CN116138365A

CN116138365A - Laying hen feed additive with water extract of strawberry tea and application of laying hen feed additive

Info

Publication number: CN116138365A
Application number: CN202310110921.2A
Authority: CN
Inventors: 刘超; 王文茂; 陈佩劍; 黄鹏; 王照洁
Original assignee: Zhangjiajie Qiankun Berry Tea Engineering Technology Research Center; Hunan Qiankun Biotechnology Co ltd
Current assignee: Zhangjiajie Qiankun Berry Tea Engineering Technology Research Center; Hunan Qiankun Biotechnology Co ltd
Priority date: 2023-02-14
Filing date: 2023-02-14
Publication date: 2023-05-23

Abstract

The invention provides a laying hen feed additive with a water extract of strawberry tea and application thereof, wherein the laying hen feed additive with the water extract of strawberry tea comprises the water extract of strawberry tea, and the water extract of strawberry tea comprises the following components: the total flavone content is 50-60%, and the dihydromyricetin content in the total flavone is 25-35%; polysaccharide content is not less than 2%; the polyphenol content is 30-38%. The water extract of the strawberry tea is a natural plant additive, has the characteristics of low residue, no drug resistance, no pollution and the like, and is reasonably added into the laying hen breeding, and the application dosage of the water extract of the strawberry tea is 50-250mg per kg of basic daily ration of the laying hen; can replace antibiotics, effectively improve the productivity, egg quality and immunity of the laying hens, improve the intestinal health and the like, and further improve the food safety.

Description

Laying hen feed additive with water extract of strawberry tea and application of laying hen feed additive

Technical Field

The invention belongs to the technical field of laying hen feed additives, relates to a laying hen feed additive and application thereof, and in particular relates to a strawberry tea water extract laying hen feed additive capable of replacing antibiotics and application thereof.

Background

In recent years, with the increase of demands of people on livestock and poultry products such as meat, eggs, milk and the like, large-scale and intensive production is becoming more and more common, and a series of animal health problems are generated. At this time, antibiotics are widely studied and applied in the fields of animal nutrition and feed science, and the use of antibiotics as growth promoters and feed enhancers brings great benefits to livestock producers. However, antibiotics are also advantageous and disadvantageous in livestock production, and abuse of antibiotics leads to problems of development of resistant pathogens, reduced immunity of livestock, drug enrichment and the like, and can seriously cause animal poisoning and even death. Although most antibiotics added in daily ration of livestock and poultry can be discharged out of the body along with excreta such as urine, the antibiotics still have the food safety problems such as medicine residues and the like caused by partial residues in the body or animal products; the part discharged outside along with the type can also have harmful effects on air, soil, water and the like without the anti-treatment, thereby endangering the health of people and livestock. Therefore, searching for an effective substitute for antibiotics in the livestock and poultry industry has become an important research point.

The berry tea (Ampelopsis grossedentata hand-Mazz.) is also called Ampelopsis grossedentata, and is prepared from tender stem and leaf of Ampelopsis grossedentata of Ampelopsis of Vitaceae, and is a wild vine. Researches show that the strawberry tea contains rich flavonoid substances, and the main components of the blueberry tea comprise dihydromyricetin, myricetin, rutin, quercetin, luteolin and the like, and also contains high-content polysaccharide. The existing modern pharmacological researches show that the strawberry tea has remarkable pharmacological activity in the aspects of resisting tumor, resisting oxidation, inhibiting bacteria, resisting inflammation, easing pain, protecting liver, preventing cough, stopping phlegm, reducing blood sugar, reducing blood pressure, regulating lipid, enhancing immunity and the like. Such as:

the Chinese patent document with publication number of CN115025165A discloses application of a strawberry tea extract in preparing a medicament for resisting porcine epidemic diarrhea virus, and the blueberry tea extract can show extremely strong effect of resisting porcine epidemic diarrhea virus infection at the concentration of 50 mug/mL. The strawberry tea extract is an ethanol extract.

The Chinese patent document with publication number of CN109548940A discloses application of a strawberry tea extract in preparing health care products with the functions of resisting oxidation, enhancing immunity, reducing blood fat, protecting liver and the like. The strawberry tea extract is a precipitated crystal after fermentation.

However, the application of the strawberry tea extract, especially the strawberry tea aqueous extract, in the laying hen feed additive is not seen at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing the strawberry tea water extract laying hen feed additive and the application thereof, wherein the strawberry tea water extract laying hen feed additive comprises a certain dose of strawberry tea water extract, can replace antibiotics, can effectively improve the production performance, egg quality, immune function and the like of laying hens, and further improves the food safety.

In order to solve the technical problems, the invention adopts the following technical scheme:

in a first aspect, the invention provides a laying hen feed additive with a water extract of blueberry tea, which comprises the water extract of blueberry tea.

Further, in the water extract of the strawberry tea, the following components are added: the total flavone content is 50-60%, and the dihydromyricetin content in the total flavone is 25-35%; polysaccharide content is not less than 2%; the polyphenol content is 30-38%.

Preferably, in the water extract of the blueberry tea, the water extract of the blueberry tea comprises: the content of total flavonoids is 57.6%, and the content of dihydromyricetin in the total flavonoids is 29.4%; the polysaccharide content was 6.83% and the polyphenol content was 35.57%.

Further, the preparation method of the water extract of the strawberry tea comprises the following steps:

(1) Adding fresh leaves of the strawberry tea, adding the fresh leaves into an extraction tank in the same day, injecting water for circulation, and adding water according to a feed-liquid ratio of 1:5;

(2) Extracting: heating by steam, carrying out reflux extraction when the water extraction temperature is 80-85 ℃ and the extraction time is 1-1.5 hours, pumping the feed liquid into an extraction liquid storage tank, carrying out rough filtration by a 60-100 mesh filter bag when the feed liquid is hot, and then carrying out fine filtration;

(3) Concentrating: concentrating the filtrate under reduced pressure at 60-64deg.C and vacuum degree of-0.07-0.08 MPa to obtain concentrate with density of 1.12, and pumping into concentrate storage tank;

(4) Spray drying: pumping the concentrated solution into a pressure spray drying tower for instantaneous drying, wherein the air inlet temperature is 135-150 ℃, and the spray drying time of each batch is 1.2-1.5 h;

(5) Collecting the dry spray extract powder, and packaging for storage.

Further, the application dosage of the blueberry tea water extract is 50-250mg per kg of basic daily ration of the laying hen.

In a second aspect, the invention provides application of the water extract laying hen feed additive of the strawberry tea.

Further, the application is the application in preparing feed additives for improving the production performance of laying hens. Specifically, the production performance of the laying hen comprises an egg laying rate, an average egg weight, a malformed egg rate and a feed-egg ratio.

Further, the application is the application in preparing feed additives for improving the immunity of the laying hens. Specifically, the immunity of the laying hen comprises the steps of protecting immune organs of the laying hen, improving biochemical indexes of serum of the laying hen, improving antioxidant capacity of livers and the like.

More specifically, the immune organs of the layer chicken comprise liver, spleen, ovary, oviduct and intestinal tissues.

More specifically, the biochemical indexes of the layer serum comprise AST, ALT, ALP, blood phosphorus, blood calcium, ALB, blood sugar and IgA, igG, igM in the layer serum.

Further, the application is the application in preparing a feed additive for improving the egg quality of laying hens. Specifically, the egg quality of the laying hen comprises egg weight, egg shape index, yolk color, egg white height, eggshell strength, eggshell thickness, yolk index and yolk proportion.

Further, the application is the application in preparing feed additives with anti-inflammatory performance of laying hens.

Further, the anti-inflammatory properties include anti-salpingitis properties, anti-ovarian inflammation properties, anti-intestinal inflammation properties.

The invention has the beneficial effects that:

the water extract of the strawberry tea adopted by the invention is a water extract of natural plant strawberry tea, and comprises a large amount of effective active ingredients with higher concentration in the strawberry tea, mainly flavonoid substances and polysaccharide. The water extract of the strawberry tea is a natural plant additive, has the characteristics of low residue, no drug resistance, no pollution and the like, and can improve the production performance of the water extract of the strawberry tea while playing the role of preventing diseases. The plant strawberry tea water extract feed additive is reasonably added into the laying hen breeding, so that the feed antibiotics can be replaced, the production performance, egg quality, immune function and the like of the laying hen can be effectively improved, and the food safety is further improved.

The existing strawberry tea extract is mainly a strawberry tea alcohol extract and is mainly applied to the aspects of porcine epidemic diarrhea, antioxidation and liver protection, the application field of the strawberry tea can be further expanded, the strawberry tea water extract with different concentrations is added into laying hen feed, and the indexes such as the production performance, reproductive function and intestinal health of laying hens are detected, so that the prepared strawberry tea water extract can obviously improve the egg quality liver index, spleen index, and the phenotypic indexes such as ovary, oviduct and intestinal morphology, and then the QPCR experiment shows that the strawberry tea water extract can relieve oviduct inflammation and intestinal oxidation reaction through an NF- κB channel and an Nrf2/Keap1 channel, thereby improving the reproductive function and improving the intestinal health. In addition, the intestinal barrier function and short chain fatty acid content can be improved and the intestinal flora can be changed. In conclusion, the water extract of the strawberry tea can improve the health state of the laying hen from multiple angles, so that the economic benefit is improved.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a graph showing the effect of adding a water extract of strawberry tea on the morphology of each organ and tissue of a layer chicken, wherein: FIG. 1 (A) is a comparison of tissue morphology of fallopian tubes, ovaries, duodenum, jejunum and ileum; fig. 1 (B) -1 (D) are index controls of the duodenum, wherein: FIG. 1 (B) is a duodenal villus height control, FIG. 1 (C) is a duodenal crypt depth control, and FIG. 1 (D) is a duodenal villus height/crypt depth control; fig. 1 (E) -1 (G) are index controls of jejunum, wherein: FIG. 1 (E) is a jejunum villus height control, FIG. 1 (F) is a jejunum crypt depth control, and FIG. 1 (G) is a jejunum villus height/crypt depth control; fig. 1 (H) -fig. 1 (J) are index controls of the ileum, wherein: FIG. 1 (H) is ileal villus height control, FIG. 1 (I) is ileal crypt depth control, and FIG. 1 (J) is ileal villus height/crypt depth control; * P <0.05, P <0.01.

Fig. 2 is a graph showing the effect of adding the water extract of the tea in the embodiment of the invention on the serum immunity and inflammation index of the laying hen, wherein: FIG. 2 (A) is an interleukin 1 beta (IL-1 beta) control, FIG. 2 (B) is a tumor necrosis factor-alpha (TNF-alpha) control, FIG. 2 (C) is an interleukin 6 (IL-6) control, FIG. 2 (D) is an interleukin 2 (IL-2) control, and FIG. 2 (E) is an immunoglobulin A (ImmunoglobulinA, igA) control; fig. 2 (F) is an Immunoglobulin M (IgM) control, fig. 2 (G) is an Immunoglobulin G (IgG) control, fig. 2 (H) is a D-lactic acid (D-Lac) control, P <0.05, P <0.01.

Fig. 3 is a graph comparing the influence of the added water extract of the strawberry tea on the oxidation resistance index of the laying hen, wherein: FIG. 3 (A) is SOD control; fig. 3 (B) is a T-AOC control with P <0.01 and P <0.001.

Fig. 4 is a graph showing the effect of adding the water extract of the strawberry tea on the expression level of the related genes of the laying hens, wherein: fig. 4 (a) to 4 (G) are controls of the oviduct-related gene expression amount, wherein: FIG. 4 (A) is a control of TNF- α, FIG. 4 (B) is a control of IL-6, FIG. 4 (C) is a control of NF- κB, FIG. 4 (D) is a control of IL-4, FIG. 4 (E) is a control of IL-10, FIG. 4 (F) is a control of COX-2, and FIG. 4 (G) is a control of PTGEs; FIG. 4 (H) is a control of ovarian Bcl 2/Bax; FIGS. 4 (I) to 4 (P) are control of ileum-related gene expression levels, wherein: fig. 4 (I) is the control of occilidin, fig. 4 (J) is the control of Claudin-1, fig. 4 (K) is the control of ZO-1, fig. 4 (L) is the control of MCU2, fig. 4 (M) is the control of Nrf2, fig. 4 (N) is the control of Keap-1, fig. 4 (O) is the control of NQO1, fig. 4 (P) is the control of HO-1, P <0.05, P <0.01.

Fig. 5 is a graph showing the effect of adding an aqueous extract of tea of a strawberry in the embodiment of the invention on short-chain fatty acid in the intestinal tract of the hindgut of a layer chicken, wherein: fig. 5 (a) is a control of acetic acid content, fig. 5 (B) is a control of propionic acid content, fig. 5 (C) is a control of isobutyric acid content, fig. 5 (D) is a control of butyric acid content, fig. 5 (E) is a control of isovaleric acid content, and fig. 5 (F) is a control of valeric acid content; * P <0.05.

Fig. 6 is a graph showing the effect of adding an aqueous extract of tea of a strawberry in accordance with the embodiment of the present invention on microorganisms in the intestinal tract of a layer chicken, wherein: FIG. 6 (A) is a control of the foregut flora alpha diversity index and beta diversity index; FIG. 6 (B) is a control of the alpha diversity index and beta diversity index of the hindgut flora; * P <0.05.

Detailed Description

The invention is further described below with reference to examples and figures, which are not intended to limit the scope of the invention.

1. Preparation of water extract of strawberry tea

(1) Adding fresh leaves of the strawberry tea, adding the fresh leaves into an extraction tank in the same day, injecting water for circulation, adding about 500kg of material into each tank, and adding tap water according to a feed-liquid ratio of 1:5;

(3) Concentrating: concentrating the filtrate under reduced pressure under the conditions of vacuum degree of-0.07 MPa to-0.08 MPa and temperature of 60-64 ℃, specifically depending on factory equipment, pumping the concentrated solution with density of about 1.12 into a concentrated solution storage tank;

(5) Collecting, subpackaging and preserving: collecting the extract powder obtained by drying and spraying, packaging into polyethylene food bags, and storing in a cardboard barrel.

And (3) detecting: the obtained water extract of tea (Waterextract of A. Grossedentata, hereinafter abbreviated as WEA) comprises: the total flavone content is 50-60%, and the dihydromyricetin content in the total flavone is 25-35%; polysaccharide content is not less than 2%; the polyphenol content is 30-38%.

Preferably, in the water extract of the strawberry tea: the content of total flavonoids is 57.6%, and the content of dihydromyricetin in the total flavonoids is 29.4%; the polysaccharide content was 6.83% and the polyphenol content was 35.57%.

2. Application of water extract of strawberry tea as feed additive for laying hens

In order to expand the application field of the water extract of the strawberry tea, the water extract of the strawberry tea prepared by adding different concentrations into the feed of the laying hens is further tested for indexes such as the production performance, the reproductive function and the intestinal health of the laying hens, so that the effect of the water extract of the strawberry tea applied to the feed of the laying hens is verified.

1 test materials

Test animals: powder chicken; test site: an country's laying hen farm in Changde city of Hunan province; the water extract of the strawberry tea comprises the following steps: the Water Extract (WEA) of the tea of the first part is prepared by the method.

2 test design

288 Beijing powder laying hens with similar laying rate and weight and age of 55 weeks are selected and randomly divided into 4 groups of 8 repeats each, and 9 chickens are in each repeat. The control group is fed with basic diet, and 50mg/kg, 150mg/kg and 250mg/kg of water extract of the strawberry tea are respectively added into the basic diet in the test I group, the test II group and the test III group. The pre-feeding period was 1 week, the positive feeding period was 8 weeks, and the specific groupings tested are shown in Table 1. The test basic ration is corn-soybean meal type and is prepared according to the nutrition requirement of laying hens in China (chicken raising Standard (NY/T33-2004)), and the composition formula and the nutrition level of the basic ration are shown in Table 2.

Table 1 test design and grouping

TABLE 2 basal ration composition and nutrient level (air dried basis)%

/>

a premix provides per kilogram of diet: fe 60mg,Cu 11mg,I 0.4mg,Zn 70mg,Mn 115mg,Se 0.30mg. VA,6000IU; VD3, 2500IU; VE,25.0mg; VK3,2.25mg; VB1,1.8mg; VB2,7.0mg; VB6,4.0mg; VB12,0.20mg; pantothenic acid,12.0mg; niacin,35.0mg; biotin,0.14mg; folic acid,0.8mg.

b the nutrition level is calculated

3 feeding management

All test laying hens are fed in a three-layer cage raising mode, 3 laying hens are separated by a partition board, the chickens are fed with food and drink water freely, the chickens are fed twice a day (6:30 and 14:30), eggs are picked up at regular time (15:00) every day, and the laying rate, the abnormal egg rate, the egg weight, the daily feed intake and the feed-egg ratio are calculated according to the repeated counting. The feeding management conditions of the layers of each group are the same, the daily illumination time is 16 hours, the layers are immunized according to the normal immunization program of the layers, the positive test period is 56d, and the conditions and the feeding condition of the chickens are observed daily during the test period.

4 sample collection

On weekends 8, the laying hens fasted for 12h. 8 chickens were randomly selected from each test group, collected from jugular vein and slaughtered. The collected blood sample is put into a 10ml centrifuge tube, kept stand at room temperature for 2 hours, centrifuged at 3000rpm and 4 ℃ for 10 minutes, and the serum is collected and put into dry ice for measuring biochemical indexes of the serum. After the killed laying hen is dissected, weighing and recording the liver, spleen, ovary and oviduct for organ index analysis; the liver is sampled and put into tinfoil paper, and stored in liquid nitrogen; taking three parts of ovaries and oviducts in total, wherein one part is placed in 4% paraformaldehyde solution for making tissue sections of ovaries and oviducts and observing tissue morphology, and the other two parts are respectively placed in tinfoil paper for preserving by liquid nitrogen for gene detection and transcriptome sequencing; rapidly separating duodenum, jejunum and ileum, washing the content of the intestinal tract with pre-cooled physiological saline, and placing about 2cm in 4% paraformaldehyde solution for making intestinal tissue slice and observing tissue morphology; respectively taking two parts of ileum and cecum, placing the two parts into a 2ml freezing tube, preserving the two parts by liquid nitrogen, and using the two parts for gene detection and transcriptome sequencing; respectively taking foregut (mixed with duodenum, jejunum and ileum) and hindgut (mixed with cecum and colorectal) chyme, placing the chyme into a freezing tube, and rapidly placing the chyme into liquid nitrogen for short chain fatty acid determination and microorganism composition analysis in the intestinal chyme; all samples were subsequently transferred to-80 ℃ for storage for subsequent assay analysis.

5 measurement index and method

5.1 measurement of production Performance

The test period is taken as a unit of repetition, the egg laying number, the total egg weight, the malformed egg number and the death and panning number of each group of test chickens are recorded every day, and the feed intake is counted every half month. And calculating the laying rate, average egg weight, abnormal egg rate, average daily feed intake and feed-egg ratio by taking the groups as units. The measuring method and the calculating method are as follows:

(1) Egg yield (%) = total number of eggs/chicken number x 100%.

(2) Average egg weight (g/piece) =total egg weight/total egg count.

(3) Abnormal egg rate (%) = total abnormal egg number/total egg production x 100%.

(4) Average daily feed intake (g) =total weekly feed consumption/9/7.

(5) Feed egg ratio (%) = total feed consumption/total egg weight x 100%.

5.2 egg quality determination

Egg samples were collected one day before the end of the test, 8 eggs were randomly selected for each group, and egg quality detection was performed.

(1) Egg weight: measurements were made using an EggAnalyzer to the nearest 0.01g.

(2) Egg-shaped index: and measuring the transverse diameter and the longitudinal diameter of each egg by using an electronic digital display vernier caliper, and then calculating. The formula: egg-shaped index = transverse/longitudinal x 100%.

(3) Eggshell strength: the whole egg is placed on an eggshell strength tester for detection.

(4) Yolk color: the eggs were broken up and placed in an EggAnalyzer for detection.

(5) Haet unit and protein height: the eggs were broken up and placed in an EggAnalyzer for detection.

(6) Eggshell thickness: measuring eggshell thickness of sharp end, blunt end and middle 3 parts of egg by using electronic digital vernier caliper

The average value was taken as eggshell thickness.

(7) Egg yolk index: two yolk pieces are selected repeatedly, and the yolk height is measured by using an electronic digital vernier caliper

The egg yolk diameter. Egg yolk index = egg yolk height/egg yolk diameter.

(8) Yolk ratio: removing egg yolk surface protein material and lacing, weighing and recording egg on analytical balance

Yellow weight, and egg weight ratio. The formula: yolk ratio = yolk weight/whole egg weight.

5.3 determination of organ index

After blood collection, spleen, liver, ovary and oviduct were rapidly separated, dried with filter paper and weighed, and index of each organ was calculated.

5.4 amino acid content in egg white

1g egg white sample is respectively and accurately weighed, placed in a bottle with a plug, and 6mol/LHCL 10ml is added into the bottle with the plug. And (5) placing the test tube with the plug in a constant temperature oven at 110 ℃ for 23 hours after the test tube with the plug is tightly covered, and taking out and cooling. The test tube was opened and poured into a 25ml volumetric flask, rinsed more than 5 times with deionized water, and the volume was set to the 25ml volumetric flask. Shaking, immediately filtering and preserving at least 10ml of hydrolysate with filter paper, sucking filtrate (hydrolysate of 1ml of liquid sample) into a crucible, drying in water bath at 95 ℃ to remove solvent, adding 1ml of deionized water, uniformly mixing, evaporating again, finally dissolving and flushing with 0.02N HCL for more than 5 times, stirring with a suction pipe uniformly, pouring into a 10ml volumetric flask, fixing the volume into the 10ml volumetric flask, filtering the solution into a sample injection bottle with a 0.22u filter membrane, and measuring by using a full-automatic amino acid analyzer.

5.5 serum related index determination

The serum of the layers was assayed for glutamic-oxaloacetic transaminase (Aspartate transaminase, AST), glutamic-pyruvic transaminase (Alanine aminotransferase, ALT), alkaline phosphatase (Alkaline phosphatase, ALP), phosphorus (P), calcium (CA), albumin (ALB), glucose (GLU), immunoglobulin a (ImmunoglobulinA, igA), immunoglobulin G (IgG), immunoglobulin M (IgM) using an automated biochemical analyzer of family Hua Quan. Serum biochemistry related kits were all purchased from the kehua organisms.

5.6 morphological detection of ovary, oviduct and intestinal tract

4% paraformaldehyde fixed layer ovary, oviduct and intestinal tract samples were prepared as paraffin sections and stained with Hematoxylin and Eosin (HE). The development of the fallopian tube and ovary was observed by an optical microscope, and the duodenal, jejunal, ileal Villus height (Villy height) and Crypt depth (Crypt depth) were measured by using CaseViewer analysis software, and the Villus height/Crypt depth (V/C) was calculated.

5.7 serum inflammation and immune index determination

Immunoglobulin A (ImmunoglobulinA, igA), immunoglobulin G (IgG), immunoglobulin M (IgM), tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), interleukin 2 (IL-2) and D-lactic acid (D-Lac) in serum are respectively determined by adopting kit related to Nanjing bioengineering institute according to kit instructions by ELISA method.

5.8 liver antioxidant index detection

Total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in the liver are detected according to the instruction of the kit, and are respectively detected by adopting related kits of Beijing box Biotechnology Co.

5.9 Gene expression level determination

(1) RNA extraction

Taking a certain amount of ileum, cecum, ovary and oviduct tissues, fully grinding the tissues in a 1.5ml centrifuge tube filled with Trizol and white magnetic beads by using a tissue grinder, adding 200ul of chloroform, shaking uniformly, standing for 10min, adding 12000g of chloroform, centrifuging for 10min, transferring 400ul of supernatant to a new EP tube, adding equal volume of isopropanol, shaking uniformly, standing for 10min at room temperature, 12000g of isopropanol, centrifuging for 10min, adding 1ml of 75% DEPC ethanol, resuspending, washing and precipitating, subsequently, 8000g of supernatant, centrifuging for 5min, washing for two times, drying for 15min by using a super clean bench, adding a proper amount of DEPC water to completely dissolve RNA, and measuring the OD260/280 value and the concentration of the RNA by using a nucleic acid analyzer. In general, the OD260/280 is guaranteed to be between 1.8 and 2.0. The extracted RNA is stored at-80 ℃ for standby.

(2) cDNA preparation

Reference RTMix Kit with gDNAClean for qPCR kit (Ai Kerui Bio Inc.). Total RNA amount was 1ug/reaction, and the reaction system (20 ul) was as shown in Table 3 below:

1) Removal of genomic DNA

TABLE 3 Table 3

Reaction conditions: 42 ℃ for 2min;4 ℃ room temperature

2) Reverse transcription reaction

TABLE 4 Table 4

Reaction conditions: 37 ℃ for 15min

85℃ 5sec

4℃ -

(3) Primer design

The primers were synthesized in the Optimago company. Primer information is shown in the following table:

TABLE 5 primer sequences

(4) Real-time fluorescent quantitation

The procedure was performed according to the instructions of the SYBR Green Pro Taq HS qPCRKit kit, which was purchased from Ai Kerui biosystems. Specific Real-time PCR reaction systems and reaction procedures are shown in the following table:

1) Reaction system

TABLE 6

2) Reaction procedure

TABLE 7

The relative expression level of each target gene was calculated using GAPDH as a reference gene.

5.10 detection of short chain fatty acids in intestinal Contents

Short chain fatty acids of the hindgut (cecum, colorectal) contents were detected using gas chromatography techniques. Accurately weighing 0.5g chyme into a 15ml centrifuge tube, adding 5ml ultrapure water, vortex shaking for 30min, standing overnight at 4 ℃ in a refrigerator, centrifuging at 10000rpm for 10min, transferring supernatant, and mixing according to V1: v2=9:1 (1350 ul supernatant +150ul 25% metaphosphoric acid) was added to a 1.5ml centrifuge tube, and after mixing, the mixture was allowed to stand at room temperature for 3 hours, and after centrifugation (to prevent precipitation, filtration was not good) was performed on the mixture, the mixture was filtered through a 25um microporous filter membrane, and the mixture was added to a liquid phase vial to be measured.

5.11 analysis of intestinal microbial composition

The microbial composition of the foregut (duodenum, jejunum, ileum) and hindgut (cecum, colorectal) contents was analyzed using 16S rDNA high throughput sequencing technology.

6 data processing

The test data are collated by Excel 2019 software; the differences among different groups are analyzed by adopting a Tukey-Kramer method through SPSS23.0 statistical software on production performance, egg quality, organ index and serum biochemical data, and single-factor analysis of variance is adopted on slice data. In summary of the data, the subsequent experiments were analyzed by using WEA-L as an independent sample T test and marked as WEA, and the results are all expressed by average number + -standard error, P <0.05 is significant, and P <0.01 is extremely significant. Data were plotted using GraphPad Prism 9.0 software, with P <0.05 and P <0.01.

7 analysis of test results

7.1 improving production performance of laying hens by using water extract of strawberry tea

As is clear from Table 8 below, there was no significant difference in the egg laying rate, average egg weight, malformed egg rate, average daily feed intake, and feed/egg ratio in the WEA-L, WEA-M and WEA-H groups as compared with the Control group, but the egg weights in the WEA-L, WEA-M and WEA-H groups tended to be higher than the Control group (P=0.098). The water extract of the strawberry tea can improve the production performance of the laying hens.

TABLE 8 influence of aqueous extract of strawberry tea on the productivity of laying hens

Note that: (1) the data were derived from the Hunan agricultural university animal nutrition genome and germplasm innovation research center. (2) The different lower case letters of the same row of data shoulder marks represent the difference is significant (P < 0.05), the different upper case letters of the same row of data shoulder marks represent the difference is significant (P < 0.01), and otherwise, the difference is not significant. (3) SEM represents standard error of mean between groups. The following is the same as

7.2 the water extract of the strawberry tea has the protection effect on immune organs

As can be seen from Table 9, the WEA-L, WEA-M and WEA-H groups each significantly improved liver index (P < 0.01) compared to the Control group; in addition, the WEA-M and WEA-H groups also significantly increased spleen index (P < 0.05), indicating that the aqueous extract of the berry tea can improve liver function and immune function. The oviduct index of the WEA-L and WEA-H groups had a higher tendency than Control (p=0.076), indicating that the aqueous extract of the berry tea has a protective effect on the oviduct tissue.

TABLE 9 influence of aqueous extract of strawberry tea on organ index of laying hen

7.3 the water extract of the strawberry tea can improve the quality of eggs

As can be seen from table 10, the egg weights (P < 0.01), egg shape indices (P < 0.01), egg yolk colors (P < 0.05) and egg yolk indices (P < 0.05) of the WEA-L group, WEA-M group and WEA-H group were significantly improved as compared with the Control group. The protein heights of the WEA-L group and the WEA-H group are obviously increased (P is less than 0.05), and the trend of increasing the Hash unit is that P=0.096, which indicates that the water extract of the strawberry tea can improve the quality of albumin. Whereas only the WEA-M group had eggshell strength significantly higher than for the Control group (P < 0.05). In addition, the eggshell thickness was significantly increased in the WEA-L (P < 0.01) and WEA-M (P < 0.01) groups, and significantly decreased in the WEA-H (P < 0.01) groups, as compared to the Control group.

In conclusion, the water extracts of the raspberries and the tea with different concentrations improve the quality of eggs.

TABLE 10 influence of aqueous extract of strawberry tea on egg quality of laying hen

7.4 influence of the aqueous extract of the tea of the strawberry on the biochemical index of the serum of the laying hen

As can be seen from table 11, the WEA-M group significantly increased IgG levels (P < 0.01) and the WEA-L group significantly increased ALB levels and decreased GLU levels (P < 0.05) compared to Control. The result shows that the water extract of the strawberry tea has the function of improving the immunity of the laying hens, increases the digestion, absorption and metabolism levels of the organism on protein, and has the function of reducing the blood fat. In addition, the WEA-L, WEA-M, WEA-H group had a trend to increase IgA (p=0.064) and CA (p=0.055), indicating that increasing eggshell quality of the water extract of the berry tea is associated with CA levels.

Table 11 influence of aqueous extract of strawberry tea on biochemical index of serum of laying hen

7.5 influence of aqueous extract of strawberry tea on histomorphology of ovary, oviduct and intestinal tract of laying hen

To further investigate the effect of WEA on ovary, fallopian tube and intestinal morphology, ovarian, fallopian tube and intestinal tissue sections were measured. The effect of addition of WEA on layer chicken tissue morphology is shown in fig. 1A: the enlarged folds of the oviduct of the WEA-L and WEA-H groups are long and thick, so that the oviduct has strong secretion capacity, the folds of the WEA-M groups are thick and leaf-shaped, the gland ducts are more, and the folds of the Control group are relatively loose; the number of the ovary granular cells of the Control group laying hen is reduced, the granular cells are loosely and irregularly arranged, and after WEA is added into the diet, the morphology structure of the ovary of the laying hen and the arrangement of the granular cells are orderly, which indicates that the aqueous extract of the strawberry tea protects the ovary by reducing the histological changes. The duodenum, jejunum and ileum morphology of WEA-L, WEA-M and WEA-H mice were more complete than Control group with less intestinal villi shedding, indicating that WEA can protect intestinal morphology. In addition, WEA-H can significantly increase villus height in the duodenum (FIG. 1B; P < 0.01), while WEA-L, WEA-M and WEA-H can both significantly decrease crypt depth (FIG. 1C; P < 0.01) and increase V/C (FIG. 1D; P < 0.01); in the jejunum, both WEA-L, WEA-M and WEA-H can significantly pile height (FIG. 1E; P < 0.01) and V/C (FIG. 1G; P < 0.01) and reduce crypt depth (FIG. 1F; P < 0.01); the morphological results of the ileum are relatively similar to those of the duodenum, WEA-H can significantly increase villus height (FIG. 1H; P < 0.05), while WEA-L, WEA-M and WEA-H can both significantly decrease crypt depth (FIG. 1I; P < 0.01), and WEA-M (P < 0.05) and WEA-H (P < 0.01) can both significantly increase V/C (FIG. 1J). In conclusion, the water extract of the strawberry tea with a certain dosage can protect the microstructure of the ovary, the oviduct and the intestinal tissues.

7.6 influence of aqueous extract of strawberry tea on serum immunity and inflammation index of laying hen

WEA-L was selected as the group of aqueous extracts of the tea and scored as WEA for subsequent multi-angle analysis by the above phenotypic data. As can be seen from fig. 2, WEA (P < 0.01) extremely significantly reduced IL-1 beta (fig. 2A) and IL-2 (fig. 2D) levels compared to Control; whereas WEA has a trend to lower TNF- α (fig. 2b; p= 0.0901), indicating that aqueous extracts of berry tea can alleviate the inflammatory response of layer chickens. In addition, WEA also significantly increased IgA (FIG. 2E; P < 0.05) levels and tended to decrease D-Lac (FIG. 2H; P= 0.0984) levels, combined with the serum biochemical data described above, the water extract of MEI tea had the effect of enhancing the immunity of the layer chicken, and D-Lac results indicated that WEA had the effect of enhancing the intestinal barrier.

7.7 influence of water extract of strawberry tea on oxidation resistance index of liver of laying hen

As can be seen from FIG. 3, compared with Control, WEA can significantly improve liver SOD (FIG. 3A; P < 0.001) and T-AOC (FIG. 3A; P < 0.01) levels, indicating that the water extract of the strawberry tea can improve the antioxidant capacity of the laying hen.

7.8 influence of aqueous extract of strawberry tea on oviduct, ovary and intestinal tract related gene expression of laying hen

As can be seen from FIG. 4, WEA significantly down-regulates the levels of the pro-inflammatory factors TNF- α (FIG. 4A; P < 0.01), NF- κB (FIG. 4C; P < 0.05) and COX-2 (FIG. 4F; P < 0.05) compared to Control; and up-regulates the expression of the anti-inflammatory factors IL-4 (FIG. 4D; P < 0.05) and IL-10 (FIG. 4E; P < 0.05), these results indicate that the aqueous extract of the tea has anti-inflammatory effect, can effectively inhibit NF- κB pathway, thereby inhibiting the expression of the downstream pro-inflammatory factors thereof, and promotes the secretion of the anti-inflammatory factors, and plays an anti-inflammatory effect to a certain extent, thereby reducing the salpingal inflammatory reaction. The up-regulation of the ovary Bcl2/Bax shows that the water extract of the strawberry tea can reduce the apoptosis of ovary cells, thereby protecting the ovary function. And WEA (figure 4I-L; P < 0.05) significantly up-regulated the expression of Occildin, claudin-1, ZO-1, MCU2, suggesting that the aqueous extract of the tea has indeed the function of enhancing intestinal barrier, similar to the result of D-Lac described above. In addition, WEA significantly up-regulates the expression of Nrf2 (FIG. 4M; P < 0.01) and NQO1 (FIG. 4O; P < 0.05), and down-regulates the expression of Keap-1 (FIG. 4N; P < 0.05), which indicates that the water extract of the strawberry tea has an antioxidant effect, and can promote the generation of downstream antioxidant enzyme NQO1 by activating Nrf2/Keap1 channels, thereby relieving oxidative stress caused by aging of laying hens.

7.9 Effect of aqueous extract of Raspberry tea on short chain fatty acids of intestinal content

As can be seen from fig. 5, the content of acetic acid, propionic acid, isobutyric acid, butyric acid, valeric acid and isovaleric acid was all increased in the WEA group compared to the Control group, wherein the difference between the content of acetic acid, propionic acid, isobutyric acid and isovaleric acid and the Control group reached a significant level P <0.05, indicating that the water extract of the berry tea was able to improve intestinal health.

7.10 influence of aqueous extract of strawberry tea on intestinal microorganisms of laying hens

According to the invention, the influence of WEA on the foregut and hindgut intestinal flora of the laying hen is determined through 16S rRNA sequencing, and alpha diversity and beta diversity analysis are carried out on intestinal contents, wherein the alpha diversity represents diversity and abundance of microbial communities. FWEA (P < 0.05) significantly reduced foregut Chaol, observed _patterns and good_coverage index compared to the control group; however HWEA (P < 0.05) significantly increases hindgut Simpson, shannon and pielou_e indices; the above results indicate that WEA has a significant effect on the alpha-diversity of the intestinal flora.

In addition, beta diversity was used to evaluate the effect of WEA on intestinal microbiota structure, and the invention also performed principal coordinate analysis (PCoA) and non-measuring multidimensional scale analysis (NMDS), which showed significant differences between the microbiota of the control and WEA groups in the foregut and hindgut, especially the foregut, which was more significant, indicating that WEA also significantly altered the Beta diversity of the intestinal flora.

In conclusion, from alpha diversity and beta diversity analysis, the water extract of the strawberry tea can obviously change the intestinal flora composition of the foreintestines and the hindintestines of the laying hens.

The above description is only of a few preferred embodiments of the present invention and should not be taken as limiting the invention, but all modifications, equivalents, improvements and modifications within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. The laying hen feed additive is characterized by comprising a water extract of the blueberry tea, wherein the water extract of the blueberry tea comprises the following components in percentage by weight: the total flavone content is 50-60%, and the dihydromyricetin content in the total flavone is 25-35%; polysaccharide content is not less than 2%; the polyphenol content is 30-38%.

2. The water extract of strawberry tea laying hen feed additive according to claim 1, wherein the preparation method of the water extract of strawberry tea comprises the following steps:

(5) Collecting the dry spray extract powder, and packaging for storage.

3. The water extract of strawberry tea layer feed additive of claim 1 or 2, wherein in the water extract of strawberry tea: the content of total flavonoids is 57.6%, and the content of dihydromyricetin in the total flavonoids is 29.4%; the polysaccharide content was 6.83% and the polyphenol content was 35.57%.

4. The water extract of strawberry tea layer feed additive according to claim 1 or 2, characterized in that the additive is applied in an amount of 50-250mg of water extract of strawberry tea per kg of basic ration of layer.

5. Use of the aqueous extract of strawberry tea as claimed in any one of claims 1 to 4 as a feed additive for laying hens.

6. The use according to claim 5, in particular for the preparation of a feed additive for improving the productivity of laying hens.

7. The use according to claim 5, in particular for the preparation of a feed additive for improving the immunity of laying hens.

8. Use according to claim 5, in particular for the preparation of a feed additive for improving the egg quality of laying hens.

9. The use according to claim 5, in particular for the preparation of feed additives with anti-inflammatory properties for laying hens.

10. The use according to claim 9, wherein the anti-inflammatory properties comprise anti-salpingitis properties, anti-ovarian inflammation properties, anti-intestinal inflammation properties.