A method for predicting the spawning period of apostichopus japonicus

Abstract

This invention discloses a method for predicting the spawning period of *Stichopus japonicus*, belonging to the field of *Stichopus japonicus* aquaculture data prediction. This invention analyzes the correlation between body weight, accumulated temperature, and changes in total saponin content in the gonads of female *Stichopus japonicus* during a period before and after spawning, constructs a model of the relationship between changes in total saponin content in the gonads and the timing of egg release, and establishes a method for accurately predicting the spawning timing of *Stichopus japonicus*. Using this prediction method, the spawning timing of *Stichopus japonicus* can be accurately determined, solving the problem of not being able to accurately predict the spawning timing for timely harvesting. Producers can then plan the water body used for seedling cultivation in their workshops.

Description

Technical Field

[0001] This invention relates to the field of data prediction in sea cucumber farming, and in particular to a method for predicting the spawning period of sea cucumbers. Background Technology

[0002] *Aposticchopus japonicus*, belonging to the phylum Echinodermata and class Holothuroidea, is an echinoderm with significant nutritional and physiological value and is currently an important aquaculture species. Aquaculture methods include factory farming, cage culture, pond culture, and bottom seeding. A large number of artificially bred seedlings are needed annually for production, and the maturity of the gonads of the parent sea cucumbers plays a crucial role in the breeding process. Parent sea cucumbers are typically harvested in spring. Current methods for determining the harvesting time generally involve technicians dissecting the parent sea cucumbers and observing the diameter of the gonadal ducts, or using a microscope to observe the state of the oocytes. Harvesting is permitted when the gonadal duct diameter reaches 1 mm or more, or when the oocytes appear free under a microscope.

[0003] In their daily practice, the inventors discovered the following problems with existing technical solutions:

[0004] The reproductive release of sea cucumbers differs from that of most mollusks. They are not sensitive to external stimuli such as dew, flowing water, and temperature increases. If the gonads do not reach the maturity required for natural release, it is difficult to collect eggs through artificial stimulation. Current methods for determining the spawning period of sea cucumbers through dissection and observation are inaccurate. After being harvested and placed in the production facility, spawning often takes 3-7 days, and sometimes up to half a month, before large-scale spawning occurs. Harvesting broodstock too early can have two adverse effects: first, it creates gaps in the production schedule, leaving the seedling-raising water idle; second, broodstock require temporary rearing and maturation under artificial conditions. Compared to the natural development of gonads, the gonads mature less synchronously, resulting in fewer eggs laid at once, which is detrimental to large-scale production.

[0005] In view of this, it is necessary to provide a new technical solution to solve the above problems. Summary of the Invention

[0006] To address the aforementioned technical problems, this application provides a method for predicting the spawning period of sea cucumber, which can accurately predict the spawning timing of sea cucumber and thus rationally arrange production time, facilitating large-scale production.

[0007] A method for predicting the spawning period of sea cucumber (Stichopus japonicus), comprising:

[0008] After the water temperature reaches the sampling threshold, female parent sea cucumbers of suitable weight are selected as samples, and continuous sampling is carried out on female parent sea cucumbers before spawning.

[0009] The collected ginseng was weighed and dissected. The total saponin content in the gonads of female ginseng in the sampled individuals was detected to determine the threshold of total saponin content at the time of ginseng spawning, and the accumulated temperature at the time of ginseng spawning was calculated.

[0010] The correlation between total saponin content in female ginseng and accumulated temperature and body weight was analyzed.

[0011] A model for the timing of ovum release in parent sea cucumbers was constructed to predict the ovum-laying timing of *Stichopus japonicus*.

[0012] Preferably, in the construction of the parent ginseng oocyte release timing model, the calculation formula for the parent ginseng oocyte release timing model is as follows:

[0013] ;

[0014] In the formula, This refers to the number of days required for the female ginseng to lay eggs; The accumulated temperature required for female ginseng to lay eggs; The average daily water temperature during the harvesting period in the sea area; Accumulated temperature starting water temperature.

[0015] Preferably, the formula for calculating the accumulated temperature required for the female parent ginseng to lay eggs is as follows:

[0016] ;

[0017] In the formula, is the accumulated temperature constant, which is the accumulated temperature during ovulation by the female ginseng parent. This is for accumulated temperature sampling.

[0018] Preferably, the analysis of the correlation between the total saponin content of female ginseng and accumulated temperature and body weight includes:

[0019] Analyze the correlation between total saponin content in female ginseng and body weight;

[0020] Analyze the correlation between total saponin content in female ginseng and accumulated temperature;

[0021] Establish a regression model for the total saponin content of female ginseng parents;

[0022] Analysis revealed that there was no direct correlation between the total saponin content of female ginseng and body weight.

[0023] Preferably, the regression model for establishing the total saponin content of female ginseng includes:

[0024] The total saponin content of female ginseng was used as the dependent variable, and accumulated temperature was used as the independent variable. Based on the sample data, the functional relationship between the variables was analyzed.

[0025] The functional equation relationship between the total saponin content of female ginseng and accumulated temperature was derived through regression analysis.

[0026] The linear regression equation for the relationship between the total saponin content of female ginseng and accumulated temperature is as follows:

[0027] ;

[0028] In the formula, The total saponin content of female ginseng in the sample; This is for accumulated temperature sampling.

[0029] Preferably, the formula for calculating the accumulated temperature is:

[0030] ;

[0031] In the formula: The daily water temperature during the harvesting period in the sea area; The initial water temperature for the harvesting area.

[0032] Preferably, the method for detecting the total saponin content of the female ginseng is as follows:

[0033] The standard solution was measured, and a standard curve was plotted.

[0034] The test solution was measured, and the saponin mass of the sample solution was calculated based on the standard curve.

[0035] Preferably, the determination of the standard solution includes: taking 1 mL of ginsenoside Re standard solution (2.0 mg / mL), accurately adding 0.2 mL of 5% vanillin glacial acetic acid solution, then adding 0.8 mL of perchloric acid, mixing well, transferring to a 5 mL stoppered graduated glass tube, heating in a 60°C water bath for 10 min, and removing; after cooling in an ice bath, accurately adding 50 mL of glacial acetic acid, and shaking well; measuring the absorbance at a wavelength of 560 nm using a 1 cm cuvette; wherein, the ginsenoside Re standard solution is 2.0 mg / mL.

[0036] Preferably, the determination of the test solution includes: weighing an appropriate amount of the well-mixed sample into a 100mL volumetric flask, adding a small amount of water, sonicating for 30min, then diluting to 100mL with water, shaking well, letting stand, filtering, taking 1mL of the filtrate, accurately adding 0.2mL of 5% vanillin-glacial acetic acid solution, then adding 0.8mL of perchloric acid, mixing well, transferring to a 5mL graduated glass tube with a stopper, heating in a 60℃ water bath for 10min, removing, cooling in an ice bath, accurately adding 50mL of glacial acetic acid, shaking well, and measuring its absorbance at a wavelength of 560nm using a 1cm cuvette.

[0037] Preferably, the formula for calculating the saponin mass of the sample solution based on the standard curve is as follows:

[0038] ;

[0039] In the formula, The total saponin content of female ginseng in the sample; The mass of the sample;

[0040] The mass of saponins in the sample solution calculated on the standard curve; This represents the extraction volume of the sample.

[0041] This refers to the volume of the sample taken during the measurement.

[0042] Compared with the prior art, this application has at least the following beneficial effects:

[0043] 1. This invention continuously samples the female gonads of *Stichopus japonicus* before spawning, and simultaneously dissects and detects the total saponin content, finds the accumulated temperature and saponin threshold, constructs a model of the relationship between the total saponin content and accumulated temperature of the gonads, and establishes a method for accurately predicting the spawning timing of *Stichopus japonicus*, so as to achieve the purpose of rationally arranging production time.

[0044] 2. This invention can accurately predict the spawning time of sea cucumbers and thus rationally arrange production time. Producers can plan the water body for seedling cultivation in the workshop. The spawning synchronization rate of the captured parent sea cucumbers is high, and the batch egg harvest is concentrated, which meets the needs of large-scale production. Attached Figure Description

[0045] The following sections will describe some specific embodiments of the invention in detail by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings:

[0046] Figure 1 This is a schematic diagram of the overall process of the present invention;

[0047] Figure 2 This is a scatter plot of the total saponin content and fresh weight of female ginseng in this invention.

[0048] Figure 3 This is a graph showing the relationship between the total saponin content of female ginseng and the accumulated temperature of sampling in this invention.

[0049] Figure 4 This is a line graph showing the distribution of total saponin content in female ginseng and the accumulated temperature during sampling in this invention. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0051] The inventors discovered through research that, in natural environments, the maturity of the gonads of *Stichopus japonicus* is mainly related to water temperature variations in the sea area where it grows, individual weight, and feeding conditions. However, in aquaculture environments, *Stichopus japonicus* are generally in a state of abundant food. Under these conditions, water temperature variations and individual weight play the main roles in gonad development. Generally, the higher the accumulated temperature and the larger the individual, the faster the gonads mature.

[0052] This application analyzes the relationship between total saponin content in the gonads of female sea cucumbers before and after spawning by detecting changes in total saponin content in the gonads, body weight, accumulated temperature, and changes in total saponin content in the gonads. A model is constructed to show the relationship between changes in total saponin content in the gonads of sea cucumbers and the timing of egg release. A method for accurately predicting the spawning timing of sea cucumbers is also established, thereby eliminating the large errors in existing methods for predicting the spawning timing of parent sea cucumbers and solving the problem of not being able to accurately predict the spawning timing of sea cucumbers for timely harvesting.

[0053] like Figure 1 As shown, a method for predicting the spawning period of sea cucumber (Stichopus japonicus) includes the following steps:

[0054] Step S1: After the water temperature reaches the sampling threshold, select female parent sea cucumbers of suitable weight as samples and continuously sample the female parent sea cucumbers before spawning.

[0055] Step S2: Weigh and dissect the collected ginseng, detect the total saponin content in the gonads of female ginseng in the sampled individuals, determine the threshold of total saponin content at the time of ginseng spawning, and calculate the accumulated temperature at the time of ginseng spawning.

[0056] Step S3: Analyze the correlation between the total saponin content of female ginseng and accumulated temperature and body weight;

[0057] Step S4: Construct a model for the timing of parent sea cucumber oocyte release to predict the oocyte release timing of sea cucumber.

[0058] Specifically, the natural reproductive activity of sea cucumbers generally begins to increase when the water temperature is above 15℃, and the selection criteria for parent sea cucumbers generally require a weight of 150g or more per head. In this embodiment, 15℃ is used as the sampling threshold. Starting from a water temperature of 15℃, sea cucumbers that have reached the parent sea cucumber size are randomly collected every few days until samples are collected from parent sea cucumbers that are currently laying eggs or have just finished laying eggs. The sampling interval is determined based on the development of the gonads of the sea cucumbers. If the gonad development activity is low, the number of days for sampling can be appropriately increased; if the gonad development activity is high, the number of days for sampling can be appropriately decreased, or even continuous sampling can be used every day.

[0059] When measuring the weight, the live sea cucumbers were weighed using an electronic balance with a sensitivity of 0.01g after the surface moisture was absorbed with absorbent paper.

[0060] The total saponin content of female ginseng was determined by separating the female gonads from the internal organs after dissection of each ginseng.

[0061] The detection instruments for the total saponin content of female ginseng included an electronic balance, a centrifuge, and a UV-Vis spectrophotometer. Reagents and standards included vanillin, perchloric acid, glacial acetic acid, and ginsenoside Re.

[0062] The determination of the standard solution includes: weighing an appropriate amount of the well-mixed sample into a 100 mL volumetric flask, adding a small amount of water, sonicating for 30 min, then diluting to 100 mL with water, shaking well, allowing to stand, filtering, and accurately adding 0.2 mL of 5% vanillin-glacial acetic acid solution to 1 mL of the filtrate, followed by 0.8 mL of perchloric acid. After mixing well, transfer the solution to a 5 mL graduated glass tube with a stopper, heat in a 60 °C water bath for 10 min, remove, cool in an ice bath, accurately add 50 mL of glacial acetic acid, shake well, and measure the absorbance at 560 nm using a 1 cm cuvette. A standard curve is plotted based on the measurement results.

[0063] The determination of the standard solution involved: accurately adding 1 mL of ginsenoside Re standard solution (2.0 mg / mL) to 0.2 mL of 5% vanillin-glacial acetic acid solution, followed by 0.8 mL of perchloric acid. After mixing, the solution was transferred to a 5 mL graduated glass tube with a stopper. The tube was heated in a 60°C water bath for 10 min, then removed and cooled in an ice bath. 50 mL of glacial acetic acid was then accurately added, and the solution was shaken well. The absorbance was measured at 560 nm using a 1 cm cuvette. The mass of saponins in the sample solution was calculated based on the standard curve.

[0064] The formula for calculating the saponin mass of the sample solution based on the standard curve is as follows:

[0065] ;

[0066] In the formula, The total saponin content of female ginseng in the sample; The mass of the sample;

[0067] The mass of saponins in the sample solution calculated on the standard curve; This represents the extraction volume of the sample.

[0068] This refers to the volume of the sample taken during the measurement.

[0069] Accumulated temperature is calculated by subtracting 15°C from the daily water temperature in the sea area where adult sea cucumbers are harvested, starting from 15°C, where the gonads of the sea cucumber are most active. The formula for calculating accumulated temperature is as follows:

[0070] ;

[0071] In the formula: For accumulated temperature sampling; The daily water temperature during the harvesting period in the sea area; The initial water temperature for the harvesting area is 15℃.

[0072] Tests showed that the accumulated temperature during ovulation in ginseng was 40.5℃, therefore the accumulated temperature constant was 40.5℃. At this time, the average total saponin content of the female gonads was 10.23 g / 100g, which was the emission threshold. After emission, the total saponin content of the gonads dropped sharply to 1.45 g / 100g. (See attached image) Figure 3 Appendix Figure 4 As shown.

[0073] Import the sample data into SPSS 26.0 software and use its Regression analysis function for calculation.

[0074] Analysis revealed that the sampled ginseng weighed between 150g and 350g. Within this weight range, no direct correlation was found between the total saponin content and weight of the female ginseng. (See attached image) Figure 2 As shown.

[0075] Using the total saponin content in female gonads as the dependent variable Y and accumulated temperature as the independent variable K, the functional relationship between the variables was analyzed based on the sample data. SPSS software was used to perform regression analysis to derive the functional equation relationship between the total saponin content Y and the accumulated temperature K, and the analysis results are shown in Tables 1-3.

[0076] Table 1. Results of ANOVA for the Regression Model

[0077]

[0078] Table 2. Results of regression model predictor variable analysis.

[0079]

[0080] Table 3. Results of Dependent Variable Analysis in the Regression Model

[0081]

[0082] From these results, the linear regression equation relating the total saponin content of female ginseng to accumulated temperature is as follows:

[0083] ;

[0084] In the formula, The total saponin content of female ginseng in the sample; The accumulated temperature is used for sampling; the correlation coefficient of the regression equation is R=0.975, which represents the percentage of the total change in the dependent variable that can be explained by the change in the independent variable. Coefficient of determination. The equation was tested, and the significance level was 0.000, which is much less than 0.01, indicating that the obtained equation is statistically significant.

[0085] From the above, the model for the timing of ovulation in parent ginseng can be derived as follows:

[0086] ;

[0087] In the formula, This refers to the number of days required for the female ginseng to lay eggs; The accumulated temperature required for female ginseng to lay eggs; The average daily water temperature during the harvesting period in the sea area; Accumulated temperature starting water temperature.

[0088] The formula for calculating the accumulated temperature required for female parent ginseng to lay eggs is as follows:

[0089] ;

[0090] In the formula, is the accumulated temperature constant, which is the accumulated temperature during ovulation by the female ginseng parent. This is for accumulated temperature sampling.

[0091] Application example:

[0092] The method for predicting the spawning period of the sea cucumber according to the present invention is applied as follows:

[0093] When the water temperature is above 16℃, sea cucumbers are harvested from the sea area to be used as parent sea cucumbers for seedling breeding. Samples are taken and dissected, and the female gonads are separated from the internal organs. The total saponin content is tested and found to be 6.75g / 100g.

[0094] Substitute the saponin content data detected in female gonads into the following formula:

[0095] ;

[0096] In the formula, The accumulated temperature constant is 40.5℃; The total saponin content of the sample's gonads was 6.75 g / 100 g. The average daily water temperature during the harvesting period in the sea area; The initial water temperature for accumulated temperature is 15℃.

[0097] If we estimate based on the average daily water temperature of 18.5℃ in this sea area during the harvesting season over the years, and substitute it into the above formula for calculation:

[0098] ;

[0099] Calculations show that... The calculated value is approximately 9, so the spawning time in the sea area of ​​the sea cucumber is about the 9th day after sampling, and the large-scale harvesting of parent sea cucumbers can be arranged to begin on the 7th day.

[0100] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0101] It should be noted that the terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in sequences other than those illustrated or described herein.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for predicting the spawning period of sea cucumber, characterized in that, include: After the water temperature reaches the sampling threshold, female parent sea cucumbers of suitable weight are selected as samples, and continuous sampling is carried out on female parent sea cucumbers before spawning. The collected ginseng was weighed and dissected. The total saponin content in the gonads of female ginseng in the sampled individuals was detected to determine the threshold of total saponin content at the time of ginseng spawning, and the accumulated temperature at the time of ginseng spawning was calculated. The correlation between total saponin content in female ginseng and accumulated temperature and body weight was analyzed. A model for the timing of oocyte release in parent sea cucumbers was constructed to predict the oviposition timing of sea cucumbers. The calculation formula for the parent ginseng oocyte release timing model is as follows: ； In the formula, This refers to the number of days required for the female ginseng to lay eggs; The accumulated temperature required for female ginseng to lay eggs; The average daily water temperature during the harvesting period in the sea area; Initial water temperature in the harvesting area; The formula for calculating the accumulated temperature required for female parent ginseng to lay eggs is as follows: ； In the formula, The accumulated temperature constant, The accumulated temperature required for female ginseng to lay eggs. For accumulated temperature sampling; The regression model for the total saponin content of female ginseng includes: The total saponin content of female ginseng was used as the dependent variable, and accumulated temperature was used as the independent variable. Based on the sample data, the functional relationship between the variables was analyzed. The functional equation relationship between the total saponin content of female ginseng and accumulated temperature was derived through regression analysis. The linear regression equation for the relationship between the total saponin content of female ginseng and accumulated temperature is as follows: ； In the formula, The total saponin content of female ginseng in the sample; For accumulated temperature sampling; The formula for calculating the accumulated temperature of the sample is: ； In the formula: The daily water temperature during the harvesting period in the sea area; The initial water temperature for the harvesting area.

2. The method for predicting the spawning period of the sea cucumber as described in claim 1, characterized in that, The analysis of the correlation between total saponin content in female ginseng and accumulated temperature and body weight included: Analyze the correlation between total saponin content in female ginseng and body weight; Analyze the correlation between total saponin content in female ginseng and accumulated temperature; Establish a regression model for the total saponin content of female ginseng parents; Analysis revealed that there was no direct correlation between the total saponin content of female ginseng and body weight.

3. The method for predicting the spawning period of the sea cucumber as described in claim 1, characterized in that, The method for detecting the total saponin content of the female ginseng is as follows: The standard solution was measured, and a standard curve was plotted. The test solution was measured, and the saponin mass of the sample solution was calculated based on the standard curve.

4. The method for predicting the spawning period of the sea cucumber as described in claim 3, characterized in that, The determination of the standard solution includes: taking 1 mL of ginsenoside Re standard solution with a concentration of 2.0 mg / mL, accurately adding 0.2 mL of 5% vanillin glacial acetic acid solution, then adding 0.8 mL of perchloric acid, mixing well, transferring to a 5 mL graduated glass tube with a stopper, heating in a 60°C water bath for 10 min, and removing; after cooling in an ice bath, accurately adding 50 mL of glacial acetic acid, and shaking well; measuring the absorbance at a wavelength of 560 nm using a 1 cm cuvette; wherein, the concentration of the ginsenoside Re standard solution is 2.0 mg / mL.

5. The method for predicting the spawning period of the sea cucumber as described in claim 4, characterized in that, The determination of the test solution includes: weighing an appropriate amount of the well-mixed sample into a 100mL volumetric flask, adding a small amount of water, sonicating for 30 minutes, then diluting to 100mL with water, shaking well, letting stand, filtering, taking 1mL of the filtrate, accurately adding 0.2mL of 5% vanillin-glacial acetic acid solution, then adding 0.8mL of perchloric acid, mixing well, transferring to a 5mL graduated glass tube with a stopper, heating in a 60℃ water bath for 10 minutes, removing, cooling in an ice bath, accurately adding 50mL of glacial acetic acid, shaking well, and measuring its absorbance at a wavelength of 560nm using a 1cm cuvette.

6. The method for predicting the spawning period of the sea cucumber as described in claim 5, characterized in that, The formula for calculating the saponin mass of the sample solution based on the standard curve is as follows: ； In the formula, The total saponin content of female ginseng in the sample; The mass of the sample; This represents the saponin mass of the sample solution calculated from the standard curve. This represents the extraction volume of the sample. This refers to the volume of the sample taken during the measurement.

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