A method for detecting the number of development stages of ovary of eriocheir sinensis

Abstract

A non-injury detection method for the ovary development stage of Scylla paramamosain, comprising the following steps: obtaining a fitting curve equation with the ovary development stage number as the variable and the cross-sectional area ratio of ovary to carapace as the dependent variable; screening individuals to be detected that meet the conditions; performing anesthesia pretreatment; placing the individual to be detected under a light source to irradiate it so that the shadow profile of the ovary appears on the carapace; obtaining an image including the carapace under the irradiation of the light source; obtaining the area value of the shadow profile of the ovary on the carapace and the image area value of the carapace in the image respectively, and calculating the ratio of the two; substituting the ratio as the dependent variable into the fitting curve equation to obtain the variable corresponding to the dependent variable, and rounding the variable to an integer to obtain the ovary development stage number of the female Scylla paramamosain individual. The present application can avoid causing body damage to the detected Scylla paramamosain, and ensure that the detected Scylla paramamosain continues to be used for production and sales and fry breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of medical instruments, and in particular relates to a method for detecting the number of developmental stages of the ovary of mud crabs. Background Art

[0002] The mud crab (Scylla paramamosain) is an important marine fishery resource and marine aquaculture crab. Its large size, rapid growth, nutritious texture, and delicious meat make it a popular choice. Once mature, the ovaries of the mud crab are rich in protein and fat and are edible tissue. The ovarian development and maturation process of the mud crab is divided into five main stages: Stages I, II, III, IV, and V. Stage V is the period of ovarian maturation, and the ovaries of the mud crab exhibit significantly different changes during these five developmental stages. In production practice, female crabs with well-developed gonads not only have high commercial value but also serve as a criterion for selecting parents for seedling breeding. Therefore, determining the ovarian development status of the mud crab is of great significance for accurately assessing the commercial value of female crabs for cultivation and screening breeding parents. Existing methods for detecting the stage of ovarian development in the mud crab involve dissecting the crab and observing the external morphology and tissue morphology of the ovaries in sections. This requires killing the crab and dissecting its ovarian tissue during the detection process. Although this detection method is relatively accurate, it requires killing the selected mud crabs. Therefore, it can only be used for sampling detection and cannot be widely popularized to every female mud crab individual in practice. Patent application number 200910054769.0 discloses a method for examining the ovarian development of crabs using a live puncture technique, in which a small amount of ovarian tissue is removed from the living crab to examine the development of the ovaries. However, this method causes trauma to the crab's ovaries during the puncture process, resulting in the crabs after the test being no longer suitable for the parent screening link for seed breeding. Therefore, how to develop a new identification method for the ovarian development period of mud crabs to overcome the above-mentioned defects in the prior art is a direction that those skilled in the art need to study. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for detecting the number of ovarian development stages of mud crabs, which can avoid causing body damage to the mud crabs being detected when performing the ovarian development stage detection, and ensure that the mud crabs being detected can continue to be used for production, sales and seed breeding.

[0004] The technical solution provided by the present invention is a method for detecting the number of ovarian development stages of mud crabs.

[0005] It includes the following steps:

[0006] Step 100: Obtaining a fitting curve equation with the number of ovarian development stages b of the female mud crab individual as a variable and the ratio of the ovarian cross-sectional area to the cephalothorax cross-sectional area of ​​the female mud crab individual a as a dependent variable;

[0007] Step 200: Screening out female mud crabs to be tested whose ovaries have entered a rapid development stage;

[0008] Step 300: performing detection pre-processing on the screened female mud crab individuals;

[0009] Step 400: placing the female mud crab that has completed the detection pre-treatment under a light source to illuminate the female mud crab so that the shadow of the ovary is projected on the cephalothorax of the female mud crab;

[0010] Step 500: Acquire an image of the cephalothorax of the female mud crab under illumination by a light source;

[0011] Step 600: obtaining the image area value CA of the cephalothorax and the area value SA of the ovarian shadow on the image, and calculating the image area ratio AR=SA / CA;

[0012] Step 700: Substitute the image area ratio AR as the dependent variable into the fitting curve equation to obtain the variable corresponding to the dependent variable, round the variable to the nearest integer, and the obtained value is the ovarian development stage number of the female mud crab individual.

[0013] Preferably, the step 100 includes:

[0014] Step 110: Select a female mud crab sample;

[0015] Step 120: Obtaining a ratio a of the ovary cross-sectional area to the cephalothorax cross-sectional area of ​​the female mud crab sample;

[0016] Step 130: dissecting a female mud crab sample and obtaining the ovarian development stage number b of the female mud crab sample;

[0017] Step 140: storing the ratio a and the number of ovarian development stages b in a sample data set, and obtaining the fitting curve equation based on the sample data set.

[0018] Preferably, the number of female mud crab samples is at least 300, and the fitting curve equation is:

[0019] y=0.50822-0.37331 / (1+(x / 3.11082) 11.56429 ), R 2 =0.97312.

[0020] Preferably, the detection method according to claim 1 is characterized in that the step 200 comprises: screening out female mud crabs whose umbilicus is dark green or black and has protruding bristles inside the umbilicus, wherein the protruding length of the bristles is 2-5 mm.

[0021] Preferably, the step 200 comprises: screening out female mud crabs with a mass of 200-500 g.

[0022] Preferably, the step 300 includes:

[0023] Step 310: subjecting the selected female mud crabs to cryo-anesthesia;

[0024] Step 320: Clean the cephalothorax of the female mud crab that has been subjected to cryo-anesthesia, and remove water stains and organic stains on the surface of the cephalothorax.

[0025] Preferably, the step 310 includes:

[0026] The screened female mud crabs were placed in an ice box with an internal temperature of 0-5°C for 8-12 minutes.

[0027] Preferably, the step 320 includes:

[0028] Wiping the cephalothorax of the female mud crab with a wet wipe to remove water stains on the surface of the cephalothorax;

[0029] The cephalothorax of the female mud crab was wiped with 75% medical alcohol cotton to remove organic stains attached to the surface of the cephalothorax.

[0030] Preferably, the step 400 includes:

[0031] The female mud crab was illuminated by an LED flashlight as a light source, wherein the luminous flux of the LED flashlight was 1000 lm and the light source area of ​​the LED flashlight was 7000-8000 mm. 2. .

[0032] Preferably, the step 500 includes:

[0033] placing a reference ruler on one side of the cephalothorax, taking photos of the entire cephalothorax including the female mud crab individual using a camera, and converting the photos into image files;

[0034] The step 600 includes:

[0035] cropping a cephalothorax image and an ovary shadow image from the image file;

[0036] The image file, the carapace picture and the ovary shadow picture are respectively input into Digimizer 6.0 software, and the image area value CA of the carapace, the area value SA of the ovary shadow and the image area ratio AR=SA / CA are calculated.

[0037] Compared with the prior art, the present application has the following advantages:

[0038] Firstly, the present application avoids causing body damage to the detected Scylla paramamosain during the detection of the ovary development period of the Scylla paramamosain, and ensures that the detected Scylla paramamosain can continue to be used for production and sales and seedling breeding.

[0039] Secondly, the present application only needs to process data of about 300-400 samples once, and a fitting curve equation with high fitting degree can be obtained to realize universal detection operation, so that large-scale full-coverage detection of Scylla paramamosain individuals is met, and the detection efficiency is greatly improved.

[0040] Finally, compared with the prior art, the operation process of the present application is shorter and the operation difficulty is lower, so that the threshold of the operation level of the detection personnel is reduced. DETAILED DESCRIPTION

[0041] Figure 1 It is a flowchart of Example 1.

[0042] Figure 2 It is a carapace diagram of the ovary period (I period) of the female Scylla paramamosain in Application Example 1.

[0043] Figure 3 It is an ovary shadow diagram of the ovary period (I period) of the female Scylla paramamosain in Application Example 1.

[0044] Figure 4 It is an ovary tissue section diagram of the ovary period (I period) of the female Scylla paramamosain in Application Example 1.

[0045] Figure 5 It is a carapace diagram of the ovary period (II period) of the female Scylla paramamosain in Application Example 1.

[0046] Figure 6 It is an ovary shadow diagram of the ovary period (II period) of the female Scylla paramamosain in Application Example 1.

[0047] Figure 7 It is an ovary tissue section diagram of the ovary period (II period) of the female Scylla paramamosain in Application Example 1.

[0048] Figure 8 It is a carapace diagram of the ovary period (III period) of the female Scylla paramamosain in Application Example 1.

[0049] Figure 9 This is a schematic diagram of the ovarian shadow of the female mud crab during the ovarian period (stage III) in Application Example 1.

[0050] Figure 10 This is a schematic diagram of the ovarian tissue section of the female mud crab in the ovarian stage (stage III) in Application Example 1.

[0051] Figure 11 This is a schematic diagram of the cephalothorax of the female mud crab during the ovarian period (stage IV) in Application Example 1.

[0052] Figure 12 This is a schematic diagram of the ovarian shadow of the female mud crab during the ovarian period (stage IV) in Application Example 1.

[0053] Figure 13 This is a schematic diagram of the ovarian tissue section of the female mud crab in the ovarian stage (stage IV) in Application Example 1.

[0054] Figure 14 This is a schematic diagram of the cephalothorax of the female mud crab during the ovarian period (stage V) in Application Example 1.

[0055] Figure 15 This is a schematic diagram of the ovarian shadow of the female mud crab during the ovarian period (stage V) in Application Example 1.

[0056] Figure 16 This is a schematic diagram of the ovarian tissue section of the female mud crab in the ovarian stage (stage V) in Application Example 1. DETAILED DESCRIPTION

[0057] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Example 1, please refer to Figure 1 :

[0059] A method for detecting the number of ovarian development stages of mud crabs comprises the following steps:

[0060] Step 100: Obtaining a fitting curve equation with the number of ovarian development stages of the female mud crab as a variable and the ratio of the cross-section of the ovary to the area of ​​the cephalothorax of the female mud crab as a dependent variable;

[0061] Step 200: Screening out female mud crabs to be tested whose ovaries have entered a rapid development stage;

[0062] Step 300: performing detection pre-processing on the screened female mud crab individuals;

[0063] Step 400: placing the female mud crab that has completed the detection pre-treatment under a light source to illuminate the female mud crab so that the shadow of the ovary is projected on the cephalothorax of the female mud crab;

[0064] Step 500: Acquire an image of the cephalothorax of the female mud crab under illumination by a light source;

[0065] Step 600: obtaining the image area value CA of the cephalothorax and the area value SA of the ovarian shadow on the image, and calculating the image area ratio AR=SA / CA;

[0066] Step 700: Substitute the image area ratio AR as the dependent variable into the fitting curve equation to obtain the variable corresponding to the dependent variable, round the variable to the nearest integer, and the obtained value is the ovarian development stage number of the female mud crab individual.

[0067] By adopting the above technical solution, the authors take advantage of the fact that after entering the rapid development stage, the size of the cephalothorax does not change significantly, while the size of the ovaries gradually increases during the development and maturation process. Furthermore, they take advantage of the fact that all organs and tissues in the shell cavity of the mud crab, except the ovaries, have a certain degree of light transmittance. By using strong light, the shadow outline of the ovaries can be mapped on the mud crab's cephalothorax, and the ratio between the two can be obtained through image processing tools. By establishing a mathematical relationship between the ratio of the area of ​​the mud crab's ovary cross-section to the cephalothorax and the numerical value of the ovarian development period, the mud crab's ovary detection process is transformed into a mathematical model calculation and prediction process based on image processing. This avoids the physical damage to the mud crabs in existing technologies, allowing the detected mud crabs to continue to be used for production, sales, and seed breeding.

[0068] In some application scenarios, step 100 includes:

[0069] Step 110: Select a female mud crab sample;

[0070] Step 120: Obtaining a ratio a of the cross-section of the ovary to the area of ​​the cephalothorax of the female mud crab sample;

[0071] Step 130: dissecting a female mud crab sample and obtaining the ovarian development stage number b of the female mud crab sample;

[0072] Step 140: storing the ratio a and the number of ovarian development stages b in a sample data set, and obtaining the fitting curve equation based on the sample data set.

[0073] By adopting the technical scheme, on the basis of the existing dissection method for detecting the ovary of Scylla paramamosain, the ratio data of the cross section of the ovary of the sample to the carapace area are further obtained, which are combined with the ovary development period obtained based on dissection to form a fitting curve equation describing the data relationship. Since the dissection method for detecting the ovary of Scylla paramamosain is fully utilized to collect data, the material cost for generating the fitting curve equation in the early stage is reduced. Meanwhile, the physiological characteristics of female Scylla paramamosain are common. Therefore, once the fitting curve equation is generated, it has universality and does not need to be repeatedly generated, thereby greatly saving the time cost of detection. It should be noted that step 120 can adopt a method similar or equivalent to steps 200-600 of the present application, and the ratio of the image area of the ovary shadow to the carapace is used as the ratio of the cross section of the ovary to the carapace area, or the data can be obtained based on the actual measurement of the carapace and ovary of the female Scylla paramamosain during dissection. In practice, the number of the female Scylla paramamosain samples is at least 300, and the obtained fitting curve equation is:

[0074] y = 0.50822 - 0.37331 / (1 + (x / 3.11082) 11.56429 ), R 2 = 0.97312.

[0075] In some application scenarios, the step 200 includes: screening out female Scylla paramamosain individuals with dark green or black umbilical region and with setae extending outward from the umbilical region, and the length of the setae extending outward is 2-5 mm. The mass of the female Scylla paramamosain individuals is 200-500 g.

[0076] In practice, the inventors found that before the ovary of the female Scylla paramamosain enters the rapid development stage, the shell of the umbilical region is mainly white, and there is no setae extending outward from the umbilical region, while after entering the rapid development stage, it gradually turns into dark green or black, and there is setae extending outward from the umbilical region. Therefore, by adopting the above technical scheme, the female Scylla paramamosain individuals with ovary entering the rapid development stage can be most directly screened out, the effectiveness of the detection object is better ensured, and the screening speed is improved.

[0077] In addition, the inventors also found through statistics that the body weight of the female Scylla paramamosain in the rapid development stage of the ovary is different from 200 g to 500 g, and only a small number of Scylla paramamosain have a body weight higher than 500 g. On the other hand, the carapace area of Scylla paramamosain with a body weight higher than 500 g is generally large, so the area ratio relationship between the ovary and the carapace is no longer applicable to the fitting curve equation. Therefore, by limiting the mass of the female Scylla paramamosain to 200-500 g, the effectiveness of the detection object can be further improved.

[0078] In some application scenarios, the step 300 includes:

[0079] Step 310: subjecting the selected female mud crabs to cryo-anesthesia;

[0080] Step 320: Clean the cephalothorax of the female mud crab that has been subjected to cryo-anesthesia, and remove water stains and organic stains on the surface of the cephalothorax.

[0081] By adopting this technical solution: in view of the need to position and film female mud crabs in the present invention, in order to avoid female mud crabs causing harm to the human body in the subsequent steps during the process, a pre-treatment relief of cryo-anesthesia for female mud crabs is set. In this example: the female mud crabs are uniformly placed in an ice box with an internal temperature of 0-5°C for 8-12 minutes. In practice, other cryo-anesthesia methods and freezing times can also be adjusted according to actual conditions. At the same time, in view of the technical need to illuminate female mud crabs so that the shadow of the ovary is visible on the cephalothorax in the present invention, a technical link for cleaning the cephalothorax of female mud crabs is set to improve the light transmittance of the cephalothorax and avoid water stains and stains affecting the imaging effect of step 400. In this example, a wet wipe was used to wipe the cephalothorax of the female mud crab to remove water stains on the surface of the cephalothorax; and a 75% medical alcohol cotton pad was used to wipe the cephalothorax of the female mud crab to remove stains attached to the surface of the cephalothorax. In practice, the cleaning method and cleaning tools can also be adjusted according to actual conditions.

[0082] In some application scenarios, an LED flashlight is used as a light source to illuminate the female mud crab, the luminous flux of the LED flashlight is 1000 lm, and the light source area of ​​the LED flashlight is 7000-8000 mm 2. .

[0083] By adopting this technical solution: flashlight irradiation has the advantages of being more convenient to operate and more adaptable to the external environment compared to fixed light source irradiation, the light source area of ​​the LED flashlight is set to 7000-8000mm 2 , which ensures that the entire cephalothorax is placed under the light source.

[0084] In some application scenarios, step 500 includes:

[0085] A reference ruler was placed on one side of the cephalothorax. A camera was used to capture a photograph of the entire cephalothorax, including that of a female Scylla parasitica. The photograph was then transferred to a computer and converted into an image file. The reference ruler image and the cephalothorax image were cropped from the image file using image processing software such as Photoshop. The ovary shadow image was also cropped from the cephalothorax image. Digimizer 6.0 software was used to calculate the cephalothorax image area (CA), the ovary shadow area (SA), and the image area ratio (AR = SA / CA).

[0086] This technical solution uses a reference scale when taking photos, allowing Digimizer 6.0 to calculate the scale using the reference scale, quickly determining the image area of ​​the cephalothorax and the image area of ​​the ovarian shadow. Digimizer 6.0 is a mature image processing software, and its built-in calculation functions will not be discussed here.

[0087] Application Example 1, please refer to Figure 2-16 :

[0088] Taking the mud crab seed breeding platform of Zhejiang Ninghai Research Center of East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences as an example, a total of 5 female mud crabs after mating were taken, and the detection operation was performed using the steps described in Example 1, and the area ratios AR of the ovarian shadow to the cephalothorax were obtained, respectively (0.134956, 0.154212, 0.293009, 0.489547, 0.507167); finally, the AR values ​​(0.134956, 0.154212, 0.293009, 0.489547, 0.507167) were substituted as the dependent variable y into the fitting curve equation y = 0.50822-0.37331 / (1+(x / 3.11082) 11.56429 ), and the x values ​​were (1.4288708, 2.418933, 3.0289568, 4.0127009, 5.1670681), which were rounded to the nearest integer. It was determined that the ovarian development stages of the five mud crabs were stages I, II, III, IV, and V, respectively.

[0089] Conclusion verification: 5 mud crabs were dissected and the ovarian tissues were fixed in Bonn test solution for 24 hours, rinsed three times with 70% alcohol and stored in 70% alcohol at -20℃. Paraffin sections were then made and the morphological characteristics of the ovarian tissue were observed after HE staining. The ovarian development stages I, II, III, IV and V were determined (e.g. Figure 4 、 Figure 7 、 Figure 10 、 Figure 13 、 Figure 16 ), the conclusion is consistent with the detection conclusion of the present invention.

[0090] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and their equivalents, they still fall within the scope of protection of the present invention.

Claims

1. A method for detecting the number of ovarian development stages of mud crab, characterized in that: The steps include: Step 100: Obtaining a fitting curve equation with the number of ovarian development stages b of the female mud crab individual as a variable and the ratio of the ovarian cross-sectional area to the cephalothorax cross-sectional area of ​​the female mud crab individual a as a dependent variable; Step 200: Screening out female mud crabs to be tested whose ovaries have entered a rapid development stage; Step 300: performing detection pre-processing on the screened female mud crab individuals; Step 400: placing the female mud crab that has completed the detection pre-treatment under a light source to illuminate the female mud crab so that the shadow of the ovary is projected on the cephalothorax of the female mud crab; Step 500: Obtain an image of the cephalothorax of the female mud crab under illumination by a light source: placing a reference ruler on one side of the cephalothorax, taking photos of the entire cephalothorax including the female mud crab individual using a camera, and converting the photos into image files; Step 600: Obtain the image area value CA of the cephalothorax and the area value SA of the ovarian shadow on the image respectively, and calculate the image area ratio AR=SA / CA: Cut out the cephalothorax image and the ovarian shadow image from the image file; input the image file, the cephalothorax image, and the ovarian shadow image into Digimizer 6.0 software respectively, and calculate the image area value CA of the cephalothorax, the area value SA of the ovarian shadow, and the image area ratio AR=SA / CA by the Digimizer 6.0 software; Step 700: Substitute the image area ratio AR as the dependent variable into the fitting curve equation to obtain the variable corresponding to the dependent variable, round the variable to the nearest integer, and the obtained value is the ovarian development stage number of the female mud crab individual.

2. The detection method according to claim 1, characterized in that The step 100 includes: Step 110: Select a female mud crab sample; Step 120: Obtaining a ratio a of the ovary cross-sectional area to the cephalothorax cross-sectional area of ​​the female mud crab sample; Step 130: dissecting a female mud crab sample and obtaining the ovarian development stage number b of the female mud crab sample; Step 140: storing the ratio a and the number of ovarian development stages b in a sample data set, and obtaining the fitting curve equation based on the sample data set.

3. The detection method according to claim 1, characterized in that The step 200 includes: selecting female mud crabs with dark green or black umbilicus and outward-extending bristles in the umbilicus, wherein the length of the bristles is 2-5 mm.

4. The detection method according to claim 3, characterized in that: The step 200 includes: screening out female mud crabs with a mass of 200-500 g.

5. The detection method according to claim 1, characterized in that: The step 300 includes: Step 310: subjecting the selected female mud crabs to cryo-anesthesia; Step 320: Clean the cephalothorax of the female mud crab that has been subjected to cryo-anesthesia, and remove water stains and organic stains on the surface of the cephalothorax.

6. The detection method according to claim 5, characterized in that: The step 310 includes: The screened female mud crabs were placed in an ice box with an internal temperature of 0-5°C for 8-12 minutes.

7. The detection method according to claim 5, characterized in that: The step 320 includes: Wiping the cephalothorax of the female mud crab with a wet wipe to remove water stains on the surface of the cephalothorax; The cephalothorax of the female mud crab was wiped with 75% medical alcohol cotton to remove organic stains attached to the surface of the cephalothorax.

8. The detection method according to claim 1, characterized in that: The step 400 includes: The female mud crab was illuminated by an LED flashlight as a light source, wherein the luminous flux of the LED flashlight was 1000 lm and the light source area of ​​the LED flashlight was 7000-8000 mm. 2 .

Images