Determination method, domestication agent, pet food, feed for domestic animals, domestication method, sociability improvement agent, and food product

Abstract

Provided is a determination method for a domesticated animal, comprising a step for detecting, in a biological sample of said animal, limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or analogs thereof.

Description

Determination method, domestication agent, pet food, livestock animal feed, domestication method, sociality improver, and food

[0001] The present invention relates to a determination method, a domestication agent, a pet food, a feed for livestock animals, a domestication method, a sociality improver, and a food.

[0002] The docility that animals show toward humans is an important behavioral characteristic for keeping livestock animals in captivity. However, in reality, many livestock animals show aggression toward humans, and there is a constant stream of accidents in the field, such as bites and contact with large livestock. Up until now, the improvement of docility in animals has been achieved through breeding techniques (see, for example, Non-Patent Document 1).

[0003] EO Price, Behavioral development in animals undergoing domestication. Applied Animal Behavior Science 65, 245-271 (1999).

[0004] However, this method requires many generations of crossbreeding, which takes a long time, and there is also the problem that genetic improvement of already established varieties is difficult due to the brand value of those varieties.

[0005] The present invention has been made in consideration of the above circumstances, and provides a technology that makes it possible to easily improve the behavior of individual animals through feed, etc., and that can be applied to a wide range of uses, such as livestock farming and companion animal breeding.

[0006] That is, the present invention includes the following aspects. [1] A method for determining whether a domesticated animal is present, the method comprising the step of detecting Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactate, oxytocin, or an analog thereof in a biological sample from the animal. [2] The method according to [1], the method comprising the step of quantifying the amount of Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactate, oxytocin, or an analog thereof present in the biological sample from the animal. [3] The method according to [1] or [2], wherein the biological sample is feces or blood. [4] The method according to any one of [1] to [3], wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvate. [5] A domestication agent containing Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof as an active ingredient. [6] The domestication agent according to [5], wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvic acid. [7] A pet food or livestock animal feed containing the domestication agent according to [5] or [6]. [8] A method for domesticating an animal, comprising a step of administering Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof to the animal. [9] The domestication method according to [8], wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvic acid.

[10] A sociality-enhancing agent comprising Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof as an active ingredient.

[11] The sociality-enhancing agent according to

[10] , wherein the improvement in sociality is at least one selected from the group consisting of improvement in sociability, improvement in communication ability, improvement in language disorders, improvement in emotional disorders, improvement in obsessive behavior, improvement in attention deficit hyperactivity disorder, and improvement in learning disabilities.

[12] The sociality-enhancing agent according to

[10] or

[11] , wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvic acid.

[13] A food product containing the sociability-enhancing agent according to any one of

[10] to

[12] .

[14] A method for determining human sociability, comprising the step of detecting Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof, in a biological sample from the human.

[15] The method according to

[14] , comprising the step of quantifying the amount of Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof present in the biological sample from the human.

[16] The method according to

[14] or

[15] , wherein the biological sample is feces or blood.

[17] The method according to any one of

[14] to

[16] , wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvate.

[18] The method according to any one of

[14] to

[17] , wherein the improvement in sociality is at least one selected from the group consisting of improvement in sociability, improvement in communication ability, improvement in language disorders, improvement in emotional disorders, improvement in obsessive behavior, improvement in attention deficit hyperactivity disorder, and improvement in learning disabilities.

[0007] According to the present invention, a technique can be provided that makes it possible to easily improve the behavior of individual animals.

[0008] 1 is a schematic diagram of the production of wild heterostock strains. (A) A diagram showing the experimental timeline. (B) A diagram showing the active obedience test. (C) A diagram showing the passive obedience test. (D) A diagram showing the stay-on-hand test. (A) Results of the heading test in the active obedience test. (B) Results of the contact test in the active obedience test. (C) Results of the movement test in the active obedience test. (D) Results of the jump test in the active obedience test. (A) Results of the heading test in the passive obedience test. (B) Results of the acceptance test in the passive obedience test. (C) Results of the movement test in the passive obedience test. (D) Results of the jump test in the passive obedience test. (A) Results of the stay-on-hand test. (B) Results of the measurement of plasma oxytocin levels in each group. (C) A graph examining the correlation between active contact time and plasma oxytocin levels. In Figures 2 to 5, N = 80 (20 animals per group, 10 males and 10 females). ＊ p<0.05); ＊＊ p<0.01; ＊＊＊p<0.001). Bar graphs show the mean ±SD of individual data points. Phylogenetic tree of the 374 MAGs generated. All 374 MAGs are arranged in this phylogenetic tree generated by the GTDB output. The innermost circle represents the different phyla. The second circle indicates the size of each MAG. The third circle indicates CheckM completeness, and the fourth circle indicates CheckM contamination. All novel MAGs are indicated by a star. Graph showing phylum-level relative abundance across all groups. The five most abundant phyla are shown. The complete dataset includes 11 phyla, 21 classes, 45 orders, 103 families, and 339 genera. The diversity of the gut microbiota is similar across groups. (A) Ven diagram of 665 bacterial species present in the gut of WHS mice. (B) Graph showing beta diversity based on Bray-Curtis dissimilarity. (A) Graph showing Chao1 diversity. (B) Graph showing Shannon diversity. Pearson correlation distance (result of random forest analysis) between the top 40 significantly different taxa and hierarchical clustering using the scores of the amenability test parameters. Significant correlations are marked with "*". (A) Result of association analysis in MaAsLin2 between S2 group and bacterial abundance. (B) Result of association analysis in MaAsLin2 between S1 group and bacterial abundance. (A-B) Graph of Limosilactobacillus reuteri generated in MaAsLin2. (A-B) Graph of Candidatus Gallimonas intestinalis generated in MaAsLin2. N=80 (20 animals per group, 10 males, 10 females). ( ＊ p<0.05); ＊＊ p<0.01; ＊＊＊p<0.001). Figure 16 shows a heat map of hierarchical clustering using Pearson correlation distance for the 70 identified metabolites. (A) Measurement of plasma pyruvate. (B) Measurement of plasma choline before FDR correction in MaAsLin2. (A) Measurement of plasma putrescine before FDR correction in MaAsLin2. (B) Measurement of plasma thymidine before FDR correction in MaAsLin2. Measurement of plasma lactate before FDR correction in MaAsLin2. Figures 16 and 17 show N=12 (6 animals per group). Maximum likelihood phylogenetic tree of the 16S rDNA region. Different species of the genus Limosilactobacillus were used for identification, with Lactobacillus helveticus used as the outgroup. 1000 bootstraps were performed, and bootstrap values ​​above 70% are indicated numerically. The bar graphs show the secretion of pyruvate and lactate in GAM medium by different colonies of L. reuteri and L. helveticus isolated in the present invention. (A) Scheme of bacterial administration to control mice via drinking water. (B) Evaluation results of active contact after bacterial administration. (A) Evaluation results of active heading after bacterial administration. (B) Evaluation results of the hand-stay test after bacterial administration. (A) Evaluation results of passive heading after bacterial administration. (B) Evaluation results of passive acceptance after bacterial administration. (A) Evaluation results of serum pyruvate levels after bacterial administration. (B) qRT-PCR quantification results of L. reuteri present in feces. (A) qRT-PCR quantification results of L. helveticus present in feces. (B) Evaluation results of serum oxytocin concentration. In Figures 19(B), 20, 21, 22(B), and 23(A), N = 48 (12 animals in each group, 6 males and 6 females), in Figure 22(A), N = 40 (10 animals in each group, 5 males and 5 females), and in Figure 23(B), N = 36 (12 animals in each group, 6 males and 6 females) ( ＊ p<0.05; ＊＊ p<0.01; ＊＊＊ p<0.001). Bar graphs show the mean±SD of individual data points.

[0009] <<Method for determining whether a domesticated animal is a domesticated animal>> In one embodiment, the present invention provides a method for determining whether a domesticated animal is a domesticated animal, the method comprising the step of detecting Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactate, oxytocin, or an analog thereof in a biological sample from the animal.

[0010] As described in the Examples below, the inventors believed that intestinal bacteria have some effect on docility and conducted an analysis using mice. Selective breeding for docility was performed over 27 generations to establish two outbred stock groups exhibiting high docility, and two control groups, which were randomly mated as controls. Metagenomic analysis of the intestinal microbiota was performed using feces obtained from these mice. The results confirmed that Limosilactobacillus reuteri was present at higher densities in the docile groups than in the control groups.

[0011] Furthermore, metabolomic analysis using plasma samples showed that pyruvate and oxytocin concentrations were significantly higher in the obedient group than in the control group.

[0012] Examples of biological samples from animals to be tested include blood, feces, urine, saliva, sweat, tissue exudate, etc., and are selected depending on the test item. For detection of Limosilactobacillus reuteri or its metabolites, the biological sample is preferably feces or urine, while for detection of pyruvic acid, lactic acid, oxytocin, or analogs thereof, the biological sample is preferably blood. Among blood samples, examples include serum and plasma, with plasma being preferred.

[0013] Methods for testing Limosilactobacillus reuteri or its metabolites are not particularly limited, and examples include methods using specific antibodies and methods analyzing bacteria-specific gene sequences. Examples of methods for analyzing bacteria-specific gene sequences include methods analyzing 16S ribosomal RNA or 16S ribosomal DNA. Specifically, a fragment of 16S ribosomal DNA or 16S ribosomal RNA may be amplified by PCR using primers and the amplified product may be analyzed. Alternatively, a probe complementary to the bacteria-specific 16S ribosomal DNA or 16S ribosomal RNA gene sequence may be used for analysis by hybridization. From the perspective of quantitative analysis, it is preferable to amplify a fragment of 16S ribosomal DNA or 16S ribosomal RNA by PCR and analyze the amplified product. Specific quantitative methods include next-generation sequencing (NGS) and real-time PCR (RT-PCR).

[0014] Furthermore, pyruvate secretion may be measured as a method for testing Limosilactobacillus reuteri or its metabolites. As described below in the Examples, L. reuteri strains were isolated from the cecal material and feces of a selected group of WHS mice, and strains secreting pyruvate were identified by biochemical assay. Furthermore, mice treated with pyruvate-secreting L. reuteri NIG-A41 showed significantly increased activity and docility. The method for measuring pyruvate secretion is not particularly limited, and examples include ELISA, mass spectrometry, chromatography, immunoassay, or a combination thereof. In other words, in this embodiment, the Limosilactobacillus reuteri used for the assessment is preferably a strain that highly secretes pyruvate. In this embodiment, a high pyruvate secretion strain preferably means a strain that secretes pyruvate at a level of 1 nmol / ml or more, more preferably 2 nmol / ml or more, and particularly preferably 5 nmol / ml or more, when cultured in GAM medium at 37°C for 24 hours under anaerobic conditions.

[0015] The method for testing pyruvic acid, lactic acid, or an analog thereof is not particularly limited, and examples thereof include mass spectrometry, chromatography, immunoassay, or a combination thereof. Examples of analogs of pyruvic acid or lactic acid include metabolites of pyruvic acid or lactic acid in vivo.

[0016] Animals that can be subjected to the determination method of this embodiment include pets and livestock animals such as cats, dogs, horses (especially racehorses), monkeys, cows, sheep, pigs, goats, rabbits, hamsters, guinea pigs, rats, mice, chickens, and quails.

[0017] In the determination method of this embodiment, it is preferable to further quantify the amount of Limosilactobacillus reuteri or its metabolites, or pyruvic acid, lactic acid, oxytocin, or their analogs in the sample. For example, by comparing the amount of Limosilactobacillus reuteri or its metabolites, or pyruvic acid, lactic acid, oxytocin, or their analogs in the sample with the control amount in a sample from a control animal known to be domesticated, the target animal can be determined to be domesticated if the amount in the sample matches or is close to the control amount in the control animal known to be domesticated. Furthermore, for example, by comparing the abundance of Limosilactobacillus reuteri or its metabolites, or pyruvate, lactate, oxytocin, or their analogs in a sample with the control abundance in a sample of a control animal known to be undomesticated, if the abundance in the sample is greater than the control abundance in the control animal known to be undomesticated, it can be determined that the target animal is domesticated or has a tendency to be domesticated. In other words, the degree of tameness of the target animal to humans can be determined.

[0018] <Domestication Agent> In one embodiment, the present invention provides a domestication agent containing Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof as an active ingredient. The Limosilactobacillus reuteri used in the domestication agent of this embodiment may be live cells, killed cells, a treated cell product, or a mixture thereof. Examples of live cells include a Limosilactobacillus reuteri culture solution, a culture solution suspension, a crude product, a purified product, or a dried cell powder thereof. Examples of killed cells include a Limosilactobacillus reuteri product that has been physically or chemically treated, such as by heat treatment or radiation treatment, or a dried cell powder thereof. Examples of the treated bacterial cell product include a homogenized product, an enzyme-treated product, a disrupted bacterial cell product, or a dried bacterial cell powder of Limosilactobacillus reuteri. The Limosilactobacillus reuteri used in the domestication agent of this embodiment is preferably a strain that highly secretes pyruvic acid.

[0019] Methods for administering the domestication agent of this embodiment include oral administration, in which the domestication agent of this embodiment is mixed with drinking water or feed, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, or intraperitoneal administration.

[0020] <Pet Food or Livestock Feed> In one embodiment, the present invention provides pet food or livestock feed containing the domestication agent. Examples of pet food or livestock feed include combinations of grains such as corn, wheat, wheat flour, wheat bran, rice, bread crumbs, barley, oats, and rye; potatoes such as sweet potato and potato; beans; starches such as wheat starch, corn starch, rice starch, potato starch, tapioca starch, sweet potato starch, sago starch, and modified starch; meat; seafood; and vegetables.

[0021] In one embodiment, the present invention provides a method for domesticating an animal, the method comprising the step of administering Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof to the animal. Furthermore, it is preferable that the Limosilactobacillus reuteri used in the domestication method of this embodiment is a strain that highly secretes pyruvic acid.

[0022] The administration method in the domestication method of this embodiment may be oral administration by mixing the domestication agent of this embodiment into drinking water or feed, or intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, or intraperitoneal administration.

[0023] <Sociality Enhancer> In one embodiment, the present invention provides a sociality enhancer containing Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof as an active ingredient. The Limosilactobacillus reuteri used in the sociality enhancer of this embodiment is preferably a strain that highly secretes pyruvic acid.

[0024] Examples of the improvement of sociality include improvement of sociability, improvement of communication ability, improvement of language disorders, improvement of emotional disorders, improvement of obsessive behavior, improvement of attention deficit disorder, improvement of hyperactivity disorder, improvement of learning disabilities, or a combination thereof. The agent for improving sociality of the present embodiment is preferably a drug that improves at least one selected from the group consisting of these improvements.

[0025] The amount of Limosilactobacillus reuteri or its metabolites, or pyruvic acid, lactic acid, or oxytocin, or an analog thereof, which is the active ingredient contained in the sociality-enhancing agent of this embodiment, can be appropriately determined taking into consideration various factors such as the sex, weight, age, and symptoms of the subject. In the case of oral administration, for example, 1 μg to 10 g of the active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of injection, for example, 0.1 μg to 1 g of the active ingredient may be administered per day, for example, 0.001 to 200 mg per day. In the case of suppositories, for example, 1 μg to 10 g of the active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. In the case of topical skin preparations, for example, 1 μg to 10 g of the active ingredient may be administered per day, for example, 0.01 to 2000 mg per day. The Limosilactobacillus reuteri used in the sociality improver of this embodiment may be live cells, killed cells, treated cells, or a mixture thereof.

[0026] The administration form of the sociality-enhancing agent of this embodiment is not particularly limited and may be appropriately selected as needed. Generally, the agent may be administered as an oral preparation such as a tablet, capsule, granule, fine granule, powder, liquid, syrup, suspension, emulsion, or elixir, or as an injection, infusion, suppository, inhalant, transmucosal absorbent, or spray. Injectable preparations are administered intravenously, either alone or mixed with a conventional fluid such as glucose or amino acids, and may also be administered intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally, as needed. Suppositories are administered rectally. External preparations for skin application are applied, patched, or sprayed onto the affected area. Inhalants are administered using a device such as a nebulizer, metered-dose inhaler, or dry powder inhaler.

[0027] The frequency of administration may be three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, etc. The administration period may be one day, two days, three days, one week, two weeks, one month, six months, a year, or longer.

[0028] The sociality enhancer of the present embodiment may be any pharmaceutically acceptable additive commonly used in the preparation of pharmaceutical compositions, without any particular limitations. More specifically, examples thereof include excipients such as starch and crystalline cellulose; binders such as gelatin, corn starch, tragacanth gum and gum arabic; swelling agents such as alginic acid; solvents for injections such as water, ethanol and glycerin; adhesives such as rubber adhesives and silicone adhesives; lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavorings such as peppermint and benzyl alcohol oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives, etc.

[0029] <<Food>> In one embodiment, the present invention provides a food containing the sociability enhancer. Examples of the food include confectioneries such as gum, candy, cookies, gummies, rice crackers, biscuits, jelly, mousse, cream caramel, soda candy, edible sheets, edible films, and lozenges; mouth fresheners such as gum, candy, gummies, edible films, and lozenges; beverages such as carbonated drinks, soft drinks, milk drinks, alcoholic drinks, fruit juice drinks, teas, and nutritional drinks; dairy products such as cheese and yogurt; bread, noodles, and cereals.

[0030] <<Method for determining human sociality>> In one embodiment, the present invention provides a method for determining human sociality, the method comprising the step of detecting Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactic acid, oxytocin, or an analog thereof in a biological sample from the human.

[0031] Examples of human biological samples to be tested include blood, feces, urine, saliva, sweat, tissue exudate, etc., and are selected depending on the test item. For detection of Limosilactobacillus reuteri or its metabolites, the biological sample is preferably feces or urine, while for detection of pyruvic acid, lactic acid, oxytocin, or their analogs, the biological sample is preferably blood. Among blood samples, examples include serum and plasma, with plasma being preferred.

[0032] Methods for testing Limosilactobacillus reuteri or its metabolites, or pyruvic acid, lactic acid, oxytocin, or analogs thereof in a biological sample include the same methods as those listed in the above section "Method for determining domesticated animals." In addition, in this embodiment, it is preferable that the Limosilactobacillus reuteri used for the determination is a strain that highly secretes pyruvic acid.

[0033] In this embodiment, the improvement in sociality to be assessed includes improvement in sociability, improvement in communication ability, improvement in language disorders, improvement in emotional disorders, improvement in obsessive behavior, improvement in attention deficit hyperactivity disorder, improvement in learning disabilities, or a combination of these, and it is preferable to assess at least one selected from the group consisting of these improvements.

[0034] In the determination method of this embodiment, it is preferable to further quantify the amount of Limosilactobacillus reuteri or its metabolites, or pyruvic acid, lactic acid, oxytocin, or their analogs in the sample. For example, by comparing the amount of Limosilactobacillus reuteri or its metabolites, or pyruvic acid, lactic acid, oxytocin, or their analogs in the sample with the control amount in a sample from a control subject who is considered to be social, it can be determined that the subject has improved prosociality if the amount in the sample matches or is close to the control amount in the control subject who is considered to be social. Furthermore, for example, by comparing the amount of Limosilactobacillus reuteri or its metabolites, or pyruvate, lactic acid, or oxytocin, or their analogs in a sample with the control amount in a sample from a control subject who is considered to be non-social, if the amount in the sample is greater than the control amount in the control subject who is considered to be non-social, it can be determined that the subject has improved sociality.

[0035] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0036] Selective breeding leads to increased active obedience. In the 27th generation of breeding, we examined obedience-related behaviors in mice in groups selected for active obedience and in groups not selected. A total of 80 mice, 10 male and 10 female, were subjected to active obedience tests, passive obedience tests, and hand-staying tests (see Figures 2(A), (B), (C), and (D)). Because S1 and C2, and S2 and C1, are closely related genetically (see Figure 1), we compared data between these two pairs to determine the effect of selective breeding on active obedience compared to the unselected control. Data were obtained for nine behavioral parameters: heading, touching, moving, and jumping in the active obedience test (see Figures 3A, 3B, 3C, and 3D); heading, receptiveness, moving, and jumping in the passive obedience test (see Figures 4A, 4B, 4C, and 4D); and median stay time in the stay-on-hand test (see Figure 5A). Given that two-way analysis of variance (ANOVA) showed no effect of sex, data from both sexes were combined for all obedience analyses. Clustering analysis of all nine parameters identified two significant clusters categorizing parameters as positively or negatively associated with obedience. For active contact, both S1 (p<0.001) and S2 (p<0.001) showed significantly longer contact times than their respective controls (see Figure 3B). Other obedience-related parameters, such as heading in the active obedience test (S1, p<0.001; S2, p<0.05) (see Figure 3(A)), heading in the passive obedience test (S1, p<0.001; S2, p<0.001) (see Figure 4(A)), and acceptance (S1, p<0.001; S2, p<0.01) (see Figure 4(B)), were higher in the selected group than in the non-selected group. In contrast, active jumping (C2, p<0.001) (see Figure 3(D)) and passive locomotion (C2, p<0.001; C1, p<0.05) (see Figure 4(C)) were significantly higher in the selected group than in the non-selected group.Parameters characteristic of wild mice, such as active contact time, passive jumping (C2, p<0.001; C1, p<0.01) (see Figure 4(D)), were higher in the unselected group than in the selected group. Plasma oxytocin levels were also measured in each group (see Figure 5(B)). As shown in Figure 5(C), a correlation was confirmed between active contact time and plasma oxytocin levels. In summary, S1 and S2 showed higher active and passive docility than the unselected group. Next, we investigated whether the changes in docility due to breeding resulted from changes in gut bacteria, or whether gut bacteria are involved in docility in a different way.

[0037] Gut Microbiome Repertoires of Selected and Unselected Mice To examine the gut microbiota of selected and unselected mice, we performed shotgun metagenomic sequencing on 80 individual fecal samples collected from each mouse after the obedience test. On average, 31 million paired reads were obtained per sample. We analyzed the gut microbiome composition of WHS mice using metagenomic-assembled genomes (MAGs). Because the gut microbiota is a complex microbiome, we employed two assemblers and multiple binning approaches to obtain MAGs and recover as many high-quality MAGs as possible. Using these approaches, we generated 14,816 MAGs. After removing redundant MAGs, we obtained 374 bacterial MAGs with completeness of 50% or more and contamination of less than 10% (see Figure 6). Of these MAGs, 226 were of high quality, with completeness of 90% or more and contamination of less than 5%. These 374 MAGs spanned 11 phyla, with 281 belonging to the Bacillota_A phylum (see Figure 6). By comparison with a comprehensive mouse microbiota genome catalog, we identified 27 novel species-level MAGs that had not previously been reported. This was further confirmed by the DFAST "taxonomy check." Of these 27 MAGs, one each belonged to Actinomycetes, Bacillota_B, and Patescibacteria, eight to the genus Bacillota, and 16 to Bacillota_A (see Figure 6). Furthermore, despite the effectiveness of machine learning binners in deriving higher-quantity and higher-quality MAGs, limitations in binning all bacterial types were identified, suggesting that the application of a hybrid approach integrating both single and coassembly techniques would be advantageous, as would the application of multiple binning tools.

[0038] Selective breeding for active docility did not affect the taxonomic diversity of the gut microbiota. For taxonomic analysis, we used the complete NCBI / RefSeq prokaryotic genome sequence database (NCBI RefSeq Complete V205) to capture the full diversity of the WHS mouse gut microbiome. We identified 665 bacterial species spanning 11 phyla, 21 classes, 45 orders, 103 families, and 339 genera (see Figure 7). Of these, 427 species were common to all four groups, contributing to 99.8% of the abundance across all samples (see Figure 8(A)). Taxonomic beta diversity, calculated using Bray-Curtis dissimilarity, did not differ between groups (see Figure 8(B)). Regarding the alpha diversity index, Chao1 was significantly higher in C1 than in S1 (p<0.001) and C2 (p<0.05). Chao1 was also more abundant in S2 than in S1 (p<0.05) (see Figure 9(A)). The Shannon diversity index was similar across all groups (see Figure 9(B)). Overall, we confirmed that host selection pressure for docile behavior does not consistently affect the overall taxonomic diversity of the mouse gut microbiota. Furthermore, no significant differences in functional diversity were found between unselected and selected mice. Using the 40 significant taxa identified from the random forest analysis, a Pearson correlation analysis was performed on the docility parameter scores. This analysis highlighted that several lactobacilli showed a significant positive correlation with both active and passive jumping behavior, but a significant negative correlation with active heading. On the other hand, Bacteroides sp. CBA7301, Ligilactobacillus animeis, and Prevotella bivia were significantly positively correlated with active contact with selection pressure (see Figure 3F).

[0039] [The selected group showed an abundance of Limosilactobacillus reuteri] To identify bacterial species enrichment in the submissive mice and further solidify the association, each selected group was compared with its respective control group. In this analysis, MaAsLin2 was used to correlate species-level gut microbiome composition with the WHS mouse group (see Figures 11(A) and 11(B)). In the MaAsLin2 analysis, only species significantly associated in both groups were considered to differ in abundance. Limosilactobacillus reuteri (see Figures 12(A) and 12(B)), formerly known as Lactobacillus reuteri, and Candidatus Gallimonas intestinalis (see Figures 13(A) and 13(B)), a type of lactic acid bacteria, were found to be abundant in both selection groups compared to their respective controls. Of the 374 MAGs generated, one high-quality MAG was obtained from L. reuteri and six MAGs from the genus Candidatus Gallimonas. To quantify the abundance of enterobacteria, reads were mapped to all MAGs using CoverM, with mapping rates ranging from 75% to 93%. Similar to Figure 7, we observed significant enrichment of L. reuteri in both selected groups compared to the unselected group (SI, p<0.05; S2, p<0.001). However, the relative abundance of the six Candidatus Gallimonas MAGs did not show significant differences between the selected and unselected groups. These results, combined with the results of Pearson correlation analysis (see Figure 10), indicate that L. reuteri is the only bacterium consistently associated with docility across our analyses. This association suggests that L. reuteri is associated with increased docility in the selected group.

[0040] The effects of the gut microbiota on animals occur through the production of metabolites that are absorbed by the host and alter its behavior. To identify metabolites that may contribute to changes in docility, metabolome analysis was performed on plasma samples obtained from WHS mice. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) measurements detected 281 peaks (179 in cation mode and 102 in anion mode) and annotated according to the HMT standard library and known / unknown peak library. Of the targeted metabolites, 70 (46 in cation mode and 24 in anion mode) were detected and quantified. Correlation clustering of the 70 metabolites showed no visible differences between the control and selected groups (see Figure 14). Of these, four metabolites were significantly higher in the selected mice (see Figures 15(A)-16(B)). To strengthen this result, MaAsLin2 analysis was performed, and after FDR correction, only pyruvate remained significantly associated with the selected mice. Plasma lactate concentrations were also observed to maintain homeostasis with endogenous pyruvate (see Figure 17).

[0041] Administration of L. reuteri Increases Submissive Behavior in Unselected Mice To examine the effects of L. reuteri and pyruvate in mice, strains that increase and decrease pyruvate secretion were isolated and administered via drinking water. We isolated bacteria from the cecal material and feces of a selected group of WHS mice. Using a special medium containing raffinose, we isolated 22 L. reuteri strains, and biochemical assays revealed that 16 colonies secreted pyruvate into GAM medium (see Figure 18). Lactobacillus helveticus JCM1120, a known bacterial species that secretes pyruvate, was used as a positive control for pyruvate-secreting strains. Furthermore, we evaluated the secretion of D-lactate and L-lactate in relation to pyruvate homeostasis (see Figure 18). In the bacterial strain administration experiment for group C1 mice, NIG-A41 (a high-pyruvate-secreting strain), NIG-23 (a low-pyruvate-secreting strain), and L. helveticus JCM1120 were selected. After administering the cultured bacterial strains via drinking water to unselected C1 mice for 21 days, an obedience test was performed (see Figure 19(A)). Mice treated with pyruvate-secreting L. reuteri NIG-A41 showed significantly increased active obedience compared with the PBS-treated group (p<0.05) and L. helveticus-treated group (p<0.05) (see Figure 19(B)). Although not significant, mice treated with NIG-23, a low-pyruvate-secreting strain, showed higher active obedience than mice treated with PBS. However, mice treated with L. helveticus did not show an increase in active submissiveness compared to mice treated with PBS. Active heading was also higher in the NIG-A41-treated group, but the difference was not significant (see Figure 20(A)). The hand-stay test, passive heading, and passive acceptance did not show significant increases in the NIG-A41-treated group (see Figures 20(B)-21(B)). Serum pyruvate levels were higher in the L. helveticus-treated group (p<0.05), but neither NIG-A41-treated nor NIG-23-treated mice showed any difference in serum pyruvate levels compared to the PBS-treated group (see Figure 22(A)).We also examined the colonization of these bacterial strains in host mice by quantitative PCR analysis of bacterial DNA obtained from feces. Compared to PBS-treated mice, significantly higher levels of L. reuteri genomes were found in the feces of both NIG-A41 (p<0.01) and NIG-23 (p<0.001)-treated mice, with an average of over 1,500-fold higher levels (see Figure 22(B)). In contrast, mice treated with L. helveticus JCM1120, originally isolated from Emmental (Swiss) cheese, showed no significant increase in feces (see Figure 23(A)). Body weight change and water intake during the treatment period were also monitored, but no significant differences were observed between groups. Because previous studies have reported that daily administration of L. reuteri increases blood oxytocin levels, we examined the oxytocin levels in these mice. Serum oxytocin levels were significantly higher in mice treated with NIG-A41 compared to the PBS-treated group (p<0.01), but not significantly different in mice treated with NIG-23 (see Figure 23(B)).

[0042] [Identification of L. reuteri strains that secrete high amounts of pyruvate] The method for identifying the L. reuteri strains that secrete high amounts of pyruvate is as follows. Cecum and fecal samples were collected from mice in the selected group (S1) that showed high docility, mixed, and serially diluted. This diluted mixture was cultured on an agar plate of L. reuteri isolation medium (LRIM) containing raffinose as the sole carbon source. The plate was cultured under anaerobic conditions (5% CO ２The plates were cultured at 45°C for 48 hours. To maintain anaerobic conditions, the plates were placed in a sealed box containing a bag of AnaeroPack (Mitsubishi Gas Chemical Company, Inc., Japan). To isolate the strains, selected colonies were first plated twice onto LRIM plates. A third isolation culture was then performed on DeMan-Rogosa-Sharpe (MRS) (Merck-Millipore, USA) agar plates. The resulting strains were cultured in Gifu Anaerobic Broth (GAM) medium (Nissui Pharmaceutical, Japan) supplemented with 1% glucose. Culture was performed at 37°C for 24 hours under anaerobic conditions. The concentrations of pyruvate, L-lactate, and D-lactate in the medium were measured using a biochemical assay kit from Cayman Chemicals (Michigan, USA) according to the manufacturer's protocol. All assays were performed in triplicate (technical replicates). These analyses revealed that all isolated strains secreted L-lactate and D-lactate. Meanwhile, the pyruvate concentration in GAM medium varied significantly among the strains. Among these, strains with high pyruvate secretion were used for administration experiments to mice. The following three kits were used to measure pyruvate, D-lactate, and L-lactate:

[0043] Company: Cayman Chemical Cat. No.: 700470 Name: Pyruvate Assay Kit Quantity: 1 kit (96 tests) Company: Cayman Chemical Cat. No.: 700520 Name: D-Lactate Assay Kit Quantity: 1 kit (96 tests) Company: Cayman Chemical Cat. No.: 700510 Name: L-Lactate Assay Kit Quantity: 1 kit(96 tests)

[0044] According to the present invention, a technique can be provided that makes it possible to easily improve the behavior of individual animals.

Claims

1. A method for determining whether a domesticated animal is a domestic animal, comprising the steps of: A method for determining whether or not Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof is present in a biological sample from the animal.

2. The method according to claim 1, comprising a step of quantifying the amount of Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactate, oxytocin, or an analog thereof present in a biological sample from the animal.

3. The method according to claim 1 or 2, wherein the biological sample is feces or blood.

4. The method according to claim 1 or 2, wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvic acid.

5. A domestication agent containing Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof as an active ingredient.

6. The domestication agent according to claim 5, wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvate.

7. 1. A method for domesticating an animal, comprising: A domestication method comprising the step of administering Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactic acid, oxytocin, or an analog thereof to the animal.

8. The domestication method according to claim 7, wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvate.

9. A method for determining human sociality, comprising: A method for determining the presence or absence of Limosilactobacillus reuteri or a metabolite thereof, or pyruvic acid, lactic acid, oxytocin, or an analog thereof in the human biological sample.

10. The method according to claim 9, comprising a step of quantifying the amount of Limosilactobacillus reuteri or a metabolite thereof, or pyruvate, lactate, oxytocin, or an analog thereof present in the human biological sample.

11. The method according to claim 9 , wherein the biological sample is feces or blood.

12. The method of claim 9, wherein the Limosilactobacillus reuteri is a strain that highly secretes pyruvate.

13. The method according to any one of claims 9 to 12, wherein the sociability is at least one selected from the group consisting of improved sociability, improved communication ability, improvement of language disorders, improvement of emotional disorders, improvement of obsessive behavior, improvement of attention deficit hyperactivity disorder, and improvement of learning disabilities.