Breeding method of high-temperature-resistant procambarus clarkii

Abstract

The method comprises two steps of large-scale breeding of temperature-resistant groups and temperature tolerance comparison of individuals, the large-scale breeding of the temperature-resistant groups comprises group temporary rearing, temperature setting, temperature tolerance testing and vigor testing, and the temperature tolerance comparison of the individuals comprises group temporary rearing, temperature setting, vigor testing, temperature stress and vigor testing. The method efficiently solves the problem of batch breeding of temperature-resistant groups in the production process of the procambarus clarkia and the problem of rapid evaluation of individual temperature-resistant character differences in a living body at low cost in the experiment process, reduces irreversible damage of traditional heat-resistant index death rate evaluation on prawn bodies, and improves the evaluation accuracy of the procambarus clarkia temperature-resistant character differences of the procambarus clarkia temperature-resistant character differences of the procambarus clarkia. The method greatly shortens the complexity and time consumption of temperature-resistant heart rate inflection point measurement, is low in cost, large in scale and high in speed, combines ecological adaptation knowledge of physiology, behavioristics and the like of crustacean, comprehensively applies multiple indexes to temperature-resistant performance evaluation, makes up for the deficiency of single-index breeding, and better conforms to natural conditions 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, has wide adaptability, omnivorous diet, strong fertility, easy breeding and rich nutrition. It is the largest economic freshwater crustacean in my country. In recent years, due to the surge in domestic consumption of Procambarus clarkii and strong market demand, the artificial breeding of Procambarus clarkii has developed rapidly, especially in Hubei, Anhui, Hunan, Jiangsu and other places.

[0003] Statistics show that since 2016, the production of crayfish farming in my country has continued to grow. At present, my country has become the world's largest producer of Procambarus clarkii. In 2023, the national Procambarus clarkii farming area will reach 29.5 million mu, the total output will reach 3.161 million tons, and the total economic output value will reach 458 billion yuan. However, in recent years, the breeding environment has changed significantly, especially under the trend of global warming, the number of high-temperature days has increased, and the average temperature has risen, which has brought a huge impact on the breeding industry of crayfish. High temperature shortens the optimal growth cycle of crayfish, affects molting, accelerates the early maturity of crayfish, and easily forms "iron shrimps" that only eat food but do not grow. With the expansion of breeding scale, disease problems are becoming more and more serious. Especially in the high temperature season every year, the disease problem is particularly prominent, causing huge economic losses. In the shrimp-rice co-cultivation and pond intensive breeding in the middle and lower reaches of the Yangtze River, Procambarus clarkii is extremely susceptible to continuous high temperature stress during summer breeding. High temperature stress can cause metabolic imbalance and immune disorder of the shrimp, which ultimately affects its growth and survival. In addition, the price of crayfish in my country is special, that is, "high at both ends and low in the middle". The high temperature has increased the breeding window period of crayfish, delayed the time to market, and missed the high dividend period from January to March. The high temperature forced crayfish to dig holes to hide, making it impossible for farmers to catch shrimp, and missed the market recovery from July to September. The crayfish population with poor temperature resistance has lost its market competitiveness, causing huge economic losses to farmers. High temperatures in summer have restricted the healthy development of my country's crayfish breeding industry. Therefore, breeding high-temperature resistant varieties is an important step to break the bottleneck of industrial development.

[0004] In the process of seedling raising, breeding and farming of Procambarus clarkii, the environmental factor "tolerance" of germplasm has become a hot topic and focus of research in recent years. Among many environmental factors, temperature has the greatest impact on the growth and survival of Procambarus clarkii, and is studied as an important environmental factor in genetic breeding. At present, the methods for evaluating temperature resistance mainly include the cumulative thermotolerance index (UTT) and the temperature resistance heart rate inflection point (ABT), but UTT will cause irreversible damage to the shrimp body, and the heart rate inflection point is time-consuming and slow, and can only evaluate adult shrimp, and the equipment cost is high. These two methods are not suitable for large-scale group breeding of high-temperature resistant Procambarus clarkii. Therefore, it is an urgent need for the healthy breeding of Procambarus clarkii to invent a breeding method for high-temperature resistant Procambarus clarkii and cultivate a new variety of high-temperature resistant Procambarus clarkii. Summary of the invention

[0005] The present invention provides a breeding method for heat-resistant Procambarus clarkii, and its purpose is to establish a new system for breeding heat-resistant parents based on the physiology and behavior of Procambarus clarkii, set the stress temperature through the average heat-resistant heart rate inflection point of the group, and design a breeding box for shrimp escape, so that the crayfish can choose the appropriate temperature by themselves, thereby quickly distinguishing the heat-resistant groups on a large scale.

[0006] A breeding method for high temperature resistant crayfish comprises two steps: large-scale breeding of high temperature resistant groups and comparison of the high temperature resistance of individuals.

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

[0008] (1) Temporary group rearing: Crawfish seedlings caught 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 habits 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, take out the crayfish that have not escaped, turn them all upside down, and select the shrimps that have turned right side up 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 evaluation of heat tolerance 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 test begins, adjust the water temperature and continue to oxygenate;

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

[0016] (d) Temperature stress: The shrimp body is fixed with a special clamp connected to a pulley and suspended in a temperature avoidance device. The upper temperature limit is set to prevent high temperature from causing irreversible damage to the shrimp body. The shrimp body is kept until it escapes by flicking its tail and the temperature is stopped. The escape temperature at this time is recorded and the stress is continued at this temperature.

[0017] (e) Vitality test: Remove the stressed shrimp, turn it upside down, record the righting time, and compare it with the initial righting time. If the righting time is significantly prolonged, it means 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 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 evaluating the temperature resistance 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 shrimps are inverted three times, 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 avoidance device, the temperature is increased by 0.5 degrees Celsius every 10 minutes, and the upper temperature limit is set to 36°C to prevent high temperature from causing irreversible damage to the shrimp body, until the crayfish escapes by flicking its tail, the temperature is stopped, the escape temperature at this time is recorded, and the stress is continued at this temperature for 30 minutes.

[0024] In the step (e), the shrimps are 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-consumingness 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. Combined with the ecological adaptation knowledge of crustacean physiology and behavior, a variety of indicators are comprehensively applied to the evaluation of heat resistance, which makes 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 group that has not escaped, thereby quickly selecting 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 the 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 performance 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 cultivation of high-temperature resistant varieties of crayfish, will 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 of Procambarus clarkii population under high temperature stress.

[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 under temperature stress.

[0030] Figure 4 This is a picture of the on-site testing of the tail-flicking escape device for Procambarus clarkii individuals under 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 a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0032] A breeding method for heat-resistant Procambarus clarkii includes a large-scale breeding method for heat-resistant Procambarus clarkii populations and an individual temperature resistance performance difference evaluation method, and the specific operating 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 Procambarus clarkii fry caught from the pond are separated and placed in different canvas pools. A thermostat is used to maintain the culture temperature at 25°C, and the pools are continuously aerated and fed with feed. The shrimps are temporarily reared for one day before use for testing.

[0035] (2) Temperature setting: Before the test, 30 male shrimps and 30 female shrimps were randomly selected from the breeding population. The heart rate changes of the crayfish individuals restricted in the water body with increasing temperature were monitored in real time using an electrocardiogram (ECG) detector, and the temperature inflection point of abnormal heart rate was recorded. The stress temperature of the high temperature avoidance device was set according to the average temperature tolerance heart rate inflection point of the female and male shrimps in the breeding population, and oxygen was continuously supplied.

[0036] (3) Temperature resistance test: Based on the nocturnal activity habits of Procambarus clarkii, the male and female Procambarus clarkii temporarily cultured 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 removed and turned upside down. The shrimps that turned right within 10 seconds were selected as temperature-resistant shrimps.

[0038] (5) Construction of temperature-resistant population: From the selected temperature-resistant shrimp, select shrimps with large size, good vitality and no damage as breeding shrimps, and release them in a 1:1 ratio of male to female for breeding.

[0039] 2. Comparison of individual temperature resistance

[0040] (a) Group culture: Procambarus clarkii seedlings were caught from ponds, and the males and females were separated and placed in different canvas pools. The culture temperature was maintained at 25°C using a thermostat, and the shrimps were continuously aerated and fed with feed. They were cultured for one day before being used for testing.

[0041] (b) 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.

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

[0043] (d) Temperature stress: The shrimp bodies were fixed with special clamps connected to pulleys and suspended in a temperature-rising avoidance device. The temperature was raised by 0.5°C every 10 min, and the upper temperature limit was set at 36°C to prevent irreversible damage to the shrimp bodies caused by high temperatures. The temperature was stopped 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, invert it 3 times, and record the time it takes to turn right. Compare it with the time before the experiment. If the time is significantly prolonged, it means that the escape temperature is the temperature tolerance limit of the shrimp.

[0045] (f) Study on temperature resistance: It 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] Embodiment 1:

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

[0048] (1) After each temperature rise was stabilized for 10 minutes, the data was measured for 5 minutes, and the waveform characteristics and heart rate data of each integer temperature point during the temperature rise process (26-36°C) were collected;

[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 group and male and female data, the average abnormal heart rhythm inflection point temperature of group 1 and group 2 male and female groups was calculated respectively. According to the heart rhythm temperature inflection point, the stress temperature was set by rounding up to 0.5°C, see 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] Embodiment 2:

[0054] The high temperature stress escape device selects temperature-resistant 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 one day of temporary rearing, they are used for testing;

[0056] (2) Temperature setting: Before the test begins, 30 male shrimps and 30 female shrimps are randomly selected from the breeding population. An electrocardiogram (ECG) monitor is used to monitor the heart rate changes of the crayfish individuals that are restricted in the water body with increasing temperature in real time, and the temperature inflection point of abnormal heart rate is recorded. The stress temperature of the high temperature stress escape device is set according to the average temperature tolerance heart rate inflection point of the female and male shrimps in the breeding population, and oxygen is continuously supplied;

[0057] (3) Temperature resistance test: Based on the nocturnal activity habits of Procambarus clarkii, the male and female Procambarus clarkii temporarily cultured 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, take out the crayfish that have not escaped, turn them upside down, and select the shrimps that turn right within 10 seconds as temperature-resistant shrimps;

[0059] (5) Heart rhythm verification: The heart rhythm of the test shrimp is tested by a heart rate detection device. The temperature is set to the stress temperature of each group, such as 32.5℃ for male shrimp in group 1, and the heart rhythm is observed to see if it is disordered. This is used to verify the accuracy of the high temperature escape device in screening temperature-resistant shrimp, see Table 2.

[0060] Table 2: Statistical table of temperature tolerance of Procambarus clarkii population 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, which can effectively screen out the 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] Embodiment 3:

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

[0066] (1) Group temporary rearing: Procambarus clarkii seed shrimps were caught from the pond, and the male and female were separated and placed in different canvas pools. The culture temperature was maintained at 25°C using a thermostat, and the shrimps were continuously aerated and fed with feed. They were temporarily reared for one day before being 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 avoidance device. The temperature was raised by 0.5 °C every 10 min, and the upper temperature limit was set at 36 °C 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 was recorded and the stress was continued at this temperature for 30 min.

[0070] (5) Vitality test: After 30 minutes of stress, remove the shrimp and invert them three times, and record the time it takes to turn right. Compare the time with the time before the experiment. If the time is significantly prolonged, it means 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 tested by a heart rate detection device, and the temperature was set to the individual tail-flicking escape temperature to observe whether the individual heart rhythm was disordered. This was used to verify the accuracy of the temperature stress tail-flicking escape device in detecting differences in shrimp temperature tolerance, see Table 3.

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

[0073]

[0074] The experimental results show that the temperature stress tail-flicking 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 advantage of this method is that it is low cost and simple to operate, and it can be used for behavioral analysis of crayfish in laboratory research.

[0075] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for breeding a heat-resistant crayfish, characterized in that: It includes two steps: large-scale breeding of temperature-tolerant populations and comparison of individual temperature tolerance. The large-scale breeding of the temperature-tolerant population includes the following steps: (1) Temporary group rearing: Crawfish seedlings caught 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 the stress, the next morning, take out the crayfish that have not escaped, turn them all upside down, and select the shrimps that have turned right side up 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 for evaluation of heat tolerance from the escaped and non-escaped Procambarus clarkii groups, place them in canvas pools, continuously aerate and feed them for temporary rearing; (b) Temperature setting: Before the test begins, adjust the water temperature and continue to oxygenate; (c) Vitality test: Before temperature stress, the shrimps were inverted and the righting time was recorded. The shrimps with the average righting time and good vitality were selected for testing; (d) Temperature stress: The shrimp body is fixed with a special clamp connected to a pulley and suspended in a temperature avoidance device. The upper temperature limit is set to prevent high temperature from causing irreversible damage to the shrimp body. The shrimp body is kept until it escapes by flicking its tail and the temperature is stopped. The escape temperature at this time is recorded and the stress is continued at this temperature. (e) Vitality test: Remove the stressed shrimp, turn it upside down, record the righting time, and compare it with the initial righting time. If the righting time is significantly prolonged, it means 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 seed shrimps 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: After 12 hours of coercion in step (4), the next morning, the crayfish that have not escaped are taken out, all are turned upside down, and the seed shrimps with a turning-right time of less than 10 seconds are selected for seed preservation.

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

5. The breeding method of a heat-resistant crayfish according to claim 1, characterized in that: In the step (b), the water temperature was adjusted to 25°C before the test started.

6. The breeding method of a heat-resistant crayfish according to claim 1, characterized in that: In the step (c), before temperature stress, the shrimps are inverted three times, and the turning time is recorded, and the average turning time is selected to be within 30 seconds.

7. The breeding method of a heat-resistant crayfish according to claim 1, characterized in that: In the step (d), the shrimp body is fixed with a special clamp connected to a pulley and suspended in a temperature avoidance device, the temperature is increased by 0.5 degrees Celsius every 10 minutes, and 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, the escape temperature at this time is recorded, and the stress is continued at this temperature for 30 minutes.

8. The breeding method of a heat-resistant crayfish according to claim 1, characterized in that: In the step (e), the shrimps are removed after being stressed for 30 minutes and inverted three times.

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