Microbial marker combination for identifying nest predisposition of chicken and application thereof

Abstract

The application discloses a microorganism marker combination for identifying nest susceptibility of chickens and application thereof, and belongs to the technical field of poultry breeding and microbiomics. The microorganism marker combination comprises relative abundance information of at least two bacterial phyla in Bacteroidetes, Firmicutes and Verrucomicrobia in cecal contents. When at least two of the following conditions are met: the abundance of Bacteroidetes is higher than 35%, the abundance of Firmicutes is lower than 35%, and the abundance of Verrucomicrobia is lower than 2%, it is determined that the chicken has nest susceptibility or is in a nest period. Based on the marker combination, the application provides a corresponding identification method, a regulation method and a regulation agent screening strategy.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary technical field of poultry reproductive physiology and microbiome, specifically relating to a combination of microbial biomarkers for identifying brood susceptibility in chickens and their applications. Background Technology

[0002] Brooding (also known as "brooding") is a natural reproductive behavior in poultry, characterized by hens remaining in their brooder for extended periods and ceasing egg production. This behavior severely reduces egg production rates, causing significant economic losses to large-scale poultry farming. Therefore, early identification of individuals susceptible to brooding and the development of effective intervention measures are crucial for improving poultry farming efficiency.

[0003] Currently, the identification and regulation of broodiness traits in chickens mainly rely on the following methods:

[0004] (1) Behavioral observation method: The behavior of hens is continuously monitored by manpower or video system to determine whether brooding characteristics (such as prolonged brooding, increased broodiness, elevated body temperature, etc.) are present. This method is highly subjective, labor-intensive, and can only be identified after brooding behavior has occurred, so it cannot provide early warning.

[0005] (2) Hormone detection method: Collect serum to detect changes in hormone levels such as prolactin (PRL) and progesterone (P4). This method requires professional blood collection techniques, causes stress damage to chickens, has high detection costs (more than 80 yuan per sample), and has large fluctuations in hormone levels with poor repeatability.

[0006] (3) Genetic marker method: Use genome-wide association analysis (GWAS) or candidate gene method to screen SNP markers associated with broodiness traits. However, broodiness in poultry is a complex trait controlled by multiple genes. The explanatory power of existing markers is generally less than 15%, and they rely on expensive gene chips (50K / 600K), which makes it difficult to promote in grassroots farms.

[0007] In the screening of probiotic regulators, traditional methods mainly adopt a phenotypic-driven blind screening model: randomly combining probiotics from a known probiotic library and verifying their effectiveness through feeding trials lasting 6-12 months. This method is time-consuming, has low throughput, a success rate of less than 5%, and lacks targeted design based on the mechanism of brood behavior, thus failing to accurately restore egg production performance.

[0008] In recent years, the association between the gut microbiome and host health and physiological status has received increasing attention. However, current technologies have not yet systematically revealed the stable and quantitative association between brood behavior in chickens and the core gut microbiota, nor have they established an early, objective warning system and targeted regulatory strategies based on microbiome characteristics. Therefore, developing an identification and regulation technology based on well-defined microbial biomarkers is a pressing technical challenge that the industry needs to address. Summary of the Invention

[0009] This invention aims to overcome the shortcomings of the prior art and provide a systematic solution for identifying brood susceptibility in chickens, screening, and applying regulatory agents based on the characteristics of cecal core microbiota imbalance.

[0010] The present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a combination of microbial biomarkers for identifying brood susceptibility in chickens, the combination of microbial biomarkers comprising Bacteroidetes from cecal contents (…). Bacteroidetes Firmicutes ( Firmicutes ) and Verrucous Microbes ( Verrucomicrobia The relative abundance information of at least two bacterial phyla in the sample.

[0012] Preferably, the relative abundance of Bacteroidetes is higher than 35%, the relative abundance of Firmicutes is lower than 35%, and the relative abundance of Verrucomicrobia is lower than 2%.

[0013] More preferably, the relative abundance of Bacteroidetes is 35.44~49.02%, the relative abundance of Firmicutes is 30.04~33.08%, and the relative abundance of Verrucomicrobia is 0.42~1.38%.

[0014] This invention discovers and verifies that changes in the relative abundance of Bacteroidetes, Firmicutes, and Verrucous microbes in the cecal contents of chickens are stably and significantly associated with brood behavior. The abundance information of these three phyla, especially their combination patterns, can serve as reliable microbial biomarkers for identifying brood susceptibility in chickens.

[0015] Secondly, the present invention provides a method for identifying brood susceptibility in chickens based on the above-mentioned combination of microbial biomarkers, comprising the following steps:

[0016] (1) Obtain a sample of the cecal contents or its metagenomic DNA from the chicken to be tested;

[0017] (2) Detect the relative abundance of at least two of the following phyla in the sample: Bacteroidetes, Firmicutes, and Verrucous Microbes;

[0018] (3) Analyze the relative abundance information of the bacterial phyla obtained in step (2). If at least two of the following criteria (a)-c) are met, the chicken is determined to be susceptible to broodiness or in the broodiness period:

[0019] a) The relative abundance of Bacteroidetes is higher than 35%;

[0020] b) The relative abundance of Firmicutes is less than 35%;

[0021] c) The relative abundance of Verrucous microbes is less than 2%.

[0022] Preferably, the detection method is metagenomic sequencing, 16S rRNA gene sequencing, real-time quantitative PCR, or gene chip detection.

[0023] Thirdly, the present invention provides the application of the above-mentioned combination of microbial biomarkers in regulating brooding behavior in chickens.

[0024] Fourthly, the present invention provides a method for regulating brooding behavior of chickens using the above-mentioned combination of microbial markers, comprising the following steps: using the above-mentioned method for identifying brooding susceptibility of chickens to identify whether chickens have brooding susceptibility or are in the brooding period, and administering to chickens identified as having brooding susceptibility or being in the brooding period a probiotic preparation, prebiotic or postbiotic composition mainly composed of Firmicutes or Verrucous microbes.

[0025] Fifthly, the present invention provides a method for screening potential regulators for controlling brood behavior in chickens using the above-mentioned combination of microbial biomarkers, comprising the following steps:

[0026] (1) Using the above-mentioned bacterial abundance characteristics as targets;

[0027] (2) Screening for candidate substances that can increase the abundance of Firmicutes and / or Verrucous microbes, and / or decrease the abundance of Bacteroidetes;

[0028] (3) Verify the effect of the candidate substance on reducing brood behavior or restoring egg production performance through animal experiments.

[0029] In a sixth aspect, the present invention provides a detection reagent or kit for identifying brood susceptibility in chickens, comprising primers, probes or antibodies for specifically detecting the characteristic sequences of the aforementioned Bacteroidetes, Firmicutes and Verrucous phyla.

[0030] In a seventh aspect, the present invention provides a feed additive comprising a microbial preparation capable of increasing the abundance of the aforementioned Firmicutes and / or Verrucous microbes, for preventing or mitigating broodiness behavior in chickens.

[0031] Preferably, the feed additive is used in poultry farming.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention is the first to clearly define the relative abundance information of at least two of the three phyla—Bacteroidetes, Firmicutes, and Verrucous—in the contents of the chicken cecum as a microbial marker related to nesting, thus providing clear biological targets for identification and regulation.

[0034] Changes in gut microbiota may precede behavioral manifestations. The combination of microbial markers provided by this invention can provide early warning of brood behavior (such as 7-14 days before a drop in egg production), creating a window of opportunity for timely intervention.

[0035] Using molecular biology detection methods, the results are digitized and reproducible, avoiding subjective errors from human observation and physiological fluctuations in hormone detection.

[0036] The combination of microbial biomarkers provided by this invention can be used to develop rapid detection kits such as qPCR, which are convenient for on-site use in farms; the regulatory targets are clearly defined, which can significantly improve the success rate and efficiency of the research and development of probiotics and other additives.

[0037] This invention not only provides new scientific insights, but can also be directly transformed into detection products for early diagnosis and functional feed additives for prevention / treatment, thus having broad market prospects. Attached Figure Description

[0038] Figure 1 Morphological differences between hens exhibiting brooding behavior (left image) and egg-laying behavior (right image);

[0039] Figure 2 Horizontal community composition of cecal microbiota in laying hens and brooding hens;

[0040] Figure 3 HE-stained sections of normal follicles (top) and atretic follicles (bottom);

[0041] Figure 4 Comparison of the number of normal follicles between the intervention group and the control group;

[0042] Figure 5 Statistical comparison of granulocytes between the intervention group and the control group. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0044] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0045] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0046] Example 1: Discovery and Threshold Establishment of Nesting-Related Microbial Markers

[0047] 1. Determination of experimental animals

[0048] Six Wuliangshan Black-boned Chicken hens in their late laying period (43 weeks) and six hens in their brooding period were selected. Their brooding behavior in the last two weeks was used to identify brooding patterns (including elevated body temperature, fluffy feathers, brooding over eggs, and cessation of egg production). Figure 1 Finally, it was confirmed that the two groups of hens were sampled correctly.

[0049] Table 1. Egg production records of the selected samples in the last two weeks.

[0050]

[0051] 2. Cecal contents sampling procedure

[0052] (1) Laboratory animal husbandry and ethics

[0053] Individually housed in cages using a mixed feed during peak egg production, all individuals were in good health. All individuals came from the same group to minimize batch variation. All procedures were performed in accordance with the experimental protocol approved by the Animal Ethics Committee of Southwest Forestry University, and humane euthanasia was carried out according to animal welfare guidelines.

[0054] (2) Preparation before the experiment

[0055] Equipment sterilization:

[0056] Prepare sterile surgical instruments: dissecting scissors, dissecting forceps, and hemostats.

[0057] Prepare the following tools for collecting the contents of the sample: 2 mL sterile screw-top cryovials and sterile scissors.

[0058] All metal instruments must be autoclaved (121°C, 20 min) or dry-heat sterilized at 180°C for at least 2 hours before use. Plastic consumables are disposable sterile products.

[0059] Pre-cooling and labeling:

[0060] Make sure a sufficient number of 2 mL sterile cryovials are uniquely labeled (e.g., sample number, date).

[0061] Prepare pre-cooled dry ice in advance.

[0062] The contents were stored in labeled 2 mL sterile cryovials using RNA later nucleic acid stabilizer.

[0063] Personnel protection: Operators must wear sterile gloves, masks, and lab coats, and change gloves between different samples or thoroughly disinfect with 75% ethanol to avoid cross-contamination.

[0064] (3) Execution and dissection

[0065] Humane euthanasia: The hen is euthanized by dislocating her neck, ensuring her death.

[0066] Expose the body cavity: Fix the chicken in a supine position to the dissecting board. Thoroughly spray the abdominal skin with 75% ethanol. Using sterile scissors and forceps, make an incision along the midline of the abdomen in the skin and peritoneum to fully expose the abdominal organs.

[0067] Locating the cecum: Gently part the intestines to locate the cecum, which is located in the posterior part of the abdominal cavity. Chickens have two well-developed cecums, which are long and sac-like.

[0068] (4) Collection of cecal contents

[0069] Separation and ligation:

[0070] Using sterile hemostatic forceps, ligate the base of the cecum (near the ileocecal junction) and the middle part of the cecum separately to form an independent intestinal segment.

[0071] Using sterile scissors, cut the cecum completely off from both ends of the ligated segment and quickly transfer it to a sterile culture dish.

[0072] Contents Collection: Use sterile scissors to make a transverse cut in the ligated cecal segment. After cutting, squeeze out the contents from the cecal mucosa. Quickly transfer the collected contents into pre-cooled 2 mL sterile cryovials. Each cecal sample should be stored separately (i.e., at least two cecal samples per chicken, which can be mixed or analyzed separately later).

[0073] Weighting and dispensing: Weigh the same amount of contents (approximately 200 mg) into new cryovials as accurately as possible to ensure consistency in subsequent extractions.

[0074] (5) Sample preservation and transportation

[0075] Stabilizer application: Since immediate flash freezing is not possible, add sufficient commercial nucleic acid stabilizer RNA later to the sample tube immediately after sampling. After stabilization at room temperature, store at -80°C. Dry ice must be used to maintain the samples frozen during transportation, and packaging must comply with biosafety regulations.

[0076] (6) Records and metadata

[0077] Record detailed information about each hen for each sample: individual ID, breed, age, egg production rate, euthanasia time, and sampling time.

[0078] 3. Metagenomic DNA Extraction and Sequencing

[0079] Total DNA was extracted using the QIAamp Fast DNA Stool Mini Kit. DNA integrity (main band >20 kb) was assessed by 1% agarose gel electrophoresis, and concentration (>50 ng / μL) and purity (A260 / A280 = 1.8–2.0) were determined using Nanodrop. 350 bp fragment libraries were constructed, and PE150 paired-end sequencing was performed on an Illumina NovaSeq 6000 platform, with each sample yielding ≥6 Gb of sequencing data.

[0080] 4. Metagenomic data analysis and identification of core microbial community imbalance characteristics

[0081] (1) Data quality control: Fastp software was used to remove adapter sequences and low-quality reads (Q<30), remove host contamination (compared to chicken reference genome GRCg6a), and obtain effective data ≥5 Gb / sample.

[0082] (2) Species annotation and quantification: After quality control, the data were compared with the RefSeq bacterial / archaic / fungal database using Kraken2 software to calculate the relative abundance of each species. Species present in at least 5% of the samples were selected and defined as core microbiota.

[0083] (3) Functional gene annotation: contigs were assembled using Megahit software, and gene prediction was performed using Prodigal. The predicted genes were compared with the KEGG database to obtain functional pathway and module annotations.

[0084] 5. Experimental Results

[0085] The results are as follows Figure 2 As shown, the average abundance of Bacteroidetes in the cecum of laying hens was 31.93% (27–34.69%), while the average abundance in the cecum of brooding hens was 43.31% (35.44–49.02%). The average abundance of Firmicutes in the cecum of laying hens was 40.87% (37.25–42.88%), while the average abundance in the cecum of brooding hens was 31.37% (30.04–33.08%). The abundance of Proteobacteria varied slightly, with an average abundance of 3.59% (3.03–4.65%) in the cecum of laying hens and 5.09% (4.19–5.54%) in the brooding period. The average abundance of Verruciformis in the cecum of laying hens was 4.34% (3.20~6.15%), but the abundance in the cecum of brooding hens was lower, with an average abundance of only 0.79% (0.42~1.38%).

[0086] Based on this data distribution, microbial abundance thresholds for determining brood susceptibility were established: Bacteroidetes >35%, Firmicutes <35%, and Verrucous Microbes <2%. Meeting at least two of these thresholds indicates a high risk.

[0087] Example 2: Validation of the identification method based on microbial biomarkers

[0088] In another breeding batch, 12 Wuliangshan black-bone hens were randomly selected, and cecal contents were collected (using the same method as in Example 1). Instead of behavioral observation, the abundance of Bacteroidetes, Firmicutes, and Verrucous microbes was directly detected using the metagenomic sequencing method described in Example 1 (or a targeted qPCR method). The detection results were then compared blinded with behavioral records over a subsequent two weeks.

[0089] The results are shown in Table 2. The results indicate that all chickens that met at least two of the above threshold criteria (e.g., hens 1-6) exhibited clear brood behavior or a sharp decrease in egg production during the subsequent observation period; while chickens that did not meet the criteria (e.g., hens 7-12) maintained normal egg production. This verification experiment demonstrates the accuracy and forward-looking nature of the identification method of this invention.

[0090] Table 2. Microbial community abundance and egg production or brood behavior in the following two weeks.

[0091]

[0092] Example 3: Biomarker-based targeted regulation of brood behavior in chickens

[0093] Twelve hens (50 weeks old) entering the late laying period were randomly divided into a control group and an intervention group. The control group was fed a basal diet, while the intervention group received a Firmicutes probiotic supplement (Lactobacillus delbrueckii, 1.2 × 10⁻⁶) added to their basal diet. 8 CFU / kg feed), with continuous intervention for 30 days.

[0094] (1) The abundance of Bacteroidetes, Firmicutes and Verrucous in the control group and intervention group was detected (the detection method is the same as in Example 1).

[0095] (2) Histological analysis of the ovaries was performed to detect changes in the number of normal follicles and granulosa cells before and after the intervention.

[0096] 1) Tissue sample collection and fixation

[0097] Dissection and sampling: After euthanizing the animal by cervical dislocation, the intact ovary was quickly removed and rinsed in pre-cooled physiological saline.

[0098] Tissue block preparation: Under a dissecting microscope, use a sharp blade to cut ovarian tissue blocks containing follicles of different sizes, controlling the size to about 5mm×5mm×3mm, to ensure that the fixative can fully penetrate.

[0099] Fixation: Immediately immerse the tissue block in 10% neutral formalin fixative. Fix for 48 hours, gently shaking during this time to ensure even fixation. This is crucial for obtaining clear tissue structures subsequently.

[0100] 2) Paraffin embedding and sectioning

[0101] Dehydration and clearing: The fixed tissue blocks were dehydrated sequentially by passing them through a gradient of alcohol (70%, 80%, 90%, 95%, 100%, 100%) for 40 minutes at each gradient, and then cleared with xylene.

[0102] Paraffin embedding: The tissue block is immersed in molten paraffin (58-60℃) 2-3 times, 1-2 hours each time, and finally embedded in paraffin.

[0103] Sectioning: Using a rotary microtome, the wax block is continuously sectioned, typically to a thickness of 4-6 micrometers. After flattening the sections in 40°C warm water, they are transferred to adhesive glass slides and dried overnight in a 60°C oven.

[0104] 3) Hematoxylin-eosin (HE) staining

[0105] Dewaxing to water: Prepare two tanks of xylene (labeled I and II respectively). First, soak the slices in tank I for 10 minutes (to wash away most of the paraffin wax), then soak them in tank II for 10 minutes (to thoroughly rinse away any remaining wax). Then, pass them through a gradient of alcohol (100%, 95%, 80%, 70%), and finally into pure water, for 2-3 minutes at each stage.

[0106] Hematoxylin staining of nuclei: Immerse in Harris hematoxylin staining solution for 5-10 minutes until the nuclei turn blue. Rinse with tap water to restore blue color.

[0107] Differentiation and blueing: Differentiate with 1% hydrochloric acid alcohol for a few seconds to remove excess staining from the cytoplasm, rinse immediately with tap water, and promote blueing with weakly alkaline water (or ammonia).

[0108] Eosin staining: Immerse in 0.5% eosin alcohol solution for 1-3 minutes to stain cytoplasm, collagen, etc., turning pink.

[0109] Dehydration and clearing mounting: The sections were sequentially immersed in 80% alcohol, 95% alcohol, 100% alcohol, and 100% alcohol for 1 minute each to achieve rapid dehydration. Then, two xylene tanks were prepared (numbered I and II respectively). The sections were first immersed in tank I for 1 minute, and then in tank II for 1 minute. The sections were then mounted using neutral resin.

[0110] 4) Observation, counting, and data analysis

[0111] Microscopic observation:

[0112] The entire ovarian follicle was scanned under low magnification (4×, 10×) to locate follicles of various stages in the ovarian cortex. High-resolution digital scan results were obtained.

[0113] 5) Key distinguishing features

[0114] Normally growing follicles: the granulosa cell layer is tightly arranged and intact, without apoptotic fragments; the oocytes have regular morphology and clear nuclei.

[0115] Atretic follicles: A large number of apoptotic cells (nuclear condensation, fragmentation) appear in the granulosa cell layer, and they are loosely arranged and broken; the oocytes atrophy or disintegrate.

[0116] Granulosa cells: located on the inner wall of the follicular cavity, surrounding the oocyte, with a round nucleus that stains dark blue (under HE staining).

[0117] 6) Quantitative analysis

[0118] Normal follicle count: On each slide, randomly select at least 5 non-overlapping fields of view (20x objective lens recommended), and count the number of all normal / atretic premature follicles within each field of view. Calculate the average number per field of view or the number per unit area, and perform intergroup comparisons.

[0119] Granulosa cell count: Select a representative growing follicle and count the number of granulosa cell nuclei per unit length (e.g., per 100 micrometers) at the same location in its granulosa cell layer (e.g., at the equator), or count the number of cells in a fixed area (e.g., 0.01 mm²). Measure 3-5 follicles for each ovarian sample and take the average.

[0120] Data processing: SPSS software was used, and independent samples t-test was employed to compare the differences between the intervention group and the control group. p<0.05 was considered statistically significant.

[0121] (3) Calculate the brooding rate and egg production of each group.

[0122] (4) Experimental results

[0123] Table 3 shows the results of bacterial abundance and broodiness incidence. The results indicate that after the intervention, the average relative abundance of Firmicutes in the cecum of hens in the experimental group was significantly higher than that in the control group (P<0.05), while the abundance of Bacteroidetes showed a decreasing trend. The broodiness incidence in the intervention group was significantly lower than that in the control group during the experimental period, and the egg production was significantly increased.

[0124] Table 3. Microbial abundance and nesting incidence in the intervention and control groups.

[0125]

[0126] Results of ovarian histological analysis are shown below Figure 3-5 The results showed that the number of normally developing follicles in the ovaries of hens in the intervention group was significantly higher than that in the control group, while the number of atretic follicles was reduced. At the same time, the number of granulosa cells around the follicles was significantly higher in the intervention group than in the control group, indicating that follicle development received more support and ovarian function was improved in the intervention group.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying the brood susceptibility of Wuliangshan Black-boned Chicken, characterized in that, Includes the following steps: (1) Obtain a sample of the cecal contents or its metagenomic DNA from the chicken to be tested; (2) Detect the relative abundance of at least two of the three phyla (Bacteroidetes, Firmicutes, and Verrucous) in the sample obtained in step (1); (3) If the relative abundance information of the bacterial phylum obtained in step (2) is analyzed and meets at least two of the following criteria (a)-c), then the chicken is determined to be susceptible to broodiness or in the broodiness period: a) The relative abundance of Bacteroidetes is higher than 35%; b) The relative abundance of Firmicutes is less than 35%; c) The relative abundance of Verrucous microbes is less than 2%.

2. The method according to claim 1, characterized in that, The method for determining the relative abundance of at least two of the three phyla (Bacteroidetes, Firmicutes, and Verrucous) in the sample obtained in step (1) is metagenomic sequencing, 16S rRNA gene sequencing, real-time quantitative PCR, or gene chip detection.

3. A method for regulating the brooding behavior of Wuliangshan black-boned chickens, characterized in that, Includes the following steps: Using the method described in claim 1, it is determined whether Wuliangshan Black-boned Chicken has broodiness or is in the broodiness period. A probiotic preparation containing Lactobacillus delbrueckii is then applied to Wuliangshan Black-boned Chicken identified as having broodiness or being in the broodiness period.

Images