Auxiliary device and method for sex determination of Chinese softshell turtle embryos and juvenile softshell turtles

Abstract

The invention provides an auxiliary device and method for sex determination of Chinese softshell turtle embryos and juvenile softshell turtles, and the sex of a sample is determined by calculating the length ratio of gonads to middle kidneys and judging whether the ratio is greater than the slope of a sex determination line, and the slope is 0.59 + / -0.01. The device comprises a bottom plate, an upper cover plate, a middle kidney limiting plate, a homogeneous shaping blade and a fixed string, and assists in sex determination. The bottom plate is used for bearing a gonad-middle kidney-adrenal compound, the upper cover plate provides fixing and measuring functions, the middle kidney position is fixed by the middle kidney limiting plate, redundant tissues are cut off by the homogeneous modification blade, and the gonad is kept stable by the fixing string. The method comprises the steps of sample preparation, fixation, treatment, measurement and sex determination. The gender is determined by measuring the length ratio of gonad to middle kidney, and the accuracy is verified by combining slice preparation, dehydration transparentizing, dyeing and microscopic observation. The method is simple in structure, standard in process, suitable for sex screening of embryos and juvenile soft-shelled turtles of different day ages, and high in efficiency, low in cost and high in applicability.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an auxiliary device and method for determining the sex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles. Background Art

[0002] The Chinese soft-shell turtle (Pelodiscus sinensis) is an important aquaculture species, and its gender has a significant impact on the economic benefits of the aquaculture process. Female Chinese soft-shell turtles usually grow faster, are larger in size, and have a stronger ability to lay eggs, so it is particularly important to prioritize female individuals in commercial aquaculture. However, the external traits of Chinese soft-shell turtles during the embryonic and juvenile stages cannot directly determine their gender, making gender screening a technical problem.

[0003] At present, the main methods used for gender identification by existing technologies include:

[0004] Karyotyping and molecular biological testing: The sex of an individual is identified through karyotyping or genetic testing (such as PCR). Although these methods are highly accurate, they have the following disadvantages:

[0005] Expensive: Requires complex experimental equipment and reagents, not suitable for large-scale farms;

[0006] Complex operation: It involves steps such as DNA extraction and amplification, which requires high technical skills of the operators;

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

[0008] Appearance observation method: Determine the sex by observing the external characteristics of the individual. This method is usually only applicable to adults with mature gonads, and cannot accurately determine the sex of embryos and juvenile turtles.

[0009] Anatomical observation method: The sex of a turtle is determined by dissecting the gonads of the individual and observing their morphology under a microscope. Although this method can accurately determine the sex of a turtle, it also has limitations:

[0010] Inefficiency: The dissection operation is time-consuming and the observation process is complicated, which makes it difficult to meet the rapid screening needs of large-scale breeding;

[0011] Highly subjective: Microscope observation requires operators to have certain experience and there is a large amount of human error;

[0012] Difficult to standardize: The complexity and diversity of the dissection steps make it difficult to form a standardized operating procedure.

[0013] In summary, the existing technology still has many shortcomings in the sex identification of Chinese soft-shelled turtle embryos and juveniles, including high cost, low efficiency, complex operation, and lack of standardization, which restricts its practical application in large-scale breeding. Summary of the invention

[0014] A technical problem to be solved by the present invention is to provide an auxiliary device for determining the sex of Chinese soft-shelled turtle embryos and juvenile turtles, so as to solve the problems of high cost, complicated operation, low efficiency and inability to standardize existing in conventional identification methods in the prior art, especially the problem that the sex cannot be directly determined by external traits in the embryonic and juvenile turtle stages.

[0015] In order to overcome the above defects of the prior art, the present invention provides an auxiliary device for determining the sex of Chinese soft-shelled turtle embryos and juvenile soft-shelled turtles, comprising:

[0016] The bottom plate is a rectangular structure located at the bottom of the device and is used to carry the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juveniles;

[0017] The upper cover plate is in a "U-shaped" structure, with an opening facing left, covering the bottom plate, and scales are provided on the right side and the bottom side of the opening;

[0018] A mid-kidney stopper, which is installed between the bottom plate and the upper cover plate, and has a height greater than the height of the "U-shaped" opening of the upper cover plate, so that it can move in the left and right directions of the "U-shaped" opening;

[0019] A homogenizing and shaping blade is installed on the right side of the opening of the upper cover plate through a rotating shaft, and is used to remove the adrenal glands and redundant tissues in the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles;

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

[0021] Compared with the prior art, the device of the present invention simplifies the complex experimental equipment in the prior art into a modular design, and the device has a reasonable structure, which is convenient for operators to quickly master the use method. The fixed and linear arrangement of the samples are ensured by the coordinated use of the mesonephric limit plate and the fixed string. The homogenizing and shaping blade can accurately remove excess tissue, standardize the samples, and improve the efficiency of gender determination. The device is low in cost and easy to promote and use, and is particularly suitable for the needs of farms for large-scale gender screening of Chinese soft-shelled turtles. It overcomes the shortcomings 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 the mesonephric kidney, and directly calculate the length ratio, avoiding the subjective error of microscopic observation. Through the above structure, the technical difficulties of high cost, low efficiency, and complex operation in the prior art are solved, and the rapid, standardized and accurate gender determination of Chinese soft-shelled turtle embryos and juveniles is achieved. The device is highly practical and economical, and provides a practical solution for the Chinese soft-shelled turtle breeding industry.

[0022] In a possible embodiment, the upper cover plate is provided with longitudinal and transverse scales for measuring the length of the gonads and mesonephros, 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.

[0023] Compared with the prior art, by adopting the above technical solution and designing a range gender determination line, the operator can more easily determine the corresponding slope, thereby making a gender determination, and the determination accuracy is higher.

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

[0025] Compared with the prior art, the above technical solution more accurately limits the slope to 0.59, which allows for more intuitive judgment. However, compared with the judgment slope of 0.59±0.01, its accuracy is slightly lower.

[0026] In a possible implementation manner, the homogenizing and shaping blade is mounted on the slot of the upper cover plate via a rotating shaft, and the starting point of the transverse scale is at the bottom of the slot.

[0027] In a possible implementation manner, a limit plate is provided on the edge of the upper surface of the bottom plate, and the limit plate is used to limit the movement of the upper cover plate.

[0028] Compared with the prior art, the above technical solution avoids the problem of incorrect slope determination after movement due to the upper cover plate not being limited, thereby reducing the determination rate, through the design of the limit plate.

[0029] Compared with the prior art, the above technical solution can significantly improve the accuracy of measurement and the convenience of operation. Specifically, by setting longitudinal and transverse scales for measuring the length of the gonads and mesonephros on the upper cover plate, the size measurement can be performed directly and accurately, avoiding errors in the manual measurement process. In addition, the homogenizing and shaping blade is installed on the card slot of the upper cover plate through a rotating shaft, and the starting point of the transverse scale is set at the bottom of the card slot, ensuring the stability of the blade and the repeatability of the measurement. This design allows the operator to easily obtain the measurement data and directly obtain the slope through the scale, thereby improving work efficiency. Finally, the advantage of setting the scale is that it greatly simplifies the measurement process, reduces human errors, and improves the accuracy and consistency of the product. It is particularly suitable for occasions requiring high-precision measurements, further ensuring the superiority of the technical solution in practical applications.

[0030] Another technical problem to be solved by the present invention is to provide a method for determining the sex of Chinese soft-shelled turtle embryos and juvenile turtles, so as to solve the problems of complex operation, low efficiency, strong subjectivity, high cost, etc. existing in the conventional methods in the prior art, especially the technical bottleneck of the difficulty in standardizing the sex determination in the embryonic and juvenile stages.

[0031] In order to overcome the defects of the above prior art, the present invention provides a method for determining the sex of Chinese soft-shelled turtle embryos and juveniles, which is implemented by the determination device and comprises the following steps:

[0032] S1: Sample preparation: Chinese soft-shell turtle embryos or juveniles were dissected, the gonad-mesonephros-adrenal complex was removed, and the samples were washed to remove blood and impurities;

[0033] S2: Sample fixation: Place the removed gonad-mesonephros-adrenal complex on the bottom plate of the measuring device, fix the gonad with a fixing string, and slide the mesonephros stop plate to fix the mesonephros, so that the long axes of the gonad and mesonephros are aligned in a straight line;

[0034] S3: Sample processing: The adrenal glands and redundant mesonephric tissues in the gonad-mesonephric-adrenal complex were removed by rotating a homogenizing and trimming blade, leaving intact gonads and mesonephric tissues;

[0035] S4: Length measurement: Use the longitudinal and transverse scales on the measuring device to measure the lengths of the gonad and mesonephros, respectively, and record the measurement data;

[0036] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephros, 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, and if it is less than 0.58, it is determined to be male.

[0037] Compared with the prior art, the present invention provides a method for determining the sex of Chinese soft-shelled turtle embryos and juveniles, which has the following advantages: the present invention directly determines the ratio of gonad to mesonephros length, replaces the cumbersome gene detection and karyotype analysis process in the prior art, is simple to operate, fast, and suitable for large-scale farms, and the method in the present invention does not rely on expensive equipment and reagents, significantly reducing the cost of sex screening, combined with the modular design of the device, more adaptable, especially suitable for the promotion and application of grassroots aquaculture, and achieves the standardization and repeatability of sex determination through the standardized process of sample fixation, homogenization and calibration measurement, reduces human errors, and avoids the problem of strong subjectivity in the prior art. The method in the present invention is suitable for the sex determination of Chinese soft-shelled turtles of different ages, whether in the embryonic stage or the juvenile stage, the method in the present invention has a high accuracy rate, and through the above steps and technical means, the method in the present invention solves the problems of cumbersome sex determination process, low efficiency, strong subjectivity, etc. in the prior art, and significantly improves the efficiency, accuracy and standardization level of sex determination of Chinese soft-shelled turtle embryos and juveniles. The method is economical and applicable, easy to promote, and can be widely used in the Chinese soft-shelled turtle breeding industry, with important economic and social value.

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

[0039] Compared with the prior art, the above technical solution can more accurately distinguish the sex of Chinese soft-shelled turtle embryos or juveniles by further clarifying the screening standard of setting a slope of 0.59±0.01, avoiding the ambiguity and misjudgment that may exist in traditional methods. This technical solution relies on the precise calculation and analysis of the morphological data of embryos or juveniles, and determines that they are female when the slope is greater than 0.60 and male when it is less than 0.58, thereby ensuring the scientificity and objectivity of gender determination. By setting this slope standard, the boundaries of gender identification are further refined, making the measurement and judgment process more intuitive and accurate, especially in applications that require high-precision screening, it can effectively improve the efficiency and success rate of determination. Ultimately, this method provides an accurate, simple and efficient technical means for gender identification in biological research and aquaculture, significantly improving the accuracy and reliability in actual operations.

[0040] In a possible embodiment, in step S1, the dissection includes: sampling the Chinese soft-shell turtle embryo or juvenile by dissection, wherein the incision is made along the midline of the abdomen, from the neck to the cloaca, and damage to the internal organs is avoided.

[0041] Compared with the prior art, the above technical solution uses the method of cutting along the midline of the abdomen during dissection to fully expose the gonad-mesonephros-adrenal complex of the Chinese soft-shell turtle while avoiding cutting the internal organs. Compared with random or other cutting paths, this path minimizes the interference with the sample structure, improves the safety and efficiency of the operation, and during the dissection process, by avoiding damage to the internal organs, ensures that the tissue structure of the gonad-mesonephros-adrenal complex remains intact, providing high-quality samples for subsequent measurement and processing.

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

[0043] Compared with the existing technology, the above technical solution, by using physiological saline or PBS buffer to wash the sample, not only protects the integrity of the gonad-mesonephros-adrenal complex, but also significantly improves the efficiency and quality of washing, ensures the accuracy of subsequent experimental steps, and provides reliable sample guarantee for gender determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the data chart of the ratio of 30-day-old young turtles;

[0045] Figure 2 This is a section of the gonad of a 30-day-old embryonic turtle;

[0046] Figure 3 This is the data chart of the ratio of 40-day-old young turtles;

[0047] Figure 4 This is a cross-sectional view of the gonad of a 40-day-old embryonic turtle;

[0048] Figure 5 This is the data chart of the ratio of 100-day-old young turtles;

[0049] Figure 6 This is a gonadal section of a 100-day-old young turtle;

[0050] Figure 7 A picture of the structure of the device of the present invention is taken;

[0051] Figure 8 The anatomical viscera diagram of the method for determining the sex of Chinese soft-shelled turtle embryos and juvenile soft-shelled turtles in the present invention;

[0052] Fig. 9 This is a rough sampling diagram of the method for determining the sex of Chinese soft-shell turtle embryos and juveniles in the present invention;

[0053] Fig.10 This is an example diagram of the standardized processing of the method for determining the sex of Chinese soft-shelled turtle embryos and juveniles in the present invention;

[0054] Fig.11It is a structural schematic diagram of the device in the present invention;

[0055] Fig.11 1. Chassis; 2. Upper cover; 3. Middle kidney limit plate; 4. Homogenizing shaping blade; 5. Slot; 6. Fixed string; 7. Horizontal scale; 8. Vertical scale. DETAILED DESCRIPTION

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

[0057] In the description of the embodiments of the present application, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific circumstances.

[0058] In the embodiments of the present application, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, a first feature being "above", "above" or "above" a second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. A first feature being "below", "below" or "below" a second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0059] The present invention provides a method for determining the sex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles, which is implemented by the determination auxiliary device and comprises the following steps:

[0060] S1: Sample preparation: Chinese soft-shell turtle embryos or juveniles were dissected, the gonad-mesonephros-adrenal complex was removed, and the samples were washed to remove blood and impurities;

[0061] S2: Sample fixation: Place the removed gonad-mesonephros-adrenal complex on the bottom plate of the measuring device, fix the gonad with a fixing string, and slide the mesonephros stop plate to fix the mesonephros, so that the long axes of the gonad and mesonephros are aligned in a straight line;

[0062] S3: Sample processing: The adrenal glands and redundant mesonephric tissues in the gonad-mesonephric-adrenal complex were removed by rotating a homogenizing and trimming blade, leaving intact gonads and mesonephric tissues;

[0063] S4: Length measurement: Use the longitudinal and transverse scales on the measuring device to measure the lengths of the gonad and mesonephros, respectively, and record the measurement data;

[0064] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephros, 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, and if it is less than 0.58, it is determined to be male.

[0065] As a preferred solution, if the slope is greater than 0.59, the animal is judged to be female, and if it is less than 0.59, the animal is judged to be male.

[0066] As a preferred solution, in step S1, the dissection includes: sampling the Chinese soft-shell turtle embryo or juvenile by dissection, wherein the incision is made along the midline of the abdomen, from the neck to the cloaca, and damage to the internal organs is avoided.

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

[0068] The present invention also provides an auxiliary device for determining the sex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles, such as Fig.11 and Figure 7 As shown, including:

[0069] The bottom plate is a rectangular structure located at the bottom of the device and is used to carry the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juveniles;

[0070] The upper cover plate is in a "U-shaped" structure, with an opening facing left, covering the bottom plate, and scales are provided on the right side and the bottom side of the opening;

[0071] A mid-kidney stopper, which is installed between the bottom plate and the upper cover plate, and has a height greater than the height of the "U-shaped" opening of the upper cover plate, so that it can move in the left and right directions of the "U-shaped" opening;

[0072] A homogenizing and shaping blade is installed on the right side of the opening of the upper cover plate through a rotating shaft, and is used to remove the adrenal glands and redundant tissues in the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles;

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

[0074] As a preferred solution, the upper cover plate is provided with longitudinal and transverse scales for measuring the length of the gonads and mesonephros, 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 solution, the homogenizing and shaping blade is mounted on the slot of the upper cover plate via a rotating shaft, and the starting point of the transverse scale is at the bottom of the slot.

[0076] As a preferred solution, in a possible implementation manner, a limit plate is provided on the edge of the upper surface of the bottom plate, and the limit plate is used to limit the movement of the upper cover plate.

[0077] The following is combined with specific embodiments to explain the above technical solution of the present invention:

[0078] Embodiment 1:

[0079] This embodiment provides a method for assisting the sex determination of Chinese soft-shelled turtle embryos and juveniles, the method comprising the following steps:

[0080] S1: Sample preparation: Place a 30-day-old embryonic turtle on the dissection table, with the abdomen facing up and firmly fixed, ensuring that the turtle remains motionless throughout the dissection process. Carefully cut the epidermis along the edge of the turtle shell, and use a pry tool to lift the carapace. Be careful not to damage the internal organs during the operation. After removing the carapace, the internal organs are exposed. At this time, be careful to prevent organ damage. Use a scalpel to separate the gonad from the adjacent tissues, and pay attention to identifying blood vessels. Use forceps to clamp the gonad and its connected mesonephros, and use scissors to cut the corresponding blood vessels and ligaments, so as to completely remove the gonad and its mesonephros, and wash the sample with saline to remove blood and impurities;

[0081] S2: Sample fixation: Place the removed gonad-mesonephros-adrenal complex on the bottom plate of the measuring device, fix the gonad with a fixing string, and slide the mesonephros stop plate to fix the mesonephros, so that the long axes of the gonad and mesonephros are aligned in a straight line;

[0082] S3: Sample processing: The adrenal glands and redundant mesonephric tissues in the gonad-mesonephric-adrenal complex were removed by rotating a homogenizing and trimming blade, leaving intact gonads and mesonephric tissues;

[0083] S4: Length measurement: Use the longitudinal and transverse scales on the measuring device to measure the lengths of the gonad and mesonephros, respectively, and record the measurement data;

[0084] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephros, 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, and if it is less than 0.58, it is determined to be male.

[0085] This embodiment also provides a device and method for assisting sex determination of Chinese soft-shelled turtle embryos and juveniles, the device comprising:

[0086] The bottom plate is a rectangular structure located at the bottom of the device and is used to carry the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juveniles;

[0087] The upper cover plate is in a "U-shaped" structure, with an opening facing left, covering the bottom plate, and scales are provided on the right side and the bottom side of the opening of the upper cover plate;

[0088] A mid-kidney stopper, which is installed between the bottom plate and the upper cover plate, and has a height greater than the height of the "U-shaped" opening of the upper cover plate, so that it can move in the left and right directions of the "U-shaped" opening;

[0089] A homogenizing and shaping blade is installed on the right side of the opening of the upper cover plate through a rotating shaft, and is used to remove the adrenal glands and redundant tissues in the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles;

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

[0091] This device is carefully designed according to the characteristics of the gonads of the Chinese soft-shelled turtle. In order to ensure the integrity of the gonads, the gonad-mesonephros-adrenal complex is first taken out during sampling, and then the device is used to accurately fix the position of the gonads and the mesonephros, and the long axis of the tissue is arranged in a straight line; at the same time, the rotating blade in the device is used to remove excess tissue in the gonad-mesonephros-adrenal complex to complete the averaging of the sample; then the device is used to accurately measure the length of the gonad and mesonephros, and the dual-coordinate scale in the device is used to directly obtain the slope corresponding to the ratio of the gonad length to the mesonephros length, thereby directly determining the sex of the individual.

[0092] S6: Verification, the verification step includes:

[0093] S61: The treated gonad-mesonephros-adrenal complex was sliced ​​and dehydrated and transparentized: 50%, 70%, 80%, 90%, and 95% ethanol were used for 10 minutes each, followed by 100% ethanol I and 100% ethanol II for 2 minutes each. The dehydrated samples were soaked in a 1:1 xylene: anhydrous ethanol buffer for 11 minutes, followed by transparentization with xylene I and xylene II, each lasting 4 minutes;

[0094] S62: Sample processing and preparation for the slices of step S62: First, wax embedding is performed, and the transparent sample is waxed at 70°C for 30 minutes in a buffer solution of paraffin: xylene 1:1, paraffin I is waxed at 70°C for 60 minutes, and paraffin II is waxed at 65°C for 2.5 hours. The treated sample is placed in preheated liquid paraffin, ensuring that the sample is completely immersed and located in the center of the mold. The paraffin is naturally cooled at room temperature or placed in a cooler to accelerate solidification. After the paraffin is completely solidified, it is carefully demolded and the edge of the embedded block is trimmed to facilitate subsequent slicing operations; then the slices are spread, and the trimmed paraffin block is cut into 5μm thick continuous slices using a precision slicer. The cut slices are placed one by one in a water bath that has been preheated to 45°C, and the operation is careful to ensure that the slices are fully unfolded, avoid any form of wrinkles or tears, and maintain the clarity of the tissue structure. Subsequently, the unfolded sections were placed on a slide drying machine 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 treatment was performed, and the slides were eluted in xylene I and xylene II for 10 minutes each using a slide holder, and then eluted in a buffer solution of 1:1 xylene: anhydrous ethanol for 5 minutes, and then eluted with 100% ethanol I and 100% ethanol II, each for 5 minutes, eluted in 90% ethanol and 80% ethanol for 3 minutes, eluted in 70% ethanol and 50% ethanol for 2 minutes, and finally soaked in distilled water for 3 minutes;

[0095] After dewaxing, the sections were stained with hematoxylin staining. Eosin staining would mask the characteristics of gonadal and mesonephric tissues, especially when the cytoplasm and extracellular matrix contained many eosinophilic substances. Hematoxylin staining alone could provide higher contrast and make the nucleus more visible. Therefore, hematoxylin staining was used. The staining process lasted for 15 seconds and then rinsed with clean water. After staining, the cell morphology of gonadal and mesonephric cells could be observed under a microscope. The sections were then transparentized again and sealed: the following solutions were used in sequence, and each step lasted for 2 minutes: distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 1:1 mixture of xylene and anhydrous ethanol, xylene I and xylene II. After placing the treated sample on a clean glass slide, use a dropper to drip an appropriate amount of neutral resin around the sample, carefully place the cover glass to cover the sample, and ensure that the resin is evenly distributed without bubbles. Place the glass slide in a ventilated place and wait for the resin to solidify naturally to complete the sealing step.

[0096] S63: The sections were observed under a microscope to confirm the cell morphology of the gonads and mesonephros. Under an optical microscope, the cell morphology of the cross-section of the gonads of 30-day-old female Chinese soft-shelled turtles was hollow ovarian tissue, while the cell morphology of the cross-section of the gonads of male Chinese soft-shelled turtles was solid tissue.

[0097] S64: Based on the microscope observation results and the slope value in step S5, the accuracy of gender determination is confirmed. Figure 1 As shown, Figure 1 This is a statistical data chart of the ratio of 30-day embryonic turtles in the present invention. By calculating the ratio of gonads to mesonephros of multiple 30-day-old turtles, the ratios of all samples are statistically calculated and a scatter plot is made to observe the difference between males and females, and the dividing line is obtained. Figure 2 This is a gonadal slice of a 30-day embryonic young turtle, corresponding to the above-mentioned 30-day embryonic young turtle, and is used to verify the accuracy of the above-mentioned method.

[0098] Through data statistics, it was found that for the gonads of 30-day-old embryos, the dividing line of the length ratio of the gonads and the mesonephros was 0.59±0.01, of which females were >0.60 and males were <0.58. The present invention performed histological sex determination on embryos incubated at 31°C for 30 days, and the accuracy of the above method was 100%.

[0099] Embodiment 2:

[0100] The device used in Example 2 is the same as that in Example 1, except that the judgment object in Example 2 is the embryonic stage of 40-day-old young turtles:

[0101] The method comprises the following steps:

[0102] S1: Sample preparation: Place a 40-day-old embryonic turtle on the dissection table, with the abdomen facing up and firmly fixed, ensuring that the turtle remains motionless during the entire dissection process. Carefully cut the epidermis along the edge of the turtle shell, and use a pry to lift the carapace, taking care to avoid damaging the internal organs during the operation. After removing the carapace, the internal organs are exposed. At this time, you need to be careful to prevent organ damage. Use a scalpel to separate the gonad from the adjacent tissues, and pay attention to identifying blood vessels. Use forceps to clamp the gonad and its connected mesonephros, and use scissors to cut the corresponding blood vessels and ligaments, so as to remove the gonad and its mesonephros completely, and wash the sample with PBS buffer to remove blood and impurities;

[0103] S2: Sample fixation: Place the removed gonad-mesonephros-adrenal complex on the bottom plate of the measuring device, fix the gonad with a fixing string, and slide the mesonephros stop plate to fix the mesonephros, so that the long axes of the gonad and mesonephros are aligned in a straight line;

[0104] S3: Sample processing: The adrenal glands and redundant mesonephric tissues in the gonad-mesonephric-adrenal complex were removed by rotating a homogenizing and trimming blade, leaving intact gonads and mesonephric tissues;

[0105] S4: Length measurement: Use the longitudinal and transverse scales on the measuring device to measure the lengths of the gonad and mesonephros, respectively, and record the measurement data;

[0106] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephros, 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, and if it is less than 0.58, it is determined to be male.

[0107] S6: Verification, the verification step includes:

[0108] S61: Dehydrate the gonads and mesonephros after standardization by soaking them in 50%, 70%, 80%, 90%, and 95% ethanol for 10 minutes each, followed by 100% ethanol I and 100% ethanol II for 2.5 minutes each. The dehydrated samples need to be soaked in a 1:1 buffer solution of xylene: anhydrous ethanol for 11 minutes, followed by transparent treatment with xylene I and xylene II, each lasting 4.5 minutes;

[0109] S62: Sample processing and preparation for the slices of step S62: First, wax embedding is performed, and the transparent sample is waxed at 70°C for 30 minutes in a buffer solution of paraffin: xylene 1:1, paraffin I is waxed at 70°C for 60 minutes, and paraffin II is waxed at 65°C for 2.5 hours. The treated sample is placed in preheated liquid paraffin, ensuring that the sample is completely immersed and located in the center of the mold. The paraffin is naturally cooled at room temperature or placed in a cooler to accelerate solidification. After the paraffin is completely solidified, it is carefully demolded and the edge of the embedded block is trimmed to facilitate subsequent slicing operations; then the slices are spread, and the trimmed paraffin block is cut into 5μm thick continuous slices using a precision slicer. The cut slices are placed one by one in a water bath that has been preheated to 45°C, and the operation is careful to ensure that the slices are fully unfolded, avoid any form of wrinkles or tears, and maintain the clarity of the tissue structure. Subsequently, the unfolded sections were placed on a slide drying machine 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, and the slides were eluted in xylene I and xylene II for 10 minutes each using a slide holder, and then eluted in a buffer solution of 1:1 xylene: anhydrous ethanol for 5 minutes, and then eluted with 100% ethanol I and 100% ethanol II for 5 minutes each, eluted in 90% ethanol and 80% ethanol for 3 minutes, 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. Because eosin staining would mask the characteristics of gonadal and mesonephric tissues, especially when the cytoplasm and extracellular matrix contained many eosinophilic substances, hematoxylin staining alone could provide higher contrast and make the nucleus more visible. Therefore, hematoxylin staining was used. The staining process lasted for 15 seconds and then rinsed with clean water. After staining, the cell morphology of gonadal and mesonephric cells could be observed under a microscope. Then, the sections were transparentized again and sealed: the following solutions were used in sequence, and each step lasted for 2 minutes: distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 1:1 mixture of xylene and anhydrous ethanol, xylene I and xylene II. After placing the processed sample on a clean glass slide, use a dropper to drip an appropriate amount of neutral resin around the sample, carefully place the cover glass to cover the sample, and ensure that the resin is evenly distributed without bubbles. Place the glass slide in a ventilated place and wait for the resin to solidify naturally to complete the sealing step.

[0111] S63: The sections were observed under a microscope to confirm the cell morphology of the gonads and mesonephros. Under an optical microscope, the cell morphology of the cross-section of the gonads of 40-day-old female Chinese soft-shelled turtles was hollow ovarian tissue, while the cell morphology of the cross-section of the gonads of male Chinese soft-shelled turtles was solid tissue.

[0112] S64: Based on the microscope observation results and the slope value in step S5, the accuracy of gender determination is confirmed. Figure 3 As shown, Figure 3 This is a data chart of the ratio of 40-day-old young turtles statistically analyzed in the present invention. By calculating the ratio of the gonads to the mesonephros of multiple 40-day-old young turtles, the ratios of all samples were statistically analyzed and a scatter plot was made to observe the differences between males and females and to obtain the dividing line. Figure 4 This is a gonadal slice of a 40-day embryonic-age young turtle, corresponding to the above-mentioned 40-day embryonic-age young turtle, and is used to verify the accuracy of the above-mentioned method.

[0113] Through data statistics, it was found that for the gonads of the 40-day-old embryos, the dividing line of the length ratio of the gonads to the mesonephros was 0.59, of which the females were >0.60 and the males were <0.58. The accuracy of the above method was 100% through histological determination of embryos incubated at 31°C for 40 days.

[0114] Embodiment 3:

[0115] The device used in Example 3 is the same as that in Example 1, except that the judgment object in Example 3 is the embryonic stage of 100-day-old young turtles:

[0116] The method comprises the following steps:

[0117] S1: Sample preparation: Place a 100-day-old young turtle on the dissection table, with the abdomen facing up and firmly fixed, ensuring that the young turtle remains motionless during the entire dissection process. Carefully cut the epidermis along the edge of the turtle shell, and use a pry to lift the carapace, taking care to avoid damaging the internal organs during the operation. After removing the carapace, the internal organs are exposed. At this time, you need to be careful to prevent organ damage. Use a scalpel to separate the gonad from the adjacent tissues, and pay attention to identifying blood vessels. Use forceps to clamp the gonad and its connected mesonephros, and use scissors to cut the corresponding blood vessels and ligaments, so as to remove the gonad and its mesonephros completely, and wash the sample with PBS buffer to remove blood and impurities;

[0118] S2: Sample fixation: Place the removed gonad-mesonephros-adrenal complex on the bottom plate of the measuring device, fix the gonad with a fixing string, and slide the mesonephros stop plate to fix the mesonephros, so that the long axes of the gonad and mesonephros are aligned in a straight line;

[0119] S3: Sample processing: The adrenal glands and redundant mesonephric tissues in the gonad-mesonephric-adrenal complex were removed by rotating a homogenizing and trimming blade, leaving intact gonads and mesonephric tissues;

[0120] S4: Length measurement: Use the longitudinal and transverse scales on the measuring device to measure the lengths of the gonad and mesonephros, respectively, and record the measurement data;

[0121] S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephros, 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, and if it is less than 0.58, it is determined to be male.

[0122] S6: Verification, the verification step includes:

[0123] The standardized gonads and mesonephros were dehydrated by soaking in 50%, 70%, 80%, 90%, and 95% ethanol for 10 minutes each, followed by 100% ethanol I and 100% ethanol II for 3 minutes each. The dehydrated samples were soaked in a 1:1 buffer solution of xylene and anhydrous ethanol for 13 minutes, followed by transparent treatment with xylene I and xylene II, each lasting 5 minutes;

[0124] S62: Sample processing and preparation for the slices of step S62: First, wax embedding is performed, and the transparent sample is waxed at 70°C for 30 minutes in a buffer solution of paraffin: xylene 1:1, paraffin I is waxed at 70°C for 60 minutes, and paraffin II is waxed at 65°C for 3 hours. The treated sample is placed in preheated liquid paraffin, ensuring that the sample is completely immersed and located in the center of the mold. The paraffin is naturally cooled at room temperature or placed in a cooler to accelerate solidification. After the paraffin is completely solidified, it is carefully demolded and the edge of the embedded block is trimmed to facilitate subsequent slicing operations; then the slices are spread, and the trimmed paraffin block is cut into 5μm thick continuous slices using a precision slicer. The cut slices are placed one by one in a water bath that has been preheated to 45°C, and the operation is careful to ensure that the slices are fully unfolded, avoid any form of wrinkles or tears, and maintain the clarity of the tissue structure. Subsequently, the unfolded sections were placed on a slide drying machine 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, and the slides were eluted in xylene I and xylene II for 10 minutes each using a slide holder, and then eluted in a buffer solution of 1:1 xylene: anhydrous ethanol for 5 minutes, and then eluted with 100% ethanol I and 100% ethanol II for 5 minutes each, eluted in 90% ethanol and 80% ethanol for 3 minutes, 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. Because eosin staining would mask the characteristics of gonadal and mesonephric tissues, especially when the cytoplasm and extracellular matrix contained many eosinophilic substances, hematoxylin staining alone could provide higher contrast and make the nucleus more visible. Therefore, hematoxylin staining was used. The staining process lasted for 15 seconds and then rinsed with clean water. The sections were then transparentized again and sealed: the following solutions were used in sequence, and each step lasted for 2 minutes: distilled water, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol I, 100% ethanol II, 1:1 mixture of xylene and anhydrous ethanol, xylene I and xylene II. After placing the processed sample on a clean slide, use a dropper to drip an appropriate amount of neutral resin around the sample, carefully place the cover glass to cover the sample, and ensure that the resin is evenly distributed without bubbles. Place the slide in a ventilated place and wait for the resin to solidify naturally to complete the sealing step.

[0126] S63: The sections were observed under a microscope to confirm the cell morphology of the gonads and mesonephros. Under an optical microscope, the cell morphology of the cross-section of the gonads of 100-day-old female Chinese soft-shelled turtles was hollow ovarian tissue, while the cell morphology of the cross-section of the gonads of male Chinese soft-shelled turtles was solid tissue.

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

[0128] Through data statistics, it was found that for the gonads of 100-day-old young turtles, the dividing line of the length ratio of the gonads to the mesonephros was 0.59±0.01, of which the females were >0.60 and the males were <0.58. The accuracy of the above method was 100% when the gonads were incubated at 31°C and 100 days after hatching.

[0129] Through the above examples, it is further proved that the present invention provides a method for determining the sex of Chinese soft-shell turtle embryos and young soft-shell turtles, which can combine the length ratio of gonads to mesonephros, and quickly determine the sex through optimized dissection and measurement processes, avoiding the high cost and low efficiency of methods such as karyotype analysis and molecular biological detection in the prior art. The method not only simplifies and standardizes the operation, but also further verifies the accuracy of the determination result through the slice verification step, ensuring the consistency of the ratio method and the microscopic observation result. The experimental results show that whether it is a Chinese soft-shell turtle individual of 30 days old, 40 days old or 100 days old, the method of the present invention can achieve a high accuracy rate (up to 100%), which fully proves its scientificity and practicality and solves the technical problems existing in the background technology.

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

[0131] In the description of the embodiments of the present application, it should be noted that in the description of the present application, terms such as "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships 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, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present application.

[0132] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "in the present embodiment", "specific example", or "some examples" etc. means that the specific features, mechanisms, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0133] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A device for assisting in determining the sex of Chinese soft-shell turtle embryos and juveniles, characterized in that: include: The bottom plate is a rectangular structure located at the bottom of the device and is used to carry the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juveniles; The upper cover plate is in a "U-shaped" structure, with an opening facing left, covering the bottom plate, and scales are provided on the right side and the bottom side of the opening; A mid-kidney limit plate, which is installed between the bottom plate and the upper cover plate, and whose height is greater than the height of the "U-shaped" opening of the upper cover plate, so that it can move in the left and right directions of the "U-shaped" opening; A homogenizing and shaping blade is installed on the right side of the opening of the upper cover plate through a rotating shaft, and is used to remove the adrenal glands and redundant tissues in the gonad-mesonephros-adrenal complex of Chinese soft-shell turtle embryos and juvenile soft-shell turtles; Fixed string: It is fixed at the rightmost end of the opening of the upper cover plate and is located above the bottom plate, and is used to fix the gonads of Chinese soft-shell turtle embryos and juvenile turtles.

2. The auxiliary device for determining the sex of Chinese soft-shell turtle embryos and juveniles according to claim 1, characterized in that: The upper cover plate is provided with longitudinal scales and transverse scales for measuring the lengths of the gonads and the mesonephros.

3. The auxiliary device for determining the sex of Chinese soft-shell turtle embryos and juveniles according to claim 2, characterized in that: A gender determination line for determining the slope is provided on the bottom plate, and the slope of the gender determination line is 0.59±0.

01.

4. The auxiliary device for determining the sex of Chinese soft-shell turtle embryos and juveniles according to claim 2, characterized in that: The slope of the gender determination line is 0.

59.

5. The auxiliary device for determining the sex of Chinese soft-shell turtle embryos and juveniles according to claim 1, characterized in that: The homogenizing and shaping blade is installed on the slot of the upper cover plate through a rotating shaft, and the starting point of the transverse scale is at the lower part of the slot.

6. The auxiliary device for determining the sex of Chinese soft-shell turtle embryos and juveniles according to claim 1, characterized in that: A limit plate is arranged on the edge of the upper surface of the bottom plate, and the limit plate is used to limit the movement of the upper cover plate.

7. A method for determining the sex of Chinese soft-shell turtle embryos and juveniles, characterized in that: The method is implemented by the determination device according to any one of claims 1 to 6, comprising the following steps: S1: Sample preparation: Chinese soft-shell turtle embryos or juveniles were dissected, the gonad-mesonephros-adrenal complex was removed, and the samples were washed to remove blood and impurities; S2: Sample fixation: Place the removed gonad-mesonephros-adrenal complex on the bottom plate of the measuring device, fix the gonad with a fixing string, and slide the mesonephros stop plate to fix the mesonephros, so that the long axes of the gonad and mesonephros are aligned in a straight line; S3: Sample processing: The adrenal glands and redundant mesonephric tissues in the gonad-mesonephric-adrenal complex were removed by rotating a homogenizing and trimming blade, leaving intact gonads and mesonephric tissues; S4: Length measurement: Use the longitudinal and transverse scales on the measuring device to measure the lengths of the gonad and mesonephros, respectively, and record the measurement data; S5: Sex determination: Calculate the ratio of the length of the gonad to the mesonephros, 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, and if it is less than 0.58, it is determined to be male.

8. The method for determining the sex of Chinese soft-shell 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-shell turtle embryo or juvenile is determined to be female, and if it is less than 0.59, the Chinese soft-shell turtle embryo or juvenile is determined to be male.

9. The method for determining the sex of Chinese soft-shell turtle embryos and juveniles according to claim 7, characterized in that: The dissection comprises: dissecting and sampling the Chinese soft-shell turtle embryo or young soft-shell turtle, wherein the incision is made along the midline of the abdomen, from the neck to the cloaca, and damage to the internal organs is avoided.

10. The method for determining sex according to claim 7, characterized in that: In the step S1, the cleaning solution used for cleaning the sample is physiological saline or PBS buffer.

Images