A method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation.

Abstract

This invention provides a method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation, relating to the field of Chinese soft-shelled turtle farming technology. The method includes the following steps: S1. Harvesting female sexually mature Chinese soft-shelled turtles after they enter hibernation and cleaning their bodies; S2. Individually feeding healthy Chinese soft-shelled turtles in a low-temperature water environment, subsequently inducing them to hibernate under low-temperature conditions; S3. Draining the pond after cleaning the pond, and disinfecting and improving the bottom mud during the hibernation season; S4. Intraperitoneally injecting the Chinese soft-shelled turtles with creatine; S5. Managing water storage and fertilization in the disinfected pond; S6. When the temperature is suitable, adding the creatine-treated Chinese soft-shelled turtles in a suitable proportion. Compared with existing technologies, this invention has the following advantages: 1. Employing... 1. Non-hormonal methods stimulate the development of Chinese soft-shelled turtle ovaries during hibernation, ensuring food safety; 2. Hibernation outside the muddy bottom environment of the breeding pond improves the hibernation environment for Chinese soft-shelled turtles; 3. Individual rearing and intensive management improve the survival rate during hibernation; 4. Re-entry into the pond the following year when the environment meets the requirements for emergence and the temperature changes are mild, with a reasonable female-to-male ratio, avoids disease outbreaks caused by the death of individual individuals; 5. While the parent stock is intensively and individually reared, the bottom mud of the external pond can be disinfected and exposed to the sun, creating an excellent environment for the following year's breeding; 6. Creatine injection can effectively stimulate the early development of Chinese soft-shelled turtle ovaries without affecting the natural hibernation process, which not only helps to shorten the sexual maturity cycle but also improves reproductive efficiency.

Description

Technical Field

[0001] This invention relates to the field of Chinese soft-shelled turtle farming technology, and more specifically, to a method for promoting ovarian development and healthy farming of Chinese soft-shelled turtles during hibernation. Background Technology

[0002] The Chinese softshell turtle (Pelodiscus sinensis) is an aquatic reptile that holds an important place in traditional Chinese medicine and culinary culture. Due to its delicious meat and medicinal value, the Chinese softshell turtle has extremely high economic value in aquaculture. However, its late gonadal development and long generation cycle limit its reproductive efficiency and economic benefits.

[0003] 1. Gonadal development and economic significance

[0004] Ovarian development in the Chinese soft-shelled turtle is a complex process influenced by genetic factors, environmental conditions, and nutritional status. Typically, gonadal development in the Chinese soft-shelled turtle is relatively slow, taking several years from juvenile to mature egg-laying. This lengthy developmental process directly leads to low reproductive rates in the population, consequently impacting yields and profits in the aquaculture industry. Therefore, exploring methods to accelerate ovarian development in the Chinese soft-shelled turtle is of significant practical importance for improving reproductive efficiency, shortening the production cycle, and increasing economic benefits.

[0005] 2. Characteristics of ovarian development during hibernation

[0006] It is noteworthy that although the Chinese soft-shelled turtle does not eat for up to six months during hibernation, its ovarian development does not cease. This continued development during this stage offers the possibility of promoting ovarian maturation through specific methods. Understanding and utilizing the unique physiological state during hibernation may become one of the key points to breaking through traditional breeding bottlenecks.

[0007] 3. The impact of the hibernation environment on health

[0008] During hibernation, Chinese soft-shelled turtles live in a relatively low-temperature, low-oxygen environment and often bury themselves in the mud and sand at the bottom of the breeding ponds. These harsh living conditions make them susceptible to various diseases, such as fungal or bacterial infections, which can negatively impact their health and even threaten their lives. Furthermore, sick individuals have poor recovery abilities, further increasing the risk of death.

[0009] 4. Current Status and Future Prospects of Research on Nutritional Supplementation During Hibernation

[0010] Currently, research on how to provide appropriate nutrition to support ovarian development in Chinese soft-shelled turtles during hibernation is very limited. If a safe and effective method can be found to enhance ovarian development without affecting the normal hibernation process, it is expected to significantly improve the reproductive capacity of Chinese soft-shelled turtles. Considering the unique metabolic patterns during hibernation, any technical solution aimed at promoting ovarian development needs to carefully consider issues such as the selection of nutrients, absorption mechanisms, and potential side effects. Summary of the Invention

[0011] The technical problem to be solved by this invention is how to utilize the hibernation period of the Chinese soft-shelled turtle to promote ovarian development and enhance its reproductive capacity.

[0012] To address the aforementioned technical problems, this invention provides a method for promoting ovarian development and healthy aquaculture of Chinese soft-shelled turtles during hibernation, the method comprising the following steps:

[0013] S1. Harvest female sexually mature Chinese softshell turtles after they enter hibernation and clean their bodies.

[0014] S2. Healthy Chinese soft-shelled turtles were individually wrapped and raised in a low-temperature water environment, and then induced to hibernate under low-temperature conditions.

[0015] S3. After cleaning the pond, drain the water and carry out deep disinfection and improvement of the bottom mud during the hibernation season.

[0016] S4. Intraperitoneal injection of creatine into the Chinese soft-shelled turtle;

[0017] S5. Manage water storage and fertilization in the disinfected pond;

[0018] S6. When the temperature is suitable, add the Chinese soft-shelled turtle treated with creatine in the appropriate proportion.

[0019] Compared with existing technologies, the present invention has the following advantages using the above method: 1. It artificially induces hibernation by removing the bottom mud environment of the breeding pond; 2. It allows for individual rearing and intensive management, and the turtles are reintroduced into the pond in the second year when the environment meets the requirements for emergence from hibernation and the temperature changes are mild; 3. It allows for a reasonable ratio of males to females, and injured or diseased individuals can be selected in time to avoid concentrated outbreaks of diseases; 4. While the parent turtles are intensively reared individually, the bottom mud of the outer pond can be disinfected and exposed to the sun, creating an excellent environment for breeding in the second year; 5. Through specific treatment methods (such as creatine injection), the early development of the ovaries of the Chinese soft-shelled turtle can be effectively stimulated without affecting the natural process of hibernation. This not only helps to shorten the sexual maturity cycle, but also increases the number and quality of eggs laid in each breeding season, thereby significantly improving the overall reproductive efficiency.

[0020] In some embodiments, in step S1, the water temperature for cleaning the turtle is 20-25°C, and the water temperature is rapidly reduced to 4-8°C 0.5 hours after cleaning.

[0021] In some embodiments, the temperature of the low-temperature water in step S2 is 0-15°C.

[0022] The Chinese soft-shelled turtle is a poikilothermic animal, meaning its body temperature changes with the ambient temperature. In nature, it hibernates in areas with lower temperatures during winter, typically around 0-15°C. By setting the water temperature in its captive environment within this range, natural hibernation conditions can be effectively simulated, reducing stress caused by sudden temperature changes and helping to maintain its normal physiological functions.

[0023] In some embodiments, in step S2, the depth of the low-temperature water is 3-10 cm above the edge of the Chinese soft-shelled turtle's skirt, and the water is changed regularly, with a frequency of 5 days per change.

[0024] The Chinese softshell turtle is an amphibious animal, but during hibernation, it prefers shallow water areas. Shallow water areas in nature (such as pond edges and gentle stream slopes) typically have relatively stable temperatures and low current speeds, which aligns with an environment designed to be 3-10 cm deeper than the edge of the water's surface. By simulating this natural habitat, the stress of adapting to a new environment can be reduced, making it easier for the Chinese softshell turtle to enter and maintain a good hibernation state. The shallow water layer helps increase the surface area between the water and the air, thereby enhancing oxygen dissolution efficiency and ensuring sufficient dissolved oxygen levels in the water. This is particularly important for the Chinese softshell turtle because even in a low metabolic state, they still require a certain supply of oxygen to maintain basic life activities. A water depth of 3-10 cm ensures sufficient dissolved oxygen while avoiding the risk of oxygen depletion that can occur in deeper water.

[0025] In some embodiments, during step S2, when raising individual soft-shelled turtles, the soft-shelled turtles are wrapped in dark nylon bags and placed in a low-temperature water environment of 0-15℃.

[0026] Although Chinese soft-shelled turtles are aquatic animals, they may leave the water for short periods under certain circumstances (such as during hibernation or after emerging from hibernation). Maintaining adequate air humidity during these times is crucial for their skin and respiratory health. The moisturizing properties of the enclosure help prevent dry skin and reduce skin damage or respiratory problems caused by dryness.

[0027] In some embodiments, during step S2, when the turtles are kept individually, the dark nylon bag has ventilation holes for breathing.

[0028] Although Chinese soft-shelled turtles are aquatic animals, they can also breathe air through their lungs. Especially during hibernation or after emerging from hibernation, ensuring a sufficient oxygen supply is crucial for their health when they are out of the water. The design of the ventilation holes allows for air circulation within the enclosure, providing fresh oxygen and helping to prevent the risk of suffocation due to oxygen deficiency.

[0029] In some implementations, during step S2, the Chinese soft-shelled turtle is in a quiet, dark environment.

[0030] The specific steps to induce Chinese soft-shelled turtles into hibernation are as follows: Start at 20-25℃, then rapidly lower the temperature to 4-8℃, and monitor the water quality daily. If the water becomes polluted, promptly perform a temperature-controlled water change. During this period, try to maintain a quiet and dark environment for the turtles.

[0031] Compared with existing technologies, the method of inducing hibernation by rapidly cooling down can quickly reduce the heart rate and blood oxygen concentration of the Chinese soft-shelled turtle, thus putting it into a hibernation state. The Chinese soft-shelled turtle gradually enters a hibernation state as shown by a heart rate of less than 10 beats per half minute and a blood oxygen concentration of less than 90%.

[0032] In some implementations, while performing steps S1 and S2, the pond bottom mud is dried and disinfected by sun exposure.

[0033] Draining the pond and disinfecting by sun exposure effectively kills pathogens such as bacteria, fungi, and parasites in the pond bottom mud, reducing the risk of disease transmission and providing a healthier living environment for Chinese soft-shelled turtles. Draining the pond removes accumulated organic waste, uneaten feed, and excrement from the bottom. These substances produce harmful substances such as ammonia nitrogen and nitrite during decomposition, affecting water quality. Disinfection by sun exposure further reduces the presence of these harmful substances, improving water quality.

[0034] In some embodiments, the specific parameters for bottom mud disinfection in step S3 are as follows: use 50-100 kg of quicklime per mu of pond, dig pits at intervals of 5-10 m in the pond, with a depth of 30-50 cm, add quicklime and mix with pond mud, and replenish water after 2 days of reaction, with the water depth controlled at 10-15 cm.

[0035] Compared with existing technologies, the present invention uses quicklime for bottom mud disinfection because quicklime is a strong alkaline substance that can neutralize acidic substances in the bottom mud and adjust the pH value to a suitable range for aquaculture. At the same time, quicklime reacts with water to generate calcium hydroxide, releasing heat. Calcium hydroxide denatures the proteins of pathogens, thereby effectively killing pathogenic microorganisms, parasites and their eggs, and reducing the occurrence of aquatic animal diseases.

[0036] In some embodiments, the creatine injection procedure in step S4 is as follows: fix the knee joint of the hind limb of the Chinese softshell turtle, expose the groin area of ​​the lower part of the plastron, and insert the needle through the hind limb muscles of the groin area parallel to the direction of the plastron; with reference to the midline of the plastron, the injection angle is between 20-60°.

[0037] Intraperitoneal injection is a method of directly injecting drugs into the body cavity, allowing for faster entry into the bloodstream compared to oral or subcutaneous injection. The injection solution passes through the muscle before entering the peritoneum, which promotes wound healing and reduces infection. This injection method also better suits the physiological characteristics of the Chinese soft-shelled turtle. For this poikilothermic animal, the digestive system slows down under low temperatures, making oral administration potentially ineffective. Intraperitoneal injection bypasses the digestive tract, ensuring rapid and efficient absorption of creatine, allowing it to act quickly on target organs such as the ovary.

[0038] In some embodiments, in step S4, the average weekly creatine injection dose of the Chinese soft-shelled turtle is 20-400 mg / kg, and the duration is more than four weeks.

[0039] The lower starting dose (20 mg / kg) provides a larger safety margin, reducing the risk of adverse reactions due to overdose; while the higher upper limit (400 mg / kg) allows for individuals requiring stronger stimulation; the weekly frequency ensures sufficient drug concentration maintenance without causing excessive stress to the Chinese soft-shelled turtle. The weekly injection interval allows creatine to gradually accumulate and exert its effects in the body, while allowing sufficient time for the body to adapt and metabolize the drug; eight weeks is a moderate treatment length, sufficient to observe significant changes in ovarian development without increasing unnecessary costs and management difficulties due to excessive duration. This period covers the key stages of the Chinese soft-shelled turtle's hibernation period, making full use of the special physiological state during hibernation to promote ovarian development.

[0040] In some embodiments, the specific steps of fertilization management of the pond after disinfection in step S5 are as follows: after restoring the pond water level to normal, ensuring that the water temperature is maintained between 15-20℃ and the pH value is stable within the range of 6.5-7.5, the EM bacteria preparation is sprayed into the pond regularly. The EM bacteria preparation is made of photosynthetic bacteria, lactic acid bacteria, yeast and actinomycetes in a ratio of 3:3:2:2, and is applied once every 15-20 days until the ambient temperature is stable above 20℃.

[0041] Compared with existing technologies, this invention achieves a more ideal state of EM bacteria activity by adjusting water temperature and pH, thus better maintaining the synergistic effect among various microorganisms. The EM bacteria preparation used is composed of photosynthetic bacteria, lactic acid bacteria, yeast, and actinomycetes: photosynthetic bacteria use light and heat as energy sources to synthesize nutrients such as sugars and amino acids; lactic acid bacteria take in sugars produced by photosynthetic bacteria and yeast to form lactic acid, which has a strong bactericidal ability, can ferment and decompose organic matter, and can inhibit the proliferation of pathogenic bacteria, thus benefiting the growth of Chinese soft-shelled turtles; yeast uses the amino acids and other organic substances synthesized by photosynthetic bacteria to provide a substrate for the proliferation of other effective microorganisms, and the single-cell protein it produces is an indispensable nutrient for Chinese soft-shelled turtles; actinomycetes can produce a variety of antibiotics and enzymes, inhibiting the growth of harmful bacteria, enhancing the immunity of Chinese soft-shelled turtles, improving their disease resistance, and promoting the circulation and transformation of nutrients in the water, providing a better living environment for the healthy growth of Chinese soft-shelled turtles.

[0042] In some embodiments, in step S6, after the creatine injection treatment is completed, when the average temperature of the air temperature is stable at or above 20°C for seven consecutive days, the 4-8°C hibernation environment of the Chinese soft-shelled turtle is rapidly heated to above 25°C, and the Chinese soft-shelled turtles are released into the pond at a female-to-male ratio of 5-30:1.

[0043] Compared to existing technologies, this invention, by controlling the male-to-female ratio (e.g., 5-30:1), ensures sufficient males participate in mating, while female Chinese soft-shelled turtles exhibit relatively docile behavior, helping to reduce injuries caused by fighting. This significantly improves fertilization and hatching success rates. Furthermore, maintaining a stable ambient temperature above 20°C creates suitable living conditions for the Chinese soft-shelled turtles, effectively promoting their healthy growth and development. Attached Figure Description

[0044] Figure 1 The image shows the histological analysis results of follicle development in female Chinese softshell turtles from Example 1 and the control group.

[0045] Figure 2 The diagram shows the analysis results of follicle development in female Chinese softshell turtles in Example 1 and the control group;

[0046] Figure 3 The figures show the analysis results of the pond before and after improvement in Example 1;

[0047] Figure 4 The image shows the histological analysis results of follicle development in female Chinese softshell turtles from Example 2 and the control group;

[0048] Figure 5 The diagram shows the analysis results of follicle development in female Chinese softshell turtles in Example 2 and the control group;

[0049] Figure 6Figure 2 shows the analysis results of the pond before and after improvement in Example 2;

[0050] Figure 7 The image shows the histological analysis results of follicle development in female Chinese softshell turtles in Example 3 and the control group;

[0051] Figure 8 The diagram shows the analysis results of follicle development in female Chinese softshell turtles in Example 3 and the control group;

[0052] Figure 9 Figure 3 shows the analysis results of the pond before and after improvement in Example 3;

[0053] Figure 10 This is a diagram showing the results of the liver transcriptome analysis of the female Chinese softshell turtle in Example 3. Detailed Implementation

[0054] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0055] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0056] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0057] It is worth mentioning that all the statistical methods used in this embodiment are summarized as follows:

[0058] I. Physiological index detection for hibernation induction: The water temperature was measured daily using a thermometer, and the heart rate and blood oxygen concentration of the Chinese soft-shelled turtle were recorded within 20 seconds by clamping the tail of the turtle with a finger-clip pulse oximeter.

[0059] II. Ovarian Development Statistics: The number and diameter of follicle granules in the ovaries of Chinese soft-shelled turtles before injection treatment and in different groups after treatment were statistically analyzed using a fiber imaging system.

[0060] III. Hematoxylin-Eosin (H&E) Staining: Ovarian tissue was embedded in paraffin according to standard methods, sectioned using a microtome, and then dewaxed. After passing through xylene, ethanol, and a series of ethanol and distilled water, hematoxylin was stained for 30 seconds, followed by eosin staining for 2 seconds, dehydration with a series of ethanol, clearing with xylene, and mounting with neutral resin. Morphological changes were observed and photographed using an optical microscope.

[0061] IV. Glycogen content determination: (1) Tissue grinding treatment: liver, skeletal muscle and ovarian tissue samples were ground; (2) Glycogen extraction: KOH was added to the ground samples and then boiled in a water bath; (3) Glycogen content determination: three centrifuge tubes were taken as blank tube, standard tube and sample tube respectively, treated with anthrone solution, boiled in a water bath, cooled and the absorbance value was measured. (4) Calculation: Glycogen (g / 100 g tissue) = (A sample / A standard) × 0.05 × (100 / 0.05) × (50 / 1000) × 1.11.

[0062] V. Determination of lactic acid content: (1) Glycolysis of tissues: Liver, skeletal muscle and ovarian tissues were taken and added to sample tubes and control tubes respectively, and homogenized. Trichloroacetic acid was added to the control tube and mixed. Starch solution was added to the sample tubes and stirred. Liquid paraffin was added and kept warm. The sample tubes were taken out and immediately trichloroacetic acid was added to each tube and mixed. The supernatant was collected by centrifugation. Saturated copper sulfate solution and calcium hydroxide powder were added to each tube and stirred thoroughly. The tubes were allowed to stand to allow the reducing sugar to precipitate completely. The supernatant was collected by centrifugation for later use. (2) Determination of lactic acid: Parallel tubes and control tubes were made for each sample tube. Concentrated sulfuric acid was added to each tube and placed in an ice bath. Then the supernatant was added, mixed, and placed in a boiling water bath. After cooling, p-hydroxybiphenyl colorimetric solution was added and kept warm in a water bath. Then the tubes were quickly placed in a boiling water bath and cooled. The absorbance of the sample tubes was measured using a UV spectrophotometer with the control tube as a blank. The results were analyzed.

[0063] VI. Creatine kinase (CK) activity assay: (1) Preparation of substrate solution: creatine solution, magnesium acetate, thymol blue, Gly-NaOH buffer, ATP, distilled water, NaOH solution. (2) Protein extraction: liver, skeletal muscle, plasma and ovary tissues were placed in centrifuge tubes, and lysis buffer was added to grind into homogenate. The homogenate was placed on ice for lysis, and the supernatant was collected by centrifugation. (3) Experimental procedure: substrate solution and enzyme solution were added to micro-cuvettes, mixed well and the absorbance value was detected. Kinetic analysis was performed.

[0064] VII. Ammonia Nitrogen Content Determination: Pipette 0.0, 0.5, 1.0, 3.0, 5.0, 7.0, and 10.0 mL of ammonium standard working solution into 50 mL colorimetric tubes, dilute to volume with pure water, add 1.0 mL of potassium sodium tartrate solution, mix well, add 1.5 mL of Nessler's reagent, mix well, and let stand for 10 min. Measure the absorbance at 420 nm, using pure water as a blank, and plot a standard curve. Take the pretreated water sample, add it to a 50 mL colorimetric tube, dilute to the mark, add 1.0 mL of potassium sodium tartrate solution, then add 1.5 mL of Nessler's reagent, mix well, and let stand for 10 min. Measure the absorbance using the same procedure as for the standard curve.

[0065] VIII. Determination of Nitrite: Take 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, and 2.5 mL of sodium nitrite standard solution into 50 mL volumetric flasks, add water to each to 25 mL, add 2 mL of 0.4% p-aminobenzenesulfonic acid solution, shake well, and let stand for 3-5 minutes. Add 1 mL of 0.2% naphthylethylenediamine hydrochloride solution, and dilute to 50 mL with pure water. Shake well and let stand for 15 minutes. Measure the absorbance at 540 nm using a spectrophotometer. Use distilled water as a blank to plot a standard curve for nitrite concentration. Take 40 mL of pretreated water sample into a 50 mL volumetric flask, add 2 mL of 0.4% p-aminobenzenesulfonic acid solution, shake well, and let stand for 3-5 minutes. Add 1 mL of 0.2% naphthylethylenediamine hydrochloride solution, measure the absorbance, and calculate the nitrite content in the sample using the standard curve.

[0066] The following description, in conjunction with specific embodiments, provides further details.

[0067] Example 1

[0068] This embodiment provides a method for promoting the development of Chinese soft-shelled turtle ovaries and healthy breeding during hibernation, including the following steps:

[0069] S1. After female sexually mature Chinese softshell turtles enter hibernation, their bodies are cleaned, and they are rapidly cooled down and stored for observation after 0.5 hours.

[0070] S2. Healthy Chinese soft-shelled turtles were individually housed in water at a depth 3 cm above the turtle's skirt, with an ambient temperature of 12℃. They were wrapped in dark nylon bags and placed in the 12℃ low-temperature water environment. The bags had ventilation holes for the turtles to breathe. During the process of rapidly inducing the turtles into hibernation, the water temperature was measured daily using a thermometer, and the turtle's heart rate and blood oxygen concentration were recorded within 20 seconds using a finger-clip pulse oximeter held against its tail.

[0071] S3. After cleaning the pond, drain the water and disinfect and improve the bottom mud. For bottom mud disinfection, use 70 kg of quicklime per mu of pond. Dig pits 7 m apart in the middle of the pond, with a depth of 40 cm. Add quicklime and mix with pond mud. After reacting for 2 days, introduce water source and control the water depth to 10-15 cm.

[0072] S4. The Chinese soft-shelled turtles were treated with creatine injections into their abdominal cavity. Specific parameters for the creatine injection were as follows: 400 mg of creatine per kg of turtle body weight, administered once weekly for eight weeks. Hibernation induction was performed as follows: starting at 20°C, the temperature was rapidly lowered to 12°C over two days, with the water changed once at the set temperature. After hibernation ended, the turtle ovaries were harvested for weight, follicle diameter and number analysis, and histological analysis.

[0073] S5. After disinfection, manage the pond by storing water and fertilizing it. The fertilization management procedure is as follows: After disinfection in step S3, restore the pond water level to normal, ensuring the water temperature is maintained between 15-20℃ and the pH value is stable within the range of 6.5-7.5. Then, regularly spray the pond with EM (Effective Microorganisms) preparation. The EM preparation is made of photosynthetic bacteria, lactic acid bacteria, yeast, and actinomycetes in a ratio of 3:3:2:2, and is applied once every 15 days. Analyze the ammonia nitrogen and nitrite content in the water using Nessler's reagent method and nylonyl ethylenediamine hydrochloride method.

[0074] Preferably, while performing steps S1 and S2, the pond bottom mud is dried and disinfected and exposed to the sun. After completing the creatine injection treatment described in step S4 and the average temperature is stable at 20°C or above for seven consecutive days, the Chinese soft-shelled turtles are placed in the pond that has undergone the drying and disinfection treatment at a certain male-to-female ratio.

[0075] Example 2

[0076] This embodiment provides a method for promoting the development of Chinese soft-shelled turtle ovaries and healthy breeding during hibernation, including the following steps:

[0077] S1. After female sexually mature Chinese softshell turtles enter hibernation, their bodies are cleaned, and they are rapidly cooled down and stored for observation after 0.5 hours.

[0078] S2. Healthy Chinese soft-shelled turtles are kept individually in water with the water depth 3 cm above the edge of the turtle's skirt. The ambient temperature is 12℃. After wrapping them in a dark nylon bag, they are placed in a low-temperature water environment at 12℃. The bag has ventilation holes for the Chinese soft-shelled turtles to breathe.

[0079] S3. After cleaning the pond, drain the water and disinfect and improve the bottom mud. For bottom mud disinfection, use 70 kg of quicklime per mu of pond. Dig pits 7 m apart in the middle of the pond, with a depth of 40 cm. Add quicklime and mix with pond mud. After reacting for 2 days, introduce water source and control the water depth to 10-15 cm.

[0080] S4. Creatine was injected intraperitoneally into the Chinese soft-shelled turtle. Specific parameters for the creatine injection were as follows: 200 mg of creatine per kg of turtle body weight, once a week for eight weeks. Hibernation induction was initiated at 20°C, rapidly decreasing to 12°C after two days, with water changed once at the same temperature. After hibernation, the ovaries of the Chinese soft-shelled turtles were harvested for ovarian weight, follicle diameter, and histological analysis. Simultaneously, creatine kinase activity in the muscles, liver, and ovaries of the Chinese soft-shelled turtles was analyzed using continuous spectrophotometry. RNA was extracted from the liver of the Chinese soft-shelled turtles using the Trizol method for transcriptome analysis.

[0081] S5. After disinfection, the pond is managed for water storage and fertilization. The fertilization management procedure is as follows: After disinfection in step S3, the pond water level is restored to normal, and the water temperature is maintained between 15-20℃ and the pH value is stable within the range of 6.5-7.5. Then, EM bacteria preparation is sprayed into the pond regularly. The EM bacteria preparation is made of photosynthetic bacteria, lactic acid bacteria, yeast and actinomycetes in a ratio of 3:3:2:2, and the frequency is once every 15 days. The ammonia nitrogen and nitrite content in the water is analyzed using Nessler's reagent method and naphthalene-ethylenediamine hydrochloride method.

[0082] Preferably, while performing steps S1 and S2, the pond bottom mud is dried and disinfected and exposed to the sun. After completing the creatine injection treatment described in step S4 and the average temperature is stable at 20°C or above for seven consecutive days, the Chinese soft-shelled turtles are placed in the pond that has undergone the drying and disinfection treatment at a certain male-to-female ratio.

[0083] Example 3

[0084] This embodiment provides a method for promoting the development of Chinese soft-shelled turtle ovaries and healthy breeding during hibernation, including the following steps:

[0085] S1. After female sexually mature Chinese softshell turtles enter hibernation, their bodies are cleaned, and they are rapidly cooled down and stored for observation after 0.5 hours.

[0086] S2. Healthy Chinese soft-shelled turtles are kept individually in water with the water depth 3 cm above the edge of the turtle's skirt. The ambient temperature is 12℃. After wrapping them in a dark nylon bag, they are placed in a low-temperature water environment at 12℃. The bag has ventilation holes for the Chinese soft-shelled turtles to breathe.

[0087] S3. After cleaning the pond, drain the water and disinfect and improve the bottom mud. For bottom mud disinfection, use 70 kg of quicklime per mu of pond. Dig pits 7 m apart in the middle of the pond, with a depth of 40 cm. Add quicklime and mix with pond mud. After reacting for 2 days, introduce water source and control the water depth to 10-15 cm.

[0088] S4. Creatine was injected intraperitoneally into the Chinese soft-shelled turtle. Specific parameters for the creatine injection were as follows: 100 mg of creatine per kg of turtle body weight, once a week for eight weeks. Hibernation induction was initiated at 20°C, rapidly decreasing to 12°C after two days, with water changed once at the set temperature. After hibernation, the turtle ovaries were harvested for histological analysis. Simultaneously, the glycogen and lactic acid content in the muscle, liver, and ovaries of the Chinese soft-shelled turtle was analyzed using the anthrone colorimetric method and the p-hydroxybiphenyl colorimetric method.

[0089] S5. After disinfection, the pond is managed for water storage and fertilization. The fertilization management procedure is as follows: After disinfection in step S3, the pond water level is restored to normal, and the water temperature is maintained between 15-20℃ and the pH value is stable within the range of 6.5-7.5. Then, EM bacteria preparation is sprayed into the pond regularly. The EM bacteria preparation is made of photosynthetic bacteria, lactic acid bacteria, yeast and actinomycetes in a ratio of 3:3:2:2, and the frequency is once every 15 days. The ammonia nitrogen and nitrite content in the water is analyzed using Nessler's reagent method and naphthalene-ethylenediamine hydrochloride method.

[0090] Preferably, while performing steps S1 and S2, the pond bottom mud is dried and disinfected and exposed to the sun. After completing the creatine injection treatment described in step S4 and the average temperature is stable at 20°C or above for seven consecutive days, the Chinese soft-shelled turtles are placed in the pond that has undergone the drying and disinfection treatment at a certain male-to-female ratio.

[0091] control group

[0092] The control group consisted of untreated female Chinese softshell turtles. Physiological indicators of the female Chinese softshell turtles in Example 1 before treatment were measured during hibernation induction, and the results are shown in Table 1.

[0093]

[0094] The analysis results are shown in Table 1. The oral temperature, heart rate in half a minute and blood oxygen concentration were at hibernation levels, indicating that the Chinese soft-shelled turtle entered hibernation at 12℃.

[0095] Histological analysis of follicular development was performed on female Chinese softshell turtles from the control group and the Example 1 group. The results are as follows: Figure 1 As shown, where Figure 1 Figures A-1 and A-2 show the histological analysis results of follicle development in female Chinese soft-shelled turtles from the control group. Figure 1 Figures B-1 and B-2 in the diagrams are histological analysis results of follicle development in the female Chinese softshell turtle of Example 1. Figure 1 The terms in ZP, TC, and Y represent the following: ZP: zona pellucida; TC: follicle sheath; → Y: yolk granules; V: vacuoles; GC: granulosa layer of follicular cells; OP: homogeneous cytoplasm; (scale bars are all 100 μm). Figure 1 It can be seen that after creatine treatment, the accumulation of yolk granules in the follicles of female Chinese soft-shelled turtles increased significantly (P<0.05).

[0096] Female Chinese softshell turtles from the control group and Example 1 group were analyzed, and the results are as follows: Figure 2 As shown, Figure 2 In the figure, A represents a comparison of the appearance of ovarian follicle development; Figure 2 B in the figure represents a comparative analysis of ovarian weight, number of follicles, and follicle diameter (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 2 It can be seen that the follicle diameter and ovarian weight of female Chinese soft-shelled turtles increased after creatine treatment (P<0.05).

[0097] Water samples from the pond in Example 1 before and after treatment were analyzed. The results are as follows: Figure 3 As shown, where Figure 3 A-1 represents a comparative analysis of ammonia nitrogen content in water samples. Figure 3 Figure A-2 shows a comparative analysis of nitrite content in water samples (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 3 It can be seen that the ammonia nitrogen and nitrite content in the pond decreased after treatment (P<0.05).

[0098] Histological analysis of follicular development was performed on female Chinese softshell turtles from the control group and the Example 2 group. The results are as follows: Figure 4 As shown, where Figure 4 Figures A-1 and A-2 show the histological analysis results of follicle development in female Chinese soft-shelled turtles from the control group. Figure 4 Figures B-1 and B-2 in the diagrams are histological analysis results of follicle development in the female Chinese softshell turtle of Example 2. Figure 3 The terms in ZP, TC, and Y represent the following: ZP: zona pellucida; TC: follicle sheath; → Y: yolk granules; V: vacuoles; GC: granulosa layer of follicular cells; OP: homogeneous cytoplasm; (scale bars are all 100 μm). Figure 3 It can be seen that after creatine treatment, the accumulation of yolk granules in the follicles of female Chinese soft-shelled turtles increased significantly (P<0.05).

[0099] Female Chinese softshell turtles from the control group and the second example group were analyzed. The results are as follows: Figure 5 As shown, Figure 5 In the text, A represents a comparison of the appearance of ovarian development; Figure 5 B in the figure represents the analysis of changes in glycogen content in muscles, liver, ovaries, and blood; Figure 5 In the figure, C represents the analysis of changes in lactate levels in muscle, liver, ovaries, and blood (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 5 It can be seen that the follicle diameter and ovarian weight of female Chinese soft-shelled turtles treated with creatine increased significantly (P<0.05), and the glycogen content in the tissues increased significantly (P<0.05).

[0100] Water samples from the pond in Example 2, before and after treatment, were analyzed. The results are as follows: Figure 6 As shown, where Figure 6 A-1 represents a comparative analysis of ammonia nitrogen content in water samples. Figure 6 Figure A-2 shows a comparative analysis of nitrite content in water samples (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 6 It can be seen that the ammonia nitrogen and nitrite content in the pond decreased after treatment (P<0.05).

[0101] Histological analysis of follicular development was performed on female Chinese softshell turtles from the control group and the Example 3 group. The results are as follows: Figure 7 As shown, where Figure 7 Figures A-1 and A-2 show the histological analysis results of follicle development in female Chinese soft-shelled turtles from the control group. Figure 7 Figures B-1 and B-2 in the image are histological analysis results of follicle development in the female Chinese softshell turtle of Example 3. Figure 7 The terms in ZP, TC, and Y represent the following: ZP: zona pellucida; TC: follicle sheath; → Y: yolk granules; V: vacuoles; GC: granulosa layer of follicular cells; OP: homogeneous cytoplasm; (scale bars are all 100 μm). Figure 7 It can be seen that after creatine treatment, the accumulation of yolk granules in the follicles of female Chinese soft-shelled turtles increased significantly (P<0.05).

[0102] Female Chinese softshell turtles from the control group and the Example 3 group were analyzed, and the results are as follows: Figure 8 As shown, Figure 8 In the text, A represents a comparison of the appearance of ovarian development; Figure 8 B in the figure represents a comparative analysis of ovarian weight and follicle diameter; Figure 8 In the figure, C represents the analysis of changes in creatine kinase activity in muscle, liver, ovaries, and blood (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 8It can be seen that after creatine treatment, the diameter of follicles and the weight of ovaries increased significantly (P<0.05), and the activity of creatine kinase also increased significantly (P<0.05).

[0103] Water samples from the ponds in Example 3, before and after treatment, were analyzed. The results are as follows: Figure 9 As shown, where Figure 9 A-1 represents a comparative analysis of ammonia nitrogen content in water samples. Figure 9 Figure A-2 shows a comparative analysis of nitrite content in water samples (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 9 It can be seen that the ammonia nitrogen and nitrite content in the pond decreased after treatment (P<0.05).

[0104] Figure 10 This study analyzed the changes in the liver transcriptome of female Chinese soft-shelled turtles treated with 100 mg / kg creatine. A is a volcano plot used for differentially expressed gene analysis; B is a bar chart showing GO enrichment of differentially expressed genes; and C is a scatter plot showing KEGG enrichment of differentially expressed genes.

[0105] from Figure 10 As can be seen, transcriptome analysis of the liver identified 4355 differentially expressed genes (Figure 10A), among which VTG, LEPR, LHR, FSHR, and CK-B genes showed significant changes. The KEGG pathway was enriched with pathways such as adipokine signaling, oxidative phosphorylation, GnRH signaling, and PPAP signaling (Figure 10C). The differentially expressed genes and enriched pathways are related to lipid metabolism and energy metabolism, which can further affect ovarian development. Therefore, creatine can have a significant positive effect on the development of the Chinese soft-shelled turtle ovary under low temperature conditions.

[0106] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for promoting ovarian development and healthy aquaculture of Chinese soft-shelled turtles during hibernation, characterized in that, The method includes the following steps: S1. After female sexually mature Chinese softshell turtles enter hibernation, they are caught and their bodies are cleaned. S2. Healthy Chinese soft-shelled turtles were individually wrapped and raised in a low-temperature water environment, and then induced to hibernate under low-temperature conditions. S3. After cleaning the pond, drain the water and carry out deep disinfection and improvement of the bottom mud during the hibernation season. S4. Intraperitoneal injection of creatine into the Chinese soft-shelled turtle; S5. Manage water storage and fertilization in the disinfected pond; S6. When the temperature is suitable, add the Chinese soft-shelled turtle treated with creatine in the appropriate proportion. In step S4, the creatine injection procedure is as follows: Fix the knee joint of the hind limb of the Chinese soft-shelled turtle, expose the groin area of ​​the lower part of the plastron, and insert the needle through the hind limb muscle of the groin area parallel to the direction of the plastron; with reference to the midline of the plastron, the injection angle is between 20-60°. In step S4, the average weekly creatine injection dose of the Chinese soft-shelled turtle is 20-400 mg / kg, and the duration is more than four weeks.

2. The method as described in claim 1, characterized in that, In step S1, the water temperature for cleaning the turtle is 20-25℃, and the water temperature is rapidly reduced to 4-8℃ within 0.5 hours after cleaning.

3. The method as described in claim 1, characterized in that, In step S2, the temperature of the low-temperature water is 0-15℃, and the depth of the low-temperature water is 3-10 cm above the edge of the Chinese soft-shelled turtle's skirt.

4. The method as described in claim 1, characterized in that, In step S2, when raising a single soft-shelled turtle, the soft-shelled turtle is wrapped in a dark nylon bag and placed in a low-temperature water environment of 0-15℃.

5. The method as described in claim 1, characterized in that, In step S2, the Chinese soft-shelled turtle is in a quiet, dark environment.

6. The method as described in claim 1, characterized in that, In step S3, the specific parameters for bottom mud disinfection are as follows: use 50-100 kg of quicklime per mu of pond, dig pits every 5-10 m in the middle of the pond, with a pit depth of 30-50 cm, add quicklime and mix with pond mud, and replenish water after 2 days of reaction, with the water depth controlled at 10-15 cm.

7. The method as described in claim 1, characterized in that, In step S5, the specific steps for fertilization and water management of the disinfected pond are as follows: After step S3, after restoring the pond water level to the normal level, while ensuring that the water temperature is maintained between 15-20℃ and the pH value is stable within the range of 6.5-7.5, the EM bacteria preparation is sprayed into the pond regularly. The EM bacteria preparation is made of photosynthetic bacteria, lactic acid bacteria, yeast and actinomycetes in a ratio of 3:3:2:2, and the frequency is once every 15-20 days.

8. The method as described in claim 1, characterized in that, In step S6, after the creatine injection treatment is completed, when the average temperature of the air temperature is stable at or above 20°C for seven consecutive days, the 4-8°C hibernation environment of the Chinese soft-shelled turtle is rapidly heated to above 25°C, and the Chinese soft-shelled turtles are released into the pond at a female-to-male ratio of 5-30:1.

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