Artificial sterile propagation method for rare fishes

Abstract

The invention discloses an artificial sterile propagation method for rare fishes, which belongs to the technical field of rare fish propagation, and comprises the following steps: artificial spawning induction: selecting rare fish parents with good gonad development, and performing combined spawning induction by adopting luteinizing hormone releasing hormone A2, carp pituitary and chorionic gonadotropin as spawning induction medicines; sterile water is added into a stainless steel basin, palm fibers are immersed in the water, female parent fish eggs are squeezed to the position above the palm fibers, male parent semen is squeezed to the positions of the fish eggs, the stainless steel basin is shaken so that the semen can make full contact with the fish eggs, artificial incubation and management are conducted, and the palm fibers are transferred into a transparent incubation film bag with sterile water; and then placing the hatching bag in an air-conditioned room, and transferring the roes to a common water environment with the same temperature for continuous hatching 5 hours before the predicted roes are demoulded until the fries are completely hatched. According to the method, a sterile environment is created, saprolegnia infection is avoided in the fertilized egg development period, and the hatching success rate of fish fries is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of rare fish breeding, and in particular relates to an artificial aseptic breeding method for rare fish. Background Art

[0002] Rare fish parents are very difficult to obtain, and the mothers carry fewer eggs, so artificial insemination has a low success rate under conventional operations. For example, the rare white snapper and small-mouthed white snapper in the Yuanshui River Basin. May to June in southern China is the breeding season for these rare fish, and it is also the active season for Saprolegnia. The biggest problem encountered through conventional artificial insemination is that Saprolegnia infects the eggs and causes hatching failure. Conventional artificial insemination and hatching are both carried out in a bacterial environment. The chance of infection with Saprolegnia during the hatching process is relatively high. Once infected, you may have to wait until next year. Therefore, the success rate of aseptic breeding methods for rare fish during the hatching process will be significantly improved, which is of great significance to the study of rare fish populations and artificial release.

[0003] Saprolegnia is a genus of fungi belonging to the subphylum Mastigomycotinae and the family Saprolegniaceae. It grows on aquatic plants and animals or their remains, and its hyphae are branched without septa. Saprolegnia is widely distributed in natural water bodies, and the probability of fish eggs being infected with Saprolegnia during fish breeding is very high. Therefore, artificial insemination of fish can effectively avoid the Saprolegnia environment and greatly improve the hatching success rate of fish seedlings. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for artificial aseptic breeding of rare fish. The method creates a sterile environment (without water mold) to avoid water mold infection during the development of fertilized eggs and improve the hatching success rate of fish seedlings.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for artificial aseptic breeding of rare fish comprises the following steps:

[0007] Step 1, artificial induction of labor: Catch rare fish in the breeding season of rare fish in the Yuanshui River Basin, select rare fish parents with well-developed gonads, and use luteinizing hormone-releasing hormone A2, carp pituitary and chorionic gonadotropin for combined induction of labor. 12-20 hours after the injection of the drug, the female fish begins to lay eggs and the male fish begins to ejaculate;

[0008] Step 2, artificial insemination: Add sterile water to the container, immerse the palm hair in the water, squeeze the female fish eggs above the palm hair first, then squeeze the male sperm to the position of the fish eggs, shake the container to make the sperm and fish eggs fully contact, then lift the palm hair steadily, discard the sterile water containing sperm, add sterile water again and wash the palm hair 2-3 times in the same way.

[0009] Step 3, artificial hatching and management: the brown hair treated in step 2 is transferred to a transparent hatching film bag added with sterile water, 8-10L of oxygen is injected, the bag mouth is tied tightly to prevent air leakage, and then the hatching bag is placed in an air-conditioned room, the development of the fish eggs is observed and recorded every day, and 5 hours before the expected demoulding of the fish eggs, the fish eggs are transferred to a common water environment at the same temperature for continued hatching until the fry are completely hatched.

[0010] As a further improvement of the above scheme, the dosage of the oxytocin drug is calculated per kilogram of female fish parent, 8-10 μg luteinizing hormone releasing hormone A2, 1 mg carp pituitary, 400-600 units of chorionic gonadotropin, and injected twice, with 1 / 3 of the drug dose in the first injection and 2 / 3 of the drug dose in the second injection, with an interval of 10-12 hours between the two injections. The dose for male fish is halved.

[0011] As a further improvement of the above scheme, the container is a stainless steel basin with a diameter of 35-40 cm, and the palm hair is a 30 cm square, which needs to be disinfected before use, specifically, boiled in water for 5 minutes, and cooled for use.

[0012] As a further improvement of the above scheme, the amount of sterile water added in step 2 and step 3 is 2L and 4-5L respectively. Sterile water preparation: boil water in a commercially available 15L large-capacity steel pot with a lid, boil for 5 minutes, and cool to room temperature without opening the lid for use.

[0013] As a further improvement of the above scheme, in step 2, the container is shaken for 1 minute and then the palm hair is lifted steadily.

[0014] As a further improvement of the above scheme, the transparent incubation film bag has a size of 30×50 cm and needs to be pretreated before use, specifically, soaked in 100 mL of 75% alcohol overnight, poured out the alcohol and then washed twice with 500 mL of sterile water for standby use.

[0015] As a further improvement of the above scheme, in step 3, the temperature of the air-conditioned room is set at 18-22° C., and the fish egg incubation time is 3-5 days.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) the whole artificial insemination process of the present invention adopts aseptic operation, after fertilization, a sterile hatching bag is used for oxygenated hatching, and the eggs are transferred to a sufficient amount of ordinary water for further hatching within 5 hours before the eggs are demolded, so that the eggs are ensured to develop in a sterile environment for most of the time, effectively avoiding infection by water molds, and the eggs are transferred to ordinary water before demolding, so that the water molds will not affect the hatching process of the eggs in a short time;

[0017] (2) The present invention takes out the fish eggs within 5 hours before demoulding and continues to incubate them in a normal water environment. First, a large amount of protein-containing substances will be released when the fry are demoulded, which is easy to cause turbidity and pollution of the water body. Second, the fry are allowed to adapt to the normal water environment in advance. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical solution, the present invention is described in detail below in conjunction with embodiments. The description in this section is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention.

[0019] The container used in the following embodiments is a stainless steel basin with a diameter of 40 cm, and the palm hair is a 30 m square. It needs to be disinfected before use, specifically boiling in water for 5 min and cooling for standby use; the sterile water preparation process used is: boiling water in a commercially available 15L large-capacity steel pot with a lid for 5 min, cooling to room temperature without opening the lid for standby use; the size of the transparent incubation film bag used is 30×50 cm, and it needs to be pretreated before use, specifically soaking in 100 mL of 75% alcohol overnight, pouring out the alcohol and then washing twice with 500 mL of sterile water for standby use.

[0020] Embodiment 1 (small mouth white soft-shelled turtle artificial breeding):

[0021] A method for artificial aseptic breeding of rare fish comprises the following steps:

[0022] Step 1, artificial induction of labor: In the middle and upper reaches of the Yuanshui River, on March 22, 2024, when the river water temperature was 13°C, 13 small-mouthed white turtles were selected. The abdomens of 10 female fish became soft and elastic, and the cloaca became slightly red and enlarged. The heads and sides of the abdomens of the three male broodstock became bright red. When the abdomens were squeezed, white semen flowed out and dispersed when it came into contact with water. At this time, 1 mg of carp pituitary, 28 micrograms of luteinizing hormone-releasing hormone A, and 400 units of chorionic gonadotropin were used per kilogram of female broodstock, and injected twice. The first injection used 1 / 3 of the drug dose, and the second injection used 2 / 3 of the drug dose. The interval between the two injections was 12 hours. The dose of male fish was halved. 16 hours after the injection, the female fish began to spawn and the male fish began to ejaculate;

[0023] Step 2, artificial insemination: add 2 liters of sterile water to a stainless steel basin, immerse the palm hair in the water, squeeze the female eggs of the small-mouthed white turtle to the top of the palm hair, then squeeze the male semen to the position of the eggs, shake the stainless steel basin to make the semen and the eggs fully contact, lift the palm hair steadily after 1 minute, discard the sterile water containing semen, add sterile water to wash, and wash the palm hair 2-3 times in the same operation;

[0024] Step 3, artificial hatching and management: transfer the brown hair to a transparent hatching film bag with 4-5L of sterile water, inject 5-8L of oxygen, tie the bag tightly to prevent air leakage, place the transparent hatching film bag in an air-conditioned room at 18-22℃, observe and record the development of the eggs every day, estimate the hatching and molting time of the eggs, transfer the eggs to a normal water environment at the same temperature 5h in advance and continue hatching until the fry are fully hatched.

[0025] On March 23, 2024, two groups of small-mouthed white turtles were artificially inseminated in a normal environment and in the sterile environment of Example 1, respectively. There were about 2000-3000 fertilized eggs in each group. After 48 hours, mold growth appeared in the fish eggs in the two groups of ordinary environment control groups, and only one group hatched seedlings, with a hatching rate of about 15% and a survival rate of 80%. After 96 hours in the sterile environment, there was no mold growth in both groups, with a hatching rate of 70% and a survival rate of 80%.

[0026] Embodiment 2 (Hunan Huaming artificial propagation):

[0027] A method for artificial aseptic breeding of rare fish comprises the following steps:

[0028] Step 1, artificial induction of labor: On April 28, 2024, when the temperature of the river water rises to 18°C, 15 Xianghua ling parents with better gonad development are selected. Ten mature female Xianghua ling have obviously swollen and elastic abdomens, posteriorly moved ovarian protuberances, and clearly visible cloaca. The tail fins of five male Xianghua ling have a rough touch, and white semen can be squeezed out by pressing the abdomen with hands. For every kilogram of female parent, 1 mg of carp pituitary, 29 micrograms of luteinizing hormone-releasing hormone A, and 600 units of chorionic gonadotropin are used. They are injected twice, with 1 / 3 of the drug dose for the first injection and 2 / 3 of the drug dose for the second injection. The interval between the two injections is 11 hours. 12 hours after the injection of the drug, the female fish begins to spawn and the male fish begins to ejaculate.

[0029] Step 2, artificial insemination: add 2L of sterile water to a stainless steel basin, immerse the palm hair in the water, first squeeze the female eggs of Xianghua ling to the top of the palm hair, then squeeze the male semen to the position of the eggs, shake the stainless steel basin to make the semen fully contact with the eggs, lift the palm hair steadily after 1 minute, discard the sterile water containing semen, add sterile water to wash, and wash the palm hair 2-3 times in the same operation;

[0030] Step 3, artificial hatching and management: transfer the brown hair to a transparent hatching film bag with 4-5L of sterile water, inject 5-8L of oxygen, tie the bag tightly to prevent air leakage, place the transparent hatching film bag in an air-conditioned room at 18-22℃, observe and record the development of the eggs every day, estimate the hatching and molting time of the eggs, transfer the eggs to a normal water environment at the same temperature 5h in advance and continue hatching until the fry are fully hatched.

[0031] On April 29, 2024, two groups of Xianghua ling were artificially inseminated in a normal environment and in the sterile environment of Example 2, respectively, with 4000-5000 fertilized eggs in each group. After 48 hours, mold growth appeared in the fish eggs in the two groups of ordinary environment control groups, and seedlings were hatched in both groups, with a hatching rate of about 35% and a survival rate of 60%. After 72 hours in the sterile environment, there was no mold growth in both groups, with a hatching rate of 90% and a survival rate of 70%.

[0032] Embodiment 3:

[0033] A method for artificial aseptic breeding of rare fish comprises the following steps:

[0034] Step 1: Artificial induction of labor. On May 12, 2024, when the temperature of the river water rises to 22°C, 14 rare white turtles are selected. The abdomens of 10 female fish become soft and elastic, and the cloaca becomes slightly red and enlarged. The abdomens of 4 male broodstock are squeezed, and white semen can flow out, which disperses when it comes into contact with water. At this time, 1 mg of carp pituitary, 10 micrograms of luteinizing hormone-releasing hormone A2, and 500 units of chorionic gonadotropin are used per kilogram of female broodstock. They are injected twice, with 1 / 3 of the drug dose for the first injection and 2 / 3 of the drug dose for the second injection. The interval between the two injections is 10 hours. The dose of male fish is halved. 18 hours after the injection, the female fish begin to spawn and the male fish begin to ejaculate.

[0035] Step 2, artificial insemination: add 2 liters of sterile water to a stainless steel basin, immerse the palm hair in the water, squeeze the female eggs of the rare white turtle to the top of the palm hair, then squeeze the male semen to the position of the eggs, shake the stainless steel basin to make the semen and eggs fully contact, lift the palm hair steadily after 1 minute, discard the sterile water containing semen, add sterile water to wash, and wash the palm hair 2-3 times in the same operation;

[0036] Step 3, artificial hatching and management: transfer the brown hair to a transparent hatching film bag with 4-5L of sterile water, inject 5-8L of oxygen, tie the bag tightly to prevent air leakage, place the transparent hatching film bag in a 24℃ air-conditioned room, observe and record the development of the eggs every day, estimate the hatching and molting time of the eggs, transfer the eggs to a normal water environment of the same temperature 5h in advance and continue hatching until the fry are fully hatched.

[0037] On May 13, 2024, two groups of rare white turtles were artificially inseminated in a normal environment and in the sterile environment of Example 3, with 5,000-6,000 fertilized eggs in each group. After 48 hours, mold growth appeared in the eggs of the two groups in the normal environment, and only one group hatched seedlings, with a hatching rate of about 15% and a survival rate of 50%. In the sterile environment control group, there was no mold growth in both groups for 80 hours, with a hatching rate of 70% and a survival rate of 80%.

[0038] It should be noted that, in this article, the terms: include, contain and any other variants are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. Specific examples are used in this article to illustrate the principle and implementation of the technical solution of the present invention. The above examples are only used to help understand the method of the present invention and its core idea. The above is only a preferred implementation of the present invention. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements, modifications or changes can be made, and the above technical features can also be combined in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the present invention to other occasions without improvement, should be regarded as the protection scope of the present invention.

Claims

1. A method for artificial aseptic breeding of rare fish, characterized in that: The following steps are involved: Step 1, artificial induction of labor: Catch rare fish in the breeding season of rare fish in the Yuanshui River Basin, select rare fish parents with well-developed gonads, and use luteinizing hormone-releasing hormone A2, carp pituitary and chorionic gonadotropin for combined induction of labor. 12-20 hours after the injection of the drug, the female fish begins to lay eggs and the male fish begins to ejaculate; Step 2, artificial insemination: Add sterile water to the container, immerse the palm hair in the water, squeeze the female fish eggs above the palm hair first, then squeeze the male sperm to the position of the fish eggs, shake the container to make the sperm and fish eggs fully contact, then lift the palm hair steadily, discard the sterile water containing sperm, add sterile water again and wash the palm hair 2-3 times in the same way. Step 3, artificial hatching and management: the brown hair treated in step 2 is transferred to a transparent hatching film bag added with sterile water, 8-10L of oxygen is injected, the bag mouth is tied tightly to prevent air leakage, and then the hatching bag is placed in an air-conditioned room, the development of the fish eggs is observed and recorded every day, and 5 hours before the expected demoulding of the fish eggs, the fish eggs are transferred to a common water environment at the same temperature for continued hatching until the fry are completely hatched.

2. The method for artificial aseptic breeding of rare fish according to claim 1, characterized in that: The dosage of the oxytocin drug is calculated per kilogram of female fish parent, 8-10 μg luteinizing hormone releasing hormone A2, 1 mg carp pituitary, 400-600 units of chorionic gonadotropin, which is injected twice, with 1 / 3 of the drug dosage for the first injection and 2 / 3 of the drug dosage for the second injection, with a time interval of 10-12 hours between the two injections, and the dosage for male fish is halved.

3. The method for artificial aseptic breeding of rare fish according to claim 1, characterized in that: The container is a stainless steel basin with a diameter of 35-40 cm, and the palm hair is a 30 cm square. It needs to be disinfected before use, specifically by boiling in water for 5 minutes and cooling for use.

4. The method for artificial aseptic breeding of rare fish according to claim 1, characterized in that: The amount of sterile water added in step 2 and step 3 is 2 L and 4-5 L respectively. Preparation of sterile water: Boil water in a commercially available 15 L large-capacity steel pot with a lid for 5 min, and cool to room temperature without opening the lid for later use.

5. The method for artificial aseptic breeding of rare fish according to claim 1, characterized in that: In the step 2, the container is shaken for 1 minute and then the palm hair is lifted steadily.

6. The method for artificial aseptic breeding of rare fish according to claim 1, characterized in that: The transparent incubation film bag has a size of 30×50 cm and needs to be pretreated before use, specifically, soaked in 100 mL of 75% alcohol overnight, poured out the alcohol and then washed twice with 500 mL of sterile water for standby use.

7. The method for artificial aseptic breeding of rare fish according to claim 1, characterized in that: In step 3, the temperature of the air-conditioned room is set at 18-22° C., and the fish egg incubation time is 3-5 days.