Method for preparing all-female fingerling of land-sealed Malus salicina by utilizing hydrostatic pressure

Abstract

The invention relates to a method for preparing all-female fingerlings of land-sealed Malus salicina by utilizing hydrostatic pressure, and belongs to the technical field of aquaculture. The method for preparing the all-female fingerling of the land-sealed Malus salicina by utilizing the hydrostatic pressure comprises the following steps: taking eggs from female land-sealed Malus salicina in a mature period, soaking the obtained eggs in an isotonic solution for 5-10 minutes, performing hydrostatic pressure treatment in the isotonic solution, taking out the obtained parthenogenetic development eggs, and putting the eggs into a hatching nest for hatching. The method is short in time consumption, can meet the requirement for one-time mass production of all-female fry of the Malus salicina, has the advantages of being low in production cost, easy to operate, time-saving, labor-saving and efficient, and can achieve the effect that the all-female rate of the hatched parthenogenetic fry is 100%.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and particularly relates to a method for preparing all-female fry of landlocked masu salmon using hydrostatic pressure, and more particularly to a method for inducing parthenogenesis of eggs of landlocked masu salmon using hydrostatic pressure, so as to produce all-female fry of the first generation. Background Art

[0002] Landlocked masu salmon belongs to precious cold-water fish. Compared with male masu salmon, female masu salmon has the characteristics of large size and fast growth rate. In addition, masu salmon eggs are rich in proteins, ω-3 fatty acids, vitamins, minerals, etc. required by the human body. A single masu salmon can lay 3,000 - 4,000 eggs, and the price of eggs can reach 1,000 yuan per catty. The meat prices of spawning female fish and male fish are the same. Therefore, female fish has greater economic value.

[0003] Masu salmon is an oviparous fish. In the natural environment, they reproduce by indirect mating. In the artificial aquaculture environment, when the physiological age and aquaculture environment meet the requirements, generally, the gonads can develop to stage V without artificial hormone induction for spawning, but they cannot spawn and fertilize by themselves, and artificial egg collection is required.

[0004] Parthenogenesis refers to the process in which eggs develop into offspring through self-replication and development without fertilization. This reproductive method does not require the participation of male individuals, and female individuals can reproduce offspring by replicating their own DNA.

[0005] If parthenogenesis of masu salmon can be achieved and applied in production, not only can the excellent traits of the female parent of masu salmon be preserved indefinitely, but also a large number of all-female masu salmon fry can be obtained. These fry can save the cost of raising male fish in production and produce more high-value female masu salmon commercial fish. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing all-female fry of landlocked masu salmon using hydrostatic pressure, which can perform parthenogenesis only relying on the eggs of female masu salmon without the participation of any male fish sperm, so as to produce a large number of all-female masu salmon fry.

[0007] The technical solution adopted by the present invention to achieve the above object is as follows.

[0008] The method for preparing all-female fry of landlocked masu salmon using hydrostatic pressure according to the present invention comprises the following steps:

[0009] Eggs are taken from mature female landlocked masu salmon. After the obtained eggs are soaked in an isotonic solution for 5 - 10 minutes, hydrostatic pressure treatment is carried out in the isotonic solution. The parthenogenetic eggs obtained are taken out and placed in an incubation nest for incubation.

[0010] Preferably, the isotonic solution is an aqueous solution containing 7.1 - 7.3 g / L of sodium chloride, 0.15 - 0.35 g / L of potassium chloride, 0.1 - 0.3 g / L of calcium chloride, 1.9 - 2.1 g / L of sodium bicarbonate, and 0.9 - 1.1 g / L of glucose.

[0011] More preferably, the isotonic solution is an aqueous solution containing 7.2 g / L of sodium chloride, 0.2 g / L of potassium chloride, 0.2 g / L of calcium chloride, 2.0 g / L of sodium bicarbonate, and 1.0 g / L of glucose.

[0012] Preferably, the conditions for the hydrostatic pressure treatment are: the hydrostatic stimulation time is 10 - 15 minutes, the pressure is 60 - 65 minutes, the pressure duration is 5 minutes, and the temperature is 6 - 11 °C.

[0013] More preferably, the conditions for the hydrostatic pressure treatment are: the hydrostatic stimulation time is 12.5 - 15 minutes, the pressure is 60 - 65 minutes, the pressure duration is 5 minutes, and the temperature is 6 - 11 °C;

[0014] Especially preferably, the conditions for the hydrostatic pressure treatment are: the hydrostatic stimulation time is 15 minutes, the pressure is 65 minutes, the pressure duration is 5 minutes, and the temperature is 6 - 11 °C.

[0015] Preferably, the conditions for the incubation are flow-through incubation, and the water temperature is maintained at 6 - 11 °C.

[0016] Preferably, the mature female landlocked masu salmon is obtained by the following method: first, healthy and disease-free female salmon are selected by population breeding technology, and then nutritional intensification cultivation is carried out, and a protein feed with a protein content of more than 50 wt% is fed to obtain mature female landlocked masu salmon.

[0017] Preferably, the method for taking eggs is artificial egg extrusion.

[0018] More preferably, the mature female landlocked masu salmon is fished out, the body surface is dried, placed on the leg, one hand holds the fish head, and the other hand kneads along the fish abdomen, and the mature eggs flow out and are placed in a clean container and stored at 12 - 14 °C.

[0019] The principle of the present invention:

[0020] 1. By utilizing the characteristics of the anadromous migration of Oncorhynchus masou and the high sensitivity of fish eggs to the liquid osmotic pressure, an isosmotic solution is prepared to stimulate the fish eggs to absorb water and swell and be activated, and then hydrostatic pressure is used to induce before the fish eggs expel the first polar body, ultimately achieving the goal of producing all-female fry of Oncorhynchus masou.

[0021] 2. By using biotechnologies such as osmotic pressure stimulation and hydrostatic pressure induction to parthenogenetically activate the eggs of Oncorhynchus masou, the unfertilized eggs undergo cleavage like fertilized eggs and ultimately develop into embryos. In this process, the eggs do not require the participation of sperm but complete development through their own gene expression and cell division.

[0022] 3. All the offspring produced by parthenogenetic development are female individuals.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The method for preparing all-female fry of landlocked Oncorhynchus masou by using hydrostatic pressure in the present invention has a short time consumption and can meet the need for mass production of all-female fry of Oncorhynchus masou at one time.

[0025] 2. The method for preparing all-female fry of landlocked Oncorhynchus masou by using hydrostatic pressure in the present invention has the advantages of low production cost, simple operation, time-saving, labor-saving and high efficiency.

[0026] 3. The method for preparing all-female fry of landlocked Oncorhynchus masou by using hydrostatic pressure in the present invention can achieve the effect that the all-female rate of the parthenogenetic fry hatched is 100%.

[0027] 4. The method for preparing all-female fry of landlocked Oncorhynchus masou by using hydrostatic pressure in the present invention is not only a major innovation in biotechnology in China, but also will have a profound impact on aspects such as gene protection and improved variety breeding of organisms. Specific Embodiments

[0028] To further understand the present invention, the preferred implementation embodiments of the present invention are described below. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention and not for limiting the claims of the present invention.

[0029] The method for preparing all-female fry of landlocked Oncorhynchus masou by using hydrostatic pressure in the present invention comprises the following steps:

[0030] S1. Selection of Broodstock

[0031] By using population selection techniques, female Oncorhynchus masou with excellent quality, stable traits, high spawning rate and rapid growth are obtained;

[0032] Select female salmon with a healthy appearance, large size, and no injuries or diseases for nutritional enrichment breeding, feed them with a protein feed with a protein content of more than 50wt%, and wait until they reach maturity. The mature salmon are characterized by a soft and slightly swollen abdomen, soft to the touch, and eggs protruding from the reproductive opening;

[0033] Take out the mature female Masu salmon, wipe off the moisture on the body surface, put it on your legs, hold the fish head with one hand, and massage the fish belly with the other hand. Then the mature eggs will flow out. Collect the flowing eggs in a plastic bag, and then put the plastic bag in a foam box for use. Do not seal the plastic bag, put an ice bag in the foam box to cool it down, and cover the foam box lid when not in use to prevent the eggs from drying out.

[0034] S2, induced activation

[0035] The eggs are placed in an isotonic solution and soaked for 5-10 minutes, and then subjected to hydrostatic pressure treatment. The conditions for the hydrostatic pressure treatment are: hydrostatic stimulation time of 10-15 minutes, pressure of 60-65 minutes, pressure duration of 5 minutes, and temperature of 6-11°C. During this process, the eggs of the salmon undergo parthenogenetic development.

[0036] S3. Egg incubation

[0037] After the eggs are taken out, they are placed in an incubation nest for running water incubation, and the water temperature is maintained at 6-11℃.

[0038] In the present invention, the isotonic solution is an aqueous solution containing 7.1-7.3 g / L of sodium chloride, 0.15-0.35 g / L of potassium chloride, 0.1-0.3 g / L of calcium chloride, 1.9-2.1 g / L of sodium bicarbonate, and 0.9-1.1 g / L of glucose; preferably, the isotonic solution is an aqueous solution containing 7.2 g / L of sodium chloride, 0.2 g / L of potassium chloride, 0.2 g / L of calcium chloride, 2.0 g / L of sodium bicarbonate, and 1.0 g / L of glucose.

[0039] In the present invention, the conditions for hydrostatic pressure treatment are: hydrostatic stimulation time is 12.5-15min, pressure is 60-65min, pressure duration is 5min, and temperature is 6-11°C; preferably, the conditions for hydrostatic pressure treatment are: hydrostatic stimulation time is 15min, pressure is 65min, pressure duration is 5min, and temperature is 6-11°C.

[0040] The technical solution of the present invention is further described below in conjunction with embodiments.

[0041] Unless otherwise specified, the reagents and experimental methods used in the following examples are conventional commercially available products or methods in the art and will not be described in detail.

[0042] Relevant experiments were conducted at the Masu Damaha Aquatic Seed Farm in Yanbian, Jilin Province.

[0043] Comparative Example 1

[0044] A method for preparing all-female landlocked salmon fingerlings using hormones + hydrostatic pressure, the steps are as follows:

[0045] S1. Broodstock breeding

[0046] First, a group breeding technique is used to select healthy female salmon, and then nutritional enrichment breeding is carried out, and protein feed with a protein content of more than 50wt% is fed to the salmon until they enter the maturity stage. The mature salmon is characterized by a soft and slightly swollen abdomen, which is soft to the touch and has eggs exposed from the reproductive opening;

[0047] Take out the mature female Masu salmon, wipe off the moisture on the body surface, put it on your legs, hold the fish head with one hand, and massage the fish belly with the other hand. Then the mature eggs will flow out. Collect the flowing eggs in a plastic bag, and then put the plastic bag in a foam box for use. Do not seal the plastic bag, put an ice bag in the foam box to cool it down, and cover the foam box lid when not in use to prevent the eggs from drying out.

[0048] S2. Divide into eight groups, recorded as experimental groups 1-8. In each group, 220 eggs were placed in the hormone corresponding to Table 1 for 5-10 minutes, and then subjected to hydrostatic pressure treatment. The conditions of hydrostatic pressure treatment were: hydrostatic stimulation time of 15 minutes, pressure of 65 minutes, pressure duration of 5 minutes, temperature of 10.2°C. The pressurized eggs were placed in an incubation nest for flowing water incubation. The water temperature was maintained at 10.2°C. After 2 months, no fry were hatched. The test results are shown in Table 1. It was observed that the fish eggs after hormone induction treatment would become opaque.

[0049] Table 1 Results of the hormone + hydrostatic pressure induction experiment on salmon eggs

[0050]

[0051] As shown in Table 1, the method of hormone stimulation + hydrostatic pressure on salmon eggs cannot induce all-female fry.

[0052] Example 2

[0053] A method for preparing all-female fingerlings of land-locked masu salmon using isotonic solution + hydrostatic pressure, the steps are as follows:

[0054] S1. Broodstock breeding

[0055] First, a group breeding technique is used to select healthy female salmon, and then nutritional enrichment breeding is carried out, and protein feed with a protein content of more than 50wt% is fed to the salmon until they enter the maturity stage. The mature salmon is characterized by a soft and slightly swollen abdomen, which is soft to the touch and has eggs exposed from the reproductive opening;

[0056] Take out the mature female Masu salmon, wipe off the moisture on the body surface, put it on your legs, hold the fish head with one hand, and massage the fish belly with the other hand. Then the mature eggs will flow out. Collect the flowing eggs in a plastic bag, and then put the plastic bag in a foam box for use. Do not seal the plastic bag, put an ice bag in the foam box to cool it down, and cover the foam box lid when not in use to prevent the eggs from drying out.

[0057] S2. Divide into 18 groups, recorded as experimental groups 9-26. In each group, 220 eggs were placed in isotonic solution (containing 7.2g / L sodium chloride, 0.2g / L potassium chloride, 0.2g / L calcium chloride, 2.0g / L sodium bicarbonate, and 1.0g / L glucose in water) for 5-10 minutes. Then, the conditions in Table 2 were used for hydrostatic pressure treatment. The temperature was 10.2℃. The pressurized eggs were placed in the hatching nest for running water incubation. The water temperature was maintained at 10.2℃. After 2 months, the hatching results of the fry are shown in Table 2.

[0058] Table 2 Experimental results of isotonic solution + hydrostatic pressure induction on salmon roe in Example 2

[0059]

[0060] Example 3

[0061] A method for preparing all-female fingerlings of land-locked masu salmon using isotonic solution + hydrostatic pressure, the steps are as follows:

[0062] S1. Broodstock breeding

[0063] Same as Example 2.

[0064] S2 was divided into six groups, recorded as experimental groups 27-32. In each group, 220 eggs were placed in isotonic solution (containing 7.1 g / L sodium chloride, 0.25 g / L potassium chloride, 0.2 g / L calcium chloride, 2.0 g / L sodium bicarbonate, and 0.9 g / L glucose) for 5-10 minutes, and then hydrostatic pressure treatment was performed using the conditions in Table 3. The temperature was 8.5±0.1℃. The pressurized eggs were placed in the hatching nest for flowing water incubation. The water temperature was maintained at 8.5±0.1℃. After 2 months, the fry hatching results are shown in Table 3.

[0065] Table 3 Experimental results of isotonic solution + hydrostatic pressure induction on salmon eggs in Example 3

[0066]

[0067] Example 4

[0068] A method for preparing all-female fry of landlocked masu salmon using isotonic solution + hydrostatic pressure, the steps are as follows:

[0069] S1. Selection of broodstock

[0070] Same as Example 2.

[0071] S2. Divide into six groups, denoted as experimental groups 33-38. Put 220 eggs in an isotonic solution (aqueous solution containing 7.3 g / L of sodium chloride, 0.15 g / L of potassium chloride, 0.1 g / L of calcium chloride, 1.9 g / L of sodium bicarbonate, and 1.1 g / L of glucose) for 5-10 minutes, and then perform hydrostatic pressure treatment under the conditions in Table 4 respectively. The temperature is 6.2 ± 0.2 °C. The pressurized eggs are placed in an incubation nest for running water incubation, and the water temperature is maintained at 6.2 ± 0.2 °C. After 2 months, the hatching results of the fry are shown in Table 4.

[0072] Table 4 Experimental results of inducing masu salmon eggs with isotonic solution + hydrostatic pressure in Example 4

[0073]

[0074] As can be seen from Tables 2-4, the combined methods of isotonic solution and hydrostatic pressure can induce parthenogenetic development of fish eggs and produce all-female fry for masu salmon, and the effects are different under different conditions. The effect is the best when the hydrostatic pressure is 65 MP and the osmotic pressure stimulation time is 15 minutes.

[0075] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing all-female fingerlings of land-locked masu salmon using hydrostatic pressure, characterized in that: Here are the steps: Eggs are collected from mature female land-locked salmon, and the obtained eggs are immersed in isotonic solution for 5-10 minutes, and then subjected to hydrostatic pressure treatment in the isotonic solution to obtain parthenogenetic eggs. After being taken out, the eggs are placed in a hatching nest for hatching to obtain all-female land-locked salmon fingerlings.

2. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 1, characterized in that: The isotonic solution is an aqueous solution containing 7.1-7.3 g / L of sodium chloride, 0.15-0.35 g / L of potassium chloride, 0.1-0.3 g / L of calcium chloride, 1.9-2.1 g / L of sodium bicarbonate, and 0.9-1.1 g / L of glucose.

3. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 2, characterized in that: The isotonic solution is an aqueous solution containing 7.2 g / L sodium chloride, 0.2 g / L potassium chloride, 0.2 g / L calcium chloride, 2.0 g / L sodium bicarbonate, and 1.0 g / L glucose.

4. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 1, characterized in that: The conditions of the hydrostatic pressure treatment are: hydrostatic stimulation time is 10-15 minutes, pressure is 60-65 minutes, pressure duration is 5 minutes, and temperature is 6-11°C.

5. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 4, characterized in that: The conditions of the hydrostatic pressure treatment are: hydrostatic stimulation time is 12.5-15 minutes, pressure is 60-65 minutes, pressure duration is 5 minutes, and temperature is 6-11°C.

6. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 5, characterized in that: The conditions of the hydrostatic pressure treatment are: hydrostatic stimulation time is 15 minutes, pressure is 65 minutes, pressure duration is 5 minutes, and temperature is 6-11°C.

7. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 1, characterized in that: The incubation condition is running water incubation, and the water temperature is maintained at 6-11°C.

8. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 1, characterized in that: The mature female landlocked masu salmon is obtained by the following method: First, the group breeding technology is used to select healthy and disease-free female salmon, and then nutritional fortification breeding is carried out, and protein feed with a protein content of more than 50wt% is fed to obtain mature female land-locked salmon.

9. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 1, characterized in that: The method for obtaining eggs is artificial egg squeezing.

10. The method for preparing all-female fingerlings of land-locked masu salmon by hydrostatic pressure according to claim 9, characterized in that: Take out the mature female landlocked salmon, wipe off the moisture on the body, put it on your legs, hold the fish head with one hand, and massage the fish belly with the other hand. The mature eggs will flow out and be stored in a clean container at 12-14℃.

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