Shrimp intestinal health state evaluation method and evaluation system

Abstract

The invention relates to the technical field of biology, and particularly discloses a shrimp intestinal tract health state evaluation method and system, and the method comprises the following steps: S1, collecting a sample of litopenaeus vannamei fed with cottonseed enzymolysis protein; s2, measuring related indexes of an intestinal physical barrier, a chemical barrier, an immune barrier and a biological barrier of the sample; and S3, performing data statistical analysis on the indexes in the step S2, performing standardization processing, endowing corresponding weights, calculating a comprehensive health score S, and determining the intestinal health level of the prawns. According to the evaluation method provided by the invention, the intestinal health or abnormal state of the prawns is accurately identified through the change of the multi-dimensional indexes, and a scientific basis is provided for precise feed formula development and disease early warning.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method and system for assessing the intestinal health status of shrimp. Background Technology

[0002] Litopenaeus vannamei is one of the most important farmed shrimp species globally. In recent years, due to changes in feed composition and increased farming intensity, intestinal health problems in shrimp have become increasingly prominent. These problems mainly manifest as impaired digestion and absorption, immune dysfunction, and intestinal flora imbalance, seriously affecting shrimp growth performance and farming efficiency.

[0003] Current assessments of shrimp gut health primarily focus on single indicators, lacking a comprehensive, systematic, quantitative, and practical evaluation method. In particular, the correlation between abnormal gut morphology, dysbiosis, and functional impairment has not been fully identified. Therefore, developing a scientific, systematic, and quantitative comprehensive assessment method for shrimp gut health has significant practical value. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the existing technology and provide a method and system for assessing the intestinal health status of shrimp. The assessment method of this application accurately identifies the healthy or abnormal state of the shrimp's intestinal tract through changes in multi-dimensional indicators, providing a scientific basis for precision feed formulation development and disease early warning.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0006] This application provides a method for assessing the intestinal health status of shrimp, the method comprising the following steps:

[0007] S1. Collect samples of Litopenaeus vannamei fed with cottonseed enzymatic hydrolysate;

[0008] S2. Determine the relevant indicators of the intestinal physical barrier, chemical barrier, immune barrier and biological barrier of the sample;

[0009] S3. Perform statistical analysis on the indicators in step S2, standardize them and assign corresponding weights, calculate the comprehensive health score S, and determine the intestinal health level of the shrimp.

[0010] Z = (X - μ) / σ;

[0011] S = 100 - ∑(weight i × |Z i| × 100);

[0012] Where X is the original data value; μ is the mean of the healthy group data; and σ is the standard error of the healthy group data.

[0013] Weight i represents the proportion of importance of the i-th indicator in the evaluation system;

[0014] |Zi| is the absolute value of the standardized Z-score of the i-th indicator;

[0015] S ≥ 80: Shrimp gut health is good; 60 ≤ S < 80: Shrimp gut health is suboptimal; S < 60: Shrimp gut health is at risk.

[0016] Cottonseed enzymatically hydrolyzed protein is obtained from cottonseed protein through complex enzymatic hydrolysis, characterized by low gossypol content and high protein content. Specifically, cottonseed enzymatically hydrolyzed protein (ECP) is a product obtained by hydrolyzing cottonseed or cottonseed protein with enzymes such as proteases and cellulases, followed by drying. Its content of soluble protein, free amino acids, and small molecule peptides is increased, thus enhancing its nutritional value. Cottonseed enzymatically hydrolyzed protein belongs to the bioactive peptide category of plant extracts, possessing functions such as promoting growth, anti-oxidation, antibacterial, antiviral, and improving intestinal health. Small molecule bioactive peptides are biochemical substances between amino acids and proteins; they are smaller in molecular weight than proteins but larger in molecular weight than amino acids, and are fragments of proteins. Peptides composed of 10-55 amino acids are called polypeptides, while peptides composed of 2-10 amino acids are small molecule bioactive peptides or oligopeptides. Bioactive peptides are characterized by 100% absorption, direct penetration through physiological barriers, maintenance of normal physiological activities in animals, enhancement of immunity, and high physiological activity in small quantities.

[0017] This application investigates the impact of feeding fishmeal replacements with cottonseed hydrolysate at varying levels on the intestinal health of Litopenaeus vannamei, aiming to provide a reference for the utilization of cottonseed hydrolysate in healthy shrimp farming. The method focuses on the four core barrier functions of the shrimp gut: physical, chemical, immune, and biological barriers, employing multi-dimensional joint detection. By setting health thresholds for key indicators of each barrier, and after normalization and weight allocation, a comprehensive health score S is calculated, thereby outputting a health level and targeted intervention recommendations.

[0018] In a preferred embodiment of the shrimp gut health assessment method described in this application, in step S1, the amount of cottonseed enzymatic hydrolysate added is 2% to 4%, preferably 2%.

[0019] This application investigates and evaluates the effects of cottonseed hydrolysate containing different amounts on the intestinal health of Litopenaeus vannamei by feeding the shrimp, providing a reference for the utilization of cottonseed hydrolysate in the healthy aquaculture of Litopenaeus vannamei.

[0020] Among them, the addition of 2% cottonseed enzymatic hydrolysate can significantly improve the intestinal health of Litopenaeus vannamei. The results of this study provide reference data for the application of cottonseed enzymatic hydrolysate in crustacean feed and the promotion of the healthy development of aquaculture.

[0021] As a preferred embodiment of the shrimp gut health status assessment method described in this application, in step S1, during sample collection, the gut of the sample is sectioned using hematoxylin and eosin staining and transmission electron microscopy; the digestive enzyme activity of the sample is measured using a standardized kit, and the expression levels of immune and antioxidant genes are assessed using real-time quantitative PCR; 16S rRNA gene sequencing is used to detect gut microbiota and perform functional prediction.

[0022] In some specific implementation schemes, the sample collection is as follows:

[0023] Sample collection was conducted in accordance with the animal collection rules of the Aquatic Animal Experiment Ethics Committee of Guangdong Ocean University. Shrimp were fasted for 24 hours before each sampling. Samples were taken after 8 weeks of culture. Ten shrimp were taken from the cultured experiment, and their intestines were dissected on ice using scissors and tweezers, placed in enzyme-free cryovials, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent enzyme activity and gene expression experiments. Two additional shrimp were taken, and their intestines were dissected and fixed in Bouin's solution and electron microscopy fixative for histological analysis.

[0024] In some specific implementation plans, methods for measuring physical barrier-related indicators include:

[0025] Approximately 1 cm of midgut tissue was taken from shrimp and fixed in Bouin's solution for 12 hours. The tissue was then washed with a gradient of 100%, 80%, and 70% ethanol and dehydrated in 70% anhydrous ethanol. Paraffin sections were prepared by resin embedding and stained with hematoxylin and eosin (H&E). The sections were then sealed on slides and observed morphologically under a microscope (ECLIPSE 90i, Nikon, Japan). The height of the midgut villi (FH) and the thickness of the muscle layer (MT) were measured using image acquisition software (NIS Elements version 4.60, Nikon, Japan). Ten different points were randomly selected within the same field of view for each measurement. Similarly, approximately 0.5 cm of midgut tissue was cut and fixed in 2.5% glutaraldehyde solution for 24 hours. The samples were then fixed in 1% osmium tetroxide at room temperature in the dark for 2 hours, bleached three times with 0.1 M phosphate buffer PB (pH 7.4), dehydrated by ethanol fractionation, bleached with acetone, and finally embedded in resin. Ultrathin sections (60-80 nm) were placed on a copper grid, stained with 2% uranium acetate saturated alcohol solution in the dark, rinsed with distilled water, and stained with 2.6% lead citrate for 8 min. They were then screened and observed using a transmission electron microscope (Hitachi, HT7700 TEM). Ten different points were randomly selected from the same field of view on each electron microscope section for measurement of microvilli length (ML).

[0026] In some specific implementation plans, methods for determining chemical barrier-related indicators include:

[0027] The activities of intestinal amylase, lipase and trypsin in shrimp were determined according to the kit instructions of Nanjing Jiancheng Bioengineering Institute (Nanjing, China) to evaluate the effect of cottonseed hydrolysate on the activity of intestinal digestive enzymes in shrimp.

[0028] In some specific implementation plans, methods for measuring immune barrier-related indicators include:

[0029] Total RNA was extracted from hepatocellular carcinoma and intestinal samples using the TransZol Up Plus RNA Kit (TransGen, Beijing), and its quality and concentration were assessed using a micro-spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA). RNA was reverse transcribed into cDNA using the Evo M-MLV Plus cDNA Synthesis Kit (Accurate Biology, Hunan). The Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, Hunan) was used in a LightCycler 480 (Roche Applied Science, Germany) to perform real-time PCR on the target gene using the following program: 40 cycles of 95℃ for 30 seconds, 95℃ for 5 seconds, and 60℃ for 30 seconds.

[0030] In some specific implementation plans, methods for measuring microbial barrier-related indicators include:

[0031] Total DNA was extracted from intestinal samples according to the HiPure Stool DNA Kits (D3141, Guangzhou Meiji Biotechnology Co., Ltd.) instructions, and DNA quality was assessed using a micro-spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA). PCR amplification was performed using primers 341F (5′CCTACGGGNGGCWGCAG3′) and 806R (5′GGACTACHVGGGTATCTAAT3′) for the V3-V4 variable region. Amplicons were collected from 2% agarose gels and purified. Sequencing libraries were constructed using the Illumina DNA Prep Kit (Illumina, CA, USA). The raw data underwent quality control to obtain representative sequences and abundance information for Operational Taxonomic Units (OTUs). Based on this information, species annotation was performed, and bioinformatics analyses were conducted, including species composition, indicator species, alpha diversity, beta diversity, and functional prediction.

[0032] As a preferred embodiment of the shrimp intestinal health status assessment method described in this application, in step S2, the intestinal physical barrier related indicators include at least one of intestinal villus length, muscle layer thickness, microvilli length, and perifecal membrane related gene expression indicators.

[0033] Chemical barrier-related indicators include at least one of the activities of digestive enzymes or gene expression levels;

[0034] Immune barrier-related indicators include at least one of the following: tumor necrosis factor-α, interferon-γ, interleukin-1β, transforming growth factor-β, myeloid differentiation factor, immunodeficiency protein, Toll-like receptor, lysozyme, antimicrobial peptide, anti-lipopolysaccharide factor, shrimp antimicrobial peptide, prophenoloxidase, superoxide dismutase, and catalase gene expression levels.

[0035] Biological barrier-related indicators include the relative abundance of gut microbiota.

[0036] In a preferred embodiment of the shrimp intestinal health status assessment method described in this application, the digestive enzyme includes at least one of amylase, lipase, trypsin, and chymotrypsin.

[0037] The intestinal flora includes at least one of Vibrio, Rugellella, Lactococcus, Cryptophyte, and Ivorymomonas.

[0038] In a preferred embodiment of the shrimp gut health status assessment method described in this application, gut health is determined to be abnormal when any of the following conditions are met by the intestinal physical barrier, chemical barrier, immune barrier, and biological barrier related indicators:

[0039] Threshold = UH group mean ± 2 × UH group standard deviation; the threshold is the critical point at which the specific indicator shows abnormalities in the intestine, and ± is determined according to the actual situation;

[0040] 1) The villus length is ≤29.48μm, the muscle layer thickness is ≤15.14μm, the microvilli length is ≤1.41μm, and the expression level of peritrophic membrane-related genes is ≤1.49;

[0041] 2) Digestive enzyme activity ≤ 0.87;

[0042] 3) Interleukin-1β expression level ≥1.36, lysozyme expression level ≥0.68, anti-lipopolysaccharide factor expression level ≥1.52, superoxide dismutase expression level ≤1.54, or myeloid differentiation factor expression level ≤0.80;

[0043] 4) The abundance of Vibrio spp. is ≥54.55%, the abundance of Rugellella spp. is ≤3.4%, the abundance of Lactococcus spp. is ≥1.0%, the abundance of Cryptophyte Aquaticus is ≤0.56%, and the abundance of Ivorymomonas molluscum is ≤0.16%.

[0044] As a preferred embodiment of the shrimp gut health status assessment method described in this application, in step S3, GraphPad Prism 9 software is used to perform differential analysis on intestinal physical barrier, chemical barrier, immune barrier and biological barrier related indicators; the data are expressed as "mean ± standard error", and P<0.05 indicates significant difference.

[0045] This application utilizes GraphPad Prism 9 software for statistical analysis, performing significance analysis on changes in slice quantification, enzyme activity, and gene expression levels. The p-value is used to determine statistical significance, enhancing the statistical validity and interpretability of the results. Compared to the simple descriptive analysis in existing technologies, the statistical methods employed in this application significantly improve the scientific rigor and precision of the data analysis.

[0046] This application also provides an intelligent assessment system for shrimp intestinal health, the assessment system comprising:

[0047] a. Data acquisition module, used to acquire data on indicators related to the intestinal physical barrier, chemical barrier, immune barrier and biological barrier of shrimp;

[0048] b. Data analysis module, used for standardizing the collected data, identifying abnormal indicators, and calculating comprehensive scores;

[0049] c. Assessment report module, used to output gut health score, diagnostic results and intervention recommendations.

[0050] Based on the aforementioned assessment methods, this application designs a corresponding intelligent system, including a data acquisition module, a data analysis module, and an assessment report module. This system can automatically collect data on various intestinal indicators of shrimp, perform data processing and anomaly identification, and output intestinal health scores and diagnostic suggestions. The system supports the rapid identification of intestinal damage risks caused by factors such as changes in feed composition or pathogen invasion.

[0051] This application provides an intelligent assessment system for shrimp gut health that integrates data acquisition, standardized analysis, identification of abnormal barrier function, and scoring. This method focuses on barrier function, featuring a comprehensive assessment structure, a scientifically sound indicator system, and high quantifiability. It is suitable for scenarios such as health monitoring, evaluation of feed intervention effects, and early disease warning in shrimp farming.

[0052] This application particularly emphasizes the core role of the unique physiological characteristics of the shrimp gut in health assessment. For example, it considers the expression of genes related to the perifecal membrane, the expression levels of shrimp immune molecules (such as anti-lipopolysaccharide factor (ALF), prophenoloxidase (propo), and superoxide dismutase (SOD),) and the abundance changes of gut microbiota (such as Vibrio and Ruegeria). Based on these multi-dimensional indicators, a comprehensive health score S is calculated by setting judgment thresholds and using a weighted scoring model, enabling the classification and judgment of the shrimp gut health status (e.g., S ≥ 80 indicates good gut health, 60 ≤ S < 80 indicates sub-health, and S < 60 indicates gut health risk). This application's solution has a clear structure, is easy to operate, and has high repeatability, providing reliable technical support for precision shrimp farming management, feed formulation improvement, and disease prevention and control.

[0053] In the technical solution of this application, a shrimp-specific indicator system is constructed, which is different from the evaluation framework of common vertebrates; four key health dimensions are integrated to establish a cross-validation mechanism to improve accuracy; a Z-score standardization + weighted scoring model is introduced to output fractional diagnostic results; and a supporting evaluation system module is developed with real-time data processing and reporting functions.

[0054] Compared with the prior art, this application has the following beneficial effects:

[0055] Existing technologies for studying the effects of cottonseed hydrolysate on the gut health of Litopenaeus vannamei during shrimp farming are rather one-sided and lack standardized evaluation procedures. This application provides a complete and scientific evaluation method through a systematic experimental design, including sample collection, enzyme activity detection, and gene expression level determination, enabling a comprehensive and accurate analysis of the multidimensional effects of cottonseed hydrolysate on the gut health of Litopenaeus vannamei. This comprehensive evaluation addresses the problem of singular evaluation methods in existing technologies and provides a scientific basis for the application of natural astaxanthin in shrimp farming.

[0056] This application systematically studies the comprehensive effects of cottonseed enzymatic hydrolysate on the intestinal health of Litopenaeus vannamei. Combining a concrete and intuitive slicing assay method, efficient enzyme activity and gene expression analysis techniques, and 16S rRNA detection technology, it comprehensively evaluates the multiple biological effects of cottonseed enzymatic hydrolysate in Litopenaeus vannamei aquaculture. This method, through innovative experimental design and data analysis, helps to improve the use of plant protein raw materials in shrimp farming, thereby increasing farming efficiency and product quality.

[0057] This application combines slide observation, enzyme activity detection, gene expression, and 16S rRNA sequencing analysis techniques to comprehensively evaluate the effects of cottonseed enzymatic hydrolysate on the gut health of Litopenaeus vannamei. This multi-level systematic evaluation method overcomes the limitations of existing technologies in the insufficient depth of research on shrimp gut health.

[0058] In summary, this application, through standardized and systematic experimental methods, solves the problem of the lack of comprehensive, accurate and scientific methods in the prior art for evaluating the effects of cottonseed enzymatic hydrolysate on the intestinal health of Litopenaeus vannamei, and achieves significant technological progress, promoting industrial application in the fields of shrimp farming and nutritional science. Attached Figure Description

[0059] Figure 1 A flowchart for a gut health assessment method; Figure 2 A schematic diagram of an H&E-stained section of shrimp intestinal tissue; Figure 3 Transmission electron microscopy (TEM) images of the ultrastructure of the shrimp intestine; Figure 4 A bar chart showing the relative abundance of the shrimp gut microbiota at the phylum level; Figure 5 A bar chart showing the relative abundance of shrimp gut microbiota at the genus level; Figure 6 Figure I shows the LefSe analysis of the gut microbiota of Litopenaeus vannamei; Figure 7 Figure II shows the LefSe analysis of the gut microbiota of Litopenaeus vannamei; Figure 8 To predict the functional map of the gut microbiota of Litopenaeus vannamei based on PICRUSt2; Figure 9 A schematic diagram illustrating the correlation between intestinal morphological parameters and bacterial genus abundance; Figure 10 A schematic diagram illustrating the correlation between intestinal digestive function indicators and bacterial abundance; Figure 11 This is a schematic diagram illustrating the correlation between intestinal immune function indicators and gut microbiota abundance. Detailed Implementation

[0060] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0061] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0062] In the following examples, the cottonseed enzymatic hydrolysate was purchased from Xinjiang Xipu Biotechnology Co., Ltd.

[0063] Example 1: A method for assessing the intestinal health status of shrimp

[0064] This embodiment provides a method for assessing the intestinal health status of shrimp, including the following steps:

[0065] S1. Collect samples of Litopenaeus vannamei fed with cottonseed enzymatic hydrolysate;

[0066] S2. Determine the relevant indicators of the intestinal physical barrier, chemical barrier, immune barrier and biological barrier of the sample;

[0067] S3. Perform statistical analysis on the indicators in step S2, standardize them and assign corresponding weights, calculate the comprehensive health score S, and determine the intestinal health level of the shrimp.

[0068] Z = (X - μ) / σ;

[0069] S = 100 - ∑(weight i × |Z i| × 100);

[0070] Where X is the original data value; μ is the mean of the healthy group data; and σ is the standard error of the healthy group data.

[0071] Weight i represents the proportion of importance of the i-th indicator in the evaluation system;

[0072] |Zi| is the absolute value of the standardized Z-score of the i-th indicator;

[0073] S ≥ 80: Shrimp gut health is good; 60 ≤ S < 80: Shrimp gut health is suboptimal; S < 60: Shrimp gut health is at risk. Flowchart as follows: Figure 1 As shown.

[0074] The detection and evaluation of intestinal physical barrier-related indicators include the following steps:

[0075] Approximately 1 cm of midgut tissue was taken from shrimp and fixed in Bouin's solution for 12 hours. The tissue was then washed with a gradient of 100%, 80%, and 70% ethanol and dehydrated in 70% anhydrous ethanol. Paraffin sections were prepared by resin embedding and stained with hematoxylin and eosin (H&E). The sections were then sealed on slides and observed morphologically under a microscope (ECLIPSE 90i, Nikon, Japan). The height of the midgut villi (FH) and the thickness of the muscle layer (MT) were measured using image acquisition software (NIS Elements version 4.60, Nikon, Japan). Ten different points were randomly selected within the same field of view for each measurement. Similarly, approximately 0.5 cm of midgut tissue was cut and fixed in 2.5% glutaraldehyde solution for 24 hours. The samples were then fixed in 1% osmium tetroxide at room temperature in the dark for 2 hours, bleached three times with 0.1 M phosphate buffer PB (pH 7.4), dehydrated by ethanol fractionation, bleached with acetone, and finally embedded in resin. Ultrathin sections (60-80 nm) were placed on a copper grid, stained with 2% uranium acetate saturated alcohol solution in the dark, rinsed with distilled water, and stained with 2.6% lead citrate for 8 min. They were then screened and observed using a transmission electron microscope (Hitachi, HT7700 TEM). Ten different points were randomly selected from the same field of view on each electron microscope section for measurement of microvilli length (ML).

[0076] After collecting shrimp intestinal segments, the sections were embedded in paraffin and stained with H&E. Figure 2 (Schematic diagram of H&E stained section of shrimp intestinal tissue). Villous length and muscle layer thickness were measured using a microscope and image analysis software; simultaneously, transmission electron microscopy (TEM) was used to observe microvilli length. Figure 3This image shows a transmission electron microscope (TEM) image of the ultrastructure of the shrimp intestine. Furthermore, the expression levels of perifecal membrane-related genes (such as chit1, proea, and sgs3) were detected by real-time quantitative PCR to assess the integrity of the intestinal perifecal membrane. The experimental groups included a control group (H1, ECP 0%), a healthy group (H2, ECP 2%), and an unhealthy group (UH, ECP 10%). Based on experimental and statistical results, the intestinal structure assessment thresholds were set as follows: villus length ≤ 29.48 μm, muscle layer thickness ≤ 15.14 μm, microvilli length ≤ 1.41 μm, and proea expression ≤ 1.49, indicating significant intestinal structural damage. This experiment also confirmed that excessive cottonseed hydrolysate (10% ECP) ​​significantly impaired shrimp intestinal health, showing a significant difference compared to the healthy group (control group H1: 0% ECP group H1 and 2% ECP group H2). In the ECP group (UH), the length of intestinal villi shrank to 28.38±0.55μm (a decrease of 28.3%, P<0.01), exceeding the pathological threshold (≤29.48μm); the muscle layer thickness decreased to 13.73±0.7μm (a decrease of 31.6%, P<0.01), below the critical value (≤15.14μm); chitinase (chit1) activity increased to 9.03±1.62U / mg (an increase of 794%, P<0.01), exceeding the warning threshold (≥5.79U / mg), indicating intestinal barrier collapse; at the same time, the expression level of peri-eating membrane factor (proea) plummeted to 1.05±0.22 (a decrease of 53.1%, P<0.01), exceeding the safety lower limit (≤1.49), while there was no difference in the above indicators between the healthy groups (H1 vs H2) (P>0.05), clearly indicating that a replacement rate of ≤2% is the safety threshold. Samples meeting these criteria indicate damage to intestinal morphology. This embodiment provides a detection procedure for morphological indicators and peri-ectic membrane-related factors, offering quantitative evidence for assessing intestinal structural integrity.

[0077] Combination Figure 7Correlation analysis of heatmap data between bacterial genus and the intestinal barrier revealed a significant association between dysbiosis and physical barrier damage: Vibrio abundance was strongly negatively correlated with villous length (r = -0.82, P < 0.01), and its excessive proliferation directly led to intestinal epithelial structure destruction; while Ruegeria (r = 0.76, P < 0.01) and Marivita (r = 0.68, P < 0.05) were positively correlated with muscle layer thickness. The depletion of these bacteria exacerbates intestinal wall muscle atrophy. Notably, *Lactococcus* was positively correlated with microvilli damage (r = 0.54, P < 0.05), confirming that its abnormal colonization induces malabsorption. Meanwhile, *Motilimonas*, a potential probiotic, showed a decrease in abundance (r = -0.61, P < 0.05), which antagonized the burst of peritrophic membrane chitinase (chit1) activity, jointly driving intestinal barrier collapse. The results are shown in Table 1.

[0078] Table 1

[0079]

[0080] Note: chit1: chitinase 1; proea: peritrophic membrane factor A; sgs3: gene silencing repressor 3. Values ​​for different superscript letters in the same row show significant differences (P<0.05), and the same applies to the following table.

[0081] The detection and evaluation of intestinal chemical barrier-related indicators include the following steps:

[0082] The activities of intestinal amylase, lipase, and trypsin in shrimp were determined according to the kit instructions from Nanjing Jiancheng Biotechnology Institute (Nanjing, China) to evaluate the effect of cottonseed hydrolysate on the activity of intestinal digestive enzymes in shrimp.

[0083] Shrimp intestinal samples were collected, and key digestive enzyme activities, such as amylase (Amy), lipase (Lip), trypsin (Tryp), and chymotrypsin (Chymo), were measured using biochemical methods. Simultaneously, quantitative real-time PCR was used to detect the expression of genes related to digestive function. Thresholds were set based on the experimental results: if trypsin activity ≤1.04, amylase activity ≤0.87, or chymotrypsin expression ≤1.24, digestive function was considered abnormal; detecting any of these abnormal indicators indicated a corresponding functional impairment. This embodiment, combining digestive enzyme activity measurement and gene expression detection, provides a specific method for the comprehensive assessment of digestive function status. This experiment also confirmed that excessive cottonseed enzymatic hydrolysate (10% ECP) ​​induced a cascade disorder of digestive and immune systems in shrimp: the expression of digestive enzyme genes was comprehensively inhibited, with α-amylase (amy) (0.69±0.09 vs 2.08±0.31 in the 2% ECP group, P<0.01) and trypsin (tryp) (0.71±0.12 vs 1.53±0.21, P<0.05) falling below their thresholds (≤0.87, ≤1.04), revealing digestive dysfunction; at the same time, immune imbalance was manifested by an increase in lysozyme (lzm) to 2.61±0.32 (161% higher than the control group, P<0.01), exceeding the warning threshold (≥0.68), while the stress response of antilipin factor (alf) increased to 2.00±0.18 (44.9% higher than the 2% ECP group, P<0.05), exceeding the safe upper limit (≥1.52). Notably, there was a dose-dependent increase in chymotrypsin (1.76±0.16a vs 1.00±0.04b) and antimicrobial peptide (pen) (1.61±0.05a vs 1.00±0.06b) between the healthy groups (H1 and H2), demonstrating that ≤2% alternative regimens can activate beneficial physiological responses.

[0084] Combination Figure 8Descriptive analysis of the association between bacterial genus and digestive function in heatmap data confirmed that dysbiosis directly inhibited digestive enzyme activity: Vibrio showed a highly significant negative correlation with trypsin (r = -0.85, P < 0.01), and when its abundance was > 54.55%, trypsin activity decreased to 0.71 ± 0.12 (falling below the threshold ≤ 1.04); Lactococcus showed a negative correlation with α-amylase (r = -0.68, P < 0.05), and its abnormal proliferation (≥ 1.0%) led to a 66.8% decrease in amylase expression (0.69 in the 10% ECP group vs. 2.08 in the 2% ECP group). Conversely, *Ruegeria* was positively correlated with chymotrypsin activity (r = 0.76, P < 0.01), and its activity decreased to 0.78 ± 0.23 when its abundance was < 3.4% (below the threshold ≤ 1.24). *Motilimonas*, on the other hand, was strongly positively correlated with lipase activity (r = 0.72, P < 0.01), and its depletion (≤ 0.16%) caused impaired fat digestion (1.09 ± 0.28 vs. 1.94 ± 0.27 in the healthy group), revealing the core mechanism by which the gut microbiota influences nutrient metabolism by regulating the expression of host digestive enzyme genes. The results are shown in Table 2.

[0085] Table 2 Note: amy : Amylase; lip Lipase; tryp Trypsin; chymo : Chymotrypsin; lzm Lysozyme; crustin Antimicrobial peptides; pen Shrimp antimicrobial peptides; alf Anti-lipopolysaccharide factor.

[0088] Note: amy: amylase; lip: lipase; tryp: trypsin; chymo: chymotrypsin; lzm: lysozyme; crustin: antimicrobial peptide; pen: shrimp antimicrobial peptide; alf: anti-lipopolysaccharide factor.

[0089] The detection and evaluation of intestinal immune barrier-related indicators include the following steps:

[0090] Total RNA was extracted from hepatocellular carcinoma and intestinal samples using the TransZol Up Plus RNA Kit (TransGen, Beijing), and its quality and concentration were assessed using a micro-spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA). RNA was reverse transcribed into cDNA using the Evo M-MLV Plus cDNA Synthesis Kit (Accurate Biology, Hunan). The Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, Hunan) was used in a LightCycler 480 (Roche Applied Science, Germany) to perform real-time PCR on the target gene using the following program: 40 cycles of 95℃ for 30 seconds, 95℃ for 5 seconds, and 60℃ for 30 seconds.

[0091] Shrimp intestinal samples were collected, and the expression of immune function-related genes, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), anti-lipopolysaccharide factor (ALF), prophenoloxidase (PropO), superoxide dismutase (SOD), and myd88, was detected using real-time quantitative PCR. Thresholds were set based on the experimental results: if IL-1β expression ≥ 1.36, ALF expression ≥ 1.52, SOD expression ≤ 1.54, or myd88 expression ≤ 0.80, it was considered an abnormality in immune function. Detection of any of these abnormal indicators suggests a dysfunction in the corresponding function. This embodiment, combining digestive enzyme activity assays and immune gene expression detection, provides a specific method for the comprehensive assessment of digestive and immune function status. This experiment also confirmed that excessive cottonseed protein (10% ECP) ​​induced systemic inflammation and immune imbalance in shrimp: the pro-inflammatory factor il-1β significantly increased to 1.59±0.11 (57.4% higher than the control group, P<0.05), exceeding the warning threshold (≥1.36); simultaneously, the expression level of the core immune pathway myd88 plummeted to 0.54±0.13 (50.5% lower than the 2% ECP group, P<0.05), falling below the safety threshold (≤0.80), indicating that Toll-like receptor signaling was blocked. The antioxidant system collapsed simultaneously, with superoxide dismutase (SOD) activity decreasing to 1.31±0.12 (43.7% lower than the mean of the healthy group, P<0.01), exceeding the pathological limit (≤1.54). Notably, the 2% ECP group exhibited beneficial immune activation: the expression level of prophenol oxidase (propo) surged to 4.39±0.43 (334% higher than the control group, P<0.01), confirming the potential of low-dose replacement therapy to enhance immunity.

[0092] Combination Figure 9Descriptive analysis of the association between bacteria genus and immune function in heatmap data revealed that gut microbiota imbalance directly regulates the immune antioxidant pathway: Vibrio showed a highly significant negative correlation with superoxide dismutase (SOD) (r = -0.86, P < 0.01), and when its abundance was > 54.55%, SOD activity decreased to 1.31 ± 0.12 (falling below the threshold ≤ 1.54), leading to the accumulation of reactive oxygen species; at the same time, Lactococcus showed a positive correlation with the pro-inflammatory factor il-1β (r = 0.62, P < 0.05), and its abnormal proliferation (≥ 1.0%) triggered a cytokine storm (il-1β increased by 57.4% in the 10% ECP group). Conversely, *Ruegeria* spp. showed a strong positive correlation with SOD expression (r = 0.78, p < 0.01), and its depletion (≤ 3.4%) exacerbated oxidative damage. *Motilimonas* spp., on the other hand, showed a positive correlation with the expression of the immune hub myd88 (r = 0.83, p < 0.01), and its deficiency (≤ 0.16%) led to a sharp drop in myd88 by 50.5% (0.54 ± 0.13 vs. 1.09 ± 0.04 in the healthy group), confirming the core mechanism by which commensal bacteria maintain immune homeostasis through the Toll-like receptor pathway. The results are shown in Table 3.

[0093] Table 3 Note: tnf-α Tumor necrosis factor-α; ifn-γ Interferon-γ; il-1β Interleukin-1β; tgf-β Transforming growth factor-β receptor protein; sod Superoxide dismutase; cat Catalase; propo : Phenoloxidaseogen; toll Toll-like receptors; myd88 Myeloid differentiation factor; imd Immunodeficiency protein.

[0095] Table 3

[0096] Note: tnf-α: tumor necrosis factor-α; ifn-γ: interferon-γ; il-1β: interleukin-1β; tgf-β: transforming growth factor-β receptor protein; sod: superoxide dismutase; cat: catalase; propo: prophenol oxidase; toll: toll-like receptor; myd88: myeloid differentiation factor; imd: immunodeficiency protein.

[0097] The detection and evaluation of intestinal biobarrier-related indicators include the following steps:

[0098] Total DNA was extracted from intestinal samples according to the HiPure Stool DNA Kits (D3141, Guangzhou Meiji Biotechnology Co., Ltd.) instructions, and DNA quality was assessed using a micro-spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA). PCR amplification was performed using primers 341F (5′CCTACGGGNGGCWGCAG3′) and 806R (5′GGACTACHVGGGTATCTAAT3′) for the V3-V4 variable region. Amplicons were collected from 2% agarose gels and purified. Sequencing libraries were constructed using the Illumina DNA Prep Kit (Illumina, CA, USA). The raw data underwent quality control to obtain representative sequences and abundance information for Operational Taxonomic Units (OTUs). Based on this information, species annotation was performed, and bioinformatics analyses were conducted, including species composition, indicator species, alpha diversity, beta diversity, and functional prediction.

[0099] High-throughput 16S rRNA sequencing was performed on shrimp gut samples to analyze the gut microbiota composition and relative abundance of each genera. Abundance data for major phyla and genera were obtained using bioinformatics methods. Based on experimental and statistical analysis, typical gut microbiota balance thresholds were established: a relative abundance of *Vibrio* ≥ 54.55%, or *Ruegeria* ≤ 3.4%, or *Lactococcus* ≥ 1.0%, or *Marivita* ≤ 0.56%, or *Motilimonas* ≤ 0.16% indicates gut microbiota dysbiosis, i.e., microbiota imbalance. Detection of any of these abnormal conditions suggests a disruption of microbiota balance. This embodiment clarified key microbiota indicators through high-throughput sequencing, providing a basis for assessing gut microbiota health status. This experiment also confirmed that excessive cottonseed protein (10% ECP) ​​led to a severe ecological imbalance in the intestinal flora of shrimp: Vibrio proliferated abnormally to 58.69±2.43% (an increase of 85.9% compared to the control group, P<0.01), exceeding the pathogenic threshold (≥54.55%); while the symbiotic bacteria Ruegeria (2.8±0.3% vs 7.1±0.88% in the 2% ECP group, P<0.01) and Marivita (0.56±0.08% vs 1.23±0.14%, p<0.01) fell below the safety threshold (≤3.4%, ≤0.56%), confirming a decline in environmental adaptability. Notably, the 2% ECP group showed targeted enrichment of probiotics: Rugellella spp. increased by 102.3% (P<0.01), and Tenacibaculum spp. remained at high abundance (1.23±0.26a), demonstrating that low-dose substitution improved the gut microbiota structure.

[0100] The relative abundance of shrimp gut microbiota at the phylum and genus levels is shown in the bar chart. Figure 4 As shown in the LefSe analysis diagram of the gut microbiota of Litopenaeus vannamei, Figure 5 As shown.

[0101] Figure 6 The study presented results predicting gut microbiota function, revealing significant differences in key biological functions between healthy and unhealthy groups. The healthy group exhibited higher abundance in multiple key biological functions, including metabolism, synthesis, and degradation, while the unhealthy group showed significant reductions. Specifically, the healthy group had significantly higher abundance in functions such as cofactor and vitamin metabolism, lipid metabolism, energy metabolism, replication and repair, nucleotide metabolism, cell motility, signal transduction, ansarmycin biosynthesis, cell chemotaxis, flagellar assembly, fatty acid biosynthesis, pantothenic acid and CoA biosynthesis, C5-branched dicarboxylic acid metabolism, thiamine metabolism, thiamine biosynthesis, glycine, serine, and threonine metabolism, D-glutamate and D-glutamine metabolism, and mismatch repair than the unhealthy group. These findings not only reveal differences in biological function between healthy and unhealthy states but also provide potential biomarkers for the diagnosis and treatment of related diseases. Further analysis showed that excessive cottonseed enzymatic hydrolysate (10% ECP) ​​led to compensatory dysfunction in the shrimp gut microbiota. In the unhealthy group (UH), the abundance of energy metabolism (such as pyruvate metabolism and the pentose phosphate pathway) and amino acid metabolism (valine / leucine / alanine pathway) was significantly lower than in the healthy group (H2), which was directly associated with the inhibition of digestive enzyme activity (trypsin activity decreased by 54.6%). Simultaneously, the aberrant activation of heterologous biodegradation pathways reflected a stress response induced by toxin accumulation. In contrast, the 2% ECP group (H2) showed significant enrichment of probiotic functions, including pantothenic acid and CoA biosynthesis, which helps support the secretion of digestive enzymes. Furthermore, enhanced peptidoglycan biosynthesis helps strengthen the intestinal mucosal immune barrier. These results confirm that ≤2% ECP replacement regimens can effectively improve the metabolic function of the gut microbiota, thereby having a positive impact on gut health. The results are shown in Table 4.

[0102] Table 4 Note: Vibrio Vibrio genus; Photobacterium Species of luminescent bacteria; Ruegeria Rugellellae; Lactococcus Lactococcus spp. Marivita Marine Bacillus genus; Shewanella Shewanella genus; Motilimonas :Aeromonas genus; Clostridium_sensu_stricto_11 Clostridium genus, group 11; Tenacibaculum genus Mycobacterium; Demequina : Demequatella genus.

[0105] Note: Vibrio; Photobacterium; Ruegeria; Lactococcus; Marivita; Shewanella; Motilimonas; Clostridium sensu stricto 11; Tenacibaculum; Demequina.

[0106] Example 2: A Smart Assessment System for Shrimp Intestinal Health

[0107] This embodiment provides an intelligent assessment system for shrimp gut health, including:

[0108] 1. Data Acquisition Module:

[0109] By integrating techniques such as microscopic imaging, electron microscopy analysis, digestive enzyme activity, immune factor ELISA, and 16S rRNA microbial sequencing, we can detect intestinal morphology, digestive capacity, immune response, and microbial community structure.

[0110] 2. Data Analysis Module:

[0111] 2.1 Data Standardization:

[0112] The original indicator values ​​are standardized using Z-scores. This involves using the mean μ and standard deviation σ of the healthy group sample as a benchmark, transforming the sample value X into Z = (X-μ) / σ, making all indicators comparable under a unified dimension. Z-score normalization eliminates the influence of differences in dimensions, testing platforms, and population groups, making the scoring results more stable and reliable. For example, Z = 0 means the sample is equal to the average level of the healthy group, while Z > 0 indicates it is higher than the healthy group mean, facilitating the assessment of health status using a unified judgment threshold.

[0113] Perform Z-score standardization on the original data:

[0114] Z = (X - μ) / σ;

[0115] Where X is the original data value; μ is the mean of the healthy group data; and σ is the standard error of the healthy group data.

[0116] Meaning of the result:

[0117] Z=0: This sample is equal to the average level of the healthy group;

[0118] Z>0: This sample is above the average level of the healthy group;

[0119] Z<0: This sample is below the average level of the healthy group.

[0120] 2.2 Multi-dimensional weight calculation:

[0121] Based on the relative correlation between each indicator and gut health, weights are assigned to different indicators or indicator groups, and a weighted calculation formula is used to obtain the comprehensive health score S. Specifically, S = 100 - ∑(weight i × |Z i| × 100); where weight i is the proportion of importance of the i-th indicator in the assessment system; and |Z i| is the absolute value of the standardized Z-score of the i-th indicator. The S value is ensured to vary between 0 and 100. This formula ensures that any deviation of an indicator from the health benchmark will linearly affect the final score according to its weight, guaranteeing that the scoring results are sensitive and scientifically sound to changes in multidimensional indicators.

[0122] The weighting values ​​should comprehensively consider the importance and variability of the indicators to reflect the contribution of each dimension to gut health. Based on experiments and literature, changes in shrimp gut morphology generally have a significant impact on health and can be assigned a high weight (40%); microbial indicators, digestive function, and immune response are assigned medium weights (30%, 20%, and 10%, respectively). The weighting process is transparent, supported by scientific data, and ensures auditability and rationality. The results are shown in Table 5.

[0123] Table 5

[0124] Dimension Weight Includes indicator examples Intestinal physical barrier Intestinal morphology 40％ villus length, muscle layer thickness biological barriers microbiome 30％ Vibrio, Ruegeria Chemical barrier Digestive function 20％ tryp, amy Immune barrier Immune response 10％ lzm, myd88, sod

[0125] Overall health score:

[0126] S = 100 - ∑(weight i × |Zi| × 100).

[0127] S: Final overall health score; the higher the score, the healthier you are.

[0128] 100: The theoretical benchmark for a perfect score.

[0129] ∑ (summation symbol): indicates that the weighted deviation of all indicators is added together to obtain the "total deduction".

[0130] Weight_i: The proportion of importance of the i-th indicator in the evaluation system.

[0131] |Z_i|: The absolute value of the standardized value (Z-score) of the i-th indicator, representing the degree to which the indicator deviates from the health benchmark.

[0132] ×100: Amplification factor that converts a decimal deviation into a larger deduction value, ensuring the final score falls within the 0-100 range.

[0133] 2.3 Assessment and Reporting Module:

[0134] The results are shown in Table 6.

[0135] Table 6

[0136]

[0137]

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for assessing the intestinal health status of shrimp, characterized in that, The method for assessing the intestinal health status of shrimp includes the following steps: S1. Collect samples of Litopenaeus vannamei fed with cottonseed enzymatic hydrolysate; S2. Determine the relevant indicators of the intestinal physical barrier, chemical barrier, immune barrier and biological barrier of the sample; S3. Perform statistical analysis on the indicators in step S2, standardize them and assign corresponding weights, calculate the comprehensive health score S, and determine the intestinal health level of the shrimp. Z = (X - μ) / σ; S = 100 - ∑(weight i × |Z i| × 100); Where X is the original data value; μ is the mean of the healthy group data; and σ is the standard error of the healthy group data. Weight i represents the proportion of importance of the i-th indicator in the evaluation system; |Zi| is the absolute value of the standardized Z-score of the i-th indicator; S ≥ 80: Shrimp gut health is good; 60 ≤ S < 80: Shrimp gut health is suboptimal; S < 60: Shrimp gut health is at risk.

2. The method for assessing shrimp intestinal health status as described in claim 1, characterized in that, In step S1, the amount of cottonseed enzymatic hydrolysate added is 2% to 4%.

3. The method for assessing shrimp gut health status as described in claim 1, characterized in that, In step S1, during sample collection, the intestinal tract of the sample is sectioned using hematoxylin and eosin staining and transmission electron microscopy; the digestive enzyme activity of the sample is measured using a standardized kit, and the expression levels of immune and antioxidant genes are assessed using real-time quantitative PCR; 16S rRNA gene sequencing is used to detect intestinal microorganisms and perform functional prediction.

4. The method for assessing shrimp intestinal health status as described in claim 1, characterized in that, In step S2, the intestinal physical barrier related indicators include at least one of the following: intestinal villus length, muscle layer thickness, microvilli length, and peritrophic membrane related gene expression indicators. Chemical barrier-related indicators include at least one of the activities of digestive enzymes or gene expression levels; Immune barrier-related indicators include at least one of the following: tumor necrosis factor-α, interferon-γ, interleukin-1β, transforming growth factor-β, myeloid differentiation factor, immunodeficiency protein, Toll-like receptor, lysozyme, antimicrobial peptide, anti-lipopolysaccharide factor, shrimp antimicrobial peptide, prophenoloxidase, superoxide dismutase, and catalase gene expression levels. Biological barrier-related indicators include the relative abundance of gut microbiota.

5. The method for assessing shrimp gut health status as described in claim 4, characterized in that, The digestive enzymes include at least one of amylase, lipase, trypsin, and chymotrypsin. The intestinal flora includes at least one of Vibrio, Rugellella, Lactococcus, Cryptophyte, and Ivorymomonas.

6. The method for assessing shrimp gut health status as described in claim 4 or 5, characterized in that, An abnormality in intestinal health is determined when any of the following conditions are met by the relevant indicators of the intestinal physical barrier, chemical barrier, immune barrier, and biological barrier: Threshold = UH group mean ± 2 × UH group standard deviation; the threshold is the critical point at which the specific indicator shows that there is an abnormality in the intestine, and ± is determined according to the actual situation; 1) The villus length is ≤29.48μm, the muscle layer thickness is ≤15.14μm, the microvilli length is ≤1.41μm, and the expression level of peritrophic membrane-related genes is ≤1.49; 2) Digestive enzyme activity ≤ 0.87; 3) Interleukin-1β expression level ≥1.36, lysozyme expression level ≥0.68, anti-lipopolysaccharide factor expression level ≥1.52, superoxide dismutase expression level ≤1.54, or myeloid differentiation factor expression level ≤0.80; 4) The abundance of Vibrio spp. is ≥54.55%, the abundance of Rugellella spp. is ≤3.4%, the abundance of Lactococcus spp. is ≥1.0%, the abundance of Cryptophyte Aquaticus is ≤0.56%, and the abundance of Ivorymomonas molluscum is ≤0.16%.

7. The method for assessing shrimp gut health status as described in claim 1, characterized in that, In step S3, GraphPad Prism 9 software was used to perform differential analysis on intestinal physical barrier, chemical barrier, immune barrier and biological barrier related indicators; data are expressed as mean ± standard error, and P < 0.05 indicates significant difference.

8. A smart assessment system for shrimp intestinal health, characterized in that, The evaluation system includes: a. Data acquisition module, used to acquire data on indicators related to the intestinal physical barrier, chemical barrier, immune barrier and biological barrier of shrimp; b. Data analysis module, used for standardizing the collected data, identifying abnormal indicators, and calculating comprehensive scores; c. Assessment report module, used to output gut health score, diagnostic results and intervention recommendations.

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