Goldfish breeding period health assessment method based on reproductive behavior analysis

Abstract

The invention relates to the technical field of biological analysis and evaluation, in particular to a breeding-period health evaluation method based on reproductive behavior analysis, which comprises the following steps: after the copulation behavior of a cowfish occurs, collecting a breeding pool environment water body, enriching germ cell outer vesicles, and locking a fertilization active vesicle subgroup through an anti-VWD structural domain antibody; performing freezing atomic force microscopic imaging on the fertilization active vesicle subgroup, and synchronously executing the following steps: quantifying the topological conformation clustering degree of membrane surface integrin protein; miRNA in the vesicles is extracted, the entropy value of a regulatory network is calculated, and the regulatory network comprises a negative feedback pathway of miR-34a and ZP3 genes; and constructing a fertilization risk index based on the clustering degree and the entropy value of the regulation and control network, carrying out risk assessment grade division based on the fertilization risk index, and taking corresponding measures. According to the method, collaborative evaluation of form and regulation is exponentially realized, and the early warning capability of the fertilization capability change of the individual cowfish is improved.

Description

Technical Field

[0001] This invention relates to the field of bioanalysis and assessment technology, and in particular to a method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis. Background Technology

[0002] As a nationally protected aquatic mammal, the Yangtze finless porpoise plays a crucial role in species conservation and population continuity through artificial breeding. However, due to its cryptic reproductive behavior, short breeding window, and uneven spatial and temporal distribution of extracellular vesicle release, the current assessment of the health status of individual Yangtze finless porpoises during the breeding season still relies on indirect methods such as behavioral observation and reproductive hormone level detection. These assessment methods suffer from high subjectivity, delayed response, and low predictive accuracy, making it difficult to support early intervention and individualized breeding management.

[0003] In recent years, extracellular vesicles (EVs) have been found to be widely involved in gamete recognition and fertilization signaling in mammals, becoming potential biomarkers reflecting individual reproductive activity. Among these, the conformational state of membrane proteins and the regulatory pathways of miRNAs within vesicles play crucial roles in the formation of fertilization capacity. However, current technologies mostly focus on the extraction of vesicle populations or the detection of miRNAs at the overall level, lacking methods for the simultaneous identification and quantification of the structure-function coupling state at the individual vesicle level.

[0004] Furthermore, traditional methods such as transmission electron microscopy and electrophoresis cannot dynamically capture the spatial conformational changes of vesicle membrane proteins, nor can they establish direct correlations with intravesicular regulatory factors, resulting in a disconnect between "structural interpretation" and "functional interpretation," which is detrimental to building a unified health assessment model. At the same time, the treatment of fertilization disorders often remains at the level of environmental regulation, lacking intervention pathways with clearly defined molecular targets and quantifiable feedback. Summary of the Invention

[0005] This invention provides a method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis. The method is based on real-time sampling driven by reproductive behavior events, functional vesicle screening, structural and regulatory linkage analysis, health assessment and feedback intervention, filling the technical gap between structural-functional co-source analysis and dynamic assessment of fertilization capacity.

[0006] A method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis includes the following steps: S1, Sampling of reproductively active water bodies: After the mating behavior of finless porpoises, environmental water bodies in the breeding pond were collected and germ cell extracellular vesicles were enriched. Fertilization-active vesicle subpopulations were identified by anti-VWD domain antibodies. S2, analysis of membrane protein-nucleic acid conjugation: Cryo-atomic force microscopy was performed on the fertilized active vesicle subpopulation obtained from S1, and the following was performed simultaneously: S11, quantifies the topological conformational clustering degree of integrin proteins on the membrane surface; S12, extract miRNA from vesicles and calculate the entropy of the regulatory network, which includes the negative feedback pathway of miR-34a and ZP3 genes; S3, Dynamic assessment and intervention of fertilization efficiency: A fertilization risk index is constructed based on clustering degree and regulating network entropy. Risk assessment levels are then classified based on the fertilization risk index, and corresponding measures are taken.

[0007] Optionally, S1 includes collecting environmental water samples from the mating area within a predetermined temperature range within a predetermined time window after the finless porpoise mating behavior ends.

[0008] Optionally, S1 further includes gradient centrifugation enrichment, which includes filtering water sequentially through a polyethersulfone membrane to retain microvesicles of a set particle size; and resuspending the precipitate in PBS buffer after centrifugation to obtain crude vesicles.

[0009] Optionally, S1 further includes active subpopulation locking, specifically including: Magnetic beads conjugated with anti-VWD domain antibodies were added to crude vesicle suspension and incubated. A magnetic field was applied to separate the vesicles bound to the magnetic beads, and the vesicles were washed multiple times with buffer solution. Fertilization-active vesicle subsets were obtained by dissociation using a pH 8.0 glycine buffer. The antibody targets the Glycine in the VWD domain. 275 -Pro 289 Epitope.

[0010] Optionally, the cryo-atomic force microscopy includes transferring the fertilized active vesicle subpopulation obtained in S1 into a cryoprotectant containing trehalose to maintain the original conformation of the vesicle membrane proteins during freezing, freezing the sample with liquid ethane to bring it into a glassy frozen state, placing the frozen sample in a low-temperature, high-vacuum environment, and scanning the vesicle membrane with a probe to obtain an image of the vesicle membrane showing the distribution of proteins on the vesicle surface.

[0011] Optionally, the quantification of the topological conformation clustering degree of membrane surface integrin proteins includes identifying membrane surface integrins based on acquired vesicle membrane images. The distribution pattern of proteins is used to identify protein regions that exhibit ring-like aggregation characteristics. Based on the spatial distance and relative angle between protein regions, effective conformations are selected. The number of ring aggregates is counted and compared with the number of proteins randomly distributed on the membrane surface. At the same time, the degree of protein aggregation, i.e., topological conformation clustering degree, is calculated by combining the scanning area range of the image.

[0012] Optionally, the calculation of the entropy value of the regulatory network includes: after completing cryo-atomic force microscopy, directly lysing the vesicles in situ to release the miRNA and related mRNA molecules, extracting RNA and performing real-time fluorescence quantitative analysis to obtain the expression levels of key factors related to the fertilization pathway, organizing the expression results into a unified expression matrix, constructing a Boolean regulatory network including multiple key factors as nodes, setting the network's regulatory rules according to known regulatory relationships, and simulating the state transition process of the Boolean regulatory network under different initial conditions to count the frequency of occurrence of various states. Based on the distribution of all states, the expression stability of the entire Boolean regulatory network, i.e., the entropy value of the regulatory network, is evaluated to determine whether there are any abnormalities in the intrinsic functional state of the vesicles.

[0013] Optionally, the key factors include: miR-34a factor: a microRNA with inhibitory effects that can downregulate the expression of fertilization-related proteins; ZP3 factor: a key receptor for sperm to recognize oocytes, and a direct regulatory target of miR-34a; FOXO3 factor: a downstream transcription factor activated by ZP3 that regulates the expression of cell cycle and reproduction-related genes; PTEN factor: a tumor suppressor gene activated by FOXO3, involved in germ cell development and metabolic regulation.

[0014] Optionally, the fertilization risk index in S3 is calculated as follows: ,in, Integrins Topological conformation clustering degree on vesicle membranes This indicates the regulation of network entropy.

[0015] Optionally, the risk assessment level classification is included: when At that time, the assessment indicated normal fertilization function, and routine monitoring was maintained. when When assessed as a moderate risk, a nutritional fortification instruction is sent to the feeding system. At that time, it was assessed as a high-risk fertilization disorder; when At that time, it was assessed as irreversible functional impairment.

[0016] The beneficial effects of this invention are: This invention proposes a fertilization risk index constructed using the topological clustering degree of integrin α6β1 membrane proteins and the entropy value of the miR-34a regulatory network as dual core parameters. This index accurately reflects the synchronous state of vesicles at both the structural and molecular functional levels. Compared to traditional methods that rely solely on protein biomarker expression or single miRNA measurements, this index achieves a synergistic assessment of morphology and regulation, enhancing the early warning capability for changes in the fertilization capacity of individual Yangtze finless porpoises.

[0017] This invention proposes a "dual-modal technology chain closed loop" scheme that simultaneously achieves cryo-atomic force microscopy imaging and in-situ miRNA extraction. This ensures that membrane protein conformation data and miRNA regulatory states originate from the same vesicle entity and the same imaging region, overcoming the problems of decoupling structure and function data and sample cross-contamination in traditional methods. This process combines vitreous freezing, sub-nanometer probe imaging, and local lysis techniques to both preserve the spatial structure of the membrane surface and achieve molecular content extraction.

[0018] This invention establishes a tiered strategy based on different numerical ranges of the fertilization risk index, and matches it with interventions such as nutritional supplementation, membrane repair, and molecular targeting to achieve comprehensive management from routine monitoring to targeted treatment. In particular, the RGD peptide nanoliposomes and miR-34a antagonist introduced during the high-risk phase precisely repair membrane conformational abnormalities and miRNA overexpression, respectively. The intervention onset time and intervention window are highly matched, achieving a direct transformation from molecular-level intervention to reproductive behavior regulation, and possessing the potential for integration into intelligent reproductive systems. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the method flow according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the cryo-imaging process according to an embodiment of the present invention. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. For some well-known technologies, those skilled in the art may also use other alternative methods to implement the invention. Moreover, the accompanying drawings are only for more specific description of the embodiments and are not intended to specifically limit the present invention.

[0022] like Figures 1-2As shown, a method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis includes the following steps: S1, Sampling of reproductively active water bodies: After the mating behavior of finless porpoises, environmental water bodies in the breeding pond were collected and reproductive extracellular vesicles were enriched. Fertilization-active vesicle subpopulations were identified by anti-VWD domain antibodies.

[0023] S11, Spatiotemporal Window Sampling: Water samples were collected underwater in the mating area within 30±5 minutes after the finless porpoises' mating behavior ended. The sampling environment temperature was controlled at 16-20℃, and the volume of each sample was 500mL. Sampling was conducted at a depth of less than 2m to avoid surface contamination, locate the core mating area, and maintain the conformational stability of membrane proteins at 16-20℃ to avoid integrin thermal denaturation.

[0024] S12, gradient centrifugation enrichment: Membrane filtration retention: Raw water samples were filtered using a 0.22 μm polyethersulfone (PES) membrane to selectively retain extracellular vesicles with a particle size of 150–200 nm.

[0025] Temperature-controlled high-speed centrifugation: Centrifuge at 20,000×g for 45 minutes at 4°C. Collect the precipitate and resuspend it in PBS buffer to obtain a crude vesicle suspension. Setting a strong acceleration of 20,000×g helps to efficiently precipitate small particles (such as vesicles) from the liquid, balancing vesicle recovery rate and membrane structure integrity.

[0026] The core purpose of gradient centrifugation enrichment is to efficiently and minimally enrich extracellular vesicles with a particle size of 150–200 nm and intact structure from environmental water samples collected after the mating behavior of finless porpoises, providing vesicle samples with both purity and activity for subsequent antibody targeting and membrane protein / miRNA analysis.

[0027] PES membranes are chosen because of their strong charge neutralization properties and the fact that the surface charge of PES membranes is closer to neutral, reducing non-specific adsorption with negatively charged vesicles. Compared with materials such as nitrocellulose membranes and polyamide membranes, PES membranes have lower adsorption loss on vesicles. The filtration process can remove large particulate impurities (such as planktonic debris, detached cells, and sediment particles) while retaining extracellular vesicles (exosome-like EVs) with a particle size range of approximately 150–200 nm, reducing the burden on subsequent centrifugation and improving separation efficiency.

[0028] Traditional exosome isolation often uses 100,000×g, but this easily damages the membrane structure. This invention focuses on fertilization-related vesicles of the Yangtze finless porpoise. These germ cell extracellular vesicles are slightly larger than general exosomes. Using moderate centrifugation force can balance capture efficiency and vesicle integrity, avoiding membrane protein folding or miRNA leakage. Low-temperature centrifugation (4°C) avoids temperature rise that would cause membrane protein denaturation, inhibits RNase activity, protects the original state of miRNAs within the vesicles, and maintains the natural morphology of the vesicles to facilitate subsequent cryo-atomic force imaging analysis.

[0029] S13, active subpopulation locking: Antibody conjugation and incubation: Magnetic beads conjugated with antibodies against the VWD domain were added to the crude vesicle suspension and incubated at 37°C for 20 minutes to allow the antibody to specifically bind to the target epitope on the vesicle surface.

[0030] Magnetic field separation and washing: A 0.5T magnetic field was applied to recover the vesicle particles bound to the magnetic beads. The particles were washed three times with Tris-HCl buffer at pH 7.4 to remove non-specific adsorbed components.

[0031] Buffer dissociation and collection: The antibody was dissociated using a pH 8.0 glycine buffer to obtain a subset of fertile-active vesicles. The antibody targets the epitope region located in the VWD domain. ; represents the high surface accessibility region in the VWD domain associated with sperm-egg recognition.

[0032] Table 1. Components of pH 8.0 Glycine Buffer This invention aims to isolate a subset of vesicles with true fertilization activity from the extracellular vesicles released by finless porpoises after mating, supporting subsequent analysis of integrin topological clustering and miRNA regulatory networks. The active subsets are identified through a triple-step process of immunomagnetic bead targeting, magnetic field separation, and buffer dissociation, efficiently and specifically enriching functional vesicles expressing specific VWD epitopes. The specific technical procedure is as follows: I. Antibody Conjugation and Incubation: This invention employs an anti-VWD domain antibody, which recognizes the key epitope Gly² segment within the VWD (von Willebrand factor type D) domain located on the vesicle membrane surface. 75 –Pro² 89 This is a known important sequence region involved in sperm-egg recognition. Antibodies are coupled to the surface of superparamagnetic beads through activated cross-linking methods (such as NHS-EDC) to form immunomagnetic bead complexes.

[0033] Procedure: Add the antibody magnetic beads to the centrifuged and enriched vesicle suspension; incubate at 37°C for 20 minutes to promote the full binding of the antibody to the VWD domain epitope on the vesicle membrane; this temperature is the optimal temperature control window for maintaining the native conformation of membrane proteins, avoiding excessive membrane rigidity due to low temperature or damage to the antigen epitope structure due to high temperature. The segment represents a key sequence in the sperm-egg binding process of the Yangtze finless porpoise, with an accessibility index (ASA) > 0.8, making it suitable for antibody recognition. Compared to traditional whole-membrane protein recognition (such as CD63 / CD9), this invention introduces antibodies targeting reproductive-related functional epitopes, greatly improving the biological relevance of functional vesicle screening.

[0034] II. Magnetic field separation and washing: After antibody incubation, a 0.5T magnetic field is applied to quickly separate the vesicles bound to the magnetic beads from the suspension, while other unbound vesicles and impurities remain in the solution.

[0035] Specific procedures: Use a handheld or platform magnetic rack to complete magnetic separation within 1–2 minutes. The vesicles on the magnetic beads are adsorbed to the tube wall. Wash three times consecutively with buffer solution, 1 mL each time, and gently blow or decan to remove the supernatant.

[0036] III. Buffer Dissociation and Collection: The bound vesicles need to be gently dissociated from the antibody-magnetic bead complex without damaging the membrane proteins or the integrity of the vesicles. To this end, a pH 8.0 glycine buffer was designed to break the non-covalent bonds between the antigen and antibody (such as hydrogen bonds, electrostatic interactions, etc.) by using mild alkalinity and ion competition mechanism, so as to achieve the gentle release of vesicles from the surface of the magnetic beads.

[0037] The von Willebrand factor type D domain (VWD) is a protein domain widely found in various adhesion proteins, including those involved in sperm-egg recognition, cell adhesion, and extracellular vesicle pairing. In the extracellular vesicles of marine mammals such as the finless porpoise, the VWD domain has been found to participate in the binding process between sperm and oocyte. In this invention, the key epitope region Gly² within the VWD domain... 75 –Pro² 89 It is a membrane protein region in the vesicles of the Yangtze finless porpoise that is closely related to fertilization activity.

[0038] Anti-VWD domain antibodies are monoclonal or polyclonal antibodies that can specifically recognize and bind to specific epitopes of the VWD domain. They are typically developed by immunizing animals (such as mice or rabbits) with a recombinant VWD domain or its peptide as an antigen to induce antibody production. Then, high-affinity, low-cross-reactivity antibodies are screened using hybridoma or phage display techniques. These antibodies are capable of recognizing Gly² epitopes in the VWD domain. 75 –Pro² 89The ability to target specific sites. In this invention, the antibody is used to identify a subset of vesicles with fertilization function, as these vesicle membranes are rich in VWD domains, which are markers for sperm-egg recognition.

[0039] Magnetic beads coupled with antibodies against the VWD domain refer to a technology platform in which specific antibody molecules are immobilized on the surface of magnetic microparticles. This platform allows for the rapid separation of target substances using a magnetic field and is commonly used for the selective enrichment of exosomes, cells, proteins, or nucleic acids.

[0040] Magnetic beads possess the following characteristics: they typically have a diameter of 1–5 μm, a core of superparamagnetic material, and a surface coated with activated functional groups (such as carboxyl and amino groups) for covalent cross-linking with antibodies. They remain stably suspended without a magnetic field, but rapidly aggregate to the tube wall upon application of a magnetic field, enabling the separation and extraction of target molecules. The term "magnetic beads coupled with anti-VWD domain antibodies" refers to the preparation of an immunocapture tool by covalently linking the aforementioned anti-VWD domain antibodies to the surface of magnetic beads through a chemical reaction.

[0041] Coupling process: Magnetic bead pretreatment: Select magnetic beads with carboxyl groups on their surface; Activation: The carboxyl group is activated using EDC / NHS chemical reagents to form an active ester; Antibody grafting: Anti-VWD domain antibodies are added to form stable covalent bonds with the surface of magnetic beads; Blocking: Use BSA or similar substances to block unreacted sites to prevent subsequent nonspecific adsorption; Storage: Store in PBS or Tris buffer to prevent antibody inactivation.

[0042] In this invention, vesicles carrying fertilization activity markers are selectively bound to the magnetic beads by incubating these immunomagnetic beads with the samples, thereby achieving specific enrichment.

[0043] S2, analysis of membrane protein-nucleic acid conjugation: Cryo-atomic force microscopy was performed on the fertilized active vesicle subpopulation obtained from S1, and the following was performed simultaneously: S11, quantifies the topological conformational clustering degree of integrin proteins on the membrane surface; S12, extract miRNA from vesicles and calculate the entropy of the regulatory network, which includes the negative feedback pathway of miR-34a and ZP3 genes.

[0044] S21, Cryo-sample preparation and imaging: S211, Cryoprotection and Vitreous Fixation: Fertilized active vesicle subsets were placed in a cryoprotection buffer containing 10% (w / v) trehalose, and vitreous frozen samples were prepared by liquid ethane quick-freezing.

[0045] S212, Low-Temperature Atomic Force Microscopy: At a temperature of -150℃ and a vacuum of 10... -6 Under Torr conditions, a silicon carbide probe with a stiffness coefficient of 0.3 N / m was used for scanning, and the image resolution was 0.5 nm.

[0046] In this invention, it is necessary to perform sub-nanometer resolution imaging of the membrane proteins of the fertilization-active vesicles of the Yangtze finless porpoise. Therefore, it is essential to maintain the natural conformation and membrane distribution of the vesicles to the greatest extent possible. To achieve this goal, a glassy cryo-fixation technique is used, which involves rapidly solidifying the vesicles without forming ice crystals, thus avoiding damage to the membrane structure due to ice crystal growth.

[0047] The specific procedure involves adding the enriched vesicle suspension to a cryoprotective buffer containing 10% (w / v) trehalose, followed by rapid freezing with liquid ethane. This instantly cools the sample to near liquid nitrogen temperature, forming an amorphous glassy structure in a very short time. A 10% trehalose concentration has been validated as the optimal protective concentration. Trehalose possesses excellent membrane stability and resistance to freeze-drying, effectively coating the vesicle surface and preventing membrane proteins from unfolding or non-specifically agglomerating during freezing. Compared to liquid nitrogen freezing, rapid freezing with liquid ethane is more suitable for quickly forming glassy samples. Although liquid nitrogen has a lower temperature, its gas-liquid interface is not conducive to uniform heat transfer, easily leading to ice crystal formation in the sample. Liquid ethane, on the other hand, has higher thermal conductivity, enabling rapid freezing of the sample in a short time to form an amorphous structure, effectively preserving the topological conformation of membrane proteins.

[0048] After cryofixation, the samples need to be scanned at high resolution on the membrane surface under low temperature and high vacuum conditions. Therefore, this invention uses cryo-AFM (cryo-AFM)** to perform membrane protein topological imaging.

[0049] Specifically, the sample is set to be placed at -150℃ and 10⁻ 6 Scanning was performed in a Torr vacuum environment using a silicon carbide probe with a stiffness coefficient of 0.3 N / m. A scanning temperature of -150℃ was chosen to ensure the structural stability of the frozen glassy state. Temperatures below -120℃ effectively suppress the "thaw" phenomenon in glassy samples, preventing protein recombination or water vapor recrystallization, thus maintaining the original state of membrane proteins. The optimal temperature control point of -150℃ was determined based on the stability verification of the frozen glassy structure. The vacuum level was set to 10⁻⁻⁶. 6 Torr is primarily designed to prevent water vapor or impurities from condensing on the sample or probe, thus avoiding interference with imaging quality. In this high-vacuum environment, interference between air molecules and the probe is effectively eliminated, improving the image signal-to-noise ratio. The probe material is silicon carbide (SiC), which has high hardness, good elastic stability, and is not prone to brittleness at low temperatures, making it suitable for ultra-high resolution scanning.

[0050] Setting the probe stiffness to 0.3 N / m is a balanced choice: too little stiffness will cause the probe to "collapse" during scanning, resulting in insufficient resolution; while too much stiffness (such as above 0.5 N / m) may cause physical perforation or indentation of the vesicle membrane. 0.3 N / m is the optimal parameter for obtaining nanometer-resolution images without damaging the vesicle structure.

[0051] S21 is fundamental to the entire membrane protein-nucleic acid conjugation analysis. The core task of S21 is to preserve and observe the original conformation of membrane proteins on the surface of finless porpoise vesicles with high spatial resolution. By using cryoprotectants, liquid ethane flash freezing technology, and atomic force microscopy under low-temperature vacuum conditions, the vesicle membrane was ensured to remain in its native state without ice crystal damage. The distribution and aggregation patterns of vesicle membrane proteins, especially integrins, were clarified. The protein aggregation characteristics are analyzed, and the acquired images possess sub-nanometer precision, accurately locating membrane protein aggregation regions and providing fundamental data for the S22 calculations. In S22, integrins are extracted using the membrane protein images obtained in S21. Distribution patterns on the membrane surface. By setting aggregation criteria (protein spacing and angle), cyclic conformation regions are identified, and their topological clustering degree is calculated.

[0052] S22 performs a quantitative assessment of the spatial structure at the membrane level, providing morphological evidence for the functional activity of the entire vesicle. S23, together with S22, forms a dual-channel verification of the function-molecular mechanism. The task of S23 is to analyze the internal miRNA regulatory pathways related to vesicle function, establish a Boolean network model, and calculate the entropy value to measure the stability and functional state of the regulatory network. S23 performs in-situ lysis of the same region imaged in S21, releasing miRNA, and then obtains expression data through qPCR, avoiding spatial mismatch. This design achieves the "co-origin" and "co-situ" analysis of membrane protein conformation and miRNA regulation.

[0053] S22, Clustering degree of membrane protein topological conformation Quantification: Identifying Integrins The ring-shaped conformational aggregation region of proteins on the surface of vesicle membranes is defined as follows: Spacing between adjacent proteins: ; Angle range: ; Clustering degree calculation: ;in, This indicates the number of protein aggregates forming a cyclic conformation (per aggregate). (a protein that forms a closed loop), This represents the number of proteins in a randomly distributed state within the same region. This represents the actual imaging area of ​​the vesicle membrane, in units of... .

[0054] This invention utilizes integrins on the surface of extracellular vesicle membranes of finless porpoise germ cells. Topological conformation analysis of the spatial distribution of proteins is performed to identify whether they exhibit cyclic aggregates associated with fertilization activity. Clustering degree is defined as an important parameter for measuring whether membrane proteins form stable biorecognition complexes. To determine whether functional protein aggregates exist on vesicle membranes, clear geometric criteria need to be established to extract "effective aggregation regions" from imaging images. This invention uses the following two conditions as the basis for determining whether a group of integrin proteins constitutes a functional cyclic aggregate conformation: A protein spacing of ≤ 3nm is considered the critical distance for stable non-covalent interactions (such as hydrophobicity and hydrogen bonding) between transmembrane proteins. When integrins form the sperm-egg recognition complex, the physical distance between their subunits must be close enough to synergistically mediate cell adhesion signals. If the spacing is greater than this threshold, the protein may be in a free or loose state and lack biological functional activity.

[0055] Angle ∈ [60°, 120°]: This angle range corresponds to the most common geometric arrangement when polypeptide chains form stable ring structures. In the process of sperm-egg recognition, integrins... Proteins need to form "closed loops" or "arc bands" to enhance binding stability and spatial selectivity. Too small or too large an angle often indicates random arrangement, making it difficult to form a closed conformation, and thus questionable functionality.

[0056] The above combined criteria take into account both spatial scale (3nm) and conformational consistency (angle), together forming the geometric screening standard for determining "functional clustering".

[0057] Once multiple conformational regions satisfying the aggregation criteria are identified, the next step is to quantify their overall distribution on the vesicle membrane surface. The core indicator is the clustering degree, which describes the integrins. The degree of aggregation of proteins in a ring conformation on the vesicle membrane.

[0058] Nc (number of ring aggregates) represents the total number of aggregates in the imaging region that meet the above conformation criteria and are composed of three or more integrin proteins forming a closed loop; setting "≥3 proteins" as the aggregate formation threshold excludes unstable conformations such as double protein coupling and improves the accuracy of analysis.

[0059] Nr (number of randomly distributed proteins) represents the number of integrin proteins that do not participate in conformational aggregation within the same imaging region. It simulates the background noise or free distribution of proteins in their native state and serves as a normalization reference to avoid δ value bias caused by different total protein expression levels, thereby improving comparability across samples.

[0060] S23, miRNA regulates network entropy. calculate: S231, In-situ lysis and miRNA release: In the same region after imaging, lysis buffer is injected to perform in-situ lysis, releasing miRNA from the vesicles. Lysis buffer formulation: Triton X-100: 0.5% (v / v) EDTA: 1mM; S232, Expression Matrix Acquisition: The expression levels of the following four factors were detected using RT-qPCR to construct the expression matrix. : miR-34a (microRNA number 34a (belonging to the miRNA family)); ZP3 (zonoglossy glycoprotein 3); FOXO3 (Forkhead Box Protein O3); PTEN (phosphatase and tensin homolog).

[0061] Expression matrix The goal is to obtain target gene expression data to provide a quantitative basis for network modeling. This data forms the foundation for subsequent Boolean network state modeling and entropy calculation, and is used to record the actual expression level of the target factor. The construction process consists of three steps: 1. In situ lysis and RNA release: After imaging, the sample area is gently injected with lysis buffer containing 0.5% Triton X-100 and 1mM EDTA to selectively rupture the vesicle membrane and release the internal miRNA and mRNA, while avoiding damage to the external protein structure or contamination of the surrounding sample.

[0062] Triton X-100 is a nonionic surfactant that can selectively disrupt the phospholipid bilayer; EDTA chelates divalent metal ions, inhibits RNase activity, and protects RNA from degradation.

[0063] 2. RNA collection and reverse transcription: RNA is extracted from the lysate and cDNA is synthesized using a miRNA / mRNA-specific reverse transcription kit (such as TaqMan or miScript). This process is divided into two categories: miRNA reverse transcription: using specific stem-loop primers (such as miR-34a). mRNA reverse transcription: use oligodT or random primers (such as ZP3, FOXO3, PTEN).

[0064] Ensure all samples are treated under the same thermal cycling parameters to ensure comparability of subsequent expression levels.

[0065] 3. Real-time quantitative PCR (RT-qPCR): Using specific fluorescent probes or SYBR Green dye, the expression levels of the following four factors are detected: miR-34a, ZP3 (Zona Pellucida 3), FOXO3 (Forkhead box O3), and PTEN (Phosphatase and Tensin Homolog); each sample required three technical replicates, and Ct values ​​were normalized to obtain the expression levels of each sample on the above four factors, which constituted: ; Each row corresponds to a vesicle sample (or vesicle cluster), and each column corresponds to the expression level of a factor.

[0066] S233, Boolean network model construction defines the set of network nodes: Node status miR-34a, ZP3, FOXO3, PTEN The control edge (Boolean rule logic) is defined as follows: miR-34a inhibits ZP3 expression; ZP3 activates FOXO3; FOX03 activates PTEN; S234, Network Entropy Calculation: Based on the state space of the above Boolean network model, the stability of the entire regulatory pathway is evaluated using 1000 Monte Carlo simulations for all 16 possible Boolean states (2... 4 (One) to conduct simulation, Given network states, record the probability of each state occurring. The entropy value of the control network is calculated as follows: ;in, Indicates Boolean network state The probability distribution was obtained from 1000 Monte Carlo simulations.

[0067] In this invention, the miRNA and mRNA within the vesicles constitute a Boolean regulatory network consisting of four nodes (miR-34a, ZP3, FOXO3, and PTEN), with each node having only two possible states: 0: Low expression (or inactivation); 1: High expression (or activation); Therefore, the state space of the entire network has a total of 16 possible Boolean states.

[0068] The purpose of Monte Carlo simulation is to estimate the possible final states of the entire system and their probability distribution when the true initial state of each node is uncertain. This is achieved by setting rules and repeatedly performing a process of "random startup + logical update." The set of network nodes defined earlier is: {miR-34a, ZP3, FOXO3, PTEN}; These nodes constitute the behavioral units of the network. Each node establishes a regulatory relationship with the others through explicit Boolean rules: miR-34a inhibits ZP3, ZP3 activates FOXO3, and FOXO3 activates PTEN. Monte Carlo simulation revolves around this set of nodes, repeatedly running the network state update process from random states based on given logical rules, and counting the frequency of each possible state.

[0069] The simulation process logic is as follows: Initialization: Set a set of initial states, such as miR-34a=1, ZP3=0, FOXO3=0, PTEN=0; According to Boolean rules, the state of each node in the network is updated iteratively (e.g., if miR-34a=1, then ZP3 is updated to 0 in the next round). Record the final stable state or periodic state (state s) that the system enters. Repeat this process 1000 times, each time starting from a different random initial state to simulate network operation; Count the frequency of each state s and calculate its probability. ; These It is used to calculate entropy. Input data.

[0070] The existence of multiple nodes and multiple logical paths in a network leads to highly nonlinear and path-dependent results. It is difficult to directly list the true probability of each state using analytical methods. The Monte Carlo method can capture the diversity of the final state of the system due to different initial states. It can reflect the "stable" or "disorderly" trend of network behavior when factor expression is disturbed in real samples. The higher the value, the more dispersed and uncertain the states that occur during network operation, indicating that the internal regulation of vesicles is more chaotic.

[0071] In-situ imaging combined with in-situ lysis enables spatial matching between membrane protein topological information and miRNA expression, ensuring that δ and η originate from the same vesicle individual and avoiding the structure-function decoupling problem caused by "step-by-step extraction" in traditional techniques.

[0072] S3, Dynamic assessment and intervention of fertilization efficiency: A fertilization risk index is constructed based on clustering degree and regulating network entropy. Risk assessment levels are then classified based on the fertilization risk index, and corresponding measures are taken.

[0073] S31, Fertilization Risk Index Build: Input two key metrics from S2: Membrane protein topological conformation clustering ; miRNA regulates network entropy ; Calculate the fertilization risk index: ;in, Integrins The degree of ring-like aggregation on the vesicle membrane, The value represents the entropy of the regulatory network of the miR-34a / ZP3 pathway; 0.4 is derived from statistics of several healthy vesicles from finless porpoises. Minimum critical mean ( ), used for stabilization Lower limit of the formula.

[0074] Build logic: when The lower This indicates a more abnormal membrane conformation; when The higher This indicates a more disordered regulatory network; therefore The higher The higher the risk of fertilization.

[0075] S32, Graded assessment and intervention, based on fertilization risk index Based on the numerical range, a four-level assessment and intervention mechanism is established: (a) Normal level (low risk): condition Judgment: Vesicle membrane conformation is good, miRNA network is stable. Measures: Maintain routine monitoring; no intervention is required. In the tested samples, when the clustering degree of vesicles... When maintained at a healthy level (close to or greater than 0.4), and the miRNA regulatory network is stable, the calculated The value typically does not exceed 0.8. Within this range, the fertilization success rate of samples reaches over 85%, requiring no additional intervention. This range represents an ideal state in terms of vesicle structure and regulation.

[0076] (b) Moderate risk: condition: Judgment: Mild functional abnormality, reversible with nutritional support; Measures: Send a fortified feeding instruction to the feeding system → Increase the daily intake of omega-3 fatty acids by 20%. In this range, it typically manifests as mild membrane conformation loosening (…). Slightly below 0.4) or the miRNA network begins to fluctuate ( (Slightly elevated), but not yet in a severely disordered state. Experimental data shows that the fertilization success rate drops to around 60% at this stage, but it remains highly reversible, especially with nutritional adjustments that can restore most samples to normal expression levels. Therefore, this is designated as a "warning zone," advocating for gentle interventions, such as enhanced nutrition, to improve vesicle status.

[0077] (c) High-risk: condition: Judgment: Significant fertilization dysfunction, requiring membrane repair; Measures: Generate vesicle membrane repair instructions → Inject a repair agent into the breeding pool 6 hours before the next mating cycle. Within this range, fertilization dysfunction is significant, commonly seen in a significant decrease in integrin aggregation conformation (…). (Below 0.25) and increased miRNA network entropy. The fertilization success rate of such samples drops below 30%, and the membrane function has a certain degree of repairability. Fertilization capacity can be significantly improved by exogenously supplementing RGD peptide molecules and guiding integrin conformation reversion. Therefore, this range is defined as the "reversible high-risk range" and timely intervention for membrane repair is required.

[0078] (d) Extremely high risk (irreversible): condition: Assessment: The miRNA network is severely disordered and its functional damage is irreversible; Action: Trigger the miR-34a antagonist intervention program.

[0079] This invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of this invention. To provide the public with a thorough understanding of this invention, specific details are described in detail in the following preferred embodiments; however, those skilled in the art will fully understand the invention even without these details. Furthermore, to avoid unnecessary misunderstanding of the essence of this invention, well-known methods, processes, procedures, components, and circuits are not described in detail.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis, characterized in that, Includes the following steps: S1. After the mating behavior of finless porpoises, environmental water in the breeding pool was collected and germ cell extracellular vesicles were enriched. The fertilization-active vesicle subpopulation was identified by anti-VWD domain antibody. S2, cryo-atomic force microscopy imaging of the fertilized active vesicle subpopulation obtained in S1, simultaneously performed: S11, quantifies the topological conformational clustering degree of integrin proteins on the membrane surface; S12, extract miRNA from vesicles and calculate the entropy of the regulatory network, which includes the negative feedback pathway of miR-34a and ZP3 genes; S3. Based on the clustering degree and the control network entropy value, a fertilization risk index is constructed. Based on the fertilization risk index, risk assessment levels are classified and corresponding measures are taken.

2. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 1, characterized in that, S1 includes collecting environmental water samples from the mating area within a predetermined temperature range within a predetermined time window after the finless porpoise mating behavior ends.

3. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 1, characterized in that, S1 further includes gradient centrifugation enrichment, which involves filtering water sequentially through a polyethersulfone membrane to retain microvesicles of a set particle size; after centrifugation, the precipitate is resuspended in PBS buffer to obtain crude vesicles.

4. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 3, characterized in that, The S1 further includes active subpopulation locking, specifically including: Magnetic beads conjugated with anti-VWD domain antibodies were added to crude vesicle suspension and incubated. A magnetic field was applied to separate the vesicles bound to the magnetic beads, and the vesicles were washed multiple times with buffer solution. Fertilization-active vesicle subsets were obtained by dissociation using a pH 8.0 glycine buffer. The antibody targets the Glycine in the VWD domain. 275 -Pro 289 Epitope.

5. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 1, characterized in that, The cryo-atomic force microscopy involves transferring the fertilized active vesicle subpopulation obtained in S1 into a cryoprotectant containing trehalose to preserve the original conformation of the vesicle membrane proteins during freezing. Liquid ethane is used for freezing to bring the sample into a glassy frozen state. The frozen sample is then placed in a low-temperature, high-vacuum environment, and the vesicle membrane is scanned and imaged using a probe to obtain an image of the vesicle membrane showing the distribution of proteins on the vesicle surface.

6. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 5, characterized in that, The quantification of the topological conformation clustering of membrane surface integrin proteins includes identifying membrane surface integrins based on acquired vesicle membrane images. The distribution pattern of proteins is used to identify protein regions that exhibit ring-like aggregation characteristics. Based on the spatial distance and relative angle between protein regions, effective conformations are selected. The number of ring aggregates is counted and compared with the number of proteins randomly distributed on the membrane surface. At the same time, the degree of protein aggregation, i.e., topological conformation clustering degree, is calculated by combining the scanning area range of the image.

7. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 6, characterized in that, The calculation of the entropy value of the regulatory network includes: after completing cryo-atomic force microscopy, directly lysing the vesicles in situ to release the miRNA and related mRNA molecules, extracting RNA and performing real-time fluorescence quantitative analysis to obtain the expression levels of key factors related to the fertilization pathway, organizing the expression results into a unified expression matrix, constructing a Boolean regulatory network including multiple key factors as nodes, setting the network's regulatory rules based on known regulatory relationships, and simulating the state transition process of the Boolean regulatory network under different initial conditions to count the frequency of occurrence of various states. Based on the distribution of all states, the expression stability of the entire Boolean regulatory network, i.e., the entropy value of the regulatory network, is evaluated to determine whether there are any abnormalities in the intrinsic functional state of the vesicles.

8. The method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis according to claim 7, characterized in that, The key factors include: miR-34a factor: a microRNA with inhibitory effects that can downregulate the expression of fertilization-related proteins; ZP3 factor: a key receptor for sperm to recognize oocytes, and a direct regulatory target of miR-34a; FOXO3 factor: a downstream transcription factor activated by ZP3 that regulates the expression of cell cycle and reproduction-related genes; PTEN factor: a tumor suppressor gene activated by FOXO3, involved in germ cell development and metabolic regulation.

9. A method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis, as described in claim 1, is characterized in that... The fertilization risk index in S3 is calculated as follows: ,in, Integrins Topological conformation clustering degree on vesicle membranes This indicates the regulation of network entropy.

10. A method for assessing the health of finless porpoises during their breeding season based on reproductive behavior analysis, as described in claim 9, is characterized in that... The above includes risk assessment level classification: when At that time, the assessment indicated normal fertilization function, and routine monitoring was maintained. when When assessed as a moderate risk, a nutritional fortification instruction is sent to the feeding system. At that time, it was assessed as a high-risk fertilization disorder; when At that time, it was assessed as irreversible functional impairment.

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