A traditional Chinese medicine composition and its application in the preparation of a drug for preventing Eimeria tenella coccidiosis in chickens and improving the growth performance of chicks.

Abstract

This invention belongs to the field of veterinary drug preparation and discloses a traditional Chinese medicine composition and its application in the preparation of drugs for preventing Eimeria tenella and improving the growth performance of chicks. The traditional Chinese medicine composition comprises the following components in parts by weight: 8-12 parts of Costus root, 8-12 parts of white hyacinth bean, 4-8 parts of immature bitter orange, 4-8 parts of peony root, 8-12 parts of milkweed root, 4-8 parts of cinnamon twig, 12-18 parts of astragalus root, 4-8 parts of ginger, and 4-8 parts of licorice root. This traditional Chinese medicine composition has the effects of warming yang and dispelling cold, strengthening the spleen and promoting diuresis, and regulating qi and relieving stagnation. It has a good preventive effect on Eimeria tenella and can improve the growth performance of chicks, without toxic side effects.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug preparation technology, and in particular relates to a traditional Chinese medicine composition and its application in the preparation of drugs for preventing Eimeria tenella coccidiosis in chickens and improving the growth performance of chicks. Background Technology

[0002] Coccidiosis in chickens is one of the most common diseases in poultry farming. It is primarily caused by Eimeria tenella and can affect chickens of all ages, with a higher infection rate and greater impact in chickens aged 15-30 days. In chicks, it causes extensive damage to the intestinal mucosa and secondary infections. Resistant chicks exhibit slow growth, poor development, and weakened immune function. Adult chickens infected with coccidiosis generally do not show obvious clinical symptoms, but it can affect weight and egg production, and carriers are prone to secondary infections. Among the seven currently recognized Eimeria species that can infect chickens, *Eimeria tenella* is the most common and most pathogenic strain. When parasitized in the cecum, it can cause hemorrhagic enteritis, bloody diarrhea, and other clinical symptoms, leading to death in severe cases.

[0003] For nearly half a century, drug-resistant strains of anticoccidial agents have been continuously isolated and reported, and many strains exhibit multidrug resistance and cross-resistance. If this continues, the control of coccidiosis will inevitably face a situation where no drugs are available. Therefore, efforts have been made to find new, highly effective, economical, and safe alternatives to anticoccidial agents.

[0004] Traditional Chinese medicine (TCM) is an important part of traditional medicine. The active ingredients in TCM are complex and exhibit multi-pathway and multi-target characteristics. Current research indicates that the anti-coccidial mechanisms of TCM active ingredients mainly focus on anti-inflammation, anti-oxidation, immune enhancement, regulation of intestinal flora, and inhibition of coccidia reproduction. Compared to single-herb TCM treatments for chicken coccidiosis, TCM formulas show more significant anti-coccidial effects. While playing a role in disease prevention and treatment, TCM formulas have advantages such as low risk of drug resistance, fewer toxic side effects, and improved immunity and growth performance. With the current trend of banning and restricting antibiotics, low-cost product-based competition is no longer sustainable. Functional, green, and healthy solutions are fundamental, and TCM preparations have become a major breakthrough in improving chicken growth performance, controlling coccidiosis, and alleviating drug resistance in coccidia. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine composition and its application in the preparation of drugs for preventing Eimeria tenella coccidiosis in chickens and improving the growth performance of chicks. The aim is to reduce or replace the addition of anticoccidial drugs in chicken feed, alleviate coccidiosis drug resistance in chickens, and contribute to the cause of "reducing and banning antibiotics" in livestock and poultry farming.

[0006] The technical solution provided by this invention is as follows:

[0007] A traditional Chinese medicine composition comprising the following components in parts by weight: 8-12 parts of costus root, 8-12 parts of white hyacinth bean, 4-8 parts of immature bitter orange, 4-8 parts of peony root, 8-12 parts of milk root, 4-8 parts of cinnamon twig, 12-18 parts of astragalus root, 4-8 parts of ginger, and 4-8 parts of licorice root.

[0008] Furthermore, the traditional Chinese medicine composition comprises the following components in parts by weight: 10 parts of costus root, 10 parts of white hyacinth bean, 6 parts of immature bitter orange, 6 parts of peony root, 10 parts of milk root, 6 parts of cinnamon twig, 15 parts of astragalus root, 6 parts of ginger, and 6 parts of licorice root.

[0009] The present invention also provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps:

[0010] Soak the raw materials in 2-4 times their volume of distilled water for 1-2 hours; boil and then simmer for 1.5-2.5 hours, then filter the liquid; add 2-4 times their volume of distilled water to the dregs and continue to simmer for 1.5-2.5 hours, then filter the liquid. Combine the two decoctions and concentrate them over a low flame, then filter to obtain an aqueous decoction.

[0011] The present invention also provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for preventing Eimeria tenella coccidiosis in chickens.

[0012] Furthermore, the application includes its use in the preparation of drugs to improve the survival rate and / or relative weight gain of chickens infected with Eimeria tenella;

[0013] Relative weight gain rate (%) = (final weight of each chicken in the group - initial weight of the chicken) / initial weight of the chicken × 100%.

[0014] Furthermore, the application includes its use in the preparation of drugs that reduce cecal lesions, cecal tissue damage, cecal tissue inflammation, and / or oxidative stress in chickens infected with Eimeria tenella.

[0015] The present invention also provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of drugs to improve the growth performance of chicks.

[0016] Furthermore, the application includes its use in the preparation of drugs that increase the height of chicken jejunal villi and / or ileal villi.

[0017] Furthermore, the application includes its use in the preparation of drugs that reduce the content of MDA in cecal tissue.

[0018] A traditional Chinese medicine composition for preventing Eimeria tenella in chickens is prepared by soaking 1000g of the composition in three times its volume of distilled water for 1.5 hours after boiling. The mixture is then simmered over low heat for 2 hours, and the liquid is filtered through an 80-mesh sieve. Three times its volume of distilled water is added to the dregs, and the mixture is simmered again for 2 hours, then filtered through an 80-mesh sieve. The two decoctions are combined and concentrated to 1000mL over low heat. After filtration, a 1g / mL aqueous decoction is obtained. The administration method is drinking water, and the dosage is 3g / kg.

[0019] The traditional Chinese medicine composition for treating Eimeria tenella in chickens consists of 10g of Aucklandia lappa, 10g of Dolichos lablab, 6g of Citrus aurantium, 6g of Paeonia lactiflora, 10g of Cassia fistula root, 6g of Cinnamomum cassia, 15g of Astragalus membranaceus, 6g of Zingiber officinale, and 6g of Glycyrrhiza uralensis. Cinnamomum cassia and Zingiber officinale have the effects of warming the meridians and promoting yang, relieving exterior syndromes and dispelling cold; Astragalus membranaceus has the effects of tonifying qi and raising yang, strengthening wei qi and consolidating the exterior; Dolichos lablab has the effects of strengthening the spleen and harmonizing the middle jiao; Cassia fistula root has the effects of stopping bleeding and removing dampness; Aucklandia lappa has the effects of promoting qi circulation and relieving pain; Citrus aurantium has the effects of breaking up qi stagnation, resolving phlegm, eliminating stagnation and bloating. Cinnamomum cassia, Astragalus membranaceus, and Zingiber officinale can enhance the overall immunity of the chicken flock and have the effects of warming the middle jiao and dispelling cold; Dolichos lablab and Cassia fistula root have the effects of strengthening the spleen and promoting diuresis; Aucklandia lappa and Citrus aurantium have the effects of promoting qi circulation and relieving stagnation.

[0020] Beneficial effects

[0021] The traditional Chinese medicine composition provided by this invention has the effects of warming yang and dispelling cold, strengthening the spleen and removing dampness, and promoting qi circulation and relieving stagnation. It has a good preventive effect on Eimeria tenella in chickens, can improve the growth performance of chicks, and has no toxic side effects.

[0022] The traditional Chinese medicine composition described in this invention can significantly inhibit inflammation and oxidative stress in the cecal tissue of E. tenella-infected chickens, reduce intestinal damage, and improve the relative weight gain and survival rate of infected chickens, thus exhibiting good preventive and therapeutic effects against E. tenella infection.

[0023] The traditional Chinese medicine composition described in this invention can significantly increase the length of jejunal and ileal villi, inhibit oxidative stress in cecal tissue, and improve the growth performance of chicks; at the same time, it has no obvious toxic side effects on the immune organs, liver, small intestine, and cecum of chicks. Attached Figure Description

[0024] Figure 1 This shows the survival rate of chickens in each experimental group;

[0025] Figure 2 This is a comparison of the body weight of chickens in each experimental group at 192 hours.

[0026] Note: Same letters indicate no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0027] Figure 3These are cecal lesions in each experimental group;

[0028] Figure 4 These are the spleen and liver indices of chickens in each experimental group;

[0029] Note: Same letters indicate no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0030] Figure 5 These are the HE staining results of the cecum of chickens in each experimental group;

[0031] Note: Red arrow: indicates coccidia oocyst; black arrow: indicates ruptured cecal villi; blue arrow: indicates vacuoles left by coccidia parasites.

[0032] Figure 6 These are the results of scanning electron microscopy observations of the cecum of chickens in each experimental group;

[0033] Note: Red arrows indicate coccidia oocysts; yellow arrows indicate damage to the intestinal villi structure.

[0034] Figure 7 The changes in the content of inflammation-related cytokines in the cecal tissue of chickens in each experimental group;

[0035] Note: Same letters indicate no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0036] Figure 8 The changes in redox indices in the cecal tissue of chickens in each experimental group;

[0037] Note: Same letters indicate no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0038] Figure 9 These are the results of changes in feed conversion ratio and body weight for chickens in each experimental group;

[0039] Figure 10 These are statistical results of some biochemical indicators in the serum of chickens from each experimental group;

[0040] Figure 11 These are the statistical results of the liver, spleen, thymus, and bursa of Fabricius indices of chickens in each experimental group;

[0041] Figure 12 These are the HE staining results of chicken liver tissue from each experimental group;

[0042] Figure 13 These are the HE staining results of chicken spleen tissue from each experimental group;

[0043] Figure 14 These are the HE staining results of chicken bursa tissue from each experimental group;

[0044] Figure 15 These are the HE staining results of the chicken duodenum from each experimental group;

[0045] Figure 16 These are the HE staining results of chicken jejunum from each experimental group;

[0046] Figure 17 These are the HE staining results of the chicken ileum from each experimental group;

[0047] Figure 18 These are the statistical results of the villus length, crypt depth, and villus-crypt ratio of the small intestine in each experimental group.

[0048] Figure 19 These are the PAS staining results of chicken cecal tissue from each experimental group;

[0049] Figure 20 These are the results of redox indices in the cecum of chickens in each experimental group; Note: *P<0.05.

[0050] Figure 21 The levels of inflammation-related cytokines in the cecal tissue of chickens in each experimental group were measured.

[0051] Note: Compared with the Control group, #P < 0.05. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0053] The traditional Chinese medicine preparation for preventing Eimeria tenella var. chrysalis provided in this embodiment of the invention consists of 10g of Costus root, 10g of white hyacinth bean, 6g of immature bitter orange, 6g of peony root, 10g of milk root, 6g of cinnamon twig, 15g of astragalus root, 6g of ginger, and 6g of licorice root.

[0054] The herbal composition of this invention has the effects of warming yang and dispelling cold, strengthening the spleen and removing dampness, and promoting qi circulation and relieving stagnation. It has a good preventive effect on Eimeria tenella in chickens, can improve the growth performance of chicks, and has no toxic side effects.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0056] Example 1

[0057] The preventive effects of different traditional Chinese medicine combinations on Eimeria tenella infection in chickens

[0058] 1. Method

[0059] 1.1 Laboratory Animals

[0060] One-day-old male yellow-feathered broiler chickens were selected and housed in the experimental animal facility of the College of Veterinary Medicine, Yangzhou University. The rearing environment was thoroughly cleaned, sprayed with ammonia, fumigated with potassium permanganate, ventilated, and then heat-treated with an alcohol flame. Cages, feed troughs, and water troughs underwent the same heat treatment to ensure the absence of coccidia and other influencing factors in the rearing environment. The chickens were provided with ample water and basic feed, with free access to feed. Drinking water was distilled water that had been autoclaved at 121℃ for 20 minutes. Feed samples were taken and examined under a microscope; no coccidia oocysts were found. A 40-watt light bulb was used for illumination throughout the rearing process, and the chickens were raised to 14 days old for experimental use. Fecal samples were collected from the flock for three consecutive days and tested for coccidia oocysts.

[0061] The feed formula should be prepared according to Table 1, referencing the chicken nutritional standards.

[0062] Table 1. Composition and nutrient levels of basal diet (air-dried basal diet)

[0063]

[0064] Note: Each 1 kg of premix provides: VA 1.25 × 10⁵ IU, VD3 5 × 10⁴ IU, VE 625 IU, VK3 50 mg, VB1 50 mg.

[0065] Vitamin B2 100mg, Vitamin B6 75mg, Vitamin B1 12 0.25mg, nicotinamide 750mg, pantothenic acid 250mg, folic acid 15mg, biotin 3.75mg, choline 11g, copper 0.03g, iron 0.87g, manganese 1g, zinc 1g, iodine 5mg, selenium 5mg, methionine 5%, calcium 15%, total phosphorus 5%, sodium chloride 8%, moisture ≤10%.

[0066] 1.2 Experimental insect strains

[0067] The *Eimeria tenella* strain used was the Nantong isolate NT6-7, which was isolated and preserved by the Department of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Yangzhou University. Previous studies have shown that this strain is completely resistant to diclazuril, chlorpyrifos, and decaoxyquin, and is a multidrug-resistant strain.

[0068] Before use, the insect strains were prepared at a ratio of 5×10 4Inoculate 14-day-old chicks with sporulated oocysts to rejuvenate them. On the 7th day post-infection, collect fresh feces, dilute them with 5 times the amount of tap water, and filter twice, first through an 80-mesh sieve and then through a 120-mesh sieve. Let the filtrate settle at room temperature for 3 hours, discard the supernatant, and then place it in a petri dish containing 2.5% potassium dichromate solution. Incubate at 29°C with aeration until the sporulation rate reaches 95% or higher. Store at 4°C for up to 30 days.

[0069] 1.3 Composition of Traditional Chinese Medicine Compounds and Preparation of Decoction

[0070] Traditional Chinese medicine composition A consists of 10g of Costus root, 10g of white hyacinth bean, 15g of Astragalus root, 6g of immature bitter orange, and 6g of licorice root, and has the effects of strengthening the spleen and replenishing qi, promoting qi circulation and relieving stagnation. Traditional Chinese medicine composition B contains 6g of cinnamon twig, 6g of peony root, 10g of milkweed root, 6g of ginger, and 6g of licorice root, and has the effects of harmonizing the ying and wei (nutritive and defensive qi), nourishing yin and promoting fluid production. Traditional Chinese medicine composition A+B, combining the above-mentioned herbs, has the effects of warming yang and dispelling cold, strengthening the spleen and promoting diuresis, and promoting qi circulation and relieving stagnation.

[0071] Weigh out 1000g each of the Chinese herbal composition A, B, and A+B according to the specified proportions, add 3 times the volume of distilled water and soak for 1.5 hours, boil and then simmer for 2 hours. Filter the decoction using an 80-mesh sieve. Add 3 times the volume of distilled water to the dregs and continue to simmer for 2 hours. Filter the decoction using an 80-mesh sieve. Combine the two decoctions and concentrate them to 1000ml over a low heat. Filter the solution to obtain a 1g / ml aqueous decoction.

[0072] 1.4 Experimental Grouping

[0073] Sixty 14-day-old experimental chickens of similar weight were randomly divided into four groups: a control group, a model group, a group receiving traditional Chinese medicine (TCM) composition A (TCM-A), a group receiving TCM composition B (TCM-B), and a group receiving TCM composition A+B (TCM-A+B), with 12 chickens in each group. The chickens were weighed at 15 days of age as their initial weight, and on the same day, they were orally administered 5 × 10 sporulated oocysts. 4 Each animal was tested for oocysts. Feces were collected 120–192 hours post-infection for oocyst counting. The animals were weighed at 23 days of age to determine their final weight, and the cecum was harvested to record intestinal lesion scores. Administration methods are shown in Table 2; the dosage was 3 g / kg body weight, and administration was via free access to drinking water.

[0074] Table 2 Grouping and treatment of experimental animals

[0075]

[0076] 1.5 Evaluation of the anticoccidioidal efficacy of traditional Chinese medicine compositions

[0077] After 192 hours of infection with Eimeria tenella, relative weight gain, survival rate, lesion count, and oocyst count were calculated using the method of Merk, Inc. (USA). The anticoccidial efficacy of the drug was evaluated by comprehensively considering the number of oocysts per gram of feces, weight gain, lesion score, and mortality rate of chickens in the experimental and control groups.

[0078] Anticoccidial Index (ACI) = (Relative weight gain rate + Survival rate) - (Lesion value + Oocyst value)

[0079] According to the anticoccidial index (ACI) value, an ACI ≥ 180 is considered highly effective, 160 ≤ ACI < 180 is considered moderately effective, 120 ≤ ACI < 160 is considered poorly effective, and ACI < 120 is considered ineffective.

[0080] 2 Experimental Results

[0081] 2.1 Anticoccidial index of each experimental group

[0082] As shown in Table 3, compared with the Control group, the Model group showed increased oocyst and lesion values, and decreased ACI, survival rate, and relative weight gain. Compared with the Model group, the ACI of the TCM-A, TCM-B, and TCM-A+B groups was significantly increased, while the oocyst and lesion values ​​were significantly decreased. The relative weight gain and ACI index of the TCM-A+B group were higher than those of the TCM-A and TCM-B groups, while the mortality rate, oocyst and lesion values ​​were lower than those of the TCM-A and TCM-B groups. This indicates that the TCM composition A+B group had the best anticoccidial effect, and the TCM composition was used for subsequent experiments.

[0083] Table 3. Anticoccidial index of each experimental group

[0084]

[0085] 3. Summary

[0086] The anticoccidial index of the traditional Chinese medicine composition A+B was higher than that of traditional Chinese medicine compositions A and B. Furthermore, traditional Chinese medicine composition A+B improved the survival rate and relative weight gain of the experimental chickens, and reduced clinical lesions. Its therapeutic effect was superior to that of traditional Chinese medicine compositions A and B. Therefore, traditional Chinese medicine composition A+B was used for subsequent experiments.

[0087] Example 2

[0088] The preventive effect of traditional Chinese medicine composition on Eimeria tenella infection in chickens

[0089] 1. Method

[0090] 1.1 Laboratory Animals

[0091] This experiment used 1-day-old male yellow-feathered broiler chickens purchased from the Poultry Research Institute of the Chinese Academy of Agricultural Sciences [Production License: Agricultural Chicken Su K050901; Usage License: SYXK(Su)2017-0044]. The experimental animals were housed in the experimental animal facility of the Veterinary College of Yangzhou University. The rearing environment was thoroughly cleaned, sprayed with ammonia, fumigated with potassium permanganate, ventilated, and then heat-treated with an alcohol flame. Cages, feed troughs, and water troughs underwent the same heat treatment to ensure the absence of coccidia in the rearing environment and the absence of other influencing factors. The chickens were provided with ample water and basic feed, with free access to food. Drinking water was distilled water that had been autoclaved at 121℃ for 20 minutes. Feed samples were taken and examined under a microscope; no coccidia oocysts were found. A 40-watt bulb was used for lighting during rearing, and the chickens were raised to 14 days old for experimental use. Fecal samples were collected from the flock for three consecutive days for examination; no coccidia oocysts were found.

[0092] All procedures in this experiment complied with the "Guiding Opinions on the Humane Treatment of Laboratory Animals" issued by the Ministry of Science and Technology of the People's Republic of China in 2006, and there were no violations of animal welfare. All animal experiments were conducted under the guidance of the Animal Protection and Utilization Committee of Yangzhou University. All experimental animal ethics were approved by the Experimental Animal Ethics Committee of Yangzhou University (No. 202103-009). The feed formulation was prepared according to Table 4, referencing the nutritional standards for chickens.

[0093] Table 4. Composition and nutrient levels of basal diet (air-dried basal diet)

[0094]

[0095] Note: Each 1 kg of premix provides: VA 1.25 × 10⁵ IU, VD3 5 × 10⁴ IU, VE 625 IU, VK3 50 mg, VB1 50 mg.

[0096] Vitamin B2 100mg, Vitamin B6 75mg, Vitamin B1 12 0.25mg, nicotinamide 750mg, pantothenic acid 250mg, folic acid 15mg, biotin 3.75mg, choline 11g, copper 0.03g, iron 0.87g, manganese 1g, zinc 1g, iodine 5mg, selenium 5mg, methionine 5%, calcium 15%, total phosphorus 5%, sodium chloride 8%, moisture ≤10%.

[0097] 1.2 Experimental insect strains

[0098] The *Eimeria tenella* strain used was the Nantong isolate NT6-7, which was isolated and preserved by the Department of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Yangzhou University. Previous studies have shown that this strain is completely resistant to diclazuril, chlorpyrifos, and decaoxyquin, and is a multidrug-resistant strain.

[0099] Before use, the insect strains were prepared at a ratio of 5×10 4Inoculate 14-day-old chicks with sporulated oocysts to rejuvenate them. On the 7th day post-infection, collect fresh feces, dilute them with 5 times the amount of tap water, and filter twice, first through an 80-mesh sieve and then through a 120-mesh sieve. Let the filtrate settle at room temperature for 3 hours, discard the supernatant, and then place it in a petri dish containing 2.5% potassium dichromate solution. Incubate at 29°C with aeration until the sporulation rate reaches 95% or higher. Store at 4°C for up to 30 days.

[0100] 1.3 Preparation of decoction of traditional Chinese medicine composition

[0101] Weigh 1000g of the herbal composition according to the specified ratio, add 3 times the volume of distilled water and soak for 1.5 hours. After boiling, simmer for 2 hours and filter the liquid using an 80-mesh sieve. Add 3 times the volume of distilled water to the dregs and continue to simmer for 2 hours. Filter the liquid using an 80-mesh sieve. Combine the two decoctions and concentrate them to 1000ml over a low heat. After filtration, prepare a 1g / ml decoction.

[0102] 1.4 Experimental Grouping

[0103] Seventy-two 14-day-old chickens of similar weight were randomly divided into four groups: a control group (Control), a model group (Model), a TCM-P prevention group, a TCM-P / T prevention group, a diclazuril prevention group (DIC-P), and a TCM-only group (TCM only), with 12 chickens in each group. The chickens were weighed at 15 days of age as their initial weight, and on the same day, they were orally administered 5 × 10⁵ sporulated oocysts. 4 Each animal was counted for oocysts by collecting feces 120–192 hours post-infection. At 23 days of age, the animals were weighed as their final weight, and the cecum was harvested to record intestinal lesion scores. Administration methods are shown in Table 5; the dosage of the traditional Chinese medicine was 3 g / kg body weight, and the dosage of diclazuril was 1 mg / kg body weight, both administered via free access to drinking water.

[0104] Table 5 Grouping and Treatment of Experimental Animals

[0105]

[0106] 1.5 Survival rate and weight gain

[0107] At 192 hours after infection, the survival rate of each group of experimental chickens was recorded. The survival rate (%) of each group was calculated as: (number of surviving chickens in the group / total number of experimental chickens in the group) × 100%.

[0108] Record the weight of the chicken flock, with the initial weight of chicks at 14 days of age and the final weight at 23 days of age, and calculate the weight gain index.

[0109] Relative weight gain rate (%) = (Final weight of each chicken in the group - Initial weight of the chicken) / Initial weight of the chicken × 100%

[0110] 1.6 Scoring and Lesion Values ​​of Chicken Cecal Lesions

[0111] At 192 hours post-infection, chickens in each experimental group were euthanized and their cecums were collected. After observing the external lesions, the cecums were cut open to observe the lesions inside the intestines. The lesion score was calculated for the cecum on the side with the most severe lesions, and the scoring criteria are shown in Table 6.

[0112] Lesion score = average score of cecal lesions within the group × 10.

[0113] Table 6. Scoring Criteria for Cecal Lesions

[0114]

[0115] 1.7 Evaluation of the anticoccidioidal efficacy of traditional Chinese medicine compositions

[0116] After 192 hours of infection with Eimeria tenella, relative weight gain, survival rate, lesion count, and oocyst count were calculated using the method of Merk, Inc. (USA). The anticoccidial efficacy of the drug was evaluated by comprehensively considering the number of oocysts per gram of feces, weight gain, lesion score, and mortality rate of chickens in the experimental and control groups.

[0117] Anticoccidial Index (ACI) = (Relative weight gain rate + Survival rate) - (Lesion value + Oocyst value)

[0118] According to the anticoccidial index (ACI) value, an ACI ≥ 180 is considered highly effective, 160 ≤ ACI < 180 is considered moderately effective, 120 ≤ ACI < 160 is considered poorly effective, and ACI < 120 is considered ineffective.

[0119] 1.8 Histological observation of chicken cecum tissue

[0120] Chickens infected with Eimeria tenella 192 hours prior to death were sacrificed in each experimental group, and their cecums were collected and the lesions were scored. One side of the cecum was fixed in 4% neutral formaldehyde and 2.5% glutaraldehyde for the preparation of routine HE staining sections and scanning electron microscopy samples, while the other side of the cecum was frozen at -80°C.

[0121] 1.9 Chicken organ index determination

[0122] At 192 hours after infection, six chickens with a weight close to the average weight were randomly selected from each experimental group. The pre-slaughter live weight of each chicken was measured. After euthanasia, the internal organs were quickly separated, and the spleen and liver were removed. Fat and connective tissue on the surface of the organs were removed, and the blood was absorbed with absorbent paper before weighing. The organ index was calculated.

[0123] Organ Index (%) = (Organ Weight / Live Body Weight) × 100%

[0124] 1.10 Determination of inflammatory factors and redox indices in chicken cecal tissue

[0125] After the experiment, six experimental chickens were randomly selected from each group, euthanized, and cecal tissue was collected, flash-frozen in liquid nitrogen, and then stored at -80°C.

[0126] Cecal tissue homogenate preparation: 100 mg of cecal tissue was divided into two equal portions. One portion was added to RIPA lysis buffer to prepare a 10% tissue homogenate for redox indices determination. The other portion was diluted with physiological saline to a 10% tissue homogenate for ELISA to determine inflammatory factor content. The levels of inflammatory factors TNF-α, IL-1β, and IL-10 in the cecal tissue of chickens in each experimental group were measured. Redox indices in the cecal tissue of chickens in each group were also measured: SOD activity, MDA content, and GSH-Px activity.

[0127] 1.11 Data Analysis

[0128] Statistical analysis was performed using IBM SPSS Statistics 17.0 software, and graphs were created using GraphpadPrism 9.5.1. All data are presented as mean ± standard deviation. This indicates that one-way ANOVA was used for comparisons between groups. The criterion for statistical significance was P < 0.05.

[0129] 2. Experimental Results:

[0130] 2.1 Traditional Chinese medicine composition improves the survival rate and relative weight gain rate of chickens infected with Eimeria tenella.

[0131] like Figure 1 As shown, at 192 hours after infection, the survival rate of the Model group was 75%, while the mortality rate of the other intervention groups was lower than that of the Model group. Compared with the Control group, the body weight of chickens in the Model group was significantly lower (P < 0.05); compared with the Model group, the body weight of chickens in the TCM-P, TCM-P / T, and DIC-P groups was significantly higher (P < 0.05); compared with the DIC-P group, the body weight of chickens in the TCM-P and TCM-P / T groups was significantly higher (P < 0.05). Figure 2 )

[0132] 2.2 Traditional Chinese medicine composition reduces cecal lesion score in chickens infected with Eimeria tenella.

[0133] like Figure 3 As shown, the cecum in the Model group was swollen, thickened, and shortened, with scattered bleeding points on its surface; the swelling and bleeding of the cecum were significantly reduced in each intervention group.

[0134] 2.3 Anticoccidial index of each experimental group

[0135] As shown in Table 7, compared with the Control group, the Model group showed increased follicle and lesion values, and decreased ACI, survival rate, and relative weight gain rate. Compared with the Model group, the ACI of the TCM-P and TCM-P / T groups was significantly increased, and both were higher than that of the DIC-P group. The follicle and lesion values ​​were significantly decreased compared with the Model group. The ACI of the TCM group was higher than that of the Control group. The results indicate that the prevention with the traditional Chinese medicine composition significantly reduced ACI, lesion values, and follicle values, and the overall effect was better than that of the DIC-P group.

[0136] Table 7. Anticoccidial index of each experimental group

[0137]

[0138] 2.4 Traditional Chinese medicine composition reduces spleen and liver indices in chickens infected with Eimeria tenella.

[0139] like Figure 4 As shown, compared with the Control group, the liver and spleen indices of chickens in the Model group were significantly increased (P < 0.05). Compared with the Model group, the liver and spleen indices of the TCM-P group, TCM-P / T group, and DIC-P group were all significantly lower than those of the Model group (P < 0.05). The TCM-P and TCM-P / T groups showed better results than the DIC-P group.

[0140] 2.5 Traditional Chinese medicine composition reduces cecal tissue damage in chickens infected with Eimeria tenella.

[0141] 2.5.1 HE staining results of chicken cecal tissue from each experimental group

[0142] like Figure 5 As shown, in the Model group, the cecal tissue was swollen, with a large number of inflammatory cells infiltrating and red blood cells exuding, and the intestinal villi were shortened; in the TCM-P group and the TCM-P / T group, the swelling of the cecal tissue of chickens was significantly reduced, the intestinal villi were relatively intact, and there were fewer inflammatory cells; in the DIC-P group, there were still a large number of inflammatory cells infiltrating and red blood cells exuding in the cecal tissue of chickens.

[0143] 2.5.2 Scanning electron microscopy results of cecum in chickens of each experimental group

[0144] like Figure 6 As shown, the cecal mucosa of chickens in the Control group was smooth and flat with intact microvilli, and no oocysts or merozoites were found. The cecal mucosa of chickens in the Model group was severely damaged, with a large number of oocysts present. The cecal mucosa of chickens in the TCM-P and TCM-P / T groups showed only local damage, and the intestinal villi were disordered due to coccidia parasitism, but no coccidia oocysts were found. The degree of cecal mucosal damage in the DIC-P group was more severe than that in the TCM-P and TCM-P / T groups.

[0145] 2.6 Traditional Chinese medicine composition reduces inflammatory response in chicken cecal tissue infected with Eimeria coccidia.

[0146] like Figure 7 As shown, compared with the Control group, the levels of IL-1β and TNF-α in the cecal tissue of chickens in the Model group were significantly increased (P<0.05), and the content of IL-10 was significantly decreased (P<0.05). Compared with the Model group, the contents of IL-1β and TNF-α in the cecal tissue of chickens in the TCM-P group, TCM-P / T group, and DIC-P group were all significantly decreased (P<0.05), and the content of IL-10 was significantly increased (P<0.05).

[0147] 2.7 Traditional Chinese medicine composition reduces oxidative stress in chicken cecal tissue infected with Eimeria tenella.

[0148] like Figure 8 As shown, compared with the Control group, the activities of SOD and GSH-Px in the cecal tissue of chickens in the Model group were significantly decreased (P<0.05), and the content of MDA was significantly increased (P<0.05). Compared with the Model group, the activities of SOD and GSH-Px in the cecal tissue of chickens in the TCM-P group, TCM-P / T group, and DIC-P group were all significantly increased (P<0.05), and the content of MDA was all significantly decreased (P<0.05).

[0149] 3. Summary

[0150] The traditional Chinese medicine composition can significantly reduce cecal damage caused by E. tenella infection in chickens, reduce the amount of oocysts excreted (P<0.01), reduce cecal mucosal damage caused by E. tenella infection, and improve the survival rate of infected chickens; its preventive and therapeutic effects are superior to diclazuril.

[0151] Example 2

[0152] Effects of Traditional Chinese Medicine Compositions on the Growth Performance of Chicks

[0153] 1. Method

[0154] 1.1 Laboratory Animals

[0155] This experiment used 1-day-old male yellow-feathered broiler chickens purchased from the Poultry Research Institute of the Chinese Academy of Agricultural Sciences [Production License: Agricultural Chicken Su K050901; Usage License: SYXK(Su)2017-0044]. The experimental animals were housed in the experimental animal facility of the Veterinary College of Yangzhou University. The rearing environment was thoroughly cleaned, sprayed with ammonia, fumigated with potassium permanganate, ventilated, and then heat-treated with an alcohol flame. Cages, feed troughs, and water troughs underwent the same heat treatment to ensure the absence of coccidia in the rearing environment and the absence of other influencing factors. The chickens were provided with ample water and basic feed, with free access to food. Drinking water was distilled water that had been autoclaved at 121℃ for 20 minutes. Feed samples were taken and examined under a microscope; no coccidia oocysts were found. A 40-watt bulb was used for lighting during rearing, and the chickens were raised to 14 days old for experimental use. Fecal samples were collected from the flock for three consecutive days for examination; no coccidia oocysts were found.

[0156] All procedures in this experiment complied with the "Guiding Opinions on the Humane Treatment of Laboratory Animals" issued by the Ministry of Science and Technology of the People's Republic of China in 2006, and there were no violations of animal welfare. All animal experiments were conducted under the guidance of the Animal Protection and Utilization Committee of Yangzhou University. All experimental animal ethics were approved by the Experimental Animal Ethics Committee of Yangzhou University (No. 202103-009). The feed formulation was prepared according to Table 8, referencing the nutritional standards for chickens.

[0157] Table 8. Composition and nutrient levels of basal diets (air-dried basal diet)

[0158]

[0159] Note: Each 1kg of premix provides: Vitamin A 1.25×10⁵ IU, Vitamin D3 5×10⁴ IU, Vitamin E 625 IU, Vitamin K3 50mg, Vitamin B1 50mg.

[0160] Vitamin B2 100mg, Vitamin B6 75mg, Vitamin B1 12 0.25mg, nicotinamide 750mg, pantothenic acid 250mg, folic acid 15mg, biotin 3.75mg, choline 11g, copper 0.03g, iron 0.87g, manganese 1g, zinc 1g, iodine 5mg, selenium 5mg, methionine 5%, calcium 15%, total phosphorus 5%, sodium chloride 8%, moisture ≤10%.

[0161] 1.2 Preparation of decoction of traditional Chinese medicine composition

[0162] Weigh 1000g of the herbal composition according to the specified ratio, add 3 times the volume of distilled water and soak for 1.5 hours. After boiling, simmer for 2 hours and filter the liquid using an 80-mesh sieve. Add 3 times the volume of distilled water to the dregs and continue to simmer for 2 hours. Filter the liquid using an 80-mesh sieve. Combine the two decoctions and concentrate them to 1000ml over a low heat. After filtration, prepare a 1g / ml decoction.

[0163] 1.3 Experimental Grouping

[0164] Eighty 14-day-old experimental chickens of similar weight were randomly divided into four groups: a blank control group (Control), a low-dose group (TCM-L), a medium-dose group (TCM-M), and a high-dose group (TCM-H), with 15 chickens in each group. The chickens were given their initial weight at 15 days of age, and medication was initiated on that day. They were weighed and their cecum was removed at 29 days of age. The TCM composition was administered according to the requirements in Table 9, with dosages of 1 g / kg, 3 g / kg, and 5 g / kg body weight, respectively, and access to drinking water was free.

[0165] Table 9 Grouping and Treatment of Experimental Animals

[0166]

[0167] 1.4 Statistics on the growth status of chickens in each experimental group

[0168] At the start of the experiment, the chickens' mental state, feed intake, and excretion were observed. During the experiment, the chickens had free access to feed, and the amount of uneaten feed and feed intake of each group was recorded twice a day, at 8:00 AM and 8:00 PM. Each group of chickens was weighed on an empty stomach at 3 days of age and again in the morning after the experiment. The initial weight at the start of the experiment was used as the initial weight, and the final weight at the end of the experiment was used as the final weight. Weight gain and feed conversion ratio were calculated.

[0169] Average weight gain = (Ending weight of each group - Beginning weight of each group) / Number of chickens in each group;

[0170] Feed conversion ratio = Average feed intake / Average daily weight gain

[0171] 1.5 Determination of serum biochemical indicators in chickens of each experimental group

[0172] On the morning of the last day of the experiment, fasting blood was collected from the heart to prepare serum. Creatinine (CREA), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in chicken serum were measured using a fully automated biochemical analyzer (Chemray-800).

[0173] 1.6 Determination of organ indices in chickens of each experimental group

[0174] At the end of the experiment, the pre-slaughter live weight of the experimental group chickens was measured on an empty stomach the following morning. Six chickens from each group were dissected, and the spleen, liver, bursa of Fabricius, and thymus were removed. Fat and connective tissue on the surface of the organs were removed, and the blood was absorbed with absorbent paper before weighing. Organ indices were calculated.

[0175] Organ Index (%) = (Organ Weight / Live Body Weight) × 100

[0176] 1.7 Histopathological observation of chicken organs in each experimental group

[0177] After the experiment, six chickens from each experimental group were weighed and euthanized. Samples were collected from the duodenum, jejunum, ileum, liver, spleen, and bursa of Fabricius. Routine HE staining sections were prepared, and PAS staining was prepared from the cecum.

[0178] 1.8 Determination of inflammatory factors and redox indices in chicken cecal tissue

[0179] Same as Example 11.12

[0180] 1.9 Data Analysis

[0181] Graphpad Prism 9.5.1 was used to create graphs and perform data analysis on the experimental data. All data are presented as mean ± standard deviation. This indicates that one-way ANOVA was used for comparisons between groups. The criterion for statistical significance was P < 0.05.

[0182] 2. Experimental Results:

[0183] 2.1 Traditional Chinese medicine composition improves chicken growth performance

[0184] like Figure 9 As shown, the growth rate of the TCM-H group was higher than that of the Control group (P < 0.05). One day after administration, the chicks in each experimental group were in good spirits, and there were no abnormal changes in feed intake and water intake. The shape and color of their feces were normal.

[0185] 2.2 Effects of Traditional Chinese Medicine Compositions on Some Biochemical Indicators in Chicken Serum of Each Experimental Group

[0186] The levels of some biochemical indicators in the serum of chicks in each experimental group are as follows: Figure 10 As shown, there were no significant differences in the levels of creatinine (CREA), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (P > 0.05).

[0187] 2.3 Effects of Traditional Chinese Medicine Compositions on Organ Indices in Chickens of Each Experimental Group

[0188] like Figure 11 As shown, there were no significant differences in spleen, thymus, bursa of Fabricius, and liver indices among the chickens in each experimental group (P > 0.05).

[0189] 2.4 Effects of Traditional Chinese Medicine Compositions on the Histopathology of Chicken Organs in Each Experimental Group

[0190] like Figure 12 , Figure 13 , Figure 14 As shown, no significant abnormalities were observed in the histopathological changes of the liver, spleen, and bursa of Fabricius in the three dosage groups: TCM-L, TCM-M, and TCM-H.

[0191] 2.5 Effects of Traditional Chinese Medicine Compositions on Chicken Intestinal Morphology

[0192] like Figure 15 , Figure 16 , Figure 17 As shown, no obvious abnormalities were found in the HE staining results of the duodenum, jejunum, and ileum of the Control group and the TCM-H group.

[0193] like Figure 18 As shown, the high-dose group of the traditional Chinese medicine composition significantly increased the height of jejunal villi (P < 0.01) and ileal villi (P < 0.05). The PAS staining results of the cecum in the TCM-L group, TCM-M group, TCM-H group, and Control group are shown below. Figure 19 As shown in the figure. The changes in glycogen and mucus components in cecal goblet cells of chicks after feeding them with three doses of the traditional Chinese medicine composition (low, medium, and high doses) were observed using PAS staining of the cecum. The results showed that the glycogen and mucus components in the cecal mucosa of the TCM-L, TCM-M, and TCM-H groups were normally distributed and orderly arranged, with no abnormalities observed.

[0194] 2.6 Effects of Traditional Chinese Medicine Compositions on Redox Indicators in Chicken Cecal Tissue

[0195] like Figure 20 As shown, there were no significant differences in SOD and GSH-Px levels between the TCM-L group, TCM-M group, TCM-H group and the Control group (P>0.05), while the MDA level in the TCM-H group was significantly lower (P<0.05).

[0196] 2.7 Inflammatory reaction of chicken cecal tissue after drinking water containing traditional Chinese medicine composition

[0197] like Figure 21As shown, compared with the Control group, the levels of IL-1β, IL-10 and TNF-α in the TCM-L group, TCM-M group and TCM-H group were not significantly different.

[0198] 3. Summary

[0199] The high-dose group of traditional Chinese medicine composition can significantly increase the length of villi in the jejunum and ileum of chickens, reduce the content of MDA in the cecal tissue, and improve the growth performance of chickens; at the same time, it has no abnormal effect on the structure, morphology and function of chicken liver, kidneys, immune organs and cecal tissue.

[0200] The traditional Chinese medicine composition can significantly reduce the amount of oocysts excreted in E. tenella-infected chickens, inhibit cecal tissue inflammation and oxidative stress, reduce intestinal damage, and improve the relative weight gain and survival rate of infected chickens, showing good preventive and therapeutic effects against E. tenella infection.

[0201] The high-dose group of traditional Chinese medicine composition can significantly increase the length of jejunal and ileal villi, inhibit oxidative stress in cecal tissue, and improve the growth performance of chicks; at the same time, it has no obvious toxic side effects on the immune organs, liver, small intestine, and cecum of chicks.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for the prevention of Eimeria tenella coccidiosis in chickens, characterized in that, The traditional Chinese medicine composition is made from traditional Chinese medicine composition A and traditional Chinese medicine composition B: traditional Chinese medicine composition A is made from 10 parts of costus root, 10 parts of white hyacinth bean, 15 parts of astragalus root, 6 parts of immature bitter orange, and 6 parts of licorice root; traditional Chinese medicine composition B is made from 6 parts of cinnamon twig, 6 parts of peony root, 10 parts of milk root, 6 parts of ginger, and 6 parts of licorice root.

2. The application according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition includes the following steps: Soak the raw materials in 2-4 times their volume of distilled water for 1-2 hours; boil and then simmer for 1.5-2.5 hours, then filter the liquid; add 2-4 times their volume of distilled water to the dregs and continue to simmer for 1.5-2.5 hours, then filter the liquid. Combine the two decoctions and concentrate them over a low flame, then filter to obtain an aqueous decoction.

3. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs to improve the survival rate and / or relative weight gain of chickens infected with Eimeria tenella; Relative weight gain rate (%) = (final weight of each chicken in the group - initial weight of the chicken) / initial weight of the chicken × 100%.

4. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs that reduce cecal lesions in chickens infected with Eimeria tenella.

5. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs that reduce cecal tissue damage in chickens infected with Eimeria tenella.

6. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs that reduce inflammation of the cecal tissue in chickens infected with Eimeria tenella.

7. The application according to claim 1, characterized in that, The applications include the use in the preparation of drugs to reduce oxidative stress in the cecal tissue of chickens infected with Eimeria tenella.

8. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs that reduce fecal oocyst values ​​in chickens infected with Eimeria tenella.

9. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs that increase the height of chicken jejunal villi and / or ileal villi.

10. The application according to claim 1, characterized in that, The application includes its use in the preparation of drugs that reduce the content of MDA in cecal tissue.

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