A method for establishing and applying a quality evaluation standard for largefin wrasse eggs

Abstract

This invention relates to a method for establishing and applying a quality evaluation standard for *Amur corvina* eggs, belonging to aquaculture and fish reproduction, and solves the problem of the lack of objective and quantifiable quality evaluation standards for *Amur corvina* eggs in existing technologies. The method includes: collecting unfertilized *Amur corvina* eggs, taking 1318g for biochemical index testing, and using the remainder for reproductive performance verification; measuring total protein concentration, vitamin A concentration, vitellin concentration, and the content of various fatty acids; artificially inseminating the eggs used for reproductive performance verification, and statistically analyzing the fertilization rate, hatching rate, and emergence rate; correlating the biochemical data with the reproductive performance data, screening core evaluation indicators, and establishing discrimination criteria. This invention achieves objective and quantitative evaluation of egg quality, can predict early before fertilization, effectively improves the efficiency of artificial breeding; and the threshold values ​​are stable and reliable after cross-validation, exhibiting good universality.

Description

Technical Field

[0001] This invention relates to the field of aquaculture and fish breeding technology, and in particular to a method for establishing and applying a quality evaluation standard for largefin bream eggs. Background Technology

[0002] The large-finned mandarin fish (Hemibagrus macropterus), also known as the stone flathead or river rat, belongs to the class Osteichthyes, order Siluriformes, family Bagridae, and genus Hemibagrus. It is naturally distributed in the main streams and tributaries of the Yangtze and Pearl River systems. Its flesh is tender and nutritious; however, due to water conservancy projects and overfishing, the wild population of large-finned mandarin fish has declined rapidly. Artificial breeding of large-finned mandarin fish is beneficial for the refined development of this resource.

[0003] Egg quality is the biological basis for determining the developmental potential of fish embryos and hatching success rates. High-quality eggs provide the embryo with sufficient nutrients, energy, and key regulatory factors, which is a prerequisite for ensuring a high hatching rate. Currently, the assessment of the quality of *Cyprinus maculatus* eggs in production still mainly relies on experience, judging by visual inspection or simple tools, observing the color, elasticity, size, and distinguishing between "normal eggs" and "overripe eggs." Although appearance characteristics can be used to assess egg quality to some extent, this method suffers from high subjectivity, low accuracy, and inability to make early predictions, severely hindering the refined development of artificial breeding techniques for *Cyprinus maculatus*.

[0004] Beyond visual assessment of external characteristics, detecting differences in the internal components of the egg can provide a more objective evaluation of egg quality. Studies have shown that the content of vitellin, total protein, lipid and fatty acid composition, and vitamin content within the egg all provide the nutritional basis and play a regulatory role during egg development. However, due to the varying physiological characteristics and nutritional needs of different fish species, a systematic and quantifiable method for assessing egg quality specifically for the largefin minnow is currently lacking. Summary of the Invention

[0005] Based on the above analysis, the present invention aims to provide a method for establishing a quality evaluation standard for largefin wrasse eggs and its application, in order to solve the problem of the lack of objective and quantifiable quality evaluation standards for largefin wrasse eggs in the prior art.

[0006] The objective of this invention is mainly achieved through the following technical solutions: This invention provides a method for establishing quality evaluation standards for the eggs of the largefin wrasse, comprising the following steps: S1: Collect unfertilized eggs of the bigfin bream. Take 13-18g of the unfertilized eggs from each parent fish for biochemical index testing, and use the remaining unfertilized eggs for reproductive performance verification. S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of various fatty acids in unfertilized eggs used for biochemical index detection to obtain biochemical index data; S3: Artificial insemination was performed on the unfertilized eggs used for reproductive performance verification, and the fertilization rate, hatching rate and hatching rate were statistically analyzed to obtain reproductive performance data; S4: Correlation analysis was performed on the biochemical index data of each unfertilized egg sample obtained in S2 and the reproductive performance data of each sample obtained in S3 to screen the core evaluation indicators of the quality of the largefin wrasse eggs and establish the discrimination criteria.

[0007] Further, step S4 includes: S41: Spearman correlation analysis was performed on the relative contents of various fatty acids and the hatching rate. The fatty acid types that were significantly correlated with the hatching rate were screened out with |R|>0.4 and P<0.05 as the criteria. Where R is the correlation coefficient, and its value ranges from -1 to 1; P represents the significance level, and a smaller P value indicates a more reliable result. S42: Principal component analysis was performed on the selected fatty acids, and the core fatty acid index was determined based on the loading diagram. The core fatty acid index, together with the total protein concentration, vitamin A concentration, and vitellin concentration, constituted the core evaluation index of the quality of the oocytes of the largefin fish. S43: Using the core evaluation indicators of the quality of the eggs of the giant finned mandarin fish as independent variables and the hatching rate as the dependent variable, a multiple linear regression model was established; S44: Diagnose and validate the multiple linear regression model; S45: With a hatching rate of ≥20% and a fertilization rate of ≥50% as the target for high-quality eggs, the thresholds of each core evaluation index of the quality of the largefin loach eggs were determined by ROC curve; S46: The threshold values ​​of each core evaluation indicator of the quality of the largefin wrasse eggs were cross-validated using the leave-one-out method.

[0008] Furthermore, the core fatty acid indicators determined in step S42 are the following 12 types: C15:0, C16:1n7, C17:0, C17:1n7, C20:1, C20:2, C20:3n6, C22:1n9, C20:4n6 (ARA), C20:5n3 (EPA), C24:1n9, and C22:6n3 (DHA).

[0009] Furthermore, the threshold values ​​for each core evaluation indicator of the quality of *Ariocarpus macrofinae* eggs determined in step S45 are as follows: Total protein concentration ≤9.83mg / mL, vitamin A concentration ≥0.348ng / mgprot, vitellin concentration ≥0.045ng / mL; Fatty acid thresholds: C15:0≥0.02, C17:0≥0.029, C20:1≤0.103, C16:1n7≤0.208, C17:1n7≥0.018, C22:1n9≤0.02, C24:1n9≤0.027, C20:2≤0.177, C20:3n6≤0.258, C20:4n6(ARA)≤0.133, C20:5n3(EPA)≤0.115, C22:6n3(DHA)≤0.864.

[0010] Further, in step S3, the fertilization rate % = number of normally developing eggs / total number of eggs × 100%, the hatching rate % = number of hatched larvae / total number of fertilized eggs × 100%, and the hatching rate % = number of hatched larvae / total number of eggs × 100%; among which, normally developing eggs are yellowish-brown and opaque, and dead eggs are pure white and opaque.

[0011] The present invention also provides a quality evaluation index system for *Ceratos maculatus* eggs established according to the above method, including: appearance characteristics of being round, full, and elastic, egg diameter ≥ 2.5 mm, and core quality evaluation indexes for *Ceratos maculatus* eggs determined according to the above method.

[0012] This invention also provides a method for assessing the quality of eggs from the large-finned mandarin fish, using the evaluation index system described in claim 6, comprising the following steps: S1: Collect unfertilized eggs of the largefin wrasse to be evaluated; S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of core fatty acids in unfertilized eggs; S3: Compare the biochemical data obtained in step S2 with the aforementioned quality evaluation index system for *Ceratos maculatus* eggs. If all the biochemical data of the *Ceratos maculatus* eggs are within the threshold range of the aforementioned quality evaluation index system, they are judged as high-quality eggs; otherwise, they are judged as low-quality eggs.

[0013] Furthermore, the evaluation method also includes step S4: verifying the accuracy of the method through fertilization rate, hatching rate and hatching rate, that is, artificial insemination and hatching are performed on batches of eggs that are judged to be of high quality. If the actual fertilization rate and hatching rate are higher than those of batches of eggs that are judged to be of low quality, it proves that the judgment result of the method is accurate.

[0014] Furthermore, in step S2, the total protein concentration, vitamin A concentration, and vitellin concentration are determined using the microplate method, and the relative content of core fatty acids is determined using gas chromatography.

[0015] Furthermore, the chromatographic conditions for the gas chromatography method are as follows: Agilent CP-81188 column with dimensions of 100m × 250μm × 0.20μm, hydrogen as carrier gas, constant flow rate of 20mL / min, and split ratio of 100:1. Temperature program: Start at 100℃ and hold for 13 min, increase to 180℃ at 10℃ / min and hold for 6 min, increase to 200℃ at 1℃ / min and hold for 20 min, increase to 230℃ at 4℃ / min and hold for 15 min; The injection port temperature is 270℃, and the injection volume is 1μL.

[0016] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: 1. This invention collects the total protein concentration, vitamin A concentration, vitellin concentration, and the content of 12 core fatty acids screened through Spearman correlation analysis and principal component analysis of unfertilized eggs from the large-finned mandarin fish. A multiple linear regression model and ROC curves are used to establish quantitative thresholds for each indicator, forming a complete evaluation index system. In application, egg quality can be determined simply by comparing the biochemical data of the eggs to be tested with this system, solving the problems of high subjectivity and low accuracy associated with traditional visual observation, and providing a quantifiable operational standard for egg quality evaluation.

[0017] 2. This invention utilizes unfertilized eggs to detect and determine various biochemical indicators, enabling the selection of high-quality eggs for production before artificial insemination, thus avoiding resource waste caused by low-quality eggs. By establishing a threshold system with a hatching rate ≥20% and a fertilization rate ≥50% as the target for high-quality eggs, the predicted results highly match the actual reproductive performance, thereby effectively improving the overall efficiency and success rate of artificial breeding of the giant wrasse.

[0018] 3. This invention uses leave-one-out cross-validation to verify the thresholds of each indicator. The coefficients of variation for each core evaluation indicator are extremely low (between 0.001 and 0.032), indicating that the determined thresholds have extremely high stability and universality. Simultaneously, diagnostic plots of the multiple regression model (residual distribution, QQ plot, scale position plot, residual and leverage value plot) verify that the model satisfies the basic assumptions of linear regression, ensuring the accuracy and repeatability of the evaluation indicator system in determining the quality of oocytes from different batches and individuals.

[0019] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0020] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0021] Figure 1 shows the PCA variable loadings obtained by principal component analysis of the fatty acids that were screened and were significantly related to the hatching rate in this invention. Figure 2 The diagnostic plots for the multiple regression model of this invention are as follows: scatter plot of residuals and fitted values, residual QQ plot, scale position plot, and residual and leverage value plot. Figure 3 This is a comparison chart of the predicted hatching rate and the measured hatching rate of the sample using the multiple linear regression model of this invention. Figure 4-1 The ROC curves for C17:0, C16:1n7, C15:0, C17:1n7, C20:5n3, and C20:4n6, which are the core evaluation indicators for the quality of the eggs of the largefin loach of this invention; Figure 4-2 The ROC curves for C20:1, C20:2, C20:3n6, C24:1n9, C22:1n9, and C22:6n3, which are the core evaluation indicators for the quality of eggs from the largefin loach of this invention; Figure 4-3 This is a ROC curve of total protein concentration, vitamin A concentration, and vitellin concentration, which are the core evaluation indicators of the quality of oocytes from the largefin loach of this invention. Figure 5-1 This is a distribution diagram of the threshold values ​​of various core evaluation indicators of the quality of largefin wrasse eggs in the leave-one-out cross-validation method of this invention; Figure 5-2 This is a graph showing the coefficient of variation of the threshold values ​​of each core evaluation index of the quality of the largefin wrasse eggs in the leave-one-out cross-validation method of this invention. Detailed Implementation

[0022] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0023] This invention provides a method for establishing quality evaluation standards for the eggs of the largefin wrasse, comprising the following steps: S1: Collect unfertilized eggs of the bigfin bream. Take 13-18g of the unfertilized eggs from each parent fish for biochemical index testing, and use the remaining unfertilized eggs for reproductive performance verification. Specifically, during the breeding season of the bigfin bream, several female parent fish in the spawning period are selected, and unfertilized eggs are collected from each parent fish. 13-18g of the unfertilized eggs from each parent fish are used for biochemical index testing, and the remaining unfertilized eggs are used for reproductive performance verification. S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of various fatty acids in unfertilized eggs used for biochemical index detection to obtain biochemical index data; Specifically, unfertilized eggs used for biochemical index detection were ground with physiological saline to prepare a 10% tissue homogenate. The supernatant was collected by centrifugation, and the total protein concentration, vitamin A concentration, and vitellin concentration in the supernatant were determined by microplate method. The relative fatty acid content of the eggs was determined by gas chromatography. Specifically, the concentrations of total protein, vitamin A, and vitellin in the supernatant were determined using the microplate method. The concentrations of total protein (Nanjing Jiancheng Bioengineering Institute, microplate method A045-4-1), vitamin A (Nanjing Jiancheng Bioengineering Institute, competitive method HT715-1-1), and vitellin (ELISA, competitive method ml890816) were all determined using the microplate method. Absorbance values ​​were measured at 562 nm, 450 nm, and 450 nm using a multi-functional microplate reader (Shanghai Flash Spectroscopy, SuperMax 3100). Specific operating procedures were performed according to the respective kit instructions, and the concentrations were calculated using the formulas provided in the kit instructions.

[0024] The relative content of various fatty acids in oocytes was determined by gas chromatography, following the standard GB 5009.168-2016. An Agilent 7890A gas chromatograph was used, with an Agilent CP-81188 column (100m × 250μm × 0.20μm), hydrogen as the carrier gas, a constant flow rate of 20 mL / min, and a split ratio of 100:1. The temperature program was as follows: initial temperature 100℃, hold for 13 min; increase to 180℃ at 10℃ / min, hold for 6 min; increase to 200℃ at 1℃ / min, hold for 20 min; increase to 230℃ at 4℃ / min, hold for 15 min; injection port temperature 270℃, injection volume 1 μL. Each sample was tested three times, and the average value was taken.

[0025] S3: Artificial insemination was performed on the unfertilized eggs used for reproductive performance verification, and the fertilization rate, hatching rate and hatching rate were statistically analyzed to obtain reproductive performance data; Specifically, the eggs used for reproductive performance verification were artificially inseminated. Under an incubation water temperature of 26℃, the embryonic development was observed under an inverted microscope 3-4 hours after fertilization. The number of normal eggs that developed to the four-cell stage was counted, and the fertilization rate was calculated. Dead eggs were aspirated and counted every 12 hours after fertilization. After the fry hatched, the total number of hatched fry was counted, and the hatching rate and hatching rate were calculated. Among them, the normally developed eggs were yellowish-brown and opaque, while the dead eggs were pure white and opaque.

[0026] Specifically, fertilization rate % = number of normally developing eggs / total number of eggs × 100; Total number of fertilized eggs % = Total number of dead eggs + Number of hatched fry; Hatching rate % = Number of hatched fry / Total number of fertilized eggs × 100; Hatching rate % = Number of hatched fry / Total number of eggs × 100.

[0027] S4: Correlation analysis was performed on the biochemical index data of each unfertilized egg sample obtained in S2 and the reproductive performance data of each sample obtained in S3 to screen the core evaluation indicators of the quality of the largefin wrasse eggs and establish the discrimination criteria.

[0028] Specifically, step S4 includes: S41: Spearman correlation analysis was performed on the content of various fatty acids measured in step S2 with fertilization rate and hatching rate to screen out fatty acids that are significantly correlated with hatching rate. Specifically, Spearman correlation analysis is a statistical method used to measure the existence and strength of a relationship between two variables. The result is a correlation coefficient (R), which ranges from -1 to 1: the larger the absolute value of R, the stronger the relationship between the two variables; a positive R indicates a positive correlation, meaning that an increase in one variable also increases the other, while a negative R indicates a negative correlation, meaning that an increase in one variable decreases the other. The analysis also provides a significance level (P-value); a smaller P-value indicates a more reliable result, typically with P < 0.05 considered statistically significant.

[0029] In this invention, the judgment criteria are set as follows: |R|>0.4 and P<0.05. Wherein, |R|>0.4 indicates a moderate or greater correlation between fatty acid content and hatching rate; P<0.05 indicates that this correlation is statistically significant and not accidental. Fatty acids that meet both conditions are judged to be significantly correlated with hatching rate and are retained for subsequent principal component analysis.

[0030] S42: Principal component analysis was performed on the selected fatty acids, and the core fatty acid index was determined based on the loading diagram. The core fatty acid index, together with the total protein concentration, vitamin A concentration, and vitellin concentration, constituted the core evaluation index of the quality of the oocytes of the largefin fish. It should be noted that Principal Component Analysis (PCA) is a data dimensionality reduction method that recombines multiple original indicators into a few composite indicators (i.e., principal components). Each principal component can explain a certain proportion of the variation (i.e., information content) in the original data. PCA identifies the original indicators that contribute the most to the variation in the data, thereby achieving the selection of core indicators.

[0031] Figure 1This is a PCA variable loading plot obtained by performing principal component analysis (PCA) on fatty acids that are significantly correlated with hatching rate obtained from the initial screening. The plot shows that the first principal component (Dim1, explaining 62.8% of the original fatty acid data variation) is strongly positively correlated with ARA (C20:4n6, loading 0.85), EPA (C20:5n3, loading 0.82), C20:1 (loading 0.95), C24:1n9 (loading 0.90), C22:6n3 (loading 0.55), C16:1n7 (loading 0.58), C20:2 (loading 0.78), and C20:3n6 (loading 0.65), and significantly negatively correlated with C15:0 (loading -0.68) and C17:0 (loading -0.62); and has a weak correlation with C17:1n7 (loading 0.02). The second principal component (Dim2, explaining 19.6% of the original fatty acid data variation) is primarily dominated by C17:1n7 (loading 0.98), showing a very strong positive correlation, and also shows significant positive correlations with C17:0 (loading 0.75) and C15:0 (loading 0.70). These two principal components collectively explained 82.4% of the original data variation, indicating that the selected fatty acid indicators can effectively represent the overall information of the original data. Based on the fact that the first principal component explained the largest proportion of variation, subsequent principal components showed decreasing proportions, and the following 12 fatty acids were selected as core evaluation indicators: C15:0, C16:1n7, C17:0, C17:1n7, C20:1, C20:2, C20:3n6, C22:1n9, C20:4n6 (ARA), C20:5n3 (EPA), C24:1n9, and C22:6n3 (DHA). At the same time, total protein concentration, vitamin A concentration, and vitellin concentration were also included in the evaluation index system.

[0032] S43: Using the core evaluation indicators of the quality of the eggs of the giant finned mandarin fish as independent variables and the hatching rate as the dependent variable, a multiple linear regression model was established; Specifically, the concentrations of 12 selected fatty acids, as well as total protein, vitamin A, and vitellin, were used as independent variables, and the hatching rate was used as the dependent variable to establish a multiple linear regression model.

[0033] S44: Diagnose and validate the multiple linear regression model; Specifically, the established multiple regression model is diagnostically verified to obtain a multiple regression model diagnostic chart. The multiple regression model diagnostic chart of this invention includes a scatter plot of residuals and fitted values ​​(denoted as 2-1), a residual QQ plot (denoted as 2-2), a scale position plot (denoted as 2-3), and a residual and leverage value plot (denoted as 2-4). From 2-1, it can be seen that the residuals are randomly distributed around the 0 line without obvious pattern, indicating that the linear relationship assumption is valid. From 2-2, it can be seen that in the QQ plot, the points are basically located near the diagonal, indicating that the residuals are approximately normally distributed. From 2-3, it can be seen that in the scale position plot, the points are evenly distributed without obvious trend, indicating good homogeneity of variance. From 2-4, it can be seen that all points in the residual and leverage value plot are within the Cook's distance contour line (dashed line), indicating that there are no abnormally strong influencing points.

[0034] Preferably, the predicted hatching rate of each sample can be compared with the actual measured hatching rate using a multiple linear regression model, and the results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the scatter points of the predicted hatching rate and the actual hatching rate are basically distributed near the diagonal, and the two are positively correlated, indicating that the overall prediction trend of the multiple linear regression model of this invention is good.

[0035] S45: With a hatching rate of ≥20% and a fertilization rate of ≥50% as the target for high-quality eggs, the thresholds of each core evaluation index of the quality of the largefin loach eggs were determined by ROC curve; Specifically, the ROC curves of each evaluation index in this invention are as follows: Figure 4-1 , 4-2 As shown in 4-3, Figure 4-1 The ROC curves for C17:0, C16:1n7, C15:0, C17:1n7, C20:5n3, and C20:4n6, which are the core evaluation indicators for the quality of the eggs of the largefin loach of this invention; Figure 4-2 The ROC curves for C20:1, C20:2, C20:3n6, C24:1n9, C22:1n9, and C22:6n3, which are the core evaluation indicators for the quality of eggs from the largefin loach of this invention; Figure 4-3This is a ROC curve of the total protein concentration, vitamin A concentration, and vitellin concentration, which are the core evaluation indicators of the quality of the eggs of the largefin loach of this invention. The ROC curves of each evaluation indicator all show good discriminative power. The calculated threshold values ​​for each indicator are as follows: C15:0 ≥ 0.02, C17:0 ≥ 0.029, C20:1 ≤ 0.103, C16:1n7 ≤ 0.208, C17:1n7 ≥ 0.018, C22:1n9 ≤ 0.02, C24:1n9 ≤ 0.027, C20:2 ≤ 0.177, C20:3n6 ≤ 0.258, C20:4n6 (ARA) ≤ 0.133, C20:5n3 (EPA) ≤ 0.115, C22:6n3 (DHA) ≤ 0.864, total protein concentration (mg / mL) ≤ 9.83, vitamin A concentration (ng / mgprot) ≥ 0.348, and vitellin (ng / mL) ≥ 0.045.

[0036] S46: The leave-one-out method was used to cross-validate the thresholds of the core evaluation indicators of the quality of the largefin wrasse eggs to ensure the stability and universality of the thresholds of each evaluation indicator.

[0037] It's important to note that leave-one-out cross-validation is a method where one sample is reserved as the validation set each time, and the threshold is calculated using all other samples as the training set. This process is repeated multiple times until every sample has been used as the validation set once. By comparing the difference between the calculated threshold and the original threshold each time, the stability and generalizability of the threshold can be evaluated. Figure 5-1 As shown in the figure, this is a distribution chart of the threshold values ​​for each indicator in leave-one-out cross-validation. The figure lists the threshold values ​​calculated for each indicator in leave-one-out cross-validation (e.g., C15:0 is 0.02, C16:1n7 is 0.03, etc.). The threshold values ​​for each indicator fluctuate very little across different validation rounds. Figure 5-2 As shown in the figure, this graph displays the coefficient of variation (CV) for each indicator threshold. The coefficient of variation is the ratio of the standard deviation to the mean, used to measure the relative dispersion of the data. The smaller the CV value, the more stable the threshold. Figure 5-2 As can be seen, the coefficients of variation for each indicator are extremely low (between 0.001 and 0.032), indicating that the thresholds determined in this invention have extremely high stability and universality. Based on the above method, this invention establishes the criteria for judging high-quality eggs of the largefin wrasse, as shown in Table 1. When the appearance characteristics and various biochemical indicators of the unfertilized eggs to be tested are all within the range shown in Table 1, they are judged to be high-quality eggs that meet the production requirements.

[0038] Table 1 Evaluation index system for high-quality eggs of *Sinocyclocheilus davidii*

[0039] This invention also provides the application of the above-mentioned *Gnaphalium affine* egg quality evaluation standard, used to assess the quality of *Gnaphalium affine* eggs, including the following steps: S1: During the breeding season of the bigfin bream, select several female parent fish in the spawning period and collect the unfertilized eggs laid by each parent fish; S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of core fatty acids in unfertilized eggs; Specifically, unfertilized eggs used for biochemical index detection were ground with physiological saline to prepare a 10% tissue homogenate, centrifuged to obtain the supernatant, and the values ​​of each evaluation index were measured. Specifically, the concentrations of total protein, vitamin A, and vitellin in the supernatant were determined using the microplate method. The concentrations of total protein (Nanjing Jiancheng Bioengineering Institute, microplate method A045-4-1), vitamin A (Nanjing Jiancheng Bioengineering Institute, competitive method HT715-1-1), and vitellin (ELISA, competitive method ml890816) were all determined using the microplate method. Absorbance values ​​were measured at 562 nm, 450 nm, and 450 nm using a multi-functional microplate reader (Shanghai Flash Spectroscopy, SuperMax 3100). Specific operating procedures were performed according to the respective kit instructions, and the concentrations were calculated using the formulas provided in the kit instructions.

[0040] The relative contents of 12 fatty acids used as evaluation indicators in unfertilized eggs were determined by gas chromatography (GC), following the standard GB5009.168-2016. An Agilent 7890A GC was used, with an Agilent CP-81188 column (100m × 250μm × 0.20μm), hydrogen as the carrier gas, a constant flow rate of 20 mL / min, and a split ratio of 100:1. The temperature program was as follows: initial temperature 100℃, hold for 13 min; increase to 180℃ at 10℃ / min, hold for 6 min; increase to 200℃ at 1℃ / min, hold for 20 min; increase to 230℃ at 4℃ / min, hold for 15 min. The injection port temperature was 270℃, and the injection volume was 1 μL. Each sample was tested three times, and the average value was taken. S3: Compare the biochemical data obtained in step S2 with the aforementioned quality evaluation index system for *Ceratos maculatus* eggs. If all the biochemical data of the *Ceratos maculatus* eggs are within the threshold range of the aforementioned quality evaluation index system, they are judged as high-quality eggs; otherwise, they are judged as low-quality eggs.

[0041] It should be noted that the accuracy of the method can be verified by fertilization rate, hatching rate, and emergence rate. That is, artificial insemination and hatching are performed on batches of eggs that are judged to be of high quality, and their fertilization rate and hatching rate are statistically analyzed. If their actual fertilization rate and hatching rate are significantly higher than those of batches judged to be of low quality, then the judgment result of the method is proven to be accurate.

[0042] This verification step can be used for verification after the method is established, or it can be selectively executed at each decision.

[0043] The "evaluation index system" in the above steps refers to the threshold ranges of each index determined earlier through steps S1-S4 (i.e., sample collection, biochemical testing, reproductive performance verification, PCA analysis, regression modeling, ROC curve analysis, and leave-one-out cross-validation). Specific values ​​are shown in Table 1. For ease of application, this evaluation index system can be pre-set, and in actual evaluation, only steps S1-S3 need to be executed to quickly determine the quality of unfertilized eggs.

[0044] Example This embodiment was conducted in the breeding workshop of the largefin catfish at the Yaowan Experimental Field of the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, to verify the effectiveness of the method of the present invention.

[0045] Includes the following steps: S1: During the breeding season of the giant bream in 2025, select several female parent fish in the spawning period and divide them into 9 groups, with 3 fish in each group; Unfertilized eggs were collected separately, mixed thoroughly, and about 10 mL (about 15 g) of eggs were aspirated with a pipette for various indicator tests. The remaining eggs were used for artificial insemination and reproductive performance was statistically analyzed. S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of core fatty acids in unfertilized eggs; Specifically, unfertilized eggs used for biochemical index detection were ground with physiological saline to prepare a 10% tissue homogenate, centrifuged to obtain the supernatant, and the values ​​of each evaluation index were measured. The concentrations of total protein, vitamin A, and vitellin in the supernatant were determined using the microplate method. Specifically, the concentrations of total protein (Nanjing Jiancheng Bioengineering Institute, microplate method A045-4-1), vitamin A (Nanjing Jiancheng Bioengineering Institute, competitive method HT715-1-1), and vitellin (ELISA, competitive method ml890816) were all determined using the microplate method. Absorbance values ​​were measured at 562 nm, 450 nm, and 450 nm using a multi-functional microplate reader (Shanghai Flash Spectrum, SuperMax 3100). Specific operating procedures were performed according to the respective kit instructions, and concentrations were calculated using the formulas provided in the kit instructions. Data are shown in Table 2.

[0046] The relative contents of 12 fatty acids used as evaluation indicators in unfertilized eggs were determined by gas chromatography (GC), following the standard GB5009.168-2016. An Agilent 7890A GC was used, with an Agilent CP-81188 column (100m × 250μm × 0.20μm), hydrogen as the carrier gas, a constant flow rate of 20 mL / min, and a split ratio of 100:1. The temperature program was as follows: initial temperature 100℃, hold for 13 min; increase to 180℃ at 10℃ / min, hold for 6 min; increase to 200℃ at 1℃ / min, hold for 20 min; increase to 230℃ at 4℃ / min, hold for 15 min. The injection port temperature was 270℃, and the injection volume was 1 μL. Each sample was tested three times, and the average value was taken. The data are shown in Table 3.

[0047] S3: Compare the biochemical data obtained in step S2 with the aforementioned quality evaluation index system for *Ceratos maculatus* eggs. If all the biochemical data of the *Ceratos maculatus* eggs are within the threshold range of the aforementioned quality evaluation index system, they are judged as high-quality eggs; otherwise, they are judged as low-quality eggs.

[0048] The accuracy of this method is verified by measuring fertilization rate, hatching rate, and emergence rate. Specifically, batches of eggs judged to be of high quality are artificially inseminated and hatched, and their fertilization rate and hatching rate are statistically analyzed. If their actual fertilization rate and hatching rate are significantly higher than those of batches judged to be of low quality, then the method's judgment results are proven to be accurate. Fertilization rate statistics: Eggs used for reproductive performance statistics were artificially inseminated. Under an incubation water temperature of 26℃, embryonic development was observed under an inverted microscope 3-4 hours after fertilization. The number of normal eggs that developed to the four-cell stage was counted, and the fertilization rate was calculated. Fertilization rate % = (Number of normally developing eggs / Total number of eggs) × 100.

[0049] Hatching rate statistics: After fertilization, dead eggs are removed and counted every 12 hours. After the fry have hatched, the total number of hatched fry is counted. Hatching rate % = (Number of hatched fry / Total number of fertilized eggs) × 100.

[0050] Hatching rate: Hatching percentage % = (Number of hatched fry / Total number of eggs) × 100.

[0051] Table 2: Changes in various indicators of eggs and corresponding fertilization rate, hatching rate and egg condition during the reproductive cycle of *Amur finned bream* in the example.

[0052] Table 3. Fatty acid content of unfertilized eggs during the reproductive cycle of *Gnaphalium affine* in the example.

[0053] Group 1: Total protein concentration 9.87 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.39 ng / mgprot (≥0.348, acceptable); Vitellin 0.06 ng / mL (≥0.045, acceptable). Fatty acid indicators: C15: 0 0.02 (≥0.02, acceptable); C16: 1n7 0.21 (requirement ≤0.208, 0.21>0.208, exceeding the standard); C17: 0 0.03 (≥0.029, acceptable); C17: 1n7 0.02 (≥0.018, acceptable); C20: 1 0.10 (≤0.103, acceptable); C20: 2 0.16 (≤0.177, acceptable); C20: 3n6 0.25 (≤0.258, acceptable); C22: 1n9 0.02 (≤0.02, acceptable); C20: 4n6 (ARA) 0.13 (≤0.133, acceptable); C20: 5n3 (EPA) 0.11 (≤0.115, acceptable); C24: 1n9 0.02 (≤0.027, qualified); C22:6n3(DHA) 0.77 (≤0.864, qualified).

[0054] Overall assessment: Total protein concentration and C16:1n7 levels exceeded standards, while other parameters were within acceptable limits; therefore, the eggs were classified as low-quality. Actual hatching rate was 43.49%, and fertilization rate was 33.88%.

[0055] Group 2: Total protein concentration 9.17 mg / mL (≤9.83, qualified); Vitamin A concentration 0.43 ng / mgprot (≥0.348, qualified); Vitellin 0.08 ng / mL (≥0.045, qualified). Fatty acid indicators: C15: 0 0.02 (≥0.02, acceptable); C16: 1n7 0.20 (≤0.208, acceptable); C17: 0 0.03 (≥0.029, acceptable); C17: 1n7 0.02 (≥0.018, acceptable); C20: 1 0.10 (≤0.103, acceptable); C20: 2 0.17 (≤0.177, acceptable); C20: 3n6 0.28 (requirement ≤0.258, 0.28 > 0.258, exceeding the standard); C22: 1n9 0.02 (≤0.02, acceptable); C20: 4n6 (ARA) 0.14 (requirement ≤0.133, 0.14 > 0.133, exceeding the standard); C20: 5n3 (EPA) 0.11 (≤0.115, qualified); C24:1n9 0.03 (requirement ≤0.027, 0.03>0.027, exceeding the standard); C22:6n3(DHA) 0.75 (≤0.864, qualified).

[0056] Based on comprehensive assessment: C20:3n6, C20:4n6(ARA), and C24:1n9 exceeded the standards, while the rest were within acceptable limits; therefore, these were determined to be substandard eggs. The actual hatching rate was 6.68%, and the fertilization rate was 50.74%.

[0057] Group 3: Total protein concentration 9.88 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.39 ng / mgprot (≥0.348, acceptable); Vitellin 0.05 ng / mL (≥0.045, acceptable). Fatty acid indicators: C15: 0 0.02 (≥0.02, acceptable); C16: 1n7 0.23 (>0.208, exceeding the standard); C17: 0 0.03 (≥0.029, acceptable); C17: 1n7 0.02 (≥0.018, acceptable); C20: 1 0.10 (≤0.103, acceptable); C20: 2 0.17 (≤0.177, acceptable); C20: 3n6 0.25 (≤0.258, acceptable); C22: 1n9 0.02 (≤0.02, acceptable); C20: 4n6 (ARA) 0.14 (>0.133, exceeding the standard); C20: 5n3 (EPA) 0.11 (≤0.115, acceptable); C24: 1n9 0.03 (>0.027, exceeding the standard); C22:6n3 (DHA) 0.82 (≤0.864, qualified).

[0058] Overall assessment: Total protein concentration, C16:1n7, C20:4n6 (ARA), and C24:1n9 exceeded standards, indicating poor egg quality. Actual hatching rate was 28.55%, and fertilization rate was 21.34%.

[0059] Group 4: Total protein concentration 13.30 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.31 ng / mgprot (<0.348, exceeding the standard); Vitellin 0.03 ng / mL (<0.045, exceeding the standard). Fatty acid indicators: C15: 0 0.02 (pass); C16: 1n7 0.23 (exceeds standard); C17: 0 0.03 (pass); C17: 1n7 0.02 (≥0.02, pass); C20: 1 0.14 (>0.103, exceeds standard); C20: 2 0.20 (>0.177, exceeds standard); C20: 3n6 0.31 (>0.258, exceeds standard); C22: 1n9 0.03 (>0.02, exceeds standard); C20: 4n6 (ARA) 0.18 (>0.133, exceeds standard); C20: 5n3 (EPA) 0.15 (>0.115, exceeds standard); C24: 1n9 0.03 (>0.027, exceeds standard); C22: 6n3 (DHA) 0.92 (>0.864, exceeding the standard).

[0060] Overall assessment: Almost all indicators exceeded the standard, indicating poor-quality eggs. Actual hatching rate: 0%, fertilization rate: 0%.

[0061] Group 5: Total protein concentration 8.77 mg / mL (≤9.83, qualified); Vitamin A concentration 0.41 ng / mgprot (≥0.348, qualified); Vitellin 0.05 ng / mL (≥0.045, qualified). Fatty acid indicators: C15: 0 0.02 (≥0.02, qualified); C16: 1n7 0.19 (≤0.208, qualified); C17: 0 0.03 (≥0.029, qualified); C17: 1n7 0.02 (≥0.018, qualified); C20: 1 0.09 (≤0.103, qualified); C20: 2 0.16 (≤0.177, qualified); C20: 3n6 0.20 (≤0.258, qualified); C22: 1n9 0.02 (≤0.02, qualified); ARA 0.12 (≤0.133, qualified); C20: 5n3 (EPA) 0.11 (≤0.115, qualified); C24: 1n9 0.02 (≤0.027, qualified); C22: 6n3 (DHA) 0.85 (≤0.864, qualified).

[0062] Overall assessment: All indicators meet the thresholds in Table 1, indicating the eggs are of high quality. Actual hatching rate: 27.55%, fertilization rate: 65.28%.

[0063] Group 6: Total protein concentration 10.14 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.42 ng / mgprot (≥0.348, acceptable); Vitellin 0.06 ng / mL (≥0.045, acceptable). Fatty acid indicators: C15: 0 0.02 (≥0.02, acceptable); C16: 1n7 0.19 (≤0.208, acceptable); C17: 0 0.03 (≥0.029, acceptable); C17: 1n7 0.02 (≥0.018, acceptable); C20: 1 0.10 (≤0.103, acceptable); C20: 2 0.18 (requirement ≤0.177, 0.18>0.177, exceeding the limit); C20: 3n6 0.23 (≤0.258, acceptable); C22: 1n9 0.02 (≤0.02, acceptable); C20: 4n6 (ARA) 0.13 (≤0.133, acceptable); C20: 5n3 (EPA) 0.12 (Requirement ≤ 0.115, 0.12 > 0.115, exceeding the standard); C24:1n9 0.03 (> 0.027, exceeding the standard); C22:6n3 (DHA) 0.87 (≤ 0.864? 0.87 > 0.864, exceeding the standard).

[0064] Overall assessment: Egg quality is poor due to excessive levels of protein, C20:2, C20:5n3 (EPA), C24:1n9, and C22:6n3 (DHA). Actual hatching rate was 3.91%, and fertilization rate was 14.93%.

[0065] Group 7: Total protein concentration 10.48 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.40 ng / mgprot (≥0.348, acceptable); Vitellin 0.05 ng / mL (≥0.045, acceptable). Fatty acid indicators: C15: 0 0.02 (≥0.02, qualified); C16: 1n7 0.20 (≤0.208, qualified); C17: 0 0.03 (≥0.029, qualified); C17: 1n7 0.02 (≥0.018, qualified); C20: 1 0.11 (requirement ≤0.103, 0.11>0.103, exceeding the standard); C20: 2 0.20 (>0.177, exceeding the standard); C20: 3n6 0.24 (≤0.258, qualified); C22: 1n9 0.02 (≤0.02, qualified); C20: 4n6 (ARA) 0.14 (>0.133, exceeding the standard); C20: 5n3 (EPA) 0.12 (>0.115, exceeding the standard); C24: 1n9 0.03 (>0.027, exceeding the standard); C22:6n3 (DHA) 0.89 (requirement ≤0.864, 0.89>0.864, exceeding the standard).

[0066] Overall assessment: Total protein concentration, C20:1, C20:2, C20:4n6 (ARA), C20:5n3 (EPA), C24:1n9, and C22:6n3 (DHA) all exceeded standards, indicating poor egg quality. Actual hatching rate was 19.37%, and fertilization rate was 18.20%.

[0067] Group 8: Total protein concentration 11.13 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.39 ng / mgprot (≥0.348, acceptable); Vitellin 0.06 ng / mL (≥0.045, acceptable). Fatty acid indicators: C15: 0 0.02 (≥0.02, acceptable); C16: 1n7 0.25 (>0.208, exceeding the limit); C17: 0 0.03 (≥0.029, acceptable); C17: 1n7 0.01 (<0.018, unacceptable); C20: 1 0.12 (>0.103, exceeding the limit); C20: 2 0.19 (>0.177, exceeding the limit); C20: 3n6 0.35 (>0.258, exceeding the limit); C22: 1n9 0.02 (≤0.02, acceptable); C20: 4n6 (ARA) 0.16 (>0.133, exceeding the limit); C20: 5n3 (EPA) 0.13 (>0.115, exceeding the limit); C24: 1n9 0.03 (>0.027, exceeding the standard); C22:6n3 (DHA) 0.93 (>0.864, exceeding the standard).

[0068] Overall assessment: Multiple indicators exceeded standards, indicating poor egg quality. Actual hatching rate: 0%, fertilization rate: 0%.

[0069] Group 9: Total protein concentration 11.41 mg / mL (>9.83, exceeding the standard); Vitamin A concentration 0.34 ng / mgprot (<0.348, unqualified); Vitellin 0.04 ng / mL (<0.045, unqualified). Fatty acid indicators: C15: 0 0.01 (<0.02, unqualified); C16: 1n7 0.21 (>0.208, exceeding the standard); C17: 0 0.02 (<0.029, unqualified); C17: 1n7 0.01 (<0.018, unqualified); C20: 1 0.13 (>0.103, exceeding the standard); C20: 2 0.21 (>0.177, exceeding the standard); C20: 3n6 0.26 (>0.258, exceeding the standard); C22: 1n9 0.02 (≤0.02, qualified); C20: 4n6 (ARA) 0.13 (≤0.133, qualified); C20: 5n3 (EPA) 0.13 (>0.115, exceeding the standard); C24: 1n9 0.03 (>0.027, exceeding the standard); C22:6n3 (DHA) 0.88 (>0.864, exceeding the standard).

[0070] Overall assessment: The vast majority of indicators are substandard, indicating poor-quality eggs. Actual hatching rate: 0%; fertilization rate: 0%.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for establishing a quality evaluation standard for the eggs of the large-finned mandarin fish, characterized in that, Includes the following steps: S1: Collect unfertilized eggs of the bigfin bream. Take 13-18g of the unfertilized eggs from each parent fish for biochemical index testing, and use the remaining unfertilized eggs for reproductive performance verification. S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of various fatty acids in unfertilized eggs used for biochemical index detection to obtain biochemical index data; S3: Artificial insemination was performed on the unfertilized eggs used for reproductive performance verification, and the fertilization rate, hatching rate and hatching rate were statistically analyzed to obtain reproductive performance data; S4: Correlation analysis was performed on the biochemical index data of each unfertilized egg sample obtained in S2 and the reproductive performance data of each sample obtained in S3 to screen the core evaluation indicators of the quality of the largefin wrasse eggs and establish the discrimination criteria.

2. The method for establishing according to claim 1, characterized in that, Step S4 includes: S41: Spearman correlation analysis was performed on the relative contents of various fatty acids and the hatching rate. The fatty acid types that were significantly correlated with the hatching rate were screened out with |R|>0.4 and P<0.05 as the criteria. Where R is the correlation coefficient, and its value ranges from -1 to 1; P represents the significance level, and a smaller P value indicates a more reliable result. S42: Principal component analysis was performed on the selected fatty acids, and the core fatty acid index was determined based on the loading diagram. The core fatty acid index, together with the total protein concentration, vitamin A concentration, and vitellin concentration, constituted the core evaluation index of the quality of the oocytes of the largefin fish. S43: Using the core evaluation indicators of the quality of the eggs of the giant finned mandarin fish as independent variables and the hatching rate as the dependent variable, a multiple linear regression model was established; S44: Diagnose and validate the multiple linear regression model; S45: With a hatching rate of ≥20% and a fertilization rate of ≥50% as the target for high-quality eggs, the thresholds of each core evaluation index of the quality of the largefin loach eggs were determined by ROC curve; S46: The threshold values ​​of each core evaluation indicator of the quality of the largefin wrasse eggs were cross-validated using the leave-one-out method.

3. The method for establishing according to claim 2, characterized in that, The core fatty acid indicators determined in step S42 are the following 12: C15:0, C16:1n7, C17:0, C17:1n7, C20:1, C20:2, C20:3n6, C22:1n9, C20:4n6 (ARA), C20:5n3 (EPA), C24:1n9, and C22:6n3 (DHA).

4. The method for establishing according to claim 2, characterized in that, The threshold values ​​for each core evaluation indicator of the quality of *Ceratos maculatus* eggs determined in step S45 are as follows: Total protein concentration ≤9.83mg / mL, vitamin A concentration ≥0.348ng / mgprot, vitellin concentration ≥0.045ng / mL; Fatty acid thresholds: C15:0≥0.02, C17:0≥0.029, C20:1≤0.103, C16:1n7≤0.208, C17:1n7≥0.018, C22:1n9≤0.02, C24:1n9≤0.027, C20:2≤0.177, C20:3n6≤0.258, C20:4n6(ARA)≤0.133, C20:5n3(EPA)≤0.115, C22:6n3(DHA)≤0.

864.

5. The method for establishing according to claim 1, characterized in that, In step S3, fertilization rate % = number of normally developing eggs / total number of eggs × 100%, hatching rate % = number of hatched larvae / total number of fertilized eggs × 100%, hatching rate % = number of hatched larvae / total number of eggs × 100%; among them, normally developing eggs are yellowish-brown and opaque, and dead eggs are pure white and opaque.

6. The quality evaluation index system for *Amur cormorant* eggs established according to any one of claims 1 to 5, characterized in that, include: The appearance characteristics are round, full, and elastic, with an egg diameter ≥2.5mm and the core evaluation indicators of the quality of the largefin loach eggs determined by the establishment method according to any one of claims 1 to 5.

7. A method for assessing the quality of eggs from the large-finned mandarin fish, characterized in that, The application of the evaluation index system described in claim 6 includes the following steps: S1: Collect unfertilized eggs of the largefin wrasse to be evaluated; S2: Determine the total protein concentration, vitamin A concentration, vitellin concentration, and relative content of core fatty acids in unfertilized eggs; S3: Compare the biochemical data obtained in step S2 with the aforementioned quality evaluation index system for *Ceratos maculatus* eggs. If all the biochemical data of the *Ceratos maculatus* eggs are within the threshold range of the aforementioned quality evaluation index system, they are judged as high-quality eggs; otherwise, they are judged as low-quality eggs.

8. The evaluation method according to claim 7, characterized in that, The method also includes step S4: verifying the accuracy of the method by fertilization rate, hatching rate and hatching rate, that is, artificial insemination and hatching are performed on batches of eggs that are judged to be of high quality. If the actual fertilization rate and hatching rate are higher than those of batches of eggs that are judged to be of low quality, it proves that the judgment result of the method is accurate.

9. The evaluation method according to claim 7, characterized in that, In step S2, the total protein concentration, vitamin A concentration and vitellin concentration were determined by microplate method, and the relative content of core fatty acids was determined by gas chromatography.

10. The evaluation method according to claim 9, characterized in that, The chromatographic conditions for the gas chromatography method are as follows: Agilent CP-81188 column with dimensions of 100m × 250μm × 0.20μm, hydrogen as carrier gas, constant flow rate of 20mL / min, and split ratio of 100:

1. Temperature program: Start at 100℃ and hold for 13 min, increase to 180℃ at 10℃ / min and hold for 6 min, increase to 200℃ at 1℃ / min and hold for 20 min, increase to 230℃ at 4℃ / min and hold for 15 min; The injection port temperature is 270℃, and the injection volume is 1μL.

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