Breeding method of high-temperature-resistant procambarus clarkii

Abstract

The application discloses a method for evaluating the heat tolerance of a population and an individual, and comprises two steps of heat tolerance population mass selection and individual heat tolerance comparison, wherein the heat tolerance population mass selection comprises population temporary culture, temperature setting, heat tolerance test and activity test, and the individual heat tolerance comparison comprises population temporary culture, temperature setting, activity test, temperature stress, and activity test. The method can efficiently solve the problems of batch selection of heat tolerance population of Procambarus clarkii in the production process and low-cost, live and rapid evaluation of individual heat tolerance trait difference in the experiment process, reduces the irreversible damage of traditional heat tolerance index mortality evaluation on the shrimp body, greatly shortens the complexity and time-consuming of heat tolerance heart rate inflection point measurement, and has the advantages of low cost, large scale, high speed, combination of crustacean physiology and behavior ecology adaptation knowledge, comprehensive utilization of multiple indexes in heat tolerance performance evaluation, and compensation of the shortage of single index selection, and is more in line with the natural situation and production practice.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture, and in particular relates to a breeding method for high-temperature resistant Procambarus clarkii. Background Art

[0002] Procambarus clarkii, commonly known as crayfish, is a highly adaptable, omnivorous species with a strong reproductive capacity, easy to raise, and nutritious. It is currently the most productive commercial freshwater crustacean in my country. In recent years, surging domestic consumption and robust market demand have led to rapid growth in artificial aquaculture, particularly in Hubei, Anhui, Hunan, and Jiangsu provinces.

[0003] High temperatures shorten the optimal growth cycle of crayfish, hindering their molting and accelerating their early maturity, leading to the formation of "iron shrimp"—those that consume food but do not grow. As aquaculture expands, disease problems are becoming increasingly serious. These problems are particularly prominent during the annual high-temperature season, causing significant economic losses. In shrimp-rice farming and intensive pond aquaculture in areas like the middle and lower reaches of the Yangtze River, Procambarus clarkii is extremely susceptible to persistent high-temperature stress during summer aquaculture. This stress can cause metabolic imbalances and immune disorders, ultimately impacting their growth and survival. Furthermore, the price of crayfish in my country is unique—high at the top and low in the middle. High temperatures increase the breeding window for crayfish, delaying their market launch and preventing them from catching the peak harvest period from January to March. High temperatures force crayfish to burrow for shelter, preventing farmers from catching them and missing the market recovery from July to September. Crayfish populations with poor heat tolerance lose their market competitiveness, resulting in significant economic losses for farmers. High summer temperatures are hindering the healthy development of my country's crayfish aquaculture industry. Therefore, breeding high-temperature resistant varieties is an important step in breaking the bottleneck of industrial development.

[0004] During the seedling cultivation, breeding, and aquaculture of Procambarus clarkii, the environmental tolerance of the germplasm has become a hot topic and focus of research in recent years. Among numerous environmental factors, temperature has the greatest impact on the growth and survival of Procambarus clarkii and is studied as a key environmental factor in genetic breeding. Currently, methods for evaluating heat tolerance primarily include the cumulative thermotolerance test (UTT) and the ablation point (ABT). However, the UTT can cause irreversible damage to the shrimp, while the ABT is time-consuming and can only be used to evaluate adult shrimp. Furthermore, the equipment costs are high. Neither method is suitable for large-scale breeding of heat-resistant Procambarus clarkii. Therefore, developing a breeding method for heat-resistant Procambarus clarkii and cultivating new heat-resistant Procambarus clarkii varieties is an urgent need for healthy crayfish farming. Summary of the Invention

[0005] The present invention provides a breeding method for heat-resistant Procambarus clarkii. The purpose of the present invention is to establish a new system for breeding heat-resistant parents based on the physiology and behavior of Procambarus clarkii. The method sets the stress temperature by measuring the average heat-resistant heart rate inflection point of the group, and designs a breeding box for shrimp escape, allowing the crayfish to select the appropriate temperature on their own, thereby quickly differentiating the heat-resistant group on a large scale.

[0006] A breeding method for heat-resistant Procambarus clarkii includes two steps: large-scale breeding of heat-resistant groups and comparison of heat resistance of individuals.

[0007] The large-scale breeding of the temperature-tolerant population includes the following steps:

[0008] (1) Temporary group rearing: Crawfish from the pond are continuously aerated and fed with feed;

[0009] (2) Temperature setting: according to the average temperature tolerance heart rate inflection point of the breeding population, set the stress temperature of the high temperature avoidance device and continuously supply oxygen;

[0010] (3) Temperature resistance test: According to the nocturnal activity of Procambarus clarkii, the temporarily cultured Procambarus clarkii were placed in a high-temperature shelter with a pre-set stress temperature at night;

[0011] (4) Vitality test: After the stress, the next morning, the crayfish that did not escape were taken out and all turned upside down, and the shrimps that turned right side up were selected for seed preservation;

[0012] The temperature resistance comparison of the individuals comprises the following steps:

[0013] (a) Temporary rearing of groups: Randomly select a number of individuals for temperature tolerance evaluation from the escaped and non-escaped Procambarus clarkii groups, place them in canvas pools, continuously aerate and feed them for temporary rearing;

[0014] (b) Temperature setting: Before the experiment begins, adjust the water temperature and continue to oxygenate;

[0015] (c) Vitality test: Before temperature stress, the shrimp were inverted and the righting time was recorded. The shrimp with the average righting time and good vitality were selected for testing;

[0016] (d) Temperature stress: The shrimp were fixed with a special clamp connected to a pulley and suspended in a temperature-elevating device. The upper temperature limit was set to prevent irreversible damage to the shrimp body caused by high temperature. The temperature was stopped until the crayfish escaped by tail flicking. The escape temperature at this time was recorded, and the stress was continued at this temperature.

[0017] (e) Vitality test: Remove the stressed shrimp, turn them upside down, and record the righting time. Compare it with the initial righting time. If the righting time is significantly prolonged, it indicates that the escape temperature is the temperature tolerance limit of the shrimp.

[0018] Preferably, in step (1), the Procambarus clarkii seedlings captured from the pond are placed in a canvas pond at 25° C. and temporarily cultured for one day.

[0019] Preferably, after 12 hours of coercion in step (4), the next morning, the crayfish that have not escaped are taken out, all of them are turned upside down, and the seed shrimps with a turning-righting time of less than 10 seconds are selected for seed preservation.

[0020] Preferably, in step (a), a number of individuals for temperature tolerance evaluation are randomly selected from the escaped and non-escaped Procambarus clarkii populations and are placed in canvas pools at 25°C.

[0021] Preferably, in step (b), the water temperature is adjusted to 25°C before the test begins.

[0022] Preferably, in step (c), before temperature stress, the shrimp is inverted three times, and the righting time is recorded, and the average righting time is selected to be within 30 seconds.

[0023] Preferably, in step (d), the shrimp body is fixed with a special clamp connected to a pulley and suspended in a temperature-rising avoidance device, and the temperature is raised by 0.5 degrees Celsius every 10 minutes. The upper temperature limit is set at 36°C to prevent high temperature from causing irreversible damage to the shrimp body. The temperature is stopped until the crayfish escapes by flicking its tail, and the escape temperature at this time is recorded. The stress is continued at this temperature for 30 minutes.

[0024] In the step (e), the shrimps were removed after being stressed for 30 minutes and inverted three times.

[0025] Beneficial effects: The present invention is a new system for breeding heat-resistant parents based on the physiology and behavior of Procambarus clarkii, which is divided into two parts, including large-scale breeding of heat-resistant groups and comparison of individual heat-resistant performance. It is characterized by high efficiency, accuracy, liveness and non-destructiveness, which solves the problem of batch breeding of heat-resistant groups in the production process of Procambarus clarkii and the problem of low-cost, liveness and rapid evaluation of individual heat-resistant trait differences in the experimental process. This method reduces the irreversible damage to the shrimp body caused by the traditional heat-resistant indicator mortality assessment, greatly shortens the complexity and time-consuming nature of the heat-resistant heart rate inflection point measurement, is not only harmless to the test group, but also low-cost, large-scale and fast. It combines ecological adaptation knowledge such as crustacean physiology and behavior, and comprehensively applies multiple indicators to the evaluation of heat-resistant performance, making up for the shortcomings of single-indicator breeding, and is more in line with natural conditions and production practices.

[0026] The present invention eliminates the situation where crayfish cannot escape due to physical weakness, and conducts a righting vitality test on the heat-resistant groups that have not escaped, thereby quickly selecting breeding shrimps that meet the breeding requirements on a large scale. In addition, combined with the unconditioned reflex behavior of crayfish to harmful temperatures, that is, the tail-flicking escape phenomenon, by continuously raising the temperature and recording the escape temperature of individual crayfish, the difference in heat resistance of crayfish between different individuals can be distinguished. The present invention, based on the physiological reaction of crayfish that things will go against the extreme and the behavioral characteristics of seeking benefits and avoiding harm, provides different methods for large-scale heat-resistant group breeding and individual shrimp heat resistance evaluation. This technology is not only non-destructive to the shrimp body, but also fast and low-cost. In production, the invention will provide a reliable technical means for the breeding of high-temperature resistant varieties of crayfish, further expand the thermal range suitable for the growth of crayfish, reduce the breeding risks of high temperatures in summer to crayfish, and increase the yield and economic benefits of crayfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the escape device for high temperature stress of Procambarus clarkii population.

[0028] Figure 2 This is a picture of the on-site test of the high temperature stress escape device for Procambarus clarkii populations.

[0029] Figure 3 This is a schematic diagram of the tail-swinging escape device for Procambarus clarkii individuals induced by temperature stress.

[0030] Figure 4 A photo of the on-site test of a tail-flicking escape device for Procambarus clarkii individuals induced by temperature stress. DETAILED DESCRIPTION

[0031] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0032] A method for breeding heat-resistant Procambarus clarkii includes a method for large-scale breeding of heat-resistant Procambarus clarkii populations and a method for evaluating individual heat-resistant performance differences. The specific steps are as follows:

[0033] 1. Large-scale breeding of heat-tolerant populations

[0034] (1) Temporary rearing of groups: According to the size of the culture device, a reasonable stocking density is set. The male and female crayfish caught from the pond are separated and placed in different canvas pools. A thermostat is used to maintain the culture temperature at 25℃, and the system is continuously aerated and fed. After one day of temporary rearing, the crayfish are used for testing.

[0035] (2) Temperature setting: Before the test began, 30 male and 30 female shrimp were randomly selected from the breeding population. The heart rate changes of the individual crayfish, which were restricted in the continuously warming water, were monitored in real time using an electrocardiogram (ECG) monitor. The temperature inflection point at which the heart rate abnormality occurred was recorded. Based on the average temperature-tolerant heart rate inflection point of the female and male shrimp in the breeding population, the stress temperature of the heat avoidance device was set, and oxygen was continuously supplied.

[0036] (3) Temperature resistance test: According to the nocturnal activity habits of Procambarus clarkii, the male and female Procambarus clarkii temporarily reared were separated at night and placed in a high temperature shelter with a pre-set stress temperature.

[0037] (4) Vitality test: After 12 hours of stress, the next morning, the crayfish that did not escape were taken out and turned upside down. The shrimps that turned right side up within 10 seconds were selected as temperature-resistant shrimps.

[0038] (5) Construction of temperature-resistant population: From the selected temperature-resistant shrimp, choose shrimps that are large, vigorous and undamaged as breeding shrimps, and release them in a 1:1 ratio of male to female for seed preservation.

[0039] 2. Comparison of individual temperature resistance

[0040] (a) Temporary rearing of Procambarus clarkii broodstock from ponds, separated by sex, were placed in separate canvas ponds. The culture temperature was maintained at 25°C using a thermostat, and the broodstock were continuously aerated and fed with feed. After one day of temporary rearing, they were used for testing.

[0041] (b) Temperature setting: Before the start of the test, the water temperature of the tail-flick escape device was adjusted to 25°C and oxygen was continuously supplied.

[0042] (c) Vitality test: Before temperature stress, the shrimp were inverted three times and the righting time was recorded. The shrimp with an average righting time of less than 30 s and good vitality were selected for testing.

[0043] (d) Temperature stress: The shrimp were fixed with a special clamp connected to a pulley and suspended in a temperature-elevating device. The temperature was raised by 0.5°C every 10 min. The upper temperature limit was set at 36°C to prevent irreversible damage to the shrimp. The temperature was kept constant until the crayfish escaped by flicking its tail. The escape temperature was recorded and the stress was continued at this temperature for 30 min.

[0044] (e) Vitality test: After 30 minutes of stress, remove the shrimp and invert them three times. Record the time it takes to right itself. Compare this time to the time it takes before the experiment. If the righting time is significantly prolonged, this indicates that the escape temperature is the temperature tolerance limit of the shrimp.

[0045] (f) Study on temperature tolerance: This can provide experimental materials for subsequent research. Shrimp with higher temperature tolerance limits and shrimp with lower temperature tolerance limits can be sampled for further in-depth molecular or physiological research.

[0046] Example 1:

[0047] The heart rate detection equipment measures the average abnormal heart rhythm inflection point temperature of group 1 and group 2 and sets the stress temperature:

[0048] (1) After each heating period, the data were measured for 5 minutes after stabilization for 10 minutes, and the waveform characteristics and heart rate data were collected at each integer temperature point during the heating process (26-36°C);

[0049] (2) Calculate the average value of the stable waveform and heart rate parameters within 5 minutes as the heart rate at each hour temperature, and obtain the inflection point temperature of abnormal heart rhythm for each crayfish based on the Arrhenius equation linear fitting;

[0050] (3) Based on the data of groups and males and females, the average abnormal heart rhythm inflection point temperature of groups 1 and 2 was calculated respectively. Based on the heart rhythm temperature inflection point, the stress temperature was set by rounding up to 0.5°C, as shown in Table 1.

[0051] Table 1: Statistics of heart rhythm abnormality inflection point temperature and stress temperature setting for different groups of Procambarus clarkii

[0052] group gender Average abnormal heart rhythm inflection point temperature (℃) Stress temperature setting (℃) Group 1 male 32.34±1.28 32.5 Group 1 female 32.85±1.82 33.0 Group 2 male 32.18±1.83 32.5 Group 2 female 32.67±1.50 33.0

[0053] Example 2:

[0054] The high temperature stress escape device selects temperature-tolerant shrimp from the group of Procambarus clarkii and verifies it with a heart rate detection device:

[0055] (1) Temporary rearing of groups: According to the size of the culture device, a reasonable stocking density is set, and the male and female crayfish caught from the pond are separated and placed in different canvas pools. The culture temperature is maintained at 25 °C using a temperature control device, and the aeration and feed are continuously fed. After temporary rearing for one day, they are used for testing;

[0056] (2) Temperature setting: Before the test begins, 30 male and 30 female shrimp are randomly selected from the breeding population. An electrocardiogram (ECG) monitor is used to monitor the heart rate changes of individual Procambarus clarkii that are restricted in the continuously warming water. The temperature inflection point of abnormal heart rate is recorded. The stress temperature of the high temperature stress escape device is set based on the average temperature-tolerant heart rate inflection point of the female and male shrimp in the breeding population, and oxygen is continuously supplied.

[0057] (3) Temperature resistance test: According to the nocturnal activity habits of Procambarus clarkii, the male and female Procambarus clarkii were separated at night and placed in a high temperature shelter with a pre-set stress temperature;

[0058] (4) Vitality test: After 12 hours of stress, the next morning, the crayfish that did not escape were taken out and turned upside down. The shrimps that turned right side up within 10 seconds were selected as temperature-resistant shrimps;

[0059] (5) Heart rhythm verification: The heart rhythm of the test shrimp was monitored using a heart rate detection device. The temperature was set to the stress temperature of each group, such as 32.5°C for male shrimp in group 1, and the heart rhythm was observed to see if there was any disturbance. This was used to verify the accuracy of the high temperature escape device in screening temperature-resistant shrimp. See Table 2.

[0060] Table 2: Statistics of temperature tolerance of Procambarus clarkii populations detected by high temperature escape device

[0061]

[0062]

[0063] The experimental results show that the high temperature stress escape device can quickly screen out most of the heat-resistant crayfish from the group, and then conduct vitality tests through individual righting movements to effectively screen out heat-resistant shrimp. This is basically consistent with the results of heart rate verification, indicating that this method is efficient and accurate, and greatly improves the screening efficiency in the selection of heat-resistant seedlings in aquaculture production.

[0064] Example 3:

[0065] The temperature stress tail-flick escape device was used to detect the temperature tolerance differences of different Procambarus clarkii individuals and verified with a heart rate monitoring device:

[0066] (1) Temporary rearing of groups: Crayfish from ponds were caught, males and females were separated, and placed in different canvas pools. The culture temperature was maintained at 25°C using a thermostat, and the culture was continuously aerated and fed. After one day of temporary rearing, they were used for testing.

[0067] (2) Temperature setting: Before the test begins, the water temperature of the tail-flick escape device is adjusted to 25°C and oxygen is continuously supplied.

[0068] (3) Vitality test: Before temperature stress, the shrimp were inverted three times and the righting time was recorded. The shrimp with an average righting time of less than 30 seconds and good vitality were selected for testing.

[0069] (4) Temperature stress: The shrimp body was fixed with a special clamp connected to a pulley and suspended in a temperature-raising device. The temperature was raised by 0.5 degrees Celsius every 10 minutes. The upper temperature limit was set at 36 degrees Celsius to prevent irreversible damage to the shrimp body caused by high temperature. The temperature was stopped until the crayfish escaped by flicking its tail. The escape temperature at this time was recorded, and the stress was continued at this temperature for 30 minutes.

[0070] (5) Vitality test: After 30 minutes of stress, remove the shrimp and invert them three times. Record the time it takes to right itself. Compare this time with the time before the experiment. If the righting time is significantly prolonged, it indicates that the escape temperature is the temperature tolerance limit of the shrimp.

[0071] (6) Heart rhythm verification: The heart rhythm of the test shrimp was monitored using a heart rate monitoring device. The temperature was set to the individual tail-flick escape temperature, and the individual heart rhythm was observed to see if it was disordered. This was used to verify the accuracy of the temperature stress tail-flick escape device in detecting differences in shrimp temperature tolerance, as shown in Table 3.

[0072] Table 3: Statistical table of individual temperature tolerance of Procambarus clarkii tested by the tail-flicking escape device under temperature stress

[0073]

[0074]

[0075] The experimental results show that the temperature stress tail-flick escape device can distinguish the differences in temperature tolerance of different individual shrimps, which is basically consistent with the results of heart rate verification. The advantages of this method are low cost and simple operation, and it can be used for behavioral analysis of crayfish in laboratory research.

[0076] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for breeding heat-resistant crayfish, characterized in that: It includes two steps: large-scale breeding of temperature-tolerant groups and comparison of individual temperature tolerance. The large-scale breeding of the temperature-tolerant population includes the following steps: (1) Temporary group rearing: Crawfish from the pond are continuously aerated and fed with feed; (2) Temperature setting: according to the average temperature tolerance heart rate inflection point of the breeding population, set the stress temperature of the high temperature avoidance device and continuously supply oxygen; (3) Temperature resistance test: According to the nocturnal activity habits of Procambarus clarkii, the temporarily cultured Procambarus clarkii were placed in a high-temperature shelter with a pre-set stress temperature at night; (4) Vitality test: After 12 hours of stress, the next morning, remove the crayfish that have not escaped, turn them upside down, and select the seed shrimp that can turn right within 10 seconds for seed preservation; The temperature resistance comparison of the individuals comprises the following steps: (a) Temporary rearing of groups: Randomly select a number of individuals from the escaped and non-escaped Procambarus clarkii groups for evaluation of heat tolerance, place them in canvas pools, continuously aerate and feed them for temporary rearing; (b) Temperature setting: Before the start of the experiment, adjust the water temperature and continuously aerate the water to 25°C; (c) Vitality test: Before temperature stress, the shrimp were inverted three times and the righting time was recorded. The shrimp with an average righting time of less than 30 seconds and good vitality were selected for testing. (d) Temperature stress: The shrimp were fixed with a special clamp connected to a pulley and suspended in a temperature-elevating device. The temperature was raised by 0.5°C every 10 minutes. The upper temperature limit was set at 36°C to prevent irreversible damage to the shrimp. The temperature was raised until the crayfish escaped by flicking its tail. The temperature was then stopped and the escape temperature was recorded. The shrimp were then subjected to this temperature for another 30 minutes. (e) Vitality test: After 30 min of stress, remove the shrimp, turn them upside down, and record the righting time. Compare it with the initial righting time. If the righting time is significantly prolonged, it indicates that the escape temperature is the temperature tolerance limit of the shrimp.

2. The breeding method of a heat-resistant crayfish according to claim 1, characterized in that: In the step (1), the Procambarus clarkii seedlings captured from the pond are placed in a canvas pond at 25° C. and temporarily cultured for one day.

3. The breeding method of a heat-resistant crayfish according to claim 1, characterized in that: In the step (a), a number of individuals for temperature tolerance evaluation are randomly selected from the escaped and non-escaped Procambarus clarkii populations and are placed in canvas pools at 25°C.

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