A method for targeted mating and egg-bearing culture of Procambarus clarkii adapted to extremely cold environments

Abstract

This invention discloses a method for targeted mating and egg-carrying culture of *Procambarus clarkii* adapted to extremely cold environments, belonging to the field of *Procambarus clarkii* aquaculture technology. This invention aims to solve the technical problems of low overwintering survival rate, asynchronous gonad development, and low mating and egg-carrying rate of *Procambarus clarkii* in extremely cold and prolonged ice-covered environments. The method includes summer and autumn energy storage and screening, overwintering low-temperature acclimatization, overwintering control during the prolonged ice period, spring gradient awakening, and spring gonad maturation and reproduction. Through gradient quantitative feeding and precise selection of broodstock, step-by-step water level raising to guide directional burrowing, ice surface snow removal to maintain light penetration for biological oxygenation, controlled water temperature gradient, and differentiated feeding of maturation-promoting feed, the method achieves safe overwintering and synchronous gonad maturation of broodstock. Once the water temperature and conditions meet the requirements, concentrated mating and egg-carrying are initiated. This invention can significantly improve the overwintering survival rate and egg-carrying rate of *Procambarus clarkii* in extremely cold regions, providing a feasible technical solution for large-scale artificial breeding of *Procambarus clarkii* in extremely cold areas.

Description

Technical Field

[0001] This invention relates to the field of red swamp crayfish farming technology, specifically to a method for directional mating and egg-bearing farming of red swamp crayfish adapted to extremely cold environments. Background Technology

[0002] The red swamp crayfish (Procambarus clarkii), commonly known as the crayfish, has become an important freshwater aquaculture species in my country due to its rapid growth, strong environmental adaptability, and high economic value. A complete industrial chain covering aquaculture, processing, and catering has been formed, and market demand remains strong. With the continuous expansion of the domestic consumer market, the demand for red swamp crayfish in northern regions has been increasing year by year, driving the development of the artificial breeding industry in northern China. However, the long, cold winters and extended ice periods in the extremely cold regions of northern my country pose severe challenges to the artificial breeding of red swamp crayfish. Although red swamp crayfish can survive in water temperatures ranging from 0℃ to 35℃, in the extremely cold and extended ice periods, they face three major technical challenges: low overwintering survival rate, asynchronous gonad development, and low mating and egg-carrying rate. These challenges severely restrict the development of large-scale artificial breeding and aquaculture of red swamp crayfish in extremely cold regions.

[0003] To address the low overwintering survival rate of *Procambarus clarkii* in high-altitude and cold regions, relevant technical solutions have been proposed. For example, invention patent CN112544515A discloses a method for overwintering broodstock of *Procambarus clarkii* in cold-region outdoor ponds under ice. This method, through measures such as selecting overwintering ponds of specific sizes, setting up two escape-proof nets, controlling the water level and ice layer changes during overwintering, and regularly clearing snow to maintain the light transmittance of the ice layer, increases the overwintering survival rate of broodstock of *Procambarus clarkii* to 60%-80%, solving the basic survival problem of *Procambarus clarkii* in high-altitude and cold regions.

[0004] However, the aforementioned existing technologies still have significant limitations: First, they focus solely on improving overwintering survival rates, neglecting the scientific energy storage and precise selection of broodstock before overwintering. This results in severe energy depletion and inconsistent physical condition among the broodstock after overwintering. Second, these technologies lack a coordinated management plan for nutritional regulation and gonadal development before and after overwintering, and lack a systematic spring gonadal maturation technique, making it impossible to achieve synchronous gonadal development and leading to dispersed mating times and low egg-carrying rates. Third, they lack a scientific spring gradient awakening mechanism; sudden increases in water temperature can easily trigger stress responses in broodstock, further reducing reproductive efficiency. These problems make large-scale artificial breeding of *Procambarus clarkii* in extremely cold regions difficult to achieve, failing to meet the local aquaculture industry's demand for seedlings.

[0005] Therefore, there is an urgent need to develop a comprehensive aquaculture technology solution that can simultaneously address the problems of low overwintering survival rate, asynchronous gonadal development, and low mating and egg-carrying rate of Procambarus clarkii in extremely cold and long ice-covered environments, providing technical support for the large-scale artificial breeding of Procambarus clarkii in extremely cold regions. Summary of the Invention

[0006] The purpose of this invention is to provide a method for directional mating and egg-bearing culture of Procambarus clarkii adapted to extremely cold environments, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides a method for targeted mating and egg-bearing culture of Procambarus clarkii adapted to extremely cold environments, comprising the following steps: S1. Summer and Autumn Energy Storage Cultivation and Screening: From late May to mid-September, use protein-specific energy storage feed to feed 1-year-old sub-adult Procambarus clarkii populations according to water temperature gradient, and use non-destructive live appearance screening method to select healthy broodstock. The male-to-female ratio of broodstock is controlled at 2:2.5-3. S2. Overwintering and low-temperature acclimatization: From late September to the end of October, replace the feed with low-protein crude fiber feed and reduce the amount of feed. Stop feeding completely 7 days before the ice freezes. At the same time, raise the water level of the breeding pond in stages and guide the parent shrimp to dig burrows in a directional manner on the slope 60cm-75cm below the ice. S3. Overwintering Management During the Long Ice Period: From November to mid-March of the following year, the ice-covered overwintering stage is entered. During this period, feeding, breaking the ice and human disturbance are prohibited throughout the entire process. Snow and ice accumulation after snowfall are cleared in a timely manner to ensure the light transmittance of the ice layer, which is used to ensure long-term biological oxygenation for photosynthesis of algae under the ice.

[0008] S4. Spring Gradual Awakening: From mid-March to the end of March of the following year, the ice layer naturally melts, controlling the daily temperature rise of the water body to ≤0.3℃, while the water level is slowly lowered and the water quality is stabilized, and the parent shrimp emerge from their burrows one after another; S5. Spring gonadal maturation and reproduction: Starting from the end of March, feed high-energy and high-protein maturation feed according to the water temperature. After the water temperature has been stable at 10℃-14℃ for more than 5 consecutive days, the water quality meets the standards, and the female shrimp gonads have matured, start centralized mating. After mating, the female parent shrimp will complete the egg laying, fertilization, and lappa egg-bearing in sequence.

[0009] Preferably, the nutritional composition of the protein-specific energy storage feed, by mass percentage, is as follows: crude protein 34%-36%, crude fat 5.0%-6.0%, calcium 1.8%-2.2%, total phosphorus 1.0%-1.3%, vitamin C 0.02%-0.025%, β-carotene 0.004%-0.006%, and aquatic compound minerals 1.2%-1.5%; the aquatic compound minerals include at least magnesium, potassium, iron, zinc, copper, manganese, and selenium.

[0010] Preferably, the standards for quantitative feeding based on water temperature gradients and differentiated feeding based on water temperature are as follows: The standard for quantitative feeding based on water temperature gradients is as follows: When the water temperature is ≥18℃, feed once a day in the evening, with the amount of protein-specific energy-storing feed being 2.0%-2.5% of the total body weight of the shrimp; when the water temperature is >12℃ and <18℃, feed once a day in the evening, with the amount of protein-specific energy-storing feed being 1.0%-1.5% of the total body weight of the shrimp; when the water temperature is <12℃, feed once every two days in the evening, with the amount of protein-specific energy-storing feed being 0.5%-0.8% of the total body weight of the shrimp. The standard for differentiated feeding based on water temperature is as follows: When the water temperature is 5℃-8℃, feed once every two days in the evening, with the amount of high-energy, high-protein growth-promoting feed being 1.0%-1.2% of the total body weight of the shrimp; when the water temperature is >8℃, feed once a day in the evening, with the amount of high-energy, high-protein growth-promoting feed being 1.8%-2.2% of the total body weight of the shrimp.

[0011] Preferably, the non-destructive live appearance screening method is as follows: select adult shrimp with vigorous activity, rapid response to external stimuli, shiny and hard shells, intact appendages without defects, no attached substances, no obvious external injuries or parasites as a candidate group of broodstock shrimp, and screen female and male broodstock shrimp from the candidate group of broodstock shrimp. The selection criteria for female parent shrimp are: weight ≥45g, body length ≥10cm, gonads in ovarian stage II, abdominal carapace above the third walking leg is light yellowish-brown and slightly swollen, genital opening is slightly convex, and there is no blackening or ulceration of the abdomen. The selection criteria for male broodstock shrimp are: weight ≥ 50g, body length ≥ 11cm, strong and powerful second walking legs with no missing chelipeds, hard, milky white, and undamaged specialized copulatory organ of the first abdominal appendage, and clearly visible genital opening.

[0012] Preferably, the low-protein crude fiber feed is prepared using corn, soybean meal, and plant crude fiber as the main raw materials, with the optional addition of animal-derived raw materials such as miscellaneous fish. Its crude protein content is 26%-29% by mass. The standard for reducing feed is: feeding once every 3 days in the evening, with the amount of low-protein crude fiber feed being 0.3%-0.5% of the total body weight of the shrimp, to gradually clear the intestinal metabolites of the broodstock shrimp and reduce the oxygen consumption load of the broodstock shrimp during the ice period of winter.

[0013] Preferably, the bank slope ratio of the aquaculture pond is 1:2.5-3.0, and the surface of the bank slope is covered with a 20cm thick heavy antifreeze and heat insulation layer, which is made by mixing and compacting deep black clay and decomposed peat in a 7:3 ratio.

[0014] Preferably, the staged, stepped water level raising is as follows: Before the expected freezing date, the water level in the breeding pond should be kept constant at 0.6m-0.8m to induce the broodstock shrimp to start burrowing behavior, so that the burrowing location of the broodstock shrimp is in the effective water layer of the bank slope under the ice later. When the ice layer in the aquaculture pond reaches a thickness of 1.5cm for the first time, raise the water level in the aquaculture pond to 1.5m. When the ice layer in the aquaculture pond reaches 2cm for the first time, raise the water level in the aquaculture pond to 2.2m-2.5m, and keep the water level in the aquaculture pond stable thereafter.

[0015] Preferably, the water quality standards for the stable water quality are: pH 7.5-8.3, dissolved oxygen ≥ 4.0 mg / L, ammonia nitrogen < 0.2 mg / L and nitrite < 0.05 mg / L.

[0016] Preferably, the high-energy, high-protein ripening feed is used for targeted ripening of broodstock gonads and repair of overwintering physical damage in broodstock. Its nutritional composition by mass percentage is: crude protein 38%-42%, crude fat 7%-9%, calcium 2.3%-2.9%, available phosphorus 1.4%-1.8%, vitamin E 0.015%-0.02%, and lecithin 0.8%-1.0%.

[0017] Preferably, the gonadal maturity of the female shrimp population refers to the gonads of more than 80% of the female shrimp in the population developing to the ovarian stage V; the conditions for the water body to meet the standards are: dissolved oxygen ≥ 5.0 mg / L, transparency 25cm-35cm, and maintaining a micro-flow of water with a daily water exchange rate of 5%-8% in the aquaculture pond.

[0018] Preferably, the method for directional mating and berried culture of Procambarus clarkii further includes disease control measures: In S1, the broodstock are soaked in 3%-5% saline solution for 3-5 minutes before being introduced into the pond; in S2, a mixture of 400 grams of salt and 400 grams of baking soda is applied to the entire pond per cubic meter of water before the pond is sealed with ice; in S3, the water quality under the ice is monitored weekly to ensure dissolved oxygen ≥3.5 mg / L; and in S4, quicklime is applied to the entire pond once a week after the broodstock emerge from their burrows, at a rate of 25 mg / L.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention establishes a scientific energy storage and precise screening system for broodstock shrimp before overwintering, solving the problems of severe energy loss and inconsistent physical condition after overwintering caused by neglecting the early-stage energy storage of broodstock shrimp in existing technologies. By combining protein-specific energy storage feed with quantitative feeding based on water temperature gradients, it avoids damage to the hepatopancreas at low temperatures and promotes stable gonadal development in female broodstock shrimp to ovarian stage II. At the same time, a non-invasive live appearance screening method is used to screen broodstock shrimp from multiple dimensions, including weight, body length, gonads, and reproductive characteristics, and to precisely control the male-to-female ratio, ensuring reproductive potential from the source.

[0020] 2. This invention constructs a systematic spring gradient awakening and gonadal synchronous maturation technology system, solving the problems of strong stress response, asynchronous gonadal development, scattered mating time, and low egg-carrying rate in broodstock shrimp caused by the lack of scientific awakening mechanisms and gonadal synchronous regulation methods in existing technologies. By strictly controlling the daily water temperature rise to ≤0.3℃ and coordinating with a gradual decrease in water level to guide broodstock shrimp to gradually emerge from their burrows, stress and ovarian absorption degeneration caused by sudden increases in water temperature are effectively avoided. At the same time, high-energy, high-protein maturation feed is used and fed according to water temperature differences to specifically repair the physical damage of broodstock shrimp during overwintering and promote gonadal development. Combined with the concentrated mating trigger mechanism that achieves 80% of female shrimp gonads reaching ovarian stage V, synchronous mating and concentrated egg-carrying of broodstock shrimp gonads are achieved, significantly improving reproductive efficiency.

[0021] 3. This invention innovates a directional overwintering management technology for ultra-long ice periods, solving the problems of poor insulation and passive oxygenation in existing overwintering burrows. By raising the water level in stages, the broodstock shrimp are guided to burrow directionally in a constant temperature layer of 60cm-75cm below the ice. Combined with a heavy antifreeze and heat insulation layer on the bank slope, the temperature of the shrimp burrows can be maintained stably under extreme low temperatures. At the same time, snow removal from the ice surface ensures light transmittance, and long-term biological oxygenation is achieved through algal photosynthesis. Human disturbance is minimized throughout the process, reducing the energy consumption of the broodstock shrimp to the greatest extent. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the overall steps of the method for directional mating and egg-bearing culture of Procambarus clarkii adapted to extremely cold environments in this invention. Figure 2 This is a schematic cross-sectional view of the aquaculture pond according to an embodiment of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] Examples, such as Figure 1 This embodiment provides a method for targeted mating and egg-bearing culture of *Procambarus clarkii* adapted to extremely cold environments. It is applicable to the large-scale artificial breeding and culture of *Procambarus clarkii* in northern high-altitude and extremely cold regions where winter temperatures are extremely low, ice thickness can reach 80cm-120cm, and the ice-covered period can last up to 5 months. The method includes summer and autumn energy storage and screening, overwintering low-temperature acclimatization, overwintering management during the extended ice period, spring gradient awakening, and spring gonadal maturation and breeding. This embodiment is applied to the extremely cold aquaculture area of ​​Northeast China, and the specific implementation steps are as follows: S1. Summer and Autumn Energy Storage and Screening (Late May to Mid-September): One-year-old subadult Procambarus clarkii (crawfish) are selected as the culture subjects. The females in these one-year-old subadults are in the ovarian stage I. The one-year-old subadults are released into the culture ponds for graded feeding to complete hepatopancreatic energy storage, physical energy accumulation, and pre-gonadal development. Afterward, broodstock are selected to enter the next overwintering low-temperature acclimatization stage. Since one-year-old subadults are not yet fully sexually mature and difficult to distinguish by external reproductive characteristics, this stage involves mixed rearing of one-year-old subadults in the culture ponds without sex separation. The mixed rearing ponds are not equipped with isolation nets.

[0025] The aquaculture pond in this embodiment is as follows: Figure 2 As shown, the bank slope ratio of the aquaculture pond is 1:2.5-3.0. A 20cm thick heavy anti-freeze and heat insulation layer is laid on the surface of the bank slope. The heavy anti-freeze and heat insulation layer is made of deep black clay and decomposed peat soil mixed in a 7:3 ratio and compacted. It can effectively prevent the permafrost layer from penetrating in winter, ensuring that the slope area where the broodstock burrows are located does not freeze through or collapse in the later stage, and improving the stability and heat preservation effect of the broodstock overwintering burrows. At the same time, the aquaculture pond is equipped with isolation nets for the separation of male and female broodstock in the later stage. During the summer and autumn energy storage and cultivation screening stage, the thickness of the silt at the bottom of the aquaculture pond should be controlled at 10cm-15cm, without blackening or odor, and the coverage rate of aquatic plants in the water body should be maintained at 40%-60%. At the same time, the water temperature of the aquaculture pond should be controlled at 18℃-28℃, pH 7.2-8.0, dissolved oxygen ≥4.5mg / L, ammonia nitrogen <0.3mg / L, nitrite <0.1mg / L, transparency 30cm-60cm, and water depth 0.8m-1.5m.

[0026] During this stage of the breeding process, a protein-specific energy-storing feed is used throughout. The nutritional composition of this protein-specific energy-storing feed, by mass percentage, is as follows: crude protein 34%-36%, crude fat 5.0%-6.0%, calcium 1.8%-2.2%, total phosphorus 1.0%-1.3%, vitamin C 0.02%-0.025%, β-carotene 0.004%-0.006%, aquatic compound minerals 1.2%-1.5%, with the remainder being food-grade binders and aquatic-specific attractants. The aquatic compound minerals contain at least magnesium, potassium, iron, zinc, copper, manganese, and selenium, which can meet the mineral requirements of broodstock shrimp during the energy-storing stage.

[0027] One-year-old subadult red swamp crayfish are fed a protein-specific energy-storing feed using a temperature gradient feeding method. The specific standards are as follows: when the water temperature is ≥18℃, feed once daily in the evening, with the protein-specific energy-storing feed accounting for 2.0%-2.5% of the crayfish's total body weight; when the water temperature is >12℃ and <18℃, feed once daily in the evening, with the protein-specific energy-storing feed accounting for 1.0%-1.5% of the crayfish's total body weight; when the water temperature is <12℃, feed once every two evenings, with the protein-specific energy-storing feed accounting for 0.5%-0.8% of the crayfish's total body weight. The protein-specific energy-storing feed should be evenly scattered around aquatic plants in the shallow water area of ​​the rearing pond. This temperature gradient feeding method allows for gradual reduction of the feeding amount as the water temperature decreases, avoiding low-temperature indigestion and hepatopancreatic congestion damage in the broodstock, ensuring stable nutrient accumulation, and promoting the natural and stable development of the gonads and ovaries of female one-year-old subadult red swamp crayfish to the ovarian stage II overwintering preparation state.

[0028] After 1-year-old sub-adult Procambarus clarkii completes hepatopancreatic energy storage, physical energy accumulation, and pre-gonadal development, healthy broodstock are selected using a non-invasive live appearance screening method. The specific screening process for the non-invasive live appearance screening method is as follows: First, select vigorous adult shrimp that respond quickly to external stimuli, have shiny and hard shells, intact appendages without defects, no attached substances, and no obvious external injuries or parasites as candidate broodstock. Then, select female and male broodstock from the candidate broodstock. The screening criteria for female broodstock are: weight ≥ 45g, body length ≥ 10cm, gonads in ovarian stage II, pale yellowish-brown and slightly swollen abdominal carapace above the third pereiopod, slightly convex genital opening, and no blackening or ulceration of the abdomen. Ovarian stage II can be determined by randomly sampling 3-5 female broodstock for dissection and calibration to ensure an accuracy rate of ≥ 90% for appearance screening. The screening criteria for male broodstock are: weight ≥ 50g, body length ≥ 11cm, strong and powerful second pereiopod with intact chelipeds, hard, milky-white, and undamaged specialized copulatory organ on the first abdominal appendage, and clearly visible genital opening. After screening, the male-to-female ratio of broodstock shrimp is controlled at 2:2.5-3, with an optimal ratio of 2:2.8, before moving on to the next overwintering low-temperature acclimatization stage.

[0029] S2. Overwintering and Low-Temperature Acclimation (Late September to End of October): After the broodstock selection is completed, the male and female broodstock are separated into different breeding ponds according to the ratio of female broodstock to male broodstock of 2:2.5-3. The preferred ratio of male to female broodstock is 2:2.8. Among them, the female broodstock are placed in the female broodstock area of ​​the breeding pond, and the male broodstock are placed in the male broodstock area of ​​the breeding pond. The two areas are completely separated by a detachable isolation net set in advance to avoid premature mating and asynchronous gonad development. Before entering the pond, the broodstock need to be soaked in 3%-5% saline for 3-5 minutes.

[0030] The first step in this stage is to replace the feed with a low-protein, high-fiber feed. This low-protein, high-fiber feed is made primarily from corn, soybean meal, and plant fiber, with optional additions of animal-derived ingredients such as miscellaneous fish. Its crude protein content is 26%-29% by weight. Simultaneously, feed reduction is implemented: feeding once every three evenings, with the low-protein, high-fiber feed amounting to 0.3%-0.5% of the shrimp's total body weight, to gradually clear intestinal metabolites from the broodstock and reduce oxygen consumption during the ice-covered winter. Feeding is completely stopped seven days before the ice seal to ensure no residual food remains in the shrimp's intestines, preventing intestinal fermentation and disease during the ice season.

[0031] Simultaneously, the water level in the breeding ponds was raised in stages to guide the broodstock shrimp to burrow 60-75cm below the ice. The specific operation was as follows: Before the expected ice-sealing date, the water level in the breeding ponds was maintained at 0.6m-0.8m to induce burrowing behavior, ensuring the burrowing locations were within the effective water layer below the ice. One day before the ice-sealing date, a mixture of 400g salt and 400g baking soda per cubic meter of water was applied throughout the pond. When the ice thickness reached 1.5cm for the first time, the water level was slowly raised to 1.5m. When the ice thickness reached 2cm for the first time, the water level was further slowly raised to 2.2m-2.5m, and the water level was kept stable thereafter. The water level increase should be gradual, with each increase not exceeding 30cm to avoid stress to the broodstock shrimp caused by sudden changes in water level. When the temperature suddenly drops below -30℃ in the later stages, the shrimp burrows are located in a constant temperature layer 60cm-75cm below the ice. Combined with the heavy antifreeze and heat insulation layer of the breeding pond, the internal temperature of the shrimp burrows can be stabilized at 1.2℃-4℃, ensuring the safety of the parent shrimp overwintering.

[0032] S3. Overwintering Management During the Extended Ice Period (November to Mid-March of the Following Year): The core management principle during this stage is to minimize the energy consumption of broodstock shrimp and ensure sufficient dissolved oxygen in the subglacial water. During this stage, broodstock shrimp remain in deep hibernation within their designated burrows, with activity and metabolism minimized. They rely on the nutrition from their summer and autumn hepatopancreas to maintain basal metabolism, and the ovaries of female broodstock shrimp remain stably locked in a stage II hibernation state. Therefore, feeding, ice breaking, and human disturbance are strictly prohibited throughout this stage to prevent broodstock shrimp from crawling out of their burrows and being exposed to the low-temperature subglacial water, which could lead to frostbite or death. Simultaneously, snow removal from the ice surface after snowfall must be completed within 24 hours to ensure ice transmittance ≥40%. This ensures long-term biological oxygenation for algal photosynthesis under the ice, meeting the oxygen requirements of the broodstock shrimp during overwintering without the need for additional artificial oxygenation.

[0033] Monitor ice thickness and sub-ice water level weekly, maintaining a stable water level between 2.2m and 2.5m. Simultaneously, monitor sub-ice water quality weekly, ensuring dissolved oxygen ≥3.5mg / L, ammonia nitrogen <0.2mg / L, and nitrite <0.1mg / L. In emergency situations where extreme continuous snowfall reduces ice transmittance to less than 10% and dissolved oxygen drops sharply below 3.0mg / L, for outdoor temperatures above -20℃, drill one 20cm diameter emergency oxygenation hole per 5 acres of ice surface and place a microporous oxygenation disc for temporary oxygenation. After oxygenation, promptly seal the oxygenation hole with insulation material. For outdoor temperatures not exceeding -20℃, drill one 20cm diameter emergency oxygenation hole per 5 acres of ice surface, spray oxygenating agent into the emergency oxygenation hole, and promptly seal the oxygenation hole with insulation material after spraying to prevent significant water temperature fluctuations.

[0034] S4. Spring Gradual Awakening (Mid-March to End of March of the following year): During this stage, it is necessary to strictly control the rate of water temperature rise and water level changes to avoid strong stress reactions in the broodstock shrimp due to sudden environmental changes, which could lead to ovarian resorption and degeneration. A combination of natural warming and shade netting should be used to strictly control the daily water temperature rise to ≤0.3℃. If strong sunlight during the day causes the water temperature to rise too quickly, shade netting can be placed over the pond to cool it down. As the ice melts, the water level in the pond should be slowly lowered in 3-5 stages, each time by 10-15cm, until it stabilizes at 1.2m-1.5m. This will guide the broodstock shrimp to gradually emerge from their burrows. At this time, the female broodstock shrimp's ovaries generally develop from the dormant state of ovarian stage II to ovarian stage III.

[0035] During this stage, it is essential to stabilize the water quality, with the following standards: pH 7.5-8.3, dissolved oxygen ≥4.0 mg / L, ammonia nitrogen <0.2 mg / L, and nitrite <0.05 mg / L. Run the impeller aerator for 2-3 hours daily to promote water exchange and prevent oxygen depletion in the bottom water. Change the water once a week, replacing 5%-10% of the total volume each time, ensuring the temperature difference between the water during water changes and the rearing pond water does not exceed 1°C. After the broodstock shrimp emerge from their burrows, apply quicklime to the entire pond once a week at a rate of 25 mg / L. Observe the emergence of broodstock shrimp every evening, recording the number and vitality of those emerging, and promptly remove and isolate any dead broodstock shrimp.

[0036] S5. Spring Gonadal Maturation and Reproduction (starting from the end of March): During this stage, the feed will be replaced with a high-energy, high-protein maturation feed. This high-energy, high-protein maturation feed is used to promote the targeted maturation of the broodstock gonads, causing the female broodstock ovaries to develop from ovarian stage III to ovarian stage IV and ovarian stage V in a mature state. At the same time, it repairs the physical damage caused by the broodstock during overwintering. Its nutritional composition by mass percentage is: crude protein 38%-42%, crude fat 7%-9%, calcium 2.3%-2.9%, available phosphorus 1.4%-1.8%, vitamin E 0.015%-0.02%, and lecithin 0.8%-1.0%, with the remainder being food-grade binders and aquatic-specific attractants.

[0037] Feeding is conducted using a temperature-differentiated feeding method. Specifically, when the water temperature is 5℃-8℃, feed once every two days in the evening, with the amount of high-energy, high-protein growth-promoting feed being 1.0%-1.2% of the total body weight of the shrimp; when the water temperature is >8℃, feed once daily in the evening, with the amount of high-energy, high-protein growth-promoting feed being 1.8%-2.2% of the total body weight of the shrimp. The feeding point is set on a platform in the shallow water area of ​​the culture pond, making it convenient for the broodstock shrimp to feed.

[0038] Every 3 days, 20-30 female broodstock shrimp are randomly selected, and ovarian development is observed using a non-invasive abdominal transillumination method. When 80% of the female broodstock shrimp reach ovarian stage V, the female shrimp population is considered to be gonadally mature. When the water temperature remains stable at 10℃-14℃ for more than 5 consecutive days, and the water body meets the conditions of dissolved oxygen ≥5.0mg / L, transparency 25cm-35cm, and maintaining a micro-flow with a daily water exchange rate of 5%-8% in the culture pond, the isolation netting between the male and female broodstock shrimp areas is removed, and centralized mating is initiated.

[0039] During mating, maintain a quiet environment in the rearing pond, avoiding human disturbance and large-scale water changes. The mating process typically lasts 3-5 days. Two to five days after mating, the female broodstock's ovaries usually develop to ovary stage VI. Subsequently, the female broodstock successively complete spawning, fertilization, and egg-carrying via their abdominal proboscis. Maintain water stability during this period, adjusting the daily feed intake to 2.0%-2.5% of the shrimp's total body weight. Add 0.01% vitamin C to the high-energy, high-protein growth-promoting feed to improve the stress resistance of the berried shrimp and the hatching rate of fertilized eggs. Once the fertilized eggs develop to the eye-spot stage, the berried shrimp can be transferred to a hatching pond for centralized incubation.

[0040] In this embodiment, the development criteria for ovarian stages I-VI are as follows: Ovary stage I: milky white and thin, closely attached to the inner wall of the cephalothorax, with eggs barely visible to the naked eye; Ovary stage II: pale yellow and thin, without clearly separated eggs, increasing in size and occupying 1 / 3 of the posterior space of the cephalothorax; Ovary stage III: light orange-yellow, further increasing in size, occupying 1 / 2 of the posterior space of the cephalothorax, with the outline of the eggs faintly visible; Ovary stage IV: orange-yellow, filling the posterior part of the cephalothorax and extending to the first abdominal segment, with clearly identifiable eggs; Ovary stage V: deep orange-red, with plump and uniform eggs, and a small number of eggs may leak out when the abdomen is gently pressed; Ovary stage VI: dark red-orange, with completely free eggs, plump and slightly movable.

[0041] Using the aquaculture method described in this embodiment, a large-scale aquaculture experiment was conducted in the extremely cold region of Northeast China (ice-covered period of 5 months, ice thickness of 80-120cm). The results showed that the overwintering survival rate of broodstock Procambarus clarkii could reach over 80%, the synchronous maturation rate of female shrimp gonads could reach over 82%, and the egg-carrying rate during concentrated mating could reach over 78%. This significantly solved the technical problems of low overwintering survival rate, asynchronous gonad development, and low egg-carrying rate of Procambarus clarkii in extremely cold and long ice-covered environments, providing a feasible technical solution for the large-scale artificial breeding of Procambarus clarkii in extremely cold regions.

[0042] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them; modifications to the technical solutions described in the foregoing embodiments, or equivalent substitutions of some of the technical features, do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.

Claims

1. A method for targeted mating and egg-bearing culture of *Procambarus clarkii* adapted to extremely cold environments, characterized in that, Includes the following steps: S1. Summer and Autumn Energy Storage Cultivation and Screening: From late May to mid-September, use protein-specific energy storage feed to feed 1-year-old sub-adult Procambarus clarkii populations according to water temperature gradient, and use non-destructive live appearance screening method to select healthy broodstock. The male-to-female ratio of broodstock is controlled at 2:2.5-3. S2. Overwintering and low-temperature acclimatization: From late September to the end of October, replace the feed with low-protein crude fiber feed and reduce the amount of feed. Stop feeding completely 7 days before the ice freezes. At the same time, raise the water level of the breeding pond in stages and guide the parent shrimp to dig burrows in a directional manner on the slope 60cm-75cm below the ice. S3. Overwintering Management During the Extended Ice Period: From November to mid-March of the following year, the ice-covered overwintering stage begins. During this period, feeding, ice breaking, and human disturbance are strictly prohibited. Snow accumulation on the ice after snowfall is promptly cleared to ensure light transmittance and support long-term biological oxygenation for algal photosynthesis beneath the ice. S4. Gradual Spring Awakening: From mid-March to the end of March of the following year, the ice naturally melts. The daily temperature rise of the water body is controlled to ≤0.3℃. Simultaneously, the water level is gradually lowered and water quality is stabilized, allowing broodstock shrimp to emerge from their burrows. S5. Spring gonadal maturation and reproduction: Starting from the end of March, feed high-energy and high-protein maturation feed according to the water temperature. After the water temperature has been stable at 10℃-14℃ for more than 5 consecutive days, the water quality meets the standards, and the female shrimp gonads have matured, start centralized mating. After mating, the female parent shrimp will complete the egg laying, fertilization, and lappa egg-bearing in sequence.

2. The method for directional mating and egg-bearing culture of Procambarus clarkii according to claim 1, characterized in that, The nutritional composition of the protein-specific energy storage feed, by mass percentage, is as follows: crude protein 34%-36%, crude fat 5.0%-6.0%, calcium 1.8%-2.2%, total phosphorus 1.0%-1.3%, vitamin C 0.02%-0.025%, β-carotene 0.004%-0.006%, and aquatic compound minerals 1.2%-1.5%; the aquatic compound minerals include at least magnesium, potassium, iron, zinc, copper, manganese, and selenium.

3. The method for directional mating and berried culture of *Procambarus clarkii* according to claim 1, characterized in that, The standards for quantitative feeding based on water temperature gradients and differentiated feeding based on water temperature are as follows: The standard for quantitative feeding based on water temperature gradients is as follows: When the water temperature is ≥18℃, feed once a day in the evening, with the amount of protein-specific energy-storing feed being 2.0%-2.5% of the total body weight of the shrimp; when the water temperature is >12℃ and <18℃, feed once a day in the evening, with the amount of protein-specific energy-storing feed being 1.0%-1.5% of the total body weight of the shrimp; when the water temperature is <12℃, feed once every two days in the evening, with the amount of protein-specific energy-storing feed being 0.5%-0.8% of the total body weight of the shrimp. The standard for differentiated feeding based on water temperature is as follows: When the water temperature is 5℃-8℃, feed once every two days in the evening, with the amount of high-energy, high-protein growth-promoting feed being 1.0%-1.2% of the total body weight of the shrimp; when the water temperature is >8℃, feed once a day in the evening, with the amount of high-energy, high-protein growth-promoting feed being 1.8%-2.2% of the total body weight of the shrimp.

4. The method for directional mating and egg-bearing culture of *Procambarus clarkii* according to claim 1, characterized in that, The non-destructive live appearance screening method is as follows: select adult shrimp with vigorous activity, rapid response to external stimuli, shiny and hard shells, intact appendages without defects, no attached substances, no obvious external injuries or parasites as the broodstock candidate group, and screen female and male broodstock from the broodstock candidate group. The selection criteria for female parent shrimp are: weight ≥45g, body length ≥10cm, gonads in ovarian stage II, abdominal carapace above the third walking leg is light yellowish-brown and slightly swollen, genital opening is slightly convex, and there is no blackening or ulceration of the abdomen. The selection criteria for male broodstock shrimp are: weight ≥ 50g, body length ≥ 11cm, strong and powerful second walking legs with no missing chelipeds, hard, milky white, and undamaged specialized copulatory organ of the first abdominal appendage, and clearly visible genital opening.

5. The method for directional mating and egg-bearing culture of *Procambarus clarkii* according to claim 1, characterized in that, The low-protein crude fiber feed is prepared using corn, soybean meal, and plant crude fiber as the main raw materials, with optional addition of animal-derived raw materials such as miscellaneous fish. Its crude protein content is 26%-29% by mass. The standard for reducing feed is: feeding once every 3 days in the evening, with the amount of low-protein crude fiber feed being 0.3%-0.5% of the total body weight of the shrimp, to gradually clear the intestinal metabolites of the broodstock shrimp and reduce the oxygen consumption load of the broodstock shrimp during the ice period of winter.

6. The method for directional mating and egg-bearing culture of *Procambarus clarkii* according to claim 1, characterized in that, The bank slope ratio of the aquaculture pond is 1:2.5-3.0, and a 20cm thick heavy antifreeze and heat insulation layer is laid on the surface of the bank slope. The heavy antifreeze and heat insulation layer is made by mixing and compacting deep black clay and decomposed peat in a 7:3 ratio.

7. The method for directional mating and egg-bearing culture of *Procambarus clarkii* according to claim 1, characterized in that, The phased, stepped water level increase is as follows: Before the pond is sealed with ice, the water level is kept constant at 0.6m-0.8m to induce the broodstock shrimp to start burrowing behavior, so that the burrowing location of the broodstock shrimp is at the effective water layer on the bank slope under the ice later. When the ice layer in the aquaculture pond reaches a thickness of 1.5cm for the first time, raise the water level in the aquaculture pond to 1.5m. When the ice layer in the aquaculture pond reaches 2cm for the first time, raise the water level in the aquaculture pond to 2.2m-2.5m, and keep the water level in the aquaculture pond stable thereafter.

8. The method for directional mating and berried culture of *Procambarus clarkii* according to claim 1, characterized in that, The high-energy, high-protein ripening feed is used for the targeted ripening of broodstock gonads and to repair the physical damage caused by overwintering. Its nutritional components, by mass percentage, are: crude protein 38%-42%, crude fat 7%-9%, calcium 2.3%-2.9%, available phosphorus 1.4%-1.8%, vitamin E 0.015%-0.02%, and lecithin 0.8%-1.0%.

9. The method for directional mating and berried culture of *Procambarus clarkii* according to claim 1, characterized in that, The term "mass gonadal maturity" refers to the gonads of more than 80% of the female shrimp in the population developing to the ovarian stage V. The conditions for meeting the water quality standards are: dissolved oxygen ≥ 5.0 mg / L, transparency 25 cm-35 cm, and maintaining a slight flow of water with a daily water exchange rate of 5%-8% in the aquaculture pond.

10. The method for directional mating and egg-bearing culture of Procambarus clarkii according to claim 1, characterized in that, It also includes disease prevention and control measures: In S1, the broodstock shrimp are soaked in 3%-5% saline solution for 3-5 minutes before being put into the pond; in S2, 400 grams of salt and 400 grams of baking soda mixture are sprinkled throughout the pond per cubic meter of water before the pond is sealed with ice; in S3, the water quality under the ice is monitored weekly to ensure dissolved oxygen ≥3.5 mg / L; in S4, quicklime is sprinkled throughout the pond once a week after the broodstock shrimp emerge from their burrows, with a sprinkling amount of 25 mg / L.

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