Method for alleviating stress response of yellowlip fish after stress and application thereof

Abstract

The present application belongs to the technical field of endangered fish germplasm resource repair and conservation, and particularly relates to a method for relieving stress response of yellowlip castraomus after stress and application thereof. In view of the problem that yellowlip castraomus has strong stress response and high mortality rate due to stress such as environment and operation, the present application proposes a synergistic anti-stress strategy of "getting rid of stress, relieving stress, adjusting metabolism, assisting respiration and observation and nursing": first, making the fish get rid of stress and restore to a suitable habitat; adding vitamin C in the water to reduce stress; using the gills and body surface of the fish to absorb and supplement glucocorticoid and adrenaline, to adjust metabolism, enhance energy supply and heart function; supplemented with low-intensity oxygen-rich water flushing to promote respiration; continuously observing until the behavior index returns to normal. The method described in the present application is standardized in operation, low in cost, can effectively reduce the mortality rate, is suitable for yellowlip castraomus conservation and wild domestication, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of endangered fish germplasm resource restoration and conservation technology, specifically involving a method for alleviating stress response in yellow croaker and its application. Background Technology

[0002] Yellow croaker ( Bahaba taipingensis This species is a rare and endangered marine fish species unique to my country, and is also listed among my country's top ten outstanding germplasm resources, possessing both extremely high ecological and economic value. As an important apex species in my country's coastal food chain, the protection of its germplasm resources is directly related to the stability of the marine ecosystem and the maintenance of biodiversity.

[0003] However, the wild population of the yellow croaker is currently extremely shrunk, facing a severe endangered situation. Besides long-term external threats such as overfishing and habitat fragmentation and degradation, its own genetic structure reveals significant vulnerability—specifically, low physiological tolerance to environmental fluctuations and insufficient stress regulation. Especially when key ecological factors such as salinity, temperature, and ambient light conditions change drastically, individuals experience severe stress responses, exhibiting significant abnormalities in behavioral indicators (such as swimming posture, schooling, and feeding frequency), which can lead to metabolic disorders or even sudden death in severe cases. These unique physiological and behavioral sensitivities not only limit its ability to adapt to natural conditions but also pose significant challenges to seedling management during artificial conservation and resource restoration. Particularly during the rewilding and domestication phase before restocking, frequent environmental changes and intensive human intervention make the yellow croaker highly susceptible to severe stress responses due to the superposition of multiple stress factors, directly restricting its survival rate and post-release adaptability in the wild.

[0004] Therefore, it is urgent to systematically study and develop scientific and effective stress-enhancing and stress-relieving technologies from the perspectives of physiological intervention and environmental optimization, in order to comprehensively improve the stress resistance and survival rate of yellow croaker during artificial domestication and wilding training. Summary of the Invention

[0005] The purpose of this invention is to provide a method for alleviating the stress response of yellow croaker after being subjected to stress and its application. The method can effectively alleviate the stress response of yellow croaker after being subjected to stress, improve the stress resistance of yellow croaker, and enhance the stress resistance performance of yellow croaker.

[0006] This invention provides a method for alleviating stress response in yellow croaker under stress, including the steps of removing the yellow croaker from stress, relieving stress, adjusting metabolism, assisting breathing, and observation and care. The removal of stress includes restoring the water temperature, salinity, ammonia nitrogen content, nitrite content, stocking density, dissolved oxygen, light intensity, and sound environment of the culture water to levels consistent with the natural habitat of the yellow croaker; The stress relief measures include adding vitamin C to the water to a concentration of 100-250 ppm; The metabolic modulation includes adding glucocorticoids to water to a concentration of 0.01 mg / L, and / or adding adrenaline to water to a concentration of 0.07 mg / L; The assisted breathing includes low-intensity oxygen-enriched water flushing of the yellow croaker. When flushing, the water flow is controlled to be directed towards the head of the fish, and the flushing intensity is appropriate to avoid triggering a startle reflex in the fish. The observation and care included observing the recovery status of the yellow croaker, and stopping the intervention when the yellow croaker exhibited one or more of the following behavioral characteristics: 1) The number of yellow croaker individuals in a state of isolation shall not exceed 2% of the total number of yellow croakers in the same aquaculture water body; 2) Individual yellow croakers that had lost their sense of balance resumed normal swimming posture; 3) The yellow croaker resumed its active feeding behavior.

[0007] Preferably, the method further includes stress assessment: intervention is carried out when the yellow croaker exhibits one or more of the following group behavioral characteristics: A: The state changes from a patrolling or undercover state to an individual floating and scattered state with no ability to swim; B: The body position changes from a normal position with the back and abdomen down to a side-rolling position due to loss of balance; C: Loss of interest in actively eating.

[0008] Preferably, when more than 10% of the yellow croaker individuals in the same aquaculture water exhibit one or more of the behavioral characteristics A to C, it indicates that the yellow croakers in the aquaculture water have experienced a stress response after being subjected to stress.

[0009] Preferably, the natural habitat of the yellow croaker has a water temperature of 20-25℃, a salinity of 13‰-17‰, dissolved oxygen >7mg / L, light intensity <100Lx, ammonia nitrogen content ≤0.10mg / L, nitrite content ≤0.02mg / L, and a stocking density of 5-10kg / m³. 3 The surrounding noise level is <20 decibels; the surrounding area refers to the area within 200-300 m around the natural habitat of the yellow croaker.

[0010] Preferably, the glucocorticoid comprises dexamethasone sodium phosphate.

[0011] Preferably, the adrenaline includes adrenaline hydrochloride.

[0012] Preferably, low-intensity oxygen-enriched water irrigation of yellow croaker involves using a variable frequency water pump to apply highly saturated oxygen water to the yellow croaker.

[0013] Preferably, the irrigation is carried out in an intermittent mode, with each irrigation lasting 15 to 30 minutes and stopping for 5 to 10 minutes; during the irrigation process, the dissolved oxygen content in the aquaculture water is not lower than 8 mg / L; and irrigation is stopped when the behavioral indicators of the yellow croaker return to the pre-stress level.

[0014] Preferably, the method is applicable to stress relief after yellow croaker is subjected to stress during the rearing and / or wild domestication of yellow croaker fry.

[0015] The present invention also provides the application of the method described in the above technical solution in the farming and / or rescue of yellow croaker.

[0016] Beneficial effects: This invention provides a method for alleviating the stress response of the yellow croaker after stress. For yellow croakers experiencing severe stress response due to stress, this invention proposes a synergistic anti-stress strategy of "removal from stress, stress relief, metabolic regulation, assisted respiration, and observation and care": First, the yellow croaker is removed from the stressful environment and gradually restored to an environment consistent with its natural habitat to avoid continuous stress. Simultaneously, vitamin C is added to the water to reduce the stress response level. Glucocorticoids and adrenaline are introduced into the fish's body through the gill filaments and skin to regulate metabolism. Glucocorticoids ensure the supply of nutrients and energy, while adrenaline increases cardiac output by strengthening myocardial contraction, accelerating heart rate, and increasing myocardial excitability, thereby increasing basal metabolic rate and blood glucose levels. Additionally, low-intensity oxygen-enriched water is used to assist the yellow croaker's respiration. Finally, continuous observation and care are provided until behavioral indicators return to pre-stress levels. The above series of steps can effectively alleviate the stress response of yellow croaker caused by stress, effectively reduce the mortality rate of yellow croaker, and improve its survival rate. It has high application value in the breeding and rescue of yellow croaker.

[0017] Secondly, the method described in this invention is standardized in operation, with clear steps, strong practicality and operability, and is easy to implement and promote. It is applicable to the breeding and rewilding of endangered protected fish, especially the yellow croaker. Furthermore, the materials and equipment selected in this invention are all conventional items in the fields of aquaculture and animal protection, with low cost, ease of implementation, and good economic and social benefits. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1The results of serum glucose measurements in Experiment Example 1 were obtained before and after crowding and manipulation of the yellow croaker, and after soaking in dexamethasone sodium phosphate for 6 hours. Figure 2 The results of serum lactate measurement in Experiment Example 1 were obtained before and after crowding and manipulation of the yellow croaker, and after soaking in dexamethasone sodium phosphate for 6 hours. Figure 3 The results of serum cortisol measurements were obtained from the two-spined yellow croakers before and after crowding and manipulation stress, and after soaking in dexamethasone sodium phosphate for 6 hours in Experiment Example 1. Figure 4 The results of serum malondialdehyde (MDA) measurements in *Scoidea dispinipes* before and after crowding and manipulation stress, and after immersion in dexamethasone sodium phosphate for 6 hours, are as follows: Figure 5 The results of serum glucose measurements were obtained for the two-spined yellow croaker before and after crowding and manipulation stress, and after soaking in adrenaline hydrochloride for 6 hours in Experiment Example 1. Figure 6 The results of serum lactate measurement in Experiment Example 1 were obtained before and after crowding and manipulation of the yellow croaker, and after soaking in epinephrine hydrochloride for 6 hours. Figure 7 The results of serum cortisol measurements were obtained for the two-spined yellow croaker before and after crowding and manipulation stress, and after immersion in epinephrine hydrochloride for 6 hours in Experiment Example 1. Figure 8 The results of serum malondialdehyde (MDA) measurements in Experiment Example 1 were obtained before and after crowding and manipulation of the yellow croaker, and after immersion in epinephrine hydrochloride for 6 hours. Figure 9 The results of serum glucose measurements were obtained for the two-spined yellow croaker before and after stress and 6 hours after the progressive treatment to relieve stress response in Experiment Example 2. Figure 10 The results of serum lactate measurement in the double-spined yellow croaker before and after stress and 6 hours after the progressive treatment to relieve stress response in Experiment Example 2; Figure 11 The results of serum cortisol measurements were obtained for the two-spined yellow croaker before and after stress and 6 hours after the progressive treatment to relieve stress response in Experiment Example 2. Figure 12 The results of serum malondialdehyde (MDA) measurement in the double-spined yellow croaker before and after stress and 6 hours after the progressive treatment to relieve stress response in Experiment Example 2; exist Figures 1-8 In the text, lowercase letters above the data columns indicate significant differences between groups. p <0.05); Uppercase letters above data columns indicate significant differences between samples within the same time period ( p <0.05); The trend line in the figure represents the trend line after 6 hours of immersion intervention with different concentrations following the cessation of stress; exist Figures 9-12 In the text, lowercase letters above the data columns indicate significant differences between groups.p <0.05); Uppercase letters above data columns indicate significant differences between samples within the same time period ( p <0.05). Detailed Implementation

[0020] This invention provides a method for alleviating stress response in yellow croaker under stress, including the steps of removing the yellow croaker from stress, relieving stress, adjusting metabolism, assisting breathing, and observation and care. The removal of stress includes restoring the water temperature, salinity, ammonia nitrogen content, nitrite content, stocking density, dissolved oxygen, light intensity, and sound environment of the culture water to levels consistent with the natural habitat of the yellow croaker; The stress relief measures include adding vitamin C to the water to a concentration of 100-250 ppm; The metabolic modulation includes adding glucocorticoids to water to a concentration of 0.01 mg / L, and / or adding adrenaline to water to a concentration of 0.07 mg / L; The assisted breathing includes low-intensity oxygen-enriched water flushing of the yellow croaker. When flushing, the water flow is controlled to be directed towards the head of the fish, and the flushing intensity is appropriate to avoid triggering a startle reflex in the fish. The observation and care included observing the recovery status of the yellow croaker, and stopping the intervention when the yellow croaker exhibited one or more of the following behavioral characteristics: 1) The number of yellow croaker individuals in a state of isolation shall not exceed 2% of the total number of yellow croakers in the same aquaculture water body; 2) Individual yellow croakers that had lost their sense of balance resumed normal swimming posture; 3) The yellow croaker resumed its active feeding behavior.

[0021] As one implementation method, this invention also includes a stress assessment of the stressed yellow croaker before intervention. Given that individual yellow croakers do not show obvious morphological changes after stress, and that invasive testing is not possible for endangered species, the assessment of stress response can only be determined by changes in group morphological characteristics. The assessment of stress response in yellow croakers after stress is usually based on a comparison of the group's morphological characteristics before and after stress: Yellow croakers are considered to have developed a stress response after stress when they exhibit one or more of the following group behavioral characteristics: A: a change from a cruising or lying-down state to a floating, scattered, and weak swimming state; B: a change from a normal dorsal and ventral position to a sideways position with loss of balance; C: loss of interest in active feeding. As one implementation method, the individual floating and dispersing mentioned in this invention refers to an individual that has broken away from the group and floats alone on the surface of the water, and is unable to swim; the loss of balance refers to the fish showing signs of rolling over or having already rolled over; the loss of feeding interest refers to the yellow croaker in the feeding period showing no interest in eating food around its mouth after being stressed, and the loss of feeding interest lasting for more than 6 hours even after the stress is lifted.

[0022] Since the conservation or rewilding of yellow croaker is carried out in groups, as one implementation method, when more than 10% of the yellow croaker individuals in the same aquaculture water exhibit one or more of the behavioral characteristics A to C, it indicates that the yellow croaker in the aquaculture water has a stress response after being stressed and needs to be relieved in time.

[0023] Following the stress assessment, the present invention removes the stress from the yellow croaker exhibiting stress response. This removal of stress includes restoring the water temperature, salinity, ammonia nitrogen content, nitrite content, stocking density, dissolved oxygen, light intensity, and sound environment of the culture water to levels consistent with the natural habitat of the yellow croaker.

[0024] In one embodiment, the water temperature of the natural habitat of the yellow croaker described in this invention is 20-25℃, more preferably 22-24℃; the salinity is 13‰-17‰, more preferably 14‰-16‰; the dissolved oxygen is >7mg / L, more preferably ≥8mg / L; the light intensity is <100Lx; the ammonia nitrogen content is ≤0.10mg / L; the nitrite content is ≤0.02mg / L; and the stocking density is 5-10kg / m³. 3 Furthermore, it can be 5~7.5 kg / m 3 That is, when the stress of yellow croakers in captivity is relieved, their stocking density should preferably be restored to 5-10 kg / m². 3 During the wild rearing process of yellow croaker fry, their weight can generally reach more than 10 kg / fish. At this time, after the stress from being subjected to stress is relieved, their stocking density is preferably restored to 1 fish / m².3 The ambient noise level is <20 decibels; the ambient noise level refers to a range of 200-300 m around the natural habitat of the yellow croaker. As one implementation method, during the stress relief process, changes in the levels of physical factors such as light, sound, and flow are prohibited within the aquaculture water body. In this invention, the stress relief operation serves to repair environmental factors, crowding, and operational stressors, and restore the environmental factors and ecological density of the natural habitat.

[0025] After removing the yellow croaker from stress, the present invention further includes a step of alleviating stress in the stressed yellow croaker; the stress relief involves adding vitamin C to the water to a concentration of 100-250 ppm, further to 200 ppm. In this invention, the vitamin C can reduce the stress response of the stressed yellow croaker. As one embodiment, the stress relief in this invention also includes increasing the oxygen content in the aquaculture water; the oxygenation is achieved by releasing pure oxygen into the aquaculture water through nano-air stones, maintaining the dissolved oxygen level in the aquaculture water at no less than 7 mg / L.

[0026] In addition to alleviating stress, this invention also includes regulating the metabolism of stressed yellow croaker. Specific steps include adding glucocorticoids to the water to a concentration of 0.01 mg / L, and / or adding adrenaline to the water to a concentration of 0.07 mg / L. As one embodiment, the glucocorticoid can be dexamethasone sodium phosphate; the adrenaline includes adrenaline hydrochloride. As one embodiment, the glucocorticoids and adrenaline can be used simultaneously or sequentially at 25-30 minutes intervals. Based on the way yellow croaker absorbs substances through their body surface and gill filaments, this invention, for the first time, addresses metabolic regulation by utilizing the pathways between the gill filaments and the skin and the outside world to allow glucocorticoids and adrenaline to enter the fish's body through the gill filaments and body surface to achieve metabolic regulation, avoiding invasive stimulation methods for endangered fish. By introducing glucocorticoids into the aquaculture water, the glucocorticoid level in the fish is increased, thereby enhancing the immune response and blood glucose levels. At the same time, adding adrenaline to the aquaculture water strengthens myocardial contraction, accelerates heart rate, increases myocardial excitability, increases cardiac output, and also increases basal metabolism and raises blood sugar.

[0027] While regulating the metabolism of the yellow croaker, this invention provides assisted respiration for the stressed yellow croaker. The assisted respiration includes low-intensity oxygen-enriched water flushing of the yellow croaker. During the flushing, the water flow is controlled to be directed towards the head of the fish, and the flushing intensity is such that it does not trigger a startle reflex in the fish.

[0028] In one implementation method, the present invention provides low-intensity oxygen-enriched water irrigation for yellow croaker, which involves using a variable frequency water pump to apply highly oxygenated water to the fish. In another implementation method, the irrigation is conducted intermittently, with each irrigation lasting 15-30 minutes and stopping for 5-10 minutes, or further, each irrigation lasting 20-25 minutes and stopping for 6-8 minutes. In one implementation method, the dissolved oxygen content in the aquaculture water is maintained at no less than 8 mg / L during the irrigation process; irrigation is stopped when the behavioral indicators of the yellow croaker return to pre-stress levels. The present invention uses a variable frequency water pump to apply highly oxygenated water at low intensity towards the direction of the fish's swimming or head, increasing the contact volume and time between the highly oxygenated water and the gill filaments, thereby increasing the amount of oxygen and stress-relieving substances dissolved in the water entering the bloodstream through the gill tissue.

[0029] During the aforementioned anti-stress operations, this invention continuously observes and cares for the yellow croaker undergoing these operations. This observation and care includes monitoring the recovery status of the yellow croaker. Intervention is stopped when the yellow croaker exhibits one or more of the following behavioral characteristics: 1) The number of yellow croakers in a state of isolation does not exceed 2% of the total number of yellow croakers in the same aquaculture water body; 2) Yellow croakers that have lost their sense of balance resume normal swimming posture; 3) Yellow croakers resume active feeding behavior. As one implementation method, the observation and care of this invention also includes ensuring that the aquaculture water is clean and has sufficient dissolved oxygen, further ensuring that the transparency of the aquaculture water is greater than 5 meters, the ammonia nitrogen concentration is less than 0.1 mg / L, the nitrite concentration is less than 0.02 mg / L, and the dissolved oxygen content is above 7 mg / L.

[0030] As one implementation method, the method described in this invention is applicable to stress relief after stress experienced by yellow croaker during conservation and / or wild rearing of yellow croaker fry. This invention is the first to propose a method for reducing the stress response level of the endangered protected fish yellow croaker during conservation rearing and pre-release wild rearing, which helps improve the yellow croaker's resistance to stress in its natural habitat. The method fills a technological gap in the conservation, rearing, and wild rearing of yellow croaker, and is highly practical and operable.

[0031] Based on the above advantages, the present invention also provides the application of the method described in the above technical solution in the farming and / or rescue of yellow croaker.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] The dexamethasone sodium phosphate and epinephrine hydrochloride used in the following experimental examples and embodiments must be stored according to specifications: dexamethasone sodium phosphate and epinephrine hydrochloride must be refrigerated at 2~8℃.

[0034] The water used for soaking in the following experimental examples and embodiments must be treated in accordance with the methods specified in the industry standard "Specifications for Harmless Treatment of Dead Aquatic Animals and Diseased Aquatic Animal Products (SC / T 7015-2022)".

[0035] Experimental Example 1 The optimal dosage of glucocorticoids and adrenaline used in regulating metabolism was explored to enhance the stress resistance of yellow croaker. The steps are as follows: 1. Experimental materials: Since the yellow croaker is a rare and endangered fish, it cannot be used as an experimental subject for dose exploration experiments of glucocorticoids and adrenaline. Therefore, the double-spined yellow croaker, a fish species in the same family as the croaker family with an average total length of 18.6±4.2cm and an average weight of 43±8.8g, was selected as the experimental material to ensure that the experimental material is similar to the yellow croaker in morphology and physiological and biochemical characteristics.

[0036] 2. Experimental Design: 1) Experimental concentration setting of glucocorticoid (dexamethasone sodium phosphate): Given that dexamethasone is an endogenous steroid hormone whose expression is controlled by the hypothalamic-pituitary-adrenal axis, it can regulate cell growth, differentiation and apoptosis. Its main role in regulating life activities is anti-inflammatory and immunosuppressive, but it also has immunosuppressive effects and high doses can lead to adverse reactions such as corticosteroid secretion disorders.

[0037] Based on the maximum value of veterinary dosage, a high limit was set, and dexamethasone sodium phosphate was set at five concentration levels of 5.00 mg / L, 1.00 mg / L, 0.2 mg / L, 0.04 mg / L and 0.008 mg / L, decreasing by a factor of 5.

[0038] 2) Experimental concentration setting of adrenaline: Adrenaline hydrochloride has the effects of stimulating the heart, dilating blood vessels, and raising blood pressure, and can take effect in a short period of 5 to 10 minutes.

[0039] Similarly, referring to the lowest dose for veterinary use, five concentration levels of epinephrine hydrochloride were set up, decreasing by a factor of 3: 0.63 mg / L, 0.21 mg / L, 0.07 mg / L, 0.023 mg / L (approximately 0.02 mg / L), and 0.008 mg / L (approximately 0.01 mg / L).

[0040] One blank control group was set up for both glucocorticoid and adrenaline experiments. Three biological replicates were set up for all concentration levels and control groups, with 15 fish in each biological replicate.

[0041] 3. Experimental steps: First, all experimental groups of yellow croaker were subjected to crowding stress for 6 hours. Specifically, 60% of the water in the pond was drained, reducing the fish density from the original 7.5 kg / m³. 3 Increased to 18.5 kg / m 3 Overcrowding stress occurs when the water volume is reduced to increase density, causing fish to react with stress, leading to increased respiratory rate, oxygen consumption, and the excretion of metabolites. This results in a drop in dissolved oxygen from 7 mg / L to 5 mg / L, and a sharp increase in ammonia nitrogen and nitrite levels from 0.10 mg / L and 0.02 mg / L to 0.55 mg / L and 0.1 mg / L, respectively, causing environmental stress. Simultaneously, the use of dip nets to catch fish and suction devices to remove waste from the pond bottom creates operational stress. Overcrowding stress lasts for 6 hours, and operational stress lasts for 30 minutes. When 10% of the fish show signs of stress, the entire pond is considered to be under stress.

[0042] At this point, the water level in the experimental pool was raised back to the pre-stress level of 7.5 kg / m³. 3 The density was then determined, and the fish were removed from the pond and placed in experimental groups with different concentrations of dexamethasone and adrenaline for immersion treatment. The immersion containers were 0.65m × 0.4m × 0.4m glass tanks, and the control group had a density of 7.5 kg / m³. 3 Density control was implemented. Simultaneously, a nano-aeration stone was used to supply highly oxygenated water to the tank, and a variable frequency water pump was used to flush water towards the fish. 200 ppm of vitamin C was added to the tank. The morphological characteristics of the experimental fish were continuously observed under different concentrations of dexamethasone and adrenaline, including whether the dispersion of the fish school changed, whether the swimming behavior of the fish slowed down, and whether the fish maintained a normal body position (dorsal-ventral). The time it took for the fish to recover to a normal state was recorded.

[0043] Given that fish exhibit a significant increase in serum glucose and lactate levels during stress responses, as these serve as energy sources, and that the stress response weakens with the action of dexamethasone or adrenaline, their concentrations also decrease. Furthermore, cortisol and malondialdehyde (MDA), biochemical indicators, are widely used as markers of oxidative stress to assess the intensity of stress responses. Therefore, in this experiment, blood samples were collected from the tail vein of the experimental fish, and serum glucose (GLU), lactate (LD), cortisol (COR), and malondialdehyde (MDA) levels were measured in both the experimental and control groups before stress, at the onset of stress, and 6 hours after stress intervention. Subsequently, multiple regression analysis was used to determine the optimal doses of dexamethasone sodium phosphate and adrenaline hydrochloride.

[0044] The test results of relevant indicators in the dexamethasone test are as follows: Figures 1-4 As shown, the test results of relevant indicators in the adrenaline test are as follows: Figures 5-8 As shown.

[0045] Depend on Figures 1-4 The results showed that there were no significant differences in glucose and cortisol levels in the low-concentration treatment groups (0.008 mg / L and 0.04 mg / L) before and after the stress intervention. P >0.05); There were no significant differences in glucose and cortisol levels in the high-concentration groups (1 mg / L and 5 mg / L) before and after the stress intervention. P >0.05); only the 0.2 mg / L experimental group showed a significant difference before and after the stress intervention ( P <0.05. Serum lactate levels showed no significant difference before and after the stress intervention. P >0.05), with only the 0.2 mg / L and 1 mg / L experimental groups showing a decrease. The malondialdehyde (MDA) index, except for the 0.008 mg / L experimental group, showed no significant difference before and after the stress intervention. P Except for >0.05, all other experimental groups showed significant differences ( P <0.05). Within the range given by dexamethasone sodium phosphate, a polynomial regression was performed to obtain the regression equations for serum glucose, serum lactate, cortisol, and malondialdehyde, respectively: y = -0.206 x 3 + 4.587 x 2 - 23.351 x +68.129, R 2 = 0.984; y = 0.028 x 3 - 0.133 x 2 + 0.0078 x +3.7585, R 2 = 0.767; y = -0.612 x 3 + 7.227 x 2 - 25.225 x +47.747, R 2 = 0.989; y = -2.156 x 3 + 24.086 x 2 - 83.876x +126.76, R 2 = 0.982.

[0046] In the four regression equations, the optimal level of dexamethasone sodium phosphate was determined by finding the extreme values. The minimum values ​​of dexamethasone sodium phosphate in the serum glucose group, serum lactate group, cortisol group, and malondialdehyde experimental group were 0.38 mg / L, 0.13 mg / L, 0.01 mg / L, and 0.01 mg / L, respectively. Considering the effectiveness in alleviating stress and the sustainability of blood glucose and lactate as energy sources in the stress response, the extreme value of 0.01 mg / L in the cortisol and malondialdehyde experimental groups was selected as the optimal dose for immersion.

[0047] Depend on Figures 5-8 The results showed that blood glucose levels continued to rise with adrenaline levels, and in the high-concentration experimental group, the blood glucose concentration even far exceeded the pre-intervention concentration, showing a significant difference. P <0.05 indicates that high concentrations of adrenaline can cause another form of stress, leading to elevated blood glucose; serum lactate levels did not reach statistically significant levels among the five adrenaline levels set in this experiment. P >0.05), indicating that adrenaline did not significantly reduce serum lactate response during stress response; both cortisol and malondialdehyde decreased with adrenaline levels, showing significant differences from pre-intervention values ​​in the median and subsequent high-concentration experimental groups in this experiment. P <0.05).

[0048] Within the range given by epinephrine hydrochloride, a polynomial regression was performed to obtain the regression equations for serum glucose, lactate, cortisol, and malondialdehyde in the epinephrine hydrochloride experimental group, respectively: y = -0.598 x 3 + 6.506 x 2 - 15.959 x +61.402, R 2 = 0.815; y = -0.023 x 3 + 0.305 x 2 - 1.120 x +4.873, R 2 = 0.987; y = 0.539x 3 - 3.783 x 2 + 3.026 x +30.375, R 2 = 0.883; y = 0.643 x 3 - 3.239 x 2 - 8.822 x +86.718, R 2 = 0.941.

[0049] Similar to the dexamethasone experiment described above, the optimal levels of adrenaline were determined for each of the four regression equations using the extreme value method. First, the optimal detection values ​​for the serum glucose and serum lactate groups were analyzed to be 0.02 mg / L and 0.06 mg / L, respectively. However, the sustained increase in blood glucose in the high-level adrenaline experimental group indicated that high levels of adrenaline had already caused stress. Furthermore, serum lactate levels did not show fluctuations in this experiment, suggesting that lactate levels were unsuitable as an evaluation indicator. Although the detection values ​​for cortisol and malondialdehyde were 0.23 mg / L and 0.24 mg / L, respectively, the differences were not significant for groups with levels above 0.07 mg / L. P (>0.05), experiments showed that 0.07 mg / L of epinephrine reached the concentration level required to relieve stress. Therefore, considering the effectiveness and rationality of stress relief and the avoidance of other stressors, 0.07 mg / L is the optimal dose for epinephrine hydrochloride immersion.

[0050] Experimental Example 2 To investigate the steps and effects of various reagents in alleviating stress responses, based on the concentrations of dexamethasone sodium phosphate and epinephrine hydrochloride obtained in Experiment Example 1, the following experiment was designed: Experimental Group 1: The *Scourgette flounder* was subjected to stress in accordance with the stress method described in Experiment 1. After a stress response was observed, the water level and water quality indicators were restored, and the density was reduced to 7.5 kg / m³, the level before overcrowding stress. 3 The density and water quality indicators also returned to the levels before environmental stress: dissolved oxygen 7 mg / L, ammonia nitrogen 0.10 mg / L, and nitrite 0.02 mg / L. High-oxygen water (≥8 mg / L) was supplied to the tank using a nano-aeration stone, and samples were taken after 6 hours.

[0051] Experimental Group 2: Based on Experimental Group 1, vitamin C was added at a concentration of 200 ppm, and samples were taken after 6 hours.

[0052] Experimental Group 3: Based on Experimental Group 2, dexamethasone sodium phosphate was added at a concentration of 0.01 mg / L, and samples were taken after 6 hours.

[0053] Experimental group 4: Based on experimental group 2, adrenaline was added at a concentration of 0.07 mg / L, and samples were taken 6 hours later.

[0054] Experimental group 5: Based on experimental group 3, adrenaline was added at a concentration of 0.07 mg / L, and samples were taken 6 hours later.

[0055] Experimental group 6 (stress control): After the stress ended, only the water level and water quality indicators were restored, and samples were taken 6 hours later.

[0056] Experimental group 7 (blank control): The water was changed synchronously, but the density of the experimental fish remained unchanged. No stress was applied, and samples were taken 6 hours later.

[0057] Serum glucose, lactate, cortisol, and malondialdehyde levels were measured before stress, after stress (i.e., at the onset of a stress response), and 6 hours after intervention. The data were compared and analyzed. The results are as follows: Figures 9-12 As shown.

[0058] Depend on Figures 9-12 The results show that stress has a significant effect on blood glucose, lactate, cortisol, and malondialdehyde. P <0.05 indicates that the experimental fish underwent a stress response.

[0059] In experimental group 1, only water level and water quality indicators were restored after the stress period ended, and high-saturation oxygen was introduced. Six hours after the intervention, there were no significant differences in the four indicators compared with the values ​​after the stress period. P >0.05), indicating that the stress response of the experimental fish was not reduced after restoring the water level and providing high saturated oxygen for 6 hours; In experimental group 2, in addition to restoring water level and quality indicators and introducing high-saturation oxygen after the stress period, a high concentration of vitamin C was also added. Six hours after the intervention, all four indicators decreased, but none were significantly different from the values ​​measured after the stress period. P >0.05), indicating that the treatment method in experimental group 2 did not alleviate the stress of the experimental fish after 6 hours; After the stress was relieved, the water level and quality in experimental group 3 were restored, high-saturation oxygen was introduced, and a high concentration of vitamin C was added. Additionally, 0.01 mg / L of dexamethasone sodium phosphate was added to the water. The values ​​measured 6 hours after the intervention were significantly different from those after the stress. P <0.05%, except for serum glucose, the other three indicators are close to the pre-stress levels ( P >0.05), indicating that the treatment method in experimental group 3 basically alleviated the stress response caused by stress; The treatment method for experimental group 4 was the same as that for experimental group 3, except that 0.01 mg / L dexamethasone sodium phosphate was replaced with 0.07 mg / L epinephrine hydrochloride. Results 6 hours after intervention showed that only serum glucose levels showed a significant difference compared to those after stress. P <0.05, and although the other three indicators decreased, none reached the level of statistical significance. P >0.05). Field observations showed that in experimental group 4, the addition of epinephrine hydrochloride resulted in all fish that had rolled over returning to their normal positions within 10 minutes, but the stress response was not alleviated after 6 hours. The treatment method for experimental group 5 was the same as that for experimental group 2, except that 0.01 mg / L of dexamethasone sodium phosphate and 0.07 mg / L of epinephrine hydrochloride were added simultaneously to the experimental caddy. The results 6 hours after the intervention showed no significant difference between the measured values ​​at 6 hours and those before the stress. P >0.05), and there were significant differences compared with those after stress ( P <0.05), indicating that the treatment method in experimental group 5 could completely alleviate the stress response caused by stress within 6 hours; Experimental groups 6 and 7 were the stress control and blank control, respectively. The results showed that without treatment after stress, all four biomarkers in the experimental fish remained elevated, indicating a state of stress. Some fish even reached a near-death state, with blood glucose levels dropping to 4.25 mg / L, significantly lower than the pre-stress normal value of 8.04 ± 0.75 mg / L. In the stress control group, cortisol and malondialdehyde concentrations remained elevated, indicating a strong stress response with no relief after 6 hours.

[0060] Based on the above experiments, the following conclusions can be drawn: When fish experience stress, the best way to alleviate this stress is to first terminate the stress and restore the pre-stress environmental conditions, reducing ammonia nitrogen and nitrite levels to below 0.1 mg / L and 0.02 mg / L, respectively. Provide a water body with a high saturated oxygen content of at least 7 mg / L. Simultaneously, add 200 ppm of vitamin C, 0.01 mg / L of dexamethasone sodium phosphate, and 0.07 mg / L of epinephrine hydrochloride reagent to the water. Observe the fish's recovery, including their dispersion, the ratio of swimming to resting, body position, and feeding behavior, while also monitoring water quality changes until the fish return to normal activity levels.

[0061] Example 1 A method for enhancing the stress resistance of yellow croaker during breeding and rearing includes the following steps: One thousand one-year-old yellow croakers, each weighing 241.5 ± 62.1 g, were raised in six cement ponds with a diameter of 6 m and a water depth of 1.2 m. The average density of the six ponds was 7.1 kg / m³.3 Due to facility maintenance, the water volume could only be maintained at half capacity for five consecutive days, leading to elevated levels of ammonia nitrogen and nitrate in the environment, reaching 3.5 mg / L and 0.4 mg / L respectively. Dissolved oxygen levels also dropped to a low of 4.8 mg / L, creating environmental stress. During water changes, to remove as much wastewater as possible, the water level needed to be significantly lowered. When the water level dropped to 0.5m, the density of yellow croaker in the pond surged to 17.1 kg / m³. 3 This created crowding stress, and the repeated dragging of the pond bottom with a vacuum cleaner created operational stress on the fish, causing a stress response in the entire pond.

[0062] Due to limited water volume, three of the six pools served as the experimental group, employing the intervention method described in this invention to alleviate stress and reduce the level of stress response; the other three pools served as the control group, with the water exchange volume reduced by half.

[0063] First, a stress assessment was conducted on the fish in the pond. Although the yellow croakers used in the rearing and conservation process are relatively large and are an endangered species, the stress response and degree still needed to be judged based on the characteristics exhibited by the group behavior under rearing conditions. Observations revealed that after the water level recovered to 1.2m, the yellow croakers in the pond generally did not exhibit the normal leisurely state of group swimming or lying still at the bottom of the pond. Instead, more than 80% of the individuals were scattered, with some individuals raising their heads and not closing their mouths; more than 5% of the individuals showed loss of balance and body rollover; after feeding during the feeding period, all the fish showed no interest in food. This indicates that the yellow croakers in the entire pond experienced a significant stress response after environmental and crowding stress.

[0064] As the water change process ended, the yellow croakers gradually escaped the state of crowding stress, but the stress response did not weaken or disappear with the cessation of crowding stress.

[0065] The experimental group operated according to the following steps: While discharging wastewater from the pond, fresh filtered water was added to gradually remove stress factors such as ammonia nitrogen and nitrite from the water, restoring the concentrations of ammonia nitrogen and nitrite to levels of 0.10 mg / L and 0.02 mg / L, respectively. Simultaneously, as the water level in the pond rose, the fish density was reduced. Through continuous water circulation, the water exchange rate reached over 400%, gradually stabilizing the water temperature at 20±1℃, salinity at 20±1, maintaining dissolved oxygen content above 7 mg / L, avoiding direct sunlight and noise interference, reducing light intensity to below 200 Lx, and noise below 20 dB. Water exchange was completed when the water quality indicators stabilized at less than 0.1 mg / L for ammonia nitrogen and less than 0.02 mg / L for nitrite.

[0066] After the water change, a high concentration of vitamin C was added to the water until the final concentration reached 200 ppm. Simultaneously, dexamethasone sodium phosphate solution was added to the water until the final concentration reached 0.01 mg / L. This dexamethasone is absorbed through the fish's skin and gills, increasing glucocorticoid levels and thus enhancing immune response and blood sugar levels. Thirty minutes later, 0.07 mg / L of adrenaline hydrochloride was added to strengthen myocardial contractility, increase heart rate, and enhance myocardial excitability. This increased cardiac output, basal metabolism, and blood sugar levels help the yellow croaker recover its stress response and regain strength.

[0067] To quickly reduce the stress response level of stressed yellow croakers and restore their normal activity, a variable frequency water pump was used to gently flush highly oxygenated water towards the direction the fish were swimming. This increased the contact area, contact time, and flow rate between the oxygenated water carrying stress-relieving substances and the gill filaments, thereby increasing the amount of oxygen and dissolved substances entering the bloodstream through the gill and epidermal tissues. The oxygenation method involved vaporizing liquid oxygen and dissolving it in the water via nano-air stones. The flow rate of the variable frequency water pump was adjusted to 0.01 m / s to ensure that the oxygen content in the water was above 7 mg / L.

[0068] Once the yellow croakers are all swimming normally or lying on the bottom without rolling over, try feeding them live bait or formulated feed. Since yellow croakers mainly feed while swimming and do not compete for food, the preferred feeding strategy is to extend the feeding process, with each feeding lasting 15-20 minutes. Feed the corresponding amount of bait based on the approximate number of yellow croakers appearing at the points along their swimming path. When more than 85% of the individuals show a desire to eat or have eaten the food, the stress response is considered to have been relieved.

[0069] The control group was operated on as follows: In the other three ponds, only half of the water volume was changed, and the ammonia nitrogen and nitrite levels remained high at 2.7 mg / L and 0.2 mg / L respectively after the water change, while the dissolved oxygen content only recovered to 5.6 mg / L. The crowding stress and operational stress caused by the water change and bottom sludge removal, combined with environmental stress, also resulted in more than 80% of the individuals exhibiting a dispersed state, 10% of the individuals raising their heads upwards with their mouths open, and more than 5% of the individuals showing loss of balance and body rollover. During the feeding period, all the fish showed no interest in food, and the stress response was obvious. After the water change, considering that ammonia nitrogen and nitrite levels did not reach the required levels of 0.1 mg / L and 0.01 mg / L respectively, and without the addition of stress-relieving agents such as vitamin C, dexamethasone sodium phosphate, and adrenaline hydrochloride, or high-saturation oxygen flushing, the dispersed condition of the yellow croakers in the three ponds did not improve within 12 hours after the end of the crowding and operational stress. While the number of fish surfacing decreased, some still showed signs of hypoxia. The tendency for fish to roll over sideways largely disappeared, and they showed no interest in food during feeding times. Based on this, it was determined that the entire pond of yellow croakers remained under stress.

[0070] For the three ponds in the control group, the water quality was consistently kept clean and oxygenated throughout the subsequent observation and care, with ammonia nitrogen ≤0.1 mg / L and nitrite ≤0.02 mg / L, and the environment was kept quiet. Two hours later, the recovery status of the yellow croaker's behavioral characteristics was observed. It was found that the number of individuals in a group swimming or resting state reached the pre-stress level, i.e., more than 95% of the individuals were in a swimming or resting state; the number of scattered individuals was less than 1%; all individuals swam at a balanced speed without rapid evasive behavior; and all individuals maintained normal body position without rolling over. When all the yellow croaker's behavioral characteristics had recovered to the pre-stress state, feeding was started, and the feeding frequency and amount also returned to the pre-stress state, indicating that the stress response had been relieved.

[0071] Example 2 A method for enhancing the stress resistance of yellow croaker during rewilding and domestication includes the following steps: Wild rearing of the yellow croaker is a crucial step in restoring its germplasm resources before restocking and release. Wild rearing improves the adaptability of artificially bred yellow croaker fry to their natural habitat, increasing their survival rate after restocking. Environmental adaptation during wild rearing involves mimicking mutations in environmental factors found in the natural habitat, particularly alterations to factors closely related to natural habitats such as resistance to current stress, noise stress, light stress, pressure stress, and predator avoidance. Such ecological changes, once stressing the yellow croaker fry, trigger stress responses. Wild rearing, through a repeated process of "stress—stress response—gradual adaptation—stress relief," ultimately enables released yellow croaker to reduce the intensity of their stress responses when encountering unsuitable environmental factors in their natural habitat.

[0072] In this embodiment, the released yellow croaker fry were first placed in a wild rearing pond. A variable frequency water pump was used to accelerate the water flow, bringing the yellow croaker to a critical equilibrium point where they neither moved with the current nor easily against it, for 60 minutes. After 10 minutes of flow stress, light stress was simultaneously applied using waterproof LED or COB lights. The light intensity was momentarily increased to 2000 Lx, then gradually dimmed over 5 minutes and turned off repeatedly. After 20 minutes of light stress, noise stress was implemented, using low-frequency noise (100-200 Hz) and high-frequency noise (1-20 kHz) alternately played for 10 minutes using a high-fidelity stereo amplifier and player. Simultaneously, an ultra-deep rearing pond was used, where the fish were continuously swam up and down within a depth range of 1-10 meters.

[0073] After a total stress period of 60 minutes, the fish's stress response was observed, and approximately 10% of the individuals showed signs of stress. Simultaneously, basic water quality indicators such as temperature, salinity, pH, ammonia nitrogen, nitrite, and dissolved oxygen concentration were measured. If the temperature difference from before stress was greater than 2°C, the salinity difference was 5‰, the pH difference was 0.5, and the ammonia nitrogen and nitrite concentrations were greater than 0.10 mg / L and 0.02 mg / L, respectively, the water quality was considered substandard and required adjustment to normal through water exchange or a circulating water system. Next, a high concentration of vitamin C was added, at 200 ppm. Simultaneously, vaporized liquid oxygen was introduced to quickly raise the dissolved oxygen content to 8 mg / L. After stopping the circulating water or performing a water exchange, 0.01 mg / L of dexamethasone sodium phosphate and 0.07 mg / L of adrenaline hydrochloride were added to the tank. Continuously observe the fish's activity. After 3 to 6 hours, when the fish's stress response disappears, that is, when more than 95% of the individuals show normal swimming or lying on the bottom, the second round of stress-repair rewilding can begin. The same pattern of 1 hour of stress followed by 6 hours of repair is adopted and repeated 3 times to ensure that the released yellow croaker basically achieves the purpose of rewilding.

[0074] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for alleviating stress response in yellow croaker under stress, characterized in that, The steps include removing stress from yellow croakers that exhibit stress responses, alleviating stress, regulating metabolism, assisting breathing, and observation and care. The removal of stress includes restoring the water temperature, salinity, ammonia nitrogen content, nitrite content, stocking density, dissolved oxygen, light intensity, and sound environment of the culture water to levels consistent with the natural habitat of the yellow croaker; The stress relief measures include adding vitamin C to the water to a concentration of 100-250 ppm; The metabolic modulation includes adding glucocorticoids to water to a concentration of 0.01 mg / L, and / or adding adrenaline to water to a concentration of 0.07 mg / L; The assisted breathing includes low-intensity oxygen-enriched water flushing of the yellow croaker. When flushing, the water flow is controlled to be directed towards the head of the fish, and the flushing intensity is appropriate to avoid triggering a startle reflex in the fish. The observation and care included observing the recovery status of the yellow croaker, and stopping the intervention when the yellow croaker exhibited one or more of the following behavioral characteristics: 1) The number of yellow croaker individuals in a state of isolation shall not exceed 2% of the total number of yellow croakers in the same aquaculture water body; 2) Individual yellow croakers that had lost their sense of balance resumed normal swimming posture; 3) The yellow croaker resumed its active feeding behavior.

2. The method according to claim 1, characterized in that, The method also includes stress assessment: intervention is performed when the yellow croaker exhibits one or more of the following group behavioral characteristics: A: The state changes from a patrolling or undercover state to an individual floating and scattered state with no ability to swim; B: The body position changes from a normal position with the back and abdomen down to a side-rolling position due to loss of balance; C: Loss of interest in actively eating.

3. The method according to claim 2, characterized in that, When more than 10% of the yellow croaker individuals in the same aquaculture water exhibit one or more of the behavioral characteristics A to C, it indicates that the yellow croakers in the aquaculture water have experienced a stress response after being subjected to stress.

4. The method according to claim 1, characterized in that, The natural habitat of the yellow croaker is characterized by a water temperature of 20-25℃, a salinity of 13‰-17‰, dissolved oxygen >7mg / L, light intensity <100Lx, ammonia nitrogen content ≤0.10mg / L, nitrite content ≤0.02mg / L, and a stocking density of 5-10kg / m³. 3 The surrounding noise level is <20 decibels; the surrounding area refers to the area within 200-300 m around the natural habitat of the yellow croaker.

5. The method according to claim 1, characterized in that, The glucocorticoids include dexamethasone sodium phosphate.

6. The method according to claim 1, characterized in that, The adrenaline includes adrenaline hydrochloride.

7. The method according to claim 1, characterized in that, Low-intensity oxygen-enriched water irrigation for yellow croaker involves using a variable frequency water pump to apply highly saturated oxygen water to the yellow croaker.

8. The method according to claim 7, characterized in that, The irrigation is carried out in an intermittent mode, with each irrigation lasting 15 to 30 minutes and stopping for 5 to 10 minutes; the dissolved oxygen content in the aquaculture water is not lower than 8 mg / L during the irrigation process; and irrigation is stopped when the behavioral indicators of the yellow croaker return to the pre-stress level.

9. The method according to claim 1, characterized in that, The method is applicable to stress relief for yellow croaker during rearing and / or wild domestication of yellow croaker fry after exposure to stress.

10. The application of the method according to any one of claims 1 to 9 in the farming and / or rescue of yellow croaker.

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