Method for efficiently and artificially inducing triploid grass carp

Abstract

The invention discloses a method for efficiently and artificially inducing triploid grass carps, which comprises the following steps: (1) collecting sperms and ova of grass carps, and carrying out artificial insemination; (2) carrying out cold shock treatment on the fertilized eggs of the grass carps, wherein the cold shock treatment is that the fertilized eggs are placed in water at the temperature of 4-6 DEG C for 10-12 minutes; and incubating the fertilized eggs to obtain the triploid grass carp.

Description

Technical Field

[0001] The invention relates to the technical field of fish genetic breeding, and in particular to a method for efficiently artificially inducing triploid grass carp. Background Art

[0002] In nature, organisms generally exist in the form of diploids. However, there are still some species in nature that contain three or more sets of chromosomes, which are called polyploids. For example, triploid individuals have been found in invertebrates and vertebrates (insects, amphibians, fish and other species). Among them, fish have the most polyploid phenomena, with at least more than 100 species; for example, in the Heilongjiang River system in my country, natural triploid silver crucian carp have been found. In addition, artificial induction technology has also been widely used in the production of sterile fish strains. Polyploid organisms have important scientific research value for understanding species formation and evolution. They are also excellent breeding materials and play an important role in production practice. Because triploids have an extra set of chromosomes in their bodies, their reproductive cells cannot effectively divide, which inhibits their gonadal development, and thus have a growth advantage over diploids. In addition, the proliferation and release of triploid fish can reduce the negative impact on local fish populations and protect the genetic diversity and integrity of local fish populations.

[0003] Grass carp (Ctenopharyngodon idella), also known as silver carp, oily silver carp, grass carp, etc., is an important freshwater economic fish in my country. It is known as the "Four Major Carps" together with black carp, silver carp and bighead carp. Grass carp has delicious meat, low fat content, high protein content, and extremely high nutritional value. In addition, due to its rapid growth and strong resistance, it has become the freshwater fish with the largest annual output. Triploid grass carp has a huge market and commercial value because of its sterility, which can shorten its time to market. Therefore, how to efficiently obtain triploid grass carp fry has become a key step in increasing grass carp production. Summary of the invention

[0004] The purpose of the present invention is to provide a method for efficiently artificially inducing triploid grass carp, so as to obtain triploid grass carp populations in batches.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] A method for efficiently artificially inducing triploid grass carp comprises the following steps:

[0007] (1) Collecting sperm and eggs from grass carp for artificial insemination;

[0008] (2) subjecting the fertilized grass carp eggs to a cold shock treatment, wherein the cold shock treatment is to place the fertilized eggs in water at a temperature of 4 to 6° C. for 10 to 12 minutes; and then incubate the fertilized eggs to obtain triploid grass carp.

[0009] Preferably, the grass carp fertilized eggs are subjected to cold shock treatment 2 minutes after fertilization.

[0010] Preferably, the cold shock treatment is to place the fertilized eggs in water at a temperature of 4° C. for 10 to 12 minutes.

[0011] Specifically, the artificial insemination can be wet insemination or dry insemination; the volume ratio of sperm to egg is 1:50-150.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The invention utilizes cold shock treatment to induce grass carp eggs and artificially cultivates triploid grass carp. The method of the invention is simple to operate, highly efficient, and highly practical, and the induction efficiency of triploid grass carp can reach 76%. The invention opens up a new technical approach for polyploid breeding of grass carp, and has great application value and broad promotion and application prospects in grass carp production and polyploid breeding of excellent varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of DNA content detected by flow cytometry in Example 1, A is diploid, and B is triploid.

[0015] Figure 2 The results of DNA content detected by flow cytometry in Example 2, A is diploid, and B is triploid.

[0016] Figure 3 The results of DNA content detected by flow cytometry in Comparative Examples 1 to 3 and 5 are shown in Figure 1, A is Comparative Example 1, B is Comparative Example 2, C is Comparative Example 3, and D is Comparative Example 5. DETAILED DESCRIPTION

[0017] Below in conjunction with the embodiment of the present invention, the technical scheme of the present invention is clearly and completely described, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. Reagents or instruments used in the present invention are all purchased from the market.

[0018] Example 1

[0019] 1. Artificial induction of triploid grass carp

[0020] (1) Selection of grass carp parents and collection of sperm and eggs: Parents are selected to be healthy, of normal body shape, strong, and weighing 10 to 15 kg each. They are placed in outdoor soil ponds for cultivation and fed with broodstock compound feed and grass every day for cultivation. Artificial insemination is carried out when the average water temperature stabilizes at 26 to 28°C.

[0021] Before artificial insemination, the female parent is first injected with luteinizing hormone releasing hormone analog (LRH-A) hormone, the dosage is 10-20 μg / kg, and 12 hours later, the male parent and female parent are injected with luteinizing hormone releasing hormone analog (LRH-A) and domperidone (DOM) hormone, the dosage is 10-20 μg / kg and 3-5 mg / kg respectively. 6-8 hours after the second injection, artificial sperm and egg squeezing begins. The fresh sperm obtained is placed at 0-4℃

[0022] Store and avoid contact with water to prevent sperm activation; place mature eggs in a dry beaker, avoid contact with water, and carry out artificial insemination as soon as possible.

[0023] Artificial insemination: dry insemination is performed by mixing sperm and eggs in a ratio of 1 mL sperm to 100 mL eggs. Specifically, first, 1 mL sperm and 100 mL eggs are fully mixed, then clean fresh water equal to the eggs is added, and stirred evenly to activate the sperm. This is the 0th hour of insemination. Then, clean fresh water is added and continued to mix and stir for 30 seconds to complete artificial insemination. If wet insemination is used, the sperm is added to clean fresh water equal to the eggs, quickly stirred, and then poured into the eggs and quickly stirred. This is the 0th hour of insemination. Then, clean fresh water is added and continued to mix and stir for 30 seconds to complete artificial insemination.

[0024] (2) Cold shock treatment: Use refrigeration equipment (such as freezers, refrigerators, etc.) to prepare aerated frozen fresh water in advance, and adjust its temperature to 6°C while performing artificial insemination; immediately transfer the fertilized eggs to the above-mentioned frozen fresh water 2 minutes after fertilization, and stir the water evenly; after 10 minutes of cold shock treatment, remove the fertilized eggs from the frozen fresh water and transfer them to a loop pool or hatching bucket for incubation.

[0025] 2. Rapid detection of triploid grass carp fry

[0026] 36 hours after fertilization, 32 grass carp fry were randomly collected for rapid DNA content detection. The specific operation was as follows: the fry were placed in 1.5 mL centrifuge tubes, 200 μL CyStain TM UVPloidy Staining Solution and grind the fry thoroughly using scissors or Tiangen Y-30 handheld tissue grinder. Then add 800μL CyStain TMUV Ploidy Staining Solution, incubate at room temperature for 30-90 seconds, and then filter the sample using a 50 μm filter. The prepared samples were tested using a Sysmex CyFlow Ploidy Analyser (parameters: Light Source: UVLED, Gain settings: 547V, Thresholds: 73%, Fluidics: 0.4 μL / sec).

[0027] As shown in Table 1 and Figure 1 As shown in the results, the triploid ratio induced by this implementation is 65.625%.

[0028] Example 2

[0029] The operation of this embodiment is the same as that of embodiment 1, except that the water temperature of the cold shock treatment is 4° C. and the time is 12 minutes.

[0030] 36h after artificial insemination, 30 grass carp fry were randomly collected for rapid detection of DNA content.

[0031] As shown in Table 1 and Figure 2 The results shown in the figure show that the triploid ratio obtained in this example is 76.667%.

[0032] Comparative Example 1

[0033] The operation of this comparative example is the same as that of Example 2, except that the cold shock treatment time is 14 minutes.

[0034] 36h after artificial insemination, 30 grass carp fry were randomly collected for rapid detection of DNA content.

[0035] As shown in Table 1 and Figure 3 The result shown in A is that no triploid was obtained in this comparative example.

[0036] Comparative Example 2

[0037] The operation of this comparative example is the same as that of Example 2, except that the water temperature for the cold shock treatment is 7°C.

[0038] 36h after artificial insemination, 30 grass carp fry were randomly collected for rapid detection of DNA content.

[0039] As shown in Table 1 and Figure 3 The result shown in B is that no triploid was obtained in this comparative example.

[0040] Comparative Example 3

[0041] The operation of this comparative example is the same as that of Example 2, except that a cold shock treatment is performed 1 minute after insemination.

[0042] 36 h after artificial insemination, 30 grass carp fry were randomly collected for rapid detection of triploid grass carp fry. Figure 3 The result shown in C is that no triploid was obtained in this comparative example.

[0043] Comparative Example 4

[0044] The operation of this comparative example is the same as that of Example 2, except that the water temperature of the cold shock treatment is 2° C. 36 hours after artificial insemination, the seedlings that can successfully hatch out of the membrane are rare and have weak vitality, so the induction conditions are not suitable for triploid breeding of grass carp.

[0045] Comparative Example 5

[0046] The operation of this comparative example is the same as that of Example 2, except that: 2 minutes after insemination, a hydrostatic shock treatment is performed in the hydrostatic pressure chamber, wherein the hydrostatic pressure is 360 bar and the action time is 4 minutes.

[0047] 36h after artificial insemination, 30 grass carp fry were randomly collected for rapid detection of DNA content.

[0048] As shown in Table 1 and Figure 3 The result shown in D is that no triploid was obtained in this comparative example.

[0049] Table 1: Conditions and triploidy rates of triploid grass carp induction

[0050]

[0051]

Claims

1. A method for efficiently artificially inducing triploid grass carp, characterized in that: The following steps are involved: (1) Collecting sperm and eggs from grass carp for artificial insemination; (2) subjecting the fertilized grass carp eggs to a cold shock treatment, wherein the cold shock treatment is to place the fertilized eggs in water at a temperature of 4 to 6° C. for 10 to 12 minutes; and then incubate the fertilized eggs to obtain triploid grass carp.

2. The method for efficiently artificially inducing triploid grass carp according to claim 1, characterized in that: Cold shock treatment was performed 2 minutes after insemination.

3. The method for efficiently artificially inducing triploid grass carp according to claim 2, characterized in that: The cold shock treatment is to place the fertilized eggs in water at a temperature of 4° C. for 10 to 12 minutes.

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