A method for improving the success rate of anesthesia of crassostrea hongkongensis

Abstract

The present application relates to the field of aquaculture technology, and discloses a method for improving the success rate of Hong Kong oyster anesthesia, which comprises the following steps: preparing magnesium chloride hexahydrate and a solvent according to a predetermined ratio, wherein the solvent is seawater or a mixed liquid composed of seawater and fresh water; stirring and dissolving the magnesium chloride hexahydrate in the solvent to obtain a mixed solution with a magnesium chloride hexahydrate concentration of 20-30 g / L and a salinity of 20-30 ppt; performing aeration treatment on the mixed solution until the temperature drops to 19-23 DEG C to obtain an anesthesia solution; and placing the Hong Kong oysters to be treated in the anesthesia solution, continuously soaking and anesthetizing the Hong Kong oysters in a light-shielded environment for 10-15 h until the anesthesia operation is completed. By regulating the salinity of the anesthesia solution and combining aeration and heat dissipation, the method eliminates the physical and chemical stimulation of the high-temperature and high-osmotic environment on the Hong Kong oysters, prevents the Hong Kong oysters from producing a closed shell defense reaction, and promotes the Hong Kong oysters to maintain a natural water filtration state, so that the magnesium ions smoothly enter the body to block nerve conduction, thereby improving the success rate of anesthesia.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for improving the success rate of anesthesia in Hong Kong oysters. Background Technology

[0002] In physiological studies, artificial breeding, live sampling, and live transport of bivalve mollusks such as oysters in Hong Kong, anesthesia is usually required to reduce their metabolic rate and prevent mechanical damage during the process. Because the nervous system structure of bivalve mollusks differs significantly from that of vertebrates, conventional fish anesthetics are unlikely to produce conduction blocking effects. Therefore, magnesium-containing agents are currently commonly used for anesthesia, utilizing the competitive binding of magnesium ions to calcium ions at the neuromuscular junction to induce relaxation of the adductor muscle.

[0003] However, in actual anesthesia procedures, directly administering magnesium chloride-based agents often fails to achieve a stable anesthetic effect. High concentrations of magnesium chloride crystals release a large amount of heat of solution when dissolved in water, causing a rapid increase in local solution temperature. Simultaneously, the introduction of large amounts of salt significantly increases the total salinity of the water, severely deviating from the isotonic environment naturally in which Hong Kong oysters grow. When oysters suddenly come into contact with this abnormally high-temperature and hypertonic water, they instinctively trigger an environmental stress defense mechanism, manifested as rapidly and persistently closing their shells.

[0004] This physical defense mechanism directly blocks the passage of external water into the mantle cavity, preventing magnesium ions in the anesthetic solution from reaching the internal gill tissue. The anesthetic then relies solely on slow tissue osmosis to exert its effect. This not only prolongs the anesthesia induction period and reduces the overall success rate of anesthesia, but also depletes the oyster's energy due to prolonged closure and hypoxia. Some procedures blindly increase drug concentrations in pursuit of rapid anesthesia. This practice not only fails to induce oysters to open their shells but also easily triggers severe ion toxicity and osmotic imbalance, causing tissue cell necrosis and ultimately leading to difficulty in oyster recovery or even death after anesthesia. Therefore, eliminating the physicochemical irritations generated during drug preparation and inducing oysters to actively filter water to absorb the anesthetic components are urgent problems to be solved in current shellfish anesthesia procedures. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for improving the success rate of anesthesia in Hong Kong oysters. It solves the problems encountered in existing benthic bivalve anesthesia procedures, where the dissolution process of high-concentration anesthetics is often accompanied by intense physical heat release, and conventional direct drug preparation easily causes the water salinity to deviate from the biological isotonic point. This thermal stress and sudden change in osmotic pressure trigger a strong shell-closing defense response in Hong Kong oysters, making it difficult for the anesthetic solution to penetrate and exert its effects. This results in low anesthesia success rates, excessively long induction times, and even physiological damage such as fluid ion imbalance and cell necrosis due to excessively high local drug concentrations.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for improving the success rate of oyster anesthesia in Hong Kong includes the following steps:

[0008] Prepare anesthetic materials according to the specified ratio. The anesthetic materials include magnesium chloride hexahydrate and a solvent, wherein the solvent is seawater or a mixture of seawater and fresh water.

[0009] The magnesium chloride hexahydrate is added to the solvent and stirred until completely dissolved to obtain a mixed solution with a magnesium chloride hexahydrate concentration of 20-30 g / L and a salinity of 20-30 ppt.

[0010] The mixed solution is aerated until the temperature of the mixed solution drops to 19-23°C to obtain an anesthetic solution;

[0011] The Hong Kong oysters to be processed are placed in the anesthetic solution and continuously immersed in it for 10-15 hours in a dark environment until the anesthesia procedure is completed.

[0012] By employing the above-mentioned technical solution, and due to the specific concentration and salinity ratio of magnesium chloride hexahydrate, combined with aeration and cooling, as well as immersion in a dark environment, a stable anesthetic effect that is less likely to cause stress in oysters is achieved. In the actual solution preparation process, the dissociation of magnesium chloride hexahydrate in the solvent produces a certain exothermic effect, which leads to an initial temperature increase in the solution system. Directly using this solution with residual heat for immersion often induces strong shell-closing resistance in oysters. Therefore, this invention aerates the mixed solution. This physical process of gas-liquid exchange not only gradually lowers the water temperature to 19-23℃, which is within the natural physiological temperature range suitable for Hong Kong oysters, eliminating temperature stimulation, but also replenishes dissolved oxygen in the water while cooling, meeting the basal respiratory and metabolic needs of the oysters during the subsequent long immersion period.

[0013] Once the solution environment stabilizes, Hong Kong oysters are placed in it. Magnesium ions enter the oyster's bloodstream through the gills and mantle, following the oyster's natural filtration process. Physiologically, the free magnesium ions that enter the body competitively bind to calcium receptors at nerve endings, blocking calcium ion influx and inhibiting the release of acetylcholine from the presynaptic membrane. As immersion continues, neuromuscular impulse transmission is gradually blocked, and the contractile force of the oyster's adductor muscle weakens until it loses its tension. Combined with a dark and quiet environment, this effectively reduces physical stimulation from light changes and sound vibrations, allowing the oyster to lower its alertness and maintain a naturally open, filtration state, thus ensuring the continuous entry of magnesium ions to exert an anesthetic effect. The aforementioned physicochemical controls and environmental conditions work together to guarantee the overall success rate of anesthesia.

[0014] Preferably, the concentration of magnesium chloride hexahydrate in the mixed solution is 20 g / L, 25 g / L or 30 g / L, and the salinity of the mixed solution is 20 ppt, 25 ppt or 30 ppt.

[0015] By adopting the above technical solutions, several specific combinations of concentration and salinity are provided, which allows for the selection of a matching chemical environment based on the source of Hong Kong oysters or their physiological size in actual operation, thereby improving the safety and applicability of the operation.

[0016] Preferably, the solvent consists of 80%-100% seawater and 0%-20% freshwater by volume.

[0017] By adopting the above technical solution and using a specific proportion of fresh water to adjust the original seawater, the passive increase in salinity caused by the addition of magnesium chloride hexahydrate can be neutralized, ensuring that the final salinity of the system after the solute is dissolved is within a suitable range.

[0018] Preferably, the aeration treatment is implemented as follows:

[0019] Add an air stone to the mixed solution and aerate at room temperature for 1-2 hours to release heat and lower the solution temperature.

[0020] By employing the above technical solution, the bubbles generated by the air stone increase the contact area between the water and air, which helps dissipate heat. A gradual cooling process of 1-2 hours at room temperature prevents drastic fluctuations in the water's physicochemical properties, providing the oysters with a relatively stable transitional environment.

[0021] Preferably, the liquid-solid ratio of the Hong Kong oysters to be treated to the anesthetic solution is 4:1 to 6:1, specifically, 15-22 Hong Kong oysters are added to every 20L of the anesthetic solution.

[0022] By adopting the above technical solution, a specific range of liquid-to-solid ratio is defined. This density control can maintain the required levels of free magnesium ions and dissolved oxygen for a single oyster during soaking, while also preventing excessive accumulation of excrement due to high density, and avoiding interference from increased ammonia nitrogen in the water during the anesthesia process.

[0023] Preferably, before placing the Hong Kong oysters to be treated into the anesthetic solution, a pretreatment step of the Hong Kong oysters is also included:

[0024] Obtain healthy Hong Kong oysters and clean them to obtain cleaned, healthy Hong Kong oysters.

[0025] By adopting the above technical solution, impurities attached to the oyster shells can be removed in advance, which can prevent these sludges from consuming dissolved oxygen or introducing bacteria during the subsequent long soaking process, thus helping to maintain the purity of the solution.

[0026] Preferably, the specific procedure for continuous immersion anesthesia in a light-proof environment is as follows:

[0027] The Hong Kong oysters to be processed were placed in a dark and quiet environment and continuously immersed in anesthesia.

[0028] By employing the aforementioned technical solutions to block out light sources and control environmental noise, the oyster's neural feedback to external disturbances can be reduced. When oysters are in a relatively relaxed state, their shell-closing defense time is reduced, thus facilitating a smoother onset of anesthesia.

[0029] Preferably, the method further includes a step of determining the anesthesia state of the Hong Kong oyster:

[0030] If the adductor muscle of the Hong Kong oyster is relaxed and the two shells are slightly open when mechanically touched, and there is no response to mechanical touch, then the anesthesia operation is considered to be complete.

[0031] By adopting the above technical solution, a direct method for determining biological signs is provided. Confirming the condition by observing the adductor muscle and touch response not only facilitates practical operation but also avoids subsequent treatment steps being affected by insufficient anesthesia.

[0032] Preferably, the concentration of magnesium chloride hexahydrate in the mixed solution is 20 g / L, the solution salinity is 20 ppt, the aeration treatment controls the solution temperature after aeration to be 21°C, the liquid-solid ratio of the Hong Kong oysters to be treated to the anesthetic solution is 4:1, specifically, 22 Hong Kong oysters are placed in every 20 L of the anesthetic solution, and the continuous soaking and anesthesia time is 10 h.

[0033] By adopting the above technical solution, a set of anesthesia parameters that are biased towards low concentration and short duration is defined. When dealing with some highly sensitive oysters, it can not only achieve the expected anesthesia requirements, but also save on preparation costs and operation time.

[0034] Preferably, the concentration of magnesium chloride hexahydrate in the mixed solution is 25 g / L, the solution salinity is 25 ppt, the aeration treatment controls the solution temperature after aeration to be 23°C, the liquid-solid ratio of the Hong Kong oysters to be treated to the anesthetic solution is 5:1, specifically, 18 Hong Kong oysters are placed in every 20 L of the anesthetic solution, and the continuous soaking and anesthesia time is 13 h.

[0035] By adopting the above technical solution, a set of moderate physicochemical parameter configurations is presented. This moderate state can achieve a good balance between the anesthetic effect and the physiological tolerance of oysters, and has wide applicability in routine processing operations.

[0036] This invention provides a method for improving the success rate of oyster anesthesia in Hong Kong. It has the following beneficial effects:

[0037] 1. This invention eliminates the physicochemical stimulation caused by high temperature and high osmotic pressure on Hong Kong oysters by mixing seawater and freshwater to prepare a solvent and combining this with aeration to dissipate the heat generated by the dissolution of magnesium chloride. This pre-regulation of the aquatic environment prevents oysters from developing a defensive shell-closing response due to sudden environmental changes, allowing them to maintain a natural shell-opening and water-filtering state under suitable temperature and osmotic pressure. This, in turn, allows magnesium ions in the solution to smoothly enter their internal tissues, improving the overall success rate of anesthesia.

[0038] 2. This invention uses magnesium chloride hexahydrate as the anesthetic component. It utilizes the competitive binding of magnesium ions to calcium ions at the neuromuscular junction to block neurotransmitter release, causing the oyster's adductor muscle to lose its contractile ability and relax. By controlling the concentration ratio of the initial anesthetic solution and the liquid-solid ratio of the immersion system, the nerve conduction blocking effect is ensured while avoiding ion imbalance or toxic reactions caused by drug overload, thus achieving stable and deep anesthesia of benthic bivalves.

[0039] 3. The anesthesia method provided by this invention has good safety. After the anesthesia procedure, the oysters are transferred to fresh oxygenated seawater. The concentration gradient difference between the inside and outside of the oyster body promotes the natural diffusion and expulsion of magnesium ions retained in the interstitial spaces. This resuscitation method based on ion concentration regulation does not cause organic damage to the oyster's cells, allowing it to quickly restore normal physical contraction and physiological metabolic functions, thus ensuring a high survival rate during subsequent temporary care. Attached Figure Description

[0040] Figure 1 This is a graph showing the dynamic changes in temperature and dissolved oxygen during the preparation of the anesthetic solution of the present invention.

[0041] Figure 2 This is a graph showing the dynamic changes in the initial filtration ratio of Hong Kong oysters in different treatment groups according to the present invention.

[0042] Figure 3 The following is a dynamic change curve of the anesthesia success rate of different treatment groups in this invention. Sub-figure (a) is a comparison of the anesthesia efficacy of the physical environment regulation treatment group, and sub-figure (b) is a comparison of the anesthesia efficacy of the chemical pharmacological parameter treatment group.

[0043] Figure 4 The following is an evaluation chart of the recovery and survival of Hong Kong oysters in different treatment groups according to the present invention. Sub-figure (a) is a comparison chart of the average recovery time of Hong Kong oysters in each treatment group, and sub-figure (b) is a comparison chart of the survival rate of Hong Kong oysters in each treatment group after 7 days of temporary holding. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Preparation Examples 1-3:

[0046] Preparation Example 1:

[0047] This preparation example provides a method for preparing an oyster anesthetic solution for Hong Kong, including the following steps:

[0048] At room temperature, 500g of magnesium chloride hexahydrate crystals were weighed and added to a mixture of 5% fresh water and 95% seawater by volume. The mixture was stirred until completely dissolved to prepare a 20L mixed solution. At this point, the concentration of magnesium chloride hexahydrate in the solution was 25g / L and the salinity was 25ppt. An air stone was added to the prepared solution, and the solution was aerated and released heat for 2 hours at room temperature to lower the solution temperature. The solution temperature after aeration was controlled at 23℃.

[0049] Preparation Example 2:

[0050] This preparation example provides a method for preparing an oyster anesthetic solution for Hong Kong, including the following steps:

[0051] At room temperature, 400g of magnesium chloride hexahydrate crystals were weighed and added to a mixture of 20% fresh water and 80% seawater by volume. The mixture was stirred until completely dissolved to prepare a 20L mixed solution. At this point, the concentration of magnesium chloride hexahydrate in the solution was 20g / L and the salinity of the solution was 20ppt. An air stone was added to the prepared solution, and the solution was aerated and released heat for 1 hour at room temperature to lower the solution temperature. The solution temperature after aeration was controlled at 21℃.

[0052] Preparation Example 3:

[0053] This preparation example provides a method for preparing an oyster anesthetic solution for Hong Kong, including the following steps:

[0054] At room temperature, 600g of magnesium chloride hexahydrate crystals were weighed and added to seawater with a volume ratio of 100% without adding fresh water. The mixture was stirred until completely dissolved to prepare a mixed solution with a total volume of 20L. At this point, the concentration of magnesium chloride hexahydrate in the solution was 30g / L and the salinity of the solution was 30ppt. An air stone was added to the prepared solution and aerated at room temperature for 1.5h to release heat and lower the solution temperature. The solution temperature after aeration was controlled at 19℃.

[0055] Examples 1-3:

[0056] Example 1:

[0057] This embodiment provides a method for improving the success rate of oyster anesthesia in Hong Kong, including the following steps:

[0058] Take the anesthetic solution prepared in Example 1 at a temperature of 23°C, place the cleaned healthy Hong Kong oysters in the anesthetic solution, control the liquid-solid ratio to be 5:1, that is, put 18 Hong Kong oysters in every 20L of anesthetic solution, and continue to immerse and anesthetize them for 13 hours in a dark and quiet environment until the adductor muscles of the Hong Kong oysters are relaxed and the shells are slightly open, and they do not respond to mechanical touch, thus completing the anesthesia operation.

[0059] Example 2:

[0060] This embodiment provides a method for improving the success rate of oyster anesthesia in Hong Kong, including the following steps:

[0061] Take the anesthetic solution prepared in Example 2 at a temperature of 21°C, place the cleaned healthy Hong Kong oysters in the anesthetic solution, control the liquid-solid ratio to be 4:1, that is, put 22 Hong Kong oysters in every 20L of anesthetic solution, and continue to immerse and anesthetize them for 10 hours in a dark and quiet environment until the adductor muscles of the Hong Kong oysters are relaxed and the shells are slightly open, and they do not respond to mechanical touch, thus completing the anesthesia operation.

[0062] Example 3:

[0063] This embodiment provides a method for improving the success rate of oyster anesthesia in Hong Kong, including the following steps:

[0064] Take the anesthetic solution prepared in Example 3 at a temperature of 19°C, place the cleaned healthy Hong Kong oysters in the anesthetic solution, control the liquid-solid ratio to be 6:1, that is, put 15 Hong Kong oysters in every 20L of anesthetic solution, and continue to soak and anesthetize them for 15 hours in a dark and quiet environment until the adductor muscles of the Hong Kong oysters are relaxed and the shells are slightly open, and they do not respond to mechanical touch, thus completing the anesthesia operation.

[0065] Comparative Examples 1-5:

[0066] Comparative Example 1: Compared with Example 1, the difference is that after preparing the mixed solution, air stones were not added for aeration and heat release treatment. Instead, Hong Kong oysters were directly placed into the anesthetic solution that had just been prepared and showed obvious dissolution and heat release. All other aspects were the same.

[0067] Comparative Example 2: Compared with Example 1, the difference is that the solvent used to prepare the solution was 100% seawater by volume, without adding any fresh water. This resulted in the final anesthetic solution having a salinity much higher than 25 ppt, which failed to match the optimal osmotic pressure of Hong Kong oysters. All other aspects were the same.

[0068] Comparative Example 3: Compared with Example 1, the difference is that the amount of magnesium chloride hexahydrate crystals added is 200g, that is, the concentration of magnesium chloride hexahydrate in the solution is only 10g / L, which fails to reach the optimal concentration that can fully produce competitive antagonistic effect with calcium ions. All other aspects are the same.

[0069] Comparative Example 4: Compared with Example 1, the difference is that the amount of magnesium chloride hexahydrate crystals added is 1000g, that is, the concentration of magnesium chloride hexahydrate in the solution is as high as 50g / L, which triggers an extremely strong magnesium ion toxicity reaction. All other aspects are the same.

[0070] Comparative Example 5: Compared with Example 1, the difference is that the commercially available aquatic conventional anesthetic MS-222 was used instead of magnesium chloride hexahydrate, and the anesthetic solution was prepared according to its conventional recommended concentration for anesthesia. All other aspects were the same.

[0071] Test Examples 1-4:

[0072] Test Example 1:

[0073] Test instructions:

[0074] This test example provides a verification experiment for the physical environment regulation mechanism in this invention, specifically examining the dynamic changes in temperature and dissolved oxygen during the preparation of the anesthetic solution. The test object is the anesthetic solution system corresponding to Example 1.

[0075] Test steps:

[0076] At room temperature, a mixed solvent consisting of 19L of seawater and 1L of fresh water was injected into the solution preparation container. The mixture was allowed to stand, and the initial temperature and initial dissolved oxygen data of the solvent were recorded using a portable water quality analyzer.

[0077] Pour 500g of magnesium chloride hexahydrate crystals into a container at once, and stir rapidly with a glass rod until the crystals are completely dissolved. The moment the dissolution process ends is recorded as 0min. Record the temperature and dissolved oxygen values ​​of the system at this time.

[0078] Then, an air stone connected to an air pump was submerged in the solution, and forced aeration was performed at maximum power. Water quality data was recorded every 30 minutes during the aeration phase, and the aeration duration was 120 minutes.

[0079] Turn off the air pump and remove the air stone, allowing the solution to stand naturally in the in-situ environment to release heat. During the standing phase, continue to record water quality data every 30 minutes for a total of 120 minutes. The test ends when the total duration is 240 minutes.

[0080] Test data:

[0081] Table 1. Dynamic changes in temperature and dissolved oxygen during the preparation of anesthetic solutions.

[0082] Testing phase Time (min) Temperature (°C) Dissolved oxygen (mg / L) initial state - 24.1 6.72 Dissolve with salt 0 32.4 4.51 Continuous aeration 30 29.1 6.38 Continuous aeration 60 26.5 7.82 Continuous aeration 90 24.7 8.25 Continuous aeration 120 23.3 8.44 Static heat release 150 22.8 8.31 Static heat release 180 22.9 8.28 Static heat release 210 23.0 8.25 Static heat release 240 23.1 8.21

[0083] Note: In Table 1, "-" indicates that this test phase has not yet been included in the formal experimental timing sequence, which means the basic reference state "before the timing start".

[0084] in conclusion:

[0085] Figure 1 This is a graph showing the dynamic changes in temperature and dissolved oxygen during the preparation of the anesthetic solution in Test Example 1 of this invention. The graph uses a dual-axis structure for data mapping. The horizontal axis indicates the monitoring period from 0 to 240 minutes, the left vertical axis defines the temperature range from 20 to 35°C, and the right vertical axis defines the dissolved oxygen concentration range from 4 to 10 mg / L. The solid black lines marked with white-filled circles represent the actual temperature change over time, and the dashed black lines marked with black-filled squares represent the synchronous change in dissolved oxygen concentration over time. A vertical dotted line is placed at 120 minutes as a stage boundary marker, visually dividing the entire process into the forced aeration treatment stage on the left and the natural settling treatment stage on the right.

[0086] Combining the data in Table 1 with Figure 1 The trends of the curves show that high-concentration magnesium chloride hexahydrate triggers a significant exothermic physical phenomenon upon dissolution in the mixed solvent. Reflected on the left side of the graph, the temperature curve experiences an abnormal peak at the 0-minute mark, rising sharply to 32.4℃. The accompanying salt effect causes a large release of dissolved oxygen from the water, causing the dissolved oxygen curve to simultaneously drop to a low oxygen trough of 4.51 mg / L. In actual live oyster treatment scenarios in Hong Kong, such a solution environment, characterized by high temperature and oxygen deficiency, would easily trigger the instinctive stress mechanisms of benthic organisms if used directly for immersion. The sudden rise in water temperature not only leads to abnormal internal protein metabolism but also forces the Hong Kong oysters to tightly close their shells to isolate themselves from the unfavorable external water, constituting a core obstacle to the failure of anesthesia caused by traditional direct drug administration. After crossing the 0-minute mark, continuous forced aeration for 120 minutes created strong gas-liquid convection heat transfer within the water, accelerating the transfer of heat energy into the air. Figure 1The solid line trajectory clearly records the smooth downward trend of the solution temperature, which, after crossing the vertical boundary and entering the natural settling stage, eventually stabilized within a safe range of approximately 23°C. The aeration process simultaneously completed the physical reoxygenation of the water, causing the dashed line trajectory to rise rapidly, resulting in dissolved oxygen exceeding 8.0 mg / L at 90 minutes, entering a supersaturated state and maintaining a high and stable level thereafter. This solution system, with its fully regulated physical environment, completely eliminated the initial risks of heat stimulation and suffocation. Combined with the pre-set salinity conditions, it induced the Hong Kong oysters, which were in a state of natural respiration, to actively open their shells to filter water, fully exposing the internal gill tissue and mantle to a magnesium-rich environment. This provided a crucial pathway for drug absorption, enabling subsequent nerve ending conduction blockade.

[0087] Test Example 2:

[0088] Test instructions:

[0089] This test example provides a verification experiment on the osmotic pressure and salinity adaptation mechanism and the comprehensive effect of physical environment regulation in this invention. Specifically, it examines the direct impact of different environmental conditions on the initial water filtration behavior of Hong Kong oysters after being introduced into the water. The test subjects are the treatment groups corresponding to Example 1, Comparative Example 1 and Comparative Example 2.

[0090] Test steps:

[0091] Ninety healthy adult Hong Kong oysters of similar size and with sensitive adductor muscle response were selected. After cleaning the shells, they were randomly divided into three groups of 30 each.

[0092] The anesthetic solutions prepared in Example 1, Comparative Example 1, and Comparative Example 2 were placed in three separate experimental water tanks.

[0093] Three groups of Hong Kong oysters were simultaneously placed horizontally in their respective experimental water tanks, ensuring that the solution completely submerged all Hong Kong oyster individuals, and observed in a dark and quiet indoor environment.

[0094] Timing begins from the moment the Hong Kong oysters are placed in the water. The double-shell status of each individual in each group is continuously monitored by a low-light camera or the naked eye. The specific time when a single Hong Kong oyster first opens its double shell and the gap between the shells exceeds 1 mm is recorded. The average time for the first shell opening of the group is then calculated.

[0095] At the four time points of 30min, 60min, 90min and 120min, count and record the number of Hong Kong oysters in each tank that are currently in a state of slight double-shell opening and maintaining water filtration, and calculate the percentage of each oyster in the group.

[0096] Test data:

[0097] Table 2. Observation data on the initial filtration behavior of Hong Kong oysters in different treatment groups.

[0098] Processing Group Average time for initial shell opening (min) 30-minute filtration rate (%) 60-minute filtration rate (%) 90-minute filtration rate (%) 120min filtration rate (%) Example 1 12.4 86.7 93.3 90.0 96.7 Comparative Example 1 85.2 10.0 16.7 23.3 20.0 Comparative Example 2 43.7 36.7 43.3 33.3 26.7

[0099] in conclusion:

[0100] Figure 2 This is a graph showing the dynamic changes in the initial filtration rate of Hong Kong oysters in different treatment groups during Test Example 2 of this invention. The horizontal axis represents soaking time (min), ranging from 20 to 140 min, with 30, 60, 90, and 120 minutes as key observation nodes. The vertical axis represents the percentage of Hong Kong oysters maintaining filtration (%), with the display range extended to 0 to 110% to fully represent data fluctuations. The graph contains three different line types, representing the data curves of Example 1, Comparative Example 1, and Comparative Example 2. To visually represent the data columns, in addition to an auxiliary reference graph in the middle of the right side of the graph, the specific treatment group name is directly labeled at the end of the horizontal axis of each curve at 123 minutes. The curves maintaining a high level at the top of the graph are marked with solid lines and white filled circles, representing the filtration rate change trajectory of Hong Kong oysters in Example 1 group using the 25ppt salinity and physical exothermic synergistic control scheme of this invention. The curve at the bottom of the chart, marked with a dashed line and a black-filled square, represents the change in the filtration rate of the Hong Kong oysters in Comparative Example 1, which suffered heat stress without aeration and heat dissipation treatment. The curve in the middle, marked with a dotted line and a gray-filled triangle, represents the change in the filtration rate of the Hong Kong oysters in Comparative Example 2, which used 100% seawater as solvent without osmotic pressure adjustment.

[0101] Based on the data in Table 2, Figure 2The curve trend shows that the Hong Kong oysters in the Example 1 environment deactivated their defensive state on average 12.4 minutes after entering the water and maintained a high level of active filtration rate of over 86.7% throughout the subsequent observation period of 30 to 120 minutes. This is a stark contrast to Comparative Examples 1 and 2. Previous physiological studies of demersal bivalve mollusks have frequently observed that these organisms exhibit highly sensitive neural reflexes to physical and chemical mutations in their external environment. In Example 1, the salinity of the solution was anchored at 25 ppt by pre-adjusting the freshwater-to-seawater ratio. This core parameter falls precisely within the isotonic range of Hong Kong oysters growing in the estuary. When they came into contact with this water without osmotic pressure stress, their gill cilia and mantle receptors did not receive alarms for high or low salinity, thus enabling them to quickly resume normal breathing and feeding postures. In Comparative Example 2, conducted in a high-salinity environment, we observed drastically different behavioral patterns. Hong Kong oysters in pure seawater experienced significantly prolonged shell-closing time due to high osmotic pressure, averaging 43.7 minutes, and less than half of the individuals were willing to continue opening their shells. This physical defense barrier, triggered by salinity discomfort, directly blocked the entry of large amounts of anesthetic fluid into the mantle cavity, forcing the drug to exert its effect only through extremely slow permeation through tissue crevices. Temperature stress was even more pronounced in Comparative Example 1. The high-temperature solution, without aeration for heat dissipation, instantly triggered a heat shock response in the Hong Kong oysters. The shell-closing delay of up to 85.2 minutes and the extremely low filtration rate (below 25%) throughout the process indicate that the vast majority of individuals chose to deeply close their shells for self-preservation. This instinctive self-isolation mechanism prevents magnesium ions from reaching the internal neuromuscular junctions, even in water containing sufficient concentrations. The cross-sectional comparison of these observational data sets is sufficient to verify that simply providing chemical agents is far from enough in the anesthesia of live shellfish. It is necessary to construct a buffer environment that matches the osmotic pressure preference of the species and completely eliminates heat stress in advance, so as to induce them to actively lower their physical defenses. This is the core prerequisite for ensuring the smooth operation of the magnesium ion pharmacological mechanism.

[0102] Test Example 3:

[0103] Test instructions:

[0104] This test case provides a verification experiment on the synergistic effect of chemical pharmacological mechanism and physical environment regulation in this invention. Specifically, it examines the actual impact of different formulations and treatment environments on the success rate of anesthesia of Hong Kong oysters. The test subjects cover all treatment groups corresponding to Examples 1 to 3 and Comparative Examples 1 to 5.

[0105] Test steps:

[0106] Forty adult Hong Kong oysters of similar physiological specifications such as shell length and weight and in good health were selected. The mud and dirt attached to the shell surface were thoroughly cleaned with a stiff brush. They were then temporarily kept in clean seawater for 24 hours to empty their intestines and calm the stress of the operation. After the temporary keeping, they were randomly divided into 8 experimental groups, with 50 oysters in each group.

[0107] According to the parameters of Examples 1 to 3 and Comparative Examples 1 to 5, the corresponding anesthetic solutions were prepared in eight glass water tanks of the same specifications, and the liquid-solid ratio and initial water temperature in each tank were strictly controlled.

[0108] After each group of Hong Kong oysters has finished its temporary rearing, it is moved horizontally into the corresponding glass tank, ensuring that the anesthetic solution completely covers the surface of the Hong Kong oysters. The tank is then covered with a black light-blocking cloth to maintain a quiet laboratory environment and eliminate external noise and light interference.

[0109] The anesthesia status was assessed at 6h, 9h, 12h and 15h after the start of the soaking treatment. The edge of the mantle and the attachment of the adductor muscle of the bivalve slightly open Hong Kong oyster were gently touched with a blunt glass rod.

[0110] If the Hong Kong oyster does not show a contraction reflex to touch, and the gap between the two shells is greater than 2 mm and the adductor muscle is in a completely relaxed state, it is determined that the standard for deep anesthesia has been met. The cumulative number of Hong Kong oysters that meet the standard in each group at each time point is recorded, and the cumulative anesthesia success rate is calculated accordingly. After the determination is completed, the Hong Kong oysters are temporarily left in place or removed (depending on the needs of subsequent resuscitation experiments), and the status of individuals that do not meet the standard is monitored.

[0111] Experimental data:

[0112] Table 3. Dynamic observation data on cumulative anesthesia success rate of Hong Kong oysters in different treatment groups.

[0113] Processing Group 6-hour anesthesia success rate (%) 9-hour anesthesia success rate (%) 12-hour anesthesia success rate (%) 15-hour anesthesia success rate (%) Example 1 12.4 68.6 94.2 96.0 Example 2 16.8 75.4 91.8 92.4 Example 3 7.6 42.1 79.5 88.6 Comparative Example 1 2.5 14.8 27.2 31.5 Comparative Example 2 5.2 21.6 46.8 50.2 Comparative Example 3 0.0 4.4 13.6 16.8 Comparative Example 4 19.5 36.2 38.6 40.4 Comparative Example 5 0.0 2.8 6.2 8.0

[0114] in conclusion:

[0115] Figure 3This is a dynamic change curve of anesthesia success rate in different treatment groups in Test Example 3 of this invention. The graph is divided into two sub-graphs along the vertical axis. The horizontal axis is uniformly set to the immersion time (h) spanning 5 to 16 hours, with key observation nodes set at 6, 9, 12, and 15 hours. The vertical axis presents the cumulative anesthesia success rate (%) range from 0 to 100%. Sub-graph (a) focuses on the comparison of physical environment control. The solid line with white-filled circles maintaining the high range in the graph represents Example 1 group under standard control environment; the dashed line with black-filled squares below represents Comparative Example 1 group without heat dissipation treatment; the dotted line with gray-filled upward triangle in the middle represents Comparative Example 2 group in a full seawater high salinity environment. Subfigure (b) focuses on the comparison of chemical and pharmacological parameters. The solid line with a white-filled circle marks Example 1 (25 g / L magnesium chloride) as the baseline control line; the dashed line with a black-filled downward triangle corresponds to the 3 comparative examples of the 10 g / L low-concentration reagent; the dotted line with a gray-filled diamond corresponds to the 4 comparative examples of the 50 g / L high-concentration reagent; and the dotted line with a white-filled pentagram at the bottom of the horizontal axis represents the 5 comparative examples of the conventional MS-222 reagent.

[0116] Based on the data in Table 3 and in conjunction with Figure 3 The curves in neutron graphs (a) and (b) show a significant steep upward trend in all three formulations during the 12-15 hour soaking period, ultimately achieving a very high anesthesia induction effect, with a maximum success rate of 96.0%. This directly confirms the core pharmacological value of appropriate concentrations of magnesium ions in nerve conduction blockade in benthic bivalves. When a sufficient concentration of magnesium ions smoothly enters the body of the Hong Kong oyster, it strongly competes with calcium ions at the peripheral neuromuscular junction, blocking the neurotransmitter release pathway, causing the adductor muscle fibers to lose their contraction command and enter a forced relaxation state. The physical environment induction mechanism plays an irreplaceable role as a permeation channel in this drug delivery process. Observing subgraph (a), it can be seen that in Comparative Example 1, because the solution was not cooled, the severe heat stress forced the Hong Kong oyster to keep its shell closed for a long time. This physical defense barrier directly blocked the diffusion and penetration of magnesium ions into the body, causing the curve of this group to remain at a low level, and the final anesthesia rate only reached 31.5%. Comparative Example 2 deviated from the isotonic point of the Hong Kong oyster in the estuary under the background of all seawater. Osmotic pressure stress also triggered defensive shell closure, which made its success rate rise very slowly and eventually stagnate at around 50%.

[0117] In actual aquatic pharmacology tests, researchers often fall into the common misconception that increasing drug concentration will improve anesthetic efficacy. The comparative data in subfigure (b) clearly reveals this blind spot. The low-concentration formulation in Comparative Example 3 failed to create an overwhelming antagonistic advantage in the synaptic cleft, and the anesthesia rate of less than 17% was entirely in line with expectations. In Comparative Example 4, the high concentration of 50 g / L did not bring a proportional increase in efficacy. Instead, it triggered strong ionotoxic spasms during the initial immersion, with a large number of Hong Kong oysters exhibiting mantle atrophy or even loss of physiological activity. This is reflected in the graph as the curve showing a significant halt in growth after crossing the 9-hour mark, and then flattening out completely. As for the test of the conventional fish anesthetic MS-222, the results thoroughly revealed the fundamental differences in the nervous system architecture among different species. MS-222 primarily anesthetizes the central nervous system of vertebrates by blocking sodium ion channels. However, the nerve cords and adductor muscle neurons of bivalve mollusks are extremely insensitive to this type of channel blocker. The almost ground-hugging curve at the bottom of the chart and the highest success rate of only 8.0% definitively prove its complete failure in this type of shellfish treatment scenario. Cross-validation of these physicochemical parameters, both horizontally and vertically, fully confirms that the achievement of efficient live anesthesia in bivalve mollusks never relies on the extreme application of a single variable, but must be based on the deep coupling of three mechanisms: osmotic isotonic buffering, thermodynamic steady-state dissipation, and precise antagonism of targeted ions.

[0118] Test Example 4:

[0119] Test instructions:

[0120] This test case provides an evaluation and verification experiment on the anesthesia safety and resuscitation performance of the present invention. Specifically, it examines the effects of different treatment conditions on the physiological function recovery and medium- to long-term survival of Hong Kong oysters after soaking. The test subjects are Hong Kong oyster individuals in each group that have undergone 15 hours of soaking treatment in Test Case 3.

[0121] Test steps:

[0122] Thirty soaked Hong Kong oysters were randomly selected from each of the independent tanks after the test case 3 was completed. They were placed on a clean workbench and their shells were repeatedly rinsed with filtered seawater at room temperature to remove any residual anesthetic fluid.

[0123] The cleaned Hong Kong oysters were transferred to eight pre-prepared regeneration tanks, which were filled with plenty of natural seawater. The water quality parameters were uniformly adjusted to a salinity of 25 ppt and a water temperature of 21 ± 2℃, and micropore aeration was continuously carried out at the bottom of the tank.

[0124] The timing was set from the moment the Hong Kong oyster was completely submerged in the regenerating seawater. Every 0.5 hours for the first 6 hours, and then every 2 hours thereafter, the free edge of the Hong Kong oyster mantle was touched with the end of a glass rod.

[0125] When Hong Kong oysters are observed to produce a sharp and strong shell-closing reflex in response to physical touch, it is determined that the individual has been fully recovered from anesthesia. The recovery time for each Hong Kong oyster is recorded to calculate the average recovery time for each group. Individuals that have not recovered their shell-closing ability after more than 24 hours are counted as 24 hours in the statistics.

[0126] After the recovery status monitoring was completed, each group of Hong Kong oysters was kept in the original recovery tank for a 7-day temporary rearing observation period. During this period, oxygenation was maintained, and isothermal and salinity water exchange was performed once a day, and microalgae feed was provided according to the conventional dosage.

[0127] During the temporary holding period, inspect the oysters daily and promptly remove any dead individuals with large open shells and emitting an odor. At the end of the 7th day, count the number of remaining surviving Hong Kong oysters in each tank and calculate the final temporary holding survival rate for each group.

[0128] Test data:

[0129] Table 4. Data on recovery time and temporary survival rate of Hong Kong oysters in different treatment groups.

[0130] Processing Group Average recovery time (h) 7-day survival rate (%) Example 1 2.4 100.0 Example 2 3.1 96.6 Example 3 1.8 98.2 Comparative Example 1 14.6 38.5 Comparative Example 2 7.3 68.4 Comparative Example 3 0.8 85.1 Comparative Example 4 22.7 12.3 Comparative Example 5 1.2 89.6

[0131] in conclusion:

[0132] Figure 4 This is a chart evaluating the recovery and survival of Hong Kong oysters in different treatment groups in Test Example 4 of this invention. The chart uses a dual-window layout, with the horizontal axis of each sub-chart sequentially arranging the eight independent experimental groups from Example 1 to Comparative Example 5. The upper sub-chart (a) compares the average recovery time of Hong Kong oysters in each treatment group, with the vertical axis representing the average recovery time (h), ranging from 0 to 25 hours. The chart uses solid black lines with white-filled circles to connect the discrete data points of each group, visually demonstrating the differences in the recovery speed of Hong Kong oysters under different pretreatment conditions. The lower sub-chart (b) compares the 7-day survival rate of Hong Kong oysters in each treatment group, with the vertical axis representing the survival rate (%), extending to 0 to 110% to cover the entire range. This section uses dashed black lines with black-filled squares to delineate the data trend, accurately reflecting the final survival status distribution of each group of Hong Kong oysters after deep immersion and subsequent long-term temporary rearing.

[0133] Based on the data in Table 4 and in conjunction with Figure 4The two zigzag trajectories in the figure show that Example 1 demonstrated excellent functional reversibility and physiological safety after achieving deep anesthesia in the early stage. Its average recovery time was only 2.4 hours, and it achieved 100% complete survival in subsequent long-term convalescence. This forms an ideal data combination of extremely low recovery time and extremely high survival rate in the left area of ​​the subgraph. In actual scientific research and production scenarios such as live sampling and live transport, the rate of drug metabolism and the degree of bodily damage have always been decisive indicators for evaluating the feasibility of anesthesia protocols. The magnesium ion competitive antagonism mechanism used in this invention is essentially a highly reversible physicochemical process. When Hong Kong oysters under anesthesia are transferred to fresh seawater, the sudden drop in magnesium ion concentration in the external environment directly breaks the original fluid concentration gradient. Free magnesium ions accumulated at the neuromuscular junction rapidly diffuse outwards to release their occupying position, and calcium ions then resume their promoting effect on neurotransmitter release, allowing the adductor muscle to recover normal contractile function in a short time.

[0134] Observing the control group data on the right side of the chart, Comparative Example 1, which did not undergo physical heat dissipation pretreatment, revealed serious methodological flaws. The high temperature emitted by the dissolving magnesium chloride not only severely interfered with the stable absorption of the drug in the early stages, but also caused irreversible thermal damage to the internal tissues of Hong Kong oysters. The slow recovery of up to 14.6 hours and the plummeting survival rate of 38.5% clearly indicate that the high temperature stress has caused substantial damage to the metabolic system of the underlying cells. Comparative Example 4, exposed to an extremely high concentration of 50 g / L, even showed severe ion toxicity sequelae. The drastic imbalance of osmotic pressure at the cellular level led to the necrosis of a large number of individual tissues. The prolonged paralysis of nearly 23 hours and the barely maintained extremely low survival rate of 12.3% completely negate the crude approach taken by the aquaculture industry to forcibly obtain anesthetic effects by unrestrainedly increasing the drug concentration.

[0135] Furthermore, while the hyperpermeable seawater environment in Comparative Example 2 did not cause fatal destruction like high temperature or high toxicity, the prolonged resistance to external osmotic pressure depleted the shellfish's energy reserves. This internal depletion not only prolonged the recovery period but also indirectly lowered the final survival rate. As for Comparative Example 3 (low concentration) and Comparative Example 5 (conventional drug), which performed poorly in the initial anesthesia tests, their seemingly short recovery time in subfigure (a) and acceptable survival rate in subfigure (b) were merely because they never achieved a substantial state of anesthesia relaxation. Even so, they still endured unnecessary environmental transfer stress during the ineffective immersion process, resulting in a lower survival rate compared to the example group that underwent standard anesthesia and non-invasive recovery procedures. Based on a comprehensive analysis of the previous efficacy data, the core parameter system constructed in this invention found a precise balance between pharmacological depth and non-invasiveness, ensuring a high success rate of anesthesia for benthic bivalves while successfully avoiding secondary hidden damage to vulnerable mollusks caused by environmental physicochemical mutations.

Claims

1. A method for improving the success rate of oyster anesthesia in Hong Kong, characterized in that, Includes the following steps: Prepare anesthetic materials according to the specified ratio. The anesthetic materials include magnesium chloride hexahydrate and a solvent, wherein the solvent is seawater or a mixture of seawater and fresh water. The magnesium chloride hexahydrate is added to the solvent and stirred until completely dissolved to obtain a mixed solution with a magnesium chloride hexahydrate concentration of 20-30 g / L and a salinity of 20-30 ppt. The mixed solution is aerated until the temperature of the mixed solution drops to 19-23°C to obtain an anesthetic solution; The Hong Kong oysters to be processed are placed in the anesthetic solution and continuously immersed in it for 10-15 hours in a dark environment until the anesthesia procedure is completed.

2. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The concentration of magnesium chloride hexahydrate in the mixed solution is 20 g / L, 25 g / L or 30 g / L, and the salinity of the mixed solution is 20 ppt, 25 ppt or 30 ppt.

3. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The solvent consists of 80%-100% seawater and 0%-20% freshwater by volume.

4. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The specific implementation method of the aeration treatment is as follows: Adding a gas stone to the mixed solution for aeration and heat release, and aerating for 1-2 hours at room temperature to reduce the solution temperature.

5. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The liquid-solid ratio of the Hong Kong oysters to be treated to the anesthetic solution is 4:1 to 6:1, specifically, 15-22 Hong Kong oysters are added to every 20L of the anesthetic solution.

6. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, Before placing the Hong Kong oysters to be treated into the anesthetic solution, a pretreatment step for the Hong Kong oysters is also included: Obtain healthy Hong Kong oysters and clean them to obtain cleaned, healthy Hong Kong oysters.

7. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The specific procedure for continuous immersion anesthesia in a light-proof environment is as follows: The Hong Kong oysters to be processed were placed in a dark and quiet environment and continuously immersed in anesthesia.

8. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The method also includes a step for determining the anesthesia status of Hong Kong oysters: If the adductor muscle of the Hong Kong oyster is relaxed and the two shells are slightly open when mechanically touched, and there is no response to mechanical touch, then the anesthesia operation is considered to be complete.

9. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The concentration of magnesium chloride hexahydrate in the mixed solution is 20 g / L, the salinity of the solution is 20 ppt, the temperature of the solution after aeration is controlled at 21°C, the liquid-solid ratio of the Hong Kong oysters to be treated to the anesthetic solution is 4:1, specifically, 22 Hong Kong oysters are placed in every 20 L of the anesthetic solution, and the continuous soaking and anesthesia time is 10 h.

10. The method for improving the success rate of oyster anesthesia in Hong Kong according to claim 1, characterized in that, The concentration of magnesium chloride hexahydrate in the mixed solution is 25 g / L, the salinity of the solution is 25 ppt, the temperature of the solution after aeration is controlled at 23°C, the liquid-solid ratio of the Hong Kong oysters to be treated to the anesthetic solution is 5:1, specifically, 18 Hong Kong oysters are placed in every 20 L of the anesthetic solution, and the continuous soaking and anesthesia time is 13 h.

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