Schizothorax fish fertilized egg low-temperature-high-temperature stimulation membrane rupture and double-high water temperature tolerance threshold experiment method in strong daily temperature difference water area

Abstract

The invention provides a low-temperature-high-temperature stimulation membrane rupture and double-high water temperature tolerance threshold experiment method for fertilized eggs of schizothorax fishes in a strong daily temperature difference water area. The method comprises the following steps: step 1, determining daily extremum and amplitude variation of hatching water temperature of the fertilized eggs of natural reproduction of the schizothorax fishes; 2, daily variable-amplitude incubation water temperature is achieved; step 3, membrane rupture key water temperature control; 4, performing large-scale membrane rupture through low-temperature-high-temperature stimulation; and step 5, determining a double-high water temperature tolerance threshold. According to the method, by determining the daily extreme value and variable amplitude of the hatching water temperature of the naturally-bred fertilized eggs, the hatching water temperature experiments with variable amplitude in different days are designed for hatching the fertilized eggs, and the natural hatching environment with the large daily water temperature difference of the Qinghai-Tibet Plateau river under climate changes is simulated; through an indoor fertilized egg water temperature gradient experiment, a low-temperature-high-temperature stimulation membrane rupture mechanism, a double-high water temperature tolerance range and a threshold value of the key fish schizopygi nojirioides from the Yangtze River source are found, and scientific support is provided for protection and repair of fish resources from the Yangtze River source under the background of subsequent climate warming.

Description

Technical Field

[0001] The invention relates to the field of fish ecological adaptation, and specifically to an experimental method for low-temperature-high-temperature stimulation membrane rupture and double-high water temperature tolerance threshold of fertilized eggs of schizothorax fish in waters with strong daily temperature differences. Background Art

[0002] Fish are typical cold-blooded animals that cannot regulate their body temperature autonomously. Their body temperature is directly affected by the ambient water temperature, and changes with the ambient water temperature. Fertilized eggs are the most vulnerable stage in the life cycle of fish, and are very sensitive to environmental changes, especially water temperature fluctuations. Fertilized eggs are excellent research materials for studying the response of fish to changes in water temperature.

[0003] The Qinghai-Tibet Plateau is a sensitive area for global climate change. Due to the polar amplification effect, the atmospheric warming in the Qinghai-Tibet Plateau is fast, twice the rate of change of the global average temperature. Climate warming affects the thermal conditions of rivers, which in turn affects the environment and aquatic life. Schizothorax fish are important species in the river and lake ecosystems of the Qinghai-Tibet Plateau. Existing studies have shown that an increase in water temperature within a certain range helps to accelerate the hatching rate of fertilized fish eggs, and the activity and efficiency of enzymes in fish are affected by temperature. However, the adaptability of fish fertilized eggs to changes in water temperature is limited. Too high or too low water temperature leads to abnormal development, developmental stagnation, and even death. The sensitivity of fertilized eggs of different species of fish to water temperature also varies. Studying the sensitivity of fertilized eggs to water temperature and controlling the water temperature within an appropriate range can promote the normal development and hatching of fertilized eggs of schizothoracic fish. At present, there are few studies on the water temperature for hatching fertilized eggs of schizothoracic fish, and only the suitable hatching water temperature for four species of fish has been reported. Researcher Wang Hongyong's team used a constant temperature system to design three groups of different incubation water temperature experiments, and found that the optimal water temperature for artificial incubation of giant beard schizothorax is 8.5-9.5℃; Researcher Liu Yanchao's team designed five groups of constant temperature incubation experiments with average water temperatures of 5℃, 8℃, 11℃, 14℃ and 17℃, and found that the suitable temperature for Lhasa naked schizothorax embryos is 14-17℃, and the suitable water temperature for hatching sharp naked carp embryos is 11-17℃; Researcher Zeng Benhe's team designed 8 groups of constant temperature fertilized egg incubation experiments at 5℃, 9℃, 12℃, 14℃, 16℃, 18℃, 20℃ and 22℃, and found that the lowest temperature limit for Lhasa schizothorax embryo development is 5℃, the highest temperature threshold is 22℃, and the suitable water temperature is 12-16℃. The water temperature process experienced by fish fertilized eggs during development affects the water temperature tolerance of fish in the later stage. Obviously, in order to incubate fish fertilized eggs with drastic daily water temperature changes in the natural environment under artificial conditions, a similar drastic change in incubation water temperature process needs to be designed. Otherwise, it will affect the water temperature tolerance of fish under artificial breeding and affect the effect of proliferation and release. However, the above studies only considered the suitable hatching water temperature for schizothorax embryos under constant temperature conditions, and did not explore the impact of drastic daily fluctuations in water temperature on embryonic development, nor did they conduct in-depth research on the low-temperature-high-temperature stimulation process. During the schizothorax breeding season, the natural water temperature of the source rivers in the Qinghai-Tibet Plateau shows drastic daily variations, with a daily fluctuation of up to 16°C, which is significantly different from the plain rivers. It is necessary to construct the water temperature requirement mechanism for the development of fertilized eggs of fish in waters with natural drastic daily temperature differences. Against the background of global warming and fish ex situ conservation research, it is urgent to construct a schizothorax hatching water temperature system.

[0004] The small-headed naked schizothorax is a fish endemic to my country and a key fish species in the Yangtze River source water ecosystem. It is crucial to maintaining the diversity of fish in the Yangtze River source and the function of the water ecosystem. It has extremely high scientific research value and is a representative fish among schizothorax. The daily water temperature in the Yangtze River source fluctuates drastically, and the water temperature system for hatching fertilized eggs of the small-headed naked schizothorax has not yet been established. In view of the extremely high ecological value of the small-headed naked schizothorax, it is of great significance to promote the protection of rare biological resources of schizothorax fish in the Qinghai-Tibet Plateau under the background of climate change and to break through the method of constructing the water temperature for hatching fertilized eggs of the small-headed naked schizothorax in the Yangtze River source. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an experimental method for exploring the membrane rupture and double high temperature extreme water temperature of schizothorax in waters with natural strong daily temperature difference, so as to provide water temperature system support for fish fertilized eggs in artificial hatching areas and to provide a scientific basis for evaluating the impact of rising water temperature on the development of fertilized eggs of regional schizothorax under climate change. The invention also provides a reference for the construction of water temperature for artificial hatching of other schizothorax.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An experimental method for membrane rupture and double high temperature extreme water temperature of schizothorax in waters with high daily temperature difference comprises the following steps:

[0008] Step 1: Determine the daily extreme value and range of water temperature for hatching fertilized eggs during natural reproduction

[0009] An underwater water temperature automatic recorder was buried in the spawning ground to record the hourly water temperature process during the fertilized egg hatching period; wavelet analysis was used to analyze the daily periodicity and determine the daily water temperature maximum value T max , minimum value T min ;

[0010] Step 2: Daily variable incubation water temperature

[0011] The incubation process adopts artificial incubation method, which is indoor basin incubation, and the incubation density is 40,000-50,000 / m 2 , the incubation water depth is 15-20cm; the incubation water source is spring water, the groundwater temperature is 5-9℃, and the incubation water temperature is achieved by blending boiled spring water with original spring water; the room temperature of the incubation room is 13-14℃, and the lighting conditions are 14L:10D, where L means daytime and D means nighttime, without direct sunlight; the incubation water temperature range is set to (13℃, T max ); During the incubation process, the temperature is raised once a day to T max After that, the water temperature automatically drops to room temperature to simulate the temperature difference between day and night, and promotes embryo development with higher water temperature; during the incubation process, the water temperature is adjusted to T maxThe groundwater at room temperature should be changed once every day at 15:00, and 4 / 5 of the water should be changed each time. Dead embryos should be cleaned in time to keep the hatching water fresh.

[0012] Step 3: Water temperature control for membrane breaking

[0013] When the embryonic tail is fully developed and reaches the head, with a length close to half of the inner perimeter of the membrane, the whole embryonic body moves violently, with the movement mode of flipping and rotating, which is the best time to break out of the membrane. Set the initial water temperature of the experiment to the minimum value T min , gradually increase the temperature by 1°C at intervals of 0.5h, and judge the critical water temperature T for the emergence of the fertilized eggs based on whether the fertilized eggs have emerged. hat (hatching temperature);

[0014] Step 4: Low-temperature-high-temperature stimulation for large-scale membrane rupture

[0015] After the peak time of film production is determined, the low temperature T min Stimulate the fertilized egg and then use high temperature T max Stimulate fertilized eggs; T min It is triggered between 9:00-10:00 am, T max Triggering at 15:00-17:00 in the afternoon. After low-temperature-high-temperature stimulation, the effect period is about 3 hours, and the fertilized eggs break the membrane on a large scale. Low-temperature-high-temperature stimulation plays a positive role in promoting the rupture of the fertilized eggs;

[0016] Step 5: Determine the double high water temperature tolerance threshold

[0017] During the incubation period, T min As the lower limit temperature, the design is based on T max As a control, the temperature was gradually increased by 1°C as a high temperature gradient experiment to explore the high temperature threshold of the high temperature zone; the number of dead fertilized eggs and the number of fertilized eggs that emerged from the membrane were observed and recorded daily, and finally the high temperature tolerance threshold T of the high temperature zone was evaluated with a hatching rate of 60% as the limit. uthr (upper thresholdtemperature); in the film-forming period, T max As the upper limit temperature, the design is based on T min The temperature was gradually increased by 1℃ as a low temperature gradient experiment to explore the high temperature threshold of the low temperature zone, and the high temperature tolerance threshold T of the low temperature zone was evaluated with a hatching rate of 60% as the limit. lthr (lowerthresholdtemperature). Further, where T min ≤12℃,T max >T hat And T max ≥22℃.

[0018] The beneficial effects of the present invention are:

[0019] The present invention determines the daily extreme value and amplitude of the incubation water temperature of fertilized eggs in natural reproduction, designs incubation water temperature experiments with different daily amplitudes to incubate fertilized eggs, and simulates the natural incubation environment with large daily water temperature differences in rivers on the Qinghai-Tibet Plateau under climate change. For the first time, the concept of double high temperature thresholds was proposed in fish ecology research. Through indoor fertilized egg water temperature gradient experiments, the mechanism that large-scale membrane rupture of small-headed naked schizothrill fish fry requires low-temperature-high temperature stimulation was revealed, the key water temperature for membrane rupture was found, and the water temperature tolerance range and double high temperature thresholds for incubation of fertilized eggs of small-headed naked schizothrill fish, a key fish in the Yangtze River source, were found. Finally, the adaptation mechanism of plateau cold-water schizothrill fish to high daily temperature difference water temperature was generally revealed. The systematic method is applicable to the research of high daily temperature difference water temperature areas and fish in the Qinghai-Tibet Plateau, and has excellent promotion value. The invention has wide applicability, high scientific feasibility and strong controllability. The high hatching rate and low dead egg rate prove that the practical application effect is good. The invention provides scientific support for the protection and restoration of fish resources in the Yangtze River source under the background of subsequent climate warming. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the change of water temperature during the experiment from low temperature 7°C to high temperature 22°C according to an embodiment of the present invention;

[0021] Figure 2 This is a diagram showing the change of water temperature during the experiment from low temperature 9°C to high temperature 24°C according to an embodiment of the present invention;

[0022] Figure 3 This is a diagram showing the change of water temperature during the experiment of low temperature 11°C to high temperature 26°C according to an embodiment of the present invention;

[0023] Figure 4 This is a graph showing the daily percentage changes of dead eggs and eggs emerging from the membrane in the low temperature 7°C to high temperature 22°C experiment of the embodiment of the present invention;

[0024] Figure 5 This is a graph showing the daily percentage changes of dead eggs and eggs emerging from the membrane in the low temperature 9°C to high temperature 24°C experiment of the embodiment of the present invention;

[0025] Figure 6 This is a graph showing the daily percentage changes of dead eggs and eggs emerging from the membrane in the low temperature 11°C to high temperature 26°C experiment of the embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The embodiment of the present invention is to analyze the water temperature characteristics of the natural incubation environment with a large daily temperature difference in the specific rivers of the Qinghai-Tibet Plateau source, and to construct the experimental method of low-temperature-high-temperature stimulation membrane rupture and double high-temperature tolerance threshold by experimental ecology. Among them, the discovery of the low-temperature-high-temperature stimulation membrane rupture mechanism and the proposal of the concept of double high-temperature tolerance threshold are both good innovations in the field of fish thermal ecology. The following is a detailed description through specific embodiments.

[0028] Example 1

[0029] Water temperature construction for hatching fertilized eggs of schizothorax at low temperature of 7℃ and high temperature of 22℃

[0030] 1. Determine the daily extreme value and range of water temperature for hatching fertilized eggs during natural reproduction

[0031] An underwater water temperature automatic recorder was buried in the spawning grounds of the small-headed naked schizont fish to record the hourly water temperature process during the hatching period of fertilized eggs. Wavelet analysis was used to analyze the daily periodicity and determine the daily water temperature maximum value T max The minimum value T is 22℃. min is 7℃.

[0032] 2. Daily variable hatching water temperature

[0033] The incubation process adopts artificial incubation method, which is indoor basin incubation, and the incubation density is 40,000 / m 2 , the incubation water depth is 15cm. The incubation water source is spring water, which ensures the excellent quality of the incubation water while taking advantage of the fact that there are fewer bacteria and viruses in groundwater in high altitude areas. The groundwater temperature is 5°C, and the incubation water temperature is achieved by blending boiled spring water with original spring water. The room temperature of the incubation room is 13°C, and the dissolved oxygen and ammonia nitrogen content are appropriate during incubation. The lighting conditions are 14L:10D, where L represents daytime and D represents nighttime, without direct sunlight; the incubation water temperature range is set to (13°C, 22°C). The 22°C experimental water temperature process change diagram is as follows Figure 1 The incubation experiment started at 16:52 on June 13 and ended at 20:50 on June 21. During the incubation process, the temperature was raised once a day. After reaching 22°C, the water temperature automatically dropped to room temperature. The temperature difference between day and night was simulated, and the embryo development was promoted with a higher water temperature. The temperature was lowered and raised 8 times during the whole experiment. During the incubation process, the water was changed once at 15:00 every day with groundwater mixed to 22°C. 4 / 5 of the water was changed each time, and the dead embryos were cleaned in time to keep the incubation water fresh.

[0034] 3. Water temperature control for membrane breaking

[0035] When the embryonic tail is fully developed and reaches the head, with a length close to half of the inner perimeter of the membrane, the whole embryonic body moves violently, with the movement mode of flipping and rotating, which is the best time to break out of the membrane. Set the initial water temperature of the experiment to the minimum value of 7℃, and gradually increase it by 1℃ at intervals of 0.5h. Determine the critical water temperature T for breaking out of the membrane based on whether the fertilized egg breaks out of the membrane. hat When the temperature rises to 18℃, no fertilized eggs have been found to break out of the membrane. When the temperature rises to 19℃, a certain number of fertilized eggs break out of the membrane. The key water temperature for judging the emergence of the membrane is T hat It is 19℃.

[0036] 4. Low-temperature and high-temperature stimulation for large-scale membrane rupture

[0037] After the peak time of film production is determined (such as Figure 1 , June 19 afternoon), first with the low temperature zone water temperature T min (7℃) to stimulate the fertilized eggs, and then the water temperature in the high temperature zone T max (22℃) stimulates fertilized eggs. min It is triggered between 9:00-10:00 am, T max The triggering time is 15:00-17:00 in the afternoon. The daily percentage changes of the 22℃ experiment eggs and dead eggs are shown in the figure below. Figure 4 After the low-temperature-high-temperature stimulation, the effect period was about 3 hours, and the daily percentage of fertilized eggs breaking the membrane reached the maximum on the evening of June 20, that is, the fertilized eggs broke the membrane on a large scale. The low-temperature-high-temperature stimulation played a positive role in promoting the fertilized egg membrane breaking.

[0038] 5. Determine the tolerance threshold of double high water temperature

[0039] During the incubation period, a high temperature gradient experiment was designed to explore the high temperature threshold of the high temperature zone, with 7°C as the lower limit temperature and 22°C as the control, with the temperature gradually increasing by 1°C (23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C). The number of dead fertilized eggs and the number of fertilized eggs that emerged from the membrane were observed and recorded daily. Fertilized eggs that developed into deformed embryos were counted as dead eggs. Finally, the high temperature tolerance threshold T in the high temperature zone was evaluated with a hatching rate of 60% as the limit. uthr (upperthresholdtemperature); The hatching rate of the control group of the Yangtze River source schizodon schizodon fertilized eggs was calculated to be 82.8% and the dead egg rate was 17.2%. When 7℃ was used as the low temperature stimulus, the hatching rates of 23℃, 24℃, 25℃, 26℃, 27℃, and 28℃ were all higher than 60%, and when the water temperature was higher than 28℃, the hatching rates were all lower than 60%, and the high temperature tolerance threshold T in the high temperature zone was determined. uthrSimilarly, in the hatching period, with 22°C as the upper limit temperature, a low temperature gradient experiment was designed to explore the high temperature threshold of the low temperature zone by gradually increasing 1°C by 7°C (8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C). The high temperature tolerance threshold T of the low temperature zone was evaluated with 60% hatching rate as the limit. lthr When 22℃ is used as high temperature stimulation, the hatching rates of 8℃, 9℃, 10℃, 11℃ and 12℃ are all higher than 60%; when the water temperature is higher than 12℃, the hatching rates are all lower than 60%. The high temperature tolerance threshold T in the low temperature zone is judged. lthr It is 12℃.

[0040] In the study on the construction of water temperature system for hatching fertilized eggs of the Yangtze River source fish, the key water temperature for breaking the membrane was 19℃; the low temperature was 7℃ (7℃ <T l <12℃) and high temperature 22℃(19℃ <T h <28℃), the fertilized eggs broke membranes on a large scale; the high temperature tolerance threshold of the fertilized eggs in the low temperature zone was 12℃, and the high temperature tolerance threshold in the high temperature zone was 28℃. The high hatching rate of 82.8% and the low death rate of 17.2% showed that the experimental effect was good and the controllability was high.

[0041] Example 2

[0042] Water temperature construction for hatching fertilized eggs of schizothorax at low temperature of 9℃ and high temperature of 24℃

[0043] 1. Determine the daily extreme value and range of water temperature for hatching fertilized eggs during natural reproduction

[0044] An underwater water temperature automatic recorder was buried in the spawning grounds of the small-headed naked fish to record the hourly water temperature process during the fertilized egg hatching period. Wavelet analysis was used to analyze the daily periodicity and determine that the maximum value of the daily water temperature was 22°C and the minimum value was 7°C. Considering the impact of climate change on the hatching water temperature of fertilized eggs naturally propagated in the spawning grounds of the small-headed naked fish, the maximum, minimum and average values ​​of the daily water temperature will increase simultaneously in the future. Experimental ecology constructs the future near-term daily water temperature maximum value T max is 24℃, minimum value T min The water temperature system for hatching fertilized eggs of the Yangtze River source fish is 9℃.

[0045] 2. Daily variable hatching water temperature

[0046] The incubation process adopts artificial incubation method, which is indoor basin incubation, and the incubation density is 45,000 / m 2, the incubation water depth is 18cm. The incubation water source is spring water, which ensures the excellent quality of the incubation water while taking advantage of the fact that there are fewer bacteria and viruses in groundwater in high altitude areas. The groundwater temperature is 7°C, and the incubation water temperature is achieved by blending boiled spring water with original spring water. The room temperature of the incubation room is 14°C, and the dissolved oxygen and ammonia nitrogen content are appropriate during incubation. The lighting conditions are 14L:10D, where L represents daytime and D represents nighttime, without direct sunlight; the incubation water temperature range is set to (13°C, 24°C). The 24°C experimental water temperature process change diagram is as follows Figure 2 The incubation experiment started at 16:45 on June 13 and ended at 15:00 on June 21. During the incubation process, the temperature was raised once a day. After reaching 24°C, the water temperature automatically dropped to room temperature. The temperature difference between day and night was simulated, and the embryo development was promoted with a higher water temperature. The temperature was lowered and raised 8 times during the whole experiment. During the incubation process, the water was changed once at 15:00 every day with groundwater mixed to 24°C. 4 / 5 of the water was changed each time, and the dead embryos were cleaned in time to keep the incubation water fresh.

[0047] 3. Water temperature control for membrane breaking

[0048] When the embryonic tail is fully developed and reaches the head, with a length close to half of the inner perimeter of the membrane, the whole embryonic body moves violently, with the movement mode of flipping and rotating, which is the best time to break out of the membrane. Set the initial water temperature of the experiment to the minimum value of 9℃, and gradually increase it by 1℃ at intervals of 0.5h. Determine the critical water temperature T for breaking out of the membrane based on whether the fertilized egg breaks out of the membrane. hat When the temperature rises to 18℃, no fertilized eggs have been found to break out of the membrane. When the temperature rises to 19℃, a certain number of fertilized eggs break out of the membrane. The key water temperature for judging the emergence of the membrane is T hat It is 19℃.

[0049] 4. Low-temperature and high-temperature stimulation for large-scale membrane rupture

[0050] After the peak time of film production is determined (such as Figure 2 , June 19 afternoon), first with the low temperature zone water temperature T min (9℃) to stimulate the fertilized eggs, and then the water temperature in the high temperature zone T max (24℃) stimulates fertilized eggs. min It is triggered between 9:00-10:00 am, T max The triggering time is 15:00-17:00 in the afternoon. The daily percentage changes of the membrane-embryo and dead eggs in the experiment are shown in the figure below. Figure 5 After the low-temperature-high-temperature stimulation, the effect period was about 3 hours, and the daily percentage of fertilized eggs breaking the membrane reached the maximum on the afternoon of June 20, that is, the fertilized eggs broke the membrane on a large scale. The low-temperature-high-temperature stimulation played a positive role in promoting the fertilized egg membrane breaking.

[0051] 5. Determine the tolerance threshold of double high water temperature

[0052] During the incubation period, a high temperature gradient experiment was designed to explore the high temperature threshold of the high temperature zone, with 9°C as the lower limit temperature and 24°C as the control, with the temperature gradually increasing by 1°C (25°C, 26°C, 27°C, 28°C, 29°C, and 30°C). The number of dead fertilized eggs and the number of fertilized eggs that emerged from the membrane were observed and recorded daily. Fertilized eggs that developed into deformed embryos were counted as dead eggs. Finally, the high temperature tolerance threshold T of the high temperature zone was evaluated with a hatching rate of 60% as the limit. uthr (upper thresholdtemperature); The hatching rate of fertilized eggs of the Yangtze River source schizodon schizodon was calculated to be 71% and the dead egg rate was 29% when the water temperature was 9℃ and 24℃ respectively. The hatching rate was higher than 60% when the water temperature was higher than 28℃ and the hatching rate was lower than 60% when the water temperature was higher than 28℃. The high temperature tolerance threshold T in the high temperature zone was determined. uthr Similarly, in the hatching period, with 24°C as the upper limit temperature, a low temperature gradient experiment was designed to explore the high temperature threshold of the low temperature zone by gradually increasing 1°C from 9°C (10°C, 11°C, 12°C, 13°C, 14°C). The high temperature tolerance threshold T of the low temperature zone was evaluated with 60% hatching rate as the limit. lthr (lower threshold temperature). When 24℃ is used as high temperature stimulation, the hatching rates of 10℃, 11℃ and 12℃ are all higher than 60%. When the water temperature is higher than 12℃, the hatching rates are all lower than 60%. The high temperature tolerance threshold T in the low temperature zone is judged. lthr It is 12℃.

[0053] In the study on the construction of water temperature system for hatching fertilized eggs of the Yangtze River source fish, the key water temperature for breaking the membrane was 19℃; the low temperature was 9℃ (9℃ <T l <12℃) and high temperature 24℃(19℃ <T h <28℃), the fertilized eggs broke membranes on a large scale; the high temperature tolerance threshold of the fertilized eggs in the low temperature zone is 12℃, and the high temperature tolerance threshold in the high temperature zone is 28℃. The high hatching rate of 71% and the low death rate of 29% indicate that the experimental effect is good and the controllability is high.

[0054] Example 3

[0055] Water temperature construction for hatching fertilized eggs of schizothorax at low temperature of 11℃ and high temperature of 26℃

[0056] 1. Determine the daily extreme value and range of water temperature for hatching fertilized eggs during natural reproduction

[0057] An underwater water temperature automatic recorder was buried in the spawning grounds of the small-headed naked fish to record the hourly water temperature process during the fertilized egg hatching period. Wavelet analysis was used to analyze the daily periodicity and determine that the maximum value of the daily water temperature was 22°C and the minimum value was 7°C. Considering the impact of climate change on the hatching water temperature of fertilized eggs naturally propagated in the spawning grounds of the small-headed naked fish, the maximum, minimum and average values ​​of the daily water temperature will increase simultaneously in the future. Experimental ecology constructs the future prospect of the maximum value of the daily water temperature T max The minimum value T is 26℃. min The water temperature system for hatching fertilized eggs of the Yangtze River source fish is 11℃.

[0058] 2. Daily variable hatching water temperature

[0059] The incubation process adopts artificial incubation method, which is indoor basin incubation, and the incubation density is 50,000 / m 2 , the incubation water depth is 20cm. The incubation water source is spring water, which ensures the excellent quality of the incubation water while taking advantage of the fact that there are fewer bacteria and viruses in groundwater in high altitude areas. The groundwater temperature is 9°C, and the incubation water temperature is achieved by blending boiled spring water with original spring water. The room temperature of the incubation room is 15°C, and the dissolved oxygen and ammonia nitrogen content are appropriate during incubation. The lighting conditions are 14L:10D, where L represents daytime and D represents nighttime, without direct sunlight; the incubation water temperature range is set to (13°C, 26°C). The 26°C experimental water temperature process change diagram is as follows Figure 3 The incubation experiment started at 15:45 on June 13 and ended at 9:45 on June 21. During the incubation process, the temperature was raised once a day. After reaching 26°C, the water temperature automatically dropped to room temperature. The temperature difference between day and night was simulated, and the embryo development was promoted with a higher water temperature. The temperature was lowered and raised 8 times during the whole experiment. During the incubation process, the water was changed once at 15:00 every day with groundwater mixed to 26°C. 4 / 5 of the water was changed each time, and the dead embryos were cleaned in time to keep the incubation water fresh.

[0060] 3. Water temperature control for membrane breaking

[0061] When the embryonic tail is fully developed and reaches the head, with a length close to half of the inner perimeter of the membrane, the whole embryonic body moves violently, with the movement mode of flipping and rotating, which is the best time to break out of the membrane. Set the initial water temperature of the experiment to the minimum value of 11℃, and gradually increase it by 1℃ at intervals of 0.5h. Determine the critical water temperature T for breaking out of the membrane based on whether the fertilized egg breaks out of the membrane. hat When the temperature rises to 18℃, no fertilized eggs have been found to break out of the membrane. When the temperature rises to 19℃, a certain number of fertilized eggs break out of the membrane. The key water temperature for judging the emergence of the membrane is T hat It is 19℃.

[0062] 4. Low-temperature and high-temperature stimulation for large-scale membrane rupture

[0063] After the peak time of film production is determined (such as Figure 3 , June 19 afternoon), first with the low temperature zone water temperature T min (11℃) to stimulate the fertilized eggs, and then the water temperature in the high temperature zone T max (26℃) stimulates fertilized eggs. min It is triggered between 9:00-10:00 am, T max The triggering time is 15:00-17:00 in the afternoon. The daily percentage changes of the membrane-embryo and dead eggs in the experiment are shown in the figure below. Figure 6 After the low-temperature-high-temperature stimulation, the effect period was about 3 hours, and the daily percentage of fertilized eggs breaking the membrane reached the maximum on the afternoon of June 20, that is, the fertilized eggs broke the membrane on a large scale. The low-temperature-high-temperature stimulation played a positive role in promoting the fertilized egg membrane breaking.

[0064] 5. Determine the tolerance threshold of double high water temperature

[0065] During the incubation period, a high temperature gradient experiment was designed to explore the high temperature threshold of the high temperature zone, with 11°C as the lower limit temperature and 26°C as the control, with the temperature gradually increasing by 1°C (27°C, 28°C, 29°C, 30°C). The number of dead fertilized eggs and the number of fertilized eggs that emerged from the membrane were observed and recorded daily. Fertilized eggs that developed into deformed embryos were counted as dead eggs. Finally, the high temperature tolerance threshold T of the high temperature zone was evaluated with a hatching rate of 60% as the limit. uthr (upper threshold temperature); The hatching rate of fertilized eggs of the Yangtze River source schizodon schizodon was calculated to be 77.6% and the dead egg rate was 22.4% when the water temperature was 11℃ and 26℃ respectively. The hatching rate was higher than 60% when the water temperature was higher than 28℃, and the hatching rate was lower than 60% when the water temperature was higher than 28℃. The high temperature tolerance threshold T in the high temperature zone was determined. uthr Similarly, in the hatching period, with 26°C as the upper limit temperature, a low temperature gradient experiment was designed to explore the high temperature threshold of the low temperature zone by gradually increasing 1°C by 11°C (12°C, 13°C, 14°C). The high temperature tolerance threshold T of the low temperature zone was evaluated with 60% hatching rate as the limit. lthr (lower thresholdtemperature). When 26℃ is considered as high temperature stimulation and 12℃ is considered as low temperature stimulation, the hatching rate is higher than 60%. When the water temperature is higher than 12℃, the hatching rate is lower than 60%. The high temperature tolerance threshold T in the low temperature zone is judged. lthr It is 12℃.

[0066] In the study on the construction of water temperature system for hatching fertilized eggs of the Yangtze River source fish, the key water temperature for breaking the membrane was 19℃; the low temperature was 11℃ (11℃ <T l <12℃) and high temperature 26℃(19℃ <T h<28℃), the fertilized eggs broke membranes on a large scale; the high temperature tolerance threshold of the fertilized eggs in the low temperature zone was 12℃, and the high temperature tolerance threshold in the high temperature zone was 28℃. The high hatching rate of 77.6% and the low death rate of 22.4% showed that the experimental effect was good and the controllability was high.

[0067] The present invention aims to explore the double-high water temperature threshold and low-temperature-high-temperature stimulation large-scale membrane rupture and experimental methods of fertilized eggs of representative schizothorax fishes on the Qinghai-Tibet Plateau under the background of climate warming, so as to study the specific water temperature adaptation of schizothorax fishes and provide support for the cultivation of fertilized eggs under artificial conditions. The specific scheme includes determining the daily extreme value and amplitude of the water temperature for hatching fertilized eggs of naturally propagated fertilized eggs, achieving the daily amplitude of hatching water temperature, controlling the key water temperature for membrane rupture, revealing the mechanism of large-scale membrane rupture stimulated by low-temperature-high-temperature stimulation, and determining the double-high water temperature tolerance threshold. The present invention can provide reference for the research on the construction of water temperature for hatching fertilized eggs of other schizothorax fishes.

[0068] In terms of results, the present invention determines that the key water temperature for hatching and breaking the membrane of fertilized eggs of small-headed naked schizont is 19°C, and the low temperature T l and high temperature T h After stimulation, the fertilized eggs broke their membranes on a large scale. The high temperature tolerance threshold of the fertilized eggs in the low temperature zone is 12°C, and the high temperature tolerance threshold of the high temperature zone is 28°C. The high hatching rate and low dead egg rate indicate that the experimental effect is good and the controllability is high.

[0069] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An experimental method for low-temperature-high-temperature stimulation membrane rupture and double-high water temperature tolerance threshold of schizothorax fertilized eggs in waters with strong daily temperature differences, characterized in that: The steps include: Step 1: Determine the daily extreme value and range of water temperature for hatching fertilized eggs during natural reproduction An underwater water temperature automatic recorder was buried in the spawning ground to record the hourly water temperature process during the fertilized egg hatching period; wavelet analysis was used to analyze the daily periodicity and determine the daily water temperature maximum value T max , minimum value T min ; Step 2: Daily variable incubation water temperature The incubation process adopts artificial incubation method, which is indoor basin incubation, and the incubation density is 40,000-50,000 / m 2 , the incubation water depth is 15-20cm; the incubation water source is spring water, the groundwater temperature is 5-9℃, and the incubation water temperature is achieved by mixing boiled spring water with original spring water; in order to shorten the incubation time, the room temperature of the incubation room is maintained at the average water temperature of natural breeding at 13-14℃, and the light condition is 14L:10D, where L means daytime and D means nighttime, without direct sunlight; the incubation water temperature interval is set to (13℃, T max ); During the incubation process, the temperature is raised once a day to T max After that, the water temperature automatically drops to room temperature to simulate the temperature difference between day and night, and promotes embryo development with higher water temperature; during the incubation process, the water temperature is adjusted to T max The groundwater at room temperature should be changed once every day at 15:00, and 4 / 5 of the water should be changed each time. Dead embryos should be cleaned in time to keep the hatching water fresh. Step 3: Water temperature control for membrane breaking When the embryonic tail is fully developed, wraps around the head, and its length is close to half of the inner circumference of the membrane, the whole embryonic body moves violently, with the movement mode of flipping and rotating, which is the best time to leave the membrane; Set the initial film-forming water temperature of the experiment to the minimum value T min , gradually increase the temperature by 1°C at intervals of 0.5h, and judge the critical water temperature T for the emergence of the fertilized eggs based on whether the fertilized eggs have emerged. hat ; Step 4: Low-temperature-high-temperature stimulation for large-scale membrane rupture After the peak time of film production is determined, the low temperature T min Stimulate the fertilized egg and then use high temperature T max Stimulate fertilized eggs; T min It is triggered between 9:00-10:00 am, T max Triggering at 15:00-17:00 in the afternoon; after low-temperature-high-temperature stimulation, the effect period is 3 hours, and the fertilized eggs break the membrane on a large scale. Low-temperature-high-temperature stimulation plays a positive role in promoting the rupture of the fertilized eggs' membranes; Step 5: Determine the double high water temperature tolerance threshold During the membrane emergence period, T min The lower limit temperature is designed with T max As a high temperature control, the temperature was gradually increased by 1°C as a high temperature gradient experiment to explore the high temperature threshold of the high temperature zone; the number of dead fertilized eggs and the number of fertilized eggs that emerged from the membrane were observed and recorded daily, and finally the high temperature tolerance threshold T of the high temperature zone was evaluated with a hatching rate of 60% as the limit. uthr ; In the membrane stage, T max As the upper limit temperature, the design is based on T min The temperature in the low temperature zone was gradually increased by 1℃ as a gradient experiment to explore the high temperature threshold of the low temperature zone. The high temperature tolerance threshold T of the low temperature zone was evaluated with a hatching rate of 60% as the limit. lthr .

2. The experimental method for low-temperature-high-temperature stimulation membrane rupture and double-high water temperature tolerance threshold of schizothorax fertilized eggs in waters with strong daily temperature differences as claimed in claim 1, characterized in that: Where T min ≤12℃,T max >T hat And T max ≥22℃.

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