Method for improving salt tolerance of grass carp

Abstract

The invention belongs to the technical field of fish genetic breeding, and discloses a method for improving salt tolerance of grass carps, in particular to a method for improving salt tolerance of fishes, which comprises the following steps: taking fish fertilized eggs as initial domestication objects, placing the fish fertilized eggs in saline water for incubation and domestication, then increasing the salinity of a water body by 0.1%-0.2% per 25-35 days, the salinity is the same as that of the target culture water body. The invention provides an imprinting method based on gradual salinity domestication to improve the salt tolerance of fishes, the method can effectively activate the expression of osmotic pressure regulation related genes of tissues such as gill and kidney, finally, the grass carp can adapt to 7 per mill of water salinity, the conventionally considered salt tolerance threshold value of the grass carp is broken through, and the application prospect is wide. The feed still has the characteristics of fast growth, healthy physique, strong ingestion and the like under the water salinity of 7 per mill.

Description

Technical Field

[0001] The invention belongs to the technical field of fish genetic breeding, and particularly relates to a method for improving the salt tolerance of grass carp. Background Art

[0002] Grass carp (Ctenopharyngodon idella) has the advantages of fast growth and delicious meat. It is one of the most important freshwater farmed fish in my country, and its output ranks first among freshwater farmed fish in my country all year round. Traditional grass carp farming is mainly concentrated in freshwater waters. However, with the growth of population and economic development, the freshwater waters available for farming are decreasing, and the continuous increase in farming density has aggravated the deterioration of water quality and the frequent occurrence of diseases. At present, there are about 500 million mu of saline-alkali waters in my country, showing different characteristics such as high salt and low alkali, low salt and high alkali, and high salt and high alkali, but none of them have been fully developed. Therefore, vigorously developing the breeding of salt-tolerant grass carp is of great significance for expanding farming space, improving economic benefits, maintaining ecological balance and ensuring food security.

[0003] At present, there are studies related to how to improve the salt tolerance of fish. For example, in tilapia and carp, by gradually increasing the salinity (increasing 1‰ per day or 2‰ every 2 weeks), tilapia and carp can adapt to 15‰ and 6‰ salinity, respectively. However, the initial salinity is freshwater, starting from juveniles, and does not involve the critical intervention period of the fertilized egg stage. For grass carp, short-term (10 days) exposure can make grass carp adapt to water salinity ≤5‰, but this study started from juveniles, did not involve the critical intervention period of the fertilized egg stage, and did not further improve the salt tolerance of grass carp, and the effect of improving salt tolerance was not obvious.

[0004] The existing salt-tolerant grass carp breeding mainly relies on natural selection or slow adaptive cultivation. On the one hand, this makes the breeding cycle extremely long; on the other hand, the existing methods mainly start from large-sized fish species, resulting in a large breeding water area and high requirements for breeding facilities. At the same time, due to the lack of clear and targeted domestication strategies, it is difficult for existing breeding methods to accurately induce grass carp to form a stable salinity adaptation mechanism at the key development stage, and the salt tolerance of the selected individuals varies greatly. The methods for breeding salt-tolerant grass carp include gene editing technology and nutritional fortification technology. Among them, gene editing technology uses gene editing technology to accurately edit key genes related to salt tolerance traits in grass carp, so that grass carp has stronger salt tolerance at the genetic level. However, although this method is efficient and accurate, it is still controversial in terms of ethics and safety. Nutritional fortification technology indirectly improves the salt tolerance of grass carp by adding specific nutrients or additives to grass carp feed to adjust the osmotic pressure balance of fish and improve the intestinal microecological environment. However, this method may be slightly worse in actual effect.

[0005] Therefore, there is an urgent need to develop a method to significantly improve the salt tolerance of grass carp, increase the survival rate of grass carp in high-salinity water, and narrow the differences in salt tolerance between selected individuals. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide a method for improving the salt tolerance of fish.

[0007] The purpose of the second aspect of the present invention is to provide application of the method of the first aspect of the present invention in fish breeding.

[0008] The purpose of the third aspect of the present invention is to provide application of the method of the first aspect of the present invention in fish farming.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a method for improving the salt tolerance of fish, comprising the following steps:

[0011] The fertilized fish eggs are used as the initial domestication objects. The fertilized fish eggs are placed in salt water for hatching and domestication. Thereafter, the salinity of the water is increased by 1‰ to 2‰ every 25 to 35 days until it reaches the same salinity as the target aquaculture water.

[0012] Taking the fertilized egg as the key developmental window and the starting point of salinity acclimation, and utilizing the epigenetic plasticity in the early developmental stage to establish lifelong physiological memory of salinity is more efficient than traditional methods.

[0013] The present invention firstly starts from the critical developmental period of fertilized eggs and adopts a method of gradual salinity domestication. This gradient domestication policy has little harm to the body, high efficiency, and greatly shortens the period of salt-tolerant breeding. Secondly, through precise gradient domestication, it is helpful to form a stable salinity adaptation mechanism, and the grass carp can be graded and selected at any time during the breeding process. The grass carp population bred is more consistent in salt tolerance, which provides a reliable basis for large-scale breeding. The method of the present invention can quickly improve the salt tolerance of grass carp on the basis of using small water bodies, which reduces the requirements for breeding water space and makes breeding work more convenient.

[0014] In some embodiments of the present invention, the salinity of the brine is 1‰ to 4‰.

[0015] In some embodiments of the present invention, the salinity of the brine is 2‰ to 4‰.

[0016] In some embodiments of the present invention, the salinity of the brine is 4‰.

[0017] The present invention constructs a model with a salinity of 4‰ to 7‰, an interval of 30 days, and a gradient salinity of 1‰. This parameter has been verified, and when the fertilized eggs are in water with a salinity higher than 4‰, the eggs will fail to hatch. In water with a salinity of 7‰, the fish can still survive and feed normally, indicating that the current parameters are the optimal solution.

[0018] In some embodiments of the present invention, the fish includes at least one of grass carp, tilapia, crucian carp, carp, sea bass, rainbow trout, and pike.

[0019] In some embodiments of the present invention, the fish fertilized eggs are freshly fertilized eggs.

[0020] In some embodiments of the present invention, the fish fertilized eggs are obtained by the following method: artificially inducing spawning of parent fish to obtain fertilized eggs. The artificial induction of spawning includes using conventional methods in the art, such as injecting oxytocin (5 μg of luteinizing hormone-releasing hormone + 5 mg of dioconazole per kg of fish body) into the parent fish, and the injection site is the muscle injection at the base of the dorsal fin.

[0021] In some embodiments of the present invention, during the domestication period, the fertilized eggs of the fish hatch and then emerge from the membranes. No additional food is provided to the fry in the first 2 to 3 days after emerging from the membranes. The fry are fed Artemia on the 4th to 7th day after emerging from the membranes, and then transition to feeding conventional fish feed.

[0022] In some embodiments of the present invention, the salt water is prepared from sea crystals.

[0023] In some embodiments of the present invention, during the domestication period, it is necessary to maintain sufficient oxygen in the water and sufficient natural light.

[0024] In some embodiments of the present invention, the feeding time of Artemia is from 8 am every day, and is provided every 3 hours until 8 pm.

[0025] Compared with the prior art, the method of the present invention is superior in terms of window period selection, domestication strategy and actual effect. Specifically, the prior art mostly starts domestication from juveniles or adult fish, while the present invention starts salinity adaptation from the fertilized egg stage, making full use of the high plasticity in the early development period, which is more efficient; secondly, the initial salinity adopted by the method of the present invention is 1‰~4‰, which has been verified to be a safe salinity by experiments. The specific process is to increase the salinity by 1‰ every 30 days. This method is relatively mild, and the stress response of the fish body is small, which can reduce the loss caused by stress during the domestication process; in addition, the method of the present invention can promote grass carp to grow and feed normally at a salinity of 7‰, breaking through the traditional salinity threshold; finally, the water body involved in the present invention is a small water body, which has smaller requirements for site facilities, avoids the difficulties of conventional large-scale breeding, and has advantages in the actual operation process.

[0026] The second aspect of the present invention provides application of the method of the first aspect of the present invention in fish breeding.

[0027] In some embodiments of the present invention, the breeding is to breed fish with higher salt tolerance.

[0028] The third aspect of the present invention provides application of the method of the first aspect of the present invention in fish farming.

[0029] In some embodiments of the present invention, the fish farming includes improving the survival rate, final body weight, body length and specific growth rate of fish in high-salinity water, and promoting the expression of related genes involved in osmotic pressure regulation in fish tissues.

[0030] In some embodiments of the present invention, the related genes involved in osmotic pressure regulation include at least one of nkaα1, nkaβ1b and sglt.

[0031] The beneficial effects of the present invention are:

[0032] The present invention provides an imprinting method based on gradual salinity domestication to improve the salt tolerance of fish (grass carp). The present invention places the fertilized fish eggs in a salinity of 1‰ to 4‰ (preferably 4‰) to start hatching, and systematically increases the salinity by 1‰ every 30 days through a gradient domestication policy, aiming to induce fish to form a physiological adaptation mechanism to salinity during the critical period of individual development. The method of the present invention can effectively activate the expression of genes related to osmotic pressure regulation in tissues such as gills and kidneys, and ultimately enable grass carp to adapt to a water salinity of 7‰, breaking through the conventional threshold of grass carp salt tolerance, which is mainly manifested in the fact that it still has the characteristics of rapid growth, good physique, and strong feeding at a water salinity of 7‰.

[0033] The method of the present invention can quickly improve the salt tolerance of grass carp based on the use of small water bodies, accelerate the breeding of salt-tolerant grass carp, and provide a feasible technical path for expanding the saline-alkali water aquaculture of grass carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention is a flow chart of the method for improving the salt tolerance of grass carp.

[0035] Figure 2 The morphology of fertilized eggs under different salinities in Example 1 of the present invention (2 hours after fertilization).

[0036] Figure 3 This is the facility bucket used for programmed imprinting in Example 2 of the present invention.

[0037] Figure 4 The effect of salinity programmed blot on serum ion concentration and NKA enzyme activity (n=4; P≤0.05 indicated significant difference).

[0038] Figure 5 The effect of salinity programmed imprinting on gene expression (n=4; P≤0.05 indicated a significant difference). DETAILED DESCRIPTION

[0039] The present invention is further described in detail below through specific examples.

[0040] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0042] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0043] Terminology explanation:

[0044] Programmed imprinting refers to the phenomenon that specific interventions at specific developmental stages (especially early life stages) induce persistent changes in the physiology, metabolism, gene expression, etc. of an organism, thereby having a long-term impact on the individual's subsequent growth, health, or ability to adapt to the environment.

[0045] qRT-PCR: Real-time fluorescence quantitative PCR is a method for quantitatively analyzing specific DNA sequences through fluorescent signals during DNA amplification. It uses fluorescent chemicals to detect the amount of DNA products during PCR cycles, thereby achieving quantitative analysis of specific genes.

[0046] nkaα1: Na + / K + -ATPase transporter alpha subunit 1 gene. Na + / K + -ATPase transporter is a membrane protein composed of α subunit and β subunit. Among them, α subunit is mainly responsible for Na + , K + Isotropic transmembrane transport.

[0047] Nkaβ1b: Na + / K + -ATPase transporter beta subunit 1b gene. Na + / K + -ATPase transporter is a membrane protein composed of α subunit and β subunit. Among them, the β subunit is mainly responsible for maintaining the stability of the α subunit.

[0048] sglt: Na + / Glucose co-transporter. Uses the electrochemical tendency of sodium ions to transport glucose against the concentration gradient. Mainly involved in glucose reabsorption.

[0049] Example 1 Determination of initial acclimatization salinity for hatching fertilized grass carp eggs

[0050] Select 4-5-year-old individuals weighing more than 5kg, with a strong physique, no obvious scars and quick reactions as broodstock. When the water temperature reaches 22-28℃, start artificial induction of spawning. The type and dosage of oxytocin used for female fish is 5μg of luteinizing hormone-releasing hormone + 5mg of dioconazole per kg of fish body, and the dosage for male broodstock is halved. The oxytocin is dissolved in normal saline. Female fish are injected twice, and the injection site is the muscle injection at the base of the dorsal fin. The first injection dose is 1 / 6 of the total dose, and the second injection is given at an interval of about 8 hours. The male fish is injected with the full dose at the time of the second injection of the female fish. About 6-8 hours after the injection, fertilized eggs will appear (keep observing during this period).

[0051] Prepare 18 petri dishes with a diameter of 10 cm, divided into 6 treatment groups (1‰~6‰), with 3 replicates in each group, and add different concentrations of salt water prepared with sea crystals to each treatment group. Use a rubber-tipped pipette to accurately draw 20 fertilized eggs into each petri dish and incubate them in a static state. After about 24 hours, the hatching rate of each treatment group was counted, and the starting concentration of salinity acclimation was determined based on this.

[0052] The results are as follows Figure 2 As shown in the figure, after being placed in salt water for about 2 hours, the fertilized eggs of the 1‰ to 4‰ treatment group can enter the cleavage stage normally, and the cell boundaries are obvious. However, the fertilized eggs of the 5‰ and 6‰ treatment groups began to necrotize and could not undergo normal cleavage. In addition, as shown in Table 1, the emergence rate of the membrane after fertilization for 24 hours was statistically analyzed. The emergence rate of the 1‰ to 4‰ treatment group was 95% to 100%, while the fertilized eggs of the 5‰ and 6‰ treatment groups almost all failed to emerge normally. Based on the purpose of the present invention (improving the salt tolerance of grass carp), 4‰ was finally selected as the initial acclimation salinity.

[0053] Table 1 The rate of fertilized eggs emerging from the membrane under different salinities

[0054]

[0055]

[0056] Example 2

[0057] A method for improving the salt tolerance of grass carp ( Figure 1 ), including the following steps:

[0058] (1) Obtaining grass carp fertilized eggs: Same as Example 1.

[0059] (2) Programmed imprinting specific operation process: According to the water salinity (4‰) determined in Example 1, the newly fertilized eggs were placed in a conical incubation bucket (1.5 m in diameter, 1.5 m in height, equipped with a filtration system, Figure 3 ) to start incubation (the salinity of the water is 4‰ at this time, and the sea crystal is prepared). After that, the salinity of the water is increased by 1‰ every 30 days until the salinity reaches 7‰, and finally maintained in 7‰ salt water for 30 days. During this period, sufficient oxygen and natural light are provided (no water change is required, and the filtration system is cleaned and disinfected every 7 days). During the programmed imprinting period (ensure sufficient oxygen, natural water temperature and light, pH 7.5±0.4mg / L, ammonia nitrogen ≤0.1±0.01mg / L), no additional food was provided in the first 3 days after hatching; from the 4th to the 7th day after hatching, Artemia (https: / / detail.com / offer / 1229794730.html) was fed every 3h from 8 am to 8 pm every day; from the 8th to the 10th day after hatching, Artemia was acclimated to commercial feed (Tongwei Feed Co., Ltd., Chengdu, China), specifically Artemia and commercial feed were fed alternately; from the 11th day after hatching, commercial feed was used for feeding, and the appropriate caliber feed was changed according to the manufacturer's recommendation as the fry grew. The feeding amount should be eaten within 10min. That is, grass carp with high salt tolerance that can withstand 7‰ salt water was obtained.

[0060] Hatching and separation of Artemia: Accurately weigh 15 g of commercial Artemia eggs, prepare 20‰ salt water with sea crystals in a 100L conical hatching bucket ( Figure 3 ) with sufficient oxygen and natural light. The eggs will hatch successfully after about 24 hours. The hatched adults are placed in a 5L large-capacity transparent beaker, with the upper part covered and the lower part exposed to light. The adults are separated by their phototaxis and used for feeding the fry.

[0061] Example 3

[0062] A method for improving the salt tolerance of grass carp, compared with Example 2, the only difference is that the initial water salinity is 1‰.

[0063] Example 4

[0064] A method for improving the salt tolerance of grass carp, compared with Example 2, the only difference is that the initial water salinity is 2‰.

[0065] Example 5

[0066] A method for improving the salt tolerance of grass carp, compared with Example 2, the only difference is that the initial water salinity is 3‰.

[0067] Example 6 Salt tolerance test of imprinted grass carp juveniles

[0068] (1) Preparation and grouping of test fish and experimental procedures

[0069] 60 grass carps with programmed imprinting (defined as the treatment group, i.e., the grass carps domesticated in Example 2) and 60 grass carps without programmed imprinting (defined as the control group, i.e., the grass carps not domesticated) with an average weight of 4.13±0.57g were randomly assigned to 6 300L circulating water culture barrels, each group was divided into 3 replicates, and each replicate had 3 barrels (20 fish / barrel). The culture water was 7‰ salt water prepared with sea crystal, and a 37-day feeding experiment was carried out. During the feeding period, 20% of the water was replaced every 3 days, sufficient oxygen and natural light were provided, the water temperature was controlled at 26.7℃±27.5℃, the pH was 7.5±0.7mg / L, and the ammonia nitrogen content was 0.25±0.03mg / L. Commercial feed was used for full feeding at 8:30 and 16:30 every morning. During this period, the fish's feed intake, mortality rate, etc. were recorded every day.

[0070] (2) Sampling

[0071] After the experiment, the test fish were fasted for 24 h and then anesthetized with the anesthetic MS-222 (100 mg / L, Sigma, China). The weight and length of each fish were accurately weighed, and the gills and kidney tissues of the test fish (4 fish / barrel) were quickly dissected and rinsed twice with pre-cooled phosphate buffer (Biyuntian Biotechnology Co., Ltd., Beijing, China), then stored in liquid nitrogen, and finally placed in a -80℃ ultra-low temperature refrigerator to extract RNA and detect the expression of related genes (nkaα1, nkaβ1b and sglt). Four fish were taken from each barrel and blood was collected from the tail vein. After the obtained blood samples were allowed to stand at 4℃ for 4 h, they were centrifuged at 2500g for 5 min at 4℃, and the upper serum was taken and stored in a -80℃ ultra-low temperature refrigerator for later use. The specific growth rate calculation formula is: 100*(ln final weight-ln initial weight) / number of breeding days.

[0072] (3) RNA extraction and qRT-PCR

[0073] RNA was extracted from gill and kidney tissues using a commercial RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China). Subsequently, the quality and content of RNA were tested by 1% agarose gel electrophoresis and nucleic acid quantification instrument (Thermo Fisher Scientific Co., Ltd., Shanghai, China). cDNA was synthesized using a commercial kit according to the kit instructions (Takara Biomedical Technology Co., Ltd., Dalian, China). The gene amplification system was 20 μL: including 20 ng of cDNA, 0.8 μL each of forward and reverse primers (primer sequences are shown in Table 2), 10 μL 2× Power SYBR TM Green PCR Master Mix (Thermo Fisher Scientific Co., Ltd., Shanghai, China) and sterile ultrapure water. The gene amplification conditions were: 95°C, 5 min; 95°C, 15 s, 61°C annealing 1 min, 40 cycles; 72°C extension 1 min. The β-actin gene was used as an internal reference, and 2 -ΔΔCt The gene expression was calculated by the method of , and the results were expressed as fold relative to the control group.

[0074] Table 2 Primer sequences used for qRT-PCR

[0075]

[0076] The experimental results are shown in Table 3. The survival rate, final body weight, body length and specific growth rate of the experimental fish that underwent salinity programmed imprinting under high salinity were significantly higher than those of the control group.

[0077] The Na content of fish serum in the two treatment groups was further tested. + , K + and Cl + The content and NKA enzyme activity of the treated fish were detected (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China). It was found that the serum ion content and NKA enzyme activity of the treated fish were significantly lower than those of the control group ( Figure 4 ). The expression of genes involved in osmotic pressure regulation in gill and kidney tissues was detected, and it was found that the expression of nkaα1, nkaβ1b and sglt in the two tissues was higher in the experimental fish with salinity programmed imprinting than in the control group ( Figure 5 ). It is suggested that the method of Example 2 can promote grass carp to grow and eat normally at a salinity of 7‰, breaking through the salinity threshold traditionally believed.

[0078] Table 3 Survival rate, final body weight, body length and specific growth rate of each group

[0079]

[0080] Note: The letters on the right shoulder of the numbers in the table represent significance.

[0081] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for improving the salt tolerance of fish, comprising the following steps: The fertilized fish eggs are used as the initial domestication objects. The fertilized fish eggs are placed in salt water for hatching and domestication. Thereafter, the salinity of the water is increased by 1‰ to 2‰ every 25 to 35 days until it reaches the same salinity as the target aquaculture water.

2. The method according to claim 1, characterized in that The salinity of the brine is 1‰ to 4‰.

3. The method according to claim 1, characterized in that The fish includes at least one of grass carp, tilapia, crucian carp, carp, perch, rainbow trout and pike.

4. The method according to any one of claims 1 to 3, characterized in that The fish fertilized eggs are freshly fertilized eggs.

5. The method according to any one of claims 1 to 3, characterized in that: During the domestication period, the fertilized eggs of the fish hatch and then emerge from the membranes. No additional food is provided to the fry in the first 2 to 3 days after emerging from the membranes. On the 4th to 7th day after emerging from the membranes, they are fed with Artemia, and then transition to feeding with conventional fish feed.

6. The method according to any one of claims 1 to 3, characterized in that: The salt water is prepared from sea crystals.

7. The method according to claim 2, characterized in that The salinity of the brine is 2‰ to 4‰.

8. Use of the method according to any one of claims 1 to 7 in fish breeding.

9. Use of the method according to any one of claims 1 to 7 in fish farming.

10. The use according to claim 9, characterized in that: The fish farming comprises improving the survival rate, final body weight, body length and specific growth rate of fish in high-salinity water, and promoting the expression of relevant genes involved in osmotic pressure regulation in fish tissues.

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