Method for evaluating salt tolerance of grass carp and application of method

Abstract

The invention belongs to the technical field of fish salt tolerance, and particularly relates to a grass carp salt tolerance evaluation method and application thereof. The serum prolactin concentration is used as the hormone molecular marker for measuring the salt tolerance of the grass carp. Under the stress of the same intensity of salinity, the higher the concentration of serum prolactin is, the stronger the salt tolerance is. In the breeding process of the grass carp salt-tolerant strain, the serum prolactin concentration of the screening material under the saline stress condition can be measured, and the salt tolerance of the grass carp can be accurately evaluated. When the method is applied to breeding practice, the breeding efficiency of the salt-tolerant grass carp new variety in the breeding process is greatly improved, and the breeding period of the salt-tolerant grass carp variety is remarkably shortened.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fish salt tolerance, and particularly relates to a method for evaluating the salt tolerance of grass carp and application thereof, and particularly relates to screening of serum molecular markers related to the salt tolerance of grass carp and application thereof. BACKGROUND

[0002] As an important factor of water environment, salinity directly affects the balance of osmotic pressure inside and outside the fish. When the osmotic pressure caused by the water environment salinity is much higher or lower than the osmotic pressure of the internal environment, the aquatic organism cannot adjust the balance between the osmotic pressure in the body and the osmotic pressure outside the body, leading to uncontrolled changes in internal environment ions and affecting the normal physiological activities of aquatic organisms. Due to the characteristics of high PH, high carbonate alkalinity, unbalanced proportion of main ions, and multiple water quality types, saline-alkali water cannot be directly drunk by humans and livestock, and cannot be directly utilized by agriculture, and most of it is in idle state.

[0003] Therefore, fish culture using saline-alkali water is one of the methods for developing and utilizing saline-alkali water areas, and the key to this method is to improve the salt tolerance of aquaculture species and cultivate new salt-tolerant fish species. Finding biomarkers to evaluate the salt tolerance of fish and then indicating the adaptation process of fish to saline-alkali environment will provide key support for the cultivation of new salt-tolerant fish species. SUMMARY

[0004] The purpose of the present application is to provide a method for evaluating the salt tolerance of grass carp and application thereof.

[0005] The first purpose of the present application is to provide a method for evaluating the salt tolerance of grass carp, which is achieved by the following technical solution: A method for evaluating the salt tolerance of grass carp, comprising the following steps: S1, collecting grass carp samples; S2, performing acute salinity stress on the grass carp in step S1; S3, performing tail vertebra blood sampling on the test grass carp to separate the upper serum; S4, determining the content of prolactin in the serum obtained in step S3; S5, distinguishing low, moderate and high salt-tolerant individuals of grass carp according to the content of prolactin in the serum.

[0006] Further, the grass carp samples in step S1 are 2-4 month old juvenile fish with a size of 10-20 g in body weight.

[0007] Further, the acute salinity stress method in step S2 is to use salt water prepared by sea water crystal to adjust the salinity, and to increase 2 ppt every 2 h until the salinity reaches 10 ppt.

[0008] Further, the method for separating the upper serum in step S3 is: after 96 h of salinity stress, 50 μL of blood is extracted by the tail vein blood extraction method, the blood is placed at 4℃ for 4 h, and then centrifuged at 5000 r / min for 20 min to separate 15-25 μL of the upper serum.

[0009] Further, the method for determining the prolactin content in step S4 is: the EILSA method is used to determine the prolactin content in the serum, the absorbance value at 450 nm is read in a multifunctional enzyme label instrument, and the content of the prolactin in the sample to be measured is calculated.

[0010] Further, the method for distinguishing the low, moderate and high salt-tolerant individuals of grass carp in step S5 is: after 96 h of salinity stress, the grass carp with a prolactin content in the serum lower than 485 mIU / L is determined as low salt-tolerant; the grass carp with a prolactin content of 485-641 mIU / L is determined as moderate salt-tolerant; and the grass carp with a prolactin content higher than 641 mIU / L is determined as high salt-tolerant.

[0011] The second object of the present application is to provide an application of the method for evaluating the salt-tolerance performance of grass carp in the selection of a salt-tolerant strain of grass carp.

[0012] Compared with the prior art, the present application has the following advantages: The present application uses the serum prolactin concentration as a hormone molecular marker for measuring the salt-tolerance performance of grass carp. Under the same intensity of salinity stress, the higher the serum prolactin concentration, the stronger the salt-tolerance performance. In the selection of a salt-tolerant strain of grass carp, the serum prolactin concentration of the screening material under the condition of salt water stress can be determined, and the salt-tolerance performance of grass carp can be accurately evaluated. When applied to breeding practice, the selection efficiency of a new salt-tolerant variety of grass carp in the breeding process can be greatly improved, and the selection cycle of a salt-tolerant variety of grass carp can be significantly shortened. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Fig. 4 is a distribution frequency of the prolactin content in the serum of grass carp under 10 ppt salinity stress for 96 h; Figure 2 Fig. 5 is the change of the prolactin content in the serum of grass carp under different acute salinity stresses. DETAILED DESCRIPTION

[0014] In order to further explain the present application, the following specific examples are used for illustration.

[0015] Example 1: Method for evaluating the salt-tolerance performance of grass carp 1.1 Experimental grass carp were purchased from Mingtao Fish Fry Farm in Dinghu District, Zhaoping City, Guangdong Province, and transported to a pond net cage for temporary feeding twice a day with fish puffed compound feed from Tongwei Co., Ltd. (9:00 am and 19:00 pm). Before the experiment, randomly selected grass carp with an average weight of 15.24 ± 4.18 g were placed in three water tanks (160 L, 0.45 m x 0.6 m x 0.6 m) in 28°C~31°C water with 24 h of pre-aeration to relieve stress, with continuous oxygen supply, and two-thirds of the water was replaced every two days. Water quality was monitored using a water quality test kit, and the dissolved oxygen ( > 8.0 mg / L), pH (7.5~8.5), total ammonia nitrogen concentration ( < 0.2 mg / L), and nitrite concentration ( < 0.05 mg / L) were maintained at appropriate levels. Water with different salinities was prepared by mixing freshwater with seawater crystals (Jiangsu Yantong Technology Co., Ltd.); 1.2 Proceed with a 4-day acute salinity stress experiment, increasing 2 ppt every 2 h until reaching the corresponding salinity (10 ppt). Each group has 40 fish, and there are three groups. Individuals that die 96 h before salinity stress are defined as low-salt-tolerant individuals, and individuals that survive 96 h of salinity stress are defined as high-salt-tolerant individuals. At the point of death of grass carp, record the time of death, and extract 50 μL of blood from each fish using the tail vein blood extraction method. Let the blood stand at 4°C for 4 h, centrifuge at 5000 r / min for 20 min, separate the upper serum, and obtain 20 μL of serum. At the end of the experiment, collect the serum of surviving individuals; 1.3 The prolactin content was measured using a prolactin ELISA kit (Shanghai Youxiao Biotechnology Co., Ltd.) Each individual was measured three times, and the average value was taken.

[0016] The results of this example show: The average prolactin content of the control group of grass carp before 10 ppt salinity stress was 721.65 ± 92.21 mIU / L, the average prolactin content in the serum of grass carp individuals that died before 10 ppt salinity stress for 96 h was 485.26 ± 105.88 mIU / L, and the average prolactin content in the serum of grass carp individuals that survived at 10 ppt salinity stress for 96 h was 641.25 ± 151.97 mIU / L.

[0017] To further distinguish the salt tolerance of grass carp according to the changes in prolactin content under salinity stress, the distribution of prolactin content in the serum of grass carp at 96 h under salinity stress was analyzed Figure 1). The results showed that the frequency distribution of the prolactin content of 96 h grass carp was in accordance with the normal distribution rule. We defined the individuals with prolactin content below 485 mIU / L as low salt-tolerant level, the individuals with prolactin content > 641 mIU / L as high salt-tolerant level, and the individuals with prolactin content between 485 and 641 mIU / L as moderate salt-tolerant level according to the average prolactin content of the dead and surviving individuals of 96 h grass carp.

[0018] In summary, prolactin can be used as a hormone molecular marker for accurately quantifying the salt stress level of grass carp, and the salt-tolerant population can be screened for the breeding of salt-tolerant strains of grass carp.

[0019] To further verify the evaluation method, 10 individuals (777.73 ± 73.88 mIU / L) from the high salt-tolerant group and 10 individuals (377.90 ± 81.98 mIU / L) from the low salt-tolerant group were subjected to 10 ppt salinity stress, and the number of dead individuals in each group was recorded, and the mortality rates at 24 h, 48 h and 96 h were calculated (Table 1).

[0020] Table 1. Statistics of the number of dead individuals of grass carp in different groups under 96 h high salinity (10 ppt) stress ; The results showed that the mortality rate of the high salt-tolerant group was 10%, and the mortality rate of the low salt-tolerant group was 90%. The accuracy of the evaluation method was more than 90% by using 10 ppt as the median lethal salinity for 96 h acute stress.

[0021] Example 2: Application of the evaluation method for salt-tolerant performance of grass carp 2.1 The grass carps used in the experiment were purchased from Mingtao Fish Fry Farm in Dinghu District, Zhaoqing City, Guangdong Province, and were transported to a pond net cage for temporary cultivation. They were fed twice a day with fish puffed compound feed from Tongwei Co., Ltd. (at 9:00 am and 19:00 pm). Before the experiment, grass carps with an average weight of 15.42 ± 0.96 g were randomly selected and placed in four water tanks (160 L, 0.45 m × 0.6 m × 0.6 m) for temporary cultivation in water with 28℃~31℃ pre-aeration for 24 h to relieve stress. Oxygen was supplied continuously during the period, and two-thirds of the water was replaced every two days. Water quality was monitored using a water quality test kit, and the dissolved oxygen (> 8.0 mg / L), pH value (7.5~8.5), total ammonia nitrogen concentration (< 0.2 mg / L), and nitrite concentration (< 0.05 mg / L) were maintained within the appropriate ranges. Water with different salinities was prepared by mixing fresh water and sea salt (Jiangsu Yantong Technology Co., Ltd.).

[0022] 2.2 To study the median lethal concentration of grass carp under salinity stress, a four-day acute salinity stress experiment was conducted, with four salinity groups set at 0 ppt, 4 ppt, 7 ppt and 10 ppt. The experimental group was increased by 2 ppt every 2 h until the corresponding salinity was reached. There were 40 fish in each group, and during the experiment, all experimental fish were not fed to avoid the influence of different feeding conditions. At 24 h, 48 h and 96 h, 50 μL of blood was taken from each group of 5 fish by tail vein blood sampling method, and the blood was placed at 4°C for 4 h, centrifuged at 5000 r / min for 20 min, and the upper serum was separated to obtain 20 μL of serum.

[0023] 2.3 The prolactin content in the serum was determined by Cytation5 multifunctional enzyme labeler (Biotek, USA). The prolactin detection kit was purchased from Shanghai Yuxiao Biotechnology Co., Ltd.

[0024] The results of this example show that: Figure 2 The prolactin content in the serum of grass carp under different salinity stress. The prolactin content in the 0 ppt group was relatively stable, and there was no significant change in its content over time. The prolactin content in the 4 ppt, 7 ppt and 10 ppt groups showed a downward trend as the stress time prolonged. In the 4 ppt group, the prolactin content at 24 h, 48 h and 96 h was significantly lower than that at 0 h. In the 7 ppt and 10 ppt groups, the prolactin content at 96 h was significantly lower than that at 0 h. According to the judgment standard of salt tolerance of grass carp, the prolactin content in the serum of grass carp under 10 ppt stress for 96 h was 531.37 ± 42.21 mIU / L, and the prolactin content was between 485 and 641 mIU / L, and its salt tolerance belonged to the moderate level.

[0025] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for evaluating the salt tolerance of grass carp, characterized in that, Includes the following steps: S1. Collect grass carp samples; S2. The grass carp described in step S1 is subjected to acute salinity stress. S3. Blood was drawn from the caudal vertebrae of the grass carp in the experiment, and the upper serum layer was separated. S4. The prolactin content in the serum obtained in step S3 is determined. S5. Differentiate grass carp individuals with low, medium, and high salt tolerance based on serum prolactin levels.

2. The method for evaluating the salt tolerance of grass carp according to claim 1, characterized in that, The grass carp sample mentioned in step S1 is a juvenile fish aged 2 to 4 months with a body weight of 10 to 20 g.

3. The method for evaluating the salt tolerance of grass carp according to claim 1, characterized in that, The acute salinity stress method described in step S2 is as follows: salinity is adjusted using saline solution prepared with sea salt crystals, increasing by 2 ppt every 2 hours until a salinity of 10 ppt is reached.

4. The method for evaluating the salt tolerance of grass carp according to claim 1, characterized in that, The method for separating the upper serum layer in step S3 is as follows: After 96 h of salinity stress, 50 μL of blood is drawn using the tail vein blood collection method. After the blood is left to stand at 4°C for 4 h, it is centrifuged at 5000 r / min for 20 min to separate 15~25 μL of upper serum layer.

5. The method for evaluating the salt tolerance of grass carp according to claim 1, characterized in that, The method for determining the prolactin content in step S4 is as follows: the prolactin content in serum is determined using the EILSA method, the absorbance value at 450 nm is read in a multi-functional microplate reader, and the prolactin content of the sample to be tested is calculated.

6. The method for evaluating the salt tolerance of grass carp according to claim 1, characterized in that, The method for distinguishing between low, moderate, and high salt tolerance individuals of grass carp described in step S5 is as follows: grass carp with a serum prolactin level below 485 mIU / L after 96 h of salinity stress are judged to have low salt tolerance; grass carp with a prolactin level between 485 and 641 mIU / L are judged to have moderate salt tolerance; and grass carp with a prolactin level above 641 mIU / L are judged to have high salt tolerance.

7. The application of the method for evaluating the salt tolerance of grass carp as described in any one of claims 1 to 6 in the breeding of salt-tolerant grass carp strains.

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