is currently a research hotspot. Earthworms are saprophytic, feeding on decaying plants or other organic matter, so chicken manure can serve as food for earthworms. Research reports on using livestock and poultry manure to raise earthworms are frequently published, but these studies mostly focus on the carbon-to-nitrogen ratio of the substrate and the breeding environment; research reports on earthworm substrate additives are rare. Based on the research of other scholars on feeding earthworms with pig manure and cow manure and the research of our team on the mixed fermentation of chicken manure and Chinese medicine residue, this experiment selected several additives and their optimal dosages. Using fermented chicken manure and Chinese medicine residue as a substrate, the effect of feeding earthworms with chicken manure was studied, providing a technical reference for the large-scale and efficient biological treatment of chicken manure.

[0027] 1.1 Experimental Design and Methods 1.1.1 Experimental animals There are 180 young earthworms and 180 adult earthworms in the Daping No. 2 variety.

[0028] 1.1.2 Test Materials Appetite enhancers: sodium citrate, potassium hydrogen phosphate, humic acid, urea, sucrose.

[0029] Substrate: chicken manure: Chinese medicine residue = 2:3, piled up and fermented for 8 weeks, dried and mixed, and the carbon-nitrogen ratio was measured.

[0030] The experimental design is shown in Table 1.

[0031] Table 1 Experimental Design

[0032] Experimental method: 1 kg of fermented chicken manure-Chinese herbal medicine residue substrate was placed in 36 foam boxes measuring 280×165×185 (mm). The control group was sprayed with drinking water, while the other experimental groups were periodically sprayed with 2000 ppm sodium citrate solution, potassium hydrogen phosphate solution, potassium humate solution, urea solution, and 5000 ppm sucrose solution, respectively. The substrate humidity was maintained at 50%–55% throughout the experimental period, and the temperature was maintained at 23–25℃. The experiment started on May 6, 2020 and ended on July 1, 2020.

[0033] Growth period experiment: 10 young earthworms (weighing 175-300 mg) were selected from each foam box, and the earthworms were weighed after 8 weeks; Reproduction experiment: Ten adult earthworms (weighing more than 500 mg) were selected from each foam box. The egg-laying situation was checked every 7 days, and the eggs were removed and stored separately. The total number of eggs was counted after 8 weeks.

[0034] 1.1.4 Measurement Indicators Earthworm weight gain: At the beginning of the experiment, the total weight of 10 earthworms in each foam box was weighed. At the end, all earthworms were separated from the substrate and weighed again. The difference in weight before and after the experiment was calculated.

[0035] Cocoon count: Earthworm eggs were separated and counted on days 7, 14, 21, 28, 35, 42, 49, and 56 of the experiment, and the total number of eggs laid during the entire experimental period was calculated.

[0036] 1.1.5 Data Analysis One-way ANOVA was performed on the experimental results using SPSS 22.0 statistical software, with P<0.05 as the criterion for statistical significance.

[0037] 1.2 Results and Analysis 1.2.1 Effects of different attractants on earthworm growth The effects of different additives on earthworm weight gain are shown in Table 2.

[0038] Table 2. Effects of different attractants on earthworm weight gain.

[0039] As shown in Table 2, the addition of different additives to the fermented chicken manure-Chinese herbal medicine residue substrate had a significant effect on the weight gain of earthworms (P<0.050). The group with 2000 ppm potassium hydrogen phosphate was the best, followed by the sodium citrate group. There was no significant difference among the humic acid group, urea group and sucrose group (P>0.05).

[0040] 1.2.2 Effects of different attractants on earthworm reproductive performance The effects of different attractants on the number of eggs laid by adult earthworms are shown in Table 2.

[0041] Reproduction experiments showed that the potassium phosphate group resulted in the highest earthworm weight gain (0.18 mg), but only 53.67 eggs were laid, significantly inhibiting reproduction. Humic acid, urea, and sucrose groups all inhibited egg production to varying degrees, with the urea group producing the lowest number of eggs (9.59). Only the sodium citrate group achieved dual promotion of growth and reproduction: a weight gain of 0.15 mg (significantly higher than the control group) and 91.49 eggs laid (a 36.6% increase compared to the control group). This solved the technical problem of existing additives that "promote growth but inhibit reproduction," demonstrating a balanced growth-reproduction effect and providing the only optimal additive solution for large-scale, efficient earthworm breeding.

[0042] Adding sodium citrate increased the levels of juvenile hormone and ecdysone in earthworms by 32.6% and 27.6%, respectively, significantly higher than the control group and other attractant groups (P<0.05). Potassium hydrogen phosphate and urea significantly reduced the levels of these two hormones (P<0.05), inhibiting reproductive-related physiological processes. The gonadal index in the sodium citrate group was 41.1% higher than that in the control group, with full gonads and a large number of mature eggs, providing a histological basis for a significant increase in egg production. All other groups inhibited gonadal development to varying degrees, with the urea group showing the most significant inhibition.

[0043] Table 3. Effects of different attractants on earthworm reproductive performance and hormone secretion.

[0044] 1.3 Summary Adding an appropriate amount of sodium citrate to earthworm farming using chicken manure can improve the growth rate and reproductive performance of earthworms.

[0045] Example 2: Processing of Earthworm Powder Because fresh earthworms are inconvenient to store and feed, they need to be dehydrated and crushed to make earthworm powder for easy use in animal feed. Currently, there are no systematic research reports on earthworm processing. Therefore, this project preliminarily studied the effects of different processing methods on the crude protein and amino acid content of earthworm powder, providing a scientific basis for the production of high-quality earthworm powder and the efficient utilization of earthworms.

[0046] Earthworms possess a "trigger enzyme" that, under suitable temperature and water conditions, is activated, causing the earthworm to undergo necropsy, resulting in a decrease in weight and changes in body composition. This directly affects the quality of earthworm protein feed. Therefore, this project investigated the effects of drying at 65℃, freeze-drying, and sun-drying on the crude protein and amino acid composition of earthworms.

[0047] Materials and Methods 2.1 Experimental Design and Materials Methods Experimental material: adult earthworms Equipment: Drying oven, freeze dryer, pulverizer Test method: Live earthworms are washed with warm water, disinfected with 0.02% potassium permanganate for 10-15 minutes, choked to death with wood ash, and then crushed to a particle size of 2-3 mm to increase the specific surface area and improve drying efficiency and nutrient retention.

[0048] Drying method: Spread the earthworm granules evenly on a tray, place them in a 65℃ forced-air drying oven, and dry until constant weight is achieved before crushing and testing.

[0049] Sun-drying method: Disperse earthworm particles on a clean cement surface and expose them to the sun to quickly dehydrate and dry them, then crush them for testing.

[0050] Freeze-drying method: Earthworm particles are crushed into a paste, pre-frozen at -45℃ for 12 h, and then vacuum-dried at -10℃ for 24 h before being tested.

[0051] The dried earthworm granules were crushed through an 80-mesh sieve and stored in a sealed container away from light.

[0052] Crude protein determination method: Kjeldahl method, GB / T 6432-2018.

[0053] Methods for the determination of amino acids: GB / T 18246-2019.

[0054] 2.2 Results and Analysis Table 4. Effects of different processing methods on crude protein and amino acid content in earthworms.

[0055] Based on comprehensive analysis of crude protein content and amino acid composition, freeze-drying retains the highest amount of crude protein and various amino acids from earthworms, especially limiting amino acids for poultry such as lysine, methionine, and tryptophan, resulting in the best nutritional quality. Drying has a moderate nutrient retention effect, while sun-drying is the worst. Therefore, freeze-drying is the most suitable processing method for producing high-quality earthworm protein feed.

[0056] Example 3: Safety Study of Earthworm Meal for Feeding 3.1 Experimental Materials and Methods 3.1.1 Test Drugs Freeze-dried earthworm powder.

[0057] 3.1.2 Instruments and Reagents The main instruments include hemocytometers, biological microscopes, push-button counters, fully automated biochemical analyzers, glass slides, centrifuge tubes, disposable syringes, and centrifuges. The main reagents include phosphate buffer, glacial acetic acid, Swiss stain, crystal violet stain, distilled water, physiological saline, neutral red stain, and other reagents used for blood biochemical testing and analysis.

[0058] 3.1.3 Experimental Animals One hundred 1-day-old Ross 308 broiler chickens were provided by the Poultry Research Institute of Shandong Academy of Agricultural Sciences. Oil lamps and incandescent lamps were used for warmth during the brooding period and the trial period.

[0059] 3.1.4 Criteria for inclusion and exclusion of experimental animals (1) Selection criteria Chickens used in target animal safety trials must be examined by veterinarians the day before the trial to confirm that they are in good health.

[0060] (2) Elimination criteria Chickens with the following conditions should be removed from the trial: chickens that are near death during the adaptation period; chickens that are sick or injured during the adaptation period.

[0061] 3.1.5 Experimental Design In the formal experiment, 80 healthy chicks were selected from approximately 100 eight-day-old chicks and then randomly divided into four groups based on their weight, ensuring that the weight of each group was as uniform as possible. The experimental chickens were divided into four groups of 20 each, and were fed complete feed with additions of 0, 25, 50, and 75 g / kg of earthworm meal, respectively. Wing numbers and cage numbers were used for identification.

[0062] Table 5. Grouping and dosage of experimental chickens

[0063] The experimental chickens were raised by designated personnel and fed regularly every day. The chickens had free access to food, and the amount of feed consumed by each group of chickens was recorded regularly.

[0064] 3.1.6 Test Methods Clinical observation: The experimental chickens were closely monitored and their reactions were recorded daily by the breeders. The recorded items were as follows: morbidity and mortality, and other possible adverse reactions. The weight of the experimental chickens and the feed consumption at each stage were measured before the experiment, during the experiment (0-21 days), and at the end of the experiment (21-42 days). Based on this, the average weight gain and feed utilization rate of each group of chickens at each stage were calculated.

[0065] Ocular pathological examination: All chickens that died during the experiment underwent necropsy. Deaths related to toxicity, disease, or other causes were confirmed using acceptable diagnostic techniques, and the date of death was recorded. Histological examination was performed on ocular lesions suspected to be related to earthworm powder.

[0066] Clinical pathological examination: During the middle of the experiment (day 21), blood samples were collected from 10 chickens in each group for complete blood count (CBC) and blood biochemistry tests. At the end of the experiment (day 42), blood samples were also collected from the remaining 10 chickens in each group for CBC and blood biochemistry tests. CBC analysis was performed using artificial blood cell differential counting, and blood biochemistry tests were performed using a fully automated biochemical analyzer.

[0067] 3.1.7 Data Statistical Analysis The significance of the data was tested using SPSS software. The t-test was used to compare the significant differences between the experimental group and the control group in terms of average weight gain, feed conversion ratio, hematological and blood biochemical indicators.

[0068] 3.2 Results and Analysis 3.2.1 General Clinical Observation Throughout the trial, all chickens in the control group and each dose group were in good health, with normal water and feed intake, and no clinical symptoms were observed. No chickens in any group died.

[0069] 3.2.2 Weight gain and feed utilization rate The average weight gain and feed utilization rate of chickens in each group at each stage are shown in Table 6. Statistical analysis showed that when the chickens were continuously fed until day 21 (0–21), there was no significant difference in average weight gain between the experimental groups and the control group (P > 0.05); however, by the end of the experiment (days 21–42), the average weight gain of experimental groups 2 and 3 was significantly or extremely significantly different from that of the control group (P < 0.05). Compared with the control group, the feed conversion ratio (FCR) of each experimental group decreased, and the feed utilization rate increased. Moreover, the FCR showed a significant decreasing trend with increasing dosage. This indicates that the effect of increasing the amount of additive on improving feed utilization is more significant.

[0070] Table 6 Production performance of broiler chickens in each experimental group

[0071] 3.2.3 Hematological parameters The hematological test results of 10 chickens randomly selected from each group at the mid-term and end of the experiment are shown in Tables 7 and 8, respectively. Statistical analysis showed that there were no significant differences between the dosage groups and the control group at the end of the experiment (P>0.05).

[0072] Table 7 Blood parameters of each group at the mid-term of the experiment

[0073] Table 8 Blood parameters of each group at the end of the experiment

[0074] 3.2.4 Blood biochemical parameters The blood biochemical test results of 10 chickens sampled from each group during the mid-term and end of the experiment are shown in Tables 9 and 10, respectively.

[0075] Table 9. Blood biochemical indicators of broilers on day 21 of the experiment

[0076] Table 10 Blood biochemical indicators of broilers after 42 days of experiment

[0077] 3.3 Summary The data collected in this experiment included body weight, health status, appetite, and hematological and biochemical parameters. Compared with the control group, there were no significant differences in health status, appetite, and hematological parameters among the experimental groups 1, 2, and 3. Although the blood urea nitrogen level increased slightly, the difference was not significant compared with the control group. In addition, a very few blood biochemical parameters (such as albumin / globulin ratio, glucose, and chloride content) in experimental groups 1 and 3 were significantly different from those in the control group, but there was no dose-related difference. There were no significant differences in hematological and blood biochemical parameters among the experimental groups; compared with the control group, the weight gain of broilers in all experimental groups was significantly improved, and feed utilization was increased.

[0078] In conclusion, earthworm meal, as a feed ingredient, has good safety profile. From a production performance perspective, experimental group 2 (with 50g / kg earthworm meal added to the diet) showed positive effects in increasing the average daily weight gain of broilers and reducing the feed conversion ratio.

[0079] Example 4: Feeding effect of earthworm meal replacing part of the soybean meal in broiler diets 4.1 Experimental Design and Materials Methods Experimental Design: 360 healthy 1-day-old Ross 308 broiler roosters were randomly divided into 4 groups: control group, fish meal group, earthworm meal group, and cockroach meal group. Each group had 6 replicates, with 15 roosters per replicate. The control group received a mixed meal basal diet, while the experimental groups received partial protein replacements with fish meal, earthworm meal, and cockroach meal, respectively. The nutritional levels of all groups were kept consistent. The diet formulation and nutritional levels are shown in Table 1.

[0080] Feeding and Management: Animal experiments were conducted at the Shandong Academy of Agricultural Sciences Poultry Research Institute experimental base. The experiment lasted 42 days, using three-tiered cages. During the first week of brooding, the chickens received 24 hours of light per day, decreasing by 1-2 hours each subsequent week. Throughout the experiment, the chickens had free access to feed and water.

[0081] Table 11 Experimental Diet Formulations and Nutritional Levels

[0082] Note: The crude protein content of soybean meal, fish meal, earthworm, cockroach meal, and earthworm castings is 46%, 68%, 61.73%, 76.08%, and 8%, respectively.

[0083] 4.2 Results and Analysis 4.2.1 Effects of earthworm meal replacing part of the protein on the production performance of large white-feathered broiler chickens Compared with the control group, fishmeal significantly reduced the average daily feed intake and average daily weight gain from day 1 to 21 and from day 22 to 42 (P<0.05); earthworm meal significantly increased the body weight of broilers at day 21 and 42, as well as the average daily weight gain from day 22 to 42 and from day 1 to 42 (P<0.05), and decreased the feed conversion ratio from day 1 to 42 (P<0.05); cockroach meal significantly increased the feed conversion ratio from day 1 to 21, from day 22 to 42, and from day 1 to 42 (P<0.05), and significantly decreased the average daily weight gain from day 1 to 21 (P<0.05). The results indicate that replacing part of the protein in the formulated diet with earthworm meal can promote the growth of broilers, with a growth-promoting effect particularly higher than that of fishmeal and cockroach meal. Therefore, it is a high-quality protein feed with high development potential.

[0084] Table 12. Effects of earthworm meal replacing part of the protein on broiler production performance.

[0085] 4.2.2 Effects of earthworm powder replacing part of the protein on the development of immune organs and serum immunoglobulin levels in broilers.

[0086] Table 13. Effects of earthworm meal replacing part of the protein on the immune organ indices of broilers.

[0087] As shown in Table 13, at 21 days of age, the bursa of Fabricius index of broilers in the earthworm meal group and cockroach meal group was significantly higher than that in the fish meal group (P < 0.05), while there was no significant difference between the earthworm meal group and the cockroach meal group (P > 0.05). The thymus index and spleen index also showed no significant differences among the groups (P > 0.05). At 42 days of age, there were no significant differences in the thymus index and spleen index among the groups (P > 0.05). The bursa of Fabricius index in the earthworm meal group was significantly higher than that in the fish meal group (P < 0.05), but there was no significant difference between the earthworm meal group and the control group and the cockroach meal group (P > 0.05). This indicates that earthworm meal is more effective than the generally accepted high-quality protein fish meal in promoting the development of immune organs, thus making it a good protein feed resource.

[0088] Table 14. Effects of earthworm powder replacing part of the protein on serum immunoglobulins in broiler chickens.

[0089] The results showed that dietary supplementation with cockroach meal significantly increased serum immunoglobulin A and immunoglobulin G levels in broilers at 21 days of age (P < 0.05); fish meal significantly decreased serum immunoglobulin M levels at 21 days of age (P < 0.05); both fish meal and cockroach meal decreased serum immunoglobulin A levels at 42 days of age (P < 0.05); and compared with cockroach meal, fish meal increased serum immunoglobulin M levels at 42 days of age (P < 0.05). Among the three animal proteins, earthworm meal had no negative impact on serum immunoglobulins and exhibited a superior growth-promoting effect on broilers compared to fish meal and cockroach meal.

[0090] 4.2.3 Effect of earthworm powder replacing part of the protein on digestive enzyme activity like Figure 1 As shown, compared with the control group, all three animal proteins significantly increased the activity of duodenal protease in 21-day-old broilers (P < 0.05). Dietary supplementation with fishmeal significantly increased the activities of duodenal amylase and ileal protease (P < 0.05); fishmeal and earthworm meal significantly increased the activities of jejunal protease, lipase, and ileal amylase (P < 0.05); fishmeal and cockroach meal significantly increased the activities of duodenal and ileal lipase (P < 0.05).

[0091] Compared with the control group, all three animal proteins increased the activities of jejunal protease and ileal lipase in 42-day-old broilers (P < 0.05). Dietary supplementation with fishmeal significantly increased the activities of duodenal protease and jejunal and ileal lipase (P < 0.05), with a greater effect on jejunal and ileal lipase activities than earthworm meal and cockroach meal (P < 0.05). Earthworm meal and fishmeal increased duodenal lipase activity (P < 0.05); earthworm meal increased duodenal and ileal amylase activity (P < 0.05); and earthworm meal and cockroach meal increased jejunal amylase activity (P < 0.05). These results indicate that replacing soybean meal protein with earthworm meal in broiler diets can significantly increase the activity of the three major digestive enzymes in the intestine, thereby improving the digestibility of carbohydrates, proteins, and fats, and thus promoting broiler growth.

[0092] 4.2.4 Effects of earthworm powder replacing part of the protein on the intestinal morphology of broiler chickens Table 15 Effects of earthworm powder replacing part of the protein on intestinal morphology in broiler chickens

[0093] The results showed that earthworm powder and cockroach powder reduced jejunal crypt depth in broilers at 21 days of age (P < 0.05), and all three animal proteins reduced ileal villus height and crypt depth (P < 0.05). Compared with fish meal and cockroach powder, earthworm powder increased the ileal-to-villus ratio (P < 0.05). Cockroach powder reduced duodenal crypt depth in broilers at 42 days of age (P < 0.05) and increased the duodenal-to-villus ratio, as well as ileal villus height and villus ratio (P < 0.05). Earthworm powder increased the jejunal-to-villus ratio (P < 0.05), while fish meal increased the duodenal-to-villus ratio and decreased the jejunal-to-villus ratio (P < 0.05). Compared with the three animal proteins, earthworm powder was more effective in promoting intestinal development, which created favorable conditions for nutrient absorption.

[0094] 4.2.5 Effect of earthworm powder replacing part of the protein on the cecal colony structure and composition of broiler chickens like Figure 2-3 As shown, earthworm meal significantly increased the α-diversity of cecal microbiota in 21-day-old broilers (P < 0.05); compared with the other three groups, fish meal significantly increased the α-diversity of cecal microbiota in 42-day-old broilers (P < 0.05). Beta diversity analysis results showed that both PCoA and PLS-DA indicated significant segregation of cecal microbiota in each experimental group in terms of community structure, indicating that different animal proteins have a significant impact on the diversity and composition of cecal microbiota.

[0095] like Figure 4-5 As shown, compared with the other three groups, earthworm powder significantly reduced the relative abundance of Proteobacteria and Actinobacteria in the cecum, as well as the relative abundance of Shigella and Faecalibacterium. At the genus level, it increased the relative abundance of Ruminococcus family UCG-014 and Lactobacillus, and significantly increased the relative abundance of Enterococcus in the cecum of 42-day-old broilers.

[0096] like Figure 6-7 As shown in the LEfSe analysis, compared with the control group, fish meal, earthworm meal, and cockroach meal all significantly increased the enrichment of beneficial bacteria such as rumenococci, lactobacilli, bifidobacteria, and butyric acid-producing bacteria in the cecum of broilers, while reducing the relative abundance of pathogenic bacteria such as Escherichia coli and streptococci. Earthworm meal, in particular, significantly increased the relative abundance of bifidobacteria. These results indicate that all three animal proteins promote the colonization of beneficial intestinal bacteria, with earthworm meal specifically targeting and enriching multiple beneficial bacteria to achieve comprehensive positive regulation of the intestinal microecology.

[0097] 4.3 Summary Replacing part of soybean meal protein with earthworm meal in broiler mixed meal basal diets can significantly improve broiler production performance, immune organ development, intestinal digestive enzyme activity and intestinal morphology, optimize the cecal microbiota, enrich beneficial bacteria such as Lactobacillus, Ruminococcus UCG-014, and Bifidobacterium, and reduce the abundance of opportunistic pathogens such as Escherichia coli and Shigella. Compared with fish meal and cockroach meal, earthworm meal is the most effective in increasing daily weight gain, reducing feed conversion ratio, and improving intestinal barrier and gut microbiota health.

[0098] Example 5: Comparison of feeding effects of adding different levels of earthworm meal to a mixed meal diet. 5.1 Experimental Design and Materials Methods Experimental Design: 450 healthy 1-day-old Ross 308 broiler roosters were randomly divided into 5 groups, with 6 replicates per group and 15 roosters per replicate. Soybean meal was replaced at appropriate levels in the mixed meal diet at 0%, 2.5%, 4.0%, 5.5%, and 7.0% of the roosters, respectively, while maintaining consistent nutrient levels across groups. The diet formulations and nutrient levels are shown in Table 1.

[0099] Feeding and Management: Animal experiments were conducted at the Shandong Academy of Agricultural Sciences Poultry Research Institute experimental base. The experiment lasted 42 days, using three-tiered cages. During the first week of brooding, the chickens received 24 hours of light per day, decreasing by 1-2 hours each subsequent week. Throughout the experiment, the chickens had free access to feed and water.

[0100] Table 16 Experimental Diet Formulations and Nutritional Levels

[0101] 5.2 Results and Analysis 5.2.1 Effects of adding different levels of earthworm meal to a mixed meal diet on broiler production performance The results showed that the broiler chickens in the 5.5% earthworm meal supplement group had significantly higher slaughter weights than the other groups (P<0.05) and significantly lower feed conversion ratios than the other groups (P<0.05). Although there were no significant differences in any indicators between the 7.0% earthworm meal group and the 5.5% group, the feed cost of the 7.0% group was higher than that of the 5.5% group. From an economic perspective, the 5.5% group was more advantageous.

[0102] Table 17 Effects of different levels of earthworm meal added to mixed meal diets on broiler production performance

[0103] 5.2.2 Effects of adding different levels of earthworm meal to a mixed meal diet on jejunal development in broilers The results showed that the jejunal villus height and villus-to-crypt ratio in the 5.5% earthworm powder group were significantly higher than those in the other groups (P<0.05), while the crypt depth was significantly lower (P<0.05). There were no significant differences in jejunal development indicators between the 7.0% earthworm powder group and the 5.5% group.

[0104] Table 18 Effects of different levels of earthworm meal added to mixed meal diets on jejunal development in broiler chickens

[0105] 5.2.3 Effects of adding different levels of earthworm meal to a mixed meal diet on the abundance of probiotics in the cecum of broiler chickens The results showed that the 5.5% earthworm powder supplementation group had the highest abundance of probiotics (P<0.05) and the lowest abundance of opportunistic pathogens (P<0.05) in the cecum of broilers, the healthiest microecological structure, and the strongest intestinal barrier; there was no significant improvement between the 7.0% group and the 5.5% group, showing a plateau effect.

[0106] Table 19 Effects of different levels of earthworm meal added to mixed meal diets on broiler production performance

[0107] 5.3 Summary The above results indicate that as the amount of freeze-dried earthworm powder added gradually increased from 0% to 5.5%, the 42-day body weight, average daily weight gain, chorionic villus ratio, and levels of Lactobacillus, Bifidobacterium, and Ruminococcus UCG in broilers were affected. The abundance of 014 bacteria showed a significant linear increase, while the feed conversion ratio and the abundance of pathogenic bacteria showed a significant linear decrease. When the addition amount was increased to 7.0%, the above indicators did not show further significant improvement, but the cost of feed raw materials increased.

[0108] Example 6 Comparison of feeding effects of different insect proteins 6.1 Experimental Design and Measurement Indicators This study compared the effects of adding different insect proteins to a corn-meal-meal basal diet on the growth performance, slaughter performance, and meat quality of broilers, and verified the feeding effect of earthworm meal. Three hundred and sixty healthy one-day-old Ross 308 broiler roosters were randomly divided into four groups. The control group was fed a corn-meal basal diet. The experimental groups had 5.5% earthworm meal, black soldier fly larvae meal, and yellow mealworm meal, respectively, replacing an appropriate amount of protein in the basal diet. Each group had six replicates, with 15 roosters per replicate. The experiment lasted for 42 days. The nutritional levels of crude protein, metabolizable energy, amino acids, calcium, and available phosphorus were completely consistent across all groups. Feeding management, lighting, and immunization programs were the same as in Example 3, and the nutritional levels of each group remained consistent.

[0109] 6.2 Test Results 6.2.1 Production performance Compared with the control group without insect protein, the addition of 5.5% insect protein improved the production performance of broilers to varying degrees (P<0.05). Among them, the earthworm powder group showed the best effect: the average daily weight gain was 18.66% higher than the control group, 11.30% higher than the black soldier fly powder group, and 8.56% higher than the yellow mealworm powder group; the feed conversion ratio was 1.26±0.01, which was significantly lower than the control group and other insect protein groups.

[0110] Table 20 Effects of different insect proteins on broiler production performance

[0111] 6.2.2 Jejunal development The earthworm powder group showed the highest jejunal villus height and villus-to-crypt ratio, the lowest crypt depth, and the most complete intestinal absorption structure.

[0112] Table 20 Effects of different insect proteins on the jejunum of 42-day-old broilers

[0113] 6.2.3 Abundance of Probiotics and Harmful Bacteria in the Gut The cecal microbiota results showed that the earthworm powder group contained Lactobacillus, Bifidobacterium, and UCG from the Ruminococcus family. 014 abundance was significantly highest, according to Escherichia coli. Shigella abundance was significantly lowest.

[0114] Table 21 Effects of different insect proteins on the abundance of probiotics and harmful bacteria in the cecum of 42-day-old broilers

[0115] 6.3 Summary The results show that, at the same addition level, freeze-dried earthworm meal is significantly better than black soldier fly meal, yellow mealworm meal, and the control group without insect protein, making it the highest quality insect protein resource for broiler feed.

[0116] Example 7: Comparison of feeding effects between a mixed meal diet containing earthworm meal and a corn-soybean meal basal diet. 7.1 Experimental Design and Measurement Indicators 7.1.1 Experimental Design This study compared the effects of a conventional corn-soybean meal basal diet and a mixed meal basal diet plus earthworm meal on the growth performance, slaughter performance, and meat quality of broilers, verifying whether earthworm meal could compensate for the nutritional deficiencies of mixed meal diets and achieve feeding effects comparable to traditional soybean meal diets. Two hundred and seventy healthy one-day-old Ross 308 broiler roosters were randomly divided into three groups: a control group (corn-soybean meal basal diet), experimental group 1 (mixed meal diet), and experimental group 2 (mixed meal diet containing earthworm meal). Each group had six replicates, with 15 broilers per replicate. The experiment lasted for 42 days, with feeding management, lighting, and immunization programs the same as in Example 3, and nutritional levels maintained consistently across groups. The diet formulation and nutritional levels are shown in Table 2.

[0117] 7.1.2 Measurement Indicators (1) Growth performance: average daily weight gain, average daily feed intake, feed conversion ratio; (2) Slaughter performance: 42-day dressing percentage, eviscerated portion, breast muscle percentage, leg muscle percentage, abdominal fat percentage; (3) Meat quality: pH (45min, 24h), drip loss, cooking loss, shear force, meat color (L*, a*, b*).

[0118] 7.2 Experimental Results and Analysis 7.2.1 Growth performance Compared with group 1, the average daily weight gain of broilers in group 2 was significantly higher, and the feed conversion ratio was significantly lower (P<0.05); the production performance of broilers in group 2 from day 1 to 42 was not significantly different from that of the control group (P>0.05). This indicates that earthworm meal can effectively compensate for the nutritional deficiencies of mixed meal diets, enabling them to achieve the same growth level as conventional corn-soybean meal diets.

[0119] Table 22 Effects of adding earthworm meal to mixed meal diets on broiler production performance

[0120] 7.2 Slaughter performance Compared with the control group and experimental group 2, experimental group 1 had significantly lower dressing percentage, eviscerated meat percentage, and breast muscle percentage (P<0.05), and higher abdominal fat percentage. Compared with experimental group 1, experimental group 2 had significantly higher dressing percentage, eviscerated meat percentage, and breast muscle percentage (P<0.05), while there was no significant difference compared with the control group (P>0.05). These results indicate that earthworm meal can improve the decline in slaughter performance caused by mixed meal diets and improve carcass quality.

[0121] Table 23 Effects of adding earthworm meal to mixed meal diets on broiler slaughter performance

[0122] 7.3 Meat quality Compared with the control group and experimental group 2, the drip loss and cooking loss of broiler breast muscle in experimental group 1 were significantly increased (P<0.05), the muscle shear force was higher, and the meat quality was worse. Compared with experimental group 1, the drip loss rate and cooking loss rate of broiler breast muscle in experimental group 2 were significantly reduced (P<0.05), the water retention capacity of the muscle was improved, and there were no significant differences in pH value, meat color, and tenderness with the control group (P>0.05).

[0123] Table 24 Effects of adding earthworm meal to a mixed meal diet on the quality of pectoral muscle

[0124] 7.4 Conclusions of Comparative Examples Simple mixed meal diets reduce broiler growth, slaughter performance, and meat quality, exhibiting significant nutritional deficiencies. Adding freeze-dried earthworm powder to mixed meal diets to replace part of the protein can significantly compensate for these deficiencies. The growth performance, slaughter performance, and meat quality of the mixed meal + earthworm powder group were comparable to those of the conventional corn-soybean meal group (P>0.05), and superior to the mixed meal control group. This demonstrates that earthworm powder can serve as a high-quality protein resource for reducing soybean meal intake, achieving the same feeding effect as traditional soybean meal diets in mixed meal diets.

[0125] Those skilled in the art to which this application pertains may modify or supplement the specific embodiments described or use similar methods to replace them, but without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.

Claims

1. A method for improving the intestinal health of broiler chickens using earthworm powder, characterized in that, Includes the following steps: (1) Earthworms were raised in a substrate formed by mixing fermented chicken manure and Chinese medicine residue at a mass ratio of 2:3, and 2000 ppm sodium citrate was added to the substrate. The substrate humidity was controlled at 50% to 55% and the temperature at 23 to 25℃. (2) After disinfection, the earthworms obtained in step (1) are prepared into earthworm powder by freeze drying. (3) In a mixed meal type broiler diet, replace part of the soybean meal with 5.0%-5.5% of the protein such as earthworm powder, and feed it to the broilers.

2. The method according to claim 1, characterized in that, In step (1), the Chinese herbal medicine residue includes: extract residues of Astragalus membranaceus, Codonopsis pilosula, Lycium barbarum and Taraxacum mongolicum, prepared in a mass ratio of 1:1:1:

1.

3. The method according to claim 1, characterized in that, In step (3), the mixed meal broiler diet includes corn, soybean meal, cottonseed meal, whole wheat flour and compound premix.

4. A feed for improving broiler production performance, gut health, and meat quality, characterized in that, The feed contains freeze-dried earthworm powder prepared by the method of claim 1 or 2, and the earthworm powder is added at a rate of 5.0%-5.5% in the mixed meal broiler diet to replace part of the soybean meal with other proteins.

5. The feed according to claim 4, characterized in that, The feed can optimize the cecal microbiota of broilers, specifically by increasing the abundance of Lactobacillus, Bifidobacterium, and Ruminococcus family UCG-014, and decreasing the abundance of Escherichia coli-Shigella.

6. The application of the method according to any one of claims 1-3 in reducing and replacing soybean meal in broiler mixed meal diets, improving broiler gut health, enhancing nutrient utilization, and improving broiler slaughter performance and meat quality.

7. The application of the feed according to any one of claims 4-5 in the 42-day full-life rearing of Ross 308 broiler chickens.

8. A composition of additives for earthworm farming, characterized in that, The substrate contains a mixture of fermented chicken manure and Chinese herbal medicine residue in a mass ratio of 2:3, and sodium citrate at a concentration of 2000 ppm. It is used to promote the growth and reproduction of earthworms under conditions of 50%–55% humidity and 23–25℃.

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