Method for evaluating alkali resistance of cyprinid fishes

Abstract

The invention discloses a cyprinid fish alkali resistance evaluation method, and relates to a fish alkali resistance evaluation method. The method solves the problem that fish bodies are easy to damage in existing fish alkali resistance evaluation. The method comprises the following steps: 1, collecting to-be-detected fish samples; 2, the fish sample is stressed by NaHCO3, and serum is separated; 3, measuring the content of glutamate dehydrogenase GDH in the serum; and 4, evaluating the alkali resistance through the GDH content in the serum. The alkali resistance of grass carp groups or individuals is evaluated based on the glutamate dehydrogenase content. The content of glutamate dehydrogenase in serum reflects the ammonia metabolism function under alkaline stress, and has the advantages of rapid detection and sensitive reaction. According to the invention, the content of GDH in serum is utilized to measure the alkali resistance of a group or an individual, and the method for evaluating the damage-free alkali resistance of the fish is established.

Description

Technical Field

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

[0002] my country has about 690 million mu of low-lying saline-alkali waters, which are widely distributed in 19 provinces, municipalities and autonomous regions in Northeast my country, North China and Northwest China. Saline-alkali water generally has the characteristics of high carbonate alkalinity, high pH and unbalanced ion coefficients. Among them, alkalinity is the key ecological factor restricting its fishery utilization. Low-alkalinity saline-alkali water (alkalinity <10mM) has basically been developed and utilized, but about 90% of medium and high alkalinity saline-alkali water is still in a state of low yield or abandonment. In recent years, the Ministry of Agriculture and Rural Affairs has issued relevant policies, emphasizing the need to gradually and steadily promote the development of saline-alkali fisheries and expand the space suitable for fishing. Medium-alkalinity saline-alkali water (alkalinity 10-30mM) is expected to be used in fishery production. Breeding salt-alkali tolerant fish is a prerequisite for the development and utilization of medium-alkalinity saline-alkali water. Carp fish have a strong tolerance to alkalinity. For example, the Dali Lake Yale fish can tolerate the extremely alkaline environment of Dali Lake with an alkalinity of up to 54mM, and the naked carp of Qinghai Lake can grow normally in lake water with an alkalinity of 32mM. Evaluation of the alkali resistance of fish is an important basis for screening and breeding salt-alkali tolerant fish. 50 ) and safe concentration (SC) to evaluate the alkali resistance of fish. However, acute stress of high alkalinity can cause organic damage to the tissues and organs of fish, thereby affecting their physiological and metabolic functions and disrupting their physiological homeostasis. Even if some individuals can survive, there are risks in continuing breeding or genetic selection due to damage to their body functions. Therefore, it is particularly important to establish a non-destructive alkali resistance evaluation technology or method. Summary of the invention

[0003] The invention provides a method for evaluating the alkali resistance of carp fish in order to solve the problem that the existing evaluation of the alkali resistance of fish easily causes damage to the fish body.

[0004] The method for evaluating the alkali resistance of carp fish of the present invention is carried out according to the following steps:

[0005] 1. Collect fish samples for inspection;

[0006] 2. Sample the fish under alkali stress, extract blood, and separate serum;

[0007] 3. Determine the level of glutamate dehydrogenase GDH in serum;

[0008] 4. The alkali resistance was evaluated by the GDH content in the serum, that is, the fish with GDH content lower than 5nmol / min / mL were judged to have high alkali resistance; the fish with GDH content between 5 and 8nmol / min / mL were judged to have moderate alkali resistance; the fish with GDH content higher than 8nmol / min / mL were judged to have low alkali resistance.

[0009] Beneficial effects of the present invention:

[0010] The present invention evaluates the alkali resistance of grass carp groups or individuals based on the glutamate dehydrogenase content. The glutamate dehydrogenase content in serum reflects the ammonia metabolism function under alkaline stress, and has the advantages of rapid detection and sensitive response. The present invention uses the GDH content in serum to measure the alkali resistance of groups or individuals, establishes a non-destructive alkali resistance evaluation method for fish, overcomes the defect of high damage to the body caused by traditional alkali resistance evaluation, can quickly realize the alkali resistance evaluation of grass carp groups or individuals, and achieves the purpose of non-destructive selection of groups or individuals with high alkali resistance.

[0011] The damage-free alkali resistance evaluation method of grass carp established by the present invention can quickly realize the alkali resistance evaluation of grass carp and select groups or individuals with high alkali resistance, providing a theoretical basis and working foundation for the breeding and promotion of new alkali-resistant grass carp varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the GDH content in serum of different grass carp individuals in Example 1;

[0013] Figure 2 The changes in GDH content in serum of grass carp at different alkalinity levels and at different time periods in Example 2 are shown. DETAILED DESCRIPTION

[0014] 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.

[0015] 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.

[0016] Specific implementation method 1: The alkali resistance evaluation method of carp fish in this implementation method is carried out according to the following steps:

[0017] 1. Collect fish samples for inspection;

[0018] 2. Sample the fish under alkali stress, extract blood, and separate serum;

[0019] 3. Determine the level of glutamate dehydrogenase GDH in serum;

[0020] 4. The alkali resistance was evaluated by the GDH content in the serum, that is, the fish with GDH content lower than 5nmol / min / mL were judged to have high alkali resistance; the fish with GDH content between 5 and 8nmol / min / mL were judged to have moderate alkali resistance; the fish with GDH content higher than 8nmol / min / mL were judged to have low alkali resistance.

[0021] 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 group or a fish individual. Other steps and parameters are the same as those in specific implementation method 1.

[0022] Specific implementation method 3: This implementation method is different from specific implementation methods 1 to 2 in that the fish to be inspected in step 1 is grass carp. Other steps and parameters are the same as those in specific implementation method 1.

[0023] Specific implementation method 4: This implementation method is different from specific implementation methods 1 to 3 in that the fish sample to be tested in step 1 is a 2-4 month old young fish with a body weight of 13-30 g. Other steps and parameters are the same as those in specific implementation method 1.

[0024] Specific implementation method 5: This implementation method is different from specific implementation methods 1 to 4 in that the fish sample is stressed with NaHCO3 in step 2. The other steps and parameters are the same as those in specific implementation method 1.

[0025] Specific implementation method 6: This implementation method is different from specific implementation method 5 in that the fish samples are subjected to alkaline stress by using NaHCO3 with a concentration of 20-30 mmol / L. The other steps and parameters are the same as those of specific implementation method 1.

[0026] Specific embodiment 7: This embodiment differs from specific embodiment 1 in that: in step 2, during the alkaline stress of the fish sample, the aeration is continued and the water body is kept circulating and filtered, 50 μL of blood is drawn, and the blood is left to stand at 4°C for 3 hours, and 18 to 23 μL of serum is separated. The other steps and parameters are the same as those of specific embodiment 1.

[0027] Specific embodiment eight: This embodiment differs from specific embodiment one in that: in step three, the glutamate dehydrogenase GDH content in serum is determined using a multifunctional microplate reader, and the absorbance value at 340nm after a reaction time of 5 minutes is read to calculate the glutamate dehydrogenase content of the sample to be tested. Other steps and parameters are the same as those in specific embodiment one.

[0028] The kit used for the glutamate dehydrogenase (GDH) content in this embodiment is from Suzhou Keming Biotechnology Co., Ltd. The instrument used is Bio-Tek Synergy 2 multifunctional microplate reader (Bio-Tech, USA), and the glutamate dehydrogenase content of the sample to be tested is calculated according to the formula in the manual.

[0029] Specific implementation method 9: The method for evaluating the alkali resistance of carp fish in this implementation method is carried out according to the following steps:

[0030] 1. Collect fish samples for inspection;

[0031] Second, stress the fish samples with alkali, extract blood, and separate serum;

[0032] 3. Determine the level of glutamate dehydrogenase GDH in serum;

[0033] Fourth, the alkali resistance is evaluated by the GDH content in the serum, that is, the fish with GDH content lower than 5nmol / min / mL are judged to have high alkali resistance; the fish with GDH content between 5 and 8nmol / min / mL are judged to have moderate alkali resistance; the fish with GDH content higher than 8nmol / min / mL are judged to have low alkali resistance;

[0034] The fish tested in step 1 is grass carp; the fish samples to be tested are 2-4 month old juveniles with a body weight of 13-30 g; 20-30 mmol / L (mM) N a The grass carp samples were subjected to HCO3 stress for 96 hours, during which aeration was continued and water circulation filtration was maintained. 50 μL of blood was drawn and allowed to stand at 4°C for 3 hours, and 18-23 μL of serum was separated. In step 3, the glutamate dehydrogenase GDH content in the serum was determined using a multifunctional microplate reader, and the absorbance value at 340 nm after a reaction time of 5 minutes was read to calculate the glutamate dehydrogenase content of the sample to be tested. The glutamate dehydrogenase (GDH) content kit used was from Suzhou Keming Biotechnology Co., Ltd., and the instrument used was Bio-Tek Synergy 2 Multifunctional microplate reader (Bio-Tech, USA), calculate the content of glutamate dehydrogenase in the sample to be tested according to the formula in the instruction manual; in step 4, the alkali resistance of the fish to be tested is evaluated by the GDH content in the serum. The alkali resistance of the group or individual with serum GDH content lower than 5nmol / min / mL after 96h stress is judged as high; the alkali resistance of the group or individual with GDH content of 5-8nmol / min / mL is judged as moderate; the alkali resistance of the group or individual with GDH content higher than 8nmol / min / mL is judged as low.

[0035] Example 1 Evaluation of the alkali resistance of individual grass carp using the method of the present invention

[0036] The specific method is as follows:

[0037] 1. Collect fish samples to be tested, i.e. grass carp, 3-month-old juveniles, from the "Husu No. 1" group bred by Shanghai Ocean University, with a body weight of (16.390±2.602) g. Randomly select 100 grass carp individuals with healthy physique, uniform size and strong activity, and temporarily raise them for 3 days;

[0038] 2. Use 25mmol / L (mM) alkalinity N a The fish samples were subjected to HCO3 stress, during which aeration was continuously maintained and water circulation and filtration were maintained. Blood was drawn and serum was separated. That is, 25mM NaHCO3 alkalinity solution was used for stress for 96 hours. 50 experimental fish were placed in each aquarium (100L water), and two parallel experiments were set up. A freshwater control group of 0mmol / L was also set up. No feeding was done during the whole process, and oxygenation was always maintained. 1 / 2 of the water was replaced daily. NaHCO3 was used to configure the corresponding alkalinity, and 0.02mmol / L (mM) hydrochloric acid standard solution was used to calibrate the alkalinity. After the experiment, 50μL of blood was drawn from each fish, and biomarkers were performed by cutting the fin rays. The blood was allowed to stand at 4℃ for 3h, centrifuged at 3500r / min for 15min, and the upper serum was separated to obtain 20μL;

[0039] 3. Determine the content of glutamate dehydrogenase GDH in serum, i.e., use the glutamate dehydrogenase (GDH) assay kit of Suzhou Keming Biotechnology Co., Ltd. to detect the content of GDH, and use the Bio-Tek Synergy 2 multifunctional microplate reader (Bio-Tech, USA) to detect the absorbance value at 340nm with an accurate reaction time of 5 minutes. Repeat the measurement 3 times for each individual and take the average value;

[0040] 4. The alkali resistance was evaluated by the GDH content in the serum, that is, the fish with GDH content lower than 5nmol / min / mL were judged to have high alkali resistance; the fish with GDH content between 5 and 8nmol / min / mL were judged to have moderate alkali resistance; the fish with GDH content higher than 8nmol / min / mL were judged to have low alkali resistance.

[0041] The results of this example show that the GDH content of the 69 grass carp individuals in the treatment group is approximately normally distributed, ranging from 1.929 to 16.718 nmol / min / ml, with an average of 5.917±2.820 nmol / min / ml (e.g. Figure 1 As shown, Figure 1The distribution of serum GDH content among different grass carp individuals). According to the criteria for determining alkalinity of grass carp, the GDH content of 30 grass carps was lower than 5nmol / min / ml, with an average of 3.879±0.776nmol / min / ml, and the alkali resistance was determined to be high; the GDH content of 24 grass carps was 5-8nmol / min / ml, with an average of 5.751±0.341nmol / min / ml, and the alkali resistance was determined to be moderate; the GDH content of 15 grass carps was higher than 8nmol / min / ml, with an average of 10.257±2.773nmol / min / ml, and the alkali resistance was determined to be low. To further verify the evaluation method, 15 fish from the high alkali resistance group (3.301±0.776nmol / min / ml) and 15 fish from the low alkali resistance group (10.257±2.773nmol / min / ml) were tested for 60mM high alkalinity tolerance. Each group was divided into 3 parallels, with 5 fish in each parallel. The aquarium was kept in 20L of water with continuous aeration. The number of deaths in each group was recorded and the 96h mortality rate was calculated (Table 1). The results showed that the average mortality rate of the high alkali resistance group was 13.3%, and the average mortality rate of the low alkali resistance group was 80%.

[0042] Table 1 Statistics of the number of grass carp individuals dying from high alkalinity (60 mM) stress for 96 h

[0043]

[0044]

[0045] 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 85%.

[0046] Example 2

[0047] This embodiment adopts the method of specific implementation method seven to evaluate the alkali resistance of individual grass carp. The grass carp is a 3-month-old fry, derived from the "Husu No. 1" group cultivated by Shanghai Ocean University, with a body weight of (19.741±6.016) g. 200 grass carp individuals with healthy physique, uniform size and strong activity were randomly selected and temporarily raised for 3 days. Previous studies have shown that the safe concentration of sodium bicarbonate alkaline tolerance of grass carp is 20mM (mmol / L), and the 96h acute stress half-lethal concentration is 60mM (mmol / L). In view of the fact that the natural aquaculture saline-alkali water body is mainly moderate alkalinity (10-30mM (mmol / L)), it is easy to cause fish alkalosis and ammonia poisoning if it is higher than 30mM (mmol / L). Therefore, in the seventh step of the specific implementation method, 20mM (mmol / L), 30mM (mmol / L), and 40mM (mmol / L) NaHCO3 alkalinity solutions were used for stress, and 60 experimental fish were placed in each aquarium (100L water body), and a freshwater control group of 0mmol / L was set. No bait was added during the experiment, and oxygenation was always maintained. 1 / 2 of the water body was replaced daily, and the corresponding alkalinity was configured using NaHCO3, and the alkalinity was calibrated with a 0.02mM hydrochloric acid standard solution. 10 samples were collected at 6h, 12h, 24h, 48h, 72h, and 96h, and 50μL of blood was extracted from each fish. The blood was kept at 4°C for 3 h and centrifuged at 3500 r / min for 15 min to separate the upper serum. The GDH content was detected using the glutamate dehydrogenase (GDH) assay kit produced by Suzhou Keming Biotechnology Co., Ltd. The absorbance at 340 nm with an accurate reaction time of 5 min was detected using the Bio-Tek Synergy 2 multifunctional microplate reader (Bio-Tech, USA). The measurement was repeated 3 times for each individual and the average value was taken.

[0048] Figure 2 Table 2 shows the changes of serum glutamate dehydrogenase (GDH) content of grass carp under different alkalinity stress over time. Figure 2 From Table 2, it can be seen that the GDH content of the control group (0mM) was relatively stable and did not change much over time. The GDH content in the serum of grass carp in different alkalinity treatment groups first increased and then decreased over time. The peak value of the 20mM treatment group appeared at 72h, and the peak values ​​of the 30mM and 40mM treatment groups appeared at 48h. The rate of decrease in the 40mM treatment group was slower. According to the criteria for the alkali tolerance of grass carp, the GDH content of grass carp in the 20mM and 30mM stress groups at 96h was (6.437±2.275)nmol / min / mL and (7.002±0.751)nmol / min / mL, respectively. The GDH content was between 5 and 8nmol / min / mL, and the alkali tolerance of the grass carp population was judged to be moderate.

[0049] Table 2 Changes of serum glutamate dehydrogenase (GDH) content in grass carp under different alkalinity stress over time

[0050]

[0051]

Claims

1. A method for evaluating the alkali resistance of carp fish, characterized in that The evaluation method of alkali resistance of carp fish is carried out according to the following steps:

1. Collect fish samples for inspection; 2. Sample the fish under alkali stress, extract blood, and separate serum; 3. Determine the level of glutamate dehydrogenase GDH in serum; 4. The alkali resistance was evaluated by the GDH content in the serum, that is, the fish with GDH content lower than 5nmol / min / mL were judged to have high alkali resistance; the fish with GDH content between 5 and 8nmol / min / mL were judged to have moderate alkali resistance; the fish with GDH content higher than 8nmol / min / mL were judged to have low alkali resistance.

2. The method for evaluating the alkali resistance of carp fish according to claim 1, characterized in that The fish samples to be tested in step 1 are fish groups or individual fish.

3. A method for evaluating the alkali resistance of carp fish according to claim 1 or 2, characterized in that The fish to be inspected in step 1 is grass carp.

4. The method for evaluating the alkali resistance of carp fish according to claim 3, characterized in that The fish samples to be tested in step 1 are 2-4 month old juveniles with a body weight of 13-30 g.

5. The method for evaluating the alkali resistance of carp fish according to claim 3, characterized in that In step 2, the fish samples were subjected to alkaline stress using NaHCO3.

6. A method for evaluating the alkali resistance of carp fish according to claim 5, characterized in that In step 2, the fish samples were subjected to alkaline stress using NaHCO3 at a concentration of 20 to 30 mmol / L.

7. The method for evaluating the alkali resistance of carp fish according to claim 1, characterized in that During the alkaline stress period of the fish samples in step 2, aeration was continued and water circulation and filtration were maintained. 50 μL of blood was drawn and allowed to stand at 4° C. for 3 h, and 18 to 23 μL of serum was separated.

8. The method for evaluating the alkali resistance of carp fish according to claim 1, characterized in that In step 3, the glutamate dehydrogenase GDH content in serum is determined by using a multifunctional microplate reader, reading the absorbance value at 340 nm after a reaction time of 5 minutes, and calculating the glutamate dehydrogenase content of the sample to be tested.

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