Method for evaluating alkali resistance of grass carp based on gill Na < + > ion flow velocity

Abstract

The invention relates to a method for evaluating alkali resistance of grass carp, in particular to a method for evaluating alkali resistance of grass carp based on gill Na < + > ion flow velocity. The method solves the problems that different concentration gradients need to be designed in existing fish alkali resistance evaluation, the workload is large, high-dose lethal is taken as a judgment standard, and repeatability and operability are poor. The method comprises the following steps: 1, collecting to-be-detected grass carp samples; 2, taking gill tissues; 3, measuring the Na < + > ion flow velocity of the gill tissue by a non-invasive micro-measurement technology; and 4, evaluating the alkali resistance of the grass carp according to the Na < + > ion flow velocity of the gill tissue. The alkali resistance of the grass carp is evaluated by utilizing the flow velocity of Na < + > ions in the gill tissue, an efficient and low-loss alkali resistance evaluation method is established, the defect that a to-be-detected sample is dead due to the fact that the alkali resistance is evaluated by a traditional 96-hour half lethal concentration is overcome, and the purpose of efficiently evaluating the alkali resistance of the grass carp in a low-loss mode is achieved.

Description

Technical Field

[0001] The invention relates to a method for evaluating the alkali resistance of grass carp. Background Art

[0002] my country has 690 million mu of low-lying saline-alkali water, mainly distributed in the arid inland and coastal areas of Northeast, Northwest and North China, with high carbonate alkalinity (C A ), high pH, ​​and unbalanced ion coefficients make it difficult to develop and utilize. Currently, less than 2% of low-salinity alkaline water (C A <10mmol / L) is used for fishery development, and more than 90% of the medium and high salinity water (C A >10mmol / L) is still in a state of low production and abandonment. High carbonate alkalinity can easily cause ammonia poisoning and alkalosis to aquatic organisms. Studies have found that HCO3 – and CO3 2– It is the main ion that affects the alkalinity of water bodies. It directly or indirectly acts on the tissues and organs of fish, causing organic damage and structural changes, which in turn leads to respiratory and metabolic alkalosis in fish, seriously affecting their growth, reproduction and survival. At present, the method for evaluating the alkali resistance of fish at home and abroad is still to estimate the median lethal concentration (LC50) by counting the number of fish deaths within 96 hours. 50 ) was used for evaluation. This method requires the design of different concentration gradients, which is labor-intensive and uses high-dose lethality as the criterion. It has poor repeatability and operability. Summary of the invention

[0003] The present invention aims to solve the problems that the existing fish alkali resistance evaluation method needs to design different concentration gradients, which is labor-intensive, uses high-dose lethality as the judgment standard, and has poor repeatability and operability. + Method for evaluating alkali resistance of grass carp by ion flow velocity.

[0004] The present invention is based on gill Na + The method for evaluating the alkali resistance of grass carp by ion flow rate is carried out in the following steps: 1. Collect the grass carp samples to be tested; 2. Soak the fish samples to be tested in 20-30 mmol / L NaHCO3 solution and take the gill tissue; 3. Determine the Na content of the gill tissue by non-destructive micro-measurement technology. + Ion flow rate, take 1 to 2 gill filaments from the gill tissue; 4. According to the gill tissue Na + The alkaline resistance of grass carp was evaluated by flow rate, that is, after immersion in alkalinity of 20-30mmol / L, the gill Na + Ion flow rate is less than 100 pmol cm -2 ·s -1 The grass carp has a high alkali resistance, Na +Ion flow rate is 100-200 pmol·cm -2 ·s -1 The grass carp has a moderate alkali resistance. + Ion flow rate greater than 200 pmol cm -2 ·s -1 The alkali resistance of grass carp is judged to be low.

[0005] Beneficial effects of the present invention:

[0006] Fish generally have to go through three stages: stress, initiation, and adaptation, and finally reach physiological homeostasis to adapt to adverse environments. To achieve physiological homeostasis, the body's acid-base balance must be ensured first, and HCO3 - It is an important ion for maintaining the acid-base balance in the body, usually with Na + or Cl - Form a buffer to regulate. Non-invasive Micro-test Technology (NMT) has been widely used in biomedicine, plant stress response, animal immunity and other fields because it can monitor the ion flow characteristics of different tissues and cells in living organisms in real time at a specific time and space. In recent years, this technology has also been applied to provide direct living evidence for the analysis of fish response mechanisms to stress such as ocean acidification and heavy metal pollution. The gills are in direct contact with water and are important tissue organs for fish respiration, osmotic pressure regulation, acid-base regulation, etc. A large number of studies on plants and animals have confirmed that Na + / HCO3 - Transporters enable Na + and HCO3 - co-transporter, so monitoring Na + Can indirectly reflect HCO3 - The invention utilizes Na in gill tissue to detect the transport status of fish, and has the advantages of high detection efficiency and little damage to the fish body. + The alkali resistance of grass carp is evaluated by ion flow rate, and an efficient and low-loss alkali resistance evaluation method is established, which overcomes the defect that the traditional 96-hour semi-lethal concentration evaluation of alkali resistance leads to the death of the sample to be tested. The method of the invention only needs to take 1 to 2 gill filaments to complete the evaluation of the alkali resistance of grass carp. After the gill filaments are taken, the fish to be tested is still in a living state. The invention achieves the purpose of evaluating the alkali resistance of grass carp with high efficiency and low loss.

[0007] The existing method for evaluating the alkali resistance of fish requires the design of different concentration gradients, resulting in a large workload, while the method of the present invention has a small workload, good repeatability and is easy to operate.

[0008] The high-efficiency and low-loss alkali resistance evaluation method for grass carp established by the present invention can quickly evaluate the alkali resistance of grass carp, select individuals with high alkali resistance, and provide technical support for the breeding of new salt-alkali resistant grass carp varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is the gill tissue Na in Example 1 + Ion flow rate detection process diagram;

[0010] Figure 2 is the gill Na of different grass carp individuals immersed in the same alkalinity in Example 1 + Ion flow rate distribution range;

[0011] Figure 3 The gill Na of grass carp at different alkalinity gradients for 96 h in Example 2 + Changes in ion flow rate. DETAILED DESCRIPTION

[0012] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0013] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0014] Specific implementation method 1: This implementation method is based on gill Na + The method for evaluating the alkali resistance of grass carp by ion flow velocity is carried out in the following steps: 1. Collect the grass carp samples to be tested; 2. Soak the fish samples to be tested in 20-30 mmol / L NaHCO3 solution and take 1-2 gill filaments from the gill tissue; 3. Use non-destructive micro-measurement technology to measure the Na + Ion flow rate; 4. According to gill tissue Na + The alkaline resistance of grass carp was evaluated by flow rate, that is, after immersion in alkalinity of 20-30mmol / L, the gill Na + Ion flow rate is less than 100 pmol cm -2 ·s -1 The grass carp has a high alkali resistance, Na + Ion flow rate is 100-200 pmol·cm -2 ·s -1 The grass carp has a moderate alkali resistance. + Ion flow rate greater than 200 pmol cm -2 ·s -1 The alkali resistance of grass carp is judged to be low.

[0015] In step 3 of this embodiment, the non-invasive micro-measurement technique is used to measure the Na of gill tissue. +The ion flux was measured using a non-invasive micro-measurement system (NMT 150-SIM-YG, Beijing Xuyue Company, China).

[0016] Specific implementation method 2: This implementation method is different from specific implementation method 1 in that the fish sample to be tested in step 1 is a fish individual or a fish group. Other steps and parameters are the same as those in specific implementation method 1.

[0017] Specific implementation method 3: This implementation method is different from specific implementation methods 1 to 2 in that the fish sample to be tested in step 1 is a 2-4 month old young fish with a body weight of 13 to 50 g. The other steps and parameters are the same as those of specific implementation methods 1 to 2.

[0018] Specific embodiment 4: This embodiment differs from the specific embodiment 1 in that: the non-invasive micro-measurement technique is used to measure the Na of gill tissue + Ion flow rate method: The detection site is 1 μm from the tip of the gill filament: The measured ion is Na + ; The filling solution is 100mmol / L NaCl; The correction slope range is 50-60mV / decade; The gill filaments adapt to the test solution for 10min; The detection time is 2min; The Na of the gill tissue of the sample to be tested is calculated + Ion flow rate.

[0019] Example 1 The method of the present invention was used to evaluate the alkali resistance of individual grass carp.

[0020] The specific method is as follows:

[0021] Collect grass carp samples to be tested. The sample used in this embodiment is "Husu No. 1" grass carp species, which was purchased from Suzhou Shenhang Ecological Technology Development Co., Ltd. Randomly select individuals with strong activity, health and no injuries, with a body weight of (43.25±4.43) g and a body length of (14.08±0.72) cm and temporarily keep them in an indoor aquarium for 1 week. Each aquarium (400L water body) has 50 experimental fish, divided into 2 parallel groups, and soaked in 30mmol / L NaHCO3 solution for 96h (stress), and a freshwater control group (0mmol / L) is set up at the same time. During the experiment, no bait was added, oxygen was continuously added, and 1 / 2 of the water body was replaced daily. NaHCO3 was used to configure the corresponding alkalinity, and the alkalinity was calibrated with 0.02mmol / L hydrochloric acid standard solution. After 96h, 1 to 2 gill filaments on the left side of each experimental fish were collected and measured at room temperature (26-28°C) using a non-destructive micro-measurement system. Among them, Na was selected + The exchanger (lix holder) recognizes Na + The filling liquid is 100mmol / L NaCl. The ion-selective microelectrode needs three-point calibration (0.1mmol / L, 1mmol / L and 10mmol / L NaCl) before use.+ The calibration slope is 55.65mV / decade (in line with the calibration range) and the test is carried out. Quickly take out 1 to 2 gill filaments on the left side of the grass carp and place them in a culture dish filled with test solution (0.2mmol / L NaCl, 0.05mmol / L KH2PO4, 0.05mmol / L, K2HPO4, 0.2mmol / L CaSO4, 0.2mmol / L MgSO4 and 300μmol / L MOPS buffer) and balance for 10 minutes. After balancing, fix the grass carp gill filaments with resin blocks and filter paper strips, place them under a microscope, and adjust the sensor to 1μm away from the fish gill filaments (such as Figure 1 , Figure 1 Na for fish gill tissue + Ion flow rate detection process diagram), select X-30 test mode to detect Na in fish gills + Ion flow rate. Each fish gill sample was tested for 2 minutes. The Na + Ion flow rate.

[0022] The results of this embodiment show that: Figure 2 As shown, Figure 2 is the gill Na of different grass carp individuals immersed in the same alkalinity + Ion flow rate distribution range, Na of 100 grass carp in the treatment group + Ion flow rate is 53.61~250.48pmol·cm -2 ·s -1 The distribution was basically normal, with a mean of 154.50±61.19pmol·cm -2 ·s -1 According to the criteria for determining the alkalinity of grass carp, 20 grass carp were tested for Na + Ion flow rate <100pmol·cm -2 ·s -1 , with an average value of 75.51±16.33 pmol·cm -2 ·s -1 , alkali resistance was judged to be high; 63 grass carp Na + Ion flow rate is 100~200pmol·cm -2 ·s -1 The mean value was 149.71±30.32pmol·cm -2 ·s -1 , alkali resistance was judged as moderate; 17 grass carp Na + Ion flow rate>200pmol·cm -2 ·s -1 , with an average value of 228.33±17.19 pmol·cm -2 ·s -1, the alkali resistance is judged to be low.

[0023] In order to further verify the evaluation method, 15 fish from the highly alkali-resistant group (75.12±12.68 pmol·cm -2 ·s -1 ) and 15 fish in the low alkali resistance group (225.41±13.36pmol·cm -2 ·s -1 ) were tested for 60mmol / L NaHCO3 high alkalinity tolerance. Three parallels were set up in each group, with 5 fish in each parallel. The aquarium contained 400L of water and sufficient oxygen was always maintained. The number of deaths in each group was recorded at 12h, 24h, 48h, 72h, and 96h, and the 96h mortality rate was calculated (Table 1). The results showed that the mortality rate of the highly alkali-resistant group was 6.7%, and the average mortality rate of the low alkali-resistant group was 73.3%.

[0024] Table 1 Statistics of the number of grass carp individuals that died in groups under high alkalinity (60 mmol / L) stress for 96 h

[0025]

[0026] The method of acute stress was verified by using a 96h half-lethal concentration of 60 mM alkalinity, and the accuracy of the evaluation of the present invention reached more than 90%.

[0027] Example 2 The method of the present invention is used to evaluate the alkali resistance of grass carp populations.

[0028] Based on individual gill Na + The alkali resistance of grass carp population was evaluated by using the method of ion flow rate to determine the alkali resistance.

[0029] It is generally believed that water bodies with an alkalinity of 10 to 30 mmol / L are medium-alkalinity water bodies. The alkalinity of most saline-alkali aquaculture waters is mainly 10 to 30 mmol / L. The safe concentration of grass carp under 96h NaHCO3 stress is 20 mmol / L, and the half-lethal concentration is 60 mmol / L. Therefore, in this embodiment, the alkalinity gradient is set in the range of ±10 mmol / L for individual determination of alkali resistance. 200 grass carp individuals with healthy physique, uniform size and strong activity were randomly selected, and subjected to 20 mmol / L, 30 mmol / L and 40 mmol / L NaHCO3 for 96h acute stress. 60 experimental fish were placed in each aquarium (100L water body), and a freshwater control group (0 mmol / L) was set. The water body configuration and detection were the same as the individual detection standards. 10 samples were collected at 6h, 12h, 24h, 48h, 72h, and 96h, and 1 to 2 gill filaments were collected from the left side of each fish. A non-destructive micro-test system was used for Na + Ion flow rate detection, the specific detection method and reagent formula are the same as above, and the Na of the gill tissue of the sample to be tested is calculated. +Ion flow rate.

[0030] Figure 3 The gill Na of grass carp at different alkalinity gradients for 96 h + The change of ion flow rate shows that the Na + The flow rate was relatively stable and hardly changed with time. The Na + The flow rate first decreases and then increases with time, and with the increase of alkalinity, Na + Change response in advance. C A After 24 hours of immersion, the Na + The ion flow rate dropped to the minimum and then returned to a stable state. The flow rate was close to that of the fresh water control group and was less than 100 pmol cm -2 ·s -1 ; C A After 12 hours of immersion, the Na + The ion flow rate drops to the minimum, and then Na + The flow rate was higher than that of the control group, with an average of 132.97±25.84pmol·cm -2 ·s -1 , between 100 and 200 pmol·cm -2 ·s -1 Between; C A The Na + The average ion flow rate was 623.66±50.79 pmol·cm -2 ·s -1 , significantly higher than 200 pmol·cm -2 ·s -1 Therefore, on the whole, grass carp can tolerate water with an alkalinity of 20 to 30 mmol / L.

Claims

1. A method based on gill Na + Method for evaluating the alkali resistance of grass carp by ion flow rate. Features Based on Na + The method for evaluating the alkali resistance of grass carp by ion flow rate is carried out in the following steps:

1. Collect the grass carp samples to be tested; 2. Use 20-30 mmol / L NaHCO 3 Soak the fish sample to be tested in the solution and take 1 to 2 gill filaments from the gill tissue; 3. Non-invasive micro-measurement technique for determination of Na in gill tissue + Ion flow rate; 4. According to gill tissue Na + The alkaline resistance of grass carp was evaluated by flow rate, that is, after immersion in alkalinity of 20-30mmol / L, the gill Na + Ion flow rate is less than 100 pmol cm -2 ·s -1 The grass carp has a high alkali resistance, Na + Ion flow rate is 100-200 pmol·cm -2 ·s -1 The grass carp has a moderate alkali resistance. + Ion flow rate greater than 200 pmol cm -2 ·s -1 The alkali resistance of grass carp is judged to be low.

2. A method based on gill Na according to claim 1 + Method for evaluating the alkali resistance of grass carp by ion flow rate. Features In step 1, the fish samples to be tested are individual fish or fish groups.

3. A method based on gill Na according to claim 2 + Method for evaluating the alkali resistance of grass carp by ion flow rate. Features The fish samples to be tested in step 1 are 2-4 month old young fish, with a body weight of 13-50 g.

4. A method based on gill Na according to claim 1 + Method for evaluating the alkali resistance of grass carp by ion flow rate. Features Non-invasive micro-measurement technique for determination of Na in gill tissue + Ion flow rate method: The detection site is 1 μm from the tip of the gill filament: The measured ion is Na + ; The filling solution is 100mmol / L NaCl; The correction slope range is 50-60mV / decade; The gill filaments adapt to the test solution for 10min; The detection time is 2min; Calculate the Na of the gill tissue of the sample to be tested + Ion flow rate.

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