Application of antrodia camphorata polysaccharide in preparation of anti-LPS (low-cholesterol solution) stress feed for yellow-feather broilers

文档序号：175577 发布日期：2021-11-02 浏览：26次中文

阅读说明：本技术 牛樟芝多糖在制备黄羽肉鸡抗lps应激饲料中的应用 (Application of antrodia camphorata polysaccharide in preparation of anti-LPS (low-cholesterol solution) stress feed for yellow-feather broilers ) 是由蒋守群叶金玲林厦菁王一冰张盛于 2021-09-09 设计创作，主要内容包括：本发明公开了一种牛樟芝多糖在制备黄羽肉鸡抗LPS应激饲料中的应用。研究了牛樟芝多糖在抗氧化、改善肠道健康和生长性能的作用机制,拓宽了牛樟芝多糖的用途,为牛樟芝多糖在养殖业的应用奠定了基础。(The invention discloses an application of antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers. The research on the action mechanism of the antrodia camphorata polysaccharide in resisting oxidation and improving the intestinal health and the growth performance widens the application of the antrodia camphorata polysaccharide, and lays a foundation for the application of the antrodia camphorata polysaccharide in the breeding industry.)

1. Application of Antrodia Camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers is provided.

2. The application of the antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide improves the activities of T-SOD, GSH-Px and T-AOC in plasma and jejunal mucosa of the LPS stress yellow-feathered broilers; the content of MDA in plasma and jejunum mucosa of LPS stress yellow feather broiler chicken is reduced.

3. The application of the antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide reduces the expression levels of inflammatory factors TNF-alpha and IFN-r in plasma of the LPS stress yellow-feathered broilers; reduces the expression level of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the intestinal mucosa of LPS stress yellow-feather broilers.

4. The application of the antrodia camphorata polysaccharide in preparing the anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide increases the height and the hiding ratio of the villus in the jejunum of the LPS stress yellow-feathered broilers, and reduces the depth of the hidden nest and the thickness of the intestinal wall.

5. The application of the antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide reduces the mRNA expression abundance of genes TLR4, NF-kappa B, TNF-alpha and IL-1 beta related to the inflammatory response of the jejunal mucosa of the LPS stress yellow-feathered broilers; improves the mRNA expression abundance of LPS stress yellow feather broiler jejunum intestinal barrier related genes MUC2, Claudin-1, Occludin and ZO-1.

6. The use of Antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the Antrodia camphorata polysaccharide improves the liver index, pancreas index and bursa of Fabricius index of the LPS stress yellow-feathered broilers.

7. The application of the antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide improves the contents of T-SOD, GSH-Px and T-AOC in livers of the LPS stress yellow-feathered broilers; the MDA content in the liver of LPS stress yellow feather broiler is reduced.

8. The application of the antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide reduces the expression levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 of livers of the LPS stress yellow-feathered broilers.

9. The application of the antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide reduces the mRNA expression abundance of TLR4, MyD88 and NF-kB in livers of the LPS stress yellow-feathered broilers.

10. The application of the antrodia camphorata polysaccharide in preparing the anti-LPS stress feed for yellow-feathered broilers according to claim 1, wherein the antrodia camphorata polysaccharide reduces the mRNA expression abundance of COX2 and BAX in the livers of the LPS stress yellow-feathered broilers; the mRNA expression abundance of Nrf2 and SOD1 is improved.

Technical Field

The invention relates to the technical field of poultry breeding, in particular to application of antrodia camphorata polysaccharide in preparation of anti-LPS stress feed for yellow-feather broilers.

Background

Under normal physiological conditions, oxidation and antioxidation of broiler chicken bodies are always in dynamic balance, but a plurality of exogenous or endogenous stimuli can cause the bodies to be in an oxidative stress state, thereby causing serious influence on the healthy breeding of the broiler chickens. The intestinal tract is the most important digestive organ in the animal body and is the first organ suffering from oxidative damage in the animal body, and pathogenic escherichia coli can destroy intestinal microbial barriers and damage intestinal mucosa structures of chickens, so that oxidative stress is caused, and the intestinal health is influenced. Meanwhile, in the course of invasion by external pathogenic microorganisms, the liver is the main tissue organ for removing endotoxin and is more sensitive to oxidative stress than other organs. Many studies have also demonstrated that liver damage is often accompanied by oxidative stress, and that oxidative stress has a promoting effect on the development and progression of liver damage or liver disease. In recent years, researchers have made important progress by establishing an oxidative stress model to investigate the influence thereof, which is of great significance for the research of oxidative stress mechanisms and the development of antioxidant stress additives. Lipopolysaccharide (LPS), also known as bacterial endotoxin, is the outer layer structure of the lipid bilayer of the cell wall of gram-negative bacteria and is a good activator for the construction of inflammatory responses. After LPS is injected into the abdominal cavity or the vein of a plurality of researchers, the researchers find that the broiler chicken has acute inflammatory reaction, and the production of inflammatory cytokines and the expression of inflammatory key genes are promoted, so that the nutrient digestion and utilization are hindered, and finally the growth performance of the broiler chicken is reduced.

The polysaccharide is mainly a polysaccharide extracted from plants, fungi and the like, has various biological activities, can improve the immunity and the oxidation resistance level of organisms, has the effects of resisting tumors, inhibiting bacteria, regulating the intestinal health of livestock and poultry and the like, and has the characteristics of safety, low toxicity, difficult generation of drug resistance, no pollution and no residue. Antrodia cinnamomea (Antrodia cinnamomea) is a specific fungus variety in Taiwan, and is also named as Antrodia cinnamomea, Antrodia camphorata, blood ganoderma lucidum, etc. The polysaccharide is the main active ingredient of antrodia camphorata, the main structure of the polysaccharide is glucose, but the active polysaccharide is a D-glucan macromolecular structure. Although relatively many studies of polysaccharides have been reported in broiler chickens, the studies mainly focus on Astragalus Polysaccharides (APS), lycium barbarum polysaccharides (lycium barbarum) and algal polysaccharides (ADP), and the like, and the studies of Antrodia Camphorata Polysaccharides (ACP) have been reported less. Because the polysaccharide has the effects of improving the oxidation resistance level and bacteriostasis of organisms, can also adjust the intestinal health of livestock and poultry, and has the characteristics of safety, low toxicity, difficult generation of drug resistance and the like, if the antrodia camphorata polysaccharide is applied to the LPS stress resistance of yellow-feathered broilers, the oxidation resistance level of the yellow-feathered broilers can be improved, the intestinal health and the growth performance of the yellow-feathered broilers can be improved, and the antrodia camphorata polysaccharide is favorable for development and utilization in the breeding production of the yellow-feathered broilers.

Therefore, how to apply antrodia camphorata polysaccharide in the breeding production of yellow-feathered broilers is a problem which needs to be solved urgently by technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides an application of antrodia camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of Antrodia Camphorata polysaccharide in preparing anti-LPS stress feed for yellow-feathered broilers is provided.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide improves the activities of T-SOD, GSH-Px and T-AOC in plasma and jejunum mucosa of LPS stress yellow feather broilers; the content of MDA in plasma and jejunum mucosa of LPS stress yellow feather broiler chicken is reduced.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide reduces the expression level of inflammatory factors TNF-alpha and IFN-r in plasma of LPS stress yellow feather broilers; reduces the expression level of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the intestinal mucosa of LPS stress yellow-feather broilers.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide increases the height and the velvet hiding ratio of the empty intestine of the LPS stress yellow feather broiler, and reduces the depth of the hidden nest and the thickness of the intestinal wall.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide reduces the mRNA expression abundance of genes TLR4, NF-kappa B, TNF-alpha and IL-1 beta related to the inflammatory response of the jejunum mucosa of LPS stress yellow-feather broilers; improves the mRNA expression abundance of LPS stress yellow feather broiler jejunum intestinal barrier related genes MUC2, Claudin-1, Occludin and ZO-1.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide improves the liver index, the pancreas index and the bursa of fabricius index of the LPS stress yellow feather broiler.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide improves the contents of T-SOD, GSH-Px and T-AOC in the livers of LPS stress yellow feather broilers; the MDA content in the liver of LPS stress yellow feather broiler is reduced.

As the preferred technical scheme of the invention, the antrodia camphorata polysaccharide reduces the expression levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 of the liver of LPS stress yellow-feather broilers.

As a preferred technical scheme of the invention, the antrodia camphorata polysaccharide reduces the mRNA expression abundance of TLR4, MyD88 and NF-kB in the liver of LPS stress yellow-feather broiler.

As the preferable technical scheme of the invention, the antrodia camphorata polysaccharide reduces the mRNA expression abundance of COX2 and BAX in the liver of LPS stress yellow-feather broiler; the mRNA expression abundance of Nrf2 and SOD1 is improved.

According to the technical scheme, compared with the prior art, the invention discloses the application of antrodia camphorata polysaccharide in preparing the anti-LPS stress feed for yellow-feather broilers, and the antrodia camphorata polysaccharide can effectively relieve growth inhibition and intestinal oxidative damage induced by LPS; can improve the liver index and the activity of antioxidant enzyme, improve the antioxidant function of the liver, influence the mRNA expression abundance of key genes, and has better effect than antibiotics and euglena powder. The action mechanism of the polypeptide is probably similar to that of astragalus polysaccharide, and through inhibiting TLR 4/NF-kB expression induced by LPS, the polypeptide further inhibits the generation of inflammatory reaction, improves the oxidation resistance of organisms, enhances the barrier function of intestinal mucosa, and fully plays the functions of repairing inflammatory injury and promoting growth performance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the effect of ACP on the morphological structure of the jejunum of LPS-stressed yellow-feathered broilers in example 1;

FIG. 2 is a graph showing the effect of ACP on the abundance of mRNA expression of response genes to enteritis of LPS-stressed yellow-feathered broilers in example 1; a is a result graph of TLR4 gene expression abundance; b is a result graph of NF-kB gene expression abundance; c is a result graph of TNF-alpha gene expression abundance; d is a result graph of the expression abundance of the IL-1 beta gene;

FIG. 3 is a graph showing the effect of ACP on the abundance of mRNA expression of the barrier-associated gene in the jejunum of LPS-stressed yellow-feathered broilers in example 1; a is a result graph of MUC2 gene expression abundance; b is a result graph of the expression abundance of the Claudin-1 gene; c is a result graph of ZO-1 gene expression abundance; d is a result graph of the expression abundance of Occludin gene;

FIG. 4 is a graph showing the effect of ACP on the expression abundance of mRNA of TLR4, MyD88 and NF- κ B in the liver of LPS-stressed yellow-feathered broilers in example 2; a is a result graph of TLR4 gene expression abundance; b is a result graph of MyD88 gene expression abundance; c is a result graph of NF-kB gene expression abundance;

FIG. 5 is a graph showing the effect of ACP on the abundance of mRNA expression of anti-oxidant and anti-injury genes in livers of LPS-stressed yellow-feathered broilers in example 2; a is a result graph of COX2 gene expression abundance; b is a result graph of expression abundance of Nrf2 gene; c is a result graph of SOD1 gene expression abundance; d is a result graph of BAX gene expression abundance.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 Effect of Antrodia camphorata polysaccharide on growth Performance and integrity of jejunal mucous Membrane of yellow-feathered broilers

Materials used in the examples: LPS (055: B5, cat # L4005) was purchased from Sigma; antrodia camphorata polysaccharide (ACP, the active ingredient is D-glucan, the content is 76.3%) is provided by Taibei Jiashikashengmu GmbH of Taiwan; astragalus polysaccharides (APS, rich in Astragalus polysaccharides and Astragaloside IV, polysaccharide content of 45%) were purchased from Beijing Sheng Tai technology corporation; gymnodinium powder (ADP, active ingredient is beta-1, 3 glucan, content 45%) was purchased from Zhuhai Jianming science and technology Co., Ltd.

Test diets used in the examples

Corn-soybean meal type basal diet, diet was prepared according to the Chinese feed ingredient table (15 th edition), and the specific experimental diet formula and nutritional level are shown in Table 1. The nutrition level of the basic feed is scientifically prepared by referring to NY/T3645-containing 2020 yellow feather broiler nutrition requirement newly written by animal science research institute of Guangdong province academy of agricultural sciences. The nutrition level of each group of diet is kept consistent, and antibiotics and other additives are added according to different water levels to replace rice bran in an equal weight mode.

TABLE 1 basal diet composition and Nutrition level (feeding base)

Item	Content (wt.)
		Raw materials
Corn (corn)	60.50
		Bean pulp	31.5
Soybean oil	1.70
		L-lysine hydrochloride	0.16
DL-methionine	0.17
		Stone powder	1.22
Calcium hydrogen phosphate	1.93
		Salt	0.30
Unite bran	1.52
		Premix compound	1.00
Total up to	100.00
		Nutritional levels
Metabolic energy/(Kcal/kg)	2960
		Crude protein	21.50
Lysine	1.29
		Methionine	0.52
Methionine + cystine	0.93
		Threonine	0.86
Tryptophan	0.21
		Isoleucine	0.86
Calcium carbonate	1.00
		Non-phytate phosphorus	0.47

The premix provides VA 15000IU and VB for each kilogram of diet at the age of 1-21 days₁1.8 mg, nicotinic acid 35mg, VB₂3.6 mg，VB₆ 3.5 mg，VB₁₂ 0.01 mg，VD₃3300 IU, VE 10IU, VK 0.5mg, biotin 0.15mg, calcium pantothenate 10mg, folic acid 0.55mg, choline chloride 2600mg, Fe 80mg, Cu 8mg, Mn 80mg, Zn 60mg, I0.35 mg, Se 0.15 mg. 2) The nutrient levels are calculated values.

Test animals and groups

The test was carried out in the animal nutrition research laboratory at the institute of animal science, academy of agricultural sciences, Guangdong province. 1200 fast and large-sized 9-day old green south yellow-feathered broilers (female chicks) with good health conditions are selected, weighed one by one, and divided into 8 treatment groups (6 times for each treatment and 25 chickens for each treatment) according to the weight consistency principle:

blank control group, LPS stress group (LPS group), LPS stress +20mg/kg virginiamycin (LPS + antibiotic group), LPS stress +100mg/kg ACP (LPS +100mg/kg ACP), LPS stress +200mg/kg ACP (LPS +200mg/kg ACP), LPS stress +400mg/kg ACP (LPS +400mg/kg ACP), LPS stress +400mg/kg APS (LPS +400mg/kg APS), LPS stress +200mg/kg ADP (LPS +200mg/kg ADP). At the age of 18 days and 20 days, the placebo group was intraperitoneally administered with 0.50mL of physiological saline, and the other treatment groups were intraperitoneally administered with 0.50mL of LPS (500. mu.g/kg body weight), respectively, for 21 days.

Feeding management

The test chickens are raised in a closed henhouse flatly, wood chips are paved on the ground, granular materials and drinking water are fed freely, feeding management and environmental conditions of all groups are consistent, and feeding and immunization are carried out according to a conventional operation procedure and an immunization flow. During the test, the illumination in the house is manually controlled, the illumination is kept for 23h every day, and the test is naturally ventilated. The chicken house temperatures and relative humidities were recorded at 08:00, 14:00, 20:00 daily.

Measurement of growth Performance

In the test process, the health condition of the chickens is observed every day, and once dead chickens appear, the dead chickens are weighed and the residual feed amount is weighed immediately so as to eliminate the influence of the dead chickens on the test result. The number of dead chickens is recorded, and the reason should be immediately found and appropriate measures should be taken. Feeding the test chickens at 19:00 days before the beginning and ending of the test, feeding water, weighing 07:00 days in the next day by taking repetition as a unit, counting the feed consumption, and calculating the average daily feed intake, the average daily gain, the feed-weight ratio and the survival rate; the results are shown in Table 2;

TABLE 2 Effect of ACP on growth Performance of LPS-stressed yellow-feathered broilers

Note: the same row of shoulder marks have significant difference (P < 0.05) in different letters, and the same letter or no letter has insignificant difference (P > 0.05); the following table is the same.

As can be seen from Table 2, LPS significantly reduced FBW, ADFI and ADG (P < 0.05) of fast and large yellow-feathered broilers compared to the blank control group, while the different polysaccharides had no significant effect on the growth performance of yellow-feathered broilers (P > 0.05). Compared with LPS, other treatments all significantly improved ADFI (P < 0.05) of yellow broilers, and ADP significantly improved ADG (P < 0.05) of yellow broilers. Compared with antibiotics, different polysaccharides had no significant effect on FBW, ADFI and ADG of yellow broilers (P > 0.05). In addition, the growth performance of the yellow-feathered broilers is not obviously different (P > 0.05) compared among different polysaccharide groups.

Sample collection and index detection

(1) Routine index detection

At the age of 30 days, repeatedly selecting 2 chicken wings with approximate average body weight for blood sampling, collecting 5mL of blood in an anticoagulation tube added with heparin sodium, centrifuging at 3500r/min for 10min, and taking supernatant. After slaughtering, the jejunum is taken out, the content is washed by precooled normal saline, longitudinally cut open, scraped to remove the inner mucous membrane and put into a 1.50mL centrifuge tube for preservation at-80 ℃. Conventionally preparing jejunum tissue homogenate, adding 9 times volume of precooled physiological saline according to the volume ratio, performing low-temperature centrifugation (4 ℃, 8000r/min, 10min) after tissue homogenate, and taking supernatant. The total protein concentration in the supernatant was determined using a BCA protein quantification kit (kaiky, Nanjing). Measuring the activity of total superoxide dismutase (T-SOD) in blood plasma by using a xanthine oxidase method; measuring the activity of plasma glutathione peroxidase (GSH-Px) by a dithio-dinitrobenzoic acid (DT-NB) colorimetric method; measuring total antioxidant capacity (T-AOC) of blood plasma by an iron (Fe) redox method; the TBA method is used for measuring the content of Malondialdehyde (MDA) in blood plasma, and the kit is purchased from Nanjing to build a bioengineering institute. A radioimmunoassay is adopted to measure tumor necrosis factor-alpha (TNF-alpha), interferon (IFN-r), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and interleukin-10 (IL-10) in the supernatant, and the required kit is purchased from the Beijing northern biotechnological research institute. The indexes are read on a multifunctional microplate reader (Spectramax M-5, molecular devices, USA), and the specific detection method and result calculation of each index are carried out according to the specification. The influence of ACP on the oxidation resistance of the plasma and the jejunum mucosa of yellow-feathered broilers under the LPS oxidative stress state is shown in a table 3; the influence of ACP on LPS stress yellow-feathered broiler plasma and jejunal mucosa inflammatory factors is shown in Table 4;

TABLE 3 Effect of ACP on the antioxidant capacity of LPS-stressed yellow-feathered broiler plasma and jejunal mucosa

As can be seen from Table 3, compared with the blank control group, the LPS significantly reduces the T-AOC activity (P is less than 0.05) in the plasma of the fast-growing yellow-feathered broilers, and significantly improves the MDA content (P is less than 0.05) in the plasma and the jejunal mucosa; antibiotics and different polysaccharides have no significant influence on the activity of T-SOD and T-AOC in plasma and jejunum mucosa of yellow-feathered broilers (P is more than 0.05); ACP (200mg/kg and 400mg/kg) and APS remarkably improve GSH-Px activity (P is less than 0.05) in plasma and jejunal mucosa of yellow-feathered broilers, but have no remarkable influence on T-AOC activity and MDA content (P is more than 0.05). Compared with LPS, ACP (200mg/kg and 400mg/kg) remarkably improves the activity of T-SOD, GSH-Px and T-AOC (P is less than 0.05) in the plasma and jejunum mucosa of yellow-feathered broilers; APS significantly improves the activity of T-SOD, GSH-Px and T-AOC in plasma (P is less than 0.05) and the activity of GSH-Px and T-AOC in jejunal mucosa; different polysaccharides significantly reduced the amount of MDA in plasma and jejunal mucosa (P < 0.05). Compared with antibiotics, ACP (200mg/kg and 400mg/kg) and APS remarkably improve GSH-Px activity (P is less than 0.05) in plasma and jejunum mucosa of yellow-feathered broilers; different polysaccharides significantly reduced the amount of MDA in plasma (P < 0.05); ADP significantly reduced the amount of MDA in plasma and significantly increased GSH-Px activity in the jejunal mucosa (P < 0.05). There was no significant difference in T-SOD activity, T-AOC activity and MDA content in plasma and jejunal mucosa (P > 0.05) between the ACP group and APS group.

TABLE 4 Effect of ACP on LPS-stressed yellow-feathered broiler plasma and inflammatory factors of jejunal mucosa

As can be seen from Table 4, compared with the blank control group, the expression of inflammatory factors TNF-alpha and IFN-r in plasma is remarkably improved by LPS (P is less than 0.05), the expression of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in jejunal mucosa is remarkably improved (P is less than 0.05), and the expression of anti-inflammatory factor IL-10 in jejunal mucosa is remarkably reduced (P is less than 0.05). Compared with LPS, the antibiotic, ACP and APS remarkably reduce the expression of inflammatory factors TNF-alpha and IFN-r in plasma (P < 0.05), the ACP and APS remarkably reduce the expression of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in jejunal mucous membrane (P < 0.05), but only 100mg/kg of ACP remarkably improves the expression of anti-inflammatory factor IL-10 in mucous membrane (P < 0.05). Compared with antibiotics, 400mg/kg ACP significantly reduced the expression of the inflammatory factor TNF-alpha in the jejunal mucosa (P < 0.05). There was no significant difference in the expression of inflammatory factors in plasma and jejunal mucosa between the different ACP groups (P > 0.05), nor was there a significant difference in the expression of inflammatory factors in plasma and jejunal mucosa between the 400mg/kg ACP group, APS group and ADP group 3 groups (P > 0.05).

(2) Jejunal tissue morphology detection

And (3) after each group takes out jejunum, selecting 1-2 cm of intestinal middle section, washing with normal saline, putting the washed intestinal section into 4% paraformaldehyde prepared in advance, storing overnight, performing conventional paraffin embedding, and performing dyeing treatment by adopting hematoxylin-eosin (HE) to prepare paraffin sections. Each set of sections was photographed at 40 Xfield using a Motic-BA210 Digital microscope. When taking a picture, the tissues are filled in the whole visual field as much as possible, and the background light of each picture is ensured to be consistent. Using Image-Pro Plus 6.0 software and using a 40 x ruler as a standard, selecting 5 most complete villi from each slice, respectively measuring the height (mum) of the villi, the depth (mum) of the crypt and the thickness (mum) of the intestinal wall, and calculating the ratio of the villi to the crypt; the results are shown in FIG. 1 and Table 5;

as can be seen from the figure 1, the jejunum mucous membrane of the yellow-feathered broilers of the basic ration group with the age of 30 days is complete in structure, clear in layer, orderly in arrangement of intestinal villi, and clear in boundary between villi and crypts. Compared with a blank control group, the yellow-feather broiler of an LPS stress group of 30-day age has the advantages that the intestinal wall thickness is increased, the structure of the mucous membrane of the jejunum is disordered and loosened, intestinal villi is shortened and damaged, epithelial cells of the intestinal villi are necrotic and shed, crypts are increased and are hypertrophied, and the situation that the LPS possibly influences intestinal stem cells located at the crypt positions and further influences cell proliferation and differentiation of the crypt positions is suggested, so that the villus structure is damaged finally. Compared to the LPS group and the antibiotic group, the different ACP group showed increased jejunal villus height, decreased crypt depth, and thinned intestinal walls, and its intestinal villus was more dense and intact. The structural integrity of the jejunal mucosa was poor in the ADP group relative to the ACP and APS groups.

TABLE 5 Effect of ACP on LPS-stressed yellow-feathered broiler jejunum tissue morphology

As can be seen from Table 5, compared with the blank control group, the height of the villus in the jejunum of the LPS stress group is remarkably reduced (P is less than 0.05), the depth of the crypt is remarkably increased (P is less than 0.05), the crypt ratio is remarkably reduced (P is less than 0.05), and the thickness of the intestinal wall is remarkably thickened (P is less than 0.05); the jejunum villus height, crypt depth and crypt ratio of the ACP group and APS group were not significantly different (P > 0.05). Compared with LPS, the antibiotic group has no significant change in both the villus height and the villus-cryptic ratio of the jejunum (P > 0.05), but has significantly reduced crypt depth (P < 0.05) and significantly reduced intestinal wall thickness (P < 0.05); the heights of the villi of the jejunum of the ACP group and the APS group are obviously increased (P is less than 0.05), the depth of the crypt is obviously reduced (P is less than 0.05), the cryptic ratio is obviously increased (P is less than 0.05), and the thickness of the intestinal wall is also obviously reduced (P is less than 0.05); the jejunal villus height of the ADP group did not change significantly (P > 0.05), crypt depth was significantly reduced (P < 0.05), crypt ratio was significantly increased (P < 0.05), and intestinal wall thickness was also significantly reduced (P < 0.05). Compared with antibiotics, the heights of the villi of the jejunum of the ACP group and the APS group are not obviously changed (P is more than 0.05), the depth of the crypt is obviously reduced (P is less than 0.05), the crypt ratio is obviously increased (P is less than 0.05), and the thickness of the intestinal wall is also obviously reduced (P is less than 0.05). Relative to the ADP group, there was no significant change in jejunal villus height (P > 0.05), a significant decrease in crypt depth (P < 0.05), a significant increase in crypt ratio (P < 0.05), and a significant decrease in intestinal wall thickness (P < 0.05) for the 100mg/kg ACP group; the intestinal wall thickness was significantly reduced (P < 0.05) for the 100mg/kg ACP group relative to the 200mg/kg ACP, APS and ADP groups; the villus height, crypt depth and villus hiding ratio of the ACP group and APS group were all not significantly changed (P > 0.05).

(3) Detection of jejunal structural integrity-related gene expression

The jejunum section is cleaned, cut into two sections and put into a 1.50mL centrifuge tube for preservation at-80 ℃. The total RNA extraction was performed according to the Trizol kit instructions, and the total concentration and purity of the RNA were determined using a Nano-Drop-2000 micro nucleic acid analyzer. Reverse transcription of RNA was performed strictly according to the kit instructions. The real-time fluorescent quantitative PCR (qRT-PCR) reaction (Bio-Rad CFX System) was a 20.0. mu.L System: iTaq Universal SYBR Green Supermix (Bio-Rad USA) 10.0. mu.L, upstream and downstream primers (10. mu. mol/L) each 1.0. mu.L, cDNA template 2.0. mu.L (10-fold dilution), ddH₂O6.0 μ L, 3 replicate wells per sample. The sequence of the gene to be tested is obtained from GenBank, a conservative region is taken to design a primer, and a housekeeping gene beta-actin gene is selected as an internal reference gene. The primers were synthesized by Shanghai Biometrics Ltd, and the sequences of the primers are shown in Table 6, and 2 was used^-ΔΔCtCalculating the relative mRNA expression amount of the target gene; the influence results of ACP on the response of LPS stress yellow-feathered broiler chicken to enteritis and the expression abundance of key genes mRNA of intestinal barriers are shown in a figure 2 and a figure 3;

as can be seen from FIG. 2, compared with the blank control group, the mRNA expression abundance (P < 0.05) of the yellow-feathered broilers, namely TLR4, NF-kappa B, TNF-alpha and IL-1 beta, is remarkably improved by LPS. ACP and APS significantly reduced mRNA expression abundance of TLR4, NF-. kappa.B and IL-1. beta. (P < 0.05) compared to LPS and antibiotics, but had no significant effect on mRNA expression abundance of TNF-. alpha. (P > 0.05). Compared with antibiotics, ADP has no significant effect on the mRNA expression abundance of the jejunal TLR4, TNF-alpha and IL-1 beta (P > 0.05), but significantly reduces the mRNA expression abundance of NF-kappa B (P < 0.05). Compared with a blank control group, ACP and APS have no significant influence on the mRNA expression abundance of yellow-feathered broiler jejunum TLR4, NF-kappa B, TNF-alpha and IL-1 beta (P is more than 0.05); compared between the ACP group and the APS group, the mRNA expression abundance of the yellow-feathered broilers, namely the TLR4, NF-kappa B, TNF-alpha and IL-1 beta, is not significantly influenced (P is more than 0.05).

As can be seen from FIG. 3, LPS significantly reduced the abundance of the expression of the mRNA of the yellow-feathered broilers, i.e., the jejunum MUC2, Claudin-1, ZO-1 and Occludin (P < 0.05), as compared with the blank control group. Compared with LPS, the antibiotics have no significant effect on the expression abundance of MUC2, Claudin-1, ZO-1 and Occludin mRNA (P > 0.05); different ACPs remarkably improve the expression abundance of MUC2, Claudin-1, ZO-1 and Occludin mRNA (P is less than 0.05); APS remarkably improves the expression abundance of MUC2, Claudin-1 and Occludin mRNA (P is less than 0.05); ADP significantly increased the expression abundance of Claudin-1mRNA (P < 0.05). Compared with antibiotics, ACP (200mg/kg and 400mg/kg) remarkably improves the expression abundance of MUC2, Claudin-1 and Occludin mRNA (P is less than 0.05); APS and ADP only significantly increased the Claudin-1mRNA expression abundance (P < 0.05). However, compared to the blank control group, the different ACPs and APSs had no significant effect on the abundance of MUC2, ZO-1, and Occludin mRNA expression (P > 0.05); only 200mg/kg ACP obviously improves the expression abundance of Claudin-1mRNA (P is less than 0.05); ADP had no significant effect on Claudin-1, ZO-1 and Occludin mRNA expression abundance (P > 0.05). However, the different ACP groups and APS groups did not have significant effect on the abundance of expression of mRNA from the yellow broiler jejunum MUC2, Claudin-1, ZO-1 and Occludin (P > 0.05) compared among the groups.

TABLE 6 qRT-PCR primer sequences

Example 2 Effect of Antrodia Camphorata polysaccharide on organ index of LPS-stressed yellow feather broiler and liver antioxidant ability

Test materials

LPS (055: B5, cat # L4005) was purchased from Sigma; antrodia camphorata polysaccharide (ACP, the active ingredient is D-glucan, the content is 76.3%) is provided by Taibei Jiashikashengmu GmbH of Taiwan; astragalus polysaccharides (APS, rich in Astragalus polysaccharides and Astragaloside IV, polysaccharide content of 45%) were purchased from Beijing Sheng Tai technology corporation; gymnodinium powder (ADP, active ingredient is beta-1, 3 glucan, content 45%) was purchased from Zhuhai Jianming science and technology Co., Ltd.

The experimental animals and design were the same as in example 1.

The feeding management was the same as in example 1.

The experimental diets were as in table 1 of example 1.

Index measurement and method

Sample collection and index determination

Collection of samples

In the test process, the health condition of the chickens is observed every day, 2 broilers in each group are randomly selected at the age of 30 days and weighed and slaughtered, livers, pancreas, spleens, thymus and bursas of Fabricius are dissected out, surface connective tissues and fat are removed, then weighing is carried out, the percentage of the weight of each broilers in the weight of the carcasses is calculated, and each organ index is obtained. The calculation formula is as follows: organ index (%) - (organ weight/carcass weight) × 100. The results are shown in Table 7;

TABLE 7 Effect of ACP on organ index (mg/g) of LPS-stressed yellow-feathered broilers

As can be seen from Table 7, compared with the blank control group, LPS and antibiotics have no significant influence on the organ index (P is more than 0.05), and different ACPs significantly improve the liver index (P is less than 0.05); only ACP (200mg/kg and 400mg/kg) significantly increased the pancreatic index (P < 0.05), and APS and ADP had no significant effect on the visceral index (P > 0.05). Compared to LPS, different polysaccharides significantly increased liver and pancreatic indices (P < 0.05), but only different ACPs significantly increased bursal index (P < 0.05). Compared with antibiotics, ACP (200mg/kg and 400mg/kg) remarkably improves liver index and pancreas index (P < 0.05). The liver index and pancreas index of 400mg/kg ACP were significantly higher than those of APS and ADP (P < 0.05). However, the organ indexes of the yellow-feathered broilers were not significantly different between the 100mg/kg ACP group and the 200mg/kg ACP group, and between the 200mg/kg ACP group and the 400mg/kg ACP group (P > 0.05).

Another 0.50g liver was placed in a 1.50mL centrifuge tube, minced, and stored at-80 ℃ for testing the following relevant indexes.

Detection of antioxidant enzymes and inflammatory factors

Conventionally preparing liver tissue homogenate, adding 9 times volume of precooled physiological saline according to the volume ratio, performing low-temperature centrifugation (4 ℃, 8000r/min, 10min) after tissue homogenate, and taking supernatant. Measuring the activity of total superoxide dismutase (T-SOD) in liver by using xanthine oxidase; measuring glutathione peroxidase (GSH-Px) activity in liver by dithio-dinitrobenzoic acid (DT-NB) colorimetric method; measuring total antioxidant capacity (T-AOC) in liver by iron (Fe) redox method; the TBA method is used for determining the content of Malondialdehyde (MDA) in liver, and the kit is purchased from Nanjing to build a bioengineering institute. Determining the total protein concentration in the supernatant by using a BCA protein quantitative kit (Kaiky, Nanjing); a radioimmunoassay is adopted to measure tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) in the supernatant, and the required kit is purchased from the institute of biotechnology in North Beijing. The indexes are read on a multifunctional microplate reader (Spectra max M-5, molecular devices, USA), and the specific detection method and result calculation of each index are carried out according to the specification. The influence of ACP on the oxidation resistance indexes of the livers of LPS stress yellow-feathered broilers is shown in Table 8; the influence of ACP on inflammatory factors of livers of LPS-stressed yellow-feathered broilers is shown in Table 9;

TABLE 8 Effect of ACP on LPS-stressed yellow-feathered broiler liver antioxidant index

As can be seen from Table 8, compared with the blank control group, the LPS significantly reduced the GSH-Px activity in the liver of yellow-feathered broilers (P < 0.05), and significantly increased the MDA content (P < 0.05); other treatments had no significant effect on T-SOD activity (P > 0.05); the activity of GSH-Px in the liver of yellow-feathered broilers is obviously improved by 400mg/kg of ACP (P is less than 0.05); different ACPs remarkably improve the activity of T-AOC in the liver (P is less than 0.05); 400mg/kg ACP and APS significantly reduced the MDA content in the liver (P < 0.05). Compared with LPS, ACP (200mg/kg and 400mg/kg) obviously improves the activity of T-SOD (P is less than 0.05) in the liver of yellow-feathered broilers; the antibiotics and different polysaccharides can obviously improve the GSH-Px and T-AOC activity (P is less than 0.05) in the liver of the yellow-feathered broiler. Compared with LPS and antibiotics, different polysaccharides significantly reduce the MDA content in the liver (P < 0.05). The different ACP groups had no significant effect on T-SOD, GSH-Px and T-AOC activity in liver when compared between groups (P > 0.05).

TABLE 9 influence of ACP on LPS-stressed yellow-feathered broilers liver inflammatory factor (ng/g protien)

As can be seen from Table 9, LPS increased the expression of TNF-. alpha.IL-1. beta.and IL-6, which are liver inflammatory factors, compared to the blank control group, wherein significant differences were achieved between TNF-. alpha.and IL-1. beta. (P < 0.05). Compared with LPS, the different polysaccharides remarkably reduce the expression of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the liver of yellow-feathered broilers (P is less than 0.05). Compared with antibiotics, the different polysaccharides remarkably reduce the expression of inflammatory factors TNF-alpha and IL-1 beta in the liver of yellow-feathered broilers (P is less than 0.05); the expression of an inflammatory factor IL-6 in the liver of the yellow-feathered broilers is obviously reduced by 400mg/kg of ACP (P is less than 0.05). However, the different ACP groups had no significant effect on the expression of the liver inflammatory factors TNF-alpha and IL-1 beta (P > 0.05) compared among the groups.

Gene expression assay

The total RNA extraction was performed according to the Trizol kit instructions, and the total concentration and purity of the RNA were determined using a Nano-Drop-2000 micro nucleic acid analyzer. Reverse transcription of RNA was performed strictly according to the kit instructions. The real-time fluorescent quantitative PCR (qRT-PCR) reaction (Bio-Rad CFX System) was a 20.0. mu.L System: iTaq Universal SYBR Green Supermix (Bio-Rad USA) 10.0. mu.L, upstream and downstream primers (10. mu. mol/L) each 1.0. mu.L, cDNA template 2.0. mu.L (10-fold dilution), ddH₂O6.0 μ L, 3 replicate wells per sample. The sequence of the gene to be tested is obtained from GenBank, a conservative region is taken to design a primer, and a housekeeping gene beta-actin gene is selected as an internal reference gene. The primers were synthesized by Shanghai Biometrics Ltd, and the sequences of the primers are shown in Table 10 and 2^-ΔΔCtCalculating the relative mRNA expression amount of the target gene; the results are shown in FIGS. 4 and 5;

as can be seen from FIG. 4, compared with the blank control group, the mRNA expression abundance of TLR4, MyD88 and NF-kappa B is remarkably improved by LPS (P is less than 0.05), and the influence of LPS on the mRNA expression abundance of TLR4, MyD88 and NF-kappa B (P is less than 0.05) is effectively relieved by different polysaccharides. Wherein, different ACP has the best effect (P is less than 0.05). But had no significant effect on the mRNA expression abundance of TLR4, MyD88 and NF- κ B (P > 0.05) compared between the antibiotic and polysaccharide groups.

As can be seen in FIG. 5, compared with the blank control group, the mRNA expression abundance of liver COX2 and BAX is significantly increased by LPS (P < 0.05), and the mRNA expression abundance of liver Nrf2 and SOD1 is significantly reduced (P < 0.05). Compared with LPS, the antibiotics and different polysaccharides remarkably reduce the mRNA expression abundance of COX2 (P < 0.05), and remarkably improve the mRNA expression abundance of SOD1 (P < 0.05); the mRNA expression abundance of liver Nrf2 is remarkably improved by different polysaccharides (P is less than 0.05), and the mRNA expression abundance of liver BAX is remarkably reduced by antibiotics and different polysaccharides (except APS) (P is less than 0.05). Compared with antibiotics, different polysaccharides have no significant influence on the mRNA expression abundance of COX2, SOD1 and BAX, but significantly improve the mRNA expression abundance of liver Nrf2 (P < 0.05). ACP (200mg/kg and 400mg/kg) and ADP have significantly higher abundance of mRNA expression to liver Nrf2 than APS (P < 0.05); the mRNA expression abundance of different ACPs to the liver apoptosis gene BAX is significantly lower than that of the APS group. But the comparison among different ACP groups did not have significant effect on the abundance of mRNA expression of COX2, Nrf2, SOD1 and BAX in liver (P > 0.05).

TABLE 10 qRT-PCR primer sequences

The test data are subjected to one-way ANOVA in SPSS 17.0 software to perform one-way analysis of variance, and Duncan's multiple comparison is performed on the basis of significant difference, wherein the significant level is P < 0.05. The results are all expressed as mean ± standard error (mean ± s.e.).

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> animal science institute of academy of agricultural sciences of Guangdong province

Application of <120> antrodia camphorata polysaccharide in preparation of anti-LPS stress feed for yellow-feather broilers

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA