Method for determining key limiting points of fish ovary development and application

Abstract

This invention belongs to the field of fish ovarian development technology and discloses a method for determining the dominant rate-limiting point of fish ovarian maturation. The method involves selecting fish ovaries at different developmental stages as materials for histological sectioning, statistically obtaining the proportion of different types of follicles during ovarian development, and, based on the proportion of different types of follicles, using spline interpolation and linear fitting data-driven modeling techniques to analyze the proportion curves, growth rates, and initial synchronous state characteristics of different types of follicles, ultimately determining the key rate-limiting point of fish ovarian development. Based on this method, the dominant rate-limiting point of fish ovarian maturation can be accurately determined, and measures can then be formulated to regulate the fish ovarian development process based on this dominant rate-limiting point. This invention also discloses the application of the method for determining the dominant rate-limiting point of fish ovarian maturation in regulating the fish ovarian development process and methods for promoting the fish ovarian maturation process.

Description

Technical Field

[0001] This invention belongs to the field of fish ovarian development technology, and relates to a method and application for modeling and determining key rate-limiting points in fish ovarian development. Background Technology

[0002] In fish ovaries, the basic functional unit for oocyte development and maturation is the follicle. The primary follicle (PF) consists of a biphasic oocyte and a single layer of follicular cells, and can be classified into four types: PF-i, PF-ii, PF-iii, and PF-iv. The secondary follicle (SF) consists of a triphasic or tetraphasic oocyte and a double layer of follicular cells. Once the yolk sac is filled with yolk, the oocyte resumes meiosis and develops into a mature follicle. The maturation and development of the fish ovary generally involves: the oogonia development stage, the primary follicle development stage (small growth phase), the secondary follicle development stage (large growth phase), and the maturation stage. A key histological feature of the fish ovary entering mature development is the appearance of the secondary follicle during the large growth phase.

[0003] In fish, the ovary takes about two months to develop from the juvenile stage to first sexual maturity in some species (such as zebrafish), while in others (such as grass carp and sturgeon), it can take several years or even more than a decade. Interestingly, the longer the fish reaches sexual maturity, the longer its ovary remains arrested in the juvenile stage during its first estrous cycle. Doroshov et al. reported that artificial breeding of the Chinese paddlefish made it very difficult for the ovary to develop from the juvenile stage to the major growth stage, and even caused it to stop developing. Artificial breeding practices of the Chinese sturgeon also show that there are reproductive dysfunctions in farmed Chinese sturgeon, and the ovary is difficult to develop to the major growth stage after reaching the juvenile stage. The sexual maturity process of female fish develops into mature follicles through different follicle types such as PF-i, PF-ii, PF-iii, PF-iv, SF-i, and SF-ii. However, the key rate-limiting points affecting the maturation and development of ovarian follicles in fish are not yet understood, especially the objective assessment strategy is still uncertain. Therefore, developing an accurate and effective method to determine the critical rate-limiting point in the development process of fish ovaries, and adjusting the aquaculture environment or feed formulation based on the growth and development characteristics of dominant follicles at the rate-limiting point, is of great significance for enhancing fish reproductive performance and improving aquaculture efficiency. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a precise modeling method and application for determining the rate limit point of ovarian development in fish.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] In a first aspect, the present invention provides a method for determining the critical rate-limiting point of ovarian development in fish, comprising the following steps:

[0007] (1) Select fish ovaries at different developmental stages as materials for histological sections, count the number of different types of follicles according to the size and morphological characteristics of the follicles, and calculate the proportion of different types of follicles during the development of fish ovaries;

[0008] (2) Based on the proportion of different types of follicles obtained in step (1), spline interpolation and linear fitting data-driven modeling techniques are used to analyze the proportion curves, growth rates and synchronous initial state characteristics of different types of follicles, and finally determine the key rate-limiting point of fish ovarian development.

[0009] The preferred method described above, specifically step (2), is as follows:

[0010] (2-1) Based on the proportion of different types of follicles obtained in step (1), spline interpolation is performed to construct a dynamic function relationship of the proportion of different types of follicles over time.

[0011] (2-2) Linear fitting was performed on the time points when all types of follicles appeared to construct linear fitting models for each type of follicle;

[0012] (2-3) Based on the linear fitting model, calculate the slope and intercept to preliminarily determine whether there is a rate-limiting relationship between different types of follicles; select the linear fitting model with a rate-limiting relationship, and then calculate the intersection of the linear relationships of different types of follicles to determine the key rate-limiting point of fish ovarian development.

[0013] Spline interpolation is a commonly used smooth curve fitting technique in numerical analysis. It approximates or interpolates data points using piecewise defined polynomials. Step (2-1) constructs the dynamic function relationship between the proportion of different types of follicles and their development over time, as follows:

[0014] Suppose there is a set of data points showing the percentage of different follicle types. In each time sub-interval [ , Construct a cubic polynomial function of the following form. :

[0015] ;

[0016] in , , , These are undetermined coefficients; , ; Ensure that the first derivative is continuous at the nodes. Ensure that the second derivative is continuous at the nodes; These are natural boundary conditions. This yields a cubic polynomial for each interval. This completes the cubic spline interpolation process.

[0017] Linear fitting, also known as linear regression, is a statistical method used to establish a linear relationship between a dependent variable and one or more independent variables. Since this invention only considers the relationship between the single variable of proportion and developmental time, i.e., simple linear regression, its basic principle is as follows:

[0018] Suppose there is a set of data points ( , ), in =1, 2, ..., n. Our goal is to determine an optimal straight line. This straight line best fits these data points. The goal of linear fitting is to minimize the sum of squared residuals (RSS) between all data points and the fitted line. The residual is defined as the difference between the actual value of a data point and the fitted value:

[0019] ;

[0020] To find the optimal parameters a and b, we need to minimize the RSS. This can be achieved by setting the partial derivatives of the RSS with respect to a and b to zero. Specifically, we solve the following system of equations:

[0021] , ;

[0022] By solving the above system of equations, we can determine the optimal parameters that minimize RSS, and thus determine the fitted line.

[0023] To calculate the angle between linear fitting models of different types of follicles, based on the fitted function expression above, we obtain the angle by calculating the inner product. The specific principle is as follows:

[0024] For two functions defined on the interval [a, b] and Their inner product (dot product) can be defined as:

[0025] ;

[0026] Two functions defined on the interval [a, b] and The included angle between them can be defined as:

[0027] .

[0028] Therefore, step (2-2) involves constructing linear fitting models for each type of follicle as follows:

[0029] For a given percentage of follicle types ( , ),in =1, 2, ..., n, the linear fitting or regression model of the dynamic changes of this follicle type is determined by minimizing the following loss function:

[0030]

[0031] The regression coefficients were obtained by performing a t-significance test.

[0032] Preferably, to further determine the coordinates of the dominant rate-limiting point, this invention calculates the intersection points of the linear relationships between different types of follicles. Step (2-3) of determining the key rate-limiting point for fish ovarian development is as follows:

[0033] Let the linear fitting models for two different follicle types be as follows: : , : Where m1 and m2 are the slopes of the two linear fitting models, and b1 and b2 are the y-intercepts of the two linear fitting models, respectively. The linear fitting model with a velocity-limiting relationship is selected, and the coordinates (x, y) of the key velocity-limiting point at a specific stage of fish ovarian development are calculated using the following formula:

[0034] , .

[0035] Secondly, the present invention provides an application of the method for determining the critical rate-limiting point of ovarian development in fish in regulating the process of ovarian development in fish.

[0036] In the above-mentioned application, preferably, the dominant follicle at the critical rate-limiting point of fish ovarian development determined by the method is the rate-limiting follicle. By improving the fish farming environment or feed formula based on the characteristics of the rate-limiting follicle at the critical rate-limiting point, the process of fish ovarian development can be regulated.

[0037] Preferably, the dominant rate-limiting follicle is purified by Percoll density gradient centrifugation, and the mRNA of the dominant rate-limiting follicle is extracted and subjected to high-throughput sequencing and transcriptome analysis to obtain the key regulatory factors for the development of the dominant rate-limiting follicle. Based on the characteristics of the key regulatory factors, the aquaculture environment or feed formulation of fish is improved.

[0038] Thirdly, the present invention provides a method for promoting the maturation and development of fish ovaries. The method is used to determine the key rate-limiting point of fish ovarian maturation and development. Combined with the growth and development characteristics of rate-limiting follicles (PF-ii), 1.7~2.5% DL-methionine and 3.4~5% L-lysine hydrochloride are added to the fish diet by mass fraction, which significantly promotes the maturation and development of fish ovaries.

[0039] Preferably, the fish is zebrafish or crucian carp.

[0040] During the feeding process, the development of the ovaries should be observed regularly. The method for observing the development of the ovaries is as follows: ovarian tissue is taken, fixed, embedded in paraffin, sectioned, dewaxed, stained with hematoxylin and eosin, and observed under a microscope.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] 1. This invention provides a model-based method for determining the rate-limiting point of ovarian development in fish. Based on this method, the dominant rate-limiting point of ovarian maturation and development in fish can be accurately determined.

[0043] 2. The present invention provides a method for regulating the development process of fish ovaries by formulating measures based on a determined dominant rate-limiting point, and for determining the dominant rate-limiting point for fish ovarian maturation. This method is applied to regulating the development process of fish ovaries and promoting the maturation and development of fish ovaries. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 The results are the analysis results of determining the rate-limiting point of ovarian maturation and development of red crucian carp using spline interpolation (A) and linear fitting (B) in Example 1.

[0046] Figure 2 The analysis results of determining the rate-limiting point of ovarian maturation in zebrafish using spline interpolation (A) and linear fitting (B) in Example 2 are shown in Figure 2 (Note: dph: number of days after hatching).

[0047] Figure 3 Example 3 shows the effect of improved crucian carp diet on the secondary cyclic ovarian development of red crucian carp; (A) ovarian histological observation results, (B) statistical results of the proportion of different types of follicles.

[0048] Figure 4Example 4 illustrates the effect of improved diet on ovarian development in zebrafish during their first estrus cycle. Detailed Implementation

[0049] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0050] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0051] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0052] Example 1:

[0053] A method for determining the critical rate-limiting point of ovarian development in red crucian carp through data-driven modeling, the specific steps of which are as follows:

[0054] (1) Tracking and statistical analysis of different types of follicles (PF-i, PF-ii, PF-iii, PF-iv, SF) at different developmental stages of the red crucian carp ovary:

[0055] (1-1) Select red crucian carp at different time stages of the estrous cycle, cut open the abdomen after anesthesia to remove the ovaries, fix them with Born's solution, embed them in paraffin, then section them at 10 mm, stain them with hematoxylin and eosin, and observe the development of red crucian carp follicles at different time stages under an optical microscope.

[0056] (1-2) The diameter and length of follicles in tissue sections of red crucian carp ovaries at different stages were measured using ImageJ software. The number of follicles was counted according to the diameter and characteristics of different types of follicles, and the proportion of different types of primary follicles and SFs during the development of red crucian carp ovaries was calculated.

[0057] (2) Modeling determination of the rate-limiting point of ovarian development in red crucian carp:

[0058] (2-1) Spline interpolation analysis was performed on the proportion of different types of follicles at different stages of red crucian carp obtained in step (1). The specific analysis is as follows:

[0059] For each sub-interval [ , The interpolation function can be expressed as:

[0060] ;

[0061] in , , , These are undetermined coefficients; in order to ensure For the first and second derivatives to be continuous at all internal nodes, the following condition must be satisfied:

[0062] ,

[0063] ,

[0064] ,

[0065] ;

[0066] In addition, boundary conditions are needed to completely determine all coefficients. The most common boundary condition is the natural boundary condition, which requires that the second derivative at the endpoints be zero, i.e., satisfying: ;

[0067] This gives us the cubic polynomial for each interval. This completes the cubic spline interpolation process.

[0068] (2-2) Linear fitting was performed on the time points when all types of primary follicles appeared to determine the slope and intercept of the fitting model, and the angle between the linear fitting models of each type of primary follicle and secondary follicle was calculated. The specific process of linear fitting is as follows:

[0069] Suppose there is a set of data points ( , ), in =1, 2, ..., n; the objective of this invention is to determine an optimal straight line. The line best fits these data points; the goal of linear fitting is to minimize the sum of squared residuals (RSS) between all data points and the fitted line; the residual is defined as the difference between the actual value and the fitted value of a data point.

[0070] ;

[0071] To find the optimal parameters a and b, we need to minimize the RSS, which can be achieved by setting the partial derivatives of the RSS with respect to a and b to zero; specifically, we solve the following system of equations:

[0072] , ;

[0073] By solving the above system of equations, we can determine the optimal parameters that minimize RSS, and thus determine the fitted line.

[0074] To further determine the dominant rate-limiting point, we calculated the intersection of the linear fitting models of primary and secondary follicles.

[0075] Let the linear model for each type of primary follicle be: : The linear model of secondary follicles is : Where m1 and m2 are the slopes of the two linear models, and b1 and b2 are their y-intercepts; (x, y) are the coordinates of the "dominant speed limit point," calculated as follows:

[0076] , ;

[0077] (2-3) The slope and intercept of the linear fitting model for each type of follicle ( Figure 1 (Table 1) It was determined that the PF-ii subtype plays a key rate-limiting role in the ovarian development process during the large growth stage of crucian carp;

[0078] (2-4) All the code of the computer program was written in MATLAB, ran on MATLAB R2024a, and executed on a PC with a Windows 10 operating system (CPU 3.10 GHz, memory 16G).

[0079] Table 1. Linear fitting relationships of different types of follicles in red crucian carp.

[0080]

[0081] Example 2:

[0082] A method for determining the critical rate-limiting point of zebrafish ovarian development through data-driven modeling, the specific steps of which are as follows:

[0083] (1) Tracking and statistical analysis of different types of follicles (PF-i, PF-ii, PF-iii, PF-iv, SF) at different developmental stages of zebrafish ovaries:

[0084] (1-1) Zebrafish at different time stages of the estrous cycle were selected. After anesthesia, the abdomen was cut open and the ovaries were removed, fixed with Born's solution, embedded in paraffin, and then sectioned into 10 mm sections. The sections were stained with hematoxylin and eosin and the development of zebrafish follicles at different time stages was observed under an optical microscope.

[0085] (1-2) The diameter and length of follicles in tissue sections of zebrafish ovaries at different stages were measured using ImageJ software. The number of follicles was counted according to the diameter and characteristics of different types of follicles, and the proportion of different types of primary follicles and SFs during the development of zebrafish ovaries was calculated.

[0086] (2) Modeling determination of the rate-limiting point of zebrafish ovarian development:

[0087] (2-1) Perform spline interpolation analysis on the proportion of different types of follicles in zebrafish at different times obtained in step (1). The specific method is the same as step (2-1) in Example 1.

[0088] (2-2) Perform linear fitting on the time points when all types of primary follicles appear, determine the slope and intercept of the fitting model, and calculate the angle between the linear fitting models of each type of primary follicle and secondary follicle. The specific method is the same as step (2-2) in Example 1.

[0089] (2-3) The slope and intercept of the linear fitting model for each type of follicle ( Figure 2 (Table 2) determined that the PF-ii subtype plays a key rate-limiting role in the ovarian development process during the large growth stage of zebrafish;

[0090] (2-4) All the code of the computer program was written in MATLAB, ran on MATLAB R2024a, and executed on a PC with a Windows 10 operating system (CPU 3.10 GHz, memory 16G).

[0091] Table 2. Linear fitting expressions for different types of zebrafish follicles

[0092]

[0093] Example 3:

[0094] A method to promote the maturation and development of red crucian carp ovaries, the specific steps of which are as follows:

[0095] (1) According to the linear fitting modeling method for determining the rate-limiting point of ovarian development in red crucian carp in Example 1, PF-ii was determined to be the key rate-limiting follicle for the mature development of ovarian red crucian carp.

[0096] (2) Through biological analysis, mTOR was identified as a developmental regulator of rate-limiting follicle PF-ii. Based on the regulatory effect of nutrition on mTOR activity and the results of preliminary experiments, the diet composition was changed. The improved feed formula for red crucian carp was: 40.0% crude protein, 4.0% crude fat, 16.0% crude ash, 8.0% crude fiber, 0.5% total phosphorus, 1.7%~3.4% DL-Met, 4.7% L-Lys·HCl, and 25.1% moisture.

[0097] (3) Collect postpartum female crucian carp and feed them with improved red crucian carp feed;

[0098] (4) Ovarian tissue after 3 months of feeding with improved feed was fixed with paraformaldehyde, embedded in paraffin, sectioned in 10 mm section, and stained with HE for observation.

[0099] The results are as follows Figure 2 As shown in Figure A, the ovaries of the control group red crucian carp were in the small growth stage, with only primary follicles observed; while the ovaries of the red crucian carp fed with the improved feed were in the large growth stage, with a large number of secondary follicles filled with yolk observed.

[0100] (5) Based on the slicing results, measure the proportion of each type of follicle.

[0101] The results are as follows Figure 2 As shown in B, the ovarian maturation and development process of red crucian carp fed with the improved feed was significantly accelerated.

[0102] Example 4:

[0103] A method to promote ovarian development during the first sexual cycle in zebrafish, comprising the following steps:

[0104] (1) According to the linear fitting modeling method for determining the rate limit of ovarian development in fish in Example 2, PF-ii was determined to be the rate limit of ovarian development in zebrafish;

[0105] (2) PF-ii was purified by Percoll density gradient centrifugation, mRNA was extracted, and high-throughput sequencing and transcriptome analysis were performed to obtain the rate-limiting follicle development regulator Notch / mTOR;

[0106] (3) Based on the regulatory effect of nutrition on mTOR activity and the results of preliminary tests, the improved feed formula obtained is: 52% crude protein, 4.5% fat, 15.0% crude ash, 5.0% crude fiber, 5.0% α-fiber, 3.0% vitamin mixture, 5% DL-Met, 6.7% L-Lys·HCl, and 5.5% moisture;

[0107] (4) Using 25-day-old zebrafish fry that have started developing from PF-ii as the starting point for feeding improved feed, after feeding for 15-20 days, zebrafish ovarian tissue was collected, fixed with paraformaldehyde, embedded in paraffin, sectioned in 10 mm, and stained with HE for observation.

[0108] The results are as follows Figure 4 The ovarian development process was significantly accelerated in the group fed the improved feed.

Claims

1. A method for determining the critical rate-limiting point of ovarian development in fish, characterized in that, Includes the following steps: (1) Select fish ovaries at different developmental stages as materials for histological sections, count the number of different types of follicles according to the size and morphological characteristics of the follicles, and calculate the proportion of different types of follicles during the development of fish ovaries; (2) Based on the proportion of different types of follicles obtained in step (1), spline interpolation and linear fitting data-driven modeling techniques are used to analyze the proportion curves, growth rates and synchronous initial state characteristics of different types of follicles, and finally determine the key rate-limiting point of fish ovarian development. The specific steps for step (2) are as follows: (2-1) Based on the proportion of different types of follicles obtained in step (1), spline interpolation is performed to construct a dynamic function relationship of the proportion of different types of follicles over time. (2-2) Linear fitting was performed on the time points when all types of follicles appeared to construct linear fitting models for each type of follicle; (2-3) Based on the linear fitting model, calculate the slope and intercept to preliminarily determine whether there is a rate-limiting relationship between different types of follicles; select the linear fitting model with a rate-limiting relationship, and then calculate the intersection of the linear relationships of different types of follicles to determine the key rate-limiting point of fish ovarian development. Step (2-1) establishes the dynamic functional relationship between the proportion of different types of follicles and development over time, as detailed below: Suppose there is a set of data points showing the percentage of different follicle types. In each time sub-interval [ , Construct a cubic polynomial function of the following form. : ； in , , , These are undetermined coefficients; , ; Ensure that the first derivative is continuous at the nodes. Ensure that the second derivative is continuous at the nodes; For natural boundary conditions; Step (2-2) involves constructing linear fitting models for each type of follicle, as detailed below: For a given percentage of follicle types ( , ),in =1, 2, ..., n, the linear fitting or regression model of the dynamic changes of this follicle type is determined by minimizing the following loss function: The regression coefficients were obtained by performing a t-significance test.

2. The method for determining the critical rate-limiting point of fish ovarian development according to claim 1, characterized in that, The process of determining the critical rate-limiting point for ovarian development in fish in steps (2-3) is as follows: Let the linear fitting models for two different follicle types be as follows: : , : Where m1 and m2 are the slopes of the two linear fitting models, and b1 and b2 are the y-intercepts of the two linear fitting models, respectively. The linear fitting model with a velocity-limiting relationship is selected, and the coordinates (x, y) of the key velocity-limiting point at a specific stage of fish ovarian development are calculated using the following formula: ， 。 3. The method for determining the critical rate-limiting point of fish ovarian development according to any one of claims 1-2, characterized in that, The fish species mentioned are zebrafish, crucian carp, blunt snout bream, grass carp, or Xianghua carp.

4. The application of the method for determining the critical rate-limiting point of ovarian development in fish as described in any one of claims 1-3 in regulating the process of ovarian development in fish.

5. The application according to claim 4, characterized in that, The dominant follicle at the critical rate-limiting point of fish ovarian development determined by the method described in any one of claims 1-3 is the rate-limiting follicle. By improving the fish farming environment or feed formulation based on the characteristics of the rate-limiting follicle at the critical rate-limiting point, the process of fish ovarian development can be regulated.

6. A method for promoting the maturation and development of fish ovaries, characterized in that, By using the method described in any one of claims 1-3 to determine the critical rate-limiting point of ovarian maturation and development in fish, and by adding 1.7-2.5% DL-methionine and 3.4-5% L-lysine hydrochloride to the fish diet by mass fraction, the process of ovarian maturation and development in fish can be promoted.

7. The method for promoting the maturation and development of fish ovaries according to claim 6, characterized in that, The fish in question is either zebrafish or crucian carp.

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