Method for inducing triploid grass carp by hydrostatic pressure

Abstract

The invention discloses a method for inducing triploid grass carp under hydrostatic pressure, which comprises the following steps: (1) mixing sperms and ova of grass carp, and carrying out artificial insemination; (2) after artificial insemination is conducted for 2 min and 50 sec to 3 min, fertilized eggs are put into a pressure device with the pressure being 63 MPa, and treatment is conducted for 70 sec; (3) after the pressure treatment is finished, the fertilized eggs are immediately put into a hatching pond to be hatched in flowing water, and the triploid grass carp is obtained. According to the method, the induction efficiency of three times of grass carp can reach 100%.

Description

Technical Field

[0001] This invention relates to the field of fish genetics and breeding, and in particular to a method for inducing triploid grass carp by hydrostatic pressure. Background Technology

[0002] Sterile strains, because they do not consume energy for reproduction, can invest more energy in individual growth, thereby improving aquaculture efficiency, and have therefore become an important research subject in the field of aquaculture. Among these methods, the preparation of triploids is considered an effective means of constructing sterile fish populations. There are two main methods for triploid preparation: one is to inhibit the release of the second polar body of the fertilized egg through physical (cold shock, hydrostatic pressure) or chemical treatments. This method has been successfully used to obtain triploid individuals in fish such as mandarin fish and blunt snout bream. Secondly, distant hybridization is also an effective method for preparing allogeneic triploids. For example, a certain proportion of triploid individuals exist in the F1 generation of blunt snout bream (♀) × snakehead (♂) and grass carp (♀) × red snapper (♂).

[0003] Grass carp (Ctenopharyngodon idella), also known as white amur, is widely distributed in major river systems of East Asia. Primarily herbivorous, it has low protein requirements and grows rapidly, making it popular among fish farmers. Furthermore, its delicious flesh, low fat content, and high protein content give it high nutritional value, making it highly sought after by consumers. In recent years, grass carp farming production has steadily increased, making it the largest freshwater fish produced annually in my country. Triploid grass carp, due to their sterility, possess significant market and commercial value, and their market time can be shortened. Therefore, inducing triploid grass carp has important practical value. Although previous reports have successfully induced triploid populations, these methods have limitations, such as unstable triploid induction rates. Therefore, establishing an efficient and stable method for inducing triploid grass carp is essential. Summary of the Invention

[0004] The purpose of this invention is to provide a method for inducing triploid grass carp by hydrostatic pressure, which can efficiently and stably obtain triploid grass carp populations.

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

[0006] A method for inducing triploid grass carp by hydrostatic pressure includes the following steps:

[0007] (1) Mix the sperm and eggs of grass carp for artificial insemination;

[0008] (2) After artificial insemination for 2 min 50 sec to 3 min, the fertilized egg is placed in a pressure device with a pressure of 63 MPa and processed for 70 sec;

[0009] (3) After the pressure treatment is completed, the fertilized eggs are immediately placed in the hatching pool for incubation with running water to obtain triploid grass carp.

[0010] The 2 min 50 sec to 3 min follow-up in the artificial insemination described in this invention refers to the 2 min 50 sec to 3 min follow-up after the sperm is activated.

[0011] Preferably, artificial insemination can be performed using wet insemination or dry insemination, with a sperm-to-egg volume ratio of 1:50 to 150.

[0012] Preferably, the fertilized egg is subjected to pressure treatment 3 minutes after artificial insemination.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention utilizes hydrostatic pressure to treat grass carp fertilized eggs, enabling efficient and stable cultivation of artificial triploid grass carp. The method is simple, efficient, and highly practical, achieving an induction efficiency of over 90%, and potentially reaching 100%, for triploid grass carp. This invention opens up a new technical avenue for grass carp polyploid breeding, possessing significant application value and broad prospects for widespread application in grass carp production and the selection of superior polyploid varieties. Attached Figure Description

[0015] Figure 1 The results of triploidity detection by flow cytometry in Example 1 are shown.

[0016] Figure 2 The results of triploidity detection by flow cytometry in Example 2 are shown.

[0017] Figure 3 The results of flow cytometry triploidy rate detection in Comparative Example 1 are shown.

[0018] Figure 4 The results of flow cytometry triploidy rate detection in Comparative Example 2 are shown.

[0019] Figure 5 The results of flow cytometry triploidy rate detection in Comparative Example 3 are shown.

[0020] Figure 6 The results of flow cytometry triploidy rate detection in Comparative Example 4 are shown.

[0021] Figure 7 The results of flow cytometry triploidy rate detection in Comparative Example 5 are shown.

[0022] Figure 8 The results of flow cytometry triploidy rate detection in Comparative Example 7 are shown.

[0023] Figure 9 The results of flow cytometry triploidy rate detection in Comparative Example 8 are shown. Detailed Implementation

[0024] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0025] Example 1

[0026] 1. Artificially induced triploid grass carp

[0027] (1) Selection of grass carp broodstock and collection of sperm and eggs: Broodstock were selected from healthy individuals with no injuries or diseases, normal body shape, and a weight of 10–15 kg / fish. They were raised in outdoor earthen ponds, fed daily with formulated feed and grass. Artificial insemination was carried out when the average water temperature stabilized at 24–28℃. Before artificial insemination, the female broodstock was injected with luteinizing hormone-releasing hormone analogue (LRH-A) at a dose of 10–20 μg / kg. Twelve hours later, the male and female broodstock were injected with LRH-A and domperidone (DOM) at doses of 10–20 μg / kg and 3–5 mg / kg, respectively. Artificial sperm and egg collection began 6–8 hours after the second injection. Fresh sperm was stored at 0–4℃, avoiding contact with water to prevent sperm activation. Mature eggs were placed in dry, light-proof beakers, avoiding contact with water, and artificial insemination was carried out as soon as possible.

[0028] Artificial insemination: Mix sperm and eggs at a ratio of 1 mL sperm to 100 mL eggs and perform dry insemination. Specifically, first mix 1 mL sperm with 100 mL eggs thoroughly, then add an equal volume of clean fresh water to the eggs and stir evenly to activate the sperm. This is designated as artificial insemination time 0. Then add clean fresh water twice to wash away any excess sperm to complete artificial insemination.

[0029] (2) Hydrostatic pressure treatment: The fresh water that has been aerated in advance is introduced into the hydrostatic pressure device. After artificial insemination for 3 minutes, the fertilized eggs are immediately transferred to the hydrostatic pressure device and a pressure of 63 MPa is applied. After treatment for 70 seconds, the fertilized eggs are taken out from the hydrostatic pressure device and transferred to the ring pool or hatching tank for incubation.

[0030] 2. Rapid detection of triploid grass carp fry

[0031] After the fertilized eggs hatched, 20 grass carp fry were randomly collected. Each fry was individually placed in a 1.5 mL centrifuge tube, and 200 μL of CyStain™ UV Ploidy Staining Solution was added. The fry were then thoroughly ground using scissors or a Tiangen Y-30 handheld tissue homogenizer. Next, 800 μL of CyStain™ UV Ploidy Staining Solution was added, and the mixture was incubated at room temperature for 30–90 s. The samples were then filtered through a 50 μm filter. The prepared samples were analyzed using a Sysmex CyFlowPloidy Analyzer (parameters: Light Source: UVLED, Gain settings: 547V, Thresholds: 73%, Fluidics: 0.4 μL / sec). See Table 1 and... Figure 1 The results show that the proportion of triploids induced in this embodiment is 100%.

[0032] Example 2

[0033] The operation of this embodiment is the same as that of embodiment 1, except that pressure is applied 2 minutes and 50 seconds after artificial insemination.

[0034] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 2 As shown in the results, the proportion of triploids induced in this embodiment is 90%.

[0035] Comparative Example 1

[0036] The operation of this comparative example is the same as that of Example 1, except that pressure is applied 2 minutes and 30 seconds after artificial insemination, and the pressure treatment time is 50 seconds.

[0037] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 3 The results shown indicate that the proportion of triploids induced in this comparative study was 35%.

[0038] Comparative Example 2

[0039] The operation of this comparative example is the same as that of Example 1, except that pressure is applied 2 minutes and 30 seconds after artificial insemination.

[0040] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 4 The results shown indicate that the proportion of triploids induced in this comparative study was 45%.

[0041] Comparative Example 3

[0042] The operation of this comparative example is the same as that of Example 1, except that pressure is applied 2 minutes and 40 seconds after artificial insemination.

[0043] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 5 The results shown indicate that the proportion of triploids induced in this comparative study was 85%.

[0044] Comparative Example 4

[0045] The operation of this comparative example is the same as that of Example 1, except that pressure is applied 3 minutes and 30 seconds after artificial insemination.

[0046] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 6 The results shown indicate that the proportion of triploids induced in this comparative study was 65%.

[0047] Comparative Example 5

[0048] The operation of this comparative example is the same as that of Example 1, except that a pressure of 48 MPa is applied.

[0049] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 7 The results shown indicate that the proportion of triploids induced in this comparative study was 50%.

[0050] Comparative Example 6

[0051] The operation of this comparative example is the same as that of Example 1, except that a pressure of 68 MPa is applied.

[0052] No surviving grass carp fry were obtained in this comparative study.

[0053] Comparative Example 7

[0054] The operation of this comparative example is the same as that of Example 1, except that the pressure treatment time is 80 seconds.

[0055] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 8 The results shown indicate that the proportion of triploids induced in this comparative study was 80%.

[0056] Comparative Example 8

[0057] The operation of this comparative example is the same as that of Example 1, except that the pressure treatment time is 60 seconds.

[0058] After the fertilized eggs hatched, 20 grass carp fry were randomly collected for ploidy testing. See Table 1 and... Figure 9The results shown indicate that the proportion of triploids induced in this comparative study was 85%.

[0059] Table 1: Conditions for hydrostatic pressure-induced triploid grass carp and triploidity rate

[0060] Group Starting time Pressure Treatment time Tritome induction rate Example 1 3 min 63 MPa 70 sec 100％ Example 2 2 min 50 sec 63 MPa 70 sec 90％ Comparative Example 1 2 min 30 sec 63 MPa 50 sec 35％ Comparative Example 2 2 min 30 sec 63 MPa 70 sec 45％ Comparative Example 3 2 min 40 sec 63 MPa 70 sec 85％ Comparative Example 4 3 min 30 sec 63 MPa 70 sec 65％ Comparative Example 5 3 min 48 MPa 70 sec 50％ Comparative Example 6 3 min 68 MPa 70 sec — Comparative Example 7 3 min 63 MPa 80 sec 80％ Comparative Example 8 3 min 63 MPa 60 sec 85％

[0061] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for inducing triploid grass carp by hydrostatic pressure, characterized in that, Includes the following steps: (1) Mix the sperm and eggs of grass carp for artificial insemination; (2) After artificial insemination for 2 min 50 sec to 3 min, the fertilized egg is placed in a pressure device with a pressure of 63 MPa and processed for 70 sec; (3) After the pressure treatment is completed, the fertilized eggs are immediately placed in the hatching pool for incubation with running water to obtain triploid grass carp.

2. The method for inducing triploid grass carp by hydrostatic pressure according to claim 1, characterized in that, Three minutes after artificial insemination, the fertilized egg was subjected to pressure treatment.

3. The method for inducing triploid grass carp by hydrostatic pressure according to claim 1, characterized in that, The volume ratio of sperm to egg is 1:50 to 150.

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