Method for promoting ovary development and healthy breeding of Chinese softshell turtles in hibernation period

Abstract

The invention provides a method for promoting ovarian development and healthy breeding of Chinese softshell turtles in a hibernation period, and relates to the technical field of Chinese softshell turtle breeding, the method comprises the following steps: S1, fishing female sexually mature Chinese softshell turtles after the Chinese softshell turtles enter the hibernation period, and cleaning the surfaces of the Chinese softshell turtles; s2, the healthy Chinese softshell turtles are subjected to single wrapping feeding in a low-temperature water environment, and then the Chinese softshell turtles are induced to enter a hibernation state under the low-temperature condition; s3, after the pond of the Chinese softshell turtles is cleaned, water in the pond is drained, and bottom mud is disinfected and improved in the hibernation season; s4, performing creatine intraperitoneal injection on the Chinese softshell turtles; s5, performing water storage and fertilizer and water management on the disinfected pond; s6, putting the Chinese softshell turtles subjected to creatine treatment in proportion when the air temperature is appropriate; compared with the prior art, the method has the following advantages that 1, ovarian development of the Chinese softshell turtles in the hibernation period is stimulated through a non-hormone method, and food safety is guaranteed; hibernation is carried out without the bottom mud environment of the culture pond, and the hibernation environment of the Chinese softshell turtles is improved; 3, single feeding and intensive management are realized, so that the survival rate of hibernation is increased; 4, when the sleeping environment is met in the second year and the temperature change is mild, pond entering treatment is carried out, the female-male proportion is reasonably matched, and disease outbreak caused by dead individuals is avoided; 5, while the parents are independently bred in an intensive manner, the bottom mud of the outer pond can be disinfected, exposed to the sun and the like, and an excellent environment is created for cultivation in the second year; and 6, through creatine injection, early development of the ovary of the Chinese softshell turtle can be effectively stimulated under the condition that the natural process of hibernation is not influenced, so that the sexual maturity period can be shortened, and the breeding efficiency can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of Chinese soft-shelled turtle breeding, and in particular to a method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation. Background Art

[0002] The Chinese soft-shell turtle (Pelodiscus sinensis) is an aquatic reptile that plays an important role in traditional Chinese medicine and food culture. Due to its delicious meat and medicinal value, the Chinese soft-shell turtle has extremely high economic value in aquaculture. However, the Chinese soft-shell turtle has late gonad development and a long generation cycle, which limits its reproductive efficiency and economic benefits.

[0003] 1. Gonadal development and economic significance The ovarian development of Chinese soft-shelled turtle is a complex process, which is affected by genetic factors, environmental conditions, and nutritional status. Under normal circumstances, the gonadal development of Chinese soft-shelled turtle is relatively slow, and it takes several years for a juvenile turtle to mature and lay eggs. This long development process directly leads to the low reproduction rate of the Chinese soft-shelled turtle population, which in turn affects the output and profits of the breeding industry. Therefore, exploring methods to accelerate the ovarian development of Chinese soft-shelled turtle is of great practical significance for improving reproduction efficiency, shortening production cycles, and increasing economic benefits.

[0004] 2. Characteristics of ovarian development during hibernation It is worth noting that although the Chinese soft-shelled turtle does not eat for six months during hibernation, its ovarian development does not stop. The continued development during this stage provides the possibility of promoting ovarian maturation through specific means. Understanding and utilizing the special physiological state during hibernation may become one of the key points to break through the bottleneck of traditional breeding.

[0005] 3. Impact of hibernation environment on health During hibernation, Chinese soft-shelled turtles live in a relatively low-temperature, low-oxygen environment and are often buried in the mud and sand at the bottom of the breeding pond. Such harsh living conditions can easily cause Chinese soft-shelled turtles to suffer from various diseases, such as fungal infections or bacterial diseases, which can have a negative impact on individual health and even threaten life safety. In addition, sick individuals have poor recovery ability, further increasing the risk of death.

[0006] 4. Research status and prospects of nutritional supplementation during hibernation There is currently very limited research on how to provide the Chinese soft-shelled turtle with proper nutrition to support ovarian development during hibernation. 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 the Chinese soft-shelled turtle. Given the special metabolic pattern during hibernation, any technical solution aimed at promoting ovarian development needs to pay special attention to the selection of nutrients, absorption mechanisms, and potential side effects. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to utilize the hibernation period of Chinese soft-shelled turtle to promote the development of ovaries and improve its reproductive capacity.

[0008] In order to solve the above technical problems, the present invention provides a method for promoting the ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation, the method comprising the following steps: S1. Catch the female sexually mature Chinese soft-shelled turtle after it enters the hibernation period and clean the surface of the turtle; S2, raising healthy Chinese soft-shelled turtles in a low-temperature water environment in a single package, and then inducing the Chinese soft-shelled turtles to hibernate under low-temperature conditions; S3. After the pond is cleaned, drain the water and perform deep disinfection and improvement of the bottom mud during the hibernation season. S4, intraperitoneal injection of creatine into Chinese soft-shell turtle; S5. Carry out water storage and fertilizer management on the disinfected pond; S6. When the temperature is suitable, release Chinese soft-shelled turtles treated with creatine in proportion.

[0009] Compared with the prior art, the present invention has the following advantages by adopting the above method: 1. It is separated from the bottom mud environment of the breeding pond and hibernation is artificially created; 2. It is individually raised and intensively managed, and when the hibernation environment is suitable and the temperature changes are mild in the second year, it is put into the pond for treatment; 3. The male-female ratio can be reasonably matched, and if there are injured or sick individuals, they can be selected in time to avoid concentrated outbreaks of diseases; 4. While the parents are intensively raised individually, the bottom mud of the pond can be disinfected and exposed to the sun in the outer pond, 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 breeding efficiency.

[0010] In some embodiments, in step S1, the water temperature for washing the turtle body is 20-25°C, and the temperature of the Chinese soft-shelled turtle is rapidly cooled to 4-8°C within 0.5 hours after washing.

[0011] In some embodiments, in step S2, the temperature of the low-temperature water is 0-15°C.

[0012] The Chinese soft-shell turtle is a cold-blooded animal, and its body temperature changes with the changes in the external environment. In nature, the Chinese soft-shell turtle will choose a place with lower temperature to hibernate in winter, and the water temperature at this time is usually around 0-15℃. By setting the water temperature of the breeding environment within this range, it can well simulate the natural hibernation conditions, reduce the stress response caused by sudden temperature changes to the Chinese soft-shell turtle, and help maintain its normal physiological functions.

[0013] In some embodiments, in step S2, the depth of the low-temperature water is 3-10 cm higher than the skirt of the Chinese soft-shell turtle, and the water is replaced regularly, with a frequency of 5 days / time.

[0014] The Chinese soft-shell turtle is an amphibious animal, but it prefers shallow water areas during hibernation. Shallow water areas in nature (such as the edge of a pond, the gentle slope of a stream, etc.) usually have relatively stable temperatures and lower water flow velocities, which is consistent with an environment set at a depth of 3-10 cm above the skirt. By simulating such a natural habitat, the pressure on the Chinese soft-shell turtle to adapt to the new environment can be reduced, making it easier for it to enter and maintain a good hibernation state; the shallow water layer helps to increase the contact area between the water surface and the air, thereby enhancing the oxygen dissolution efficiency and ensuring sufficient dissolved oxygen in the water. This is especially important for the Chinese soft-shell turtle, because even in a low metabolic state, they still need a certain oxygen supply to maintain basic life activities. A water flow depth of 3-10 cm can ensure sufficient oxygen dissolution while avoiding the risk of hypoxia that may be caused by deep water.

[0015] In some embodiments, in step S2, when the Chinese soft-shell turtle is raised individually, it is wrapped in a dark nylon bag and placed in a low-temperature water environment of 0-15°C.

[0016] Although Chinese soft-shell turtles are aquatic animals, they will leave the water for a short period of time in certain circumstances (such as during hibernation or after waking up). At this time, maintaining appropriate air humidity is essential for the health of their skin and respiratory tract. The moisturizing properties of the wrapping bag help prevent the skin of Chinese soft-shell turtles from drying out and reduce skin damage or respiratory problems caused by dryness.

[0017] In some embodiments, in step S2, when the soft-shell turtle is raised individually, a ventilation hole is left in the dark nylon bag for the Chinese soft-shell turtle to breathe.

[0018] Although Chinese soft-shell turtles are aquatic animals, they can also breathe air through their lungs. Especially during hibernation or after waking up, when they leave the water, ensuring adequate oxygen supply is essential to maintaining their health. The design of the vents ensures air circulation in the cage, providing fresh oxygen and helping to prevent the risk of suffocation due to lack of oxygen.

[0019] In some embodiments, in step S2, the Chinese soft-shelled turtle is in a quiet and dark environment.

[0020] The specific steps to induce the Chinese soft-shelled turtle to enter hibernation are as follows: start at 20-25℃, quickly lower the temperature to 4-8℃, and pay attention to the water quality every day. If the water quality is polluted, perform isothermal water changes in time. During this period, try to keep a quiet and dark environment for the Chinese soft-shelled turtle.

[0021] Compared with the existing technology, the method of inducing hibernation by rapid cooling can quickly reduce the heart rate and blood oxygen concentration of the Chinese soft-shelled turtle, making the Chinese soft-shelled turtle enter a hibernation state. The Chinese soft-shelled turtle gradually enters a hibernation state, which is manifested as: the heart rate is less than 10 times in half a minute, and the blood oxygen concentration is less than 90%.

[0022] In some embodiments, while performing step S1 and step S2, the pond bottom mud is dried and disinfected by exposure to the sun.

[0023] Pond drying and disinfection and sun exposure can effectively kill bacteria, fungi, parasites and other pathogens in the pond mud, reduce the risk of disease transmission, and provide a healthier living environment for Chinese soft-shelled turtles. Pond drying can remove organic waste, leftover bait and excrement accumulated at the bottom of the pond. These substances will produce harmful substances such as ammonia nitrogen and nitrite during the decomposition process, affecting water quality. Disinfection and sun exposure further reduce the presence of these harmful substances and improve water quality.

[0024] In some embodiments, in step S3, the specific parameters for sludge disinfection are: 50-100 kg of quicklime is used per acre of pond, pits are dug in the pond at intervals of 5-10 m and a depth of 30-50 cm, quicklime is added and mixed with pond mud, water is added after reaction for 2 days, and the water depth is controlled to be 10-15 cm.

[0025] Compared with the prior art, the present invention adopts quicklime to disinfect the bottom mud, because quicklime is a strong alkaline substance, which can neutralize the acidic substances in the bottom mud and adjust the pH value to a range suitable for aquaculture. At the same time, quicklime reacts with water to generate calcium hydroxide, releasing heat. Calcium hydroxide denatures the protein of pathogens, thereby effectively killing pathogenic microorganisms, parasites and their eggs, and reducing the occurrence of aquatic animal diseases.

[0026] In some embodiments, in step S4, the operation method of creatine injection treatment is as follows: fix the knee joint of the hind limb of the Chinese soft-shelled turtle to expose the inguinal part below the plastron, and insert the needle through the hind limb muscle of the inguinal part parallel to the plastron; refer to the midline of the plastron, and the injection angle is between 20-60°.

[0027] Intraperitoneal injection is a method of directly injecting drugs into the body cavity. Compared with oral or subcutaneous injection, it can enter the blood circulation system faster. The injection solution passes through the muscles and then enters the abdominal cavity, which is conducive to wound healing and reduces infection. At the same time, this injection method is more in line with the physiological structure characteristics of the Chinese soft-shelled turtle. For cold-blooded animals such as the Chinese soft-shelled turtle, its digestive system slows down under low temperature conditions, and oral administration may not be effectively absorbed. Intraperitoneal injection can bypass the digestive tract, ensuring that creatine is absorbed quickly and efficiently, and quickly acts on target organs, such as the ovaries.

[0028] In some embodiments, in step S4, the average weekly creatine injection amount of the Chinese soft-shelled turtle is 20-400 mg / kg, and the duration is more than four weeks.

[0029] The lower starting dose (20 mg / kg) provides a larger safety margin and reduces the risk of adverse reactions due to overdose; while the higher upper limit (400 mg / kg) provides the possibility for individuals who need stronger stimulation; the frequency of once a week ensures that the drug concentration is maintained for a sufficient time without causing stress to the Chinese soft-shelled turtle too frequently. The weekly injection interval allows creatine to gradually accumulate and take effect in the body, while allowing the body to have enough time to adapt and metabolize the drug; eight weeks is a moderate treatment length, which is enough to observe obvious changes in ovarian development, and will not increase unnecessary costs and management difficulties due to excessive time. This period covers the key stages of the hibernation period of the Chinese soft-shelled turtle, and can make full use of the special physiological state during the hibernation period to promote ovarian development.

[0030] In some embodiments, in step S5, the specific steps of fertilizer and water management of the pond after disinfection are: after the water level in the pond is restored to a normal level and the water temperature is maintained between 15-20°C and the pH value is stabilized within the range of 6.5-7.5, the EM bacteria preparation is regularly sprayed into the pond. The EM bacteria preparation is composed of photosynthetic bacteria, lactic acid bacteria, yeast, and actinomycetes in a ratio of 3:3:2:2, with a frequency of once every 15-20 days, until the ambient temperature stabilizes at above 20°C.

[0031] Compared with the prior art, the present invention adjusts the water temperature and pH value to make the activity of EM bacteria in a more ideal state, and can better maintain the synergistic effect between 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 and can synthesize nutrients such as sugars and amino acids; lactic acid bacteria absorb sugars produced by photosynthetic bacteria and yeast to form lactic acid, which has a strong bactericidal ability and can also ferment and decompose organic matter, and can inhibit the proliferation of pathogenic bacteria, which is beneficial to the growth of Chinese soft-shelled turtles; yeast uses organic substances such as amino acids synthesized by photosynthetic bacteria to provide a matrix for the proliferation of other effective microorganisms, and the single-cell protein produced by it is an indispensable nutrient for Chinese soft-shelled turtles; actinomycetes can produce a variety of antibiotics and enzyme substances, inhibit the growth of harmful bacteria, enhance the immunity of Chinese soft-shelled turtles, improve their disease resistance, and promote the circulation and transformation of nutrients in the water body, providing a better living environment for the healthy growth of Chinese soft-shelled turtles.

[0032] In some embodiments, in step S6, the creatine injection treatment is completed, and when the average temperature is stable at 20°C or above for seven consecutive days, the 4-8°C hibernation environment of the Chinese soft-shelled turtle is quickly heated to above 25°C, and Chinese soft-shelled turtles are released into the pond at a male-to-female ratio of 5-30:1.

[0033] Compared with the prior art, the present invention can ensure that there are enough males to participate in mating by controlling the male-female ratio (such as 5-30:1), and at the same time, the growth behavior of female Chinese soft-shelled turtles is relatively docile, which helps to reduce the injuries of the turtles caused by fighting. This can significantly improve the fertilization rate and hatching success rate. At the same time, when the ambient temperature is stable at above 20°C, it can create suitable living conditions for Chinese soft-shelled turtles, thereby effectively promoting their healthy growth and good development. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure is the result of histological analysis of follicle development of female Chinese soft-shelled turtle in Example 1 and the control group; Figure 2 The figure is the analysis result of the follicle development of female Chinese soft-shelled turtle in Example 1 and the control group; Figure 3 The figure is the analysis result before and after the improvement of the pond in Example 1; Figure 4 The figure is the result of histological analysis of follicle development of female Chinese soft-shelled turtle in Example 2 and the control group; Figure 5 The figure is the analysis result of the follicle development of female Chinese soft-shelled turtle in Example 2 and the control group; Figure 6 The figure is the analysis result before and after the improvement of the pond in Example 2; Figure 7The figure is the result of histological analysis of follicle development of female Chinese soft-shelled turtle in Example 3 and the control group; Figure 8 The figure is the analysis result of the follicle development of female Chinese soft-shelled turtle in Example 3 and the control group; Fig. 9 The figure is the analysis result before and after the improvement of the pond in Example 3; Fig.10 This is a graph showing the results of the analysis of changes in the liver transcriptome of the female Chinese soft-shelled turtle in Example 3. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the 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 method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0036] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0037] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0038] It is worth mentioning that all statistical methods in this embodiment are summarized as follows: 1. Detection of physiological indicators of hibernation induction: Use a thermometer to measure the water temperature every day, clamp the turtle's tail with a finger clip oximeter, and record the heart rate and blood oxygen concentration of the Chinese soft-shelled turtle within 20 seconds.

[0039] 2. Ovarian development statistics: The ovaries of Chinese soft-shelled turtles before injection and the ovaries of different groups after injection were statistically analyzed for the number and diameter of follicle particles using a fiber imaging system.

[0040] 3. Hematoxylin-eosin (H&E) staining: Ovarian tissue was embedded in paraffin according to standard methods, sliced ​​with a microtome, then unfolded and dewaxed, and then stained with xylene, ethanol, gradient ethanol and distilled water, followed by hematoxylin staining for 30 seconds, eosin staining for 2 seconds, and then dehydrated with gradient ethanol, xylene transparent, and neutral gum sealing. Morphological changes were observed using an optical microscope and photographed.

[0041] IV. Glycogen content determination: (1) Tissue grinding: Grind liver, skeletal muscle and ovarian tissue samples; (2) Glycogen extraction: Add KOH to the ground samples and place in a boiling water bath; (3) Glycogen content determination: Take three centrifuge tubes, namely blank tube, standard tube and sample tube, treat them with anthrone solution, place in a boiling water bath, cool and measure the absorbance. (4) Calculation: Glycogen (g / 100 g tissue) = (A sample / A standard) × 0.05 × (100 / 0.05) × (50 / 1000) × 1.11.

[0042] V. Determination of lactic acid content: (1) Glycolysis of tissues: Take liver, skeletal muscle and ovarian tissues and add them to sample tubes and control tubes respectively for homogenization. Add trichloroacetic acid to the control tube and mix well, then add starch solution to the sample and control tubes respectively, stir well, add liquid paraffin, keep warm, take out and immediately add trichloroacetic acid to each sample tube and mix well, centrifuge and take the supernatant. Add saturated copper sulfate solution and calcium hydroxide powder to each tube, stir well, let stand to allow the reducing sugar to precipitate completely, centrifuge and take the supernatant for later use. (2) Determination of lactic acid: Make parallel tubes for each sample tube and control tube, add concentrated sulfuric acid to each tube and put them in an ice bath, then add supernatant, mix well, put them in a boiling water bath, cool, add p-hydroxybiphenyl colorimetric solution, keep warm in a water bath, then quickly put them in a boiling water bath, cool, use the control tube as a blank and use an ultraviolet spectrophotometer to measure the absorbance of the sample tube and analyze the results.

[0043] VI. Creatine kinase (CK) activity assay: (1) Preparation of substrate solution Required reagents: creatine solution, magnesium acetate, thymol blue, Gly-NaOH buffer, ATP, distilled water, NaOH solution. (2) Protein extraction: Take liver, skeletal muscle, plasma and ovary tissues and place them in a centrifuge tube, add lysis solution and grind them into a homogenate. Place them on ice for lysis, centrifuge and take the supernatant. (3) Experimental operation: Add substrate solution and enzyme solution to a micro-cuvette, mix well and detect the change in absorbance, and perform kinetic analysis.

[0044] VII. Determination of ammonia nitrogen content: Pipette 0.0, 0.5, 1.0, 3.0, 5.0, 7.0 and 10.0 mL of ammonium standard solution into a 50 mL colorimetric tube, dilute with pure water, add 1.0 mL of potassium sodium tartrate solution, mix well, add 1.5 mL of Nessler's reagent, mix well, let stand for 10 min, measure the absorbance at a wavelength of 420 nm with pure water as blank, and draw 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, add 1.5 mL of Nessler's reagent, mix well, let stand for 10 min, and measure the absorbance in the same way as the standard curve.

[0045] 8. Determination of nitrite: Take 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5 mL of sodium nitrite standard solution in a 50 mL volumetric flask, add water to 25 mL each, 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, dilute to 50 mL with pure water, shake well and let stand for 15 min, measure the absorbance at a wavelength of 540 nm with a spectrophotometer, use distilled water as a blank, and make a standard curve of nitrite concentration. Take 40 mL of pretreated water sample in a 50 mL volumetric flask, add 2 mL of 0.4% p-aminobenzenesulfonic acid solution, shake well and let stand for 3-5 minutes, then add 1 mL of 0.2% naphthylethylenediamine hydrochloride solution, measure the absorbance, and calculate the nitrite content in the sample through the standard curve.

[0046] The following is further described in conjunction with specific embodiments.

[0047] Example 1 This embodiment provides a method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation, comprising the following steps: S1. Catch the female sexually mature Chinese soft-shelled turtle after it enters the hibernation period, clean the surface of the turtle, quickly cool it down for 0.5 h, and store it for observation; S2. Healthy Chinese soft-shelled turtles were raised individually, with the water depth 3 cm higher than the edge of the turtle's skirt and the ambient temperature at 12°C. They were wrapped in dark nylon bags and placed in a low-temperature water environment at 12°C. There were vents in the bag for the turtle to breathe. During the process of rapidly inducing the Chinese soft-shelled turtle to enter hibernation, the water temperature was measured daily using a thermometer, and the turtle's tail was clamped with a finger-clip oximeter to record the turtle's heart rate and blood oxygen concentration within 20 seconds.

[0048] S3, After the Chinese soft-shell turtle pond is cleaned, the pond water is drained, and the bottom mud is disinfected and improved. For the bottom mud disinfection, 70 kg of quicklime is used per mu of pond. Pit holes are dug in the pond at intervals of 7 m and the depth is 40 cm. Quicklime is added and mixed with pond mud. After reacting for 2 days, water is introduced and the water depth is controlled to be 10-15 cm. S4. The Chinese soft-shell turtle was injected with creatine into the abdominal cavity. The specific parameters of the creatine injection treatment were as follows: 400 mg of creatine was injected per kg of Chinese soft-shell turtle body weight, once a week, and for eight weeks. The hibernation induction treatment was as follows: starting from 20°C, the temperature was rapidly reduced to 12°C in 2 days, and the isothermal water was replaced once. After the hibernation ended, the ovaries of the Chinese soft-shell turtle were taken for weight, follicle diameter, number analysis, and histological analysis.

[0049] S5. The pond after disinfection is stored with water and fertilized. The fertilization and water management operation is as follows: after the disinfection in step S3 is completed, the water level of the pond is restored to a normal level, and the water temperature is maintained between 15-20°C, and the pH value is stabilized within the range of 6.5-7.5, and then the EM bacteria preparation is sprayed regularly in the pond. The EM bacteria preparation is composed 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 body are analyzed using the Nessler reagent method and the naphthalene-ethylenediamine hydrochloride method.

[0050] Preferably, while carrying out steps S1 and S2, the pond bottom mud is dried and disinfected and exposed to the sun. After the creatine injection treatment described in step S4 is completed and the average temperature is stable at 20°C or above for seven consecutive days, the Chinese soft-shelled turtles are placed in a pond that has been dried and disinfected and exposed to the sun at a certain male-female ratio.

[0051] Example 2 This embodiment provides a method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation, comprising the following steps: S1. Catch the female sexually mature Chinese soft-shelled turtle after it enters the hibernation period, clean the surface of the turtle, quickly cool it down for 0.5 h, and store it for observation; S2. Healthy Chinese soft-shell turtles were raised individually in a water environment 3 cm deeper than the edge of the turtle's skirt. The temperature of the environment was 12°C. The turtles were wrapped in a dark nylon bag and placed in a low-temperature water environment at 12°C. There were ventilation holes in the bag for the turtles to breathe. S3, After the Chinese soft-shell turtle pond is cleaned, the pond water is drained, and the bottom mud is disinfected and improved. For the bottom mud disinfection, 70 kg of quicklime is used per mu of pond. Pit holes are dug in the pond at intervals of 7 m and the depth is 40 cm. Quicklime is added and mixed with pond mud. After reacting for 2 days, water is introduced and the water depth is controlled to be 10-15 cm. S4. The Chinese soft-shell turtle was injected with creatine into the abdominal cavity. The specific parameters of the creatine injection treatment were as follows: 200 mg of creatine was injected per kg of Chinese soft-shell turtle body weight, once a week, and the duration was eight weeks. The hibernation induction treatment was as follows: starting at 20°C, the temperature was rapidly reduced to 12°C in 2 days, and the isothermal water was replaced once. After hibernation, the ovaries of the Chinese soft-shell turtle were taken for ovarian weight, follicle diameter, and histological analysis; at the same time, the continuous photometry method was used to kinetically analyze the creatine kinase activity in the muscles, liver, and ovaries of the Chinese soft-shell turtle; the Trizol method was used to extract the liver RNA of the Chinese soft-shell turtle for transcriptome analysis.

[0052] S5. The pond after disinfection is stored with water and fertilized. The fertilization and water management operation is as follows: after the disinfection in step S3 is completed, the water level of the pond is restored to a normal level, while ensuring that the water temperature is maintained between 15-20°C and the pH value is stabilized within the range of 6.5-7.5. Then, the EM bacteria preparation is regularly sprayed in the pond. The EM bacteria preparation is composed 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 are analyzed using the Nessler reagent method and the naphthalene-ethylenediamine hydrochloride method.

[0053] Preferably, while carrying out steps S1 and S2, the pond bottom mud is dried and disinfected and exposed to the sun. After the creatine injection treatment described in step S4 is completed and the average temperature is stable at 20°C or above for seven consecutive days, the Chinese soft-shelled turtles are placed in a pond that has been dried and disinfected and exposed to the sun at a certain male-female ratio.

[0054] Example 3 This embodiment provides a method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation, comprising the following steps: S1. Catch the female sexually mature Chinese soft-shelled turtle after it enters the hibernation period, clean the surface of the turtle, quickly cool it down for 0.5 h, and store it for observation; S2. Healthy Chinese soft-shell turtles were raised individually in a water environment 3 cm deeper than the edge of the turtle's skirt. The temperature of the environment was 12°C. The turtles were wrapped in a dark nylon bag and placed in a low-temperature water environment at 12°C. There were ventilation holes in the bag for the turtles to breathe. S3, After the Chinese soft-shell turtle pond is cleaned, the pond water is drained, and the bottom mud is disinfected and improved. For the bottom mud disinfection, 70 kg of quicklime is used per mu of pond. Pit holes are dug in the pond at intervals of 7 m and the depth is 40 cm. Quicklime is added and mixed with pond mud. After reacting for 2 days, water is introduced and the water depth is controlled to be 10-15 cm. S4. Inject creatine into the abdominal cavity of Chinese soft-shelled turtles. The specific parameters of creatine injection are as follows: the creatine injection volume is 100 mg per kg of Chinese soft-shelled turtle body weight, once a week, and the duration is eight weeks. The hibernation induction treatment is as follows: start at 20℃, quickly drop to 12℃ in 2 days, and change isothermal water once. After hibernation, the ovaries of Chinese soft-shelled turtles were taken for histological analysis; at the same time, the anthrone colorimetric method and the p-hydroxybiphenyl colorimetric method were used to analyze the glycogen and lactic acid content in the muscles, liver, and ovaries of Chinese soft-shelled turtles.

[0055] S5. The pond after disinfection is stored with water and fertilized. The fertilization and water management operation is as follows: after the disinfection in step S3 is completed, the water level of the pond is restored to a normal level, while ensuring that the water temperature is maintained between 15-20°C and the pH value is stabilized within the range of 6.5-7.5. Then, the EM bacteria preparation is regularly sprayed in the pond. The EM bacteria preparation is composed 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 are analyzed using the Nessler reagent method and the naphthalene-ethylenediamine hydrochloride method.

[0056] Preferably, while carrying out steps S1 and S2, the pond bottom mud is dried and disinfected and exposed to the sun. After the creatine injection treatment described in step S4 is completed and the average temperature is stable at 20°C or above for seven consecutive days, the Chinese soft-shelled turtles are placed in a pond that has been dried and disinfected and exposed to the sun at a certain male-female ratio.

[0057] Control group The control group used female Chinese soft-shelled turtles that had not been treated in any way. The female Chinese soft-shelled turtles before treatment in Example 1 were tested for physiological indicators during the hibernation induction process, and the analysis results are shown in Table 1.

[0058]

[0059] The analysis results are shown in Table 1. Indicators such as oral temperature, heart rate in half a minute and blood oxygen concentration are at hibernation levels, indicating that the Chinese soft-shelled turtle entered a hibernation state at 12°C.

[0060] The female Chinese soft-shelled turtles of the control group and the Example 1 group were subjected to histological analysis of follicular development. The analysis results are shown in FIG. Figure 1 As shown, Figure 1 A-1 and A-2 are histological analysis results of follicle development of female Chinese soft-shelled turtles in the control group. Figure 1 B-1 and B-2 in the figure are histological analysis results of follicular development of female Chinese soft-shelled turtle in Example 1, Figure 1 The designations in the figure are as follows: ZP: zona pellucida; TC: follicular sheath; → Y: yolk granules; V: vacuoles; GC: granulosa layer of follicular cells; OP: homogeneous cytoplasm; (scale bars are 100 μm). Figure 1It 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).

[0061] The female Chinese soft-shelled turtles of the control group and the Example 1 group were analyzed. The analysis results are as follows: Figure 2 As shown, Figure 2 A in the figure is a comparison of the appearance of ovarian follicle development; Figure 2 B in the figure is 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 ovary weight of female Chinese soft-shelled turtles also increased after creatine treatment (P<0.05).

[0062] The water samples from the pond in Example 1 were taken before and after treatment and analyzed. The results are as follows: Figure 3 As shown, Figure 3 A-1 is a comparative analysis of ammonia nitrogen content in water samples. Figure 3 A-2 is 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 content and nitrite content in the pond decreased after treatment (P<0.05).

[0063] The female Chinese soft-shelled turtles of the control group and the Example 2 group were subjected to histological analysis of follicular development. The analysis results are shown in FIG. Figure 4 As shown, Figure 4 A-1 and A-2 are histological analysis results of follicle development of female Chinese soft-shelled turtles in the control group. Figure 4 B-1 and B-2 in the figure are the histological analysis results of the follicle development of the female Chinese soft-shelled turtle in Example 2, Figure 3 The designations in the figure are as follows: ZP: zona pellucida; TC: follicular sheath; → Y: yolk granules; V: vacuoles; GC: granulosa layer of follicular cells; OP: homogeneous cytoplasm; (scale bars are 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).

[0064] The female Chinese soft-shelled turtles of the control group and the Example 2 group were analyzed. The analysis results are as follows: Figure 5 As shown, Figure 5 A in the figure is a comparison of the appearance of ovarian development; Figure 5 B in it is the analysis of changes in glycogen content in muscle, liver, ovary, and blood; Figure 5The C in the figure is the analysis of changes in lactic acid content in muscle, liver, ovary, 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 after creatine treatment, the follicle diameter and ovarian weight of female Chinese soft-shelled turtles increased significantly (P<0.05), and the glycogen content in the tissue increased significantly (P<0.05).

[0065] The water samples from the pond in Example 2 were taken before and after treatment and analyzed. The results are as follows: Figure 6 As shown, Figure 6 A-1 is a comparative analysis of ammonia nitrogen content in water samples. Figure 6 A-2 is 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 content and nitrite content in the pond decreased after treatment (P<0.05).

[0066] The female Chinese soft-shelled turtles of the control group and the Example 3 group were subjected to histological analysis of follicular development. The analysis results are shown in FIG. Figure 7 As shown, Figure 7 A-1 and A-2 are histological analysis results of follicle development of female Chinese soft-shelled turtles in the control group. Figure 7 B-1 and B-2 in the figure are the histological analysis results of the follicle development of the female Chinese soft-shelled turtle in Example 3, Figure 7 The designations in the figure are as follows: ZP: zona pellucida; TC: follicular sheath; → Y: yolk granules; V: vacuoles; GC: granulosa layer of follicular cells; OP: homogeneous cytoplasm; (scale bars are 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).

[0067] The female Chinese soft-shelled turtles of the control group and the Example 3 group were analyzed. The analysis results are as follows: Figure 8 As shown, Figure 8 A in the figure is a comparison of the appearance of ovarian development; Figure 8 B in the figure is the comparative analysis of ovarian weight and follicle diameter; Figure 8 The C in the figure is the analysis of changes in creatine kinase activity in muscle, liver, ovary, and blood (in the same figure, different lowercase letters indicate statistically significant differences between the two groups, P < 0.05). Figure 8 It can be seen that after creatine treatment, the follicle diameter and ovarian weight increased significantly (P<0.05), and the creatine kinase activity also increased significantly (P<0.05).

[0068] The water samples of the pond in Example 3 were taken before and after treatment and analyzed. The results are as follows: Fig. 9As shown, Fig. 9 A-1 is a comparative analysis of ammonia nitrogen content in water samples. Fig. 9 A-2 is 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). Fig. 9 It can be seen that the ammonia nitrogen content and nitrite content in the pond decreased after treatment (P<0.05).

[0069] Fig.10 The following is an analysis of the changes in the liver transcriptome of female Chinese soft-shelled turtles after treatment with 100 mg / kg creatine. A is a volcano plot for differentially expressed gene analysis; B is a bar chart of GO enrichment of differentially expressed genes; and C is a scatter plot of KEGG enrichment of differentially expressed genes.

[0070] from Fig.10 It can be seen that the transcriptome analysis of the liver identified 4355 differentially expressed genes (Figure 10A), among which genes such as VTG, LEPR, LHR, FSHR and CK-B changed significantly. KEGG pathways were enriched in adipose cell factor signaling pathways, oxidative phosphorylation pathways, GnRH signaling pathways and PPAP signaling pathways (Figure 10C). The differentially expressed genes and enriched pathways are related to lipid metabolism and energy metabolism, which can further affect the development of the ovary. Therefore, creatine can have a significant positive effect on the development of the ovary of Chinese soft-shelled turtle under low temperature conditions.

[0071] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for promoting ovarian development and healthy breeding of Chinese soft-shelled turtles during hibernation, characterized in that: The method comprises the following steps: S1. Catch the female sexually mature Chinese soft-shelled turtle after it enters the hibernation period and clean the surface of the turtle; S2, raising healthy Chinese soft-shelled turtles in a low-temperature water environment in a single package, and then inducing the Chinese soft-shelled turtles to hibernate under low-temperature conditions; S3. After the pond is cleaned, drain the water and perform deep disinfection and improvement of the bottom mud during the hibernation season. S4, intraperitoneal injection of creatine into Chinese soft-shell turtle; S5. Carry out water storage and fertilizer management on the disinfected pond; S6. When the temperature is suitable, release Chinese soft-shelled turtles treated with creatine in proportion.

2. The method according to claim 1, characterized in that In step S1, the water temperature for washing the turtle body is 20-25°C, and the temperature of the Chinese soft-shelled turtle is rapidly reduced to 4-8°C within 0.5 h after washing.

3. The method according to claim 1, characterized in that In step S2, the temperature of the low-temperature water is 0-15°C, and the depth of the low-temperature water is 3-10 cm higher than the skirt of the Chinese soft-shelled turtle.

4. The method according to claim 1, characterized in that In step S2, when the soft-shell turtle is raised individually, it is wrapped in a dark nylon bag and placed in a low-temperature water environment of 0-15°C.

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

6. The method according to claim 1, characterized in that In step S3, the specific parameters for the disinfection of the bottom mud are: 50-100 kg of quicklime is used per mu of pond, pits are dug in the pond at intervals of 5-10 m, the pit depth is 30-50 cm, quicklime is added and mixed with the pond mud, water is added after reaction for 2 days, and the water depth is controlled to be 10-15 cm.

7. The method according to claim 1, characterized in that In step S4, the operation method of creatine injection treatment is as follows: fix the knee joint of the hind limb of the Chinese soft-shelled turtle to expose the groin under the plastron, and insert the needle through the hind limb muscle of the groin parallel to the plastron; refer to the midline of the plastron, and the injection angle is between 20-60 degrees.

8. The method according to claim 1, characterized in that In step S4, the average weekly creatine injection amount of the Chinese soft-shelled turtle is 20-400 mg / kg, and the duration is more than four weeks.

9. The method according to claim 1, characterized in that In step S5, the specific steps of fertilizer and water management of the pond after disinfection are as follows: after step S3, after the water level of the pond is restored to a normal level and the water temperature is maintained between 15-20°C and the pH value is stabilized within the range of 6.5-7.5, the EM bacteria preparation is regularly sprayed into the pond. The EM bacteria preparation is composed 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.

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

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