Method for improving salt tolerance of grass carp

Abstract

The present application belongs to the technical field of fish genetic breeding, and discloses a method for improving salt tolerance of grass carp, and specifically discloses a method for improving salt tolerance of fish, which comprises the following steps: taking fish fertilized eggs as initial domestication objects, and placing the fish fertilized eggs in salt water for hatching and domestication; thereafter, the salinity of the water body is increased by 1‰-2‰ every 25-35 days until the salinity is the same as that of the target aquaculture water body. The present application provides a footprint method based on salinity gradual domestication to improve the salt tolerance of fish. The method can effectively activate the expression of tissue osmotic pressure regulation related genes such as gills and kidneys, and ultimately can enable grass carp to adapt to 7‰ water body salinity, breaking the conventional salt tolerance threshold of grass carp. The main performance is that the grass carp still has the characteristics of rapid growth, healthy body, and strong feeding under 7‰ water body salinity.

Description

Technical Field

[0001] This invention belongs to the field of fish genetic breeding technology, specifically relating to a method for improving the salt tolerance of grass carp. Background Technology

[0002] Grass carp (Ctenopharyngodon idella) is one of the most important freshwater aquaculture fish in my country, boasting rapid growth and delicious flesh, and its production has consistently ranked first among freshwater aquaculture fish in the country. Traditional grass carp farming has primarily focused on freshwater areas. However, with population growth and economic development, the available freshwater areas for aquaculture are decreasing, and the increasing stocking density is exacerbating problems such as water quality deterioration and frequent disease outbreaks. Currently, my country has approximately 500 million mu (about 33 million hectares) of saline-alkali water areas, exhibiting different characteristics such as high salinity and low alkalinity, low salinity and high alkalinity, and high salinity and high alkalinity, but none of these have been fully developed. Therefore, vigorously developing the breeding of salt-tolerant grass carp is of great significance for expanding aquaculture space, improving economic benefits, maintaining ecological balance, and ensuring food security.

[0003] Currently, there are studies on how to improve the salt tolerance of fish. For example, in tilapia and carp, gradually increasing salinity (1‰ daily or 2‰ every two weeks) allows tilapia and carp to adapt to salinities of 15‰ and 6‰, respectively. However, these studies started with freshwater salinity, began with juvenile fish, and did not address the critical intervention period during the fertilized egg stage. For grass carp, short-term (10 days) exposure can help them adapt to water salinities of ≤5‰, but this study also started with juvenile fish, did not address the critical intervention period during the fertilized egg stage, and did not further improve the grass carp's salt tolerance; the improvement effect was not significant.

[0004] Current methods for breeding salt-tolerant grass carp mainly rely on natural selection or slow adaptive breeding. This results in an extremely long breeding cycle and, moreover, primarily starts with large-sized fry, requiring large breeding areas and demanding facilities. Furthermore, the lack of clear and targeted domestication strategies makes it difficult to precisely induce stable salinity adaptation mechanisms in grass carp during critical developmental stages, leading to significant differences in salt tolerance among the bred individuals. Methods for breeding salt-tolerant grass carp include gene editing and nutritional fortification. Gene editing precisely edits key genes related to salt tolerance in grass carp, thus enhancing their genetic salt tolerance. However, while highly efficient and precise, this method remains controversial due to ethical and safety concerns. Nutritional fortification indirectly improves salt tolerance by adding specific nutrients or additives to grass carp feed to regulate osmotic pressure balance and improve the intestinal microecological environment. However, this method may be less effective in practice.

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

[0006] The first objective of this invention is to provide a method for improving the salt tolerance of fish.

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

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

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

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

[0012] Using the fertilized egg as a critical developmental window and the starting point for salinity acclimatization, and leveraging the epigenetic plasticity of the early developmental stage, a lifelong physiological memory of salinity can be established, which is more efficient than traditional methods.

[0013] This invention first addresses the critical developmental stage of fertilized eggs by employing a gradual salinity acclimatization method. This gradient acclimatization policy minimizes harm to the organisms, is highly efficient, and significantly shortens the salt tolerance breeding cycle. Secondly, precise gradient acclimatization helps establish a stable salinity adaptation mechanism, allowing for graded selection at any time during the breeding process. The resulting grass carp population exhibits greater consistency in salt tolerance, providing a reliable foundation for large-scale aquaculture. Furthermore, this invention's method can rapidly improve the salt tolerance of grass carp even with limited water volume, reducing the space requirements for aquaculture and making the breeding process 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] This invention constructs a model with a salinity range of 4‰ to 7‰, an interval of 30 days, and a gradient salinity of 1‰. These parameters have been verified; fertilized eggs fail to hatch when exposed to water with a salinity higher than 4‰. At a salinity of 7‰, the fish can still survive and feed normally, indicating that the current parameters are optimal.

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

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

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

[0021] In some embodiments of the present invention, during the domestication period, after the fertilized fish eggs hatch, the fry are not given any additional food for the first 2 to 3 days after hatching, and are fed artichokes from the 4th to 7th day after hatching, and then transitioned to conventional fish feed.

[0022] In some embodiments of the present invention, the brine is prepared from sea salt 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 brine shrimp are fed starting at 8:00 a.m. every day, once every 3 hours, until 8:00 p.m.

[0025] Compared with existing technologies, the method of this invention is superior in terms of window period selection, domestication strategy, and actual results. Specifically, existing technologies mostly start domestication from juvenile or adult fish, while this invention starts salinity adaptation from the fertilized egg stage, making full use of the high plasticity in the early developmental stage, resulting in higher efficiency. Secondly, the initial salinity used in this invention is 1‰ to 4‰, which has been verified as a safe salinity through experiments. The specific process involves increasing the salinity by 1‰ every 30 days. This method is relatively gentle, with less stress response in the fish, reducing losses caused by stress during domestication. In addition, this invention can enable grass carp to continue to grow and feed normally at a salinity of 7‰, breaking through the traditionally considered salinity threshold. Finally, the water body involved in this invention is a small body of water, requiring less site and facility, avoiding the difficulties of conventional large-scale breeding, and has advantages in practical operation.

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

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

[0028] A third aspect of the present invention provides the 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, as well as promoting the expression of genes involved in osmotic pressure regulation in fish tissues.

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

[0031] The beneficial effects of this invention are:

[0032] This invention provides an imprinting method based on gradual salinity acclimation to enhance the salt tolerance of grass carp. The invention involves placing fertilized fish eggs in a salinity of 1‰ to 4‰ (preferably 4‰) for incubation, and then systematically increasing the salinity by 1‰ every 30 days in a gradient acclimation policy. This aims to induce the fish to develop a physiological adaptation mechanism to salinity during the critical period of individual development. This method effectively activates the expression of genes related to osmotic pressure regulation in tissues such as gills and kidneys, ultimately enabling grass carp to adapt to a water salinity of 7‰, breaking through the conventionally accepted salt tolerance threshold for grass carp. This is mainly manifested in rapid growth, robust health, and vigorous feeding even at a salinity of 7‰.

[0033] The method of this invention can rapidly improve the salt tolerance of grass carp by using small water bodies, accelerate the breeding of salt-tolerant grass carp, and provide a feasible technical path for expanding grass carp aquaculture in saline-alkali water. Attached Figure Description

[0034] Figure 1 This is a flowchart of the method for improving the salt tolerance of grass carp according to the present invention.

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

[0036] Figure 3 This refers to the facility bucket used for the procedural imprint in Embodiment 2 of the present invention.

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

[0038] Figure 5 The effect of salinity programmed imprinting on gene expression (n=4; P≤0.05 indicates significant difference). Detailed Implementation

[0039] The present invention will be further described in detail below through specific embodiments.

[0040] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0042] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0043] Terminology Explanation:

[0044] Programmed imprinting: The phenomenon in which organisms undergo persistent changes in their physiology, metabolism, gene expression, etc., through specific interventions at a specific developmental stage (especially in the early life stage), thereby having a long-term impact on the individual's later growth, health, or environmental adaptability.

[0045] qRT-PCR, or quantitative real-time PCR, is a method for quantitatively analyzing specific DNA sequences using fluorescence signals during DNA amplification. It utilizes fluorescent chemicals to detect the amount of DNA product during PCR cycles, thereby enabling quantitative analysis of specific genes.

[0046] nkaα1: i.e., Na + / K + - ATPase transporter α subunit 1 gene. Na + / K + -ATPase transporter is a membrane protein composed of α and β subunits. The α subunit is primarily responsible for Na+ transport. + K + Transmembrane transport of plasma.

[0047] nkaβ1b: i.e., Na + / K + - ATPase transporter β subunit 1b gene. + / K + -ATPase transporter is a membrane protein composed of an α subunit and a β subunit. The β subunit is mainly responsible for maintaining the stability of the α subunit.

[0048] sglt: i.e. Na + Glucose cotransporters. They utilize the electrochemical tendency of sodium ions to transport glucose against its concentration gradient. They are primarily involved in glucose reabsorption.

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

[0050] Select healthy, robust individuals aged 4-5 years, weighing over 5 kg, without obvious injuries, and exhibiting agile reflexes as broodstock. Artificial spawning induction begins when the water temperature reaches 22-28℃. For female broodstock, the spawning induction agent is 5 μg of luteinizing hormone-releasing hormone (LH) + 5 mg of dioxin per kg of body weight; the dosage for male broodstock is halved. The spawning induction agent is dissolved in physiological saline. Female broodstock are injected twice into the muscle at the base of the dorsal fin. The first injection is 1 / 6 of the total dose, with a second injection approximately 8 hours later. Male broodstock receive the full dose during the second injection. Fertilized eggs typically appear approximately 6-8 hours after the injection (continuous observation is necessary).

[0051] Eighteen petri dishes with a diameter of 10 cm were prepared and divided into 6 treatment groups (1‰–6‰), with 3 replicates per group. Different concentrations of saline solution prepared with sea salt were added to each treatment group. Twenty fertilized eggs were accurately transferred to each petri dish using a pipette and incubated under static conditions. The hatching rate of each treatment group was recorded after approximately 24 hours, and this was used to determine the initial concentration for salinity acclimatization.

[0052] The results are as follows Figure 2 As shown, after approximately 2 hours in saline solution, fertilized eggs in the 1‰–4‰ treatment groups were able to normally enter the cleavage stage, with clear cell boundaries. However, fertilized eggs in the 5‰ and 6‰ treatment groups began to die and could not undergo normal cleavage. Furthermore, as shown in Table 1, the hatching rate 24 hours after fertilization was statistically analyzed. The hatching rate in the 1‰–4‰ treatment groups was 95%–100%, while almost all fertilized eggs in the 5‰ and 6‰ treatment groups failed to hatch normally. Considering the overall objective of this invention (to improve the salt tolerance of grass carp), 4‰ was ultimately selected as the initial salinity for acclimatization.

[0053] Table 1. Fertilized egg emergence rate under different salinity levels

[0054]

[0055]

[0056] Example 2

[0057] A method to improve the salt tolerance of grass carp Figure 1 The process includes the following steps:

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

[0059] (2) Specific operational procedures for programmed imprinting: Based on the water salinity (4‰) determined in Example 1, the newly fertilized eggs were placed in a conical incubation tank (1.5m in diameter, 1.5m in height, equipped with a filtration system). Figure 3 Incubation begins in a solution with a salinity of 4‰ (prepared from sea salt crystals). Subsequently, the salinity is increased by 1‰ every 30 days until it reaches 7‰, and then maintained at 7‰ salinity for 30 days. During this period, ample oxygen and natural light are provided (no water changes are needed; the filtration system is cleaned and disinfected every 7 days). During the programmed imprinting period (ensuring sufficient oxygen, natural water temperature and light, pH 7.5±0.4mg / L, ammonia nitrogen ≤0.1±0.01mg / L), no additional food is provided for the first 3 days after hatching. From the 4th to the 7th day after hatching, feed with artemia (https: / / detail.com / offer / 1229794730.html), starting at 8:00 AM and providing it every 3 hours until 8:00 PM daily. From the 8th to the 10th day after hatching, begin the transition from artemia to commercial feed (Tongwei Feed Co., Ltd., Chengdu, China), specifically alternating between artemia and commercial feed. From the 11th day after hatching, feed exclusively with commercial feed, and according to the manufacturer's recommendations, change to feed with appropriate mouth size as the fry grow. The amount of feed should be such that it is consumed within 10 minutes. This method yields grass carp with high salt tolerance, capable of withstanding 7‰ salinity.

[0060] Hatching and separation of the above-mentioned artichokes: Accurately weigh 15g of commercial artichoke eggs, and prepare a 20‰ saline solution using sea salt in a 100L conical hatching tank. Figure 3 Provide ample oxygen and natural light, and the eggs will hatch successfully in about 24 hours. Take the hatched adult insects and place them in a 5L large-capacity transparent beaker. Cover the top part and place the bottom part in the light. Use the phototaxis of the adult insects to separate them for feeding the fish fry.

[0061] Example 3

[0062] A method to improve the salt tolerance of grass carp, which differs from Example 2 only in that the initial water salinity is 1‰.

[0063] Example 4

[0064] A method to improve the salt tolerance of grass carp, which differs from Example 2 only in that the initial water salinity is 2‰.

[0065] Example 5

[0066] A method to improve the salt tolerance of grass carp, which differs from Example 2 only in that the initial water salinity is 3‰.

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

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

[0069] Sixty grass carp with an average weight of 4.13 ± 0.57 g (defined as the treatment group, i.e., grass carp domesticated in Example 2) and 60 grass carp without a programmed imprint (defined as the control group, i.e., grass carp not domesticated) with an average weight of 5.17 ± 0.45 g were randomly assigned to six 300 L recirculating aquaculture tanks, with each group having three replicates and three tanks per replicate (20 fish / tank). The culture water was a 7‰ saline solution prepared by Haijing Crystal, and the feeding experiment lasted for 37 days. During the feeding period, 20% of the water was replaced every three days, and sufficient oxygen and natural light were provided. The water temperature was controlled at 26.7℃ ± 27.5℃, the pH at 7.5 ± 0.7 mg / L, and the ammonia nitrogen content at 0.25 ± 0.03 mg / L. Commercial feed was given for satiation at 8:30 AM and 4:30 PM daily. Feed intake and mortality were recorded daily.

[0070] (2) Sampling

[0071] After the experiment, the fish were fasted for 24 hours and then anesthetized with MS-222 (100 mg / L, Sigma, China). The weight and length of each fish were accurately measured, and gill and kidney tissues (4 fish / bucket) were quickly dissected. The tissues were rinsed twice with pre-cooled phosphate buffer (Beyotime Biotechnology Co., Ltd., Beijing, China), then stored in liquid nitrogen, and finally placed in a -80°C ultra-low temperature freezer for RNA extraction and expression detection of related genes (nkaα1, nkaβ1b, and sglt). Additionally, blood was collected from the tail veins of 4 fish from each bucket. The blood samples were allowed to stand at 4°C for 4 hours, then centrifuged at 2500g at 4°C for 5 minutes. The supernatant serum was collected and stored at -80°C for later use. The specific growth rate was calculated using the formula: 100 * (ln(final weight - ln(initial weight)) / number of days of rearing.

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

[0073] RNA was extracted from gill and kidney tissues using a commercial RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). The quality and content of the RNA were then determined by 1% agarose gel electrophoresis and nucleic acid quantification using a 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 consisted of 20 μL of cDNA, 0.8 μL each of forward and reverse primers (primer sequences shown in Table 2), and 10 μL of 2×Power SYBR. TM Green PCR Master Mix (Thermo Fisher Scientific Co., Ltd., Shanghai, China) and sterile ultrapure water were used. Gene amplification conditions were: 95℃ for 5 min; 95℃ for 15 s, annealing at 61℃ for 1 min, 40 cycles; extension at 72℃ for 1 min. β-actin was used as an internal control. -ΔΔCt Gene expression was calculated using a method that expressed the results as a multiple of the control group.

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

[0075]

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

[0077] Further analysis of the Na+ levels in the serum of the fish from the two treatment groups... + K + and Cl + The content and NKA enzyme activity of the serum ions in 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 osmolarity regulation in gill and kidney tissues was detected, and it was found that the expression of nkaα1, nkaβ1b, and sglt in both tissues was higher in the salinity-programmed imprinted fish than in the control group. Figure 5 The method in Example 2 can enable grass carp to continue growing and feeding normally at a salinity of 7‰, exceeding the traditionally accepted salinity threshold.

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

[0079]

[0080] Note: The letter above the right shoulder of the number in the table indicates significance.

[0081] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for improving the salt tolerance of grass carp, comprising the following steps: Using grass carp fertilized eggs as the initial domestication target, the grass carp fertilized eggs are placed in salt water for incubation and domestication. Then, the salinity of the water is increased by 1‰ to 2‰ every 25 to 35 days until it is the same as the salinity of the target culture water, and finally grass carp with high salt tolerance can be obtained that can tolerate 7‰ salt water. The concentration of the brine used in the incubation and domestication process in brine is 2‰ to 4‰. The grass carp fertilized eggs mentioned are newly fertilized eggs; During the domestication period, after the grass carp fertilized eggs hatch, the fry are not given any extra food for the first 2-3 days after hatching. They are fed artichokes from the 4th to the 7th day after hatching, and then transitioned to regular fish feed. Artichokes are fed every 3 hours starting at 8 am until 8 pm. The brine was prepared from sea salt crystals.

2. The application of the method described in claim 1 in grass carp breeding.

3. The application of the method described in claim 1 in grass carp farming.

4. The application according to claim 3, characterized in that, The grass carp farming method includes improving the survival rate, final weight, body length and specific growth rate of grass carp in high salinity water, as well as promoting the expression of genes involved in osmotic pressure regulation in grass carp tissues.

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