A pig breed disease resistance breeding screening method based on taurine cholate metabolism markers

Abstract

The application discloses a pig breed disease resistance breeding screening method based on taurine cholate metabolic markers, and belongs to the technical field of animal breeding. The application effectively screens the breeding pigs with stronger intestinal barrier function by establishing a breeding standard based on the intestinal barrier function difference, especially taking the taurine cholate level as a key index, and improves the health level and production benefit of pigs. Compared with the prior art, the ileum villus height to crypt depth ratio of the breeding pig is increased by 25.3%, the mRNA relative expression amount of ZO-1 and other tight junction proteins is increased by 32.7%, the SIgA and IgG levels are increased by 38.5%, the health level of the pig is significantly improved, and the production benefit is increased by 15.6%.

Description

Technical Field

[0001] This invention belongs to the field of animal breeding technology, specifically relating to a method for screening pig breeds for disease resistance based on taurine bile salt metabolism markers. Background Technology

[0002] Currently, pig breeding mainly focuses on traditional indicators such as growth rate, lean meat percentage, and reproductive performance, while systematic evaluation of intestinal barrier function is insufficient. The intestinal barrier, as a key component in maintaining the intestinal and overall health of pigs, comprises four parts: mechanical barrier, chemical barrier, immune barrier, and microbial barrier. The strength of its function directly determines the health level and production efficiency of pigs.

[0003] While some studies in the field have focused on the relationship between gut microbiota composition and health in pigs, there is a lack of breeding standards that link intestinal barrier function to specific metabolites (such as taurine bile salts). Current pig breeding standards do not consider differences in intestinal barrier function, leading to potential risks to gut health in selected breeding pigs, thus impacting production efficiency and health levels.

[0004] Therefore, how to establish a breeding standard for pigs based on differences in intestinal barrier function has become an important issue that urgently needs to be addressed. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for screening pigs for disease resistance based on taurine bile salt metabolic markers. By using taurine bile salt levels as a key indicator, this method can screen for breeding pigs with stronger intestinal barrier function, thereby improving the health and production efficiency of pigs.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for screening disease resistance in pig breeds based on taurine bile salt metabolism markers, comprising the following steps: S1, Collect ileum and colon tissue samples from pigs; S2, measure intestinal barrier function-related indicators and taurine bile salt levels in the samples; S3. Based on the intestinal barrier function-related indicators and the taurine bile salt level, establish a breeding standard scoring system; S4. Based on the breeding standard scoring system, pig breeds are selected.

[0008] Based on the above technical solution, the intestinal barrier function related indicators further include the ratio of ileal villus height to crypt depth, expression levels of atresia 1, atresia 2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, mucin-2, secretory immunoglobulin A, and immunoglobulin G.

[0009] Among them, the following proteins are abbreviated as ZO-1, ZO-2, Claudin-1, Claudin-4, Occludin, Mucin-1, Mucin-2, SIgA, IgG, FXR, and TGR-5.

[0010] Based on the above technical solution, the specific method for determination in S2 is as follows: using tissue sectioning technology, performing HE staining, q-PCR and ELISA.

[0011] Based on the above technical solution, the breeding standard scoring system further includes a taurine cholate level ≥15.2 μmol / g.

[0012] Based on the above technical solution, the breeding standard scoring system further includes a taurine cholate level of 12.5-15.1 μmol / g.

[0013] Based on the above technical solution, the breeding standard scoring system further includes the taurine cholate level <12.5 μmol / g.

[0014] Based on the above technical solution, the breeding standard scoring system further includes: the ratio of ileal villus height to crypt depth ≥ 3.5; the relative mRNA expression levels of atresia 1, atresia 2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, and mucin-2 ≥ 1.8 times; and the expression levels of secretory immunoglobulin A and immunoglobulin G ≥ 250 pg / mg.

[0015] Based on the above technical solution, the breeding standard scoring system further includes: the ratio of ileal villus height to crypt depth is 3.0-3.4; the relative mRNA expression levels of atresia zona 1, atresia zona 2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, and mucin-2 are ≥1.2-1.7 times; and the expression levels of secretory immunoglobulin A and immunoglobulin G are 200-249 pg / mg.

[0016] Based on the above technical solution, the breeding standard scoring system further includes: the ratio of ileal villus height to crypt depth <3.0; the relative mRNA expression levels of atresia 1, atresia 2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, and mucin-2 <1.2-fold; and the expression levels of secretory immunoglobulin A and immunoglobulin G <200 pg / mg.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The method for screening pigs for disease resistance based on taurine bile salt metabolism markers provided by this invention establishes breeding standards based on differences in intestinal barrier function, especially using taurine bile salt level as a key indicator, to effectively screen breeding pigs with stronger intestinal barrier function, thereby improving the health level and production efficiency of pigs.

[0018] 2. The method provided by this invention, when used for screening, increases the ratio of ileal villus height to crypt depth in breeding pigs by 25.3%, increases the relative expression level of mRNA of tight junction proteins such as ZO-1 by 32.7%, increases SIgA and IgG levels by 38.5%, significantly improves the health level of pigs, and increases production efficiency by 15.6%. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0020] Figure 1 The flowchart for the detection of intestinal barrier function evaluation indicators provided by this invention; Figure 2 A schematic diagram of the breeding standard scoring system provided by this invention. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0022] Example 1 This embodiment verifies the effectiveness of the disease resistance screening of breeding pigs based on the method of the present invention.

[0023] I. Experimental Materials and Design 1. Laboratory animals Sixty breeding pigs (30±2.0 kg, half male and half female) of the Dahe pig × Dahe black pig crossbred at the Yunnan Agricultural University Experimental Pig Farm were selected. This crossbred combination was identified in previous systematic studies as having significant differences in intestinal barrier function, with well-defined baseline physiological indicators and a clear genetic background. All pigs were clinically confirmed to be healthy, with no history of digestive tract diseases, and their immunization program complied with national standards such as the "Technical Specifications for the Prevention and Control of Swine Fever".

[0024] 2. Experimental Grouping The animals were randomly divided into two groups of 30 each using a completely randomized design. There were no significant differences in body weight or sex ratio between the groups (P>0.05). Control group: Conventional breeding screening methods were used, and selection was based solely on growth rate (daily weight gain of 20-90 kg), body appearance (body sturdiness, body symmetry) and backfat thickness. Experimental group: The "taurine bile salt + intestinal barrier function index" combined screening method described in this invention was used.

[0025] 3. Feeding and Management Both groups of pigs were housed in the same standardized pigsty, in individual pens (2.5m × 1.8m). The ambient temperature was controlled at 20-24℃, and the relative humidity at 50-60%. Ventilation was carried out twice daily (1.5 hours each time), and disinfection was performed regularly (three times a week). All pigs were fed the same basal diet (formulated according to GB / T39235-2020 Nutritional Standards for Fat-Type Pigs), with a dry matter content of 88.5%, crude protein of 16.2%, and metabolizable energy of 13.8 MJ / kg. They had free access to feed and water, and reached a weight of 120 ± 5.6 kg at the end of the feeding period.

[0026] II. Screening Steps (corresponding to S1-S4 of this invention) 1. S1 Sample Collection After reaching 120kg, six animals (half male and half female) were randomly selected from each group for slaughter. Following the established standardized sampling procedure: 2g of tissue was aseptically collected from the mid-ileum (10cm from the ileocecal valve) and mid-colon (avoiding the mesenteric attachment), along with 1g of colonic contents. Samples were flash-frozen in liquid nitrogen within 10 minutes of collection and stored at -80℃ for later use to prevent tissue autolysis from affecting test results. The sampling procedure has passed animal experiment ethics review (No.: YN Agricultural University Animal Ethics 2024-032).

[0027] 2. S2 Index Measurement Ileal villus height to crypt depth ratio: Ileal tissue was fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, and then prepared into 5μm thick sections. After HE staining, the sections were mounted with neutral resin. Ten complete fields of view were selected under a microscope, and villus height (VH) and crypt depth (CD) were measured using ImageJ software. The VH / CD ratio was calculated, and the average value was taken as the detection result. This method was verified by three previous repeated experiments, and the measurement error was ≤3.2%.

[0028] Relative expression levels of tight junction proteins and mucin mRNAs: Total RNA was extracted from tissues using the Trizol method. After the RNA purity was tested and found to be acceptable (A260 / A280 = 1.8-2.0), cDNA was synthesized by reverse transcription. Using GAPDH as an internal reference gene, the relative expression levels of ZO-1, Claudin-1, Occludin, and Muc-2 mRNAs were determined by q-PCR. The primer sequences were standardized sequences that had been optimized and validated in the previous period (specific amplification efficiency ≥95%), and three technical replicates were set up for each sample. SIgA and IgG expression levels: Ileal tissue was homogenized with physiological saline at a ratio of 1:10, centrifuged at 12000 r / min for 15 min at 4℃, and the supernatant was collected. ELISA kits were used for detection, strictly following the instructions. Absorbance values ​​were read at 450 nm using a microplate reader. Concentrations were calculated based on the standard curve. The intra-assay coefficient of variation was ≤5%, and the inter-assay coefficient of variation was ≤8%. Taurine bile salt levels: 0.5 g of colon contents were taken and 1 mL of methanol / acetonitrile / water (2:2:1, v / v) extraction buffer was added. The mixture was ground in a high-throughput tissue homogenizer for 10 min, allowed to stand at -20℃ for 20 min, and then centrifuged at 4℃ and 12000 r / min for 15 min. The supernatant was filtered through a 0.22 μm filter membrane and analyzed by untargeted metabolomics using ultra-high pressure liquid chromatography-mass spectrometry. Data processing and quantification were performed using Progenesis QI software, and the detection limit was 0.01 μmol / g.

[0029] 3. Application of the S3 Breeding Standard Scoring System According to the three-level scoring rules of claims 4-9 of this invention (Excellent = 3 points, Good = 2 points, Average = 1 point), the scores are determined in conjunction with the indicator thresholds determined in previous experiments: Taurine cholate: ≥15.2μmol / g (excellent), 12.5-15.1μmol / g (good), <12.5μmol / g (fair), weight 30%; this threshold was determined based on statistical analysis of metabolomics data from 60 experimental pigs, corresponding to the characteristic values ​​of breeding pigs with excellent intestinal barrier function; Ileal villus-crypt ratio: ≥3.5 (excellent), 3.0-3.4 (good), <3.0 (average), weight 25%; Tight junction protein / mucin mRNA expression levels: ≥1.2-fold control group (excellent), 0.8-1.1-fold control group (good), <0.8-fold control group (fair), weight 25%; SIgA / IgG levels: ≥250pg / mg (excellent), 200-249pg / mg (good), <200pg / mg (fair), weight 20%; Overall score = score of each indicator × corresponding weight. A total score of ≥85 indicates an excellent breeding pig, which will be included in the breeding program.

[0030] 4. S4 breeding pig selection and breeding verification Ten breeding pigs (3 males and 7 females) with a comprehensive score ≥85 (excellent) were selected for breeding in the experimental group. Ten breeding pigs (3 males and 7 females) were selected using conventional methods. Both groups were bred simultaneously (artificial insemination was used, with an insemination volume of 30 mL / pig and a sperm density ≥3×10⁻⁶). 8 (number / mL). Statistical analysis of health indicators (diarrhea rate, morbidity) and production performance (average daily weight gain, feed conversion ratio) of offspring during the fattening period (30-120kg) was conducted to calculate the improvement in production efficiency.

[0031] III. Test Results Table 1

[0032] IV. Verification Conclusion This embodiment verifies the scientific validity and practicality of the screening method of the present invention through standardized testing: The results are shown in Table 1: The experimental group of pigs showed significantly better results than the control group in terms of ileal villus crypt ratio, tight junction protein expression, immunoglobulin level and taurine bile salt content, which is in complete agreement with the core conclusions of previous intestinal barrier function studies. The diarrhea rate of offspring during the fattening period was significantly reduced by 74.7%, the average daily weight gain increased by 15.6%, the feed conversion ratio decreased by 15.6%, and production efficiency increased by 15.6% simultaneously, which is consistent with the effects claimed in this invention. The entire experimental process (sample collection, index detection, scoring and screening) is repeatable and quantifiable. The differences in each data were statistically significant (P<0.05). The precision and accuracy of the detection methods meet the requirements of the technical specifications for breeding pigs. This method, through the combined screening of core metabolic markers and intestinal barrier function indicators, solves the problem of insufficient precision in disease resistance selection caused by conventional breeding relying solely on growth performance, and provides efficient and feasible technical support for the selection of disease-resistant breeding pigs.

[0033] Example 2 This embodiment uses taurine bile salt metabolism markers to screen for disease resistance in pig breeds.

[0034] This embodiment provides an example of screening for disease resistance in pig breeds using a method based on taurine bile salt metabolism markers. The research object is a "Dahe pig × Dahe black pig hybrid replacement breeding pig". The specific process is as follows. All operations comply with the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35892-2018) and completely correspond to the technical solution described in the claims of this invention.

[0035] I. Experimental Design and Material Preparation 1. Selection and grouping of pig breeds to be evaluated Sixty healthy Dahe pig × Dahe black pig hybrid breeding pigs (30±2.0 kg, half male and half female) bred at the Yunnan Agricultural University experimental pig farm were selected. Clinical examination confirmed that they had no history of digestive tract diseases, normal body temperature (38.5-39.5℃), and a complete immunization schedule (swine fever and porcine reproductive and respiratory syndrome (PRRS) vaccination completed according to standard procedures). They were randomly assigned to two groups of 30 pigs each using a completely randomized design. There were no significant differences in initial body weight (P=0.68) or sex ratio (15 males and 15 females) between the groups, ensuring the fairness of the experiment. Control group: Using conventional breeding screening methods, the selection of breeds was based solely on growth rate (average daily weight gain in the 20-90kg stage), body shape (body firmness and body symmetry), and backfat thickness (measured by ultrasound at 100kg body weight), which served as the control benchmark for the method of this invention. Experimental group: The "taurine bile salt + intestinal barrier function index" combined screening method described in this invention was used.

[0036] 2. Feeding and Management Conditions Both groups of pigs were housed in the same standardized pigsty, in single pens (2.5m × 1.8m). Environmental parameters included: temperature 20-24℃, relative humidity 50-60%, ventilation twice daily (1.5 hours each time), and disinfection three times weekly with 0.3% peracetic acid spray. Both groups were fed the same basal diet (formulated according to GB / T 39235-2020 Nutritional Standards for Fat-Type Pigs). The diet formula consisted of: corn 64.2%, soybean meal 16.1%, rice bran 10.0%, wheat bran 7.1%, limestone powder 1.08%, dicalcium phosphate 0.32%, salt 0.18%, and premix 1.0%. Nutritional levels were: digestible energy 13.81 MJ / kg, crude protein 15.17%, lysine 0.63%, calcium 0.55%, and total phosphorus 0.53%. Pigs had free access to feed and water. The total weight reached 120 ± 6.8 kg.

[0037] 3. Main Instruments and Reagents Instruments: Real-time PCR instrument (Bio-rad CFX connect), microplate reader (Multiskan FC), ultra-high pressure liquid chromatography-mass spectrometry (AB Triple TOF 6600), pathological slide machine (RM2016), optical microscope (Nikon Eclipse E100), high-speed refrigerated centrifuge (Eppendorf 5430R); Reagents: Trizol Total RNA Extraction Kit (Invitrogen, catalog number 15596026), Reverse Transcription Kit (TaKaRa, catalog number RR047A), Porcine SIgA ELISA Kit (Nanjing Jiancheng, catalog number H109), Porcine IgG ELISA Kit (Nanjing Jiancheng, catalog number H106), HE Staining Kit (Servicebio, catalog number G1003), Methanol / Acetonitrile (chromatographic grade, Sigma), q-PCR primers (synthesized by Sangon Biotech, sequences shown in Table 2).

[0038] Table 2. Primer sequences for q-PCR detection

[0039] II. Detailed Screening Process (corresponding to S1-S4 of claim 1) 1. S1 Sample Collection (to be performed when the animals reach a weight of 120±6.8kg) Six animals (half male and half female) were randomly selected from each group for slaughter. Sampling was carried out according to standardized procedures, with aseptic operation throughout. Tissue samples: 2g each of mid-ileum (10cm from the ileocecal valve) and mid-colon (avoiding the mesenteric attachment site) were aseptically collected. 1g of the tissue was placed in 4% paraformaldehyde fixative (fixed at 4℃ for 48h for HE staining), and the other 1g was flash-frozen in liquid nitrogen and stored at -80℃ (for q-PCR detection). Contents samples: 1g of ileal and colonic contents were aseptically collected, flash-frozen in liquid nitrogen, and stored at -80℃ (for non-targeted metabolomics detection of taurine bile salt levels). Fixation or freezing should be completed within 10 minutes after sampling to avoid tissue autolysis affecting test results.

[0040] 2. S2 index determination (corresponding to the determination method in claim 3) (1) Determination of the ratio of ileal villus height to crypt depth (HE staining method) Operating procedures: The fixed ileum tissue was subjected to gradient dehydration (70% ethanol for 1 h → 80% ethanol for 1 h → 90% ethanol for 1 h → 95% ethanol for 30 min × 2 → anhydrous ethanol for 30 min × 2), xylene clearing (15 min × 2), and paraffin embedding (60℃, 2 h) to prepare 5 μm thick continuous sections. Dewax sections to water (xylene I 10 min → xylene II 10 min → anhydrous ethanol 5 min × 2 → 95% ethanol 5 min → 90% ethanol 5 min → 80% ethanol 5 min → 70% ethanol 5 min → rinse with tap water 5 min); Hematoxylin staining for 5 min → rinse with tap water for 2 min → differentiate with 1% hydrochloric acid ethanol for 30 s → rinse with tap water for 2 min → stain with 0.5% eosin for 2 min → rinse with tap water for 1 min; Gradient dehydration (80% ethanol 30s → 90% ethanol 30s → 95% ethanol 30s × 2 → anhydrous ethanol 1min × 2), xylene clearing (1min × 2), neutral resin mounting; Under an optical microscope (10×40x), 10 complete and unbroken villi were observed. The villi height (VH, from the villi tip to the crypt opening) and crypt depth (CD, from the crypt opening to the crypt bottom) were measured using ImageJ software. The VH / CD ratio was calculated, and the average value was taken as the test result for the pig.

[0041] Results: Table 3 shows the data on ileal villus height, crypt depth and ratio between the control group and the experimental group. The VH / CD ratio in the experimental group was significantly higher than that in the control group (P<0.05).

[0042] Table 3. Results of ileal villus height, crypt depth, and ratio (x±s, n=6)

[0043] (2) Determination of relative expression levels of tight junction proteins and mucin mRNA (q-PCR method) Operating procedures: Take 0.1g of ileal tissue stored at -80℃, add 1mL of Trizol reagent, grind and break it up with a high-throughput tissue homogenizer (60Hz, 3min), and extract total RNA according to the kit instructions; RNA purity (A260 / A280 = 1.8-2.0) and concentration (≥50 ng / μL) were detected using a NanoDrop 2000 micro-spectrophotometer, and RNA integrity (1.5%) was verified by agarose gel electrophoresis (clear 28S and 18S bands, no degradation). Following the instructions of the reverse transcription kit, cDNA was synthesized using 1 μg of total RNA as a template (reaction conditions: 37℃ for 15 min → 85℃ for 5 s → storage at 4℃). q-PCR reaction system (20 μL): 10 μL 2×SYBR Green Mix, 0.8 μL upstream primer (10 μmol / L), 0.8 μL downstream primer (10 μmol / L), 2 μL cDNA template, 6.4 μL RNase-free water; Reaction conditions: 95℃ pre-denaturation for 30s → 95℃ denaturation for 5s → 60℃ annealing and extension for 30s (40 cycles) → melting curve analysis (95℃ for 15s → 60℃ for 1min → 95℃ for 15s); Using GAPDH as an internal reference gene, 2 - The relative expression level of the target gene mRNA was calculated using the ΔΔCt method (with the average expression level of the control group as a reference of 1, i.e., the control group data is "1.00").

[0044] Results: The relative expression levels of each target gene mRNA in the experimental group were significantly higher than those in the control group (P<0.05), as shown in Table 4.

[0045] Table 4. Relative expression levels of tight junction proteins and mucin mRNAs (x±s, n=6, 1.00 times that of the control group).

[0046] (3) Measurement of SIgA and IgG expression levels (ELISA method) Operating procedures: Take 0.1g of ileal tissue stored at -80℃, add 1mL of physiological saline, grind in an ice bath to make a homogenate, centrifuge at 12000r / min for 15min at 4℃, and take the supernatant; Follow the instructions of the ELISA kit, dilute the supernatant 10 times and add it to the wells of the microplate (100 μL / well), and incubate at 37°C for 60 min; Discard the liquid in the wells, wash the plate 3 times (30s each time) with washing buffer, add enzyme-labeled secondary antibody (100μL / well), and incubate at 37℃ for 30min; Wash the plate 3 times, add colorimetric reagent (100 μL / well), and incubate at 37°C in the dark for 15 min; Add stop solution (50 μL / well), read absorbance (OD value) at 450 nm wavelength using an ELISA reader, and calculate SIgA and IgG concentrations (unit: pg / mg) based on the standard curve.

[0047] Results: The levels of SIgA and IgG in the experimental group were significantly higher than those in the control group (P<0.05), as shown in Table 5.

[0048] Table 5. Results of SIgA and IgG expression levels (x±s, n=6, pg / mg)

[0049] (4) Taurine bile salt level determination (non-targeted metabolomics method) Operating procedures: Take 0.5g of colon contents, add 1mL of methanol / acetonitrile / water (2:2:1, v / v) extraction solution, vortex mix for 1min, sonicate in an ice bath for 30min, and let stand at -20℃ for 20min; Centrifuge at 4℃ and 14000r / min for 20min, collect the supernatant, freeze dry under vacuum, add 100μL of acetonitrile / water (1:1, v / v) to reconstitute, vortex for 1min, and centrifuge at 4℃ and 14000r / min for 15min; The supernatant was filtered through a 0.22 μm filter membrane and analyzed using ultra-high pressure liquid chromatography-mass spectrometry (HPLC-MS / MS) (column: Waters ACQUITY UPLC BEH Amide 1.7 μm, 2.1 mm × 100 mm; mobile phase A: water + 25 mM ammonium acetate + 25 mM ammonia; mobile phase B: acetonitrile; gradient elution: 0-0.5 min 95% B, 0.5-7 min B 95%-65%, 7-8 min B 65%-40%, 8-9 min B 40%, 9-9.1 min B 40%-95%, 9.1-12 min B 95%; flow rate 0.5 mL / min; column temperature 25 ℃; injection volume 2 μL; mass spectrometry ion source: ESI, positive and negative ion mode; scan range m / z 60-1000). Data were processed using Progenesis QI software, qualitative analysis was performed using Mass Bank database matching, and quantitative analysis was performed using external standard method (taurine bile salt standard, Sigma, catalog number T0875).

[0050] Results: The taurine cholate level in the experimental group was significantly higher than that in the control group (P<0.05), as shown in Table 6.

[0051] Table 6. Results of taurine bile salt levels in colonic contents (x±s, n=6, μmol / g)

[0052] 3. Application of the S3 breeding standard scoring system (corresponding to claims 4-9) (1) Definition of control group and data source The control group was a "breeding pig population selected through conventional breeding". The data of each indicator were the average values ​​of the actual measurements obtained during the same period of feeding and using the same testing methods (i.e., the control group data in Table 2-5). This data served as the reference benchmark for the "fold value" in the scoring system of this invention (e.g., "mRNA relative expression level ≥ 1.8 times" means 1.8 times the average value of the control group).

[0053] (2) Scoring rules and weight allocation The scoring method adopts a "four-level indicator + weight" method, with a total score of 100 points. The weights and scoring standards of each indicator are shown in Table 7. Among them, excellent = 3 points, good = 2 points, average = 1 point. The score of a single indicator = grade score × weight ratio, and the overall score = the sum of the scores of each single indicator.

[0054] Table 7. Breeding Standard Scoring System (Total score: 100 points)

[0055] (3) Example of calculating the overall score The total score is 100 points. The grade score is "the full score of the corresponding weight", that is, Excellent = weight percentage score, Good = weight percentage score × 2 / 3, and Average = weight percentage score × 1 / 3.

[0056] Taking a superior breeding pig in the experimental group as an example, the test results and scores of various indicators are as follows: Taurine cholate level: 14.3 μmol / g (Good, 3 points) → Taurine cholate level (20 points): Excellent = 30 points, Good = 20 points, Average = 10 points; Ileal villus height / crypt depth ratio: 3.9 (Excellent, 3 points) → Ileal villus height / crypt depth ratio (25 points): Excellent = 25 points, Good = 17 points, Average = 8 points; Relative expression levels of tight junction proteins and mucin mRNA: both 1.85-fold (excellent, 3 points) → Relative expression levels of tight junction proteins and mucin mRNA (25 points): Excellent = 25 points, Good = 17 points, Average = 8 points; SIgA and IgG levels: 245 pg / mg, 242 pg / mg (good, 2 points) → SIgA and IgG levels (13 points): Excellent = 20 points, Good = 13 points, Average = 7 points; The overall score is the sum of the scores of each indicator. A score of ≥85 is considered excellent, 70-84 is considered good, and <70 is considered average.

[0057] Calculation example: Taurine cholate: 14.3 μmol / g (Good, 20 points); Fiber crypt ratio: 3.9 (Excellent, 25 points); mRNA expression level: 1.85-fold (excellent, 25 points); SIgA / IgG: 245 / 242 pg / mg (good, 13 points); Overall score = 20 + 25 + 25 + 13 = 83 points (good breeding pig).

[0058] 4. S4 Breeding Pig Screening Results After screening through the above process, 12 excellent breeding pigs (overall score ≥85 points), 23 good breeding pigs (70-84 points), and 25 average breeding pigs (<70 points) were finally selected from 60 pigs to be evaluated. The specific indicator data of the excellent breeding pigs are shown in Table 8. All indicators meet the "excellent standard" and are consistent with the improvement ratio claimed in the abstract of this invention (25.3% increase in villus crypt ratio, 32.7% increase in tight junction protein mRNA expression, and 38.5% increase in SIgA / IgG level).

[0059] Table 8. Test results of various indicators of excellent breeding pigs (x±s, n=12)

[0060] III. Screening Effect Verification The 12 selected superior breeding pigs were bred simultaneously with 12 conventionally selected breeding pigs from the control group. The production performance and health indicators of the offspring during the fattening period (30-120kg) were statistically analyzed. Average daily weight gain of offspring: 838±42g / d in the experimental group and 725±35g / d in the control group, representing an increase of 15.6%. Feed conversion ratio of offspring: 2.71±0.09 in the experimental group and 3.21±0.11 in the control group, a decrease of 15.6%; Diarrhea rate in offspring during fattening period: 2.1±0.8% in the experimental group and 8.3±1.2% in the control group, a decrease of 74.7%; the verification results show that the breeding pigs obtained by the screening method of the present invention have significantly improved offspring production efficiency and health level, which is consistent with the technical effects claimed by the present invention.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening disease resistance in pig breeds based on taurine bile salt metabolism markers, characterized in that, Includes the following steps: S1, Collect ileum and colon tissue samples from pigs; S2, measure intestinal barrier function-related indicators and taurine bile salt levels in the samples; S3. Based on the intestinal barrier function-related indicators and the taurine bile salt level, establish a breeding standard scoring system; S4. Based on the breeding standard scoring system, pig breeds are selected.

2. The method for screening disease resistance in pig breeds based on taurine bile salt metabolism markers according to claim 1, characterized in that, The intestinal barrier function-related indicators include the ratio of ileal villus height to crypt depth, and the expression levels of atresia bar protein-1, atresia bar protein-2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, mucin-2, secretory immunoglobulin A, and immunoglobulin G.

3. The method for screening disease resistance in pig breeds based on taurine bile salt metabolism markers according to claim 1, characterized in that, The specific methods for determining the intestinal barrier function-related indicators in S2 include: using tissue sectioning techniques, performing HE staining, q-PCR, and ELISA; the specific method for determining the taurine bile salt level in S2 is a non-targeted metabolomics analysis method.

4. The method for screening disease resistance in pig breeds based on taurine bile salt metabolism markers according to claim 2, characterized in that, The breeding standard scoring system includes a taurine cholate level ≥15.2 μmol / g.

5. The method for screening pig breeds for disease resistance based on taurine bile salt metabolism markers according to claim 2, characterized in that, The breeding standard scoring system includes a taurine cholate level of 12.5-15.1 μmol / g.

6. The method for screening disease resistance in pig breeds based on taurine bile salt metabolic markers according to claim 2, characterized in that, The breeding standard scoring system includes a taurine cholate level of <12.5 μmol / g.

7. A method for screening pig breeds for disease resistance based on taurine bile salt metabolism markers according to any one of claims 4 to 6, characterized in that, The breeding standard scoring system also includes: the ratio of ileal villus height to crypt depth ≥3.5; the relative mRNA expression levels of atresia zona 1, atresia zona 2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, and mucin-2 ≥1.8 times; and the expression levels of secretory immunoglobulin A and immunoglobulin G ≥250 pg / mg.

8. A method for screening pig breeds for disease resistance based on taurine bile salt metabolism markers according to any one of claims 4 to 6, characterized in that, The breeding standard scoring system also includes: the ratio of ileal villus height to crypt depth is 3.0-3.4; the relative mRNA expression levels of atresia 1, atresia 2, tight junction protein 1, tight junction protein 4, closure protein, mucin 1, and mucin 2 are ≥1.2-1.7 times; and the expression levels of secretory immunoglobulin A and immunoglobulin G are 200-249 pg / mg.

9. A method for screening disease resistance in pig breeds based on taurine bile salt metabolism markers according to any one of claims 4 to 6, characterized in that, The breeding standard scoring system also includes: the ratio of ileal villus height to crypt depth <3.0; the relative mRNA expression levels of atresia 1, atresia 2, tight junction protein-1, tight junction protein-4, closure protein, mucin-1, and mucin-2 <1.2-fold; and the expression levels of secretory immunoglobulin A and immunoglobulin G <200 pg / mg.

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