日糧添加廢棄蛹蟲草培養基對蛋雞生產性能及腸道菌群多樣性的影響

摘要：試驗旨在探討廢棄蛹蟲草培養基作為蛋雞生產飼料的可行性。在蛋雞基礎飼糧中分別添加100g/kg、200g/kg、300g/kg的廢棄蛹蟲草培養基。結果表明，廢棄蛹蟲草培養基在蛋雞日糧中的最佳添加量為10%。根據雞蛋常規指標的測定結果，用廢棄的蛹蟲草培養基喂養的蛋雞產蛋重量高于用普通蛋雞喂養的蛋雞。試驗組白細胞介素1含量顯著高于對照組，白細胞介蛋白1濃度增加141.5pg/mL，表明廢棄蛹蟲草培養基的應用有效提高了蛋雞的免疫力。對兩組蛋雞腸道內容物的高通量分析表明，實驗組腸道微生物種群豐度大于對照組，廢棄蛹蟲草培養基的應用增加了蛋雞腸道細菌的多樣性。此外，雞腸道中的一些致病菌對藥物的敏感性也有所提高，從而減少了抗生素的使用。蛹蟲草廢棄培養基的二次利用具有很大的開發利用前景，為蛹蟲草廢棄培養基在蛋雞生產中作為飼料的開發提供了科學參考和基本理論依據。

關鍵詞：蛹蟲草; 廢棄培養基; 藥物敏感性; 腸道菌群; 蛋雞

In recent years，problems such as antibiotic abuse have also received great attention in the aquaculture industry.In order to maintain the laying rate of laying hens，some chicken farms add a large number of additives to the feed，and the components are mainly antibiotics，resulting in drug resistance of laying hens，and a large number of antibiotics will also circulate to the environment with the excrement of laying hens，causing environmental pollution and endangering human health［1］.Therefore，some researchers are eager to explore alternatives to antibiotics［25］.Therefore，it is urgent to replace the use of antibiotics to improve the immune regulation system of laying hens.

Our previous experiments found that the content of crude protein，inorganic Ca，P and other nutrients in discarded nutrient medium of Cordyceps militaris increased after fermentation.If we can conduct indepth research on discarded nutrient medium，it is of great significance to develop natural，green and diseaseresistant new feed，reduce the abuse of antibiotics and environmental pollution［6］.This experiment is to cofeed discarded nutrient medium with laying hens diet，and use some antibacterial substances in discarded nutrient medium to reduce and replace the use of antibiotics and improve the immune regulation system of laying hens.

1Material and methods

1.1Animals and experimental diets

Several laying hens，laying hens feed consists of basic laying hens diet （control） and three diets supplemented with discarded nutrient medium of Cordyceps militaris.100g/kg，200g/kg，300g/kg of discarded nutrient medium of Cordyceps militaris were added to the basal diet of laying hens.The discarded nutrient medium was provided by Key Laboratory of Cordyceps militaris with Functional Value of Liaoning Province，and the diet of laying hens was provided by Heishan，Liaoning Province.

1.2Performance measurements

1.2.1Nutrients of discarded nutrient medium of Cordyceps militaris

Agilent 1100 high performance liquid chromatograph was used to detect cordycepin and adenosine，with methanol ：water=25：75 isocratic elution.The injection volume was 10 μL，and the mass concentration of cordycepin and adenosine （mg/g） was calculated by peak area.Selenium was determined by ICP atomic emission spectrometry.The measured spectral intensity was brought into the standard curve formula to calculate the selenium content in the sample.The atomic fluorescence spectrometer was used to detect the wavelength at 620nm，and the measured absorbance Y was brought into the standard curve regression equation to calculate the content of polysaccharides.According to the international standard method （GB/T 188682002），the indexes of crude protein，crude ash，water and fat were detected by near infrared spectrometer.

1.2.2Production performance of laying hens

The remaining discarded nutrient medium of Cordyceps militaris was taken out，dried in a drying oven at 55 °C and ground into powder，and the excess impurities were screened out with a sieve，leaving fine powder for feed addition.64 chickens were divided into 4 groups，16 chickens in each group.In the first group，10% of the diet of laying hens was added with discarded nutrient medium，20% of the diet of laying hens was added with discarded nutrient medium in the second group，30% of the diet of laying hens was added with discarded nutrient medium in the third group，and the other group was only fed with the diet of laying hens without discarded nutrient medium.A set of data was recorded every 2 days，and the feeding period was 30 days.The indicators measured daily were body weight，feed intake，mental state，and egg production rate.

1.3Egg quality measurements

The feeding cycle was 30 days.From the 30th day of feeding，the eggs of three groups of laying hens were collected every day and marked as 10 ordinary eggs and 10 functional eggs respectively，and routine indicators were detected.Record the number of eggs laid per day.The average laying rate was calculated，and the eggs were weighed by electronic balance to detect egg density，yolk weight and eggshell weight.The egg shape index and eggshell thickness were measured by vernier caliper.The determination of egg yolk index can be calculated by the formula egg yolk index=egg yolk longitudinal diameter / egg yolk transverse diameter.Egg yolk color with egg yolk and Roche colorimetric fan color comparison［7］.

1.4Determination of resistance to disease

After 30 days of feeding，3mL of wing venous blood was taken into anticoagulant tubes and kept in cold storage for 1～2h［8］.After returning to room temperature，the blood was transferred to centrifuge tubes and centrifuged for 20min at 2，000×g.The required plasma was obtained.The resistance to disease was measured using chicken IL1 kit purchased from Shanghai ，Enzymelinked Biochem Co.，LTD.

1.5Determination of drug susceptibility to intestinal pathogens

Macconkey Agar （MAC），Chromogenic Coliform amp; Escherichia coli Agar （ECC），Nutrient Agar Medium （NA），Enterococcus Agar （Bile Esculin Azide Agar） （EB），Enterococcus faecal Agar （EF Agar），CaryBlair Transport Medium （CB），Columbia blood plate were purchased from Beijing Luqiao Co.，LTD.

1.5.1Separation and purification

The experimental group and control group of laying hens were selected，and cotton swabs were used to sample the cloaca of laying hens，15 in each group，a total of 30 samples.The samples were streched on MAC medium and cultured at 36℃ for 18～24 h，and single colonies were selected and streched on ECC medium and cultured at 36℃ for 16～24h［9］.Cotton swabs with bacteria were placed in EB broth and incubated at 36℃ for 48h.After 48h，the culture medium was selected and strewed on EF Agar，and the culture was incubated at 36℃ for 48h［10］.

1.5.2Biochemical identification

The green single colonies on ECC medium were selected and streched on NA plate.After 18～24h of culture at 36℃，4～6 colonies were selected and prepared in normal saline to make bacterial suspension.The purity was identified by bacterial identification instrument.A proper amount of single colonies on LB medium was selected and streated on the blood plate.After 24h of culture at 37℃，the single colonies were selected and prepared in normal saline to make bacterial suspension，and the bacterial identification instrument VITEK2 was used for identification.

1.5.3Drug sensitivity test

The single colonies of Escherichia coli（E.coli） on NA medium and Enterococcus faecalis（E.faecalis） on blood plate were respectively selected and prepared into 0.5 McGrath′s turbidities solution.0.01mL of turbidities solution was diluted 100times with 0.99mL nutrient broth and mixed.350 μL of bacterial solution was accurately absorbed by pipetting gun and added into ATB series antimicrobial sensitive plate，respectively.The samples were incubated at 37℃ for 24h，and the results were read by ATB drug sensitivity analyzer.S was sensitive，I was generally sensitive，and R was insensitive［11］.

1.6Determination of diversity of intestinal flora

The laying hens in the experimental group and the control group were randomly selected，and the intestinal contents were scraped and stored in sterile closed centrifuge tubes at -80℃.Qubit 3.0 DNA extraction kit was used to extract and quantify genomic DNA.Gene sequencing was commissioned from Shanghai Shenggong Technology Co.，LTD.Illumina Miseq highthroughput sequencing platform was used to amplify the V3V4 region of intestinal microbial 16s rRNA gene in two groups of laying hens，and then analyzed by highthroughput sequencing.The number of OTUs was determined by clustering，and similar species were found in the database for matching，and the phylum and genus of samples were statistically analyzed to obtain the result of microbiota analysis［1213］.

1.7Statistical analysis

SPSS 20.0 statistical software was used to analyze the data，and the chisquare value x2 was used to indicate whether there was a statistical difference.

2Results and discussion

2.1Performance

2.1.1Nutritional components of discarded nutrient medium of Cordyceps militaris

As shown in Table 1 and Table 2，the discarded nutrient medium of Cordyceps militaris was tested according to the feed standard of the laying hens diet.Compared with the proportion of nutrients required for the laying hens diet，discarded nutrient medium of Cordyceps militaris contained cordycepin，nucleoside，polysaccharide and selenium unique to discardednutrient medium of Cordyceps militaris，and there were many nutrients such as crude protein and inorganic substances.

2.1.2Production performance of laying hens

As shown in Table 3，the average laying rate of laying hens in M 3 group decreased by 36.28%，and the difference was not significant compared with the control group.The average laying rate of laying hens in M 2 group decreased by 29%.The average laying rate of laying hens in the M1 group decreased by 8.4%，and the difference was not significant.Therefore，it can be seen from the data that feeding a certain proportion of discarded nutrient medium of Cordyceps militaris will reduce the laying rate of laying hens.

2.2Egg quality

The eggs added with discarded nutrient medium of Cordyceps militaris were more important than ordinary eggs，and the weight increased by about 8.13%，and the difference was not significant.There was no significant difference in egg yolk weight gain of 10% and egg density of 1% between the eggs added with discarded nutrient medium and ordinary eggs.

The results showed that the egg weight of the diet added with discarded nutrient medium was larger than that of ordinary eggs，and the yolk weight was significantly larger than that of ordinary eggs，and the difference was significant.

The eggshell weight of the diet supplemented with discarded nutrient medium increased by 1.02% compared with that of ordinary eggs.The eggshell thickness and egg shape index of the two kinds of eggs were basically unchanged，and the difference was not significant.

The results showed that laying hens have no significant effect on the eggshell index of eggs by feeding discarded nutrient medium.There was no significant difference in yolk color between eggs supplemented with discarded nutrient medium and ordinary eggs.It indicated that the addition of discarded nutrient medium in laying hens feed has little effect on egg yolk.Through the detection of the routine indexes of three kinds of eggs，it can be preliminarily concluded that the weight of eggs added with discarded nutrient medium was larger than that of ordinary eggs produced by the control group，and the yolk weight was also significantly larger than that of ordinary eggs.In addition，there was no significant change in eggshell thickness before and after the experiment，and the yolk color value decreased slightly compared with the ordinary eggs.

2.3Resistance to disease

The OD value displayed by microplate reader was used as the ordinate，and the concentration of IL1 standard was used as the abscess to establish the curve.The IL1 concentration of hens in each group were 303.1pg/mL，445.3pg/mL，444.9pg/mL，443.6pg/mL，respectively.The IL1 concentration of hens was significantly increased.The results proved that feeding discarded nutrient medium can effectively improve the immunity of laying hens.

2.4Drug susceptibility to intestinal pathogens

After biochemical identification，16 strains of E.coli and 10 strains of E.faecalis were finally obtained for drug sensitivity test.According to Fig.1，compared with the control group，the resistance of the experimental group to the following drugs was reduced to varying degrees，including amoxicillin，amoxicillin/clavulanic acid，piperacillin，ticarcillin，ticarcillin/clavulanic acid，cefalotin，cefoxitin，ceftazidime，cefuroxime，indicating that the E.coli in the experimental group was more sensitive to these drugs.

2.5Diversity of intestinal flora

The intestinal microorganisms of laying hens were classified at the genus level，and 50 genera were detected.Compared with the control group，the genus Bacteroides in the experimental group increased by 6.79%，the genus Lactobacillus increased by 391.7%，and the genus Faecalibacterium decreased by 66.22%，which indicated that feeding with discarded nutrient medium of Cordyceps militaris had a selective effect on the improvement of beneficial bacteria.In addition，some bacteria that did not appear in the control group were detected in the experimental group，such as Blautia and Acetobacteroides，etc.，and some of the original bacteria in the control group disappeared，such as Helicobacter pylori，Campylobacter Olsenella，etc.Helicobacter is a identified pathogen.

3Conclusions

By exploring the best addition ratio of discarded nutrient medium of Cordyceps militaris in the process of feeding laying hens，and taking the detection of egg production rate and feed intake as indicators，the best feeding scheme of discarded nutrient medium in laying hens was 10%.Feeding discarded nutrient medium has a certain effect on effectively improving the immunity of laying hens，which not only improving the species diversity of intestinal flora of laying hens，but also improving the sensitivity of intestinal E.coli to drugs.

References：

［1］KIM W H，LILLEHOJ H S.Immunity，immunomodulation，and antibiotic alternatives to maximize the genetic potential of poultry for growth and disease response［J］. Anim Feed Sci Technol，2019，250：4150.

［2］WANG H S，JIANG Z Y，XIAO M，et al.The effect of adding traditional Chinese medicine to feed instead of antibiotics on the sensitivity of intestinal Salmonella and Escherichia coli in largescale chicken farms［J］.Plateau Agr，2022，6（6）：542546.

［3］QI R S.Observation on the effect of traditional Chinese medicine instead of antibiotics in the treatment of pig asthma［J］.The Farmers Cons，2022（23）：143145.

［4］PARK Y，PARK M，HAMIDOGHLI A，et al.Optimum dietary processed sulfur （ImmunoF） level has antibiotic effects on the growth，hematology and disease resistance of juvenile olive flounder，Paralichthys olivaceus［J］.Anim Feed Sci，2021，279：115035.

［5］LI N，MU S Q，YAN.Effects of antibiotic replacement by Bacillus licheniformis on piglet health［J］.Food Feed Ind，2018（12）：5256.

［6］TANG H R，HAN Y X，LIU X J.Advances in pharmacological effects of Cordyceps militaris polysaccharides and adenosine［J］.Biochem Eng，2022，8（1）：164167.

［7］WANG Y，LI X N，HAN J Z.Tracking detection and analysis of drug resistance of Escherichia coli in a broiler farm［J］.Chi Anim Husbandry Vet，2015（42）：33293337.

［8］HAN Y L，SUN Z B.Technique and precautions of blood collection from subwing vein of chicken［J］.The Chi Livest Poultry Breeding，2015，11（1）：133.

［9］XU H，WANG L X，LI L S，et al.Isolation，identification and drug sensitivity test of Escherichia coli［J］.Anhui Agr Sci，2020，48（19）：8488.

［10］LI D S，LI X N，HAN X Z，et al.Surveillance and analysis of drug resistance of Enterococcus faecalis from animals in Northeast China［J］.Modern J Anim Husbandry Vet Med，2018（2）：4247.

［11］SUN Z，ZHEN Y G，ZHAO W，et al.To analyze the effect of yeast culture on cecal microbiota of broilers by highthroughput sequencing［J］.Chin J Anim Sci，2017（53）：6772.

［12］XIAO Y P，YANG C M，DAI B.Effect of Clostridium butyricum on cecal microflora structure of broilers based on highthroughput sequencing［J］.Zhejiang Agr J，2017，29（3）：373379.

［13］TANG H R，HAN Y X，LIU X J.Advances in pharmacological effects of Cordyceps militaris polysaccharides and adenosine［J］.Biochem Eng，2022，8（1）：164167.

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