A medium for insemination of goldfish and a method for artificial insemination and hatching of goldfish

Abstract

The present application belongs to the technical field of aquaculture, and particularly relates to a goldfish insemination medium and a method for artificial insemination and hatching of goldfish. The specific implementation steps are as follows: selecting female and male parent fish which are in the process of tail chasing reproduction, squeezing the abdomen of the fish to discharge the eggs and sperm into the insemination medium, and stirring and mixing the artificial insemination; transferring the eggs to a mucus removal medium to remove the mucus of the eggs; placing the mucus-removed eggs into a hatching bag after simple rinsing; and transferring the hatched fry into a rearing pond for feeding. The suitable osmotic pressure and egg white protein environment provided by the insemination medium can significantly prolong the duration of the activity of goldfish eggs and sperm, and improve the effect of increasing the fertilization rate of the eggs; the mucus removal medium is used to remove the mucus of the eggs, so as to avoid the mutual adhesion of the eggs into groups or clusters, improve the hatching rate of the goldfish, and enable the hereditary ornamental traits of the goldfish to fully develop, while preventing pathogenic microorganisms from being transmitted from the parent generation to the offspring generation. The selection of the hatching bag provides a good hatching environment for the eggs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquaculture, and relates to a method for artificial insemination and hatching of fish, in particular to a goldfish insemination medium and a method for artificial insemination and hatching of goldfish. BACKGROUND

[0002] Goldfish is a popular ornamental fish, and has been bred in China for more than 1,000 years, having important ornamental value and economic and cultural value. In the past, goldfish breeding was usually carried out by natural reproduction in an artificial environment, and ordinary breeding water was used as the insemination medium. The fertilization activity duration of goldfish sperm in a low-osmotic water body is very short, and the fertilization rate and hatching rate cannot be guaranteed. At the same time, goldfish eggs are sticky eggs, that is, after being activated by water, the surface of the fish eggs produces sticky substances, which can randomly adhere to the surrounding objects, and the fish eggs can also adhere to each other, often resulting in the situation that the fish eggs are stuck together, making it very difficult to remove unfertilized dead eggs. Unfertilized dead eggs often rot and deteriorate in the late hatching period, breeding water mold and bacteria, causing the surrounding normally developing embryos to be infected and die, resulting in a decrease in the hatching rate, and even the whole destruction. The sticking of fertilized eggs also reduces the surface area of fertilized eggs in contact with water to obtain dissolved oxygen in water, causing the fertilized eggs to lack oxygen during development, resulting in slow, stagnant, deformed, and even dead embryos.

[0003] Chinese patent application (CN1742688A) relates to a method for artificial insemination of Chinese sturgeon and a diluent used therein, and discloses a diluent as an insemination medium, which is prepared by adding the following substances and their contents to 1000ml of distilled water: NaCl 6.78g, KCl 1.56g, CaCl2·2H2O 0.20g, MgCl·6H2O 0.22g, NaHCO3 0.30g, Na2CO3 0.14g, NaOH 0.18g, citric acid 0.10g, penicillin 800,000 units, and streptomycin 800,000 units.

[0004] However, the above-mentioned diluent is prepared by referring to the blood composition of Chinese sturgeon, and cannot be applied to artificial fertilization and hatching of goldfish. SUMMARY

[0005] In order to solve the problems existing in the current artificial breeding process of goldfish, the present application provides a goldfish insemination medium and a method for artificial insemination and hatching of goldfish, so as to significantly prolong the activity duration of goldfish eggs and sperm, avoid the mutual adhesion of fish eggs into groups or clusters, and thus improve the fertilization rate and hatching rate.

[0006] The present application adopts the following technical solutions:

[0007] An artificial insemination medium for goldfish, which is an aqueous solution containing sodium chloride (55-65 mmol / L), potassium chloride (1.0-1.5 mmol / L), calcium chloride (0.3-0.5 mmol / L), sodium bicarbonate (5.0-10 mmol / L), glycine (2.0-5.0 mmol / L), glucose (2.0-5.0 mmol / L), urea (3.0-5.0 mmol / L), glycerol (1.0-3.0 mmol / L) and egg white (5-20 mL / L).

[0008] Preferably, the artificial insemination medium is prepared by the following method: oxygenate tap water or purified water for more than 20 minutes, then add appropriate amounts of sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, glycine, glucose, urea, glycerol and egg white, and mix them evenly while adding, until the substances are completely dissolved.

[0009] Preferably, the artificial insemination medium further contains 50,000 units of penicillin sodium and 50,000 units of streptomycin sulfate.

[0010] An artificial insemination method for goldfish, which comprises the following steps:

[0011] Separating the female and male parent goldfishes to be ovulated and to release sperm, and placing them separately;

[0012] Taking out the female and male parent goldfishes respectively, and squeezing the abdomen of the fish to release the eggs and sperm in the artificial insemination medium described above, while stirring the artificial insemination medium;

[0013] After 5-15 minutes, take out the eggs and put them into a desmucification solution for 3-5 minutes, wherein the desmucification solution is an aqueous solution containing tannin (0.2-1.0 mmol / L), gallic acid (2.0-4.0 mmol / L), sodium chloride (50-70 mmol / L), sodium bicarbonate (10-15 mmol / L) and glucose (1.0-2.0 mmol / L); wherein tannin and gallic acid can rapidly react with proteins at room temperature to denature the proteins on the surface of the eggs, thereby achieving the effect of desmucification, and the simultaneous presence of tannin and gallic acid makes the desmucification effect better; sodium chloride, sodium bicarbonate and glucose mainly play the role of maintaining osmotic pressure, thereby ensuring that the desmucification of the eggs is complete. In addition, tannin and gallic acid also have the function of killing viruses, bacteria, molds and protozoa, thereby preventing pathogenic microorganisms from being transmitted from the parent to the offspring

[0014] Taking out the eggs, rinsing them and obtaining the fertilized eggs of goldfish.

[0015] Preferably, the desmucification solution is prepared by the following method: oxygenate tap water or purified water for more than 20 minutes, then add appropriate amounts of tannin, gallic acid, sodium chloride, sodium bicarbonate and glucose in sequence, and mix them evenly while adding, until the substances are completely dissolved.

[0016] Preferably, the rinsing is performed in pre-oxygenated tap water or purified water.

[0017] A method for hatching goldfish, comprising the steps of:

[0018] The goldfish eggs as described above are placed in a hatching bag, the bag is sealed, the oxygen content in the air inside the bag is above 40%, the volume ratio of water to the eggs is above 5:1, and the volume ratio of air to water is above 2:1.

[0019] Preferably, the water flowers obtained after hatching are transferred to a rearing pond, and feeding is performed after the water flowers swim.

[0020] Preferably, after all the water flowers swim, the larvae of the abundant year shrimp hatched from the abundant year shrimp fine eggs are used as the opening feed, and the larvae of the abundant year shrimp hatched from the abundant year shrimp large red eggs are used for feeding after two days.

[0021] Preferably, the hatching bag contains methylene blue with a concentration of 1-3 mg / L, and the water in the hatching bag is changed once a day, and the dead eggs with blue color or opaque yolk are removed during the water changing until the eggs are hatched.

[0022] Preferably, the hatching temperature of the eggs of the egg type goldfish is 18-22 degrees Celsius, and the hatching temperature of the eggs of the text type and the grass type goldfish is 20-25 degrees Celsius.

[0023] The beneficial effects of the present application are as follows:

[0024] The sperm medium composition for fish egg fertilization provided by the present application refers to the chemical composition of the goldfish ovary fluid, provides a suitable osmotic pressure, and contains egg white protein environment, which can significantly prolong the duration of goldfish egg and sperm activity, and improve the effect of fish egg fertilization rate. In the sperm medium, the parent fish eggs and sperm are squeezed, the time of the parent fish leaving the water surface stress is reduced, and the egg white lysozyme in the sperm medium can kill bacteria and avoid postpartum infection of the parent fish, thereby improving the survival rate of the parent fish. The fish eggs are de-mucilaged by the de-mucilaging liquid, which can prevent the fish eggs from adhering to each other to form groups or clusters, prevent the fish eggs from adhering to each other and other objects, increase the egg surface in contact with the dissolved oxygen in water, significantly improve the hatching rate of goldfish, make the heritable ornamental traits of goldfish fully develop, and make the hatching time more synchronized and the malformation rate of the fry significantly reduced. In addition, the de-mucilaging liquid also has the functions of killing viruses, bacteria, molds and protozoa, and preventing pathogenic microorganisms from being transmitted from the parent to the offspring. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The de-mucilaged fish eggs of the TAI SHI goldfish taken from the sperm medium

[0026] Figure 2 The fertilized eggs in the hatching bag placed indoors are hatching

[0027] Figure 3 Guppy water flower hatched out of shell in incubation bag

[0028] Figure 4 Lionhead goldfish water flower swimming on the third day after hatching DETAILED DESCRIPTION

[0029] In order to make the above features and advantages of the present application more obvious and easy to understand, the core steps and examples are specifically given below, and are described in detail as follows with the aid of drawings.

[0030] The present application mainly includes the following core steps and principles

[0031] Preparation of insemination medium: 10 liters of tap water is prepared, and is fully aerated for more than 20 minutes, and then sodium chloride (55-65 mmol / L), potassium chloride (1.0-1.5 mmol / L), calcium chloride (0.3-0.5 mmol / L), sodium bicarbonate (5.0-10 mmol / L), glycine (2.0-5.0 mmol / L), glucose (2.0-5.0 mmol / L), urea (3.0-5.0 mmol / L), glycerol (1.0-3.0 mmol / L) and egg white (5-20 ml / L) are added, and are stirred and mixed until the substances are completely dissolved, and then are left for standby.

[0032] Preparation of mucus removal medium: 5 liters of tap water is prepared, and is fully aerated for more than 20 minutes, and then tannin (0.2-1.0 mmol / L), gallic acid (2.0-4.0 mmol / L), sodium chloride (50-70 mmol / L), sodium bicarbonate (10-15 mmol / L) and glucose (1.0-2.0 mmol / L) are added, and are stirred and mixed until the substances are completely dissolved, and then are left for standby.

[0033] Preparation of incubation bag: the incubation bag is made of PP material plastic bag with good sealing property, no water leakage and no air permeability. The tap water which is fully aerated in advance is injected into the incubation bag, 3 mg of methylene blue is added into the tap water, and the incubation bag is continuously aerated for standby. After each hatching is completed, the incubation bag is cleaned and disinfected.

[0034] Isolation of parent fish: the parent fish which is chasing the tail is taken out for isolation, and the male and female fish are respectively placed in different isolation nets to avoid the male fish continuing to chase the female fish.

[0035] Artificial insemination: the fish is placed into the insemination medium, the abdomen of the fish is gently squeezed with the thumb, the fish eggs are fully released, 20 microliters of sperm are released, and the fish eggs and sperm are stirred and mixed, and the process is continued until the female fish no longer releases mature fish eggs. After the fish eggs are completed in the insemination medium for 5 minutes, the next mucus removal step is entered. The female fish after artificial ovulation needs to be isolated and fed in the original pool on the same day of breeding, and is released into the fish group near the night.

[0036] De-adhesion: The fertilized eggs in the fertilization medium are scooped out with a skimmer and put into the de-adhesion liquid, and gently mixed and stirred for 5 minutes to make the eggs fully de-adhesion.

[0037] Hatching: The de-adhesion eggs are scooped out with a skimmer, rinsed in pre-oxygenated tap water, and then put into a pre-prepared hatching bag for hatching. After the eggs are put in, the bag is filled with oxygen from an oxygen cylinder, and the oxygen content in the bag is controlled to be more than 40%, the volume ratio of water to eggs is more than 5:1, the volume ratio of gas to water is more than 2:1, and the bag opening is tied with a rubber band; during the hatching process, the eggs are distributed as single layer as much as possible, and the water is changed once a day. The hatching bag is placed in a room with air conditioning for hatching. On the 2nd to 4th day after fertilization, dead eggs, including unfertilized eggs and eggs that have developed too maturely or not maturely in the mother's body, are selected and removed during each water change. Dead eggs appear as methylene blue is immersed in the eggs, the color of the eggs turns blue, or the yolk becomes opaque. Normally developed fertilized eggs are full of granules, transparent and clear, and methylene blue cannot be immersed in the eggs. During the egg picking process, transparent glassware can be used to hold the eggs, white paper is used as the background, and the eggs are carefully selected in batches. There are very few new dead eggs in the following days.

[0038] Rearing: After all the goldfish fry are hatched out of the shell, they are transferred to the rearing pond for feeding. The water flower opening feed of goldfish is preferably the Daphnia magna larvae hatched from Daphnia magna fine eggs, and then Daphnia magna larvae hatched from Daphnia magna large red eggs are used after 1-2 days, and then powder feed or small granular feed, and live water flea and other goldfish feeds are gradually added.

[0039] The present application is further illustrated by the following examples, but the scope of protection of the present application is not limited thereto.

[0040] Example 1: Efficient breeding of traditional red lionhead goldfish

[0041] Select will be tailing two-year-old traditional red lionhead goldfish female 3, male 2, put into the insemination medium, extrusion abdominal ovulation and sperm, artificial insemination. 10 minutes later, with a strainer fish eggs in the insemination medium out, put into the desmoplastic, gently mixed and stirred 3 minutes, so that the eggs fully desmoplastic, pre-oxygenated tap water rinse, count 32506 eggs. Select a 90 cm * 50 cm ornamental fish PE bag, add 2.5 liters of fully oxygenated tap water, drop a small amount of methylene blue (final concentration of about 3 mg / L), put into the desmoplastic eggs, tighten the bag, oxygen, the proportion of oxygen in the bag to 80%, with a rubber band tied the bag, the incubation bag flat in the room, air conditioning settings temperature 22 degrees Celsius. The next day, all the eggs into a plastic basin, change the new water, batch selection and remove dead eggs, count 32089 fertilized eggs, the fertilization rate of 98.7%. The eggs back to the incubation bag continue to incubate. The third day, according to the second day of operation to change the water, no new dead eggs, the eggs back to the incubation bag continue to incubate. The fourth day, the eggs hatch, all the fry into a plastic basin, count 32078 tail, the hatching rate of fertilized eggs close to 100%. The fry into the plastic basin, two days later, the fry all flat swim (no body deformity), using the Daphnia magna fine eggs hatched Daphnia magna larvae as the opening bait, two days later using Daphnia magna red egg hatched Daphnia magna larvae feeding, feeding a week later, the fry grow well, no fry died. Compared with the existing traditional red lionhead goldfish breeding method, the method of traditional red lionhead goldfish fertilization rate, hatching rate and fry flat swim rate significantly increased 8-22%.

[0042] Example 2: Efficient breeding of tigerhead goldfish

[0043] Two female and one male two-year-old red veiltail goldfishes which were going to be rear-ended were selected and put into the insemination medium. The ovum and sperm were squeezed from the abdomen, and the insemination was performed by stirring the mixture. After 10 minutes, the fish ovum in the insemination medium was taken out with a strainer, put into the desmucosa, and stirred gently for 3 minutes to make the fish ovum fully desmucosa. The fish ovum was then rinsed in the pre-oxygenated tap water, and the number of the fish ovum was counted to be 15832. A 70cm*40cm ornamental fish PE packaging bag was selected, 1.5 liters of fully aerated tap water was added, a small amount of methylene blue (final concentration about 3mg / L) was added, and the desmucosa ovum was put into the bag. The bag was tightly closed, and the oxygen ratio in the bag was made to be 70%. The bag was placed horizontally in the room, and the temperature was set to be 18 degrees Celsius. The next day, all the fish ovum was poured into a plastic basin, and new water was added. The dead ovum was removed by batch selection, and the number of the fertilized ovum was counted to be 15680, with a fertilization rate of 99.0%. The fish ovum was put back into the incubation bag for further incubation. On the third and fourth days, the water was replaced according to the operation of the second day, and there was no new dead ovum. The fish ovum was put back into the incubation bag for further incubation. On the fifth day, the fish ovum was hatched, and all the fish fry was poured into a plastic basin, and the number of the fish fry was counted to be 15639, with a hatching rate of more than 99.7%. The fish fry was transferred to a breeding pond, and after three days, 15455 fish fry swam horizontally. The fine ovum hatched shrimp larvae of the abundant year shrimp was used as the opening feed, and after two days, the large red ovum hatched shrimp larvae of the abundant year shrimp was used as the feed. After one week of feeding, the fish fry grew well. Compared with the traditional goldfish breeding method, the fertilization rate, hatching rate and horizontal swimming rate of the fish fry were increased by about 10-25%.

[0044] Example 3: Efficient breeding of goldfish with goose head

[0045] Select will be tailgating one-year-old goose head red goldfish 2 female, male fish 1, put into the insemination medium, squeeze the abdomen ovulation and sperm, uniform stirring while extruding. 10 minutes later, the fish eggs in the insemination medium were separated by a strainer, put into the desmoplastic, gently mixed and stirred for 3 minutes, so that the fish eggs were fully desmucilaged, rinsed in pre-oxygenated tap water, counted 4424 fish eggs. Select a 60 cm * 40 cm ornamental fish PE packaging bag, add 1 liter of fully aerated tap water, drop a small amount of methylene blue (final concentration about 3 mg / L), put into the desmoplastic egg, oxygen, make the oxygen ratio of the bag to reach 80%, tighten the bag mouth, with a rubber band tied tightly, the incubation bag is placed in the room, air conditioning set temperature 20 degrees Celsius. The next day, all the fish eggs were poured into a plastic basin, replaced with new water, selected and removed dead eggs, counted 4280 fertilized eggs, the fertilization rate was more than 96.7%. The fish eggs were put back into the incubation bag for further incubation. The third day, according to the operation of the second day to replace the water, there is no new dead eggs, the fish eggs were put back into the incubation bag for further incubation. The fourth day, fry hatched, all water flowers were transferred into a plastic basin, counted 4274 water flowers, the hatching rate of fertilized eggs was close to 100%. The water flowers were transferred into the breeding pond, 4225 water flowers swam after three days, the fine eggs of the shrimps hatched as the opening feed, two days later, the shrimps hatched from the large red eggs were fed, after a week of feeding, the fry grew well. Compared with the traditional breeding method of goose head red goldfish, the method improved the fertilization rate, hatching rate and water flower swimming rate of goose head red goldfish by about 10-25%.

[0046] Example 4: Express delivery test of spermatozoon of TAI SUI goldfish

[0047] Selecting two female and one male two-year-old General Tai-Shi Goldfish that are being tailgated, place them in the insemination medium, squeeze the abdomen to assist in ovulation and sperm, and mix while squeezing. Use a strainer to separate the eggs in the insemination medium, place them in the desiccated mucus, mix for 3 minutes to fully desiccate the eggs, rinse them in pre-oxygenated tap water, and count 20246 eggs. Select a 60cm*40cm ornamental fish PE packaging bag, add 2 liters of pre-aerated tap water, add a small amount of methylene blue (final concentration of about 3mg / L), place the desiccated eggs, tighten the bag opening, aerate to 80% oxygen, use a rubber band to tighten the bag opening, and place the incubation bag horizontally in the room with the air conditioning set to 20 degrees Celsius. The next day, pour the eggs into a plastic basin, replace the water, remove the dead eggs, count 20195 fertilized eggs, and the fertilization rate is over 99.7%. Randomly select 10000 fertilized eggs and place them in a 34cm*63cm ornamental fish PE packaging bag, add 1 liter of water, aerate to more than 60% oxygen, use a rubber band to tighten the bag opening, wrap it with another packaging bag, use a rubber band to tighten the bag opening, and place the packaging bag containing the fertilized eggs in a foam box, send it from Changping, Beijing to Zibo, Shandong by express, and return it directly from Zibo after arrival. The transportation time is 3 days and 14 hours. After returning, open the foam box and incubation bag, and all the eggs have hatched, counting 9999 live fry, with only one loss during the entire transportation process. The live fry are transferred to the breeding pond, and after three days, all 9999 live fry are swimming, using Daphnia magna eggs hatched from Daphnia magna larvae as starter feed for two days, and then using Daphnia magna larvae hatched from Daphnia magna large red eggs for a week, the fry grow well without any deaths. This experiment shows that using desiccated eggs for fry transportation not only saves transportation costs, but also has a very high survival rate.

Claims

1. A goldfish insemination medium, which is an aqueous solution containing sodium chloride at a concentration of 55-65 mmol / L, potassium chloride at a concentration of 1.0-1.5 mmol / L, calcium chloride at a concentration of 0.3-0.5 mmol / L, sodium bicarbonate at a concentration of 5.0-10 mmol / L, glycine at a concentration of 2.0-5.0 mmol / L, glucose at a concentration of 2.0-5.0 mmol / L, urea at a concentration of 3.0-5.0 mmol / L, glycerol at a concentration of 1.0-3.0 mmol / L, and egg white at a concentration of 5-20 ml / L.

2. The insemination medium according to claim 1, characterized in that, The insemination medium is prepared by the following method: A suitable amount of tap water or purified water is aerated for more than 20 minutes, and then a suitable amount of sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, glycine, glucose, urea, glycerol, and egg white are added while stirring until the substances are completely dissolved.

3. A method for artificial insemination of goldfish, comprising the following steps: Separating female and male parent goldfishes to be ovulated and to release sperm; Separately taking out the female and male parent goldfishes, and squeezing the abdomen of the fish to release the eggs and sperm in the insemination medium of claim 1, while stirring the insemination medium; After 5-15 minutes, taking out the eggs, and stirring in a de-mucus solution for 3-5 minutes, wherein the de-mucus solution is an aqueous solution containing tannin at a concentration of 0.2-1.0 mmol / L, gallic acid at a concentration of 2.0-4.0 mmol / L, sodium chloride at a concentration of 50-70 mmol / L, sodium bicarbonate at a concentration of 10-15 mmol / L, and glucose at a concentration of 1.0-2.0 mmol / L; Taking out the eggs, and obtaining the fertilized eggs of goldfish after rinsing.

4. The method of artificial insemination of goldfish according to claim 3, wherein The de-mucus solution is prepared by the following method: A suitable amount of tap water or purified water is aerated for more than 20 minutes, and then a suitable amount of tannin, gallic acid, sodium chloride, sodium bicarbonate, and glucose are added while stirring until the substances are completely dissolved.

5. The method of artificial insemination of goldfish according to claim 3, wherein The rinsing is performed in pre-aerated tap water or purified water.

6. A method for hatching goldfish, comprising the following steps: Placing the fertilized eggs of goldfish obtained by the artificial insemination method of goldfish according to claims 3-5 into a hatching bag, wherein the hatching bag is closed, the oxygen content in the air inside the bag is more than 40%, the volume ratio of water to the fertilized eggs is more than 5:1, and the volume ratio of air to water is more than 2:

1.

7. The method of hatching goldfish according to claim 6, wherein Transferring the water flowers obtained after hatching into a rearing tank, and feeding after the water flowers swim normally.

8. The goldfish hatching method according to claim 7, wherein After all the water flowers swim normally, using the Daphnia magna larvae hatched from the fine eggs of Daphnia magna as the initial feed, and using the Daphnia magna larvae hatched from the large red eggs of Daphnia magna after two days.

9. The goldfish hatching method according to claim 6, wherein The hatching bag contains methylene blue at a concentration of 1-3 mg / L, and the water in the hatching bag is changed once a day, and the dead eggs with blue color or opaque yolk are removed during the water changing until the fertilized eggs are hatched.

10. The method of hatching goldfish according to claim 6, wherein The hatching temperature of the fertilized eggs of egg-type goldfish is 18-22°C, and the hatching temperature of the fertilized eggs of fancy-type and grass-type goldfish is 20-25°C.

Images