Breeding method for army grazing No.1 backup boars based on intestinal flora

Abstract

The invention discloses a breeding method for military grazing No.1 backup boars based on intestinal flora, and belongs to the technical field of animal breeding. By comparing the intestinal flora difference and testis development and production performance parameters of the boar individuals with low testis index and high testis index in the same batch, a specific flora related to testis development and production performance is screened out, and the specific flora is used as the breeding index of the army pasture No.1 backup boar. Compared with a conventional breeding method, the method has the advantage that the speed is greatly increased. Research results provide a new way for rapid selection of the army grazing No.1 backup boars based on the intestinal flora.

Description

Technical Field

[0001] The invention discloses a method for breeding Junmu No. 1 reserve boar based on intestinal flora, and belongs to the technical field of animal breeding. Background Art

[0002] The Junmu No. 1 white pig, my country's first independently bred lean pig breed, boasts remarkable characteristics such as rapid growth, high feed conversion rate, high carcass leanness, excellent meat quality, and strong disease resistance and adaptability. It passed national breed approval in March 1999. High-quality Junmu No. 1 boars play a key role in improving the production performance of fattening pig herds, which is directly related to the ability to efficiently produce sufficient offspring to cover costs in modern pig production systems. Therefore, breeding high-quality Junmu No. 1 boars is an effective way to enhance the genetic quality and production efficiency of pig herds. Research has shown that testicular development is closely linked to sufficient sperm reserves required for reproductive efficiency. Excellent reproductive and production performance are the core goals of Junmu No. 1 boar breeding. This not only helps select excellent breeding pigs and promote the improvement and development of boar breeds, but also provides high-quality offspring for the market and promotes pork production. Excessive subcutaneous fat reduces lean meat percentage, reduces edible meat, and thus affects pig farm profitability. Excessive subcutaneous fat can also negatively impact boar reproductive performance. Studies have shown that testicular volume is related to fat deposition, and excessive fat deposition may impair testicular development and function, thereby reducing sperm quality and quantity. Therefore, reducing fat deposition and increasing lean meat percentage are key goals in pig breeding.

[0003] The mammalian gut is home to trillions of microorganisms, which play key roles in reproduction, metabolism, immunity, and behavior. Recent studies have revealed correlations between gut microbial profiles and various parameters in pigs, including reproduction and production performance. For example, certain gut microbes can influence the synthesis and metabolism of testosterone. Testosterone is the primary male sex hormone produced by Leydig cells in the testes and is essential for testicular development and maintenance, reproductive organ growth, and spermatogenesis. Insufficient testosterone secretion can lead to testicular hypoplasia, manifested as reduced testicular size. Furthermore, a healthy gut microbiome facilitates nutrient absorption and utilization, providing essential nutritional support for growth. Conversely, an imbalance in the gut microbiome can lead to nutrient malabsorption, indirectly impacting production performance. Therefore, analyzing the gut microbiome is a key research area for improving pig production efficiency. However, the impact of the gut microbiome on testicular development and production performance in the Junmu No. 1 boar remains unclear. In practical breeding, individuals with larger testicles are often selected for replacement breeding by visual inspection. However, accurate selection of production performance indicators, such as fat and lean meat percentage, is difficult to achieve by visual inspection alone. This study analyzed the intestinal flora of two groups of 170-day-old Junmu No. 1 boars with different testicular indexes under the same feeding conditions, and found the specific differential flora that affect the testicular development and production performance of Junmu No. 1 boars, providing theoretical support for the rapid selection of Junmu No. 1 reserve boars based on intestinal flora. Summary of the Invention

[0004] The present invention aims to provide a method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora (using fecal samples for flora testing). This method is simple to operate, fast, efficient, scientific, and convenient. It can determine whether Junmu No. 1 boars have good testicular development and production performance without damaging them. This method can be widely used in the rapid selection and breeding of subsequent reserve boars, providing strong support for pig production.

[0005] A method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora, wherein the steps of selecting and breeding Junmu No. 1 reserve boars include: comparing differences in testicular development, comparing differences in production performance, analyzing characteristics of intestinal flora, analyzing differences in intestinal flora composition, screening for specific flora, and selecting and breeding Junmu No. 1 reserve boars based on intestinal flora.

[0006] By analyzing the differences in testicular development and production performance, we distinguished between individuals with high and low testicular indexes. Then, by comparing the intestinal flora characteristics and intestinal flora composition differences between individuals with high and low testicular indexes, we screened out specific flora related to production performance and testicular development, and ultimately achieved the selection and breeding of Junmu No. 1 reserve boars based on intestinal flora.

[0007] The specific methods for screening specific bacterial communities are as follows:

[0008] SA, boar fecal samples were collected in 5 mL sterile polypropylene tubes and stored at -80 °C for subsequent DNA extraction;

[0009] SB, DNA was extracted from boar fecal samples using the DDNeasy PowerSoil Kit;

[0010] SC, assess the quality of the DNA extracted in step SB by 1% agarose gel electrophoresis, and quantify the DNA concentration using a Nanodrop instrument;

[0011] SD, bacterial 16S rDNA gene high-throughput sequencing

[0012] The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified by PCR using the extracted DNA as a template. The primers used are as follows:

[0013] Primer 341F: 5′-CCTAYGGGRBGCASCAG-3′;

[0014] Primer 806R: 5′-GGACTACNNGGGTATCTAAT-3′;

[0015] PCR amplification conditions were as follows: predenaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min;

[0016] The PCR reaction system contains 15 High-Fidelity PCR Master Mix, 0.2 μM concentration of each primer, and 10 ng of target DNA extracted in step SB;

[0017] SE, adopt Ultra TM II DNA library preparation kit was used according to the manufacturer's instructions to construct libraries from PCR amplification products. The construction process included fragmentation, end-repair, adapter ligation, and purification, ultimately creating a library suitable for high-throughput sequencing. The library was then sequenced on the Illumina NovaSeq platform to obtain 250 bp paired-end sequences. The relevant sequencing data have been deposited in the NCBI SRA database and analyzed using QIME2 software.

[0018] SF, primer sequences and low-quality reads were denoised using the DADA2 v1.16 plug-in to remove possible sequencing errors. High-quality paired-end clean reads were merged into complete tag sequences using FLASH v1.2.11. Chimeric sequences were then removed using the VSEARCH tool to obtain high-quality amplicon sequence variants, ASVs. Reference: DADA2: high-resolution sample inference from illumina amplicon data. Nat. Methods 13, 581–583. doi: 10.1038 / nmeth.3869; ASV sequences were annotated and classified based on the Silva database. SG, analysis of boar fecal sequencing data:

[0019] SG-1, Alpha diversity analysis:

[0020] The alpha diversity of fecal microbiota was calculated using QIME2 (version 2019.4) and evaluated using Chao1, Shannon, Pielou_e, and Simpson indices;

[0021] SG-2, Beta diversity analysis:

[0022] Beta diversity was calculated based on the Unweighted_unifrac distance, and the differences between samples were displayed using principal coordinate analysis (PCoA) in R software. Based on the Unweighted_unifrac distance, the anosim function was used to analyze whether the differences in community structure between groups were significant.

[0023] SG-3, species difference analysis and specific bacterial group screening:

[0024] MetaStat R software was used to analyze species differences at each classification level (top 10 phyla, genera, and species). Simper was used to quantify the contribution of each species to the difference between the two groups, and specific bacterial groups were screened. Finally, three specific bacterial groups, Campilobacterota, Lactobacillus, and Streptococcus, were selected based on their contribution.

[0025] Preferably, the comparison of testicular development differences is assessed by measuring testicular weight, testicular volume, serum and testicular testosterone levels.

[0026] Preferably, the comparison of production performance differences is evaluated by daily weight gain, feed conversion rate, fat rate, and lean meat rate.

[0027] Preferably, the NCBI SRA database project number in step SE is: PRJNA1070801.

[0028] Preferably, the identification of the intestinal flora of the Junmu No. 1 reserve boar is carried out by high-throughput sequencing of 16S amplicon and identification of specific flora.

[0029] Preferably, the identification of the intestinal flora of the reserve boar is carried out by dissecting the intestinal contents of the reserve boar or analyzing the collected feces of the reserve boar.

[0030] The beneficial effects of the present invention are:

[0031] 1. By comparing the differences in intestinal flora between individuals with high and low testicular indexes, specific flora associated with production performance and testicular development can be screened and used as indicators for the selection of reserve boars. Compared with traditional breeding methods, the breeding method of this invention is simple to operate and significantly faster.

[0032] 2. In actual breeding, usually only individuals with larger testicles can be selected as reserve boars by the naked eye, while production performance indicators such as fat rate and lean meat rate are difficult to accurately select by the naked eye alone. The present invention provides a rapid breeding method for reserve Junmu No. 1 boars based on intestinal flora (fecal sample flora detection), which is simple to operate and has the advantages of being fast, efficient, scientific and convenient. It can judge whether the boar has good testicular development and production performance without damaging the boar. This method can be widely used in the subsequent rapid breeding of reserve boars, providing strong support for pig production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the testicular development index of the boars in the low and high testicular index groups of the present invention; wherein Figure 1 a is the testicular weight; Figure 1 b is the testicular index; Figure 1 c is the volume of the left testicle; Figure 1 d is the volume of the right testicle; Figure 1 e is the total testicular volume.

[0034] Figure 2 is the testosterone level of boars in the low and high testicular index groups of the present invention; wherein Figure 2 a is the relative level of serum testosterone; Figure 2 b is the relative level of testosterone in testicles.

[0035] Figure 3 are the production performance indicators of the low and high testicular index groups of boars in the present invention; Figure 3 a Average daily weight gain; Figure 3 b is feed conversion ratio; Figure 3 c is lean meat percentage; Figure 3 d is the fat percentage.

[0036] Figure 4 The alpha diversity of the intestinal flora of the boars in the low and high testicular index groups of the present invention; Figure 4 a is the Chao1 index; Figure 4 b is the Shannon index; Figure 4 c is the Pielou_e index; Figure 4 d is the Simpson index.

[0037] Figure 5 This is the beta diversity of the intestinal flora of boars in the low and high testicular index groups of the present invention.

[0038] Figure 6 This is an analysis of the intestinal flora composition of boars in the low and high testicular index groups of the present invention; Figure 6 a is the abundance of bacterial communities at the main phylum level; Figure 6 b is the abundance of the main genus level bacteria; Figure 6 c is the abundance of bacterial communities at the main species level.

[0039] Figure 7 is the abundance of different intestinal flora in the low and high testicular index groups of boars in the present invention; Figure 7 a is the relative abundance of Campilobacterota; Figure 7 b is the relative abundance of Lactobacillus; Figure 7 c is the relative abundance of Streptococcus; Figure 7 d is the relative abundance of Anaerovibrio sp.

[0040] Figure 8 The top 10 bacterial groups that contribute the most to the difference in intestinal flora between the low and high testicular index groups of the present invention; Figure 8 a is the bacterial community at the phylum level; Figure 8 b is the bacterial community at the genus level; Figure 8 c is the bacterial community at the species level. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0042] Example 1 Comparison of testicular development differences

[0043] 1. Experimental Materials and Methods

[0044] 1.1 Experimental animals: The boar breed was Junmu No. 1 white pig, raised at the Agricultural Experiment Base of Jilin University.

[0045] 1.2 Main test reagents: see Table 1.

[0046] Table 1 Test reagents

[0047]

[0048] 1.3 Main test instruments: see Table 2.

[0049] Table 2 Test instruments

[0050]

[0051] 1.3 Experimental Methods: Forty-one healthy Junmu No. 1 boars, 80 days old and of similar initial weight, were randomly assigned to five pens, with 8 to 9 boars per pen. These boars were reared for 90 days at the Agricultural Experimental Base of Jilin University, China. During this period, the boars were fed a standard commercial diet. At the conclusion of the experiment, the boars were transported to a commercial slaughterhouse for slaughter. The slaughter process followed standard commercial procedures, including electric shock, bleeding, scalding, depilation, head and foot removal, evisceration, cleaning, and cooling.

[0052] 1.4 Index Measurement: At the end of the experiment, the boars' final body weight (kg) was recorded. Immediately after slaughter, testicular weight (kg), testicular length (cm), and testicular width (cm) were measured. Based on the testicular index, the General Mu No. 1 boars were divided into a low testicular index group and a high testicular index group.

[0053] Testicular index (%) = testicular weight (kg) / final body weight (kg) × 100;

[0054] Testicular volume (cm 3 )=4×π×(a×b 2 ) / 3;

[0055] Where a is half of the testicle length,

[0056] b is half the width of the testicle.

[0057] 2. Test results:

[0058] like Figure 1 As shown, the testicular index and testicular weight of boars in the high testicular index group were significantly higher than those in the low testicular index group (P<0.0001, P<0.0001) ( Figure 1 Similarly, the left testicular volume (P = 0.003), right testicular volume (P = 0.001) and total testicular volume (P < 0.0001) of the boars in the high testicular index group were significantly higher than those in the low testicular index group ( Figure 1 ce). This study showed that boars with a high testicular index had larger testicular volume. Studies have shown that the seminiferous tubules account for over 80% of the testicular volume and are highly correlated with the production of mature sperm, suggesting that boars with a high testicular index may have better testicular development.

[0059] Example 2 Determination of boar serum testosterone levels

[0060] 1 Experimental materials and methods

[0061] 1.1 Experimental animals: The boar breed was Junmu No. 1 white pig, raised at the Agricultural Experiment Base of Jilin University.

[0062] 1.2 Main test reagents: see Table 3.

[0063] Table 3 Test reagents

[0064]

[0065] 1.3 Main test instruments: see Table 4.

[0066] Table 4 Test instruments

[0067]

[0068] 1.4 Experimental Methods: Forty-one healthy Junmu No. 1 boars, 80 days old and of similar initial weight, were randomly assigned to five pens, with 8 to 9 boars per pen. These boars were reared for 90 days at the Agricultural Experimental Base of Jilin University, China. During this period, the boars were fed a standard commercial diet. At the conclusion of the experiment, the boars were transported to a commercial slaughterhouse for slaughter. The slaughter process followed standard commercial procedures, including electric shock, bleeding, scalding, depilation, head and foot removal, evisceration, cleaning, and cooling.

[0069] 1.5 Grouping: At the end of the experiment, the final body weight (kg) of the boars was recorded; after slaughter, the testicle weight (kg) was measured immediately. Based on the testicular index, the General Mu No. 1 boars were divided into a low testicular index group and a high testicular index group;

[0070] Testicular index (%) = testicular weight (kg) / final body weight (kg) × 100.

[0071] 1.6 Sample collection: Testis and serum samples were collected from boars immediately after slaughter and stored at -80°C for testosterone level analysis.

[0072] 1.7 Test parameters: Testosterone levels were determined using the Testosterone EIA Kit in strict accordance with the manufacturer's protocol.

[0073] Testicular tissue samples were first homogenized in 1.15% KCl solution at a solvent-to-tissue ratio of 3:1 (weight / volume). The homogenate was then centrifuged at 9,000 × g for 20 minutes at 4°C and then at 105,000 × g for 60 minutes at the same temperature. Testosterone was isolated from the supernatant obtained from the second centrifugation by two rounds of ether extraction and quantified. Testosterone content in testicular tissue samples was expressed per gram of testicular tissue. Serum samples were analyzed directly for testosterone levels without any pretreatment.

[0074] 2. Experimental results:

[0075] Compared with the boars in the low testicular index group, the serum testosterone level (P = 0.049) and testicular testosterone level (P = 0.039) of the boars in the high testicular index group were significantly increased. Figure 2 a and Figure 2 Testosterone is essential for testicular development and maintenance, reproductive organ growth, and spermatogenesis. Insufficient testosterone secretion can lead to testicular hypoplasia, as further demonstrated by the improved testicular development seen in boars with a high testicular index.

[0076] Example 3 Production performance measurement

[0077] 1 Experimental materials and methods

[0078] 1.1 Experimental animals: The boar breed was Junmu No. 1 white pig, raised at the Agricultural Experiment Base of Jilin University.

[0079] 1.2 Main test reagents: see Table 5.

[0080] Table 5 Test reagents

[0081]

[0082] 1.5 Main test instruments: see Table 6.

[0083] Table 6 Test instruments

[0084]

[0085]

[0086] 1.4 Experimental Methods: Forty-one healthy Junmu No. 1 boars, 80 days old and of similar initial weight, were randomly assigned to five pens, with 8 to 9 boars per pen. These boars were reared for 90 days at the Agricultural Experimental Base of Jilin University, China. During this period, the boars were fed a standard commercial diet. At the conclusion of the experiment, the boars were transported to a commercial slaughterhouse for slaughter. The slaughter process followed standard commercial procedures, including electric shock, bleeding, scalding, depilation, head and foot removal, evisceration, cleaning, and cooling.

[0087] 1.5 Grouping: At the end of the experiment, the final body weight (kg) of the boars was recorded; after slaughter, the testicle weight (kg) was measured immediately. Based on the testicular index, the General Mu No. 1 boars were divided into a low testicular index group and a high testicular index group;

[0088] Testicular index (%) = testicular weight (kg) / final body weight (kg) × 100.

[0089] 1.6 Testing indicators:

[0090] A: Record before slaughter: initial body weight, final body weight and total feed intake of the boar.

[0091] B: Measurements after slaughter: carcass weight (kg), fat weight (kg), lean meat weight (kg).

[0092] C: Calculation indicators:

[0093] Body weight gain (kg) = final body weight (kg) - initial body weight (kg);

[0094] Average daily weight gain (kg / day) = body weight gain (kg) / 90 (days);

[0095] Feed conversion rate (kg / kg) = total feed intake (kg) / body weight gain (kg);

[0096] Fat rate (%) = fat weight (kg) / carcass weight (kg) × 100;

[0097] Lean meat rate (%) = lean meat weight (kg) / carcass weight (kg) × 100.

[0098] 2. Experimental results:

[0099] Compared with the boars in the low testicular index group, the lean meat rate of the boars in the high testicular index group was significantly increased and the fat rate was significantly reduced (P = 0.014, P = 0.014) (see Figure 3 c and Figure 3 d). However, there were no significant differences in average daily gain and feed conversion rate between the high testicular index group and the low testicular index group (P = 0.338, P = 0.640, P = 0.458, P = 0.454, P = 0.614) (see Figure 3 a and Figure 3b) Research has shown that backfat thickness is a key economic trait affecting the efficiency and profitability of modern pig production. Excess subcutaneous fat is considered "waste fat" of low commercial value. Reducing subcutaneous fat deposition in pigs has always been a key goal in breeding. In this study, we found no significant differences in average daily weight gain and feed conversion rate between boars with a high and low testicular index. However, boars with a high testicular index had a lower fat percentage and a higher lean meat percentage. These results indicate that boars with a high testicular index are more efficient at converting feed into lean meat production than those with a low testicular index.

[0100] Example 4 Screening of specific bacterial groups

[0101] 1. Experimental Materials and Methods

[0102] 1.1 Experimental animals: The boar breed was Junmu No. 1 white pig, raised at the Agricultural Experiment Base of Jilin University.

[0103] 1.2 Main test reagents: see Table 7.

[0104] Table 7 Test Reagents

[0105]

[0106] 1.3 Main test instruments: see Table 8.

[0107] Table 8 Test instruments

[0108]

[0109]

[0110] 1.4 Experimental Methods: Forty-one healthy Junmu No. 1 boars, 80 days old and of similar initial weight, were randomly assigned to five pens, with 8 to 9 boars per pen. These boars were reared for 90 days at the Agricultural Experimental Base of Jilin University, China. During this period, the boars were fed a standard commercial diet. At the conclusion of the experiment, the boars were transported to a commercial slaughterhouse for slaughter. The slaughter process followed standard commercial procedures, including electric shock, bleeding, scalding, depilation, head and foot removal, evisceration, cleaning, and cooling.

[0111] 1.5 Grouping: At the end of the experiment, the final body weight (kg) of the boars was recorded; after slaughter, the testicle weight (kg) was measured immediately. Based on the testicular index, the General Mu No. 1 boars were divided into a low testicular index group and a high testicular index group;

[0112] Testicular index (%) = testicular weight (kg) / final body weight (kg) × 100.

[0113] 1.6 Sample collection: At the end of the experiment, boar fecal samples were collected in 5 mL sterile polypropylene tubes and stored at -80 °C for subsequent DNA extraction.

[0114] 1.7 Extraction of DNA from boar fecal microbiome and high-throughput sequencing of bacterial 16S rDNA genes: as follows.

[0115] 1.7.1 DNA extraction and quality assessment

[0116] A: DNA extraction: DNA was extracted from boar fecal samples using the DNeasy PowerSoil Kit according to the manufacturer's instructions. The fecal sample was first added to the bead tube provided with the kit, followed by lysis buffer and mechanical homogenization to ensure complete cell lysis. PCR inhibitors were then removed using the kit's inhibitor removal technology. Finally, DNA was purified using a silica-membrane spin column and eluted with elution buffer.

[0117] B: Quality Assessment: DNA samples were electrophoresed on a 1% agarose gel at 100 V for 30 minutes along with a 1 kb DNA ladder. After electrophoresis, the DNA bands were observed under UV light for clarity and position to determine if the DNA was degraded or contaminated. The absorbance of the DNA was then measured at 260 nm and 280 nm using a Nanodrop analyzer. The DNA concentration and purity ratio (A260 / A280) were calculated. Based on the concentration, the DNA was diluted to 1 ng / μL with sterile water for later use.

[0118] 1.7.2 PCR amplification and sequencing

[0119] A: PCR amplification:

[0120] PCR amplification of the V3 and V4 hypervariable regions of the 16S rRNA gene was performed using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). The PCR reaction system contained 15 μL High-Fidelity PCR Master Mix, 0.2 μM of each primer, and 10 ng of target DNA extracted in step 1.7.1B.

[0121] PCR amplification conditions were as follows: initial denaturation at 98°C for 1 min, followed by 30 cycles, each cycle consisting of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 5 min.

[0122] B: Library construction and sequencing: Ultra TM II DNA Library Preparation Kit was used to construct a library from the PCR product according to the manufacturer's instructions. The construction process included fragmentation, end-repair, adapter ligation, and purification, ultimately generating a sequencing library suitable for high-throughput sequencing. The constructed library was then sequenced on the Illumina NovaSeq platform, obtaining 250 bp of paired-end sequences. The relevant sequencing data have been deposited in the NCBI SRA database (Project No. PRJNA1070801).

[0123] 1.7.3 Data Processing and Analysis

[0124] A: Data processing: Primer sequences and low-quality reads were denoised using the DADA2 v1.16 plugin. High-quality paired-end clean reads were merged into complete tag sequences using FLASH v1.2.11. Chimeric sequences were then removed using the VSEARCH tool to obtain high-quality amplicon sequence variants (ASVs). This step is referenced in: DADA2: high-resolution sample inference from illumina amplicon data. Nat. Methods 13, 581–583. doi:10.1038 / nmeth.3869.

[0125] B: Species annotation: ASV sequences were annotated and classified into species based on the Silva database.

[0126] 1.8 Analysis of boar feces sequencing data: as follows.

[0127] 1.8.1 Alpha diversity analysis: The alpha diversity of fecal microbiota was calculated using QIME2 (version 2019.4) and evaluated using the Chao1, Shannon, Pieloué, and Simpson indices.

[0128] 1.8.2 Beta diversity analysis: Beta diversity was calculated based on the Unweighted_unifrac distance, and the differences between samples were displayed using principal coordinate analysis (PCoA) in R software. Based on the Unweighted_unifrac distance, the anosim function was used to analyze whether the differences in community structure between groups were significant.

[0129] 1.8.3 Species Difference Analysis and Specific Bacterial Group Screening: MetaStat (R software) was used to analyze species differences at each taxonomic level (top 10 phyla, genera, and species). Simper was used to quantify the contribution of each species to the differences between the two groups and to screen for specific bacterial groups.

[0130] 2. Experimental results:

[0131] 2.1 Analysis of alpha diversity of intestinal flora

[0132] Chao1 index in fecal samples of boars in the low testicular index group (P=0.008) ( Figure 4 a) were significantly higher than those in the fecal samples of the boars in the high testicular index group, but there were no significant differences in Pielou_e (P=0.315), Shannon (P=0.197) and Simpson (P=0.334) indices between the low testicular index group and the high testicular index group ( Figure 4 bd).

[0133] 2.2 Analysis of beta diversity of intestinal flora

[0134] PCoA and differential distance analysis ( Figure 5 ) showed that there was a significant difference in the intestinal microbial composition between the low testicular index group and the high testicular index group (P = 0.005).

[0135] 2.3 Analysis of differences in intestinal flora composition

[0136] To analyze whether there were significant differences in intestinal microbial species between different groups, MetaStat difference tests were performed on the relative abundances at the phylum, genus, and species levels. The results showed that at the phylum level, the top five phyla in relative abundance were Firmicutes, Bacteroidota, Proteobacteria, Campilobacterota, and Euryarchaeota; at the genus level, the top ten genera in relative abundance were Clostridium sensu stricto 1, Lactobacillus, Streptococcus, Prevotella, Terrisporobacter, Escherichia-Shigella, Listeria, p-251-o5, Pasteurella, and T34. At the species level, the top five species in relative abundance were Pasteurella aerogenes, Prevotella copri, Actinobacillus rossii, Eubacterium coprostanoligenes, and Megasphaeraelsdenii ( Figure 6ac). The dominant bacteria in the intestines of boars in the high testicular index group were Lactobacillus and Anaerovibrio sp., and their relative abundances were significantly higher than those in the low testicular index group (P=0.044, P=0.028); the dominant bacteria in the intestines of boars in the low testicular index group were Campilobacterota and Lactobacillus, and their relative abundances were significantly higher than those in the high testicular index group (P=0.046, P=0.003) ( Figure 7 ad).

[0137] 2.4 Screening of specific intestinal flora

[0138] To screen for specific bacterial communities with high contributions, Simper was used to quantify the contribution of each species to the difference between the low and high testicular index groups. The results show the top 10 bacterial communities that contributed to the difference between the low and high testicular index groups. Among them, Campilobacterota, Lactobacillus, and Streptococcus had the highest contributions and were significantly different between the two groups ( Figure 8 ac). Studies have shown that Lactobacillus is associated with changes in semen parameters, while Streptococcus has a negative impact on sperm quality parameters such as sperm concentration, total motility, forward motility, activity, DNA integrity, and relative mRNA expression. In addition, Campilobacterota is associated with a variety of inflammatory diseases. Inflammation may cause damage to the testicles and epididymis, thereby affecting sperm production and maturation. This may explain why, in Examples 1 and 2, boars in the high testicular index group had higher testicular weight, testicular volume, and testosterone levels compared to boars in the low testicular index group. In addition, Lactobacillus was significantly positively correlated with feed efficiency. Excessive fat accumulation is believed to reduce the efficiency of feed conversion into lean meat production. This finding is consistent with the results in Example 3 that boars in the high testicular index group had a higher lean meat percentage and a lower fat percentage than boars in the low testicular index group.

[0139] In summary, the results of Examples 1-4 show that the boars in the high testicular index group have less fat deposition, a higher lean meat rate, and more mature testicular development. In addition, the abundance of Lactobacillus, a beneficial bacteria group related to nutrient absorption and utilization, in the intestines of the boars in the high testicular index group is relatively high, while the abundance of Streptococcus and Campilobacterota, bacteria groups related to inflammation and health problems, is relatively low. Therefore, in the rapid breeding of the Reserve Military Pasture No. 1 boar, these three bacteria groups, namely Lactobacillus, Streptococcus and Campilobacterota, can be used as indicators for the selection of reserve boars. The results of this study provide strong support for the rapid breeding of the Reserve Military Pasture No. 1 boar based on the intestinal microbiome.

[0140] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0141] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for breeding Junmu No. 1 reserve boar based on intestinal flora, characterized by: The steps of selecting and breeding the Junmu No. 1 reserve boar include: comparison of testicular development differences, comparison of production performance differences, analysis of intestinal flora characteristics, analysis of intestinal flora composition differences, screening of specific flora, and selection and breeding of the Junmu No. 1 reserve boar based on intestinal flora. By analyzing the differences in testicular development and production performance, we distinguished between individuals with high and low testicular indexes. Then, by comparing the intestinal flora characteristics and intestinal flora composition differences between individuals with high and low testicular indexes, we screened out specific flora related to production performance and testicular development, and ultimately achieved the selection and breeding of Junmu No. 1 reserve boars based on intestinal flora. The specific methods for screening specific bacterial communities are as follows: SA, boar fecal samples were collected in 5 mL sterile polypropylene tubes and stored at -80 °C for subsequent DNA extraction; SB, DNA was extracted from boar fecal samples using the DDNeasy PowerSoil Kit; SC, assess the quality of the DNA extracted in step SB by 1% agarose gel electrophoresis, and quantify the DNA concentration using a Nanodrop instrument; SD, bacterial 16S rDNA gene high-throughput sequencing The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified by PCR using the extracted DNA as a template. The primers used are as follows: Primer 341F: 5′-CCTAYGGGRBGCASCAG-3′; Primer 806R: 5′-GGACTACNNGGGTATCTAAT-3′; The PCR reaction system contains 15 μL High-Fidelity PCR Master Mix, 0.2 μM concentration of each primer, and 10 ng of target DNA extracted in step SB; PCR amplification conditions were as follows: predenaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min; SE, adopt Ultra TM II DNA library preparation kit was used according to the manufacturer's instructions to construct libraries from PCR amplification products. The construction process included fragmentation, end-repair, adapter ligation, and purification, ultimately creating a library suitable for high-throughput sequencing. The library was then sequenced on the Illumina NovaSeq platform to obtain 250 bp paired-end sequences. The relevant sequencing data have been deposited in the NCBI SRA database and analyzed using QIME2 software. SF, primer sequences and low-quality reads were denoised using the DADA2 v1.16 plug-in to remove possible sequencing errors, and high-quality paired-end clean reads were merged into complete tag sequences using FLASH v1.2.

11. Chimeric sequences were then removed using the VSEARCH tool to obtain high-quality amplicon sequence variants and ASVs. The ASV sequences were annotated and classified into species based on the Silva database. SG, boar feces sequencing data analysis: SG-1, Alpha diversity analysis: The alpha diversity of fecal microbiota was calculated using QIME2 and evaluated using Chao1, Shannon, Pieloué, and Simpson indices; SG-2, Beta diversity analysis: Beta diversity was calculated based on the Unweighted_unifrac distance, and the differences between samples were displayed using principal coordinate analysis in R software. Based on the Unweighted_unifrac distance, the anosim function was used to analyze whether the differences in community structure between groups were significant. SG-3, species difference analysis and specific bacterial group screening: MetaStat tool of R software was used to analyze species differences at each classification level. Simper was used to quantify the contribution of each species to the difference between the two groups, and specific bacterial groups were finally selected based on their contribution. Campilobacterota, Lactobacillus and Streptococcus were finally selected.

2. The method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora according to claim 1, characterized in that: The comparison of testicular development differences was evaluated by measuring testicular weight, testicular volume, serum and testicular testosterone levels.

3. The method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora according to claim 1, characterized in that: The comparison of production performance differences is evaluated by daily weight gain, feed conversion rate, fat rate, and lean meat rate.

4. The method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora according to claim 1, characterized in that: The NCBI SRA database project number in the step SE is: PRJNA1070801.

5. The method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora according to claim 1, characterized in that: The classification levels in step SG-3 are specifically the top 10 phyla, genus, and species.

6. The method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora according to claim 1, characterized in that: The identification of the intestinal flora of the Junmu No. 1 reserve boar is carried out by utilizing 16S amplicon high-throughput sequencing and identification of specific flora.

7. The method for selecting and breeding Junmu No. 1 reserve boars based on intestinal flora according to claim 6, characterized in that: The identification of the intestinal flora of the reserve boar is carried out by dissecting the intestinal contents of the reserve boar or analyzing the collected feces of the reserve boar.

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