Seed production method of channa maculata

Abstract

The invention belongs to the technical field of fish germplasm resource improvement, and discloses a breeding method of channa maculata, which comprises the following steps: selecting mature channa maculata parent female fish and male fish, carrying out spawning induction treatment, and then stimulating by running water to collect ova and seminal fluid; diluting the semen, carrying out shake culture, and irradiating by adopting an ultraviolet lamp during the shake culture to obtain inactivated semen; adding the inactivated seminal fluid into the ovum, stirring to activate fertilization, and adding aerated water to obtain a fertilized ovum; carrying out cold shock treatment and hatching to obtain newly hatched larva fish; after the newly hatched fries open mouths, fish fries are obtained and fed with biological bait for breeding, when the fish fries grow to 2 cm, the bodies start to become yellow gradually, and when the fish fries grow to 5 cm, the whole bodies show obvious and stable golden body lines, and the channa maculata is obtained. According to the method, artificial insemination is carried out in the mode that the semen is irradiated by the ultraviolet lamp to be inactivated, the channa maculata is obtained through natural incubation, the method is simple, convenient and rapid, guarantees are provided for production and breeding of the channa maculata, and the method has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of improving fish germplasm resources, and particularly relates to a method for breeding golden snakehead fish (Channa maculata). Background Art

[0002] Snakehead fish (Channa maculata) belongs to Perciformes, Channoidei, Channidae, and Channa in taxonomy, also known as snakehead, and is a fierce carnivorous freshwater fish. Snakehead fish has a delicious taste and rich nutrition, and is widely welcomed in the market. In addition, snakehead fish can be cultured as an ornamental fish, and is loved by some enthusiasts because of its golden or red body color. This color-varied snakehead fish is called "golden snakehead". At present, in order to meet the market demand for snakehead fish as an ornamental fish, by studying the genomic structure and function of snakehead fish and understanding its genetic diversity, artificial selective technical means are used to cultivate a snakehead fish variety with a golden body color. As an effective means to obtain excellent varieties, accelerate population breeding, and control gender, the core technologies of artificial induction of fish gynogenesis include inactivation of sperm genetic material and chromosome doubling of fertilized eggs. Both the method of inactivating sperm genetic material and the method of chromosome doubling of fertilized eggs have physical and chemical methods, and the physical method is more commonly used.

[0003] Golden snakehead fish has certain requirements for water quality and is prone to problems such as cloudy eyes. Therefore, good water quality management needs to be maintained. From the selection of broodstock to hatching and then to the cultivation of fry, the whole process involves multiple links, and each link needs to be strictly controlled, increasing the technical difficulty. Despite taking various measures, the survival rate of fry is still an urgent problem to be solved, especially in the early stage. Although the breeding technology of golden snakehead fish has been relatively mature, there are still some technical and environmental challenges that need to be overcome. Therefore, it is urgent to study new technologies for cultivating "golden snakehead" and establish an artificial breeding method for "golden snakehead". Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a method for breeding golden snakehead fish to solve the problems in the artificial reproduction and breeding technology of golden snakehead fish and meet the market demand for golden snakehead fish.

[0005] To achieve the above object, the present invention provides a method for breeding golden snakehead fish, comprising the following steps:

[0006] Step (1): Select mature female and male snakehead fish parents. The female and male snakehead fish parents are respectively subjected to induced spawning treatment. After induced spawning, the female and male snakehead fish parents are stimulated by running water, and then eggs and semen are collected;

[0007] Step (2): Dilute the semen obtained in step (1), shake and culture the diluted semen, and irradiate it with an ultraviolet lamp during the period. Observe that more than 90% of the inactivated sperm lose their motility, and thus the inactivated semen is obtained;

[0008] In step (3), the inactivated semen obtained in step (2) is added to the eggs obtained in step (1), stirred to activate fertilization, and aerated water is added to obtain fertilized eggs.

[0009] In step (4), the fertilized eggs obtained in step (3) are subjected to cold shock treatment and then hatched to obtain newly hatched larvae.

[0010] In step (5), after the newly hatched larvae described in step (4) start feeding at 96 - 120 h, fry are obtained. They are fed with biological bait and artificial feed for cultivation. When the fry grow to 2 cm, yellow color gradually appears on their bodies. When they grow to 5 cm, obvious and stable golden body patterns appear all over their bodies, and golden snakehead fish are obtained.

[0011] Preferably, the specific oxytocin treatment for the female parent fish in step (1) is as follows: The female parent fish is injected with luteinizing hormone - releasing hormone A2, chorionic gonadotropin, and domperidone maleate into the body cavity. The injection amount of luteinizing hormone - releasing hormone A2 is 4 - 6 μg / kg, the injection amount of chorionic gonadotropin is 400 - 500 IU / kg, and the injection amount of domperidone maleate is 2 - 3 mg / kg.

[0012] The specific oxytocin treatment for the male parent fish in step (1) is as follows: The male parent fish is injected with luteinizing hormone - releasing hormone A2, chorionic gonadotropin, and domperidone maleate into the body cavity. The injection amount of luteinizing hormone - releasing hormone A2 is 2 - 3 μg / kg, the injection amount of chorionic gonadotropin is 200 - 250 IU / kg, and the injection amount of domperidone maleate is 1 - 1.5 mg / kg.

[0013] Preferably, the time of the flowing - water stimulation in step (1) is 32 - 40 h. The flowing - water stimulation is to promote the male parent fish to chase the female parent fish.

[0014] Preferably, the dilution of the semen obtained in step (1) in step (2) is specifically as follows: The semen obtained in step (1) is mixed with the sperm preservation solution according to a volume ratio of 1:2 - 6.

[0015] The specific shaking culture of the diluted semen in step (2) and the ultraviolet lamp irradiation during this period are as follows: The diluted semen is smeared on a culture dish with a thickness ≤ 1 mm, placed on ice, and shaken at 100 - 140 r / min for culture. During this period, in a light - proof environment, 4 - 8 ultraviolet lamps with a power of 20 - 40 W are used to irradiate for 20 - 40 s, and the vertical distance between the ultraviolet lamp and the semen surface is 20 - 30 cm.

[0016] More than 90% of the inactivated sperm in step (2) lose their motility, specifically: > 90% of the sperm move in circles or in curves, and ≤ 10% of the sperm move in a straight line.

[0017] Preferably, in step (3), a goose feather rod is used for stirring, and the stirring time is 2 - 4 min.

[0018] Preferably, in step (4), the environment for cold shock treatment is an ice-water mixture at 0-4°C, and the time for cold shock treatment is 20-40 min.

[0019] Preferably, in step (4), the environment for incubation is water at 25-27°C, and the incubation time is 28-32 h.

[0020] Preferably, in step (5), the environment for waiting for the newly hatched larvae to start feeding is water at 25-27°C until the newly hatched larvae swim horizontally.

[0021] Preferably, the specific method of feeding biological bait in step (5) is: feeding biological bait 4 times a day, the biological bait is freshwater rotifers and / or cladocerans, and after feeding biological bait for 7-8 days, artificial feed is fed.

[0022] Compared with the prior art, the present invention has the following advantages and technical effects:

[0023] The present invention provides a complete artificial breeding method for golden snakehead. Artificial induction of spawning is carried out on mature parent snakehead, sperm is irradiated with ultraviolet lamp, artificial insemination is carried out, fertilized eggs are treated by cold shock, and golden snakehead is cultivated. By using the method of inactivating semen by ultraviolet lamp irradiation for artificial insemination and then incubating naturally to obtain golden snakehead, the method is simple, the equipment is common, convenient and fast, which provides a guarantee for the production and reproduction of golden snakehead and has good application prospects. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 It is a flow chart for breeding golden snakehead. In the figure, ① represents putting parent snakehead into a grid for artificial induction of spawning with running water stimulation, ② represents taking the ovary to collect semen of parent male fish, ③ represents inactivating semen by ultraviolet lamp irradiation, ④ represents artificial insemination of inactivated semen and eggs, ⑤ represents the newly hatched larvae obtained by incubation, and ⑥ represents the golden snakehead obtained by breeding in the present invention. Detailed Embodiments

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0031] The female and male parent snakehead fish were purchased from Haozhen Breeding Farm in Zhongshan City; luteinizing hormone-releasing hormone A2 (LRH-A2), human chorionic gonadotropin (HCG), and domperidone maleate (DOM) were purchased from Ningbo No. 2 Hormone Factory; the sperm preservation solution was the sperm preservation solution for hybrid snakehead fish in the patent with the authorization number CN105941391B; freshwater rotifers and cladocerans were purchased from Haozhen Breeding Farm in Zhongshan City, and the artificial feed was purchased from Wanghai Fine Biological Technology Group.

[0032] Example 1

[0033] (1) Select healthy and mature parent female and male snakehead fish. The parent female fish is subjected to induced spawning treatment by coelomic injection of LRH-A 25 μg / kg, HCG 450 IU / kg and DOM 2.5 mg / kg. The parent male fish is subjected to induced spawning treatment by coelomic injection of LRH-A 22.5 μg / kg, HCG 225 IU / kg and DOM 1.25 mg / kg. The post-induced-spawning parent female fish and the post-induced-spawning parent male fish are stocked in a grid and stimulated by running water for 36 h. The male fish chases the female fish, then the testis is taken, cut into pieces, added to the sperm preservation solution and ground, and the semen is collected. Gently press the abdomen of the female fish (the abdomen is swollen, soft, and the genital pore is red and swollen). When egg grains flow out, egg collection is carried out. Gently hold the female fish with its abdomen facing up, and start from the tail with fingers and gently push forward along the abdomen. This can promote the movement of eggs towards the genital pore. When reaching near the genital pore, apply a little more force, but keep the movement gentle to avoid hurting the fish. Repeat several times until all the eggs are discharged, and then collect the eggs.

[0034] (2) Mix the semen with the sperm preservation solution according to a volume ratio of 1:4 for dilution. Apply the diluted semen on a culture dish with a diameter of 15 mm at a thickness of 1 mm, place it on ice and shake it at 120 r / min for cultivation. During this period, in a light-proof environment, irradiate it with 6 30-W ultraviolet lamps for 30 s. The vertical distance between the ultraviolet lamp and the semen surface is 25 cm. Check the sperm motility every 15 s. When it is found that the sperm motility significantly weakens, if the sperm is in a static state, add another drop of water. When more than 90% of the sperm rotates or moves in a curve and ≤10% of the sperm moves in a straight line, it is regarded that more than 90% of the inactivated sperm loses its motility, and thus the inactivated semen is obtained.

[0035] (3) Add the inactivated semen to the eggs, and gently stir for 3 min with a goose feather rod to activate fertilization, and then add aerated water to obtain fertilized eggs.

[0036] (4) The fertilized eggs are subjected to cold shock treatment in an ice-water mixture at 0 - 4 °C for 30 min, and then hatched in water at 26 °C for 30 h to obtain newly hatched larvae.

[0037] (5) The newly hatched larvae are in aerated water at 26 °C for 108 h until they open their mouths. When the newly hatched larvae swim horizontally to obtain fry, feed rotifers and cladocerans 4 times a day, at 7:00, 10:00, 14:00 and 17:00 respectively. After feeding the biological bait for 7 d, feed artificial feed. One week later, put them into the breeding pond for breeding. The breeding pond is equipped with a water pool filter box, connected to an 80-W water pump for 24-hour circulation. When the fry grow to 2 cm, yellow color gradually appears on the body. When they grow to 5 cm, obvious and stable golden body patterns appear all over the body, and thus the golden snakehead fish is obtained.

[0038] As Figure 1As shown in the figure, it is the flow chart for breeding golden snakehead fish. ①Put the parental snakehead fish into the grid and stimulate them artificially with running water for induced spawning. ②Take the ovarian tissue and collect the semen of the parental male fish. ③Inactivate the semen by irradiating it with an ultraviolet lamp. ④Artificially inseminate the inactivated semen with the eggs. ⑤Hatch the obtained newly hatched larvae. ⑥The golden snakehead fish obtained by the breeding method of the present invention.

[0039] Example 2

[0040] (1) Select healthy and mature female and male parental snakehead fish. The female parental fish is subjected to induced spawning treatment, with an intracoelomic injection of LRH-A 24 μg / kg, HCG 400 IU / kg, and DOM 2 mg / kg. The male parental fish is subjected to induced spawning treatment, with an intracoelomic injection of LRH-A 22 μg / kg, HCG 200 IU / kg, and DOM 1 mg / kg. The induced spawning female and male parental fish are stocked in the grid and stimulated with running water for 32 h. The male fish chases the female fish. Then, take the testis, cut it into pieces, add sperm preservation solution, and grind it to collect semen. Gently press the abdomen of the female fish (the abdomen is enlarged, soft, and the genital pore is red and swollen). When egg grains flow out, collect the eggs. Gently hold the female fish with its abdomen facing up, and start from the tail with your finger, gently push forward along the abdomen. This can promote the movement of the eggs towards the genital pore. When reaching near the genital pore, apply a little more force, but keep the movement gentle to avoid hurting the fish. Repeat several times until all the fish eggs are discharged, and then collect the eggs.

[0041] (2) Mix the semen with the sperm preservation solution at a volume ratio of 1:3 for dilution. Apply the diluted semen on a culture dish with a diameter of 15 mm at a thickness of 1 mm, place it on ice, and shake it at 100 r / min for cultivation. During this period, in a light-proof environment, irradiate it with 4 ultraviolet lamps of 40 W for 40 s. The perpendicular distance between the ultraviolet lamp and the semen surface is 30 cm. Check the sperm motility every 15 s. It is found that the sperm motility is significantly weakened. If the sperm is in a static state, add a drop of water. When more than 90% of the sperm rotate or move in a curve and no more than 10% of the sperm move in a straight line, it is considered that more than 90% of the inactivated sperm lose their motility, and thus the inactivated semen is obtained.

[0042] (3) Add the inactivated semen to the eggs, and gently stir it with a goose feather rod for 2 min to activate fertilization, and then add aerated water to obtain fertilized eggs.

[0043] (4) The fertilized eggs are subjected to cold shock treatment in an ice-water mixture at 0 - 4 °C for 20 min, and then hatched in water at 25 °C for 32 h to obtain newly hatched larvae.

[0044] (5) The newly hatched larvae start to open their mouths in aerated water at 25°C after 96 hours. After the newly hatched larvae swim horizontally to become fry, rotifers and cladocerans are fed to them 4 times a day, at 7:00, 10:00, 14:00, and 17:00 respectively. After 7 days of feeding biological bait, artificial feed is fed. One week later, they are placed in a culture pond for breeding. A water filtration box is equipped in the culture pond, connected to an 80W water pump, and circulated 24 hours. When the fry grow to 2 cm, yellow color gradually appears on their bodies. When they grow to 5 cm, obvious and stable golden body patterns appear throughout their bodies, and golden snakehead fish are obtained.

[0045] Example 3

[0046] (1) Select healthy and mature snakehead parent female fish and parent male fish. The parent female fish is subjected to induced spawning treatment, with an intracoelomic injection of LRH-A 26 μg / kg, HCG 500 IU / kg, and DOM 3 mg / kg. The parent male fish is subjected to induced spawning treatment, with an intracoelomic injection of LRH-A 23 μg / kg, HCG 250 IU / kg, and DOM 1.5 mg / kg. The induced-spawning parent female fish and the induced-spawning parent male fish are stocked in a grid and stimulated by running water for 40 hours. The male fish chases the female fish, then the testis is taken, cut into pieces, added to sperm preservation solution and ground, and semen is collected. Gently press the abdomen of the female fish (the abdomen is enlarged, soft, and the genital pore is red and swollen). When egg grains flow out, eggs are collected. Gently hold the female fish with its abdomen facing up, and gently push forward along the abdomen from the tail with fingers. This can prompt the eggs to move towards the genital pore. When reaching near the genital pore, apply a little more force, but keep the movement gentle to avoid hurting the fish. Repeat several times until all the eggs are discharged, and the eggs are collected.

[0047] (2) Take the semen and mix it with sperm preservation solution at a volume ratio of 1:5 for dilution. The diluted semen is smeared on a culture dish with a diameter of 15 mm at a thickness of 1 mm, placed on ice and shaken at 140 r / min for cultivation. During this period, in a light-proof environment, 8 ultraviolet lamps of 20W are used to irradiate for 20 s. The vertical distance between the ultraviolet lamp and the semen surface is 20 cm. The sperm motility is checked every 15 s. When it is found that the sperm motility significantly weakens, if the sperm is in a static state, add a drop of water. When more than 90% of the sperm rotate or move in a curve, and no more than 10% of the sperm move in a straight line, it is considered that more than 90% of the inactivated sperm lose their motility, and the inactivated semen is obtained.

[0048] (3) Add the inactivated semen to the eggs, and gently stir for 4 min with a goose feather rod to activate fertilization, then add aerated water to obtain fertilized eggs.

[0049] (4) The fertilized eggs are subjected to cold shock treatment in an ice-water mixture at 0 - 4°C for 40 min, and then hatched in water at 27°C for 28 h to obtain newly hatched larvae.

[0050] (5) The newly hatched larvae start to open their mouths in aerated water at 27°C for 120 hours. After the newly hatched larvae can swim horizontally to become fry, they are fed rotifers and cladocerans 4 times a day, at 7:00, 10:00, 14:00, and 17:00 respectively. After 7 days of feeding biological bait, artificial feed is fed. One week later, they are put into the breeding pond for breeding. The breeding pond is equipped with a water filter box and connected to an 80W water pump for 24-hour circulation. When the fry grow to 2 cm, yellow color gradually appears on their bodies. When they grow to 5 cm, obvious and stable golden body patterns appear all over their bodies, and the golden snakehead is obtained.

[0051] Table 1 Comparison between the production method of the golden snakehead of the present invention and the production method of the common snakehead

[0052]

[0053] The golden snakehead is obtained for the first time in the present invention, and only the common snakehead can be obtained by conventional breeding techniques.

[0054] The above embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for producing seeds of Channa chrysogenum, characterized in that: The following steps are involved: Step (1) selecting mature female and male parent fish of Channa maculata, respectively inducing spawning of the female and male parent fish, subjecting the female and male parent fish to water flow stimulation after induced spawning, and then collecting eggs and semen; Step (2) diluting the semen obtained in step (1), shaking and culturing the diluted semen, irradiating it with ultraviolet light, and observing that more than 90% of the inactivated sperm lose their motility, thereby obtaining inactivated semen; Step (3) adding the inactivated semen obtained in step (2) to the eggs obtained in step (1), stirring to activate fertilization, and adding aerated water to obtain fertilized eggs; Step (4): cold shock treatment of the fertilized eggs obtained in step (3), followed by hatching to obtain newly hatched fry; Step (5) The newly hatched fry of step (4) are obtained after opening for 96 to 120 hours, and are fed with biological bait and artificial feed for breeding. When the fry grow to 2 cm, their bodies begin to gradually turn yellow. When they grow to 5 cm, they show obvious and stable golden body patterns all over their bodies, thus obtaining golden spotted snakehead.

2. The seed production method according to claim 1, characterized in that: The induced spawning treatment of the parent female fish in step (1) is specifically as follows: injecting luteinizing hormone releasing hormone A2, chorionic gonadotropin and dioxigenin maleate into the body cavity of the parent female fish, wherein the injection amount of luteinizing hormone releasing hormone A2 is 4 to 6 μg / kg, the injection amount of chorionic gonadotropin is 400 to 500 IU / kg, and the injection amount of dioxigenin maleate is 2 to 3 mg / kg; The induced spawning treatment of the parent male fish in step (1) is specifically as follows: the parent male fish is injected with luteinizing hormone releasing hormone A2, chorionic gonadotropin and dioxigenin maleate into the body cavity, the injection amount of luteinizing hormone releasing hormone A2 is 2-3 μg / kg, the injection amount of chorionic gonadotropin is 200-250 IU / kg, and the injection amount of dioxigenin maleate is 1-1.5 mg / kg.

3. The seed production method according to claim 1, characterized in that: The duration of the water stimulation in step (1) is 32 to 40 hours.

4. The seed production method according to claim 1, characterized in that: The step (2) of diluting the semen obtained in step (1) is specifically as follows: the semen obtained in step (1) is mixed with a sperm preservation solution in a volume ratio of 1:2 to 6; The step (2) of shaking and culturing the diluted semen and irradiating with ultraviolet light during the process is as follows: smearing the diluted semen on a culture dish with a thickness of ≤1 mm, placing it on an ice surface and shaking and culturing it at 100-140 r / min, during which time, irradiating it with 4-8 20-40 W ultraviolet lamps for 20-40 seconds in a light-proof environment, with the vertical distance between the ultraviolet lamp and the surface of the semen being 20-30 cm; More than 90% of the sperm after inactivation in step (2) lose their motility, specifically: >90% of the sperm move in circles or curves, and ≤10% of the sperm move in a straight line.

5. The seed production method according to claim 1, characterized in that: The stirring in step (3) is performed using a goose feather rod, and the stirring time is 2 to 4 minutes.

6. The seed production method according to claim 1, characterized in that: The cold shock treatment environment in step (4) is an ice-water mixture at 0 to 4° C., and the cold shock treatment time is 20 to 40 minutes.

7. The seed production method according to claim 1, characterized in that: The incubation environment in step (4) is 25-27° C. water, and the incubation time is 28-32 hours.

8. The seed production method according to claim 1, characterized in that: The environment in which the newly hatched fry are waited for to hatch in step (5) is water at 25 to 27°C.

9. The seed production method according to claim 1, characterized in that: The biological feed breeding described in step (5) specifically includes: feeding biological feed 4 times a day, wherein the biological feed is freshwater rotifers and / or cladocerans, and feeding artificial feed after feeding the biological feed for 7 to 8 days.

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