KR20250088843A - Microbial gene marker (glmS) for predicting nitrogen excretion in broilers and its use - Google Patents

Abstract

The present invention relates to glmS as a microbial genetic marker for predicting nitrogen excretion in broilers and to uses thereof. By varying the breeding stage and nutritional level of broilers, the nitrogen excretion level was changed, and then the microbial diversity and distribution in the rectum or cecum of broilers under each condition were analyzed. Dominant microorganisms in the rectum and cecum were each discovered 13 species, and common dominant microorganisms in the rectum and cecum were discovered 7 species. Among them, the glmS gene was identified among the genes commonly found in representative beneficial bacteria, such as Lactobacillus crispatus, Lactobacillus johnsoni, and Faecalibacterium fraunici, and since the glmS gene was confirmed to contribute to nitrogen metabolism, it is thought that it can be used as a genetic marker for predicting nitrogen excretion, and through this, it will be possible to contribute to increasing feed utilization, improving productivity and digestibility, and reducing environmental load caused by nitrogen excretion by predicting nitrogen excretion and energy metabolism efficiency.

Description

Microbial gene marker (glmS) for predicting nitrogen excretion in broilers and its use

The present invention relates to a microbial genetic marker (glmS) for predicting nitrogen excretion in broilers and its use, and can be usefully used for predicting nitrogen excretion and energy metabolic efficiency.

The greenhouse gas emissions from the agricultural sector in our country amount to 147 million _CO2 tons, accounting for about 25% of the total greenhouse gas emissions in our country. Of these, about 39% are emitted during the livestock farming process. Nitrogen dioxide ( _N2O ) emitted from livestock manure is the second largest global warming gas after methane. Nitrogen dioxide is produced when the nitrogen component of livestock manure decomposes in the absence of oxygen. Therefore, when oxygen is supplied under anaerobic conditions, the amount of methane produced decreases, but the amount of nitrogen dioxide produced increases.

Accordingly, in order to reduce greenhouse gases in the livestock sector, methods are being implemented to reduce methane emissions through high-capacity livestock breeding, use of production enhancers, or feed management such as pH and digestion time control or fat supply. In addition, efforts are being made to reduce methane and nitrogen dioxide emissions by avoiding anaerobic decomposition of organic matter and decomposing it in an aerobic state in the treatment of livestock manure.

As they are in excess, the excess elements contaminate groundwater. These excess elements can leach through the soil, contaminating groundwater.

Nitrogen is usually supplied through protein in the feed, and excess nitrogen is transferred to the liver and converted to uric acid for excretion. Nitrogen in broiler feces is excreted as uric acid (approximately 50%) and undigested protein, and _NH3 is formed in the process of microorganisms decomposing uric acid in broiler feces.

Feed costs account for 70% of broiler breeding costs. The growth of broilers is related to feed efficiency, which is measured in units of weight gain through feed intake. In order to increase feed efficiency by improving the bioavailability of nutrients for each broiler, it is necessary to accurately measure the amount of feed required for growth. If the protein requirement (approximately 20% of the feed weight) that affects feed price is exceeded, the function of the digestive organs (small intestine, ileum, etc.) decreases and the utilization of protein, amino acids, and nitrogen decreases. As a result, nitrogen excretion increases.

An effective way to reduce nitrogen excretion in the poultry industry is to reduce the level of crude protein (CP) or amino acids in the feed (Bregendahl et al., 2002; Hernandez et al., 2012; Lemme et al., 2019; Attia et al., 2021). In the case of adult animals, there is a tendency to balance the protein in the current state, so excessive protein is decomposed and excreted as nitrogen. Therefore, research is being conducted on methods to reduce urinary nitrogen in livestock by adjusting the protein component of the feed composition.

In previous studies related to this, Korean Patent Publication No. 10-2008-0088128 entitled “Livestock Feed Composition” discloses the effect of reducing feed costs and nitrogen emissions by reducing crude protein in pig feed, and Kamiya et al. discloses the effect of reducing urinary nitrogen emissions by lowering the crude protein content and adding amino acid additives to cattle feed.

In addition, a paper analyzing the expression of genes related to energy metabolism in the cecum of 1-week-old broilers according to the addition of phosphatase (Journal of the Korean Academia-Industrial Cooperation Society, Vol. 24, No. 6, 2023) disclosed that transcriptome sequence analysis was performed using cecum samples of 1-week-old broilers with controlled nutritional levels and the functions of differentially expressed genes were analyzed. In addition, Korean Patent Publication No. 10-2022-0069626 analyzed the genetic information of microorganisms existing in the cecum of poultry and described that this can be used to predict information about the habitat, but there is no mention of the glmS gene. It is already widely known that the glmS gene is involved in the hexosamine synthesis process, but there has been no study on the correlation between the glmS gene and prediction of nitrogen excretion in livestock.

Accordingly, the inventors of the present invention discovered common dominant microorganisms in the cecum and rectum (feces) of broilers according to a decrease in the amount of crude protein added to the feed at each breeding stage (1, 3, and 5 weeks of age) of broilers, and completed the present invention by confirming a microbial gene (glmS), which is a nitrogen metabolism-related gene, commonly existing in Lactobacillus crispatus, Lactobacillus johnsonii , and Faecalibacterium prausnitzii , which are representative beneficial bacteria among the seven common dominant microorganisms discovered. Therefore, the microbial gene glmS for predicting nitrogen excretion in broilers of the present invention is expected to contribute to increasing feed utilization, improving productivity and digestibility, and reducing environmental load caused by nitrogen excretion by predicting nitrogen excretion and energy metabolism efficiency.

The present invention relates to a microbial gene glmS for predicting nitrogen excretion in broilers and a use thereof. By utilizing the microbial gene glmS, which is commonly possessed by common dominant microorganisms existing in the cecum and rectum of broilers, it can contribute to reducing the feed efficiency, productivity, and environmental load caused by nitrogen excretion of broilers in the future.

To achieve the above purpose,

The present invention provides a genetic marker composition for predicting nitrogen excretion in broilers including the glmS gene.

In addition, the present invention provides a composition for predicting nitrogen excretion in broilers, comprising a preparation that amplifies a microbial gene, glmS.

In addition, the present invention provides a kit for predicting nitrogen excretion comprising a composition comprising an agent that amplifies the above gene.

The present invention also provides a method for screening feed or additives that reduce nitrogen excretion in broilers.

The present invention relates to a genetic marker for predicting nitrogen excretion in broilers including the glms gene, and specifically, the microbial gene glmS, which is commonly present in common dominant microorganisms in the caecum and rectum according to a decrease in the crude protein content in the feed at each breeding stage of broilers, was discovered, and this is a gene related to nitrogen metabolism and was confirmed to affect nitrogen excretion in broilers, thereby contributing to predicting nitrogen excretion in advance, thereby improving feed efficiency, productivity, and reducing environmental load caused by nitrogen excretion.

Figure 3 shows the results of microbial distribution in the cecum and rectum after a feeding experiment with reduced nutritional levels at each breeding stage of broilers.
Figure 4 is a diagram showing the results of differential microbial excavation according to nutritional level at 1 week of age after a feeding experiment with reduced nutritional level in each breeding stage of broilers.
Figure 5 shows the results of differential microbial excavation according to nutritional level at 3 weeks of age after a feeding experiment with reduced nutritional level feed for each breeding stage of broilers.
Figure 6 is a diagram showing the results of differential microbial excavation according to nutritional level at 5 weeks of age after a feeding experiment with reduced nutritional level in each breeding stage of broilers.
Figure 7 shows the results of common differential microbial excavation in the cecum after a feeding experiment with reduced nutritional levels at each breeding stage of broilers.
Figure 8 shows the results of common differential microbial excavation in the rectum after a feeding experiment with reduced nutritional levels at each stage of broiler breeding.
Figure 9 shows the results of differential microbial excavation in the cecum and rectum after a feeding experiment with reduced nutritional levels at each breeding stage of broilers.
Figure 10 shows the results of analyzing genes in Lactobacillus crispatus, one of the common dominant microorganisms in broilers, using the KEGG database.
Figure 11 shows the results of analyzing genes in Lactobacillus johnsonii , one of the common dominant microorganisms in broilers, using the KEGG database.
Figure 12 shows the results of analyzing genes in Faecalibacterium prausnitzii , one of the common dominant microorganisms in broilers, using the KEGG database.

The present invention provides a genetic marker composition for predicting nitrogen excretion in broilers including the glmS gene.

The above gene is a gene consisting of a sequence described by sequence number 1.

The above gene can be expressed in microorganisms present in the cecum, rectum, or feces of broilers.

The above microorganism may be a common dominant microorganism according to the breeding stage and nutritional level of the broiler, preferably a beneficial bacterium, more preferably a lactic acid bacterium, and may be at least one selected from the group consisting of Lactobacillus crispatus, Lactobacillus johnsonii , and Faecalibacterium prausnitzii .

The above glmS is a gene that expresses glutamine-fructose-6-phosphate aminotransferase, and the protein expressed by the gene converts fructose-6P into glucosamine-6P, and uses glutamine as a nitrogen source in the process.

The above nitrogen may be, but is not limited to, nitrogen in urine or nitrogen in feces.

The above nitrogen excretion is that some of the protein consumed in feed is absorbed and used to synthesize body protein, and the unabsorbed nitrogen is excreted through feces and urine, and is discharged in the form of uric acid (C ₅ H ₄ N ₄ O ₃ ). Uric acid is decomposed by microorganisms in the wet litter inside the chicken house and converted to ammonia, causing negative effects such as environmental pollution.

The above nitrogen excretion is calculated by calculating the feed intake by calculating the remaining feed amount compared to the feed provided. A manure collector is installed in the broiler cage to collect the manure, measure the weight, and analyze the feed components (moisture, fat, protein, and ash) and the nitrogen content in the manure using an AOAC-approved analysis method. The nitrogen excretion rate can be measured by calculating the nitrogen content in the manure compared to the nitrogen consumed.

The above broiler refers to chicken for meat, and as long as it is chicken used for food, the breed is not limited, but may be Cornish, Cochin, Brahma, Bresse, Pitou de Calella, Goum chicken, Ross 308, and preferably Ross 308.

The above broiler may be 1 to 5 weeks old, 1 to 3 weeks old, or 3 to 5 weeks old, but is not limited thereto.

In addition, the present invention provides a composition for predicting nitrogen excretion comprising a preparation that amplifies the glmS gene.

As agents for amplifying the above glmS gene, primers, probes, and antisense nucleotides that can specifically detect organic biomolecules such as nucleic acids can be used.

In the present invention, the primer refers to a sequence of 7 to 50 nucleic acids that can form complementary base pairs with the template strand and function as a starting point for copying the template strand. The primer is usually synthesized, but can also be used from naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but it is sufficiently complementary so that it can hybridize with the template. It is preferable that the primer specifically binds to and specifically detects the gene of the present invention and does not bind to the sequence of other genes. Additional features that do not change the basic properties of the primer can be incorporated.

The above primers are complementary to the sequence of glmS and any primer designed to amplify it can be used.

The above probe refers to a nucleic acid fragment, which is short, several bases or long, several hundred bases, and can specifically bind to a target substance to be detected in a sample, and refers to a substance that can specifically confirm the presence of a target substance in a sample through said binding. The type of the probe is not limited to a substance commonly used in the art, and may be, for example, PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA.

The above probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc., and can be labeled with biotin, FITC, rhodamine, DIG (digoxigenin), etc., or labeled with a radioactive isotope, etc.

In the present invention, the antisense nucleotide means an oligomer having a sequence of nucleotide bases and a backbone between subunits that allows the antisense oligomer to hybridize with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.

The composition may further comprise a ligand capable of specifically binding to the agent that amplifies the gene.

The above ligand may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent substance, a radioactive isotope or a colloid, and a ligand treated with streptavidin or avidin.

The above composition may contain, in addition to the amplifying agent and ligand as described above, distilled water or a buffer to maintain their structure stable.

The composition may include a reverse transcriptase polymerase, a DNA polymerase, a cofactor such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP. Various DNA polymerases can be used in the amplification step of the present invention to amplify the reverse transcribed cDNA, examples of the DNA polymerase include the Klenow fragment of E. coli DNA polymerase I, thermostable DNA polymerase or bacteriophage T7 DNA polymerase. The polymerase can be isolated from the bacteria themselves, purchased commercially or obtained from cells expressing high levels of the cloned gene encoding the polymerase.

In addition, the present invention provides a kit for predicting nitrogen excretion comprising a composition comprising an agent that amplifies the above gene.

The above composition may have the characteristics as described above.

The kit of the present invention can be manufactured by a conventional manufacturing method known to those skilled in the art, and may further include a buffer, a stabilizer, an inactive protein, etc.

The above kit can use a high throughput screening (HTS) system using a fluorescence method performed by detecting the fluorescence of a fluorescent substance attached as a detector to search for the amount of a detection reagent, a radiological method performed by detecting the radiation of a radioisotope attached as a detector, a surface plasmon resonance (SPR) method that measures the change in surface plasmon resonance in real time without labeling the detector, or a surface plasmon resonance imaging (SPRI) method that confirms by imaging the SPR system.

In the present invention, the kit may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (multiple reaction monitoring) kit.

The kit of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

For example, the kit of the present invention may further include essential components necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a primer pair specific for a genetic marker. The primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene and may have a length of about 7 bp to 50 bp, for example, a length of about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may include a test tube or other appropriate container, a reaction buffer (having various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

In addition, the kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, etc. for producing a fluorescent label probe. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In the present invention, the level of nitrogen excretion of broilers can be predicted using the kit, and further, broilers with high energy metabolism efficiency can be predicted at an early stage.

In addition, the present invention

(a) a step of feeding general feed according to the rearing stage to the control group broilers and feeding test feed to be screened or feed with the test target additive added to general feed according to the rearing stage to the treatment group broilers;

(b) a step of isolating total RNA from biological samples of control and treated broilers;

(c) a step of amplifying the glmS gene from the isolated total RNA; and

(d) a step of determining the difference in the glmS gene level in the treatment group by comparing it with the glmS gene level in the control group;

Provided is a method for screening feed or additives capable of reducing nitrogen excretion in broilers, including:

The above feed may be a general poultry breeding feed, and preferably may be a pure feed composition without antibiotics added. The above feed contains essential nutrients that must be supplied for the maintenance and growth of poultry, and may contain ingredients that are generally added to feed, such as carbohydrates, proteins, fats, minerals, vitamins, minerals, and water, and all feed raw materials used in general poultry feed, such as soybean meal, soybeans, corn, potatoes, rye, and wheat, may be used.

The above poultry refers to birds raised by humans to obtain useful livestock products for humans, such as meat, eggs, hair, and feathers, and may include chickens, turkeys, geese, domestic ducks, hen's hen, pigeons, quail, etc., and preferably refers to edible poultry, and most preferably refers to broiler chicken.

The above additive is intended for feeding to poultry, and refers to a substance added to feed other than feed, and is additionally included in feed for the purpose of improving digestibility, promoting growth, or improving the intestinal environment.

The above control group may be broilers fed feed with added crude protein and phosphatase in the feed according to the feeding standards for each rearing stage. According to the feeding standards for each rearing stage, 22% of crude protein is to be fed to 1-week-old broilers, 20% of crude protein is to be fed to 3-week-old broilers, and 18% of crude protein is to be fed to 5-week-old broilers, and phosphatase can be fed at a concentration of 1000 ppm to broilers in all rearing stages.

The above biological sample is any material obtained from or derived from a broiler, which may be a rectum, cecum, or feces.

In the present invention, if the glmS gene level of the microorganisms in the test target broiler is higher than the glmS gene level of the microorganisms in the control group, it can be determined that nitrogen excretion is low. Here, the criterion for determining 'high' means that the glmS gene level observed in the treatment group is 1.2 times, 1.5 times, 1.8 times, or 2.0 times higher than the glmS gene level observed in the control group.

In addition, molecular and immunological methods widely used in the art can be used to detect the microbial genes of the present invention.

In a specific embodiment of the present invention, as a result of analyzing microorganisms according to nutritional levels (crude protein and phosphatase) in each growth stage of broilers, the results of microbial diversity by growth stage were 455 species at 1 week of age, 322 species at 3 weeks of age, and 491 species at 5 weeks of age (Fig. 2). It was found that there were more microorganisms in the rectum than in the cecum of broilers (Fig. 3), and it was found that microorganisms in the cecum and rectum of broilers were abundant in the order of Sclerotia (70%), Bacteroides (20%), and Proteobacteria (4%).

In addition, as the crude protein content in broiler feed decreased, the dominant bacteria, the bacterium Fusarium, increased and the ratio of Bacteroides/Fusarium decreased. Bacteroides is a genus of Gram-negative, anaerobic bacteria known to mainly perform nitrogen fixation in the roots of legume crops.

As a result of discovering differential microorganisms according to nutritional level at each stage of broiler breeding, it was confirmed that 1-week-old broilers had low digestion, absorption, and utilization rates of feed nutrients due to underdeveloped digestive organs and insufficient secretion of digestive enzymes, and 71 species of differential microorganisms were discovered (Fig. 4).

At 3 weeks of age, the digestive tract of broilers was fully developed, and the microbial species that dominated the cecum during the early growth stage were the most diverse, and 57 species of differential microorganisms were identified (Fig. 5). At 5 weeks of age, the growth was over 80%, the microbial diversity was reduced, and 28 species of differential microorganisms were identified (Fig. 6).

According to the breeding stage and nutritional level of broilers, samples were collected from the cecum of broilers to identify the dominant microorganisms. As a result, 13 species of dominant microorganisms were identified : Alistipes putredinis, Lactobacillus crispatus, Lactobacillus johnsonii, Ligilactobacillus salivarius, Flintibacter butyricus, Blautia glucerasea, Merdimonas faecis, Anaerobacterium chartisolvens, Faecalibacterium prausnitzii, Fournierella massiliensis, Papillibacter cinnamivorans, Ruminococcoides bili, and Clostridium spiroforme (Fig. 7). As a result of identifying the dominant microorganisms by collecting samples from the rectum (feces) of broilers, 13 species of dominant microorganisms were identified : Bacteroides fragilis, Lactobacillus crispatus, Lactobacillus johnsonii, Butyricicoccus pullicaecorum, Clostridium oryzae, Blautia glucerasea, Clostridium hylemonae, Anaerobacterium charisolvens, Faecalibacterium prausnitzii, Fournierella massiliensis, Romboutsia timonensis, Clostridium spiroforme, and Escherichia fergusonii were discovered (Figure 8).

As a result of discovering common dominant microorganisms in the cecum and rectum according to the above results, a total of 7 types of microorganisms were discovered, including Lactobacillus crispatus, Lactobacillus johnsonii, Blautia glucerasea, Anerobacterium chartisolvens, Faecalibacterium prausnitzii, Fournierella massiliensis, and Clostridium spiroforme (Fig. 9).

Among the seven types of microorganisms mentioned above, Lactobacillus crispatus, Lactobacillus johnsonii , and Faecalibacterium prausnitzii, which are known as representative beneficial bacteria, were found to have the glmS gene, which is a microbial gene involved in the process of producing antibacterial compounds that inhibit protein synthesis in bacteria through the N-acetylglucosamine and sialic acid amine-containing sugar derivative (amino sugar) reaction pathway in the carbohydrate derivative metabolism process (Figs. 10 to 12). The glmS gene is known to affect nitrogen excretion by removing nitrogen from amino acids through its involvement in the transamination reaction.

Accordingly, the inventors of the present invention discovered common dominant microorganisms in the cecum and rectum (feces) of broilers according to a decrease in the amount of crude protein added to the feed at each breeding stage (1, 3, and 5 weeks of age) of broilers, and confirmed that the glmS gene, a gene involved in nitrogen metabolism, was commonly included in the major beneficial bacteria among these, thereby completing the present invention.

Hereinafter, the present invention will be described in detail through examples.

However, the following examples are only intended to specifically illustrate one aspect of the present invention, and the present invention is not limited to the following examples.

<Example 1> Analysis of cecum and rectal (fecal) microorganisms according to feed with reduced nutritional level at each breeding stage of broilers

To investigate the distribution of microorganisms according to nutritional levels (crude protein and phosphatase) at each rearing stage of broilers, a total of 144 male broilers (Ross 308 breed) were divided into four groups by age (1, 3, and 5 weeks old), and each group was divided into 12 broilers to conduct a test (Fig. 1).

As a treatment group, the 1-week-old group was fed a feed composition containing 20% crude protein content and 500 ppm phosphatase, the 3-week-old group was fed a feed composition containing 18% crude protein content and 500 ppm phosphatase, and the 5-week-old group was fed a feed composition containing 16% crude protein content and 500 ppm phosphatase. The control group was fed a feed composition containing 2% more crude protein added than the treatment group at each breeding stage and containing 1000 ppm phosphatase.

We obtained cecum and rectal (feces) samples according to the breeding stage and nutritional level of broilers, analyzed the 16S rRNA base sequence information (hypervariable region) and the microbial diversity of each treatment group, and utilized the existing QIIMEII and MG-RAST pipelines for the analysis.

Specifically, each read was separated into sample units, demultiplexed, and noise and duplicate sequences were removed (denosing, ASVs). Then, they were compared and analyzed with 16S rRNA databases such as SILVA, Greengenes, and RDP. The relative abundance was converted to determine the distribution within the microbial community through taxonomic arrangement at the phylum and family levels. Alpha-diversity was used to measure species richness and diversity, and beta-diversity was used to measure the similarity between different groups, and the mapped sequence information was used to analyze evolutionary relationships and compare microbial diversity (OTUs, ASVs, etc.).

As a result of analyzing the microorganisms according to the nutritional level (crude protein and phosphatase) of each growing stage of broilers, the microbial diversity results by growth stage were 455 species at 1 week of age, 322 species at 3 weeks of age, and 491 species at 5 weeks of age (Fig. 2). It was found that there were more microorganisms in the rectum than in the cecum of broilers (Fig. 3), and the microorganisms in the cecum and rectum of broilers were abundant in the order of Sclerotia (70%), Bacteroides (20%), and Proteobacteria (4%).

In addition, as the crude protein content in broiler feed decreased, the dominant bacteria, the bacterium Fusarium, increased and the ratio of Bacteroides/Fusarium decreased. Bacteroides is a genus of Gram-negative, anaerobic bacteria known to mainly perform nitrogen fixation in the roots of legume crops.

As a result of discovering differential microorganisms according to nutritional level in each rearing stage of broilers, it was confirmed that 1-week-old broilers had low digestion, absorption, and utilization rates of feed nutrients due to underdeveloped digestive organs and insufficient secretion of digestive enzymes, and 71 species of differential microorganisms were discovered (Fig. 4). 3-week-old broilers had sufficiently developed digestive tracts, and the most diverse microorganism species that dominated the cecum during the early growth stage were discovered, and 57 species of differential microorganisms were discovered (Fig. 5). 5-week-old broilers had grown more than 80%, had a reduced diversity of microorganisms, and 28 species of differential microorganisms were discovered (Fig. 6).

According to the breeding stage and nutritional level of broilers, samples were collected from the cecum of broilers to identify the dominant microorganisms. As a result, 13 species of dominant microorganisms were identified : Alistipes putredinis, Lactobacillus crispatus, Lactobacillus johnsonii, Ligilactobacillus salivarius, Flintibacter butyricus, Blautia glucerasea, Merdimonas faecis, Anaerobacterium chartisolvens, Faecalibacterium prausnitzii, Fournierella massiliensis, Papillibacter cinnamivorans, Ruminococcoides bili, and Clostridium spiroforme (Fig. 7). As a result of identifying the dominant microorganisms by collecting samples from the rectum (feces) of broilers, 13 species of dominant microorganisms were identified : Bacteroides fragilis, Lactobacillus crispatus, Lactobacillus johnsonii, Butyricicoccus pullicaecorum, Clostridium oryzae, Blautia glucerasea, Clostridium hylemonae, Anaerobacterium charisolvens, Faecalibacterium prausnitzii, Fournierella massiliensis, Romboutsia timonensis, Clostridium spiroforme, and Escherichia fergusonii were discovered (Figure 8).

As a result of discovering common dominant microorganisms in the cecum and rectum according to the above results, a total of 7 types of microorganisms were discovered, including Lactobacillus crispatus, Lactobacillus johnsonii, Blautia glucerasea, Anerobacterium chartisolvens, Faecalibacterium prausnitzii, Fournierella massiliensis, and Clostridium spiroforme (Fig. 9).

Therefore, it is thought that the above seven common dominant microorganisms are related to the nitrogen metabolism process in broilers.

<Example 2> Identification of common genes in common dominant microorganisms and analysis of metabolic pathway network

To identify genetic markers found in common dominant microorganisms, databases such as KEGG biochemical pathway (KEGG, Reaction pathway, WIT/EMP, Biochemical pathway, Ecocyc MetPath, MetaCyc, and Biocyc) were utilized to extract information on genes, intermediates, enzymes, ligands, and reactions related to amino acid and nitrogen metabolic pathways. The web application KEGG mapper was used to network map and visualize the extracted data, KEGG pathComp tabulates information on reaction pathways applying gene information of each microbial community, and K-vit is used to compare metabolic pathways in other species. In addition, Blast2Go, Cytoscape, and Pathview (R/Bioconductor) were utilized.

Among the seven common dominant microorganisms, representative beneficial bacteria, Lactobacillus crispatus , Lactobacillus johnsonii , and Faecalibacterium prausnitzii , were confirmed to have the glmS gene in common, which is involved in the process of producing antibacterial compounds that inhibit protein synthesis in bacteria through the N-acetylglucosamine and sialic acid amine-containing sugar derivative (amino sugar) reaction pathway in the carbohydrate derivative metabolism process (Figs. 10 to 12). It is thought that the microbial gene glmS affects nitrogen excretion by removing nitrogen from amino acids through a transamination reaction, and it was determined that the glmS gene can be used as a marker for predicting nitrogen excretion in broilers.

Accordingly, the inventors of the present invention discovered common dominant microorganisms in the cecum and rectum (feces) of broilers according to a decrease in the amount of crude protein added to the feed at each breeding stage (1, 3, and 5 weeks of age) of broilers, and confirmed that representative beneficial bacteria among them commonly possess the glmS gene and that the gene is a gene involved in nitrogen metabolism, thereby completing the present invention. Therefore, the microbial gene glmS marker for predicting nitrogen excretion in broilers of the present invention can be usefully used to increase feed utilization, improve productivity and digestibility, and reduce environmental load caused by nitrogen excretion by predicting nitrogen excretion and energy metabolism efficiency.

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Claims (15)

A genetic marker composition for predicting nitrogen excretion in broilers comprising the glmS gene.
In the first paragraph,
A genetic marker composition, wherein the glmS gene is composed of a sequence described in sequence number 1.
In the first paragraph,
A genetic marker composition, wherein the glmS gene is expressed in microorganisms present in a biological sample of broiler chickens.
In the third paragraph,
A genetic marker composition, wherein the biological sample is at least one selected from the group consisting of cecum, rectum, and feces of broiler chickens.
In the third paragraph,
A genetic marker composition, wherein the microorganism is at least one selected from the group consisting of Lactobacillus crispatus, Lactobacillus johnsonii , and Faecalibacterium prausnitzii .
In the first paragraph,
A genetic marker composition, wherein the broiler is at least one selected from the group consisting of Cornish, Cochin, Brahma, Bresse, Pitou de Calella, Goum, and Ross 308.
In Article 6,
A genetic marker composition, wherein the above broiler is Ross 308.
A composition for predicting nitrogen excretion in broilers, comprising a preparation that amplifies a microbial gene, glmS.
In Article 8,
A composition wherein the above formulation is a primer, a probe, or an antisense nucleotide.
In Article 8,
A composition, wherein the composition further comprises a ligand capable of specifically binding to an agent that amplifies a gene.
A kit for predicting nitrogen excretion in broilers, comprising the composition of claim 8.
(a) a step of feeding general feed according to the rearing stage to the control group broilers and feeding test feed to be screened or feed with the test target additive added to general feed according to the rearing stage to the treatment group broilers;
(b) a step of isolating total RNA from biological samples of control and treated broilers;
(c) a step of amplifying the glmS gene from the isolated total RNA; and
(d) a step of determining the difference in the glmS gene level in the treatment group by comparing it with the glmS gene level in the control group;
A method for screening feed or additives for reducing nitrogen excretion in broilers, comprising:
In Article 12,
A method wherein the biological sample is cecum, rectum, or feces.
In Article 12,
A method for determining that a higher level of glmS gene in a treated broiler compared to the level of glmS gene in a control group reduces the level of nitrogen excretion in the broiler.
In Article 14,
A method in which a judgment criterion is set in advance in the step (d) above, and the nitrogen excretion level is judged to be reduced if the glmS gene level in the treated broiler is 1.2 times or more compared to the glmS gene level in the control group.