Method for determining key speed limit point of fish ovarian development and application

Abstract

The invention belongs to the technical field of fish ovarian development, and discloses a method for determining a fish ovarian maturation advantage speed limit point. The method comprises the following steps: selecting fish ovaries at different development stages as materials, carrying out histological slicing, and carrying out statistics to obtain proportions of different types of follicles in a fish ovarian development process; according to the proportion conditions of different types of follicles, the proportion curve, growth rate and synchronous initial state characteristics of different types of follicles are analyzed by using a spline interpolation and linear fitting data driven modeling technology, and finally the fish ovarian development key speed limit point is determined. Based on the method, the dominant speed limit point of fish ovarian maturation development can be accurately determined, and then measures are made according to the dominant speed limit point to regulate and control the fish ovarian development process. The invention further discloses application of the method for determining the fish ovarian maturation advantage speed limit point to regulation and control of the fish ovarian development process and a method for promoting the fish ovarian maturation development process.

Description

Technical Field

[0001] The invention belongs to the technical field of fish ovary development, and relates to a method for modeling and measuring key rate-limiting points of fish ovary development and its application. Background Art

[0002] In fish ovaries, the basic functional unit of oocyte occurrence and development is the follicle. The primary follicle (PF) is composed of two-phase oocytes and a peripheral single-layer follicular cell, and can be divided into four types: PF-i, PF-ii, PF-iii and PF-iv. The secondary follicle (SF) is composed of three-phase or four-phase oocytes and a peripheral double-layer follicular cell. When the oocyte is filled with yolk, it immediately resumes meiosis and develops into a mature follicle. The maturation and development of fish ovaries generally go through: oogonia development period, primary follicle development period (small growth period), secondary follicle development period (large growth period) and maturation period. The important histological feature of the mature development of fish ovaries is the appearance of secondary follicles in the large growth period.

[0003] In fish, it takes about two months for the ovaries of some species (such as zebrafish) to develop from the small growth period to the first sexual maturity, while other species (such as grass carp and sturgeon) take several years or even more than ten years. Interestingly, the older the age of sexual maturity of fish, the longer the ovaries stagnate in the small growth period during the first sexual cycle. Doroshov et al. reported that artificial breeding makes it very difficult for the ovaries of white sturgeon to develop from the small growth period to the large growth period, and even stagnates; the practice of artificial breeding of Chinese sturgeon also shows that there are reproductive dysfunctions in farmed Chinese sturgeon, and it is difficult for the ovaries to develop to the large growth period after the small growth period. The sexual maturation process of female fish develops to mature follicles through different follicle types such as PF-i, PF-ii, PF-iii, PF-iv, SF-i and SF-ii. However, the key speed-limiting points that affect the maturation and development of fish ovaries have not been mastered, especially the objective evaluation strategy is still uncertain. Therefore, developing a method to accurately and effectively determine the critical speed-limiting point in the development process of fish ovaries and adjusting the breeding environment or feed formula according to the growth and development characteristics of the dominant follicles at the development speed-limiting point is of great significance for enhancing fish reproductive performance and improving breeding efficiency. Summary of the invention The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide an accurate modeling determination method and application of the rate-limiting point of fish ovarian development.

[0004] In order to solve the above technical problems, the technical solution proposed by the present invention is: In a first aspect, the present invention provides a method for determining a critical rate-limiting point of fish ovarian development, comprising the following steps: (1) The ovaries of fish at different developmental stages were selected as materials for histological sectioning. The number of different types of follicles was counted according to the size and morphological characteristics of the follicles, and the proportion of different types of follicles in the development of fish ovaries was calculated; (2) Based on the proportion of different types of follicles obtained in step (1), the spline interpolation and linear fitting data-driven modeling technology are used to analyze the proportion curves, growth rates and synchronous initial state characteristics of different types of follicles, and finally determine the critical rate-limiting point of fish ovarian development.

[0005] In the above method, preferably, the specific operation of step (2) is as follows: (2-1) performing spline interpolation based on the proportions of different types of follicles obtained in step (1) to construct a dynamic functional relationship between the proportions of different types of follicles and their development over time; (2-2) Perform linear fitting on the time points at which all types of follicles appear, and construct a linear fitting model for each type of follicle; (2-3) Calculate the slope and intercept based on the linear fitting model to preliminarily determine whether there is a speed-limiting relationship between the various types of follicles; select a linear fitting model with a speed-limiting relationship, and then calculate the intersection of the linear relationship equations of the various types of follicles to determine the key speed-limiting point of fish ovarian development.

[0006] Spline interpolation is a smooth curve fitting technique commonly used in numerical analysis. It approximates or interpolates data points by using piecewise defined polynomials. Step (2-1) constructs a dynamic functional relationship between the proportion of different types of follicles and their development over time as follows: Suppose there is a set of data points with different follicle types , in each time subinterval [ , ], construct a cubic polynomial function of the following form : ; in , , , is the coefficient to be determined; , ; Ensure that the first-order derivatives are continuous at the nodes, Ensure that the second-order derivatives are continuous at the nodes; is the natural boundary condition. This gives us a cubic polynomial on each interval , thus completing the cubic spline interpolation process.

[0007] 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. Because the present invention only considers the relationship between the single variable of proportion and development time, that is, simple linear regression, its basic principle is as follows: Suppose there is a set of data points ( , ), in =1, 2, ..., n. Our goal is to determine an optimal straight line , the straight line fits these data points to the greatest extent. The goal of linear fitting is to minimize the residual sum of squares (RSS) between all data points and the fitted line. The residual is defined as the difference between the actual value of the data point and the fitted value: ; To find the optimal parameters a and b, we need to minimize the RSS. This can be achieved by setting the partial derivatives of RSS with respect to a and b to zero. Specifically, we solve the following system of equations: , ; By solving the above system of equations, we can determine the optimal parameters that minimize the RSS and thus determine the fitted line.

[0008] In order to calculate the angle between the linear fitting models of different types of follicles, based on the above fitting function expression, we calculated the inner product to obtain the angle. The specific principle is as follows: For two functions defined on the interval [a,b] and , their inner product (dot product) can be defined as: ; Two functions defined on the interval [a,b] and The angle between them can be defined as: .

[0009] Therefore, step (2-2) constructs the linear fitting model of each type of follicle as follows: For a given follicle type percentage data point ( , ),in =1, 2, ..., n, the linear fitting or regression model of the dynamic change of the follicle type is to determine the regression coefficient by minimizing the following loss function:

[0010] The regression coefficient was obtained by performing a t significance test.

[0011] Preferably, in order to further determine the coordinates of the dominant speed-limiting point, the present invention calculates the intersection of the linear relationship equations of each type of follicle. The process of step (2-3) determining the key speed-limiting point of fish ovarian development is as follows: Assume that the linear fitting models of two different follicle types are : , : , where m1 and m2 are the slopes of the two linear fitting models, b1 and b2 are the intercepts of the two linear fitting models on the y axis, respectively. The linear fitting model with a speed-limiting relationship is selected, and the coordinates (x, y) of the key speed-limiting point at a specific stage of fish ovarian development are calculated by the following formula: , .

[0012] In a second aspect, the present invention provides an application of the method for determining the critical rate-limiting point of fish ovarian development in regulating the development process of fish ovaries.

[0013] In the above application, preferably, the dominant follicle at the critical rate-limiting point of fish ovarian development determined by the method is the rate-limiting follicle, and the fish breeding environment or feed formula is improved according to the characteristics of the rate-limiting follicle at the critical rate-limiting point, thereby achieving the regulation of the fish ovarian development process.

[0014] Preferably, the dominant rate-limiting follicle is purified by Percoll density gradient centrifugation, the mRNA of the dominant rate-limiting follicle is extracted, high-throughput sequencing and transcriptome analysis are performed to obtain the key regulatory factors for the development of the dominant rate-limiting follicle, and the breeding environment or feed formula of the fish is improved according to the characteristics of the key regulatory factors for development.

[0015] In a third aspect, 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, and combined with the growth and development characteristics of the rate-limiting follicle (PF-ii), 1.7-2.5% DL-methionine and 3.4-5% L-lysine hydrochloride are added to the fish feed by mass fraction to significantly promote the maturation and development of fish ovaries.

[0016] Preferably, the fish is zebrafish or crucian carp.

[0017] During the feeding process, the ovarian development status was observed regularly. The method for observing the ovarian development status was as follows: fix the ovarian tissue, embed it in paraffin and then slice it, dewax it, stain it with hematoxylin and eosin, and observe it under a microscope.

[0018] Compared with the prior art, the present invention has the following beneficial effects: BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The results of the analysis of the rate-limiting point of ovarian maturation and development of red crucian carp determined by spline interpolation (A) and linear fitting (B) in Example 1.

[0020] Figure 2 The results of the analysis of the rate-limiting point of zebrafish ovarian maturation development determined by spline interpolation (A) and linear fitting (B) in Example 2 (Figure legend: dph: days after hatching).

[0021] Figure 3 This is the effect of the improved crucian carp diet on the secondary cycle ovarian development of red crucian carp in Example 3; (A) ovarian histological observation results, (B) statistical results of the proportion of different types of follicles.

[0022] Figure 4 This is Example 4, the effect of the improved diet on the ovarian development of the first sexual cycle of zebrafish. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0025] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0026] Embodiment 1: A method for determining the critical rate-limiting point of ovarian development of red crucian carp through data-driven modeling, the specific steps are as follows: (1) Tracking statistics of various types of follicles (PF-i, PF-ii, PF-iii, PF-iv, SF) at different developmental stages of the ovary of red crucian carp: (1-1) Red crucian carp at different time stages of the sexual cycle were selected, the abdomen was cut open after anesthesia, the ovaries were removed, fixed with Bonn's solution, embedded in paraffin, and then cut into 10 mm sections, stained with hematoxylin and eosin, and the development of ovarian follicles of red crucian carp at different time stages was imaged and tracked under an optical microscope; (1-2) Image J software was used to measure the diameter and length of follicles in tissue sections of the ovary of red crucian carp at different stages, and the number of different types of follicles was counted according to their diameters and characteristics, and the proportion of different types of primary follicles and SFs in the development of the ovary of red crucian carp was calculated; (2) Modeling determination of the rate-limiting point of ovarian development in red crucian carp: (2-1) The proportion of different types of follicles in different stages of red crucian carp obtained in step (1) was subjected to spline interpolation analysis. The specific analysis is as follows: For each subinterval [ , ] The interpolation function can be expressed as: ; in , , , is an undetermined coefficient; in order to ensure The first and second order derivatives at all internal nodes are continuous, and the following conditions need to be met: , , , ; In addition, boundary conditions are required to fully determine all coefficients. The most common boundary conditions are natural boundary conditions, which require the second-order derivatives at the endpoints to be zero, that is, satisfying: ; This gives us a cubic polynomial on each interval , thus completing the cubic spline interpolation process; (2-2) Perform linear fitting on the time points at which 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 process of linear fitting is as follows: Suppose there is a set of data points ( , ), in =1, 2, ..., n; The goal of the present invention is to determine an optimal straight line , the straight line can best fit these data points; the goal of linear fitting is to minimize the residual sum of squares (RSS) between all data points and the fitted line; the residual is defined as the difference between the actual value of the data point and the fitted value: ; 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, solve the following system of equations: , ; By solving the above system of equations, we can determine the optimal parameters that minimize the RSS and thus determine the fitted line; To further identify the dominant rate-limiting point, we calculated the intersection of the linear fitting models of primary and secondary follicles.

[0027] Suppose the linear model of each type of primary follicle is : , the linear model of secondary follicles is : , where m1 and m2 are the slopes of the two linear models, b1 and b2 are their intercepts on the y-axis; (x, y) is the coordinate of the "dominant speed limit point", and the calculation formula is as follows: , ; (2-3) The slope and intercept size of the linear fitting model of each type of follicle ( Figure 1 , Table 1 ), determined that the PF-ii subtype plays a key rate-limiting role in the progression of ovarian development in the large growth stage of crucian carp; (2-4) All codes of the computer program were written in MATLAB, run on MATLAB R2024a, and executed on a PC (CPU 3.10 GHz, memory 16G) under the Windows 10 operating system environment.

[0028] Table 1 Linear fitting relationship expressions of various types of follicles of red crucian carp

[0029] Embodiment 2: A method for determining the critical rate-limiting point of zebrafish ovarian development through data-driven modeling, the specific steps are as follows: (1) Tracking statistics of various types of follicles (PF-i, PF-ii, PF-iii, PF-iv, SF) at different developmental stages of zebrafish ovaries: (1-1) Select zebrafish at different time stages of the sexual cycle, cut open the abdomen after anesthesia, remove the ovaries, fix them with Bonn's solution, embed them in paraffin, then slice them into 10 mm slices, stain them with hematoxylin and eosin, and image and track the development of zebrafish follicles at different time stages under an optical microscope; (1-2) Image J software was used to measure the diameter and length of follicles in zebrafish ovarian tissue sections at different stages, and the number of different types of follicles was counted according to their diameters and characteristics, and the proportion of different types of primary follicles and SFs during zebrafish ovarian development was calculated; (2) Modeling determination of the rate-limiting point of zebrafish ovarian development: (2-1) performing spline interpolation analysis on the proportions of different types of follicles at different stages of zebrafish obtained in step (1), the specific method being the same as step (2-1) of Example 1; (2-2) performing linear fitting on the time points at which all types of primary follicles appear, determining the slope and intercept of the fitting model, and calculating 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) of Example 1; (2-3) The slope and intercept size of the linear fitting model of each type of follicle ( Figure 2 , Table 2 ), confirming that the PF-ii isoform plays a key rate-limiting role in the progression of ovarian development during the large growth stage of zebrafish; (2-4) All codes of the computer program were written in MATLAB, run on MATLAB R2024a, and executed on a PC (CPU 3.10 GHz, memory 16G) under the Windows 10 operating system environment.

[0030] Table 2 Linear fitting relationship expressions of various types of zebrafish follicles

[0031] Embodiment 3: A method for promoting the maturation and development of the ovary of red crucian carp, comprising the following specific steps: (1) According to the linear fitting modeling determination method of the rate-limiting point of ovarian development of red crucian carp in Example 1, PF-ii was determined to be the key rate-limiting follicle for the maturation and development of the ovary of red crucian carp; (2) Through biological analysis, mTOR was screened as the developmental regulator of the rate-limiting follicle PF-ii. The dietary components were changed according to the regulatory effect of nutrition on mTOR activity and the results of the preliminary test. 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% water. (3) Collect postpartum female crucian carp and feed them with modified red crucian carp feed; (4) After 3 months of feeding with the modified diet, the ovarian tissue was fixed with polyformaldehyde, embedded in paraffin, sliced ​​into 10 mm sections, and stained with HE for observation.

[0032] The results are as follows Figure 2 As shown in A, the ovaries of red crucian carp in the control group were in the small growth stage, with only primary follicles observed; while the ovaries of red crucian carp in the group fed with the improved feed were in the large growth stage, with a large number of secondary follicles filled with yolk observed.

[0033] (5) Based on the slice results, measure the proportion of each type of follicle.

[0034] The results are as follows Figure 2 As shown in B, the ovarian maturation and development process of red crucian carp in the improved feed feeding group was significantly accelerated.

[0035] Embodiment 4: A method for promoting the ovarian development process of zebrafish in the first sexual cycle, the specific steps are as follows: (1) According to the linear fitting modeling determination method of the rate-limiting point of fish ovarian development in Example 2, PF-ii was determined to be the rate-limiting point of zebrafish ovarian development; (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 regulatory factor Notch / mTOR; (3) Combining the regulatory effect of nutrition on mTOR activity and the results of the preliminary test, the improved feed formula was obtained: 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% water; (4) The 25-day-old zebrafish fry that began to develop from PF-ii were used as the starting point for feeding with the improved feed. After 15-20 days of feeding, the zebrafish ovarian tissues were collected, fixed with polyformaldehyde, embedded in paraffin, sliced ​​into 10 mm sections, and stained with HE for observation.

[0036] The results are as follows Figure 4 The ovarian development process of the group fed with improved feed was significantly accelerated.

Claims

1. A method for determining the critical rate-limiting point of fish ovarian development, characterized in that: The steps include: (1) The ovaries of fish at different developmental stages were selected as materials for histological sectioning. The number of different types of follicles was counted according to the size and morphological characteristics of the follicles, and the proportion of different types of follicles in the development of fish ovaries was calculated; (2) Based on the proportion of different types of follicles obtained in step (1), the spline interpolation and linear fitting data-driven modeling technology are used to analyze the proportion curves, growth rates and synchronous initial state characteristics of different types of follicles, and finally determine the critical rate-limiting point of fish ovarian development.

2. The method for determining the critical rate-limiting point of fish ovarian development according to claim 1, characterized in that: The specific operations of step (2) are as follows: (2-1) performing spline interpolation based on the proportions of different types of follicles obtained in step (1) to construct a dynamic functional relationship between the proportions of different types of follicles and their development over time; (2-2) Perform linear fitting on the time points at which all types of follicles appear, and construct a linear fitting model for each type of follicle; (2-3) Calculate the slope and intercept based on the linear fitting model to preliminarily determine whether there is a speed-limiting relationship between the various types of follicles; select a linear fitting model with a speed-limiting relationship, and then calculate the intersection of the linear relationship equations of the various types of follicles to determine the key speed-limiting point of fish ovarian development.

3. The method for determining the critical rate-limiting point of fish ovarian development according to claim 2, characterized in that: Step (2-1) constructs a dynamic functional relationship between the proportion of different types of follicles and their development over time as follows: Suppose there is a set of data points with different follicle types , in each time subinterval [ , ], construct a cubic polynomial function of the following form : ； in , , , is the coefficient to be determined; , ; Ensure that the first-order derivatives are continuous at the nodes, Ensure that the second-order derivatives are continuous at the nodes; is a natural boundary condition.

4. The method for determining the critical rate-limiting point of fish ovarian development according to claim 3, characterized in that: Step (2-2) constructs the linear fitting model of each type of follicle as follows: For a given follicle type percentage data point ( , ),in =1, 2, ..., n, the linear fitting or regression model of the dynamic change of the follicle type is to determine the regression coefficient by minimizing the following loss function: The regression coefficient was obtained by performing a t significance test.

5. The method for determining the critical rate-limiting point of fish ovarian development according to claim 4, characterized in that: The process of determining the critical rate-limiting point of fish ovarian development in step (2-3) is as follows: Assume that the linear fitting models of two different follicle types are : , : , where m1 and m2 are the slopes of the two linear fitting models, b1 and b2 are the intercepts of the two linear fitting models on the y axis, respectively. The linear fitting model with a speed-limiting relationship is selected, and the coordinates (x, y) of the key speed-limiting point at a specific stage of fish ovarian development are calculated by the following formula: ， 。 6. The method for determining the critical rate-limiting point of fish ovarian development according to any one of claims 1 to 5, characterized in that: The fish is zebrafish, crucian carp, bighead carp, grass carp or Hunan bream.

7. Application of the method for determining the critical rate-limiting point of fish ovarian development as claimed in any one of claims 1 to 6 in regulating the development process of fish ovaries.

8. The use according to claim 7, 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 to 6 is the rate-limiting follicle. The fish breeding environment or feed formula is improved according to the characteristics of the rate-limiting follicle at the critical rate-limiting point, thereby regulating the fish ovarian development process.

9. A method for promoting the maturation and development of fish ovaries, characterized in that: The critical rate-limiting point of the maturation and development of the fish ovaries is determined by the method described in any one of claims 1 to 6. At this development stage, 1.7-2.5% DL-methionine and 3.4-5% L-lysine hydrochloride are added to the fish feed by mass fraction to promote the maturation and development process of the fish ovaries.

10. The method for promoting the maturation and development of fish ovaries according to claim 9, characterized in that: The fish is zebrafish or crucian carp.

Images