A device and method for determining the sex of Chinese soft-shelled turtle embryos and juveniles.

Abstract

This invention provides an auxiliary device and method for sex determination of Chinese soft-shelled turtle embryos and juveniles. The sex of the sample is determined by calculating the ratio of the length of the gonad to the mesonephric fossa and whether this ratio is greater than the slope of a sex determination line, with a slope of 0.59 ± 0.01. The device includes a base plate, a top cover plate, a mesonephric fossa positioning plate, a homogenized trimming blade, and a fixing string. The base plate supports the gonad-mesonephric-adrenal gland complex, the top cover plate provides fixation and measurement functions, the mesonephric fossa positioning plate fixes the mesonephric fossa position, the homogenized trimming blade removes excess tissue, and the fixing string maintains gonadal stability. The method includes sample preparation, fixation, processing, measurement, and sex determination. Sex is determined by measuring the ratio of the length of the gonad to the mesonephric fossa, and its accuracy is verified by section preparation, dehydration and clearing, staining, and microscopic observation. This invention has a simple structure and standardized process, is suitable for sex screening of Chinese soft-shelled turtle embryos and juveniles of different ages, and possesses high efficiency, low cost, and strong applicability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an auxiliary device and method for determining the sex of Chinese soft-shelled turtle embryos and juvenile turtles. Background Technology

[0002] The Chinese softshell turtle (Pelodiscus sinensis) is an important aquaculture species, and its sex has a significant impact on economic benefits during the farming process. Female Chinese softshell turtles typically grow faster, are larger, and have a stronger egg-laying capacity, making it particularly important to prioritize the selection of female individuals in commercial aquaculture. However, during the embryonic and juvenile stages, external characteristics cannot be directly used to determine the sex of the Chinese softshell turtle, making sex selection a technical challenge.

[0003] Currently, the main methods used for sex determination include:

[0004] Karyotype analysis and molecular biological testing: This involves identifying an individual's sex through karyotype analysis or genetic testing (such as PCR). While these methods are highly accurate, they have the following drawbacks:

[0005] High cost: It requires complex experimental equipment and reagents, making it unsuitable for large-scale farm applications;

[0006] The operation is complex, involving steps such as DNA extraction and amplification, which requires a high level of technical skill from the operators.

[0007] It takes a long time: from sample collection to gender determination, it is difficult to meet the needs of rapid screening.

[0008] External observation method: This method determines sex by observing the external characteristics of an individual. This method is usually only applicable to adults with mature gonads and cannot accurately determine sex during the embryonic and juvenile stages.

[0009] Dissection and observation method: Sex is determined by dissecting the individual's gonads and observing their morphology under a microscope. While this method can determine the sex of juvenile turtles relatively accurately, it also has limitations:

[0010] Inefficient: Dissection is time-consuming and the observation process is complex, making it difficult to meet the rapid screening needs of large-scale farming.

[0011] Highly subjective: Microscopic observation requires operators to have certain experience, and there is a significant possibility of human error;

[0012] Difficult to standardize: The complexity and diversity of dissection procedures make it difficult to establish standardized operating procedures.

[0013] In summary, existing technologies for sex determination of Chinese soft-shelled turtle embryos and juveniles still have many shortcomings, including high cost, low efficiency, complex operation, and lack of standardization, which restricts their practical application in large-scale farming. Summary of the Invention

[0014] One technical problem this invention aims to solve is to provide an auxiliary device for determining the sex of Chinese soft-shelled turtle embryos and juveniles, in order to address the problems of high cost, complex operation, low efficiency, and lack of standardization in conventional identification methods in the prior art, especially the difficulty of directly determining the sex based on external characteristics during the embryonic and juvenile stages.

[0015] To overcome the shortcomings of the prior art, the present invention provides an auxiliary device for determining the sex of Chinese soft-shelled turtle embryos and juveniles, comprising:

[0016] The bottom plate, which is rectangular in shape, is located at the very bottom of the device and is used to support the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles.

[0017] The top cover has a U-shaped structure with an opening facing left, covering the bottom plate. Scales are provided on the right and bottom sides of the opening.

[0018] The mid-kidney limiting plate is installed between the bottom plate and the top cover plate. Its height is greater than the height of the "U-shaped" opening of the top cover plate, so that it can move in the left and right directions of the "U-shaped" opening.

[0019] A homogeneous trimming blade, mounted on the right side of the opening in the upper cover plate via a pivot, is used to remove the adrenal glands and excess tissue from the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles.

[0020] Fixed string: It is fixed at the rightmost end of the opening of the upper cover plate and located above the bottom plate, and is used to press down the gonads of Chinese soft-shelled turtle embryos and juvenile turtles.

[0021] This application discloses an auxiliary device for determining the sex of Chinese soft-shelled turtle embryos and juveniles, which has the following advantages compared with the prior art: The device simplifies the complex experimental equipment in the prior art into a modular design. The device has a reasonable structure, making it easy for operators to quickly master the usage method. The combination of the mesonephric ligament limiting plate and the fixing string ensures the fixation and linear arrangement of the sample. The homogenizing trimming blade can accurately remove excess tissue, standardize the sample, and improve the efficiency of sex determination. Moreover, the device is low in cost and easy to promote and use. It is particularly suitable for the needs of large-scale Chinese soft-shelled turtle sex screening in aquaculture farms, overcoming the disadvantages of high cost and difficulty in promotion in the prior art. The scales set on the right and lower sides of the opening of the upper cover plate can quickly measure the length of the gonad and mesonephric ligament and directly calculate the length ratio, avoiding the subjective error of microscopic observation. Through the above structure, the technical problems of high cost, low efficiency and complex operation in the prior art are solved, realizing the rapid, standardized and accurate sex determination of Chinese soft-shelled turtle embryos and juveniles. The device has high practicality and economy, providing a practical solution for the Chinese soft-shelled turtle farming industry.

[0022] In one possible implementation, the upper cover plate is provided with longitudinal and transverse scales for measuring the length of the gonads and mesonephrum, and the bottom plate is provided with a sex determination line for determining the slope, wherein the slope of the sex determination line is 0.59±0.01, specifically 0.58 and 0.60.

[0023] Compared with existing technologies, the above technical solution, through the design of a range-based gender determination line, allows operators to more easily determine the corresponding slope, thereby making gender judgments with higher accuracy.

[0024] In one possible implementation, the slope of the gender determination line is 0.59.

[0025] Compared with existing technologies, the above technical solution more precisely limits the slope to 0.59, which allows for more intuitive judgment. However, its accuracy is slightly lower than that of a judgment slope of 0.59±0.01.

[0026] In one possible implementation, the homogeneous trimming blade is mounted on a slot in the upper cover plate via a pivot, and the starting point of the transverse scale is at the lower part of the slot.

[0027] In one possible implementation, a limiting plate is provided at the edge of the upper surface of the base plate, the limiting plate being used to restrict the movement of the upper cover plate.

[0028] Compared with existing technologies, the above technical solution, through the design of the limiting plate, avoids the problem of incorrect slope determination after movement due to the unlimited position of the upper cover plate, thus reducing the judgment rate.

[0029] Compared with existing technologies, the above-mentioned technical solution significantly improves measurement accuracy and ease of operation. Specifically, by setting longitudinal and transverse scales on the upper cover plate for measuring the length of the gonads and mesonephroid glands, dimensional measurements can be performed directly and accurately, avoiding errors in manual measurement. Furthermore, the homogeneous trimming blade is mounted on a slot in the upper cover plate via a rotating shaft, with the starting point of the transverse scale set at the lower part of the slot, ensuring blade stability and measurement repeatability. This design allows operators to easily obtain measurement data and directly obtain the slope from the scale, thereby improving work efficiency. Ultimately, the advantage of setting the scale is that it greatly simplifies the measurement process, reduces human error, and improves product accuracy and consistency, making it particularly suitable for applications requiring high-precision measurements, further ensuring the superiority of this technical solution in practical applications.

[0030] Another technical problem that this invention aims to solve is to provide a method for determining the sex of Chinese soft-shelled turtle embryos and juveniles, in order to address the problems of complex operation, low efficiency, strong subjectivity, and high cost of conventional methods in the prior art, especially the technical bottleneck of difficulty in standardizing sex determination during the embryonic and juvenile stages.

[0031] To overcome the shortcomings of the prior art, the present invention provides a method for determining the sex of Chinese soft-shelled turtle embryos and juveniles. The method is implemented using the determination device and includes the following steps:

[0032] S1: Sample preparation: Dissect the Chinese soft-shelled turtle embryo or juvenile turtle, remove the gonad-mesonephric-adrenal complex, and wash the sample to remove blood and impurities;

[0033] S2: Sample fixation: Place the extracted gonad-mesonephric-adrenal complex on the base plate of the measuring device, fix the gonad with a fixing string, and at the same time slide the mesonephric limiting plate to fix the mesonephric, so that the long axis of the gonad and the mesonephric are aligned in a straight line.

[0034] S3: Sample processing: Using a rotating, homogeneous trimming blade, the adrenal gland and excess mesonephric tissue in the gonad-mesonephric-adrenal complex are removed, while the gonad and mesonephric tissue are preserved intact.

[0035] S4: Length measurement: Measure the length of the gonads and mesonephritis using the longitudinal and transverse scales on the measuring device, and record the measurement data;

[0036] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephrium, and determine the sex of the sample based on whether the ratio is greater than the slope of the sex determination line. If the slope is greater than 0.60, it is determined to be female; if it is less than 0.58, it is determined to be male.

[0037] This application presents a method for sex determination of Chinese soft-shelled turtle embryos and juveniles, which, compared with existing technologies, has the following advantages: This invention directly determines the sex by the ratio of gonadal to mesonephric length, replacing the cumbersome gene testing and karyotype analysis processes of existing technologies. It is simple and fast to operate, suitable for large-scale farms. Furthermore, the method does not rely on expensive equipment and reagents, significantly reducing the cost of sex selection. Combined with the modular design of the device, it is more adaptable, especially suitable for widespread application in grassroots aquaculture. Through standardized procedures of sample fixation, homogenization, and calibration measurement, it achieves standardization and repeatability of sex determination, reducing human error and avoiding the strong subjectivity of existing technologies. The method of this invention is applicable to sex determination of Chinese soft-shelled turtles of different ages, exhibiting high accuracy in both the embryonic and juvenile stages. Through the above steps and technical means, this invention solves the problems of cumbersome procedures, low efficiency, and strong subjectivity in sex determination in existing technologies, significantly improving the efficiency, accuracy, and standardization of sex determination for Chinese soft-shelled turtle embryos and juveniles. This method is economical, easy to promote, and can be widely applied to the Chinese soft-shelled turtle farming industry, possessing significant economic and social value.

[0038] In one possible implementation, in step S5, if the slope is greater than 0.59, the Chinese soft-shelled turtle embryo or juvenile is determined to be female; if it is less than 0.59, the Chinese soft-shelled turtle embryo or juvenile is determined to be male.

[0039] Compared to existing technologies, the above-mentioned technical solution, by further clarifying the screening standard with a slope of 0.59±0.01, can more accurately distinguish the sex of Chinese soft-shelled turtle embryos or juveniles, avoiding the ambiguity and misjudgment that may exist in traditional methods. This technical solution relies on precise calculation and analysis of the morphological data of embryos or juveniles, classifying them as female when the slope is greater than 0.60 and male when it is less than 0.58, thus ensuring the scientific and objective nature of sex determination. By setting this slope standard, the boundaries for sex identification are further refined, making the measurement and judgment process more intuitive and accurate. Especially in applications requiring high-precision screening, it can effectively improve the efficiency and success rate of determination. Ultimately, this method provides a precise, simple, and efficient technical means for sex identification in biological research and aquaculture, significantly improving the accuracy and reliability in practical operation.

[0040] In one possible implementation, step S1, the dissection includes: taking samples of Chinese soft-shelled turtle embryos or juvenile turtles through dissection, wherein the incision is made along the midline of the abdomen, from the neck to the cloaca, while avoiding damage to the internal organs.

[0041] Compared to existing technologies, the above-mentioned technique, which involves dissecting along the midline of the abdomen, fully exposes the gonad-mesonephric-adrenal complex of the Chinese soft-shelled turtle while avoiding the cutting of internal organs. Compared to random or other incision paths, this approach minimizes interference with the sample structure, improves operational safety and efficiency, and ensures the integrity of the gonad-mesonephric-adrenal complex tissue structure by avoiding damage to internal organs during dissection, thus providing a high-quality sample for subsequent measurements and processing.

[0042] In one possible implementation, in step S1, the cleaning solution used to clean the sample is physiological saline or PBS buffer.

[0043] Compared with existing technologies, the above-mentioned technical solution, by washing the sample with physiological saline or PBS buffer, not only protects the integrity of the gonad-mesonephric-adrenal complex, but also significantly improves the efficiency and quality of washing, ensuring the accuracy of subsequent experimental steps and providing a reliable sample guarantee for sex determination. Attached Figure Description

[0044] Figure 1 A graph showing the ratio of juvenile turtles aged 30 days;

[0045] Figure 2 Image of a section of the gonads of a 30-day-old turtle embryo;

[0046] Figure 3 A graph showing the ratio of juvenile turtles aged 40 days;

[0047] Figure 4 Image of a section of the gonads of a 40-day-old turtle embryo;

[0048] Figure 5 A graph showing the ratio of juvenile turtles at 100 days old;

[0049] Figure 6 A section of the gonads of a 100-day-old juvenile turtle;

[0050] Figure 7 This is a structural photograph of the device in this invention;

[0051] Figure 8 This is an anatomical diagram of the internal organs of the Chinese soft-shelled turtle embryo and juvenile turtle sex determination method in this invention;

[0052] Figure 9 This is a rough sample image of the method for determining the sex of Chinese soft-shelled turtle embryos and juvenile turtles in this invention;

[0053] Figure 10 This is a standardized processing example diagram of the sex determination method for Chinese soft-shelled turtle embryos and juvenile turtles in this invention;

[0054] Figure 11This is a schematic diagram of the device in the present invention;

[0055] Figure 11 In the middle section, 1. chassis; 2. top cover plate; 3. mid-kidney limiting plate; 4. homogeneous trimming blade; 5. slot; 6. fixing string; 7. horizontal scale; 8. vertical scale; 9. limiting plate; 10. gender determination line. Detailed Implementation

[0056] First, those skilled in the art should understand that these embodiments are merely used to explain the technical principles of the embodiments of this application and are not intended to limit the scope of protection of the embodiments of this application. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0057] In the description of the embodiments of this application, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application based on the specific circumstances.

[0058] In the embodiments of this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "on top of," and "over" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0059] This invention provides a method for determining the sex of Chinese soft-shelled turtle embryos and juveniles. The method is implemented using the aforementioned determination auxiliary device and includes the following steps:

[0060] S1: Sample preparation: Dissect the Chinese soft-shelled turtle embryo or juvenile turtle, remove the gonad-mesonephric-adrenal complex, and wash the sample to remove blood and impurities;

[0061] S2: Sample fixation: Place the extracted gonad-mesonephric-adrenal complex on the base plate of the measuring device, fix the gonad with a fixing string, and at the same time slide the mesonephric limiting plate to fix the mesonephric, so that the long axis of the gonad and the mesonephric are aligned in a straight line.

[0062] S3: Sample processing: Using a rotating, homogeneous trimming blade, the adrenal gland and excess mesonephric tissue in the gonad-mesonephric-adrenal complex are removed, while the gonad and mesonephric tissue are preserved intact.

[0063] S4: Length measurement: Measure the length of the gonads and mesonephritis using the longitudinal and transverse scales on the measuring device, and record the measurement data;

[0064] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephrium, and determine the sex of the sample based on whether the ratio is greater than the slope of the sex determination line. If the slope is greater than 0.60, it is determined to be female; if it is less than 0.58, it is determined to be male.

[0065] As a preferred option, if the slope is greater than 0.59, it is determined to be female; if it is less than 0.59, it is determined to be male.

[0066] As a preferred embodiment, in step S1, the dissection includes: taking samples from the embryo or juvenile Chinese soft-shelled turtle through dissection, wherein the incision is made along the midline of the abdomen, from the neck to the cloaca, while avoiding damage to the internal organs.

[0067] As a preferred embodiment, in step S1, the cleaning solution used for cleaning the sample is physiological saline or PBS buffer.

[0068] This invention also provides an auxiliary device for determining the sex of Chinese soft-shelled turtle embryos and juveniles, such as... Figure 11 and Figure 7 As shown, it includes:

[0069] The bottom plate, which is rectangular in shape, is located at the very bottom of the device and is used to support the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles.

[0070] The top cover has a U-shaped structure with an opening facing left, covering the bottom plate. Scales are provided on the right and bottom sides of the opening.

[0071] The mid-kidney limiting plate is installed between the bottom plate and the top cover plate. Its height is greater than the height of the "U-shaped" opening of the top cover plate, so that it can move in the left and right directions of the "U-shaped" opening.

[0072] A homogeneous trimming blade, mounted on the right side of the opening in the upper cover plate via a pivot, is used to remove the adrenal glands and excess tissue from the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles.

[0073] Fixed string: It is fixed at the rightmost end of the opening of the upper cover plate and located above the bottom plate, and is used to press down the gonads of Chinese soft-shelled turtle embryos and juvenile turtles.

[0074] As a preferred embodiment, the upper cover plate is provided with longitudinal and transverse scales for measuring the length of the gonads and mesonephrum, and the bottom plate is provided with a gender determination line for determining the slope, and the slope of the gender determination line is 0.59±0.01, specifically 0.58 and 0.60.

[0075] As a preferred embodiment, the homogeneous trimming blade is mounted on a slot in the upper cover plate via a rotating shaft, and the starting point of the transverse scale is at the lower part of the slot.

[0076] As a preferred embodiment, in one possible implementation, a limiting plate is provided on the upper surface edge of the base plate, the limiting plate being used to restrict the movement of the upper cover plate.

[0077] The following specific embodiments are used to explain the above-mentioned technical solutions of the present invention:

[0078] Example 1

[0079] This embodiment provides a method for determining the sex of Chinese soft-shelled turtle embryos and juvenile turtles using an auxiliary device, the method comprising the following steps:

[0080] S1: Sample Preparation: Place the 30-day-old juvenile turtle on the dissection table, keeping its abdomen facing upwards and securely fixed to ensure it remains still throughout the dissection process. Carefully cut along the edge of the shell to open the epidermis, and use a pry tool to lift the carapace, taking care to avoid damaging the internal organs. After removing the carapace, the internal organs are exposed; proceed with caution to prevent organ damage. Use a scalpel to separate the gonads from adjacent tissues, while carefully identifying blood vessels. Use forceps to grasp the gonads and their connected mesonephroid kidneys, and use scissors to cut the corresponding blood vessels and ligaments to completely remove the gonads and mesonephroid kidneys. Wash the sample with physiological saline to remove blood and impurities.

[0081] S2: Sample fixation: Place the extracted gonad-mesonephric-adrenal complex on the base plate of the measuring device, fix the gonad with a fixing string, and at the same time slide the mesonephric limiting plate to fix the mesonephric, so that the long axis of the gonad and the mesonephric are aligned in a straight line.

[0082] S3: Sample processing: Using a rotating, homogeneous trimming blade, the adrenal gland and excess mesonephric tissue in the gonad-mesonephric-adrenal complex are removed, while the gonad and mesonephric tissue are preserved intact.

[0083] S4: Length measurement: Measure the length of the gonads and mesonephritis using the longitudinal and transverse scales on the measuring device, and record the measurement data;

[0084] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephrium, and determine the sex of the sample based on whether the ratio is greater than the slope of the sex determination line. If the slope is greater than 0.60, it is determined to be female; if it is less than 0.58, it is determined to be male.

[0085] This embodiment also provides an auxiliary device and method for determining the sex of Chinese soft-shelled turtle embryos and juvenile turtles, the device comprising:

[0086] The bottom plate, which is rectangular in shape, is located at the very bottom of the device and is used to support the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles.

[0087] The top cover plate has a "U" shaped structure with an opening facing left, covering the bottom plate. Scales are provided on the right and bottom sides of the opening of the top cover plate.

[0088] The mid-kidney limiting plate is installed between the bottom plate and the top cover plate. Its height is greater than the height of the "U-shaped" opening of the top cover plate, so that it can move in the left and right directions of the "U-shaped" opening.

[0089] A homogeneous trimming blade, mounted on the right side of the opening in the upper cover plate via a pivot, is used to remove the adrenal glands and excess tissue from the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles.

[0090] Fixed string: It is fixed at the rightmost end of the opening of the upper cover plate and located above the bottom plate, and is used to press down the gonads of Chinese soft-shelled turtle embryos and juvenile turtles.

[0091] This device is meticulously designed to address the characteristics of the gonads of the Chinese soft-shelled turtle. To ensure the integrity of the gonads, the gonad-mesonephric-adrenal complex is first removed during sampling. Then, this device is used to precisely fix the positions of the gonads and mesonephric kidneys, aligning the long axes of the tissues in a straight line. Simultaneously, the rotating blade in the device removes excess tissue from the gonad-mesonephric-adrenal complex, completing the sample averaging process. Afterward, the device accurately measures the lengths of the gonads and mesonephric kidneys. Using the dual-coordinate scale in the device, the slope corresponding to the ratio of gonad length to mesonephric kidney length is directly obtained, thus directly determining the sex of the individual.

[0092] S6: Verification, which includes the following steps:

[0093] S61: The processed gonad-mesonephric-adrenal complex was sliced ​​and dehydrated and cleared: It was sequentially soaked in 50%, 70%, 80%, 90%, and 95% ethanol for 10 minutes each, followed by dehydration with 100% ethanol I and 100% ethanol II for 2 minutes each. The dehydrated sample was then soaked in a 1:1 xylene:anhydrous ethanol buffer solution for 11 minutes, followed by clearing with xylene I and xylene II for 4 minutes each time.

[0094] S62: Sample processing and preparation for the sections from step S62: First, paraffin embedding is performed. The cleared samples are embedded in a 1:1 paraffin:xylene buffer solution at 70°C for 30 minutes, paraffin I at 70°C for 60 minutes, and paraffin II at 65°C for 2.5 hours. The processed samples are placed in preheated liquid paraffin, ensuring complete immersion and placement in the center of the mold. The paraffin is allowed to cool naturally at room temperature or placed in a cooler to accelerate solidification. After complete solidification, the samples are carefully demolded and the edges of the embedding block are trimmed to facilitate subsequent sectioning. Sectioning is then performed using a precision microtome to cut the trimmed paraffin block into continuous sections 5 μm thick. Each section is placed individually in a preheated 45°C water bath, carefully ensuring complete unfolding and avoiding any wrinkles or tears to maintain the clarity of the tissue structure. Subsequently, the unfolded sections were placed on a slide dryer preheated to 60°C and dried evenly for 10 minutes to remove moisture from the sections, enhance their adhesion and toughness, and lay a good foundation for subsequent staining and microscopic observation. Then, the slides were dewaxed and eluted in xylene I and xylene II for 10 minutes each using a slide holder. Then, they were eluted in a 1:1 xylene:anhydrous ethanol buffer solution for 5 minutes. Next, they were eluted in 100% ethanol I and 100% ethanol II for 5 minutes each. Then, they were eluted in 90% ethanol and 80% ethanol for 3 minutes, and in 70% ethanol and 50% ethanol for 2 minutes. Finally, they were soaked in distilled water for 3 minutes.

[0095] After dewaxing, the sections were stained using hematoxylin staining. Eosin staining can mask the characteristics of gonads and mesonephric tissue, especially when the cytoplasm and extracellular matrix contain many eosinophilic substances. Hematoxylin staining alone provides higher contrast, making the cell nuclei more clearly visible. Therefore, hematoxylin staining was used, with the staining process lasting 15 seconds, followed by rinsing with water. This staining process allowed for microscopic observation of the cell morphology of the gonads and mesonephric tissue. The sections were then cleared again and mounted using the following solutions, each lasting 2 minutes: distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, a 1:1 mixture of xylene and anhydrous ethanol, xylene I, and xylene II. After placing the prepared sample on a clean glass slide, a suitable amount of neutral resin was added around the sample using a dropper. A coverslip was carefully placed to cover the sample, ensuring the resin was evenly distributed without air bubbles. The slide was then placed in a ventilated area to allow the resin to cure naturally, completing the mounting process.

[0096] S63: By observing the sections under a microscope, the cell morphology of the gonads and mesonephroides was confirmed. Under an optical microscope, the cross-section of the gonads of 30-day-old female Chinese softshell turtles showed that the cells were hollow, while the cross-section of the gonads of male Chinese softshell turtles showed that the cells were solid.

[0097] S64: Based on the microscopic observation results and the slope value in step S5, confirm the accuracy of the gender determination. Figure 1 As shown, Figure 1 This is a statistical data chart of the ratio of 30-day-old juvenile turtles. By calculating the ratio of gonads to mesonephric fossa in multiple 30-day-old juvenile turtles, the ratios of all samples were statistically analyzed and a scatter plot was created to observe the differences between males and females and to derive the dividing line. Figure 2 The gonads of 30-day-old juvenile turtles were used to verify the accuracy of the above method, corresponding to the 30-day-old juvenile turtles mentioned above.

[0098] Statistical analysis revealed that for gonads in 30-day-old embryos, the ratio of gonadal length to mesonephric length was 0.59 ± 0.01, with females having a ratio > 0.60 and males < 0.58. This invention uses histological sex determination of embryos incubated at 31°C for 30 days, and the accuracy rate of the above method is 100%.

[0099] Example 2

[0100] The apparatus used in Example 2 is the same as that in Example 1, except that the object of judgment in Example 2 is a 40-day-old juvenile turtle embryo.

[0101] The method includes the following steps:

[0102] S1: Sample Preparation: Place the 40-day-old juvenile turtle on the dissection table, keeping its abdomen facing upwards and securely fixed to ensure it remains still throughout the dissection process. Carefully cut along the edge of the shell to open the epidermis, and use a pry tool to lift the carapace, taking care to avoid damaging the internal organs. After removing the carapace, the internal organs are exposed; proceed with caution to prevent organ damage. Use a scalpel to separate the gonads from adjacent tissues, carefully identifying blood vessels. Use forceps to grasp the gonads and their connected mesonephroid kidneys, and use scissors to cut the corresponding blood vessels and ligaments to completely remove the gonads and mesonephroid kidneys. Wash the sample with PBS buffer to remove blood and impurities.

[0103] S2: Sample fixation: Place the extracted gonad-mesonephric-adrenal complex on the base plate of the measuring device, fix the gonad with a fixing string, and at the same time slide the mesonephric limiting plate to fix the mesonephric, so that the long axis of the gonad and the mesonephric are aligned in a straight line.

[0104] S3: Sample processing: Using a rotating, homogeneous trimming blade, the adrenal gland and excess mesonephric tissue in the gonad-mesonephric-adrenal complex are removed, while the gonad and mesonephric tissue are preserved intact.

[0105] S4: Length measurement: Measure the length of the gonads and mesonephritis using the longitudinal and transverse scales on the measuring device, and record the measurement data;

[0106] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephrium, and determine the sex of the sample based on whether the ratio is greater than the slope of the sex determination line. If the slope is greater than 0.60, it is determined to be female; if it is less than 0.58, it is determined to be male.

[0107] S6: Verification, which includes the following steps:

[0108] S61: Dehydrate the standardized gonads and mesonephroides by sequentially soaking them in 50%, 70%, 80%, 90%, and 95% ethanol for 10 minutes each, followed by dehydration with 100% ethanol I and 100% ethanol II for 2.5 minutes each. After dehydration, the samples are soaked in a 1:1 xylene:anhydrous ethanol buffer solution for 11 minutes, then cleared with xylene I and xylene II for 4.5 minutes each time.

[0109] S62: Sample processing and preparation for the sections from step S62: First, paraffin embedding is performed. The cleared samples are embedded in a 1:1 paraffin:xylene buffer solution at 70°C for 30 minutes, paraffin I at 70°C for 60 minutes, and paraffin II at 65°C for 2.5 hours. The processed samples are placed in preheated liquid paraffin, ensuring complete immersion and placement in the center of the mold. The paraffin is allowed to cool naturally at room temperature or placed in a cooler to accelerate solidification. After complete solidification, the samples are carefully demolded and the edges of the embedding block are trimmed to facilitate subsequent sectioning. Sectioning is then performed using a precision microtome to cut the trimmed paraffin block into continuous sections 5 μm thick. Each section is placed individually in a preheated 45°C water bath, carefully ensuring complete unfolding and avoiding any wrinkles or tears to maintain the clarity of the tissue structure. Subsequently, the unfolded sections were placed on a slide dryer preheated to 60°C and dried evenly for 10 minutes to remove moisture from the sections, enhance their adhesion and toughness, and lay a good foundation for subsequent staining and microscopic observation. Then, dewaxing was performed. The slides were eluted in xylene I and xylene II for 10 minutes each using a slide holder, then eluted in a 1:1 xylene:anhydrous ethanol buffer for 5 minutes, then eluted in 100% ethanol I and 100% ethanol II for 5 minutes each, then eluted in 90% ethanol and 80% ethanol for 3 minutes, then eluted in 70% ethanol and 50% ethanol for 2 minutes, and finally soaked in distilled water for 3 minutes.

[0110] After dewaxing, the sections were stained. Eosin staining can mask the characteristics of gonadal and mesonephric tissues, especially when the cytoplasm and extracellular matrix contain many eosinophilic substances. Hematoxylin staining alone provides higher contrast, making the cell nuclei more clearly visible. Therefore, hematoxylin staining was used, lasting 15 seconds, followed by rinsing with water. This staining process allowed for microscopic observation of the cell morphology of the gonads and mesonephroides. The sections were then cleared again and mounted using the following solutions, each lasting 2 minutes: distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, a 1:1 mixture of xylene and anhydrous ethanol, xylene I, and xylene II. After placing the prepared sample on a clean glass slide, a suitable amount of neutral resin was added around the sample using a dropper. A coverslip was carefully placed to cover the sample, ensuring the resin was evenly distributed without air bubbles. The slide was then placed in a ventilated area to allow the resin to cure naturally, completing the mounting process.

[0111] S63: By observing the sections under a microscope, the cell morphology of the gonads and mesonephroides was confirmed. Under an optical microscope, the cross-section of the gonads of 40-day-old female Chinese softshell turtles showed that the cells were hollow, while the cross-section of the gonads of male Chinese softshell turtles showed that the cells were solid.

[0112] S64: Based on the microscopic observation results and the slope value in step S5, confirm the accuracy of the gender determination. Figure 3 As shown, Figure 3 This is a statistical data chart of the ratio of 40-day-old juvenile turtles in this invention. By calculating the ratio of gonads to mesonephric fossa of multiple 40-day-old juvenile turtles, the ratio of all samples was statistically analyzed and a scatter plot was made to observe the differences between males and females and to obtain the dividing line. Figure 4 The gonadal sections of 40-day-old juvenile turtles were used to verify the accuracy of the above method, corresponding to the 40-day-old juvenile turtles mentioned above.

[0113] Statistical analysis revealed that for 40-day-old embryos, the ratio of gonadal length to mesonephric length was 0.59, with females having a ratio >0.60 and males <0.58. Histological analysis of 40-day-old embryos incubated at 31°C showed that the above method had 100% accuracy.

[0114] Example 3

[0115] The apparatus used in Example 3 is the same as that in Example 1, except that the object of judgment in Example 3 is a 100-day-old juvenile turtle embryo.

[0116] The method includes the following steps:

[0117] S1: Sample Preparation: Place the 100-day-old juvenile turtle on the dissection table, keeping its abdomen facing upwards and securely fixed to ensure it remains still throughout the dissection process. Carefully cut along the edge of the shell to open the epidermis, and use a pry tool to lift the carapace, taking care to avoid damaging the internal organs. After removing the carapace, the internal organs are exposed; proceed with caution to prevent organ damage. Use a scalpel to separate the gonads from adjacent tissues, carefully identifying blood vessels. Use forceps to grasp the gonads and their connected mesonephroid kidneys, and use scissors to cut the corresponding blood vessels and ligaments, thus completely removing the gonads and mesonephroid kidneys. Wash the sample with PBS buffer to remove blood and impurities.

[0118] S2: Sample fixation: Place the extracted gonad-mesonephric-adrenal complex on the base plate of the measuring device, fix the gonad with a fixing string, and at the same time slide the mesonephric limiting plate to fix the mesonephric, so that the long axis of the gonad and the mesonephric are aligned in a straight line.

[0119] S3: Sample processing: Using a rotating, homogeneous trimming blade, the adrenal gland and excess mesonephric tissue in the gonad-mesonephric-adrenal complex are removed, while the gonad and mesonephric tissue are preserved intact.

[0120] S4: Length measurement: Measure the length of the gonads and mesonephritis using the longitudinal and transverse scales on the measuring device, and record the measurement data;

[0121] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephrium, and determine the sex of the sample based on whether the ratio is greater than the slope of the sex determination line. If the slope is greater than 0.60, it is determined to be female; if it is less than 0.58, it is determined to be male.

[0122] S6: Verification, which includes the following steps:

[0123] The standardized gonads and mesonephric samples were dehydrated by sequentially soaking in 50%, 70%, 80%, 90%, and 95% ethanol for 10 minutes each, followed by dehydration with 100% ethanol I and 100% ethanol II for 3 minutes each. After dehydration, the samples were soaked in a 1:1 xylene:anhydrous ethanol buffer solution for 13 minutes, and then cleared with xylene I and xylene II for 5 minutes each time.

[0124] S62: Sample processing and preparation for the sections from step S62: First, paraffin embedding is performed. The cleared samples are embedded in a 1:1 paraffin:xylene buffer solution at 70°C for 30 minutes, paraffin I at 70°C for 60 minutes, and paraffin II at 65°C for 3 hours. The processed samples are placed in preheated liquid paraffin, ensuring complete immersion and placement in the center of the mold. The paraffin is allowed to cool naturally at room temperature or placed in a cooler to accelerate solidification. After complete solidification, the samples are carefully demolded and the edges of the embedding block are trimmed to facilitate subsequent sectioning. Then, sections are spread using a precision microtome to cut the trimmed paraffin block into continuous sections 5 μm thick. Each section is placed individually in a water bath preheated to 45°C, handled carefully to ensure complete unfolding and avoid any wrinkles or tears, maintaining the clarity of the tissue structure. Subsequently, the unfolded sections were placed on a slide dryer preheated to 60°C and dried evenly for 10 minutes to remove moisture from the sections, enhance their adhesion and toughness, and lay a good foundation for subsequent staining and microscopic observation. Then, dewaxing was performed. The slides were eluted in xylene I and xylene II for 10 minutes each using a slide holder, then eluted in a 1:1 xylene:anhydrous ethanol buffer for 5 minutes, then eluted in 100% ethanol I and 100% ethanol II for 5 minutes each, then eluted in 90% ethanol and 80% ethanol for 3 minutes, then eluted in 70% ethanol and 50% ethanol for 2 minutes, and finally soaked in distilled water for 3 minutes.

[0125] After dewaxing, the sections were stained. Eosin staining can mask the characteristics of gonads and mesonephric tissue, especially when the cytoplasm and extracellular matrix contain many eosinophilic substances. Hematoxylin staining alone provides higher contrast, making the cell nuclei more clearly visible. Therefore, hematoxylin staining was used, lasting 15 seconds, followed by rinsing with water. The sections were then cleared again and mounted using the following solutions, each lasting 2 minutes: distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, a 1:1 mixture of xylene and anhydrous ethanol, xylene I, and xylene II. After placing the prepared sample on a clean glass slide, a suitable amount of neutral resin was added around the sample using a dropper. A coverslip was carefully placed to cover the sample, ensuring the resin was evenly distributed without air bubbles. The slide was placed in a ventilated area to allow the resin to cure naturally, completing the mounting process.

[0126] S63: By observing the sections under a microscope, the cell morphology of the gonads and mesonephroides was confirmed. Under an optical microscope, the cross-section of the gonads of 100-day-old female Chinese softshell turtles showed that the cells were hollow, while the cross-section of the gonads of male Chinese softshell turtles showed that the cells were solid.

[0127] S64: Based on the microscopic observation results and the slope value in step S5, confirm the accuracy of the gender determination. Figure 5 As shown, Figure 5 This is a statistical data chart of the ratio of 100-day-old juvenile turtles in this invention. By calculating the ratio of gonads to mesonephric fossa of multiple 100-day-old juvenile turtles, the ratio of all samples was statistically analyzed and a scatter plot was made to observe the differences between males and females and to obtain the dividing line. Figure 6 The gonadal sections of 100-day-old juvenile turtles were used to verify the accuracy of the above method, corresponding to the 100-day-old juvenile turtles mentioned above.

[0128] Statistical analysis revealed that for 100-day-old juvenile turtles, the dividing line between the length ratio of the gonad and mesonephric fossa was 0.59 ± 0.01, with females > 0.60 and males < 0.58. The method was found to have 100% accuracy in determining the gonads of turtles incubated at 31℃ for 100 days after hatching.

[0129] The above embodiments further demonstrate that this invention provides a method for determining the sex of Chinese soft-shelled turtle embryos and juveniles. This method combines the length ratio of the gonads to the mesonephric fossa and uses an optimized dissection and measurement process to quickly determine sex, avoiding the high cost and low efficiency of existing methods such as karyotype analysis and molecular biological detection. This method not only simplifies and standardizes the operation but also further verifies the accuracy of the determination results through a section verification step, ensuring consistency between the ratio method and microscopic observation results. Experimental results show that this invention achieves a high accuracy rate (up to 100%) for Chinese soft-shelled turtles of 30, 40, and 100 days old, fully demonstrating its scientific validity and practicality, and solving the technical problems existing in the background art.

[0130] In summary, this invention effectively solves the technical problems existing in the prior art and has significant promotional value and application prospects.

[0131] In the description of the embodiments of this application, it should be noted that the terms "inner" and "outer" and other terms indicating direction or positional relationship are based on the direction or positional relationship shown in the drawings. This is only for the convenience of description and does not indicate or imply that the device or component must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this application.

[0132] In the description of this application, the references to terms such as "an embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0133] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A device for determining the sex of Chinese soft-shelled turtle embryos and juveniles, characterized in that, include: The bottom plate, which is rectangular in shape, is located at the very bottom of the device and is used to support the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles. The top cover has a U-shaped structure with an opening facing left, covering the bottom plate. Scales are provided on the right and bottom sides of the opening. The mid-kidney limiting plate is installed between the bottom plate and the top cover plate. Its height is greater than the height of the "U-shaped" opening of the top cover plate, so that it can move in the left and right directions of the "U-shaped" opening. A homogeneous trimming blade, mounted on the right side of the opening in the upper cover plate via a pivot, is used to remove the adrenal glands and excess tissue from the gonad-mesonephric-adrenal complex of Chinese soft-shelled turtle embryos and juvenile turtles. Fixed string: It is fixed at the rightmost end of the opening of the upper cover plate and located above the bottom plate, and is used to fix the gonads of Chinese soft-shelled turtle embryos and juvenile turtles.

2. The sex determination auxiliary device for Chinese soft-shelled turtle embryos and juvenile turtles according to claim 1, characterized in that, The upper cover plate is provided with longitudinal and transverse scales for measuring the length of the gonads and mesonephrium.

3. The sex determination auxiliary device for Chinese soft-shelled turtle embryos and juvenile turtles according to claim 2, characterized in that, The base plate is provided with a gender determination line for judging the slope, and the slope of the gender determination line is 0.59±0.

01.

4. The sex determination auxiliary device for Chinese soft-shelled turtle embryos and juvenile turtles according to claim 3, characterized in that, The slope of the gender determination line is 0.

59.

5. The sex determination auxiliary device for Chinese soft-shelled turtle embryos and juvenile turtles according to claim 2, characterized in that, The homogeneous trimming blade is mounted on a slot in the upper cover plate via a rotating shaft, and the starting point of the transverse scale is at the lower part of the slot.

6. The sex determination auxiliary device for Chinese soft-shelled turtle embryos and juvenile turtles according to claim 1, characterized in that, A limiting plate is provided on the upper surface edge of the base plate, and the limiting plate is used to restrict the movement of the upper cover plate.

7. A method for determining the sex of Chinese soft-shelled turtle embryos and juveniles, characterized in that, The method is implemented by the determining device according to any one of claims 1-6, and includes the following steps: S1: Sample preparation: Dissect the Chinese soft-shelled turtle embryo or juvenile turtle, remove the gonad-mesonephric-adrenal complex, and wash the sample to remove blood and impurities; S2: Sample fixation: Place the extracted gonad-mesonephric-adrenal complex on the base plate of the measuring device, fix the gonad with a fixing string, and at the same time slide the mesonephric limiting plate to fix the mesonephric, so that the long axis of the gonad and the mesonephric are aligned in a straight line. S3: Sample processing: Using a rotating, homogeneous trimming blade, the adrenal gland and excess mesonephric tissue in the gonad-mesonephric-adrenal complex are removed, while the gonad and mesonephric tissue are preserved intact. S4: Length measurement: Measure the length of the gonads and mesonephritis using the longitudinal and transverse scales on the measuring device, and record the measurement data; S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephrium, and determine the sex of the sample based on whether the ratio is greater than the slope of the sex determination line. If the slope is greater than 0.60, it is determined to be female; if it is less than 0.58, it is determined to be male.

8. The method for determining the sex of Chinese soft-shelled turtle embryos and juveniles according to claim 7, characterized in that, In step S5, if the slope is greater than 0.59, the Chinese soft-shelled turtle embryo or juvenile turtle is determined to be female; if it is less than 0.59, the Chinese soft-shelled turtle embryo or juvenile turtle is determined to be male.

9. The method for determining the sex of Chinese soft-shelled turtle embryos and juveniles according to claim 7, characterized in that, The dissection includes: taking samples from Chinese soft-shelled turtle embryos or juvenile turtles through dissection, wherein the incision is made along the midline of the abdomen, from the neck to the cloaca, while avoiding damage to the internal organs.

10. The gender determination method according to claim 7, characterized in that, In step S1, the cleaning solution used to clean the sample is physiological saline or PBS buffer.

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